NEUROGLIA

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 NEUROGLIA
THIRD EDITION

Helmut Kettenmann Bruce R. Ransom

MAX DELBRÜCK CENTER FOR MOLECULAR MEDICINE DE PA RTME NT OF N EUR OLO GY

BERLIN, GERMANY U N I V E R S I T Y O F WA S H I N G T O N S C H O O L O F M E D I C I N E

S E AT T L E , WA

1
 3
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Library of Congress Cataloging-in-Publication Data

Kettenmann, Helmut.
Neuroglia / [edited by] Helmut Kettenmann, Bruce R. Ransom. –3rd ed.
p. ; cm.
Includes bibliographical references and index.
ISBN 978–0–19–979459–1 (hardcover : alk. paper)
I. Kettenmann, Helmut. II. Ransom, Bruce R. III. Title.
[DNLM: 1. Neuroglia. 2. Nervous System Diseases—physiopathology. WL 102]
612.8c1045—dc23
2012016025

9 8 7 6 5 4 3 2 1
Printed in the United States of America
on acid-free paper
To our children, Lucas and Georg Kettenmann, and Rebecca, Christopher and Cole Ransom,
who often missed their busy Dads.
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 PREFACE TO THE FIR ST EDITION
Nonneuronal cells, termed neuroglia, were recognized as inde-
pendent elements of the nervous system nearly a century and a
half ago by Virchow. These cells are present in primitive nervous
systems and, undoubtedly driven by positive evolutionary pres-
sures, have persisted in high density and acquired greater diver-
sity in mammals. Knowledge about glial cells has accumulated
at a phenomenal rate in the past 30 years, and has become rel-
evant to all fields of neurobiology. With so much new informa-
tion at hand, we felt that this was an important time to assemble
the facts about these cells as we presently understand them.
Historically, glial cells were viewed as a type of central
nervous system connective tissue whose main function was to
provide support to the true functional cells of the brain, the
neurons. This firmly entrenched concept remained virtually
unquestioned for the better part of a century. But glial cells
are neither connective tissue nor mere supportive cells. In con-
trast to early beliefs, glial cells are now recognized as intimate
partners with neurons in virtually every function of the brain
and as participants in the pathophysiology of the dysfunc-
tional or diseased brain. These cells have been challenging to
study, however, because their functions are not associated with
easily recorded electrical signals, as is the case with neurons.
While books about the nervous system have grown in size
and complexity, attempting to accommodate the frantic pro-
duction of new neuroscience information, the incorporation of
new facts about glia has not kept pace. One simply cannot learn
about glial cells by turning to the typical neuroscience text-
book (From Neuron to Brain by Nicholls, Martin, and Wallace,
is a notable exception). This curious fact has also been a moti-
vation for bringing together in the present volume a detailed
summary of what is currently known about these cells. It will,
we hope, also encourage better integration of the glial and neu-
ronal information bases, which each suffer in the absence of the
other. The brain cannot be understood as the functional sum of
two isolated cellular compartments; it must, we think, be seen
as a single entity containing neurons and glial cells working in
seamless harmony with one another. Somehow, this essential
message has gone too long undelivered.
Glial research is at a particularly exciting point in its
evolution. Great advances in our knowledge about nervous
system diseases have opened the door for thinking about
the role of glial cells in the pathogenesis of these conditions
and in their treatment. Therapies that would literally have
been the stuff of science fiction only a decade ago are now in
advanced stages of testing. Patients with Parkinson’s disease
who no longer respond to our best medicines, for example,
have received brain tissue transplants, whose effectiveness
vii
may be enhanced by including glial cells as factories for the
production of trophic substances. Glial transplants to refur-
bish areas of demyelination may also be possible in the near
future. Our capacity to measure the brain’s functional mol-
ecules and determine their cellular topography has revealed
a baffling array of neurotransmitters, receptors, ion chan-
nels, adhesion molecules, and trophic factors associated with
glial cells. These findings are stimulating and broadening the
field of glial research. They provide critical insights into how
neurons and glial cells might communicate with each other,
and reveal an astonishing overlap between the features of the
brain’s two principal cell types that would have been heresy
not long ago.
The many experts who wrote chapters for this volume
contributed in other valuable ways as well. Before the writ-
ing began, they provided invaluable advice about what topics
should be covered. In an unselfish manner, they adjusted the
scope of their individual contributions so that they fit the con-
text of the book as a whole. To enhance the quality and utility
of the chapters, each underwent a stage of peer review and this
was cheerfully provided by other authors. The editorial burden
was significantly lightened by the satisfaction of dealing with
this uniquely talented and energetically committed group of
authors. They share our view of the importance of develop-
ing a compendium volume about glial cells and continuously
reinforced our enthusiasm for the project as it moved forward.
We acknowledge their essential partnership in the making of
Neuroglia and thank them for their efforts.
One point should be made in concluding. As impressive as
our gains in glial cell knowledge have been, the best is yet to
come. Glial researchers have struggled with our own version
of the Heisenberg uncertainty principle: how to study the role
of glial cells in the multicellular actions of the nervous system
without interfering with the very functions we wish to under-
stand. Our initial efforts were a compromise. We retreated
somewhat from the immense complexity of the intact nervous
system in favor of simplified preparations that allowed more
rigorous study. This reductionistic approach has produced a
mountain of provocative information, as detailed here, but
few definitive answers. Consequently, we have long lists of
glial cell properties, while the list of proven functions is small.
But starting with these demonstrated properties, testable
hypotheses of glial cell function can now be formulated with
improved precision, taking full advantage of new or refined
research technologies. A rich yield of vital new insights into
the functions of neuroglia should follow, and future editions
of this book will survey those benefits.
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 PREFACE TO THE SECOND EDITION
Almost 10 years have passed since the first edition of Neuroglia
was published. Neuroglia was warmly received and we were
pleased that it soon became a standard reference work in this
field. The pace of glial research has continued to accelerate,
however, and we could not ignore the obvious need for a fresh
assessment and summary of this field.
The need for a comprehensive and contemporary book
on glial cells has never been greater. All of the motivating fac-
tors mentioned in the preface to the first edition continue to
operate, including the lack of glial coverage in general neuro-
science textbooks. In fact, the practical impact of the second
edition of Neuroglia may be greater than the first because it
is offered at a time when a majority of neuroscientists would
acknowledge that neuroglia are elemental to most, if not all,
brain functions. This was certainly not the case when the first
edition appeared.
This book is not simply an edited version of the first edi-
tion. We started with the proverbial “clean sheet” of paper in
planning the second edition. Based on advice solicited from
many of our colleagues, the book was entirely reorganized to
more logically assemble current information about glial cells.
We recruited many new contributors, reflecting the emer-
gence of new topics and new experts in the field. Less than
a fourth of the original chapters were retained, and most of
these were revised so extensively that they are essentially new
contributions themselves. Novel topics appearing in the sec-
ond edition include: transmitter release by exocytosis from
glia, glial derived stem cells, glia and synaptic transmission,
glia and axon guidance, an entire section on mechanisms of
glial injury, and several new chapters about the roles of glia in
different diseases.
ix
In many ways the second edition of Neuroglia was more
challenging to conceive and produce than the first edition.
With affordability in mind, we were committed to a second
edition that was smaller than the first edition. We also wanted
a greater degree of uniformity in terms of subject treatment
than was true for the first edition. We struggled in the planning
stages with how to condense topic areas without sacrificing
crucial content. Accordingly, we urged authors to be concise,
and we limited the number of citations and edited each chap-
ter based on reviews provided by other contributors. But the
universe of relevant knowledge about glial cells is much larger
today and the need to include new topics while adhering to a
strict page budget came at a price. Some “classic” topics covered
in detail in the first edition such as glial anatomy and ion chan-
nel expression were significantly condensed, and the first edi-
tion of Neuroglia will remain useful for exactly that reason.
We predicted in the first edition preface that “As impres-
sive as our gains in glial cell knowledge have been, the best is
yet to come.” The second edition of Neuroglia thoroughly doc-
uments our progress in understanding these cells and vindi-
cates this statement, but we readily admit that this prediction
remains true for the future. Not so long ago it was imagined
that glia represented a functionally uniform cell population.
This concept was discarded as we recognized that glia are a
diverse and complex cell family whose only common feature
is that they are not neurons. Situational plasticity and regional
variability in these cells are emerging themes that promise to
further enlarge their range of functions. But the path to the
future always starts with a current and accessible base of infor-
mation. We believe the second edition of Neuroglia provides
this important starting point.
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 PREFACE TO THE THIRD EDITION
Good reasons must exist to do a third edition of a book.
After two editions, the project’s novelty has worn off and any
delusions about the glamorous life of a book-editor are dead,
replaced by the grinding reality of assembling and editing a
creation with more than a hundred ‘parents’. We looked hard
at the past editions of Neuroglia and asked ourselves if a new
edition could possibly be worth the effort. ‘Sprucing up’ the
second edition was not a sufficient reason, even if this might
prove commercially successful. We were simply not motivated
by the notion of a renewable textbook franchise. What finally
pushed us to action were two undeniable facts: the exciting,
upward trajectory of knowledge about glial cells, and the con-
tinued void of comprehensive treatises on this subject.
Glial biology as a field has entered a period of extraordi-
nary growth and evolution. The number of glial research arti-
cles published annually continues to increase, and, while there
is no precise metric for judging their scientific impact, it is safe
to say that this is rising as well. Some recent developments pro-
vide evidence of this growing vitality. It is now established that
in addition to astrocytes, microglia and oligodendrocytes,
a fourth major class of glial cells exists in the brain, termed
NG2 cells. The roles of glia in disease are becoming clearer,
and remarkably there are no apparent exceptions to the rule
that glial cells participate in all brain pathologies. This is a
particularly fast-paced area of glial research and one likely to
yield unexpected therapeutic targets for diseases with no pres-
ent cures and few treatments. Glia may even play important
mechanistic roles in psychiatric diseases. New findings tell us
that glial cells modulate complex brain-mediated behaviors
like memory, learning and sleep. These few examples illustrate
the wide range and compelling novelty of the field, and help
explain why we acceded to the challenge of creating this third
edition of Neuroglia. Collectively, this scientific progress has
led to a state of current knowledge that is transformative. Many
traditional concepts about glial cells will need to be discarded
or revised in response to this flood of new information. An
overarching principle that is now unassailable is that nervous
system function is best characterized as a partnership between
glia and neurons.* We hope that this book will faithfully trans-
mit the powerful sense among ‘gliologists’ of forward momen-
tum in understanding glial functions, and in so doing that it
will attract and inspire the next generation of glial scientists.
* The long-held practice of referring to glia as ‘support cells’, therefore, should be
abandoned in favor of the term ‘partner cells’.
** We especially want to recognize the superb work of Birgit Jarchow, Cheryl
Hutton and Lisa Leukel.
xi
We are calling this book ‘Neuroglia, the third edition’, but
the massive revision and redesign from the second edition
could have justified a new name altogether. The changes are
too numerous to detail but some general comments will serve
to illustrate the point. The number of individual chapters has
been expanded from 47 to 71, a 50% increase, and very few of
the chapters have been spared a new name and altered content
range. At the same time, we pushed for more concise chap-
ters with a consistent level of presentation. More than 50 new
authors contributed to this edition guaranteeing the incorpo-
ration of new perspectives and knowledge. An unanticipated
benefit of so many new contributors was the lift provided by
their genuine and contagious enthusiasm for this project.
What is now known about glial cells will not conve-
niently fit in a single volume. In planning the third edition
of Neuroglia, difficult decisions had to be made about what
to include, and at what level of detail to present information.
Compromises were made. We chose to include more topic
areas treated generally rather than a narrower range of topics
treated at greater depth. The number of illustrations and refer-
ences had to be limited. Our contributors accepted these con-
straints with good humor. We believe the resulting volume has
achieved the desired balance between breadth, accuracy and
depth to satisfy the broad range of users we had in mind.
Edited volumes vary greatly in terms of style, tone, and
content value. Our contributors graciously agreed to more
stringent standards and oversight than in previous editions in
the interest of a high quality end product. All of the chapters
were commissioned and written within about nine months, a
very ambitious timetable and one that ensured that the con-
tent would be up-to-date and relevant when the book was
published. Preliminary outlines were solicited and posted on
a website for review by all participants in an effort to identify
and eliminate redundancies. Each of the 71 chapters was peer-
reviewed by two knowledgeable experts from among our con-
tributors, and by the editors. Substantial revisions were often
required and no chapter was accepted in final form without
further review. The logistical issues that arose when there were
71 ‘balls in the air’, so to speak, surprised us. The Berlin office**
bore the brunt of the onerous record keeping and communica-
tion, and without their efforts, which were far beyond reason-
able, the third edition would not have appeared on time or in
the well organized form that you see before you.
Craig Panner at Oxford University Press (OUP) believed
in the value of a new edition and was a true partner in its
creation. He did not shy from contentious issues that we
confronted, sought consensus with us on all the main points
regarding production and worked to bring Neuroglia up to
a modern standard. Consequently, the book is destined to
appear as a fully electronic volume as part of OUP’s electronic
publishing program. This feature will appeal to contemporary
users who will value the greatly enhanced access. It also holds
open the possibility that over time important content might
be added as appendices, without the need for formal republi-
cation. Reader feedback will influence future decisions on this
option.
Past prefaces spoke to the importance of glial cells in neu-
roscience. Unconsciously, they offered arguments and justifi-
cations for attention to this subject. We hope you agree that
this is no longer necessary. To do so, in fact, would disrespect
the enlightened perspective of most neuroscientists that glial
xii • P R E FAC E 3 R D E D I T I O N
cells partner with neurons in all brain functions. This point of
view constitutes a paradigm shift. While the first edition of
Neuroglia largely focused on glial properties, the theme of the
third edition, 17 years later, is more on dynamic interactions
and functions. The myriad roles of glia in brain function are in
better focus than ever before, but there are still a vast number
of unsolved issues in this infinitely satisfying puzzle. The suc-
cess of the third edition of Neuroglia will be measured by the
further progress it inspires in our understanding of the harmo-
nious partnership between glia and neurons underlying brain
function, and the partnership discords that produce diseases.
Helmut Kettenmann Berlin, Germany
Bruce R. Ransom Seattle, USA
 CONTENTS

Contributors xvii Physiological Properties

16. Physiology of Astrocytes: Ion Channels
and Ion Transporters 185
SECTION 1 Christian Steinhäuser, Gerald Seifert,
P R O P E RT I E S O F N EU R O G L I A L C E L L S and Joachim W. Deitmer
17. Release of Gliotransmitters and Transmitter
Evolution of Glial Cells: Insights from Non-Mammalian Glia Receptors in Astrocytes 197
Helmut Kettenmann and Robert Zorec
1. Evolution of Glial Cells 5
Christian Klämbt 18. Storage and Release of Nontransmitter Signaling
Molecules from Macroglia 212
2. Invertebrate Glia 12
Oliver von Bohlen und Halbach and Klaus Unsicker
Marc R. Freeman
19. Physiology of Microglia 223
3. Nonmammalian Vertebrate Glia 24
Mami Noda and Alexei Verkhratsky
Bruce Appel
20. Physiology of Oligodendrocytes 238
Morphology, Ultrastructure, and Identification
Vittorio Gallo and Jean-Marie Mangin
4. Astrocytes and Ependymal Glia 35
21. Physiological Properties of NG2+ Glial Cells 254
Andreas Reichenbach and Hartwig Wolburg
Dwight E. Bergles
5. Radial Glial Cells 50
22. Cytokine, Chemokine, and Growth Factor
Magdalena Götz
Receptors and Signaling 266
6. Structure and Function of Oligodendrocytes 62 Erik W. G. M. Boddeke, Bart J. L. Eggen,
Arthur M. Butt and Knut P. H. Biber
7. Schwann Cells and Myelin 74 23. Lipids, Lipid Mediators, and Other
Rudolf Martini and Ágnes Patzkó Signaling Molecules 281
8. Microglial Cells 86 Hideki Hayashi
Wolfgang J. Streit 24. Gap Junctions and Hemichannels 292
9. Pericytes of the Central Nervous System 98 Bruce R. Ransom and Christian Giaume
Martin Krüger and Ingo Bechmann 25. Purinergic Mechanisms in Glial Cells 306
10. NG2 Cells (Polydendrocytes) 109 Margaret S. Ho and Shumin Duan
Akiko Nishiyama 26. Calcium Signaling in Neuroglia 320
11. Glial Cells in Autonomic and Sensory Ganglia 122 Alexei Verkhratsky and Vladimir Parpura
Menachem Hanani and David C. Spray 27. The Central Role of Astrocytes
Lineage and Development in Neuroenergetics 333
12. Astrocyte Development 137 Pierre J. Magistretti and Luc Pellerin
James E. Goldman Genomic Profiles
13. Lineage and Development: Oligodendrocytes 148 28. The Astrocyte Transcriptome 347
Katsuhiko Ono and Kazuhiro Ikenaka Ditte Lovatt and Maiken Nedergaard
14. The Schwann Cell Lineage: Cellular Transitions 29. Gene Expression Patterns of Oligodendrocyte
During Development and After Injury 159 Progenitor Cells and Oligodendroglia 358
Kristján R. Jessen, and Rhona Mirsky Fraser J. Sim and Steven A. Goldman
15. Microglia Lineage and Development 172
Marco Prinz

xiii
 SECTION 2 48. Factors Controlling Microglial Activation 614
F U N C T I O N S O F N EU R O G L I A L C E L L S Uwe-Karsten Hanisch
49. Roles of Activated Microglia 626
Astrocytes Kelly R. Miller, Stefan Prokop, and Frank L. Heppner
30. Neurogenesis and Outer Subventricular Zone Radial 50. Immune Functions of Microglia 638
Glial Cells 379 Trevor Owens
Xiaoqun Wang and Arnold R. Kriegstein
31. Glial Control of Synaptogenesis 388 SECTION 3
Nicola J. Allen ROLE OF GLIAL CELLS IN DISEASE
32. Neuron Migration and Axon Guidance 402
Andreas Faissner
Mechanisms of Glial Injury
33. The Role of Glia in the Formation and Function of the
51. Astrocyte Responses to Central Nervous System Injury
Blood-Brain Barrier 417
and Disease 653
Istvan Krizbai, Imola Wilhelm, Hans-Christian Bauer,
Michael V. Sofroniew
and Hannelore Bauer
52. Metabolic Injury of Oligodendrocytes and Myelin 665
34. Control of the Extracellular Ionic Environment
Peter K. Stys
and Volume 430
Eva Syková 53. Interaction of Microglia with Neurons and Astrocytes
Under Lesioned Neuronal Conditions 677
35. Amino Acid Neurotransmitter Synthesis
Kazuyuki Nakajima and Shinichi Kohsaka
and Removal, 443
Arne Schousboe, Lasse K. Bak, Karsten K. Madsen, 54. Schwann Cells and Injury 687
and Helle S. Waagepetersen Violetta Zujovic and Alexandros A. Lavdas
36. Glycogen and Energy Metabolism 457 Recovery of Neural Function
Angus Brown 55. Nerve Regeneration in the Peripheral
37. Astrocyte Regulation of Neurovascular Control 470 Nervous System, 701
Clare Howarth, Grant R. J. Gordon, Tessa Gordon and Olawale A. R. Sulaiman
and Brian A. MacVicar 56. Nerve Fiber Regeneration in the Central Nervous
38. Astrocytes: Modulation of Synaptic Function and System of Higher Vertebrates 715
Network Activity 481 Anita D. Buchli and Martin E. Schwab
Andrea Volterra 57. Glial Cell Transplantation for Central Nervous
39. Astrocytic Modulation of Mammalian Synapses: System Repair 728
Circuits and Behaviors 494 Anne Baron-Van Evercooren and Rebecca Matsas
Michael M. Halassa and Philip G. Haydon Ischemia
40. Adult Neurogenesis 504 58. Focal Cerebral Ischemia: The Multifaceted Role
Gerd Kempermann of Glial Cells 745
41. Modulation of Neuroendocrine Systems 515 Ulrich Dirnagl, Bruce R. Ransom, and Josef Priller
Stéphane H. R. Oliet Gliomas
Oligodendrocytes/Schwann Cells 59. Malignant Glioma 759
42. Myelin, Impulse Conduction, and the Shannon Donnola Rebecca Bish, and Dolores
Pathophysiology of Demyelination 529 Hambardzumyan
Lakshmi Bangalore and Stephen G. Waxman 60. Glial Tumors in Neurofibromatosis and Tuberous
43. Transcription Factors in Myelinating Cells 543 Sclerosis Complex 772
Michael Wegner Anthony J. Apicelli and David H. Gutmann
44. Factors Controlling Myelin Formation 555 Demyelinating Diseases
Ruth Stassart, Sandra Goebbels, 61. Multiple Sclerosis 785
and Klaus-Armin Nave Monika Bradl and Hans Lassmann
45. Regulation of Myelination by Functional Activity 573 62. Genetic Mutations Affecting Myelin Formation 798
R. Douglas Fields Steven S. Scherer, M. Laura Feltri, and Lawrence Wrabetz
46. Iron and Glia 586 Neurodegenerative Diseases
James R. Connor 63. Amyotrophic Lateral Sclerosis 811
Microglia Rita Sattler and Jeffrey Rothstein
47. Role of Microglia in the Normal Brain 605 64. Alzheimer Disease 825
Frank Kirchhoff Andrew Kraft and Jin-Moo Lee

xiv • CONTENTS
 65. Huntington Disease, Parkinson Disease, and Other 68. Microglia and Pain 876
Neurodegenerative Diseases 837 Simon Beggs, Tuan Trang, and Michael W. Salter
Thomas Möller 69. Genetic Disorders Affecting Astrocytes 884
Infectious Diseases Albee Messing and Michael Brenner
66. Glia in Bacterial and Viral Central Nervous 70. Epilepsy 896
System Infections 847 Detlev Boison
Gwenn Garden 71. Psychiatric Disorders 906
Miscellaneous Diseases Josef Priller
67. Neuroglia in Hepatic Encephalopathy 863
Mireille Bélanger, Javier Vaquero, and Roger F. Butterworth Index 917

CONTENTS • xv
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 CONTRIBUTOR S

Nicola J. Allen Ingo Bechmann

Molecular Neurobiology Laboratory Institute for Anatomy
The Salk Institute for Biological Studies University of Leipzig
La Jolla, CA Leipzig, Germany

Anthony J. Apicelli Simon Beggs

Department of Radiation Oncology Program in Neurosciences & Mental Health
Washington University School of Medicine Hospital for Sick Children
St. Louis, MO Toronto, Canada

Bruce Appel Mireille Bélanger

Departments of Pediatrics and Cell and Developmental Neuroscience Research Unit
Biology Centre Hospitalier de l’Université de Montréal
University of Colorado School of Medicine Montreal, Canada
Aurora, CO
Dwight E. Bergles
Lasse K. Bak The Solomon H. Snyder Department of Neuroscience
Department of Drug Design The Johns Hopkins University School of Medicine
and Pharmacology Baltimore, MD
University of Copenhagen
Copenhagen, Denmark Knut P. H. Biber
Department of Psychiatry and Psychotherapy
Lakshmi Bangalore University of Freiburg Medical Center
Department of Neurology and Center for Neuroscience Freiburg, Germany
Research
Yale University School of Medicine Rebecca Bish
New Haven, CT Center for Genomics and Systems Biology
New York University
Anne Baron-Van Evercooren New York, NY
Centre de Recherche de l’Institut du Cerveau et de Moelle
Epinière Erik W. G. M. Boddeke
Paris, France Department of Neuroscience
University Medical Center Groningen
Hannelore Bauer Groningen, The Netherlands
Department of Organismic Biology
University of Salzburg Detlev Boison
Salzburg, Austria Legacy Research Institute
Portland, OR
Hans-Christian Bauer
Department of Organismic Biology Monika Bradl
University of Salzburg Department of Neuroimmunology
Salzburg, Austria Medical University of Vienna
Vienna, Austria

xvii
Michael Brenner Andreas Faissner
Department of Neurobiology Department of Cell Morphology and Molecular
University of Alabama Birmingham Neurobiology
Birmingham, AL Ruhr-University of Bochum
Bochum, Germany
Angus Brown
School of Biomedical Sciences M. Laura Feltri
University of Nottingham Hunter James Kelly Research Institute and Department
Nottingham, United Kindom of Biochemistry
SUNY at Buffalo, School of Medicine
Anita D. Buchli Buffalo, NY
Brain Research Institute
University of Zurich and Federal Institute R. Douglas Fields
of Technology Zurich Nervous System Development and Plasticity Section
Zurich, Switzerland National Institutes of Health, NICHD
Bethesda, MD
Arthur M. Butt
Institute of Biology and Biomedical Sciences Marc R. Freeman
University of Portsmouth Department of Neurobiology
Portsmouth, United Kingdom University of Massachusetts Medical School
Worcester, MA
Roger F. Butterworth
Neuroscience Research Unit Vittorio Gallo
Centre Hospitalier de l’Université de Montréal Center for Neuroscience Research
Montreal, Canada Children’s National Medical Center
Washington, DC
James R. Connor
Department of Neurosurgery Gwenn Garden
Pennsylvania State University Department of Neurology
Hershey, PA University of Washington
Seattle, WA
Joachim W. Deitmer
Department of General Zoology Christian Giaume
University of Kaiserslautern Centre Interdisciplinaire de Recherche en Biologie
Kaiserslautern, Germany Collège de France
Paris, France
Ulrich Dirnagl
Center for Stroke Research Sandra Göbbels
Charité-Universitätsmedizin Berlin Department of Neurogenetics
Berlin, Germany Max Planck Institute of Experimental Medicine
Göttingen, Germany
Shannon Donnola
Department of Stem Cell Biology and Regenerative James E. Goldman
Medicine Department of Pathology and Cell Biology
Cleveland Clinic Columbia University College of Physicians and Surgeons
Cleveland, OH New York, NY

Shumin Duan Steven A. Goldman

Institute of Neurobiology Department of Neurology and the Center for Translational
Zhejiang University School of Medicine Neuromedicine
Hangzhou, China University of Rochester Medical Center
Rochester, NY
Bart J. L. Eggen
Department of Neuroscience Grant R. J. Gordon
University Medical Center Groningen Department of Physiology and Pharmacology
Groningen, The Netherlands University of Calgary
Calgary, Canada

xviii • C O N T R I B U TO R S
Tessa Gordon Clare Howarth
Department of Surgery Department of Psychiatry and the Brain
The Hospital for Sick Children Research Centre
Toronto, Canada University of British Columbia
Vancouver, Canada
Magdalena Götz
Institute of Stem Cell Research Kazuhiro Ikenaka
Munich University Division of Neurobiology and Bioinformatics
Munich, Germany National Institute for Physiological Sciences
Okazaki, Japan
David H. Gutmann
Department of Neurology Kristján R. Jessen,MSc
Washington University School of Medicine Department of Cell and Developmental Biology
St. Louis, MO University College London
London, United Kingdom
Michael M. Halassa
Department of Psychiatry Gerd Kempermann
Massachusetts General Hospital Center for Regenerative Therapies (CRTD)
Boston, MA Technical University of Dresden
Dresden, Germany
Dolores Hambardzumyan
Department of Stem Cell Biology and Helmut Kettenmann
Regenerative Medicine Max Delbrück Center for Molecular Medicine (MDC)
Cleveland Clinic Cellular Neurosciences
Cleveland, OH Berlin, Germany

Menachem Hanani Frank Kirchhoff

Laboratory of Experimental Surgery Department of Molecular Physiology
Hadassah-Hebrew University Medical Center University of Saarland
Jerusalem, Israel Homburg, Germany

Uwe-Karsten Hanisch Christian Klämbt

Institute of Neuropathology Institute of Neuro- and Behavioural Biology
University of Göttingen University of Münster
Göttingen, Germany Münster, Germany

Hideki Hayashi Arnold R. Kriegstein

Priority Organization for Innovation and Excellence Department of Neurology
Kumamoto University University of California San Francisco
Kumamoto, Japan San Francisco, CA

Philip G. Haydon Istvan Krizbai

Department of Neuroscience Institute of Biophysics
Tufts University School of Medicine Hungarian Academy of Sciences
Boston, MA Szeged, Hungary

Frank L Heppner Shinichi Kohsaka

Department of Neuropathology Department of Neurochemistry
Charité-Universitätsmedizin Berlin National Institute of Neuroscience
Berlin, Germany Tokyo, Japan

Margaret S. Ho Andrew Kraft

Department of Anatomy and Neurobiology Department of Neurology
Tongji University School of Medicine Washington University School of Medicine
Shanghai, China St. Louis, MO

C O N T R I B U TO R S • xix
Martin Krüger Albee Messing
Institute for Anatomy Waisman Center and Department of Comparative
University of Leipzig Biosciences
Leipzig, Germany University of Wisconsin-Madison
Madison, WI
Hans Lassmann
Center for Brain Research Kelly R Miller
Medical University of Vienna Department of Neuropathology
Vienna, Austria Charité-Universitätsmedizin Berlin
Berlin, Germany
Alexandros A. Lavdas
Laboratory of Cellular and Molecular Neurobiology Rhona Mirsky
Hellenic Pasteur Institute Department of Cell and Developmental Biology
Athens, Greece University College London
London, United Kingdom
Jin-Moo Lee
Department of Neurology Thomas Möller
Washington University School of Medicine Lundbeck Research USA
St. Louis, MO Neuroinflammation Disease Biology Unit
Paramus, NJ
Ditte Lovatt
Center for Translational Neuromedicine Kazuyuki Nakajima
University of Rochester Medical School Department of Neurochemistry
Rochester, NY National Institute of Neuroscience
Tokyo, Japan
Karsten K. Madsen
Department of Drug Design and Pharmacology Klaus-Armin Nave
University of Copenhagen Department of Neurogenetics
Copenhagen, Denmark Max Planck Institute of Experimental Medicine
Göttingen, Germany
Brian A. MacVicar
Department of Psychiatry and the Brain Maiken Nedergaard
Research Centre Center for Translational Neuromedicine
University of British Columbia University of Rochester Medical School
Vancouver, Canada Rochester, NY

Pierre J. Magistretti Akiko Nishiyama

Brain Mind Institute Department of Physiology and Neurobiology
Ecole Polytechnique Fédérale de Lausanne University of Connecticut
Lausanne, Switzerland Storrs, CT

Jean-Marie Mangin Mami Noda

INSERM U952 Laboratory of Pathophysiology
Université Pierre et Marie Curie Kyushu University
Paris, France Fukuoka, Japan

Rudolf Martini Stéphane H. R. Oliet

Department of Neurology INSERM U862 - Neurocentre Magendie
University of Würzburg Université de Bordeaux
Würzburg, Germany Bordeaux, France

Rebecca Matsas Katsuhiko Ono

Laboratory of Cellular and Molecular Neurobiology Department of Biology
Hellenic Pasteur Institute Kyoto Prefectural University of Medicine
Athens, Greece Kyoto, Japan

xx • C O N T R I B U TO R S
Trevor Owens Rita Sattler
Department of Neurobiology Research Department of Neurology
University of Southern Denmark Johns Hopkins University
Odense, Denmark Baltimore, MD

Vladimir Parpura Steven S. Scherer

Department of Neurobiology Department of Neurology
University of Alabama The Perelman School of Medicine at the University
Birmingham, AL of Pennsylvania
Philadelphia, PA
Ágnes Patzkó
Department of Neurology Arne Schousboe
University of Würzburg Department of Drug Design and Pharmacology
Würzburg, Germany University of Copenhagen
Copenhagen, Denmark
Luc Pellerin
Department of Physiology Martin E. Schwab
University of Lausanne Brain Research Institute
Lausanne, Switzerland University of Zurich and Federal Institute
of Technology Zurich
Josef Priller Zürich, Switzerland
Department of Neuropsychiatry and Laboratory of
Molecular Psychiatry Gerald Seifert
Charité-Universitätsmedizin Berlin Institute of Cellular Neurosciences
Berlin, Germany University of Bonn
Bonn, Germany
Marco Prinz
Department of Neuropathology Fraser J. Sim
University of Freiburg Department of Pharmacology and Toxicology
Freiburg, Germany State University of New York at Buffalo School
of Medicine
Stefan Prokop Buffalo, NY
Department of Neuropathology
Charité-Universitätsmedizin Berlin Michael V. Sofroniew
Berlin, Germany Department of Neurobiology
David Geffen School of Medicine
Bruce R. Ransom University of California, Los Angeles
Department of Neurology Los Angeles, CA
University of Washington School of Medicine
Seattle, WA David C. Spray
Dominick P. Purpura Department
Andreas Reichenbach of Neuroscience
Paul Flechsig Institute for Brain Research Albert Einstein College of Medicine
University of Leipzig Bronx, NY
Leipzig, Germany
Ruth Stassart
Jeffrey D. Rothstein Department of Neurogenetics
Department of Neurology Max Planck Institute of Experimental Medicine
Johns Hopkins University Göttingen, Germany
Baltimore, MD
Christian Steinhäuser
Michael W. Salter Institute of Cellular Neurosciences
Department of Physiology University of Bonn
University of Toronto Centre for the Study of Pain Bonn, Germany
Toronto, Canada

C O N T R I B U TO R S • xxi
Wolfgang J. Streit Helle S. Waagepetersen
Department of Neuroscience Department of Drug Design and Pharmacology
University of Florida College of Medicine University of Copenhagen
Gainesville, FL Copenhagen, Denmark

Peter K. Stys Xiaoqun Wang

Department of Clinical Neurosciences Department of Neurology
University of Calgary University of California San Francisco
Calgary, Canada San Francisco, CA

Olawale A. R. Sulaiman Stephen G. Waxman

Ochsner Health Systems Department of Neurology and Center for Neuroscience
New Orleans, LA Research
Yale University School of Medicine
Eva Syková New Haven, CT
Department of Neuroscience
Charles University Michael Wegner
Prague, Czech Republic Institute of Biochemistry
Friedrich-Alexander-University of Erlangen-Nürnberg
Tuan Trang Erlangen, Germany
Department of Physiology
University of Toronto Centre for Imola Wilhelm
the Study of Pain Institute of Biophysics
Toronto, Canada Hungarian Academy of Sciences
Szeged, Hungary
Klaus Unsicker
Department of Molecular Embryology Hartwig Wolburg
University of Freiburg Institute of Pathology and Neuropathology
Freiburg, Germany University of Tübingen
Tübingen, Germany
Javier Vaquero
Neuroscience Research Unit Lawrence Wrabetz
Centre Hospitalier de l’Université de Montréal Department of Neurology and Biochemistry
Montreal, Canada SUNY at Buffalo, School of Medicine
University of Buffalo
Alexei Verkhratsky Buffalo, NY
Faculty of Life Sciences
The University of Manchester Robert Zorec
Manchester, United Kingdom Institute of Pathophysiology
University of Ljubljana
Andrea Volterra Ljubljana, Slovenia
Department of Cell Biology and Morphology
University of Lausanne Violetta Zujovic
Lausanne, Switzerland INSERM, CR1
Centre de Recherche de l’Institut du Cerveau et de la
Oliver von Bohlen und Halbach Moelle Epinière
Institute of Anatomy and Cell Biology Paris, France
University of Greifswald
Greifswald, Germany

xxii • C O N T R I B U TO R S
NEUROGLIA
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 SECTION 1
P R O P E RT I E S O F N E U R O G L I A L C E L L S
This page intentionally left blank
EVOLUTION OF GLIAL CELLS: INSIGHTS FROM
NON-MAMMALIAN GLIA
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 1.
EVOLUTION OF GLIAL CELLS
Christian Klämbt

Nothing in Biology makes sense, except in the light of evolution. THEODOSIUS DOBZHANSKY

A B B R E VI AT I O N S instruct us about general aspects of glial biology, but will help to

identify the functional constrains sculpting non-neuronal cells in
APC/C anaphase-promoting complex/cyclosome the nervous system. In contrast, if we assume that evolution has
C. elegans Caenorhabditis elegans invented glial cells only once, this provides us with the opportu-
CNS central nervous system nity to deduce the core functions of glia and indeed learn from
GABA gamma-aminobutyric acid studying more simple, genetically amenable organisms.
gcm glial cells missing
GFP green fluorescent protein
GPI glycosylphosphatidylinositol 2 W H AT A R E G L I A L C E L L S ?
GS glutamine synthetase
NCAM neural cell adhesion molecule Although it is fairly straightforward to define a neuronal cell,
PLP proteolipid protein it is still not satisfactorily clear how we can recognize glial
PNS peripheral nervous system cells without any doubt. In fact, particularly in more simple
PSA polysialic acid invertebrates, cells are quite often named glial because they are
not obviously neurons (Bullock and Horridge 1965; Hartline
2011; Radojcic and Pentreath 1979). These classical studies
1 E S S AY: W H Y S H O U L D W E C A R E provide a minimal set of criteria that qualify a cell as a glial cell:
A B O U T T H E E VO LU T I O N A RY (1) Glial cells should have an intimate morphological associa-
ORIGIN OF GLIAL CELLS? tion with neurons or they should separate neuronal elements
from mesodermal layers; and (2) glial cells should originate
Most animals are able to respond to external stimuli by mov- from the embryonic ectoderm and are referred to collectively
ing. To do so, many phyla have developed complex neural as macroglia.
structures allowing more and more sophisticated computing Microglial cells are not covered here. These cells origi-
of information. As the nervous system changed from a simple nate from the mesoderm and enter the vertebrate brain during
netlike structure to condensed ganglia and centralized brains, embryogenesis to become resident immunologically compe-
a new cell type could be recognized in morphological stud- tent cells acting as macrophages during infection and injury
ies: glial cells (Bullock and Horridge 1965; Hartline 2011). (Kettenmann et al. 2011; Saijo and Glass 2011). In Drosophila,
The importance of this new cell type for a functional brain no mesodermal cells are known to migrate into the nervous sys-
is reflected by the increase in the relative number of glial cells tem and macrophage functions are taken over by macroglial cells
during evolution. Although quantifying glial cell numbers in (Awasaki and Ito 2004; Doherty et al. 2009; Watts et al. 2004).
larger mammalian brains is difficult, there are roughly equal There are many examples in which the preceding criteria
numbers of glial cells and neurons in these species (Azevedo allow an easy definition of glial cells, and in some cases even
et al. 2009; Herculano-Houzel 2011; Hilgetag and Barbas obvious similarities between the mammalian and invertebrate
2009). By contrast, only 10% of invertebrate neural cells as in glia can be seen. Good examples are the peripheral glial cells
Drosophila or C. elegans are glial cells (Beckervordersandforth in insects. The wrapping glial cells of the peripheral nerves
et al. 2008; Hilchen et al. 2008; Oikonomou and Shaham in Drosophila are very large cells, stretching their cytoplasm
2011; Pereanu et al. 2005). about 1 mm around axon fascicles (Sieglitz and Klämbt
But what are glial cells, and are they really comparable in all unpublished). Morphologically and functionally these cells
phyla? Are they the many non-neuronal cells of the brain that very much resemble Remak fibers of the mammalian nervous
we consider as glia truly homologous, or have they appeared system (Nave and Trapp 2008; Rodrigues et al. 2011; Stork
independently several times during evolution? If the latter is the et al. 2008). Likewise, cells with an astrocytic morphology
case, the analysis of different animal species will not necessarily are not confined to the mammalian central nervous system

5
(CNS), but can also be identified in Drosophila (Awasaki et al.
2008; Stork and Freeman personal communication; Volterra
and Meldolesi 2005) (see chapter 2). Moreover, these cells
express a related set of genes controlling neurotransmitter
homeostasis (Stacey et al. 2010).
In general, these morphological criteria clearly define
glia in most animal phyla with a condensed nervous system.
However, sometimes it remains difficult to decide whether or
not a cell can be classified as glial cell. Here, the perineurial
glial cells or the “myelin” forming glial cells of invertebrates
may serve as a paradigm.
Myelination has been considered to be a clear glial func-
tion found in vertebrates and invertebrates (Bullock 2004;
Hartline and Colman 2007; Nave 2010a). Interestingly, two
recent studies cast some doubt on this central dogma of glial
biology. In calanoid copepods myelin-like structures appeared
before glial cells became associated with axons and their for-
mation was initiated internal to the axolemma. These find-
ings suggest a nonglial origin of myelinated structures; thus,
a typical glial structure is made by nonglial cells (Wilson and
Hartline 2011a,b).
A reverse example can be seen in the insect perineurial glia
that abuts the blood-brain barrier forming subperineurial glia
(Stork et al. 2008). The perineurial glial cells do not form a
tight sheath around the nervous system and appear only rel-
atively late during development. Significantly, the perineurial
glia lack any contact with neuronal cells. Over a long time
the glial nature of these cells has been highly controversial
(Radojcic and Pentreath 1979). The perineurial glia has been
carefully analyzed in Drosophila. There is now broad consent
to consider these cells as glial, based on the neural lineage of
these cells and the expression of several glial specific markers,
although proliferation of perineurial glial cells during pupal
stages does not appear to depend on the regulatory gene glial
cells missing (Gcm) (Awasaki et al. 2008; Stork et al. 2008).
Thus, the assignment of glial identity should not be based
solely on histological arguments, but rather requires the inclu-
sion of as much molecular data as possible to faithfully define
a glial cell type.
3 E VO LU T I O N O F N E U R O N S
Any serious discussion regarding the origin of glial cells has
to be linked to the question about the origin of neurons,
which are found in most multicellular animals. The inven-
tion of multicellularity set the stage for a division of labor,
and organs devoted to feeding, reproduction, or movement
could be formed. In addition, individual ectodermal cells were
equipped with sensory filters to allow the detection of changes
in the outside world and subsequent response to them. These
properties must have evolved in very early metazoa because
Porifera, which are considered to have not formed neuronal
cells (Bullock and Horridge 1965; Hartline 2011; Radojcic
and Pentreath 1979), use well-conserved molecular mecha-
nisms (proneural genes encoding atonal related proteins and
neurogenic genes such as Notch and Delta) to generate an
ancient sensory cell type (Richards et al. 2008; Srivastava et
6 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
al. 2010). Once the sensory neuron was defined, it had to spe-
cialize more and more and therefore became dependent on the
support of its neighbors. Such simple isolated nerve cells still
exist in coelenterates, in which the molecular networks con-
trolling neurogenesis in bilateria are all in place (Galliot et al.
2009; Marlow et al. 2009). In Hydrozoa, for example, sensory
cells and nematocytes are concentrated in the spherical end
bulbs of the tentacles. Interestingly, the four types of sensory
cells present in these bulbs are separated by distinct support-
ing cells, which resemble accessory cells of complex peripheral
sense organs in bilateria (Holtmann and Thurm 2001a, b).
Thus, neurons may have evolved with supporting cells or glial
cells from the very beginning.
4 PA I RW I S E E VO LU T I O N O F
NEURONS AND GLIAL CELLS
A pairwise evolution of a neuronal cell and a support cell (the
pigment cell) has been very well documented for the light
sensing organs. Photoreceptor neurons are able to receive sig-
nals and are generally accompanied by pigment cell(s), which
provide numerous accessory functions (Arendt 2003; Arendt
and Wittbrodt 2001). Charles Darwin speculated that the eye
contains, as minimal equipment, a neuron to detect light and
a pigment cell for support and shading. Indeed, such simple
eyes can be found in planarians and some polychaete larvae
(Gehring and Ikeo 1999). Over the centuries, however, the
evolution of eyes has been a matter of quite some debate. The
traditional view, backed by numerous anatomical studies, sug-
gested that the eye has evolved independently several times.
However, modern molecular genetic tools have clearly favored
the monophyletic origin of all photoreceptor cells by unravel-
ing the surprisingly well-conserved network of transcriptional
regulators directing eye development (Arendt 2003; Bullock
and Horridge 1965; Erclik et al. 2008, 2009; Gehring, 2002,
2005; Gehring and Ikeo 1999; Nilsson 2004).
In all bilateria, pax6-related genes are core components of
the genetic circuitry orchestrating eye development (Gehring
and Ikeo 1999). In Cnidaria, which are the earliest metazoans
with a well-developed nervous system, Pax6-like transcription
factors do not exist. Nevertheless, many Cnidaria, including
hydrozoa and cubozoa form highly elaborated eyes with cor-
nea, lens, and retina (Martin 2002). Interestingly, in the cubo-
zoan jellyfish Tripedalia cystophora Pax-B has been implicated
in eye development (Kozmik 2008; Kozmik et al. 2003),
whereas in the hydrozoan jellyfish Cladonema radiatum Pax-A
rather than Pax-B controls eye formation (Suga et al. 2010).
These data suggest that the origin of eyes occurred before the
duplication of the Pax genes and further supports the hypo-
thesis of the monophyletic origin of all animal eyes.
How can the likely monophyletic origin of animal eyes
help to address the origin of glia? In this respect it is impor-
tant to consider that eye formation always implies the pres-
ence of a receptor and a pigment cell; indeed, all pax genes do
exactly this, they specify receptor cells as well as pigment cells.
Pigment cells serve a number of metabolic functions necessary
for normal vision. Vision requires the detection of a photon
by a receptor that comprises a protein (opsin) coupled to
11-cis-retinal, which isomerizes to all-trans-retinal on activation
of the receptor. In vertebrates the regeneration of 11-cis-retinal
occurs in pigments cells. A similar visual cycle between pho-
toreceptor cells and pigment cells recently has been identified
in Drosophila (Arshavsky 2010; Fain et al. 2010; Wang et al.
2010). This underscores the intricate interaction of these two
cell types, one being a sensory cell, and the other one being clas-
sified as a support cell—in other words—as a glial cell.
The interaction of a photoreceptor cell and a pigment cell
is not confined to the re-isomerization of all-trans-retinal.
Neurons generally need a lot of energy and much debate is on
how neurons and in particular how long axons are metaboli-
cally supported by glial cells for the maintenance of axonal
transport and long-term survival (Brown and Ransom 2007;
Nave 2010b; Nave and Trapp 2008). Metabolic coupling is
not only needed between ensheathing glia and long axons, but
is likely to reflect a much more ancestral function in which
support cells (glial cells) feed the accompanying neurons. In
this way, it is not surprising that pigment cells in the retina
of bees take up glucose and supply metabolic products (in
this case alanine) to the receptor cells. Likewise, mammalian
astrocytes are able to provide lactate to fuel neuronal activity
(Brown and Ransom 2007; Coles 1989; Pellerin et al. 2007;
Tsacopoulos and Magistretti 1996; Tsacopoulos et al. 1988).
Assuming that at least sensory neurons have evolved as cell
pairs—a sensory specialist and a support specialist—it appears
likely that glial-like cells should exist in the most simple organisms
with a fairly well developed nervous system, Cnidaria. However,
the prevailing view in the literature is that Cnidaria lack glial
cells (Bullock and Horridge 1965; Hartline 2011; Radojcic and
Pentreath 1979). Within the different Cnidaria classes (Antozoa,
Hydrozoa, Cubozoa, Scyphozoa, and Staurozoa) an amazing
spectrum of eyes has developed during evolution. The complex
lens eyes of cnidarian species are organized together with other
sensory organs in the so-called rhopalia (Martin 2002; Nilsson
2004). Sensory input then is integrated in a radial symmetrical
central nervous system, which is arranged in a ring form and allows
in part complex behavior of jellyfish, such as courtship behavior
in some medusae (Galliot et al. 2009; Garm et al. 2007; Lewis
and Long 2005; Marlow et al. 2009). The ring nerve of a 1-cm
large Tripedalia cystophora medusa is generated by about 10.000
neurons that process and transmit information in several distinct
subsystems (Garm et al. 2007). Within the ring nerve, special
epithelial cells provide compartmentalization by cellular exten-
sions that encircle groups of axons, and thus might provide some
ancestral glial function by isolating the axonal bundles (Garm et
al. 2007; Mackie and Meech 1995). Here, more work is needed to
address the question whether these cells can be considered as glial
cells or not, and in the author’s view the chances are high to find
these typical characteristics.
5 M O L E C U L A R C R I T E R I A TO D E F I N E
G L I A : A D H E S I VE P R O P E RT I E S
Important insights in the evolution of glial cells originate from
analyzing their molecular signatures. A prominent example
1. E VO LU T I O N O F G L I A L C E L L S
is seen in the septate junctions in insects and the related
septate-like junctions seen in the paranodes of the mammalian
peripheral nervous system (Banerjee and Bhat 2007; Girault
and Peles 2002; Sherman and Brophy 2005). In both cases
morphologically similar structures are formed by homologous
sets of interacting transmembrane proteins. In mice, Caspr,
Contactin, and Neurofascin155 were found to be incorpo-
rated into the septate-like junctions (Einheber 1997; Peles et
al. 1997; Poliak et al. 1999). In Drosophila the homologous
proteins NeurexinIV, dContactin, and Neuroglian form the
base of septate junctions that are essential to establish the
blood-brain barrier (Banerjee et al. 2006; Baumgartner et
al. 1996; Stork et al. 2008). In addition, genetic analyses in
Drosophila have unraveled 12 additional membrane-associated
proteins that associate with the septate junctions (Genova and
Fehon 2003; Hijazi et al. 2009; Nilton et al. 2010; Oshima
and Fehon 2011; Syed et al. 2011; Tiklová et al. 2010; Wu and
Beitel 2004). Although we still do not know how septate junc-
tions are initially assembled and then maintained, it is certain
that deeper knowledge on the biology of septate junctions in
invertebrates will help to decipher how septate-like junctions
are formed in the mammalian nervous system.
Adhesion systems are not only working to ensure glial–
glial cell contact, but are also needed to ensure the close
association of glial cells with neurons. Neuron–glia inter-
action is obvious and evolution has selected astonishingly
well-conserved adhesion molecules to guarantee this specific
interaction (Silies and Klämbt, 2011). The neural cell adhesion
molecule (NCAM) gene is a quite remarkable example and
demonstrates not only the evolutionary conservation of gene
functions, but also the conservation of specifically spliced iso-
forms. In mammals and insects, NCAM encodes three distinct
isoforms, which share an identical extracellular domain with
five immunoglobin (Ig) domains and two fibronectin (FN)
domains, but differ in the way they are linked to the cell mem-
brane. Two NCAM isoforms carry a transmembrane domain
but differ in their cytoplasmic domains, and one NCAM iso-
form is linked to the outer cell membrane by a GPI anchor. In
vertebrates and invertebrates, the transmembrane-bound iso-
form is expressed by neurons, whereas the GPI-linked protein
form is made by glial cells (Higgins et al. 2002; Maness and
Schachner 2007; Noble et al. 1985; Silies and Klämbt 2010;
Wright and Copenhaver 2000, 2001).
The neural cell adhesion molecule and its orthologous
proteins function as homophilic adhesion proteins and
among others control glial migration in the developing nerv-
ous system (Grenningloh et al. 1991; Hoffman and Edelman
1983). During this process, modulation of NCAM-mediated
adhesiveness is of clear relevance. Interestingly, although the
expression of specific isoforms has been highly conserved dur-
ing evolution, distinct mechanisms evolved to control the
modulation of NCAM-mediated cell stickiness. In the ver-
tebrate CNS, adhesiveness is altered by the addition of poly-
sialic acid (PSA) moieties to the NCAM protein (Rutishauser
2008; Weinhold et al. 2005). Whereas NCAM mutants are
viable and fertile and have only relatively minor defects during
nervous system development (Chazal et al. 2000; Cremer et
al. 1994), loss of the two enzymes that transfer PSA to NCAM
• 7
causes severe defects during brain development, which can be
rescued by loss of NCAM (Weinhold et al. 2005). Drosophila
follows a quite different molecular approach to reach the
same functional result. During fly development, no polysialic
acid moieties are attached to extracellular proteins. Instead,
the NCAM homolog Fasciclin2 is removed from axons in a
graded manner through APC/C dependent regulatory endo-
cytotic processes (Silies and Klämbt 2010).
These data demonstrate that on the one hand, too much
adhesion is worse than not enough. On the other hand, these
molecular studies suggest that during evolution glial cells and
neurons were equipped with different NCAM proteins before
the split of Protostomia and Deuterostomia. The genomes of
several cnidarian species have been sequenced in the mean-
time (Hydra, Nematostella, Clytia) and Ig-domain proteins
resembling NCAM have been identified (Chapman et al.
2010; Houliston et al. 2010; Marlow et al. 2009; Putnam et al.
2007). However, we will have to wait for more sophisticated
annotation tools to learn whether several isoforms of NCAM
are generated in these simple metazoan as well and whether
the expression of specific isoforms can be linked to individual
neural cell types.
Neuron–glial interaction cumulates most spectacularly
in the formation of myelin sheaths in the vertebrate nervous
system. Here specific adhesion systems have evolved that may
be considered as evolutionary new inventions as invertebrates
lack several of the “myelin” genes. Of the ten human genes
known to be required for myelin production, only one gene
encoding a proteolipid protein was found in the Drosophila
genome (Cravchik et al. 2001; Stecca et al. 2000). The fly
proteolipid protein (PLP)–like protein M6 is expressed and
required during oogenesis. A GFP enhancer/gene trap inser-
tion into the promoter of the M6 gene suggests that there is
additional expression in the developing nervous system and
shows accumulation of the M6-GFP fusion protein along
axon tracts (Zappia et al. 2011). However, it is presently
unknown whether this protein is made by glial cells or neu-
rons and whether any phenotypic abnormalities are found in
mutant glial cells.
In conclusion, glial cells use a variety of conserved adhe-
sion systems. As a consequence, glial biology became more
complex during evolution by recruiting additional adhesion
systems to perform specific tasks such as myelination.
6 M O L E C U L A R C R I T E R I A TO
D E F I N E G L I A : S U P P O RT O F
SY N A P T I C T R A N S M I S S I O N
A key function of glial cells resides in the support and modulation
of synaptic function. Astrocytes cooperate with the presynapse
and postsynapse to maintain synaptic fidelity. Indeed, a synapse
can be viewed as a tripartite structure that is found in complex
animals such as leech, Drosophila, or mammals (Danjo et al.
2011). In the mammalian system it has been shown that astrocytes
express several neurotransmitter transporters such as glutamate,
gamma-amino butyric acid (GABA), and glycine transporters
(Halassa and Haydon 2010). Several of these transporters are also
8 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
found in the Drosophila CNS (Altenhein et al. 2006; Besson et
al. 1999; Soustelle et al. 2002; Stacey et al. 2010). Interestingly,
not only the uptake of neurotransmitters appears evolutionary
conserved, but also the support of the presynaptic terminals with
metabolic intermediates that allow an efficient and fast resynthesis
of these transmitters. This has been well studied for the uptake of
glutamate by astrocytes. On intake of glutamate, the astrocytic
enzyme glutamine synthetase (GS) converts it to glutamine,
which is then released to the neuron. The Drosophila genome
encodes two glutamine synthase genes, one of which (GS2) is
expressed in cells associated with the neuropile, which are likely
to exert astrocytic functions (Freeman et al. 2003; Stacey et al.
2010; Thomas and van Meyel 2007). Indeed, GS is an evolution-
ary well-conserved protein and is found in the nervous system of
many animal species (Niva et al. 2008; Roots 1981). The genomes
of Caenorhabditis elegans and Hydra magnipapillata also encode
several well-conserved glutamine synthetase genes, but currently
no studies have disclosed the expression of these genes. Glutamate
and GABA receptors are known even in Cnidaria, and thus the
close metabolic coupling of neurons and glial cells exemplified in
the tripartite synapse may reflect an ancient evolutionary devel-
opment and glutamine synthetase thus serves as an evolutionary
early marker of astrocytic cells (Delgado et al. 2010; Scappaticci
and Kass-Simon 2008).
7 M O L E C U L A R C R I T E R I A TO D E F I N E
GLIA : TR ANSCRIPTION
The evidence collected in the preceding indicates that glial
cells have indeed been invented very early during the evolution
of metazoa. In particular, when we include molecular criteria,
glia appears as a very ancient cell type. However, molecular
biology also provides us with some arguments against this
hypothesis. Gliogenesis in the mammalian nervous system is
linked to a complex network of transcription factors such as
olig or sox10 (Britsch 2001; Richardson et al. 2006; Rowitch
2004; Rowitch and Kriegstein 2010; Zhou et al. 2001). In
contrast, gliogenesis in Drosophila is based on the presence
of a single transcription factor, Gcm, which is expressed and
required in almost all glial cells (Hosoya et al. 1995; Jones
et al. 1995; Kammerer and Giangrande 2001; Vincent et al.
1996). Glial cells missing activates the transcription of several
downstream factors that mediate glial differentiation (Giesen
et al. 1997; Granderath et al. 2000; Shandala et al. 2003;
Yuasa et al. 2003). Although gcm is prominently involved in
specifying glial cell fate, it is not exclusively expressed by glial
cells. Besides additional expression domains outside the nerv-
ous system, gcm is also expressed in the neuronal cells, where
it exerts some by now unknown functions (Bernardoni et al.
1997; Chotard et al. 2005; Soustelle and Giangrande 2007;
Soustelle et al. 2004, 2007). Although the gliogenic functions
of gcm have not been conserved during evolution, gcm appears
to trigger the activation of a conserved epigenetic pathway by
regulating histone acetylation (Flici et al. 2011; Wegner and
Riethmacher 2001). Thus, common regulatory mechanisms
underlying glial development may affect the general tran-
scriptional profile of glial cells and during evolution different
regulatory transcription factors have learned to restrain the
activity of these epigenetic modulators.
8 S U M M A RY A N D P E R S P E C T I VE S
Clearly, evolution has provided us with a bewildering diver-
sity of neuronal and glial cell types. Yet molecularly, all these
cells are deviations from a simple basic ground plan, suggest-
ing that quite likely this cell type has appeared just once. Thus,
the analysis of simple and highly derived glial cell types in
many different species will contribute to our understanding
of human glial cells. Furthermore, the integration of results
obtained in Cnidaria and genetically amenable organisms
such as Drosophila, C. elegans, zebrafish, or mice is needed to
define what the first glial cells may have looked like and what
their first functions may have been.
AC K N OW L E D G M E N T S
The author is thankful to S. Limmer, S. Thomas, and
S. Sasse for comments and discussions. The work in the lab
of C. Klämbt is funded through grants of the Deutsche
Forschungsgemeinschaft and the EC.
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• 11
 2.
INVERTEBRATE GLIA
Marc R. Freeman

A B B R E VI AT I O N S and are now poised to contribute in similar ways to our under-

standing of fundamental properties of glial cells (see chapter 1).
BBB blood-brain barrier This chapter reviews the features of key invertebrate preparations
CEP cephalic used to study glial cell biology, provides a short overview of what
CEPsh cephalic sheath has been learned from these organisms, compares their biol-
CNS central nervous system ogy to that of mammals, and explores how invertebrate systems
DCC deleted in colorectal cancer might be used in the future to probe deeply into the biology of
DRG dorsal root ganglia glial cell types such as the astrocyte.
Flp flippase
FRT flip recombinase target site 2 N O N G E N ET I C I N VE RT E B R AT E
GPR G protein–coupled receptor M O D E L SYS T E M S
ISN intersegmental nerve
MARCM mosaic analysis with a repressible cell marker The first evidence that neurons could signal to glia through
MG midline glia neurotransmitters came from studies of the squid (Loligo
MN motor neuron pealei) giant axon preparation, which includes surrounding
NMJ neuromuscular junction Schwann cells. In contrast to mammalian Schwann cells found
NT neurotransmitter in the peripheral nervous system, Schwann cells surrounding
PG perineurial glia the squid giant axon lay in parallel to the giant axon, and highly
PNS peripheral nervous system interdigitated, and many together form a single sheath that
pSJ pleated septate junction envelopes the axon shaft (Brown et al. 1991). Schwann cells were
RNAi RNA interference found to exhibit a rapid membrane hyperpolarization follow-
SBC segment boundary cell ing stimulation of giant axons (Villegas 1972), which resulted
SPG subperineurial glia from axonal release of glutamate (Lieberman et al. 1989) that
acted on Schwann cell metabotropic glutamate receptors
(Evans et al. 1985). Interestingly, following activation by gluta-
1 INTRODUCTION mate, Schwann cells activated a glial-glial signaling event: They
released acetylcholine, which in turn increased Schwann cell
The study of invertebrate preparations has illuminated a number hyperpolarization (Evans et al. 1985; Villegas 1974; Villegas
of fundamental principles of neuronal function, including the et al. 1987). Glial control of neuronal electrophysiology was
electrophysiological and chemical basis of the action potential, also established using the snail Helix pomatia. Ca2+-dependent
mechanisms of synaptic vesicle release, and pathways governing long-term changes in glial membrane K+ conductance after
neuronal cell fate specification and growth. Invertebrate prepa- neuronal activity was shown to alter K+ tone in local circuits
rations offer a number of advantages for studying the nervous and thereby affect local firing of neurons (Gommerat and Gola
system, including ease of culture, rapid growth, high experimen- 1994). In addition, basic aspects of metabolic coupling between
tal accessibility, and the opportunity to uniquely identify and neurons and glia were first detailed in invertebrates. In studies
manipulate individual cells in vivo. Historically such preparations, of the honeybee, drone retina glia were shown to be respon-
especially larger preparations such as the squid, grasshopper, or sible for metabolizing and storing extracellular glucose in the
Aplysia, were used for detailed studies of single neuron physiol- form of glycogen (Tsacopoulos et al. 1988, 1994). Local neu-
ogy, morphological analysis, or the effects of cell transplanta- ronal activity was found to induce rapid glycogen metabolism
tion. In the past three decades as the focus has shifted toward in glia (Evequoz-Mercier and Tsacopoulos 1991) (see chapter 36),
understanding the molecular basis of neuronal development which resulted in glial release of alanine for use as metabolic
and function, smaller invertebrates amenable to rapid molecular fuel by neurons (Tsacopoulos et al. 1994).
genetic analysis such as Caenorhabditis elegans and Drosophila The nervous system of the medicinal leech (Hirudo medici-
have become extremely popular experimental systems. Worms nalis) houses two uniquely identifiable astrocyte-like glial cells
and flies have since made profound contributions to delineating per segmental ganglion that are enormous: their cell bodies
ancient and conserved pathways governing neuronal biology, are approximately 100 μm across (Lohr and Deitmer 1997,

12
1999) and their fine processes that fill the synaptic neuropil
cover an area of 300 to 350 μm in diameter (Kuffler and Potter
1964). For comparison, a hippocampal mouse astrocyte cell
body is approximately10 μm and their processes cover a spatial
domain of approximately100 μm in diameter (Bushong et al.
2004), whereas the average size of human cortical astrocyte
ranges from 100 to 400 μm in diameter (Oberheim et al.
2009). Leech giant astrocyte–like glia, like mammalian astro-
cytes, are coupled electrically through gap junctions, thereby
allowing for coordination of electrical responses (Coggeshall
1974; Kuffler and Potter 1964). Because of their large size, one
can perform direct electrophysiological recordings, micro-
fluorometry, or microinjections (e.g., pharmacological agents,
or RNAi constructs) of astrocyte-like glia, while simultane-
ously stimulating specific neurons or inducing simple behav-
iors such as fictive swimming in largely intact preparations.
The leech astrocyte–like glial cell is an electrophysiologically
active cell that responds to neuron signaling with changes in Ca2+
signaling and membrane potential. These glia exhibit a selective
K+ conductance and can act as sinks for K+ in spatial buffering
(Kuffler and Nicholls 1966). They actively regulate the pH of
the intracellular and likely extracellular compartments through
mechanisms including a Na+-HCO3 cotransporter (Deitmer
1991; Deitmer and Schlue 1987, 1989; Szatkowski and Schlue
1994). By controlling extracellular pH these glia could play a
direct role in regulating neuronal electrophysiology (Deitmer
and Schneider 1995; Deitmer et al. 1999). Leech astrocyte–like
glia exhibit dramatic Ca2+ fluxes that are regulated potently by
membrane potential (Nett and Deitmer 1998). Ca2+ increases
are mediated largely by entry of extracellular Ca2+ (Deitmer et al.
1999), although glutamate application can elicit Ca2+ release from
intracellular stores (Lohr and Deitmer 1997, 1999). Ca2+ spikes
can occur throughout the soma, but also occur at the local level
and in a heterogeneous fashion (Lohr and Deitmer 1999), poten-
tially responding to and regulating neural activity. Astrocyte-like
glia indeed respond with rapid changes in membrane potential
to a number of neurotransmitters, including glutamate, acetyl-
choline, serotonin, or GABA (Deitmer and Rose 1996; Deitmer
et al. 1998; Lohr and Deitmer 1999; Muller et al. 2000; Schmidt
and Deitmer 1999). This responsiveness likely represents normal
glial function in the leech, because stimulation of even a single
cell, the neuromodulatory Leydig neuron, promotes hyperpolar-
ization of the giant astrocyte–like glia with the amplitude and rate
of hyperpolarization increasing with the frequency of neuronal
stimulation (Britz et al. 2002; Schmidt and Deitmer 1999).
Additional invertebrate preparations have made important
contributions to our understanding of how glial cells coordi-
nate their developmental program with neurons, or how glia
guide key steps in neuronal development. Glia in the antennal
lobe of the moth Manduca sexta have proved quite useful in
understanding the cellular basis of how neuronal contact initi-
ates glial organization of antennal lobe glomeruli (Tolbert and
Oland 1990), or how glia guide the sorting of olfactory recep-
tor neuron axon fibers to the correct location (Rossler et al.
1999). In some cases even single axon–glia interactions have
been examined in insects. For example, during normal embry-
onic development of the grasshopper, the segment boundary
cell (SBC), a glial cell, establishes a position that will ultimately
2. I N VE RT E B R AT E G L I A
form the intersegmental peripheral nerve route. The growth
cones of axons that pioneer the intersegmental nerve (ISN)
show a strong affinity for the SBC; at the point at which they
meet the SBC they exit longitudinal axon tracts and pioneer the
ISN. Ablation of the SBC glial cells in vivo results in a striking
failure of these growth cones to enter the ISN tract (Bastiani
and Goodman 1986).
A major shortcoming of the invertebrate prepara-
tions discussed in the preceding is the absence of incisive
molecular-genetic approaches that can be used to explore how
specific genes promote glial development, function, or neuron-
glia interactions. However, genome sequence databases are
becoming available for some of these organisms (e.g., Apis is
complete, Hirudo and Loligo are underway), and the recent
revolution in the use of RNA interference (RNAi) has opened
the door for a limited amount of genetic manipulation.
3 C A E N O R H A B D I T I S E L E GA N S G L I A
3.1 H I S TO L O GY A N D E X P E R I M E N TA L U T I L IT Y
O F C A E NO R H A B D I T I S E LEGA N S G L I A
The nervous system of the nematode Caenorhabditis elegans
consists of only 302 neurons and 50 glial-like cells based on
morphological and functional criteria (Shaham 2005, 2006).
All worm glia are closely associated with peripheral sensory
structures; none are associated exclusively with the nerve
ring (which is the worm brain). As such, it has been argued
that C. elegans glia may represent a good example of the evo-
lutionarily ancient glial subtype, which might have appeared
first in the peripheral nervous system (Reichenbach and
Pannicke 2008). Glia in the worm can be divided into two
broad classes: 24 are sheath glia, which ensheathe a subset
of sensory neuron dendrites; and 26 are socket glia, which
surround sensory neuron dendrite tips to form a pore to
allow for direct exposure of dendrites to the environment
(Fig. 2.1). A subset of four sheath glia, the cephalic sheath
(CEPsh) glia, are bipolar and not only ensheathe sensory
dendrite tips but also extend flattened membrane processes
toward the nerve ring. Each of the 50 C. elegans glia are
uniquely identifiable, associate with well-defined subsets of
sensory neurons, and one can therefore study neuron–glia
interactions in single-neuron pairs.
Worms are completely transparent, thereby allowing for
imaging and manipulation of all glial cells in the intact organ-
ism. Promoters have been identified that can be used to drive
expression of any transgene of interest in very specific sub-
sets of glia or neurons. Available lines of experiments range
from simple imaging of glia during development, through
opto-genetic activation of specific neurons in mature circuits
while imaging glial Ca2+ transients in glia with genetically
encoded Ca2+ indicators. Because the entire cell lineage of
C. elegans is well defined one can perform ablations of single
cells or precursors early in development and then explore
developmental roles for glia in nervous system assembly, or
ablate single glial cells at adult stages and examine glial func-
tion in the mature nervous system (see later for examples).
• 13
 Sensory
A Sheath
Socket neuron Nerve ring
B Single cell ablation studies in C. elegans have revealed that worm
Dendrites/processes Cell
bodies
B in the nerve ring (Yoshimura et al. 2008). Ablation of amphid
(Environment)
Cuticle
Socket
Glia
Sheath
Cilia
Sensory
neuron
Dendrite
Figure 2.1 Caenorhabditis elegans Glial Subtypes and Associations with
Neurons. A. Anterior tip of the adult stage of C. elegans. Caenorhabditis
elegans glia are highly polarized and extend processes from the cell bodies
to the tips of sensory neurons (blue). Socket glia (green) are unipolar and
only ensheathe sensory neuron tips. Most sheath glia are also unipo-
lar, but a subset termed CEPsh glia (red) are bipolar and also extend
processes posteriorly that ensheathe the nerve ring. B. At sensory neuron
tips (box region enlarged from A) socket glia form a pore through which
sensory neuron ciliated endings extend into the environment. Sheath
glia enwrap the tips of sensory neurons: Some sensory neuron ciliated
endings extend into the environment, whereas others are fully or partially
embedded within the sheath glial cell.
Caenorhabditis elegans is also remarkable with respect to the
genetic approaches available. Worms offer the opportunity
to perform powerful, rapid, and unbiased forward genetic
screens to identify mutations affecting any aspect of glial
cell biology that can be observed in vivo. Unlike more com-
plex organisms, C. elegans can survive even when the worm
harbors extremely deleterious mutations—as long as the pha-
ryngeal pump is working (which is controlled by a very small
number of neurons), the worm is able to feed and survive to
late stages even when otherwise paralyzed. In addition because
hermaphrodites self-fertilize they can self-cross and generate
additional progeny without the need for mating. As such, one
can explore the in vivo function of a number of genes that
would cause early lethality in other animals. This is especially
important for C. elegans researchers because genetic mosaic
approaches, although available, are not as straightforward as
in Drosophila or mice. Finally, in recent years collections of
whole-genome RNAi libraries that contain constructs that
target nearly every C. elegans gene can be used for RNAi-based
screens in which RNAi constructs carried by bacteria are fed
to animals (Kamath et al. 2003).
14 • NEUROGLIA
3.2 G L I A L C O N T RO L O F N EU RO NA L
D EV E L O PM E N T I N T H E WO R M
glia modulate diverse aspects of neuronal development from
neurite extension, dendrite morphogenesis, axon pathfinding,
synaptogenesis, to nerve ring assembly. For example, ablation of
CEPsh glia, which are closely associated with cephalic (CEP)
sensory neurons from early developmental stages, led to defects
in CEP neuron dendrite extension and wiring of sensory axons
sheath glia (those sheath glia that do not extend processes to
the nerve ring) had little affect on dendrite extension, but led
to many sensory neurons elaborating a very simple dendritic
architectures compared with controls. Interestingly, a sub-
set of sensory neurons appeared unaffected by glial ablations,
indicating that some neurons can extend and elaborate den-
drites properly in the absence of glial input (Bacaj et al. 2008).
Finally, because dendrites and glial processes are organized into
mature sensory structures, glial cells control the precise size of
the sensory compartment that houses dendrite tips through
the opposing actions of the Patched-related molecule DAF-6,
which restricts the expansion of the sensory compartment, and
LIT-1, which promotes compartment growth (Oikonomou
et al. 2011). Notably, C. elegans sensory neurons survive in the
absence of glia, arguing against a role for worm glia in sustain-
ing neuronal survival.
C. elegans glia also play an important role in synapto-
genesis in the nerve ring, and this appears to extend to the
level of individual synapse placement. The AIY interneu-
ron forms synaptic contacts with the RIA interneuron in
the nerve ring, and these are in close proximity to CEPsh
glia. UNC-40 (worm DCC, deleted in colorectal cancer), a
receptor for UNC-6, is expressed in AIY and important for
proper positioning of AIY-RIA synapses (Colon-Ramos et
al. 2007). Interestingly, UNC-6 is expressed in CEPsh glia
(Yoshimura et al. 2008), where it acts to cluster neuronal DCC
and promote AIY-RIA synapse formation (Colon-Ramos
et al. 2007). In unc-6 mutants, AIY-RIA synaptic connectivity is
defective. Strikingly, simply repositioning of CEPsh glial mem-
branes more posteriorly in the nerve ring is sufficient to pro-
mote the formation of ectopic AIY-RIA synapses within these
domains, arguing that AIY-RIA form synapses in response to
instructive cues from CEPsh glia (Colon-Ramos et al. 2007).
3.3 G L I A L RO L E S I N B E H AVI O R
Ablation of glial cells in the adult allows for direct assessment
of their roles in sensory-driven behavior and neuronal signal-
ing. A particular strength of the worm is behavioral analysis—
the sensory-driven responses driven by uniquely identifiable
neurons are well described and can be used to directly assess
the function of specific neurons. Bacaj et al. laser ablated
adult sheath glia and assayed the function of three uniquely
identifiable neurons AWC, AWA, and AWB. Strikingly, glial
ablation dramatically reduced AWC- and AWA-mediated
chemotaxis toward odors, but had no effect on AWR func-
tion (Bacaj et al. 2008). Because AWR functions normally
without glia, behavioral defects in AWC and AWA cannot
be explained by a simple structural change in sensory organ
morphology and the roles for glia in neuronal function
appear to be neuron specific. Moreover, neuronal signaling
molecules (e.g., odorant receptors and G proteins) localized
normally in the absence of glia, arguing against a defect in
maintenance of sensory machinery. Interestingly, Ca2+ imag-
ing of osmosensory neurons in the same glial-ablated prepa-
rations revealed a loss of stimulus-induced increases in Ca2+
levels in neurons that are normally seen within seconds in
controls. Thus glia appear essential even at the earliest stages
of sensory neuron signaling (Bacaj et al. 2008). The molecu-
lar basis by which glia modulate neuronal physiology remains
unclear, but recent work has revealed that glial ACD-1
(acid-sensing channel degenerin-like) coordinates with the
neuronal DEG/ENaC channel DEG-1 to modulate acid and
lysine chemotactic behaviors (Wang et al. 2008, 2012).
4 DROSOPHILA GLIA
The Drosophila nervous system is far more complex than that
of C. elegans—the adult fly nervous system houses approx-
imately150,000 to 200,000 neurons. The precise number of
glia remains unclear, but likely represents approximately10%
of the total population of cells in the nervous system. The
Drosophila brain is quite sophisticated structurally and func-
tionally, and compared with C. elegans bears much more
resemblance to the mammalian nervous system. Distinct brain
regions are subdivided into lobes, with each lobe coordinating
specific neurophysiological processes, and many brain regions
are interconnected by well-defined nerves. Fly circuits contain
neurons with electrophysiological properties remarkably sim-
ilar to mammalian neurons: They modulate a diverse behav-
ioral repertoire and exhibit both electrophysiological and
behavioral plasticity. As discussed in the following, an array of
functional classes of glia exist in the fly, each participating in
its own way in nervous system development and function, and
many Drosophila glia appear to perform functions remarkably
similar to their mammalian counterparts.
4.1 MO R P H O L O G I C A L C O M P L E X I T Y
O F D RO SO PH I L A G L I A
In the embryo, larva, and adult, the Drosophila nervous system
can be divided into two major histological regions: the neu-
ronal cell cortex, where neuronal and glia cell bodies reside;
and the neuropil, where axons and dendrites project to form
neural circuits (Fig. 2.2A). Each of these domains contains
multiple classes of glial cells (Fig. 2.2B). The entire Drosophila
nervous system is surrounded by a blood-brain barrier (BBB)
composed of subperineurial glia (SPGs). They exhibit a flat-
tened morphology, cover the entire surface of the CNS, and
seal the BBB by forming pleated septate junctions (pSJs)
with one another (Auld et al. 1995; Baumgartner et al. 1996;
Schwabe et al. 2005). At late larval and adult stages an addi-
tional layer of glia, termed perineurial glia (PGs), forms on the
surface of SPGs (Awasaki et al. 2008; Pereanu et al. 2005; Stork
2. I N VE RT E B R AT E G L I A
A Larval brain-neurons B Larval brain-glia
(dorsal view) (cross section) wrapping
astrocyte ensheathing
(peripheral
nerve)
neuropil
motorneuron
neuronal
cell cortex interneuron cell body
perineurial
(cortex) subperineurial
C neuromuscular junction D sensory organ
wrapping
subperineurial dendrite shaft
socket sheath cell
cell
muscle
sensory neuron glia (axonal)
Figure 2.2 Subtypes, Positions, and Morphology of Drosophila Glia.
A. Overview of the histology of the Drosophila larval central nervous
system. The neuronal cell cortex (gray) houses all neuronal and most
glial cell bodies. All CNS synaptic contacts between neurons are found
within the neuropil (light gray). B. Position and morphology of glial
subtypes (green) in the Drosophila larval CNS. Glial morphology in the
adult brain is essentially the same. See text for details for B, C, and D.
Midline glia are not shown. C. Glia at the Drosophila larval neuromuscu-
lar junction. Motoneuron (MN) terminals are buried in the muscle, but
subperineurial glia (light green) invade the space between the MN and
muscle. D. Sensory organs in Drosophila contain at least three glial-like
cell types: the socket cell, the sheath cell, and an axon-associated glial cell.
A mechanosensory organ is shown as an example.
et al. 2008), and these are thought to be involved in deposi-
tion of a carbohydrate-rich neural lamella that surrounds
the CNS and peripheral nerves (Leiserson et al. 2000) that
serves as an additional physical and chemical barrier to entry
of molecules into the CNS (Carlson et al. 2000). Peripheral
nerves, although covered by the BBB composed of SPGs and
PGs, also house a population of glia termed wrapping glia that
directly interact with and wrap motor and sensory neuron
axons (Beckervordersandforth et al. 2008; Stork et al. 2008).
At the neuromuscular junction, SPGs dynamically invade the
space between the motor neuron terminal and postsynaptic
muscle cells and interact closely with growing synaptic fields
(Fig. 2.2C) (Fuentes-Medel et al. 2009).
In the CNS, cortex glia are found among neuronal cell
bodies, and by the late embryonic stages appear to ensheathe
each neuronal cell body. Impressively, a single cortex glial cell
can surround as many as 100 or more neuronal cell bodies
(Awasaki et al. 2008). The neuronal cell cortex is separated
from the neuropil by ensheathing glia, which compartmental-
izes different regions of the CNS synaptic neuropil and wrap
nerves (Ito et al. 1995). Within the synaptic neuropil are also
astrocytes (Awasaki et al. 2008; Doherty et al. 2009), which
have a highly tufted morphology, have an approximate size
of 20 to 30 μm in diameter, and associate closely with most
synapses.
• 15
 Adult Drosophila sensory organs also house glia that are
quite similar to those found in C. elegans (Fig. 2.2D). For
example, mechanosensory organs house socket glial cells,
which form the socket through which sensory dendrites
extend, and sheath glial cells that enwrap the neuron and prox-
imal dendrites (Walker et al. 2000). An additional glial cell is
also born during development that migrates down the axons
and ensheathes it (Gho et al. 1999).
4.2 T EC H N I C A L A D VA N TAG E S O F
S T U DY I N G D RO SO PH I L A G L I A
Drosophila offers a number of powerful genetic and molec-
ular approaches with which to study glial cell development
and function in vivo. As with C. elegans, the lineages of
Drosophila embryonic glia are fully characterized in both the
CNS and PNS with near single-cell resolution. A number
of molecular markers have been characterized that uniquely
label all, or even specific subtypes of Drosophila glia (Stork
et al. 2012). Impressively, multiple binary expression systems
are now available in the fly: Gal4/UAS (Brand and Perrimon
1993), LexA/LexAOp (Lai and Lee 2006), and QF/QUAS
(Potter et al. 2010) (Fig. 2.3A). Binary expression systems
allow one to use cell type specific promoters (e.g., astrocyte
gene promoters) to drive Gal4 expression, which in turn acti-
vates the tissue specific expression of any transgene placed
under UAS control (e.g., a mis-expression transgene, markers
such as gfp or GCaMP, or optogenetic molecules including
ChR2). In essence one can carefully control the spatiotem-
poral expression of any gene of interest in any cell type for
which there is a defined promoter. Multiple Gal4 driver lines
are available for nearly all subtypes of Drosophila glia (Stork
et al. 2012). Because each of the binary expression systems
function independently, one can combine them in the same
animal to label or manipulate multiple cell types simultane-
ously in vivo. For example, astrocytes can be labeled using
the alrm-Gal4 driver (Doherty et al. 2009), whereas one
labels neurons with 201Y-QF (Potter et al. 2010) and astro-
cyte–neuron interactions can be assayed in live preparations.
Multiple whole-genome UAS-driven RNAi collections are
now available in Drosophila and it is therefore possible to
knockdown almost any gene with high cellular specificity
and assay phenotypic consequences.
Mosaic analysis is a particular strength of Drosophila and
has improved dramatically over the last decade. Gene function
can be analyzed in the fly using mosaic approaches that allow
for the specific labeling of small, or even single homozygous
mutant cell clones. Flippase (Flp)-mediated recombination
works extremely well in the Drosophila nervous system with
flip recombinase target (FRT) sites and can be used to acti-
vate, or inactivate, transgene expression in large groups of cells,
or even in single-cell clones (Fig. 2.3B) (Stork et al. 2012).
One can generate a “Flp-out” cassette, in which Flp-mediated
recombination leads to a change in a gene’s placement relative

A Drosophila binary expression sysytems B Flp/FRT-mediated transgene regulation

glial prometer +++ glial

Gal4 prometer stop reporter gene
UAS (OFF)
FRT FRT
gene X
Flp-medicated
glial prometer recombination
+++
LexA LexAOp
glial
gene Y prometer reporter gene
(ON) FRT
glial prometer +++
QF QUAS
gene Z

C Mosaic analysis with a repressible cell marker (MARCM) D astrocyte MARCM

glial nuclei
mut neuropil
mut mut
mut
astro-Gal4, UAS-GFP
Gal80 Gal4/UAS active
Gal80 Gal80
Gal80
astro-Gal4, UAS-GFP
astro-Gal4, UAS-GFP
Gal4/UAS repressed by Gal80 Gal4/UAS repressed by Gal80

Figure 2.3 Genetic Mosaic Approaches in Drosophila. A. The Gal4/UAS, LexA/LexAOp, and QF/QUAS binary expression systems. Each
functions independently (e.g., Gal4 cannot activate LexAOp or QUAS) in Drosophila. B. Flp-mediated excision of FRT-flanked regions can activate
or inactivate transgene expression. C. Mosaic analysis with a repressible cell marker (MARCM) allows for the production of homozygous mutant,
GFP-labeled clones in an otherwise wild-type nervous system. Gal80 normally represses Gal4/UAS activation. In a MARCM clones after cell division
Gal80 is lost in one daughter cell where the trans chromosomal arm is also made homozygous and Gal4/UAS is de-repressed. D. A MARCM clone in
a Drosophila adult brain astrocyte. Courtesy of Ozge Tasdemir.

16 • NEUROGLIA
to a promoter (ON, putting it near the promoter; or OFF, sep-
arating it from the promoter). Alternatively, one can use Flp
to remove the Gal80 repressor in a cell- and/or developmental
stage-specific manner.
Mosaic analysis with a repressible cell marker (MARCM)
combines flippase Flp/FRT mediated recombination events
with Gal4/UAS and the Gal4 repressor Gal80 to tightly
control marker gene (e.g., GFP) expression only in mutant
cells (Fig. 2.3C,D) (Lee and Luo 2001). Briefly, FRT sites are
available at the base of all five major chromosomal arms. In
MARCM, a mutation of interest is placed distal to an FRT site
and in trans over a chromosome arm contain the same FRT
with a distal Gal80 element. Also included in the background
is the Gal4 driver of interest (e.g., a glial Gal4) and a marker
gene (e.g., membrane-tethered GFP). Before recombination
the Gal4/UAS system is constitutively repressed by Gal80. If a
Flp source is supplied at mitosis, this promotes recombination
at FRT sites, and at some frequency clones are generated that
are homozygous mutant for a gene of interest, and have lost the
Gal80 repressor. The Gal4/UAS system is therefore derepressed
in homozygous mutant clones (but not in other genetic types),
and cells are labeled with GFP. This has the important advan-
tage of allowing one to examine homozygous mutant clones
(that are marked) in an otherwise wild-type nervous system.
Genetic mosaics can also be generated using Gal4/UAS alone.
For example, one can knockdown genes specifically in glial
cells by driving glial-specific expression of UAS-RNAi con-
structs. Alternatively, in mutant backgrounds one can resupply
A late embryo/
1st instar larva
axons
subperineurial glia
wrapping
glia
perineurial
glia
perineurium
B C
Wrapping
(hemolymph) glia
subperineurial
glia
perineurium
axon
(solute
exchange)
Figure 2.4 Formation of Nerve Bundles, Axon Sorting, and Blood-Brain Barrier in Drosophila. A. Morphology of peripheral nerves in the larva
immediately after hatching (first instar) and 3 to 4 days later at third instar larval stages. Note the dramatic sorting of axons (red) into small bundles
by wrapping glia by third instar. B. Morphology of the blood-brain barrier in Drosophila (enlarged from A). Entry into the CNS requires passage
through the carbohydrate-rich perineurium, subperineurial glia (SPG), and often wrapping glia. C. Molecular components required for assembly of
the SPG-SPG pleated septate junction (enlarged from B). See text for details.
2. I N VE RT E B R AT E G L I A
a rescuing transgene only in cells of interest to demonstrate
autonomy of gene function.
It should be emphasized that each of the preceding tech-
niques can be further combined with the powerful forward
genetic approaches available in Drosophila to study virtually
any glial function in vivo. In addition, as the wiring diagram of
the Drosophila nervous system becomes clearer, one can more
specifically manipulate glial genes or glial function and deter-
mine how perturbation of glial physiology affects the process-
ing of information by neural circuits.
4.3 D EVE L O PM E N TA L A N D F U N C T I O NA L
P RO P E RT I E S O F D RO SO PH I L A G L I A
4.3.1 Axon Sorting and Wrapping
The ensheathment of axons by wrapping glia in peripheral
nerves during Drosophila larval stages is remarkably similar
at the cellular level to radial sorting of axons by oligodendro-
cytes or Schwann cells in mammals (see chapters 42 and 44).
Drosophila motor neuron and sensory neuron axons extend
projections out of, or into the CNS, respectively, by late embry-
onic stages through the peripheral nerves. At early larval stages
axons are initially clustered into large bundles that are in close
contact with wrapping glia that exhibit a relatively simple mor-
phology (Fig. 2.4A). Impressively, during larval stages these
wrapping glia do not divide but grow 10- to 20-fold in length,
3rd instar larva
axon sorting
B
10-20X growth of
nerve length
(~3 days)
Dcont NrxIV Nrg
Moody
Claudin
Cora
Ga Gγ
Gβ loco pSJ assembly
cortical action assembly
membrane interdigitation
• 17
and by the third instar larval stage axons are efficiently sorted
by wrapping glia into smaller clusters of a few or even sin-
gle axon fibers (Leiserson et al. 2000; Stork et al. 2008). The
molecular mechanisms underlying this sorting event remain
poorly defined, but axonal protein Vein seems to signal to the
EGF receptor in glia to coordinate activation of glial genes and
nerve morphogenesis (Sepp and Auld 2003), much like axonal
Neuregulin1 signals to ErbB2/B3 in Schwann cells in mam-
mals to modulate Schwann cell survival and myelination of
axons (Garratt et al. 2000).
4.3.2 Formation of the Blood-Brain Barrier
The blood-brain barrier (BBB) of Drosophila is first estab-
lished in the embryo and insulates the CNS and periph-
eral nerves from the surrounding hemolymph (see chapter
33). The fly CNS appears to be immune privileged in that
peripheral macrophages fail to enter the CNS under nor-
mal conditions. The first layer of the BBB surrounding the
brain (from inside to out) are the subperineurial glia (SPGs)
that form pleated septate junctions (pSJs) with one another
(Fig. 2.4B,C). The injection of dye-coupled dextrans into
the hemolymph revealed that the BBB becomes impenetra-
ble to relatively large dextrans (10kDa) by mid-embryonic
stages, a time point that coincides with the SPGs taking on a
sheet-like morphology and forming pSJs with their neighbors
(Schwabe et al. 2005). The infolding of SPG membranes with
adjacent cells and formation of pSJs is mediated by the Moody
receptor, an SPG-expressed 7TM GPR (Bainton et al. 2005;
Schwabe et al. 2005) (Fig. 4.4C). In moody mutants the inter-
digitation of pSJs at points at which pSJs will form is signifi-
cantly decreased, and pSJs do not form properly (Schwabe et
al. 2005). Dye-dextran injections demonstrate the embryos
lacking Moody have a permeabilized BBB at both embry-
onic (Schwabe et al. 2005) and adult stages (Bainton et al.
2005), but not to the point at which animals are paralyzed
(as is the case in other mutants with more significantly per-
meabilized BBBs), indicating that significant sealing of the
BBB has still occurred. Maintenance of the BBB is an active
and Moody-dependent process throughout the fly life cycle:
Activation of a moody-RNAi construct in adults after nor-
mal BBB formation led to permeabilization, and, strikingly,
turning off expression of the moody-RNAi element resulted in
recovery of BBB function (Bainton et al. 2005).
The components of Drosophila pSJs are molecularly
related to components of the mammalian axon–glial sep-
tate junctions found at paranodes. Molecular components
include Drosophila contactin (Dcont), NeurexinIV (NrxIV),
Neuroglian (Nrg), Coracle (Cora), and Claudin (Fig. 4.4C)
(Banerjee et al. 2006), and mutations in any of these molecules
compromise pSJ function and permeabilization of the BBB.
The consequences of pSJ breakdown are thought to include
influx of K+ from the hemolymph (which is K+-rich), per-
turbed neuronal firing, and animal paralysis (Auld et al. 1995).
It is likely that the BBB is the key site of regulatory exchange
between the hemolymph and the CNS. For any molecule to
gain entry into or to exit the CNS, it must pass through the
carbohydrate-rich perineurium, a layer of SPGs that are sealed
18 • NEUROGLIA
by pSJs, and in many cases also wrapping glia (or in the CNS
cortex glia). Based on their close contact with SPGs, it may
be that wrapping glia and cortex glia act as a direct conduit
for exchange of material into and out of the CNS. Likewise,
Drosophila glial cells likely also play a key role in the exchange
of O2/CO2 in the nervous system since tracheal elements (i.e.,
the fly breathing apparatus) are closely associated with multi-
ple glial cell types in the CNS (Pereanu et al. 2005).
4.3.3 Neural Circuit Assembly, Synaptic Growth,
and Trophic Support
Glial cells in Drosophila exert powerful control of neural cir-
cuit assembly during early axon guidance, and later as circuits
undergo activity-dependent modification. Perhaps the most
famous example is Drosophila midline glia (MG). Early in
CNS development these cells are arranged along the midline
and express potent guidance cues including Netrin and Slit
that govern nearly all longitudinal and commissural axon
path finding decisions at the Drosophila embryonic mid-
line, in many ways acting similarly to the mammalian floor
plate ( Jacobs 2000). At late embryonic stages (initially 12)
MG compete for trophic factors on commissural axons, most
undergo programmed cell death, and three MG per segment
migrate dorsally and ultimately ensheathe commissural axons
(Bergmann et al. 2002). Axonal target layer selection is also
modulated by specific classes of glia during visual system
development. Photoreceptor axons emanating from the lar-
val eye disk project into the CNS optic lobe where they must
decide which target layer to innervate. Photoreceptors 1 to 6
(R1–6) target the laminar compartment, which is defined by
layers of glial cells, while photoreceptors 7 and 8 (R7 and R8)
project through this glial layer into the medulla. Perturbation
of the layer of glia separating the lamina from medulla dra-
matically affects axon targeting. If glia are not present R1 to 6
fail to innervate the lamina and instead project more deeply
into the medulla compartment, indicating that glial cells pro-
vide a spatial cue that selectively promotes the termination
of R1 to 6 growth, while not affecting R7 or R8 (Poeck et al.
2001). The molecular identity of this cue remains undefined.
Neural circuit assembly in mammals often entails an initial
phase of overproduction of neurons and hyper-innervation of
the target areas, followed by selective elimination of exuberant
connections and superfluous neurons. Similar events occur in
Drosophila during both embryonic and pupal stages of nervous
system development and glia play an essential role in the removal
of neuronal debris. For example, approximately 450 neurons are
generated per embryonic hemisegment, but roughly one-third
of these undergo programmed cell death (Rogulja-Ortmann et
al. 2007). Multiple subtypes of glial cells rapidly engulf these
cell corpses to clear them from the CNS (Sonnenfeld and Jacobs
1995). At larval stages, Drosophila mushroom body gamma neu-
rons initially project both medial and dorsal processes during
larval stages, but at metamorphosis both the medial and dorsal
axonal projections and dendrites of gamma neurons are pruned
to rewire the mushroom body with adult-specific axon mor-
phologies. Glial cells invade the mushroom body lobes at the
initiation of axon pruning (Fig. 2.5A) and secrete the TGF-β
molecule Myoglianin, which is essential for activation of axonal
degeneration (Awasaki et al. 2011). As axons fragment glial cells
engulf and degrade degenerating axonal debris, genetic block-
ade of glial engulfing activity potently blocks axonal pruning
(Awasaki and Ito 2004) and genetically labeled axon fragments
can be found within phagocytic glial cells (Watts et al. 2004).
Precisely how much axonal and dendritic material is pruned
during Drosophila metamorphosis remains unclear, but it seems
likely that glial cells are the primary cell type responsible for
clearing most neuronal debris in the CNS. Interestingly, recent
work has shown that glia can also play a protective role during
the pruning of neurites. In the larval peripheral nervous system
proximal sensory dendrites are ensheathed by glial membranes
and dendrite degeneration for pruning initiates precisely where
the glial sheath ends, suggesting a novel neurite protective role
for glial membranes (Fig. 2.5B) (Han et al. 2011).
There is increasing interest in the role of glial cells in the
formation and plasticity of synapses. An excellent preparation
in which to study glial–synapse interactions in Drosophila is
the larval neuromuscular junction (NMJ). Glial cells dynami-
cally invade the NMJ and associate closely with synaptic
contacts between the motoneuron (MN) and postsynaptic
muscle cell (Fuentes-Medel et al. 2009). The morphology and
size of the Drosophila NMJ allows for excellent microscopic
examination, which has led to the discovery that this rapidly
growing synaptic field is quite wasteful. In mutants defective
in engulfment function (e.g., draper, or dCed-6, see the follow-
ing) MN terminals were found to accumulate large amounts of
presynaptic debris in the form of shed membrane fragments or
aborted synaptic boutons. Closer examination of MN termi-
nals in controls revealed that wild-type growing synapses nor-
mally shed large amounts of debris in an activity-dependent
manner. At the NMJ shed presynaptic debris is normally
phagocytosed and degraded by the combined activity of glia
and the postsynaptic muscle cell (Fig. 5C). When efficient
clearance of presynaptic debris by glia was blocked, its accu-
mulation potently blocked synaptic growth, arguing that effi-
cient clearance is essential for the addition of new synaptic
contacts (Fuentes-Medel et al. 2009). Under some conditions
it also appears that glia can also actively promote synapse loss
and MN retraction from the NMJ. In mutants defective in the
spectrin/ankyrin cytoskeleton NMJ terminals initially form,
but later exhibit significant retraction of MN synaptic bou-
tons. During this retraction event glial cells secrete the TNF-α
molecule Eiger, which binds to the TNF-α receptor Wengen
and activates a caspase-dependent pro-degenerative signal-
ing pathway in the MN terminal to promote retraction. Loss
of Eiger, Wengen, or other components of this neuron → glia
signaling pathway significantly suppressed retraction, arguing
that glia instruct MN terminals during this example of pro-
gressive denervation (Keller et al. 2011).
In mammals, glial neurotrophins provide critical support
for neuronal survival. Obvious components of the neurotro-
phin family long eluded discovery in Drosophila, and it was
therefore proposed that glia–neuron trophic support might
not be essential for construction of the fly nervous system.

A CNS axon/dendrite pruning B PNS neurite protection

dendrites
neurites degenerate growth of adult-
glia engulf debris specific neurites
dendrites
protected

dendrites
glia degenerate
3rd Instar early pupa mid/late pupa
larva

C clearance of shed neuronal debris D NT uptake/recycling

glia shed debris Gln

Gs
shed bouton
Glu Glu

Glu EAAT

GAT GABA
degradation
motor GABA
muscle cell neuron
glial cell

Figure 2.5 Functional Roles for Glial Cells in Neural Circuit Assembly, Growth, and Function. A. Drosophila glial cells actively engulf axons and
dendrites early in pupal stages during mushroom body gamma neuron pruning. B. In the peripheral nervous system dendritic processes that are housed
within glial sheaths are protected from degeneration at metamorphosis. C. Muscle growth requires significant expansion of the synaptic field at the
Drosophila neuromuscular junction (NMJ). During larval stages glia invade the growing NMJ and, working coordinately with muscle cells, engulf shed
synaptic debris (membrane fragments and shed synapses). D. In mature neural circuits Drosophila glia express channels and neurotransmitter metabo-
lizing enzymes to take up and recycle glutamate and GABA from the synaptic cleft.

2. I N VE RT E B R AT E G L I A • 19
However, multiple studies have demonstrated that ablation of
Drosophila glia results in a dramatic loss of neurons, and more
recently a number of molecules with neurotrophic properties
have been identified in Drosophila. The Drosophila neurotro-
phin–like family of molecules includes Drosophila neurotro-
phin 1 (DNT1), 2 (DNT2), Spatzle (Spz) (Zhu et al. 2008),
and dmMANF (Palgi et al. 2009). Each of these molecules,
based on loss of function studies, appears to signal to neurons
to promote maintenance of axonal fascicles and neuronal sur-
vival. Spz appears to act through its receptor Toll (Zhu et al.
2008), but how the additional fly neurotrophin–like mol-
ecules signal remains to be determined.
4.3.5 Neurotransmitter Uptake and Recycling,
Maintenance of Ionic Balance
The neurotransmitters (NTs) glutamate and GABA are widely
used in signaling in the invertebrate nervous system. After
synaptic release, rapid clearance of these NTs is essential to
allow for subsequent signaling events to occur properly, and
avoid excitotoxicity or synaptic spillover. Drosophila CNS
glia express NT transporters for both glutamate and GABA:
EAAT1 or EAAT2 (Freeman et al. 2003), or GAT (Thimgan
et al. 2006) (Fig. 2.5D). Loss of EAAT1 function results in
defects in motor behavior in adult flies, and age-dependent
neurodegeneration. Intriguingly, the onset of degeneration
and motor defects could be suppressed by application of drugs
used to treat excitotoxicity in humans (Rival et al. 2004). Glia
also express enzymes required for the subsequent metabo-
lism of glutamate, including glutamine synthetase (Freeman
et al. 2003), which converts glutamate to glutamine for sub-
sequent redelivery to neurons. Drosophila glia also appear to
closely maintain the ionic balance of the nervous system. fray
mutants were initially identified as having large bulges in larval
peripheral nerves. Fray was later identified to be a Drosophila
PASK molecule, which in subsequent work was shown to inter-
act with the Na-K-Cl cotransporter Ncc69 and regulate water
volume and likely osmotic homeostasis (Leiserson et al. 2011).
4.3.6 Activation of Glia After Neural Injury,
Glial Phagocytic Activity
Axotomy induces potent responses from Drosophila glia
in the adult brain. Within hours after axon injury, local
glial cells upregulate expression of the engulfment receptor
Draper, extend membranes to regions of the brain housing
degenerating axons, and phagocytose axonal debris (Fig. 2.6A)
(MacDonald et al. 2006). Draper encodes the Drosophila
ortholog of the cell corpse engulfment receptor CED-1,
which is required in the engulfing cell for the recognition and

A
Reactive responses after neural injury
glial activation
“resting” phagocytosis of axonal debris termination of response
glomerulus

axotomy ~1 week

axons

ensheathing glia
B C

Cell corposes

apoptotic
cell corpose
Draper
-6
ed
ark
dC
Sh

Simu
Draper
P-

Src42a

engulfment and
degradation

Figure 2.6 Phagocytic Roles for Drosophila Glial Cells During Development and Activation After Axonal Injury. A. In response to axon (red)
injury glial cell membranes (green) invade regions containing axonal debris, internalize axon fragments through phagocytosis, and ultimately
return to a resting state. For clarity, only a subset of axons are shown. B. Glial cells engulf and degrade developmentally produced CNS neuronal
cell corpses at embryonic and pupal stages through activation of the Draper signaling pathway. C. Draper (green) is expressed on embryonic glial
membranes and surrounds engulfed neuronal cell corpses (red).

20 • NEUROGLIA
phagocytosis of cell corpses (Zhou et al. 2001). Activation
of the Draper signaling pathway is essential for glial respon-
siveness to axotomy (Fig. 2.6B), and is thought to occur by
clustering of the Draper receptor on engulfment targets
through its extracellular domain. Draper clustering has been
proposed to activate a Src-family signaling cascade composed
of Src42a and Shark, which, together with the PTB-domain
protein dCed-6, promote engulfment (Doherty et al. 2009;
Ziegenfuss et al. 2008). Draper is also important for glial
engulfment of pruned axons in the mushroom body (Awasaki
et al. 2006; Hoopfer et al. 2006), and embryonic neuronal cell
corpses (e.g., Fig. 2.6C). In the context of cell corpse engulf-
ment in the embryo Draper requires the co-receptor/bridg-
ing molecule Six-microns-under (Simu) for activity (Kurant
et al. 2008). Recent work has shown that DRG satellite cells
also use MEGF10/Jedi signaling (the mammalian orthologs
of Draper/CED-1) to engulf cell corpses during mammalian
nervous system development (Wu et al. 2009), arguing that
the glial Draper/MEGF-10/Jedi signaling pathway is a con-
served feature mediating glial phagocytic activity.
5 S U M M A RY A N D P E R S P E C T I VE S
Invertebrate model organisms seem well positioned to help
move the field of glial biology forward in the coming years
in major ways. As discussed in this chapter there are a num-
ber of well-conserved features of glial cells when one com-
pares mammals with Drosophila and even C. elegans. We can
therefore anticipate studies of glia in these organisms, which
have only recently been undertaken in a serious way by many
groups, will yield fundamental insights into glial biology that
are broadly applicable to the more complex nervous system
of mammals. However, there are notable exceptions impor-
tant to the field: (1) proper myelin sheaths are not found in
Drosophila nor in C. elegans (although atypical myelin-like
structures are indeed found in invertebrate species); (2)
radial glial cells appear to be a vertebrate specific cell type;
and (3) the mechanisms of glial cell fate specification have
yet to show significant overlap. Therefore, it seems unlikely
that these topics will be a focus of studies in invertebrates.
Nevertheless, molecular-genetic approaches in invertebrates
are beginning to yield exciting insights into how glia develop,
interact with neurons, regulate synaptic signaling, and mod-
ulate even complex behavioral output. In the future it seems
likely that even larger questions will be incisively addressed in
invertebrates including: (1) Are there “glial circuits” impor-
tant for information processing? (2) What is the in vivo rele-
vance of glial transmission? (3) What are the precise roles of
astrocyte Ca2+ waves? (4) What role do glia play in the pro-
gression of neurodegenerative disease?
AC K N OW L E D G M E N T S
The author would like to thank A. Nicole Fox for critical
reading of the manuscript and excellent suggestions, and all
2. I N VE RT E B R AT E G L I A
members of the Freeman laboratory for helpful discussions.
Figure 2.3D was generously provided by Ozge Tasdemir. The
author apologizes to colleagues in the field whose work he was
unable to cite because of space limitations.
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• 23
 3.
NONMAMMALIAN VERTEBRATE GLIA
Bruce Appel

A B B R E VI AT I O N S 2 C H O R DAT E P H Y L O G E N Y

3V third ventricle Chordates, which include urochordates, cephalochordates,

aATEN anterior apical trunk epidermal neuron and vertebrates, are characterized by a dorsal neural tube.
BBB blood-brain barrier Different members of the chordate phylum have neural tubes
ca capillaries that vary greatly in size and complexity, resulting from differ-
CC coronet cell ences in the numbers, types, and associations of neural cells.
CE ciliated ependymal cell Chordate evolution is also typified by gains in body size,
CNS central nervous system requiring a peripheral nervous system capable of transmitting
DCEN dorsocaudal epidermal neurons motor and sensory information rapidly over long distances.
ep ependymal glia Glial cells vary greatly in number, morphology, and function
ES endodermal strand among chordates, indicating that glial cell evolution goes hand
fp floor plate in hand with the evolution of increasingly large and complex
GFAP glial fibrillary acid protein nervous systems.
GI group I photoreceptor Most current phylogenies, drawing on molecular evi-
GIII group III photoreceptor dence, place urochordates, represented by ascidians, as a
GP gut primordium sister group to the vertebrates, and cephalochordates, rep-
GS glutamine synthetase resented by amphioxus, as a more distantly related, basal
LC lens cell group in the chordate lineage (Bourlat et al. 2006) (Fig.
M meningeal surface 3.1). However, extant ascidians have highly derived mor-
MC mesenchyme cell phologies and life histories and their nervous systems have
mg midline glia been reduced in size, cell type, and functional complexity
MS muscle cell relative to other chordates. Therefore, among extant mod-
NC notochord els examined to date, amphioxus might provide the best
NT neural tube insight to the starting point of evolutionary processes that
pATEN posterior apical trunk epidermal neuron produced the vertebrate nervous system (Lacalli 2008).
PC pigment cup Among the vertebrate groups, increasing size, metabolic
PNS peripheral nervous system demands of the brain, and vascularization of the central
rp roof plate nervous system (CNS) appear to be accompanied by glial
VCEN ventrocaudal epidermal neurons cell diversification.
VT tectal ventricle

1 INTRODUCTION
Glial cells are remarkably varied in morphology, distribu-
tion, and function. This variety reflects evolutionary adapta-
tion, and it has contributed to the evolution of increasingly
complex nervous systems and behaviors (see chapter 1).
Although glial cells are not preserved in fossilized specimens,
ideas about how the evolution of glia have contributed to the
evolution of nervous systems can be gathered from investi-
gations of glial cell form, gene expression, and intercellular
relationships in diverse species representing the major chor-
date groups.
3 E P E N DY M A L G L I A , R A D I A L G L I A ,
AND ASTROCY TES
Among the most striking features of nervous system evolu-
tion among chordates are differences in the number and
diversity of ependymal glia, radial glia, and astrocytes. In
fact, numerous subtypes of these glia have been described
and often given different names such as ependymocytes,
ependymoglia, tanycytes, and radial astrocytes (Cuoghi
and Mola 2009). Whether these apparent subtypes reflect
functionally distinct types of glia is not clear. Therefore,
for simplicity, in this chapter cells lining the ventricles are
ependymal glia, bipolar cells that extend long processes from
the ventricle to the pial surface of the nerve cord are radial

24
 ancestral
chordate

cephalochordates (amphioxus), radial glia

urochordates (tunicates), ependymal glia

hagfish, astrocytes

lamprey, radial glia

placoderms (extinct jawed fish)

sharks, radial glia

skates, rays, astrocytes
bony fish, radial glia
myelin
amphibians, radial glia

reptiles, radial glia

birds, astrocytes

mammals, astrocytes

Figure 3.1 Chordate Phylogeny and Glial Cell Evolution. The tree represents a simplified phylogeny highlighting chordates discussed in this chapter
and their glial cell composition. For each group the predominant, but not necessarily exclusive, cell type among ependymal glia, radial glia, and
astrocytes is indicated. Astrocytes appear to have arisen multiple times in chordate evolution. Compact myelin is a common feature of all jawed
vertebrates.

glia and nonependymal, multiprocess cells that express glial
fibrillary acid protein (GFAP) are astrocytes. Many, but not
all, ependymal glia and radial glia also express GFAP. In many
organisms, the cell bodies of radial glia line the ventricles. In
these cases the terms ependymal glia and radial glia are syn-
onymous. Characteristics of ependymal glia and astrocytes
are described in greater detail in chapter 4 and radial glia are
the subject of chapter 5.
Ependymal glia, cells that line the ventricular system of
the nerve cord, are evident throughout the chordate group.
Thus, ependymal cells might represent a basal glial cell type
from which other glia arose during evolution. The ventricu-
lar lumens of neural tubes in tadpoles of the ascidian Ciona
intestinalis are lined by cuboidal ependymal glia that extend
cilia into the lumen (Katz 1983; Konno et al. 2010). The
basal surface of these cells form the exterior of the neural
tube and are surrounded by basal lamina (Fig. 3.2A, B).
Therefore, ependymal glia comprise most of the neural tube,
with a few neurons embedded among the ependymal glia.
No other types of glial cells have been described for ascid-
ians. During metamorphosis, the ascidian nervous system is
dramatically remodeled. Fate mapping experiments showed
that larval ependymal glia give rise to ependymal glia and
neurons of the adult nervous system, indicating that ascid-
ian ependymal glia have stem cell characteristics (Horie
et al. 2011).
Anatomical and molecular investigations of the amphioxus
larval and adult nervous system have revealed clear homologies

A B C rp
pATEN
DCEN
eg eg
CE NT
PC eg eg
GI
CC GIII LC
MS NC
GP mg fp
MC ES
EP

VCEN

Figure 3.2 Glia of Tunicates and Amphioxus. Transverse section views at the level of the brain (A) and nerve cord (B) of tadpole larvae of the ascidian
Ciona intenstinalis. Cells comprising the neural parenchyma are green. aATEN, anterior apical trunk epidermal neurons; CC, coronet cell; CE,
ciliated ependymal cell; DCEN, dorsocaudal epidermal neurons; ES, endodermal strand; GI, group I photoreceptor; GIII, group III photoreceptor;
GP, gut primordium; LC, lens cell; MC, mesenchyme cell; MS, muscle cell; NC, notochord; NT, neural tube; pATEN, posterior apical trunk
epidermal neuron; PC, pigment cup; VCEN, ventrocaudal epidermal neurons. C. Transverse section through the nerve cord of larval amphioxus. eg,
ependymal glia; fp, floor plate; mg, midline glia; rp, roof plate. (A,B) From Konno, et al. 2010. (C) Modified from Lacalli TC and Kelly SJ. 2002.
with permission.

3. N O N M A M M A L I A N VE RT E B R AT E G L I A • 25
with vertebrate nervous systems (Lacalli 2008). Unlike sessile
adult ascidians, adult amphioxus remain motile throughout
life. Therefore, compared with ascidians, the amphioxus nerv-
ous system is quite complex (Fig. 3.2C). However, relative to
most vertebrates, amphioxus larvae and adults are small and
the CNS is not vascularized, which are important differences
relevant to glial cell functions.
Amphioxus have a prominent ventricular system
(Brocklehurst 1979), which in larvae is surrounded by
ependymal glia (Wicht and Lacalli 2005). The ependymal
glia extend processes to the limiting membrane of the nerve
cord and therefore have radial glia character (Bone 1960;
Lacalli and Kelly 2002). Small glial cells, called Schneider’s
glia, line the ventricle near the dorsal roof of the nerve cord
(Bone 1960). These cells have short processes, and therefore
could be considered as astrocyte-like, but the processes do
not appear to terminate within the limiting membrane of
the cord. However, several types of neurons lie within close
proximity to Schneider’s glia, raising the possibility that
these glial cells provide some sort of support to neurons. A
distinct floor plate and roof plate is evident in amphioxus
larvae, as well as a small number of other non-neuronal cells
described as midline glia and axial glia, which extend long
membrane processes along the ventral nerve cord (Lacalli
2000; Lacalli and Kelly 2002). In the periphery, groups of
very small cells called Müller’s glia are clustered with the dor-
sal nerves (Bone 1960). Neither the origin nor function of
Müller’s glia are known.
Agnathans, or jawless fish, represented by lampreys and
hagfish, comprise the most basal group of vertebrates (see
Fig. 3.1), having diverged from the lineage that gave rise to
jawed vertebrates about 600 million years ago. Unlike ascid-
ians and amphioxus, the hagfish CNS is vascularized (Cecon
et al. 2002). Hagfish and lampreys have a blood-brain bar-
rier with similar characteristics to mammals, and the BBB
is formed by endothelial cells (Bundgaard 1982; Bundgaard
and Cserr 1981). GFAP+ cells are prominent throughout the
A B
Figure 3.3 A. Drawing of GFAP+ astrocyte-like cells and processes in hagfish. In the center a multiprocess cells (2) makes contacts on capillaries (ca).
B. Section obtained from optic tectum of dogfish. Glial fibrillary acid protein labeling reveals radial fibers spanning the distance between the tectal
ventricle (VT) and the meningeal surface (M). C. Cross-section through the diencephalon of skate showing distribution of GFAP. In the center, radial
fibers stretch from the third ventricle (3V) to the basal surface. Only nonependymal astrocyte-like cells are evident laterally. (A) From Wicht et al.
1994. (B,C) from Kálmán M and Gould RM. 2001, with permission.
26
hagfish CNS (Wicht et al. 1994). However, GFAP+ radial
glia do not occupy the entire CNS but are found only in por-
tions of the spinal cord. By contrast, numerous, multiprocess
GFAP+ cells that do not contact the ventricle are distributed
throughout the CNS (Fig. 3.3A). These cells have processes
that are fairly short, irregular, and with few branches, and
they make contacts on both blood vessels and neurons.
Antibody to glutamine synthetase (GS), a commonly used
marker of astrocytes, also labels nonependymal cells with
numerous fine processes that form meshworks around blood
vessels and neurons (Wicht et al 1994). Therefore, the glial
cell architecture of hagfish represents a departure from
that of amphioxus, with a transition from a predominantly
ependymal, radial glial organization to a predominantly non-
ependymal, astrocyte-like organization. Very little is known
about glia in lamprey. However, antibodies to GFAP and
keratin-like proteins detect numerous radial fibers extending
from the ventricles to the limiting membrane of the nerve
cord (Merrick et al. 1995; Wasowicz et al. 1994), indicat-
ing that in contrast to hagfish, lamprey has a predominantly
radial glial organization.
Elasmobranchs, a subclass of cartilaginous fish repre-
sented by sharks, skates, and rays, first appear in the fossil
record about 400 million years ago (see Fig. 3.1). Notably,
the BBB of elasmobranchs is not endothelial, as in other ver-
tebrates, but composed of glia cells (Bundgaard and Abbott,
2008; Bundgaard and Cserr 1981). GFAP labeling of dogfish
shark revealed that radial glia are prominently distributed
throughout the CNS with little or no evidence of stellate
astrocyte-like cells (Kálmán and Gould 2001; Wasowicz
et al. 1999) (Fig. 3.3B). Radial glia make en passant contacts
onto blood vessels and radial glia endfeet make up the menin-
geal glia limitans of the nerve cord (Kálmán and Gould
2001). By contrast, GFAP+ radial glia are limited to portions
of the CNS of skates and rays, with numerous astrocyte-like
cells evident in some parts of the nervous system (Fig. 3.3C).
These cells appear to form a meshwork around blood vessels,
C
• NEUROGLIA
consistent with a glial BBB, and they form the meningeal glia
limitans (Kálmán and Gould 2001; Wasowicz et al. 1999).
Therefore, among the elasmobranchs there is an apparent
transition from nervous systems having predominantly radial
glia (sharks) to nervous systems having mixtures of radial glia
and astrocytes (skates and rays). Kálmán and Gould (2001)
noted that that the difference in radial glia and astrocyte
composition between sharks and skates is similar to the dif-
ferences between reptiles, which have predominantly radial
glia, and mammals and birds, which have both radial glia and
astrocytes. This indicates that radial glia in different verte-
brate lineages have undergone independent and parallel evo-
lution toward astrocyte characteristics.
The glia of several species of bony fish have been surveyed,
and although distinctions of possible subclasses of cell type
can be made (Cuoghi and Mola 2009), bony fish clearly have
predominantly radial glia and relatively few nonependymal,
stellate or radial astrocyte-like glia (Fig. 3.4A). The adult
barbel has numerous radial glia throughout the CNS, with
a few GFAP+ cells, described as radial astrocytes, located
just under the pial surface of the nerve cord with their pro-
cesses contributing to the glia limitans (Bodega et al. 1993).
Radial glia in barbel seem to be heterogeneous with respect
to intermediate filament expression. Although most radial
glia express GFAP some do not and a small subset of radial
glia is labeled by antibody to vimentin (Bodega et al. 1993).
Similarly, Iberian barb has numerous GFAP+ radial glia, a few
vimentin+ radial glia and some GFAP+, nonependymal radial
astrocytes (Rubio et al. 1992). Heterogeneity of radial glia is
also revealed by NADPH-diaphorase histochemistry, which
in sunfish labels a subset of radial glia described as tanycytes
as well as nonependymal, multiprocess cells that contact
blood vessels, neurons, and the pial surface (Ma 1993). Carp
have numerous GFAP+ radial glia, some of which appear
to extend long processes along blood vessels, and very few
astrocyte-like cells (Kálmán 1998). Both GFAP immunohis-
tochemistry and transgenic reporter gene expression reveal
numerous radial glia in zebrafish (Bernardos and Raymond
2006; Marcus and Easter 1995). Additionally, nonependy-
mal star-shaped cells that make contacts on blood vessels and
the subpial surface of the nerve cord are apparent in adult
zebrafish (Kawai et al. 2001). Many radial glia in zebrafish
also express GS, as well as aquaporin-4, which in mammals
functions as the major astrocyte water channel (Grupp et al.
2010). Unlike mammals, in which aquaporin-4 channels are
mainly localized in astrocyte processes around blood vessels
and at the subpial surface, in zebrafish aquaporin-4 is distrib-
uted along the length of radial glial fibers and not concen-
trated in glial endfeet. Therefore, zebrafish radial glia share
some characteristics with mammalian astrocytes, indicating
that radial glia provide some basic astrocyte functions in
nonmammalian vertebrates.
Amphibians are similar to the elasmobranchs, in that
species with primarily radial glia or mixtures of radial glia
and nonependymal astrocyte-like cells can be identified. For
example, GFAP antibody labeling reveals widespread radial
glia that project endfeet onto blood vessels and the glia limi-
tans in salamander but does not detect nonependymal glia
(Naujoks-Manteuffel and Roth 1989). By contrast, in adult
toad GFAP expression marks both radial glia and nonependy-
mal radial astrocyte-like cells, both of which extend processes
on to blood vessels and the subpial glia limitans (Bodega et al.
1990) (see Fig. 3.4).
The glia of several reptile species have been examined,
mostly by using GFAP immunohistochemistry. In turtle,
GFAP antibody labels mostly radial glia and only a relatively
small number of radial astrocytes in the spinal cord (Lazzari
and Franceschini 2006). Geckos and lizards have numerous
GFAP+ radial glia throughout the CNS, fewer radial astro-
cytes in the spinal cord, and rare stellate astrocyte-like cells in
the brain (Lazzari and Franceschini 2001, 2005a,b). In addi-
tion to radial glia, stellate GFAP+ astrocytes occupy the snake
brain (Onteniente et al. 1983). In crocodile, both GFAP+
radial glia and widely distributed astrocyte-like cells are evi-
dent, with radial glia much more numerous than astrocytes
(Fig. 3.4B) (Kálmán and Pritz 2001).

A B C

Figure 3.4 A. Drawing of GFAP+ structure in the forebrain of carp. Only radial glia are evident. B. Drawing of cross-section through telencephalon
of crocodile, showing GFAP+ features. Radial glia are predominant, with some irregular fibers evident in lateral regions (4b). C. GFAP labeling of
chicken tectum. Radial fibers at the subventricular zone are evident (small arrows) as well as prominent astrocytes (curved arrows). (A) From Kálmán
1998. (B) from Kálmán and Prinz 2001. (C) from Kálmán et al. 1998, with permission.

3. N O N M A M M A L I A N VE RT E B R AT E G L I A • 27
 With birds, the relative contributions of radial glia and
astrocytes to the CNS become more like mammals than other
nonmammalian vertebrates (Fig. 3.4C). Numerous GFAP+
and vimentin+ radial glia are evident in many, but not all
regions of the brain of chicken, including adults, but non-
ependymal astrocytes, which extend processes on to blood
vessels, neurons and the glia limitans of the nerve cord, are
much more pronounced in number than other nonmam-
malian vertebrates (Alvarez-Buylla et al. 1987; Kálmán et al.
1993, 1998).
4 GLIAL PHYLOGENY AND
I M P L I C AT I O N S F O R A S T R O C Y T E
E VO LU T I O N
Surveys of glia raise the possibility that astrocytes arose
independently from radial glia in several vertebrate lineages
through evolutionary history. In particular, the agnathan lam-
preys and hagfish, which may have arisen from the vertebrate
lineage independently (see Fig. 3.1), have distinct glial archi-
tectures, with lamprey having primarily radial glia and hag-
fish having relatively few radial glia but many astrocyte-like
cells. Among elasmobranchs, sharks have primarily radial glia,
whereas skates and rays have both radial glia and astrocyte-like
cells. Bony fish, amphibians, and reptiles have mostly radial
glia, with relatively small numbers of astrocytes evident in
some species. Birds and mammals are similar in having rela-
tively few radial glia and large numbers of nonependymal,
stellate astrocytes. However, birds are more closely related
to alligators, which have primarily radial glia, than to mam-
mals, indicating that the astrocyte-predominant patterns of
birds and mammals evolved independently. An important
caveat to the multiple, independent origin hypothesis is that
investigations of radial glia and astrocyte in nonmammalian
vertebrates have been mostly limited to the use of GFAP as a
marker, which might fail to uncover primitive astrocyte-like
cells. Application of a broader set of histochemical stains
known to reveal glial cell morphologies might help to clarify
the question of astrocyte origins.
If astrocytes arose multiple times from radial glia, what
might have driven the transition? One possible hint is that the
transition of radial glia to astrocytes parallels the transition of
thin- to thick-walled brains, which reflects increasing brain
complexity (Kálmán 2002). The formation of thick-walled
brains might place metabolic demands on glial cells that can-
not be supported by very long radial glia. In particular, it has
been speculated that extremely elongated radial glia cannot
maintain normal potassium equilibrium and, consequently,
transform into stellate cells (Reichenbach 1989; Reichenbach
et al. 1987). However, the correlation between astrocyte appear-
ance and brain wall thickness or complexity is not a perfect one
among vertebrates (Kálmán and Gould 2001). Furthermore,
astrocyte-like cells are evident in the very small and relatively
simple nervous systems of fruit flies (see chapter 2). Perhaps
the repeated transition of radial glia to astrocytes facilitated
increasingly complex neural functions, for example, by modu-
lating synaptic activity, as has been proposed for evolution of
28 • NEUROGLIA
astrocyte form and function among mammals (Oberheim
et al. 2012).
5 G L I A , A D U LT N E U R O G E N E S I S ,
A N D I N J U RY R E S P O N S E
One characteristic that distinguishes many nonmammalian
vertebrates from mammals is widespread neurogenesis in the
adult brain (Kaslin et al. 2008). For example, bony fish, which
have a predominantly radial glia architecture, maintain high
levels of neural cell proliferation and neurogenesis in adult-
hood. Cell proliferation is widespread across the brains of
adult teleost fish, centered in about 16 distinct proliferation
zones (Adolf et al. 2006; Grandel et al. 2006; Zupanc 2001).
In the adult zebrafish brain, approximately 6000 cells are born
within any 30-minute period, representing about 0.06% of
total cells, a substantially higher proportion than in mammals
(Hinsch and Zupanc 2007). Numerous studies show that
radial glia are the sources of new neurons that can persist for
long periods (Adolf et al. 2006; Grandel et al. 2006; Hinsch
and Zupanc 2007; Zupanc et al. 2005). In adult fish, both the
numbers and volume of individual muscle fibers increase over
time (Zimmerman and Lowery 1999), whereas in mammals
postembryonic growth results from an increase in size but not
in the number of muscle fibers (Rowe and Goldspink 1969).
Additionally, adult fish increase the number of sensory recep-
tor cells and organs with age ( Johns and Easter 1977; Nuñez
et al. 2009). The continuous addition of peripheral motor
and sensory elements could promote continuous addition of
central neurons that process the associated motor and sensory
functions (Zupanc 2001).
Close examination of cycling characteristics and molec-
ular marker expression reveals heterogeneity among neural
precursors in adult zebrafish. Some precursors do not express
markers common to radial glia, but instead seem to retain
characteristics of embryonic neuroepithelial precursors
(Ganz et al. 2010; Ito et al. 2010; Kaslin et al. 2009; Marz et
al. 2010). Whether adult glial and nonglial neural precursors
represent distinct stages in progression of a precursor cell lin-
eage, neural precursors can alternate between glial and non-
glial states, and glial and nonglial precursors have different
roles in homeostasis and response to disease and injury are
areas of investigation that might provide important insights
into the biology of mammalian neural stem cells.
Another distinguishing feature of many nonmamma-
lian vertebrates is a robust ability to regenerate neural tis-
sue following injury. In adult zebrafish, a stab wound to the
brain or spinal cord transection prompts a large increase in
cell division by radial glia and formation of new neurons
(Baumgart et al. 2011; März et al. 2011; Reimer et al. 2008;
Zupanc 2001). Transgenic fate mapping experiments show
that radial glia are the source of new neurons following
injury (Kroehne et al. 2011). Furthermore, although wound-
ing induces features of reactive gliosis, glial scarring is not
evident as in mammals (Baumgart et al. 2011; Kroehne et al.
2011; März et al. 2011), which might also contribute to the
regenerative capacity of fish.
 6 M Y E L I N AT I N G G L I A
Glial cells wrap axons in many invertebrates (see chapter 2),
and some invertebrates such as copepods and earthworms
have axons ensheathed by myelin-like membrane (Davis et al.
1999; Roots 2008). Although invertebrate myelin enhances
nerve conduction velocity (Lenz et al. 2000), similar to verte-
brate myelin, invertebrate and vertebrate myelin have distinct
characteristics, indicating that they evolved independently
(Roots 2008).
Among chordates, CNS axons of ascidians and amphi-
oxus lack compact myelin and, in fact, appear not to be
ensheathed by glia (Katz 1983; Wicht and Lacalli 2005).
Axons are similarly unmyelinated in lamprey and hagfish,
although in hagfish many central and peripheral axons are
ensheathed by a single turn of glial cell membrane and bun-
dled axons are often separated by glial processes (Bullock et
al. 1984). The elasmobranchs, bony fish, amphibians, reptiles,
and birds have compact myelin with characteristics similar
to that of mammals (Kitagawa et al. 1993; Schweigreiter
et al. 2006; Waehneldt et al. 1986), indicating that myelin
likely evolved with the placoderms, jawed fish that appeared
about 400 million years ago. Indeed, inference of peripheral
nerve length from examination of fossilized skulls suggests
that nerves of placoderms but not of contemporaneous jaw-
less ostracoderm fish must have been myelinated, raising the
possibility that jaws and myelin evolved coordinately (Zalc
et al. 2008).
All extant vertebrates have two populations of myelinat-
ing glial cells: oligodendrocytes for the CNS and Schwann
cells for the peripheral nervous system (PNS). This raises
fascinating questions about whether myelinating glia arose
once in evolution and then diverged into oligodendrocyte
and Schwann cell populations or two distinct types of glial
cells independently gained the ability to myelinate axons,
and if so, when they evolved relative to each other. Schwann
cells arise from the neural crest, a multipotent precursor
population that forms at the boundary between neural and
non-neural ectoderm (Knecht and Bronner-Fraser 2002).
The evolution of neural crest appears to have accompanied
and enabled the transition from filter feeding to active pre-
dation at early stages of vertebrate evolution (Northcutt
and Gans 1983) consistent with the idea that myelination of
peripheral nerves first occurred among the placoderms. By
contrast, oligodendrocytes have a different embryonic ori-
gin, with most produced by neural precursors that occupy
the ventral neural tube (Rowitch and Kriegstein 2010), well
separated from neural crest. Oligodendrocytes and Schwann
cells are specified from their precursors by distinct molecular
regulatory mechanisms, and the manner in which they wrap
axons is very different. Therefore, the ability to form compact
myelin on axons likely arose twice at a very early stage in ver-
tebrate evolution.
Many axons cross the nerve cord boundary and are myeli-
nated by both oligodendrocytes and Schwann cells. Because
absence of myelin on one portion of an axon causes a conduc-
tion block, it has been proposed that myelinating Schwann
3. N O N M A M M A L I A N VE RT E B R AT E G L I A
cells and oligodendrocytes must have appeared at the same
time, in placoderms (Zalc 2006). An alternative possibility is
that one population of glia initially myelinated both central
and peripheral axons and the second population arose later
to enable evolution of increasingly large animals with increas-
ingly complex motor activities. Consistent with this possi-
bility, in the absence of Schwann cells in zebrafish and mice,
oligodendrocyte progenitor cells migrate from the spinal cord
through motor axon exit points to wrap and myelinate periph-
eral motor axons (Coulpier et al. 2010; Kucenas et al. 2009).
Similarly, Schwann cell apparently migrate into the nerve cord
and myelinate central axons in multiple sclerosis patients and
demyelinated rodents (Duncan and Hoffman 1997; Itoyama
et al. 1983), although recent fate mapping experiments show
that Schwann cells arise from CNS progenitors following
demyelination (Zawadzka et al. 2010). Oligodendrocytes are
produced by neural precursors that also give rise to motor
neurons and oligodendrocytes myelinate the proximal por-
tions of motor axons before they exit the nerve cord. The
selective advantage of myelin likely first had bearing on motor
functions, by permitting rapid escape behaviors. Therefore, it
seems plausible that oligodendrocytes evolved first and that
their initial function was to myelinate motor axons along their
entire length. Subsequently, the ability of neural crest-derived
glia to myelinate axons might have provided additional selec-
tive advantage, thereby leading to their replacement of oligo-
dendrocytes as the peripheral myelinating cells.
7 S U M M A RY A N D P E R S P E C T I VE S
Two features of glia in nonmammalian vertebrates stand
out as being markedly different from mammalian glia and
therefore potentially instructive for our understanding of
glial cell evolution and function. First, a predominantly
radial glial organization is widespread throughout extant
chordates with evidence of multiple and independent tran-
sitions to astrocytes, which are smaller and less extensively
branched than astrocytes in mammals, particularly humans.
Investigation of astrocyte properties in nonmammalian spe-
cies might provide insights to basic astrocyte roles in mam-
mals and a better understanding of how further elaboration
of astrocyte functions contribute to human brain function.
Second, many radial glia have stem cell characteristics in
adult nonmammalian vertebrates and respond to neural
injury by elevating cell division and neuron production.
Investigation of radial glia precursors in normal and injured
brains of genetically tractable organisms such as zebrafish
might yield information that can be used to promote repair
and injury of human nervous systems damaged by disease
or injury.
AC K N OW L E D G M E N T S
Thanks to Robert M. Gould for discussion and comments on
the manuscript.
• 29
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MORPHOLOGY, ULTRASTRUCTURE, AND IDENTIFICATION
This page intentionally left blank
 4.
ASTROCY TES AND EPENDYMAL GLIA
Andreas Reichenbach and Hartwig Wolburg

A B B R E VI AT I O N S principle, radial glia cells are involved in brain development,

by acting as stem and progenitor cells generating neurons and
AQP aquaporins (water channels) glia, as well as by guiding migrating neurons and thereby con-
BMI basal membrane infoldings tributing to the organized arrangement of neurons (Götz and
CA1 region region 1 in the cornu ammonis (hippocampus) Huttner 2005; Howard et al. 2008) (see chapter 5). Although
CNS central nervous system most radial glia cells differentiate into neurons and all macro-
CVO circumventricular organs glial classes, some radial glia cells maintain a radial arrange-
GFAP glial fibrillary acid protein ment in the adult brain; these cells are then called tanycytes
GFP green fluorescent protein (see chapter 12) (in brain: Fig. 4.1I) or Müller cells (in retina:
GLAST glutamate aspartate transporter Fig. 4.2A).
GS glutamine synthetase There are two types of astroglia-like cells in the central
NG2+ cells glial/progenitor cells expressing the chondroi- nervous system (CNS) that will not be subject of this chapter.
tin sulfate proteoglycan, NG2 First, the olfactory ensheathing cells partially occupy the brain
Kir4.1 inwardly rectifying potassium channel type 4.1 but are descendants of the peripheral nervous system. Second,
NKCC1 Na+/K+/2Cl– cotransporter the immature astrocytes/glioblasts (Fig. 4.1, IX) will be sub-
OAP orthogonal arrays of particles ject of chapter 12.
RPE retinal pigment epithelium
SVZ subventricular zone

pia mater
1 M O R P H O L O GY O F T H E P E C U L I A R
III
CELL TYPES molecular
layer
As shown in Figure 4.1, the morphology of astroglia and VIII
ependymoglia is very diverse. In particular, the soma of the II
cells may give rise to one or several “primary” or “stem” pro- blood grey
cesses, from which secondary branches may begin. Much of vessel matter
this diversity is related to structural and functional interac- IV
tions of a given cell with its microenvironment, which includes granular
on the one side the neurons and on the other side, blood ves- V
VII layer
sels, the pia mater, and/or the ventricular space (Wolburg
et al. 2009). Macroglial cells may form a “border sheath” against blood white
the ventricular space, the pia, or blood vessels. This is observed la
vessel matter
blood
in ependymocytes (Fig. 4.1, X), choroid plexus cells (Wolburg vessels
lb VI
subepen-
basal membrane
and Paulus 2010) (Fig. 4.1, XI), and retinal pigment epithelial IX labyrinth(s) dymal zone
cells (Fig. 4.4C) but also, although less obvious, in marginal
XI X ependyma
astrocytes (Fig. 4.1, III), perivascular astrocytes (Fig. 4.1, VII),
and pecten glial cells (Fig. 4.4A, B). Within the brain paren- cilium ventricle
microvilli
chyma proper, typical astrocytes are more or less star-shaped
(Fig. 4.1, IV and VIII), but this may be modified by adjacent Figure 4.1 Semi-schematic survey of the main types of astroglial and
neuronal cell bodies (e.g., Fig. 4.1, V) or axons (Fig. 4.1, VI), or ependymoglial cells, and their localization in different layers/specialized
peculiar relationships to the pial surface (Fig. 4.1, II). The term regions of the central nervous system tissue. I, tanycyte (a, pial; b, vascular);
radial glia should be restricted to bipolar ependymoglial cells II, radial astrocyte (Bergmann glial cell); III, marginal astrocyte; IV,
protoplasmic astrocyte; V, velate astrocyte; VI, fibrous astrocyte; VII,
that extend long processes throughout (most of ) the thickness perivascular astrocyte; VIII, interlaminar astrocyte; IX, immature
of the tissue from one surface (the outer pial surface) to the astrocyte/glioblast; X, ependymocyte; XI choroid plexus cell. From
other surface (the inner ventricular surface) of the brain. In Reichenbach and Wolburg 2005, with permission.

35
 1.1 R A D I A L G L I A L C E L L S I N A D U LT C E N T R A L
N E RVO US SYS T E M ( TA N YC Y T E S )
Throughout the literature, the terms radial astrocyte and
radial glia are often used as synonyms. As the primary (fetal)
radial glia consists of bipolar ependymoglial cells, we propose
to reserve this term in adult CNS to persistent ependymoglial
cells such as tanycytes and Müller cells, whereas Bergmann
glial cells and similar astrocytic cells (e.g., in the hippocam-
pus), which lack an ependymal process, should be termed
radial astrocytes (or radially oriented astrocytes).
Radial glial cells, often referred to as tanycytes (Fig. 4.1, Ia
and Ib) are the most common type of macroglia in the CNS of
lower vertebrates (and even of deuterostomic invertebrates).
In adult mammals, they are restricted to certain brain regions
where the tissue is rather thin, such as some circumventricular
organs (circumventricular organs [CVOs], e.g., the subcom-
missural organ, subfornical organ, area postrema), the stalk
of the hypophysis, and the velum medullare, and to the raphe
region of the spinal cord. In the CVOs of all vertebrates except
sharks, the capillaries are fenestrated. In these regions, the
tanycytes (as well as the choroid plexus epithelial cells within
the choroid plexus) constitute the blood-cerebrospinal fluid
barrier by expressing extensive tight junctions. Some of these
tanycytes are specialized for the secretion of signaling mol-
ecules, and of the material constituting Reissner’s fiber. In the
adult subventricular zone (SVZ), short tanycytes are found
which extend a cilium into the ventricle, and form endfeet
at blood vessels; these cells were shown to function as stem
cells (see chapter 30). More about tanycytes and the choroid
plexus can be found in Wolburg et al. (2009) and Wolburg
and Paulus (2010).
1.2 M Ü L L E R C E L L S
Müller cells are the radial glia of the retina. In many verte-
brates, including some mammals (i.e., those with avascular
retinae), they are the only cells representing the macroglia fam-
ily. They contact virtually every neuronal and non-neuronal
element of the retina with specialized branches of their pro-
cesses. In the nuclear layers of the retina (particularly, in the
outer nuclear layer containing the somata of the photorecep-
tor cells) the Müller cell processes assume the shape of velate
astrocytes, whereas in the plexiform layers (where the synapses
of the retinal neurons are located) the Müller cell processes
resemble those of protoplasmic astrocytes (see Fig. 4.1). In the
central retina close to the optic nerve head, in many species
the nerve fiber layer becomes very thick; there, the Müller cell
processes are thin and smooth like those of fibrous astrocytes.
Müller cells occupy a variable volume fraction of the retinal
tissue, from about 3% (most lower vertebrates), 5% to 8%
(most mammals, i.e., those with vascularized retinae) up to
about 20% (mammals with avascular retinae, such as the rab-
bit and guinea pig). The volume of individual Müller cells may
vary from 400 μm3 (mouse) to greater than 2,000 μm3 (rabbit,
retinal periphery); their surface area is in the range of 6,000 to
12,000 μm2. Müller cells constitute a fairly uniform “lattice”
36 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
in the retinal tissue; their densities per mm2 retinal surface
area range between 1,550 and 2,000 (frog, salamander), 5,000
to 12,000 (most mammals), and greater than 25,000 (in the
fovea centralis of primates). Each Müller cell ensheathes and
supports a columnar group of retinal neurons. The number of
neurons per Müller cell ranges between 7 (tree shrew), about
16 (human nonfoveal retina, rabbit, and other herbivorous
mammals), about 30 (rodents and carnivores with strongly
rod-dominant retinae), up to 80 (frog), and even more than
200 (deep sea teleosts with lifelong generation of new rod
photoreceptor cells). The size and shape of Müller cells depend
on animal species and, within a given retina, on retinal topog-
raphy. The total length of a Müller cell is determined by the
(local) thickness of the retina, with a few exceptions in reti-
nae with thick blood vessels in which individual Müller cells
may end at the scleral surface of such a vessel, rather than the
inner limiting membrane facing the vitreous body. Generally,
in rod-dominant retinae, a Müller cell extends just one stem
process from soma to the inner limiting membrane. This is the
case in most fish and mammals. In the cone-dominant retinae
of most reptiles and birds (as well as of the tree shrew), the
vitread stem processes of Müller cells are split into several
branches. In species with a native polyploidy (e.g., lungfish and
salamander) not only the cell nucleus, but also the entire cell
is huge. This may be advantageous for experiments on single
cells such as intracellular electrophysiological recordings, dye
injections, etc. Literature about Müller cells can be found in
Reichenbach and Bringmann (2010).
1.3 R A D I A L A S T RO C Y T E S
Radial astrocytes (Fig. 4.1, II) are common in the spinal cord
and brain of lower vertebrates. As they cross white and gray
matter, the properties of their cell processes may change from
“protoplasmic” to “fibrous.” In some lower vertebrates such
as fish, radial astrocytes possess “velate” processes. Typical
examples of radial astrocytes are shown in Figure 4.2A. Some
radial astrocytes are also found in the optic nerve of mammals,
where they are intermingled with the more abundant longitu-
dinally aligned fibrous astrocytes (Fig. 4.2F).
In the mammalian hippocampus, radially oriented astro-
cytes occur that do not abut the pia with their processes;
rather, they are confined to the stratum granulare of the den-
tate gyrus (Feig and Haberly 2011) and the stratum oriens
of the CA1 region. Examples of these cells are shown in
Figure 4.2B, C (see chapter 5). They should be considered as
a unique cell type, clearly different from the radial astrocytes
of lower vertebrates (and from Bergmann glial cells), which
all contact the pia mater.
1.4 B E RG M A N N G L I A
According to our definition, Bergmann glial cells (also termed
Golgi epithelial cells) are radially oriented astrocytes of the cer-
ebellum in all vertebrates. However, because their cell bod-
ies reside in the layer of the Purkinje cell somata, and their
processes (usually, 3–6 per cell) cross the molecular layer,
 A
f
f
nerve
p p

radially oriented
astrocytes:
p mouse optic nerve

v axon

Bergmann glial cells: radially oriented

chick cerebellum astrocytes:
Müller cells: cow retina turtle spinal cord
50μm 25μm
B C
SM

SG

MCE

MCE

MCE

D E 20μm

100μm

fibrous astrocytes:
F cat optic nerve mouse optic nerve

Figure 4.2 A–C. Radially oriented glial cells. (A) Camera-lucida drawings of Golgi-stained or dye-injected cells. Müller cells represent bona fide radial
glial cells (as the “tanycytes of the retina”), whereas cerebellar Bergmann glial cells and radially oriented astrocytes in other central nervous system
regions miss a contact to the ventricular surface and thus are specialized astrocytes. Note that the character of the processes is determined by their local
microenvironment and may change from complex (“protoplasmic,” p) to smooth (“fibrous,” f ) when they pass from gray to white matter. In nuclear
layers with many small neuronal somata they are velate (v). (B, C) Glial fibrillary acid protein immunofluorescence of the dentate gyrus of
the hippocampus of an adult rat. Astroglial cell processes are radially aligned in the stratum granulare (SG), whereas typical “star-shaped” astrocytes
are found in the stratum moleculare (SM). D–F. Non–radially oriented fibrous astrocytes in the mammalian retina (D, E) and optic nerve (F).
(D, E) Glial fibrillary acid protein-labeled fibrous astrocytes in the flat-mounted murine retina, immunofluorescence. Close to the optic nerve (D) the
astrocytic processes are aligned in parallel to the axon bundles that run between rows of Müller cell endfeet (MCE). In the retinal periphery (E), the
cells are more or less star-shaped and form a regular pattern that is only modified by the contacts to retinal blood vessels (asterisks). (F) Camera-lucida
drawings of fibrous astrocytes from cat (Golgi stained cell) and murine optic nerve (dye-injected cell). The cell processes are mainly aligned
longitudinally (i.e., parallel to the axons). The cells extend fingerlike perinodal processes; an artist’s reconstruction of one of them is shown in
(A, bottom right), based on electron microscopical serial sections of the dye-labeled cell. (B,C) Original confocal microphotographs (courtesy of Gert
Brückner, Leipzig). Modified after Reichenbach (1989a) and Reichenbach and Wolburg. 2005, in which the references can also be found.

4 . A S T R O C Y T E S A N D E P E N DY M A L G L I A • 37
but do not reach the ventricular surface, they do not belong A stem fiber
to radial glial cells in the strictest sense. Rather, they resem-
ble protoplasmic astrocytes. Viewed from the pial surface, the
processes of Bergmann glial cells form rows or palisades that
are aligned parallel to the long axis of the folium, therefore
topologically resembling radial glia cells. Their many elabo-
rate side branches (Fig. 4.3) display complex morphological
and functional interactions with the synapses on the dendrites
of the Purkinje cells (see Fig. 4.3); they are characterized by stem fiber
a high surface-to-volume ratio of up to greater than 20 μm–1
(Grosche et al. 1999, 2002). In rodent cerebellum, there are
about 8 Bergmann glial cells per Purkinje cell (i.e., about 8000
mm–1 cerebellar surface area). It has been estimated that each
Bergmann glial cell ensheathes several thousands (2,000–
6,000) Purkinje cell synapses (Reichenbach et al. 2010).
Bergmann glial cells occupy about 15% to 18% of the volume
of the molecular layer of the cerebellum. An average rodent
Bergmann glial cell has a volume of about 3600 μm3. The size
and shape of Bergmann glial cells differ in dependence on ani-
mal species. The total length of the processes is determined by
the thickness of the molecular layer. Generally, in small spe-
cies (e.g., in shrews) the Bergmann glial cell processes are short
and densely covered with lateral appendages, whereas in large
species (e.g., humans) these processes are much longer but stem fiber
show less dense lateral outgrowths. Figure 4.5B gives a survey
of the Bergmann glial cell morphology in several vertebrates.
Fañana’s cells constitute a subtype of short Bergmann glial B D Bergmann
cells, with their somata being located in the molecular layer glia cell
processes
rather than at the level of the Purkinje cell somata. stalk

1.5 P ROTO P L A S M I C A S T RO C Y T E S
Astrocytes are the “prototypical” macroglial cells of the mam-
malian brain, although they occur also in lower vertebrates.
Protoplasmic astrocytes (Fig. 4.1, IV; Fig. 4.5C, D) are found
in the gray matter. Their numerous processes are spread more
or less radially from the soma, usually occupying a spheroid
volume, and extend many fine and complex lamellar side
branches. In rodent brain astrocytes, these surface extensions
occupy about 50% of the volume but as much as 80% of the
surface area of an average cell (Vcell ~5,500 μm3; Scell ~80,000
μm2) (Chao et al. 2002), resulting in high surface-to-volume
ratios of 10 to 20 μm–1. Thus, although the volume fraction of
astroglia in the cortical tissue amounts only to 10% to 20%,
the astrocytic processes and side branches contact much of
the neuronal surfaces present in a given brain volume com-
partment (Chao et al. 2002). Human astrocytes are larger
and even more complex (Oberheim et al. 2006, 2009) (Fig.
4.5C). Independent on species, at least one of the cell pro-
cesses is bearing one or several perivascular endfeet such that
the surfaces of the blood vessels in the CNS are virtually com-
pletely ensheathed by astroglial endfoot plates (Mathiisen et
al. 2010). The density of astrocytes in the cerebral cortex is
high (layers III/IV: 4,000–10,000 mm–3 in lissencephalic
cortices of insectivores [Stolzenburg et al. 1989]; 12,000
≥ 30,000 mm–3 in the rat cortex [Distler et al. 1991]). The
cortical glia-to-neuron index (largely determined by proto-
plasmic astrocytes) increases with the thickness of the tissue;
head
(stem fiber) neuronal
Purkinje cell spine element
C
mito-
chondrion
stalk
parallel
stem
fiber head fibers
Figure 4.3 Three-Dimensional Reconstruction of a Part of a Bergmann
Glial Cell Process in the Murine Cerebellum. A. The living cell was
dye-injected in a perfused cerebellar slice. Then, after fixation and
dye-conversion, about 600 consecutive serial ultrathin sections were
photographed in the electron microscope, and the images of the
dye-labeled profiles were reconstructed by a computer program. The
inset shows a substructure labeled in blue; this part was quantitatively
analyzed (B, C). B,C. A microdomain of the Bergmann glial cell process
shown in (A); reconstruction (B) and schematic drawing of such a
glial microdomain and its relationships to the neuronal elements (C).
D. Three-dimensional reconstruction of a group of neighbored cerebellar
synapses (yellow; synaptic clefts: orange) together with the surrounding
leaflets provided by the injected Bergmann glial (blue-green). The
arrowheads point to neuronal surfaces not covered by glial sheaths from
the labeled cell. Modified after Reichenbach (1989a) and Reichenbach
and Wolburg 2005, in which the references also can be found.
from about 0.1 in shrew to about 5 in whale, with interme-
diate values of about 2 in humans (for a survey of the rich
literature, see Reichenbach and Wolburg 2009).

38 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
 1.6 FI B RO US A S T RO C Y T E S
Fibrous astrocytes (Fig. 4.1, VI; Fig. 4.2D–F) are found in
white matter tracts, the optic nerve, and the retinal nerve fiber
layer of mammals with vascularized retinae. Their somata are
often arranged in rows between the axon bundles. Their pro-
cesses are comparatively smooth, and frequently oriented in
parallel to the axons. For the mouse optic nerve it was shown
that every astrocyte possesses several perivascular and/or sub-
pial endfeet (see Fig. 4.2F). The fibrous astrocytes in the ret-
ina may bear endfeet at both the intraretinal blood vessels and
the vitreal surface. As a characteristic feature, the processes of
fibrous astrocytes extend multiple fingerlike outgrowths into
the perinodal space of adjacent axons (Figs. 4.2A, 4.4G). A
density of about 200,000 fibrous astrocytes mm–3 has been
estimated for the anterior commissure of the mouse (Sturrock
et al. 1977). The processes of fibrous astrocytes are generally
longer (mouse: up to 300 μm) than those of protoplasmic
astrocytes (mouse: < 50 μm) but their surface-to-volume ratio
is significantly smaller (~5 μm-1). Examples of fibrous astro-
cytes from different locations are shown in Fig. 4.2D–F.
1.7 VE L AT E A S T RO C Y T E S
Velate astrocytes (see Fig. 4.1, V) were described in the granule
layer of the cerebellum, where each of them surrounds several
small neuronal granule cells with velate sheaths (for summary
of the literature, see Reichenbach and Wolburg 2009). Similar
cells occur in the olfactory bulb, where they ensheathe several
periglomerular neurons and dendritic segments. Obviously,
this cell type develops at sites where many small, densely
packed neurons occur. Morphometric data on velate astro-
cytes are not available, but by comparison with the “velate”
parts of Müller cell processes in the outer nuclear layer, it can
be estimated that their surface-to-volume ratio is very high
(20–30 μm–1).
1.8 I N T E R L A M I NA R A S T RO C Y T E S
Interlaminar astrocytes (see Fig. 4.1, VIII) have been found in
the supragranular layers of the cerebral cortex of higher pri-
mates, including humans; they do not occur in lower mam-
mals. They are similar to protoplasmic astrocytes in the upper
cortical layers (I–III), but are characterized by a long (up to 1.0
mm in humans) process arising from the internal (deep) side
of the cell soma usually located in lamina I, and descending
over at least two laminae (down to lamina IV, where it ends in
a small bulb). Collectively they form a visible “palisade” that
has been found to be severely disrupted in cases of Alzheimer
disease and in response to experimental mechanical lesions in
monkeys. Although their functional relevance is still unclear,
it is speculated that they may optimize the modular (colum-
nar) organization of the cortex (Colombo 2001).
1.9 M A RG I NA L G L I A A N D P E R I VA S CU L A R
A S T RO C Y T E S
Close to the pia mater, specialized astrocytes can be found
(see Fig. 4.1, III) that may form several layers of endfoot
4 . A S T R O C Y T E S A N D E P E N DY M A L G L I A
plates. Usually they extend several long, smooth processes
down into the neuropil (not unlike the interlaminar astro-
cytes), but their main function is thought to constitute
a glial “limiting zone” below the pia mater. Recently, such
surface-associated, gap junction–interconnected astrocytes
have been described in the posterior piriform cortex of the
rat, and were discussed in terms of neurovascular regulation,
interstitial homeostasis, and neuromodulation (Feig and
Haberly 2011).
In the human and rabbit retina, perivascular astrocytes
have been described (Fig. 4.1, VII) that are virtually devoid of
neuron-contacting processes but form extensive endfoot con-
tacts to retinal blood vessels. They seem to occur also elsewhere
in the brain (e.g., the neurohypophysis), but their occurrence
and possible functions are poorly studied so far. In analogy to
the marginal astrocytes, they may constitute a peculiar “glial
coating” around the blood vessels.
1.10 E P E N DY M O C Y T E S , C H O RO I D
P L E XUS C E L L S , A N D R ET I NA L P I G M E N T
E P IT H E L I A L C E L L S
Ependymocytes, choroid plexus cells, and retinal pigment
epithelial cells are specialized glial cells lining the ventricle
(or the subretinal space, respectively). At their basal pole,
most mature ependymocytes (see Fig. 4.1, X) contact rem-
nants of embryonic blood vessels (so-called “basement mem-
brane labyrinths”) rather than intact blood vessels. On their
other (i.e., the ventricular) pole, they possess, in addition to
microvilli, kinocilia to support the stream of the cerebrospi-
nal fluid. The latter is mainly secreted by the choroid plexus
cells (see Fig. 4.1, XI), characterized by a high density of
Na+/K+-ATPase molecules at their microvillous membrane.
This secretion requires a high permeability of the fenes-
trated plexus endothelial cells; thus, the blood-cerebrospinal
fluid barrier is formed by the choroid plexus epithelium
(Wolburg and Paulus 2010). Retinal pigment epithelial
(RPE) cells (Fig. 4.4C) line the subretinal space opposite
to the neuroretina. Their apical surface extends two types
of microvilli: long (5–7 μm) thin microvilli maximizing the
membrane area available for transepithelial transport, and
specially arranged shorter microvilli termed photoreceptor
sheaths. The basal surface of the RPE cells contains numerous
invaginations (basal membrane infoldings, BMI, Fig. 4.4C)
to increase the surface area. The size and shape of the RPE
cells depend on their location in retina; within the human
macula, the cells measure about 14 μm in diameter (12 μm
height) but they become wider (up to 60 μm diameter) and
flatter in the periphery. Like the choroid plexus cells, the
RPE cells (1) are in close apposition to many blood vessels,
(2) are specialized for transmembrane transport, and (3)
form the blood-cerebrospinal (or, in this case, -subretinal)
fluid barrier by their tight junctions (Fig. 4.4E). Like chor-
oid plexus epithelial cells the RPE cells express the water
channel protein aquaporin 1 (AQP1), which is present in
the apical membrane opposite to the photoreceptor outer
segments. An important difference between pigment and
choroid plexus epithelial cells is the direction of water flux:
• 39
 A B
P
vit vit bv
R
R vit
RPE
RPE vit c direction of the Na+/K+/2Cl– cotransporter (NKCC1) and
ONH
500 μm 50 μm
C D
0.5 μm
E
0.2 μm
F sible. Even in these instances, however, melanin granules
G choroid plexus cells, and RPE cells can be found in Wolburg
0.25 μm 100 nm
Figure 4.4 A–C. Specialized ependymoglia of the eye. A. Survey of the
fundus of an avian (blue-and-yellow macaw) eye. Close to the optic nerve
head (ONH), two peculiar forms of ependymoglia can be found: in the
pecten (P), the pecten glial cells, and below the retina (R), the retinal
pigment epithelial (RPE) cells. B. Higher magnification of an area of the
pecten shown in (A). Many capillaries (c) and larger blood vessels (bv) are
embedded in a tissue that contains endothelial cells and pigmented pecten
glial cells (black arrowheads) that contact the vitreous (vit). The pecten glial
cells contain pigment granules but do not form tight junctions (D, freeze
fracture replica). C. The RPE cells (e.g., from rabbit) contain pigment
granula (P). Apically (i.e., toward the outer segments of the photoreceptor
cells, ROS) they extend microvilli, which may enclose the shed tips of
ROS (asterisk) as a first step of phagocytosis. Basally, the cells face a basal
lamina (Bruch’s membrane, between the arrows) and display an enlarged
surface area resulting from basal membrane enfoldings (BME). Because
the capillaries of the underlying choroid possess a fenestrated endothelium
(black arrowheads), the RPE cells from the blood-retina barrier by the
expression of tight junctions (white arrowheads in C and “networks” in E).
D–F. Freeze-fracture replicas demonstrating specific astroglial membrane
features of pecten glial cells (D), RPE cells (E), and a subpial astrocytic
endfoot membrane of the adult rat optic nerve (F). The tight junctions
form a regular network in the lateral membranes of chicken RPE cells
(E), but are rudimentary in pecten glial cells (arrowhead in D). At the line
between the white arrowheads in (E) as indicated by the white arrowheads,
the fracture level switches from the protoplasmic (pf ) to the external face
(ef ) of the cell membrane. In addition, gap junctions (encircled) also occur
between RPE cells (and, typically, between astrocytes; not shown). F.
Both Müller cell (not shown) and astrocytic endfoot membranes display
orthogonal arrays of particles (OAPs; encircled). G. Transmission electron
micrograph of a corona of fingerlike astrocytic processes around a nodelike
specialization of an unmyelinated axon (asterisk) in the rat retinal nerve
fiber layer (see Fig. 4.2A). Modified after Reichenbach (1989a) and
Reichenbach and Wolburg 2005, in which the references also can be found.
40 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
Choroid plexus cells release water across the apical mem-
brane into the ventricle. However, if RPE cells would release
water into the analogous space, that is, the subretinal space
(representing an obliterated ventricular cleft), detachment
of the neuroretina from the pigment epithelium would be
the disastrous consequence. This is avoided by the inward
the Na+/bicarbonate cotransporter in the apical membrane,
followed by AQP1-mediated water flux from the subretinal
space toward the choroid vasculature. This even supports
the apposition of the retina to the pigment epithelium
(Strauss 2005). Another specific feature of RPE cells is the
presence of black (melanin) pigment granules aimed at the
absorption of light that passed the photoreceptor cells, and
thus at the avoidance of back-scattering light. By contrast, in
some vertebrates adapted to dark environments (e.g., fishes
living in the deep sea or turbid water), the RPE cells may
contain guanidine crystals to reflect as much light as pos-
are present; probably, they serve an important role as sinks
for free radicals and excited oxygen species such as singlet
oxygen. A comprehensive overview about ependymocytes,
et al. (2009).
1.11 P EC T E N E A L G L I A L C E L L S
The pecten oculi is a peculiar vascular structure of the avian
eye, where it bulges from the optic nerve head into the vitre-
ous body (Fig. 4.4A,B). It is comprised of two types of cells,
endothelial cells and specialized glial cells. The latter (like
the RPE cells) originate in the outer leaflet of the optic cup.
They contain pigment granules (like RPE cells) but lose their
tight junctions during differentiation (unlike RPE cells) such
that the blood-retina barrier is maintained by the endothe-
lial cells of the pecten. Immunocytochemical localization of
the enzyme glutamine synthetase (GS) in these cells is weak
during embryonic development but increasingly strong after
hatching. Although in astrocytes and Müller cells, GS expres-
sion is thought to be involved in transmitter recycling, in the
pecteneal glia it may participate in the pH-regulation of the
avian eye (Wolburg et al. 1999). Very probably, the pecten
glial cells play important roles in the nutrition and detoxifica-
tion of the avian retina (Wolburg et al. 1999).
In addition to the Müller cells in the avian retina and
pecteneal glial cells that are developmentally related to the
RPE, there is a further population of retinal glial cells lin-
ing the base of the pecten oculi; it has been described in the
chicken retina as the peripapillary glia, specifically expressing
R-cadherin (Schuck et al. 2000). This retina-specific type of
glia retains characteristics of radial glia by spanning the dis-
tance from the vitreous to the ventricular cleft. Furthermore,
it represents the border between the vascularized optic
nerve head and the adjacent avascular retina, suggesting
that these cells demarcate the outer limit of vascularization
and prevent the ingrowth of vessel sprouts into the retina.
However, after injection of R-cadherin antibodies and pre-
absorbed complement to lyse the R-cadherin-positive glial
Table 4.1 MARKER ANTIGENS
CELL TYPE ANTIGEN DEVELOPING ADULT REACTIVE

Astroglia GFAP (+) ++ +++

Vimentin +++ – ++
Nestin ++ – ++
CytokeratinF,A ++
Glutamine synthetase ++
iNOS + +++
RC1/2 (antibodies) ++ –
RAN-2 + ++
BLBP* ++ –
S-100β – ++
3CB2C
R-cadherinC + ++
Müller glia GFAPF – – +++
Vimentin + ++ +
RAN-2 ++
3CB2 ++
nNOS/iNOS + +++
CA ++ ++
S-100β ++
3CB2 ++
B-cadherinC + ++
F1* ++ ++
Glutamine synthetase ++
CRALBP ++
Bergmann glia GFAP ++
Vimentin ++ +
Nestin ++
Glutamine synthetase ++
S-100β ++
Ependymoglia GFAP (+) ++
Vimentin ++
Cytokeratin +++
RAN-2 ++
Retinal pigment Vimentin ++
epithelium cells CRALBP +++
R-cadherinC ++ –
Pecten glia Vimentin + ++
(only avian) B-cadherin + ++
Glutamine synthetase ++
Choroid plexus Cytokeratin ++
epithelium GFAP – ++
Vimentin ++
Neurofilament* ++

List of “marker antigens” suitable to visualize and/or identify the various types of astroglial and ependymoglial cells during ontogenetic development, in the normal
mature CNS, and during reactive changes in cases of pathology
Whereas the list basically reflects the situation in mammals, many of the antigens can also be found in the particular cell types of other vertebrates. In cases in which an
antigen is only found in non-mammalian cells, it is labeled by an exposed letter: F, fishes; A, amphibians; C, chicken. An asterisk indicates that the listed antigen (or anti-
body, respectively) labels not selectively glial cells, but (in other regions of the CNS) also neurons. Antigens expressed in the cytoplasmic membranes such as ion chan-
nels, receptors, or adhesion molecules are excluded from the list because immunocytochemistry for these antigens usually results in “diffuse” labeling of the neuropile, at
the light microscopical level.
BLBP, brain lipid–binding protein; CA, carbonic anhydrase; CRALBP, cellular retinaldehyde-binding protein; GFAP, glial fibrillary acidic protein; NOS, nitric oxide
synthase (n neuronal, i inducible form); RAN-2, rat neural antigen-2.

4 . A S T R O C Y T E S A N D E P E N DY M A L G L I A • 41
cells, blood vessels were not observed to enter the retina
(Gerhardt et al. 2000).
2 I M MU N O C Y TO C H E M I C A L
VI S UA L I Z AT I O N A N D
I D E N T I F I C AT I O N O F A S T R O G L I A L
A N D E P E N DY M O G L I A L C E L L S
Astrocytic and ependymoglial cells can be visualized (and,
in many instances, even identified) by immunocytochemical
labeling of certain antigens that are, at least within the CNS,
restricted to these cells. The expression of these molecules by
certain cell types may change with differentiation, or dur-
ing pathological processes (Table 4.1). Furthermore, not all
members of a given cell population (otherwise considered
homogeneous) must express the same antigen (at detectable
levels); for instance, although it may be safely stated that
every glial fibrillary acid protein (GFAP)-immunopositive
cell in brain is an astrocyte, there seem to be many astro-
cytes in brain that are not labeled by antibodies directed to
GFAP. It should also kept in mind that some of the antigens
(e.g., the intermediate filament proteins) allow mainly an
immunocytochemical visualization of the larger stem pro-
cesses, whereas antibodies directed to cytoplasmic proteins
(e.g., glutamine synthetase and S-100β) may stain fine side
branches, and may be used at the electron microscopical
level to identify even very thin cytoplasmic leaflets of glial
sheaths. By contrast, antibodies against membrane proteins
(e.g., ion channels, ligand receptors, transporter proteins)
may label (parts of ) the cell surface. A survey of the com-
monly used immunolabeling procedures, and their results
on specific types of astroglial and ependymoglial cells, is
presented in Table 4.1. It should also be mentioned here
that ependymoglial and astroglial cells have the capability
to accumulate exogenously applied fluorescent dyes such as
sulfo-rhodamine, Fura-2, MitoTracker Orange, and others.
Thus, such dyes can be used to monitor Ca2+, glutathione,
pH, and so on, specifically in living glial cells, and simul-
taneously, to study their morphology. Microscopically con-
trolled intracellular injections of fluorescent dyes can also
be used to visualize individual glial cells. Finally, astroglial
cells can be induced to express the green fluorescent protein
(GFP) or similar fluorescent proteins by coupling this gene
to a glia-specific promoter (e.g., for GFAP, GS, or GLAST)
in transgenic mice. In any of these cases, fluorescent dyes are
desirable because they can be used with the high-resolution
confocal microscopy. More about suitable staining proce-
dures in the case of Müller cells can be found in Reichenbach
and Bringmann (2010).
3 U LT R A S T RU C T U R A L F E AT U R E S
3.1 C E L L S O M A A N D N U C L EUS
The soma of astrocytes is usually rather poor in organelles if
compared with that of neurons. In most cases, characteristic
42 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
bundles of intermediate filaments can be found that may even
be used as “markers” of astroglial cells in electron microscopic
sections. The somata of some types of ependymoglial cells
contain conspicuous melanin pigment granules (e.g., RPE
cells, pecteneal glial cells, and choroid epithelial cells). In most
of these epithelioid glial cells (including the ependymocytes
but not the pecteneal glial cells), the lateral membranes of the
somata are interconnected by zonulae adherentes and tight
junctions, thus forming the blood-brain (or -retina) barrier
(Wolburg et al. 2009).
In astroglial and ependymoglial cell nuclei, the nucleo-
plasm is rather evenly distributed if compared with that in
oligodendrocytes and microglial cells. In some ependymoglial
cells such as in many tanycytes, the cell nuclei are very irregu-
larly shaped and may display deep incisions. The nuclei (and
somata) of Müller cells seem to be “indented” by neighbor-
ing neurons. It can be shown by atomic force microscopy that
Müller cell somata are “softer” (i.e., possess a lower module of
elasticity) than the somata of the neighboring bipolar neurons
(Lu et al. 2006).
3.2 S T E M P RO C E S S E S
Stem processes are those cellular processes that directly arise
from soma. Typically they contain bundles of intermediate
filaments. Particularly high densities of intermediate filaments
are found in the basal (i.e., endfoot-bearing) processes of tany-
cytes and Müller cells, and in processes of fibrous and radial
astrocytes including Bergmann glia. Microtubules are rarely
found in astroglial cell processes. One of the few exceptions
are the apical (i.e., microvilli-bearing) processes of Müller
cells. The stem processes usually contain numerous mito-
chondria. An interesting exception are Müller cell processes
in species with avascular retinae that contain mitochondria
only at their apical pole (i.e., close to the choroid, which is the
only source of oxygen supply), whereas their stem processes
are devoid of these organelles. It has been argued that because
of their dominant glycolytic energy metabolism, Müller cells
(and perhaps other astroglial cells) are free to move and place
their mitochondria toward sites of high pO2 (Germer et al.
1998a,b; Wolburg et al. 1999) rather than to sites of high
energy demand, as observed in the neurons with their domi-
nant aerobic metabolism.
The stem processes of astrocytes, tanycytes, and Müller
cells do not show the rather regular dichotomised branching
pattern characteristic of neuronal dendrites, but rather are the
origins of specialized endings or side branches described in the
following sections.
3.3 E N D FE ET
Astrocytic endfeet almost completely cover all basal lami-
nae within the CNS (along the blood vessels, pia mater, and
vitreous body in the eye, here together with the Müller cell
endfeet). They are often coupled to each other by gap junc-
tions (and sometimes by zonulae adherentes) (Iadecola and
Nedergaard 2007; Tam and Watts 2010) constituting the
basis of dynamic interactions between astrocytes, neurons,
and vascular structures (Figley and Stroman 2011; Giaume et
al. 2010). In the case of tanycytes and Müller cells, the end-
feet are densely filled with smooth endoplasmic reticulum.
At the ventral surface of the rat brain, near the hypothala-
mus, astrocytes form lamellar stacks of astrocyte processes
(Holen 2011). Bundles of intermediate filaments extend into
the endfeet, but fail to occupy the cytoplasm close to the
basal lamina–contacting endfoot membrane. These filaments
consist primarily of vimentin when the endfoot is in contact
with cerebrospinal fluid or vitreous humor and glial fibrillary
protein when a blood vessel is contacted (Pixley and DeVellis
1984); however, this rule may be modified in some cases.
With the exception of Müller cells in species with completely
avascular retinae (i.e., missing both intraretinal and suprareti-
nal blood vessels), the endfeet are rich in mitochondria. The
occurrence of caveolae, coated pits, and vesicles concerned
with endo-/exo-/or pinocytosis indicates active material
exchange with the compartment behind the basal lamina
(i.e., blood plasma, vitreous body, or subarachnoidal fluid). A
secretory function has been ascribed to the tanycytes of some
circumventricular organs.
The most prominent and distinctive ultrastructural
property of endfoot membranes of all astroglial cells in
higher vertebrates is the occurrence of orthogonal arrays of
intramembranous particles (OAPs) (Fig. 4.4F), which can be
visualized in the transmission electron microscope by means
of the freeze-fracture technique. The OAPs are accumulated
in those parts of the endfoot membrane that directly contact
the perivascular or superficial basal lamina, whereas within
the neuropil, there are few if any OAPs. This polarity devel-
ops concomitantly with the maturation of the blood-brain
barrier, and is lost or severely reduced under pathological
conditions such as tumors or inflammatory diseases, as well
as in cultured glial cells (for a recent overview, see Wolburg
et al. 2011). The molecular identity of the particle-forming
protein(s) has been a matter of debate for many years, but now
the water channel–forming protein, aquaporin-4 (AQP4) is
considered the main constituent of the OAPs (Wolburg et al.
2011). However, in zebrafish telencephalon, radial glial cells
have been described that express AQP4 without forming
arrays (Grupp et al. 2010), whereas generally, teleost glial cells
can form OAPs (Wolburg et al. 2011). Still, the preconditions
for OAP formation are not completely known. Noteworthy,
in mammalian glial cells the inwardly rectifying K+ channel,
Kir4.1, shows a very similar polarized distribution (Nagelhus
et al. 1999) and may be another constituent of the OAPs,
but this remains to be demonstrated. An important role
of OAP/AQP4 for the water homeostasis of the brain and
maintenance of the blood-brain barrier has been shown (e.g.,
Amiry-Moghaddam et al. 2004; Benfenati and Ferroni 2010;
King et al. 2004; Tait et al. 2009; Wolburg et al. 2009; Yool
2007; Yukutake and Yasui 2010).
Given that astroglial cells are highly polarized, it is an
important question how cellular polarity is organized at the
molecular level. The mere morphological observation that the
occurrence (or at least density) of OAPs crucially depends on
basal lamina contact suggested a pivotal role of the extracellu-
lar matrix for the molecular arrangement of the endfoot mem-
brane. Indeed, the OAP/AQP4-related polarity of astrocytes
as well as the array formation from AQP4 tetramers seems to
4 . A S T R O C Y T E S A N D E P E N DY M A L G L I A
depend on the presence of agrin and the dystrophin–dystro-
glycan complex (Noell et al. 2009, 2011). Agrin is a heparan
sulfate proteoglycan of the extracellular matrix, originally dis-
covered as being essential for the clustering of acetylcholine
receptors in the postsynaptic membrane of the motor end-
plate; it is also present around brain microvessel (Barber and
Lieth 1997). Agrin also binds to α-dystroglycan, a member of
the dystrophin–dystroglycan complex that localizes at glial
endfeet membranes in a similar way as OAP/AQP4 (Noell
et al. 2011).
When, under pathological or experimental condi-
tions, apicolateral membranes of RPE cells, Müller cells,
or ependymocytes are confronted with the mesenchymal
compartment, they lose their original features and develop
an endfoot-like structure. This response suggests that mes-
enchymal contact (i.e., collagen/laminin/agrin and/or other
molecules) stimulates the insertion of potassium channels
of the Kir4.1 type (Ishii et al. 1997). Although astroglial
and ependymoglial cells are thought to contribute to basal
lamina formation, this seems to require the presence of adja-
cent mesenchymal cells; mature Müller cells cannot restore
the vitreal basal lamina when it is destroyed by enzymatic
digestion (Halfter 1998) or microsurgical peeling (Miller
et al. 1986).
3.4 V E N T R I C U L A R C O N TAC T S
Cell processes contacting the ventricular (or subretinal)
space occur in ependymoglial cells such as tanycytes and
Müller cells, but never in astrocytes. They always display an
enlarged surface area, by extending microvilli into the fluid.
Furthermore, the apical pole contains abundant mitochon-
dria, features that indicate a high metabolic activity that is
presumably related to an active exchange of substances with
the luminal fluid.
Neighboring glial ventricular contact processes (and
adjacent neuronal cell processes, if present) are connected by
various types of apicollateral junctions. These (in particular,
desmosomes) are general “markers” of virtually all epithelial
cells and occur very early in development. However, their
nature varies considerably depending on the local microen-
vironment. In regions in which no endothelial blood-brain
barrier exists (e.g., in most circumventricular organs and the
RPE) but not elsewhere, ependymoglial cells form a cerebro-
spinal fluid–brain barrier by expressing tight junctions (Fig.
4.4E). Apicollateral gap junctions normally occur between
Müller cells in frogs but not mammals. When, however, rab-
bit Müller cells form homogeneous cultures in vitro, gap and
even tight junctions can be observed (Wolburg et al. 1990).
Likewise, when the basal pole of Müller cells is directly
exposed to the vitreous humor (because of basal lamina
defects in retinal wounds) the cells may send microvilli into
the vitreous cavity (Foos and Gloor 1975). Retinal pigment
epithelium cells proliferating under areas of retinal detach-
ment may lose their basal lamina contact and face the sub-
retinal fluid round about; in this case, their entire surface is
covered by microvilli (Anderson et al. 1983).
• 43
 The apical surface of typical ependymocytes is character-
ized by the presence of 12 to 60 kinocilia, which vary in num-
ber according to the species. The cilia are 10 to 20 μm long,
and are of the 9 x 2 + 2 type. These cilia beat rhythmically at
a frequency of about 200 per minute, and appear to assist the
rostro-caudad flow of cerebrospinal fluid. All these observa-
tions suggest that the polarity of ependymoglial cells strongly
depends on the microenvironment with which the different
cellular surface domains are contacted.
3.5 L A M E L L A R N EU RO N- C O N TAC T P RO C E S S E S :
G L I A L M I C RO D O M A I N S
The end branches of neuropilar astroglial cell processes are the
site of glia–neuron interactions. These processes are character-
ized by the formation of flat or lamellar sheaths (Chao et al.
2002; Wolff 1968) that enclose neuronal somata, synapses, or
bundles of axonal internodes, or by fingerlike extensions that
contact the nodal membrane of myelinated axons (Fig. 4.2A).
Such fingerlike astroglial processes also contact nodelike spe-
cializations of unmyelinated axons in the retinal nerve fiber
layer (Fig. 4.4G). They may origin not only from astrocytes
but also from Müller cells (Reichenbach et al. 1988) and from
the NG2+ cells. In contrast to the stem processes, the lamel-
lae are virtually devoid of organelles (with the exception of
actin filaments), but contain ezrin and radixin (Derouiche
and Frotscher 2001), two actin-binding proteins that link
the cell membrane to the actin cytoskeleton and may medi-
ate the formation and stabilization of the very complex, thin
side branches with their large surface-to-volume ratios of up
to more than 20 μm–1 (see Fig. 4.3). Whereas generally a large
part of all neuronal compartments is ensheathed by such lamel-
lar processes, the degree (or even the presence) of ensheathing
may vary considerably even within the same area of the CNS.
There is an obvious tendency to ensheathe the synapses. In rat
neocortex for example, about 56% of all synaptic perimeters
are covered by astroglia, whereas astroglial membranes make
up only 22% of all membranes in the neuropil (Chao et al.
2002 and references therein). However, on synaptic glomeruli
or “complex synapses” (i.e., specialized subcortical structures
in which multiple synaptic junctions are enclosed in a com-
mon glial sheath) the glial coverage is very high (there are even
multilamellar sheaths) but the glia does not penetrate the
interior of the complexes, and thus cannot seal the individual
synaptic clefts. As an extreme, there are astroglia-free neuropil
compartments, such as in Rolando’s substantia gelatinosa of
the spinal cord and the cochlear nucleus, in which thin sensory
axons terminate in “synaptic nests” that lack intrinsic glia.
In Bergmann glial cells, the existence of microdomains has
been demonstrated (Grosche et al. 1999, 2002). These occur
as “repetitive units” on the stem processes (or as appendages of
another microdomain). Each glial microdomain consists of a
thin stalk and a cabbagelike, very complex head structure that
bears the lamellar perisynaptic sheaths for about five synapses
(see Fig. 4.3). It has been shown that these microdomains
may interact with “their” (about five) synapses, independent
of each other, and also of the stem process. Stimulation of
the axons ending in the ensheathed synapses causes transient
44 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
intracellular Ca2+ rises in individual microdomains (Grosche et
al. 1999). Furthermore, mathematical simulation of the cable
properties reveals that even large (e.g., glutamate-induced)
depolarizations of the perisynaptic membranes are not con-
ducted over the stalks toward neighboring microdomains
or the stalk (Grosche et al. 2002). The energetic demands of
each individual microdomain may be supported by the mito-
chondria found in the “head” structures (Grosche et al. 1999,
2002). The glial microdomains overlap; in every given volume
unit of the molecular layer, at least two microdomains, origi-
nating from different Bergmann glial cells, interdigitate. This
may fit with the observation that Purkinje cells express two
functionally distinct populations of synaptic spines, and indi-
vidual spines are capable of independent activation (Denk et
al. 1995), as the glial microdomains may be adjusted to meet
this functional diversity of Purkinje cell synapses.
4 I N T E R S P E C I E S VA R I AT I O N,
O N TO G E N ET I C / P H YS I O L O G I C A L
P L A S T I C I T Y, A N D H I E R A R C H Y
OF ASTROGLIA
Despite of displaying a very complex structure and ultra-
structure (Figs. 4.1 to 4.4), it is now clear that astroglial cell
processes are by no means unchangeable, static structures.
After the general shape of an astroglial cell has been estab-
lished in ontogenesis (i.e., after at least one endfoot-bearing
stem process has grown), the neuropilar processes, particu-
larly the lamellar perineuronal sheaths, develop in (mutual)
dependence on the developing neuronal cell processes and
synapses (Reichenbach et al. 2010). Furthermore, it has been
shown that there occurs a lifelong, activity-dependent plastic-
ity of glial cell processes (Henneberger et al. 2010; Hirrlinger
et al. 2004). It should be kept in mind, however, that most of
the available data were obtained from studies on small labo-
ratory rodents, often at very young stages (to facilitate work
on slice preparations) and must not necessarily apply to adult
humans. Thus, some comparative and developmental aspects
are discussed here.
4.1 G E N ET I C VE R S US A DA P T I VE C O N T RO L
O F A S T RO G L I A L C E L L S I Z E
It has been pointed out that the total size of a mammal cor-
relates with the size of its brain as well as with the size of
its neurons (Purves 1988). This has been explained by the
facts that in a big body: (1) more cells exist and need inner-
vation (which requires increasing numbers of neurons and
of axon collaterals), and (2) the nerves have to bridge larger
distances (which requires an increase of not only length but
also thickness of axons, to accelerate impulse propagation,
and enforces an increasing size of the somata to maintain
the voluminous axons). In parallel, not only the absolute
and relative number of glial cells (Reichenbach 1989b)
but also their size increases (Fig. 4.5B, C). This appears to
be genetically controlled, at least partially. One example is
constituted by animals (e.g., lungfish and some amphibians)
with polyploidy. The increasing DNA content causes not
only an enlargement of the cell nuclei but also of the cells
themselves (Fig. 4.5A). This has been used by electrophysi-
ologists because a large size facilitates recordings from a cell.
Another recent argument was provided by Nedergaard and
colleagues, who transplanted human astrocytes into mouse
brains and found that the human astrocytes were much larger
than the neighboring murine host astrocytes (Nedergaard
2011) (see chapter 28). With these few exceptions, it is hard
to say how much of the different size of astrocytes in dif-
ferent animals (Fig. 4.5C) results from genetic instruction
or adaptation to the environment. For instance, cerebellar
Bergmann glial cells have to span the entire thickness of the
molecular layer. Depending on the number and thickness
of neuronal cell processes (see the preceding, and Purves
1988), this layer is much thinner in small animals than in
larger ones. Accordingly, the Bergmann glial cell processes
are much elongated in large species (Fig. 4.5B). Of note, the
brain and its layers are growing during ontogenetic develop-
ment, such that young Bergmann glial cells of a large species
may be smaller than adult cells from a small species (Hanke
and Reichenbach 1987). The adaptive growth of astroglial
cell processes is discussed in the following.
4 .2 O N TO G E N ET I C D EV E L O PM E N T
A N D A D U LT P L A S T I C I T Y O F A S T RO G L I A L
CELL SIZE AND SHAPE
The number, length, and complexity of astrocytic side
branches grow rapidly during the early ontogenesis (Grosche
et al. 2002; Hanke and Reichenbach 1987; Senitz et al. 1995)
(Fig. 4.5D). This may be triggered by the overall growth of
the neural tissue, ingrowth of blood vessels, and specific neu-
ron–glia interactions (for an overview of such interactions,
see Figley and Stroman 2011; Giaume et al. 2010). Specialized
glia–neuron contacts cannot be elaborated until neurons
have completed their differentiation (Waxman et al. 1983).
Indeed, the formation of such processes may be triggered by
the onset of neuronal activity, and their growing filopodia
may be attracted (or repelled) by signals from active neurons
(e.g., K+ ions, neurotransmitter molecules, and/or growth
factors). The performance of these mechanisms during onto-
genesis may be modulated by the strength and/or pattern of
neuronal activity that, in turn, is triggered by sensory inputs
and behavioral requirements. For instance, dependent on
whether rats are kept in enriched environments (Sirevaag
and Greenough 1991) or complete darkness (Stewart et al.
1986) the complexity of their glial cell processes may differ
significantly. The same (or very similar) mechanisms seem
to be maintained in the mature CNS, in which they may
modify the structure of glial cells even in the short-term
range (Henneberger et al. 2010, Hirrlinger et al. 2004) so
as to meet the changing needs of their neuronal partners. A
recent discussion of adult astroglial plasticity and the tech-
nical problems to monitor it unequivocally can be found in
Reichenbach et al. (2010). The role of astrocytic plasticity
in the modulation of neuroendocrine systems is discussed
further in chapter 41.
4 . A S T R O C Y T E S A N D E P E N DY M A L G L I A
4.3 VA R I A B I L IT Y A N D H I E R A RC H Y
O F G L I A L D O M A I NS
It has been shown that astroglial cell processes may form
repetitive microdomains, each interacting (more or less auton-
omously) with a small group of synapses (Grosche et al. 1999)
(see Fig. 4.3). It has also been speculated that the cell processes
of each astrocyte occupy a neuropilar territory that constitutes
the domain of its (autonomous) interactions with neuronal
(and vascular) compartments. In several studies, the territories
of neighboring astrocytes were found to abut each other with
minimal overlap (Bushong et al. 2002, 2004; Wilhelmsson
et al. 2006). It has also been demonstrated that in fact, indi-
vidual astrocytes may specifically interact with the neuronal
synapses within their territory (Henneberger et al. 2010).
These individual domains are competitively organized during
early postnatal development (Bushong et al. 2004) and may
even be demarked by molecules such as chondroitin sulfate
(Horii-Hayashi et al. 2010). However, electron microscopi-
cal studies have observed a substantial overlap of astrocytic
domains in murine cerebellum (Grosche et al. 1999, 2002) as
well as in rat cortex (Wolff 1968). This may depend on the spe-
cies and/or brain compartment studied, but also on late post-
natal development. We have found that the domains of murine
cortical astrocytes do not overlap in young (2-month-old) ani-
mals, but show considerable overlap (with a factor of about 2)
in 2-year-old animals (Fig. 4.5E).
Generally, it is clear that neither the microdomains nor the
cellular domains are always autonomously interacting with their
neuronal partners. Astrocytes form gap junction–coupled syn-
cytial networks (see chapters 24 and 26) such that rather large
numbers of cells may form bigger, multicellular domains that
concertedly interact with the neurons in this territory. Recently,
we have proposed a concept of variable, hierarchically organized
domains at many levels, from substructures at individual synapses
(nanodomains) to entire cortical columns or even areas (macro-
domains) (Reichenbach and Wolburg, 2009). Depending on
the degree and distribution of neuronal excitation, the whole
repertoire of glial arrangements from nanodomains and micro-
domains up to macrodomains or even “superdomains” (cortical
gyri or fields) may be switched on in series, within the very same
part of the CNS (Fig. 4.6). At the (pathological) end of this
scale, phenomena such as spreading depression may spread over
the entire cortex, thus involving the entire glial population.
At the end of this chapter, the authors hope to leave the
reader with the view that much of the morphological diversity
of astrocytes and ependymoglial cells results from the differ-
ent local microenvironments into which a given cell is born (or
migrating). Inevitably, mesenchymal contact induces the for-
mation of endfeet with OAP-rich membranes, whereas contact
to the cerebrospinal fluid induces the outgrowth of microvilli,
and the formation of stabilizing cell–cell junctions. Where
neuronal elements are contacted, the glial cells form delicate
side branches that end in lamellar sheaths or fingerlike branch-
lets. The number, size, and shape of these glial “end structures”
are precisely adjusted to the morphological and functional
features of the adjacent neuronal elements. This adjustment
continues after ontogeny as a lifelong process of plasticity. In
addition, rapid, reversible changes may occur that involve both
• 45
 A Müller cells: carp frog C 50 μm
salamander protoplasmic
astrocytes:

mouse man
50 μm

D human cortical
teraploid diploid teraploid diploid astrocytes:
neonatal
B

miniature 50 μm
shrew
adult
rat

Bergmann glial cells:

overlap factor of
E territories:

cortex
young mouse
ca.1

100 μm cortex
man monkey old mouse>>1 50 μm

Figure 4.5 The size of (astro-)glial cells and their occupied territories depends on several factors. A. As exemplified by Müller cells of fish and amphibian
retinae, a species-specific tetraploidy (i.e., three sets of chromosomes) causes not only an enlargement of the cell nucleus, but also of the entire cell.
B,C. As exemplified by cerebellar Bergmann glial cells (B) and brain astrocytes (C), bigger animal species not only have bigger brains, but also bigger
glial cells. D. As shown for human cortical astrocytes, there occurs a significant postnatal increase of the number, length, and complexity of cell
processes. E. The overlap factor (OF) of neuropilar territories occupied by the processes of astroglial cells may vary with age. In young animals the
territories may just touch each other (OF ~1), but in aged mice considerable overlap of astrocytic territories (OF ~2) was found (own unpublished
data). (A,B,D) Modified after Reichenbach (1989a) and Reichenbach and Wolburg (2005), in which the references also can be found; (C) from
Oberheim et al. 2006, with permission.

the neurovascular unit. One important task of future glial

morphological alterations of glial cell processes and a modifica-
tion of the gap junction–mediated coupling of the astrocytic
networks, allowing for fast switches among subcellular, cellular,
and multicellular glio–neuronal interactions. Further studies
of astrocytic plasticity at all these levels are supposed to provide
novel insights into the complexity of astrocytic functions.
5 S U M M A RY A N D P E R S P E C T I VE S
The multitude of glial cell types in all areas of the CNS reflects
the wealth of glial roles, including such crucial functions as,
for instance, guidance in the development of cortical lay-
ering, regulation of extracellular homeostasis, support of
neuronal energy and neurotransmitter metabolism, and reg-
ulatory processes at the blood-brain barrier. This last example
underlines that astrocytes do not only interact with neurons
and other glial cells, but also with vascular structures, forming
cell research will be to elucidate the molecular interactions
between glial cells and blood vessels with similar emphasis
as is currently dedicated to the glial contributions to synap-
tic and cognitive processes. For instance, an increase of brain
capillary permeability must not necessarily be restricted to
pathological processes (stroke, inflammation, tumor, neuro-
degenerative diseases), but may give the healthy brain access
to hematogenous factors in the circulation, so as to modulate
neuronal circuits. Another promising future research area will
be the biomechanics of brain cells and tissue, because it has
been shown that the viscoelastic properties of neurons, glial
cells, and the extracellular matrix are important for the growth
of neural cell processes. Summarizing the current state of
(functional-) morphological knowledge given in this chapter,
the authors want to leave the reader with the view that much
of the morphological diversity of astrocytes and ependymo-
glial cells results from the different local microenvironments
into which a given cell is born (or migrates).

46 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
 I mesodomain
II
ni
cellular
domain 1 III
microdomain
IV
n1
nii
networks
V
nano- VI macrodomain
cellular
domain
domain 2
superdomain
“neuron” striate cortex occipital cortex
Figure 4.6 Schematic Representation of the Various Types of Coexisting
Glial Domains Showing the Transition (Arrows) Between the Striate and
the Occipital Cortex in a Human Brain. At increasing levels of spatial
organization, the glial cells provide nanodomains up to superdomains
for their interaction with neurons and blood vessels. With the possible
exception of the nanodomains, which probably interact with “their”
neuronal partner structures as long as they exist, the domains are not
only determined by the (ultra-)structural features of the glial cells, but
also by the properties of the signal (or stimulus): (I) (a few) individual
synapses are associated with their ensheathing glial microdomains but
parallel/strong stimulation of related inputs may finally integrate; and
(II) (?oligo-)cellular domains involving the whole (or a few) glial cell and
their neuronal partners (see Fig. 4.4). It depends on the shape of the glial
cells involved whether these domains are columnar (“type 1,” for example,
Bergmann glial cells; probably radial astrocytes in hippocampal stratum
oriens, and hypothetically interlaminar astrocytes in primate cortex), or
rather spherical or ellipsoid (“type 2,” star-shaped astrocytes). Appropriate
(i.e., strong, frequent, or synchronized) stimulation may then activate, via
gap-junctional coupling, networks consisting of more than 50 astrocytes
(types n1 and n2 depend on the shape of the constituting glial cells).
These networks, however, cannot be considered as static; any member
of the coupled network at its margin is per se coupled to other cells that
are outside the first coupling range but that would be belong to another
(overlapping) network if the dye would have been injected into such a
cell. Thus, if a neuronal stimulus arrives at such a cell later, or if a Ca2+
wave was triggered by the first “excited” astrocyte, (III) macrodomains
will develop; this mechanism may proceed either radially (n1–nii) or
tangentially (n1–ni). The size of these macrodomains may vary from small
(macrodomain 1, corresponding for example to the orientation columns
of Mountcastle 1957) to large (e.g., ocular dominance columns or barrel
fields; macrodomain 2). A further progression of integration will result
in the generation of very large functional units. (IV) superdomains,
corresponding to entire cortical areas or gyri. Eventually, even whole
hemispheres associated with huge astrocytic populations may transiently
be involved, putatively mediating events such as spreading depression,
seizures, and/or widespread neuronal degeneration. I–VI, layers of the
cortex. From Reichenbach and Wolburg 2009, with permission.
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4 . A S T R O C Y T E S A N D E P E N DY M A L G L I A • 49
 5.
RADIAL GLIAL CELLS
Magdalena Götz

A B B R E VI AT I O N S A B C

Aldh1L1 aldehyde dehydrogenase 1 family, member L1

B-FABP brain-fatty acid binding protein
BLBP brain lipid binding protein
BM basement membrane
BMP bone morphogenic protein
ChSPG heparan sulphate proteoglycans
CNS central nervous system
CSF cerebrospinal fluid
ECM extracellular matrix
FACS fluorescence-activated cell sorting
FGFR fibroblast growth factor receptor
GFAP glial-fibrillary acidic protein
GFP green fluorescent protein Adherens Basal Body
GLAST glutamate transporter astrocyte-specific D junction of cilium
GLT-1 glutamate transporter 1
GS glutamine synthase
IGF insulin-like growth factor
3-PGDH 3-phosphoglycerate dehydrogenase
SCO subcommissural organ
E β-catenin
SHH sonic hedgehog
SEZ subependymal zone F-Actin α-catenin
SVZ subventricular zone Cadherin G-Actin
TN-C Tenascine-C
WNT released proteins equivalent to the wingless and Cilium with
Par
INT1 proteins in Drosophila Prominin
Complex
Apical/Ventricular surface

Figure 5.1 Radial Glial Cell Polarity. A. Schematic drawing depict-

1 D E F I N I T I O N A N D O VE RVI EW
Radial glial cells were first identified in the embryonic chick
spinal cord (Bentivoglio and Mazzarello 1999) and named
according to their elongated radial morphology. As their name
implies, besides their radial morphology “radial glial cells” are
also glial in nature. Radial glial cells are widespread in the
developing central nervous system (CNS) of all vertebrates
examined so far and are the first glial cell type to develop per-
sisting to different extent into adulthood. They line the ven-
tricle and extend their radial processes throughout the entire
radial thickness of the neural tube (Fig. 5.1). Radial glial cells
are true epithelial cells with an apical side in contact with the
ventricle and a basal endfoot connected to the basement mem-
brane (BM). Radial glial cells are coupled with neighboring
cells by gap junctions as well as adherens or tight junctional
complexes (Götz and Huttner 2005; Pinto and Götz 2007).
ing the epithelial nature of radial glial cells with their main contacts at
the apical and basal side. B. Freeze-fracture depicting radial glia endfeet
at the pial surface from a mouse embryo. C. Schematic drawing of the
molecular composition of the radial glia endfeet attachment to the
basement membrane underlying the meninges. Note the anchoring
of the radial glia endfoot to the basement membrane components by
dystroglycan and integrin complexes. D. Electron microscopy depict-
ing the junctional complex linking radial glial cells at the apical side.
E. Schematic drawing of the molecular composition of apical adherens
junction complexes and their dynamic anchoring to the cytoskeleton
by shuttling of α-catenin. F-actin, filamentous actin; G-actin, globular
actin. (A) Courtesy of Hartwig Wolburg, University of Tübingen, in
collaboration with Benedicte Dehouc, University of Lille; (D) courtesy
of Michaela Wilsch-Bräuninger and Wieland B. Huttner.
These junctions delineate the apical membrane domain fac-
ing the ventricular lumen with characteristic sorting of apical
membrane components (e.g., the glycoprotein prominin1) to
this compartment and a distinct basolateral membrane domain

50
Table 5.1a COMPARISON BETWEEN RADIAL GLIAL CELLS AND OTHER CELL TYPES IN THE MAMMALIAN BRAIN
NEURO-
EPITHELIAL RADIAL GLIA RADIAL GLIA MATURE EPENDYMAL REACTIVE ADULT NEURAL
PROTEIN CELLS EARLY LATE ASTROGLIA CELL ASTROGLIA STEM CELL

GFAP – –/+ +/++ –/++ + +++ +++

GLAST(Slc1a3) – ++ ++ +++ ++ +++ ++

GLT1(Slc1a2) – – + +++ ++ ++ ++

Glutamine – – + +++ +++ ++

synthetase

S100-β – – + ++ +++ +++ +

Connexin 43 – + ++ +++ ++ +++ +++

(Gja1)

Aquaporin 4 Nd Nd Nd +++ + +++ ++

KIR 4.1/2.1 – + ++ +++ ++ ++ +++

Aldlhl1 – – + +++ ++ +++ +

Nestin +++ +++ +++ – ++ +++ +++

(RC1/RC2)

Vimentin – + ++ – +++ +++ +++

BLBP ++ +++ +++ – – +++ +++

TN-C – +++ +++ – – +++ ++

Sox2 +++ +++ +++ +++ ++ +++ +++

Phosphacan/ – +++ +++ – – ++ +++
DSD-1

Note that cell heterogeneity is not incorporated in this table.

Based on Doetsch et al. 1997; Pinto and Götz, 2007; Mori et al. 2005; Liu et al. 2006 and references therein.

containing typically integrins in contact with the BM or extra-
cellular matrix components (Götz and Huttner 2005).
Besides their radial morphology, these cells also possess
glial hallmarks. These distinguish radial glial cells from the
earlier epithelial cell type in the developing neural tube, the
neuroepithelial cells (Table 5.1) (Götz and Huttner 2005).
The transition between neuroepithelial cells and radial glia is
a gradual process with some hallmarks coming up earlier than
others (see Table 5.1). The glial features shared between radial
glia and other glial cells comprise ultrastructural, cell biolog-
ical, and molecular aspects (Götz and Huttner 2005; Pinto
and Götz 2007). Radial glial cells contain glycogen granules
and a high density of 9-nm intermediate filaments reminiscent
of astrocytes, especially in their basal endfeet (Pinto and Götz
2007). These filaments comprise nestin, vimentin, and in
many species the glia-fibrillary acidic protein (GFAP), a hall-
mark of many, although not all, astrocytes in the adult brain
(see chapter 4; Table 5.1). Although GFAP is low in rodent
radial glial cells increasing only at the time of astrocyte genera-
tion from these cells, it is already prominent in radial glial cells
at embryonic stages in developing brains of many other verte-
brates (Mori et al. 2005). In addition, radial glial cells contain
also astrocyte-specific glutamate-aspartate transporters, such
as GLAST and GLT-1, as well as glutamine synthase (GS),
S100β, and metabolic enzymes that are shared in expression
between astroglia and radial glia (Beckervordersandforth
et al. 2010; Lovatt et al. 2007; Pinto et al. 2008), including
3-phosphoglycerate dehydrogenase (3-PGDH), an essen-
tial enzyme for L-serine biosynthesis (Yamasaki et al. 2001),
the aldehyde dehydrogenase family member AldhL1 (Cahoy
et al. 2008) or brain lipid–binding protein (BLBP, also
B-FABP). Significantly, radial glial cells share particularly
many aspects with activated or reactive astrocytes, which often
reactivate the expression of proteins previously downregu-
lated during maturation. These comprise nestin, vimentin, the
extracellular matrix protein tenascin-C, BLBP, and others
(see Table 5.1). Thus, among the “typical” astroglial proteins,
no difference can be detected between activated astrocytes
after brain injury and radial glial cells in the developing brain
(see Table 5.1). Genomewide expression analysis, however,
yields important differences (Beckervordersandforth et al.,
2010) (see chapter 28) also related to profound differences in
function as detailed in the following.
Radial glia also share many hallmarks with ependymal
cells, such as their contact to the ventricle, glycogen storage,
and the expression of the described proteins, which are not

R ADIAL GLIAL CELLS • 51

Table 5.1b COMPARISON BETWEEN RADIAL GLIAL CELLS AND OTHER CELL TYPES IN THE MAMMALIAN BRAIN
ADULT
NEUROEPITHELIAL RADIAL RADIAL MATURE EPENDYMAL REACTIVE NEURAL
FUNCTION CELLS GLIA EARLY GLIA LATE ASTROGLIA CELL ASTROGLIA STEM CELL

Glutamate uptake – + ++ +++ ++ +++ +++

K-conductance – – ++ +++ ++ ++ ++
at rest

Glycogen storage – + ++ +++ +++ +++ ++

Gap-junctions/ Nd +++ +++ +++ ++ ++ +++

Hemichannels/
Ca-waves

Blood vessel – + ++ +++ – +++ +++

contact/blood flow
regulation

Cell division +++ +++ ++ – – ++ ++

Multipotency +++ +++ ++ – – + +++

Self-renewal ++ ++ + – – + +++

Based on Doetsch et al. 1997; Pinto and Götz, 2007; Mori et al. 2005; Liu et al. 2006 and references therein.

only contained in astrocytes, but also in ependymal cells and A Pia mater B OB cortex
differ only at the quantitative level among these cell types (see LV
Table 5.1; chapter 4) (Beckervordersandforth et al. 2010).
Radial glial cells thus share hallmarks with both astrocytes RMS
and ependymal cells not only at the morphological, but also C
at the molecular level. These commonly expressed proteins
SEZ
indeed confer key functional properties shared by astrocytes,
ependymal cells, and radial glia, such as glutamate uptake, cilia
K+-buffering and water transport, Ca2+-waves mediated by
gap junctions and hemichannels (see Table 5.1; chapters 16,
24, 34 and 35). Also adult neural stem cells, which likewise
possess radial glial hallmarks with apical contact and a short- blood
ened basal process (Kriegstein and Alvarez-Buylla 2009), vessels
share these functional hallmarks with astrocytes and ependy- LV
mal cells (Fig. 5.2; see Table 5.1).
According to their similarity to ependymal cells, includ- ventricle
ing ventricular contact, radial glial cells are often referred to
as ependymoglia. In the mammalian CNS, radial glial cells are D
largely transient and disappear at early postnatal stages (see
Fig. 5.2). The ventricle is largely lined by cuboid ependymal
cells, lacking a long radial process. However, in some regions E
ependymal cells with an extended radial morphology are pres- OPC
Radial glia/adult neural stem cell
ent (see chapter 4), oftesn referred to as tanycytes (a term used
for glial cells with access to the ventricle and a longer radial Figure 5.2 Radial Glial Cells in Postnatal Mouse
process), such as in the hypothalamus and the subcommis- Forebrain. A. Fluorescence micrograph depicting GFP-labeled radial
sural organ (SCO) (Rodríguez et al. 1998). Thus, tanycytes are glial cells in the dorsal telencephalon of neonatal mouse. Note that radial
glial cells still span the entire width of the brain parenchyma from the
included in the described morphological definition of radial ventricle to the pial surface (pia mater) at this stage. Scale bar: 100 μm.
glial cells with ventricular access, apico-basal polarity, and an B. Schematic drawing of a sagittal section through the adult mouse brain
extended radial process. In most cases these subtypes of radial depicting the areas indicated in panels A and C with a red square. The
glia possess highly specialized (e.g., secretory) functions, as lower panel depicts the subependymal zone (SEZ). In this region adult
in the SCO, releasing a glycoprotein-rich substance forming neural stem cells have hallmarks of radial glial cells. C. Schematic drawing
depicting the cellular composition of the adult mouse SEZ according to
the Reissner’s membrane in the ventricle. Interestingly, radial Fischer et al. 2011. D,E. Examples of radial glial cells (labeled with GFP
glial cells releasing the same protein have also been observed in hGFAP-eGFP mice) from the wall of the lateral ventricle of adult mice
in the lancelet and the ectoneural system of echinodermata (D, lateral wall; E, medial wall). Scale bars: (A) 100 μm; (D,E) 20 μm.

52 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
 A B
C D
E F f
e in contrast with the multiciliated ependymal cells.) These hall-
Figure 5.3 Examples of Radial Glial Cells in the Zebrafish
Telencephalon. A. Micrograph of transgenic GFP+ radial glial cells in
48 hours post fertilization zebrafish forebrain with Dsred transgenic
neurons (NBT-DsRed line). B. Micrograph of GFAP-immunostained
section of an adult zebrafish telencephalon hemisphere. Note the radial
GFAP+ processes traversing the entire thickness of the telencephalon.
C. Micrograph of GFAP-GFP+ radial glial cells with somata lining the ven-
tricle and processes extending through the everted adult zebrafish telenceph-
alon line. D. Micrograph of S100β+ radial glia somata lining the ventricle of
the everted adult zebrafish telencephalon. E. Micrograph of a single GFP+
radial glial cell from the adult zebrafish telencephalon 6 days after lipofec-
tion. F. Micrograph of a single GFP+ radial glial cell from the adult zebrafish
telencephalon 5 days after transduction with a viral vector. Note the differ-
ence in branching between radial glial cells in (E) and (F). Scale bars:
(A) 16 μm; (B,D) 100μm; (C,E,F) 20 μm.(C) From Bernardos et al. 2006.
(Viehweg et al. 1998), suggesting their early emergence in the
deuterostome lineage. Thus, radial glial cells are not only the
first glial cells to appear in ontogeny, but seemingly also appear
at early stages in deuterostome phylogeny (Hartline, 2011).
In many if not most nonmammalian and nonavian ver-
tebrates, radial glial cells persist in a widespread manner
into adulthood. Glial cells with a radial morphology and an
apical contact to the ventricle line the adult CNS as during
development with a notably longer radial process extending
all the way to the pial surface (Fig. 5.3). Many of these radial
glial cells divide in most CNS regions, such as in the zebrafish
telencephalon (Chapouton et al. 2007), and can resume cell
division in regions where they normally do not divide, such
as the zebrafish spinal cord (Reimer et al. 2008). Likewise,
R ADIAL GLIAL CELLS
many radial glial cells are present in the adult amphibian,
reptilian, and avian CNS (Cuoghi and Mola 2009).
1.1 A D U LT N EU R A L S T E M C E L L S A R E
RADIAL GLIA
Also in the adult mammalian brain dividing radial glial cells
are present in very few niches in the forebrain where they
act as adult neural stem cells (Fig. 5.2b; see chapters 30, 40)
(Kriegstein and Alvarez-Buylla 2009). This is the case at the
wall of the lateral ventricle, where radial glial cells persist in a
widespread manner (Gubert et al. 2009). These cells possess a
small apical process in contact with the ventricle, junctional cou-
pling to neighboring ependymal cells or other stem cells, and a
radial process contacting the basement membrane surrounding
blood vessels (see Fig. 5.2). Similar to radial glial cells during
development, these cells also have a distinct apical membrane
domain with, for example, prominin1 sorted to microvilli or
cilia. (Note that radial glial cells typically bear a single cilium,
marks (Prominin1 and glial expression) allow enrichment of
adult neural stem cells (Beckervordersandforth et al. 2010) as
for radial glial cells in the developing brain (Pinto et al. 2008)
by fluorescence-activated cell sorting (FACS). Notably, radial
glial cells in other niches of the ventricular lining in the adult
rodent brain (e.g., in the hypothalamus) can also resume pro-
liferation and seemingly generate young neurons under some
conditions (Kokoeva et al. 2005; Pérez-Martín et al. 2010).
1.2 R A D I A L A S T RO C Y T E S
In addition, cells with astroglial properties and radial morphol-
ogy are present in some regions of the adult mammalian CNS,
such as the spinal cord, dentate gyrus, the cerebellum with the
Bergmann glia, and the retina with the Müller glia (see chapter
4). All of these cells lack contact to the ventricle and all except
the Müller glia (see chapter 4) also lack epithelial hallmarks
with an apical and basolateral membrane domain. Therefore,
they are referred to as radial or radially oriented astrocytes.
This terminology also applies to the so-called outer or basal
“radial glia” (Borell and Reillo 2012), a secondary type of glial
cells in some regions of the developing brain that lack access
to the ventricle but are connected by a long radial process to
the basement membrane. These cells appear in the develop-
ing cerebral cortex of rodents and become even more frequent
during phylogeny, in carnivores and primates (see chapter 30)
(Borell and Reillo 2012; Lui et al. 2011).
2 R A D I A L G L I A I N D E VE L O PM E N T
During development, radial glial cells perform various key
functions. They mediate architectonic stability and hence
allow appropriate morphogenetic movements. This includes
formation of boundary structures limiting cell migration. The
radial processes of radial glial cells act as guidance structures for
migrating neurons and radial glial cells act as lineage-restricted
progenitors as well as multilineage stem cells.
• 53
 2.1 R A D I A L G L I A A S S T E M A N D P RO G E N ITO R generate neuronal as well as glial progeny (Malatesta et al.
C E L L S —H ET E RO G E N E IT Y O F R A D I A L G L I A 2000; Miyata et al. 2001; Noctor et al. 2001). Radial glial cells
isolated by FACS comprise different sets of progenitors and
Historically, when radial glial cells were first discovered in
stem cells generating only neurons, only glia, or glial cells and
the developing nervous system, they were suggested to act
neurons (Fig. 5.5) (Malatesta et al. 2000). Live imaging then
as progenitor cells, including a contribution to neurogen-
allowed monitoring directly how neurons are generated from
esis (Bentivoglio and Mazzarello 1999; García-Marin et al.
radial glia with apparent differences in the zebrafish embryo
2007). These suggestions were then neglected because of the
in vivo (Alexandre et al. 2010) and slice preparations of the
misconception of considering cells in different phases of the
forebrain from rodent embryos (see chapter 30) (Kriegstein
cell cycle as distinct cell types (interkinetic nuclear migration
and Alvarez-Buylla, 2009; Miyata et al. 2001; Noctor et al.
causes cells in S-phase to be located at abventricular locations,
2001). Genetic fate mapping further substantiates the contri-
whereas M-phase occurs at the ventricular surface) (Fig. 5.4).
bution of rodent radial glia to neurogenesis as well as other
His proposed in 1902 that the neural tube was composed of
glial lineages (Pinto and Götz 2007). The heterogeneity of
two kinds of committed progenitor cells, neuroblast produc-
radial glial cells in lineage and potential relates to differences in
ing germinal cells (dividing at the ventricular surface) and
gene expression (Pinto et al. 2008, 2009) distinguishing radial
nonproliferative glia, the “spongioblasts” (located at abven-
glia generating neurons directly from radial glial cells generat-
tricular positions). Only with the discovery of the cell cycle
ing glial cells and neurons indirectly via an intermediate set of
phases and their occurrence at different positions (Fujita
progenitors (see Fig. 5.5; chapter 30) (Pinto et al. 2008, 2009).
2003) was this concept questioned, and the “matrix cell the-
These distinct modes of radial glia–derived neurogenesis were
ory” was proposed with a homogeneous set of bilineage or
revealed by live imaging (Haubensak et al. 2004; Miyata et al.
multilineage progenitors or stem cells generating first neurons
2001, 2004; Noctor et al. 2001, 2004) as well as FACS allow-
and later glia (Fujita 2003). However, in the 1960s the glial
ing to separate directly from indirectly neurogenic radial
hallmarks of these cells were also discovered, which stabilized
glial cells (Pinto et al. 2008, 2009). Interestingly, direct and
the concept of distinct sets of neural progenitors and radial
glial cells, the latter serving support functions and the former
generating neurons (Bentivoglio and Mazzarello 1999). In
addition, transitional forms between radial glia and astrocytes A
(Pinto and Götz 2007) further supported the link between
radial glial cells and the generation of glia, namely, astrocytes.
Following the initial misconception of His a century longer,
many authors also suggested that the spongioblast equivalent
radial glial cells would not divide.
Now it is known that virtually all radial glial cells in the
developing brain of mammals and other vertebrates (except
for cells in the floor plate and roof plate because of par-
ticularly high levels of Notch signaling) divide and undergo Pure neuronal Mixed Pure glial
interkinetic nuclear migration (Götz and Huttner 2005). progeny progeny progeny
The concept of nonproliferating radial glia was overcome at
the end of the 1990s, when virtually all radial glial cells were B
shown to divide (Götz et al. 1998; Hartfuss et al. 2001) and

apical radial glia subapical radial glia

SVZ
VZ

Direct Indirect
AS

t t
S- G2- M- M- G1- S- G2- M- M- G1-
phase phase
Figure 5.4 Radial Glial Cell Division and Regionalization. Schematic
drawing of interkinetic nuclear migration of radial glial cells with exam-
ples of GFP electroporated cells next to it. Note that subapically dividing
radial glia (right side) are a subtype specific to a few forebrain regions in
mice, whereas apically dividing radial glial cells are widespread (left side).
neurogenesis neurogenesis
Figure 5.5 Schematic Drawing of Radial Glia Lineage. A. Single cell
progeny of radial glial cells isolated from the cerebral cortex of embryonic
day 14 mice (see Malatesta et al. 2000). B. Different modes of neurogene-
sis from radial glial cells either by intermediate progenitors in an indirect
mode (right) or direct neurogenesis (left). Note that these subtypes can
be separated on the basis of the level of GFAP-driven GFP and their api-
cal prominin1 (see Pinto et al. 2008).

54 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
indirect generation of cells has also been observed in astroglio-
genesis (see chapter 12) with astrocytes derived from glial pro-
genitors in the postnatal brains, but also by direct conversion
of a radial glial cell to an astrocyte (see sections 2 and 3; see
chapter 12). Likewise, oligodendrogliogenesis (see chapter 13)
occurs by intermediate highly proliferative progenitor migrat-
ing throughout the brain in a first wave as well as from radial
glial cells upregulating oligodendroglial fate determinants
(Pinto et al. 2008) at late stages (Kessaris et al. 2006) suppos-
edly in a more direct manner. Taken together, the concept of
direct and indirect modes of cell type generation may serve to
expand cell numbers at specific stages of development.
2.2 T H E MU LT I P L E RO L E S O F R A D I A L
G L I A I N PAT T E R N I N G T H E D EV E L O P I N G
C E N T R A L N E RVO US SYS T E M
Besides their lineage heterogeneity in one brain region, the
cerebral cortex of mice, radial glial cells also differ profoundly
in different regions of the developing CNS. For example, in
the spinal cord they appear only at the onset of gliogenesis
and hence hardly contribute to neurogenesis (Pinto and Götz
2007). In the ventral telencephalon indirect neurogenesis (see
Fig. 5.5) is largely increased and radial glial cells seem to con-
tribute only indirectly to neurogenesis, with daughter cells
amplifying first in the ventricular zone and then in the sub-
ventricular zone, thereby expanding their progeny in number
(Pilz et al. 2012). Part of this amplifying progenitor cascade
is a special type of radial glial cells not undergoing interki-
netic nuclear migration but dividing at subapical positions
still in the ventricular zone (see Fig. 5.4). Significantly, radial
glial cells generate—indirectly or directly—very distinct
types of neurons and glia in different brain regions, thereby
contributing essentially to patterning of the brain (Götz and
Campbell 2002). For example, throughout the ventral regions
of the developing CNS, radial glial cells generate oligoden-
drocyte progenitor cells that migrate extensively distributing
throughout the CNS, whereas radial glial cells in the dorsal
telencephalon contribute to oligodendrogliogenesis only at
later stages (see chapter 13 and the preceding) (Kessaris et
al. 2006; Pinto et al. 2008). In some regions radial glial cells
generate largely GABAergic neurons and in others exclusively
glutamatergic neurons during development (Malatesta et al.
2003). Intriguingly, this regionalization is inherited to the
radial glial cells persisting in these regions as adult neural stem
cells (Brill et al. 2009; Merkle et al. 2007). Likewise, regional
differences in radial glial cells are at the source of generating
regional diversity in astrocytes in the developing spinal cord
(Hochstim et al. 2008) further supporting the importance of
radial glia diversity for patterning and function in the adult
CNS.
Radial glial cells contribute further to patterning and
regionalization of the developing CNS by forming unique
signaling centers and boundary regions. Radial glial cells form
fascicles condensing their radial processes into tight bound-
ary structures, for example, at the pallial–subpallial bound-
ary delineating the dorsal and the ventral telencephalon, the
mid-hindbrain boundary or the rhombomere boundaries,
R ADIAL GLIAL CELLS
distinct anlagen in the developing hindbrain. These bound-
aries are of functional significance because their absence in
mutant mice results in increased cell mixing between these
regions and ectopic neurogenesis (Chapouton et al. 1999;
Takahashi and Osumi 2011). Moreover, the roof plate and
floor plate, key signaling centers in the developing neural tube,
consist of specialized radial glial cells. These radial glial cells
hardly proliferate because of particularly high levels of Notch
activity that inhibits proliferation (Baek et al. 2006). Radial
glial cells in the floor plate contribute to neurogenesis only in
the ventral midbrain, where they generate the dopaminergic
neurons of the substantia nigra (Bonilla et al. 2008; Ono et al.
2007).
2.3 S TA B I L I Z I N G T H E D EVE L O P I N G
B R A I N: T H E E S S E N T I A L F U N C T I O N O F
A P I C O -BA S A L P O L A R IT Y A N D E P IT H E L I A L
HALLMARKS OF RADIAL GLIA
In addition to the described region-specific functions, bound-
ary structures formed by radial glia also regulate morphogen-
esis and often constrict the neural tube in radial dimension
at the sites of boundary formation leading to an apical sul-
cus. Thus, the length of the radial glia fiber determines the
radial extension of the growing neural tube. Radial glial cells
further provide tangential restrictions in tissue extension by
their tight junctional coupling, thereby limiting the tangen-
tial expansion of a given brain region. Therefore, the num-
ber of radial glial cells at the apical surface defines the size
of a brain region. The total number of radial glial cells lining
the ventricular surface and restricting the expansion of this
surface is largely achieved by earlier symmetrical divisions of
apical progenitors at the neuroepithelial stage regulating the
pool of self-renewing apical progenitor cells, including radial
glia. In addition, the self-renewing capacity of radial glial cells
is required to maintain this pool (Cappello et al. 2006). Thus,
as radial glial numbers regulate the size of the progenitor pool
in a brain region they determine the size of this region and
simultaneously supply the respective brain region with the
appropriate number of guiding structures for radial neuronal
migration.
2.4 A P I C A L A N C H O R I N G A N D S I G NA L I N G
Defects in anchoring of radial glia at the apical surface affect-
ing the junctional complexes and/or their connections to
the cytoskeleton result in severe defects in morphogenesis.
Junctional complexes (see Fig. 5.1C) are composed by dense
clusters of the transmembrane proteins cadherins whose extra-
cellular domains tightly bind two neighboring cells together (see
Fig. 5.1D). The intracellular domains of cadherins interact with
catenins (β- and α-catenin) connecting the cadherin complexes
in a dynamic mode to the actin cytoskeleton (see Fig. 5.1D).
The actin cytoskeleton stability is regulated by various signal-
ing molecules, including small Rho-GTPases influencing the
tight balance among individual actin monomers, the globular
form of G-actin, and their assembly into larger filaments, the
filamentous F-actin. Defects in either of these components in
• 55
radial glia, such as defects in cadherin clustering, β- or α-catenin
and F-actin formation, result in severe aberrations in neuro-
architecture of the affected brain region. For example, the lami-
nated structure of the cerebral cortex with neurons arranged
in horizontal layers is abolished into a random or concentric
arrangement of neurons when radial glia anchoring is disrupted
by affecting either of the preceding components (Cappello et al.
2006, 2012; Lien et al. 2006; Yokota et al. 2009). Notably, these
defects observed after conditional gene deletion in the mouse
model reflect neuronal dysplasias observed in patients (Bielas
and Gleeson 2004); for example, formation of a double-cortex
with a second cerebral cortex underlying the white matter
(Cappello et al. 2012). Besides providing essential anchors for
tissue architecture, adherens junctions also act as signaling cen-
ters enriching components of, for example, Notch-, Par-, and
Wnt-mediated signaling pathways (see Fig. 5.1D), and thereby
regulate proliferation and differentiation of radial glial cells
(Johansson et al. 2010; Zhang et al. 2010). In addition, gap junc-
tional coupling of radial glial cells is important for communica-
tion mediated by Ca2+ or other ions and small molecules (Elias
and Kriegstein 2008). Ca2+-signaling in radial glial cells can also
be elicited by depolarizing via neurotransmitter receptors (e.g.,
GABA or glutamate receptors), which positively regulate radial
glia proliferation (Elias and Kriegstein 2008). Taken together,
the function of radial glial cells as stabilizing structures and stem
and progenitor cells is integrated at the points of adhesion and
coupling.
Apical junctions of radial glial cells are also critical to delin-
eate the contact of the ventricular fluid to the apical membrane
domain. Indeed, access to the cerebrospinal fluid (CSF) is a
key hallmark of radial glial cells allowing direct access to key
signaling molecules in the CSF, such as insulin-like growth
factor (IGF), sonic hedgehog (SHH), bone morphogenic
protein (BMP), or WNT ( Johansson et al. 2010; Lehtinen
and Walsh 2011). In addition, vesicles of distinct size contain-
ing apical membrane components are released into the CSF
by radial glial cells as so-called prominosomes (Marzesco et al.
2009) and may constitute a further signaling component via
CSF. Notably, during embryonic development the ventricular
system is still closed, with no connection to the subarachnoid
space and circulation (also differentiated ependymal cells with
beating cilia are not yet present). Thus localized release of sig-
naling molecules into the CSF may form gradients depend-
ing on the diffusion of the respective protein. Significantly,
the special junctions coupling radial glial cells (Mollgoard
and Saunders, 1975) restrict diffusion of molecules within the
CSF into the neural tube. Because the apical side of radial glial
cells, however, has access to these components, radial glial cells
act as key mediator of signals to and from the CSF.
2.5 BA S A L A N C H O R I N G A N D S I G NA L I N G
The basal endfoot of radial glial cells (see Fig. 5.1B, C) is like-
wise important for anchoring and neuronal histogenesis as
well as mediating key signaling pathways. Disruption of the
radial glia basal endfeet attachment to the BM underlying the
meninges results in rupture of the BM and subsequent migra-
tion of neurons into these ectopic sites, reflecting the human
56 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
brain disorder of cobblestone lissencephaly, in which neurons
assemble in cobblestone-like extrusions atop the brain sur-
face (Bielas and Gleeson 2004). Thus, many disorders with
mislocalization of neurons at ectopic sites do not result from
defects within the migrating neurons themselves, but rather
arise as a secondary consequence of loss of radial glial anchor-
ing (Halfter et al. 2002). Rupture of the BM caused by defec-
tive anchoring of the radial glia endfeet reveals their key role
in stabilizing the BM. This function is mediated by integrins
in the radial glia endfeet anchoring extracellular matrix com-
ponents (ECM) of the BM (see Fig. 5.1B). Accordingly, loss
of major integrin subunits in radial glial cells leads to BM rup-
ture and subsequent migration of neurons through these rup-
tures (Bielas and Gleeson 2004; Halfter et al. 2002; Haubst
et al. 2006). Anchoring of integrins to the actin cytoskeleton
is likewise critical to avoid BM rupture and neuronal ectopia.
Integrins binding to ECM components also mediate signaling
promoting proliferation (Fietz et al. 2010; Loulier et al. 2009).
Accordingly, maintenance of the basal process after radial glial
cell division plays a key role in the decision to continue pro-
liferation as revealed by live imaging in zebrafish and mouse
radial glia (Alexandre et al. 2010; Shitamukai et al. 2011).
Thus, like the apical anchoring by junctional complexes, also
the basal anchoring mediates key signaling pathways, thereby
linking progenitor functions and stabilizing functions in an
almost inseparable manner. Taken together, the epithelial
properties of radial glial cells are not only a defining feature,
but also exert essential roles at both apical and basal sides for
all the key functions of radial glial cells.
2.6 R A D I A L G L I A A S GU I D E S F O R
M I G R AT I N G N EU RO NS
Apico-basal contacts of radial glia are also critical to maintain
the radial process, which fails to extend in a normal radial
manner when anchoring of radial glial cells is lost. These radial
processes provide important guiding structures along which
some neurons migrate from their place of birth to their final
position. Consistent with first observations (Bentivoglio and
Mazzarello 1999), ultrastructural analysis revealed the tight
alignment of neurons to radial glial processes (Chapter 32),
and live imaging revealed their mode of migration in a salta-
tory manner (Cooper 2008). Guidance of neurons along the
radial process depends on a multitude of molecules expressed
by radial glial cells, including the extracellular domains of gap
junctional proteins, the connexins, forming hemichannels
along the radial process (Elias and Kriegstein 2008). Molecules
regulating radial glia–guided migration are specific and often
not involved in other modes of migration, such as tangential
migration or somal translocation (Nadarajah and Parnavelas
2002), which are influenced by other factors (Cooper 2008).
3 R A D I A L G L I A I N T H E A D U LT
C E N T R A L N E RVO U S SYS T E M
Although radial glial cells are the first and the predomi-
nant glial cell type in the developing nervous system of all
vertebrates analyzed so far, in most regions of the mamma- Table 5.2A COMPARISON BETWEEN RADIAL GLIAL
lian brain they are transient and differentiate into diverse cell CELLS IN THE DEVELOPING AND ADULT ZEBRAFISH
types at the end of neurogenesis and neuronal migration (e.g., BRAIN
shortly after birth in the rodent cerebral cortex). Most radial RADIAL GLIA IN RADIAL GLIA
glia disappear by symmetrical neurogenic divisions, that is, ZEBRAFISH IN ADULT
generating two postmitotic neurons at the end of neurogen- PROTEIN LARVAE ZEBRAFISH
esis, by differentiating into ependymal cells or differentiat-
GFAP ++ +++
ing into astrocytes translocating via their radial process from
the ventricular surface (see chapters 12 and 30) (Kriegstein GLAST(Slc1a2) Nd Nd
and Alvarez-Buylla 2009). In few regions, however, such as GLT1(Slc1a3) Nd Nd
the wall of the lateral ventricle, radial glial cells persist inte-
grated into the otherwise differentiating ependyma. The Glutamine synthetase Nd +++
radial glial soma is located above the ependyma, where also S100-β – +++
all the progenitors, the transit-amplifying progenitor and the Connexin 43 Nd Nd
neuroblasts, comprise the “subependymal zone” (SEZ) (see (Gja1)
Fig. 5.2). In the developing brain, where no ependymal cells
Aquaporin 4 Nd +++
are present, radial glial cells line the ventricle and hence other
progenitors that are not located apically (Götz and Huttner KIR 4.1/2.1 Nd +
2005) form the “subventricular zone” (SVZ). However, most Aldlhl1 Nd Nd
of the SEZ radial glial cells do not maintain a radial process
contacting the pial surface, but rather have a shorter radial Nestin ++ +++
process to the BM surrounding neighboring blood vessels Vimentin + +
(see Fig. 5.2B, C) (Kriegstein and Alvarez-Buylla 2009).
BLBP ++ +++
According to the hallmark of radial glial cells, these cells also
possess access to the ventricle with a small apical endfoot, TN-C Nd Nd
which contains apical membrane proteins, such as promi- Sox2 ++ +++
nin1 (Beckervordersandforth et al. 2010). These radial glial
Aromatase B ++ +++
cells are the adult neural stem cells as isolation of cells with
glial expression (high levels of GFAP-driven GFP) (see Fig. FGFR2/3 ++ ++
5.2C) and the apical membrane protein prominin1 by FACS ChSPG4 Nd Nd
allows enrichment of self-renewing and multipotent stem cells
(Beckervordersandforth et al. 2010). Indeed, genetic fate map- According to Zfin database and Alexandre et al. 2010; Chapouton et al. 2007,
2010; Ganz et al. 2010 ; Grupp et al. 2009; März et al. 2010; Rothenaigner et al.
ping using the split-Cre technology also confirms the stem cell 2011; Tong et al. 2009; Topp et al. 2008.
identity of cells coexpressing GFAP and prominin1 in vivo
(Beckervordersandforth et al. 2010). These stem cells gener- Table 5.2B COMPARISON BETWEEN RADIAL GLIAL
ate a series of progenitors that then migrate to the olfactory CELLS IN THE DEVELOPING AND ADULT ZEBRAFISH
bulb generating diverse types of interneurons life long (see BRAIN
chapter 40) (Brill et al. 2009; Kriegstein and Alvarez-Buylla
RADIAL GLIA RADIAL
2009). Notably, interneurons of the olfactory bulb originate AFTER 48 HPF GLIA IN
during development from radial glia of the lateral wall of the IN ZEBRAFISH ADULT
lateral ventricle, and neurogenesis of these neurons contin- FUNCTION EMBRYOS ZEBRAFISH
ues into adulthood in almost all vertebrates analyzed so far
(Doetsch and Scharff 2001; Kishimoto et al. 2011). Glutamate uptake Nd Nd
K-conductance at rest Nd Nd
3.1 WI D E S P R E A D R A D I A L G L I A I N T H E A D U LT Glycogen storage Nd Nd
B R A I N O F M A N Y V E RT E B R AT E S
Gap-junctions/ Nd Nd
Although radial glial cells persisting in the adult brain are the Hemichannels/
exception in the mammalian and avian brains, this is the rule Ca-waves
in most other vertebrates (Cuoghi and Mola 2009; Kalman Blood vessel contact/blood ++ ++
2002). Most vertebrates maintain GFAP+ cells with long flow regulation
radial processes lining the ventricle, which are also coupled Cell division +++ +++
by junctional complexes delineating apical from basolateral
membrane (Grupp et al. 2009). Notably, these cells have a sin- Multipotency +++ +++
gle cilium, such as radial glial cells, and they also express other Self-renewal +++ +++
typical astrocyte/ependyma hallmarks such as S100β, glu- According to Zfin database and Alexandre et al. 2010; Chapouton et al. 2007,
tamine synthase and BLBP (Table 5.2). However, as for radial 2010; Ganz et al. 2010 ; Grupp et al. 2009; März et al. 2010; Rothenaigner et al.
glial cells in the developing brain, there is regional and subtype 2011; Tong et al. 2009; Topp et al. 2008.

R ADIAL GLIAL CELLS • 57

heterogeneity among radial glial cells. For example, in the
adult zebrafish telencephalon (Ganz et al. 2010; Grupp et al.
2009; März et al. 2010), a subset of radial glial cells proliferates
and nonproliferating radial glia can be activated by blocking
Notch-signaling or injury (Ayari et al. 2010; Baumgart et al.
2012; Chapouton et al. 2010; Kroehne et al. 2011; Reimer
et al. 2008). Thus, similar to embryogenesis, high levels of
Notch signaling block radial glia proliferation. In some CNS
regions, such as the spinal cord, radial glial cells do not prolif-
erate (Reimer et al. 2008), but can also be activated by injury
(Echeverri and Tanaka 2002; Reimer et al. 2008, 2009). In
both cases, with or without prior proliferation, these cells
are able to replace the damaged neurons, as clearly demon-
strated in the telencephalon and spinal cord. In the absence
of injury, adult neurogenesis occurs in regions with radial glia
proliferation in a much more widespread manner compared
with the mammalian or avian brain (Chapouton et al. 2007;
García-Verdugo et al. 2002).
Radial glial cells persist in most CNS regions of the adult
teleost CNS, as well as in Chondrichtyes, other Actinopterygii,
Amphibia, and reptiles (Cuoghi and Mola 2009; Kalman
2002). With the exception of the optic nerve, virtually no free
stellate–shaped astrocytes or cuboid ependymal cells are pre-
sent in most of the species analyzed in these vertebrate classes.
Therefore, radial glial cells comprise both functions of astro-
cytes and ependymal cells in the adult CNS. Accordingly, they
also possess Aquaporin channels, even though these are not
sorted toward the polarized BM interface, as is the case for
astrocytes (see chapter 4) (Grupp et al. 2009). Interestingly,
however, in brain regions with increased complexity and
thickness, parenchymal astrocytes appear in all these groups,
and these stellate cells reduce GFAP expression (Kalman
2002). For example, the squalomorph shark brain has GFAP+
radial glia, whereas large areas in the skate brain are popu-
lated by stellate astrocytes mostly lacking GFAP expression
(Kalman 2002). Taken together, astrocytes seemingly evolved
in parallel in Agnathi, Chondrichtyes, Actinopterygii, and
Sarcopterygii-Amniotes in correlation to increased complexity
and thickness of respective brain regions. Increased size appar-
ently required support by parenchymal glial cells prompting
delamination of radial glial cells and astrocyte formation dur-
ing phylogeny (Cuoghi and Mola 2009; Kalman 2002).
4 R A D I A L G L I A L C E L L R E AC T I O N
TO I N J U RY
Reactive astrogliosis is a major hallmark of the injury reaction
in the mammalian brain. Thus, gliosis differs profoundly after
injury in vertebrate classes with persistent radial glial cells
lacking stellate astrocytes. After an invasive injury in the adult
zebrafish forebrain, most radial glial cells do not delaminate
from the ventricle and do not migrate toward the injury site
(Baumgart et al. 2012), except in very large injury conditions
(Kroehne et al. 2011). Likewise, the reaction of oligodendro-
cyte progenitors in the adult brain, prominent in the mam-
malian brain (Robel et al. 2011), largely fails to occur in the
injured zebrafish forebrain (Baumgart et al. 2012), except in
58 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
very large injuries (Kroehne et al. 2011). This distinct glial reac-
tivity may at least contribute to the scar-less wound healing
in the adult zebrafish CNS (Ayari et al. 2010; Baumgart et al.
2012; März et al. 2011). Only after very large injuries, some
scarlike lesion remains in correlation to migration of radial
glia–derived astrocytes to the injury site as well as oligoden-
drocyte progenitor activation (Kroehne et al. 2011). Notably,
injuries damaging the ventricular surface of radial glial cells
evoke a reaction more similar to reactive astrogliosis (März
et al. 2011).
Most distinct from the situation after brain injury in mam-
mals, however, radial glial cells increase neurogenesis after
injury not only in regions of normal neurogenesis (Ayari et al.
2010; Baumgart et al. 2012; Kroehne et al. 2011), but also in
the spinal cord where no adult neurogenesis occurs normally
(Reimer et al. 2009). A similar neurogenic repair reaction of
a radial glial-like glia cell, the Müller glia, also occurs in the
zebrafish retina (see chapter 4) (Raymond et al. 2006; Yurco
and Cameron 2005). Thus, the loss of radial glial cells in adult
brains of mammals and birds required by increased size and
complexity is accompanied by a rather dramatic side effect
leading to differences in reactive gliosis involved in scar forma-
tion and the loss of regenerative response (Robel et al. 2011).
5 S U M M A RY A N D P E R S P E C T I VE S
Taken together, radial glial cells are the first and major glial
cell type in ontogeny and phylogeny. They combine hallmarks
of ependymal cells (ventricular access, epithelial polarity) and
astrocytes (blood vessel contact; water, glutamate-transport
and K+-buffering) with a progenitor and stem cell function.
Accordingly, their delineation from astrocytes and ependymal
cells by so-called “markers” (see Table 5.1) has been difficult
because these are largely shared with only quantitative differ-
ences between these glial cell types (Beckervordersandforth
et al. 2010). Although expression of all of these genes clearly
delineates radial glial cells from neuroepithelial cells (see
Table 5.1A), difficulties delineating radial glia from astrocytes
and ependymal cells have led to a rather confused nomencla-
ture, especially when referring to adult brains. Especially radial
glial cells in the adult CNS, for example, in teleosts, are either
referred to as astroglia (Grupp et al. 2009) or ependymoglia
(Cuoghi and Mola 2009), and sometimes as radial glia (Robel
et al. 2011). This highlights (1) the need for a clear nomen-
clature, and (2) a more comprehensive expression analysis
beyond the use of a few marker proteins.
In regard to a clear nomenclature, glial cells with ventricu-
lar access, a radial morphology, and epithelial characteristics
should be referred to as radial glial cells. This definition clearly
comprises cells of diverse functions ranging from neural stem
and progenitor cells to cells with other specialized functions,
such as secretory radial glia often referred to as tanycytes in
adult brains. The epithelial hallmarks of apico-basal polarity
are central to this definition consistent with their functional
roles in radial glial cells, providing radial tissue stability as well
as influencing their proliferation, fate, and stable guidance and
boundary structure. In regard to extending expression analysis
of distinct glial cells to a genomewide expression level we now
have access to genomewide expression profiles of various types
of glia (see chapters 28, 29) (Beckervordersandforth et al.
2010; Cahoy et al. 2008; Lovatt et al. 2007), including adult
and embryonic radial glial cells (Beckervordersandforth et al.
2010; Pinto et al. 2008) revealing shared and unique expres-
sion patterns of these cells. This comparative analysis also
suggests new selective “markers” that still require appropriate
antibodies to be generated, but in the future will allow more
unequivocal delineation of these cells types, besides morpho-
logical and functional criteria.
An essential functional difference between radial glial cells
and their astrocyte and ependymal cell relatives is their role
as stem and progenitor cells contributing to neurogenesis and
gliogenesis in a widespread manner in developing and adult
vertebrate CNS, and a few niches in the adult mammalian
forebrain. Obviously, beyond gaining new candidates for spe-
cific “marker” proteins, comparative analysis of the glial cells
with and without stem cell properties will allow unravelling
the core network regulating this key functional property, as
well as the genetic regulation of other core functions of dis-
tinct glial cells (see chapters 28, 29).
AC K N OW L E D G E M E N T S
Foremost the author would like to thank the DFG for the
Leibniz Award, which has provided the freedom to pursue
exciting new avenues in research, such as the stem cell function
of glial cells. The author is also particularly grateful to Joana
Barbosa, Emily Baumgart, Ruth Beckervordersandforth, Judith
Fischer, Wieland Huttner, Jovica Ninkovic, Gregor Pilz, Stefanie
Robel, Franziska Weinandy, Michaela Wilsch-Bräuninger, and
Hartwig Wolburg for beautiful examples of radial glia and their
polarity aspects for the figures; and would also like to thank
Laure Bally-Cuif, Angelique Bordey, and Andreas Reichenbach
for valuable comments and suggestions.
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• 61
 6.
STRUCTURE AND FUNCTION OF OLIGODENDROCY TES
Arthur M. Butt

A B B R E VI AT I O N S integrate historical studies such as these with more recent

findings using immunohistochemistry, modern imaging tech-
CAII carbonic anhydrase II niques, and innovative experimental paradigms, such as tissue
CNS central nervous system culture, transgenic mice, and zebrafish.
CNP 2′,3′-cyclic nucleotide 3′-phosphodiesterase
Cx connexin
eGFP enhanced green fluorescent protein 2 F R O M R I O H O RT E G A TO T H E
EM electron microscopy P R E S E N T DAY
GFAP glial fibrillary acidic protein
GFP green fluorescent protein
2.1 R I O H O RT EG A’S O L I G O D E N D RO C Y T E
Kv voltage-gated potassium channel
P H E N OT Y P E S I TO I V
MAG myelin associated glycoprotein
MBP myelin basic protein Del Rio Hortega classified oligodendrocytes into types I to
MOG myelin oligodendrocyte glycoprotein IV, based on their morphological characteristics of the num-
MRI magnetic resonance imaging ber and orientation of their cellular processes, shape and
MS multiple sclerosis size of their somata, size of the axons with which they were
Nav voltage-gated sodium channel associated, and their distributions within the CNS (Penfield
Nfasc155 neurofascin 155 1932). Oligodendrocyte phenotypic diversity is largely
OPC oligodendrocyte progenitor cell neglected, but has been confirmed by EM, intracellular dye
PDGFR platelet-derived growth factor receptor injection, immunohistochemistry, and more recently expres-
PLP proteolipid protein sion of reporter genes (Berry et al. 1995; Bjartmar et al.
1994; Butt et al. 1994, 1998a; Murtie et al. 2007; Remahl
and Hildebrand 1990a,b; Stensaas and Stensaas 1968a,b;
1 INTRODUCTION Vinet et al. 2010; Weruaga-Prieto et al. 1996a,b). Our studies
indicated that oligodendrocytes can be broadly subdivided
Oligodendrocytes are defined by their singular function— into two distinct phenotypes defined by the caliber of axons
they produce the myelin sheaths that insulate axons in the within the unit, respectively, below and above a diameter of
central nervous system (CNS). Myelin is one of the most 2 to 4 μm, and corresponding to Del Rio Hortega’s types I/II
complex biological structures and is absolutely essential for and III/IV (Butt et al. 1998a). Type I and II units have small
rapid neuronal communication that underpins the mas- rounded somata, 10 to 12 μm in diameter, from which four or
sive integrative computing power of the human brain. more fine primary processes extend and branch to myelinate
Consequently, the loss of myelin has devastating effects on 10 to 30 small axons, less than 2 μm in diameter (Fig. 6.1A,B).
CNS function. In addition to myelin-forming oligodendro- Type I and II oligodendrocytes are similar but can be dis-
cytes, the CNS contains significant populations of “adult tinguished by the alignment of axons they myelinate, which
oligodendrocyte progenitors” (OPCs) and non-myelinating pass in multiple directions and are more widely distributed in
“perineuronal” or “satellite” oligodendrocytes, which can type I units (see Fig. 6.1A), whereas type II units have paral-
generate myelin-forming oligodendrocytes throughout life lel arrays of myelin segments situated within a short distance
and following injurious insults. This chapter deals with the of a centrally located cell body (see Fig. 6.1B). Type I/II oli-
morphology of oligodendrocytes and their myelinating func- godendrocytes are the most generally studied, because they
tion and is indebted to authoritative articles that regretfully are the main population throughout white and gray matter
are slowly disappearing from the literature. Del Rio Hortega with axons of less than or equal to 2 μm. Three-dimensional
is credited with first identifying oligodendrocytes—naming analyses of immunostained and dye-injected type II oligo-
them from the Greek for cell with few processes (Penfield dendrocytes in the optic nerve, cerebellum, spinal cord, and
1932), and the cellular connection between oligodendrocytes cortex has demonstrated a relative uniformity in their over-
and myelin sheaths was demonstrated by electron microscopy all appearance, but this disguises a high degree of heteroge-
(EM) (Hirano 1968; Peters 1964). The emphasis here is to neity in respect to the number of myelin segments per unit

62
(5–50) and the internodal lengths (50–300 μm) (Berry et al. A C E
1995; Butt et al. 1994,1998a; Weruaga-Prieto et al. 1996a,b).
Similarly, studies in mice expressing green fluorescent protein
(GFP) under the control of the proteolipid protein (PLP)
or CNPase (2′,3′-cyclic nucleotide-3′-phosphodiesterase)
gene promoters, in combination with confocal microscopy
and computerized cell tracing systems, have characterized
the morphology of type I oligodendrocytes in the mouse
frontal cortex and hippocampus (Murtie et al. 2007; Vinet
et al. 2010). In mice expressing CNPase-eGFP, three variants
of type I oligodendrocytes were identified in the hippocam- B D
pus, as ramified, stellar, or smooth oligodendrocytes, distin-
guished by decreasing complexity, and that may represent
stages of a maturation process (Vinet et al. 2010). In mice
expressing PLP-eGFP, type I oligodendrocytes in the frontal
cortex were remarkably homogeneous and appeared more
complex with much shorter myelin internodes than the pat-
terns described for type II oligodendrocytes (Butt et al. 1994;
Murtie et al. 2007). Unlike earlier studies using dye injection
and the Rip antibody, studies using the eGFP reporter did
not demonstrate by EM that the reporter completely fills
the entire internodal length, and this may explain the short
internodes observed. However, short internodes are a char-
acteristic of late-forming oligodendrocytes and of remyelina-
tion, and variants of type I oligodendrocytes in the cortex Figure 6.1 Immunolabeling of oligodendrocyte phenotypes with the
Rip antibody in whole mounts of adult rat anterior medullary velum.
and hippocampus may reflect the high degree of remodelling A,B. Multipolar oligodendrocytes supporting numerous myelin sheaths
that continues long into adulthood in these regions. Type for small-caliber fibers, corresponding to Rio Hortega’s type I (A) and
III oligodendrocytes are localized to areas in which axon type II (B). Type I oligodendrocytes have multiple branching processes
diameters are greater than 2 to 4 μm, such as the cerebral and supporting numerous radially oriented myelin sheaths, whereas type II
cerebellar peduncles, the medulla oblongata, and the spinal oligodendrocytes have fewer branching processes that support parallel
myelin sheaths, but otherwise cell bodies of type I and II are difficult to
cord. Type III oligodendrocytes have larger ovoid, irregular, distinguish. C. Type III oligodendrocyte with a large cell body and small
or elongated cell bodies, 12 to 15 μm diameter, which are number of stout processes (arrows) that engage large-caliber fibers; as in
often applied directly to an axon, with one or more thick this case, type III oligodendrocytes often have a cell body directly applied
primary processes that rarely branch and myelinate a small to a large-caliber fiber, which is a phenotypic characteristic of type IV oli-
number of axons, usually less than five, with external sheath godendrocytes. D. A transitional oligodendrocyte with features of type II
and III phenotypes, with a large stout process extending to a large-caliber
diameters ranging from 4 to 15 μm (Fig. 6.1C). There are also fiber, and multiple fine branching processes that myelinate small-caliber
transitional forms between oligodendrocyte phenotypes I/II fibers. E. Type IV oligodendrocyte with cell body directly applied to a
and III units that contain both small and large-caliber axons single large caliber fiber (asterisk), with a long single internode (nodes
(Fig. 6.1D). Finally, type IV oligodendrocytes are restricted indicated by large arrows). Arrowheads in (B) show Rip staining at points
to tracts containing the largest diameter fibers greater than of engagement of oligodendrocyte processes with internodal myelin
sheaths; this cell appears to extend processes to consecutive internodal
10 μm in diameter, and occur near the entrance of nerve myelin segments (arrowheads) either side of a node (arrow). Spiraling of
roots into the CNS. Type IV unit somata do not have pro- the cytoplasmic tongue process is clear in larger fibers (arrows in D and
cesses and they form a single long myelin sheath over a large arrowheads in E). The number of fibers engaged by each unit is inversely
diameter fiber (Fig. 6.1E). The rarity of type IV oligodendro- proportional to fiber diameters, but diameters vary within individual units
cytes in the rodent brain may be related to the modest size of (C). Scale bar = 25 μm in A–D and 50 μm in E. (A–E) From Berry et al.
1995, with permission.
the largest diameter fibers, compared with humans and other
vertebrates where they are more common (Anderson et al.
1999; Hildebrand and Hahn 1978; Hildebrand et al. 1993;
Remahl and Hildebrand 1990a,b; Stensaas and Stensaas
1968a,b). Hence, oligodendrocyte phenotypes differ in the or small numbers of long myelin sheaths for large diameter
number of axons within the unit, the diameters of their axons. Thus, in rodents, type I oligodendrocytes support 30
myelinated fibers, and their internodal lengths (Berry et al. or more small caliber fibers with short internodal lengths
1995; Bjartmar et al. 1994; Butt et al. 1998a; Hildebrand and of 10 to 50 μm, type II oligodendrocytes support 10 to 30
Hahn 1978; Hildebrand et al. 1993; Remahl and Hildebrand small-caliber fibers with internodal lengths between 50 and
1990a,b). These morphological parameters are related, 350 μm, type III oligodendrocytes support 2–5 large-caliber
whereby oligodendrocytes tend either to support a large fibers with internodal lengths of around 400 μm, and type
number of short myelin sheaths for small diameter axons, IV units support a single large-caliber fiber with internodal

S T RU C T U R E A N D F U N C T I O N O F O L I G O D E N D R O C Y T E S • 63
length of up to 1,000 μm. However, there does not appear to
be a strict phenotypic segregation, type I to IV units are the
main variants of what appears to be a morphological contin-
uum, and transitional forms are unexceptional (Berry et al.
1995; Butt et al. 1998a). The phenotypic differences between
oligodendrocytes are not trivial, because fiber diameter and
internodal length determine axonal speed of conduction.
Hence, axons within type III/IV units conduct the fastest,
with conduction velocities of up to 80 to 120 m/second,
whereas those in type I/II units have much slower conduc-
tion velocities, generally less than 20 m/second.
2.2 U LT R A S T RU C T U R E O F
O L I G O D E N D RO C Y T E S
The ultrastructural characteristics of oligodendrocytes were
defined many years ago and have been reviewed in consider-
able detail (Peters et al. 1991). Ultrastructurally, oligoden-
drocytes can be identified by their generally round, dark
nuclei, which are surrounded by dark cytoplasm contain-
ing short granular endoplasmic reticulum, polyribosomes,
short mitochondria, and Golgi apparatus (Sandell and
Peters 2002). The nuclei contain dense patches of chroma-
tin and there is a dense uneven layer of chromatin beneath
the nuclear envelope. Hildebrand et al. provided EM
three-dimensional reconstruction of oligodendrocyte phe-
notypes I/II (Fig. 6.2A,B) and III/IV (Fig. 6.2C,D), and
identified no outstanding differences in their ultrastructure
(Remahl and Hildebrand 1990a).
2.3 T H E G E N E R A L I Z E D
O L I G O D E N D RO C Y T E
The generalized view is based on EM, immunohistochem-
istry, and intracellular dye-filling of the type II oligo-
dendrocyte phenotype. Visualization of the cytoplasmic
compartments in oligodendrocytes and myelin using intra-
cellular dye-filling showed that the inner and outer cyto-
plasmic ridges (tongue processes or mesaxons) of the myelin
sheath “corkscrew” around the axon (Fig. 6.3A) (Berry et al.
1995; Butt and Ransom 1989). Dye-filling of oligodendro-
cytes demonstrated distinct networks of cytoplasmic inter-
connections (reticulations) or “pockets” within the myelin
sheath that included Schmidt-Lanterman clefts and provide
cytoplasmic access to the compacted myelin (Fig. 6.3B)
(Berry et al. 1995; Butt and Ransom 1989; Ransom et al.
1991; Velumian et al. 2011). It takes more than 1 hour for
small (<500 Da) molecules to diffuse along the whole
myelin sheet (Velumian et al. 2011). If unwrapped, each
myelin sheath would appear as an extraordinarily large trap-
ezoid sheet that is an extension of the oligodendroglial cell
membrane (Ransom et al. 1991). The compacted myelin
sheet is circumscribed by a continuous cytoplasmic ridge
that is connected to the cell body via the processes and pro-
vides a conduit for transport of the myelin constituents from
the cell body to the myelin. The myelin sheath is wrapped
around the axon to form concentric lamellae and the cyto-
plasmic ridges stack up to form the paranodal loops at nodes
of Ranvier (Fig. 6.3C,D). Oligodendroglial paranodal loops

A C

B D

Figure 6.2 Electron Micrograph Reconstructions of Oligodendrocytes. A,B. Type II oligodendrocyte from feline corpus callosum, 21 days postnatal.
The oligodendrocyte myelinates 11 nearby axons (A), indicated by asterisks in the electron micrograph (B), which corresponds to the plane marked in
(A). C,D. Type IV oligodendrocyte from feline spinal cord of 47-day-old fetus. The oligodendrocyte is attached to a single axon that it myelinates (A),
indicated by the asterisk in the electron micrograph (B). There are no ultrastructural distinguishing features between cell bodies and nuclei of type II and
IV units (indicated by O in B and D). From Remahl and Hildebrand 1990a, with permission.

64 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
form complex axoglial junctions that appear as “transverse
bands” in EM (Fig. 6.3D), which are important in the separa-
tion of ion channels at nodes of Ranvier (Charles et al. 2002;
Hinman et al. 2006; Mierzwa et al. 2010; Zonta et al. 2008)
and involves a neurofascin155 (Nfasc155)-Caspr-Contactin
axoglial adhesion complex.
A and both the longitudinal (internodal lengths, L) and radial
B
1μm
C and Hoekstra 2010), and by extrapolation myelin biogenesis
1μm
D and oligodendrocyte polymorphism signifies the greater met-
40 μm 0.1μm
Figure 6.3 Oligodendroglial Cytoplasmic Compartments. Confocal
microscopy of oligodendrocyte intracellularly dye filled with lysinated
rhodamine dextran (A,B) and freeze-fracture EM (C) of oligodendro-
cyte in the whole-mounted adult rat anterior medullary velum, and
transmission EM in mouse spinal cord (D). A. Intracellular dye-filling
resolves the oligodendroglial cytoplasmic compartments of this large
type III oligodendrocyte unit, which has five axons with external
sheath diameters of 10 to 15 μm and internodal lengths of around
400 μm. Myelin sheaths have prominent cytoplasmic reticulations
and the dye-filled cytoplasmic tongue processes spiral along the axons
(large arrows) and delineate the paranodal loops (small arrows). B.
High-contrast zoom illustrating internodal cytoplasmic compartments
forming pockets between compacted myelin, which is occluded to the
dye and appears black. C. Scanning electron micrograph of myelinat-
ing fiber in the postnatal anterior medullary velum of a 15-day-old rat
showing the paranodal loops (between arrows) spiraling toward the node
of Ranvier (asterisk). D. Electron micrograph showing transverse bands
at paranodes (arrowheads). (A–C) From Berry et al. 1995; (D) from
Mierzwa et al. (2010), with permission.
S T RU C T U R E A N D F U N C T I O N O F O L I G O D E N D R O C Y T E S
3 F U N C T I O N A L I M P L I C AT I O N S
OF OLIGODENDROCYTE
P O LY M O R P H I S M
3.1 MY E L I N VO LUM E , SY N T H E S I S ,
A N D M ETA B O L I S M
There are direct relationships between fiber diameter (D) and
the number of myelin sheaths per oligodendrocyte unit (n),
(number of lamellae, N) dimensions of the myelin sheaths
(Butt et al. 1998a). The relationship between axon diam-
eter and the number of myelin lamella is a strict 1:10 ratio,
defined as the g-ratio (Friede 1972). Each myelin sheath has
dimensions of approximately π.D.N.L. and multiplying this
by the number of myelin segments within the oligodendro-
cyte unit (n) provides the total volume of myelin supported
by oligodendrocytes. There is a clear demarcation between
type I/II and type III/IV units, which respectively support
myelin volumes of approximately 500 and 30,000 μm3 (Butt
et al. 1998a). However, this is not an absolute segregation,
because transitional type II/III units contain both small-
and large-caliber axons and support intermediate volumes of
myelin. As noted, type I units in the cerebral cortex have 30
or so very short internodal lengths for small diameter axons
and so support the smallest volume of myelin (Murtie et al.
2007). In contrast, type IV units in which internodal lengths
attain 1,000 μm support the largest volumes of myelin, as
great as 1,50,000 μm3 (Butt et al. 1998a). During active myeli-
nation, myelin biosynthesis in type II oligodendrocytes has
been calculated to be 5 to 50 × 103 μm2/cell per day (Baron
would be more than 100 times greater in type III/IV oligoden-
drocytes. Greater myelin metabolism and turnover has been
noted in large diameter CNS fibers (Hildebrand et al. 1993),
abolic demand of supporting a much larger mass of myelin in
type III/IV oligodendrocytes compared with type I/II units.
Hence, type III/IV units have large cell bodies, thick radial
processes, and prominent Schmidt-Lantermann incisures,
which facilitate the substantial distribution of myelin gene
products. In contrast, type I/II units have small somata and
long fine processes, reflecting the lower mass of myelin they
support.
3.2 MY E L I N B I O C H E M I S T RY
AND FUNCTION
The main constituents of myelin are lipids (70% of its dry
weight) and proteins (30% of the dry weight), many of which
are specific to myelin and are used to identify oligodendro-
cytes by immunohistochemistry, or their genes are used to
drive expression of fluorescent reporter proteins (see chap-
ters 13 and 44). Central nervous system myelin lipids are rich
in the glycosphingolipids galactocerebroside (GalC) and the
sulfated derivative recognized by the O4 antibody, which
are used as specific markers for oligodendrocytes (Raff et al.
1978; Sommer and Schachner 1981). The major CNS myelin
• 65
proteins are proteolipid protein (PLP, 50% of myelin pro-
tein) and myelin basic protein (MBP, 30% of myelin protein),
which are not specific to CNS myelin (Griffiths et al. 1998a;
Sternberger et al. 1978). The enzyme CNPase (2′,3′-cyclic
nucleotide-3′-phosphodiesterase) is expressed in the CNS
specifically by oligodendrocytes and immunohistochemi-
cally is localized to the cell body and processes rather than
compacted myelin (Lappe-Siefke et al. 2003). The myelin
glycoproteins MOG (myelin oligodendrocyte glycoprotein)
and MAG (myelin-associated glycoprotein) are also used to
identify oligodendrocytes; MAG is expressed in both CNS
and PNS myelin (Schachner and Bartsch 2000), whereas
MOG is CNS-specific and a key autoantigen for primary
demyelination in multiple sclerosis (MS) (Clements et al.
2003). Oligodendrocytes also express many other proteins,
such as oligodendrocyte-specific protein OSP/claudin-11
(third most abundant protein in myelin), gap junction con-
nexins (Cx32 and Cx47), Nogo, isoform II of carbonic
anhydrase (CAII), and transferrin, which have multifarious
functions (see chapters 13, 43, 44).
The majority of studies have been on type II oligoden-
drocytes, which express all of the aforementioned proteins,
and there have been few studies on biochemical differences
between oligodendrocyte phenotypes I/II and III/IV (Butt
and Berry 2000). Proteolipid protein (PLP) expression may
vary as a function of axon caliber, the smallest myelinated
fibers containing higher levels of PLP relative to MBP than
the largest myelinated fibers (Hartman et al. 1982). There
is evidence that PLP serves a distinct function in sup-
porting the integrity of type II units, whereby there was
greater disruption in small-diameter axons in the absence
of PLP-DM20 (Griffiths et al. 1998b). Similarly, CNP
appeared to be more critical for the formation of a normal
inner tongue process in small diameter axons that are myeli-
nated by type II oligodendrocyte units (Edgar et al. 2009).
Studies on oligodendrocyte subtypes have indicated differ-
ential phenotypic expression of the small isoform of MAG
(S-MAG) and CAII, with type I/II units being S-MAG –/
CAII+ and type III/IV being S-MAG+/CAII– (Butt et al.
1998b, 1995). S-MAG is essential for normal myelination
in the PNS (Schachner and Bartsch 2000), suggesting a
specific role for this isoform in maintaining the integrity
of type III/IV oligodendrocyte units (Butt et al. 1998b);
the large isoform of MAG (L-MAG) is the most important
functionally in the CNS (Schachner and Bartsch 2000),
and is expressed by all oligodendrocyte phenotypes (Butt
et al. 1998b). No major abnormalities were observed in the
brains of CAII-deficient mice (Ghandour et al. 1989), and
the functional significance of CAII, specifically in type I/II
units, is unclear. Carbonic anhydrase is a key enzyme in cel-
lular pH regulation, and fluorescence correlation spectros-
copy analysis of CAII microinjected into oligodendrocytes
revealed it exists both as a freely diff using protein through-
out the cell as well as in microdomains associated with Na+/
H+ exchangers in the somata and Na+/HCO3– cotransporter
in the processes (Ro and Carson 2004). Specific expres-
sion of CAII in type I/II oligodendrocytes may reflect a
greater capacity for intracellular pH regulation in the face
66 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
of the large acid shifts in extracellular pH during axonal
activity in CNS white matter (Kettenmann et al. 1990;
Sykova 1989), and would provide a protective mechanism
for intracellular pH regulation in hypoxic stress and during
lactate uptake (Ransom et al. 1992; Rinholm et al. 2011).
Oligodendrocytes also express acid-sensing ion channels
that may contribute to their vulnerability to CNS ischemia
(Feldman et al. 2008) (see chapters 20 and 52). In addition,
oligodendrocytes express the highest levels of iron in the
brain, where it is a basic requirement for oxidative metabo-
lism, and differences in iron-mediated oxidative stress are
most likely involved in susceptible oligodendrocyte popu-
lations (Todorich et al. 2009) (see chapter 46). A number
of studies indicate that type I/II oligodendrocytes may be
more susceptible to injury and demyelination than type III/
IV oligodendrocytes. Late developing type I/II oligoden-
drocyte units are most susceptible in periventricular white
matter injury caused by perinatal ischemic insult (Riddle
et al. 2006), and in tauopathies and metachromatic leu-
kodystrophy (Hildebrand et al. 1993). In the taiep mutant
rat, tauopathy more severely affected type I/II oligodendro-
cytes than type III/IV oligodendrocytes (Song et al. 2001).
These studies indicate that regional predilections to injury
are related to differences in susceptibility of oligodendro-
cyte phenotypes.
4 D E VE L O PM E N T O F
O L I G O D E N D R O C Y T E P H E N OT Y P E S
4.1 O L I G O D E N D RO C Y T E P H E N OT Y P I C
D I VE RG E N C E
The mechanisms determining axon growth and oligoden-
drocyte phenotype divergence are unresolved, but they are
entirely interdependent (see chapters 13, 44, and 45). There
are direct relationships among the age at which axons are
myelinated, their final diameter in the adult, and the develop-
mental divergence of oligodendrocyte phenotypes (Bjartmar
et al. 1994; Butt et al. 1997; Hildebrand et al. 1993; Remahl
and Hildebrand 1990a,b). Thus, prospective large-diameter
fibers (>4 μm) are myelinated early in development by type
III/IV oligodendrocytes that diverge before birth in rodents,
whereas prospective small diameter fibers (<2 μm) are myeli-
nated later in development, by type I/II oligodendrocytes
that differentiate perinatally in rodents (Butt et al. 1997).
However, oligodendrocytes are not inherently programmed
to myelinate a specific size of axon and there are no evident
morphological or physiological differences between early and
late developing OPCs (Butt et al. 1997; Tripathi et al. 2011).
Oligodendrocyte progenitor cells that normally develop into
type I/II oligodendrocytes will myelinate large diameter
fibers when transplanted into experimentally demyelinated
spinal cord funiculi of the rat (Fanarraga et al. 1998), and in
zebrafish, oligodendrocytes that normally myelinate numer-
ous smaller caliber axons readily myelinate much larger cali-
ber supernumerary axons (Almeida et al. 2011). The existence
of transitional type II/III oligodendrocyte units containing
both small and large caliber axons clearly points to local con-
trol of myelinated fiber dimensions (Berry et al. 1995; Butt
et al. 1998a; Friede 1972).
4.2 I N T E R D E P E N D E N T D EV E L O PM E N T O F
O L I G O D E N D RO C Y T E -AXO N U N I T S
Oligodendrocyte phenotypic divergence and myelination
within units occurs in a series of distinct axoglial interde-
pendent phases (Fig. 6.4) (Butt and Berry 2000; Butt et al.
cGFP PLP/DM20 SMI31 20μm
10μm
Figure 6.4 Axon-Oligodendrocyte Relations in Developing Oligodendrocyte
Units In Vivo (A,B,E) and In Vitro (C,D). A,B. Confocal z-stacks of intact
anterior medullary vela from postnatal day 10 rat, double immunofluo-
rescence labeled with NG2 (green) for OPC and neurofilament for axons
(red). A. Two OPC lie serially along an axon that they both contact, with
the OPC on the left extending a process that on contact with the axon
bifurcates and this “initiator” process coils along the axon in a bidirectional
manner before ensheathment, whereas the OPC on the right forms multiple
contacts with the same axon via fine processes that extend short lamel-
lipodia on the axon. B. Single OPC forming multiple initiator processes
along numerous axons within its domain. C,D. Cocultures of neurons with
cyto-GFP labeled oligodendrocytes immunostained for PLP, illustrating
a z-stack (C) and serial single z-section analysis (D), showing the spiral-
ing cytoplasmic tongue process extending around the PLP+ myelin that
ensheathes a segment of the axon. E. Transitional oligodendrocyte in vivo
in the postnatal day 10 rat anterior medullary velum immunolabeled with
Rip, illustrating the cytoplasm-filled processes forming ensheathing cuffs
around the axon. (A,E) From Butt, Ibrahim, and Berry 1997; (C,D) from
Ioannidou et al., with permission. (B) Unpublished data from Butt, with
permission.
S T RU C T U R E A N D F U N C T I O N O F O L I G O D E N D R O C Y T E S
1997; Hardy and Friedrich 1996): (1) axonal contact and
recognition by OPCs; (2) induction phase, in which OPCs
extend initiator processes along receptive axons to form short
ensheathing segments, triggering the differentiation of OPCs
into a premyelinating oligodendrocyte phenotype and initia-
tion of axonal ion channel clustering; (3) remodeling phase,
in which oligodendrocyte-axon unit phenotype is established
by the loss of non-myelinating processes within the unit, and
radial and longitudinal growth of myelinating processes forms
incipient internodal segments, induces axon radial growth
and establishes nodes of Ranvier; and (4) maturation phase,
in which axons and myelin sheaths within units undergo
interdependent growth to establish adult dimensions of axon
diameter, myelin sheath g-ratios, and intermodal lengths.
Differentiation of oligodendrocyte phenotypes, and there-
fore the size of the myelin sheaths and speeds of conduction
of axons within the axoglial units, are driven by complex axo-
glial interactions that are only now being unraveled (Baron
and Hoekstra 2010) (see chapters 44 and 45). The develop-
mental onset of myelination involves a number of axoglial rec-
ognition and adhesion events that regulate the production of
myelin-related gene products and radial axon growth, thereby
governing phenotypic changes in oligodendrocyte-axon units
(see chapter 44). However, axon contact per se is not the
myelin inducting factor, because many OPCs that contact
axons do not differentiate into myelin-forming oligodendro-
cytes, and within individual OPCs and premyelinating oli-
godendrocyte units, many processes that contact axons do
not go on to form myelin (see Fig. 6.4A–C) (Bjartmar et al.
1994; Butt and Ransom 1993; Butt et al. 1997; Hardy and
Friedrich 1996). It is not known why some axons trigger dif-
ferentiation and myelination in certain OPCs, and the rules
of competition between processes within individual units are
unknown. All axons are submicron at the time of myelin ini-
tiation, and the mechanisms governing the divergent growth
of oligodendrocyte phenotypes and axons within the units are
mystifying. Furthermore, developmental myelination along
individual axons is multifocal and asynchronous (Butt et al.
1997; Remahl and Hildebrand 1990b; Skoff 1978), and it is
not known how axoglial recognition signals are spatiotempo-
rally regulated along the length of individual axons.
4.3 AXO N- O L I G O D E N D RO C Y T E
R EC O G N IT I O N S I G NA L S
The first step in myelination is a recognition event between
OPC processes and an axonal segment that is receptive for
myelination. There is a role for β1 integrin in the axoglial
interactions that initiates myelination and the loss of integ-
rin signaling leads to a delay in myelination of small-diameter
axons (Camara et al. 2009). Interactions between axonal
Caspr and oligodendroglial Nfasc155 are also indicated
in early myelination, axonal Caspr distributing as a helical
coil that winds around the axon to interact with Nfasc155
on the overlying myelinating process (Pedraza et al. 2009).
Three-dimensional imaging of whole oligodendrocyte units
in the anterior medullary velum and live cell imaging in
brain slices and in cultures have verified that the initiator
• 67
cytoplasmic processes run in a bidirectional coil along the
axon (Fig. 6.4A–D) (Butt et al. 1997; Ioannidou et al. 2012;
Sobottka et al. 2011). Subsequently, growth of the ensheath-
ing process around an individual axon does not occur in a uni-
form and synchronous manner, but there are focal expansions
of the corkscrew process to form short cytoplasm-filled cuffs
around the axon (Fig. 6.4C–E) (Butt et al. 1997; Ioannidou
et al. 2012; Remahl and Hildebrand 1990b). A current “liquid
croissant” model of myelination envisages concentric wrap-
ping of multiple membrane layers around axons (Sobottka et
al. 2011). The myelin sheath appears to wrap around the axon
in a concertina fashion and, as the number of wraps increases
and internodal myelin compaction expands to establish an
incipient internode, the spiraling cytoplasmic processes are
squeezed to the paranode and stack up at the developing node
of Ranvier (see Fig. 6.3C) (Berry et al. 1995; Hildebrand and
Waxman 1984; Pedraza et al. 2009; Remahl and Hildebrand
1990a,b). Within oligodendrocyte units, secondary pro-
cesses that do not form myelin are retracted during remod-
eling (Butt et al. 1997; Butt and Ransom 1993; Hardy and
Friedrich 1996; Remahl and Hildebrand 1990a). Myelinating
processes engage their axons through interactions involving
the oligodendroglial ligands integrin α6β1 and/or dystrogly-
can and their respective axonal receptors L1 and laminin-2
(Baron and Hoekstra 2010; Camara et al. 2009; Laursen et al.
2009). This is followed by growth of the axon and myelin
sheath, which studies in rodents and zebrafish clearly point
to being interdependent (Buckley et al. 2010; Colello et al.
1994). Myelin growth depends on laminin2-α6β1integrin
and neuregulin1-ErbB signaling between axons and oligo-
dendrocytes (Chun et al. 2003; Roy et al. 2007). L1 binding
to oligodendrocytes and interactions between oligodendro-
glial integrin and axonal contactin regulate myelin forma-
tion by controlling Fyn activity (Laursen et al. 2009; White
et al. 2008). Neurons stimulate the transport of PLP to the
plasma membrane (Trajkovic et al. 2006), and axonal elec-
trical activity stimulates localized synthesis of MBP through
Fyn kinase-dependent signaling (Wake et al. 2011) (see chap-
ter 45). Likewise, myelin proteins are essential for axon radial
growth. For example, when both OSP/claudin-11 and PLP
genes are knocked out, mice exhibit markedly smaller axon
diameters (Chow et al. 2005). In addition to controlling axon
growth, oligodendrocytes are also essential for axon integ-
rity (Edgar and Nave 2009). Mice developed widespread
axon degeneration in the absence of PLP-DM20 (Griffiths
et al. 1998b) or Cnp1 (Lappe-Siefke et al. 2003), and mice
deficient in both CNP and PLP display more severe axonal
degeneration (Edgar et al. 2009). A study in sulfatide-null
mice indicated an interesting function for this myelin lipid,
which played a limited role in myelin development, but was
essential for myelin maintenance and axonal integrity in aged
animals; in the absence of sulfatide displayed degenerating
myelin sheaths, deteriorating nodal/paranodal structure and
decreased axonal caliber (Marcus et al. 2006). Thus, oligoden-
drocytes and axons are entirely interdependent and should be
considered functional units rather than separate functional
entities per se.
68 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
4.4 O L I G O D E N D RO C Y T E -AXO N
I N T E R AC T I O NS A N D N O D E S O F R A N VI E R
The formation and maintenance of nodal structure are key
functions of oligodendrocytes. During development, solu-
ble factors secreted by oligodendrocytes promote the clus-
tering of Nav1.2 α subunits at immature nodes of Ranvier,
and myelin ensheathment induces the clustering of Nav1.6
α subunits, which are characteristic of mature nodes (Boiko
et al. 2001; Kaplan et al. 2001). Mature nodes of Ranvier
are further characterized by the formation of axoglial inter-
cellular “transverse bands,” which anchor oligodendroglial
cytoplasmic paranodal loops to the axon (Rasband and
Trimmer 2001; Rosenbluth 2009). The paranodal axoglial
junctions are formed by an adhesion complex between oli-
godendroglial Nfasc155 and the axonal proteins Caspr and
Contactin (Charles et al. 2002; Poliak et al. 2003; Rios
et al. 2003). Although not essential for the initial cluster-
ing of sodium channels, paranodal axoglial junctions are
essential for the maintenance of the nodal complex, and
mice lacking either Caspr or contactin have everted para-
nodal loops and absent transverse bands, resulting in dis-
placement of potassium and sodium channels (Poliak et al.
2003; Rios et al. 2003). The periodicity of nodes of Ranvier
along axons is directly and positively related to axon cali-
ber (Ibrahim et al. 1995). Internodal lengths are relatively
uniform within oligodendrocyte units, generally less than
50 μm in cortical type I oligodendrocytes, 100 to 200 μm
in type II units, and 400 to 500 μm in type III units (Butt
et al. 1998a; Murtie et al. 2007), and nodal periodicity is
established developmentally before myelin compaction
(Butt et al. 1997; Butt and Ransom 1993). This raises the
question of how the regular spacing of oligodendrocytes
along axons is determined. As axons grow in length, unmy-
elinated gaps are myelinated by late developing oligoden-
drocytes, and this continues well into adulthood (Butt
et al. 1997; Murtie et al. 2007; Peters and Sethares 2004;
Rivers et al. 2008), which might explain the extremely short
internodes observed in oligodendrocytes of late myelinated
areas such as the cortex.
4.5 AG E -R E L AT E D C H A N G E S I N
O L I G O D E N D RO C Y T E S
Electron microscopy studies have provided evidence of
age-related degenerative changes in myelin, such as split-
ting of sheaths, and blebbing or ballooning of sheaths, and
both short internodes and thin sheaths can be found, as
occurs in remyelination (Peters 2002; Peters et al. 2008).
The conclusion is that with age some internodal lengths
of myelin sheaths degenerate and are replaced by newer
and shorter internodal lengths by newly generated oligo-
dendrocytes, which originate from endogenous OPCs and
OPCs derived from the subventricular zone (Peters and
Sethares 2004; Rivers et al. 2008). Age-related changes
in paranodal structure have also been noted, and affect
axonal protein localization at nodes of Ranvier, including
Kv1.2 and Caspr, resulting in changes in axonal conduction
(Hinman et al. 2006). The advent of magnetic resonance
imaging (MRI) provides a noninvasive method for examin-
ing age-related changes in myelin, and these studies suggest
that metabolically vulnerable late-differentiating oligoden-
drocytes may contribute to the cognitive deficits observed
in normal aging and dementias through disruption of rapid
transmission and the loss of synchronization of higher cog-
nitive functions (Bartzokis et al. 2004). Oligodendrocyte
and myelin dysfunction are also implicated as primary
changes in schizophrenia, resulting from disruption of syn-
aptic formation and function (Takasaki et al. 2010) (see
chapter 71). Neuregulin 1 and its receptor erbB4 are genet-
ically linked with susceptibility to schizophrenia and bipo-
lar disorder, and the loss of erbB signaling leads to reduced
myelin thickness and slower conduction velocity in CNS
axons, as well as alterations in dopaminergic function
and behavior relevant to neuropsychiatric disorders (Roy
et al. 2007). Ultrastructural analysis has confirmed a 20%
decrease in the number of myelinated nerve fibers of cere-
bral white matter fiber tracts associated with frontal lobe
areas critical in cognitive processing in the normal aging
brain (Bowley et al. 2010). Thus, normal aging, cognitive
decline, and several psychiatric disorders are associated
with white matter defects, suggesting that oligodendrocyte
abnormalities underlie some aspects of these diseases (see
chapter 71).
5 N O N M Y E L I N AT I N G
OLIGODENDROCYTES
5.1 A D U LT O L I G O D E N D RO C Y T E
P RO G E N I TO R C E L L S
Adult oligodendrocyte progenitor cells (OPC) are identified
by their expression of the platelet-derived growth factor recep-
tor alpha (PDGFRαα) and the NG2 chondroitin sulfate pro-
teoglycan (Levine and Card 1987; Levine and Stallcup 1987;
Stallcup 1981; Stallcup and Beasley 1987) (see chapters 10 and
21 for further details). The sheer number of OPC throughout
adult gray and white matter (Fig. 6.5A) has led many to refer
to them as a distinct fourth glial cell type, called NG2-glia
or polydendrocytes (Butt et al. 2005; Nishiyama et al. 2009;
Peters 2004) (see chapter 10). There has been some debate
about whether all NG2-glia in the adult brain are OPCs (Butt
et al. 2002; Richardson et al. 2011), but fate-mapping studies
in transgenic mice show that NG2-glia have an oligodendro-
glial lineage and provide a pool of slowly proliferating cells that
generate oligodendrocytes throughout life (Kang et al. 2010;
Rivers et al. 2008). In gray matter, the cell bodies of NG2-glia
are often applied directly to neuronal somata (Fig. 6.5B), like
perineuronal oligodendrocytes (Fig. 6.5F), but the two cell
types are readily distinguished by their differential expression
of NG2 (Takasaki et al. 2010). Ultrastructurally, NG2-glia have
elongate nuclei and a thin rim of pale organelle-poor cytoplasm
containing some short rough endoplasmic reticulum, small
mitochondria, and few free polyribosomes (Fig. 6.5E) (Butt
S T RU C T U R E A N D F U N C T I O N O F O L I G O D E N D R O C Y T E S
et al. 1999; Levine and Card 1987; Peters 2004; Peters and
Sethares 2004). Electron microscopy and physiological stud-
ies show NG2-glia form functional synapses with neurons and
axons and respond to glutamate and GABA released at synapses
(Fig. 6.5B–E) (Bergles et al. 2000; Butt et al. 1999; Haberlandt
et al. 2011; Hamilton et al. 2010; Kukley et al. 2007; Velez-Fort
et al. 2010) (see chapter 21). There is no direct evidence of a
function for NG2-glia at synapses, but it is presumed that syn-
aptic signaling plays a role in sustaining the adult population
or regulating their differentiation into oligodendrocytes (De
Biase et al. 2010; Kukley et al. 2010; Velez-Fort et al. 2010). The
density and distribution of NG2-glia in adult gray matter areas
such as the hippocampus and cerebral cortex highlight their
importance in cognitive function by maintaining oligodendro-
cytes and myelination throughout life (Bartzokis et al. 2004;
Peters et al. 2008). There is increased frequency of degenerative
changes in myelin in the normal aging primate cortex, and this
is partly offset by a 50% increase in the oligodendrocyte num-
ber between young (4–10 years of age) and old (25–35 years
of age) monkeys (Peters and Sethares 2004). Additional oli-
godendrocytes are generated from NG2-glia and are required
to remyelinate nerve fibers whose sheaths have broken down,
which leads to cognitive decline (Peters and Sethares 2004;
Peters et al. 2008). In human brains, myelination extends to age
50 in cortical regions, and MRI studies suggest myelin break-
down accelerates as aging progresses and underlies age-related
cognitive decline (Bartzokis et al. 2004). It is clear the gen-
eration of oligodendrocytes in adult gray and white matter
necessitates a major population of OPC and their diminished
capacity for regeneration in the aging brain is likely to be criti-
cal in cognitive decline.
5.2 P E R I N EU RO NA L O L I G O D E N D RO C Y T E S
Perineuronal oligodendrocytes are apposed directly to neu-
ronal cell bodies in various gray matter regions (Fig. 6.5F)
(Szuchet et al. 2011; Takasaki et al. 2010). Perineuronal oli-
godendrocytes are the progeny of A2B5+/O4+ OPC, but
have an antigenic phenotype distinct from both OPC and
myelin-forming oligodendrocytes, expressing CNPase and
A2B5, but not expressing NG2 and having low expression of
Sox10, and using PDGFRαβ in contrast with PDGFRαα in
OPCs (Fig. 6.5Fi) (Szuchet et al. 2011; Takasaki et al. 2010).
Ultrastructurally, perineuronal oligodendrocytes are oval or
polygonal cells, with a round nucleus rich in dark hetero-
chromatin, forming a concave impression on the neuron to
which they are tightly attached, with parallel alignment of
two plasma membranes and a narrow intercellular space (Fig.
6.5Fii) (Takasaki et al. 2010). Like astrocytes, perineuronal
oligodendrocytes are immunopositive for glutamine syn-
thase, but they do not express glial fibrillary acidic protein,
or the glutamate transporters GLAST and GLT-1 (Takasaki
et al. 2010). The functions of perineuronal oligodendrocytes
are unclear, but they appear to fulfill a neuron metabolic sup-
port function (Takasaki et al. 2010) and protect against neu-
ronal apoptosis (Taniike et al. 2002). A recent study provided
• 69
Figure 6.5 NG2-Glia and Perineuronal Oligodendrocytes. A. Immunoperoxidase NG2 labeling illustrating the dense distribution of NG2-glia in gray
matter of the adult rat cerebral cortex. B. Immunolabeling for NG2 (red) and calbindin (blue) in the cerebral cortex of GFAP-GFP mice, illustrated
in z-stack (Bi) and single z-section (Bii), showing astrocytes and NG2-glia forming overlapping networks over the neuronal somata. C. Serial electron
micrographs illustrating the processes of an NG2-glial cell (black, peroxidase reaction) receiving a synapse (arrow) from a bouton (b) that also gives a
synapse to a dendritic spine(s) in rat hippocampus. D. Confocal microscopic reconstructions in whole-mounts of rat anterior medullary vela triple
immunolabeled for NG2 (green), MBP (blue), and ankyrin G (red), an axoskeletal protein that define nodes of Ranvier, illustrating NG2-glial cell
contacting multiple nodes (Di) and three-dimensional isoformed reconstruction of a cell process that terminates at a node of Ranvier and partly
ensheathes the axon (Dii). E. Pre-embedding EM immunocytochemistry shows the surface labeling of the NG2 membrane-embedded chondroitin
sulfate proteoglycan in a cell that sends a process (filled arrows) that wraps around the axolemma at a nearby node of Ranvier (asterisk), defined by the
paranodal loops of the oligodendroglial myelin sheath (open arrows). F. Perineuronal oligodendrocytes in the adult rat cerebral cortex, identified by
immunolabeling (Fi) and EM (Fii). Fi. CNP+ perineuronal oligodendrocyte (green, arrowhead) attached to a MAP2-positive neuron (blue, asterisk).
Fii. Electron micrograph of a perineuronal oligodendrocyte (PO) tightly contacting the soma of a neuron (Neu). (Bi,Bii) From Wigley and Butt
2009. (Ci–Ciii) from Bergles et al. 2000; (Di, Dii) from Butt et al. 2005; (E) from Butt et al. 1999; (Fi, Fii) from Takasaki et al. 2010, with permission.

evidence for a deficit of perineuronal oligodendrocytes in
the prefrontal cortex in schizophrenia, bipolar disorder, and
major depression, suggesting they may play a key role in the
pathophysiology of these severe mental disorders (Vostrikov
et al. 2007).
6 S U M M A RY A N D P E R S P E C T I VE S
Oligodendrocyte phenotypic differences determine the
conduction properties of axons within oligodendrocyte
units and susceptibility to damage. Oligodendrocyte phe-
notype divergence is regulated by axon-derived factors,
and developmentally the growth of axons and myelin
sheaths are interdependent. In recent years, major advances
have been made in the study of adult OPCs and perineu-
ronal oligodendrocytes, but knowledge of their functions
remains scanty. It is not clear why the CNS requires two
significant populations of cells that are capable of regen-
erating myelin-forming oligodendrocytes throughout life,
but their sheer number reflects the profound importance
of maintaining oligodendrocytes and makes them worthy
of greater study. A wide array of new markers and genetic
models will enable us to unravel the mechanisms of axo-
glial interactions that control the interdependent growth
of myelin and axons within units, and how myelination is
essential for axon integrity and axoglial specialization at
nodes of Ranvier. In addition, modern imaging techniques
will greatly facilitate our understanding of the significance
of oligodendrocytes and their progenitors in the aging
brain and pathological conditions. These new techniques
will enable us to begin to understand the profound impor-
tance of oligodendrocytes and the devastating effects of
their loss on integrative brain function, as occurs in MS,
cerebral palsy, leukodystrophies, psychological diseases,
and cognitive decline in the aging brain.

70 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
 AC K N OW L E D G M E N T S
The author would like to thank the funding bodies who have
supported this work, and all coworkers past and present, with
special thanks to Bruce Ransom and Martin Berry.
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 7.
SCHWANN CELLS AND MYELIN
Rudolf Martini and Ágnes Patzkó

A B B R E VI AT I O N S addition, progress in live imaging technologies, combined

with immunocytochemical and transgenic labeling approaches
CAM cell adhesion molecule extended our knowledge of the spatio-temporal dynamics
cAMP cyclic adenosine monophosphate of these cells and their related derivatives, such as the termi-
Caspr contactin-associated protein nal Schwann cells at the neuromuscular junction (Brill et al.
CNP-1 2′,3′-cyclic nucleotide 3′-phosphodiesterase 2011). However, even the more “traditional” approaches, such
isoform 1 as transmission electron microscopy, have been essential for
CNS central nervous system understanding basic mechanisms; a representative example
Cx32 connexin 32 is the systematic analysis of the position and putative roles of
DRP2 dystroglycan-dystrophin-related protein 2 Schwann cell precursors during nerve growth (Wanner et al.
ECM extracellular matrix 2006b). Moreover, the roles of the nonmyelinating Schwann
EGR-2 early growth response protein 2 cells, also called Remak Schwann cells, the usually less consid-
FAK focal adhesion kinase ered Schwann cell population, have also been studied in detail
GABA gamma-aminobutyric acid (Griffin and Thompson 2008).
GAP-43 growth associated protein 43 For many years the nodes of Ranvier and particularly the
Gpr126 G protein–coupled receptor 126 role of Schwann cells in directing the relevant ion channels
MAG myelin-associated glycoprotein have been in the focus of interest; depending on the techno-
MBP myelin basic protein logical approaches applied, these studies led to various, some-
MPZ myelin protein zero times even controversial models describing how axons and the
N-Cadherin neural cadherin glia synergistically interact to warrant the function of nerve
N-CAM neural cell adhesion molecule fibers (Feinberg et al. 2010; Thaxton et al. 2011).
Necl nectin-like The present chapter focuses on the major structural
NGF nerve growth factor characteristics of Schwann cells (Fig. 7.1). Although dis-
NF neurofascin cussed more in detail in other chapters, molecular aspects
Nr-CAM neuron glial-related cell adhesion are also considered, especially where they help to under-
molecule stand the cytoarchitecture and function of these fascinat-
Nrg1 neuregulin 1 isoform ing cells.
N-WASP Neural-Wiskott-Aldrich syndrome
protein
Omgp oligodendrocyte myelin glycoprotein 2 E A R LY D E VE L O PM E N T O F
P0 myelin protein zero S C H WA N N C E L L S
PDZ postsynaptic density protein—disk large—
zonula occludens protein
2.1 S C H WA N N C E L L P R EC U R S O R S
PLP proteolipid protein
PMP22 peripheral myelin protein 22 Being derivatives of the neural crest ( Jessen and Mirsky 2005),
PNS peripheral nervous system embryonic Schwann cells are associated with axon bundles
SCP Schwann cell precursor during development (Billings-Gagliardi et al. 1974). These
TAG-1 transient axonal glycoprotein-1 cells are Schwann cell precursors (i.e., prospective Schwann
cells that still lack the ability to survive in the absence of axons)
( Jessen and Mirsky 2005). They show immunoreactivity for
1 INTRODUCTION the low-affinity NGF receptor p75, and the cell adhesion mol-
ecules L1 and N-CAM (Dong et al. 1999; Martini 1994), but
Recent years have brought a dramatic increase in our knowl- the glial marker S100 is not detectable unless the sensitivity of
edge about the molecular components of Schwann cells in conventional immunohistochemical techniques is improved
development and disease (Berti et al. 2011; Jessen and Mirsky ( Jessen and Mirsky 2005). They also express low levels of the
2005; Salzer et al. 2008; Sherman and Brophy 2005). In myelin components P0, PMP22, and PLP ( Jessen and Mirsky

74

A
B C
Figure 7.1 Two principal forms of peripheral nerve fibers: a myelinated
nerve fiber (A) and two Remak fibers associated with nonmyelinating
Schwann cells (B, C). Saphenus nerve of an adult mouse. The
myelinating Schwann cell is surrounded by a basement membrane
(arrows). Note that the inner Schwann cell loop contains cytoplasm
(arrowheads). The “outer” cytoplasmic domains (asterisks) appear as
“Cajal bands” in longitudinal views (compare with Fig. 7.4). Double
arrowheads indicate the outer mesaxon on the right hand site. Bar =
0.5 μm. (B) Note that most, but not all small-caliber axons are separated
from each other by slender processes of the nonmyelinating Schwann cell.
Bar = 1 μm. (C) This micrograph probably displays the border region of
two adjacent nonmyelinating Schwann cells (SC 1, SC 2). Bar = 0.5 μm.
2005). Schwann cell precursors are not only the likely source
for mature Schwann cells but also pivotal cellular components
for axon survival ( Jessen and Mirsky 2005; Riethmacher et al.
1997).
Because of the position of HNK-1-positive cells “ahead”
of growing axons, it was suggested that Schwann cell precur-
sors may guide axons to their targets during development
(Noakes and Bennett 1987). Indeed, recent studies in rats
using electron microscopy, confocal three-dimensional (3D)
reconstructions, and immunohistochemistry against p75
neurotrophin receptor and N-cadherin, identified Schwann
cell precursors as components of the growing nerve front,
but not stringently ahead of the growth cones (Wanner et al.
2006a,b). There, growth cones make preferential contacts
with precursors rather than with extracellular space (Fig. 7.2)
(Wanner et al. 2006b). In this position the precursor cells
show lamellipodia and filopodia-like, slim processes, called
gliopodia (Wanner et al. 2006b). These combined observa-
tions suggest that the Schwann cell precursors serve as cellular
chaperons (Wanner et al. 2006b) leading to nerve compac-
tion and may facilitate navigation when axons approach
S C H WA N N C E L L S A N D M Y E L I N
A B
Figure 7.2 Electron Micrographs of Schwann Cell Precursors
(SCP). A. Cross-section of an embryonic nerve displaying axons and
growth cones (gc) in association with the SCPs. Schwann cell processes
(asterisks), gliopodia (arrows), and adherens junctions (aj; white arrows)
are indicated. ECS: extracellular space devoid of axons. Bar = 1 μm.
B. Longitudinal section of a growth cone (gc) with actin filaments (a) and
neurofilaments (nf ) at its proximal region. Note tight association with
SCPs. Bar = 1 μm. Reprinted from Wanner et al. 2006, with permission
from Wiley.
their peripheral targets. The view that glial precursor cells
may at least “shape” the nerve is supported by an elegant
report on the developing lateral organ of the zebra fish in
which genetic ablation of these cells lead to defasciculation
(Gilmour et al. 2002). Fasciculation may be mediated by
the polarized secretion of the repulsive extracellular matrix
molecule tenascin-C that has been found previously to sur-
round the abaxonal surfaces of the “mantling” (Wanner et al.
2006b) precursor cells (Martini and Schachner 1991; Wehrle
and Chiquet 1990).
Based on electron microscopic data (Martini 1994;
Wanner et al. 2006b), the most important morphological fea-
ture of Schwann cell precursors is the absence of a basal lamina
(Fig. 7.2). They intermingle with fasciculating axons proximal
to the growing nerve tip and extend processes in between of
the axon bundles (Fig. 7.3). In addition, these cells are found at
the margin of the prospective nerve or nerve fascicle and face
the mesenchyme with their abaxonal surface (see Fig. 7.2).
2.2 I M M AT U R E S C H WA N N C E L L S
When development proceeds, Schwann cell precursors start
to form a basal lamina, proliferate, and collectively ensheathe
fasciculating axons forming so called “Schwann cell families”
as initially described by Webster and colleagues (see Fig. 7.3)
(Peters et al. 1991). The respective glial cells reached the stage
of “immature Schwann cells,” a term that characterizes the
ability of the Schwann cells to survive independently from
axons resulting from an autocrine survival mechanism ( Jessen
and Mirsky 2005).
At the same time when the immature Schwann cells acquire
their basal lamina, additional connective tissue elements and
• 75
A B C 3.1 BA S A L L A M I NA F O R M AT I O N
D Laminin 211, 411
1,5 4,6
Integrin αβ1 dystroglycan
3 2
NrgTypell Akt1 ILK, Rac1 p38 MAPK
radial axonal sorting
αβ1 integrins myelination
dystroglycan
Figure 7.3 Radial Sorting (A–C; 4-Day-Old Mouse) and the Underlying
Molecular Mechanisms (D). A–C. Immature Schwann cells associated
with a basement membrane are in contact with smaller caliber axons
(asterisks) and with larger axons (double asterisks) in the process of sorting.
Note that some axons have already reached the promyelinating stage (PM)
or are in the process of myelination (M). Bar = 1 μm. D. Morphogenetic
steps comprising the formation of Schwann cell families (1), Schwann
cell process formation (2, requiring αβ1 integrins), and radial sorting to
promyelinating stages (steps 4–6, requiring dystroglycan) are shown. The
presumed involvement of signaling pathways is also indicated (see also
text). Reprinted from Berti et al. 2011, with permission from the Society
for Neuroscience.
blood vessels appear to be generated and intermingle between
the prospective nerve fibers ( Jessen and Mirsky 2005). In this
context, it is worthwhile to consider recent views on the ori-
gin of endoneurial fibroblasts. Although there are some argu-
ments favoring the idea that endoneurial fibroblasts might be
derivatives of neural crest cells or even Schwann cell precur-
sors ( Jessen and Mirsky 2005; Joseph et al. 2004), there are
also strong arguments supporting the more traditional idea,
namely the mesodermal origin of fibroblasts (Mäurer et al.
2003).
3 E S TA B L I S H I N G T H E M AT U R E S TAG E
Immature Schwann cells develop into two directions. Those
Schwann cells associated with larger caliber axons (>1 μm)
eventually differentiate into the myelinating phenotype (see
section 3.3). The Schwann cells remaining associated with
small caliber axons collectively ensheathe them. This bidirec-
tional development is mediated by Neuregulin-1 (Nrg1) type
III (see chapter 44).
The multistep process by which Schwann cells segregate
axons is called radial sorting (Fig. 7.3D). Typically, prospec-
tive myelinated axons of larger caliber expressing higher
levels of Nrg1 are “moved” to the periphery of axon bun-
dles and become associated with a prospective myelinating
Schwann cell, generated by mitosis of immature Schwann
cells.
76 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
AND FUNCTION
An important prerequisite for radial sorting and the follow-
ing processes is the generation of a basal lamina around the
Schwann cell. Thus, the Schwann cell gains polarity (Ozcelik et
al. 2010). In longitudinal sections, the Schwann cell basal lam-
ina forms a continuous tube that even bridges the nodal gaps
(Fig. 7.5). The major Schwann cell basal lamina constituents
are the extracellular matrix components laminin-2 (merosin),
heparan sulfate proteoglycans (e.g., perlecan, agrin), fibronec-
tin (Chernousov and Carey 2000), collagen type IV, and the
recently discovered type XXVIII (Grimal et al. 2010). For the
first time the fundamental role of basal lamina during myeli-
nation was presented by the Bunge laboratory (Eldridge et al.
1989). An additional hint reflecting the importance of basal
lamina is provided by the dysmyelinating phenotype of dys-
trophic mice, which are deficient in laminin-2 (Matsumura
et al. 1997).
Laminin-2 is not only crucial in radial sorting and devel-
opment of myelinating Schwann cells, but also participates in
nonmyelinating Schwann cell development and Remak bun-
dle formation (Yu et al. 2009).
Many studies confirm the importance of cell substrate sig-
naling mediated by laminin-2 and the corresponding receptors
of the abaxonal Schwann cell membrane, including integrin
heterodimers such as α6/β1 and α6/β4, dystroglycan and
dystroglycan-dystrophin-related protein 2 (DRP2, Fig. 7.3D).
The latter forms a complex with αβ-dystroglycan (Sherman
and Brophy 2005). Both dystroglycan and integrins play an
essential and distinct role in radial sorting. Deletion of dystro-
glycan in Schwann cells revealed an arrest in radial sorting that
was most evident in spinal roots (Berti et al. 2011).
Schwann cell–specific gene inactivation of β1-integrin
leads to a severe dysmyelinating phenotype with impaired
axonal sorting (Feltri et al. 2002). β4-integrin appears to be
redundant during myelination, because absence of this com-
ponent does not interfere with initial myelin formation in
vivo or in vitro (Frei et al. 1999). Nevertheless, in adult nerves,
α6/β4 integrin-null myelin is more prone to abnormal fold-
ing with aging. When the laminin receptor dystroglycan is
also ablated, major folding abnormalities occur, associated
with acute demyelination and macrophage activation. These
data indicate that α6/β4 integrin confers stability to myelin
in peripheral nerves (Nodari et al. 2008). Another molecule
that has lately been linked to α6β1 integrin and ErbB recep-
tor signaling is focal adhesion kinase (FAK). In case of con-
ditional FAK inactivation, Schwann cells could interdigitate
among axon bundles, but radial sorting and Schwann cell
proliferation were impaired leaving large unsorted bundles
(Grove et al. 2007). Growing evidence indicates that collagens
perform important functions during the development of the
peripheral nervous system. Collagen XXVIII is almost exclu-
sively expressed in the peripheral nervous system and is local-
ized at the basal lamina associated with unmyelinated fibers.
It is worth noting that collagen XXVIII is also present at the
nodes of Ranvier and in terminal Schwann cells in sensory end
organs (Grimal et al. 2010) (i.e., in subdomains of myelinated
fibers lacking true myelin). All in all, the Schwann cell basal
lamina components and their cognate Schwann cell–related
receptors are crucial elements for maturation.
3.2 N O N MY E L I NAT I N G S C H WA N N C E L L S
Nonmyelinating Schwann cells typically ensheathe axons
smaller than 1 μm. Nociceptors, postganglionic sympathetic,
and certain preganglionic sympathetic and parasympathetic
fibers represent this entity (Griffin and Thompson 2008).
These axons are separated from each other by slender Schwann
cell processes (see Fig. 7.1 B, C) being embedded in pocket-like
structures. Of note, one single nonmyelinating Schwann
cell can “home” thin-caliber axons of distinct modalities, as
for instance, sensory and sympathetic axons (Griffin and
Thompson 2008). In the region of the Schwann cell nucleus,
cross-cut axons are found at the periphery of the Schwann cell
soma and show a rosette-like organization around the nucleus.
In this position, the axons are often only partially covered by
Schwann cell processes and are in direct apposition with the
Schwann cell basal lamina. Mature nonmyelinating Schwann
cells share a couple of cell surface molecules with immature
Schwann cells that are not found on myelinating Schwann
cells, such as the cell adhesion molecules L1, N-CAM, and the
NGF-receptor p75 ( Jessen and Mirsky 2005; Martini 1994).
Although nonmyelinating Schwann cells are morphologi-
cally and molecularly distinct from myelinating Schwann cells
(see Fig. 7.1), they can adopt a myelinating phenotype under
experimental conditions (Weinberg and Spencer 1975).
The biological function of nonmyelinating Schwann cells
may be best understood when compared with molecular fea-
tures of myelinating Schwann cells (Griffin and Thompson
2008). Nonmyelinating Schwann cells lack inhibitory com-
ponents, such as MAG and Omgp, which are constituents of
myelin sheaths and nodes of Ranvier, respectively (Griffin and
Thompson 2008). Because nonmyelinating Schwann cells are
associated with the highly competent and important warning
system against noxious insults, the nociceptive fibers, it might
be of advantage to be embedded in an environment that allows
rapid sprouting on injury, thus preventing that extended areas
remain irresponsive to pain after injury (Griffin and Thompson
2008).
3.3 MY E L I NAT I N G S C H WA N N C E L L S
AND THE INTERNODE
As a result of radial sorting and Schwann cell prolifera-
tion, large-caliber axons achieve a so called 1:1 ratio with
immature Schwann cells (see Fig. 7.3). Schwann cells of this
stage are called “promyelinating.” In rodents, the genera-
tion of promyelinating Schwann cells occurs around birth.
However, myelin formation in the PNS is not a highly syn-
chronized event. Axons with large calibers receive their
myelin sheath earlier than smaller axons so that some pro-
myelin stages can still be found in mice of approximately 3
weeks of age.
Around the promyelinating stage, many cell surface mol-
ecules related to immature or nonmyelinating Schwann cells
S C H WA N N C E L L S A N D M Y E L I N
are downregulated, whereas typical myelin components, such
as P0, MAG, PMP22, MBP, Cx32, and periaxin are strongly
upregulated (Martini 1994). Important regulators of such mol-
ecules are krox20, which promotes and maintains myelination
(Svaren and Meijer 2008) and c-Jun that is upregulated upon
dedifferentiation ( Jessen and Mirsky 2008). Cross-inhibition
of these transcription factors serves to switch Schwann cells
between the mature and dedifferentiated phenotype (Salzer
et al. 2008).
The pro-myelin stage is followed by spiral formation of
one of the Schwann cell processes engulfing the axon (see
Fig. 7.3C). The apposition of Schwann cell membranes at the
inner, axon-related site of the Schwann cell process is called
“inner mesaxon,” whereas the contact of Schwann cell mem-
branes at the endoneurial site is termed “outer mesaxon” (see
Fig. 7.1A). These cell contacts are characterized by the forma-
tion of E-cadherin-positive adherens (desmosome-like) junc-
tions (Fannon et al. 1995) and tight junctions (Scherer and
Arroyo 2002).
Pioneering work by the Bunge-laboratory identified in
Schwann cell–dorsal root ganglion cocultures that the inner
lip of the Schwann cell is turning around the axon (Bunge et
al. 1989). However, in vivo studies revealed the myelinating
process to be more complicated than just a spiral formation of
the inner Schwann cell process around the axon (Peters et al.
1991).
In myelinating Schwann cells, specialized cytoplasmic
structures are “Bands of Cajal” which are channel-like domains
“on top” of the outer aspects of the myelin sheathes that
alternate with DRP2/periaxin rich “patches” or plaques of
Schwann cell membrane apposition with basal lamina without
underlying cytoplasm (Fig. 7.4) (Court et al. 2009; Sherman
and Brophy 2005). Most importantly, both Cajal bands and
the alternating patches are lost in mice deficient in periaxin,
which is a central component of dystroglycan complexes, that
are important receptors for the basement membrane com-
ponent laminin-2 (see the preceding). The disruption of this
component results in the dysregulation of internodal length,
which might be related to a disruption of microtubule based
transport within the cytoplasmic Schwann cell channels
(Sherman and Brophy 2005). Taking into account that choles-
terol is essential for correct myelinogenesis (Saher et al. 2011),
the Cajal bands could have important functions in addition to
those mentioned, because they harbor the cholesterol trans-
porter ABCA1 (Albrecht et al. 2008).
Which other molecules are involved in the process of mye-
lin formation? mTor, a core kinase, that regulates cell growth
and differentiation in various mammalian cells, proved to be
essential for myelin formation following the promyelinating
stage, but also mediates axon caliber adjustment and determi-
nation of internodal length (Sherman et al. 2012). Radial sort-
ing, however, is not mediated by this kinase (Sherman et al.
2012). Among the recently identified myelin-related proteins
G protein–coupled receptor Gpr126 emerged as an important
regulator as well. In gpr126 mutants, Schwann cells failed to
express oct6 and krox20 and were arrested at the promyeli-
nating stage. Elevation of cAMP in gpr126 mutants, but not
krox20 mutants, could restore myelination (Monk et al. 2011).
• 77
 A them mediates Schwann cell adhesion and the disruption of
B spiral (see Figs. 7.1A and 7.3). In rodent Schwann cells this
Figure 7.4 A. Bands of Cajal and transverse trabeculae (A, Phalloidin
in green) and complementary expression of DRP2 (red). B. Electron
micrograph with artificially red-colored domains of presumed DRP-2
deposition. Cytoplasmic compartments between the red-colored areas
represent Cajal bands (compare with Fig. 7.1A). Reprinted from Court
et al. 2009.
Another important mediator of the initiation of myelination
is Dicer, a regulatory protein that is responsible for the gen-
eration of micro-RNAs and subsequently for gene expression
regulation. It is abundantly expressed at the promyelinating
stage, when some micro-RNAs are substantially upregulated,
others downregulated, reflecting a complex regulatory sce-
nario at this stage (Gokey et al., 2012). Schwann cell–specific
inactivation or reduction of Dicer resulted in impaired krox20
expression and the reduction of myelin proteins, arrest-
ing of most fibers at the promyelinating stage (Bremer et al.
2010; Dugas and Notterpek 2011; Pereira et al. 2010; Verrier
et al. 2010). The transcription factor nuclear factor-kappa
B—which is also required for myelin formation—is activated
through cAMP-dependent protein kinase A that phosphory-
lates the p65 subunit of nuclear factor–kappa B (Yoon et al.
2008). Similarly, Neural Wiskott-Aldrich syndrome protein
(N-WASP) appears to be necessary for the transition from the
promyelinating to the myelinating stage (Novak et al. 2011).
Many studies identified several molecules that play an
important role in Schwann cell–axon interactions during
myelin formation. Both in vitro and in vivo evidence sug-
gests that β-catenin–N-cadherin interaction contributes to
establishing Schwann cell polarity and the timely initiation
of axon-induced Schwann cell proliferation (Gess et al. 2008;
Lewallen et al. 2011). Additional molecules that have been
shown to mediate Schwann cell–axon interaction are cell
adhesion molecules (CAMs) of the nectin-like (Necl) fam-
ily. Necl4 is the main Necl expressed by myelinating Schwann
cells and is located along the internodes in direct apposition
to Necl1, which is localized on axons. The interaction between
78 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
the interaction by their soluble extracellular domains, or the
expression of a dominant-negative Necl4 in Schwann cells,
inhibits myelination (Spiegel et al. 2007).
A striking subcellular feature of myelinating glial cells is
the compaction of the apposing membranes of the myelin
starts after completion of several turns of the Schwann cell
process around the axon. Two morphogenetic events occur
nearly simultaneously, namely (1) the narrowing of the spiral-
ing cell surface membranes from approximately 12 to 2 nm,
and (2) the loss of cytoplasm. The collapsed cytoplasmic sites
of the Schwann cell membranes fuse and form the 3.5-nm
“major dense line”; the membrane leaflets facing the extracel-
lular space of the spiral form the “intraperiod line,” which is
double-stranded because of the 2-nm wide gap separating the
extracellular leaflets (Fig. 7.1B) (Peters et al. 1991).
A pivotal molecular mediator for myelin compaction in
the PNS is the cell adhesion molecule P0, also designated
myelin protein zero (MPZ). It mediates homophilic adhesion
leading to the close apposition of the extracellular aspects of
the spiraling Schwann cell membrane forming the intraperiod
line (reviewed in Martini and Schachner 1997). This model
received strong support by the determination of the 3D struc-
ture of the extracellular domain of P0 by X-ray crystallography
(Shapiro et al. 1996). The intracellular domain of P0 contains
predominantly basic residues that have been suggested to
interact with negatively charged phospholipids of the adjacent
cytoplasmic parts of the Schwann cell membrane leading to the
formation of the major dense line (Ding and Brunden 1994;
Kirschner and Ganser 1980; Lemke et al. 1988). Final proof
that P0 mediates myelin development and compaction was
provided by mice deficient in this molecule that show severe
dysmyelination with disturbed myelin compaction (Giese et
al. 1992), with MBP as a partial compensator for major dense
line formation (Martini et al. 1995a).
During growth of the myelin sheath, the axon plays a piv-
otal role in the initiation of myelination and the regulation
of myelin thickness (see chapter 44). Additionally, a constant
ratio exists between axon diameter and the outer aspect of the
myelin sheath (G-ratio) and the axon diameter and internodal
length in the adult nerve. Quantitative studies in various species
revealed that a normal internode of a mature peripheral nerve
is usually 100 times as long as the diameter of the correspond-
ing axon (Friede and Beuche 1985). However, developmental
studies in nerves with a robust growth in length during matura-
tion in the absence of Schwann cell proliferation modified this
simple view (Friede and Beuche 1985; Schröder et al. 1988).
4 S P E C I A L M Y E L I N C O M PA RT M E N T S
4.1 N O D E O F R A N VI E R , PA R A N O D E S ,
A N D JUX TA-PA R A N O D E S A N D T H E I R
M O L ECU L A R CO M P O N E N TS
The molecular and cytological architecture of the node of
Ranvier has attracted the attention of neurobiologists for
decades. It became clear that this structure, together with its
neighboring compartments, is a highly organized compart-
ment that warrants the clustering of ion channels and the
rapid transmission of action potentials and may seal impor-
tant functional compartments against pathogens (Rosenbluth
2009).
4.1.1 The Node of Ranvier Proper
The nodal compartment consists not only of the exposed axo-
lemma, but also of multiple slender Schwann cell protrusions
abutting the axolemma, called microvilli (Figs. 7.5A,B, 7.6A).
They “dive” into a nodal gap substance (Landon and Williams
1963) composed of tenascin-C, syndecan-3, collagen V, col-
lagen XXVIII, and versican V1 (Grimal et al. 2010; Martini
et al. 1990; Melendez-Vasquez et al. 2005). Possibly, some of
these ECM substances may have nerve growth–repulsive char-
acteristics. The CNS myelin component Omgp has been iden-
tified as possible inhibitor of axon “out”-growth at the PNS
node (Huang et al. 2005).
A pivotal task of Schwann cell components at the node of
Ranvier is their contribution to the clustering of voltage-gated
A Moreover paranodal domains may even invade the putative
B C
Figure 7.5 Nodes of Ranvier, Paranodes, and Juxtaparanodes in the Adult
Mouse. A. Longitudinal section of a node (arrow), flanked by paranodal
loops (asterisks). Arrowheads mark Schwann cell microvilli abutting the
nodal axolemma. Bar = 1 μm. B. Cross-section of a node of Ranvier.
Note Schwann cell microvilli (arrowheads) abutting the nodal axolemma,
which is typically undercoated by electron dense material. Bar = 0.5
μm. C. Cross-section of a juxtaparanode of a larger-caliber fiber with
the typical trefoil shape of the myelin. Outer Schwann cell cytoplasm
“fills” the grooves of the folded myelin and shows an accumulation of
mitochondria. Bar = 2 μm.
S C H WA N N C E L L S A N D M Y E L I N
Na+ channels (Salzer et al. 2008; Schafer and Rasband
2006). Prominent molecular players in this context are dis-
tinct axon-related cell adhesion molecules, such as NF186
and Nr-CAM (Koticha et al. 2006). Furthermore, axonal
cytoskeletal elements, such as ankyrin G, which binds to the
cytoplasmic (axoplasmic) domain of the voltage-gated Na+
channels and its “anchor” protein βIV-spectrin are important
(Dzhashiashvili et al. 2007; Yang et al. 2007). On the site of
the Schwann cell microvilli Nr-CAM and the gliomedins
interact with the extracellular domains of axonal Nr-CAM
and particularly with NF186 and, in this way, appear to direct
and fix a molecular complex consisting of the two CAMs and
the nodal cytoskeletal and Na+ channel components (Eshed et
al. 2005, 2007; Feinberg et al. 2010). A proof of principle dem-
onstrating the pivotal role of neurofascins has initially been
provided by investigating corresponding knock-out mice that
show defective Na+ channel clustering at nodes (Sherman et al.
2005). Further studies implicated not only the gliomedin-Nr-
CAM-NF186-axis during clustering, but—by in vitro myeli-
nation approaches—showed that supplementary paranodal
junction mechanisms can also rescue lack of axolemmal NF186
(Feinberg et al. 2010). Subsequent studies in vivo are at some
variance to this model, showing that paranodal domains can-
not compensate for the loss of axonal Nf186 (Thaxton et al.
2011), as it has been initially described (Sherman et al. 2005).
nodal compartment in the absence of Nf186 (Thaxton et al.
2011). One explanation for the different findings by Feinberg
and Thaxton might be the different experimental approach
(in vitro versus in vivo models) (Thaxton et al. 2011). Recently,
a study focusing on the origin of nodal components revealed
two distinct sources of nodal molecular components. NF186
is initially a ubiquitous and freely diffusible axonal compo-
nent that can thus be “collected” from local sources via inter-
action and diffusion trapping by Schwann cell microvilli. Na+
channels and the cytoskeletal elements, however, need to be
transported to the node (Zhang et al. 2012). This model is
summarized in Figure 7.6F–H.
Other Schwann cell–derived molecules involved in correct
ion channel clustering include laminin 2 and its receptor com-
ponent, dystroglycan (Occhi et al. 2005; Saito et al. 2003).
The tight interaction of the myelinating Schwann cell and
nodal Na+ channels is impressively reflected by spatial abnor-
malities of the channels in myelin mutant mice. Both in the
myelin mutants, P0–/– and trembler mice (carrying a spon-
taneous PMP22 missense mutation), clustering appears pre-
mature (Devaux and Scherer 2005; Ulzheimer et al. 2004).
Moreover, in P0–/–, but not trembler mice, the Nav1.8 iso-
form is ectopically expressed in the abnormal nodes, leading
to pathological conduction properties (Moldovan et al. 2011)
(Fig. 7.6A–C).
4.1.2 The Paranodes
The nodes of Ranvier proper are flanked by the paranodal loops
(see Fig. 7.5A), that are, cytoplasmic pockets of the respective
myelinating glial cells. Similar to the Schmidt-Lanterman inci-
sures (see section 4.2), they form a helix-shaped cytoplasmic
• 79
 A

F NaCh NF186 NrCAM G Forming node

AnkG Gliomedin MV
B
PNJ
Premyelinating
Schwann cell

C Axon

H Mature node

Figure 7.6 Teased fiber preparations from wild-type (A–D) and P0–/– mice (B, C, E), prepared for immunocytochemistry using antibodies
against voltage-gated Na+ channels (A–C) and K+ channels (D–E). Figure F–H displays the current view of mechanisms leading to deposition
of Na+ channels at the node of Ranvier. A–C. Clusters of Na+ channels at the nodes of adult P0–/– mice display an immature feature (B,C) in
comparison to the nodes of wild-type mice (A). D,E. In wild-type mice (D) K+ channels are confined to the juxtaparanodes, whereas in the P0–/–
mice, they are shifted to paranodes. Kv1.2 is also located along the internodal, inner mesaxon (black arrows), and Schmidt-Lanterman incisures
(white arrows). Bar = 10 μm. F. Before myelination, components of the node are diffusely expressed along the axon. G. During node assembly
NF186 redistributes via diffusion to the node, where it is “trapped” by interactions with the gliomedin/NrCAM complex on Schwann cell
microvilli (MV). Sodium channels are delivered for exocytosis. H. In mature nodes, all node components are being transported by carrier vesicles,
replenishing components that slowly turn over; NF186 is linked to sodium channels via ankyrin G. Note that paranodal junctions are providing a
diffusion barrier. (A–E) Reprinted from Ulzheimer et al. 2004. Altered expression of ion channel isoforms at the node of Ranvier in P0-deficient
myelin mutants. Mol Cell Neurosci 25:83–94, with permission from Elsevier; (F–H) reprinted from Zhang et al. 2012, with permission from
Elsevier.

band that connects the periaxonal with the abaxonal Schwann
cell cytoplasm. The paranodal loops are linked to the associ-
ated axolemma by specialized septate junctions. Laterally,
Schwann cell paranodes are connected by adherens junctions
(Fannon et al. 1995). Depending on the size of the axon, there
are in principle two different forms of paranodal regions
(Berthold 1996; Phillips et al. 1972). In smaller-caliber fibers,
the cytoplasmic pockets approach tangentially to the axon,
and each pocket reaches the axolemma (see Fig. 7.5A). They
generally appear well organized. In larger-caliber fibers, the
pockets approximate the axolemma in a steep and almost per-
pendicular fashion and not all pockets reach the axolemma.
They are usually smaller and often appear much more electron
dense than the pockets of the smaller-caliber axons. Because
of these characteristics they often appear less organized than
paranodes of the smaller fibers. This is of particular impor-
tance when investigating pathological alterations in nodal and
paranodal aspects, because the high variability of paranodes of
larger-caliber fibers make a thorough quantification of patho-
logical “alterations” necessary.
The typical molecular features of paranodal loops include
the presence of Nf155, the putative ligand of the axonal com-
ponents contactin and Caspr, contributing to the organiza-
tion of the septate junctions (Salzer et al. 2008; Schafer and
Rasband 2006). Mice lacking one of these molecular compo-
nents fail to form paranodal junctions (Sherman and Brophy
2005). Abnormal paranodes develop in the absence of protein
4.1.B that interacts with Caspr (Cifuentes-Diaz et al. 2011).
Recently, the paranodal loops have been suggested to control
the nodal molecular arrangement by forming a molecular bar-
rier preventing the “escape” of nodal molecules into the wrong
compartment; however, this model is presently under debate
(Feinberg et al. 2010; Thaxton et al. 2011; see the preceding).
By using fluorescent tracers of different sizes Rosenbluth
et al. investigated the tightness of the paranodal junction of
myelinated fibers (Mierzwa et al. 2010). Interestingly, 3- and
even 70-kDa tracers have been found to be able to pass from
the nodal space into the paranodal compartment, most prob-
ably “using” the intercellular “pathway” between the heli-
cally arranged paranodal terminal loops. By this pathway an

80 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
exchange of water soluble nutrients and metabolites to and
from the internodal axon is conceivable. Furthermore, this
might be an ionic connection from the node to the juxtaparan-
odal potassium channels. Last, antibodies and other detrimen-
tal molecules could reach the internodal axon under various
pathological conditions (Mierzwa et al. 2010). An obviously
important intracellular pathway is realized by reflexive gap
junctions formed by Cx32 molecules that may allow rapid
radial connection between the axonal and abaxonal Schwann
cell cytoplasm (Balice-Gordon et al. 1998). This radial path-
way is also found in Schmidt-Lanterman incisures.
4.1.3 Juxtaparanodes
Another region of interest is the compartment neighboring
the paranodes, the juxtaparanodal region. From the morpho-
logical point of view, this compartment is particularly con-
spicuous in larger-caliber fibers (see Fig. 7.5C). Because of a
tapering of larger-caliber axons, the myelin sheath appears
fluted so that in cross-section the myelin sheath and axon are
cross-shaped or acquire the form of a trefoil (see Fig. 7.5C).
Here, the outer Schwann cell cytoplasm “fills” the grooves of
the folded myelin and shows an accumulation of mitochon-
dria. The axon–Schwann cell apposition often shows a com-
plicated organization because of a profound interdigitation
of inner Schwann cell loop and axolemma, called the axon–
Schwann cell network.
Initially the juxtaparanodal region was not distinguished
from the paranode, but regarded as a component of this com-
partment (Berthold 1996). However, when focusing on the
molecular characteristics, paranodal and juxtaparanodal sites
became clearly distinct. The juxtaparanodes are molecularly
defined by the presence of TAG-1, on both the glial and axonal
site and by Caspr2 and voltage-gated Kv1.1 and Kv 1.2 chan-
nels on the axonal site (Salzer et al. 2008). Caspr2 binds to the
cytoskeletal adaptor protein 4.1B that has pivotal functions for
the organization of paranodes and juxtaparanodes, including
Caspr2-enrichment (Cifuentes-Diaz et al. 2011). Interestingly,
in myelin mutants devoid of P0, K+ channels and Caspr2 are
shifted to the paranode (Ulzheimer et al. 2004) (Fig. 7.6D,E),
and in other myelin mutants of the PNS K+ channels are disor-
ganized (Groh et al. 2010; Kohl et al. 2010). Caspr2 also binds
with its PDZ-binding sequence to other Caspr2-molecules
that anchor the K+ channels to their characteristic position
(Salzer et al. 2008).
A typical ultrastructural feature is the juxtaparanodal net-
work between axonal and glial membranes, which is most
obvious in larger-caliber fibers. In longitudinal sections, a
striking asymmetry is often detectable in that the axon–
Schwann cell network is much more conspicuous at the distal
aspect of the nodal region than at the proximal one. In addi-
tion, axonal lysosomes containing acid phosphatase are more
frequent in the distal regions (Gatzinsky 1996). Interestingly,
this compartment appears particularly vulnerable to molecu-
lar changes in Schwann cells, such as reduced expression of P0
(Martini et al. 1995b), absence of Cx32 (Groh et al. 2012),
or lack of β4 integrin and dystroglycan (Nodari et al. 2008).
Of note, these conditions lead to macrophage-related myelin
S C H WA N N C E L L S A N D M Y E L I N
destruction. The disorganization of the juxtaparanodal struc-
tures in myelin-related mutants might be a pathological con-
sequence of a labile, molecularly specialized structure that not
only comprises the K+ channel compartment and Caspr2 mol-
ecules, but also the juxtaparanodal network between axonal
and glial membranes.
4 .2 S C H M I DT-L A N T E R M A N I N C I S U R E S
These are cytoplasmic clefts that connect the periaxonal with
the perinuclear (abaxonal) Schwann cell cytoplasm. They are
believed to mediate communication of adaxonal and abaxonal
Schwann cell cytoplasm, thus fulfilling partially overlapping
functions with paranodal loops. This may be supported by
the finding that both structures are expressing gap junctions
that are composed of Cx32 molecules (Scherer and Arroyo
2002). In longitudinal sections of osmium tetroxide–fixed
tissue, they can be identified by light microscopy as slim,
bright lines (“incisures”). In teased fiber preparations labeled
for markers of noncompacted myelin (e.g., MAG, Cx32),
Schmidt-Lanterman incisures appear as funnel-like profiles
(Scherer and Arroyo 2002). Moreover, immunohistochemical
studies indicated that Caspr and the voltage-gated K+ chan-
nels Kv1.1 and 1.2 demarcate the axonal domain underlying
the inner loop of the Schmidt-Lanterman incisures (Scherer
and Arroyo 2002). Electron microscopy reveals that the inci-
sures consist of slender pockets of cytoplasm that form a heli-
cal funnel. Similarly to paranodal loops, Schmidt-Lanterman
incisures contain E-cadherin–positive adherens junctions
which interact with p120 catenin. This interaction is neces-
sary for their formation (Tricaud et al. 2005).
Membranes of these Schmidt-Lanterman incisures con-
tain the myelin-associated glycoprotein (MAG) but not P0.
Paradoxically, however, the latter molecule appears to be
essential for their formation. When in oligodendrocytes the
major CNS myelin component PLP is replaced by P0, these
cells express ectopic incisures (Yin et al. 2008). Why this oli-
godendrocytic myelin, however, is detrimental for the myeli-
nated axons, is presently unclear. Moreover, absence of MBP
appears to foster the formation of Schmidt-Lanterman inci-
sures (Gould et al. 1995).
5 S P E C I A L I Z E D ( N O N -R E M A K )
S C H WA N N C E L L S D E VO I D O F M Y E L I N
5.1 T E R M I NA L S C H WA N N C E L L S O F T H E
N EU RO MUS C U L A R J U N C T I O N
Both in the CNS and PNS, glial cells have been identified to
perform pivotal functions during synapse formation, prun-
ing, maintenance, and modulation of activity (Eroglu and
Barres 2010). In the PNS, specialized Schwann cells that do
not myelinate fulfill this task at the neuromuscular junction.
Based on their position they are synonymously called terminal
Schwann cells, perisynaptic Schwann cells, and teloglia (Griffin
and Thompson 2008). In the adult stage, these cells form an
• 81
ensheathing structure around terminal axon branches and
synaptic boutons and are covered by a basal lamina that fuses
with that of the muscle fiber and that of the motor endplate.
Interestingly, the presynaptic membranes of the boutons are
preserved from being ensheathed, whereas the axon terminals
connecting the boutons are mostly entirely enwrapped by the
cells. Although terminal Schwann cells do not form myelin,
they express typical myelin-related molecules, such as galacto-
cerebroside, CNP-1, MAG, and P0 (Georgiou and Charlton
1999). In addition, terminal Schwann cells express cell adhe-
sion molecules that are usually confined to nonmyelinating
Schwann cells, such as L1 and N-CAM (Sanes and Lichtman
2001) and collagen XXVIII (Grimal et al. 2010). Recently the
question about the size of the territory occupied by a terminal
Schwann cell has been investigated during development, in
the adult normal state and axonal degeneration. By labeling
individual terminal Schwann cells and performing time-lapse
imaging experiments on them, adult cells—as expected—
appeared to be arranged in a static fashion, whereas young
terminal Schwann cells intermingled dynamically (Brill et al.
2011) (Fig. 7.7).
Terminal Schwann cells appear to be necessary for the
maintenance of presynaptic terminals under normal con-
ditions in adults (Reddy et al. 2003). They contribute to
the maintenance of ionic homeostasis caused by buffer-
ing high concentrations of K+ released as a consequence of
high-frequency activities (Griffin and Thompson 2008).
Interestingly, they regulate the efficacy of synaptic transmis-
sion, as they express purinergic and muscarinic acetylcholine
receptors that can trigger the release of internal Ca2+ stores
and thus modulate perisynaptic Ca2+ concentration (Griffin
and Thompson 2008).
On nerve injury, the perisynaptic Schwann cells upregu-
late GAP-43 and nestin and extend multiple processes along
the muscle fiber that might guide regrowing motor axons
toward their denervated endplates (Griffin and Thompson
2008; Kang et al. 2007). Additionally, denervated perisyn-
aptic Schwann cells upregulate the active form of the extra-
cellular matrix component agrin and can induce aggregation
A B C principal phenotypes emerge from the immature Schwann
Figure 7.7 A–C. In vivo imaging of the same neuromuscular junction in a
mouse at 6.5 (A) and 10 months (B,C) of age. Note the increased number
of terminal Schwann cells at the later time point (2–4) that are demarcating
their territory. Territory reconstruction of individual terminal SCs is based on
photobleaching in the living animal. Axons are labeled green, acetylcholine
receptors red. Bar = 5 μm. Reprinted from Brill et al. 2011.
82 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
of acetylcholine receptors at extrasynaptic sites of denervated
muscle fibers (Griffin and Thompson 2008).
Another interesting function of terminal Schwann cells
occurs during early postnatal development when polyneuronal
innervation is eliminated. Combining in vivo imaging and
electron microscopy, terminal Schwann cells have been iden-
tified to phagocytose competing motor axon terminals that
have to be pruned (Bishop et al. 2004). Of note, similar obser-
vations have been made in P0-null-mutant peripheral nerves
in which nerve Schwann cells also phagocytose end bulbs of
retrogradely dying axons (Ey et al. 2007), possibly reflecting a
general program in axon clearance by Schwann cells.
5.2 S C H WA N N C E L L S AT S E NS O RY T E R M I NA L S
Schwann cell–like cells devoid of myelin and associated with
terminal regions of myelinated axons are also found in the
sensory part of the nervous system. One example is the mul-
tilamellar cells of the inner core of Pacinian corpuscles that
express the Schwann cell marker S100. Similar to nonmyeli-
nating Schwann cells, they constitutively express N-CAM, but
L1-expression is confined to the developmental stages (Nolte
et al. 1989). Thus, these cells might share molecular features
of both myelinating (L1-negativity in adulthood) and nonmy-
elinating Schwann cells (maintenance of N-CAM positivity).
Furthermore, neuregulin receptors of the erb-family have been
described on them (Gonzalez-Martinez et al. 2007). Recently,
these cells have been identified to potentially play a role in sen-
sory transduction and stimulus modification; implicating neu-
ronal glutamate and Schwann cell–derived GABA, possibly
involved in adapting permanent pressure (Pack and Pawson
2010; Pawson et al. 2009). The lamellar cells of Meissner cor-
puscles also express S100 and transiently the low-affinity NGF
receptor p75 also, which might be reminiscent of the down-
regulation of p75 in myelinating Schwann cells (Albuerne et
al. 2000).
6 S U M M A RY A N D P E R S P E C T I VE S
Derived from the neural crest, the Schwann cell precursor cells
not only give rise to the true Schwann cells in the adult stage,
but may also be involved in shaping embryonic nerves. Two
cells: the myelinating and nonmyelinating Schwann cells. The
nonmyelinating Schwann cells probably provide an environ-
ment for permanent axonal plasticity, supporting functional
recovery after injury. The myelinating Schwann cells have a
pivotal role in the saltatory propagation of action potentials
and maintenance of axonal structure. The node of Ranvier is
the myelin-related compartment responsible for the genera-
tion and propagation of the action potential. As opposed to
the nonmyelinating Schwann cells, the nodes are “sealed” by
molecules repulsive to nerve growth to prohibit unwanted
axonal sprouting. Nodal, paranodal, and juxtaparanodal sites
are highly specialized, comprising the functionally impor-
tant clustering of Na+ and K+ channels. Special cell contacts
between axon and Schwann cells might attenuate the access of
noxious components to the internode. Future studies should
consider alterations of nodal structure and function as part of
neuromuscular disorders.
The internode consists mainly of compacted myelin. In
addition to the well-known, noncompacted structures, para-
nodal loops, and Schmidt-Lanterman incisures, special cyto-
plasmic channels of the myelinated PNS fiber, the Cajal bands,
have recently been identified as important structures for fiber
development and disease. Future studies will certainly con-
tinue to address these issues with regard of demyelinating or
dysmyelinating disorders. Both the myelinating and nonmy-
elinating Schwann cells are surrounded by a basal lamina that
not only forms the demarcation to the mesenchymal environ-
ment within the nerve, but also contains components that are
important for Schwann cell differentiation. Less conspicuous
cells, such as terminal Schwann cells, might fulfill important
functions during the formation of neuromuscular junction
cytoarchitecture and neuromuscular transmission. Applying
live-imaging microscopy and other sophisticated techniques,
more knowledge will certainly emerge about this important
component of the “tripartite synapse” (Eroglu and Barres
2010; Parpura et al. 2012). Thus, Schwann cells are essential
determinants of the functional integrity of the peripheral
part of the axon and it is not surprising that disorders pri-
marily affecting Schwann cells have serious consequences for
axon structure and survival, nodal organization and synaptic
integrity.
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• 85
 8.
MICROGLIAL CELLS
Wolfgang J. Streit

A B B R E VI AT I O N S 2 H I S TO R I C A L P E R S P E C T I VE S

AD Alzheimer disease Following early descriptions of neuroglia by Virchow in the

CNS central nervous system mid nineteenth century, other contemporary pathologists
CX3CL1 fractalkine and psychiatrists, including Nissl and Alzheimer, commented
CX3CR1 fractalkine receptor on the possibility that the developing CNS was populated by
Iba1 ionized calcium binding adaptor molecule 1 cells of non-neuroectodermal origin. Speculation abounded as
LPS lipopolysaccharide to the source of these invading cells, but attention was focused
MHC major histocompatibility complex with increasing consistency on the possibility that mesoder-
NSE nonspecific esterase mally derived cells were the invaders. Eventually this led to the
RhIC rhodamine isothiocyanate formulation by Cajal of el tercer elemento, the third element of
TPPase thiamine pyrophosphatase the CNS, referring to a group of cells that was morphologi-
cally distinct from both first and second elements (neurons
and astrocytic neuroglia). Cajal’s third element, defined by
1 INTRODUCTION
him strictly in morphological terms, received further distinc-
tion into oligodendrocytes and microglia by del Rio-Hortega,
Microglial cell numbers are thought to make up 5% to 20% of
the Spanish neuroanatomist who provided the first systematic
the entire central nervous system (CNS) glial cell population,
investigation on microglial cells. Del Rio-Hortega’s detailed
and, assuming that the brain is composed roughly of equal num-
cytological observations, which remain quite relevant even
bers of neurons and glia, one must conclude that 2.5% to 10%
today, gave rise to a longstanding controversy over the ori-
of all brain cells are microglia. This simple calculation is aston-
gin of microglia that dominated microglial research for sev-
ishing only insofar as the very existence of microglial cells was
eral decades into the 1990s. In recent history, the debate over
questioned by some as recently as 20 years ago (Graeber 2010).
microglial ontogeny has been succeeded by another fervent
Research on microglial cells has grown significantly during
discussion, this one focusing on the functional significance of
the past 25 years. This positive trend can be attributed largely to
activated microglial cells, that is, whether activation of micro-
the development of reliable histological methods for identifying
glial cells is a beneficial or harmful process. The significance
the cells in CNS tissue sections using light microscopy. Most of
of this discussion pertains primarily to the issue of bystander
these methods, which are described in this chapter, can also be
damage, a phenomenon that is thought to be important in
carried to the electron microscopic level allowing verification of
both acute and chronic CNS injury and disease. For exam-
microglial identity through direct comparison of the presence
ple, regarding the pathogenesis of Alzheimer disease (AD),
of a specific marker with ultrastructural morphology. Before
many scientists believe that microglial activation is a critical
the advent of microglia-specific markers, electron microscopy
component in the development of AD reflecting a type of
was almost always needed for positive identification using solely
chronic glial inflammation that is triggered by the presence of
morphological criteria. Modern neurobiology now has available
amyloid-beta proteins and causes neurodegenerative changes,
an assortment of reliable light microscopic techniques that read-
such as neurofibrillary tangles through excessive production
ily enable identification of microglial cells by neuroscientists.
of neurotoxic molecules by activated microglia (Akiyama et
Regarding the terminology used in this chapter, it is impor-
al. 2000). However, treatments with antiinflammatory drugs
tant to point out that microglial cells in the adult CNS can
have failed to show clear benefits, and a new view on AD
assume at least four clearly identifiable states: (1) resting (rami-
pathogenesis focusing on the aging-related, structural dete-
fied) microglia, which are distributed ubiquitously throughout
rioration of microglia (cell senescence) has emerged (Streit
the normal and nonpathological CNS; (2) activated (reactive)
2006) (see final section of this chapter).
microglia, which occur in pathological states, but are not always
phagocytic; (3) phagocytic microglia, which appear as rounded
brain macrophages; and (4) dystrophic microglia, which are
senescent cells. For additional descriptions of these microglial 3 O R I G I N A N D L I N E AG E O F M I C R O G L I A
states the reader is referred elsewhere (see chapter 48) (Morioka
et al. 1992; Streit and Kreutzberg 1988; Streit and Xue 2009; Two related issues were at the heart of a longstanding debate
Streit et al. 2004). over the origin of microglia: (1) Are microglia derived from

86
mesoderm or from neuroectoderm? (2) When and how do
microglia populate the CNS? As discussed elsewhere (Streit
2001), it appears that both issues have been largely resolved
and it is clear that (1) microglia are of myelomonocytic
lineage and therefore likely derived from hemangioblastic
mesoderm, and (2) microglia become part of the CNS paren-
chyma early during embryonic development at about the
time neurulation has been completed. Microglial precursor
cells are an integral component of the CNS during embry-
onic and postnatal development. Cells that are most aptly
described as “fetal macrophages” (Takahashi et al. 1989)
populate the developing neuroectoderm as early as embry-
onic day 8 in rodents and late during the first trimester in
humans (Alliot et al. 1999; Ginhoux et al. 2010; Monier et al.
2007). These fetal macrophages can be visualized using lec-
tin histochemical markers that also label microglia (Sorokin
et al. 1992) and therefore they are considered the earliest
detectable microglial precursor cells. Significantly, fetal
macrophages can be found in the primitive neuroectoderm
before it becomes vascularized (Chan et al. 2007; Monier
et al. 2007) (Fig. 8.1), which eliminates the possibility that
blood-borne monocytes serve as direct microglial precursors.
It is likely that yolk sac–derived fetal macrophages are direct
precursors for microglia, whereas blood monocytes are bone
marrow–derived (Prinz et al. 2011) (see also chapter 15). As
the embryonic CNS develops toward the perinatal stage and
various neural cell types mature and differentiate, fetal mac-
rophages also metamorphose from rounded cells to more
differentiated embryonic microglia with short processes.
A Embryonic Neuroepithelium
Fetal macrophages
B Perinatal Brain
Cluster of ameboid
microglia in corpus
callosum
C Adult Brain
Ramified microglia
Figure 8.1 Summary of Microglial Ontogeny During Three
Developmental Stages A. Fetal macrophages are found in the developing
neuroectoderm as early as embryonic day 8 in rodents. B. In the
perinatal brain, clusters of dividing ameboid microglia are found in the
supraventricular corpus callosum. The cells migrate from the clusters into
the cerebral cortex and differentiate into ramified microglia. C. Ramified
microglia have colonized throughout the adult brain.
8. M I C R O G L I A L C E L L S
These process-bearing embryonic microglia are at an inter-
mediate stage of differentiation and have not yet matured to
the fully ramified morphology that is characteristic of adult
microglia.
During perinatal stages in rodents at about embry-
onic day 20, unique accumulations of so-called ameboid
microglial cells become apparent (Ling and Wong 1993) as
aggregated clusters of rounded cells in specific anatomical
locations, most prominently in the supraventricular cor-
pus callosum (Hurley et al. 1999; Ling and Wong 1993).
In the postnatal CNS, ameboid microglia within these
clusters undergo mitosis, and these prominent supraven-
tricular clusters of proliferating cells were recognized by
early microglial researchers who termed them fountains of
microglia (Kettenmann et al. 2011). Contemporary neuro-
biologists might be inclined to apply the term microglial
progenitor cells instead of ameboid microglia to emphasize
their status as immature precursor cells. Microglial pro-
genitor cells in the corpus callosum persist through the first
two postnatal weeks and during that time the cells migrate
into the overlying cerebral cortex, differentiating into fully
ramified microglia. This perinatal burst of microgliogen-
esis occurs to facilitate microglial colonization of the fore-
brain, which undergoes its most expansive growth during
the postnatal period (see Fig. 8.1). Ameboid microglial pro-
genitor cells can be distinguished antigenically from rami-
fied microglia in that ameboid cells are readily labeled with
the ED1 antibody (directed against rat CD68), whereas
ramified microglia are not (Milligan et al. 1991). At birth,
ED1-positive cells are abundant in the CNS but they disap-
pear completely by the third postnatal week (Milligan et al.
1991), indicating that differentiation of ameboid cells into
ramified microglia is complete at that point in time. ED1 is
a macrophage marker that recognizes an intracytoplasmic,
lysosomal antigen whose expression increases during phago-
cytosis (Dijkstra et al. 1985); thus, disappearance of ED1
immunoreactivity during postnatal development shows that
fully differentiated ramified microglia are in a phagocytoti-
cally quiescent state.
During adult life there is little if any replacement of
microglia from exogenous sources, such as the bone marrow,
as shown in parabiotic studies (Ajami et al. 2011). Microglia
have the greatest mitotic potential of all parenchymal cells in
the CNS and are therefore capable of self-renewal. Microglial
mitosis in the normal CNS occurs at a very low rate, indi-
cating low turnover and a long lifespan of cells (Lawson et
al. 1990). Nonetheless, a small fraction of microglial cells
may undergo replacement by bone marrow–derived pre-
cursors via perivascular cells. The latter are mononuclear
phagocytes that reside in the Virchow-Robin (perivascular)
spaces surrounding medium and small-sized cerebral vessels.
Perivascular cells, which are replaced continuously by bone
marrow–derived progenitors, on occasion may penetrate the
perivascular basement membrane, enter the parenchyma and
differentiate into process-bearing microglia. Studies using
bone marrow chimeras and localization of major histocom-
patibility antigens support this idea (Hickey and Kimura
1988; Streit et al. 1989).
• 87
4 M ET H O D S F O R S TA I N I N G M I C R O G L I A
4.1 S I LVE R C A R B O NAT E M ET H O D
The first selective stain for microglia was the weak silver car-
bonate method developed by del Rio-Hortega. Despite its
capriciousness, this method remained the only useful histo-
chemical procedure for at least 50 years. As with many other
histochemical techniques involving metallic silver impregna-
tions, Hortega’s weak silver carbonate method for microglia
has very specific fixation requirements that do not, however,
guarantee reproducible results in every preparation. The results
obtained are quite variable in terms of numbers of microglia
stained, and vary also with the animal species used. For rea-
sons unknown, the method seems to work reliably only in
rabbit brain. Although it can be carried to the electron micro-
scopic level, its usefulness is limited because of poor structural
preservation and the deposition of metallic precipitates that
can obscure much of the cellular detail.
4.2 E N ZY M E H I S TO C H E M I C A L M ET H O D S
Thiamine pyrophosphatase (TPPase) and nucleoside diphos-
phatase (NDPase) are the most reliable and specific enzyme
histochemical methods for staining resting microglial cells
in a variety of species. These methods have been used suc-
cessfully to localize microglia at both light and electron
microscopic levels (Murabe and Sano 1981; Schnitzer 1989).
TPPase activity, originally described to be localized to the
Golgi apparatus in a variety of cell types, including neurons,
was later found to be associated specifically with the plasma
membrane of microglial cells and with blood vessels in the
CNS (Murabe and Sano 1981). Nonspecific esterase (NSE)
is an enzyme used to identify microglia in mixed brain cul-
tures (Sawada et al. 1990), but is of little use for detecting
resting microglial cells in tissue sections. Actively prolifer-
ating, reactive microglial cells in the hypoglossal nucleus
after peripheral nerve transection do not show any stain-
ing for this enzyme (Schelper and Adrian 1980). However,
nonspecific esterase can be found in microglia-derived and
other brain macrophages that are prevalent in stab wounds.
Nonspecific esterase enzyme histochemistry thus supports
the view that microglia in vitro are, in fact, microglia that
have transformed into brain macrophages as a consequence
of having been placed into cell culture. Activated and/or
phagocytic microglia in cell culture or the pathological brain
show increased activities for a number of other enzymes that
are absent from resting microglial cells in situ. These include
acid phosphatase, 5′-nucleotidase, and oxidoreductase.
Other studies have shown the presence of nitric oxide syn-
thase, cyclooxygenase, lysosomal proteinases, plasminogen
activator, lysozyme, purine nucleoside phosphorylase, and
elastase (Banati et al. 1993; Castellano et al. 1990; Nakajima
et al. 1992). It is worth noting that some of these enzymatic
activities are also found in other glial cells types and there-
fore are not always useful as selective histochemical markers
for microglial cells.
88 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
4.3 I M MU N O H I S TO C H E M I C A L D ET EC T I O N
O F M I C RO G L I A
The microglial plasma membrane is complex and studded
with a large variety of receptor and adhesion molecules in
addition to enzymatic activities. Because of this large reper-
toire of surface antigens, numerous antibodies are now avail-
able to facilitate immunohistochemical staining of microglia.
Interestingly, many of these monoclonal antibodies were not
produced with the intention of specifically marking microg-
lia, but were meant to target differentiation antigens found
on cells of the immune system, such as macrophages, thymo-
cytes, and lymphocytes. Following initial failures of demon-
strating presence of monocytic and lymphoid antigens on
human microglia (Oehmichen et al. 1979), it was found later
that a mouse macrophage–specific antigen could be localized
on resting microglia with a monoclonal antibody designated
F4/80 (Hume et al. 1983; Perry et al. 1985). These investigators
also succeeded in showing the presence of Fc and complement
receptors on resting mouse microglia using antibodies 2.4G2
and Mac-1, respectively. Analogously, ramified microglia in rat
and mouse brain can be demonstrated reliably using antibod-
ies against the CD11b antigen, also known as the CR3 com-
plement receptor (Graeber et al. 1988). It is important to note
that these receptors are also found on macrophages in non-
neural tissues, and their presence on microglia underscores the
phagocytic potential of these cells, as well as their close rela-
tionship to the myelomonocytic cell lineage. Crossreactivity
of macrophage-specific antibodies with microglia and blood
monocytes has been taken as evidence that microglia are
derived from monocytes. However, a direct lineage relation-
ship between monocytes and microglia is not likely because
both microglia and monocytes are fully differentiated cell
types that may arise from different precursor cells, as dis-
cussed. A distinction between microglia and so-called “other
brain macrophages” has also been made using flow cytometric
analyses, which have shown that “other CNS macrophages”
are phenotypically distinct (CD11b/c + and CD45hi) from
parenchymal microglia (CD11b/c + and CD45low) (Ford et al.
1995).
In the human brain, microglia can also be localized using
antibodies against macrophage surface receptors (Akiyama
and McGeer 1990). In addition to the expression of Fc and
complement receptors, other cell adhesion molecules are
expressed constitutively on resting microglia in normal brain.
Belonging to the integrin superfamily of adhesion molecules,
these include typical lymphocytic antigens, such as lympho-
cyte function antigen, CD4 antigen, as well as leukocyte com-
mon antigen (Akiyama and McGeer 1990; Perry and Gordon
1987). Species differences among mouse, rat, and human
in the constitutive expression of these molecules on resting
microglia have been observed, and these are likely caused by
both antibody specificities, as well as variations in tissue pro-
cessing techniques. B-lymphocyte antigens are detectable on
human microglial cells using monoclonal antibodies LN-1 and
LN-3, the latter recognizing HLA-DR antigens (Dickson and
Mattiace 1989; Miles and Chou 1988). Although the LN-1
antibody may label both astrocytes and microglia depending
on fixation and tissue processing techniques, antibody LN-3 A
has an exclusive specificity for microglia in both normal and
pathologic human brain. Figures 8.2 and 8.3 provide examples
of LN-3 staining of resting and activated microglia in human
brain. Significantly, staining with LN-3 is fixation-sensitive
and optimal preparations require lightly fixed tissues (Streit
and Sparks 1997).
It is apparent then that the microglial surface membrane
bears molecules usually associated with white blood cells,
including antigens of the major histocompatibility complex
(MHC). Although it was once thought that MHC antigens B
were entirely absent from brain, supporting the notion of the
brain as an immunologically privileged organ, it is now well
established that MHC antigens are expressed in normal brain,
and the principal parenchymal cell type expressing MHC anti-
gens is the microglial cell (Craggs and Webster 1985; Hayes et
al. 1987; Streit et al. 1989). The expression of MHC antigens
in normal brain also includes endothelial and perivascular
cells, and there are considerable species differences in the levels
of constitutive MHC antigen expression on these various cell
types. Major histocompatibility antigen expression on micro- C
glia is increased dramatically under pathological conditions,
but also increases with normal aging in rodents, non-human
primates, and humans (Finch et al. 2002; Perry et al. 1993;
Sheffield and Berman 1998; Streit and Sparks 1997). To date a
truly specific marker for microglia, namely, one that does not

Figure 8.3 Resting and Activated Microglia Stained with LN-3 Antibody
in Human Brain. A. Activated microglia stain more strongly than resting
ones and have a bushy appearance in a 41-year-old individual (arrows).
B. Microglial rod cells in the cerebral cortex of a 94-year-old individual.
C. Bushy microglia in the 94-year-old, possibly representing two or more
cells that have fused to form a microglial cluster. Bar = 100 μm.

cross-react with other macrophages, has not been generated.

B cells also label activated microglia and microglia-derived brain
Figure 8.2 Ramified Resting Microglial Cells. A. Ramified resting
microglial cell in the nonpathological brain of a 64-year-old human
visualized with monoclonal antibody LN-3. Note extensive branching of
cytoplasmic processes. Bar = 20 μm. B. Ramified resting microglial cell in
perivascular position. The blood vessel (BV) is seen coursing horizontally.
LN-3 immunohistochemistry in non-pathological brain of a 68-year-old
individual. Bar = 20 μm.
All antibodies and lectins that react with resting microglial
macrophages, as well as peripheral macrophages. Conversely,
not all antibodies that react with brain or other macrophages
label resting microglia, suggesting that the antigenic repertoire
of resting microglia is smaller than that of activated microglia
and brain macrophages, and/or that the level of expression of
certain antigens in resting cells is below the detection limit of
immunohistochemistry (Table 8.1). One antibody that has
proved to be particularly useful because its reactivity is rela-
tively unaffected by prolonged fixation or the species being
studied (notably mouse, rat, and human) is the one raised
against the ionized calcium binding adaptor molecule 1, Iba1
(Ito et al. 1998). Immunostaining with Iba1 antibody can be
used to visualize all morphological states of microglia (see
Table 8.1).
Immunohistochemical localization of ramified micro-
glia has been achieved through the use of phosphotyrosine
antibodies (Griffith et al. 2000). This procedure detects the

8. M I C R O G L I A L C E L L S • 89
Table 8.1 IN VIVO STATES OF MICROGLIAL BIOLOGY AND ASSOCIATED PHENOTYPIC CHARACTERISTICS

RESTING ACTIVATED PHAGOCYTIC DYSTROPHIC

Proliferation −/+ + + −

Iba1 + + + +

Griffonia simplicifolia + + + ND
B4-isolectin (rat) and Ricinus
communis lectin (mouse)

Vimentin − + + ND

Macrophage markers (CD68) − −/+ −/+ −

CR3 complement receptor + + + +

(CD11b)

MHC class I antigen − + + ND

MHC class II (Ia) antigen −/+ + + +

CD4 antigen −/+ + + ND

CD8 antigen − −/+ + ND

Leukocyte common antigen − + + ND

(CD45)
Antigens listed are relevant for rat, mouse, and human.
Explanation of symbols: − absent; −/+ weak; + strong; ND not determined.

products of an enzymatic reaction carried out by tyrosine
kinase. There are functional implications for this observa-
tion, because it is known that tyrosine kinases are commonly
associated with cell surface receptors, which are plentiful on
the microglial membrane. Various other immunohistochemi-
cal methods aimed at detecting somewhat unconventional
antigens, such as vaults, ferritin, and lipocortin-1, have also
been described (Chugani et al. 1991; Kaneko et al. 1989;
McKanna 1993). Vaults, which are multiarched ribonucleo-
protein particles of unknown function, appear to be enriched
in microglia during ontogeny, but mostly disappear in adult
cells. Ferritin, on the other hand, is a well-known iron-storage
protein, and its detection in microglia suggests that the cells
actively participate in the trafficking and sequestration of
iron. Lipocortin-1 is a Ca2+-binding protein that is thought
to function as an antiinflammatory or immunosuppressive
molecule.
In summary, remarkable progress has been made in the
development of immunohistochemical procedures used for
the detection and identification of microglia. Given the great
variety of receptor molecules known to cover the microglial
cell surface, it is likely that additional antibodies will be devel-
oped in the future, and undoubtedly these will be important
for further defining microglial phenotypes and advancing
understanding of their functional roles.
4.4 L EC T I N H I S TO C H E M I C A L D ET EC T I O N
O F M I C RO G L I A
While investigating the distribution of complex carbohydrates
in nervous tissue using lectin histochemistry, it was noted that
the B4-isolectin derived from Griffonia simplicifolia resulted
in the selective visualization of a population of rat glial cells
that were identified as microglia (Streit et al. 1985). These ini-
tial observations were confirmed soon thereafter in human
tissue, where it was shown that the lectin from Ricinus com-
munis could be used as a histochemical marker for microglia
(Mannoji et al. 1986). Both lectins have similar sugar-binding
characteristics in recognizing anomeric forms of galactose, with
Griffonia simplicifolia binding to α-D-galactose and Ricinus
communis recognizing ß-d-galactose residues. An additional
ß-d-galactose–binding lectin derived from mistletoe has
been shown to preferentially stain human over rat microglia
(Suzuki et al. 1988), emphasizing the subtle difference in gly-
cocalyx composition between rodent and human microglial
cells, being one of anomeric configuration. The galactose sugar

90 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
residues occur as terminal sugars in the oligosaccharide side
chains of nervous system glycoproteins that are embedded
in the microglial plasma membrane, as revealed by electron
microscopy (Streit and Kreutzberg 1987). Interestingly, the
lectin from Lycopersicum esculentum (tomato), which has an
affinity for poly-N-acetyl lactosamine residues can also be used
for staining rat microglia (Acarin et al. 1994), indicating diver-
sity of carbohydrate domains present on the microglial cell
surface. The specific nature and function of lectin-binding gly-
coproteins on microglial cells has not yet been resolved; how-
ever, it is likely that the carbohydrate domains of many of the
surface receptors, described in the preceding section, account
in large part for lectin binding. Lectin staining is perhaps the
quickest and most resilient method for visualizing microglia
in tissue sections because the carbohydrate epitopes, unlike
most proteins, are largely unaffected by cross-linking through
aldehyde-based fixation and tissue processing techniques.
4.5 OT H E R M ET H O D S F O R L A B E L I N G
M I C RO G L I A
Among all the parenchymal cell types in the mature CNS,
microglia are the cells with the greatest potential for mitosis.
Their ability to divide and proliferate makes them amenable
to labeling with 3H-thymidine and other markers of dividing
cells, such as 5-bromo-2′-deoxyuridine or proliferating cell
nuclear antigen. Microglial cell division is usually triggered by
perturbations in CNS homeostasis, such as neuronal injury,
but there is also evidence that microglial cell division occurs
normally in the rodent brain, albeit at a low rate (Korr et al.
1983). Microglia may also be labeled directly or indirectly
using various dyes and tracer substances. Following intrap-
eritoneal injection of the fluorescent dye, rhodamine isothio-
cyanate (RhIC), labeled ameboid microglia, were observed
in the corpus callosum. Subsequently, the ameboid cells were
observed to transform into RhIC-labeled ramified microglial
cells (Leong and Ling 1992), confirming earlier observations
using colloidal carbon introduced in the form of India ink.
An indirect method for labeling microglia makes use of their
ability to phagocytose dead or dying neurons. Following injec-
tion of the appropriate tracer substance into axons, the tracer
is retrogradely transported toward the parent neuron cell bod-
ies. If the injected nerve is also axotomized, in some instances
this will cause degeneration of the parent neurons, followed by
removal of dead neurons by local microglia that phagocytose
not only the neuronal debris, but also the tracer substance and
thus become labeled. Such experiments have been successfully
carried out in various systems, including the visual system,
resulting in the labeling of retinal microglia with the carbocya-
nine dye DiI (Thanos 1991), the dorsal motor nucleus of the
vagus where the neural tracer fluorogold was used (Rinaman
et al. 1991), and also the rat facial nucleus where fluorogold
was used in conjunction with toxic ricin to induce motor
neuron degeneration (Streit and Graeber 1993). Interestingly,
direct injection of fluorogold into the brain does not label
ramified microglial cells, but if the cells are maintained in cul-
ture, where they undergo macrophage transformation, they do
take up fluorogold rather avidly (Pennell and Streit 1998).
8. M I C R O G L I A L C E L L S
5 M I C R O G L I A A N D R E L AT E D
CELL TYPES
5.1 D E FI N IT I O NS
Microglial cells, because they can change their morphology
and appearance in certain pathological and developmental
states, have been given various descriptive names and attri-
butes, such as rod cells, gitter cells, globoid, and ameboid cells,
to name a few. Even though these terms accurately reflect the
cells’ changed appearance, the descriptive terminology has
somewhat obscured the true identity of the cells associated
with the term microglia. In the normal adult brain, resting
microglia can be defined both in terms of morphology and
phenotype. They are highly branched (ramified) glial cells
with a small amount of perinuclear cytoplasm and a small,
dense, and heterochromatic nucleus (Figs. 8.2–8.5). They can
be distinguished easily from other glial cells by their surface
immunophenotype; that is, they are the only glial cell type
that constitutively expresses the CR3 complement recep-
tor (CD11b antigen) and binds lectins with a specificity for
galactose residues. Furthermore, at the ultrastructural level
microglia are recognizable as true parenchymal constituents
of the CNS because they are located outside of the vascular
basement membrane. At the same time, they may be consid-
ered part of the perivascular glia limitans, because microglial
cytoplasmic processes are found incorporated intermingled
with the layer of astrocytic foot processes (Lassmann et al.
1991). The observation that microglia are frequently found
in the vicinity of blood vessels has resulted in the use of the
term perivascular microglia, which is yet another descriptive
term referring to parenchymal microglial cells, as defined,
which happen to be located near a cerebral blood vessel (see
Fig. 8.2). Perivascular microglia are not to be confused with
so-called perivascular cells, which, unlike microglia, are not
Figure 8.4 Ultrastructural Appearance of a Perineuronal Microglial
Cell in the Rat Brain. The cell has a heterochromatic nucleus (Nu) and
prominent cisternae of rough endoplasmic reticulum (arrowheads). Its
plasma membrane, which is accentuated by lectin staining, is directly
apposed to the neuronal plasma membrane (arrows). Bar = 2 μm.
• 91

Figure 8.5 Electron Micrograph. The electron micrograph shows a
microglial cell (M), an oligodendrocyte (O), and a large dendrite (D).
Both cell types have a heterochromatic nucleus that is larger in the
oligodendrocyte. Bar = 2 μm.
part of the CNS parenchyma but are separated from it by a
perivascular basement membrane (see chapter 9). Perivascular
cells are components of the vascular wall and located in the
perivascular spaces. They fit the morphological definition
of a pericyte (Graeber and Streit 1990b; Mato et al. 1986).
Perivascular cells are phagocytic and typically express MHC
antigens and macrophage antigens (CD68) constitutively
(Graeber et al. 1989b; Mato et al. 1986; Streit et al. 1989),
which has made it difficult in certain pathological situations
to distinguish between perivascular cells and perivascular
microglia. However, in the normal brain these two cell types
are readily distinguished by their morphology and surface
immunophenotype. Perivascular cells are seen only in asso-
ciation with blood vessels, they are not ramified but have an
elongate shape, and they can be specifically labeled in the rat
with ED1 and ED2 antibodies (Graeber et al. 1989a,b). Thus,
there are at least two clearly definable and indigenous sources
of brain macrophages present in normal brain: microglia
and perivascular cells. The term brain macrophage is generic
and encompasses all phagocytic cells in the CNS, including
blood-derived monocytes, which may enter the CNS follow-
ing lesions that disrupt the blood-brain barrier.
5.2 FAC TO R S A FFEC T I N G D I S T R I BU T I O N,
MO R P H O L O GY, A N D P H E N OT Y P E
O F M I C RO G L I A
Microglia are distributed ubiquitously throughout the nor-
mal CNS with regional differences having been reported in
mouse brain (Lawson et al. 1990). According to these authors,
the highest microglial densities are encountered in the
92 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
hippocampal formation, olfactory telencephalon, portions
of the basal ganglia, and substantia nigra. A total number of
3.5 × 106 microglia is estimated to reside in the adult mouse
brain (Lawson et al. 1990), although that estimate is probably
too conservative. Individual microglial cells typically occupy
a distinct territory; that is, neighboring cells do not contact
each other with their cytoplasmic processes. The morphology
and branching patterns of microglial cells show heterogeneity
among different brain regions, which is perhaps most remark-
able when comparing cells in gray and white matter.
Although microglia in gray matter tend to be profusely
ramified with processes extending into all directions, cells in
the white matter often align their cytoplasmic extensions in
parallel, but also at right angles to nerve fiber bundles. Thus,
the cell shape of microglia adapts to the geometry of the brain
region they populate. The microglial immunophenotype is
heterogeneous and appears to be influenced by the chemical
composition of the microenvironment. For example, MHC
class II–positive, as well as CD4-positive microglia are local-
ized preferentially in white matter of normal brain (Hayes
et al. 1987; Perry and Gordon 1987; Streit et al. 1989). Brain
regions lacking a blood-brain barrier, such as the circumven-
tricular organs, do show microglia and microglia-like cells,
such as the Kolmer cells of the choroid plexus, with a different
immunophenotype, suggesting that the chemical milieu influ-
ences microglial and macrophage phenotypes. This is sup-
ported further by in vivo studies showing profound changes
in microglial phenotype after brain lesions, such as forebrain
ischemia and kainic acid injections that compromise the
blood-brain barrier. Similar changes in microglial immuno-
phenotype occur also when the cells are maintained in vitro
using serum-containing culture medium.
5.3 M I C RO G L I A I N C E L L CU LT U R E
Although the maintenance of microglia/brain macrophages
in cell culture was used and described in the 1930s, possibly
even earlier, the procedure did not gain widespread popular-
ity until the 1980s. The technique described by Giulian and
Baker (1986) has been widely used with numerous modifica-
tions. When culturing microglial cells, perhaps more so than
with any other neural cell type, it is apparent that microglia in
vitro are quite different from microglia in vivo. The prepara-
tion of primary mixed brain cultures from which microglia are
isolated causes the generation of large amounts of tissue debris
that, together with a high serum content of the growth media,
promotes rapid transformation of microglial cells into brain
macrophages. Isolated microglia plated onto plastic culture
dishes take on a rounded cell shape resembling immature ame-
boid microglial precursor cells, and it was once widely accepted
that cultured microglia are the same as ameboid microglia.
Because isolated microglia in vitro are essentially brain mac-
rophages, it is important to distinguish this advanced func-
tional (phagocytic) state from the precursor state that defines
ameboid microglial cells in the developing CNS. Brain mac-
rophages, like cultured microglia, secrete a variety of cytokines
and growth factors, whereas ameboid microglial progenitor
cells in situ do not (Hurley et al. 1999).
 Another important consideration in the context of using
cultured microglial cells to better understand the cells’ functions
concerns the issue of microglial activation. Typically, microglia
in vitro are activated by exposing them to potent immunos-
timulatory agents, such as lipopolysaccharide (LPS) and/or
interferon-gamma, which means that cells already transformed
into brain macrophages by the culturing process itself become
superactivated when exposed to LPS. This in vitro stimulation
produces a different kind of activated microglia than what is seen
in vivo when resting microglia are activated by neuronal injury.
Microglia in vivo progress to become brain macrophages only
if debris from degenerating cells needs to be phagocytosed; in
the absence of cell death microglia may become activated in vivo
but do not necessarily become brain macrophages. Accordingly,
cultured microglia/brain macrophages, before LPS stimulation,
are already at an activation state that is equivalent to what is
perceived as maximal microglial activation in vivo, that is, the
brain macrophage stage. Therefore, it is important not to equate
microglial activation in vitro with microglial activation in vivo.
Techniques for inducing microglial ramification in vitro, includ-
ing the use of organotypic or slice cultures, co-culturing micro-
glia with astrocytes or exposing them to astrocyte-conditioned
medium, or treating them with ramifying agents, such as vitamin
E and thapsigargin (Eder et al. 1999; Heppner et al. 1998; Kloss
et al. 1997; Mertsch et al. 2001; Sievers et al. 1994; Tanaka and
Maeda 1996; Yagi et al. 1999) may allow researchers to study
ramified microglia more directly. It remains to be seen, however,
whether ramified microglia in vitro show the same gene expres-
sion patterns as ramified microglia in vivo and can therefore be
considered equivalent.
Microglia activated in vitro with LPS or other immunos-
timulants can produce potentially neurotoxic molecules, such
as nitric oxide, glutamate, reactive oxygen and nitrogen spe-
cies, and proinflammatory cytokines. These observations have
been extrapolated to mean that activated microglia in vivo are
harmful and could be responsible for exacerbating damage in
the injured or diseased CNS by producing neurotoxic com-
pounds that cause neurodegeneration secondarily. However,
this inference from cell culture studies is difficult to reconcile
with in vivo observations that show that microglial activa-
tion is the result of neural tissue damage rather than its cause,
underscoring the basic concept of inflammation, namely, that
inflammation is the cellular response to tissue injury. The idea
of microglia as instigators of bystander damage also clashes
with studies showing that cultured microglial cells can produce
neurotrophic factors and other neuroprotective substances,
and that increasingly the cells’ primary function is being
viewed as one of neuroprotective glia (Nakajima and Kohsaka
2004; Polazzi and Monti 2010; Streit 2002). Microglia in vitro
behave much like other tissue macrophages in a dish, and as
such are capable of performing the full repertoire of immune
functions in vitro, including phagocytosis, antigen presenta-
tion, and cellular cytotoxicity. However, this type of immu-
nological activity does not necessarily reflect what occurs in
the normal or injured CNS where inhibitory influences and
cell–cell interactions may dampen immune responses and
inflammation. Investigators have worked on differentiating
in vitro functionally distinct microglial phenotypes classified
8. M I C R O G L I A L C E L L S
as M1 (cytotoxic), M2 (reparative), and even a third, “deac-
tivated” form (Colton 2009; Michelucci et al. 2009; Moon
et al. 2011), generating data analogous to those obtained from
studying peripheral macrophages in vitro (Gordon 2003).
With reference to the foregoing discussion in this section, to
generalize and assume that the M1/M2 in vitro classification
applies to the microglial population in situ would be a rather
indiscriminate and indeed misleading. Neurotoxic (M1) and
neuroprotective (M2) microglial phenotypes cannot be dis-
tinguished histopathologically in the brain, and it is a matter
of common sense to state that purified cells in a petri dish do
not accurately reproduce the cells’ functions in the complex,
normal, or pathologically altered CNS microenvironment.
6 MICROGLIA IN THE NORMAL
A D U LT B R A I N
Microglia are ubiquitous in the CNS, where they are spaced
evenly in a networklike fashion throughout the brain and
spinal cord. Unlike astrocytes, microglia are not known to
form connections with each other normally, and each cell
occupies its own individual plot of three-dimensional space,
which is approximately 50,000 μm3 in volume. In the normal
uninjured CNS the cells are referred to as resting microglia
to set them apart from the activated or reactive microglia
that appear after brain injury. Resting microglia have a char-
acteristic cell shape marked by finely branched, ramified cell
processes that extend into all directions (see Fig. 8.2) reflect-
ing the cells’ recognized function as “sensors of pathology”
(Kreutzberg 1996). Apparently, microglia are constantly on
the lookout for biochemical or bioelectric changes in their
microenvironment that may signal ongoing perturbations
in brain homeostasis and require them to jump into action
(Davalos et al. 2005; Nimmerjahn et al. 2005; Petersen and
Dailey 2004). This role as a sentry is very much analogous
to the roles served by cells of the immune system in the rest
of the body. The concept of microglia as “the brain’s immune
system” (Graeber and Streit 1990a), thus reconciles the dis-
crepancy between the absence of leukocytes in the brain and
the brain’s ability to defend itself against infection, injury,
and disease. With microglia, evolution has found a way to
achieve compatibility between the destructive power of the
immune system and the relative vulnerability of the CNS to
injury and disease. Functionally speaking, one might there-
fore view microglia as a hybrid cell type that combines char-
acteristics of a neuroprotective glial cell with some of the
attributes of macrophages and lymphocytes. In line with
their role as sensors of pathology, the microglial cell surface is
covered with an abundance of receptor molecules that range
from ion channels to immunological recognition molecules
to neurotransmitter receptors. Low levels of a unique com-
position of many cytokines and their receptors in the normal
CNS may contribute to an immunologically quiescent CNS
microenvironment, although precise functional roles of most
cytokines/chemokines and their respective receptors in the
normal CNS are largely unknown, and may actually be differ-
ent there than in the periphery.
• 93
 For the normal CNS, one chemokine, termed fractalkine is a promising area of investigation, not only in terms of an
(CX3CL1), is of some interest because both it and its recep- enhanced understanding of normal brain plasticity, but also
tor (CX3CR1) are expressed constitutively in relatively for advancing knowledge about neurodegenerative diseases in
high amounts (Harrison et al. 1998; Nishiyori et al. 1998). which the loss of synaptic connections is a major and consis-
Fractalkine, which is present in both membrane-bound and tent correlate of diminished cognitive function.
secreted forms in neurons of the CNS, is bound by the frac-
talkine receptor, which is present on microglial cells. The
distinct separation in cellular localization of CX3CL1 and 7 MICROGLIAL SENESCENCE,
CX3CR1 suggests a role for fractalkine in mediating neuron– DYS T R O P H Y, A N D AG I N G -R E L AT E D
microglia interactions normally, as well as after injury. It is N E U R O D E G E N E R AT I O N
currently thought that high levels of fractalkine in uninjured
CNS neurons function in a constitutively inhibitory fashion The idea that microglia are subject to cell senescence stems
to help maintain microglia in their resting state. Constitutive from histopathological observations in the human brain
inhibition of microglia in the normal CNS is thought to be showing that with aging an increasing proportion of micro-
mediated by other neuronal molecules as well. The CD200 glial cells display abnormal morphological features, such as
molecule, which is present in neurons, has been implicated in shortened, gnarled, beaded, or fragmented cytoplasmic pro-
suppressing microglial activation through interactions with cesses, as well as loss of fine ramifications and formation of
the CD200 receptor thought to be present on the microglial spheroidal swellings, changes that were designated collectively
cell surface (Hoek et al. 2000). as microglial dystrophy (Streit et al. 2004). Although in the
The examples of fractalkine and CD200 as neuronal mol- normally aged human CNS dystrophic microglia appear only
ecules that may regulate microglial cell activity and activation sporadically and seem to be distributed at random through-
provide not only an illustration of how biochemical signaling out various brain regions, the situation changes dramatically
occurs between neurons and microglia, but also underscores in Alzheimer disease and in Down syndrome where severely
the necessity for neuron-microglia signaling to occur con- dystrophic microglial cells are abundant and appear preferen-
stantly within the CNS. Structural observations of perineu- tially in regions marked by neurofibrillary degeneration, that
ronal microglial satellite cells in the normal CNS support is, tau-positive neurofibrillary tangles, pretangles, neuropil
these molecular studies. Perineuronal microglial satellites are threads, and neuritic plaques (Figs. 8.6 and 8.7) (Streit et al.
microglia that are located vis-à-vis to CNS neurons in such a
way that the glial processes are partially wrapped around the
neuronal somata (see Figs. 8.4 and 8.5). There is close physical A
proximity between microglial satellites and neurons, a spatial
arrangement that is ideal for facilitating specific cell–cell inter-
actions involving the targeted exchange of minute quantities
of signaling molecules. Because not all neurons have perineu-
ronal microglial satellites, it is likely that those neurons that
do may have attracted the cells for a reason. The presence of
perineuronal microglial satellites could signify the need for
increased trophic support from microglia because of height-
ened metabolic demands or increased physiological activity.
In addition to providing trophic support, perineuronal micro-
glial satellites could also be involved in the remodeling of
synaptic contacts on these neurons. Microglia have long been
known to engage in synaptic remodeling, a phenomenon first B
reported more than 40 years ago (Blinzinger and Kreutzberg
1968). This potentially important role of microglia in synap-
tic plasticity received very little attention until quite recently
when a number of laboratories started to reexamine and delve
deeper into this phenomenon. It now appears that microglial
regulation of neuronal connectivity is important during
development, as well as in the normal and injured adult brain
(Paolicelli et al. 2011; Tremblay et al. 2010; Wake et al. 2009).
In fact, microglia are well equipped to participate in synap-
tic remodeling because they generate a number of enzymatic
activities, including matrix metalloproteinase, elastase, and
plasminogen activator. They also produce extracellular matrix Figure 8.6 Comparison of Normal (Ramified) and Degenerating
(Dystrophic) Microglia Using Iba1 Immunostaining in Human Cerebral
molecules, such as laminin, thrombospondin, and keratan sul- Cortex. A. A 22-year-old male subject reveals cells with normal morphology.
fate, as well as neurotrophic factors. Continued research into B. A 48-year-old female subject with Down syndrome shows cells displaying
the possible role of microglia in regulating synaptic plasticity dystrophic cytoplasmic fragmentation (cytorrhexis). Scale bar: 50 μm.

94 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
 A that immunological responses generally wane in the elderly,
B
Figure 8.7 Colocalization of Microglia with Hallmark
Neuropathological Features of Alzheimer Disease. A. Neurons expressing
hyperphosphorylated tau as they are undergoing neurofibrillary
degeneration (black reaction product) are surrounded by degenerating
(fragmented) microglial cells (brown reaction product). Immunostaining
with antibodies AT8 and iba1 in a 92-year-old male subject with
Alzheimer disease. B. Deposits of amyloid-β protein (red fluorescence)
are surrounded and infiltrated by ramified microglia (green fluorescence).
Immunostaining with antibodies 10D5 and iba1 in a 49-year-old male
subject with Down syndrome. Scale bars: 50 μm.
2009; Xue and Streit 2011). This colocalization of neurode-
generative and gliodegenerative changes has cast the patho-
genesis of neurodegeneration in a new and different light,
suggesting that a decline rather than an upsurge of immuno-
logical activity within the CNS may be responsible for the
development of aging-related neurodegenerative changes
(Croisier and Graeber 2006; Fellner et al. 2011; Streit 2010;
Streit and Xue 2009). Clearly, this idea stands in contrast to
the longstanding and popular notion that an overly active,
chronic neuroinflammatory response accounts for neuro-
degeneration in Alzheimer’s and other neurodegenerative
diseases (Eikelenboom et al. 2002; Lee et al. 2009; McGeer
and McGeer 2001), but aligns with the fact that clinical trials
designed to suppress chronic neuroinflammation with anti-
inflammatory drugs have failed to show clear benefits of such
treatments (Martin et al. 2008). This failure of attempted
immunological suppression is perhaps not surprising given
8. M I C R O G L I A L C E L L S
rendering geriatric patients more susceptible to all kinds of
infections, cancers, and other ailments because of compro-
mised immune functions. The hypothesis that is currently
being pursued claims that microglial senescence is a key fac-
tor contributing to aging-related neurodegenerative changes.
Thus, neurodegeneration occurs because progressively neu-
roprotective microglial cells are being lost, and Alzheimer
disease might be the consequence of a rapidly deteriorating
immune system in the brain.
8 S U M M A RY A N D P E R S P E C T I VE S
Tremendous advances in our understanding of microglial
biology have been made in the last two decades, which result
in large part from the availability of numerous markers for
microglia that now make it routine procedure for laborato-
ries to visualize the cells in vivo and in situ. Because of the
abundance of cell surface receptors of every imaginable type
on the microglial cell membrane, continued exploration of
molecular phenotypic changes will help to better define the
dynamic functional states of these cells and thus provide
novel insights into their involvement in diverse processes,
including (but certainly not limited to) neuroprotection,
neuroinflammation, repair, synaptic plasticity, and other tis-
sue remodeling. The recognition of dystrophic microglia in
the aging brain has provided a new and different perspec-
tive on the pathogenesis of aging-related neurodegenerative
diseases that is reshaping our views on neuroinflammation
and neuroprotection and may thus influence how future
approaches toward the treatment and/or prevention of these
conditions are constructed.
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• 97
 9.
PERICY TES OF THE CENTRAL NERVOUS SYSTEM
Martin Krueger and Ingo Bechmann

A B B R E VI AT I O N S 2 TO P O G R A P H Y O F P E R I C Y T E S W I T H I N
T H E N E U R O VA S C U L A R U N I T
Ang-1 angiopoietin 1
BBB blood-brain barrier Although pericytes have been repeatedly under investigation
CNS central nervous system during the last decades, still relatively little facts are known about
GFAP glial fibrillary acid protein their roles in health and disease. Furthermore, much of the data
GFP green fluorescent protein concerning pericytes are difficult to interpret and compare with
MMP matrix metalloproteinase other studies. This problem mainly derives from the fact that
NVU neurovascular unit pericytes are often mixed up with other cell types of neighboring
pAPN pericytic aminopeptidase N compartments, such as vascular smooth muscle cells, perivascular
PDGF-β platelet-derived growth factor β cells, or juxtavascular microglia (Table 9.1). Indeed, pericytes are
PDGFR-β platelet-derived growth factor receptor β difficult to address because they are only defined by their unique
SMA alpha smooth muscle actin position in the outermost vascular basement membrane, whereas
TGF-β transforming growth factor β an unambiguous pericyte marker is lacking. Therefore, three dif-
VEGF vascular endothelial growth factor ferent compartments within the NVU must be distinguished
VRS Virchow-Robin-Spaces (Bechmann et al. 2007). The first is constituted by the vascular
wall consisting of endothelial cells, pericytes, and if present, vas-
cular smooth muscle cells. The second compartment represents
1 INTRODUCTION the perivascular space (Virchow-Robin-Space [VRS]), which is
localized between the outermost vascular basement membrane
More than a century ago the French scientist C. M. Rouget and the basement membrane of the glia limitans. The third is the
was the first to describe a population of cells residing in the juxtavascular parenchyma, which is delineated by the glia limi-
vascular wall of capillary vessels. After a long period of neglect tans and its basement membrane (Fig. 9.1).
and misconception, pericytes have turned into focus of scien- The best way to define pericytes within the NVU is given
tific interest again as they prove to be key players in matura- by their name (peri-, meaning around; cyto-, meaning cell),
tion and regulation of the neurovascular unit (NVU). Being which describes their location around endothelial cells in
part of the vascular wall of central nervous system (CNS) microvessels (Rhodin 1968; Zimmermann 1923). With
capillaries, they are perfectly positioned to conduct criti- their long processes, pericytes follow the vessels in longitu-
cal aspects of proper functioning of the blood-brain barrier dinal direction, whereas smaller, radial arms can also encircle
(BBB), and therefore have become of interest in a variety of the capillary wall. Thus, one might have the idea that blood
cerebral disorders. (see chapter 33) vessels are engirdled or cradled by pericytes. In vitro stud-
Pericytes are positioned on the abluminal surface of the ies demonstrated that both endothelial cell and pericytes
endothelial layer, being ensheathed in their “own” basement are capable of contributing to the production of the base-
membrane. Therefore, they are located in an intermediate posi- ment membrane in which they are situated (Cohen 1980;
tion between two compartments. On the one hand pericytes Mandarino 1993), and often their association is so intimate
are in direct contact to endothelial cells as part of the vascular that the intercellular distance is narrowed down to less than
wall, and on the other hand they are juxtaposed to the astro- 20 nm (Sims 1986). In fact, the vascular and glial basement
cytic endfeet that give rise to the glia limitans and its basement membranes of the NVU are structurally and functionally dis-
membrane. Proper functioning and the ability to respond to tinct (Bechmann et al. 2007; Sixt et al. 2001) and provide
critical systemic and neural demands involve a functional clear-cut morphological borders that can be used for pericyte
network not only consisting of endothelial cells, but also detection. Often pericytic processes are found to interdigi-
pericytes, vascular smooth muscle cells, astrocytes, microglia tate with endothelial cells, thereby forming peg-socket con-
and neurons, thus making up the NVU. Therefore, this chap- tacts that consist of N-cadherin, adherens junction protein,
ter addresses pericytes as part of the NVU, highlights recent and connexin-43 hemichannels (Gerhardt et al. 2000; Li
advances in pericyte research, and illustrates current concepts et al. 2011; Winkler et al. 2011). The latter readily form gap
of their function. junctions with endothelial cells, thus allowing exchange of

98
Table 9.1 DISTINCTION BETWEEN PERICYTES AND ADJACENT CELL TYPES
CELL TYPE LOCATION MARKERS ORIGIN

Pericytes Vascular wall, ensheathed in the PDGFR-β, pAPN, (SMA) Neuroectoderm and mesoderm, turnover by
outermost vascular basement membrane blood-derived cells suggested

Smooth muscle Vascular wall, surrounded by own SMA, desmin Mesoderm

cells basement membranes

Perivascular Inside perivascular spaces CX3CR1hi, Iba-1, F4/80, Blood, high turnover by blood derived myeloid
macrophages CD11b, CD45hi, IL-B4, CD163 cells

Juxtavascular Parenchyma proper, behind and closely CX3CR1hi, Iba-1, F4/80, Yolk sac macrophages, self renewal, no exchange
microglia associated to the glia limitans CD11b, CD45lo, IL-B4 with blood-derived cells during life time

Adopted from Graeber and Streit 1990, Prinz et al. 2011.

A Pericytes and dura mater

the compartments arachnoidea
of the NVU subarachnoid
space
pia mater

glia limitans subarachnoid

vessel

neuropil

non capillary vessel capillary

perivascular space

perivascular space
endothelial cells
smooth muscle cells of the tunica media
pericytes
perivascular macrophages
astrocytes
juxtavascular microglia
dura mater
arachnoidea and pia mater
B

neuropil neuropil neuropil

capillary non capillary vessel subarachnoid vessel
vascular wall: endothelial vascular wall:endothelial cells vascular wall: endothelial cells
cells + pericytes + pericytes+smooth muscle + smooth muscle cells+
cells (if present) pericytes
basement membranes

Figure 9.1 Pericytes and the compartments of the NVU. A. This figure represents a topographic overview of the NVU along the different segments
of CNS vessels, where three distinct compartments can be identified. These compartments are the vascular wall, the perivascular (Virchow-Robin)
space, and the adjacent parenchyma proper, all of which are delineated by distinct basement membranes (Bechmann et al. 2001a; Sixt et al. 2001). B.
In the capillary segment, the vascular wall consists of endothelial cells and pericytes only. Here, the perivascular space is mostly occluded by the fused
gliovascular basement membrane, which allows direct contact of astrocytic endfeet to pericytes and endothelial cells. In noncapillary vessels such
direct contact is hampered by presence of perivascular spaces and smooth muscle cells of the tunica media. Subarachnoid vessels commonly exhibit a
continuous layer of smooth muscle cells, at least in the arterial branch of the vascular tree. In contrast, venous vessels may also show a rather irregular
covering by smooth muscle cells, which are then difficult to distinguish from pericytes. In these areas, pericytes may also be regarded as a transitional
form of smooth muscle cells. Adopted from Krüger and Bechmann 2010.

metabolites, second messengers, and ions between both cell 3 M ET H O D S F O R P E R I C Y T E D ET E C T I O N

types (Bobbie et al. 2010). Pericytes and endothelial cells also
contact the glial basement membrane within the microvascu- Pericytes express several antigens (PDGFR-ß, pAPN/CD13,
lature, whereas in noncapillary vessels such direct contact is α-smooth muscle actin, NG-2, RGS5) allowing their identi-
inhibited by Virchow-Robin-Spaces (VRS). fication (Bondjers et al. 2003; Cho et al. 2003; Krause et al.

P E R I C Y T E S O F T H E C E N T R A L N E RVO U S SYS T E M • 99
1992; Lindahl et al. 1997; Nehls and Drenckhahn 1991;
Ozerdem et al. 2001; Ruiter et al. 1993). However, none of
these markers is specific for this population. First, they may
also be present in adjacent cell types. Second, their expression
can be upregulated in diverse cell types under experimental
conditions. Precise and unambiguous identification is still
only possible by means of morphology at the ultrastructural
level and in semi-thin sections. These techniques are indeed
the only tools to demark their unique position in the vascular
wall. Although technically feasible, these methods are rarely
combined with immunocytochemistry. Unfortunately, light-
and fluorescence microscopy do not provide the necessary
resolution to accurately determine the location of putative
pericytes within the compartments of the neurovascular unit,
which is by definition a prerequisite for pericyte identification
(Figs. 9.2 and 9.3).
An antigen that already proved its specificity at the ultra-
structural level is the pericytic aminopeptidase N (pAPN/
CD13), which belongs to the family of matrix metalloprotei-
nases (Alliott et al. 1999; Kunz et al. 1995). These enzymes are
shown to be involved in zinc dependent cleavage of extracellu-
lar matrix- and non-matrix components such as growth factors
or neuropeptides (Sato 2004). The specificity of other markers
often has not been tested at the ultrastructural level. Moreover,
the expression pattern of pericytes seems to vary strongly
A
Figure 9.2 Detection of pericytes. These photos show representative
stainings for the pericyte marker pAPN, which proved its specificity at
the ultrastructural level. In the brain, pericytes are regularly found in the
capillary segment, but also at larger vessels. Their morphology can vary
depending on their position in the vascular tree. The respective shape
ranges from elongated cells, which are longitudinally oriented along the
vascular axis to a form also surrounding the vascular circumference in ves-
sels of larger diameters (arrowheads). Scale bars: (A) 50 μm, (B) 25 μm.
100 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
depending on the species, tissue, developmental state, and posi-
tion within the vascular tree. In chicken embryos, pericytes of
angiogenic vessels are regularly positive for α smooth muscle
actin (Gerhardt et al. 2000), whereas mice and rats do not show
any immunoreactivity for this marker (Hellström et al. 1999).
Electron microscopy, at least in mature vessels, still rep-
resents the most specific tool to identify pericytes (Fig. 9.4).
Unfortunately, this technique cannot be applied to every study
design. In experimental conditions of altered basement mem-
branes or during pericyte recruitment toward and away from
the vascular wall, detached pericytes are even ultrastructurally
Figure 9.3 Pericytes and their localization in the vascular wall. A. This
picture represents a confocal three-dimensional reconstruction of a
typical capillary pericyte stained for pAPN. The cell body is widely
elongated showing three major processes in longitudinal orientation.
B. These images show confocal single scans of double fluorescence stain-
ing for pAPN (pericytes; red) and laminin (basement membrane; green).
The merged picture demonstrates the close association of the basement
membrane sheaths in relation to the pericyte body and its processes.
Furthermore, it illustrates the difficulty to differentiate between distinct
vascular compartments, even using confocal microscopy. C. This image is
obtained by standard fluorescence microscopy and depicts a staining for
pericytes (pAPN; red) in red and bone marrow–derived perivascular cells
in green (a mouse chimera grafted with gfp-expressing bone marrow;
green). Again, it is often impossible to distinguish between the different
compartments of the NVU. In fact, standard fluorescence microscopy
does not offer the necessary resolution to differentiate between perivascu-
lar cells and pericytes (arrows). The arrowhead points to a bone marrow–
derived cell, which is clearly “behind” pericytes and thus could be located
within the perivascular space or the juxtavascular parenchyma.
difficult to differentiate from cells within VRS, such as cells
of the leptomeninges or other perivascular cells.
4 O R I G I N O F C E N T R A L N E RVO U S
SYS T E M P E R I C Y T E S
Initial vascularization of the CNS occurs via invading, sprout-
ing angiogenesis from a vascular plexus being juxtaposed to
the neural tube. The latter is penetrated by ingressing endothe-
lial cells that migrate toward the ventricles. The endothelial
cells are pursued by pericytes sensing cues which recruit these
cells toward the nascent vessels (Bautch and James 2009). In
this early angiogenic phase pericytes of the CNS are shown
to have two distinct origins. On the one hand, several stud-
ies using avian chimeras repeatedly demonstrated that trans-
planted neuroectoderm gives rise to pericytes of the forebrain,
and on the other hand transplanted mesoderm gives rise to
pericytes of the mid brain, brainstem, spinal cord, and periph-
eral organs (Etchevers et al. 2001; Korn et al. 2002; Kurz
2009). These studies became possible after the discovery of an
antibody selectively labelling quail angioblasts and endothe-
lial cells without cross-reactions to other species. Therefore,
it was possible to trace graft derived quail tissue in develop-
ing quail-chick chimeras. In areas of vessel formation during
angiogenesis, endothelial cells are suggested to drive migration
and attachment of pericytes along the nascent capillary tubes
(Abramsson et al. 2007; Hellström et al. 1999; Ozerdem and
Stallcup 2003, 2004; Stratman et al. 2010; Stenzel et al. 2009).
However, whether these events also take place during vascular
remodeling in the adult CNS is still under investigation.
Figure 9.4 Identification of pericytes by electron microscopy. This photo
depicts a pericyte (P) adjacent to an endothelial cell (E) of a capillary
vessel. Here, the basement membrane of the glia limitans is directly
attached to the outermost vascular basement membrane thereby forming
a fused gliovascular basement membrane (dotted line), which occludes
the perivascular space in the capillary segment. The pericyte also shares
a basement membrane (dashed line) to the endothelial layer. Therefore,
pericytes are by definition part of the vascular wall (first compartment).
Clear-cut identification is only possible at this ultrastructural level.
Arrow, tight junction complex; L, vascular lumen; N, neuropil.
P E R I C Y T E S O F T H E C E N T R A L N E RVO U S SYS T E M
Other studies describe the bone marrow to constitute
a reservoir for pericyte precursors (Kokovay et al. 2006;
Lamagna and Bergers 2006). Using green fluorescent protein
(GFP)-positive bone marrow mouse chimeras, Kokovay et al.
investigated whether pericytes are recruited from the periph-
ery to stabilize blood vessels in the model of experimental
stroke. Indeed, blood-derived GFP-positive cells were found
in areas of robust angiogenic response after stroke. However,
these cells were insufficiently determined to be pericytes by
their expression of desmin, vimentin, and angiogenic factors
(Kokovay et al. 2006). Ultrastructural identification is lack-
ing in these studies. Moreover, investigating recruitment of
blood-derived cells in models of bone marrow transplanta-
tion strictly depends on lethal irradiation of host animals.
Today, there are several lines of evidence suggesting that irra-
diation itself may condition the brain to attract myeloid lin-
eages. On the one hand this may result from the upregulation
of chemoattractant factors, and on the other hand result from
the loss or downregulation of inhibitory signals (Mildner
et al. 2007). Therefore, the physiological turnover of CNS
pericytes by blood-derived cells remains to be discussed.
In the past, pericytes were also repeatedly reported to derive
from the monocyte/macrophage lineage. Several studies dem-
onstrated the expression of myeloid markers as ED1 or CD11b
on pericytes (Balabanov et al. 1996; Graeber et al. 1989). These
findings were supported by in vitro studies demonstrating the
ability of major histocompatibility class II antigen (MHC-II)
expression on stimulation with interferon γ (Dore-Duff y
and Balabanov 1998). Further studies also addressed their
phagocytic potential using antibody-coated zymosan and
fluorochrome-conjugated polystyrene beads (Balabanov et al.
1996). However, the methods used for identification were rather
imprecise in these studies; thus, the findings may likely relate to
perivascular macrophages. Indeed, this population was shown to
be capable of phagocytosis, act as antigen-presenting cells, and
is supplemented by hematogenous precursors (Bechmann et al.
2001a, b; Greter et al. 2005; Hickey and Kimura 1988; Priller
et al. 2001). Furthermore, the question of whether pure peri-
cyte cultures can be established is not yet answered. Therefore,
potential pericyte precursors remain a subject of further inves-
tigation, and their ability to be recruited to the CNS so as to
renew the resident population during injuries or under normal
conditions remains to be determined.
5 PERICY TE SIGNALING
Central nervous system pericytes are involved in a variety of dif-
ferent signaling cascades. These include platelet-derived growth
factor β (PDGF-β), transforming growth factor β (TGF-β),
Notch, and angiopoietin. Endothelial cells have been shown to
secrete PDGF-β, whereas its receptor is expressed on vascular
mural cells, including pericytes (Bell et al. 2010; Hellström et al.
1999; Lindahl et al. 1997; Winkler et al. 2010). The secreted
ligand readily interacts with heparin sulfate proteoglycans
within the extracellular matrix, thereby establishing a steep
concentration gradient by local retention of PDGF-β (Andrae
et al. 2008). This gradient was suggested to be pivotal for proper
pericyte recruitment and attachment to the capillary tube
• 101
(Abramsson et al. 2003, 2007; Armulik et al. 2010; Lindblom
et al. 2003). In contrast, pericyte proliferation was dependent
on the freely diffusible ligand (Abramsson et al. 2003, 2007).
Nevertheless, the detailed mechanism behind the different
PDGF-β responses remains to be demonstrated. It is impor-
tant to note that the density of pericytes within the develop-
ing neural tube correlates with the levels of PDGFR-β, thereby
emphasizing the importance of this signaling cascade (Tallquist
et al. 2003). Moreover, recent studies also suggested a role for
PDGFR-β signaling in pericyte survival (Bell et al. 2010). Mice
lacking the ability of PDGF-β/PDGFR-β signaling by homozy-
gous deletion of the respective genes are shown to develop lethal
phenotypes by a complete absence of pericytes in CNS vessels
(Hellström et al. 1999; Leveen et al. 1994; Soriano 1994).
The source of TGF-β variants in the CNS is commonly
known to be rather widespread. Cellular sources of this
ligand are represented by glial cells, neurons, endothe-
lial cells, and pericytes, all of which produce TGF-β pri-
marily as protein-bound forms (Gaengel et al. 2009). On
activation by thrombospondin or integrins, TGF-β binds
to TGF-β receptor type 2, resulting in activation of the
activin like kinase 5 (ALK 5)/Smad pathway, leading to
differentiation, migration, and/or proliferation (Lebrin
et al. 2005). Vascular development strictly depends on the
function of these signaling cascades. Therefore, knocking
out one of the respective genes (TGF-β, ALK 5, endoglin,
or Smad4 and Smad 5) results in an embryonically lethal
phenotype in mice, which exhibit defective vascular devel-
opment (Gaengel et al. 2009). Thus, the current concept
suggests that vascular development critically depends on an
intact bidirectional signaling between endothelial cells and
pericytes via TGF-β. On the one hand, TGF-β binding to
TGF-β receptor type 2 on pericytes induces production of
extracellular matrix as well as contractile elements, whereas
proliferation is inhibited (Sieczkiewicz and Herman 2003;
van Geest et al. 2010). On the other hand, the TGF-β signal-
ing cascade in concert with Notch signaling in endothelial
cells enhances firm pericyte attachment by upregulation of
N-cadherin in peg-socket contacts (Gerhardt et al. 2000; Li
et al. 2011; Winkler et al. 2011). Moreover, pericyte attach-
ment and vessel stabilization are also shown to be driven
by sphingosine-1-phosphate signaling (Gaengel et al. 2009;
Paik et al. 2004). To our current understanding, recruited
pericytes attach to the endothelial layer and promote sta-
bilization of the capillary tube by TGF-β signal transduc-
tion via the ALK 5—Smad2/3 pathway and inhibition of
endothelial proliferation (Li et al. 2011) Furthermore, sev-
eral lines of evidence suggest this signaling cascade to be
crucial for vascular maturation, basement membrane forma-
tion, and BBB establishment (Dohgu et al. 2005; van Geest
et al. 2010; Walshe et al. 2009) (Fig. 9.5).
A variety of data additionally suggest Notch to be a key
player during vascular development and angiogenesis, which
also implies crucial roles for pericytes. Still there are very lit-
tle available data addressing this issue. At least recent studies
demonstrate the importance of Notch signaling for pericyte
attachment and survival (Li et al. 2011; Liu et al. 2009; Regan
and Majesky 2009; Stewart and Wiley 2011; Walshe et al. 2011;
Winkler et al. 2011). In mammals, there are four described
102 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
receptors, Notch1-Notch4, and five ligands, delta-like (DLL)
1, 3, and 4, and jagged ( JAG) 1 and 2. Because all these pro-
teins, receptors as well as ligands, represent transmembrane
proteins, proper cell contacts between endothelial cells
and pericytes are necessary to induce signal transduction
by binding of ligand and receptor on directly opposing cell
membranes. After binding, the Notch intracellular domain
(NICD) is released by proteolytic cleavage and translocates to
the nucleus, where it binds the transcription factor RBP-Jк.
The putative role of this pathway for proper vascular stabiliza-
tion is underlined by studies using a specific knockout model
of RBP-Jк in endothelial cells, which is shown to display peri-
natal hemorrhages. Indeed, morphological analysis revealed
impaired pericyte adhesion and reduced pericyte coverage of
the vasculature characterizing this phenotype (Li et al. 2011;
Winkler et al. 2011). The potential role of Notch signaling
in vessel maturation is further substantiated by the finding
that endothelial contact to mural cells upregulates Notch3 in
pericytes and vascular smooth muscle cells (Liu et al. 2009).
Notch3 has also been suggested to convey survival of mural
cells (Walshe et al. 2011). Therefore, it is not surprising that
Notch may also be involved in the regulation of PDGFR-β
signaling ( Jin et al. 2008). Thus, it is likely that PDGFR-β in
concert with Notch and its ligands functions in vascular sta-
bilization and pericyte differentiation, which has already been
postulated for DLL4 (Stewart et al. 2011).
ECM production
+differentiation
inhibititon of
proliferation
pericyte recruitment
TGF βR
TGF β
N-cadherin
PDGFR- β
Notch ligand
TGFβR
TGFβ N-cadherin
PDGF- β Ang1 TGF β
Notch1
Tie-2
TGF βR2
S1P1
S1P
endothelial maturation
BBB formation
Figure 9.5 Pericyte Signaling. Endothelial cells (yellow) secrete
PDGF-β, which is retained in the extracellular matrix. Binding to the
receptor PDGFR-β on pericytes (green) leads to receptor dimerization
and activation of signaling cascades, resulting in recruitment toward
endothelial cells. Firm attachment is further driven by binding of TGF-β
to its receptor TGFβR2. In pericytes TGFβR2-mediated signaling results
in an inhibition of proliferation and production of ECM components. In
endothelial cells, binding of TGF-β to its receptor also leads to inhibi-
tion of proliferation and acts in concert with Notch and S1P signaling
in upregulation of N-cadherin, which mediates formation of so called
peg-socket contacts. Pericytes can also contribute to BBB formation in
the endothelium by angiopoietin 1 (Ang-1)/Tie-2 and TGF-β/TGFβR2
signaling. Adapted from Winkler et al. 2011.
 6 F U N C T I O N S O F C E N T R A L N E RVO U S paracellular leakage as a consequence of decreased expression
SYS T E M P E R I C Y T E S of tight junction-associated proteins (Armulik et al. 2010; Bell
et al. 2010). In pericyte deficient mice, Bell and colleagues
demonstrated the progressive reduction of pivotal tight junc-
6.1 P E R I C Y T E S : R E GU L ATO R S O F T H E tion proteins such as occludin, claudin-5, and zonula occludens
B L O O D -B R A I N BA R R I E R 1 (Bell et al. 2010). In fact, increased paracellular leakage of
hydrophilic tracers correlated with an age-dependent decrease
Since the discovery of pericytes, their putative functions in
of pericyte density on CNS vessels. Increased paracellular leak-
the CNS have been discussed repeatedly. The NVU is unique,
age and subsequent accumulation of vasculotoxic and neuro-
not only by means of its architecture, but also by means of its
toxic blood-derived molecules in the vascular wall or adjacent
tight, barrier-forming function. Therefore, it has been tempt-
perivascular spaces may likely result in reduced microvascular
ing to link Paul Ehrlich’s early finding that the brain vascu-
perfusion and therefore result in secondary neurodegeneration
lature proved tight to certain hydrophilic molecules with
(Bell et al. 2010; Winkler et al. 2011).
another feature of CNS vessels, that is, the high density of
The contribution of pericytes to vascular stability was
pericytes within the CNS. Indeed, the number of pericytes
further substantiated by addressing the angiopoietins Ang-1,
covering the vascular wall was found to be significantly higher
Ang-2, and their receptor Tie-2 in the model of PDGF-β–
compared with peripheral organs (Shepro and Morel 1993).
deficient mice. Pericytes and perivascular cell populations are
Thus, a cardinal role for pericytes in the maintenance of the
responsible for the production of Ang-1 and Ang-2, whereas
BBB has been suggested, but has also been attributed to astro-
their receptor is expressed on endothelial cells (Davis et al.
cytes (Arthur et al. 1987; Janzer and Raff 1987; Stewart et al.
1996; Suri et al. 1996). Here, Ang-1 restored the vascular struc-
1981). However, later studies succeeded to demonstrate the
ture and function in models of PDGF-β deficiency (Uemura
establishment of tight junction complexes in the absence of
et al. 2002). Therefore, Ang-1 is proposed to act as a stabiliz-
astrocytes (Felts and Smith 1996; Jaeger and Blight 1997).
ing factor, whereas Ang-2 is a merely destabilizing cue, as it
Using mice deficient for the astrocytic protein glial fibril-
binds to its receptor without inducing signal transduction. The
lary acidic protein (GFAP) Balabanov et al. demonstrated
antagonistic model of both ligands has been confirmed by sev-
an increased coverage of pericytes in CNS vessels (Balabanov
eral other studies (Maisonpierre et al. 1997; Sato et al. 1995;
and Dore-Duff y 1998). This finding was interpreted as a pos-
Suri et al. 1996). There is also evidence that in ischemic models
sible counter-regulation against vascular leakage. Later stud-
Ang-1 is capable of reducing BBB leakage (Zhang et al. 2002).
ies substantiated this theory in that addition of pericytes to
Vascular stability is also shown to depend on sphingosine-
endothelial cell monolayers led to an increased barrier func-
1-phosphate (S1P) signaling as described, which involves
tion for hydrophilic molecules and augmented transendothe-
regulation and expression of N-cadherin and VE-cadherin
lial resistance (Dente et al. 2001). This implies that different
in endothelial cells (Armulik et al. 2005). Expression of both
populations of cells of the NVU are involved in maintaining
molecules is induced via binding of S1P to its receptor (Paik
barrier function and, in case of functional impairment of one,
et al. 2004). VE-cadherin is found in interendothelial junc-
the other can take over to prevent further damage.
tions, whereas N-cadherin is engaged in forming peg-socket
The importance of pericytes in the formation of the BBB
contacts between endothelial cells and pericytes. Interfering
mostly derives from studies using mice lacking PDGF-β or its
with S1P signaling regularly results in decreased pericyte and
receptor. These mice display the same vascular phenotypes of
smooth muscle cell coverage of blood vessels, again causing
pericyte-depleted vessels with hemorrhages and microvascu-
vascular abnormalities and neonatal lethality (Liu et al. 2000).
lar leakage consecutively leading to neonatal lethality. Recent
Interestingly, S1P signaling is currently focused as a putative
studies using depleted and hypomorphic alleles for PDGFR-β
drug target for the therapy of multiple sclerosis by Fingolimod
have demonstrated an early impact of pericytes on the devel-
(FTY720). This drug is suggested to downregulate lymphocyte
opment of the BBB. Vascular alterations were found at the
egress from lymphoid tissues, which is thought to reduce infil-
known time of the appearance of pericytes, an event markedly
tration of autoreactive lymphocytes to the CNS. However, also
preceding the formation of astrocytes (Daneman et al. 2010).
the potential (beneficial) influence for the modulation of neu-
This study also provided evidence that pericyte coverage of the
roinflammation at the CNS vasculature should be discussed.
endothelial wall regulates the formation of tight junctions and
transendothelial vesicle trafficking.
6.2 P E R I C Y T E S A N D A N G I O G E N E S I S
It is important to note that viable pericyte-deficient mice
with disturbed PDGFR-β signaling do not lack the expression In general, pericytes are commonly seen as a rather static
of BBB associated genes (Armulik et al. 2010; Li et al. 2011). In population, which is shown to be critically involved in vessel
contrast, they lack genes downregulating vascular leakage such as maturation and stabilization. However, pericytes are recruited
angiopoietin-2 and plasmalemma associated protein. Moreover, to newly forming vascular tubes by sensing PDGF-β, which
pericytes suppress the expression of genes increasing endothelial is secreted by endothelial cells. There are also several lines of
permeability and infiltration of blood-borne leukocytes already evidence strongly suggesting a complex and more dynamic
at a time of a rather immature BBB (Daneman et al. 2010). function during angiogenesis because pericytes are described
However, the crucial role of pericytes in regulating the to express matrix metalloproteinases (MMP-2, MMP-3, and
BBB is not limited to embryogenesis, but remains essential MMP-9) (Candelario-Jalil et al. 2009; Virgintino et al. 2007)
throughout adulthood and aging. In the aging brain, loss of and urokinase plasminogen activator receptor (Dore-Duff y
CNS pericytes has been demonstrated to accompany increased et al. 2000). On the other hand, and quite contrary to the
P E R I C Y T E S O F T H E C E N T R A L N E RVO U S SYS T E M • 103
function of these proteins they also contribute to the synthesis
of extracellular matrix components (Diaz-Flores et al. 2009;
Stratman et al. 2009; van Geest et al. 2010) and tissue inhibi-
tor of metalloproteinase 3 (TIMP-3), which is a potent inhib-
itor of a number of MMPs (Saharinen et al. 2008).
Ultrastructural studies indeed demonstrated that pericytes
are the first population within the NVU to respond to brain
hypoxia and traumatic brain injury, which are strong stimuli for
angiogenesis (Dore-Duff y et al. 1999, 2000; Gonul et al. 2002).
As early as 2 hours after brain hypoxia pericytes showed first
morphological alterations, whereas no other populations of the
NVU were visibly affected. The authors demonstrated a thick-
ening of the luminal basement membrane, whereas the ablu-
minal one was evidently thinned out. Moreover, characteristic
“peaks” in the sense of pericytic processes were detectable point-
ing toward the parenchyma. These findings were interpreted as
initial signs of migration from the vascular wall (Gonul et al.
2002). Comparable observations were reported after traumatic
brain injury. In this study the authors observed elongated peri-
cytes and confirmed the disappearance of the abluminal base-
ment membrane at leading pericyte processes. Additionally,
migration of pericytes was confirmed by a decreased density of
pericyte on affected vessels (Dore-Duff y et al. 2000).
Activation and migration of cells can be linked to the expres-
sion of urokinase plasminogen activator (uPA) and its receptor
(uPAR) on migrating cells (Blasi 1999; Washington et al. 1996).
Indeed, pericytes are reported to express uPAR on leading pro-
cesses (Dore-Duffy et al. 2000). It is important to note that the
complex of ligand and receptor represents an active protease and
its expression on migrating pericytes might be crucial to over-
come the barrier of the ensheathing basement membrane to clear
a way for other cell types, such as endothelial cells. Of note, peri-
cytes remaining in position exhibited cytoplasmic and nuclear
alterations suggesting degeneration of nonmigratory pericytes
(Dore-Duffy et al. 2000). Moreover, leading tips of migratory
pericytes are shown to interfere with synaptic complexes. This was
interpreted as an initial sign of synaptic stripping originally relat-
ing to the concept of an active displacement of synaptic terminals
by microglial cells (Blinzinger and Kreutzberg 1968; Trapp et al.
2007). Whether this is indeed an active displacement by pericytes
or a neuronal process that further leads to appearance of pericytic
processes in a second event is yet to be elucidated.
Furthermore, hypoxia is reported to stimulate production
of vascular endothelial growth factor (VEGF) in pericytes
(Yamagishi et al. 1999). This molecule is reported to increase
pericyte density on newly formed vessels in a dose-dependent
manner (Benjamin et al. 1998). With the expression of VEGF,
pericytes do also have an impact on endothelial cells by enhancing
endothelial survival, proliferation, and formation of angiogenic
sprouts (Darland et al. 2003; Ozerdem and Stallcup 2004).
In summary, pericytes appear to have a dual role in angio-
genesis. On the one hand, they seem to be capable of inducing
endothelial cell migration and proliferation during early phases
(Darland et al. 2003; Ozerdem and Stallcup 2003, 2004). On
the other hand, they promote vascular stability and reduction
of proliferation in later stages (Hellström et al. 2001; Li et al.
2011). Therefore, it may be important to clarify whether peri-
cytes initiate vascular sprouting and guide endothelial tube
formation or if they are recruited into tubes by endothelial
104 • M O R P H O L O GY, U LT R A S T RU C T U R E , A N D I D E N T I F I C AT I O N
cells (Dore-Duff y et al. 2000; Gerhardt and Betsholtz 2003;
Ozerdem and Stallcup 2003, 2004; Virgintino et al. 2007).
6.3 P E R I C Y T E S C O N T RO L C A P I L L A RY
D I A M ET E R A N D B L O O D FL OW
In contrast with vascular smooth muscle cells, the major pro-
cesses of pericytes are oriented in parallel to the longitudinal
axis of the vessels. Therefore, they should hardly be capable of
constricting capillary vessels. However, pericytes were initially
described as contractile elements by Rouget in 1873. He distin-
guished this population from migratory and phagocytic leuko-
cytes (Rouget 1874, 1879), and later investigators confirmed his
assumption (Mayer 1902; Vimtrup 1922; Zimmermann 1923).
Mayer and Zimmermann even suggested pericytes and smooth
muscle cells of the tunica media to be a continuous population
with transitional forms between the two extremes (Mayer 1902;
Zimmermann 1923). Applying an early form of live imaging tech-
nique, Vimtrup observed capillary contractions, which began at
single pericytes and spread onward in both directions from the
cell body (Vimtrup 1922). This study was performed in young
living larvae, which were constantly rinsed with fresh water at the
body, whereas the transparent tail was observed on a microscope
slide. To ensure that what he observed really were capillaries, he
restricted his studies to areas in which the afferent arteriole as
well as the efferent venule were visible. That way, he was able to
identify the capillary bed in between (Vimtrup 1922).
Since then, the discussion of whether pericytes contribute to
the regulation of blood flow by contracting capillaries has always
been alive. More recent studies corroborated the view on pericytes
as regulators of capillary blood flow by describing their potential
ability to respond to vasoactive substances, such as nitric oxide,
prostacyclin, angiotensin II, and endothelin-1, as they are reported
to express corresponding receptors (Chakravarthy and Gardiner
1999; Dehouck et al. 1997; Healy and Wilk 1993). Another
prerequisite for active contraction is the presence of contractile
filaments; in fact, both smooth muscle and non–smooth muscle
isoforms were found in CNS pericytes (Herman and D’Amore
1985), albeit with species-dependent variations of the expression
of alpha smooth muscle actin (SMA). For example, in embryonic
chicken, SMA can be detected in virtually all pericytes, whereas
in mice and rats the situation is quite different (Gerhardt et al.
2000; Hellström et al. 1999). In mice, the expression of SMA
in cerebral pericytes is restricted to the ones positioned on ves-
sels of a diameter larger than 10 μm. Vessels with a diameter of
less than 10 μm are on the other hand negative for SMA (Alliott
et al. 1999; Nehls and Drenckhahn 1991). These findings raise
the question of whether or not pericytes are indeed structurally
capable of contracting capillaries because these proteins are lack-
ing in the respective segment. Another explanation is that com-
mon methods simply fail to detect low levels of SMA in pericytes,
which again would raise the question of whether such low expres-
sion under the detection level is functionally relevant.
However, the differential expression of contractile elements is
nevertheless a remarkable finding because it suggests functional
differences of CNS pericytes along the vascular tree. A possible
explanation may be given by the morphology of the NVU within
the respective vascular segment. At the level of capillaries, astro-
cytic endfeet of the glia limitans are separated only by a basement
membrane. In non–capillary vessels, direct and specific interac-
tion is hampered by the presence of PVS (see Fig. 9.1). Thus, the
loss of astrocytic contact may change the expression pattern to
a rather smooth muscle-like pericyte isoform. However, this
potential influence needs to be determined.
Current textbooks comprehensively claim that blood flow
is regulated by precapillary arterioles. Therefore, it is notewor-
thy that the majority of the noradrenergic innervation of CNS
blood vessels is reported to terminate in the vicinity of capillaries
(Cohen et al. 1997). Indeed, two recent studies using live imaging
techniques confirmed Rouget’s original concept of pericytes as
contractile elements in the capillary bed. In 2006, Peppiatt et al.
demonstrated pericyte-driven capillary constriction by ATP and
noradrenalin in rat retinal and cerebellar slices. Also, administra-
tion of GABA blockers led to a visible reduction of the capillary
diameter, which was readily abrogated by addition of glutamate
(Peppiatt et al. 2006). The constriction of capillary vessels by
pericytes was further demonstrated in vivo (Fernández-Klett et
al. 2010). Application of intravital two-photon laser scanning
microscopy in mice did not only allow visualization of constric-
tions of the capillary wall in response to thromboxane analogs, but
also allowed determination of red blood cell perfusion changes
in constricted capillaries. Furthermore, the authors described a
significantly higher change of the capillary diameter adjacent to
pericyte bodies compared with areas only reached by pericytic
processes (Fernández-Klett et al. 2010). This finding may reflect
the unique morphology of pericytes, which better enables them
to retract their longitudinal processes than to constrict the vas-
cular circumference. Thereby, contraction may lead to shortened
processes and consequently thickening of the body, which again
narrows the capillary lumen. However, the authors also dem-
onstrated that capillary dilatation is not required for functional
hyperemia (Fernández-Klett et al. 2010). Thus, the functional
impact of pericytes on cerebral blood flow under physiological
conditions still remains to be determined, but methods are now
available to continue on this path.
7 PERICY TES IN DISEASES OF THE
C E N T R A L N E RVO U S SYS T E M
Diabetic retinopathy represents a widespread complication of
type I and type II diabetes in which early pericyte loss is a com-
mon pathological hallmark. Here, a nonproliferative phase can
be distinguished from a proliferative phase (Hammes et al. 2011).
The nonproliferative phase is characterized by vessel regression,
occluded and pericyte-deprived capillaries, and microaneurysms,
whereas the proliferative phase exhibits massive proliferation of
abnormal, rupture-predisposed vessels. The latter often give rise
to hemorrhages and retinal detachment. Interestingly, mouse
models of PDGF-β deficiency or Ang-2 overexpression have
demonstrated a phenotype almost mimicking nonproliferative
diabetic retinopathy (Hammes et al. 2004; Pfister et al. 2010).
However, in a study selectively deleting PDGF-β in endothelial
cells the authors also demonstrated a switch toward a prolifera-
tive phenotype as pericyte coverage dropped below 50%, thereby
suggesting a threshold for pericyte loss either leading to nonpro-
liferative or proliferative vascular pathologies (Enge et al. 2002).
The critical role of PDGF-β pathway was further confirmed by
P E R I C Y T E S O F T H E C E N T R A L N E RVO U S SYS T E M
the finding that hyperglycemia- mediated dephosphorylation of
PDGFR-β led to resistance to endothelially secreted PDGF-β,
which led to pericyte apoptosis in this model (Geraldes et al.
2009). Therefore, it is possible that the PDGFR-β pathway may
be target for future pharmacological inventions.
Recently, pericytes have also become drug targets in tumor
therapy because they are vitally involved in angiogenesis. Of
note, antiendothelial tumor therapy alone by using VEGF
inhibitors proved insufficient because pericytes often remained
in position (Baluk et al. 2005). They are believed to provide a
scaffold for rapidly regrowing tumor vessels after removal of
VEGF inhibition. Therefore, a synergistic therapy was suggested
affecting both proliferation of endothelial cells and survival of
pericytes. In respective studies the positive effect in tumor ves-
sel reduction was confirmed in animal models by combining
anti-VEGF and anti-PDGF-β/PDGFR-β signaling (Bergers et
al. 2003; Erber et al. 2004). However, inhibition of PDGFR-β
alone does not result in endothelial regression (Abramsson et
al. 2003), but augments the effect of VEGF inhibitors (Bergers
et al. 2003) or antimitotic drugs such as Taxol (Pietras et al.
2002). Recently, also aminopeptidase N (APN) has become of
interest in tumor therapy as combined treatment with antien-
dothelial therapies prolonged survival of human neuroblastoma
bearing mice (Loi et al. 2010). Therefore, it may be possible that
future tumor therapies include antipericyte drugs and hopefully
enhance outcome by deprivation of tumor angiogenesis.
Neuronal function critically depends on an intact NVU;
consequently, neurovascular dysfunction is often associated with
neurodegeneration (Zlokovic 2008). Therefore, the function of
the vascular wall and its proper architecture and signaling, which
also involve pericytes, is essential to maintain a healthy neuronal
microenvironment. Pericyte-deficient mice have recently been
reported to express a neurodegenerative phenotype as a conse-
quence of BBB breakdown, leading to toxic extravasation of plasma
proteins and chronic hypoperfusion with consequential hypoxia
(Bell et al. 2010). Because vascular lesions in pericytes-deficient
mice precede inflammation and neuronal damage, it is possible
that this secondary inflammation also contributes to the observed
neuronal damage (Quaegebeur et al. 2010).
Nevertheless, the exact role of pericytes in specific neu-
rodegenerative disorders such as Alzheimer disease remains
to be determined. Accumulations of amyloid-β are known
to occur around capillaries and pericytes (Vinters et al. 1994;
Wisniewski et al. 1992) and addition of amyloid-β peptides to
isolated human brain pericytes has proved to lead to pericyte
death in vitro (Wilhelmus et al. 2007). Furthermore, mouse
models of amyloid-β overexpression display BBB breakdown
and reduced vascular density before neuronal loss or amyloid
deposition occurs (Paul et al. 2007; Zlokovic 2005). Because
several lines of evidence suggest vascular basement membranes
to be drainage pathways for solutes (Carare et al. 2008; Hawkes
et al. 2011), impaired pericyte function and contractility by
vascular and perivascular amyloid beta deposition may cause
reduced clearance of soluble amyloid peptides, thus perpetuat-
ing a vicious cycle. This hypothesis may be corroborated by the
finding that experimentally induced hypercontraction of cere-
bral arterioles resulted in reduced amyloid clearance (Bell et al.
2009). However, the precise role of pericytes and their potential
role as targets in clinical therapies are yet to be established.
• 105
 8 S U M M A RY A N D P E R S P E C T I VE S
Although several lines of evidence now confirm a role for peri-
cytes in regulating capillary diameters, the lack of a specific
marker for these cells still causes confusion. Even though under
normal conditions pericytes can be identified by their topo-
graphic localization within the vascular wall, it is still impos-
sible to unequivocally determine their fate once they detach
from the endothelial basement membrane. This is illustrated
by the recent description of pericytes playing a role in scar for-
mation after spinal cord injury (Göritz et al. 2011). Although
cells expressing pericyte markers were clearly identified within
the scar, one cannot exclude that this expression is induced on
cells that never had been pericytes based on the topographic
definition. Unless both, basement membranes and pericytes
can be visualized in vivo over long periods of time, this issue
will remain a matter of debate. Eventually it may turn out that
the term pericytes is misleading, because it only describes a sta-
tionary phase of a highly versatile and mobile cell.
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 10.
NG2 CELLS (POLYDENDROCY TES)
Akiko Nishiyama

A B B R E VI AT I O N S and other glial cell populations. They are the primary source
of myelinating and nonmyelinating oligodendrocytes and are
BrdU 5-bromo-2′-deoxyuridine hence often equated with oligodendrocyte progenitor cells
CNP 2′,3-cyclic nucleotide 3′-phosphodiesterase (OPCs), although some questions remain as to whether all
CNS central nervous system polydendrocytes generate oligodendrocytes. Polydendrocytes
Cspg-4 chondroitin sulfate proteoglycan-4 are distributed throughout both gray and white matter and
EAE experimental autoimmune encephalomyelitis are distinct from neural stem/progenitor cells or radial glia.
Ecrg4 esophageal cancer–related gene 4 This chapter summarizes the current knowledge of polyden-
EGF epidermal growth factor drocytes based primarily on morphological observations,
EGFP enhanced green fluorescent protein and highlights some unsolved questions related to their iden-
GFAP fibrillary acidic protein tity and lineage. A more extensive review of their development
GFP green fluorescent protein and electrophysiological properties is provided in chapters 13
GLAST glutamate aspartate transporter and 21.
H2B histone 2B
HDAC histone deacetylase
Hes-5 hairy and enhancer of split-5 2 I D E N T I F I C AT I O N O F
ID2/4 inhibitor of differentiation 2/4 P O LY D E N D R O C Y T E S
MCSP melanoma chondroitin sulfate proteoglycan
MS multiple sclerosis
OPC oligodendrocyte precursor cell 2.1 I M MU N O H I S TO C H E M I C A L
PDGF platelet-derived growth factor I D E N T I FI C AT I O N
Pdgfra alpha receptor for platelet-derived growth
factor 2.1.1 Cell Surface Antigens
PDZ domain postsynaptic density 95/disk large/zonula-
occludens-1 domain Polydendrocytes are identified by their typical multiprocessed
PLP proteolipid protein morphology and the expression of two defining integral
PSA-NCAM polysialylated form of neural cell adhesion plasma membrane antigens, NG2 and Pdgfra (Table 10.1).
molecule Pdgfra belongs to the family of receptor tyrosine kinases with
SVZ subventricular zone a split intracellular tyrosine kinase domain and transduces sig-
YFP yellow fluorescent protein nal when bound by its cognate dimeric ligands, which include
PDGF-AA, PDGF-BB, and PDGF-CC homodimers and
PDGF-AB heterodimers. Pdgfra mediates survival, prolifera-
1 INTRODUCTION tion and migration of polydendrocytes (Andrae et al. 2008;
Vora et al. 2011; see also chapter 22).
Studies conducted over the last three decades have revealed a NG2 is a product of the Cspg-4 (chondroitin sulfate
glial progenitor cell population in the developing and mature proteoglycan-4) gene, also known as the melanoma chon-
mammalian central nervous system (CNS), which are com- droitin sulfate proteoglycan (MCSP) in humans. It exists as
monly known as NG2 cells. The term polydendrocyte is pro- a 300-kDa single membrane-spanning core glycoprotein or as
posed as a formal name for NG2 cells in recognition of their a heterogeneous high molecular weight proteoglycan. NG2
existence as a normal resident cell population in the adult interacts with many extracellular and intracellular proteins
CNS and to match the nomenclature of other macroglial and has been implicated in the regulation of cell migration and
cells. The name reflects their highly branched morphology proliferation (Nishiyama et al. 2009; Stallcup 2002; Sugiarto
and lineal kinship to oligodendrocytes (Nishiyama 2007). et al. 2011; Trotter et al. 2010).
Polydendrocytes are defined as nonvascular cells in the CNS In the oligodendrocyte lineage, Pdgfra and NG2 are highly
parenchyma that express NG2 and the alpha receptor for expressed on proliferating progenitor cells, and the expression
platelet-derived growth factor (Pdgfra). They comprise 2% of these genes is downregulated as the cells differentiate into
to 9% of the cells in the CNS and are distinct from neurons oligodendrocytes (Fig. 10.1). Neither protein is expressed

109
Table 10.1 SPECIFICITY OF COMMONLY USED ANTIGENS EXPRESSED BY POLYDENDROCYTES
ANTIGEN EXPRESSION

NAME DESCRIPTION NSCs PDs ODs ASTROCYTES NEURONS MICROGLIA

NG2 Cell surface – + – – – – On resting microglia

proteoglycan + On some activated
macrophages

Pdgfra Receptor tyrosine – + – – – –

kinase

Olig2 Transcription factor + In some + + Weaker – On most – –

Sox10 Transcription factor – + + – – –

CNPase Cytosolic protein – – On most + on some + – – –

PLP Membrane protein – – On most + on some + – – –

O4 Membrane – + On most (rat) + – – –

glycolipid + on some (mouse)

S100β Cytosolic calcium- – + In some + In some + – –

binding protein

NSC, neural stem cell; OD, oligodendrocyte; PD, polydendrocyte; – not detected; + detected.

exclusively on polydendrocytes, and outside the CNS they are
expressed on cycling lineage committed progenitor cells of the
mesenchyme and skin. In the CNS, NG2 is also detected on
vascular mural cells, most commonly on pericytes and smooth
muscle cells (Nishiyama et al. 2009). Pdgfra is also expressed
by vascular cells (Richardson et al. 2011).
One caveat to using these cell surface antigens to identify
polydendrocytes is that both NG2 and Pdgfra are sensitive
to overfixation with aldehydes (Mori et al. 2009). However,
with careful control of tissue processing conditions, NG2
and Pdgfra have been successfully detected in postmortem
human CNS tissue, where they share many of the character-
istics of rodent polydendrocytes (Chang et al. 2000; Jennings
and Carroll 2010; Reynolds et al. 2002; Wolswijk 1998). In
a microarray analysis, the Cspg-4 and Pdgfr transcripts were
identified as the two most highly enriched in acutely iso-
lated glial progenitor cells from human white matter (Sim
et al. 2006) (see chapter 29). Similarly, Cspg-4 and Pdgfra tran-
scripts were among the five most enriched in polydendrocytes
isolated from P16 mouse brain by immunopanning for Pdgfra,
compared with mature oligodendrocytes (Cahoy et al. 2008).
2.1.2 Transcription Factors
Oligodendrocyte lineage transcription factors such as Olig2
and Sox10 are expressed in polydendrocytes and are often
used in conjunction with the aforementioned cell surface
antigens for their identification (see Fig. 10.1; Table 10.1).
The basic helix-loop-helix transcription factor Olig2 and the
product of the adjacently located gene Olig1 are implicated

Neuroepithelial NG2 cells Immature

= polydendrocytes Oligodendrocytes
cells oligodendrocytes
germinal zone parenchyma parenchyma

Olig2
Sox10
NG2, Pdgfra
CNP
PLP

Figure 10.1 Relationship Between Polydendrocytes and Oligodendrocyte Lineage Cells. Box indicates polydendrocytes that are process-bearing cells that
express NG2 and Pdgfra. Thick gray lines indicate the duration of expression of the indicated antigens.

110 • NEUROGLIA
in oligodendrocyte specification and differentiation (Peru
et al. 2008). Sox10 belongs to the high mobility group tran-
scription factors and is required for terminal differentiation
of polydendrocytes into oligodendrocytes (see chapter 43).
The expression of Sox10 precedes that of Pdgfra, and the onset
of Pdgfra expression is dependent on Sox10 (Finzsch et al.
2008). Although Sox10 expression in the CNS is confined to
the oligodendrocyte lineage, Olig2 is expressed more broadly
in cells fated to become neurons or astrocytes (see Fig. 10.1)
(Ono et al. 2008; Tekki-Kessaris et al. 2001). Within the oli-
godendrocyte lineage, Olig2 is expressed abundantly in the
nucleus of all polydendrocytes (Ligon et al. 2006). The level
of Olig2 declines after they terminally differentiate into oligo-
dendrocytes (Kitada and Rowitch 2006), whereas the level of
Sox10 is sustained throughout all stages of the oligodendro-
cyte lineage.
2.2 T R A NS G E N I C MO US E L I N E S T H AT
E X P R E S S R E P O RT E R P ROT E I N S I N
P O LY D E N D RO C Y T E S
Immunohistochemical methods of cell identification rely on
the availability of specific antibodies and adequate preserva-
tion of the antigen through tissue processing. As an alter-
native method of imaging and isolating polydendrocytes,
particularly in live tissue, a number of transgenic mouse
lines that express various fluorescent reporter genes in NG2
cells have become available (Table 10.2). None of the lines
express the reporter gene exclusively in polydendrocytes,
but with an accurate knowledge of the pattern of reporter
expression of each line, these mouse lines can be used to
mark polydendrocytes in a certain anatomical region at the
desired age.
2.2.1 NG2 Transgenic Mouse Lines
The two transgenic mouse lines in which the expression of
a fluorescent protein occurs most faithfully in polydendro-
cytes throughout all stages of development are the NG2-YFP
knock-in mice (Karram et al. 2008) and the NG2-DsRed
BAC mice (Zhu et al. 2008; Ziskin et al. 2007). In NG2-YFP
transgenic mice, despite the loss of one endogenous NG2
allele because of insertion of the YFP expression cassette,
there is no discernible developmental or behavioral change
in heterozygous mice. Additional mouse lines that express
GFP (green fluorescence protein) in the majority of poly-
dendrocytes include Pdgfra-H2B-GFP and Olig2-EGFP
mice (Table 10.2; Fig. 10.2C).
2.2.2 Transgenic Mice with
Oligodendrocyte-Specific Promoters
There are transgenic mouse lines that were designed to
express reporter genes in mature oligodendrocytes but also
express them in polydendrocytes. In CNP-EGFP mice that
express EGFP (enhanced green fluorescent protein) driven
by a 3.7-kb promoter of the mouse 2′,3′-cyclic nucleotide
3′-phosphodiesterase (CNP) gene, EGFP is expressed
robustly in differentiated oligodendrocytes and weakly
in polydendrocytes (Yuan et al. 2002). The regulatory
sequences of the myelin proteolipid protein (PLP) gene
have been used to generate various lines of PLP transgenic
mice (see Table 10.2). Proteolipid protein appears slightly
after CNP in oligodendrocyte lineage cells during devel-
opment (see Fig. 10.1). In mice with less sensitive reporter
constructs, the reporter is detected primarily in mature oli-
godendrocytes, whereas in mice with enhanced sensitivity
and reporter expression, the reporter is detected in 50% of

Table 10.2 TRANSGENIC MOUSE LINES THAT EXPRESS FLUORESCENT REPORTER IN POLYDENDROCYTES
GENE MODE OF PERCENT OF EXPRESSION IN OTHER
EXPRESSED INSERTION PDs MARKED CNS CELL TYPES REFERENCES

NG2-DsRed DsRed BAC transgenic ~100% Pericytes Zhu et al. 2008

JAX 008241

NG2-YFP YFP Knock-in ~100% Pericytes Karram et al. 2008

Pdgfra-H2BGFP GFP-H2B Knock-in Vascular cells Hamilton et al. 2003

fusion Homozygous lethal JAX 007669

CNP-EGFP EGFP Promoter Mature oligodendrocytes Yuan et al. 2002

PLP-EGFP GFP DsRed Promoter Mature oligodendrocytes Fuss et al. 2000

PLP-DsRed Hirrlinger et al. 2005

PLP-EGFP EGFP Promoter and 50% Mature oligodendrocytes Mallon et al. 2002
(3′UTR) 3c-UTR

Olig2-EGFP EGFP BAC 100% Neural stem cells Gensat Project

Radial glia
Oligodendrocytes

PD, polydendrocyte.

N G 2 C E L L S ( P O LY D E N D R O C Y T E S ) • 111
Figure 10.2 Morphology of Polydendrocytes in the Developing and Mature Rodent Central Nervous System. A,B. Morphology of polydendrocytes
in adult mouse neocortex. (A) and corpus callosum (B) revealed by membrane-anchored EGFP (mGFP) expressed on isolated polydendrocytes in
NG2creER:mTmG double transgenic mice. C. A polydendrocyte in the adult mouse corpus callosum from Olig2-EGFP transgenic mouse (Gensat
Project) immunolabeled for NG2 (red). The majority of the NG2-negative EGFP+ cells are oligodendrocytes. (Inset) A reactive polydendrocyte in
the adult corpus callosum stained for NG2 following a demyelinating lesion. D. Relationship between polydendrocyte processes immunolabeled for
NG2 (green) and processes of a premyelinating oligodendrocyte immunolabeled for DM20/PLP (red) in the hippocampus of a P9 mouse. Arrowheads
indicate close apposition of polydendrocyte and oligodendrocyte processes. E. Relationship between a polydendrocyte immunolabeled for NG2
(red) and mGFP in an isolated astrocyte (green) in the neocortex of a P8 GFAPcreER:mTmG mouse. Only half of the astrocyte is shown. Astrocyte
processes decorate the long slender processes of the polydendrocyte and a nearby blood vessel (bv). Asterisks indicate adjacent cell bodies of the
astrocyte and polydendrocyte. F. Relationship between polydendrocytes (NG2, red) and axon bundles (L1 axonal cell adhesion molecule, green) in a
longitudinal section of a P11 rat spinal cord. Many polydendrocyte processes encircle bundles of L1+ axons (arrows). Asterisk marks the cell body of
a polydendrocyte. G. Relationship between polydendrocytes (EGFP+ NG2+, blue) and radial glia (GLAST+, red) in the ventral forebrain of E18.5
NG2cre:zeg mouse. Polydendrocytes are process-bearing cells that are distinct from GLAST+ radial glia. bv: blood vessel. H. Relationship between
polydendrocytes (EGFP+ Olig2+, arrowheads) and radial glia/immature astrocytes (GLAST+) around the SVZ of a P0 NG2cre:zeg mouse. Olig2
is expressed in some cells with GLAST+ processes that are EGFP-negative (arrows). bv: blood vessels that are EGFP+. LV: lateral ventricle. Scale bar,
50 μm. I.Morphology of a polydendrocyte in the neocortex of a P21 NG2cre:zeg mouse stained for NG2 ( blue) and Aldh1L1 (red). All scale bars
except (H) are 10 μm. (D) Kindly provided by Dr. Dirk Dietrich, University of Bonn. GFAPcreER mice were obtained from Dr. Frank Kirchhoff
(Universitaet des Saarlandes, Germany); (G) modified from Zhu et al. 2008.

112 • NEUROGLIA
polydendrocytes as well as in oligodendrocytes (Mallon
et al. 2002).
2.2.3 Transgenic Mice with
Astrocyte-Specific Promoters
Fluorescent reporter expression has also been detected in
polydendrocytes in transgenic mouse lines that were gen-
erated with the original intent of targeting astrocytes. In
GFAP-EGFP transgenic mice generated with a 2.2-kb human
glial fibrillary acidic protein (GFAP) promoter, some poly-
dendrocytes with the morphological, antigenic, and elec-
trophysiological characteristics of polydendrocytes express
EGFP, although GFAP protein is not detected in those
NG2+ EGFP+ cells (Matthias et al. 2003). Caution must be
exercised when interpreting expression studies based on tran-
scriptional activity, because promoter activation may occur
in the absence of stable protein accumulation. Moreover,
in transgenic mouse lines generated with short promoter
sequences, the level and fidelity of transgene expression varies
from line to line and is dependent on the site of integration
of the transgene.
3 D I S T R I B U T I O N A N D M O R P H O L O GY
O F P O LY D E N D R O C Y T E S
3.1 MO R P H O L O GY
In the mature CNS, polydendrocytes characteristically have
small, irregularly shaped elongated cell bodies and long slen-
der processes (Peters 2004). The cell body often tapers into
a primary process at one end of the soma, creating an asym-
metry. Although their morphology varies slightly in different
anatomical regions and different planes of section (Dawson
et al. 2003), polydendrocytes in both gray and white matter
extend their processes in multiple directions (Fig. 10.2A–C).
Ultrastructurally, polydendrocytes have a pale,
bean-shaped nucleus with an irregular contour and has
clumped chromatin beneath the nuclear envelope. The
nucleus is surrounded by relatively light cytoplasm that is
devoid of intermediate filaments (Peters 2004). Their fine
structure resembles that of previously reported prolifera-
tive cells (Carroll et al. 1998; Reyners et al. 1986; Vaughn
1969). Ultrastructurally polydendrocytes most closely
resemble astrocytes but differ from the latter by their irreg-
ularly shaped nuclei and thin mitochondria (Peters 2004).
Pre-embedding immunoelectron microscopy performed
with anti-NG2 antibody reveals clusters of thin profiles of
labeled processes in the neuropil, indicative of their complex
branching or looping (Fig. 10.3, arrowheads).
3.2 D I S T R I BU T I O N
In the mature CNS, polydendrocytes are uniformly distrib-
uted throughout the gray and white matter. They appear
to be tiled with their processes occupying nonoverlapping
territories. The estimated density of polydendrocytes in
N G 2 C E L L S ( P O LY D E N D R O C Y T E S )
Figure 10.3 Ultrastructural Profiles of Polydendrocyte
Processes. Pre-embedding immunolabeling for NG2 showing clusters
of multiple thin profiles of NG2-labeled polydendrocyte processes
(arrowheads) in the neuropil outside the SVZ in an adult mouse brain,
suggesting a convoluted nature of the processes. Scale bar, 2 μm.
the adult CNS varies from 10 to 140/mm2 (reviewed in
Nishiyama 2007). Polydendrocytes constitute 8% to 9% of
total cells in the white matter and 2% to 3% of total cells
in the gray matter (Dawson et al. 2003). The ratio of poly-
dendrocytes to oligodendrocytes ranges from 1:1 in the
rat hippocampus to 1:10 in the cat spinal cord. In a recent
study in which simultaneous labeling and quantification of
all glial cells was performed, 99.5% and 100% of all the glial
cells in the cat and human optic nerve, respectively, could
be accounted for as polydendrocytes, oligodendrocytes,
astrocytes, or microglia ( Jennings and Carroll 2010). Thus,
polydendrocytes do indeed make up the fourth major nor-
mal resident glial population in the mature CNS, and it is
unlikely that there are additional previously overlooked cell
populations.
3.3 E A R LY A P P E A R A N C E O F
P O LY D E N D RO C Y T E S
In the embryonic forebrain, Pdgfra mRNA+ and Sox10
mRNA+ cells are detected on embryonic day 13.5 (E13.5) in
the anterior entopeduncular area (Tekki-Kessaris et al. 2001).
At this stage NG2 is expressed only in the vasculature. Cells
that express the NG2 and Pdgfra proteins do not appear
until after E14.5 in the mouse forebrain. They are found in
the parenchyma outside the ventricular zone and do not
express radial glial antigens such as GLAST (see Fig. 10.2G).
Thus, polydendrocytes represent cells that have become
committed to a glial lineage and have begun their migration
away from the germinal zones. By E18.5, many of them have
already acquired several long slender processes. They are to
be distinguished from Olig2+ cells in the germinal zones
that represent a heterogeneous population of progenitor
• 113
cells that give rise to neurons, oligodendrocytes, and astro-
cytes (Ono et al. 2008) (see Fig. 10.2H). Polydendrocytes
in the forebrain are generated from three distinct sources,
each of which originates from a distinct domain marked
by its location and transcription factor code (Kessaris et al.
2006) (see chapter 13). The neocortex is the last forebrain
region to be populated by polydendrocytes. By the end of
the first postnatal week, polydendrocytes have reached their
peak density, become uniformly distributed, and acquired
a highly branched morphology resembling of those in the
mature CNS (see Fig. 10.2I).
4 R E L AT I O N S H I P B ET W E E N
P O LY D E N D R O C Y T E S A N D OT H E R
CELL TYPES IN THE CENTRAL
N E RVO U S SYS T E M
Morphological, immunohistochemical, and functional stud-
ies have shown that polydendrocytes are distinct from neurons
and other glial cells. Following is a summary of the distinction
and spatial relationship between polydendrocytes and other
CNS constituents.
4.1 N EU RO N S
Polydendrocytes are functionally distinct from neurons, but
are intimately associated with them. One such mode of inter-
action involves responding to neurotransmitters released
from neuronal axons via ionotropic receptors expressed by
polydendrocytes in both gray and white matter (see chapter
21). Ultrastructurally, polydendrocyte processes are found
apposed to neuronal presynaptic terminals in gray matter and
surrounding unmyelinated axons in white matter. Synaptic
input onto polydendrocytes disappears as they differenti-
ate into oligodendrocytes (De Biase et al. 2010; Kukley et al.
2010).
In addition, polydendrocytes in gray matter exist in a sat-
ellite position adjacent to neuronal soma (Karram et al. 2008;
Mangin et al. 2008), and electron-dense patches have been
observed on the adjacent plasma membranes (Peters 2004).
In myelinated fiber tracts, polydendrocyte processes contact
the nodes of Ranvier (Butt et al. 1999). Although the precise
function of such polydendrocyte–axonal contacts remains
unknown, they could allow axonally derived signals to reg-
ulate polydendrocyte proliferation and differentiation, and
reciprocally polydendrocytes could provide neurotrophic sup-
port for neurons.
It has been debated as to whether polydendrocytes that
interact with neurons are a distinct postmitotic popula-
tion from those that can proliferate and differentiate into
oligodendrocytes. However, the observations that polyden-
drocytes can divide while retaining their synaptic inputs
(Kukley et al. 2008) and that polydendrocytes lose their
synaptic input as they differentiate into oligodendrocytes
(De Biase et al. 2010; Kukley et al. 2010) do not support this
view. Instead, these observations suggest that such neuron–
polydendrocyte interaction could be a normal physiological
114 • NEUROGLIA
function of polydendrocytes in their oligodendrocyte pro-
genitor capacity.
4.2 O L I G O D E N D RO C Y T E S
When polydendrocytes terminally differentiate into oligo-
dendrocytes, they lose the expression of NG2 and Pdgfra and
acquire the expression of oligodendrocyte antigens (see Fig.
10.1). As polydendrocytes differentiate into oligodendrocytes,
the size of the soma and the volume occupied by their processes
increase significantly (Kukley et al. 2010). Polydendrocytes
often contact oligodendrocyte processes (see Fig. 10.2D).
However, the functional significance of such contacts remains
unknown.
4.3 A S T RO C Y T E S
Polydendrocytes are antigenically distinct from protoplasmic
or fibrous astrocytes (Peters 2004) or the velate astrocytes in
the cerebellar cortex (Chan-Palay and Palay 1972). NG2 and
Pdgfra are never detected in cells that express GFAP or the
astrocyte antigen aldehyde dehydrogenase 1 L1 (Aldh1L1),
which was recently discovered by microarray analysis (Cahoy
et al. 2008). Conversely, GFAP or Aldh1L1 proteins are
never detected in polydendrocytes (Nishiyama 2007 or 2009;
Richardson et al. 2011; Zhu et al. 2008). However, GFAP tran-
scription seems to occur in some polydendrocytes (reviewed
in Nishiyama et al. 2009). Although polydendrocytes never
express markers of astrocytes, a subpopulation of polyden-
drocytes in the embryonic forebrain generates protoplasmic
astrocytes in a region-specific manner (see section 5.2.2).
Polydendrocytes can be morphologically distinguished
from protoplasmic and fibrous astrocytes by their long, slen-
der processes that are less densely branched in their proximal
regions than those of astrocytes, thus lacking the characteristic
“bushy, fuzz ball appearance” of astrocytes (see Fig. 10.2E) (see
chapters 4 and 12). The morphology of polydendrocytes may
be more suited for point-to-point communication with other
cellular entities rather than the global homeostatic function of
astrocytes, such as the clearance of extracellular potassium ions
or neurotransmitters that require them to cover a large volume
of the neuropil. Although astrocytes extend their endfeet onto
blood vessels, polydendrocyte processes terminate on the vas-
cular wall in a small punctum or do not end on the vasculature
at all (Zerlin and Goldman 1997). Polydendrocyte processes
are extensively intertwined with those of astrocytes (see Fig.
10.2E; Hamilton et al. 2010), suggestive of some functional
interaction. Future studies could be directed toward elucidat-
ing the nature of such interactions.
4.4 M I C RO G L I A
Polydendrocytes are distinct from resting ramified microglia.
In the normal developing and mature CNS, NG2 and Pdgfra
are not expressed on resting ramified microglia that express
CD11b and F4/80. Although both cell types have branched
processes, the processes of microglia are barbed with regularly
occurring short side branches, and those of polydendrocytes
are longer, smoother, and branch more distally. In response to
various types of insult to the CNS, both polydendrocytes and
microglia undergo rapid morphological changes characterized
by shortening and thickening of their processes. Often the cell
bodies of activated polydendrocytes and microglia are seen
adjacent to each other, and their processes are intimately inter-
twined (Bu et al. 2001; Dawson et al. 2003; Nishiyama et al.
1997), suggestive of functional interactions between the two
cell types (Wu et al. 2010). Under certain pathological condi-
tions involving extensive neuronal damage, NG2 is detected
on a subpopulation of round macrophages that invade the
lesion (Bu et al. 2001; Kucharova et al. 2011).
4.5 N EU R A L S T E M C E L L S
There have been conflicting reports about the relationship
between polydendrocytes and multipotential neural stem
cells and other progenitor cells in the germinal zones, par-
ticularly those in the subventricular zone (SVZ) around the
lateral ventricle where neurogenesis persists in adulthood.
Polydendrocytes are very sparsely scattered in the SVZ,
and they are distinct from GFAP+ neural stem cells, transit
amplifying cells, or migrating neuroblasts (Chojnacki et al.
2011; Komitova et al. 2009; Platel et al. 2009; Richardson
et al. 2011). This is consistent with the lack of evidence that
polydendrocytes give rise to neurons in the olfactory bulb.
The parenchyma outside the germinal zones is more densely
populated by regularly spaced polydendrocytes, giving the
appearance that polydendrocytes are lined up at the outer
border of the SVZ. In the SVZ along the dorsal wall of the
lateral ventricles, polydendrocytes appear to be more abun-
dant, and they may provide a source of new oligodendrocyte
lineage cells that populate the corpus callosum (Etxeberria
et al. 2010).
5 T H E FAT E O F N G 2 C E L L S
5.1 H I S TO R I C A L P E R S P E C T I V E
Our understanding of the fate of polydendrocytes has evolved
over the past three decades after many twists and turns.
A2B5+ cells isolated from rodent optic nerves generate oli-
godendrocytes when grown in serum-free medium and stel-
late GFAP+ type 2 astrocytes when grown in the presence of
serum (Raff et al. 1983; Stallcup and Beasley 1987). This led
to the hypothesis that polydendrocytes represent bipotential
glial progenitor cells. However, numerous attempts made in
the 1990s failed to unequivocally demonstrate that polyden-
drocytes can give rise to astrocytes as well as oligodendrocyte
in vivo. Thus, the concept of bipotential glial progenitor cells
was gradually dismissed as a cell culture artifact, and the cells
began to be called oligodendrocyte precursor cells (OPCs) by
the end of the 1990s.
Just as the debate on their oligodendrocyte-astrocyte
bipotentiality was winding down, another controversy was
sparked by the discovery that polydendrocytes from perinatal
N G 2 C E L L S ( P O LY D E N D R O C Y T E S )
rat optic nerves could be reprogrammed to become neurons
after a long period in culture (Kondo and Raff 2000). This
led to an intensive search by many labs for evidence that poly-
dendrocytes are multipotent cells that can generate neurons
as well as oligodendrocyte lineage cells. However, subsequent
genetic fate mapping studies described in the following sec-
tion revealed that polydendrocytes do not generate neurons
under normal physiological conditions, and that their fate in
the postnatal CNS is restricted to the oligodendrocyte lin-
eage, although some polydendrocytes in some embryonic
brain regions generate astrocytes as well as oligodendrocyte
lineage cells.
5.2 G E N ET I C FAT E M A P P I N G O F
P O LY D E N D RO C Y T E S
Mouse genetic tools were developed over the past decade
to directly follow the fate of polydendrocytes. This involves
Cre-loxP–mediated site-specific recombination to perma-
nently turn on the expression of a reporter gene specifi-
cally in polydendrocytes and examining the phenotype of
reporter-expressing (reporter+) cells (Fig. 10.4) (Nishiyama
2007). Temporal control of reporter activation can be added
to this system by replacing constitutively active Cre with
tamoxifen-inducible Cre (CreER).
5.2.1 Polydendrocytes in the Postnatal Central
Nervous System Are Committed to the
Oligodendrocyte Lineage
In mice that were double transgenic for NG2-cre, which
expresses constitutively active Cre in NG2-expressing cells,
and a Cre reporter, the majority of oligodendrocytes in both
gray and white matter expressed the reporter (Zhu et al.
2008). This provided the first direct evidence that polyden-
drocytes generate oligodendrocytes. Several ensuing studies
used transgenic mice that express CreER regulated by the gene
encoding Pdgfra (Kang et al. 2010; Rivers et al. 2008), NG2
(Zhu et al. 2011), or Olig2 (Dimou et al. 2008) and exam-
ined the fate of polydendrocytes at different postnatal time
The fate of polydendrocytes in the forebrain
prenatal postnatal
DG oligodendrocytes
WM
polydendrocytes
VG astrocytes
Figure 10.4 The Fate of Polydendrocytes in Different Forebrain
Regions. Right. Postnatal brain. Left. Embryonic brain. Polydendrocytes
in all the regions of the postnatal forebrain generate only polydendro-
cytes or oligodendrocytes. Polydendrocytes in the ventral gray matter
(VG) of the embryonic brain consist of those that generate astrocytes
(dark gray) and those that generate oligodendrocyte lineage cells
(oligodendrocytes and polydendrocytes). Polydendrocytes in the embry-
onic white matter (WM) and dorsal gray matter (DG) generate only
polydendrocytes and oligodendrocytes.
• 115
points. Collectively, the common findings from these studies
are that the vast majority of polydendrocytes in the postnatal
CNS generate only oligodendrocyte lineage cells, and their
progeny are either polydendrocytes or oligodendrocytes (Fig.
10.5). There are small variances among these reports regarding
whether other cell lineages are derived from polydendrocytes
in the postnatal brain. Rivers et al. (2008) observed a small
number of neurons in the piriform cortex, whereas the other
studies did not find any neuronal progeny of polydendro-
cytes (Dimou et al. 2008; Kang et al. 2010; Zhu et al. 2011).
Dimou et al. (2008) observed a small number of astrocytes
that expressed the Cre reporter, whereas astrocyte differentia-
tion was not detected from postnatal polydendrocytes in the
other studies (Kang et al. 2010; Rivers et al. 2008; Zhu et al.
2011). The reason for this discrepancy is not clear at present,
but the majority of evidence indicates that polydendrocytes in
the postnatal forebrain are restricted to the oligodendrocyte
lineage under normal physiological conditions.
The rate of oligodendrocyte differentiation from poly-
dendrocytes is higher in white than gray matter and declines
with age (Dimou et al. 2008; Kang et al. 2010; Rivers et al.
2008; Zhu et al. 2011). Single polydendrocytes at all postnatal
stages generate two daughter oligodendrocytes, two daughter
polydendrocytes, or one of each, but those in the mature brain
generate two daughter polydendrocytes more frequently than
oligodendrocytes (Zhu et al. 2011). Further studies are needed
to determine the precise mechanism of symmetric and asym-
metric division of polydendrocytes and their self-renewal abil-
ity (see section 7.2) (Sugiarto et al. 2011).
Inducible fate mapping of polydendrocytes
NG2creER:YFP
double tg mouse
NG2-creER mouse
NG2 creER
40HT
NG2+(immunolabeled)
NG2– YFP+(progency of NG2+ cells)
NG2+/YFP+
CA p YFP
e.g. ROSA-YFP mouse
CA p Stop-pA YFP
NG2+/YFP+ NG2–/YFP+
loxP
polydendrocyte oligodendrocyte
Figure 10.5 Fate Mapping of Polydendrocytes Using Inducible Cre-loxP
Recombination. Left. A double transgenic mouse line generated by cross-
ing a Cre driver (e.g., NG2-creER) and a Cre reporter (e.g., ROSA-YFP).
The Cre driver line expresses inducible Cre (CreER) under the control of a
polydendrocyte-specific promoter. The Cre reporter contains a constitu-
tively active promoter (CAp) followed by a loxP-flanked transcriptional
stop sequence, and the cDNA encoding YFP. When Cre is activated
by 4-hydroxytamoxifen (4OHT), the sequence between the two target
loxP sites is excised and YFP expression is permanently turned on. Right.
Examples of Cre activation in polydendrocyte (NG2+ and YFP+) and its
progeny that no longer expresses NG2 (oligodendrocyte). Modified from
Nishiyama 2007.
116 • NEUROGLIA
A significant number of oligodendrocytes continue to be
generated in the adult CNS. It has been estimated that 17% to
30% of oligodendrocytes are generated de novo from polyden-
drocytes within a period of 2 to 3 months in young adult mice
(Rivers et al. 2008; Zhu et al. 2011). Whether they replace
existing or degenerating oligodendrocytes or contribute to a
gradual rise in the total number of oligodendrocytes (Peters
and Sethares 2004) remains to be determined. It is also unclear
whether all polydendrocytes are endowed with the ability to
differentiate into oligodendrocytes, or there is a subpopulation
that permanently remains as polydendrocytes throughout life
and is incapable of generating oligodendrocytes.
5.2.2 A Subpopulation of Polydendrocytes in the
Embryonic Brain Generates Astrocytes
Fate mapping studies using constitutively active NG2-cre mice
revealed that more than 40% of protoplasmic astrocytes in the
ventral forebrain but not those in the white matter or dorsal
forebrain are generated from polydendrocytes (Zhu et al.
2008). Using NG2-creER mice it was found that astrocyte dif-
ferentiation from polydendrocytes occurs only in the embry-
onic brain and does not occur after birth (see Fig. 10.5) (Zhu
et al. 2011). However, contrary to the initial oligodendrocyte–
astrocyte bipotential glial progenitor theory based on optic
nerve cultures, polydendrocytes in the white matter do not
generate astrocytes at all ages. Single polydendrocytes in the
embryonic ventral gray matter generate either all astrocytes or
all oligodendrocyte lineage cells, and clusters of polydendro-
cyte progeny that contain both astrocytes and oligodendro-
cyte lineage cells have not been found. Thus, the divergence
of the astrocyte and oligodendrocyte fates of polydendrocytes
must occur early, and the embryonic ventral forebrain con-
tains an intermingled population of polydendrocytes that are
fated to become astrocytes and those committed to become
oligodendrocytes (see Fig. 10.5). Olig2 is initially expressed in
polydendrocytes, but is downregulated as they develop into
astrocytes. The mechanism why only polydendrocytes in ven-
tral and not dorsal forebrain generate astrocytes is not known.
Polydendrocytes originating from different domains of the
embryonic germinal zone may not be equivalent in their astro-
gliogenic potential.
5.2.3 Polydendrocytes Do Not Contribute
to Neurogenesis
Initially different findings were reported regarding the neu-
rogenic fate of polydendrocytes. Rivers et al. (2008) found
that a small subpopulation of Pdgfra+ cells in the piriform
cortex generated cells that morphologically resembled pro-
jection neurons, and a similar observation was made using
PLP-creER mice (Guo et al. 2010), although the PLP pro-
moter can be activated in some neurons (Miller et al. 2009).
By contrast, other studies did not to find any neuronal prog-
eny of polydendrocytes (Kang et al. 2010; Zhu et al. 2008,
2011). Because additional studies using Pdgfra-creER have
not revealed a significant number of neuronal progeny of
polydendrocytes (Tripathi et al. 2010; Zawadzka et al. 2010),
the current consensus is that polydendrocytes do not contrib-
ute to the generation of new neurons under normal physiolog-
ical conditions (Richardson et al. 2011).
6 P R O L I F E R AT I O N A N D R E S P O N S E O F
P O LY D E N D R O C Y T E S TO I N J U RY
Polydendrocytes continue to cycle throughout life and
comprise 70% to 90% of the proliferating population in the
adult CNS (Horner et al. 2000; Polito and Reynolds 2005).
This is consistent with the earlier reports on the existence
of a radiosensitive proliferative cell population in the adult
CNS (Carroll et al. 1998; Reyners et al. 1986). Throughout
all stages of postnatal development, 50% or more polyden-
drocytes remain in cell cycle. The cell cycle time increases
from 2 to 3 days in the early postnatal brain to greater than
70 days in the adult brain (Kukley et al. 2008; Psachoulia et
al. 2009), and there appears to be heterogeneity in the cell
cycle time among polydendrocytes in the same anatomical
region (Polito and Reynolds 2005). In addition to various
factors that are known to regulate polydendrocyte prolifera-
tion (Franklin and ffrench-Constant 2008), voluntary exer-
cise precipitates their cell cycle exit in the neocortex (Simon
et al. 2011). Polydendrocytes also have been implicated as the
cell of origin of glial tumors (Liu et al. 2011; Persson et al.
2010).
Polydendrocytes undergo increased proliferation in
response to a variety of insults to the CNS. They proliferate
robustly in response to changes in the density of myelin or
oligodendrocytes, and their numbers can increase severalfold
(Nishiyama 2007). By contrast, the total number of mature
oligodendrocytes appears to be under tighter regulation, and
increased polydendrocyte numbers are often not accompanied
by a corresponding increase in oligodendrocyte numbers. Thus,
polydendrocytes could be serving as a buffer for the homeo-
static regulation of oligodendrocyte density. Polydendrocytes
also dramatically alter their morphology in response to a wide
variety of insults, which is often seen as shortening and thick-
ening of their processes and a strong upregulation of NG2
expression (see Fig. 10.2C, inset), although the morphology
varies with the nature of the insult.
There has been a considerable debate as to whether
polydendrocytes contribute to reactive astrocytes. Earlier
studies that used pulse-chase labeling with BrdU (5-bromo-
2′-deoxyuridine) led to claims that reactive astrocytes are
generated from proliferated polydendrocytes. However,
more recent genetic fate mapping studies did not reveal a
significant number of reactive astrocytes that were derived
from polydendrocytes (Dimou et al. 2008; Komitova et al.
2011; Tripathi et al. 2010; Zawadzka et al. 2010). By contrast,
Tatsumi et al. (2008) observed Cre reporter+ astrocytes fol-
lowing cryoinjury in the brain. Olig2-fate mapping is com-
plicated by the fact that Olig2 is expressed by some reactive
astrocytes (Chen et al. 2008). Further studies are needed to
determine whether the extent of astrocyte differentiation
from polydendrocytes differs in response to different types
of injury and the precise relationship between NG2-negative
N G 2 C E L L S ( P O LY D E N D R O C Y T E S )
astrocyte precursor cells and polydendrocytes that contribute
to the glial scar.
7 P O LY D E N D R O C Y T E S P R O VI D E
AN ENDOGENOUS SOURCE OF
R E M Y E L I N AT I N G C E L L S
7.1 P O LY D E N D RO C Y T E S P RO L I FE R AT E A N D
D I F F E R E N T I AT E I N TO R E MY E L I NAT I N G
C E L L S A F T E R D E MY E L I NAT I O N
Remyelination is often incomplete in multiple sclerosis (MS)
and other types of CNS insults, such as spinal cord injury,
that are accompanied by extensive demyelination. Thus strate-
gies to improve myelin repair should include identifying the
endogenous source of remyelinating cells and enhancing their
ability to produce more oligodendrocytes and myelin. Before
the recognition of polydendrocytes as a normal resident glial
cell population, unidentified “small glial progenitor cells”
were seen to proliferate around experimentally demyelinated
lesions in the corpus callosum and optic nerve and generate
remyelinating cells (Carroll et al. 1998; Gensert and Goldman
1997). Subsequently, proliferating cells were identified as
NG2-expressing polydendrocytes in an acutely demyelinated
lesion in the rat spinal cord (Keirstead et al. 1998).
Oligodendrocytes that are likely to have been gener-
ated from proliferated polydendrocytes appear in acute and
chronic experimentally demyelinated lesions (Mason et al.
2004; Watanabe et al. 2002). Genetic fate mapping studies
using Pdgfra-creER have provided more direct evidence that
polydendrocytes in the spinal cord generate new oligodendro-
cytes that remyelinate acute chemically demyelinated lesions
(Zawadzka et al. 2010) or demyelinated lesions in experimen-
tal autoimmune encephalomyelitis (EAE), which is often used
as a rodent model of inflammatory demyelinating diseases
such as MS (Tripathi et al. 2010). In addition to generating
oligodendrocytes, polydendrocytes in astrocyte-depleted
areas of acute chemically demyelinated spinal cord also gen-
erate remyelinating Schwann cells (Zawadzka et al. 2010).
However, neither Schwann cell remyelination nor Schwann
cell differentiation from polydendrocytes has been observed
in the forebrain.
7.2 S E L F-R E N EWA L O F P O LY D E N D RO C Y T E S
A F T E R R E MY E L I NAT I O N
For polydendrocytes to provide a continued source of remy-
elinating cells, they must be able to replenish themselves.
Studies to determine whether polydendrocytes become
depleted after successful remyelination have yielded con-
flicting results. Some suggest that polydendrocytes become
depleted (Keirstead et al. 1998; Mason et al. 2004), whereas
others indicate efficient repopulation from neighboring cells
(Chari and Blakemore 2002) or efficient remyelination of
repeated demyelinating lesions created after a 10-week inter-
val (Penderis et al. 2003). The conflicting observations may
reflect the slow kinetics of polydendrocyte proliferation and
• 117
differentiation after myelin repair has been achieved in adult
CNS. Repopulation of differentiation-competent polyden-
drocytes may not occur efficiently if there is a chronic demand
for remyelinating cells.
7.3 P O LY D E N D RO C Y T E S A N D N EU R A L
S T E M C E L L S I N MY E L I N R E PA I R
In the normal adult brain, immature cells in the SVZ do
not significantly contribute to oligodendrocyte production
in the corpus callosum (Marshall et al. 2003). However,
when the corpus callosum is demyelinated, increased
migration of proliferated cells from the adult SVZ into the
lesioned corpus callosum has been observed (Menn et al.
2006). The polysialylated form of neural cell adhesion mol-
ecule (PSA-NCAM) is expressed on such migrating cells
(Nait-Oumesmar et al. 2008). Proliferating GFAP+ type B
cells have been suggested as a source of remyelinating cells
in the corpus callosum (Menn et al. 2006). Enhancing epi-
dermal growth factor (EGF) signal stimulates their recruit-
ment, although reports differ as to whether EGF enhances
their differentiation into myelinating oligodendrocytes
(Aguirre et al. 2007; Gonzalez-Perez et al. 2009; Ivkovic
et al. 2008). Further quantitative studies are needed to deter-
mine the relative contribution of local polydendrocytes and
SVZ-derived cells toward remyelination. Identification of
the most efficient endogenous source of remyelinating cells
will facilitate the design of strategies to promote myelin
repair.
7.4 FAC TO R S T H AT I N F LU E N C E
R E MY E L I NAT I O N E FFI C I E N C Y FRO M
P O LY D E N D RO C Y T E P RO G E N Y
A number of studies have identified cell intrinsic and extrin-
sic mechanisms that promote oligodendrocyte differentiation
and are discussed in many excellent review articles (Chong
and Chan 2010; Franklin and ffrench-Constant 2008; Peru
et al. 2008) (see chapters 13 and 57). A sampling of some new
developments is given in the following.
7.4.1 Old and Young Brain
The efficiency of remyelination declines with age. In old ani-
mals, remyelination occurs, but the rate of polydendrocyte
accumulation in the lesion and their differentiation into oli-
godendrocytes is decreased (Franklin and ffrench-Constant
2008). Both cell-intrinsic and -extrinsic mechanisms have
been implicated in the age-dependent decline of remyelination
efficiency. Intrinsically, an age-dependent decline in histone
deacetylase (HDAC) activity causes de-repression of the tran-
scription of differentiation inhibitors such as the Sox2, Hes5,
and ID2/4, and compromises the ability of polydendrocytes
to differentiate into remyelinating oligodendrocytes (Shen et
al. 2008; Ye et al. 2009). Cell senescence may also contribute
to reduced remyelination efficiency. Polydendrocytes in the
aged mouse brain express the inducer of senescence esopha-
geal cancer–related gene 4 (Ecrg4), which causes G1 arrest
118 • NEUROGLIA
by proteasome-mediated degradation of cyclin D1 and D3
(Kujuro et al. 2010).
Cell extrinsic mechanisms also play a major role in
the decline of remyelination efficiency with age. The level
of growth factors necessary for polydendrocyte recruit-
ment is not significantly altered with age (Franklin and
ffrench-Constant 2008). However, a recent parabiosis
experiment indicates that macrophages from young animals
can stimulate polydendrocyte proliferation and differentia-
tion and significantly promote remyelination in old animals
(Ruckh et al. 2012).
7.4.2 Gray Matter Versus White Matter
Genetic fate mapping has shown that polydendrocytes in the
adult white matter differentiate into oligodendrocytes at a
greater rate than those in the gray matter (Dimou et al. 2008;
Kang et al. 2010; Rivers et al. 2008; Zhu et al. 2011). Multiple
sclerosis affects not only white matter but also gray matter,
and recently extensive cortical remyelination was observed
in patients with chronic MS (Albert et al. 2007) (see chap-
ter 61). Further studies are needed to determine if there are
differences in the ability of polydendrocytes in white and gray
matter to generate myelinating oligodendrocytes under nor-
mal and demyelinated conditions.
8 S U M M A RY A N D P E R S P E C T I VE S
It has now become widely accepted that polydendrocytes repre-
sent a fourth resident glial cell population in the normal CNS.
Polydendrocytes in the postnatal CNS generate only oligo-
dendrocyte lineage cells and are often equated with oligoden-
drocyte precursor cells, although it is not known whether all
polydendrocytes differentiate into oligodendrocytes. During
embryonic development, a subpopulation of polydendrocytes
in the ventral forebrain, which are distinct from radial glia,
downregulates oligodendrocyte lineage antigens and differ-
entiates into protoplasmic astrocytes. Polydendrocytes in the
postnatal CNS that receive synaptic inputs are not a separate
terminally differentiated cell population, as previously hypoth-
esized, but are capable of proliferating and differentiating into
oligodendrocytes.
Why is there a need for the adult brain to maintain a popula-
tion that can only generate oligodendrocytes? The mammalian
CNS has become critically dependent on proper myelination
of its axons and may have developed a cytoarchitecture that
ensures the maintenance of the correct ratio of oligodendro-
cytes to axons. The rapid proliferation of polydendrocytes that
occurs in response to deviation from the normal number of oli-
godendrocytes or the amount of myelin may be a part of the
homeostatic mechanism. It is paradoxical that demyelinated
lesions in MS and spinal cord injury are often incompletely
repaired. Polydendrocytes, together with astrocytes, may have
evolved in the CNS to carry out the role of nonmyelinating
Schwann cells that coexist with myelinating Schwann cells in
the peripheral nervous system. It is possible that polydendro-
cytes retain some degree of lineage plasticity and can alter their
fate in response to manipulation of key regulatory switches.
Future studies may be directed toward understanding the fun-
damental mechanisms that regulate their fate and differentia-
tion and their role in the neural network.
AC K N OW L E D G M E N T S
The author thanks Dr. Dirk Dietrich (University of Bonn)
for providing the image shown in Fig. 10.2D, Dr. Frank
Kirchhoff (Universitaet des Saarlandes) for his generous gift
of GFAPcreER mice, and David Serwanski for the electron
micrograph in Fig. 10.3. The author also thanks graduate stu-
dents Bobby Hill, Jelena Medved, Kiran Patel, Alex Reiss,
and Hao Zuo for their helpful discussions and tissue prepara-
tions that were used to generate the figures. The author regrets
that space limitation did not permit the inclusion of many
important references that are not cited here. This work was
supported by funds from the NIH and the National Multiple
Sclerosis Society.
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• 121
 11.
GLIAL CELLS IN AUTONOMIC AND SENSORY GANGLIA
Menachem Hanani and David C. Spray

A B B R E VI AT I O N S 1 I N T R O D U C T I O N : T H E O R G A N I Z AT I O N
OF PERIPHERAL GANGLIA
ANS autonomic nervous system
AQP4 aquaporin 4 The peripheral nervous system (PNS) consists of nerves ema-
ATP adenosine triphosphate nating from brain and spinal cord, as well as sensory and auto-
BMP bone morphogenetic proteins nomic ganglia (Fig. 11.1). Sensory ganglia contain neuronal cell
cAMP cyclic adenosine monophosphate bodies of afferent nerves responsible for mechanical, thermal,
CD Crohn disease painful, and proprioceptive input (mechano-, thermo-, noci-,
CNS central nervous system and proprioceptors). These neurons have T-shaped axons,
with one branch forming nerve endings in the periphery that
Cx connexin
can be either anatomically specialized or nonspecialized, and
DRG dorsal root ganglion the other branch synapsing onto interneurons within the dor-
dsRNA double stranded RNA sal horn of the spinal cord. The main types of sensory ganglia
EGCs enteric glial cells are the dorsal root ganglia (DRG), which innervate most of
ENS enteric nervous system the body and internal organs; trigeminal ganglia (TG), which
ErbB3 avian erythroblastic leukemia viral innervate the face, head and teeth; and nodose ganglia, which
oncogene homolog3 receive sensory inputs from internal organs such as heart, res-
ET endothelin piratory tract, and stomach through the vagal nerves.
GABA gamma-aminobutyric acid The three main types of glia in the PNS are the satellite
GAT GABA transporter glial cells (SGCs) in sensory, parasympathetic, and sympa-
GDNF glia cell line-derived neurotrophic thetic ganglia that share certain properties with astrocytes,
factor the enteric glia, which seem to be even more similar to CNS
GFAP glial fibrillary acidic protein astroglia, and the myelinating and nonmyelinating Schwann
GGF glial growth factor (now termed cells (see chapters 7 and 14). Schwann cells are dealt with in
neuregulin1, NRG1) detail in other chapters in this volume. The PNS also contains
macro-phages, which are similar to microglia, and are not
GLAST glial glutamate aspartate transporter
discussed here. For a description of more specialized types of
IL interleukin peripheral glia, see Hanani (2010).
NO nitric oxide Satellite glial cells share many properties with astrocytes,
NRG1 neuregulin1 including expression of glutamine synthetase and a variety of
NTPDase ecto-nucleotidase receptors (including purinergic receptors) and transporters
P2 purinergic receptor type 2 characteristic of astrocytes; like astrocytes they are mutually
P2Y4 purinergic receptor type P2Y4 coupled by gap junctions, although the coupling is less exten-
P2X7 purinergic receptor type P2X7 sive in SGCs (Hanani et al. 1999; Hanani et al. 2002). A unique
PAR protease activated receptor feature of SGCs that distinguishes them from astrocytes is that
PNS peripheral nervous system they surround the cell bodies of the sensory, sympathetic, or
S100 calcium binding protein S100 parasympathetic neurons, having an anatomical relationship to
SGC satellite glial cells neurons that is similar to that of Schwann cells. Table 11.1 sum-
marizes some of the main characteristics of glia in the PNS.
SK3 calcium activated small conductance
potassium channel KCNN3
Sox10 transcription factor Sox 10 2 I D E N T I F Y I N G S AT E L L I T E
TTX tetrodotoxin GLIAL CELLS
TG trigeminal ganglion
TNF tumor necrosis factor Identifying SGCs may pose difficulty because these cells form
UTP uridine triphosphate a thin sheath around the neurons, which at places may be
too thin to be viewed under the light microscope (Fig. 11.2).

122
 A Dorsal root ganglion When these cells are selectively labeled, their thin profile
and close proximity to the neurons may lead to the mistaken
Periphery conclusion that the label is present in or near the neuronal
plasma membrane. A close look will usually reveal that the
SGC envelope is not smooth as expected from the neuronal
Sympathetic ganglion membrane, but has several thickenings, especially where the
B C nucleus is located (which may be missed when viewing sec-
SGC SGC
tions). In general, confirmatory immunohistochemical label-
ing is recommended for identifying these cells. Satellite glial
N N
cells and Schwann cells may share several proteins (e.g., S100
and laminin); therefore, these markers are not definitive in
cases in which ambiguity may exist (e.g., in tissue culture,
D
Submucosal plexus
where structural relations are disrupted). More selective SGC
Longitudinal SM
markers are glutamine synthetase and several other enzymes
Myenteric plexus involved in cell metabolism (Miller et al. 2002; Weick et al.
Circular SM
Submucosa
2003) (Fig. 11.3A), the glutamate transporter GLAST (Ohara
et al. 2008), the K+ channel SK3 (Ohara et al. 2009), or the
P2 receptor P2X7R (Belzer et al. 2010) (Fig. 11.3B). SGCs in
Figure 11.1 The Organization of Peripheral Ganglia. A. Schematic sympathetic and parasympathetic ganglia can be identified by
diagram of spinal cord, nerve roots, paravertebral sympathetic ganglion
and a dorsal root ganglion. B. The arrangement of satellite glial cells
staining for S100 (Cocchia and Michetti 1981; Hanani et al.
(SGCs) around a neuron in a sensory ganglion. C. A similar arrangement 1999); however, this marker can also be found in neurons and
is found in sympathetic ganglia, except that the neurons receive synapses. Schwann cells (Gonzalez-Martinez et al. 2003). Satellite glial
D. Schematic diagram of a cross section in the intestine showing the cells in urinary bladder ganglia (parasympathetic) are positive
arrangement of the myenteric plexus between the two layers of smooth for glutamine synthetase (Hanani et al. 1999), but no similar
muscle (SM), and of the submucosal plexus. (D) Modified from Heanue
and Pachnis 2007.
information is available for sympathetic ganglia.

Table 11.1 COMPARISON AMONG THE MAIN TYPES OF PERIPHERAL GLIAL CELLS
NONMYELINATING
CELL TYPE/PROPERTY AUTONOMIC SGCS SENSORY SGCS SCHWANN CELLS ENTERIC GLIA

Neurotransmitter transporters
Vimentin, S100
GFAP
Glutamine synthetase
Schwann cell myelin protein
Coupling by gap junctions
P2 receptors
Ecto-ATPase
Calcium waves
Cytokine expression
Cell processes
+
Inward rectifying K channels
Contacts with blood vessels
Relation with neuronal somata
+ +
+ +
+ ±
?1,2 + +
− −
+ +
P2Y1,2,6 P2X7, P2Y1,2,4,6,12,13
+ +
? +
LIF IL-1β, TNFα
− −
+ +
− −
Form a complete cover Form a complete cover
+ +
+ +
+ +
+3
+ −
+ +++
P2X7, P2Y1,2 P2X7, P2Y2,4
+ +
? +
IL-1β, IL-10, TNFα IL-1β, IL-6, TNFα
− +++
+ +
− +
Do not contact somata Form a partial cover

Engagement in phagocytosis + + + ?4

Abbreviations: Vim, Vimentin; BMP, bone morphogenetic proteins; LIF, leukemia inhibitory factor
Notes:
1. The question mark indicates a lack of information in the literature.
2. Glutamine synthetase was found in parasympathetic SGCs (Hanani et al. 1999; Sha et al. 2001), but there is no similar information in sympathetic SGCs.
3. Several authors identified glutamine synthetase in enteric glia, but Rühl (2005) reported a failure to reproduce this result.
4. The only report on phagocytosis by enteric glia is an abstract (Hollenbach E. et al. Gastroenterology 2000;118:A184).
Modified from Hanani (2010). See this reference for further details.

G L I A L C E L L S I N AU TO N O M I C A N D S E N S O RY G A N G L I A • 123

Figure 11.2 A Low-Power Electron Micrograph Showing the
Neuron-Satellite Glial Cell Unit in Sensory Ganglia Satellite glial
cell(s) (red). The small empty spaces in the SGC sheath are neuronal
protrusions. Ct, connective tissue space; N, neuronal nuclei; v, blood
vessels. Calibration bar, 4 μm.
Using GFAP to identify SGCs is problematic. Under rest-
ing conditions SGCs in sensory ganglia express very low levels
of GFAP, but after a variety of insults such as axotomy and local
inflammation, GFAP expression is greatly increased. In fact,
the increase in the number of neurons that are surrounded by
GFAP-positive SGCs has been used to quantify the extent of glial
activation (Hanani 2005). Satellite glial cells are quite sensitive to a
variety of insults, and even treatment with a drug such as lidocaine
causes the level of GFAP to increase in them (Puljak et al. 2009).
The expression of GFAP is detectable in sympathetic
ganglia SGCs under resting conditions, and it is strongly
upregulated after injury (for review, Hanani 2010). An inter-
esting common feature of sensory and sympathetic ganglia
is that following injury, GFAP is elevated not only in SGCs
surrounding the injured neurons, but also increases around
noninjured neurons, indicating a spread of activation signals
within the ganglion (Hanani 2010; Stephenson and Byers
1995).
A B
Figure 11.3 Immunohistochemical Identification of Satellite Glial Cells
in Sensory Ganglia A. Satellite glial cells are labeled for glutamine
synthetase (red). The green dots are gap junctions that were labeled for
connexin 43. B. Labeling of SGCs for the purinergic receptor P2X7
(red). Satellite glial cell nuclei are labeled with DAPI (blue). The neuronal
nuclei were also labeled, but are very faint. Note that in both (A) and (B),
the neurons are not labeled and are seen as empty spaces.
124 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
Little is known regarding GFAP expression in SGCs in
parasympathetic ganglia. Hanani et al. (1999) reported an
absence of this marker in the intrinsic ganglia of the guinea pig
bladder, whereas SGCs in ganglia of cat pancreas are GFAP
positive (Sha et al. 2001).
3 G L I A I N S E N S O RY G A N G L I A
In sensory ganglia, individual neurons are generally covered by
a single layer of SGCs (for detailed reviews, see Hanani 2005;
Ohara et al. 2009; Pannese 1981). The space between SGCs
and neuron is about 20 nm, similar to the extracellular space
in the central nervous system (CNS). Despite the absence
of synapses, the SGCs synthesize a number of neuroactive
compounds and express a variety of neurotransmitter recep-
tors (see Table 11.1), implying that they can modify and be
modified by neurons and other SGCs.
Gap junctions are likely to have important implications
for the function of glial cells because they allow the passage of
ions (electrical currents) and also metabolites of up to molec-
ular weight of 1,000 (see chapter 24). Extensive coupling of
glial cells by gap junctions enables astrocytes to control the
extracellular concentration of potassium ions (K+ buffering).
The SGCs covering an individual neuron are well coupled,
which has been extensively evaluated using Lucifer yellow
injection to measure gap junction-mediated intercellular
communication (Fig. 11.4). This coupled network of SGCs,
together with the neuron that they surround, seems likely
A B
N6
N5 N4
N2
N
N3
N1
C D Control
Inflammation
Coupling incidence (%)
50 20
Coupling incidence (%)
+CBX
+MFA
40 +PA
30
10
20
10
0 0
Figure 11.4 Changes in Dye Coupling Between Satellite Glial Cells of
Mouse S1 DRG 10 to 12 Days After Induction of Colonic Inflammation
with Dinitrobenzoate Sulfonate Inflammation A. A Lucifer yellow
(LY)−injected SGC is coupled to other SGCs only around the same
neuron. B. Dye coupling between SGCs around different neurons,
observed in dinitrobenzoate sulfonate (DNBS)-treated mouse. The
asterisks indicate the LY-injected SGCs. Scale bars, 20 μm. C,D. The
histograms show the effect of gap junction blockers: carbenoxolone
(CBX, 50 μM), meclofenamic acid (MFA, 100 μM), and palmitoleic acid
(PA, 30 μM), on the augmented coupling among SGCs after inflamma-
tion. C. Incidence of coupling between SGCs around the same neuron.
D. Incidence of coupling between SGCs around different neurons.
to form a functional unit. Bidirectional intercellular signal-
ing between sensory neurons and SGCs is apparent in Ca2+
wave studies in dissociated cell culture, in which the Ca2+ wave
was partially inhibited by purinergic receptor antagonists and
completely blocked when cocultures were treated with a com-
bination of suramin (broad spectrum P2 receptor antagonist)
and heptanol (gap junction blocker) (Suadicani et al. 2010).
The issue of which gap junction proteins (connexins)
form the coupling pathway in sensory ganglia has only been
addressed relatively recently. Connexin43 (Cx43) was iden-
tified in the perineuronal SGCs of rat trigeminal ganglion
(Ohara et al. 2009) and mouse DRG (Procacci et al. 2008)
(see Fig. 11.3). Both Cx43 and Cx26 were also reported to
be present in SGCs of rat trigeminal ganglion (Damodaram
et al. 2009; Garrett and Durham 2008), whereas trigeminal
ganglion neurons were reported to express Cx36 and Cx40
(Garrett and Durham 2008). The expression of gap junction
proteins and the strength of gap junction−mediated coupling
display extraordinary plasticity in the sensory SGCs, one
example being the increase in abundance and strength that
occur with age in DRG of rabbits (Martinelli et al. 2004), and
mice (Huang et al. 2006). In contrast, the expression of Cx43
was found to decline in old mice (Procacci et al. 2008), sug-
gesting that the increased amount of gap junctions observed
in aged animals results from the augmentation of a gap junc-
tion protein other than Cx43. Thus, the plasticity in gap junc-
tions likely also extends to changes in the type of connexins
expressed. Even more profound changes occur following
axotomy, with SGCs extending processes that form bridges
connecting previously separate perineuronal sheaths, and
number of gap junctions between SGCs increasing markedly
(Hanani et al. 2002). The incidence of dye coupling among
SGCs also increases, with the increase in the number of gap
junctions correlating with this functional increase (Hanani
et al. 2002). Thus, SGCs that are ordinarily coupled only
to SGCs surrounding the same neuron become extensively
coupled to SGCs enveloping other neurons following axo-
tomy (see Fig. 11.4). Similar results were obtained in studies
of trigeminal ganglion following infraorbital nerve section
(Cherkas et al. 2004). Other conditions under which gap
junction-mediated coupling among SGCs increases include
compression injury of the DRG (Zhang et al. 2009), intesti-
nal obstruction or inflammation (Huang and Hanani 2005;
Huang et al. 2010), paw inflammation (Dublin and Hanani
2007) and neuritis of the sciatic nerve (Ledda et al. 2009).
3.1 T H E RO L E O F S AT E L L IT E G L I A L
C E L L S I N C H RO N I C PA I N
There is considerable evidence that glia contribute to chronic
pain (see chapter 68). As mentioned, there are increases in gap
junctions and dye coupling among sensory SGCs in diverse
types of pain models and it was found that inhibition of cou-
pling can decrease the hypersensitivity as measured behavior-
ally. Several studies report that the hypersensitivity is reversed
by treatment of the animals with gap junction inhibitors. For
example, Ohara et al. (2008) knocked down Cx43 expression
in rat trigeminal ganglion by injecting Cx43 dsRNA into the
G L I A L C E L L S I N AU TO N O M I C A N D S E N S O RY G A N G L I A
ganglion, thereby achieving analgesia in a model of neuro-
pathic facial pain. However, these authors also reported that
treatment with Cx43 dsRNA could lead to tactile allodynia
in control animals (Ohara et al. 2009), raising concerns about
whether there is actually a cause-effect relationship between
coupling or gap junction expression and pain sensitivity.
Pharmacological approaches using the gap junction inhib-
itor carbenoxolone have been reported to be quite effective in
producing analgesia in several pain models in which the tar-
get was presumed to be sensory ganglia (Dublin and Hanani
2007; Hanstein et al. 2010; Huang et al. 2010). Finally, it has
been reported that the antiepileptic drug tonabersat relieved
hypersensitivity in a rodent orofacial pain model and blocked
both the pain-related enhancement of dye spread between
neurons and SGCs and the upregulation of Cx26 in trigem-
inal ganglion (Garrett and Durham 2008). These authors
reported that the increased dye coupling between neurons and
SGCs was associated with upregulation of the neuronal gap
junction proteins Cx40 and Cx36; notably, Cx43 upregula-
tion was not affected.
The increased coupling following axon injury or inflamma-
tion is likely to increase communication between SGCs, and
our studies on dissociated ganglia from an orofacial pain model
showed enhanced SGC-SGC and SGC-neuronal signaling
(Suadicani et al. 2010). An important underlying concept when
discussing SGCs is that these cells are rarely subject to direct
injury. Rather, it is the neurons that are injured by damage to the
central or peripheral part of the axon. Thus, any change in SGCs
must be a secondary change driven by alterations to the neuron,
implying signaling mechanisms between neurons and SGCs.
There are a large number of molecules that could be involved in
such signaling, and many substances are released from injured
(and noninjured) neurons, such as nitric oxide, tumor necro-
sis factor-α (TNF-α), and adenosine triphosphate (ATP)
(Bradman et al. 2010; Hanani 2005; Takeda et al. 2009).
Adenosine triphosphate appears to be involved in pain-
related processing through the activation of metabotropic (P2Y
family) or ionotropic (P2X) purinergic receptors. Satellite glial
cells express a variety of purinergic receptors (Gu et al. 2010;
Kushnir et al. 2011; Villa et al. 2010; Weick et al. 2003), and
there is evidence that these receptors are involved in neuron-
SGC communication, and in particular in nociception-related
processes. The P2Y4 receptor has been shown to be exclusively
expressed by SGCs in sensory ganglia (Vit et al. 2006). Adenosine
triphosphate released following nerve injury may activate P2Y4
receptors on SGCs, resulting in an increase in intracellular Ca2+,
which in turn can trigger activation of K+ channels. The result-
ing change in extracellular K+ may increase nociceptive neuron
excitability through altered membrane potential or activation of
the inflammasome (Silverman et al. 2009).
4 O R G A N I Z AT I O N O F G L I A L C E L L S
I N SY M PAT H ET I C A N D
PA R A SY M PAT H ET I C G A N G L I A
The peripheral autonomic nervous system (ANS) is made up
of three functionally and structurally discrete components,
• 125
the sympathetic and parasympathetic ganglia, and the enteric
nervous system (ENS) (for detailed review see Hanani 2010).
4.1 G L I A I N SY M PAT H ET I C G A N G L I A
Preganglionic neuronal somata of the sympathetic nervous sys-
tem are located in the interomediolateral columns of the T1 to
L2 spinal cord and synapse onto paravertebral neurons of the
sympathetic chain, adrenal chromaffin cells, and prevertebral
ganglia consisting primarily of principal neurons that supply
the ENS, and abdominal and pelvic viscera. Preganglionic
parasympathetic somata are located in the brainstem and sacral
spinal cord and synapse upon parasympathetic ganglia of the
head (ciliary, submandibular, otic, and pterygopalatine gan-
glia) and on cell bodies in tissues that are targets of vagus and
pelvic nerves. The postganglionic autonomic ganglia contain
efferent nerves directly innervating smooth muscle or glands,
whereas the ENS is totally embedded in the gastrointestinal
wall. Sympathetic and parasympathetic ganglia are largely con-
trolled by the CNS, whereas the ENS is more autonomous.
Figure 11.5 shows the basic organization of a neuron in
sympathetic ganglia and its attending SGCs, which is simi-
lar to that of sensory ganglia (Matthews, 1983; Pannese 1981,
2010). Each neuron is surrounded by its own glial cover, and
together they form a distinct unit, largely isolated from other
similar units in the ganglion.
Unlike sensory neurons, sympathetic neurons receive syn-
apses, and SGCs cover axon terminals that make synaptic con-
tacts on or near the neuronal somata (Elfvin 1971; Matthews
1983). Usually most of the synapses in sympathetic ganglia
(at least in the paravertebral ones) are between preganglionic
axons and dendrites or small protrusions from the neuronal
A B Syn
n Figure 11.6 Characterization of the Satellite Glial Cell Envelope in the
nuc Syn
n same neuron. C. An example of an SGC that makes a nearly complete
Ax
Figure 11.5 The Organization of the Neuron−Satellite Glial Cell Unit
in Sympathetic Ganglia A. A low-power electron micrograph showing a
sympathetic neuron (n) surrounded by an envelope consisting of satellite
glial cells. The borders of the SGCs are traced by a thick black line. Note
that the contour of the SGC is very thin at certain points. The nucleus
of a SGC is indicated with an arrow. Nuc, nucleus of the neuron. Mouse
superior cervical ganglion. Calibration bar, 2 μm. B. Schematic diagram
of a sympathetic neuron (n) surrounded by SGCs (arrows). Note that
the synapses (Syn) are covered by SGC processes and that these processes
extend beyond the neuronal soma and ensheathe an axon (Ax) and
a dendrite (d). (A) Courtesy of M. Egle De Stefano and Paola Paggi,
La Sapienza University, Rome; (B) from Hanani 2010.
126 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
somata, and only a minority are axosomatic. Nevertheless,
ultrastructural studies have shown that the synapses are close
to the soma, and that they are enclosed by SGC processes that
wrap around dendrites emerging from the neuronal cell body
(Elfvin 1971; Matthews 1983). Dye injection experiments
reveal that SGCs send tubelike structures having lengths of at
least 30 to 40 μm, which enwrap neuronal processes in mouse
superior cervical ganglion (Fig. 11.6E). Thus, it can be con-
cluded that SGCs cover most synapses in sympathetic ganglia,
and are likely to influence synaptic transmission in these gan-
glia, just as astrocytes are partners in most aspects of synaptic
function in the CNS (Perea et al. 2009).
Nevertheless, the organization of glial cells in sympathetic
ganglia is quite different than in the CNS. Whereas astrocytes
contact numerous neurons, oligodendrocytes, and the vascu-
lature (see Dermietzel and Spray 2012), these connections are
absent in the sympathetic ganglia, where each neuron is sur-
rounded by several SGCs that are in close contact with each other,
and are separated from SGCs surrounding other neurons.
Elfvin and Forsman (1978) identified gap junctions in
SGCs in paravertebral and prevertebral ganglia of rabbits and
guinea pigs. Using intracellular dye injections (see Fig. 11.6),
we showed that SGCs in mouse superior cervical ganglion
A B C
D E F
N2
N1
N3
Mouse Superior Cervical Ganglion with Intracellular Injection of the
Fluorescent Dye Lucifer Yellow All images were obtained on live tissue
during the injection experiments; the injected cell is marked with an
asterisk. A. A single SGC makes a complete envelope around the neuron.
Here, and in the rest of the images, neurons were not stained. B. The
dye has passed from the injected SGC into another SGC around the
sheath around a neuron, but a small neuronal region appears not to
be covered by the injected SGC (arrow). This may result from a great
attenuation of the glial envelope in this region, or an incomplete invest-
ment of the neuron by this cell. It is quite possible that another SGC
(which was not coupled to the injected cell) covers this region. D. An
example of an SGC making a partial envelope around a neuron. In this
case it is very likely that another SGC covers the left part of the neuron,
and again is not coupled to the injected cell. E. The injected SGC fully
surrounds the neuron and also wraps around two neuronal processes.
Note the tubelike glial cover of these processes (arrows). F. An example
of interenvelope dye coupling. An SGC around neuron N1 was injected
with dye, which spread to another SGC around this neuron, and also to
SGCs around two adjacent neurons (N2 and N3). This type of coupling
is rare (in about 3% of the cells) under normal conditions, but its inci-
dence increases eightfold following peripheral injury. Calibration bars,
20 μm. From Hanani 2010.
are coupled around a given neuron. Coupling between SGCs
surrounding different neurons is normally very rare, but
increases after peripheral inflammation (Hanani et al. 2010)
(see Fig. 11.6). The connexin type responsible for gap junction-
mediated intercellular communication between sympathetic
SGCs remains to be determined, as does the degree to which
coupling strength can be modulated by agents affecting cou-
pling in sympathetic neurons (Kessler et al. 1984).
A major change in sympathetic ganglia following axo-
tomy is the detachment of the presynaptic terminals from the
postsynaptic membrane (“synaptic stripping,” see Kreutzberg
1995). Using extracellular electrical recordings, Matthews and
Nelson (1975) found that after axotomy, synaptic transmission
in the ganglion was greatly depressed. Similarly, intracellular
recordings from guinea pig superior cervical ganglion after
axotomy demonstrated a 70% reduction in synaptic poten-
tial amplitude (Purves 1975). Matthews and Nelson (1975)
also noted that sympathetic neurons lost most or all of their
synaptic inputs after axotomy and became physically separated
from them. Significantly, presynaptic terminals appeared
normal. These findings indicate that the reduction in synap-
tic transmission was not caused by changes in the presynap-
tic compartment. Matthews and Nelson (1975) noted that
glial processes become interposed between the retracting ter-
minals and the postsynaptic cells and therefore apparently
played a role in synaptic stripping. Detached presynaptic
profiles were often wrapped by one or more narrow lamella
of SGC cytoplasm, which enveloped the specialized presyn-
aptic region. Thus it appears that after injury SGCs formed
new extensions. The presence of glial processes between pre-
synaptic and postsynaptic elements after injury was confirmed
by more recent ultrastructural studies (DeStefano et al. 2007).
Overall, this phenomenon of interposed glial process between
presynaptic and postsynaptic neuronal elements is similar to
that studied extensively in supraoptic hypothalamus (Oliet
et al. 2008).
4.2 G L I A I N PA R A SY M PAT H ET I C G A N G L I A
Much less research has been conducted on SGCs in parasym-
pathetic ganglia than in sympathetic ganglia. This is caused in
part by most parasympathetic ganglia being relatively inacces-
sible. Also, in contrast with sympathetic ganglia, which are
compact and well-defined structures that are located at a large
distance from their targets, parasympathetic ones are more
diffuse, and therefore more difficult to isolate.
The general organization of parasympathetic ganglia is very
similar to that seen in sympathetic ganglia. The glial cells wrap
around the neurons, and thus are classified as SGCs. The intrin-
sic ganglia of the urinary bladder are typical for this system
(Gabella 1990), and SGCs in them make a thin sheath around
individual neurons, which is attenuated in some regions, but
is still continuous. In the guinea pig tracheal ganglia, neurons
and their processes are almost always covered by a sheath con-
sisting of several SGCs, which can be extremely thin (about
0.2 μm) (Baluk et al. 1985). This sheath is interrupted in
only very small areas, in which a thin neuronal process pro-
trudes between two SGCs, making contact with the basal
G L I A L C E L L S I N AU TO N O M I C A N D S E N S O RY G A N G L I A
lamina and allowing the possibility that the neural protrusions
might play a functional role in sensing the ganglionic environ-
ment. The neuronal plasma membrane and SGC membranes
display complex interdigitations, which greatly increase
the surface area of both cells, and can promote intercellular
interactions.
There is only scarce information on gap junctions in
SGCs of parasympathetic ganglia. There is electron-micro-
scopic evidence for gap junctions in SGCs in the chicken
ciliary ganglion (Forsman et al. 1989). Hanani et al. (1999)
studied dye coupling among SGCs in the intramural gan-
glia of the guinea pig urinary bladder and found only a small
degree of dye coupling among SGC forming the envelope of
individual neurons.
5 ENTERIC GLIAL CELLS
Since the 1970s there has been intense interest in the ENS,
as investigators recognized that it is the main element in the
control of all functions of the digestive system (Goyal and
Hirano 1996). Although most of the research in this field
was devoted to enteric neurons, there has been a fair amount
of work on enteric glial cells (EGCs), and these cells are the
most characterized among the glial cells in peripheral ganglia.
The mere name enteric glia indicates that these cells are unique
to this system, in accord with the special properties of the ENS
within the ANS. It should be noted that most of the current
knowledge on the ENS is derived from work on the guinea
pig small intestine; therefore, generalizations to other species,
especially humans, should be made with caution.
The ENS contains more than 100 million neurons and 4
to 10 times as many glia. (Neunlist et al. 2008). These gan-
glia are organized as submucosal (Meissner’s) plexus in the
submucosa, which control mucosal activity, and myenteric
(Auerbach) plexus between circular and longitudinal layers
that control these muscles. Markers for these glial cells include
Sox10, GFAP, and S100b. It is believed that EGCs are major
regulators of barrier and neuronal functions in the gut. Enteric
glial cells decrease intestinal mucosal barrier permeability via
release of S-nitrosoglutathione and regulation of expression
of the tight junction proteins ZO1 and occludin (Savidge
et al. 2007).
The depth of understanding of molecular and develop-
mental neurobiology of the gut is extensive, with Sox10 and
Notch signaling pathways being critical to gliogenesis in the
ENS (see chapters 14 and 43) (Ngan et al. 2011; Taylor et al.
2007). Sequences of transcription factor activation have been
proposed to underlie differentiation of both neurons and the
EGCs (Chalazonitis and Kessler 2011; Chalazonitis et al.
2011). In this scheme of development of the bowel ganglia,
bone morphogenetic proteins (BMP2 and BMP4) play criti-
cal roles in differentiation of both neurons at early embryonic
times and later glia. The early effect is BPM enhancement
of GDNF, thereby favoring neurogenesis, and suppress-
ing NRG1 (formerly GGF2) and the development of glia.
Later, the BMPs increase expression of ErbB3 and enhance
NRG1-driven enteric gliogenesis with decreased neurogenic
• 127
 A1 A3
A4
A2
A5
50 μm
B C D quantified by a fractal dimension of around 1.5, which is simi-
E F
30 μm 20 μm
Figure 11.7 Some Morphological, Histochemical, and Functional
Characteristics of Enteric Glial Cells A1−A5. Morphology of EGCs in
the myenteric plexus of the guinea pig small intestine that were injected
intracellularly with horseradish peroxidase (HRP). A1. A stellate EGC,
located within the ganglion; several processes of the cell make endfeet
with a blood vessel, and other processes contact the borders of the gan-
glion. A2. Another example of a stellate EGC, showing its relationship
to a neuron (asterisk). A3. An example of SGC located in the fiber tract
connecting the ganglia. Note the elongated shape of the processes, and
the thickenings that contact the border of the fiber tract. A4. Another
example of an EGC in a fiber tract. A5. An EGC whose right part, which
is located within the ganglion is symmetrical (stellate shape), whereas its
left side is in a fiber tract. B. Myenteric ganglion in the guinea pig small
intestine, where a single EGC (asterisk) was injected with the fluorescent
dye Lucifer yellow. The dye diffused (most likely through gap junctions)
to numerous EGCs in the ganglion. The neurons are not labeled with the
dye, and are seen as dark circles. C. Injection of an EGC in a submu-
cosal ganglion shows dye spread even to EGCs in neighboring ganglia
(arrowheads). D. An EGC in the submucosal plexus injected with HRP.
Some processes contact the border of the ganglion. E. Double immuno-
histochemical labeling of mouse colonic myenteric plexus for aquaporin
4 (AQP4) and the glial marker GFAP. Some neurons (arrowheads)
and nerve fibers (arrows) are labeled for AQP4. Enteric glial cells are
GFAP-positive, and do not label for AQP4. Similar results were obtained
in the small intestine of mice and rats. F. Immunostaining for AQP4
and GFAP in the mouse colonic submucosal plexus. (E,F) From Thi
et al. 2008.
response to GDNF. In the context of interplay between tran-
scription factor determination of fates of neurons and glia,
it should be noted that recent reports have indicated that
EGCs can differentiate into neurons under specific conditions
(Gershon 2011).
128 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
5.1 S T RU C T U R E O F E N T E R I C G L I A L C E L L S
Enteric glial cells are similar in many respects to astrocytes
of the CNS (Gabella 1981; Jessen and Mirsky 1980). Unlike
SGCs, EGCs do not envelop neurons, but each EGC sends
processes that make contact with several neurons, as seen in
astrocytes. Hanani and Reichenbach (1994) studied the mor-
phology of glial cells in the myenteric plexus of the guinea
pig small intestine using intracellular injection of horseradish
peroxidase, and found a great complexity of structure, resem-
bling that found in astrocytes (Fig. 11.7). The complexity was
lar to that of astrocytes (Reichenbach et al. 1992). Two main
morphological EGC types were found: stellate cells that send
processes in a symmetrical manner and are found within the
ganglia, and cells sending processes in an elongated pattern
that are found within the fiber bundles connecting the ganglia
(see Fig. 11.7). These types roughly correspond to protoplas-
mic and fibrillary astrocytes, respectively.
Satellite glial cells can make contacts with blood vessels
and/or with the surface of the ganglia via thickenings of the
glial processes that resemble astrocytic endfeet (Gabella 1981;
Hanani and Reichenbach 1999). Some EGC processes form
“windows,” which wrap around nerve bundles (Gershon and
Rothman 1991; Hanani and Reichenbach 1994). Enteric glial
cells share other features with astrocytes such as the pres-
ence of glutamine synthetase, Ran-2 (ceruloplasmin) ( Jessen
and Mirsky 1983), apolipoprotein E (Bolyes et al. 1985). See
Gershon and Rothman (1991) and Rühl (2005) for reviews.
Enteric glial cell processes (apparently from the submu-
cosal plexus) extend into the mucosa to the villous tips, and
thus might affect mucosal functions such as secretion and
influence mucosal permeability (Bush et al. 1998; Cornet
et al. 2001; Neunlist et al. 2008). These projections might
sense the luminal contents and send appropriate signals to
the ENS. It has been claimed that a population of glial cells is
located in the intestinal mucosa (Bernstein and Vidrich 1994).
This would require that these cells are not associated with
neuronal somata of the ENS, which would be an exception
for EGCs, because there is no evidence for neurons within the
mucosa proper. This potentially important observation has
not been confirmed.
5.1.1 Gap Junctions
Gap junctions in EGCs were described by electron micros-
copy (Gabella 1981). Dye injection studies demonstrated that
EGCs are extensively coupled to each other in both myenteric
and submucosal plexuses (Hanani et al. 1989; Maudlej and
Hanani 1992) (see Fig. 11.7). As can be seen in Figure 11.7B,
the dye coupling in myenteric ganglia can extend over a dis-
tance of several hundred μm, and may cover the whole gan-
glion. In the case of submucosal plexus, coupling even extends
between ganglia (see Fig. 11.7C). The spatial extent of dye cou-
pling seen in these studies probably exceeds that observed in
the CNS, which may be caused by numerous gap junctions,
but can also be explained by the essentially two-dimensional
structure of the enteric ganglia, in which no dilution of the
dye occurs in three dimensions, as in the CNS. These observa-
tions suggest that EGCs engage in spatial buffering of K+ and
possibly in clearing other potentially harmful substances.
5.2 P H YS I O L O GY A N D P H A R M AC O L O GY
OF ENTERIC GLIAL CELLS
A well-established function of glial cells is to regulate their
environment. There is evidence that EGCs contain the GABA
transporter GAT2 (Fletcher et al. 2002), whereas astrocytes
contain GAT1,3. There is evidence for glutamatergic trans-
mission in the ENS (Kirchgessner 2001), and for glutamate
receptors in EGCs (Nasser et al. 2007), but there is so far no
evidence for glutamate transporters in EGCs, which are abun-
dant in astrocytes.
The small size of EGC is one reason the physiology of
these cells has received little attention. Patch clamp record-
ing is clearly the method of choice for studying the physiol-
ogy of these cells. Broussard et al. (1993) developed a tissue
culture preparation containing a nearly pure population of
EGC from the myenteric plexus of guinea pig ileum. The
cells retained expression for the glial markers S100 and glu-
tamine synthetase, and for GD3 ganglioside. Broussard et
al. (1993) made patch clamp recordings from these cells and
found that the major ionic conductance is for outward recti-
fying K+ channels. Surprisingly, 8% of the cells had tetrodo-
toxin (TTX)-sensitive Na+ channels. Hanani et al. (2000)
made patch recordings from glia in isolated myenteric gan-
glia, a preparation that is closer to the intact situation than
cultures. They also identified outward rectifying K+ channels.
However, when the gap junctions were blocked, the K+ chan-
nel blocker Ba2+ (1 mM) had a strong inhibitory effect on the
currents (particularly the inward ones) indicating that inward
rectifying channels are also present in EGCs. These channels
are believed to be important for K+ buffering in central glia
(Verkhratsky and Steinhäuser 2000), and these results provide
indirect evidence for such a function of EGCs. These experi-
ments show that various ionic conductances may normally
be masked by dominant “passive” currents, which are largely
caused by gap junctions among EGCs (Hanani et al. 1989).
An elegant series of experiments on cultured EGCs
from the myenteric plexus of the newborn guinea pig ileum
employed Ca2+ imaging to characterize responses to a num-
ber of putative neurotransmitters. EGCs were found to
respond to ATP and UTP by an elevation of intracellular
Ca2+ concentration ([Ca2+]in), and apparently possess what
are now termed P2Y2 and/or P2Y4 receptors (Kimball et al.
1996). These results correlate with the presence of the ecto-
nucleotidase (NTPDase2) in EGCs (Braun et al. 2004), an
enzyme that breaks down ATP. Endothelin increased [Ca2+]in
by IP3-dependent release from intracellular stores, appar-
ently via ETB receptors (Zhang et al. 1997). Responses to
both ATP and endothelin were followed by capacitative Ca2+
influx (Sarosi et al. 1998; Zhang et al. 1998). In addition to
P2Y receptors, which are metabotropic, ionotropic P2X7
receptors were identified in EGCs by immunohistochem-
istry (Vanderwinden et al. 2003). These authors suggested
that, as ATP levels increase under pathological conditions
G L I A L C E L L S I N AU TO N O M I C A N D S E N S O RY G A N G L I A
(e.g., inflammation), these receptors may participate in the
response of EGCs. Also, EGCs contain protease-activated
receptors (PAR), which are thought to participate in tissue
responses to injury and inflammation Activation of these
receptors by trypsin and thrombin induced a rise in [Ca2+]in in
cultured myenteric EGCs (Garrido et al. 2002). Enteric glial
cells display additional receptors, among which are receptors
for glutamate (Nasser et al. 2007), interleukin beta 1 and 6
(Rühl, 2005), lysophosphatidic acid (Segura et al. 2004a),
and sphingosine-1-phosphate (Segura et al. 2004b).
A study on intact segments of guinea pig ileum showed
that ATP indeed is a messenger between neurons and EGCs
(Gulbransen and Sharkey 2009). Neuronal stimulation-
evoked Ca2+ increase in EGCs, which were blocked by P2
receptor blockers, and EGCs responded to exogenous ATP.
These authors concluded that EGCs have P2Y4 receptors.
In summary, EGCs are endowed with a wide spectrum of
membrane receptors that enable them to interact with their
environment. The presence of receptors that are activated with
ligands associated with inflammation (ATP, PAR) supports the
reports on the potential protective role of EGCs against inflam-
mation (Bush 2002). Therefore, EGCs might be an important
target for medical therapy for gastrointestinal disorders, and
inflammatory bowel diseases in particular (see section 5.3).
5.3 E N T E R I C G L I A A N D I N T E S T I NA L D I S E A S E
Crohn disease (CD) is a debilitating and unexplained inflam-
matory bowel disease. Enteric glial cells from CD patients
display abnormal features; they are strongly positive for major
histocompatibility class II antigens (Geboes et al. 1992). Thus,
EGCs may serve as antigen presenting and/or target for T
cells. The functional significance of these observations is not
clear, because so little is known about EGCs in humans.
Animal studies suggest that EGCs may be essential for
the integrity of the digestive tract. In two separate studies,
transgenic mice were used to deplete or reduce the numbers
of EGCs (Bush et al. 1998; Cornet et al. 2001). This resulted
in intestinal inflammation leading to the animals’ death. The
clinical and pathological findings indicate an inflammatory
bowel disease resembling CD. Interestingly, in CD patients
both involved and uninvolved intestinal segments showed a
diminished EGC network, suggesting that EGCs control the
integrity and permeability of submucosal blood vessels and
intestinal mucosa (Cornet et al. 2001). Thus, when EGCs are
not present, vascular and mucosal barriers break down, lead-
ing to inflammation. Glial depletion was proposed to lead
to mucosal inflammation (Bush 2002). Bush (2002) empha-
sized the structural similarities between EGCs and astrocytes,
which may indicate common functions. Reactive astrocytes
release a number of cytokines, growth factors, and other active
compounds (Watkins and Maier 2002). These substances
can affect the neurons directly, by acting on receptors, or
indirectly, for example by altering blood flow. These responses
can be beneficial to the survival of neurons, but in some cases
they may be detrimental.
Further evidence on the role of EGCs in inflammatory
bowel disease (Rühl et al. 2001), demonstrated that EGCs
• 129
from rat myenteric plexus produce the proinflammatory
cytokine IL-6, and this process is regulated by the proinflam-
matory cytokines IL1-β and IL-6 but not TNF-α. This again
supports the notion that EGCs play an important role in intes-
tinal inflammatory reactions. The authors argued that myen-
teric glia are strategically located to amplify and perpetuate
the inflammatory response in the ENS because of their close
anatomical association with enteric neurons and their location
at the edge of the ganglia. This provides them with the ability
to intervene between the extraganglionic and intraganglionic
compartments. Thus they can receive signals and produce the
proinflammatory cytokine IL-6.
Because the submucosa is more closely associated with the
mucosa, glial cells in the submucosal plexus are even more
likely than myenteric plexus glia to be involved in the path-
ological processes discussed in the preceding. However, the
studies mentioned in the previous sections were mostly done
on glial cells of the myenteric plexus.
Chagas disease, caused by infection with the intracellu-
lar protozoan parasite Trypanosoma cruzi, is a major public
health problem in Latin America (Tanowitz et al. 2009). It
is characterized by a brief acute phase, followed by a chronic
phase that can last several decades. Although the most severe
pathology associated with the disease is its cardiac manifesta-
tion, the gastrointestinal tract is involved in 8% to 10% of
infected patients. The main pathology of the gut is the devel-
opment of a megaesophagus and/or megacolon with severe
defects of motility (de Souza et al. 2010). Although modu-
lated tone of the gut wall has been widely attributed to loss
of enteric neurons, it has recently been shown that dilated
intestines are characterized by reduced EGCs (da Silveira
et al. 2009), whereas GFAP-positive EGCs are increased in
nondilated portions (Nascimento et al. 2010). Thus, it now
appears that chagasic megacolon may also represent a gliopa-
thy, in which loss of EGC reduces secretion of neurotrophic
factors. Similarly to CD, it has been suggested that EGCs
could also act as antigen-presenting cells in Chagas disease
(da Silveira et al. 2011). Trypanosoma cruzi infection of car-
diac muscle and other cell types leads to decreased gap junc-
tion-mediated intercellular signaling (Adesse et al. 2011), and
whether this is also the case in EGCs will be most interesting
to determine.
There is evidence that in another inflammatory bowel
disease in humans—ulcerative colitis—EGCs are activated
and release increased amounts of nitric oxide (NO), which
can contribute to mucosal damage (Cirillo et al. 2011). Celiac
disease is a common inflammatory disease in which the mucosa
in the small intestine is disrupted (Schuppan et al. 2009). It
has been found that NO production (via S100B upregula-
tion) by EGCs participates in the inflammatory process in this
disease as well (Esposito et al. 2007). Bassotti and Villanacci
(2011) have reviewed the literature and argue that constipa-
tion should be considered a neurogliopathy because functional
impairment is associated with reduced EGC number.
In summary, the idea that SGCs are involved in gastroin-
testinal disease is quite recent, but the available information
indicates key roles for these cells in a variety of inflammatory
and other gastrointestinal disorders.
130 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
6 B L O O D − N E RVO U S SYS T E M B A R R I E R S
I N T H E P E R I P H E R A L N E RVO U S SYS T E M
The question of whether a barrier exists between the blood and
neural cells in the periphery has been controversial. On the
one hand, peripheral nerves are surrounded by a highly imper-
meable perineurium, creating the so-called blood-nerve bar-
rier, and both enteric and sensory ganglia are also encased in
an impermeable sheath. However, in contrast to most regions
of the CNS, the vasculature within the ganglia is fenestrated
and leaky, so that the capillaries provide relatively free access
to neurons and glia. This arrangement of a connective tissue
sheath surrounding sensory and sympathetic ganglia providing
an impermeable covering, yet penetration by proteins from the
blood creates the interesting paradox in which removal, rather
than delivery, may be impeded in the ganglia. The ENS is avas-
cular, leading to the proposal that there may be a blood-brain
barrier in that tissue (Gershon 1981). However, subsequent
studies indicated that EGCs and neurons are in contact with
circulating factors (Allen and Kiernan 1994). There have also
been reports that penetration within the ganglia of injected
probes may be different in sensory and sympathetic ganglia.
Thus, a tight junction barrier was proposed by Ten Tusscher
et al. (1989) to be present in SGCs in autonomic but not in
sensory ganglia. As pointed out by Kiernan (1996), more recent
studies using a variety of probes have indicated that in the gan-
glia of the PNS, neurons and glia are exposed to blood-borne
agents. The absence of a blood neural barrier in PNS offers the
opportunity for exposure of cells to therapeutic agents.
Astrocytes in the CNS send endfoot processes to the vessel
wall, both signaling for local autoregulation of blood flow and
establishing a tight barrier through induction of various genes
(see Dermietzel and Spray 2012). A marker of this specialized
astrocyte domain is the water channel protein aquaporin 4
(AQP4) (Nicchia et al. 2004). Aquaporin 4 is located in astro-
cytic endfeet in membranes that contact capillaries and pia,
in which water can be exchanged between inside and outside
the brain. Therefore, it was suggested that AQP4 may play a
role in the regulation of water homeostasis in the central ner-
vous system (Nicchia et al. 2004). Our immunohistochemi-
cal studies reveal that AQP4 expression is confined to a small
number of myenteric neurons and a larger fraction of submu-
cosal neurons, but was absent in EGCs (Thi et al. 2008) (see
Fig. 11.7E,F). This is a further example of a difference between
EGCs and astrocytes.
7 S U M M A RY A N D P E R S P E C T I VE S
Peripheral ganglia are much more than relay stations between
the CNS and sensory terminals or target organs such as the
heart, smooth muscle, and glands. It is now recognized that
these ganglia are the site of processing of neural information,
which takes place mainly via chemical messengers. Other
key players in these ganglia are specialized glial cells—SGCs
in sensory, sympathetic, and parasympathetic ganglia, and
EGCs in the ENS. Research on most aspects of this topic is
only at the very beginning, and we still lack information on
the basic physiology and pharmacology of these cells and how
their function is altered under pathological conditions. The
central question concerning glial cells in peripheral ganglia is
the nature of neuron−glia interactions, and in the ENS, also
between EGCs and the intestinal mucosa. The chemical mes-
sengers mediating these interactions include ATP, but addi-
tional molecules are very likely to be present. Also, much needs
to be learned about the role of gap junctions among the glia,
and possibly between them and the neurons. In the CNS, glial
cells communicate with blood vessels, but in the periphery vir-
tually nothing is known about this topic. It is hoped that this
chapter will stimulate additional interest in these cells, and
that both ongoing and new research in the area will provide
answers to these questions.
AC K N OW L E D G M E N T S
Work done in the authors’ laboratories was supported by the
European Community’s 7th Framework Programme through
the Marie Curie Initial Training Network Edu-GLIA, the
Israel Cancer Association, the Israel Science Foundation (grant
no. 212/08), by the US−Israel Binational Science Foundation
(grant no. 2007311) and by the National Institutes of Health
(NS041282).
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 SECTION 2
L I N E AG E A N D D E VE L O PM E N T
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 12.
ASTROCY TE DEVELOPMENT
James E. Goldman

A B B R E VI AT I O N S different regions varies (e.g., the white matter of the spinal

cord is at its periphery, whereas the white matter of the
bHLH basic helix-loop-helix transcription factor hemispheres is more centrally located). There seem to be
BLBP brain lipid binding protein several general rules, however: (1) Astrocytes, along with
BMP bone morphogenetic protein neurons and oligodendrocytes, originate from ventricu-
CNTF ciliary neuronotropic factor lar zone (VZ) cells in the embryonic CNS. (2) Astrocytes
CT1 cardiotrophin 1 have at least two different origins—one directly from radial
DNMT DNA (cytosine-5)-methyltransferase glial cells and the other indirectly, through a proliferative
EGF epidermal growth factor and migratory population that populates the subventricular
FGF fibroblast growth factor zone (SVZ). (3). A series of soluble and cell surface mole-
IL-6 interleukin 6 cules regulate astrocyte genesis. (4) These molecules induce
LIF leukemia inhibitory factor different combinations of transcription factors that specify
NF1 nuclear factor 1 astrocyte fate and progressively restrict the fate of immature
NGF nerve growth factor cells.
OSM oncostatin M
PACAP pituitary adenylate cyclase activating
polypeptide 2.1 A S T RO C Y T E D EV E L O PM E N T
SVZ subventricular zone IN THE FOREBRAIN
TGF-ß transforming growth factor-ß
2.1.1 Some Astrocytes Arise Directly from
VZ ventricular zone
Radial Glial Cells
The VZ is a pseudostratified epithelium composed of the
1 INTRODUCTION earliest neuroectodermal cells. At about the time when cor-
tical neurons begin to develop (around E11 in mouse), elon-
The developing central nervous system (CNS) gener- gated, radially oriented cells appear in the VZ that share
ates a wealth of astrocytes of different forms and func- many characteristics in common with astrocytes, such as
tions. Therefore, any model of astrocyte development must the GLAST type of glutamate transporter, glycogen gran-
account both for the origins of astrocytes from the imma- ules, brain lipid–binding protein (BLBP), and GFAP (see
ture neuro-epithelium and the wide variety of astrocytes that chapter 5). Radial glia, the historical term for these cells,
populate the adult CNS (see chapter 4). This chapter sum- span the width of the neural tube from ventricular to pial
marizes astrocyte development in several CNS regions and surface, and are found in all regions of the developing CNS.
discusses the molecular regulation of the commitment to an In the cerebral cortex, radial glia generate neurons, primar-
astrocyte fate. ily projection neurons (Malatesta et al. 2000, 2003; Noctor
et al. 2001) (see chapter 5). At a later time, after neuronal
migration has ceased, some of the radial glial cells trans-
2 PAT T E R N S O F A S T R O C Y T E form into astrocytes (Fig. 12.1). We do not know, however,
D E VE L O PM E N T I N D I F F E R E N T R E G I O N S what proportion of astrocytes are generated in this way or if
O F T H E C E N T R A L N E RVO U S SYS T E M : I S those astrocytes have different properties from those gener-
A S T R O C Y T E D E VE L O PM E N T I D E N T I C A L ated from SVZ cells (see section 2.1.2). The generation of
E VE RY W H E R E I N T H E B R A I N ? astrocytes from radial glia likely occurs in all areas of the
CNS, although a direct visualization of this transformation
It is unlikely that the details of astrocyte development are has been made in forebrain (Gaiano et al. 2000; Ventura
identical in every region of the CNS. Development needs and Goldman 2007; Voigt 1989). Fate tracing of radial
to be placed in an appropriate anatomical context, because glia into astrocytes shows that they can generate both gray
different types of astrocytes need to be generated at differ- matter and white matter astrocytes (Ventura and Goldman
ent times in different regions and because the anatomy of 2007).

137
 Glial Precursor Migration and Differentiation in Forebrain

AS
OL

OL

OL(NG2)

AS

AS
RG

Radial Glia Tranform into Astrocytes

Postanatal Glial Precursor Migration Paths

Figure 12.1 Near the end of gestation and into postnatal life, glial precursors migrate from the forebrain SVZ into white matter and cortex to become
astrocytes and oligodendrocytes (myelinating oligodendrocytes, NG2+ cells, and immature oligodendrocytes). NG2+ cells reside in white matter
(not shown) as well as cortex. Some of the radial glia transform directly into astrocytes. AS, astrocyte; OL, oligodendrocyte; OL(NG2) NG2+ glia;
RG, radial glia.

2.1.2 Some Astrocytes Do Not Arise Directly from

Radial Glia, but from Intermediate, Highly
Migratory Precursor Cells
Other astrocytes arise from immature cells that emigrate
from the SVZ in the perinatal period and migrate extensively
through forebrain to colonize both gray and white matter (see
Fig. 12.1) (Levison and Goldman 1993; Marshall et al. 2003).
Although these cells initially must originate from radial glial
cells, how are they generated and from where do they colonize
the SVZ?
The forebrain SVZ at this time is populated by a large
number of highly migratory and proliferating cells, whose
fates include neurons and oligodendrocytes and astrocytes
(Levison and Goldman 1993; Luskin and McDermott 1994;
Luskin et al. 1993). Although SVZ cells do not appear to
express well-known astrocyte markers within the SVZ itself,
they belong to the olig2+, gliogenic population, which gener-
ates astrocytes and oligodendrocytes ( Jang and Goldman 2011;
Marshall et al. 2005). Those gliogenic SVZ cells that develop
along an astrocyte lineage begin to express early markers such
as Zebrin II (aldolase C), vimentin, nestin, and GFAP after
they exit from the SVZ (Staugaitis et al. 2001; Zerlin et al.
1995). The astrocyte precursors migrate extensively and con-
tinue to divide as they migrate (Fig. 12.2), and then acquire
further markers depending in part on whether they settle in
white matter or gray matter (see section 2.1.3).
One of the earliest interactions of astrocyte precursors is
with blood vessels (Zerlin and Goldman 1997), which sug-
gests they already express molecules that recognize endothelial
cells or basal laminae (Fig. 12.3). Astrocytes then develop far
more complex shapes with multiple branched processes until
they acquire the bushy morphology of mature, protoplasmic
astrocytes. Astrocyte precursors do not appear to develop syn-
chronously, because during early postnatal development, astro-
cyte morphologies are heterogeneous and sometimes overlap
(Fig. 12.4) (see Bushong et al. 2004 for a study of astrocyte
maturation in the hippocampus). It takes about 1 month to
reach mature shapes and mature cellular domain boundaries
(Bushong et al. 2004) (see chapter 4).
These migratory SVZ cells arise from both dorsal and ven-
tral areas of the developing forebrain. Ventrally derived SVZ
cells migrate dorsally along the ventricle to form the large SVZ
at the dorsolateral angle of the lateral ventricle in perinatal life
(Marshall and Goldman 2002). This dorsolateral SVZ popu-
lation gives rise to astrocytes and oligodendrocytes of the cor-
tex, white matter, and striatum, as well as to interneurons of
the olfactory bulb (Levison and Goldman 1993; Luskin and
McDermott 1994; Luskin et al. 1993). The ventral origins of
some interneurons, oligodendrocytes, and some astrocytes
raise the question of whether glia and interneurons might
arise from common progenitors. This is difficult to answer
now, although cells isolated from the embryonic lateral gang-
lionic eminence and placed in culture will generate clones
composed of neurons and oligodendrocytes (He et al. 2001),
but interestingly, not astrocytes, at least under those particular
conditions. In the neonatal period, forebrain SVZ cells placed
in culture give rise to clones containing neurons and glia, but
under these conditions the glia are both astrocytes and oligo-
dendrocytes (Levison and Goldman 1997). Thus, at least some
of the immature cells from these regions have the potential to
differentiate into both neurons and glia—whether they actu-
ally do in vivo is not known.

138 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
 ZII
GLAST, GLT-I
Vimentin (low)
Nestin (low)
ASTROCYTE DEVELOPMENT FROM FOREBRAIN SVZ CELLS GFAP(low)
S100β
YbGST
OLIGODENDROCYTES Aquaporin4
lon Channels
Aldh1

“Protoplasmic”
TO GRAY MATTER
SVZ

PSA-NCAM
ZII
GLAST
OLIG1,2 “Fibrous”
ZII
TO WHITE MATTER
GLAST, GLT-I (low)
Other Transporters
Vimentin (low)
Nestin (low)
GFAP(high)
S100β
YbGST
Aquaporin4
lon Channels
CD44
Aldh1

Figure 12.2 Astrocyte precursors migrate from the neonatal SVZ into white matter and gray matter. Early steps include contact with blood vessels (see
Fig. 12.3). Astrocytes that settle in white matter become “fibrous” astrocytes, and those in gray matter “protoplasmic” astrocytes, to use the classical
terminology. The latter interact with both blood vessels and neurons, including synapses. Some of the proteins found in the cells are given in red. SVZ,
subventricular zone

Astrocytes contact blood vessels at an early stage

of astrocyte development

A B

BV

Figure 12.3 Astrocyte precursors contact blood vessels early in their
development. Images are from retroviral labeled cells that migrated from
neonatal forebrain SVZ into the cortex (see Zerlin and Goldman 1997).
Glia lineages in vivo have been studied using
replication-deficient retroviruses, an approach that can deter-
mine lineage relationships by defining the set of cells that
arise from the viral infection of a single, dividing progeni-
tor. In some cases, both neurons and glia arise from the same
progenitor. For example, single progenitors in the embryonic
retina give rise to neurons and Muller glia (Turner and Cepko
1987). The developmental potential of retinal cells becomes
restricted over time, however, and in the late embryonic and
Figure 12.4 Astrocyte precursors in the cortex do not differentiate
synchronously. Image taken 7 days after X-gal–expressing retrovirus was
injected into the neonatal forebrain SVZ. 2 astrocytes have become more
mature and protoplasmic (*); precursor contacting a blood vessel (BV)
(arrow); two precursors next to each other, each with a limited arborization
(arrowhead). Bar: 100 μ.
early postnatal period, dividing progenitors give rise largely to
Muller glia and photoreceptor cells. Individual progenitors in
the embryonic neocortex and the striatum can generate both
astrocytes and neurons (Halliday and Cepko 1992; Walsh
and Cepko 1993), possibly through radial glial intermedi-
ates. Most studies of gliogenesis find that retroviral-labeled

A S T R O C Y T E D E VE L O PM E N T • 139
cells accumulate in groups, or clusters, which are in most 2.2 A S T RO C Y T E D EVE L O PM E N T I N T H E
cases homogeneous—either astrocytic or oligodendrocytic. C E R E B E L LU M
However, a small proportion of clusters, about 15%, are The cerebellum contains a variety of astrocyte forms, includ-
mixed, either containing astrocytes and oligodendrocytes, or ing the fibrous astrocytes of white matter, velate astrocytes of
more rarely, neurons and glia (Grove et al. 1993; Levison and the internal granule cell layer, and Bergmann glia, which send
Goldman 1993; Luskin and McDermott 1994; Luskin et al. radially directed processes from the cell bodies in the Purkinje
1988, 1993; Parnevales 1999; Price and Thurlow 1988). Most cell layer to end at the pial surface (Palay and Chan-Palay
if not all of these clusters are clonal (Zerlin et al. 2004). The 1974).
presence of mixed astrocyte–oligodendrocyte clusters tells Early morphological and 3H-thymidine studies suggested
us that a small proportion of the gliogenic SVZ cells may
that some Bergmann glia are generated from other Bergmann
be specified to a glial lineage, but not necessarily to either
glia (because they incorporate thymidine) and/or some pro-
an astrocyte or an oligodendrocyte lineage as they emigrate
genitor, of an unknown type (Basco et al. 1977; Choi and
from the SVZ.
Lapham 1980). It is likely that some Bergmann glia as well as
Analysis of gliogenesis is complex because of spatial dis-
other astrocytes arise from embryonic radial glia of the cer-
persion of members of a clone. Note that immature astrocytes
ebellum. Some astrocytes, including Bergmann glia, share a
and oligodendrocytes continue to divide as they migrate, but
common lineage with Purkinje cells, as determined by retro-
the two progeny of a dividing cell may not necessarily con-
viral tracing in chick (Lin and Cepko 1999).
tinue to migrate together, and therefore would not necessarily
During the perinatal period, astrocyte progenitors, along
end up in the same cluster. Indeed, members of the same clone
with oligodendrocyte and interneuron progenitors, arise
can disperse widely (Zerlin et al. 2004). A direct visualization
in the base of the cerebellum, just dorsal to the fourth ven-
of migrating glia shows that cells cease migration before they
tricle, and migrate through the white matter in a largely
divide, and recommence migration after they divide, the two
radial direction (Miyake et al. 1995; Zhang and Goldman
progeny moving off in different directions. Thus related astro-
1996) (Fig. 12.5). At least some of these progenitors in the
cytes can become spatially separated.
white matter begin to differentiate into astrocytes as they
migrate, because they express astrocyte characteristics, such
2.1.3 Astrocyte Development in the Optic Nerve as GLAST (Milosevic and Goldman 2002). Astrocyte pre-
cursors in the cerebellum express CD44, a surface recep-
In the optic nerve astrocytes and oligodendrocytes are gener- tor for hyaluronan and osteopontin (Cai et al. 2011), in this
ated around the end of gestation and for the first week or two respect similar to astrocyte precursors in the spinal cord (Liu
of life in rodents. Optic nerve astrocytes appear to be intrinsic
to the nerve, and likely arise from radial glia, which are derived
in turn from the initial optic nerve neuroepithelium. The con-
version of the precursors into astrocytes goes through a stage
during which they express the vimentin type of intermediate
filament and the gangliosides recognized by the monoclonal
antibody, A2B5, but do not express either GFAP or S-100beta,
markers of more mature astrocytes in rodents (Mi and Barres SC
1999). In vivo the vimentin+ cells will eventually also express
GFAP. In culture, the astrocyte progenitors can be induced
bc oI
to express GFAP by ciliary neuronotropic factor (CNTF) or
leukemia inhibitory factor (LIF). as gc
Oligodendrocyte precursors, which originate in the SVZ
at the base of the third ventricle and migrate into and along bg
the nerve (reviewed in Miller 2002), display a glial develop- as
mental plasticity. If removed from the neonatal optic nerve
and cultured in the presence of serum or IL-6/LIF/CNTF
family members they develop into a stellate astrocyte type,
the so-called “type 2 astrocytes” (Raff et al. 1983). The “bipo- oI
tential” nature of these oligodendrocyte precursors reveals
that the environment of the developing optic nerve either Migration and Differentiation of
Neuronal and Glial Precursors
promotes their differentiation into oligodendrocytes and/or from Cerebellar White Matter to
Pro
inhibits their differentiation into astrocytes. This pattern of Cortex
immature glia differentiating largely into oligodendrocytes in
white matter also seems to be followed elsewhere in the fore- Figure 12.5 During late gestation and into postnatal life, glial and
brain, because the large majority of SVZ cells that settle in neuronal precursors (Pro) generated at the base of the cerebellum migrate
through white matter into cortex and differentiate into oligodendrocytes
subcortical white matter in the neonatal period differentiate (ol), several astrocyte forms (including Bergmann glia (bg) and velate
into oligodendrocytes, not astrocytes (Levison and Goldman astrocytes (as), and interneurons. bc, basket cells; gc, granule cells; sc,
1993). stellate cells. From Zhang L, Goldman JE 1996.

140 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
et al. 2004) and chick cord (Alfei et al. 1999). Interestingly,
hyaluronan in the early postnatal cerebellum is concentrated
in white matter and arranged in fiberlike structures (Baier
et al. 2007), possibly providing migratory guides for astrocyte
precursors.
2.3 A S T RO C Y T E D EV E L O PM E N T
I N T H E S P I NA L C O R D
The ventricular zone of the embryonic mammalian spinal cord
is divided into a number of discrete domains along the dor-
soventral axis, defined by molecular markers, including tran-
scription factors and growth factor receptors. These markers
have allowed genetic studies to trace the fates of immature
cells originating in each of these domains. Thus, the spinal
cord has been the major CNS area in which to approach the
question of whether an astrocyte fate is specified by the initial
position of a precursor within the ventricular zone. Pringle
et al. (2003) inferred a widespread generation of astrocytes
from both dorsal and ventral cord, observing a widespread
expression of fibroblast growth factor receptor type 3 (Fgfr3)
in the embryonic neuroepithelium. Fgfr3+ cells, which do not
represent oligodendrocyte precursors and apparently not neu-
ronal precursors either, likely correspond to very early astrocyte
precursors. However, other observations suggest a positional
determination of astrocyte fate. Thus, Muroyama et al. (2005)
show a generation of astrocytes from the ventral p2 domain,
which also generates spinal interneurons. Astrocyte develop-
ment in this domain depends on the expression of Scl (Stem
cell leukemia, a bHLH transcription factor). Further evidence
for positional specificity in astrocyte development comes from
the observation that subpopulations of astrocytes in the white
matter of the ventral cord can be distinguished by the combina-
tions of Reelin and Slit expression (Hochstim et al. 2008). The
expression patterns are identical to the patterns of these genes
in the embryonic neuroepithelium, indicating a topographi-
cal representation of neuroepithelium onto its final astrocyte
products. Reelin expression is dependent on the expression of
Pax6, and the deletion of Pax6 greatly reduces the numbers of
Reelin+ astrocytes, but does not alter the numbers of astro-
cytes. Thus, Pax6 specifies one characteristic of a set of white
matter astrocytes, but does not disrupt astrocyte development
per se. These findings indeed link initial positional specifica-
tion to astrocyte subtypes. It is not known, however, whether
these astrocyte subtypes differ in any other ways.
Other evidence for a separation of astrocyte from oligo-
dendrocyte lineages includes the observation that oligoden-
drocytes in the ventral cord require sonic hedgehog (Shh) for
their development (Orentas et al. 1999), whereas astrocytes
do not (Pringle et al. 2003). However, in olig1/olig2 double
knockout (–/–) mice, in which oligodendrocytes do not
develop, fate mapping studies show astrocytes arising from
cells that had originated in the domain in which olig2 would
have been normally expressed (Zhou and Anderson 2002).
This observation raises the possibility that the Olig factors
normally repress astrocyte differentiation in a population that
develops into oligodendrocytes and neurons. Neither olig1
nor olig2 appears to be expressed in astrocytes in the normal
A S T R O C Y T E D E VE L O PM E N T
spinal cord, consistent with the idea that these genes inhibit
astrocyte development (Lu et al. 2000; Zhou et al. 2000).
However, developing astrocytes in other regions of the CNS
do express olig2 (Marshall et al. 2005).
How astrocytes arise in cord is not fully understood. By
analogy to the radial glial–astrocyte transformation in the
forebrain (see the preceding), it seems likely that at least some
of the cord astrocytes arise from VZ cells via radial glia.
The preceding considerations argue for a common lineage
of oligodendrocytes with some neurons and a separate lineage
for astrocytes in the cord. Nevertheless, Rao et al. (1998) have
isolated a progenitor from embryonic spinal cord that appears
restricted to glial lineages, able to generate both astrocytes and
oligodendrocytes in culture, but not neurons. These cells bind
the monoclonal antibody A2B5 and express the hyaluronan
receptor, CD44 (Liu et al. 2004). They can be isolated from both
dorsal and ventral regions of the cord. These observations do not
appear to coincide with observations in vivo, as noted. There are
at least two ways to harmonize these findings. First, one can argue
that development is highly regulated in spatial and temporal pat-
terns and thus immature cells are prevented from assuming all of
their potential fates. Removing progenitors from their normal
environment relieves fate restrictions to some degree and allows
progression through lineages not otherwise taken. Second, one
could argue that there are indeed three lineages for oligodendro-
cytes (which might in fact give rise to different oligodendrocyte
populations), one in common with motor neurons, one from the
p3 domain, and the other in common with astrocytes.
3 A S T R O C Y T E D E VE L O PM E N T I N T H E
A D U LT C E N T R A L N E RVO U S SYS T E M
Astrocytes are generated in the adult CNS, although at a low
rate. Early studies used 3H-thymidine to estimate glial turn-
over in the adult rodent brain, concluding that astrocytes
and oligodendrocytes continue to be generated. However, it
appears as if the numbers of oligodendrocytes increase slowly,
while the numbers of astrocytes remain approximately con-
stant (Hommes and Leblond 1967; Kaplan and Hinds 1980;
Korr et al. 1973; Paterson 1983). A similar conclusion was
drawn from retroviral labeling, which showed an increase in
the size of oligodendrocyte clonal clusters but not astrocyte
clusters over time (Levison et al. 1999).
New astrocytes may arise either from the proliferation
of mature astrocytes or the differentiation of progenitors.
The bulk of evidence strongly favors the latter possibility.
Although astrocytes divide in pathological states (Norton
1999), there is little evidence that mature astrocytes divide in
the unperturbed brain. A variety of immature glial cells, how-
ever, populate the adult CNS. Thymidine-labeling studies (see
the preceding) all described proliferating cells that did not
have characteristics of mature astrocytes or oligodendrocytes,
based on nuclear morphology and ultrastructural characteris-
tics. It was difficult at the time to place all of these cells into
specific lineages, however, and the more recent use of antigenic
markers has begun to sort out the nature of these progenitors.
The thymidine studies estimated that cycling cells constituted
• 141
a small but significant proportion of the total cell number. For
example, proliferating cells in the adult rodent white matter
make up as much as 2% of the total (Paterson 1983).
Populations isolated directly from adult CNS (Gensert and
Goldman 2001; Nunes et al. 2003) and enriched for immature
cells are heterogeneous, expressing various combinations of mark-
ers, including A2B5, O4, and vimentin, although not mature
glial markers. In culture or after transplantation into brain, these
cells can generate glia and neurons, although the majority appear
to belong to the oligodendrocyte lineage. Whether astrocytes are
generated from O4+ cells seems less likely, although Armstrong
et al. (1992) isolated the rare cell from adult human white mat-
ter that became both O4+ and GFAP+ in culture. Similarly, the
A2B5+ cells isolated from the adult CNS differentiate into oli-
godendrocytes in culture, although they can develop into astro-
cytes under the appropriate culture conditions (Wolswijk and
Noble 1989, 1992). Thus, most immature cells isolated from the
adult CNS appear to be oligodendrocyte precursors, not astro-
cyte precursors, although changes in culture environment reveal
developmental plasticity. Whether this developmental plasticity
occurs in vivo under pathological circumstances is not clear.
3.1 G E N E R AT I O N O F A S T RO C Y T E S
FRO M N G2+ G L I A
Glia expressing a chondroitin sulfate proteoglycan recognized
by the NG2 antibody populate the developing and adult CNS,
displaying a lacy shape with many delicate processes (see chapters
10, 13, and 21). Although the NG2 marker is found on oligo-
dendrocytes during their early development (Nishiyama et al.
1996), the NG2+ glia in the adult brain do not myelinate and do
not display mature oligodendrocyte or astrocyte characteristics.
In terms of lineage, most investigators believe that the NG2+
population is related to oligodendrocytes, rather than astrocytes.
Fate mapping of NG2+ cells in the postnatal brain reveals that
they generate either more NG2+ cells or more mature oligoden-
drocyte precursors (Zhu et al. 2011). However, NG2+ cells in
the embryonic brain produce oligodendrocytes and astrocytes
that populate the ventral forebrain gray matter (Zhu et al. 2011).
This and other studies in the developing and lesioned CNS indi-
cate that NG2+ cells largely belong to the oligodendrocyte lin-
eage (Komitova et al. 2011; Zhu et al. 2008). It is possible that
the population of NG2+ cells is a heterogeneous one, especially
during the embryonic development of the CNS.
To make matters more complex, there is a population of
NG2+/vimentin+ cells in the adult rat spinal cord (Horner
et al. 2000). These appear to be relatively simple cells that
assume a radial orientation and do not look at all like the lacy
cells in the brain. Thus, cells expressing NG2 may differ in dif-
ferent regions of the adult CNS.
4 TEMPOR AL CHANGES IN ASTROCY TE
GENE TR ANSCRIPTION DURING
D E VE L O PM E N T
Astrocyte precursors change their gene expression patterns
as they differentiate. The sequence has best been described
142 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
to date in gene expression profiling from astrocytes isolated
from the developing and adult mouse forebrain (Cahoy et al.
2008). In general, genes promoting or allowing cell prolifera-
tion are decreased and genes referable to astrocytes’ functions
in neurotransmitter uptake and processing, lipid synthesis and
other metabolic pathways, and secretion are more strongly
expressed. Much of the changes in gene expression occur before
p17 in mouse forebrain, a time at which astrocytes are consid-
ered to have reached a mature state. The Cahoy et al. (2008)
paper makes the additional point that some genes expressed by
astrocytes in culture do not match those expressed in acutely
isolated cells and vice versa.
5 ASTROCY TES ARE
M O R P H O L O G I C A L LY A N D
F U N C T I O N A L LY H ET E R O G E N E O U S .
H OW I S T H I S H ET E R O G E N E I T Y
G E N E R AT E D ?
Astrocytes comprise a highly heterogeneous population, rang-
ing from radial forms such as Bergmann glia to the bushy pro-
toplasmic astrocytes of gray matter to the less complex fibrous
astrocytes of white matter (see chapter 4). The molecular het-
erogeneity of astrocytes includes electrophysiological charac-
teristics, neurotransmitter transporters and receptors, levels of
enzymes that catabolize neurotransmitters, gap junction cou-
pling, degree and frequency of calcium transients, GFAP levels,
and levels of receptors that interact with extracellular matrix
(see, for example, chapters 16, 17, 24, and 28; and as reviewed in
Kimelberg 2010; Zhang and Barres 2010). Transcriptional pro-
filing that compared astrocyte cultures with astrocytes derived
from neurospheres to total transcripts from different regions of
the CNS (Bachoo et al. 2004) showed transcripts that many
astrocytes expressed in common as well as transcripts expressed
in a heterogeneous way. More recent transcriptional analyses
using astrocyte cultures established from different CNS regions
(Yeh et al. 2009) or astrocytes acutely isolated from the adult
mouse neocortex by fluorescence-activated cell sorting (Lovatt
et al. 2007), or astrocyte ribosome–associated mRNAs from
the ALDHL1-BAC-TRAP mice (Doyle et al. 2008) have also
demonstrated gene expression common to astrocytes, patterns
distinctly different from neurons and oligodendrocytes, as well
as region-specific expression patterns (see chapter 28). The
differences in astrocyte phenotypes are critical to understand
astrocyte function, but from a developmental point of view
the studies do not tell us whether the astrocyte progenitors are
programmed because of their initial location to acquire spe-
cific characteristics or whether the local environment regulates
some of the characteristics, and if so, which ones.
How is this heterogeneity established and when is it estab-
lished during the development of astrocytes? There are two
general ways of thinking about the development of astrocyte het-
erogeneity. In one model, the specification of astrocyte fate and
the specific characteristics of a given astrocyte are determined
early, during the patterning of the neuroepithelium. Positional
information, determined by dorsoventral gradients and interac-
tions among transcription factors regulate an astrocytes fate and
type. In another model, the neuroepithelium generates astrocyte
precursors and the specific type(s) of astrocytes are determined
later, either during their migration or even where they finally
reside. It is likely that both mechanisms exist.
As noted, spinal cord astrocytes are heterogeneous in some
ways that reflect positional information and combinatorial
codes of transcription factors in the embryonic neuroepithe-
lium. Thus, in cord, some degree of astrocyte subtype specifi-
cation is determined early. The heterogeneity in Reelin and Slit
expression in cord astrocytes may or may not be accompanied
by heterogeneity in other astrocyte phenotypes, which could
be regulated in their initial position in the neuroepithelium or
during precursor migration or at their final destinations.
Other observations in other parts of the CNS suggest that the
fate decision to differentiate along an astrocyte lineage is made
well before the decision to develop into a specific astrocyte sub-
type. Thus, there may be a basic astrocyte type, on which is then
layered a series of transcriptional changes to provide astrocytes
functionally appropriate for the area in which they reside. In fact,
the final decision of form and function may not be made until
an astrocyte precursor has reached its final destination. Several
observations suggest this is the case during some astrocyte devel-
opment in the forebrain and possibly cerebellum. Astrocyte
colonization of the forebrain from migratory precursors in the
SVZ appears to be a more random situation than one sees in the
patterned neuroepithelium of the spinal cord (see the preced-
ing). In neonatal rodent forebrain a clonal analysis of SVZ cells
revealed radial migration of glial precursors into white matter
and cortex, with some degree of tangential migration in cortex
(Zerlin et al. 2004) and the generation of astrocytes in white
matter, cortex, at the pial surface and in the striatum. Thus, a
single precursor can generate different types of astrocytes that
reside in different areas. Fate tracing of dorsal radial glial cells
in the neonatal mouse forebrain showed that these generated
astrocytes in both cortex and subcortical white matter (Ventura
and Goldman 2007). A dramatic display of astrocyte plastic-
ity comes from experiments in which SVZ cells from neonatal
rats were transplanted into the neonatal cerebellar white mat-
ter, where they generated (morphologically) fibrous astrocytes
in white matter, velate astrocytes in the internal granule layer,
and Bergmann glia (Milosevic et al. 2008). Thus, forebrain glial
precursors can give rise to cerebellar-specific astrocyte forms.
The transplanted astrocytes were not analyzed for other charac-
teristics, however, so it is not known whether the transplanted
cells were able to generate cerebellar astrocytes with full fidelity.
Finally, astrocyte precursors derived from the neonatal rat SVZ
(A2B5+, expanded in vitro with FGF) transplanted into neona-
tal rat forebrain SVZ and white matter generate astrocytes with
a variety of morphologies (Lin and Goldman 2009). The final
phenotype of a given astrocyte thus may be regulated both by its
initial position and by the local environment in which it comes
to reside. All of this speaks to the high degree of developmental
plasticity of astrocyte precursors.
Given that (some) astrocyte precursors in spinal cord and cer-
ebellum express CD44, it is interesting that astrocytes in the adult
CNS vary considerably in their CD44 levels. In general white
matter and subpial astrocytes express far higher amounts of CD44
than do gray matter astrocytes (Akiyama et al. 1993; Girgrah et al.
A S T R O C Y T E D E VE L O PM E N T
1991; Kaaijk et al. 1997), but how adult levels are regulated and
how they are related to precursor levels is not known.
6 A S T R O C Y T E D E VE L O PM E N T I S
R E GU L AT E D BY S E VE R A L C L A S S E S O F
M O L E C U L E S A N D I N T R AC E L LU L A R
PAT H WAYS
For astrocytes, attention has focused on the IL-6/LIF fam-
ily of cytokines and the LIF receptor/gp130 pair, the TGF-β
growth factor family, particularly bone morphogenetic pro-
teins (BMPs) and BMP receptors, fibroblast growth factor,
and the Notch and Notch ligand pairs.
6.1 IL-6 Family Members
The IL-6 family of proteins includes ciliary neuronotropic fac-
tor (CNTF), leukemia inhibitory factor (LIF), cardiotrophin 1
(CT1), and oncostatin M (OSM). All of these stimulate astro-
cytic differentiation, usually defined as the induction of GFAP,
in cells cultured from the embryonic CNS or from the neo-
natal optic nerve (Bonni et al. 1997; Gard et al. 1995; Hughes
et al. 1988; Johe et al. 1996; Ochiai et al. 2001; Yanagisawa et al.
1999). These ligands signal through the LIF receptor/gp130
complex (Nakashima et al. 1999a), to activate the JAK-STAT
intracellular signaling pathway (Bonni et al. 1997; Kahn et al.
1997). This pathway is linked to GFAP regulation, because
phosphorylated JAK then phosphorylates STAT3, a transcrip-
tion factor, which then binds to CBP/p300 complex to bind
to and activate the S3BE sequence in the GFAP promoter
(Yanagisawa et al. 2001) (Fig. 12.6). IL-6 family members act
synergistically with the extracellular matrix of mesenchymal
cells (Lillien and Raff 1990) in inducing GFAP expression. As
noted, astrocytes have extensive interactions with basal lami-
nae of blood vessels and the pial surface of the brain. In fact,
cerebral endothelial cells do express LIF (Mi et al. 2001). Such
an interaction might help induce astrocyte differentiation, a
mechanism that could appropriately match the numbers of
astrocytes with vessels (Zerlin and Goldman 1997).
However, bear in mind that astrocyte differentiation must
involve the induction of many genes other than GFAP. Indeed,
some of the GFAP-negative progenitors that migrate through
the forebrain and cerebellum express astrocyte markers such as
the glutamate transporter, GLAST (Milosevic and Goldman
2002) or zebrin II (Staugaitis et al. 2001) before contacting
basal laminae. Thus, early stages of astrocyte development may
begin before mesenchymal interactions.
6.2 Transforming Growth Factor-β Family Members
The TGF-β family of proteins, particularly BMP2 and BMP7,
promote astrocyte development from cells of the embryonic
telencephalon (Gross et al. 1996). These ligands bind to BMP
receptors to activate Smad transcription factors (see Fig. 12.6).
The BMPs can act synergistically with IL-6 family members
(Nakashima et al. 1999c), inducing a complex that has both
Smad and STAT3 (Nakashima et al. 1999b). In addition to
• 143
 Induction of Astrocyte Genes by IL-6, TGF-β and Notch Pathways
IL-6/LIF/CNTF BMP Notch Ligand
pSmad Notch Receptor
P-JAK
P-STAT
Hes activation
CBP/p300
NF1 Transcription
Me Me
Me Me Demethylation
Astrocyte gene promoter Astrocyte Gene Promoter
Figure 12.6 Members of the IL-6, TGF-E (BMP) families induce
astrocyte genes through Jak/Stat and Smad activation pathways. Notch
activation regulates NF1 binding. Promoter methylation (Me) prevents
transcription factor binding. NF1 binds to a different site than does the
CBP/p300 complex.
inducing astrocyte differentiation, BMP2 may be responsible
for inhibiting neuronal differentiation—that is, controlling
a switch from one fate to the other, or promoting astrocyte
development at the expense of neurogenesis. In favor of this
idea is that BMP2 upregulates Id1, Id3, and Hes-5 in embry-
onic brain cells in culture (Nakashima et al. 2001). The inhibi-
tor of differentiation genes (“Id” genes) plays important roles
in repressing specific differentiation pathways, in this case,
neurogenic pathways. These two Ids and Hes-5, which lie in
the Notch signaling pathway, inhibit the expression of the
neurogenic bHLH genes, mash1 and neurogenin.
An interesting question is whether IL-6 and TGF-β
ligands promote the transcription of identical genes in devel-
oping astrocytes. This in fact may not be the case, because
treatment of glial restricted precursors with CNTF or BMPs
results in astrocytes with different protein profiles and differ-
ent abilities to promote axonal growth and neuronal survival
after spinal cord injury (Davies et al. 2011). Bone morphoge-
netic proteins induce GFAP, N-CAM, and CD44 expression
in mouse embryo cells to a higher degree than does TGF-β1
(D’Alessandro et al. 1994).
6.3 N OTC H A N D N OTC H L I G A N D S
Members of the Notch family of transmembrane receptors and
their ligands, jagged and delta, play an important role in the devel-
opment of radial glia and astrocytes. Notch 1, when constitutively
expressed in an activated form in VZ cells of the E9.5 mouse CNS,
strongly promotes an astrocyte fate (Gaiano et al. 2000), instead
of the normal neuronal and astrocyte fates. Similarly, expressing
Notch 1 in cells of the embryonic retina strongly promotes dif-
ferentiation of Muller glial cells, apparently at the expense of rod
photoreceptors (Furukawa et al. 2000). Thus, as in invertebrates,
Notch activation appears to promote a glial fate, while inhibiting
a neuronal fate. Indeed, the source of the Notch ligands appears
144 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
to be early committed neurons (Namihira et al. 2009). This find-
ing provides a rationale for the neocortical developmental pattern
in which projection neurons are generated first from radial glia,
and then followed by astrocytes.
An example of Notch pathway interactions with proglial
transcription factors is given by the transcription factor, nuclear
factor 1 (NF1), which binds to the GFAP promoter (Cebolla
and Vallejo 2006) and appears in the cortex at about the stage of
astrocyte development. In spinal cord, NF1A and B appear ear-
lier, at E10 in the VZ and thereafter serve to promote gliogenesis
and repress neurogenesis (Deneen et al. 2006). Nuclear factor
1A function requires Notch activation and the expression of the
downstream Hes genes (Deneen et al. 2006), thus coordinating
NF1A and Notch promotion of astrocyte genesis.
6.4 F I B RO B L A S T G ROW T H FAC TO R S I G NA L I N G
Members of the fibroblast growth factor (FGF) family have
important effects on astrocyte development. They appear
to have multiple effects, in some contexts regulating the dif-
ferentiation and proliferation of astrocyte precursors, Thus,
FGF1 can upregulate astrocyte functions such as ATP release
and coupling via gap junctions (Garré et al. 2010) and FGF2
upregulates glutamate transport in cultured forebrain astro-
cytes (Figiel et al. 2003). Figiel et al. (2003) also found
increases in glutamate transport with other growth factors,
including EGF, PACAP, and TGF-α, suggesting final com-
mon pathways for growth factor regulation of transporters.
Fibroblast growth factor 1 changes astrocyte morphology to
a stellate shape and increases the release of NGF in cultured
astrocytes (Cassina et al. 2005). Fibroblast growth factor 2 has
a strong proliferative effect on astrocyte precursors cultured
from neonatal rat forebrain, although these precursors turn
off the proliferative effect even in the continued presence of
FGF2 (Lin and Goldman 2009). Other studies suggest that
FGF2 can promote an astrocyte differentiation from imma-
ture cortical progenitor cells and astrocyte proliferation, the
latter through MAP kinase signaling (Kang and Song 2010).
Clearly, progenitors are subjected to a large variety of sig-
nals and the interactions among these signals results in fate
decisions. For example, when neurogenin1 is overexpressed
in embryonic neural cells, it not only promoted neurogenesis,
but also inhibited the cells from differentiating into astrocytes,
even when stimulated by LIF (Sun et al. 2001). If neurogenin 1
competes with STATs for binding to the CBP/p300 complex,
then one can think of a model of fate determination in which
the relative levels of transcription factors are the critical vari-
ables (Sun et al. 2001). Consistent with this idea is the finding
that the mouse double knockout of neurogenin 2 and Mash 1
did not contain large numbers of astrocytes at the expense of
neurons (Nieto et al. 2001).
6.5 C YC L I C A D E N O S I N E MO N O P H O S P H AT E
R EGU L AT I O N O F A S T RO C Y T E D EVE L O PM E N T
Another astrogenic pathway may function through cAMP,
because the induction of cAMP in embryonic forebrain cells
induces an astrocytic fate (McManus et al. 1999). Cyclic
adenosine monophosphate is well known to increase the tran-
scription of GFAP in astrocytes (Masood et al. 1993; Shafit-
Zagardo et al. 1988), possibly via the CREB sequence in the
GFAP promoter (Besnard et al. 1991; Masood et al. 1993).
Pituitary adenylate cyclase-activating polypeptide (PACAP)
promotes GFAP transcription through PAC1 receptor, which
is coupled to adenylate cyclase (Vallejo and Vallejo 2002).
7 E P I G E N ET I C R E GU L AT I O N O F
A S T R O C Y T E D E VE L O PM E N T
The demethylation of astrocyte genes appears to be an impor-
tant prerequisite to the onset of astrocyte differentiation. Thus, a
number of astrocyte genes are methylated early in development,
including GFAP, Aldolase C, and Kir4.1 (Hatada et al. 2008,
who compared methylation status of neural precursors from
E11.5 with those at E14.5 and with P1 astrocytes). Methylation
of astrocyte genes in the early embryonic CNS is regulated by
the methyl transferase, DNMT1, and serves to repress astrocyte-
specific gene transcription (Fan et al. 2005). Demethylation,
which occurs at the beginning of astrocyte development, allows
LIF and TGF-β ligands to upregulate astrocyte genes by allow-
ing transcription factors downstream of these signaling pathways
to bind to astrocyte gene promoters (see Fig. 12.6) (Fan et al.
2005; Hatada et al. 2008; Namihira et al. 2009; Takizawa et al.
2001). As another example of epigenetic control, FGF2 changes
histone methylation at the STAT binding site on the GFAP pro-
moter, allowing STAT to bind and activate GFAP transcription
by CNTF (Song and Ghosh 2004).
8 S U M M A RY A N D P E R S P E C T I VE S
Among a number of outstanding questions concerning astro-
cyte development are the following:
1. Some genes are transcribed early in astrocyte development
and some later. What accounts for the temporal sequence
of transcriptional changes during astrocyte development?
Do astrocytes develop first as cells expressing a basic stable
of astrocyte characteristics and then regulate other genes
to provide for a match to their local environments?
2. Astrocytes interact intimately with blood vessels and
neurons. How is astrocyte development matched to vascu-
lar development? How is astrocyte development linked to
neuronal development, particularly the development
of dendrites and synapses?
3. How is the molecular and morphological heterogeneity
of astrocytes generated? In what ways are astrocytes
functionally heterogeneous?
4. How can we characterize the population of immature cells
in the adult CNS that have the potential to develop into
astrocytes? Why is their development normally inhib-
ited? Do adult astrocyte progenitors play any role in the
response of the CNS to neurological diseases and how?
5. Could adult astrocyte precursors be sources of astrocytomas?
A S T R O C Y T E D E VE L O PM E N T
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• 147
 13.
LINEAGE AND DEVELOPMENT: OLIGODENDROCY TES
Katsuhiko Ono and Kazuhiro Ikenaka

A B B R E VI AT I O N S mature into OLs. In this chapter, lineage-tracing experiments

of OLs are discussed for this purpose. However, to understand
BMP bone morphogenetic protein the references cited, it is important for the reader to know the
CTGF connective tissue growth factor meaning of markers that are used to label OL lineage cells, and
CNS central nervous system these are described at the beginning of the chapter. The same
CDK2 cyclin-dependent kinase 2 marker could label cells of different developmental stages when
ET-1 endothelin-1 different species are analyzed or even when different regions are
GalC galactocerebroside analyzed. In the second part of this chapter, the mechanisms
GFAP glial fibrillary acidic protein underlying OL development are dissected. Many intrinsic and
GPR G protein–coupled receptor extrinsic factors are known to play a role in OL development:
GRP glia restricted progenitor Some regulate OL specification from neural stem cells, some
hEGFR human epidermal growth factor receptor affect migration, some regulate exit from cell cycle, and oth-
HMG high mobility group ers regulate maturation from OPCs to OLs. Regulation of OL
IGF-1 insulin-like growth factor I development at each of these steps is discussed.
LRR leucin-rich repeat
MAG myelin-associated glycoprotein
MBP myelin basic protein 2 D E VE L O PM E N TA L S TAG E S O F
MRF myelin gene regulatory factor O L I G O D E N D R O C Y T E L I N E AG E C E L L S
NGF nerve growth factor C A N B E VI S UA L I Z E D BY U S I N G S TAG E
O-2A oligodendrocyte-type2 astrocyte SPECIFIC MARKER S
OL oligodendrocyte
OPC oligodendrocyte progenitor cell 2.1 G LYC O L I P I D
PDGF platelet-derived growth factor
Oligodendrocytes generate myelin membranes that are rich in
PDGFRD Platelet-derived growth factor receptor D
lipids. In fact, lipids account for 70% to 85% of the dry weight
PLP proteolipid protein
of compact myelin. Probably because of this, OL lineage cells
pMN motoneuron progenitors
express cell type and cell stage–specific glycolipids even before
PSA-NCAM Poly-Sialated Neural Cell Adhesion Molecule
myelin formation. A2B5 antigen, O4 antigen, sulfatide, and
SRF serum response factor
galactocerebroside (GalC) are often used as stage-specific
VZ ventricular zone
markers for OL lineage cell in vitro and in vivo, including in
YY1 Yin Yang 1
the human fetal tissues (Hajihosseini et al. 1995; Wilson et al.
2003; Zhang et al. 2000). The sequence of the onset of lineage
marker expression is summarized in Figure 13.1.
1 INTRODUCTION
2.1.1 A2B5 Antigen
Oligodendrocytes (OLs) are generated from neural stem cells
present in the ventricular zone (VZ) surrounding the ventricle. A monoclonal antibody, A2B5 (Eisenbarth et al. 1979), was
Although neurons and astrocytes are generated from most, if originally reported as a neuron-specific antibody. Later however,
not all, regions of the central nervous system (CNS), OLs are Raff et al. (1983) reported that in the newborn rat optic nerve
generated from only several restricted areas. Thus, after specifi- cell culture, devoid of neurons, a special type of glial progenitor
cation to OL lineage cells from multipotent neural stem cells, cell was labeled with A2B5 antibody. This special cell differen-
OL progenitor cells (OPCs) need to migrate a long distance tiates in vitro into either an astrocyte (GFAP+/A2B5+; type
in a nonradial fashion before they reach their final destination, 2 astrocyte) or OL depending on culture condition, and was
which is mostly the white matter. There, OPCs cease prolifer- called an oligodendrocyte-type 2 astrocyte (O-2A) progeni-
ation and mature into myelinating OLs. Therefore, to under- tor cell. O-2A progenitor cells at the initial stage of culture are
stand the development of OLs, it is important to know where bipolar in shape and highly motile (Small et al. 1987). When
and when OPCs are generated, and where they migrate to and purified O-2A progenitor cells were transplanted into newborn

148
 neuroepithelial OPC late progenitor pre-myelinating myelinating
procursor pro-oligodendrocyte oligodendrocyte oligodendrocyte

morphology migratory
proliferative
process-bearing
bipolar
(rodent/human)

MBP
A2B5 04/OPA 01/GalC
in vitro*

PLP
NG2
PDGFRα

01/GalC
O4(/OPA?)
Chick

Sox 10 MBP
Olig2 PLP
PDGFRα
NKx2.2
Rodent

Olig2/Olig1 NKx2.2 PLP

O4 01/GalC MBP
PDGFRα (O4/OPA)**
Sox 10 NG2
spinal cord

O4(45dpc)
Human

O1/GalC(45dpc) MBP(52dpc)***
PDGFRα(49dpc)

Figure 13.1 Scheme of Sequential Expression of Lineage Markers for Oligodendrocyte Progenitor Cells Details are explained in the main text. *Sequence
of lineage marker expression on human OPC was examined in neurosphere-derived OPCs. **When O4 was applied to open-book cultures of neural
tube or hindbrain, presumed migratory OPCs were labeled with O4. ***First myelin was found in the human spinal cord in a 10-week-old fetus under
TEM; therefore, the first MBP+ cells in the human spinal cord were probably premyelinating oligodendrocytes. From Zhang et al. 2000; Wada
et al. 1982.

rat brain, nearly all cells became OLs (Espinosa de los Monteros
et al. 1993), and thus the in vivo equivalent of O-2A progenitor
cells are believed to be OPCs with some competence to astrocyte
differentiation. A2B5 antibody also recognizes glia restricted
progenitor (GRP) cells that differentiate not only into OLs
and type 2 astrocytes, but also into type 1 (GFAP+/A2B5–)
astrocytes (Rao et al. 1998). Although GFAP+/A2B5+ astro-
cytes were reported to be in the developing optic nerve (Miller
et al. 1985), the presence of two distinct astrocyte lineages was
not confirmed in vivo (Skoff 1990). A2B5 has been reported to
react with a variety of antigens, including multiple ganglioside
and sulfatide (Kundu et al. 1983), and thus the specificity of the
antibody to the OPC or OL lineage cells is not high.
2.1.2 O4 Antigens
A monoclonal antibody O4 was generated by immuniz-
ing mice with homogenates of the bovine corpus callosum
(Sommer and Schachner 1981). O4 antibody labels OL lin-
eage cells at a pro-oligodendroblast stage (see Fig. 13.1), at the
GalC-negative stage and even before the cells express sulfatide
(Bansal et al. 1992). Therefore, the antigen(s) recognized by
O4 was designated as pro-oligodendroblast antigen (POA).
When O4 antibody was applied to chick embryo CNS, O4
labels OPCs in a restricted region of the ventral VZ (Ono
et al. 1995, 1997), and also OL lineage cells at migratory and
proliferative stages (Ono et al. 2001). Therefore, it is probable
that O4 antibody identifies a more immature, migrating, and
mitotically active stage of OL lineage cells in chick embryo
than in the rodent. O4 antibody recognizes sulfatide, semino-
lipid, sulfated and nonsulfated cholesterol, and POA. In spite
of much improvement in the technology to analyze lipid anti-
gens, the nature of POA has not been identified yet (Bansal
et al. 1992).
2.1.3 O1 Antigen and GalC
A monoclonal antibody O1 was generated in the same
series of experiments as the O4 antibody. O1+ cells in vitro
are multiple process bearing and no longer proliferative or
migratory. Therefore, the O1+ cell stage is a relatively mature
stage in OL lineage cells, although early O1+ cells do not
show heavy myelination. O1 antibody recognizes GalC and
monogalactosyldiglyceride.
2.2 M E M B R A N E P ROT E I NS
Proteins localized at the cell surface are also frequently used to
identify OL lineage cells and judge their developmental stages.
There have been lots of arguments about the specificity for
each of these proteins described in the following. It was very
difficult to follow the fate of cells that expressed a particular

L I N E AG E A N D D E VE L O PM E N T: O L I G O D E N D R O C Y T E S • 149
marker protein at one stage in their differentiation, but then
downregulated it on further differentiation. However, the use
of the Cre-loxP system, especially tamoxifen inducible Cre, has
allowed precise fate mapping of cells that are expressing the
marker protein at the time of interest (Brocard et al. 1997). We
now have better understanding of which kind of cells at which
developmental stage express particular marker proteins.
2.2.1 NG2
NG2 antibody was generated using B49 cells as antigen.
Antisera were absorbed with neuronal cell lines, B103 and
B65 cells. Absorbed antisera recognized both pseudo-glia and
pseudo-neuron cell lines, and also tetanus toxin-positive neu-
rons and GFAP+ astrocytes in primary culture. Therefore, the
putative antigen (NG2 antigen) was believed to be expressed
by common progenitor cells for neurons and astrocytes
(Stallcup 1981). Later, NG2 antibody was shown to react with
O-2A progenitor cells in vitro (Stallcup and Beasley 1987),
and in vivo NG2+ cells coexpress PDGFRα (see section
2.2.2). Thus, NG2+ cells were elucidated to recognize OPCs
both in vivo and in vitro (Nishiyama et al. 1996). NG2 antigen
is a chondroitin sulfate proteoglycan (cspg4) (Stallcup et al.
1983). NG2+ cells may be more immature than A2B5+ OPC,
because, at least in vitro, NG2+ cells appear before the expres-
sion of A2B5+ cells and generate A2B5+ cells (Baracskay et al.
2007). As described in chapter 10, fetal NG2+ progenitor cells
in the rodent give rise to OLs and astrocytes in the gray matter.
By contrast, those in newborn animals differentiate into OLs
exclusively. Therefore, NG2+ cells in early development may
be bipotential glial progenitor cells, whereas postnatal NG2+
cells are OPC. Details of fate mapping study of NG2+ cells
are discussed in chapter 10.
2.2.2 Platelet-Derived Growth Factor
Alpha-Receptor
Platelet-derived growth factor alpha-receptor PDGFRα tran-
scripts first appear at the VZ of pMN domain in the cervi-
cal spinal cord on embryonic day 12.5 (E12.5) in the mouse
(E14 in rat, E7 in chick) (Pringle and Richardson 1993; Pringle
et al. 1996). PDGFRα+ cells proliferate and migrate away from
the VZ and by E17 (in the mouse) the number of PDGFRα+
cells reaches a steady state, and they are distributed evenly
throughout the cord. The onset of PDGFRα expression coin-
cides with the initiation of OL generation and several other
lines of evidence have demonstrated that PDGFRα+ cells are
OPCs (Richardson et al. 2000). Moreover, colocalization of
PDGFRα and NG2 has been shown in various studies (see
the preceding).
2.2.3 Proteolipid Protein/DM20
Myelin proteolipid protein (PLP) is one of the major pro-
teins present in the CNS myelin membrane and is found in
the mature OL. Therefore, immunohistochemical detec-
tion of PLP unambiguously identifies mature OLs (mostly
150 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
myelinating OLs). However, PLP gene transcripts, especially
its alternative-splicing product DM20, can be found from very
early stages of development (Ikenaka et al. 1992; Timsit et al.
1992) even from E9.5 in the basal plate of the diencephalon
(Timsit et al. 1995). However, PLP/DM20 producing cells at
this very early stage have been shown to differentiate mainly
into neurons (Delaunay et al. 2008); thus, identifying the cell
type expressing PLP/DM20 early in development is contro-
versial. It is noteworthy that PLP/DM20 expressing cells in a
certain region, such as olfactory bulb, may be OPCs that are
not expressing PDGFRα (Spassky et al. 2001).
2.3 T R A NS C R I P T I O N FAC TO R S
Because OL development proceeds through regulated gene
expression mainly carried out by transcription factors, it is not
surprising that several transcription factors are used to mark
OL lineage cells. In this chapter transcription factors com-
monly used as OL lineage markers are described. The function
and precise description of these factors is provided later.
2.3.1 Olig1
Olig1 expression commences at E9.0 in the motoneuron
progenitor (pMN) domain of the mouse spinal cord, then is
downregulated at around E11.0, and restarts at E12.5. From
this time on, it is expressed by OL lineage cells. However, early
expressing cells generate motor neurons (Lu et al. 2002) and later
they also generate astrocytes or radial glial cells (Liu and Rao
2004).
2.3.2 Olig2
Olig2 also starts its expression in the pMN domain of mouse
spinal cord at E8.5 and is continuously expressed to adulthood
by the OL lineage cells. At E9.5 30% of Olig2 expressing cells
generate motor neurons, but after E12.5 they stop producing
motor neurons and only produce glial cells, including OLs
and astrocytes (Masahira et al. 2006).
2.3.3 Sox10
In contrast to Olig1/2, Sox10 is more specific to OL lineage
cells and starts its expression in the pMN domain from E11.5
(Stolt et al. 2002; Tripathi et al. 2011).
3 O L I G O D E N D R O C Y T E D E VE L O PM E N T
IN ANIMAL MODELS
Developmental study of OLs became more popular after
O-2A progenitors were identified in vitro (see the preceding).
A great number of researchers uncovered characteristics and
behavior of O-2A progenitor cells in vitro, some of which
were later elucidated to reflect, at least partly, those of OPCs
in vivo. Here, we summarize the in vivo development of OL
lineage cells in the optic nerve, spinal cord and forebrain.
 3.1 O P T I C N E RV E of neural cell differentiation (Richardson et al. 2000). Such
restricted appearance of OPC was later confirmed with CNP,
O-2A progenitor cells in vitro were first identified in cul-
Sox10, PLP, Olig1, Olig2, and Nkx2.2. These origins of OPCs
tures of optic nerve glia. This finding was the start point of
may be divided into two separate groups based on slight dif-
cellular and molecular analyses of OL development. Based
ferences in dorsoventral position; namely, one group includes
on this culture system, many aspects of OPCs were under-
Olig+, PDGFRα+, and Sox10+ cells, corresponding to cells
stood, which led to finding of lineage markers and behavioral
in the pMN domain, and the other are Nkx2.2+ and O4+
characters of OPC. Oligodendrocyte progenitor cells in the
cells slightly ventral to the pMN domain (dorsal part of p3
optic nerve were believed to be immigrants from the ventral
domain; Fig. 13.3). The latter group of OPC might be avian
forebrain. This was first suggested by isolated explant culture
specific (Fu et al. 2002). OPCs go on to coexpress both Olig2
(Small et al. 1987); the optic nerve in the developing rat was
and Nkx2.2 in the parenchymal region in later development
divided into chiasmal end, middle part, and retinal end, and
(Cai et al. 2010). Oligodendrocyte progenitor cells gradually
each fragment was cultured separately for several days. GalC+
disperse from the ventral part to the dorsal spinal cord, and
OLs appeared sequentially from cultures of the chiasmal end
finally they are evenly distributed throughout the spinal cord.
to the retinal end. In vivo migration of OPCs from the fore-
The initial ventral origin and subsequent ventral-to-dorsal
brain was elucidated in the chick embryo (Ono et al. 1997).
developmental gradient seem to be conserved in the verte-
O4+ OPCs initially appear in the floor of the third ventricle
brate from fish to human spinal cord (Hajihosseini et al. 1996;
around E5 as a cell cluster, and subsequently O4+ cells gradu-
Kirby et al. 2006).
ally spread into the optic nerve from the chiasmal to retinal
Initial ventral OPCs are induced by Shh from notochord
end. When chick embryos received DiI in the third ventri-
and floor plate, which was demonstrated by notochord graft-
cle at E5 or E6, O4+ progenitor cells that had incorporated
ing (Orentas et al. 1999). Later, OPCs also rise from the dor-
DiI were observed in the optic nerve 2 days later (arrow in
sal ventricular zone in a Shh independent manner (Fig. 13.3)
Fig. 13.2). This was the first direct evidence for OPC migra-
(Cai et al. 2005; Vallstedt et al. 2005). Lineage analysis with
tion in vivo. Migratory OPCs in vivo were unipolar or asym-
Msx3-Cre mice demonstrated that approximately 20% of
metrical bipolar in shape and aligned along retinal ganglion
OLs are generated from the dorsal ventricular zone in the
cell axons (see Fig. 13.2).
spinal cord (Tripathi et al. 2011). Regulatory mechanisms
of OPC migration and proliferation are summarized in the
3.2 S P I NA L C O R D following.
Oligodendrocyte progenitor cells in the spinal cord have been
most extensively studied in vivo with respect to their sites of
origin, and regulatory mechanisms of migration and prolifera-
tion. At early stages of development, spinal cord OPCs were
reported to originate in the ventral part by split culture and
dP1
DiI tracing (Warf et al. 1991). Then, after the identification
of PDGFRα as a lineage marker for OPC, the focal restricted dP2
origin of OPCs was demonstrated in the ventral ventricu-
lar zone of the fetal rat spinal cord (Pringle and Richardson Msx3 dP3
Dorsal OPC
1993). Later, similar results were described for O4+ OPCs dP4
in chick embryo spinal cord (Ono et al. 1995). These obser-
dP5
vations strongly indicated focal restricted origin of OPCs in
the spinal cord, which led to the dorsoventral domain theory dI P6
Dbx1
p0
p1
p2
04/Dil
Olig2, Sox10 pMN
Ventral OPC
Nkx2.2 p3

Notochord

Figure 13.3 Scheme of Ventrally and Dorsally Derived Oligodendrocyte

Figure 13.2 Basal Forebrain–Derived O4+ Oligodendrocyte Progenitor
Cells in the Optic Nerve. DiI was injected into an E5 chick embryo, and an
O4+ cell incorporating DiI (arrow) was observed in the optic nerve 2 days after
the injection. The asterisk indicates an O4-migratory cell.
Progenitor Cells in the Spinal Cord. Ventral OPCs originate from Olig2+/
Sox10+ pMN domain in the rodent spinal cord. In addition to this, the dorsal part
of the Nkx2.2+ p3 domain generates OPCs in avian embryo. Dorsal OPCs are
generated from Dbx1+ and Msx3+ domains, although the number is much less
compared with ventral OPCs.

L I N E AG E A N D D E VE L O PM E N T: O L I G O D E N D R O C Y T E S • 151
 3.3 F O R E B R A I N
As is the case for the spinal cord, OL generation is initiated
in the ventral part of the forebrain in a Shh-dependent man-
ner and later converts to the dorsal part. Using chick-quail
chimera, it was shown that OLs have a mono-focal origin
at anterior entopeduncular area, from which OPCs migrate
throughout the forebrain in the chick (Olivier et al. 2001).
Also, in rodents OL generation starts from a restricted ven-
tricular foci, the anterior entopeduncular area and medial gan-
glionic eminence (starting from E12.5). At later stages (a few
days later), sources for OLs change to the lateral ganglionic
eminence and cerebral cortex (mainly after birth), as defined
by the expression of Emx1 (Kessaris et al. 2006). This dorsal
induction of OL lineage cells may be Shh-independent, as in
the spinal cord.
3.4 OT H E R B R A I N R E G I O N S
Oligodendrocyte development in other brain regions, such
as diencephalon and cerebellum, is still controversial. Several
areas expressing Olig2 can be found but lineage tracing experi-
ment of those Olig2 expressing cells (discriminating from
Olig2 cells in other areas) are difficult to perform with cur-
rently existing techniques. Recently, at least a certain fraction
of OPCs in the cerebellum are reported to be derived from
mesencephalon (Mecklenburg et al. 2011), suggesting multiple
origins of cerebellar OLs.
4 R E GU L AT I O N O F O L I G O D E N D R O C Y T E
D E VE L O PM E N T
As mentioned, OLs develop through their specification from
multipotent neural stem cells, migration, exit from the cell cycle,
and terminal differentiation/maturation to myelin-forming
OLs. Many intrinsic and extrinsic factors affect these pro-
cesses. In this chapter the regulation of OL development at
each of these steps is discussed. Unless otherwise noted, OL
development in the rodent spinal cord is described.
4.1 S P E C I FI C AT I O N
4.1.1 Sonic Hedgehog Dependent Early
Generation of Ventral Oligodendrocytes
Oligodendrocyte induction in the ventral spinal cord is heav-
ily dependent on Shh that is secreted from the notochord.
Addition of Shh resulted in ectopic induction of OL lineage
cells and inhibition of Shh signaling blocked their emergence
(Orentas et al. 1999; Pringle et al. 1996). Oligodendrocyte
specification is also dependent on Shh in the telencephalon
(Nery et al. 2001; Spassky et al. 2001) and therefore it seems to
be a general phenomenon. Shh induces expression of several
genes in a dose-dependent manner. These include Nkx and
Olig, both of which positively regulate OPC specification and
development (see Fig. 13.3) (Ericson et al. 1997; Lu et al. 2000,
2002; Takebayashi et al. 2002; Zhou and Anderson 2002).
Especially, Nkx6 and Olig2 are known to be essential for OPC
152 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
development. In addition, a high mobility group (HMG) fac-
tor, SRY-box containing gene 10 (Sox 10) and Nkx 2.2 (Liu et
al. 2007; Zhou et al. 2000) are important factors for differen-
tiation of OLs as well as specification. Another HMG factor,
Sox 9, plays a more direct role than Sox 10 in specification of
OLs. Ablation of Sox9 results in defects in the specification of
OLs and astrocytes, which recovers at later stages of develop-
ment (Stolt et al. 2003).
Mash1/Ascl1 is broadly expressed by brain and spinal cord
progenitors and is also expressed in OPCs (Kondo and Raff
2000). Mash1 cooperates in vivo with Olig2 in OL specifica-
tion, demonstrating an essential role for Mash1 in the genera-
tion of a subset of OLs in the mouse forebrain (Parras et al.
2007; Sugimori et al. 2007).
4.1.2 Sonic Hedgehog Independent Late
Generation of Dorsal Oligodendrocytes
The discovery of the dorsal origin of OLs questioned the gener-
ality of Shh-dependent appearance of OLs. Oligodendrocyte
progenitor cell emergence from the dorsal spinal cord was
shown to be Shh independent and probably FGF2 dependent
(Cai et al. 2005; Chandran et al. 2003). Recently, it was shown
that even in the ventral forebrain FGF2 signaling cooperates
with Shh signaling and is equally important in OL specifica-
tion (Furusho et al. 2011; Kessaris et al. 2004). FGF2 signaling
induces expression of Olig2 and Sox9 independently of each
other in zebrafish (Esain et al. 2010).
In contrast with the inducing signal by Shh and FGF2 in
the ventral spinal cord, specification of OLs from neural stem
cells is inhibited by factors secreted from the dorsal spinal
cord (Wada et al. 2000). Wnts (Langseth et al. 2010; Shimizu
et al. 2005; Ye et al. 2009) and bone morphogenetic proteins
(BMPs) (Miller et al. 2004; Samanta et al. 2004) are shown to
be the factors inhibiting generation of OPCs. Both proteins,
at least partially, induce expression of the inhibitor of differen-
tiation 2 and 4 (Id 2 and 4) that repress Olig1/2 function. It
is also suggested that Wnt signaling exerts its effects through
the BMP signaling pathway (Feigenson et al. 2011; Kasai et al.
2005).
4.2 M I G R AT I O N
Cells specified for the OL lineage migrate from the VZ and
spread out widely in the parenchyma. Molecular mechanisms
by which OPC migration is regulated have been uncovered
first in the optic nerve (Sugimoto et al. 2001). Oligodendrocyte
progenitor cell expressing unc5a or neuropilin-1 were repelled
by sema3a and netrin1, both of which are expressed in the
chiasmal region of the ventral forebrain (Spassky et al. 2002).
Migration of OPCs in the spinal cord is also regulated by
netrin1 secreted by floor plate cells, which inhibits pro-
cess extension by OPCs and functions as a chemo repellent
( Jarjour et al. 2003; Tsai et al. 2003). Thus, these repellent
molecules apparently facilitate OPC migration. In addition,
OPCs were reported to cease migration through CXCL1/
CXCR2 signaling (Tsai et al. 2002).
 Endothelin-1 (ET-1) is an astrocyte-derived signal that
also regulates migration and differentiation of OPCs, which
express functional ET(A) and ET(B) receptors (Gadea et al.
2009). Endothelin-1 exerts both chemotactic and chemoki-
netic effects on OPCs to enhance cell migration.
4 .3 C E L L P RO L I FE R AT I O N A N D E X IT
FRO M T H E C E L L C YC L E
Platelet-derived growth factor promotes the expansion of
OPCs and allows their timely differentiation into OLs in vitro
(Noble et al. 1988). Proliferation of OPCs in vivo is mainly
regulated by PDGF-A as observed in vitro; mice lacking
PDGF-A show a lack of PDGFRα+ OPCs and a decreased
number of PLP+ OL (Fruttiger et al. 1999). In addition, trans-
genic mice over expressing PDGF-A resulted in hyperprolif-
eration of OPCs, although superfluous OPCs are eliminated
by programmed cell death postnatally (Calver et al. 1998).
FGF-2 up-regulates PDGFRα on OPCs and, in combination
with PDGF, supports their long-term proliferation (Bögler
et al. 1990; McKinnon et al. 1990).
Oligodendrocyte progenitor cells stop proliferating before
terminal differentiation. It was originally proposed that OPC
differentiation is intrinsically regulated by a “timing” mecha-
nism that links the number of cell divisions to growth arrest
and the initiation of differentiation (Temple and Raff 1986).
This mechanism was regulated by mitogens (Calver et al.
1998) and molecularly characterized by the progressive accu-
mulation of the cell cycle inhibitor, p27/Kip1 (Durand et al.
1997). It was shown that only a fraction of OPCs derived from
p27-knockout mice differentiated successfully but continued
proliferation (Casaccia-Bonnefil et al. 1997; Durand et al.
1998). However, overexpression studies with viral vectors
SRF
Neuron CTGF
ATP
IGF-1
Wnt
OPC
β -Catenin Sox17
Glu
NGF
Notch1
Jagged1 LINGO1 TrkA
PSA-CAM
Electrical Activity
Figure 13.4 Factors Affecting the Terminal Differentiation of Oligodendrocytes After migrating into the white matter, most OPCs differentiate into
myelinating OLs. There are various environmental factors affecting this process. Factors promoting differentiation are depicted in red, whereas those
inhibiting differentiation are in blue. Various transcription factors affect this process but are not listed here. Please see chapter 43 for detail.
L I N E AG E A N D D E VE L O PM E N T: O L I G O D E N D R O C Y T E S
expressing p27 that interacted with cyclin-dependent kinase
2 (CDK2) did not induce differentiation of progenitors,
even though cells arrested at the G1/S transition (Tikoo et al.
1998). Together, these studies suggested that cell cycle exit was
necessary but not sufficient to induce differentiation of pro-
genitors into OLs.
4.4 T E R M I NA L D I FFE R E N T I AT I O N/
M AT U R AT I O N/MY E L I NAT I O N
4.4.1 Electrical Activity
In mammals, myelin membrane is inhibitory for axonal exten-
sion (Fig. 13.4). Therefore, myelination has to be initiated only
after neuronal circuit formation has been completed. Thus, strict
regulation of myelination by the axon is necessary. Electrical
activity passing through the axon could be easily considered as
one of the candidates because electrical activity should increase
after circuit formation. Indeed, the proliferation of OPCs
depended on electrical impulse activity in neighboring axons
(Barres and Raff 1993) and myelination was induced by elec-
trical activity (Demerens et al. 1996). The mediator between
the electrical active axon and OPCs seems to be ATP (and its
degraded product, adenosine) and glutamate secreted from
axons through vesicular release (Ishibashi et al. 2006; Wake
et al. 2011). ATP first acts on astrocytes and induces release of
leukemia inhibitory factor (LIF), which then stimulates termi-
nal differentiation of OL (Ishibashi et al. 2006).
4.4.2 Axonal Membrane Proteins
Several different receptor-ligand pairs that connect OPCs and
axons are induced or reduced by the electrical activity and are
Astrocyte
LIF
Wnt
myelin
genes OL
Notch1
Contactin
Axon
• 153
also important for OPC/OL differentiation and/or myelina-
tion, including ErbB2 and neuregulin, TrkA and NGF, and
Notch and Jagged or F3/contactin.
ErbB2 functions in the transition of OPC to OL by trans-
ducing a terminal differentiation signal both in vitro (Park
et al. 2001) and in vivo (Kim et al. 2003).
Nerve growth factor (NGF) is a potent regulator of the
axonal signals that control myelination and reduces myelina-
tion by OLs (Chan et al. 2004). NGF and its cognate recep-
tor, TrkA, induce the expression of leucin-rich repeat (LRR)
and Ig domain-containing, Nogo receptor–interacting pro-
tein (LINGO-1) (Lee et al. 2007) that inhibits OL differen-
tiation (Mi et al. 2005).
Oligodendrocytes differentiation is regulated by activation
of the Notch pathway as well. OPCs/OLs in the developing
rat optic nerve express Notch1 receptors and retinal ganglion
cells express Jagged1, a ligand of the Notch1 receptor, along
their axons. Jagged1 expression decreases with a time course
that parallels myelination in the optic nerve (Wang et al.
1998). Notch1 also interacts with F3/contactin clustered at
the axonal paranodal junction and this interaction signals via
Deltex1 to promote OL differentiation (Hu et al. 2003). Thus,
the timing of OL differentiation and myelination is controlled
by the Notch pathway.
PSA-NCAM is first expressed on all growing fibers and
negatively regulates myelin formation. Then axonal expression
is downregulated and myelin deposition occurs only on PSA-
NCAM–negative axons (Charles et al. 2000).
4.4.3 Humoral Factors
Terminal differentiation/myelination is also affected by vari-
ous humoral factors that are secreted from cells surrounding
the OPCs, such as astrocytes.
Although Wnt proteins inhibit differentiation of OPC as
well as their specification, at more mature stages, it directly
drives myelin gene expression (Tawk et al. 2011). Thus, Wnt
proteins affect multiple developmental stages of OL from
specification to myelination. The Wnt pathway itself is under
fine control by several factors, including Sox17. Sox17 is maxi-
mally expressed in pro-oligodendrocytes (see Fig. 13.1), and
its downregulation increases OPC proliferation and decreases
lineage progression after mitogen withdrawal (Sohn et al.
2006). It suppresses cyclin D1 expression and cell prolif-
eration by directly antagonizing β-catenin, whose activity in
OPCs is stimulated not only by Wnt3a, but also by PDGF
(Chew et al. 2011).
Insulin-like growth factor I (IGF-1) increases brain growth
and CNS myelination in a transgenic mouse line that over-
expresses IGF-1 (Carson et al. 1993). On the contrary, Igf1
gene disruption results in reduced brain size, and CNS hypo-
myelination in young animals (Beck et al. 1995). Insulin-like
growth factor I directly affects OLs and myelination in vivo
via type 1 IGF receptor (Zeger et al. 2007), and thus IGF-1
signaling in the cells of OL lineage is required for normal OL
development and myelination. Insulin-like growth factor I sig-
naling is inhibited by connective tissue growth factor (CTGF)
produced in neurons, whose expression is repressed by serum
154 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
response factor (SRF). Thus, neuron-restricted SRF ablation
in mice increased the production of CTGF and elevated
OPCs, while inhibiting terminal OL differentiation (Stritt
et al. 2009).
BMP signaling through type I BMP receptors also affects
the maturation/myelination steps of OLs. In the BMPR1a
and b double mutants, the number of OPCs and the timing
of their emergence was unchanged compared with wild-type;
however, myelin protein expression and mature OL num-
bers were significantly reduced (See et al. 2007). These data
indicate that BMP signaling promotes the generation of
mature, myelinating OLs in vivo but does not affect OPC
development.
Overexpression of human epidermal growth factor recep-
tor (hEGFR) in OL lineage cells contributes to the earlier
maturation of OLs in the corpus callosum. The opposite
occurs in a mouse with reduced EGFR signaling (wa2 mouse),
as demonstrated by the number of differentiated, myelinating
oligodendrocytes and myelin basic protein (MBP) expression
levels (Aguirre et al. 2007). Therefore, EGF signaling is also
important in maturation/myelination step.
4.4.4 Other Membrane Proteins/Extracellular Matrix
Laminins are likely to regulate CNS myelination by interact-
ing with both integrin receptors and dystroglycan receptors
(Colognato et al. 2007), and the normal role for CNS laminin
is to promote the development of OPCs into myelin-forming
OLs via modulation of Fyn regulatory molecules (Relucio
et al. 2009).
G-protein–coupled receptor 17 (GPR17) is regulated
by Olig1 and its function is to oppose the action of Olig1. G
protein–coupled receptor 17 is restricted to OL lineage cells,
but is down regulated during the peak period of myelination
and in adulthood. G protein–coupled receptor 17 overexpres-
sion inhibited OL differentiation and maturation. Conversely,
Gpr17 knockout mice showed early onset of OL myelination
(Chen et al. 2009). Thus, GPR17 orchestrates the transition
between immature and myelinating oligodendrocytes via an Id
protein–mediated negative regulation.
4.4.5 Transcription Factors
The presence of various transcription factors is required for nor-
mal differentiation of OPCs into mature OLs and for the for-
mation of myelin. In the brains of Olig1-null mice, expression of
myelin-specific genes is abolished and they develop widespread
progressive axonal degeneration and gliosis. It has been shown
that Olig1 regulates transcription of the major myelin-specific
genes, MBP, PLP, and myelin-associated glycoprotein (MAG),
and suppresses expression of a major astrocyte-specific gene,
glial fibrillary acid protein (GFAP). In zebrafish, Olig1 asso-
ciates physically with another myelin-associated transcription
factor, Sox10, and the Olig1/Sox10 complex activates MBP
gene transcription via conserved DNA sequence motifs in the
MBP promoter region (Li et al. 2007). Zfp488, an OL-specific
zinc-finger transcription regulator, can interact with Olig2
to promote precocious and ectopic OL differentiation.
Knockdown of Zfp488 in an OL cell line leads to the down-
regulation of myelin gene expression. Thus, Zfp488 functions
as a transcription coregulator important for OL maturation
and Zfp488/Olig2 cooperation can serve as a mechanism for
OL differentiation (Wang et al. 2006).
Myelin gene regulatory factor (MRF) is specifically
expressed by postmitotic OLs within the CNS. Overexpression
of MRF within cultured OPCs or the chick spinal cord pro-
motes expression of myelin genes. In mice lacking MRF within
the OL lineage, premyelinating OLs are generated but display
severe deficits in myelin gene expression and fail to myelinate
(Emery et al. 2009). These findings establish MRF as a criti-
cal transcriptional regulator essential for OL maturation and
CNS myelination.
Yin Yang 1 (YY1) is another transcription factor that is an
essential component of the transcriptional network regulating
the transition of OPCs from cell cycle exit to differentiation.
Yin Yang 1 acts as a lineage-specific repressor of transcrip-
tional inhibitors of myelin gene expression (Tcf4 and Id4),
by recruiting histone deacetylase-1 to their promoters during
OL differentiation (He et al. 2007). More detailed description
of transcriptional control of OL maturation can be found in
chapter 43.
5 S U M M A RY A N D P E R S P E C T I VE S
Several questions remain elusive in OPC development. First,
recent evidence suggests that OPCs migrate for long dis-
tances, sometimes across regional boundaries (Mecklenburg
et al. 2011), and thus OLs in a certain region may have mul-
tiple origins. Most of these studies were performed in the
chick embryo. It is of interest to examine whether such long
distance migration of OPCs is observed in the developing
rodent brain (Lachapelle et al. 1984). Second, whereas the
origin of OPC in the chick optic nerve may be a focal O4+
cell cluster in the suprachiasmal neuroepithelium (Ono et al.
1997), the origin of rodent or mammalian OPCs in the ventral
forebrain has not yet been elucidated. Lineage tracing stud-
ies with Cre–loxP system are needed to identify the origin of
OPC in the optic nerve. Third, the mechanisms by which dor-
sally derived OPCs are induced are not fully understood. It is
probable that dorsally derived OPCs are induced in an FGF-
dependent manner. However, the dorsal spinal cord releases
factors inhibiting OPC differentiation. Wnt and BMP family
molecules are strong candidates for these inhibitory activities
(Mekki-Dauriac et al. 2002; Shimizu et al. 2005). How these
negative and positive regulators in the dorsal spinal cord coor-
dinate OPC differentiation may be future issues of this field.
Finally, how do OPCs regulate themselves in cell number? As
mentioned, although PDGF-A is a strong mitogen for OPC,
excess OPCs are eliminated and their number becomes nor-
mal even in PDGF-A overexpressing mice. Oligodendrocyte
progenitor cells might recognize their own density by some
form of cell–cell interaction as suggested in zebrafish (Kirby
et al. 2006). Better understanding OL development by uncov-
ering these issues is clearly important not only for glial biol-
ogy, but also for the translational study of OLs and myelin.
L I N E AG E A N D D E VE L O PM E N T: O L I G O D E N D R O C Y T E S
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 14.
THE SCHWANN CELL LINEAGE: CELLULAR TRANSITIONS
DURING DEVELOPMENT AND AFTER INJURY
Kristján R. Jessen and Rhona Mirsky

A B B R E VI AT I O N S 2 O VE RVI EW O F T H E L I N E AG E

ADAM A disintegrin and metalloprotease protein Schwann cell precursors are the earliest glial phenotype in devel-
BACE1 (β-secretase) beta-site APP-cleaving oping spinal nerves (Jessen et al. 1994). They derive from neu-
enzyme ral crest cells that break free from the closing neural tube and
BDNF Brain derived neurotrophic factor migrate through the extracellular matrix before they convert
CHO Chinese hamster ovary to the Schwann cell precursor phenotype as they take up posi-
CNS central nervous system tions among the axons of early nerves. In mouse sciatic nerves,
Dhh desert hedgehog Schwann cell precursors are found at embryo day (E)12–13 (rat
DRG dorsal root ganglia E14–15). In contrast to crest cells, Schwann cell precursors lie
ERK Ras-extracellular signal-regulated kinas in intimate association with neurons (axons), a feature that is a
FAK focal adhesion kinase distinguishing characteristic of glial cells throughout the unper-
GDNF Glia derived neurotrophic factor turbed central nervous system (CNS) and peripheral nervous
GFAP Glia fibrillary acidic protein system (PNS) (Fig. 14.3). Schwann cell precursors generate
Ilk integrin linked kinase immature Schwann cells that populate mouse nerves from E15–
JNK c-Jun N terminal kinase 16 (rat E17–18). Starting around birth (E20–21), the process of
Lgi leucine-rich glioma-inactivated radial sorting results in some immature Schwann cells adopt-
LIF leukemia inhibitory factor ing a 1:1 relationship with single large diameter axons, form-
LPA lysophosphatidic acid ing promyelin cells. In small mammals, the large majority of the
MAPK mitogen-activated protein kinases cells that achieve a 1:1 relationship with axons progress to form
MBP myelin basic protein myelin sheaths. Other immature Schwann cells do not take part
MCP1 Monocyte chemoattractant protein1 in radial sorting, but later associate with small-diameter axons,
NCAM Neural cell adhesion molecule accommodating each axon in a separate pocket along their sur-
NRG1 β -NEUREGULIN 1 face, thus forming mature Remak fibers; in rat nerves they are
P0 myelin protein zero first seen during the third postnatal week (Diner 1965; Jessen
p75NTR p75 neurotrophin receptor and Mirsky 2005; Webster and Favilla 1984).
PNS peripheral nervous system The development of Schwann cell precursors and immature
RBPJ recombining binding protein suppressor of Schwann cells is controlled by axon-associated signals. These sig-
hairless nals are essential for the survival and differentiation of Schwann
TACE tumor necrosis factor α-converting enzyme cell precursors, determine whether immature Schwann cells myeli-
nate or form Remak cells, and control the elaborate molecular and
morphological architecture of myelinating cells. Additionally,
1 INTRODUCTION axonal signals drive the proliferation of Schwann cell precur-
sors and immature Schwann cells throughout early development
The Schwann cell lineage in rodents exhibits four main cel- (Jessen and Mirsky 2005; Stewart et al. 1993; Yu et al. 2005).
lular transitions ( Jessen and Mirsky 2005). These are: (1) the Although Schwann cells in adult nerves are quiescent, they retain
generation of Schwann cell precursors; (2) the generation of the ability to re-enter the cell cycle following injury and in patho-
immature Schwann cells, both of which take place in embry- logical conditions (Jessen and Mirsky 2005, 2008).
onic nerves; (3) the largely postnatal differentiation of myelin Neural crest cells, Schwann cell precursors and immature
and mature nonmyelin (Remak) cells; and (4) in injured Schwann cells are each faced with a fate choice during devel-
nerves, the generation of dedicated repair Schwann cells opment. This is well established for crest cells, and also for
(Figs. 14.1 and 14.2). This chapter describes the cellular immature Schwann cells because they can form both myelin
changes that characterize these four main transitions, and dis- and Remak cells. It is only recently, however, that Schwann
cusses the signals that drive cells between differentiation states cell precursors have been shown to generate endoneurial fibro-
in developing and injured adult nerves. blasts and skin melanocytes in addition to immature Schwann

159
Main transitions in the Schwann cell lineage
Myelin
Axon Schwann cell
Büngner cell
Remak cell
Immature
Neural Schwann cell
crest precursor (SCP) Schwann cell
(iSch)
E 12/13 E 15/16- ADULT
EARLY POSTNATAL
Figure 14.1 Main Transitions in the Schwann Cell Lineage. Schematic
illustration showing the main cell types and developmental and
injury-induced transitions. Black uninterrupted arrows: normal
development. Red arrows: the Schwann cell injury response. Stippled
arrows: post-repair formation of myelin and Remak cells.
cells (Adameyko et al. 2009; Joseph et al. 2004). This unex-
pected diversity of options open to Schwann cell precursors
is reminiscent of that seen in early CNS glia such as radial
glia, and indicates that a broad developmental potential is a
characteristic of early glial cells both in the CNS and PNS
(Adameyko et al. 2009; Jessen and Mirsky 2005; Joseph et al.
2004; Mirsky et al. 2008).
Our detailed knowledge of Schwann cell development is
derived mainly from studies on spinal nerves and in particu-
lar the sciatic nerve. The process is not identical in dorsal and
ventral nerve roots, where the Schwann cells have a distinct
molecular characteristics and origin, because they develop
from boundary cap cells (Coulpier et al. 2009; Maro et al.
2004). Other distinct groups of PNS glia include satellite cells
in ganglia and enteric glial cells (see chapter 11).
A small population of progenitor/precursor cells commonly
involved in tissue regeneration and repair is found in many
adult tissues. Although such “standby” cells do not appear to be
present in uninjured nerves, damaged rodent nerves regenerate
successfully. A main reason for this is that injury triggers a strik-
ing transformation of the myelin and Remak cells to generate
denervated Schwann cells that form regeneration tracks (Bands
of Büngner) distal to the injury (Fig. 14.4). These Büngner cells
are specialized to support neuronal survival, axon regenera-
tion and target reinnervation. At the completion of the repair
process they are re-specified to form myelin and Remak cells
around the regenerated axons ( Jessen and Mirsky 2008).
3 D I F F E R E N C E S B ET W E E N T H E N E U R A L
C R E S T S C H WA N N C E L L P R E C U R S O R S ,
A N D I M M AT U R E S C H WA N N C E L L S
Schwann cell precursors within embryonic nerves differ from
migrating neural crest cells, first, because they express glial dif-
ferentiation markers and other factors not found on migrating
160 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
crest cells (see Fig. 14.2) (Buchstaller et al. 2004; D’Antonio et al.
2006b; Li et al. 2007; Mirsky et al. 2008). Second, as mentioned,
Schwann cell precursors exhibit the diagnostic glial phenotype
of intimate association with nerve cells, because they are found
among axons within nerves, whereas crest cells migrate through
mesenchymal connective tissue. Third, Schwann cell precursors
and crest cells respond differently to survival signals and mitogens
(Woodhoo et al. 2004). Last, compared with crest cells Schwann
cell precursors are insensitive to the neurogenic action of BMP2
and strongly biased toward the generation of immature Schwann
cells (Kubu et al. 2002; Woodhoo and Sommer 2008).
The next transition, the generation of immature Schwann
cells from Schwann cell precursors, also involves substantial
changes in gene expression (see Fig. 14.2) (Buchstaller et al.
2004; D’Antonio et al. 2006b; Dong et al. 1999; Frank et al.
1999). Schwann cell precursors and immature Schwann cells
also show a marked difference in survival regulation. Schwann
cell precursors are acutely dependent on axonal survival sig-
nals, chiefly β-neuregulin 1 (NRG1) (Dong et al. 1995),
whereas immature Schwann cells can prevent their own death
by secretion of autocrine survival factors, a mechanism that
Schwann cell precursors lack (Dong et al. 1999; Meier et al.
1999). Because Schwann cell precursors lack a basal lamina,
formation of basal lamina by immature Schwann cells repre-
sents another difference between these cells.
Cadherin 19 is first activated when Schwann cell precur-
sors are formed from the crest, but is downregulated in imma-
ture Schwann cells. It is therefore the only known gene that
selectively marks the Schwann cell precursor stage.
4 GLIOGENESIS: THE APPEARANCE
O F S C H WA N N C E L L P R E C U R S O R S
Transcription factors that specify glial cell differentiation
from crest cells have not been identified (Cheli et al. 2010;
Marmigère and Ernfors 2007). Similarly, cell-extrinsic factors
required for initiation of PNS glial differentiation in vivo have
not been clearly determined (Woodhoo and Sommer 2008).
Three major factors that have been shown (Sox10), or sug-
gested (NRG1 and Notch), to be involved in Schwann cell
precursor generation are discussed in the following.
4.1 S OX10
The transcription factor Sox10 is required for the generation of
glia from the crest, but because it is expressed by essentially all
migrating crest cells before the appearance of glial cells and at
the earliest stages of the melanocyte lineage, it does not specify
glial cells. An important role of Sox10 may be to uphold expres-
sion of ErbB3 receptors for NRG1, a signal with key functions
in Schwann cell development (Britsch et al. 2001).
4.2 E -N EU R EGU L I N 1
NRG1 does not appear to be required for glial cell formation
from the crest for the following reasons. First, in zebrafish,
Schwann cells are generated despite inactivating mutations in
 Associate Associate Associate
with ECM with axons with axons
NRG1 survival NRG1 survival NRG1 survival
signaling is signaling is signaling is
ECm dependent ECM independent ECM independent

Neural crest SCP iSch

ErbB3 ErbB3 ErbB3
NO CHANGE L1 L1 L1
p75NTR p75NTR p75NTR
Sox10 Sox10 Sox10
BFABP
Dhh
Po
GAP43
PMP22
PLP
Connexin 29
PrPc
UPREGULATION Astrotactin
Serpin2 GFAP
NFIB S100
Cad19 Oct6
O4
MAL
Galectin
Desmoyokin
Reelin
Decorin

α4integrin
DOWNREGULATION AP2
Ncad
Cad19

Figure 14.2 The Phenotype of the Main Stages of the Embryonic Schwann Cell Lineage. Below the lineage drawing some of the molecular markers that
characterize the lineage are shown. Four groups are shown: (1) markers that show no significant change between the three stages; (2) markers that are
upregulated at the crest/Schwann cell precursor transition; (3) markers that are upregulated at the Schwann cell precursor/immature Schwann cell
transition; and (4) markers that are downregulated at the Schwann cell precursor/immature Schwann cell transition. For references see text and Jessen
and Mirsky 2005.

the NRG1 receptor ErbB3 (Lyons et al. 2005). Second, in rat 4.3 N OTC H
neural crest cultures, cells expressing P0 mRNA, a Schwann cell
Notch increases the number of Schwann cells in rat neural
precursor marker, appear readily without addition of NRG1
crest cultures, using the late glial marker GFAP to monitor
(Woodhoo et al. 2009), and this is also true of GFAP-positive
Schwann cell appearance (Kubu et al. 2002). Other studies
Schwann cells (Shah et al. 1994). Last, the satellite cells in
on mouse and chick crest cells using the earlier differentia-
DRG, a major category of PNS glia, appear to form normally
tion marker P0 mRNA, which detects the initial appearance
in mouse mutants in which NRG1 or functional ErbB2 or 3
of Schwann cell precursors, rather than GFAP that identifies
receptors are missing (Garratt et al. 2000). Taken together,
the subsequent generation of Schwann cells, were unable to
this shows that NRG1 signaling is unlikely to be obligatory
detect this effect. Notch activation in neural crest cells in ovo
for crest cells to adopt a glial fate.
also failed to promote Schwann cell generation (Wakamatsu
Nevertheless, NRG1 may bias crest cells toward glial dif-
et al. 2000; Woodhoo et al. 2009). In mice lacking Hes1 and
ferentiation indirectly. For instance, one of the most striking
Hes 5, the effectors of canonical Notch signaling, the appear-
effects of NRG1 on neural crest cultures is inhibition of neu-
ance of Schwann cell precursors was also unaffected (Woodhoo
rogenesis. This effect could increase gliogenesis by extend-
et al. 2009). Similarly, in mice lacking the transcription factor
ing the exposure of crest cells to gliogenic signals (Shah et al.
RBPJ, a key mediator of canonical Notch signaling, Schwann
1994). After the onset of glial development, NRG1 pro-
cells were generated, although formation of satellite glia, and to
motes the Schwann cell precursor/immature Schwann cells
a lesser extent neurons, in sensory ganglia was impaired (Taylor
transition and the generation of Schwann cells by promoting
et al. 2007). Therefore, it is unlikely that the generation of
their survival and proliferation.

T H E S C H WA N N C E L L L I N E AG E : C E L LU L A R T R A N S I T I O N S D U R I N G D E VE L O PM E N T A N D A F T E R I N J U RY • 161
 Schwann cell precursors in spinal nerves is significantly regu-
lated by Notch signaling. On the other hand, Notch accelerates
the subsequent step in Schwann cell development, the Schwann
cell precursor/immature Schwann cell transition, discussed in
the following.

5 E -N E UR E GUL IN 1 A N D N OTC H
S I G N A L IN G IN S C H WA N N C E L L
P R E C UR S O R S

In mouse mutants in which NRG1 or the NRG1 receptors

ErbB2 or ErbB3 have been inactivated, there is a conspicuous
loss of Schwann cell precursors (Birchmeier 2009; Birchmeier
and Nave 2008; Garratt et al. 2000). As discussed, it is proba-
ble that crest cells can initiate glial fate without NRG1 signals.
Therefore, it is likely that the loss of Schwann cell precursors
in these embryos results from the role of NRG1 in controlling
Schwann cell precursor survival and migration.

Figure 14.3 Schwann cell precursors and immature Schwann cells in
embryonic nerves. Upper panel shows a transverse section through E14 rat
sciatic nerve. Schwann cell precursors are embedded among the axons and
at the surface of the nerve (big arrows). A dividing Schwann cell precursor
is also seen (small arrow). Connective tissue (turquoise) is not found inside
the nerve. Lower panel shows a transverse section through E18 rat sciatic
nerve. One or a few immature Schwann cells together surround a number
of axons, forming compact groups (families; asterisk). A dividing immature
Schwann cell is seen (double arrows). Connective tissue (turquoise) contain-
ing blood vessels (large arrow) is present throughout the nerve surrounding
the families. Bracket indicates the developing perineurium. Adapted from
Jessen and Mirsky 2005.
5.1 S U RVI VA L
Unlike Schwann cells, Schwann cell precursors cannot be grown
in vitro without the addition of survival factors. This is because
Schwann cell precursors, but not Schwann cells, depend on sur-
vival signals from axons. In cell culture, Schwann cell precursor
death can be prevented by contact with axons, and the axonal
signal responsible for this contact-mediated rescue of Schwann
cell precursors has been identified as NRG1. Soluble NRG1 is
also a potent survival factor for these cells (Dong et al. 1995).
In vivo, the Schwann cell precursor death that results from
neuronal degeneration is prevented by application of NRG1
(Winseck and Oppenheim 2006). NRG1 is present on axonal

Myelin cells Cells that accelerate the

conduction of action potentials

The Schwann cell

injury response

Büngner cells
(forming Büngner bands)

c-Jun-dependent Dedifferentiation
repair program program

Upregulation of Myelin breakdown

molecules that Formation of Downregulation Upregulation
regeneration tracks directly and by of myelin genes of iSch markers
promote neuronal macrophage
survival (Bands of Büngner)
recruitment
and growth

Cells that control nerve repair

Figure 14.4 The Schwann Cell Injury Response Main events during the transdifferentiation of myelin cells to form Büngner repair cells.

162 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
surfaces and is expressed in DRG and motor neurons at the
developmental stage when Schwann cell precursors are found
in nerves (Birchmeier and Nave, 2008; Taveggia et al. 2005).
Therefore, NRG1 is expressed at the right time and place to con-
trol Schwann cell precursor survival. These observations indicate
that axonal NRG1 is an essential survival factor for Schwann cell
precursors in embryonic nerves. Therefore, a major reason for
the lack of Schwann cell precursors in NRG1 mutants is likely to
be cell death owing to the absence of NRG1 signaling.
5.2 M I G R AT I O N
NRG1 stimulates migration of developing Schwann cells along
zebrafish nerves in vivo and the migration of rat Schwann
cells in vitro (Lyons et al. 2005; Meintanis et al. 2001; Yamauchi
et al. 2008). In mutants without NRG1 signaling, the crest fails
to migrate to form sympathetic ganglia, although the forma-
tion of DRG appears relatively unaffected (Britsch et al. 1998).
Therefore, failure of cell migration in early nerves is likely to
contribute to the lack of Schwann cell precursors in NRG1
mutants (Birchmeier 2009).
5.3 N RG1 O N D EV E L O P I N G AXO N S
Most of the known effects of axonal NRG1 on Schwann
cell precursors or Schwann cells are mediated by isoform III.
Following proteolytic cleavage, the EGF-like NRG1 domain
is presented on the axonal surface and signals in a juxtacrine
fashion by binding to ErbB2/3 receptors on adjacent glial
cells (Birchmeier 2009; Taveggia et al. 2005). The membrane-
associated protease BACE1 (β-secretase) is involved in activa-
tion of NRG1 signaling from the axonal surface, whereas its
opposing enzyme TACE inhibits myelination (Hu et al. 2006;
La Marca et al. 2011; Willem et al. 2006).
5.4 β -N EU R EGU L I N 1 A N D N OTC H I N T E R AC T
TO P RO MOT E S C H WA N N C E L L P R ECU R S O R
S U RVI VA L A N D I M M AT U R E S C H WA N N
C E L L G E N E R AT I O N
Notch ligands, like NRG1, are expressed on axons. Unlike
NRG1 signaling, activation of Notch signaling alone in
Schwann cell precursors does not promote survival. But
Notch promotes the survival effects of NRG1, particularly at
low NRG1 concentrations (Woodhoo et al. 2009). Another
effect of Notch on Schwann cell precursors is to accelerate the
generation of immature Schwann cells. Thus the appearance of
immature Schwann cells is delayed in mice with conditional
activation of Notch1 or the transcription factor RBPJ, which
transduces canonical Notch signaling. Conversely, immature
Schwann cells are generated ahead of schedule if Notch1 is
overexpressed (Woodhoo et al. 2009).
Both of these effects of Notch are probably because Notch
elevates expression of the NRG1 receptor ErbB3 in Schwann
cell precursors (Woodhoo et al. 2009). This and the fact that
NRG1 promotes Schwann cell survival and differentiation
allows these functions to be regulated indirectly by Notch
(Brennan et al. 2000; Woodhoo et al. 2009).
T H E S C H WA N N C E L L L I N E AG E : C E L LU L A R T R A N S I T I O N S D U R I N G D E VE L O PM E N T A N D A F T E R I N J U RY
6 S C H WA N N C E L L G E N E R AT I O N
AND THE ARCHITECTUR AL
R E O R G A N I Z AT I O N O F
P E R I P H E R A L N E RVE S
The Schwann cell precursor/immature Schwann cell transition
is accompanied by a striking reordering of cell and tissue relation-
ships within nerves (Wanner et al. 2006b). At E12/13 in mouse
(rat E14/15) embryonic nerves are compact, consisting of tightly
packed axons and flattened sheetlike processes of Schwann cell
precursors separating large groups of axons. The Schwann cell
precursor cell bodies are found at the nerve surface, where these
flattened cells separate the axons from surrounding connective
tissue and, in larger nerves, also embedded among the axons (see
Fig. 14.3). Blood vessels and connective tissue, including fibro-
blasts and extracellular spaces containing collagen, are excluded
from these early nerves. This architecture transforms during the
next 2 to 3 days, at the same time as Schwann cell precursors
convert to immature Schwann cells. An E18 nerve is a collection
of axon-immature Schwann cell bundles (“families”) (Webster
and Favilla 1984) lying in connective tissue spaces containing
blood vessels and collagen, whereas basal lamina is forming at
the immature Schwann cell surfaces (Wanner et al. 2006b).
At this time the developing perineurial sheath can first be dis-
cerned (Parmantier et al. 1999). The signals that control these
changes are unknown, although it has been suggested that they
are promoted by the downregulation of glial N-cadherin expres-
sion that takes place at the Schwann cell precursor/immature
Schwann cell transition (Wanner et al. 2006b).
We know more about the regulation of the Schwann cell
precursor/immature Schwann cell transition, in particular
the positive role of Notch and NRG1 (previous section). The
Schwann cell precursor/immature Schwann cell transition is
negatively regulated by two signals, endothelin and the tran-
scription factor AP2α. Endothelin delays immature Schwann
cell generation from Schwann cell precursors in vitro, and
inactivation of endothelin B receptors in vivo results in prema-
ture appearance of immature Schwann cells, confirming that
endothelin signaling negatively regulates immature Schwann
cell generation (Brennan et al. 2000).
Enforced AP2α expression in Schwann cell precursors in
vitro retards the conversion of these cells to immature Schwann
cells, whereas in vivo this transcription factor is strongly down-
regulated at the Schwann cell precursor/immature Schwann
cell transition, suggesting that AP2 α is involved in maintaining
the Schwann cell precursor phenotype (Stewart et al. 2001).
7 T H E MU LT I P OT E N T S C H WA N N
CELL PRECUR SOR : A SOURCE OF
MEL ANOCY TES AND FIBROBL ASTS
Although the broad developmental potential of Schwann cell
precursor was first detected in vitro ( Jessen and Mirsky 2004),
there is now evidence that Schwann cell precursors generate
both melanocytes and fibroblasts during normal development
in vivo ( Joseph et al. 2004). In mice a significant number of
skin melanocytes originate from Schwann cell precursors,
• 163
and migrate from nerves to skin. Although melanogenesis
is restricted to Schwann cell precursors during development
(Takahashi and Osumi 2005), adult myelin Schwann cells
appear capable of melanogenesis after injury, corroborating a
previous finding (Adameyko et al. 2009; Rizvi et al. 2002).
In principle, this confirms the ability of PNS glia to generate
melanocytes, and provides a striking example of the plasticity
of the Schwann cell phenotype.
Lineage tracing indicates that endoneurial fibroblasts orig-
inate from cells in the nerve that express Dhh, a gene expressed
by Schwann cell precursors and immature Schwann cells, but
not crest cells (Bitgood and McMahon 1995; Jaegle et al.
2003; Joseph et al. 2004; Parmantier et al. 1999). The notion
that both fibroblasts and immature Schwann cells originate
from Schwann cell precursors, accords well with the fact that
the disappearance of Schwann cell precursors dovetails with
the appearance of both immature Schwann cell and fibroblasts
during nerve development (previous section).
8 S C H WA N N C E L L P R E C U R S O R S A N D
E A R LY S C H WA N N C E L L S C O N T R O L
N E U R O N A L S U RVI VA L , FA S C I C U L AT I O N,
A N D SY N A P S E F O R M AT I O N
8.1 N EU RO NA L S U RV I VA L
Studies on embryonic nerves provide compelling support
for the idea that neuronal survival depends on glia. Because
Sox10, ErbB2, or 3 or NRG1(isoform III) are important for
glial development, mutants lacking these genes all have in com-
mon that early glia in embryonic nerves are absent or much
reduced in numbers (Britsch et al. 2001; Garratt et al. 2000;
Woldeyesus et al. 1999; Wolpowitz et al. 2000). Interestingly,
all the mutants show a striking loss of spinal cord and DRG
neurons at cervical and lumbar levels late in embryonic devel-
opment, although these neurons are initially generated in
normal numbers. This suggests that the survival of distinct
of CNS and PNS neuronal populations depends on signals
from Schwann cell precursors and perhaps also immature
Schwann cell and developing satellite cells in sensory ganglia.
It is possible that in these situations neuronal survival is medi-
ated by back-signaling through the intracellular domain of
axon-associated NRG1, cleaved as a result of binding to glial
ErbB2/B3 receptors (Birchmeier and Nave 2008).
Because axonal signals such as NRG1 are needed for
Schwann cell precursor survival (earlier section), neurons and
glia appear to be mutually dependent on each other for sur-
vival during early nerve development.
8.2 FA S C I CU L AT I O N A N D S Y NA P S E
F O R M AT I O N
The broad pattern of limb innervation is determined by
outgrowing axons, and is not significantly altered in mouse
mutants lacking Schwann cell precursors. This unexpected
observation was first made in Splotch (Pax2) mutants, and
later confirmed in mice lacking ErbB2 receptors (Britsch et al.
164 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
2001; Grim et al. 1992; Woldeyesus et al. 1999). A comparable
observation has been made in zebrafish (Raphael and Talbot
2011).
The early stages of synapse formation are also normal with-
out Schwann cell precursors or Schwann cells, although the
establishment of a normal, functioning relationship between
nerve and muscle ultimately fails. This is owing to neuronal
death (discussed earlier), defective fasciculation and abnormal
axon growth and branching within the target tissue (Lin et al.
2000; Morris et al. 1999; Woldeyesus et al. 1999; Wolpowitz
et al. 2000).
The finding that glial cells are required for the transforma-
tion of growing nerve tips into fully formed synapses on muscle
is not surprising when the architecture of growing nerve endings
is considered (Wanner et al. 2006a). Quantitative, ultrastruc-
tural analysis shows that Schwann cell precursors form com-
plex scaffolds among the axonal growth cones, and the amount
of membrane contact between Schwann cell precursors and
growth cones is remarkably constant from nerve front to nerve
front (Wanner et al. 2006a). Clearly, this intricate arrangement
between neurons and glia is essential for normal nerve fascicu-
lation, branching, and interactions with target tissues.
9 E VE N T S L E A D I N G TO S C H WA N N C E L L
D I VE R S I F I C AT I O N : P R O L I F E R AT I O N,
D E AT H , A N D R A D I A L S O RT I N G
In late embryonic nerves, before myelination starts, immature
Schwann cells ensheathe bundles of axons to form axon–
Schwann cell families (previous section). The key developmen-
tal events at this stage are: (1) cell proliferation; (2) apoptotic
cell death, which together with proliferation matches the
number of immature Schwann cell and axons during develop-
ment; (3) radial sorting of axons from the bundles to gener-
ate a 1:1 relationship between axons and Schwann cells; and
(4) the transition from the 1:1 stage to myelination.
Although these events are obviously coordinated, in prin-
ciple they can be regulated independently. Notch signaling,
for instance, drives proliferation but does not control sur-
vival (Woodhoo et al. 2009). TGFβ, through TGFβ type II
receptors, controls proliferation and death, but not sorting
or myelination (D’Antonio et al. 2006a). Conversely, the
transcription factor Sox10 controls sorting and myelination,
but is not required for proliferation (Finzsch et al. 2010).
Control of sorting and myelination can also be separated.
This is seen in Oct6 and Krox-20 mutants, in which myeli-
nation is inhibited, but sorting remains essentially normal
(Svaren and Meijer 2008; Topilko et al. 1994). Last, prolifera-
tion and cell numbers can be controlled without impact on
sorting. This is seen in Notch mutants, in which proliferation
is impaired, resulting in about 30% reduction in cell num-
bers, yet the ratio of sorted to nonsorted cells remains normal
(Woodhoo et al. 2009). Eventually, however, further reduc-
tion in the number of Schwann cells would doubtless affect
the sorting process.
Although these events can be independently controlled,
the same molecule often takes part in regulating more than
one event. For instance, laminin directly or indirectly stimu-
lates immature Schwann cell proliferation, but it also binds to
β1 integrin in the Schwann cell membrane to regulate sorting
(Feltri et al. 2008; Yang et al. 2005; Yu et al. 2005).
9.1 P RO L I F E R AT I O N
It is widely accepted that axonal signals promote Schwann cell
proliferation in perinatal nerves. Early evidence for this came
from neuron-Schwann cell cocultures and the in vivo observa-
tion that cell proliferation in neonatal nerves drops if cells are
deprived of axonal contact by nerve transection (Komiyama
and Suzuki 1992; Salzer et al. 1980). At present, two cell–
cell signaling systems are known to drive Schwann cell divi-
sion in perinatal nerves in vivo. They are the Notch pathway
and transforming growth factor beta (TGFβ)/TGFβ type
II receptor (D’Antonio et al. 2006a; Woodhoo et al. 2009).
In the case of Notch, there is direct evidence that the signaling
represents axon–Schwann cell interactions, but the cellular
source of TGFβ in peripheral nerves is unclear. In vivo, basal
lamina components, the small Rho GTPase cdc42 and focal
adhesion kinase (FAK) also stimulate Schwann cell DNA
synthesis (Benninger et al. 2007; Grove et al. 2007; Yu et al.
2005).
9.1.1 Notch
Notch ligands are expressed on axons in perinatal nerves,
suggesting that Notch signaling is involved in axon–Schwann
cell communication (Woodhoo et al. 2009). Inactivation of
Notch in Schwann cells results in a substantial reduction in
Schwann cell DNA synthesis and cell numbers (see the preced-
ing) (Woodhoo et al. 2009). Activation of Notch signaling in
Schwann cell cultures is also strongly mitogenic. Together this
shows that Notch mediated signaling from axons to Schwann
cells is a major driver of Schwann cell proliferation.
9.1.2 Transforming Growth Factor β
In vivo, conditional inactivation of TGFβ type II receptor in
Schwann cells, substantially reduces proliferation (D’Antonio
et al. 2006a). Because Schwann cell death is also decreased,
Schwann cell numbers are not altered. This suggests that TGFβ
stimulates both proliferation and death in perinatal Schwann
cells. This dual action is also seen in vitro (Einheber et al.
1995; Jessen and Mirsky 2004; Parkinson et al. 2001). Because
TGFβ stimulated proliferation in the presence of NRG1 but
death in the absence of NRG1, it was suggested that TGFβ
amplifies the proliferation of cells with tight axonal contact
and strong NRG1 input, while promoting the death of super-
numerary cells (Parkinson et al. 2001).
9.1.3 Neuregulin
Although it is often assumed that axonal NRG1 drives
Schwann cell division in developing nerves, direct in vivo evi-
dence for this is still missing. In vitro NRG1, on the surface of
axons, in the membrane of CHO cells or in soluble form, is a
T H E S C H WA N N C E L L L I N E AG E : C E L LU L A R T R A N S I T I O N S D U R I N G D E VE L O PM E N T A N D A F T E R I N J U RY
potent Schwann cell mitogen, and in zebrafish ErbB2 receptor
blockers reduce Schwann cell proliferation (Lyons et al. 2005;
Nave and Salzer 2006; Taveggia et al. 2005).
It is intriguing, however, that inactivation of ErbB2 NRG1
receptors results in increased Schwann cell proliferation.
Similarly, in Sox10 mutants, Schwann cell proliferation in
postnatal nerves is unaffected or increased in spite of substan-
tial reduction in mRNA levels for the Schwann cell NRG1
receptor ErbB3. Schwann cells also proliferate normally in
cyclin D1–/– mice, although NRG1 is reported to be unable
to drive proliferation of cyclin D1–/– Schwann cells in vitro
and in regenerating nerves (Atanasoski et al. 2001; Finzsch
et al. 2010; Garratt et al. 2000; Kim et al. 2000; Syroid et al.
2000). In sum, the notion that axonal NRG1 is an important
Schwann cell mitogen in rodent nerves is plausible, but awaits
confirmation in vivo.
9.1.4 Laminin
Removal of laminin from Schwann cells in vivo (γ1
chain mutants or dy2J/α double mutants) decreases prolif-
eration but increases death in perinatal nerves (Chernousov
et al. 2008; Yu et al. 2005). In vitro laminin can promote
Schwann cell survival independently of proliferation (Meier
et al. 1999), and other studies confirm that laminin increases
Schwann cell numbers, but fail to determine whether
this is owing to proliferation or survival (McGarvey et al.
1984; Yang et al. 2005; Yu et al. 2005). The mechanisms
underlying these effects are via the activation β1 integrin
receptors, PI3K, FAK and cdc42 (Benninger et al. 2007;
Berti et al. 2011; Grove et al. 2007; Yang et al. 2005; Yu
et al. 2005).
9.2 D E AT H
There is in vivo evidence for two signals that potentially kill
Schwann cells, TGFβ signaling through the TGFβ type II
receptor, and NGF signaling through p75NTR . Normal devel-
opmental death in perinatal nerves is mediated by TGFβ type
II receptors, not p75NTR , presumably acting in concert with
proliferation and survival signals to generate correct imma-
ture Schwann cell numbers. Cell death is also seen following
injury (D’Antonio et al. 2006a; Grinspan et al. 1996). Like
developmental death, injury-induced death results from sig-
naling through TGFβ type II receptors but in this case there
is an equally significant contribution by p75NTR, presumably
following activation by NGF.
The function of laminin in supporting immature Schwann
cell survival was mentioned earlier. Evidence that NRG1,
which is an indispensable survival signal for Schwann cell pre-
cursors, also contributes to survival of iSc (immature Schwann
cells) in neonatal nerves, comes from the observation that
exogenous application of NRG1 reduces cell death induced
by axotomy (Grispan et al. 2006).
Cell culture studies show that Schwann cell numbers
are also regulated by autocrine mechanisms. The survival of
immature Schwann cell from E18 and older nerves is density
dependent. This indicates the existence of autocrine survival
• 165
circuits that enable Schwann cells to live without axonal con-
tact or neuronal signals. The autocrine signal consists of a
cocktail of factors, including IGF2, NT3, PDGF-B, LIF, and
LPA ( Jessen and Mirsky 2005).
Autocrine survival signals are absent from Schwann cell
precursors, representing one of the most clear-cut phenotypic
differences between Schwann cell precursors and immature
Schwann cell. Because of this, Schwann cell precursors are
entirely dependent on axonal NRG1 type III. This arrange-
ment is likely to help match the number of Schwann cell pre-
cursors and axons (Dong et al. 1995; Meier et al. 1999). On
the other hand, the ability of most postnatal Schwann cells to
survive in the medium term without axons (although some
cells die when axons are cut as described in the preceding) is
essential for repair, because effective regeneration depends on
living Schwann cells in the nerve stump distal to the injury.
9.3 R A D I A L S O RT I N G
In addition to its other functions, axonal NRG1 type III is
involved in radial sorting and the establishment of normal
axon–Schwann cell relationships. This applies both before
myelination, and during generation of mature Remak cells.
This function of NRG1 is distinct from its role in activating
the myelination program and controlling myelin thickness
(Fricker et al. 2011; Raphael and Talbot 2011; Taveggia et al.
2005). Signals in the opposite direction also control sorting,
from Schwann cells to axons. This is seen in studies on Lgi4, a
protein that is secreted from Schwann cells to bind to ADAM
22, a transmembrane protein on axons (Özkaynak et al. 2010).
The transcription factor Sox10, a protein like NRG1with
many functions in Schwann cells including the control of
myelination, is also essential for normal sorting. Some of the
sorting defects in Sox10 mutants may be caused by reduced
NRG1 signaling because ErbB3 receptor expression is reduced
in these mice (Britsch et al. 2001).
Radial sorting is profoundly affected by signaling to
Schwann cells by components of the extracellular matrix,
particularly laminin. Genetic inactivation of laminins, or
β1-integrin or dystroglycan laminin receptors, results in
impaired sorting (Chernousov et al. 2008; Yu et al. 2005).
There is good evidence that laminin, signaling via β1 integrin,
activates the small Rho GTPase Rac1, and that this controls
the formation of Schwann cell lamellipodia associated with
early steps of the sorting process (Chernousov et al. 2008;
Feltri et al. 2008; Grove et al. 2007; Nodari et al. 2007; Yu
et al. 2005). The Rho GTPase cdc42, activated by NRG1, also
participates early in sorting (Benninger et al. 2007). Signaling
through dystroglycan, however, regulates later stages of the
process (Berti et al. 2011). In culture NT3 stimulates Schwann
cell migration by activating Rac1 and may also contribute to
sorting (Yamauchi et al. 2005).
Two β1 integrin-associated kinases, FAK and Ilk, also con-
trol radial sorting (Grove et al. 2007; Pereira et al. 2009). The
major reason for the sorting defects seen in Ilk mutants is fail-
ure of Schwann cell process extension.
166 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
Although a major role of Lgi4/ADAM22, Rac1 and Ilk is
to promote sorting, the data indicate that these systems also
promote myelination proper; that is, wrapping and the for-
mation of the myelin sheath by cells that have already sorted
to form the 1:1 axon–Schwann cell relationship. So far, less
attention has been paid to this aspect of their function.
10 F R O M S O RT I N G TO M Y E L I N AT I O N
In rodents, attainment of a 1:1 axon–Schwann cell relation-
ship is usually a step on the way to myelination, although some
5% to 15% of sorted axons (<1–1.5 μm in diameter) do not
progress further and remain unmyelinated. And in humans
most unmyelinated axons have undergone radial sorting
and exist in a 1:1 relationship with Remak Schwann cells
(Berthold et al. 2005; Sharghi-Namini et al. 2006). Radial
sorting to attain the 1:1 axon–Schwann cell relationship is
therefore not a component of a myelination-specific sequence
of events.
Because radial sorting is needed before Schwann cells can
form myelin, the proteins needed for sorting are indirectly
required for myelination. Accordingly, the sorting mutants
discussed in the preceding show severe lack of myelin. In con-
trast, many of the proteins that control the myelin program,
including the transcription factors Oct6 and Krox-20, and the
transcription regulators NAB1/2 do not exert significant con-
trol over sorting (Svaren and Meijer 2008).
Another regulatory mechanism that works primarily by
promoting the myelin program rather than sorting, involves
micro RNAs (miRNAs) and RNA binding proteins. Mice
with selective deletion of Dicer1, a major miRNA process-
ing enzyme, from Schwann cells primarily arrest after the 1:1
promyelin stage has been reached, although a relatively minor
delay in sorting is also seen (Gokey et al. 2012; Iruarrizaga-
Lejarreta et al. 2012; Pereira et al. 2010; Yun et al. 2010).
11 P O S I T I VE A N D N E G AT I VE
R E GU L ATO R S C O N T R O L M Y E L I N AT I O N
Schwann cells in a 1:1 relationship with larger diameter axons,
myelinate if they are induced to do so by cell-extrinsic sig-
nals (discussed in chapter 44). These extrinsic signals activate
intracellular pathways and transcription factors that drive the
myelination program (discussed in chapter 43). The existence
of these cell-intrinsic positive regulators of myelination is well
established. This applies in particular to the transcription
factors Krox-20, Sox10, Oct6, NAB1/2, NFkB, and NFAT
(Svaren and Meijer 2008).
Recently it has become clear that Schwann cell myelina-
tion is also subject to negative regulation ( Jessen and Mirsky
2008). For instance, in certain experimental settings, activa-
tion of any of the major MAPK pathways ERK, JNK, and p38
can suppress myelin gene expression. The activation of certain
transcription factors or transcriptional regulators also inhibits
or reverses myelination. At present, the evidence is strongest
for c-Jun and Notch, but related observations have been made
for a number of other factors, including Sox2, Id2, and Pax3
( Jessen and Mirsky 2008; Le et al. 2005).
What is the function of these novel negative myelin regu-
lators? Clearly, it is possible that they help prevent premature
myelination during development or regeneration, or counter-
act positive regulators to ensure that myelination proceeds
at an appropriate rate. A timing role of this kind has been
shown in principle for Notch signaling in vivo (Woodhoo
et al. 2009).
But the most important function for some of the negative
regulators may not be developmental, but be carried out in
the adult, following injury. Nerve cut or crush triggers perhaps
the most remarkable phenotypic transition in the Schwann
cell lineage. This is the transformation of myelin and Remak
cells distal to the injury into an alternative Schwann cell phe-
notype, the denervated Schwann cell, a cell that is specialized
to support regeneration. Recent studies indicate that some of
the signals referred to as negative regulators of myelination, in
particular c-Jun and Notch, are essential for one or both of the
two processes that are the hallmark of this transition, namely,
the dismantling of the myelin phenotype (dedifferentiation)
and the activation of the alternative repair phenotype.
12 N E G AT I VE R E GU L ATO R S O F
M Y E L I N AT I O N C O N T R O L T H E S C H WA N N
C E L L I N J U RY R E S P O N S E A N D T H E
G E N E R AT I O N O F A D E D I C AT E D R E PA I R
C E L L I N I N J U R E D N E RVE S
12.1 T H E S C H WA N N C E L L I N JU RY R E S P O NS E
Nerve cut or crush results in axon death and triggers a cas-
cade of changes distal to the injury that are collectively called
Wallerian degeneration (discussed in chapters 54 and 55). This
process has mainly been studied in the context of myelin cells,
and this is reflected in the discussion that follows, but it is
likely that the same principles apply to Remak cells.
Central to these events is a radical alteration in the pheno-
type of Schwann cells. This transformation, the Schwann cell
injury response, has two components (see Fig. 14.4). One is a
change in gene, protein, and lipid expression by myelinating
cells that is the reverse of that seen when immature Schwann
cells myelinate. This includes downregulation of components
associated with myelin cells, such as Krox-20, P0, and MBP, and
reappearance of the classical markers of immature Schwann
cells, including p75NTR, NCAM, L1, and GFAP. Schwann
cells of the injured distal stump also re-enter the cell cycle and
apoptotic cells are again seen in the Schwann cell population.
Because these changes broadly represent a reversal to a previ-
ous state that resembles immature Schwann cells before myeli-
nation, they are appropriately referred to as dedifferentiation.
The other component of the Schwann cell injury response,
in contrast, can be represented as alternative differentiation,
T H E S C H WA N N C E L L L I N E AG E : C E L LU L A R T R A N S I T I O N S D U R I N G D E VE L O PM E N T A N D A F T E R I N J U RY
namely the reprogramming of the dedifferentiated state to
generate a dedicated repair supportive cell. This includes: (1)
upregulation of surface molecules and trophic factors that
prevent excessive death of injured neurons and promote axon
growth, such as N-cadherin, GDNF, BDNF, artemin, and
sonic hedgehog; (2) formation of cellular regeneration tracks
(Bands of Büngner) that guide axons from the injury site to
the reinnervation target; this involves adoption of a distinc-
tive cell morphology that remains to be fully determined, but
appears to include the formation of several parallel processes
projecting to each side of the perikaryon; and (3) activation of
a poorly characterized process of endogenous myelin break-
down, that enables Schwann cells to breakdown their own
myelin, and activation of factors, including MCP1 and LIF,
that recruit blood-borne macrophages to complete the clear-
ance of myelin, believed to inhibit axon growth.
In this way, the Schwann cell injury response transforms
the myelin cells to cells that are specialized for supporting
repair. This represents an unambiguous change in cell func-
tion, brought about by the combination of dedifferentiation
and the activation of an alternative differentiation program,
the Schwann cell repair program. Transformations that share
this set of features have been described in other systems, where
they are often referred to as transdifferentiation ( Jopling et al.
2011).
Although the original definition of the term transdiffer-
entiation was narrower than that implied in much current
usage, the broader understanding of the term is useful, because
it defines a set of biological phenomena of great interest and
importance for understanding how some tissues respond to
injury and disease, namely, the reorganization of differenti-
ation programs to allow a cell to abandon one function and
adopt another. The Schwann cell injury response represents a
clear example of an event of this kind.
This response can be clearly differentiated from injury-
induced dedifferentiation/proliferation, a response that is
characteristic of situations in which the primary need is to
replace lost tissue. This is not the case in nerves, because cut
or crush involves axon death, but very little Schwann cell
loss except at the actual injury site. Therefore, the adaptive
value of the Schwann cell injury response is not primarily
to replace lost cells, but to generate a novel Schwann cell
variant specialized for supporting neuronal survival, axon
regrowth, and pathfinding. Because these cells form the clas-
sical Bands of Büngner it is appropriate to refer to them as
Büngner cells.
12.2 C-JU N, N OTC H, A N D
M ITO G E N-AC T I VAT E D P ROT E I N K I NA S E
AC T I VAT I O N D R I VE T H E S C H WA N N
C E L L I N J U RY R E S P O NS E
Although a number of transcriptional regulators are likely to
control Schwann cell transdifferentiation, the evidence is most
extensive for c-Jun and Notch. There is also strong evidence
for the involvement of MAPK pathways.
• 167
 12.2.1 c-Jun
Although the transcription factor c-Jun is expressed at very
low levels in unperturbed adult nerves, injury results in a rapid
and strong expression of this protein in the Schwann cells dis-
tal to the injury site. If this is prevented, by genetic inactiva-
tion of c-Jun in Schwann cells, nerve regeneration is severely
impeded, although c-Jun remains normally expressed in the
neurons and other cells in the nerve.
This is owing to the key role of c-Jun in controlling both
components of the Schwann cell injury response. c-Jun drives
dedifferentiation, which therefore proceeds more slowly in
c-Jun–/– cells (Parkinson et al. 2008). But more importantly,
c-Jun is required for the normal activation of the repair pro-
gram. The denervated c-Jun–/– Schwann cells fail to upregulate
key genes for trophic factors that promote neuronal survival
and regeneration, form abnormal regeneration tracks, and show
long-term defects in myelin clearance. The generation of func-
tional Büngner repair cells therefore fails in the absence of c-Jun,
resulting in a dramatic failure of regeneration and functional
recovery (Jessen and Mirsky 2010; Arthur-Farraj et al. 2012).
12.2.2 Notch
Like c-Jun, Notch signaling is activated in Schwann cells of cut
or crushed nerves. Notch is likely to have more than one func-
tion, but the best characterized of these is the promotion of the
dedifferentiation component of the injury response, and myelin
clearance. Even in uninjured adult nerves, activation of Notch
in myelin Schwann cells is sufficient to trigger demyelination
(Woodhoo et al. 2009). In line with this, myelin breakdown is
accelerated in genetically modified mice in which Notch signal-
ing is overactivated after injury. Conversely, in mice with genetic
inactivation of Notch signaling in Schwann cells (genetic inac-
tivation of Notch1 or the major downstream transcription fac-
tor RBPJ) injury-induced demyelination is delayed (Woodhoo
et al. 2009). Interactions between Notch and c-Jun are likely to
have a major bearing on how these factors control Schwann cell
dedifferentiation (DK Wilton, RM, and KRJ, unpublished).
12.2.3 The MAPK Pathways: ERK, JNK, and p38
ERK, c-Jun N-terminal kinase ( JNK), and p38 are all strongly
activated in the distal stump of injured nerves. Although ERK
signaling is required for myelination (Newbern et al. 2011),
strong activation of Ras/Raf/ERK suppresses myelin genes
and induces dedifferentiation and myelin breakdown even
in myelinating Schwann cells in contact with axons in vitro
(Harrisingh et al. 2004; Napoli et al 2012). ERK is also involved
in another component of the Schwann cell injury response, the
upregulation of the macrophage attractant MCP1 (Fischer
et al. 2008). Activation of JNK similarly inhibits myelin gene
expression, and reverses established myelin differentiation in
purified Schwann cells and in neuron-Schwann cell cocultures
(Parkinson et al. 2008). Last, p38 activation, although involved
in establishing normal axon–Schwann cell relationship early
in myelination, can suppress myelin genes and drive myelin
breakdown ( Jessen and Mirsky 2008; Yang et al. 2012).
168 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
13 S U M M A RY A N D P E R S P E C T I VE S
The development of Schwann cell precursors from the neural
crest, the subsequent transition to immature Schwann cells and
finally to myelin Schwann cells or Remak cells are described in
terms of the major molecular and cellular changes that occur.
In addition, the radical response of Schwann cells to nerve
injury, resulting in the generation of Büngner cells that con-
trol nerve repair is discussed and compared with the changes
in differentiation states that take place during development.
In the near future there is likely to be rapid increase in stud-
ies on the epigenetic control of the Schwann cell lineage dur-
ing development and after injury. There is also a need for better
integration between studies on cell extrinsic signals, including
trophic factors and matrix molecules, studies on intracellu-
lar signaling cascades, such as MAPkinase and cAMP path-
ways, and studies on transcription factors, during Schwann
cell development. Some fundamental instances of neuroglial
signaling in peripheral nerves also remain obscure in molecu-
lar terms. This includes the glial-derived survival signaling to
developing neurons in embryonic nerves, the neuronal signals
that, following injury, inform Schwann cells that axons are
dying, and signals that cooperate with neuregulin to control
myelination. In terms of technical advances, the development
of three-dimensional electron microscopy using focussed ion
beam or field emission techniques is likely to open up radical
new ways of understanding the complex interactions between
axons and Schwann cells during embryonic development,
radial sorting, myelination, and nerve regeneration.
AC K N OW L E D G M E N T S
The authors would like to thank the present and past mem-
bers of their laboratory for allowing them to cite work in prog-
ress, and funding from the Wellcome Trust (program grant)
and the EU Seventh Framework Program (FP7/2007–2013)
under grant agreement No. HEALTH-F2–2008–201535.
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 15.
MICROGLIA LINEAGE AND DEVELOPMENT
Marco Prinz

A B B R E VI AT I O N S
AGM aorta-gonad-mesonephros
BBB blood-brain barrier
BM bone marrow
BM-DP Bone Marrow–Derived Phagocyte
CCR2 chemokine receptor 2
CNS central nervous system
Csf-1 colony stimulating factor
Csf-1R colony stimulating factor 1 receptor
dpc days post conception
GFP green fluorescent protein
HSC hematopoietic stem cells
MPS mononuclear phagocyte system
SOD superoxide dismutase
1 INTRODUCTION
Several reports propose that microglia are specialized cells
of the mononuclear phagocyte lineage. Indeed, it has been
shown that they share many features with other myeloid cells
in the body. For example, microglia express Fc and comple-
ment receptors CD11b and F4/80 epitopes typically found
on myelomonocytes (Hanisch 2002; Perry et al. 1985).
Therefore, it appears that brain microglia, like other resident
macrophages, derive from myeloid precursors that originate
during embryonic development. Aside from these similari-
ties, microglia also display specific molecular differences when
compared with their myeloid cousins. For instance, it has been
shown that microglia produce significantly lower levels of
superoxide dismutase (SOD) compared with splenic or bone
marrow (BM) macrophages (Enose et al. 2005; Glanzer et al.
2007). Furthermore, during lipopolysaccharide/interferon-γ–
induced central nervous system (CNS) inflammation micro-
glia behave differently than CNS-infiltrating phagocytes,
for example, as is reflected by the expression levels of C1qA,
Trem2, and CXCL14 (Schmid et al. 2009).
In addition to the microglia that invade the brain dur-
ing early embryogenesis, it has been postulated that myeloid
progenitors can penetrate into the brain even in normal adult
mice to replace decaying microglial cells. Moreover, it has been
reported that during CNS diseases phagocytes with morpho-
logical features of endogenous microglia can be derived from
BM cells or circulating monocytes. These cells subsequently
become an integral part of the pathology and can be incorpo-
rated into the local cellular networks (Mildner et al. 2007, 2011;
Priller et al. 2001; Prinz and Mildner 2011; Simard et al. 2006).
However, conflicting data have been published in recent years
that contribute to the complexity of microglia research. This
chapter summarizes and discusses the latest findings on the ori-
gin and developmental fate of myeloid cells in the CNS during
health and disease (see also chapters 8, 19, 47, and 48).
2 M I C R O G L I A A S PA RT O F T H E
M O N O N U C L E A R P H AG O C Y T E FA M I LY
In 1908, the Russian Elie Metchnikoff (1845–1916),
together with the German Paul Ehrlich (1854–1915), was
awarded the Nobel Prize for Medicine and Physiology for
his ground-breaking work on phagocytosis (Kaufmann
2008). Metchnikoff coined the term phagocytes (named
after the Greek phago, meaning devour, and cytes, meaning
cells) and divided them into microphages (smaller cells with
polymorphonuclear-shaped nuclei now known as granulocytes)
and macrophages (large eaters). He suggested that both types of
phagocytes played an important role in host resistance against
germs such as bacteria and fungi. The scientist Metchnikoff also
recognized the close relationship between mononuclear phago-
cytic cells in the bone marrow, spleen, lymph nodes, and the
connective tissue, excluding the brain, leading him to introduce
the term macrophage system (Gordon and Taylor 2005). Later
on, the German pathologist Karl Albert Ludwig Aschoff from
Freiburg im Breisgau developed this concept further and grouped
several cell types into what he called the reticulo-endothelial
system in 1924 and subsequently the reticulo-histiocyte system
(Aschoff 1924). This system included reticular cells (or fixed
macrophages) of the spleen and lymph nodes as well as endothe-
lial cells, monocytes, and histiocytes as mobile macrophages. All
these cells were grouped on the basis of their ability to be labeled
by a vital dye in vivo, an assay assumed to measure phagocytotic
activity. However, some cells with poor phagocytotic capacity,
such as endothelial cells, also stained positively because of their
ability to perform pinocytosis and were subsequently falsely
categorized. Therefore, this method of labeling is an unreliable
criterion for the identification of mononuclear phagocytes.
Hence, a new classification was introduced in 1969 to group
only highly phagocytic cells and their precursors in one system
called the mononuclear phagocyte system (MPS) a term that is
still used (Geissmann et al. 2010; van Furth et al. 1972).
Nowadays this broad family comprises numerous mono-
nuclear cells in the body, namely, circulating monocytes in
the bloodstream, dendritic cells in the secondary lymphoid

172
organs, and tissue macrophages (Geissmann et al. 2010; Prinz
et al. 2011). As such, macrophages are resident phagocytic
cells in lymphoid (spleen, lymph nodes) and nonlymphoid
tissues such as the brain (microglia), liver (Kupffer cells), skin
(Langerhans cells), lung (alveolar macrophages), bone (osteo-
clasts), and kidney (kidney macrophages). At these sites mac-
rophages are generally believed to be involved in steady state
tissue homeostasis, via the clearance of apoptotic cells and
the production of growth factors. Therefore, macrophages
are equipped with a broad range of pathogen-recognition
receptors that make them efficient at phagocytosis and induce
the production of inflammatory cytokines (Gordon 2002).
Although the term MPS has created a framework that helped
our understanding of tissue phagocyte biology, it also led to
the assumption that all tissue macrophages are identical in
origin and function. However, there is still a lack of clear-
cut genetic studies, such as fate mapping studies, that could
provide unequivocal evidence that microglia and tissue mac-
rophages such as Kupffer cells in the liver are identical twins
but have grown up in distinct chambers of the body.
3 THE ORIGIN OF MICROGLIA
F R O M T H E YO L K S AC
The crucial finding that the brain in the developing mouse
embryo already contains microglia at 9.5 dpc (Alliot et al.
1991, 1999) forces the developmental neuroscientist to dive
deeply into the complex situation of hematopoiesis. Our cur-
rent understanding of the multifaceted process of hematopoi-
esis was extensively reviewed recently by Ana Cumano and
Isabelle Godin (2007).
Microglia appearance in the neuroepithelium at 9.5 days
post conception (dpc) suggests that microglia precursors may
originate from the yolk sac rather than hematopoietic stem
cells (HSC) in the fetal liver or bone marrow. As shown in
Figure 15.1, HSC are the founders of the hematopoietic sys-
tem, responsible for blood production. Starting at 10.5 dpc
they emerge from ventral aortic hematogenic endothelial
cells in the aorta-gonad-mesonephros (AGM) region of the
embryo. At 10.5 dpc HSC are only found in the embryonic
AGM region, and HSC engrafting activity only becomes
detectable at 11.0 dpc (Bertrand et al. 2005; Cumano and
Godin 2007). Hematopoietic stem cell–derived myeloid cells
are then produced abundantly in the fetal liver by 12.5 dpc.
In contrast, microglia are present in the embryonic brain
before their production from HSC. A population of mater-
nally derived macrophages can be found in the yolk sac of the
embryo as early as 7.5 dpc (not shown). This population, how-
ever, subsequently decreases in number, becomes almost unde-
tectable at 9.0 dpc, and is later absent in the embryo (Bertrand
et al. 2005). A second wave of extraembryonic hematopoietic
cells of zygotic origin differentiates into anucleated red blood
cells and macrophages in the yolk sac (Bertrand et al. 2005).

CNS
8.5-9.0 dpc
haematopoiesis
primitive

hsc and macrophages

?
in yolk sac
7.5–8.0 dpc

hsc in
fetal liver
12.5 dpc
haematopoiesis
definitive

hsc in hsc in
AGM region bone marrow
10.5 dpc 15.5 dpc

Figure 15.1 Different Regions of Hematopoietic Stem Cell Location in the Mouse. A population of transient maternally derived macrophages enters
the yolk sac between 7.5 and 9.0 days post conception (dpc, not shown). The first hematopoietic cells form as blood islands in yolk sac tissues at 8.0
dpc and present the transient and early primitive hematopoiesis (see also Fig. 15.2). As early as 10.5 dpc the aorta-gonad-mesonephros (AGM) region
serves as the main the hematopoietic organ until 12.5 dpc, when the fetal liver contains most uncommitted hematopoietic stem cells (HSC). Around
15.5 dpc bone marrow starts its activity as the hematopoietic compartment, giving rise to long-lived and definitive hematopoiesis. The bone marrow
now becomes the principal organ that sustains an adult-type hematopoiesis. Central nervous system colonization by yolk sac progenitors begins at 9.5
dpc. It is still unclear if there is a contribution from cells of the fetal liver to the CNS. Figure by Markus Knust.

M I C R O G L I A L I N E AG E A N D D E VE L O PM E N T • 173
These yolk sac macrophages invade the developing embryo
between 9.5 and 10.5 dpc (Figs. 15.2 and 15.3) (Bertrand et al.
2005). Thus, because tissues such as the CNS contain yolk
sac–derived macrophages, but not HSC or maternal mac-
rophages, it is reasonable to hypothesize that microglia may
originate from yolk sac macrophages rather than from HSC
in the fetal liver or bone marrow.
As stated, the first macrophage-like cells with an amoe-
boid shape appear in the rodent neuroepithelium as early as
day 8.5 to 9.0 of embryogenesis (Ashwell, 1990, 1991; Chan
et al. 2007) independently of a developed circulatory system
(Kurz and Christ 1998) (see Fig. 15.1). At this early time point,
the first immature macrophages can already be found in the
yolk sac (Takahashi et al. 1996) and were already suggested
to be the precursors of microglial cells a long time ago (Alliot
et al. 1999), which then develop through a nonmonocyte
pathway (Lichanska and Hume 2000; Takahashi et al. 1996).
At embryonic stage 13.5 dpc, when the fetal liver is already
the primary hematopoietic organ and the main site of HSC
expansion and differentiation (Lichanska and Hume 2000),
microglial precursors can be detected in significant numbers
within the ventricular lining of the fourth ventricle (Chan
et al. 2007). However, a 20-fold increase of CD11b+ F4/80+
microglia cell numbers can be observed during the early post-
natal period (P0–P11) in rodents (Alliot et al. 1999). It is still
unclear whether this increase is caused by the proliferation
of embryonic microglial precursors, a phenomenon that has
frequently been observed in the developing brain (Cuadros
and Navascues 1998; Mander and Morris 1996), or whether
an additional recruitment of monocyte-derived microglial
precursors occurs. The latter hypothesis is supported by the
fact that the absence of microglia in mice deficient for the
transcription factor PU.1 can be rescued by the injection of
wild-type bone marrow into newborns, leading to a complete

8.0 dpc 10.5 dpc

umbilical cord

yolk sac blood islands

yolk sac

placenta

embryo embryo

vitelline duct

Figure 15.2 Different Locations of Hematopoiesis During Rodent Embryonic Development. During early embryogenesis (8.0 dpc) first hematopoietic
cells form extraembryonically in a band of primitive erythroid cells, most often described as “blood islands.” Hematopoietic cells properly enter the
embryo via a primitive capillary plexus. At later stages of development hematopoiesis occurs within different tissues of the embryo, as depicted in
Figure 15.1. Figure by Markus Knust.

C
B
A

yolk sac 8.5 dpc 9.5 dpc P1

yolk sac precursors
A B C

meninges

neuroepithelium

Figure 15.3 Embryonic and Postnatal Development of Microglia in the Mouse. A. Early stem cells from yolk sac tissues migrate to embryonic CNS
structures and first gather on neuroectodermal surface structures in the meninges at 8.5 dpc, where they differentiate into primitive macrophage-like
cells (yellow cells) in situ. B. These early embryonic microglia (yellow cells) migrate into the neuroepithelium where they are proliferative and phago-
cytotically active. C. During postnatal (P) development, microglia lose their phagocytic activity, become quiescent, and gain their typical ramified
morphology (yellow cells). Figure by Markus Knust.

174 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
repopulation of the CNS by donor-derived microglia (Beers
et al. 2006). However, it remains to be proved whether this
postnatal recruitment of microglial precursors also occurs in
wild-type animals.
A recent study sheds light on the origin of microglia
(Ginhoux et al. 2010). By inducing Cre recombinase activ-
ity in the Runx locus through injections of tamoxifen into
pregnant mice between 7.0 to 7.5 dpc, when embryonic
hematopoiesis is restricted to the yolk sac, the authors iden-
tified immature yolk sac macrophages as the predominant
source of microglia. Interestingly, myeloid progenitors from
the blood did not significantly contribute to the pool of
adult microglia after birth, at odds with previous studies and
strongly suggesting that the expansion of microglial numbers
in the postnatal period depends on the proliferation of the
resident microglia population. Indeed, local proliferation
of microglia rather than the entry of circulating monocytes
into the CNS during autoimmune inflammation was shown
just recently (Ajami et al. 2011). Therefore, the vast major-
ity of adult microglia appears to be yolk sac–derived (from
a remarkably restricted time period during early embryogen-
esis). It remains an open question whether adult microglia
could also be derived in part from the embryonic liver or
other hematopoietic organs during embryogenesis, such as
the AGM. One limitation of this seminal work (Ginhoux et
al. 2010) is that only one third of the yolk sac macrophages
could be labeled genetically.
4 PAT H WAYS O F M I C R O G L I A I N TO
T H E D E VE L O P I N G B R A I N
It is remarkable that key features of microglia ontogeny are
obviously preserved among vertebrates and quite similar in
humans, mouse, rat, chicken, and fish (Cuadros et al. 1993;
Herbomel et al. 2001; Pont-Lezica et al. 2011; Sorokin
et al. 1992; Takahashi et al. 1989). This strongly indicates that
microglia might have an essential role in the development of
the CNS throughout these species.
The phases of macrophage colonization of the neuroecto-
derm in rodents and humans (Sorokin et al. 1992; Verney et al.
2010) are similar to those observed in the chick and zebrafish
(Cuadros et al. 1993; Herbomel et al. 2001). Although there
are very few macrophages in the mouse embryo at 8.5 dpc,
most are found in the mesenchyme of the head and neck.
These macrophages are thought to enter the mesenchyme by
crossing the pial surface of the meninges (see Fig. 15.3). They
subsequently migrate from the mesenchyme through the base-
ment membrane of the neuroepithelium toward the ventricle.
Once inside the neuroepithelium, the macrophages rapidly
proliferate and colonize the brain from the dorsal to the ventral
side and rostrally to caudally (Pont-Lezica et al. 2011). Within
the next hours microglia move deeper into the developing
parenchyma and begin to differentiate, become branched, and
express markers of mature microglia, thereby losing features
of immature macrophages (Fig. 15.4) (Herbomel et al. 2001).
In the developing mouse brain, microglia numbers increase
steadily and reach approximately 60% of proliferating micro-
glia (Alliot et al. 1999).
Although microglia can be found throughout the brain,
they are not uniformly distributed during development, but
tend to preferentially dwell in specific locations (Cuadros
et al. 1993; Verney et al. 2010). Microglia therefore preferen-
tially concentrate at specific sites in the growing brain such
as in: (1) areas of increased neuronal apoptosis; (2) close
proximity to sprouting vessels; (3) close relation to radial
glial cells; and finally (4) the marginal layer that contains
developing axon bundles (Dalmau et al. 1997; Rezaie and
Male 1999; Rigato et al. 2011). Therefore, microglia are obvi-
ously required for proper neuronal and vessel development
in the brain, as described in more detail in the following
paragraphs.
Although the kinetics of microglia engraftment into
the developing brain have been described in great detail by

embryonic microglia adult resting microgila

phagocytosing non-phagocytosing
CD11b+, CD45high, CX3CR1+, Iba-1+, CD11b+, CD45low, CX3CR1+, Iba-1+,
F4/80+, LAMP-2+ F4/80low

Figure 15.4 Different Morphological Appearance of Embryonic Versus Adult Rodent Microglia. Embryonic microglia (12,5 dpc, left) exhibit a
macrophage-like appearance with round soma and no processes and are phagocytically active. In contrast, adult microglia (P60, right) lose their phago-
cytic activity and undergo dramatic morphological changes giving rise to typical microglia-like cells with roundish to fusiform cell bodies and fine
delineated processes. Typical markers expressed in the respective developmental stages are indicated. Pictures by Katrin Kierdorf.

M I C R O G L I A L I N E AG E A N D D E VE L O PM E N T • 175
several groups, we do not yet know the guidance cues that
direct microglia to their final target. In addition, the poten-
tially protease-mediated signals that may facilitate microglia
migration into the depth of the neuroepithelium are still
unknown.
5 T H E I M P O RTA N C E O F M I C R O G L I A
F O R B R A I N D E VE L O PM E N T
Why are macrophages recruited to the developing brain
and what might be their functional relevance for this organ?
Several reports have shown that during development, the
arrival of microglia in the brain is correlated with the pres-
ence of apoptotic cells, mostly neurons (Ashwell 1991;
Cuadros and Navascues 1998; Wakselman et al. 2008).
In fact, association of microglia and neurons undergoing devel-
opmental death has been described in various brain regions
during development such as the hippocampus (Dalmau et al.
1997; Wakselman et al. 2008), the cerebellum (Marin-Teva
et al. 2004), the spinal cord (Chan et al. 2007; Rigato et al.
2011; Sedel et al. 2004), and the retina (Ashwell et al. 1989;
Cuadros et al. 1992). Transgenic zebrafish expressing GFP in
the macrophage-specific ApoE locus were recently used to
investigate microglia interaction with dying neurons in the
living animal (Peri and Nusslein-Volhard 2008). Engulfment
of apoptotic neurons by microglia occurred in compartments
arising from the progressive fusion of vesicles and was depen-
dent on changes of the intracellular pH. This engulfing capac-
ity might also explain that in the rodent retina, the peak of
microglia density coincides with a period of retinal ganglion
cell and optic nerve axon reduction (Ashwell et al. 1989). In
addition to their phagocytic and thereby clearing role, micro-
glia have an instructive role in developmental cell death. It
has been shown that in the embryonic chick and mouse retina
as well as in the neonatal mouse cerebellum and hippocam-
pus microglia can instruct cells to apoptosis by production of
nerve growth factor or induction of a respiratory burst (Frade
and Barde 1998; Marin-Teva et al. 2004; Wakselman et al.
2008).
Therefore, the ability of microglia to phagocytose and
induce neuronal death grant microglia an important role in
CNS development. Interestingly, microglia can also be found
in large numbers even in the absence of apoptotic cells (Rezaie
and Male 1999; Wakselman et al. 2008), suggesting additional
roles during CNS development.
It has long been recognized that during development of
the neuroepithelium, microglia are found in close proximity
to the developing vasculature (Chan et al. 2007; Cuadros
et al. 1993; Herbomel et al. 2001; Rigato et al. 2011).
It was recently suggested that microglia enter the brain
parenchyma through blood vessels (Ginhoux et al. 2010).
Indeed, microglia were absent in the brains of Ncx mutant
mice (Ginhoux et al. 2010), but a complete lack of heart-
beat in these animals could also impair vessel-independent
microglia engraftment into the CNS. In line with this idea,
early microglia were identified within the brain paren-
chyma already before the onset of vascularization (Cuadros
176 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
et al. 1993; Herbomel et al. 2001; Rigato et al. 2011). This
strongly suggests that early macrophages colonize the CNS
independently of blood vessels; however, the possibility
that, at later time points, microglia may enter via blood ves-
sels cannot be excluded.
In turn, microglia may even support angiogenesis in a chap-
erone-like manner. Recent in vivo studies elegantly showed
that microglia apparently facilitate tip cell fusion, thus induc-
ing vessel anastomosis (Fantin et al. 2010; Rymo et al. 2011).
Communication between microglia and vascular sprouts
appears to be collaborative because microglia quickly migrate
toward the developing vessels, and their angiogenic activity is
enhanced in growing vessels (Rymo et al. 2011). As a matter of
fact, mice with reduced numbers of microglia such as Csfop/op,
PU.1 (Table 15.1) or after depletion using clodronate lipo-
somes have a sparser vascular network (Checchin et al. 2006;
Fantin et al. 2010; Rymo et al. 2011).
The association of microglia with developing neuronal
axons throughout brain development has been reported in
several species (Ashwell 1990; Cuadros et al. 1993; Herbomel
et al. 2001; Verney et al. 2010). In fact, as the first marginal
zones of the brain develop, microglia are consistently found in
close association with the developing axon fascicles (Cuadros
et al. 1993).
The remarkable and recurring association of microglia
with developing axons also raises the question of whether
microglia may have a role in neurite development. Indeed,
microglia are positioned at the right place and appear at the
proper time to influence the setup of axon tracts, axon remod-
eling, and putatively, synaptogenesis. For example, the elegant
combination of 2-photon imaging and high resolution trans-
mission electron microscopy showed that microglia processes
in the juvenile visual cortex of the mouse permanently con-
tact synaptic elements and that this apposition is regulated by
sensory experience (Tremblay et al. 2010). In another recent
study using CX3CR1GFP/GFP mice, microglia were shown to
be able to actively engulf synapses in young postnatal animals
(Paolicelli et al. 2011). These effects were remarkably tran-
sient because microglia loss observed in microglia-specific
CX3CR1GFP/GFP mice, surprisingly was restricted to a short
period of time, namely postnatal days 8 to 18. In light of
recent reports that the chemokine receptor CX3CR1 also
essentially regulates cellular activation in microglia (Prinz et
al. 2011) it is tempting to speculate that the observed effects
on neuronal circuit maturation in CX3CR1GFP/GFP mice might
also be partially owing to CX3CR1-mediated effects on other
cells, such as astrocytes or even neurons directly. Interestingly,
developing microglia express the complement receptor CR3
and it has been proposed that they eliminate unwanted
synapses marked by the complement protein C1q (Stevens
et al. 2007). Furthermore, an essential role for microglia in
postnatal neurogenesis has now been demonstrated in 2- to
3-day-old postnatal mice (Sierra et al. 2010). Determining
whether microglia are important for adult neurogenesis that
takes place months later will need to be the objective of future
research.
Taken together, these data suggest that microglia are poten-
tial modifiers of synapse physiology during development.
Table 15.1 SOME ESSENTIAL MOLECULES REGULATING MICROGLIA DEVELOPMENT
MORPHOLOGY
MOLECULE OF MICROGLIA NUMBER OF MICROGLIA
PU.1 NA ppp
Csf-1 p p
Csf-1R ppp ppp
DAP12 ND pp
Thymosin α1 p p
Microglia phenotypes in knockout animals lacking specific transcription factors or cytokines. The p indicates a reduction in knockout animals. The number of arrows
indicates the relative strength of the effect.
ND, not determined; NA, not allocable.
6 R E GU L AT I O N O F M I C R O G L I A
D E VE L O PM E N T
The transcriptional program that controls macrophage–mi-
croglia differentiation is only poorly understood. Indeed, most
studies on the transcriptional control of macrophage differen-
tiation in general were done in vitro with progenitor-enriched
populations. Thus, the precise role of these factors in driv-
ing the differentiation of yolk sac macrophages into typical
microglia in vivo remains to be determined. This is particularly
important, given that functional macrophage specialization is
likely to be regulated at tissue level, such as in the brain. Some
essential molecules regulating microglia development have
already been described (see Table 15.1). A dramatic reduction
of tissue macrophages including microglia has been observed
in Csf1r–/– mice that lack the colony stimulating factor (Csf-1)
receptor (Dai et al. 2002; Erblich et al. 2011; Ginhoux et al.
2010) and in Csf-1op/op mutant mice (Wegiel et al. 1998;
Yoshida et al. 1990), that have a natural null mutation in the
Csf 1 gene, establishing the key role of Csf-1 and its receptor
in microglia homeostasis in vivo (Pixley and Stanley 2004).
The precise role of Csf-1 and its receptor in microglia com-
mitment remains controversial. One hypothesis suggests that
Csf-1 drives the differentiation of phagocytic yolk sac mac-
rophages entering the embryo into microglia (Metcalf 1985),
whereas a different theory proposes that Csf-1 provides a sur-
vival signal for the differentiating macrophages and that sur-
viving cells use an intrinsic developmental program to become
mature microglia (Lagasse and Weissman 1997; Nakahata et
al. 1982). Interestingly, microglia are more profoundly affected
in the absence of Csf-1R than in the absence of its ligand Csf-1
(Ginhoux et al. 2010). Of note, a second Csf-1R ligand called
interleukin 34 (IL-34) has been identified recently (Lin et al.
2008). Indeed, IL-34 is highly expressed in the brain of post-
natal mice (Wei et al. 2010), but the exact role of IL-34 in
the development and homeostasis of microglia remains to be
determined. Therefore, we are eagerly awaiting the generation
of IL-34–deficient mice. A comparable phenotype of reduced
microglia cell number could be detected in mice deficient for
an adaptor protein for Csf-1R, DAP12 (Otero et al. 2009).
DAP12 contains an ITAM in its cytoplasmic domain and is
M I C R O G L I A L I N E AG E A N D D E VE L O PM E N T
REFERENCES
(Beers et al. 2006; Herbomel et al. 2001)
(Wegiel et al. 1998)
(Erblich et al. 2011; Ginhoux et al. 2010)
(Otero et al. 2009)
(Htain et al. 1997)
highly expressed in Natural Killer (NK) cells cells and myeloid
cells.
A transcription factor that is expressed exclusively in
hematopoietic cells and is involved in microglia development
as well is PU.1. The PU.1 (Sfpi-1) gene is a member of the ets
family of transcription factors (Rosenbauer and Tenen 2007).
Its target disruption leads to multiple hematopoietic abnor-
malities, including a lack of mature B cells and macrophages
(McKercher et al. 1996). In fact, PU.1-deficient mice are devoid
not only of circulating monocytes and tissue macrophages
(McKercher et al. 1996), but also of parenchymal microglia
in the brain (Beers et al. 2006). Similar data have been
obtained in zebrafish microglia. In the zebrafish, yolk sac–
derived macrophages enter the developing brain where they
develop into immature microglia with high endocytotic activ-
ity in a PU.1-dependent fashion because the Panther muta-
tion of PU.1 leads to a complete loss of brain macrophages
(Herbomel et al. 2001).
Further studies investigated microglia development in
athymic mice and demonstrated a significant reduction of
CD11b+ microglia in various brain regions (Htain et al. 1997).
This effect was attributed to a disturbed production of the
hormone thymosin by the thymus gland, which is thought to
be essential for the maturation of myeloid progenitors.
In contrast, only few researchers still believe that microglia
are derived from neuroectodermal matrix cells that differen-
tiate locally into microglia (Chan et al. 2007). Others have
suggested that microglia originate from pericytes (Baron and
Gallego 1972) or from the subependyma adjacent to the lat-
eral ventricles (Lewis 1968). Although the myelomonocytic
origin of microglia has now been widely accepted, the neu-
roectodermal hypothesis remains interesting from a historical
point of view.
7 B O N E M A R R OW–D E R I VE D M I C R O G L I A
D U R I N G A D U LT H O O D
One of the most important questions in the field of micro-
glia research during the last years has been, whether “bone
marrow–derived microglia” in the adult brain exist and if
• 177
so, whether they are functional. The answer to this question
could have tremendous clinical implications for the treatment
of many important diseases of the human CNS such as amyo-
trophic lateral sclerosis, Alzheimer, and Parkinson disease,
because specific peripheral microglia precursors could be used
as carriers to deliver neuroprotective or immune relevant genes
into the diseased CNS to modulate pathology.
The first seminal cell transplantation experiments in rats
demonstrated that only perivascular macrophages, but not cells
with ramified microglia characteristics in the parenchyma, could
be observed in the CNS after BM transplantation (Hickey and
Kimura 1988; Hickey et al. 1992). Similar results were obtained
in humans, in female patients who underwent sex-mismatched
BM transplantation and were examined for the engraftment of
Y-chromosome–positive microglial cells (Unger et al. 1993).
Importantly, only donor-derived perivascular macrophages but
no parenchymal microglia could be detected in this study. It
is important to emphasize that all these studies are based on
immunohistochemical approaches and therefore lack the sensi-
tivity of cell transfer experiments with genetically labeled cells.
Priller et al. (2001) were among the first who used green fluo-
rescent protein (GFP)–marked hematopoietic cells transduced
with retroviral vectors to examine the long-term fate of myeloid
cells in the murine CNS after BM transplantation in an experi-
mental setting, including whole body irradiation. Similar to
other groups they were able to demonstrate GFP-expressing
parenchymal microglia deep in the cerebellum, striatum and
hippocampus several weeks after transplantation (Eglitis and
Mezey 1997; Priller et al. 2001). Despite the differences to the
other studies mentioned, the concept of bone marrow–derived
phagocytes in the CNS was firmly established. In the follow-
ing years, a plethora of publications appeared which examined
the assumed function and fate of BM-derived mononuclear
phagocytes (BM-DP) in different neurological models using
similar experimental paradigms. Remarkably, CNS infiltration
of BM-DP was demonstrated in animal disease models with
no obvious BBB damage, such as amyotrophic lateral sclerosis
(Solomon et al. 2006), Alzheimer disease (Malm et al. 2005;
Mildner et al. 2011; Simard et al. 2006), scrapie (Priller et al.
2006), and many more (Djukic et al. 2006; Priller et al. 2001).
However, all these studies used irradiation of the recipients fol-
lowed by whole BM transplantation to discriminate between
the labeled hematopoietic cells from the donor and the resi-
dent microglia in the hosts. An alternative strategy was used
by Massengale and colleagues. They investigated the fate of
hematopoietic cells in the brain by using parabiotic mice in
which the bloodstream of a GFP-positive partner was con-
nected to a GFP-negative mouse (Massengale et al. 2005).
Although the comparison of data from different laboratories
using divergent protocols is problematic, the parabiosis model
always resulted in dramatically less BM-DP in the brain com-
pared with irradiated chimeras (Massengale et al. 2005). What
might have been the reason for these discrepancies in microglia
engraftment between the parabiosis model and BM transplan-
tation studies using irradiation protocols? Two possibilities
were conceivable. First, lethal irradiation could have significant
influences on the CNS tissue. Indeed, it had been reported
that irradiation affected the integrity of the BBB (Diserbo
et al. 2002; Yuan et al. 2003) and the expression of tight junc-
tion proteins (Kaya et al. 2004), and also induced apoptosis
of endothelial cells (Li et al. 2004). An alternative explanation
could have been the injection of BM cells by which myeloid
precursors gain nonphysiological access to the circulation and
thereby facilitate phagocyte generation from the bloodstream.
To clarify these questions, we and others performed experi-
ments to shed light onto the mystery of BM-DP (Ajami et al.
2007; Mildner et al. 2007). An experimental setup was used
in which the recipient mice received only partial irradiation
by excluding the head from the irradiation field and thus cir-
cumventing any irradiation-induced changes of the brain
(“protected” irradiation). Importantly, de novo generation of
BM-DP from the circulation was strongly diminished in the
brains of mice in which this tissue was not irradiated before
transplantation and also when the chemokine receptor (CCR)
2 was absent from the BM (see Fig. 15.5) (Mildner et al. 2007,
2011). CCR2 is required for BM cells to egress from the bone
marrow (Mildner et al. 2009; Serbina and Pamer 2006).
However, GFP-positive CCR2+ BM-DP exhibiting microglia
morphology could be detected in unprotected and thus irra-
diated parts of the CNS such as the spinal cord. These data

CCR2
B

bone marrow precursors transport via blood stream conditioning of CNS (irradiation + disease) bone marrow derived phagocytes in the CNS

Figure 15.5 Formation of Bone Marrow–Derived Phagocytes (BM-DP) in the Adult Mouse Brain. Postnatal BM-DP form only under defined host
conditions in the CNS. Bone marrow precursors (left) are released into the bloodstream in a chemokine receptor (CCR) 2–dependent fashion and
may enter the conditioned CNS. Local conditioning of the CNS can occur via irradiation and neurodegeneration that both lead to disturbances of the
blood-brain barrier allowing engraftment of BM-DP. A. Yolk sac–derived microglia (orange). B. Bone marrow–derived phagocyte (green). Figure by
Markus Knust.

178 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
clearly support the idea that irradiation itself has a significant
influence on the engraftment of microglial precursors in the
CNS under physiological as well as under pathological con-
ditions. Ajami et al. (2007), on the other hand, investigated
the recruitment of peripheral myeloid precursors to the CNS
in the parabiosis model. The bloodstream of a GFP-positive
mouse was connected with a GFP-negative animal and the
presence of GFP-expressing mononuclear cells was examined
during steady state as well as pathogenic conditions in the CNS
of the GFP-negative recipient. Unlike Massengale et al. (2005),
Ajami et al. (2007) failed to detect BM-DP cells in the CNS of
the GFP-negative partner under any tested conditions. These
findings indicate that the engraftment of bone marrow–derived
myeloid cells in the CNS is an extremely rare event, which is
strongly influenced by the experimental design, for example,
irradiation. Furthermore, these experiments also point to the
fact that endogenous microglia exhibit a high potential for
self-renewal and proliferation that was confirmed by a recent
publication (Ajami et al. 2011). However, there are also some
discrepancies between the studies of Mildner and Ajami and
questions remain. In one experiment, Ajami et al. irradiated the
GFP-negative partner of a parabiotic pair and analyzed the brain
6 weeks after irradiation for the presence of GFP-expressing
CNS mononuclear phagocytes. However, despite the irra-
diation and high levels of chimerism, they still did not detect
any GFP-positive cells in the recipient brain. Was this because
the mice did not receive an intravenous injection of BM cells,
which contain potential myeloid precursors, as was the case in
BM-transplanted mice? This is an interesting possibility, but it
cannot explain the fact that we failed to detect donor-derived
cells in the brains of protected (head shielded but body irra-
diated) animals that also received an intravenous injection of
BM cells (Mildner et al. 2007). It is most likely that irradia-
tion as a precondition, together with the injection of BM cells
synergistically facilitated BM-DP development. One concern
that arose from these studies is the supposed low chimerism of
the examined mice (Soulet and Rivest 2008). In fact, the para-
biosis model results in a chimerism rate of about 40% to 50%,
which is well comparable to the rate observed in protected
(head shielded but body irradiated) animals, in which the skull
bone marrow also contributes to hematopoiesis. Nevertheless,
both studies undoubtedly support the hypothesis that the rate
of microglia turnover from the circulation under physiological
conditions in experiments using irradiation is overestimated
because of irradiation-induced changes and artificial BM cell
injections into the bloodstream. On the other hand, an under-
estimation of microglia turnover in parabiosis animal models
or head-shielded irradiation methods because of low chime-
rism is also possible. In any case, much of the previous work on
chimeric mouse models which involved whole body irradiation
has to be re-evaluated under these aspects.
Despite all this complexity one point is indisputable. Under
specific conditions, BM-DP precursors can engraft into the
brain and become an integral part of the cellular network in
the CNS. These specific conditions include irradiation but
might also occur in certain diseases with no obvious primary
BBB damage, such as X-linked adrenoleukodystrophy. A recent
publication indeed reports of successful autologous stem cell
M I C R O G L I A L I N E AG E A N D D E VE L O PM E N T
transplantation with gene-modified HSC in nonirradiated
humans (Cartier et al. 2009). Adrenoleukodystrophy is a fatal
demyelinating disease of the CNS caused by mutations of the
adenosine triphosphate–binding cassette transporter (ABCD1)
gene. Cartier et al. (2009) purified CD34+ HSC of two patients
with mutated ABCD1 genes. After isolation, the cells were trans-
duced ex vivo with a lentivirus encoding for a functional ABCD1
gene and transplanted back into the patients after they received
myeloablative treatment with cyclophosphamide and busulfan.
Surprisingly, despite low levels of ABCD1-expressing periph-
eral blood cells (only 15% of leukocytes produced ABCD1),
progressive cerebral demyelination stopped in both patients
and the levels of very long-chain fatty acids in the plasma as
indicators of disease activity decreased. Whether the BBB was
intact in these patients even on a microscopic level remains to
be determined, but at least gross measurements by magnetic
resonance imaging (MRI) indicated no major disturbances of
this physiological barrier. However, the authors attributed the
beneficial outcome in the treated patients to “the recruitment of
BM-derived cells to CNS macrophages/microglia” because they
were able to observe precisely this treatment effect in an animal
model using irradiated mice. The authors therefore speculated
that this approach “may provide a new avenue for cell-base gene
therapy in . . . CNS diseases” (Cartier et al. 2009). Consequently,
the identification of a microglial precursor for the therapy of
CNS diseases is of great importance.
These findings clearly indicate that the engraftment of bone
marrow–derived myeloid cells in the CNS is an extremely rare
event, which is strongly influenced by the experimental design
(e.g., cranial irradiation and intravenous transfer of femoral
bone marrow–enriched for hematopoietic progenitors and
stem cells). Furthermore, these results underscore the fact that
endogenous microglia are of yolk sac origin and exhibit a high
potential for self-renewal and proliferation. Nevertheless, in
my personal view, bone marrow–derived phagocytes might be
capable of exploitation for potential therapeutic application
in neurodegenerative settings, although the requirement for
cranial irradiation to achieve CNS engraftment might limit
their utility.
8 S U M M A RY A N D P E R S P E C T I VE S
Although long underestimated, microglia nowadays comprise
an attractive target for accessing the diseased CNS. Their
presence has been confirmed extensively in countless reports
describing their involvement in virtually all neuropathologies.
Furthermore, their role for ensuring normal brain homeostasis
is undoubted. However, the question as to their origin is still
controversial, but recent achievements shed some light on the
mystery of microglia ancestry.
Given the fact that endogenous microglia are not replen-
ished by the periphery at all under normal CNS conditions
and only sparsely during neurodegenerative disease, if the
BBB integrity is broken, it will be a major challenge for future
research to facilitate the use of this ambivalent Trojan horse
to access the brain for putative therapeutic approaches. On
the other hand, it is now believed that localized self-renewal
• 179
can sustain microglia maintenance and probably also func-
tion throughout adult life as a main mechanism of micro-
gliosis under conditions of preserved BBB. Therefore, further
research is necessary to understand key mechanisms and mole-
cules involved in microglia development, local expansion, and
migration to the CNS during development.
AC K N OW L E D G M E N T S
The author thanks people working at the Department of
Neuropathology for the continuous and lively support of
“microglia,” especially Alexander (“Akki”) Mildner (now in
Rehovot, Israel) and Katrin Kierdorf, who has made excep-
tional investigations in the field of microglia biology in the
last years. Moreover, the author is especially grateful to Markus
Knust (“Knust”) for his gifted pictures illustrating this chapter,
and thanks Stefanie Brendecke for critical reading and edit-
ing. Without the enthusiasm of these and other co-workers of
the Department of Neuropathology the work of the last years
would not have been possible.
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PHYSIOLOGICAL PROPERTIES
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 16.
PHYSIOLOGY OF ASTROCY TES: ION CHANNELS
AND ION TRANSPORTER S
Christian Steinhäuser, Gerald Seifert, and Joachim W. Deitmer

A B B R E VI AT I O N S is, to act as direct communication partners of neurons. Although

various types of ion channels, including voltage-gated Na+ (Nav)
AE anion exchanger and Ca2+ (Cav) channels, have been described in the cell culture,
4-AP 4-aminopyridine clear evidence for their expression in astrocytes of acute prepa-
AQP aquaporin rations or in vivo is still lacking. This might partially be caused
ATP adenosine triphosphate by the fact that the identification of small current components
BAPTA 1,2-Bis(o-aminophenoxy)ethane- of astrocytes in situ is hampered by the cells’ very low input
N,N,N′,N′-tetraacetic acid) resistance (usually <2 M:). Moreover, earlier work describing
CA carbonic anhydrase Nav and Cav channels in presumed immature astrocytes par-
Ca2+ pump calcium ATPase tially has to be reconsidered because many of those cells might
CAAC Ca2+-activated anion channel actually have been NG2 cells that have now been identified as
cAMP cyclic adenosine monophosphate a separate glial cell type. Astrocyte current phenotypes are domi-
CCC cation-chloride cotransporter nated by K+ conductances that are important for the regulation
CCE capacitive Ca2+ entry of K+ homeostasis. In addition, water and anion channels are
CNS central nervous system expressed, allowing for the control of the volume of the extra-
EK K+ equilibrium potential cellular space and the release of transmitters from astrocytes.
ER endoplasmic reticulum Astrocytic ion transporters such as the electrogenic Na+/K+
GABA γ-aminobutyric acid pump, the electrogenic Na+/Ca2+ exchanger, or the electrogenic
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesul- Na+/HCO3–-cotransporter are activated in response to changes
fonic acid in extracellular ion concentrations and/or membrane potential.
KCC potassium-chloride cotransporter Depending on the stoichiometry of the transporters, consider-
MCT monocarboxylate transporter able currents can be generated on their activation. In neurons,
Na+-K+-pump sodium-potassium ATPase this can lead to significant changes in excitability: Activation of
NBC sodium-bicarbonate cotransporter the Na+/K+-ATPase generates an outward current and hence
NCX sodium-calcium exchanger counteracts excitation, whereas activation of Na+/Ca2+ exchange
NHE sodium-hydrogen exchanger generates an inward current that depolarizes the cell membrane
NKCC sodium-potassium-2 chloride cotrans-porter and promotes excitation. Because of the low input resistance
NPPB 5-nitro-2-(3-phenylpropylamino)-benzoic of astrocytes, transporter currents have less influence on their
acid membrane potential. Ion transporters are often dependent
OGD oxygen/glucose deprivation on the transmembrane Na+ gradient, and its perturbation can
PEPT peptide transporter result in modulation of transport of various ions and metabolite
PMCA plasma membrane ATP-driven Ca2+ pump substrates. This chapter gives an update about current knowl-
RVD regulatory volume decrease edge of expression and function of astroglial ion channels and
SERCA sarco-endoplasmic reticulum Ca2+ ATPase ion transporters, and discusses its impact on the regulation of
SOCE store operated Ca2+ entry neuronal excitability. Neurotransmitter-activated channels (see
SR101 sulforhodamine 101 chapter 17) and transmitter-activated and metabolic substrate–
TEA tetraethylammonium dependent transporters (see chapters 35 and 27, respectively) are
TTX tetrodotoxin dealt with in other chapters of this book. The focus here is on
VRAC volume-regulated anion channel the properties of astrocytes in acute preparations rather than cell
VSOR volume-sensitive outwardly rectifying culture, whenever possible.
anion channel
2 ION CHANNELS
1 INTRODUCTION
2.1 VOLTAG E - G AT E D Na + A N D Ca 2+ CH A N N E L S
Ion channels and ion-coupled membrane carriers in astrocytes Nav channels represent a prerequisite for initiation and propa-
enable these cells to sense and modulate neuronal activity, that gation of action potentials. The Nav channel family comprises
185
nine different α subunits, Nav1.1 to Nav1.9, although not all
are expressed in the CNS. The principal, pore-forming α sub-
unit associates with membrane-spanning auxiliary β-subunits
(Navβ1–4). Whether or not astrocytes in situ express functional
Nav channels is still under debate. Earlier work reported tetro-
dotoxin (TTX)-sensitive Nav currents in presumed astrocytes
of rat hippocampus (“complex cells”) (Bordey and Sontheimer
1997; Kressin et al. 1995) or in the rabbit retina (Clark and
Mobbs 1994), but from today’s perspective many of these cells
actually might have been NG2 cells rather than astrocytes. In
line with this assumption is a recent study that did not detect
Nav currents in SR101-positive astrocytes of the hippocampus
(Kafitz et al. 2008). However, the latter study did not use TTX
or K+ channel blockers for current separation so that small Nav
currents might have been overlooked. Nav currents have been
recorded from Müller cells freshly isolated from mouse ret-
ina (Pannicke et al. 2002). Immunohistochemistry detected
Nav1.6 in Bergmann glial cells (Schaller and Caldwell 2003),
but whether these cells carry functional Nav channels remains
uncertain. In conclusion, in the context of the increasing evi-
dence that NG2 cells in gray matter represent a distinct glial
cell population (Bergles et al. 2010), there is need to recon-
sider previous conclusions as to the presence of functional Nav
channels in astrocytes in acute preparations.
By mediating Ca2+ influx on membrane depolarization,
Cav channels constitute an important route for Ca2+ delivery
necessary to regulate a multitude of intracellular processes, for
example, cellular metabolism, secretion, and gene expression.
According to their functional properties, the high-voltage acti-
vated L-type (Cav1.1–1.4) and P/Q-, N-, R-type (Cav2.1–2.3)
channels are distinguished from low-voltage activated T-type
(Cav3.1–3.3) channels. Cav channels are formed by a princi-
pal α subunit and auxiliary subunits (Cavα2G1–3, Cavβ1–4,
Cavγ2–8). Cultured astrocytes express Cav channels, but their
functional expression in situ or in vivo is still unclear. Cav cur-
rents could be activated in presumed immature hippocampal
astrocytes (“complex cells”) (Akopian et al. 1996), but prob-
ably these recordings were obtained from NG2 cells (see the
preceding). Immunogold labeling detected Cav1.2 in bona
fide hippocampal astrocytes (Tippens et al. 2008), and there
is some indirect hint at its functional expression, but no direct
proof has yet been presented. This is in contrast to Müller
cells of the retina, which express L- and T-type Cav channels,
both in situ and after acute isolation (Bringmann et al. 2000b;
Newman 1985).
2.2 VOLTAG E -ACT I VAT E D A N D Ca 2+ -AC T I VAT E D
K+ CHANNELS
So far, 12 subfamilies of the Kv α subunits have been identi-
fied, Kv1 to Kv12, which mediate delayed rectifier (KD) and
transient (KA) K+ currents. Astrocytes in situ are endowed with
KD and KA currents, although they are masked by much larger
time- and voltage-independent K+ currents mainly mediated
by Kir and K2P conductances (see section 2.3), which hampers
analysis of their kinetics, stationary properties, and develop-
mental regulation. To improve voltage control, astrocytes were
freshly isolated from the hippocampus, which revealed TEA
186 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
and 4-AP sensitive KD and KA currents, respectively (Seifert
et al. 2009; Zhou and Kimelberg 2000). KD currents were
also recorded from immature Bergmann glial cells, but not at
later (several weeks) developmental stages (Müller et al. 1994).
Developmental changes were also observed in Müller cells of
rabbit retina, where KA currents dominated early after birth
and declined during maturation, whereas KD currents reached
their maximum in adulthood (Bringmann et al. 2000a). Little
is known about the subunits encoding astrocytic Kv currents.
Kv1.5 has been detected immunocytochemically in endfeet of
hippocampal astrocytes and Bergmann glia (Roy et al. 1996),
and its modulation by Src tyrosine phosphorylation regulates
cell differentiation in culture (MacFarlane and Sontheimer
2000). Astrocytes in spinal cord white matter express Kv1.4
(Edwards et al. 2002). Kv11.1 (erg1) mediated K+ currents were
observed in hippocampal astrocytes in situ, and the authors
speculated that they contribute to the clearance of extracellular
K+ (Emmi et al. 2000).
The activity of large conductance Ca2+ activated K+ chan-
nels (BK channels, also termed KCa1.1), depends on membrane
potential and [Ca2+]i. The α subunit can co-assemble with
β-subunits (BKβ1–4), which changes pharmacological prop-
erties and the Ca2+-sensitivity of the channels. Small conduc-
tance Ca2+ activated K+ channels (SK channels; KCa2.1–2.3,
KCa3.1) are voltage-independent and possess a smaller single
channel conductance. BK channels are ubiquitously expressed
in astrocytes in culture. In brain slices of rat hippocampus and
cerebellum, immunohistochemistry and ultrastructural analy-
sis revealed co-localization of BK channels with aquaporin4
(AQP4) water channels in perivascular astrocytic endfeet,
suggesting the involvement of BK channels in K+ redistribu-
tion and regulation of cerebral blood flow (Price et al. 2002).
Recent work confirmed that these channels (KCa1.1) in end-
feet mediate arterial dilation and constriction (Filosa et al.
2006; Girouard et al. 2010) (Fig. 16.1). This α subunit might
co-assemble with BKβ4 or BKβ1 (Seidel et al. 2011). In addi-
tion, evidence was provided supporting the involvement
of KCa3.1 in neurovascular coupling (Longden et al. 2011).
The latter study did not find evidence, however, for functional
expression of KCa2.1–2.3 in astrocytes. Interestingly, the activ-
ity of BK and SK channels in perivascular astrocytes is con-
trolled by metabotropic glutamate receptors (mGluRs) and
epoxyeicosatrienoic acid release (Higashimori et al. 2010).
Similarly, BK channel activity in Müller cells is regulated by
metabotropic ATP receptors (P2Y receptors) (Bringmann
et al. 2002). Thus, although neuronal BK and SK channels
couple with Cav channels, their activity in astrocytes is regu-
lated by Ca2+ release from intracellular stores following stimu-
lation of G-protein–coupled receptors (GPCRs).
2.3 IN WA R D LY R E CT IF Y IN G A N D T WO
P OR E -D OM A IN K + CH A N N E L S
Inwardly rectifying K+ (Kir) channels belong to the superfam-
ily of two-membrane domain channels and lack an intrinsic
voltage sensor. Seven subfamilies have been identified, Kir1 to
Kir7, which form homomeric or heteromeric tetramers. Inward
rectification results from positively charged polyamines and
 A Control
4

Current (nA)
IbTX
3
Electrode
2
Parenchymal IbTX-sensitive
arteriole current
1

20 μm

–120 –80 –40 40 80

Voltage (mV)

B Baseline channel activity

C
O

Channel activity following ES

C
O

10 pA
100 ms

Figure 16.1 BK Channels in Astrocytic Endfeet. A. I–V relationship of whole-cell currents before and 7 minutes after exposure to the specific BK chan-
nel inhibitor IbTX (200 nM). Currents were recorded in response to 200 ms voltage ramps, and the holding potential was –80 mV. The IbTX-sensitive
current is shown in gray. The inset depicts positioning of the patch pipette on an endfoot in a brain slice. B. Representative traces showing single BK
channel currents in cell-attached patches from an endfoot before (top) and after (bottom) electrical stimulation (ES). The holding potential was 0 mV,
[K+]o 6 mM. C, closed; O, open state. Reproduced with permission from Filosa et al. 2006.

Mg2+, which plug the channel from the intracellular site in a
voltage-dependent manner. Kir channels are sensitive to sub-
millimolar extracellular Ba2+ concentrations, intracellular pH,
and adenosine triphosphate (ATP), and are modulated by G
proteins. Kir6 subunits co-assemble with sulfonylurea recep-
tors to form channels that couple electrical activity to the met-
abolic state of the cell. Kir channels are abundantly expressed
by astrocytes of various CNS regions and upregulated dur-
ing postnatal development (Verkhratsky and Steinhäuser
2000), whereas decreased expression is seen in the diseased
CNS (Seifert et al. 2006). They are predominantly respon-
sible to set the resting potential close to the K+ equilibrium
potential (EK). Kir channels in astrocytes represent a prereq-
uisite for the regulation of the K+ homeostasis (Orkand et al.
1966). In the retina, unequal distribution of K+ conductances
along the Müller cell surface allows redistribution of locally
elevated K+, a process termed K+ siphoning (Newman 1986),
and similar spatial buffering of K+ has been suggested in the
cortex (Holthoff and Witte 2000). Among the Kir subunits
identified in the CNS, Kir4.1 is special because of its selec-
tive expression in glia where it is mainly localized to astroglial
processes wrapping synapses and blood vessels. In Müller
cells, the strongly rectifying Kir2.1 subunit predominates in
direct apposition to neuronal membranes, whereas the weakly
rectifying Kir4.1 was identified in endfeet at the capillaries.
This segregated expression suggests a model of K+ siphoning
in which K+ influx is mediated by Kir2.1, whereas K+ leaves
via Kir4.1 channels (Kofuji et al. 2002). The significance of
Kir4.1 for spatial buffering and control of excitability has been
confirmed through general (Kofuji et al. 2000) or conditional
knockout of Kir4.1 (Chever et al. 2010). Astrocytes are het-
erogeneous with respect to Kir subunit expression patterns.
In the spinal cord, the K+ buffer capacity differs between sub-
regions (Olsen et al. 2007). Heteromeric Kir4.1/Kir5.1 chan-
nels were identified in neocortex and olfactory bulb, whereas
in the hippocampus Kir4.1 mainly forms homomeric channels
(Hibino et al. 2004; Seifert et al. 2009) (Fig. 16.2). Highly
pH sensitive Kir4.1/Kir5.1 channels in astrocytes of the ret-
rotrapezoid nucleus contribute to chemoreception and the
control of breathing (Mulkey and Wenker 2011). Antibody
staining identified Kir6.1 in astrocytes of almost all brain
regions, including the retina, preferentially in perisynaptic and
peridendritic processes (Thomzig et al. 2001), and Kir6-like
currents were recorded from Bergmann glia (Brockhaus and
Deitmer 2000). Downregulation of astrocytic Kir currents
is commonly observed after injury or in the diseased CNS
(Olsen and Sontheimer 2008; Seifert et al. 2006).
Astrocytes display prominent passive or background cur-
rents largely lacking time- and voltage-dependency. Recent
data suggest that this current pattern might result from

P H YS I O L O GY O F A S T R O C Y T E S : I O N C H A N N E L S A N D I O N T R A N S P O RT E R S • 187
 A B C
control 100 μM Ba2 I
Inorm 1
100

Membrane conductance
80

at –130 mV[%]
60
V[mV]
–160 –120 –70 –40 20 40
control –Ba2+ 20
–0.5
0
0.01 0.1 1 10 100
–1
1 nA Ba2+[μM]
20 ms

D E
(39)
Relative Frequency [%]

Kir4.1 5.1 2.1 2.2 2.3 bp 100

p3 p10 p21 p42 p60
612 80
495 170 kDa
60 (33) (26) (26) (26) 130 kDa Kir4.1
392 40
297 56 kDa
20 43 kDa β-actin
210 0
Kir4.1 Kir5.1 Kir2.1 Kir2.2 Kir2.3

Figure 16.2 Astrocytes Acutely Isolated from the CA1 Region of the Hippocampus Display Kir Channels. A. Membrane currents were elicited
between −160 and +20 mV (10-mV increments; holding potential, −70 mV; dashed line, zero current). After adding 100 μm Ba2+, currents were
reduced. Ba2+-sensitive currents were determined by subtracting respective current families (control—100 μM BaCl2). B. Mean I–V relationships of
Ba2+-sensitive currents (n = 9). In each cell, data were normalized to maximum inward current. C. Mean dose–inhibition curve of membrane conduc-
tance (n = 7). Half-maximal inhibition occurred at a Ba2+ concentration of 7.1 ± 3.2 μm, with a Hill coefficient of nH = 0.7 ± 0.1. D. After recording,
the cell content was harvested, and RT-PCR was performed. Kir4.1 was coexpressed with other Kir subunits, as shown in the agarose gel. Summary
of Kir detection in single astrocytes. E. Roentgen films of Western blot analysis of Kir4.1 tetramers (160 kDa) and β-actin (42 kDa). Protein content
of lysates prepared from whole hippocampi was analyzed at the ages indicated. The β-actin signal served as a loading control (60 μg of protein each).
Data in (B,C) are given as mean ± SD; cell numbers in parentheses. Reproduced with permission from Seifert et al. 2009.

superposition of currents through Kir and two-pore–domain
K+ (K2P) channels. Similar to Kir, K2P channels are open at
rest and drive the membrane potential toward EK Channel
activity is modulated by pH, polyunsaturated fatty acids,
temperature, volatile anesthetics, phospholipids and mechani-
cal stress. Currently, 15 members of the K2P channel family
have been cloned that are active as dimers. Antibody staining
localized K2P3.1 (TASK1) and K2P10.1 (TREK2) protein in
astrocytes of different brain regions, including radial-glia like
precursors of the subventricular zone (Kindler et al. 2000;
Pruss et al. 2011). In acutely isolated astrocytes from mouse
hippocampus, small K2P2.1 (TREK1)-mediated currents
have been identified with combined functional and transcript
analysis, which were sensitive to polyunsaturated fatty acids,
volatile anesthetics, and quinine (Seifert et al. 2009). The low
surface expression of K2P1.1 channels might result from its
G-protein–mediated endosomal internalization (Feliciangeli
et al. 2010). Müller cells express pH- and bupivacaine-sensitive
K2P3.1 (TASK1) channels that may inhibit cellular swelling
under ischemic conditions (Skatchkov et al. 2006). How
K2P channels in astrocytes influence ion homeostasis and
neuron-glia signaling has still to be elucidated.
Patch-clamp recordings from astrocytes in hippocampal
or cortical brain slices usually reveal linear current–voltage
relationships, because of the predominating Kir4.1 and K2P
conductances. However, before postnatal day 10, the current
patterns often show voltage- and time-dependent current
components (probably because of coexpression of Kv chan-
nels), indicating delayed upregulation of Kir4.1 and/or K2P
channels during postnatal development (Seifert et al. 2009).
2.4 WAT E R C H A N N E L S
Water flux across cell membranes is mediated by homote-
trameric transmembrane channels called aquaporins. Eleven
aquaporins, AQP0 to AQP10, are known, of which AQP1,
AQP4 and AQP9 are expressed in the brain. AQP4 is pre-
dominantly found in astrocytes and Müller cells where it is
primarily localized to the endfeet contacting blood vessels,
the pial surface, and the vitreous body, respectively (Nagelhus
et al. 2004; Oshio et al. 2004). AQP4 colocalizes with Kir4.1
and TRPV4 (Benfenati et al. 2011), and water flux follows
the osmotic gradient during activity-dependent K+ redistri-
bution. These channels are involved in maintaining extracel-
lular ion gradients and regulation of the extracellular and
intracellular volume. In acute cortical slices, neuronal stimu-
lation generated a radial water flux that was mediated through
astrocytic AQP4 channels and facilitated by activation of
vasopressin-1a receptors expressed by astrocytes (Niermann
et al. 2001). Knock-out or downregulation of AQP4 entailed

188 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
reduced water permeability of astrocytes (Nicchia et al.
2003; Solenov et al. 2002). Stimulation-induced increase
and recovery of extracellular K+ concentration was slower
in AQP4–/– mice, probably attributable to an increase of
the extracellular space volume and impaired astrocytic K+
uptake (Binder et al. 2006). On the other hand, astrocytes in
AQP4–/– mice displayed enhanced gap junction coupling,
which obviously counteracted the deficits in K+ buffering
and may explain the relatively weak phenotype of AQP4–/–
mice (Strohschein et al. 2011). In the hippocampus, AQP4
is upregulated during the first 6 postnatal weeks (Hsu et al.
2011), mimicking the alterations seen for Kir4.1 in the same
subregion (Seifert et al. 2009). Maximal expression of AQP4
was observed in the CA1 stratum lacunosum-moleculare and
stratum moleculare of the dentate gyrus (Fig. 16.3). These
layers possess the highest density of microvessels in the hip-
pocampus, and thus identify them as potential sites of K+ and
water regulation. AQP4 expression in the cerebellum follows
A cytes in Cl– redistribution and an increase in efficacy of GABA
50 μm
B such as the excitatory amino acids glutamate and aspartate.
*
80
*
* However, the molecular identity of VRACs is still elusive.
% difference in gray value
70
relative to P9 SP
60
50 *
* *
* *
40 * *
30
20 *
3w
6w
*
P9
10
0 cells, although there is no work to date on the existence of
SO SP SR SLM
Figure 16.3 AQP4 Expression in the Hippocampus. A. AQP4 immuno-
reactivity from a 6-week-old mouse hippocampus demonstrates laminar
specificity. Although blood vessels are labeled well in all laminae, paren-
chymal AQP4 immunoreactivity is strongest in CA1 stratum lacunosum
moleculare (SLM), dentate gyrus molecular layer (ML), and along the
hippocampal fissure. Scale bar, 50 μm. DGC, dentate granule cell layer;
H, hilus; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radia-
tum. B. Developmental and laminar-specific quantitation of hippocam-
pal AQP4 immunoreactivity. Values are expressed as percent difference
in gray value compared with P9 stratum pyramidale (SP). P9, black bars;
3 week, red bars; 6 week, blue bars. Higher values indicate more intense
immunoreactivity. **P < .01 and ***P < .001 compared with SP at same
time point. With permission from Hsu et al. 2011.
P H YS I O L O GY O F A S T R O C Y T E S : I O N C H A N N E L S A N D I O N T R A N S P O RT E R S
a similar time course and is predominantly localized to glial
membranes facing the pia mater and blood vessels (Wen et al.
1999).
2.5 A N I O N C H A N N E L S
Voltage-gated anion channels of the ClC-family function
as homomeric or heteromeric dimers. Up to now, nine ClC
subunits have been cloned. ClC1–1,2, ClC-Ka, ClC-Kb,
and the β-subunit barttin are localized to the plasma mem-
brane, whereas ClC3–7 together with the β-subunit Ostm1
are expressed in membranes of intracellular organelles (endo-
somes, lysosomes). These proteins function as chloride chan-
nels or Cl–/H+ exchangers. The voltage-sensitive inwardly
rectifying chloride channel ClC-2 is broadly expressed in
glial cells. These channels are activated after hyperpolariza-
tion of the cell membrane or by hypotonic stress. In astrocytes
of the hippocampus, ClC-2 is found in endfeet contacting
small blood vessels, primarily in strata oriens, pyramidale,
and lacunosum-moleculare and in the outer molecular layer
of the DG. The coincidence of ClC-2–positive astrocytes and
GABAergic presynaptic terminals suggests a role for astro-
receptor–mediated inhibition (Sik et al. 2000). Detection
SO of ClC-2–mediated transmembrane currents in astrocytes
SP in situ is difficult because they are masked by the large rest-
ing membrane conductance of these cells (Kimelberg et al.
SR
2006; Makara et al. 2003). CLC-2 has also been localized
to Bergmann glial cells in the cerebellum (Blanz et al. 2007).
SLM
ClC-2–/– mice showed astrocyte activation and neurodegen-
eration in the hippocampus (Cortez et al. 2010).
ML Outwardly rectifying Cl– currents are activated when cells
are exposed to hypotonic solutions. These currents are medi-
DGC ated through volume-regulated anion channels (VRACs),
also termed volume-sensitive outwardly rectifying (VSOR)
H
anion channels. These channels are ubiquitously expressed by
virtually all cells and are permeable to small organic anions,
* Volume-regulated anion channels and amino acid release
* are inhibited by NPPB, DCPIB, tamoxifen, and phloretin.
A physiological role of VRACs in astrocytes is thought to be
* the regulatory volume decrease (RVD) by extrusion of Cl–,
and osmo-active anions such as taurine, glutamate, and aspar-
tate. However, so far analyses have been performed on cultured
ML DGC Hilus VRACs on astrocytes in acute preparations (Kimelberg et al.
2006). A recent in vivo study found some indirect evidence for
swelling-induced VRAC-mediated release of glutamate and
aspartate from astrocytes in rat neocortex (Haskew-Layton
et al. 2008). Clearly, the experimental data available so far are
insufficient to draw any sound conclusions as to the existence
and physiological role of VRACs in astrocytes.
Ca2+ activated Cl– channels (CAACs) are activated on
increases in [Ca2+]i, display outward rectification and are sen-
sitive to NPPB, Zn2+ and chlorotoxin. Astrocytes acutely iso-
lated from mouse brain have recently been shown to express
functional CAACs encoded by bestrophin-1, a member of
• 189
the bestrophin family. Activation of Gq/11-protein–coupled
receptors by thrombin, ATP, and bradykinin led to increases in
[Ca2+]i and evoked large membrane currents that were inhib-
ited by the Ca2+ chelator BAPTA or depletion of internal Ca2+
stores by thapsigargin. 5-Nitro-2-(3-phenylpropylamino)-
benzoic acid and niflumic acid blocked the thrombin-activated
currents, confirming their anion selectivity (Park et al. 2009).
Bestrophin-1 was identified on the mRNA and protein level
in hippocampal astrocytes. In the cerebellum, bestrophin-1
channels in Bergmann glial cells mediate release of GABA
and, thereby contribute to tonic inhibition. GABA release was
even observed at resting [Ca2+]i (100 nM), but was profoundly
enhanced on [Ca2+]i elevation (Lee et al. 2010). These data add
to the increasing evidence identifying astrocytes as important
partners of neurons in brain signaling.
2.6 T R A NS I E N T R E C E P TO R P OT E N T I A L
CHANNELS
The transient receptor potential (TRP) channel family is
subdivided into various subfamilies of which the canoni-
cal (TRPC), vanilloid (TRPV), melastatin (TRPM), and
ankyrin (TRPA) TRP subfamilies are found in the nervous
system. Transient receptor potential channels are cationic
channels that have 6 TM regions, similar to voltage-gated
ion channels, but TM4 lacks a voltage sensor. In astrocytes,
Ca2+ release from internal stores activates transmembrane
TRPC channels, to replenish Ca2+ deficits in the endoplasmic
reticulum (Venkatachalam and Montell 2007). This process
is termed capacitive Ca2+ entry (CCE) or store operated Ca2+
entry (SOCE), and probably is mediated by heteromeric
TRPC1, TRPC4, and TRPC5 channels. Glutamate recep-
tor activation or depletion of internal stores lead to [Ca2+]i
increase and Ca2+ oscillations in cultured astrocytes, which are
blocked by La3+ and RT-PCR identified transcripts encoding
TRPC1, 3, 4, and 6 in these cells (Pizzo et al. 2001). In cul-
tured astrocytes, SOCE is confined to microdomains in the
plasma membrane that are located in close proximity to the
endoplasmic reticulum and involve TRPC1, as shown by an
antisense technique (Golovina 2005). TRPC1, TRPC4, and
TRPC5 protein expression has been detected in cultured and
freshly isolated cortical astrocytes, and TRPC1-dependent
[Ca2+]i increase was shown to regulate the release of glutamate
from these cells (Malarkey et al. 2008). Long-term administra-
tion of fluoxetine reduced TRPC1-mediated SOCE in astro-
cytes (Li et al. 2011).
A structural analog of DAG, oleyl-acetyl-glycerol (OAG),
induces Ca2+ oscillations in cultured astrocytes, probably
through TRPC3, but here SOCE seems not to be involved
(Grimaldi et al. 2003). Oleyl-acetyl-glycerol-sensitive TRP
channels have also been observed in freshly isolated astro-
cytes (Beskina et al. 2007). The latter study suggested that the
inflammatory cytokine IL-1β dysregulates astroglial Ca2+ sig-
naling through an upregulation of TRPC6 channels.
The TRPV subfamily consists of six members. In cul-
tured astrocytes, TRPV4 was activated by the specific agonist
4αPDD or hypotonic cell swelling, leading to [Ca2+]i increase
and transmembrane currents (Benfenati et al. 2007). The
authors also reported TRPV4 immunoreactivity in astrocytes
of the superficial layer of the cerebral cortex, particularly in
astrocytic endfeet facing the pial surface and blood vessels.
Together with AQP4, TRPV4 forms a complex that func-
tions as an osmosensor because of the mechanosensitivity of
TRPV4. Actually, astrocytes respond to hypoosmotic swell-
ing with [Ca2+]i elevations and initiate regulatory volume
decrease only if AQP4 is coexpressed (Benfenati et al. 2011).
The relatively high Ca2+ permeability of TRPV4 channels was
suggested to induce cell death through generation of reactive
oxygen species (Bai and Lipski 2010). The TRPV1 channels
seem also to be expressed by astrocytes (Toth et al. 2005) and
might be activated on acidification (Huang et al. 2010).
3 I O N T R A N S P O RT E R S
3.1 Na + -K + -AT Pase
The maintenance of ion gradients across plasma membranes
is a prerequisite for the establishment of cellular membrane
potentials, electrical signaling, and metabolite transport
driven by ion gradients (Fig. 16.4). Here, the Na+/K+-ATPase,
which exchanges intracellular Na+ for K+ with a stoichiometry
of 3:2, is of fundamental importance. A wealth of secondary
and tertiary active transporters uses the inwardly directed Na+
gradient to generate gradients for other ions such as K+, Mg2+,
H+, bicarbonate and, at least partly, Ca2+ and Cl–, and are thus
dependent on proper function of the Na+/K+-ATPase. Na+/
K+-ATPases are ubiquitous and are composed of α, β, and γ
subunits. In the brain, the α1 subunit is distributed in all cells,
whereas the ouabain-affine isoforms α2 and α3 subunits have
[Na+]e = 158 mM
[K+]e = 2.5 mM
[Cl–]e = 125 mM
pHe = 7.2 is [H+]e = 63 nM
(blood: pH = 7.4 or 40 nM [H+]e )
[HCO3–]e = 17 mM (blood: 25 mM]
CO2 = 1.2 mM (5%)
Extracellular
[K+]i = 120 mM
[Na+]i = 8 – 20 mM
[Cl–]i = 20 – 40 mM
pHi = 7.2 is [H+]i = 63 nM
[HCO3–]i CO2 (5%) = 17 mM
CO2 = 1.2 mM (5 %)
Intracellular
Figure 16.4 Presumed Values of Intracellular Na+, K+, pH (H+), HCO3–,
and CO2 During Hypothetical Steady State Conditions in Astrocytes and
the Extracellular Space.

190 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
been found in astrocytes and neurons, respectively ( Juhaszova
and Blaustein 1997). The α subunit is required for the cata-
lytic and transport properties of the enzyme and contains
binding sites for cations, ATP, and digitalis-like compounds
like ouabain. One of the most prominent sources of [Na+]i
increase in astrocytes is linked to the high-affinity uptake of
glutamate at excitatory synapses. Hence, rapid glutamate clear-
ance is dependent on a large Na+ gradient across the astrocyte
membrane, which is provided by the Na+/K+-ATPase. A well-
documented consequence of glial Na+ elevations is increased
ATP hydrolysis by the Na+/K+-ATPase, followed by increased
aerobic glycolysis, glucose uptake, and glycogen breakdown
(Barros and Deitmer 2010; Magistretti 2006). Thus, astro-
cyte metabolism might be linked to neuronal activity via
the ATP-consuming Na+/K+-ATPase. Spread of intracellu-
lar Na+ from one hippocampal astrocyte to another was dis-
turbed by pharmacological inhibition of gap junctions, and
virtually omitted in Cx30/Cx43 double-deficient mice, and
Cx30/Cx43-mediated Na+ diffusion between astrocytes was
speculated to represent a signal indicating increased metabolic
needs, independent of concomitant Ca2+ (Langer et al. 2012).
Cell volume responses of astrocytes in hypo-osmotic
medium involve the net movement of osmoles, a mechanism
that is energy dependent and tightly coupled to the Na+/K+-
ATPase. It is noteworthy that Kir4.1, previously found to bind
MLC1, is also present in the macromolecular complex of the
Na+/K+-ATPase, thus indicating that the two proteins may
be functionally coupled. Syntrophin and dystrobrevin might
mediate the link between different components of the Na+/
K+-ATPase transmembrane complexes (Brignone et al. 2011).
See sections 2.3 and 2.4 for discussion of the role of Kir4.1 in
the control of K+ homeostasis.
3.2 Na + /H + E XC H A N G E R A N D
Na + /H C O 3 – C OT R A NSP O RT E R
The intracellular, cytosolic pH (pHi) of neurons and glial cells
is actively maintained at a value usually between 7.0 and 7.4,
which is similar to the extracellular pH value (pHo) in nervous
tissue (7.1–7.3). Thus, “resting” pHi and pHo range between 40
and 100 nM free [H+]. This means that there is often only a
small chemical gradient of [H+], if any, across the membranes
of “resting” neurons and astrocytes. The main driving force of
H+ from outside to inside of a cell is the negative membrane
potential. Any conductance of the cell membrane for H+ and/
or HCO3– at negative membrane potentials would result in
a gain of acid or loss of base, and hence lead to intracellular
acidification. Therefore, in living cells, pHi regulation requires
extrusion of acid or uptake of base. The link between CO2
production and pH is the CO2/HCO3– buffer system, which
plays a major role for the maintenance of both pHi and pHo. To
understand the mechanisms underlying acid and alkaline tran-
sients, it is often necessary to monitor pH in neurons, astrocytes,
and the extracellular spaces under the same conditions. Net
acid flux across astroglial and neuronal membranes may occur
in opposite directions, in different cells, however. Thus, during
neural activity, cytosolic alkalization of astrocytes, cytosolic
acidification of neurons, and a transient interstitial alkalization
P H YS I O L O GY O F A S T R O C Y T E S : I O N C H A N N E L S A N D I O N T R A N S P O RT E R S
may occur simultaneously (Rose and Deitmer 1995a,b). Glial
cells have been ascribed a special role for acid-base regulation in
the brain, in particular of the extracellular spaces.
Major pH regulating systems in astrocytes and other glial
cells are the Na+/H+ exchanger (NHE) and the Na+-HCO3–
co-transporter (NBC). The NHE extrudes H+ out of the cell
at the expense of Na+ influx, is electroneutral and operates
similarly as described for neurons and many other cell types.
The NBC has been described in several types of macroglia,
including astrocytes, oligodendrocytes, and retinal Müller cells
(Deitmer and Chesler 2009), but may also be present in neu-
rons (Svichar et al. 2011). The NBC in astrocytes is electrogenic
and cotransports one Na+ with two HCO3– (Deitmer 2007).
The reversal potential of NBCs lies near the resting potential,
and can be adjusted to the actual membrane potential by driv-
ing NBC in the inward or outward direction. Activation of the
NBC in the inward direction results in intracellular alkaliniza-
tion, rise in [Na+]i, and outward currents leading to a hyperpo-
larization of the membrane, whereas activation of the NBC in
the outward direction acidifies the cytosol, drives Na+ out of
the cell, and depolarizes the cells. In addition, when HCO3– is
carried outwardly, buffering capacity is added to the extracel-
lular spaces at the expense of the cell interior. In Müller cells,
the NBC is localized in endfoot processes and has a stoichiom-
etry of 3 HCO3–: 1 Na+, suggesting a role in the transport of
CO2/HCO3– from the retina to the vitreous humor (Newman,
1996). The direction in which the NBC operates across the
plasma membrane depends on pHi and pHo (and [HCO3–]i/o),
[Na+]i, and the cell membrane potential. Depolarizations and
hyperpolarizations are expected to result in an intraglial alka-
linization and acidification, respectively. The cotransporter
appears to have a remarkably high affinity for HCO3–, because
it can be active even in the nominal absence of CO2/HCO3–,
when [HCO3–]o is less than 0.3 mM, attributable to air-CO2
(Brookes and Turner 1994). Expressed in frog oocytes, the
NBC significantly contributes to the apparent cytosolic buffer
capacity, which becomes voltage dependent and may enhance
the efficacy of other acid-base transport systems (Becker and
Deitmer 2004). Carbonic anhydrase activity renders activ-
ity of NBCs significantly more efficient by forming a “trans-
port metabolon” (Becker and Deitmer 2007; Schueler et al.
2011). In situ hybridization revealed NBC mRNA expression
throughout the CNS, with particularly high levels in the olfac-
tory bulb, dentate gyrus, and cerebellum.
The anion Cl–/HCO3– exchanger (AE), which is expressed
in most cells, has not yet been studied in astrocytes, but it is
likely that astrocytes are also equipped with this membrane
carrier (Fig. 16.5). This carrier exchanges intracellular HCO3–
with extracellular Cl–, and is required by most cells to recover
from intracellular alkalosis. We have observed recovery from
alkalosis in cultured cortical astrocytes, which is affected by
the presence of extracellular HCO3– (Theparambil, Alt, and
Deitmer, unpublished observations). A detailed study of this
process in astrocytes, however, is still lacking.
Rapid alkalosis has been observed in astrocytes of rat cortex
following adjacent neuronal activity (Chesler and Kraig 1987).
Numerous studies have suggested that this response results
from activation of an electrogenic Na+-HCO3– cotransporter
• 191
 K+
Na+ Na-K-P Na+
ATP
ADP
NHE
NCX Na+
Ca2+ H+
Cl–
CA
KCC
K+
HCO3– AE
K+ Na+
NKCC
Cl–
NBC
Ca2+
ADP
ATP
Na+
Ca-P
Figure 16.5 Some of the Major Ion-Transporting Membrane Carriers of
Astrocytes Discussed in This Chapter. The Na+-K+ pump (Na-K-P) and the
plasmalemmal Ca2+ pump (Ca-P) require energy from ATP degradation.
Because of their stoichiometry, the Na-K-P, Ca-P, NCX, and NBC are
electrogenic, that is, they depolarize or hyperpolarize the cell membrane
when activated. See the text for further details.
caused by glial depolarization, presumably attributable to an
increase in [K+]o, as was first described in giant glial cells of
the leech (Rose and Deitmer 1994). Glutamate can induce
intracellular acidosis via the glutamate uptake carrier, which is
not only driven by the inward gradient of Na+, but also takes
up one H+ per cycle. Glial pHi decreases of several tenths of
pH units have been reported during exposure to glutamate or
D-aspartate. As astrocytes convert glutamate to glutamine, a
reaction catalyzed by the pH-sensitive glutamine synthetase,
this may in turn stimulate glutamine export from astrocytes
using neutral amino acid transporters, which are coupled to
H+, such as SNAT3 and SNAT5. Hence, glutamate uptake
and glutamine export by astrocytes, as described by the gluta-
mate/glutamine cycle hypothesis, can cause intraglial acidosis,
and can modulate each other by concomitant pHi changes.
3.3 AC I D -BA S E – C O U P L E D M ETA B O L I T E
T R A N S P O RT E R S
H+ and HCO3– are coupled to a variety of metabolite carrier
systems in glial cells. These are either inorganic ion transport-
ers, such as NHE, anion exchanger (AE) or NBC, which
are related to pH regulation (see the preceding), or might
be employed to drive acid-base–coupled metabolite trans-
porters, such as the monocarboxylate transporter (MCT) or
some amino acid and peptide transporters. In addition, cou-
pling of H+, OH–, and/or HCO3– renders pH a regulator of
these membrane transporters (Becker and Deitmer 2004).
Although the Na+ gradient plays the dominant role for most
carrier systems, some transporters are driven by, or coupled to,
the H+/HCO3– gradient.
192 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
Monocarboxylate transporters belong to a family of trans-
porters including multiple isoforms, which carry mono-
carboxylate anions, such as lactate, pyruvate, and ketone
bodies, in cotransport with an H+ in an electroneutral manner.
Monocarboxylate transporter isoforms 1 and 4, as found in astro-
cytes, can mediate both uptake and release of lactate or other
monocarboxylates. According to the lactate shuttle hypothesis,
monocarboxylic acids are released by astrocytes via MCT1 and
4, and taken up by neurons via neuronal MCT2, converted to
CO2 pyruvate, and consumed to generate ATP (Magistretti 2006)
(see chapters 27 and 36). In synaptic domains, in which energy
Cl– requirements are high, astroglial lactate may substitute or com-
plement the supply of glucose to synapses (Barros and Deitmer
2010). Possible functional relationships between some of the
Na+ and H+/HCO3––coupled carriers, as discussed, with ion-
coupled metabolic substrate carriers are outlined in Figure 16.6.
In this scheme, both NBC and MCTs interact with intracellu-
lar and extracellular carbonic anhydrases (CA), and support the
flow of lactate from astrocytes to neurons, as suggested by the
astrocyte-to-neuron lactate shuttle hypothesis.
The neutral amino acid transporter SNAT3, which
exchanges Na+ and H+ and the amino acid substrate cotrans-
ported with Na+, has been shown to mediate glutamine trans-
port in astrocytes (Deitmer et al. 2003), and may be involved
in the glutamate-glutamine cycle between neurons and astro-
cytes. During extrusion of glutamine via SNAT3, Na+ would
be carried out of the cell, which would require either a large
H+ gradient into, or a large glutamine gradient out of the cell.
Astrocyte uptake of peptides (glycylsarcosine) is mediated
by the high-affinity H+-peptide transporter PEPT2 (Wada et
al. 2005). Because PEPT2 seemed to cooperate with NHE,
peptide transport was not only dependent on the transmem-
brane H+ gradient, but also on [Na+] and modulated by NHE
inhibitors. It remains to be studied, if PEPT2 activity is also
modulated by direct interaction with NHE1 and/or NHE2.
3.4 Ca 2+ -AT Pases A N D Na + /Ca 2+ E XCH A N G E R
Ca2+ signaling is based on the low [Ca2+]i (50–100 nM), and
large Ca2+ concentration gradients between the cytosol and
the extracellular space and the lumen of intracellular organ-
elles, respectively (e.g., endoplasmic reticulum, with [Ca2+]
reaching up to a few mM). Cytosolic Ca2+ transients have been
identified to evoke transmitter release from astrocytes (see
chapters 17, 26, and 38). It is the interplay of the different cel-
lular mechanisms (Ca2+ influx and release, Ca2+ extrusion and
uptake, Ca2+ buffering) that control the [Ca2+li levels, which
is crucial for the generation and shape of Ca2+ signaling in
astrocytes and other cell types. Mechanisms that are involved
in regulating cytosolic Ca2+ contribute to shaping kinetics
and amplitude of Ca2+ transients. Ca2+ binding proteins such
as parvalbumin, calcineurin, and calmodulin, which are pres-
ent in the cytoplasm, may serve to buffer cytosolic [Ca2+], and
may also be involved in Ca2+-dependent signaling. Energy-
consuming mechanisms are involved in Ca2+ clearance from
the cytosol, either by transport out of the cell or sequestering
Ca2+ into organelle stores, such as endoplasmic reticulum (ER),
lysosomes, or mitochondria, in astrocytes as in most other cell
Figure 16.6 Acid-Base–Coupled Transporters as They May Interact Between Astrocytes and Neurons. Intracellular H+ regulation and net CO2 flux
from neurons to astrocytes can drive HCO3– and Na+ via the NBC, and lactate/pyruvate via the monocarboxylate transporter MCT isoforms 1 and
4 out of astrocytes. Neurons could then take up these substrates via MCT2 and extrude H+ primarily via NHE. Note that some of the carriers may
interact with intracellular carbonic anhydrase II (CAII) and/or with extracellular carbonic anhydrase IV (CAIV).

types. Sarcoendoplasmic reticulum Ca2+-ATPases (SERCAs)
pump Ca2+ ions into the endoplasmic reticulum. Plasma
membrane ATP-driven Ca2+ pump (PMCA) and Na+/Ca2+
exchangers (NCX) use ATP or the electrochemical gradient
of Na+ across the plasma membrane, respectively, to extrude
Ca2+ out of the cell. Finally, mitochondria and lysosomes are
dynamic Ca2+ stores that can also sequester and release Ca2+
through the activity of the Ca2+ uniporter and/or channels,
respectively. In cultured astrocytes, most of the Ca2+ influx
was reported to be mediated by Na+/Ca2+ exchange, which
required tyrosine kinase to operate. The NCX is linked to the
Na+ gradient and hence to the activity of the Na+/K+ pump,
the α2 subunit of which might be involved in the regulation
of NCX and hence subcellular Ca2+ domains (Golovina et al.
2003a,b). In experimental ischemia, NCX was also suggested
to act in concert with the Cl–-coupled Na+-K+-cotransporter
(NKCC1) to cause perturbation of mitochondrial Ca2+
homeostasis and dysfunction (Kintner et al. 2007).
3.5 Cl – -DE PE N DE N T Na + - A N D
K + - C OT R A NSP ORT E R S
Cation-Cl– cotransporters (CCC), encoded by the SLC12
gene family, are ubiquitous and involved in the regulation of
[Cl–]i. [Cl–]i has been determined with ion-selective micro-
electrodes in cultured cerebral astrocytes to be between 20
and 40 mM, that is, higher than expected from electrochemi-
cal equilibrium, resulting in a Nernst potential for Cl– of
between –30 to –50 mV, which compared well with the rever-
sal potential of GABA-evoked membrane response of –52 mV
(Kettenmann et al. 1987). Cation-Cl– cotransporter activity
is therefore important for GABAergic and glycinergic inhibi-
tory neurotransmission and for cell volume homeostasis in
the developing and mature nervous system. NKCC, which
cotransports Na+, K+ and Cl– into cells, has been identified in
a large variety of cell types. In the brain, the NKCC isoform
1 (NKCC1) is expressed in neurons, oligodendrocytes, and
astrocytes. NKCC1 was found in astrocytes of the cortex,
cerebellum, hippocampus, and perivascular glia, presumably
mostly in endfeet. In astrocytes, NKCC1, mainly driven by
the Na+ gradient, contributes to K+ uptake, Cl– accumula-
tion, cell swelling, and swelling-induced glutamate release, for
example, when [K+]o is increased (Su et al. 2000; Walz and
Hertz 1984). High-K+ stimulation of NKCC1 was reported
to be Ca2+-dependent, and may affect the intracellular level
of other ions, which are dependent on the Na+ gradient, such
as Ca2+ and H+ (see the preceding). Expression of NKCC1 is
upregulated in cultured astrocytes with dibutyryl cAMP (Su
et al. 2000), and NKCC1 activity was found to be stimulated
following phosphorylation during oxygen-glucose deprivation
(OGD) (Lenart et al. 2004). Inhibition or genetic ablation of
NKCC1 reduced OGD-mediated Na+ load, Cl– influx, and
cell swelling. It has been suggested that NKCC1 may con-
tribute to cerebral ischemic injury, as for example, causing cell
swelling and glutamate release, whereas inhibition of NKCC1
was found to be neuron-protective under these conditions.
Activation of NKCC1 is an important player in the process
of astrocyte swelling during brain edema under various neu-
rological conditions, and its contribution to cell swelling may
assist in the development of new therapeutic strategies for the
treatment of brain edema after ischemia, trauma, and acute
liver failure (for review, see Jayakumar and Norenberg 2010).
The K+-Cl– cotransporter (KCC), encoded by four genes
(KCC1–4), is electroneutral and, in contrast to NKCC, medi-
ates Cl– efflux from cells. In the brain, several KCC isoforms
have been reported, including the neuron-specific KCC2. In
astrocytes and C6 glioma cells, KCl uptake has been studied,
but with variable Na+ dependence (Gagnon et al. 2007; Walz
and Hinks 1985). Thus, the differential contribution of Na+-
dependent NKKC1 and Na+-independent KCC for cell swell-
ing and Cl– regulation has been poorly studied in astrocytes,
and its physiological impact has still to be defined.

P H YS I O L O GY O F A S T R O C Y T E S : I O N C H A N N E L S A N D I O N T R A N S P O RT E R S • 193
 4 S U M M A RY A N D P E R S P E C T I VE S
Astrocytes are equipped with a large number of plasma
membrane ion channels and ion carriers. Research in recent
years has concentrated on their functional aspects, and their
regulation and modulation by intracellular and extracel-
lular messengers. Although K+ channels are subject to, and
important causes of, membrane potential changes, carriers are
often related to the Na+ gradient across the cell membrane.
Although the chemical gradient of K+ and Na+ are both large
and opposite, the electrochemical gradient of K+ is usually
maintained small by maintaining a membrane potential close
to the K+ equilibrium potential, primarily attributable to a
relatively large conductance for K+. The large Ca2+ gradients
between the cytosol and the extracellular space and intracel-
lular Ca2+-storing organelles, respectively, are widely used for
signaling, including the modulation of some K+ channels. In
the future, an important strategy will be to study functional
relations between ion channels or transmembrane carriers
in astrocytes and information processing, development, and
metabolism in the brain.
AC K N OW L E D G M E N T S
The work of the authors is supported by Deutsche Forschungs-
gemeinschaft (grants SFB/TR3 C1, C9, STE 552, DE 231)
and European Commission (FP7–202167 NeuroGLIA). The
authors thank I. Nauroth for comments on the manuscript,
and apologize to all those whose work could not be discussed
because of space constraints.
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 17.
RELEASE OF GLIOTRANSMITTER S AND TRANSMITTER
RECEPTOR S IN ASTROCY TES
Helmut Kettenmann and Robert Zorec

A B B R E VI AT I O N S was considered an exclusive property of neurons. This view was

challenged by a series of studies on cell culture demonstrating
Ach acetylcholine that astrocytes responded to the neurotransmitters glutamate
AMPA D-amino-3-hydroxy-5-methyl-4-isoxazole- and γ-aminobutyric acid (GABA) with membrane depolariza-
propionic acid tion. Although astrocytes remove neurotransmitters from the
ANP atrial natriuretic peptide extracellular medium, they are also able to release neuroactive
ANP.emd emerald green fluorescent protein ANP.emd substances such as glutamate. This led to the concept that the
ATP adenosine triphosphate synapse consists of three elements: the presynaptic and post-
BDNF brain-derived neurotrophic factor synaptic neuronal compartments and the astrocyte compart-
cAMP cyclic adenosine monophosphate ment, first called the synaptic triad (Kettenmann et al. 1996)
cGMP cyclic guanosine monophosphate and subsequently the tripartite synapse (Araque et al. 1999).
c-NP C-type natriuretic peptide
CNS central nervous system
DMCM methyl-6,7-dimethoxy-4-ethylbeta- 2 M O D E S O F G L I OT R A N S M I T T E R
carboline-3-carboxylate RELEASE
eNOS endothelial nitric oxide synthase
GABA γ-aminobutyric acid Several mechanisms of gliotransmitter release appear to coexist
GAD glutamic acid decarboxylase in a single astrocyte (Fig. 17.1A) (Hamilton and Attwell 2010).
GDNF glial-derived neurotrophic factor In addition to the vesicle-based mechanisms, non–vesicle-based
GFAP glial fibrillary acidic protein release is mediated through channels, including volume-regulated
5HT 5-hydroxytryptamine anion channels, connexon/pannexon hemichannels; ionotropic
IL-6 interleukin 6 purinergic receptors; and reversal uptake of membrane transport-
mACh muscarinic ACh ers (Parpura and Zorec 2010). Although all of these mechanisms
MAP mitogen-activated protein require a concentration gradient along which chemical messen-
mGluR metabotropic glutamate receptor gers are transported to their targets, the vesicle-based mechanism,
NMDA N-methyl-d-aspartate currently actively debated (Smith 2010), is advantageous for cells.
NP natriuretic peptide First, relatively small structures (e.g., secretory vesicle), in
NPR natriuretic peptide receptor which chemical messengers can be stored, are more economi-
PCR polymerase chain reaction cal. Transport of molecules across the membrane against a
SNARE soluble N-ethyl maleimide-sensitive fusion concentration gradient, such as l-glutamate entering a vesi-
protein attachment protein receptor cle, consumes energy in the form of adenosine triphosphate
TCA tricarboxylic acid (ATP). It was estimated that the budget for recycling 4,000
TNF tumor necrosis factor glutamate molecules into a vesicle consists of 11,000 ATP mol-
TRH thyrotropin-releasing hormone ecules (Attwell and Laughlin 2001). In contrast, the budget
UV ultraviolet for concentrating glutamate into the whole cytosol must be
VAMP vesicle-associated membrane protein much higher, because the volume of a vesicle (a sphere 50 nm
VGLUT vesicular glutamate transporter in diameter) is at least nine orders of magnitude smaller than
VIAAT vesicular inhibitory amino acid transporter the volume of a cell (a sphere 15 μm in diameter).
VIP vasoactive intestinal peptide Second, secretory vesicles represent movable compart-
VNUT vesicular nucleotide transporter ments that can be placed in sites within the cell, where a high
concentration gradient of chemical messengers is required for
diffusional delivery to the targets. Vesicles can be considered as
1 INTRODUCTION signal driving modules, which can be strategically positioned
within the cell. Such regulation cannot be attained with the
Until about 30 years ago, the release of transmitters and the nonvesicle-based modes of messenger release unless these are
expression of neurotransmitter receptors in the nervous system integrated into the plasma membrane by targeted exocytosis.
197
 A may be caused by the slow delivery of vesicles to the plasma
Ca2+-dependent
channel/vesicle
Glutamate
transporters
3 Na+ + H+
ca2+
K+
Cystine
Glutamate
Cell swelling
Volume-sensitive
organic anion channel
B
hemifusion fluctuations of full fusion
a narrow fusion pore
A B C
Figure 17.1 A. Mechanisms of glutamate release from astrocytes.
Glutamate may be released in a calcium-dependent manner in response
to receptor-mediated increases in cytosolic calcium. Some studies indi-
cate that glutamate is released by exocytosis, by channel-mediated efflux
of cytosolic glutamate, through volume-sensitive channels, by a reversal
of membrane glutamate transport mainly under pathological conditions.
B. Stages vesicles undergo to accomplish the regulated release of chemical
messengers by regulated exocytosis. This process is thought to begin with
the vesicle delivery to the plasma membrane, followed by the formation
of a hemi-fusion stalk (transition A), an intermediate structure con-
necting the outer leaflets of fusing membranes. Hemi-fusion stalk then
proceeds into a fusion pore (transition B), an aqueous channel connect-
ing a spherical vesicle and the plasma membrane, through which cargo
molecules diffuse from the vesicle lumen into the cell exterior. Fusion
pore can reversibly vary its diameter (transitions C). During the inter-
mediate state (C), which is energetically stable, the fusion pore diameter
may be too narrow to permeate luminal cargo molecules and therefore
this state could be considered release incompetent. Following the delivery
of a stimulus, vesicles in state C may transit into a state with a wider
fusion pore diameter, leading into a state of full fusion. (A) Modified
from Nedergaard et al. 2002; (B) modified from Jorgačevski et al. 2010.
3 R E GU L AT E D E XO C Y TO S I S I N
A S T R O C Y T E S I S S L OW E R T H A N
IN NEURONS
The properties of vesicle-based release mechanisms have been
studied at the cellular level by monitoring changes in the
plasma membrane area; merging the vesicle membrane with
the plasma membrane affects the plasma membrane area,
which can be monitored by measuring membrane capacitance
(Cm), a parameter linearly related to the membrane area (Kreft
et al. 2004) (Fig. 17.2). In cultured astrocytes, increase in cyto-
solic calcium ([Ca2+]i) by photolysis of caged calcium elicited
an increase in Cm. Half-maximal response in Cm increase exhib-
ited a similar Ca2+-sensitivity to that in neurons. However,
the kinetics of this response in astrocytes was at least two
orders of magnitude slower compared with the rate of regu-
lated exocytosis in neurons (see Fig. 17.2A) (Kreft et al. 2003,
2004). The relatively slow responsiveness of glial exocytosis
198 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
membrane and/or distinct (slow) vesicle–plasma membrane
fusion mechanisms (see stages of exocytosis, Fig. 17.1B). The
Ca2+-dependent increase in Cm was sensitive to tetanus toxin,
indicating a SNARE-based vesicular mechanism; however, it
is unclear which vesicles contribute to the Cm increases.
As for neurotransmitters, the criteria to define a chemi-
cal as a gliotransmitter have been reviewed (Parpura and
Verkhratsky 2011; Parpura and Zorec 2010) and include:
(1) synthesis by and/or (vesicular) storage in glia; (2) regu-
lated release triggered by physiological and/or pathological
stimuli; (3) activation of paracrine or autocrine responses; and
(4) a role in (patho)physiological processes.
4 G L I OT R A N S M I T T E R S
4.1 G LU TA M AT E A S G L I OT R A NS M IT T E R
In astrocytes, glutamate is synthesized de novo as a byprod-
uct of the tricarboxylic acid (TCA) cycle, involving the
astrocyte-specific enzyme pyruvate carboxylase. Glutamate is
converted from the TCA cycle intermediate, α-ketoglutarate,
usually via transamination of another amino acid, aspartate.
All three known isoforms of vesicular glutamate transporters
(VGLUTs) 1, 2, and 3, which use the proton gradient created
by vacuolar (V)-ATPases to package glutamate into vesicles,
have been detected in astrocytes and stimuli that increase
astrocytic [Ca2+]i all evoked release of glutamate (Parpura and
Verkhratsky 2011; Parpura and Zorec 2010).
Vesicle-based Ca2+-dependent release of transmitters
requires the presence of an exocytotic secretory machinery. As
neurons, astrocytes express the proteins of the soluble N-ethyl
maleimide-sensitive fusion protein attachment protein recep-
tor (SNARE) complex: synaptobrevin 2 (Sb2), also referred to
as vesicle-associated membrane protein 2 (VAMP 2); syntaxin
1, synaptosome-associated protein of 23 kDa (SNAP-23), as
well as several ancillary proteins to this complex, includ-
ing synaptotagmin 4 (Montana et al. 2006; Parpura and
Verkhratsky 2011). The use of clostridial, tetanus, and various
types of botulinum toxins, which cleave exocytotic SNARE
proteins, caused a reduction of Ca2+-dependent glutamate
release in astrocytes (Montana et al. 2006; Parpura and Zorec
2010). Similarly, the expression of mutated synaptotagmin 4,
acting in a dominant-negative manner, caused the reduction
of Ca2+-dependent glutamate release from astrocytes (Zhang
et al. 2004a). Often the question is asked whether glutamate
release from astrocytes occurs in vivo under normal conditions.
To carry out experiments with the resolution obtained in cul-
tured cells also at the level of tissue slices or even in the whole
brain would be difficult, if not impossible, because glutamate
and ATP are rather common metabolites and identifying the
cellular source and mechanism of release with subcellular reso-
lution would be technically most challenging.
Astrocytic secretory vesicles are essential morphological
elements for regulated Ca2+-dependent exocytosis, and range
from 30 to more than 1,000 nm in diameter; they exhibit clear
and dense cores (Parpura et al. 2010).
 A B

–kexot
C = A(1–e )

10 μM
[Ca2+]

1 pF
1s 500 fF
Cm

k ~ 2 s–1
G 5 nS
kf ~ 400 s–1

1s
C= A1(1–e–k1exot )+A2(1–e–k2exot )

Figure 17.2 Comparison of time-dependent changes in membrane capacitance (Cm) recorded in a neuronal cell (A, photoreceptor) and an astrocyte (B).
A. Two types of Ca2+-induced increases in Cm in a photoreceptor have been recorded. Top trace was best fitted by a single exponential function (dot-
ted line). The bottom trace was best fitted to a sum of two exponential functions as shown by the equation below the horizontal line. The fastest rate
constant (kf ) was around 400 s–1. B. Top trace shows time-dependent changes in cytosolic calcium activity [Ca2+]i, elicited by ultraviolet (UV) light
flash photolysis of caged Ca2+ compound dialyzed into the cytosol of the cell (Kreft et al. 2004). UV flash was applied at the time indicated by the
arrow. Note that the rapid increase in [Ca2+]i following the UV flash application induced an exponential increase in Cm with a rate constant (k) of 2 s–1.
G denotes the real part of the admittance trace. (A) Modified from Kreft et al. 2003; (B) modified from Kreft et al. 2004.

In addition to the vesicle-based mechanisms, nonvesicle
mechanisms of glutamate release are also present in astro-
cytes (Hamilton and Attwell 2010). Functional hemichannels
were confirmed by passage of extracellular Lucifer Yellow into
astrocytes in divalent cation-free solution and the ability to
block this passage with gap junction blocking agents (Ye et al.
2003). Orellana et al. (2011) studied the release of ATP and
glutamate from connexion 43 hemichannels, which mediate
neuronal death under inflammatory conditions.
4.2 G A BA A S G L I OT R A N S M I T T E R
Recent studies indicate that astrocytes can create tonic inhi-
bition by the release of GABA. Astrocytes can accumulate,
synthesize and release GABA (Angulo et al. 2008; Vélez-Fort
et al. 2011). Astrocytes have the capacity to accumulate GABA
as shown with anti-GABA antibodies in the brain stem or cere-
bellum. Astrocytes express transporters for GABA as reviewed
in chapter 35. GABA can be produced by two distinct path-
ways: by conversion of glutamate to GABA by the enzyme glu-
tamic acid decarboxylase (GAD), or by the monoacetylation
of putrescine. Glutamic acid decarboxylase expression in glial
cells, however, is significantly lower than in neurons.
Gallo et al. (1986) demonstrated that GABA can be
released from astrocytes. The release could be triggered by acti-
vating glutamate receptors, and they used the release of GABA
from the astrocytes as a tool to characterize the pharmacology
of the glutamate receptors. In Bergmann glia, an electro-
genic GABA efflux has been reported, activating neighbor-
ing GABA receptors. This release was considered because of
reversed GABA transport via GAT-1, leading to a tonic eleva-
tion of GABA in the extracellular space and thereby to tonic
inhibition (Barakat and Bordey 2002). A similar observation
was made in the olfactory bulb, in which spontaneous slow
synaptic currents were observed in mitral cells. These currents
were mediated by GABA and depended on astrocyte activity
because they disappeared when astrocytes were silenced. The
GABA release from astrocytes in the olfactory bulb serves to
synchronize neuronal activity. These slow GABA-mediated
transient currents were also observed in hippocampal pyrami-
dal neurons. Astrocytes can also control the frequency of min-
iature inhibitory postsynaptic currents in pyramidal neurons.
Stimulation of astrocytes led to an increase in these currents,
and potentially this could be mediated by GABA release from
astrocytes (Kang et al. 1998). In the barrel cortex, astrocyte
activity resulted in a tonic- and stimulation-induced inhibi-
tion of pyramidal cells and spiny stellate cells. Silencing astro-
cytes by dialysis with BAPTA increased the excitability of the
neurons (Benedetti et al. 2011).
A novel mechanism for GABA release has recently been
identified in the cerebellum. Astrocytes chronically release
GABA through an anion channel, bestrophin-1, and thereby
mediate tonic inhibition. Bestrophine-1 can be modulated
by changes in intracellular Ca2+ and cell volume, but is even

R E L E A S E O F G L I OT R A N S M I T T E R S A N D T R A N S M I T T E R R E C E P TO R S I N A S T R O C Y T E S • 199
tonically active at resting Ca2+ levels. The permeability of
GABA through the channel is considerable, about four
times that of Cl–. The importance for bestrophine to main-
tain tonic inhibition was demonstrated by downregulating
bestrophin-1 by an shRNA approach. This defect could be
rescued by selectively expressing bestrophin in (glial fibrillary
acidic protein [GFAP]-positive) astrocytes (Lee et al. 2010).
Although a significant body of data shows that GABA
uses non–vesicle-based modes of release, there is evidence that
the vesicular inhibitory amino acid transporter (VIAAT) is
expressed in astrocytes (Echigo and Moriyama 2004), indicat-
ing that GABA may be stored in vesicles. However, unequivo-
cal evidence to support regulated exocytosis of GABA from
astrocytes is yet to be provided.
4 .3 d -S E R I N E A S G L I OT R A N S M I T T E R
Astrocytes can also release d-serine (Schell et al. 1995),
a ligand to the glycine modulatory binding site of the
N-methyl-d-aspartate (NMDA) receptor. d-Serine is con-
verted from l-serine by the action of serine racemase, an
enzyme found in astrocytes (Rosenberg et al. 2010; Wolosker
et al. 1999). Astrocytes are thought to be the key metabolic
provider of l-serine, which is shuttled to neurons, likely via
membrane transporters, for d-serine production in neurons
(Wolosker 2011). In addition to the non–vesicle-based mecha-
nisms of release, astrocytes release d-serine by regulated exo-
cytosis in an activity-dependent manner (Henneberger et al.
2010; Oliet and Mothet 2006).
Mothet et al. (2005) investigated the mechanism of d-ser-
ine release using an enzyme-linked assay to measure extracel-
lular d-serine concentration and established that astrocytes
release d-serine in a Ca2+-dependent manner; extracellular Ca2+
was required; it was reduced by concanamycin A, a V-ATPase
inhibitor, and tetanus toxin, implicating the involvement of
a vesicular mechanism. How d-serine is loaded into vesicles
remains an open question.
4.4 P E P T I D E S A S G L I OT R A N S M I T T E R
Unlike amino acids, which can get loaded into vesicles via
membrane transporters, peptides enter vesicles via the syn-
thetic secretory pathway. These peptides are typically made
as pro-peptides in the endoplasmic reticulum, transit Golgi
compartments, where they are concentrated and sorted into
organelles; then, they are processed in vesicles to their final
form before release. The classic view holds that vesicles car-
rying peptidergic transmitters seem to have relatively larger
diameters compared with the synaptic-like vesicles, and
exhibit electron-dense cores (dense-core vesicles). A diam-
eter of approximately 100 nm was reported for secretogranin
II–positive dense-core vesicles (Calegari et al. 1999). This
study also reported that astrocytes contain fewer smaller and
less dense secretory granules containing secretogranin II, indi-
cating that peptidergic vesicles in astrocytes are not uniform
in morphological appearance. Neuropetide Y was shown to
be contained in vesicles distinct from synaptic-like vesicles in
astrocytes (Ramamoorthy and Whim 2008).
200 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
Atrial natriuretic peptide (ANP) is expressed in astrocytes
and plays a role in cerebral blood flow regulation (McKenzie
et al. 2001) and as an autocrine regulator, because all natriu-
retic peptide receptors are expressed by astrocytes. To study
the discharge of ANP, Krzan et al. (2003) transfected astro-
cytes with a construct to express pro-ANP fused with
the emerald green fluorescent protein (ANP.emd). The num-
ber of fluorescent ANP.emd puncta was reduced on stimulat-
ing the cells by ionomycin, which strictly depended on the
extracellular Ca2+. Vesicles containing ANP also appear to
contain ATP (Pangrsic et al. 2007), which is consistent with
the report that ATP is stored in secretorgranin II–containing
vesicles (Coco et al. 2003). ATP and peptides could be
differentially released from a single vesicle by regulating
fusion-pore diameter (Stenovec et al. 2004). The mobil-
ity of recycling ANP vesicles was one order of magnitude
slower than the ANP vesicles trafficking to the plasma mem-
brane sites (Potokar et al. 2005, 2007) and was reduced on
stimulation of cells (Potokar et al. 2008, 2010), differing from
the stimulation-increased mobility of anti-VGLUT1 anti-
body capturing vesicles in astrocytes (Stenovec et al. 2007).
This indicates that vesicles with different cargo may traffic by
distinct mechanisms.
Recycling vesicles may not only carry lumenal cargo but
also membrane-associated signaling molecules which partici-
pate in contact cell–cell interactions (Kopan and Ilagan 2009;
Soos et al. 1998) or in determining the density of plasma
membrane transporters (Robinson 2002), such as the gluta-
mate transporter EAAT2 (Stenovec et al. 2008).
Recycling vesicles may serve bidirectional communica-
tion between neurons and glia. When studying the activity-
dependent secretion of brain-derived neurotrophic factor
(BDNF) and its extracellular availability, Bergami et al. (2008)
provided evidence that BDNF, which is synthesized de novo
in neurons, is secreted in its pro-form into the extracellular
medium, and is taken up via endocytosis into astrocytes, where
it is processed and released by a SNARE-dependent secretory
pathway.
4.5 AT P A S G L I OT R A NS M IT T E R
Intracellular ATP is produced via glycolysis and oxidative
phosphorylation to reach more than 10 mM in the cytosol,
establishing a concentration gradient favoring the exit of ATP
from cells. Once released into the extracellular space, ATP can
be used in intercellular signaling, acting directly on purinergic
receptors. Alternatively, on hydrolysis by membrane-bound
ecto-nucleotidases, the extracellular degradation products
ADP and adenosine can activate different plasma membrane
receptors (Fields and Burnstock 2006).
ATP can be released via non-vesicle and vesicle-based
mechanisms (Parpura and Zorec 2010). Several lines of evi-
dence support the latter mechanism. ATP seems to be present
in peptide-containing vesicles and in lysosomes (Hamilton
and Attwell 2010; Parpura and Zorec 2010). ATP is loaded
into vesicles by SLC17A9 (Sawada et al. 2008), a vesicular
nucleotide transporter (VNUT) that appears to be present in
astrocytes (Larsson et al. 2011).
 To study ATP release from astrocytes, Pangrsic et al.
(2007) used glutamate stimulation of astrocytes and showed
quantal release of ATP as recorded by ATP reporter cells.
The exocytotic release of ATP stored in astrocytic lysosomes
could be detected only with unphysiologically long stimula-
tion (Zhang et al. 2007), exhibiting different sensitivity com-
pared with other types of vesicles (Liu et al. 2011), indicating
that distinct exocytotic mechanisms control release from vesi-
cle subtypes. Consistent with this, only lysosomes carrying the
VAMP7/TI-VAMP seem to be fusion competent (Verderio
et al. 2011). Distinct vesicle fusion mechanisms are consistent
with distinct mobility properties of astrocytic vesicles, which
are in part determined by the intermediate filament cytoskel-
eton (Potokar et al. 2011).
5 T R A N S M I T T E R R E C E P TO R S
5.1 I O N OT RO P I C G LU TA M AT E R E C E P TO R S
About 28 years ago, two groups provided the first evidence that
not only neurons but also astrocytes in cell culture express func-
tional glutamate receptors (Fig. 17.3) (Bowman and Kimelberg
1984; Kettenmann et al. 1984). Cultured astrocytes express
2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid
(AMPA) (assembled from subunits GluR1–GluR4) and kain-
ate receptors (GluR5–GluR7, KA1, KA2), but do not respond
to NMDA. Functional AMPA receptors have been described
in astrocytes in situ in various brain regions, including cortex,
spinal cord, brainstem, cerebellum, optic nerve, nucleus ruber,
and retina. In the hippocampus, AMPA receptor expression
is restricted to the NG2 cells; the classic astrocyte (with high
GFAP promoter activity) is characterized by high glutamate
transporter activity (Matthias et al. 2003).
Although in most brain regions astrocyte AMPA receptors
display low Ca2+ permeability, Bergmann glial cells in the cere-
bellum lack the GluR2 subunit and hence allow passage of diva-
lent cations (Müller et al. 1992). The physiological relevance
of this subunit combination has been demonstrated by experi-
mentally inducing the expression of GluR2 in Bergmann glial
cells, which results in significant neuronal rearrangement (Iino
et al. 2001). The pharmacological properties and single chan-
nel conductances of the AMPA receptors of the hippocampal
NG2 cells mimic those of their neuronal counterparts. In the
hippocampus, fast application techniques, Ca2+ imaging, and
single cell reverse transcription (RT)–polymerase chain reac-
tion (PCR) revealed that the astroglial receptors possess an
intermediate Ca2+ permeability and are primarily assembled
from the subunits GluR1, GluR2, and GluR4. Molecular and
functional changes in astrocyte receptor expression occur in
epilepsy, and these alterations have been proposed to contrib-
ute to the generation and spread of seizure activity in human
temporal lobe epilepsy (Seifert and Steinhäuser 2011). After
ischemic insult to the adult striatum, a novel astrocyte subtype
appears with distinct properties: Cells with high GFAP pro-
moter activity express physiological properties of NG2 cells,
namely, voltage-gated channels, complex responses to kainate,
and a high rate of gap junction coupling (Wang et al. 2008).

Presynaptic terminal

Astrocyte Astrocyte

Glu Glu
Glu
Gln Gln Glu
Glu
Gln
Gln
ER
ER Glu Ca2+
Ca2+ Glu
Glu
Glu

Postsynaptic neurone

Metabotropic Glutamate av
GluR transporter
AMPA, NMDA Glu Glutamate
ionotropic GluRs Gln Glutamine

Figure 17.3 Glutamate-Mediated Neuronal–Glial Signaling. Synaptically released glutamate activates glial ionotropic (AMPA and NMDA) and
metabotropic receptors. Activation of group I metabotropic receptors initiates phospholipase C–dependent synthesis of InsP3, which in turn triggers
Ca2+ release from the endoplasmic reticulum (ER) Ca2+ store. The majority (~80%) of glutamate released during synaptic transmission is taken up by
astroglial Na+/glutamate transporters; subsequently, glutamate is converted into glutamine, which is transported back to neurons, where it acts as a
main source of newly synthesized glutamate (“glutamate–glutamine shuttle”). From Verkhratsky and Kirchhoff 2007.

R E L E A S E O F G L I OT R A N S M I T T E R S A N D T R A N S M I T T E R R E C E P TO R S I N A S T R O C Y T E S • 201
 Although antibody staining and in situ hybridization
demonstrated widespread distribution of kainate receptors
in brain, clear evidence for functional expression of these
receptors in astrocytes is lacking. Recently it has been estab-
lished that astrocytes in situ can express functional NMDA
receptors in contrast to cultured astrocytes (Fig. 17.4)
(Verkhratsky and Kirchhoff 2007). Responses of astrocytes
to NMDA have been reported for astrocytes in the spinal
cord and cortex and Bergmann glial cells of the cerebel-
lum. Bergmann glial cells express mRNAs for NR2B, and a
physiological study on acute brain slices reported NMDA-
induced membrane currents in Bergmann glial cells. The
responses were best studied in cortical astrocytes in acute
slices or in freshly isolated cells (Lalo et al. 2006; Schipke
et al. 2001). In contrast to neurons, the responses could be
recorded at a physiologic Mg2+ concentration of 1.3 mM.
Only at concentrations of 4 mM and higher did the channels
A (Bezzi et al. 1998). These responses are likely to occur under
EGFP
0.1 nA
50 s
PDC,CNQX
NMDA
B Because group I mGluRs induce glial Ca2+ oscillations and
5F/Fo
CNQX, TTX, Cd2+
NMDA
0.1 nA
30 s
Figure 17.4 NMDA receptors. A. Astrocytes in the cortex respond to
NMDA. Astrocytes were identified in cortical slices using a transgenic
mouse line that expressed the green fluorescent protein under the
control of the GFAP promoter (left). NMDA was applied (100 μM)
to the same cell in the presence of 50 μM PDC and 50 μM CNQX. B.
NMDA triggered larger Ca2+ responses at distal processes. On the left
is a micrograph of an astrocyte filled with Fluo-4. Note the recording
pipette approaching the cell from top. Fluorescence changes (F/Fo) were
analyzed in the areas demarked by the squares and are displayed in the
top three traces. The lower trace is the simultaneously recorded current
response. NMDA (100 μM) and CNQX, TTX, Cd2+ were applied as
indicated by bars. From Schipke et al. 2001.
202 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
exhibit a voltage-dependent block. There is also evidence
that the NG2 cell population in the hippocampus expresses
NMDA receptors (Serrano et al. 2008).
5.2 M ETA B OT RO P I C G LU TA M AT E R EC E P TO R S
The metabotropic glutamate receptor (mGluR) family con-
sists of eight members, which couple to G-proteins. mGluR3
and mGluR5 are the predominant subtypes expressed by
astrocytes and have been described in cultured cells and in situ
(D’Antoni et al. 2008); these subtypes are also found in human
hippocampal astrocytes. mGluR1 has been reported in hip-
pocampal astrocytes and the spinal cord. Activation of these
receptors led to an increase in intracellular Ca2+ and inhibition
of cAMP accumulation, although Gs-coupled cAMP stimula-
tion has also been reported. Stimulation of astroglial mGluRs
leads to intracellular Ca2+ oscillations and Ca2+ wave propaga-
tion within the astrocyte network, activates Ca2+-dependent
K+ channels, and induces prostaglandin-mediated glutamate
release from astrocytes that activates neuronal receptors
physiological conditions because astroglial mGluR activa-
tion and subsequent Ca2+ responses could be evoked by elec-
trical stimulation of fiber tracts, causing neuronal glutamate
release in acute brain slices. Activation of mGluRs induced
other astrocyte responses, including swelling, activation of
phospholipase D and glutamine synthetase, release of arachi-
donic acid, cAMP-dependent block of K+ currents, modula-
tion of proliferation, and regulation of the expression of the
glutamate transporter GLAST. Dramatic changes in mGluR
expression occur under pathophysiological conditions, and in
spinal cord injury, epilepsy, and amyotrophic lateral sclerosis.
Ca2+ wave propagation and influence neuronal excitability
(see the preceding), their upregulation as observed in epilepsy
might indicate an astroglial involvement in seizure generation
or spread (Seifert and Steinhäuser 2011; Seifert et al. 2006).
5.3 G A BA A R EC E P TO R S
Ionotropic GABAA receptors are expressed by astrocytes
both in culture and in situ (Vélez-Fort et al. 2011). As in neu-
rons, astrocytic GABAA receptors form Cl– channels with a
conductance of about 30 pS (Fig. 17.5). In contrast to mature
neurons, GABAA receptor activation leads to a large depo-
larization in astrocytes studied in culture (Kettenmann et al.
1984). The Cl– reversal potential can be as positive as –40
mV, indicating that GABA can lead to a substantial depo-
larization from the normal resting membrane potential of
about –80 mV. In astrocytes of the pituitary gland, postsyn-
aptic potentials were activated by neuronally released GABA
(Mudrick-Donnon et al. 1993). GABAA receptor activation
in astrocytes also triggers a long-lasting blockade of K+ con-
ductances, thereby augmenting depolarization, for example,
in Bergmann glial cells (Müller et al. 1994) and activating
voltage-gated Ca2+ channels with subsequent increase in
cytosolic Ca2+ (in freshly isolated astrocytes from hippocam-
pus) (Fraser et al. 1995).
 A 50 μM GABA B
Astrocyte in culture
Astrocyte
20 μM GABA 20 μM GABA + 10 μM Diazepam
100 pA
1 sec

50 μM GABA 0.2 nA
1 sec

1 pA Bergmann glial cell in situ

1 sec

Vm, mV
70
50 pA
50 15 s

30 GABA GABA
0 Diazepam
–30

–50
0.25 nA
–70 GABA
30 s
1 pA
50 msec GABA
Pentobarbital

Figure 17.5 GABA receptors. A. Single GABAA receptor channel recording from astrocytes. The upper panel shows single channel activity in response
to application of GABA. Below is single channel activity displayed at different membrane potentials ranging from +70 to –70 mV. B. GABA receptor
pharmacology in cultured astrocytes and Bergmann glial cells in situ. The upper trace shows the GABA response from a cultured astrocyte that is
enhanced by coapplication of diazepam. Below is a response from a Bergmann glial cell in an acutely isolated cerebellar brain slice. Note that diazepam
did not significantly change the GABA-induced current. In contrast, pentobarbital significantly increased the GABA response. From Bormann et al.
1988; Müller et al. 1994.

The GABAA receptor has a complex pharmacology. As
in neurons, barbiturates and steroids enhance the astrocytic
GABA response. There is a difference between the responses
in neurons and cultured astrocytes. Although typical benzo-
diazepines such as diazepam enhanced the GABA response
in both neurons and cultured astrocytes, the inverse benzo-
diazepine agonist, DMCM (methyl-6,7-dimethoxy-4-ethyl-
beta-carboline-3-carboxylate), also enhanced the response in
astrocytes indicating a different subunit composition of the
GABAA receptor in astrocytes. In contrast, Bergmann glial
cells studied in cerebellar slices showed GABA responses that
were insensitive to benzodiazepine due to the expression of
δ subunits (see Fig. 17.5) (Müller et al. 1994). In freshly iso-
lated astrocytes from the hippocampus, the benzodiazepine
sensitivity was as described for neurons (Fraser et al. 1995).
This indicates that GABAA receptor subunit composition is
heterogeneous in astrocytes, resulting in different populations
of astrocytes with respect to their GABAA receptor profile. In
Bergmann glial cells, the GABAA receptors were prominently
expressed in immature cells, but were downregulated in the
mature cerebellum. In addition, an uneven distribution was
reported in these cells: Immunolabeling preferentially identi-
fied receptors along the processes. There is evidence from both
cell culture and in situ studies that GABAA receptor activation
promotes differentiation of astrocytes. In culture, GABA trig-
gers formation of processes and leads to astrocytes with a more
complex morphological shape.
5.4 G A BA B R EC E P TO R S
GABAB receptors in astrocytes play an important role for sens-
ing the activity of interneurons. In the hippocampus, interneu-
ronal firing elicits a GABAB-receptor–mediated increase of
calcium in surrounding astrocytes, which in turn potentiates
inhibitory postsynaptic currents in pyramidal neurons (Kang
et al. 1998). It also mediates heterosynaptic depression by acti-
vating GABAB receptors in the astrocytes resulting in release
of ATP (Serrano et al. 2006). In the hippocampus, astrocytic
GABAB receptors are part of a functional circuit involv-
ing inhibitory neurons, astrocytes, and pyramidal neurons.
All three subtypes of GABAB receptors (GABAB1a, GABAB1b,
and GABAB2) are expressed by astrocytes as revealed by

R E L E A S E O F G L I OT R A N S M I T T E R S A N D T R A N S M I T T E R R E C E P TO R S I N A S T R O C Y T E S • 203
immunohistochemistry. At the ultrastructural level, GABAB
receptor subunits were expressed on astrocytic processes sur-
rounding symmetrical and asymmetrical synapses in the CA1
subregion of the hippocampus (Charles et al. 2003). Functional
GABAB receptor expression changes during development.
Only 10% of astrocytes in hippocampal slices responded at P3
or P32–34 to the application of the GABAB receptor agonist
baclofen, but 60% of the astrocytes responded between P11
and P15, indicating that GABAB receptor–mediated calcium
signaling in astrocytes occurs preferentially during postnatal
development when hippocampal networks are established
(Meier et al. 2008).
5.5 G LYC I N E R E C E P TO R S
Functional strychnine-sensitive glycine receptors have been
detected in astrocytes and glial precursor cells (most likely
NG2 cells) in spinal cord (Pastor et al. 1995). A combined
patch clamp and RT-PCR approach revealed the expression
of α1 and β subunits (Kirchhoff et al. 1996). Glycine receptor
expression is not a common property of astrocytes: Neither
cultured astrocytes nor Bergmann glial cells express functional
glycine receptors.
In the supraoptic nucleus, clusters of glycine receptors are
associated with glial fibrillary acidic protein positive astroglial
processes. This finding suggests that the astrocytic glycine
receptors could mediate a paracrine communication between
astrocytes and neurons in this brain region (Deleuze et al.
2005).
5.6 AC ET Y L C H O L I N E R E C E P TO R S
There is evidence for the expression of the α7 subunit of nico-
tinic acetylcholine receptors in astrocytes in culture and in
situ. In cultured astrocytes, α-bungarotoxin–sensitive nico-
tinic acetylcholine receptors containing the α7 subunit led
to Ca2+ influx through the receptor channels and Ca2+-induced
Ca2+ release from caffeine-sensitive stores (Sharma and
Vijayaraghavan 2001). This subunit was also localized to
astrocytes of human cerebellum in situ (Graham et al. 2002).
In the prefrontal cortex, electron microscopic immuno-
labeling of α7 nicotinic acetylcholine receptor revealed
expression on astrocyte processes, which were frequently
located close to terminals colocalized with vesicular acetyl-
choline transporters.
The expression of muscarinic ACh (mACh) receptors by
astrocytes is well established (Porter and McCarthy 1997;
Verkhratsky et al. 1998). There is evidence for the expression
of transcripts for the mACh receptor subtypes, M1 to M5, in
cultured astrocytes. Acetylcholine induces IP3 formation and
release of Ca2+ from cytoplasmic stores, inhibits adenylate
cyclase, and induces an increase in astroglial proliferation.
Neuronal activity can stimulate astrocytic mACh receptors as
shown in the hippocampus (Araque et al. 2002). In the barrel
cortex, muscarinic acetylcholine receptor–dependent synap-
tic plasticity depends on astrocyte Ca2+ signaling. This in vivo
study suggests a role of astrocytes as a gate for cholinergic plas-
ticity in the cortex (Takata et al. 2011). During development,
204 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
astrocytes promote neurite outgrowth mediated by acetylcho-
line-mediated signaling. Exposure of astrocytes to carbachol,
a muscarinic agonist, increased the expression of the extra-
cellular matrix proteins fibronectin and laminin-1 indicating
that muscarinic stimulation of astrocytes induces the release
of permissive factors that accelerate neuronal development
(Guizzetti et al. 2008).
5.7 OX Y TO C I N A N D VA S O P R E S S I N R EC E P TO R S
Vasopressin and oxytocin can trigger Ca2+ responses in cultured
astrocytes, indicating the presence of their receptors (Panatier
2009). Neuronal production and release of TGF-β led to an
increase in astroglial oxytocin receptor mRNA in astrocytes,
indicating that neurons were able to upregulate the astrocyte
receptor (Mittaud et al. 2002). Astrocytes isolated from the
hippocampus predominantly express vasopressin1b receptors,
but astrocytes isolated from the cerebral cortex of neonatal
rats solely expressed vasopressin1a receptors. Activation of
both receptors triggers glutamate release from the astrocytes
(Syed et al. 2007).
5.8 VA S OAC T I VE I N T E S T I NA L
P O LY P E P T I D E R EC E P TO R S
Vasoactive intestinal polypeptide (VIP) receptors are
expressed by astrocytes and activation leads to Ca2+ signaling
(Masmoudi Kouki et al. 2007). Expression of the VIP recep-
tor, VPAC2, increases in astrocytes after injury (Nishimoto
et al. 2011). Vasoactive intestinal polypeptide receptor acti-
vation induces the release of interleukin 6 (IL-6) and neu-
rotrophic factors, and stimulates proliferation. Vasoactive
intestinal polypeptide receptor activation stimulates glutamate
uptake into astrocytes by upregulation of GLT-1 and GLAST
expression (Figiel and Engele 2000), indicating that VIP can
increase the strength of glutamate-mediated neurotransmis-
sion. In addition, VIP receptors may play an important role in
regulating energy metabolism. Vasoactive intestinal polypep-
tide depletes astrocyte glycogen initially, followed by delayed
reaccumulation to a level beyond baseline. These effects are
mediated by regulating a number of related genes such as
glycogen synthase via the transcription factor family C/EBP.
However, there is not yet convincing evidence of the presence
of VIP receptors in astrocytes in situ.
5.9 A D R E N E RG I C R EC E P TO R S
Both α- and β-adrenergic receptors have been described in
astrocytes in culture and in situ (Porter and McCarthy 1997;
Verkhratsky et al. 1998). α1 Receptors affect astrocyte func-
tion by inhibiting gap junction coupling via PLC. This is in
line with the finding that mechanically induced Ca2+ waves in
hippocampal astroglial cells are inhibited after stimulation of
α1 receptors (Muyderman et al. 1998). Moreover, α1b receptor
activation stimulated glutamate uptake in cultured astrocytes.
There is also a hint that metabolic activity may be controlled
by α receptors because receptor activation induced lactate for-
mation and glycogen turnover (see chapters 27 and 36). In the
hippocampus, α2 receptors are located on astroglial processes,
near terminals forming asymmetrical excitatory synapses
(Milner et al. 1998). Neuronal stimulation in acute cerebellar
slices led to the release of noradrenaline from afferent fibers
and activation of α1 receptors in Bergmann glial cells (Kulik
et al. 1999).
Both subtypes of β receptors, β1 and β2, are expressed by
astrocytes in situ; β2 are most prominently expressed under
pathological conditions (Laureys et al. 2010). In the injured
brain, adrenergic receptors are differentially regulated.
Although α1 receptors decrease in areas of neuronal degen-
eration and gliosis, β receptors are upregulated. Blockade of
β receptors suppressed glial scar formation, indicating that
adrenergic receptors are part of the cascade leading to astro-
cyte activation (Griffith and Sutin 1996). This suggestion is
substantiated by the finding that β2 receptors are activated or
upregulated in the transected optic nerve in vivo, confirm-
ing that β-adrenergic signaling is an important feature of the
astrocyte response to injury. Further support comes from the
observation that stimulation of β receptors leads to astroglio-
sis and cell proliferation in the optic nerve in vivo. This effect
might be mediated via the control of growth factor expres-
sion (e.g., FGF1, FGF2, BDNF, CNTF). Stimulation of β1
receptors is also accompanied by enhanced glycogen levels,
and stimulation of β2 and β3 adrenoceptors increases glucose
uptake in astrocytes (Catus et al. 2011) and increases cytoso-
lic glucose concentration in astrocytes (Prebil et al. 2011). The
observation that β adrenergic receptor stimulation leads to a
cAMP-mediated inhibition of astroglial inwardly rectifying
K+ channels (Roy and Sontheimer 1995) points to a role in
the regulation of extracellular K+ homeostasis.
5.10 A N G I OT E NS I N R E C E P TO R S
Expression of angiotensin receptors in astrocytes in situ
seems to be restricted to white matter (Sumners et al. 1994;
Verkhratsky et al. 1998). Antibody staining identified AT1 and
AT2 receptors in astrocytes of white matter tracts in cerebel-
lum and subcortical regions, in the optic nerve and in the cor-
pus callosum; no immunoreactivity was found in gray matter.
The diversity in expression is also reflected when astrocytes
are harvested from different brain areas: AT1 receptors were
found in astrocytes from medulla oblongata and cerebellum,
whereas astrocyte cultures from hypothalamus and cortex
did not express functional receptors. Receptor activation as
studied in culture-stimulated proliferation, increased glucose
uptake and induced prostaglandin release. Moreover, astro-
cytes regulate leukocyte infiltration into the central nervous
system (CNS) mediated by angiotensin II signaling involving
AT1 receptors (Füchtbauer et al. 2011).
5.11 S O M ATO S TAT I N R E C E P TO R S
Prominent expression of somatostatin receptors in astrocytes
of hippocampus, amygdala, and hypothalamus in situ has
been identified in binding studies on brain slices (Porter and
McCarthy 1997). The subtypes have been more stringently
identified in culture, and it is evident that astrocytes express
R E L E A S E O F G L I OT R A N S M I T T E R S A N D T R A N S M I T T E R R E C E P TO R S I N A S T R O C Y T E S
the sst2A splice variant (Viollet et al. 1997). Receptor activa-
tion is linked to inhibition of cAMP accumulation, leading
to enhanced proliferation rates and a reduction in forskolin-
induced IL-6 release.
5.12 S E ROTO N I N R EC E P TO R S
RT-PCR in human and rat brain cultures revealed that astro-
cytes can express various serotonin receptors (Azmitia 2001;
Verkhratsky et al. 1998). Distinct expression of 5HT subtypes
also occurs in situ because 5HT1A, 5HT2A, and 5HT5A sub-
types have been identified in different brain areas. In astrocytes
of the brainstem, 5-HT triggers Ca2+ responses (Härtel et al.
2009). 5HT receptors have been speculated to be involved in
pathogenesis. The 5HT5A subtype is upregulated in gliosis and
the 5HT2A subtype is enhanced in schizophrenia. Stimulation
of 5HT receptors leads to the release of S100β from astro-
cytes, and this release was hypothesized to have an impact on
the ongoing pathological event. 5HT5A transcripts are devel-
opmentally regulated: Receptor mRNA is detected before
birth and expression peaks at postnatal day 20 in the rat.
5.13 NAT R IU R ET I C P E P T I D E R EC E P TO R S
All three subtypes, ANP, brain natriuretic peptide, and C-type
natriuretic peptide (c-NP) are present in the brain (Potter
et al. 2006). Natriuretic peptides exert their actions by bind-
ing to natriuretic peptide receptors (NPRs): ANP binds pref-
erentially to NPR-A, and brain natriuretic peptide and c-NP
to NPR-B (Lucas et al. 2000). All NPs bind with equal affinity
to NPR-C (Lucas et al. 2000). NPR-A and NPR-B are par-
ticulate, cell membrane–bound guanylyl cyclase receptors that
mediate the signal by increasing cyclic guanosine monophos-
phate (cGMP). NPR-C, earlier ascribed the role of a clearance
receptor removing the peptides to the periphery, does not pos-
sess guanylyl cyclase activity (Potter et al. 2006; Rose and Giles
2008) and appears to have a complex signaling mechanism of
its own. The consequence of NPR-C activation is inhibition
of adenylyl cyclase (cAMP) via guanine nucleotide inhibitory
proteins (Gi proteins) (Potter et al. 2006). However, NPR-C
is also coupled to endothelial nitric oxide synthase (eNOS)–
dependent signal transduction, in which NO activates a solu-
ble form of guanylate cyclase leading to production of cGMP
(Murthy et al. 1998). In astrocytes, NPs have been reported to
elicit downstream signals by interacting with NPR-A (Borán
and García 2007; Prado et al. 2010) or NPR-B (Zielińska et al.
2007). The presence of NPR-C in astrocytes was identified on
the basis of ligand displacement analysis (Sumners and Tang
1992) and its functional role by showing that agonists of this
receptor attenuate the accumulation of reactive oxygen spe-
cies and nitric oxide synthesis in ammonia-treated astrocytes
(Skowrońska et al. 2010).
5.14 TAC H Y K I N I N R EC E P TO R S
Astrocytes express all subtypes of tachykinin receptors, NK1 to
NK3. In cell culture, activation of NK1 receptors leads to mem-
brane depolarization because of blockade of the constitutive
• 205
Table 17.1 TRANSMITTER RECEPTORS AND GLIOTRANSMITTER RELEASE MECHANISMS
NON–VESICLE-BASED
GLIOTRANSMITTER GLIOTRANSMITTER RECEPTORS VESICLE-BASED RELEASE RELEASE

l-Glutamate Ionotropic receptors: Yes Yes

AMPA (GluR1–4)
Kainate (GluR5–7)
NMDA (NR2B)
Metabotropic receptors:
mGluR 1, 3–5
γ-Amino butyric acid (GABA) GABAA, GABAB ? Yes
Glycine Not common, subunits ? ?
α1 β of strychnine-sensitive
receptor
Acetylcholine (ACh) Nicotinic AChR (α7 subunit); ? ?
muscarinic AChR (M1–M5)
Oxytocin, vasopressin Oxytocin receptor (mRNA); ? ?
vasopressin receptor 1a; vaso-
pressin receptor 1b
Vasoactive intestinal peptide (VIP) VPAC2 ? ?
Adrenaline, noradrenaline α1-, α2-, β1-, β2-, ? ?
β3-adrenoreceptors
Angiotensin AT1 and AT2 ? ?
Somatostatin Somatostatin receptor ? ?
(sst2A splice variant)
Serotonin (5HT), 5HT receptor subtypes: ? ?
5-hydroxytryptamine 5HT1A, 5HT2A, 5HT5A
Atrial natriuretic peptide (ANP) NPR-A, NPR-B, NPR-C Yes ?
Tachykinin peptides NK1 to NK3 ? ?
Bradykinin B1 and B2 ? ?
Thyrotropin-releasing hormone Receptor subtype TRH1 ?Yes ?
(TRH)
Opioid peptides μ, δ, κ ? ?
Histamine H1, H2, and H3 ? ?
Dopamine D1 to D5 ? ?
Endothelin ETB
Adenosine-5′-triphosphate (ATP) Ionotropic and metabotropic: Yes Yes
see chapter 25

206 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
K+ conductance and opening of Cl– channels (Backus et al.
1991). Receptor localization at the light and electron micro-
scopic level in different species, including humans, has iden-
tified NK2 and NK3 receptors in astrocytes of various areas
of the CNS, for example, spinal cord, cortex, and hippocam-
pus. NK2 receptors have been found to cluster close to axon
terminals in spinal cord. Substance P, as a prominent ligand,
enhances secretion of IL-6 and PGE2 on IL-1β stimulation in
cultured spinal cord astrocytes (Palma et al. 1997). Substance
P augments glutamate-triggered ATP release from cultured
spinal cord astrocytes via activation of neurokinin recep-
tors (Werry et al. 2006). Moreover, LPS-induced secretion
of tumor necrosis factor α (TNFα) and IL-1 from cultured
astrocytes was augmented by the ligand, substance P (Luber-
Narod et al. 1994). A proinflammatory function of tachykinin
receptors in reactive astrocytes has been suggested because a
lesion-induced upregulation of the receptors was observed in
astrocytes of transected optic nerve in vivo.
5.15 B R A DY K I N I N R E C E P TO R S
Functional bradykinin receptors (B1 and B2 receptors) have
only been identified in cultured astrocytes. Receptor activation
leads to an increase in intracellular Ca2+, induced by release
from intracellular stores, and to a blockade of a constitutive
K+ conductance mediated by B2 receptors (Gimpl et al. 1992).
B2 receptor–induced increase in intracellular Ca2+ leads to the
release of glutamate and aspartate from astrocytes stimulating
neuronal glutamate receptors; this substantiates that astro-
cytes can directly modulate neuronal activity (Parpura et al.
1994). Bradykinin is also involved in astrocyte volume control
because it directly activates volume-sensitive outwardly recti-
fying anion channels, even without an increase in cell volume
(Akita and Okada 2011). In rat brain cultured astrocytes, bra-
dykinin leads to upregulation of matrix metalloproteinase-9
and promotes cell migration (Hsieh et al. 2008).
5.16 T H Y ROT RO P I N-R E L E A S I N G
H O R MO N E R E C E P TO R S
Thyrotropin-releasing hormone (TRH) receptors have been
identified in culture and in situ by immunocytochemistry
and Western blot. Astrocytes express the TRH1 subtype.
Expression in situ is stronger in white matter than gray mat-
ter as studied in the adult rat spinal cord. The receptors are
present in TRH-synthesizing astrocytes, indicating that
this hormone acts in an autocrine and paracrine fashion
(Fernández-Agulló 2001).
5.17 O P I O I D R E C E P TO R S
There is evidence for the expression of all three subtypes of
the opioid receptors, μ, δ, and κ, in cell culture and in situ
(Ruzicka et al. 1995). The μ and δ receptors were shown to
be preferentially expressed on astrocytic processes in situ.
Although μ receptors are more frequently present on imma-
ture astrocytes, the expression of δ and κ receptors is increased
in adult tissue.
R E L E A S E O F G L I OT R A N S M I T T E R S A N D T R A N S M I T T E R R E C E P TO R S I N A S T R O C Y T E S
5.18 H I S TA M I N E R EC E P TO R S
In cultured astrocytes, binding sites for H1 and H2 subtypes of
histamine receptors have been identified (Inagaki and Wada
1994), which couple to Gq and Gs types of G-proteins, respec-
tively. H1 receptors couple to PLC leading to IP3 production
and Ca2+ release, whereas H2 receptor activation in astrocytes
is linked to adenylate cyclase and intracellular cAMP accu-
mulation. H1 receptors were mainly found on the processes
of astrocytes. Histamine acts as a mitogen in cortical astro-
cytes. Activation of H1 receptors has been shown to stimu-
late glycogen breakdown. Histamine triggered an increase in
[Ca2+]i in acute rat hippocampus because of an upregulation
of functional H1 receptors after postnatal day 8 (Shelton and
McCarthy 2000). Based on ultrastructural studies suggest-
ing glial apposition to histaminergic neurons, astrocytes were
believed to respond to histamine released from hippocampal
neurons. In a recent study on cultured astrocytes, all three
types of histaminergic receptors (H1, H2, H3) were identi-
fied by RT-PCR and a pharmacological approach. All three
receptors control the release of neurotropin-3 and converge at
the level of mitogen-activated protein (MAP) kinase activity
( Jurič et al. 2011).
5.19 D O PA M I N E R EC E P TO R S
Cultured astrocytes from the striatum express D1 to D5 sub-
types of dopamine receptors as shown by RT-PCR (Miyazaki
et al. 2004). D1 receptor activation leads to PTX-sensitive
cAMP formation via protein kinase A stimulation. Prolonged
receptor activation led to a reduction of astroglial dopamine
sensitivity (Reuss et al. 2001). D2 receptors in astrocytes are
linked to the actin cytoskeleton via interaction with filamin
A. Ligand binding and ultrastructural analysis in vivo found
strong expression of D2 receptors in GFAP-positive corti-
cal astrocyte processes surrounding interneurons. In the pre-
frontal cortex, they were found to be colocalized with alpha7
nicotinic acetylcholine receptors (Duff y et al. 2011). These
receptors were indeed functional because their activation
induced increases in intracellular Ca2+ in the astrocytes (Khan
et al. 2001). Activation of the classic dopamine receptor D1 or
D2 and the phosphatidylinositol-linked D1-like receptor elicits
FGF-2 biosynthesis and secretion in striatal astrocytes, which
may play a role in neuroprotection (Zhang et al. 2009).
5.20 E N D OT H E L I N R EC E P TO R S
The predominant receptor for endothelin in astrocytes is
endothelin B (ETB). Activation of the receptors triggers an
increase in Ca2+ and opens charybdotoxin-sensitive, calcium-
activated K+ channels (Supattapone and Ashley 1991). It
stimulates proliferation and promotes activation of astro-
cytes, thus inducing reactive gliosis (Ishikawa et al. 1997). It
also affects glutamate levels by downregulating the glutamate
transporter GLAST in the astrocytes (Matsuura et al. 2002).
Endothelin stimulates the release of glial cell line–derived
neurotrophic factor (GDNF) (Koyama et al. 2003), increases
production of 2-arachidonoyl glycerol, the most abundant
• 207
endocannabinoid in the CNS (Walter and Stella 2003), and
increases mRNA and extracellular release of neurotrophin-3
(Koyama et al. 2005). Endothelin also controls the commu-
nication between astrocytes. Both endothelin-1 and endothe-
lin-3 inhibit the extent of coupling between astrocytes by
decreasing the expression of phosphorylated connexin 43,
the gap junction protein prominently expressed by astrocytes
(Blomstrand et al. 2004).
6 S U M M A RY A N D P E R S P E C T I VE S
It has become clear over the last years that astrocytes are an
integral functional part of synapses, which led to the formu-
lation of the tripartite synapse concept (see chapter 38). As
summarized in this chapter, astrocytes have the capacity to
express essentially all transmitter and hormone receptors that
are known to be relevant for signaling in the nervous system
(for summary see table 17.1). Thus, they have the capacity to
sense any activity in the brain, including the synaptic activity.
Astrocytes are also able to release neuroactive substances in
an activity-dependent manner. Most prominent is the release
of glutamate, ATP, GABA, and D-serine. Thereby, astrocytes
are able to modulate synaptic function and network activ-
ity (see chapter 38). As a consequence, astrocytes modulate
behavior (see chapter 39). Moreover, by their ability to take
up neurotransmitters (see chapter 35), they have a second
way to affect the neuronal network. Although neuronal
activity is decoded in a millisecond time frame, the astrocyte
activity is slower rather in the range of seconds or tenth of
seconds. Moreover, astrocytes are more part of the volume
than the wiring transmission system; thus, extracellular space
plays an important role for this type of communication (see
chapter 34). Thus, brain function is not solely determined by
neurons, but is a harmonious collaboration between these
cells and their glial partners .
AC K N OW L E D G M E N T S
RZ is supported by grants (P3 310, J3 4051, J3 3632, and
J3 4146) from the Slovenian Research Agency (ARRS) and
the EduGlia ITN EU grant, HK is supported by Deutsche
Forschungsgemeinschaft (TR 43).
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 18.
STORAGE AND RELEASE OF NONTRANSMITTER
SIGNALING MOLECULES FROM MACROGLIA
Oliver von Bohlen und Halbach and Klaus Unsicker

A B B R E VI AT I O N S on synaptic connectivity, myelination and support of axonal

integrity, to implications in neurode- and regeneration, just to
AAV adeno-associated vectors name a few. The vast array of functions performed by glial cells
AR amphiregulin crucially depends on signaling molecules, which they release
ART artemin and to which they respond. This chapter focuses on macroglia-
BDNF brain-derived neurotrophic factor derived protein growth factors acting on neurons and their
BMP bone morphogenetic protein processes, synapses, vascular cells, and, in both autocrine and
BTC betacellulin heterocrine modes, on glial cells. The literature selected is
CNS central nervous system mostly from the past decade, with a focus on articles that have
CNTF ciliary neurotrophic factor appeared since the second edition of Neuroglia. Each subchap-
EGF epidermal growth factor ter discussing a growth factor or growth factor family (arranged
EGFR epidermal growth factor receptor in alphabetical order), respectively, is introduced by a brief
EPR epiregulin general outline followed by specific details of their functions
FGF fibroblast growth factor related to Schwann cells, astrocytes, and oligodendrocytes.
FGFR fibroblast growth factor receptor
GDF growth differentiation factor
GDNF glial cell–derived neurotrophic factor 2 E P I D E R M A L G R OW T H FAC TO R FA M I LY
GFR growth factor receptor
HB-EGF heparin-binding EGF-like growth factor Epidermal growth factor (EGF), as well as transforming
IGF insulin-like growth factor growth factor-α (TGF-α), heparin-binding EGF-like growth
IGFBP IGF-binding protein factor (HB-EGF), amphiregulin (AR), epiregulin (EPR), epi-
IL-6 interleukin 6 gen, betacellulin (BTC), and the neuregulins 1–4 (NRG1–4)
LIF leukemia inhibitory factor belong to the EGF family. Epidermal growth factor signals via
MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydro EGF receptors, known as ErbB1–ErbB4. The ErbB protein
pyridine family or epidermal growth factor receptor (EGFR) family is
NGF nerve growth factor a family of four structurally related receptor tyrosine kinases.
NRG1–4 neuregulin 1–4 ErbB1 is activated, for example, by EGF, TGF-α, HB-EGF,
NT-3 neurotrophin 3 amphiregulin, betacellulin, epiregulin, and epigen, whereas the
NT-4 neurotrophin 4 ErbB4 receptor is mainly activated, for example, by HB-EGF,
NTN neurturin betacellulin, and the neuregulins 1–4.
PAR protease-activated receptor
PDGF platelet-derived growth factor
2.1 S C H WA N N C E L L S
PNS peripheral nervous system
PSPN persephin During development of the peripheral nervous system (PNS),
TβR TGF-β receptor Schwann cells require neuregulin (NRG1) provided by neu-
TGF-α transforming growth factor-α rons for growth, migration, and myelination; moreover, NRG
TGF-β transforming growth factor-β and ErbB also control Schwann cells during axon regeneration
(Joung et al. 2010). Neuregulin acts as a potent chemoattractive
and kinetic molecule, as revealed by analyzing Schwann cell pre-
1 INTRODUCTION cursors (Cornejo et al. 2010). Neuregulin (type III) is essential for
myelination in the PNS. Interestingly, NRG1/ErbB signaling dif-
Since the second edition of Neuroglia in 2005 our knowledge fers between Schwann cells and oligodendrocytes (Brinkmann et
on macroglial cells (Schwann cells, astrocytes, and oligoden- al. 2008). Nave et al. generated a series of conditional null mutants
drocytes) has enormously expanded. This applies to almost completely lacking NRG. Surprisingly, these mice had normal
all fields of glia biology, from developmental genetics, impact amounts of CNS myelin. Furthermore, double mutants deficient

212
for ErbB3 and ErbB4 in oligodendrocytes were myelinated with-
out stimulation by neuregulins, indicating that signaling through
NRG1 and ErbB differs between Schwann cells and oligodendro-
cytes. Activation of NF-kappaB seems to be critical for Schwann
cell myelination (Limpert and Carter 2010). Its transcriptional
activity can be significantly increased by forskolin. Both ErbB2
and ErbB3 serve as the transducers of the NRG1 signal. Schwann
cell–derived tumors of the neurofibromatosis type 1 express the
EGF-R and respond to EGF (DeClue et al. 2000), suggesting
that the EGF-R plays an important role in Schwann cell transfor-
mation. EGF is also involved in reprogramming Schwann cells to
multipotent adult neural crest cells (Widera et al. 2011).
2.2 A S T RO C Y T E S
Transforming growth factor-α and EGF-R immunoreactiv-
ity are found in both neurons and astrocytes in the developing
and adult brain of humans and different species of animals. In
addition, TGF-α and EGF-R colocalize in most neurons and
maturing astrocytes. Heparin-binding EGF-like growth fac-
tor stimulates the proliferation of astrocytes and multipotent
progenitors. Neuregulin 1 seems to have a specific role for the
generation of astrocytes, because NRG1–ErbB2 signaling is
required for the establishment of radial glia and their transfor-
mation into astrocytes in cerebral cortex (Schmid et al. 2003).
EGF-induced proliferation of astrocytes requires the transcrip-
tion factor Egr-1 (Mayer et al. 2009). Several pharmacologically
relevant stimuli, as for example by adrenoceptor agonists and
angiotensin II, have been shown to transactivate the EGF-R in
astrocytes (Clark and Gonzalez 2007; Li et al. 2008). Epidermal
growth factor receptor activation also downregulates collagens
in cultured astrocytes, thereby probably providing an explana-
tion for the fact that astrocytes do not secrete collagens in vivo
(Heck et al. 2007) and that therefore the extracellular matrix
of the CNS lacks fibrillar elements. Similarly, EGF-R ligands
regulate injury-induced growth factor and matrix production
by astroglia, as for example chondroitin sulfate proteoglycans
(Smith and Strunz 2005) and prostaglandin (Zhang and Neufeld
2005). Astroglia-derived EGF and TGF-α also promote their
own calcium oscillations (Morita et al. 2005). Together, there is
robust evidence now that astrocytic ErbB signaling is crucially
implicated in the autocrine and paracrine regulation of astro-
glial function and integration and processing of neuronal inputs
by astrocytes (Sharif and Prevot 2010).
2.3 O L I G O D E N D RO C Y T E S
Neonatal oligodendrocytes contain and secrete neuregulins
in vitro. Furthermore, it is known that oligodendrocytes express
ErbB-3 as well as ErbB-4; moreover, HB-EGF mRNA and protein
are expressed by oligodendrocytes in vivo (Du and Dreyfus 2002).
3 F I B R O B L A S T G R OW T H
FAC TO R FA M I LY
A substantial fraction of the 23 fibroblast growth factor (FGF)
family members are expressed by neurons and glial cells (Reuss
S TO R AG E A N D R E L E A S E O F N O N T R A N S M I T T E R S I G N A L I N G M O L E C U L E S F R O M M AC R O G L I A
and von Bohlen und Halbach 2003). The majority of FGFs
possess amino-terminal signal peptides; therefore, they are
assumed to be readily secreted from cells, whereas some FGFs,
including FGF-2, lack conventional signal peptides, but can be
secreted nevertheless (Okada-Ban et al. 2000). Although most
FGFs act in a paracrine and/or autocrine manner, some have
potential roles in metabolism, acting in an endocrine man-
ner. In the adult CNS, high levels of the receptors FGFR-1,
FGFR-2, and FGFR-3 are expressed within the diencephalon
and telencephalon, whereas their expression levels are rela-
tively low in other areas. Fibroblast growth factor-1 is widely
expressed, but nevertheless confined to specific neuronal
populations in the adult CNS; however, FGFR-1 has also
been detected in astrocytes of white matter tracts. Fibroblast
growth factor-2 and -3 are primarily found on glial cells. The
fourth member of the FGF receptors (FGFR-4), shows robust
expression only during early stages of development, and is not
detectable in the adult CNS outside the habenular complex.
3.1 S C H WA N N C E L L S
Schwann cells express FGF-1, -2, and -5, and the FGFR-1, -2,
and -3 (Grothe and Nikkhah 2001) expression is increased
following nerve crush. Lack of FGF-2 leads to poor func-
tional recovery after peripheral nerve injuy (Seitz et al. 2011).
Although faster regeneration after sciatic nerve injury in mice
overexpressing FGF-2 has been reported ( Jungnickel et al.
2006), consistent with enhanced axonal regeneration across
long gaps mediated by FGF-2 (high molecular form) overex-
pressing Schwann cells (Timmer et al. 2003), in a facial nerve
paradigm FGF-2 overexpressing Schwann cells apparently
failed to promote functional recovery (Haastert et al. 2009).
Fibroblast growth factor-1 and -2 have been implicated in
stimulation of Schwann cell proliferation and inhibition of
myelination at the lesion site, whereas activation of FGFR-3
likely promotes neurite formation (Grothe et al. 2006).
3.2 A S T RO C Y T E S
Astrocytes are a prominent source of FGF-2. Autocrine and
paracrine actions of FGF-2 on astrocytes include regulation
of gap junction (connexin43) expression and function (see
also chapter 24), neurotransmitter sensitivity, and intermedi-
ate filament density (Unsicker et al. 2006). Similar to FGF-2,
FGF-1 is also implicated in gap junction regulation (Garré
et al. 2010). Interestingly, FGF-2 affects astrocyte differentia-
tion selectively in the gray matter and in a brain region-specific
manner, for example, in the hindbrain (Irmady et al. 2011), as
revealed by monitoring GFAP expression and blood-brain
barrier integrity. (More information concerning the blood-
brain barrier is provided in chapter 33.) Mechanistically, it
was shown that methylation of histone H3 at lysine 4 resi-
due associated with the STAT binding site on the GFAP
promoter was significantly decreased in the gray matter of
the FGF-2 null mouse, suggesting a role for FGF-2 in the
epigenetic regulation of astrocyte differentiation in vivo.
Astrocyte specification as well as proliferation is medi-
ated through diverse FGFRs (Kang and Song 2010). FGF-2
• 213
selectively induces A2B5-positive astrocyte precursor cells
to differentiate into GFAP-positive cells (Lin and Goldman
2009). A clinically relevant aspect concerns the activation of
FGFR through antidepressants, an effect that also implicates
glial cell–derived neurotrophic factor (GDNF) production
(Hisaoka et al. 2011); however, conclusive clinical studies
have not yet been done. Brain injury leads to upregulation
of FGF-2 in astrocytes and of FGFR-1 in both astrocytes and
neurons. Association of specific heparin sulfate proteoglycans
to the FGF-2–FGFR-1 complex accounts for the regulation
of FGF-2 storage, nuclear trafficking, and cell-specific injury
responses (Leadbeater et al. 2006).
3.3 O L I G O D E N D RO C Y T E S
Oligodendrocytes are further important target cells for
FGF-actions. Thus, FGF signaling is required for the genera-
tion of oligodendrocyte progenitors from the embryonic fore-
brain (Furusho et al. 2011). The 120-kDa isoform of FGFR-2
is the most abundant tyrosine-phosphorylated protein in
oligodendrocytes and phosphorylated even in the absence of
FGF-2. Fibroblast growth factor receptor-2, but not FGFR-1
is associated with lipid raft domains in oligodendrocytes and
myelin, but not in astrocytes (Bryant et al. 2009). In addition
to FGF-2, FGF-9 is expressed by both astroglial cells and oli-
godendrocytes. Using double immunofluorescence and in situ
hybridization, specific FGF-9 signals in GFAP-positive white
matter astrocytes of adult rat spinal cord and brainstem as well
as in CNPase-positive cerebellar and callosal oligodendrocytes
have been detected (Nakamura et al. 1999).
4 G L I A C E L L L I N E –D E R I VE D
N E U R OT R O P H I C FAC TO R FA M I LY
The GDNF family, consisting of GDNF, neurturin (NTN),
artemin (ART), and persephin (PSPN), are distant members
of the transforming growth factor-beta (TGF-β) superfam-
ily. The members of the GDNF family differentially promote
survival and regulate differentiation in peripheral and central
neuronal populations. In contrast with the other members
of the TGF-β superfamily, which signal through receptor
serine-threonine kinases, the GDNF family ligands acti-
vate intracellular signaling cascades via the receptor tyrosine
kinase Ret. The GDNF family members bind first to the
GDNF receptor α (GFRα), a GPI-anchored membrane pro-
tein, forming a heterodimer with the receptor tyrosin kinase
c-ret. This complex activates the ras/raf and PI3 kinase sig-
naling pathways. GFRα1, GFRα2, GFRα3, and GFRα4
are the co-receptors for GDNF, NT, ART, and PSPN,
respectively (Peterziel et al. 2007).
4.1 S C H WA N N C E L L S
Glial cell–derived neurotrophic factor, GFRα1, GFRα2,
and the alternative Ret coreceptor NCAM are expressed by
Schwann cells (Iwase et al. 2005). Growth factor receptor α1
214 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
is localized adjacent to the outermost myelin sheath and in the
endoneurium. Schwann cells contain GFRα1 in both soluble
and membrane fractions; it is localized on the outer surface
of the Schwann cell plasma membrane (Hase et al. 2005).
In peripheral nerve and spinal cord lesions substituted with
Schwann cell grafts, GDNF has been shown to promote axon
regeneration. In lesioned nerves, an autocrine loop of BMP-2
upregulates GDNF mRNA in Schwann cells (Kinameri and
Matsuoka 2003). After long-term denervation, a decline in
Schwann cell–derived GDNF may account for impaired
regeneration (Höke et al. 2002). Specific expression patterns
of GDNF family members have been reported during periph-
eral nerve development and in cultured Schwann cells (Piirsoo
et al. 2010). Artemin has been shown to influence neural dam-
age in chronic pancreatitis (Ceyhan et al. 2007).
4.2 A S T RO C Y T E S
Glial cell–derived neurotrophic factor is also expressed by astro-
cytes. Levels of GDNF are increased by FGF-2 and bone mar-
row stromal cells (Shen et al. 2010; Shin et al. 2009), and release
of GDNF from astroglia is stimulated by dexmedetomidine
(Yan et al. 2011). Fibroblast growth factor-2–mediated induc-
tion requires the transcription factor Egr-1. Glial cell–derived
neurotrophic factor is likely secreted both with and without pro-
cessing by furin-like proteases; the prodomain and C-terminal
cysteines play important roles in GDNF processing and secre-
tion in cultured astrocytes and C6 glioma cells (Oh-hashi
et al. 2009). Reactive, nestin-positive astrocytes induced by
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) express
prominent levels of GDNF, thereby probably contributing to
the neurotrophic support of neurons (Chen et al. 2006).
4.3 O L I G O D E N D RO C Y T E S
Glial cell–derived neurotrophic factor is expressed by oligo-
dendroglia and decreased in alpha-synuclein transgenic oligo-
dendroglial cells (Ubhi et al. 2010). Further, NTN and PSPN
mRNAs have been reported to occur in oligodendroglial cell
lines (Strelau and Unsicker 1999).
5 I N S U L I N -L I K E G R OW T H FAC TO R S
The insulin-like growth factors (IGFs) are proteins with high
sequence similarity to insulin. Currently, two IGFs are known
(IGF-1 and IGF-2), along with a family of six high-affinity
IGF-binding proteins (IGFBP 1–6). The IGFs are known to
bind to IGF-1 and IGF-2 receptors, as well as to insulin recep-
tors, insulin-related receptors, and possibly to further receptors.
Insulin-like growth factor-1R is a cell surface heterotetrameric
tyrosine kinase receptor coupled to numerous intracellular
second messenger pathways, including Raf/Ras-mitogen–
activated protein kinase and phosphatidylinositol-3 kinase sig-
naling cascades; hence, IGF-1R is thought to be crucial for cell
survival, whereas IGF-2 appears to have a tumor suppressor role,
because mutated or deleted forms have been reported in several
forms of cancers (Capoluongo 2011). Expression of IGF-1 and
-2, IGF-IR, and the IGFBPs is well documented during devel-
opment of the CNS (Sullivan et al. 2008). Cultured embry-
onic neurons and glia synthesize IGF-1 mRNA; however, only
glia seems to produce IGF-2 mRNA (Rotwein et al. 1988).
5.1 S C H WA N N C E L L S
Schwann cells are a prominent source of IGF-1, which has
been shown to promote the survival of Schwann cells, for
example, after serum withdrawal (Rodrigues et al. 2010).
Insulin-like growth factor-1 also stimulates the early events
in myelination; the fatty acid biosynthetic pathway is a major
target of IGF-1 (Liang et al. 2007).
5.2 A S T RO C Y T E S
Evidence is emerging for a role of the IGF system in astro-
glial cell biology. Astroglial cells express IGF-1 and IGFBP-2,
which are upregulated by progesterone, but downregulated
by dexamethasone (Chesik and De Keyser 2010). Insulin-like
growth factor-1 reduces cAMP levels through a beta2-adrener-
gic receptor–dependent process (Chesik et al. 2008). Reactive
astrocytes in the spinal cord of amyotrophic lateral sclerosis
transgenic rats upregulate IGF-2-R (Dagvajantsan et al. 2008).
Insulin-like growth factor-1 signaling in astrocytes is modulated
by PTEN, a phosphatase which inhibits Akt activation, and in
turn is positively regulated by PI3K (Fernandez et al. 2008).
5.3 O L I G O D E N D RO C Y T E S
Oligodendrocytes express IGF-1 receptors as well as IGF-1
mRNA and IGF-1 protein, which elicit distinct effects on
developing oligodendrocytes (Du and Dreyfus 2002). Insulin-
like growth factor-1 increases proliferation, inhibits apoptosis,
and promotes differentiation of oligodendrocytes and their
precursor cells, suggesting an important function of IGFR-1
in myelination. Vice versa, deficits in IGF-1 cause decreased
cell proliferation in the CNS white matter and decreased sur-
vival of newborn oligodendrocytes (Hua et al. 2009). Hence,
IGF-1 holds a therapeutic potential in demyelinating and neu-
rodegenerative diseases (Chesik et al. 2008). An additional
“side effect” may be the neurotrophic effect of oligodendro-
cyte-derived IGF-1 on neurons (Wilkins et al. 2001).
6 N E U R OT R O P H I N S
The family of neurotrophins consists of nerve growth factor
(NGF), brain-derived neurotrophic factor (BDNF), neu-
rotrophin 3 (NT-3), and neurotrophin 4 (NT-4). The neu-
rotrophins bind to specific receptors that belong to the class
of the Trk family of tyrosine protein kinase receptors. NGF
specifically recognizes trkA, whereas BDNF and NT-4 spe-
cifically activate trkB receptors. NT-3 primarily activates trkC
receptors. In addition, all neurotrophins can signal through a
low-affinity receptor (which is structurally unrelated to the trk
S TO R AG E A N D R E L E A S E O F N O N T R A N S M I T T E R S I G N A L I N G M O L E C U L E S F R O M M AC R O G L I A
receptors), known as p75NTR receptor, which is a member
of the TNF receptor/Fas/CD40 superfamily. The p75NTR
receptor appears to modify Trk signaling when the two recep-
tor types are coexpressed. When expressed in the absence of
Trks, p75NTR seems to mediate responses to neurotrophins,
including promotion of apoptotic death. The neurotrophins
have long been implicated in neuronal survival during develop-
ment, but they also play prominent roles within the postnatal
brain. Thus, there is evidence that neurotrophins are involved
in the maintenance of the postnatal brain and neuronal plas-
ticity (von Bohlen und Halbach 2010).
6.1 S C H WA N N C E L L S
Schwann cells have long been known to promote nerve growth
and regeneration by the secretion of trophic molecules and
supportive extracellular matrix. Neurotrophin family members
and their receptors exhibit distinct expression patterns dur-
ing Schwann cell development and regeneration as well as in
cultured Schwann cells (Piirsoo et al. 2010). Moreover, recent
studies have identified neurotrophins as important regulators of
peripheral (and CNS!) myelination (Xiao et al. 2009). Because
Wallerian degeneration is now being reinvestigated with
molecular perspectives on inflammatory events, neurotrophins
secreted by Schwann and other cells are once again gaining
momentum (Gaudet et al. 2011). Very recently, Schwann cells
have also been targeted by injection of adeno-associated vector
(AAV) pseudotypes as a gene therapy strategy for peripheral
nerve regeneration (Homs et al. 2011). Schwann cells express
the pan-neurotrophin receptor p75NTR, which is massively
upregulated following peripheral nerve lesions, thereby limit-
ing neurotrophin availability (Scott and Ramer 2010).
6.2 A S T RO C Y T E S
Astrocytes from several brain areas, including the hippocam-
pus, are capable of synthesizing NGF and NT-3. Developing
and lesioned astrocytes have been shown to express p75, but
not TrkA. Activation of p75NTR by NGF induces cell cycle
arrest of dividing astrocytes (Cragnolini et al. 2011). BDNF–
expressing astroglia cells have been detected in the spinal cord
and the brain (Du and Dreyfus 2002). In the hippocampus,
BDNF has been reported to restore astroglial S100 in a rat
model of depression (Ye et al. 2011). Likewise, treatment of
astrocytes with the antidepressants fluoxetine and paroxetine
upregulates BDNF in astrocytes (Allaman et al. 2011).
6.3 O L I G O D E N D RO C Y T E S
Oligodendrocytes have been identified to express NGF,
BDNF, NT-3, and NT-4 mRNA. Concerning BDNF, it
was shown that it is expressed by oligodendrocytes myelinat-
ing the central processes of sensory neurons. Recent studies
support the concept that BDNF plays a role in demyelinat-
ing lesions by regulating the number of oligodendrocyte pro-
genitors and the abilities of demyelinating and differentiating
cells to express myelin proteins (VonDran et al. 2011). In
• 215
addition to expressing neurotrophins, oligodendrocytes also
express neurotrophin receptors (trkA, trkB, trkC, as well as
p75NTR), suggesting possible autocrine signaling (Du and
Dreyfus 2002).
7 P L AT E L ET-D E R I VE D G R OW T H FAC TO R
Platelet-derived growth factor (PDGF) signals through two
types of the alpha- and beta-type. Currently, five different iso-
forms of PDGF are known that can activate the two different
receptors: PDGFA, PDGFB, PDGFC, PDGFD, and a PDGFAB
heterodimer. The different isoforms seem to be expressed dif-
ferently within the brain and it is thought that many neurons
express the A and/or B chains of PDGF, whereas astrocytes
express the A chain.
7.1 S C H WA N N C E L L S
Cultured Schwann cells secrete PDGF and express PDGF
receptors. In neonatal rats, Schwann cells in the DRGs and
sciatic nerve synthesize PDGF, but the expression of PDGF
protein declines postnatally. In adult rats, PDGF is low in
myelinating Schwann cells, but is upregulated in nonmyelinat-
ing Schwann cells (Eccleston et al. 1993). Responses to PDGF
are not seen until the precursor/Schwann cell transition
(Woodhoo et al. 2004). PDGFR is prominently upregulated
in crushed nerves (Oya et al. 2002; Yamazaki et al. 2009).
7.2 A S T RO C Y T E S
Information on PDGF and PDGFRs in astrocytes is scarce.
Levels are apparently low, but can be induced, for example, by
the HIV protein Tat (Bethel-Brown et al. 2011). In a subpopu-
lation of astroglia located in the optic nerve head, light expo-
sure has been reported to induce PDGF (Kernt et al. 2010).
When overexpressed in brain under the control of the human
GFAP promoter, PDGFB induces glioblastomas (Hede et al.
2009). For further details concerning gliomas, see chapter 59.
7.3 O L I G O D E N D RO C Y T E S
In 1988, PDGF was discovered to promote division and
motility and inhibit premature differentiation of the oligo-
dendrocyte/type-2 astrocyte progenitor cell. Oligodendroglia
express PDGFRβ protein, and PDGF is capable of stimulating
proliferation of oligodendrocytes and their precursors, respec-
tively. Stimulation of proliferation is accompanied by inhibi-
tion of myelination (Wang et al. 2007b). Oligodendrocyte
precursors have been shown to migrate toward a PDGF
source (Zhang et al. 2004).
8 R E GU L ATO RY C Y TO K I N E S
The term neuroregulatory cytokines is often used for a large
number of different growth factors, including the members of
the ciliary neurotrophic factor (CNTF) family, comprising,
216 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
inter alia, leukemia inhibitory factor (LIF), cardiotrophin-1,
interleukin 6 (IL-6), and oncostatin-M. In the context of this
chapter, data are also incorporated on cytokines and growth
factors unrelated to the CNTF family, often termed inflam-
matory cytokines (see chapter 22). Ciliary neurotrophic factor
is a potent survival factor for neurons and oligodendrocytes
and is relevant in reducing tissue destruction during inflam-
matory attacks. LIF regulates the differentiation of myeloid
leukemic cells, but also acts as a regulatory cytokine in the
CNS. Cardiotrophin-1 is associated with the pathophysiology
of heart diseases, but also relevant to neuron survival. IL-6 acts
as both a proinflammatory and antiinflammatory cytokine,
which is secreted by T cells and macrophages to stimulate
immune response. However, aside from this well-known func-
tion, IL-6 acts as a regulatory cytokine within the CNS. The
members of the family of neuroregulatory cytokines share,
in part, receptor and transduction components. CNTF, for
example, binds to a complex consisting of a glycophosphati-
dylinositol anchored α receptor, which is not involved in sig-
nal transduction, the LIF receptor β (LIFRβ) and gp130. LIF
binds to a complex consisting of LIFRβ and gp130.
8.1 S C H WA N N C E L L S
CNTF is a classic growth factor produced by Schwann cells
both in vivo and in vitro and is induced following lesion-
ing. Schwann cells are also a prominent source of IL-6 and
IL-6R. Interestingly, factors belonging to the class of IL-6-
related regulatory cytokines, including CNTF, LIF, car-
diotrophin-1, and oncostatin M, have been found to exert
strong promyelinating effects (Stankoff et al. 2002). Human
fetal Schwann cells constitutively express IL-1β, -6, -8, -11,
-12, -15, and the receptors for IL-1, -4, -6, -8, 13, TNFα,
and gp130 (Ozaki et al. 2008). Tumor necrosis factor has
been shown to inhibit Schwann cell proliferation by upregu-
lating Src-suppressed protein kinase C substrate expression
(Tao et al. 2009). Moreover, TNF-α plays pivotal roles in the
interactions of Schwann cells with axons. Apparently, TNF-α
mediates its effects by orchestrating axon guidance. Schwann
cells in TNF-deficient peripheral nerve bundles fail to envelop
axons efficiently, an effect that can be rescued by exogenous
TNF-α. A large number of diverse pathological conditions
can be affected by cytokines from Schwann cells, making these
molecules potential targets for treating peripheral neuropa-
thies. The gene CXCL14, for example, is exclusively expressed
by Schwann cells and very prominently upregulated in PMP22
overexpressing mouse mutants, which model the pathogenesis
of Charcot-Marie-Tooth disease type 1 (Barbaria et al. 2009).
8.2 A S T RO C Y T E S
Ciliary neurotrophic factor, LIF, and IL-6 have long been
known to be expressed by astroglial cells. These factors
promote astroglial differentiation, and CNTF is known to
induce an activated astrocyte phenotype. Certain astrocyte
phenotypes may also promote myelination under certain
conditions (Nash et al. 2011). A large number of cytok-
ines synthesized and released by astrocytes are important
players in immunological and inflammatory events in the
CNS. Factors include IL-1β, -6, -8, -10, -17, -27, TNF-α,
IFN-γ, IFN-β, CCL-2, -3, -5, and CXCL-10 and -12 (Qin
and Benveniste 2012). These factors are expressed at low
levels in the healthy brain, but are significantly elevated in
multiple sclerosis, Alzheimer and Parkinson disease, and
amyotrophic lateral sclerosis (for more details on neurode-
generative diseases, see chapters 63–65). An understanding
of molecular mechanisms underlying the regulation of astro-
glial cytokines is gradually emerging. Apparently, astrocytes
regulate the chemokine network in a pathogen-specific man-
ner (McKimmie and Graham 2010). Bacterial-associated
molecules induce preferentially CCL2, CXCL1, CCL20,
and CCL3, whereas virus-associated RNAs preferentially
upregulate CXCL10 and CCL5. Astrocytes themselves can-
not respond to these chemokines; they lack most chemokine
receptors, but express CXCR4, CXCR6, and CXCR7 at
rest. Astroglia-derived cytokines are also implicated in the
regulation of protease-activated receptors (PAR) on astro-
glia, which play important roles during development and
in various inflammatory and neurodegenerative disorders
(Sokolova et al. 2012). Cytokines from astroglia, for exam-
ple, IL-1β and TNF-α, also regulate the expression of the
astroglial (and neuronal) p75 receptors (Choi and Friedman
2009).
8.3 O L I G O D E N D RO C Y T E S
Ciliary neurotrophic factor and LIF promote differentia-
tion of oligodendrocytes and oligodendrocyte progenitors.
Overexpression of CNTF in oligodendrocytes has been
exploited for remyelination and successful functional recovery
after spinal cord injury (Cao et al. 2010). Interleukin-11 has been
described as a potent factor for promoting survival, maturation,
and myelin formation in oligodendrocytes (Zhang et al. 2006).
For inducing apoptosis of oligodendroglial progenitors, exem-
plified by the OLI-neu cell line, TNF-α and TGF-β cooperate
(see TGF-β in section 9).
9 T R A N S F O R M IN G G R OW T H FAC TO R - Β
Transforming growth factor-β (TGF-β) comprises a group
of more than 30 proteins that regulate many developmental
processes, tissue homeostasis and repair (Krieglstein, 2006).
TGF-βs are divided into two major subgroups. The first
group consists of the TGF-βs, activins, nodal and myosta-
tin. The second group comprises the bone morphogenetic
proteins (BMPs) and growth differentiation factors (GDFs).
Within this superfamily, TGF-βs proper build a subfamily
of three members, TGF-β1, -β2, and -β3. TGF-βs are syn-
thesized as pre-proproteins containing a signal peptide and
a C-terminally located mature part. TGF-β1, TGF-β2, and
TGF-β3 bind to heterodimers of TGF-β receptors (TβRs)
I and II. After activation, TβR II phosphorylates and
recruits TβR I into a heterotetrameric complex that acti-
vates itself as well as downstream elements, such as Smads
(Krieglstein 2006). In addition, TGF-βs activate non-Smad
S TO R AG E A N D R E L E A S E O F N O N T R A N S M I T T E R S I G N A L I N G M O L E C U L E S F R O M M AC R O G L I A
signaling pathways, as for example p38, Jun N-terminal
kinase, and mitogen-activated protein kinase pathways (Mu
et al. 2012).
9.1 S C H WA N N C E L L S
Schwann cells are well-established sites for the synthesis of
TGF-β2 and -β3 and also carry the TGF-βRs (D’Antonio et al.
2006). TGF-β has been implicated in the control of myeli-
nation and response of adult nerves to injury. TGF-β1 defi-
ciency results in gross abnormalities in myelination, whereas
axons are normal, suggesting that TGF-β1 may directly act
on Schwann cells (Day et al. 2003). Lesioning downregulates
TGF-β2 (Stoll et al. 2004), which is involved in the regulation
of myelin phagocytosis by macrophages.
A landmark study has recently shown that Schwann cells
of nerves traversing the hematopoietic niche are responsible
for activating latent TGF-β, thereby maintaining hematopoi-
etic stem cell hibernation (Yamazaki et al. 2011). With regard
to further mechanisms that regulate TGF-β signaling, the
protooncogene Ski, an inhibitor of TGF-β signaling, is cru-
cially involved in the control of Schwann cell proliferation and
myelination (Atanasoski et al. 2004).
Schwann cells are also the source of a novel member of the
TGF-β superfamily, GDF-15, which is required for postnatal
maintenance of motoneurons in the spinal cord and sensory
neurons in spinal ganglia (Strelau et al. 2009). In addition,
Schwann cells produce BMP-6, which is axonally transported
by motoneurons and supports their survival in vitro (Wang et
al. 2007a). Cell-specific elimination of the TβR II on Schwann
cells has revealed reduced proliferation and ontogenetic cell
death (D’Antonio et al. 2006).
9.2 A S T RO C Y T E S
TGF-β1, -β2, and -β3 are expressed by astrocytes, and astro-
cytes respond to TGF-β (Krieglstein 2006). Astroglia-derived
TGF-βs execute multiple functions in the healthy and dis-
eased brain. An autocrine/paracrine loop with TGF-β
regulates one of the most prominent markers of astroglia,
GFAP, involving cooperation between canonical and non-
canonical TGF-β pathways (Stipursky and Gomes 2007).
During development, TGF-β has been hypothesized to foster
radial glia-astrocyte transformation (Stipursky and Gomes
2007). A neurotrophic role for TGF-β is well established
(Krieglstein 2006). For example, TGF-βs from astroglia
promote the survival of midbrain dopaminergic and other
neurons. Interestingly, astrocytes from different brain regions
seem to have different trophic efficacies and differ with
respect to their content or releasable pool of TGF-β. Release
of TGF-β from astrocytes can be markedly induced by FGF-2
(Dhandapani et al. 2002). The effect of FGF-2 is mediated
by MEK/ERK signaling and activation of AP-1. Similarly,
S100B, when increased in the extracellular space, has the
capacity to upregulate TGF-β in astrocytes (Reali et al.
2011). Angiotensin II induces TGF-β expression in astro-
cytes via activation of thrombospondin-1 (Lanz et al. 2010),
• 217
 Astrocytes:
TGF-α, EGF, FGF-2, FGF-9, GDNF,
IGF-1, IGF-2, NGF, NT-3, BDNF,
PDGF, LIF, CNTF, interleukins, TGF-
β1, TGF-β2, TGF-β3, BMPs
Oligodendrocytes:
Neuregulin, FGF-9, GDNF,
neurturin, persephin, IGF-
1, BDNF, NGF, NT-3, NT-4,
TGF-β1, TGF-β2, TGF-β3,
BMPs
Schwann cells:
FGF-1,FGF-2, FGF-5, GDNF,
IGF-1, NGF, BDNF,
persephin, PDGF, CNTF,
interleukins, TGF-β2, TGF-
β3, GDF-15, BMP
Figure 18.1 Schematic Overview of the Expression of Growth Factors of
Macroglial Origin. A tight network of bidirectional neuron–glial and
interglial interactions is present in central and peripheral nervous tissue.
BDNF, brain-derived neurotrophic factor; BMP, bone morphogenetic
protein; CNTF, ciliary neurotrophic factor; EGF, epidermal growth
factor; FGF, fibroblast growth factor; GDF, growth differentiation fac-
tor; GDNF, glia cell line–derived neurotrophic factor; IGF, insulin-like
growth factor; LIF, leukemia inhibitory factor; NGF, nerve growth
factor; NT, neurotrophin; PDGF, platelet-derived growth factor; TGF,
transforming growth factor.
thereby sustaining brain inflammation in a mouse model
of multiple sclerosis (concerning multiple sclerosis, see also
chapter 61). Consequently, inhibitors of the angiotensin II
type 1 receptor (which are prescribed as antihypertensives)
can interrupt this TGF-β–mediated proinflammatory path-
way. As in nonneural tissues, TGF-β regulates extracellular
matrix proteins and metalloproteinases (Hsieh et al. 2010),
and serves as a molecular link between vascular permeabil-
ity and astroglial scar formation (Schachtrup et al. 2010).
Upregulation of chondroitin sulfate proteoglycans in astro-
cytes, which are inhibitory to axonal regeneration, are promi-
nently upregulated by stimulation with TGF-β (Susarla et
al. 2011). There is, however, a differential dependence of the
synthetic machinery on Smad2 and Smad3: TGF-β induc-
tion of neurocan and xylosyl transferase 1 requires Smad2,
but not Smad3. Smad3 knockdown selectively reduces induc-
tion of chondroitin-4-sulfotransferase 1 and amounts of
218 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
4-sulfated proteoglycans released from astrocytes. Moreover,
TGF-β specifically upregulates CXCR6 expression in astro-
cytes (McKimmie and Graham 2010). High glucose induces
the expression of multiple growth factors and cytokines in
astrocytes; surprisingly, however, TGF-β does not seem to be
induced (Wang et al. 2012). TGF-β released from astrocytes
is also an important means of communication with microglia
(Liu et al. 2011). In the lesioned brain, inhibition of TGF-β,
for example, by small molecule inhibitors of the TβR-I, sup-
presses formation of the fibrotic scar and promotes regen-
eration of nigrostriatal dopaminergic axons (Yoshioka et al.
2011). TβR-I immunoreactivity has been reported to be
induced in astrocytes of patients with temporal lobe epilepsy
(Lu et al. 2009). TGF-β is increased with age, most notably
in astrocytes (together with activated microglia and mac-
rophages), and is upregulated following stroke (Doyle et al.
2010).
In addition to TGF-βs, BMP-2, -4, the BMP scavenger
noggin, and the BMPRs IA, IB, and II are detected on both
neurons and astrocytes. BMP-7 within the locus coeruleus is
most prominently expressed in astrocytes relative to neurons
and oligodendrocytes, and cell-specifically reduced in both
human subjects with major depression disorder and in rats
exposed to chronic social defeat (Ordway et al. 2011).
9.3 O L I G O D E N D RO C Y T E S
All three TGF-β isoforms are expressed by oligodendrocytes
(Du and Dreyfus 2002). For the generation of new mature oli-
godendrocytes, TGF-β is crucial (Gao et al. 2006) and has an
important impact on the regulation of the balance between
oligodendrocyte progenitor proliferation and differentiation,
employing a Notch-Jagged1 mediated pathway and astrocytes
as an intermediate (Zhang et al. 2010).
TGF-β also acts as a key proapoptotic molecule in oli-
godendroglial progenitor cells and the oligodendroglial cell
line OLI-neu (Schulz et al. 2009). The underlying mecha-
nism implies a Bcl-xl interaction with Fractin. Interestingly,
TGF-β1 and the superfamily member ActivinA induce apop-
tosis in oligodendrocytes by different pathways (Schulz et
al. 2008). ActivinA acts autonomously, without cooperating
with TGF-β. In the TGF-β death pathway, downregulation
of Bcl-xl is involved, whereas Bcl-xl in the Activin A–induced
pathway is sequestered by the BH3-only protein Puma. In the
apoptotic TGF-β pathway, caspase-3 is activated, whereas in
the ActivinA pathway, apoptosis-inducing factor is released
to trigger DNA fragmentation. An intermediate in TGF-β
signaling in oligodendroglial progenitors is the zinc finger
protein Tieg3/Klf11, which enhances TGF-β signaling by
transcriptional repression of the inhibitory Smad7. Loss of the
N-terminal repression domains of Tieg3/Klf11 abrogates the
proapoptotic effect of this transcription factor and abolishes
the enhancement of Smad-mediate TGF-β responses (Bender
et al. 2004; Gohla et al. 2008). Following a spinal cord con-
tusion, BMP-2/4 is elevated in oligodendrocytes—and astro-
cytes around the injury site (Matsuura et al. 2008). The BMP
scavenger noggin permits enhanced locomotor activity and
regrowth of the corticospinal tract, suggesting that BMP plays
an inhibitory role during axon regrowth and limits functional
recovery. Similarly, BMP-4, 6, and 7 are upregulated in spinal
cord autoimmune encephalitis in oligodendrocytes as well as
macrophages and astrocytes (Ara et al. 2008).
10 S U M M A RY A N D P E R S P E C T I VE S
The past decade has seen a great increase in the number of
growth factors, cytokines, and respective receptors produced
by and acting on macroglial cells. Although much has been
learned about regulation of their expressions (Fig. 18.1) and
implications for development and disease, significant gaps
remain on both molecular/cellular and systems levels. For
example, modes of intracellular storage and mechanisms of
release are enigmatic for many of these proteins. We also have
to admit that our knowledge on their regulatory and inter-
active networks is fragmentary—it almost does not exist.
Comprehensive analyses based on systems biology approaches
have hardly been conducted. And, if properly planned, the
respective strategies would definitely have to include contri-
butions from neurons. Thus, hopefully the coming years will
see an expanded perception of glial cell biology with implica-
tions of glia-derived growth factors both on the molecular and
systems level.
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 19.
PHYSIOLOGY OF MICROGLIA
Mami Noda and Alexei Verkhratsky

A B B R E VI AT I O N S NFAT nuclear factor of activated T cells

NK-1 neurokinin-1
Aβ amyloid β NMDA N-methyl-d-aspartate, N-methyl-d-aspartic
ADP adenosine diphosphate acid
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazole NO nitric oxide
propionic acid PAF platelet-activating factor
AQ4 aquaporin4 PPADS pyridoxal-phosphate-6-azophenyl-2′,
ATP adenosine triphosphate 4′-disulfonate
AT2 angiotensin II receptors type 2 PrP prion protein
Best bestophine RANTES regulated on activation, normal T cell
BzATP 2′,3′-(benzoyl-4-benzoyl)-ATP expressed and secreted
CaRs plasmalemmal calcium receptors RyRs ryanodine receptors
C3a and C5a receptors to complement fragments TNF-α tumor necrosis factor-α
CB cannabinoid TGF-β transforming growth factor-β
CCR chemokine (C-C motif ) receptor Trk-B1 truncated tropomyosin-related kinase B-T1
CCL C-C motif chemokine ligand TRP transient receptor potential
CD200Rs CD200 receptors TRPM transient receptor potential cation channel
CLIC-1 chloride intracellular channel-1 subfamily M
COX-2 cyclooxygenase-2 TRPV transient receptor potential vanilloid
CREB cAMP response element binding protein TTX tetrodotoxin
CSF-1R colony stimulating factor-1 receptor VIP vasoactive intestinal peptide
CXCR C-X-C chemokine receptor Xc glutamate-cystine antiporter
CysLT cysteinyl leukotriene
EGFR epidermal growth factor receptor
ET endothelin 1 INTRODUCTION
FPR formyl peptide receptor
GABA gamma-aminobutyric acid Fetal macrophages/ameboid microglia, which migrate into
GLUT5 glucose transporter 5 the central nervous system (CNS) (see chapters 8 and 15),
GPCR G-protein–coupled receptors undergo fundamental phenotypic changes and become the
HIV-1 human immunodeficiency virus-1 innate immune cells generally referred to as microglia (Del Rio-
ICRAC Ca2+-release activated Ca2+ current Hortega 1932; Hanisch and Kettenmann 2007; Kettenmann
INa sodium currents et al. 2011). These changes in phenotype include a profound
IFN-γ interferon-gamma morphological metamorphosis that turns ameboid microglia
IKDR delayed rectifying K+ currents into highly specialized surveillance cells with small somata and
IK IR inward rectifier K+ currents long highly motile processes (“ramified” microglia), which are
IL-1 interleukin-1 accompanied with similarly profound changes in cell physiol-
InsP3 inositol 1,4,5-trisphosphate ogy. Microglia residing in the nervous system acquire numer-
LPA lysophosphatidic acid ous receptors to neurotransmitters, neuromodulators, and
LPS lipopolysaccharide neurohormones that allow them to monitor the normal activ-
MAPK mitogen-activated protein kinase ity of neural cells in search for possible signs of damage; mul-
MCP-1 monocyte chemotactic protein-1 tiple chemical factors originating from neurones, astrocytes
M-CSFR macrophage colony-stimulating factor and oligodendrocytes control microglial activation (Biber et al.
receptor 2007). The physiology of microglia is surveyed in this chapter,
mGluR metabotropic glutamate receptor including their membrane properties, expression of receptors
nAChRs nicotinic acetylcholine receptor and transporters; with specific focus on receptors related to
NCX Na+/Ca2+ exchanger CNS context. For many other aspects of microglial physiology,

223
including specific expression of immune-related molecules (e.g.,
pattern recognition receptors) as well as for microglial surveil-
lance–related motility, the reader is referred to other chapters
in this volume (see chapters 8, 15, 47, 48, 49, 50 and 53).
2 M E M B R A N E P R O P E RT I E S
OF MICROGLIAL CELLS
The general membrane properties of microglial cells very
much depend on the experimental preparation and activa-
tion status of microglia. In primary cultures from rodents and
humans (in which microglial cells acquire certain activation
status) the resting membrane potential is approximately –50
mV (Kettenmann et al. 1990; Norenberg et al. 1994b). The
membrane permeability of these cultured cells is dominated
by inward rectifying K+ currents. In contrast, microglial cells
(identified by tomato lectin vital staining) in acutely isolated
slices from cortex, striatum, and facial nucleus, obtained from
8- to 12-week-old rats, have very low membrane potential
(on average approximately –20 mV), high input resistance,
and very small (if any) voltage-gated currents (Boucsein et
al. 2000). Similar properties were observed in microglia in
forebrain slices from 6- to 8-week-old mice; these cells lack
voltage-gated currents and had a relatively low membrane
potentials (with two subpopulations having an average of Vm
approximately –52 mV and –29 mV) (Boucsein et al. 2003).
Incidentally, holding microglial cells in situ at hyperpolar-
ized potentials (–70 mV) lead to their rapid disintegration
(Boucsein et al. 2000; Brockhaus et al. 1993).
Ca2+-permeable
channels
CRAC
Orai/Stim1
TRPM2, TRPM6,
TRPM7,TRCPC6,
TRPV1
Na+ channels?
Nav1.1
Nav1.5
Nav1.6
Aquaporins
AQP4
Proton channels
Hv1
Figure 19.1 Ion Channels Expressed in Microglia.
224 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
The membrane properties of ameboid microglial cells
studied in corpus callosum slices from early postnatal (6- to
9-day-old) mice were more similar to those obtained in cul-
tured microglia: these ameboid cells had more hyperpolarized
resting membrane potential (on average approximately –42
mV) and expressed a prominent inward rectifier potassium
current (Brockhaus et al. 1993).
Activation of microglial cells by pathological stimuli
induces substantial changes in their membrane properties.
In primary cultures, exposure to bacterial lipopolysaccharide
(LPS) or interferon-gamma (IFN-J) induced the expression
of an outward K+ current (Norenberg et al. 1992). Similarly,
in experiments in situ, microglial activation in facial nucleus
following a surgical lesion to the facial nerve led to substan-
tial increase in both the inward rectifier and outward delayed
rectifier K+ currents. This increase was maximal 24 hours
after surgery (Boucsein et al. 2000). Ischemic insult as well as
experimental status epilepticus caused similar upregulation in
K+ currents 24 to 48 hours after the surgery/kainate injection
(Avignone et al. 2008; Lyons et al. 2000).
3 ION CHANNELS IN MICROGLIA
Microglial cells express multiple sets of ion channels
(Fig. 19.1), and this expression is often altered after micro-
glial activation. Functional expression of voltage-gated
sodium channels in microglia is debatable. Small sodium
currents (INa) with peak amplitudes of approximately 50 pA
were described in cultured rat and human microglial cells
K+ channels
Inward rectifier
Kir2.1
Ca2+-dependent
BK/KCa1.1,
KCNN1-4/SK1-4
Delayed rectifier
Kv1.1,Kv1.2,
Kv1.3,Kv1.5
Anion channels
CLIC-1, Best1-4
(Korotzer and Cotman 1992; Norenberg et al. 1994a). The
INa in cultured microglia was TTX-sensitive (complete inhi-
bition at 2–5 PM), and had a very rapid kinetics and charac-
teristic voltage dependence (threshold approximately –40 mV
and peak approximately 0 mV). Sodium currents were found
only in 20% of cultured rat microglial cells. Incidentally, the
cells with ramified morphology had a higher incidence of INa
detection (Korotzer and Cotman 1992). In human microglia
(which were cultured from explants obtained during sur-
gery on patients suffering from brain tumors; hence, these
cells could have been pathologically modified), the majority
of cells (30 out of 32 recorded) expressed TTX-sensitive INa
(Norenberg et al. 1994b). On a molecular level expression of
TTX-sensitive Nav1.1 and Nav1.6 and TTX-resistant Nav1.5
channels was identified by specific antibodies in rat microglial
cultures (Black et al. 2009). The sodium channel Nav1.6 was
also found in activated microglia in autoimmune encepha-
lomyelitis (Craner et al. 2005). Nonetheless, the relevance
for Na+ channels in microglial physiology remains unclear
because many laboratories failed to detect INa in resting as well
as in activated microglia cells (see Kettenmann et al. 2011 for
further details).
The functional expression of voltage-gated Ca2+ channels,
which were occasionally recorded from cultured microglia,
remains similarly questionable. There are also evidence indi-
cating upregulation of L-type Ca2+ channels in microglial
cultures exposed to β-amyloid Aβ25–35, prion protein PrP 106–
126 (Silei et al. 1999), HIV-1 regulatory protein Tat, or CCR5
cytokine receptor agonist RANTES (CCL5) (Shideman et al.
2006). However, these observations remain isolated and most
of the laboratories have not confirmed the presence of func-
tional voltage-gated Ca2+ channels in microglia (Kettenmann
et al. 2011).
Microglial cells express another type of Ca2+-selective plas-
malemmal channel responsible for the Ca2+-release activated
Ca2+ current, ICRAC. The ICRAC was identified in whole-cell
patch-clamp experiments on cultured microglia (Norenberg
et al. 1997). Subsequently, ICRAC was measured from both
resting and activated mouse microglial cells. In the resting con-
ditions ICRAC amplitude was approximately 1.2 to 1.3 pA/pF;
whereas LPS-induced activation significantly reduced the
amplitude of the current to approximately 0.5 pA/pF 48 hours
after exposure to LPS (Beck et al. 2008). The molecular sub-
strate for ICRAC is represented by Orai proteins, which were
identified at the transcriptional level in cultured neonatal rat
microglia. Electrophysiological and pharmacological studies
indicated that highly Ca2+-selective (PCa/PNa > 1,000) Orai1/
CRAC channels are responsible for ICRAC generation in micro-
glia (Ohana et al. 2009).
Microglial cells are in possession of several types of tran-
sient receptor potential (TRP) channels. At a transcriptional
level (in cultured neonatal rat microglia) the following rank
order of mRNA expression was found: TRPM7 > TRPC6 >
TRPM2 > TRPC1 > TRPC3 ≥ TRPC4 > TRPC7 > TRPC5
> TRPC2 (Ohana et al. 2009). Activation of TRPM2 by
H2O2 or by cADP ribose resulted in Ca2+ influx and cationic
currents that were significantly potentiated by LPS (Kraft et
al. 2004). The TRPM4- and TRPM7-mediated currents were
P H YS I O L O GY O F M I C R O G L I A
also recorded from rodent-cultured microglia (Beck et al.
2008). There is evidence that TRPV1 channels initiate micro-
glial cell death, whereas TRPM2 channels somehow can be
involved in microglial responses to ischemia (see Kettenmann
et al. 2011 for further details).
Potassium channels are widely expressed by microglial cells
at various activation states. The inward rectifier K+ currents
(IKIR) are present in all types of activated and cultured micro-
glial cells. They are absent in resting microglia in situ. At the
single channel level two unitary conductances (30 and 43 pS)
with inward rectification have been identified (Kettenmann
et al. 2011 and references therein). The IKIR can be consid-
ered as an early hallmark of microglial activation. Activation
of microglia by in vivo ischemia or facial nerve lesions results
in upregulation of inward rectifier current accompanied with
a hyperpolarizing shift in the resting membrane potential
(Boucsein et al. 2000; Lyons et al. 2000). Similarly, in post-
natal (5- to 7-day-old) mice approximately 75% of resting
microglial cells recorded in hippocampal slice had a relatively
small (mean current density approximately 3.6 pA/pF) IKIR .
In activated microglia from the same organotypic slices cul-
tured for 3 to 7 days the current was expressed in 90% of cells
and its density increased threefold (to ~9.6 pA/pF) (Schilling
and Eder 2007).
Activation of microglia (both in vitro and in situ) is also
accompanied by an appearance of delayed rectifying K+ cur-
rents (IK DR), which most likely results from de novo synthesis
of channel molecules (Boucsein et al. 2000; Lyons et al. 2000;
Norenberg et al. 1992). Cultured rat microglia express mRNA
specific for Kv1.2, Kv1.3, and Kv1.5 channels; LPS-induced acti-
vation upregulates expression of Kv1.3 (Fordyce et al. 2005).
In contrast, in vivo microglial activation resulted in an increase
in immunoreactivity for Kv1.5 but not for Kv1.3 channels ( Jou
et al. 1998). Incidentally, treatment of microglial cultures
with “calming” signaling agent transforming growth factor-β
(TGF-β) induced a fivefold increase in the Kv1.3 mRNA level
and a sixfold increase in delayed rectifying K+ current density
(Schilling et al. 2000). Amplitudes of IKDR and expression of
Kv1.3/Kv1.5 channels were also increased following treatment
of cultured microglia with pneumococcal cell walls, β-amyloid
protein fragments 25 to 45, or experimentally induced (sys-
temic kainate injection) status epilepticus (see Kettenmann et
al. 2011 for further details). Manipulation with expression or
function of delayed rectifier K+ channels affects various aspects
of microglial function. For example, antisense or knock-out
deletion of Kv1.5 channels resulted in inhibition of NO release
from LPS activated microglia; deletion of Kv1.3 and Kv1.5
channels increased microglial proliferation. Pharmacological
inhibition of Kv1.3 channels reduce respiratory burst in cul-
tured microglia (see Kettenmann et al. 2011 for review).
As for other voltage-gated K+ channels, human ether-
a-go-go–related gene product (HERG)-like K+ channel was
reported in microglial cell line (Pennefather et al. 1998; Zhou
et al. 1998) and was also confirmed in primary cultured rat
microglia (Noda et al. unpublished data) as well as the expres-
sion of Kv7 (KCNQ) channels (Noda et al. 2007b).
Cultured microglia express several types of Ca2+-dependent
+
K channels represented by: (1) high-conductance (BK)
• 225
channels, with reported single channel conductances of 106
pS, 149 pS, 187 pS, and 240 pS, depending on experimen-
tal preparation (Bordey and Spencer 2003; McLarnon et al.
1997); and (2) small conductance Ca2+/calmodulin acti-
vated K+ channels of KCNN4/KCa3.1/SK4/IK1 type (pre-
dominant type) and KCNN2/SK2 and KCNN3/SK3 types
(Kaushal et al. 2007; Khanna et al. 2001). The importance of
BK channels in microglia in bradykinin- and galanin-induced
migration (Ifuku et al. 2007, 2011) and nerve injury–induced
neuropathic pain (Hayashi et al. 2011) was deduced based on
various pharmacological assays. The immunoreactivity for
KCNN3/SK3 was found in microglial cells in healthy adult
rat striatum. This immunoreactivity increased after ischemic
lesion (Schlichter et al. 2010). The SK3 and SK4 channels are
reportedly involved in the control of microglia activation and
contribute to the upregulation of iNOS and the production
of NO and peroxynitrite, which exert neurotoxic effects. The
G-protein–activated K+ currents were also described in cul-
tured murine microglia (Ilschner et al. 1995).
Outwardly rectifying, voltage- and time-independent
volume-sensitive Cl– currents were detected in cultured murine
microglial cells, where they were suggested to regulate prolif-
eration and to be involved in setting the resting membrane
potential. These channels have a very small unitary conduc-
tance (1–3.5 pS) and are possibly represented by Bestophine
(Best) channel family; expression of Best 1–4 mRNAs have
been detected in rat microglial cultures (Ducharme et al.
2007). In addition, microglial cells were reported to express
chloride intracellular channel-1 (CLIC-1) in the plasma mem-
brane (unitary conductance 6.5–8 pS). Expression and activ-
ity of CLIC-1 channels were potentiated by Aβ1–42 or Aβ25–35
peptides; inhibition of CLIC-1 channels reduced neurotoxic-
ity (Kettenmann et al. 2011; Milton et al. 2008).
The proton channels, characterized by an exceptional selec-
tivity to H+ ions, were detected in primary microglial cultures
from mice, rats, and humans (Eder and DeCoursey 2001).
These channels have a very small (fS range) unitary conduc-
tance and their function is inhibited following LPS-induced
microglial activation. It is generally assumed that microglial
H+ currents play a role in the “respiratory burst” associated
with phagocytosis (Eder and DeCoursey 2001). Activated
microglial cells have also been reported to express aquaporins
of AQP4 type as well as Cx36 and Cx43 connexins; func-
tional role of these channels remain unknown (Kettenmann
et al. 2011).
4 R E C E P TO R S I N M I C R O G L I A
4.1 N EU ROT R A NS M IT T E R R EC E P TO R S I N
M I C RO G L I A
Acquisition of neurotransmitter receptors (Fig. 19.2) argu-
ably represents the most profound phenotypical digression of

Purinoceptors

P2X4, Glutamate
P2Y2
P2X7 receptors
P2Y6 GluR1-4
P2Y12 Glu R5
A1, A2A, P2Y13
A2B, A3 mGluR5

InsP3 InsP3
Dopamine K+ mGluR2,3
receptors cAMP mGluR4,6,8
D1, D2 cAMP
D3, D4 cAMP
K+
InsP3
GABAB(1a),
InsP3 GABAB(1b),
GABAB(1c),
α1A,α2A
cAMP InsP3
GABA
β1, β2 receptors

5-HT2
Adrenoreceptors 7-TM
nAChR receptors
α3,α5,α7 Serotonin
β4 Ionotropic
receptors
receptors
Cholinoreceptors
Ion
channels

Figure 19.2 Neurotransmitter Receptors in Microglia.

226 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
microglia from their fetal macrophage ancestors. By express-
ing neurotransmitter receptors, microglia adapt to the CNS
environment and attain the ability to sense chemical signals
employed in communications between neural cells. Microglial
neurotransmitter receptors can either trigger microglial acti-
vation (“on” signals) or promote maintenance of resting, sur-
veillance state (“off ” signals) (Biber et al. 2007; van Rossum
and Hanisch 2004).
Probably the most widespread microglial neurotransmitter
receptors are purinoceptors activated by adenosine triphos-
phate (ATP), adenosine, and related nucleotides. ATP acts as
a widespread neurotransmitter and is also an ancient “danger”
signal because massive ATP release invariably accompanies
cell death. Numerous observations in vitro, in situ, and in vivo
have demonstrated that ATP and its analogs trigger rapid
functional responses of microglial cells, comprising fast con-
verging movement of microglial processes toward the lesion,
membrane ruffling, the outgrowth of microglial processes,
and the release of various biologically active substances, such
as cytokines and inflammatory proteins (Davalos et al. 2005;
Kettenmann et al. 2011; Nimmerjahn et al. 2005). Microglial
cells usually express several types of purinoceptors and expres-
sion pattern of these receptors changes with microglial activa-
tion (Moller et al. 2000b).
The ionotropic P2X purinoceptors are classic cati-
onic (Na+/K+/Ca2+) ligand-gated channels assembled as
homo-trimers or hetero-trimers from seven different subunits
classified as P2X1 to P2X7. The P2X receptors have substantial
Ca2+ permeability, which may vary, depending on the subunit
composition (PCa/Pmonovalent), between 1 and greater than 10
(Pankratov et al. 2002, 2009), and could be even greater for
the pore-forming P2X7 receptors (Egan et al. 2006). The main
type of ionotropic purinoceptors expressed in mature microg-
lial cells are P2X4 and P2X7 receptors.
The P2X4 receptors are fundamental for microglial activa-
tion and in particular have been found to be instrumental for
pathogenesis of neuropathic pain. The genesis of neuropathic
pain is associated with long-lasting activation of microglia in
the spinal cord (Inoue and Tsuda 2009). The specific role for
P2X4 receptors in mediating microglial activation in neuro-
pathic pain was initially suggested following experiments on
the rat spinal nerve injury model in which L5 spinal nerve was
surgically severed. The tactile allodynia (the symptom of neu-
ropathic pain) which developed following nerve lesion was
specifically alleviated following intrathecal injection of P2X
receptor antagonist TNP-ATP, while being not affected by
another antagonist PPADS. This peculiar sensitivity of tac-
tile allodynia to P2 receptor antagonists (TNP-ATP inhibits
P2X1–4 receptors, whereas PPADS blocks all P2X receptors
except P2X4) indicated the role for P2X4 receptors (Tsuda et
al. 2003). Further investigations revealed a significant increase
in P2X4 receptors expression in the spinal cord microglia fol-
lowing nerve lesion. Similarly, increase in P2X4 receptors in
spinal cord microglia was observed in rats with experimental
autoimmune neuritis and following intraperitoneal injection
of LPS (which triggered hyperalgesia and microglia activa-
tion). An increased P2X4 immunoreactivity in the spinal cord
was confined to OC42 positive microglial cells; moreover,
P H YS I O L O GY O F M I C R O G L I A
intrathecal injection of activated cultured microglia expressing
P2X4 receptors triggered allodynia without the need of surgi-
cal nerve lesion (see Inoue and Tsuda 2009 and Kettenmann
et al. 2011, for details).
The pore-forming P2X7 receptors represent another type of
purinoceptors specifically relevant for microglial function. The
P2X7 receptors have several unique features, which include:
(1) exceptionally low sensitivity to ATP; several mM of ATP
are needed to activate the receptor; (2) modulation of ATP
sensitivity by extracellular divalent cations; and (3) ability of
P2X7 receptors to form large membrane pores (permeable to
molecules with mw up to 900 Da) on strong stimulation. The
pore formation may result from the dilatation of the channel
permeation path or from the activation of other pore-forming
proteins (Pelegrin and Surprenant 2009). It has to be noted
that 2′,3′-(benzoyl-4-benzoyl)-ATP (BzATP) often used
as a specific activator of P2X7 receptors, activates other
P2X subunits with high potency. P2X7 receptors are widely
expressed in the cells of the immune system and mediate many
immune reactions, including the processing and the release of
various cytokines. The P2X7 mediated currents were for
the first time identified in ameboid microglial cells in situ
(Haas et al. 1996); simultaneously the P2X7-mediated [Ca2+]i
increases were found in freshly isolated mouse microglia
(Ferrari et al. 1996).
In the healthy brain P2X7 receptors are present in micro-
glial cells in all regions; brain damage triggers massive
upregulation of microglial P2X7 expression (see Sperlagh
et al. 2006, for a review). Similarly, microglial P2X7 recep-
tors expression is significantly upregulated in various neuro-
pathologies, including multiple sclerosis, amyotrophic lateral
sclerosis, and Alzheimer disease (see Kettenmann et al. 2011
for a detailed review). Activation of P2X7 receptors controls
multiple microglial functions. Stimulation of P2X7 receptors
can trigger microglial cell death via apoptosis and autophagy.
Overexpression of P2X7 receptors triggers microglial activa-
tion in the in vitro system; similarly P2X7 receptors are nec-
essary to launch microglial activation in response to in vivo
injections of Aβ protein (Monif et al. 2009; Sanz et al. 2009).
Finally, activation of P2X7 receptors can induce both neuro-
toxicity and neuroprotection in a context-dependent manner.
These functional responses are mediated through a variety of
intracellular transcription factors (e.g., CREB, NFAT, MAP
kinases, etc.) to which P2X7 receptors are linked (Kettenmann
et al. 2011). In addition, P2X7 receptor signaling controls basal
and TNF-α–stimulated glial cell proliferation by regulating
AQP4 (Zou et al. 2012).
Microglial cells express a variety of metabotropic P2Y
receptors, among which P2Y2, P2Y6, P2Y12, and P2Y13 seems
to be dominating. Activation of these receptors induces Ca2+
signaling, including [Ca2+]i oscillations and often triggers sec-
ondary store–operated Ca2+ influx. In ramified microglial cells
in acute slices from adult mice activation of P2Y receptors
trigger an activation of outward rectifying K+ conductance
(Boucsein et al. 2003). The P2Y receptors regulate micro-
glial release of cytokines and expression of immediate early
genes. The UDP-preferring P2Y6 receptors regulate micro-
glial Ca2+ signaling and induction of phagocytosis (Koizumi
• 227
et al. 2007). The ADP-preferring P2Y12 receptors (which in
the brain are predominantly expressed in microglia) control
early microglial responses to injury, being critical for induc-
ing the morphological activation, membrane ruffling, and
chemotaxis (Kettenmann et al. 2011). The P2Y12 receptor con-
trol integrin-β1 signaling cascade, which regulates extension
of microglial processes (Ohsawa et al. 2010). In the spinal cord
P2Y12 receptors are involved in the genesis of neuropathic pain
(Inoue and Tsuda 2009).
Microglia also possess adenosine receptors. All four sub-
types (A1, A2A, A2B, and A3) were detected in microglial cells
from various species. Adenosine receptors control microglial
proliferation, K+ currents, release of prostaglandins and neu-
roprotective cascades (Kettenmann et al. 2011).
Microglial cells contain ionotropic glutamate receptors pre-
dominantly of AMPA/Kainate type; expression of all 4 GluR
subunits for AMPA was detected in microglial cells in vitro. A
minor subpopulation of cultured spinal microglia expressed
functional GluR5 kainate receptors; whereas GluR6,7 recep-
tors were found only in microglial cell line (Noda et al. 2000;
Yamada et al. 2006). Functional expression of NMDA recep-
tors in microglia remains questionable. In addition microglia
express several types of metabotropic glutamate receptors
including mGluR5 linked to inositol 1,4,5-trisphosphate
(InsP3) production and Ca2+ signaling (Biber et al. 1999) and
mGluR2,3 (Group II) and mGluR4,6,8 (Group III) recep-
tors linked to cAMP signaling cascade; activation of group II
receptors triggered microglial activation, neurotoxicity and
promoted the release of TNF-α, whereas activation of group
III receptors reduced microglial neurotoxicity following treat-
ment with LPS or chromogranin A (Kettenmann et al. 2011;
Taylor et al. 2002).
Functional GABAB receptors (in all three splice variants
GABAB(1a), GABAB(1b) and GABAB(1c)) were found in cul-
tured microglia and in about 50% of tomato-lectin positive
cells in brain slices; stimulation of GABAB receptors triggered
Ca2+ signaling and activated an outwardly rectifying K+ cur-
rent (Kuhn et al. 2004). Microglial cells also expressed several
subunits of nicotinic acetylcholine receptors, including neu-
ronal α7nAChR subunit. Activation of nAChRs generally
inhibits the immune response of microglial cells thus repre-
senting an endogenous “cholinergic antiinflammatory path-
way” (Shytle et al. 2004). There are also some indication that
cultured microglia may possess muscarinic cholinoreceptors
linked to InsP3-mediated Ca2+ signaling (Kettenmann et al.
2011). Furthermore, microglial cells express α1A, α2A, β1,
and β2 adrenoreceptors (Kettenmann et al. 2011; Tanaka et
al. 2002). The β2 receptors are positively coupled to adeny-
lyl cyclase, whereas α adrenoreceptors are coupled to Ca2+
signaling. Selective stimulation of α1 or β1 or β2 receptors sup-
pressed the expressions of IL-6 and TNF-α Microglial cells
also contain D1,2,3,4 dopamine receptors involved in negative
regulation of inward rectifier and positive regulation of out-
ward K+ currents and also involved in chemotaxis (Kettenmann
et al. 2011). Finally, functional expression of 5-HT2 sero-
tonin receptors modulating K+ currents and triggering Ca2+
signaling was recently discovered in murine microglial cells
(Krabbe et al. 2011). Some microglial neurotransmitter
228 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
receptors are linked to microglial superoxide production via
Nox activation to promote neuroprotection or neurotoxicity
(Mead et al. 2012).
4.2 R EC E P TO R S F O R N EU RO H O R M O N E S A N D
N EU RO M O D U L ATO R S I N M I C RO G L I A
Microglial expression of receptors for neurohormones and
neuromodulators is summarized in Figure 19.3; the majority
of these receptors belong to 7-transmembrane G-protein–
coupled receptors (7-TM GPCRs), which are linked either to
PLC/InsP3 metabolism and Ca2+ signaling or coupled (via G
proteins) to adenylate cyclase, K+ channels, MAP kinases, or
other signaling cascades.
The platelet-activating factor (PAF) receptors mediate
strong chemotactic response of microglial cells which involves
G-protein and mitogen-activated protein kinase (MAPK)
signaling pathways. PAF receptors also induce robust
[Ca2+]i transients originated from both ER Ca2+ release and
store-operated Ca2+ entry (Aihara et al. 2000). Bradykinin
receptors of B2 subtype are constitutively expressed in micro-
glia. These receptors trigger InsP3-mediated Ca2+ signaling
with subsequent activation of K+Ca channels. Activation of
microglia induces synthesis of B1 bradykinin receptors and
upregulation of B2 receptors expression. The B1 receptors in
activated microglia positively modulate Na+/Ca2+ exchanger
as well as K+Ca channels and induce chemotaxis (Kettenmann
et al. 2011; Noda et al. 2003). The role of microglial B1 recep-
tor is contradictory, either harmful as reported in diabetic pain
neuropathy (Talbot and Couture 2011; Talbot et al. 2010) or
neuroprotective (Noda et al. 2007a; Yasuyoshi et al. 2000).
Expression of B1 receptors in the spinal cord microglia is also
upregulated following activation of TRPV1 channels induced
by capsaicin (Talbot et al. 2012). The role of kinins in the CNS
is reviewed in Noda (2011a). Cultured microglial cells have
histamine receptors linked to InsP3-mediated Ca2+ signaling
(Kettenmann et al. 2011). The ETB endothelin receptors were
identified in mouse primary microglia. Stimulation of these
receptors induced InsP3-mediated Ca2+ release from the ER
with subsequent store-operated Ca2+ entry in a subpopulation
of microglia (Moller et al. 1997a). Microglial cells respond to
cannabinoids, although activation of CB1 and CB2 receptors
coupled to Gi/o and Gi proteins, respectively; expression of
these receptors is strongly upregulated following microglial
activation. Generally, the activation of cannabinoid recep-
tors increases microglial proliferation (Carrier et al. 2004)
and reduces microglial neurotoxicity and neuroinflammation
(Stella 2008).
The resting microglia express angiotensin II recep-
tors type 2 (AT2), whereas microglial activation induces
additional expression of type 1 angiotensin receptors AT1.
Pharmacological inhibition of AT1 receptors suppresses mor-
phological activation of microglia, LPS-induced activation
of NF-NB and reduces production of nitric oxide (NO) and
IL-1β (Miyoshi et al. 2008). Cultured rat microglial cells were
reported to express sst2, sst3 and sst4 types of somatostatin
receptors, stimulation of which inhibits microglial prolifera-
tion (Feindt et al. 1998). Several types of opioid receptors were
 orexin
Platelet-activating receptors
factor(PAF) OX1R
receptor

Bradykinin Neurotrophin
receptors, receptors,
B1, B2 Trk-B1
InsP3/Ca2+ cAmP,
ruffling

InsP3/Ca2+
InsP3/Ca2+ VIP
Endothelin NCX receptors,
receptors, chemotaxis – VPAC1
ETB
InsP3/Ca2+ activation

NF-κB Neurokinin
(Substance P)
InsP3/Ca2+ receptors,
Histamine
chemotaxis NK-1
receptors,
migration
ETB ?
Proliferation
+ Opioid
receptors,
Angiotensin II KOR, MOR
receptors,
AT1, At2

Cannabinoid Somatostatin
receptors, receptors,
CB1, CB2 sst2,sst3,sst4 7-TM
receptors
Tyrosine
kinase
receptors

Figure 19.3 Receptors for Neurohormones and Neuromodulators in Microglia.

identified in primary human and feline microglia. The kappa-
opioid receptors (KOR) regulate microglial immune response
(specifically in HIV-1 dependent encephalopathies), whereas
mu-opioid receptor facilitate microglial activation and nega-
tively regulate chemotaxis (Chao et al. 1997; Dobrenis et al.
1995). Neurokinin-1 (NK-1 or substance P) receptors are pres-
ent in cultured murine and fetal human microglia; they control
activation of transcriptional factor NF-NB, regulate produc-
tion of cytokines, and may enhance inflammatory responses
induced by bacterial infections of CNS (Kettenmann et al.
2011). The VPAC1 type of vasoactive intestinal peptide (VIP)
receptors identified in cultured rat microglia, inhibit TNF-α
IL-1β and NO production and release in LPS-activated cells,
reduce microglial neurotoxicity and suppress microglial acti-
vation in vivo (Delgado and Ganea 2003; Kettenmann et al.
2011). Cultured rat microglia also express the subtype of Trk
neurotrophin receptors, the truncated tropomyosin-related
kinase B-T1 (Trk-B1) receptors. Activation of these receptors
by BDNF triggers sustained [Ca2+]i elevation mediated by an
initial PLC/InsP3-driven Ca2+ release from the ER followed
by a long-lasting activation of the store-operated Ca2+ entry.
In addition, exposure of activated microglial cells to BDNF
decreased release of NO (Mizoguchi et al. 2009). Although
direct evidence of the expression of specific type of benzodiaz-
epine receptor has not been reported, benzodiazepine recep-
tor agonists decreased the ATP-induced increase of COX-2
gene expression and release of IL-1β and TNF-α (Choi et al.
2011). Likewise, neuropeptide Y receptors (Y1 receptor) are
supposed to inhibit interleukin-1β–induced phagocytosis in
microglial cell line (Ferreira et al. 2011).
4.3 C Y TO K I N E S A N D C H E M O K I N E
R EC E P TO R S I N M I C RO G L I A
Microglial cells are in possession of several types of cytokine
and chemokines receptors (Fig. 19.4), which regulate a multi-
tude of immune responses. The chemokines are chemoattrac-
tive cytokines involved in regulation of cell migration, which
act as diffusable messengers able to create chemotactic gradi-
ents for cell migration. A distinction as to inflammatory and
homeostatic chemokines is based on the inducible versus con-
stitutive expression. The chemokines are small proteins with
mw between 8 and 12 kDa that are classified into four groups
of C, CC, CXC, and CX3C chemokines (see Laing and
Secombes 2004 for a review). Chemokines are expressed in the
CNS in neurons, macroglial cells, and microglia. Microglial

P H YS I O L O GY O F M I C R O G L I A • 229
cells in particular express CCR1, CCR2, CCR7 and CCR5
(rodents), and CXCR1, CXCR3, and CCR3 (humans)
(Boddeke et al. 1999a,b; Flynn et al. 2003). Chemokine recep-
tors are 7-transmembrane domain G-protein–coupled recep-
tors linked to multiple signaling cascades that include adenyl
cylase, phospholipases, GTPases (Rho, Rac, and Cdc42),
and some kinases such as mitogen-activated protein kinase
(MAPK) or phosphatidyl inositol-3 kinase (PI3-K) (Biber
et al. 2008). Stimulation of cultured naïve and LPS treated
microglia with β-chemokines MCP-1 (or C-C chemokine
CCL2; agonist of CCR2 receptors) or MIP-1α and RANTES
(agonists of CCR5 receptors) resulted in Ca2+ signals. These
[Ca2+]i responses were larger in activated cells. The CCR5-
dependent MIP-1α and RANTES-induced Ca2+ signals most
likely involve InsP3-dependent Ca2+ release, whereas MCP-1
induced Ca2+ signaling was also sensitive to ryanodine and
plasmalemmal Ca2+ influx (Boddeke et al. 1999b). In addition,
activation of chemokine receptors stimulated Ca2+-dependent
K+ currents and activated volume-regulated Cl– channels
linked to microglial migration (see Kettenmann et al. 2011 for
details). Expression of chemokine receptors is modified during
microglial activation and during neuropathology (Kremlev
et al. 2004). For example, the levels of CCR5 receptors (on
transcriptional level) were increased following hypoxic-isch-
emic insults and nerve injury. Microglial cells from post-mor-
tem Alzheimer disease brains had increased levels of CCR3
and CCR5 receptors and increased levels of CCR5 receptors
were also found in biopic material from patients with early
stages of multiple sclerosis. Likewise, CCL2 (MCP-1) levels
in the cerebrospinal fluid (CSF) at baseline correlated with
TNF-α receptors
TNFR1,2
Inhibition of
activation,
neuroprotection
IFNγR
Induction of
Interferon receptors immunoproteosome
neuroprotective
phonotype
IFNAR
7-TM
receptors
Cytokine IL-18R
receptors IL-15Rα
Figure 19.4 Cytokines and Chemokines Receptors in Microglia.
230 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
a faster cognitive decline in Alzheimer disease (Westin et al.
2012).
Receptors for cytokines include tumor necrosis factor-α
receptors, interleukin receptors, and receptors to interferon
β and J. Microglial cells express two types of TNF-α recep-
tors, TNFR1 and TNFR2, which positively modulate
microglial activation and phagocytosis (Kettenmann et al.
2011). The interleukin-1 (IL-1) receptors are represented by
IL-1 type-I receptor (IL-1RI), IL-1 type-II receptor (IL-1RII),
and IL-1 receptor accessory protein (IL-1RAcP). At mRNA
level resting microglia express IL-1RII, and microglial activa-
tion upregulates expression of IL-1RI, IL-1RII, and IL-1RacP.
In human microglial cells mRNA transcripts for multiple
interleukin receptors (IL-1RI, IL-1RII, IL-5R, IL-6R, IL-8R,
IL-9R, IL-10R, IL-12R, IL-13R, and IL-15R) were identified
(Lee et al. 2002).
4.4 OT H E R R EC E P TO R S Y S T E M S I N M I C RO G L I A
Microglial cells, studied in vitro, in situ, and in vivo, express
many other receptors systems (Fig. 19.5) (see also Kettenmann
et al. 2011, for a detailed description and relevant references).
Microglia are endowed with glucocorticoid and mineralocor-
ticoid receptors mediating multiple pleiotropic effects (Tanaka
et al. 1997). Cultured microglia also express plasmalemmal
calcium receptors CaRs (the 7-transmembrane GPCRs acti-
vated by extracellular Ca2+), which are involved in regulation
of Ca2+-dependent K+ channels (Chattopadhyay et al. 1999).
Cysteinyl leukotrienes receptors of CysLT1 and CysLT2
types induced [Ca2+]i rise and ATP release from cultured rat
Chemokine receptors
CCR1,2
CCR5
Ca2+
K+ channels CXCR3
Volume-regulated
CI– channels
Regulation of
inflammatory
phenotype, and IL-1R1/R2
production of
proinflammatory
factors IL-2R
IL-4R
IL-10R
IL-13R
Interleukin receptors
microglia. Notch-1 receptors are present in ameboid micro-
glial cells in the early postnatal brain and contribute to regula-
tion of production/release of NO and inflammatory cytokines
(Grandbarbe et al. 2007). Receptors to complement fragments
C3a and C5a (which are 7-TM GPCRs) are present in cul-
tured microglial cells and in microglia in situ and are coupled
to generation of Ca2+ signals, stimulation of K+ currents and
control of microglial motility; both receptors types are linked
with pertussis-toxin sensitive G proteins (Moller et al. 1997b).
The receptors for C5a for example induce membrane ruffling,
extension of lamellipodia, and the rearrangement of the actin
cytoskeleton (Nolte et al. 1996). The C5a receptors are also
involved in pathogenesis of neuropathic pain.
The receptors to thrombin or proteinase-activated recep-
tors, PARs 1–4 are detected in microglia. Exposure to throm-
bin triggers cytoplasmic Ca2+ signaling and the release of
NO, various cytokines, and chemokines (e.g., TNF-α IL-6,
IL-12[p40] CXCL, monocyte chemoattractant protein 1
[MCP-1, CCL2], macrophage inflammatory protein 1α and
1β [MIP-1α/-1β, CCL3/CCL4], etc.) (Moller et al. 2000a).
All four PAR receptor subtypes were detected in rodent
microglia (Moller et al. 2000a). The activation of PAR-1
receptors was suggested to contribute to microglial inflamma-
tory response in Parkinson disease. Activated microglial cells
express the macrophage colony-stimulating factor receptors
(M-CSFRs), also known as colony stimulating factor-1 recep-
tor (CSF-1R), which stimulate release of NO and proinflam-
matory cytokines, activate phagocytosis, uptake of Aβ and
promote neuroprotection (Mitrasinovic and Murphy 2003).
More importantly, absence of M-CSFRs results in loss of
microglia and these receptors therefore were considered to be
required for the development of microglia (Erblich et al. 2012).
On the other hand, blockade of M-CSFRs (CSF-1R) signaling
completely inhibited microglial enhancement of glioblastoma
invasion (Coniglio et al. 2012). Receptors to epidermal growth
factor (EGFRs) are members of ErbB family of receptor protein
kinases. Their functional expression was identified in cultured
mouse microglia, where they positively modulated K+ chan-
nels through PTX-sensitive Gi proteins (Ilschner and Brandt
1996). Pharmacological inhibition of EGFR strongly inhib-
ited microglia-stimulated invasion of glioblastoma (Coniglio
et al. 2012). Microglial cells also express CD200 receptors
(CD200Rs), activated by surface glycoprotein CD200, the
lysophosphatidic acid receptors LPA1 and LPA3 (which trigger
Ca2+ signaling) and formyl peptide receptors FPR1 (high affin-
ity) and FPR2 (low affinity). These latter receptors also induce
microglial Ca2+ signaling. Finally, microglial cells express intra-
ER sigma receptors that regulate Ca2+ release.

Calcium
receptors,
CAR Leukotriene
Notch-1
receptor receptors,
CysLT1, CysLT2

Glucocorticoid Ca2+-activated
mineralcorticoid Complement
Release of K+ channels
receptors NO and 2+ receptors
Ca
cytolines C3a, C5a

Ca2+
pleiotropic motility
effects
Thrombin
Sigma-1 receptors,
receptors, ER InsP3/Ca2+
PAR1,3,4
σ1R
Delayed-rectifyer
K+ channels Ca2+
Release of
NO and Formyl peptide
Epidermal Ca2+ receptors,
cytolines
growth factor
FPR1, FPR2
receptors,
EGFR/Erb
Lysophophatidic
receptors,
Macrophage colony-
CD200 LPA1, LPA3
stimulating factor
receptors, M-CSFR receptors

7-TM Colony-stimulatung Gluco/mineralo

receptors factor receptors corticoid
intracellular
Tyrosine receptors
kinase DAP12 receptors DAP12 receptors
receptors
Intracellular
Sigma-1 receptors

Figure 19.5 Other Receptor Systems in Microglia.

P H YS I O L O GY O F M I C R O G L I A • 231
 5 MICROGLIAL PLASMALEMMAL
T R A N S P O RT E R S
The summary of microglial plasmalemmal transporters is
shown in Figure 19.6. Microglial potassium gradients are
mainly regulated by H+/K+ ATPase; whereas the Na+/K+
ATPase, in contrast, is not functionally active. The KD for
microglial H+/K+ pump is approximately 3.7 mM, which is
within the physiological range of extracellular K+ concentra-
tions (Shirihai et al. 1998). In addition microglial cells possess
the proton pump. Microglial cells express all three isoforms of
Na+/Ca2+ exchanger, NCX1, NCX2, and NCX3, with NCX1
being the dominant subtype (Nagano et al. 2004). Because of
low levels of microglial resting membrane potential the NCX
is probably always operating in a reverse mode favoring Ca2+
entry. The reverse mode of the NCX is involved in microglial
migration (Ifuku et al. 2007). Chloride transporters expressed
in microglia include the K+/Cl– cotransporters of KCC1,
KCC2, KCC3, and KCC4 types. Microglia also have opera-
tional Na+/HCO3– cotransporter and/or Na+-dependent Cl–/
HCO3– exchanger (Faff et al. 1996). Anion exchange–medi-
ated chloride/bicarbonate transporter is a major component
in the regulation of intracellular pH.
Microglial cells possess several amino acid transporters.
In particular, microglial cells express high levels of glutamate-
cystine antiporter Xc, which is involved in glutathione meta-
bolism and may provide neurotoxic glutamate release, which
for example causes oligodendroglial death in rat optic nerve
preparations (Domercq et al. 2007). Activated microglia
acquire glutamate transporters of EAAT-1/GLAST and
EAAT2/GLT types. Rapid de novo expression of both
GLAST and GLT-1 transporters was identified in rat micro-
glia following controlled cortical impact injury. The EAAT-1
transporters were also found in microglial cells of HIV-1 posi-
tive patients. The glutamate transporters in activated microglia
can serve neuroprotective function participating in removal of
toxic glutamate loads (see Kettenmann et al. 2011 for details).
Microglial cells specifically express glucose transporter
5 (GLUT5) (Sasaki et al. 2004). Activated microglia also
express monocarboxylate transporters MCT1 and MCT2,
which may be involved in the supply of lactate (Moreira et al.
2009). The expression of ATP-binding cassette ABC trans-
porters was reported in cultured rat microglia, where they can
be involved in the release of purines and cysteinyl leukotrienes
(Ballerini et al. 2005).
6 CALCIUM SIGNALING IN MICROGLIA
Microglial calcium signaling, similarly to other neuroglial cells
(see chapter 26) is primarily regulated by endoplasmic reticu-
lum Ca2+ store. Microglial cells contain both InsP3 receptors
and ryanodine receptors (RyRs). The InsP3-dependent route
usually dominates, although activation of RyRs by cyclic
ADP ribose was found to trigger Ca2+ release in human cul-
tured microglia (Shideman et al. 2006). The status of ER Ca2+
store can be affected in pathological conditions, for example,
the InsP3-mediated Ca2+ release (following the stimulation of

Na+/K+ Glutamate transporters

ATPase EAAT-1/EAAT-2
+ 2+
Na /Ca exchanger, activated microglia
NCX 2K+ 3N +
a

1G
lu –
2+ 1H +
1Ca
+
a +
3N 3Na
1K +
2+
a
1C
+
a
3N erse
Rev de
1K

mo
ect Chloride
+

Dir de
1Na

Bicarbonate mo
transporters
+
–
3

transporters
2/3HCO

2CI
–
1Na +

Glucose transporter
H+ pump,
GLUT5 glucose
Na+/H+ pump
H+/K+ pump

Cys
Glu
lactate

Glutamate-cystine ABC cassette transporters

transporter Xc

Monocarboxylate transporter
MCT-1, MCT-2
activated microglia

Figure 19.6 Microglial Plasmalemmal Transporters.

232 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
purinoceptors or PAF receptors) was reduced by more than
50% in microglial cells obtained from the brains of patients
with Alzheimer disease, possibly indicating chronic depletion
of the ER store (McLarnon et al. 2005). The store-operated
Ca2+ entry also plays important role in shaping microglial Ca2+
signals being mainly responsible for plateau phase of [Ca2+]i
transients (Moller et al. 1997a, 2000b). The maximal deple-
tion of ER Ca2+ store in cultured microglia by overstimula-
tion of P2Y2/4 metabotropic purinoceptors triggers chronic
activation of store-operated Ca2+ entry (Toescu et al. 1998).
This chronic activation may account for an increased resting
[Ca2+]i levels observed in microglial cells in various forms of
neuropathology, such as for example in Alzheimer disease (see
Kettenmann et al. 2011 for details).
7 M I C R O G L I A L M I G R AT I O N
A N D M OT I L I T Y
Microglial cells exhibit two types of movement activity. In
the ramified form, they actively move their processes without
translocation of the cell body. In the ameboid form, in addition
to the movement of the processes, the entire cell can migrate
within brain tissue, including translocation of their soma.
It is not yet explored whether these forms of motility,
migration, and process movement are distinctly regulated.
Moreover, a systematic study comparing the migratory
behavior of microglia during development and in response
to pathology has not been performed. There are many
candidate molecules that serve as signals for pathological
events to microglia, including ATP (Davalos et al. 2005;
Honda et al. 2001; Ohsawa and Kohsaka 2011), cannabi-
noids (Walter et al. 2003), morphine (Takayama and Ueda
2005), the chemokine CCL21 (Rappert et al. 2002), lyso-
phosphatidic acid (Schilling et al. 2004), bradykinin (Ifuku
et al. 2007), galanin (Ifuku et al. 2011), and other neuro-
peptides (Noda et al. 2011b). Effects of various neuropep-
tides on microglial motility are shown in Figure 19.7A,
with additional information that neuropeptide Y inhibits
IL-1β–induced microglial motility, although this was shown
only in cell line (Ferreira et al. 2012). In addition, ion chan-
nels and transporters play an important role in cell migration
(Fig. 19.7B). This is true in particular for K+ channels, Cl–
channels, the Na+/H+ exchanger, Cl–/HCO3– exchanger,

A 200

** **
150 **
Total distance (μm)

** **

100

50

0
n l
yn I
en mb in
lth in
1
ur nin

ox sin
ato in
bs in

P
so H
sin

P
in
ste tro

n-

VI
br sin-

bo kin
do es

m oc
su stat

ex
Va TR
ce
en

es
ne ala
eli
gio on

or
tan
so yt

pr
ot
g
An C

ad

B K(Ca)(BK,IK,SK)

Ca2+
K+
Gi

Gq/11 Ca2+
Na+
PLC CI–
–
HCO3 Na+
InsP3 CI–
Na+
InsP3R
DAG
HCO3– H+
PKC actin

Retraction Protrusion

Figure 19.7 Microglial Migration and Motility A. Effects of various neuropeptides on microglial motility. B. Model summarizing the role of ion
channels and transporters in controlling microglial migration. (A) Modified from Noda M, Ifuku M, Okuno Y, Beppu K, Mori Y, Naoe S. 2011b.
Neuropeptides as attractants of immune cells in the brain and their distinct signaling. Adv Neuroimmune Biol 1:53–62. (B) Reproduced with permis-
sion from Kettenmann H, Hanisch UK, Noda M, Verkhratsky A. 2011. Physiology of microglia. Physiol Rev 9:461–553.

P H YS I O L O GY O F M I C R O G L I A • 233
and Na+/HCO3– cotransporter, which all are linked to actin
cytoskeleton (Schwab 2001a,b). In addition, the reverse mode
of Na+/Ca2+ exchanger might be involved in regulation of
migration through inducing Ca2+ influx (Ifuku et al. 2007).
For precise information on the control of microglial pro-
cess motility in vivo or migration in vitro induced by various
signals and their inhibitors, see Kettenmann et al. (2011).
8 S U M M A RY A N D P E R S P E C T I VE S
Recent studies indicate that in the normal brain microglia have
highly motile processes by which they seem to scan their ter-
ritorial domains. Microglial cells can communicate with mac-
roglial cells and neurones and with cells of the immune system
by a large number of signaling pathways. Likewise, microglial
cells express receptors classically described for brain-specific
communication such as receptors for neurotransmitter and
neurohormones and neuromodulators. In addition, microglial
cells also express ion channels, transporters, and receptors ini-
tially discovered as immune cell-specific such as receptors for
cytokines. In addition, as one of the most characteristic fea-
tures of microglia, microglial migration such as motility and
chemotaxis is controlled by various signals. Nonetheless, we
have no physiological recordings from microglial cells in vivo
so far. The majority of studies on microglial physiology were
performed on cultured microglial cells or using brain slices.
There are also many investigations performed on various types
of microglial cell lines. None of the preceding may be consid-
ered as a proper model of microglial cells because of substan-
tial and inconsistent modifications in physiological properties.
Therefore, true physiology of microglia, for example, resting
membrane properties or developmental change, requires fur-
ther investigations.
AC K N OW L E D G M E N T S
MN was supported by Grants-in Aid for Scientific Research of
Japan Society for the Promotion of Science; and AV’s research
was supported by BBSRC UK, Royal Society, Alzheimer
Research Trust (UK) and INTAS.
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• 237
 20.
PHYSIOLOGY OF OLIGODENDROCY TES
Vittorio Gallo and Jean-Marie Mangin

A B B R E VI AT I O N S 1 INTRODUCTION

AMPA 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) Oligodendrocytes (OLs) are myelinating cells of the central ner-
propanoic acid vous system (CNS), closely associated with neurons and astro-
ASIC acid sensing ion channel cytes in both developing and adult brain. Oligodendrocytes
4-AP 4-Aminopyridine wrap axons in extremely large membrane expansions, and form
CC corpus callosum direct cellular contacts among themselves and with astrocytes
CFTR cystic fibrosis transmembrane conductance through gap junctions. These contacts with other neural cell
regulator types identify OLs as active participants in cellular networks
CIC voltage-gated chloride channels of the mature CNS and raise important questions about their
CNP cyclic nucleotide phosphodiesterase physiological properties.
CNS central nervous system Oligodendrocytes are found in both gray and white mat-
CNTF ciliary neurotrophic factor ter of the CNS, and undergo significant molecular, morpho-
D3R Dopamine D3 receptor logical, and physiological changes during early postnatal brain
DTI diffusion tensor imaging development. In white matter, OLs represent a major fraction
EAE experimental autoimmune (or allergic) of all cells. Because white matter function is involved in many
encephalomyelitis cognitive and behavioral processes, defining the physiologi-
EGFP enhanced green fluorescent protein cal properties of OLs is fundamental to understanding higher
FGF fibroblast growth factor brain functions, learning disabilities, and psychiatric disorders.
GABA gamma-aminobutyric acid Oligodendrocytes express a variety of ligand- and
GFP green fluorescent protein voltage-gated ion channels, as well as G-protein–coupled
GlyR glycine receptor Cl– channels neurotransmitter receptors, maintaining this expression
HVA high voltage–activated throughout the OLs’ lives (Tables 20.1 and 20.2). However,
LVA low voltage–activated their physiological role—clearly established in excitable
MBP myelin basic protein cells such as neurons—is not yet completely defined in OLs.
MS multiple sclerosis Heterogeneous receptor and channel expression has been
nAchR Nicotinic acetylcholine ionotropic receptors demonstrated in OLs, and physiological differences between
NMDAR N-methyl-d-aspartic acid (NMDA)- gray and white matter OLs suggest that, much like neurons
sensitive iGluRs and astrocytes, distinct OL subpopulations with different
OL oligodendrocyte physiological properties may exist. Moreover, many recep-
OPC oligodendrocyte progenitor cell tors and channels in OLs display remarkable plasticity during
PDGF platelet-derived growth factor development or on exposure to different environmental sig-
PI-3 phosphoinositide-3 nals, hinting at the complexity of OL physiology.
PLP proteolipid protein The anatomical relationships of OLs with neurons and
PNS peripheral nervous system astrocytes suggest that neurotransmitter receptors and ion
RMP resting membrane potential channels are actively involved in mediating cell–cell communi-
ROMK1 renal outer medullary potassium channel cation between OLs and other neural cells, in addition to the
RT-PCR Reverse transcriptase polymerase chain direct roles in developmental myelination and myelin mainte-
reaction nance. Recent studies also reveal a likely role for OLs in regu-
STX saxitoxin lating functional activity in axons (Yamazaki et al. 2007).
SUR sulfonylurea receptor The chapter attempts to integrate current information
SVZ subventricular zone about these receptors and channels into the general scheme
TEA tetraethylammonium of OL physiology, and associate this complex array of func-
TTX tetrodotoxin tional proteins with OL functions in normal and pathologi-
VDAC voltage-dependent anion-selective channel cal brains. Because of space constraints, this chapter focuses
VOCC voltage-operated calcium channel exclusively on the molecular and functional properties of

238
neurotransmitter receptors and voltage-gated ion channels in
OLs, although membrane transporters or specific enzyme sys-
tems such as carbonic anhydrase are also legitimately part of
OL physiology. Readers are referred to chapters 21 and 52 for
further considerations of OL physiology and pathology.
2 VO LTAG E - G AT E D I O N C H A N N E L S
The overall electrophysiological properties of OLs drastically
change during their development from oligodendrocyte pro-
genitor cells (OPCs) to mature, myelinating OLs (Fig. 20.1).
These changes result from modifications of their total mem-
brane size, as well as specific regulation of voltage-gated ion
channels, including a shift from Kv to Kir expression, and
downregulation of Na+ and Ca2+ channels during OL matura-
tion (see Fig. 20.1 and Table 20.1).
2.1 P OTA S S I U M C H A N N E L S
Voltage-gated K+ channels play a crucial role in repolarizing
the membrane and maintaining the resting membrane poten-
tial (RMP). These proteins represent the largest and most
diverse class of voltage-dependent ion channels (Berkefeld
et al. 2010; Bichet et al. 2003), and are widely expressed in
CNS and peripheral nervous system (PNS) glia, including
OLs. Electrophysiological studies combined with molecu-
lar analysis have identified expression of channel subtypes
based on single channel conductance, voltage-dependence
of activation/inactivation, whole-cell current properties, and
pharmacological agents that either selectively block or acti-
vate these channels.
2.1.1 Inwardly Rectifying Potassium Channels
Oligodendrocytes have a very negative RMP (approxi-
mately –80mV) and a high resting permeability for K+ ions.
Pharmacological and biophysical studies suggest that the
activity of inwardly rectifying, Ba2+ sensitive, weakly rectify-
ing Kir channels establishes the RMP. The classical view of Kir
channels in glia is that their main function is to buffer extra-
cellular K+ ions (see also chapter 16). However, their molecu-
lar diversity and their distinct expression pattern in different
types of glia suggest additional functions for these channels in
gliogenesis and glial pathology. For example, Kir channels are
implicated in regulating OL proliferation, differentiation, and
survival (Gallo et al. 1996; Knutson et al. 1997; Neusch et al.
2001; Olsen and Sontheimer 2008; Sontheimer et al. 1989).
Weakly rectifying currents associated with Kir channels have
been recorded in OLs (Chittajallu et al. 2005; Sontheimer
et al. 1989; Steinhauser et al. 1992). Molecular and electrophys-
iological studies have demonstrated heterogeneity in expression
of these channels in OLs from distinct brain regions and differ-
ent developmental stages; that is, OPCs, immature pre-OLs,
and mature OLs (Chittajallu et al. 2004; Soliven et al. 1989).
In OPCs, upregulation of Kir channels is associated with
hyperpolarization of the RMP, and short-term changes in the
RMP are counterbalanced by activation of Kir channels (Knutson
et al. 1997). Genetic deletion studies, combined with molecular
and functional identification of the channels, demonstrated that

Table 20.1 VOLTAGE-GATED CHANNELS

VOLTAGE-GATED SUBTYPES/ MODE OF DEVELOPMENTAL
CHANNELS SUBUNITS ACTIVATION PHYSIOLOGICAL ROLE REGULATION

Kir potassium channels Heterogenous but Open at Maintenance of hyperpolarized Upregulated in

Kir4.1 is predominant hyperpolarized membrane potential myelinating OLs
potentials Extracellular K+ buffering
OPC: Initiation of cell cycle exit
KD potassium channels Kv1.2, Kv1.5, Kv1.4, Depolarization OPC: Upregulated by mitogens High expression at OPC
and Kv1.6 (PDGF, FGF). Promote OPC stage. Downregulated in
proliferation and differentiation mature OLs
OLs: Unknown
KA potassium channels Unknown Depolarization Unknown Downregulated during
OL differentiation
KCa potassium channels BK Depolarization Regulation of intracellular Ca2+ Decreased during OPC
IK? + intracellular Ca2+ OLs maturation? maturation
SK?
KATP potassium channels Kir6.1, Kir6.2, Intracellular ATP Unknown Unknown
and SUR2 OLs development and
regeneration?
Sodium channels Unknown Depolarization Unknown Decreased during
OL maturation. Not
expressed in mature OLs
Calcium channels L-, N-, and R-type LVA: Low Unknown, but regulate Ca2+ entry, Expressed throughout the
depolarization thus likely to influence many processes OL lineage
HVA: High from proliferation to myelination
depolarization

P H YS I O L O GY O F O L I G O D E N D R O C Y T E S • 239
 A B
OPC pre-myelinating

persistant K current (pA)

300 600

peak K current (nA)

2

Na current (pA)
200

0.1 nA

0.1 nA
300
1
0.3 ms 0.3 ms 100

0 0 0

mature
pre-myelin.

OPC

mature
pre-myelin.
OPC

mature
pre-myelin

OPC

young

young
young
*
young mature
C
300 3
–80

Cm (pF)
200 2

Rin (GΩ)
Vrest (mV)
100 –40
1

0 0 0
0.5 nA

OPC
pre-myelin.

mature

OPC
pre-myelin.

mature

OPC
pre-myelin.

mature
young

young

young
20 ms

Figure 20.1 Changes in Electrophysiological Properties of Oligodendrocytes During Their Maturation. A. Whole-cell current pattern of four different
developmental stages of oligodendroglial cells. From a holding potential of –80 mV, cells were depolarized in steps of 10 mV up to +20 mV. Only in
OPCs a transient fast inward current—resembling an Na+ current—was detected soon after the beginning of the voltage step (top row insets). Mature
oligodendrocytes show a pure ohmic current response. B. Summary bar graphs of the peak amplitudes of the three current components analyzed at
each developmental stages. Voltage-activated sodium currents are lost as soon as OPCs differentiate, whereas voltage-activated potassium currents
remain present in young oligodendrocytes and are only lost once a cell has fully matured. C. Summary of the passive membrane properties quantified
in each of the four developmental stages. Error bars indicate SEM. From Kukley and Dietrich 2009.

resting conductance in OLs is mostly caused by Kir4.1 (Butt and
Kalsi 2006; Neusch et al. 2001). Oligodendrocytes are strongly
immunopositive for Kir4.1 in situ, suggesting that they might
be involved in buffering K+ ions released during propagation of
action potentials (Kalsi et al. 2004).
Kir channels—in particular Kir4.1 in OLs—were shown to act
as critical regulators of cell division: Kir function correlates with
cell cycle exit and initiation of OL differentiation. Conversely,
loss of functional Kir channels is associated with cells’ re-entry
into the cell cycle. Upregulation of Kir4.1 expression and a sub-
sequent negative shift in RMP are believed to be critical for
differentiation and initiation of developmental myelination in
OLs (Butt and Kalsi 2006). Therefore, Kir expression and func-
tion play important roles in coupling intrinsic molecular mech-
anisms of cell cycle regulation with myelinogenesis.
This hypothesis has been confirmed by the generation
of mice with a null mutation in Kir4.1 (Neusch et al. 2001).
Kir4.1–/– OLs in culture display a more immature morphol-
ogy and mutant mice display significant OL apoptotic death
during critical phases of oligodendrogenesis and initiation
of myelination, likely because of chronic cell depolarization.
Axonal degeneration is also observed in these mice, causing
further OL damage and death at later developmental stages.
Because of these cellular defects, Kir4.1–/– mice display a severe
hypomyelinating phenotype.
2.1.2 Outwardly Rectifying Potassium Channels
This section focuses on the subfamilies of these channels
expressed in OLs, particularly the Kv family, including the
delayed rectifying K+ channels (KD) and the fast-inactivating
or transient A-type K+ channels (KA). Calcium-activated K+
channels (KCa) are also discussed briefly. KD, KA, and KCa chan-
nels are distinguished by their molecular identity and func-
tional and pharmacological properties. Besides their activation
threshold and channel conductance, these channel types differ
significantly in terms of channel kinetics and selective ion per-
meability. However, most of these channels are sensitive to both
tertraethylammonium (TEA) and 4-amino-pyridine (4-AP).
2.1.2.1 Delayed Rectifier Potassium Channels
In neurons, these channels are responsible for repolariza-
tion after action potential propagation and regulating neu-
rotransmitter release at synapses. Studies in OL lineage cells
identified expression of specific Kv subunits; however, some
discrepancies were also generated in different cell prepara-
tions. Attali et al. (1997) identified Kv1.5 as a major compo-
nent of K+ currents in A2B5+ and O4+ OPCs and in GalC+
OLs, although expression of subunits Kv1.2, Kv1.4, and Kv1.6
was also detected. Further studies (Schmidt et al. 1999) dem-
onstrated that all Kv1.1 to Kv1.6 channel transcripts were
expressed in OPCs, but only Kv1.4, Kv1.5, and Kv1.6 proteins
were detected. Consistent with the previous study, functional
evidence was found for either homomeric Kv1.5, or hetero-
meric Kv1.4/Kv1.5 and Kv1.5/Kv1.6 channels. Analysis of K D in
OL lineage cells in situ confirmed the findings in culture, with
KD currents found in both NG2+ and O4+ cells in subcortical
white matter (Chittajallu et al. 2005). In addition to the Kv1
subunits, a recent study revealed Kv7/KCNQ channel expres-
sion in cells of the OL lineage (Wang et al. 2011).
KD channels are highly expressed at immature (NG2+ and
+
O4 ), proliferative stages of the OL lineage, but significantly

240 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
downregulated in GalC+ OLs (Chittajallu et al. 2005; Knutson
et al. 1997; Sontheimer et al. 1989). Actively dividing NG2+
cells in white matter displayed a higher density of KD channels
than did slowly dividing/postmitotic NG2+ cells in cerebral cor-
tex (Chittajallu et al. 2004). Thus, indications that expression of
these channels tightly correlates with cell cycle activity prompted
further molecular and electrophysiological analysis of KD chan-
nels during OL development. Chittajallu et al. (2002) showed
that upregulation of these currents occurs in the G1 phase of the
OPC cycle and is caused by RNA synthesis-dependent increase
in Kv1.3 and Kv1.5 expression. In situ analysis demonstrated
large KD currents in proliferating OPCs of the subventricular
zone (SVZ) and white matter; these currents were attenuated
by Kv1.3 channel blockers. These results indicate that Kv1.3 (at
variance with Attali et al. 1997) and perhaps Kv1.5 form KD
channels in proliferating OPCs in vivo.
Consistent with these findings, the OL mitogens
platelet-derived growth factor (PDGF) and fibroblast growth
factor (FGF) were found to be important in inducing KD
expression in OPCs, particularly in upregulating Kv1.5 and
Kv1.6 mRNAs and increasing the density of KD currents
(Soliven et al. 2003). PDGFαR-mediated upregulation of K D
current expression in NG2+ cells of subcortical white matter
was prevented by tyrosine kinase inhibition (Chittajallu et al.
2005), demonstrating that endogenous mitogens enhance K D
channel expression at early stages of the OL lineage through
tyrosine kinase activity.
These findings raise questions about the functional role of
KD channels in OL development. Several studies investigated
this issue by (1) using channel blockers in various developmen-
tal assays, and (2) analyzing the relationship between cellular
signals that modulate cell proliferation and differentiation, and
their influence on KD channel expression. These studies found
that all KD channel blockers strongly inhibit OP cell cycle pro-
gression and proliferation (Attali et al. 1997; Chittajallu et
al. 2002; Gallo et al. 1996; Ghiani et al. 1999; Soliven et al.
2003; Tiwari-Woodruff et al. 2006). Glutamate activation of
2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid
(AMPA) receptors in OPCs induced Na+-dependent blockage
of KD channels and inhibited OPC proliferation (Gallo et al.
1996). KD channels formed by Kv3.1 are expressed both in OPCs
and OLs and are associated with the OL-specific tight junction
protein (OSP)/claudin-11 (Tiwari-Woodruff et al. 2006). These
channels appear to be involved not only in OPC proliferation
and migration, but also in myelination, because Kv3.1-null mice
exhibited decreased axonal diameter and myelin thickness.
KD channels may also be important in OL response to
pathological insults. A recent study investigated Kv subunit
expression in an animal model of multiple sclerosis (MS)
and experimental autoimmune encephalomyelitis (EAE)
(Herrero-Herranz et al. 2007). Subunit Kv1.4 was re-expressed
at high levels in proliferating immature OLs of EAE mice
around demyelinated lesions. Kv1.4 expression was greatly
reduced in a ciliary neurotrophic factor (CNTF)-null mouse,
whose myelin lesion repair is impaired. In a separate study,
Kv1.3 was identified as the important subunit in comple-
ment-induced cell cycle re-entry of immature OLs (Tegla et
al. 2011). Complement-induced myelin basic protein (MBP)
P H YS I O L O GY O F O L I G O D E N D R O C Y T E S
and proteolipid protein (PLP) mRNA decay was blocked by
inhibiting Kv1.3 expression, and this subunit was expressed in
immature OLs in MS tissue. These results indicate that cyto-
toxicity, mediated by complement via antibody-independent
activation of the classical pathway, regulates Kv1.3 channel
expression with concomitant activation of the cell cycle and
OL de-differentiation.
2.1.2.2 Rapidly Inactivating A-Type Potassium
Channels
In neurons, the main function of KA channels is to regulate
interspike intervals. KA currents display such a broad range of
inactivation rates that, in some cases, a clear distinction between
KA and KD currents is challenging. The Kv channel family also
includes subunits that give rise to currents with biophysical
properties representing a continuum between classical KD and
KA currents. These channels are less sensitive to TEA and more
sensitive to 4-AP than are other outwardly rectifying channels.
KA channels are expressed in cells of the OL lineage at dif-
ferent developmental stages, but—like KD channels—their
functional expression is downregulated as OPCs differenti-
ate to mature OLs (Knutson et al. 1997). KA currents were
detected in OL lineage cells in situ, although significant het-
erogeneity was observed in the levels of KA current expression
in OPCs. Cortical NG2+ cells displayed twice the density of
KA channels seen in their white matter counterparts, although
biophysical properties were identical (Chittajallu et al. 2004).
Because of similarities in the functional properties of KA and
KD channels and in their developmental regulation in the OL
lineage, their roles in OL maturation are still poorly defined.
2.1.2.3 Calcium-Activated Potassium Channels
All KCa channels require Ca2+ binding for activation, but three
subtypes—BK (large conductance; >100 pS and iberiotoxin/
charybdotoxin/TEA sensitive), IK (intermediate conductance;
30–70 pS and apamin insensitive), and SK (small conductance;
5–20 pS and apamin sensitive)—have been identified based on
their biophysical properties, and sensitivity to specific pharma-
cological reagents and selective toxic channel blockers.
A crucial physiological role of Ca2+-activated K+ channels
is to couple Ca2+ metabolism and membrane potential to K+
flux and membrane excitability. A recent study first inves-
tigated the expression and function of BK channels in OLs
(Buttigieg et al. 2011). In OPCs, outward currents blocked by
BK channel blocker iberiotoxin were observed together with
immunofluorescence labeling of BK channels. Fura-2AM
microscopy showed that these channels are directly involved
in regulating intracellular Ca2+ levels. Furthermore, BK chan-
nel currents and RNA and protein levels decreased with OPC
development, being lower in mature OLs. Thus, BK channels
appear to be involved in regulating Ca2+ influx in cells of the
OL lineage, and might be involved in OL maturation.
2.1.2.4 Adenosine Triphosphate (ATP)–Dependent
Potassium Channels
ATP-sensitive K+ channels (KATP) are gated by ATP and
formed by Kir6.x-type subunits combined with sulfonylurea
receptor (SUR) subunits and with auxiliary components of
• 241
the channel. Channels are further classified based on their sub-
cellular localization into sarcolemmal (sarcKATP), mitochon-
drial (mitoKATP), and nuclear (nucKATP). The Kir subunits have
two transmembrane domains and form the main channel pore,
whereas SUR subunits have five transmembrane domains with
two nucleotide-binding domains on the cytoplasmic region of
the channel. These domains play a crucial physiological role,
sensing changes in a cell’s metabolic activity.
Expression of different KATP channel subtypes has been
detected in OLs, but their characterization in OLs is still
incomplete—particularly the relationship between molecu-
lar subtypes and channels’ functional properties. An inward
rectifying K+ channel with Walker type-A ATP-binding
domain (KAB-2), predominantly expressed in glial cells, was
isolated and functionally characterized (Takumi et al. 1995).
Expression of KAB-2 was detected in cerebellar and forebrain
OLs using in situ hybridization.
A recent study demonstrated expression of Kir6.1 and
Kir6.2 proteins, together with SUR2, in cultured OPCs and
OLs (Fogal et al. 2011). When a series of KATP channel acti-
vators, including diazoxide, was tested in cultured OPCs, all
these compounds enhanced cell proliferation. Furthermore,
diazoxide enhanced myelination in vitro and attenuated the
effects of perinatal chronic hypoxia on white matter, as dem-
onstrated by increased myelination. Thus, KATP channels might
regulate OL development and regeneration, and activators of
the channels might have therapeutic value in periventricular
white matter injury.
2.2 S O D IU M C H A N N E L S
Na+ channels were first demonstrated in Schwann cells and
astrocytes (Bevan et al. 1984; Chiu et al. 1984), prompting
studies to investigate the expression and regulation of Na+
channels in cells of the OL lineage.
Compared with K+ channels, voltage-gated Na+ channels
in developing OLs are less well characterized and their func-
tional role is intensely debated (Fields 2008). Oligodendrocyte
progenitor cells are known to express voltage-gated Na+ cur-
rents displaying rapid activation/inactivation kinetics and
tetrodotoxin (TTX) sensitivity (Sontheimer et al. 1989;
Williamson et al. 1997). Because these findings were in
recordings from slices (Berger et al. 1992; Chittajallu et al.
2004; Paez et al. 2009a), it was concluded that voltage-oper-
ated Na+ channels are functionally expressed in OPCs in vivo
(Fig. 20.2).
Several studies have shown that expression of voltage-gated
Na+ channels is downregulated between the NG2+ OPC and
the O4+ pre-OL stage, and further between the O4+ and
the O1+ OL stages (Berger et al. 1992; Paez et al. 2009a).
Analysis of voltage-operated Na+ channels in cortical white
matter in situ using whole cell patch-clamp in OLs demon-
strated functional expression only at early postnatal develop-
mental stages.
Availability of transgenic mice selectively expressing
green fluorescent protein (GFP) in OLs (PLP-GFP and
CNP-EGFP mice) in situ demonstrated that O4+ pre-OLs
in corpus callosum lack inward Na+ currents at early
242 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
postnatal ages (Chittajallu et al. 2004; Paez et al. 2009a),
confirming previous findings that voltage-operated Na+
channels are not functionally expressed in premyelinating
OLs. Conversely, Sox9-expressing OPCs located near the
lateral ventricles displayed sizable Na+ currents (Paez et al.
2009a).
In summary, mature OLs do not express functional Na+
channels, but these channels are expressed during early devel-
opmental stages of the OL lineage. These findings raise inter-
esting questions about regulation and physiological function
of these channels. One possibility is that voltage-operated Na+
channels are expressed in OPCs because these cells are devel-
opmentally closer to multipotential neural progenitors capa-
ble of generating neurons as well as glia.
2.3 C A LC IUM C H A N N E L S
The entry route of Ca2+ across the OL membrane and the
changes occurring in intracellular Ca2+ levels are factors that
determine the physiological or pathological responses of OLs.
Ca2+ influx in OLs can occur through routes involving distinct
molecular mechanisms: (1) through ligand-gated channels
that display differential permeability to this cation (Kastritis
and McCarthy 1993; Kirchhoff and Kettenmann 1992;
Patneau et al. 1994); (2) through voltage-operated Ca2+ chan-
nels (VOCCs) (Paez et al. 2009a; Patneau et al. 1994); and
(3) through other channels also identified in OLs (Alberdi et
al. 2005; Simpson et al. 1997). Transmembrane Ca2+ influx also
occurs in OLs through acid-sensing ion channels (Feldman
et al. 2008) (see section 2.4).
Voltage-operated Ca2+ channels are divided into two
classes: (1) high-voltage–activated (HVA) Ca2+ channels,
displaying an activation threshold around –30 mV; and
(2) low-voltage–activated (LVA), displaying a lower threshold
of approximately –60 mV and therefore requiring a smaller
depolarizing stimulus for activation. Six classes of HVA
VOCCs have been characterized based on molecular, electro-
physiological, and pharmacological properties: the P/Q, N, L,
R, and T types.
Several studies demonstrated expression of L-, N-, and
R-type VOCCs in cells of the OL lineage in culture and in
situ (Berger et al. 1992; Butt 2006; Williamson et al. 1997).
Unlike voltage-gated Na+ channels, VOCCs are expressed
throughout the entire OL lineage. Although OPCs isolated
from distinct tissues produced different results depending
on developmental stage and type of preparation, the stud-
ies concluded that Ca2+ currents are present in OPCs. These
currents have also been examined in mature OLs by electro-
physiological recordings in early postnatal corpus callosum,
revealing expression of both HVA and LVA Ca2+ channels in
OLs of white matter regions (Fig. 20.3) (Fulton et al. 2010;
Paez et al. 2010). More recent studies characterized these
channels in corpus callosum and demonstrated expression of
verapamil-sensitive L-type Ca2+ channels in immature OLs
(see Fig. 20.3) (Paez et al. 2010). Functional expression of
VOCCs appears to be progressively attenuated as OPCs
mature into myelinating OLs (Fulton et al. 2010; Paez et al.
2010).
 A C1 cNG2+ D cNG2+
wmNG2+

40 mV
16/16 19/54 5/19

200 ms

B cNG2+ C2 cNG2+ E cNG2+

35/54 4/4 2/56

+1μM TTX

I J K *
–25
0
wmNG2+
–200 –20 ns
INa density(pA/pF)
INa amplitude(pA)

cNG2+ no spike –400 –15

–600 –10
cNG2+ spike wmNG2+
–800 –5
500 pA

wmNG2+ no spike
cNG2+ no spike
–1000 0
5 ms –60 –40 –20 0 20 wmNG2+ cNG2+ cNG2+
Vtest(mV) no spike spike

Figure 20.2 A Subpopulation of Cortical NG2+, but Not White Matter NG2+ Cells, Display TTX-Sensitive Spikes in Response to Depolarizing
Current Pulses. A. Representative current clamp traces from a single white matter (wm) NG2+ cell in response to membrane depolarization. B,C.
From a total of 56 cortical (c) NG2+ cells tested, 35 showed no membrane response (B), and 19 displayed a single spike occurring at the beginning
of the test pulse (C1). In all four of these 19 cells tested, the spike was abolished by 1 μM TTX (C2). D. Five out of the 19 spike-producing cortical
NG2+ cells also clearly displayed an after depolarization (arrow). E. Single example of a cortical NG2+ cell that displayed multiple spikes (two cells
were found with this response). G,H. Morphology and confirmation of NG2+ expression in single and multiple spiking cells, respectively. I. Single
trace examples of INa in a white matter NG2+ cell (top trace), a cortical NG2+ cell not displaying a spike (middle trace), and a cortical NG2+ cell with
spike (bottom trace). J. Voltage/current relationships of INa for white matter NG2+ cells (open circle; n = 5), and for cortical NG2+ cells without and
with spike (filled circles and filled triangles; n = 10 and 9, respectively). K. Corresponding pooled INa densities (n = 5–10). All data were obtained from
P5-P8 CNP-EGFP mice. Scale bars = 20 μM. *P < .05, ns—not significant (i.e., P > .05), Mann-Whitney U-Test. Chittajallu et al. 2004.

P H YS I O L O GY O F O L I G O D E N D R O C Y T E S • 243
 A –60 –40 –20 0 20 40
–0.0 mV
Vh-120 Vh-50

LVA pA/pF
–0.5

–1.0
LVA HVA
–1.5 OL
OPC * **
50 pA **
–2.0
20 ms
0.4 0.8
0.2
–60 –40 –20 0 20 40 –60 –40 –20 0 20 40

HVA pA/pF
–0.0 mV mV
pA/pF

–0.2
–0.2
–0.4
–1.2
–0.6 HVA OL
LVA OPC
–2.2 *** ***
***
100
B PDGF+bFGF 100

Intracellular C++ (% change)

80

Intracellular Ca++ (% change)

K+(20mM) 80
–0.18
60 60
–0.17
Fura-2 ratio

NG2+
O1+ mN2
–0.16 40 40

–0.15 20 20

0 2 4 6 8 10 0 0

α
2

BP
Time (min)

4
1
IV

IV

IV

IV

G
O
O
Fr

M
N
1D

2D

3D

4D
C

G
PD
0 min D
80
K+(20mM)
LV
SVZ Intracellular Ca++ (% change)
60 SVZ OPCs

GFP Fura-2 ratio Fura-2 ratio 7 min

40
0 min 7 min

20 ** ***
***
CC 0
p

+
l
sa

ra

ife

a+
Ba

Ve

–C

GFP Fura-2 ratio Fura-2 ratio

E F SVZ OPCs
120
Amplitude of Ca++ uptake (%)

CC Ols
0.18 K+ (20mM) 0.18 K+ (20mM)
P4 P8
0.17 SVZ OPCs 0.17 CC OLs 80
Fura-2 ratio

Fura-2 ratio

0.16 0.16 **
40 * **
0.15 0.15 *

0.14 0.14 0
0 5 10 15 0 5 10 15
au

au

au

au
Pl eak

Pl eak

Pl eak

Pl eak
ate

ate

ate

ate

Time (min) Time (min)

P

Figure 20.3 Oligodendrocyte Lineage Cells Express Different Types of Voltage-Activated Ca2+ Channels. A. Low-voltage–activated and high-voltage–
activated currents in immature oligodendrocytes. Left. Recordings of Ca2+ currents in immature OLs. Currents were activated from a holding
potential of –120 mV and –50 mV. Note inactivation of the transient component when currents are activated from –50 mV. Right. Current density/
voltage graphs for LVA and HVA currents. Both types of current are reduced in immature OLs (mean ± SEM). *P < .05, **P < .01, ***P < .001. B.
Left. NG2 and O1 staining were used after Ca2+ imaging. NG2+ OPCs responded to high K+ with large increases in intracellular Ca2+ than O1+ cells.
Center. Oligodendrocyte progenitor cells were cultured for 1 to 2 days in vitro (1–2 DIV) with PDGF and bFGF, or in mitogen-free medium (mN2)
for 2 more days (3–4DIV). Ca2+ influx amplitudes after high K+ treatment are shown. Right. Staining for PDGFrα, NG2, O4, O1, and MBP was
used after Ca2+ imaging. Average Ca2+ influx amplitude (50 cells) for each OL marker is shown. C. Time lapse series of GFP-expressing OPCs in the
dorsolateral subventricular zone (SVZ) and corpus callosum (CC) (P4). An increased Fura-2 fluorescence ratio is indicated by warmer colors. Time is
denoted in minutes. LV: lateral ventricle. Scale bar (Top) 100 μm, (Bottom) 50 μm. D–E. Voltage-operated Ca2+ channel activity in GFP-expressing
OPCs from the SVZ and in OLs from the CC. Each trace corresponds to a single cell. D. K+-induced Ca2+ uptake in SVZ OPCs was abolished in 25
μM verapamil, 25 μM nifedipine and in the absence of external Ca2+ (–Ca2+). F. Voltage-operated Ca2+ channel activity was examined in SVZ OPCs
and in CC OLs. Graphs show the average maximal peak values and plateau values, expressed as percentage of change of the emission intensities (mean
± SEM). *P < .05, **P < .01, ***P < .001 versus basal (D) and versus SVZ OPCs (F). From Fulton et al. 2010; Paez et al. 2010.
 The functional role of VOCCs in the OL lineage remains 2.4 C H L O R I D E A N D OT H E R C H A N N E L S
largely unknown. In OPCs these channels are thought to
Voltage-activated Cl– channels play an important physiologi-
regulate early developmental processes, including gene expres-
cal role, because Cl– is the most abundant anion present in
sion, cell proliferation, and cell migration (Butt 2006; Paez et all physiological cellular environments. So far, four distinct
al. 2009a,b). In mature, myelinating OLs, VOCCs might be classes of Cl– channels have been cloned, including: (1) Ca2+
involved in initiating and maintaining myelination. Moreover,
activated Cl– channels; (2) the cystic fibrosis transmembrane
VOCCs are thought to regulate process extension and retrac-
conductance regulator (CFTR); (3) voltage-dependent
tion of membrane sheets in mature OLs (Kirischuk et al.
anion-selective channels (VDACs); and (4) voltage-gated
1995; Paez et al. 2009b). Consistent with this notion, and
chloride channels (CICs) ( Jentsch et al. 1999).
with the hypothesis that VOCCs might be involved in modu-
Oligodendrocytes display significant resting Cl– con-
lating initiation of myelination, Kirischuk et al. (1995) found
ductances, and Cl– currents were first recorded in excised
heterogeneous distribution of these channels in subcellular
inside-out patches from OLs (Barres et al. 1988). Further stud-
OL domains. High-voltage–activated channels were primarily
ies in culture demonstrated the presence of voltage-activated
located on cell bodies, whereas LVA channels were mostly on
Cl– channels in cells of the OL lineage by whole-cell record-
cell processes, suggesting differential activation as a signal for
ings (Williamson et al. 1997). These outwardly rectifying
initiation of myelination.
currents were identified as arising from voltage-gated rather
A study by Paez et al. (2007) revealed a functional inter-
than Ca2+-activated Cl– channels. The function and molecu-
action between golli proteins and Ca2+ channels in OLs.
lar identity of Cl– channels in OLs is still unknown, but they
Golli proteins are encoded by the myelin basic protein
might be involved in cell shape or cell volume changes that
(MBP) gene, which also encodes MBP constituents of myelin
accompany Cl– efflux through these channels.
(Campagnoni et al. 1993). Overexpression of golli proteins
Acid-sensing ion channels (ASICs) are formed by different
enhanced transmembrane Ca2+ influx and intracellular Ca2+
combinations of six molecularly distinct subunit proteins that
levels in OLs, and triggered remodeling of OL processes and
associate to form either homomeric or heteromeric channels
membrane sheets. Binding of golli to the plasma membrane is
with different physiological properties. Acid-sensing ion chan-
important for modulating Ca2+ homeostasis—supporting the
nel 1a channels are Ca2+ permeable and likely to be important
hypothesis that these myelin proteins form a protein complex
in CNS pathology, as their inactivation significantly attenu-
in membrane subdomains and modulate Ca2+ entry along OL
ates ischemic brain damage (Xiong et al. 2006).
processes. The first study identifying acid-sensing Na+ channels in
Voltage-operated Ca2+ channels are also engaged in axo-
cells of the OL lineage demonstrated large Na+ currents in
glial signaling between neurons and OLs. Propagation of
response to acidification in OPCs and in more mature cells of
action potentials and axonal activity cause enough extra-
the lineage (Sontheimer et al. 1989). Their functional proper-
cellular K + accumulation to depolarize surrounding glial
ties were similar to their neuronal counterpart. A decrease in
cells and activate their VOCCs. This could be a physi-
functional expression of these proton-activated channels was
ological mechanism that couples electrical activity in
observed from OPCs to mature OLs.
neurons with axonal myelination. In fact, enhanced extra-
Feldman et al. (2008) demonstrated ASIC expression in
cellular K + levels within the normally observed physiolog-
white matter in situ, and ASIC1a was specifically found in
ical range have been shown to induce OL depolarization
white matter OL lineage cells. ASIC1a, 2a, and 4 mRNAs
and a rise in intracellular Ca 2+ (Kirischuk et al. 1995).
were expressed in OL lineage cells in culture, but mRNA levels
Furthermore, the microstructure of white matter changes
for these channels decreased during OPC maturation to OLs
continuously during OL maturation and myelination, as
(Feldman et al. 2008). Patch-clamp recordings demonstrated
demonstrated by direct diffusion tensor imaging (DTI)
predominance of homomeric ASIC1a in OLs, whose activa-
measurements showing progressive compaction of the
tion caused robust membrane depolarization and a transient
extracellular space (Schmithorst et al. 2002). Reduced
increase in intracellular Ca2+.
volume would cause larger changes in extracellular K+
Based on their functional properties and activation owing
concentration within the local axoglial microenviron-
to acidification of the extracellular environment, ASICs in OLs
ment when K+ is released from axons, so even small axon
might play a role in pathology, particularly ischemic damage.
fibers could produce fluctuations in extracellular K + able
to depolarize surrounding OLs.
Immunohistochemical analysis of VOCC expression
in white matter in situ supports this mechanism, as robust 3 L I G A N D - G AT E D I O N C H A N N E L S
expression of R-type Ca2+ channels was transiently detected
in OL cell bodies and processes, as well as in paranodal myelin Ligand-gated ion channels are a highly important family of
wraps, during the peak of myelination (Chen et al. 2000). proteins in the brain, underlying almost all neurotransmission
R-type channel immunoreactivity was closely associated with the exception of electrical synapses. Expression of a vari-
with OL membranes in direct contact with axons, allowing ety of neurotransmitter-receptor channels and metabotropic
rapid activation of these VOCCs and initiation of myeli- receptors has been recognized in glial cells (OLs among them)
nation when extracellular K+ accumulated in the axoglial for at least 20 years, yet their function in these cells remains in
microenvironment. many cases elusive.

P H YS I O L O GY O F O L I G O D E N D R O C Y T E S • 245
Table 20.2 NEUROTRANSMITTER RECEPTORS
IONOTROPIC MODE OF PHYSIOLOGICAL PHYSIOLOGICAL DEVELOPMENTAL
RECEPTORS SUBUNITS TRANSDUCTION MODE OF ACTIVATION ROLE REGULATION

AMPAR Glur2, Glur3, and Depolarization, OPC: Synaptic OPC Inhibit prolif- Peak of expression at
GluR4 Calcium entry OL: Unknown (spillover, eration, favor differen- the OPC stage.
astrocytes) tiation, and increase Downregulated in
migration. mature OLs
OL: Unknown, but
excitotoxic at high
concentration

KAR GluR6, GluR7, KA1, and Depolarization OPC: Unknown but Unknown Unknown. Low expres-
KA2 nonsynaptic sion in OPC
OL: Unknown
(spillover, astrocytes)

NMDAR NR1, NR2A, NR2B, Depolarization, OPC: Unknown but No physiological role? Unknown. Low
NR2C, NR2D, and calcium entry nonsynaptic Absence of phenotype expression in OPC
NR3A OL: Unknown in
(spillover, astrocytes, NR1
glycine) KO but could mediate
excitotoxicity

GABAAR Unknown OPC: OPC: Synaptic and/or OPC: Increase Peak of expression at
Depolarization. extrasynaptic. proliferation. the OPC stage
OL: Unknown OL: Unknown (spillover, OL: Unknown Strongly downregulated
(hyperpolarizing ?) astrocytes) in mature OLs

Purinergic P2XR P2X7 Depolarization, Mostly unknown, but Unknown, but excito- Robust expression in
calcium entry via activated by ATP release toxic OPCs and mature OLs
pore formation from astrocytes at high concentration

GlyRs α2 and β OPC: Unknown (synapses?) Unknown Peak of expression at

Depolarization. the OPC stage.
OL: Unknown Downregulated in
mature OLs
Nicotinic AchRs α3, α4, α5, α7, β2, OPC: Unknown. Synapses Unknown Unknown
and β4 Depolarization. could not be detected in
OL: Unknown hippocampal OPCs
METABOTROPIC SUBTYPES/ MODE OF PHYSIOLOGICAL PHYSIOLOGICAL DEVELOPMENTAL
RECEPTORS SUBUNITS TRANSDUCTION MODE OF ACTIVATION ROLE REGULATION

mGluRs Group I,II and III IP3-Induced Unknown (synapses?) OPC: Regulate Ca2+- Downregulated during
calcium release permeable AMPARs OL maturation
expression. Regulate
the release of BDNF.
GABABRs B1 and B2 Adenylate cyclase Unknown (synapses?) OPC: Promote prolif- Downregulated during
Inhibition eration and migration OL maturation
Purinergic P2Y P2Y1 IP3-induced Mostly unknown, but OPC: Decrease Robust expression in
calcium release activated by ATP release proliferation, increase OPCs and mature OLs
from astrocytes migration and
differentiation
OL: Unknown
Muscarinic AchRs M3>M4>M2>M1>M5 IP3-Induced Unknown OPC: Increase prolif- Downregulated during
calcium release eration and survival, OL maturation
inhibit differentiation
Dopamine D2 and D3 Not yet defined Unknown OPC: Regulate OPC D3 expressed in OPC
in OL proliferation but absent from
OL: Unknown, but mature OL. D2/D3
protective against ratio increases during
excitotoxicity maturation

246 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
 Oligodendrocyte lineage cells express functional ionotro-
pic receptors for most, if not all, known neurotransmitters
(Table 20.2). Except for 5-HT and ATP, the expression level
of neurotransmitter receptors appear to decrease once OPCs
begin differentiating into myelinating OLs—suggesting that
neurotransmitters may play specific roles during early stages of
OL lineage, particularly the OPC stage, characterized by pro-
teoglycan (NG2) expression (see chapter 21). However, the
physiological function of neurotransmitter receptor remains
unresolved in both OPCs and oligodendrocytes.
3.1 I O N OT RO P I C G LU TA M AT E R E C E P TO R S
Ionotropic glutamate receptors (iGluR) have a central role
in brain physiology, being fundamental to most excitatory
neurotransmission between neurons. The three main types
of iGluRs are: AMPA- and kainate-sensitive iGluRs (respec-
tively, AMPARs and KARs), and N-methyl-d-aspartic acid
(NMDA)-sensitive iGluRs (NMDARs). In neurons, the
types of iGluRs tend to have distinct functions: AMPARs
and KARs enable fast synaptic excitatory neurotransmission,
whereas NMDARs are crucial in synaptic plasticity—mod-
ulating the strength of excitatory synapses, notably via their
Ca2+ permeability. Although NMDARs and AMPARs have
complementary functions in neurons with respect to synap-
tic transmission, their functions and interactions in OLs are
largely undetermined.
3.1.1 AMPA- and Kainate-Sensitive
Glutamate Receptors
AMPARs and KARs are tetrameric, cationic, receptor
channels that can be composed from four distinct subunits
for AMPAR (GluA1–GluA4) and five subunits for KAR
(GluK1–GluK5). AMPARs can assemble either as homo-
tetramers or heterotetramers, with functional properties
dictated by their subunit composition and the presence
of auxiliary transmembrane AMPAR regulatory proteins
(TARPs). Most AMPARs are composed of a dimer of GluA2
subunits and a dimer of GluA1, GluA3, or GluA4. KARs can
either form homomers from subunits GluK1, GluK2, GluK3,
or heteromers where GluK1–3 subunits combine with GluK4
or GluK5.
Reverse transcriptase polymerase chain reaction (RT-PCR)
and Western blot experiments have detected AMPAR subunits
GluA2, GluA3, and GluA4 in OLs (De Biase et al. 2010; Itoh et
al. 2002). AMPA receptors in most mature neurons include a
RNA-edited GluA2 subunit, which conveys poor Ca2+ perme-
ability. By contrast, AMPARs in OPCs were shown to exhibit
a significant fraction of Ca2+-permeable AMPARs (Bergles
et al. 2000). It is unclear whether this property stems from
the presence of a nonedited GluA2 subunit or the complete
absence of GluA2 subunits in the Ca2+-permeable AMPARs
(Li and Stys 2000). However, biochemical evidence suggests
that Ca2+-permeable AMPARs in OLs lack the GluA2 subu-
nit, with immmunoprecipitation experiments demonstrating
the presence of protein complexes in OLs formed exclusively
of GluA3 and GluA4 (Itoh et al. 2002).
P H YS I O L O GY O F O L I G O D E N D R O C Y T E S
Rat OLs in culture and in vivo are known to express KAR
subunits GluK2, GluK3, GluK4, and GluK5 (De Biase et al.
2010; Sánchez-Gómez and Matute 1999), but OPCs appar-
ently express only a small number of functional KARs. Indeed,
kainate applied to OPCs in brain slices induces only small per-
sistent inward currents (Kukley and Dietrich 2009).
Cellular consequences of AMPAR/KAR activation in
OLs are not yet clearly defined. A current hypothesis regard-
ing transduction of GluR activation in OLs involves entry
of Ca2+, the most common intracellular second messenger;
AMPA receptors expressed by OPCs are often Ca2+-permeable
(Bergles et al. 2000). Ca2+ entry might also involve the abil-
ity of glutamate to depolarize OL membranes enough to
activate voltage-operated Ca2+ channels (Berger et al. 1992).
Depolarization induced by glutamate has also been shown
to activate Na+ channels, inducing a large enough increase
in intracellular Na+ to allow Ca2+ entry via the Na+/Ca2+
exchanger (NCX1) (Chen et al. 2007); and GluR activation
could influence OL physiology in a Ca2+-independent man-
ner. Indeed, Na+ entry via GluR can block voltage-gated K+
channels independently of Ca2+ and G-protein (Borges and
Kettenmann 1995). This blockade, for example, has been
shown to prevent OPC proliferation and their differentiation
into myelinating OLs (Gello et al. 1996).
As in neurons, it is now clear that OPCs receive functional
glutamatergic synapses from neurons, both in white and gray
matter (see chapter 21); however, these synapses are transient
(De Biase et al. 2010; Etxeberria et al. 2010) and disappear as
OLs mature into myelinating OLs: KARs do not appear to
be recruited during neuron-to-OPC synaptic transmission
(Kukley and Dietrich 2009). Therefore, if AMPARs/KARs
expressed by mature OLs are activated at all, it is likely to occur
in a nonsynaptic manner such as volume transmission, spill-
over, or release from astrocytes (Volterra and Meldolesi 2005).
Because in vitro studies showed that AMPAR activation
can regulate OPC differentiation into myelinating OLs, it
was proposed that axons can regulate their own myelination
by releasing glutamate onto surrounding OLs (Yuan et al.
1998). Axonal glutamate release could allow surround-
ing OPCs and myelinating OLs to be constantly informed
about their relative conduction speeds and degree of
synchrony with surrounding axons, allowing myelination
to occur in an orderly manner (Mangin and Gallo, 2011).
In mature OLs, the importance of AMPAR is less certain
since neuron-to-OPC synapses and AMPAR expression are
both downregulated when OLs begin myelination. Because
overactivation of AMPARs can mediate OLs’ cell death in
pathological conditions such as ischemia (Matute 2011),
AMPARs might also regulate physiological cell death in
mature OLs occurring during normal myelin turnover in
adulthood.
3.1.2 NMDA-Sensitive Glutamate Receptors
NMDARs are heterotetramers formed through combinations
of NR1 with NR2 or NR3 subunits. The NR1 subunit con-
tains the binding site for glycine/d-serine and is necessary for
forming a functional NMDAR.
• 247
 Both OPCs and mature OLs express functional NMDARs
(De Biase et al. 2011; Káradóttir et al. 2005) that can induce
intracellular Ca2+ transients when activated (Micu et al.
2006). Patch-clamp recordings on rat cerebellar OLs and
co-immunoprecipitation experiments with rat purified myelin
have shown that functional NMDARs in OLs are most likely
composed of NR1, NR2C, NR2D, and NR3A subunits
(Burzomato et al. 2010; Micu et al. 2006).
Unlike AMPARs in OPCs, NMDARs are apparently not
significantly activated by the synaptic release of glutamate
from neurons (De Biase et al. 2010). Interestingly, a recent
study showed that NMDARs composed of NR1 and
NR3 subunits in optic nerve myelin could be activated by
non-glutamatergic ligands such as glycine and D-serine
(Piña-Crespo et al. 2010).
Several years ago, some high profile studies showed
that glutamate excitotoxicity in OLs during ischemia was
mediated not only by AMPAR/KAR, but also by NMDAR
activation during development (Karadottir et al. 2005;
Micu et al. 2006). However, the pathophysiological and
physiological importance of NMDAR expression in the
OL lineage was recently undermined by two studies analyz-
ing the consequences of a specific NMDAR genetic invali-
dation in OLs. After a specific NR1 knockout in the OL
lineage, researchers failed to detect any physiological
or pathophysiological consequences of the absence of
functioning NMDAR in OLs (De Biase et al. 2011; Guo
et al. 2012); only upregulation of Ca2+-permeable AMPARs
was noted (De Biase et al. 2011). Moreover, it has been
shown that NMDAR blockade during white matter isch-
emic injury could worsen outcome (Baltan et al. 2008), sug-
gesting that NMDAR activation may not mediate glutamate
excitotoxicity.
3.2 I O N OT RO P I C G A M M A-A M I N O BU T Y R I C
AC I D R E C E P TO R S
Gamma-aminobutyric acid (GABA) receptor Cl– channels
(GABAAR) are members of the nicotinic ligand–gated ion
channel superfamily and are important in brain function for
mediating fast synaptic inhibition between neurons.
GABAARs are pentamers usually made of two α subunits,
two β subunits, and one γ subunit. They exhibit great com-
binatorial diversity because of a large number of GABAAR
subunits: six different α subunits (α1–α6), three different β
subunits (β1–β3), and three different γ subunits (γ1–γ3).
Expression of functional GABAARs in OLs was first
demonstrated in spinal cord organotypic culture (Gilbert et
al. 1984) and OL cell culture (Von Blankenfeld et al. 1991),
and later confirmed in brain slice recordings. The exact com-
position of GABAARs in OLs remains unclear. For example,
GABAARs containing the γ subunit are identified based on
their sensitivity to benzodiazepines, and initial study of OLs
in cell culture showed that GABAAR-mediated responses
were enhanced by applications of barbiturates and benzodi-
azepines and diminished by the inverse benzodiazepine ago-
nist DMCM (Von Blankenfeld et al. 1991). However, a study
248 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
examining GABA responses in rat OPCs in culture failed to
detect modulation by the benzodiazepine flunitrazepam or
the inverse agonist DMCM (Williamson et al. 1998). Recent
experiments in brain slices showed that GABAARs expressed
by OPCs can be activated via neuron-to-OPC synapses and
the resulting synaptic currents can be modulated by applying
benzodiazepines (Lin and Bergles 2004). Because γ2 is nec-
essary for synaptic targeting of GABAARs in neurons, these
observations support functional expression of γ2-containing
GABAARs in mouse OPCs. Interestingly, in mouse soma-
tosensory cortex, GABAARs expressed by OPCs switched
from a synaptic to an extrasynaptic localization during devel-
opment, suggesting that expression of γ2 subunits may be
physiologically regulated in OPCs (Vélez-Fort et al. 2010).
GABAAR activation in cultivated OLs can lead to intracel-
lular Ca2+ increases that are inhibited by the N-type VOCC
blocker nifedipine (Kirchhoff and Kettenmann 1992). Recent
studies in brain slices confirmed the ability of GABAAR ago-
nists to induce Ca2+ entry into OLs (Vélez-Fort et al. 2010).
As in immature neurons, GABAAR activation in OPCs had an
excitatory effect because of a depolarized reversal potential of
Cl–, between –30 and –40 mV (Lin and Bergles 2004).
GABAergic synapses from neurons are known to contact
OPCs (Lin and Bergles 2004). Neuron-to-OPC GABAergic
synapses tend to be lost when OPCs start differentiating into
myelinating OLs. However, GABAARs can also be activated in
cortical OPCs via extrasynaptic release of GABA (Vélez-Fort
et al. 2010).
As to physiological function, GABAAR activation is known
to regulate the growth rate of different types of neural precur-
sors by inhibiting or promoting their proliferation, depending
on the neural cell type and/or culture conditions. GABAAR
activation by exogenous application of GABA on perina-
tal OPCs in cultured organotypic cerebellar slices inhibits
their proliferation (Yuan et al. 1998). However, treating the
slices with GABAAR antagonist bicuculline had no effect
(Yuan et al. 1998), suggesting that endogenous activation of
GABAAR may not be a major control mechanism for OL cell
cycle progression, at least in the cerebellum.
3.3 AT P S E NS IT I VE P U R I N E RG I C
R EC E P TO R S
ATP is often released by neurons along with other neurotrans-
mitters and contributes to their excitability. ATP responses
are mediated by both ionotropic (P2X) and metabotropic
(P2Y) receptors (see section 4.3). Seven mammalian P2X
subunits (P2X1–7) assemble as trimers to form cationic
homomeric and heteromeric receptor channels with diverse
properties. Much like NMDA receptors, ATP-gated P2X
channels have marked Ca2+ permeability. P2X receptors are
expressed in CNS neurons, where they participate in fast syn-
aptic transmission and modulation; P2X7 receptors mediate
immunomodulatory responses in microglia and neurodegen-
eration after ischemia.
Oligodendrocyte lineage cells express P2X receptors in
vitro and in vivo (Agresti et al. 2005a,b; Matute et al. 2007).
The electrophysiological, pharmacological, and molecular
profile of P2XR expressed by OPCs and mature OLs mostly
corresponds to the P2X7 subtype. P2X7 receptors are acti-
vated at high concentrations of ATP (0.1–1 mM range), and
are capable of pore formation, resulting in sustained influx
of Ca2+. ATP and adenosine released from axons during elec-
trical impulse activity are known to regulate OPC migra-
tion, proliferation, and differentiation (Agresti et al. 2005).
Although this effect seems mostly mediated via metabotro-
pic purinergic receptor (see section 4.3), P2X7R may also
be involved. ATP released by astrocytes can also evoke a
rapid and transient rise in intracellular Ca2+ in OPCs involv-
ing metabotropic purinergic receptor and P2X7 receptors
(Hamilton et al. 2010).
Unlike many other ligand-gated ion channels in the OL lin-
eage, the robust expression of P2x7R by mature OLs suggests
a specific physiological function in these cells, possibly related
to myelin formation and preservation, and allowing mature
OLs to sense electrical activity in the axons they insulate.
3.4 G LYC I N E R E C E P TO R S
Similar to GABAARs, glycine receptor Cl– channels (GlyRs)
belong to the nicotinic ligand-gated ion channel superfam-
ily. Their main known function is to mediate fast synaptic
inhibition between neurons, mostly in the spinal cord and
brainstem. Structurally, GlyRs are pentameric transmembrane
proteins that can comprise two types of subunits: a 48-kDa α
subunit with four variants (α1, α2, α3, and α4) and a 58-kDa
β subunit. Two main types of GlyRs are distinguished: a pen-
tameric configuration of five α subunits and a heteromeric
configuration of two α and three β subunits.
Functional GlyRs have been described in newborn rat OL
lineage cells and OPCs in spinal cord slices (Belachew et al.
1998; Kirchhoff et al. 1996). Although the exact structure and
composition of GlyRs expressed in OLs is not determined,
RT-PCR using total RNA extracted from cultivated OPCs
of newborn rat cortex demonstrated only the presence of α2
and β subunits (Belachew et al. 1998). However, GlyR subunit
expression is known to be both developmentally regulated and
region specific, so extensive analysis of GlyR subunit expres-
sion is still needed in several regions of developing mouse
brain.
As for most other ligand-gated ion channels, GlyR expres-
sion seems to peak at the OPC stage and decrease thereafter
(Belachew et al. 1998).
3.5 N I C OT I N I C AC ET Y L C H O L I N E R E C E P TO R S
Nicotinic acetylcholine ionotropic receptors (nAchRs)
have numerous functions in the brain. First, they are funda-
mental to locomotion and behavior, mediating excitatory
neurotransmission between motor neurons and their target
muscles. In adult brain, nAChRs participate in neuromodula-
tion by influencing release of various neurotransmitters from
neurons. During development, nAchRs participate in early
activity-dependent network patterning.
P H YS I O L O GY O F O L I G O D E N D R O C Y T E S
nAChRs are pentameric transmembrane proteins and per-
meable to cations. In vertebrates, nAChR subtypes are broadly
classified into muscle and neuronal subtypes. In mammalian
CNS, neuronal nAChR can consist of eight α subunits (α2,
α3, α4, α5, α6, α7, α9, and α10) and three β subunits (β2,
β3, and β4). nAChRs are either heteromers made of two α
and three β or homomers made of five α subunits. Receptors
composed of different subunits exhibit diverse pharmacologi-
cal, regulatory, and functional properties.
Oligodendrocyte progenitor cells express different sub-
types of functional nAChRs. RT-PCR analysis and immu-
nocytochemistry in OPCs cultured from rat corpus callosum
have detected expression of nAChR subunits α3, α4, α5,
α7, β2, and β4. Nicotine application can induce increased
intracellular Ca2+ in OPCs cultivated from rat corpus cal-
losum (Rogers et al. 2001), an increase sensitive to the Ca2+
VOCC blocker nifedipine, demonstrating that nAChR
activation can depolarize OPCs enough to activate VOCCs
(Rogers et al. 2001). Recently, the presence of Ca2+-permeable
α7-containing nAChRs has been demonstrated in OPCs
recorded from mouse hippocampus slices (Vélez-Fort et al.
2009). Importantly, this study could not detect any nAChR-
mediated synaptic current in OPCs.
4 M ETA B OT R O P I C R E C E P TO R S
Metabotropic neurotransmitter receptors are G-protein–
coupled proteins and important in neuromodulation. Acting
indirectly on neuronal behavior, they modulate the function
of ligand- and voltage-gated ion channels or influence diverse
intracellular processes via second messengers such as Ca2+ and
cAMP.
4.1 M ETA B OT RO P I C G LU TA M AT E R EC E P TO R S
Metabotropic glutamate receptors (mGluR) can belong to one
of three groups. Group I includes mGluR1 and mGlur5; their
activation stimulates phospholipase C (PLC) to produce inosi-
tol 1,4,5-triphosphate (IP3), inducing the release of Ca2+ from
intracellular stores. Group II, comprising mGluR2 and mGluR3,
and Group III (mGluR4, mGluR6–8) have a stimulating influ-
ence on adenylate cyclase, leading to cAMP production.
Although OL cells can express all three groups of mGluRs
(Bagayogo and Dreyfus 2009; Luyt et al. 2006), their expres-
sion level is apparently developmentally regulated, becoming
very low in mature OLs. Group I mGluRs were recently shown
to regulate expression of Ca2+-permeable AMPARs in OPCs
via elevation of Ca2+ and release of IP3 (Zonouzi et al. 2011).
Group I mGluRs may also be involved in regulating normal
physiological OL cell death, as they offer protection against
kainate-induced excitotoxicity (Luyt et al. 2006). Although
the physiological role of mGluRs in OLs has not been eluci-
dated, a recent study showed that group I mGluRs can regulate
release of growth factor BDNF in OL cell cultures (Bagayogo
and Dreyfus 2009). By cultivating cortical neurons with OLs,
the authors showed that OL-derived BDNF could regulate
• 249
the number of glutamatergic neurons, suggesting that OL
may release trophic factors in an activity-dependent manner
by activating group I mGluRs, and regulating neuron survival
and synapse formation in vivo.
4.2 M ETA B OT RO P I C G A BA B R E C E P TO R S
GABAB receptors (GABABRs) are G-coupled metabotropic
receptors known to mediate slow inhibition at central synapses.
They can be composed of two major types of subunits, B1 and
B2, among which different splice variants (GABAB1a–g) are
described. Most neurons coexpress both subunits, which form
functional GABABRs via heterodimeric assembly, although
GABAB1 subunits may be able to form functional homodi-
mers. In neurons, presynaptic GABABRs are known to sup-
press neurotransmitter release by inhibiting VOCCs. At the
postsynaptic membrane, GABABR activation usually inhibits
adenylyl cyclase (AC) and activates Kir3-type K+ channels,
hyperpolarizing the membrane.
A study reported functional expression of GABABRs in
OLs (Luyt et al. 2007). Using RT-PCR and in situ hybridiza-
tion, this study showed the presence of RNA for GABAB1 and
GABAB2 subunits in OPCs and, to a modest extent, in a CD4-
derived OL cell line. Reduced expression of GABAB subunits in
mature OLs is compatible with the lack of immunoreactivity for
GABAB1 in white matter myelinating OLs (Charles et al. 2003).
In a physiological context, it is unknown whether GABABRs in
OLs are activated by synaptic or extrasynaptic release of GABA
from neurons and/or by a glial source of agonist.
4.3 P U R I N E RG I C M ETA B OT RO P I C R E C E P TO R S
Purinergic metabotropic receptors comprise both adenosine-
sensitive P1 and ATP-sensitive P2Y receptors. Four subtypes
of adenosine receptors have been described: P1A1 and P1A3
receptors inhibit cAMP via Gi/o, whereas P1A2A and P1A2B
receptors stimulate cAMP via the protein Gs. Although all
four subtypes have been detected in OPC culture by RT-PCR
(Stevens et al. 2002), their presence in more mature OLs is
still undetermined.
Eight subtypes of P2Y receptors have been cloned in mam-
mals; they are sensitive either to adenine nucleotides ATP/
ADP (P2Y1, 11, 12, 13), uracil nucleotides UTP/UDP (P2Y4,
6), both (P2Y2), or UDP-glucose (P2Y14). P2Y receptors can
activate PLC and induce Ca2+ release via Galpha(q/11) (P2Y1,
P2Y2, P2Y4, P2Y6, and P2Y11). They can also stimulate or
inhibit AC via Galpha(s) and Galpha(i/o) proteins (P2Y12,
P2Y13, and P2Y14).
Oligodendrocytes in cell culture and in acute corpus
callosum slices respond to ATP with Ca2+ elevation.
Oligodendrocytes seem to predominantly express P2Y1R, its
activation leading to IP3-mediated release of Ca2+ from intra-
cellular stores (Agresti et al. 2005a,b). It was suggested that
P2Y responses are developmentally regulated, because only
late OL precursors and mature OLs appeared to exhibit sig-
nificant ATP-induced Ca2+ elevation. However, recent stud-
ies showed that P2Y1R activation in OPCs can stimulate cell
migration, inhibit mitogenic response to PDGF, and promote
OL differentiation (Agresti et al. 2005a,b).
250 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
4.4 MUS C A R I N I C AC ET Y L C H O L I N E
R EC E P TO R S
As in other metabotropic neurotransmission, mAchRs play
various neuromodulatory functions. So far, five mAchR sub-
types have been identified (M1–M5).
Oligodendrocytes express functional mAchRs whose acti-
vation by muscarine, or the specific agonist carbachol, triggers
intracellular signals such as MAPK, IP3, and Ca2+ mobili-
zation (Kastritis and McCarthy 1993). At the RNA level,
expression of all mAChR subtypes has been reported in both
OPCs and OLs (De Angelis et al. 2011; Ragheb et al. 2001).
The predominant mAChR subtype expressed in OLs is M3,
followed by M4, M2, M1, and M5 (Ragheb et al. 2001). As for
most other neurotransmitter receptors, all mAchR subtypes
are downregulated in mature OLs (De Angelis et al. 2011).
In OPCs, M3, M1, and M4 activation significantly increases
OPC proliferation and survival (De Angelis et al. 2011),
and mAchR activation inhibits OPC differentiation into
myelinating OLs.
4.5 D O PA M I N E R EC E P TO R S
Dopamine D3 receptor (D3R) is known to be expressed by
precursors and immature OLs, but is absent from mature OLs
(Bongarzone et al. 1998; Niu et al. 2010). D3R expression cor-
related with the peak of myelination in corpus callosum. The
presence of D2R was also reported in a subset of mature inter-
fascicular OLs in rat corpus callosum (Howard et al. 1998),
suggesting a model in which OLs switch from D3 to D2 as they
mature (Niu et al. 2010). Applying the D2/D3 specific agonist
quinpirole was shown to increase the number of OL precur-
sors in cell culture, an effect blocked by dopamine antagonist
haloperidol (Bongarzone et al. 1998). However, another study
showed that haloperidol increased proliferation of rat OPCs in
culture (Niu et al. 2010). These contradictory results may stem
from the inability of quinpirole and haloperidol, acting on both
D2R and D3R, to discriminate between these receptors, whose
ratio may vary with culture conditions and duration. Finally,
D2/D3 activation has been shown to protect OLs against glu-
tamate excitotoxicity (Rosin et al. 2005), suggesting that dop-
amine receptors may also regulate OL physiological cell death.
4.6 OT H E R L I G A N D S
Oligodendrocytes are also reported to express receptors to
a variety of other neuromodulators such as adrenaline α1
receptors (Cohen and Almazan 1993), bradykinin recep-
tors (Stephens et al. 1993), opioid μ and κ receptors (Knapp
et al. 2009), and cannabinoid CB1 and CB2 receptors (Gomez
et al. 2011; Molina-Holgado et al. 2002).
5 S U M M A RY A N D P E R S P E C T I VE S
This chapter demonstrates that the study of OL physiology is
still a work in progress. Oligodendrocytes have been clearly
shown to express a broad variety of membrane proteins involved
in cell-to-cell signaling in the CNS. The major challenge now
is to elucidate and define the functional roles of these channels
and receptors in: (1) OL development; (2) neuron-OL and
astrocyte-OL signaling; (3) participation in neuronal net-
work function; and (4) OL pathology. Elucidating OL physi-
ology will depend on optimizing the methods by which these
cells are identified, and investigating their physiology in situ
to analyze changes related to their development, morphology,
and survival. Many of the crucial questions still pending will
be investigated using more advanced molecular and biophysi-
cal approaches to determine the subcellular location of ionic
channels and receptors.
Central to understanding OL physiology is the question
of whether these myelinating cells have additional functions,
such as maintaining axonal integrity and intercellular com-
munication. It will be important to define the distinct roles of
receptors and channels throughout the various stages of OL
development, as well as during OL turnover and regeneration
in the brain. A second question crucial to understanding OL
pathology—as well as normal physiology—is whether these
signaling proteins regulate myelin formation directly, and
whether their engagement in myelin maintenance continues
in adults. A comparative analysis of the physiological proper-
ties of OLs in white and gray matter would be a valuable con-
tribution to these efforts. Finally, researchers must determine
when, and whether, the physiological properties of OLs are
terminally established and the degree to which OL channel
and receptor plasticity in the adult and aging brain is respon-
sive to changing patterns of neuronal activity.
In the past 20 years, perception of OLs has evolved from
images of relatively passive insulation providers for CNS
axons to excitable cells sharing many membrane and conduc-
tive properties with the neurons they support. Now, as new
questions are asked and new technologies become available to
answer those questions, it should surprise no one if the next
two decades see even more rapid gains in understanding OL
physiology—not only piling up facts, but applying the knowl-
edge with ingenuity to OL pathologies in the human CNS.
AC K N OW L E D G M E N T S
The authors thank Li-Jin Chew for critically reading parts of this
chapter. They also thank Pablo Paez and Ramesh Chittajallu
for providing figures. VG was supported by R01NS045702,
R01NS056427, P01NS0626860, MS Society RG 4019 and
P30HD40677. JMM was supported by INSERM, CNRS,
and ANR.
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 21.
PHYSIOLOGICAL PROPERTIES OF NG2 + GLIAL CELLS
Dwight E. Bergles

A B B R E VI AT I O N S is known about the physiological characteristics of NG2+ glial

cells, with a focus on their membrane properties and the changes
AMPA 2-amino-3-(5-methyl-3-oxo-1, that these cells undergo during development and differentiation.
2-oxazol-4-yl) propanoic acid The expression of a diverse array of voltage-gated ion channels
CaV voltage-gated Ca2+ channel and neurotransmitter receptors, and their interaction with neu-
CNS central nervous system rons through direct synapses, raise new questions about the role
Cx32 Connexin32 of these enigmatic glial cells in CNS physiology.
EPSCs excitatory postsynaptic currents
GABA gamma-aminobutyric acid
MNI-L-glutamate 4-methoxy-7-nitroindolinyl-caged- 2 T E R M I N O L O GY
L-glutamate
NAV voltage-gated Na+ channel The development of antibodies that recognize NG2 made it pos-
NBQX 2,3-dihydroxy-6-nitro-7-sulfamoyl- sible to identify NG2+ glial cells in fixed CNS tissue (Levine and
benzo(f )quinoxaline Card 1987), and provided the impetus to develop transgenic mice
NMDA N-methyl-d-aspartate that enable visualization of these cells in living tissue for targeted
OPC oligodendrocyte precursor cell physiological studies in different brain regions (Karram et al.
PCR polymerase chain reaction 2008; Mallon et al. 2002; Zhu et al. 2008; Ziskin et al. 2007).
pre-OL premyelinating oligodendrocytes However, pioneering investigations into the physiological prop-
RS-CPP (RS)-3-(2-carboxypiperazin-4-yl)- erties of glial cells occurred long before these tools were available,
propyl-1-phosphonic acid and thus the identity of NG2+ cells was established. Although
SR-95531 6-Imino-3-(4-methoxyphenyl)- it is difficult to achieve certainty through retrospective analysis,
1(6H)-pyridazinebutanoic acid prior studies of cells defined as “β astrocytes,” “complex astrocytes
hydrobromide (Gabazine) or complex cells,” “GluR astrocytes,” and “glial progenitors,” are
TARP transmembrane AMPAR regulatory likely to represent evaluations of NG2+ cells, based on similarities
protein of their properties to those exhibited by unambiguously defined
TBOA DL-threo-β-benzyloxyaspartic acid NG2+ cells. NG2+ glial cells have the ability to differentiate into
TEA tetraethylammonium oligodendrocytes, and therefore they are routinely referred to as
oligodendrocyte precursor cells (OPCs); the term polydendro-
cytes has more recently been adopted to avoid the implication that
1 INTRODUCTION NG2+ cells only serve as oligodendrocyte progenitors. As new
roles for these ubiquitous cells are uncovered, the terms used to
The mammalian central nervous system (CNS) contains a popu- describe these cells may expand further. Moreover, the possibility
lation of stellate-shaped glial cells that express the chondroitin exists that there is further diversity within this broad grouping, a
sulfate proteoglycan NG2 (CSPG4) and the alpha receptor for topic that is discussed in the following. The studies described in
platelet derived growth factor (PDGFDR). These NG2+ glial this chapter were performed exclusively in brain tissue isolated
cells are widely distributed throughout the gray and white matter from rats and mice, although NG2+ cells (OPCs) also appear to
regions of the adult CNS, accounting for approximately 5% to be abundant in the CNS of humans and other primates (Chang
8% of all cells, and exhibit tiling behavior, with each cell occupy- et al. 2000; Peters 2004).
ing a unique domain. Although these cells were initially classified
as a type of astrocyte, based on their similar morphology, it is
now clear that they belong to the oligodendrocyte lineage and 3 M E M B R A N E P R O P E RT I E S
are physiologically distinct from typical protoplasmic astrocytes.
These cells serve as progenitors for oligodendrocytes during 3.1 M E M B R A N E C A PAC ITA N C E , R E S T I N G
both early development and adulthood, and retain the capac- P OT E N T I A L , A N D R E S T I N G M E M B R A N E
ity to divide throughout life. The recent development of trans- C O N D U C TA N C E
genic mice in which fluorescent proteins are selectively expressed
by NG2+ cells has provided unprecedented access to this glial In the mature CNS, NG2+ cells have elongated, small somata
population for physiological studies. This chapter discusses what approximately 10 Pm in diameter from which numerous,
254
 A B

Hippocampus Cerebellum (ml)

C D

Optic Nerve Corpus Callosum

Figure 21.1 Morphology of NG2+ Cells. A–D. NG2 immunostaining in brain tissue from an adult mouse. NG2+ glial cells have small cell bodies from
which numerous processes extend into the surrounding neuropil. The number of processes, their length and extent of ramification, and their orienta-
tion varies between brain regions. Scale = 20 Pm.

highly branched processes extend (Fig. 21.1). Although simi-
lar, their morphology varies significantly among brain regions:
in the hippocampus and cortex their processes extend radially,
while in the corpus callosum their processes are closely aligned
to the orientation of callosal axons. Within these regions, vari-
ations are seen in the number of processes, their length, and
their extent of ramification within the neuropil.
Whole-cell recordings from NG2+ cells in brain slices iso-
lated from young adult mice indicate that they have a total
cell capacitance of approximately 20 pF, consistent with their
modest size. By comparison, most mature neurons have capac-
itances in excess of 50 pF (Tyzio et al. 1999), because of their
larger somata and extensive dendritic arbors. Unlike neurons,
NG2+ cells do not show obvious polarity or morphological
specializations, such as axons or dendrites that would suggest
that there are defined input and output regions of the cell. In
physiological conditions, NG2+ cells exhibit a highly negative
resting potential of approximately –90 to –100 mV (Bergles
et al. 2000; De Biase et al. 2010; Lin and Bergles 2004), close
to the calculated equilibrium potential for K+, suggesting that
the primary ion channels open at rest flux K+. In mature tissue
significant numbers of channels are open at rest in NG2+ cells,
as the membrane resistance averages 300 MΩ, a value compa-
rable to most neurons, but approximately tenfold higher than
astrocytes or oligodendrocytes. Among the channels that are
likely to contribute to the resting potential are inwardly rec-
tifying K+ channels, in particular Kir4.1, a channel that has
been implicated in establishing the resting potential in vari-
ous glial cell types (Djukic et al. 2007; Neusch et al. 2001).
P H YS I O L O G I C A L P R O P E RT I E S O F N G 2 + G L I A L C E L L S
It is also likely that NG2+ cells express members of the two-
pore K+ channel (K2P) families, as the membrane resistance
of many cells remains low in the presence of internal Cs+ and
tetraethylammonium (TEA), which are effective at blocking
most voltage-dependent K+ channels, but have limited ability
to inhibit K2P channels (Kim, 2005).
3.2 VO LTAG E - G AT E D I O N C H A N N E L S
3.2.1 Voltage-Gated K+ Channels
When positive current is injected into NG2+ cells from the
resting potential during whole-cell current-clamp record-
ings, their membrane depolarizes, revealing nonlinear
behavior that is indicative of the presence of voltage-gated
ion channels. This behavior contrasts with that exhibited by
astrocytes, which exhibit passive responses because of their
much higher resting conductance (see chapter 16). Notably,
it was the “complex” voltage waveform exhibited by NG2+
cells that resulted in their designation as “complex cells”
or “complex astrocytes”. On depolarization, the membrane
potential rapidly moves to a new voltage and then contin-
ues to rise slowly when current is maintained (Fig. 21.2).
Both A-type and delayed-rectifier K+ channels contribute
prominently to this behavior. In voltage-clamp conditions,
rapidly activating and inactivating A-type K+ channels can
be observed in the initial response to a voltage step, whereas
the sustained current is mediated by delayed-rectifier chan-
nels, both of which are inhibited by internal Cs+ and TEA.
• 255
 A NG2+ cell B Astrocyte
10 mV 0.5 mV
100 ms 100 ms
C D
500 pA 2 nA
50 ms 50 ms
E F
Figure 21.2 Comparison of the Physiological Properties of NG2+ Cells
and Astrocytes. A. Current-clamp recording from an NG2+ cell located
in the stratum radiatum region of area CA1 in a hippocampal brain
slice. Injection of positive current injection elicited a complex mem-
brane response due to activation of NaV, CaV, and Kv. B. Whole-cell
current-clamp recording from an astrocyte located in the same region.
The same current amplitudes used as shown in (A). Note that these cur-
rents only induced small deviations of the membrane potential because
of the high resting conductance. Vm = −90 mV. C. Whole-cell voltage
clamp recording from the same cell shown in (A, B). Hyperpolarizing
voltage steps revealed the presence of Kir currents, while depolarizing
voltage steps reveal the presence of A-type and delayed-rectifier type K+
currents. Steps: −60 pA to 300 pA, 40 pA increments from −90 mV
in (A, B), and −130 mV to 20 mV, 20-mV increments in (C, D) from
−90 mV. (E,F) Tracer injection (neurobiotin) into an NG2+ cell (E)
does not lead to transfer to other cells, whereas tracer injection into
an astrocyte (F) results in transfer to many neighboring cells. Scale =
50 Pm. Adapted from Lin et al. 2004.
It is likely that Kv.1.3 and Kv.1.5 channels contribute to these
conductances because they are expressed in NG2+ cells in
early postnatal tissue (Chittajallu et al. 2002). In response
to hyperpolarizing steps, an inward current develops nega-
tive to the K+ reversal potential. This current is blocked by
low concentrations of extracellular Ba2+, and is absent in
glial-specific Kir4.1 knock-out mice (Djukic et al. 2007).
256 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
In Kir4.1 knock-out mice, NG2+ cells (complex glia) exhib-
ited resting membrane potentials approximately 40 mV
more depolarized and much higher membrane resistances,
(~500 MΩ), suggesting that this K+ channel plays a domi-
nant role in controlling both the resting potential and rest-
ing membrane conductance of NG2+ cells, although an
indirect effect caused by removal of Kir4.1 from surround-
ing astrocytes cannot be excluded.
3.2.2 Voltage-Gated Na+ Channels and
Membrane Excitability
NG2+ cells also express voltage-gated Na+ channels (NaVs) that
are sensitive to block by tetrodotoxin. The density of these chan-
nels varies over a wide range among the population (Chittajallu
et al. 2004; De Biase et al. 2010; Tong et al. 2009); however, the
current density (current normalized to cell capacitance) is sig-
nificantly lower than the density observed in neurons, and the
kinetics of these channels is slower (Tong et al. 2009). Voltage-
gated Na+ channels perform the crucial role of generating the
current necessary for the depolarizing upstroke of the action
potential in neurons. Given that NG2+ cells express the neces-
sary repertoire of channels involved in action potential genera-
tion, they may also be excitable. However, in most NG2+ cells
in the mature CNS, injection of depolarizing current does
not elicit a regenerative, Na+-dependent spike in membrane
voltage characteristic of action potentials (Chittajallu et al.
2004; De Biase et al. 2010). Action potential generation is
dependent on the relative abundance of Na+ and K+ channels,
their activation and inactivation kinetics, as well as intrinsic
membrane properties (membrane capacitance and resistance).
In NG2+ cells, the relatively low density of NaV channels rela-
tive to Kv channels, and the slower kinetics of the NaV chan-
nels expressed help to limit generation of action potentials.
However, during early postnatal periods (< postnatal day 14),
when K+ channel density is lower, one or more Na+ spikes can
be elicited in some NG2+ cells on depolarization (Chittajallu
et al. 2004; De Biase et al. 2010; Karadottir et al. 2008)
(Fig. 21.3). In comparison with neurons, these spikes have
higher threshold for initiation, smaller amplitude, and the
waveform is not as tightly stereotyped, varying with the volt-
age at which the spikes are generated. NG2+ cells also typically
cannot generate prolonged trains of Na+ spikes in response to
continued depolarization, perhaps because of the slow recov-
ery of NaV channels from inactivation and the presence of a
persistent NaV current (but see Karadottir et al. 2008). The
incidence of this rudimentary form of excitability varies among
the population even at young ages (Chittajallu et al. 2004),
but progressively declines during the first postnatal month,
as K+ channel expression increases (De Biase et al. 2010). A
subset of NG2+ cells may still exhibit excitable behavior in
adults, and the proportion of cells with this capability appears
to differ among mammalian species (Karadottir et al. 2008).
Because of their highly negative resting potential, NG2+ cells
must experience a larger depolarization to reach threshold for
generation of these Na+ spikes, and spontaneous spiking is
rarely observed.
 A
Early Postnatal
B
Young Adult
* transfer of ions, metabolites, and signaling intermediates among
* hypothesis that there is a “pan-glial” syncytium in the CNS to
30 mV
CC HC CC HC
200 ms
Figure 21.3 Age-Dependent Changes in the Membrane Properties of
NG2+ Cells Whole-cell current-clamp recordings from NG2+ cells in the
corpus callosum (CC) and hippocampus (stratum radiatum, area CA1,
HC) in brain slices prepared from early postnatal (P5–P8) or mature
mice (P40–P45). The red trace shows responses to 160 pA currents. The
red asterisk highlights a response where depolarization triggered a small
NaV-mediated spike. The black asterisk highlights a response where the
largest current injection was 70 pA. Responses to two cells are shown
for each region to highlight the variability that is seen within brain
regions. From De Biase et al. 2010.
3.2.3 Ca2+ Channels
The influx of Ca2+ through membrane channels is a crucial
step in signal transduction pathways involved in regulating
growth, transformation, and functional plasticity. Whole-
cell recordings from NG2+ cells in conditions appropriate for
isolating Ca2+ currents have revealed the presence of inward
currents sensitive to inhibition by L- and T-type Ca2+ channel
antagonists (Haberlandt et al. 2011). These currents are rather
small (peak current = –100 pA in 5 mM Ca2+), indicating that
the overall density is low. Additional evidence for expression
of different voltage-gated Ca2+ channel (CaV) isoforms has
been obtained through PCR of mRNA extracted from sin-
gle cells in tissue slices, indicating that transcripts for L-type
(CaV 1.2, 1.3) and T-type (CaV 3.1, 3.2) are most abundant
(Haberlandt et al. 2011). These channels appear to have con-
ventional voltage dependencies, with a half-maximal current
activation observed near –50mV. Intracellular Ca2+ dynamics
in NG2+ cells may be modified by Ca2+ induced Ca2+ release
(Haberlandt et al. 2011), Na+-dependent Ca2+ exchange (Tong
et al. 2009), and plasma membrane store refilling channels,
such as TRPC1 (Paez et al. 2011). In vitro studies of isolated
NG2+ cells (OPCs) indicate that they undergo spontaneous
Ca2+ transients while migrating, events that are inhibited by
the L-type Ca2+ antagonist nifedipine. Elevation of Ca2+ in
these cells through CaVs triggers process retraction, suggest-
ing that these channels are involved in regulated the directed
movement of NG2+ cells during early development. Although
NG2+ cells rest more than 30 mV more negative than the acti-
vation threshold of CaVs, the higher membrane resistance and
more depolarized membrane potential of NG2+ cells at this
age may promote CaV activation.
4 GAP JUNCTIONAL COUPLING
One of the hallmarks of astrocytes is their extensive coupling
through gap junctions. By forming these intercellular junctions,
P H YS I O L O G I C A L P R O P E RT I E S O F N G 2 + G L I A L C E L L S
astrocytes assemble into a functional syncytium, enabling the
connected cells. Gap junctions also facilitate heterotypic inter-
actions among different cell types (Robinson et al. 1993). It is
this coupling among distinct glial cell types that has led to the
enable signaling and homeostasis among distinct glial networks.
NG2+ cells, by contrast, do not appear to form gap junctions
with one another: Injection of a small molecular weight marker
into one NG2+ cell does not lead to its transfer to adjacent cells
(Bergles et al. 2000; Wallraff et al. 2004), unlike the extensive
intercellular transfer that occurs when one astrocyte is loaded
with tracer (see Fig. 21.1E, F). This lack of direct coupling among
NG2+ cells is consistent with the physical separation often pres-
ent between the processes of adjacent cells (see Fig. 21.2E, F).
However, dye coupling is not the most sensitive measure of gap
junctional coupling, and electrical coupling can occur in the
absence of observable dye transfer. Although paired recordings
from NG2+ cells and neighboring neurons have failed to detect
electrical coupling (Lin et al. 2005; Mangin et al. 2008; Muller
et al. 2009), recent studies indicate that some NG2 cells are
coupled to oligodendrocytes in the corpus callosum (Maglione
et al. 2010), indicating that they are capable of forming hetero-
typic junctions (see also chapter 24).
Mutations in Connexin32 (Cx32) are responsible for
Charcot-Marie-Tooth type X1 disease, a peripheral neu-
ropathy associated with demyelination (see also chapter 62).
Connexin32 appears during first and second week of postnatal
development and is expressed by mature oligodendrocytes (and
Schwann cells), which by electron microscopy has been local-
ized to paranodal loops of myelin, Schmidt-Lanterman incisures
and oligodendrocyte processes, rather than at contacts with
astrocytes (Kamasawa et al. 2005), suggesting that is involved
in autologous or within-cell oligodendrocyte coupling. Some
NG2+ cells in the dentate gyrus express Cx32, and in Cx32
null mice, the turnover (proliferation and apoptotic removal)
of NG2+ cells in this region is enhanced (Melanson-Drapeau
et al. 2003), suggesting that early expression of this channel in
the oligodendrocyte lineage is important for the proper differ-
entiation of some NG2+ cells. At present, it is unclear whether
this channel is involved in cell-cell coupling or functions as an
unpaired connexin hemichannel to enable signal transduction.
Mature oligodendrocytes and CNS myelin are formed in Cx32
null mice (Menichella et al. 2003), indicating that global expres-
sion of this channel among the NG2+ cells is not an absolute
requirement for oligodendrogenesis and myelination.
5 N E U R OT R A N S M I T T E R R E C E P TO R
EXPRESSION
5.1 G LU TA M AT E R EC E P TO R S
5.1.1 AMPA and Kainate Receptors
In vitro studies of oligodendrocyte lineage cells, in particular
so-called “O-2A” progenitors that have the capacity to dif-
ferentiate into either oligodendrocytes or type-2 astrocytes,
• 257
demonstrated that these glial cells express functional iono-
tropic receptors for glutamate (Barres et al. 1990; Wyllie et al.
1991). Concurrent analysis of glial progenitors in brain slices
revealed that the presence of glutamate-gated currents in this
subset of glial cells was not merely an aberration of isolation
and maintenance in culture ( Jabs et al. 1994; Steinhauser
et al. 1994). Application of glutamate or kainate, an agonist
of AMPA and kainate receptors that is not a substrate for glu-
tamate transporters, to NG2+ cells voltage clamped at their
normal resting potential in brain slices triggers an inward cur-
rent that is blocked by selective AMPA receptor antagonists
and enhanced by cyclothiazide, an inhibitor of AMPA recep-
tor desensitization (Fig. 21.4A). Although the conclusion that
such responses are produced by direct effects on NG2+ cells,
this assessment is complicated by the certain activation of
receptors on surrounding neurons. However, similar responses
can be induced by focal application of agonists, through tech-
niques such as local photolysis of caged glutamate, and by per-
forming experiments in the presence of TTX to limit neuronal
activation and depolarization of NG2+ cells through elevation
of extracellular K+ (see Fig. 21.7). A small number of kainate
receptors also appear to be expressed by NG2+ cells (Kukley
and Dietrich 2009), consistent with previous in vitro studies
(Patneau et al. 1994).
AMPA receptors are multisubunit complexes assem-
bled from four different subunits encoded by distinct genes
(GluA1–4); alternative splicing and interaction with acces-
sory subunits (TARPs) can modify their properties further.
These receptors are cation channels that enable the flux of
Na+ and K+, and when formed without the GluA2 subunit
also allow Ca2+ influx. The hallmark of GluA2 lacking, Ca2+
permeable AMPA receptors is sensitivity to inhibition by
polyamine toxins (e.g., philanthotoxin) and inward rectifica-
tion in current-voltage (I–V) plots, a phenomenon caused by
intracellular block by polyamines. AMPA receptor currents
in NG2+ cells show some inward rectification (Fig. 21.5C)
(Bergles et al. 2000; Ge et al. 2006; Lin et al. 2005; Seifert
and Steinhauser 1995; Ziskin et al. 2007) and partial sensitiv-
ity to polyamine toxins (see Fig. 21.5C); however, the relative
proportion of these receptors varies with development and
between brain regions, with the highest proportion observed
in NG2+ cells in the molecular layer of the cerebellum (Lin
et al. 2005).
Analysis of mRNA expression by single cell PCR and gene
array analysis (OPCs) has confirmed that all four AMPA
receptor subunits are expressed in NG2+ cells (complex astro-
cytes/GluR cells) (Seifert and Steinhauser 1995; Seifert et al.
1997a,b), with GluA4 relatively enriched compared to most
mature neurons.
5.1.2 NMDA Receptors
NMDA receptors play a central role in the induction of
activity-dependent changes in synaptic strength. Although it
has long been thought that NMDA receptor expression was
restricted to neurons, recent physiological studies and gene
expression analysis has revealed that these receptors also are
expressed by NG2+ cells (Karadottir et al. 2005; Ziskin et al.
2007) as well as astrocytes (Lalo et al. 2006) and oligodendro-
cytes (Karadottir et al. 2005; Micu et al. 2006). Bath applica-
tion of NMDA (Karadottir et al. 2005) or focal application
of NMDA elicits currents in voltage-clamped NG2+ cells that
are blocked by NMDA receptor antagonists (see Fig. 21.4A)
(De Biase et al.; Ziskin et al. 2007). There are several aspects

A B C
(+40 mV)
+ CPP –25 mV

–45 mV
6 I

–65 mV
NMDA
4
+ Picrotoxin/SR-95531
–
ECI = –43mV
+ GYKI 53655

(–80 mV) 1
–120 –80 –20
Kainate THIP (10 mM) V 40
–1

Figure 21.4 AMPA, NMDA, and GABAA Receptor Currents Recorded from NG2+ Cells A. Focal application of kainate (200 mM) to an NG2+ cell
in the corpus callosum elicited an inward current (lower traces) that was blocked by the AMPA receptor selective antagonist GYKI 53655 (100 PM).
Focal application of NMDA (100 PM) resulted in an outward current (Vm = 40 mV) that was blocked by the NMDA receptor selective antagonist
D,L-CPP (10 PM). B. Focal application of the GABAA receptor agonist THIP to an NG2+ cell in a hippocampal slice elicited transient currents that
reversed near the predicted Cl– reversal potential (ECl– = ~0 mV, CsCl-based electrode solution) and were blocked by the GABAA receptor antagonists
picrotoxin (100 PM) and SR-95531 (5 PM). Voltage steps were: −30 to 20 mV, 10 mV/step. C. Plot of the peak amplitudes (normalized) of responses
to focal THIP application recorded at different voltages in perforated-patch (•) or whole-cell ({) recording configurations. The reversal potential of
GABAA receptor responses was determined to be –43 mV (◊), indicating that they maintain a high level of intracellular Cl–. Adapted from Lin et al.
2004.

258 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
of NMDA receptor responses in NG2+ cells that differ from
neurons. Physiological studies suggest that these receptors
exhibit reduced sensitivity to block by external Mg2+. The volt-
age-dependent block by external Mg2+ ensures that Ca2+ influx
occurs only when the postsynaptic partner has been sufficiently
depolarized. Because of the lower sensitivity to Mg2+, NMDA
receptors in NG2+ cells can contribute significant current and/
or Ca2+ influx without the need for substantial depolarization,
although this property also may vary among brain regions and
species (Hamilton et al. 2009). NMDA receptors are formed
by the assembly of two GluN1 and two GluN2, or less often
GluN3 subunits. Receptors formed from GluN2C and 2D sub-
units have lower Mg2+ sensitivity, and in accordance with the
physiological studies, mRNA encoding 2C subunits have been
observed in NG2+ cells (OPCs) (Cahoy et al. 2008). However,
NMDA receptors are expressed at a much lower level than in
neurons, and some NG2+ cells appear to lack these receptors
entirely (De Biase et al. 2010; Ziskin et al. 2007).
NG2+ cells exhibit a pharmacological profile consistent with
the presence of the D-5 subunit, a subunit that confers slow
deactivation kinetics. Pyramidal neurons undergo a develop-
mental switch from D-5 to D-1, a transition that accelerates
the decay of GABAA receptor-mediated synaptic currents
(Fig. 21.6). Measurements of GABAA receptor-mediated cur-
rents in perforated patch recordings, which preserve the nor-
mal internal Cl– concentration, revealed that these currents
reverse at –43 mV, about 30 mV more positive than GABAA
reversal potential in neurons at this age, indicating that NG2+
cells maintain a high intracellular Cl– concentration (see Fig.
21.4C) (Lin and Bergles 2004; Tanaka et al. 2009; Tong et al.
2009). NG2+ cells in the cortex also express GABAA recep-
tors, suggesting that this is a feature common to these cells
(Tanaka et al. 2009; Velez-Fort et al. 2010). However, the
expression of GABAA receptors appears to be much lower in
NG2+ cells in the corpus callosum, perhaps because there are
few GABAergic fibers in this region.

5.2 G A BA A R E C E P TO R S 5.3 OT H E R N EU ROT R A NS M IT T E R

GABAA receptors help limit neuronal excitation by hyperpo-
larizing the membrane potential and by increasing the mem-
brane conductance (shunting), and have been shown to play
important roles in regulating neuronal development. NG2+
cells in the hippocampus and barrel cortex express func-
tional GABAA receptors that exhibit characteristics similar to
those expressed by young pyramidal neurons. Currents medi-
ated by these receptors have been observed in outside-out
patches removed from physiologically defined NG2+ cells (see
Fig. 21.4B) (Lin and Bergles 2004), providing unequivocal
evidence for the expression of GABAA receptors by these pro-
genitors. In the hippocampus, GABAA–mediated responses in
R EC E P TO R S
The breadth of neurotransmitter receptor expression by NG2+
cells is slowly being uncovered. Recent studies suggest that
NG2+ cells in the hippocampus express nicotinic acetylcho-
line receptors (Velez-Fort et al. 2009), metabotropic glutamate
receptors (Haberlandt et al. 2011), D-1 adrenergic receptors
(Papay et al. 2004), and P2 purinergic receptors (Hamilton
et al. 2009). Indeed, gene expression studies (Cahoy et al.
2008) suggest that receptors for most if not all neurotransmit-
ters are expressed by NG2+ cells. However, a systematic, func-
tional assessment of the effects of different neurotransmitters
in different brain regions remains to be performed.

A B C
mEPSCs control
+ GYKI 53655 +spermine

Control
0.5

Vm(mV)

–80 –40 40
10 pA
τ =1.64 ms
+CTZ 500 ms –0.5
200 pA 2 pA
4 ms INORM
10 ms
–1

Figure 21.5 Glutamatergic Synaptic Currents in NG2+ Cells in the Cerebellum. A. Whole-cell voltage-clamp recording from an NG2+ cell in the
molecular layer of the cerebellum (cerebellar slice) showing EPSCs elicited by stimulation of a climbing fiber. The response was potentiated by cycloth-
iazide (CTZ; 100 PM), a compound that reduces desensitization of AMPA receptors, and blocked by GYKI 53655 (100 PM) (Vm = −80 mV). B.
Spontaneous miniature EPSCs recorded in the presence of tetrodotoxin (1 PM). C. Current-voltage plot showing the voltage dependence of climbing
fiber-evoked EPSCs recorded from NG2+ cells with or without the polyamine spermine included. Black circles and solid lines indicate responses
recorded with internal spermine; the reduced outward current in this configuration indicates the contribution of Ca2+-permeable AMPA receptors.
Error bars are ± SEM. From Lin et al. 2005.

P H YS I O L O G I C A L P R O P E RT I E S O F N G 2 + G L I A L C E L L S • 259
 6 N E U R O N –N G 2 + C E L L SY N A P S E S
Synaptic junctions have long been considered to be the sole
provenance of neurons. These structures enable rapid com-
munication between distinct elements in neural circuits and
provide a substrate for neural plasticity. However, a wealth of
studies now indicate that NG2+ cells represent an exception
to this rule, as axons form functional synaptic junctions with
these glial cells in both white and gray matter regions of the
CNS. The existence of these neuron–NG2+ cell synapses has
been established through a variety of physiological and ana-
tomical criteria (described in the following), and this mecha-
nism for rapid communication appears to be one of the most
defining physiological features of these progenitors. NG2+
cells are targets of innervation, that is, they are postsynaptic
elements. It is not yet known if NG2+ cells also act as presyn-
aptic elements capable of transmitting signals to surrounding
neurons through NG2+ cell–neuron synapses.
6.1 G LU TA M AT E RG I C SY NA P S E S
AMPA receptors have a relatively low affinity for glutamate
and are desensitized by low glutamate concentrations, plac-
ing tight constraints on the manner in which these receptors
can be activated. In neurons, rapid activation of ionotropic
receptors is achieved through exposure to high concentra-
tions of glutamate that are released from transmitter-laden
vesicles into the small volume of the synaptic cleft. The
near-synchronous activation of receptors results in excitatory
postsynaptic currents (EPSCs) that reach a peak in less than
a millisecond, whereas rapid dilution through diffusion and
buffering by transporters ensures that such responses are
transient. EPSCs mediated by AMPA receptors are also vis-
ible in whole-cell recordings from NG2+ cells in brain slices
following stimulation of surrounding axons (see Fig. 21.5A)
(Bergles et al. 2000; Jabs et al. 2005). These responses reach a
peak in a few hundred microseconds and decay exponentially,
similar to EPSCs in neurons, occur with minimal delay after
stimulus onset, and are blocked by AMPA receptor antago-
nists. Moreover, miniature EPSCs of varying amplitude and
time of occurrence occur in these glial cells, consistent with
the stochastic, spontaneous vesicle fusion events that occur at
synapses between neurons (see Fig. 21.5B). In further support
of a direct fusion of vesicles opposite NG2+ cell membranes,
these responses are not dramatically potentiated by cycloth-
iazide, as would be expected if they resulted from activa-
tion by glutamate that spills over from neighboring synapses
between neurons (Dzubay and Jahr 1999). In contrast with

A1 B
* * *
+TTX

20 pA
A2 1s
5 pA
25 ms
control
20 pA
C
20 ms

A3

τ =21.9 ms
+SR 95531
5 pA 1 mV
20 ms 200 ms

Figure 21.6 GABAergic Synaptic Currents Recorded From NG2+ Cells in the Hippocampus. A1. Spontaneous miniature GABAA receptor-mediated
currents recorded from an NG2+ cell in the presence of tetrodotoxin (1 PM) to block action potentials and NBQX (5 PM) to block AMPA receptors.
A2. Overlay of three spontaneous GABAA receptor-mediated currents (highlighted by asterisks in A1). Note the slow decay kinetics. A3. Average time
course of 64 events recorded from this NG2+ cell. These responses decay approximately 50% more slowly than IPSCs recorded from CA1 pyramidal
neurons. B. Evoked responses recorded from an NG2+ cell in voltage-clamp showing the pronounced synaptic depression with repetitive stimulation.
These responses were blocked by tetrodotoxin (red trace). C. Spontaneous GABAA receptor mediated depolarizations recorded from an NG2+ cell in
the perforated-patch configuration following stimulation of interneurons with carbachol. Note the small depolarizations produced by activation of
these synapses. Recordings were performed in 5 PM NBQX and 5 PM RS-CPP, and all events were blocked by subsequent application of the GABAA
receptor antagonist SR-95531. All recordings were from cells in the stratum radiatum region of area CA1 in the whole-cell voltage-clamp configura-
tion. From Lin et al. 2005.

260 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
most neuronal glutamatergic synapses, NMDARs contribute
little to spontaneous or evoked EPSCs (De Biase et al. 2010),
suggesting that they are either predominantly extrasynap-
tic or there are only a small number of receptors present at
each synapse. The contribution of Ca2+ permeable receptors
to these EPSCs varies, with the highest level observed in the
molecular layer of the cerebellum (see Fig. 21.5C).
Anatomical evidence for the existence of axon–NG2+ cell
synapses has been obtained from post hoc analysis of physi-
ologically defined cells and in brain sections. Examples of
mitochondria-rich presynaptic boutons containing small ves-
icles approximately 30 nm in diameter are observed in direct
apposition to the processes of NG2+ cells in electron micro-
graphs (Bergles et al. 2000; Haberlandt et al. 2011; Kukley
et al. 2007). The membranes of the cells are rigidly aligned at
these sites, and the space between the cells is filled with elec-
tron dense material, as seen at neuronal synapses. However,
the postsynaptic density is less well defined, perhaps because
of the lower number of NMDA receptors and associated regu-
latory machinery.
Neuron–NG2+ cell synapses have been observed in all brain
regions that have been examined, including the hippocam-
pus (Bergles et al. 2000), cerebellum (Lin et al. 2005), cortex
(Chittajallu et al. 2004), and brainstem (Muller et al. 2009),
suggesting that this mode of communication is a conserved
property of these glial cells. Moreover, these axoglial synaptic
junctions are not limited to gray matter, but also occur in the
white matter of the cerebellum (Karadottir et al. 2005) and
corpus callosum (Kukley et al. 2007; Ziskin et al. 2007).
Estimates of the connectivity of NG2+ cells with axons,
measured using hypertonic solution to force the release of
docked and primed vesicles (Fig. 21.7B), indicate that the
highest degree of connectivity occurs in the corpus callosum,
a region that was thought to be essentially devoid of synapses
(De Biase et al. 2010). However, even within a particular brain
region, the connectivity varies over a tenfold range. Although
far fewer events are seen than in neurons, when the smaller size
of these cells is taken into account, the overall density per unit
area is remarkably similar, in keeping with the comparable
density of expression of AMPA receptors.
NG2+ cells do not appear to be innervated by a unique
population of fibers, rather they receive inputs appropri-
ate to the region they are located; in the CA1 region of the
hippocampus they form synapses with Schaffer collateral–
commissural fibers, in the dentate gyrus with granule cells,
the medial nucleus of the trapezoid body with axons from the
cochlear nucleus, and the molecular layer of the cerebellum
with parallel and climbing fibers. Moreover, these inputs arise
from the same axons that form axodendritic synapses with
surrounding neurons, as shown through paired recordings in
the cerebellum (Lin et al. 2005), hippocampus (Mangin et al.
2008), and brainstem (Muller et al. 2009).
These inputs exhibit other characteristics of neuronal
synapses, such as facilitation and depression with repetitive
stimulation, and release can be altered through activation of
presynaptic receptors (Ziskin et al. 2007), indicating that
these junctions have assembled many of the components
required for rapid modulation. Changes in the composition
P H YS I O L O G I C A L P R O P E RT I E S O F N G 2 + G L I A L C E L L S
of AMPA receptors at neuron–NG2+ cell synapses have been
observed in response to theta-burst stimulation of axons (Ge
et al. 2006), group I mGluR stimulation (Zonouzi et al. 2011),
and NMDA receptor deletion (De Biase et al. 2011).
6.2 G A BA E RG I C SY NA P S E S
+
NG2 cells also form synaptic junctions with GABAergic
interneurons. Spontaneous, miniature, and evoked GABAA
receptor-mediated currents have been observed in recordings
from NG2+ cells in the hippocampus ( Jabs et al. 2005; Lin
and Bergles 2004; Mangin et al. 2008) and cortex (Tanaka
et al. 2009; Velez-Fort et al. 2010) in the presence of gluta-
mate receptor antagonists (see Fig. 21.6). Paired stimulation of
these inputs with a short interval induces paired-pulse depres-
sion, suggesting that these inputs have a high initial release
probability, and decay kinetics are slower than events observed
in neurons at the same age, in accordance with the expression
of GABAA receptors containing the D-5 subunit (Lin and
Bergles 2004).
In the cortex, there is an age-dependent decrease in the
amplitude and frequency of miniature and GABAA–mediated
responses, and a lengthening of the decay of evoked responses
(Velez-Fort et al. 2010), a phenomenon caused by a shift from
direct activation of these receptors at synapses to indirect acti-
vation through spillover from presumed neuronal synapses.
Thus, direct GABAergic synapses, but not GABAergic signal-
ing entirely, are restricted to the first postnatal month in corti-
cal NG2+ cells, in contrast with glutamatergic inputs, which
remain prominent into adulthood.
In the cortex, GABAergic inputs to NG2+ cells appear to
arise from parvalbumin-containing interneurons (basket and
chandelier cells) (Tanaka et al. 2009), whereas the identity
of inputs in other regions is less certain. NG2+ cells often are
located in close proximity to neuronal somata, conforming
to the satellite glial cell classification. Despite this proximity,
paired recordings from interneurons and satellite NG2+ cells
in the dentate gyrus indicated that these satellite glia were
never innervated by the neighboring interneuron, although
GABAergic synaptic inputs were visible in these NG2+ cells
(Mangin et al. 2008). These findings suggest that GABAergic
innervation patterns are not random or based simply on
proximity.
7 C H A N G E S IN T HE P H YS I O L O G I C A L
P R O P E RT IE S O F N G 2 + C E L L S W IT H
D IF F E R E N T I AT I O N
In vivo genetic fate tracing studies indicate that NG2+ cells
serve as progenitors for oligodendrocytes throughout the CNS.
Although this capacity is realized most frequently during early
postnatal life, NG2+ cells retain the ability to differentiate into
oligodendrocytes in the adult CNS (Kang et al. 2010; Rivers
et al. 2008), a phenomenon that is enhanced following oligo-
dendrocyte death (Traka et al. 2010; Tripathi et al. 2010) and
following exercise (Simon et al. 2011). Thus, at any one time,
and particularly in the developing CNS when the majority of
• 261
 A1 NG2 + cell B Pre-OL C OL

20 μm 20 μm 20 μm

D E F

20 pA
2.5 s
G H I

CPP
+40 mV +40 mV
CPP CPP
+NBQX/GYKI

+NBQX/GYKI TBOA
+NBQX/GYKI
–80 mV –80 mV
+NBQX/GYKI

25 pA/ 25 pA/
100 pA
100 pA 100 pA
250 ms 250 ms 250 ms

Figure 21.7 NG2+ Cell Differentiation Is Accompanied by Synapse Removal and Receptor Downregulation A–C. Representative NG2+ cells, premyeli-
nating oligodendrocytes (pre-OL), and oligodendrocytes (OL) recorded from cells in the corpus callosum. Insets show the responses to current injec-
tion, illustrating the different membrane properties exhibited by oligodendrocyte lineage cells in different stages of maturation. The red traces show
responses to 160 pA current injection; scale bars: 25 mV/100 ms. D–F. Responses of cells in these stages to focal application of hypertonic solution, a
manipulation that induces the release of docked, primed vesicles from synapses. Note the absence of inward currents from cells in the Pre-OL and OL
stages. G. Currents recorded from an NG2+ cell in response to local photolysis of MNI-L–glutamate (black dots). Lower traces show that responses
recorded at –80 mV were blocked by AMPA receptor antagonists (NBQX/GYKI53655). In these blockers, NMDA receptor-mediated responses
recorded at +40 mV were blocked by RS-CPP (upper traces). H. Responses recorded from a pre-OL in similar conditions as in (G). Note the reduced
amplitude of the AMPA receptor current and the absence of an NMDA receptor current. I. Responses from OLs recorded at –80 mV, showing the
effects of NBQX/GYKI53655, RS-CPP (upper trace, 0 Mg2+ ACSF/30 PM D-serine), and TBOA (lower trace, normal ACSF). From De Biase et al.
2010.

physiological studies have been performed, there are oligoden-
drocyte lineage cells in different states of maturation, creat-
ing challenges for defining the characteristics of this dynamic
population. Using genetic labeling techniques it is possible to
identify cells in distinct stages of differentiation (De Biase et al.
2010). Studies of these cells have revealed that NG2+ cells rap-
idly alter their physiological properties as they begin to differ-
entiate, consistent with the analysis of glial cells in developing
white matter (Berger et al. 1991). Transition to the premyeli-
nating stage is accompanied by a tripling of the membrane
capacitance reflecting the elaboration of additional processes,
and a doubling of the membrane resistance caused by a reduc-
tion in K+ and Na+ channel expression (De Biase et al. 2010;
Kukley et al. 2010) (Fig. 21.7A–C). Mature oligodendrocytes,
in contrast, exhibit nearly tenfold lower membrane resistance
and a lower (more positive) resting potential, suggesting that
the relative K+ conductance is reduced. Thus, maturation is
accompanied by a transient downregulation of ion channels.
These changes are accompanied by a dramatic reduction in
connectivity with surrounding neurons, as few synaptic cur-
rents are visible in premyelinating oligodendrocytes, and none
are observed in oliogdendrocytes (Fig. 21.7D–F) (De Biase
et al. 2010; Kukley et al. 2010). Accompanying this decrease
in innervation, there is a tenfold reduction in AMPA receptor
density, and NMDA receptor–mediated currents are rarely
observed (Fig. 21.7G–I). Gene expression analysis further
substantiates these findings, as this transition is accompanied
by a reduction in abundance of mRNAs that encode AMPA

262 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
receptor subunits GluA1 to 4 and the NMDA receptor sub-
unit GluN1 (NR1) (Cahoy et al. 2008), which is required
for the formation of NMDA receptors that are activated by
glutamate. These studies suggest that within the oligodendro-
cyte lineage, NG2+ cells are uniquely positioned to engage
in rapid synaptic communication with surrounding neurons.
At present, less is known about the changes in expression of
other receptors during this transition period.
8 P OT E N T I A L R O L E S O F
N E UR OT R A N S M IT T E R S I G N A L IN G
W IT H N G 2 + C E L L S
Synapses enable the rapid transmission of information
through neural circuits, and provide a substrate for modifi-
cation of these networks in response to changing patterns of
activity. The establishment of glutamatergic synapses with
NG2+ cells early in postnatal development, the high incidence
of these inputs among the population in diverse regions of the
CNS, and the persistence of these connections in adults, raise
the possibility that they play a prominent role in promoting
changes in the behavior of these cells in response to neural
activity. Because these cells have the capacity to differentiate
into oligodendrocytes, such input may be used to control their
maturation. Indeed, previous in vivo studies have shown that
proliferation of these progenitors and myelination are influ-
enced by neural activity (Barres and Raff 1993). In support
of this hypothesis, in vitro studies have shown that glutamate
receptor signaling has diverse effects on these progenitors,
inhibiting proliferation and differentiation (Gallo et al. 1996;
Gudz et al. 2006; Yuan et al. 1998), enhancing migration
(Gudz et al. 2006), and controlling expression of myelin pro-
teins (Wake et al. 2011). GABAA receptor activation also has
been shown to promote NG2+ cell migration in vitro (Tong
et al. 2009). Evaluation of the role of neurotransmitter signal-
ing in these glial cells in vivo will require specific manipulation
of NG2+ cells, rather than pharmacological approaches, to
exclude the possibility that effects are indirectly mediated by
actions on neurons or other glial cells. Recent studies in which
the requisite NMDA receptor subunit NR1 (GluN1) subunit
was selectively deleted from NG2+ cells indicate that NMDA
receptor signaling is not required for NG2+ cell proliferation,
migration, oligodendrogenesis, or myelination (De Biase et al.
2011), suggesting that there are redundant pathways to control
NG2+ cell behavior or that in vitro conditions do not accu-
rately reproduce in vivo conditions.
Glutamatergic and GABAergic synaptic inputs produce
only minimal depolarization of the membrane of NG2+ cells
(Bergles et al. 2000; Jabs et al. 2005; Ziskin et al. 2007), with
unitary responses averaging less than 1 mV in amplitude. How
then would these inputs influence behavior, given that their
resting potential is 20 to 30 mV more hyperpolarized than the
activation range of NaV and CaV channels? As these depolariza-
tions were measured at the soma, it is possible that greater volt-
age changes occur in their fine processes, which could engage
T-type CaV channels that are activated in a more negative
voltage range. Alternatively, the expression of Ca2+ permeable
P H YS I O L O G I C A L P R O P E RT I E S O F N G 2 + G L I A L C E L L S
AMPA and NMDA receptors, and the weak sensitivity of their
NMDA receptors to Mg2+ block, could provide the means to
link neuronal activity to Ca2+ elevation without the need for
significant depolarization, an effect that could be potentiated
by depolarizing GABAergic inputs. Therefore, the effects of
these synaptic inputs may be very local, and perhaps influence
the dynamics of their processes (Haberlandt et al. 2011; Kirby
et al. 2006) or trigger secretion of neurotransmitters, growth
factors, or matrix molecules (Hunanyan et al. 2010).
Our ever-increasing knowledge about the characteristics
of NG2+ cells indicate that they express many “neural” genes,
and in vitro studies indicate that these cells can develop into
neurons in response to particular growth factors (Kondo and
Raff 2000). However, genetic fate mapping studies in vivo
indicate that NG2+ cells are primarily, if not exclusively, lin-
eage restricted progenitors in the normal (uninjured) CNS,
that either remain in the progenitor state or differentiate into
oligodendrocytes. Given the large number of proteins that are
required to assemble functional synaptic junctions, it seems
likely that these structures provide some advantage for sur-
vival. It is possible that rapid synaptic communication with
NG2+ cells controls aspects of their behavior that is unrelated
to their potential to develop into oligodendrocytes. Moreover,
although it is clear that functional receptors are present at
these synapses, perhaps the primary role of these junctions is
to establish areas of contact to enable transduction through
other signaling pathways.
9 S U M M A RY A N D P E R S P E C T I VE S
The relatively recent discovery that the mammalian CNS is
populated by an abundant, widely distributed population of
NG2+ glial cells that are physiologically distinct from other glia
and neurons, has raised many new questions about their roles
in the development and function of CNS circuits. Although
these cells have the capacity to differentiate into oligodendro-
cytes, and thus can be considered OPCs, it is unlikely that this
designation fully encompasses their role in the adult CNS.
Indeed, the presence of these cells in regions that lack oligo-
dendrocytes (e.g., molecular layer of the cerebellum), their
persistence throughout life, and their relatively uniform distri-
bution and highly ramified morphology in gray and white mat-
ter, suggest that they may be involved in homeostasis or active
regulation of neural circuits. NG2+ cells exhibit a number of
intriguing physiological characteristics, including the expres-
sion of a diverse array of ion channels and neurotransmitter
receptors, the formation of synapses with axons in gray and
white matter, and the ability to proliferate and differentiate in
the adult CNS. Among the questions that remain to be fully
answered are the functions of the synaptic junctions that are
formed with NG2+ cells, the extent to which these cells exhibit
excitability, their fate in the context of injury and disease, and
the pathways that control their proliferation and differentia-
tion. Investigations into these questions will help answer why
these presumed progenitor cells persist in the adult CNS.
Because it is necessary to study these cells in the complex
environment of the CNS, it is difficult to exclude the possibility
• 263
of indirect effects mediated by surrounding neurons and glia,
particularly when the membrane resistance of these cells is low
and can be influenced by changes in extracellular ion concen-
trations. In addition, most physiological studies have relied on
single cell recording in tissue slices, necessitating small sample
sizes, and raising the possibility of cell perturbation and selec-
tion bias. Notably, there have been few physiological studies
of NG2+ cells in vivo. It also has been difficult to obtain com-
plete, high-resolution reconstructions of these cells, because
of the poor preservation of their fine processes in tissue pro-
cessed for transmission electron microscopy. These data could
reveal additional morphological specializations and the struc-
ture of junctions formed with different types of neurons and
glial cells, providing further insight into their roles in CNS
circuits.
The next decade will undoubtedly provide answers to some
of the essential questions associated with these enigmatic cells.
The development of new genetic tools, such as inducible
Cre lines, that enable unambiguous identification, fate trac-
ing, and manipulation of gene expression, will help evaluate
how particular ion channels and signaling pathways influence
the behavior of NG2+ cells in vivo. When used in combina-
tion with mouse strains that enable cell-specific expression
of genetically encoded indicators of voltage and Ca2+, as well
as light activated ion channels, it will be possible to develop-
ing a broader understanding of the properties of NG2+ cells
in the intact CNS, the extent of diversity within the popula-
tion, and their interactions with other neurons and glial cells.
Information about the physiological properties and functions
of NG2+ cells may lead to new approaches to enhance remy-
elination, promote axonal regeneration, and perhaps stimulate
replacement of other cell types following injury or disease.
AC K N OW L E D G M E N T S
The author thanks Shih-chun Lin, Jennifer Ziskin, and Lindsay
DeBiase for contributing many of the results illustrated in this
chapter, Michele Pucak for her assistance with NG2 immuno-
labeling, and past and present members of his laboratory for
their insights. These studies were supported by grants from the
National Institutes of Health.
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• 265
 22.
CY TOKINE, CHEMOKINE, AND GROW TH FACTOR
RECEPTOR S AND SIGNALING
Erik W. G. M. Boddeke, Bart J. L. Eggen, and Knut P. H. Biber

A B B R E VI AT I O N S MyD88 myeloid differentiation primary response

gene (88)
AAF IFN-α–activated factor NADE NT-associated cell death executor
AcP accessory protein NF-κB nuclear factor κB
B7 costimulatory molecule NGF nerve growth factor
BDNF brain-derived neurotrophic factor NIK NFκβ inducing kinase
CBP CREB binding protein NNT-1 novel neurotrophin-1
CCL chemokine (C-C motif ) ligand NRAGE NT-receptor–interacting MAGE homolog
CD cluster of differentiation NRIF NT-receptor–interacting factor
Cdc42 cell division control protein 42 NT neurotrophin
CDK cyclin-dependent kinase OAZ O/E-associated zinc finger protein
CLC cardiotrophin-like cytokine OSM oncostatin-M
CNS central nervous system P38 P38 mitogen–activated protein kinase
CNTF ciliary neurotrophic factor p300 E1A binding protein p300
COX-2 cyclooxygenase-2 PI3K phosphatidylinositol-3-OH kinase
CT 1 cardiotrophin-1 PDGF platelet-derived growth factor
CrkL v-CRK avian sarcoma virus CT10- PKB protein kinase B (also known as Akt)
homolog-like PLC phospholipase C
CXCL chemokine (C-X-C motif ) ligand RIP receptor interacting protein
CX3CL1 fractalkine RUNX runt-related transcription factor
ERK extracellular signal-regulated kinases SAPK stress activated protein kinase
FADD fas-associated death domain protein Shc SH2 containing proto-oncogene
FOXH1 homolog of Xenopus forkhead activin signal sIL-6R soluble IL-6 receptor
transducer-1 SODD silencer of death domain
GAS interferon-γ activating site STAT signal transducer and activator of
GDP guanosine diphosphate transcription
Gp130 glycoprotein 130 TGF transforming growth factor
GTP guanosine triphosphate TNF tumor necrosis factor
ICAM intercellular adhesion molecule TMEV Theiler’s Murine Encephalomyelitis Virus
IFN interferon TRADD tumor necrosis factor receptor-1–associated
IFNAR interferon-α/β receptor death domain protein
IKK IκB kinase TRAF tumor necrosis factor receptor–associated
IL interleukin factor
IL-1RA IL-1 receptor antagonist Trk tropomyosin-receptor-kinase
iNOS inducible nitric oxide synthase TYK2 tyrosine kinase-2
IRAK IL-1 receptor-associated kinase VCAM vascular cell adhesion protein
IRF interferon regulatory factor (ISGF3G) WNT acronym for wingless integrated
IRS-2 insulin receptor substrate-2
ISRE IFN-stimulated regulatory elements
JAK Janus kinase 1 INTRODUCTION
JNK c-jun N-terminal kinase
LIF leukemia inhibitory factor The various types of glia cells are of paramount importance
MAPK mitogen-activated protein kinase for homeostasis and maintenance of nervous tissue. They play
MHC major histocompatibility complex an essential role in processes that support survival, growth,

266
differentiation, and repair. For these tasks glial cells make
abundant use of cytokines, chemokines, and growth factors.
Via a large number of different receptors these signaling mole-
cules convey information to specific populations of target cells,
which in turn switch on appropriate intracellular signaling
cascades that lead to adaptation of cell function and gene tran-
scription activity. A diverse group of cytokines, chemokines,
and growth factors activate specific receptors expressed in glia
cells. Notwithstanding the diversity of cytokine-, chemokine-,
and growth factor receptors, the signaling cascades that are
initiated after receptor activation are conserved. Receptor
activation is initiated by heteromerization of receptor subunits
that is induced on cytokine/growth factor binding. In many
cases, downstream signaling is initiated by receptor tyrosine
phosphorylation by receptor-associated kinases, followed by
recruitment of subsequent signaling factors. These include
signal transducers and activators of transcription (STAT),
mitogen-activated protein kinase (MAPK), the transcrip-
tion factor complex NF-κB, and phosphoinositide-3 kinase/
protein kinase B (PI3K/Akt) (Fig. 22.1). This chapter reviews
the specific signaling pathways of cytokines, chemokines and
growth factors that are involved in glia signaling.
2 T H E I N T E R F E R O N R E C E P TO R FA M I LY
Interferons (IFNs) are antiviral cytokines that are released
from numerous cell types in response to a variety of stimuli
including viral infection or Toll-like receptor activation. The
original discovery of IFN goes back to 1957, making IFNs the
first cytokines discovered, and has set the starting point of
cytokine research.
Three forms of IFN (type I and II IFN and the recently dis-
covered type III IFN) activate different IFN receptors. Type
I IFN has been subdivided in different classes (INF-α, β, δ,
ε, κ, τ, ϖ), which are not all found in humans. IFN-γ is the
only type II IFN. The type III form is IFN-λ, of which three
subtypes are known: IFN-λ1 (IL-29), IFN-λ2 (IL-28A), and
IFN-λ3 (IL-28B) (Takaoka and Yanai 2006). Because little is
known about the potential role of type I IFN-δ, ε, κ, τ, and ϖ
and type III IFNs in brain, the biology of these factors is not
discussed further.
Type I IFNs all signal through type I IFN receptors that
are composed of a single α-chain (IFNAR1) and a single
β-chain (IFNAR2), that are coupled to the Janus tyrosine
kinases TYK2 and JAK1, respectively (Fig. 22.2A). On

Figure 22.1 Receptor Signaling Cascades. A. Receptor dimerization caused by ligand binding: Example of receptors that dimerize because of recep-
tor binding. The two kinase domains cross-phosphorylate each other and thereby activate kinase domains. B. JAK/STAT signaling pathway: After
receptor activation and dimerization the associated JAKs now transphosphorylate tyrosine residues in the cytoplasmic domain of the receptor.
Phosphorylated tyrosine residues function as docking sites for STATs, which are then phosphorylated by JAKs. This results in STAT dimerization,
nuclear translocation, and target gene activation. C. PI3K signaling pathway: Receptor activation and subsequent phosphorylation of the regulatory
subunit for PI 3-kinase. D. Receptor signaling pathway resulting in MAPK-activation through a signaling cascade involving JAK/SHP2, RAS,
and RAF.

C Y TO K I N E , C H E M O K I N E , A N D G R OW T H FAC TO R R E C E P TO R S A N D S I G N A L I N G • 267
Figure 22.2 IFN Signaling. The IFN signaling pathways differ between type I and II IFN. All type I IFNs act through a heteromerized cell-surface
receptor composed of an α-chain (IFNAR1) and a β-chain (IFNAR2) and two associated cytoplasmic tyrosine kinases, JAK1 and Tyk2. A. Type I
IFN binds as a monomer to the two-chain IFNAR complex and induces binding of STAT2 to IFNAR2; subsequently, STAT1 is recruited to STAT2.
On tyrosine phosphorylation, STAT1 and STAT2 are released and associate with IRF-9/p48/ISGF3-γ to form the heterotrimeric IFN-stimulated gene
factor 3 (ISGF3) complex. ISGF3 translocates to the nucleus and binds to IFN-stimulated regulatory elements (ISREs). At the same time, STAT1
homodimers (AAF); other STAT complexes such as STAT3 or STAT5 homodimers, and STAT1/STAT3 and STAT5/CrkL heterodimers might
also form. AAF and STAT5/CrkL translocate to the nucleus and bind to IFN-γ activated sequences (GAS). B. Homodimers of type II IFN (IFN-γ)
activate a receptor complex that consists of two IFN-γR1 and two IFN-γR1 subunits, each of which is associated with JAK1 and JAK2, respectively.
On activation, two STAT1 molecules are recruited that form a dimer, which translocates to the nucleus. The activation of GAS via STAT1/STAT1
homodimers is the major route of IFN-γ signaling. Both type I and type II IFN may also activate NF-κB, ERK/MAPK, and PI3/AKT pathways.

activation by IFN-α/β, STAT2 is recruited to IFNAR2 in a
JAK1-dependent manner and subsequently STAT1/STAT2
heterodimers are formed and released from the receptor com-
plex (Fig. 22.2A). These heterodimers form together with
cytoplasmic interferon regulatory factor-9 (IRF-9), the het-
erotrimeric IFN-stimulated gene factor 3 (ISGF3) complex,
which translocates into the nucleus in order to activate gene
transcription at IFN-stimulated regulatory elements (ISREs)
(Fig. 22.2A). Other STAT complexes such as STAT5/CrkL
(Crk-like protein) or STAT1/STAT3 or STAT1/STAT1 can
also be formed on IFNAR activation. These latter STAT com-
plexes, however, activate distinct genes via binding to IFN-
γ–activated sequences (GAS), which indicates that GAS are
major targets of IFN-γ–activated signaling. Homodimers of
IFN-γ activate a receptor complex that consists of two IFN-
γRI/IFN-γRII pairs, each of which is associated with JAK1
and JAK2, respectively (type II IFN signaling) (Fig. 22.2B).
The activation of GAS via STAT1/STAT1 homodimers is the
major route of IFN-γ signaling. However, to a lesser extent
also STAT1/STAT2 heterodimers are formed that together
with IRF-9 constitute ISGF3, which activates ISREs in the
nucleus (see Fig. 22.2B). Thus, the signaling pathways of IFN-
α/β and IFN-γ show major overlap (Takaoka and Yanai 2006).
Next to the canonical JAK-STAT pathways, both type I and II
IFN may also activate other signaling routes such as NF-κB,
PI3/Akt, and ERK/MAPK pathway (Gough et al. 2008) (see
Fig. 22.2B).
Interferons are crucial for a general antiviral response.
Consequently, also viral infections of the CNS are controlled
by IFNs. This has become evident in IFN receptor knock-out
studies that provide evidence for a vital role of IFNAR, but
not IFN-γ-R in several viral infection models, indicating a piv-
otal role of type I IFN in clearing CNS viral response (Paul
et al. 2007).
The exact source of type I IFN in the CNS in vivo is cur-
rently not very well understood (Paul et al. 2007). Most likely,
microglia, astrocytes, and infiltrating blood cells are the main
sources given the fact that the evidence that also neurons
release type I IFN is mostly based on in vitro data (Paul et al.
2007). Stimulation of cultured astrocytes, oligodendrocytes,
neurons, and microglia with type I IFN causes induction of
several interferon-stimulated genes, indicating that all cells
of the CNS respond to type I IFN. Supporting this notion is
the observation that Theiler’s murine encephalomyelitis virus
infection in mice causes an induction of MHCI in all CNS
cell types (Paul et al. 2007). These data indicate that IFNARs
are expressed broadly in CNS cells, which makes it difficult to
relate IFN actions to specific cell types.

268 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
 Because type I IFN is widely used in the treatment of mul-
tiple sclerosis, it is important to understand its function in the
CNS. Recent data using cell type–specific IFNAR deficient
animals show that the beneficial effect of type I IFN signal-
ing in demeylinating disease requires IFNAR expression in
myeloid cells (Prinz and Kalinke 2010), which puts infiltrating
macrophages and endogenous microglia in focus. This study
shows that various aspects of myeloid cell immune activity
are more pronounced in the absence of type I IFN signaling,
indicating an antiinflammatory function of this cytokine in
demyelinating disease (Prinz and Kalinke 2010). In fact, there
is little evidence that type I IFN affects other brain cells than
microglia during demyelinating disease in vivo (Prinz and
Kalinke 2010). Thus despite the observation that all endog-
enous brain cells respond to type I IFN, many details about
type I IFN activity in vivo remain to be established.
Astrocytes and microglia are the prime source of IFN-γ
in the CNS (Dafny and Yang 2005) and both cell types are
also targets of IFN-γ signaling. In contrast with type I IFN,
which mainly downregulates the proinflammatory capacity
of microglia, IFN-γ signaling in astrocytes and microglia pre-
dominantly induces the release of proinflammatory cytokines
and chemokines (Dafny and Yang 2005). It has been shown
recently that IFN-γ stimulates CXCL10 expression in astro-
cytes and microglia, whereas CXCL9 is induced in microglia
but not astrocytes (Ellis et al. 2010). This cell type–specific
effect of IFN-γ is caused by the transcription factor PU.1 that
is specifically expressed in myeloid cells, showing that next to
the canonical IFN-γ signaling pathway (see Fig. 22.2B), the
presence of other transcription factors may determine the
outcome of IFN-γ signaling (Ellis et al. 2010). Moreover, an
important role for IFN-γ in driving microglia-activation in a
mouse neuropathic pain model was reported, indicating that
this cytokine may control microglia function in vivo (Tsuda
et al. 2009). The cellular source of IFN-γ, however, has not
been elucidated in this study.
3 I L - 6 –T Y P E C Y TO K I N E S A N D
G P 13 0 -M E D I AT E D S I G N A L I N G
The family of IL-6–type cytokines consists of seven pleio-
tropic molecules, which are structurally and functionally
related, namely, IL-6, IL-11, leukemia inhibitory factor (LIF),
ciliary neurotrophic factor (CNTF), oncostatin M (OSM),
cardiotrophin-1 (CT 1), and novel neurotrophin-1 (NNT-1)
or cardiotrophin-like cytokine (CLC) (Heinrich et al. 2003).
IL-6–type cytokines exert their effects via different receptor
complexes that all include glycoprotein 130 (gp130) and addi-
tional ligand receptor subunits (Heinrich et al. 2003). Most
ligand recognition subunits (IL-6R, IL-11R, and CNTFR) rely
for their intracellular signaling function on the gp130 subunit
(Heinrich et al. 2003). Because of this fact, numerous redun-
dant properties have been reported for IL-6–type cytokines,
and this cytokine family is also referred to as gp130 cytokines.
IL-6–type cytokines are important for normal develop-
ment of the brain by regulating proliferation and maintenance
C Y TO K I N E , C H E M O K I N E , A N D G R OW T H FAC TO R R E C E P TO R S A N D S I G N A L I N G
of stem cells, neurogenesis, gliogenesis, and cell survival (Bauer
et al. 2007; Lee et al. 2008). Apart from their role in develop-
ment, IL-6–type cytokines regulate a variety of proinflamma-
tory and antiinflammatory processes and therefore are widely
implicated in the pathology of the CNS.
IL-6–type cytokines bind to their corresponding ligand
recognition subunit (IL-6R or respectively OSMR, CNTFR,
IL-11R, or LIFR) and recruit one or two gp130 subunits to
activate downstream signaling pathways (Fig 22.3A) (Heinrich
et al. 2003). Activation occurs via phosphorylation events of
gp130-associated tyrosine kinases ( JAK kinases: JAK1, JAK2,
JAK3, and/TYK2) and subsequent phosphorylation and acti-
vation of signal transducers and activators of transcription
(STAT), mainly STAT3 and STAT1 (Heinrich et al. 2003). In
addition, phosphorylation of gp130 by JAKs may also result in
the activation of mitogen-activated protein kinases (MAPKs),
including extracellular signal regulated kinase (ERK1/2), p38
and c-jun N-terminal kinases/stress-activated protein kinases
( JNK/SAPK). In addition to JAK/STAT and MAPK cas-
cades, IL-6–type cytokines may also activate the PI3K/Akt
signaling pathway (Kamimura et al. 2003) (see Fig. 22.3A).
Furthermore, IL-6 and its receptor display a remarkable prop-
erty called trans-signaling (Rose-John et al. 2007). IL-6R is
not only found as a membrane bound receptor, but also can be
released and thus occurs in soluble form (sIL-6R). In stark con-
trast to the effect of other soluble cytokine receptors, binding
of IL-6 to its soluble receptor is not inhibitory. Instead, binding
IL-6 to its soluble receptors forms a ligand–receptor complex
that is able to activate gp130 in the same way as the membrane-
bound IL-6R (Fig. 22.3B). This unique process enables cells to
respond to IL-6 even in the absence of endogenous IL-6R.
The shared receptor subunit of all IL-6–type cytokines
gp130 is ubiquitously expressed and is therefore found in all
types of brain cells. In contrast to gp130, the ligand recogni-
tion subunits are expressed more selectively. Leukemia inhibi-
tory factor receptor and CNTFR are predominantly expressed
in cells of the oligodendrocyte lineage, rendering these cells
sensitive to LIF and CNTF. Accordingly, numerous findings
of LIF and CNTF in the maturation of oligodendrocytes dur-
ing development and demyelinating diseases have been pub-
lished. It was described that exogenously administered LIF
limited cuprizone-induced demyelination and LIF-deficient
mice showed both potentiated demyelination and oligoden-
drocyte loss after cuprizone challenge. Both were ameliorated
by exogenous LIF, arguing for a direct beneficial effect of
endogenous LIF receptor signaling. The numbers of oligo-
dendrocyte progenitor cells in cuprizone-challenged mice
were not influenced by LIF, arguing for a direct effect of this
cytokines in differentiated oligodendrocyte. Endogenous LIF
receptor signaling may thus not only be protective for oligo-
dendrocytes but can also enhance remyelination making LIF
a potentially therapeutic cytokine for limiting oligodendro-
cyte damage (Marriott et al. 2008). Oncostatin-M regulator
is highly expressed in astrocytes, and its stimulation controls
the expression and release of proinflammatory cytokines like
TNF-α and IL-1β (Tanaka and Miyajima 2003). Microglia
differentially respond to IL-6, OSM, and CNTF. Whereas
IL-6 and OSM induce proinflammatory cytokine production
• 269
Figure 22.3 IL-6 Signaling. A. IL-6 signals via two IL-6R subunits that on binding associate with two gp130 subunits. Because IL-6 receptors lack
signal-transduction capacity, signaling occurs via phosphorylation events of gp130-associated tyrosine kinases ( JAK kinases: JAK1, JAK2, JAK3, and/
TYK2) and subsequent phosphorylation and activation of signal transducers and activators of transcription (STAT), mainly STAT3 and STAT1. In
addition, phosphorylation of gp130 by JAKs may also result in the activation of mitogen-activated protein kinases (MAPKs), including extracellular
signal regulated kinase (ERK1/2), p38, and c-jun N-terminal kinases/stress-activated protein kinases ( JNK/SAPK). In addition, PI3K/Akt signal-
ing pathway may also be activated. B. IL-6R is not only found as a membrane bound receptor, but can be released and thus occurs in soluble form
(sIL-6R). Binding IL-6 to its soluble receptors forms a ligand–receptor complex that is able to activate gp130 in the same way as the membrane-bound
IL-6R (trans-signaling).

and thus activate these cells, treatment with CNTF in micro-
glia results in downregulation of cyclooxygenase-2 (COX-2)
without affecting TNF-α and IL-1β expression (Baker et al.
2010; Krady et al. 2008). It should be noted, however, that
these results are solely based on cultured cells and that very
little is yet known about the expression of IL-6–type cytokine
receptors in astrocytes and microglia in vivo.
4 THE TUMOR NECROSIS
R E C E P TO R FA M I LY
The tumor necrosis factor (TNF) family contains approx-
imately 20 members that are involved in the regulation of
inflammation, immune responses, development of lym-
phoid organs, and tissue homeostasis. These proteins act in
membrane bound form and as soluble cytokines. Most
studies on TNF-related cytokines in the central nervous
system (CNS) have addressed TNF-α (and to a lesser extent
TNF-β/lymphotoxin). Also in the brain TNF-α is considered
a key inflammatory regulator that induces cytokine produc-
tion, inflammation, gliosis, blood-brain barrier damage, demy-
elination, and neural damage (Montgomery and Bowers 2011).
In the CNS, TNF-α is primarily produced by microglia, astro-
cytes, and neurons (Hanisch 2002; Montgomery and Bowers
2011). TNF-α not only drives inflammatory responses, but is
also involved in tissue support, neuronal development, and
restoration of homeostasis.
Two related receptors for TNF-α are known, TNFR1 and
TFNRII. TNFRI contains a death domain, which TNFRII
lacks. Accordingly, TNFRI is designated as a death receptor.
Both receptors are expressed by astrocytes, microglia, and
oligodendrocytes (Dopp et al. 1997). In glia cells, TNFRI is
constitutively expressed, whereas TNFRII expression is trig-
gered under inflammatory conditions and is found in hemato-
poietic lineage cells. It has thus been demonstrated that under
inflammatory conditions microglia and Schwann cells express
TNFRI and TFNRII, whereas astrocytes and oligodendro-
cytes primarily express TNFR1 (Dopp et al. 1997; Qin et al.
2008). Both TNRF1 and TNRFII do not contain an intracel-
lular signaling domain and thus signal via intracellular adaptor
molecules (Aggarwal et al. 2011). Receptor activation occurs
by oligomerization and requires internalization of the ligand–
receptor complex.
As mentioned, TNFRI contains a cytoplasmic death
domain that mediates induction of apoptosis and NF-κB–
mediated signaling cascades. Thus, on activation of TNFR1,
dissociation of silencer of death domain (SODD), which
prevents death domain signaling, takes place and recruit-
ment of TRADD (TNF receptor–associated death domain)
initiates downstream signaling (Fig. 22.4). This involves sub-
sequent binding of FADD (Fas-associated death domain),

270 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
Figure 22.4 Tumor Necrosis Factor-α Receptor Signaling. Binding of the TNF-α homotrimeric ligand induces trimerization of membrane-associated
tumor necrosis factor receptor 1 (TNFR1) subunits. Subsequently, recruitment of tumor necrosis factor receptor-1–associated death domain protein
(TRADD) to the cytoplasmic domain of TNFR1 occurs. TNFR1-TRADD recruits tumor necrosis factor receptor-associated factor-2 (TRAF2)
and receptor interacting protein (RIP). This trimeric complex activates c-jun N-terminal kinase ( JNK) and mediates activation of the inhibitory
κB kinase complex (IKK) that phosphorylates IκB. Phosphorylation of IκB releases and activates NF-κB. Additionally, the TNFR1-TRADD
binds Fas-associated death domain protein (FADD), which recruits caspase-8. The subsequent signaling cascade mediated by caspase-8 cleavage and
activation leads to apoptosis.

TRAF2 (TNF-receptor–associated factor 2) and RIP (recep-
tor interacting protein). This receptor–signaling complex
induces a phosphorylation cascade, leading to activation of
the transcription factor NF-κB, which is essentially involved
in the regulation of expression of proinflammatory cytokines,
chemokines, adhesion molecules, and inducible effector mol-
ecules including iNOS (inducible nitric oxide synthase) and
cyclooxygenase-2 (COX-2). TNFRII directly associates with
TRAF2 in conjunction with TRAF1 (see Fig. 22.4) and elic-
its intracellular cross-talk with TNFRI via RIP. Furthermore,
TNFRII activates the MEKK-JNK signaling pathway.
Clearly, TNFRI and TNFRII have opposing roles in cyto-
toxicity and cytoprotection (Fontaine et al. 2002). TNFRI is
responsible for the majority of known biological properties of
TNF-α and involves inflammation, immune responses, and
cytotoxicity, which are primarily induced through induction
of adhesion molecules, cytokines, and chemokines. TFNRII
is considered protective against neurodegenerative diseases by
the activation of PI3K (phosphoinositol 3 kinase), PKB/Akt
(protein kinase B/Akt) phosphorylation, and long-term acti-
vation of NF-κB (Marchetti et al. 2004). As mentioned, in
various types of neurodegenerative diseases (Haase et al. 2008),
TNF-α is expressed primarily by microglia (Hanisch 2002)
and astrocytes. Glia-derived TNF-α is involved in a variety of
neurodegenerative diseases including multiple sclerosis (MS),
Parkinson disease (PD), and Alzheimer disease (AD); for
review see Montgomery and Bowers (2011). Generally, TNF-α
is believed to have a direct cytotoxic effect on oligodendrocytes
but is also important for remyelination (Arnett et al. 2001)
and is a potent inducer of cytokines, chemokines, and adhe-
sion molecules in microglia and astrocytes. At peripheral nerve
injury TNF-α is produced by Schwann cells and may act as a
factor involved in neuropathic pain (Campana et al. 2007).
5 I N T E R L E U K I N -1 R E C E P TO R S
AND SIGNALING
Interleukin-1 (IL-1) is a pleiotropic cytokine and is essentially
involved in host-defense responses to injury and infection,
including fever, sickness behavior, and metabolic and immune
changes, and is associated with many CNS disorders, includ-
ing brain trauma, stroke, and epilepsy, as well as many forms
of chronic neurodegenerative disorders such as Alzheimer
and Parkinson disease or multiple sclerosis (Allan et al. 2005).
IL-1 induces the expression of a large variety of proinflam-
matory mediators, including cytokines, cytokine receptors,
acute-phase reactants, growth factors, tissue remodeling

C Y TO K I N E , C H E M O K I N E , A N D G R OW T H FAC TO R R E C E P TO R S A N D S I G N A L I N G • 271
enzymes, extracellular matrix components, and adhesion mol-
ecules (Pinteaux et al. 2009).
The primary receptor for IL1, IL-1R1 belongs to a large
Toll/IL-1R (TIR) superfamily and is defined by the Toll/
IL-1 receptor (TIR) domain, which occurs in the cytosolic
region of these receptors (Bowie and O’Neill 2000). IL-1R1
has shown to be expressed in astrocytes, oligodendrocytes,
microglia, and Schwann cells (Skundric et al. 1997; Wang et
al. 2006). The activity of IL-1 is mediated by the two agonists
IL-1α and IL-1β that induce activation of IL-1R1. IL-IRI con-
tains an extracellular ligand-binding domain that comprises
three immunoglobulin-like domains (Ig domains). IL-IRI
also contains an intracellular signal-transducing domain that
bears homology to the Toll-like receptor family members
(Bowie and O’Neill 2000). The effects of IL-1R1 are coun-
teracted by the endogenously expressed receptor antago-
nist (IL-1RA), and by a nonsignaling type 2 IL-1 receptor
(IL-1R2), a soluble-binding protein that acts as a suppres-
sor of IL-1 signaling. Furthermore, it has become clear that
IL-1β is involved in the inflammasome signaling cascade. In
the last few years, inflammasomes (protein signaling com-
plexes involved in cytokine release) have emerged as a crucial
intracellular pathway mediating inflammation (Strowig et al.
2012). Activated inflammasomes give rise to cytokine release
and inflammation. It is thus well established that caspase-1
is activated by the inflammasome complex and subsequently
mediates processing of IL-1β (Trendelenburg 2008 and refer-
ences therein). Inflammasomes are powerful sensors of patho-
logical factors such as protein aggregates including amyloid-β.
Indeed, recent work showed in vitro evidence for activation of
the NALP3 inflammasome pathway in microglia in response
to amyloid-β (A-β) (Halle et al. 2008). It has thus been sug-
gested that inappropriate oligomerization of A-β protein is
sensed by the glial inflammasome complex and thus gives rise
to neuroinflammation (Masters and O’Neill 2011).
On IL-IRI signaling IL-1R accessory protein (AcP), a struc-
tural homolog of IL-IRI, forms a heterodimer with IL-IRI, that
is essential for IL-IRI–induced signal transduction. Owing to
IL-1 binding, the IL-lRI:IL-lRAcP heterodimer assembles
and serves as a docking site for a variety of signaling proteins.
These signaling proteins are primarily kinases, including IL-1
receptor-associated kinases 1 and 2 (IRAK-1, IRAK-2), which
bind to IL-lRAcP and IL-IRI, respectively (Fig. 22.5).
In IL-1 signaling the transcription factor NF-kB is a cen-
tral element that expression regulation of most IL-1–induced
genes. IL-1–mediated activation of NF-κB yields a hetero-
meric complex that leads to phosphorylation and subsequent
degradation of the inhibitor I-κB, thus allowing nuclear trans-
location of NF-κB where it affects gene transcription. In addi-
tion, MyD88, a member of the IL-1 receptor family, associates
with both IL-lRAcP and IRAK-2. Subsequently, tumor necro-
sis factor receptor–associated factor 6 (TRAF6) binds to the

Figure 22.5 Interleukin-1 Receptor Signaling. Following binding of IL-lα or IL-1β to IL-1 receptor-I (IL-IRI), IL-lRI dimerizes with IL-lRAcP.
At this scaffold the IL-1 receptor-associated kinases IRAK-1 and IRAK-2 are recruited to IL-lRAcP and IL-IRI, respectively. Additionally, MyD88
associates with IL-lRAcP and IRAK-2. This complex recruits tumor necrosis factor receptor–associated factor-6 (TRAF6), which binds and activates
NF-κB inducing kinase (NIK). NIK activates the I-κB kinase complex, which phosphorylates I-κB. IκB is then targeted for proteosomal degradation
and the now active NF-κB enters the nucleus and affects transcription of IL-1 target genes.

272 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
IL1-RI complex and recruits NF-κB-inducing kinase (NIK)
that activates the I-κB kinase complex. This causes phosphory-
lation of I-κB, which ultimately leads to its proteosomal deg-
radation. After disposal of I-κB, NF-κB enters the nucleus and
regulates gene transcription, as mentioned.
Also as mentioned, IL-1 is essentially involved in CNS
disorders, including brain trauma, stroke and epilepsy, and
chronic neurodegenerative disorders, including Alzheimer
and Parkinson disease and multiple sclerosis (Allan et al.
2005). The mechanism underlying this effect is the activa-
tion of astrocytes that produce neurotoxic, neuroprotective
and inflammatory mediators. It has been suggested that IL-1–
induced neurotoxicity is dependent on induced production of
astrocytic neurotoxic factors. IL-1–induced neurotoxicity was
found to be dependent on free radical- and caspase activity and
production of TNF-α (Pinteaux et al. 2009). Most likely, the
toxic effect of IL-1 on oligodendrocytes also occurs through
actions on astrocytes. Furthermore, it has been suggested that
the neuroprotective effect of IL-1 is mediated by NGF release
from astrocytes (Pinteaux et al. 2009). Thus, it is likely that
low levels of IL-1 act on astrocytes to produce neurotoxic fac-
tors, which can be counteracted by neuroprotective agents
secreted by astrocytes at higher IL-1 levels. Furthermore, IL-1
appears to control neuronal cell death directly in response to
injury, which may be mediated by enhancement of NMDA-
induced toxicity (Viviani et al. 2003).
6 T H E I N T E R L E U K I N -2
R E C E P TO R FA M I LY
Interleukin-2 receptor (IL-2R) together with receptors for
other cytokines including IL-4, IL-7, IL-9, IL-13, IL-15, and
IL-21 makes up the IL-2 receptor family (O’Shea et al. 2002).
These receptors contain a ligand-specific binding chain (the
α-chain) and a general γ-chain. Additionally, IL-2 and IL-15
contain a β-chain, whereas the IL-13 receptor contains the
IL-4 receptor α-chain and a ligand binding chain specific
for IL-13. The receptors for IL-2, IL-7, IL-9, IL-15, and IL-21
transduce signals through activation of STAT-5, whereas the
IL-4 and IL-13 receptors activate STAT-6.
The IL-2 and IL-4 signal transduction cascades are discussed
as examples for signaling of the IL-2 receptor superfamily.
7 I N T E R L E U K I N -2
As mentioned, IL-2R is a heterotrimeric protein complex that
is activated by IL-2. Three receptor chains (α, β, and γ) associ-
ate to form IL-2R (Wang et al. 2005). Both α- and β-chains
are involved in binding IL-2, whereas the γ-chain together
with the β subunit is involved in signal transduction. Whereas
the β-chain is associated the tyrosine kinase Janus Kinase 1
( JAK1), the γ-chain associates with JAK3. Consequently,
IL-2 activates three signaling cascades, the MAP kinase cas-
cade, the phosphoinositide kinase 3 (PI3K) cascade, and the
JAK-STAT cascade (Ellery and Nicholls 2002; Moon et al.
2004). After IL-R2–induced activation of JAK1/3 kinases
C Y TO K I N E , C H E M O K I N E , A N D G R OW T H FAC TO R R E C E P TO R S A N D S I G N A L I N G
STAT5a/b molecules are activated and promote transcrip-
tional activation of D (2 and 3) cyclins. In addition, on IL-R2
activation of PI3K signaling, p27, an inhibitor of cyclin-D/
CDK activity, is downregulated (Nourse et al. 1994). These
events stimulate progression through G1 of the cell cycle and
thus trigger DNA synthesis and replication. Activated micro-
glia express IL-2R as well as IL-2 and increase their prolifera-
tion rate upon stimulation with IL-2 and support production
of NO (Girard et al. 2008; Hanisch 2002; Sawada et al. 1995).
Also oligodendrocytes express IL-2R and respond to exposure
to IL-2 by increased proliferation (Otero & Merril, 1997).
Furthermore, several lines of evidence suggest that IL-2 is an
important neuroregulatory cytokine independent of its role in
inflammation and immunity (Hanisch and Quirion, 1995).
8 I N T E R L E U K I N -4
The IL-4 receptor (IL-4R) is expressed by astrocytes, microg-
lia, and oligodendrocytes (Brodie et al. 1998; Cannella and
Raine 2004; Hulshof et al. 2002; Wei and Jonakait 1999),
and is commonly considered an antiinflammatory mediator in
these cells. IL-4 has an immunosuppressive effect on microg-
lia by suppressing expression of factors of the immune system,
including MHC-II, CD40, and B7 costimulatory molecules
(Nguyen and Benveniste 2000; O’Keefe et al. 1999; Wei and
Jonakait 1999). Furthermore, IL-4 inhibits NO production,
secretion of TNF-α and expression of ICAM-1 and addi-
tionally induced secretion of NGF by astrocytes (Brodie et
al. 1998). As mentioned, IL-4R consists of a high-affinity
α-chain and a γ-chain. In addition, another form of IL-4R
exists and contains an IL-4Rα chain and an IL-13–binding
protein denoted IL-13Ra (Liu et al. 2000).
IL-4R–mediated activation of JAKI and JAK3 has been
shown to occur in many cell types. Activation of JAKs results
in recruitment and activation of STAT6 and STAT5. After
recruitment and phosphorylation, the STAT molecules
dimerize, translocate to the nucleus and activate transcription
of IL-4 inducible genes.
In addition to STAT signaling, IRS-2 plays an impor-
tant role in IL-4–induced cellular proliferation. This protein
named insulin receptor substrate 2 (IRS-2) has been shown
to become activated in response to IL-4 and IL-13. Insulin
receptor substrate molecules play a role in signal transmis-
sion to downstream pathways, including PI3K/Akt- and ERK
MAP kinase cascades. Finally, activation of IL-4R induces an
increase in PI3-kinase activity. Cell type–specific modulation
of the PI3-kinase pathway by IL-4 may differentially influence
cell proliferation or gene induction, respectively.
9 CHEMOKINES AND
C H E M O K I N E R E C E P TO R S
Chemokines (chemotactic cytokines) constitute the larg-
est cytokine family in humans and consist of structur-
ally related molecules that are important regulators of the
• 273
Figure 22.6 The Complex Pharmacology of the Chemokine System. There are approximately 50 different chemokines that are classified into four
subfamilies because of conserved cysteine residues (CC, CXC, CX3C, and C-chemokines). The classification of chemokine receptors is related to
their ligands; thus, chemokine receptors are also classified into four subfamilies: CCRs (CCR1–10), CXCRs (CXCR1–6), XCR1, and CX3CR1. The
pharmacology of chemokines shows a considerable amount of promiscuity; thus, many chemokines activate more than one receptor, and numerous
receptors are activated by more than one chemokine. Only seven nonredundant chemokine–chemokine receptor pairs are known. Moreover, the
chemokine system contains four atypical receptors: DARC, D6, CXCR7, and CCX-CKR that lack classical signaling properties. These atypical recep-
tors may bind a large variety of chemokines (e.g., DARC and D6) and act as decoy and scavenger receptors.

peripheral immune response and therefore are involved
in numerous diseases. There are approximately 50 differ-
ent chemokines which are classified into four subfamilies
due to conserved cysteine residues (CC, CXC, CX3C, and
C-chemokines) (Fig. 22.6). Chemokines are small (8- to
14-kDa) proteins that share a highly conserved tertiary struc-
ture, the so-called “chemokine-scaffold,” despite relatively low
sequence homology (Mantovani et al. 2010). Unlike other
cytokines, chemokines activate specific G-protein–coupled
receptors with seven transmembrane spanning domains, of
which 18 have been described. The classification of chemo-
kine receptors is related to their ligands; thus, chemokine
receptors are also classified into four subfamilies: CCRs
(CCR1–10), CXCRs (CXCR1–6), XCR1, and CX3CR1 (see
Fig. 22.6). The pharmacology of chemokines displays a con-
siderable amount of promiscuity, thus many chemokines
activate more than one receptor, and numerous receptors
are activated by more than one chemokine. Only few non-
redundant chemokine–chemokine receptor pairs are known,
of which the CX3CL1-CX3CR1 pair is the most established
example for the brain (Mantovani et al. 2010). An interest-
ing property of the chemokine system is the existence of four
receptors: DARC, D6, CXCR7, and CCX-CKR that lack
classical signaling properties. These so-called atypical or silent
receptors lack crucial structural domains that are required for
G-protein activation, including the DRY motif in the second
intracellular loop (Mantovani et al. 2010). Nevertheless, these
receptors bind a variety of chemokines and act as decoy and
scavenger receptors, thereby fulfilling multiple important
roles in the regulation of chemokine function (Mantovani
et al. 2010).
Chemokine receptors are coupled to Gi/o-proteins, which
primarily inhibit adenylate cyclase activity; however, it is clear
that Gi/o-proteins additionally activate other intracellular tar-
gets, including phospholipases, GTPases such as Rho, Rac,
and Cdc42, and signaling pathways of major kinases such as
mitogen-activated protein kinase (MAPK) and phosphati-
dyl inositol-3 kinase (PI3-K) (Fig. 22.7) (Neves et al. 2002;
Rodríguez-Frade et al. 2001). This diversity in intracellu-
lar signaling shows that chemokine receptors, in addition to
pathways involved in cell migration, also activate other path-
ways and control a large spectrum of cellular functions.
Similar to other organs, the infiltration of the inflamed brain
by blood leukocytes is regulated to a large extent by chemok-
ines (Prinz and Priller 2010). However, also most endogenous
brain cells (astrocytes, oligodendrocytes, NG2 cells, microglia,

274 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
Figure 22.7 Chemokine Receptor Signaling. All chemokine receptors are coupled to Gi/o-proteins. On binding the GDP is exchanged for GTP and
the activated α-subunit dissociates to inhibit adenylate cyclase activity and cAMP formation. The βγ-subunit can further activate other intracellular
targets, including phospholipases, GTPases such as Rho, Rac, and Cdc42, and signaling pathways of major kinases such as mitogen-activated protein
kinase (MAPK) and phosphatidyl inositol-3 kinase (PI3-K). Moreover, there is evidence that dimerization of activated chemokine receptors may
change gene transcription via activation of the JAK/STAT pathway.

and neurons) express functional chemokine receptors and are
able to release chemokines (see chapter 18). This indicates
that the functions of endogenous chemokines in the brain
go beyond the control of infiltration (Biber et al. 2006). For
example, chemokines regulate the migration, maturation, and
survival of oligodendrocyte precursor cells via the chemokine
receptors CXCR2, CXCR4, and CXCR7 (Banisadr et al.
2011). Generally, the basal expression of chemokines in the
healthy brain is low, but increases considerably under patho-
logical conditions. The only exception is the membrane-bound
chemokine CX3CL1 (previously known as fractalkine), that
is highly expressed in healthy neurons. Deficient CX3CL1-
CX3CR1 signaling has long been thought to have little impact
on healthy brain function; however, recent findings have
shown small developmental and electrophysiological abnor-
malities in the brain of CX3CR1-deficient animals that point
toward a function of CX3CR1 in microglia in pruning of syn-
apses during development (Paolicelli et al. 2011). Despite these
recent results, CX3CL1 exerts its function mainly in pathol-
ogy, where it regulates the response of microglia after neuronal
injury (Cardona et al. 2006). Next to CX3CL1, CCL21 is also
an important player in the communication between damaged
neurons and microglia, which is crucial for the development of
neuropathic pain after peripheral nerve injury (Biber et al. 2011;
Old and Malcangio 2011). Interestingly, whereas CX3CL1 in
the brain is a neuronal signal that dampens microglia activity,
it is a microglia-activating signal in the spinal cord (Old and
Malcangio 2011). After spinal nerve injury, microglia release
cathepsin S, which causes the shedding of neuronal CX3CL1
and the subsequent further activation of microglia Src-kinase
pathway (Old and Malcangio 2011). Such an activating activ-
ity of CX3CL1 was never found in the central brain and
may point toward a region-specific function of this neuron–
microglia signaling system. Neuronal CCL21 is unique among
the neuronal chemokines, as it is expressed exclusively in dam-
aged neurons, where it is sorted into large dense core vesicles
and transported to the axon ends (Biber et al. 2011; de Jong
et al. 2008). Mice that lack functional CCL21 do not develop
neuropathic pain in response to peripheral nerve injury, high-
lighting the functional importance of these chemokines in
neuron–microglia communication (Biber et al. 2011). Next to
the large number chemokine functions confined to astrocytes
(Biber et al. 2006), it was recently found that CXCR4-activation
by CXCL12 regulates astrocytic glutamate release and thus
influences synaptic transmission (Calì and Bezzi 2010).
10 T R A N S F O R M IN G G R OW T H
FAC TO R - β
The superfamily of transforming growth factors-β (TGF-β)
has a broad spectrum of biological effects and comprises

C Y TO K I N E , C H E M O K I N E , A N D G R OW T H FAC TO R R E C E P TO R S A N D S I G N A L I N G • 275
Figure 22.8 The TGF-β Signaling Pathway. Members of the TGF-β superfamily bind to type I (TGF-β-RI) or type II (TGF-β-RII) receptors.
TGF-β-RI is phosphorylated by TGFβ-RII, resulting in an activated receptor complex. R-SMADS are recruited to the receptor complex and are phos-
phorylated at their C-terminus. Phosphorylated SMADs then form a trimeric complex with common SMAD4. The SMAD complex then translocates
to the nucleus, where it interacts with DNA and transcription factors, including a large variety of DNA-binding transcription factors (cofactors) in a
target gene-dependent manner. FKBP12, SARA, SMURF1/2, and SMADs 6/7 are negative regulators of TGF-β signaling.

at least 30 members that include various forms of TGF-β,
bone morphogenetic protein, and the factors activin and
nodal. During embryogenesis, TGF-β family members play
a critical role in axis formation, left-right organization, and
tissue patterning and disruptions in TGF-β signaling in
humans often leads to tumorigenesis (Massagué 2008). In
the nervous system, all three TGF-β isoforms (β1–3) are
ubiquitously expressed (Kitisin et al. 2007) and are impor-
tant for neuronal and astrocytic differentiation (McKinnon
et al. 1993; Stipursky and Gomes 2007; Yi et al. 2010). In the
CNS, the effect of TGF-β is different for different cell types
and has been reported to act as a neurotrophic factor as well
as an inducer of apoptosis. TGF-β inhibits proliferation of
astrocytes in vitro (Vergeli et al. 1995) and induces apopto-
sis in oligodendrocyte precursors (Schulz et al. 2009), but
promotes neuronal survival. This is supported by knock-out
studies in mice, in which a loss of TGF-β1 leads to micro-
gliosis and reduces neuronal survival (Brionne et al. 2003).
The neuroprotective effect of TGF-β on neurons occurs
most likely indirectly through induction of neurotrophic
factors in glial cells. A role for TGF-β has been suggested in
many CNS pathologies, including ischemia and excitotoxic-
ity as well as a range of neurodegenerative conditions such
as Parkinson’s disease, Alzheimer’s disease, and multiple
sclerosis (reviewed in Pratt and McPherson 1997). In general,
an increase in TGF-β expression has been observed under
these conditions both in neurons and glial cells. Whether
this elevated expression of TGF-β reflects a “wound healing”
and antiinflammatory response or contributes to pathology
is unclear. In animal models, it has been shown that TGF-β
contributes to the pathology caused by β-amyloid accumu-
lation by increasing its production, but on the other hand
TGF-β enhances microglial clearance of β-amyloid deposits.
In autoimmunity studies, TGF-β has been reported to have
antiinflammatory and neuroprotective functions (Owens
et al. 2001).
The TGF-β superfamily signals through TGF-β and
BMP receptors that can be subdivided into type I (TGF-βI)
and type II (TGF-βII) serine/threonine kinase receptors.
(Fig. 22.8). After ligand binding, TGFβRII receptors phos-
phorylate TGF-β-RI receptors, which in turn phosphorylate
and activate specific SMAD proteins. These SMADs form a
complex with common SMAD4, translocate to the nucleus,
and activate target gene transcription. The composition of
these complexes is dependent on the type of TGF-β recep-
tor activated. In the CNS, TGF-β is constitutively expressed
(isoforms 1–3) and expression of TGF-β-RI and TGF-β-RII
is detected on neuronal as well as glial cells.

276 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
Figure 22.9 Neurotrophin Signaling. Signaling by members of the neurotrophins can be mediated by two receptor types, TrkA-D and p75. Each Trk
receptor controls three major signaling pathways. Activation of Ras results in activation of the MAP kinase–signaling cascade, activation of PLC-γ1
results in activation of Ca2+- and protein kinase C–regulated pathways and activation of the PI3-Akt pathway. In general, these signaling pathways acti-
vate the expression of genes involved in differentiation and/or survival. The p75 receptor activates three major signaling pathways. NF-κB activation
results in induction of prosurvival genes. Activation of the Jun kinase pathway results in activation of proapoptotic genes. And neurotrophin binding
to p75 regulates the activity of Rho, affecting growth cone motility.

The SMADs (an acronym for SMA [the small protein in
C. elegans] and MAD [mothers against decapentaplegic in D.
melanogaster]) mediate intracellular signaling by TGF-β fam-
ily members. The SMADs are subdivided in three functionally
distinct groups: (1) receptor-regulated SMADs (R-SMADs),
(2) the co-SMAD, SMAD4, and (3) antagonistic SMADs.
SMAD4 participates in signal transduction of all TGF-β
family members, interacts with activated R-SMADs and,
after nuclear translocation, activates target gene transcription.
R-SMAD–SMAD4 complexes have been reported to interact
with various cofactors, these include forkhead family members
like P300, CBP, FOXH1, OAZ, RUNX family members, and
AP1 transcription factors, and different combination bind to
different cognate binding sites (Massagué et al. 2005).
Interferon-γ and TNF-α regulate TGF-β signaling indi-
rectly: interferon-γ (via JAK/STAT1) and TNF-α (via NF-κB)
can induce the expression of antagonistic SMAD7 protein and
thereby inhibit TGF-β receptor signaling. However, TNF-α
is not exclusively antagonistic to TGF-β, TNFα can also acti-
vate STAT3, which has been reported to be able to coopera-
tively with SMAD1 to induce astrocyte differentiation.
The primary effect of TGF-β signaling on astrocytes and
microglia is immune suppressive. The expression of MHC
class II, B7, CD40, ICAM-1, VCAM-1, and TNF-α is inhib-
ited by TGF-β (O’Keefe et al. 2002).
11 N E U R OT R O P H I N S
The family of neurotrophins (NTs) consists of four members:
nerve growth factor (NGF), brain-derived neurotrophic factor
(BDNF), neurotrophin-3 (NT-3), and NT-4. Neurotrophins
are essential factors that play a critical role in survival, differ-
entiation, and myelination of neurons during neuronal devel-
opment (Chao 2003). Recently, NTs have also been reported
to be important regulators of myelination, both in the periph-
eral and CNS (Rosenberg et al. 2006; Xiao et al. 2009).
Nerve growth factor and BDNF are ubiquitously expressed
by all glial cell types. Neurotrophins can signal through two
classes of transmembrane receptors: receptor tyrosine kinases
TrkA, -B, -C, and the P75 NT receptor (a member of the
tumor necrosis factor [TNF] receptor superfamily), which
all are expressed by glial cells (Fig. 22.9). Nerve growth fac-
tor preferably binds TrkA, BDNF TrkB, NT-3 TrkC, and
NT-4 TrkB, and all NTs can bind p75. Neurotrophins bind
their receptors with relatively low affinity, which is regulated
by receptor dimerization resulting in the formation of a high
affinity complex. Trk receptor dimerization leads to trans-
phosphorylation of tyrosine residues in the cytoplasmic por-
tion of the receptor (see Fig. 22.9). These phosphorylated
tyrosine residues then function as docking sites for down-
stream signaling cascades. Trk receptors recruit and activate
PLCγ and the adapter protein Shc, which in turn leads to
activation of phosphatidylinositol 3-kinase (PI3K) Akt and
Ras/ERK1/2. Ultimately, Trk signaling mediates a plethora
of activities, which include neuronal survival, neurite out-
growth, and myelination and differentiation of Schwann cells.
Additionally, NTs have been reported to stimulate microglial
proliferation in vitro (Cosgaya et al. 2002).
The role of p75 in NT signaling is complex and is dif-
ferent in the presence or absence of Trk receptors (Nykjaer
et al. 2005). The affinity of p75 for NTs is similar to that of

C Y TO K I N E , C H E M O K I N E , A N D G R OW T H FAC TO R R E C E P TO R S A N D S I G N A L I N G • 277
monomeric Trk. The Trk-A receptor can dimerize with p75
and this heterodimer has a higher affinity for NGF than both
homodimeric receptors and allows for receptor activation at
lower NGF concentrations. Additionally, the specificity of Trk
receptors for NTs is affected by heterodimerization with p75.
The intracellular domain of the p75 receptor activates various
intracellular signaling pathways, different from the pathways
activated by Trk receptors. This suggests that the signaling
properties of Trk homodimers and Trk/p75 heterodimers
are different with different biological activity as a result. For
instance, TrkA dimers sustain neurite outgrowth, but TrkA/
p75 heterodimers are required for maturation and long-term
neuronal survival.
The p75 receptor also mediates Trk-independent NT signal-
ing. Genetic studies in mice have convincingly shown that p75
can promote apoptosis, most likely through the activation of
Jun N-terminal kinase ( JNK). The intracellular domain of p75
has been shown to couple to downstream signaling pathways via
several adaptor proteins, including NT-receptor–interacting
factor (NRIF), NT-associated cell death executor (NADE),
NT-receptor–interacting MAGE homolog (NRAGE) and TNF
receptor–associated factors (TRAFs). All these interacting pro-
teins (alone or in complexes) have been shown to promote p75-
dependent apoptosis. Additionally, p75 has been shown to be
involved in Schwann cell migration (involving factor Schwann
cell 1 [SC1]) or survival (involving receptor-interacting protein
2 [RIP2]) by activating NF-κB signaling, resulting in reduced
NGF-p75–induced Schwann cell death. With respect to astro-
cytes, NGF inhibits their proliferation through the p75 recep-
tor (Cragnolini et al. 2009). Oligodendrocytes express NTs as
well as all receptors: TrkA-C and p75, suggesting autocrine sig-
naling by NTs (Du and Dreyfus 2002). Oligodendrocytes that
myelinate sensory neurons express BDNF, and a potential role
BDNF in the myelination process is further corroborated by
the observation that BDNF regulates the number of oligoden-
drocyte precursors and their differentiation in demyelinating
lesions (VonDran et al. 2011).
12 P L AT E L ET-D E R I VE D G R OW T H
FAC TO R S I G N A L I N G
Platelet-derived growth factor (PDGF) is one of the first iden-
tified mitogens, and stimulates proliferation of cells of mes-
enchymal origin, mainly smooth muscle cells and glial cells.
PDGF signaling is mediated by four ligands (PGDF-A, B, C,
and D) and two receptors (PDGF-Rα and -β). Expression of
PDGFRs seems to be restricted to cells derived from O2A
precursors, mainly type 2 astrocytes and oligodendrocytes
(Hart et al. 1989). All PDGF ligands activate the PDGFRs as
disulfide-linked homodimers or as PDGF-A-B heterodimers.
PDGFR is a transmembrane receptor tyrosine kinase, and on
ligand binding, two receptors dimerize, cross-phosphorylate
tyrosine residues in their cytoplasmic tails, which then serve
as docking sites for SH2-containing adaptors, Shc and Grb2.
Signaling pathways acting downstream of the PDGFR include
Ras-MAPK, PI3K, Src, STATs, and PLCγ (Heldin and
Westermark 1999).
278 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
Signaling through PDGFR has been implicated in
glioma-genesis and glial development. During embryonic
development (E15), PDGF is expressed by neurons, in which
the PDGFR is expressed in glial precursors, mainly of the
oligodendrocyte lineage. PDGF-A–deficient mice show
reduced oligodendrocyte numbers and display hypomyelina-
tion, indicating a lack of trophic effect of neuronal PDGF on
glial precursors. This is further supported by the observation
that PDGF overexpression in neurons resulted in increased
oligodendrocyte precursor number, but normal mature oli-
godendrocytes, presumably owing to loss of neuronal contact.
Additional experiments targeting tyrosines in the cytoplas-
mic domain of the PDGFR required for downstream signal-
ing molecules has shown that these pathways are required for
proper expansion of oligodendrocyte precursors and chemo-
taxis to migrate and populate other areas of the brain.
Aberrant PDGF signaling has also been implicated in glial
tumorigenesis. PDGF and PDGFR have been found to be
overexpressed in glial tumors as well as glial tumor cell lines.
Enhanced expression of PDGF or PDGFR was associated
with a higher tumor grade and hence a poorer prognosis. The
expression of both the ligand and its receptor by these cells
allows for autocrine stimulation. However, a causal relation
between PDGF signaling and glial tumorigenesis is unclear as
well as the mechanism of PDGF/PDGFR overexpression
13 S U M M A RY A N D P E R S P E C T I VE S
In this chapter an outline of the common signaling pathways
and functions of cytokine-, chemokine-, and growth factor
receptors in glia cells has been provided. This signaling gen-
erally involves activation of membrane-associated receptors
induced by agonist binding, followed by biochemical signal-
ing cascades that result in functional responses and induced
gene transcription. Because these receptor families are very
extensive, this outline is far from complete, but clearly delin-
eates the known response patterns of glia and their relation-
ship to glia functions.
Clearly, the wide variety of specific receptor subtypes
expressed in glia allows detailed responses to neuroimmune sig-
nals that convey stress, inflammation, or regenerative processes.
The integration of the diverse immune signals culminates in
dedicated activated glia phenotypes and unified responses to
situations of maintenance/regeneration, neural damage, and
aging. Particularly the effects of cytokines and chemokines
range from neuroprotection to severe neurodamage and are
tightly regulated with respect to strength and duration.
A better understanding of the synchronized glial response
to these complex signal patterns may reveal basal mechanisms
underlying neuropathology and neurorepair and maintenance,
and may provide possible targets for therapeutic intervention.
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 23.
LIPIDS, LIPID MEDIATOR S, AND OTHER
SIGNALING MOLECULES
Hideki Hayashi

A B B R E VI AT I O N S 1 INTRODUCTION

AA arachidonic acid Lipids in the central nervous system (CNS) play various roles as
ABC ATP-binding cassette components of membrane structures and biological regulators
AD Alzheimer disease of cell functions. Their metabolites also act as bioactive lipid
apo apolipoprotein mediators in brain functions. The brain contains the second
ATX autotaxin highest lipid content next to adipose tissue (Leskovjan et al.
Aβ amyloid β peptide 2010) and is the most cholesterol-rich organ in the body. In con-
BBB blood-brain barrier trast with the concentration of cholesterol in most other organs
CETP cholesteryl ester transfer protein of the adult mouse (in the range of 2–6 mg/g wet weight), the
CNS central nervous system brain contains at about 15 mg/g tissue (Dietschy 2009). Because
COX cyclooxygenase the CNS is segregated from the peripheral circulation by the
CSF cerebrospinal fluid blood-brain barrier (BBB), lipid homeostasis in the CNS is dis-
DHA docosahexaenoic acid tinctively different from that in peripheral tissues.
FFA free fatty acid A lipoprotein is a soluble complex of proteins and lipids
FLAP 5-lipoxygenase–activating protein that carries lipids in the circulation and the cerebrospinal fluid
GPCR G protein–coupled receptor (CSF). In contrast with the circulation, which contains very low
HDL high density lipoproteins density lipoproteins (VLDL), low density lipoproteins (LDL),
HIV human immunodeficiency virus and high density lipoproteins (HDL), the CNS contains only
HPGDS hematopoietic prostaglandin HDL-like particles of size approximately 15 nm and density
D synthase 1.06 to 1.12 g/mL (LaDu et al. 1998). Under normal conditions
IL interleukin these lipoproteins are secreted mainly from astrocytes, but it
LCAT lecithin-cholesterol acyltransferase is known that neurons and microglia are also able to express
LDL low density lipoproteins some amounts of apolipoproteins in response to injury (Xu
LOX lipoxygenase et al. 2006). The lipoproteins secreted from glia not only trans-
LPA lysophosphatidic acid port lipids, but also transduce signals via receptors of the LDL
LPCAT acetylCoA:1-o-alkyl-2-lysophosphatidylcho- receptor family. The members of the LDL receptor family are
line acetyltransferase widely and abundantly expressed in cells of the CNS, including
LPS lipopolysaccharide neurons, astrocytes, microglia, and oligodendrocytes.
LRP low density lipoprotein receptor-related Lipid mediators are the bioactive messengers formed mostly
protein from phospholipids, sphingolipids, and cholesterol of the cell
LT leukotriene membrane. The brain is rich in long-chain polyunsaturated
MCAO middle cerebral artery occlusion fatty acids (PUFAs) such as arachidonic acid (AA) (20:4 n-6)
NF-κB nuclear factor-κB and docosahexaenoic acid (DHA) (22:6 n-3) (Wainwright
NPD1 neuroprotectin D1 2002). Hence, AA- and DHA-derived lipid mediators such
PAF platelet-activating factor as eicosanoids and docosanoids act as key endogenous regu-
PG prostaglandin lators of cell proliferation, differentiation, inflammation, and
PLA phospholipase A survival in neurons and glial cells. Because PUFAs cannot be
PS phosphatidylserine synthesized from 2-carbon fragments in mammalian cells,
PUFA polyunsaturated fatty acid the brain needs to take them up directly through the BBB.
TNF-α tumor necrosis factor-α They can be found in dietary sources such as eggs, fish, and
tPA tissue plasminogen activator meat, or can be synthesized from dietary essential fatty acid
TXA thromboxane A precursors of AA and DHA such as linoleic acid (18:2 n-6) and
VLDL very low density lipoproteins α-linolenic acid (18:3 n-3), respectively (Rapoport 2003). The

281
majority of these free fatty acids (FFAs) in the CNS are incor-
porated into phospholipids of membranes and used as struc-
tural components and signaling molecules. A small amount of
FFAs is β-oxidized, after activation to acyl-CoA by acyl-CoA
synthetases (Hamilton and Brunaldi 2007) (Fig. 23.1).
2 L I P I D H O M E O S TA S I S
The regulation of lipid homeostasis is distinct between the
inside and outside of the CNS because of the separation of
these compartments by the BBB. Although lipoproteins in
Drosophila can cross the BBB, which is functionally like that
in vertebrates (Brankatschk and Eaton 2010), lipoproteins
in the bloodstream of mouse, rat, and human do not cross
the BBB (Dietschy 2009). It was reported that the apolipo-
protein (apo) E genotype was changed to that of the donor
in the plasma of a patient who had liver transplantation,
whereas the genotype of the recipient was retained in CSF
(Linton et al. 1991). Furthermore, radiolabeled cholesterol
administrated into the circulation was not detected in the
CNS, and the rate of accumulation of sterols, such as choles-
terol and desmosterol, was similar to that of newly synthe-
sized sterols in the brain ( Jurevics and Morell 1995). Thus,
brain cells control their own lipid homeostasis, especially for
cholesterol, in the CNS.
The human brain contains approximately 25% of the total
body cholesterol, although the brain accounts for only 2% of
total body mass. It has been estimated that approximately 70% of
CNS cholesterol is present in myelin, 20% is present in astrocytes
and microglia, and the remainder is present in neurons. Quan
et al. (2003) demonstrated that the cholesterol concentration
in brains of LDL receptor-, Apo E-, or Apo A1-deficient mice
was not altered, although outside the brain these deficient mice
showed significant changes. This evidence suggests that the cho-
lesterol concentration within the brain is strictly regulated by the
brain cells. Because adult neurons produce only small amounts
of cholesterol (Nieweg et al. 2009), it is thought that glial cells
play a central role in lipid/cholesterol homeostasis in the CNS.
2.1 A S T RO C Y T E S
Astrocytes play crucial roles in maintaining lipid homeostasis
in the CNS. In a coculture system for neurons and astrocytes,
cholesterol is synthesized predominantly in astrocytes. When
astrocytes are present in the same culture environment as neu-
rons, the neurons downregulate squalene synthase, which cat-
alyzes the biosynthesis of a key cholesterol precursor, squalene
(Nieweg et al. 2009). Thus, neurons do not efficiently synthe-
size cholesterol and rely on the provision of cholesterol from
astrocyte-derived lipoproteins associated with apo E, which is
a main apolipoprotein secreted from astrocytes.

Plasma
VLDL
Albumin LDL
HDL
DHA, AA
BBB

HDL Astrocyte HDL

Neuron CNS
CoA
HDL

HDL

Figure 23.1 Model of Fatty Acid Transport into the CNS and Lipoprotein Environment Between the Plasma and CNS. PUFAs such as arachidonic acid
(AA) and docosahexaenoic acid (DHA) in the plasma disassociate from albumin and cross the BBB. The PUFAs are activated by acyl-CoA synthetase
for making membrane phospholipids. There are several types of lipoproteins such as VLDL, LDL, and HDL in the plasma, whereas only HDL-like
particles, derived mainly from astrocytes, are in the CNS. The system of lipid metabolism and transport in the CNS is different from that in the
peripheral circulation. One of the reasons is that these lipoproteins including cholesterol are not able to cross the BBB.

282 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
 Although the essential PUFAs such as AA and DHA are
highly enriched in neurons of the brain, cultured neurons
synthesize almost no DHA and do not desaturate PUFA
precursors (Moore et al. 1991). In contrast, astrocytes read-
ily synthesize AA and DHA from their precursors. This evi-
dence indicates that astrocytes elongate and desaturate PUFA
precursors, then supply the preformed PUFAs to neurons
(Moore 2001). These PUFAs are an important source for lipid
mediators in neurons and glial cells.
Lecithin-cholesterol acyltransferase (LCAT) is local-
ized on the surface of HDL and converts cholesterol and
phosphatidylcholine to cholesteryl ester and lysophosphati-
dylcholine, respectively. In the CNS, LCAT is secreted pri-
marily from astrocytes, although neurons express LCAT
mRNA at approximately threefold higher levels than astro-
cytes (Hirsch-Reinshagen et al. 2009). Apo A1 is known as
the principal activator for plasma LCAT. However, although
astrocytes do not synthesize apo A1, apo E activates LCAT
in conditioned medium of primary cultured astrocytes
(Hirsch-Reinshagen et al. 2009). This finding indicates that
apo E secreted from astrocytes is probably the principal acti-
vator of LCAT in the CSF. Cholesteryl ester transfer pro-
tein (CETP), which mediates the exchange and transfer of
cholesteryl ester and triglycerides between lipoproteins, is
present and active in the human CSF (Albers et al. 1992).
However, mice, rats, and some other species do not express
CETP, and the levels of cholesteryl ester and triglycerides are
low in the brain. CETP was detected in fibrillary astrocytes
of white matter in normal conditions, whereas reactive astro-
cytes were strongly positive in the white matter and also gray
matter of the Alzheimer diseased (AD) brain (Yamada et al.
1995). These observations suggest that astrocytes play signif-
icant roles in lipid homeostasis under physiological as well as
pathological conditions in the CNS.
2.2 O L I G O D E N D RO C Y T E S
Because this hydrophobic membrane wraps the adjacent
axons with many layers that are rich in cholesterol, the concen-
tration of unesterified cholesterol in the spinal cord reaches
40 mg/g, whereas the cholesterol concentration of hepato-
cytes is only approximately 3 mg/g in mice (Dietschy and
Turley 2004). Thus, the rate of cholesterol synthesis is high-
est during the first few weeks after birth, corresponding to
the period of active myelination. The cholesterol content of
the mouse whole brain markedly increases from 1.5 to 10.6
mg during myelinogenesis. This is also seen in the human
brain, in which cholesterol increases from approximately 360
g in the newborn to approximately 1,400 g in the adult. The
rate of cholesterol accumulation then declines dramatically
with maturation of the animals (Dietschy and Turley 2004).
Cholesterol synthesis continues in mature animals, primarily
in astrocytes, at a very low level.
Demyelination, that is, the loss of oligodendrocytes, is a
pathological process with implications for diseases causing
serious disability such as multiple sclerosis (see chapter 61).
Studies with squalene synthase conditional knock-out mice,
in which cholesterol biosynthesis is inactivated specifically in
L I P I D S , L I P I D M E D I ATO R S , A N D OT H E R S I G N A L I N G M O L E C U L E S
oligodendrocytes, revealed a severe disruption of CNS myeli-
nation as well as ataxia and tremor. The studies in these mutant
mice demonstrate that a high level of cholesterol in oligoden-
drocytes is essential for growth of the myelin membrane and
normal motor function (Saher et al. 2005).
2.3 M I C RO G L I A
The immune defense system in the CNS is maintained pri-
marily by microglia (see chapter 50). Apo E has been reported
to downregulate the activation of microglia and reduce the
secretion of inflammatory molecules such as tumor necrosis
factor-α (TNF-α), interleukin (IL)-1β and IL-6 (Lynch et al.
2001). The lack of apo E potentiates microglial activation and
worsens neuronal death in the hippocampus of kainic acid–
treated mice (Duan et al. 2006).
Phosphatidylserine (PS) is normally sequestered in the
inner leaflet of the plasma membrane of cells. In the early
stage of apoptosis, PS becomes exposed on the outer leaflet so
that the apoptotic cells are recognized and ingested by phago-
cytes. Macrophages/microglia actively limit the production
of proinflammatory cytokines after the ingestion of apoptotic
cells. Administration of PS, as PS-containing lipoproteins, can
imitate the microglial response against apoptotic cells thereby
inhibiting the secretion of TNF-α, nitric oxide and superox-
ide from activated microglia (Hashioka et al. 2007). Some
clinical trials showed that oral administration of PS improved
the behavior and cognitive functions of dementia patients
(McDaniel et al. 2003), but the efficacy of PS has not been
conclusively demonstrated.
3 L I P O P R OT E I N S A N D
T H E I R R E C E P TO R S
Because the only lipoproteins in the CNS are HDL-like par-
ticles, their functions and regulatory mechanisms must differ
from those in the peripheral circulation and tissues. In addi-
tion, the composition of apolipoproteins in the CNS is similar
to, but not the same as, that in plasma. The CSF contains:apo
AI, apo AII, apo CI, apo CII, apo CIII, apo D, apo E, and apo
J, but not apo B (Ladu et al. 2000). Primary cultures of astro-
cytes secrete apo E, apo J, and apo D, but not apo AI (LaDu
et al. 1998). Thus, although apo A1 is the most abundant apo-
lipoprotein in plasma, apo E is the major apolipoprotein con-
stituent of the CSF. Members of the LDL receptor family are
abundantly expressed in neurons and glial cells of the CNS.
In recent years, extensive research has demonstrated that these
lipoprotein receptors act not only as endocytotic receptors
for lipoproteins, but also act as functionally diverse signaling
receptors in the CNS.
3.1 L I P O P ROT E I N F O R M AT I O N BY
A D E N O S I N E T R I P H O S P H AT E –B I N D I N G
C A S S ET T E T R A NS P O RT E R S
Adenosine triphosphate–binding cassette (ABC) transport-
ers are members of a protein superfamily that use ATP to
• 283
 apoE-LP
apoA1
OH
OH
chol PC
OH
OH
ABCA1
Figure 23.2 A Model of Lipid Transport by ABC Transporters. ABCA1 mediates the ATP-dependent efflux of cholesterol (chol) and phosphatidyl-
choline (PC) to lipid-poor apo A1 and apo E-containing lipoproteins. ABCG1 preferentially mediates the efflux of chol and sphingomyelin (SM) to
HDL and apo E-containing lipoproteins. ABCG4, expressed in the eye and brain, mediates the efflux of chol to HDL.
translocate solutes across membranes. An important function
of ABC transporters in the CNS is to mediate the efflux of cel-
lular cholesterol and phospholipids for the formation of apo
E-containing lipoproteins from astrocytes. Approximately
50 ABC transporters have been identified in humans
(Kimura et al. 2007). ABCA1 and ABCG1 are widely
expressed in multiple tissues, whereas ABCG4 is expressed
primarily in the CNS (Fig. 23.2). Because neurons do not
efficiently produce cholesterol and apo E (Nieweg et al.
2009; Xu et al. 2006), these ABC transporters mainly func-
tion in astrocytes.
In mice, the deficiency of ABCA1 demonstrated a remark-
able reduction of the cholesterol concentration, as well as the
apo E level, in the CSF and the brain, whereas the amount
of apo J was not altered (Hirsch-Reinshagen et al. 2004).
Furthermore, brain-specific ABCA1 deficiency not only
decreased cholesterol and apo E in the CSF and the brain, but
also resulted in abnormal motor activity and synaptic struc-
tures (Karasinska et al. 2009). These findings suggest that
ABCA1 is required for the formation of apo E-, but not apo
J- containing lipoproteins in the CNS, and that ABCA1 can
alter the functions of the CNS.
Although a deficiency of either ABCG1 or ABCG4
in mice resulted in essentially normal brain sterol levels.
A deficiency of both ABCG1 and ABCG4 significantly
increased brain sterol intermediates such as desmosterol,
lanosterol, and lathosterol. The brain and primary astrocytes
from Abcg1–/–/Abcg4–/– mice showed a marked increase in
ABCA1. In addition, the deficiency of both ABCG1 and
ABCG4 increased the secretion of apo E from primary
astrocytes (Wang et al. 2008). These findings indicate that
ABCG1 and ABCG4 have some overlapping functions in
sterol efflux from astrocytes, and that ABC transporters in
astrocytes tightly cooperate with each other to maintain the
CNS sterol environment.
284 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
apoE-LP
HDL
OH
chol SM
ABCG1/ABCG4
3.2 A P O E - C O N TA I N I N G L I P O P ROT E I N S
A N D T H E I R R EC E P TO R S
3.2.1 Functions of Apo E-Containing Lipoproteins
Apo E is recognized as the quantitatively and functionally
major apolipoprotein in the CNS and is a component of apo
E-containing lipoproteins secreted mainly from astrocytes
(LaDu et al. 1998). In addition to previous studies, transgenic
knock-in mice that expressed green fluorescent protein regu-
lated by the endogenous apo E promoter demonstrated that
the main source of apo E in the CNS is astrocytes, although
neurons and microglia expressed minor amounts of apo E in
response to nerve injury (Xu et al. 2006).
The expression and the secretion of apo E from glial cells
are markedly upregulated in response to nerve injury (Ignatius
et al. 1986). Studies with apo E-deficient mice revealed signif-
icant neurodegeneration in the brain during aging (Masliah
et al. 1995), deficits of learning and memory (Gordon et al.
1996), and enhanced susceptibility of hippocampal neurons
to endoplasmic reticulum stress caused by transient ischemia
(Osada et al. 2009). Furthermore, apo E-containing lipopro-
teins promote axon extension (Hayashi et al. 2004) and also
protect CNS neurons from apoptosis in a receptor-mediated
manner (Hayashi et al. 2007). Thus, it is thought that
astrocyte-derived apo E-containing lipoproteins have impor-
tant roles in the protection and/or repair of neurons.
The major role of apo E-containing lipoproteins secreted
from astrocytes is thought to be the supply of lipids to neurons
in the brain. It has been reported that cholesterol associated
with apo E-containing lipoproteins stimulates synaptogenesis
in CNS neurons (Mauch et al. 2001) (see chapter 31) and also
alters synaptic plasticity and neurotransmission in the CNS
(Pfrieger 2003). Apo E-containing lipoproteins are taken up
by members of the LDL receptor family and astrocyte-de-
rived cholesterol can be used as a component of membrane
structures, a source for neurosteroids and lipid mediators in
neurons. Excess cholesterol in the CNS is converted to 24S-
hydroxycholesterol, a cholesterol-derived lipid mediator, by
cholesterol 24-hydroxylase. 24-Hydroxlase is expressed only
in certain types of neurons, such as pyramidal neurons of the
hippocampus and cerebral cortex and Purkinje neurons of
the cerebellum (Ramirez et al. 2008). This oxysterol can cross
the neuron membrane and the BBB and diffuse into the plasma
for delivery to the liver and subsequent elimination from the
body in bile. 24S-Hydroxycholesterol can also diffuse to astro-
cytes and act as a ligand for liver X receptors. These receptors
are the members of the nuclear hormone receptor superfamily
and regulate the expression of ABCA1, ABCG1 and apo E
(Fig. 23.3) (Abildayeva et al. 2006). Therefore, it is thought
that astrocytes contribute to brain functions by regulating
cholesterol transport and metabolism in the CNS.
3.2.2 Roles of Apo E Receptors
Receptors for apo E are members of the LDL receptor fam-
ily, which are expressed widely and abundantly in the CNS.
Apo E-containing lipoproteins can bind to all members of this
receptor family, which consists of seven core members—the
LDL receptor, the VLDL receptor, the LDL receptor–related
protein 1 (LRP1), LRP1B, megalin/LRP2, LRP4/MEGF7,
and apoER2/LRP8, as well as three distant members—
LRP5, LRP6, and SorLA/LR11 (Bu, 2009) (Fig. 23.4). In the
CNS, the VLDL receptor and apoER2/LRP8 play impor-
tant roles in neuron migration during development and also
modulate synaptic plasticity and neurite outgrowth via the
Figure 23.3 Cholesterol Homeostasis in Neurons and Astrocytes. Since
neurons synthesize cholesterol (Chol) at low level in the adult brain,
astrocytes provide cholesterol associated with apo E-containing lipo-
proteins (Apo E-LP) in processes mediated by ABCA1 and ABCG1
in astrocytes. Apo E-LP bind to receptors of the LDL receptor family
(LDLRs) in neurons, are endocytosed and used for cell structure and
functions. Any excess cholesterol is converted to 24S-hydroxycholesterol
(24-OH chol), which can diffuse from neurons, cross the BBB, and go
into the bloodstream. This oxysterol also diffuses to astrocytes, stimu-
lates liver X receptor (LXR), and enhances the expression of apo E and
ABCA1, thereby promoting cholesterol efflux.
L I P I D S , L I P I D M E D I ATO R S , A N D OT H E R S I G N A L I N G M O L E C U L E S
Ligand binding repeat
β- propeller domain
EGF repeat
O-linked sugar domain
NPxY motif
LDLR VLDLR apoER2 LRP4 LRP1 LRP1B Megalin
/LRP8 /MEGF7 /LRP2
Figure 23.4 The Structural Organization of Seven Core Members of the
LDL Receptor Family. All members have common characteristic motifs
such as ligand binding repeats, epidermal growth factor (EGF) repeats, and
β-propeller domains, and also contain NPxY motif(s) in the cytoplasmic tail.
N-methyl-d-aspartate receptor (Herz et al. 2009). In addition,
the LDL receptor and LRP1 are the major receptors for lipid
metabolism in the CNS (Bu 2009). This is because knock-
out of the LDL receptor and conditional knock-out of the
forebrain neuron–specific LRP1 in mice increases the level of
apo E in the brain (Fryer et al. 2005; Liu et al. 2007). The
latter conditional knock-out mice, but not the LDL receptor
knock-out mice, also showed a decrease in brain cholesterol. In
addition, apo E-containing lipoproteins secreted from astro-
cytes have a higher affinity for the LDL receptor compared
with LRP1 (Fryer et al. 2005). Whereas HDL-like particles
isolated from the CSF prefer to bind to LRP1 than the LDL
receptor (Bu 2009; Fagan et al. 1996). Thus, the LDL recep-
tor and LRP1 might have different roles in lipid metabolism
in the brain.
The multifunctional receptor LRP1 (also known as the
α2-macroglobulin receptor and CD91) is expressed abun-
dantly in neurons, astrocytes, oligodendrocytes, and microglia
(Gaultier et al. 2009). At least 40 different ligands for LRP1
have been identified, including apo E, α2-macroglobulin, tis-
sue plasminogen activator (tPA), amyloid β peptide (Aβ),
and amyloid precursor protein. LRP1 acts as an endocytotic
receptor for apo E-containing lipoproteins and other ligands
and also functions as a transmembrane signaling receptor.
In Schwann cells, LRP1 activates the major anti-apoptotic
enzyme, phosphatidylinositol 3-kinase, and antagonizes the
unfolded protein response-activated proapoptotic signaling
• 285
pathway after nerve injury (Mantuano et al. 2011). LRP1 inter-
acts with myelin basic protein and contributes to phagocyto-
sis of degraded myelin in astrocytes and microglia (Gaultier
et al. 2009). Ischemic brain injury increases the expression of
LRP1 in astrocytes and induces an interaction between LRP1
and tPA followed by activation of nuclear factor-κB (NF-κB)
(Zhang et al. 2007). In primary cultured microglia, the acti-
vation of LRP1 by apo E suppressed c-jun N-terminal kinase
activation induced by lipopolysaccharides (LPS) (Pocivavsek
et al. 2009), and the interaction of microglial LRP1 and tPA
increased matrix metalloproteinase-9 expression and activity,
which was associated with the development of brain edema,
after middle cerebral artery occlusion (MCAO) in mice
(Zhang et al. 2009). These data suggest that LRP1 not only
contributes to lipid metabolism, but also plays a crucial role in
inflammation, survival, and other signaling events in glia.
3.3 A P O J- C O N TA I N I N G L I P O P ROT E I N S
A N D T H E I R R E C E P TO R S
3.3.1 Functions of Apo J-Containing Lipoproteins
Apo J (also known as clusterin) is a heterodimeric, sulfated gly-
coprotein that is expressed at high levels in the brain, ovary, tes-
tis, and liver (de Silva et al. 1990). Similar to apo E, apo J exists in
the form of lipoproteins that are secreted from astrocytes in the
CNS. The levels of apo J mRNA and protein are also increased
in astrocytes after nerve injury. Cultured astrocytes isolated
from apo E-deficient mice secrete lipoprotein particles that are
lipid-poor compared with the particles secreted in wild-type
mice. However, the lipoprotein particles express comparable
amounts of apo J. Moreover, primary cultured astrocytes from
apo J-deficient mice secrete lipoproteins similar to those from
wild-type mice (Ladu et al. 2000). Thus, apo J and apo E are
associated with distinct lipoproteins in the CSF.
The deficiency of apo J does not remarkably alter the mor-
phology of the CNS during development, but worsens the
susceptibility to ischemic brain injury (Imhof et al. 2006).
Cytosolic apo J can bind to Bax, a pro-apoptotic protein, and
inhibit cytochrome c release from mitochondria and the sub-
sequent activation of a caspase cascade (Zhang et al. 2005).
These findings indicate that apo J might be a neuroprotective
molecule. However, it is also reported that the lack of apo J
attenuated neurodegeneration after ischemic injury in vitro
and in vivo (Han et al. 2001). Furthermore, the translocation
of truncated apo J to the nucleus induces cell death (Yang
et al. 2000). Apo J also stimulates the proliferation of primary
cultured astrocytes through an extracellular signal-regulated
kinase signaling pathway. Moreover, astrocyte-derived apo J
promotes neuronal differentiation in human neuronal precur-
sor cells (Cordero-Llana et al. 2011). Thus, apo J may perform
diverse roles in the CNS but further research is required to
establish these functions.
3.3.2 Roles of Apo J Receptor
Apo J interacts with megalin (also known as LRP2), which is a
multiligand endocytic cell-surface receptor that is expressed in
286 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
oligodendrocytes and astrocytes. Megalin in astrocytes mediates
the uptake of albumin, which stimulates the synthesis of oleic
acid; oleic acid can act as a neurotrophic factor (Bento-Abreu
et al. 2008). The expression of metallothionein, which is released
from astrocytes and has been shown to be a neuroprotective met-
al-binding protein, is markedly increased in reactive astrocytes
of sporadic Parkinson diseased brains, and megalin expression is
also upregulated (Michael et al. 2011). Because metallothionein
is a ligand of megalin, this finding indicates that megalin might
have a neuroprotective role in the brain.
Apo J directly binds to Aβ and to apo E, which is also a
ligand for megalin. Because megalin is expressed in the epithe-
lium and ependymal cells at the choroid plexus, the apo J- or
apo E-Aβ complex is thought to be removed from the brain
into plasma by megalin and other receptors such as LRP1
(Nuutinen et al. 2009). Although the functions of megalin as
an apo J receptor on glial cells and in the adult CNS is still not
clearly defined, this receptor seems to be an important media-
tor between the CNS and the peripheral circulation.
3.4 A P O A1- C O N TA I N I N G L I P O P ROT E I N S
Apo A1 is the most abundant apolipoprotein in plasma, but
in the CSF the concentration of apo A1 is only 0.5% of that in
plasma (Pitas et al. 1987). Exogenously administrated apo A1
can induce the translocation of newly synthesized cholesterol
and phospholipids to the lipid-protein particles and gener-
ate HDL-like particles from primary cultured astrocytes (Ito
et al. 2004). This result indicates that apo A1 is functional in
the CNS, although this apolipoprotein is not made within the
CNS. Overexpression of human apo A1 in a mouse model of
AD suppressed the activation of astrocytes and microglia and
improved spatial learning and memory functions in vivo. In
addition, exogenous administration of human apo A1 attenu-
ated the activation of cultured microglia and the secretion of
proinflammatory cytokines induced by Aβ from hippocampal
slice cultures (Lewis et al. 2010). It has also been reported that
the concentration of serum apoA1 is inversely associated with
the risk of AD (Saczynski et al. 2007). Moreover, the level of
apo A1 is reduced in the CSF, the brain and peripheral tissues
of subjects with schizophrenia (Huang et al. 2008). Thus, apo
A1 might play important roles in brain functions. However,
more investigations are required.
4 L I P I D M E D I ATO R S A N D
T H E I R R E C E P TO R S
Lipid mediators are bioactive regulators that are enzymatically
formed from membrane glycerophospholipids, sphingolipids,
and cholesterol in response to cell stimulation or injury. Lipid
mediators are involved in inter-cell and intra-cell communi-
cations and responses such as proliferation, differentiation,
adhesion, migration, oxidative stress, inflammation, and cell
survival. Phospholipases A2 (PLA2s) belong to a superfam-
ily of lipolytic enzymes that catalyze the hydrolysis of fatty
acids from the sn-2 position of glycerophospholipids to
produce lysophospholipids and FFAs such as AA and DHA
(Fig. 23.5). More than 30 different types of PLA2s have been
found in mammalian cells and have been classified into five
major groups: cytosolic PLA2, Ca2+-independent PLA2, lyso-
somal PLA2, secretory PLA2, and platelet-activating factor
(PAF) acetyl hydrolases (Schaloske and Dennis 2006). These
PLA2s are not only important for regulating the levels of cell
membrane phospholipids, but they also play crucial roles
in regulating the production of a variety of lipid mediators,
which have been implicated in diverse events of physiological
and pathological states (Liu and Xu 2010).
The enzyme 5-LOX converts AA into 5-hydroxyeicosatetr
aenoic acid and leukotriene A4 (LTA4). In humans, the expres-
sion of 5-LOX was upregulated in astrocytes at damaged foci
after ischemic injury, whereas COX-2 was increased in neurons
in the peri-infarct area (Tomimoto et al. 2002). In a rat model
of focal cerebral ischemia, a 5-LOX inhibitor reduced infarct
volume and dampened the increase in 5-LOX expression in
neurons and microglia after cerebral ischemia. Furthermore,
the 5-LOX inhibitor also blocked LPS-mediated activation of
NF-κB in primary cultured microglia ( Jatana et al. 2006).

4 .1 A R AC H I D O N I C AC I D –D E R I V E D 4.1.1 Functions of Prostaglandins and

E I C O S A N O I D S A N D T H E I R R E C E P TO R S
Eicosanoids are generated from AA by cyclooxygenases
(COXs), lipoxygenases (LOXs), and epoxygenases. The two
COXs (COX-1 and COX-2) are rate-limiting enzymes in the
conversion of AA to prostaglandins (PGs), such as PGD2,
PGE2, PGF2α, and PGI2, and thromboxane A2 (TXA2). Both
COX-1 and COX-2 are expressed in the brain. COX-1 is gen-
erally considered to be constitutively expressed in most tissues
and mainly responsible for homeostatic states. In contrast,
COX-2 is primarily induced in response to inflammatory stim-
uli and mitogens in numerous pathological states (Milatovic
et al. 2011). However, in the CNS, COX-2 is also constitu-
tively expressed in cortical and hippocampal glutamatergic
neurons, which are important for learning and memory (Yang
et al. 2008). COX-1 is predominantly localized to microglia
and perivascular cells, and the expression level of COX-1 is
robustly stimulated by inflammatory stimuli (Garcia-Bueno
et al. 2009). Moreover, COX-1-positive microglia were found
surrounding amyloid plaques in the AD brain (Yermakova
et al. 1999). These findings indicate that COX-1 in microglia
might play important roles in inflammation, whereas COX-2
might contribute to neurotransmission and synaptic plasticity
in the CNS.
Their Receptors
In the CNS, activated microglia and reactive astrocytes
secrete large amounts of proinflammatory molecules includ-
ing cytokines, chemokines and PGs in response to neuroin-
flammation, which is a diverse response to brain injury. PGs
and TXA2 are critical lipid mediators of neuroinflammation
induced by traumatic brain injury, stroke, and AD (Hein and
O’Banion 2009). The receptors for PGD2, PGE2, PGF2α,
PGI2, and TXA2 are named DP, EP, FP, IP, and TP, respec-
tively. Among these receptors, the EPs (EP1–4) are the most
well-characterized. PGE2 plays major physiological and path-
ological roles in the brain (Miller 2006). EPs are expressed
in almost all organs at various levels, and the brain expresses
all of the EPs. Astrocytes express EP2 and EP4, whereas
microglia have EP1 and EP2. EP3 is inducibly expressed in
astrocytes and microglia. Neurons also express EP1, EP2,
and EP3 in multiple areas of the brain, whereas EP4 localizes
in hypothalamic nuclei (Cimino et al. 2008). It is reported
that exogenous PGE2 administration to primary cultured
hippocampal neurons induced caspase-dependent apop-
tosis (Takadera et al. 2004). In primary cultured microglia
isolated from EP2-deficient mice, LPS-mediated paracrine
neurotoxicity was reduced. The presence of EP2 in microglia

Phospholipids

PLA 2s

Docosahexaenoic acid Arachidonic acid Lysophospholipids

15-LOX LPCAT ATX

Neuroprotectins Resolvins Platelet activating factor Lysophosphatidic acid

COX 5-LOX

Thromboxanes Prostaglandins Leukotrienes

Figure 23.5 Pathways for Synthesis of Lipid Mediator. Phospholipids in cell membranes are hydrolyzed by phospholipases A2 (PLA2s) to liberate
PUFAs, such as arachidonic acid and docosahexaenoic acid, and lysophospholipids. Lysophospholipids are further converted to platelet activating
factor by acetyl CoA:1-O-alkyl-2-lysophosphatidylcholine acetyltransferase (LPCAT), or lysophosphatidic acid by autotaxin (ATX). Arachidonic
acid is converted to prostaglandins and thromboxanes, and leukotrienes by cyclooxygenase (COX) and 5-lipoxygenase (5-LOX), respectively.
Docosahexaenoic acid is converted to resolvins and neuroprotectins by 15-lipoxygenase (15-LOX).

L I P I D S , L I P I D M E D I ATO R S , A N D OT H E R S I G N A L I N G M O L E C U L E S • 287
was shown to be necessary to induce COX-2 and inducible
nitric oxide synthase for this neurotoxicity (Shie et al. 2005).
In primary cultured cortical astrocytes, exogenous LPS treat-
ment upregulated the expression of various enzymes involved
in PG synthesis, such as cPLA2, COX-2, and PGE synthase,
whereas downregulating the expression of PGE-degrading
enzymes ( Johann et al. 2008). Thus, astrocytes also partici-
pate in the innate immune response by providing PGE2 in the
CNS. Moreover, the level of PGE2 in the CSF was increased
in patients with neurological disorders such as AD, amyo-
trophic lateral sclerosis, Creutzfeldt-Jakob disease, ischemic
stroke, and human immunodeficiency virus (HIV)–associated
dementia (Cimino et al. 2008).
PGD2 is the most abundant prostanoid in the mamma-
lian brain (Narumiya et al. 1982) and plays a variety of roles
in the immune system. Studies with the demyelinating mouse
twitcher, a model of human Krabbe disease, demonstrated
that activated microglia express hematopoietic prostaglandin
D synthase (HPGDS), and that reactive astrocytes expressed
DPs for PGD2. Deficiency of HPGDS or DP in this disease
model markedly suppressed astrogliosis and demyelination
(Mohri et al. 2006). These findings indicate that PGD2 is the
key factor for promoting these neuroinflammatory responses
in the brain.
4.1.2 Functions of Leukotrienes and
Their Receptors
Leukotrienes, a family of eicosanoids, are made from AA by
5-LOX. An integral membrane protein, 5-LOX-activating
protein (FLAP), is required for this conversion. FLAP selec-
tively transfers AA to 5-LOX and enhances the production
of LTA4. LTA4 is further converted into LTB4 or cysteinyl
leukotrienes, LTC4, LTD4, and LTE4. These leukotrienes are
proinflammatory lipid mediators. Their G protein–coupled
receptors (GPCRs) have been identified; BLT1 and BLT2
are receptors for LTB4, and CysLT1, CysLT2, and CysLT3 are
receptors for LTC4, LTD4, and LTE4. It is reported that the
production of cysteinyl leukotrienes was increased in the rat
brain after MCAO. Treatment of these animals with a 5-LOX
inhibitor decreased the levels of cysteinyl leukotrienes and
the size of infarct area (Ciceri et al. 2001). Furthermore, the
expression of CysLT1 was upregulated in neurons and microg-
lia of the ischemic core and reactive astrocytes of the bound-
ary zone after MCAO in rats. Moreover, a CysLT1 antagonist
reduced infarct area, neurological deficits, and astrocyte pro-
liferation (Fang et al. 2006). These findings demonstrated that
cysteinyl leukotrienes and CysLT1 in microglia and astrocytes
play significant roles in causing neuronal damage after cerebral
ischemia.
4.2 LY S O P H O S P H O L I P I D, LYS O P H O S P H O L I P I D
D E R I VAT I VE S , A N D T H E I R R E C E P TO R S
Lysophosphatidic acid (LPA) is a metabolite of glycero-
phospholipids. Several potential pathways produce LPA in
cells. One example is the production of LPA extracellularly
by the action of a lysophospholipase D called autotaxin
288 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
(ATX) (Savaskan et al. 2007). ATX has been detected in
oligodendrocytes, choroid plexus epithelial cells, and lep-
tomeningeal cells under normal conditions. However, neu-
rotrauma led to a significant increase in the level of ATX
specifically in reactive astrocytes (Savaskan et al. 2007). To
date, GPCRs for LPA have been classified into five sub-
types: LPA1–5 (Tigyi 2010). It is possible that two or four
more receptors will be recognized as LPA receptors. In the
nervous system, LPA1 was originally identified in neuronal
progenitor cells of the ventricular zone and subsequently
found in oligodendrocytes. Low expression of LPA2–5
has been detected in the brain by Northern blot analy-
sis. Exogenous administration of LPA increases the area
of oligodendrocyte’s process network and the expression
of myelin basic protein during oligodendrocyte matura-
tion (Nogaroli et al. 2009). The expression of LPA recep-
tors appears to be low in a normal human brain, whereas
LPA is increased on reactive astrocytes following traumatic
brain injury (Frugier et al. 2011). These findings indicate
that LPA and ATX may play important roles in the brain
under normal and pathological conditions, particularly in
oligodendrocytes.
4.3 D O C O S A H E X A E N O I C AC I D A N D
IT S M ETA B O L IT E S
DHA (22:6 n-3) is one of the most abundant PUFAs in
the brain; another is AA. In the adult rat, 2% to 8% of
esterified brain DHA and 3% to 5% of esterified brain AA
are replaced daily by plasma unesterified PUFAs. In the
human brain, 0.3% of AA is replaced daily (Rapoport
et al. 2001). DHA and its metabolites are antiinflam-
matory, wheres AA and its metabolites are generally
proinflammatory. In vitro cell culture studies showed
that astrocytes readily produce DHA from their precur-
sors, whereas neurons synthesize little DHA and have no
desaturation activity. Thus, neurons rely on the uptake
of preformed DHA from external sources in the CNS
(Moore, 2001). DHA is converted to the resolvins and
the neuroprotectins by 15-LOX. Resolvins downregulate
TNF-α–stimulated IL-1β expression in human glial
cells (Hong et al. 2003). DHA stimulates neuroprotec-
tin D1 (NPD1) biosynthesis and attenuates the secretion
of Aβ, which can cause neuronal apoptosis. NPD1 also
upregulates the antiapoptotic proteins, Bcl-2 and Bcl-xL,
and downregulates the proapoptotic proteins, Bax and Bad,
during cerebral ischemia (Bazan 2005). These findings dem-
onstrate that DHA and its metabolites—the resolvins and
NPD1—facilitate neuroprotection by inducing the expres-
sion of neuroprotective molecules and reducing the secretion
of neurotoxic molecules.
4.4 OT H E R L I P I D M E D I ATO R S A N D
T H E I R R EC E P TO R S
Platelet-activating factor (PAF), 1-O-alkyl-2-acetyl-sn-
glycero-3-phosphorylcholine, is a potent proinflammatory
phospholipid mediator in infectious and inflammatory
diseases. PAF is generated from lysophospholipids by ace-
tyl CoA: 1-o-alkyl-2-lysophospatidylcholine acetyltrans-
ferase. PAF is released from many cell types, including
neurons and glia. In the CNS, sustained PAF activation
has been observed in ischemia, encephalitis, meningitis,
and HIV infection. PAF synthesis and secretion in cul-
tured neurons were upregulated by glutamate treatment,
and cultured microglia showed a remarkable chemotactic
response to PAF. Furthermore, this chemotaxis was not
found in microglia isolated from PAF receptor–deficient
mice (Aihara et al. 2000). PAF receptors from various spe-
cies possess a structure typical of GPCRs with seven trans-
membrane helices. PAF receptors are expressed primarily in
neurons and microglia under normal conditions. However,
the expression of the PAF receptor is markedly upregulated
in activated microglia and reactive astrocytes for targeting
apoptotic debris after kainic acid treatment (Bennett et al.
1998). Thus, PAF receptors potentially mediate the cere-
bral phagocytic response by activated glia.
5 S U M M A RY A N D P E R S P E C T I VE S
Lipid homeostasis in the CNS is tightly regulated under
physiological conditions by a partnership between neurons
and glia, especially astrocytes. In recent years, critical roles
have been elucidated for many players associated with lipid
metabolism in the CNS, such as ABC transporters and mem-
bers of the LDL receptor family. However, many specific func-
tions of these molecules in neurons and each type of glia under
physiological and pathological conditions have not yet been
defined.
Dietary PUFAs, such as AA and DHA, are important
sources for structural membrane components and lipid
mediators, as discussed. In many cases, pathological stimuli
recruit activated microglia, reactive astrocytes, and inflamma-
tory response; including the secretion of lipid mediators, and
induction of neuroprotective and/or neurotoxic responses by
lipid mediators dependent on how stimuli are produced. To
define these responses in glia, further research is required.
AC K N OW L E D G M E N T S
The author would like to thank Jean E. Vance for critical
reviewing of the manuscript during its preparation and Mie
Moriyama for help in preparing the figures. This work was
supported in part by a grant-in-aid for Young Scientists B
(No. 22790254) and the Special Coordination Funds for
Promoting Science and Technology from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
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• 291
 24.
GAP JUNCTIONS AND HEMICHANNELS
Bruce R. Ransom and Christian Giaume

A B B R E VI AT I O N S one another, leaving a narrow “gap” of only about 2.5 nm.

Each HC is composed of six Cxs (Goodenough 1976) and a
A astrocyte GJC is formed when a HC in one cell aligns with another HC
A:O astrocyte and oligodendrocyte coupling in an adjacent cell (Fig. 24.1). A gap junction is an aggregate
AMPA D-amino-3-hydroxy-5-methylisoxazole- of many, often hundreds, of tightly packed GJCs. Unlike Cxs,
4-propionic acid Panxs (homologs to innexin proteins in invertebrates) form
ATP adenosine-triphosphate stable membrane channels but not GJCs.
FGF fibroblast growth factor Each HC consists of 24 “rods” that correspond to the
CBX Carbenoxolone transmembrane spanning, alpha-helical portions of the six
GFAP glial fibrillary acid protein constituent Cxs (see Fig. 24.1). The extracellular loops of
GFP green fluorescent protein two HCs mesh to form an extracellular docking area con-
GJC gap junction channel sistent with a structural “tight seal” guaranteeing that the
HC hemichannel extracellular portion of the channel will not leak. The limit-
O oligodendrocyte ing diameter of the central pore is about 1.5 nm, setting the
O:O oligodendrocyte and oligodendrocyte upper limit for permeable ions and small molecules at about
coupling 1 to 1.2 kDa (Harris 2007).
Panx pannexin

1 INTRODUCTION
Gap junctions are membrane specializations consisting of dense
aggregates of large pore channels that extend from one cell into
an adjacent cell and mediate direct cytoplasm-to-cytoplasm
communication. Gap junction channels (GJCs) in vertebrates
are formed by a family of proteins, called connexins (Cxs).
These channels are poorly selective for ions and small molec-
ular weight signaling molecules, and allow extensive ionic and
biochemical exchanges between cells. In the mammalian brain,
glia express Cxs most abundantly and in the greatest variety.
Under certain conditions Cxs can also operate as “half ” a GJC,
or a hemichannel (HC), that represents another functional
state providing a pathway suitable for autocrine as well as para-
crine interactions. Membrane channels may also be formed by
proteins called pannexins (Panxs) that have some properties
that are similar to Cx HCs. The characteristics and functions
of these channels in glial cells are the focus of this chapter.
2 G E N E R A L F E AT U R E S O F C O N N E X I N /
PA N N E X I N C H A N N E L S
2.1 G A P J U N C T I O N A N D H E M I C H A N N E L
S T RU C T U R E
In electron micrographs, gap junctions appear as discrete areas
where the cell membranes of adjacent cells closely approach
2.2 C O N N E X I NS A N D PA N N E X I NS
Connexins are a family of proteins with 20 and 21 members in
rodents and humans, respectively. In the brain, at least 11 dif-
ferent Cxs have been detected, including: Cx26, Cx29, Cx30,
Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx46, and Cx47 (Sohl
and Willecke 2004; Theis et al. 2005). Connexins, named on
the basis of their molecular weight, are transmembrane pro-
teins with four transmembrane domains, two extracellular
loops, and two cytoplasmic tails (see Fig. 24.1). Within the Cx
protein family, the most conserved parts are the NH2 terminal
and the two extracellular loops. Differences in Cx molecular
weights are mainly reflected in the length of the C-terminal
end. The functional gating of GJCs is primarily mediated
by the cytoplasmic loop and the C-terminal segment. The
extracellular loops mediate the end-to-end docking of paired
HCs, and this union is stabilized by hydrophobic interactions
involving conserved cysteine residues (Harris 2001). All types
of glia express more than one type of Cx and HCs of differ-
ent protein composition may form functional GJCs with one
another (Table 24.1). The six Cxs forming a HC can be identi-
cal, forming a homomeric HC, or consist of more than one Cx
isoform, forming a heteromeric HC. A GJC formed from two
different homomeric or heteromeric HCs is called heterotypic.
Astrocytes and oligodendrocytes necessarily form heterotypic
GJCs because they express different Cxs (astrocytes—Cx43
and Cx30; oligodendrocytes—Cx29, Cx32, and Cx47) (Nagy
et al. 2003). Not all heterotypic “pairings” lead to functional
GJCs (Bukauskas and Verselis 2004). For example, Cx32

292
 Pannexin
Pannexin channel
Extracellular
E1 E2
Cell 1
M1 M2 M3 M4

NH2
Intracellular CL
COOH

Gap junction plaque

Intracellular 1

Extracellular

Intracellular 2

Connexin hemichannel
Connexin
Extracellular
E1 E2

M1 M2 M3 M4
Cell 2
NH2 CL
Intracellular COOH

Figure 24.1 Gap Junction and Hemichannel Molecular Organization. Diagrammatic cells show how Cx GJCs form a gap junction plaque at a point
of close contact. Each GJC is formed by two Cx HCs docked together and rotated 30° with respect to one another. The center diagrams show the
topology of a Panx channel (top, Panx channels do not form GJCs; see text) and a Cx HC (bottom) in the plasma membrane. Both have four trans-
membrane domains (M1–4) with amino (NH2) and carboxy (COOH) termini on the cytoplasmic side, two extracellular loops (E1 and E2), and one
cytoplasmic loop (CL). Modified from Orellana et al 2009.

Table 24.1 GAP JUNCTION COUPLING AND CONNEXIN EXPRESSION IN MAMMALIAN GLIAL CELLS
GLIAL CELL PAIR RELATIVE COUPLING STRENGTH CONNEXIN PAIRING

Astrocytes ++++ (Sontheimer et al. 1991) Cx43-Cx43, CX43-Cx30, Cx30-Cx30, Cx30-Cx26, Cx26-Cx26
(Dermietzel et al. 1991; Giaume et al. 1991) (Dermietzel et al. 1991; Nagy et al. 1999; see also Giaume and Theis
2010; Giaume et al. 2010; Rash 2010; Theis et al. 2005)
Small amount of Cx40, Cx45, Cx46 (Dermietzel et al. 2000)

Astro-Oligo + (Ransom and Kettenmann 1990) Cx43-Cx47, Cx30-Cx32, CX26-Cx32 (Maglione et al. 2010; Nagy
et al. 2003)

Oligodendrocytes ++ (Von Blankenfeld et al. 1993) Cx32-Cx32 (Bergoffen et al. 1993),

(Butt and Ransom 1989; Kettenmann and Cx29-Cx29 (Altevogt et al. 2002; but see Ahn et al. 2008)
Ransom 1988) Cx47 (Maglione et al. 2010)

Schwann cells ++ Cx32-Cx32 (Altevogt et al. 2002)

Microglia + (Upregulated by cytokines) Cx43-Cx43 (Eugenin et al. 2001)

Muller cells ++ (Ceelen et al. 2001) Cx43-Cx43, Cx43-Cx45, Cx45-Cx45 (Zahs et al. 2003)
(Dermietzel et al. 2000)

Muller cell-astrocyte ++ (Ceelen et al. 2001) Cx43-Cx43, Cx43-Cx45, Cx45-Cx45

Ependymal cells +++ Cx43-Cx43 (Ochalski et al. 1997)

Astrocyte-neuron + Cx43-Cx36 (Alvarez-Maubecin et al. 2000; Froes et al. 1999; but see
Rash et al. 2001)

GAP JUNCTIONS AND HEMICHANNELS • 293

HCs form functional GJCs with Cx30 and Cx26 HCs, but
not with Cx43 HCs (Elfgang et al. 1995). Coupling between
different HCs is primarily determined by the compatibility of
Cx extracellular loops (Harris 2001).
Another class of membrane proteins, the Panxs, forms
membrane channels in mammalian brain with some similarity
to Cx channels (Phelan and Starich 2001). Pannexin channels
are expressed primarily in neurons (Sosinsky et al. 2011), but
glia also express them (Bruzzone et al. 2003). Pannexin chan-
nels have functional properties similar to Cx HCs, and three
Panx isoforms (1–3) have been found in mouse and human;
Panx1 and Panx2 have been detected in the brain (MacVicar
and Thompson 2010). The structural topology of Panxs and
Cxs is similar (see Fig. 24.1), but there is only 16% overall
amino acid sequence identity (Panchin et al. 2000).
2.3 B I O P H YS I C A L P RO P E RT I E S O F
G A P JU N C T I O N C H A N N E L S
The permeability and biophysical properties of GJCs have been
studied using electrophysiological and imaging techniques
(Giaume et al. 2012). The extent of GJC-coupling among cells
can be detected using low molecular weight fluorescent dyes
injected into single cells. The electrophysiological profile of a
given GJC (i.e., unitary conductance and voltage-dependent
properties) can indicate which protein(s) form the channels of
interest. Using such techniques, GJCs formed by Cx43 were
identified between pairs of astrocytes (Dermietzel et al. 1991;
Giaume et al. 1991) and Cx36 between pairs of microglial cells
(Dobrenis et al. 2005), respectively. Dye injections can visual-
ize the shape and spatial distribution of coupled cells in whole
tissue such as acute brain slices or in vivo (Ball et al. 2007;
Houades et al. 2006).
Electrophysiological analysis at the single channel level
indicates that GJC abruptly open and close in a manner sim-
ilar to that of other ion channels (Bennett et al. 1991; Harris
2001). Each of the different Cx isoforms “produces GJCs
with distinct unitary conductance, molecular permeability,
and electrical and chemical gating sensitivities” (Harris 2001).
For example, the unitary conductance of GJCs varies from
15 pS to greater than 300 pS and charge selectivity varies from
slightly anion- to highly cation-selective. The rank order of
unitary conductance, highest to lowest, according to Cx com-
position of homomeric channels is: Cx37 > Cx40 = Cx46 >
Cx43 = Cx26 > Cx32 > Cx45. GJCs formed by distinct Cxs
have different limiting pore diameters and differ in their per-
meability to cytoplasmic molecules, but not in a manner con-
sistently predicted by pore size (Harris 2007). For example,
Cx43 channels are much more permeable to ATP and ADP
than Cx32 channels, but Cx43 and Cx32 channels are simi-
larly permeable to glutamate and glutathione (Harris 2001).
This suggests that the pore may “chemically” interact with
cytoplasmic molecules in a manner that selectively affects
permeability.
The permeability of GJCs is influenced by physiological
variables, including transjunctional voltage, intracellular ionic
concentrations, protein kinases and phosphatases, eicosanoids
and nitric oxide. Junctional conductance is maximal when
294 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
transjunctional voltage is 0, whereas it decreases with hyper-
polarization or depolarization of either cell (Harris 2001).
Increases in intracellular concentrations of H+ or Ca2+ rapidly
decrease junctional conductance and permeability to large mol-
ecules (Bennett et al. 1991; Harris 2001). Gating by pH oper-
ates in the physiological range (i.e., 7 ± 0.5 pH) and depends
on the C-terminal cytoplasmic tail. The increases in intracel-
lular Ca2+ that result in decreased junctional conductance are
high (e.g., t10 μM). A site sensitive to near-millimolar calcium
is located on the extracellular portion of the pore (Gomez-
Hernandez et al. 2003). This explains how HCs are held in the
closed position by high extracellular calcium, which maintains
cell integrity. However, the calcium-sensitive site is blocked
in GJCs because the HC extracellular loops mesh tightly and
prevent access. Signaling molecules such as neurotransmitters,
bioactive lipids and cytokines have complex effects on GJCs,
implying plasticity in the control of events mediated by GJCs
(Bennett et al. 1991; Giaume et al. 2010).
2.4 P H Y S I O L O GY O F C O N N E X I N
H E M I C H A N N E L S A N D PA N N E X I N C H A N N E L S
As expected from Ohm’s law, the unitary conductance of an
HC is double that of a GJC (Bennett et al. 2003). Connexin
HCs have been identified in astrocytes after treatment with
proinflammatory cytokines (Retamal et al. 2007) or in amyloid
treated astrocytes and microglia (Orellana et al. 2011c). They
can be gated open in several ways, including reducing external
divalent cations (mainly Ca2+ and Mg2+), cell depolarization,
and in some cases, elevation of intracellular Ca2+ (Fig. 24.2)
(Ye et al. 2003, 2009). Pannexin channels have much higher
unitary conductance compared with Cx HCs (but see Ma et al.
2012) with rather similar permeability properties. However,
Panx channels are not sensitive to extracellular divalent cations
and may open at resting membrane potential (Scemes et al.
2009) a property that is also found for Cx HCs activated by
proinflammatory treatment (Retamal et al. 2007).
2.5 P H A R M AC O L O GY A N D G E N ET I C TO O L S
Great effort has been applied to the discovery and character-
ization of molecules and pharmacological strategies to selec-
tively block Cx and Panx channels ( Juszczak and Swiergiel
2009; Rozental et al. 2001a), with mixed results (Ye et al.
2009). Long chain alcohols, such as octanol and heptanals,
and volatile anesthetics, such as halothane, block GJCs in
glial cells (Dermietzel et al. 1991; Giaume et al. 1991; Mantz
et al. 1993). However, these compounds are not specific and
unwanted side effects have been reported including neurotox-
icity (see Juszczak and Swiergiel 2009; Rozental et al. 2001b).
The most extensively used GJC inhibitor is carbenoxolone
(CBX), a water-soluble glycyrrhetinic acid derivative, that
rapidly (<1 minute) and reversibly blocks astrocyte GJCs
(Meme et al. 2009). Unfortunately, this compound alters
neuronal properties by affecting voltage-dependent potas-
sium (Rouach et al. 2003) and calcium channels (Vessey et al.
2004), swelling activated anion channels (Ye et al. 2009) and
NMDA-evoked currents (Chepkova et al. 2008). Mefloquine
 low Ca2+. +Vm, quinine, dephosphorylation
A A
increased ROS, cytoplasmic signals.
Closed Open
high Ca2+, Mg2+, Ba2+, Sr2+, Ni2+, La3+, Gd3+, H+,
–Vm, phosphorylation, most gap junction blockers.

Pannexin channel
Connexin gap junction channel
B C
1.0 4 DCFS + EGTA Connexin hemichannel
glutamate release
LY loading or

glutamate release

0.8 LY
3 Ctrl
B
0.6 CBX
Glu 2
0.4
A A
0.2
1
0.0
0.0 1 10 100 0 10 20 30 40
carbenoxolone (μM) minutes

Figure 24.2 Features of Cx Hemichannels. A. Cartoon showing some of

compact myelin
the factors that influence gating of HCs. B. Astrocytes show LY loading
and glutamate release in divalent cation free solution (DCFS), which is
blocked in a concentration dependent manner by carbenoxolone (CBX). periaxonal space
C. Glutamate is released from freshly isolated adult mouse optic nerve Cx30 Cx43 Cx32 Cx47 Cx29 juxtaparanode
paranode
in DCFS (plus EGTA). Consistent with release from HCs, release is
node
blocked by CBX, a gap junction blocker. (A) Modified from Saez
et al. 2003b; (B) Modified from Ye et al. 2003.
Figure 24.3 Summary of Cx and Panx Channels in and Between
Glial Cells and Neurons. A. The size of the cell symbol represents
the magnitude of protein expression and functional communication
between the different cell types (A: astrocytes; O: oligodendrocytes; M:
at low concentration (3 μM) completely blocks Cx36 GJCs microglial cells; N: neurons). The black symbols refer to Cx-based HCs
and has no effect on Cx26, Cx32, and Cx43 GJCs; at higher and white symbols to Panx channels. Question marks indicate where the
concentration (30 μM) it inhibits almost completely Cx43 contribution of Cx or Panx channels are either controversial or remain
to be established. B. Diagram of the distribution and Cx-composition
GJCs (Cruikshank et al. 2004). Interestingly, Panx1 channels of GJCs and HCs in brain white matter. Note the presence of “reflex-
are 1,000- to 10,000-fold more sensitive to mefloquine than ive” GJCs between the oligodendrocyte paranodal loops. From
Cx43 GJCs (Cruikshank et al. 2004; Iglesias et al. 2008). Orthmann-Murphy et al. 2007, with permission.
Not surprisingly, HCs are sensitive to many of the regula-
tory factors and compounds listed for GJCs. However, unlike
GJCs, the extracellular loops of HCs are far more accessible
pharmacological agents is the specificity for selected Cxs
to extracellular drugs. For instance, when applied extracellu-
and defined cell types. Genetically altered mice deficient for
larly La3+ blocks HCs but not GJCs in astrocytes (Contreras
all the main glial Cxs as well as Panx1 and Panx2 are pres-
et al. 2002). The sensitivity of Panx channels to CBX occurs
ently available. Because global deficiency for Cx26 and Cx43
at lower concentrations (5- to 20-fold less) than with Cx
is lethal, conditional knock-out mice have been engineered
HCs, and niflumic acid inhibits Cx HCs but does not affect
whereby cell-type restricted deletion is achieved using the
Panx channels (Bruzzone et al. 2005), whereas probenecid
Cre/loxP system. A further improvement is mouse lines
does the opposite. Panx1 forms a protein–protein associa-
permitting timed gene inactivation of Cxs and Panxs to
tion with purinergic P2X7 receptors. When astrocyte P2X7
avoid the confounding factor of developmental disturbances
receptors are activated by adenosine triphosphate (ATP), a
(Giaume and Theis 2010).
cationic channel opens within milliseconds followed seconds
later by activation of a large pore permeable to molecules up
to 900 Da (Pelegrin and Surprenant 2006; Surprenant et al. 3 EXPRESSION OF CONNEXINS AND
1996). Consequently, antagonists of P2X7 receptors such PA N N E X I N S I N G L I A L C E L L S
as brilliant blue G also affect Panx channels (Qiu and Dahl
2009). 3.1 D I S T R I BU T I O N A N D A NATO MY
The major advantage of genetically modified animals Gap junction coupling and Cx expression in mammalian
with deletion of gap junction proteins compared with glial cells is summarized in Table 24.1 (see also Fig. 24.3).

GAP JUNCTIONS AND HEMICHANNELS • 295

The ultrastructure of glial gap junctions are similar to what is
described in other cells. Astrocytes are the most robustly cou-
pled glial cells and form homotypic GJCs with other astro-
cytes and heterotypic junctions with oligodendrocytes and
ependymal cells. Astrocytes, as well as other glial cells, express
several different Cxs that are regulated during development
(Kunzelmann et al. 1999) and affected by injury (Saez et al.
2003a). There are some reports indicating that astrocytes form
GJCs with neurons (Alvarez-Maubecin et al. 2000; Froes et al.
1999). Except in the case of coupling between Purkinje neu-
rons and Bergmann glia (Pakhotin and Verkhratsky 2005),
these junctions may occur only during early developmental
stages (Rash et al. 2001).
3.1.1 Astrocytes
Mammalian astrocytes in both gray and white matter show
widespread dye-coupling with each other (Butt and Ransom
1993; Gutnick et al. 1981). Dye injected into a single corti-
cal astrocyte stains as many as 100 adjacent cells. Astrocytes
express Cx43, Cx30, and possibly Cx26 (Lynn et al. 2011;
Wasseff and Scherer 2011). Interestingly, Cx30/Cx30 and
Cx43/Cx43 form functional channels, whereas Cx30/Cx43
does not (Orthmann-Murphy et al. 2007). Connexin43 is
predominant, but its expression varies by brain region and
stage of development. White matter astrocytes express mini-
mal or no Cx30 (Nagy et al. 1999) and are less coupled than
those from the gray matter (Maglione et al. 2010). Coupling
between astrocytes is reduced by half in cells lacking Cx43
and abolished when both Cx43 and Cx30 are absent (Rouach
et al. 2008; Roux et al. 2011; Wallraff et al. 2006). Connexin43
appears immediately after birth, whereas Cx30 develops sev-
eral weeks after birth (Kunzelmann et al. 1999). Patch-clamp
recordings of pairs of coupled astrocytes (Giaume et al. 1991)
reveal a unitary conductance with similar biophysical prop-
erties to Cx43 GJCs described in other tissues (Spray and
Burt 1990). Cultured astrocytes express Cx43, whereas Cx30
appears only after 10 weeks, however, the addition of neurons
induces expression of Cx30 after a week (Koulakoff et al. 2008).
Neurotransmitters, endogenous active molecules (peptides,
endocannabinoids), and second messenger pathways regulate
gap junctional communication between astrocytes (for review
see Giaume et al. 2010). In cultured astrocytes dye-coupling is
increased by application of glutamate (Enkvist and McCarthy
1994), but in acute cerebellar slices glutamate, through the
activation of D-amino-3-hydroxy-5-methylisoxazole-4-
propionic acid (AMPA) receptors, reduces ionic coupling
between Bergmann glial cells (Muller et al. 1996). Glutamate
may contribute to neuronal activity–dependent control of
Cx43 expression in astrocytes with important consequences
for astrocyte coupling (Rouach et al. 2000). High [K+]-
induced upregulation of astroglial GJCs involves the calmod-
ulin kinase pathway (De Pina-Benabou et al. 2001) and also
occurs in the olfactory bulb, where Cx30 GJCs are regulated
by neuronal activity (Roux et al. 2011). Glutamate or high [K+]
solution causes membrane depolarization, and this might be
linked to changes in coupling via changes in intracellular pH.
Depolarization produces an alkaline shift in astrocytes that
296 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
could enhance coupling (Ransom and Sontheimer 1992). In
contrast with the effect of neurons, brain macrophages reduce
Cx43 expression in astrocytes (Faustmann et al. 2003; Rouach
et al. 2002). Other naturally occurring molecules, including
endothelins and proinflammatory cytokines (Duff y et al.
2000; Meme et al. 2006), have also been shown to downregu-
late astroglial GJC expression and function.
3.1.2 Oligodendrocytes
Based on ultrastructural studies, few gap junctions are found
between oligodendrocytes (O:O) in rat brain, although they
are abundant between astrocytes and oligodendrocytes (A:O),
suggesting that GJC communication between oligodendro-
cytes is mediated by A:O connections (see Fig. 24.3) (Rash
et al. 2001) (see chapter 62). However, O:O gap junctions
occur between oligodendrocytes, and coupling is seen in vitro
(Ransom and Kettenmann 1990; Von Blankenfeld et al. 1993)
and in situ (Butt and Ransom 1993; Robinson et al. 1993). This
apparent conflict might be explained by recent studies show-
ing marked regional differences in O:O and O:A coupling:
The former predominates in white matter and the latter in gray
matter (Maglione et al. 2010; Wasseff and Scherer 2011). The
significance of regional differences in intercellular coupling
patterns is not known. In corpus callosum slices from animals
older than 2 weeks, the majority of injected oligodendrocytes
show coupling to other glial cells, mainly oligodendrocytes,
but also some astrocytes. Direct O:O coupling was confirmed
by persistent O:O coupling in animals lacking the astrocyte-
specific connexins Cx43 and Cx30 (Maglione et al. 2010).
The paranodes of oligodendrocytes have Cx32 homomeric
reflexive junctions, that is, GJCs between compartments of
a given cell (see Fig. 24.3B). Oligodendrocytes express Cx29,
Cx32, and Cx47 (see Table 24.1). By electrophysiological test-
ing, Cx pairs that could mediate O:O coupling are Cx32/
Cx32 and Cx47/Cx47, while the heterotypic pair Cx30/Cx47
is not functional (Orthmann-Murphy et al. 2007). Current
evidence is in conflict about whether Cx32/Cx32 GJCs are
functionally more important than Cx47/Cx47 GJCs in medi-
ating O:O coupling (Maglione et al. 2010; Wasseff and Scherer
2011). There is complete agreement, however, that the absence
of Cx32 and Cx47 eliminates O:O coupling. As to A:O cou-
pling, the pairings that produce functional GJCs are Cx30/
Cx32 and Cx43/Cx47. Interestingly, the most abundant Cxs
in these two cell types, Cx43 and Cx32, do not pair with each
other (Elfgang et al. 1995).
3.1.3 Schwann Cells
Proliferating Schwann cells, during early development, or after
peripheral nerve crush injury, primarily express Cx46 and are
coupled to one another. Cytokines may be involved in the
injury-related switch from Cx32 to Cx46 (Chandross 1998).
Nonmyelinating Schwann cells in vitro exhibit dye-coupling
that disappears when they begin to make myelin (Konishi
1990). Myelinating Schwann cells stop expressing Cx46 but
increase expression of Cx32, which is confined to paranodal
regions and Schmidt-Lanterman incisures. At these sites
“reflexive” gap junctions connect the paranodal folds, facilitat-
ing ion and small molecule movements to and from the tight,
periaxonal space (Balice-Gordon et al. 1998). The X-linked
form of the hereditary neuropathy known as Charcot-Marie-
Tooth disease is associated with mutations in the Cx32
gene (Bergoffen et al. 1993). In this condition, Cx32 is also
depressed in the brain but oligodendrocytes appear normal as
they express Cx29 (Altevogt et al. 2002). These observations
indicate that Cxs expressed in Schwann cells play a critical role
in peripheral nerve integrity and function.
3.1.4 Microglial Cells
Microglia in their resting state express little or no Cx. Activated
microglia, however, express Cx43 and show dye coupling
in vitro (Eugenin et al. 2001); however, so far there is no in situ
evidence of GJC-mediated coupling. Interestingly, activated
microglia can downregulate Cx43 expression and functional
coupling in nearby astrocytes (Faustmann et al. 2003), suggest-
ing that astrocyte communication may be reduced at sites of
brain injury in which microglia would assume their activated
phenotype. Expression of Cx43 and functional GJCs can be
induced among cultured microglia by interferon-J plus tumor
necrosis factor-D. Induction of functional GJCs between
microglia is blocked in Cx43-deficient mice (Eugenin et al.
2001). Amyloid E peptide induces expression of Cx43 and
Panx1 in microglia within 72 hours. Microglia show enhanced
HC activity with release of glutamate and ATP that initiates
a pathological sequence leading to neuronal injury (Orellana
et al. 2011b). This is hypothesized to be an important step in
the pathogenesis of Alzheimer disease.
4 O R G A N I Z AT I O N O F C O U P L E D
G L I A L AG G R E G AT E S
Astrocytes coupled together by GJCs were initially assumed
to form a nonspecific glial syncytium (Mugnaini 1986; Theis
et al. 2005), but recent evidence suggests they are organized in
a more complex and restricted manner forming networks of
communicating cells with rules governing their functional sta-
tus and plasticity (Giaume and McCarthy 1996; Giaume et al.
2010). This feature has functional consequences depending on
the nature of the signals that are exchanged through GJCs.
4.1 G L I A L N ET WO R K S
Dye injected into a single astrocyte in the cortex, hippocam-
pus, olfactory bulb, and optic nerve diffuses via GJCs to other
cells, 80% to 95% of which are astrocytes, identified by spe-
cific astrocyte markers or by expression of a fluorescent pro-
tein in genetically altered mice (Binmoller and Muller 1992;
Blomstrand et al. 2004; Butt and Ransom 1993; Houades
et al. 2008; Rouach et al. 2008; Roux et al. 2011; Schools et al.
2006). This established the notion of astroglial “networks”
(Giaume and McCarthy 1996; Giaume et al. 2010), although
weak coupling to oligodendrocytes and, very rarely, to neurons
may be seen (see the preceding). Interestingly, astrocytes are
GAP JUNCTIONS AND HEMICHANNELS
not universally coupled; using the hGFAP-eGFP mouse, some
eGFP positive astrocytes located within the coupling domain
are not dye coupled hinting at the possibility that astrocytes
may couple in a way that is purposeful and not dictated solely
by proximity (Giaume et al. 2010; Houades et al. 2006).
Selectivity in astrocyte coupling is also supported by double
patch-clamp recordings in hippocampal slices, in which 18%
of neighboring pairs were not coupled (Meme et al. 2009).
Finally, in brain areas characterized by strong anatomical-
functional compartments, astroglial networks may overlap
with functional units of neurons such as in the somatosensory
cortex (Houades et al. 2008) and the glomerular layer of the
olfactory bulb (Roux et al. 2011).
4.2 R EGU L AT I O N O F C O N N E X I N E X P R E S S I O N
AND CHANNEL FUNCTION
Understanding how Cx expression and function are regu-
lated by normal and pathological brain environments is of
fundamental importance. Glial Cx channels are regulated
by a number of releasable bioactive molecules, and their
expression is modified by brain pathologies (Giaume et al.
2010). Indeed, like in other tissues, in the brain Cx-based
communication is subject to long- and short-term regula-
tion (Bruzzone et al. 1996). Long-term regulation occurs
over hours or days, and operates at the transcriptional level.
This is likely associated with changes in the number of junc-
tional plaques and/or GJCs. Moreover, changes in the rate
of gap junction internalization and Cx degradation may also
occur. Short-term regulation occurs in minutes and deals
with changes in opening probability, time of opening/clo-
sure, and/or unitary conductance of functional GJCs already
in place. Interestingly, Cx expression in astrocytes is a target
for a number of neurotransmitters, growth factors, peptides,
cytokines, and endogenous bioactive lipids, most of them
associated with intracellular signaling pathways (Giaume
et al. 2010). This indicates that glial networks are subject to
plasticity being tightly modulated by neuronal products and
secretions from other brain cells types, such as glia (astrocytes,
microglia), endothelial cells, and inflammatory cells such as T
cells (Eugenin et al. 2001). An example of activity-dependent
plasticity of astroglial networks was demonstrated in olfac-
tory glomeruli in which extracellular K+ plays an essential
role (Roux et al. 2011).
4.2.1 What Passes Through Astroglial Gap
Junction Channels
To understand the functions of glial GJCs, we must know what
substances pass through them. Ions clearly pass through GJCs,
and they have long been thought to contribute to K+ home-
ostasis during neuronal activity (Kuffler and Nicholls 1966;
Wallraff et al. 2006), and assist in the intracellular homeostasis
of Na+ (Rose and Ransom 1997). Biochemical coupling also is
likely to be very important in glial functions and is expected
to depend on Cxs identity. At present, however, most of the
information available comes from astrocytes, or cell lines
selectively expressing Cx43, the most common astrocyte Cx.
• 297
Signaling molecules that pass through these GJCs are listed
in Table 24.2.
Interestingly, in astrocytes the high number of GJCs can
be used as a tool to deliver pharmacological or molecular
agents in a specific manner to a group of communicating cells.
Whole-cell recording of a single astrocyte allows dialysis of
molecules included in the patch pipette and these can diffuse
within the network. This strategy was used to deliver the Ca2+
chelator BAPTA, showing that calcium signaling in astro-
glial networks affects hippocampal heterosynaptic depression
(Serrano et al. 2006). Likewise, IP3 delivered via the coupled
astrocyte network induced glutamate release, triggering tran-
sient depolarizations, and epileptiform discharges in CA1
pyramidal neurons (Kang et al. 2005).
5 HEMICHANNELS IN GLIAL CELLS
Unlike GJCs, Cx HCs are directly exposed to extracellu-
lar ions and are gated by extracellular, as well as intracellular,
ionic changes. A wide range of factors influence HC gating
(Contreras et al. 2002; Saez et al. 2003b; Ye et al. 2003) (see Fig.
24.2). Open HCs are detected by their “signature” unitary con-
ductance and uptake or release of appropriate marker dyes, and
both events should be blocked by GJC blockers. Hemichannels
have been primarily studied in cultured cells, but functional
HCs are seen in acute brain slices (Karpuk et al. 2011; Orellana
et al. 2011a) and in situ (Ye et al. 2003). Pathological condi-
tions may predispose HC activation (Contreras et al. 2002). It
also seems likely that a small fraction of these channels may be
open under normal physiological conditions, or be gated open
in a controlled manner during normal activity, for instance, by
decrease in extracellular Ca2+ (Torres et al. 2012). The implica-
tions for brain physiology, ranging from synaptic transmission
to development, are obvious but remain largely unexplored.
5.1 A S T RO C Y T E S
Normally, HCs open when extracellular divalent cations are
removed (Ye et al. 2003), but opening can occur in the presence
of divalent cations during metabolic inhibition (Contreras
et al. 2002), after oxygen and glucose deprivation (Orellana
et al. 2011b) or after exposure to proinflammatory cytok-
ines (Karpuk et al. 2011; Orellana et al. 2011a; Retamal et al.
2007). In addition, the activity of HCs composed of Cx43 or
Cx45, but not Cx26, is enhanced by FGF-1 mainly owing to
an increase in the surface level of HCs mediated by a rise in
intracellular Ca2+ concentration (Schalper et al. 2008). The
patterns of Cx expression and activation in glia are modified
by pathological conditions (Koulakoff et al. 2012; Orellana
et al. 2009). These changes depend on the nature and sever-
ity of insult, distance from the lesion site, and elapsed time.
Both deleterious and protective effects have been reported, in
particular after ischemia, stroke, or trauma. However, in most
studies, the relative contribution of GJCs and HCs was not
discriminated because, in general, the pharmacological agents
used block both channel types (see the preceding). In neu-
rodegenerative diseases, changes in astroglial Cx expression
have been observed, but changes in their channel functions,
HCs, and/or GJCs, have been tested in only a few of them

Table 24.2 WHAT PASSES THROUGH ASTROGLIAL GAP JUNCTION CHANNELS

BLOCK OF GAP
JUNCTIONAL
CELL MODELS TECHNIQUES PERMEANT MOLECULES COMMUNICATION REFERENCES

Cultured cortical astrocytes SL/DT, radiolabeled Glucose, Octanol, Tabernero et al. 1996
compounds Glucose-6-phosphate, beta-glycyrrhetinic acid,
lactate Arachidonic acid,
Endothelin-1

Cultured cortical astrocytes SL/DT, radiolabeled Glutamate, Octanol Giaume et al. 1997
compounds glutamine

Cultured cortical astrocytes Calcium imaging Inositol-triphosphate NT Leybaert et al. 1998

C6 transfected with Cx43 Cell viability Unidentified death signals Nontransfected C6 cells Lin et al. 1998
co-cultured with astrocytes (TUNEL)
ATP cell content

C6 transfected with Cx43 Capture of radiolabeled ADP, ATP, Nontransfected C6 cells Goldberg et al. 1999
compounds Glutathione
Glutamate

C6 transfected with Cx43 Layered culture system, ATP, ADP, AMP, glutathione, Nontransfected C6 cells Goldberg et al. 2002
radiolabeled compounds glutamate, glucose

Cultured hippocampal SL/DT 2-NBDG Carbenoxolone, Blomstrand and

astrocytes Endothelin-1 Giaume 2006

Hippocampal acute slices Patch-clamp loading Inositol-triphosphate NT Kang et al. 2005

Hippocampal acute slices Patch-clamp loading 2-NBDG Cx43 KO, Cx30 KO, Rouach et al. 2008
6-NBDG carbenoxolone

298 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
(Koulakoff et al. 2012). Interestingly, proinflammatory cytok- from analysis of diseases associated with Cx mutations (this
ines (i.e., IL-1E and TNF-D) increase function of Cx43 HCs large topic is beyond the scope of our review, for example, see
and simultaneously decrease function of Cx43 GJCs (Retamal Abrams and Scherer 2011; Koulakoff et al. 2011; Saez et al.
et al. 2007). Recently, an opposite effect has also been reported 2003b) and from studies on specific Cx knock-out.
following Staphylococcus aureus infection in the striatum with
uncoupling and Cx43 HC/Panx1 channel activation, depend-
6.1 [ K + ] O H O M E O STA SI S
ing on time after injection and location within the infected
area (Karpuk et al. 2011). Panx1 was found in cultured astro- Neural activity generates transient increases in [K+]o that
cytes (Bianco et al. 2009; Iwabuchi and Kawahara 2009) and would disrupt orderly information processing if there are no
in situ (Santiago et al. 2011). Activation of P2X7 receptors in mechanisms to minimize these increases. The “spatial buffer
these cells induces ATP release via Panx1 channels, but not hypothesis” states that glial cells participate in moving extra-
Cx43 HCs (Iglesias et al. 2009). In contrast, cultured astro- cellular K+ from areas in which it accumulates secondary to
cytes subjected to hypoxia-reoxygenation or treated with
proinflammatory cytokines or the amyloid peptide exhibit HC A Homeostasis
activity mediated by Cx43, but not Panx1 (Froger et al. 2010;
Orellana et al. 2010; Retamal et al. 2007). To make matters Mem. Pot. Mem. Pot. Mem. Pot.
more complicated, two recent studies report Cx43 and Panx1 [Na+] [Na+] [Na+]
HC activity in astrocytes provoked by fibroblast growth fac- [Ca2+] [Ca2+] [Ca2+]
[K+] [K+] [K+]
tor (FGF) in one case (Garre et al. 2010) and proximity to a Amino acids Amino acids Amino acids
brain abscess in the other (Karpuk et al. 2011). At present, it Metabolites Metabolites Metabolites
appears that Panxs and Cxs may both form astrocyte HCs and
channels under certain conditions, the details dependent on
the conditions triggering their activation. Further evidence of B stimulus Response amplification
Cx43 HC expression in astrocytes is provided by the obser- receptor
vation that HC-mediated ATP release persists in the Panx1/ cAMP cAMP
cAMP
Panx2 double knock-out mouse (Bargiotas et al. 2011). IP3 IP3 IP3
Ca2+ Ca2+ Ca2+

5.2 M I C RO G L I A Response Response Response

In vivo, only 5% of resting microglia express low levels of Cx43.
Brain trauma in the form of a stab wound robustly induces Cx43
expression in microglia (Eugenin et al. 2003). Cultured microglia C Signaling: astrocyte calcium waves stimulus
(e.g. glutamate)
express little or no Cx43 (Meme et al. 2006; Rouach et al. 2002),
but can be induced to do so by certain proinflammatory agents glutamate
or the amyloid peptide, leading to glutamate release via HCs receptors
(Takeuchi et al. 2006). The resultant neurotoxicity is inhibited glutamate
release PLC
by CBX (D’Hondt et al. 2009). Treatment of microglia with PLC
ATP
amyloid peptide induces Cx43 and Panx1 HCs that release ATP IP3
and glutamate (Orellana et al. 2011b). Over time, the microglia- [Ca2+]i Ca2+
altered
mediated neurotoxicity is progressive and could contribute to excitability Ca2+
the progressive nature of diverse neurodegenerative diseases. astrocyte stores
neuron

6 FUNCTIONS OF GLIAL GAP
JUNCTION CHANNELS AND
HEMICHANNELS
Several functions have been proposed for GJCs based on their
physiological properties, and this list is growing (Bennett et al.
1991; Harris 2001; Loewenstein 1981; Saez et al. 2003b). They
may serve to coordinate the electrical and metabolic activities
of cell populations, act to amplify the consequences of signal
transduction, control intrinsic proliferative capacity, and help
to orchestrate the complex events of embryonic morpho-
genesis (Fig. 24.4). In addition, Cxs can have functions in
astrocytes that are unrelated to channel formation such as cell
adhesion (Elias et al. 2007), modulation of purinergic recep-
tor responses (Scemes 2008) and influencing cell survival (Lin
et al. 2003). Further insights about Cx functions are emerging
Figure 24.4 Schematic Illustration of Possible Functions of GJCs and
HCs. A. In strongly coupled cell aggregates, the intracellular concentra-
tions of ions and molecules less than approximately 1,000 kDa (including
amino acids, sugars, and second messenger molecules) will equilibrate
because of free exchange across gap junctions. B. Coupling can amplify
the physiological consequences of a chemical stimulus (i.e., hormone,
neurotransmitter) acting on a single cell. Such stimuli often cause the
production of second messenger molecules (i.e., Ca2+, IP3, cAMP) that
can move easily into adjacent cells via gap junctions; the coupled cells
are then recruited to respond. C. GJCs and HCs participate in astrocyte
Ca2+ waves. Astrocytes can be provoked to exhibit Ca2+ waves in many
ways, such as the application of glutamate. Effective stimuli activate phos-
pholipase C (PLC) and inositol (1,4,5)-triphosphate (IP3) dependent
pathways leading to Ca2+ release from Ca2+ stores. IP3 and Ca2+ can
diffuse to adjacent cells through GJCs. Astrocytes also release ATP on
activation, probably through HCs. In turn, ATP activates purinergic
receptors on nearby astrocytes and this is the predominant mode by
which Ca2+ waves spread. Increase in astrocyte [Ca2+]i can cause gluta-
mate release that modulates the excitability of neighboring neurons.

GAP JUNCTIONS AND HEMICHANNELS • 299

neural activity, to distant areas in which the K+ is not elevated
(Orkand et al. 1966). Strong astrocytic coupling would extend
the distance over which this K+ distribution system could
operate and, perhaps, increase its efficiency (Mobbs et al.
1988). Glial cells also minimize the extent of K+ accumula-
tion occurring with neural activity by uptake and sequestra-
tion of K+ (Rose and Ransom 1996). Glial coupling might
benefit this process by creating a functionally larger intracel-
lular volume that would tend to minimize changes in intracel-
lular K+ concentration that occur with K+ uptake. Coupling
between astrocytes and oligodendrocytes (i.e., A:O) might also
participate in [K+]o homeostasis. In fact, K+ may accumulate
to very high levels in the paranodal area. Astrocyte processes
that project to nodes of Ranvier, called perinodal astrocytes,
are frequently connected by gap junctions to the terminal
paranodal loops of nearby myelinating oligodendrocytes
(see Fig. 24.3B). This specialized arrangement could medi-
ate an axon-specific type of K+ spatial buffering. The impor-
tance of A:O coupling for the integrity of myelinated axons is
undeniable based on the pathological changes in myelin
seen when A:O coupling is genetically altered (Abrams and
Scherer 2011).
6.2 I N T R AC E L LU L A R M ETA B O L I C
T R A F F I C K I N G A N D H O M E O S TA S I S
Glial gap junctions may be important for metabolic coordina-
tion within the coupled aggregate. For example, coupled astro-
cytes tend to have similar intracellular [Na+] ([Na+]i), but [Na+]i
diverges between neighboring cells when coupling is blocked
(Rose et al. 1997). Gap junctions, therefore, can cause a group
of individual cells to function almost as one in terms of metab-
olism and membrane potential (see Fig. 24.4A). This offers
a theoretical advantage for the operation of the high-affinity
glutamate uptake system in glia. The rate of glutamate uptake
is affected by the transmembrane gradients of glutamate and
the ions that are cotransported or countertransported. Strong
glial coupling, by providing a larger effective intracellular vol-
ume, prevents large changes in the intracellular concentration
of the transmitter and associated ions from occurring with
uptake, maximizing the efficiency of this process.
The energy substrate glucose and its metabolites, including
lactate, can pass through GJCs between astrocytes and allow
astroglial networks to serve as a supply line for neurons under
certain circumstances (Blomstrand and Giaume 2006; Rouach
et al. 2008). Infusion of glucose and lactate within an astroglial
population was shown to sustain neuronal activity in the absence
of external glucose (Rouach et al. 2008). These observations sup-
port the idea that astrocytes support neuron energy metabolism
by providing lactate to them (Pellerin and Magistretti 2011) (see
chapter 27). Aspects of this process seem to depend on GJCs
and are disrupted by GJC inhibitors and in the Cx43–/–/Cx30–
/–
double knock-out mouse (Rouach et al. 2008).
6.3 S I G NA L T R A N S FE R
Another functional consequence of intercellular coupling is
the divergence of signal transmission (see Fig. 24.4B). Consider
300 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
the case of a cell stimulated to produce an intracellular sec-
ond messenger molecule (e.g., cAMP, IP3, Ca2+) by binding a
specific ligand. If the cell is not connected to its neighbors by
GJCs, it will act in isolation, but if the cell is widely coupled to
other cells, the diffusible, GJC-permeable second messenger
has the potential of affecting many cells (Loewenstein 1981).
Traveling waves of increased cytoplasmic [Ca2+] are elicited
in astrocytes by a variety of stimuli including glutamate appli-
cation (Cotrina et al. 1998; Finkbeiner 1992). As the wave
passes through astrocytes it can elicit glutamate release and
alter the behavior of adjacent neurons; in essence this event
is a slow form of signaling (Haydon 2001). Ca2+ wave propa-
gation is dependent on both GJCs and extracellular signal-
ing pathways, including HC-mediated ATP release (Cotrina
et al. 1998; Giaume and Venance 1998; Guthrie et al. 1999);
the relative contribution of these two mechanisms may vary
in different brain regions (Scemes and Giaume 2006) (see
Fig. 24.4C). Recent in vivo confirmation of intercellular cal-
cium waves in astrocytes has generated heightened interest
in this mode of signaling between glia in normal (Hoogland
et al. 2009; Kuga et al. 2011) as well as in pathological situa-
tions (Kuchibhotla et al. 2009).
6.4 P RO L I FE R AT I VE AC T I VIT Y
A N D D EV E L O PM E N T
The role of gap junctional communication during develop-
ment has been discussed at length (Levin 2002). Embryos
exhibit widespread gap junctional communication, which may
be important in establishing developmental “fields” critical for
morphogenesis. Coupling in glia, however, develops as these
cells mature.
Considerable evidence indicates that gap junctions can
restrict cell proliferation. Most primary brain tumors derive
from glial cells, and the possibility that loss of gap junctions
contributes to their neoplastic transformation is intriguing.
The absence of gap junctions in C6 glioma cells and highly
malignant human gliomas (Tani and Higashi 1973) supports
this concept. Moreover, proliferation is reduced in glioma cells
induced to express Cx43 (Sanchez-Alvarez et al. 2001). How
gap junctions suppress mitotic activity is not clear; however,
Cx43 can act as a substrate of c-Src reducing the rate of glioma
cell proliferation (Herrero-Gonzalez et al. 2010).
6.5 R EGU L AT I O N O F SY NA P T I C AC T I VI T Y
Astrocytes dynamically interact with neurons to regulate
synaptic transmission (Halassa and Haydon 2010), and astro-
cyte coupling may play a role in this process. Inactivation of
the Cx30 and Cx43 genes increase hippocampal synaptic
transmission and impairs long-term synaptic plasticity in
CA1 pyramidal neurons due to astrocyte hypertrophy (Fig.
24.5) (Pannasch et al. 2011). Indeed, in normal conditions
the astroglial network facilitates glutamate and K+ removal
during synaptic activity by regulating astrocyte contribution
to the rate of clearance and extracellular space volume. Thus,
astroglial Cxs play an important role in the control of synap-
tic strength by modulating extracellular homeostasis. These

C +/+ –/– D RESTING STATE
TMA+-ISM
Neuron
Glia
–0.5
fEPSP slope (mV/ms)
ACTIVITY
–0.4
–0.3
–0.2
–0.1
0.0
0.05 0.10 0.15 0.20 0.25 0.30
Fiber volley amplitude (mV)
Figure 24.5 Hippocampal Synaptic Activity Is Enhanced in the Absence
of Astrocyte Cxs. A,B. Astrocytes are hypertrophic in the hippocampal
CA1 region in Cx30 –/–/Cx43–/– mice compared with wild-type. (A)
Sulforhodamine 101 cell labeling and (B) anti-GFAP immunoreactivity
(Scale bars: 10 mm). C. Excitatory postsynaptic field potentials, and their
input/output slope, are enhanced in hippocampal slices from Cx30 –/–/
Cx43–/– mice (upper traces, scale bars: 0.2 mV, 10 ms; empty symbols:
wild-type; filled symbols: Cx30–/–/Cx43–/–). D. Schematic representation
of the changes of extracellular concentrations owing to increased astro-
glial morphology as the result of cell swelling in the Cx30–/–/Cx43–/–
mouse during neuronal activity compared with resting state. Changes
in extracellular space volume were detected by the monitoring of
time-dependent concentration of the extracellular marker tetramethylam-
monium (TMA+) recorded in the stratum radiatum of CA1 region dur-
ing Schaffer collateral stimulations. Modified from Pannasch et al. 2011.
results help establish glial Cxs as critical proteins for extracel-
lular homeostasis, important for the formation of functional
synapses.
6.6 P O S S I B L E C O N T R I BU T I O N S O F C O N N E X I N
CHANNELS IN BRAIN DISEASES
In general, in most neurodegenerative diseases and in isch-
emia changes in Cx43 expression were detected in reactive
astrocytes. Moreover, following brain injury, changes in the
level of Cx expression are dependent on the proximity of the
reactive astrocytes to the damaged site. What are the conse-
quences of such altered Cx expression on the status of astro-
glial networks? In situ models start to provide expression and
function correlation for astroglial Cxs (Koulakoff et al. 2008;
Ochalski et al. 1995; Theriault et al. 1997). What are the con-
sequences of astroglial network changes on neuronal func-
tion and/or survival? For that purpose, two strategies have
been adopted. One is a pharmacological approach using GJC
blocking agents. However, the results obtained are difficult to
interpret because of a lack of specificity of these agents. Based
on these limitations, alternative strategies using Cx knockout
mice or molecular tools were developed to examine the role
played by astroglial Cxs in pathological conditions. Most
of these experiments concern acute injury (ischemia and
trauma), but so far have led to controversial results in respect
to the role of Cxs (see chapter 58).
GAP JUNCTIONS AND HEMICHANNELS
Glial Cx HCs have been implicated in paracrine and auto-
crine cellular communication in several normal and patholog-
ical conditions (Saez et al. 2010). Many brain diseases, both
acute and chronic, induce variable degrees of inflammation,
and some general features of this process are emerging. Both
astrocytes and microglia participate in this pathological cas-
cade and can respectively exhibit Cx HC and Panx1 channel
activation leading to neurotoxicity. Thus, these cells can be
“bad neighbors” for neurons. Normalization of Cx- and Panx-
based channel dysfunctions should confer tissue protection,
and this goal should be considered a future therapeutic target
(Orellana et al. 2011c).
7 S U M M A RY A N D P E R S P E C T I VE S
Connexins and the special forms of transmembrane commu-
nication that they mediate have been extensively investigated
in the half-century since their discovery. This is especially so
in the brain, in which these channels are widely expressed in
glial cells. Although we lack final insight about how they par-
ticipate in brain functions and pathologies, new information
strongly implicates glial cell GJCs and HCs in extracellular
ion homeostasis, neural metabolic support, and disease path-
ogenesis, both acute and chronic. Research prospects in this
area have never been brighter given the powerful experimental
techniques currently available and the intriguing progress of
the last decade.
AC K N OW L E D G M E N T S
This work was supported by NIH grants NS 15589 (BRR),
INSERM/CNRS, and ANR grants (CG). The authors wish
to thank N. Rouach and J. A. Orellana for providing them
with illustration material and Kass H. Klemz for her help in
finalizing this chapter.
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• 305
 25.
PURINERGIC MECHANISMS IN GLIAL CELLS
Margaret S. Ho and Shumin Duan

A B B R E VI AT I O N S for the first time that the synaptic currents in the brain are
mediated by ATP receptors (Edwards et al. 1992). This pio-
ABC ATP-binding cassette neering work, together with abundant others, provide clues
AC adenylyl cyclase on the physiological significance of extracellular ATP and its
ADA adenosine deaminase breakdown products in the brain, further promoting in-depth
ADK adenosine kinase investigation on purinergic mechanisms in nervous systems.
ADP adenosine diphosphate Widely recognized as neurotransmitters, neuromodula-
AMP adenosine monophosphate tors, or gliotransmitters, ATP and adenosine have been shown
AK adenylate kinase to mediate intercellular signaling in the brain. Not only does
AP alkaline phosphatase ATP promote intercellular calcium wave propagation among
ATP adenosine triphosphate astrocytes (Bowser and Khakh 2007; Butt 2011; Scemes and
cAMP cyclic adenosine monophosphate Giaume 2006), ATP and adenosine-mediated cell–cell com-
CBF cerebral blood flow munication is also crucial in modulating various neuronal
DPM dipyridamole and glial functions. Notably, ATP and adenosine are essential
E-NDPK ectonucleoside diphosphokinase components that modulate synaptic transmission and plas-
E-NPP ecto-nucleotide pyrophosphatase/ ticity in both long- and short-term scales. In this chapter, we
phosphodiesterase provide an overview on purinergic mechanisms in glial cells.
E-NTPDase ecto-nucleoside triphosphate diphospho- Following an introduction on the basic properties of purines
hydrolase and purinergic receptors, we discuss the functions and mecha-
EPSC excitatory postsynaptic current nisms of purinergic signaling in glial cells. In particular, we
ER endoplasmic reticulum focus on the roles of P2X7 receptor activation in glial cells and
GPCR G protein–coupled receptor modulation of synaptic transmission and plasticity by astro-
IL-β interleukin-1β cyte-derived purines. Finally, we briefly describe other types
IP3 inositol triphosphate of purine-mediated intercellular communications in the brain,
mGluR metabotropic glutamate receptor including astrocyte calcium signaling, gliovascular coupling,
5′-NT ecto-5′-nucleotidase axon-myelinating cell interactions, and microglial responses,
OPC oligodendrocyte progenitor cell topics that are addressed in more details in other chapters.
PLC phospholipase C purinoceptors purinergic
receptors
RB-2 reactive blue 2 2 S TO R AG E , R E L E A S E , A N D
SC Schwann cell D E G R A DAT I O N M E C H A N I S M S O F
SNARE Soluble N-ethylmaleimide-sensitive factor A D E N O S I N E T R I P H O S P H AT E
attachment protein receptor
Being a widespread route for cell–cell communication, puri-
nergic signaling mediates a variety of functions during neu-
1 INTRODUCTION rons–glia or glia–glia cross-talk. The major signaler ATP
in this pathway is known to be stored in and released from
Purines are chemical substances that have been regarded as the both neurons and glia, and its impact upon releasing has been
major intracellular energy sources and drive most of the reac- immensely studied. Previous studies revealed that in neurons,
tions in the animal body. Over the years of research, discoveries ATP is often coreleased with other neurotransmitters such as
have been made on functions of these substances in various cel- acetylcholine (Redman and Silinsky 1994) or noradrenaline
lular contexts and new ideas about how they act as extracellular (von Kugelgen et al. 1998). In glial cells, ATP was reported
signaling molecules are emerging. Although some of the earli- to be stored and released from organelles that associate with
est work showed that adenosine triphosphate (ATP) acts as a components of the soluble N-ethylmaleimide-sensitive fac-
neurotransmitter in the peripheral nervous system (Burnstock tor attachment protein receptor (SNARE) complex in cul-
1972), a plethora of evidence has suggested its importance in tured astrocytes in a Ca2+-dependent manner (Coco et al.
the central nervous system. For instance, Edwards et al. showed 2003; Maienschein et al. 1999). More intriguingly, by directly

306
targeting the expression of a dominant-negative SNARE
domain in astrocytes in transgenic mice, adenosine-mediated
heterosynaptic suppression was shown to depend on
SNARE-mediated vesicle fusion (Pascual et al. 2005). These
results suggest that astrocyte release of ATP, like glutamate
(Araque et al. 2000; Bezzi et al. 2004; Jourdain et al. 2007;
Pasti et al. 1997, 2001), is vesicular and SNARE-dependent.
Nonetheless, other venues for astrocyte ATP release have
been continuously uncovered. For instance, molecular com-
ponents such as connexin hemichannels (Cotrina et al. 1998a;
Stout et al. 2002), P2X7 receptors (Suadicani et al. 2006), and
ATP-binding-cassette (ABC) proteins (Ballerini et al. 2002)
have all been reported to be involved in ATP release (for details
on the mechanisms of astrocyte gliotransmitter release and
hemichannels, please refer to chapters 17 and 24). Surprisingly,
lysosomes in astrocytes were found to exhibit Ca2+-dependent
exocytosis in response to various stimuli (Jaiswal et al. 2007; Li
et al. 2008; Verderio et al. 2011; Zhang et al. 2007) and serve
as the prominent exocytosis machineries for releasing ATP in
astrocytes (Verderio et al. 2011; Zhang et al. 2007). Similar ATP
release mechanisms have been shown in micro-glia (Dou et al.
2012). Multiple lines of evidence indicated that physiological
stimulation by neurotransmitters evokes partial lysosomal exo-
cytosis (kiss-and-run) that allows the release of a small amount
ATP to mediate intercellular communication, whereas patho-
logical stimulation by ischemia induces full lysosomal exocytosis
that enables the release of all lysosomal content including large
amount of ATP together with lysosome enzymes, contribut-
ing to secondary neural injury (Fig. 25.1). Recently, the tetanus
neurotoxin-insensitive vesicle-associated membrane protein
TI-VAMP/VAMP7 was identified as the vesicular SNARE
mediating lysosome exocytosis of both ATP and cathepsin B
from astrocytes (Verderio et al. 2011). Furthermore, VAMP7-
and Rab 27-dependent lysosomal exocytosis of myelin proteins
have also been shown to play an important role in myelin bio-
genesis and remyelination in oligodendrocytes (Feldmann et al.
2011) and Schwann cells (SCs) (Chen et al. 2012), respectively. It
will be of great interest to examine whether lysosomal exocytosis
of ATP also occurs in these types of glial cells.
High concentration of ATP in synaptic vesicles has been
reported to depend on the activity of vesicular proton pump that
maintains an inside-positive potential of the vesicle membrane for
accumulating negative-charged ATP through the nucleotide trans-
porter (Sperlágh and Vizi 1996). Although it is not clear whether
lysosomes also express nucleotide transporters, expression of puta-
tive ATP-binding cassette transporters and multidrug-resistant
proteins permeable to ATP (Abraham et al. 1993; Ballerini et al.
2002) on the lysosomal membrane have been reported (Cabrita
et al. 1999; Eskelinen et al. 2003). Thus, via a similar mechanism,
lysosomes may accumulate higher amounts of ATP because their
higher proton pump activity and more inside-positive potential
compared with synaptic vesicles (Fig. 25.2).
Unlike classical neurotransmitters, which action is
achieved by being released into and subsequently cleared
from the synaptic cleft, ATP, once released from either neu-
rons or glia, is degraded by various enzymes into products
such as nucleoside diphosphates or adenosine. The nucleotide
hydrolysis pathway includes a series of events that convert
ATP or intermediate substrates to different end-products
(Yegutkin 2008; Zimmermann 2000) (Fig. 25.3). The break-
down of ATP into ADP (adenosine diphosphate) or ADP to
AMP (adenosine monophosphate) is mediated by enzymes

A B
Physiological stimulation Pathological stimulation
(Glutamate, ATP) (Ischemia, injury)

Full lysosome exocytosis

Partial lysosome exocytosis

Release of low level ATP Release high release lysosome

levelATP enzymes

A1/P2Y receptors in P2Y receptors in Propagating P2X7 pore

presynaptic terminals astrocytes ATP-induced
ATP release

Modulation of Astrocyte glutamate and

synaptic activity Ca2+ wave Microglia ATP release
and plasticity migration

Neural network stability Neuronal damage

Figure 25.1 ATP-Accumulated Lysosomes in Astrocytes Exhibit Partial and Full Exocytosis in Response to ATP and Ischemic Stimuli, Respectively
Schematic models for lysosomal exocytosis in glial cells. (A) physiological stimulation with neurotransmitters evokes transient moderate elevation of intra-
cellular Ca2+ in glial cells and induces partial lysosomal exocytosis (kiss-and-run) that allows the release of small amount ATP to activate high affinity
P2Y or A1 receptors, resulting in the heterosynaptic suppression of neuronal activity or inter-astrocyte Ca2+ signaling propagation. (B) Pathological
stimulation with ischemia or injury induces sustained dramatic increase in intracellular Ca2+ in glial cells and evokes full lysosomal exocytosis that
enables the release of all content inside lysosomes, including large amount of ATP together with lysosome enzymes. High level of extracellular ATP
activates low affinity P2X7 receptors that may form large pores to permeate molecules as large as 900 KDa. Furthermore, ATP-induced ATP exocytosis
from lysosomes in microglia may occur under such pathological condition to evoke and maintain microglia migration toward the injury site. All these
elements may contribute to the secondary neural injury.

PURINERGIC MECHANISMS IN GLIAL CELLS • 307

 ATP accumulation in synaptic vesicle ATP accumulation in lysosome
ATP (~1mM) ATP (~1mM)
H+ H+
nucleotide
ABC transporter
vATPase H+ transporter H+
proton pump

H+ H+ +
(ATP~100mM) (ATP>100mM) H
PH=5-6 + PH=4-5 +
H H
VAMP1 or 2 VAMP7

Figure 25.2 Mechanisms of ATP Accumulation in Synaptic Vesicles and Lysosomes ATP has been reported to be released or coreleased with some
classical transmitters from some synapses. The concentration of ATP in these synaptic vesicles has been estimated to be ~100mM. The intravesicular
high concentration of the positive-charged proton produced by proton pump (vATPase) activity maintains an inside-positive membrane potential
of synaptic vesicles, which provides a driving force for the negative-charged ATP to be accumulated from cytoplasm (~1mM) into synaptic vesicles
through nucleotide transporters. In lysosomes, the components responsible for accumulating ATP are vATPase and ABC transporters. The concen-
tration of ATP in lysosome is speculated to be greater than 100 mM, because lysosomes are more acidic and have a more inside positive membrane
potential than synaptic vesicles.

of the ecto-nucleoside triphosphate diphosphohydrolase
(E-NTPDase) family (Robson et al. 2006). Among the eight
different NPTDase family members, NPTDase1, also known
as the cell activation antigen CD39, is highly expressed in
microglial cells and regulates migration behaviors (Braun
et al. 2000; Farber et al. 2008). The breakdown of ATP into
AMP is mediated by enzymes of the ecto-nucleotide pyro-
phosphatase/phosphodiesterase (E-NPP) family (Goding
et al. 2003; Stefan et al. 2006), whereas the enzyme ecto-
5′-nucleotidase (5′-NT) is responsible for degrading AMP into
adenosine. One of the 5′-NT, CD73, is known to work in con-
cert with CD39 to stimulate the immunosuppressive adenos-
ine production during inflammatory responses (Deaglio et al.
2007). Other ecto-enzymes involved in the ATP breakdown
process include alkaline phosphatase (AP), which exhibits
broad substrate specificity and adenosine deaminase (ADA)

AP
E-NPP

Ecto-5’-NT
E-NTPDase E-NTPDase AP
Adenosine ADA Inosine
ATP ADP AMP
E-NDPK ADK
AK
AP
ATP Adenosine
Ca2+
Na+ ADP

+
K
α α
β β P2X receptors (P2X1-7)
Gs γ γ
+ -Gi Gq Gs Gi
+ + - P2Y receptors
(P2Y1,2,4,6,11-14)
AC
AC
PLC P1 receptors
+ - (A1, A2A, A2B, A3)
+ -
cAMP levels
+
cAMP levels
IP3 ER Ca2+ release

Figure 25.3 ATP Degradation, Purinergic Receptors, and Their Signaling Pathways ATP undergoes degradation and is converted into various break-
down products including ADP, AMP, adenosine, and inosine. Enzymes responsible for the conversion include: ecto-nucleoside triphosphate diphos-
phohydrolase (E-NTPDase), ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP), adenosine deaminase (ADA), adenylate kinase (AK),
adenosine kinase (ADK), alkaline phosphatase (AP), ectonucleoside diphosphokinase (E-NDPK), and ecto-5′-nucleotidase (5′-NT). Purinergic
receptors are categorized into P1 (responded to adenosine), P2X (responded to ATP) and P2Y (responded to ATP and ADP) receptors. P1 and
P2Y receptors are seven transmembrane proteins coupled to G proteins, whereas P2X receptors are ligand-gated ion channels. Depending on the
G proteins P1 and P2Y receptors are coupled to, distinct cellular responses are activated such as intracellular endoplasmic reticulum (ER) release of
Ca2+ and the stimulation or inhibition of cAMP levels.

308 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
responsible for further inactivation of adenosine into inosine.
In addition, ectonucleoside diphosphokinase (E-NDPK) and
adenylate kinase (AK) are enzymes for nucleotide rephos-
phorylation and extracellular ATP synthesis, respectively.
These ecto-nucleotidases and their relatives are distributed in
brains and other tissues, often with overlapping expressions.
Yet, among these enzymes, adenosine kinase (ADK) is promi-
nently expressed in astrocytes (Studer et al. 2006) and serves
as a key regulator for adenosine metabolism. ADK eliminates
and converts adenosine into AMP and its astrocytic expres-
sion has further hinted the importance of glial purinergic sig-
naling in regulating various synaptic events.
It is very crucial to maintain a working balance between
ATP and adenosine in extracellular milieu and subtle changes
during storing, release, and degradation processes of ATP
could possibly affect its turnover and ambient adenosine lev-
els. Multiple and complex regulatory mechanisms are placed
upon these processes in order to coordinate the final outcome
of purinergic signaling. One such regulation comes from the
ability of these ATP metabolites to differentially activate cor-
responding purinergic receptors (purinoceptors). Diversity of
purinoceptors, especially their differential expressions in dis-
tinct neuronal and glial cell populations, adds an extra layer of
complexity in their ultimate actions such as modulations on
synaptic transmission and plasticity, glial calcium wave propa-
gation, and glial pathological responses.
3 P U R I N E R G I C R E C E P TO R S
On the receiving end of purinergic signaling, two major types
of purinoceptors are activated by ATP metabolites: P1 and P2
(see Fig. 25.3). P1 receptors respond to mainly adenosine and
include four subtypes: A1, A2A, A2B, and A3. Early in the nam-
ing history of purinoceptors, P1 receptors were categorized
into inhibitory (A1) and stimulatory (A2) types (van Calker
et al. 1979). Importantly, A1 and A3 receptors activate the Gi
family of G proteins and inhibit cyclic adenosine monophos-
phate (cAMP) formation via inhibiting the adenylyl cyclase
(AC), whereas A2A and A2B receptors activate the Gs family of
G proteins and stimulate AC and cAMP formation (Ralevic
and Burnstock 1998).
The P2 receptors respond to ATP and ADP and belong
to two major subfamilies, P2X and P2Y (Boison et al. 2010;
Fredholm et al. 2001; Ralevic and Burnstock 1998). At pres-
ent, seven P2X receptors and twelve P2Y receptors have
been named and cloned (Brake et al. 1994; Lustig et al. 1993;
Ralevic and Burnstock 1998; Valera et al. 1994; Webb et al.
1993). Ionotropic P2X receptors belong to the family of cat-
ionic ligand-gated ion channels, whose pore openings are
mediated by ATP binding and become permeable to Na+, K+,
and Ca2+ ions. These P2X receptors form trimers of subunits
encoded by P2X1 to P2X7 genes and express in different brain
regions (Nicke et al. 1998; North 2002; Roberts et al. 2006).
P2Y receptors are classical seven transmembrane-spanning
metabotropic receptors coupled to families of G proteins
including Gi, Go, or Gq/11 (GPCRs). Among the twelve cloned
P2Y receptors, eight of them have been shown to respond
PURINERGIC MECHANISMS IN GLIAL CELLS
to specific purines and pyrimidines. P2Y1, P2Y6, P2Y12, and
P2Y13 receptors are activated by nucleoside diphosphates
(ADP or UDP), whereas P2Y2, P2Y4, and P2Y11 receptors
are activated by ATP or UTP. Depending on the downstream
G proteins with which they interact, P2Y receptors activate
either the phospholipase C (PLC)/inositol triphosphate (IP3)
endoplasmic reticulum (ER) Ca2+-release pathway (Golovina
and Blaustein 2000; Scemes 2000; Sheppard et al. 1997;
Verkhratsky 2005) via coupling with Gq/11 proteins (P2Y1,
P2Y2, P2Y4, P2Y6, and P2Y11), or modulate the activity of AC
and cAMP levels via coupling with Gs (stimulatory, P2Y11) or
Gi (inhibitory, P2Y12, P2Y13, P2Y14) protein (Abbracchio et al.
2006) (see Fig. 25.3).
Purinoceptors have widespread expressions in neurons
(Illes and Ribeiro 2004) and glial cells. Broadly speaking,
most P1, P2X, and P2Y receptors are expressed in astrocytes
and microglia, whereas some of the purinoceptors are also
expressed in different subtypes of glial cells like SCs or oli-
godendrocytes (Table 25.1). mRNAs of all four P1 receptors
have been detected in cultures of oligodendrocyte progenitor
cells (OPCs) by RT-PCR (Stevens et al. 2002; Verkhratsky
et al. 2009) and functional assays have been done to con-
firm their expressions in astrocytes and microglia (Dare et al.
2007; Verkhratsky et al. 2009). In particular, mRNA and pro-
tein expressions of A1 adenosine receptor have been revealed
in both astrocytes and murine retina glial cells. Evidence also
implicates a function in the volume homeostasis of retina glial
cells by A1 adenosine receptor (Iandiev et al. 2007; Wurm et al.
2009). Activities of another adenosine receptor, A2A, have also
been implicated in SCs and microglia. Adenosine activates
A2A receptors and stimulates MAP kinase, thereby regulating
SC proliferation (Stevens et al. 2004). On the other hand, A2A
receptors are expressed in microglia and crucial for the repul-
sive effect on microglial motility (Gyoneva et al. 2009).
Expressions of most P2 receptors have been detected
in various glial cell types including astrocytes, microglia,
and OPCs in both transcriptional and protein levels (see
Table 25.1). Analyses of different sample preparations revealed
the expressions of most P2X receptor mRNAs and proteins in
astrocytes and Müller glia (Verkhratsky et al. 2009), whereas
immunoreactive results of P2X receptors have also been
shown in OPCs (Agresti et al. 2005; James and Butt 2001).
In microglia, mRNA and protein expressions of P2X1, P2X4,
and P2X7 receptors have been revealed (Xiang and Burnstock
2005). Evidence for functional expressions of P2X receptors
in most glial cells, however, is not conclusive. Significantly,
P2X7 receptor-mediated currents and Ca2+ signaling events
have been identified in astrocytes both in cultures and in-situ,
suggesting a functional role of P2X7 receptor during synaptic
activity (Duan et al. 2003; Fumagalli et al. 2003; Nobile et al.
2003). In addition, in mouse cortical astrocytes, assays have
been done to detect ATP-induced currents with properties
unique for heterodimers of P2X1 and P2X5 subunits (Lalo
et al. 2008), further demonstrating the functional existence of
P2X1/P2X5 heterodimers.
On the other hand, transcriptional and protein expressions
of most P2Y receptors including P2Y1, P2Y2, P2Y4, and P2Y6
have been shown in astrocytes, Müller glia, and microglia
• 309
Table 25.1 PURINERGIC RECEPTORS IN GLIAL CELLS
DOWNSTREAM
SIGNALING
RECEPTOR COMPONENTS SCHWANN CELLS OLIGODENDROCYTES

MÜLLER ENTERIC
ASTROCYTE (EYE) GLIA MYELIN NON-MYELIN TERMINAL PROGENITOR MYELIN MICROGLIA

P1(Adenosine)
A1 GipcAMP R,P,F P,F F R P,F F
A2A GsncAMP F R,P,F R F
A2B GsncAMP F R R F
A3 GipcAMP F R F
P2X(ionotropic)
P2X1 R,P,F R P R,P
P2X2 R,P P
P2X3 R,P R P
P2X4 R,P R P R,P,F
P2X5 R,F R P
P2X6 R,P P
P2X7 R,P,F R,P,F P F P,F P,F R,P,F
P2Y(metabotropic)
P2Y1 Gq/11nPLC R,P,F R,P F P,F R,F
P2Y2 Gq/11nPLC R,P,F R,P F F F F P,F F
P2Y4 Gq/11nPLC R,P,F R,P F P,F R,F
P2Y6 Gq/11nPLC R,P,F R,P F R,F
P2Y11 Gs, Gq/11 R F
nPLC
ncAMP
P2Y12 GipcAMP R,P,F F P R,F
P2Y13 GipcAMP R R F R
P2Y14 GipcAMP R,F

R, mRNA evidence; P, protein evidence; F, functional evidence. Functional evidence includes calcium imaging, protein kinase activation, responses to selective agonists and antagonists, and electrophysiological studies. The down-
stream signaling components of purinergic receptors are also integrated.
Modified with permission from Fields RD, Burnstock G. 2006. Purinergic signalling in neuron-glia interactions. Nat Rev Neurosci 7:423–436 and updated to include recent research data on purinergic receptor expressions.
(Abbracchio and Ceruti 2006; Fields and Burnstock 2006;
Fries et al. 2005). Immunoreactive and functional studies
also showed the expressions of most P2Y receptors in OPCs
(Agresti et al. 2005). Especially, P2Y12 receptors are expressed
in OPCs, myelin, and astrocytes (Amadio et al. 2006; Laitinen
et al. 2001). Proper P2Y12 receptor expression is required for
pathological conditions such as multiple sclerosis and signal-
ing between axons and myelinating glia (Amadio et al. 2010).
In summary, P1 and P2 receptors are generally expressed in
most glial cell types, yet differential expression patterns were
found to be associated with physiological or pathological
changes. Together with a precise balance of extracellular ATP
over adenosine, these factors diversify the downstream intrac-
ellular signaling events and pattern glial purinergic responses
to modulate synaptic transmission, glia-glia communication,
and pathological consequences.
4 P 2 X 7 R E C E P TO R - M E D I AT E D R E L E A S E
O F G L I OT R A N S M IT T E R S
A N D C Y TO K IN E S
Among all P2X receptors, the P2X7 subtype, also known as
P2Z receptor, functions either as a cation channel or a large
nonselective pore, depending on the concentration of ATP
and the duration of stimulation. In response to low extracel-
lular ATP levels, P2X7 receptors serve as channels for the per-
meabilization of small ions like Ca2+, K+, and Na+, and result
in the elevation of intracellular Ca2+. Upon induction by high
levels of ATP, P2X7 receptors are transformed into large non-
selective pores for the passage of molecules up to 900 kDa.
The opening of P2X7 pores results in the influx of extracel-
lular Ca2+, large cations such as N-methyl-D-glutamine, and
the uptake of fluorescent dyes. Ultimately, the sustained acti-
vation could lead to cell death (Di Virgilio et al. 1998; North
2002; Surprenant et al. 1996). Although P2X7 receptors are
expressed in various neural cells including astrocytes and neu-
rons, their prominent presence in microglia and high expres-
sion in the monocyte/macrophage cell lineage have indicated
their essential function during microglia-mediated inflamma-
tion, innate immunity, and the release of cytokines and other
inflammatory factors (Ballerini et al. 1996; Brandle et al. 1998;
Deuchars et al. 2001; Ferrari et al. 1996).
In addition to exhibit amino-acid sequence homology with
other members of the P2X family, P2X7 receptors contain a
distinct long C-terminus tail which interacts with numerous
cytoskeleton and lipid proteins, leading to the activation of
multiple intracellular signaling pathways. Variants of P2X7
receptors with C-terminus tail deletions have been shown to
exhibit impaired ability of large pore formation, yet are still
capable of mediating small ion influxes (Cheewatrakoolpong
et al. 2005; Surprenant et al. 1996). Moreover, C-terminal
deleted P2X7 receptors are expressed in different cell types,
suggesting that the cell-type specific actions of P2X7 recep-
tors are in some ways associated with their cytoplasmic tail
formation.
To date, it remains unclear how P2X7 receptors response to
high levels of ATP and transform into large pores for differential
PURINERGIC MECHANISMS IN GLIAL CELLS
actions on ion influx and the release of various factors. Insights
are gained from two hypotheses proposed. Direct dilation and
the secretory lysosome hypothesis state that ATP-mediated
activation of P2X7 receptors transform directly the receptor
itself into a large pore by activating the downstream second
messenger systems and rearranging cytoskeleton elements via
the cytoplasmic tail so that the receptor is now permeable to
large molecules. In conjunction with this change, the secre-
tory lysosomes release cytokine factors such as interleukin-1β
(IL-β) via exocytosis. On the other hand, separate pore and
membrane bleb hypothesis explains how an unidentified sec-
ond pore is formed for the passage of large molecules instead
of transforming the P2X7 receptor itself. Also, microvesicles
formed by the budding of the plasma membrane are respon-
sible for pinching off cytokines into the extracellular space in
this model (Duan and Neary 2006).
The findings that P2X7 pores may directly mediate efflux
of cytosolic glutamate and ATP in astrocytes demonstrate a
novel mechanism of gliotransmitter release during both physi-
ological intercellular communication and pathological neural
injury (Duan et al. 2003). In addition to glutamate release,
P2X7 receptors have been reported to play a key role in medi-
ating ATP-induced ATP release in astrocytes (Suadicani et al.
2006). Surprisingly, P2X7 receptor-mediated ATP release
is also blocked by gap junction inhibitors (Suadicani et al.
2006). Connexin hemichannels frequently have been reported
to mediate the efflux of intracellular large molecules includ-
ing ATP and amino acids (chapter 24) (Parpura et al. 2004;
Ye et al. 2003). Interestingly, some properties of P2X7 pores
resemble that reported for gap junction hemichannels, such as
inhibition by divalent cations and some anion channel inhibi-
tors. In this respect, it should be noted that P2X7 receptors
have been suggested to interact with connexins (Fortes et al.
2004) or pannexin 1, an ortholog to invertebrate innexin gap
junctions (Pelegrin and Surprenant 2006). In particular, acti-
vation of the P2X7 receptor-pannexin 1 complex has been
related to the processing of the cytokine IL-β in immune cells
(Pelegrin and Surprenant 2006) and the release of ATP from
astrocytes (Scemes et al. 2007). Thus, pannexin 1 may function
as a downstream effector of P2X7 receptor activation to medi-
ate transmembrane efflux/influx of large molecules and dyes.
5 P U R I N E R G I C S I G N A L I N G M O D U L AT E S
SY N A P T I C T R A N S M I S S I O N A N D
PL ASTICIT Y DURING NEURON–GLIA
I N T E R AC T I O N
One major impact of astrocyte-released ATP is to modulate
synaptic transmission of nearby synapses. For instance, a form
of short-term plasticity, heterosynaptic suppression, is mediated
by such ATP release (Pascual et al. 2005; Zhang et al. 2003).
Pioneer studies by Zhang et al. have thoroughly examined the
role of ATP in hippocampal cultured neurons and slices. First,
by applying different purinergic receptor antagonists, the effect
of endogenous ATP on synaptic transmission was examined.
Specifically, the addition of P2Y receptor antagonist, reactive
blue 2 (RB-2), reduced ATP-mediated synaptic suppression in
• 311
hippocampal cultures. On the other hand, direct perfusion of
exogenous ATP caused a reduction in the recorded excitatory
postsynaptic current (EPSC) amplitudes. In the presence of an
ectonucleotidase inhibitor, dipyridamole (DPM), the EPSC
amplitudes were suppressed and subsequent RB-2 addition
has blocked this suppression. In toto, these results suggest that
endogenous ATP tonically suppresses synaptic transmission
and this suppression is mediated through P2Y receptors. In
addition, this ATP-mediated suppression is modulated pre-
synaptically, as concluded from the decrease in the frequency
rather than the amplitude of spontaneous mini EPSCs (mEP-
SCs) when ATP is present (Zhang et al. 2003).
Because of its close proximity to neurons, astrocytes are
readily considered to be the source of released ATP. To sup-
port this hypothesis, the authors further investigated whether
astrocytes are required for ATP-mediated synaptic suppres-
sion. Neither high-frequency stimulation nor RB-2 treat-
ment increased the amplitudes of spontaneous mEPSCs in
hippocampal neurons grown in the absence of astrocytes
(GCM cultures), indicating a requirement for astrocytes dur-
ing synaptic transmission. Also, perfusion of a glia-specific
metabolic inhibitor, fluoroacetate, or gap junction inhibitor,
octanol, abolished heterosynaptic suppression detected in an
experimental setting in which synaptic activities of two nearby
neurons with reciprocal synaptic connections were measured
by dual patch recordings. Such recordings provide precise
measurements of heterosynaptic activities between two neu-
rons and clarify a number of points in the proposed model.
First, consistent with results that ATP concentrations in the
medium were markedly increased when supplementing the
pure astrocyte cultures with exogenous glutamate, heterosyn-
aptic suppression in two nearby neurons was only induced
when synapses were glutamatergic. Moreover, in accord with
previous observations that P2Y receptors were implicated in
synaptic suppression via pharmaceutical means, both RB-2
and 6,7-dinitroquinoxaline-2,3(1H,4H)-dione, the antago-
nist of non-NMDA receptors, blocked heterosynaptic sup-
pression. Taken together, these results support a model that
glutamate released on high-frequency stimulation activates
astrocyte non-NMDA receptors, which in turn trigger ATP
release and induce heterosynaptic suppression (Zhang et al.
2003) (Fig. 25.4A).
Several lines of evidence suggested that ATP-mediated syn-
aptic suppression by astrocytes is physiological relevant. First,
in hippocampal slice preparation, treatment with the adenos-
ine A1 receptor antagonist, cyclopentyltheophylline, instead

Figure 25.4 Purinergic Signaling in Astrocytes. A. Heterosynaptic suppression is mediated by the release of ATP from the astrocyte (blue).
Non-NMDA receptors in astrocytes are activated by neuronal activity (the glutamate release, brown dots), hence triggering the release of ATP
(pink dots) to activate the presynaptic A1 receptor and suppress synaptic transmission in a nearby synapse. B. ATP released by astrocytes mediate glio-
vascular coupling and cause arteriole dilation via the activation of A2 receptor on the blood vessel (red) by the degradation product of ATP, adenosine
(green dots). On the other hand, the P2Y1 receptor on either astrocyte or blood vessel can be activated by ADP, and promotes vasodilation.
C. Calcium wave propagation is mediated by purinergic signaling in astrocytes. Calcium concentration arises when triggered by the activity-dependent
release of ATP or glutamate from presynaptic terminals. ATP is then released upon intracellular calcium levels increase and this elevation propagates as
wave from one astrocyte to another.

312 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
of RB-2, was found to block the heterosynaptic suppression
mediated by direct perfusion of ATP. Furthermore, in the
presence of DPM, the addition of RB-2 significantly enhances
the EPSP amplitudes, suggesting that because of high activ-
ity of ectonucleotidases in brain tissue (not in cultured cells),
heterosynaptic suppression detected in hippocampal slices is
mediated by endogenous adenosine derived in part from extra-
cellularly degraded ATP (Zhang et al. 2003). This idea was
further supported by results from transgenic mice expressing
a dominant-negative SNARE domain that blocks gliotrans-
mitter release selectively in astrocytes. In this experimental
paradigm, endogenous adenosine, rather than ATP, is respon-
sible for heterosynaptic suppression (Pascual et al. 2005). In
summary, these results conclude that astrocyte ATP release
is activity-dependent and requires the activation of astrocyte
non-NMDA receptors. Once released, ATP gets hydrolyzed
to adenosine, which activates presynaptic A1 receptors and
mediates heterosynaptic suppression of nearby synapses.
On the other hand, astrocytes release ATP to activate post-
synaptic P2X7 receptors and modulate a long-lasting form of
plasticity at central hypothalamic glutamate synapses (Gordon
et al. 2005, 2009). Different from classical long-term potentia-
tion that requires the coincidence of presynaptic activity and
postsynaptic depolarization, this excitatory form of plastic-
ity is distributed among all synapses and synaptic strength is
increased in a multiplicative and scaled fashion. Triggered by
afferent activities mimicked with electrical stimulation in phys-
iological patterns or direct activation of astrocytes via uncag-
ing glutamate or a membrane-permeable caged IP3 compound,
astrocyte [Ca2+]i is elevated; consequently, ATP is released to
activate postsynaptic P2X7 receptors, leading to the scaling in
synaptic responses. In summary, glial purinergic signaling plays
essential roles to modulate different forms of synaptic plastic-
ity, in short and long time scales, and in inhibitory and excit-
atory modes. (Please see chapter 38 for further details.)
In addition to the heterosynaptic suppression effects dem-
onstrated in the hippocampus, similar inhibitory effects were
also reported in the retina, where light stimulation of glial cells
evokes Ca2+ waves, causing the release of ATP to inhibit neuronal
activity (Newman 2003, 2004). Focal application of ATP in the
retina glial cells, Müller cells, hyperpolarizes the neuronal mem-
brane conductance and produces outward currents in a subset
of retinal ganglion cells, thereby reducing the firing rate of these
neurons. Treatment of the A1 adenosine receptor antagonist
8-cyclopentyl-1,3-dipropylxanthine, the ecto-ATPase inhibi-
tor 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP, or
the ectonucleotidase inhibitor adenosine-5′-O-(alpha,beta-
methylene)-diphosphonate abolishes this glial-evoked inhibi-
tion, suggesting that glial released ATP is degraded into ade-
nosine extracellularly, which in turn activates the A1 adenosine
receptor to inhibit neuronal activity (Newman 2003, 2004).
The activation of GPCRs, which triggers the PLC/IP3 ER
Ca2+-release (Golovina and Blaustein 2000; Scemes 2000;
Sheppard et al. 1997; Verkhratsky 2005), has long been con-
sidered to be the major pathway mediating astrocyte Ca2+
signaling. However, controversies arise when genetic analy-
sis of a transgenic mice line altered in astrocyte GPCR Ca2+
signaling was reported (Agulhon et al. 2008; Fiacco et al.
PURINERGIC MECHANISMS IN GLIAL CELLS
2007). These findings indicate that in hippocampus, expres-
sion of the foreign receptor MrgA1 that selectively increases
the GPCR-mediated Ca2+ signaling fails to modulate syn-
aptic transmission and plasticity, raising the concern of
whether GPCR-mediated Ca2+ signaling is indeed involved in
gliotransmitter release by astrocytes.
To investigate the controversies further, it is of great inter-
est to note that [Ca2+]i microdomain produced by Ca2+ influx
is likely to be more efficient in inducing gliotransmitter release
than the GPCR-coupled [Ca2+]i elevation, because the former is
closer to the plasma membrane where exocytosis occurs. In sup-
port of this idea, Ca2+ influx through TRPC channels has been
reported to be essential for astrocyte gliotransmitter release
(Malarkey et al. 2008). Other players such as gap junction
hemichannels, mechanical sensitive channels, and the reverse
Na+/Ca2+ exchange triggered by intracellular Na+ elevation
accompanying glutamate and GABA transporter activity or
other stimuli also participate in mediating Ca2+ influx in astro-
cytes (Doengi et al. 2009; Rojas et al. 2007, 2008; Tong et al.
2009). Findings that propose two distinctive forms of astrocyte
calcium excitability (Shigetomi et al. 2008), and recent work on
that spontaneous Ca2+ microdomain near plasma membrane in
astrocytes was blocked by extracellular Ca2+ free solution instead
of inhibitors for intracellular Ca2+ store release (Shigetomi et al.
2010) have further supported the above conclusion. Notably,
based on this model, it is worthy to reconsider the exact nature
of the previously described metabotropic glutamate receptors’
(mGluRs) agonist-induced responses because many agonists
also serve as substrates for glutamate transporters (Duan et al.
1999; Harris et al. 1987; Ye et al. 2001) and might as well induce
Ca2+ responses from such transporter activities.
6 ASTROCY TE PURINERGIC SIGNALING
M E D I AT E S G L I O VA S C U L A R C O U P L I N G
The importance of glial purinergic signaling is exemplified
again in the scenario of gliovascular coupling. Astrocytes, as
the core component of the neurovascular unit, are ideally situ-
ated in a setting in which their endfeet connect with blood
vessels such as arteries/arterioles and on the other end they are
in close contact with synaptic compartments. This structural
organization allows astrocytes to couple the neuronal activity
with the cerebral blood flow (CBF) via purinergic signaling.
(Details on how astrocytes regulate gliovascular coupling in
other aspects are described in chapter 37.) Increased neuronal
activity triggers localized increases in CBF via promoting
vasodilation, an action of blood vessel widening mediated by
the Ca2+-dependent ATP release from astrocytes (Fig. 25.4B).
Increased synaptic activity, represented by the release of
neurotransmitters glutamate and ATP, activates the mGluRs
and P2YRs on astrocytes respectively and triggers the tran-
sient elevations of astrocyte [Ca2+]I, which lead to the release
of ATP. Gliovascular coupling process mediated by the
Ca2+-dependent ATP release exhibits similar features with
heterosynaptic modulation on nearby synapses. Findings sug-
gested that, the dilation of arterioles requires ATP hydroly-
sis. Once released, the actions of enzymes like E-NTPDases,
• 313
E-NPPs, or 5′-NT are required to degrade ATP into products
such as AMP, ADP, or adenosine at the astrocyte endfeet
(Zimmermann 2000, 2006). These degradation products all
possess strong vasodilation properties. For instance, degrada-
tion products AMP and adenosine are capable of activating
the A2Rs, which causes arteriole dilation (Pelligrino et al. 2011;
Simard et al. 2003). On the other hand, ADP activates the
P2Y1R on both glial membrane and the vascular endothelium
as an alternative pathway to dilate arterioles (Duarte-Araujo
et al. 2009; Xu et al. 2005).
7 P U R I N E R G I C S I G N A L I N G R E GU L AT E S
N E U R O N -M Y E L I N AT I N G G L I A
I N T E R AC T I O N
One of the earliest evidence on how ATP modulates synaptic
transmission comes from the observation of the perisynaptic
SCs at the frog neuromuscular junction (Robitaille 1995).
Purinergic receptors were detected in the perisynaptic SCs
and the release of endogenous ATP from the synaptic termi-
nal triggers the release of Ca2+ from intracellular stores in SCs.
Intriguingly, in a nonsynaptic setting, axons firing bursts of
action potentials also cause the release of ATP, which regulates
SC myelination through their trophic functions. In summary,
ATP activates receptors on SCs and regulates gene expressions
to inhibit SC proliferation, differentiation, and myelination.
For further details on activity-dependent signaling between
axons and myelinating glia, please refer to chapter 45.
8 P U R I N E R G I C S I G N A L I N G M E D I AT E S
A S T R O C Y T E C A L C I U M WAVE
P R O PAG AT I O N
Glial cells have been considered as the silent components
until calcium wave propagation within astrocytes, a form of
information processing and signaling transduction, was dis-
covered in the 1990s (Cornell-Bell et al. 1990). In addition
to the modulation of synaptic transmission by ATP released
from glia, ATP released from astrocyte is also important for
propagating calcium waves among astrocytes network (Fig.
25.4C). Activity-dependent triggers from neurons induce
an increase in the astrocyte intracellular [Ca2+]I (Dani et al.
1992), which in turn causes the release of ATP. To date,
there has been great interest in understanding the intrinsic
properties of astrocyte Ca2+ signaling and how it serves as an
efficient way for information processing (see chapter 26). In
addition to gap junctions, known as the common route for
intercellular calcium waves propagation (Boitano et al. 1992;
Charles 1994; Sanderson et al. 1994; Sneyd et al. 1994), ATP
released by astrocytes was demonstrated to be the crucial
extracellular molecule that propagate the Ca2+ rise in a wave-
like fashion from one astrocyte to another (Cotrina et al.
2000; Cotrina et al. 1998b; Guthrie et al. 1999; Wang et al.
2000).
A plethora of evidence showed that ATP is responsible
for calcium wave propagation. First, focal application of ATP
314 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
induces glial calcium wave propagation and the addition of
purinergic receptor antagonists blocked the propagation of
evoked calcium waves. Moreover, in the experimental setting
where the medium during astrocyte calcium wave propaga-
tion can be collected, the collected medium was found to con-
tain ATP. Application of purinergic receptor antagonists or
pretreatment with apyrase, an enzyme for ATP degradation,
blocked the evoked calcium wave propagation (Guthrie et al.
1999). In addition, both P2X and P2Y receptors contribute
to the calcium wave propagation; particularly, the P2Y1 recep-
tor responses to micromolar ATP levels and generates fast
and rapid Ca2+ changes, whereas the P2X7 receptor responds
to millimolar ATP levels and generates long and sustained
Ca2+ changes ( James and Butt 2002). Together with other
work, purinergic signaling has been identified as the essential
pathway mediating calcium wave propagation during glia–
glia cross-talk (Bowser and Khakh 2007; Guthrie et al. 1999;
Hassinger et al. 1996).
9 PURINERGIC SIGNALING IN
MICROGLIA
Microglia are considered the resident immune cells in the brain.
During pathological states induced by brain injury, ischemia, or
hypoxia, ATP and adenosine are crucial substances that possess
dual roles. A great body of work has suggested that in addition
to the neuroprotection effects provided by ATP and adenos-
ine, ATP is also an important factor mediating the inflamma-
tory action of microglia. As a danger signal, high levels of ATP
were observed in the extracellular space during inflammation
(Idzko et al. 2007; Pellegatti et al. 2008) and upon patho-
logical release, ATP activates different purinergic receptors on
microglia, leading to morphological change and proliferation
of microglia, and the release of cytokines and growth factors
during inflammation (Fig. 25.5). For a general description on
the physiology of microglia, please refer to chapter 19.
As mentioned, the role of purinergic signaling in micro-
glia is exemplified in the scenario of P2X7 receptor-mediated
IL-1β processing and secretion (Ferrari et al. 1996; Perregaux
and Gabel 1994). Over the years of research, secretions of
other cytokines, chemokines, and the activated oxygen spe-
cies have also been associated with P2X7 receptor, suggesting
that P2X7-mediated microglia responses in general mediate
major actions during inflammation. In addition to P2X7, other
receptors such as P2X4, P2Y6, and P2Y12 are also implicated
in microglia-mediated inflammation responses. For instance,
elevated expression of P2Y6R during brain damage triggers
phagocytosis in response to exogenously added or endoge-
nously released UDP, whereas the P2Y12R expression is altered
during ipsilateral to peripheral nerve injury in the spinal cord
(Kobayashi et al. 2008; Koizumi et al. 2007; Tozaki-Saitoh
et al. 2008; Tsuda et al. 2003).
Observations on reciprocal changes in expression levels of
P2Y12R and P2X4R (together with A2AR) upon microglial acti-
vation have linked these purinoceptors to the ATP-mediated
microglial process extension, migration, and activation during
inflammation. In-depth analysis provided by Ohsawa et al.
 A ATPγS 1 mM C ATP γ S
0 min 10 min ATP γ S+Apyrase
2.5

Oriented Migration Rate

2.0

(μm/min)
1.5

1.0 *
*

0.5
*
0.0
<70μm 70~140μm 140~250μm
B Apyrase + ATPγS 1 mM
D
0 min 10 min 1.4
1.2

Oriented Migration Rate

1.0 *
0.8

(μm/min)
0.6
0.4
*
0.2
0.0

PγS N
GP S ove
ry
AT Pγ Rec
T
+A

Figure 25.5 Lysosomal Released ATP Mediates Microglia Migration A,B. Example microscopic images showing cultured microglia migrating
toward the pipette tip (indicated by the white arrow) at the center of the field before (0 min) and 10 min (right panels) after exposure to a gradient
of non-hydrolyzable ATPγS created by pulsatile application through a pipette (at the center of the image field) in the absence (A) and presence (B)
of the ATP degradation enzyme apyrase. Scale bars, 50μm. C. Averaged migration rates of microglia located in three areas at different distances from
the tip of the pipette in conditions shown in A and B. Note that greater inhibition by apyrase was seen for the migration of remote microglia (located
between 140 and 250 μm from the pipette tip). *p < .01 compared with the corresponding control group (without apyrase). D. Averaged data showed
that the ATPγS-induced microglia migration was reversibly inhibited by treatment with 100μM GPN, a substrate of the lysosomal exopeptidase
cathepsin C that selectively induces lysosome osmodialysis. *p < .001 compared with control (before GPN treatment) or between any two groups
indicated. Modified from Dou et al. 2012.

(2007), et al. showed that low levels of extracellular ATP
mediate microglial chemotaxis sufficiently and recruit cells
to the injured site. The activation of P2Y12R is also required
to mediate such microglial process extension (Davalos et al.
2005). Upon microglial activation, P2Y12R expression is down-
regulated while P2X4R and A2AR expressions are upregulated.
Also at this point, high levels of ATP are present because of
brain damage and the ATP/adenosine balance is disturbed.
Activations of P2X4R and A2AR by ATP and adenosine then
allow subsequent microglial migration and process retrac-
tion. Additional microglial functions such as phagocytosis
and cytokine secretion are also triggered, correlating with
all changes. Thus, in addition to the differential activation of
purinoceptors, an ATP concentration gradient presents also
a key mechanism to achieve such graded action of microglial
activation (Farber et al. 2008; Haynes et al. 2006; Honda et al.
2001; Ohsawa et al. 2007).
Although ATP released from damaged neural cells has
been considered the major chemokine for inducing rapid
process extension and cell body migration of microglia, ATP
leaking from the local injured cells may not be able to diffuse
extensively to induce migration of remote microglia, because
extracellular nucleotides are rapidly degraded by ecto-ATPases
in the brain. Similar to that discovered in astrocytes, lysosomes
in microglia are found to contain abundant ATP and exhibit
Ca2+-dependent exocytosis (Dou et al. 2012). Collective evi-
dence indicated that microglia respond to extracellular ATP
by releasing ATP themselves through lysosomal exocytosis
(Dou et al. 2012). This type of regenerative ATP-induced ATP
release from surrounding cells may thus provide a positive
feedback mechanism to generate and maintain a long-range
extracellular ATP gradient required for the sustained chemot-
axis of remote microglia (Dou et al. 2012) (see Fig. 25.5).
Recent studies suggest that neuron-microglia interactions
mediated by purinergic signaling are essential in the pathogen-
esis of neuropathic pain, a chronic pain following peripheral
nerve injury. Pharmacological, genetic, and behavioral find-
ings indicate that, among the multiple P2 receptor subtypes
expressed in microglia, the P2X4, P2X7, and P2Y12 recep-
tors are responsible for the neuropathic pain after peripheral

PURINERGIC MECHANISMS IN GLIAL CELLS • 315

nerve injury. Release of brain-derived neurotrophic factor and
proinflammatory cytokines including IL-1β form microglia
have been linked to the activation of P2X4 and P2X7 recep-
tors, respectively, to induce neuropathic pain. Interestingly,
p38 MAPK activation has been suggested to be a common
downstream signaling pathway for P2X4, P2X7, and P2Y12
receptors in neuropathic pain. (Relevant topics are also dis-
cussed in chapter 68.)
10 S U M M A RY A N D P E R S P E C T I VE S
Purines-mediated intercellular communication plays impor-
tant roles in various aspects of physiological and pathological
conditions and is recognized in almost every type of glial cell.
Thus, astrocyte-derived ATP and its degradation products par-
ticipate not only in inter-astrocyte Ca2+ wave propagation, but
also heterosynaptic modification of neuronal activity and glio-
vascular coupling. Furthermore, action potential–induced ATP
release from axons regulates myelination of both oligodendro-
cytes and peripheral SCs. Finally, purines induce complicated
microglial responses including chemotaxis, phagocytosis, and
cytokine release, functions that play critical roles in various
pathological activities such as inflammation and neuropathic
pain. Altogether, glial purinergic signaling nurtures a variety of
functions during neurons–glia or glia–glia cross-talk.
The mechanisms of purines release and their potential roles
in mediating various glial cell functions have just begun to be
explored. In particular, regulated ATP release through lyso-
some exocytosis has been shown as a prominent route in astro-
cytes and microglia. Yet, signaling molecule release through
this action may not be limited to glial cells. In fact, lysosomes
in melanocytes and in cells derived from the haematopoietic
lineage have been long known as secretory lysosomes because
of their dual functions of protein degradation and signaling
molecules secretion (Blott and Griffiths 2002). Furthermore,
lysosomes can fuse with the plasma membrane for membrane
repair in response to membrane injury (Reddy et al. 2001),
a typical behavior of exocytosis. Thus, lysosomes seem to be
conserved organelles for “general” exocytosis of signaling mol-
ecules in conventional cells, whereas synaptic vesicles are newly
evolved organelles for efficient release of neurotransmitters in
specialized neurons.
As a signaling molecule, ATP is unique for its extracellular
degradation by different enzyme systems into products that
activate distinct subtypes of purinergic receptors coupling to
different downstream signaling pathways. Whereas ATP acti-
vates both ionotropic P2X receptors and metabotropic P2Y
receptors but not P1 receptors, its degradation products ADP
and adenosine activate only metabotropic P2Y receptors
and P1 receptors, respectively. Thus, the degradation scheme
in extracellular space mediated by collections of enzymes
is central to the paradigm of purinergic receptor activation.
To understand the functional roles of purines-mediated sig-
naling in a particular brain region, the cellular source of
ATP release, distribution pattern of purinergic receptor sub-
types, and the expression level of different extracellular ATP-
degradation enzymes in the local neural network need to be
316 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
identified. In the future, it will be of great interest to study
the distribution patterns and dynamic changes of extracellular
ATP-degrading enzyme expressions in various brain regions
during developmental or pathological stages.
AC K N OW L E D G M E N T S
The authors apologize for not being able to include all the rel-
evant research work because of limitations on chapter length.
Thanks to Nature Publishing Group, Dr. R. Douglas Fields,
and Dr. Geoffery Burnstock for permission with Table 25.1.
Thanks also to Tianming Yu for help with reproducing the
table.
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 26.
CALCIUM SIGNALING IN NEUROGLIA
Alexei Verkhratsky and Vladimir Parpura

A B B R E VI AT I O N S activation of these receptors triggers distinct secondary path-

ways that form the basis for excitability of nervous tissue. The
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazole- neuronal excitability is mainly caused by their plasma mem-
propionic acid brane, which represents an excitable media because of expres-
α7AChR α7 acetylcholine receptor sion of high densities of voltage-gated ion channels leading
[Ca2+]i cytosolic free Ca2+ concentration to generation of action potentials. Glial cells (as a rule) do
[Ca2+]l intra-ER (or intraluminal) free Ca2+ not generate plasmalemmal action potentials and hence are
concentration classified as electrically nonexcitable. The excitability of neu-
CBP Ca2+ binding proteins roglia is of a different kind, being associated with intracellular
CGP37157 7-chloro-5-(2-chlorophenyl)-1,5-dihydro- movements of ions and second messengers. The intracellular
4,1-benzothiazepin-2(3H)-one signaling system mediated through highly controlled move-
CICR Ca2+-induced Ca2+ release ments of Ca2+ ions is particularly important for regulation
ER endoplasmic reticulum of glial Ca2+ responses and is generally believed to provide a
iGluR ionotropic glutamate receptor substrate for glial excitability (Verkhratsky and Kettenmann
InsP3R inositol 1,4,5 trisphosphate (InsP3)–gated 1996).
Ca2+ channel/receptor
MCU mitochondrial Ca2+ uniporter
mGluRs metabotropic glutamate receptors
MPTP mitochondrial permeability transition pore 2 PRINCIPLES OF CALCIUM SIGNALING
NCX Na+/Ca2+ exchanger
NMDA N-methyl-d-aspartate Control over intracellular concentration of inorganic ions is
PCD paroxysmal depolarization shift the fundamental property of life; movements of ions across
PLC phospholipase C plasma membrane and membranes of organelles underlie the
PMCA plasmalemmal Ca2+ ATPase most basic processes of cellular energetics and function. The
RyR ryanodine receptor monovalent ions (Na+, K+, H+, and Cl–) generally provide for
SERCA sarco(endoplasmic) reticulum Ca2+ ATPase electrical potentials across biological membranes, which drive
SOCE store-operated Ca2+ entry electrical excitability and energy production. The divalent
SPCA secretory pathway Ca2+ ATPase cations (Ca2+, Mg2+, Zn2+) are critical for cellular biochemis-
TRP transient receptor potential try through binding to enzymes and regulating their activity.
TRPC canonical type TRP channel Calcium because of its unique biophysical properties (e.g.,
VDAC voltage-dependent anion channel flexible coordination chemistry, high affinity for carboxylate
VGCC voltage-gated Ca2+ channel oxygen, which is the most frequent motif in amino acids, rapid
binding kinetics, and effects on fluidity and fusion of cellular
membranes) ( Jaiswal 2001) is central for controlling a wide
1 I N T R O D U C T I O N : G L I A L E XC I TA B I L I T Y variety of enzymatic cascades and therefore acts as a univer-
sal signaling molecule (Heilbrunn 1943; Petersen et al. 2005;
The intricate cellular web created by excitable (neurons) and nonex- Toescu and Verkhratsky 1998). This role was assumed by Ca2+
citable (neuroglia) elements provides the structural basis for infor- from the very dawn of evolution, as specificities of ATP-based
mation processing in the nervous system. The cell-to-cell signaling energetics require tight control over Ca2+ concentrations in the
that underlies information flow within this complex network is cytosol and within cellular organelles. Excess of Ca2+ inside the
achieved either by ions fluxing through the plasma membrane, cell is toxic because high Ca2+ induces aggregation of proteins
molecules diffusing through the extracellular space (neurotrans- and nucleic acids, affects the integrity of lipid membranes,
mitters, neuromodulators, and hormones), or direct intercellular and triggers precipitation of phosphates (Case et al. 2007).
flow of ions and second messengers via gap junctions. As a result, cells developed a highly sophisticated system for
Neurons and neuroglia express multiple receptors for Ca2+ homeostasis. This system includes numerous intracellular
neurotransmitters and neuromodulators (see chapter 17) Ca2+ binding proteins and several sets of Ca2+ transporters and
that allow intercellular information exchange. However, exchangers (Berridge et al. 2003; Carafoli 2004).

320
 Concentration of free Ca2+ ions in the cytosol ([Ca2+]i)
of all cells from most primitive prokaryotes to highly special-
ized eukaryotes is maintained at an exceedingly low level of
approximately 50 to 100 nM, which is approximately 20,000
times lower than that in the extracellular space. The Ca2+ bind-
ing proteins (CBPs; Ca2+ buffers) present in the cytosol have
very high affinity to Ca2+ (KD in a low nanomolar range) (Ikura
et al. 2002), which limits cytosolic Ca2+ diffusion and facili-
tates localization of cytosolic Ca2+ signals, which often appear
in a form of microdomains or even nanodomains of very high
(in excess of 10–100 μM) [Ca2+]i. These highly localized
[Ca2+]i domains are fundamental for regulation of fast local
cellular responses (e.g., exocytosis). Similar concentration
difference also exists between cytosol and some intracellular
organelles (e.g., the ER, Golgi complex, some acidic organelles
such as secretory vesicles), which may contain up to 1 mM of
free Ca2+. These Ca2+ concentration gradients always directed
toward the cytosol act as a backbone for Ca2+ signaling sys-
tem by creating a driving force underlying Ca2+ diffusion into
the cytosol (Fig. 26.1) (Berridge et al. 2003; Carafoli 2004;
Verkhratsky 2006). The combination of Ca2+ movements
between intracellular compartments and the cell and extracel-
lular space produce spatiotemporally organized fluctuations
of free Ca2+ in different parts of the cell and within intracel-
lular organelles. These fluctuations are generally referred to as
Ca2+ signals.
Various environmental signals (physical or chemical)
initiate Ca2+ diffusion through several sets of plasmalemmal
and intracellular (endomembrane) Ca2+ channels that trigger
[Ca2+]i rise. It is shaped in space and time by cytoplasmic Ca2+
buffering (CBP) and active Ca2+ transport by plasmalem-
mal and intracellular Ca2+ pumps and exchangers (see Fig.
26.1). The number of Ca2+ signaling/homeostatic molecules
is relatively limited; the most diverse being plasmalemmal
Ca2+-permeable channels represented by the voltage- and
ligand-gated channels, mechanosensitive and store-operated
channels, and extensive family of cationic transient recep-
tor potential (TRP) channels. Transmembrane Ca2+ trans-
port is achieved by plasmalemmal Ca2+ ATPases (PMCAs),
sarco(endoplasmic) reticulum Ca2+ ATPases (SERCAs), and
secretory pathway Ca2+ ATPases (SPCAs) of the Golgi com-
plex and other acidic compartments (Brini and Carafoli 2009;

iGluR
P2XR SOC Connexins
[Ca2+] ~ 2 mM NCX α7AChR PMCA GPCR TRPC Exocytosis Pannexins

[Ca2+] ~ 100 nM ADP ATP

SOCE
Astrocyre InsP3R
Calcium gradients

InsP3R

[Ca2+] ~
0.2-1 mM
Neurone

Ca2+ microdomain
RyR InsP3R
ADP ATP
[Ca2+] ~ 100 nM

VGCC/LGCC
[Ca2+] ~ 2 mM neurotransmitter NCX PMCA iGluR GPCR
release P2XR
α7AChR

Figure 26.1 Calcium Signaling Cascades in Neurons and Glia. The force driving calcium signaling is formed by Ca2+ concentration gradients, which
in turn are created by energy-dependent Ca2+ transport across cellular membranes. Physiological stimulation opens plasmalemmal or intracellular
Ca2+ channels, thus creating Ca2+ fluxes affecting [Ca2+] in various intracellular compartments. In neural cells, calcium signaling events occur either
in the form of localized [Ca2+]i microdomains or global [Ca2+]i elevation. The Ca2+ microdomains regulate various localized functional responses, for
example, being responsible for the initiation of exocytosis. Conceptually, neuronal Ca2+ signals mainly rely on Ca2+ entry via voltage- and ligand-gated
plasmalemmal Ca2+ channels. This Ca2+ entry creates short-lived [Ca2+]i microdomains that determine fast and focal neurotransmitter release from
the synaptic terminals. In glial cells, Ca2+ signals are usually initiated by Ca2+ release from the ER that creates more widespread and longer-lasting
[Ca2+]i elevation, thus determining slower and more sustained release of gliotransmitters. The domains of elevated [Ca2+]i are sensed by mitochondria,
which are able to accumulate Ca2+ via uniporter. Mitochondrial Ca2+ entry acts as the main link between neural cell activity and energy production
by regulating mitochondrial electron transport and ATP synthesis. The Ca2+ signaling cascades are tightly integrated in overall cellular homeostatic
system. Particularly in astrocytes, the NCX is colocalized with NMDA receptors, Na+-dependent glutamate transporter, and Na+/K+ ATPase. This
arrangement allows rapid reversal of the transporter in response to [Na+]i elevation associated with ionotropic receptors and glutamate transport,
which (1) maintains Na+ gradients needed for glutamate transporter function, and (2) creates local Ca2+ signals by Ca2+ entry through the exchanger.
Abbreviations: α7AChR, α7 acetylcholine receptor; CBP, Ca2+-binding protein; ER, endoplasmic reticulum; GPCR, G protein–coupled recep-
tor; InsP3, inositol(1,4,5)trisphosphate receptor; LGCC, ligand-gated Ca2+ channel; NCX, sodium-calcium exchanger; PLC, phospholipase C;
PMCA, plasmalemmal Ca2+ ATP-ase; RyR, ryanodine receptor; SERCA, sarco(endo)plasmic reticulum Ca2+ ATP-ase; SOC, store-operated Ca2+
channel; VGCC, voltage-gated Ca2+ channel. Connexins/pannexins may create high-permeability plasmalemmal channels (which also can include
pore-forming P2X7 receptors or volume-regulated anion channels) that can act as a pathway for nonexocytotic release of neurotransmitters. The role of
Ca2+ signals in controlling the activation and permeability status of these channels remains unknown.

CALCIUM SIGNALING IN NEUROGLIA • 321

Pizzo et al. 2011), as well as by Na+/Ca2+ exchangers or NCXs
(Guerini et al. 2005). Further, cellular Ca2+ signaling involves
mitochondria that accumulate and release Ca2+ through the
uniporter channel, permeability transition pore, and mito-
chondrial NCX (Bernardi et al. 2006; De Stefani et al. 2011).
These Ca2+ signaling/homeostatic cascades are tightly regu-
lated by Ca2+ ions themselves, so that changes in Ca2+ concen-
trations in different cellular compartments immediately feed
back to Ca2+ channels and Ca2+ transporters regulating their
function (Burdakov et al. 2005). Additionally, by altering
expression of various components of the Ca2+ homeostatic
system the cells create context-dependent “Ca2+ signaling
toolkits” that tailor Ca2+ signals to environmental challenges
(Berridge et al. 2003). Altogether these many levels of control
and regulation create a versatile and robust system that ensures
the variability and adaptability of Ca2+ signaling machinery.
The Ca2+ signals acquire distinct spatiotemporal parameters
depending on the cell type, the nature of the stimuli, or
the cell metabolic state. These [Ca2+]i signals can appear as
local microdomains that control neurotransmitter release
or regulate cell process guidance. Alternatively, global Ca2+
signals are instrumental for excitation-contraction and
-secretion coupling, gene expression, and tissue development
can be mounted (Bolsover 2005; Bregestovski and Spitzer
2005; Parpura et al. 2011; Toescu and Verkhratsky 1998;
Verkhratsky 2006). In the cellular syncytia (e.g., astroglial
networks) Ca2+ signals can spread over long distances in the
form of propagating Ca2+ waves. The Ca2+ signals also con-
trol cell survival and death. Ca2+-dependent pathways initi-
ate programmed cell death, which is fundamental for normal
tissue development and homeostasis, whereas massive Ca2+
influx can compromise Ca2+ homeostasis and induce necrosis
(Nicotera et al. 2007).
Ca2+ signals are targeted to “Ca2+ sensors” represented
by Ca2+-sensitive proteins, in many cases enzymes. Binding/
unbinding of Ca2+ ions to “Ca2+ sensors” affects their activity.
The specificity of Ca2+ signaling is determined by variability
in affinities of these proteins to Ca2+ ions and by their intra-
cellular localization. Activation/deactivation of Ca2+ sensors
triggers or discontinues diverse biochemical processes, thus
regulating cellular reactions; the spatio-temporal organization
of Ca2+ signals determines the timing/localization of “Ca2+
sensors” reactions, hence providing for spatial and temporal
coding of Ca2+ signaling (Burgoyne et al. 2004; Toescu and
Verkhratsky 1998).
3 CALCIUM SIGNALING IN ASTROGLIA
3.1 Ca 2+ PE R M E A B L E PL A SM A LE M M A L
CHANNELS
Astrocytes in vitro, in situ, and in vivo express several types of
Ca2+ permeable cationic channels which have different lev-
els of Ca2+ permeability. These channels include ionotropic
receptors, store-operated channels, TRP channels, and pos-
sibly mechanosensitive cationic channels. The properties of
store-operated Ca2+ influx are discussed in the later sections;
here the focus is on voltage- and ligand-gated channels.
322 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
3.1.1 Voltage-Gated Ca2+ Channels
There is little evidence for functional expression of voltage-gated
Ca2+ channels (VGCCs) in astrocytes in situ/in vivo. VGCCs
have been identified in astrocytes in vitro (see chapter 16), in
particular cultured astrocytes were found to express mRNA
for L- (Cav1.2), N- (Cav2.2), P/Q- (Cav2.1), R- (Cav2.3), and T
(Cav3.1)- types of calcium channels. Activation of VGCCs results
in inward Ca2+ currents and [Ca2+]i transients (Barres et al. 1990;
D’Ascenzo et al. 2004; Duff y and MacVicar 1994; Latour et al.
2003; see also Verkhratsky and Steinhauser 2000 and references
therein). Quite often, the VGCCs expression required trophic
manipulations with astrocytes, for example, incubation with
dibutyryl-cyclic adenosine monophosphate, coculturing with
neurons, or acute oxidative stress (Corvalan et al. 1990; MacVicar
and Tse 1988). VGCCs reported for immature hippocampal
astrocytes in brain slices (Akopian et al. 1996) most likely repre-
sent channel expression in the NG2 glial cells. The VGCCs were
considered to participate in spontaneous [Ca2+]i oscillations in
astrocytes in slices from the ventrobasal thalamus. These [Ca2+]i
oscillations were inhibited by nifedipine and enhanced by Bay
K8644 (Parri and Crunelli 2003). The immunoreactivity for
N (Cav2.2) and R (Cav2.3) channels subtypes was detected in
pituicytes (hypophyseal astrocytes) in situ; water deprivation for
24 hours induced a significant increase in immunoreactivity of L
(Cav1.2)-type channels in these cells (Wang et al. 2009). These
observations, however, remain sporadic; as a rule mature astro-
glial cells in situ do not demonstrate measurable voltage-gated
Ca2+ currents. There are some indications about upregulation
of L- and P/Q- channels in cells undergoing reactive astroglio-
sis induced by pilocarpine-induced status epilepticus (Xu et al.
2007). Incidentally, Ca2+ may also enter astrocytes through Kir4.1
inward rectifier channels after lowering extracellular K+ concen-
tration below 2 mM, although the mechanism of this Ca2+ influx
remains unknown (Hartel et al. 2007).
3.1.2 Ionotropic Ca2+-Permeable Receptors
Astroglial cells express several families of ligand-gated Ca2+-
permeable channels (see chapter 17), which can be differentially
present in various brain regions (Lalo et al. 2011; Verkhratsky and
Steinhauser 2000). Most of the astrocytes in vitro and in situ,
express the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA) glutamate receptor (GluR). The AMPA recep-
tors have been found in astrocytes from cerebellum and cor-
tex (Seifert and Steinhauser 2001). In some brain regions
(e.g., in cerebellar Bergmann glial cells) the AMPA receptors
lack GluR2 subunit, which makes them moderately perme-
able to Ca2+ (PCa/Pmonovalent ~1, with fractional Ca2+ currents
~4%) (Burnashev et al. 1992, 1995). Entry of Ca2+ through
these receptors is additionally limited by fast desensitization.
In the cortex and spinal cord protoplasmic astrocytes express
functional N-methyl-d-aspartate (NMDA) receptors, which
demonstrate weak Mg2+ block at –80 mV and thus are avail-
able for glutamate activation at the resting membrane potential
(Lalo et al. 2006; Verkhratsky and Kirchhoff 2007). The Ca2+
permeability of astroglial NMDA receptors is about three
times less compared with the neuronal form of the receptor
(PCa/Pmonovalent approximately 3 (Palygin et al. 2010) (Fig. 26.2).
 A B
P2X receptors

Imem(r.u.)

Imem(r.u.)
NMDA receptors

0.5 0.5
2.5 mM [Ca2+]o 2.5 mM [Ca2+]o
10 mM [Ca2+]o 10 mM [Ca2+]o
Vm(mV) Vm(mV)
–80 –60 –40 –20 –10 10 20 30 –80 –60 –40 –20 –10 10 20 30
NMDA ATP
+20 mV
2.5 mM [Ca2+]o +20 mV 2.5 mM [Ca2+]o
–0.5 –0.5
0 mV 0 mV

–20 mV –20 mV 30 pA
–1.0 25 pA –1.0 500 ms
500 ms 2+
+20 mV 10 mM [Ca2+] +20 mV 10 mM [Ca ]o
o
0 mV 0 mV
–1.5 –1.5
–20 mV –20 mV

C D

Fura-2 ratio Fura-2 ratio

(F340/F380) image (F340/F380) image

1.2 Control Single stimulus 1.2

F340/F380 Control Single stimulus
F340/F380

1.0 1.0

0.8 0.8
50 pA 50 pA
0.6 0.6
1s 1s
Control HFS train Control HFS train
1.2 1.2
F340/F380
F340/F380

1.0 1.0

0.8 0.8

0.6 0.6

1.2 HFS train

1.2 D-AP5 HFS train NF449
F340/F380
F340/F380

1.0 1.0

0.8 0.8

0.6 0.6

0 10 20 30 0 10 20 30
time (s) time (s)

Figure 26.2 Calcium Signaling in Cortical Astrocytes Induced by Activation of Ionotropic Receptors. A,B. The I–V curves and examples of current
recordings (insets) in response to stimulation with (A) NMDA (30 μM) and (B) ATP (10 μM) at 2.5 and 10 mM [Ca2+]o. Increase in [Ca2+]o shifts the
reversal potentials for both NMDA- and ATP-induced currents, indicating their Ca2+ permeability. The I–V curves were constructed from 8 (NMDA)
and 7 (P2X1/5) independent experiments. The amplitudes of responses were normalized to the value measured at -40 mV; data are presented as mean
± SD. Solid lines show the results of the best polynomial fit (least squares routine), intersection with zero current axis gives the following values of
reversal potential in 2.5 and 10 mM [Ca2+]o: 1.9 and 5.1 mV for NMDA-evoked currents and 4.6 mV and 6.7 mV for ATP-evoked currents. The permeabil-
ity ratio PCa/PK calculated by extended Goldman-Hodgkin-Katz equation is 3.1 for NMDA receptors and 2.2 for P2X receptors. C,D. Cortical layer
II astrocytes were loaded with Fura-2 in situ via patch pipette. Fluorescent images were recorded following neuronal afferent stimulation in continuous
presence of CNQX (control) and after application of NMDA receptor blocker D-AP5, 30 μM (A) or 10 nM of NF-449, selective antagonist of P2X1
receptors (B). Representative images (pseudo-color, pipette image subtracted; warmer colors correspond to higher [Ca2+]i levels) and [Ca2+]i transients
were recorded from two different cells. [Ca2+]i-transients (middle columns) are expressed as F340/F380 ratio. Modified from Palygin et al. 2010.

CALCIUM SIGNALING IN NEUROGLIA • 323

Astroglial NMDA receptors are sensitive to memantine and
GluR NR2C/D subunit-selective antagonist UBP141. This
pharmacological profile taken together with low Ca2+ perme-
ability and weak Mg2+ block indicates that astroglial NMDA
receptors can possible represent trimers assembled from NR1,
NR2C/D, and NR3 subunits (Palygin et al. 2011).
Ionotropic purinoceptors (see also chapter 25) repre-
sent another type of Ca2+ permeable ligand-gated channels.
Cortical astrocytes express functional P2X1/5 heteromeric
purinoceptors, which are characterized by exceptionally high
affinity to ATP (EC50 approximately 50 nM) and slow desen-
sitization (Lalo et al. 2008). The P2X1/5 receptors are Ca2+
permeable with P Ca/Pmonovalent approximately 2 (see Fig. 26.2)
(Palygin et al. 2010). Cortical astrocytes in situ were also
reported to express functional P2X7 receptors activated by
millimolar ATP concentrations (Oliveira et al. 2011). These
receptors can have a substantial Ca2+ permeability, especially
after pore formation following extensive stimulation. The
P2X7 receptors are likely to be activated in pathological con-
ditions. Calcium permeable α7 nicotinic cholinoreceptors
(P Ca/Pmonovalent ~6) were also identified in cultured hippocam-
pal astrocytes (Sharma and Vijayaraghavan 2001); however,
their expression in situ remains to be confirmed. Stimulation
of astroglial NMDA and P2X1/5 receptors by appropriate
agonists or electrical stimulation of neuronal inputs resulted
in generation of Ca2+ signals in both cultured astrocytes and
astrocytes in brain slices (see Fig. 26.2) (Palygin et al. 2010).
These Ca2+ signals can be functionally relevant, especially
when generated in perisynaptic processes in which they can
produce local [Ca2+]i microdomains, which can be important
for spatially segregated functional responses.
3.2 E N D OPL A SM I C R ET I CULUM A S A PR IM A RY
S OUR CE OF AST R O G LI A L Ca 2+ SI G NA L IN G
The ER possesses its own Ca2+ homeostatic machinery which
determines its participation in cellular Ca2+ signaling. The
intra-ER (or intraluminal) free Ca2+ concentration ([Ca2+]L)
varies between 0.2 and 1 mM (Verkhratsky 2005). Levels
of [Ca2+]L this high are achieved by energy-dependent Ca2+
uptake provided by SERCA pumps. The ER lumen also con-
tains a set of specific Ca2+ binding proteins (e.g., calreticulin
and calsequestrin) characterized by low affinity to Ca2+ (K D
~0.5–1 mM). This arrangement helps maintaining high [Ca2+]L
and allows unhindered Ca2+ diffusion through the lumen of
the ER, which is instrumental for formation of intra-ER “Ca2+
tunnels” that contribute to long-range Ca2+ transport in polar-
ized cells (Verkhratsky 2005).
The ER membrane also contains several families of intra-
cellular Ca2+ channels represented by: (1) Ca2+-gated Ca2+
channels or ryanodine receptors (RyRs); (2) the inositol
1,4,5-trisphosphate (InsP3)–gated Ca2+ channels/recep-
tors (InsP3Rs); and (3) the nicotinic acid adenine dinucle-
otide phosphate receptors (see Berridge et al. 2003; Petersen
et al. 2005; Verkhratsky 2005 for relevant references).
Activation of these intracellular Ca2+ channels results in Ca2+
release from the ER lumen into the cytosol, thus creating
cytoplasmic Ca2+ signals.
324 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
The InsP3-induced Ca2+ release from the ER plays the
leading role in astroglial Ca2+ signaling. Incidentally, in the
astrocytes in situ the ER is localized close to plasma mem-
brane, whereas in cultured cells it tends to be closer to the
nuclear envelope (Pivneva et al. 2008). Astrocytes express a
wide variety of metabotropic receptors coupled to phospho-
lipase C (PLC); indeed, stimulation of astrocytes in vitro,
in situ and in vivo by various neurotransmitters and neuro-
modulators routinely results in production of InsP3 and sub-
sequent Ca2+ release from the ER (Fig. 26.3) (Agulhon et al.
2008; Finkbeiner 1993; Verkhratsky et al. 1998). These InsP3-
mediated Ca2+ signals have the following common features:
(1) astroglial Ca2+ signals often do not require extracellular
Ca2+; (2) they are blocked following inhibition of SERCAs
with thapsigargin or with cyclopiazonic acid; (3) they are
inhibited by intracellular injections of InsP3R blocker hepa-
rin; and (4) they are absent in transgenic mice lacking type 2
InsP3Rs (Kirischuk et al. 1999; Petravicz et al. 2008). These
InsP3Rs type 2 seems to be predominantly expressed in astro-
glia and they are frequently concentrated in distal processes
(Holtzclaw et al. 2002). Astrocytes also express types 2 and 3
RyRs, although their contribution to Ca2+ signaling genera-
tion remains unclear. The RyR-mediated CICR has not been
detected in hippocampal astrocytes. On the contrary, [Ca2+]i
transients in response to caffeine (which opens RyRs in mM
concentrations) were described in astrocytes in thalamus
(Parri and Crunelli 2003).
The InsP3-mediated ER Ca2+ release in astrocytes regu-
lates several functional responses. First, ER originated Ca2+
signals were found to activate Ca2+-regulated exocytosis and
Ca2+-dependent release of glutamate from cultured astrocytes
(Malarkey and Parpura 2009; Parpura et al. 2011). Inhibition
of ER Ca2+ release by exposure of cultured astrocytes to
thapsigargin almost completely obliterated Ca2+-dependent
secretion of glutamate following activation of metabotropic
purinoceptors (Innocenti et al. 2000; Jeremic et al. 2001;
Malarkey and Parpura 2009). Similarly, thapsigargin inhib-
ited glutamate release in response to mechanical stimulation
(Hua et al. 2004). The same inhibition of glutamate release
was observed after incubation of astroglial cultures with
membrane-permeable inhibitor of InsP3Rs diphenyl boric
acid 2-aminoethyl ester (Hua et al. 2004). In this context it
is interesting to note that glutamate release in cultured astro-
glial cells was also inhibited by ryanodine and preincubation
with caffeine, suggesting a certain role for RyRs in mediating
relevant Ca2+ signals (Hua et al. 2004). Subsequently, the ER
Ca2+ release was shown to activate the release of glutamate and
D-serine from astrocytes in acute hippocampal slices (Angulo
et al. 2004; Rusakov et al. 2011; Shigetomi et al. 2008).
However, the functional role and importance of ER Ca2+
release in astroglial physiology remains under debate. Some
experiments in transgenic animals in which the ER release was
occluded (the InsP3R knock-out mice) found no changes in
synaptic transmission or plasticity in hippocampus. Similarly,
targeted stimulation of astrocytes overexpressing peripheral
(i.e., nonexistent in the CNS) metabotropic receptor did not
alter synaptic transmission or plasticity in Schaffer collater-
als–CA1 neuron synapses (Agulhon et al. 2010; Fiacco et al.
 A B
250

30 s 200 30 s

[Ca2+]i(nM)

[Ca2+]i(nM)
65 5 min 80 5 min

ATP ATP ATP ATP ATP ATP

2+
Ca - free 500 nM thapsigargin

C D
180
Control Heparin
>3
[Ca2+]i(nM)

50 s 25 s

F/F0
1
50

ATP ATP

Figure 26.3 ATP-Induced Ca2+ Signaling in Bergmann Glial Cells Results Exclusively from Inositol 1,4,5-Trisphosphate (InsP3)–Mediated Ca2+ Release
from ER Ca2+ Stores. A. ATP-induced [Ca2+]i transients were measured from Bergmann glial cells “bulk-loaded” by incubating cerebellar slices in fura-2
acetoxymethyl ester (AM)–containing solutions. Addition of ATP triggered an increase in [Ca2+]i that persisted in Ca2+-free extracellular solution. B.
In a similar experiment, incubation of slice with 500 nM thapsigargin completely and irreversibly blocked ATP-induced Ca2+ signaling. C. Intracellular
administration of heparin via intracellular dialysis with a patch pipette inhibited [Ca2+]i increase induced by ATP. Control [Ca2+]i transient was
recorded from fura 2-AM–loaded cells before commencing intracellular dialysis. D. Illustration of spatial distribution of [Ca2+]i at time of maximum
ATP response. Note higher levels of [Ca2+]i in Bergmann glial cell processes as compared with cell body. Modified from Kirischuk et al. 1995a.

2007; Petravicz et al. 2008). However, other authors found
that under specific conditions, InsP3-induced Ca2+ release in
astrocytes resulted in modulation of synaptic plasticity in neu-
ronal networks (Haydon and Lee 2011). Obviously, functional
heterogeneity and intrinsic plasticity of astroglia may account
for these seemingly contradictory results.
The InsP3-mediated Ca2+ release is a key factor for astro-
glia-dependent regulation of local blood flow. Astroglial Ca2+
signals, triggered following activation of metabotropic recep-
tors, induce release of vasoactive substances (e.g., derivatives
of arachidonic acid or carbon monoxide) from perivascular
endfeet. These substances regulate the tone of cerebral arte-
rioles and are the leading mechanism of functional hyper-
emia (Iadecola and Nedergaard 2007). Glial Ca2+ signaling
of ER origin also plays numerous trophic roles and is directly
involved in initiation of Bax translocation and astroglial
apoptosis (Morales et al. 2011).
3.3 T HE STOR E - OPE R AT E D Ca 2+ E N T RY:
A R OLE F OR T R A NSIE N T R E CE PTOR
P OT E N T I A L CH A N N E L S
The ER Ca2+ store is linked to the plasmalemmal Ca2+ influx
pathway generally known as a “capacitative” or a store-operated
Ca2+ entry (SOCE). This link, initially proposed by Jim Putney
(1986), has been identified in a majority of nonexcitable and
some excitable cells; in all of which depletion of the ER from
releasable Ca2+ triggers secondary Ca2+ influx through the spe-
cific set of plasmalemmal channels (Parekh and Putney 2005).
Activation of the SOCE facilitates replenishment of the ER
store (capacitative function) and contributes to the plateau
phase of [Ca2+]i transients that may outlast the period of stimu-
lation. Molecular pathways involve either specific Ca2+-release,
activated Ca2+ channels known as CRAC (Hoth and Penner
1992) or certain types of transient receptor potential (TRP)
channels (Smyth et al. 2006). The molecular nature of CRAC
channels has been recently deciphered. This Ca2+-permeation
pathway is created by plasmalemmal pore-forming Orai pro-
teins, activation of which is controlled by the ER resident
sensor Stim1. When the ER store is depleted, the Stim1 is
redistributed to the near-plasmalemmal portion of the retic-
ulum, where it signals to Orai proteins and opens CRAC
channels (Putney 2007).
The SOCE is operative in all types of neuroglial cells,
including astrocytes, oligodendrocytes, microglia, and patho-
logically modified gliomas (Hartmann and Verkhratsky 1998;
Kettenmann et al. 2011; Tuschick et al. 1997). Whether astro-
glial SOCE involves Stim1/Orai complex remains unknown.
The expression of Orai and Stim1 proteins was detected
in the astroglial cell line U373 MG (Barajas et al. 2008);
however, this was not yet confirmed in experiments on pri-
mary astrocytes. In general the Ca2+-release activated Ca2+
currents (ICRAC) indicative of Orai/Stim1 Ca2+ permeation
have not been detected in astrocytes. At the same time astro-
cytes widely express canonical type TRP (TRPC) channels
tetramerically assembled from an obligatory TRPC1 subunit
in combination with ancillary TRPC4 and/or TRPC5 sub-
units (Golovina 2005; Malarkey et al. 2008). The TRPC1/4/5
channels can act as a store-operated pathway and have rela-
tively high Ca2+ permeability (PCa/Pmonovalent ~1–9) (Nilius

CALCIUM SIGNALING IN NEUROGLIA • 325

et al. 2007). Downregulation of TRPC1 expression by the
antisense knock down of the TRPC1 gene or its inhibition by
a blocking antibody directed at an epitope in the pore-forming
region of the TRPC1 protein significantly suppressed SOCE
in cultured astrocytes (Golovina 2005; Malarkey et al. 2008).
The antibody-induced inhibition of TRPC1-containing chan-
nels specifically suppressed plateau phase of the ATP-induced
[Ca2+]i transients in astrocytes as well as mechanically induced
Ca2+ signals and Ca2+-dependent glutamate release (Malarkey
et al. 2008).
3.4 T HE Na + /Ca 2+ E XC H A N G E R
IN A ST R O G L I A
The Na+/Ca2+ exchangers, NCXs, belong to the SLC8 fam-
ily of solute carriers. The mammalian NCX family is encoded
by three genes—NCX1, NCX2, and NCX3—all of which
are expressed in astrocytes. The NCX proteins are mainly
localized in perisynaptic astroglial processes, with particu-
larly prominent appearance in processes covering excitatory
synapses, where they colocalize with NMDA receptors and
glutamate transporters (Minelli et al. 2007). According to
the stoichiometry of 3 Na+: 1 Ca2+ the NCX can operate in
both forward (Ca2+ extrusion in exchange for Na+ influx) and
reverse (Ca2+ entry in exchange for Na+ extrusion) modes.
The switch between forward/reverse operational modes is
controlled by Na+ and Ca2+ transmembrane ion gradients and
the level of membrane potential. The cytoplasmic Na+ con-
centration in astrocytes is relatively high, around 17 to 20 mM
(approximately twice the level of Na+ concentration in neurons)
(Reyes et al. 2012), which sets the reversal potential for NCX
(approximately –70 to –90 mV) rather close to the character-
istic resting membrane potential of astrocytes (Vm approxi-
mately –80 mV). As a result, the NCX in astrocytes dynamically
fluctuates between forward and reverse modes and participates
in both Ca2+ entry and clearance (Kirischuk et al. 1997; Reyes
et al. 2012). In cultured cortical astrocytes, NCX operates in
reverse mode even in resting conditions (Reyes et al. 2012).
The Ca2+ entry mediated by the reverse mode of NCX
were detected and analyzed in primary cultured astrocytes
and in astroglial cells in situ (Goldman et al. 1994; Kirischuk
et al. 1997; Reyes et al. 2012). The NCX can amplify Ca2+
signals following activation of astroglial ionotropic receptors.
The latter carry Na+ currents and may significantly increase the
cytosolic Na+ concentration, which can produce additional
Ca2+ influx by reversing the exchanger (Goldman et al. 1994;
Kirischuk et al. 1997; Lalo et al. 2011; Reyes et al. 2012). The
same cascade can be initiated by activation of the plasmalemmal
glutamate transporter, which induces substantial Na+ influx
(the stoichiometry of transporter is 3 Na+: 1 glutamate). The
resulting elevation in [Na+]i (by up to 20 mM) rapidly turns
NCX into the reverse mode and produces substantial Ca2+
influx (Kirischuk et al. 2007). The activation of reverse mode
of the NCX was also achieved by moderate depolarization of
cultured astrocytes (Paluzzi et al. 2007). The NCX-mediated
[Ca2+]i increases can be functionally relevant and were shown,
for example, to trigger exocytotic glutamate release (Paluzzi
et al. 2007; Reyes et al. 2012).
326 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
3.5 M ITO C H O N D R I A IN A ST R O G L I A L
Ca 2+ SI G NA L IN G
The mitochondrion is a cellular organelle directly involved
in many aspects of Ca2+ signaling and Ca2+ homeosta-
sis in all eukaryotic cells (Nicholls 2005). Mitochondrial
Ca2+ accumulation is mediated by ion channels located in
their double membrane. The outer membrane contains the
voltage-dependent anion channels (VDAC), which have con-
siderable Ca2+ permeability, whereas the inner mitochondrial
membrane possesses the highly selective Ca2+ channel histori-
cally known as a Ca2+ uniporter. This comprises the channel
protein of mitochondrial Ca2+ uniporter (MCU) and auxil-
iary EF-hand–containing protein that regulates the uniporter
(MICU1/CBARA1) (De Stefani et al. 2011). The driving
force for Ca2+ accumulation is created by mitochondrial
electron transport through inner mitochondrial membrane,
which makes the mitochondrial interior highly electrone-
gative. On average, mitochondrial membrane potential, the
Δψ, is set at about –160 to –200 mV. This electronegativity
provides the electric force that drives Ca2+ toward the mito-
chondrial matrix when cytosolic Ca2+ exceeds the set point
of approximately 300 to 400 nM (Nicholls 2005). The Ca2+
influx into the mitochondrial matrix depolarizes the mito-
chondrial membrane and stimulates ATP production; thus,
it is the main mechanism for excitation-energetics coupling.
In addition, mitochondrial Ca2+ accumulation contributes to
overall cellular Ca2+ buffering and limits large cytosolic Ca2+
loads. When too much of Ca2+ is accumulated in mitochon-
dria the pathological developments may ensue. Mitochondrial
Ca2+ overload may trigger the opening of mitochondrial per-
meability transition pore (MPTP) that can dissipate Δψ;thus
terminating ATP production and facilitating the release of cell
death–promoting factors (Nicholls 2005).
According to the Ca2+ handling described in the preced-
ing, mitochondria play a dual role in astroglial Ca2+ signaling,
acting either as a Ca2+ buffer or a Ca2+ source. Cessation of
mitochondrial Ca2+ accumulation by inhibiting mitochon-
drial uniporter by Ruthenium 360 significantly enhanced
the amplitude of mechanically stimulated [Ca2+]i transients
in primary cultured astrocytes (Reyes and Parpura 2008). At
the same time, when mitochondrial Ca2+ release was blocked
by inhibiting the mitochondrial Na+/Ca2+ exchanger with
7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiaze-
pin-2(3H)-one (CGP37157), the mechanically induced Ca2+
signals were reduced (Reyes and Parpura 2008). Similarly, the
mechanically induced Ca2+ signals were attenuated by treat-
ment with cyclosporin A, which inhibits the MPTP, indicat-
ing that flickering of MPTP with subsequent Ca2+ efflux into
the cytosol may have a physiological role in astroglial cells
(Reyes and Parpura 2008).
3.6 A ST R O G LI A L Ca 2+ WAVE S
Astroglial Ca2+ signals have complex spatiotemporal and
hierarchical organization, ranging from local Ca2+ micro-
domains to [Ca2+]i oscillations and propagating Ca2+ waves.
Highly localized microdomains of [Ca2+]i can be instrumental
in controlling various astroglial functions, such as exocytosis
(Parpura et al. 2011; Shigetomi et al. 2011). In situ the initial
[Ca2+]i rise often occurs locally in a form of Ca2+ microdo-
mains in the distal perisynaptic astrocytic processes. These
Ca2+ microdomains may stay localized or may spread toward
the soma in a form of propagating wave of ER Ca2+ release (the
intracellular Ca2+ wave) (Grosche et al. 1999; Kirischuk et al.
1995a). At the same time, the single-cell Ca2+ signals often
do not terminate at the astrocyte cell border but spread into
neighboring astrocytes; thus creating an intercellular Ca2+
wave, which may convey Ca2+ signal over the long distance
(~300–400 μm from the site of original excitation) and can
excite many tens or even hundreds of cells within the path of
its propagation (see Scemes and Giaume 2006 for details and
further references).
The intercellular Ca2+ waves were discovered in confluent
astroglial cell cultures (Cornell Bell et al. 1990). This was a
seminal discovery that demonstrated that focal stimulation
with glutamate triggers propagating Ca2+ wave in the glial
syncytia. These Ca2+ waves had a complex path, crossed cell
borders without appreciable delay, and propagated with a
velocity of approximately 15 to 20 μm/second (Cornell Bell
et al. 1990). Further experiments have found that the intercel-
lular Ca2+ waves in cultured astroglia could be evoked by focal
mechanical stimulation, although these waves were somewhat
different in that they demonstrated delay at cell borders. The
intercellular Ca2+ wave required functional Ca2+ store and can
be irreversibly blocked by thapsigargin. Similarly, Ca2+ wave
propagation in cultured cells required functional PLC and
can be blocked with the PLC inhibitor U73122 (see Scemes
and Giaume 2006 and references therein). The initial observa-
tions on cultured cells were subsequently confirmed in experi-
ments in situ in acute brain slices and in vivo (Fig. 26.4) (Dani
et al. 1992; Scemes and Giaume 2006; Schipke et al. 2002).
Propagating Ca2+ waves, which can be blocked by thapsigargin,
were also found in astrocytes in the acutely isolated rat retina
in response to local electrical stimulation or local applications
of ATP, carbachol or phenylephrine (but not of glutamate)
(Newman and Zahs 1997). The synchronized propagating
Ca2+ waves that spread through several hundreds of astrocytes
were also recorded in vivo in mice hippocampus. These Ca2+
waves were sensitive to inhibition of purinoceptors and gap
junctions (Kuga et al. 2011).
Astroglial Ca2+ wave induction and maintenance is
achieved through several, often complementary, mechanisms
(Fig. 26.5). First, the propagation of the wave can result from
diffusion of InsP3 through gap junctions (see Fig. 26.5A). It
is important to remember that heavy cytosolic Ca2+ buffering
greatly limits diffusion of Ca2+ ions themselves and propagat-
ing Ca2+ wave is not an actual movement of Ca2+ ions, but
rather a propagating wave of excitation of ER Ca2+ channels.
The importance of gap junctions in Ca2+ wave propagation
was initially demonstrated by blocking the wave propaga-
tion by broad spectrum gap junction inhibitors octanol and
halothane, or by inhibiting gap junctions following activation
of protein kinase C. Subsequently it was shown that in rat C6
gliomas intercellular Ca2+ waves could be generated only in cul-
tures transfected with connexins. The gap junction–mediated
CALCIUM SIGNALING IN NEUROGLIA
A Control 18 s B
4.5 s 22.5 s C 0.5
Stim
F/F0
30 s
1
2
9s 27 s
3
6
13.5 s 62.5 s
4
5
7
Figure 26.4 Propagation of Ca2+ Waves in an Acute Brain Slice. A. A
series of fluorescence images just before (control) and at defined times
(as indicated) after electrical stimulation illustrate the spread of the Ca2+
signal within a slice. The position of the stimulation electrode is marked
by the asterisk. The cartoon in B outlines the anatomical structures of
the fluorescence images. Moreover, the transient changes in Ca2+ were
measured at the indicated areas (B) and are displayed in C. In area 1, close
to the stimulation pipette, the increase in fluorescence (F/F0) occurred
right after stimulation (vertical line). At more distant areas, the delay
amounted to several seconds. Note that cells in the ventricle wall respond
with an intense signal. Reproduced from Schipke et al. 2002.
connectivity, controlling spread of astroglial Ca2+ waves, can
be regulated by physiological stimuli, such as depolarization,
exposure to glutamate, or neuronal firing (see Arcuino et al.
2002; Cotrina et al. 1998; Scemes and Giaume 2006 for
details and references).
The alternative mechanism for intercellular Ca2+ waves
involves extracellular diffusion of transmitters (usually
ATP or glutamate) released by astrocytes (Fig. 26.5B,C).
This mechanism was initially discovered in vitro in conflu-
ent astroglial cultures. In these cultures the cell-free line was
mechanically drawn. It turned out that a Ca2+ wave can “jump”
over the cell-free space of up to 120 μm in width, thus suggest-
ing the role of diffusible extracellular messenger (Hassinger
et al. 1997). This mechanism were subsequently confirmed and
it was found that the transmitter can be released from astro-
glia either through Ca2+-regulated exocytosis or diffusion via
plasmalemmal pores associated with unpaired connexin hemi-
channels, pannexins, pore-forming P2X7 receptors, or a vari-
ety of ATP-permeable anion channels (Arcuino et al. 2002;
Cotrina et al. 1998; Scemes and Giaume 2006).
These two main mechanisms of Ca2+ wave propagation
may coexist, or be differentially employed in astroglial syncytia
• 327
 A chapter 24). At the same time the extent of propagation and
exact pathways carrying Ca2+ waves in the brain tissue in vivo
InsP3 remain to be characterized.
Ca2+ InsP3
Ca2+
InsP3 Ca2+ InsP3 4 C a 2+ S I G N A L IN G IN
OLIGODENDROGLIA AND NG2 GLIA

Activity dependent Ca2+ signaling was detected in oligoden-

B
drocytes and their precursors in vivo and in vitro (see chapter
20 and Verkhratsky et al. 1998 for early works and relevant
InsP3
ATP ATP references). These oligodendroglial Ca2+ signals are medi-
Ca2+
InsP3
Ca2+
ated through both Ca2+ entry via plasmalemmal channels and
InsP3 2+
through ER Ca2+ release. Immature oligodendrocytes express
Ca
high- and low- threshold VGCCs; importantly low-threshold
ATP VGCCs (T-type) are predominantly localized at the very tips
of the processes on oligodendroglial precursors. The channels
C
can be activated by rather small (up to ~10 mM) elevations in
extracellular K+ concentration, which induced highly localized
InsP3 [Ca2+]i elevations in the tips of the processes (Kirischuk et al.
ATP
InsP3 1995b). This idiosyncratic localization of T-type Ca2+ channels
Ca2+ Ca2+ in oligodendroglial precursors may help them to identify active
InsP3 Ca 2+
axons (which electrical activity causes local increase in extracel-
lular K+ concentration) and initiate the myelination program.
Oligodendrocytes are also endowed with several families
of Ca2+-permeable ionotropic receptors. In particular mature
Gap junction ER Ca2+ release oligodendrocytes express NMDA receptors, which are quite
Ca2+
similar to astroglial receptors in having weak Mg2+ block and
therefore are fully operative at physiological resting potential
Metabotropic (P2Y) Vesicular release of
receptor neurotransmitter (ATP) (see Stys and Lipton 2007; Verkhratsky and Kirchhoff 2007 for
ATP references and details). In addition, oligodendrocytes express
Non-vesicular release of Ca2+-permeable AMPA receptors and P2X7 purinoceptors (and
neurotransmitter (ATP)
maybe other P2X types as well) (Matute et al. 2007). Finally,
ATP through connexins/pannexins all oligodendrocytes demonstrate robust InsP3-mediated Ca2+
release from the ER stores (Verkhratsky et al. 1998).
Figure 26.5 Mechanisms of Propagating Astroglial Ca2+ Waves. A. Ca2+ The NG2 glial cells (see chapter 21), besides being oligo-
waves can be maintained by diffusion of InsP3 through the gap junction
and secondary initiation of InsP3-induced Ca2+ release. B. Ca2+ waves
dendroglial progenitors and perhaps pluripotent neural pre-
can be maintained by regenerative Ca2+-dependent exocytotic release of cursors, are involved in neural networking by forming contacts
neurotransmitters (e.g., ATP or glutamate) or release of neurotransmit- with both astrocytes and neuronal afferents (from which they
ters through high permeable membrane channels (connexins, pannexins, actually receive synaptic contacts). Therefore, the NG2 glia
P2X7 receptors, or volume-regulated anion channels) acting on neighbor- (also called polydendrocytes or “contact” cells) may respond to
ing cells through extracellular diffusion. C. Ca2+ waves can result from
a focal release of a neurotransmitter, which then diffuses over a long
both neuronal and astroglial inputs. Mechanisms of Ca2+ sig-
distance. Modified from Verkhratsky and Butt 2007. naling in the NG2 glia remains mostly unexplored; however,
several experiments demonstrated that NG2 glia respond with
[Ca2+]i elevations to ATP and glutamate; these signals being
in different brain regions. In neocortex, for example, propa- mediated through Ca2+ permeable AMPA receptors (Ge et al.
gating astroglial Ca2+ waves are mediated mainly through 2006), P2X7 receptors and metabotropic P2Y1 receptors and
gap junctions and completely depend on the expression of group I metabotropic glutamate receptors (Haberlandt et al.
connexin Cx43. In hippocampus and corpus callosum, in 2011; Hamilton et al. 2010). Ca2+ entry through reversed NCX
contrast, spread of Ca2+ waves is mediated by ATP release and were reported to modulate migration of NG2 glia (Tong et al.
subsequent activation of metabotropic P2Y receptors (Haas 2009). In addition, NG2 glial cells were found to express Ca2+
et al. 2006; Schipke et al. 2002). In retina both mechanisms permeable ASIC1a channels (Lin et al. 2010).
are in operation, and gap junctions are involved in propa-
gating Ca2+ wave between astrocytes, whereas ATP release
mediates Ca2+ wave spread between astrocytes and Müller 5 C a 2+ S I G N A L IN G IN M I C R O G L I A
cells (Newman and Zahs 1997). Similarly in hippocampus in
vivo astroglial Ca2+ waves were sensitive to blockade of both In microglial cells, Ca2+ signals are ultimately important for
gap junctions and purinoceptors (Kuga et al. 2011) (see also controlling their activation and multiple functional responses

328 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
(see chapter 19) (Kettenmann et al. 2011). Ca2+ signaling in
microglia is controlled by many metabotropic receptors,
including purinoceptors, adrenoceptors, thrombin, chemo-
kine, and complement receptors (see chapter 19), which induce
Ca2+ release from the ER. In addition, microglial Ca2+ signal-
ing is controlled by Ca2+-permeable P2X4 and P2X7 receptors,
which are particularly important for microglial activation in a
variety of pathological conditions (see Kettenmann et al. 2011
for details and all relevant references). Finally, microglial cells
express SOCE mediated by Orai/Stim1 molecular complex.
This SOCE is important for generation of long-lasting [Ca2+]i
elevations and can be constantly activated following overstim-
ulation of microglia (Toescu et al. 1998).
6 PAT H O L O G I C A L P OT E N T I A L O F
A ST R O G L I A L C a 2 + S I G N A L IN G
Neuroglial cells, being the primary homeostatic and defensive
cells of the nervous system are intimately involved in all forms
of neuropathology, and their reactions determine the progres-
sion and outcome of neurological diseases to a very large extent.
Glial Ca2+ signals participate in pathological developments by
regulating both neuroprotective and neurotoxic responses.
In ischemia and stroke, astrocytes are specifically impor-
tant for infarction spread through the ischemic penumbra
surrounding the area of pan-necrosis. Astrocytes are gener-
ally resilient to the ischemic stress and they quite often outlive
neurons in the damaged areas. Furthermore, they provide the
major metabolic support to neurons through anaerobic gly-
colysis and lactate shuttle. Aberrant astroglial [Ca2+]i waves,
however, are involved in the spread of damage by inducing
propagating wave of astroglial glutamate release, which in turn
triggers distant excitotoxicity (see Nedergaard et al. 2010 for
details and references). Pathological Ca2+ signaling are espe-
cially important for ischemic damage to white matter (e.g., in
periventricular leukomalacia or Binswanger disease), which is
mainly associated with the rapid death of oligodendrocytes
and oligodendroglial precursors that are particularly vulner-
able to ischemia. The oligodendroglial death shows all hall-
marks of Ca2+-dependent excitotoxicity with Ca2+ entering
oligodendrocytes through P2X7 and NMDA glutamate recep-
tors (Matute 2010; Nedergaard et al. 2010). Similarly, Ca2+
excitotoxicity resulting from enhanced Ca2+ entry through
P2X7 receptors may be involved in oligodendrocyte damage
in multiple sclerosis (Matute 2010).
Pathological Ca2+ signaling can be also involved in the
pathogenesis of epileptic seizures (see chapter 70). Astroglial
Ca2+ signaling was affected in cells from patients suffering
from the autoimmune form of child epilepsy, Rasmussen
encephalitis. These pathologically remodeled astrocytes
demonstrated spontaneous Ca2+ oscillations, probably aris-
ing from antibody-induced alterations in the GluR3 subunit
of AMPA glutamate receptor. Experimental epilepsy also
upregulates metabotropic glutamate receptors (mGluRs),
particularly mGluR5 linked to PLC and InsP3-induced Ca2+
release. Pathologically increased glial Ca2+ signaling can be
involved in generating epileptiform seizures. For example,
CALCIUM SIGNALING IN NEUROGLIA
the neuronal paroxysmal depolarization shift (PCDs, which
is an electrophysiological correlate of synchronous neuronal
discharge) resulted from massive Ca2+-dependent release of
glutamate from astroglia; the antiepileptic drugs suppressed
astroglial Ca2+ waves and inhibited PCDs (Nedergaard et al.
2010 and references therein). Finally, glial Ca2+ dyshomeo-
stasis can be involved in altered neurotransmission in psychi-
atric disorders and various neurodegenerative pathologies.
For example, significant elevation in resting [Ca2+]i levels
and enhanced spontaneous [Ca2+]i oscillations were found
in transgenic model of Alzheimer disease (Kuchibhotla et al.
2009).
7 S U M M A RY A N D P E R S P E C T I VE S
It is generally acknowledged that excitability of neuroglia is
mediated by intracellular Ca2+ signals resulting from Ca2+
movements between intracellular compartments and the
cell and extracellular space. Neuroglial cells express highly
sophisticated and intrinsically regulated molecular cascades
responsible for cellular Ca2+ homeostasis and Ca2+ signal-
ing. Stimulation of neuroglial cells triggers coordinated Ca2+
signals that can appear in a form of local Ca2+ microdomains,
global Ca2+ signals, [Ca2+]i oscillations, or propagating Ca2+
waves. This complex organization of Ca2+ signals in both
space and time determines functional outcome, which ranges
from local signaling at the level of single synapse (regulation
of ion/neurotransmitter release or sequestrating, local meta-
bolic support) to regulation of long-lasting adaptive responses
(myelination, cell survival, cell death, and differentiation
or defensive reactions such as microglial activation or reac-
tive astrogliosis). The main future challenge lies in detailed
description of these Ca2+ signals in the cells in vivo and in
establishing the links between Ca2+ dynamics and function
of neuroglial cells. Furthermore, the interplay between neu-
roglial Ca2+ dynamics and inherently linked intracellular
Na+ dynamics will have to be detailed and cross-correlated
in respect to in functional contribution of neuroglia to the
operation of the brain.
AC K N OW L E D G M E N T S
The authors’ research was supported by Alzheimer’s Research
Trust (UK) Programme Grant (ART/PG2004A/1) to AV;
an Ikerbasque grant to A. V.; a National Science Foundation
(CBET 0943343) grant to V. P.; and a Grant Agency of the
Czech Republic (GACR 305/08/1384) grant to A. V.
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 27.
THE CENTRAL ROLE OF ASTROCY TES IN
NEUROENERGETICS
Pierre J. Magistretti and Luc Pellerin

A B B R E VI AT I O N S state that after all the term neuroglia as it has been used in the
past is well adapted to describe a type of tissue which, while
ATP adenosine-5′-triphosphate being connective in nature as it connects elements of different
BDNF brain-derived neurotrophic factor nature and subserves the function of distributing nutrients,
CBF cerebral blood flow still is different from the connective tissue because of mor-
CREB cAMP response element binding protein phological and chemical characteristics as well as because of
DAB 1,4-dideoxy-1,4-imino-d-arabinitol its fundamentally different embryonic origin” [Dichiaro anzi
EAAT excitatory amino acid transporter che, dopo tutto, la parola nevroglia adoperata nel senso passato in
GABA gamma-aminobutyric acid uso mi sembra abbia titoli di preferenza, valendo ad indicare un
GDH glutamate dehydrogenase tessuto, che sebbene sia connettivo, perch connette elementi d’altra
GLAST glutamate aspartate transporter natura e alla sua volta serve alla distribuzione del materiale
GLT-1 glutamate transporter 1 nutritizio, pure si differenzia dal connettivo comune per carar-
GLUT glucose transporter reri morfologici, chimici, e quasi certamente, come diròin seguito,
LDH lactate dehydrogenase anche pel carattere fondamentale della diversa origine embrion-
MCT monocarboxylate transporter ale.@. Despite such an early suggestion for a role of astrocytes
mGluR metabotropic glutamate receptor in metabolic control, it was not until recently that the experi-
NMR nuclear magnetic resonance mental evidence supporting this early intuition was obtained.
PGE2 prostaglandin E2 Additional features such as the presence of specific glucose
PPP pentose phosphate pathway transporters (GLUT1) at the surface of astrocytes (Morgello
TCA tricarboxylic acid cycle et al. 1995; Yu and Ding 1998) favor the notion that these cells
VIP vasoactive intestinal peptide represent an active uptake site for circulating glucose into the
brain parenchyma. Other astrocytic processes ensheath syn-
apses, and express receptors and reuptake sites for a variety of
1 INTRODUCTION neurotransmitters (see chapters 16, 17, and 35) providing astro-
cytes with the capacity of sensing synaptic activity in order to
As highlighted in other chapters of this book, astrocytes come couple it with glucose utilization (Pellerin and Magistretti
in close contact with several cellular components of the brain 1994) and, as recently revealed, with the local regulation of
parenchyma including blood vessels, pial surfaces, neurons, blood flow (see chapter 37).
and other glial cells. Although astrocytes have been consid- This chapter reviews the mechanisms that have been dis-
ered traditionally as structural elements within the central covered during the last two decades supporting a central role
nervous system with the main function of maintaining the of astrocytes in brain energy metabolism, particularly as such
nervous tissue in place, hence their designation as neuroglia regulation occurs in register with synaptic activity.
or nerve glue, studies over the last two decades have high-
lighted a much more dynamic role for these cells. Already at
the end of the 19th century anatomists noticed the strategic 2 D I F F E R E N T A N D C O M P L E M E N TA RY
position occupied by astrocytes between neurons and blood M ETA B O L I C P R O F I L E S O F
vessels. It was also realized that astrocytes were sending spe- ASTROCY TES AND NEURONS
cific processes, called endfeet, in close contact with capillaries,
covering almost their entire surface as recently unequivocally There is now considerable evidence gathered from both in vitro
documented (Kacem et al. 1998). This arrangement sug- and in vivo studies that indicate clear differences in the meta-
gested that astrocytes might dynamically regulate the entry bolic profiles of astrocytes and neurons. Early studies on enzy-
and distribution of energy substrates to neurons, providing matic analyses of individually isolated cells (Hamberger and
an early argument in favor of their role in the regulation of Hydén 1963; Hydén and Lange 1962) as well as the most recent
brain energy metabolism (Andriezen 1893). This hypothesis gene expression analyses (Cahoy et al. 2008; Lovatt et al. 2007;
was eloquently formulated by Golgi (1886): “I would like to Rossner et al. 2006), indicate a predominance of glycolytic

333
and glycogen pathways in astrocytes and of lactate utilization
in neurons. For example, key enzyme and transporter isoforms
such as lactate dehydrogenase and monocarboxylate transport-
ers are selectively expressed in each cell type (Bittar et al. 1996;
Laughton et al. 2007; O’Brien et al. 2007; Pellerin et al. 2005).
Functional studies in each cell type have consistently supported
the view of a predominant glycolytic activity in astrocytes
and an oxidative profile in neurons (Boumezbeur et al. 2010a;
Bouzier-Sore et al. 2006; Itoh et al. 2003; Lebon et al. 2002.). It
also appears that neurons can efficiently use lactate as an energy
substrate (Boumezbeur et al. 2010b; Bouzier et al. 2000; Qu
et al. 2000; Serres et al. 2005; Schurr et al. 1997) and even pref-
erentially oxidize lactate over glucose when both substrates are
present (Bouzier-Sore et al. 2006; Itoh et al. 2003).
Recent evidence provides an explanation for the limited
capacity of neurons to sustain glycolysis. Indeed, in con-
trast to astrocytes, the enzyme 6-phosphofructose-2-kinase/
fructose-2,6-bisphosphatase-3, which is a key positive modu-
lator of glycolysis, is virtually absent in neurons due to its
constant proteasomal degradation (Almeida et al. 2004;
Herrero-Mendez et al. 2009). Consequently, neurons display a
slower glycolytic rate than astrocytes and are unable to upregu-
late this pathway (Almeida et al. 2004; Herrero-Mendez et al.
2009). It appears that in neurons, the glucose flux is predomi-
nantly through the pentose phosphate pathway (PPP)—which
is essential for the production of NADPH and therefore for
the maintenance of the cellular antioxidant potential. This
profile is consistent with the preferential use of lactate as an
oxidative substrate by neurons, because it allows the sparing of
glucose for the PPP, while providing, after conversion to pyru-
vate, the fuel for the production of adenosine-5′-triphosphate
(ATP) by oxidative phosphorylation (Bolanos et al. 2010).
In terms of their specific metabolic profile, astrocytes
are endowed with the capacity for the rapid removal of
synaptically-released glutamate, a process primarily achieved
by the astrocyte-specific sodium-dependent high affinity gluta-
mate transporters GLT-1 and glutamate aspartate transporter
(GLAST) corresponding to human excitatory amino acid
transporter EAAT2 and EAAT1, respectively (Bak et al. 2006).
Glutamate is converted to glutamine by the astrocyte-specific
enzyme glutamine synthetase. After release and transfer to neu-
rons, glutamine is converted back to glutamate via deamination
by the neuron-specific enzyme phosphate-activated glutami-
nase in a metabolic pathway known as the glutamate-glutamine
cycle (Bak et al. 2006; McKenna 2007).
In addition to the glutamate/glutamine cycle, other path-
ways have been proposed to replenish the neuronal glutamate
pool; these mechanisms involve de novo synthesis of glutamate.
One of these pathways is based on the ability of astrocytes to
provide the carbon backbone for the synthesis of glutamate
from glucose as α-ketoglurate. Then, α-ketoglutarate formed
in the glial tricarboxylic acid cycle (TCA) cycle could be con-
verted to glutamate upon addition of an amino group. This
amino group would be provided by either free ammonia
brought from the circulation in a reaction catalyzed by glu-
tamate dehydrogenase (GDH) or from leucine also imported
from the circulation via a transamination mediated by leucine
transaminase leading to the formation of α-ketoisocaproate
334 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
in addition to glutamate (Yudkoff 1997). Glutamate formed
through this mechanism would simply reintegrate the gluta-
mate/glutamine cycle via its conversion to glutamine in astro-
cytes and its subsequent release. Another possibility is that
astrocytes could export α-ketoglutarate directly to neurons
(Peng et al. 1993; Shank and Campbell 1984). Neurons would
themselves resynthesize glutamate either via a GDH-catalyzed
reaction or by a transamination of alanine by ALAT (alanine
aminotransferase). Notice that in these last two pathways,
use of α-ketoglutarate generated by astrocytes to resynthesize
glutamate could eventually lead to a depletion in TCA cycle
intermediates. In order to avoid such a situation, it is neces-
sary for the astrocyte to form new TCA cycle intermediates
downstream of α-ketoglutarate. This is done by an anaplerotic
reaction catalyzed by the enzyme pyruvate carboxylase that is
found specifically in astrocytes (Shank et al. 1985). In this
reaction, CO2 is fixed to pyruvate in order to form oxaloac-
etate. In this manner, a complete astrocytic TCA cycle can be
maintained despite the loss of α-ketoglutarate for glutamate
synthesis. Finally, neurons were also shown to have the possi-
bility of synthesizing glutamate via TCA cycle intermediates
and replenish their intermediate pool by an anaplerotic reac-
tion of CO2 fixation (Hassel and Brathe 2000).
The important difference in their metabolic profiles, nota-
bly as far as glycolysis is concerned, is reflected in a larger
glucose utilization by astrocytes than neurons (Barros et al.
2009; Bouzier-Sore et al. 2006; Waagepetersen et al. 1998),
in particular during activation. Indeed, an increase in glucose
uptake into astrocytes, mainly induced by glutamate, has been
widely confirmed since the original description by Pellerin and
Magistretti in 1994 (Bittner et al. 2011; Chuquet et al. 2010;
Keller et al. 1996; Takahashi et al. 1995). In contrast, glucose
transport into neurons is decreased upon glutamate exposure
(Loaiza et al. 2003; Porras et al. 2004).
A final note on cell-specific metabolism: as discussed in
chapter 36 and in paragraph of this chapter, astrocytes are the
exclusive site of glycogen metabolism in the brain.
3 G LU TA M AT E -M E D I AT E D
T R A N S M I S S I O N D R I VE S E N E R GY
U T I L I Z AT I O N I N T H E B R A I N
Nuclear magnetic resonance (NMR) studies have suggested
that there is a particular mechanistic link between the recy-
cling of glutamate that occurs essentially in astrocytes, and
glucose metabolism that provides the energy to maintain neu-
ronal activity (Hyder et al. 2006; Sibson et al. 1998). From
these experiments, it was also concluded that restoration of
ion gradients that occurs in parallel with glutamate/glutamine
cycling as a consequence of glutamatergic neurotransmis-
sion is responsible for approximately 80% of oxidative glu-
cose consumption under normal unanesthetized conditions.
In other words, processes not involved in neurotransmission
but rather in maintenance of cell structure and function like
protein and membrane turnover account for a rather small
fraction of the total energy consumption. The same can be
said for other neurotransmitter systems. In the specific case of
gamma-aminobutyric acid (GABA), which can be included in
the glutamate/glutamine cycle rate because GABA is partly
recycled in astrocytes via glutamine synthesis by a pathway
known as the GABA shunt (McGeer and McGeer 1989), the
estimate is that it would not account for more than 11% of
the total glutamine flux in the rat (Rothman et al. 1999) or
ten times less than for glutamate in human cortex (Shen et al.
1999). The essential conclusion is that the majority of energy
consumption (which consists almost entirely of glucose oxida-
tion) is a direct reflection of the level of glutamatergic neuro-
transmission and its associated recycling involving astrocytes.
Interestingly, using a theoretical bottom-up approach,
Attwell and Laughlin estimated the relative energy cost of
different cellular processes involved in brain activity both for
rodents and primates. Attwell and Laughlin (2001) came to
the conclusion that the large majority of energy consumption
would be caused by excitatory signaling, mostly the reestablish-
ment of ion gradients following action potential propagation
and postsynaptic currents. Their value of 81% to 84% of energy
cost attributed to excitatory (essentially glutamatergic) signal-
ing is in good agreement with NMR studies mentioned in the
preceding. Moreover, estimation of glial energy cost, including
glutamate recycling, would account for approximately 6% of
total energy usage. These data offer guidelines to assign energy
consumption to specific processes and fix certain limitations
as well as proportions. What they do not provide, however, is
the mechanism(s) linking these energy consuming processes to
energy generation and they do not take into account the pos-
sibility of cooperation between neurons and astrocytes.
4 TOWA R D A U N I F Y I N G C O N C E P T:
T H E R ET I N A M O D E L
Experiments performed in the honeybee retina had already
suggested that a marked metabolic compartmentation occurs
between glial cells and photoreceptors. Thus, it was observed
that upon exposure to light, an increase in 2-deoxyglucose
accumulation was taking place exclusively in glial cells while an
increase in oxygen consumption, indicative of increased oxi-
dative metabolism, occurred in photoreceptors (Tsacopoulos
et al. 1988). These data suggested two important points: (1)
There must exist a neuronal signal released to trigger the
increased glucose utilization in glial cells. (2) A substrate must
be released by glial cells to serve as an oxidative fuel in photo-
receptors. These questions were investigated in both honey-
bee and mammal retinal preparations by the group of Marcos
Tsacopoulos at Geneva University. It was observed that gluta-
mate (together with ammonia) constitutes the signal released
by photoreceptors to induce the metabolic response in Müller
glial cells of the retina. The critical step in the mechanism of
activation is the conversion of glutamate into glutamine by
glutamine synthetase and its associated ATP consumption
(Poitry et al. 2000). In the honeybee retina, it was observed
that alanine was the metabolite released by glial cells to fuel
photoreceptor oxidative metabolism (Tsacopoulos et al.
1994). In contrast, it was shown that in mammalian retina, lac-
tate was the most likely candidate (Poitry-Yamate et al. 1995).
T H E C E N T R A L R O L E O F A S T R O C Y T E S I N N E U R O E N E R G ET I C S
5 A S T R O C Y T E -N E U R O N L AC TAT E
S H U T T L E : T H E M A I N N E U R O M ETA B O L I C
COUPLING MECHANISM
Astrocytes have a prominent capacity to spontaneously con-
sume glucose and produce lactate under normoxia (Walz
and Mukerji 1988). Such a metabolic characteristic was first
observed on tumor cells and was termed aerobic glycolysis
by Otto Warburg in the 1920s (Warburg et al. 1924). Since
that time, it was demonstrated that aerobic glycolysis is not
restricted to cancer cells but it can be exhibited by normal cells
like astrocytes. Interestingly, it can be further enhanced under
specific conditions in this cell type. Thus, it has been shown that
glutamate, via a mechanism that involves its uptake through
astrocyte-specific, high affinity Na+-dependent transporters
followed by an activation of the Na+/K+ ATPase, enhances
both glucose utilization and lactate production (Pellerin and
Magistretti 1994). Based on this observation, it was suggested
that astrocytes might be endowed with the capacity to detect
synaptic activity at glutamatergic synapses and consequently
metabolize glucose into lactate that would be provided to
neurons (Fig. 27.1). Evidence for such a central function, that
would be critically dependent on glial glutamate transporters,
has been provided in vivo. Injection of antisense oligonucle-
otides directed against GLAST, one of the two glial gluta-
mate transporters, led to a decrease of the glucose utilization
response triggered by whisker stimulation in the somatosen-
sory cortex (Cholet et al. 2001). Similarly, a reduction of the
glucose utilization response upon activation of the whisker-
to-barrel pathway in knockout mice for either GLAST and
GLT-1, the two glial glutamate transporters, was observed in
animals at postnatal day 10 (Voutsinos-Porche et al. 2003a),
whereas a persistent decrease in metabolic response was
seen in adult GLT-1 knockout mice (Voutsinos-Porche et al.
2003b). Furthermore, such findings were confirmed in both
the visual cortex (Herard et al. 2005) as well as in the olfactory
bulb (Martin et al. 2012).
Several observations made in vitro as well as in vivo have
provided further insights into the intracellular mechanism
associated with the metabolic response of astrocytes. In par-
allel with the increase in glucose use triggered by glutamate
uptake, it was demonstrated that glutamate causes a rapid
stimulation of glucose transport in astrocytes (Loaiza et al.
2003). The observed increase developed within 10 seconds.
Such a fast time course would be consistent with the concept
of a rapid uptake and conversion of glucose into a metabolic
intermediate (e.g., lactate) to be released for neuronal use after
a brief activation typically encountered in the central ner-
vous system. Based on the known stoichiometry of glutamate
transporters that co-transport every glutamate with three Na+
ions, it was predicted that intracellular sodium concentration
changes should be a critical factor involved in the metabolic
response of astrocytes. Indeed, it was shown that glutamate
uptake caused a significant increase of intracellular Na+ con-
centration in astrocytes (Chatton et al. 2000), and this in turn
was an essential condition for enhanced aerobic glycolysis
(Voutsinos-Porche et al. 2003a). As a consequence of the ele-
vation in intracellular Na+ concentrations, a direct activation
• 335
 A
Glutamatergic synapse Astrocyte Capillary

GLUTAMINE
ATP
LACTATE
Synaptic vesicles
ADP
GLUCOSE
GLUTAMATE GLUCOSE
H+
K+

Neuronal Na+ Glycolysis

ATP
glutamate K+
receptors ADP
NaK
LACTATE ATPase
a2

Na+

B GLAST +/+ GLAST –/–

650 700

20 20
nCulg

Figure 27.1 Astrocyte–Neuron Metabolic Interactions. A. Schematic representation of the astrocyte-neuron lactate shuttle (ANLS). Glutamate (Glu)
released at the synapse activates glutamatergic receptors (GluR) and is associated with important energy expenditures in neuronal compartments.
A large proportion of the glutamate released at the synapse is taken up by astrocytes via excitatory amino acid transporters (EAATs, more specifically
GLT-1 and GLAST) together with 3 Na+ ions. This Na+ is extruded by the action of the Na+/K+ ATPase, consuming ATP. This triggers nonoxida-
tive glucose utilization in astrocytes and glucose uptake from the circulation through the glucose transporter GLUT1 expressed by both capillary
endothelial cells and astrocytes. Glycolytically derived pyruvate is converted to lactate by lactate dehydrogenase 5 (LDH5; mainly expressed in astro-
cytes) and shuttled to neurons through monocarboxylate transporters (mainly MCT1 and MCT4 in astrocytes and MCT2 in neurons). In neurons,
this lactate can be used as an energy substrate following its conversion to pyruvate (Pyr) by LDH1 (mainly expressed in neurons). Neurons can also
take up glucose via the neuronal glucose transporter 3 (GLUT3). Concomitantly, astrocytes participate in the recycling of synaptic glutamate via the
glutamate-glutamine cycle. Following its uptake by astrocytes, glutamate is converted to glutamine (gln) by the action of glutamine synthetase (GS)
and shuttle to neurons where it is converted back to glutamate by glutaminases (GLS). B. Representative pseudocolored digi-tized autoradiograms
were obtained either from antero-posterior coronal sections at the level of the somatosensory barrel field or from tangential sections through layer IV
of the primary somatosensory cortex. The level of 2-DG uptake is color coded according to the respective colored scales. Unilateral left or bilateral
C1C2 whisker stimulation in GLAST+/+ and GLT-1+/+ mice produced a local increase in 2-DG uptake in the right somatosensory cortex (white
square) in control, but not GLAST deficient mice. Modified from Pellerin and Magistretti 1994 and Voutsinos-Porche et al. 2003a.

of the Na+/K+ ATPase was evidenced (Pellerin and Magistretti
1997) together with a decrease of its association with acety-
lated tubulin (Casale et al. 2003), an indication of a switch
from inactive to activated state. Moreover, it was suggested
from in vitro experiments that a particular catalytic subunit
of the Na+, K+ ATPase, akin to the α2 subunit, is specifically
activated upon glutamate uptake in astrocytes (Pellerin and
Magistretti 1997). In vivo, the distribution of the α2 subunit
as observed both at the light and electron microscopic lev-
els revealed not only that it is expressed more specifically by
astrocytes but that it is found on processes that ensheath glu-
tamatergic but not GABAergic synapses (Cholet et al. 2002).
Moreover, a strong colocalization between the Na+/K+ ATPase
α2 subunit and both glial glutamate transporters GLAST and
GLT-1 was observed, suggesting a close association aimed
at maximizing the efficiency of glutamate transport and its
associated metabolic response. These data clearly identify glu-
tamate uptake and the subsequent activation of a particular
subunit of the Na+/K+ ATPase as key elements in the coupling
mechanism involving astrocytes, although other signals and
mechanisms (e.g. potassium) could also contribute to rapid
and reversible glycolytic activation (Bittner et al. 2011).
6 L AC TAT E A S T H E P R E F E R R E D E N E R GY
S U B S T R AT E F O R N E U R O N S
Since the pioneering work of Henry McIlwain in the 1950s,
evidence for the use of lactate as an energy substrate by neu-
rons has been accumulating (for review, see Bouzier-Sore et al.
2002; Pellerin 2003). Such evidence was provided by different
approaches ranging from oxidation rates measured in cultured

336 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
cells to NMR spectroscopy in vivo. One consistent and system-
atic finding is the fact that lactate is efficiently oxidized to CO2
in different neuronal preparations and is usually preferred to
glucose for this purpose. Experiments performed with brain
slices (Fernandez and Medina 1986; Ide et al. 1969), cul-
tured telencephalic neurons (McKenna et al. 2001; Tabernero
et al. 1996; Vicario et al. 1991), aggregated neuronal cultures
(Honegger et al. 2002), sympathetic ganglia (Larrabee 1983,
1992, 1995, 1996) as well as synaptic terminals (McKenna et al.
1993, 1994, 1998) have all reached the same conclusion. Lactate
was also able to sustain the same respiration rate attained with
glucose as determined by measurement of mitochondrial dehy-
drogenase activity (Pellerin et al. 1998a). Recent studies have
further confirmed and extended these findings. Sokoloff and
colleagues have demonstrated that neurons in culture display
a kinetic preference for oxidation of extracellular lactate over
lactate/pyruvate produced intracellularly from glucose via gly-
colysis (Itoh et al. 2003). In contrast, they showed that it was
not the case for cultured astrocytes that exhibit an intense gly-
colytic activity with a predominance of lactate release rather
than use. Use of NMR spectroscopy to investigate this ques-
tion has also provided unequivocal answers. Both GABAergic
and glutamatergic neurons in culture were shown to actively
metabolize lactate through the TCA cycle as determined by
the labeling from lactate of numerous TCA cycle intermedi-
ates and several related amino acids (Schousboe et al. 1997;
Waagepetersen et al. 2000). It was also estimated that com-
pared to glucose, lactate must have an equivalent access to the
TCA cycle (Waagepetersen et al. 1998). A quantitative assess-
ment of the concomitant oxidative use of lactate and glucose
was performed by NMR spectroscopy on cultured telenceph-
alic neurons (Bouzier-Sore et al. 2003, 2006). When both
substrates were present at an equimolar concentration (either
1.1 or 5.5 mM), it was determined that 79% of neuronal oxi-
dative metabolism was supported by lactate while only 21%
relied on glucose-derived pyruvate. Such observations leave no
doubt that at least in vitro, lactate represents a prominent and
preferential oxidative energy substrate for neurons.
Some indications have been provided to suggest that it is
also the case in vivo. Measurements performed using either
microdialysis or lactate-sensitive microelectrodes have shown
enhanced lactate utilization following either electrical or
behavioral activation (Fellows et al. 1993; Hu and Wilson
1997). Observations in humans have revealed a reduction in
cerebral glucose utilization upon raising plasma lactate levels
(Smith et al. 2003). More direct evidence of lactate utilization
by the brain was provided by NMR spectroscopy. It was shown
that following intravenous injection in rat, labeled [U-(13C)]
lactate was readily metabolized once it had entered the brain
in a compartment deprived of pyruvate carboxylase activity
(Bouzier et al. 2000; Hassel and Brathe 2000). Because pyru-
vate carboxylase expression is restricted to astrocytes, it was
inferred that lactate utilization must take place principally
in neurons. Furthermore, from the labeling pattern of key
compounds such as glutamine, glutamate, and GABA, it was
concluded that lactate is prominently used in glutamatergic
neurons (Qu et al. 2000). Similarly, it was shown that plasma
lactate is readily taken up by the human brain and oxidized
T H E C E N T R A L R O L E O F A S T R O C Y T E S I N N E U R O E N E R G ET I C S
by neurons in a manner analogous to glucose (Boumezbeur
et al. 2010b). In addition, a direct demonstration that lactate is
a preferential neuronal energy substrate in vivo has been pro-
vided in rat (Wyss et al. 2011).
Functional evidence has also been provided to support
the notion that lactate is used, in conjunction with low levels
of glucose, to support neuronal activity. Several studies per-
formed on hippocampal slices have highlighted not only the
neuroprotective action of lactate but also its ability to main-
tain synaptic function under conditions where glucose avail-
ability is reduced (Cater et al. 2001; Fowler, 1993; Ivanov et al.
2011; Izumi et al. 1994, 1997a,b; Rouach et al. 2008; Schurr
et al. 1988, 1999). Similarly, it was shown that lactate restores
excitability of glucoresponsive neurons from hypothalamic
and brainstem nuclei following glucose deprivation (Ainscow
et al. 2002; Himmi et al. 2001; Mobbs et al. 2001). In vivo,
the usefulness of lactate to preserve cognitive functions dur-
ing controlled hypoglycemic episodes has been demonstrated
(King et al. 1998; Maran et al. 1994, 2000), thus further
strengthening the concept of lactate as a significant and valu-
able fuel to sustain neuronal activity and brain function. As
further discussed in paragraph 7, lactate transfer from astro-
cytes to neurons is required for long-term memory (Suzuki
et al. 2011).
7 M O N O C A R B OXY L AT E T R A N S P O RT E R S :
M A I N G AT E S F O R L AC TAT E
TRAFFICKING
Lactate, together with pyruvate and the ketone bodies
acetoacetate and E-hydroxybutyrate, belongs to a group of
compounds known as monocarboxylates. Because of their
hydrophilic nature, these substances require a transporter to
cross cellular membranes. A family of proton-linked mono-
carboxylate transporters has been described. It contains
14 members sharing sequence homologies and identified as
monocarboxylate transporter (MCT)1 to 9 and MCT11
to 14 as well as another member known as TAT1 (Drewes
2003; Halestrap and Meredith, 2004). Of those nine trans-
porters, only MCT1, MCT2, MCT3 and MCT4 have been
functionally characterized and proven to act as monocar-
boxylate carriers. Kinetically, MCT2 was found to have the
lowest Km and thus the highest affinity for its substrates while
MCT4 displayed a very high Km. In the central nervous sys-
tem, the presence of MCT1, MCT2 and MCT4 has been
detected both at the mRNA and protein levels (Bergersen
et al. 2001; Jackson et al. 1997; Koehler-Stec et al. 1998;
Pellerin et al. 1998b). Studies on the cellular distribution of
these MCTs by immunohistochemistry, both in vitro and
in vivo, revealed a specific pattern. Thus, in addition to a
strong expression on blood vessels (Gerhart et al. 1997, 1998)
and on oligodendrocytes (Rinholm et al. 2011), MCT1
was found to be highly expressed by astrocytes, both in cul-
tures (Bröer et al. 1997; Debernardi et al. 2003; Hanu et al.
2000) and in vivo (Hanu et al. 2000; Pierre et al. 2000). A
small but significant expression was also found in cultured
cortical neurons (Debernardi et al. 2003) and by some neurons
• 337
in vivo (Leino et al. 1999). The predominant neuronal mono-
carboxylate transporter both in vitro and in vivo was found to
be MCT2 (Bergersen et al. 2001; Bröer et al. 1997; Debernardi
et al. 2003; Pierre et al. 2000, 2002). Finally, MCT4 is
expressed by Bergmann glia in the cerebellum (Bergersen et al.
2001) as well as by astrocytes in numerous brain areas includ-
ing the hippocampus (Pellerin et al. 2005; Rafiki et al. 2003).
Such a cell-specific distribution is paralleled by an enriched
expression of particular lactate dehydrogenase (LDH) iso-
forms displaying different kinetic characteristics. Isoforms
containing the LDHB subunits are more abundant in neurons
while isoforms enriched in LDHA subunits are enriched in
astrocytes (Bittar et al. 1996; O’Brien et al. 2007). Association
of the high affinity transporter MCT2 with LDH isoforms
enriched in LDHB subunits creates kinetic conditions highly
favorable for lactate uptake and utilization by neurons. The
presence of LDH isoforms predominantly exhibiting LDHA
subunits together with the high capacity monocarboxylate
transporters MCT1 and MCT4 rather provide optimal set-
tings to favor lactate production and release from astrocytes.
Considering the preferential metabolic profile exhibited by
each cell type as described above, these characteristics rein-
force the concept that lactate is shuttled preferentially from
astrocytes to neurons, at varying degrees depending on the
activation state.
Energy substrate utilization can be limited by the trans-
port capacity. The possibility of shuttling lactate between
brain cell types is determined by the expression of specific
transporters exhibiting different kinetics. Indeed, modeling
studies have demonstrated the key role that monocarboxy-
late transporters play in regulating lactate influx and efflux
(Aubert et al. 2005). It was previously shown that this could
be the case for neuronal lactate utilization since MCT2 over-
expression in cultured neurons using viral vectors promoted
lactate utilization in these cells when they were stimulated
with kainate (Bliss et al. 2004). It became of interest to deter-
mine whether the expression of monocarboxylate transport-
ers could be regulated in brain cells. In astrocytes, it was
recently shown that MCT4 expression is under the control
of the neuromodulator nitric oxide (Marcillac et al. 2011).
In parallel, it was found that MCT2 expression in neurons
is under the regulation of several neuroactive substances.
Noradrenaline, insulin, insulin-like growth factor-1, and
brain-derived neurotrophic factor (BDNF) were all shown to
enhance MCT2 expression in cultured neurons (Chenal and
Pellerin 2007; Chenal et al. 2008; Robinet and Pellerin 2010).
The mechanism involves a stimulation of translation via the
PI3K/Akt/mTOR/S6 pathway. Moreover, it was shown that
such activation occurs at the synaptic level (Robinet and
Pellerin 2010). The effect of BDNF on MCT2 expression
was recently confirmed in vivo after its injection into the
hippocampus (Robinet and Pellerin 2011). In addition to
changes in the overall expression levels, evidence was provided
that the amount of MCT2 proteins present at the cell sur-
face can be modified. It was shown that translocation of
MCT2 from an endogenous pool to the cell surface can be
induced in cultured cortical neurons by exposing them to a
combination of glutamate and glycine (Pierre et al. 2009).
338 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
Quite importantly, it was shown that a doubling of MCT2
expression at the cell surface leads to an increase of approxi-
mately 80% in lactate transport (Pierre et al. 2009). Thus,
changes in expression and localization of MCT2 induced by
neuroactive signals cause significant modifications of neuronal
energetics. Since such adaptations appear to occur quite rapidly
(<1 minute measured with static approaches), it is likely that
they represent the most appropriate process by which neurons
could adapt their substrate supply and concomitant energy
production to face changing needs associated with fluctuating
activity. It is expected that in the case of synaptic plasticity,
changes in synaptic efficacy will be accompanied by adapta-
tions in energy supply. The AMPA type glutamate receptor
subunit GluR2 is known to participate to the mechanism of
synaptic plasticity at glutamatergic synapses (Isaac et al. 2007).
Observations that MCT2 not only interacts with GluR2 but
modifies its subcellular distribution and expression levels sup-
port the idea that energy supply might be tightly regulated as
part of the mechanism of synaptic plasticity (Maekawa et al.
2009; Pierre et al. 2009). Indeed, it was recently demonstrated
that lactate supply to neurons via MCT2 is critical for both
short-term and long-term memory formation (Newman et al.
2011; Suzuki et al. 2011(see section 8).
8 A S T R O C Y T I C G LYC O G E N : I T S R O L E
I N N E U R OT R A N S M I S S I O N A N D
SY N A P T I C P L A S T I C I T Y
In addition to glutamate-stimulated aerobic glycolysis as
described by the ANLS, another astrocyte-based metabolic
pathway can produce lactate, namely glycogenolysis. Glycogen
is almost exclusively localized in astrocytes (Magistretti 2008).
Glycogen is not found in adult neurons, except occasionally in
large neurons in the brainstem or in the peripheral nervous sys-
tem (Sotelo and Palay 1968) (see also chapter 36). Surprisingly,
however, glycogen synthase—the enzyme responsible for gly-
cogen synthesis—is present in many neurons; it is however
permanently degraded by proteasome-dependent mechanism,
mediated by the malin–laforin complex (Vilchez et al. 2007).
This complex regulation is vital for neurons, as shown by the
fact that interventions aimed at maintaining glycogen syn-
thase function and therefore glycogen synthesis in neurons,
lead to neuronal apoptosis. A clinical condition, progressive
myoclonus epilepsy (also known as Lafora disease) is caused
by a loss-of-function mutation in the malin/laforin complex,
and is characterized by accumulation of glycogen-containing
deposits in neurons.
Astrocytic glycogen is subject to a tight regulation
by a restricted number of neurotransmitters/modulators
(Magistretti et al. 1981; Magistretti and Morrison 1988). Thus
noradrenaline (NA), vasoactive intestinal peptide (VIP) and
adenosine promote glycogenolysis in astrocytes. Because of
the particular morphology of VIP-containing neurons in the
cerebral cortex, the metabolic action of VIP is restricted to
cortical columnar modules, while that of noradrenaline, which
is released from noradrenergic intracortical horizontally-
oriented fibers, can prime metabolically the neocortex globally
(Magistretti and Morrison 1988) (Fig. 27.2). Noradrenaline and
VIP interact synergistically to produce radially-oriented corti-
cal “metabolic hot-spots” (Magistretti and Schorderet 1984).
Thus, the astrocyte-based glycogenolysis mediated by VIP,
NA, or adenosine (Magistretti et al 1986) represents another
mechanism to couple neuronal activity to energy metabolism
by providing additional energy substrates to active neurons
(Bélanger et al. 2011; Magistretti 2008). Consistent with this
view, stimulation of the whisker-to-barrel pathway promotes
glycogenolysis in the barrel field of layer IV somatosensory
cortex (Swanson et al. 1992). Maintenance of sustained axonal
spiking in mouse optic nerve, depends on adequate glycogen
content and lactate shuttling between astrocytes and the axonal
fibers (Tekkök et al. 2005).
Recent in vivo data have demonstrated a central role of
glycogenolysis and the resulting release of lactate from astro-
cytes in synaptic plasticity in a learning paradigm (Suzuki et al.
2011). Indeed, inhibition of glycogenolysis in the hippocampus
by stereotaxic administration of the glycogen phosphorylase
inhibitor DAB (1,4-dideoxy-1,4-imino-d-arabinitol) (Walls
et al. 2008) prevents the establishment of long-term memory
in an inhibitory avoidance learning paradigm. Since glycog-
enolysis results in lactate production by astrocytes, rescue of
the DAB effect was attempted by local administration of lactate;
under these conditions long-term memory was reestablished. As
I
II
& ters, adenosine, arachidonic acid metabolites and nitric oxide
III
VIP
IV
? nisms entail glutamate signaling on astrocytes for vasomotor
NA SA
V et al. 2010; Carmignoto and Gomez-Gonzalo 2010; Iadecola
VI
Pyr
SP
AF ECIF
FE IC
(SARE
)
W.M. AFFEREN
T Takano et al. 2006; Zonta et al. 2003). Arachidonic acid per
REN ERGIC
NORAD
(NA)
EFFERENT
Figure 27.2 Functional Domains for Glycogenolysis Induced by VIP and
NA in Astrocytes. The bipolarly oriented VIP neurons promote glycog-
enolysis locally within cortical columns, while the noradrenergic fibers
which have a tangentially organized trajectory promote glycogenolysis
globally across functionally-distinct regions of the cortex. The metabolic
effect of VIP and NA is exerted in astrocytes, the only brain cells that
contain glycogen, resulting in lactate release. From Magistretti and
Morrison. 1988.
T H E C E N T R A L R O L E O F A S T R O C Y T E S I N N E U R O E N E R G ET I C S
a corollary, downregulation of the expression of the astrocyte-
specific lactate transporter MCT4 also resulted in memory
inhibition, which was rescued by local administration of lactate
but not by equicaloric glucose. In contrast, inhibition of expres-
sion of the neuron-specific lactate transporter MCT2 although
also leading to amnesia was not subject to rescue by exogenous
lactate, indicating that lactate import into neurons is necessary
for long-term memory formation. Furthermore, in parallel
with the behavioral effects observed, the in vivo induction of
long-term potentiation and of genes known to be required for
memory formation such as phospho-CREB, activity regulated
cytoskeletal-associated protein, and phospho-cofilin was pre-
vented by inhibition of glycogenolysis and MCT4 expression
in a lactate-reversible manner (Suzuki et al. 2011). These data
clearly demonstrate that astrocyte to neuron lactate transfer is
required for long-term memory formation (Suzuki et al. 2011).
Similar results have been obtained in a different (working
memory) learning paradigm (Newman et al. 2011).
9 ROLE OF ASTROCY TE IN THE
R E GU L AT I O N O F L O C A L C E R E B R A L
B L O O D F L OW
As noted in the first paragraph of this chapter, astrocytes are
ideally positioned to sense synaptic activity and provide the
appropriate metabolic supply notably by interacting with
intraparenchymal capillaries. Accordingly, several lines of
experimental evidence indicate that astrocytes play a key role
in neurovascular coupling (see also chapter 37).
Since the original observation made by Sherrington reveal-
ing neurovascular coupling (Roy and Sherrington 1890), a
variety of vasoactive agents, including H+, K+, neurotransmit-
have been implicated in the activity-dependent increase in
cerebral blood flow (CBF) (Attwell et al. 2010; Carmignoto
and Gomez-Gonzalo 2010; Gordon et al. 2008; Iadecola and
Nedergaard 2007). It is now clear that several of these mecha-
responses, both vasoconstriction and vasodilation (Attwell
and Nedergaard 2007; Takano et al. 2006).
Glutamate receptors of the metabotropic type (mGluR)
activate Ca2+ transients in astrocytes resulting in activation of
cytosolic phospholipase A2, triggering the formation of vaso-
dilating agents such as epoxyeicosatrienoic acids and prosta-
glandin E2 (PGE2) from arachidonic acid (Attwell et al. 2010;
se, can diffuse to smooth muscle cells and cause vasoconstric-
tion after its conversion to 20-hydroxyeicosatetraenoic acid
(Metea and Newman 2006; Mulligan and MacVicar 2004).
Energy metabolism seems to also play a modulatory role in the
effects of prostanoids (Gordon et al. 2008). Thus, the direc-
tionality of the vasomotor effect of the astrocyte-derived vaso-
active molecule PGE2 depends on the activity of astrocytes.
Indeed extracellular lactate produced by astrocytes inhibits the
clearance of PGE2, thus promoting vasodilation. In contrast,
when extracellular lactate is low vasoconstriction prevails. In
• 339
addition, adenosine released by astrocytes under low-oxygen
inhibits astrocyte-mediated vasoconstrictions at the level of
smooth muscle cells by blocking the effect of arachidonic acid.
Interestingly, it was recently observed that increased blood
flow in activated brain regions is strongly correlated with
elevations in lactate concentrations, but negatively correlated
with oxygen consumption, further suggesting that increased
CBF is driven by glycolytic, as opposed to oxidative metabo-
lism under normal physiological conditions (Lin et al. 2010).
10 S U M M A RY A N D P E R S P E C T I VE S
This brief overview highlights the emerging role of astrocytes
not only in neurometabolic but also in neurovascular coupling
and strongly supports the notion that astrocytes are impor-
tant elements in the production of functional neuroimaging
signals in relation to synaptic activity (Figley and Stroman
2011; Magistretti and Pellerin 1996). The main challenges for
the future concern the exploration of the importance of this
role in neuroenergetics for astrocytes in the context of vari-
ous physiological and pathological situations. Indeed, it has
been suggested that astrocytes via their neurometabolic cou-
pling role could be important for various functions of the
central nervous system such as regulation of blood glucose
levels (Lam et al. 2005), arousal (Parsons and Hirasawa 2010),
respiratory control (Erlichman et al. 2008), sodium homeo-
stasis (Shimizu et al. 2007), or memory formation (Newman
et al. 2011; Suzuki et al. 2011). Moreover, targeting astrocytes
through an enhancement of their metabolic capacities could
represent a valuable therapeutic strategy in various neurode-
generative diseases for which a deficit in energetics has been
reported. Such an approach has been shown to be effective
in the case of excitotoxicity (Bliss et al. 2004) and metabolic
insult (Escartin et al. 2007), but other possibilities exist and
could be already indirectly exploited, as is the case for cogni-
tive enhancers (Pellerin and Magistretti 2005).
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GENOMIC PROFILES
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 28.
THE ASTROCY TE TRANSCRIPTOME
Ditte Lovatt and Maiken Nedergaard
A B B R E VI AT I O N S
Aldh1l1 10-formyltetrahydrofolate dehydrogenase
AQP aquaporin
FACS fluorescence-activated cell sorting
FISH fluorescent in situ hybridization
GFAP glial fibrillary acid protein
GFP green fluorescent protein
hGFAP human glial fibrillary acid protein
LCM laser capture microdissection
qPCR quantitative polymerase chain reaction
RNA-Seq Next Generation RNA sequencing
TRAP translating ribosome affinity purification
1 INTRODUCTION
Although astrocytes are the most abundant cell type in the
mammalian brain, surprisingly little is known with regard
to the functional properties of astrocytes in situ (Agulhon
et al. 2008; Ransom et al. 2003). Studies of astrocytic energy
metabolism, receptor expression, Ca2+ signaling, release of
neuroactive compounds, and interactions with other cell
types are traditionally based on observations of cultured cells
or acute slices prepared from rodent pups younger than three
weeks old (Nedergaard and Verkhratsky 2012; Theodosis et al.
2008). The inability to selectively stimulate astrocytes and
detect a functional output has prevented a basic analysis of the
functional properties of astrocytes in the adult intact central
nervous system. The first analysis of adult astrocytes in situ
first emerged five to six years ago, when several laboratories
pioneered the use of two-photon Ca2+ imaging combined with
electrophysiological recordings of synaptic activity (Dombeck
et al. 2007; Schummers et al. 2008; Takano et al. 2006; Wang
et al. 2006). These in vivo imaging studies showed that cor-
tical astrocytes display complex patterns of calcium signaling
in response to sensory stimulation and spontaneous calcium
spikes during locomotion in awake behaving head-restrained
mice. Nevertheless, it is not known whether astrocytic cal-
cium signaling plays a functional role in processing of sensory
information. Similarly, it is well documented from histologi-
cal studies that astrocytes display remarkable regional differ-
ences in morphology, spanning from rounded, polyhedral
cells in cortex to elongated asymmetrical cells in the neuro-
genic area of the hippocampal dentate region (Bushong et al.
2002; Oberheim et al. 2012). However, it is not established
whether the distinct morphological features of astrocytes
347
in different brain regions indicate that their functions dif-
fer. Another heavily debated topic is astrocyte heterogeneity
(Matyash and Kettenmann 2010; Zhang and Barres 2010). Are
there subtypes of astrocytes within the same region, and can
these be distinguished molecularly and functionally? In the
diseased brain, how do reactive astrocytes differ from healthy
ones? This chapter compares existing transcriptome databases
and evaluates whether these unique sets of information can be
used as a discovery tool to gain insight into the function of
astrocytes in the intact adult brain (Cahoy et al. 2008; Doyle
et al. 2008; Lovatt et al. 2007). Specifically, it discusses the use
of the astrocyte transcriptome in defining whether the physio-
logical characteristics, regional variations, and transformation
in reactive gliosis can be identified in the molecular profile of
astrocytes.
2 M ET H O D S U S E D TO P R O D U C E
T R A N S C R I P TO M E S F R O M A S T R O C Y T E S
I N I N TAC T T I S S U E
To date, only a few studies have reported on the transcriptome
from astrocytes in situ or in vivo (Cahoy et al. 2008; Doyle
et al. 2008; Lichter-Konecki et al. 2008; Lovatt et al. 2007;
Yang et al. 2011). In comparison, the transcriptomes of numer-
ous neuronal subtypes from striatal, cerebellar, cortical, and
hippocampal regions have already been published (Cahoy
et al. 2008; Doyle et al. 2008; Dugas et al. 2008; Heiman et al.
2008; Lobo et al. 2006; Molyneaux et al. 2005; Sugino et al.
2006). The technical aspects of transcriptomics procedures,
such as microarray and RNA-seq, have not prevented progress
in astrocyte transcriptomics. Rather, it is the technical aspects
of harvesting enriched populations of astrocytes in vivo or in
situ as a source of mRNA for transcriptomics that have pre-
vented progress mainly due to the limited number of trans-
genic animals with fluorescently labeled astrocytes. To date,
both microarray and RNA-seq can produce the transcriptome
from minute amounts of RNA with relative ease (Tang et al.
2009; Wang et al. 2009). The processing of RNA for microar-
ray or sequencing, as well as transcriptome data analysis, are
reviewed in detail elsewhere (Wang et al. 2009). However, there
are significant technical challenges in isolating mRNA from
astrocytes in vivo. As opposed to cultured astrocytes, which
are flat epithelioid-appearing cells, astrocytes in intact tissue
have highly branched processes and are intermingled with
neighboring cells, making it difficult to isolate mRNA from
astrocytes without mRNA contamination from surrounding
cell types (Bushong et al. 2002; Simard et al. 2003). This ana-
tomical feature is not unique to astrocytic preparations from
intact tissue. Isolating mRNA from neurons and other brain
cells has been equally difficult. However, many of the recently
developed techniques used to isolate mRNA from cells in situ
rely on the availability of transgenic lines with fluorescently
labeled cells of interest. To this end, far more transgenic lines
with fluorescently labeled neuronal subtypes exist than lines
labeling astrocytes (Feng et al. 2000; Gong et al. 2003; Regan
et al. 2007; Sugino et al. 2006; Zhuo et al. 1997).
Over the past decade, several experimental approaches have
been developed to harvest mRNA from single cells or single
populations of cells isolated from intact brain tissue. These
methods include fluorescence-activated cell sorting (FACS)
(Cahoy et al. 2008; Lobo et al. 2006; Lovatt et al. 2007),
immunopanning (panning) (Cahoy et al. 2008), translat-
ing ribosome affinity purification (TRAP)/Ribo-tag (Doyle
et al. 2008; Heiman et al. 2008; Sanz et al. 2009), laser cap-
ture microdissection (LCM) (Espina et al. 2006; Tang et al.
2009), patch pipette aspiration (Martina et al. 1998; Surmeier
et al. 1996), and manual sorting of reporter labeled cells in dis-
sociates (Hempel et al. 2007; Sugino et al. 2006) (Fig. 28.1).
Each method has advantages and disadvantages (Table 28.1).
Although LCM is capable of isolating mRNA from a single
cell for which the location and morphology is known, it has
to be done in fixed or frozen brain tissue, which may compro-
mise the quality of the mRNA. It is also technically difficult to
dissect out the cells without cross-contaminating with mRNA
from neighboring cells. Because of the small size of the astro-
cytic cell body, LCM is rarely used to isolate astrocytic mRNA
(Ferraiuolo et al. 2011). Refinement in LCM, including use of
vibrating piezoelectric driven micro-chisel, may enable isola-
tion of small astrocytic cell bodies in the future. FACS sort-
ing, panning, and manual sorting all use acutely dissociated
brain tissue for which the location and information about the
morphology of the sorted cells are lost during the dissociation
procedure. The standard dissociation protocol uses enzymatic
treatment with papain followed by mechanical dissociation
to achieve a single cell suspension. The cell types identified in
these suspensions include astrocytes, oligodendrocyte precur-
sors, microglia, neurons, and endothelial cells (Daneman et al.
2010; Dugas et al. 2008; Lobo et al. 2006; Lovatt et al. 2007).
Depending on the quality of the dissociation and the age of the
animal, astrocytes account for 30% to 50% of the dissociated

Start material: Brain tissue XFP expressing brain tissue TRAP BAC-transgenic brain

Freeze & Fix Dissociate Dissociate Lyze tissue

Procedure:

Label with antibody

LCM: Immunopanning: FACS: Manual sorting: TRAP:

mRNA isolation method: Dissect cell of interest Isolate cells Isolate cells Isolate cells and Affinity purify mRNA:ribosome
and extract mRNA and extract mRNA and extract mRNA extract mRNA complexes with GFP antibody

RNA amplification
and processing

Microarray RNA-seq

Transcriptome
analysis

Figure 28.1 Overview of methods used for transcriptome profiling of cells isolated from intact tissue. The type of cells to be isolated depends upon the
availability and type of transgenic mouse lines. If no transgenic line is available, the desired cell population can be isolated either by: i) surface-labeling
with a specific antibody, and then isolating the cells by FACS or immunopanning, or ii) by dissecting single cells or brain regions out from frozen and
fixed whole brain tissue selected either by morphology or antibody-labeling. If transgenics lines that label the desired cells fluorescently, are available,
then FACS or manual sorting can isolate these labeled cells. If BAC-transgenic lines, that label the translating ribosomes, are available, then the TRAP
method can be used to isolate the desired cell type. See table 28.1 for more details on these methods. Once the desired cell or cells are isolated by any
of the above methods, mRNA is extracted and linearly amplified to achieve μg levels of cDNA. The cDNA is then either labeled for microarray or is
used for library construction for RNA-seq. The resulting transcriptome is then analyzed and mined to identify, for instance, novel biomarkers, disease
related transcripts, cell-type specific alternative splicing and signaling pathways. (LCM) Laser Capture Microdissection, (FACS) Fluorescent Activated
Cell Sorting, (TRAP) Translating Ribosome Affinity Purification, (BAC) Bacterial Artificial Chromosome.

348 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
Table 28.1 mRNA COLLECTION METHODS FROM INTACT TISSUE PREPARATIONS
mRNA
COLLECTION CROSS- SINGLE CELL
METHOD CONTAMINATION HETEROGENEITY TIME (H)
FACS-array* Low + 5–67
Immunopanning† Low – 4
Manual sorting‡ Low + 3–5
LCM§ High + 1
Patch pipette¶ Medium + 1
TRAP# High – 3
Five hours using fluorescent reporter mice and six hours using antibody labeling of cells.
*Lovatt et al. 2007; †Cahoy et al. 2008; ‡Hempel et al. 2007; §Espina et al. 2006; ¶Martina et al. 1998; Surmeier et al. 1996; #Doyle et al. 2008.
cells (Lovatt et al. 2007). Astrocytes and other cell types can
be isolated from the dissociate by FACS or manual sorting
based on their expression of fluorescent reporters, such as
GFAP-GFP, or by either FACS or immunopanning based on
their expression of surface markers that can be tagged with an
antibody, such as anti-GLT1. Astrocytic mRNA has also been
harvested using the TRAP method, in which astrocyte-specific
expression of BAC transgenic GFP-ribosome chimeras serve as
a tag for affinity purifying mRNA–ribosome complexes from
tissue lysates using a GFP antibody (Doyle et al. 2008). Like
the methods using cell dissociates, information about cellular
morphology and subcellular location is lost using the TRAP
method. Further, some investigators have used a patch pipette
approach to aspirate cytosolic mRNA from whole-cell patched
cells in intact tissue (Martina et al. 1998; Surmeier et al. 1996);
however, a global transcriptome from brain cells using this
technique remains to be published. The development of meth-
ods being able to isolate purer sources of mRNA from single
astrocytes in intact tissue will facilitate the correlation of the
transcriptome to other types of single cell information, such as
morphology, chemical profile and physiological response pat-
terns (see Table 28.1). For instance, profiling single astrocytes
of different morphologies could provide insights into the mol-
ecules facilitating such morphological differences, and profil-
ing single astrocytes from distinct microenvironments could
provide important insights into how astrocytes interact with
the local environment.
A comparison of the transcriptomes produced by the
FACS, panning, TRAP, LCM, and manual harvesting meth-
ods showed that all of them demonstrated comparably high
levels of reproducibility (Okaty et al. 2011). Further com-
parison showed that the data from LCM and TRAP exhib-
ited significantly higher levels of mRNA contamination from
neighboring cells (see Table 28.1). In the TRAP transcrip-
tomes, contamination by other cell types was apparent for
astrocytes, GABAergic cells, and oligodendrocytes, suggest-
ing that contamination may be a general phenomenon of this
method. Although some degree of contamination is always
present, at least at trace levels, higher levels become a problem
T H E A S T R O C Y T E T R A N S C R I P TO M E
CELL SELECTION PARAMETERS
MOLECULAR CELL SUBREGIONAL
MARKER MORPHOLOGY LOCATION
+ – –
+ – –
+ – –
+ + +
+ + +
+ – –
when quantifying low abundance transcripts, or attempting to
identify cell specific transcripts.
Induction of immediate early genes is an important con-
cern when using time-consuming harvesting methods to iso-
late mRNA from intact tissue for transcriptome profiles. To
this end, both LCM and TRAP have the advantage of using
fixed and lysed tissue preparations in which biological activi-
ties can be inhibited. FACS, panning, and manual sorting use
live dissociated cells; therefore, timing becomes an important
factor because the majority of mature astrocytes in prepara-
tions of dissociated adolescent and adult brain tissue undergo
apoptosis within 40 hours (Foo et al. 2011). To generate
genomic profiles that pass quality control, it is crucial to use
high-quality mRNA that is not derived from dying cells with
increased RNAse activities. The tissue dissociation procedure
of adult tissue takes about three hours, and cell isolation and
subsequent mRNA extraction add an additional 2 hours. Most
published dissociation protocols attempt to keep the prepara-
tion cold whenever possible, and some even add inhibitors to
prevent excitoxicity when isolating neurons. RNA synthesis
or RNA degradation can also be inhibited by the addition of
pharmacological inhibitors, for instance, by storing sorted cells
in products such as RNA later (Belgard et al. 2011). An analysis
of inflammatory and stress-related transcripts in astrocytes and
other cell types did not indicate that the dissociation procedure
and subsequent FACS procedure induced any such responses
(Lobo et al. 2006; Lovatt et al. 2007), suggesting that these
procedures allow harvesting of RNA from nonstressed cells as
long as the procedure is carried out within a 6-hour time frame
at low temperatures and dying cells, identified by propidium
iodide uptake, are excluded.
3 T H E T R A N S C R I P TO M E O F A D U LT
A S T R O C Y T E S F R O M I N TAC T T I S S U E
The use of transcriptome profiling was first applied to astro-
cytes in situ in 2007 (Lovatt et al. 2007). Using FACS, two
distinct populations of astrocytes were isolated from acutely
• 349
dissociated adult, cortical tissue from mice based on their
transgenic GFAP-GFP expression and surface GLT1 protein
expression. Following isolation, RNA was immediately har-
vested for transcriptome profiling. Analysis of the transcrip-
tome of these astrocytes confirmed that they expressed several
expected astrocyte specific markers such as AQP4 (aquaporin
4), GFAP, Gjb6 (connexin 30), Gja1 (connexin 43), S100b,
and Slc1a2 (GLT1). In contrast, they did not express enriched
levels of markers characteristic of other cell types, suggesting
that the harvested RNA was from a pure population of astro-
cytes. The importance of pure population becomes significant
when considering low abundance transcripts and specificity
determination. Even a very low level of contaminating RNA
can obscure the transcriptome profile. Currently the only ways
to evaluate contamination are by immunolabeling the purified
cells with an astrocyte specific antibody, and inspecting the
transcriptome data for expression of cell type–specific mark-
ers (Lovatt et al. 2007). Besides the expression of anticipated
markers, the population of astrocytes expressed about 9,400
transcripts. Among these, roughly 3,000 transcripts were astro-
cyte enriched when compared with the astrocyte-depleted
pool. This is in line with the number of transcripts expressed
by populations of other cell types (Cahoy et al. 2008; Lobo
et al. 2006).
Once the in situ astrocyte transcriptome had been pro-
duced, one of the first questions was, how different are in situ
astrocytes from their cultured counterparts? Cultured astro-
cytes from neonatal rodent brain tissue have been the preferred
model to study the physiological properties of astrocytes since
the 1980s (McCarthy and de Villis 1980). Classically cultured
astrocytes are made from dissociated neonatal brain tissue
that is then cultured in high serum-containing media. At this
point in development, the potential for glia progenitors to dif-
ferentiate into astrocytes is high, and cultured astrocytes rep-
resent mostly progenitor cells that are differentiated in vitro
into a cell type expressing high of levels GFAP and vimentin
(Steindler and Laywell 2003). However, GFAP and vimentin
are both markers of reactive gliosis in vivo (Pekny and Nilsson
2005), questioning whether cultured astrocytes is a model
of reactive gliosis rather than a model of healthy astrocytes
in vivo (see chapter 51). However, more recent studies using
intact tissue have led to several discoveries suggesting that the
in vivo counterpart of cultured astrocytes differed morpho-
logically (Bushong et al. 2002) and functionally. For example,
voltage-gated Ca2+ channels in cultured astrocytes are not
present in vivo (MacVicar 1984). This is consistent with our
analysis of cultured astrocytes that, when compared with astro-
cytes in situ, express several transcripts encoding voltage-gated
calcium channels, including Cacna1d, Cacna2d1, Cacnb3,
and Cacnb4 (see data analysis in section 5). These discoveries
have left little support, indicating that cultured astrocytes suf-
ficiently modeled astrocytes in vivo, and pushed for the devel-
opment of novel methods that can be applied to the study of
astrocyte function both in intact tissue and under more physi-
ological conditions than the use of cultured astrocytes.
A comparison between in situ FACS-isolated GFAP-
GFP+ astrocytes and cultured FACS-isolated GFAP-GFP+
astrocytes prepared according to the classical protocols of
McCarthy and de Vellis (1980) revealed that the two types of
astrocytes are indeed very different (Lovatt and Nedergaard,
unpublished). A simple comparison of the correlation coef-
ficient showed a very high similarity among samples within
the same biological group (0.98–0.99) (Table 28.2). In con-
trast, the correlation coefficient between cultures and in situ
astrocytes (0.78) was in the same range as that of in situ astro-
cytes and the astrocyte-depleted pool (0.72), suggesting that
cultured astrocytes are as different from astrocytes in situ, as
astrocytes in situ are different from the average of all other
cells in the brain. A statistical comparison revealed that 17%
of the genes were significantly enriched in astrocytes in situ
as compared with cultures, and 24% of the genes were signifi-
cantly enriched in cultures as compared with in situ astrocytes.

Table 28.2 SIMILARITY OF IN SITU AND CULTURE ASTROCYTE TRANSCRIPTOMES

COMPARISON GROUPS R^2

Within groups GFAP-GFP+/GLT1+ in situ 0.98

GFAP-GFP–/GLT1+ in situ 0.98

GFAP-GFP+ culture 0.99

GFAP-GFP–/GLT1– in situ 0.99

Between groups GFAP-GFP+/GLT1+ in situ vs. GFAP-GFP–/GLT1+ in situ 0.93

GFAP-GFP+ culture vs. GFAP-GFP+/GLT1+ in situ 0.78

GFAP-GFP+/GLT1+ in situ vs. GFAP-GFP–/GLT1– in situ 0.72

GFAP-GFP+ culture vs. GFAP-GFP–/GLT1– in situ 0.80

In situ cell populations are FACS-purified and from Lovatt et al. (2007).
GFAP-GFP+ cultures were grown according to the procedure described by McCarthy and de Villis (1980), and then
FACS-purified for GFP after two in culture.
Lovatt et al. 2007.
McCarthy and de Vellis J. 1980.

350 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
For instance, only astrocytes in situ expressed the NMDA
receptor Grin2c, while only cultured astrocytes expressed the
AMPA receptor Gria3. Likewise, cultures expressed Adora2a
(adenosine receptor 2A), whereas in situ astrocytes expressed
Adora2b (adenosine receptor 2B). Cultures also expressed
a number of laminin subunits, suggesting that their surface
composition is different from astrocytes in situ. Other dif-
ferences included significant enrichment of the synaptic exo-
cytosis proteins Syt1 (synaptotagmin 1) and Syn1 (synapsin
1) in cultured astrocytes but not in situ astrocytes. Several
studies have found that astrocytes in culture possess the
same machinery as neurons for regulated fast exocytosis, but
whether astrocytes in vivo also exhibit fast exocytosis is heav-
ily debated. Our findings are consistent with previous stud-
ies showing that although astrocytes in vivo do express many
proteins involved in exocytosis, they lack the ones involved
in fast exocytosis usually observed in synapses (Cahoy et al.
2008; Wilhelm et al. 2004). These and other gene expression
differences may elicit distinct responses to ligands present in
the neuropil. Nevertheless, this finding questions the use of
cultured astrocytes as models of astrocytes.
In line with our findings, a recent study found that isolat-
ing astrocytes from P1 to P8 animals by immunopanning and
growing them in serum-free media led to a high rate of apop-
tosis 40 hours after plating. The survival rate was improved
after the addition of selected growth factors that matched the
receptor profile of the acutely isolated cells (Foo et al. 2011).
Subsequent transcriptional comparison of acutely isolated
astrocytes with classically cultured astrocytes and acutely
isolated astrocytes grown in serum-free media with heparin-
binding EGF-like growth factor as the growth factor for 7
days in vitro suggested that astrocytes grown in a select media
maintained a transcriptome profile that largely mimicked
that of acutely isolated astrocytes, whereas classically cultured
astrocytes differed dramatically from both acutely isolated
astrocytes and those cultured in serum-free medium for 7
days. Notably, it has not yet been validated whether astrocytes
cultured using this promising novel approach remain imma-
ture (i.e., similar to the day of harvest) or if they differentiate
and display a transcriptome profile that more closely mimics
the transcriptome of differentiated astrocytes isolated from
intact adult brain.
4 N O VE L G E N E S A N D PAT H WAYS
I D E N T I F I E D I N A S T R O C Y T E S BY
T R A N S C R I P TO M E P R O F I L I N G
Transcriptome profiling serves as a discovery tool to identify
novel transcripts expressed by a target cell population as well
as a tool to compare quantitative expression data among sev-
eral groups of cells. To this end, significant differences were
detected between distinct populations of FACS isolated cells.
GFAP-GFP+/GLT1+ and GFAP-GFP–/GLT1+ astrocytes
isolated from 10- to 12-week-old animals only displayed a
subtle difference of approximately 1% in significantly enriched
genes, whereas age-matched GLT1+ astrocytes and Thy1+
neurons had 14% of all present genes significantly expressed
T H E A S T R O C Y T E T R A N S C R I P TO M E
between them. Among the genes that differed between astro-
cytes and neurons were many related to metabolic pathways.
For instance, both cell types expressed genes involved in
the enzymatic processes of glycolytic and oxidative glucose
metabolism, suggesting that both astrocytes and neurons have
self-sufficiency with respect to anerobic and oxidative glucose
metabolism. In contrast, metabolic pathways that entered or
exited either glycolysis or the TCA cycle, such as those for
lactate or glutamate metabolism, were clearly compartmental-
ized between astrocytes and neurons (Lovatt et al. 2007). A
functional validation of these data using qPCR and mass spec-
troscopy of the glucose metabolites confirmed the findings of
the transcriptome analysis, and indicates that transcriptome
profiling is a powerful tool to gain insight into biological path-
ways and their differences among different cell types. A simi-
lar study used a combination of FACS and immunopanning
to purify RNA from developing astrocytes, oligodendrocyte
precursors, and neurons from acutely dissociated 17-day-old
mouse brain (Cahoy et al. 2008). This study also found that
purification by FACS or immunopanning yielded highly
pure populations of astrocytes, as indicated by significantly
enriched levels of Slc1a2 (GLT1), Gja1 (connexin 43), and
AQP4 (aquaporin 4), while being devoid of markers labeling
oligodendrocyte precursors and neurons.
Transcriptome profiling can also be used to identify novel
biomarkers in astrocytes. This is done by comparing the astro-
cyte transcriptome to the transcriptome of other cell types or
the transcriptome of unsorted cells, and then extracting genes
that are significantly enriched in astrocytes above a certain
threshold. Lovatt et al. identified by transcriptome profiling
924 genes that were highly enriched and presumably only
expressed in GLT1+ astrocytes as compared with GLT1– cells
and Thy1+ neurons. These astrocyte-enriched genes included
Cbs (Cystathionine β-synthase), Slc14a1 (solute carrier fam-
ily 14 member 1, an urea transporter), Mlc1 (Megalencephalic
leukoencephalopathy 1), and Aldh1l1 (10-formyltetrahydro-
folate dehydrogenase) in addition to the already-mentioned
biomarkers. Cahoy et al. also identified Aldh1l1 and validated
it as a new astrocyte-specific biomarker. Aldh1l1 is a cytosolic
enzyme that has several advantages over the well-characterized
astrocyte marker GFAP, which is a filament protein localized
mainly to larger, but not finer, astrocytic processes. In con-
trast, Aldh1l1 labels both large and fine processes, making it
a preferred marker to use for histology studies, because the
entire astrocyte can be visualized using a simple staining. An
independent study (Yang et al. 2011) followed up on the dis-
covery of Aldh1l1 and found that postnatal expression in cor-
tex peaks at P2 and then declines to a lower steady level for the
remainder of the rodent’s life. However, Aldh1l1 expression
in spinal cord appears to continuously decline from a peaked
level at P2. Yang et al. also found that Aldh1l1 is upregulated
in reactive astrocytes, similarly to GFAP. Thus, the expres-
sion of Aldh1l1 is developmentally regulated, and pathologi-
cal conditions also change its expression pattern. Exploring
the transcriptome profile of astrocytes in vivo (Table 28.3,
described in the following) hopefully will lead to discovery
of other novel cell-specific markers for which expression is
not regulated developmentally or by pathological processes.
• 351
Table 28.3 Enriched Genes in Cortical Astrocytes

1110020G09Rik Appl2 Ddah1 Fgfr1 Heph Mtmr11

1200009O22Rik Aqp4 Dio2 Fgfr3 Hes5 Myo6
1700019G17Rik Aqp9 Dlg5 Fjx1 Hhatl Nat1
1810014B01Rik Arhgef26 Dmp1 Fkbp10 Hopx Nat8
2310022B05Rik Asrgl1 Dmrta2 Fzd1 Hrsp12 Ndp
2610017I09Rik Atp13a4 Dpy19l3 Fzd2 Hsd11b1 Ndrg2
2610034M16Rik Atp1a2 Dtna Gabrg1 Htra1 Nkain4
2810459M11Rik Atp1b2 E130114P18Rik Gas1 Id4 Nkx2–2
2900052N01Rik Baalc E130304F04Rik Gdf10 Igdcc4 Npas3
4933431E20Rik Bcan Ednrb GFAP Igfbp2 Nr2e1
5730469M10Rik Bmpr1b Efhd1 Ghr Il18 Nrarp
A930005H10Rik Car8 Efs Gja1 Il33 Ntrk2
Aass Cbr3 EGFR Gjb6 Itih3 Ntsr2
Abcd2 Cbs Elmod3 Gldc Kcnj16 Nwd1
Abhd3 Cdh19 Elovl2 Gli2 Kcnn2 Olfml1
Abtb2 Chi3l1 Emx2 Gli3 Kctd14 Oplah
Acad11 Chpt1 Enho Glis3 Klf15 Pacrg
Acot1 Chrdl1 Entpd2 Glud1 Lcat Padi2
Acot11 Chst2 Ephx2 Gm11627 Lgi4 Papss2
Acsbg1 Cideb Eps15 Gm2115 Lgr4 Paqr8
Acsl3 Cldn10 Eps8 Gm266 Lhx2 Pax6
Acsl6 Clmn Eya1 Gm3932 Lrig1 Pdk4
Acss1 Cml1 Ezr Gm5089 Lsamp Pdlim4
Acss2 Cml5 F3 Gm715 Lxn Pdpn
Adhfe1 Cmtm5 Fabp5 Gm7901 Maob Pex11a
Adk Crot Fabp7 Gnao1 Masp1 Pfn4
Adora2b Cspg5 Fads1 Gpam Meis1 Pgcp
Agl Cth Fads2 Gpc5 Metrn Pgm2
Agpat5 Ctso Fam107a Gpld1 Mfge8 Phgdh
Agt Cxcl14 Fam176a Gpr37l1 Mfrp Phka1
Agxt2l1 Cyp2j6 Fam181b Gprc5b Mgst1 Phkg1
AI464131 Cyp2j9 Fam20a Gramd3 Mlc1 Pla2g7
AI848258 Cyp4f14 Fam55d Grin2c Mmd2 Plcd4
Aldh1l1 Cyp4f15 Fam59b Gstm1 Mras Pld2
Aldoc Cyp7b1 Fars2 Gstm5 Msi1 Plekhb1
Aox1 Daam2 Fbxo2 Hapln1 Mt1 Pm20d1
Apoe Dbi Fgf1 Hepacam Mt2 Polr3h

352 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
Table 28.3 (Cont’d )

Ppap2b Pygb S100b Slc1a3 Sox9 Tspan7

Ppil6 Pygm Sc4mol Slc25a18 Spag1 Tst

Ppp1r3c Rab30 Scara3 Slc27a1 Spag5 Ttpa

Ppp1r3d Rab34 Scd1 Slc39a12 Spire1 Ttyh1

Ppp1r3g Ranbp3l Scg3 Slc41a1 Spsb4 Tubb2b

Prdm16 Rftn2 Scrg1 Slc48a1 Srebf1 Tulp3

Prdx6 Rfx4 Sdc4 Slc4a4 Svip Ugp2

Prkd1 Rgma Selenbp1 Slc6a11 Sypl2 Vav3

Proca1 Rgs20 Sfxn5 Slc7a10 Timp4 Vcam1

Prodh Rhobtb3 Sh3pxd2b Slc7a11 Tlcd1 Vegfa

Prrx1 Rlbp1 Slc13a3 Slc9a3r1 Tmem229a Wnt7a

Psat1 Rnf182 Slc13a5 Smpd2 Tmem47 Wscd1

Psd2 Rorb Slc14a1 Smpdl3a Tom1l1 Zfp521

Ptplb Rpe65 Slc15a2 Sox2 Tprkb Zfp783

Pxmp2 S100a1 Slc1a2 Sox21 Tsc22d4

This list was produced by extracting the intersection of astrocyte-enriched genes defined by a fold change ≥log2 2 and a FDR-corrected p-value ≤.05 among three previ-
ously published datasets (Cahoy et al. 2008; Doyle et al. 2008; Lovatt et al. 2007). Astrocyte-enriched genes from Lovatt et al. were produced by comparing adult cortical
GFAP-GFP+/GLT1+ astrocytes with GFAP-GFP–/GLT1– cells (N = 3). Astrocyte-enriched genes from Cahoy et al. were produced by comparing P16–17 FACS
and panning-isolated astrocytes (N = 5) to P16–17 panning-isolated neurons (N = 3). Astrocyte-enriched genes from Doyle et al. were produced by comparing TRAP
affinity-purified mRNA from BAC-transgenic Aldh1l1 mice. From Doyle et al., each sample (N = 4) derived from several 8- to 10-week-old mice, and was compared with
unbound affinity-purified samples (N = 2). Each of the three datasets was normalized individually using the RMA algorithm in R/Bioconductor. A representative probe set
was chosen for cases where a gene had more than one probe set on the platform. This was done by choosing the probe set with the lowest p-value for that gene. When there
was a p-value tie, the probeset with the higher value fold change was chosen.

Identification of such genes are essential for objective analy-
sis of disease-related changes in the astrocytic transcriptome
because it will allow unbiased collection of mRNA across vari-
ous stages of the disease of interest.
5 C O M PA R I S O N O F C O RT I C A L
A S T R O C Y T E T R A N S C R I P TO M E S
To date, three studies have produced the transcriptome from
cortical astrocytes (Cahoy et al. 2008; Doyle et al. 2008;
Lovatt et al. 2007). Each of these studies produced the astro-
cyte transcriptome with slight differences in the experimental
design. For instance, Cahoy et al. used adolescent brain tissue
(P17), whereas Doyle et al. and Lovatt et al. used adult brain
tissue from 6- to 8- and 10- to 12-week-old animals, respec-
tively. Also, Lovatt et al. used FACS isolation of GFAP-GFP+/
GLT1+ cells, Cahoy et al. used a combination of FACS and
immunopanning, and Doyle et al. used TRAP technology
to isolate astrocytes from the Aldh1l1 BAC transgenic line
(see Fig. 28.1). However, all of these data sets used the same
microarray platform, and a direct comparison of them provides
important information with regard to astrocyte-enriched tran-
scripts. A direct comparison resulted in a list of 311 astrocyte-
enriched transcripts that are shared among all three data sets
(see Table 28.3; and see legend for analysis details). Notably,
the transcripts on this list are enriched in astrocytes, but may
also be present in other cells. In addition, among the tran-
scripts in Table 28.3 that are truly astrocyte-specific, these
might be expressed only by a subset of cortical astrocytes as
opposed to all cortical astrocytes. Therefore, validation at the
transcript or protein level is necessary before final conclusions
can be drawn about their expression pattern in the brain. The
list can be used as a guide to identify novel astrocyte markers
because many of them are likely to be expressed above the aver-
age mRNA and protein levels. Given higher relative expres-
sion, it is likely that the protein products of the transcripts
in Table 28.3 may be identified with ease using antibody
markers or the promotors of these genes to drive expression
of reporter genes, such as GFP. The list can also be used to
confirm existing functions, as well as define new tasks of astro-
cytes. Validation of the list confirmed the expression of genes
expected in astrocytes, such as Aldh1l1, AQP4 (aquaporin
4), GFAP, Gjb6 (connexin 30), Gja1 (connexin 43), S100b,
and Slc1a2 (GLT1), as well as the absence of marker genes
of other cell types. A gene ontology (GO) analysis of these
311 genes revealed that 113 were assigned the GO term cata-
lytic activity and 36 was assigned transporter activity, in agree-
ment with previous reports suggesting that astrocytes express
many transporters. Among genes with receptor activity were,

T H E A S T R O C Y T E T R A N S C R I P TO M E • 353
for instance, EGFR (epidermal growth factor receptor),
GABAARG1 (gamma-aminobutyric acid (GABA) A receptor,
gamma 1), Fgfr1 (fibroblast growth factor receptor 1), Fgfr3
(fibroblast growth factor receptor 3), Rorb (RAR-related
orphan receptor beta), Nr2e1 (nuclear receptor subfamily
2, group E, member 1), Ntsr2 (neurotensin receptor 2), and
Eps15 (epidermal growth factor receptor pathway substrate
15). Other genes found on the list included Sox2, which is a
transcription factor previously characterized in self-renewing
progenitor cells and has been used in directed reprogramming
of somatic cells to produce induced pluripotent stem (iPS)
cells (Graham et al. 2003; Nakagawa et al. 2008). When Sox2
expression ceases or is inhibited in progenitor cells, they exit
the cell cycle and differentiate into neuroblasts. Forced expres-
sion of a battery of trasncription factors, including Sox2,
induced “stemness” and the potential to divide. However,
radial glia cells from adult brain, which are known to express
GFAP and stem cell–like properties, also express Sox2 (Marko
et al. 2011). Whether cortical protoplasmic astrocytes, or even
a small subset of them, express Sox2 at the protein level and
harbor the potential to self-renew remains to be explored. The
finding of Sox2 expression in astrocytes is interesting because
astrocytes have previously been suggested to maintain the
potential to divide into adulthood (Steindler and Laywell
2003). Other genes with transcription factor activity included
Srebf1 (sterol regulatory element binding transcription factor
1), Glis3 (GLIS family zinc finger 3), Emx2 (empty spiracles
homolog 2), and Pax6 (paired box gene 6) just to name a
few. Tables 28.3 and tables of the complete transcriptome of
cortical astrocytes from 3 published data sets can be found
on http://www.networkglia.eu/en/genomics_screens may
serve as tools to discover novel astrocyte functions and per-
haps identify novel markers labeling subsets of cortical pro-
toplasmic astrocytes. In addition to Table 28.3, we have also
included the entire transcriptome from the same analysis in
http://www.networkglia.eu/en/genomics_screens.
6 H ET E R O G E N E I T Y A M O N G
ASTROCY TES IN THE
MAMMALIAN BRAIN
Astrocytes can be found throughout the brain, and have been
proposed to participate in a number of functions including
metabolic support of neurons, potassium buffering, synapse
development, and regulation of synaptic transmission as
reviewed in detail in other chapters. But do all astrocytes carry
out the same functions? Are there differences among astro-
cytes from different brain regions? And how similar are astro-
cytes within the same region? To date, astrocyte heterogeneity
remains a largely unexplored area with only a few published
gene-profiling studies comparing astrocytes across various
regions (inter-regional heterogeneity) or within regions
(intra-regional heterogeneity).
Among the various regions in the mammalian brain,
several types of astrocytic glia have been described, includ-
ing tanycytes, Müller cells, radial glia cells, Bergmann glia,
protoplasmic astrocytes, fibrous astrocytes, marginal glia,
354 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
perivascular glia, and ependymal glia. The classification of
these cell subtypes is primarily based on location and to some
extent on morphology. How these cell types differ functionally
is only beginning to be deciphered, and is reviewed in detail
in other chapters and reviews (Matyash and Kettenmann
2010; Oberheim et al. 2012; Zhang and Barres 2010) (see
also chapter 4). However, the global set of information gath-
ered by transcriptome profiling makes it an ideal method to
qualitatively assess astrocytic heterogeneity. Although several
examples of astrocyte heterogeneity exist in the literature
(Bordey and Sontheimer 2000; Emsley and Macklis 2006;
Houades et al. 2006; Matthias et al. 2003; Nimmerjahn et al.
2009; Takata and Hirase 2008; Walz 2000; Walz and Lang
1998; Zhou and Kimelberg 2000; Zhu and Kimelberg 2004),
none of them is capable of simultaneously addressing the large
number of genes that transcriptome profiling does. Still, stud-
ies showing that single transcripts or proteins differ among
astrocytes motivate global profiling studies that will establish
and characterize transcriptional differences, and hopefully
establish astrocytic subtypes that were previously unknown.
A split-Cre technology have recently been used to enriched
adult neural stem cells by isolating cells with coincident activ-
ity of the hGFAP and prominin1 promoters in mouse sub-
ependymal zone in vivo (Beckervordersandforth et al. 2010.
A similar approach could be utilzed to isolaed subpopupation
of astrocytes. An example of heterogeneity is the expression
of the astrocyte marker glial fibrillary acid protein (GFAP),
which can vary significantly among astrocytes from various
regions in the adult mouse brain. Glial fibrillary acid protein
is a good marker of protoplasmic and fibrous astrocytes in the
developing neocortex, but in adulthood cortical astrocytes in
the deeper layers downregulate GFAP (Lovatt et al. 2007).
Lovatt et al. found that cortical astrocytes that were positive
for GLT1 could be grouped as either GFAP positive or nega-
tive. However, analysis of the transcriptome between these
two populations only demonstrated that a subtle difference
of 1% of all the expressed genes was significantly different
between the two groups, and the majority of these were not
astrocyte specific genes. In comparison, 14% of all expressed
genes were differentially expressed by GLT1+ astrocytes and
Thy1+ neurons. This finding suggests that the two popula-
tions of cortical astrocytes distinguished by GFAP expression
are very similar at the transcriptional level. However, one has
to keep in mind that posttranscriptional regulation may play
a role in determining levels, location and activity of protein
products, and that level of a transcript and its protein prod-
uct is not always directly correlated. Moreover, heterogeneity
among astrocytes also tends to decrease detectable differ-
ences in gene expression among the populations of cells to
be compared.
This is supported by a gene expression study investi-
gating regional differences among whole tissue samples
from different cortical layers. This study found that
cortical layer-specific gene expression paired to known
neuron-specific genes are 63% more likely to exhibit lay-
ered patterning than being unpatterned. In contrast,
astrocyte-specific genes are 17% less likely to exhibit layered
patterning than being unpatterned. Although this finding
suggests that neurons exhibit more heterogeneity with
respect to cortical layers than astrocytes, it still demon-
strates that astrocytic gene expression differs across layers,
calling for studies that decipher subpopulations of astro-
cytes in cortical layers (Belgard et al. 2011).
Doyle et al. used the TRAP-technology to generate the
transcriptome of cortical Aldh1l1+ astrocytes and cerebellar
Sept4+ Bergmann glia (Doyle et al. 2008). A reanalysis of this
data (Lovatt and Nedergaard, unpublished data) showed that
approximately 62% of the 2,777 Bergmann glia enriched tran-
scripts were shared by cortical astrocytes, and approximately
51% of the 3,354 cortical astrocyte–enriched transcripts were
shared by Bergmann glia. Among the intersecting transcripts
were several that would be expected to be found in both cell
types, including Aldh1l1, S100b, Slc1a2 (GLT1), Slc1a3
(GLAST), Kcnj10 (Kir4.1), Kcnj16 (Kir5.1), Gja1 (con-
nexin 43), Gjb6 (connexin 30), Glul (glutamine synthetase),
GFAP, EGFR (epidermal growth factor receptor), AQP4
(aquaporin 4), and AQP9 (aquaporin 9). Both cell types also
expressed Adk (adenosine kinase) and Adora2b (adenosine
receptor 2B), suggesting that both are capable of responding
to adenosine and phosphorylating adenosine after reuptake.
In addition, the relative high discrepancy between the corti-
cal astrocytes and Bergmann glia transcriptomes suggests that
despite the fact that both are “astrocytic glia,” they may carry
out significantly different functional roles in their respective
brain regions. For instance, Bergmann glia are known to be
intimately associated with Purkinje cells and can respond to
both purinergic and glutaminergic signals with ion channel–
activated Ca2+ signals. In agreement with previous litera-
ture, the transcriptome data showed that Bergmann glia,
but not cortical astrocytes, expressed Gria1 (GluR1) and
Gria4 (GluR4) ionotropic glutamate receptors (Matsui et al.
2005), and the ionotropic ATP-gated P2rx7 (P2X7) receptor
(Habbas et al. 2011). Other ligand-gated receptors expressed
exclusively by Bergmann glia included P2ry1 (P2Y1), Grin2b
(NMDA2B), Grin3a (NMDA3A), and Grm3 (mGluR3).
In contrast, cortical astrocytes expressed another set of
receptors, including P2rx4 (P2X4), P2ry5 (P2Y5), and
Grin2c (NMDA2C) (Lovatt et al. 2007). Of note, BEST1,
a Ca2+-activated Cl– channel that may play a role in sus-
tained GABA release from Bergmann glial cells (Lee et al.
2010), according to the transcriptional analysis of cerebellum
(Doyle et al. 2008), is not selectively expressed by Bergmann
glial cells because it also present in the unbound TRAP
samples that contain both other glial types and Purkinje neu-
rons. Cortical astrocytes, as opposed to Bergmann glia, also
expressed enriched levels of Ldhb (lactate dehydrogenase 2),
suggesting that the metabolic compartmentalization of corti-
cal astrocytes and neurons versus Bergmann glia and Purkinje
cells may differ. Ldhb functions in cortical astrocytes to syn-
thesize lactate for shuttling to neurons (Lovatt et al. 2007).
Further studies exploring the repertoire of receptors and their
functional role will deepen our understanding of astrocytes
in different brain regions, and how astrocytes interact with
their environment.
Although only a few studies to date have explored
interregional heterogeneity of astrocytes, intraregional
T H E A S T R O C Y T E T R A N S C R I P TO M E
heterogeneity or single astrocyte heterogeneity remains
an unexplored area (Doyle et al. 2008; Lovatt et al. 2007).
Future studies and improvements in methods capable of
producing transcriptome profiles from single astrocytes
will provide exciting insight into the diversity among astro-
cytes, and perhaps reveal whether intraregional heterogene-
ity among single protoplasmic astrocytes is as great as their
numerous neighboring neuronal subtypes. Surprisingly, only
a single publication has used transcriptome analysis to study
astrocytes in the diseased brain so far (Lichter-Konecki et al.
2008). In this study, a mouse with the X-linked UCD orni-
thine transcarbamylase (OTC) deficiency was crossed with
the hGFAP-EGFP mouse, and FACS was used to purify
astrocytes from the brains of hyperammonemic and healthy
Otcspf/GFAP-EGFP mice following microarray analyses
and qRT-PCR. The major observation was significant down-
regulation of the gap-junction channel connexin 43 and
aquaporin 4 genes, concomitantly with a reduced expression
of astrocytic inward-rectifying potassium channels Kir4.1
and Kir5.1 in hyperammonemic mice (Lichter-Konecki et al.
2008). Thus, ammonia toxicity may suppress the expression
of proteins involved in astrocyte-mediated water and potas-
sium homeostasis.
7 T E C H N I Q U E S U S E D TO VA L I DAT E
T R A N S C R I P TO M E F I N D I N G S
Once transcriptome data have been mined and novel pat-
terns of gene expression extracted, a necessary next step is to
validate the findings using an independent method. For the
majority of the cases, validation assesses the cellular expres-
sion pattern of candidate genes. First, validation can occur
either at the RNA or protein level. In addition, functional
validation at the activity or behavioral level may also be able
to add supporting evidence to a particular expression pattern
(Lovatt et al. 2007). To validate at the RNA level, one can per-
form in situ hybridization or fluorescent in situ hybridization
(FISH), which with single cell resolution can detect down to
single molecules (Cahoy et al. 2008; Daneman et al. 2010;
Itzkovitz et al. 2012). If large amounts of RNA are available,
then one can evaluate the candidate genes using quantitative
polymerase chain reaction (qPCR) (Lobo et al. 2006; Lovatt
et al. 2007). The advantage of FISH over qPCR is that FISH
can be performed in intact tissue and thus is independent of
the mRNA isolation method used for the transcriptome. On
the other hand, whereas qPCR may use the same mRNA iso-
lation method employed for the transcriptome, qPCR may
be better at detecting low abundant transcripts that would be
difficult to detect by regular in situ hybridization. If valida-
tion takes place at the protein level, then immunohistochem-
istry may be an option if specific antibodies are available for
the candidate transcript. In addition, if information about the
promoter region of the candidate gene is available, one can
generate promoter-driven reporter mice, such as GFAP-GFP
or BAC-transgenic eGFP mice. Altogether, validation of tran-
scriptome data is necessary to confirm the correctness of the
data-mining methodology used.
• 355
 8 L I N K TO T R A N S C R I P TO M E DATA
Tables of the complete transcriptome of cortical astrocytes
from 3 published data sets can be found on http://www.net-
workglia.eu/en/genomics_screens.
9 S U M M A RY A N D P E R S P E C T I VE S
Transcriptome profiling of astrocytes is a novel experimental
tool in glial research that has just begun to unravel the roles of
astrocytes in health and disease. Future studies will explore and
functionally validate the astrocyte transcriptome. Such studies
may lead to a better classification of astrocyte subtypes and dis-
covery of novel roles executed by astrocytes. Gene expression
studies will also explore the transcriptome of reactive astro-
cytes, which remain largely unexplored by global transcriptom-
ics. Such studies could lead to a better classification of diseased
astrocytes and possibly to medical interventions to treat neuro-
logical diseases in which reactive gliosis is a hallmark. Studies are
also needed to explore the transcriptomes of single astrocytes to
gain insight into single cell heterogeneity, and how gene expres-
sion differences correlate with astrocyte location, morphology,
and physiological characteristics. Such studies could lead to a
better classification of neocortical astrocytes beyond the proto-
plasmic and fibrous types.
Although methods to isolate mRNA from cells in vivo
and in situ are continuously being improved, recent develop-
ments in RNA-seq techniques now allow an unprecedented
global view of the transcriptome and its organization, includ-
ing identification of introns, and novel 5′ and 3′ boundaries.
To date, the application of RNA-seq techniques to astrocytes
in situ or in vivo remains unpublished, and future studies
are likely to reveal novel insights into gene transcription in
astrocytes.
The true power of transcriptome analysis has only just
begun to be unraveled. Further inspection and functional vali-
dation of the overwhelming amount of data in the astrocytic
transcriptome will discover novel markers and functional roles
of astrocytes that would have been difficult or perhaps impos-
sible to discover without transcriptome profiling.
AC K N OW L E D G M E N T S
The authors thank Adam Cornwell for technical assistance
with the bioinformatics.
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• 357
 29.
GENE EXPRESSION PATTERNS OF OLIGODENDROCY TE
PROGENITOR CELLS AND OLIGODENDROGLIA
Fraser J. Sim and Steven A. Goldman

A B B R E VI AT I O N S MBP myelin basic protein

MERTK Mer tyrosine kinase protooncogene
BAC bacterial artificial chromosome MOG myelin oligodendrocyte glycoprotein
BAMBI BMP and activin membrane-bound inhibitor NKX2–2 NK2 homeobox 2
BMP bone morphogenic protein O-2A oligodendrocyte and type-2 astrocyte
CD11b cluster of differentiation 11b OLIG1 oligodendrocyte lineage transcription
CD133 cluster of designation 133 (encoded by factor 1
PROM1 gene) OLIG2 oligodendrocyte lineage transcription
CD9 cluster of designation 9 factor 2
CHRDL1 chordin-like 1 OPC oligodendrocyte progenitor cell
CNP 2′,3-cyclic nucleotide 3′- OTP oligodendrocyte transmembrane protein
phosphodiesterase (CLDN11)
CNS central nervous system PDGFRA platelet-derived growth factor alpha
CNTN1 contactin/F3 receptor
Cre Cre recombinase PLP1 proteolipid protein 1
CSPG4 chondroitin sulfate proteoglycan-4 PPARγ peroxisome proliferator-activated receptor-
CSRP1 cysteine and glycine-rich protein 1 gamma
DEX differentially expressed PROM1 prominin 1 (encodes the CD133 antigen)
EGF epidermal growth factor PSA-NCAM polysialylated form of neural cell adhesion
EGFR epidermal growth factor receptor molecule
FACS fluorescence-activated cell sorting PTN pleiotrophin
GalC galactocerebroside C PTPRZ1 protein-tyrosine phosphatase, receptor-
GAS6 growth arrest-specific 6 type, zeta-1
GFAP glial fibrillary acidic protein Ran-2 rat neural antigen-2
GFP green fluorescent protein RISC RNA-induced silencing complex
GLT1 glutamate transporter 1 (SLC1A2) RT-PCR reverse transcription polymerase chain
GLTP glycolipid transfer protein reaction
GPR17 G protein–coupled receptor 17 S100E S100 calcium-binding protein, beta
GRB14 growth factor receptor-bound protein 14 SEPP1 selenoprotein P
GRP glia restricted precursor shRNAi short hairpin RNA
GSEA gene set enrichment analysis siRNA small interfering RNA
HAT histone acetyltransferase SOX10 Sry-Box 10
HDAC histone deacetylase SOX2 Sry-Box 2
HES-5 hairy and enhancer of split-5 ST8SIA1 ST8 alpha-N-acetyl-neuraminidase alpha-
HMGCR HMG-CoA reductase 2,8-siayltransferase 1
ID4 inhibitor of differentiation 4 STAT signal transducer and activator of transcription
IL6 interleukin 6 T3 triiodothyronine
INSIG1 insulin-induced gene 1 TCF7L1/2 transcription Factor 7-Like 1/2
JAG1 jagged 1 TF transcription factor
JAK janus kinase 1 TGFD transforming growth factor, alpha
LNX1 ligand of numb protein X1 TMEM10 transmembrane protein 10
MAPK mitogen-activated protein kinase TSA trichostatin A
MATN4 matrilin 4 ZB4 zinc finger BED domain-containing protein 4

358
 1 INTRODUCTION
Oligodendrocyte progenitor cells (OPCs) pervade both
the gray and white matter parenchyma of the brain.
Oligodendrocyte progenitor cells have typically been noted
to be intrinsically bipotential for astrocytes as well as oligo-
dendrocytes, and have thus been interchangeably called glial
progenitor cells. Yet recent studies have found that both
during development and following injury, the principal role
of these cells is to generate new myelinogenic oligodendro-
cytes (Kang et al. 2010; Rivers et al. 2008). As a result, these
cells are most typically referred to as oligodendrocyte progeni-
tor cells (OPCs), and are referred to as such in this chapter.
Nonetheless, it is important to note that in culture, OPCs
remain competent to generate astrocytes as well as oligoden-
drocytes, and under some conditions neurons as well (Nunes
et al. 2003; Richardson et al. 2011). The molecular basis of
their largely oligodendrocytic differentiation in vivo remains a
mystery. Indeed, it is unclear what the function of these cells is
in the normal adult brain; it is difficult to understand why 3%
to 4% of all cells in the human white matter persist as resident
progenitors, absent a need for significant turnover. As such, it
is critical for us to better understand both the natural history
and molecular regulation of these cells. It is also incumbent on
us to assess these cells using human OPCs, because substantial
differences exist in both the biology and molecular control of
human and rodent OPCs. Therefore, this chapter focuses on
the molecular regulation of the homeostatic maintenance, self-
renewal, lineage specification, and differentiation of human
OPCs. Each of these topics has become a focus of investiga-
tion over the past several years, as new technologies for inves-
tigating cell-type specific gene expression and transcriptional
regulation have become available. Whole genome analysis of
gene expression, chromatin organization, and miRNA regula-
tion represent unbiased approaches to better understand both
the natural history and disease-associated responses of OPCs.
In particular, microarray technologies and next-generation
high throughput sequencing have accelerated the analysis of
gene expression on a genome-wide scale, allowing us to assess
OPC gene expression as a function of age, developmental
stage, and differentiation, as well as to compare the expression
signatures of human OPCs to both their rodent counterparts,
and their differentiated astrocytic and oligodendrocytic deriv-
atives. The focus of this chapter is to discuss the insights that
genomic approaches have provided to our understanding of
the signal control and environmental responsiveness of human
OPCs, and to our understanding of how best to mobilize and
instruct human OPCs for therapeutic purposes.
2 L I N E AG E A N D FAT E P OT E N T I A L O F
G L I A L P R O G E N I TO R C E L L S
Genomic approaches of cells in tissue are subject to the same
limitations of other biochemical analyses in that they rely on
the specificity and homogeneity of the samples used for analy-
sis. The genomic analysis of unfractionated whole tissue rarely
provides coherent information regarding cell type–specific
G E N E E X P R E S S I O N PAT T E R N S O F O L I G O D E N D R O C Y T E P R O G E N I TO R C E L L S A N D O L I G O D E N D R O G L I A
mechanisms, as the gene expression profiles are derived from
an admixture of different cell types, so that cell type–specific
or autonomous regulation can rarely be inferred. Recognizing
this issue, the majority of extant databases addressing neural
and glial gene expression were generated from either sorted
cells profiled immediately following isolation, or from cells
cultured for extended periods of time (>2 days) (see Table 29.1
for a summary of studies).
As such, it is important to note the differences in lineage
potential between OPCs in tissue culture, and those in vivo,
as revealed by recent fate mapping studies using transgenic lin-
eage tracing (for review, Richardson et al. 2011) (see chapters
10 and 13). Isolated glial cells from rodent adult and neonatal
optic nerve had previously been found to generate both oligo-
dendrocytes and astrocytes in culture (ffrench-Constant and
Raff 1986; Wolswijk and Noble 1989). This led to the termi-
nology of O-2A progenitor or glial restricted precursor (GRP)
(discussed in Noble et al. 2004). Subsequent studies both in
vitro and following transplantation, revealed that at least a
fraction of these precursors could be directed to generate
neurons, whether by serum exposure–associated reprogram-
ming, or culture as neurosphere in the presence of mitogens
(Belachew et al. 2003; Kondo and Raff 2000; Nunes et al.
2003). As such, the more generic term glial progenitor cell was
introduced, to include the less-restricted nature of these cells.
Indeed, some studies have suggested that on activation, that
parenchymal glial progenitor cells might acquire a neural stem
cell–like character (Costa et al. 2010; Richardson et al. 2011).
However, data from adult human OPCs indicated that these
cells could not undergo unlimited self-renewal, and did not
express detectable telomerase activity, suggesting that while
these cells might have multilineage competence, that do not
behave as stem cells (Nunes et al. 2003). Rather, these data sug-
gested that OPCs comprise a multipotent transit-amplifying
progenitor, whose lineage choices are profoundly affected by
the in vitro environment (Goldman 2003).
In contrast to the multilineage potential of OPCs in
vitro, a number of recent lineage tracing studies in vivo, using
Cre-lox technology with BAC-driven reporters for NG2,
PDGFRA, and OLIG2, have largely concluded that in post-
natal brain OPCs generate only oligodendrocytes in vivo
(Kang et al. 2010; Rivers et al. 2008) (see chapters 10 and
13 for a detailed description). The lack of astrocytic differ-
entiation in the adult mouse stands in direct contrast to the
observations made using isolated cells. What is the basis for
the difference? What factors restrict cell lineage in vivo? Or
are OPCs comprised of an admixture of phenotypes of dif-
ferent lineage competencies? How do these differ between
the developing and adult brain? These questions have con-
siderable potential clinical importance. Might OPC trans-
plants for the purpose of remyelination be associated with
ectopic astrocytic or neuronal generation, and their atten-
dant clinical complication of epileptogenesis? Do the gene
expression profiles of these cells, as obtained from sorted
isolates, accurately predict their responses to environmental
signals in vivo? These issues are serially addressed in the con-
text of describing current knowledge of gene expression by
human OPCs.
• 359
Table 29.1 GENE AND miRNA EXPRESSION STUDIES OF OLIGODENDROCYTE PROGENITOR CELLS
METHOD OF OPC
PAPER ACCESSION PLATFORM ARRAYS SPECIES AGE PURIFICATION DESCRIPTION

Sim et al. (2011) GSE29368 Aff ymetrix 10 Human Fetal CD140a CD140a+ OPCs isolated from fetal human brain
HG-U133_Plus_2 21–22 weeks FACS
Sim et al. (2009) GSE36634 Aff ymetrix 24 Human Adult A2B5 A2B5+ adult human OPCs from white matter and cor-
HG-U133_Plus_2 30–46 years MACS tex, GLT1+ astrocytes, CD11b+ microglia
Sim et al. (2006) GSE26535 Aff ymetrix 6 Human Adult A2B5 A2B5-sorted adult human white matter-derived OPCs
HG_U95Av2 17–56 years MACS
Shankar et al. (2003) NA cDNA array Research 3 Human Fetal O4 Human O4+ immunopanned cells from spinal cord at
Genetics 18–23 weeks Immunopanning 18 and 22 gestational weeks
Cahoy et al. (2008) GSE9566 Aff ymetrix 48 Mouse Postnatal Serial Oligodendrocyte lineage cells isolated at p7 by immu-
Mouse430_2 day 7 Immunopanning nopanning and S100β-EGFP astrocytes and depleted
using PDGFαR, neurons
MOG and GalC
Gobert et al. (2009) GSE14406 Aff ymetrix 54 Mouse Cell line NA Mouse OPC cell line (Oli-neu) treated with various
Mouse430_2 compounds and profiled at 10, 24, and 72 h
Budde et al. (2010) GSE21797 Agilent mRNA 2 Mouse Embryonic Shaking method Comparison of cultured OLs and astrocytes and time
GSE21799 Exiqon miRNA 2 day 14–16 course in vitro
GSE21798 Agilent miRNA 8
Zhao et al. (2010) NA Rodentia miRNA NA Mouse Not None, tissue extracts Dicer1 conditional knock-out and Olig1 null optic
specified from Dicer1 cko mice nerve and spinal cord tissue compared with wild-type
Dugas et al. (2006) NA Aff ymetrix 96 Rat Postnatal A2B5 and GalC Isolation of A2B5+ and GalC+ cells, and time course of
day 7 (OPC) rat OPC differentiation in vitro
day 10–12 (OLs)
Lau et al. (2008) GSE11218 Aff ymetrix 8 Rat Postnatal A2B5 and GalC A2B5 and GalC-sorted cells at postnatal day 7
Rat230_2 day 7 FACS
Nielsen et al. (2006) GSE5940 Aff ymetrix 18 Rat Postnatal A2B5 and O4 FACS isolated A2B5+ OPCs and O4+ oligodendro-
RAE230A/B day 7 FACS cytes from day 7 pups
Dugas et al. (2010) NA miRNA 4 Rat Postnatal PDGFαR and GalC Immunopaned OPC and oligodendrocytes
not stated day 7 Immunopanning
Lin et al. (2009) NA Aff ymetrix 3 Rat Postnatal O4 Immunopanned O4+ cells isolated from neonatal fore-
RG-U34A day 2 Immunopanning brain (p2) and adult subcortical white matter (200–250
and adult g).
Lyssiotis et al. (2007) NA Aff ymetrix 8 Rat Postnatal Serial immunopanning Rat OPCs (p6) treated with either TSA (20 nM) or
Rat230_2 day 6 BMP-2 (20ng/ml) for 6, 12, 24, and 48 h.

This table summarizes the study design of expression studies referred to in this chapter. Several studies have examined gene expression in human, mouse, and rat development. The profile of oligodendrocyte progenitor cells has been
determined by isolation using FACS, MACS, and immunopanning with various cell-type selective markers.
 A B

MBP

O4
CD140a– CD140a+
C D Chondroitin sulfate biosynthesis
Glycan structures - biosynthesis 1
Axon guidance
CD140a– cells CD140a+ cells Glycerolipid metabolism
Taste transduction
TCF7L1 Thyroid cancer
WNT pathway

Sphingolipid metabolism
TCF7L2 Cell adhesion molecules (CAMs)
Glycine, serine and threonine metabolism
PPAP2B Melanoma
Focal adhesion
CCND1 Glutamate metabolism
Vibrio cholerae infection
CNTN1 Lysine degradation
Notch pathway

ECM-receptor interaction
JAG1 TGF-beta signaling pathway
Olfactory transduction
MAML2 Fatty acid metabolism
Urea cycle and metabolism of amino groups
Glycerophos pholipid metabolism
HEY2 Propanoate metabolism
Tyrosine metabolism
EGFR Homologous recombination
EGFR pathway

Ether lipid metabolism

ERBB3 Glycolysis / Gluconeogenesis
Arachidonic acid metabolism
GRB14 Nicotinate and nicotinamide metabolism
Long-term depression
TGFA Pyruvate metabolism
Notch signaling pathway
CD9 Type II diabetes mellitus
Cell cycle
Phenylalanine metabolism
Nitrogen metabolism
−3 −1 1 3 Valine, leucine and isoleucine degradation
LogFC Ubiquitin mediated proteolysis
Glycos phingolipid biosynthesis - neo-lactoseries
GS

Figure 29.1 Determination of Fetal Human CD140a-Sorted Oligodendrocyte Progenitor Cell Expression Profile. A. Fetal human CD140a+ cells were
plated onto substrate and allowed to differentiate following removal of exogenous growth factors. Four days after FACS, CD140a+ cells had developed
characteristic immature oligodendrocyte morphology and expressed the sulfatide antigen O4. To determine myelin competence, CD140a+ cells were
transplanted into shiverer/rag2 hypomyelinating mice, which lack endogenous MBP. B. Photomicrograph of the corpus callosum and fimbria of an
engrafted shiverer mouse at 12 weeks; stained for myelin basic protein (MBP, green), showing substantial donor-derived myelin (scale, 200 μm).
C. Differential gene expression analysis of CD140a sorted cell profiles identified several functionally relevant genes. This heatmap shows significantly
differential expressed genes comprise members of WNT, Notch and EGFR pathways (>3 FC, and <5% FDR) (heatmap: red, high expression; green,
lower expression; color key indicates expression levels in log base 2). D. Parametric gene set enrichment analysis of CD140a+ OPCs identified several
KEGG cell-signaling and metabolic pathways as either enriched or depleted in human fetal OPCs. Significant pathways were selected based on a
moderated t-test statistic and 5% FDR cutoff. The results are illustrated in decreasing order of significance in a heatmap whereby each sorted cell
sample is represented by a single cell and relative enrichment compared to the CD140a– fraction is indicated by deepening red color, and depletion in
blue. Modified from Sim et al. 2011. Complete data available from NCBI GEO as GSE29368, and at www.findDB.org.
 3 T H E P H E N OT Y P I C H ET E R O G E N E I T Y
O F G L I A L P R O G E N I TO R S
Oligodendrocyte progenitor cells may arise from several
distinct sites during development in vivo (Richardson et al.
2006). In the forebrain, OPCs and oligodendrocytes may
arise not only from classically described sources in the ventral
germinal zones and ganglionic eminences, but also dorsally in
the cortical subventricular zone (Cai et al. 2005; Levison and
Goldman 1993; Vallstedt et al. 2005). Indeed, cortical ven-
tricular zone–derived progenitors appear to play a major role
in the generation of oligodendrocytes in the corpus callosum
and other cortical white matter tracts, even replacing a fraction
of the earlier-generated medial ganglionic eminence-derived
OPCs (Kessaris et al. 2006). The developmental pattern of
oligodendrocyte generation has yet to be determined in the
human brain, but these data suggest that there may be mul-
tiple routes to oligodendrocyte fate.
Indeed, recent evidence suggests significant phenotypic het-
erogeneity among OPCs in embryonic rodent brain and cell cycle
kinetics in adult brain (discussed in chapter 10). Importantly,
this heterogeneity may be lost to analysis when gene expression
profiles are based on cells isolated using single antigens or from
multiple tissue regions. However, one advantage of gene expres-
sion profiling is that high expression of genes encoding surface
proteins—such as receptors—provides additional candidates by
which to dissect heterogeneous populations. This has now been
achieved for fetal human OPCs, as described next.
4 G E N E E X P R E S S I O N BY F ETA L H U M A N
O L I G O D E N D R O C Y T E P R O G E N I TO R S
Oligodendrocyte progenitor cells have been isolated from the
fetal human brain using a number of techniques. The monoclo-
nal antibody A2B5 was first identified as a selective marker for
OPCs from isolates of rodent optic nerve, which do not contain
neuronal cell bodies. Although many groups have used A2B5
to identify human OPCs from fetal brain (Ruffini et al. 2004;
Windrem et al. 2004), A2B5 is also expressed on the surface of
neuroblasts. As a result, concurrent sorting with depletion of
PSA-NCAM–defined neuroblasts is needed to enrich OPCs
form the fetal brain using A2B5 (Windrem et al. 2004).
In contrast to the lack of cell-type selectivity exhibited by
the gangliosides recognized by the A2B5 antibody, the PDGFα
receptor appears to be a more specific marker by which to iden-
tify and isolate oligodendrocyte progenitor cells. In the mouse
brain, nearly all PDGFαR+ cells coexpress NG2, and vice versa
(Rivers et al. 2008). On that basis, PDGFαR+ cells were isolated
from the fetal human forebrain using an antibody that recog-
nizes the CD140a extracellular epitope of the PDGFαR pro-
tein (Sim et al. 2011). CD140a+ cells comprised less than 2.5%
of cells in whole brain isolates before 18 weeks gestation, and
just under 5% of cells in 21 to 23 weeks gestation cortical and
subcortical tissue. Importantly, CD140a+ cells rapidly differen-
tiated as oligodendrocytes in vitro (Fig. 29.1A). Quantitative
real-time RT-PCR revealed that these cells expressed high lev-
els of rodent OPC-expressed genes, such as PDGFRA, CSPG4
362 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
(the gene encoding NG2), OLIG2, SOX10, and NKX2–2.
Compared with A2B5+/PSA-NCAM– defined fetal OPCs,
CD140a+ myelinated the hypomyelinated shiverer brain more
rapidly and efficiently (Fig. 29.1B). Interestingly, although only
a fraction of A2B5+/PSA-NCAM– defined cells expressed
CD140a antigenicity, only CD140a+ cells were capable of oli-
godendrocyte differentiation in vitro.
To determine the whole genome expression profile of
PDGFαR/CD140a-defined fetal OPCs, Aff ymetrix microar-
rays were performed immediately following isolation on sev-
eral human brain isolates. The genomic profile of CD140a+
cells shared many characteristics of OPCs in human and
rodent brain (Table 29.2). CD140a-sorted cells expressed sig-
nificantly high levels of the prototypic OPC markers OLIG1
(56-fold higher in PDGFαR+), OLIG2 (24-fold), NKX2.2
(20-fold), PDGFRA (31-fold), SOX10 (31-fold), CSPG4/
NG2 (11-fold), and ST8SIA1, the enzyme responsible for
synthesis of the gangliosides recognized by the A2B5 anti-
body (3.3-fold). The high expression of PDGFRA, NG2, and
ST8SIA1/SIAT8A resembles the phenotype of adult human
OPCs (Sim et al. 2006) (discussed in detail in the following).
Similarly, S100ß, claudin 11 (OTP/CLDN11) and CNP were
also significantly upregulated in CD140a-sorted cells. Markers
of other neural phenotypes, including oligodendrocytes, astro-
cytes, neurons, and neural stem cells were not highly expressed
by CD140a+ cells.
By comparing CD140a+ cells to the rest of the tissue dis-
sociate, more than 400 genes whose expression was relatively
restricted to human OPCs could be identified (Sim et al. 2011).
Pathway analysis was then applied to identify functionally
related genes that were coordinately regulated. We identified
evidence for the differential regulation of WNT, NOTCH, and
EGFR pathways (see Figs. 29.1C,D). Active WNT signaling
was suggested by the presence of high levels of two TCF tran-
scription factor isoforms, TCF7L1 and TCF7L2. Furthermore,
gene set enrichment analysis (GSEA) revealed that WNT tar-
get genes were significantly induced in CD140a+ cells, suggest-
ing active WNT signaling. Four notch regulators were found
among the CD140a+ selective genes. These included the notch
ligands contactin/F3 (CNTN1) and jagged 1 ( JAG1), which
may have opposing effects on OPC differentiation in rodents
(Hu et al. 2003). The epidermal growth factor (EGF) recep-
tor family EGFR and erbB3 were highly enriched in CD140a+
cells. In addition, the SH3-adaptor protein GRB14, which
interacts with EGFR and the EGF-ligand TGFα, was signifi-
cantly overexpressed. Interestingly, membrane-bound TGFα
may be primed to activate EGFR via the tetraspanin protein
CD9, which was also highly enriched in CD140a+ cells. CD9
can strongly enhance EGFR activation via binding TGFα sug-
gesting an OPC-selective mechanism for CD9-potentiated sig-
naling through EGFR (Shi et al. 2000). CD9 was not expressed
by all CD140+ OPCs, thus permitting their further categorical
subdivision (Sim et al. 2011). Indeed, this latter observation
highlights the power of combining cell isolation with micro
array as a means by which to both define and immunopheno-
type progressively more discrete cell lineages.
Besides A2B5- and CD140a/PDGFαR–defined OPCs,
later-stage progenitors and oligodendrocytes have also been
Table 29.2 FETAL HUMAN OLIGODENDROCYTE PROGENITOR CELL–EXPRESSED GENES
TYPE SYMBOL DESCRIPTION FC Q-VALUE TYPE SYMBOL DESCRIPTION FC Q-VALUE

Ligand BMP8B OP2 4.92 2.34E-08 Transcription SOX10 31.08 7.80E-12

TGFA TGF-alpha 5.49 4.35E-06 Factor SIX4 SIX homeobox 4 7.12 7.80E-12
BMP7 OP-1 4.41 3.27E-05 OLIG1 Oligodendrocyte transcription factor 1 55.52 3.46E-11
JAG1 Jagged1 3.31 5.76E-05 OLIG2 Oligodendrocyte transcription factor 2 24.15 9.93E-11
PDGFA PDGF-A polypeptide 3.18 6.13E-05 NKX2–2 NK2 related, locus 2 19.74 2.86E-09
DLK1 Delta-like 1 homolog 4.53 7.15E-05 TCF7L2 TCF4 3.09 2.64E-07
PTN Pleiotrophin; HB-GAM 3.65 1.65E-04 TCF7L1 TCF3 4.66 4.04E-07
IL8 Interleukin 8 5.86 2.51E-04 RFX4 Regulatory factor X, 4 4.21 4.34E-05
FGF1 Acidic FGF 4.69 1.84E-03 SOX8 3.11 1.65E-04
BMP2 3.49 3.10E-03 Enzyme B3GNT7 UDP-GlcNAc:betaGal 14.62 3.86E-12
Receptor BAMBI BMP pseudoreceptor 15.76 2.27E-12 B4GALT6 Beta 1,4- galactosyltransferase, 6 4.65 7.24E-10
PDGFRA PDGF receptor, alpha 30.80 3.26E-10 CSGALNACT1 Beta4GalNAcT 13.01 6.52E-07
polypeptide
CHRM3 Cholinergic receptor, mus- 17.08 1.71E-09 XYLT1 Xylosyltransferase I 3.06 8.44E-07
carinic 3
SEMA5A Semaphorin 5A 9.92 1.67E-07 ST8SIA1 Responsible for A2B5 synthesis 3.25 1.28E-06
PTPRE RTPε 5.16 1.29E-06 B3GAT2 Beta-1,3-glucuronyltransferase 2 4.37 1.60E-04
ERBB3 9.74 1.59E-06 HS6ST2 HS 6-O-sulfotransferase 2 3.58 3.38E-04
NTRK2 TrkB 7.02 2.37E-06 HS3ST3A1 HS 3-O-sulfotransferase 3A1 4.29 1.99E-03
PTPRG RTPγ 4.63 3.56E-04 HAS2 Hyaluronan synthase 2 4.40 2.78E-06
GPR17 G protein–coupled 3.65 5.83E-04 Other/Novel CCND1 Cyclin D1 13.18 2.82E-09
receptor 17
EGFR EGF receptor 3.30 6.63E-03
ECM/ CNTN1 Contactin 1 30.41 4.01E-15
Cell adhesion CSPG4 NG2 11.11 2.19E-09
MMP2 4.36 2.53E-06
OMG Oligodendrocyte myelin 9.66 2.62E-05
glycoprotein
GPC6 Glypican 6 4.40 3.19E-05
PROM1 CD133/prominin 1 6.48 5.53E-05
FBLN7 Fibulin 7 4.15 1.18E-04
HAPLN1 Hyaluronan and 5.26 4.34E-04
proteoglycan link 1
CD9 CD9 antigen (p24) 7.44 1.10E-03

Human CD140a+ OPCs were isolated using FACS from fetal brain between 21 and 22 weeks gestational age (n = 5) (Sim et al. 2011). Microarray profiles of CD140a+ and CD140a– cells were generated and compared using a
moderated t-test statistic with a 5% false-discovery rate cutoff. Individual differentially expressed transcripts were annotated by Gene Ontology and selected genes in each major category are shown here. The expression ratio of
positive: negative sorted cells are shown along with the FDR-corrected q-values to indicate significance. A complete list of fetal CD140a+ specific genes can be found at www.findDB.org.
isolated from the fetal spinal cord, using O4 sulfatide expres-
sion (Shankar et al. 2003). Although O4 is expressed exclu-
sively by postmitotic oligodendrocytes in human cells in vitro
(Armstrong et al. 1992), O4 is commonly used as a marker of
progenitor cells in rodent brain (Mason and Goldman 2002).
Shankar and colleagues used O4-based immunopanning to
isolate O4+ cells from an 18- to 22-week gestational age spinal
cord. Microarray analysis was solely focused on the expres-
sion of receptors, and data were presented only for kinases
expressed by O4+ cells. Nonetheless, the resultant data were
illuminating; among those highly expressed transcripts, the
GAS6 receptors, AXL, and MERTK stood out, suggesting
the attractiveness of these kinases as targets for oligodendro-
glial fate modulation and support. Indeed, the GAS6 ligand
was then found to potentiate human oligodendrocyte survival
in vitro, via an AKT-dependent pathway (Shankar 2006). On
that basis, GAS6 infusion was subsequently found to reduce
oligodendrocyte death, and possibly promote remyelination,
in response to cuprizone-induced demyelination (Tsiperson
et al. 2010).
5 C O M P L E M E N TA RY PAT T E R N S O F
G E N E E X P R E S S I O N BY A D U LT H U M A N
O P C S A N D W H I T E M AT T E R
In the human brain, OPCs were first identified using
PDGFαR or A2B5 antibodies (Scolding et al. 1998). Mitotic

A2B5+ OPC CD11b+ GLT1+ unsorted B

A CD36
Microglia
CD68
CD86
A2B5 +OPC vs. GLT1+astrocyte vs.
FCGR2A
ITGAM
unso rted unso rted
CDH5
Endothelial TEK
VWF
CLDN11
MAL 552 455 6435
Mature MBP 215 3137
Oligodendrocyte MOBP 343
MOG
PLP1
CSPG4 2
OPC PDGFRA
ST8SIA1
CNP
2 137 229
MAG 45 225
Oligodendrocyte NKX2−2
Lineage OLIG1
OLIG2
SOX10
AQP4 up
GFAP 829
Astrocyte GLUL
505 down
SLC1A2
TNC
ASCL1
NPC DCX
SOX1
FUT4 CD11b +microglia
NSC HES1
HES5
−5 0 5 vs. unso rted
SOX2
LogFC
C
Type Symbol Description Ratio q-value Type Symbol Description Ratio q-value
Ligands DKK1 dickkopf 1 5.44 4.22E-05 Transcription SOX11 11.03 5.89E-09
BMP2 4.06 5.57E-05 Factors ASCL1 MASH1 18.28 1.19E-08
WNT5A 4.72 1.11E-04 SIX4 SIX homeobox 4 4.85 4.13E-08
Receptors GFRA1 GDNF family receptor alpha 1 9.63 2.04E-08 MYT1 myelin transcription factor 1 8.91 6.34E-08
PDGFRA PDGF receptor, alpha 11.70 4.72E-08 SOX6 9.18 1.33E-07
CNR1 cannabinoid receptor 1 (brain) 12.65 4.84E-07 SOX4 3.89 4.98E-07
SEMA5A semaphorin 5A 4.38 5.34E-06 MYCN 3.82 2.54E-05
BAMBI BMP pseudoreceptor 3.81 5.40E-06 Enzymes XYLT1 xylosyltransferase I 4.69 7.22E-09
EPHA5 3.36 5.71E-06 HS6ST2 HS 6-O-sulfotransferase 2 7.48 9.50E-09
SEMA5B semaphorin 5B 4.00 8.06E-06 CASK 3.06 7.37E-08
PTPRZ1 RTPβ/ζ 4.94 2.83E-05 B3GAT2 glucuronosyltransferase S 10.44 7.84E-08
PTPRE RTPε 3.33 2.45E-04 HAS2 hyaluronan synthase 2 4.23 8.34E-07
SEMA3E semaphorin 3E 6.52 2.05E-03 B4GALT6 UDP-Gal:betaGlcNAc 9.70 9.33E-05
GPR17 G protein-coupled receptor 17 3.19 1.70E-02 ST8SIA1 responsible for A2B5 synthesis 5.52 5.47E-03
ECM TNR tenascin R (restrictin, janusin) 11.59 6.07E-09 Other FHL1 four and a half LIM domains 1 3.40 1.48E-06
CNTN1 contactin 1 7.73 9.06E-09 FRZB frizzled-related protein 3.54 1.39E-04
FBLN7 fibulin 7 5.10 2.27E-08
HAPLN1 hyaluronan and proteoglycan link 1 18.31 1.32E-07 Ratio and q-value vs. unsorted dissociate
CSPG4 chondroitin sulfate proteoglycan 4 7.16 1.95E-07
MMP16 11.39 2.19E-07
COL15A1 collagen, type XV, alpha 1 6.45 2.86E-07
OMG oligodendrocyte myelin glycoprotein 3.41 3.61E-05
PROM1 CD133/prominin 1 3.42 1.72E-02

Figure 29.2 Determination of Adult Human A2B5-Sorted Oligodendrocyte Progenitor Cell Expression Profile. Adult human OPCs were isolated on
the basis of A2B5-immunoreactivity using MACS. The Aff ymetrix U133+2 microarray profiles of A2B5+ OPCs were combined with expression
profiles generated from matched unsorted white matter and gray matter dissociates, and GLT1-sorted astrocytes and CD11b-sorted microglial cells.
A. The expression of cell type–specific marker genes illustrates the relative enrichment of OPC, microglial and astrocytic genes, in A2B5, CD11b,
and GLT sorted cells, respectively (heatmap: red, high expression; green, lower expression. Color key indicates expression levels in log base 2).
B. Differential gene expression was performed using linear modeling and an empirical Bayes test statistic with more than threefold change and 5%
FDR cutoffs. The gene expression of each cell type was compared with unsorted white and gray matter dissociates. The number of individual dif-
ferentially expressed probe sets are shown in the Venn diagram. Although there is some overlap of A2B5+ OPC and GLT1+ astrocyte profiles, the
three cell populations are clearly distinct from one another. C. Differentially expressed genes were categorized according to Gene Ontology Biological
process categories and selected genes in each functionally relevant category are shown in the table (ratio vs. unsorted cells, and FDR corrected q-value).
Modified from Sim et al. 2009. Complete data available from NCBI GEO as GSE36634 and at www.findDB.org.

364 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
OPCs were first identified from human subcortical white
matter using CNP promoter-driven GFP-based fluores-
cence–activated cell sorting (FACS) (Nunes et al. 2003; Roy
et al. 1999). As A2B5-expression is not present in postmitotic
neurons in the adult CNS, A2B5-based sorting was found as
a surrogate, providing greater yield and allowing immediate
isolation following tissue dissociation by obviating the need
for transfection and GFP expression (Nunes et al. 2003).
Similarly, to CD140a-sorted fetal OPCs, A2B5-defined
OPCs were abundant comprising between 3% and 4% of
all cells in both adult subcortical white and cortical gray
matter (Nunes et al. 2003; Sim et al. 2009). Indeed, in the
adult mammalian brain, OPCs represent the most abundant
progenitor cell population far exceeding numbers of neural
stem cells. Adult A2B5+ OPCs retain the capacity to gener-
ate oligodendrocytes and are capable of extensive myelina-
tion when transplanted into a shiverer brain (Windrem et al.
2004).
The regulation of OPC fate and homeostasis is under
strict control in the intact adult brain, as OPCs give rise
largely to oligodendrocytes and only rarely astrocytic
phenotypes (see preceding discussion and chapter 10).
However, when removed from their local environment,
adult A2B5-defined OPCs give rise to both astrocytes
and neurons, both in vitro and following transplantation
into embryonic rat brain (Nunes et al. 2003). This sug-
gests that the human OPC retains a neurogenic capacity
that is repressed by the adult parenchymal environment.
To define the in vivo signaling pathways that thus both regu-
late the turnover of adult OPCs while restricting their differ-
entiation, A2B5+ OPCs were sorted from adult human brain
tissue and profiled, and their gene expression patterns com-
pared with the tissue from which they were derived, initially
using earlier generation Aff ymetrix U95 arrays (Sim et al.
2006), and subsequently using contemporary U133+2 arrays
(Sim et al. 2009). Statistically rigorous analysis identified a
cohort of more than 100 shared with fetal CD140a+ cells,
such as the recognized OPC marker genes CSPG4 (NG2),
PDGFRA, and ST8SIA1 (Fig. 29.2A).
A striking feature of these data was the number of genes
encoding cell surface receptors that were expressed along
with their cognate ligands, such as PDGF and its receptor
PDGFRA, and PTN and its receptor PTPRZ1, suggest-
ing a prominent pattern of homeostatic autocrine regula-
tion (Sim et al. 2006). In addition, a number of paracrine
pathways supporting both the homeostatic maintenance
of OPCs and their oligodendrocytic or astrocytic differen-
tiation have been identified (Fig. 29.3). Bone morphogenic
protein (BMP) signaling appears to mediate both autocrine
and paracrine pathways by which oligodendrocytic differ-
entiation is regulated; adult OPCs express high relative lev-
els of the BMP ligands, BMP2 and BMP7, which may limit
both expansion and oligodendrocytic fate. Indeed, addition
of the BMP antagonist noggin, which relieves OPCs from
endogenous BMP signaling, potentiated the expansion of
OPC in vitro, and prevented their astrocytic commitment
(Sim et al. 2006). At the same time, OPCs expressed high
levels of the BMP antagonists, BAMBI, a dominant-nega-
tive BMP receptor 1–like protein, and CHRDL1, a secreted
BMP antagonist, which together appear to serve to protect
the OPC from the effects of BMPs, especially from BMP4,
which serves as a potent stimulus to astrocytic differentia-
tion. Thus, BMP signaling in OPCs appears to be the prod-
uct of the tightly regulated extrinsic and intrinsic regulatory
pathways, the net output of which may determine whether
parenchymal OPCs are maintained as such, or whether they

RTPβ /ζ
(a)

TN-R syndecan-3
PTN
Adult glial
PTN progenitor cell
cadherin FGF
(CDH11/18) NrCAM
FGFR3
PDGF-A
Jagged1 γ-secretase
P CASK
Y PDGF α R CHRDL1 BMP4
βCatenin
Notch1 P
Y P
βCatenin P BMP2
P BMPR2 BMP7
P
Numb Y
βCatenin BMPR1
RBP-J
TCF NRCAM, JUN,
MSI1 MYC
PTN PP
BAMBI
FHL1B CASK
? HES1
RBP-J Tbr-1 target genes
unknown

Figure 29.3 Signaling Pathways Differentially Expressed by Adult Human Oligodendrocyte Progenitor Cells. The annotation and analysis of differen-
tially expressed genes by adult human A2B5-defined OPCs allow the prediction of coherent signaling pathways, which may regulate OPCs at steady
state in the adult tissue environment. The signaling pathways predominant in this model include those initiated by PTPRZ1, as modulated by pleiotro-
phin, syndecan-3, and tenascin (purple); syndecan-3 cleavage and CASK translocation (yellow); notch activation (blue); PDGFRA signaling (green);
and BMP signaling and inhibition thereof (purple). Genes shown in color were found to be selectively and significantly enriched in A2B5-defined
OPCs, relative to unsorted white matter.

G E N E E X P R E S S I O N PAT T E R N S O F O L I G O D E N D R O C Y T E P R O G E N I TO R C E L L S A N D O L I G O D E N D R O G L I A • 365
instead restrict to astrocytic or oligodendrocytic fate, and if
so, to which lineage they commit.
6 O L I G O D E N D R O C Y T E P R O G E N I TO R
C E L L P R O F I L E S R E L AT I VE TO
ASTROCY TES AND MICROGLIA IN THE
A D U LT W H I T E M AT T E R
In gene expression analysis from microarray data, as in any
relative quantitation methodology, the selection of point
of comparison is vital and has a profound effect on the
identification of differentially regulated genes. As such, by
comparing the profile of human OPCs to other defined cel-
lular phenotypes it is possible to better define those genes
and pathways that distinguish OPCs from other cells in
the human brain (Fig. 29.2B). In addition to A2B5-sorted
OPCs, profiles of CD11b+ microglia and GLT1+ astrocytes
were obtained following magnetic bead–based sorting. This
larger database included profiles obtained from both subcor-
tical white matter and cortical gray matter–derived A2B5-
sorted OPCs and from unsorted cell dissociates for control.
Using a linear model approach to reliably detect genes spe-
cific to each cell population, partially overlapping gene sets
were identified that were selectively regulated by A2B5+
OPCs, GLT1+ astrocytes, and CD11b+ microglia. These
data thus included the phenotype-selective patterns of gene
expression of each major human glial subpopulation, with
the exception of mature oligodendrocytes (Fig. 29.2C) (Sim
et al. 2009).
7 D I S T I N C T PAT T E R N S O F G E N E
E X P R E S S I O N BY F ETA L A N D
A D U LT O L I G O D E N D R O C Y T E
P R O G E N I TO R C E L L S
Age-dependent differences in the behavior of OPCs were
first described in rodents, in whom A2B5-isolated cells
from postnatal and adult animals behave differently in vitro
with respect to both their proliferation kinetics and dif-
ferentiation rate (Wolswijk and Noble 1989). Subsequent
work revealed that A2B5-defined progenitors isolated from
human fetal brain and adult subcortical white matter, and
then transplanted into the hypomyelinated shiverer brain,
similarly exhibited differences in phenotype (Windrem
et al. 2004). In humans as well as rodents, fetal OPCs are
more highly and actively proliferative, and manifest shorter
cell cycle times and slower maturation as myelinating oli-
godendrocytes than do adult-derived OPCs, which exhibit
slower proliferative expansion but markedly more rapid
myelination.
The differences between fetal and adult rodent OPCs
have been examined using O4-based sorting. Monoclonal
O4 antibody recognizes an oligodendrocyte specific sul-
fatide that is expressed by late OPCs and immature oligo-
dendrocytes in rodents (Armstrong et al. 1992; Gogate et al.
1994), as well as by postmitotic oligodendrocytes in humans
366 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
(Kirschenbaum et al. 1994). Lin and colleagues isolated O4+
cells from neonatal rat forebrain and adult subcortical white
matter using immunopanning (Lin et al. 2009). Like their
human counterparts, adult rodent O4-defined OPCs differ-
entiated as oligodendrocytes more rapidly than did neona-
tal O4+ cells. Using Aff ymetrix RG_U24A microarrays, Lin
et al. identified several hundred transcripts whose expression
differed by at least twofold in neonatal and adult O4+ cells.
Adult-derived OPCs expressed markers of mature oligoden-
drocyte lineage cells, and included several myelin protein
genes, whereas neonatally derived OPCs expressed higher
levels of PDGFRA and other earlier glial lineage transcripts.
Interestingly, pathway analysis of adult-selective genes using
the Ingenuity database revealed a surprising number of cell
death–related genes. In contrast, cell cycle–related genes
were relatively enriched in neonatal progenitors, consistent
with their greater proliferative activity index and mitotic
competence. Yet although these data provided some insight
into the transcriptional distinctions between adult and neo-
natal progenitors, the selection of OPCs using O4-based
sorting identifies a relatively heterogeneous pool of late
OPCs and committed immature oligodendrocytes. As such,
additional antigenic markers will be needed to isolate more
homogeneous pools of stage-synchronized OPCs, whose
expression profiles might permit more discrete and informa-
tive investigation of the ontogeny of oligodendroglial gene
expression.
The analogous age-related changes in gene expression by
human OPCs have not yet been formally assessed, as pub-
lished studies to data have used different marker antigens
for isolating OPCs from fetal and adult human brain tissue.
Nonetheless, the profiles thus far reported indicate that a
large number of differentially expressed genes are shared by
human fetal and adult OPCs. The prototypical OPC marker
genes, CSPG4 (NG2) and PDGFRA, as well as the A2B5
synthetic enzyme ST8SIA1, were among genes shared by
both fetal and adult OPCs. In addition, their shared expres-
sion of BMP2 and BMP pseudoreceptor BAMBI indicate
that the BMP signaling cascade is actively regulated by
human OPCs throughout development. In addition, the
fetal human neural stem cell marker CD133/PROM1 was
highly expressed by fetal and adult human OPCs alike, sug-
gesting that CD133 exhibits promiscuous expression by
progenitors of varying stages in the human brain, and is
not a specific marker of stem cells as previously described
(Uchida et al. 2000). Additional pathway analysis of shared
transcripts revealed the enrichment of genes involved in gly-
cosaminoglycan biosynthesis, cell adhesion, neurogenesis,
and nervous system development.
8 C E L L -AU TO N O M O U S PAT T E R N S
O F G E N E E X P R E S S I O N BY
RODENT OLIGODENDROCYTE
P R O G E N I TO R C E L L S
Initial reports of the gene expression profiles of rodent OPCs
were perforce limited by the restricted coverage of the rat
 A Mouse cell isolate
Neuron Astrocyte OPC GalC MOG
Microglia Cd68
Fcgr3
Cdh5
Endothelial Tek
Vwf
Cldn11
Mal
Mbp
Mature Mobp
Oligodendrocyte Mog
Nkx6−2
Plp1
Ugt8a
Cspg4
GPC Pdgf ra
St8sia1
Cnp
Mag
Oligodendrocyte Nkx2−2
Lineage Olig1
Olig2
So x10
Aqp4
Gfap
Glul
Astrocyte S100b
Slc1a2
Tnc
Ascl1
NPC Dcx
S o x1
Hes1
Hes5
NSC Nes
S o x2
Ela vl3
Ela vl4
Nefh
Nefl
Neuron Nefm
Neurod6
−5 0 5 Snap25
LogFC Tuba1a
Tubb3

B
Type Symbol Description Ratio q-value Type Symbol Description Ratio q-value
Liga nd Dll3 delta-like 3 (Drosophila) 19.86 1.03E-13 Transcription My t1 myelin transcription fa ctor 1 21.76 2.30E-16
Nxp h 1 neurexophilin 1 13.90 4.32E-14 Factor Ascl1 MAS H1 10.05 8.58E-11
Dll1 delta-like 1 (Drosophila) 8.99 2.36E-10 Sox6 S RY-box containing gene 6 9.28 7.82E-13
Inhbb inhibin beta -B 7.57 4.43E-09 Gsx1 GS homeobox 1 7.49 4.95E-15
Bm p 7 bone morphogenetic protein 7 6.46 4.62E-09 Sox5 S RY-box containing gene 5 5.07 8.50E-08
Wn t4 5.80 3.07E-10 Enzyme Chst5 GlcNAc-6-O-S ulfotransferase 14.42 8.60E-16
Receptor Sema5 b semaphorin 5B 9.80 1.35E-05 Galnt10 N-acetylgalactosaminyltransferase 10 14.08 3.37E-14
Cxcr7 chemokine (C-X-C motif) receptor 7 8.15 1.17E-04 Ccnd1 cyclin D1 10.96 1.06E-08
Ntrk3 Neurotrophin 3 receptor 7.48 9.69E-09 Chst11 carbohydrate sulfotransferase 11 10.18 5.96E-12
Lrp 1 low density lipoprotein receptor 7.35 4.90E-10 B3gnt5 beta3Gn-T5 9.39 7.00E-04
Gfra2 GDNFfamily receptor alpha 2 6.14 7.65E-09 Car8 carbonic a nhydrase 8 8.95 2.07E-08
Oprl1 opioid receptor-like 1 6.04 9.76E-11 St8sia1 responsible for A2B5 synthesis 7.36 7.14E-09
Ptprn RTPn 5.58 1.77E-09 Chst7 carbohydrate sulfotransferase 7 6.72 2.79E-08
ECM / Pcdh15 protocadherin 15 25.20 1.07E-13 Chst15 carbohydrate sulfotransferase 15 5.71 1.55E-06
Cell Adhesion Ntn 1 netrin 1 15.84 2.13E-10 Other /Novel Mki6 7 Ki67 a ntigen 25.68 8.17E-06
Mmp2 matrix metallopeptida se 2 11.22 3.60E-09 Cspg4 chondroitin sulfate proteoglyca n 4 22.98 3.22E-13
Cntn6 contactin 6 6.27 1.47E-09 Tmem100 tra nsmembrane protein 100 13.75 2.06E-11
Vcan versican 6.05 1.55E-08 Ccnb2 cyclin B2 13.67 6.97E-07
Efs embryonal Fyn-associated substrate 6.00 9.78E-08 Cspg5 chondroitin sulfate proteoglyca n 5 9.83 4.34E-11
Chl1 close homolog of L1CAM 5.10 1.58E-05
Ratio and q-value vs. mean of other cell types

Figure 29.4 Mouse Cell Type–Specific Profiles. A. Mouse cell-type enriched populations were isolated using a combination of serial immunopan-
ning and FACS techniques (see Cahoy et al. 2008 for details). Gene expression of cell-type specific marker genes was examined across each of the
five populations and shows strong relative enrichment for OPC and oligodendrocyte genes in the relevent profiles (heatmap: red = high expression;
green = lower expression). To determine OPC specific genes, raw data were reanalyzed using the same analysis pipeline described in Sim et al. (2011)
with a linear model approach. Oligodendrocyte progenitor cell genes were defined by comparison of PDGFαR+ profiles against the profiles of GalC+
and MOG+ oligodendrocytes as well as neuronal and astrocytic defined profiles (>fivefold change, 1% FDR). B. Differentially expressed genes were
categorized according to GO Biological process categories, and selected genes in functionally relevant categories are shown (enrichment relative to
other cell types, and associated FDR corrected q-values; Pdgfra and Cspg4 not shown). Data from Cahoy et al. 2008; available from NCBI GEO as
GSE9566.

genome; these early studies were able to examine less than 10%
of known genes (Cohen et al. 2003; Hu et al. 2004; Scarlato
et al. 2000). The whole genome expression profile of rat OPCs
was first studied using a combination of FACS and Aff ymetrix
analysis (Nielsen et al. 2006). In this study, the expression
profiles of sorted A2B5+O4 – OPCs were compared with
those of A2B5–O4+-sorted oligodendrocytes from 7-day-old
postnatal rat forebrain. However, because the O4+ fraction
is heterogeneous in rodents, and contains both immature
GalC– and more mature GalC+ oligodendrocytes, this study

G E N E E X P R E S S I O N PAT T E R N S O F O L I G O D E N D R O C Y T E P R O G E N I TO R C E L L S A N D O L I G O D E N D R O G L I A • 367
 A B

1.0
Human
Human GPC Mouse OPC

0.8
genes genes
Mouse

sensitivity
0.4 0.6
244 92 465

0.2
targets missed by mouse-specific
mouse analysis genes AUC = 0.74

0.0
pAUC = 0.09
0.0 0.2 0.4 0.6 0.8 1.0
(1 − specificity)
C
Type S ymbol De s cription Type S ymbol De s cription
Lig a nd NXP H1 ne ure xophilin 1 Tra ns cription Fa ctor AS CL1 MAS H1
S CG2 s e cre tog ra nin II (chromog ra nin C) S OX11
CHGB chromog ra nin B (s e cre tog ra nin 1) S OX6
LINGO1 le ucine rich re pe a t ne urona l 6A MYT1 mye lin tra ns cription fa ctor 1
Re ce ptor CNR1 ca nna binoid re ce ptor 1 (bra in) Enzyme HS 6S T2 HS 6-O-s ulfotra ns fe ra s e 2
P DGFRA P DGF re c e ptor, a lpha polype ptide S T8S IA1 re s ponible for A2B5 s ynthe s is
GHR g rowth hormone re ce ptor CHS T11 ca rbohydra te s ulfotra ns fe ra s e 11
S EMA5B s e ma phorin 5B Othe r /Nove l CA10 ca rbonic a nhydra s e X
EP HA5 EphA5 FAM19A2 TAFA2 prote in
EC M /C e ll a dhe s ion DS CAM Down s yndrome CAM GAP 43 growth a s s ocia te d prote in 43
HAP LN1 ca rtila g e linking prote in 1
VCAN ve rs ica n
CS P G4 NG2
P CDH15 protoca dhe rin-re la te d 15
FLRT3 fibrone ctin le ucine rich prote in 3
NTN1 ne trin 1

Figure 29.5 Human and Mouse Comparison Reveals Conserved Oligodendrocyte Progenitor Cell–Expressed Genes. A. Venn analysis of the overlap of
human (A2B5-sorted) and mouse (PDGFαR-sorted) OPC-specific genes. Five hundred fifty-seven unique human homologs of mouse OPC-specific
genes were found in the human array data. Although 92 genes were significantly expressed by human A2B5+ OPCs, there were a large number, more
than 200 genes, whose expression was significant in human OPCs but not present in mouse cells. B. Receiver Operating Characteristic (ROC) curve
analysis was used to compare human and mouse profiles. The red line indicates the line of no discrimination, representing random gene selection.
At 20% FDR (vertical dashed line), the original human U95-derived gene list (Sim et al. 2006) (accessible via NCBI GEO:GSE26535) is greater
than 80% sensitive (green line), whereas the mouse OPC profile showed only approximately 50% sensitivity (black line). These analyses suggests that
although the mouse OPC profile broadly resembled that of human OPCs, there were substantial differences between both expression profiles and
evidence for species-specific gene expression in oligodendroglial progenitors. C. To examine the genes whose expression is highly conserved between
species, we identified genes expressed in both adult human A2B5 and mouse PDGFαR-sorted cells (overlap in Venn diagram). These genes were cat-
egorized according to GO Biological process categories and selected genes in each functionally relevant category are shown. Modified from Sim
et al. 2009.

only identified OPC and mature oligodendrocyte-selective
transcripts. Using a small sample size that precluded rigor-
ous analysis, a fold-change cutoff was used to identify dif-
ferentially expressed (DEX) transcripts. More than 1,000
genes were differentially expressed by the two antigenically
defined stages. Consistent with the cessation of proliferation
by postmitotic oligodendrocytes, A2B5+ OPCs differentially
expressed several genes involved in cell proliferation, whereas
genes involved in the metabolism of fatty acid and cholesterol
were increased in the O4+ fraction. Indeed 17 of 18 cholesterol
biosynthetic genes were upregulated in O4+ cells. This con-
trasted with adult human OPCs, which exhibit high relative
expression of cholesterol regulatory and synthetic genes such
as HMG-CoA reductase (Sim et al. 2006). This difference
may represent an important species divergence, or perhaps
instead reflects differences in gene expression between neona-
tal and adult OPCs.
In a similar study, serial immunopanning for O4+/Ran-2–/
GalC cells was used to select later stage OPCs from 7 day -old
rat forebrain (Dugas et al. 2006). Over 90% of these O4+
OPCs coexpressed NG2+, suggesting that most were still pro-
genitors. Of note, earlier-stage O4 –/A2B5+ OPCs were not
examined in this study, and were hence unavailable for com-
parison. Rather, these O4+ OPCs were compared to GalC+/
Ran-2–/A2B5– oligodendrocytes isolated from day 10 to 12
postnatal brains. By this means, Dugas and colleagues identi-
fied several novel oligodendrocyte-expressed transcripts, such
as GLTP, TMEM10, SEPP1, CSRP1, that serve to distinguish
later stages of oligodendrocytic lineage maturation (see Dugas
et al. 2006, for a more complete listing).
To study mouse oligodendrocyte development, the same
group subsequently used serial immunopanning to isolate three
stages of oligodendrocyte lineage from postnatal day 16 juvenile
mouse forebrain (Cahoy et al. 2008), a time point at which all

368 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
stages of oligodendrocyte maturation coexist in vivo. These stages
included PDGFαR+ OPCs, PDGFαR–/MOG+ myelinating
oligodendrocytes, and PDGFαR–/MOG–/GalC+ cells, as a
presumptive intermediate oligodendrocyte population. In the
same study, Cahoy et al. (2008) prospectively isolated astrocytes
on the basis of S100ß-driven GFP-based FACS, and used a sub-
tractive approach to exclude the majority of glial populations, so
as to enrich for neurons. Aff ymetrix microarray gene expression
profiling revealed a strong relationship between the three oligo-
dendrocyte lineage populations. Surprisingly, the authors found
that on a genome-wide scale, oligodendroglial lineage cells were
more closely related to neurons than astrocytes. More than 2,000
genes were identified as differentially regulated by each major lin-
eage. Components of several canonical signaling pathways were
enriched in mouse oligodendrocytes, including MAPK, AKT,
JAK/STAT, IL6, Wnt, Notch, and several tyrosine kinase receptor
pathways including PDGF, IGF, neuregulin, and EGF signaling
(Fig. 29.4). Interestingly, unlike rat and human cells, fatty acid
metabolism was not enriched in the oligodendrocyte lineage but
rather astrocytes.
Although their data were limited by a small sample size (N
= 2), Cahoy et al. (2008) identified a number of OPC expressed
transcripts, including markers PDGFαR and NG2/CSPG4.
The G protein–coupled receptor GPR17 was identified as a
mouse OPC-specific gene, and likewise was found to be highly
expressed by both fetal and adult human OPCs. GPR17 has since
been shown to negatively regulate oligodendrocyte differentia-
tion and myelination (Chen et al. 2009). Surprisingly several of
these genes, including MATN4, LNX1, and ZBED4, were not
identified in either fetal (Sim et al. 2011) or adult (Sim et al. 2009)
human cells.
9 C O M PA R AT I VE D I S T I N C T I O N S
B ET W E E N M O U S E A N D H U M A N
O L I G O D E N D R O C Y T E P R O G E N I TO R
C E L L G E N E E X P R E S S I O N PAT T E R N S
The analysis of cross-species conservation and divergence via
whole genome techniques is complicated by differences in both
tissue processing and cell isolation techniques. Notwithstanding
that caveat, cross-study comparison of genes identified as dif-
ferentially expressed by A2B5+ mouse (Cahoy et al. 2008) and
human OPCs identified at least 92 shared genes that were over-
expressed by the OPCs of both species (Sim et al. 2009). These
shared transcripts notwithstanding, substantial differences were
noted in the gene expression patterns of human and mouse OPCs
(Fig. 29.5). The importance of these differences remains unclear,
but suggests caution in the interpretation of both pharmacologi-
cal and genetic data obtained in the assessment of murine OPCs,
at least as extended to their human counterparts.
10 O N TO G E N ET I C C H A N G E S I N
GLIAL GENE EXPRESSION
Oligodendrocyte development from purified OPCs has been
studied in vitro for more than two decades (Barres et al. 1992,
G E N E E X P R E S S I O N PAT T E R N S O F O L I G O D E N D R O C Y T E P R O G E N I TO R C E L L S A N D O L I G O D E N D R O G L I A
1993). Dugas et al. first isolated O4+/GalC – OPCs and char-
acterize their gene expression profile during oligodendro-
cyte differentiation in vitro (Dugas et al. 2006). O4+ OPCs
isolated from rat neonatal forebrain (day 7) were cultured
in medium lacking mitogens and containing T3 for up to 9
days and profiled every other day. The authors found that the
transcriptional processes underlying oligodendrocyte differ-
entiation were largely complete by 7 days in vitro, with less
than 10% of genes regulated at longer time points. These data
may have been confounded somewhat by the concurrence of
astrocytic generation in these cultures, since several astrocytic
genes including GFAP appeared in the O4+GalC– gene set,
yet were not apparent in acutely isolated GalC+ oligodendro-
cytes. Yet once these astroglial genes were excluded, the gene
expression pattern of oligodendrocyte differentiation in vitro
was strikingly similar to that of acutely isolated oligodendro-
cytes. At least two stages of oligodendrocytes differentiation
were suggested, by the distinct clustering of known myelin
genes into those either rapidly induced in vitro in the first 2
to 3 days, or those later-appearing transcripts first expressed
after day 5. The multistep nature of oligodendrocyte differ-
entiation was also reflected in the profiles of transcription
factor expression, with both early- and late-stage regulation
observed. Indeed, the pattern of expression of selected TFs
correlated with the effect of knockdown on oligodendrocyte
differentiation, such that knockdown of the early TF SOX10
reduced the appearance of differentiating MBP+ oligoden-
drocytes, whereas knockdown of the later-expressed ZFP536
blocked the development of later stage MOG+ oligodendro-
cytes (Dugas et al. 2006).
In an alternative approach to assessing differential gene
expression during oligodendroglial ontogeny, Gobert et al.
characterized the response of Oli-Neu cells, a line of trans-
formed mouse OPCs, following treatment with drugs that
trigger oligodendrocyte differentiation (Gobert et al. 2009).
Using the resultant gene expression profiles to identify can-
didate genes capable of modulating oligodendrocyte dif-
ferentiation, several siRNAs were identified that induced
oligodendrocyte differentiation. The murine homologs of
the early and late oligodendrocyte differentiation genes
identified during rat OPC differentiation segregated in an
analogous manner during Oli-Neu oligodendrocytic differ-
entiation, as induced by the EGFR tyrosine kinase inhibitor
PD174265. Interestingly, the time course of oligodendro-
cytic differentiation was accelerated in PD174265-treated
Oli-Neu cells, such that with PLP1 expression became
asymptotic within days, rather than over the course of over
more than a week. As such, the distinction between early
and late expressed transcripts was severely compressed, sug-
gesting the potential flexibility of the time frame of OPC
maturation, and by extension, the potential malleability of
the “fetal” and “adult” OPC phenotypes.
Of note, Cahoy et al. (2008) similarly assessed gene
expression during astrocytic ontogeny, using cells selected
from an S100β-driven GFP transgenic mouse. Interestingly,
the majority of genes most strongly downregulated during
postnatal astrocyte development were also downregulated
during oligodendrocyte progression, from OPCs to MOG+
• 369
oligodendrocytes (Cahoy et al. 2008). In both phenotypes,
a downregulation of cell cycle genes accompanied differen-
tiation, with the expression of several G2/M phase transition
and M phase progression genes initially peaking then being
rapidly downregulated as differentiation proceeded. Thus, the
maturation of both glial phenotypes appears to be associated
with the repression of a common set of genes involved in cell
cycle and mitogen-activated signaling pathways.
11 HI STO N E D E AC ET Y L A S E –
M E D I AT E D E P I G E N ET I C
R E GUL AT I O N O F T R A N S L AT I O N IN
OLIGODENDROCYTE
P R O G E N ITO R C E L L S
Epigenetic regulation plays a critical role in the specification
of oligodendrocyte fate and differentiation (for a review see
Liu and Casaccia 2010). Histone acetylation is regulated by
the balance of histone acetyltransferase (HATs), and histone
deacetylases (HDACs). Histone deacetylase activity may be
required for oligodendrocytic fate commitment, in that phar-
macological inhibition of HDAC activity prevents oligoden-
drocyte differentiation by both rat (Marin-Husstege et al.
2002) and mouse OPCs (Shen et al. 2008), while increasing
the multilineage competence of these cells (Shen et al. 2008).
Similarly, HDAC inhibition has been correlated with the
induction of SOX2, and with increased neurogenic capacity of
treated OPCs (Kondo and Raff 2004; Lyssiotis et al. 2007).
Accordingly, HDAC activity has been confirmed as required
for oligodendrocyte differentiation in vivo; in oligodendrocyte
lineage-specific (Olig1Cre) HDAC1/2 double mutants, normal
myelin fails to form, as oligodendroglial lineage cells fail to
appear. These animals exhibit the complete absence of either
PDGFRA-expressing OPCs or mature oligodendrocytes
(MBP or PLP) (Ye et al. 2009).
Class I HDACs form complexes that repress the expression
of several inhibitors of oligodendrocyte differentiation such
as SOX2, TCF7L2, ID4, HES5 (Lyssiotis et al. 2007; Shen
et al. 2008; Ye et al. 2009). To determine additional targets of
HDAC regulation, whole genome microarray was performed
following treatment of rat OPCs with trichostatin A (TSA)
(Lyssiotis et al. 2007), a potent HDAC inhibitor ( Johnstone
2002). Trichostatin treatment repressed several known OPC
and oligodendrocyte-expressed transcripts while inducing
several known markers of neural stem cells. Interestingly, the
expression profiles were found to closely resemble the pro-
file of OPCs treated with BMP-2, which has been reported
to trigger the reversion of OPCs to a multilineage compe-
tent neural stem cell-like phenotype in vitro (Kondo and
Raff 2000). Together, these data support the restriction of
oligodendroglial lineage by HDACs, while highlighting the
potential therapeutic modulation of this effect, such that
HDAC inhibition might act not only to preserve the pheno-
typic plasticity of early OPCs, but also to potentially recover
the neurogenic competence of yet earlier stem and progenitor
phenotypes.
370 • P R O P E RT I E S O F N E U R O G L I A L C E L L S
12 miR N A R E GU L AT I O N O F
T R A N S L AT I O N I N O L I G O D E N D R O C Y T E
P R O G E N I TO R C E L L S
MicroRNAs (miRNAs) are short, noncoding RNA molecules
between 19 and 21 nucleotides that regulate gene expression,
typically via control of mRNA translation. miRNAs are ini-
tially transcribed as pre-miRNAs which fold into stem-loop
structures before processing by Drosha and Dicer1 and incor-
poration into the RNA-induced silencing complex (RISC).
Several groups have recently assessed the expression and func-
tional roles of miRNAs in rodent oligodendrocyte develop-
ment (Budde et al. 2010; Dugas et al. 2010; Lau et al. 2008;
Zhao et al. 2010). By conditionally deleting Dicer1 in Olig1,
Olig2, and CNP-driven Cre transgenics, a number of authors
found that Dicer1 deleted mice developed tremor and other
myelin defects (Budde et al. 2010; Dugas et al. 2010; Zhao
et al. 2010). In addition, CNPCre mice developed peripheral
myelin defects that prevented the analysis of older animals
(Budde et al. 2010; Dugas et al. 2010). In the CNS, Dicer1
mutant animals exhibited a delay in normal myelination
(Dugas et al. 2010; Zhao et al. 2010), which was resolved in
part by late-recruited OPCs, in which recombination had not
occurred (Dugas et al. 2010). The number of PLP-expressing
oligodendrocytes was significantly reduced in Olig2Cre/Dicer1
mutants, but not in CNPCre/Dicer1 animals, suggesting that
later timing of Dicer1 deletion in the latter failed to block oli-
godendrocytic development. Together, these data suggested
that the generation of miRNAs in OPCs is required for their
oligodendrocytic differentiation and maturation.
On that basis, miRNA profiling was used to identify those
specific miRNAs regulated during oligodendrocyte differen-
tiation (Dugas et al. 2010; Lau et al. 2008), as well as to define
those specific to oligodendrocytic lineage restriction relative to
astrocytes (Budde et al. 2010). miRNA microarray analysis of
rat postnatal day 7 A2B5- and GalC-sorted cells revealed that
the miRNAs most significantly upregulated with differentia-
tion were miR-223, miR-338, and miR-219 (Lau et al. 2008).
Similarly, the top three candidates expressed by differentiat-
ing mouse oligodendrocytes relative to OPCs were miR-219,
miR-138, and miR-338 (Dugas et al. 2010), whereas each
of these were expressed at higher levels in OPCs than in
astrocytes (Budde et al. 2010). In that same vein, Dicer1
oligodendrocyte-specific deletion yielded the significant
downregulation of miR-219 and miR-338 in both the optic
nerve and spinal cord (Zhao et al. 2010). Together, these data
suggested that these miRNAs were most highly expressed in
oligodendroglial lineage cells.
Subsequent anatomical studies revealed that miR-219 is
selectively expressed by oligodendroglial lineage cells in post-
natal brain, whereas miR-338 was restricted to spinal cord
white matter by p14 (Zhao et al. 2010). In contrast, miR-138
was also expressed by neurons or other cell types (Dugas
et al. 2010; Zhao et al. 2010). Transfection with miR-219 or
miR-338 induced oligodendrocytic maturation in vitro in
the presence of PDGF (Dugas et al. 2010; Zhao et al. 2010),
yet interestingly did not affect maturation in the absence
of PDGF (Dugas et al. 2010). However, transfection with
miR-219 was able to only partially rescue the effect of Dicer1
deletion, suggesting that multiple miRNA pathways might
be concurrently required for normal differentiation. In con-
trast, miR-138 appeared sufficient to potentiate the transition
to CNP and MBP-expressing cells in the presence of PDGF.
Interestingly, the predicted targets of these miRNAs were
coordinately downregulated during oligodendrocyte differen-
tiation (Cahoy et al. 2008). Specific inhibitors of oligoden-
drocyte differentiation, including both Sox6 and Hes5, were
found to be targets of both miR-219 and -338, strongly sug-
gesting the direct regulation of oligodendrocyte differentia-
tion by these miRNAs.
Microarray analysis comparing primary cultures of OPCs
and astrocytes identified miRNAs specific to each cell type.
In addition to miR-138, miR-338, and miR-219, the miR-17–
92 cluster was identified as highly expressed by both OPCs
and oligodendrocytes, relative to astrocytes (Budde et al.
2010). Notably, this cluster was found to contribute to oli-
godendrogenesis in vivo, as CNP-Cre mediated deletion of
miR-17–92flox/flox mice resulted in a reduced number of Olig2-
expressing cells at birth (Budde et al. 2010). miR-17–92 over-
expression and antisense respectively increased and decreased
proliferation in cultures of mouse primary OPCs, by a path-
way that appeared mediated via the downregulation of PTEN
translation and hence de-repression of Akt signaling.
13 G E N E E X P R E S S I O N –B A S E D D RU G
D I S C O VE RY ( RT P Z I N H I B I TO R S ,
S TAT I N S , P PA R S )
Adult human OPCs have been found to differentially express
several pathways able to regulate OPC fate. One of the most
highly differentially expressed transcripts of human OPCs,
PTPRZ1, encodes the receptor tyrosine phosphatase RTPβ/ζ.
PTPRZ1 expression was at high levels along with a number
of its modulators, including chondroitin-sulfate proteogly-
cans, pleiotrophin, NrCAM, and tenascin R. This pattern
of concurrent gene expression by a host of PTPRZ1 signal
modulators suggested the importance of PTPRZ1 to the
regulatory control of the phosphoproteomes of human OPC.
Accordingly, both pharmacological and shRNAi inhibition of
PTPRZ1 promoted the expansion of oligodendroglial-biased
progenitors in cultures of fetal human OPCs, while potenti-
ating oligodendrocyte differentiation by adult human OPCs
(McClain et al. 2012; Sim et al. 2006).
The expression profiles of oligodendrocyte progenitor cells
have predicted other attractive targets for their potential ther-
apeutic modulation. The cells express differentially high levels
of the rate-limiting enzyme in cholesterol biosynthesis, HMG-
CoA reductase (HMGCR) (Sim et al. 2008). In addition, they
exhibit high differential expression of INSIG1, a regulator of
intracellular cholesterol homeostasis, indicating active sterol
response pathway activity. Accordingly, adult OPCs express
low levels of PPARγ (PPARG), which is negatively regulated
in a sterol-response element–dependent fashion. These obser-
vations have potentially profound significance for both the
G E N E E X P R E S S I O N PAT T E R N S O F O L I G O D E N D R O C Y T E P R O G E N I TO R C E L L S A N D O L I G O D E N D R O G L I A
therapeutic modulation of endogenous progenitor cells, and
for the effects of a variety of common pharmacological agents
on these cells. The statin drugs, such as atorvastatin, simvasta-
tin, and pravastatin act as strong inhibitors of HMGCR, and
simvastatin or pravastatin were each found to induce oligoden-
drocyte differentiation in a dose-dependent manner (Miron
et al. 2007; Sim et al. 2008). Importantly, they appeared to do
so via the potentiating PPARG signaling, and PPARG antago-
nism prevented stain-associated oligodendrocytic differentia-
tion (Sim et al. 2008). Accordingly, direct PPARG agonists
have also been shown to regulate oligodendrocyte differ-
entiation in rodents (De Nuccio et al. 2012; Leisewitz et al.
2008; Saluja et al. 2001). The differentiative actions of both
the statins and PPAR agonists are of potentially great clinical
significance, given the common use of the statins for the treat-
ment of hyperlipidemia, and that of PPARG agonists such as
rosiglitazone in the treatment of type II diabetes, in which
they are used as insulin sensitizers. Indeed, we may specu-
late that the induction of terminal differentiation by both
the statins and thiazolidinediones may contribute to some of
the toxicities noted in patients on long-term treatment with
each, in whom a variety of musculoskeletal, cardiovascular,
and cognitive sequelae have been noted (Evans and Golomb
2009; Friedland et al. 2012). Significantly, both the effects of
these agents upon OPCs, and at least some of their toxicity
profiles, were predictable on the basis of their gene expression
signatures, highlighting the importance of these types of data
to drug development and experimental therapeutics, as well as
to our further understanding of oligodendrocyte progenitor
cell biology.
14 S U M M A RY A N D P E R S P E C T I VE S
Several gene expression studies of isolated glial and oligoden-
drocyte progenitor cells have revealed a complex series of cell
signaling cascades that appear to regulate both the homeo-
static maintenance and differentiation of OPCs. The relative
confluence of gene expression of human, mouse, and rodent
OPCs has not yet been formally assessed, but it is clear that a
number of core conserved pathways are active in the OPCs of
each species. That said, individual components of these path-
ways may prove to be distinct in each species, and it is already
clear that a number of species-specific transcripts are differ-
entially expressed in mouse and human OPCs. Although it is
too early to assess the importance of these species-dependent
differences, we can nonetheless speculate that they may
underlie the distinctions both the rate and extent of remy-
elination observed in rodents and humans (Franklin and
ffrench-Constant 2008).
Human OPCs have been isolated and profiled from both
the fetal and adult brain, and their expression profiles have pro-
vided critical insight into the molecular distinctions between
actively myelinating and quiescent progenitors. These differ-
ences in gene expression may prove of critical importance, in
that they may predict those pathways by which quiescent pro-
genitors are prevented from oligodendrocytic differentiation
and myelinogenesis in the context of chronic demyelination.
• 371
In addition, the advent of genome-scale studies of both
microRNA expression by, and epigenetic regulation of human
OPCs should provide a systems-level understanding of these
processes. Together, these data should provide us sufficient
information to both predict and modulate pivotal hub genes
and their regulated signaling pathways, by which the mobiliza-
tion, expansion, and directed differentiation of human OPCs
may be regulated for therapeutic ends.
AC K N OW L E D G M E N T S
Studies in the Goldman lab described in this chapter were
supported by NINDS R01NS039559 and R01NS075345,
the Adelson Medical Research Foundation, the Mathers
Charitable Foundation, the National Multiple Sclerosis
Society, and the New York State Stem Cell Research Board
(NYSTEM).
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 SECTION 2
FUNCTIONS OF NEUROGLIAL CELLS
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ASTROCY TES
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 30.
NEUROGENESIS AND OUTER SUBVENTRICULAR
ZONE RADIAL GLIAL CELLS
Xiaoqun Wang and Arnold R. Kriegstein

A B B R E VI AT I O N S exist in the ferret, a nonprimate species with a gyrencephalic

brain; the agouti, a rodent with a gyrencephalic brain; and
BLBP brain lipid-binding protein lissencephalic species such as marmoset, mouse, and rat (Fietz
bRG basal radial glia et al. 2010; Garcia-Moreno et al. 2012; Kelava et al. 2012;
CNS central nervous system Martínez-Cerdeño et al. 2012; Reillo et al. 2011; Shitamukai
GFAP glial fibrillary acidic protein et al. 2011; Wang et al. 2011). This chapter reviews recent data
GLAST glutamate transporter concerning these newly described progenitor cells, their role
INM interkinetic nuclear migration in cortical development, and how they may have contributed
IP intermediate progenitor to evolutionary changes in cortical size and architecture.
ISVZ inner subventricular zone
IZ intermediate zone
MST mitotic somal translocation 2 H I S TO R I C A L P E R S P E C T I VE O F
NES Nestin N E U R A L P R O G E N I TO R S I N T H E
oRG outer radial glial-like D E VE L O P I N G N E O C O RT E X
OSVZ outer subventricular zone
Pax6 paired box gene 6 Radial glial (RG) cells and intermediate (IP) or basal progeni-
RG radial glia tor cells are the two principal subtypes of neuronal progeni-
Sox2 SRY (sex determining region Y)-box 2 tor cells that have been extensively explored in the embryonic
SVZ Subventricular zone rodent neocortex (Haubensak et al. 2004; Malatesta et al.
Tbr2 T-box transcription factor 2000; Miyata et al. 2004; Noctor et al. 2001, 2004) (see
VIM vimentin chapter 5). Approximately at the onset of neurogenesis, neu-
VZ ventricular zone roepithelial cells transform into radial glial cells, and their
numbers swell as the progenitor region known as the ven-
tricular zone (VZ) increases in thickness. Radial glial-like
1 INTRODUCTION cells are ubiquitously present during neurogenic phases in the
central nervous system (CNS) of all vertebrates. Radial glial
The subventricular zone (SVZ) is significantly expanded in cells were long known to function as a scaffold for neuronal
the developing primate cortex, and the outer subventricular migration (Kriegstein and Alvarez-Buylla 2009; Kriegstein
zone (OSVZ) in particular, had been suggested as a major and Noctor 2004; Noctor et al. 2004; Rakic 1974, 1995)
site of cortical neurogenesis (Lukaszewicv et al. 2005; Rakic (see chapter 32), but they are now thought to represent the
1974; Smart et al. 2002). Cells within the OSVZ were found major population of neural progenitor cells in the develop-
to express markers of radial glia and intermediate progenitor ing mammalian neocortex (Malatesta et al. 2000; Miyata
cells (Bayatti et al. 2008; Mo and Zecevic 2008; Zecevic et al. et al. 2001; Noctor et al. 2001). Because they produce neu-
2005), but it was not until time-lapse studies were performed rons as well as a variety of glial cell types, they have recently
that the neurogenic potential of these cells and the lineage been referred to as neural stem cells. Time-lapse imaging
relationship between them was demonstrated (Hansen et al. studies have shown that RG cells divide extensively at the
2010). In the developing human neocortex, the outer radial apical surface of the VZ and undergo interkinetic nuclear
glial-like (oRG) cells were observed to undergo asymmetrical migration (INM) as they progress through the cell cycle
division to self-renew and generate intermediate progenitor (Chenn et al. 1998; Wang et al. 2009). The cell cycle–depen-
cells that undergo subsequent rounds of symmetrical divi- dent nuclear oscillations, whereby RG nuclei in G1 phase
sions. Therefore, the oRG cells may be termed neural progen- ascend to the upper VZ to undergo S phase and then descend
itor cells, and oRG daughter cells in human brain are better to the ventricular surface during G2 to undergo mitosis (Gotz
termed transit amplifying cells (Lui et al. 2011). It has been and Huttner 2005; Miyata et al. 2004; Noctor et al. 2001)
suggested that the OSVZ may be a primate specific feature serve to pseudostratify the VZ, and is believed to maxi-
and a hallmark of primate corticogenesis. But recent studies mize the number of mitoses that can occur at the apical sur-
have shown that OSVZ progenitor cells (i.e., oRG cells) also face (Taverna and Huttner 2010). During the peak phase of

379
neurogenesis, RG cells predominantly divide asymmetrically
to self-renew while simultaneously producing either a neuron,
or more commonly, an IP cell (Haubensak et al. 2004; Noctor
et al. 2004).
Intermediate progenitor cells reside within the VZ and often
divide at the ventricular surface at early stages of neurogenesis
(Noctor et al. 2004). However, as neurogenesis proceeds, the
IP cells migrate to a distinct proliferate layer adjacent to the
VZ, the SVZ. Retroviral labeling and time-lapse imaging in
embryonic rodent cortical slice cultures as well as neuronally
expressed marker expression was used to demonstrate that IP
cells most often undergo one round of symmetrical division to
produce two neurons (Attardo et al. 2008; Haubensak et al.
2004; Noctor et al. 2004, 2008). In contrast with RG cells,
IP cells seem to lack apical-basal polarity (Attardo et al. 2008;
Kriegstein and Noctor 2004; Miyata et al. 2004; Noctor et al.
2004). The two-step pattern of neurogenesis, involving RG
cells and IP cells, appears to be the predominant pathway
for cortical neurogenesis in rodents (Haubensak et al. 2004;
Miyata et al. 2004; Noctor et al. 2004). It has been suggested
that the emergence of the SVZ and its constituent IP cells may
have been responsible for the evolutionary increase in cortical
thickness and layering that presumably occurred in the inter-
val between a reptile-like mammalian ancestor and early mam-
mals (Cheung et al. 2007).
3 OUTER RADIAL GLIAL CELLS IN
T H E D E VE L O P I N G H U M A N F ETA L
N E O C O RT E X
In the evolution of the mammalian neocortex, neuron number
reaches a peak in the human brain (Herculano-Houzel 2009;
Rakic 2009). Homo sapiens distinguish themselves from other
mammalian species by the enormous expansion in cortical sur-
face area, coupled with a high density of neurons per cortical
volume, and an associated alteration of cortical architecture,
that together form the substrate for the unique cognitive abili-
ties of humans (Kriegstein and Alvarez-Buylla 2009; Rakic
2009). However, the processes regulating cortical surface area
expansion during development and evolution are still not very
clear. One way to explore for answers is to examine the pro-
cess of neurogenesis in the developing neocortex of a variety
of mammalian species.
The substrate for neurogenesis in the developing human
cortex lies within the fetal progenitor zones. Autoradiographic
analysis of 3H-thymidine labeled cells in the embryonic mon-
key cortex suggested that although early generated neurons arise
from the VZ, the SVZ was a more important site for neurogen-
esis at later stages, and that the SVZ was likely the major source
of cortical neurons by the end of neurogenesis (Rakic 1975).
Recently, the expanded SVZ of the monkey was examined in
detail and was structurally subdivided into an inner subven-
tricular zone (ISVZ) and an outer subventricular zone (OSVZ)
separated by an inner fiber layer (Fish et al. 2008; Howard et al.
2006; Smart et al. 2002). Glial fibrillary acid protein (GFAP)
staining revealed that RG cells are present in the OSVZ as well
as in the VZ during periods of peak neurogenesis (Levitt et al.
380 • FUNCTIONS OF NEUROGLIAL CELLS
1981; Schmechel and Rakic 1979), but these were thought at
the time to be glial restricted astrocyte precursor cells. With the
appreciation that RG cells are neuronal precursors (Malatesta
et al. 2000; Miyata et al. 2001; Noctor et al. 2001), the presence
of radial glial-like cells in the primate OSVZ was additional evi-
dence in support of the designation of the OSVZ as a major site
of neuron production (Smart et al. 2002).
The characterization of specific proteins expressed by pro-
genitor cells in the developing mouse cortex has led to the use
of these cell markers to examine cellular expression patterns in
the primate OSVZ. Cells in the human OSVZ were described
that expressed RG markers such as nestin, vimentin, Pax6,
BLBP, Glast, and GFAP, as well as an alternate cell type that
expressed the IP cell maker, Tbr2 (Abdel-Mannan et al. 2008;
Bayatti et al. 2008; Howard et al. 2006; Mo et al. 2007). These
observations supported the conclusion that two or more pop-
ulations of progenitor cells may be present in the developing
human OSVZ (Fig. 30.1), but how OSVZ progenitor cells
contributed to neurogenesis was not understood.
Recent studies have begun to illuminate the diversity of
progenitor cell types involved in human cortical develop-
ment and highlight the importance of the OSVZ as a region
of neurogenesis. Attention has focused on a cell type found in
the human OSVZ that expresses RG markers including Pax6
and Sox2. DiI-coated beads applied to the pial surface of fixed
human cortical tissue revealed radial glia-like cells with radial
basal processes but without apical processes and therefore
lacking contact with the ventricle (Fietz et al. 2010; Hansen
et al. 2010). Unlike ventricular RG cells, the OSVZ progeni-
tors lack apico-basal polarity in that they do not express apical
membrane proteins prominin-1 or Par3, or ZO-1 that marks
apical adherens junctions, but they do make contact with the
basal lamina thereby establishing them as epithelial cells (Fietz
et al. 2010; Hansen et al. 2010). Like ventricular RG they also
depend on notch activation to maintain identity, as well as inte-
grin signaling mediated through their basal process (Fietz et al.
2010; Hansen et al. 2010). These cells have been termed oRG
cells (OSVZ radial-glia like cells) as well as OSVZ progenitors,
intermediate radial glia, and basal radial glia (Fietz et al. 2010;
Hansen et al. 2010; Reillo et al. 2011; Wang et al. 2011). They
are referred to as oRG cells here. Double labeling of oRG cells
in slice cultures using pulses of tagged thymidine analogs sug-
gested that oRG cells undergo self-renewing division, but direct
evidence for asymmetrical self-renewing division was obtained
through time-lapse imaging of fluorescently labeled human fetal
brain slices (Hansen et al. 2010). These studies also revealed a
lineage relationship between oRG cells and Tbr2 cells in the
OSVZ; namely, that the oRG cells generate IP-like, Tbr2+, cells
through asymmetrical division (Hansen et al. 2010), not unlike
the way ventricular RG produce IP cells in the mouse.
At the time OSVZ cells were characterized in the develop-
ing human cortex, they were also described in the developing
cortex of the ferret (Fietz et al. 2010; Reillo et al. 2011). The
progenitor cells in the ferret SVZ were studied by immunohis-
tochemistry and shown to fall into two classes; one that labeled
with markers of RG such as Pax6, BLBP, and GLAST and were
morphologically similar to human oRG cells, and another class
that were Pax6 – but Tbr2+ (Fietz et al. 2010; Reillo et al. 2011;
 TexasRed- GFP p-vitementin Sox2
dextran Merge Sox2 PAX6 percentrin Dil
A B C D
Pia

OSVZ
180º

OSVZ
VZ
Ventricle

Figure 30.1 The Human Outer Subventricular Zone Is Populated with Outer Radial Glial-Like Cells. A. Five OSVZ cells in gestational week (GW)
15.5 cortex labeled by focal dye electroporation followed by immunostaining for SOX2 (blue). Cell bodies were retrogradely labeled 100 μm from the
injection site through their basal processes. A three-dimensional rendering is shown. B. A representative GFP-retrovirus–labeled cell in the OSVZ
at GW14 coexpressing PAX6 (red). Scale bar, 15 μm. C. GW17 cortex stained for SOX2 (blue), phospho vimentin (p-vimentin; green, cytoplasm of
M-phase cells), and pericentrin (red, centrosomes), demonstrating the morphology and centrosome location of oRG cells during mitosis. Scale bar, 15
μm. D. Fixed GW15 cortex labeled with DiI-coated beads (DiOlistics) applied to the pia. Inset demonstrates dye diffusion along radial processes from
the pia terminating at oRG cell bodies in the OSVZ (stars) amid coexistent radial fibers that traverse the OSVZ to the ventricle (arrowheads), and
autofluorescent blood vessels (x). Scale bars, 40 μm (d) and 20 μm (inset). Reprinted from Hansen et al. 2010.

Wang et al. 2011). These two progenitor cell types were similar
to the progenitor cells found in the primate OSVZ, and a close
examination of the architecture of the ferret SVZ suggested
that OSVZ and ISVZ regions could be distinguished, in part
owing to a radial organization of progenitor cells in the ferret
OSVZ (Fietz et al. 2010; Reillo et al. 2011). Thus, oRG cells,
also termed intermediate radial glial cells in the ferret, and an
enlarged SVZ (OSVZ) are not primate-specific features, but
are also found in nonprimates such as the ferret.
the developing mouse cortex. Therefore, they were identified
as mouse oRG cells based on their oRG-like morphology and
expression of molecular markers characteristic of RG cells. The
mouse oRG cells were anatomically distinct from the bipolar
RG cells located in the VZ, and from the multipolar IP cells in
the VZ and SVZ (Shitamukai et al. 2011; Wang et al. 2011).
5 M I TOT I C S O M A L T R A N S L O C AT I O N

One of the defining features of oRG cells is their mitotic behav-

4 OUTER RADIAL GLIAL CELLS IN
T H E D E VE L O P I N G M O U S E E M B RYO N I C
N E O C O RT E X
The finding of oRG cells in the developing human and ferret
cortex prompted a search for similar embryonic cortical pro-
genitor cells in other species. GFP-labeled oRG-like cells with
a long basal but no apical process were observed in developing
mouse cortex when low-titer GFP-expressing retrovirus was
injected into the lateral ventricle (Fig. 30.2) (Shitamukai et al.
2011; Wang et al. 2011). These cells could be triple-labeled with
the mitotic marker phosphohistone H3, and the RG markers
Sox2 and Pax6, and were located in the IZ and outer SVZ of
ior, in which the cell body translocates very quickly up the
basal fiber before cell division, as first described in human fetal
brain (Hansen et al. 2010). Time-lapse imaging of mouse oRG
cells demonstrated that, like human oRG cells, the mouse cells
also undergo mitotic somal translocation (MST) before divi-
sion (Wang et al. 2011). In mice, the GFP-retrovirus–labeled
cell body was observed to move rapidly along the basal pro-
cess, usually migrating to a swelling within the process, and
traveling an average of around 25 μm (Fig. 30.3) (Wang et al.
2011). This is a shorter distance than that of human oRG cells,
in which the MST averages approximately 60 μm (Hansen
et al. 2010). The duration of MST is less than 1 hour in mouse.
Thus MST appears to be a highly specific mitotic behavior

N E U R O G E N E S I S A N D O U T E R S U BVE N T R I C U L A R Z O N E R A D I A L G L I A L C E L L S • 381
 A B
Adeno-GFP GFP/ DNA

E12.5-E14.5

RG Cell

ORG Cell
IZ
IP Cell

Neuron
SVZ
VZ

Figure 30.2 Mouse Outer Radial Glial Cell Unipolor Polarity. A. Labeling of mouse RG cells and oRG-like cells with Adeno-GFP. Images of E14.5
cortices transfected with Adeno-GFP (green) at E12.5. High magnification images of representative cells (1 and 2) are shown to the right. Note that
the oRG-like cell (cell 1) located at the outer (basal) side of the SVZ has a long basal process (open arrowhead) but no apical process. Pairs of cells
were seen adjacent to each other in which only one cell has a long basal process (cell 2, white arrow), whereas the other has none (cell 2, red arrow)
suggesting that the oRG-like cell might have divided. Scale bars: 50 μm and 15 μm. B. A representative GFP-retrovirus–labeled cell counterstained
with a DNA dye (DAPI, blue). Scale bars: 25 μm. C. Schematic drawing depicting the asymmetrical division of radial glial cells and oRG cells. (A,B)
Reprinted from Wang et al. 2011.

characteristic of oRG cell types in the developing cortex of
multiple species.
Apical anchoring of the centrosome within the ventricular
endfoot is required for the maintenance of RG cells in the VZ
(Chenn et al. 1998; Wang et al. 2009). The relationship of cen-
trosome position to the dynamic mitotic behavior of oRG cells
has been examined in mouse oRG cells. In utero electropora-
tion was used to label the centrosome with Dsredex-centrin
and the cell body with pCAG-GFP (Wang et al. 2011). At
interphase, the centrosome was observed to move into a vari-
cosity within the basal process, with the nucleus then following
(Wang et al. 2011). In migrating neurons, a similar transloca-
tion of the centrosome into the leading process ahead of the
nucleus has been considered crucial for coordinated neuronal
migration (Chenn et al. 1998; Elias et al. 2007; Higginbotham
and Gleeson 2007), and the similar dynamic behavior of the
centrosome and nucleus in MST (Wang et al. 2011) suggests a
possible conserved mechanism. The spatial arrangement of the
centrosome in oRG cells is unique compared with other cor-
tical precursor cells because the centrosome anchors the ven-
tricular endfeet of RG cells during division, and remains in the
cell body of dividing IP cells (Wang et al. 2011). Interestingly,
the basal motion that oRG cells make with each cell division
likely contributes to a progressive developmental expansion of
the OSVZ (Hansen et al. 2010).
6 OUTER RADIAL GLIAL CELL ORIGINS
Recent time-lapse imaging of RG cells in embryonic mouse
cortex demonstrated that 5% to 10% of divisions produced a
self-renewed RG cell and a daughter cell with oRG cell morphol-
ogy (Wang et al. 2011). Moreover, oRG-like daughter cells of
RG cell divisions have been observed to undergo characteristic

382 • FUNCTIONS OF NEUROGLIAL CELLS

 A B

Translocation Distance
Retrovirus-GFP
60

40

(μm)
20

Figure 30.3 Outer Radial Glial Cells Undergo Mitotic Somal Translocation. A. Outer radial glial cells undergo mitotic somal translocation, shown
here in the mouse. A GFP-labeled oRG cell imaged at 10-m intervals beginning 48 hours after infection. Arrows indicate oRG cells (white) and a
non-oRG daughter (red). An asterisk indicates the characteristic swelling in the proximal basal process. A dashed line indicates the cleavage plane.
Scale bar: 20 μm. B. Quantification of mitotic somal translocation distances. Average distance = 23.56 ± 1.56 μm. C. Time-lapse images of cen-
trosome dynamics in an oRG cell. High magnification images from the outlined regions are shown in the following. The time is indicated at the top
(in hours and minutes). Arrows indicate centrosomes. Scale bars: 10 μm and 2.5 μm. Reprinted from Wang et al. 2011.

MST-associated cell division in the SVZ (Wang et al. 2011).
These observations suggest that oRG cells can be generated
through the asymmetrical divisions of RG cells. Cleavage
plane orientation has been suggested to govern the fate of RG
cell progeny (Chenn et al. 1998; Farkas and Huttner 2008;
Konno et al. 2008; Kosodo et al. 2004; Noctor et al. 2008).
Although most RG cell divisions are parallel to the ventricular
surface, a minority of divisions occur with oblique or vertical
divisions in which one daughter cell inherits the basal process
and one the apical membrane (Fishell and Kriegstein 2003;
Gotz and Huttner 2005; Haubensak et al. 2004; Kosodo et al.
2004). By experimentally manipulating spindle orientation,
Shitamukai et al. (2011) were able to induce large numbers of
oblique RG cell divisions, and observed that the cell inheriting
the basal fiber became an oRG-like cell. It thus appears that
oRG cells arise from the asymmetrical division of RG cells,
and that they correspond to daughter cells that inherit the
basal process but not the apical membrane.
7 TR ANSIT AMPLIFYING CELLS
IN THE HUMAN OUTER
S U BVE N T R I C U L A R Z O N E
An important feature of RG cells is their ability to undergo
multiple self-renewing divisions. Time-lapse imaging of fluo-
rescently labeled cells in slice cultures demonstrates that human
oRG cells similarly undergo multiple rounds of self-renewing
division (Hansen et al. 2010). Nearly all oRG cells divide with a
horizontal or oblique cleavage plane such that the basal daugh-
ter inherits the basal fiber and maintains oRG morphology.
These behaviors are consistent with asymmetrical self-renewing
divisions of the oRG cells (Hansen et al. 2010; Lui et al. 2011).
Some oRG divisions produce daughter cells that grow their
own basal processes and appear to be oRG cells, but these are
infrequently observed. Most often, the apical oRG daugh-
ter, which is itself a progenitor cell, initially extends an apical
process directed toward the ventricle as well as a shorter basal
process, but retracts the processes before undergoing its own
division. Long-term time-lapse imaging demonstrates that
oRG cells undergo successive self-renewing divisions, inherit-
ing the radial fiber at each division, and undergoing repeated
MST movements. The oRG cells thus move progressively in
the basal direction with each cell cycle (Hansen et al. 2010).
Although the parent human oRG cells inherit the basal
process and remain SOX2+, TBR2–, the daughter cells become
TBR2+ and divide, indicating that the daughters of human oRG
cells are progenitor cells in a neurogenic lineage. Cells with
the bipolar morphology of oRG daughter cells were observed
to undergo multiple divisions. These observations have been
interpreted as evidence of asymmetrical oRG cell divisions that
yield a self-renewed oRG cell and a neuronally committed IP
cell. But unlike in the rodent, in which the IP cell daughters of
RG cells divide only once to produce two neurons, the human

N E U R O G E N E S I S A N D O U T E R S U BVE N T R I C U L A R Z O N E R A D I A L G L I A L C E L L S • 383
oRG daughter cells undergo multiple rounds of division. For
this reason they have been referred to as transit amplifying cells
rather than IP cells (Hansen et al. 2011; Lui et al. 2011). As
mentioned, mouse oRG cell divisions appear to generate neu-
rons directly (Shitamukai et al. 2011; Wang et al. 2011), high-
lighting an important difference in terms of overall neuron
production between these species (Fig. 30.4).
Evidence from mouse indicates that oRG cells arise from
RG cells, and this is likely how oRG cells arise during early
stages of outer subventricular zone (OSVZ) development
in animals with larger cortices, such as human and ferret.
However, in human and ferret, oRG cells themselves can pro-
liferate to expand the number of ORG cells in the OSVZ.
For example, using GFP-expressing retrovirus to label OSVZ
progenitors in the neonatal ferret led to a preponderance
of labeled oRG cells in the subsequent cell cycle, suggesting
that a majority of divisions in the OSVZ produced two oRG
cells (Reillo et al. 2011). This is not the case in human cortical
development, in which time-lapse imaging studies suggest that
only a minority of oRG cell divisions lead to two oRG cells,
with one daughter inheriting the basal fiber and one growing a
new one (Hansen et al. 2010). The ability of proliferating oRG
cells to expand oRG number may be a feature of animals with
an expanded OSVZ.
8 A SY M M ET R I C A L I N H E R I TA N C E O F
THE BASAL FIBER
Intermediate progenitor (IP) cells in the mouse SVZ are non-
polarized, and their divisions appear to be symmetrical and do
not require control of cleavage plane orientation. In contrast,
RG cells exhibit extreme apical-basal polarity, and divide with
a cleavage plane parallel to the apical-basal axis. When RG
cell divisions produce IP cells, the cleavage furrow is essen-
tially vertical, so that the self-renewed RG cell inherits both
the basal fiber and the apical membrane (Noctor et al. 2008;
Shitamukai et al. 2011). Recent time-lapse imaging experi-
ments in the mouse indicate that when RG cells produce oRG
cells, the cleavage plane is perpendicular to the apical-basal
axis or oblique, so that the oRG cell inherits the basal fiber
but not the apical membrane (Shitamukai et al. 2011; Wang
et al. 2011). However, even though oRG cells lack apical mem-
brane, they maintain epithelial identity through the basal fiber
that makes contact with the basal lamina (Fietz et al. 2010;
Hansen et al. 2010). Thus the retention of the basal process in
oRG cells may convert them from dividing symmetrically (as
rodent IP cells) to dividing asymmetrically (as RG cells do).
The self-renewal mechanism may involve integrin signaling via
the basal process (Fietz et al. 2010), as well as notch activation

oRG
Daughter

Daughter
Daughter

B C
oRG
Daughter oRG D
Daughter
oRG D D D
Daughter IP
Daughter
IP IP
D D D D
N e
+D erg
ge

A
n
x6
FP

eu

er

D E
M
Pa

M
N
G

Figure 30.4 Outer Radial Glial Cells Self-Renew and Produce Transit Amplifying Cells in Human and Generate Neurons in Mice. A. A human oRG cell
at GW15 undergoes two self-renewing divisions followed by division of the daughter cell (intermediate progenitor [IP]). Red and white arrowheads fol-
low the lineages of oRG cell and oRG daughter, respectively. Scale bar, 10 μm. B. Intermediate progenitor cell resembling an oRG daughter undergoes two
rounds of proliferative division. As a result, they are classified as transit amplifying cells. Red and white arrowheads follow the lineages of the two IP cells.
Scale bar, 20 μm. C. Lineage relationships between cells in (A) and (B). D. Asymmetrical division of a mouse oRG cell (arrows) generates a self-renewed
oRG cell (arrows) and a daughter neuron (arrowheads). E. The oRG daughter is Pax6+ (a neuronal stem cell marker, blue), and the non-oRG daughter is
NeuN+ (a neuronal marker, red) after 12 hours more in culture. Scale bar: 10 μm. (A–C) Reprinted from Hansen DV, Lui JH, Parker PR, Kriegstein AR.
2010. Neurogenic radial glia in the outer subventricular zone of human neocortex. Nature 464(7288):554–561. (D,E) Reprinted from Wang et al. 2011.

384 • FUNCTIONS OF NEUROGLIAL CELLS

(Hansen et al. 2010). For example, functional disruption of
integrin signaling in ferret oRG cells markedly decreases the
OSVZ progenitor population size (Fietz et al. 2010), whereas
drug-induced inhibition of notch signaling drives human oRG
cells to a neuronal fate (Hansen et al. 2010). Additionally,
the centrosome, which is located in the basal process during
interphase (Wang et al. 2011), may likewise be involved in
the maintenance of oRG cell polarity. Therefore, inheritance
of the basal fiber may be a key factor contributing to RG and
oRG self-renewal.
9 OUTER RADIAL GLIAL CELLS AND
T H E D E VE L O PM E N T O F GY R E N C E P H A LY
Gyrencephalic brain development is highly correlated with
overall brain and body size, but not with phylogeny, as exam-
ples of lissencephalic and gyrencephalic brains can be found
among most mammalian orders, including marsupials that
separated from Eutheria 160 million years ago (Hansen et al.
2010). Among primates, although most have a large folded
cortical surface, very small primates such as the Senegal bush
baby and marmoset are lissencephalic. Similarly, although
most rodents have a lissencephalic cortex, larger rodents such
as agouti and capybara are gyrencephalic.
It has been hypothesized that cortical expansion may have
occurred through evolution because of an increase in the
Direct Transformation
Asymmetric Division
Symmetric Division
Immature
neuron
Blood
vessels
Immature
neuron
nIPC
MZ
NE
Neuroepithelial
cells
First Trimester
Figure 30.5 The Mode of Neurogenesis During Cortical Development. Radial glia (RG) in cortex generate neurons (a) directly through asymmetrical
division. that generates a neurogenic intermediate progenitor cell (nIPC) that undergoes one round of amplification. Outer radial glial cells generate
neurons indirectly through transit amplifying cells (TACs) that go through multiple divisions before terminal symmetrical neurogenic division via
nIPCs. CP, cortical plate; IZ, intermediate zone; MZ, marginal zone; SVZ, subventricular zone; VZ, ventricular zone. Modified from Kriegstein and
Alvarez-Buylla 2009.
N E U R O G E N E S I S A N D O U T E R S U BVE N T R I C U L A R Z O N E R A D I A L G L I A L C E L L S
number of neuroepithelial or RG founder cells, before the
onset of neurogenesis, thus increasing the number of radial
units while keeping the number of neurons per unit of cortical
thickness the same (Rakic 1995). Although such a mechanism
may contribute to evolutionary cortical expansion, the obser-
vation that the VZ, and therefore the number of founder cells,
becomes very small in primates at stages when neuron dense
mid to upper cortical layers are still being generated (Hansen
et al. 2010; Smart et al. 2002) suggests that an increase in
founder cells alone is not sufficient to account for cortical
expansion. Recent studies suggest that the development of
oRG cells and their transit amplifying daughter cells (i.e.,
IP-like cells) may be key cellular steps underlying expansion in
primate corticogenesis (Hansen et al. 2010). Given that pro-
genitor cells located in the SVZ may contribute to the expan-
sion of the overlying cortex, it has been proposed that SVZ size
may be able to predict the future locations of gyri and sulci,
respectively, in the developing primate cortex (Kriegstein et al.
2006). In the developing ferret, regions of future gyral forma-
tion contain more proliferating SVZ cells during development
than regions of sulcus formation, and in these areas prolif-
eration is more pronounced in the OSVZ than in the VZ or
ISVZ (Reillo et al. 2011). The concept that oRG cells expand
in number within the OSVZ, and that OSVZ proliferation
correlates with gyrus size has been further substantiated by
experimental evidence in the ferret. Enucleation at embryonic
stages reduces OSVZ proliferation in the contralateral ferret
MZ
Astrocyte
CP
Mature
neuron
IZ
TAC TAC nIPC
oRG
oRG
astrocyte
OSVZ
oRG
SVZ
VZ
Radial glial cells
Second Trimester Third Trimester
• 385
visual cortex and leads to local reduction in the size of the neo-
cortical gyrus (Reillo et al. 2011). These findings confirm the
importance of oRG cell proliferation in the tangential expan-
sion of the neocortex, but as described, oRG cells are not only
found in animals that have gyrencephalic cortex, but are also
found in the lissencephalic mouse.
In the marmoset, a near-lissencephalic primate, oRG cells
expressing Pax6 and Sox2 (but not Tbr2) and retaining a
basal process at mitosis, have been found in the OSVZ and
occur at similar relative abundance as in human and ferret
(Garcia-Moreno et al. 2012; Kelava et al. 2012). The finding
of oRG cells in both gyrencephalic mammals such as human
and ferret, as well as non-gyrencephalic mammals such as the
mouse and marmoset, suggests that oRG cells, although per-
haps necessary, are not sufficient for gyrencephaly. Possibly,
the presence of transit amplifying daughter cells, observed in
large numbers in the developing human cortex but not at all
in the mouse, may be a more sensitive indicator of degree of
cortical expansion. It is likely that changes in multiple param-
eters, including progenitor diversity and abundance as well as
cell cycle kinetics and features associated with neuronal dif-
ferentiation and circuit formation that occur later, including
the physical tension exerted by axons (Van Essen 1997), may
determine whether the neocortex becomes lissencephalic or
gyrencephalic.
10 S U M M A RY A N D P E R S P E C T I VE S
The finding of oRG cells and transit amplifying progenitor
cells in the OSVZ of the developing cortex across a range of
mammalian species is changing our concepts of neurogenesis,
neuronal migration, and the model of cortical expansion in
neocortical development and evolution (Fig. 30.5). Further
exploration of the number and types of progenitor cells in the
developing cortices of additional mammalian species, along
with analysis of their lineages, will lead to a better understand-
ing of the features that may be unique to human cortical devel-
opment. An increase in progenitor cell diversity is consistent
with the evolutionary increase in neuronal diversity between
species, and underscores the importance of developing mark-
ers for specific progenitor cell types, and unraveling the tran-
scription programs that determine the cell fate of progenitor
cells as well as the neurons they produce.
The finding that the OSVZ contains RG-like cells with
basal processes that could support neuronal migration has
potential implications for our understanding of neuronal
migration as well as migration disorders. Although the basal
fibers of oRG cells can provide additional guides for radial
migration, evidence suggests that clonally related neurons do
not form ontogenetic cortical columns. Lateral dispersion of
clonally related neurons has been observed in rodent, ferret,
and primate cortex (Reillo and Borrell 2011; Reillo et al. 2011;
Walsh and Cepko 1993) and may be particularly prominent
in large-brained species. Characterizing the migration tra-
jectories of neurons generated from RG, IP, and oRG cells
may lead to new insights into cortical organization as well as
386 • FUNCTIONS OF NEUROGLIAL CELLS
development, and may lay the groundwork for a better under-
standing of neurodevelopmental disorders ranging from major
cortical malformations to more subtle disorders such as autism
and schizophrenia.
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 31.
GLIAL CONTROL OF SYNAPTOGENESIS
Nicola J. Allen

A B B R E VI AT I O N S fundamental functional units of the nervous system (Scheiffele

2003; Waites et al. 2005; Yamagata et al. 2003). During devel-
ACM astrocyte conditioned media opment, nascent synapses form between an axonal growth cone
ADNF activity-dependent neurotrophic factor and a postsynaptic target, which is normally the dendritic region
AMPA alpha-amino-3-hydroxy-5-methyl-4-isoxa of another neuron. This initial contact induces the sequential
zolepropionic acid recruitment of specialized components to both the presynaptic
ApoE apolipoprotein E and postsynaptic contact site. For example, neurotransmitter
BG Bergmann glia vesicles and the machinery to release and replenish them accu-
C1q complement component 1, q sub- mulate in the axon. In the dendrite the postsynaptic specializa-
component tion recruits scaffolding proteins and forms the postsynaptic
C3 complement component 3 density, and recruits neurotransmitter receptors to the postsyn-
CNS central nervous system aptic membrane, thus rendering the postsynaptic cell responsive
CR3 complement receptor 3 to neurotransmitter release from the presynaptic cell.
CSPG chondroitin sulfate proteoglycan There are billions of synapses in the adult mammalian
DS Down syndrome brain, but what factors control the formation of synapses and
E embryonic day ensure that the correct synapses form in the right place at the
FX fragile X syndrome right time? Astrocyte processes are closely associated with
GABA γ-aminobutyric acid synapses throughout development and in the mature brain,
GFAP glial fibrillary acidic protein and emerging evidence has demonstrated that astrocytes
GFP green fluorescent protein play an active role in synapse formation via a combination of
HNS hypothalamo-neurohypophysial system contact-mediated and secreted signals. In addition to inducing
HSPG heparan sulfate proteoglycan new synapse formation astrocytes enhance synaptic strength,
KO knock-out stabilize synaptic structure, and modulate the elimination of
mEPSC miniature excitatory postsynaptic current preexisting synapses.
NMDA N-methyl-d-aspartate This chapter reviews the evidence for an active role of glia
NMJ neuromuscular junction in synapse formation, describes the glial-derived synaptogenic
NT-3 neurotrophin 3 factors that have been identified, demonstrates that glia are
P postnatal day also involved in synaptic stabilization and elimination, dis-
PC Purkinje cell cusses how glial synaptogenic factors interact with neuronal
PKC protein kinase C pathways, and finally, looks at how glial-induced synaptogen-
PNS peripheral nervous system esis goes awry in neurodevelopmental disorders.
RGC retinal ganglion cell
SPARC secreted protein acidic and rich in cysteine
TGF-β1 transforming growth factor beta 1 2 G L I A I N D U C E SY N A P S E F O R M AT I O N
TNFα tumor necrosis factor alpha B ET W E E N N E U R O N S
TSP thrombospondin
TTX tetrodotoxin A close spatial relationship exists between astrocytes and syn-
VIP vasoactive intestinal polypeptide apses, with 60% of synapses being associated with an astrocytic
WT wild-type process in the adult rodent hippocampus (Fig. 31.1) (Ventura
and Harris 1999; Witcher et al. 2007). Indeed, it has been esti-
mated that one astrocyte interacts with as many as 140,000
1 INTRODUCTION synapses in the mature brain (Bushong et al. 2002; Kirov et al.
1999). This close spatial relationship has led to the hypothesis
Synapses are specialized asymmetrical cell adhesions that permit that astrocytes actively contribute to synapse formation and
communication between neurons and target cells, and are the modulation of synaptic function.

388
A Rouget et al. 1993). Synapses only formed when astrocytes
B C
D F O R M AT I O N B ET WE E N P U R I FI E D R ET I NA L
Figure 31.1 Location of Perisynaptic Astrocyte in Area CA1 of
Hippocampus from Mature Rat. Astroglial processes at the axon–spine
interface (astroglial process, blue); psd, postsynaptic density (red); sp,
dendritic spine head (yellow); ax, axonal bouton (green). Reproduced
with permission from Witcher et al. 2007.
2.1 IN VI VO C LU E S O F A RO L E F O R
A S T RO C Y T E S I N SY NA P S E F O R M AT I O N
An indication that astrocytes play a role in neuronal synapse
formation comes from the temporal correlation between the
timing of astrocyte generation and the timing of synapse for-
mation. During development neurons are generated before
astrocytes (Miller and Gauthier 2007), yet few synapses form
until after astrocytes are born (see chapter 12 for more details
on the timing of astrocyte generation). This has been dem-
onstrated in the superior colliculus of both the possum and
the rodent (Correa-Gillieron and Cavalcante 1999; Ullian
et al. 2001). There is a delay between target innervation and
synaptogenesis, during which time astrocytes are generated,
which suggests that glial derived signals are required for neu-
rons to form synapses. Another line of evidence comes from
studies of explant cultures. Embryonic striatal neurons were
cultured alone or on striatal astrocytes, and their ability to
form synapses with neighboring explants of mesencephalon
examined by electron microscopy (Autillo-Touati et al. 1993;
G L I A L C O N T R O L O F SY N A P TO G E N E S I S
were present, and interestingly the astrocytes had to be from
the same brain region—when striatal neurons were cul-
tured with mesencephalic astrocytes no synaptogenesis was
observed.
These in vivo temporal observations offer a compelling
case for an active role of astrocytes in neuronal synapse
formation, but direct investigation has been hampered by
the fact that astrocytes produce trophic factors that are
necessary for neuronal survival, thus making it difficult to
study synapse formation in the absence of astrocytes. This
has been demonstrated using in vitro cultures of rodent
hippocampus—in the absence of astrocytes embryonic
hippocampal neurons die within a few days of isolation
(Banker 1980; Kaech and Banker 2006). In vivo approaches
to delete mouse astrocytes, for example, by targeting deletion
of cells expressing the astrocyte-specific protein GFAP, caused
the loss of Bergmann glia in the cerebellum, which led to
the death of cerebellar granule neurons and the atrophy of
Purkinje cells, and subsequently ataxic mice (Cui et al. 2001;
Delaney et al. 1996).
2.2 A S T RO C Y T E S I N D U C E SY NA P S E
GANGLION CELLS
The inability to dissociate the effects of astrocytes on neuronal
survival from specific effects on synapse formation have been
overcome by the development of a neuronal culture system
using retinal ganglion cells (RGCs). Retinal ganglion cells can
be purified away from all of the other cells they are normally in
contact with in the retina by immunopanning, and maintained
in culture for weeks in defined serum-free media containing
peptide growth factors. (Meyer-Franke et al. 1995). This
culture system enables high levels of neuronal survival along
with extensive axonal and dendritic growth, all in the absence
of astrocytes. Retinal ganglion cells cultured in isolation have
little synaptic activity, as assessed by electrophysiological
recording. By contrast, RGCs cultured in the presence of
astrocytes possess significantly more synaptic activity (Pfrieger
and Barres 1997). This increase in synaptic activity is caused by
astrocytes inducing the formation of new structural synapses in
addition to enhancing the efficacy of existing synapses (Ullian
et al. 2001). Physical contact between astrocytes and neurons
is not required as the same effect is seen when astrocytes are
placed in a feeding layer above the neurons as when grown
in contact, demonstrating that a soluble signal released from
astrocytes increases the number of synapses (Fig. 31.2A).
Addition of astrocyte conditioned media (ACM) is equally
effective in inducing synapses as a feeder layer of astrocytes,
suggesting synaptogenic factors are constitutively released
from astrocytes grown in isolation in culture, and do not
require a neuronal signal to stimulate release.
Extensive characterization has demonstrated that the
synapses induced in RGCs by astrocytes are fully functional,
showing induction and maturation of multiple aspects
of synapse formation. Structural synapse number can be
analyzed by immunostaining for presynaptic and postsynaptic
• 389
proteins, and counting the clustering and colocalization (a)
RGC alone RGC + astrocyte
of these markers as evidence of synapse formation, and
demonstrated a sevenfold increase in the number of synapses
in RGCs cultured in the presence of astrocytes (Fig. 31.2B). (b)
Synaptic strength can be determined by electrophysiological
recording from RGCs. Astrocyte exposure causes an increase
in presynaptic release probability, as well as an increase in the
frequency and amplitude of mEPSCs indicative of an increase
in postsynaptic strength (Fig. 31.2C, D). In addition, FM
dye-uptake can be used to study presynaptic vesicle cycling,
which is also enhanced by astrocytes. These results have been
validated by electron microscopy analysis of the synapses
induced between RGCs by astrocytes, which showed them
to be ultrastructurally normal, exhibiting presynaptic vesicle
clusters and an electron-dense postsynaptic density. Taken Pre
together these findings demonstrate that synapses induced to
form between neurons by glial cells possess similar structural Post
and functional aspects as synapses observed in vivo.
Merge
2.3 G L I A I N D U C E SY NA P S E S I N MU LT I P L E
N EU RO N C L A S S E S (c)

200 pA
Following this initial study on RGCs numerous groups have
demonstrated a role for glial cells in inducing synaptogenesis 10 sec

in multiple types of neurons (Allen and Barres 2005, 2009;

Eroglu and Barres 2010; Pfrieger 2010). Glutamatergic RGCs
(Nägler et al. 2001; Pfrieger and Barres 1997; Ullian et al.
2001), glutamatergic and GABAergic spinal motor neurons
(Li et al. 1999), glutamatergic and GABAergic hippocampal

400 pA
neurons (Elmariah et al. 2005b; Hughes et al. 2010; Tournell 200 msec
et al. 2006), glycinergic spinal cord neurons (Cuevas et al.
2005), cortical subplate neurons (McKellar and Shatz 2009), (d)
cortical neurons (Hu et al. 2007), and cerebellar neurons
(Buard et al. 2010; Steinmetz et al. 2006) all show enhanced
40 pA

synapse formation and function in the presence of astrocytes. 5 sec

In addition, synaptogenic effects can also be induced by other
types of glial cells—oligodendrocytes and Schwann cells can
induce neuronal synaptogenesis (Peng et al. 2003; Pfrieger
40 pA

and Barres 1997; Ullian et al. 2004), as well as support cells in

the cochlea (Gómez-Casati et al. 2010). As well as enhancing
synapse formation and function between individual neurons,
astrocytes can enhance neuronal connectivity across networks
of neurons (Boehler et al. 2007; Geissler and Faissner 2012;
Pannasch et al. 2011; Pyka et al. 2011a).
2.4 G L I A L-I N D U C E D SY NA P TO G E N E S I S I S
C O NS E RVE D AC RO S S M A N Y S P EC I E S
The effects of astrocytes on synaptogenesis are not restricted
to rodents, but conserved across multiple species. Glial cells
enhance synaptogenesis in human, rodent, Xenopus, and C.
elegans neurons (Bacaj et al. 2008; Colón-Ramos et al. 2007;
Hartley et al. 1999; Peng et al. 2003) (see chapter 2 for more
details on invertebrate glia). In addition, studies using neurons
derived from human embryonic stem cells and rodent neural
stem cells have shown that the presence of astrocytes accel-
erates synapse formation (Hartley et al. 1999; Johnson et al.
2007; Song et al. 2002).
5 msec
Figure 31.2 Astrocytes Increase Synapse Number and Function in
Retinal Ganglion Cells. The left column represents data from RGCs
cultured in isolation. The right column represents RGCs cultured in
the presence of an astrocyte feeder layer. A. Schematic representation
of the experimental setup showing RGCs in the presence and absence
of an astrocyte feeder layer. B. Immunostaining for synaptic markers
demonstrates that few synapses are present when RGCs are grown in
isolation, and that many synapses are induced by the presence of astro-
cytes (red, presynaptic marker bassoon; green, postsynaptic marker
homer; colocalization, synapse). C. Electrophysiological record-
ing demonstrates that RGCs grown alone have little synaptic activity,
whereas RGCs grown in the presence of an astrocyte feeder layer have
large amounts of spontaneous synaptic activity. D. Electrophysiological
recording in the presence of TTX to isolate mEPSCs demonstrates
an increase in mEPSC frequency and amplitude when RGCs are
cultured in the presence of astrocytes. Example data from Allen
et al. 2005.

390 • FUNCTIONS OF NEUROGLIAL CELLS

 3 M E C H A N I S M S O F G L I A L -I N D U C E D
SY N A P TO G E N E S I S
The preceding data demonstrate that astrocytes enhance
synapse formation and function, and there have been mul-
tiple studies aimed at identifying the molecular mechanisms
responsible. In this section evidence is presented that astro-
cytes induce synapse formation via a combination of con-
tact-mediated and secreted signals, and that different signals
are used depending on the class of synapse being induced.
Interestingly, contact-mediated signals appear to be required
for synaptogenesis between embryonic neurons, whereas post-
natal neurons are receptive to secreted signals, suggesting dis-
tinct signals are required for each stage of development (see
Table 31.1 for a summary of factors).
3.1 C O N TAC T-M E D I AT E D S Y NA P TO G E N E S I S
3.1.1 Integrin-Mediated Protein Kinase C Signaling
Autaptic embryonic hippocampal neurons cultured in the
presence of ACM (to enhance survival) form few synapses;
however, addition of an astrocyte that physically contacts the
neuron induces multiple synapses to be formed (Hama et al.
2004). Astrocyte contact induces an increase in both synapse
number and function, as shown by a combination of immunos-
taining and electrophysiological recording. Interestingly, even
when the astrocyte only contacts a local region of the neuron,
there is enhancement of synaptogenesis throughout the whole
cell, suggesting upregulation of a global signaling cascade. The
mechanism of this enhancement is owing to integrin-mediated
contact, leading to a spreading enhancement of PKC signaling
throughout the neuron, and subsequent synapse formation.
3.1.2 Synaptic Receptivity Caused by Neuronal
Neurexin Relocation
In contrast to the postnatal RGCs described in the previous
section, when embryonic RGCs are isolated at E17 and cul-
tured in the presence of soluble astrocyte-derived factors,
they are unable to receive synapses but are capable of forming
presynaptic specializations (Barker et al. 2008). This was ele-
gantly demonstrated by mixing RGCs isolated from E17 and
P5 rats in vitro, and showing that P5 RGCs received many
presynaptic inputs onto their dendrites from E17 RGCs,
whereas E17 RGCs received few presynaptic inputs from P5
RGCs. It was hypothesized that contact with another cell
type may be responsible for the switch to occur, as many other

Table 31.1 SUMMARY OF GLIAL-SECRETED SIGNALS THAT REGULATE SYNAPSE FORMATION

GLIA/NEURON
MOLECULE SPECIES TYPE SYNAPSE CLASS ACTION REFERENCE

Thrombospondin Rodent Astrocyte; RGC, Glutamatergic Structural synapse formation Christopherson et al.
1 and 2 hippocampal 2005; Hughes et al. 2010;
Xu et al. 2009

Cholesterol Rodent Astrocyte; RGC Glutamatergic Presynaptic differentiation Mauch et al. 2001

Hevin Rodent Astrocyte; RGC Glutamatergic Structural synapse formation Kucukdereli et al. 2011

Glypican 4 and 6 Rodent Astrocyte; RGC Glutamatergic Enhancement of AMPA receptor synapse Allen et al. 2012
delivery and postsynaptic differentiation

SPARC Rodent Astrocyte; RGC, Glutamatergic Negative regulator of synapse formation Jones et al. 2011;
hippocampal and AMPA receptor synapse delivery Kucukdereli
et al. 2011

TNFα Rodent Astrocyte; Glutamatergic, Homeostatic synaptic scaling: increases Beattie et al. 2002;
hippocampal GABAergic synaptic AMPA and decreases synaptic Stellwagen and Malenka
GABAA receptors 2006; Stellwagen et al.
2005

CSPGs Rodent Astrocyte; Glutamatergic Stabilization of synaptic AMPA receptors Frischknecht et al. 2009;
hippocampal Pyka
et al. 2011b

Estrogen Rodent Astrocyte; Glutamatergic Enhancement of synapse formation Hu et al. 2007

cortical and function

ADNF Rodent Astrocyte; Glutamatergic Enhancement of synapse formation and Blondel et al. 2000
hippocampal function, increase in NMDA receptors

TGF-β1 Xenopus, Schwann cell; spi- Cholinergic Synapse formation and acetylcholine Feng and Ko 2008
rodent nal neurons receptor clustering
BDNF Rodent Support cells; Glutamatergic Synapse formation Gomez-Casati et al. 2010
hair cells

G L I A L C O N T R O L O F SY N A P TO G E N E S I S • 391
cell types are generated in the retina during this period of
embryonic development. When the embryonic neurons were
grown in contact with astrocytes, but not amacrine cells from
which they normally receive innervation in the retina, they
acquired the ability to receive synapses. Surprisingly, it was
shown that embryonic neurons had high levels of the presynap-
tic transmembrane molecule neurexin in their dendrites (where
it is not normally expressed in mature neurons), and the den-
dritic neurexin level decreased when embryonic neurons were
contacted by astrocytes. Thus, one explanation for the switch in
synaptic receptivity is that embryonic neurons express neurexin
in their dendrites that inhibits synapse formation, and astrocyte
contact downregulates dendritic neurexin (through an as yet to
be determined mechanism, which is not dependent on PKC
signaling), therefore allowing synapses to be formed.
3.1.3 Gamma-Protocadherins in Perisynaptic
Astrocyte Processes
Gamma-protocadherins are a family of 22 cell adhesion mol-
ecules that are encoded by a single gene cluster and present in
both neurons and astrocytes, and in astrocytes are localized
to the perisynaptic astrocyte processes that enwrap the syn-
apse (Garrett and Weiner 2009). Mice globally deficient in
γ-protocadherins die at birth and exhibit neuronal apoptosis
and decreased synapse density in the spinal cord (Wang et al.
2002; Weiner et al. 2005), raising the question of whether
neuronal or astrocytic γ-protocadherins are responsible for
synaptogenesis. This was addressed with a combination of
in vitro and in vivo experiments in which cell-specific deletion
of γ-protocadherins was conducted (either astrocytic or neu-
ronal), and the effect on synaptogenesis assessed in the spinal
cord (Garrett and Weiner 2009). When either astrocytes or
neurons lack γ-protocadherins then synaptogenesis is greatly
delayed, although if just the astrocytes lack γ-protocadherins
then the number of synapses eventually catches up to the
WT level with a delay. This demonstrates that astrocyte
γ-protocadherins are involved in neuronal synapse formation,
presumably through a contact-mediated mechanism via the
perisynaptic astrocyte processes (ACM was unable to rescue
synaptogenesis). This study raises the possibility that a distinct
γ-protocadherin code exists, whereby the diversity enabled by
22 potential γ-protocadherins enables astrocytes to specify
when and where particular synapses form.
3.2 G L I A I N D U C E SY NA P TO G E N E S I S VI A
S EC R ET E D FAC TO R S
3.2.1 Thrombospondins Induce Structural
Synapse Formation
Thrombospondins comprise a family of high molecular weight
secreted matricellular proteins with five known members,
that mediate both cell–cell and cell–matrix interactions. TSP-1 and
-2 are expressed by astrocytes in vitro and in the developing
brain during the peak period of synaptogenesis, and their
levels decrease by adulthood when synaptogenesis has largely
finished. Addition of TSP to cultured postnatal RGCs
392 • FUNCTIONS OF NEUROGLIAL CELLS
increases the number of structural synapses that form, to the
same extent as that induced by astrocytes, whereas depletion
of TSP from ACM reduces its synapse-inducing effects
(Christopherson et al. 2005). Thrombospondin-induced
synapses are presynaptically active but postsynaptically
silent—lacking the AMPA subtype of the ionotropic
glutamate receptor but containing extrasynaptic NMDA
receptors. In vivo studies demonstrated that mice lacking
both TSP-1 and -2 form 30% fewer excitatory synapses in
the cerebral cortex after 3 weeks of development, suggesting
prolonged defects in synapse formation. In addition to RGCs,
TSPs induce excitatory, but not inhibitory, synapse formation
between cultured hippocampal neurons (Hughes et al. 2010;
Xu et al. 2009).
3.2.2 Cholesterol Enhances Presynaptic Maturation
Astrocyte-derived cholesterol bound to ApoE enhances
synapse formation and function between autaptic RGCs
in vitro (Mauch et al. 2001). Cholesterol is necessary for
synaptogenesis and can be supplied to neurons by astrocytes
when neurons are deficient in cholesterol, such as in clonal-
density cultures. Cholesterol enhances presynaptic function
and neurotransmitter release by increasing quantal content,
and also increases dendritic out growth (Christopherson et al.
2005; Goritz et al. 2005).
3.2.3 Hevin and Secreted Protein Acidic and Rich in
Cysteine Are Positive and Negative Regulators
of Structural Synapse Formation
Hevin and SPARC are secreted matricellular proteins that
are expressed at high levels by astrocytes both in vitro and
in vivo, and unlike thrombospondin remain expressed
in the adult brain (Kucukdereli et al. 2011). Addition of
hevin to postnatal RGCs in vitro induces the formation of
structural synapses, which like synapses induced by TSP are
presynaptically active and postsynaptically silent. SPARC by
itself has no effect on synapse formation between RGCs, but
SPARC is a potent and specific inhibitor of hevin-induced
synapse formation (SPARC does not inhibit synapse
formation induced by TSP). In vivo analysis of synapse
formation in the superior colliculus, the target region of
RGC synapses, demonstrated that mice lacking hevin have
significantly fewer excitatory synapses, whereas mice lacking
SPARC have significantly more excitatory synapses. These
studies identify hevin as a positive and SPARC as a negative
regulator of synaptogenesis.
3.2.4 Glypicans Enhance Postsynaptic Function
and AMPA Glutamate Receptor Clustering
at Synapses
Both thrombospondin and hevin induce postsynaptically silent
synapses that lack AMPA receptors, suggesting that astrocytes
secrete additional signals that can recruit AMPA receptors
to synapses or induce fully functional synapse formation.
When RGCs are cultured in the presence of astrocytes
there is an increase in the strength of individual excitatory
synapses which is accompanied by a threefold increase in
the surface levels of all of the four different AMPA receptor
subunits (GluA1–4) and increased clustering of AMPA
receptors at synaptic sites, demonstrating that astrocytes
regulate glutamate receptors (Allen et al. 2012). Glypicans
4 and 6, which are gpi-linked HSPGs, have been identified
as astrocyte-derived proteins that are sufficient to induce
functional synapses, and act by increasing surface levels of
the GluA1 AMPA receptor subunit and clustering AMPA
receptors at synapses. Glypican 4 is expressed by astrocytes
in the hippocampus during early postnatal development, and
mice lacking glypican 4 have weaker excitatory synapses in the
hippocampus in vivo, shown by a decrease in the amplitude of
individual excitatory currents and a decreased recruitment of
GluA1 to synapses.
3.2.5 SPARC Is a Negative Regulator of AMPA
Glutamate Receptor Synaptic Delivery
Interestingly, in addition to releasing glypican, which enhances
synaptic AMPA receptor levels, astrocytes also release SPARC,
which acts as a negative regulator of AMPA receptor deliv-
ery to synapses ( Jones et al. 2011). SPARC is expressed by
astrocytes in the hippocampus during early postnatal devel-
opment and is regulated by neuronal activity—when activ-
ity is enhanced in hippocampal slices, then SPARC protein
is increased in astrocytes. Mice lacking SPARC have stronger
excitatory synapses in the hippocampus during development,
with an increase in the frequency and amplitude of mEPSCs.
Studies on cultured neurons in the presence of wild-type
(WT) or SPARC knock-out (KO) astrocytes demonstrated
that there is an increase in neuronal surface AMPA recep-
tor levels in the absence of SPARC, and that this difference
can be rescued by the addition of SPARC protein to the KO
astrocytes. This suggests that astrocytes sense and respond to
neuronal activity levels by altering the expression of SPARC,
with high levels of activity increasing SPARC which may act
back on neurons to reduce synaptic AMPA receptor levels and
hence help maintain synaptic balance.
3.2.6 Tumor Necrosis Factor Alpha Regulates AMPA
and GABAA Receptor Levels and Homeostatic
Synaptic Scaling
In addition to regulating synaptic strength during develop-
ment, astrocytes have been shown to release factors that reg-
ulate the recruitment of AMPA and GABAA receptors to
synapses in the mature brain. Glia release the cytokine TNFα,
which modulates synaptic strength by increasing surface
AMPA receptors and decreasing surface GABAA receptors,
thus leading to an overall strengthening of synaptic transmis-
sion—known as homeostatic synaptic scaling (Beattie et al.
2002; Stellwagen et al. 2005; Stellwagen and Malenka 2006).
Addition of TNFα to cultured hippocampal neurons causes a
rapid increase (within 15 minutes) in surface GluA1 AMPA
receptor levels, whereas blockade of the actions of endogenous
TNFα lead to a decrease in surface AMPA receptors. When
G L I A L C O N T R O L O F SY N A P TO G E N E S I S
TNFα KO astrocytes were cultured with WT neurons activ-
ity blockade no longer caused synaptic scaling, showing a glial
source of TNFα (see chapter 38 for additional details).
3.2.7 Chondroitin Sulfate Proteoglycans
Modulate AMPA Glutamate Receptor
Stability at Synapses
Astrocytes and neurons both produce a variety of CSPGs,
including brevican, neurocan, phosphacan, and versican
(Faissner et al. 2010) (see chapter 32 for additional details).
Chondroitin sulfate proteoglycans contribute to the extracel-
lular matrix and are concentrated at synapses in the mature
brain forming perineuronal net structures. Chondroitin
sulfate proteoglycans also accumulate in perineuronal nets
in an astrocyte–neuron coculture system as synaptogenesis
progresses. Enzymatic digestion of these nets increases the
number of structural synapses that form and decreases the
amplitude of excitatory synaptic currents (Pyka et al. 2011b).
These results suggest that the build-up of CSPGs leads to the
stabilization of mature synapses, thus regulating the num-
ber of synapses that are present. Indeed, CSPGs stabilize
AMPA receptors at synaptic sites by limiting the diffusion
of these receptors within the membrane, suggesting a mecha-
nism for the observed decrease in synaptic current amplitude
(Frischknecht et al. 2009).
3.2.8 Estrogen Enhances Synapse Formation
and Function
Cortical astrocytes enhance synapse formation and transmis-
sion between cortical neurons in vitro which can be attenu-
ated by treatment with tamoxifen, an antagonist of estrogen
receptors (Hu et al. 2007). Astrocytes do not normally express
aromatase enzymes in vivo, which are necessary to synthesize
estrogen, but these enzymes are induced in reactive astrocytes
following injury. This suggests that estrogen secretion from
astrocytes may be induced in injury, and could contribute to
the recovery from damage.
3.2.9 Activity-Dependent Neurotrophic Factor
(ADNF) and N-Methyl-d-aspartate
Receptors
Astrocyte-neuron cocultures isolated from E16 rat hippo-
campus have few synaptic connections, whereas those isolated
from E18 rats have numerous synaptic connections (Blondel
et al. 2000). Vasoactive intestinal peptide is released from
embryonic hippocampal neurons when isolated at E18, but
not E16, and its addition to E16 cultures is able to enhance
synaptic connectivity. Vasoactive intestinal peptide acts on
astrocytes to cause the release of ADNF, which acts back on
neurons to stimulate release of neurotrophic factors such as
NT3. The eventual outcome following 2 weeks of culture is an
increase in the number of presynaptic terminals, an increase
in synaptic event frequency and amplitude, and an increase
in NMDA receptor subunits (NR2A and NR2B) within
neurons.
• 393
 3.2.10 Perisynaptic Schwann cells Enhance Synapse
Formation at the Neuromuscular Junction via
Transforming Growth Factor-β1
Perisynaptic Schwann cells are specialized Schwann cells at the
NMJ synapse, which enhance synapse formation and function
between cultured spinal neurons and target muscle in Xenopus
and rodent, and stabilize synaptic connections in vivo (Cao and
Ko 2007; Feng et al. 2005; Peng et al. 2003; Reddy et al. 2003).
Schwann cells act to enhance synapse formation and clustering
of acetylcholine receptors in neuron–muscle cocultures (Peng
et al. 2003). Interestingly, it appears that Schwann cells induce
synapse formation by overcoming the inhibitory effects of neu-
rotrophic factors that are necessary for neuronal survival. High
levels of neuronal death are observed in the absence of neu-
rotrophic factors and Schwann cells, but the neurons that sur-
vive make synapses. The addition of neurotrophic factors to the
cultures greatly enhances neuronal survival, but few synapses
form, suggesting synaptogenesis has been inhibited. The addi-
tion of Schwann cells to the coculture, in the continuous pres-
ence of neurotrophic factors, enables synapses to form between
neurons and muscle cells. The molecular basis for this has been
determined—neurotrophic factors inhibit the production of
agrin in neurons, and Schwann cells overcome this inhibition
and reinduce the expression of agrin. Agrin is essential for the
stabilization of acetylcholine receptor clusters at NMJs and
hence for NMJ function and stability. Transforming growth
factor-β1 has been identified as the factor secreted from
Schwann cells that is sufficient and necessary to upregulate
agrin levels in presynaptic neurons (Feng and Ko 2008).
3.2.11 Support Cell–Induced Synaptogenesis in
the Vestibular Organ Mediated by Brain-Derived
Neurotrophic Factor
In the vestibular system, supporting cells have many of the
characteristics of astrocytes and Schwann cells; therefore,
their effect on synaptogenesis between hair cells and vestibu-
lar neurons in the inner ear was investigated (Gómez-Casati
et al. 2010). A series of in vivo experiments using a variety of
transgenic and KO mice demonstrated that neurons signal
to support cells via the tyrosine kinase receptor erbB, leading
to the expression of BDNF in support cells that feeds back
onto neurons to induce synapse formation between hair cells
and vestibular neurons. Blockade of erbB signaling in sup-
port cells, or loss of BDNF specifically from support cells,
led to severe defects in synapse number at P21. Interestingly,
the defect in synapse number was less severe at early postnatal
ages, and total synapse number actually decreased with age in
mutant mice, suggesting a failure of synapse maintenance.
4 G L I A I N F LU E N C E SY N A P T I C
S T RU C T U R E , E L I M I N AT I O N,
A N D S TA B I L I T Y
In addition to inducing initial synapse formation, astrocytes
are actively involved in determining the fate of newly formed
394 • FUNCTIONS OF NEUROGLIAL CELLS
synapses. Whether a synapse matures, is stabilized and main-
tained, or even eliminated can be influenced by astrocytes.
4.1 A S T RO C Y T E –SY NA P S E P H YS I C A L
I N T E R AC T I O NS
Physical contact between astrocytes and neurons can have
multiple outcomes; for example, stabilization of synaptic
contacts, elimination of synapses, or even preventing syn-
apses from forming by blocking potential sites. Each of these
outcomes has been observed, suggesting that the outcome of
astrocyte–neuron contact is context dependent; for example,
based on developmental stage or neuronal activity.
4.1.1 Astrocyte–Dendritic Spine Physical
Interactions Stabilize Synapses
Astrocyte–dendritic spine contacts have been observed to be
more stable at large mature spines compared to weak filopodia,
leading to the hypothesis that astrocyte–spine interactions are
involved in stabilizing synaptic connections (Lippman and
Dunaevsky 2005; Lippman et al. 2008; Witcher et al. 2007).
Indeed, when the actin cytoskeleton is disrupted, which dis-
rupts astrocyte process protrusion, there is an increase in den-
dritic filopodia, a decrease in mature spines, and more synapses
are observed (Nishida and Okabe 2007).
4.1.2 Astrocyte Coverage Limits
Synapse Formation
In the cerebellum, climbing fibers release ectopic vesicles
of glutamate directly onto BG (Matsui and Jahr 2003),
and BG sense this via calcium permeable AMPA receptors,
which lack the GluA2 subunit. Expression of GluA2 in BG
(making AMPA receptors calcium impermeable) causes the
retraction of glial processes from the PC with which they
are normally in close association (Iino et al. 2001), lead-
ing to aberrant innervation of the PC by multiple climb-
ing fibers instead of the single climbing fiber that innervates
each PC in the mature cerebellum. This shows that release
of glutamate directly onto the BG signals the cell to remain
in close association with the PC it is surrounding, thus lim-
iting the number of synapses that can form onto the PC. In
the HNS astrocytes respond to hormonal signals by fully
retracting from synapses in a reversible manner (Theodosis
et al. 2008). Neurons with retracted astrocytes receive more
synaptic inputs suggesting astrocytes were previously block-
ing these areas and stopping neuronal innervation (see chap-
ter 41 for more details).
4.1.3 Physical Coupling Between Astrocyte Processes
and Dendritic Spines Via EphrinA3–EphA4
Interactions
Dendritic spines express the receptor tyrosine kinase EphA4,
which interacts with its ligand ephrin-A3 on neighboring
Figure 31.3 Example of Physical Coupling Between an Astrocyte Process and a Synapse via Adhesion Molecules Triple labeling shows ephrin-A3 on
astrocytic processes (red) encompassing a synaptophysin-positive presynaptic terminal (blue) and YFP (green) to label the dendritic spine of a CA1
pyramidal neuron where EphA4 is localized. Scale bar, 0.5 μm. Reproduced with permission from Mura et al. 2003.

astrocytic processes (Fig. 31.3) (Klein 2009; Murai and
Pasquale 2011; Murai et al. 2003). EphA4 levels in the
hippocampus are high during early postnatal development
when synapses are actively forming, and it is present in
an inactive nonphosphorylated form in the adult brain.
Reactivation of EphA4 in the adult hippocampus collapsed
20% of dendritic spines and decreased the length of other
spines, causing spine retraction and an overall reduction in
spine density. On the other hand, when EphA4 was inhibited
this caused disorganization of spines and an overall increase
in spine length. These results suggest that during development
(when EphA4 is active), contact between astrocytic processes
and dendritic spines may be repulsive, and that the level
of signaling plays a role in the localization of synapses by
determining whether a spine is eliminated or stabilized.
4.2 G L I A I N FLU E N C E SY NA P S E
E L I M I NAT I O N
In the developing brain excess synapses are formed, and
elimination processes remove these synapses to generate the
final adult number. Possible mechanisms of synapse elimina-
tion include retraction of presynaptic terminals and axons,
degeneration of axons and subsequent phagocytosis of the
debris by surrounding cells e.g. glial cells, or active pruning
of excess synapses by neighbouring glia. There is evidence

Astrocyte factors Immature Mature Mature synaptic unit

Presynaptic maturation Presynaptic terminal

Cholesterol
Thrombospondin 1,2
Hevin
Glypican 4,6

Structural synaptogenesis
Thrombospondin 1,2
Hevin
Glypican 4,6
Astrocyte process

Postsynaptic maturation
Glypican 4,6 Key
Neurotransmitter vesicle
Sparc
Neurotransmitter receptor
Tumour necrosis factor alpha
Postsynaptic density
CSPGs
Postsynaptic terminal

Figure 31.4 Schematic Representation of Astrocyte Regulation of Distinct Aspects of Synapse Formation During Development, via the Secretion of
Multiple Factors. Presynaptic maturation is defined as increased neurotransmitter vesicle recruitment to presynaptic sites and increased neurotransmit-
ter release probability. Structural synaptogenesis is defined as clustering of presynaptic and postsynaptic density proteins and the alignment of presyn-
aptic and postsynaptic sites. Postsynaptic maturation is defined as insertion of postsynaptic receptors into the postsynaptic density membrane.

G L I A L C O N T R O L O F SY N A P TO G E N E S I S • 395
from studies of Drosophila that glia do indeed phagocytose
axonal debris. In rodents, astrocytes induce synapse elimina-
tion pathways in neurons, and microglia phagocytose syn-
apses, demonstrating that glia are actively involved in synapse
elimination.
4.2.1 Drosophila Glia Engulf Axonal Debris
During Development
In the developing Drosophila nervous system, glial cells
invade regions of axonal degeneration and engulf axonal
varicosities by phagocytosis (Awasaki and Ito 2004; (Walts
et al. 2004). Blocking glial phagocytosis significantly per-
turbs axon pruning. The glial phagocytosis receptor Draper
is essential for this process—when Draper is absent from
glial cells (and muscle) the removal of axonal debris is greatly
reduced, causing defects in synapse growth (Fuentes-Medel
et al. 2009; MacDonald et al. 2006) (for more information
see chapter 2).
4.2.2 Axosome Shedding at the
Neuromuscular Junction
As axons are removed at the developing NMJ they produce
axosomes, which are small membrane bound particles, and
time-lapse imaging and EM have shown that these axosomes
can be seen within neighbouring Schwann cells. (Bishop
et al. 2004). Thus, a similar process of removing axonal
debris from the developing nervous system exists in both
Drosophila and mammals.
4.2.3 C1q Induction in Neurons by Astrocytes
The immune system molecule C1q is induced in RGCs when
they are cultured in the presence of astrocytes (Stevens et al.
2007). C1q acts as an opsonin in the immune system, mark-
ing damaged cells and debris for removal by phagocytosis
or lysis by activation of the complement cascade. Mice defi-
cient in C1q have more functional synapses in vivo, suggest-
ing that C1q has a similar role in the developing brain and
plays an active role in neuronal synapse elimination. The
excess of synapses in C1q deficient mice has functional con-
sequences, as these mice are spontaneously epileptic (Chu
et al. 2010).
4.2.4 Synapse Elimination and Synaptic
Stripping by Microglia
A role for microglia in synapse elimination has been proposed
in the mammalian CNS, both during development and in
injury situations. The process of synaptic stripping, whereby
microglia displace axonal inputs from damaged motoneu-
rons, was first described in 1968 (Blinzinger and Kreutzberg
1968; Cullheim and Thams 2007). Synaptic inputs onto
damaged neurons are eliminated, and it is hypothesized that
microglia actively denervate the damaged postsynaptic cell
by intervening between the presynaptic and postsynaptic
396 • FUNCTIONS OF NEUROGLIAL CELLS
side and pushing off the axon. Recent studies have shown
an essential role for microglia in synapse elimination in the
healthy developing CNS (Paolicelli et al. 2011; Schafer et al.
2012). Microglia phagocytose both presynaptic and postsyn-
aptic elements, as revealed by immunoelectron microscopy
and high resolution confocal imaging, both of which show
synaptic elements completely within microglia and in the
phagocytic degradation pathway. Notably, when the ability
of microglia to phagocytose is inhibited by genetic removal
of the microglia-specific phagocytic pathway CR3/C3, then
more synapses are present in the adult brain, demonstrating
that microglia are actively involved in synapse elimination in
development (Schafer et al. 2012) (see chapters 15, 47, and 53
for more details).
4 .3 A R O L E F O R G L I A I N SY N A P S E
M AINTENANCE
The preceding data demonstrate that glial signals power-
fully induce synapse formation and remodeling, but do glia
act as a switch to enable neurons to form synapses, or are
glial signals constantly required so as to maintain synapses?
Initial studies suggest that glia are indeed necessary for syn-
apse maintenance.
4.3.1 Constant Astrocyte Signals Are Required
for Synapse Maintenance In Vitro
Retinal ganglion cells in vitro require a continuous signal
from astrocytes to maintain the astrocyte-induced increase
in synapse formation and function. When synapses are
induced by astrocytes, and the astrocytes removed, the num-
ber of synapses decreases over a period of days to a similar
level as that observed in RGCs that have never been exposed
to astrocytes (Ullian et al. 2001). The identity of the soluble
maintenance signal is unknown, and it is not clear whether
it is the same signal that induces synapse formation (e.g.,
thrombospondin) or a novel signal.
4.3.2 Perisynaptic Schwann Cells Maintain
Neuromuscular Junction Synapses In Vivo
The effect of removing synaptic glia from a mature synaptic
contact in vivo has been investigated at the Xenopus NMJ
(Reddy et al. 2003). Monoclonal antibodies were used to
label perisynaptic Schwann cells in an intact adult frog
NMJ and the cells were lysed via the complement cascade,
selectively killing Schwann cells and leaving the presynap-
tic motor neuron terminal and the postsynaptic muscle cell
intact. Loss of perisynaptic Schwann cells had no immedi-
ate effect on the structure or function of the synapse, but 1
week after ablation presynaptic function decreased by half,
and there was a tenfold increase in the retraction of presyn-
aptic terminals from the muscle.
 5 I N T E R AC T I O N S B ET W E E N
A S T R O C Y T E FAC TO R S A N D N E U R O N A L
SY N A P TO G E N I C PAT H WAYS
There has been much work on identifying the astrocyte-derived
signals that induce synaptogenesis, but less is known about how
these signals interact with neurons to induce synaptogenesis.
Neuronal receptors for astrocyte factors are beginning to be iden-
tified, with α2δ1 identified as the TSP receptor and TrkB shown
to be involved in GABAergic synapse formation. The question
of whether astrocyte synaptogenic signals act locally to induce
individual synapses or globally to enhance the synaptogenic
state of the whole neuron (e.g., via effects on gene transcription)
is still unanswered. Although physical astrocyte contact can
enhance global synaptogenesis in embryonic neurons (Hama
et al. 2004), it is not known whether this is the case for secreted
signals. Neuronal activity plays an important role in synaptic
remodeling in the developing brain, and studies are beginning
to show that activity is able to regulate the release of some astro-
cyte synaptogenic factors. Whether all factors are modulated by
activity or are constitutively released or developmentally con-
trolled will be an important point for further study.
5.1 N EU RO NA L R E C E P TO R S F O R
A S T RO C Y T E S I G NA L S
5.1.1 Thrombospondin Receptors
The synaptically localized α2δ1, first identified as an acces-
sory subunit of voltage-gated calcium channels, has been
identified as a neuronal receptor necessary for TSP-induced
synapse formation (Eroglu et al. 2009). Overexpression of
α2δ1 causes an increase in synapse formation both in vitro
and in vivo. Conversely, inhibition of α2δ1 with gabapentin
blocks TSP-induced synapse formation in vitro and greatly
reduces developmental synapse formation in vivo. A study
in hippocampal neurons showed that TSP can interact with
postsynaptically localized neuroligin (which interacts with
presynaptic neurexin to stabilize synaptic contacts), suggest-
ing another potential mechanism by which TSP can regulate
synapse formation (Xu et al. 2009).
5.1.2 TrkB in GABAergic Synapse Formation
Hippocampal neurons undergo enhanced GABAergic synap-
togenesis in the presence of astrocytes, and as for RGCs, the
astrocytic effect is via a soluble factor. Astrocytes induce an
increase in inhibitory synapse number, increase surface levels
of GABAA receptors, and enhance inhibitory synaptic trans-
mission and GABA currents (Elmariah et al. 2005a,b; Liu
et al. 1996). The identity of the astrocyte signal that induces
GABAergic synapse formation in hippocampal neurons is
currently unknown, but the downstream effectors have been
identified. Astrocytes modulate neuronal BDNF and TrkB
signalling, which leads to increased synaptogenesis by modu-
lating postsynaptic maturation and synaptic recruitment of
GABAA receptors (Elmariah et al., 2005b).
G L I A L C O N T R O L O F SY N A P TO G E N E S I S
5.2 D OWNS T R E A M S I G NA L I N G C A S C A D E S
I N D U C E D I N N EU RO NS BY A S T RO C Y T E
SY NA P TO G E N I C S I G NA L S
5.2.1 Gene Transcription
The question of whether astrocytes induce synapse forma-
tion via global effects on gene transcription was addressed
using microarrays to identify genes altered in RGCs following
30 hours of exposure to ACM (Göritz et al. 2007). Surprisingly
very few genes were changed, with 28 genes being upregulated
and 46 genes downregulated, and the majority of the genes
were not known to be associated with synaptogenic pathways.
This suggests that regulation of gene expression does not play
a major role in astrocyte-induced synaptogenesis, although it
would be interesting to know whether more genes would be
regulated following a longer exposure to astrocytes, or if the
neurons and astrocytes were grown in contact.
5.2.2 Neuronal Activity
Neuronal activity is involved in synaptic remodeling in the
developing nervous system, but is it required for neurons to
respond to astrocyte-derived synaptogenic factors? The effect
of activity blockade was investigated in RGCs by culturing
them alone or with ACM in the presence or absence of TTX
to block action potential-mediated synaptic transmission
(Nägler et al. 2001). Astrocyte conditioned media caused a
significant increase in the frequency and amplitude of synap-
tic events, even in the presence of TTX, showing that action
potentials are not necessary for neurons to form synapses in
response to astrocyte factors. Whether synapses would still
form if all synaptic activity was blocked, for example, by pre-
venting glutamate release or blocking postsynaptic glutamate
receptors, remains to be addressed.
5.3 T R I G G E R S F O R R E L E A S E O F
SY NA P TO G E N I C FAC TO R S FRO M A S T RO C Y T E S
5.3.1 Developmental Stage
Many of the identified astrocyte synaptogenic factors are
developmentally regulated, being highly expressed in early
postnatal stages during the peak period of synaptogenesis, and
at low levels or absent in the adult brain when very little synap-
togenesis is occurring. Examples of this include thrombospon-
din 1 and 2, glypican 4 and 6, and SPARC (Allen et al. 2012;
Christopherson et al. 2005; Jones et al. 2011; Kucukdereli et al.
2011). Indeed, the transplantation of immature astrocytes to
adult cat cortex reinduces ocular dominance plasticity after
the end of the critical period, suggesting that immature astro-
cytes release more prosynaptogenic compounds than mature
astrocytes (Hensch 2005; Muller and Best 1989). Intriguingly,
there is a correlation between the formation of perineuronal
nets, which are composed of astrocyte and neuron-derived
CSPGs, around synapses in the visual cortex and the end of
the critical period. Removal of these nets in adult rat brain via
chondroitinase treatment, which degrades the CSPGs that
• 397
make up the nets, reactivated ocular dominance plasticity,
linking astrocyte-derived CSPGs with the closure of the criti-
cal period and thus as factors that limit synaptic plasticity (see
section 3.2.7) (Pizzorusso et al. 2002).
5.3.2 Neuronal Signals and Activity
Is there feedback between neurons and astrocytes to regulate
the release of synaptogenic factors, so generating the final cor-
rect pattern of synapse number and strength? When neuronal
activity is enhanced or astrocytes are stimulated with glutamate,
then astrocytes increase SPARC secretion, which negatively
regulates glutamate receptor levels at synapses, hence reducing
overall synaptic activity ( Jones et al. 2011). Conversely, stimula-
tion of astrocytes with glutamate causes a decrease in release of
TNFα, which would normally increase glutamate receptor lev-
els at synapses, so acting as a negative feedback loop to prevent
synaptic strength from increasing by too much (Stellwagen
and Malenka 2006). Treatment of cultured astrocytes with
ATP increases TSP1 secretion (Tran and Neary 2006), as does
mechanical trauma, suggesting that in an injury situation TSP1
may contribute to new synapse formation. Astrocyte condi-
tioned media, however, is able to induce active synapse forma-
tion in neurons, demonstrating some constitutive release of
synaptogenic factors from astrocytes (Ullian et al. 2001).
6 A R O L E F O R D E F E C T I VE
A S T R O C Y T E -I N D U C E D SY N A P S E
F O R M AT I O N I N N E U R O D E VE L O PM E N TA L
DISORDER S
Many neurodevelopmental disorders are characterized by
defective synapse formation or function, and there is emerg-
ing evidence that defects in astrocyte-induced synaptogenesis
can play a role in the pathogenesis of these diseases.
6.1 D OWN SY N D RO M E
Down syndrome (DS), the most common genetic form of
mental impairment, is caused by the trisomy of chromosome
21 and is characterized by a reduced number of dendritic
spines on neurons along with disordered spine morphology.
To address whether astrocytes could be contributing to this
phenotype, WT rodent hippocampal neurons were cultured
in contact with human astrocytes, derived either from DS
or unaffected embryonic brains (Garcia et al. 2010).
Neurons grown on DS astrocytes have fewer spines and fewer
mature spines than those grown on unaffected astrocytes.
Down syndrome astrocytes secrete less TSP1, and addition
of TSP1 protein to DS astrocytes rescues spine number and
morphology, suggesting that reduced secretion of this synap-
togenic protein from astrocytes may contribute to DS.
6.2 FR AG I L E X S Y N D RO M E
Fragile X syndrome (FX) is the most common inherited form
of mental impairment and is caused by a mutation in the Fmr1
398 • FUNCTIONS OF NEUROGLIAL CELLS
gene, which encodes a protein that modulates translation of
mRNA into protein. Culturing Fmr1 KO astrocytes with WT
neurons and vice versa revealed that Fmr1 KO astrocytes cause
defective dendrite development in neurons—dendrites are
more highly branched and the total dendritic area is reduced.
The number of synapses appears to be decreased when the total
synapse number per neuron is analyzed, and increased when
synaptic density is analyzed, perhaps because of the reduced
dendritic arbor size causing the synapses to be more closely
spaced ( Jacobs and Doering 2010; Jacobs et al. 2010).
6.3 R ET T SY N D RO M E
Rett syndrome is an X-linked autism spectrum disorder caused
by a loss of function of the transcription factor MeCP2.
Non-cell autonomous effects from mutant astrocytes are
responsible for some of the features of the disorder (Ballas
et al. 2009; Lioy et al. 2011). Culturing of WT neurons in the
presence of ACM from KO astrocytes causes a severe stunt-
ing of dendritic outgrowth, suggesting that Rett’s astrocytes
are either deficient in the secretion of a pro-dendrite growth
factor or secreting a toxic factor. Furthermore, re-expression of
Mecp2 specifically in astrocytes in vivo on an MeCP2 deficient
background partially rescues the behavioral defects observed
in Rett syndrome, as well as rescuing dendritic defects and
increasing presynaptic terminal numbers.
7 S U M M A RY A N D P E R S P E C T I VE S
Glia are intimately associated with synapses at all stages of
development and adult life, and induce the formation of syn-
apses as well as modulating presynaptic and postsynaptic func-
tion. Glia contribute to the maintenance of synaptic structure
and arrangement, ensuring that neurons receive the correct
pattern of innervation and hence maintain synaptic balance
and nervous system function.
A growing list of astrocyte-derived synaptogenic factors
have been identified, with multiple aspects of synapse for-
mation and maturation being modulated by distinct signals
from astrocytes (Figure 31.4). Current evidence suggests that
embryonic neurons first require physical contact with an astro-
cyte so as to become receptive to astrocyte-secreted signals
that are present during postnatal periods. Different factors
are required for excitatory and inhibitory synapse formation,
in the CNS compared with the PNS, and for structural syn-
apse formation compared with presynaptic differentiation and
neurotransmitter receptor clustering. Further in vitro studies
examining the effects of combinations of each of these synap-
togenic factors on neuronal synapse formation will greatly aid
in assessing the role that each of them has to fully functional
synapse formation. Investigating the precise spatiotemporal
control of when and where each astrocyte synaptogenic factor
is expressed in vivo in the developing brain, and whether there
is regional heterogeneity in expression, will be an essential part
in understanding how each of these factors contributes to syn-
aptogenesis, as well as to how their dysfunction may contrib-
ute to neurological disorders
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 32.
NEURON MIGRATION AND AXON GUIDANCE
Andreas Faissner

A B B R E VI AT I O N S SVZ subventricular zone

TN-C tenascin-C, extracellular matrix
BMPs bone morphogenetic proteins glycoprotein
CAMs cell adhesion molecules TN-R tenascin-R, extracellular matrix glycoprotein
CD44 hyaluronan receptor, many isoforms known Wnt wingless proteins, morphogens in embry-
CNTN contactin, CAM of the IgSF onic development
CSF cerebrospinal fluid
CSPGs chondroitin sulfate proteoglycans
1 E VI D E N C E F O R C O N D U C I VE
DCC “deleted in colorectal cancer,” receptor of
F U N C T I O N S O F A S T R O G L I A–N E U R O N A L
the IgSF
M I G R AT I O N
DRG dorsal root ganglion
ECM extracellular matrix
1.1 N EU RO G E N E S I S EVO LVE S I N D I S T I N C T
EGF epidermal growth factor
S T E P S D U R I N G C E N T R A L N E RVO US SYS T E M
EGL external granular layer
D EVE L O PM E N T A N D I N VO LVE S R A D I A L G L I A
FGF fibroblast growth factor
A S N EU R A L S T E M C E L L
GAG glycosaminoglycan
Gcm glial cell missing The CNS originates from a layer of neuroepithelial cells that
GPI glycosylphosphatidylinositol-anchor of CAMs expand by symmetrical division. With increasing thickness
HSPGs heparan sulfate proteoglycans of the walls of the neural tube the neuroepithelium evolves to
IGL internal granular layer radial glia (RG) (see chapter 5). These proliferate and self-renew
IgSF immunoglobulin superfamily by symmetrical division, but progressively give rise to neurons
KS keratan sulfate that are generated in an asymmetrical division mode (Kriegstein
KSPGs keratan sulfate proteoglycans and Alvarez-Buylla 2009; Merkle and Alvarez-Buylla 2006) (see
L1-CAM cell adhesion molecule L1 chapter 30). Thereby, radial glia cells do give rise to neurons in
Mab monoclonal antibody vitro (Hartfuss et al. 2001; Malatesta et al. 2000) and serve as
MAG myelin associated glycoprotein neural stem cells (NSCs), as evidenced by fate mapping stud-
N-CAM neural cell adhesion molecule ies in vivo (Anthony et al. 2004; Malatesta et al. 2003; Miyata
NG2 chondroitin sulfate (part-time) proteoglycan et al. 2001; Noctor et al. 2001, 2002). A subpopulation of neu-
Nogo regeneration inhibitory glycoprotein rons called intermediate progenitors proliferates by a restricted
NSPCs neural stem/progenitor cells number of symmetrical divisions and can be viewed as a reser-
OmGP oligodendrocyte myelin glycoprotein voir for ongoing cortical expansion. In species with substan-
PDGF platelet-derived growth factor tially enlarged cortical width, a second zone of neurogenesis
PGs proteoglycans has recently been localized in a second subventricular zone,
PNN perineuronal net closer to the cortical surface (Fietz and Huttner 2011; Hansen
PSA polysialic acid et al. 2010). Following neurogenesis, oligodendrocyte precur-
PSA-N-CAM N-CAM derivatized with PSA (formerly sors are formed and migrate to target regions to myelinate the
“embryonic” N-CAM) axonal connections (Nave 2010). Finally, the radial glia recedes
RG radial glia and transforms into astrocytes, with two well-known excep-
RPTP receptor protein tyrosine phosphatase tions, the Bergmann glia of the cerebellum (Fig. 32.1) and the
Robo “roundabout,” slit-receptors of the Ig- Müller glia of the retina. Progeny of the radial glia is thought
superfamily to reside as radial-type astrocytes in the subventricular zone
Shh sonic hedgehog of the lateral ventricle and the subgranular zone of the dentate
Slits chemorepellent proteins expressed by mid- gyrus of the hippocampus, where these cells serve as stem cells
line glia, ligands of Robo-receptors for neurogenesis in the adult CNS (Kriegstein and Alvarez-
SGZ subgranular zone Buylla 2009).

402
 A Basolateral B

EGL
ML

Apical
IGL
Radial migration Tangential Cerebellar migration
migration

C Phase 3-channel Hoechst GLAST βIII-

contrast Tubulin

Figure 32.1 Modes of Glia-Guided Migration. A. Neurons (red) arise from dividing (curved green arrow) radial glia by asymmetrical division and use
the glia scaffold for migration toward the outer cortical layers, where they differentiate. An independent wave of tangential migration is orthogonally
oriented (curved yellow arrow) and conveys progenitors from the median ganglionic eminence to the telencephalic cortex (Hatten 2002). B. In the
cerebellum, neuronal progenitors (red) divide in the external granular layer (EGL). After a final round of division, a leading process and then the nucleus
follow the radially oriented process of the Bergmann glia (blue). The neurons finally lodge in the growing internal granule cell layer (IGL), leaving a trail-
ing process that forms a T-shaped axon that apposes itself to the growing molecular layer (ML). C. Under particular culture conditions, neurospheres
extend radial glia processes (open arrowheads) that express the marker GLAST (green fluorescence) and guide the migration of neurons (filled arrow-
heads; ßIII-tubulin–positive, red fluorescence) that are also visible in phase contrast (Sirko et al. 2010). Micrographs courtesy of Dr. U. Theocharidis.

1.2 S H I F T FRO M SY M M ET R I C A L TO by P. Rakic and his school. The iconic reconstruction of electron
A SY M M ET R I C A L D I VI S I O N, R A D I A L micrographs represents a neuronal cell that closely apposes the
G L I A– GU I D E D N EU RO N M I G R AT I O N radial glia fiber and follows its trail toward the surface of the grow-
ing cortex (Rakic 2007). Biological clocks determine the assign-
As has been pointed out, the transition from the symmetri-
ment of the migrating neurons to discrete layers of the cortex,
cal to the asymmetrical division mode is of crucial importance
which are formed by neurons of similar birth date (Rakic et al.
for the diversification of neural cell lineages (Kriegstein and
2009). The radial unit hypothesis postulated that these neurons
Alvarez-Buylla 2009) (see chapter 30). On theoretical grounds,
would form the columnar networks of the cortex that would be
this switch of division mode can either be caused by an asym-
patterned by the trajectories of the radial glia guiding fiber (Rakic
metrical distribution of specific cellular determinants to only
2007). Recent findings have shown that a wave of neurons migrat-
one of the daughters, as has been paradigmatically proven in the
ing tangentially from the median ganglionic eminence would be
Drosophila nervous system (Gotz and Huttner 2005); alterna-
superimposed onto the pattern of radially migrating cells (see
tively, it could be caused by dispatching the daughters to distinct
Fig. 32.1). These cells are thought to give rise to GABAergic
microenvironments that might differentially instruct their fur-
interneurons of cortical networks (Hatten 2002).
ther fate (Scadden 2006).
The neuronal progenitors that arise in the course of the asym-
metrical division in the telencephalon use the radial glia cells as
1.3 C O RT I C A L L AY E R I N G A N D
migration support to reach their final destination in the form-
R E E L I N-M E D I AT E D N EU RO N M I G R AT I O N
ing layers of the emerging cortex (see Fig. 32.1). The radial glia–
guided migration represents a paradigmatic representation of At early stages of neural development, radial glia cells span
neuron–glia interactions that has been worked out in great detail the width of the growing cortex in the telencephalon and the

N E U R O N M I G R AT I O N A N D AXO N GU I DA N C E • 403
external granular layer (EGL) in the cerebellum. In the latter
case, the cell type is referred to as Bergmann glia. Evidence
for a genetic cause of migration deficits was obtained as early
as in the 1970s with the description of the mouse mutant
reeler. In this model, a general mal-positioning of neurons
could be determined that leads to an inversion of cortical
layering. The gene responsible was discovered as an unfore-
seen target of insertional mutagenesis and subsequently
named Reelin. This glycoprotein of the extracellular matrix
(ECM) is released by the Cajal-Retzius (CR) neurons of the
developing cortex that vanish after development has ceased
(Frotscher 1998). On the molecular level, the glycoprotein
presents extensive egf-type repeats that are similar to those
detected in the ECM glycoprotein Tenascin-C (Tnc) (Feng
and Walsh 2001).
Beyond its developmental function, Reelin seems to be
important for the adult CNS as well. Thus, several gene screens
have revealed that it is associated with or implicated in neu-
rological or psychiatric diseases such as temporal lobe epilepsy
(TLE), schizophrenia, or bipolar disorder (Frotscher 2010). In
light of its roles in the regulation of cortical neuron migration,
receptor systems and downstream signaling mechanisms have
been extensively studied. The major Reelin receptors pertain
to the broad family of lipoprotein receptors, that is, VLDLR
and ApoER2, and involve downstream signaling proteins such
as mDab1 and abl that were also detected in genetic screens
aiming at the bases of cortical malformations.
Extensive screens in the human have meanwhile provided
evidence for the occurrence of cortical malformations such
as lissencephaly, a condition characterized by a reduction of
the volume of gyri (microgyria) and sulci that is accompanied
by a smoothing of the cortical surface and a smaller brain size
(microcephaly). This pathology is associated with neuron
migration deficits. Linkage analysis has yielded several genes
of interest that are involved in these processes, for example,
centrosome-associated proteins that are implicated in progen-
itor cell division, and translocation of the nucleus and soma
in the course of neuronal migration (Ross and Walsh 2001).
These anatomical malformations are generally causing mental
retardation of varying degrees of severity (Manzini and Walsh
2011). A recent study highlights that the small GTPase RhoA
expressed by radial glia plays a crucial role in the neuron migra-
tion process (Capello et al. 2012).
1.4 N EU RO N M I G R AT I O N I N T H E C E R E B E L LUM
The cerebellum is a neuroanatomical system of the CNS with
comparatively well-defined cellular composition. The neurons
of the cerebellum are generated in two distinct developmental
phases. In the first wave, the Purkinje cells and deep cerebel-
lar nuclei neurons, appear at the bottom of the future fourth
ventricle, around embryonic day (E) 12/13 in the rodent.
Thereafter, in a second wave the granule cell progenitors are
born. These proliferate and migrate from the rhombic lip into
an archlike structure that expands over the rhombic groove
of the future forming fourth ventricle. There, the small diam-
eter neurons constitute the external granule cell layer (EGL)
(Fig. 32.1). The granule cell neurons, as they are called,
404 • FUNCTIONS OF NEUROGLIAL CELLS
proliferate extensively using mechanisms that involve sonic
hedgehog (Shh)–dependent signaling and cell cycle genes
such as D2-cyclin and PP2A. After a final round of division,
progenitors detach from the EGL and begin to grow two
neurites in opposed orientation that elongate along the form-
ing molecular layer. Thereafter, a process is extended into the
depth of the developing cerebellum that is oriented orthog-
onally with respect to the cerebellar surface (see Fig. 32.1).
The specification of granule neurons involves fate determin-
ing receptors such as Notch2, Jagged2, and their downstream
targets HES and E(spl), as well as the transcription factors
Math1 and NeuroD (Hatten and Roussel 2011).
The leading process extends along a glial process that spans
the whole width of the cerebellar cortex and is produced by
the so-called Bergmann glia cells. This represents another
famous and extensively studied model of neuron migration, as
this phase is prolonged for more than two weeks after birth in
the rodent. Therefore, it was amenable to experimental manip-
ulation both in vivo and in ex vivo tissue explants. When the
leading process has reached the layer of the Purkinje cells,
the nucleus of the granule neuron migrates along the leading
process and finally trans-locates the granule cell body toward
the growing internal granule cell layer, beyond the Purkinje
cell layer. The trailing process finally matures to an axon that
constitutes the growing parallel fiber layer of the cerebellum
(see Fig. 32.1). This layer develops synaptic contacts with the
dendrites of the Purkinje cells.
The analysis of the migration process has revealed many
regulating factors, including the cytoskeletal elements
doublecortin (DCX) and Tuj1 with regard to nuclear
trans-location, the cell interaction molecules TAG1 (Cntn2)
of the Ig-superfamily and the astrotactin genes (Wilson et al.
2010). A particular role has been assigned to myosin II motors
and F-actin dynamics that coordinate movements of the cen-
trosome and the soma during this process (Solecki et al. 2009).
Also Rho GTPases have been implicated in migration control
in the cerebellum (Govek et al. 2011).
1.5 N EU RO G E N E S I S I S L I M IT E D I N T H E
A D U LT C E N T R A L N E RVO US SYS T E M : T H E
RO S T R A L M I G R ATO RY S T R E A M
In the adult CNS, neurogenesis is limited to two canonical
regions, that is the subventricular zone (SVZ) of the lateral
ventricle and the subgranular zone (SGZ) of the hippocampus,
where a subclass of slowly dividing astrocytes are considered as
stem cells (Ming and Song 2005). The radial astrocytes that
presumably descend from radial glia serve as neural stem/pro-
genitor cells (NSPCs) and sustain olfactory bulb neurogenesis
(Ming and Song 2011) (see chapter 40). The granule neurons
of the hippocampus are sequentially born from NSPCs in the
subgranular layer of the hippocampal dentate gyrus of the
adult forebrain (Kempermann et al. 2004) (see chapter 40).
It is widely accepted that these neurogenic territories
harbor specialized environments that sustain NSCs and are
regarded as niches that function as integrative entities for a
large number of physiological stimuli (Scadden 2006; Zhao
et al. 2008).
 The privileged growth and survival environment of the
adult stem cell niche is constructed by astrocytes, endothelia
of neighboring blood vessels, leptomeningeal cells, and cere-
brospinal fluid in the case of the subventricular zone (SVZ).
A variety of morphogens, cytokines, and extracellular matrix
constituents are released into the niche environment (Ihrie
and Alvarez-Buylla; Kazanis and ffrench-Constant 2011), as
well as neurotransmitters (Platel et al. 2010).
The SVZ is starting point of a migration pathway con-
cealed by astrocytes. Sonic hedgehog contributes to the main-
tenance and proliferation of progenitors that migrate toward
the olfactory bulb in the so-called rostral migratory stream.
The neuronal progenitors express PSA-NCAM in the migra-
tory chain, and colonize the olfactory glomeruli, where they
differentiate into interneurons and contribute to the regen-
eration of the local olfactory network (Zhao et al. 2008). On
their way to the olfactory bulb, the neurons are concealed by a
subpopulation of specialized astrocytes that form the walls of
a guiding astroglial tunnel. This astroglial channel is enriched
with extracellular matrix components, in particular tenascin-C
glycoproteins. These may exert inhibitory, repellent properties
when presented as sharp gradients (see section 2.4) (Fig. 32.5),
and thereby contribute to prevent the evasion of neuronal
progenitors ( Jankovski and Sotelo 1996). On the other hand,
in the olfactory bulb the related gene tenascin-R is required to
lure the neurons toward the olfactory bulb (Saghatelyan et al.
2004). Hence, also the rostral migratory stream illustrates the
concept of glial-guided migration. Here, however, the astro-
cytes seem to play more a deterrent rather than a supportive
role (see section 2 for further examples of inhibitory astroglial
functions).
1.6 A S T RO G L I A L C E L L S C O N S T I T U T E
AXO NA L G ROW T H PAT H WAY S IN VI VO
The Müller cell glia spans the width of the retina and extends
the glial endfeet toward its inner surface. After generation
Axon Guidance by Astroglia
Support of outgrowing axons
by a glial bridge (e.g. the glial sling in
the developing corpus callosum)
Figure 32.2 Axon Guidance by Glial Surfaces. Many examples have been provided for axon guidance by astroglial cells in the developing nervous
system. Both supportive properties—as shown for the developing corpus callosum—and boundary properties, for example in the rhombomeres, have
been discussed.
N E U R O N M I G R AT I O N A N D AXO N GU I DA N C E
of the retinal ganglion cells, the axons forming the prospec-
tive optic nerve elongate along this specialized structure. The
growth substrate is provided by a basal lamina that lines the
endfeet and comprises the extracellular matrix (ECM) com-
ponents laminin-1, collagen IV, heparan-sulfate proteogly-
can (HSPG), and nidogen (Halfter et al. 2000). The growth
response of retinal ganglion cells to laminin-1 is regulated by
integrins, consistent with this expression pattern.
Another example of the support of axon growth by astro-
cytes is provided by the development of the corpus callosum.
The emergence of this structure depends on the formation of
a transient bridge of astrocytes that connects the left and the
right hemisphere of the developing telencephalon. This glial
bridge, also called the glial sling, supports the reciprocal growth
of cortical axons (Fig. 32.2). The experimental interruption of
the sling leads to the elimination of the corpus callosum. In
this situation, the connecting cortical axons role up on either
side of the cerebral midline, forming longitudinal fascicles of
misdirected axons, designated the bundle of Probst. Growth
promotion of cortical axons can be restored by the implanta-
tion of nitrocellulose filters that are covered with embryonic
astrocytes or membranes derived therefrom, consistent with a
promoting role in vivo (Katz et al. 1983).
Along these lines, channels outlined by astrocyte surfaces
have been revealed in the developing spinal cord and inter-
preted as growth conduits for advancing corticospinal axons.
The blueprint hypothesis of axon growth pledges that channels
walled by astrocyte surfaces might provide a mechanical growth
and guidance substrate for growth cones. The mechanistic con-
cept of axon guidance has been modified and substituted by an
interpretation that emphasizes molecular signals in the growth
environment, the readout by specific growth cone–based recep-
tors, and the integration of these influences by signal transduc-
tion cascades that eventually modulate growth cone movements.
Thus, a conduit function of astroglia based on the chemorepel-
lent slit-2 has recently been proposed as an additional guidance
principle in the corpus callosum (Lemke 2001).
Growth along and deflection by
a glial boundary (stimulatory abutt onto
Inhibitory astrocytes)
• 405
 1.7 A S T RO C Y T E S C O N S T I T U T E A genes in wiring and synaptogenesis. In some cases, protocad-
G ROW T H-P RO MOT I N G S U B S T R AT E herins are expressed by astrocytes and contribute to synapse
F O R M A N Y N EU RO NA L C E L L T Y P E S formation (Williams et al. 2010). The cytoplasmic domains
of classical cadherins interact with catenins that are required
It is well known that astrocyte monolayers constitute an excel-
for functional activation. β-Catenin is a key component of the
lent growth substrate for axons in vitro. These growth-promoting
Wnt-signaling pathway and involved in signal transduction to
properties are consistent with the conclusions derived from
the cell nucleus, also in radial glia.
the in vivo studies and are age- and lineage dependent. Thus,
The Immunoglobulin (Ig-) superfamily (IgSF) is char-
astrocytes obtained from embryonic or perinatal CNS are
acterized by the canonical Ig-domain, a structure consist-
more efficient than those obtained from postnatal tissues, exert
ing of 90 to 100 amino acids arranged in seven antiparallel
their strongest effect after short culture periods, and tend to
beta-pleated sheets that form a globular domain. In many
lose supportive properties with time in culture. Interestingly,
members, the Ig-domain is combined with fibronectin-type
Ca2+ oscillations in astrocytes condition their neurite growth
three (FNIII-, first discovered in fibronectin) domains, and
promoting properties (Kanemaru et al. 2007).
a transmembrane domain (see Fig. 32.3). In some cases,
The neurite growth promoting properties also depend on
CAMs of the Ig-superfamily are linked to the membrane by
the spatial organization of the cells. Indeed, astrocytes sus-
a glycosyl-phosphatidyl-inositol (GPI) anchor, which con-
tain axon growth when presented as monolayers, but may
fers particular mobility within the membrane. Functionally,
inhibit neurite growth when contained in a tube. In this para-
Ig-superfamily members serve calcium-independent adhe-
digm, astrocytes are grown in a cellulose acetate tube in three
sion mechanisms of the homophilic and heterophilic type
dimensions and confronted to a dorsal root ganglion that is
(Rougon and Hobert 2003). Several neuronal adhesion mol-
apposed to one end of the tube. Axons from early postnatal
ecules with strong axonal expression such as L1/Ng-CAM/
or perinatal DRGs grow readily into tubes that are filled with
neuroglia, contactin/F11/F3 (Cntn1), and TAG-1/Axonin-1
embryonic astrocytes, but less well into tubes with aged or
(Cntn2) have been grouped as Ax-CAMs, referring to their
matured astrocytes. Also, DRGs from later stages are less effi-
prominent role in axon fasciculation. This involves the activa-
cient and do not penetrate tubes with elder astrocytes. This
tion of downstream signaling mechanisms, including modula-
model hence mimics properties of the dorsal root entry zone
tion of intracellular calcium in the growth cone, converging
that represents a stop zone for centripetal axons in the adult
with those elicited via N-cadherin, and the basic FGF-receptor
(Fawcett 1997).
(Walsh and Doherty 1997).
With regard to neuron–glia interactions, selected iso-
1.8 M E M B R A N E -BA S E D A D H E S I O N SYS T E M S forms of the homophilic adhesion molecule N-CAM medi-
M E D I AT E P RO M OT I N G I N T E R AC T I O N S O F ate the binding of neurons to astrocyte surfaces (see Fig. 32.3)
N EU R A L C E L L T Y P E S (Keilhauer et al. 1985). Concerning heterophilic interactions,
the small isoform of the phosphotyrosine phosphatase recep-
The promoting and guiding effects of astrocytes discussed pre-
tor RPTP-β/ζ is expressed by astrocytes, a transmembrane
viously are mediated by specialized components such as cell
component that can interact with the neuronal adhesion
adhesion molecules (CAMs) or constituents of the extracel-
molecule contactin/F11/F3 (Cntn1) and other CAMs of the
lular matrix (ECM). In view of their functional importance,
Ig-superfamily (Faissner et al. 2006). These examples illus-
the following sections will briefly introduce the major gene
trate the functional involvement of Ig-CAMs in supportive
families involved and discuss their roles with special refer-
neuron–astrocyte interactions.
ence to astrocytes. Cell adhesion molecules were originally
The third prominent gene family of membrane based
discovered in the course of aggregation experiments per-
CAMs is represented by the integrins. These are heterodimers
formed by Holtefreter with dissociated sponges in the 1950s.
composed of α and β-subunits, which primarily mediate the
Subsequently, a large body of evidence has been accumulated
interactions of neural cells with extracellular matrix compo-
that suggests that the concept of preferential cell adhesion is
nents (see Fig. 32.3 and section 2). In some cases, interactions
also valid in the nervous system. Operationally, it is possible
between integrins and Ig-superfamily members have also been
to distinguish calcium-dependent and -independent adhesion
reported (Denda and Reichardt 2007).
mechanisms. The calcium-dependent adhesion is mediated
by the cadherin gene family, which includes the classical cad-
herins and the protocadherins. Classical cadherins comprise 2 A S T R O G L I A AT C H O I C E A N D
five cadherin repeat motifs, calcium binding sites, and a trans- DECISION POINTS
membrane domain that result in an overall molecular mass of
about 100 kDa (Fig. 32.3). The classical N-cadherin mediates 2.1 G L I A L B O U N DA R I E S ( S U B D I VI S I O N O F
neuron-astrocyte or neuron-neuron interactions. The classical R H O M B O M E R E S , N EU RO M E R E S AT T H E M O R E
cadherins underlie a homophilic, calcium-dependent adhe- A N T E R I O R , RO S T R A L C E N T R A L N E RVO US
sion mechanism that is strong enough to induce sorting-out of S Y S T E M–T H A L A MUS )
cells, as shown for E-cadherin (Takeichi 2007). A large num-
ber of cadherin superfamily genes are expressed in the CNS. Beside their function as a supportive growth substrate, also
There, the expression patterns are confined to distinct net- the formation of tissue boundaries by astrocytes has been dis-
works, and recent concepts propose a functional role of these cussed (Steindler 1993). One extensively studied example for

406 • FUNCTIONS OF NEUROGLIAL CELLS

 Structures of CAMs and Recognition Molecules

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Immunoglobulin domain EGF-type domain
Cadherin ectodomain Glycosyl-Phosphatidyl-Inositol
Calcium binging site Transmembrane domain
SCR consensus motif Intracellular domain
Heparan sulfates Globular domain
EF-Hand motif Cysteine-rich region
Tyrosine phosphatase

Figure 32.3 Structures of Cell Adhesion Molecules. Many molecules have been described that mediate various kinds of intercellular adhesive interac-
tions in neural tissues. These can be grouped into large gene families according to key structural features, such as the immunoglobulin type C2 domain.

segmented systems is macro-anatomically visible in 3-day-old
chicken embryos: the rhombencephalon of the brainstem,
which is subdivided into rhombomeres. The rhombomere
pattern has been described in higher vertebrates, including
mammals, and can be identified as a series of eight swellings
along the neuraxis, which are separated by inter-rhombomeric
glial boundaries.
Thus, in rhombomere boundary regions the interkinetic
nuclear migration of neuroepithelial cells seems significantly
reduced and intercellular adhesion is probably increased, as
deduced from the finding that PSA-NCAM is enriched within
rhombomeres, whereas the more adhesive non-sialylated vari-
ant is expressed in the boundary. Neuroepithelial cells in this
area form a less motile and more coherent group of cells 4 to
10 cell diameters wide, which might constitute a barrier to the
movement of epithelial cells from one rhombomere to the
next. Interestingly, the cells defining the inter-rhombomeric
boundary display a reduced spread of currents or diffusing
dyes such as Lucifer Yellow or biocytin between themselves or
from one rhombomere to the next. In contrast, the neuroepi-
thelia within the rhombomere are extensively electrically
coupled. The boundary cells exhibit an unusual fan-shaped
array and abut with their endfeet on both the pial and the
ventricular surfaces. Comparably, in zebrafish a serial arrange-
ment of glial cells denominated the glial curtain separates
individual rhombomeres. It has been envisaged that the spe-
cialized boundary cells construct a privileged pathway for
outgrowing axons (Faissner and Steindler 1995; Kiecker and
Lumsden 2005).
For example, studies on axonogenesis have demonstrated
that the first neurons emerge in the even-numbered and
one stage later in odd-numbered rhombomeres. The axons
of motor neurons of the brainstem emerge from the lips of
even-numbered rhombomeres to innervate specific branchial
arches. For example, the first branchial arch is innervated by
the trigeminal nerve, whose axons originate in rhombomeres
2 and 3 and exit in r2 at defined exit points. Lineage tracing
studies have shown that the neuronal progeny of these nuclei
displays lineage restriction in individual segments and are pre-
vented to cross to the neighboring rhombomere after the glial
boundary has formed.
The segmentation of the hindbrain is mirrored by remark-
able expression patterns of transcription factors. For example,
at embryonic day 9.5 (E9.5) the mouse genes of the Hox-B

N E U R O N M I G R AT I O N A N D AXO N GU I DA N C E • 407
cluster homologues of the Drosophila antennapedia complex
are aligned and transcribed on the mouse chromosome 2 in a
5′ to 3′ direction. The limits of expression of these genes in the
mouse spinal cord progress along the rostrocaudal axis and in
register with boundaries of rhombomeric pairs in ascending
order. To illustrate the case, Hox-a2 expression terminates at
the boundary between r1 and r2, Hox-b2 at the r2/3, and so
forth. These expression boundaries coincide in many cases with
those of paralogous genes of the Hox-A and Hox-C complexes.
The resulting Hox-code in the hindbrain can be modified by
treatment with retinoic acid, which results in the reprogram-
ming of rhombomere identity. These experiments underline
the concept that the Hox-code determines regional neural
identities in segments. A more detailed discussion of the role
of transcription factors in developing neural and other tissues
is beyond the scope of this chapter, and is available elsewhere
(Kiecker and Lumsden 2005; Pasini and Wilkinson 2002).
The functions of glial boundaries are illustrated by the find-
ing that motor fibers migrating from odd- to even-numbered
rhombomeres to their exit points preferentially elongate along
boundary structures, as do neurofilament expressing reticular
axons. A directing influence is supported by transplantation
experiments in which axon tracts follow ectopic glial bound-
aries. The mechanism of the hypothesized boundary functions
for axon guidance most probably involves specialized recogni-
tion molecules.
2.2 M I D L I N E G L I A AT D EC I S I O N P O I N T S
F O R G ROW T H C O N E S ( T H E O P T I C C H I A S M ,
RO O F P L AT E , FL O O R P L AT E , A N D M I D L I N E
G L I A I N DROSOPH I L A )
In addition to the boundaries present between rhombomeres
and prosomeres, a second class of boundaries associated with
glial cell types has been described in the midline of developing
nervous systems of vertebrates. Thus, an assembly of glial cells
separates the left and right axonal projection systems at the
optic chiasm. It seems that growing axons interact with this
glial structure and are directed either to the ipsilateral or the
contralateral cortex, as required in the context of binocular
vision. Several genes have been examined as potential candi-
dates in mediating the choice decision. Among these are the
glycoproteins L1-CAM and CD44, and also chondroitin sul-
fate proteoglycan(s) (CSPGs), as visualized by the expression
of chondroitin sulfate epitopes in the glial boundary territory
(Petros et al. 2008; Rasband et al. 2003).
Comparable decisions are also observed in the roof and
the floor plates of the developing spinal cord, prominent glial
midline structures (Fig. 32.4). In the rat, the emerging com-
missural axons of the dorsal horn first migrate to the ventral
half of the cord, toward the floor plate, under the influence
of the chemo attractant netrin-1. The dorsal glial midline cells
of the cord form a boundary that is not traversed by the com-
missural axons (Faissner and Steindler 1995). This structure

Open Book Preparation

Developing spinal cord Open book preparation

Roof plate Midline

Commissural neuron
Keratansulfate TAG-1/axonin-1 (cis-midline)
Proteoglycan (KSPG) L1/Ng-CAM (trans-midline)
DCC (netrin-1 receptor)
NP1/NP2 (Sema3A receptors)
Plexins (Sema receptors) Rostral
Robos (slit-receptors)
Shh

Caudal

Attractive Floor plate

(Netrin-1) Floor plate

- Netrin-1
- Nr-CAM
- Slits
Repulsive - Ephrin B3
(Slits)

Figure 32.4 The Open Book Preparation. Commissural neurons send their axons toward the floor plate in the developing rat spinal cord, following a
netrin gradient. After crossing the midline at the floor plate, responsiveness to netrins is lost and substituted by sensitivity to other types of cues, such
as slit-proteins. In response to these, the axons turn away from the midline and grow along the rostrocaudal axis, fasciculating onto existing longitudi-
nal fiber pathways. These events can be partially monitored and perturbed in vitro in the so-called open book preparation, an ex vivo explant that is
produced by cutting the developing E13 spinal cord along the midline.

408 • FUNCTIONS OF NEUROGLIAL CELLS

expresses inhibitory components, including a keratan sulfate
(KS) proteoglycan, and morphogenic signaling proteins of
the Wnt- and BMP families. In this context, Bmp7 seems to
exert repulsive effects on a subgroup of dorsal interneurons
(Chizhikov and Millen 2005).
On the ventral side of the cord, the glial floor plate cells
separate the left and right half of the neural tube. The growth
and decision events of the ventral side can be monitored in
the so-called open-book in the culture dish, a preparation that
lends itself to antibody perturbation assays (see Fig. 32.4). The
glial floor plate cells release the extracellular (ECM) chemoat-
tractant netrin-1, which attracts the commissural fibers toward
the glial midline, where they cross and subsequently turn to
elongate along the rostrocaudal orientation within the cord.
The netrin-1 signal is decoded by the receptor DCC (deleted
in colorectal cancer), an Ig superfamily member (Colamarino
and Tessier-Lavigne 1995). The commissural fibers destined
to cross the midline express the GPI-linked axonin-1/TAG-1
(also referred to as contactin-2, Cntn2), an IgSF-member that
is downregulated after the crossing has occurred. In parallel,
the IgSF-molecule L1-CAM appears on the longitudinally ori-
ented fibers, an Ig-CAM involved in fasciculation. Antibodies
to the IgSF-member axonin-1/TAG-1 inhibit the crossing step
at the glial midline, which concomitantly expresses Nr-CAM,
a heterophilic Cntn2 ligand. Concerted interactions of these
IgSF-members hence seem required to regulate the cross-
ing step. Subsequently, the axons lose responsiveness to the
attractant netrin-1, yet do not progress to the lateral part of
the cord. This might reflect the action of selected semaphorins
of the sema3-class and the complementary neuropilin and
plexin receptor complexes. The re-crossing of the glial mid-
line is prevented by the slit-proteins of the ECM, repulsive
chemodiffusible signals released by the glial midline (Dickson
and Gilestro 2006). Three IgSF-proteins of the robo-type (for
roundabout) interact with slits and integrate the guidance sig-
nals in a complex choreography involving downstream signal-
ing (Ypsilanti et al. 2010). In addition, the inhibitory ephrins
and their Eph-kinase receptors (see section 2.3) contribute to
prevent illegitimate axonal crossings (Kullander et al. 2001).
Thus, ephrin-B3 is expressed by the glial midline and prevents
re-crossing of these axons when they enter the gray matter
after having migrated to the contralateral side of the spinal
cord (Quinn and Wadsworth 2006). After successful cross-
ing, the axons are guided in the rostrocaudal direction by the
chemodiffusible signal Shh (sonic hedgehog) (Salinas 2003).
Both slit and robo, which occur as several homologues
in the vertebrate, have originally been discovered in droso-
phila, where the glial midline of the ladder-like nervous sys-
tem involves the so-called midline-glia (see chapters 1 and 2).
Complex genetic analysis in drosophila has revealed a battery
of genes that are required for midline-glia generation (Klambt
et al. 2001). Upstream, the fate determination of midline
cells in general is controlled by single-minded. Glial cells
emerge under the regulatory influence of spitz, a drosophila
homologue of TGF-α (transforming growth factor alpha),
and the transcription factor pointed, which intervenes in the
expression of gcm (glial cell missing), a further gene required
for the generation of glial cells in general. Elimination of the
N E U R O N M I G R AT I O N A N D AXO N GU I DA N C E
midline-glia prevents the separation of commissural fibers
and eliminates the longitudinal connections of the ladder-like
nervous system in drosophila. The commissural fibers of the
system begin to extend between the three pairs of midline
glia cells on the one hand and the pair of MP1 neurons on
the other hand. Separation of the commissural fiber systems
involves migration of the midline glia in the correct direction
(Silies and Klambt 2011). Recently, a genetic screen has led to
the identification of a number of additional genes that con-
trol this migratory behavior, one of which, called klötzchen,
seems to implicate the spectrin cytoskeleton of midline-glia.
In all cases, deficits in midline–glia migration lead to errors
in the separation of the connecting commissural fiber systems
(Klambt 2009).
2.3 M E M B R A N E BA S E D G E N E FA M I L I E S
I N VO LVE D I N C H O I C E P O I N T D EC I S I O N S
( E P H-K I NA S E S A N D E P H R I NS , RO B O,
A N D S L IT, S E M A P H O R I NS )
The investigation of neural cell interactions had first resulted
in the identification of the immunoglobulin and cadherin
superfamilies, and of growth- and motility-promoting ECM
constituents. But within the 1980s it became clear that in addi-
tion to growth promotion, growth inhibitory molecules also
contribute to the regulation of cell migration and growth cone
movement (Goodman 1996; Tessier-Lavigne and Goodman
1996). In particular, the phenomenon of growth cone col-
lapse had been described in the context of co-culture models
in which growth cones from sympathetic neurons encounter
retinal neurites in the culture dish, or retinal neurites engage
central myelin preparations. These interactions invariably
resulted in collapse and retraction of the growth cone, which
stayed paralyzed for 30 to 60 minutes in vitro before resum-
ing growth and exploratory behavior (Kapfhammer and
Schwab 1992; Raper and Mason 2010). It was realized that
this inhibitory effect might as well affect guidance and inhibi-
tion of regeneration.
With regard to the guidance aspect, the systematic analy-
sis of the innervation of the tectum in the visual system led
to the identification of novel mechanisms of axon guidance
based on gradients of ephrin molecules and their complemen-
tary Eph-kinase receptors. One distinguishes the GPI-linked
ephrins-A that interact with Eph-A type tyrosine kinases from
the transmembranous ephrins-B that link to the Eph-B type
tyrosine kinases. Both groups contain several members of
ephrin- and Eph-type kinase genes, and a certain degree of pro-
miscuity in the mutual pairing combinations has been recorded
(Egea and Klein 2007; Pasquale 2008). In many cases, a recip-
rocal gradient–like expression of the components in neural
tissues has been documented. Thus, ephrin-A5 is expressed as
gradient in the tectum and so are the complementary recep-
tors EphA3 and EphA5 in the retinal neurons (Simpson et al.
2009). Conversely, the ephrin-B1 and ephrin-B2 ligands of
the retinal EphB1-kinase are expressed by radial glia of the tec-
tum during the period of axon ingrowth (Braisted et al. 1997).
It is believed that these complementary ligand receptor combi-
nations encode positional information in the nervous system.
• 409
Meanwhile, it seems clear that Eph-kinases and ephrins are
expressed in neuronal and glial lineages, respectively, and that
they mediate the regulatory effects of glia on synapse forma-
tion and plasticity, in which ephrin-A3 ligand in astrocytes is
found close to EphA4 receptors that are exposed on dendrites
(Faissner et al. 2010; Murai and Pasquale 2011; Sloniowski and
Ethell 2011). Furthermore, they contribute to the emergence
of the rhombomeric compartments mentioned in the preced-
ing and are involved in the choice decision of the growing cor-
ticospinal projection, where ephrin-B3 is expressed by midline
glia as a ligand and stop signal for EphA4-kinase receptors (see
section 2.2) (Wilkinson 2001).
Following the avenue of growth cone collapse induction in
sympathetic neurons, the distinct gene family of semaphorins
has been identified that comprises homologous members in
mouse and human. Some semaphorins can induce growth
cone collapse mediated by the plexin and neuropilin (NP1
and NP2) receptor complexes in the growth cone membrane
(Raper 2000). The signal transduction pathways downstream
of receptor activation involve small GTP-binding proteins,
such as Rho and Rac1 (Raper and Mason 2010). Sema3a is
expressed in the midline glia and regulates axon guidance at
the optic chiasm (Sakai and Halloran 2006).
Furthermore, several constituents of the myelin sheath
that inhibit axon growth by inducing growth cone collapse
have been identified, in particular Nogo-A. Nogo-A contains
a region that induces growth cone collapse by interacting
with the complementary Nogo receptor NgR (Schwab 2004;
Schwab et al. 1993). NgR is GPI anchored to the growth cone
membrane and part of a receptor complex. Interestingly, two
other myelin components inhibitory to axon growth have
been detected, the IgSF-member MAG (myelin-associated
glycoprotein), and OMGP (oligodendrocyte-myelin glyco-
protein). Both also are able to activate the Nogo–receptor
complex, which suggests a common downstream pathway
of myelin-dependent inhibition (Buchli and Schwab 2005;
Rossignol et al. 2007) (see chapter 56 for detailed treatment).
2.4 E X T R AC E L LU L A R M AT R I X
G LYC O P ROT E I NS ( T E NA S C I NS , N ET R I N S ,
A N D R E C E P TO R S )
The pericellular space is structured by macromolecules of the
extracellular matrix (ECM), which consists of glycoproteins
and proteoglycans (Barros et al. 2011; Dityatev et al. 2010;
Garwood et al. 2001a). It was rapidly realized that astrocytes
in vitro produce many of the ECM glycoproteins originally
described in other tissues, namely, fibronectin, laminin-1,
vitronectin, thrombospondins, and tenascin-C. Laminin-1
is a functional component of astroglial endfeet in limiting
membranes, for example, in the developing retina and forms
an excellent growth substrate for axon extension of many
neuronal cell types (Fig. 32.5). The structurally related genes
netrin-1 and netrin-2 are chemodiffusible chemoattractants,
which guide outgrowing commissural axons toward the floor-
plate glia of the midline in the spinal cord. This mechanism
is highly conserved because it has already been evolved in
the nematode, in which unc-5 guides circumferential axons
410 • FUNCTIONS OF NEUROGLIAL CELLS
(Lai Wing Sun et al. 2011). Fibronectin has been found in
association with blood vessels, a structure in which astrocytes
contribute to the formation of the blood-brain barrier that
isolates the CNS from the blood stream.
Tenascin-C has been studied to some detail because it is
transiently expressed by immature astrocytes in the developing
CNS in vivo. There, it has been found in numerous glial bound-
aries, for example, in the somatosensory barrel field (Faissner
and Steindler 1995). The glycoproteins of the tenascin gene
family are characterized by structural motifs that are shared
between tenascin-C (TN-C), tenascin-R (TN-R), tenascin-X
(TN-X), and tenascin-W (TN-W). In TN-C, a cysteine-rich
amino-terminus is followed by a series of egf-type repeats,
fibronectin type III modules, and finally, homologies to
fibrinogen β and -γ ( Joester and Faissner 2001) (see Fig. 32.5).
This structural organization is maintained in most members
of the gene family, with the exception of a tenascin-like gene in
drosophila, which contains the characteristic egf-type repeats
but is devoid of other structural elements. The egf-type repeats
of tenascins show a particular arrangement of cysteines that
has also been found in the extracellular matrix molecule ree-
lin (see section 1.3). This motif is distinct from the egf-type
repeat modules present in Notch or in the laminins. The
amino-terminus links tenascin monomers to multimers in
several cases, for example, TN-R to trimers and TN-C to hex-
amers under nonreducing conditions. The hexamer appears
under the electron microscope as hexabrachion in rotary shad-
owed preparations (Chiquet-Ehrismann and Tucker 2011).
Two isoforms that are distinguished by one FNIII-motif have
been described in TN-R, a gene that is expressed in oligoden-
drocytes at later stages of development (Czopka et al. 2009,
2010). TN-C possesses an alternative splice site between the
fifth and sixth FNIII-module of the basic structure. As many
as six and nine additional FNIII-repeats can be inserted at
this position in mouse and human TN-C, respectively. These
modules are highly conserved at their respective positions, but
independently spliced. Thus, an amplicon profiling performed
in the mouse CNS has revealed up to 30 alternatively spliced
variants, about 50% of the theoretically possible number of
64 isoforms ( Joester and Faissner 1999). In the human, the
number of isomers could reach up to 512 possible variants,
assuming that the modules are freely exchangeable. In light of
this result, the glycoprotein seems suited to specify pericellu-
lar microenvironments, or to distinguish glial lineages ( Joester
and Faissner 2001).
Furthermore, TN-C is associated with various pathologi-
cal conditions, including cancers and glial tumors (Orend
and Chiquet-Ehrismann 2006). It represents an interesting
possibility that TN-C could serve diagnostic purposes in this
context.
With regard to function, the tight association of expres-
sion with neuroanatomical boundaries, developmental events
and sites of plastic changes have motivated numerous experi-
mental studies in vitro. It has been shown that the glycopro-
tein is antiadhesive for a large variety of cell types and deflects
growth cones and neuronal cell bodies at boundaries in choice
situations in vitro in which TN-C alternates with laminin-1
(Faissner 1997). On the other hand, homogeneous substrates
 A – Neurite growth promotion B – Anti-adhesion

(a) (b) A B Fluorescence

Phase

p-Orn tenascin-C

(c) (d)

Laminin-1 Fibronectin Tnc

EGF domains Fibronectin type III domains Fibrinogen domain

NH2 1 2 3 4 5 A1 A2 A4 B C D 6 7 8 COO–

EGFR αvβ3 RPTP-β Cntn1 α8β1 α2β1

α9β1 Annexin II
Syndecan

Figure 32.5. Structure-Function Model of Tenascin-C Glycoproteins. A. A homogenous tenascin-C substrate promotes neurite outgrowth from E18
hippocampal neurons. B. When presented as stripe (green fluorescence), tenascin-C boundaries deflect both neuronal cell bodies and axons. C. The
scheme depicts the structural organization of mouse tenascin-C and details functional regions that have been worked out on the basis of bioassays
using antibody perturbation and recombinant domains. Various receptors have been mapped to distinct domains. Micrograph by courtesy of
Dr. M. Michele.

of TN-C promote neurite outgrowth of most neuronal cell
types studied so far (see Fig. 32.5). Several receptors have been
described (Faissner 1997), among these the IgSF-member
Cntn1 (Rigato et al. 2002), which mediates TN-C–dependent
stimulation of neurite outgrowth in embryonic hippocam-
pal neurons, and various integrins (Myers et al. 2011) such as
αvβ3, α1β8, and α1β9. In the extracellular matrix, TN-C
interacts with proteoglycans, for example, phosphacan or neu-
rocan. Although the knock-out mice appeared viable and able
to reproduce at first sight, recent studies demonstrated modi-
fied behaviors in response to stress and lesions and deviations
in the generation and number of NSPCs, suggesting roles in
synaptic plasticity and the neural stem cell niche (Faissner
et al. 2010; Karus et al. 2011).
2.5 H E PA R A N S U L FAT E - A N D C H O N D RO I T I N
S U L FAT E P ROT E O G LYC A N S
Various proteoglycans are expressed by embryonic radial
glia, and both adult and reactive astrocytes and constitute
the second class of ECM components expressed in the CNS.
Proteoglycans are characterized as glycoproteins that comprise
at least one additional, covalently linked glycosaminoglycan
chain. One distinguishes heparan sulfate (HSPGs), chon-
droitin sulfate (CSPGs), and keratan sulfate proteoglycans
(KSPGs) of the nervous system. Tissue fractionation studies
performed with rat brain revealed that most HSPGs are tightly
associated with cell membranes, whereas chondroitin sulfate
proteoglycans (CSPGs), which represent the major popula-
tion of PGs in the CNS, are recovered in detergent-free salt
extracts (Bandtlow and Zimmermann 2000). In many cases,
the HSPGs are membrane-bound and are cofactors that syn-
ergize the signaling of growth factors such as FGF2, PDGF,
or Wnt-proteins, which bind to specific motifs in heparan
sulfate chains (Yamaguchi 2001) in the glial stem cell com-
partment. Both members of the syndecan gene family and the
GPI-linked HSPG glypican have been detected in the CNS
(Yamaguchi et al. 2010; Yanagisawa and Yu 2007).
With regard to CSPGs, the members of the lectican family
brevican, neurocan, versican, and aggrecan have been identi-
fied in the CNS (Bandtlow and Zimmermann 2000). These
CSPGs, which display a specific pattern of structural motifs,
associate with distinct lineages. Thus, versican is expressed
by mature oligodendrocytes, whereas neurocan and aggre-
can have been found associated with neurons (Zimmermann
and Dours-Zimmermann 2008). Interestingly, several of the

N E U R O N M I G R AT I O N A N D AXO N GU I DA N C E • 411
core glycoproteins carry the HNK-1-epitope, a carbohydrate
structure also expressed by neural recognition molecules,
or other N-linked carbohydrates, for example, of the Lewis
X-type, which are recognized by specific monoclonal antibod-
ies (Hennen et al. 2011). These reports indicate a substantial
heterogeneity of CSPGs in the CNS. Several MAbs have been
described that react specifically with individual PGs, such as
CAT 301, which identifies a variant of aggrecan expressed in
neurons, and NG2, which recognizes the CSPG named NG2
that is expressed by oligodendrocyte precursors and in wound
regions of neural tissues (Morgenstern et al. 2002; Richardson
et al. 2011). The use of MAbs specific for epitopes on keratan
sulfate (KS) chains has shown that this carbohydrate polymer
is transiently detectable as a boundary in the roof plate of the
developing spinal cord, where it displays inhibitory properties
(see section 2.2) (Chizhikov and Millen 2005). Experiments
performed with versican documented inhibitory effects on
the migration of neural crest cells and of DRG axons in a
laminin-1-rich territory. Finally, the neuronal CSPG neurocan
binds to CAMs of the Ig-superfamily, inhibits homophilic L1-
or N-CAM-mediated cell adhesion, and interferes with both
neuron adhesion to and neurite outgrowth on substrates con-
sisting of combinations of CAMs. In light of findings such
as these, CSPGs were discussed as inhibitors of neurite out-
growth, an aspect that has since then obtained considerable
attention in the situation of CNS lesions (Fawcett 2009; Kwok
et al. 2008; Morgenstern et al. 2002; Properzi and Fawcett
2004). The receptor tyrosine phosphatase RPTP-σ has been
proposed as receptor of inhibitory CSPGs (Shen et al. 2009).
On the other hand, glycosaminoglycans per se have not
proved inhibitory to neurite outgrowth in each situation
tested, and chondroitin sulfate epitopes have been found
upregulated in the regenerating peripheral nerve. In some
cases, chondroitin sulfate epitopes have displayed neurite
outgrowth–stimulating properties. The functional properties
of axon growth hence need to be considered in the context
of overall matrix composition and the lineage and age of the
neurons involved (Purushothaman et al. 2012).
2.6 R P T P -β/ζ, P H O S P H AC A N, A N D R E L AT E D
I S O F O R M S I N N EU RO N– G L I A I N T E R AC T I O NS
Embryonic radial glia cells and adult neurogenic niches
strongly express DSD-1-PG/phosphacan, one of the more
abundant soluble CSPGs in the postnatal mouse brain and
mouse homolog of the CSPG phosphacan from rat tissues
(Faissner et al. 2006). The GAG-composition of DSD-1-PG/
phosphacan is characterized by the chondroitin sulfates CS-A
and CS-C, a keratan sulfate moiety that has been detected
with the MAb 3H1, and the DSD-1 epitope. The latter is rec-
ognized by the MAb 473HD and requires (at least) a chain of
seven disaccharides, sulfation of the carbohydrate backbone, a
significant proportion of CS-D dimers, and dermatan sulfate
motifs in its sequence. The DSD-1 epitope can be enriched
by affinity chromatography and promotes neurite outgrowth
from E18 hippocampal neurons. Thus, it represents an exam-
ple of a chondroitin sulfate with neurite outgrowth–promot-
ing properties. The secreted proteoglycan phosphacan is a
412 • FUNCTIONS OF NEUROGLIAL CELLS
splice variant of the transmembrane receptor protein tyrosine
phosphatase beta (RPTP-β/ζ). It corresponds to the entire
extracellular region of the largest isoform of RPTP-β/ζ,
which is extensively glycosylated with chondroitin sulfate gly-
cosaminoglycan chains. The large variant and a short isoform
derived therefrom possess a transmembrane domain and two
phosphotyrosine phosphatase modules oriented toward the
cytoplasm. The different isoforms of RPTP-βζ display devel-
opmental regulation and lineage-restricted expression.
Neural stem/progenitor cells, glial precursor cells, radial
glia, Golgi cells, and astrocytes of different developmental
stages and from various parts of the CNS all express
RPTP-β/ζ isoforms. For example, the large transmembrane
variant is expressed by NSPCs in the subventricular zones of
the developing and the adult CNS, and by oligodendrocyte
precursors. The short transmembrane form is found in
astrocytes, whereas both lineages seem to release the soluble
variant phosphacan to some extent. Although the mRNA is
mostly in the neuroepithelium of the embryonic brain and
spinal cord, the protein is more widely distributed in these
tissues, presumably following transport along glial processes,
local secretion, and/or redistribution as a consequence of
cell migration. Expression in neuronal subpopulations has
also been observed. The spatiotemporal expression pattern of
the RPTP-β/ζ isoforms during development, maintenance,
and pathology of the CNS has been correlated with a
range of developmental processes, which involve cell–cell
signaling, cellular proliferation, migration, differentiation,
axon outgrowth, synaptogenesis, synaptic activity, and
tissue regeneration (Garwood et al. 2001b). Based on the
preponderant glial expression of phosphacan/RPTP-ζ/β, the
effects on neuronal behavior of extracellular signals presented
by RPTP-ζ/β have been considered, whether as protein
sequences or domains or associated with the CS-GAG chains,
with which they are modified.
In the adult rat brain, it has been shown that phosphacan is
expressed by a selected subpopulation of neurons that express
the calcium-binding protein parvalbumin. It has been sug-
gested that CSPGs associate with hyaluronic acid in pericel-
lular matrices designated perineuronal nets (PNNs). Different
neuronal subsets display different complements of CSPGs
such that perineuronal CSPGs could regulate the extracellu-
lar milieu of neurons in cell type–specific ways. For example,
late in development the mature ECM may be an important
element in limiting synaptic plasticity (Faissner et al. 2010;
Galtrey and Fawcett 2007; Kwok et al. 2011).
With regard to function, phosphacan interacts with IgSF
members such as Cntn1 and Cntn2, Nr-CAM, and Ng-CAM,
and hence might intervene in both homophilic and het-
erophilic CAM interactions. Binding of Cntn1 with the
amino-terminal domain of RPTP-β/ζ expressed in eucaryotic
cells promotes neurite outgrowth. Therefore, it has been pro-
posed that the transmembrane variants expressed in glia could
serve as receptors for CAMs expressed in the neuronal growth
cone in the framework of neuron–glia interactions. Because
both RPTP-β/ζ and the Ig-CAMs are linked to signal trans-
duction pathways, these interactions might involve reciprocal
signaling mechanism pathways (Stoker 2001).
 Within the extracellular matrix, DSD-1-PG/phosphacan
interacts with various ligands, for example, the ECM glyco-
protein tenascin-C. The purified proteoglycan promotes neu-
rite outgrowth by E 18 rat hippocampal and cortical neurons,
an effect that involves the DSD-1 epitope chondroitin sulfate
chains. On the other hand, DSD-1-PG/phosphacan blocks
the neurite outgrowth promoting effect of laminin-1, suggest-
ing that the inhibitory effect of the CSPG is context depen-
dent (Garwood et al. 1999). A similar result has been obtained
with embryonic cortical neurons. Also in this case, blockade
of laminin-1-dependent neurite growth promotion was still
detectable after removal of the chondroitin sulfates and, there-
fore, related to the ECM context and the lineage of the respon-
sive cells. The integration into ECM superstructures might
explain to some extent why the elimination of the phosphacan
gene does not result in serious impairment of development in
mice. However, on the other hand it results in delayed regen-
eration of inflammatory myelin lesions (Harroch et al. 2002).
3 THE ASTROGLIAL SCAR
3.1 S C A R I N G I N R E S P O NS E TO L E S I O N,
S T RU C T U R E O F T H E S C A R , R E AC T I V E
A S T RO C Y T E S , A N D L E P TO M E N I N G E A L C E L L S
For decades the dogma prevailed that the regeneration of
severed central nervous system (CNS) axons is impossible.
The seminal experiment by Albert Aguayo, which showed
that CNS neurons can extend vigorously growing axons into
peripheral nerve tubes, challenged this view and stirred a con-
siderable interest in cellular and molecular aspects of wound
reactions in the CNS. In particular, the growth capacity of
neurons strongly suggested that environmental factors in the
lesion were responsible for the abortion of axon regrowth, and
not an intrinsic lack of capacity of the axon. Meanwhile, it is
well established that the incapacity of the CNS to regenerate
is at least in part caused by inhibitory factors released by glial
cells into the lesion environment (Schwab et al. 1993; Silver
and Miller 2004) (see chapter 56). Following lesion, microg-
lia activation leads to the release of numerous cytokines and
the removal of cellular debris. Furthermore, leptomeningeal
cells invade the wound territory, as a consequence of mechan-
ical penetration or migration processes. This cell lineage con-
tributes to the release of collagen deposits into the lesion site.
Concomitantly, the astrocytes in the lesion territory change
their shape, with strong starlike projections, cellular hyper-
trophy, and upregulation of the glial fibrillary acidic pro-
tein (GFAP). The reactive astrocytes tend to form a palisade
of cellular constituents destined to shield the wound from
the surrounding tissue (see chapters 51 and 53). The reac-
tive gliosis constitutes a central cellular element of the scar
that is stimulated by the lesion stimulus (Asher et al. 2001;
Fitch and Silver 2008). Finally, myelin debris of degenerating
sheaths litters the lesion zone and exposes inhibitory factors.
Thus, oligodendrocytes express the inhibitory proteins
Nogo-A, MAG, and OmGP, which block axon growth by
induction of growth cone collapse, in a manner dependent
N E U R O N M I G R AT I O N A N D AXO N GU I DA N C E
on calcium modulation and G-protein activation (Giger et al.
2010; Schwab 2004) (see chapter 56).
3.2 R E AC T I VE A S T RO C Y T E S U P R EGU L AT E
E X T R AC E L LU L A R M AT R I X C O NS T IT U E N T S
Independently of myelin, the reactive astrocytes by themselves
inhibit neurite outgrowth in vitro and in vivo (Asher et al.
2001). The relevance of the cellular compartment in CNS
lesion territories is emphasized by the observation that sig-
nificant regrowth of axonal connections can be obtained by
implanting olfactory ensheathing and other cell types to foster
axonal growth through lesion structures. Likewise, Schwann
cells promote regeneration and are able to span bridge struc-
tures in the CNS. The molecular components that underlie
the bridge properties are currently unknown, but a functional
effect of ECM is also probable in this regard (Gardiner 2011).
From this point of view, the ECM might exert positive effects
on axon outgrowth in some cases. The functional ambivalence
of the ECM is reflected under various circumstances. Thus, an
upregulation of CSPGs in the regenerating peripheral nerve
has been observed. In contrast, upregulation of CSPGs in CNS
structures clearly correlates with inhibition of axon growth in
some territories. Numerous studies have emphasized, that the
reactive astrocytes upregulate CSPGs, and these components
are sufficient to override the beneficial influence of laminin-1,
thus impeding the axon growth process (Kwok et al. 2008;
Morgenstern et al. 2002). Along these lines, it has been
reported that neurons implanted by a nontraumatic technique
into the corpus callosum are able to regenerate long fibers in
the presence of intact myelin, provided the fibers escape a ring
of reactive astrocytes emerging around the implantation site
that express tenascin-C and CSPGs (Silver and Miller 2004).
In addition, tenascin-C is detected in stab wounds of rodent
and human CNS, the tissue of human hippocampal sclero-
sis, and hippocampal structures of rodents subject to experi-
mental seizure. Furthermore, the glycoprotein is significantly
enhanced in human glial CNS tumors. In view of the numer-
ous effects on axon growth, a functional role in the scar tissue
seems plausible (Faissner 1997). These results have strongly
stimulated further investigations into the molecular identity
of the CSPGs expressed by reactive astrocytes. It could be
shown that inhibitory activities are associated with NG2, a
CSPG that is expressed by a subclass of glial cells, upregulated
in lesions and inhibiting neurite outgrowth in several in vitro
assays. The functional significance of CSPGs in CNS lesions is
highlighted by the observation that axon sprouting and recov-
ery of function are enhanced following treatment with chon-
droitinase ABC in vivo (Fawcett 2009).
3.3 T H E M EC H A N I S M S O F I N H I B IT I O N O F
AXO N G ROW T H M I G HT I N VO LVE T H E
E X T R AC E L LU L A R M AT R I X S U P E R S T RU C T U R E
The mechanism of inhibition enacted by CSPGs is cur-
rently not well understood. One possibility is that the puri-
fied proteoglycans by themselves have inhibitory properties.
In this context, it is significant that indirect evidence for the
• 413
existence of defined structural motifs in glycosaminoglycan
chains of proteoglycans has been obtained (Purushothaman
et al. 2012). Also, degradation of a CSPG by matrix metallo-
proteinase-2 neutralizes its inhibitory properties. On the other
hand, several reports suggest that the core proteins of proteo-
glycans as expressed in target cells are able to deter advanc-
ing growth cones, and a receptor has been discovered (Shen
et al. 2009). Alternatively, it is conceivable that inhibitory,
growth cone collapse–inducing components are associated
with proteoglycan structures. For example, semaphorins com-
prise charged carboxyterminal domains that are well suited
to interact with proteoglycans in lesions. Evidence has been
reported that netrin-1 protein binds to HSPGs, an interaction
that may restrict diffusion in vivo. Furthermore, treatment of
ex vivo CNS tissue slices with high salt solutions, hyaluroni-
dase, or chondroitinase reduced the inhibitory or antiadhesive
properties of otherwise inhospitable sections. These findings
are consistent with the view that the emergence of inhibitory
properties might result from matrix assembly. Along these
lines, recent experiments have documented that interference
with basal lamina formation in vivo favors the regeneration
of the transected fimbria–fornix system. In this context, col-
lagens have attracted increased attention. Indeed, collagen
type IV is the major collagenous component of basal laminae,
which seemed to form the obstacle to successful regeneration
in that case. This raises the question whether also fibrillar col-
lagens are upregulated by astrocytes, and whether collagen-
like gene products of astroglial origin exist.
4 S U M M A RY A N D P E R S P E C T I VE S
Migration of neuronal progenitors and growth cones repre-
sent key processes in the developing nervous system. A sub-
stantial body of research over the past decades has provided
evidence that beyond chemotactic signaling systems also
membrane-mediated haptotaxis plays a pivotal regulatory
role. In this context, the immature and mature astrocytes
serve manifold instructive morphogenetic functions. Radial
glia and astrocytes form transient spatiotemporal guidance
structures that direct neurons or growth cones to the desti-
nation. On the other hand, the astroglial structures reveal
an ambivalent character because they may also act as barriers
that conceal neuronal assemblies and deter advancing growth
cones. A large number of gene products that distribute into
distinct gene families of CAMs, ECM, and recognition mol-
ecules partake in neuron–glia interactions and sculpture the
connectome of the CNS. Yet, although the wide variety of
neurons can be classified progressively thanks to combinato-
rial arrangements of transcription factors and neurotransmit-
ter receptors, much less is known about the corresponding
astrocyte compartments. Much remains to be achieved both
with regard to developmental markers and the identification
of fate-determining signaling pathways and transcriptional
regulators. It is conceivable that finally an extensive tem-
poral and topological astroglial heterogeneity may emerge,
in which astrocyte subpopulations define functionally tailored
microenvironments for their neuronal counterparts.
414 • FUNCTIONS OF NEUROGLIAL CELLS
AC K N OW L E D G M E N T S
The author apologizes to all colleagues whose valuable contri-
butions could not be cited because of editorial limitations.
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 33.
THE ROLE OF GLIA IN THE FORMATION AND FUNCTION
OF THE BLOOD-BRAIN BARRIER
Istvan Krizbai, Imola Wilhelm, Hans-Christian Bauer, and Hannelore Bauer

A B B R E VI AT I O N S 2 T H E N E U R O VA S C U L A R U N I T

ABC ATP-binding cassette A large number of experimental data suggested that brain
AJ adherens junction functions are largely determined by a complex interaction
apoE apolipoprotein E of different cell types including neurons, glial cells, brain
AQP4 aquaporin-4 endothelial cells, and pericytes, which led to the development
BBB blood-brain barrier of the neurovascular unit (NVU) concept. One of the princi-
bFGF basic fibroblast growth factor pal functions of the NVU is the formation of the BBB. Key
CNS central nervous system cellular components of the BBB are cerebral endothelial cells,
CSF cerebrospinal fluid astrocytes, and pericytes (Fig. 33.1).
EC endothelial cell
GDNF glial-derived neurotrophic factor
GLUT-1 glucose transporter-1 2.1 E N D OT H E L I A L C E L L S
GFAP glial fibrillary acidic protein
IL interleukin Although the concept of a blood-brain barrier was conceived
LDL low density lipoprotein nearly a hundred years ago, the role of capillary endothe-
lial cells (ECs) representing this barrier was only appreci-
LIF leukemia inhibitory factor
ated much later. Detailed electron microscopical analysis
NVU neurovascular unit
using horseradish peroxidase as a tracer revealed that the
P-gp P-glycoprotein BBB is located in the cerebral endothelium (Reese and
SLC solute carrier Karnovsky 1967). Permeability studies of cerebral and non-
TGF-β transforming growth factor-β cerebral endothelia affirmed the special role of brain capillary
TJ tight junction endothelial cells, demonstrating that they undergo an addi-
TNF-α tumor necrosis factor-α tional step of differentiation that results in a specific cel-
VEGF vascular endothelial growth factor lular phenotype. The most prominent feature of cerebral
ZO zonula occludens endothelial cells is the occurrence of continuous tight
junctions (TJs), which seal the paracellular passage for
1 INTRODUCTION macromolecules and cells from the blood stream. Other char-
acteristics include the absence of fenestrations, few pinocytotic
Proper neuronal function within the vertebrate brain requires a vesicles, a high number of mitochondria and a variety of
strictly regulated microenvironment and a steady-state level of hor- transport systems in the luminal and abluminal cellular
mones, ions, transmitters and other biologically active substances. membrane. Tight junctions are a key structure to the barrier
In providing this “milieu intérieur” two barrier systems play impor- function of cerebral endothelial cells and have been a focus of
tant roles. The blood-cerebrospinal fluid (CSF) barrier is the site intense research throughout the last decades. This has led
between the blood in the choroid plexus and the CSF in the ven- to the elucidation of an unanticipated complexity of the
tricles. It consists of a monolayer of epithelial cells without interac- molecular architecture of TJs, together with the emer-
tion with cerebral cells. The second barrier system, the blood-brain gence of new functional aspects of TJ-associated proteins
barrier (BBB), represents an active interface between the central (reviewed in Bauer et al. 2011).
nervous capillaries and the extracellular fluid of neurons and glial However, endothelial cells are not the only cell type
cells. The BBB is formed by brain capillary endothelial cells in close involved in BBB function. There is sufficient experimental
association with pericytes and macroglial cells or astrocytes cov- evidence to suggest a relationship among endothelial cells,
ering the majority of the cerebral surface of the endothelium (see pericytes, glial cells, and neurons to create and/or support
Fig. 33.1A and B). This suggests a possible influence of astrocytes the BBB. Neurons are supposed to influence astroglial mor-
on capillary endothelial cells. The present chapter focuses on the phology, differentiation, and proliferation whereas pericytes
role of glial cells in the formation and function of the BBB. The and astrocytes, in turn, directly or indirectly contribute to
blood-CSF barrier will not be dealt with further in this survey. the maintenance of the BBB.

417
 2.2 A S T RO C Y T E S
Originally, astrocytes were considered to create the BBB in
mammals, comparable to lower vertebrates like primitive fish
(Bundgaard and Abbott 2008). Astrocytes nearly completely
ensheath the capillary walls with their foot processes, thereby
covering not only endothelial cells but also the intimately asso-
ciated pericytes. In this way, astrocytes are the main mediators
between endothelial cells and the surrounding neuronal tis-
sue. Astrocytes contact the capillaries by specialized structures
called endfeet. Astrocytic endfeet are characterized by a high
expression level of several specific proteins at their luminal
surface, like glucose transporter-1 (GLUT-1), P-glycoprotein,
aquaporin-4 (AQP4), connexin-43, and Kir 4.1 K+ channel. In
addition to their role in transport, astrocytes are also required
for structural support of the BBB—an issue discussed more in
detail in the following.
2.3 P E R I C Y T E S
Pericytes (PCs) (described in chapter 9)—which originate
from the mesoderm or from the neural crest–derived neu-
roectoderm, depending on their location in the central ner-
vous system (CNS) (Winkler et al. 2011)—are located in close
proximity to cerebral endothelial cells, separated only by a
common basement membrane (see Fig. 33.1). Migrating PCs
guide endothelial cells during angiogenesis in the develop-
ing brain (Virgintino et al. 2007). Interestingly, the number
of PCs associated with cerebral barrier-forming capillaries is
higher than the number of PCs found at nonbarrier capillar-
ies, the pericytic coverage of brain capillaries being in the range
of 22% to 32% (Sims 1991). Pericytes are phagocytic cells but
have also been suggested to be involved in capillary contrac-
tion, regulation of endothelial proliferation, and angiogenesis.
The role of PCs in BBB formation is illustrated by the find-
ing that absence of pericytes leads to endothelial hyperpla-
sia, abnormal vasculogenesis (Hellström et al. 2001), and an
increased BBB permeability (Armulik et al. 2010).
2.4 OT H E R C E L L S
In the adult mammalian brain, few neurons come in contact
with cerebral endothelial cells. However, during early devel-
opment, when astrocytes are still absent, neuroblasts and
undifferentiated neurons are the only partners of endothelial
cells. Thus, it is not unreasonable to suggest that early neurons
may play a critical role in the initial induction of the BBB in
cerebral endothelial cells. It is unclear whether there are sig-
nals from endothelial cells to neurons and vice versa which
could be important for the brain homeostasis or for neuronal
function. Such communication could be accomplished by the
presence of neurotransmitter receptors on endothelial cells, as
observed previously.
Microglia (described in chapter 8 and 47) is another cell
type neighboring cerebral vessels. The role of microglia in
the NVU is still poorly understood. Microglial cells control
immune responses in the brain and their activation has been
shown to potentiate BBB damage during neuroinflammation
(Nishioku et al. 2009). Possible beneficial actions of micro-
glial cells on BBB integrity have also been described, especially
in response to ischemic brain injury (Denes et al. 2007); how-
ever, contradictory results have also been published (Yenari
et al. 2006).
3 S T RU C T U R E A N D F U N C T I O N S O F
T H E B L O O D -B R A I N B A R R I E R
The general function of the BBB is to protect the homeostasis
within the brain from changes arising from the vascular sys-
tem and to serve the nutritional demands of the CNS. Thus,
the BBB has a dual role: it is a barrier for cells, solutes, and cer-
tain xenobiotics, and harbors a multitude of transporters for
the selective transport of various substances, e.g., for nutrients
essential to the brain (blood to brain direction) and poten-
tially harmful metabolic products (brain to blood direction)
(Fig. 33.2).

A B
Astrocyte

Basement
membrane

Endothelial Pericyte
cell

Nerve
ending

Tight junction

Figure 33.1 Structure of a Brain Capillary. Besides endothelial cells—which form the structural basis of the BBB—the main cellular components of
brain capillaries are astrocytes and pericytes. A. Schematic structure of a brain capillary. B. Electron micrograph of a cross-sectioned brain capillary.
The image shows an endothelial cell (EC) separated from a pericyte (PC) by the basement membrane (arrowheads) and ensheathed by astrocyte foot
processes (Ap).

418 • FUNCTIONS OF NEUROGLIAL CELLS

 BBB functions
Transport function Barrier function

Endocytosis: Efflux Enzymatic Transcellular

Carrier
Receptor transporters barrier barrier
mediated
mediated
Adsorbtive
Paracellular
barrier
EC ABC TJs
SLC transporters
transporters enzymes

Pericyte
Astrocyte

Figure 33.2 Scheme of a Brain Capillary Showing the Main Functions of the Blood-Brain Barrier. Barrier functions: paracellular barrier provided by
tight junctions sealing the interendothelial cleft, transcellular barrier provided by the low level of pinocytosis, metabolic barrier represented by dif-
ferent enzymes, and efflux transporters (ABC transporters) excreting xenobiotics. Transport function provided by SLC transporters, which are often
asymmetrically distributed in the membrane, adsorbtive endocytosis and receptor-mediated endocytosis via receptors such as LDL-, transferrin- or
insulin-receptor.

3.1 BA R R I E R F U N C T I O N there are differences as well. The barrier properties of

The barrier function of the BBB is to restrict the transport of
potentially harmful substances and cellular elements from the
blood to the brain. This is achieved by a fourfold defense line,
as follows:
1. The BBB constitutes a strong paracellular barrier that limits
the free movement of solutes and cellular elements between
adjacent cells. The molecular basis of the paracellular
barrier is formed by intercellular junctions that seal the cleft
between neighboring endothelial cells (Fig. 33.3).
The junctional complex is formed by the most apically
located tight junctions (TJs) or zonulae occludentes and
the adherens junctions (AJs) (zonulae adherentes), which
support the formation and maintenance of TJs. Junctional
proteins associate with each other to constitute a
branching network of fibril-like structures (TJ strands) in
the cell membrane, which forms a continuous belt in each
cell. Tight junctions not only seal the intercellular way
of transport but they play a crucial role in maintaining
cellular polarity by preventing lateral diffusion of
membrane proteins between the apical and basolateral
surfaces (“fence function”).
Both TJs and AJs are composed of transmembrane
proteins and cytoplasmic plaque proteins that directly
contact the actin cytoskeleton (see Fig. 33.3) (reviewed in
Bauer et al. 2011). Tight junctions of brain endothelial cells
share structural similarities with epithelial TJs; however,
the TJs are directly linked to the four-span integral
membrane proteins. In this respect, brain endothelial
cells express occludin, members of the claudin family, and
MarvelD3. Moreover, junctional adhesion molecules,
which are single-span transmembrane proteins with two
extracellular Ig-like domains, are believed to mediate
homotypic cell adhesion and transmigration of leukocytes.
The key components of the barrier are claudins, actually
a superfamily of 27 members. Brain endothelial cells
express mainly claudin-5, which forms a selective barrier
for molecules smaller than 800 Da (Nitta et al. 2003);
however, the expression of other claudins (-1, -3, -12, and
possibly others) has also been reported in the cerebral
endothelium (Ohtsuki et al. 2008). Plaque proteins of
the TJs include proteins containing PDZ domains like
zonula occludens proteins ZO-1 and ZO-2, and non-PDZ
proteins like cingulin or junction-associated coiled-coil
protein/paracingulin. In addition, a considerable number
of proteins locating at tight intercellular contacts have been
discovered, the role of which has just partly been unraveled.
A recent study, in which a transcriptional profile of CNS
endothelial cells was generated, raised the possibility of
the enrichment of other TJ molecules at the BBB (e.g.,
tricellulin, PARD3) (Daneman et al. 2010a).
AJs consist of a single-span transmembrane protein
belonging to the cadherin family, mainly vascular
endothelial cadherin, which is linked to the cytoskeleton
through catenins (α, β, γ, and p120). The second major

T H E R O L E O F G L I A I N T H E F O R M AT I O N A N D F U N C T I O N O F T H E B L O O D -B R A I N B A R R I E R • 419
 OCCLUDIN
ITCH
TJ
LIN
GU
CIN

P1
UP
JAM AF6

M
CASK
actin

CLAUDIN
ZO2
ZO1

ZO2

ZO1
AJ
tenin
β-ca
VE-cadherin α-catenin
γ-catenin

Figure 33.3 Schematic Structure of Interendothelial Junctions: Tight Junctions and Adherens Junctions. The transmembrane proteins of tight junctions
are claudins (claudin-5, presumably the main constituent of tight junction strands, but claudin-1,-3 and -12 are also present), occludin (assumed to
function as a regulatory tight junction protein), and JAMs (junctional adhesion molecules, possibly interacting with leukocytes). At the inner mem-
brane surface one can find ZO-proteins (zonula occludens proteins, especially ZO-1 is thought to recruit other tight junction proteins, and ZO-2) and
MUPP-1 (multi-PDZ protein 1, possibly involved in the formation of junctional strands).

protein complex at AJs consists of the transmembrane
nectin proteins linked to afadin/AF6.
ZO proteins are scaffolding proteins that bind directly to
transmembrane proteins of TJs and also interact with AJs and
signaling molecules. Moreover, they act not only as structural
components, but are involved in signaling pathways leading
to alterations of gene expression and cell behavior (migration,
proliferation) (reviewed in Bauer et al. 2011).
2. The transcellular barrier (i.e., inhibition of the transport
of substances through the cytoplasm) is based on the low
level of endocytosis and transcytosis characteristic for
brain endothelial cells. A reduced number of caveolae in
the cerebral endothelium compared with other endothelia
has also been described (Nag 2011), whereas increase
in the level of caveolin-1 and the number of caveolae is
associated with BBB breakdown.
3. The enzymatic barrier is provided by a complex set
of enzymes, including acetylcholinesterase, alkaline
phosphatase, γ-glutamyl transpeptidase, monoamine
oxidases, and other drug metabolizing enzymes capable of
degrading different chemical compounds.
4. In addition to these, the barrier function is supported
by the expression of a large number of efflux transporters
(ATP-binding cassette [ABC] transporters), like ABCB1
(P-gp—P-glycoprotein, MDR—multidrug resistance
protein), ABCC1, ABCC4 (MRPs—multidrug
resistance-associated proteins) and ABCG2 (BCRP—breast
cancer resistance protein) (reviewed in Shen and Zhang
2010). These transporters are mainly expressed on the
apical membrane of endothelial cells and excrete different
xenobiotics from the endothelium into the blood stream.
The most important is P-gp, which contributes to the brain-to-
blood transport of several potentially harmful substances such
as amyloid-β and also many drugs including antiepileptic,
psychotropic, antiviral, and chemotherapeutic drugs.
3.2 T R A NS P O RT F U N C T I O N
Because of the relative impermeability of the BBB water solu-
ble substances cannot pass freely from blood to the brain and
vice versa. Different transport systems localizing at the BBB
(carrier systems, transport ATPases, and receptor-mediated
transport processes) (see Fig. 33.2) are responsible for both the

420 • FUNCTIONS OF NEUROGLIAL CELLS

uptake of substances, mediators, and regulators essential for the
brain and the release of products or factors from the brain. The
large majority of these transporters belong to the solute carrier
(SLC) family of membrane transporters. The tetrameric glu-
cose transporter SLC-A2 (GLUT-1) is specific for endothelial
cells and together with other glucose transporters of the CNS
allows for an enormous uptake of substrate to satisfy the aero-
bic energy demand of the brain (reviewed in Bauer 1999).
In addition, several other SLC transporters are also
expressed in brain endothelial cells including heterodimeric
amino acid transporters formed by the members of the SLC3
and SLC7 families (LAT-1, y+L b0,+ transporters), and mem-
bers of the SLC1 (glutamate transporters), SLC6 (sodium/
neurotransmitter symporters), SLC15 (proton/oligopeptide
transporter), SLC16 (monocarboxylate transporter), SLC21
(organic anion transporter), SLC22 (organic anion/cation
transporter), and SLC44 (choline transporter) families. The
SLC-mediated transport processes can be passive or active,
unidirectional, or bidirectional. Furthermore, the BBB medi-
ates an active ion transport through the Na+/K+-ATPase, the
Na+/K+/Cl– transporter, the Na+/H+-antiporter, and the
H+-ATPase as well.
Further transport possibilities across the BBB include
adsorptive endocytosis; moreover, receptor mediated endo-
cytosis through the insulin receptor, transferrin receptor, or
the low density lipoprotein (LDL) receptor also have a special
role (reviewed in Abbott et al. 2010).
4 O N TO G E N I C D E VE L O PM E N T O F T H E
B L O O D -B R A I N B A R R I E R
Vascularization of the mammalian CNS starts from a pre-
existing perineural plexus (at embryonic day 12 in the rat)
(Daneman et al. 2010b), from where endothelial cells invade
the neurectoderm (Fig. 33.4A and B) and—by fusion and
branching—establish the first vascular system in the develop-
ing neocortex (Risau 1997). This angiogenic phase is regulated
by VEGF-A and angiopoietin-1 and results in a leaky endothe-
lium. In this process activated/angiogenic pericytes play an
important role by guiding endothelial cells and organizing the
growing vessel wall (Virgintino et al. 2007). Gain of barrier
properties (development of complex TJs) and upregulation
of specific transporter systems (including GLUT-1, various
amino acid and efflux transporters) is induced by the brain
environment during a differentiation phase. In this process the
Wnt/β-catenin signaling and G-protein coupled receptor 124
may play an important role (reviewed in Liebner et al. 2011).
In the embryonic brain astrocytes are almost absent and
radial glia are the only cells that have contact with cerebral
endothelial cells. Radial glia not only provide scaffolding for
migration and placement of neurons but exhibit neurogenic
properties themselves. In this light, the earlier findings by
Tontsch and Bauer (1991) showing that a neuronal membrane
fraction from 14-day-old mouse embryos induces BBB proper-
ties in cloned cerebral capillary endothelial cells even to a higher

A C E 12.5

Figure 33.4 Developmental Aspects of the Blood-Brain Barrier: Microscopic Images of Two Stages of Ontogenic BBB-Formation. A,B. At embryonic day
10 the intraneural domain gets vascularized from an already existing perineural plexus. Mesenchymal cells (endothelial cells and pericytes) invade the
neuroectoderm (large arrows). Small arrows indicate cross-sectioned blood vessels containing blood cells. Bars = 10 Pm and 20 Pm, respectively.
C. Medially sectioned 12-day-old mouse embryo, after perfusion with 0.5% Trypan Blue solution. The dye (bound to proteins) was excluded from
most parts of the CNS. A weak blue staining is observed in the frontal cortex and in the area of the developing choroid plexus (arrow), probably
reflecting a gradual establishment of the BBB.

T H E R O L E O F G L I A I N T H E F O R M AT I O N A N D F U N C T I O N O F T H E B L O O D -B R A I N B A R R I E R • 421
extent than do glial cells, may be considered an important step
toward a new understanding of the mechanism(s) underlying
BBB induction (reviewed in Bauer and Bauer 2000).
During postnatal development astroglia becomes the pre-
dominant cell population in the CNS and astrocytic endfeet
get intimately attached to the capillary walls during the first
postnatal week (Daneman et al. 2010b). Several data suggest
that the bidirectional interaction of the endothelium with
developing astrocytes is crucial. In astrocytes upregulation
of Src-suppressed C-kinase substrate was observed inducing
downregulation of vascular endothelial growth factor (VEGF)
and upregulation of angiopoietin-1, resulting in blockage of
angiogenesis and increase in TJ formation in endothelial cells
(Lee et al. 2003). Moreover, astrocytic α(v)β8 integrin has
been shown to activate TGF-β in endothelial cells promoting
vessel differentiation and stabilization (Cambier et al. 2005).
Recently it has been shown that Sonic hedgehog secreted
by astrocytes interacts with Hedgehog receptors of brain
endothelial cells, promoting BBB formation and integrity
(Alvarez et al. 2011). Astrocyte-derived fibronectin was also
shown to promote angiogenesis; however, hitherto this has
been demonstrated only in the retina (Stenzel et al. 2011). On
the other hand, endothelial cells release leukemia inhibitory
factor (LIF), which facilitates differentiation of astrocytes
(Mi et al. 2001).
Taken together, neural progenitors are the primary inducers
of BBB-specific genes in cerebral endothelial cells, while BBB
permeability is controlled by pericytes during embryonic devel-
opment and astrocytes postnatally (Daneman et al. 2010b).
It is generally agreed that the BBB matures gradually dur-
ing development. The exclusion of dyes from large parts of the
embryonic and neonatal CNS is a strong indication that there
EC
Migration
enzymes
Proliferation
Astrocyte
Figure 33.5 Schematic Representation of the Interaction of Endothelial Cells and Astrocytes. There is a bidirectional communication between astrocytes
and brain endothelial cells. Astrocytes promote formation of tight junctions and increase the activity of efflux transporters, SLCs, and enzymes; more-
over, they inhibit proliferation and migration of endothelial cells. Endothelial cells induce differentiation and polarization of astrocytes.
422 • FUNCTIONS OF NEUROGLIAL CELLS
is already a functional BBB at least for proteins and macromol-
ecules at early stages of embryonic development (Fig. 33.4C)
(Bauer et al. 1995; reviewed in Saunders et al. 2000). Smaller
molecules, such as inulin (5 kDa) are less rigorously excluded
by the BBB in developing animals, which may be explained
by structural and functional differences between TJs in the
developing and in the adult brain. Moreover, BBB maturation
during development seems to correlate with the development
of polar heterogeneity of astrocytic endfeet.
5 I N VO LVE M E N T O F G L I A L C E L L S I N
B L O O D -B R A I N B A R R I E R F U N C T I O N
Several lines of evidence suggest that glial cells have a cru-
cial role both in the induction and in the maintenance of
the BBB. In this context, astroglia-dependent induction
of barrier-associated characteristics, such as increase in
BBB-related marker enzyme activities (e.g., γ-glutamyl trans-
peptidase, Na+/K+-ATPase, alkaline phosphatase), elevation
of glucose transporter (GLUT) protein levels, enhancement
of transendothelial electrical resistance, as well as increased
expression of LDL receptors and P-gp in cultured cerebral
endothelial cells has been documented (reviewed in Nag
2011). Furthermore, astrocytes suppress endothelial prolif-
eration and regulate tubulogenesis by reducing the number
of tubes formed and increasing their diameter and length.
Astrocytes induce localization of junctional proteins to cell
borders, contribute to the development of tube polarity, and
induce P-gp activity (Al Ahmad et al. 2011). Barrier properties
of the capillary brain endothelium influenced by astrocytes are
summarized in Figure 33.5.
Efflux transporters
SLC
ABC TJs
transporters
Differentiation
Polarization
 The potential of astroglia to determine the barrier charac-
teristics of the central nervous capillaries has manifold confir-
mation, although the situation is more complex than originally
expected (reviewed in Abbott et al. 2006).
Several lines of evidence indicate that induction of BBB
features in cerebral endothelial cells depends on a close and
long lasting contact between astrocytes and the capillary
endothelium. Moreover, a physical contact between endothe-
lial cells and astrocytes has been found to be essential (Tontsch
and Bauer 1991). The exact nature of this physical contact is
not known but it is unlikely that a heterotypic gap junction
between endothelial cells and astrocytes can be established
due to the presence of the basal lamina. The crucial role of
the neural environment in the induction of the BBB is also
illustrated by the finding that BBB properties are inducible in
nonneural endothelial cells by close apposition to cocultured
astrocytes (Hayashi et al. 1997).
Experimental results from different in vitro studies have
also demonstrated that astrocyte-conditioned medium might
influence cerebral endothelial function (e.g., can increase
γ-glutamyl transpeptidase activity), but the experimental
results were not always consistent. Further, the susceptibil-
ity of cerebral endothelial cells to astroglial induction of
blood-brain barrier enzymes was shown to depend on their
proliferative state (Meyer et al. 1991). Considerable efforts
have been made to characterize a soluble factor responsible
for inducing junctional development; however, its nature is
still unclear. Inhibitors of protein synthesis or trypsin were
shown to prevent astrocyte-related actions on the BBB, which
pointed to a protein or peptide as the inductive factor.
Astrocytes are able to synthesize a large number of biologi-
cally active molecules (see chapter 18), which can contribute
to the induction of BBB phenotype. These include TGF-β
(transforming growth factor-β), GDNF (glial-derived neu-
rotrophic factor), bFGF (basic fibroblast growth factor), and
IL-6 (Haseloff et al. 2005). More recently Sonic hedgehog
secreted by astrocytes has been shown to play a critical role in
the formation and integrity of the BBB (Alvarez et al. 2011).
In addition, not only secreted factors but also the extracellular
matrix produced by astrocytes can induce BBB characteristics
in brain endothelial cells (Hartmann et al. 2007).
Despite intense investigative efforts, the role of astrocytes
in the induction of barrier properties of the cerebral endothe-
lium is far from being clear. A recent proteomic analysis has
found upregulation of 55 distinct genes in cerebral endothelial
cells following astroglial induction (Pottiez et al. 2011). These
quantitative changes mainly affected proteins involved in cell
structure and motility, protein metabolism and protein modi-
fications, indicating a broad target spectrum. The number of
affected genes may be even higher but their identification is
difficult out of technical limitations: usually proteomic analy-
ses are less suitable for the analysis of membrane proteins,
not to mention the possible posttranslational modifications.
Tools of systems biology will help to understand the biological
meaning of the activation of the given set of genes.
A very important issue in determining the effect of astro-
cytes on the cerebral endothelium is their functional status.
In glial fibrillary acidic protein (GFAP) knockout mice,
T H E R O L E O F G L I A I N T H E F O R M AT I O N A N D F U N C T I O N O F T H E B L O O D -B R A I N B A R R I E R
having—as a consequence—astrocytes with impaired func-
tionality, the BBB proved to be also compromised (Liedtke
et al. 1996) and these astrocytes were unable to induce BBB
properties in vitro (Pekny et al. 1998).
Astrocytes and brain endothelial cells mutually influence
each other. In this respect, astrocytes show a strong polariza-
tion in adult brains where the BBB is well differentiated, but
are rather unpolarized in neonates with poorly developed
BBB (Wolburg 1995). Astrocytic endfeet oriented toward
blood vessels consist of well-defined rosette-like structures,
the so-called orthogonal arrays of particles which are rich in
AQP4 (Wolburg et al. 2011). The number of these particles
can be increased by co-cultivation with brain endothelial cells
(Wolburg et al. 2011). When the glial cell membrane loses
contact with the basal lamina, the number of these particles
is significantly reduced. Astrocytes in coculture with endothe-
lial cells exhibit a morphologically differentiated appear-
ance and have more developed signal transduction systems
(Hansson et al. 2008). Moreover, cerebral endothelial cells
were shown to induce the expression of plasminogen activa-
tor inhibitor-1 mRNA in astrocytes (Hultman et al. 2010),
whereas endothelium-derived LIF has been shown to induce
astrocytic differentiation (Mi et al. 2001).
The antioxidative activity of astrocytes and endothelial
cells kept in coculture has been reported to be significantly
higher than in mono-cultures of astrocytes (Schroeter et al.
1999). Moreover, endothelial cells can regulate growth
(Estrada et al. 1990), glutamate synthetase activity (Spoerri
et al. 1997), and laminin production (Wagner and Gardner
2000) of astrocytes.
6 T H E B L O O D -B R A I N B A R R I E R U N D E R
PAT H O P H YS I O L O G I C A L C O N D I T I O N S :
ROLE OF GLIAL CELLS
Lesions of the BBB are associated with different patholo-
gies such as brain tumors, stroke, degenerative diseases
(e.g., Parkinson’s disease, Alzheimer’s disease), inflammatory
processes (e.g., meningitis, multiple sclerosis), or even mood
disorders. Because astrocytes play a crucial role in the main-
tenance of the BBB, an altered endothelial-glial interaction
may significantly contribute to BBB disturbance. Although
the mechanisms are not yet completely unraveled, different
factors produced by astrocytes under pathological conditions
(IL-6, TNF-α, endothelin, or VEGF) may have important
roles in this process, causing deterioration of endothelial and
BBB functions. In other cases astrocytes proved to protect the
integrity of the BBB. Effects of astrocytes on the BBB in dif-
ferent CNS disorders are summarized in Table 33.1.
6.1 B R A I N T UMO R S
Malignant processes of the brain include primary and meta-
static brain tumors. Among the primary brain tumors the large
majority are gliomas (see chapter 59). The status of the BBB
in glial tumors is of great therapeutic relevance and thus has
attracted considerable research interest. In general, the BBB
• 423
Table 33.1 EFFECT OF ASTROCYTES ON BBB FUNCTIONALITY IN CENTRAL NERVOUS SYSTEM DISORDERS

CNS DISORDER EFFECT OF ASTROCYTES MECHANISMS, MEDIATORS REFERENCE

Brain tumors BBB permeability increase VEGF release Machein et al. 1999

Modification of basal lamina Lee et al. 2009

Cytokine release (TNF-α, IL-1β, IL-6)? Seike et al. 2011

Cerebral ischemia/ Protective effects on BBB Induction of radical defense enzymes in ECs Schroeter et al. 1999
oxidative stress
Decrease of hypoxia-induced VEGF Fischer et al. 2000
expression in ECs

Ischemic preconditioning Gesuete et al. 2011

Maintenance of claudin-5 localization in ECs Al Ahmad et al. 2011

Increase in endothelial GLUT-1 expression Regina et al. 2001

BBB permeability increase? VEGF release Kaur et al. 2006

Cytokine release (IL-1β) Zhang et al. 2000

HIV encephalitis Induction of endothelial apoptosis, Dependent on gap junctions Eugenin et al. 2011
alteration of BBB integrity

Multiple sclerosis, BBB permeability increase Release of inflammatory mediators, loss of Wolburg-Buchholz
EAE polarized localization of AQP4 et al. 2009

Downregulation of TJ proteins in ECs VEGF release Argaw et al. 2009

Immune cell migration through the BBB Astrocytic ABC transporters Kooij et al. 2011

Alzheimer’s disease Decrease of barrier properties apoE4 release Nishitsuji et al. 2011
Subarachnoidal Protective effect Osteopontin Suzuki et al. 2011
hemorrhage

in gliomas is impaired: expression of TJ-associated proteins is
decreased, the paracellular pathways are partially opened, and
edema is formed in the surrounding brain because of the failure
to clear excess fluid. For example, the TJ protein claudin-1 is not
found, while claudin-5 and occludin expression is extremely low
in vessels of glioblastoma multiforme (Liebner et al. 2000).
In small early-stage tumors the BBB is still intact; how-
ever, the microvascular network becomes more and more
aberrant as we move to more advanced-stage tumors. In large
astrocytomas the tumor core contains scarce, dilated vessels
expressing GLUT-1 and the border area displays glomeruloid
vessels. The vessels are positive for VEGF (Bulnes et al. 2009).
Currently, it is not clear why the BBB in gliomas becomes
defunct. Apparently, glioma cells lack the signals which are
necessary to maintain the BBB in cerebral endothelial cells.
Moreover, excessive VEGF production may have an impor-
tant role by over-stimulating endothelial cell proliferation
and simultaneously suppressing BBB properties (Bulnes et al.
2009; Machein et al. 1999). In addition, glioma cells are able
to modify the composition of the basal lamina of capillaries
of advanced tumors: a decrease in laminin and agrin, and an
increase in tenascin were described (Lee et al. 2009).
The majority of cerebral tumors, however, are not primary
tumors but metastases. Comparably to gliomas, the BBB is
not compromised in small metastatic tumors; however, in
metastases larger than 0.25 mm in diameter the BBB is leaky.
It has been suggested that survival and proliferation of meta-
static cells largely depends on the organ microenvironment
(reviewed in Fidler 2011). In this respect astrocytes and tumor
cells may influence each other directly or by secreting factors
affecting the vasculature as well.
It has been shown that tumor cells and astrocytes stimulate
each other through the secretion of inflammatory cytokines.
Metastatic lung cancer cells secrete macrophage migration
inhibitory factor IL-8 and plasminogen activator inhibitor-1,
whereas astrocytes activated by these factors produce IL-6,
TNF-α, and IL-1β (Seike et al. 2011). All these factors may
influence the BBB as well; however, no direct evidence exists
so far.
6.2 B L O O D -B R A I N BA R R I E R F U N C T I O N
D U R I N G I S C H E M I A/H Y P OX I A
Hypoxia/reoxygenation and consequent oxidative stress
induce severe BBB dysfunction (reviewed in Lehner et al.
2011). Brain endothelial cells are vulnerable to oxidative stress,
which may result in genotoxic damage, apoptosis and—of spe-
cial importance for the barrier function of the BBB—loss of

424 • FUNCTIONS OF NEUROGLIAL CELLS

TJ-related proteins and increased number of pinocytotic vesi-
cles. Thus ischemia/hypoxia leads to an increased permeability
that allows plasma proteins to extravasate into the brain tissue
and results in brain edema (Plateel et al. 1997).
In general, astrocytes cope more readily with hypoxic
insults than do endothelial cells and neurons (Bresgen et al.
2006), which could be explained by their ability to upregu-
late their glycolytic capacity, allowing anaerobic glycolysis to
provide sufficient ATP for cell survival or glutamate
uptake during hypoxic conditions. Furthermore, astrocytes
show higher antioxidant activity than endothelial cells as
revealed by in vitro studies using cell culture models (Schroeter
et al. 1999).
Astrocytes may play an important role in the regulation of
the BBB in response to oxidative insults. Generally, astrocytes
have been considered to protect the BBB during ischemic and
oxidative stress conditions. There are indications that cerebral
endothelial cells co-cultured with glial cells are less suscep-
tible to hypoxic insult, i.e., they restore membrane structures
more quickly and do not exhibit hypoxia-induced paracellu-
lar permeability changes (Fischer et al. 2000; Schroeter et al.
1999). Astrocytes obviously induce the expression of radical
defense enzymes in endothelial cells, thereby improving the
overall defense against oxidative stress (Schroeter et al. 1999,).
Other results suggest that astrocytes protect the BBB from
hypoxia-induced paracellular permeability changes by decreas-
ing hypoxia-induced VEGF expression in microvascular
endothelial cells (Fischer et al, 2000). The protective effect of
astrocytes is demonstrated by the finding that astrocytes play
an important role in ischemic preconditioning-induced BBB
protection (Gesuete et al. 2011). Furthermore, under hypoxic
conditions astrocytes were shown to maintain the proper local-
ization of claudin-5 in brain endothelial cells (Al Ahmad et al.
2011). During cerebral ischemia, hypoxia is accompanied by
hypoglycemia, which aggravates BBB disruption. Astrocytes
might exert a protective role against hypoglycemia as well,
because experimental data show that treatment of cerebral
endothelial cells with conditioned medium obtained from
glucose-deprived astrocytes increased endothelial GLUT-1
expression and glucose uptake (Regina et al. 2001).
However, acute traumatic injury and/or ischemia/hypoxia
induce “reactive astrocytes.” Interestingly, stem/precursor
cell–related proteins such as the intermediate filament protein
nestin and the RNA-binding protein Musashi-1—normally
not present in mature astrocytes—were found to be expressed
in reactive astrocytes (Oki et al. 2010). Hypoxia was shown
to enhance expression of VEGF and AQP4 in astrocytes,
which may contribute to increased permeability of blood ves-
sels and edema formation (Kaur et al. 2006). Oxidative stress
may cause inflammatory reactions of cerebral endothelial cells
and astrocytes. Locally secreted chemokines such as IL-8 and
the monocyte chemoattractant protein-1 (MCP-1) are impor-
tant signals for leukocyte recruitment to an inflammatory site.
Using in vitro systems it has been further shown that hypoxia/
reoxygenation induced an increase in the release of IL-1β into
the culture medium of astrocytes. Endothelial cells treated
with that medium responded with a pronounced elevation of
chemokine expression (Zhang et al. 2000).
T H E R O L E O F G L I A I N T H E F O R M AT I O N A N D F U N C T I O N O F T H E B L O O D -B R A I N B A R R I E R
6.3 OT H E R C E N T R A L N E RVO US
SYS T E M D I S O R D E R S
Besides cerebral ischemia and brain tumors, the BBB is
involved in the pathogenesis of a large number of neurologi-
cal disorders including inflammatory and neurodegenerative
processes of the CNS, infections, or epilepsia. Astrocytes are
considered to play an important role in the induction of BBB
dysfunction in these pathologies. Activated astrocytes are
able to produce and release different substances (e.g., IL-1β),
which in turn might affect the functional integrity of the BBB
(Didier et al. 2003).
It has been shown that HIV-infected astrocytes can induce
apoptosis in cerebral endothelial cells and BBB integrity was
shown to be compromised by a gap-junction-mediated mech-
anism (Eugenin et al. 2011). A disturbed crosstalk between
brain endothelial cells and astrocytes has been suggested to
play a role in brain edema formation during experimental
autoimmune encephalitis (EAE)—an animal model of mul-
tiple sclerosis. In this process loss of β-dystroglycan and—as
a consequence—loss of polarized AQP4 localization in astro-
cytic endfeet was observed (Wolburg-Buchholz et al. 2009).
Moreover in EAE, astrocyte-derived VEGF-A was shown to
be involved in the downregulation of claudin-5 and occludin
in the cerebral endothelium (Argaw et al. 2009). An unex-
pected role of astrocytic ABC transporters in immune cell
migration across the BBB has been reported recently: block-
ing this transporter activity on reactive astrocytes inhibited
immune cell migration across the BBB, which was explained
by the reduction of astrocytic release of the chemokine (C-C
motif ) ligand 2 (Kooij et al. 2011).
During epileptogenesis the BBB breakdown precedes and
induces astrocyte dysfunction: primary BBB lesion leads to
extravasation of albumin which binds to TGF-β-receptor 2 in
astrocytes and induces rapid transcriptional modifications, astro-
cytic transformation and dysfunction (Friedman et al. 2009).
It has also been shown that apolipoprotein E (apoE)-
containing particles secreted by astrocytes regulate TJ integ-
rity in an apoE isoform-dependent manner. In this respect
apoE4—which is a major risk factor in Alzheimer disease—
impairs the integrity of the BBB, and this may be involved in
the pathogenesis of the disease (Nishitsuji et al. 2011). The
vascular component of Alzheimer’s disease has been character-
ized in details in a mouse model. Decreased GLUT-1 expres-
sion was observed in both endothelial cells and astrocytes,
together with signs of neurovascular uncoupling (astrocyte
endfeet retraction and swelling), and loss of expression of the
astrocyte endfeet β-dystroglycan (Merlini et al. 2011).
Astrocytes are also able to express TJ-related proteins
(Bauer et al. 1999) and expression of occludin, claudin-2, and
claudin-11 was found to be significantly higher in astrocytes of
Alzheimer’s disease and vascular dementia brains (Romanitan
et al. 2010). However, the importance of this phenomenon has
not been clarified so far.
Astrocyte-derived molecules may have neuroprotective
effects as well. For instance, osteopontin synthesized by astro-
cytes was shown to attenuate BBB dysfunction caused by sub-
arachnoidal hemorrhage (Suzuki et al. 2011).
• 425
 7 I N VI T R O M O D E L S U S E D F O R
T H E S T U DY O F T H E B L O O D -B R A I N
B A R R I E R : C O C U LT U R E O F C E R E B R A L
E N D OT H E L I A L C E L L S A N D A S T R O C Y T E S
The enormous scientific and industrial interest in the physiology
and pathology of brain barriers led to the development of several
BBB models. The large number of these suggests that there is no
“perfect” model so far and—depending on the respective pur-
pose—one or the other model can be more advantageous. Efflux
transport of drug candidates can be easily and effectively tested on
epithelial cell-based models; however, real in vitro BBB models
are based on the culture of brain endothelial cells. In vivo models
are the most complex, but low-throughput and labor-intensive,
and therefore are used mainly for validation of data.
Cerebral endothelial cells used in in vitro BBB models are
especially primary cells of rat, mouse, pig, and bovine origin. The
relatively high costs and special skills required for the isolation
of brain endothelial cells led to the development of several cell
lines mainly of murine, rat, and human origin. Although these
cells maintain important BBB characteristics, their barrier prop-
erties are significantly inferior compared with primary cells.
Although cerebral endothelial cells are the principal com-
ponents of the BBB, several other cell types play important
regulatory roles in the induction and maintenance of a prop-
erly functioning BBB. This led to the inclusion of glial cells,
pericytes, and even neurons in different BBB models, mimick-
ing the in vivo structure of the BBB.
B
Figure 33.6 In Vitro Blood-Brain Barrier Model System. A. Schematic representation of the triple coculture system. Cerebral endothelial cells are
cultured on semipermeable filter inserts, pericytes are placed on the lower side of the filters, whereas astrocytes are seeded into the wells of the culture
plate. B. Immunofluorescence staining of markers of brain endothelial cells (ZO-1 tight junction protein), pericytes (α- smooth muscle actin), and
astrocytes (GFAP).
426 • FUNCTIONS OF NEUROGLIAL CELLS
The most widely used in vitro models are cocultures of
cerebral endothelial cells and glial cells. For this purpose,
either primary cells or stable cell lines—such as the C6 astro-
cytoma cell line—are used. Syngeneic mouse or rat models are
the most widespread, but allogenic models, using cells of dif-
ferent origin (e.g., bovine endothelial cells and rat astrocytes)
can also be applied. Endothelial cells are cultured on semiper-
meable filter inserts, whereas glial cells can either be cultured
on the bottom of the filter (in direct contact mode) or on the
bottom of the wells (noncontact mode) (Fig. 33.6). Addition
of astrocyte-conditioned medium instead of astroglial cells is
frequently chosen.
It is generally accepted that the presence of astrocytes
significantly improves barrier characteristics of the cerebral
endothelial monolayer, resulting in high transendothelial
electrical resistance and low permeability values (reviewed in
Wilhelm et al. 2011).
Coculture of brain endothelial cells with pericytes was also
shown to enhance barrier properties (Nakagawa et al. 2009);
therefore, triple coculture models consisting of endothelial
cells, astrocytes and pericytes are considered the most suitable
and efficient in vitro models characterized up to the present
(see Fig. 33.6).
There is increasing evidence that shear stress is able to affect
endothelial function. This led to the development of dynamic
in vitro models. For this purpose in general hollow fibers are
used, which mimic capillaries and allow coculture of other cell
types (astrocytes) as well.
Endothelial cells
green: ZO-1 staining
blue: nuclei
Pericytes
α-SMA staining
Astrocytes
red: GFAP staining
blue: nuclei
 8 S U M M A RY A N D P E R S P E C T I VE S
In this chapter we describe the basic characteristics of the
structure and function of the BBB. Although the barrier
itself is located at the level of brain capillary endothelial cells,
other cellular elements like astrocytes or pericytes also play an
important role in the development and maintenance of the
BBB. Here special attention is given to the influence of astro-
cytes and astrocytic factors on BBB function under physiolog-
ical and pathological conditions as well. Furthermore, some
developmental aspects of the BBB and the in vitro models are
also discussed.
In the last few years important scientific developments and
discoveries have been made with respect to the mutual inter-
action of brain endothelial cells and astrocytes in physiologi-
cal and pathological conditions. However, the picture is still
incomplete. Despite many known details, the mechanisms of
how glial cells may intervene with BBB properties of endothe-
lial cells are far from being clear. Recent proteomic analyses
have opened new directions, and tools of systems biology are
expected to help in understanding the role of different genes
and molecules that seem to be involved in the induction of
endothelial barrier characteristics by astrocytes.
Despite a significant progress made in the elucidation of
BBB physiology and function during the last decades, two
major problems—concerning its clinical relevance—have
remained unresolved. First, it is still unknown whether BBB
dysfunction—which is observed under neuropathological
conditions—is the cause or the consequence of the disease.
Second, because of its relative impermeability, the BBB repre-
sents a major impediment in the therapy of a large number of
disorders affecting the brain. At present, drug delivery strate-
gies are far from satisfying all therapeutic needs. Elucidation
of the molecular mechanisms underlying BBB function is an
indispensable need to help to design successful therapies and
prophylactic strategies.
AC K N OW L E D G M E N T S
The work of I. K. and I. W. was supported by the Hungarian
Scientific Research Fund (OTKA K-100807 and PD-100958,
respectively). H. C. B. and H. B. are supported by the European
Union (HEALTH-F2–2009–241778, Neurobid).
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• 429
 34.
CONTROL OF THE EXTRACELLULAR IONIC
ENVIRONMENT AND VOLUME
Eva Syková

A B B R E VI AT I O N S brain tissue volume. Later, it was shown by a number of differ-

ent techniques (rapid freezing, which preserves the ECS for
ADC apparent diffusion coefficient electron microscopy, the use of radiotracers and, in particular,
ADCW apparent diffusion coefficient of water studying the diffusion of ions using ion-selective microelec-
APP amyloid precursor protein trodes) that in the adult brain, the average ECS volume under
CNS central nervous system physiological conditions is about 18% to 24% of the total brain
CSPG chondroitin sulfate proteoglycan volume (Nicholson and Sykova 1998). Nevertheless, the ECS
Cx30 connexin 30 volume and composition change dynamically every second
Cx43 connexin 43 throughout one’s entire life during neuronal activity, develop-
DW-MRI diffusion-weighted magnetic resonance ment and aging as well as in pathological states. The size and
imaging geometry of the ECS affect the concentration, movement, and
ECM extracellular matrix clearance of substances that are released into it. The ECS is
ECS extracellular space also an important communication channel. Substances that
GABA gamma-aminobutyric acid are released by neurons and glia diffuse through the ECS and
ISM ion-selective microelectrode thus reach receptors located on nearby cells or synapses. This
[K+]e extracellular potassium concentration means of communication is called extrasynaptic or volume
mM millimolar transmission because the substances are diffusing through the
pHe extracellular pH volume of the ECS.
pHi intracellular pH
RVD regulatory volume decrease
TEA tetraethylammonium 2 E C S I O N I C A N D VO LU M E
TMA tetramethylammonium H O M E O S TA S I S
VT volume transmission
WHO World Health Organization Increases in the extracellular concentration of K+ ([K+]e),
decreases in extracellular Ca2+ and alkaline-acid shifts in extra-
cellular pH (pHe) accompany neuronal activity in different
1 T H E E X T R AC E L LU L A R S PAC E brain regions (Sykova, 1992). Glial cells play an important role
in the maintenance of ionic homeostasis.
The extracellular space (ECS) is the microenvironment of
neurons and glia, and its chemical and biophysical properties
2.1 T H E F U N C T I O N O F G L I A I N
are crucial for the proper functioning of neurons and also for
E X T R AC E L LU L A R P OTA S S IUM H O M EO S TA S I S
signal transmission. It is a porous structure with uneven pore
sizes (Sykova and Nicholson 2008). It includes ions, trans- Measurements with K+-selective microelectrodes revealed
mitters, metabolites, peptides, neurohormones, and other the local changes in extracellular K+ activity during sponta-
neuroactive molecules as well as various macromolecules of neous discharges (Sykova et al. 1974) as well as after stimula-
the extracellular matrix. Neurons, astrocytes, and oligoden- tion (Kriz et al. 1975; Svoboda and Sykova 1991). However,
drocytes release ions, neurotransmitters, and many other neu- even during intensive stimulation, [K+]e does not exceed a
roactive substances into the ECS, which then diffuse to the certain steady state, the so-called “ceiling” level, which is
nearby neuronal elements. The actual structure and volume of about 8 to 10 mM in the adult mammalian cortex and spinal
the ECS are therefore of crucial importance. cord (Czeh et al. 1981; Heinemann and Lux 1977; Kriz et al.
The first estimates of ECS volume were made in the sec- 1974; Sykova and Svoboda 1990). After the activity ends, the
ond half of the last century from data obtained by electron original [K+]e is quickly reestablished through redistribution
microscopy. However, the true ECS volume was altered by by several mechanisms localized particularly in neighboring
conventional preparation procedures, leading to the erroneous astrocytes (Fig. 34.1). Potassium ions are cleared from the ECS
conclusion that the ECS represents less than 5% of the total by glial cells through: (1) KCl uptake through Cl- channels

430
 Ref.
– MRF
+ K+

ISM [K+]e
ion exchanger

action potentials

striatum spinal cord

5
+
[K ]
+ mM
+ K
K 3

7.3
pH
pH pH
7.4

light 60 s 100 Hz

Figure 34.1 Schema of a double-barreled potassium-selective microelectrode for measuring extracellular neuronal activity together with K+ changes
in the extracellular space. MRF: Spontaneous neuronal action potential bursts are accompanied by an increase in [K+]e in the rat mesencephalic
reticular formation. Striatum: Increases in [K+]e and pHe in the chick striatum as a result of light stimulation of the contralateral eye. Spinal cord:
Stimulation-evoked changes in [K+]e and pHe in the adult rat spinal cord. The change in pH was biphasic: the fast “initial” alkaline shift was followed
by a dominant acid shift. Note the poststimulation undershoots in both [K+]e and pHe. Adapted from Sykova and Chvatal 2000, with permission from
Elsevier.

activated by membrane depolarization (Walz and Hertz 1983; 2.2 T H E RO L E O F G L I A I N E X T R AC E L LU L A R

Walz and Hinks 1986); (2) the opening of Ca2+-activated K+
channels (MacVicar 1984); (3) Na+/K+-ATPase transport,
present on the membranes of both neurons and glial cells;
and (4) K+ spatial buffering (Orkand et al. 1966) (Fig. 34.2).
Potassium spatial buffering is based on the high permeability
of the glial membrane for K+ and astrocytic coupling into
the syncytium, wherein cells are interconnected by gap junc-
tions, allowing for the movement of K+, Ca2+, and other
ions (Coles and Orkand 1983; Kettenmann et al. 1983). The
part of the syncytium that is depolarized by a local increase
in [K+]e acts as a cathode, whereas the distant regions with a
normal membrane potential represent the anode. A current is
carried by K+ inside the glial syncytium and by Na+ and Cl-
outside the syncytium in the ECS. In the regions with a nor-
mal extracellular K+ concentration and membrane potential,
K+ returns back to the ECS. This redistribution of K+ does
not require energy and depends only on the existence of a K+
gradient. Gap junction proteins, connexin 30 (Cx30) and
connexin 43 (Cx43), mediate the extensive network of astro-
cytes (see chapter 24). Gap junctional networking facilitates
extracellular potassium and glutamate removal during synap-
tic activity through the modulation of the astroglial clearance
rate and ECS volume (Pannasch et al. 2011). It has been shown
that the inactivation of the Cx30 and Cx43 genes in mice
increases hippocampal synaptic transmission and impairs
long-term synaptic plasticity.
P H H O M EO S TA S I S
Extracellular space pH changes—alkaline as well as acid
shifts—result from both neuronal and glial cell activity. It was
demonstrated that activity-related alkaline shifts are of neu-
ronal origin, whereas acid shifts are of glial origin ( Jendelova
and Sykova 1991; Sykova 1992). During early development
when glia precursor cells dominate, glial homeostatic function
is incomplete, and activity-related pHe and [K+]e changes are
substantially different from those in the mature central ner-
vous system (CNS) (Chvatal et al. 1995). Alkaline shifts dom-
inate in newborn rats with incomplete gliogenesis. During
development they become smaller and are finally overtaken
by the acid shifts characteristic of mature glial homeostatic
function ( Jendelova and Sykova 1991). Stimulation-evoked
alkaline shifts are of neuronal origin because they are blocked
by the synaptic transmission blockers Mn2+ and Mg2+, whereas
acid shifts are unaffected.
Changes of pHe and pHi (intracellular pH) are caused
by the movement of H+, OH- and their equivalents NH4+
and HCO3- through the membranes of neurons and glia.
Some of the membrane transport mechanisms regulat-
ing pHe and pHi, such as Na+/H+ exchange (Astion et al.
1989; Chesler 1987; Deitmer and Schlue 1987) or Na+/H+/
Cl–/HCO3– cotransport (Thomas 1977), are common to
both neurons and glial cells, whereas others are specific for
either neurons, for example voltage-dependent H+ channels

C O N T R O L O F T H E E X T R AC E L LU L A R I O N I C E N VI R O N M E N T A N D VO LU M E • 431
 K+ homeostasis pH homeostasis
H2O
Na+
Na+ K+ Na+ Na+
K+ H+ H+
ATP
K+
ATP
Na-K pump
Na+ H+
Na cycle Cl– CO
Na+ H+ Na+ HCO3– 2
+
glia 1 Cl– HCO3– H2O
Na+
K+ spatial HCO3–
buffering Cl–
neuron mechanism HCO3– HCO3–
H2CO3
K+ K+
Cl–
neuron glia
Ca2+ Ca2+
H+
K+ H+
Lac–
Cl– KCI uptake
H+
Cl– +
HCO3–
NT Cl– HCO3–
glia 2 GABA GABA
H2O

extracellular space extracellular space

Figure 34.2 Schema of ECS K+ and pH Homeostasis. Note the differences in the membrane transport processes controlling ionic homeostasis in neu-
rons and glia. Potassium ions are cleared from the ECS by glial cells owing to KCl uptake through Cl– channels activated by membrane depolarization,
the opening of Ca2+-activated K+ channels, Na+/K+-ATPase transport, and K+ spatial buffering. Glial membrane transport mechanisms regulating pHe
and pHi are Na+/H+ exchange, Na+/H+/Cl–/HCO3– cotransport, voltage-dependent Na+- HCO3– cotransport, and H+-lactate extrusion.

(Meech and Thomas 1987; Sykova and Svoboda 1990), or
for glia, such as voltage-dependent Na+- HCO3– cotransport
(Astion et al. 1991) or H+-lactate extrusion (Siesjo et al. 1985)
(see Fig. 34.2). The membrane transporters resulting in extra-
cellular alkaline shifts (acid loaders) predominate in neurons,
whereas those resulting in extracellular acid shifts (acid extrud-
ers) prevail in glial membranes ( Jendelova and Sykova 1991).
Active neurons release K+, which accumulates in the ECS
and is taken up by glia. An alkaline shift is evoked in depolar-
ized glial cells, for example by the activation of Na+- HCO3–
cotransport. This alkaline shift in glial pHi causes an acid shift
in pHe. Extracellular acidosis suppresses neuronal activity.
Glial swelling resulting in an ECS volume decrease leads to a
greater accumulation of ions and to a further decrease of neu-
ronal excitability (see Fig. 34.4). This phenomenon has been
termed a nonspecific feedback mechanism suppressing neu-
ronal activity (Sykova, 1997).
2.3 T H E RO L E O F A S T RO C Y T E S I N
E X T R AC E L LU L A R VO LU M E H O M E O S TA S I S
All ions cross the cell membrane in a hydrated form. This
transmembrane water movement leads to cellular (particu-
larly astrocytic) swelling and to compensatory ECS volume
shrinkage. An increased cell volume activates transport mech-
anisms that decrease the concentration of osmotically active
substances in cells, which results in a decrease in the volume
of the swollen cells. This process is called regulatory volume
decrease (RVD) (Mongin and Orlov 2001; Pasantes-Morales
et al. 2000) and is dependent on stretch-activated channels.
The mechanisms of cell volume regulation are modulated
by the intracellular concentration of Ca2+ (McCarthy and
O’Neil 1992) and intracellular pH (Kempski et al. 1990).
In addition, the release of glutamate, taurine, aspartate, and
other amino acids from cells into the ECS is also involved in
RVD (Kimelberg et al. 1990; Pasantes-Morales and Schousboe
1988). Recent data indicate an important role for aquaporin
4 in sensing osmolarity changes and in regulatory volume
decrease. Aquaporin 4 is mainly expressed by astrocytes and
may affect ECS homeostasis during pathological states such as
the outcome of brain edema (Benfenati et al. 2011).
Astrocytic swelling is a major clinical problem after trau-
matic brain injury. Moreover, various studies have demon-
strated that astrocytic swelling is an early event in numerous
pathological states, accompanied by an elevation of [K+]e
and a decrease of extracellular osmolality (Kimelberg 1991;
Kimelberg et al. 1990). In isolated spinal cords of 4 to 21-day-
old rats, the application of an isotonic solution containing
50 mM K+ or hypotonic solution (235 mmol/kg) evoked an
initial decrease in ECS volume of about 50% (Sykova et al.
1999). Because the total water content remained stable, the
changes were attributed to cell swelling. The observed changes
in ECS volume were blocked in Cl– free solution and slowed
down by furosemide and bumetanide, blockers of KCl uptake,
suggesting the involvement of glial swelling. In animals older
than 10 days, during the continuous application of 50 mM K+

432 • FUNCTIONS OF NEUROGLIAL CELLS

or hypotonic solution, the ECS volume started to return to
control values because of the shrinkage of previously swollen
cells caused by RVD (Cserr et al. 1991; Gullans and Verbalis
1993). The mechanisms of RVD are almost exclusively related
to the homeostatic regulation of volume by glial function,
because RVD is not observed in immature animals with
incomplete gliogenesis and is also blocked by the gliotoxin
fluoroacetate (Sykova et al. 1999).
3 E X T R AC E L LU L A R ( VO LU M E )
TR ANSMISSION
Neuroactive substances such as ions, neurotransmitters (see
chapter 17), neurohormones, neurotrophic factors and regu-
latory cytokines (see chapter 22) and metabolites diffuse
through the ECS and affect nerve and glial cells distant from
their release site (Sykova, 1997). Diffusion in the ECS is the
underlying mechanism of extrasynaptic volume transmis-
sion (VT). It has been shown that VT is far more common
than classical synaptic transmission. Synaptic transmission is
a precise communication through fixed synapses, for exam-
ple cell to cell or one to one communication, whereas VT
communication involves a population of cells at the same
time (long-distance communication), that is, one to many
(Fig. 34.3). This type of communication is the only way in
which nerve cells can communicate with glial cells, because
glia have no synapses. The existence of functional interac-
tions between nerve cells without any synaptic contacts, “mis-
matches” between release sites and the location of receptors,
and the widespread existence of high-affinity nonsynaptic
receptors led to the conclusion that VT is a distinct and impor-
tant alternative mode of signal transmission (Agnati et al.
1995; Fuxe and Agnati 1991; Kiss and Vizi 2001; Nicholson
and Sykova 1998; Sykova 1997, 2001, 2003, 2004, Sykova and
Nicholson 2008; Vizi 1980, 1984, 2000; Zoli et al. 1999).

EXTRASYNAPTIC “VOLUME” TRANSMISSION (VT)

A LONG-DISTANCE B SHORT-DISTANCE

neu presynaptic
g ron

volume fraction
tortuosity
uptake g
Glu K+
g
glia
g Ca2+
glia glia
GLT1/GLAST
glia
ATP

extracellular
space
postsynaptic cell
postsynaptic

C D
SPILLOVER SYNAPTIC PLASTICITY
Presynaptic
terminal New
synapse

Glutamate

Dendritic
spine Glutamate
Astrocyte receptor

Astrocyte
Extrasynaptic
receptors

Figure 34.3 Concept of Long- and Short-Distance Communication by Diffusion (i.e., Extrasynaptic Volume Transmission). A. The CNS architecture is
composed of neurons, axons, glial cells (glia), cellular processes (G), molecules of the extracellular matrix, and intercellular channels between cells. This
architecture slows the long-distance movement (diffusion) of substances in the brain, which is critically dependent on the ECS diffusion parameters
volume fraction (α), tortuosity (λ) and nonspecific uptake (k'). B. This synapse is tightly ensheathed by glial processes and the extracellular matrix,
forming perisynaptic nets. C. An open synapse is typical of volume transmission. It allows the escape of transmitters (e.g., glutamate, GABA) from
the synaptic cleft (spillover), diffusion through the ECS and binding to receptors on nearby synapses. This phenomenon is known as “cross-talk”
between synapses. D. The spillover may also lead to plastic changes, inducing the formation of new synapses or eliciting the rearrangement of astro-
cytic processes around the synapse. Adapted from Sykova, 2004; and Sykova and Nicholson, 2008, with permission from Elsevier and The American
Physiological Society.

C O N T R O L O F T H E E X T R AC E L LU L A R I O N I C E N VI R O N M E N T A N D VO LU M E • 433
 Diffusion is characterized by the random Brownian motion
of molecules. In comparison with diffusion in a free medium,
described by Fick´s laws, diffusion in the ECS is restricted
by: (1) the size of the extracellular pores; (2) diffusion barri-
ers such as membrane infoldings, fine neuronal and glial pro-
cesses, macromolecules of the extracellular matrix (ECM), and
charged molecules; and (3) nonspecific cellular uptake. The
diffusion parameters in the CNS were described by Nicholson
and Phillips (1981), who modified the original diffusion equa-
tions by introducing three diffusion parameters: (1) extra-
cellular volume fraction (α), sometimes also called porosity;
(2) tortuosity (λ), representing the hindrances to the move-
ment of molecules in the ECS that are absent in a free
medium; and (3) nonspecific concentration-dependent or
independent uptake, (k´) (see Fig. 34.3). The ECS volume
fraction α is a dimensionless quantity defined as the ratio
between the volume of the ECS and the total volume of the
tissue:
α VECS/V Total
It is now well established that the ECS of adult brain tissue
represents about 20% to 25% of the total tissue volume, which
means that α = 0.20–0.25. It is also evident that the ECS is
not uniform; nevertheless, a study using quantum dot nano-
crystals estimated the average width of the ECS in the rat cor-
tex to be between 38nm and 64nm (Thorne and Nicholson
2006).
The free diffusion coefficient in the brain is reduced by
the presence of diffusion barriers by the tortuosity factor λ
(Nicholson and Sykova, 1998), which is defined as:
λ  D/ADC
where D is the diffusion coefficient in free medium and ADC
is the apparent diffusion coefficient of a substance in the ner-
vous tissue. The tortuosity value, which reflects the number
and extent of diffusion barriers, is about 1.5 in healthy tissue,
which means that diffusion in the brain is about 2.5× slower
than in a free medium.
The advent of ion-selective microelectrodes (ISMs) made
it possible to introduce a technique for diffusion measure-
ments in situ. Both real-time iontophoretic and also real-time
pressure ejection methods use micropipettes to deliver mol-
ecules to which cell membranes are relatively impermeable
(tetramethyl- or tetraethylammonium—TMA+ or TEA+)
into the ECS. The concentration changes of these mol-
ecules are sensed by ISMs at a known distance (Sykova and
Nicholson 2008), and the recorded diffusion curves serve for
mathematical analysis (see Figs. 34.4, 34.5, 34.6, and 34.7).
The values of α, ADCTMA, λ and k´ are extracted by a nonlin-
ear curve-fitting simplex algorithm operating on the diffusion
curve (Nicholson and Phillips 1981). This is the most suitable
method for measuring the absolute values of the ECS diffu-
sion parameters and their dynamic changes in nervous tissue
in vitro as well as in vivo.
The other methods used to study ECS volume fraction
and tortuosity are less comprehensive because they can either
measure only one of the parameters, determine only relative
434 • FUNCTIONS OF NEUROGLIAL CELLS
changes in the ECS volume fraction, or detect changes that
are only partially related to ECS volume changes (Nicholson
and Sykova 1998). An important noninvasive method that
can also be used in humans is measuring the ADC of water
(ADCW ) by diffusion-weighted magnetic resonance imaging
(DW-MRI), which reveals the inhomogeneous and aniso-
tropic diffusion of water, similar to the diffusion of TMA+
(Mamata et al. 2002; Pierpaoli et al. 1996; Vorisek et al.
2002). It has been found that under some experimental con-
ditions, a decrease in ADCW (i.e., lower diffusibility) can be
related either to a decrease in α, typically during fast acute
changes such as ischemia (Van der Toorn et al. 1996), or to
an increase in the number of diffusion barriers, represented
by an increase in λ without substantial changes in α such as
occur during chronic states after injury (Vorisek et al. 2002).
3.1 D I FF US I O N I N T H E EC S I S
I N H O M O G E N EO US A N D A N I S OT RO P I C
The ECS has a complicated and uneven geometry, which
depends on the type and number of cells in the local tissue,
the density and orientation of cellular processes, and the
actual macromolecular content of the tissue, particularly
that of the extracellular matrix. The ECS therefore cannot be
homogeneous; its properties vary around different cells and
also in different brain regions (Sykova and Nicholson 2008).
Inhomogeneity as a result of different tissue structures affects
both αandλfor example in the hippocampus inhomogene-
ity exists between the CA1 and CA3 layers and the dentate
gyrus (Vorisek and Sykova 1997a).
Whereas the volume fraction is a scalar quantity and
therefore has only one value in a given tissue, tortuosity is
a vector, so its values differ in anisotropic regions; that is,
they differ along different axes in the tissue as a consequence
of differences in the local structure (Fig. 34.5). Diffusion
anisotropy has been found in the white matter such as the
corpus callosum, as well as in the gray matter of the cerebel-
lum, striatum, hippocampus, and supraoptic nucleus (for
review, see Sykova and Nicholson 2008). Substances diffuse
more freely along axon bundles than across them, for example
in the myelinated corpus callosum (Vorisek and Sykova 1997a)
or along astrocytic processes that are organized in parallel,
such as those in the hippocampal dentate gyrus (Mazel
et al. 1998; Sykova et al. 1998) (see Fig. 34.5). Glial cells can
influence both α and λ under physiological as well as
pathological conditions, a fact that is sometimes used for
diagnostic purposes, for example in demyelinating dis-
eases. Besides α and λ diffusion in the ECS is affected by
nonspecific, concentration-dependent uptake (kc), bet-
ter characterized as the irreversible loss of a given sub-
stance across the blood-brain-barrier or owing to its
binding to receptors, enzymatic degradation or cellular
uptake, for example to astrocytes. In many cases substances
released extrasynaptically such as glutamate and GABA
(see chapters 38 and 41) are transported by energy-dependent
uptake systems that obey nonlinear kinetics.
 A Resting state C Δ[K+]e
7
5 [K+]
Diffusion curves mM
Iontophoresis TMA+-ISM [TEA+]e 3
α = 0.20 10
λ = 1.55 I = 40 nA 30 Hz
+
5 [TEA ]
mM
0
[TMA+]
TMA+ 7.2
pH
80 nA pHe 7.4
5 min
30 Hz 60 s

B Neuronal activity D

neuron pHe
α = 0.12
glia λ = 1.70

pHi excitability

[TMA+] HCO3–
TMA + Na+ –
Cl +
K
H2O K+
80 nA
GLIA NEURON

Figure 34.4 A,B. Experimental setup, TMA+ diffusion curves and typical ECS diffusion parameters α (volume fraction) and λ (tortuosity) obtained
in the brain before (A, resting state) and during (B, activity) neuronal activity, evoked by the same iontophoretic current of 80 nA. A TMA+-selective
double-barreled ISM was glued to a bent iontophoresis microelectrode. The ECS in the unstimulated brain is 20% (volume fraction α = 0.20) and λ is
1.55. The ECS is smaller because of cell swelling during stimulation-evoked neuronal activity, therefore the diffusion curves are larger. The ECS volume
decreased to about 12% (α = 0.12), whereas λ increased to 1.70. C. The effect of repetitive stimulation of afferent input on TEA+ diffusion curves,
extracellular K+ and pH in the isolated frog spinal cord. Higher diffusion curves indicate a decrease in α due to cell swelling outlasting the 1 min
repetitive stimulation (30 Hz) of the sciatic nerve for 30 minutes. Note the extracellular K+ increase and the acid shift evoked by stimulation. The time
course of the acid shift correlates with the decrease in α. D. Schematic of the mechanism of nonspecific feedback suppressing neuronal excitability.
Active neurons release K+, which accumulates in the ECS and depolarizes glial cells. This causes an alkaline shift in glial pHi and an acid shift in pHe.
Extracellular acidosis further suppresses neuronal activity. Transmembrane ionic movements result in glial swelling, an ECS volume decrease and the
increased accumulation of ions and neuroactive substances in the ECS. From Sykova 2003, with permission from John Wiley and Sons.

4 THE ECS AROUND ASTROCY TES
AND OLIGODENDROCYTES
The existence of regional differences in the extracellular space
volume around glial cells affects the membrane currents gener-
ated in response to voltage steps applied through a patch-clamp
pipette, because of K+ accumulation just outside the cell. In
spinal cord oligodendrocytes the currents decay and large tail
currents appear after the offset of the voltage command (Berger
et al. 1991; Chvatal et al. 1999). The current decay and the tail
currents are related to the accumulation of K+ in the vicinity
of oligodendrocytes because of barriers that prevent the fur-
ther diffusion of K+ that has escaped from the cell. Because
glial cells are exclusively permeable for K+, knowing the val-
ues of the tail current and the reversal potential, the Nernst
equation can be used to calculate the value of [K+]e in the
vicinity of the cell membrane. Osmotically induced cell swell-
ing or cell shrinkage has revealed that the extracellular space
in the close vicinity of astrocytes is larger than that around
oligodendrocytes (Vargova et al. 2001). The application of a
hypotonic solution resulted in a 306% increase in K+ accu-
mulation around astrocytes but only a 110% increase around
oligodendrocytes. The different effects of an osmotic chal-
lenge on tail currents in astrocytes and oligodendrocytes can
be explained by a more “compact” space around oligodendro-
cytes. As tail currents appear in oligodendrocytes during their
maturation and correspond temporally with the myelination
of the tissue (Chvatal et al. 1997), we can assume that the dif-
fusion barriers around oligodendrocytes are formed mainly by
myelin sheaths or by extracellular matrix molecules produced
by mature oligodendrocytes.
5 D I F F U S I O N PA R A M ET E R S I N
P H YS I O L O G I C A L S TAT E S
5.1 D EV E L O PM E N T
During development, the diffusion parameters in the CNS
are different from those found in adulthood. In rats, the
volume fraction decreases throughout the animal’s entire

C O N T R O L O F T H E E X T R AC E L LU L A R I O N I C E N VI R O N M E N T A N D VO LU M E • 435
 A Young Aged

GFAP GFAP 200 μm

C D

α = 0.27 α = 0.16
Δ [TMA+] = 1 mM

λx = 1.50 λx = 1.53
λy = 1.60 λy = 1.55
λz = 1.70 λz = 1.59

60 s 60 s
(I = 80 nA) (I = 80 nA)

E F >1000
900

800
700
600
500
400
ADCW = 631 μm s 2 –1 2 –1
ADCW = 452 μm s <300
ADCW = [μm2s–1]

Figure 34.5 Structural Changes in the Hippocampus Gyrus Dentatus Region of Young and Aged Rats. A. Astrocytes stained for GFAP in a young adult
rat; note the radial organization of the astrocytic processes between the pyramidal cells (not stained). B. In an aged rat, the radial organization of the
astrocytic processes is lost. C. Anisotropic diffusion in the dentate gyrus of a young adult rat. TMA+ diffusion curves (concentration-time profiles)
were measured along three orthogonal axes: x—mediolateral, y—rostrocaudal, z—dorsoventral. The slower rise in the z than in the y direction and in
the y than in the x direction indicates a higher tortuosity and more restricted diffusion. The amplitude of the curves shows that the TMA+ concentra-
tion, at approximately the same distance from the tip of the iontophoresis electrode, is much higher along the x-axis than along the y-axis and even
higher than along the z-axis (lx, ly, lz). Note that the actual ECS volume fraction α can be calculated only when measurements are done along the x, y,
and z axes. D. Anisotropy is almost lost in an aged rat. E,F. Pseudocolor images showing typical ADCW maps of a young (E) and an aged
(F) rat. The scale at right shows the relation between the intervals of the ADCW values and the colors used for visualization Adapted from Sykova
2003, with permission from John Wiley and Sons.

life. A steep fall occurs in the first 2 postnatal weeks, during
which α is reduced by approximately half in the rat cortex
and corpus callosum (Lehmenkuhler et al. 1993; Prokopova
et al. 1997; Vorisek and Sykova 1997a). This decrease can
be attributed to changes in ECM composition, neuronal
migration, the development of dendritic arborization, rapid
myelination, and the proliferation of glia. The larger volume
fraction in immature tissue (0.35 to 0.40) slows down any
increase in the concentration of neuroactive substances to
neurotoxic levels and represents a protective factor in the
immature brain during pathological states such as ischemia
(Vorisek and Sykova 1997b) or epilepsy (Kilb et al. 2006).
The connection between gliogenesis and the ECS diffusion
parameters is probably most evident in the white matter.
The period of the gradual myelination of the corpus callo-
sum closely corresponds with the decrease in the extracellu-
lar volume fraction. Moreover, the tortuosity values, which
during the first postnatal week are the same along all three
orthogonal axes (i.e., diffusion is isotropic) start to differ
during the second week—a period of intensive myelination.
After myelination is fully completed at the end of the third
postnatal week, diffusion in the corpus callosum is strictly
anisotropic with preferential diffusion along the myelinated
fibers (Vorisek and Sykova 1997a).

436 • FUNCTIONS OF NEUROGLIAL CELLS

 5.2 AC T I VIT Y-R E L AT E D C H A N G E S
Because the ions crossing the cell membrane during neuronal
activity are accompanied by water molecules, their transmem-
brane transport causes cell swelling and compensatory ECS
shrinkage. Repetitive electrical stimulation of the dorsal root
of the rat or frog spinal cord causes a substantial increase in
[K+]e and a decrease in α from about 0.24 to between 0.17
and 0.12, that is, a decrease of 30% to 50% (see Fig. 34.4).
A 20% to 50% decrease in ECS volume also occurs after
injury of the ipsilateral hind paw evoked by a subcutaneous
injection of turpentine or after a thermal injury that produces
chronic pain and long-lasting interneuronal activity (Svoboda
and Sykova 1991). The changes in the ECS diffusion param-
eters persist after stimulation ends (for 30 minutes after 1
minute of electrical stimulation or even 120 minutes after 3
minutes of stimulation), suggesting long-term glial swell-
ing and subsequent long-term changes in neuronal excitabil-
ity, neuron–glia communication, and extrasynaptic volume
transmission.
5.3 L AC TAT I O N
In rat males and virgin females, the hypothalamic supraoptic
nucleus is an anisotropic structure with a relatively large extra-
cellular volume of 32% (Piet et al. 2004). The retraction of
glial processes toward the basal lamina under specific physi-
ological conditions, such as lactation or dehydration, leads to a
significant reduction in the astrocytic coverage of the magno-
cellular neurons (Hatton 1997; Theodosis and Poulain 1993)
(see also chapter 41). A relative absence of glial processes
around glutamatergic synapses results in the insufficient clear-
ance of glutamate and thus increases its level in the ECS (Oliet
et al. 2001). During lactation, the ECS volume decreases to
about 20%, and the retraction of astrocytic processes results
in the disappearance of diffusion barriers. Facilitated diffu-
sion accompanied by the retraction of astrocytic processes
results in glutamate-mediated cross-talk between synapses and
enhanced heterosynaptic depression of GABA-ergic transmis-
sion (Piet et al. 2004). Depressing the inhibitory effect of
GABA-ergic neurons can increase hormone release during
suckling and thus represents one of the mechanisms regulat-
ing milk ejection in the rat supraoptic nucleus.
6 A S T R O C Y T E S , T H E T R I PA RT I T E
SY N A P S E A N D D I F F U S I O N PA R A M ET E R S
The depolarization of astrocytes leads to the activation of ion
channels, second messengers, and intercellular metabolic path-
ways and subsequently to changes in glial volume, structural
changes of the astrocytic processes and, eventually, to the pro-
duction of extracellular matrix molecules. Therefore, in addi-
tion to their role in the maintenance of extracellular ionic and
volume homeostasis, glial cells may play an important role in
regulating synaptic as well as extrasynaptic volume transmis-
sion. Signal transmission in the brain results from the activation
of receptors located in synapses or situated extrasynaptically.
C O N T R O L O F T H E E X T R AC E L LU L A R I O N I C E N VI R O N M E N T A N D VO LU M E
Neurons interact both by synapses as well as by the diffusion
of molecules and neuroactive substances in the ECS. Neurons
also release ions and other substances in synapses and from the
extrasynaptic regions of their membranes. Since glial cells have
no synapses, neuron–glia communication is only achieved by
diffusion in the ECS, and glia release substances only nonsyn-
aptically. This type of extrasynaptic communication acts over
both short and long distances (see Fig. 34.3) and provides a
mechanism for synchronizing the activity of a population of
neurons and long-range information processing in functions
such as vigilance, hunger, sleep, chronic pain, depression, lac-
tation, long-term potentiation and depression, and memory
formation.
Synaptic transmission, which is characterized by fixed syn-
aptic contacts, usually allows only one to one cell communi-
cation. The classical concept of a tripartite synapse (Haydon
2001) involves a presynaptic terminal, a postsynaptic element,
and astrocytes (see chapter 39). Certain synapses (“private”
synapses) or even whole neurons are tightly ensheathed by
astrocytic processes and by extracellular matrix molecules
produced by astrocytes; these components form perineuronal
nets (Celio et al. 1998). Other synapses are left more “open,”
and those can be easily reached by diffusible signals in the ECS
(Sykova 2001). Transmitters such as glutamate and GABA
can thus escape from a nearby synapse and reach the neigh-
boring one. This frequent phenomenon is called transmitter
spillover and results in synaptic “cross-talk” (Asztely et al.
1997; Kullmann et al. 1996). Cross-talk could be involved
in long-term potentiation and long-term depression, which
involve both ionotropic and metabotropic receptors (Isaacson
et al. 1993; Piet et al. 2004) (see Fig. 34.3).
7 E C S D I F F U S I O N PA R A M ET E R S I N
PAT H O L O G I C A L S TAT E S
Many pathological states that are accompanied by structural
changes in the neuronal and astrocytic populations, changes
in the ECM, or the deposition of macromolecules such as
βamyloid substantially affect the diffusion parameters in the
brain and therefore information processing as well. Astrocytic
rebuilding and nerve cell loss are typical features of damaged
tissue. Brain injury of any kind elicits reactive gliosis, involv-
ing both the hyperplasia and hypertrophy of astrocytes. Active
astrocytes change their morphology; their processes become
shorter and thicker, and qualitative and quantitative changes
in their production of extracellular matrix molecules can be
detected. Changes in the ECS diffusion parameters have been
found during pathological states such as ischemia, epilepsy,
injury, hydrocephalus, multiple sclerosis, Parkinson’s disease,
Alzheimer’s disease, and tumors.
7.1 I S C H E M I A/A N OX I A , B R A I N I N J U RY
A N D A S T RO G L I O S I S
CNS ischemia, anoxia or hypoxia is accompanied by large
ionic shifts in the ECS, for example an increase in [K+]e to 50
• 437
to70 mM and an acid shift to 6.4 to 6.6. The ionic changes are
accompanied by the redistribution of water from the ECS to
intracellular compartments. Therefore, both neurons and glia
swell, and ECS volume decreases from about 20% (α = 0.2) to
as low as 5% of total tissue volume (α = 0.05), while tortuosity
processes including a decreased number and efficacy of syn-
apses, a decrease in transmitter release, neuronal loss, astro-
gliosis, demyelination, deposits of βamyloid, changes in the
extracellular matrix and others, resulting in behavioral changes
and a cognitive deficit, particularly memory and learning
(λ increases fromabout 1.5 to 2.0 to 2.2 (Homola et al. 2006;
Sykova et al. 1994; Vorisek and Sykova 1997b). A decrease
in ADCW is revealed by DW-MRI (Sykova and Nicholson
2008). In experiments in which the animals are resuscitated
and recover to normoxic conditions, the diffusion parameters
return to control values, and the volume fraction often reaches
25% to 30% owing to the formation of postischemic edema.
In models of brain injury, changes in α and λ are often inde-
pendent and result from the structural remodeling of the tissue
(cell death, astrogliosis) (Roitbak and Sykova 1999; Vorisek et al.
2002). The formation of gliotic tissue caused by local brain dam-
age is responsible for a long-lasting increase of diffusion barriers
in the ECS, preventing or slowing down the diffusion of neu-
roactive substances and growth factors and leading to impaired
extrasynaptic transmission, cell-to-cell communication, and
regenerative processes. In the formation of diffusion barriers,
not only are hypertrophied astrocytic processes involved, but
also the enhanced production of extracellular matrix molecules.
Changes in the diffusion parameters in areas remote from the
site of injury possibly lead to functional deficits.
7.2 D E MY E L I NAT I N G D I S E A S E S
Changes in the ECS diffusion parameters, including a loss
of the anisotropy typical of white matter, have been found
in patients with demyelinating diseases such as multiple scle-
rosis. In an animal model of multiple sclerosis, experimental
autoimmune encephalomyelitis, typical morphological fea-
tures of multiple sclerosis can be observed in the CNS tissue,
including demyelination, an inflammatory reaction, astroglio-
sis, blood-brain-barrier damage, and clinical signs of paraly-
sis (see chapter 61). Diffusion measurements have revealed
an increase in ECS volume fraction α and a decrease of
tortuosity in both the white and the gray matter of the spinal
cord. There was a close correlation between the changes in the
ECS diffusion parameters and the manifestation of clinical
signs (paraparesis, paraplegia), which were preceded and out-
lasted by astrogliosis and an inflammatory reaction (Simonova
et al. 1996).
Changes in ECS diffusion have also been found during
acute inflammation. In an experimental model, inflammation
was evoked by intracerebral inoculation with a weakly patho-
genic strain of Staphyloccocus aureus. The evoked acute inflam-
mation and an increase in blood-brain barrier permeability in
the abscess region resulted in a rather mild increase in ECS
volume fraction and a lower tortuosity (Lo et al. 1993).
7.3 AG I N G A N D A L Z H E I M E R’S D I S E A S E
Aging is a normal physiological process of the organism that
has many features in common with degenerative diseases of the
CNS, including Alzheimer’s disease. Both aging and degener-
ative brain diseases are accompanied by various pathological
438 • FUNCTIONS OF NEUROGLIAL CELLS
impairment. These changes affect the biophysical and diffu-
sion properties of the ESC in the cortex, corpus callosum and
hippocampus of senescent rats (CA1, CA3 and dentate gyrus).
In all three brain regions, the mean ECS volume fraction α is
significantly reduced in aged rats compared to young adults,
and the anisotropy typical of the hippocampus decreases
(Mazel et al., 1998). Morphological changes observed in the
hippocampal tissue include the disorganization of astrocytic
processes (Fig. 34.5) and a loss of the extracellular matrix mol-
ecules that form perineuronal nets, for example fibronectin
and chondroitin sulfate proteoglycan (CSPG). This accounts
for both the decrease in α and the disappearance of anisot-
ropy. Rats with severe morphological changes also display a
significant learning impairment that closely correlates with
changes in the ECS diffusion parameters (Sykova et al., 2002).
It is therefore reasonable to assume that diffusion anisotropy,
which leads to a certain degree of specificity in extrasynaptic
communication by channeling the flux of substances in a pref-
erential direction, may also play an important role in memory
formation and learning (Mazel et al., 1998; Sykova et al., 2002;
Wieshmann et al., 1999). The decrease in ECS volume during
aging not only impairs extrasynaptic transmission in the cor-
tex and hippocampus, but is also responsible for the greater
susceptibility of the aged brain to pathological insults, the
poorer outcome of clinical therapy and limited regeneration.
Changes in the ECS diffusion parameters have also been
found in transgenic APP23 mice—a model of Alzheimer’s
disease. The deposition of β-amyloid is associated with an
increase in ECS volume fraction and tortuosity (Fig. 34.6).
It is assumed that the volume fraction occupied by extracel-
lular amyloid plaques is not included in the value of α. The
deposition of macromolecules or ECS matrix can thus prevent
the ECS volume fraction decrease normally observed in aging
animals (Sykova et al. 2005a). Changes in the ECS diffusion
parameters have been found in mice lacking tenascin-R, a large
extracellular glycoprotein (Sykova et al. 2005b). Decreases in
both α and λ were found in the entire cortex and hippocam-
pus in the knockout mice as compared to wild-type animals.
Measurements using DW-MRI showed a reduction in ADCW
in the cortex, hippocampus, and thalamus in the knockouts
(see Fig. 34.6).
Extracellular matrix molecules play a role in tissue cyto-
architecture by maintaining the optimal size of the intercel-
lular spaces (Sykova et al. 2005b). This role of the ECM in
determining the ECS volume can be demonstrated by the
changes seen during aging, when a decrease in the amount of
CSPG and fibronectin in the hippocampus of old rats with
a severe learning disability coincides with a decrease in ECS
volume (Sykova et al. 2002). The explanation for the rela-
tionship between the size of the ECS and the ECM content
is simple: the side chains of ECM macromolecules, especially
those of glycosaminoglycans, carry a large number of nega-
tively charged groups that repel one another. Concomitantly,
 Tenascin R-/- control - aged

α = 0.12 α = 0.11
λ = 1.53 λ = 1.43

1 mM TMA+
α = 0.19

1 mM TMA+
λ = 1.63 α = 0.23
λ = 1.60
APP23
Tenascin R+/+

60 s 60 s
(I = 180 nA) (I = 180 nA)

Tenascin R-/- aged

ADCW = 0.61 ADCW = 0.45

Tenascin R+/+ APP23

ADCW = 0.52 ADCW = 0.55

<0.3 0.4 0.5 0.6 0.7 0.8 0.9 >1.0

ADCW × 105cm2s–1

Figure 34.6 (Left) TMA+ diffusion curves with the corresponding values of volume fraction (α) and tortuosity (λ) obtained in the cortex and
pseudocolor ADCW maps obtained throughout the whole brain of tenascin-R knockout (-/-) and control (+/+) mice. (Right) TMA+ diffusion curves
with the corresponding values of volume fraction (α) and tortuosity (λ) obtained in the cortex and pseudocolor ADCW maps obtained throughout
the whole brain of aged control mice and aged APP23 mice (17–25 months old); the APP23 mice showed excessive extracellular amyloid deposition.
The larger ECS volume fraction demonstrates that diffusion from any given source will lead to a lower concentration of the diffusing substance in the
surrounding tissue and a smaller action radius in APP23 mice than in age-matched controls. The scale at the bottom shows the relation between the
intervals of the ADCW values and the colors used for visualization. Adapted from Sykova et al., 2005a and Sykova et al., 2005b, with permission from
the National Academy of Sciences USA and John Wiley and Sons.

they attract osmotically active cations such as Na+, causing a
large amount of water to be drawn into the matrix. This cre-
ates a turgor that enables the matrix to withstand compres-
sive forces and leads to the expansion of the ECS (Alberts
et al. 1994).
7.4 G L I A L T U MO R S
The impact of glial remodeling and rebuilding on the biophys-
ical properties and diffusion parameters of the ECS is also
manifested in glial tumors. Studies using human glioma sam-
ples obtained from patients during surgery showed that the
migratory abilities of tumor cells might be strongly affected by
changes in the ECS volume fraction and the content of ECM
molecules in tumors (Vargova et al. 2003; Zamecnik et al.
2004). The tumor slices were histopathologically classified
according to WHO grading (WHO grade I-IV) following
diffusion measurements, and the changes in the values of the
ECS diffusion parameters correlated with tumor malignancy.
The proliferative activity of the tumors was assessed by the

C O N T R O L O F T H E E X T R AC E L LU L A R I O N I C E N VI R O N M E N T A N D VO LU M E • 439
labeling indices MIB-1 and anti-topo-II α. The measurements
showed that the proliferative activity and malignancy grade
of astrocytomas are directly proportional to increasing val-
ues of α and λ (Fig. 34.7). In the cellular regions of the most
malignant glioblastomas (WHO grade IV), tortuosity was
about 1.8 and α was twice as high (0.44) as in normal cortex
(Vargova et al. 2003). The increase in α and λ in high-grade
tumors strongly correlated with an increased accumulation
of ECM molecules, particularly of tenascin and vitronectin
(Zamecnik et al. 2004). It has been shown that the overex-
pression of certain types of ECM molecules corresponds with
tumor malignancy (Camby et al. 2002; Hayen et al. 1999;
Zhang et al. 1998), as these molecules may serve as “ropes for
climbing” for migrating tumor cells and thus facilitate their
infiltration into the surrounding tissue. In addition, a large
ECM content holds cells apart and thus creates a larger space
for migrating cells.
8 S U M M A RY A N D P E R S P E C T I VE S
It is evident that glial cells play an important role in differ-
ent brain functions. Voltage- and ligand-activated channels
and a number of transport mechanisms on the glial mem-
brane (Sontheimer et al. 1989) allow for the maintenance of
ionic and volume homeostasis, which in turn affect neuronal
function. A link between ionic and volume changes has been
proposed in a model of a nonspecific feedback mechanism
suppressing neuronal activity (Sykova 1997). Glial cells con-
trol not only the ionic composition of the ECS, but also its
biophysical properties that determine the movement of ions,
neuroactive substances, neurohormones, growth factors, and
metabolites. Changes in the ECS diffusion parameters have
a strong impact on modulating both synaptic and extrasyn-
aptic volume transmission, the synchronization of neuronal
activity in complex functions, and neuron–glia communica-
tion. Astrocytes are highly sensitive to stimuli accompanying
both physiological and pathological states such as increased
potassium levels, osmolality changes, neuromediators, or sig-
nals from damaged tissue, and promptly react by cell swelling,
proliferation, and hypertrophy. The production of extracellu-
lar matrix molecules by activated astrocytes further hinders
already impaired diffusion and contributes to an increase in
tortuosity. Astrocytes can thus be viewed as active elements
in signal transmission as they affect both synaptic as well as
extrasynaptic volume transmission.

TMA+ - iontophoresis
Human cortex:
α = 0.24
[TMA+]e λ = 1.45
k′ = 3.2 × 10–3 s–1
Δ [TMA+] = 0.5 mM

Glioblastoma:
α = 0.37
TMA+ - ISM λ = 1.86
k′ = 9.3 × 10–3 s–1

60 s
(I = 200 nA)

Cortex Glioblastoma

100 μm

Figure 34.7 Schema of the Experimental Arrangement for Measuring the ECS Diffusion Parameters in a Glioblastoma. A TMA+-selective
double-barreled microelectrode was glued to a bent iontophoresis microelectrode. Diff usion curves were recorded in acute brain slices obtained
during neurosurgery. Typical diffusion curves in the normal cortex and in the glioblastoma show that the volume fraction (α) and tortuosity
(λ) were increased in the tumor. This increase correlated with the increased deposition of extracellular matrix and with the mitotic index of the
tumor cells.

440 • FUNCTIONS OF NEUROGLIAL CELLS

 AC K N OW L E D G M E N T S
The author is grateful for the support provided by the Grant
Agency of the Czech Republic (309/09/1597).
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 35.
AMINO ACID NEUROTRANSMITTER SYNTHESIS
AND REMOVAL
Arne Schousboe, Lasse K. Bak, Karsten K. Madsen, and Helle S. Waagepetersen

A B B R E VI AT I O N S analog, taurine, which is present in the brain in large amounts

(Huxtable 1992), is sometimes referred to as a neurotransmitter
AAT aspartate aminotransferase or neuromodulator because it can activate both GABA and gly-
ALAT alanine aminotransferase cine receptors (Oja and Saransaari 2007), but this amino acid
AOAA aminooxyacetic acid will not be dealt with in this chapter. Interestingly, the amino
BCAA branched chain amino acid acid neurotransmitters include a D-amino acid namely D-serine
BCAT branched chain aminotransferase which is functionally closely related to glycine (Wolosker et al.
BGT betaine-GABA transporter 2008). The amino acid neurotransmitters mediate via activa-
CIT citrate tion of specific receptors either excitatory or inhibitory signals.
EAAC excitatory amino acid carrier Hence, glutamate and aspartate function as excitatory neu-
EAAT excitatory amino acid transporter rotransmitters (Danbolt 2001) whereas glycine and GABA
GABA gamma-aminobutyric acid function as inhibitory transmitters (Curtis and Johnston 1974;
GABA-T GABA transaminase Schousboe and Waagepetersen 2008). Glycine, however, has
GAD glutamate decarboxylase a more complex mode of action because it, in addition to its
GAT GABA transporter action on specific glycine receptors, also acts as a cotransmitter
GC glutamate hydroxyl carrier (coagonist) with glutamate on a separate glycine receptor site
GDH glutamate dehydrogenase on the N-methyl-d-aspartate (NMDA) subtype of the iono-
GLAST glutamate aspartate transporter tropic glutamate receptors (Zafra and Gimenez 2008). This
GLN glutamine way glycine can be regarded as an excitatory neurotransmitter
GLU glutamate as well. However, because this action is strictly dependent on
GlyT glial glycine transporter glutamate binding to the NMDA receptor, glycine is normally
GS glutamine synthase considered to be inhibitory in its own right. In relation to the
GLT-1 glutamate transporter 1 glycine site on the NMDA receptors, D-serine is of interest
ME malic enzyme because it turns out that it binds to the glycine site, thereby
NMDA N-methyl-d-aspartate replacing glycine as the coagonist. This aspect will be discussed
OAA oxaloacetate in a separate section because astrocytes appear to be directly
PAG phosphate activated glutaminase involved in this action of D-serine (Wolosker et al. 2008).
PC pyruvate carboxylase The major excitatory amino acid neurotransmitter glutamate
PDH pyruvate dehydrogenase exhibits a complex metabolism involving an obligatory neu-
PKC protein kinase C ronal–glial interaction (Waagepetersen et al. 2009). The neu-
SLC solute carrier rotransmitter activity of glutamate is terminated by binding to
SSA succinic semialdehyde and transport by a number of specific, high affinity transporters,
SSADH SSA dehydrogenase the majority of which in terms of transport capacity are located
TCA tricarboxylic acid on astroglial cells (Danbolt 2001; Gegelashvili and Schousboe
TMD transmembrane spanning domain 1997). The metabolism of the inhibitory neurotransmitter
GABA is also complex, involving both neuronal and astrocytic
elements and, in analogy to glutamate, its inactivation is medi-
1 INTRODUCTION ated by a number of high affinity transporters in neurons and
astrocytes (Schousboe and Waagepetersen 2008). However,
Among the amino acids present in the central nervous system, contrary to glutamate, the GABA transporter capacity has a
only glutamate, aspartate, and glycine are normally considered more prominent expression in GABAergic neurons compared
to act as neurotransmitters in addition to being part of meta- with that in astrocytes (Schousboe and Waagepetersen 2008).
bolic processes and protein synthesis. In addition to these, gam- This chapter will deal in detail with the previously men-
ma-aminobutyric acid (GABA) also acts as a transmitter and tioned aspects of glutamatergic, GABAergic, and glycinergic
a metabolite but it is not found in proteins. Its closely related neurotransmission, emphasizing the role of astroglial cells.

443
 2 G LU TA M AT E
2.1 B I O SY N T H E S I S A N D M ETA B O L I S M
Astrocytes are key elements in the maintenance of glutamater-
gic neurotransmission because only astrocytes express the
enzymes that are mandatory for homeostasis of glutamate,
such as glutamine synthetase (GS) and pyruvate carboxylase
(Norenberg and Martinez-Hernandez 1979; Yu et al. 1983).
In contrast, neurons are metabolically handicapped and are
not equipped to perform de novo synthesis of their own neu-
rotransmitter. The concept of metabolic interaction between
neurons and astrocytes necessary to sustain glutamate home-
ostasis was founded on studies of glutamate and glutamine
metabolism in brain tissue preparations. The early studies
indicated distinct cellular compartments of glutamate and
glutamine with different turnover rates (van den Berg and
Garfinkel 1971).
Glutamate neurotransmission (Fig. 35.1) is terminated
by diffusion of glutamate away from the receptor, binding to
high affinity glutamate transporters primarily in the astrocyte
plasma membrane and eventually uptake (see section 2.3). In
the cytosol of the astrocyte, glutamate may be converted to
glutamine by the enzyme GS, which is exclusively expressed
in astrocytes (Norenberg and Martinez-Hernandez 1979).
Glutamate is irreversibly amidated using free ammonium
and energy is provided by the cleavage of ATP to ADP. The
Km values for GS in brain have been reported to be 0.2 mM
for ammonia, 2.5 mM for glutamate, and 2.3 mM for ATP
(Pamiljans et al. 1962). Intracellular ATP levels in brain are
approximately 2.4 mM, and the ammonia concentration in
the astrocyte compartment is estimated to be less than 0.2
mM (Cooper and Plum 1987). The glutamate concentra-
tion in astrocytes is low because of rapid metabolism, and
the microenvironment around the glutamate transporter is
probably of great functional importance. A phenomenon

Glucose

PYR
PDH
PC
ME
OAA
TCA CIT
Cyele
MAL

Glutamatergic α–KG
neuron ASP
Gln Gln
NH4+ AAT
+
SNAT3 + GDH OAA
NH4+ Na H GS
GLU
Gln Gln
GLU GLN SNAT1 LAT2 GLN
Na+ AA
PAG
Gln Gln
ASCT2
Na+ GS Astrocyte
NH4+

GLU GLU
GLUTAMATE

Figure 35.1 Neurotransmitter glutamate is to a large extent taken up by the glial transporters, GLT-1 or GLAST, subsequent to postsynaptic receptor
interaction. Glutamate (GLU) may be amidated to glutamine (GLN) via glutamine synthetase (GS) and glutamine is subsequently released from the
astrocyte via specific transporters (i.e., SNAT3, LAT2, or ASCT2), and taken up by the neuron via SNAT1. In the neuron, glutamine is deamidated by
phosphate activated glutaminase (PAG) to glutamate, completing the glutamate–glutamine cycle. Alternatively, glutamate may be oxidatively metabo-
lized after entrance into the mitochondria. In this organelle glutamate is deamidated by glutamate dehydrogenase (GDH) or converted by aspartate
aminotransferase to α-ketoglutarate (α-KG). For complete oxidation of the carbon skeleton, pyruvate (PYR) recycling is required via the concerted
action of malic enzyme (ME) and pyruvate dehydrogenase (PDH). Glutamate and glutamine can be de novo synthesized from glucose in astrocytes
via the carboxylation of pyruvate catalyzed by the mitochondrial enzyme pyruvate carboxylase (PC). CIT, Citrate; MAL, malate; OAA, oxaloacetate;
TCA, tricarboxylic acid.

444 • FUNCTIONS OF NEUROGLIAL CELLS

that is only sparsely investigated but worth noting is the
recent finding that GS was not one of the enzymes immu-
noaffinity isolated with the glutamate transporter GLT-1
(Genda et al. 2011).
Glutamine is released from astrocytes and taken up by
nearby neurons via specific transporters as described later (see
section 2.2). The subsequent deamidation of glutamine form-
ing glutamate by phosphate activated glutaminase, an enzyme
enriched in neurons, completes the glutamate–glutamine
cycle (see Fig. 35.1). Phosphate activated glutaminase is asso-
ciated with the inner mitochondrial membrane, a localization
that may have functional consequences facilitating oxidative
metabolism of glutamine in neurons (Waagepetersen et al.
2005). As an alternative to the simple amidation of glutamate
into glutamine in astrocytes, glutamate may enter the mito-
chondria for oxidative metabolism, which is likely promoted
by a close association of the glutamate transporter, GLT-1,
to mitochondria (Genda et al. 2011). Oxidative metabolism
of glutamate in astrocytes and that of glutamine in neurons
disrupts a simple stoichiometric view of the glutamate–glu-
tamine cycle but provides the cells with a dynamic system
ready to respond to changes in neurotransmission as well as
energy requirements.
One of the ways of glutamate entrance into the mitochon-
dria is the glutamate hydroxyl carrier (GC). Another entry via
the aspartate/glutamate carrier Aralar1 was found plausible
from the results of a transcriptomic analysis of acutely iso-
lated astrocytes (Lovatt et al. 2007) but translation into the
protein level has been questioned in astrocytes (Ramos et al.
2003). Aralar1 is an antiporter transporting aspartate out
accompanied by the simultaneous import of glutamate to the
mitochondria, which has to be taken into account considering
the purpose or metabolic fate of glutamate inside the mito-
chondria. In contrast to Aralar1, GC enables transfer of gluta-
mate either into or out of the mitochondria dependent on the
concentration gradient of glutamate and protons. This allows
coupling of the transport to the activity of the electron trans-
port chain and subsequent ATP yield. Two distinct isoforms
of the glutamate hydroxyl carrier, GC1 and GC2, have been
identified with equal expression level in the brain (Fiermonte
et al. 2002). This is in contrast to other organs, which pre-
dominantly express GC1. Primarily, GC1 has been found in
an enriched glial fraction (Berkich et al. 2007) whereas no
information is available on the cellular localization of GC2
in brain. Distinct kinetic characteristics are exhibited by
GC1 and 2, the Km value for GC1 being 5 mM glutamate and
that for GC2 only approximately 0.26 mM. The capacity for
transport, that is the Vmax, which is dependent on pH, ranges
from 12 to 63 μmol/minute per gram for GC1 and from 4
to 16 μmol/minute per gram for GC2. For comparison the
glutamate-glutamine cycling rate has been estimated to be 0.5
μmol/minute per gram in awake animals (Oz et al. 2004).
Interestingly, Aralar1 as well as GC1 were immunoaffinity
isolated with GLT-1 (Genda et al. 2011), strongly indicating
that Aralar1 is translated in astrocytes. A close association of
mitochondria to plasma membrane glutamate uptake may
support the energy requirement of the linked Na+/K+ ATPase
activity as suggested by Genda et al. (2011). Additionally, a
A M I N O AC I D N E U R OT R A N S M I T T E R SY N T H E S I S A N D R E M O VA L
rapid removal of glutamate away from the microenvironment
around the transporter may continuously preserve the plasma
membrane gradient of glutamate and efficient uptake (see
section 2.3.3).
Glutamate is to a considerable extent metabolized oxida-
tively (Fig. 35.2) and the significance of this pathway is appar-
ently increasing with the extracellular glutamate concentration,
whereas the flux of glutamate converted into glutamine is inde-
pendent of this (McKenna et al. 1996). Moreover, the synthesis
Glucose
PYR
LDH
LAC
PC PDH
ME
OAA
TCA CIT
cycle
MAL
α–KG
NH4+ ASP
AAT
GDH OAA
GLU
Astrocyte
GLU
Figure 35.2 The extent to which glutamate (GLU) is oxidized in astrocytes
seems to increase particularly during higher glutamate concentrations. A
net synthesis of tricarboxylic acid (TCA) cycle intermediates occurs
when the initial reaction is catalyzed by glutamate dehydrogenase
(GDH), which paves the way for the complete oxidation of the carbon
skeleton of glutamate. This requires pyruvate recycling via the concerted
action of malic enzyme (ME) and pyruvate dehydrogenase (PDH)
converting malate into acetyl CoA producing NADPH. Acetyl CoA is
oxidized completely in one turn of the TCA cycle. A partial oxidation of
glutamate is acquired when pyruvate (PYR) is reduced to lactate instead
of being oxidized to acetyl CoA. The redox state of the cell is likely
important in the regulation of the destiny of the glutamate molecule.
Alternative to the activity of GDH, aspartate aminotransferase (AAT)
facilitates the formation of α-ketoglutarate (α-KG) from glutamate at
the expense of oxaloacetate (OAA); thus no net synthesis of TCA cycle
intermediates is obtained. In contrast to the complete oxidation initiated
by the activity of GDH, AAT enables the truncated TCA cycle, which
refers to the net synthesis of aspartate from glutamate, a pathway shown
to accelerate during hypoglycemic conditions. All enzymes except ME
and LDH are located in the mitochondria. CIT, Citrate; PC, pyruvate
carboxylase.
• 445
of glutamine seems resistant to energy deprivation in spite of
the energy requirement of the reaction (Morland et al. 2004).
The concentration dependency can on one side reduce a poten-
tial excitotoxic level of glutamate in the synaptic area because of
oxidation of glutamate and reduction of glutamate-glutamine
cycle activity. On the other hand, it may lead to depletion of
the neuronal pool of tricarboxylic acid (TCA) cycle intermedi-
ates and compromise neuronal energy metabolism. The initial
reaction for glutamate oxidation is either an oxidative deami-
nation by glutamate dehydrogenase (GDH) or a transamina-
tion by an aminotransferase. Of the transaminases, aspartate
aminotransferase (AAT) is the one exhibiting the highest
activity in brain, although the branched chain aminotrans-
ferase (BCAT) and alanine aminotransferase (ALAT) also
have to be taken into account. Only the mitochondrial iso-
form of BCAT seems to be expressed in astrocytes (Bixel et al.
2001). The relative importance of GDH and AAT has been
debated and GDH seems to be primarily active in the deami-
nation of glutamate whereas AAT catalyzes the conversion of
α-ketoglutarate to glutamate, concomitantly forming oxaloac-
etate from aspartate (Westergaard et al. 1996). It is important
to realize the metabolic consequences of the different enzymes
that can catalyze the formation of α-ketoglutarate from gluta-
mate, such as AAT, ALAT, BCAT, and GDH. Glutamate is
converted to α-ketoglutarate by AAT at the expense of oxalo-
acetate conversion to aspartate. It should be noted, however,
that the activity of AAT enables the truncated TCA cycle,
that is, the net conversion of glutamate to aspartate, forming
75% of the ATP that is generated from oxidation of acetylCoA
(Yudkoff et al. 1994). In contrast, the activity of the three latter
enzymes ALAT, BCAT, and GDH leads to a net synthesis of
TCA cycle intermediates, and potentially a complete oxida-
tion of glutamate. Pyruvate recycling, which is the concerted
action of malic enzyme and pyruvate dehydrogenase catalyz-
ing the oxidative decarboxylations of malate into acetyl CoA
via pyruvate, is required for such complete oxidation of gluta-
mate (see Fig. 35.2). Alternatively, pyruvate may be reduced to
lactate for release. The extent to which this occurs is probably
regulated by the energy and redox state of the cell (McKenna
et al. 1996; Sonnewald et al. 1996). Pyruvate recycling activity
has been demonstrated in cultured astrocytes repeatedly but
based on in vivo data the activity is limited.
Several studies have shown that GDH is predominantly
expressed in astrocytes, especially in regions exhibiting high
glutamatergic activity (Aoki et al. 1987). There are two iso-
forms of GDH, GDH1 and GDH2. Only great apes and
humans possess the gene, GLUD2, from which GDH2 is
expressed, mainly in retina, brain, and testis (Zaganas et al.
2009). In brain GDH is mainly active in the direction of
oxidative deamination although the thermodynamic equilib-
rium favors reductive amination. The reason for this is a high
NAD+/NADH ratio in astrocytes and a high Km value (in
the range of 10 mM) of ammonia (Zaganas et al. 2009). Both
GDH1 and 2 are activated by ADP and leucine. However,
the effect is 10-fold higher for GDH2 than for GDH1 and
GDH2 is barely active in the absence of ADP. In contrast to
GDH1, GDH2 is insensitive to an inhibitory effect of GTP
(Plaitakis et al. 2003; Zaganas et al. 2009). Thus, both GDH1
446 • FUNCTIONS OF NEUROGLIAL CELLS
and GDH2, but likely especially GDH2, may have a particu-
lar role in glutamate oxidation when astrocytes are experienc-
ing either a global or local elevation of ADP.
Carboxylation of pyruvate to oxaloacetate is catalyzed by
pyruvate carboxylase (see Fig. 35.2), the most important ana-
plerotic enzyme in brain and it is like GS confined to astrocytes
(Yu et al., 1983). Anaplerosis, for example de novo synthesis
of TCA cycle intermediates, is the opposite of pyruvate recy-
cling, but the activity of anaplerosis, which has been estimated
to account for 25% of glutamate–glutamine cycle activity is
high in contrast to that of pyruvate recycling (Oz et al. 2004).
Pyruvate carboxylation increases in response to hyperammone-
mia, providing the carbon skeleton necessary for assimilation
of ammonia into glutamine (Sibson et al. 2001).
The glutamate–glutamine cycle does not provide any spe-
cific mechanism by which ammonia generated in the gluta-
matergic neurons reaches the astroglial compartment to allow
amidation of glutamate catalyzed by GS. Although some
ammonia will be cycled from neurons to astrocytes by passive
diffusion as suggested by Provent et al. (2007), an amino acid
shuttle mechanism would decrease the concentration of free
ammonia (Fig. 35.3). Two different mechanisms have been
proposed based on nitrogen transfer via alanine or one of
the branched chain amino acids (BCAAs) (Bak et al. 2006).
Glutamine is deamidated in the neuron and the liberated
ammonia is suggested to be used in the reductive amination
of α-ketoglutarate by glutamate dehydrogenase that works in
concert with either BCAT or ALAT, transferring the nitrogen
into one of the BCAAs or alanine, respectively. Alanine or the
BCAA is released from the neuron and taken up by the astro-
cyte, thereby transferring the nitrogen, which through the
concerted action of the respective transaminases and GDH
is dissimilated and available for GS. The alanine shuttle was
shown to operate in co-cultures of glutamatergic neurons and
cerebellar astrocytes; however, the activity was not increasing
in parallel with the glutamate-glutamine cycle as would have
been expected (Bak et al. 2005). The GDH reaction has to
operate in both directions for these shuttles to operate, that
is, reductive amination in neurons and oxidative deamina-
tion in astrocytes; however, GDH is predominantly located
in astrocytes and the deamination is favored except during
high levels of ammonia (Zaganas et al. 2009). Additionally,
the BCAAs have been suggested to provide the amino nitro-
gen for de novo synthesis of glutamate via pyruvate carboxy-
lation in astrocytes followed by amidation of glutamate with
free ammonia by GS and transfer of glutamine to the neurons.
The BCAA is thought to be regenerated in the neuron via
BCAT using glutamate formed via GDH catalyzed reductive
amination of α-ketoglutarate and returned to the astrocyte.
It should be noted that this is the unfavored direction of the
GDH catalyzed reaction (Lieth et al. 2001).
2.2 RO L E O F G LU TA M I N E T R A NS P O RT E R S
I N G LU TA M AT E H O M EO S TA S I S
Efflux of glutamine from astrocytes must be met by influx in
neurons for the glutamate/GABA-glutamine cycle to func-
tion. To achieve this, one would need two sets of transport
 Glutamatergic
neuron Astrocyte
ka ka
Glu Glu

AT AT
α-KG α-KG
aa aa
GDH
GDH
NH4+

GLU GLN GLN

PAG

GS NH4+

GLU GLU
GLUTAMATE

Figure 35.3 Neurotransmitter glutamate is primarily taken up by the glial transporters, GLT-1 or GLAST, subsequent to postsynaptic receptor interaction.
Glutamate (GLU) may be amidated to glutamine (GLN) via glutamine synthetase (GS) and glutamine is subsequently released from the astrocyte via
specific transporters followed by uptake into the neuron. In the neuron glutamine is deamidated by phosphate activated glutaminase (PAG) to gluta-
mate, completing the glutamate–glutamine cycle. Although often neglected, the cycling of glutamate and glutamine is accompanied by a net transfer
of nitrogen from the astrocyte to the neuron carried by glutamine. We have suggested that this nitrogen transfer is counteracted by transport of a
neuroinactive amino acid (aa) likely either alanine or one of the branched chain amino acids. The glutamate–glutamine cycle is coupled to the aa–keto
acid (ka) cycle via the concerted action of glutamate dehydrogenase (GDH) and the relevant aminotransferase (AT).

systems, one in astrocytes and one in neurons that may or may Table 35.1 NOMENCLATURE OF CLONED GLUTAMINE
not be the same (see Fig. 35.1). It would intuitively make sense TRANSPORTERS
if the astrocytic transport system is coupled closely to the syn- SYSTEMATIC NAME ALTERNATIVE NAMES
thesis of glutamine in astrocytes and if the neuronal transport
system is concentrative; this would create ideal conditions System A SNAT1 ATA1, GInT, SA2, SAT1,
for a transcellular flux of glutamine. Indeed, the current con- (neurons) SNAT2 NAT2
sensus is that the neuronal transporter is the sodium-coupled SNAT4 ATA2, SA1, SAT2
ATA3, NAT3
system A, whereas the astrocytic transport is mediated by sys-
tem N, a glutamine/Na+/H+ antiporter, albeit systems L and System N SNAT3 SN1, NAT1
ASC also may be involved as discussed below (see Fig. 35.1). (astrocytes) SNAT5 SN2
The system A transporter has been suggested by a number of
Nomenclature for systems A and N, two of the transport systems suggested as
authors as the neuronal transporter (see Bak et al. 2006 and being involved in neuronal and astrocytic glutamine cycling, respectively.
references) and it is comprised of three isoforms (Table 35.1):
SNAT1 (ATA1, GlnT, SA2, SAT1, NAT2), SNAT2 (ATA2, See text and Bak et al. 2006.
SA1, SAT2), and SNAT4 (ATA3, NAT3). Both SNAT1 and
2 are expressed in the brain, with SNAT1 being fairly brain-
and neuron-specific and found in the membrane of both glu- 230 μM compared with 1.65 mM for SNAT2 (references in
tamatergic and GABAergic neurons (see Bak et al. 2006 for Bak et al. 2006); the extracellular concentration of glutamine
references). On the other hand, SNAT2 seems to be more in rat brain is about 400 μM (Kanamori and Ross 2004).
widely distributed and is found on both neurons and astro- However, some controversy remains since Rae et al. (2003)
cytes (references in Bak et al. 2006). The previously men- found that a transport system different from system A is
tioned requirements for concentrative glutamine transport involved in glutamine cycling and Conti and Melone (2006)
into neurons are exhibited by SNAT1, being a Na+-glutamine were not able to localize system A transport molecules to syn-
symporter (i.e., electrogenic) with a Km for glutamine of aptic regions employing immunocytochemistry. System N

A M I N O AC I D N E U R OT R A N S M I T T E R SY N T H E S I S A N D R E M O VA L • 447
(see Table 35.1) comprising SNAT3 (SN1, NAT1) and
SNAT5 (SN2) has been suggested to mediate the glutamine
efflux from astrocytes, as reviewed by Bak et al. (2006).
System N is a Na+-glutamine symporter/H+ antiporter, mak-
ing it sodium-dependent, yet electroneutral (see Bak et al.
2006 for references). Interestingly, this transporter mediates
glutamine flux in both directions under physiological condi-
tions probably governed by fluctuations in intracellular Na+
levels and pH as shown in Xenopus laevis oocytes expressing
SNAT3; Na+ levels and pH are parameters that are likely to
vary at glial microdomains during neurotransmission because
glutamate uptake may affect both (see section 2.3). The Km of
system N (SNAT3) transport was lowered in cultured astro-
cytes by incubation in the presence of glutamate (30–60
minutes, 10–100 μM; Bröer et al. 2004) further substantiat-
ing the possible involvement of this transport system in efflux
of neurotransmitter glutamate-derived glutamine. However,
the possibility that efflux of glutamine from astrocytes is
mediated by other transport systems in addition to system N
should not be neglected. Thus, Deitmer et al. (2003) found
that glutamine efflux was mediated by no less than four dif-
ferent transport systems, namely system N (SNAT3), system
L (LAT2), system ASC (ASCT2), and an unidentified trans-
port system mediating as much as 40% of the efflux. System L
is an amino acid antiporter comprising two isoforms, LAT1
and 2, of which LAT2 shows affinity for glutamine (see Bak
et al. 2006 for references). System L mediates glutamine release
in the presence of extracellular amino acid substrates such as
alanine and leucine, which might be important for shuttling
nitrogen from neurons to astrocytes. System ASC is a sodium-
dependent antiporter, which exists as two different isoforms
termed ASCT1 and ASCT2, with ASCT2 having affinity
for glutamine, although this transport activity is probably of
minor importance in the adult mammalian brain (Bak et al.
2006 and references therein). Several regulatory mechanisms
have been proposed for astroglial glutamine transport activ-
ity including protein kinase C-mediated phosphorylation of
SNAT3 and ASCT2 and subsequent ubiquitin-mediated deg-
radation as well as a possible rapid caveolin-dependent inter-
nalization of SNAT3 protein induced by protein kinase C
(PKC) activity (Balkrishna et al. 2010; Sidoryk-Wegrzynowicz
et al. 2011). However, much work is still needed to ascertain
which amino acid transporters are involved in the glutamate/
GABA-glutamine cycle and their regulation.
2.3 H I G H A FFI N IT Y T R A NS P O RT E R S
The concept of an efficient and concentrative, energy-
dependent transport system for glutamate in the brain origi-
nates from studies by sir Hans Krebs and coworkers of brain
slices (Stern et al. 1949). These and numerous subsequent
studies using brain slice preparations have convincingly pro-
vided evidence for high affinity glutamate transporters, a dem-
onstration instrumental for the concept of glutamate acting as
a neurotransmitter to be accepted (for references, see Danbolt
2001; Schousboe 1981)). Based on the demonstration of high
affinity glutamate transport in synaptosomes (Danbolt, 2001)
and on the finding that degeneration of neuronal pathways led
448 • FUNCTIONS OF NEUROGLIAL CELLS
to decreased glutamate uptake (Divac et al. 1977), glutamate
transport was originally believed to be associated primarily
with glutamatergic neurons. The demonstration of a high affin-
ity glutamate uptake in bulk-prepared glial cells (Schousboe
1981) and primary cultures of astrocytes (Schousboe 1981)
showed, however, that astrocytes must play an important role
in glutamate transport.
2.3.1 Cloning of Transporters
The purification of a glutamate transporter from a synapto-
somal preparation (Danbolt et al. 1990) paved the way for the
cloning of a series of glutamate transporters (for references, see
Danbolt 2001; Gegelashvili and Schousboe 1997). The names
of the transporters have been somewhat confusing because two
different systems have been used. The original acronyms and
the more systematic names of the transporters as well as a list
of selective synthetic substrates and inhibitors are provided in
Table 35.2 as well as in a recent review by Gether et al. (2006).
2.3.2 Cellular Localization of Transporters
The transporters GLAST (EAAT-1) and GLT-1 (EAAT-2) are
by far the most highly expressed glutamate transporters in the
brain, with GLAST being more highly expressed in cerebel-
lum compared with forebrain and GLT-1 comparatively more
highly expressed in forebrain (Danbolt 2001; Gegelashvili
and Schousboe 1998). The transporter EAAC-1 excitatory
amino acid carrier (EAAT-3), on the other hand, is primarily
expressed in neuronal spines and EAAT-4 seems to be located
in spines of particularly Purkinje cells (Danbolt 2001). The
present review therefore will deal only with the transporters
GLT-1 and GLAST.
2.3.2.1 GLT-1
Using highly specific antibodies against GLT-1, Danbolt
et al. (1992), Levy, (1993), and Rothstein et al. (1994) all have
found this glutamate transporter to be expressed only in astro-
cytic processes (see Figs. 35.1 and 35.2). However, the mRNA
for GLT-1 has been found in neurons (Danbolt 2001) but if
GLT-1 protein is expressed in neurons, it must be at a very low
level or alternatively it may be a variant that is not recognized
by the currently available antibodies (Danbolt 2001). A recent
immunocytochemical study using immunogold labeling at the
EM level and a D-aspartate specific antibody has convincingly
shown that D-aspartate is taken up into presynaptic nerve end-
ings (Furness et al. 2008) as indicated in Figure 35.1. It should
also be noted that in early development, GLT-1 appears to
be expressed in neuronal populations and also tissue culture
Table 35.2 NOMENCLATURE OF CLONED GLUTAMATE
TRANSPORTERS
TRANSPORTER SUBTYPE
Systematic EAAT-1 EAAT-2 EAAT-3 EAAT-4 EAAT-5
name
Original GLAST GLT-1 EAAC-1 EAAT-4 EAAT-5
name
preparations of neurons have been reported to express GLT-1
(Danbolt 2001). It may be of interest that in spite of the fact
that GLT-1 is the most prevalent glutamate transporter in fore-
brain (Danbolt 2001), cultured astrocytes from cerebral cortex
generally have a higher expression of GLAST compared with
that of GLT-1 (Gegelashvili et al. 1997; Skytt et al. 2010). This
likely reflects the finding that the expression of GLT-1 in cul-
tured astrocytes, and perhaps also in the brain, is dependent
on neuronal signaling (Gegelashvili et al. 1997; Schlag et al.
1998; Swanson et al. 1997) as well as other signaling molecules
(O’Shea et al. 2006; Schlag et al. 1998). It should also be noted
that the surface expression of the glutamate transporters is reg-
ulated by trafficking from the cytoplasm to the membrane and
vice versa (for references, see Robinson 2002).
2.3.2.2 GLAST
As mentioned (section 2.3.2), GLAST exhibits a higher
expression level in cerebellum than in forebrain, that is, the
opposite of that of GLT-1 but it should be emphasized that
astrocytes throughout the brain express both GLAST pro-
tein and mRNA (for references, see Danbolt 2001). Cultured
embryonic neurons seem to express GLAST in analogy to
their expression of GLT-1 (Plachez et al. 2000). It should be
noted that the highest expression level of GLAST has been
found in the cerebellar Bergmann glia (Danbolt 2001), which
also have a high density of Ca2+-permeable AMPA receptors
(Müller et al. 1992; Parpura et al. 2012).
2.3.3 Role of GLAST and GLT-1 in Termination
of the Neurotransmission Process
Because of the extremely high density of glutamate trans-
porters in the astrocytic plasma membrane (Danbolt 2001),
it is clear that termination of the interaction of glutamate
with its receptors is functionally related to these transporters
(Danbolt 2001; Robinson and Dowd 1997). It is, however,
important to note that the cycling time for the transporters
of 50 to 100 milliseconds (Wadiche et al. 1995) is too slow
to effectively terminate the transmission process which should
operate at a micro-to-millisecond scale (Tong and Jahr 1994).
The high density of the transporters does, however, allow effi-
cient binding of a large number of glutamate molecules to take
place prior to transport, thereby securing the process to occur
within the required time scale (Tong and Jahr 1994). Hence,
the concerted action of binding to and transport by the glial
transporters leads to an extremely efficient clearance of gluta-
mate from the synaptic cleft, preventing accumulation of neu-
rotoxic extracellular concentrations to occur (Choi 1988).
It may be interesting to note that because of the cotrans-
port of three sodium ions and possibly a proton and the
counter-transport of one potassium ion, which makes the
transporters highly electrogenic (Levy et al. 1998), the trans-
port process leads to a depolarization of the astrocytes which,
without a pharmacological characterization, may be indistin-
guishable from that seen after activation of ionotropic gluta-
mate receptors (Bowman and Kimelberg 1984; Kettenmann
et al. 1984). Interestingly, it was shown that in cultured hip-
pocampal astrocytes, kainate receptors as well as glutamate
A M I N O AC I D N E U R OT R A N S M I T T E R SY N T H E S I S A N D R E M O VA L
transporters mediate changes in the intracellular concentra-
tions of Na+ and protons (Rose and Ransom 1996). Because
a tight functional coupling appears to exist between the
glutamate transporters and the Na+/K+-ATPase in the astro-
cytic membrane (Bauer et al. 2012; Cholet et al. 2002; Genda
et al. 2011; Rose et al. 2009; Schousboe et al. 2011), it may be
assumed that the dissipation of the sodium gradient associated
with glutamate transport is rapidly recovered.
2.3.4 Regulation of Expression of Glutamate
Aspartate Transporter and Glutamate Transporter 1
The first indication that expression of glutamate transporters
in astrocytes may be regulated by neuronal stimuli came from
a study by Drejer et al. (1983) demonstrating that exposure of
cultured cerebellar astrocytes to a conditioned medium from
cultured cerebral cortical neurons led to a dramatic increase
in glutamate transport activity. It was subsequently shown
by several investigators that this upregulation of transport
capacity in astrocyte cultures induced either by coculturing
with neurons or by exposure to neuronal conditioned cul-
ture medium is explained by selective upregulation of the
GLT-1 transporter (Gegelashvili et al. 1997; Schlag et al.
1998; Swanson et al. 1997). Other factors such as arachidonic
acid, the redox state, the pH, and availability of zinc ions also
seem to play important roles (Levy et al. 2002). Agents that
affect the phosphorylation state have repeatedly been shown
to enhance the transport capacity of astrocytes, and it appears
that both astrocytic transporters are regulated by phosphory-
lation (Levy et al. 2002). It may be of interest that GLAST
and GLT-1 are expressed in microglial cells after cortical injury
(van Landeghem et al. 2001). It is important to note that there
is a rapid and highly regulated trafficking of the transporters
between the cytoplasm and the plasma membrane because this
can lead to rapid changes in the transport capacity in response
to changes in the cellular milieu (Robinson 2002).
2.3.5 Failure of Glial Glutamate Uptake Results
in Neurodegeneration
As mentioned previously (section 2.3.3) it has been known
for a long time that high extracellular concentrations of the
excitatory transmitters glutamate and aspartate almost cer-
tainly result in excitotoxic neuronal damage (Choi 1988).
Under physiological conditions the capacity of the glutamate
transporters expressed in astrocytes is high enough to clear
the extracellular or extrasynaptic space for glutamate and thus
prevent any neuronal damage (Choi 1988; Danbolt 2001).
During energy failure, which is associated with reduced oxy-
gen and/or glucose supply, the ATP level is reduced leading to
dramatic reduction of the activity of the Na+/K+-ATPase and
dissipation of the sodium gradient needed for maintenance of
the glutamate transporter efficiency. This results in reversal of
the transporters and an increase in the extracellular glutamate
concentration (Phillis et al. 2000; Rossi et al. 2000). It should,
however, be noted that the glycogen store in astrocytes (see
chapter 36) can sustain sodium homeostasis in the absence of
glucose and oxygen at least for a limited period of time (Rose
• 449
et al. 1998). This is in keeping with the demonstration that
glycogen metabolism and turnover is important for astrocytic
glutamate transport (Schousboe et al. 2011). Inhibition of the
glutamate transporters using a nontransportable inhibitor
such as DL-threo-beta-benzyloxyaspartate (Shimamoto et al.
1998) has been shown to effectively prevent neurodegenera-
tion in hippocampal slices induced by oxygen-glucose depriva-
tion (Bonde et al. 2003) clearly demonstrating the importance
of glutamate transporters. This latter notion is underlined by
the finding that exposure of hippocampal slices to a variety
of glutamate transporter inhibitors under physiological con-
ditions will lead to extensive neurodeneration (Bonde et al.
2003; O’Shea et al. 2002).
3 GABA
enzyme glutamate decarboxylase (GAD; Schousboe and
Waagepetersen 2008). Degradation of GABA requires GABA-
transaminase (GABA-T) to convert GABA to succinic semial-
dehyde (SSA) by transamination with the cosubstrates glutamate
and α-ketoglutarate (Schousboe and Waagepetersen 2008).
Succinic semialdehyde is subsequently oxidized by SSA dehy-
drogenase (SSADH) to succinate, a constituent of the TCA
cycle. These latter processes can take place both in neurons and
astrocytes because the enzymes involved are expressed in both
cell types (Schousboe and Waagepetersen 2008). As discussed
(sections 2.1 and 2.2), glutamate homeostasis involves an active
exchange of substrates between neurons and astrocytes. The
same is the case for GABA because its de novo synthesis requires
glutamine (see Fig. 35.4), which is generated in astrocytes and
transferred to GABAergic neurons (Schousboe and
Waagepetersen 2008).

3.1 B I O SY N T H E S I S A N D M ETA B O L I S M 3.2 M ETA B O L I S M A N D E P I L E P SY

The metabolism of GABA (Fig. 35.4) may be less complex than GABA-T is a pyridoxal phosphate dependent enzyme and
that of glutamate (see section 2.1) and its synthesis is restricted thus, GABA-T is sensitive to carbonyl trapping agents such
to GABAergic neurons expressing the GABA synthesizing as aminooxyacetic acid (AOAA), which potently inhibits the

Glucose

PYR
PDH
PC
NH4+
GLU GLN OAA
GLN TCA CIT
PAG cycle
GS suc
NH4+ SSADH
GAD GABAergic α–KG
neuron SSA
CO2 GLU GABA-T

GABA
GABA
GABA
Na+

Astrocyte
GABA
GAT1
Na+
GAT1

GABA
GAT3
GABA GABA Na+

Figure 35.4 Neurotransmitter GABA is to a large extent taken up by the neuronal GAT1 and to a minor extent by glial GAT1 and GAT3, subsequent to
postsynaptic receptor interaction. GABA taken up by neurons is primarily reused as neurotransmitter whereas GABA entering astrocytes is metabolized
via succinic semialdehyde (SSA) to succinate by the concerted action of GABA-transaminase (GABA-T) and SSA dehydrogenase (SSADH). The
astrocytic part of the synapse provides a net synthesis of glutamine (gln) via the concerted action of pyruvate carboxylase (PC) and pyruvate dehydro-
genase (PDH) generating OAA and acetyl-CoA, the combination of which leads to synthesis of CIT. The presynaptic neuron shows the biosynthetic
machinery for GABA comprising conversion of glutamine (gln) to GABA via glutamate (glu) catalyzed by the two enzymes phosphate activated
glutaminase (PAG) and glutamate decarboxylase (GAD), respectively. CIT, Citrate; GS, glutamine synthetase; OAA, oxaloacetate.

450 • FUNCTIONS OF NEUROGLIAL CELLS

enzyme (Schousboe et al. 1974). In addition, AOAA increases
GABA levels and displays anticonvulsant activity in a variety
of seizure models (Sarup et al. 2003), but since it is nonselec-
tive it is not a valuable tool in this regard. To circumvent this
problem the GABA analog, γ-vinyl GABA was synthesized
(Lippert et al. 1977). This compound is a GABA-T substrate
that displays irreversible covalent binding to the active site of
GABA-T upon transamination and thus acts as a suicide, cat-
alytic site-directed inhibitor (Lippert et al. 1977). This action
explains the remarkable specificity displayed by γ-vinyl GABA.
Because of its high selectivity toward GABA-T it was devel-
oped as a clinically active antiepileptic drug named vigabatrin
(Gram 1990). It currently represents the only antiepileptic
agent whose mechanism of action specifically relies on inhibi-
tion of GABA-T. It may not be clear to what extent inhibition
of the astroglial GABA-T may be involved in the anticonvul-
sant activity of vigabatrin, but from a study of the potency of
vigabatrin as a GABA-T inhibitor in neurons and astrocytes
it appears that the action on the neuronal enzyme is the more
important in this regard (Larsson et al. 1986). In GABAergic
neurons, glutamine released from astrocytes appears to be an
important precursor for GABA (Walls et al. 2011 and refer-
ences therein) via formation of glutamate that is subsequently
decarboxylated. It should be noted that recently a novel spe-
cific inhibitor of GABA-T has been developed and because
this compound appears to have fewer side effects than vigaba-
trin it could serve as a model compound for development of a
new antiepileptic drug (Pan et al. 2012). For a further discus-
sion of this topic, please see chapter 70.
3.3 T R A NS P O RT E R S , T H E I R L O C A L I Z AT I O N
A N D F U N C T I O NA L A N D P H A R M AC O L O G I C A L
C H A R AC T E R I Z AT I O N
Termination of GABAergic neurotransmission is facilitated
via high affinity GABA transporters (GATs) embedded in the
plasma membrane of both neurons and astrocytes (Iversen and
Neal 1968). However, this discovery was preceded by Elliott
and van Gelder (1958) who were the first to show that GABA
in the incubation media accumulated in slices of cerebral corti-
ces. Much attention has been devoted to describe the neuronal
and glial transport systems and it was shown that most of the
exogenous [3H]GABA applied to slices and synaptosomes of
rat cerebral cortices accumulated into nerve terminals; using
[3H]β-alanine it was demonstrated that this is a substrate for
the glial GAT, but not for the neuronal GAT (Schousboe 1981,
2000). Investigations using glial preparations have demon-
strated that uptake of GABA into these cell types could account
for a substantial portion of the vesicular released GABA esti-
mated to approximately 20% (Schousboe 2000). As described
above the concerted action of GABA-T and SSADH degrades
GABA to succinate in both neurons and astrocytes. Only a
minor fraction of GABA degradation takes place in neurons
due to recycling of neurotransmitter, whereas all GABA taken
up by astrocytes is degraded (see Fig. 35.4). Because of a signif-
icant loss of neurotransmitter via GABA uptake in astrocytes
and subsequent degradation, along with the discovery that
the neuronal GABA uptake inhibitor diaminobutyric acid is
A M I N O AC I D N E U R OT R A N S M I T T E R SY N T H E S I S A N D R E M O VA L
acting as a proconvulsant, it was hypothesized that selective
inhibitors of the glial GAT would be promising agents for sei-
zure management (Schousboe 2000).
3.4 C L O N I N G O F T R A NS P O RT E R S
Radian et al. (1986) isolated from rat brain what was believed
to be the neuronal GAT. Subsequently, 4 years later this trans-
porter was cloned revealing a 67 kDa protein consisting of
599 amino acids with an apparent Km for GABA of 7μM
(Guastella et al. 1990) compared with 32 and 8 μM, which
is the apparent Km for GABA for the astrocytic and neuronal
GAT, respectively (Schousboe 2000). The pharmacological
studies conducted confirmed this to be comparable to the
neuronal subtype and it was designated GAT-1 (Guastella
et al. 1990). Following this initial discovery a total of four
GATs were cloned in several species which unfortunately has
resulted in a rather confusing nomenclature. The nomencla-
ture in rat, canine, and human is identical, namely GAT-1,
BGT-1, GAT-2, and GAT-3, whereas the nomenclature in
mouse is mGAT1, mGAT2, mGAT3, and mGAT4, respec-
tively. BGT-1 (rat)/mGAT2 (mouse) is the betaine-GABA
transporter that transports GABA with an apparent Km of 79
μM and betaine with an apparent Km of 398 μM and encodes
a protein of 614 amino acids. GAT-2 (rat)/mGAT3 (mouse)
encodes a protein of 602 amino acids with an apparent Km for
GABA of 18 μM and an apparent Km for β-alanine of 28 μM.
Last, GAT-3 (rat)/GAT4 (mouse) encodes a protein of 627
amino acids that also transports GABA and β-alanine with an
apparent Km of 0.7 and 99 μM, respectively (Liu et al. 1993).
To avoid confusion, the nomenclature GAT1, BGT1, GAT2,
and GAT3 as proposed by the HUGO gene nomenclature
committee (Table 35.3) will be used hence forth (please note
that it corresponds to the rat nomenclature without hyphen.
GABA transport by GAT2 and GAT3 is strongly inhibited
by β-alanine and certain taurine analogs such as hypotaurine,
suggesting that these two transporters most likely resemble
the astroglial GAT. On the other hand, BGT-1 seems not to
resemble either the neuronal or astroglial GAT but it is, how-
ever, the only GAT inhibited by betaine (Liu et al. 1993).
3.5 S T RU C T U R E A N D F U N C T I O N
O F T R A NS P O RT E R S
The GABA transporters belong to the Solute Carrier 6 (SLC6)
superfamily, which is also comprised of the monoamine
Table 35.3 NOMENCLATURE OF CLONED GABA
TRANSPORTERS
TRANSPORTER SUBTYPE
Systematic GAT1 BGT1 GAT2 GAT3
name
Mouse mGAT1 mGAT2 mGAT3 mGAT4
nomenclature
Rat/human GAT-1 BGT-1 GAT-2 GAT-3
nomenclature
• 451
transporters for serotonin, dopamine, and noradrenaline and
transporters for amino acids like proline, glycine, and tau-
rine (Kristensen et al. 2011; Madsen et al. 2007). The energy
demanding uptake of these transmitters is coupled to an
inward cotransport of sodium ions which fuels the process.
The four GABA transporters show an absolute requirement
for two sodium and one chloride ion per transported GABA
molecule (Kristensen et al. 2011). The sodium driving force is
capable of generating a concentration gradient in the order of
magnitude of 105 between the intracellular and extracellular
GABA (Schousboe 1981). The GATs share similar structural
features with the other transporters of the SLC6 gene fam-
ily in that they have twelve transmembrane spanning domains
(TMD) with a large extracellular loop between TMD3 and
4 containing three glycosylation sites and intracellular facing
carboxy and amino terminals (Kristensen et al. 2011). Recently,
a crystal structure of a bacterial homologue of a sodium- and
chloride-dependent neurotransmitter transporter has brought
about critical knowledge regarding the structure and function
of these transporters (Yamashita et al. 2005). As seen with the
pharmacological profile of the GATs, GAT1 shares the least
amount ~50% of amino acid sequence homology with the
other GATs whereas BGT1, GAT2, and GAT3 have an iden-
tity of 65% to 70% (Miller et al. 2002).
3.6 R EGU L AT I O N O F T R A N S P O RT E R AC T I VIT Y
The regulation of GAT surface expression has been shown
to be influenced by PKC, tyrosine kinase/phosphatase activ-
ity and the presence of extracellular substrate all of which
can exert changes in the rates of endocytosis and exocytosis
and/or the number of readily available transporters for recy-
cling (Hu and Quick 2008; Madsen et al. 2007). Activation
of PKC-dependent phosphorylation decreases the function
of GAT1 by an increase in the rate of endocytosis and direct
tyrosine phosphorylation of GAT1 decreases the rate of endo-
cytosis whether it is activated by brain derived neurotrophic
factor or short exposures to carrier substrates (Hu and Quick
2008; Madsen et al. 2007; Vaz et al. 2011). Brain-derived neu-
rotrophic factor affects the function of GAT1 in both neurons
and astrocytes but not the function of GAT3 (Vaz et al. 2011).
Prolonged exposure for hours rather than minutes to higher
concentrations of GAT substrates causes an increase in the
rate of internalization and a decrease in the recycling pool of
GAT1 mediated via a GABA receptor effect through serine
phosphorylation (Hu and Quick 2008).
3.7 C E L LU L A R L O C A L I Z AT I O N
O F T R A N S P O RT E R S
The GABA transporters GAT1 and GAT3 which are respon-
sible for the majority of GABA uptake in the brain are also
restricted to this organ, whereas BGT1 and GAT2 are found
in multiple other organs like liver and kidney. Expression of
GAT1 is along GABAergic pathways primarily on presynaptic
GABAergic neurons but also on distal astrocytic processes (see
Fig. 35.4) adjacent to GABAergic synapse formations (Madsen
452 • FUNCTIONS OF NEUROGLIAL CELLS
et al. 2007). Compared with GAT1, the expression of GAT3
is restricted to distal astrocytic processes (see Fig. 35.4) that
are in direct contact with GABAergic synapses and perhaps
to a much lesser degree on neuronal elements (Madsen et al.
2007). The highest degree of expression of GAT2 is in the
neonatal brain suggesting a role in fetal development. In the
adult brain GAT2 is strongly expressed in the leptomeninges
and ependyma. However, GAT2 is only weakly expressed on
cortical neurons close to both symmetric and asymmetric syn-
apses but away from areas with synaptic specialization, and on
distal and proximal astrocytic processes and cell body (Madsen
et al. 2007). BGT1 has also been found in the leptomeninges
(Takanaga et al. 2001) and on pyramidal neurons in the cere-
bral cortex and in the CA field of the hippocampus. It local-
izes to small diameter dendrites or dendritic spines making
asymmetric synapses but to an extrasynaptic region away from
synaptic specialization (Zhu and Ong 2004). It should be
noted, however, that a recent study by Lehre et al. (2011) has
demonstrated a low expression level of BGT1 in the brain.
3.8 P H A R M AC O L O GY O F T R A NS P O RT E R S
The development of the GAT1 selective inhibitor tiagabine by
Novo Nordisk underlines that inhibitors of GABA transporters
can be developed as anticonvulsants (Nielsen et al. 1991). Many
compounds were developed inhibiting GABA transporters;
however, only a few preferentially inhibited the glial GAT. In
a series of compounds based on exo-THPO the most selective
compound to this date is N-methyl-exo-THPO, displaying a
10-fold selectivity toward the glial carrier compared to the neu-
ronal GAT (Madsen et al. 2007). The knowledge obtained from
the cloning of the four GATs and their localization suggests
that GAT1 should be responsible for the majority of neuronal
reuptake, whereas GAT3 should primarily be responsible for
the uptake into astrocytes. Therefore, the finding that the glial
preferring inhibitors selectively inhibited GAT1 was surpris-
ing. However, as indicated by Vaz et al. (2011) as much as 40%
of GABA uptake in astrocyte cultures is facilitated by GAT1,
offering an explanation to the GAT1 and glial selective inhibi-
tors. A question remains as to what mechanism is responsible
for the glial selectivity when considering that the majority of
GAT1 is expressed on neurons? To obtain glial selective GAT
inhibitors rational drug design approaches also suggest target-
ing the GAT3 subtype. Unfortunately, such an endeavor has
not yet been successful. The compound SNAP-5114 is almost
equipotent at GAT2, GAT3, and BGT1 (Dalby 2000), which
makes this drug less optimal to investigate the importance of
the GAT3 subtype in relation to seizure management.
4 G LYC I N E A N D D -S E R I N E A S
TR ANSMITTER S AND THE ROLE
OF ASTROCY TES
Glycine serves as the major inhibitory transmitter in the spi-
nal cord (Curtis and Johnston 1974) and in keeping with this,
a high affinity transport system was described in spinal cord
( Johnston and Iversen 1971). It is also clear that glycine trans-
port is associated with astrocytes and the cloning and struc-
tural characterization of the high affinity glycine transporters
have identified specific transporters in neurons and astrocytes
(for references, see Eulenburg and Gomeza 2010; Zafra and
Gimenez 2008). It appears that the glial glycine transporter 1
(GlyT1) is of vital importance because GlyT1 knockout mice
die shortly after birth (Gomeza et al. 2003; Eulenburg et al.
2010). This glycine transporter also appears to be an interesting
pharmacological target because it plays a role in neuropsychi-
atric disorders and dementia (Gether et al. 2006; Kristensen
et al. 2011). It is of particular interest that glycine in addition
to acting as an inhibitory neurotransmitter in its own right
functions as a coagonist at NMDA receptors (Eulenburg and
Gomeza 2010) and the glycine transporters therefore have a
functional role in the regulation of the activity of excitatory
neurotransmission (Eulenburg and Gomeza 2010; Zafra and
Gimenez 2008).
It is of considerable interest that the D-amino acid, D-serine
has been identified as a coagonist at NMDA receptors (for
references, see Oliet and Mothet 2009; Pollegioni and Sacchi
2010; Wolosker 2007; Wolosker et al. 2008;). Originally,
D-serine was believed to be primarily if not exclusively synthe-
sized in astroglia by the enzyme serine racemase (Pollegioni and
Sacchi 2010), but there is considerable evidence that neurons
also play a role in this context (Wolosker 2007) and the origi-
nal term characterizing D-serine as a gliotransmitter probably
needs to be modified (Wolosker 2007). Nevertheless, there is
no question that astrocytes play an important role in regulat-
ing NMDA receptor activity via the two coagonists glycine
and D-serine, albeit the release mechanism for the two amino
acids from astrocytes may not be fully understood and char-
acterized (Hamilton and Attwell 2010). It may be of interest
to note that although glycine can be effectively removed from
the synaptic cleft by the high affinity transporters, no such sys-
tems have been identified for D-serine (Wolosker et al. 2008).
For this reason, it is believed that D-serine more efficiently
compared to glycine can reach the synaptic receptors be evad-
ing the transport systems (Wolosker et al. 2008).
Further discussions of gliotransmitters may be found in
chapter 17.
5 S U M M A RY A N D P E R S P E C T I VE S
Because neurons are incapable of performing a de novo synthe-
sis of glutamate and GABA from glucose because of the lack of
pyruvate carboxylase (PC), a symbiotic relationship between
neurons and astrocytes exists allowing transfer of glutamine,
the main precursor for these amino acids, from astrocytes to
neurons. Astrocytes, which express PC as well as glutamine
synthetase, that is, the key enzymes allowing de novo synthesis
of glutamine from glucose, are therefore of paramount impor-
tance for the maintenance of excitatory and inhibitory neu-
rotransmission. Regulatory mechanisms related to this will
be in focus for future studies of the metabolism of glutamate
and GABA. It should be kept in mind that the high affinity
A M I N O AC I D N E U R OT R A N S M I T T E R SY N T H E S I S A N D R E M O VA L
transporters for glutamate and GABA are intimately associ-
ated with the metabolic processes and the exchange between
neurons and astrocytes of the transmitters and their precursor.
As the transport processes are also coupled to energy metabo-
lism due to the dependency of ATP supply it will be important
to establish a better understanding of the mechanisms involved
in regulation of this coupling between transport and energy
metabolism. In this context it appears that astrocytic glycogen
metabolism is particularly interesting (Obel et al. 2012).
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 36.
GLYCOGEN AND ENERGY METAB OLISM
Angus Brown

A B B R E VI AT I O N S in both plants and higher organisms developing mechanisms

of storing glucose by polymerization of this ubiquitous car-
aCSF artificial cerebrospinal fluid bohydrate into polysaccharides. In plants this polysaccharide
AMP adenosine monophosphate is amylose and amylopectin, whereas in higher animals it is
ATP adenosine triphosphate the more complex molecule, glycogen (Voet and Voet 2011).
CAP compound action potential Glycogen evolved to act as protection against the unreliable
cAMP cyclic adenosine monophosphate food sources encountered by our hunter-gatherer ancestors.
CNS central nervous system Fat deposits serve a similar purpose in storing excess fat in sub-
DAB 1,4-dideoxy-1,4-imino-D-arabinitol cutaneous depots, whereas glycogen is stored primarily in the
EPSP excitatory postsynaptic potential liver and skeletal muscle.
Hz Hertz The glycogen stores in the body are far exceeded by the
LDH lactate dehydrogenase fat stores (rough estimates are that fat stores provide energy
MCT monocarboxylate transporter for ~35 days during starvation, whereas total body glyco-
mM milli moles per liter gen provides <1 days worth of energy). Glycogen does not
MON mouse optic nerve contain as much energy as the more reduced fat; therefore,
mRNA messenger ribonucleic acid why does the body store glycogen at all? The answer is that:
ms millisecond (1) glycogen is much more rapidly metabolized than fat,
NMR nuclear magnetic resonance and can thus provide energy substrate at very short notice;
OGI oxygen uptake ratio (2) glycogen can be metabolized in the absence of oxygen,
Pa active phosphorylase a a key consideration for exercising muscle, whereas fat can-
Pb inactive phosphorylase b not; and (3) animals cannot convert fat to glucose; thus, for
PNS peripheral nervous system glucose-dependent organs such as the brain, fat cannot pro-
PP1 protein phosphatase 1 vide essential glucose (Voet and Voet 2011). The dependence
TCA tricarboxylic acid of the brain on glucose provides an explanation of the advan-
T2D type 2 diabetes tages of glycogen as an energy store. As the body’s most vora-
UDP uridine diphosphate cious glucose consumer, per unit weight, the brain takes up
UTP uridine triphosphate 20% of available glucose, although it represents only 2% of
ZDF Zucker diabetic fatty body weight (McKenna et al. 2006).
ZL Zucker lean The brain can receive glucose from three main sources:
ZOb Zucker obese (1) blood-borne glucose provided by dietary intake of carbo-
hydrates, (2) gluconeogenesis, whereby nonglucose energy
substrates are converted to glucose in the liver and kidney, and
(3) metabolism of glycogen. Given our ancestors’ unpredict-
1 INTRODUCTION able supply of food, and the slow rate of substrate conversion
associated with gluconeogenesis in response to falling blood
The main energy support of the brain is glucose, which must glucose levels, brain glycogen is the ideal energy reserve to
be delivered continuously and in excess of demand for the provide for a sudden shortfall in glucose. A curious feature
brain to function normally. Interruption of blood-borne glu- of brain glycogen is its exclusive localization to astrocytes in
cose and oxygen results in brain dysfunction within seconds, adult brain (Cataldo and Broadwell 1986). This feature, and
highlighting the extreme dependency of the mammalian cen- the astrocyte’s inability to enzymatically cleave phosphate
tral nervous system (CNS) on these energy substrates (Rossen from glucose residues, could well have contributed to the
et al. 1943). Drastic reduction of glucose alone, in the con- early prejudice that brain glycogen was not important. The
tinued presence of oxygen, induces neural dysfunction more importance of astrocyte glycogen and the unique fashion of
slowly, and the presence of brain glycogen is a major reason its metabolism in support of brain energy needs are the sub-
for this, as we shall see. Evolutionary pressures have resulted ject of this chapter.

457
 2 G LYC O G E N B I O C H E M I S T RY
2.1 G LYC O G E N A S A C A R B O H Y D R AT E
S TO R AG E D E P OT
Glycogen is formed from molecules of D-glucose, a hexose,
and is a branched homopolysaccharide. D-glucose is a pyranose
closed structure with the six carbon atoms arranged in a hex-
agonal formation (Berg et al. 2006). The main bonds formed
between glucose molecules in glycogen are D(1→4) glycosidic
bonds, which result in a linear structure. In plants, the primary
storage form of glucose, amylose, is formed entirely of glucose
molecules connected by D(1→4) bonds. This results in a linear
structure with the disadvantages of being poorly water-soluble
and having only a limited number of locations onto which glu-
cose molecules can be attached. Glycogen has D(1→6) branch
points that defeat these problems. Glycogen molecules can be
up to 108 Daltons and contain 120,000 glucose units (Voet
and Voet 2011), their structure a discrete self-contained gran-
ule, between 10 and 40 nm in diameter, located in the cyto-
plasm. The enzymes responsible for metabolism and synthesis
of glycogen are also present in the granule (Voet and Voet
2011; Wender et al. 2000) (Fig. 36.1).
2.1.1 Synthesis and Degradation
Glycogen synthesis is a three step process, requiring:
(1) formation of UDP-glucose via the combination of
glucose-1-phosphate and uridine triphosphate (UTP); (2)
transfer of glucosyl residues from UDP-glucose to the gly-
cogen molecule forming D(1→4) glycosidic bonds; and (3)
Figure 36.1 Glycogen Granules Are Present in Astrocytes in Mouse Optic
Nerve. From Wender et al., 2000.
458 • FUNCTIONS OF NEUROGLIAL CELLS
formation of branch points via D(1→6) glycosidic bonds
(Voet and Voet 2011). Glycogen synthesis is initiated by an
existing glycogen molecule, or by glycogenin, a protein that
can accept glucose molecules, but only after tyrosine binds to
the glycogenin and activates the site of glucose attachment.
The process of glucose attachment liberates 1 molecule of H2O
per glucose molecule, and thus the MW of each glucosyl resi-
due is 162 compared with 180 for glucose. After attachment of
a few glucosyl residues to glycogenin, the structure can act as a
primer that glycogen synthase can act on. A first step in glyco-
gen formation is the combination of glucose-1-phosphate with
uridine triphosphate (UTP), to form UDP-glucose. This mol-
ecule is the substrate for glycogen synthase, which can only
act on existing glycogen molecules. Increasing the size of the
glycogen molecule requires the movement of glucose from
the UDP-glucose molecule to the growing chain, forming an
D(1→4) glycosidic bond between the hydroxyl group on car-
bon 4 of the terminal glucosyl residue, and the carbon 1 of
the new glucosyl residue to be added. The UDP released in
this action is converted back to UTP, an adenosine triphos-
phate (ATP)–requiring step. Repetition of this reaction adds
increasing numbers of glucosyl residues to the glycogen mol-
ecule. If this were the only reaction that occurred, then the
linear molecule amylose would be formed. However, in mam-
mals at about every eighth to tenth glucosyl residue a D(1→6)
bond is inserted that results in branching of the molecule.
The branches are formed by the action of the enzyme amylo-
(1,4→1,6)-transglycolase, which attaches a branch of up to a
dozen glucosyl residues to the end of the chain by breaking
an D(1→4) bond. Glycogen synthase then adds glucosyl res-
idues to each of the ends of the chain. A key action of this
branching process is to create multiple ends to which, or from
which, glucosyl residues can be attached or removed, respec-
tively. This makes glycogen a very dynamic molecule, which
in time of need can release multiple glucosyl residues simulta-
neously, or to which multiple glucosyl residues can be added
simultaneously.
Glycogen metabolism requires the coordinated action of
three enzymes to complete the following steps: (1) release of
glucose-1-phosphate from glycogen; (2) restructuring of the
glycogen molecule so it is available for further metabolism; and
(3) conversion of glucose-6-phosphate to glucose-1-phosphate
(Berg et al. 2006). Glycogen is degraded, or metabolized,
by the enzyme glycogen phosphorylase. A key aspect of
glycogen metabolism is that degradation is not simply a rever-
sal of synthesis using the same enzymes, but rather is a com-
pletely separate biochemical pathway using a separate set of
enzymes. Thus, degradation and synthesis can occur simul-
taneously endowing glycogen with an increased flexibility
depending on prevailing conditions. The product of glycogen
breakdown in the periphery is either glucose-1-phosphate,
the result of breakdown of D(1→4) bonds, or free glucose,
the result of breakdown of D(1→6) bonds. In the brain each of
these compounds will form glucose-6-phosphate via the actions
of phosphoglucomutase or hexokinase, respectively. Given that
astrocytes do not possess the enzyme glucose-6-phosphatase
(Wiesinger et al. 1997) glucose cannot be formed, a simi-
lar situation occurs in skeletal muscle (Voet and Voet 2011),
but in the liver free glucose is formed as a result of glyco- (a) Glycogen
gen metabolism, for subsequent liberation into the systemic Glycogen phosphorylase
circulation. In astrocytes the glucose-6-phosphate is Glycogen(n=1)
glycolytically metabolized ultimately to lactate (see later) Glucose-1-phosphate
(Dringen et al. 1993). Glycogen phosphorylase breaks the Phosphoglucomutase
D(1→4) bonds until only four glucosyl residues extend past
the nearest D(1→6) bond. This short four-residue sec- Glucose-6-phosphate

tion is called limit dextran and is the substrate for

oligo-D(1→4)→D(1→4) glucan transferase, which trans-
fers the last three residues to the end of the chain, where
glycogen phosphorylase removes the individual residues as
glucose-1-phosphate. The single residue remaining at the
branch point is removed by amylo-D(1→6)-glucosidase
and released as free glucose. The relative proportion of
glucose-1-phosphate to glucose released as a result of glyco-
gen metabolism depends on the number of glucosyl residues
between each branch point, but a widely accepted value is
90%. Details of glycogen biochemistry are covered in detail in
standard biochemistry textbooks, of which the following are
recommended (Berg et al. 2006; Garrett and Grisham 1999;
Voet and Voet 2011).
Once glucose enters cells it is phosphorylated by hexoki-
nase to glucose-6-phosphate, a step that requires ATP. As
previously described, glycogen breakdown ultimately releases
glucose-6-phosphate; thus, it is natural to ask what is the cost
of storing excess glucose as glycogen? Only 1 ATP is required
because, depending on the frequency of branch points, about
90% of glycogen is released as glucose-1-phosphate, which is
converted to glucose-6-phosphate by phosphoglucomutase
in a reaction that does not require ATP. Conversion of UDP
to UTP requires ATP, thus oxidation of glucose-6-phosphate
produces 31 molecules of ATP, whereas storage as glycogen
only requires 1 ATP per molecule of glucose-6-phosphate, an
efficiency of about 97% (Fig. 36.2).
2.1.2 RO L E O F G LYC O G E N I N L I VE R A N D
S K E L ETA L MUS C L E
The two main depots of glycogen stores in the body are the
liver and skeletal muscle. Glycogen is present at higher con-
centrations in the liver compared with skeletal muscle (10%
vs 1%–2%), but given the greater mass of skeletal muscle in
the body skeletal muscle contains about 400 g of glycogen and
the liver 100 g (Champe and Harvey 2008). Limits on the
amount of glycogen stored are set such that during extended
periods of plenty glycogen content remains fixed within this
range. However, pathology resulting from the family of gly-
cogen storage diseases can result in excess glycogen stored in
both liver and muscle (Voet and Voet 2011). The role of liver
glycogen is to buffer fluctuations in blood glucose levels such
that during periods of excess, glucose is stored as glycogen
in the liver, but during periods of falling glucose (systemic
blood glucose < 5 mM) glycogen metabolism is mobilized to
release glucose into the systemic circulation to maintain eug-
lycemia (Voet and Voet 2011). Glucose inhibits pancreatic D
cells from releasing glucagon; thus, when blood glucose lev-
els fall, this inhibition is released. The glucagon acts on liver
cells, resulting in increased cyclic adenosine monophosphate
Pyruvate
Muscle Liver
brain Glucose-6-phosphatase
Lactate H2O+CO2 Glucose
(b)
Adrenaline Glucagon Insulin
ATP cAMP
PKAb PKAa GSK GSK
PKb PKa
G-1-P
GPb GPa GSa GSb
Glycogen
PP1
Figure 36.2 Metabolism of Glycogen. A. Pathways of glycogen metabo-
lism highlighting the tissue-dependent fate of glycogen-derived metabo-
lites. Key enzymes are in blue. Adapted from Figure 21.3 (Berg et al.
2006). B. Regulation of glycogen metabolism indicating the reciprocal
modulation of glycogen phosphorylase and glycogen synthase via (de)
phosphorylation of key enzymes. Glycogen metabolism is initiated by
adrenaline or glucagon increasing cAMP levels via binding to metabotro-
pic receptors. cAMP phosphorylates protein kinase A (PKA), which
in turn phosphorylates phosphorylase kinase (PK) to the active a form.
PKa phosphorylates glycogen phosphorylase (GP) to its active a form,
and additionally phosphorylates glycogen synthase (GS) to its inactive
phosphorylated b form. Phosphorylated GPa promotes glycogen break-
down to glucose-1-phosphate (G-1-P). Glycogen synthesis is promoted by
insulin via the action of insulin receptor substrates converting glycogen
synthase kinase (GSK) from its dephosphorylated to its inactive phos-
phorylated state. Dephosphorylated GSK maintains GS in its phosphory-
lated inactive state; thus, insulin acts to release GS from this resting level
of inhibition and promotes incorporation of glucosyl residues into the
glycogen molecule. Protein phosphatase 1 (PP1) promotes conversion of
GS to its active a form, whereas dephosphorylating PK inhibits glycogen
metabolism. Glycogen phosphorylase is also allosterically modulated by
AMP, ATP, glucose-6-phosphate, and glucose (not shown).
(cAMP) in the hepatocytes, causing glycogen breakdown,
which leads to elevated glucose-6-phosphate, the substrate
for glucose-6-phosphatase and ultimately released as glucose.
In periods of excess glucose insulin is released, which acts
on liver cells to trans-locate the Glut-4 transporter from the
cytosol to the cell membrane to facilitate influx of glucose

G LYC O G E N A N D E N E R GY M ETA B O L I S M • 459

and subsequent storage as glycogen (Voet and Voet 2011). In
the liver, glucose and glucose-6-phosphate act to inhibit the
action of glycogen phosphorylase.
In muscle, glycogen content is controlled by insulin and
adrenaline. Adrenaline acts to promote glycogen breakdown
by activating a cAMP-dependent cascade involving phospho-
rylation, thus resulting in inhibition of glycogen synthesis
but promoting glycogen metabolism. Insulin acts to stimulate
glycogen synthesis via action on protein phosphatase 1 (PP1).
In muscle, glycogen is metabolized to glucose-6-phosphate,
which is subsequently glycolytically metabolized to lactate
because muscle cells lack glucose-6-phosphatase. In liver gly-
cogen phosphorylase is sensitive to glucose, which deacti-
vates the enzyme. There is 90% homology between liver and
skeletal muscle phosphorylase, with a key difference being
that liver glycogen phosphorylase is not sensitive to adenos-
ine monophosphate (AMP), because AMP does not exhibit
the large concentration fluctuations in liver that occur in
skeletal muscle.
An emerging aspect of muscle glycogen metabolism
is that the excess lactate liberated during this process, which was
previously considered to be a waste product and converted by
gluconeogenesis in the liver or kidneys, now may have a more
interesting role. Recent human in vivo experiments showed
that during severe exercise the systemic concentrations of lac-
tate can rise tenfold, from 1 to 10 mM, whereas blood glucose
levels fall to hypoglycemic levels of less than 4 mM. This blood
glucose concentration would normally result in symptoms of
hypoglycemia, but this does not occur. Evidence suggests that
under these circumstances the brain takes up and metabolizes
systemic lactate, sparing glucose for muscle metabolism. This
metabolic cooperation suggests that the brain may be more
versatile in its use of energy substrates, and the muscle may
be more altruistic in providing usable energy substrate than
previously thought.
2.1.3 I N C R E A S E D AWA R E N E S S O F G LYC O G E N
I N T H E C E N T R A L N E RVO US SYS T E M :
T H E G LYC O G E N S H U N T
An unexplained phenomenon in muscle physiology is that
under conditions of increased tissue energy demand the oxy-
gen usage does not match the glucose uptake; thus, the glu-
cose uptake:oxygen uptake ratio (OGI) falls below the value
of 6 expected for total oxidation of glucose to CO2 and H2O
(Shulman and Rothman 2001). In addition to the OGI falling
as low as 3, excess lactate is produced. This was conventionally
explained as insufficient oxygen availability in muscle during
exercise, resulting in incomplete oxidative metabolism of glu-
cose with excess lactate produced as the waste product. The
“glycogen shunt” model proposed by Shulman and Rothman
seeks to explain the experimental contradictions obtained in
muscle by suggesting a key role for glycogen during muscle
contraction (Shulman and Rothman 2001). In this scenario
glycogen is required to provide energy substrate very rapidly
(on the order of milliseconds) to fuel muscle contraction. The
first phase of muscle contraction is fueled by phosphocrea-
tine breakdown to supply ATP, followed by a second phase
460 • FUNCTIONS OF NEUROGLIAL CELLS
in which glycogen is metabolized to provide ATP to sustain
muscle contraction. In the third phase, the intercontraction
phase, glycogen is resynthesized. Stoichiometric calculations
(Shulman and Rothman 2001) indicate that metabolism
of glucose to fuel muscle contraction is more efficient than
the glycogen shunt, producing 2 molecules of ATP versus
1. However, it is the time scale that is important, with the
very rapid enzyme glycogen phosphorylase producing ATP
more rapidly than glucose metabolism, with glycogen resyn-
thesized between periods of muscle contraction (Shulman
and Rothman 2001). The glycogen shunt also has been pro-
posed for metabolism in the brain. The resting OGI in the
brain is about 5.5, indicative of incomplete glucose oxidation
(Fox and Raichle 1986). Sensory stimulation of various brain
areas leads to an increase in glucose metabolism not matched
but oxygen uptake (Fox and Raichle 1986), with excess lac-
tate produced (Prichard et al. 1991). These results, as in mus-
cle, were explained as glycolytic metabolism of glucose with
lactate produced as a waste product. These data suggest that
activated brain requires little energy above the basal needs of
the tissue, which is in disagreement with data showing the
majority of energy in the nonstimulated state supports func-
tional processes (Shulman et al. 2001). The glycogen shunt
proposes that glycogen, as in muscle, acts to provide energy
substrate to fuel rapidly firing actions potentials, that is, in
the millisecond timescale, and that glycogen is replenished
during the interspike interval (Shulman et al. 2001). The
glycogen-derived energy substrate is lactate, which is shut-
tled out of the astrocyte to the neuron where it is oxidatively
metabolized. However, lactate in excess of requirements is
produced, and is recorded as an increase in the extracellular
lactate concentration. Based on the stoichiometric analysis
(Shulman et al. 2001) the net ATP produced relative to the
proportion of glucose metabolized via the glycogen shunt is
illustrated in Figure 36.3 (Shulman et al. 2001), indicating
that in terms of ATP production, efficiency decreases as the
glycogen shunt contribution increases. However, the same
amount of lactate is produced irrespective of its source. This
“glycogen shunt” bears many similarities to the astrocyte
neuron lactate shuttle hypothesis proposed in 1994 (Pellerin
and Magistretti 1994), in that astrocyte production of lac-
tate fuels neural elements in response to increase neuronal
activity.
3 G LYC O G E N I N T H E C E N T R A L
N E RVO U S SYS T E M
3.1 A S T RO C Y T I C L O C AT I O N O F
G LYC O G E N I N A D U LT B R A I N
It has been known for many years that the brain does con-
tain glycogen (Nelson et al. 1968). However, it is present in
such low quantities relative to liver and skeletal muscle glyco-
gen, that a role as an energy reserve has been dismissed. This
is primarily owing to calculations demonstrating that in the
absence of glucose brain glycogen could only support func-
tion for several minutes (Dienel 2009). Recent interest has
been rekindled in brain glycogen for reasons described in this
 (a)
2 of glycogen in distinct brain regions in commonly used rodent
ATP produced
1 with both age and disease state (see later). It has been found
0
0 0.5 1
Fraction to glycogen
(b)
2
Lactate produced
1
0 sis, glycogen phosphorylase, and glycogen synthase, respec-
0 0.5 1 tively, given the absence of a reliable antibody for glycogen.
Fraction to glycogen
Figure 36.3 The ATP and Lactate Production via the Brain Glycogen
Shunt Is Dependent of the Proportion of Glucose Metabolized via Glycogen
(Fraction to Glycogen) A. As the net fraction of glucose metabolized
via the glycogen shunt (x) increases the net ATP production (solid line)
decreases. Net ATP production from glycolysis (dashed line) is 2(1-x) and
via the glycogen shunt (dotted line) is x. B. The total lactate produced
(solid line) is unaffected by glucose flux via the glycogen shunt as lactate
production via glycolysis is 2(1-x) and via the glycogen shunt (dotted line)
is 2x. Data adapted from Shulman et al. 2001.
chapter. It is generally accepted that glycogen is present exclu-
sively in astrocytes in the adult brain (Cataldo and Broadwell
1986), but this must be qualified. It is certainly true that gly-
cogen is expressed in neurons in embryologic tissue, but this
expression decreases with maturity. Glycogen is expressed
in some neurons in the brainstem, ependyma, and choroid
plexus cells (Bloom and Fawcett 1968), and our recent experi-
ence (see later) suggests that it may well be expressed in other,
as yet unidentified cell types. A key area worthy of investiga-
tion is the hypothalamus, given its role in energy regulation.
It is interesting to note that glycogen is expressed in tanycytes
in Siberian hamster (Nilaweera et al. 2011), but its expres-
sion varies depending on the duration of photoperiod the
hamsters were exposed to, suggesting a specialized role that
does not apply to astrocytic glycogen located elsewhere in the
brain. That glycogen is located predominantly in astrocytes is
beyond question, although this broad description of cell types
based on morphology gives no clue as to any specific function
carried out by glycogen in particular subtypes of astrocytes.
Different brain areas have different concentrations of glyco-
gen, but whether this reflects astrocyte density is unknown.
Even among different strains of rat glycogen content of the
same brain area varies, illustrated in a recent study, which
showed similar values for glycogen in the cortex, hippocam-
pus, and cerebellum of Zucker Lean and Zucker Diabetic
fatty rats, but with much lower glycogen levels measured
in Sprague-Dawley rats (Sickmann et al. 2010). This rather
G LYC O G E N A N D E N E R GY M ETA B O L I S M
confused picture strongly suggests establishing baseline levels
strains, a priority that would clarify the variation of glycogen
expression. In addition it is likely that glycogen content varies
that glycogen content is lowered in T1 and T2 diabetes, but
the effects of other metabolic and neurological conditions are
mostly unknown. Aside from a handful of studies (Dalsgaard
et al. 2006), this is an entirely neglected field and worthy of
exploration. Indeed, our recent result of robust glycogen con-
tent in peripheral nerve Schwann cells is the first account of
the presence of glycogen in this tissue.
3.2 E X P R E S S I O N O F R E L EVA N T E N ZY M E S
The presence of glycogen can be deduced by the presence
of the enzymes responsible for its metabolism and synthe-
Glycogen synthase is present in three isoforms, associated
with muscle, liver, or brain (Pellegri et al. 1996). The brain
and liver isoforms are specific for those tissues, but the mus-
cle isoform is present in most tissues. Brain tissue labeling was
highest in the cerebellum, hippocampus, and olfactory bulb,
and at the cellular level, although both astrocytes and neurons
expressed the mRNA, expression was greatest in astrocytes
(Pellegri et al. 1996). As previously discussed, glycogen is pre-
dominantly expressed in astrocytes; thus, its expression in
neurons is puzzling. The expression of glycogen synthase does
not necessarily predict glycogen expression, and indeed only
under pathological conditions is robust neuronal glycogen
expression found (Vilchez et al. 2007). Glycogen phospho-
rylase exists as liver, muscle, and brain isoforms. Astrocytes
express only the muscle and brain isoform, but not the liver
isoform (Pfeiffer-Guglielmi et al. 2003), suggesting brain gly-
cogen phosphorylase is regulated in a manner similar to mus-
cle (see section 3.3); that is, it is regulated allosterically and
requires AMP for full activation, thus it is sensitive to energy
changes within the cell.
3.3 R EGU L AT I O N O F G LYC O G E N C O N T E N T
Glycogen metabolism is strictly regulated and involves mul-
tiple pathways. Although the details of glycogen regulation
have not yet been fully described for the brain, given the simi-
larities of glycogen structure and the homology between the
isoforms of glycogen synthase (Pellegri et al. 1996) and glyco-
gen phosphorylase (Pfeiffer-Guglielmi et al. 2003) it is a fairly
safe assumption that the mechanisms of glycogen regulation
in the periphery also apply to the brain. Glycogen content is
regulated by the hormones glucagon, adrenaline, and insu-
lin, but also allosterically by glucose, glucose-6-phosphate,
AMP, and ATP, indicators of the cellular energy status.
Broadly speaking, the factors that promote glycogen metabo-
lism also inhibit glycogen synthesis and vice versa, highlight-
ing the advantages of two separate pathways for metabolism
and synthesis. Glucagon and adrenaline activate glycogen
phosphorylase and promote glycogen metabolism, whereas
• 461
insulin activates glycogen synthase and promotes storage of
glucose-6-phosphate as glycogen.
Glycogen metabolism requires the combined actions of
several enzymes, which (1) degrade glycogen, (2) remodel
glycogen so it remains viable for degradation, and (3) con-
vert glycogen breakdown product into a suitable form for fur-
ther metabolism. Glycogen phosphorylase exists as an active
phosphorylase, a (Pa) and an inactive phosphorylase b (Pb).
Phosphorylase kinase phosphorylates the inactive Pb to active
Pa. Phosphorylase kinase itself is converted to its active form
(phosphorylase kinase a) from phosphorylase kinase b by
phosphorylation by protein kinase. The active form of protein
kinase a is produced by phosphorylation of protein kinase b by
cAMP. Protein kinase a also acts to covert the active a form
of glycogen synthase to the inactive b form. Thus, a cascade
of phosphorylation initiated by production of cAMP results
in glycogen phosphorylase activation and glycogen metabo-
lism. The cAMP is produced by the binding of glucagon and
adrenaline to metabotropic receptors and activation of G
proteins. Thus, a signaling cascade mediated by hormone bind-
ing initiates glycogen metabolism (Berg et al. 2006; Voet and
Voet 2011).
Glycogen synthesis is catalyzed by glycogen synthase,
which is converted from its inactive b form to the active a form
by protein phosphatase 1 (PP1). PP1 binds to the glycogen–
glycogen synthase complex and results in production of gly-
cogen synthase a by dephosphorylating glycogen synthase a,
promoting glycogen synthesis. PP1 also dephosphorylates Pa
to Pb, thereby inhibiting glycogen metabolism, and addition-
ally dephosphorylates phosphorylase kinase a to phosphorylase
kinase b, thereby inhibiting activation of glycogen phosphory-
lase (Berg et al. 2006; Voet and Voet 2011).
Thus, the general pattern of regulation is that cAMP
results in a phosphorylation cascade mediated by kinases,
which results in glycogen metabolism, whereas PP1 causes
dephosphorylation of key enzymes promoting glycogen syn-
thesis. Insulin and adrenergic receptors are highly expressed
in liver and muscle cells, whereas glucagon receptors are more
prevalent in the liver than in muscle.
Allosteric modulation of glycogen metabolism is mediated
by compounds that are indicative of the energy status of the
cell, namely, glucose, glucose-6-phosphate, AMP, and ATP. In
muscle cells, AMP activates glycogen phosphorylase, and ATP
and glucose-6-phosphate inhibit glycogen phosphorylase,
whereas glycogen synthase is activated by glucose-6-phosphate.
This demonstrates that indicators of increased energy statues
(glucose-6-phosphate and ATP) inhibit glycogen phosphory-
lase and activate glycogen synthase; conversely, indicators of
decreased energy status (AMP) promote glycogen metabolism.
In liver, glucose inhibits glycogen phosphorylase and activates
glycogen synthase, because liver cells express glucose-6-phos-
phatase and convert glycogen-derived glucose-6-phosphate
to glucose. However, muscle cells do not express glucose-6-
phosphatase; thus, glucose-6-phosphate is used as the muscle
energy substrate. In muscle cells, Ca2+ is an important modula-
tor of phosphorylase kinase. The muscle phosphorylase kinase
consists of four subunits, one of which, calmodulin, is sensitive
to Ca2+. Binding of Ca2+ to calmodulin causes conformational
462 • FUNCTIONS OF NEUROGLIAL CELLS
changes, resulting in phosphorylase kinase activation. Thus
muscle contraction, which causes increases in intracellular
Ca2+ via release of Ca2+ from the sarcoplasmic reticulum, thus
leads to glycogen metabolism, providing ATP for the energy-
dependent contraction process (Berg et al. 2006; Garrett and
Grisham 1999; Voet and Voet 2011).
4 FUNCTION IN THE CENTR AL
N E RVO U S SYS T E M
4 .1 WH I T E M AT T E R : G LYC O G E N S U P P O RT S
AXO N C O N D U C T I O N I N AG LYC E M I A A N D
I N C R E A S E D E N E RGY D E M A N D
Initial studies that drew attention to a potential functional
role for glycogen suggested that active neurons utilized
astrocytic glycogen. In a tissue culture study, the reliance of
neuronal survival on the presence of astrocytes was clearly
demonstrated. More detailed analysis revealed that the key
factor that sustained the neurons was the glycogen that was
present in the astrocytes; the greater the glycogen content the
longer the duration of neuronal survival (Swanson and Choi
1993). This was an important study for several reasons. First,
it corroborated an earlier study, which had demonstrated
that cultured neurons incubated in the presence of glial cells
survived longer than neurons grown in the absence of glia
cells, although no mechanism was proposed (Whatley et al.
1981). Second, it directly correlated neuronal survival with
the metabolic status of astrocytes. For more than 100 years
it has been hypothesized that there was an intimate meta-
bolic link between neurons and astrocytes, in which neurons
were dependent on astrocytes for the supply of metabolic
substrate (Andriezen 1893). Third, it suggested an intimate
cell-to-cell metabolic signaling pathway between astrocytes
and neurons, in which astrocytes supplied an energy sub-
strate to neurons. This implies a great degree of functional
cooperation between the two cells types, such that the appro-
priate transporters and enzymes must be present in each cell
type. In addition, appropriate signaling systems must be pre-
sent for the neuron to signal the astrocyte when substrate is
required (see also chapter 27).
In a study using autoradiographic techniques, a link was
made between sensory stimulation and glycogen use in the
associated brain area. Under in vivo conditions, stimulation
of whiskers in the rat resulted in a decrease in glycogen in
the barrel cortex (Swanson et al. 1992). This demonstrated a
metabolic relationship between neuronal activity and glyco-
gen content under in vivo conditions, but more importantly
showed that there was a relationship between physiological
function, in this case afferent sensory input, and glycogen
breakdown in the associated cortical area. The contemporary
experiments in the Magistretti lab demonstrating the regula-
tion of glycogen content by neurotransmitters (Hof et al. 1988;
Magistretti 1988; Magistretti et al. 1986; Sorg and Magistretti
1991), suggested CNS glycogen was not a vestigial throwback,
but rather a dynamic compound intimately involved in brain
function.
 We took these experiments as the basis for studies we car-
ried out in rat, and subsequently in mouse optic nerve, as the
rodent optic nerve offered a simplified structure, a represen-
tative central white matter tract that was composed of myeli-
nated axons, astrocytes, and oligodendrocytes, but that lacked
the complications of glutamatergic synapses and neuronal
cell bodies (Ransom et al. 1994). The optic nerve affords the
opportunity to probe cell-to-cell metabolic communication
in a reduced, simplified model, but that is physiologically rel-
evant, because white matter comprises 50% of human adult
brain volume (Wang et al. 2008). The tissue is minimally
disrupted during dissection and can be maintained for many
hours in a superfusion bath while recording axon conduction.
Initial studies in rat optic nerve showed that in the absence of
oxygen the CAP area, an index of axon conduction, falls to
zero within a few minutes (Stys et al. 1992), but in the absence
of glucose, with oxygen present, the CAP area took more than
30 minutes before it began to fail (Ransom and Fern 1997).
These results suggested that the tissue contained an endoge-
nous energy reserve that could support axon conduction in
the absence of exogenously supplied glucose. Upregulation or
downregulation of glycogen content was achieved by incubat-
ing the nerves at various concentrations of glucose, or in the
presence of noradrenaline, indicative of glycogen as a labile
energy reserve (Wender et al. 2000). Because glycogen is
stored in astrocytes in the adult, a transportable conduit must
be present for axons to benefit from astrocyte glycogen. The
ability of blockers of monocarboxylate transporters (MCTs)
to impede the ability of glycogen to support axon conduction
is strong evidence that the transportable conduit was lactate
(Wender et al. 2000). Subsequent rodent optic nerve studies
were carried out on the mouse optic nerve for compatibility
with future transgenic models, and for the decreased diffusion
distance (Baltan Tekkök et al. 2003) in studies of this sort in
which energy substrates and drugs are applied exogenously in
the superfusate.
Complementary data from the mouse optic nerve (MON)
reinforced the capacity of rodent optic nerve to conduct
action potentials in the absence of glucose (Brown et al.
2003), although only for about 20 minutes compared to 30
minutes in the rat. Biochemical assays of glycogen carried out
in parallel with electrophysiological recordings showed that
the onset of aglycemia coincided with a decrease in glycogen
content, and once glycogen levels reached their nadir CAP
conduction failed, establishing a direct relationship between
glycogen and axon conduction (Brown et al. 2003). Elevating
glycogen content before aglycemia prolonged latency to CAP
failure; conversely, decreasing glycogen content before agly-
cemia accelerated CAP failure (Brown et al. 2003). Indeed,
there was a linear relationship between glycogen content at
the onset of aglycemia and the latency to CAP failure, such
that accurate predictions about latency to CAP failure could
be made based on the glycogen content of the tissue (Brown
et al. 2003) (Fig. 36.4).
Given the rarity of spontaneous hypoglycemia, and the
role of liver glycogen in maintaining euglycemia during sys-
temic glucose decrease, it seems unlikely that the primary
role for glycogen is supporting function during periods of
G LYC O G E N A N D E N E R GY M ETA B O L I S M
A 30
Failure latency
20
(mins)
10
0
0 2 4 6 8 10
Glycogen (pmoles/μg protein)
B
1
CAP area
0.5 1 2 3
0
0 60 120 180 240
Latency (mins)
Figure 36.4 Glycogen Prolongs Latency to CAP Failure During Glucose
Withdrawal. A. Latency to stimulus-evoked CAP failure in MON dur-
ing aglycemia is linearly related to the glycogen content of the tissue at
onset of aglycemia according to the relationship Latency = 3.87*
[Glycogen] – 8.32. B. Latency to CAP failure of the A peak (3) and C
peak (1) of the sciatic nerve during aglycemia showing the A peak failure
is significantly delayed relative to C peak failure, an effect that is lost
when glycogen metabolism is inhibited by DAB (2). (A) Data adapted
from Brown et al. 2003.
glucose decrease (although this does suggest that glycogen
might be targeted as a clinically relevant therapeutic target
to stave off the injury that results from iatrogenic systemic
insulin-induced hypoglycemia) (Suh et al. 2007). We sought
to determine if glycogen played a role in more physiological
function in the form of high frequency firing, which is an
experimental correlate for increased tissue energy demand
resulting from increased frequency of axon action poten-
tials. High frequency stimulus (up to 100 Hz) was imposed
on the MONs under euglycemic conditions and the result-
ing CAPs recorded. Parallel experiments measuring glycogen
content at the end of the stimulus period showed that there
was a significant decrease in glycogen during the period of
stimulus (Brown et al. 2003). This indicated that glycogen was
metabolized, even though the tissue was perfused with 10 mM
glucose aCSF, implying that euglycemic concentrations of glu-
cose are insufficient to support function, and glycogen must
be metabolized to provide a supplemental energy substrate for
axon conduction to be fully maintained (Brown et al. 2003).
However, an alternate explanation is that the glucose supplied
to the nerve partly supports axon conduction and partly sup-
ports glycogen synthesis. During periods of increased tissue
energy demand, the glucose is preferentially used to support
axon conduction and thus less or none is used to support gly-
cogen synthesis. However, the experiment lasted 4 minutes, a
time period considered too rapid for the effect to result from
reduction in glycogen turnover.
• 463
 The ability of glycogen to support function implies a
metabolic pathway from astrocytes to axons that is capable of
metabolizing glycogen to lactate in the astrocyte, transporting
the lactate out of the astrocyte, followed by the axon having
the ability to take up the lactate and metabolize it oxidatively.
The enzyme lactate dehydrogenase, which interconverts lac-
tate and pyruvate, comprises several isoforms. The isoform
that preferentially converts lactate to pyruvate (LDH1) is
found in neural elements, whereas the isoform that converts
pyruvate to lactate (LDH5) is found in astrocytes, consistent
with lactate production in astrocytes and metabolism in axons
(Bittar et al. 1996). For lactate transport to occur between the
two cell types, the appropriate transporter proteins must also
be present. Lactate is cotransported with H+ via monocarbox-
ylate transporters (MCTs). There are four isoforms of MCTs
that can transport lactate in mammals (Halestrap 2012), but
the isoform that exports lactate (MCT1) is found on astro-
cytes, and the isoform that imports lactate (MCT2) is located
on neural elements (Halestrap and Price 1999; Koehler-Stec
et al. 1998; Pierre et al. 2000). Immunohistochemical studies
in MON confirm the appropriate cellular location for MCT
subtypes (Baltan Tekkök et al. 2005). Experiments with pH-
sensitive microelectrodes showed that the onset of aglycemia
resulted in alkalization of the extracellular fluid consistent
with decreased lactate transport out of astrocytes into the
ECF in the face of decreased glucose availability, succeeded
by a second stage in which extracellular pH increased to bath
pH of 7.45, indicative of a total loss of lactate efflux (Brown
et al. 2001). Recent experiments with lactate biosensors have
illuminated these data. Recording lactate in the vicinity of the
nerve registers a lactate concentration of about 400 μM under
unstimulated baseline conditions. These results imply that lac-
tate is constantly produced in the tissue and exported to the
extracellular fluid. Given the rate of our perfusion system and
the superfusion of the tissue with aCSF, this lactate is washed
out of the tissue and into the bath. These data certainly suggest
that under resting conditions an excess of lactate is produced
relative to the energy requirements of the tissue, but more
importantly suggests that lactate is present in the extracel-
lular fluid in sufficiently large concentrations to be used as a
source of energy during periods of need. The cellular source
(s) of lactate has not been identified, but astrocyte metabo-
lism is almost certain to contribute. Whether glycogen
metabolism contributes to this lactate signal was investigated
by adding 1 mM 1,4-dideoxy-1,4-imino-D-arabinitol (DAB),
a compound that inhibits the action of glycogen phosphory-
lase (Walls et al. 2008). The lactate signal fell by 30% in the
presence of DAB and was reversible on DAB washout, dem-
onstrating that under baseline resting conditions glycogenoly-
sis contributes to the lactate production of the tissue, implying
that glycogen is constantly being metabolized, and thus syn-
thesized, indicating that glycogen turnover is very active in
this tissue. This flies in the face of conventional attitudes
that glycogen is a minor energy reserve that is only called on
in times of glucose depletion or periods of increased energy
demand (Fig. 36.5) (Wender et al, 2000).
4.2 G R AY M AT T E R : G LYC O G E N S U P P O RT S
SY NA P T I C F U N C T I O N I N AG LYC E M I A
Pioneering studies seeking the location of glycogen noted a
high expression in synaptic regions (Koizumi 1974; Koizumi

Blood Astrocyte Neural element

cap
astrocyte glycogen
end foot
pyruvate
e
tat

glucose glucose ATP

lac

endfeet
E CO2 & H2O
n
d glucose
o
t
h
e
l
i
u
m

Figure 36.5 Model of Metabolic Substrate Relationship Between Astrocytes and Neural Elements. Astrocytic endfeet almost completely ensheathe
capillary walls. Glucose present in the blood can be transported into the extracellular fluid for uptake into neural elements, or alternatively, may be
taken up into astrocytes, where it can be converted to glycogen. Glycogen metabolism yields lactate, which is transported into the extracellular fluid
for uptake and metabolism by neural elements. Under baseline conditions in which glucose is available in excess of demand, lactate accumulates in the
extracellular fluid because not all is taken up by axons. In the absence of glucose, glycogen is metabolized to lactate, which is shuttled out of astrocytes
and taken up by neural elements as their sole metabolic substrate. Because lactate would not accumulate in the extracellular fluid under these
conditions, its concentration is low.

464 • FUNCTIONS OF NEUROGLIAL CELLS

and Shiraishi 1970a,b; Phelps 1972). Given the high meta-
bolic cost of synaptic activity (Attwell and Laughlin 2001),
such a location seems logical. Indeed, a central role for glyco-
gen is likely given the recent proposal of the tripartite synapse
(Araque et al. 1999; Perea et al. 2009) in which the astro-
cytes play a key role as sensors for neurotransmitter release.
In hippocampal slice preparations, in which stimulus-evoked
CAPs recorded from the CA1 region are monitors of
synaptic efficacy, removal of glucose from perfusing aCSF
leads to a delayed fall in the response. Depleting glycogen
before aglycemia leads to an accelerated fall in the excitatory
postsynaptic potential (EPSP) (Izumi et al. 1997), whereas
preincubation in 30 mM glucose, a maneuver that leads to
increased glycogen content, resulted in prolonged latency to
EPSP failure (Cater et al. 2001). These results strongly sug-
gested that hippocampal glycogen supports synaptic activity
during aglycemia.
4.3 G LYC O G E N A N D L E A R N I N G :
G LYC O G E N S U P P O RT S L E A R N I N G D U R I N G
D EV E L O PM E N T I N C H I C K
Studies carried out on memory acquisition and consolidation
in 1-day-old chicks demonstrates the importance of glycogen
in this higher brain function. DAB was injected either focally
into the brain or systemically to inhibit glycogenolysis. A train-
ing paradigm using avoidance techniques were used as a proxy
for memory. In the chick there are several stages in the laying
down of memory. Phase A lasts for 10 to 25 minutes, and phase
B lasts from 30 to 55 minutes after the training event. DAB
was found to affect memory when applied 5 minutes before
training, with testing of memory at 120 minutes after train-
ing. The effects of DAB were dependent on when DAB was
applied. The greatest effects were seen 5 minutes before train-
ing or between 25 and 40 minutes after training, implying that
glycogen is involved in the memory processing at these two
distinct time periods (Gibbs et al. 2006). Additional experi-
ments determined that injection of DAB 5 minutes before
training caused deficits in memory when tested 5 to 10 min-
utes after training and for 40 minutes onward after training.
Similarly, DAB injected 25 minutes after training with mem-
ory tested at 35 to 60 minutes after training, showed deficits
in memory. However, memory could be restored if glutamine,
or acetate and aspartate, were injected immediately after
training in DAB-treated chicks. These experiments showed
for the first time that glycogen is actively involved in higher
brain function in vivo (Gibbs et al. 2006), albeit in chick. The
experiments suggested that glycogen is important in memory
consolidation at several stages of the process. As glycogen is
metabolized to pyruvate, which can then be converted to glu-
tamate via TCA cycling, supplemental glutamine, presumably
a precursor to glutamate, circumvents the inhibitory effects
of DAB. Fueling the astrocyte TCA cycle with acetate and
aspartate also restored memory, probably because aspartate
compensates for pyruvate carboxylation and acetate compen-
sates for pyruvate dehydrogenase to supply glutamate (Gibbs
et al. 2006).
G LYC O G E N A N D E N E R GY M ETA B O L I S M
4.4 G LYC O G E N A N D L E A R N I N G : G LYC O G E N
S U P P O RTS M E M O RY AC Q U I S IT I O N
I N T H E A D U LT RO D E N T
Recently Suzuki et al. (2011) have used the model described
in the preceding section as a template to examine the role of
glycogen in memory formation in the rodent. Using an in vivo
rat preparation in which field potentials were recorded from
the CA1 region of the hippocampus, along with a dialysis
probe for introduction of DAB or lactate, they investigated
the effect of DAB on the ability of rats to retain memory in
the form of aversion therapy, which serves as a proxy for mem-
ory. Rats were housed in a white room with a door leading
to a dark room. When the rats entered the dark room they
received an electric shock. Rats were then re-housed in the
white room and the duration before they re-entered the dark
room was taken as an index of memory, the longer they took
to enter the room, the better the memory was consolidated.
The study showed that DAB was only effective in abolishing
memory when administered 15 minutes before the training
period. DAB injection after the training had no effect on the
memory. After DAB injection the memory was absent for up
to 7 days after the training period, showing that DAB had effi-
ciently prevented memories being formed. Injecting lactate in
the region around the CA1 in the presence of DAB restored
the ability to lay down memories, convincingly showing
that glycogen-derived lactate is the metabolic conduit that sup-
ports laying down of memory (Suzuki et al. 2011). Interestingly,
the study found that glucose was not as effective as lactate in
laying down memories. This appears initially puzzling, but
if the memory mechanism requires an immediate energy
supply, then perhaps glycogen is more suited to deliver this
than an increase in glucose delivery mediated by increase
blood flow, and only lactate rather than glucose can act as the
preferred energy substrate. These results support a physiologi-
cal role for glycogen predicted by previous experiments in
white matter (Brown et al. 2003), namely, that it is an energy
buffer to provide energy substrate during periods of increased
tissue energy demand that cannot be met by ambient glu-
cose. These experiments predict that glycogen is a vital energy
source in the brain, supporting a critical aspect of higher brain
function.
5 G LYC O G E N I N T H E P E R I P H E R A L
N E RVO U S SYS T E M : G LYC O G E N I S
L O C A L I Z E D TO S C H WA N N C E L L S
A N D S E L E C T I VE LY S U P P O RT S A
FIBER CONDUCTION
The previous data have described glia in the CNS as possessing
glycogen and supplying glycogen derived substrate to supply
neural elements, via astrocyte release of lactate into the extra-
cellular fluid. Immunohistochemical studies in the periph-
eral nervous system (PNS) suggested that this might not be
the case in peripheral nerve (Pfeiffer-Guglielmi et al. 2007).
In sciatic nerve, antibodies to glycogen phosphorylase revealed
its presence exclusively in axons with none apparent in the
• 465
Schwann cells (Pfeiffer-Guglielmi et al. 2007). This was a tan-
talizing result because it suggested that if glycogen was pres-
ent in peripheral nerves it was located in the axons rather than
the glial cells, in this case the Schwann cell, and its function
must be different from that in the CNS. The authors under-
took a series of experiments to determine the cellular location
of glycogen in the sciatic nerve, a peripheral nerve. Previously
there had been no reports of glycogen in peripheral nerve,
only the appearance of glycogen granules as a result of pathol-
ogy (Kalichman et al. 1998; Katsuragi et al. 1988; Powell et al.
1985). Our initial studies using biochemical assay detected
glycogen in the sciatic nerve at concentrations about double
that of optic nerve; thus, our next objective was to locate
the cellular domain of the glycogen. Immunohistochemical
studies identified glycogen phosphorylase in both axons and
Schwann cells. It is important to note at this point that the
presence of glycogen phosphorylase does not necessarily sig-
nify the presence of glycogen. We used electron microscopy to
localize glycogen predominantly in the Schwann cells myeli-
nating large A fibers, but found no evidence of glycogen in the
Schwann cells bundling unmyelinated C fibers. We also found
no evidence of glycogen in axons. As such these data par-
tially refute the initial data from the PNS (Pfeiffer-Guglielmi
et al. 2007) and demonstrate selective glial expression of gly-
cogen. Experiments were then carried out to identify a role(s)
for glycogen.
We investigated the ability of glycogen to support the sci-
atic nerve action potential conduction. The stimulus-evoked
CAP in sciatic nerve is comprised of two separate elements:
a fast unipolar peak that occurs within a millisecond of the
stimulus artifact and is contributed to by large myelinated
axons (A peak), followed by a smaller bipolar peak 5 to 15 ms
later that is caused by small unmyelinated C axons (C peak).
On exposure to aglycemia the A peak was sustained for 2.5
hours, whereas the C peak lasted only 0.5 hour. Measurement
of glycogen during aglycemia showed that it falls and reaches
its nadir temporally correlated with the A peak failure, con-
sistent with glycogen metabolism during aglycemia, but once
the glycogen is exhausted the A peak fails. The C peak did not
appear correlated with glycogen content. These data suggest
that glycogen does not afford the C axons any particular pro-
tection during aglycemia, consistent with the apparent lack of
glycogen in Schwann cells surrounding C fibers. To test this
further we bathed the sciatic nerve in DAB before exposure to
aglycemia. Under these conditions the A peak fell at the same
time as the C peak, increasing the evidence that glycogen sup-
ports the A axons, but not the C axons, and in the absence of
glycogen-derived substrate there is no difference between the
A and C peaks.
These data differ from the CNS optic nerve, in which glyco-
gen appears to support all axons equitably. The obvious reason
for this difference is in the morphology of the tissue, with clear
differences between the large A fibers, which are myelinated
by single Schwann cells, and the C fibers, which are bundled
with several others such that multiple C axons are myelinated
by a single Schwann cell. Thus, Schwann cells myelinating C
axons that lack glycogen are not protected by glycogen. An
additional consideration is the energy requirements of the
466 • FUNCTIONS OF NEUROGLIAL CELLS
axons. Small myelinated axons have a higher energy demand
per unit volume than do large axons (Nicholls et al. 2001).
Thus it could well be that the axons do not benefit from what-
ever glycogen is available. We carried out experiments in which
we upregulated glycogen by 2 hours before incubation in 30
mM glucose and found that the latency to C fiber CAP failure
was delayed during aglycemia relative to control, results that
appear to highlight that C axons are not incapable of benefit-
ing from glycogen-derived substrate, but that under physio-
logical conditions, insufficient substrate is available. Given the
parallels between the optic and sciatic nerve, we determined
whether the transportable conduit was likely to be lactate. We
showed that lactate can support both the A and C fibers in
the absence of glucose, and additionally can rescue A and C
fibers during aglycemia; thus, lactate is capable of supporting
axon conduction, and is most likely the conduit. Experiments
with lactate-sensitive biosensors showed that although the
tissue does release lactate, the concentration recorded was
much lower than that recorded from the optic nerve (120 vs
400 μM). The reasons for this are unclear, but may be related
to the decreased metabolic demand of peripheral versus cen-
tral tissue. As was the case in optic nerve, DAB reversibly
decreased lactate concentration by about 25%, indicating that
glycogen-derived lactate is constantly produced by the tissue,
and this lactate is surplus to the demands of the tissue under
resting conditions. Thus, it appears peripheral and central
nerve share a similar debt to glycogen in providing lactate to
the extracellular fluid that can be taken up and metabolized by
axons as required.
6 G LYC O G E N A N D D I S E A S E
6.1 D I A B ET E S
For glycogen to play a role in diabetes in humans, the presence
of glycogen in the human must be established. This has been
done using nuclear magnetic resonance (NMR) spectroscopy
(Oz et al. 2003, 2007) and biochemical assay of resected human
brain tissue (Dalsgaard et al. 2006). The effects of type-2 diabe-
tes (T2D) were studied in Zucker obese rat (ZOb) and Zucker
Diabetic fatty rat (ZDF) models of T2D, and the general trend
was that in models of T2D model, glycogen levels were lower
than in the cerebellum and hippocampus of control mice.
However, this was not found in the cortex (Sickmann et al.
2010). The results were confused by the differences in brain
glycogen levels among the control animals, namely, that in the
control rats, Sprague-Dawley, Zucker lean (ZL), and Zucker
Diabetic lean (ZDL), there was considerable variation in gly-
cogen levels among the cortex, cerebellum, and hippocampus.
The Zucker lean (ZL) is the genetic control for the ZOb, and
had a higher glycogen content in cortex; however, in the other
two brain regions the glycogen was lower in ZOb than in ZL.
This could be caused by a variety of factors. In these animal
models of diabetes there is insensitivity to insulin accompa-
nied by ambient hyperglycemia. It is known that CNS glyco-
gen is sensitive to ambient glucose (Brown et al. 2003), and
also to insulin (Banks et al. 1997), but the balance between
the two is unknown under control or pathological conditions.
The situation is different from the periphery in which the
insulin-sensitive Glut-4 transporter is the major route of cel-
lular glucose entry (Voet and Voet 2011). In the CNS the main
routes of cellular glucose access are via the insulin-insensitive
Glut-1 or Glut-3 receptors (Vannucci et al. 1997); thus, insu-
lin does not play a key role in glucose uptake in CNS tissue,
as it does in the periphery. It is interesting to note that in the
cortex the glycogen levels are higher than control, as would be
expected if ambient glucose was the sole factor in determining
glycogen content. That glycogen levels were lower in the cere-
bellum and hippocampus in T2D suggests that diabetes may
affect brain regions selectively. A possible explanation is the
selective expression of the insulin-sensitive Glut-4 transporter
predominantly in neurons in the olfactory bulb, hippocam-
pus, and cerebellum (El Messari et al. 1998, 2002; Vannucci
et al. 1998); however, these issues require further study.
The first measurement of glycogen in the brains of patients
with T1D has recently been published (Oz et al. 2011). When
compared to control subjects, newly synthesized glycogen
was greater in control subjects than T1D patients, results that
could be explained as higher glycogen content or increased
glycogen turnover. Subsequent metabolic modeling of the
data, when taking into account prevailing glucose and insu-
lin levels, indicated that patients with T1D did indeed have
lower glycogen levels. The issue is complex, however, because
if glucose uptake is both glucose and insulin dependent, then
the contribution of each may lead to unpredictable values,
depending on prevailing insulin and glucose levels. In addi-
tion, Glut-1 glucose transporters in the blood brain barrier are
upregulated relative to control in T1D patients, which would
augment glucose influx into the brain. The method of mea-
suring glycogen could also lead to problems of interpretation
(Oz et al. 2003), because the method requires long periods of
infusion of 13C glucose; however, the results for control sub-
jects were consistent with biochemical assay of glycogen from
biopsied human brain tissue (Dalsgaard et al. 2006). A second
aim of this study was to determine if glycogen played a role in
hypoglycemia unawareness. This is a condition in which expo-
sure to hypoglycemia results in a decrease in the autonomic
warning signals of impending hypoglycemia (Frier and Fisher
2007). The argument for glycogen involvement proposes that
post-hypoglycemia levels of glycogen super-compensate to
higher levels than pre-hypoglycemia (Oz et al. 2009). It has
thus been hypothesized that this increased glycogen, and the
release of glycogen-derived substrates in the brain leads to the
brain being fooled into thinking there is sufficient energy sub-
strate in the face of hypoglycemia. However, the time course of
the glycogen concentration changes did not correlate with the
time course of the faulty signaling, which argues against post
hypoglycemia super-compensation playing a role in hypogly-
cemia unawareness.
6.2 G LYC O G E N S TO R AG E D I S E A S E S
Glycogen storage diseases are a family of conditions resulting
from deficient expression of a variety of enzymes involved in
glycogen metabolism and synthesis. There are at least 10 types
G LYC O G E N A N D E N E R GY M ETA B O L I S M
of hereditary glycogen storage disease associated with deficient
expression of glucose-6-phosphatase (type I), D(1→4) glucosi-
dase (type II), glycogen debranching enzyme (type 3), glycogen
branching enzyme (type 4), glycogen phosphorylase (types V
and VI), phosphofructokinase (type VII), phosphorylase kinase
(types VIII and IX) and glycogen synthase (type 0) (Voet and
Voet 2011). All of these diseases result in an overabundance of
glycogen in liver and/or muscle, except type 0, which results in
decreased glycogen content, suggesting that the presence of gly-
cogen is a prerequisite for life. The effects of glycogen storage
diseases on brain glycogen have been little studied, and there
are only a few scattered accounts in the literature, reporting
pathology associated with the disease, rather than any puta-
tive effect of the disease on brain glycogen content (Garty
et al. 1992; Lamperti et al. 2009; Martin et al. 1973; Melis et al.
2004; Middleditch et al. 1998; Weghuber et al. 2007).
7 S U M M A RY A N D P E R S P E C T I VE S
In conclusion, interest in glycogen has recently been reawak-
ened after a period when interest in its physiological impor-
tance was neglected. Recent advances have seen in vivo
measurements of glycogen in human brain (Oz et al. 2003,
2007), demonstration of the ability of glycogen to support
neural function in the absence of glucose in both gray (Cater
et al. 2001; Izumi et al. 1997) and white matter (Wender et al.
2000), and its ability to provide supplemental energy sub-
strate during increased tissue energy demand in white matter
(Brown et al. 2003). Glycogen has also been shown to support
higher brain function under in vivo conditions (Suzuki et al.
2011), and recent reports suggest diabetes is associated with
brain glycogen control dysfunction (Oz et al. 2011; Sickmann
et al. 2010). Thus, these are exciting times for brain glycogen
research, in which a multitude of experimental approaches in
both animal models and humans are gradually elucidating key
roles in fundamental brain function.
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 37.
ASTROCY TE REGULATION OF NEUROVASCULAR
CONTROL
Clare Howarth, Grant R. J. Gordon, and Brian A. MacVicar

A B B R E VI AT I O N S 1 INTRODUCTION

AA arachidonic acid It is critical for the maintenance of normal brain function that
ASL arterial spin labeling cerebral blood flow (CBF) is matched to neuronal metabolic
ATP adenosine triphosphate needs. Blood flow is increased to areas in which neurons are
BK channels big potassium channels more active (a response termed functional hyperemia), and it
BOLD blood oxygen level dependent is this blood flow increase that forms the basis of functional
[Ca2+]i intracellular calcium concentration imaging techniques such as blood oxygen level dependent
(BOLD) functional magnetic resonance imaging (fMRI).
CBF cerebral blood flow
Although a coupling between cerebral energy consumption
cGMP cyclic guanosine monophosphate and neuronal activity was originally suggested more than 100
COX cyclooxygenase years ago (Roy and Sherrington 1890), the exact relationship
CSD cortical spreading depression remains an active area of research. Over the past two decades
CYP4A cytochrome P450 4A there has been extensive research into the role astrocyte Ca2+
EETs epoxyeicosatrienoic acids signals play in CBF regulation. Astrocytes, which are the most
EP4 receptor prostaglandin 4 receptor numerous cells in the central nervous system (CNS), are situ-
FITC fluorescein isothiocyanate ated at the synaptic cleft, and every astrocyte has at least one
fMRI functional magnetic resonance endfoot, which is in contact with the vasculature. Therefore,
imaging astrocytes are ideally positioned to relay information regard-
GABA gamma-aminobutyric acid ing changes of synaptic activity to blood vessels so that neu-
GFAP glial fibrillary acidic protein ronal energy demands can be met. In response to glutamate
20-HETE 20-hydroxyeicosatetraenoic acid release by active neurons, astrocyte [Ca2+]i increases and dif-
fusible messengers are released from astrocytic endfeet, which
IOS intrinsic optical signals
are opposed to smooth muscle cells. In this way, astrocytes
[K+]o extracellular potassium communicate with the smooth muscle cells, evoking changes
concentration in arteriole diameter and thus regulating CBF. This chapter
Kir channel inward rectifier potassium channel focuses on the specializations of astrocytes, which make these
LDF laser Doppler flowmetry cells well suited to vascular control, the evidence for synap-
mGluRs metabotropic glutamate receptors tic activity–evoked Ca2+ signals in astrocytes, how such Ca2+
MPEP 2-methyl-6-(phenylethynyl)pyridine signals may evoke changes in vascular diameter, thereby regu-
MRI magnetic resonance imaging lating blood flow, and how this neurovascular coupling can
NADH nicotinamide adenine dinucleotide change in pathology.
NMR nuclear magnetic resonance
nNOS neuronal nitric oxide synthase
NO nitric oxide 2 T H E I M P O RTA N C E O F R E GU L AT I N G
PET positron emission tomography C E R E B R A L B L O O D F L OW
PGE2 prostaglandin E2
2.1 O VE RVI EW: T H E I M P O RTA N C E
PLA2 phospholipase A2 O F S U P P LY I N G S U F F I C I E N T E N E RGY
pO2 oxygen partial pressure FOR THE BRAIN
RBC red blood cells
siRNA small interfering RNA The human brain accounts for only 2% of the body’s weight,
TBOA threo-β-benzyloxyaspartic acid yet uses 20% of the body’s resting energy use (Clarke and
Sokoloff 1999). This high energy demand occurs largely
UV ultraviolet
because the neural processing of information is metabolically

470
expensive. Most of the energy is expended on signaling, with
the majority being used on the reversal of ion fluxes underlying
synaptic potentials and action potential propagation (Attwell
and Laughlin 2001; Clarke and Sokoloff 1999). When neural
activity increases, the demand for oxygen and glucose by those
neurons increases. Because the total brain energy supply is
essentially constant, energy resources must be allocated flexi-
bly among regions according to neuronal demand. Therefore, a
tight spatial and temporal coupling exists between local blood
flow regulation and neuronal metabolic need. Understanding
the mechanisms of neurovascular coupling is essential for
the development of therapies to correct problems with CBF
that occur during disorders such as ischemic stroke, subarach-
noid hemorrhage, and cerebral palsy after perinatal asphyxia,
in which CBF and neuronal metabolic demands can be
mismatched, leading to neuron and glia damage and death.
Furthermore, it is essential so as to correctly interpret data
from experiments applying high resolution functional imag-
ing techniques such as BOLD fMRI.
2.2 R EVI EW O F S T R AT E G I E S TO M E A S U R E
C E R E B R A L B L O O D F L OW I N H U M A N S A N D
F U N C T I O NA L H Y P E R E M I A
Positron emission tomography (PET) can be used to quanti-
tatively measure CBF. A radioactive tracer isotope is injected
into or inhaled by the subject. The isotope decays by emitting
a positron, which travels a short distance before encountering
an electron. As the positron and electron annihilate each other,
a pair of photons is created. These photons move in opposite
directions and are detected by a coincidence detector.
A noninvasive technique commonly used for brain imaging
is magnetic resonance imaging (MRI). In arterial spin labeling
(ASL), endogenous water proton nuclear spins are labeled in
the arterial blood by inverting them in the neck region, before
entering the brain. The difference between images acquired
with and without ASL can be modeled, allowing calculation
of a quantitative blood flow image (Williams et al. 1992).
Alternatively, BOLD contrast fMRI uses changes in deoxy-
hemoglobin levels as an indirect measure of CBF and neural
activity (Ogawa et al. 1990). The iron in deoxyhemoglobin is
paramagnetic, creating local inhomogeneities in the magnetic
field and a faster decay of the MRI signal. During an increase
in neuronal activity, the oxygen use increases followed within
a few seconds by a larger fractional increase in blood flow,
resulting in a net decrease in the concentration of deoxyhemo-
globin (Fox and Raichle 1986).
Laser Doppler flowmetry (LDF) allows continuous measure-
ment of local, relative, CBF. Laser Doppler flowmetry is based on
the principle of the Doppler shift: When laser light is reflected
from moving blood cells, the light undergoes a frequency shift;
therefore, this reflected light can be used to measure the move-
ment of red blood cells (Frerichs and Feuerstein 1990).
Blood flow within capillaries can be visualized and
even quantified by observing the passage of red blood cells
(RBCs) using two photon laser scanning microscopy in vivo
(Kleinfeld et al. 1998). Injection of a fluorescein isothiocya-
nate (FITC)–conjugated dextran into the bloodstream results
A S T R O C Y T E R E GU L AT I O N O F N E U R O VA S C U L A R C O N T R O L
in a fluorescent serum containing nonfluorescent (dark)
RBCs. The passage of RBCs within an individual capillary can
be imaged, allowing determination of RBC velocity, flux, and
linear density (Kleinfeld et al. 1998). Alternatively, capillary
diameter changes can be measured to estimate the impact on
flow using Poiseuille’s law.
3 T H E R E L AT I O N S H I P O F T H E
A S T R O C Y T E E N D F E ET W I T H C E R E B R A L
B L O O D VE S S E L S
3.1 D E S C R I P T I O N O F T H E E N D F O OT
M O R P H O L O GY A N D C A P I L L A R I E S ,
A RT E R I O L E S , A N D VE I N S T RU C T U R E S
More than 150 years ago (1858), Virchow commented on the
presence of glia around blood vessels, and almost a century
ago (1913), Ramon y Cajal suggested that astrocytes are ide-
ally situated to relay information between neurons and the
microvasculature. Single astrocytes extend processes that con-
tact a large number of synapses (Ventura and Harris 1999),
other neighboring astrocytes (Massa and Mugnaini 1982),
and the cerebrovasculature (Simard et al. 2003). Contacts
between astrocytes and brain microvessels are made by end-
feet, enlarged compartments at the distal end of astrocytic
processes, which ensheathe microvessels. Collectively, all
astrocyte endfeet completely circumscribe the brain microvas-
culature. The thin barrier of astrocyte endfeet enclosing the
blood vessels within the brain is known as the glia limitans
perivascularis. Structural studies have suggested that astrocyte
endfeet could allow direct exchanges of signaling molecules
and metabolites between neurons and microvessels (Kacem
et al. 1998).
The microvasculature consists of arterioles, veins, and cap-
illaries. Arterioles are covered by a continuous layer of smooth
muscle formed of annuli around the vessel. The constriction
and dilation of these smooth muscle cells cause alterations
in the diameter of arterioles and, thereby, blood flow. Veins
have only a thin layer of smooth muscle cells surrounding the
endothelial cell layer. By contrast, capillaries lack a continuous
layer of smooth muscle; instead, contractile cells called peri-
cytes are located along the capillary wall. Pericytes have recently
been shown to alter capillary diameter in vitro (Peppiatt et al.
2006) and to constrict brain capillaries in response to ischemia
in vivo (Yemisci et al. 2009). However, their role in functional
hyperemia remains unclear because in vivo studies failed to
find evidence of pericyte-mediated increases in blood flow in
response to neural activity (Fernandez-Klett et al. 2010).
3.2 K N OWN S P EC I A L I Z AT I O NS O F E N D FE ET
T H AT A R E R E L AT E D TO VA S C U L A R C O N T RO L
One of the first suggestions that astrocytes have a dynamic
and active role in brain function, rather than simply being
structural support, came from evidence that glutamate could
induce [Ca2+]i oscillations in cultured astrocytes that propa-
gated as waves between adjacent astrocytes (Cornell-Bell et al.
• 471
1990). [Ca2+]i waves in brain slices were also elicited by neu-
ronally released glutamate that acted via astrocytic metabotro-
pic glutamate receptors (mGluRs), suggesting that astrocyte
networks could act as a signaling system in response to syn-
aptic activity (Dani et al. 1992; Pasti et al. 1997; Porter and
McCarthy 1995).
Astrocytic endfeet have many specializations thought to
be important for vascular control, including ion channels (see
chapter 16), receptors (see chapter 17), and signaling path-
ways, allowing astrocytes to detect changes in neural activity
and convey this information to the vasculature. The gap junc-
tion protein connexin-43 and the purinergic receptors P2Y2
and P2Y4 are highly expressed in astrocyte vascular endfeet
in situ (Simard et al. 2003). Calcium signaling is mediated by
ATP release from astrocytes via the activation of astrocytic
purinergic receptors (Arcuino et al. 2002; Guthrie et al. 1999),
whereas connexin-43 is thought to regulate Ca2+ signaling by
controlling ATP release (Cotrina et al. 1998). Hence, vascular
endfeet are well equipped for the generation and propagation
of Ca2+ waves.
Therefore, astrocytes are able to respond with an increase
in [Ca2+]i to various neurotransmitters released by synaptic
activity. How this information is communicated to the vascu-
lature, and results in arteriole diameter change is discussed in
section 5.
4 SY N A P T I C M O D U L AT I O N O F
C E R E B R A L B L O O D F L OW VI A
ASTROCY TES
4.1 I N VI VO EVI D E N C E T H AT A S T RO C Y T E
C A L C IUM S I G NA L S A LT E R C E R E B R A L B L O O D
FL OW A N D/O R A RT E R I O L E D I A M ET E R
In vivo work into the role that astrocyte [Ca2+]i signals play
in functional hyperemia was sparked by early in vitro find-
ings suggesting that astrocytes were critically involved in the
dynamic matching of CBF to neuronal energy demands.
One of the first pieces of evidence that astrocytes may
participate in neurovascular coupling was presented by the
Carmignoto lab in 2003. In acutely isolated cortical slices, fol-
lowing mGluR activation or synaptic stimulation, Zonta et al.
(2003a) observed astrocyte soma and endfeet [Ca2+]i signals
that were correlated with the time course of vasodilation.
Similar responses were seen when individual astrocytes were
stimulated with patch pipettes. Pharmacologically inhibiting
cyclooxygenase (COX) blocked the vasodilation, implying
that COX products (e.g., prostaglandin E2 [PGE2]) played a
role in the vasodilation. Zonta et al. (2003a) extended their
findings to the in vivo case, using LDF to demonstrate that
the CBF increase in the contralateral somatosensory cortex
following forepaw stimulation was reduced by an mGluR
antagonist. This suggested that mGluRs play a key role in
functional hyperemia in vivo. A year later, in hippocampal
slices, the MacVicar lab used two-photon fluorescence micros-
copy and uncaging to evoke [Ca2+]i transients directly within
an astrocyte soma. [Ca2+]i waves spread through the network
472 • FUNCTIONS OF NEUROGLIAL CELLS
of adjacent astrocytes and their endfeet. Although both these
groups evoked a [Ca2+]i increase in astrocytes, opposite vas-
cular responses were observed with Zonta et al. (2003a)
observing vasodilation, whereas Mulligan and MacVicar
(2004) observed vasoconstriction. Pharmacological inhibi-
tion of 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis
implicated the arachidonic acid (AA) metabolite, 20-HETE
in the arteriolar constriction. By using ultraviolet (UV) light
to uncage Ca2+ in astrocyte endfeet, Takano et al. (2006)
examined the role of astrocyte signals in arteriole diameter
control in vivo. Similar to the in vitro findings of Mulligan
and MacVicar (2004), when Ca2+ was uncaged in the astro-
cyte endfoot, a [Ca2+]i rise was triggered within the astrocyte;
however, in this case the [Ca2+]i rise was followed by dilation
of the adjacent arteriole.
Combining all these data, it seems that astrocytes relay
information to the vasculature via a Ca2+-evoked AA pathway
and there may be other factors that determine whether vaso-
dilation or vasoconstriction occurs. These issues are discussed
in greater detail later.
There is a growing body of evidence that astrocytic glu-
tamate uptake may provide an alternative pathway by which
astrocytes can control CBF. In the olfactory bulb in vivo,
odor-evoked intrinsic optical signals (IOS, used as a proxy for
CBF changes) were found to be coupled to action potential–
evoked glutamate release, but surprisingly, were independent
of ionotropic or metabotropic glutamate receptors (Gurden
et al. 2006). However, a broad-spectrum glutamate trans-
porter blocker, threo-β-benzyloxyaspartic acid (TBOA),
inhibited the odor-evoked increase in IOS, suggesting that
glial uptake of glutamate mediates the increase in CBF. More
recently, Schummers et al. (2008) combined 2-photon imag-
ing with IOS to investigate the link between the response of
astrocytes to a visual stimulus and hemodynamic signals in the
visual cortex. In agreement with the earlier study in the olfac-
tory bulb, 2-photon imaging of cells loaded with a calcium
indicator dye revealed that both the increase in astrocytic
[Ca2+]i and the IOS response evoked by a visual stimulus were
significantly reduced in the presence of TBOA. Although
the astrocytic [Ca2+]i transients observed here demonstrated
a longer latency than the neuronal responses, Ca2+ signals
within endfeet were not examined; hence, no conclusion can
be drawn about their timescale. Intrinsic optical signals are
commonly assumed to reflect a change in hemoglobin oxygen-
ation and/or blood volume; however, changes in IOS can also
be caused by changes in light scattering owing to astrocyte cell
volume changes. Nonetheless, further evidence supporting a
role for astrocyte uptake of glutamate in the control of CBF
was presented by Petzold et al. (2008), who used multipho-
ton microscopy to reveal that odor-evoked [Ca2+]i elevations
in astrocytic endfeet were strongly correlated, both tempo-
rally and spatially, with increases in arteriolar cross-sectional
area. Pharmacological experiments implicated two indepen-
dent pathways by which glutamate could mediate functional
hyperemia. The first was via mGluR5, which in the olfactory
glomerulus is exclusively expressed by astrocytes. Injection
of 2-methyl-6-(phenylethynyl)pyridine (MPEP), a selective
mGluR5 antagonist, into the bulb resulted in a large reduction
in both odor-evoked astrocytic [Ca2+]i increases and functional Membrane phopholipids
hyperemia. The second was via astrocytic glutamate uptake.
Blocking glial glutamate transporters reduced odor-evoked Phospholipase A2 Ca2+
increases in blood flow, but in contrast to the results in the
visual cortex (Schummers et al. 2008), had no effect on the Arachidonic Acid
astrocyte calcium response. These studies imply that astrocytic
glutamate uptake is an important pathway contributing to the

Cyclooxygenases
regulation of arteriole diameter. However, the cellular mecha-

Lipoxygenase
nisms underlying this pathway are as yet unresolved.
After a decade of research investigating astrocytes as poten-

Epoxygenases
tial mediators of functional hyperemia, the idea remains contro-

ω–hydroxylase
versial. The presence, prevalence, and timing of astrocyte [Ca2+]i

(CYP4A)
signaling in response to neural activity and its role in vivo with HPETE
PgH2
respect to control of CBF is still hotly debated. Using in vivo
2-photon imaging of astrocytes within the mouse whisker bar- EETs
rel cortex, Wang et al. (2006) reported only delayed increases
in astrocyte [Ca2+]i in response to whisker stimulation. It has
20-HETE Leukotrienes
been suggested that these [Ca2+]i transients, which peak at 9
seconds, are too slow to mediate functional hyperemia, which
can occur within hundreds of milliseconds after the onset of
neuronal activity (Kleinfeld et al. 1998; Zonta et al. 2003a).

Thromboxane
Prostacyclin
However, in contrast to these observations, other groups have

synthase

synthase

synthase

synthase

synthase
PgD
PgE

PgF
found evidence consistent with a role for astrocytes in func-
tional hyperemia. Using two-photon in vivo imaging, Winship
et al. (2007) reported that approximately 5% of astrocytes in
the hindlimb area of the mouse primary somatosensory cortex
displayed short-latency, limb-specific [Ca2+]i transients in their PgI2 PgE2 PgD2 PgF2a TxA2
soma and endfeet. The kinetics of these Ca2+ signals are on a
similar timescale to neuronal activity and are correlated with Figure 37.1 Following the activation of PLA2, arachidonic acid is formed
the onset of functional hyperemia, measured by IOS. from membrane phospholipids. Arachidonic acid is metabolized by the
enzymes shown to form vasodilators (EETs, PGE2, PgD2, and PgI2) or
vasoconstrictors (PgH2, 20-HETE, PgF2α, TxA2, and leukotrienes).

5 BIDIRECTIONAL CONTROL OF
A RT E R I O L E D I A M ET E R BY A S T R O C Y T E
CALCIUM
5.1 A R AC H I D O N I C AC I D M ETA B O L I T E S
A N D VA S C U L A R C H A N G E S
Increases in astrocyte [Ca2+]i have been reported to evoke both
vasodilation (Petzold et al. 2008; Takano et al. 2006; Zonta
et al. 2003a) and vasoconstriction (Chuquet et al. 2007;
Mulligan and MacVicar 2004). Both responses can result from
neuronally released glutamate acting on astrocyte mGluRs,
increasing astrocyte [Ca2+]i (Pasti et al. 1997). Elevations
in astrocyte [Ca2+]i activate phospholipase A2 (PLA2), a
Ca2+-sensitive enzyme that is highly expressed in astrocytes
(Cahoy et al. 2008), resulting in arachidonic acid (AA)
release from the plasma membrane. Arachidonic acid can be
metabolized within the astrocyte to produce COX-generated
derivatives such as PGE2 and cytochrome P450 (CYP) epox-
ygenase-derived epoxyeicosatrienoic acids (EETs) (Fig. 37.1).
Arachidonic acid is highly diffusible and can be released from
the astrocyte and metabolized within the smooth muscle cell
to form 20-HETE. Hence, astrocytes are capable of produc-
ing and releasing several vasoactive compounds in response
to synaptic activity, evoking vasodilation or vasoconstriction
depending on which AA metabolism pathway dominates.
5.2 P RO S TAG L A N D I N E2 A N D
E P OX Y E I C O S AT R I E N O I C AC I D S
C AUS E D I L AT I O NS
Immunohistochemistry using antibodies against COX-1 and
GFAP revealed that astrocytes in situ express COX-1 in their
endfeet (Gordon et al. 2008; Takano et al. 2006). Notably,
astrocyte processes far from vessels are negative for COX-1
(Takano et al. 2006), suggesting that COX-1 is localized to
the vascular region. In cultured astrocytes, COX activity
triggered by glutamate agonists resulted in the generation of
PGE2 (Zonta et al. 2003b), a vasodilator (Niwa et al. 2000;
Takano et al. 2006; Zonta et al. 2003a). Following its release
from astrocyte endfeet, PGE2 can bind to EP4 receptors on
smooth muscle cells, evoking hyperpolarization and vasodi-
lation. Based on results using COX inhibitors (Takano et al.
2006; Zonta et al. 2003a), PGE2 was suggested to mediate
a significant component of the dilatory response to raised
astrocyte [Ca2+]i. However, controversy remains regarding
the role of COX-1 in astrocyte-mediated vasodilation in
response to increased neural activity in vivo. Although a
high dose of the COX-1 specific inhibitor SC560 (500μM)
can inhibit the increase in CBF in response to odorant
stimulation of the olfactory bulb (Petzold et al. 2008) or

A S T R O C Y T E R E GU L AT I O N O F N E U R O VA S C U L A R C O N T R O L • 473
uncaging of Ca2+ in astrocytes (Takano et al. 2006), several
groups have found that lower doses have no effect on the
CBF response to whisker stimulation (Liu et al. 2011a; Niwa
et al. 2001). In addition, using genetic knock-out of COX-1
had no effect on functional hyperemia, whereas attenuating
hypercapnia-evoked CBF increases (Niwa et al. 2001). In
contrast, pharmacological inhibition or genetic knock-out of
COX-2 leads to a reduction in the CBF response to neuronal
activation (Liu et al. 2011a; Niwa et al. 2000). As COX-2 is
more highly expressed in neurons than astrocytes, neuronal
COX activity and release of COX products may underlie
functional hyperemia.
Astrocytes express CYP epoxygenase, CYP 2C11, which
can metabolize AA to form vasodilatory epoxyeicosa-
trienoic acids (EETs) (Alkayed et al. 1996; Ellis et al. 1990).
Epoxyeicosatrienoic acids activate K+ channels and hyper-
polarize smooth muscle cells, thus inhibiting voltage-gated
Ca2+ channels and evoking vasodilation (Alkayed et al. 1997;
Gebremedhin et al. 1992; Hu and Kim 1993). In cultured
astrocytes, the production of EETs can be stimulated by gluta-
mate agonists (Alkayed et al. 1997). In vivo evidence has sug-
gested that the cortical increase in CBF in response to both
mGluR activation (Liu et al. 2011b) and whisker stimulation
(Liu et al. 2011a) is largely dependent on EET production.
Furthermore, in ex vivo retina, pharmacological inhibition
of EET synthesis reduced the magnitude of vasodilations
observed in response to either light stimulation or uncaging
of Ca2+ within glial cells (Metea and Newman 2006), whereas
inhibition of COX activity had no effect, suggesting that
glial-evoked vasodilations in the retina are mediated by EETs
rather than COX products.
5.3 20-H Y D ROX Y E I C O S AT ET R A E N O I C
AC I D C AUS E S C O NS T R I C T I O NS
Downstream of Ca2+-evoked activation of PLA2, AA can dif-
fuse from the astrocyte endfoot to the smooth muscle layer
surrounding the arteriole, producing 20-HETE (Lange et al.
1997; Mulligan and MacVicar 2004) via ω-hydroxylases
(Roman 2002). 20-hydroxyeicosatetraenoic acid inhibits K+
channels, thus evoking more depolarization activated Ca2+
entry, and hence, arteriole constriction (Lange et al. 1997)
(Fig. 37.2).
5.4 RO L E S F O R P OTA S S IUM E FFLUX FRO M
A S T RO C Y T E S I N C H A N G I N G A RT E R I O L E TO N E
Increases in extracellular potassium concentration ([K+]o)
evoke vasodilation (Kuschinsky and Wahl 1978) by hyperpo-
larizing smooth muscle cells through the action of inward rec-
tifier K+ (Kir) channels, leading to a decrease in calcium entry
through voltage-gated channels and vasodilation (Knot et al.

Synapse
glu
mGluR
NMDAR
Ca2+ Astrocyte

Ca2+ PLA2*

AA
Neuron nNOS

NO

EETs PgE2
NO

Smooth NO AA 20HETE Basement

muscle cell cGMP* dilation dilation membrane
constriction

Red
blood cell
Endothelial
cells

Figure 37.2 Glutamate-Mediated Pathways of Cerebral Blood Flow Regulation. Synaptically released glutamate can act on neuronal NMDA recep-
tors (NMDAR), increasing [Ca2+]i, activating neuronal nitric oxide synthase (nNOS) and leading to the release of nitric oxide (NO). Nitric oxide can
act directly on the smooth muscle cells, evoking vasodilation via cGMP activation. Glutamate can also act on astrocytic mGluRs, increasing astrocyte
[Ca2+]i, activating PLA2 resulting in the release of AA from the plasma membrane. Arachidonic acid can be metabolized within the astrocyte to form
PGE2 or EETs, which are released and act on the smooth muscle cell, evoking vasodilation. Alternatively, AA can be released and act on smooth
muscle cells, where it is metabolized to the vasoconstrictor 20-HETE.

474 • FUNCTIONS OF NEUROGLIAL CELLS

1996). The high potassium conductance of astrocyte endfeet
(Newman 1986) led to the hypothesis that astrocytic “siphon-
ing” of potassium in response to neuronal activity plays a role
in local blood flow regulation (Paulson and Newman 1987).
Following neuronal activity, a local increase in [K+]o leads to
an influx of K+ into glial cells, depolarizing the glial cells and,
in addition, causing K+ efflux from quiescent regions of the
cell. It was hypothesized that most K+ would be “siphoned”
through the endfeet (where the highest density of Kir 4.1
occurs) (Brew et al. 1986; Newman 1986) onto blood vessels.
However, recent work by the Newman lab found that direct
depolarization of astrocytes with patch pipettes, which should
provide driving force for K+ efflux, did not cause vasodilation,
thus concluding that glial K+ siphoning does not contribute
significantly to neurovascular coupling in the retina (Metea
et al. 2007).
An alternative pathway involving the efflux of K+ through
BK channels (expressed in astrocytes) (Price et al. 2002) has
been proposed (Filosa et al. 2006), although it is difficult to
imagine how such a scenario would occur because glial mem-
brane potentials are close to the equilibrium potential for
potassium (Kuffler et al. 1966), meaning that increasing K+
conductance would not increase the net efflux of K+.
5.5 R E L AT I O NS H I P TO OT H E R K N OWN
FAC TO R S T H AT H AVE VA S CU L A R AC T I O NS
Adenosine triphosphate (ATP), which acts as an extracellular
messenger to propagate astrocytic [Ca2+]i waves in response to
stimulation (Guthrie et al. 1999), is also a potent vasomodu-
lator (Peppiatt et al. 2006). Adenosine, produced when ATP
is broken down by ectoenzymes during neuronal activation,
has been shown to be vasodilatory in both the cerebral cortex
and cerebellum, and is thought to be involved in the coupling
of CBF to neuronal activity (Akgoren et al. 1997; Dirnagl
et al. 1994). Not only does adenosine play a significant role
in the functional hyperemia response in vivo (Shi et al. 2008),
but also it can potentiate the [Ca2+]i increase in both retina
(Newman 2003) and cerebellum ( Jimenez et al. 1998). Hence,
the fast and transient ATP-evoked increase in astrocyte [Ca2+]i
is potentiated by the ectoenzymatic breakdown product of
ATP, adenosine.
In the parenchyma, both the partial pressure of oxygen
(pO2) (Offenhauser et al. 2005), and extracellular lactate
concentration (Hu and Wilson 1997), change during neural
activity. Recently, Gordon et al. (2008) demonstrated that
the metabolic state of brain tissue determines the polarity of
the response to increased astrocyte [Ca2+]i. Clamping O2 at a
high or low level evoked vasoconstrictions and vasodilations,
respectively, in response to uncaging Ca2+ within astrocytes.
Low O2 was achieved by bubbling ACSF with 20% O2, 5%
CO2, resulting in a pO2 of approximately 19 mmHg at 50
μm below the surface, where many imaging and electrophysi-
ology experiments are performed, which is similar to physi-
ological pO2 levels (12.7–64.4 mmHg in vivo) (Offenhauser
et al. 2005). At these lower O2 levels, the metabolic
state of the tissue was altered, resulting in raised levels of
A S T R O C Y T E R E GU L AT I O N O F N E U R O VA S C U L A R C O N T R O L
lactate and adenosine. Under these conditions, vasodilation by
PGE2 was facilitated because extracellular lactate can inhibit
prostaglandin uptake (Chan et al. 2002), whereas adenosine
can act directly on smooth muscle A2A receptors, blocking
Ca2+ channels (Murphy et al. 2003) and preventing vaso-
constriction. These findings are supported by several studies
in vivo in animals and humans, demonstrating that changing
the external lactate/pyruvate ratio, and hence directly con-
trolling the intracellular NADH/NAD+ ratio, regulates CBF
(Ido et al. 2004; Mintun et al. 2004; Vlassenko et al. 2006).
More recently, Lin et al. (2010) used NMR spectroscopy to
demonstrate a strong correlation between the lactate signal
and CBF increases. However, the role of O2 levels in deter-
mining functional hyperemia remains controversial. In one
study, changing tissue pO2 by altering the level of O2 breathed
in resulted in a change in basal CBF, the direction of which
matched the predictions of the in vitro work (Lu et al. 2009).
However, in more recent work the functional hyperemia
response to sensory stimulation was not altered in animals
in a hyperbaric chamber breathing high O2 (Lindauer et al.
2010). It is interesting to note that although forepaw stimu-
lation evokes an increase in CBF and glucose metabolism in
the contralateral hemisphere as expected, there is a decrease
in CBF but an increase in glucose metabolism in the ipsilat-
eral hemisphere (Devor et al. 2008), indicating that a complex
interaction occurs between metabolic and neurogenic CBF
signaling factors.
Blanco et al. (2008) hypothesized that there is a vascu-
lar tone set point that modulates the polarity of the arte-
riole’s response to astrocyte signals. As vascular tone was
increased, larger vasodilation responses were evoked by
increased [K+]o, whereas the polarity of the response to
mGluR activation was found to be dependent on initial levels
of tone. The importance of this pathway in vivo remains to be
determined.
It is likely that all the elements described in this chapter act
together to ensure that information about increased synaptic
activity is relayed to the vasculature via the astrocyte network
so as to match blood flow with neuronal energy demands.
This feed forward model allows for faster alterations of CBF
in response to neural activity compared with the previously
suggested negative feedback models that rely on the buildup
of metabolic products such as CO2 or an increase in the use
of O2 or glucose.
6 I N F LU E N C E O F N E U R O N S O N
A S T R O C Y T E –VA S C U L A R C O U P L I N G
6.1 T R A NS M IT T E R AC T I VAT I O N O F
A S T RO C Y T E C A L C IUM S I G NA L S L E A D I N G TO
C A L C IU M WAVE S A N D VA S C U L A R C H A N G E S
Local blood flow control is achieved by fast neurotransmitter-
mediated signaling (Attwell and Iadecola 2002). Both glu-
tamate and NMDA elicit vasodilatory responses in neocor-
tical and hippocampal microvessels (Fergus and Lee 1997b).
• 475
Astrocytes are ideally located because of their close proximity
to synaptic clefts, to receive synaptic information via released
neurotransmitters. Appropriately, astrocytes in situ and in vivo
express several neurotransmitter receptors, including gluta-
matergic, adrenergic, GABAergic, and purinergic receptors
(see chapters 17 and 25) (see review by Porter and McCarthy
1997, and references therein). These receptors are coupled to
intracellular signaling pathways, which can lead to changes in
vascular diameter and/or cerebral blood flow. Astrocytes in situ
express mGluRs, in particular mGluR5 (van den Pol et al. 1995).
During neuronal activity, glutamate is released, which can act
on astrocyte mGluRs, raising astrocyte [Ca2+]i (Pasti et al. 1997)
resulting in AA-mediated changes in arteriole diameter.
In the brain, astrocytes are a major target for noradrenergic
innervations. Astrocytes in situ have shown immunoreactiv-
ity for β-adrenergic receptors in the cortex and neostriatum
(Aoki et al. 1987; Liu et al. 1992), whereas astrocytes in the
hippocampus, trigeminal motor nucleus, cerebral cortex,
striatum, and cerebellum express α1-adrenoceptors (Duff y
and MacVicar 1995; Shao and Sutin 1992). In vivo, CBF is
decreased following activation by noradrenaline (Raichle
et al. 1975). In addition to its direct effects on smooth muscle
cells, noradrenaline can evoke Ca2+ transients in astrocytes in
hippocampal slices (Duff y and MacVicar 1995) and the cortex
in vivo (Bekar et al. 2008). This increase in astrocyte [Ca2+]i
can result in the release of AA from astrocyte endfeet and its
conversion to 20-HETE.
Functional GABA receptors are expressed by astrocytes in
the hippocampus (Fraser et al. 1995) and Bergmann glia in the
cerebellum (Muller et al. 1994). The inhibitory neurotransmit-
ter, GABA, has been implicated in neurovascular signaling in
the hippocampus (Fergus and Lee 1997a), where activation of
GABAA receptors (Fraser et al. 1995) results in an efflux of Cl–
and depolarization of astrocytes, activating voltage-gated Ca2+
channels and increasing astrocyte [Ca2+]i. In addition, GABA
released from hippocampal interneurons causes GABAB
receptor–mediated elevations of [Ca2+]i in astrocytes (Kang
et al. 1998), in a mechanism analogous to that proposed for
glutamate (Zonta et al. 2003a), and dilates arterioles (Fergus
and Lee 1997a).
6.2 R E L E A S E O F N I T R I C OX I D E F RO M N I T R I C
OX I D E SY N T H A S E AC T I VAT I O N
Synaptically released glutamate can act on NMDA/AMPA
receptors to raise neuronal [Ca2+]i, activating neuronal nitric
oxide synthase (nNOS) (Fergus and Lee 1997b). The release
of nitric oxide (NO) activates guanylate cyclase in vascular
smooth muscle, producing cyclic guanosine monophosphate
(cGMP). Cyclic guanosine monophosphate activates K+
channels, producing a hyperpolarization that reduces calcium
entry into the muscle, resulting in vasodilation and increased
blood flow (Akgoren et al. 1994; Robertson et al. 1993).
In the cortex, inhibition of nNOS reduced the increase in
blood flow evoked by neuronal activity (Ma et al. 1996). The
response can be restored by NO donors, suggesting that NO is
a modulator rather than a mediator of the blood flow response
(Lindauer et al. 1999). In addition to the direct effects of NO
476 • FUNCTIONS OF NEUROGLIAL CELLS
on the vasculature, many enzymes responsible for the produc-
tion of vasoactive molecules from AA are sensitive to NO.
Pharmacological experiments in vivo suggested that increased
NO production by nNOS during functional activation in the
whisker barrel cortex may act to suppress 20-HETE synthesis
or its downstream signaling and so promote EETs-dependent
vasodilation (Liu et al. 2008). In contrast to the cortex, in the
cerebellum NO donors do not reverse the effect of inhibiting
nNOS; hence, NO is a mediator of the functional hyperemia
response in cerebellum (Akgoren et al. 1996).
Nitric oxide levels have been hypothesized to dictate
whether vasodilation or vasoconstriction occurs in response
to increased glial [Ca2+]i in the retina (Metea and Newman
2006). Constrictions within the retina were caused by AA
conversion to 20-HETE, in agreement with work in brain
slices (Mulligan and MacVicar 2004), whereas vasodila-
tions were caused by conversion of AA into EETS. Nitric
oxide can inactivate CYP2C11 (Roman 2002), thus inhibit-
ing synthesis of EETs and vasodilation. As NO levels in the
retina rose, the frequency of vasodilations diminished, reveal-
ing only vasoconstrictions (Metea and Newman 2006). The
MacVicar group had also observed that vasoconstrictions in
response to astrocyte stimulation were switched to vasodila-
tions in the presence of the NO synthase inhibitor, L-NAME
(Mulligan and MacVicar 2004). Initially this was thought to
be caused by changes in resting vessel tone, because L-NAME
causes preconstriction of vessels, thereby predisposing them to
dilate (Blanco et al. 2008). Differences in the basal level of the
NO of the two preparations may account for the differences
seen, that is, the NO level of brain slices may be higher than
in the retina, explaining the prevalence of vasoconstrictions
observed by the MacVicar group, as the EET pathway could
be blocked by the basal level of NO. In addition to the effect
of NO on the CYP2C11 enzyme, the enzymes responsible for
producing 20-HETE are also sensitive to NO (Roman 2002),
suggesting that a complex relationship may exist between NO
levels and vasomotor responses. Several groups have demon-
strated that interactions exist among the NO, cyclooxygenase,
and epoxygenase pathways. Combining inhibition of these
pathways does not result in a greater block of CBF increase
than inhibiting them individually (Liu et al. 2011a; Peng et al.
2004) (Fig. 37.3).
7 A LT E R AT I O N S O F A S T R O C Y T E A N D
N E U R O VA S C U L A R C O U P L I N G I N
PAT H O L O G I C A L C O N D I T I O N S
Failure to provide sufficient blood flow to match the energy
demands of neurons can lead to cell damage or death follow-
ing several pathological conditions, including ischemic stroke,
secondary ischemia after spinal cord injury, and vasospasm
after subarachnoid hemorrhage. Hence, alterations in neu-
rovascular coupling may underlie neurological deficits that
occur following such conditions.
Ischemic stroke occurs when there is a failure of the brain
blood flow because of a blockage (e.g., a blood clot in an arte-
riole). Following clearance of the blockage there is an initial
 Synapse
glu
NMDAR
Ca 2+
Neuron nNOS
NO
NO
Smooth NO
cGMP* dilation
muscle cell
Red
blood cell
Figure 37.3 Nitric Oxide Effects on Glutamate-Mediated Retinal Blood Flow Changes. As seen in Figure 37.2, glutamate can lead to the release of NO
from neurons. Nitric oxide can evoke vasodilation via cGMP activation. Alternatively, NO can inhibit the conversion of AA to 20-HETE by the
enzyme ω-hydroxylase, hence promoting vasodilation. Nitric oxide can also inhibit dilation by inhibiting CYP450 epoxygenase, which converts AA to
EETs. These enzymes have different sensitivities to NO.
transient increase in blood flow followed by a decrease that
can last for several hours (Ames et al. 1968; Hauck et al. 2004;
Nelson et al. 1992). It was originally hypothesized that this
long-lasting decrease in blood flow, which may result in fur-
ther damage to cells after the initial stroke damage, was caused
by astrocyte endfeet swelling, causing capillary diameters to
be reduced and capillaries to become blocked by blood cells
(Ames et al. 1968). However, the importance of this effect is
debated (Theilen et al. 1994), and it has been suggested that
a failure of arteriole vasodilatory mechanisms underlies the
CBF decrease (Leffler et al. 1989; Nelson et al. 1992). Because
it is difficult to tell these two situations apart experimentally, it
remains unclear which pathway is more important. Recently,
pericyte constrictions were proposed to play a key role in
the long-lasting decrease in blood flow (Peppiatt et al. 2006;
Yemisci et al. 2009).
Cortical spreading depression (CSD) is a phenomenon that
occurs during migraine and following stroke, brain trauma or
subarachnoid hemorrhage. During CSD, there is a transient rise
in [K+]o from approximately 3 mM to approximately 50 mM,
leading to depolarization of neurons and astrocytes (resulting
in increased [Ca2+]i) and a rise in glutamate (Vanharreveld
and Kooiman 1965) because of release by neurons and a fail-
ure of uptake by glia (Barbour et al. 1988). Although energy
use by the brain increases during CSD, after an initial transi-
ent increase, CBF is decreased for hours (Fabricius et al. 1995;
Takano et al. 2007). This mismatch between energy supply and
demand may result in increased neuronal damage following
A S T R O C Y T E R E GU L AT I O N O F N E U R O VA S C U L A R C O N T R O L
mGluR
Ca2+ Muller cell
or
PLA2* Astrocyte
AA
EETs
20HETE AA Basement
dilation
constriction membrane
Endothelial
cells
subarachnoid hemorrhage (Dreier et al. 2006). The transient
increase in blood flow is thought to be caused by the release
of calcitonin gene–related peptide (CGRP) by trigeminal
neurons (Wahl et al. 1994), although the target cell of CGRP
remains to be confirmed. In addition, vasodilation may, in part,
be caused by the release of NO (Fabricius et al. 1995; Wahl
et al. 1994), possibly because of NO inhibition of the synthesis
of 20-HETE. In vivo, two-photon imaging of astrocytes loaded
with a calcium indicator dye combined with vascular labeling
with FITC dextran revealed that vasoconstrictions of penetrat-
ing cortical arterioles occurred during CSD at the onset of a
fast astrocyte [Ca2+]i wave (Chuquet et al. 2007). The evoked
vasoconstriction was inhibited in the presence of thapsigargin,
which inhibits the refilling of internal calcium stores, and
Methyl arachidonyl fluorophosphonate (MAFP), which inhib-
its PLA2, suggesting that astrocytes mediate the CSD-induced
vasoconstriction that occurs via the PLA2-mediated release of
AA, in agreement with previous in vitro findings (Mulligan and
MacVicar 2004). However, this study only investigated the ini-
tial vasoconstriction that occurs before the transient increase in
CBF. Impairments in the NO system (Fabricius et al. 1995) are
partly responsible for the disruption in functional hyperemia
that occurs after CSD (Wahl et al. 1987). A decrease in NO
levels could result in either less dilation of vessels because of a
direct effect of NO on smooth muscle cells or less inhibition of
20-HETE production by NO.
Finally, in Alzheimer disease, which is characterized by the
deposition of amyloid-β peptide in the neuropil and blood
• 477

vessels and the accumulation of neurofibrillary tangles in
neurons, resting CBF is reduced and functional hyperemia is
impaired (Girouard and Iadecola 2006). Amyloid-β peptide
promotes oxidative stress, which inhibits the production of
vasodilating messengers; hence, decreasing functional hyper-
emia (Sun et al. 2008; Zou et al. 1999). Furthermore, mice
with amyloid-β plaques had an enhanced occurrence of spon-
taneous astrocyte [Ca2+]i waves (Kuchibhotla et al. 2009) and
increased contractility of vascular smooth muscle cells (Chow
et al. 2007), these alterations could underlie changes in neuro-
vascular coupling.
8 S U M M A RY A N D P E R S P E C T I VE S
Over the past two decades much progress has been made in
understanding the role of astrocytes and their Ca2+ signals in
the regulation of arteriole diameter, and thereby blood flow in
response to changes in synaptic activity. It is clear that astro-
cytes are ideally positioned to sense changes in synaptic activity
and relay this information to the cerebrovasculature, releasing
gliotransmitters from their endfeet onto smooth muscle cells
and thus evoking changes in arteriole diameter. However, sev-
eral questions remain. Do astrocyte Ca2+ signals reliably occur
under physiological conditions and, if so, is their timing relevant
to the regulation of CBF? What determines whether a vaso-
constriction or vasodilation response dominates in vivo? Does
this factor depend on the physiological conditions or stimulus?
What role does glial glutamate uptake play in the regulation
of CBF and how is this effect mediated? The use of new tech-
niques such as genetic manipulation (including optogenetics,
gene knock-out, and siRNA techniques), which enable specific
manipulation of astrocytes, their Ca2+ signaling, and the down-
stream pathways, which result in changes in arteriole diameter,
will allow researchers to address these questions. The combina-
tion of optogenetics with BOLD fMRI will be a powerful tool
to allow researchers to specifically target activation of astro-
cytes while using genetic or pharmacological manipulation to
alter the pathways underlying functional hyperemia.
AC K N OW L E D G M E N T S
CH is a Sir Henry Wellcome Postdoctoral Fellow, a
Government of Canada Postdoctoral Fellow, and a Michael
Smith Foundation for Health Research Postdoctoral Fellow.
BAM is a Canada Research Chair Tier 1 in Neuroscience.
GRJG is a Canada Research Chair Tier 2 and a Heart and
Stroke Foundation of Canada McDonald Scholar. BAM is
funded by CIHR, the Fondation Leducq, Human Frontiers
Science Program, and Heart and Stroke Foundation.
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 38.
ASTROCY TES: MODULATION OF SYNAPTIC FUNCTION
AND NET WORK ACTIVIT Y
Andrea Volterra

A B B R E VI AT I O N S the previously unknown roles of these cells in the control of

synapse formation, function, and plasticity. The communi-
AMPA 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) cation properties of astrocytes were discovered only recently
propanoic acid because astrocytes differ in their excitation mechanisms from
BAPTA 1,2-bis(o-aminophenoxy)ethane- neurons: They do not generate action potentials and have
N,N,N′,N′-tetraacetic acid “passive” membrane properties, which for many years led
CB Cannabinoid researchers to consider them nonexcitable cells, and there-
DHPG (S)-3,5-dihydroxyphenylglycine fore unable to communicate. However, in the 1990s, the
dnSNARE dominant negative SNAP (soluble NSF development of cellular imaging approaches changed this
attachment protein) receptor protein view. Application of Ca2+ imaging to the study of astrocytes
GABA gamma-aminobutyric acid revealed that these cells display intracellular Ca2+ ([Ca2+]i)
GFAP glial fibrillary acidic protein elevations in response to chemical signals coming from
GPCR G protein–coupled neighboring cells, including from synapses (Dani et al. 1992;
HFS high-frequency stimulation Pasti et al. 1997; Porter and McCarthy 1996). In turn, [Ca2+]i
LTD long-term depression rises were found to trigger release of transmitters from astro-
LTP long-term potentiation receptors cytes able to modulate neuronal functions (Bezzi et al. 1998;
IP3R2 inositol 1,4,5-trisphosphate receptor type 2 Kang et al. 1998; Parpura et al. 1994). Initial data suggested
mEPSC miniature excitatory postsynaptic current the existence of communication loops between synapses and
2MeSADP 2-methylthio-adenosine-5′-diphosphate astrocytes, by which astrocytes can feedback break/accel-
mGluR metabotropic glutamate receptor erator inputs to synapses. This gave rise to the concept of
MrgA1 mas-related gene receptor type A1 gliotransmission (Bezzi and Volterra 2001), subsequently
NMDA N-methyl-d-aspartate expanded to encompass feedforward astrocytic actions in
NPY neuropeptide Y which an astrocyte responds to synaptic activation by releas-
NR2B NMDA receptor subtype 2B ing a transmitter on a synaptic circuit distinct from the one
PP-GC perforant path-granule cell responsible for its activation. The next studies then clarified
SIC slow inward currents that astrocytes are able to produce multiple synaptic effects
SNAP25 synaptosomal-associated protein 25kDa and exert both inhibitory and stimulatory influences. Several
SOC slow outward current gliotransmitters were identified, together with multiple
TNFα tumor necrosis factor-alpha release pathways and a variety of synaptic targets, providing a
TNF-R1 tumor necrosis factor receptor type 1 first hint of the complexity of gliotransmission, governed by
TTX tetrodotoxin astrocytic Ca2+ encoding, whose properties still are not well
VAMP vesicle-associated membrane protein understood. Based on all the evidence, a provocative para-
v-SNARE vesicle SNAP (soluble NSF attachment digm shift in synaptic function was proposed at the end of the
protein) receptor protein 1990s, summarized as the tripartite synapse concept (Araque
et al. 1999; Kettenmann et al. 1996; Volterra et al. 2002). This
suggests that communication at the synapse is not limited to
1 INTRODUCTION presynaptic and postsynaptic compartments, but extends to
neighboring astrocytic processes endowed with receptors for
The last 20 years have brought about a potential conceptual synaptic mediators. It must be emphasized that the tripartite
revolution about the role of astrocytes in synaptic and net- synapse concept, although vividly illustrating the inclusion
work function. In the early 1990s, it was discovered that astro- of astrocytes in synaptic communication, does not define a
cytes possess active properties—notably a competence for specific anatomical unit, because the morphological relations
releasing transmitters, dubbed gliotransmitters as opposed to between astrocytic and synaptic elements differ significantly
neurotransmitters of neuronal origin. Recognizing that astro- in different brain circuits (Ventura and Harris 1999) and are
cytes communicate has been instrumental for understanding subject to dynamic changes (Oliet et al. 2001).

481
 As a counterpart to the advances in the functional under-
standing of astrocytes, parallel new observations were made
on their structural organization. Dye injection of neighboring
astrocytes revealed that these cells have an ordered arrange-
ment with minimal overlap (Bushong et al. 2002), which
allows each astrocyte to cover a specific territory comprising
a few neuronal cell bodies, hundreds of dendrites, and several
thousands of synapses (Halassa et al. 2007). Consequently, it
was proposed that an individual astrocyte might oversee all the
activities undergoing in its territory, providing an extra layer
of intercellular coordination. However, an emerging concept
in parallel is that astrocytes possess specialized microdomains
in their perisynaptic processes able to control fractions of the
astrocytic territory autonomously.
2 A S T R O C Y T E S A R E AC T I VAT E D
BY SY N A P T I C F U N C T I O N
Astrocyte activation during synaptic transmission was first
observed in brain slices. Tetrodotoxin (TTX)-sensitive astro-
cytic [Ca2+]i elevations were reported to occur in several brain
areas, including the hippocampus, cerebral cortex, cerebellum,
nucleus accumbens, olfactory bulb, and retina, in response to
release of a variety of transmitters and factors, such as gluta-
mate, adenosine triphosphate (ATP), gamma-aminobutyric
acid (GABA), acetylcholine, noradrenaline, nitric oxide, and
endocannabinoids (Perea et al. 2009; Volterra and Meldolesi
2005). Most recently, studies were extended to living ani-
mals and confirmed that astrocytes show neuronal activity–
dependent [Ca2+]i elevations triggered by physiological sen-
sory stimuli (Petzold et al. 2008; Schummers et al. 2008;
Wang et al. 2006) or locomotor behavior (Nimmerjahn et al.
2009) (see section 11). Transfer of information from neurons
to astrocytes is thought to occur mostly by spillover of synap-
tically released transmitters, particularly during intense activ-
ity. However, examples of direct synapse–glia communication
do exist, such as the one received by cerebellar Bergmann glia
from “ectopic” presynaptic release sites of climbing fibers
(Matsui and Jahr 2006). Moreover, astrocytes can be acti-
vated by postsynaptic retrograde messengers such as endocan-
nabinoids (Navarrete and Araque 2008, 2010). Synaptically
induced astrocytic [Ca2+]i elevations are thought to be evoked
in most cases by binding of mediators to G protein–coupled
receptors (GPCR) linked to inositol 1,4,5-trisphosphate (IP3)
signaling and Ca2+ release from the endoplasmic reticulum. In
some circuits, activation of Ca2+-permeated ligand-gated recep-
tors, such as AMPAR, was also reported, with direct influx of
Ca2+ from the extracellular medium. Moreover, other types of
Ca2+ channels or exchangers could operate to increase [Ca2+]i
in astrocytes in response to synaptic activity (see chapter 26).
[Ca2+]i elevation in astrocytes is not an “on-off ” type of
signal. On the contrary, there are multiple and varied patterns
of [Ca2+]i elevation, which probably underlie different types of
function, including generation of diverse output signals to syn-
apses. The relevance of differences in amplitude, frequency, and
spatiotemporal properties of [Ca2+]i events generated in astro-
cytes by neuronal activity is under intense investigation. Initial
studies suggested a relation between neuronal input intensity
and astrocytic Ca2+ response (Matyash et al. 2001; Pasti et al.
1997). For example, in the cerebellum, low-frequency firing of
parallel fibers generated [Ca2+]i rises in Bergmann glial cells
restricted to small peripheral cell protrusions, possibly rep-
resenting independent functional microdomains (Grosche
et al. 1999). However, when parallel fibers discharged at high
frequency, several such microdomains were activated and
coordinated, and the ensuing [Ca2+]i rise became generalized
and propagated up to the cell body (Matyash et al. 2001).
The functional difference between the two types of Ca2+ sig-
nal was not defined, but the fact that neuronal activity can
generate highly localized Ca2+ responses in glial processes
suggests that astrocytes contain privileged zones for local
communication with neurons (Grosche et al. 1999), allowing
them to distinguish and integrate incoming activity gener-
ated by different axonal pathways (Perea and Araque 2005).
Recent work (Di Castro et al. 2011; Panatier et al. 2011)

WS
0.5
∆F/F

10 s
2MeSADP
0.3
∆G/R
2s

Figure 38.1 Ca2+ Signals in Astrocytic Processes and Cell Body Have Different Properties and May Underlie Distinct Biological Phenomena. (Blue)
Cell body Ca2+ signal, recorded in the somatosensory cortex in vivo following whisker stimulation (Wang et al. 2006). (Red) Ca2+ signal in an astro-
cytic process, recorded in hippocampal slices in response to local P2Y1R stimulation with 2MeSADP (Santello et al. 2011). Both signals were recorded
using Fluo-4 Ca2+ dye and are scaled in time and amplitude for comparison. Note that the Ca2+ signal in the process is spatially confined (∼4 μm),
occurs immediately on stimulation, is relatively fast (time-to-peak: hundreds milliseconds), short-lasting (∼3 seconds), and of small amplitude,
whereas the signal in the cell body is spatially larger, occurs with a few-second delay from stimulation, is slower (time-to-peak: ∼6 seconds),
longer-lasting (∼30 seconds), and of larger amplitude. Adapted from Wang et al. 2006. Santello et al. 2011.

482 • FUNCTIONS OF NEUROGLIAL CELLS

has strengthened this idea, by showing that individual random
synaptic events or single-action potentials induce highly con-
fined, small, and fast local Ca2+ responses in astrocytic pro-
cesses, distinct from somatic [Ca2+]i rises (Fig. 38.1). These
local phenomena are capable of triggering gliotransmission
and modulating basal transmission at individual synapses.
3 A S T R O C Y T E S R E L E A S E N E U R OAC T I VE
G L I OT R A N S M I T T E R S
Evidence exists for the release of several neuroactive gliotrans-
mitters from astrocytes in situ, including glutamate, D-serine,
ATP (often rapidly metabolized to adenosine), and GABA, as
well as eicosanoids, including arachidonic acid, prostaglandins,
epoxyeicosatrienoic acids (EETs), and the cytokine TNFα (see
chapters 17 and 18). Concerning the mechanisms of release,
Ca2+-dependent exocytosis is the best characterized. There is
direct evidence for Ca2+-dependent secretion of glutamatergic
vesicles in cultured astrocytes (Bezzi et al. 2004; Marchaland
et al. 2008). Such organelles at the EM level appear small
(40- to 100-nm diameter), clear, and round, resembling synap-
tic vesicles in nerve terminals; for this reason they were dubbed
synaptic-like microvesicles (SLMV). There is also evidence for
secretion of D-serine from a similar vesicle population, as well
as for release of ATP from lysosomes and peptides (NPY)
from secretory granules (reviewed in Hamilton and Attwell
2010). Synaptic-like microvesicles have been ultrastructur-
ally identified also in perisynaptic astrocytic processes in the
adult hippocampus, in which immunogold labeling indicates
that they contain glutamate (Bergersen et al. 2011) and express
vesicular glutamate transporters (VGLUTs) and proteins sus-
taining regulated exocytosis, such as the v-SNARE VAMP3/
cellubrevin (Bezzi et al. 2004). The presence of VGLUT
transcripts in adult hippocampal astrocytes is confirmed by
single-cell RT-PCR experiments, and a third, functional line of
evidence supporting SNARE-dependent astrocytic glutamate
release in situ comes from experiments in which tetanus toxin
(TeNT), a protease that specifically inactivates VAMP2 and
VAMP3, was selectively infused in hippocampal astrocytes (see
section 4), leading to the abolishment of glutamate-dependent
synaptic modulation ( Jourdain et al. 2007; Perea and Araque
2007). The same effect was obtained with TeNT when study-
ing astrocyte D-serine (Henneberger et al. 2010) and ATP/
adenosine release (Panatier et al. 2011). At variance with
this evidence, a transcriptome study of forebrain astrocytes
from juvenile mice failed to find transcripts for VGLUTs
and SNARE proteins such as VAMP2 and SNAP25 (Cahoy
et al. 2008). However, VGLUT transcripts in astrocytes are
expected to be much less abundant than in synaptic terminals
(and, therefore, elusive for transcriptomics), given that astro-
cytes lack the large “reserve pool” that accounts for approxi-
mately 90% of synaptic vesicles in neurons. Moreover, adult
hippocampal astrocytes express SNARE protein isoforms
distinct from canonical synaptic ones (Schubert et al. 2011).
Finally, astrocytic exocytosis seems to have peculiar prop-
erties distinguishing it from synaptic exocytosis, such as
TNFα-dependent regulation (Santello et al. 2011). Of course,

Blocking gliotransmission

Ins(1,4,5)P3R
Inhibit TCA cycle No release BAPTA [Ca2+]i knockout
Fluoroacetate or fluorocitrate

Cleaves No Binds No
TeNT VAMP2 dn SNARE
and VAMP3 exocytosis SNARES exocytosis

Stimulating gliotransmission

+ ++
[Ca2+]i I +mV [Ca2+]i

Light

[Ca2+]i Ca2+ [Ca2+]i

P2Y1 MRGA1
2MeSADP RF amide

Figure 38.2 Experimental Strategies Used to Reveal the Role of Gliotransmission in Synaptic Function. (Upper) Methods and genetic models used
to selectively block gliotransmission, including metabolic poisoning of astrocytes, chelation of internal Ca2+, deletion of key astrocyte Ca2+ signal-
ing proteins (e.g., IP3R2), and pharmacological (TeNT) or transgenic (dnSNARE) blockade of exocytosis. (Lower) Methods and genetic models to
selectively stimulate [Ca2+]i elevation and gliotransmission, including mechanical or electrical astrocytic stimulation, activation of endogenous (P2Y1)
or transgenic (MrgA1) GPCR, and Ca2+ uncaging in astrocytes. Adapted from Hamilton and Attwell 2010.

A S T R O C Y T E S : M O D U L AT I O N O F SY N A P T I C F U N C T I O N A N D N ET WO R K AC T I VI T Y • 483
much more work is needed to clarify the many pending aspects
concerning astrocytic gliotransmitter exocytosis (Hamilton
and Attwell 2010).
Adenosine triphosphate, glutamate, GABA, and other
gliotransmitters are apparently released by astrocytes also
via additional Ca2+-dependent and -independent processes,
mediated by large-pore ion channels, such as gap-junction
hemichannels, P2X7 receptor-channels, and transmitter car-
riers or exchangers (Volterra and Meldolesi 2005). In several
cases operation of these release pathways in physiological con-
ditions remains to be demonstrated. However, recent work
has provided evidence that Bestrophin-1 anion channels in
cerebellar glia are a physiological source of GABA release
underlying tonic inhibitory control of cerebellar granule cells
(Lee et al. 2010) (see section 10).
4 E X P E R I M E N TA L PA R A D I G M S TO
S T U DY G L I OT R A N S M I S S I O N
One of the main challenges associated with the emerging idea
that astrocytes play active roles at synapses concerns the meth-
odological approaches to study integrated synapse-astrocyte
functioning and the design of experiments appropriate for
revealing specific astrocytic contributions within established
paradigms of synaptic function. Two complementary strate-
gies have been used to date, based on either selectively acti-
vating or inactivating astrocyte signaling (Fig. 38.2). They are
presented here, and their advantages and limitations are dis-
cussed so as to provide readers with a critical appraisal of the
quality of the evidence presented by different studies.
4.1 S U BT R AC T I VE S T R AT EGY: A P P ROAC H E S
TO S E L EC T I VE LY I NAC T I VAT E
G L I OT R A NS M I S S I O N
These approaches aim at perturbing and abolishing signaling
steps involved in gliotransmission, while in parallel measuring
the consequences on synaptic or network function. Multiple
methods are used to achieve this goal. Some of them rely on
broadly perturbing astrocyte physiology, thereby secondarily
affecting gliotransmission pathways. This can be achieved
with toxins that are selectively taken up by astrocytes, such
as α-amino adipate (Khurgel et al. 1996), or with metabolic
poisons such as fluorocitrate and fluoroacetate, which target
metabolic enzymes selectively present in astrocytes (Swanson
and Graham 1994). These are quite crude approaches that end
up by intoxicating the cells and fully compromising their func-
tion. However, more selective and preferable approaches exist.
In some cases, one can use pharmacological blockers of recep-
tors whose activation triggers gliotransmission, or of enzymes
involved in the synthesis of specific gliotransmitters (e.g.,
D-serine), but only if parallel evidence exists that the target
receptors or enzymes are selectively expressed in astrocytes in
the brain region investigated ( Jourdain et al. 2007; Panatier
et al. 2006). It must be emphasized that extracellular applica-
tion of pharmacological agents in brain slices or in vivo does not
provide per se evidence that they act specifically on astrocytes.
484 • FUNCTIONS OF NEUROGLIAL CELLS
In contrast, an astrocyte-specific pharmacological approach
consists in infusing nonpermeant agents in an individual
astrocyte or a gap junction–connected network of astrocytes
via the patch-pipette used for whole-cell clamping the astro-
cyte. This approach was successfully used to block astrocytic
[Ca2+]i elevations via infusion of the Ca2+ chelator BAPTA
( Jourdain et al. 2007; Serrano et al. 2006), the Ca2+ clamp
solution (Henneberger et al. 2010), or the G protein inhibitor,
GDP-beta-S, which blocks signal transduction downstream
of GPCR (Robitaille 1998). Infusion of the activated TeNT
light-chain, which blocks SNARE protein–dependent vesicle
fusion involved in gliotransmitter release (see section 3), also
has been used successfully ( Jourdain et al. 2007; Perea and
Araque 2007). The internal dialysis approach is significantly
more selective than use of gliotoxins because it affects only spe-
cific pathways, just for the limited time of the experiment and
only in the patched astrocyte or in those directly connected to
it. However, only low MW agents (<1.2 kDa) can diffuse within
minutes via gap junctions to a network of connected astrocytes
and block large communication domains. Selective enrich-
ment in astrocytes over other brain cells of key signaling mol-
ecules such as IP3R2 (Holtzclaw et al. 2002), one of the three
IP3 receptor isoforms, has led to the use of IP3R2ko mice in
gliotransmission studies (Di Castro et al. 2011; Navarrete et al.
2012; Petravicz et al. 2008; Takata et al. 2011). These mice repre-
sent a powerful tool, particularly for bringing analyses from the
cellular/network level to the behavioral level. However, correct
interpretation of the results requires accurate verification that
IP3R2 is knocked-out only in astrocytes and that its permanent
deletion does not trigger compensatory mechanisms. A more
refined genetic approach consists in the conditional, cell-specific
inactivation of gliotransmission signaling steps. In principle, this
was achieved with the creation of the so-called dnSNARE mice,
in which the SNARE domain of SNARE proteins is condition-
ally expressed under the astrocytic GFAP promoter (Pascual
et al. 2005). Expression of this domain has a dominant negative
effect, trapping endogenous SNARE partners and preventing
formation of SNARE complexes necessary for vesicle fusion. An
expected consequence is blockade of gliotransmitter exocytosis,
although the selective occurrence of this event in dnSNARE
mice has not yet been unequivocally demonstrated.
4.2 A D D IT I VE S T R AT EGY: A P P ROAC H E S TO
S E L EC T I VE LY AC T I VAT E G L I OT R A NS M I S S I O N
The opposite approach, selective stimulation of gliotransmis-
sion, has also been used, verifying in parallel its impact on syn-
aptic and network functions. One conceptual problem with
this approach is that it may overlap with ongoing endogenous
gliotransmission. Thus, subtractive approaches often show that
blockade of endogenous gliotransmission leads to synaptic
changes. Therefore, by adding exogenous astrocyte stimulation,
one may not simply amplify the endogenous mechanisms, but
also produce complex interactions with them, which compli-
cates the interpretation of the results. The approaches initially
used privileged selectivity of astrocyte stimulation over repro-
duction of physiological stimuli. Quite harsh approaches such
as mechanical stimulation or strong electrical depolarization of
patched astrocytes were shown to produce [Ca2+]i elevations
that trigger gliotransmission (Araque et al. 1998; Kang et al.
1998). Pharmacological stimulation of GPCR expressed in
astrocytes was also used, although the cell selectivity of these
agents remains an issue. In some cases, use of a GPCR agonist
was coupled to a subtractive approach, showing that the agent
lost its activity when a Ca2+ chelator was selectively introduced
in astrocytes (Santello et al. 2011). A much more targeted
approach consists in locally uncaging glutamate or other astro-
cyte receptor agonists to stimulate gliotransmission (Panatier
et al. 2011; Poskanzer and Yuste 2011). Even if one can restrict
the target area to approximately 200 nm by using two-photon
uncaging, this area is still too large to assure astrocyte speci-
ficity. Thus, a tight contiguity (often only a few nm) exists
between perisynaptic astrocytic processes and synaptic ele-
ments expressing glutamate receptors. This observation is also
valid in the opposite direction, and in interpreting their results,
studies using glutamate uncaging to evaluate the role of presyn-
aptic or postsynaptic glutamate receptors should not exclude
the involvement of astrocytic mechanisms. A more physiolog-
ical and cell-selective approach consists in introducing caged
Ca2+ (or IP3) in the astrocyte via the patch pipette (Fiacco
and McCarthy 2004; Perea and Araque 2007). Although this
approach can generate [Ca2+]i elevations of physiological ampli-
tude, it hardly reproduces the spatiotemporal features of [Ca2+]i
elevations induced by physiological stimuli, features that may
be important in shaping the downstream gliotransmission (see
section 2). A genetic model was created to overcome this issue,
in which a GPCR not endogenously present in brain, MrgA1,
is conditionally expressed only in astrocytes using the GFAP
promoter, and activated on demand by applying its natural
agonists, RF amides (Fiacco et al. 2007). Theoretically, this ele-
gant approach should mimic with high-fidelity the endogenous
[Ca2+]i elevations driving gliotransmission. In practice, MrgA1
activation leads to massive astrocytic [Ca2+]i elevations last-
ing several minutes (Agulhon et al. 2010; Fiacco et al. 2007),
which are far from mimicking the spatiotemporal patterns of
[Ca2+]i elevations elicited by physiological stimuli (see Fig.
38.1) (see section 2). This may be caused by overexpression
of MrgA1 receptors, possibly at sites different than those of
endogenous GPCR. Another elegant approach recently used
to activate astrocytic signaling in vivo is optogenetics, via selec-
tive viral transfection of channel rhodopsin (ChR) in astro-
cytes (Gourine et al. 2010). UV light–evoked ChR activation
is used in neurons to trigger physiological patterns of action
potentials on demand. In astrocytes, ChR activation was found
to induce [Ca2+]i elevations, but the underlying mechanism
and its physiological relevance remain uncertain, given that
these cells normally lack Na+ channels and do not fire action
potentials. In conclusion, critical analysis indicates that sev-
eral clever approaches have been developed to study the role of
gliotransmission in synaptic and network function. However,
no perfect approach exists. Therefore, with the present tools,
researchers should strengthen their conclusions by using more
than one approach in their studies.
5 C O N T R O L O F P R E SY N A P T I C
TR ANSMITTER RELE ASE
Multiple observations indicate that gliotransmission can
modulate presynaptic excitability by controlling the probabil-
ity of synaptic transmitter release directly in nerve terminals
(Fiacco and McCarthy 2004; Jourdain et al. 2007; Navarrete
and Araque 2010; Perea and Araque 2007). Surprisingly,
recent data indicate that astrocytic inputs can also modulate

5
1

4b

NR2B
[Ca 2+]i
3
NMDAR 2
AMPAR 1

GluT Glu
P2Y1R ATP
TNFR TNFα

Figure 38.3 Mechanism and Ultrastructural Correlate of Gliotransmission Inducing Presynaptic Modulation at Perforant Path–Granule Cell
Hippocampal Synapses. (Left) Adenosine triphosphate released from the synapse or astrocytes as a result of synaptic transmission (1) activates P2Y1R (2),
raising astrocyte [Ca2+]i (3), and triggering glutamate release from SLMV (4) facing NR2B-containing pre-NMDAR. N-methyl-d-aspartate receptor
activation increases synaptic transmitter release (5). Ambient TNFα is permissive on astrocyte glutamate release (4b) ( Jourdain et al. 2007; Santello
et al. 2011). (Right) Immunogold EM showing preferential NR2B expression (arrows) in the extrasynaptic region of an excitatory nerve terminal (ter)
facing an astrocytic process (ast) carrying SLMV (arrowheads, magnified in inset). Adapted from Jourdain et al. 2007.

A S T R O C Y T E S : M O D U L AT I O N O F SY N A P T I C F U N C T I O N A N D N ET WO R K AC T I VI T Y • 485
action potentials (Sasaki et al. 2011). Thus, astrocytes would
send inputs to CA3 pyramidal cell axons (e.g., by releasing glu-
tamate acting on axonal AMPA/kainate receptors), thereby
causing action potentials to broaden and resulting in enhanced
synaptic transmitter release. A direct local modulatory action
at the level of excitatory nerve terminals was described in the
hippocampal dentate gyrus (Di Castro et al. 2011; Jourdain
et al. 2007; Santello et al. 2011). There, astrocytes sense syn-
aptic activity at perforant path-granule cell (PP-GC) syn-
apses, respond with local [Ca2+]i elevations in their processes,
mediated by GPCR such as P2Y1 purinergic receptors, and
increase the probability of transmitter release at individual GC
synapses. This effect depends on astrocyte glutamate release
activating ifenprodil-sensitive NMDA receptors present on
the excitatory terminals (pre-NMDAR). Pre-NMDAR acti-
vation increases synaptic release probability via an unknown
mechanism, as indicated by the observed effects: An increase
in mEPSC frequency (without parallel change in amplitude
and kinetics of the events), and an increase in the amplitude
of evoked EPSCs accompanied by changed paired-pulse ratio.
This neuromodulatory action of astrocytes is the only one for
which a precise ultrastructural correlate has been established
(Fig. 38.3). Thus, Jourdain et al. found that excitatory nerve ter-
minals in the dentate molecular layer express NR2B subunits,
which are particularly abundant in the extrasynaptic terminal
membrane apposed to astrocytic processes, where SLMVs can
be observed within tens of microns from the NR2B subunits.
Intriguingly, the presence of ambient levels of TNFα was
found to be a critical factor controlling pre-NMDAR activa-
tion by astrocyte glutamate (Santello et al. 2011).
A similar stimulatory action on synaptic transmitter
release by astrocyte glutamate was reported at CA3 Schaffer
collateral–CA1 pyramidal cell (CA3-CA1) synapses. The glu-
tamate release was found to be Ca2+ dependent and blocked
by infusion of TeNT in the astrocytes (Perea and Araque
2007), such as the release at PP-GC synapses. However, the
astrocytic control at CA1 synapses was initially observed only
in artificial conditions, that is, on uncaging IP3 (Fiacco and
McCarthy 2004) or Ca2+ (Perea and Araque 2007) in stratum
radiatum astrocytes. Actually, when the McCarthy group sub-
sequently used MrgA1 and IP3R2ko mice (see section 4) to
stimulate and block gliotransmission, respectively, they failed
to reproduce their own initial observations, and argued that
the neuromodulatory gliotransmission previously observed
was a methodological artifact resulting from the unphysiolog-
ical stimuli used (Fiacco et al. 2007). However, more recent
data by the Araque group demonstrate that this is not the case,
showing that astrocyte glutamate is released following physi-
ological stimuli that activate endogenous astrocyte GPCR.
Thus, this occurs when astrocyte CB1 receptors are stimulated
by endocannabinoids released as result of CA1 pyramidal cell
activity (Navarrete and Araque 2010) or when astrocytic mus-
carinic acetylcholine receptors are stimulated by activity of
cholinergic afferents coming from the septum. Moreover, in
the latter case, the synaptic consequences of astrocyte gluta-
mate release are abolished in IP3R2ko mice (Navarrete et al.
2012). The increase in EPSC frequency induced by astrocyte
glutamate at CA3-CA1 synapses is blocked by group I mGluR
486 • FUNCTIONS OF NEUROGLIAL CELLS
antagonists, not by NMDAR antagonists as seen at PP-GC
synapses, suggesting the existence of presynaptic stimulatory
mGluR on CA3 afferents. However, no ultrastructural corre-
late of the functional data has been provided to date.
Studies at CA3-CA1 synapses also identified a puriner-
gic inhibitory presynaptic control by astrocytes, via adenos-
ine acting on A1 receptors. Thus, use of dnSNARE mice (see
section 4), in which ATP/adenosine release from astrocytes
is abolished, demonstrated that astrocyte adenosine is largely
responsible for tonic suppression of transmission at these syn-
apses (Pascual et al. 2005). Intriguingly, a recent study, in iden-
tifying local synapse–astrocyte communication at individual
CA3-CA1 synapses, found that synaptic glutamate stimulated
Ca2+-dependent and TeNT-sensitive ATP/adenosine release.
This astrocyte adenosine, however, was found to activate A2,
not A1 receptors, and increase the probability of synaptic glu-
tamate release (Panatier et al. 2011). The conditions leading
TeNT-sensitive ATP/adenosine release from astrocytes to
privilege activation of A2 versus A1 receptors (or vice versa)
remain to be understood.
Astrocyte-dependent presynaptic modulation is not
restricted to excitatory circuits. Evidence for such a con-
trol exists also for inhibitory synapses made by GABAergic
interneurons onto CA1 pyramidal cells. The astrocytic
mechanism is responsible for physiological potentiation of
the inhibitory input to pyramidal cells observed in response
to repetitive firing of the interneurons (Kang et al. 1998).
Interneuron firing activates GABAB receptors on astrocytes,
inducing elevation of astrocytic [Ca2+]i and glutamate release.
It is likely that astrocytic glutamate induces feedback potenti-
ation by targeting excitatory GluR5-containing kainate recep-
tors present on interneurons (Liu et al. 2004).
6 C O N T R O L O F P O S T SY N A P T I C
E XC I TA B I L I T Y
Another proposed consequence of gliotransmission is stim-
ulation of postsynaptic neuronal excitability (Araque et al.
1998). In hippocampal slices, peculiar transient inward
currents, often of very large amplitude (up to 1 nA) and
much slower than synaptic currents, were observed in CA1
pyramidal neurons (Angulo et al. 2004; Fellin et al. 2004)
(Fig. 38.4). Such slow inward currents (SICs) occur at low
frequency, particularly in Mg2+-containing medium, are
TTX-insensitive, sustained by NMDAR activation, and
have been attributed to glutamate released from astrocytes in
response to evoked or intrinsic [Ca2+]i elevations. Like its pre-
synaptic effects, astrocyte-released glutamate directly activates
ifenprodil-sensitive NMDAR, this time located extrasynapti-
cally on dendrites. Consequently, the large SICs evoked can
significantly depolarize the postsynaptic cell and even trigger
its firing. Importantly, astrocyte-evoked SICs have been found
to occur in strict temporal correlation in two or more neigh-
boring CA1 pyramidal cells, which could lead the cells to fire
synchronously. Generation of synchronous SICs has been
proposed to depend on a single episode of glutamate release
in an individual astrocyte, sensed simultaneously by different
Figure 38.4 Astrocytic Glutamate Release Triggers Synchronous Slow Inward Currents in Neighboring Neurons, Coordinately Increasing Their
Excitability. (Left) Proposed mechanism for synchronous slow inward current (SIC) generation. Glutamate released from an astrocyte in a single
event is sensed by receptors on dendrites belonging to two CA1 pyramidal neurons sitting within the astrocytic release territory. (Right) Recordings
from two neighboring CA1 pyramidal cells show occurrence of synchronous SICs (enlarged in insets). Slow inward currents occur both spontaneously
and on mGluR agonist (DHPG) stimulation. Scale: 200 pA, 50 s; insets: 200 pA, 400 ms. Adapted from (left) Volterra and Meldolesi 2005. (right)
Fellin et al. 2004.

pyramidal cells sitting in its territory. Thereby, astrocytes
would contribute to synchronization of neuronal activity.
Astrocyte-evoked SICs have been observed in several brain
areas (D’Ascenzo et al. 2007; Parri et al. 2001), but it is unclear
whether they represent a relevant phenomenon at all or only at
some synapses. For instance, SICs were not observed at PP-GC
hippocampal synapses ( Jourdain et al. 2007). This could be
explained by differences existing between the excitatory cir-
cuits in the CA1 region and dentate gyrus. For instance, at
PP-GC synapses, pre-NMDAR activation seems to prevail
over extrasynaptic NMDAR activation (Dalby and Mody
2003; Jourdain et al. 2007). Expression of NR2B subunits is
fourfold higher in presynaptic than dendritic membrane–fac-
ing astrocytes ( Jourdain et al. 2007). Several additional aspects
about SICs remain to be clarified; for example, the conditions
under which they occur. Slow inward currents were found to
be generated on activation of GPCR that induce astrocytic
[Ca2+]i elevation such as mGluR5 (D’Ascenzo et al. 2007),
CB1 (Navarrete and Araque 2008), or PAR1 (Shigetomi
et al. 2008). However, other GPCR that produce astrocytic
[Ca2+]i elevations, such as P2Y1R, were shown not to gener-
ate SICs ( Jourdain et al. 2007; Shigetomi et al. 2008), sug-
gesting that GPCR-induced astrocytic [Ca2+]i elevations need
to have specific characteristics to produce SICs. On the other
hand, some authors reported that they could produce SICs
in conditions not involving GPCR-evoked [Ca2+]i elevation,
such as hypoosmotic conditions (Fiacco et al. 2007), or could
trigger spontaneously occurring SICs in CA1 pyramidal cells
on repeatedly applying high concentrations (50 mM) of exog-
enous glutamate (Xu et al. 2007). The latter were found to
cause de novo formation of large-diameter (>1-μm) organelles
in astrocytes, compatible with ensuing release of large gluta-
mate amounts that induce large-amplitude SICs, as observed
by most authors. However, the Araque group reported SICs of
much smaller amplitude (<20 pA) (Perea and Araque 2005),
compatible with glutamate release from SLMV, raising the
question of whether “small” and “large” SICs represent the
same phenomenon mechanistically.
Another phenomenon similar to SIC generation, but with
opposite functional consequences, has been described in mitral
cells of the olfactory bulb, where low-frequency, slow, and large
outward currents (SOCs) occur spontaneously in response to
mechanical stimulation of astrocytes. Such currents have been
attributed to release of GABA from the astrocytes and shown
to occur synchronously in adjacent mitral cells, leading to
blockade of their firing activity (Kozlov et al. 2006).
Another form of postsynaptic inhibition by astrocytes has
been reported in the retina, where Müller cells release ATP, then
transformed to adenosine, which causes postsynaptic hyperpo-
larization of ganglion cells via A1 receptors, an effect that reduces
their action potential firing frequency (Newman 2003).
Astrocyte-released transmitters seem to act mainly at extra-
synaptic receptors on dendrites. The case of astrocyte-released
D-serine is different, because most of the effects reported to
date for this gliotransmitter appear to be caused by a direct
action at synaptic NMDAR (Henneberger et al. 2010;
Panatier et al. 2006; Stevens et al. 2003; Yang et al. 2003). In
several brain circuits, D-serine, not glycine, was shown to be
the endogenous ligand of the coagonist binding sites of synap-
tic NMDAR. Such coagonist binding sites were found to be

A S T R O C Y T E S : M O D U L AT I O N O F SY N A P T I C F U N C T I O N A N D N ET WO R K AC T I VI T Y • 487
not tonically saturated, allowing phasic D-serine release from
astrocytes to exert important physiological controls on synap-
tic transmission and plasticity (see section 8). The reason why
D-serine activates mainly synaptic instead of extrasynaptic
NMDAR is presently unclear. Possibly, the coagonist binding
sites on extrasynaptic NMDAR are tonically saturated, and
diffusion of D-serine from its astrocytic release sites to the
cleft is not restricted by potent uptake systems, as is probably
the case for glutamate diffusion.
7 H ET E R O SY N A P T I C R E GU L AT I O N
Astrocyte signaling can functionally interconnect synaptic cir-
cuits. This feedforward action was first described as part of the
phenomenon known as heterosynaptic depression, a form of
intersynaptic communication in which active synapses decrease
the efficacy of neighboring, inactive synapses. At CA3-CA1
synapses high-frequency activity of a Schaffer collateral fiber
can trigger heterosynaptic depression of another, adjacent
fiber. This process is probably not caused by direct intersyn-
aptic cross-talk via neurotransmitter spillover, as originally
thought, but by the feedforward modulation of an intermedi-
ate astrocyte that senses the level of activity in the first fiber and
consequently turns down the activity of a second one by releas-
ing inhibitory adenosine (Zhang et al. 2003). An intermedi-
ary GABAergic interneuron, stimulated by glutamate released
from the first fiber, is apparently responsible for astrocyte acti-
vation and enhanced astrocytic [Ca2+]i via GABAB receptors
(Serrano et al. 2006). Heterosynaptic depression is abolished
in dnSNARE mice lacking astrocyte ATP/adenosine release,
confirming that this form of synaptic adaptation requires
astrocytic signaling (Pascual et al. 2005). Gliotransmission
also was recently found to mediate the opposite phenomenon,
heterosynaptic facilitation. At CA3-CA1 synapses, release of
endocannabinoids from CA1 pyramidal neurons was shown
to have a dual action on proximal and more distant synapses.
Although proximal synapses are homosynaptically depressed
via the classical direct action of cannabinoids on presynaptic
CB1 receptors, more distant ones are heterosynaptically poten-
tiated via activation of astrocytic CB1 receptors, followed by
[Ca2+]i elevation in the astrocyte and glutamate release at dis-
tal sites, resulting in the activation of presynaptic stimulatory
group I mGluRs (Navarrete and Araque 2010).
8 ASTROCY TES IN
AC T I VI T Y-D E P E N D E N T SY N A P T I C
PL ASTICIT Y
Astrocyte signaling participates to the establishment of
Hebbian forms of synaptic plasticity such as long-term poten-
tiation (LTP) and long-term depression (LTD), although this
has been disputed by some studies (discussed at the end of
this section). An initial indication came from the observation
that Ca2+ uncaging in a stratum radiatum astrocyte, which per
se produces short-lasting synaptic facilitation at CA3-CA1
synapses, produced long-lasting potentiation when paired to
488 • FUNCTIONS OF NEUROGLIAL CELLS
postsynaptic depolarization (Perea and Araque 2007). This
implies that coincident astrocytic and synaptic activity, like
coincident presynaptic and postsynaptic activity, can induce
long-term synaptic plasticity. Recently, astrocyte-dependent
LTP of CA3-CA1 synapses was shown to occur physiologi-
cally in situ and in vivo when cholinergic afferents from the
septum coincidentally stimulate stratum radiatum astrocytes
and CA1 pyramidal cells (Navarrete et al. 2012). A similar
finding was made in the barrel cortex in vivo, where combined
stimulation of mouse whiskers and cholinergic afferents from
the nucleus basalis of Meynert produced NMDAR-mediated
LTP, requiring muscarinic receptor–evoked [Ca2+]i eleva-
tion and D-serine release from astrocytes (Fig. 38.5) (Takata
et al. 2011). An involvement of astrocyte D-serine in the
modulation of NMDAR-dependent LTP was initially pro-
posed via pharmacological studies at CA3-CA1 synapses
(Yang et al. 2003). More recent work (Henneberger et al.
2010) confirmed that astrocytes respond to the CA3-CA1
LTP induction protocol with transient [Ca2+]i elevation
and D-serine release, which boosts NMDAR activation.
The D-serine–dependent component of NMDAR activa-
tion is apparently necessary for LTP induction because its
selective blockade is sufficient to prevent LTP. Interestingly,
astrocyte-released D-serine contributes to LTP within a
defined spatial range. Thus, it appears that any individual
astrocyte provides D-serine for inducing LTP at all the stim-
ulated synapses lying within or near its anatomical volume,
whereas it is unable to influence synapses that are greater than
or equal to 200 μm away. Availability of astrocytic D-serine
crucially also controls NMDAR-dependent synaptic plastic-
ity in other circuits, notably in the supraoptic hypothalamic
nucleus (SON) (Panatier et al. 2006). There, astrocytes
retract from synapses on SON neurons during lactation,
thereby reducing D-serine availability for NMDAR activa-
tion. Consequently, stimulations that induce LTP in virgin
animals instead induce LTD in lactating rats. This metaplas-
ticity is likely explained by the fact that reduced coagonist
site occupancy by D-serine reduces NMDAR activation to
levels sufficient to induce LTD but not LTP (see chapter 41).
Another astrocyte-released mediator, lactate, was recently
found to be critical for the maintenance of hippocampal LTP
and the formation of long-term memory in vivo (Suzuki et al.
2011). However, it is unclear if lactate sustains memory for-
mation metabolically or by stimulating relevant neuronal sig-
naling. On the other hand, very recent work has implicated
astrocyte CB1 signaling in an opposite in vivo effect, that is,
induction of LTD reducing spatial working memory (Han
et al. 2012). This effect was mediated by glutamate release
from astrocytes. Consistently, CB1-dependent astrocyte
glutamate exocytosis was recently found to underlie spike
timing–dependent depression (t-LTD) in cortical slices via
activation of pre-NMDAR (Min and Nevian 2012).
Despite the preceding evidence supporting the involve-
ment of gliotransmission in activity-dependent synaptic plas-
ticity, the work of the McCarthy group provided negative
results (Agulhon et al. 2010). This was particularly surprising
because these authors obtained results opposite to those of
Henneberger et al. (2010), although the two groups studied
 Air Puff+NBM
160

140

Relative LFP slope (%)

Air Puff
120

NBM 100

80

0
–20 0 20 40 60
Time (min)

Air Puff+NBM Air Puff+NBM+D-Ser

160 160
IP3R2-KO IP3R2-KO
140 140
Relative LFP slope (%)

Relative LFP slope (%)

120 120

100 100

80 80

0 0
–20 0 20 40 60 –20 0 20 40 60
Time (min) Time (min)

NBM axon

Astrocyte mAchR Ach

NMDAR Glu
[Ca2+]i
D-Ser

[Ca2+]i LTP

Presynapse Postsynapse

Figure 38.5 Cholinergic Long-Term Potentiation in the Somatosensory Cortex In Vivo Requires Gliotransmission. (Upper Panels, Left) Experimental
setup for paired air puff whisker stimulation and electrical stimulation of nucleus basalis of Meynert (NBM). (Right) Paired stimulation induces
long-term potentiation of the local field potentials (LFP). (Middle Panels, Left) Long-term potentiation is abolished in IP3R2ko mice lacking Ca2+
signaling in astrocytes. (Right) Long-term potentiation is rescued by D-serine infusion in the same mice. (Lower Panel) Proposed mechanism for the
astrocytic involvement. Acethylcholine released from NBM axonal terminals activates muscarinic receptors in astrocytes, increasing [Ca2+]i and induc-
ing D-serine release necessary for LTP induction at the cortical excitatory synapses. Adapted from Takata et al. 2011.

LTP in the same circuit (CA3-CA1 synapses) and used the
same induction protocol (classical high-frequency stimulation
[HFS]). However, the experimental strategies were totally dif-
ferent. In the Henneberger et al. study, HFS of afferent fibers
was delivered after having dyalized astrocytes with Ca2+ chela-
tors or exocytosis blockers to abolish endogenous signaling.
In this condition, HFS failed to induce LTP because astro-
cytes were unable to release D-serine. In the Agulhon et al.
(2010) study, the authors used MrgA1 mice (see section 4),
and via stimulation of MrgA1 receptors, induced large astro-
cytic [Ca2+]i elevation just before HFS. However, the resulting
LTP was identical to that produced by HFS alone. They also
tested HFS in IP3R2ko mice lacking GPCR-mediated Ca2+
signaling in astrocytes (Petravicz et al. 2008) (see section 4),
but in this case too HFS-evoked LTP was unaffected, which
led the authors to conclude that astrocyte Ca2+ signaling and
gliotransmission do not modulate LTP. More recently, how-
ever, other authors observed an astrocytic modulation of LTP
in MrgA1 mice using the same protocol as Agulhon et al.
(2010). The only difference was that they induced LTP by
using theta-burst stimulation, a milder stimulation than HFS
(Lee et al. 2010). In complement, two recent studies reported
disruption of cholinergic LTP in hippocampus and cerebral
cortex in vivo in IP3R2ko mice (Navarrete et al. 2012; Takata
et al. 2011). Overall, these data suggest that gliotransmission
participates in synaptic plasticity in defined conditions that
depend on specific types of activity and/or particular circuits
and are not reproduced by any type of stimulus in any circuit.

A S T R O C Y T E S : M O D U L AT I O N O F SY N A P T I C F U N C T I O N A N D N ET WO R K AC T I VI T Y • 489
 9 A S T R O C Y T E S I N H O M E O S TAT I C
SY N A P T I C P L A S T I C I T Y
Astrocyte-released factors such as TNFα in the hippocampus
(Beattie et al. 2002; Stellwagen and Malenka 2006; Stellwagen
et al. 2005) and ATP in the hypothalamus (Gordon et al. 2005,
2009) are involved in synaptic scaling, a form of homeostatic
plasticity independent of coincident changes in presynap-
tic and postsynaptic activity. Thus, unlike LTP or LTD, syn-
aptic scaling affects all the synapses in a neuron and consists
in a proportional adaptation of their strength to changes in
network activity with the goal of stabilizing the neuron’s fir-
ing. Postsynaptic scaling is classically observed in response
to persistent blockade (e.g., with TTX) of network activ-
ity in vitro, or to sensory deprivation in vivo, although other
adaptive mechanisms probably exist that operate over differ-
ent temporal and spatial scales (Pozo and Goda 2010). Both
TNFα, acting via TNF-R1, and ATP, acting via P2X7, induce
synaptic potentiation (scaling up) by promoting trafficking of
postsynaptic AMPAR subunits and increasing their insertion
at the postsynaptic membrane. Tumor necrosis factor alpha
also decreases postsynaptic GABAA receptors. According to
the mechanistic model initially proposed, astrocytes sense
the reduction in presynaptic activity and neuronal glutamate
release and react by increasing TNFα release. Tumor necrosis
factor alpha then acts on neurons to scale excitatory activity
up and inhibitory activity down via its effects on receptor traf-
ficking. Recent data suggest, however, that TNFα is not the
active signal mediating synaptic scaling; rather it is a permis-
sive factor for expression or maintenance of the phenomenon
(Steinmetz and Turrigiano 2010). Initially described in cell
cultures, TNFα-dependent scaling may take place also in vivo,
as a component of the visual cortex plasticity that follows mon-
ocular deprivation in the critical period (Kaneko et al. 2008).
Synaptic scaling induced in the hypothalamus by astrocyte ATP
is different from TNFα-dependent scaling: It is triggered by
increased (not decreased) presynaptic activity, develops rapidly
(minutes), and is mediated by glutamate activating mGluRs on
a local astrocyte. This triggers a [Ca2+]i elevation in the astro-
cyte, spreading to its processes and resulting in a release of ATP
that invests the dendritic arbor of a neighboring magnocellular
neuron and induces strengthening of all its synapses.
10 TO N I C SY N A P T I C C O N T R O L S
BY A S T R O C Y T E S
Tonic activation of high-affinity glutamate (NMDAR) and
GABA (α6 and δ subunit-containing GABAA) receptors has
been described at cerebellar and hippocampal synapses and
appears to play an important developmental role. Moreover,
there is a clear role for tonic activation of GABA receptors in
the adult nervous system, where they contribute to informa-
tion processing in the cerebellum and probably in the hip-
pocampus. The importance of tonic activation of glutamate
receptors in the adult is less certain (Cavelier et al. 2005).
There are indications that astrocytes are the source of tonic
glutamate and GABA release. A main mechanism proposed
490 • FUNCTIONS OF NEUROGLIAL CELLS
for tonic glutamate release is via astrocytic cystine–glutamate
exchange (Baker et al. 2002), although the available evidence
relies on use of rather unselective pharmacological inhibi-
tors. Recent work has provided more compelling evidence for
tonic GABA release from cerebellar Bergmann glia and astro-
cytes via bestrophin-1 channels (Lee et al. 2010). Such chan-
nels have the intriguing property of being both leakage anion
channels, therefore tonically active, and channels regulated by
[Ca2+]i or cell swelling. However, it is unclear when and how
the two functional modes take place. Bestrophin-1–dependent
tonic (Ca2+ independent) GABA release from glia is the main
source of GABA responsible for tonic inhibition of cerebellar
granule cells.
11 OT H E R F O R M S O F A S T R O C Y T E
SY N A P T I C C O N T R O L : G LU TA M AT E
U P TA K E A N D M O R P H O L O G I C A L
PL ASTICIT Y
Forms of astrocyte–synapse interaction other than direct
release of neuroactive gliotransmitters have important modu-
latory influences on synaptic functions. A well-recognized
mode is via the activity of glutamate transporters expressed on
the plasma membrane of perisynaptic astrocytic processes. By
binding and taking up synaptic glutamate, such transporters
dynamically control the extracellular level of the transmitter
and therefore determine its capacity to activate or desensitize
synaptic and extrasynaptic glutamate receptors, as well as to
spillover to neighboring synapses and glial cells (Bergles and
Jahr 1998; Kullmann and Asztely 1998). Because the extent
of astrocytic coverage differs from synapse to synapse and not
all synapses, even in the same area, are directly contacted by
astrocytic processes (Ventura and Harris 1999), this mode of
control is synapse specific. Thus, its functional consequences
depend on the reciprocal distribution of glutamate transport-
ers and receptors that, in turn, depends on the morphological
arrangement of the astrocyte around the synapse (Rusakov
and Lehre 2002). It is noteworthy that such an arrangement
is not static but plastic, and specific physiological conditions
induce synaptic modulation via a dynamic change of the astro-
cytic coverage of synapses. For instance, in the hypothalamic
SON, lactation triggers retraction of astrocytic processes sur-
rounding magnocellular neurons, and causes repositioning of
glutamate transporters far away from the synapses (see section
8). Consequently, synaptically released glutamate can diffuse
to presynaptic metabotropic receptors at the same excitatory
or neighboring inhibitory synapses, inducing homosynaptic or
heterosynaptic depression, a phenomenon not seen in nonlac-
tating animals (Oliet et al. 2001; Piet et al. 2004) (see chapter
41). Increased sensory stimulation has an opposite effect in the
barrel cortex, enhancing coverage of synapses by astrocytic pro-
cesses and increasing expression of glutamate transporters which
most likely increase clearance and reduce spillover of synaptic
glutamate out of the cleft (Genoud et al. 2006). Morphological
plasticity of astrocytic processes also influences the positioning
of gliotransmitter release sites, as already discussed for D-serine
release in the hypothalamus (see section 8).
 12 C O O R D I N AT E AC T I VI T Y O F
ASTROCY TIC AND NEURONAL
N ET WO R K S
Increasing evidence suggests that astrocytic signaling plays
modulatory roles not only at the level of individual syn-
apse, but also of macroscopic networks. In vivo studies
show the occurrence of coordinated [Ca2+]i elevations in
vast astrocytic populations. This is particularly evident in
unanesthetized, behaving animals because anesthesia dra-
matically suppresses astrocytic Ca2+ signaling (Schummers
et al. 2008). During locomotor behavior [Ca2+]i elevations
involving hundreds of Bergmann glial cells are seen in the
cerebellum (Nimmerjahn et al. 2009). Such [Ca2+]i eleva-
tions, never observed in resting animals, strictly depend
on neuronal excitatory activity. Their function is unclear:
They arise at the same time when blood flow increases in the
same region, but the overall time courses of the two events
do not match. Moreover, glial [Ca2+]i elevations are sup-
pressed by anesthetics, whereas the blood response is not or
to a lower extent. Population [Ca2+]i elevations are observed
also in visual cortex astrocytes in response to visual stimuli
(Schummers et al. 2008). Importantly, Ca2+ responses in
astrocytes occur according to specific functional maps over-
lapping with neuronal ones and share many receptive-field
characteristics with neuronal responses. Functional organ-
ization of astrocytic networks according to maps reflecting
the neuronal maps is confirmed by dye-coupling experi-
ments showing that astrocytes within an olfactory glomer-
ulus are gap junction–coupled among them, but not with
astrocytes present in neighboring glomeruli (Roux et al.
2011). The appearance of coordinated waves of [Ca2+]i ele-
vation in hundreds of astrocytes is another interesting phe-
nomenon recently described in the hippocampus in vivo
(Kuga et al. 2011). These responses, called glissandi, depend
on neuronal activity and have been proposed to specifically
emerge in response to network state changes. In this con-
text, a recent study provided the first evidence that astrocyte
Ca2+ signaling actively controls neuronal networks, notably
the number of their UP states (Poskanzer and Yuste 2011).
UP states consist of membrane potential depolarizations
occurring synchronously in an entire neuronal network,
and are thought to underlie behaviorally relevant cortical
states. According to Poskanzer and Yuste, when Ca2+ activ-
ity is triggered in the astrocytic network, the frequency of
neuronal UP states increases, and when astrocytic Ca2+ sig-
naling is blocked, neuronal UP state frequency decreases.
Although these fascinating observations in cortical slices
await confirmation in the living animal, other in vivo stud-
ies already have shown that blocking Ca2+ signaling in astro-
cytes affects complex network activity–sustaining behavior.
This is the case for circadian rhythms in Drosophila (Ng
et al. 2011) and respiratory activity in rodents (Gourine
et al. 2010). In the latter study, brainstem astrocytes were
found to be chemosensitive and respond to decreased blood
pH/increased Pco2 with [Ca2+]i elevations expanding in the
astrocytic network to reach respiratory neurons and modify
breathing patterns.
A S T R O C Y T E S : M O D U L AT I O N O F SY N A P T I C F U N C T I O N A N D N ET WO R K AC T I VI T Y
13 S U M M A RY A N D P E R S P E C T I VE S
Twenty years of research outline a role for astrocytes in the
control of synaptic circuits, providing a new conceptual frame
for understanding brain function. A variety of neuromodula-
tory effects of astrocytes have been reported, forming a com-
plex and sometimes contradictory preliminary picture. A
number of experimental paradigms and genetic models have
been developed to study astrocyte–synapse interactions and
define their relevance. Given the incomplete understanding
of the underlying biology, such methods and models are not
fully validated and the resulting data need to be interpreted
with some caution. For instance, if it is well established that
astrocytes respond to physiological neuronal activity with
[Ca2+]i elevations triggering astrocytic responses, the full
range of synaptically evoked astrocytic Ca2+ signals and their
individual roles remain at present undefined, although it is
likely that different types of signal have distinct biological
function. Recent work suggests that fast local signals confined
to astrocytic processes are relevant for neuromodulation, but
most astrocyte studies, notably in vivo studies, mainly record
the larger and slower cell body signals, whose functional role
and neuromodulatory relevance are unclear. The other side
of the same issue is that distinct paradigms used to exper-
imentally increase [Ca2+]i in astrocytes most likely trigger
biologically nonequivalent responses. Moreover, the effect
produced by increasing astrocytic [Ca2+]i can be different at
different brain locations depending on the synaptic circuit
to which the astrocyte is apposed, and on local differences in
the structural and molecular determinants of astrocyte–syn-
apse communication. Moreover, even at the same brain site,
such determinants can change depending on the animal’s
age; therefore, data obtained in developing animals do not
necessarily match those in adults. Studies to date have not
carefully considered all these emerging complexities, which
are probably the cause of discrepant results. Future studies
need to take all of these points into full account, and address
critical pending issues, such as the specific mechanisms by
which gliotransmission modulates neuronal functions and
the time-course of astrocytic actions. Future studies need to
also bring more clarity to the existence of different modes
of gliotransmission that sustain distinct astrocytic actions,
for example, some modes that are activity dependent and in
register with synaptic transmission, and others that are tem-
porally uncorrelated, and set the background of neuronal
circuit function or its long-term modulation. Indeed, there
is need of a better understanding of the many and appar-
ently contradictory modes of astrocytic action reported to
date. How do astrocytes control individual synapses inde-
pendently, and all the synapses in their territory collectively,
and all the synapses in a neuron independent of astrocytic
territories?
AC K N OW L E D G M E N T S
Research by AV is supported by the Swiss National Science
Foundation, grants 3100A0 120398 and 31003A 140999
• 491
NCCR “Synapsy” and NCCR “Transcure.” The author is
grateful to Nicolas Liaudet for the artwork and to Hajime
Hirase and the Journal of Neuroscience for granting permission
to reproduce figures from the 2011 Takata et al. study.
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• 493
 39.
ASTROCY TIC MODULATION OF MAMMALIAN
SYNAPSES: CIRCUITS AND BEHAVIOR S
Michael M. Halassa and Philip G. Haydon

A B B R E VI AT I O N S transmission as well as for the normal control of sleep-wake

cycles. Because sleep disorders are prevalent and comorbid
A1R adenosine 1 receptor with many other neurological and psychiatric disorders, it is
ATP adenosine triphosphate hypothesized that astrocytes play important roles in the gen-
cAMP cyclic adenosine monophosphate esis and expression of these conditions.
CPT cyclopentyl theophylline
EEG electroencephalograph
EMG electromyograph 2 ASTROCY TIC TR ANSMITTER
GABA gamma-aminobutyric acid R E C YC L I N G I S E S S E N T I A L F O R
GLT-1 glutamate transporter 1 NORMAL COGNITION
GS glutamine synthetase
HFS high frequency stimulation Synapses are now recognized to be tripartite structures. In
L-LTP late long-term potentiation addition to the presynaptic and postsynaptic terminals, the
LTP long-term potentiation surrounding astrocytic process is considered to be an integral
NREM non–rapid eye movement part of the synapse (Araque 2006; Haydon, 2001; Kettenmann
REM rapid eye movement et al. 1996). In contrast to the neural elements of the synapses,
SCN suprachiasmatic nucleus however, astrocytes operate on a slower timescale (>six orders
SNARE soluble NSF attachment protein receptor of magnitude) to modulate synaptic transmission (Halassa
TNF-α tumor necrosis factor alpha et al. 2007a). For example, astrocytes are known to express
TTX tetrodotoxin inward rectifier potassium channels (e.g., Kir4.1) (Olsen and
VAMP2 vesicle-associated membrane protein 2 Sontheimer 2008) whose function is essential for maintain-
ing normal ionic homeostasis at the synapse. Loss of astrocytic
potassium buffering is associated with a number of neuro-
1 INTRODUCTION logical illnesses, including epilepsy (Hinterkeuser et al. 2000;
Steinhauser and Seifert 2002) and tuberous sclerosis ( Jansen
Synapses are tripartite structures in which the astrocyte is a et al. 2005).
third element along with the presynaptic and postsynaptic ter- One of the most recognized roles of astrocytes at the syn-
minal. The tripartite synapse is recognized as playing essential apse is neurotransmitter uptake and recycling (Sattler and
roles to sustain synaptic transmission and as a consequence Rothstein 2006) (see chapter 35). Astrocytes express high
the function of neuronal circuits that underlie behavior. This levels of the glutamate transporter GLT-1, which constitutes
chapter highlights how the astrocytic component can influ- about 1% of total brain protein (Rothstein et al. 1994). This
ence synapses through the supply of neurotransmitter precur- transporter uses the sodium concentration gradient to drive
sors, cytokines as well as gliotransmitters, which modulate the influx of glutamate into astrocytes following synaptic
synaptic transmission. In particular, this chapter pays special transmission. Astrocytes subsequently convert glutamate to
attention to the intact animal, in which it is critical to under- glutamine, which they release back onto presynaptic termi-
stand how sensory experiences influence astrocytes and as a nals. In excitatory terminals, glutamine is converted to gluta-
consequence how the astrocyte provides feedback regulation mate, completing the “glutamate-glutamine” cycle, whereas
of the behavioral state. This chapter also pays special attention it is used in inhibitory terminals for the synthesis of GABA
to the authors’ work, which provided the first demonstra- (Schousboe et al. 1991, 1992). Interestingly, GABAergic ter-
tion of behavioral roles for astrocytes in the control of sleep minals have a limited stored pool of glutamine and thus are
homeostasis through the regulation of the level of the extracel- highly dependent on astrocytic glutamine synthesis com-
lular purine adenosine. Although astrocytes are not electrically pared with glutamatergic terminals (Liang et al. 2006). In
excitable and are thus relatively slow signaling systems, it is fact, acute inhibition of astrocytic glutamine synthetase (GS)
now known that they exert powerful actions in brain function in rodents precipitates seizures (Eid et al. 2008; Wang et al.
and are essential for the maintenance of GABAergic synaptic 2009), likely because of a failure of inhibition. Furthermore,

494
astrocytes express the GABAergic transporter GAT-3 that
has been recently shown to be regulated by submembrane
Ca2+ changes mediated by TRPA1 channels (Shigetomi et al.
2011). Inhibition of these ion channels results in a significant
decrease in the surface expression of GAT-3 in astrocytes and
a subsequent attenuation of miniature inhibitory postsynaptic
potentials of nearby neurons.
Astrocytic impact on synaptic inhibition is critical to
understanding systems-level processes given the emerging
roles of inhibition in neural circuit function. In addition to
other functions, fast synaptic inhibition is critical for sensory
function (Swadlow 2003), gating of modulatory input (Friedel
and van Hemmen 2008), and the emergence of particular neu-
ronal oscillations (Bazhenov et al. 2000; Hajos et al. 2004).
Neocortical gamma oscillations, for example, are thought to
be generated by rhythmic activity of parvalbumin-positive fast
spiking interneurons (Cardin et al. 2009). Gamma oscillations
are thought to be essential for normal cognitive function,
likely because of the impact it has on regulating the timing of
pyramidal cell firing (Cardin et al. 2009; Sohal et al. 2009).
As a result, failure of gamma oscillations is seen in a number of
different neuropsychiatric diseases, including schizophrenia
and autism (Uhlhaas and Singer 2006, 2007; Uhlhaas et al.
2008), and is often linked to a primary dysfunction in inhibi-
tory interneurons. Given the important modulatory impact
astrocytes have on inhibition at multiple levels (neurotrans-
mitter synthesis and uptake), a disruption in these processes
is likely to lead to major pathology. This is supported by the
example of major depressive disorder, in which parallels have
been found between decreases in cortical astrocytes (Ongur
et al. 1998) and decreases in neocortical GABA (Sanacora
et al. 1999). Further research is needed to determine whether
these processes are causally linked.
One area in which such a linkage has been found concerns
epilepsy (see chapter 70). In the epileptic temporal lobe, astro-
cytes enter a state of reactive astrocytosis with an associated
reduction in the expression of the astrocyte-enriched enzyme
glutamine synthetase. Given that acute pharmacological inhi-
bition of this enzyme leads to an attenuation of GABAergic
inhibition and seizures, it would not be surprising if reactive
astrocytosis initiates certain neuronal dysfunctions. In agree-
ment with this possibility, selective transduction of astrocytes
with adeno-associated virus serotype 5 leads to reactive astro-
cytosis and a loss of glutamine synthetase (Ortinski et al. 2011).
Coincident with this change is a reduction in synaptic inhibi-
tion, which is restored by addition of exogenous glutamine,
and enhanced excitation viewed with voltage-sensitive dyes
throughout brain slices (Fig. 39.1).
3 A S T R O C Y T E -D E R I VE D
C Y TO K I N E S M O D U L AT E C O RT I C A L
D E VE L O PM E N TA L P L A S T I C I T Y
Tumor necrosis factor alpha (TNFα) is a proinflammatory
cytokine that is known to mediate crucial signaling functions

A B +10 ms +100 ms +600 ms

CA1
Control

CA2
SC
EC
TA
DG 0.2
dF/F (%)
AAV2/5

CA3 0.1
stim
0

–0.1

C
AAV2/5
Control
SO

SO

0.12 0.12
dF/F (%)

dF/F (%)

0.08 0.08
SR

SR

0.04 0.04
SLM

SLM

0 0
0 0.3 0.7 0 0.3 0.7 0 0.3 0.7 0 0.3 0.7
Time (s) Time (s) Time (s) Time (s)

Figure 39.1 Selective Induction of Astrocytic Gliosis Results in Hippocampal Hyperexcitability. A. Schematic of the hippocampal slice preparation.
B. Voltage-sensitive dye imaging at different time points following the stimulation of entorhinal cortex input to CA1. C. Induction of astrogliosis via
selective transduction of astrocytes with high-titer AAV2/5 harboring green fluorescent protein results in enhanced network activity in response to
input at the EC to CA1 synapses. From Ortinski et al. 2011.

A S T R O C Y T I C M O D U L AT I O N O F M A M M A L I A N SY N A P S E S : C I R C U I T S A N D B E H AVI O R S • 495
across multiple organs (Heller and Kronke 1994). In the brain,
this molecule is thought to be released by astrocytes, based
on cell culture experiments in which astrocyte-enriched glial
cultures released this molecule, and recently has been shown
to mediate excitatory synaptic scaling (Stellwagen and
Malenka 2006). This form of non-Hebbian plasticity occurs
after prolonged periods of neuronal inactivity, such as experi-
mental incubation of cell or slice cultures with an activ-
ity blocker such as the voltage-sensitive channel blocker
tetrodotoxin (TTX). After a 24-hour incubation with
TTX, cultured neurons exhibit an increase in the miniature
excitatory postsynaptic currents (mEPSCs), a process that
requires release of TNFα from nearby astrocytes. Co-culturing
neurons derived from wild-type mice with astrocytes
derived from TNFα knockout mice failed to result in syn-
aptic scaling when neuronal activity was blocked, providing
direct mechanistic support for an astrocytic source of TNFα
in synaptic scaling. Interestingly, astrocytic TNFα does not
appear to be essential for Hebbian forms of plasticity (LTP
and LTD), at least under a limited number of stimulation
paradigms.
Experience-dependent plasticity in primary sensory neo-
cortical areas is critical for the development of normal percep-
tion (Buonomano and Merzenich 1998). In the visual system,
monocular deprivation during a critical period (28 days
postnatal in mice) results in rapid cortical plasticity (4 days
in mice) (Gordon and Stryker 1996). Recordings in the bin-
ocular region of mouse primary visual cortex (V1) normally
show a predominant representation of the contralateral eye
(Gordon et al. 1996). This representation shifts to a primar-
ily ipsilateral presentation on closure of the contralateral eye.
Recent studies have shown that two dissociable processes may
underlie this rapid shift; a weakening of visual responsiveness
to the deprived eye and a delayed strengthening of the nonde-
prived eye (Frenkel and Bear 2004). Although the first pro-
cess is thought to be mediated by Hebbian plasticity rules that
mediate synaptic depression, non-Hebbian synaptic scaling
processes are thought to underlie the second process (Desai
et al. 2002). Guided by the discovery that astrocytic TNFα
mediates synaptic scaling, Kaneko et al. (2008) made intrigu-
ing observations on the role of this process in developmental
visual plasticity. Tumor necrosis factor alpha knock-out mice
showed overall attenuated monocular deprivation plastic-
ity response compared with wild-type littermates. However,
intrinsic optical imaging of the cortical response to visual
stimulation of the deprived eye 3 days after lid closure revealed
an equal attenuation in TNFα knock-out and wild-type ani-
mals. After 5 days of lid closure, wild-type animals showed an
increased response to the nondeprived eye, whereas TNFα
knockouts failed to show an increase, concluding that a syn-
aptic scaling–like process in vivo is essential for mediating the
later phase of monocular deprivation plasticity. Although an
astrocyte-specific manipulation was not performed in these
experiments, it is possible that astrocytes control this process
based on aforementioned culture data. Overall, this finding
strengthens the concept that astrocytes act as modulators
of neuronal activity, mediating processes over a timescale of
hours to days.
496 • FUNCTIONS OF NEUROGLIAL CELLS
3.1 B E H AVI O R M O D U L AT E S A S T RO C Y T I C
S T RU C T U R E A N D F U N C T I O N
Astrocytic morphology is known to be modulated by neu-
ronal activity resulting from behavioral changes. Because the
greatest majority of astrocytic volume is in tertiary processes
that contact synapses (Bushong et al. 2002; Halassa et al.
2007b), astrocytic morphological changes have profound
impact on synaptic physiology. This process has been observed
in multiple brain regions, including the hypothalamus, supra-
chiasmatic nucleus (SCN), and sensory cortex. In the hypo-
thalamus, astrocytic coverage of oxytocin neurons has been
shown to be modulated by lactation, parturition, and chronic
dehydration, conditions associated with massive neurohypo-
physial hormone secretion (Oliet et al. 2008; Panatier and
Oliet 2006) (see chapter 41). Although the functional conse-
quence of this process is unclear, it is associated with increased
juxtaposition of neuronal processes and a rise in extracellular
glutamate because of a decrease in glutamate transport (Oliet
et al. 2001). In the suprachiasmatic nucleus, astrocytic mor-
phology changes as a function of the circadian phase (Gerics
et al. 2006). Recent molecular investigations have shown
that expression of the cytoskeletal astrocytic protein ezrin is
modulated by the circadian cycle in the SCN (Lavialle et al.
2011). Ezrin expression is linked to astrocytic filopodia motil-
ity and activity of astrocytic metabotropic glutamate recep-
tors, suggesting that astrocytic volumetric changes in the SCN
are driven by photic stimulation. The impact of this process
on circadian rhythms is unclear, although a general role for
astrocytes in regulating circadian rhythms is emerging. In pri-
mary somatosensory cortex, chronic vibrissal stimulation by
remotely controlled piezo stimulation in mice results in a two-
fold increase of astrocytic coverage of synapses and upregula-
tion of GLT-1 expression (Genoud et al. 2006).
4 S E N S O RY S T I MU L AT I O N S E VO K E
C A L C I U M S I G N A L S I N C O RT I C A L
ASTROCY TES
Astrocytes respond to transmitter spillover from nearby syn-
apses by intracellular Ca2+ elevation (see chapter 26). In the
anesthetized mouse, repetitive 10-Hz single vibrissal stimu-
lation results in astrocytic Ca2+ signals in the corresponding
cortical barrel. This response, which has a latency of approx-
imately 3 seconds, is dependent on activation of astrocytic
metabotropic glutamate receptors (Wang et al. 2006). In
the anesthetized ferret, visual gratings that trigger neuronal
activity results in similar activation of nearby astrocytes,
suggesting a spatial visual alignment between these two cell
types (Schummers et al. 2008). Interestingly, astrocytes show
sharper tuning to orientation and spatial frequency than
neurons, and a group of two to fewer than ten astrocytes
responds to a particular one-stimulus orientation, suggesting
a network organization of visual cortical astrocytes. Because
sensory tuning of cortical neurons is known to be shaped by
the interplay between excitation and inhibition (Higley and
Contreras 2006; Liu et al. 2010), this experiment suggests that
astrocytes may be tracking the output rather than the input
of neurons. Inhibiting the astrocytic visual response inhibits
the hemodynamic response in visual cortex (Schummers et al.
2008). Similarly, mouse olfactory bulb astrocytes respond to
odor-evoked synaptic input, and their response coincides with
the vascular response (Petzold et al. 2008). These converging
findings from different brain regions and species strongly link
astrocytic Ca2+ signaling to activity-dependent neurovascular
coupling. Consistent with this conclusion are recent findings
showing decoupling of cerebral blood flow from local neu-
ronal activity (Mishra et al. 2011) under certain conditions,
suggesting a non-neuronal mediator of the cerebrovascular
signal, and that under such conditions the vascular response
might play an anticipatory role in brain state changes, rather
than act as a follower for neuronal activity. This suggests that
one function of astrocytic sensory drive is mediating an antic-
ipatory hemodynamic response to alter local circuit function
(Moore and Cao 2008).
5 P H C H A N G E S E VO K E C A 2 + S I G N A L S
IN M E D UL L A RY A ST R O C Y T E S
Ca2+ imaging using the genetically encoded indicator Case12
revealed that astrocytes of the medulla chemosensitive zone are
sensitive to changes in pH (Gourine et al. 2010). A pH drop
from 7.4 to 7.2 resulted in a robust increase in intracellular Ca2+
of several astrocytes. These Ca2+ signals were independent of
neuronal activity as application of TTX or muscimol (a potent
GABA receptor agonist known to silence local neural activity)
had no impact on astrocytic Ca2+ response to changes in pH.
Furthermore, pH sensitivity appeared to be a feature of astro-
cytes of the ventral medulla because cortical or dorsal brain-
stem astrocytes failed to show a Ca2+ response to a pH drop.
Although the precise mechanism by which changes in pH are
linked to astrocytic Ca2+ signals is unknown, hydrolyzing extra-
cellular ATP by application of apyrase results in abolishment
of the astrocytic Ca2+ response. Further, optogenetic elevation
of astrocytic Ca2+ in medullary astrocytes augments breathing
rate, causally demonstrating the astrocytic drive of behavior.
5.1 T H E S L E E P -WA K E C YC L E I S A S S O C I AT E D
WI T H C H A N G E S I N A S T RO C Y T E -
D E R I VE D A D E N O S I N E
Since 1994 when we first observed that gliotransmitters can
be released from astrocytes (Parpura et al. 1994), there has
been a constant pursuit of identifying the conditions that
control when these gliotransmitters are released. Although
this initial observation identified glutamate as a gliotransmit-
ter, understanding the regulation of this gliotransmitter has
been the most difficult, probably because of the presence of
avid glutamate transporters, which clamp extracellular gluta-
mate at around 1 μM. In contrast to glutamate, the regula-
tion of the gliotransmitter adenosine has yielded much more
readily, probably because it is less tightly controlled. Much of
the uptake is regulated by equilibrative transporters rather
than concentrative Na+-dependent uptake, as is the case for
glutamate.
A S T R O C Y T I C M O D U L AT I O N O F M A M M A L I A N SY N A P S E S : C I R C U I T S A N D B E H AVI O R S
One of the first demonstrations that ATP is a gliotransmit-
ter was made by Kater’s laboratory when they performed cell
culture experiments examining the mechanism underlying the
propagation of Ca2+ waves between astrocytes in cell culture
(Guthrie et al. 1999). In culture an elevation of astrocytic Ca2+
in one cell can propagate to neighbors as a wave of elevated
Ca2+. Although the relevance in vivo is unclear, at the time
these studies were performed there was great interest in identi-
fying the intercellular signaling pathways in culture to identify
the potential mechanisms that may operate in vivo. Kater’s lab-
oratory made two critical observations that initiated a series of
studies on the role of purinergic signaling by astrocytes. First,
they asked whether Ca2+ waves propagate between neighbor-
ing cells via a cell contact–mediated mechanism, such as gap
junctions, or whether there was an extracellular route of signal
propagation. To achieve this objective, they physically iso-
lated astrocytes by making a scratch in the culture. Strikingly,
they observed that when a Ca2+ wave approached the edge of
a monolayer of astrocytes, after a brief pause, the Ca2+ wave
could continue at a distance in physically isolated astrocytes
(Fig. 39.2). This clearly pointed to the presence of an extra-
cellular signal in Ca2+ wave propagation. Further support was
provided by a clever experiment in which they sucked the
extracellular saline from around astrocytes when their Ca2+
level elevated. They then moved the pipette to a different cel-
lular region with low Ca2+ and were able to evoke a Ca2+ signal
on ejection. To identify the nature of the extracellular signal
they used apyrase, an enzyme that metabolizes ATP as well as
purinergic antagonists that both blocked the ability of the col-
lected medium to evoke a Ca2+ signal. These elegant studies
clearly identified ATP as a potential extracellular signal in the
CNS that is used by astrocytes.
Making the transition from cell culture studies to in situ
and in vivo approaches has been more challenging, largely
because of the difficulty of providing selective stimulation and
inhibition of astrocytes. However, the advent of cell-specific
molecular genetics has provided a powerful tool to investi-
gate the regulation of gliotransmission in vivo. In 2005 we
developed a conditional astrocyte-selective tool that impaired
the astrocyte-derived accumulation of extracellular purines
(Pascual et al. 2005). There has been much debate about how
gliotransmitters are released from astrocytes. Although space
will not be used here to rehash the pros and cons of different
release mechanisms, the authors used a powerful and highly
selective tool to impair regulated exocytosis (Fig. 39.3).
Vesicle fusion requires the formation of a SNARE com-
plex. The authors interfered with SNARE complex formation
using a well-characterized approach of expressing the SNARE
domain of a vesicle protein: The expressed protein prevents
the formation of the endogenous complex and thereby pre-
vents that particular SNARE-mediated membrane fusion
event. It should be noted that there is a high degree of speci-
ficity between different SNARES. For example, the SNARE
domain of VAMP2 does not inhibit VAMP8 events and visa
versa (Scales et al. 2000a,b).
The authors used the tetracycline-off system together
with an astrocyte-selective promoter to conditionally con-
trol the expression of the SNARE domain of VAMP2 only in
• 497
 A B

Figure 39.2 Astrocytic Ca2+ Waves Spread Through an Extracellular Messenger. Pioneering work by Kater’s group using fluo-3 AM imaging of Ca2+
in cultured astrocytes. Astrocytic Ca2+ wave can spread throughout the culture despite physical discontinuity. From Guthrie et al. 1999.

astrocytes. Independent studies have shown that this pertur-
bation impairs regulated membrane cycling, but expression of
the SNARE domain does not affect trafficking of channels or
receptors, for example (Fellin et al. 2009). That this is a highly
selective approach is also provided by the observation that
astrocytes maintain their physiological integrity even follow-
ing months of continued expression of the SNARE domain.
Once the authors developed mice expressing dominant
negative SNARE only in astrocytes (termed dnSNARE mice),
it was asked whether they changed the extracellular concen-
tration of chemical transmitters. Initially neurons were used
as sensors of gliotransmitters, but more recently these results
have been validated using biosensors of purines, which directly
measure purines in the extracellular space. Initial studies were
performed using hippocampal brain slices in which the effect of
astrocytic expression of dnSNARE on basal synaptic transmis-
sion was monitored (Pascual et al. 2005). Input/output curves
of Schaffer collateral CA1 synaptic transmission was moni-
tored in the presence and absence of dnSNARE, and a strik-
ing result was observed: When dnSNARE was expressed in
astrocytes, the magnitude of basal excitatory transmission was
augmented (Fig. 39.4A,B). It is well known that there is a tonic
adenosine-mediated presynaptic inhibition of excitatory trans-
mission in hippocampus and cortex. Therefore, the authors
asked whether this source of adenosine was derived from the
astrocyte. Initial studies determined the magnitude of enhance-
ment of synaptic transmission caused by addition of adenosine
1 receptor (A1R) antagonists. In dnSNARE mice there was a
significant attenuation of the basal adenosine tone, indicating
that astrocytes supply the source of adenosine that tonically
suppresses excitatory synaptic transmission (Fig. 39.4C).
There are two prominent mechanisms that could give rise
to extracellular adenosine: the release through equilibrative
transporters or the release of ATP that is metabolized in the
extracellular space to adenosine. Data supported the concept
that this arises from the latter because luminescence imaging
demonstrated that dnSNARE expression also reduced extra-
cellular ATP and the inhibition of equilibrative nucleoside
transporters did not lead to a depletion of adenosine, but
rather to an increase in adenosine. Together with other data
it was concluded that astrocytes release ATP that is hydro-
lyzed in the extracellular space to adenosine. Because of the
selectivity of the dnSNARE manipulation for inhibiting
exocytosis, the authors conclude that the most likely mecha-
nism underlying this release is through vesicle fusion with the
plasma membrane. However, there is also evidence indicating
that connexins can contribute to ATP release. Whether these
two pathways are linked or independent parallel mechanisms
remains to be revealed.
Perhaps the most surprising aspect of this dnSNARE study
was that much of the adenosine was found to be derived from
a glial source. Given that neurons can release ATP during syn-
aptic transmission, one might have thought that adenosine
would accumulate from neuronal release of purines. Clearly,
neurons can provide purines. However, they release adenosine
in a pulsatile, activity-dependent manner providing fast neu-
romodulation, while astrocytes control the tonic accumula-
tion of adenosine.

498 • FUNCTIONS OF NEUROGLIAL CELLS

 A Doxycycline B
hGFAP tTA tTA

Doxycycline No Doxycycline

TetO Lac-Z TetO Lac-Z

C D
TetO EGFP TetO EGFP

TetO SNARE TetO SNARE

Astrocyte-specific
No expression
expression dn-SNARE + Dox dn-SNARE – Dox

E F G

β-Gal/EGFP GFAP Merge

H I J

β-Gal/EGFP NeuN Merge

Figure 39.3 Temporally Regulated, Astrocyte-Specific Expression of a Dominant Negative SNARE Domain. A. Outline of the genetic strategy used:
two lines of transgenic mice are made in which the first uses the human GFAP promoter to drive the expression of the tetracycline transactivator
(tTA), the second harbors the tetracycline operator (tetO) driving the expression of the dominant negative SNARE (dnSNARE), and two reporter
proteins, GFP and LacZ. When the two mice are mated, astrocyte specific expression of these transgene is achieved and can be inhibited by including
doxycycline in the mice’s diet. B–E. Astrocyte-specific expression that is suppressed by doxycycline. From Pascual et al. 2005.

Adenosine is known to be regulated during sleep-wake
cycles. In basal forebrain, for example, microdialysis and
HPLC have revealed that during wakefulness adenosine rises
and during sleep adenosine falls (Porkka-Heiskanen et al.
1997). The source of adenosine has been presumed to be neu-
ronal. However, given that astrocytes have been demonstrated
to powerfully control extracellular adenosine, the question
that begs to be answered is whether these sleep wake cycles
of adenosine depend on the astrocyte source of adenosine. To
address this question two levels of experimentation have been
performed. First, the authors monitored adenosine in brain
slices that are isolated at different times of day during the sleep
cycle (Schmitt et al. 2012). Second, a time of day experiment
was performed in vivo in which the astrocytic contribution
to the neuromodulatory purine pool could be assessed. Brain
slices were isolated at 4-hour intervals throughout the 24-hour
day and the basal adenosine tone was monitored using A1R
antagonists to relieve the basal presynaptic inhibition of
synaptic transmission. Striking diurnal rhythms of adenosine
tone were revealed. When brain slices were isolated at the end
of the dark phase (the animals’ subjective daytime) adenosine
tone was high. When isolated during the light phase (subjective
night time) adenosine tone was low. Importantly, when mice
were sleep deprived during the light phase, they maintained an
elevated level of adenosine. When dnSNARE and wild-type
mice were compared, the dark phase and wakefulness depen-
dent elevation of adenosine was shown to be derived from an
astrocytic source. In addition to using synaptic transmission to
monitor extracellular adenosine, enzymatic/amperometric bio-
sensors of adenosine were also used that directly measured ade-
nosine and validated all of the synaptic bioassay results. These
results provide striking insight into the control of adenosine.
Wakefulness regulates the extracellular level of adenosine from
an astrocyte-dependent dnSNARE sensitive pathway.
One concern about these studies is that they were per-
formed in situ. Could they be reproduced in vivo? To ask this

A S T R O C Y T I C M O D U L AT I O N O F M A M M A L I A N SY N A P S E S : C I R C U I T S A N D B E H AVI O R S • 499
 A 3 B 2.5 C **
** 100
dn-SNARE
2.5 2 80
wt

Δ fEPSP slope (% of control)

fEPSP slope (-mV/ms)

fEPSP slope (-mV/ms)

dn-SNARE
2 60 wt
1.5
40
1.5
1 20
1
0
0.5 0.5
–20
0 0 –40 D-AP5 Bicuculline RB-2 DPCPX
0 5 10 15 20 25 wt dn-SNARE wt dn-SNARE (n=4) (n=5) (n=4) (n=4)
Stim (V) Dox + + – – –60 Ifenprodil PPADS DPCPX
(n=9) (n=5) (n=6)

Figure 39.4 Astrocytic dnSNARE Expression Attenuates Tonic Adenosine in Hippocampal Slices. A. dnSNARE-derived slices show an increased input/
output relation of CA3-CA1 synaptic transmission. B. This effect is inhibited by doxycycline-mediated suppression of dnSNARE expression. C.
Enhanced synaptic transmission can be explained by an absent suppressive adenosine tone, evident by a blunted impact of A1-R antagonist on synaptic
transmission in the dnSNARE-derived slices. From Pascual et al. 2005.

question local field potential recordings in anesthetized mice
were performed. Under urethane anesthesia, the cortex exhib-
its slow oscillations of network activity that are modulated by
adenosine. Therefore, slow oscillations were recorded in vivo
and the magnitude of A1R modulation was determined. Just
as observed in brain slice studies, it was found that adenos-
ine was controlled by wakefulness and from an astrocytic
source. When dnSNARE was expressed in astrocytes, sleep
deprivation no longer elevated extracellular adenosine
(Schmitt et al. 2012).
Taken together, these results provide a powerful example
of how behavior regulates via an astrocyte intermediate the
extracellular level of a gliotransmitter that in turn modulates
synaptic transmission and neuronal network activity (slow
oscillations). During wakefulness, astrocytes lead to elevated
adenosine, but during sleep this source of gliotransmitter
declines to low levels. These results also provide an important
warning to scientists in the field. The time at which the brain
is studied is an extremely important variable that affects the
ability to observe astrocyte-dependent events. Most animal
facilities have their light phase coincident with our light phase.
However, this means that in vivo studies or brain slice experi-
ments performed during the light phase (subjective nighttime
for mice) will have minimal activation of this gliotransmitter
pathway. Thus, it becomes imperative to record zeitgeber time
when studies are performed so that clarity can return to a field
in which there has recently been conflicting results.
5.2 G L I OT R A N S M I S S I O N MO D U L AT E S
B E H AVI O R—S L E E P H O M E O S TA S I S
Given that sleep-wake cycles regulate astrocyte-derived ade-
nosine, a natural resulting question is whether this nucleoside
provides a feedback regulation of behavior given that it exerts
powerful modulatory actions on synaptic transmission and
neural networks. The first hint that astrocytes might affect
sleep was provided by a review of the literature that showed
that adenosine rises during wakefulness and falls during sleep,
and that adenosine promotes sleep and adenosine antagonists
promote wakefulness (Basheer et al. 2000). To examine this
issue, the dnSNARE mouse was used in EEG and EMG stud-
ies of sleep-wake cycles to determine whether their patterns
were perturbed. Initially it was asked whether the amount of
time spent in the three vigilance states—wake, rapid eye move-
ment sleep (REM), and non-REM (NREM) sleep—were dif-
ferent in dnSNARE mice. Initial results showed no distinction
between wild-type and dnSNARE mice (Halassa et al. 2009).
Sleep is controlled by at least two processes: the circadian
oscillator, which controls the timing of sleep and wakefulness,
and the sleep homeostat, which integrates the amount of time
one has been awake and delivers the drive to sleep (Borbely and
Achermann 1999). To discriminate between the two a simple
test is performed in which one sleep deprives mice for 4 to 6
hours and determines whether there is a homeostatic increase
in the delta power of NREM sleep. In dnSNARE mice there
is an attenuation of the sleep deprivation–dependent increase
in delta power, indicating that the astrocyte modulates the
sleep hemostat (Fig. 39.5A–C). Further support is provided
by the observation that the compensatory increase in sleep
time that normally follows sleep deprivation is no longer
present in dnSNARE mice (Fig. 39.5D–G). Using osmotic
minipumps to deliver A1R antagonists, it was also found
that the dnSNARE phenotype was phenocopied by inhibit-
ing A1R. Consequently, these data indicate that wakefulness
stimulates the extracellular accumulation of astrocyte-derived
adenosine, and that this adenosine, acting through A1R, pro-
vides the pressure to sleep that normally follows a day of wake-
fulness (Halassa et al. 2009).
5.3 A S T RO C Y T E S A FFEC T T H E CO G N IT I V E
CONSEQUENCES OF SLEEP LOSS
It is well known that sleep is critical for the consolidation of
long-term memories. Sleep deprivation can prevent memory
formation. Because astrocyte-derived adenosine is necessary
for compensatory responses to sleep loss (increased sleep time
and NREM bout duration), it is intriguing to ask if the astro-
cyte actively prevents memory consolidation. Therefore, the

500 • FUNCTIONS OF NEUROGLIAL CELLS

 A 140 B 160 * Wildtype C 260 Recovery after SD
Wildtype
* dnSNARE Wildtype
dnSNARE

Normalized Power
Normalized Power

Normalized Power
140 * * 220 * * dnSNARE
*

(0.5-1.5 Hz)
(0.5-0.4 Hz)

(0.5-1.5 Hz)
120
* *
120 180

100 140
100

80 100
0 4 8 12 0 4 8 12 6 8 10 12
Time (Zeitgeber) Time (Zeitgeber) Time (Zeitgeber)

Baseline Wildtype Baseline Wildtype

D Recovery E dnSNARE F Recovery G dnSNARE
** * *
60 N.S. 10 300 N.S. 120
(% Recording Time)

(% Recording Time)
** ΔTotal Sleep Time
Total Sleep Time

ΔNREM bouts
NREM bouts

(seconds)
(seconds)
40 200 80
5
20 100 40

0 0 0 0
Wildtype dnSNARE Wildtype dnSNARE

Figure 39.5 Astrocytes Control Sleep Homeostasis. A. dnSNARE mice show an attenuated expression of slow wave activity under baseline conditions.
B. This effect is amplified if the low-frequency component of SWA activity is examined. C. The impact of the dnSNARE on SWA is accentuated by
sleep deprivation. D,E. Although wild-type mice respond to sleep deprivation with an increase in recovery sleep time, dnSNARE mice do not. F,G.
A similar effect is seen when examining the increased NREM bout duration following sleep deprivation. From Halassa et al. 2009.

authors performed studies in which a period of sleep depriva- potentiation (LTP) transitions to a Late-LTP that is protein
tion was interjected between a training session and probe test. synthesis dependent. Late long-term potentiation is thought
Initially, novel object recognition and later spatial object rec- to be a cellular correlate of long-term memory. dnSNARE
ognition was studied with similar results. In the novel object mice and wild-type littermate mice exhibit similar L-LTP.
recognition task mice were allowed to explore two identical However, a striking result is observed when mice are sleep
objects at the beginning of the light phase, and then were deprived before testing L-LTP. Sleep deprivation prevents
allowed to sleep and were re-exposed to objects 24 hours later. the induction of L-LTP unless brain slices are obtained from
However, one of the familiar objects was replaced by a novel dnSNARE mice (Florian et al. 2011) (Fig. 39.6). Moreover,
object. If mice have a recognition memory of the object, they the infusion of the A1R antagonist CPT similarly protects
were exposed to in the training session they will spend more synapses from the negative consequences of sleep deprivation.
time exploring the novel object. Indeed, this was the case in Thus during sleep deprivation the wakefulness dependent
both dnSNARE mice and wild-type littermates. However, if source of astrocyte-derived adenosine acts on A1R to pre-
a 6-hour period of sleep deprivation followed the training ses- vent cellular (L-LTP) and behavioral memory consolidation.
sion, wild-type mice did not show a preferential exploration Because an elevation of cAMP is necessary for memory con-
of the novel object on the next day: They had no recognition solidation and A1R couple through the G protein Gi, which
memory. In contrast, dnSNARE mice had equivalent recogni- negatively regulates adenylyl cyclase, it is presumed that during
tion memory regardless of whether they were left undisturbed enforced wakefulness (sleep deprivation) astrocytes attenuate
or were sleep deprived (Halassa et al. 2009). neuronal elevations of cAMP that are required for memory
Because it is known that dnSNARE prevents the accumu- consolidation (Vecsey et al. 2009).
lation of extracellular adenosine, it was then asked whether
this sleep deprivation–dependent cognitive impairment was
phenocopied by A1R antagonist. In wild-type mice introduc- 6 S U M M A RY A N D P E R S P E C T I VE S
tion of the A1R antagonist CPT prevented the negative con-
sequences of sleep deprivation. Since the first demonstration of gliotransmission in 1994,
How could an astrocyte actively inhibit memory consoli- the field has made considerable progress toward appreciat-
dation? To answer this question use was made of the fact that ing roles that gliotransmitters can play in behavior. During
brain slices maintain the adenosine tone present at the time development gliotransmitters play influential developmen-
the animal is euthanized, and it was asked whether cellular tal roles in synaptic scaling that is important for the for-
long-term memory was affected by prior sleep deprivation. mation of experience-dependent sensory maps. Later in
Tetanic high frequency stimulation (HFS) of the Schaffer col- adulthood, the environment modulates astrocytic structure
laterals leads to long-term potentiation of synaptic transmis- and function. In addition to structural change the astrocyte
sion. When the HFS is repeated four times, this long-term responds to sensory inputs with Ca2+ signals that operate on

A S T R O C Y T I C M O D U L AT I O N O F M A M M A L I A N SY N A P S E S : C I R C U I T S A N D B E H AVI O R S • 501
 450
NSD SD
400
fEPSP Slope (% baseline)
350
300
250
200
150
100
WT-NSD (n=5)
50
WT-NSD (n=7)
0 0
0 25 50 75 100 120 150
Time (min)
Figure 39.6 Gliotransmission Is Required for the Effect of Sleep Deprivation on the Late Phase of Hippocampal Long-Term Potentiation. Although sleep
deprivation abolishes L-LTP in wild-type slices, it has no impact on this process in slices derived from dnSNARE mice.
the timescale of seconds. On one hand, these signals modu-
late blood flow with the potential to promote anticipatory
hemodynamic changes that may impact neural response. On
the other hand, sensory activation of the astrocyte influences
feedback control of neural networks and behavior. This is
particularly well highlighted by the observation that wake-
fulness signals to astrocytes to in turn regulate the level of
extracellular adenosine. As a consequence of this elevated
adenosine, an astrocyte and adenosine-dependent pressure
to sleep accumulates during the daytime and contributes to
the drive to sleep. Because sleep disorders are comorbid with
so many neurological and psychiatric conditions, it will be
intriguing to determine whether there exist primary dysfunc-
tions in astrocytes that initiate these disorders of brain func-
tion. Two areas of compelling interest are: major depressive
disorder, in which there is a correlated change in astrocyte
structure together with sleep dysfunction; and epilepsy, in
which changes in glutamine synthetase expression in astro-
cytes contribute to reduced inhibition and epilepsy. As one
looks back on the progress in the field of gliotransmission of
the past 18 years and how its pervasive nature and dominant
influence on synapses, circuits, and behavior is now appre-
ciated, one cannot resist looking forward to what is in the
future. Perhaps in the next 18 years the first treatment for a
major brain dysfunction will be through small molecules or
biologics targeted to glia!
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• 503
 40.
ADULT NEUROGENESIS
Gerd Kempermann
A B B R E VI AT I O N S
BMP bone morphogenic protein
DCX doublecortin
GFAP glial fibrillary acidic protein
LTP long-term potentiation
NG2 neuron-glia 2 (proteoglycan)
RGL radial glia-like (cells)
SEZ subependymal zone
SGZ subgranular zone
shh sonic hedgehog
SVZ subventricular zone
1 I N T R O D U C T I O N : W H AT I S
A D U LT N E U R O G E N E S I S ?
Adult neurogenesis is the developmental process of generating
new neurons under the conditions of the adult brain. Adult
usually stands for sexually mature, but when exactly and how
postnatal neurogenesis transgresses into adult neurogenesis
remains a matter of discussion (Rakic 2002a). Despite many
similarities, adult neurogenesis differs from fetal and postna-
tal neurogenesis in a number of issues. Significantly, although
the early forms of neurogenesis are primarily dependent on
genetic programs, adult neurogenesis is characterized by mas-
sive regulatory influence of extrinsic factors, whether physio-
logical activity or pathology. Adult neurogenesis is a particular
form of brain plasticity in which structural and functional
adaptation to functional demands occurs at the level of cells
rather than neurites and synapses. This is an exceptional situa-
tion and in adult rodents and primates is essentially limited to
two “canonical” neurogenic regions, the subventricular zone
(SVZ) of the lateral ventricles that produces several types of
interneurons in the olfactory bulb, and the subgranular zone
(SGZ) of the dentate gyrus in the hippocampus, in which new
excitatory granule cells are generated (Fig. 40.1) (Kempermann
2011a). Other species have more or fewer neurogenic zones.
Zebrafish, for example, have 16 (Grandel et al. 2006; Kaslin
et al. 2008), drosophila none (Siegrist et al. 2010), most bats
only one in the olfactory system (Amrein et al. 2007), and in
humans neurogenesis in the olfactory bulb appears to dry out
in late childhood or adolescence (Curtis et al. 2007; Sanai et al.
2011), whereas neurogenesis in the adult human hippocampus
is maintained into old age (Eriksson et al. 1998; Knoth et al.
2010). Adult neurogenesis is an extremely complex, polygenic
504
process that is regulated at numerous levels (Kempermann
2011d). As such it allows particularly sensitive adaptations,
but adult neurogenesis comes at high cost if the regulation is
going to be specific. In many “lower” animals in contrast, adult
neurogenesis might be widespread and not specific to particu-
lar functional and structural contexts.
Neurogenesis, like all development, originates from stem
cells, which are defined as cells with the ability to self-renew.
Dividing stem cells always produce one (in an asymmetrical
division) or two (in a symmetrical division) new stem cells.
Whereas symmetrical divisions allow expansion of the stem
cell pool, only asymmetrical divisions generate differentiating
progeny, while still maintaining the stem cell pool. The differ-
entiating daughter cell of a stem cell is called a progenitor cell,
whose ability to self-renew is limited, and that may undergo
a terminal “neurogenic symmetrical division” in which both
progeny are differentiating cells and the precursor cell is thus
consumed. Although stem cells are usually rare, progenitor
cells are numerous and highly reactive to regulation. Expansion
can occur at the progenitor cell stage. The term precursor cell
is used here as an umbrella term for stem and progenitor cells
as well as neuroblasts (an ill-defined and problematic category
of more advanced progenitor cells), if details are not known or
all cells are meant. Strictly speaking, the term stem cell should
be reserved for cases in which self-renewal and multipotency
have been demonstrated, but the common use of the term is
much looser, and this has infiltrated the scientific literature.
Besides self-renewal, stem cells are defined by their ability
to differentiate along at least two different lineages (known as
multipotency). Different stem cells differ in the width of their
potential to produce differentiated cells. Throughout this
book (see chapters 5 and 30), slightly different nomenclatures
for stem and progenitor cells are used, which reflects the state
of the different subfields of neurogenesis research. The termi-
nology used here is widely used but not commonly accepted
and many details remain unresolved.
2 R E L AT I O N S H I P W I T H F ETA L A N D
E A R LY P O S T N ATA L N E U R O G E N E S I S
Among the key similarities between embryonic and adult
neurogenesis, the fact stands out that neurogenesis is a glial
process to a large degree (Morrens et al. 2012). Radial glial
cells serve as stem cells both in the embryo and the adult
(Chojnacki et al. 2009; Doetsch et al. 1999a; Kriegstein and
 Subgranular zone (SGZ)
of the hippocampal dentate gyrus
Immature
neuron
Type-3 Immature
neuronally
and
determined
neuroblasts mature,
new
granule cell
Type-1
radial glia-like Type-2b postmitotic
stem cells Type-2a neuronally maturation stages
glia-like determined CA3
in the granule cell
intermediate progenitor cells
Glia-like progenitor cells layer
Neuronal
precursor cells
precursor cells
in the SGZ in the SGZ

3.

1E
leehwniP
1E lacipa :buh
1E sessecorp
2.
Ventricle

sllec 1B fo
1E B1
1E
B1
New neurons in the
B1 granule cell layer and
1.
Migratory neuroblasts periglomerular zones
eht no elcirtnev eht morf weiV
llaw ralucirtnev eht fo ecafrus Precursor cells (A cells) of the olfactory bulb
B1 cell C cell A cell in the SVZ in the rostral migratory stream

E1
E1
Subventricular zone (SVZ) / Olfactory bulb
Pinwheel pattern
(at ventricular surface)

Figure 40.1 The Neurogenic Regions of the Adult Rodent Brain. Most mammals have two “canonical” neurogenic regions, the dentate gyrus in the
hippocampus and the subventricular zone (SVZ)/olfactory bulb, sharing certain features of neurogenesis.

Alvarez-Buylla 2009; Noctor et al. 2001). However, several
definitional and semantic issues arise if it comes to describing
radial glia in a way that satisfies the particular demands of the
conditions of both the fetal and the adult brain. Therefore,
the stem cells of the adult brain are often referred to as radial
glia-like (Bonaguidi et al. 2011). The alternative has been a
neutral nomenclature with letters in the SVZ (Doetsch et al.
1997) and numbers in the SGZ (Kempermann et al. 2004).
Adult hippocampal neurogenesis is a process in the dorsal
brain; therefore, the same or similar patterns of transcription fac-
tors and regulatory principles that govern dorsal neurogenesis in
the embryo also appear in adult hippocampal neurogenesis. For
example, there is a signature sequence of transcription factors
from Pax6 in the stem cells over Neurogenin2 and Tbr2 in the
intermediates to Tbr1 in the neurons that is shared by fetal cor-
tical neurogenesis and adult hippocampal neurogenesis (Hodge
et al. 2008). Excitatory neurons and astrocytes are generated.
Neurogenesis in the adult SVZ and olfactory bulb, in
contrast, essentially follows a ventral pattern and produces
interneurons and oligodendrocytes. Here, neurogenesis is in
continuation of embryonic neurogenesis originating from
the ganglionic eminences. Because of the different types of
interneurons that are produced, the pattern of transcription
factors is less uniform. There is even one lineage showing the
dorsal sequence from Pax6 to Tbr1 intermingled with the
otherwise ventral process (Brill et al. 2009).
Despite the many similarities, adult neurogenesis is also
fundamentally different from embryonic and fetal neurogen-
esis in that it represents an exception in an environment that
is otherwise fully developed and has turned nonneurogenic.
Other than in the fetal brain, adult neurogenesis is also not
as much synchronized as it is during embryonic brain devel-
opment, but all stages of development are found next to each
other at any given time (Fig. 40.2).

A D U LT N E U R O G E N E S I S • 505
Figure 40.2 Adult-Generated Neurons and Glia in the Granule Cells Layer (GCL) of the Murine Dentate Gyrus. Within each panel the antigens
investigated are listed in the order red, green, and blue. A,B. Two types of astrocytes can be distinguished in the granule cell layer of the adult dentate
gyrus. One type has a radial or vertical morphology and expresses GFAP (blue) and Nestin (Nestin-GFP, green) and represents the radial glia-like stem
cell (type-1 cells; left panel in B). The second type is more horizontally oriented and expresses GFAP (blue) together with S100β (red), is postmitotic
and does not have precursor cell functions. Scale bar (in M for all images) 40 μm for (A). C. Four hours after a single BrdU injection newly generated
BrdU-positive cells (green) in the SGZ express doublecortin (DCX), which here identifies neuroblasts and immature postmitotic neurons (blue). Scale
bar 25 μm. D. One week after BrdU an intermediate stage of neuronal maturation in the SGZ is detected in most newborn neurons. Newly generated
BrdU-positive cells (green) coexpress DCX (blue) and postmitotic neuronal marker NeuN (red). Scale bar 30 μm. E. DCX (blue) expressing “neu-
roblast” (type-3 cells) in the SGZ co-express Ki67 (green) a marker detecting cells during active cell cycle. Scale bar 25 μm. F. Four weeks after BrdU
almost all BrdU-positive cells (green) coexpressed the mature neuronal marker NeuN (red) and have lost DCX expression (blue). Scale bar 60 μm. G.
Four hours after BrdU, a subset of the labeled cells (red) coexpress GFAP (blue), indicating the proliferative activity of the stem cells. Scale bar 25 μm.
H. Three days after BrdU GFAP-positive (blue) cells coexpressed BrdU (red) and Ki67 (green), indicating these cells were still mitotically active in this
phase of maturation or have re-entered the cell cycle. Scale bar 25 μm. I. Four weeks after BrdU almost all GFAP-positive (blue)/BrdU-positive (red)
cells coexpressed S100β (green), indicating these cells were classical astrocytes in terms of antigen expression. Scale bar 25 μm. K. At all investigated
time points newly generated BrdU-positive (red) ramified microglia cells expressing Iba-1 (blue) were detected in the SGZ. Scale bar 40 μm.
L. NG2 cells (green) were detected in the SGZ and hilus. Only the cells in the hilus incorporated BrdU. No BrdU-positive (red) NG2-positive cells
were found in the SGZ. Scale bar 30 μm. M. CNPase-positive (green) mature oligodendrocytes did not incorporate BrdU (red) at any investigated
time point. Scale bar 30 μm. Reprinted from Steiner et al. 2006.

3 A S T R O C Y T E -L I K E O R R A D I A L
G L I A-L I K E S T E M C E L L S ?
Accordingly, the fact justifying that adult neurogenesis
is featured in a book on neuroglia is that a population of
astrocyte- or radial glia-like cells produces the new neurons
of the adult brain. On the basis of GFAP expression, stem
cells can be isolated from the brain and genetic ablation of
GFAP-expressing cells eliminated adult neurogenesis (Garcia
et al. 2004; Giachino et al. 2005; Imura et al. 2003; Morshead
et al. 2003). The stem cells that are found in the adult SVZ
and SGZ share many glial properties besides GFAP expres-
sion (see Fig. 40.2A,B). Their basic morphology and marker
profile (GLAST, BLBP, Pax6) is highly similar to radial glia
in the developing brain, even though they do not span out
between the ventricular and pial surface and do not serve as
guidance structure to radially migrating newborn neurons
(Merkle et al. 2004). In the hippocampus, they also have lost
contact with the ventricular surface, whereas in the SVZ their
apical process reaches the ventricular lumen through a central

506 • NEUROGLIA
opening of ependymal cells that is ordered in a rosette-like
arrangement (Mirzadeh et al. 2008). In the SGZ they are
referred to as type-1 cells or radial glia-like (RGL) stem cells;
in the SVZ as B cells. In analogy to the role of radial glia dur-
ing cortical development (see chapters 5 and 30) they serve as
the stem cell of the two neurogenic zones. Consequently, they
express stem cell markers such as Sox2 and Nestin (Komitova
and Eriksson 2004; Steiner et al. 2006; Suh et al. 2007). In
the SGZ they generate one lineage of granule cells plus
astrocytes (Fig. 40.2C–F), in the SVZ there are at least
seven (probably more) distinct types of interneurons in the
olfactory bulb (Lledo et al. 2008). A separate lineage, prob-
ably originating from a specific population of B cells is respon-
sible for producing NG2 cells and oligodendrocytes. These do
not migrate to the olfactory bulb as do the neurons, but below
the corpus callosum and into the cortex. The radial glia-
like stem cells have low proliferative activity but produce
fast dividing intermediate progenitor cells, referred to as
type-2 cells in the hippocampus and C cells in the SVZ.
At the type-2 cell stage, the progenitor cells go through a
transition from glial to early neuronal marker expression.
A characteristic marker that is expressed at this stage is
doublecortin (DCX) (Fig. 40.2C–F), a cytoskeleton-associated
protein involved in migration and neurite extension
(Brown et al. 2003; Couillard-Despres et al. 2005; Rao and
Shetty 2004).
Quiescent subpopulations exist at the stem cell level that
can be recruited into the process of neurogenesis. Studies
using different methods to study the mode of division of the
stem cells in the hippocampus came to opposing conclusions
(Bonaguidi et al. 2011; Encinas et al. 2011). Possibly, in the
absence of activating stimuli, the stem cells exhaust them-
selves, ultimately turning into astrocytes (Encinas et al. 2011).
However, both symmetrical and asymmetrical divisions seem
principally possible (Bonaguidi et al. 2011), so that activating
situations are conceivable, in which exhaustion is prevented
(Kempermann 2011c).
4 ASTROCY TES AS NICHE CELLS
The stem cell niche is the functional unit of the stem cells and
their microenvironment. Stem cell cultures have to provide
the particular niche factors and often still do so insufficiently.
Cell culture conditions are biased toward secreted humoral
factors and extracellular matrix, but generally do not faithfully
represent the cellular composition of the niche. The so-called
neurospheres agglomerate cultures of neural precursor cells
in which spontaneous clusters of cells develop, and probably
represent intrinsic attempts for self-organizing niches that in
this case drive the cells toward differentiation (Reynolds and
Rietze 2005). Monolayer cultures are more homogenous,
but are essentially devoid of cell–cell contacts, which are an
important component of the niche.
Niche cells comprise the different precursor cells, endothe-
lial cells, astrocytes, and microglia (Fig. 40.2K). In addition,
the SVZ niche also contains ependymal cells, whereas in the
SGZ neurons are also found. The different niche cells make
A D U LT N E U R O G E N E S I S
different contributions to niche functionality, maintenance of
the precursor cells, and regulation of adult neurogenesis.
Astrocytes play a dual role, as stem cells and niche cells,
and at present it is not fully clear whether the astrocytes per-
forming these tasks are always identical or subpopulations
exist (Fig. 40.3). In the SGZ a population of nonproliferating,
S100E-positive astrocytes exists (and is produced in the course
of adult hippocampal neurogenesis) (Steiner et al. 2004),
which has a “horizontal” morphology, as opposed to the radial
appearance of stem cells (Seri et al. 2001). How much they
contribute to niche functions is not quite clear. Niche astro-
cytes from neurogenic regions are different from astrocytes
in nonneurogenic zones (Song et al. 2002), which supports
the idea that the niche provides a critical neurogenic permis-
siveness. Astrocytes exert numerous niche functions (besides
their role as precursor cells) in that they are a main source of
secreted factors. Despite concrete evidence for a number of
factors and plausible hypotheses for several others (Morrens
et al. 2012), it is still unresolved what exactly renders a neuro-
genic region neurogenic.
Key examples of soluble niche signals are the paracrine fac-
tors Notch, Wnt, Shh, and BMP. All of these have well-studied
functions in embryonic neurogenesis and play similar but
adjusted roles in adult neurogenesis (Bonaguidi et al. 2005;
Breunig et al. 2008; Lie et al. 2005; Lim et al. 2000; Lugert
et al. 2010; Palma et al. 2005). By and large, they are all
involved in maintaining the stem cell pool and balancing this
maintenance function with effects on later stages of develop-
ment. We still know little about how the four factors interact
and how far their signaling is combinatorial, but much para-
crine signaling originates from astrocytes, supporting their
role as key orchestrators.
5 OT H E R G L I A L C O N T R I B U T I O N S
TO A D U LT N E U R O G E N E S I S
Glial cells play numerous roles related to adult neurogen-
esis (Fig. 40.4). Microglia are prime candidates as source of
cytokine signaling and mediators of immune effects on adult
neurogenesis. However, astrocytes have certain immunologi-
cal functions, too, so that the precise individual contributions
have not yet been identified. Microglia have a double-edged
influence on adult neurogenesis in that microglia might both
support and damage adult neurogenesis, presumably depend-
ing on their state of activation. Evidence for this dual role,
however, so far largely comes from studies on ischemia-in-
duced reactive neurogenesis in the striatum. An initial inflam-
matory response directly following the injury downregulated
precursor cell activity in the SVZ, but was prevented when
microglia were inhibited with the drug minocycline (Ekdahl
et al. 2003). At the same time, the very low but long-lasting
neurogenic response in the striatum also seems to be a con-
sequence of the immunological response (Ekdahl et al. 2003;
Thored et al. 2009). Microglia in physiological neurogenesis
have been far less studied, but it is assumed that even in a
resting state they contribute to niche effects, for example, by
phagocytosis of cells produced in excess (Sierra et al. 2010).
• 507
 astrocyte-like
precursor cell
generates
NICHE
influences
generates
Figure 40.3 Astrocytes and the Concept of the Stem Cell Niche. Astrocytes play a particular role in the “stem cell niches” of the adult brain.
Astrocyte-like precursor cells give rise to new neurons and produce other astrocytes without precursor cell functions (see Fig. 40.1). Both types of
astrocytes are constituents of the niche, but their relative contribution has not yet been elucidated. The depicted morphologies reflect the situation
in the hippocampus. Reprinted from Morrens et al. 2012.
6 N E U R O G E N E S I S I N T H E A D U LT
S U BVE N T R I C U L A R Z O N E A N D
O L FAC TO RY B U L B
In neurogenesis of the adult SVZ and olfactory bulb, neuronal
development is spread out over a long distance between the
ventricular wall and periphery of the olfactory bulb, and hence
involves extensive migration (see Fig. 40.1). Proliferation
of SVZ precursor cells is much more prominent than in the
hippocampus.
The radial glia-like stem cells (B cells) give rise to tran-
siently amplifying C cells, which in turn generate the migra-
tory neuroblasts (A cells), which embark on the long journey
to the olfactory bulb (Doetsch et al. 1999a,b). For this migra-
tion through the rostral migratory stream, they use a partic-
ular type of migration, called chain migration, in which the
migrating cells use each other as guidance structures (Lois
and Alvarez-Buylla 1994). The rostral migratory stream, at
least in rodents, is lined by a tube of astrocytes, which serve
as a guidance structure to the migratory neuroblasts and also
have precursor cell functions. New migratory A cells and
thus new neurons can arise from the stem cells of the RMS as
well (Gritti et al. 2002). However, migration can also occur
without this glial tube ( Jankovski et al. 1998). Blood vessels
and possible the obliterated olfactory ventricles seem to be
involved in route finding (Whitman et al. 2009). In the bulb,
the cells switch to a radial mode of migration and reach their
final destination in the granule cell layer of the periglomerular
zones.
Precursor cells in the SVZ are subregionalized, and differ-
ent parts of the walls of the lateral ventricles give rise to a large
508
other astrocyte
• cell-cell contact
• paracrine/autocrine (short-range)
• modulation of ambient
neurotransmitters (mid-range)
• growth factors, hormones,
cytokines (long-range)
number (at least seven) of different populations of interneu-
rons in the olfactory bulb (Merkle et al. 2007). These are func-
tionally distinct, and it is unclear whether specific functional
demand might trigger the production of the subtypes.
7 N E U R O G E N E S I S I N T H E A D U LT
HIPPOCAMPUS
Adult hippocampal neurogenesis differs from neurogenesis
in the adult olfactory system in numerous ways: Only one
type of neuron is produced and this is an excitatory principal
cell, not an interneuron. The entire process of hippocampal
neurogenesis takes place in a very confined space, with migra-
tion from the SGZ into the granule cell layer amounting
to a few hundred microns at most. The new neurons in the
hippocampus feature a very elaborate morphology with a
large dendritic tree and a long axon that becomes part of
the mossy fiber projection between the dentate gyrus and
CA3 region. The mossy fiber tract shows an amazing degree
of plasticity, and adult neurogenesis explains part of this
adaptiveness.
In the SGZ, the radial glia-like stem cells (type-1 cells)
have many astrocytic features (Filippov et al. 2003; Seri et al.
2001) and give rise to transient amplifying type-2 cells, which
are highly proliferative and respond to many external regula-
tory influences (Kronenberg et al. 2003) (see Fig. 40.1). At the
level of type-2 cells, a transition from a glia-like (type-2a) to an
early neuronal phenotype (type-2b) takes place (Steiner et al.
2006). The following “neuroblast” stage is referred to as type-3.
Type-3 cells might still proliferate but their rate of proliferation
• NEUROGLIA
 Subgranular zone Subventricular zone

Precursor cells
• self-renewal, expansion
• gap-junctional coupling
• guidance structure
Type-1 • auto- and paracrine signals
• ECM production B1 cell
Type-2a
Type-2b C cell
Type-3 A cell
Non-precursor cell
astrocytes
• modulation of
neurotransmitter effects B2 cell
• ECM production
Horizontal astrocyte • secretion of trophic factors Other astrocyte (?)
• paracrine signals

NG2 cells
• modulation of
neurotransmitter effects (?) NG2+ C cell

Microglia
• cytokine signaling
• phagocytosis
Microglial cell Ependyma Microglial cell
• paracrine and
juxtacrine signaling (?)
• back-up precursor cell
function Ependymal cell

Vasculature
• secretion of trophic factors
Capillaries • general nutrient supply
• guidance structure
Capillaries
(not in SVZ proper but
reached by B cell processes)

Neurons
• secretion of trophic factors
• ambient (and synaptic)
neurotransmitters
• guidance structure
Granule cells
and No neurons in the
interneurons SVZ proper

Figure 40.4 The Cellular Components of the Neurogenic Niches. Reprinted from Morrens et al. 2012.

is much lower than at the type-2 level. Type-3 cells also migrate
their short distance into the granule cell layer (but the major-
ity stays in the lower third), and at this stage also the first
neurites might appear. After exit from the cell cycle, a wave
of cell death occurs while the new neurons mature. Within
about 10 days (in rodents) the axon has reached CA3 (Hastings
and Gould 1999; Stanfield and Trice 1988), where the new
terminals compete with existing synapses at the mossy
fiber boutons, the characteristic input structures to CA3
(Toni et al. 2008). In the following period, the new neurons
go through a phase of increased synaptic plasticity (Garthe
et al. 2009; Saxe et al. 2006; Schmidt-Hieber et al. 2004;
Snyder et al. 2001), which not only results in a particular
functionality during this time, but is also instrumental for
their ultimate recruitment and lasting integration into the
network.
8 R E GU L AT I O N O F A D U LT
NEUROGENESIS
Adult neurogenesis responds to a very large number of factors
and stimuli at diverse conceptual levels, from behavioral down
to genetic and epigenetic (see discussion in Kempermann
2011d). This hierarchy is immensely complex. Because adult
neurogenesis is a process involving many stages, regulation is
also spread out over time. In the course of development, late
regulatory events build on the preceding events.
Importantly, neurogenesis in the adult hippocampus is
strongly regulated by behavioral activity and learning (Gould
et al. 1999a; Kee et al. 2007; Kronenberg et al. 2003; Tashiro
et al. 2007). This aspect seems less prominent in the olfactory
bulb (Lledo et al. 2006; Rochefort et al. 2002), but in both
situations adult neurogenesis is part of adaptive plastic events.

A D U LT N E U R O G E N E S I S • 509
Adult hippocampal neurogenesis appears to be the brain’s way
to cope with novelty and complexity in learning experiences
and is a way to master the tradeoff between stability and flex-
ibility in a neuronal network that is supposed to learn. In this
sense, regulation of neurogenesis and its function are closely
linked.
At the cellular level the regulatory factors converge on the
niche. Cell–cell contacts, extracellular matrix, and humoral
factors make up a complex machinery. A large number of
secreted molecules including autocrine and paracrine signals,
growth and neurotrophic factors, and neurotransmitters have
been studied and yield the picture of a process that is sensitive
to a broad range of extrinsic stimuli and builds on the integra-
tion of numerous intrinsic signaling pathways.
9 F U N C T I O N A L R E L E VA N C E O F
A D U LT N E U R O G E N E S I S
In the hippocampus, the new neurons contribute to basic
functionality of the dentate gyrus. All detectable enhanced
postsynaptic potentials (long-term potentiation) originate
from the newborn neurons, which go through a postmitotic
period of enhanced synaptic plasticity. Mature granule cells
are heavily inhibited by the local interneurons. If one elimi-
nates adult neurogenesis, dentate gyrus LTP disappears, unless
one also blocks the inhibition (Garthe et al. 2009; Saxe et al.
2006). Then, a strong LTP from the mature cells can be seen.
This plasticity provided by the new neurons seems to be
important for the functions of the dentate gyrus, especially
pattern separation (Aimone et al. 2006; Clelland et al. 2009)
and the avoidance of catastrophic interference (Wiskott et al.
2006). Pattern separation is the ability to memorize two stim-
uli as distinct and is a cardinal feature underlying higher-or-
der cognitive functions. New neurons, one theory goes, are
involved in providing a “time-stamp” to incoming information
that needs to be learned (Aimone et al. 2009). Related to this
is the finding that adult-born neurons are required to form the
association between a shock stimulus and the environment, in
which this shock was applied (contextual fear conditioning)
(Imayoshi et al. 2008). This function also bridges between the
cognitive and affective functions of the hippocampus (Revest
et al. 2009). The hypothesis that adult neurogenesis might play
a role in the development and maintenance of major depres-
sion is supported by this involvement (Sahay and Hen 2007).
Route finding and spatial memory are a particular case of
episodic memory in which a sequence of combinations of land-
marks has to be memorized. Consequently, there is also a mea-
surable effect of adult neurogenesis on the acquisition of the
classical water maze task, a test for hippocampus-dependent
learning (Garthe et al. 2009). In addition, however, and more
importantly, new neurons are critically involved in perfor-
mance in the “reversal” situation, in which the target to which
the animals have to navigate, is secretly moved to a new posi-
tion and the flexibility of the animals to adjust to the new situ-
ation and learn the new target location is assessed.
The function of new neurons in the adult olfactory bulb is
less clear. An involvement of adult neurogenesis in olfactory
510 • NEUROGLIA
memory has been proposed, but this is likely to be a down-
stream effect of some functionality in the olfactory bulb itself.
Adult-generated olfactory interneurons might help to opti-
mize the network (Lledo and Lagier 2006), also with respect
to the challenge that occurs, because owing to massive lifelong
neurogenesis in the olfactory epithelium that constantly alters
the input to the olfactory bulb.
10 N E U R O G E N E S I S O U T S I D E T H E
NEUROGENIC REGIONS OF THE
A D U LT B R A I N ?
Numerous studies have dealt with the question, whether new
neurons might also be constitutively found in additional
regions of the rodent (and primate) brain. Adult cortical neu-
rogenesis received most attention and controversy (Gould
et al. 1999b; Rakic 2002b). Examples of the two imaginable
cases of neurogenesis originating from the SVZ and local
resident precursor cells within the cortical parenchyma have
been reported (Dayer et al. 2005; Gould et al. 1999b; Ohira
et al. 2010). But ultimately most claims about constitutive
adult cortical neurogenesis have not withstood the most rig-
orous testing or at least still await independent confirmation.
Mostly methodological issues caused fundamental concern
(Nowakowski and Hayes 2000; Rakic 2002b). One exception
is the case of an extremely low level of neurogenesis in corti-
cal layer VI at least in young-adult animals (Dayer et al. 2005;
Inta et al. 2008), but the numbers are extremely low. The case is
different for reactive neurogenesis in the case of ischemia and
after boosting with growth factor infusions. The best known
example is neurogenesis in a very precise neuronal ablation
model, in which no inflammatory response is evoked (Magavi
et al. 2000). However, for region CA1 of the hippocampus a
case of massive regenerative neurogenesis after ischemia and
growth factor infusion has been claimed but never been con-
firmed and substantial questions about the proposed mecha-
nism remain (Nakatomi et al. 2002). Nevertheless, it cannot
be excluded that pathological situations might uncover a neu-
rogenic potential outside the “canonical” neurogenic regions.
Possibly many examples of “neurogenesis” actually repre-
sent signs of neuronal differentiation in NG2 cells (see chapter 10).
The problem is that, interestingly, neuronal markers can also
appear in NG2-expressing glia, even though these cells never
turn into neurons devoid of coexisting glial features and signs
of neuronal maturity. Especially the expression of doublecor-
tin (DCX) in NG2 cells can be misleading (Ehninger et al.
2011; Guo et al. 2010; Tamura et al. 2007). Doublecortin is
characteristically expressed in precursor cells and new neurons
of SGZ and SVZ; it is often used as a surrogate marker of neu-
rogenesis, even though the generalization from neurogenesis
in SGZ and SVZ is not justified.
The hypothalamus stands out among noncortical regions
for which neurogenesis has been reported (Kokoeva et al.
2005; Migaud et al. 2010; Perez-Martin et al. 2010). Here,
radial glia-like precursor cells of the wall of the third ventricle
are thought to be the origins of adult-generated neuroendo-
crine cells. The appropriate developmental potential of these
precursor cells has demonstrated ex vivo (Markakis et al.
2004). Apparently, neurogenesis can be elicited by CNTF
(Kokoeva et al. 2005) or IGF1 (Perez-Martin et al. 2010), but
methodological concerns remain.
Generally, the absence of a phenomenon is difficult to
prove, and interesting observations should not be prema-
turely dismissed, but should be rigorously tested before they
are accepted. It might turn out that a too-rigid definition of
neurogenesis, excluding the neuronlike properties of NG-cells
(see chapter 10) is not justified. On the other hand, departure
from very stringent criteria of what a neuron is in relation to a
glial cell will enhance confusion rather than reducing it. After
all, adult neurogenesis in the hippocampus and olfactory bulb,
in which neurons lifelong originate from astrocyte-like cells,
calls many classical distinctions into question.
11 G L I O G E N E S I S I N T H E C O N T E X T
O F A D U LT N E U R O G E N E S I S
Gliogenesis and neurogenesis are intricately linked, not only
because cells with glial properties produce new neurons. Both
neurogenic regions produce glia alongside neurons: oligoden-
drocytes and interneurons in the SVZ, and astrocytes and
excitatory granule cells in the SGZ. These astrocytes are not
precursor cells, but have a distinct morphology and antigen
profile. They are found throughout the granule cell layer. In the
hippocampus, gliogenesis and neurogenesis are coregulated to
a certain extent (Steiner et al. 2004). One of the population
models describing the dynamics of the different precursor cells
and developmental stages in the adult hippocampus suggests
an even tighter coupling, ultimately leading to an exhaustion
of the precursor cell pool and their conversion into astrocytes
(Encinas et al. 2011). Other studies suggest more flexibility in
self-renewal and the proportions within the lineage tree, but
similarly suggest a very close interdependency (Bonaguidi
et al. 2011; Suh et al. 2007).
Self-renewal of the astrocyte-like stem cells itself is also
a particular form of gliogenesis present in both neurogenic
regions. The adult SGZ produces very few oligodendrocytes
(Kempermann et al. 2003), and it is not fully clear whether
these originate from the same population of precursor cells
as the astrocytes and neurons. Ex vivo, the precursor cells are
indeed tripotent, and overexpression of transcription fac-
tor Ascl1 (Mash1) in precursor cells of the hippocampus in
vivo induced oligodendrogenesis ( Jessberger et al. 2008).
Conversely, a low number of astrocytes without involvement
in neurogenesis might be produced in the SVZ, but there is
limited information on this aspect.
12 A D U LT N E U R O G E N E S I S I N
B R A I N PAT H O L O GY
Adult neurogenesis is relevant for medicine for several reasons.
First, adult neurogenesis inspires fundamental research on
regenerative medicine because here nature demonstrates what
bioengineering tries to achieve: how to make neurons under
A D U LT N E U R O G E N E S I S
the conditions of the adult brain. Second, adult neurogenesis
itself has medical relevance if it fails to deliver the particular
type of plasticity for which it is responsible. Several specific
hypotheses have been raised linking adult hippocampal neu-
rogenesis to age-related cognitive decline and dementia, major
depression and posttraumatic stress disorder, and schizophre-
nia and temporal lobe epilepsy (Kempermann 2011b). These
theories can obviously only relate to the contribution that
hippocampal function makes to the disease. Therefore, failing
adult neurogenesis does not explain major depression in its
entirety, but might represent an important facet at the inter-
face of affective functions and cognition as it takes place in
the hippocampus. In the case of temporal lobe epilepsy, the
central idea is that seizure induces proliferation of precursor
cells, including those at later stages of development, leading
to increased numbers of immature neurons with ectopic posi-
tions and aberrant neurites (Bengzon et al. 1997; Jessberger
et al. 2005; Parent et al. 2006). These might contribute to the
propagation of the disease and the transition from individual
seizures to epilepsy. Seizure activity also affects the SVZ, but
the pathological consequences of this activation are not yet
clear (Young et al. 2011).
Failing neurogenesis in the olfactory bulb has been brought
into connection with the anosmia found in neurodegenerative
disease (Galvan and Bredesen 2007) and depression (Negoias
et al. 2010), but given the limited importance of the sense of
smell for greater function of the human brain, neurogenesis in
the adult olfactory bulb has received less medical attention.
The potential contribution to SVZ-derived precursor cells to
regenerative processes throughout the brain, whether regener-
ative neurogenesis or other forms of cellular plasticity, has had
a significant impact on regenerative neurobiology (Lindvall
and Kokaia 2006). Much of this interest has been sparked by
the observation of regenerative neurogenesis after ischemia in
the striatum. Here, after experimental stroke, neural precur-
sor cells migrated from the SVZ into the striatum, where they
differentiated into neurons, albeit at extremely low numbers
(Arvidsson et al. 2002). However, the effect was very long last-
ing (Thored et al. 2006).
The presence of neural precursor cells in the adult brain
and their astrocyte-like properties have also affected con-
cepts about the origin of brain tumors (Park and Rich 2009;
Siebzehnrubl et al. 2011; Stiles and Rowitch 2008). Generally,
the stem cell hypothesis of cancerogenesis has fundamentally
changed oncology, but the influence on neurooncology has
been particularly strong. If gliomas originate from precursor
cells rather than differentiated glial cells, then several features
of gliomas, especially the patterns of differentiation within
them, can be explained. This particularly applies to features of
neuronal differentiation in gliomas, which have always been
difficult to understand, and to neuronal tumors, such as gangli-
ocytomas and neurocytomas. Thus, the differences among the
known types of brain tumors might reflect their origin from
different precursor cell types in the neurogenic zones or brain
parenchyma. At the same time, tumors seem to attract precur-
sor cells, and precursor cells exert a certain antitumorigenic
activity that could be consistent with their role in the niche to
keep proliferative activity controlled (see also chapter 30).
• 511
 Anticancer treatments aiming at proliferative tumor cells
might do collateral damage to proliferating precursor cells,
possibly explaining cognitive deficits after chemotherapy and
irradiation.
13 S U M M A RY A N D P E R S P E C T I VE S
Adult neurogenesis is a process that adds a particular type of
functionality and plasticity to the adult brain. Rather than
being an atavism, as was presumed for some time after the
initial description, adult neurogenesis is a highly specialized
mechanism that adds flexibility and adaptation to very par-
ticular types of networks. Outside the two canonical neuro-
genic regions in the hippocampus and olfactory system, the
interpretation of the often-conflicting results and potential
functional benefit is more difficult. However, there might be
more examples of neurogenesis than hippocampus and olfac-
tory system, even in mammals, but under physiological con-
ditions they seem to range on a minute scale, if they exist at
all. Nevertheless, some neurogenesis might be inducible under
pathological conditions, although if and how this reactive
neurogenesis contributes to regeneration is not known.
At the cellular level, the fact that glia makes neurons and
even normal astrocytes can be experimentally reprogrammed
to become neurogenic has overturned many traditional con-
cepts about the relationship between the cell lineages of the
brain. But below this deceiving simplicity lies an immensely
complex process, controlled by numerous genes and regulated
by behavioral activity and a large number of extrinsic factors.
The key questions in the field that await answers in the
years to come are the following. How does adult neurogenesis
contribute to brain plasticity in humans? How is the recipro-
cal interaction of regulation and function in adult neurogen-
esis established? What exactly is the specific function of new
neurons? And what exactly is the nature of the glia-like stem
cells that drive adult neurogenesis?
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 41.
MODULATION OF NEUROENDOCRINE SYSTEMS
Stéphane H. R. Oliet

A B B R E VI AT I O N S within the PVN and SON (Ludwig 1998). Peripheral and cen-
tral secretion of these hormones is dependent on the electrical
AMPA D-amino-3-hydroxy-5-méthylisoazol-4- activity of magnocellular neurons that is itself under the influ-
propionate ence of afferent synaptic inputs. The SON is a very homog-
ATP adenosine triphosphate enous nucleus, composed essentially of the soma and dendrites
ECM extracellular matrix of OT and VP neurons, astrocytes, and vascular elements. The
ECS extracellular space majority of glial cells in this nucleus are radial astrocytes whose
GABA J-aminobutyric acid cell bodies are located in the ventral portion of the SON, or
GnRH gonadotropin-releasing hormone ventral glia limitans, from which arise long processes oriented
HNS hypothalamo-neurohypophysial system ventrodorsally that enwrap neuronal elements (Bonfanti et al.
LTD long-term depression 1993; Israel et al. 2003b). These astrocytes are reminiscent of
LTP long-term potentiation radial glia in the developing central nervous system. The main
mGluR metabotropic glutamate receptor afferents to the SON and the PVN use GABA and glutamate
NCAM Neural cell adhesion molecule as inhibitory and excitatory transmitters, respectively (Wuarin
NMDA N-methyl-d-aspartate and Dudek 1993). In the SON, about 40% of all synapses are
NO nitric oxide GABAergic, whereas 25% are glutamatergic (Theodosis 2002).
OT oxytocin At the level of the neurohypophysis, at which axon terminals of
PSA polysialic acid OT and VP neurons contact the blood vessels together, one can
PVN paraventricular nucleus find specialized astrocytes known as pituicytes.
SON supraoptic nucleus Another hypothalamic area that has received a lot of
VP vasopressin attention in the context of neuronal–glial interactions is the
gonadotropin-releasing hormone (GnRH) system that is made
of GnRH neurons whose cell bodies are located in the arcuate
1 INTRODUCTION nucleus and which project their axon terminals in the median
eminence, where GnRH can be directly released in the portal
The hypothalamus is a brain structure controlling several vital system to mediate key reproductive functions. Astrocytes can be
functions such as reproduction, lactation, food intake, drink- found in both the arcuate nucleus and the median eminence. In
ing, circadian rhythms, autonomic functions, body fluid, and the latter, specialized glial cells known as tanycytes are also pres-
temperature homeostasis. It is comprised of many heteroge- ent. These radial-like glial cells extend from the third ventricle to
neous nuclei that are themselves composed of neurons and the portal system where they enwrap GnRH axon terminals.
glial cells. Hypothalamic neurons are neuroendocrine cells that One of the most remarkable properties of the
synthesize and release peptides as a function of their electrical hypothalamo-neurohypophysial system is its ability to
activity. Such peptides can be released locally, where they serve undergo a reversible anatomical remodeling that is character-
as signaling molecules, and/or secreted in the bloodstream, ized by a reduction of the glial coverage of magnocellular neu-
where they act as neurohormones to regulate distant targets. rons and synapses (Bobak and Salm 1996; Hatton et al. 1984;
One of the most extensively studied hypothalamic areas is the Theodosis and Poulain 1993). This occurs during physiologi-
hypothalamo-neurohypophysial system (HNS). It is made of cal conditions such as parturition, lactation, and dehydration.
magnocellular neurons that synthesize the hormones oxytocin A similar process takes place at the level of the neurohypo-
(OT) and vasopressin (VP) and whose cell bodies are located in physis, where the pituicytes retract from between the axon
the supraoptic (SON) and paraventricular (PVN) nuclei of the terminals (Hatton 1999). This phenomenon has permitted
hypothalamus. These neurons project their axons in the neuro- the study of neuronal–glial interactions by comparing cel-
hypophysis where their hormonal content is released directly in lular processes under different conditions of glial environ-
the blood stream. OT is essential for vital functions such as lac- ment. As such, it was proved to be a unique model to establish
tation and parturition, whereas VP is the antidiuretic hormone the contribution of glial cells to neuronal functions. Similar
critical to body fluid and cardiovascular homeostasis. In addi- neuronal–glial anatomical changes have since been reported
tion to their peripheral release, it is now clear that OT and VP in other hypothalamic centers of rodents and nonhuman pri-
can also be released centrally in different brain regions, including mates, as well as in nonhypothalamic structures.

515
 2 CONTRIBUTION UNDER
BASAL CONDITIONS
Because of their position in the neuropile, astrocytic processes
represent a physical barrier restricting spillover and diffusion of
signaling molecules that are released locally (Piet et al. 2004).
Moreover, because of their position, these glial cells are known
to control the local environment in which neurons develop and
function. In particular, they maintain a tight control on local
ion concentrations and pH. Through their coupling with blood
vessels, astrocytes provide metabolic substrates to neurons
while clearing away toxic waste. The concentrations of extracel-
lular K+ or glutamate, that are produced as a result of neuronal
activity for example, need to be tightly regulated because their
accumulation in the extracellular space (ECS) can alter neu-
ronal excitability dramatically (Boudaba et al. 2003; Oliet et al.
2001). The clearance of glutamate, the major excitatory neu-
rotransmitter in the brain, is ensured by GLT-1 and GLAST,
two glutamate transporter subtypes expressed at the glial mem-
brane (see chapter 35). This control is essential to preserve the
spatiotemporal profile of the glutamate transient and regulate
the synaptic efficacy through the activation of presynaptic glu-
tamate receptors coupled to transmitter release. This includes
metabotropic glutamate (mGluR), as well as kainate receptors,
whose activation inhibits and facilitates synaptic transmission
in the SON, respectively (Bonfardin et al. 2010; Boudaba et al.
2003; Oliet et al. 2001).
Glial cells also release active substances termed gliotrans-
mitters by analogy to neurotransmitters (see chapter 17).
In the hypothalamus, several gliotransmitters have been identi-
fied, including taurine, d-serine, and ATP (Gordon et al. 2005;
Hussy et al. 2000; Panatier et al. 2006). Taurine is released
through volume-sensitive ion channels in response to hypo-
tonic stimulation. It activates strychnine-sensitive glycinergic
receptors expressed by magnocellular neurons. These receptors
are permeable to chloride ions so that they mediate membrane
hyperpolarization when they are stimulated at resting mem-
brane potential. This hypotonic-induced release of taurine is
believed to be part of the osmoregulatory process that enables
the control of osmolality by VP cells (Hussy et al. 2000).
When osmolality is reduced, glial cells swell, causing the release
of taurine. Glycinergic receptors are in turn activated, leading
to hyperpolarization of VP cells and inhibition of their firing.
This ultimately leads to reduced secretion of VP in the general
circulation. As a consequence, diuresis is increased, thereby
contributing to the recovery of normal osmolality.
d-Serine is also released from astrocytes in the SON
(Panatier et al. 2006). It serves as an endogenous coagonist
of N-methyl-d-aspartate receptors (NMDARs). These glu-
tamate receptors contribute to excitatory neurotransmission
and mediate the most common forms of activity-dependent
synaptic plasticity, long-term potentiation, and long-term
depression. In the SON, NMDARs are also responsible for
generating oscillatory activity (Hu and Bourque 1992). In the
absence of such a coagonist, NMDA receptors cannot operate,
as revealed by the use of D-amino acid oxidase, an enzyme that
selectively degrades d-serine without affecting glycine (Panatier
et al. 2006). Conversely, degrading glycine with glycine oxidase
516 • NEUROGLIA
that does not affect d-serine levels has no effect on NMDAR
activity in the SON. Thus, it appears that astrocytes are essen-
tial for all the processes that depend on NMDA receptor activ-
ity. Similarly, adenosine triphosphate (ATP) originating from
astrocytes has been shown to induce the insertion in the mem-
brane of D-amino-3-hydroxy-5-méthylisoazol-4-propionate
(AMPA) receptors in PVN magnocellular neurons (Gordon
et al. 2005). Such a release is triggered by noradrenaline, and
ATP action is mediated by P2X7 subtype of receptors. This
phenomenon is believed to be important for controlling syn-
aptic strength under conditions in which adrenaline-releasing
A1 inputs onto PVN are recruited.
In the neurohypophysis, as well as the external layer of the
median eminence, processes of astrocytic-like cells (pituicytes,
tanycytes) enclose neurosecretory axons, thereby limiting the
release of their products of secretion and their possible paracrine
and/or autocrine actions. In addition, these glial processes lie
alongside perivascular spaces, thereby physically restricting the
access of neurosecretory terminals to the perivascular basal lam-
ina. Such a physical barrier affects neurosecretion by limiting dif-
fusion of neuropeptides released at the terminals into the general
circulation. In the median eminence, where capillaries are fenes-
trated, enwrapping of blood vessels by tanycyte processes appears
to contribute to the blood-brain barrier (Prevot et al. 2010).
Under control conditions, these tanycytes also prevent access of
most neuroendocrine terminals to the pericapillary space.
3 G L I A L S T RU C T U R A L P L A S T I C I T Y
3.1 C H A N G E I N G L I A L C OVE R AG E
O F N EU RO NS
It is now more than two decades ago that evidence was
reported for the first time that the morphology of glial cells
in specific neuroendocrine systems undergoes remarkable
dynamic changes in relation to the activity of adjacent neu-
rons. It is the case of the HNS during conditions associated
with enhanced secretion of VP and OT, such as chronic dehy-
dration, lactation, or parturition (Theodosis 2002). These
changes are completely reversible on cessation of stimulation
and are characterized by a progressive hypertrophy of the neu-
rons, an increased number of excitatory and inhibitory syn-
apses, and a pronounced reduction in the astrocytic coverage
of magnocellular neurons (Fig. 41.1). As a result, neuronal
surfaces no longer separated by glial processes become directly
juxtaposed. Similar changes occur at the level of the neurohy-
pophysis, in which neuro-hemal contacts increase as a result
of pituicyte retraction. These changes are similar to those
that were reported later in the suprachiasmatic (Gerics et al.
2006; Lavialle and Serviere 1993; Lavialle et al. 2001), arcu-
ate (Garcia-Segura et al. 1994b), and preoptic (Gerhold and
Wise 2006; Jansen et al. 2003) nuclei in response to circadian
rhythms and fluctuating levels of sexual steroid, respectively.
In the hypothalamus, OT neurons usually occur in tightly
packed clusters intermingled with VP neurons. Electron
microscopy shows unambiguously that in spite of such close
packing, OT cells remain separated by fine astrocytic processes
 unstimulated
dendrite
soma1
soma2
soma3
stimulated
~10 nm 0.1 μm
soma2
soma1
soma3
Figure 41.1 Anatomical Glial Remodeling in the Supraoptic Nucleus.
Electron microscopy pictures taken in the SON under control conditions
(top; unstimulated) and in lactating rats (bottom; stimulated). Thin astro-
cytic processes (blue) separate neuronal somatic and dendritic elements
in unstimulated conditions. The glial withdrawal that accompanies the
structural remodeling of the SON in stimulated conditions such as
lactation results in directly juxtaposed neuronal elements. Adapted
from Montagnese et al. 1988.
that also enwrap synapses. In contrast, during parturition,
lactation, chronic dehydration, or in response to elevated
ambient levels of OT, there is a significant reduction in the
astrocytic coverage of the neurons and their synapses. Under
normal conditions, astrocytic processes cover about 90% of
any OT somata, which is reduced to 70% during lactation or
chronic dehydration (Chapman et al. 1986; Theodosis et al.
1986a). It is of note that the astrocytic coverage of VP neurons
is not altered and remains around 90% even after strong and
persistent stimulation, such as chronic salt loading (Chapman
et al. 1986). At the same time, part of the membrane surface
of OT somata and dendrites become directly juxtaposed (see
Fig. 41.1). Although VP neurons display similar juxtapositions,
their incidence and extent are low and show no variations with
conditions that modify VP secretion (Chapman et al. 1986;
Theodosis et al. 1986a). Concomitantly, glial remodeling in
the neurohypophysis results in a reduced glial ensheathment
of neurosecretory terminals, leading to reduced gliovascular
and increased neurovascular contact zones (Theodosis and
MacVicar 1996). Under basal conditions of neurosecretion,
about 40% of the perivascular basal lamina is contacted by
M O D U L AT I O N O F N E U R O E N D O C R I N E SYS T E M S
neurosecretory terminals and about 60% by pituicytes. These
proportions are reversed during conditions that stimulate
VP and/or OT secretion (Hatton et al. 1984; Monlezun
et al. 2005).
Glial–neuronal changes occur within a few hours of
the onset of parturition or osmotic stimulation. They can
be reproduced in vitro, in acute slices of adult hypothala-
mus that include the SON (Langle et al. 2003; Theodosis
et al. 2006), or isolated neurohypophysis (Monlezun et al.
2005; Perlmutter et al. 1984). In such preparations, the glial
remodeling takes place within 1 hour after the onset of stimu-
lated neurosecretion. When the stimulation of OT neurons
ends and circulating OT concentrations come back to basal
values, the astrocytic coverage of neurons returns to its ini-
tial level, a process that can be very rapid (Langle et al. 2003)
depending on how long the system has remained activated
(Theodosis 2002).
Rapid activity-dependent astrocytic plasticity is detectable
in other hypothalamic areas. In the rat arcuate nucleus, com-
posed of neurons that control growth hormone, luteinizing
hormone, and prolactin secretion, the proportion of neuronal
somatic surface covered by astrocytic processes fluctuates with
changing sexual steroid levels. This proportion is high when
plasma estrogen is elevated, as in the afternoon of proestrus
and morning of estrus, and is low 24 hours later at metestrus
when estrogen levels have diminished (Garcia-Segura et al.
1994a). Administration of estrogen to ovariectomized animals
mimics the effects of the estrous cycle on the glial coverage of
neurons (Garcia-Segura et al. 1994a). A similar type of struc-
tural reorganization has been reported in the infundibulum
and preoptic area of nonhuman primates (Garcia-Segura et al.
1994a; Witkin et al. 1991), in which fluctuation of steroid
levels causes a variation of the glial coverage of the neurons
synthesizing GnRH, a hormone that controls the secretion of
gonadotropin from the pituitary. Such variations can be mim-
icked when estrogen is given to ovariectomized animals.
The axons of GnRH neurons project to the external layer
of the median eminence, where changing steroid levels result
in glial changes similar to those observed in the neurohypo-
physis (De Serrano et al. 2004; King and Rubin 1995), includ-
ing retraction of tanycyte processes from the perivascular
zone and access of GnRH terminals to fenestrated capillar-
ies. Therefore, in neurohemal structures glial rearrangements
ultimately promote the access of neurosecretory terminals
to perivascular zones. This is likely to favor the secretion of
neurohormone into the bloodstream, while at the same time
allowing exposition of neuroendocrine terminals to signaling
molecules circulating in the general circulation.
3.2 P E R M I S S I VE FAC TO R S
The structural plasticity that occurs in different neuroendocrine
structures must involve several permissive and inductive factors,
including cell surface and extracellular matrix (ECM) molecules,
as well as soluble agents, some of which have been identified in
developing systems undergoing similar glial and neuronal struc-
tural remodeling. It is not surprising then that neural systems
displaying remodeling properties in the adult continue to express
• 517
many of these embryonic factors. This includes cell adhesion
molecules such as cadherins, neurexins, and Neural cell adhe-
sion molecule (NCAM). Complex ECM glycoproteins such
as chondroitin sulfate proteoglycans and tenascins also appear
to participate (Kleene and Schachner 2004; Theodosis et al.
2004a; Waites et al. 2005). NCAM, and in particular its highly
sialylated isoform PSA-NCAM (polysialic acid-NCAM), seem
to play an important role in glial–neuronal structural plastic-
ity. Whereas PSA-NCAM is abundant in developing tissues,
it is not the case in adult structures that express NCAM with
little PSA. However, it continues to be expressed strongly in
adult systems that are able to undergo morphological plastic-
ity (Bonfanti 2006; Bonfanti et al. 1992; Seki and Arai 1993).
In the HNS, PSA-NCAM is made by both astrocytes and neu-
rons throughout life (Kiss et al. 1993; Theodosis et al. 1991,
1999). Whereas in some systems, such as the hippocampus,
PSA-NCAM expression depends on synaptic activity (Kiss and
Muller 2001), its expression in the HNS is constitutive and not
very sensitive to neuronal activity.
PSA-NCAM appears to be a prerequisite for the struc-
tural plasticity of the HNS. The specific enzymatic removal
of PSA from NCAM in situ in the SON inhibits glial and
neuronal remodeling associated with lactation and chronic
dehydration (Theodosis et al. 1999). Similarly, morphological
changes occurring at the levels of axon and glia in the neuro-
hypophysis are compromised by the PSA enzymatic removal
(Monlezun et al. 2005). This manipulation, however, has no
effect once the remodeling has already occurred. PSA-NCAM
is also very highly expressed in the GnRH system of rodents
(Bonfanti et al. 1992; Hoyk et al. 2001), sheep (Viguié et al.
2001), and primates (Perera et al. 1993). The mechanism by
which PSA-NCAM plays a role in morphological plasticity
is unknown, but it is very likely that large quantities of car-
bohydrate on cell membranes reduces adhesion (Rutishauser
and Landmesser 1996), thereby favoring, if not enabling,
cells’ detachment from each other or the ECM. PSA-NCAM
is then considered a permissive factor for morphological
plasticity because its expression is required for the changes
to happen.
3.3 I N D U C T I V E FAC TO R S
There must be other factors, specific to each plastic system,
that act as specific stimuli, triggering cascades of intracellular
events and ultimately resulting in glial and neuronal reorga-
nization. There are many potential candidates for such func-
tions, including peptides, transmitters, steroids, and trophic
factors that are released by neurons and/or glial cells. Presently,
however, not much is known about these signaling pathways
and how they could operate to affect neural cell morphol-
ogy. In the HNS, the conditions in which plasticity is most
striking, lactation and parturition, are associated with the
massive peripheral release of OT, whose secretion within the
hypothalamic nuclei is also augmented. Central OT acts as a
local modulator, affecting presynaptic and postsynaptic activ-
ity of OT neurons (de Kock et al. 2003; Israel et al. 2003a;
Jourdain et al. 1998). Most important, intracerebroventricular
518 • NEUROGLIA
infusion of this peptide induces morphological neuronal–
glial changes in the SON similar to those detected during lac-
tation (Theodosis et al. 1986b). These effects are also observed
in vitro in acute hypothalamic slices obtained from gestating
female rats. That this neuropeptide acts specifically via its
receptors was confirmed by using close analogs that mimicked
OT action on plasticity, whereas specific antagonists inhibited
it (Langle et al. 2003). Furthermore, VP, which is structurally
related to OT, had no effect. The receptor-mediated action of
the neuropeptide may explain why morphological changes in
the HNS are restricted to its OT system because OT receptors
occur on OT but not VP neurons in the HNS (Barberis and
Tribollet 1996). Interestingly, OT receptors are also expressed
by astrocytes (Guenot-Di Scala and Strosser 1992).
It is likely that OT does not act alone when considering the
induction of the structural plasticity of the HNS. Among the
cofactors that could be involved are estrogens. When given
alone or together with OT, estrogens facilitate morphological
plasticity in vivo and in vitro (Langle et al. 2003; Montagnese
et al. 1990; Theodosis et al. 2006). The action of these steroids
appears to be rapid, in agreement with the involvement of a
membrane receptor. It is of note that steroids have a facilitat-
ing effect on the electrical activity of OT neurons (Israel and
Poulain 2000), their secretory activity, and their gene expres-
sion (Crowley and Amico 1993). Whether this is related to
their reported effect on HNS morphological neuronal–glial
remodeling remains to be elucidated. Although OT, with or
without steroids, appears essential for inducing the structural
plasticity in the HNS, it is also possible that other factors are
involved in such signaling. One of them is glutamate, which
can serve as a bidirectional signal to transfer information
between excited neurons and adjacent astrocytes. In vitro, the
remodeling induced in the SON by OT and estrogen is indeed
inhibited with a cocktail of metabotropic and ionotropic glu-
tamate receptor antagonists (Langle et al. 2003), suggesting a
role for the excitatory amino acid in mediating hypothalamic
activity–dependent structural changes. Another candidate
signaling molecule is nitric oxide (NO). Electron micro-
scopic analysis of the external layer of the median eminence
indicates that NO regulates neuronal–glial structural plastic-
ity (De Serrano et al. 2004). Activation of endogenous NO
release induced rapid structural remodeling, resulting in the
withdrawal of tanycyte endfeet and favoring access of GnRH
terminals to the basal lamina. Other putative signaling can-
didates for hypothalamic structural plasticity are purines,
especially in the neurohypophysis, in which ATP is packed in
secretory granules (Sperlagh et al. 1999). Once released from
neurohypophysial terminals, ATP could then act on pituicytes
that express purinergic receptors (Loesch and Burnstock
2001; Rosso et al. 2002). Furthermore, adenosine, which is
produced from the metabolic breakdown of ATP, has been
shown to affect glial morphology, triggering stellation of cul-
tured pituicyte (Miyata et al. 1999; Rosso et al. 2002) (see also
chapter 25).
Finally, there is strong evidence indicating that steroids
themselves trigger the onset of morphological changes.
As noted, estrogen can substitute for OT, at least in vitro,
to induce neuronal–glial changes in the SON (Langle et al.
2003; Theodosis et al. 2006). In the arcuate nucleus, admin-
istration of estradiol to match plasma levels measured at
proestrus causes glial rearrangements similar to those detected
at that specific period of the estrous cycle (Garcia-Segura
et al. 1994a). Because GnRH neurons do not express estrogen
receptors, such a steroid action has to be mediated either by
presynaptic terminals and/or astrocytes (Witkin et al. 1991).
Indeed, in this area of the hypothalamus, astrocytes are not
only affected by estrogen action, but they also release factors
that could affect neuronal–glial interactions. Thus, gonadal
steroids facilitate the astrocytic expression of growth fac-
tors of the epidermal growth factor family and their tyrosine
kinase receptors, thereby contributing to neuronal–glial sig-
naling occurring at the beginning of puberty (Ojeda and Ma
1999). Furthermore, astrocytes and tanycytes of the median
eminence release factors such as PGE2, which contribute to
the estrogen-induced structural plasticity of GnRH terminals
in the external layer of the median eminence ( Garcia-Segura
and McCarthy 2004; Ojeda and Ma 1999).
4 CONSEQUENCES OF THE GLIAL
S T RU C T U R A L P L A S T I C I T Y
Whereas some factors involved in the anatomical remodeling
of the SON have been identified (Theodosis 2002), the func-
tional consequences of such changes have remained hypotheti-
cal for a long time. It was proposed that the increased number of
synapses represented a compensatory mechanism to maintain
a stable level of synaptic density for hypertrophied neurons.
Electrophysiological recordings performed in the SON at dif-
ferent stages of the reproductive cycle confirmed this hypoth-
esis (Brussaard et al. 1999). Regarding the relative absence of
astrocytic processes in the vicinity of neuronal elements and
synapses, this has important functional consequences in view
of the various roles played by glia in the CNS.
real time iontophoretic TMA+ method
TMA+
[TMA+]e
Δ [TMA+] = 1 mM
SON
optic chiasm
x z
y (I = 200 nA)
Figure 41.2 Real-Time Measurements of Diffusion Parameters. Real-time measurements of SON diffusion properties can be obtained through ionto-
phoresis applications of TMA+ in acute slices (left panel) and its detection by an ion-sensitive electrode. In virgin rats (middle panel), the concentration
profiles varies according to the axis (x, y, and z), yielding different tortuosity (O) values. Such anisotropy disappears in the SON of lactating rats, in
which tortuosity become equivalent in all direction (right panel). The volume fraction (D) is reduced once structural neuronal–glial remodeling has
occurred. Adapted from Piet et al. 2004.
M O D U L AT I O N O F N E U R O E N D O C R I N E SYS T E M S
4.1 D I FF US I O N I N T H E
E X T R AC E L LU L A R S PAC E
Modification of the glial environment of neurons results in
important changes in the ECS volume and geometry. As a
consequence, extrasynaptic transmission mediated by the
diffusion of molecules is profoundly altered. Similarly, the
absence of fine astrocytic processes around neuronal elements
and synapses in the SON of lactating rats produces changes
in ECS properties. This hypothesis was tested in the SON
of virgin and lactating rats (Piet et al. 2004) using the real-
time tetramethylammonium (TMA+) iontophoretic method
(Nicholson and Sykova 1998). This approach permits mea-
surement of diffusion parameters such as tortuosity, which is a
measure of restriction on diffusion in the tissue in comparison
with an obstacle-free medium, volume fraction which corre-
sponds to the volume of tissue occupied by the ECS, and non-
specific uptake. In virgin rats, diffusion in the SON was not
equivalent in all directions, a condition known as anisotropy,
with tortuosity being hindered less along the ventrodorsal
axis (Fig. 41.2). This situation probably reflects the particular
ventrodorsal orientation of most astrocytic processes in this
structure (Bonfanti et al. 1993; Israel et al. 2003b). Glial with-
drawal in the SON of lactating animals caused a significant
reduction of volume fraction and tortuosity. As a result, dif-
fusion became equivalent in all the planes (isotropy), indicat-
ing that the glial remodeling not only facilitated diffusion in
the ECS, but also modified the geometry of the nucleus. The
reduction in volume fraction can be explained by the hypertro-
phy of the neurons filling the empty space resulting from glial
withdrawal. Such modifications of the diffusion parameters of
the SON should enhance largely the range of action of diffus-
ing substances and their local concentrations (Fig. 41.3).
4.2 G LU TA M AT E U P TA K E
Because glial cells play a key role in glutamate clearance, with-
drawal of astrocytic processes in the vicinity of glutamatergic
Virgin rat Lactating rat
λx = 1.39 λx = 1.35
λy = 1.49 λy = 1.38
λz = 1.53 λz = 1.36
α = 0.30 α = 0.20
60 s
• 519
 unstimulated
SON neuron
glutamate
GLT-1
terminal
astrocytic process
mGluR GABA
terminal
SON neuron
stimulated
SON neuron
glutamate
terminal
GABA
terminal
SON neuron
Figure 41.3 Structural Plasticity in the Supraoptic Nucleus and Its
Consequences On Transmitter Concentrations and Diffusion. Diagrams
illustrating the glial structural remodeling in the SON of stimulated
rats and its consequences on synaptic and extrasynaptic transmission.
In unstimulated animals (virgin or normally hydrated rats), astrocytic
processes are enwrapping synapses and neurons, thereby preventing
neurotransmitters such as glutamate to diffuse in the ECS. In stimulated
animals (lactating or chronically dehydrated), glial withdrawal promotes
an elevation of glutamate levels around glutamatergic synapses and
enhanced activation of presynaptic autoreceptors (mGluRs). Under con-
ditions of intense glutamatergic fiber stimulation, glutamate could diffuse
away from its release sites and affect remote receptors such the mGluRs
located on GABAergic terminals. This extrasynaptic form of communi-
cation is likely to be favored by the facilitated diffusion resulting from
morphological remodeling. Adapted from Oliet and Piet 2004.
release sites may have important consequences on the extra-
cellular levels of the excitatory amino acid. This in turn may
cause change in synaptic transmission and neuronal excitabil-
ity. Because astrocytes play a major role in the uptake of glu-
tamate (see chapter 35), removal of fine astrocytic processes
should result in deficient or delayed glutamate clearance that
could have a tremendous impact on synaptic transmission.
Accordingly, inhibition of glutamate transporters in the SON
of virgin rats yielded augmented levels of glutamate in the
ECS, enhanced tonic activation of presynaptic mGluRs and
reduced synaptic efficacy (Boudaba et al. 2003; Oliet et al.
2001). To assess whether the removal of fine glial processes
enveloping synapses and neurons indeed affected the local
concentration of glutamate, the tonic activity of presynaptic
mGluRs was evaluated under conditions of reduced astrocytic
coverage of neurons in the SON of lactating (Oliet et al. 2001;
520 • NEUROGLIA
Piet et al. 2003) and chronically dehydrated (Boudaba et al.
2003) animals in which a similar neuroglial reorganization
occurs (Theodosis 2002). As expected from delayed glutamate
clearance, mGluR tonic activity is significantly enhanced under
conditions of reduced glial coverage, resulting in reduced
synaptic efficacy at glutamatergic inputs.
Conversely, the elevation in glutamate concentrations
associated with anatomical remodeling is apparently not suffi-
cient to modulate mGluRs at GABAergic synapses (Piet et al.
2003). At GABA synapses, tonic activation of mGluRs by
ambient glutamate was detected neither in virgin rats nor lac-
tating animals. It seems then that basal glutamate release is not
sufficient to activate glutamate receptors located at a distance
from excitatory inputs. This is consistent with previous stud-
ies describing the regulation of GABAergic transmission by
synaptically released glutamate, a process known as heterosyn-
aptic modulation. Such modulation depends on the activity
of glutamatergic inputs, so that glutamate levels in the ECS
have to reach sufficient concentrations to saturate glutamate
transporters, thereby enabling diffusion to remote receptors.
In the SON of virgin rats, such heterosynaptic depression
of GABAergic transmission occurs when short condition-
ing trains of stimulation were applied to glutamatergic fibers
(Oliet et al. 2004). This inhibition has a presynaptic origin
and is caused by the activation of mGluRs present on inhibi-
tory terminals. Because this type of modulation relies on the
diffusion of glutamate in the ECS, it should be sensitive to
anatomical remodeling of the SON in lactating animals that
impairs glutamate uptake. In agreement with this hypothesis,
intersynaptic cross-talk between glutamate and GABA syn-
apses is greatly facilitated in the SON of lactating rats (see
Fig. 41.3) under conditions of reduced glial coverage of neu-
rons and synapses (Oliet et al. 2004).
4.3 G L I OT R A NS M I S S I O N
Anatomical changes in the neuronal–glial relationship mod-
ify the concentration and diffusion in the ECS of the various
gliotransmitters released from astrocytes (see chapters 34 and
35). The retraction of glial processes occurring in the SON
and paraventricular (PVN) nuclei of the hypothalamus during
lactation and dehydration, respectively, results in remarkable
changes in astrocyte–neuron communication. In the SON of
lactating rats, the concentration of d-serine within the synap-
tic cleft is dramatically reduced (Fig. 41.4), as revealed by the
reduced activity of NMDA receptors (Panatier et al. 2006).
This has severe consequences on the NMDA receptor-depen-
dent forms of synaptic plasticity such as long-term potentiation
(LTP) and depression (LTD). In the SON of lactating rats,
the activity dependence of these processes is shifted toward
higher values, so it takes a much stronger synaptic activity to
induce LTP in lactating rats compared with virgin animals (see
Fig. 41.4). This is because fewer NMDA receptors are available
for activation and, consequently, less Ca2+ enters the postsyn-
aptic neurons. Both NMDA receptor activity and the ability
to induce LTP and/or LTD in lactating rats are completely res-
cued when exogenous d-serine is applied to the tissue. Because
d-serine release from astrocytes requires the activation of glial
 UNSTIMULATED
synaptic terminal
astrocyte
glutamate
D-serine
NMDAR
OT neuron
Figure 41.4 Gliotransmission in Lactating Rats. Under unstimulated conditions (left panel), astrocytes provide d-serine to the glutamatergic
synapses of OT neurons, enabling NMDA receptor activation. In lactating rats (stimulated; right panel), the glial withdrawal causes a deficiency in
d-serine supply, impairing NMDA receptor activation.
glutamatergic receptors (Mothet et al. 2005), it is likely that
such release is less stimulated under these conditions because
of the increased distance between glial and neuronal elements.
Because of this anatomical change, even if d-serine release
occurs, concentrations of the d-amino acid reaching the syn-
aptic cleft would be largely reduced compared with control
conditions. As a result, the activity dependence of long-term
synaptic changes is shifted toward higher values (Fig. 41.5).
A similar observation has been made in the PVN of dehy-
drated rats in which the release of ATP from glial cells no
longer activates P2X receptors on magnocellular neurons,
thereby compromising synapse strengthening through AMPA
receptor insertion (Gordon et al. 2005). Here again, this form
of synaptic plasticity is rescued by providing exogenous ATP
to the tissue. Taken together, these data indicate that remodel-
ing of glial processes has tremendous consequences on excit-
atory transmission and long-term synaptic plasticity (Bains
and Oliet 2007).
4.4 SY NA P TO G E N E S I S
In the magnocellular nuclei of rodents, variations in astro-
cytic coverage of neuronal somata and dendrites are always
accompanied by synapse turnover. The most obvious change is
an increased number of terminals impinging onto more than
one postsynaptic element simultaneously. They are referred
to as multiple or shared synapses. In the magnocellular nuclei,
their incidence increases during conditions of enhanced OT
release coincident with the reduction of astrocytic coverage of
neuronal elements (Theodosis et al. 2005). In the OT system,
augmentation of synaptic densities also concerns terminals
making single synaptic contact. Ultrastructural morphometric
M O D U L AT I O N O F N E U R O E N D O C R I N E SYS T E M S
STIMULATED
synaptic terminal
astrocyte
glutamate
D-serine
NMDAR
OT neuron
analyses demonstrated that GABAergic, glutamatergic, and
noradrenergic inputs undergo remodeling in relation to stim-
ulation of OT secretion, with the most important increases
occurring for GABAergic inputs (El Majdoubi et al. 1997;
Gies and Theodosis 1994). In the SON of lactating rats, 50%
of all axosomatic and axodendritic synapses are GABAergic,
compared with 35% in virgin animals. Such synaptogen-
esis was reproduced in vitro in acute hypothalamic slices
that include the SON. In this model, synapse formation was
found to be very rapid, occurring within 1 hour and resulting
in the formation of inhibitory synapses that were functional
(Theodosis et al. 2006). In the arcuate nucleus of adult female
rats, there are also important synaptic changes accompanying
the astrocytic remodeling (Garcia-Segura et al. 1994a). The
number of axosomatic GABAergic synapses on arcuate neu-
rons falls significantly between the morning and afternoon
of proestrus, remains low throughout estrus, and comes back
to baseline levels on metestrus, to fall again at proestrus. The
inverse correlation between the level of glial ensheathment and
the number of synaptic inputs in relation to steroid levels has
been reported not only in rats but in nonhuman primates as
well (Garcia-Segura et al. 1994a; Witkin et al. 1991). Whether
such synapse turnover is directly related to the glial remodel-
ing occurring in those different hypothalamic areas remains to
be determined.
5 C O N C LU S I O N S
The hypothalamus is made of several neuroendocrine sys-
tems that intervene in the regulation of many vital processes.
Their morphological reorganization is now a well-established
• 521
 A Virgin Lactating B
Control Control D-serine
DAAO D-serine LTP + –
250 250 Pairing
Pairing
200 200
amplitude (%)

amplitude (%)
Virgin
150 150

100 100
Lactating
50 50

0 0 LTD
–10 0 10 20 30 –10 0 10 20 30 low high
time (min) time (min) synaptic stimulation

Figure 41.5 Glia-Dependent Plasticity. A. Pairing synaptic stimulation with membrane depolarization causes long-term potentiation (LTP) in
the supraoptic nucleus of virgin rats (left panel; control; black dots), whereas the same protocol applied in lactating animals causes long-term depres-
sion (LTD) (right panel, control; black dots). Long-term potentiation can be restored in lactating rats by supplying d-serine to the slices (right panel;
d-serine; empty dots) and is switched into LTD in virgin animals when d-serine is degraded with D-amino acid oxidase (left panel; DAAO; empty
dots). B. At these synapses, the induction of plasticity and its magnitude depends on the rate of synaptic stimulation according to the model described
by Biennenstock, Cooper, and Munro (black curve; virgin). Glial withdrawal in the SON (red curve, lactating) causes a rightward shift of the activity
dependence of synaptic plasticity. This relationship between plasticity and synaptic stimulation is governed by the availability of d-serine to NMDA
receptors, a process that depends on the glial environment of synapses. From Bains and Oliet 2007.

phenomenon. Because these systems, especially the HNS and
median eminence, are easily accessible in vivo and in vitro,
they have been used as model to analyze the importance of
neuronal–glial remodeling not only at the level of synaptic
function, as discussed, but in the more general context of sys-
tem physiology as well. This is particularly clear in the case
of the OT system, during its highly characteristic activity at
lactation.
Peripheral information during suckling is transmitted to
OT neurons through proximal glutamatergic afferents. During
the milk ejection reflex, for example, OT neurons display high
frequency bursts of action potentials (Poulain and Wakerley
1982) that are triggered by bursts of glutamatergic postsynap-
tic potentials (Israel et al. 2003a; Jourdain et al. 1998). As dis-
cussed, patch clamp recordings provided convincing evidence
that excitatory information transmitted by glutamatergic
inputs is affected by glial remodeling (Oliet et al. 2004). Thus,
the reduced glial coverage of OT neurons and their synapses
during lactation results in increased levels of glutamate in the
ECS, which in turn augment the tonic inhibition exerted by
presynaptic mGluRs on glutamate release (Oliet et al. 2001).
On first sight, it seems paradoxical that these inputs exhibit
a lower probability of release at a time when OT neurons are
strongly activated. However, presynaptic inhibition is not a
linear process in the sense that its efficiency depends on pre-
synaptic activity. A tonic presynaptic inhibition mediated by
mGluR activation is likely to be overcome by the facilitation
of probability of release associated with the intraterminal
Ca2+ strong increase that occurs during high-frequency trains
of action potentials in glutamatergic terminals at the time of
milk ejection. Information transmitted via high-frequency
excitatory synaptic activity, therefore, would be less affected
by a negative glutamate feedback than information transmit-
ted by low or moderate frequency activities. This may serve as
a high-pass filter to increase signal-to-noise ratio for informa-
tion carried by high-frequency activities. On the other hand,
accumulating glutamate in the extracellular environment,
via its heterosynaptic actions, can inhibit GABAergic trans-
mission in the vicinity of glutamatergic inputs (Piet et al. 2003,
2004), leading to a local disinhibition of the somatodendritic
compartment (Mitchell and Silver 2000).
Also as described, the presence or absence of astrocytic
processes has important consequences on the concentration
of gliotransmitters such as d-serine, which affect NMDA
receptor activation, intracellular Ca2+ concentrations, and
ultimately phenomena of synaptic plasticity such as LTP and
LTD (Panatier et al. 2006). During lactation, when fewer
NMDA receptors are occupied by d-serine, the threshold for
inducing LTP is elevated, making it more difficult for syn-
apses to become potentiated. Making potentiation harder to
achieve may favor very active synapses, such those communi-
cating the suckling stimulus to OT neurons, whereas at others
less active, synaptic strength would rather be decreased or left
unchanged by the afferent drive activity. It should also be kept
in mind that the increased number of synapses associated with
reduced glial coverage in the magnocellular nuclei at lactation
(El Majdoubi et al. 1997) facilitate enhanced OT firing as well.
It is obvious that an increased number of glutamatergic inputs
potentiate such firing, just like the additional noradrenergic
synapses (Michaloudi et al. 1997) that provide an excitatory
input to OT neurons, both on their own (Day et al. 1984) and
through their synergistic action with glutamate (Daftary et al.
1998; Parker and Crowley 1993). As for the highly significant
increase in the number of inhibitory synapses, their action,
at first glance, might appear incompatible with enhanced
excitability. A likely possibility is that the increase number
of inhibitory inputs may serve to prevent the neurons from
being stimulated by factors other than those relevant to partu-
rition and lactation, attenuating the response of OT neurons
to stimuli other than suckling (Fénelon et al. 1994; Lightman
and Young 1989). This mechanism could also contribute to
maintain membrane potential of OT neurons at a depolarized

522 • NEUROGLIA
level, thereby facilitating their subsequent synchronous activa-
tion just before milk ejection (Moos 1995; Voisin et al. 1995).
In addition to these phenomena in the hypothalamus,
glial–axonal changes in the neurohypophysis and the median
eminence appear to favor neurosecretion. Retraction of glial
processes from around neurosecretory axons and the perivas-
cular space promote paracrine actions of the secreted peptides.
Moreover, as noted, by removing a physical barrier from the
perivascular spaces, such plasticity facilitates diffusion of the
secreted hormones into fenestrated capillaries and thus the
general circulation.
Although the consequences of the glial remodelings
occurring in different neuroendocrine centers have not been
completely determined, and the detailed molecular processes
underlying such forms of structural plasticity remain to be
identified, these phenomena have served as remarkable mod-
els to improve our understanding of glial cells and their contri-
bution to neuronal functions.
6 S U M M A RY A N D P E R S P E C T I VE S
Investigations on neuronal–glial interactions carried
out in the hypothalamus have provided very impor-
tant insights regarding the contribution of glial cells
to the physiology of neurons and their dynamic inter-
actions. The hypothalamo-neurohypophysial sys-
tem has proved to be an excellent model to study these
processes because of the remarkable activity-dependent
remodeling that occurs during physiological responses such
as lactation, parturition, and dehydration. The magnitude of
this remodeling is so important that it was possible to study
it a time when structural analysis was limited to electron
microscopy. The physiological changes that affect the glial
environment of neurons during such structural plasticity
has revealed the crucial roles played by astrocytes, including
the regulation of glutamate concentration and diffusion in the
extracellular space and the supply of key signaling molecules
such as ATP and serine for controlling synaptic strength.
The ongoing development of imaging techniques has per-
mitted to observe similar types of structural neuronal–glial
remodeling, although of smaller extent, in other major
brain areas such as the brainstem, hippocampus, or cortex.
Availability techniques such as super resolution microscopy
permitting the visualization of thin glial processes should
allow in the near future the study of the morphofunctional
plasticity of the tripartite synapse at the nanoscopic level. This
should provide unexpected details of the intricate and dynamic
association among glia, neurons, and the extracellular matrix.
The time courses of these remodelings as well as the intracel-
lular cascades involved await description and identification.
At a more macroscopic level, the functional consequences
of the glial remodeling on the physiology of the neuroen-
docrine centers are still debated. Whether such forms of
structural plasticity are essential processes for lactation, dehy-
dration, the estrous cycle, or the circadian rhythm remains to
be answered.
M O D U L AT I O N O F N E U R O E N D O C R I N E SYS T E M S
AC K N OW L E D G M E N T S
The author wishes to thank Dionysia Theodosis and
Dominique Poulain for their continual support, wise advice,
and persistent encouragement throughout the last 15 years.
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 42.
MYELIN, IMPULSE CONDUCTION, AND THE
PATHOPHYSIOLOGY OF DEMYELINATION
Lakshmi Bangalore and Stephen G. Waxman

A B B R E VI AT I O N S 2 S T RU C T U R A L C O M P O N E N T S O F
T H E M Y E L I N AT E D AXO N
CNS central nervous system
EAE experimental autoimmune A myelinated nerve fiber consists of an axon ensheathed in seg-
encephalomyelitis ments of compact extensions of the cell membranes of myeli-
FGF fibroblast growth factor nating glia. In the peripheral nervous system (PNS), where
FHF FGF homologous factor axoglial interactions are much simpler than in the central ner-
GFAP glial fibrillary acidic protein vous system (CNS), myelinated fibers derive their myelin from
hESC human embryonic stem cells myelin-forming Schwann cells. A single Schwann cell can form
Kv voltage-gated potassium channels only one myelin segment, thus it takes multiple Schwann cells
md myelin-deficient to form successive segments of myelin sheath along a peripheral
Nav voltage-gated sodium channels axon (see also chapters 7, 44 and 45). In contrast, myelin in the
NCX sodium-calcium exchanger CNS is produced by oligodendrocytes that extend numerous
PLP proteolipid protein processes, each of which forms a segment of myelin around mul-
PNS peripheral nervous system tiple axons (Bjartmar et al. 1994; Hildebrand 1971). The oligo-
MS multiple sclerosis dendrocyte cell body typically does not circumferentially hug
its myelin sheaths but instead, maintains contact with its myelin
sheaths through thin cytoplasmic bridges (Bunge et al. 1961;
Hirano et al. 1968). This topographically tenuous connection
1 INTRODUCTION between the genomic and biosynthetic machinery within the
oligodendrocyte cell body and the myelin has been suggested to
Myelinated axons are essential for nervous system function underlie the scarcity of remyelination within the CNS. Studies
and represent examples of exquisite biological design. In have suggested that there is local biosynthesis of myelin in dis-
this chapter, we build upon our chapter in a prior edition tal parts of the oligodendroglial processes close to the regions
and discuss implications of the biological design of myeli- in which polyribosomes are present (Waxman 1984; Waxman
nated axons in normal and pathological conditions. Rapid et al. 1988). This is functionally important because it permits
transmission of nerve impulses confers a selective advan- a single oligodendrocyte to produce myelin sheaths of differ-
tage to species. Rapid impulse transmission can be achieved ent thickness around axons of different diameter in its vicinity
either by increasing axonal diameter or by myelination. In (Waxman et al. 1988).
axons without myelin, the speed of impulse conduction is The advantages of myelination are twofold. First, compact
proportional to the square root of axonal inner diameter. lipid-rich myelin provides high electrical resistance and low
Species such as cephalopods that lack myelin enlarged capacitance, which prevents current loss and enables rapid and
their axons substantially in order to achieve a faster rate efficient action potential conduction. Second, increased con-
of impulse conduction. The squid giant axons evolved to duction velocity allows for computational complexity within
be as large as 400 to 900Pm in diameter to support rapid a compact nervous system. The nodes of Ranvier that punctu-
conduction velocities—this specialization enabled early ate myelinated regions are the primary sites of ionic exchange
electrophysiologists to study the nerve impulse through between the interior and exterior of the nerve fiber. Internodal
recordings on single nerve fibers. The price of axonal distances that range from less than 100 Pm (small-diameter
gigantism, however, is spatial expansion, unaffordable fibers) to slightly over 1 mm (larger-diameter fibers) optimize
in higher species that have more axons within the same conduction velocity. Myelin, nodes of Ranvier, and internodal
cross-sectional area of one squid giant axon. Higher species distance specializations enable action potentials generated in
thus evolved another mechanism for increasing conduction the axon initial segment to propagate from node of Ranvier to
velocity by wrapping small-diameter axons in multiple lay- node of Ranvier in a saltatory manner, resulting in rapid and effi-
ers of myelin that enable nerve impulses to transmit in a cient impulse conduction at a conduction velocity that is pro-
rapid “saltatory” manner. portional to fiber diameter (Huxley and Stämpfli 1949; Tasaki

529
1959). Moreover, whereas conduction velocity in myelinated
fibers has a linear dependence on axon diameter, nonmyelinated
fibers conduct with slower velocity that varies with the square
root of axon diameter (Waxman and Bennett 1972). Therefore,
above a critical diameter wherein the conduction velocity–
diameter relationships intersect, myelinated fibers conduct
impulses more rapidly than nonmyelinated fibers of the same
diameter. This critical diameter of intersection is approximately
0.2 Pm. Within the CNS, this is the diameter at which myelina-
tion is first seen (Waxman and Bennett 1972) (Fig. 42.1).
3 M O L E C U L A R C H A R AC T E R I S T I C S
O F T H E M Y E L I N AT E D AXO N
Although nonmyelinated axons generally display a uniform
membrane structure that does not vary from one region to
another (Black et al. 1981), the myelinated axon membrane is
highly specialized with a spatially heterogeneous distribution
of voltage-sensitive ion channels and other proteins (Fig. 42.2).
High densities (approximately 1,000/Pm2) of voltage-gated Na+
channels (Nav) cluster in the axon membrane at the nodes of
Ranvier (Ritchie and Rogart 1977; Waxman and Quick 1977).
The internodal axon membrane beneath the myelin sheath has
a much lower density of Na+ channels (<25/Pm2) (Ritchie and
Rogart 1977; Waxman and Quick 1977), and thus is unable to
support conduction at high frequencies. Nine different sodium
channel subtypes have been discovered to date, with distinct
molecular structures superimposed on an invariant overall motif
(four similar domains, each containing six membrane-spanning
segments). The identities of sodium channel subtypes within
various compartments of the myelinated axon are well under-
stood. Sodium channel Nav 1.6 is the predominant sodium
channel at the nodes of Ranvier (Caldwell et al. 2000). Recent
8
myelinated
6 4-aminopyridine (4-AP), have a reciprocal distribution to that
Conduction velocity (m/sec)
4 and Ritchie 1980, 1981; Foster et al. 1982; Kocsis et al. 1982).
non-myelinated
2 this paranodal density within the node and internode (Röper
0 1 2
3 4
Fiber diameter (μm)
Figure 42.1 The relationships between conduction velocity (Y-axis) and
diameter (X-axis), for myelinated (solid line) and nonmyelinated (dashed
line) axons are superimposed. Above a diameter of 0.2Pm, at which the
two relationships cross, myelinated axons conduct more rapidly than
nonmyelinated axons of the same size. 0.2 Pm is, in fact, the diameter of
the smallest myelinated axon within the CNS. Modified from Waxman
and Bennett 1972.
530
studies using a highly sensitive, quantitative electron micro-
scopic immunogold technique (SDS-digested freeze-fracture
replica-labeling) estimated the density of Nav1.6 at the nodes
to be approximately 350/Pm2, nearly twice that observed at the
axon initial segment (Lorincz and Nusser 2008).
Myelinated axons also express several proteins that modu-
late the availability and activity of Nav channels at the nodes.
A member of the fibroblast growth factor (FGF) homologous
factor (FHF) subfamily, FHF2B, colocalizes with Nav1.6
at mature nodes of Ranvier in myelinated sensory fibers in
the dorsal root of the sciatic nerve (Wittmack et al. 2004).
However, retinal ganglion cells and spinal ventral horn motor
neurons have low expression of FHF2B, and no nodal FHF2B
staining within the optic nerve and ventral root, respectively
(Wittmack et al. 2004). Electrophysiological studies show
that the association of FHF2B with Nav1.6 results in increased
current density and a small depolarizing shift in channel avail-
ability (Rush et al. 2006; Wittmack et al. 2004). Wittmack
et al. (2005) show association of p38 MAP kinase with Nav1.6
in rat brain tissue and demonstrate modulation of Nav1.6 cur-
rent in vitro by activated p38 MAP kinase via phosphoryla-
tion of the L1 loop of Nav1.6 at serine 553. Recent studies
have reported that the ubiquitin ligase Nedd4–2 interacts
with a Pro-Ser-Tyr motif and a Pro-Gly-Ser-Pro motif in the
C terminus of Nav1.6, and reduces Nav1.6 current density in
a p38-MAP kinase dependent manner. Interestingly, block-
ing Nedd4–2 from binding to the Pro-Ser-Tyr motif in the
C terminus results in stress-mediated increase in Nav1.6 cur-
rent density. Thus, Nav1.6 can be differentially modulated by
Nedd4–2 depending on the availability of the C-terminal
Pro-Ser-Tyr motif to bind Nedd4–2 (Gasser et al. 2010).
Myelinated axons display a heterogeneous distribu-
tion of several types of voltage-dependent K+ channels (Kv).
Electrophysiological studies have revealed the expression of at
least three distinct K+ channel currents in myelinated axons—
the “fast” K+ current, a “slow” K+ current, and an inward rec-
tifier current (Röper and Schwarz 1989; Waxman and Ritchie
1993). Fast K+ channels, which can be blocked by exogenous
of the clustered nodal Na+ channels. They are present at rela-
tively low densities along the nodal axon membrane and high-
est density in the axon membrane beneath the myelin (Chiu
Voltage-clamped experiments suggest that the density of fast K+
channels is maximal in the paranode, decreasing to one-sixth of
and Schwarz 1989), but this study may not have accurately dif-
ferentiated between the paranodal and juxtaparanodal regions.
Immunocytochemical studies have shown high-density
clusters of Kv1 channels (Kv1.1 and Kv1.2; Shaker/KCNA
subfamily of voltage-dependent K+ channels) at the juxtapara-
nodes of myelinated axons (Rasband et al. 1998; Vabnick and
Shrager 1998; Wang et al. 1993). More recently, studies have
identified Kv1 channels enriched at the axon initial segments
wherein they form macromolecular complexes with a variety
of cell-adhesion molecules and cytoskeletal proteins (Goldberg
et al. 2008; Kole et al. 2007; Ogawa and Rasband 2008). Recent
studies have also identified Kv channels KCNQ2 and KCNQ3
•
 g g
Na
AXON
OLIGO./S.C
Figure 42.2 Schematic model of ion channel organization of the myelinated fiber. gNa+ sodium conductance; gK fast potassium conductance. Sodium
channels gNa are clustered at the node of Ranvier. In contrast, fast potassium channels, responsible for repolarization of the action potential,
are present in the internodal axon membrane.
of the Kv7 (KCNQ) subfamily, which are slowly activated by
depolarization, are present at axon initial segments and nodes
of Ranvier (Devaux et al. 2004; Pan et al. 2006). The seques-
tration of KCNQ2 and KCNQ3 to the axon initial segments
and nodes of Ranvier is dependent on their binding to the
cytoskeletal scaffolding protein ankyrin G, which is essential
for the assembly of the axon initial segment and maintenance
of neuronal polarity (Pan et al. 2006). Another subfamily
of Kv channels, the Kv3 channels also present within myeli-
nated axons and contribute to high-frequency repetitive firing
(Devaux et al. 2003; Rudy and McBain 2001). A unique splice
variant of the Kv3.1 gene, Kv3.1b, is found in a subset of CNS
and PNS nodes of Ranvier although it is more highly expressed
in CNS nodes wherein it colocalizes with Nav channels, partic-
ularly in large myelinated axons (Devaux et al. 2003). In con-
trast to other nodal proteins that have a presence at the axon
initial segments, Kv3.1b is not present at axon initial segments
and many smaller nodes unlike other nodal proteins that also
have a presence at the axon initial segments, and it does not
account for the effects of 4-AP on compound action potentials
recorded from central myelinated tracts (Devaux et al. 2003).
Furthermore, nodal localization of Kv3.1b is maintained,
at least temporarily, even in the absence of myelin sheath, as
shown in myelin-deficient (md) rats with severe CNS demyeli-
nation (Devaux et al. 2003) and paralleling the persistence of
node-like clusters of Nav channels after oligodendrocyte death
in md rats (Arroyo et al. 2002).
The specialized organization of myelinated axonal elements
poses severe problems in demyelinated axons. Damage to myelin
disrupts action potential electrogenesis because of loss of capaci-
tative shield and low density of inward Na+ current within the
exposed intermodal axon membrane, which is normally scarce
in Na+ channels (Waxman 1982). Loss of myelin also unmasks
fast K+ channels (normally covered by the myelin) that clamp
the demyelinated resting membrane potential close to the K+
equilibrium potential Ek, opposing depolarization and further
impeding action potential conduction (Chiu and Ritchie 1981).
Demyelination also imposes a significant energy burden as Na+,
K+–ATPases attempt to restore the ionic gradient and maintain
neuronal excitability (Ames 2000; Benarroch 2011).
Molecular plasticity of the axon membrane contributes to
the recovery of conduction following demyelination. Expression
of higher-than-normal density of Na+ channels in some
M Y E L I N, I M P U L S E C O N D U C T I O N, A N D T H E PAT H O P H YS I O L O GY O F D E M Y E L I N AT I O N
K
demyelinated (former internodal) axon regions may restore the
capability to support action potential conduction in the absence
of myelin (Black et al. 1987; Dugandzija-Novakovic et al.
1995; England et al. 1990; Foster et al. 1980). Unfortunately,
this may have deleterious consequences in terms of placing an
increased metabolic load on axons. Pharmacological blockade
of fast K+ channels that are unmasked by demyelination is also
expected to increase the safety factor (i.e., reliability) for con-
duction in demyelinated axons (Bowe et al. 1987; Kocsis et al.
1987).
4 AC T I O N P OT E N T I A L
E L E C T R O G E N E S I S I N M Y E L I N AT E D
A N D D E M Y E L I N AT E D AXO N S
In contrast to nonmyelinated axons, in which action poten-
tials are conducted in a continuous manner, myelinated fibers
exhibit saltatory conduction. In mammalian myelinated axons
at 370C, the time between excitation of one node and the
next (internodal conduction time) is approximately 20Psec
(Rasminsky and Sears 1972a, 1972b). Action potential con-
duction is usually unidirectional in nature because sodium
channels close and remain inactive for a short period soon
after activation. This refractory period, during which the axon
is unable to support a second action potential, has a duration
of milliseconds.
Because myelin functions as an insulator, the action cur-
rent from each active node of Ranvier is shunted to subsequent
nodes via the relatively low-resistance axoplasm (Fig. 42.3A).
The first few wraps of tight myelin seem to be the most impor-
tant because they provide a capacitative shield (Funch and
Faber 1984). Safety factor (the ratio between current avail-
able to stimulate a node of Ranvier and current required to
stimulate the node) is 5 to 7 in normal myelinated fibers, so
that there is a high degree of reliability for impulse conduction
(Tasaki 1959).
Capacitative and resistive shunting causes a fall in density
of action current, following damage to myelin (Fig. 42.3B).
If the safety factor is decreased but still greater than 1.0, the
charging time for the nodal membrane will be increased
so that it will take longer than normal for the axon to reach
threshold, and conduction will continue but conduction
• 531
 A
Figure 42.3 Diagrammatic representation of current flow associated with conduction through normally (A) myelinated and (B) demyelinated regions
of an axon. The action potential is conducted from left to right (arrow). This idealized diagram represents the myelin as a perfect insulation. Dashed
arrows illustrate current flow resulting from an action potential that is located at the cross-hatched node. Current is lost in demyelinated regions as a
result of capacitative and resistive shunting.
velocity will be reduced. In demyelinated spinal root fibers,
internodal conduction time can be increased nearly 20-fold,
to about 500Psec (Rasminsky and Sears 1972a). Thus conduc-
tion velocity is reduced. In more severely demyelinated axons,
the safety factor can fall to less than 1.0, reaching threshold,
and thus lead to conduction failure (Waxman 1982).
From a descriptive point of view, demyelinated axons
can display a spectrum of conduction abnormalities under-
lying signs and symptoms of pathology. Conduction abnor-
malities that are negative in the clinical or Jacksonian sense
include slowed, desynchronized, or blocked conduction
Figure 42.4B-E. Such abnormalities are confined to the
zone of demyelination, with normal conduction proximal
and distal to the zone (Fig. 42.5). Slowed conduction appears
to be less important than conduction block in producing
clinical deficits (McDonald 1963; McDonald and Sears
1970). Loss of synchrony in tracts, in which different
fibers exhibit unequal degrees of conduction slowing, also
known as temporal dispersion, can occur as a consequence
of decreased conduction velocity and reflects the variabil-
ity in internodal conduction times in demyelinated axons
(see Fig. 42.4C). Temporal dispersion can interfere with
functions such as the stretch reflex that require synchronous
discharge.
Changes in passive and active characteristics of demyeli-
nated fibers both contribute to conduction block. In addition
to capacitative current loss through injured myelin sheaths,
conduction in focally demyelinated axons can also fail because
of impedance mismatch between the normally myelinated
and demyelinated regions (Sears et al. 1978; Waxman and
Brill 1978). Low Na+ channel density in the demyelinated
axon membrane also contributes to conduction block. In
addition, fast K+ channels (normally covered by the myelin)
are unmasked after demyelination and tend to clamp the
532
demyelinated membrane close to the potassium equilibrium
Ek, further impeding action potential electrogenesis (Bostock
et al. 1981; Ritchie et al. 1981).
Conduction failure need not be all-or-none. It can be
frequency-related; with high-frequency impulse trains fail-
ing to propagate while low-frequency trains conduct reliably
(see Fig. 42.4D). In more severely affected fibers, conduction
failure can be complete, with even single action potentials
failing to propagate beyond the region of demyelination (see
Fig. 42.4E). It has been hypothesized that hyperpolarization
of the axon membrane, owing to electrogenic pump (Na+/
K+-ATPase) activity, may contribute to conduction block
of high-frequency impulse trains (Bostock and Grafe 1985).
There is also evidence suggesting that increased intracellular
Na+ at the “driving node” (Rasminsky and Sears 1972a) and
depolarization of demyelinated axons because of increases
in extracellular K+ concentration may also contribute to
high-frequency block.
Conduction abnormalities that are positive in a Jacksonian
sense are also important from a clinical perspective, and are
illustrated in Figure 42.4F-I. Ectopic impulse generation (Fig.
42.4F) has been observed, for example, in demyelinated dor-
sal column axons (Smith and McDonald 1980). Abnormal
cross-talk, also termed ephaptic interaction, may occur between
abnormally myelinated axons (see Fig. 42.4H). Increased
mechanosensitivity (Fig. 42.4G) has been experimentally
observed in demyelinated axons and probably accounts for
clinical phenomena such as Tinel’s and Lhermitte’s sign
(Smith and McDonald 1980). Impulse reflection may occur
in some focally demyelinated fibers (Burchiel 1980) and may
result in extraneous activity (e.g., paresthesia, pain, or tonic
spasms). Because reflected (antidromic) impulses can collide
with and abolish orthodromic impulses, impulse reflection
may also interfere with normal impulse traffic.
• NEUROGLIA
 A NORMAL CONDUCTION
F ECTOPIC IMPULSE GENERATION

B DECREASED CONDUCTION VELOCITY

G INCREASED MECHANOSENSITIVITY

C TEMPORAL DISPERSION

H CROSS-TALK

D FREQUENCY-RELATED BLOCK

I IMPULSE REFLECTION

E TOTAL CONDUCTION BLOCK

Figure 42.4 Conduction Abnormalities in Demyelinated Axons. Demyelinated regions of the axon are diagrammatically shown as dashed lines. Cell bodies
are located to the left, and axon terminals to the right. The direction of normal conduction is indicated by the arrow; see text for further explanation.

A
1 2

2.0
1.0 mV
msec
prox. demyel. dist.

1 2

0.2 mV

C
1 2

0.5 mV

Figure 42.5 Recording obtained proximal to (A), across (B), and distal (C) to a focally demyelinated region (injected with lysophosphatidylcholine) from
rat sciatic nerve. Conduction is relatively normal in proximal and distal nerve segments in which myelin is intact (A2, C2). However, conduction
slowing, block, and temporal dispersion (B2) are present when action potentials are conducted through the lesion site. Modified from Kocsis and
Waxman 1985.
 4 .1 C O N T I NU O US C O N D U C T I O N
F O L L OWI N G D E MY E L I NAT I O N
Continuous action potential conduction, similar to that of
normal unmyelinated axons, has been observed in some demy-
elinated axons (Bostock and Sears 1976). Some demyelinated
axons can conduct with a velocity that can fall to as low as 5% of
normal saltatory conduction velocity, over continuous lengths
of demyelination exceeding 2 mm (several internodes) (Felts
et al., 1977). Sequestration of sodium channels at nodes along
normal myelinated axons raises the question of how continuous
conduction can occur along previously internodal parts of the
axon following demyelination. Computer simulations provide
a partial answer in demonstrating that, in some small-caliber
demyelinated axons, the density of preexisting Na+ channels in
the demyelinated region may approach the density required to
support conduction (Hines and Shrager 1991; Waxman and
Brill 1978). In small-diameter premyelinated axons (diameter
<0.25Pm) in the optic nerve, the conduction of single action
potentials can be supported by Na+ channel densities of less
than 10/Pm2 (Waxman et al. 1989). The diameter of some
demyelinated axons is reduced (Prineas and Connell 1978;
Smith et al. 1983), possibly as a result of decreased neurofila-
ment phosphorylation and increased neurofilament packing
density (De Waegh et al. 1992). These results are applicable to
demyelinated axons with small diameters but may not apply to
larger axons, because of their lower input impedance. Because
of this, the acquisition of a higher-than-normal Na+ channel
density appears to be required for restoration of conduction.
Experimental evidence indicates that this does in fact occur in
some demyelinated axons.
Electron microscopy studies on experimentally demy-
elinated axons demonstrated the development of regions
of axon membrane with cytochemical (Foster et al. 1980)
and freeze-fracture (Black et al. 1987) characteristics similar
to those of nodal membrane, suggesting the acquisition of a
higher-than-normal sodium channel density. Early immuno-
cytochemical studies also provided evidence for the acquisi-
tion of increased Na+ channel densities in the demyelinated
parts of the axon membrane. These studies, on fish lateral
line nerves, demonstrated the development of relatively high
densities of Na+ channels in previously internodal regions
(England et al. 1990). Immunocytochemical observations
2 to 3 weeks following injection of the demyelinating toxin
doxorubicin also suggested the expression of sodium channels
at newly formed nodes along mammalian remyelinated axons
(Dugandzija-Novakovic et al. 1995). These early investiga-
tions, however, utilized pan-sodium channel antibodies that
did not differentiate between channel subtypes.
It is possible that acquisition of higher-than-normal densi-
ties of Na+ channels in demyelinated axon regions is because of
dedifferentiation of the axon membrane (see later). Although
the mature internodal membrane is incapable of secure con-
duction (Ritchie and Rogart 1977; Waxman 1977), the pre-
myelinated axon membrane, (including regions that will
develop into internodal membrane), is electrically excitable
(Foster et al. 1982; Waxman et al. 1989). Studies on the devel-
oping internodal axon during normal development indicate
that suppression of Na+ channel expression reduces axonal
534 • NEUROGLIA
excitability and suggest that this occurs in a time-dependent
manner after axons are covered by myelin. From a functional
standpoint, this insures that conduction is not compromised
owing to premature loss of sodium channels during develop-
ment (Black and Waxman 1986).
Immunocytochemical studies utilizing subtype specific
antibodies to sodium channels have provided evidence that
supports the suggestion of dedifferentiation of the axon
membrane in some demyelinated axons. Craner et al. (2003,
2004a) observed a switch from Nav1.6 to Nav1.2 expression
at nodes (Fig. 42.6) along demyelinated CNS axons in experi-
mental allergic encephalomyelitis and multiple sclerosis (MS)
tissue. Some demyelinated axons exhibited continuous immu-
nostaining for Nav1.2 and less frequently for Nav1.6 chan-
nels, while extending for tens of micrometers along the fiber
trajectory (i.e., as far as the axons could be followed within
sections). A similar pattern of continuous Nav1.2 immunos-
taining has been reported in premyelinated axons (Boiko et al.
2001), which may provide a substrate for continuous conduc-
tion of impulses. The functional implications of a reversion to
Nav1.2 expression (rather than Nav1.6) are not fully under-
stood. Evidence from patch-clamp studies (Rush et al. 2005)
indicates that different functional properties may permit
Nav1.6 channels to support higher firing rates. Substitution
of Nav1.2 for Nav1.6 may permit conduction to continue in
demyelinated axons even if they are depolarized, because the
voltage dependence of inactivation of Nav1.2 is depolarized
compared with that of Nav1.6 (Rush et al., 2005).
4.2 N O NU N I F O R M C O N D U C T I O N F O L L OWI N G
D E MY E L I NAT I O N
Smith et al. (1982) have argued that nonuniform impulse prop-
agation may also contribute to recovery of conduction in some
demyelinated axons. In this mode of conduction, action poten-
tials propagate nonuniformly between isolated foci of inward
current generation (termed phi-nodes). These seem to consist of
focal aggregations of Na+ channels that are established before
remyelination (Smith et al. 1982). Phi-nodes develop several
days before remyelination, and it has been suggested (Smith
et al. 1982) that they are the precursors of nodes.
5 BASIS FOR MOLECULAR REMODELING
O F T H E D E M Y E L I N AT E D AXO N
It has been clearly established that, in some demyelinated
axons, sodium channel density increases so that it can support
impulse invasion into the demyelinated region and continuous
conduction through it (Bostock and Sears 1978; Foster et al.
1980). There is evidence (Hines and Shrager 1991) that chan-
nel diffusion from nearby nodes is not sufficient to support
action potential conduction through demyelinated regions.
Former nodes of Ranvier in demyelinated axons, when stud-
ied by patch-clamp, display sharp gradients in channel density,
which have been interpreted as suggesting that Na+ channels
do not diffuse away from the node in large numbers follow-
ing demyelination (Shrager 1989). Radioimmunoassay studies
Figure 42.6 E-APP-positive spinal cord axons coexpress NCX and Nav1.6 over extensive regions in acute MS lesions. Digital images demonstrate axons
in MS spinal cord white matter immunostained for E-APP (E and F; blue), Nav1.6 (A; red) or Nav1.2 (B; red), and NCX (C and D; green). G and H
correspond to merged images. A, C, E, and G show coexpression of Nav1.6 and NCX within axons displaying E-APP, a marker of axonal injury. In
contrast, B, D, F, and H demonstrate NCX-immunopositive staining but an absence of Nav1.2 immunostaining within E-APP-positive axons, and
coexpression of NCX and Nav1.2 within E-APP-negative axons. From Craner et al. 2004b.

demonstrate a significant increase in Na+ channel concentra-
tion per weight of tissue (approximately three-fold at 21 to
28 days after peripheral nerve demyelination), supporting the
idea that new Na+ channels are inserted into the demyelinated
axon membrane (England et al. 1991). Quantitative autora-
diography reveals a fourfold increase in STX-binding sites in
demyelinated white matter in MS patients, compared with
normal white matter (Moll et al. 1991). The site of produc-
tion of the Na+ channels in demyelinated axons has not been
firmly identified. Synthesis of channels in the neuronal cell
body, with translocation by the axonal transport, is a clear pos-
sibility. Electrophysiological experiments have demonstrated
the production of new, functional Na+ channels by neurons
after axonal transection, and there is evidence for axonal
transport of Na+ channels in peripheral nerves (Lombet et al.
1985). Craner et al. (2003) noted upregulated transcription of
Nav1.2 mRNA in cell bodies of demyelinated axons, implying
that new channels continue to be produced by the neuron.
Another potential source of sodium channels that has been
proposed is synthesis in glial cells, with subsequent transfer of
these glial-synthesized channels to the axon (Bevan et al. 1985;
Gray and Ritchie 1985). Astrocyte processes and Schwann cell
microvilli contact the axon membrane at the node in a highly
specific manner (Hildebrand 1971; Waxman and Black 1984).
Similar glial processes are apposed to demyelinated axons at
sites of Na+ channel clustering (Black et al. 1984; Rosenbluth
1985; Rosenbluth and Blakemore 1984). The two special-
izations (glial contact and Na+ channel clustering) usually
are juxtaposed—an observation that has been interpreted
as suggesting that glial cell contact may contribute to the
development of Na+ channel clusters in the axon membrane.
Voltage-sensitive Na+ currents are present in astrocytes (Bevan
et al. 1985) and in some cases the density of sodium channels in
astrocytes is sufficient to support production of over-shooting
action potentials, if resting inactivation is removed by hyperpo-
larizing the cell. Black et al. (2010) have recently demonstrated
a striking upregulation of sodium channel Nav1.5 within
reactive astrocytes in MS plaques (Fig. 42.7). Physiological
properties of tetrodotoxin-resistant sodium channels in astro-
cytes are consistent with these channels being of the Nav1.5

M Y E L I N, I M P U L S E C O N D U C T I O N, A N D T H E PAT H O P H YS I O L O GY O F D E M Y E L I N AT I O N • 535
subtype. However, there is no evidence for expression of Nav1.5
within either normal myelinated or demyelinated axons. The
functional role of Nav1.5 channels in reactive astrocytes is under
investigation but, at this time, there is no evidence that these
astrocyte channels are transferred to axons. Although the glial
sodium channel synthesis hypothesis attracted much attention,
and more than 20 years have passed since Ritchie and collabo-
rators proposed this hypothesis, there is no demonstration of
channel transfer from glial cells to axons. Alternative functions
for astrocyte Na+ channels have been proposed (e.g., providing
a return pathway for Na+ ions that maintains astrocyte Na+ and
K+ -ATPase activity) (Sontheimer et al. 1994). Astrocytes may
also secrete extracellular molecules that target or anchor Na+
channels at sites of aggregation (Waxman et al. 1992).

6 S O D I U M C H A N N E L S A N D AXO N A L
D E G E N E R AT I O N

Axonal degeneration occurs early in the course of MS

and is a strong contributor to acquisition of persistent
(non-remitting) deficits (Trapp et al. 1999). Studies suggest
that sodium channels are crucial to this degeneration process.
The sodium-calcium exchanger (NCX) is present along mam-
malian myelinated axons within the CNS (Steffensen et al.
1997), consistent with the presence of Nav1.6 at nodes. Stys
et al. (1993) showed that, in energetically stressed axons, a
persistent Na+ influx, via sodium channels, can drive reverse
Na+-Ca2+ exchange which, in turn, imports deleterious lev-
els of Ca2+ into the axonal cytosol, triggering axonal injury.
Craner et al. showed that Nav1.6 sodium channels and NCX
are both present, and are colocalized, along injured axons in
experimental allergic encephalomyelitis (EAE) (Craner et al.
2004a) and in MS (Craner et al. 2004b) (see Fig. 42.6). It has
been shown that Nav1.6 produces substantial persistent cur-
rent (Rush et al. 2005) and it appears likely that high levels
of expression of Nav1.6 along demyelinated axons, in regions
that also express NCX, can trigger axonal degeneration.
The expression of Nav1.2 along demyelinated axons in EAE
(Craner et al. 2004a) and MS (Craner et al. 2004b) is not
colocalized with NCX, suggesting that expression of Nav1.2
is an adaptive change, supporting conduction along demy-
elinated axons while not contributing to their degeneration
(Waxman 2006).
7 I M P E DA N C E M I S M ATC H I N
D E M Y E L I N AT E D AXO N S
Figure 42.7 Nav1.5 Expression in Astrocytes Within MS Tissue.
A montage of grayscale images of R. communis agglutinin I (RCA1)
positive macrophages (white) throughout a portion of a section of
MS tissue was constructed to provide orientation for localization of
astrocytes within the tissue. Two regions of substantial macrophage
infiltration (top and bottom), are separated by white matter that exhibits
limited macrophage infiltration. In a serial section to that stained for
RCA I, labeling for Nav1.5 (red), GFAP (green) and PLP (blue) was
performed and high-magnification images acquired (right-hand panels).
An astrocyte within a normal-appearing white matter region of the sec-
tion not included in the montage and which exhibits strong PLP labeling
displayed minimal Nav1.5 labeling (top right panel). In contrast, an astro-
cyte in the white matter between two areas of macrophage infiltration
(panel 2) is hypertrophic and also shows an upregulation of Nav1.5. Note
that the PLP labeling (blue) is more disrupted compared with that in
normal-appearing white matter (top right panel). Astrocytes at the border
of (panel 3) and within (panel 4) the active lesion display intense Nav1.5
immunolabeling. Note the substantial disruption of PLP labeling at the
border of the lesion (panel 3) and the absence of PLP labeling within the
lesion (panel 4). PLP, blue; GFAP, green; Nav1.5, red; co-localization of
GFAP and Nav1.5, yellow. From Black et al. 2010.

A high Na+ channel density in demyelinated axon regions does
not, in itself, ensure reliable conduction. Electrical loading,
largely as a result of increased membrane capacitance, imposes
impedance mismatch that can cause conduction block at
sites of axonal inhomogeneity such as the junction between
myelinated and demyelinated regions (Waxman 1978).
Computer simulation studies of action potentials (Waxman
and Brill 1978) in a fiber with a single focally demyelinated
internode, in which the demyelinated axon membrane has
developed a high Na+ channel density (similar to that at
nodes) show that despite the high Na+ channel density in
the demyelinated area, conduction block occurs at the junc-
tion between normal and demyelinated axon region because
of impedance mismatch which prevents threshold from being
reached (Waxman and Brill, 1978).
Impedance matching can be overcome through the devel-
opment of relatively short myelinated segments proximal to the
demyelinated area (Waxman and Brill 1978), decrease in axon

536 • NEUROGLIA
diameter within the demyelinated region (Sears and Bostock
1981), the development of increased Na+ channel density at
the node proximal to the demyelinated area, or the develop-
ment of a specialized transition zone (with relatively high Na+
channel densities or relatively low K+ channel densities) at
the edge of the region of demyelination (Waxman and Wood
1984). Remyelinated myelin segments are known to be shorter
than normal internodes, and this serves as an adaptive func-
tion to facilitate impedance matching.
8 E N E R G ET I C A S P E C T S O F
D E M Y E L I N AT I O N
As a result of larger capacitative load and increased overall
number of sodium channels per unit length, the energetic
load imposed by impulse conduction is substantially higher
in non-myelinated compared to myelinated axons. Wang et al.
(2008) suggest that energetic cost per action potential per
centimeter of axon is 10-fold higher in nonmyelinated, com-
pared with myelinated axons. Similar considerations appear
to apply to demyelinated axons. Thus, the observation of
increased number and activity of mitochondria along axons in
MS lesions (Witte et al. 2009) might be viewed to be a com-
pensatory change. Energetically stressed mitochondria, how-
ever, produce ROS, which can injure axons and, moreover,
show increased susceptibility to mutations within mitochon-
drial DNA.
Campbell et al. (2011) observed an increased number of
respiratory deficient neurons within brain tissue from patients
with secondary progressive MS and noted high levels of clon-
ally expanded mitochondrial DNA deletions within these
neurons. Dutta et al. (2006) observed reduced mitochon-
drial gene expression within axons in MS lesions and sug-
gested that reduced ATP production might contribute to
impaired ionic homeostasis, thereby playing a role in axonal
degeneration in MS. Studies also show a reduced number of
Na+/K+-ATPase positive axons in chronic MS lesions (Young
et al. 2008) and impaired mitochondrial complex IV activity
within Na+/K+-ATPase positive chronic MS lesions (Mahad
et al. 2009), suggesting that mitochondrial dysfunction may
contribute to axonal degeneration in active demyelinating
lesions. Interestingly, chronic inactive areas of demyelination
from the same tissue display increased mitochondrial mass
and complex IV activity, possibly reflecting a compensatory
response to increased energy demand (Mahad et al. 2009).
Current studies are exploring the detailed nature of the mito-
chondrial contribution to pathophysiology of axonal injury in
MS (see, e.g., Campbell and Mahad 2011).
9 R E M Y E L I N AT I O N
Computer simulations predict that remyelination with even
thin, or short, myelin sheaths should provide a capacitative
shield that promotes conduction through previously demy-
elinated fibers, as long as sodium channel densities similar
to those in normal fiber develop at the remyelinated nodes
M Y E L I N, I M P U L S E C O N D U C T I O N, A N D T H E PAT H O P H YS I O L O GY O F D E M Y E L I N AT I O N
of Ranvier (Koles and Rasminsky 1972; Waxman and Brill
1978). Conduction velocity approaches normal in demyeli-
nated peripheral nerves, and the refractory period and the
ability to conduct high-frequency impulse trains is improved
as remyelination occurs (Smith et al, 1980; Smith et al. 1981,
1983). Moreover, clinical recovery in rats with EAE is cor-
related with remyelination and restoration of conduction
(Stanley and Pender, 1991). Reduced internode distances in
remyelinated fibers result in conduction velocities that, while
higher than in demyelinated axons, are lower than in normally
myelinated axons (Brill et al. 1977). However, reduced con-
duction velocity, in itself, does not necessarily produce clini-
cal deficits. Restoration of conduction due to remyelination is
expected to lead to clinical improvement. Both oligodendro-
cyte and Schwann cell-mediated remyelination of CNS axons
can enhance conduction. Remyelination of dorsal column
axons by Schwann cells, for example, has been shown to restore
secure action potential conduction with a normal refractory
period of transmission (but with decreased conduction veloc-
ity) and the capability to conduct the high-frequency impulse
trains in these fibers (Blight and Young 1989; Felts and Smith
1991).
10 T R A N S P L A N TAT I O N O F
M Y E L I N -F O R M I N G C E L L S
Demyelinating diseases with a genetic etiology, such as leu-
kodystrophies, can cause dysmyelination (abnormal myelin
formation), hypomyelination (insufficient myelin), and demy-
elination (myelin degradation) (Biffi et al. 2011; Kohlschutter
et al. 2010; Nave 2010; Pastores 2009). Inflammatory
demyelinating diseases, exemplified by MS in the CNS
and Guillain-Barre syndrome in the PNS, involve immune
system-mediated attack of components of myelin, which leads
to the progressive loss of axonal integrity (Makowska et al.
2008; Steinman et al. 2002). Despite the inherent capacity of
the nervous system to remyelinate, persistent demyelination
is a major problem in leukodystrophies, inflammatory demy-
elination, and spinal cord trauma, and results in a spectrum
of pathologies including irreversible neurological damage.
Potential approaches to myelin restoration include methods
to improve endogenous remyelination as well as methods to
deliver exogenous sources of myelin to demyelinated lesions.
Numerous studies have demonstrated the feasibility of remy-
elination through transplantation of central or peripheral
derived myelin-forming cells in experimental animal models
of demyelination (Einstein et al. 2006; Learish et al. 1999;
Pluchino 2003).
Some of the earliest proof-of-principle studies of
transplantation-based myelin repair utilized Schwann cells
(Aguayo et al. 1977; Blakemore 1977) and grafts containing
oligodendrocyte precursor cells (Blakemore and Crang 1988;
Duncan et al. 1988; Gansmuller et al. 1986; Lachapelle et al.
1983). Subsequent studies demonstrated the ability of a variety
of cell types including O2A+ progenitor cells from optic nerve
cultures (Groves et al. 1993), olfactory ensheathing cells (Sasaki
et al. 2006), cultured neurospheres (Pluchino et al. 2003),
• 537
A2B5+ glial progenitor cells (Windrem et al. 2004), and oli-
godendrocyte precursor cells (Crang et al. 2004; Franklin
and Blakemore 1997) to differentiate into myelin forming
cells upon transplantation. However, the ability of these cell
types to restore myelin varies widely and a direct compar-
ison of their efficiency to remyelinate has not been studied.
For example, fetal progenitor cells myelinate more extensively
than adult cells, although with somewhat delayed onset of
myelin production (Windrem et al. 2004), whereas oligoden-
drocyte precursor cells from higher order mammals migrate
more extensively and exhibit more robust remyelination com-
pared to their rodent counterparts (Windrem et al. 2008).
Furthermore, the highly migratory nature of oligodendrocyte
precursor cells enables them to expand and terminally differen-
tiate differently in response to varying regional cues (Windrem
et al. 2008). Although the primary assay for the outcome of
transplantation studies is structural, that is, whether or not
morphologically normal myelin is formed, does not, in itself,
ensure improved conduction, because impedance mismatch
or failure to form nodes with adequate Na+ channel densities
can lower the safety factor.
In the first physiological study of conduction properties
in axons myelinated by transplanted cells, Utzschneider et al.
1994) examined myelin-deficient axons in the md rat spinal cord
after transplantation of oligodendrocytes, and demonstrated a
fourfold increase in conduction velocity, which approached
myelinated control values, after formation of myelin by
transplanted glial cells. The ability to follow high-frequency
stimulation returned to almost normal. Moreover, action
potentials initiated outside the transplant region could invade
and propagate into the region of demyelination, and could
then propagate beyond the demyelinated region. Similar
improvements in action potential conduction have been
observed following transplantation of Schwann cells
(Honmou et al. 1996), olfactory ensheathing cells (Imaizumi
et al. 1998),”humanized” olfactory ensheathing cells from the
pig (Imaizumi et al. 2000) and bone marrow stromal cells
(Akiyama et al. 2002a,, 2002b) to demyelinated lesions within
the spinal cord of adult rats.
More recent studies of transplanted olfactory ensheathing
cells from green fluorescent protein-expressing donor rats into
a region of spinal cord demyelination demonstrated exten-
sive remyelination, including development of mature nodes,
paranodes, and juxtaparanodes, and restoration of impulse
conduction, as assessed by ultrastructural, immunocytochem-
ical, and electrophysiological analyses (Sasaki et al. 2006).
Furthermore, remyelinated axons display clusters of Nav1.6
at the nodes and Kv1.2 at the juxtaparanodes, demonstrat-
ing that in addition to forming compact myelin, transplanted
cells provide an environment that supports the restoration
of the molecular organization of a myelinated axon (Sasaki
et al. 2006). Black et al. (2006) demonstrate that spontaneous
remyelination of central axons by endogenous Schwann cells
recapitulates the nodal clustering of Nav1.6 and juxtaparan-
odal distribution of Kv1.2, consistent with findings by Schafer
et al. (2006) that Nav1.6, but not Nav1.2, is detected at newly
formed nodes in remyelinated axons in a lysolecithin-induced
model of PNS demyelination.
538 • NEUROGLIA
Several studies have provided evidence for transplantation-
based restoration of myelin and improved action potential
conduction in demyelinated and dysmyelinated axons in ani-
mal models. Human embryonic stem cells (hESC) committed
to an oligodendrocyte lineage (induced pluripotent stem cells)
are among the cell-types currently being studied (Keirstead
et al. 2005; McDonald et al. 1999). Induced pluripotent stem
cells, in addition to providing a robust supply of oligodendro-
cyte precursor cells with a uniform differentiation capacity,
avoid the possibility of immunological rejection when gen-
erated from autologous tissue (see review by Hu et al. 2009;
Potter et al. 2011). Highly purified populations of oligoden-
drocyte precursor cells have been generated and demonstrated
to be capable of differentiating into myelinating oligodendro-
cytes upon transplantation into the spinal cord of shiverer
mice (Nistor et al. 2005). Encouraged by these results, Geron
Corporation initiated a phase 1 clinical trial in 2009 to evalu-
ate the safety of transplanting oligodendrocyte precursor cells
derived from hESC into people with acute spinal cord injury.
Two years later, however, Geron announced that it will not
pursue its spinal cord injury trial using oligodendrocyte pre-
cursor cells derived from hESC. Thus the safety and efficacy
of transplantation-based remyelinating therapies remains to
be determined. A recent report of tumor development many
years after transplantation of poorly characterized human
fetal brain-derived cells into a patient with ataxia telangiecta-
sia has increased awareness about the potentially malignant
side-effects of stem cell transplants (Amariglio et al. 2009).
11 S U M M A RY A N D P E R S P E C T I VE
Rapid action potential conduction in vertebrates relies heav-
ily on the functional architecture of the myelinated axon. A
myelinated axon is more than just a bare axon with myelin
wrapped around it. It is a complex and intimate interaction
between two cells that defines the regional heterogeneity
characteristic of a myelinated axon. The high resistance and
low capacitance of myelin, combined with nodes of Ranvier
between segments of myelinated internodes support saltatory
conduction while restricting action potential and ion currents
to less than 0.5% of the axon’s surface and reducing energy con-
sumption. Strategic subdomain localization of voltage-gated
ion channels and associated molecules in the myelinated axon
membrane governs action potential conduction. The specific
assortment of molecules present within subsets of neurons,
further adds to the complexity of action potential initiation
and conduction. The complexity of the myelinated fiber is
matched by the complexity of demyelination. Passive and
active properties of the axon interact to shape the conduction
properties of a demyelinated axon and a variety of cellular and
molecular mechanisms contribute to these properties. The
multifactorial determinants of altered axonal conduction after
demyelination have made it more difficult to study, but also
have provided multiple therapeutic opportunities that may
improve function in demyelinated axons. These include phar-
macological manipulation of K+ channels, promotion of Na+
channel expression, manipulation of cellular characteristics to
correct impedance mismatch at the edge of the plaque, promo-
tion of endogenous remyelination, and remyelination through
transplantation of myelin-forming cells.
AC K N OW L E D G M E N T S
The authors’ research has been supported in part by grants
from the Rehabilitation Research Service and Medical
Research Service, Department of Veterans Affairs, and by gifts
from the Paralyzed Veterans of America.
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 43.
TRANSCRIPTION FACTOR S IN MYELINATING CELLS
Michael Wegner

A B B R E VI AT I O N S network that exceed its buffering capacity, it will move from its
old metastable state to a different one and thereby establish a new,
BMP bone morphogenetic proteins characteristic pattern of gene expression. In other words: a gene
cAMP 3c,5ccyclic adenosine monophosphate regulatory network is robust enough to guarantee the expression
CNS central nervous system pattern that is characteristic for a cell in a particular stage of its
Gpr G-protein–coupled receptor development, but also allows the ordered changes in gene expres-
HDAC histone deacetylase sion that underlie lineage progression and maturation.
HMG high mobility group To satisfy the need for stability and at the same time allow
MRF myogenic regulatory factor change, gene regulatory networks usually contain transcrip-
MSE myelinating Schwann cell enhancer tion factors that are present at all times and those that are only
NFAT nuclear factor of activated T cells recruited at specific times or under certain circumstances.
OPC oligodendrocyte precursor Those that are constitutively present function as determinants
PDGFRα platelet-derived growth factor receptor α of cell type identity and lineage, whereas the facultative and
PNS peripheral nervous system transiently present ones mark a specific stage. The network
POU Pit1-Oct1/2-Unc86 also needs checks and balances. Reinforcing and antagonizing
SCAN Srezbp, Ctfin51, AW-1, and number 18 activities, stabilizing and destabilizing activities, transcriptional
YY Yin Yang activators and repressors, and pro- and anti-differentiation
Zfp zinc finger protein factors are present at all times. This means that transcription
factors are expected in myelinating glia with the following
functions: (1) factors that establish competence for myelina-
tion and impart cell identity, (2) factors that initiate the myeli-
1 INTRODUCTION nation program and maintain it, and (3) factors that actively
counteract this program at various stages of cell development.
Transcription factors are the immediate effectors of gene Transcription factors have two main modes of action.
expression. Considering the special gene expression profile They promote or suppress recruitment of the basal transcrip-
of myelinating glia (see chapter 29), it has long been assumed tion machinery to genes by binding to their regulatory regions
that the transcription factors in charge are also unique and (i.e. promoter, enhancer, and silencer elements). Contacts
restricted in their occurrence to Schwann cells and oligoden- with components of the basal transcription machinery can be
drocytes. However, such factors are few, and none is completely direct or indirect through transcriptional cofactors such as the
restricted in occurrence and function to myelinating glia. Gene Mediator complex. Alternatively, transcription factors recruit
expression in myelinating glia is thus caused to a large extent by chromatin modifying enzymes and chromatin remodeling
transcription factors with limited specificity. Conceptionally, complexes, and by modifying the epigenetic state of chromatin
this is feasible because gene expression is not a readout of the change accessibility of their target genes for the transcriptional
action of single transcription factors, but of transcription factor apparatus. It follows that many transcription-related proteins
combinations and their synergistic, additive, and antagonistic including components of the basal transcription machinery,
interactions in the context of a gene regulatory network. chromatin modifiers, and remodeling complexes are tightly
The advantages of regulating gene expression through such linked to the gene regulatory network.
networks are manifold. Considering that many different gene Various types of small RNAs are also part of the network.
regulatory networks with unique characteristics can be gen- MicroRNAs in particular play a role in fine tuning network activ-
erated from relatively few building blocks in a combinatorial ity. Considering the multitude of transcription factors involved,
manner, a network is resource-efficient in evolutionary terms. the intricate relationships among them and the many interactions
Additionally, a gene regulatory network has substantial homeo- between transcription factors, basal transcription machinery,
static capacity because of the many redundant, reinforcing, syn- chromatin modifiers, remodelers, and microRNAs, this chapter
ergistic, and antagonistic relationships among its constituting focuses on key transcription factors in myelinating glia and their
transcription factors. As a consequence, it is optimally suited to interactions within the network. Other comprehensive accounts
maintain expression patterns. At the same time, a gene regulatory of transcription factors in myelinating glia have recently been
network remains receptive to input. If changes are induced in the published (e.g., Li et al. 2009; Svaren and Meijer 2008).

543
 2 TR ANSCRIPTIONAL CONTROL
O F P E R I P H E R A L N E RVO U S SYS T E M
M Y E L I N AT I O N BY S C H WA N N C E L L S
2.1 S C H WA N N C E L L S P E C I FI C AT I O N
Schwann cells as the myelinating glia of the Peripheral
Nervous System (PNS) are neural crest derivatives (see chap-
ter 14). Therefore signals and regulatory mechanisms must
exist that commit neural crest progenitor cells to the Schwann
cell lineage. This first step in lineage development is still
poorly understood on the transcriptional level. Although
loss of several transcription factors leads to a reduction in
Schwann cell numbers, it has rarely been analyzed whether
Schwann cell development is directly influenced or indirectly
through effects on early neural crest development. To date the
best studied example of a transcription factor with influence
on Schwann cell specification is Sox10 (Fig. 43.1). This fac-
tor belongs to the SoxE subgroup of the Sox protein family
and contains a DNA-binding high mobility group (HMG)-
domain as defining feature (Wegner 2010). It has been shown
in Sox10-deficient mice and zebrafish that neural crest cells
find their way to the nerve roots, but then fail to convert
into Schwann cell precursors that express the ErbB2/ErbB3
receptor, which in turn establishes neuregulin dependence
of this cell type (Britsch et al. 2001; Dutton et al. 2001) (see
also chapter 14). In fact ErbB3 expression in Schwann cells is
under direct control of a Sox10-responsive enhancer (Prasad
Schwann cell development
NC cell SCP
NEP cell OPC
Oligodendroglial development
Figure 43.1 Overview of Transcription Factors with Restricted Occurrence and Prodifferentiation Function in Myelinating Glia. Expression during
Schwann cell development (upper part) is indicated by red bars, expression during oligodendroglial development (lower part) by green bars. Axons
are in blue. NC, Neural crest cell; SCP, Schwann cell precursor; iSC, immature Schwann cell; pSC, promyelinating Schwann cell; mSC, myelinating
Schwann cell; NEP, neuroepithelial precursor; OPC, oligodendrocyte precursor; pOL, pro-oligodendrocyte; mOL, myelinating oligodendrocyte.
544
et al. 2011). Although required for Schwann cell specification,
Sox10 alone cannot be sufficient because it is expressed in neu-
ral crest precursors long before, and because it is also involved
in other neural crest specification events including melanocyte
specification. This is not surprising because HMG-domain
containing, minor groove binding transcription factors gen-
erally cooperate in gene activation with other transcription
factors that bind the major groove of DNA (Wegner 2010).
The identity of these cooperating transcription factors during
Schwann cell specification is currently unknown.
2.2 T H E S C H WA N N C E L L O N T H E WAY
TO MY E L I NAT I O N
Sox10 expression does not extinguish after specification (see
Fig. 43.1). Instead it continues in Schwann cells at all times
(Kuhlbrodt et al. 1998). This expression is ensured by mul-
tiple enhancers in the vicinity of the Sox10 gene that respond
to different sets of transcription factors and autoregulation
(Wahlbuhl et al. 2011; Werner et al. 2007). Conditional
deletion of Sox10 at various stages of Schwann cell develop-
ment and the analysis of hypomorphic mouse mutants has
confirmed that Sox10 remains functional and contributes
to Schwann cell development during the consecutive stages
of the immature Schwann cell, the promyelinating and
the myelinating Schwann cell (see chapter 14) and is even
required in the mature Schwann cell for myelin maintenance
(Bremer et al. 2011; Finzsch et al. 2010; Fröb et al. 2012;
iSC pSC mSC
Oct6 (& Brn2) Krox20
Sox10
Sox10
Nkx2.2 MRF
Olig2 (& Olig1)
pOL mOL
• NEUROGLIA
Schreiner et al. 2007). Sox10 can therefore be regarded as a
marker for Schwann cell lineage and identity. It represents one
of the invariant building blocks of the gene regulatory network
in this cell type. Intriguingly, Sox10 activates different target
genes during various developmental stages. Sox10-dependent
ErbB3 activation occurs already in the Schwann cell precur-
sor (Britsch et al. 2001; Prasad et al. 2011), whereas activa-
tion of Desert Hedgehog starts in the immature Schwann cell
(Finzsch et al. 2010) and activation of myelin genes such as
Myelin Associated Glycoprotein, Connexin-32, Myelin Protein
Zero, Peripheral Myelin Protein 22, Periaxin, and Myelin Basic
Protein (see chapter 7) is delayed until the onset of myelina-
tion (Bondurand et al. 2001; Denarier et al. 2005; Jones et al.
2007, 2011, 2012; LeBlanc et al. 2007; Peirano et al. 2000).
This further emphasizes the need for cooperating tran-
scriptions factors that modulate the function of Sox10 in a
stage-specific manner. One way of identifying these factors
is the detailed dissection of Schwann cell–specific regulatory
regions which have been identified for most of the mentioned
Sox10-responsive genes.
At least some of the cooperating transcription factors should
be responsive to signals that drive Schwann cell development
and myelination such as increased intracellular cAMP levels
(Monuki et al. 1989). In zebrafish and mammals the rise in
cAMP levels is caused by activation of the G-protein–coupled
orphan receptor Gpr126 (Monk et al. 2009, 2011). The axonal
ligand for the receptor is unknown, but the POU-domain
A Oct6
Sox10
iSC pSC
B
MSE
N
FA
T
YY
AC Sox10
1
Oct6
HD
Pol ll
P Krox20
Figure 43.2 Essential Regulatory Events During Schwann Cell Differentiation. A. Genetic and functional interactions of the prodifferentiation
factors Sox10, Oct6, and Krox20 in Schwann cells. B. Model of MSE-dependent Krox20 gene activation in myelinating Schwann cells. P, Promoter;
PolII, RNA polymerase II.
T R A N S C R I P T I O N FAC TO R S I N M Y E L I N AT I N G C E L L S
transcription factor Oct6 (also known as Pou3f1, Scip, or
Tst-1) is the key effector at the downstream end of this signal-
ing pathway (Monuki et al. 1989) (see also chapters 7 and 14).
In fact, Oct6 expression cannot be detected in zebrafish in
the absence of Gpr126 (Monk et al. 2009). Another fac-
tor required for Oct6 expression in Schwann cells is Sox10
(Fig. 43.2A), which binds to and activates the Schwann
cell-specific enhancer downstream of the Oct6 gene ( Jagalur
et al. 2011; Schreiner et al. 2007). It is unknown whether
Sox10 activity is itself regulated by cAMP or rather cooperates
with a cAMP-responsive transcription factor to regulate Oct6
expression. One such cAMP-responsive factor in Schwann
cells is the broadly expressed p65 subunit of NF-NB (Yoon
et al. 2008). PKA-dependent phosphorylation of NF-NB in
response to high cAMP concentrations increases Oct6 levels.
Expression of Oct6 initiates late during the immature
Schwann cell stage and peaks during the promyelinating
stage in which Schwann cells prepare for myelination and
are in a one-to-one relationship with large caliber axons (see
Fig. 43.1) (see chapter 14). In fact, Oct6 is one of the major
driving forces in the transition from immature into myelinat-
ing Schwann cells. This is documented by the transient arrest
of Schwann cell development in the promyelinating stage in
Oct6-deficient mice (Bermingham et al. 1996; Jaegle et al.
1996). This relatively mild phenotype is caused by functional
redundancy between Oct6 and the closely related and coex-
pressed POU-domain transcription factor Brn2 (also known
Krox20
myelin
genes
mSC
• 545
as Pou3f2). In accord, mice with deficiency for both Oct6
and Brn2 exhibit a dramatically enhanced arrest at the pro-
myelinating stage and severe hypomyelination ( Jaegle et al.
2003). This is just one example for the frequently existing
functional redundancy between transcription factors in myeli-
nating cells.
In addition to the cAMP pathway, there is a strong influence
on Schwann cell development by neuregulins through activa-
tion of the ErbB2/ErbB3 receptor. Downstream signaling
involves PI3 kinase activation, increased intracellular calcium,
and the MAP kinase pathway (see chapter 14). These pathways
in turn influence the function of a number of transcriptional
regulators that are present in Schwann cells around the time of
myelination. The DNA-binding activity of the p65 subunit of
NF-NB is enhanced (Limpert and Carter 2010). Additionally,
the broadly expressed transcription factor YY1 becomes
phosphorylated (He et al. 2010), and calcineurin-dependent
dephosphorylation of NFATc3/c4 leads to nuclear transloca-
tion and activation (Kao et al. 2009).
All these signal-dependent changes in the gene regula-
tory network contribute to the initiation of the myelination
program in Schwann cells. This is closely connected with the
induction of the zinc-finger transcription factor Krox20 (also
known as Egr2), which is often referred to as the master regula-
tor of peripheral myelination (see Fig. 43.1) because Schwann
cell development is stalled permanently at the promyelinat-
ing stage in Krox20-deficient mice (Topilko et al. 1994) and
Schwann cells fail to form myelin.
Activation of Krox20 requires the Oct6 protein (and/
or its relative Brn2) to bind to the myelinating Schwann cell
enhancer (MSE) of the Krox20 gene (Ghislain and Charnay
2006) (see Fig. 43.2B). The presence of Oct6 alone is, how-
ever, not sufficient to activate the MSE. It rather depends on
the synergistic support of Sox10 (Ghislain and Charnay 2006;
Reiprich et al. 2010), the very same transcription factor that has
already been involved in activating Oct6 expression (see Fig.
43.2A,B). Additionally, the MSE is targeted and activated by
dephosphorylated NFATc3/c4 and phosphorylated YY1 (He
et al. 2010; Kao et al. 2009) (see Fig. 43.2B). At least NFATc3/
c4 again requires the synergistic support of Sox10 for Krox20
activation (Kao et al. 2009). A picture thus emerges in which
several signaling pathways converge through their downstream
effectors on the MSE and combine with Schwann cell identity
factors such as Sox10 to synergistically activate Krox20 and
kick-start the myelination program.
Myelination not only requires transcription factor activity
but also relies on microRNAs. This is evident from deletion of
the microRNA-processing enzyme Dicer in Schwann cells and
the resulting myelination block (e.g., Dugas et al. 2010; Pereira
et al. 2010; Yun et al. 2010). Numerous microRNAs are differ-
entially expressed during Schwann cell development and thus
represent candidates for stage-specific regulators (Gokey et al.
2012). Intriguingly, at least nine of these differentially regu-
lated microRNAs are under transcriptional control of Sox10.
While microRNAs participate in the regulation of myeli-
nation, Krox20 is the decisive regulator. Schwann cell-specific
regulatory regions of myelin genes and genes for lipid
546 • NEUROGLIA
biosynthetic enzymes are directly bound by Krox20 in vivo
(Denarier et al. 2005; Jones et al. 2007, 2011, 2012; LeBlanc
et al. 2006, 2007), and expression of the genes depends on
Krox20 (Nagarajan et al. 2001) and its cofactors Nab1 and
Nab2 (Le et al. 2005b). Additionally, there is support from
SREBP transcription factors in the regulation of lipid produc-
tion (Verheijen et al. 2003) and from Sox10 in the regulation
of myelin genes. In fact, Krox20 and Sox10 often target the
same regulatory regions in myelinating Schwann cells and
cooperate to induce high levels of myelin gene expression.
This cooperativity is further strengthened by direct interac-
tions between the two proteins and cross-stimulatory effects
on their respective expression (Ghislain and Charnay 2006;
Reiprich et al. 2010; Wahlbuhl et al. 2011; Wissmüller et al.
2006). The multiple links between Sox10 and Krox20 are
quite remarkable and likely indicate the central role of this
pair of transcription factors in the gene regulatory network of
myelinating Schwann cells.
2.3 C O U N T E R AC T I N G MY E L I NAT I O N
I N S C H WA N N C E L L S
Krox20 and Sox10 are functional both during induction and
maintenance of myelin gene expression. This is not the case for
Oct6. In fact Oct6 has to be downregulated after the initial
phases for myelination to proceed properly. Experimentally
prolonged Oct6 expression leads to hypomyelination and loss
of axons in the PNS (Ryu et al. 2007). It has been proposed
that Krox20 plays a role in the eventual shut-down of Oct6
expression (Zorick et al. 1999). It is known that Oct6 itself
is also a negative regulator of its own expression ( Jaegle et al.
2003). Why Oct6 affects early and late phase of myelination
differently is not yet clear. It shows however, that Oct6 has two
distinct functions: it promotes initiation and the early steps of
the myelination program, but at the same time prevents transi-
tion into the fully mature state.
Such a behavior is typical for many transcription fac-
tors with transient expression during Schwann cell develop-
ment. They function both as positive and negative regulators.
Immature Schwann cells, for instance, express the transcrip-
tion factors cJun and Sox2, and contain nuclear complexes
of the Notch intracellular domain and RBPJ as a result of
active Notch signaling (Le et al. 2005a; Parkinson et al.
2008; Woodhoo et al. 2009). These factors establish and
define the immature Schwann cell state. Active Notch sig-
naling, for instance promotes the generation of immature
Schwann cells from Schwann cell precursors, controls prolif-
eration and regulates the size of the Schwann cell pool (see
chapter 14). Additionally, however, these factors also actively
repress the myelination program by inhibiting Krox20 expres-
sion, and thereby prevent precocious Schwann cell differentia-
tion. As a consequence, prolonged expression leads to delayed
myelination. Their deletion, in contrast, causes premature myeli-
nation. Once inhibition is overcome in developing Schwann
cells and the myelinated state is established, Krox20 similarly
suppresses cJun and Sox2 expression. These transcription factors
are therefore not only functionally antagonistic and expressed
 cJun

iSC Sox2 Krox20

RBPJ/NICD

mSC

Figure 43.3 Cross-Regulatory Interactions Between the Differentiation Inhibitors cJun, Sox2, and RBPJ, and the Prodifferentiation Factor Krox20 in
Schwann Cells.

in a mutually exclusive manner, they also cross-inhibit each A PNS

other’s expression (Fig. 43.3). This implies that even mature
Schwann cells will start to re-express cJun, Sox2 and acti-

HD
vate Notch signaling if Krox20 levels drop to sufficiently low CBP

AC
levels. It grants Schwann cells a certain degree of plasticity and Ac p65
permits them to dedifferentiate in cases of nerve injury as a p65
precondition for the ensuing demyelination and remyelina-
tion processes (see chapter 14).
There is also evidence that genes coding for activators and
inhibitors of the myelination program carry opposite histone
marks in their chromatin at any given time, with trimethylated myelination
H3K4 being found on the active, and trimethylated H3K9
being found on the repressed gene (Chen et al. 2011). In myeli-
nating Schwann cells, prodifferentiation genes are marked by
trimethlyated H3K4, whereas antidifferentiation genes prefer-
entially carry H3K9. In immature Schwann cells, the situation
is reversed. Ordered changes in histone marks and chromatin
thus contribute to Schwann cell differentiation and myelina- TCF4
βcat

H
tion. So far, the effects of histone methylases and demethylases TCF4

on Schwann cell development have not been studied. The
only group of chromatin modifying enzymes studied
are histone deacetylases (HDACs), in particular HDAC1
and HDAC2. Their deletion stalls Schwann cell development
in vivo (Chen et al. 2011; Jacob et al. 2011). The mechanism
is, however, less straightforward than expected. The histone
deacetylases seem to predominantly target transcription fac-
tors. One prime target is the p65 subunit of NF-NB, which by
exchanging histone acetylase CBP for HDACs as interaction
partners, becomes deacetylated (Fig. 43.4A). This in turn causes
the replacement of inhibitory by activating histone marks on
prodifferentiation genes such as Sox10 and the reverse on
anti-differentiation genes (Chen et al. 2011). A second
HDAC target appears to be the Sox10 protein whose prod-
ifferentiation activity is increased in complex with HDAC2
( Jacob et al. 2011). These results show that chromatin changes
occur during differentiation and that the machinery involved
in these changes is part of the gene regulatory network in
Schwann cells.
DA
Groucho
C
B CNS
Figure 43.4 Cofactor Dependency of Transcription Factors in
Myelinating Glia. A. By switching CBP for HDAC, the p65 subunit
of NF-NB turns from a differentiation inhibitor in Schwann cells into a
pro-differentiation factor. B. Similarly, TCF4 changes activity in prooli-
godendrocytes by exchanging E-catenin for Groucho and HDACs. Ac,
Acetylated lysine residue.
2.4 T R A NS C R I P T I O NA L C O N T RO L O F
C E N T R A L N E RVO US SYS T E M MY E L I NAT I O N BY
O L I G O D E N D RO C Y T E S
2.4.1 Oligodendroglial Specification
In contrast to Schwann cells, oligodendrocytes are central
nervous system (CNS) cells. Oligodendroglial cells therefore
have to be specified from pluripotent neuroepithelial pre-
cursor cells in the ventricular zone of the embryonic CNS.

T R A N S C R I P T I O N FAC TO R S I N M Y E L I N AT I N G C E L L S • 547
The oligodendroglial specification process is much better
understood than Schwann cell specification (see chapters 13,
14). Oligodendrocyte precursors (OPCs) are generated in
defined, frequently ventrally located areas of the ventricular
zone, usually after local neurogenesis has already been com-
pleted (Rowitch 2004) (see also chapter 13). In the spinal cord,
which serves as a simple model system, OPCs are first gener-
ated ventrally in the so-called pMN domain, and only later
in the more dorsal dP3-dP5 domains (Fogarty et al. 2005).
The pMN domain first gives rise to motoneurons, and then to
the majority of spinal cord OPCs (Fig. 43.5). This argues that
oligodendrocyte specification is dependent on at least two
different sets of transcription factors: one that defines pMN
domain identity and a second that allows a switch from neu-
ron to glia production.
The pMN domain identity depends on the sonic hedge-
hog (Shh) gradient along the dorsoventral axis of the spinal
cord and the resulting concentration-dependent induction
of different transcription factors (see chapter 13). Some tran-
scription factors are only induced in a narrow window of Shh
concentrations; others have a wider window. The Nk home-
odomain transcription factor Nkx2.2 is, for instance, only
expressed under highest concentrations in the most ventral
region, whereas the Iroquois homeodomain transcription fac-
tor Irx3 requires lower Shh concentrations (see Fig. 43.5). The
closely related Nkx6.1 and Nkx6.2 proteins in contrast can be
induced over a wider range of Shh concentrations so that they
are coexpressed with both Nkx2.2 and Irx3 in their respec-
tive domain as well as in the domain between them (Cai et al.
2005; Vallstedt et al. 2005). One of the target genes of Nkx6.1
and Nkx6.2 is the gene for bHLH transcription factor Olig2.
Considering that Nkx2.2 and Irx3 both repress Olig2 expres-
sion, Nkx6.1 and Nkx6.2 will activate Olig2 expression spe-
cifically in the region between the Nkx2.2 and Irx3 domains.
Neurogenesis
(early)
ventricular zone
mantle
Nkx6.1
MN Olig2 Nkx6.2
+ Neuro-
genin
ventral spinal cord
Figure 43.5 Expression of signaling molecules and transcription factors in the ventricular zone of the ventral spinal cord that lead to formation of the
pMN domain, and consecutive production of motoneurons and oligodendrocyte precursors in the mantle zone. MN, Motoneurons; OPC oligodendrocyte
precursor; Shh, sonic hedgehog.
548
This corresponds to the pMN domain (see Fig. 43.5). Olig2
is thus a specific marker for the pMN domain and its pluri-
potent neuroepithelial precursor cells. It is required for the
production of both motoneurons and OPCs (Lu et al. 2000,
2002; Zhou and Anderson 2002; Zhou et al. 2000). The pro-
duction of both from the pMN domain is severely impaired
in Olig2-deficient mice. However, OPCs do not completely
disappear in all CNS regions. This is because of the remain-
ing activity of the closely related Olig1 protein. A complete
OPC loss is only observed in mice deficient for both Olig1
and Olig2.
Considering that neurons and glia have to be pro-
duced at different times, there must be factors that promote
Olig2-dependent motoneuron production and prevent OPC
production at early times. These include the bHLH transcrip-
tion factors Neurogenin-1 and Neurogenin-2 (Mizuguchi
et al. 2001; Nieto et al. 2001; Sun et al. 2001) (see Fig. 43.5). At
later stages, however, Olig2 function has to be modulated such
that glia instead of neurons are produced. This is achieved by
induction of gliogenic transcription factors including NFIA,
Sox9, and activated Notch (Deneen et al. 2006; Park and
Appel 2003; Stolt et al. 2003) (see Fig. 43.5). The Neurogenins
are simultaneously down-regulated (Mizuguchi et al. 2001;
Nieto et al. 2001; Sun et al. 2001). As a consequence of the
combined action of Olig2 and gliogenic factors, OPCs
are specified. Intriguingly, Olig2 undergoes different phos-
phorylation events at multiple sites (Li et al. 2011; Sun et al.
2011). One of these phosphorylations changes its ability
to interact with neurogenins (Li et al. 2011) arguing that
Olig2 function is not only determined by the availability of
interacting transcription factors. Posttranslational modifica-
tions additionally modulate the process. Such modifications
may also determine interplay with transcriptional cofactors
and chromatin modifying enzymes such as HDACs that
Gliogenesis
(late)
mantle
Irx3
pMN Olig2 OPC
Shh Nkx2.2 + Sox9
NFIA
Notch
• NEUROGLIA
are required for oligodendroglial specification and as much
involved in oligodendrocyte as in Schwann cell development
(Cunliffe and Casaccia-Bonnefil 2006; Shen et al. 2005;
Ye et al. 2009).
Oligodendroglial specification is marked by the induction
of Sox10 expression (see Fig. 43.1). Analysis of the regulatory
regions of the Sox10 gene furthermore indicates that Sox10 is
a direct target gene of Olig2 in OPCs (Küspert et al. 2011).
Whereas motoneurons lose Olig2 expression after specifica-
tion, cells of the oligodendrocyte lineage stay positive for
Olig2 throughout development and in the mature state (Lu
et al. 2000; Zhou et al. 2000). The same holds for Sox10 (Stolt
et al. 2002). In areas of the ventricular zone where Olig2 is not
expressed in the pluripotent precursors, and OPCs arise inde-
pendent of Olig2 such as the dP3-dP5 domains of the spinal
cord, Olig2 comes on as soon as the cells are specified (Cai
et al. 2005; Vallstedt et al. 2005). Again, Olig2 is immediately
followed by Sox10. Considering the expression of Olig2 in a
subset of neuroepithelial precursors, motoneurons and some
astrocytes, Olig2 is not an unambiguous marker of oligoden-
drocyte lineage and identity. Rather it is the combination of
Olig2 and Sox10. Both the similarities and the differences to
Schwann cells are striking. Only oligodendrocyte specifica-
tion depends on Olig2, and only Schwann cell specification
depends on Sox10. Both types of glia, however, depend for
their identity at all times on Sox10.
2.4.2 Maintaining the Oligodendroglial Precursor
State and Preventing Differentiation
Whereas Schwann cells express Sox10 as the only SoxE pro-
tein, oligodendroglial cells additionally contain the closely
related Sox9 and Sox8 for most of their development (Stolt
et al. 2005). The ensuing functional redundance makes it
much more difficult to determine SoxE function during oli-
godendrocyte than Schwann cell development. Considering
that both SoxE and Olig proteins are expressed in OPCs
as they proliferate, migrate, and distribute throughout the
parenchyma, it is expected that these factors are relevant for
OPC development. In agreement, SoxE factors are required
Zfp488
Sox10
Olig1/2
Nkx2.2
Promoter
Id2/4 Sox5/6
Hes5
Figure 43.6 Summary of Transcription Factors That Influence Oligodendroglial Myelin Gene Expression and CNS Myelination. Activating factors are
in dark green if their direct influence on myelin gene expression was shown, and in light green if postulated. Inhibiting factors are in red.
T R A N S C R I P T I O N FAC TO R S I N M Y E L I N AT I N G C E L L S
to maintain expression of the PDGFRD receptor (Finzsch
et al. 2008), the main oligodendroglial receptor for PDGF as
an important survival factor, mitogen and migratory cue (see
chapters 13, 20, and 22).
Similarly, Notch signaling is active in OPCs (Genoud
et al. 2002). One of the genes induced by Notch in OPCs
is the bHLH transcription factor Hes5 (Liu et al. 2006),
which has been mainly described as a differentiation inhibi-
tor (Fig. 43.6). It prevents premature terminal differentiation
and myelination by binding directly to the regulatory region
of myelin genes and by repressing their expression following
HDAC recruitment, histone deacetylation, and changes in
chromatin structure. Additionally, Hes5 interacts with Sox10
and thereby sequesters it in a complex that prevents Sox10
from stimulating myelin gene expression.
In this inhibitory function, Hes5 is not alone. The OPCs
also express high levels of the Id2 and Id4 inhibitors (see
Fig. 43.6) as downstream effectors of BMP signaling. Part of
the Id proteins’ inhibitory function is exerted by heterodi-
merization with Olig proteins (Kondo and Raff 2000; Samanta
and Kessler 2004; Wang et al. 2001). Additionally, high lev-
els of Sox6 and its close relative Sox5 can be found in OPCs
(Stolt et al. 2006). These members of the SoxD subgroup of
the Sox protein family also counteract premature differen-
tiation (see Fig. 43.6). They bind to the regulatory regions
of myelin genes, recruit HDACs and block the binding
of Sox10. In addition to these anti-differentiation activi-
ties, many of the mentioned transcription factors probably
also have genuine functions in actively maintaining the pro-
genitor state of OPCs, although these aspects are less well
studied. For instance, Id2 and Id4 do not only inhibit myelin
gene expression but also promote proliferation (Kondo and
Raff 2000; Marin-Husstege et al. 2006; Samanta and Kessler
2004; Wang et al. 2001). Similarly, evidence from chondro-
cyte development indicates that Sox5 and Sox6 do not only
counteract SoxE activity but can also enhance it (Wegner
2010). Thus it is utterly plausible that Sox5 and Sox6 prevent
Sox10 from activating myelin genes prematurely, and at the
same time help it to activate genes whose products are required
in OPCs.
CNS myelination
MRF activators
YY1
Mash1
Myelin Basic Protein Gene
repressors
• 549
 2.4.3 Converting Precursors to Oligodendrocytes
Around the time when OPCs start to enter the final phase
of differentiation that leads to a myelinating phenotype,
a group of novel transcription factors appears in these
pro-oligodendrocytes. These include Sox17 (Sohn et al. 2006).
This member of the SoxF subgroup of the Sox protein fam-
ily is only distantly related to either SoxE or SoxD proteins.
Sox17 appears to induce cell cycle exit by cyclin D upregula-
tion and by counteracting E-catenin (Chew et al. 2011), which
as the downstream effector of the Wnt signaling pathway
promotes OPC maintenance and proliferation (Fancy et al.
2009; Shimizu et al. 2005). Another modulator of Wnt and
E-catenin activity at the transition from OPC to oligoden-
drocyte is the Tcf/Lef family transcription factor TCF4 (also
known as TCF7L2). This E-catenin interacting transcription
factor has been reported to function as an inhibitor of myeli-
nation (He et al. 2007). Its deletion in the mouse, however,
leads to a delay in myelination (Ye et al. 2009), arguing that
it functions also as a prodifferentiation factor. Likely, this is
due to an exchange of interacting cofactors from E-catenin to
Groucho corepressors and HDACs at this critical time of oli-
godendroglial development (Ye et al. 2009) (see Fig. 43.4B).
Another transcription factor with essential functions dur-
ing this transition period is YY1 (He et al. 2007). Shortly
after cell cycle exit, it starts to repress factors such as Hes5 and
Id4 that maintain the progenitor state and counteract differ-
entiation. Also, YY1 recruits HDAC-containing repressor
complexes to the corresponding genes, which lead to tran-
scriptional inhibition. In addition to its function as a repressor
of myelination inhibitors, YY1 may also function as a direct
activator of the myelination program as indicated by its ability
to stimulate PLP expression (see Fig.43.6).
NEP cell OPC
Sox10
Olig2
Figure 43.7 Summary of the Regulatory Network in Myelinating Oligodendrocytes with Focus on the Genetic Interactions Among Participating
Transcription Factors and Their Effect on Myelin Gene Expression. Proposed interactions are marked by stippled lines.
550 • NEUROGLIA
Myelination inhibitors are not only removed by the action
of antagonistic transcription factors. MicroRNAs also play a
role. As in Schwann cells, there is a plethora of microRNAs
with differential expression during oligodendrocyte develop-
ment (Lau et al. 2008) and oligodendrocyte-specific dele-
tion of the microRNA processing Dicer enzyme proves the
requirement of microRNAs for terminal differentiation of
oligodendrocytes and myelination (Dugas et al. 2010; Zhao
et al. 2010). Among these differentially regulated microRNA
are miR-219 and miR-338. As YY1, they are upregulated dur-
ing the transition from late OPC to pro-oligodendrocyte and,
among other targets, recognize the 3c-UTR of Sox6 and Hes5
transcripts. By inhibiting the translation of transcripts for
myelination inhibitors, these microRNAs help cells to enter
the final phase of differentiation.
Additionally, activators of the myelination program need
to be induced as late OPCs turn into prooligodendrocytes.
Among them are the bHLH transcription factor Mash1 (also
known as Ascl1) and the Nk homeodomain transcription
factor Nkx2.2 as indicated by the severe delay of terminal
oligodendrocyte differentiation and myelination in the cor-
responding mouse mutants (Qi et al. 2001; Sugimori et al.
2008) (see Fig. 43.1). These factors combine with Sox10
and the Olig proteins to establish the gene regulatory net-
work that initiates terminal differentiation and myelination
(Fig. 43.7). Many of the transcription factors that are part of
this network influence each other’s expression. For instance,
Nkx2.2 is downstream of Olig2, Sox10 (Liu et al. 2007), and
Mash1 (Sugimori et al. 2008). In return, Nkx2.2 positively
influences both Olig2 and Sox10 expression (Liu et al. 2007).
At least in case of Sox10, this effect may be direct (Küspert
et al. 2011).
pOL mOL
Nkx2.2
Maxh1 myelin
genes
Zfp488
MRF
 2.4.4 Terminal Differentiation and Myelination
in Oligodendrocytes
Both Nkx2.2 and Mash1 are transiently expressed and dis-
appear during later phases of the myelination process (see
Fig. 43.1). Their prodifferentiation function must therefore
be restricted to the initial phase of myelination. This is differ-
ent for the myogenic regulatory factor (MRF) transcription
factor (see Fig. 43.1), which appears in prooligodendrocytes
shortly before the onset of myelin gene expression, but con-
tinues to be expressed from then on (Emery et al. 2009).
Prooligodendrocytes undergo apoptosis in the absence of
MRF, thus arguing that MRF is required for survival at
this stage. Expression profiling furthermore showed that
there is no myelin gene expression in MRF-deficient oli-
godendrocytes. Its function may therefore be analogous
to the role of Krox20 in myelinating Schwann cells (see
Figs. 43.6 and 43.7). Similar to Krox20 in the PNS, MRF
seems to activate CNS myelin gene expression in synergy with
Sox10 (Emery et al. 2009). Considering that Sox10 regulates
expression of many myelin genes in the CNS (Bondurand et al.
2001; Schlierf et al. 2006; Stolt et al. 2002), that Olig proteins
do the same, and that Sox10 not only cooperates with MRF,
but also with Olig proteins (Li et al. 2007; Wissmüller et al.
2006), MRF, Sox10, and Olig proteins seem to jointly acti-
vate and maintain the myelination program (see chapter 13).
It also deserves mentioning that Olig1-deficient mice exhibit
severe myelination defects throughout the CNS. Specification
defects in the same mice were minimal. This argues that the
function of Olig1—different from Olig2—is much more
prominent during terminal differentiation and myelination
than during oligodendroglial specification (Arnett et al. 2004;
Lu et al. 2002; Xin et al. 2005; Zhou and Anderson 2002).
In their activation of the myelination program MRF, Sox10
and Olig proteins receive support from additional transcription
factors. An example is the zinc finger protein Zfp488 (Wang
et al. 2006). In the CNS, Zfp488 is selectively induced in the oli-
godendrocyte lineage at the same time as the major myelin genes
(see Fig. 43.1). Its expression depends on the Olig proteins with
which it then interacts to maintain high levels of myelin gene
expression in differentiating oligodendrocytes (see Figs. 43.6
and 43.7). A similar effect on myelin gene expression and the
myelination program has also been observed for the SCAN zinc
finger transcription factor Zfp191 (Howng et al. 2010). In con-
trast to Zfp488, it is also present in other CNS cell types and ear-
lier stages of oligodendrocyte development so that its functions
are likely not restricted to differentiating oligodendrocytes.
As mentioned for Schwann cells, gene expression in oli-
godendrocytes has to be adjusted once cells transit from the
active state of myelination into the steady state of myelin main-
tenance. This again requires changes of the gene regulatory
network. In mature oligodendrocytes the majority of Olig1 is,
for instance, removed from the nucleus (Arnett et al. 2004).
Additionally, Nkx2.2 and Nkx6.2 become reexpressed (Cai
et al. 2010). Analysis of Nkx6.2-deficient mice reveals subtle
myelin abnormalities and altered expression of paranodal pro-
teins, arguing that regulation of paranodal axon–glia interac-
tions may be part of its function (Southwood et al. 2004).
T R A N S C R I P T I O N FAC TO R S I N M Y E L I N AT I N G C E L L S
The role of Nkx2.2 is currently unclear, but likely different
from its function in the initiation phase of myelination.
3 S U M M A RY A N D P E R S P E C T I VE S
Recent years have seen the identification and characteriza-
tion of many transcription factors with regulatory functions
in myelinating glia. It has become increasingly clear that com-
mon principles govern the development of Schwann cells and
oligodendrocytes. However, the emerging picture also suggests
that similar concepts have been implemented differently in the
two cell types with few of the major players being conserved
at their position between the two gene regulatory networks.
This includes Oct6 and Krox20 as much as MRF and the Olig
proteins. This may be attributed to the fact that Schwann cells
and oligodendrocytes are of different developmental origins
and likely acquired their capacity to myelinate independently.
Myelination of the CNS and PNS clearly represent convergent
processes. On this background, the central comparable role of
Sox10 in both gene regulatory networks is striking and identi-
fies Sox10 as a key factor and cornerstone in myelination.
The concept of gene regulatory networks has opened up
new perspectives on the processes of transcriptional regula-
tion in myelinating glia. Interactions between participating
transcription factors allow for cross-regulation, cooperativ-
ity, and antagonism and explain how specificity is generated
and how transcription factors can acquire new functions or
loose previous ones depending on the context of the network.
It is probably fair to assume that most of the key transcrip-
tional regulators in myelinating glia have been identified by
now. Thus, it will be the next challenge to understand how
they interact and influence each other, and how this will be
modulated by posttranslational modifications. Several critical
circuits between key factors have already been identified in the
gene regulatory networks of Schwann cells and oligodendro-
cytes. Nevertheless, it is still a long way to a complete picture.
The more pieces are added to the puzzle, the better will be
the understanding of the myelination process and of myelin
diseases, including those that are caused by mutations in glial
transcription factors such as Krox20 and Sox10 (Inoue et al.
2004; Warner et al. 1998) (see also chapter 62). It will also be
easier to selectively and systematically manipulate the system
for treatment of myelin diseases and traumatic injuries.
Finally there is no doubt that for a full understanding of
these gene regulatory networks it will be necessary to integrate
the action of small RNAs, in particular microRNAs, and of
chromatin modifiying and remodeling machineries. Although
the few existing studies prove the relevance of these factors
in myelinating glia, the current picture is at best patchy with
many areas waiting to be filled in.
AC K N OW L E D G M E N T S
Work in the author’s laboratory on the subject is sup-
ported by grants from the Fonds der Chemischen Industrie,
ForNeuroCell and the Deutsche Forschungsgemeinschaft.
• 551
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 44.
FACTOR S CONTROLLING MYELIN FORMATION
Ruth Stassart, Sandra Goebbels, and Klaus-Armin Nave

A B B R E VI AT I O N S MAPK mitogen-activated protein kinase

MBP myelin basic protein
ADAM A disintegrin and metalloprotease MTMR2 myotubularin-related protein 2
AKT V-Akt murine thymoma viral oncogene mTOR mammalian target of rapamycin
homolog 1 NCAM neural cell adhesion molecule
ATP adenosine triphosphate NECL nectin-like protein
BACE1 E-site amyloid precursor protein cleaving NFAT nuclear factor of activated T cells
enzyme 1 NGF nerve growth factor
BDNF brain-derived neurotrophic factor NgR1 nogo receptor 1
cAMP cyclic adenosine monophosphate NICD notch intracellular domain
Cdc42 cell division control protein 42 homolog NMDA N-methyl-d-aspartate
CnB1 calcineurin NRD1 N-arginine dibasic convertase/Nardilysin
CNS central nervous system NRG1 neuregulin-1
CNTF ciliary neurotrophic factor NT3/NT4/5 neurotrophin-3/4/5
CRB crumbs complex N-WASP neuronal Wiskott–Aldrich syndrome
CREB cAMP response element-binding protein
Dcc deleted in colorectal carcinoma Oct6 octamer transcription factor 6
Dlg1 disk-large homolog 1 Olig1/Olig2 oligodendrocyte transcription factor 1/2
DRG dorsal root ganglia OPC oligodendrocyte precursor cell
EAE experimental autoimmune encephalomyelitis p75NTR P75 neurotrophin receptor
EGF-like domain epidermal growth factor–like domain Pals1 protein associated with lin seven 1
EGR-2 early growth response protein 2 Par complex partitioning-defective protein polarity
ErbB erythroblastic leukemia viral oncogene complex
homolog PDGF platelet-derived growth factor
ERK extracellular signal-regulated kinase PH domain pleckstrin homology domain
FGF fibroblast growth factor PI3K phosphatidylinositol 3-kinase
FGFR FGF receptor tyrosine kinase PIP2 phosphatidylinositol (4,5)-bishosphate
GDNF glial cell line derived neurotrophic factor PIP3 phosphatidylinositol (3,4,5)-trisphosphate
GH growth hormone PLC phospholipase C
GPCR/GPR G protein–coupled receptor PLP proteolipid protein
GPI glycosylphosphatidylinositol Pmp22 peripheral myelin protein of 22kDa
HDAC histone deacetylase PNS peripheral nervous system
hnRNP heterogeneous nuclear ribonucleoprotein PrPc prion protein cellular
ID2/ID4 inhibitor of DNA-binding protein PSA-NCAM polysialylated form of the neural cell
L1 cell adhesion molecule L1 adhesion molecule
Ig immunoglobulin PTEN phosphatase and tensin homolog
IGF-1 insulin-like growth factor 1 P2Y purinergic receptors, G protein–coupled
IGFR1 IGF receptor tyrosine kinase receptor
ILK integrin linked kinase Rac1 Ras-related C3 botulinum toxin substrate 1
Kif13b kinesin family member 13B Rho Ras homolog gene family
Lgi4 leucine-rich repeat LGI family, member 4 SCRIB scribble complex
LIF leukemia inhibitory factor SHP2/PTPN11 protein tyrosine phosphatase, non-receptor
Lingo1 leucine rich repeat and Ig domain type 11
containing NOGOreceptor interacting SREBP sterol responsive element binding proteins
protein TACE tumor necrosis factor-D–converting
MAG myelin-associated glycoprotein enzyme (ADAM17)

555
TCF T-cell factor/lymphoid enhancer factor or 2009), and hormones (Calzà et al. 2010; Schumacher et al.
TCF/LEF 2012).
TGF-E transforming growth factor beta
TGN trans Golgi network
TrkA/B/C tropomyosin-related kinase receptors A/B/C 2 F O R M AT I O N O F T H E
AXO N –M Y E L I N U N I T

1 INTRODUCTION Myelination in the peripheral nervous system (PNS) is in

The last steps in the cellular evolution of the nervous system
included the invention of myelin, some 300 million years
ago in ancestors of lower vertebrates. This was a prerequi-
site for the subsequent evolution of complex yet compact,
quickly operating, and thus powerful nervous systems. The
importance of myelinated fibers becomes obvious in the con-
text of severe neurological diseases, in which the formation
or maintenance of myelin is perturbed (see chapters 54, 61,
62, and 71).
This chapter gives a brief overview of the factors that
have been identified as regulators of myelin formation,
mostly non–cell autonomous protein ligands and their glial
receptors (Table 44.1), and intracellular second messengers.
Other systemic factors that influence myelin biogenesis (and
remyelination) cannot be adequately covered here, and the
reader is referred to articles on the role of nutrition and vita-
mins (Steinfeld et al. 2009), trace elements (Todorich et al.
many ways simpler than in the central nervous system (CNS),
although there is no evidence that it originated here in ner-
vous system evolution. As detailed in chapter 14, Schwann
cells that are derived from neural crest are associated with
axons throughout PNS development. However, before myeli-
nation, myelinating Schwann cells sort out a single axon into
a stable one-to-one relationship. Enwrapping this axon with
a myelin membrane establishes the axon–myelin unit that
then grows in size, becoming up to 1 mm or more in inter-
nodal length (Hildebrand et al. 1994). In contrast, myelinat-
ing oligodendrocytes of the CNS develop from multipolar
oligodendrocyte progenitor cells (OPC). These precursors
have no such tight association with axonal membranes before
myelination. In contrast, oligodendrocytes interact with mul-
tiple axons that they individually ensheath. Internodal length
in the CNS is significantly shorter but measures up to sev-
eral hundred micrometers (Remahl and Hildebrand 1990).
A combination of imaging and recording techniques led to

Table 44.1 NON–CELL AUTONOMOUS FACTORS CONTROLLING MYELINATION

SIGNAL, LIGAND,
AND/OR REFERENCES AND FURTHER
RECEPTOR PNS CNS READING

Direct Axonal electrical Inhibits Schwann cell Promotes differentiation and myeli- Barres et al. 1993
axon–glia activity proliferation/differentia- nation in vitro Demerens et al. 1996
signaling tion in vitro Stevens et al. 2002
Wake et al. 2011
Fields 2008 (Rev)

NRG1 typeIII Promotes Schwann cell Dispensable for Michailov et al. 2004
ErbB2/3/4 survival, differentiation, myelination in vivo Taveggia et al. 2005,
myelin growth control Brinkmann et al. 2008
Taveggia et al. 2010 (Rev)

Nectin-like Required for Schwann Contribute to the initiation of Maurel et al. 2007
proteins cell myelination in vitro myelination Spiegel et al. 2007
Park et al. 2008
Pereira et al. 2012 (Rev)

Prion protein Required for myelin Dispensable in vivo Bremer et al. 2010
(axonal) maintenance Pereira et al. 2012 (Rev)

Lingo1 Inhibits oligodendrocyte differentia- Mi et al. 2004, 2005

tion and myelination Lee et al. 2007
Kremer et al. 2011 (Rev)

Jagged Inhibits myelin Inhibits oligodendrocyte differentia- Givogri et al. 2002

Notch1 formation tion and myelination Woodhoo et al. 2009
Taveggia et al. 2010 (Rev)
Piaton G et al. 2010 (Rev)
PSA-NCAM Inhibits myelination Charles et al. 2000
in vitro Fewou et al. 2007
Piaton G et al. 2010 (Rev)

(Continued)

556 • NEUROGLIA
Table 44.1 (Cont’d)
SIGNAL, LIGAND,
AND/OR REFERENCES AND FURTHER
RECEPTOR PNS CNS READING

Orphan receptors GPR126 Required for Schwann Dispensable for Monk et al. 2009, 2011
cell differentiation and myelination in vivo Pereira et al. 2012 (Rev)
myelination Taveggia et al. 2010 (Rev)

GPR17 Not expressed Inhibits oligodendrocyte Chen et al. 2009

differentiation Taveggia et al. 2010 (Rev)
Neurotrophins NGF Promotes myelination Inhibits myelin formation Chan et al. 2004
in vitro in vitro Lee et al. 2007
Rosenberg et al. 2006 (Rev)
Xiao et al. 2009 (Rev)
BDNF Promotes myelination via Promotes myelination, Xiao et al. 2010
p75NTR, inhibits it via but controversial reports Vondran et al. 2010
TrkB receptors Rauskolb et al. 2010
Taveggia et al. 2010 (Rev)
NT-3 Inhibits myelination Cosgaya et al. 2002
in vitro; controversial Xiao et al. 2009 (Rev)
in vivo data
Other IGF-1 Promotes myelination Important for oligodendrocyte Carson et al. 1993
extracellular differentiation/myelination in Ye et al. 1995, 2002
factors development Russel et al. 2000
Zeger et al. 2007
Taveggia et al. 2010 (Rev)
FGF Promotes myelin formation in Bansal et al. 2002
vivo; FGF2 inhibits differentiation Fortin et al. 2005
in vitro Mason et al. 2007
Furusho et al. 2012
Wnt Promotes myelination Inhibits oligodendrocyte Fancy et al. 2009
differentiation Feigenson et al. 2009
Promotes myelination Ye et al. 2009
Tawk et al. 2011
Rosenberg and Chan 2009 (Rev)
GDNF Positive role for myelina- Höke et al. 2003
tion suggested Xiao et al. 2009
Extracellular Laminin Required for Schwann Important for oligodendrocyte Previtali et al. 2003
matrix Dystroglycan cell differentiation, basal differentiation Benninger et al. 2006
Integrin lamina production, Barros et al. 2009
myelination Camara et al. 2009
Colognato and Tzvetanova 2011
(Rev)
Hormones Progesterone Promotes myelination Promotes oligodendrocyte Schumacher et al. 2012 (Rev)
differentiation
Thyroid hormone Promotes oligodendrocyte Calza et al. 2010 (Rev)
differentiation/myelination Taveggia et al. 2010 (Rev)

Compilation of signaling molecules that either promote or inhibit myelination in the peripheral nervous system and central nervous system, or are dispensable based on
genetic experiments.
For details see references (right, reviews indicated as ‘rev’) and text.

the discovery that in the developing corpus callosum many
OPC receive a transient synaptic input from unmyelinated
axons before myelination (Ziskin et al. 2007). Whether these
glutamatergic synapses induce (or alternatively prevent)
subsequent maturation of OPC and myelin assembly is not
known.
Glial cell processes and their membranes that interact with
myelination-competent axons in trans become polarized, which
is required for the initial ensheathment and determines the site of
membrane growth (Bauer and ffrench-Constant 2009; Simons
and Trotter 2007). The assembly of large quantities of myelin
components occurs in the secretory path and is regulated within

FAC TO R S C O N T R O L L I N G M Y E L I N F O R M AT I O N • 557
distinct subcellular compartments. It involves both high-level
lipid biosynthesis and translation and proper trafficking of
myelin proteins. The generation of myelin membranes further
requires a tight coordination of gene expression and membrane
growth control (Emery 2010a; Nave 2010) to maintain the
unique stoichiometry of myelin proteins and lipids (<30% pro-
teins and >70% lipids, with about 50% cholesterol). Even when
developmental myelination has come to a halt, maintaining the
fine balance of continued exocytosis and endocytosis of myelin
membranes seems essential. Minor deviations of the steady state
can theoretically lead to significant demyelination over time.
3 AXO N – G L I A I N T E R AC T I O N I N
THE PERIPHERAL AND CENTRAL
N E RVO U S SYS T E M
Historically, the demonstration that glial cell membranes were
the source of myelin (Bunge 1968; Geren and Raskind 1953)
was a starting point for the investigation of interactions between
axons and their ensheathing glial cells. Studies involving the
addition of purified neurite membrane fractions to cultured
Schwann cells were the first to demonstrate that axonal sig-
nals stimulate Schwann cells to survive and proliferate (Salzer
et al. 1980a,b; Wood and Bunge 1975). In turn, in the absence
of axonal signals Schwann cells are unable to differentiate into
myelinating cells (Mirsky et al. 1980). Finally, determining the
axon caliber of cultured dorsal root ganglia (DRG) neurons
revealed that the addition of Schwann cells triggers myelina-
tion, but also an increase of the axon diameter (Windebank
et al. 1985), establishing bidirectional axon–glial signaling.
In the PNS only axons greater than a diameter of about 1
μm are myelinated, and the axonal caliber maintains a rather
constant ratio to the myelin sheath thickness (Friede and
Bischhausen 1982), also quantified as the g-ratio (Fig. 44.1A).
Experimental support for the idea that axons regulate myeli-
nation came from nerve graft experiments. Schwann cells
originating from unmyelinated fibers can differentiate into
myelinating Schwann cells once transplanted into a myeli-
nated nerve with larger axons (Aguayo et al. 1976). Thus,
Schwann cells change their myelination fate depending on
axonal signals. Axon caliber itself poses a crucial variable as
demonstrated by Voyvodic (1989), who found that normally
unmyelinated sympathetic fibers become (de novo) myeli-
nated by resident Remak cells following an increase of the
axon caliber (experimentally induced here by a nerve hemisec-
tion, which increased the size of the neurotrophic target tis-
sue, resulting in radial axonal growth) (Voyvodic 1989).
This response of Schwann cells to the diameter of the axon
is compatible with a model of an axon-resident myelination sig-
nal that is integrated by Schwann cells as a measure of axon size,
and that controls myelination quantitatively once a threshold
level has been reached. In the PNS, this signal is provided by
the axonal growth factor Neuregulin 1 (Michailov et al. 2004;
Taveggia et al. 2005). Myelination in the CNS is less well
understood, although the same axons are often myelinated by
Schwann cells and oligodendrocytes as they course from the
PNS into the CNS. Unlike Schwann cells, oligodendrocytes
558 • NEUROGLIA
can differentiate into myelinating cells in culture without
direct contact with neurons or axons (Dubois-Dalcq et al.
1986; Mirsky et al. 1980). Indeed, oligodendrocytes are capa-
ble of ensheathing paraformaldehyde-fixed axons or synthetic
fibers in vitro, indicating that only limited bidirectional sig-
naling is required (Lee et al. 2012; Rosenberg et al. 2008). On
the other hand, oligodendrocytes only target axonal processes,
never dendrites (Lubetzki et al. 1993), and synthesize myelin
appropriate to axon calibre (although g-ratios in the CNS
vary considerably more than in the PNS). This suggests that
oligodendrocytes also myelinate in response to axonal signals.
However, these signals are not necessarily “instructive,” and an
alternative hypothesis holds that oligodendrocyte differentia-
tion and myelin membrane synthesis is ultimately by default,
determined by an intrinsic transcriptional network (see chap-
ter 43) and guided by inhibitory extracellular factors. Indeed,
potent inhibitory axonally expressed signals, including ligands
such as Lingo-1, Jagged, and PSA-NCAM, have been impli-
cated in either inhibiting OPC differentiation or preventing
myelination by oligodendrocytes (Kremer et al. 2011; Piaton
et al. 2010).
Growth factors and cytokines (e.g., PDGF, FGF-2, IGF-1,
NT3, CNTF, BMP and LIF) have been studied in detail as
potent regulators of OPC proliferation and oligodendrocyte
differentiation however, the source of these factors is not nec-
essarily axonal (see chapters 6 and 13). The specific require-
ment of axons for oligodendrocyte survival was indicated by
lesion studies, in which optic nerve transections reduced the
number of oligodendrocytes (Barres et al. 1993). By contrast,
optic nerves of Bcl2-transgenic mice, in which the number of
ganglion cell axons was increased, harbored correspondingly
more oligodendrocytes (Burne et al. 1996). In other experi-
ments, it was the axonal electrical activity that promoted
OPC proliferation and supported myelination (Barres and
Raff 1993; Demerens et al. 1996), possibly as a response to the
axonal release of adenosine (Stevens et al. 2002). However,
unlike in the PNS, no specific axonally presented signaling
molecule has been identified to be essential for CNS myelina-
tion, as is axonal Neuregulin-1 for the myelination of periph-
eral nerves.
4 N E U R E GU L I N 1/ E R B B R E C E P TO R
S I G N A L I N G I N P E R I P H E R A L N E RVO U S
SYS T E M M Y E L I N AT I O N
Neuregulin-1 (NRG1) comprises a family of growth factors,
encoded by one of the largest mammalian genes (~1.6 Mb).
Expressed in many cell types, more than 16 NRG1 isoforms are
generated by different promoter usage and alternative RNA
splicing (Nave and Salzer 2006). By their different N-termini,
neuregulins are currently subdivided into six subtypes (Nave
and Salzer 2006), with NRG1 types I, II, and III most widely
studied. All isoforms are synthesized as transmembrane pro-
teins and further processed by proteases; however, NRG1 type
III (with a C-terminal cystein-rich domain) is anchored twice
in the membrane and therefore not shed from the membrane
by a single processing step (Fig. 44.1B).
 A a NRG1 type III +/– wildtype NRG1 type III tg
g-ratio =
b

a Axonal NRG1
b

NRG1 type III +/- NRG1 +/+ NRG1 type III tg

(viable) (wildtype) (viable)

Figure 44.1 Neuregulin-1 Is an Axonal Growth Factor That Regulates Myelination by Schwann Cells. A. Myelin sheath thickness is roughly propor-
tional to the axon diameter, a relationship quantified as the “g-ratio” between the axonal caliber (a) and the total fiber diameter (b), as illustrated on
the electron micrograph of a myelinated axon (left). Schwann cells “measure” the axon caliber by integrating NRG1 type III signaling at the axonal
surface as a surrogate marker, so as to wrap the corresponding numbers of myelin membranes and reach the normal g-ratio of about 0.67 (in the
PNS). Neuregulin-1 type III–dependent control of myelination can be demonstrated genetically (Michailov et al. 2004; Taveggia et al. 2005) with
heterozygous Nrg1 mouse mutants, in which axons expressing 50% NRG1 are thinly myelinated (left) compared with wild-type (middle). In contrast,
transgenic mice overexpressing NRG1 type III in projection neurons exhibit hypermyelinated axons caused by elevated NRG1 density (right).
B. Activation of axonal NRG1 type III by proteolytic cleavage. The major NRG1 isoforms (depicted are types I, II, and III) differ at their N-terminus
as a result of alternative transcription and RNA splicing, but share the same EGF-like signaling domain necessary for ErbB receptor activation. NRG1
type III is anchored by a second membrane spanning region, the cysteine rich domain (CRD). Proteolytic processing of NRG1 by the protease
BACE1 activates this growth factor, but only NRG1 types I and II are also released as paracrine signaling molecules. BACE1 cleavage of type III cre-
ates a paracrine signal that promotes myelination (Hu et al. 2006; Velanac et al. 2012; Willem et al. 2006). In contrast, the protease TACE/ADAM17
cleaves NRG1 in the EGF-like domain, thereby inhibiting NRG1 signaling (La Marca et al. 2011). The conserved intracellular domain of NRG1 is
processed by gamma-secretases that thereby create a “retrograde” signaling molecule within neurons. The temporal order of these cleavage steps and
their intracellular localization are not known. (A) Adapted from ffrench-Constant et al. 2004. (B) Adapted from Nave and Salzer 2006.

All NRG1 isoforms share an epidermal growth factor
(EGF)–like domain, which is required and sufficient to acti-
vate erythroblastic leukemia viral oncogene homolog (ErbB)
receptor tyrosine kinases. Binding of axonal NRG1 to glial
ErbB proteins induces receptor homodimerization and
heterodimerization, with autophosphorylation of tyrosine
residues for the recruitment of adaptor proteins and the acti-
vation of various downstream kinases. For NRG1/ErbB sig-
naling in Schwann cells, the PI3K/AKT pathway, the Ras/
MAPK/ERK pathway, and the NFAT/Calcineurin signaling

FAC TO R S C O N T R O L L I N G M Y E L I N F O R M AT I O N • 559
pathway are most important (Newbern and Birchmeier 2010).
Because ErbB3 lacks a potent kinase domain and ErbB2 lacks
receptor functions, NRG1 signaling to Schwann cells requires
ErbB2/3 heterodimerization. In oligodendrocytes also ErbB4
is abundantly expressed.
Because NRG1 is involved in heart development, mouse
mutants completely lacking NRG1 die embryonically. To
genetically analyze this growth factor in nervous system devel-
opment, NRG1 isoform-specific conditional mouse mutants
and transgenic mice were instrumental (Birchmeier and
Nave 2008). In the PNS, axonal NRG1 type III emerged as
a key regulator of virtually all steps of Schwann cell develop-
ment and myelination (Birchmeier and Nave 2008; Newbern
and Birchmeier 2010) (see also chapter 14). At early devel-
opmental stages, NRG1 promotes Schwann cell precursor
proliferation and migration, and ablation of NRG1/ErbB3
signaling results in the near complete absence of Schwann
cell progenitors in the nerve. Later, NRG1 switches from a
pro-proliferation to a pro-differentiation signal (Birchmeier
and Nave 2008), which is at least partially mediated by the ele-
vation of cAMP in Schwann cells (Arthur-Farraj et al. 2011).
In general, NRG1 modulates the Schwann cell phenotype in
a dose-dependent fashion, at least in vitro. Low concentra-
tions promote myelin formation, whereas high concentra-
tions inhibit differentiation, possibly caused by an altered
balance between the activated second messenger pathways
(Syed et al. 2010).
NRG1 serves as an instructive signal for myelination and
provides Schwann cells with the information about axons
that have reached radial size (~1 μm) to be myelination
competent. This information is encoded as a threshold level
of NRG1 type III on the axonal surface. Indeed, overex-
pression of NRG1 type III in normally unmyelinated fibers
results in their de novo myelination in vitro (Taveggia et al.
2005) and in vivo (Schwab and Nave in preparation). First
evidence that NRG1/ErbB signaling regulates myelination
came from hypomyelinated mouse mutants lacking ErbB2
selectively from EGR2-positive Schwann cells (Garratt
et al. 2000). Direct proof that axonal NRG1 type III reg-
ulates myelin sheath thickness involved mouse mutants
with different NRG1 gene dosages (Michailov et al. 2004).
Heterozygous mice exhibit a reduced content and presum-
ably density of NRG1 in PNS axons, which are only thinly
myelinated (see Fig. 44.1A). In contrast, transgenic mice
that overexpress NRG1 type III in neurons exhibit a hyper-
myelination phenotype (see Fig. 44.1A) (Michailov et al.
2004; Taveggia et al. 2005). This increase in myelin sheath
thickness is specific for NRG1 type III, because no periph-
eral hypermyelination is induced by neuronal overexpres-
sion of NRG1 type I (Michailov et al. 2004). Interestingly,
mice heterozygous for the ErbB2 gene are fully myelinated,
demonstrating that only the ligand, not the receptor, is
rate-limiting for myelination (Michailov et al. 2004). Once
myelination has been completed, continued NRG1/ErbB
signaling is mostly dispensable for myelin maintenance
in adult life (Atanasoski et al. 2006), and would only be
required for remyelination, for example after peripheral
nerve injury (Fricker et al. 2011).
560 • NEUROGLIA
The role of NRG1 in the CNS is more complex and pos-
sibly more relevant for neuron-to-neuron signaling. Although
some experiments suggested that NRG1/ErbB signaling stim-
ulates the proliferation of OPC and oligodendrocyte survival,
and possibly myelination, the detailed analysis of mice lack-
ing NRG1 from cortical neurons, or both ErbB3 and ErbB4
receptors from oligodendrocytes, revealed normal oligoden-
drocyte numbers and intact myelination of subcortical white
matter tracts (Brinkmann et al. 2008). In contrast, trans-
genic overexpression of NRG1 type III (as well as NRG1
type I) in cortical neurons induces some hypermyelination
(Brinkmann et al. 2008). Thus, oligodendrocytes are respon-
sive to NRG1, but NRG1/ErbB signaling is not required for
CNS myelination.
5 P O L A R I Z AT I O N A S A P R E R E Q U I S I T E
F O R M Y E L I N AT I O N
NRG1/ErbB signaling activates glial PI3K, and elevated
phosphatidylinositol (3,4,5)-trisphosphate (PIP3) has been
recognized as a key regulator of cellular polarity in other sys-
tems. This strongly suggests that altered glial cell polarity is
an important step in initiating axonal ensheathment, which is
supported by morphological data. Myelin sheaths assembled by
oligodendrocytes and Schwann cells are polarized, longitudi-
nally from node to node, and radially from the inner adaxonal
membrane (and compact myelin) to the outer abaxonal mem-
brane. In analogy to polarized transport in epithelial cells, the
biogenesis of myelin requires correct spatiotemporal sorting
of cargo proteins and the recruitment of proteins, lipids, and
mRNA to subcellular domains, which requires a tightly regu-
lated intracellular trafficking machinery (Simons and Trotter
2007). For example, major myelin lipids and structural pro-
teins are delivered in vesicules from the trans Golgi network
(TGN) to the plasma membrane, and are later internalized by
cholesterol-dependent and clathrin-independent endocyto-
sis, transported to a late endosome/lysosomal compartment
(Trajkovic et al. 2006) or directly targeted to the growing
myelin compartment. This balance of endocytosis and exocy-
tosis at the cell membrane is thought to regulate myelin bio-
genesis (Simons and Trotter 2007).
The key principles of establishing the basolateral-apical
polarity in epithelial cells are also found in myelinating glial
cells (Baron and Hoekstra 2010; Pereira et al. 2012). In epi-
thelial cells, apical and basolateral membrane domains exhibit
different compositions of membrane proteins and lipids. The
major regulatory protein complexes are the crumbs (CRB)
complex localizing to the apical domain, the scribble (SCRIB)
complex defining the basolateral membrane domain, and the
partitioning-defective protein (Par) polarity complex that pro-
motes the formation of the apical-basolateal membrane bor-
der (Martin-Belmonte and Perez-Moreno 2012; Mellman and
Nelson 2008). Also, the signaling lipids phosphatidylinositol
(4,5)-bishosphate (PIP2) and PIP3 are asymmetrically distrib-
uted, with PIP3 (and its synthesizing enzyme PI3Kinase) being
enriched in the basolateral domain and PIP2 (and the lipid
phosphatase and tensin homolog [PTEN]) predominantly
found in the apical domain (Mellman and Nelson 2008).
How does this cellular architecture translate to myelinating
glial cells?
In peripheral myelin, the epithelial basolateral markers
are enriched in the abaxonal domain, whereas apical mark-
ers define the adaxonal domain and Schmidt-Lanterman
incisures. These observations suggest that the control of
Schwann cell polarization is similar to that of epithelial
cells (Pereira et al. 2012). Indeed, the polarity protein Par3,
by directly interacting with p75NTR , is required for the
polarization of Schwann cells before myelination (Chan
et al. 2006). Moreover, interfering with the uneven distri-
bution of PIP2 and PIP3 in Schwann cells induces strik-
ing nerve pathology, with myelin outfoldings and focal
hypermyelination in mutant mice (Goebbels et al. 2012).
Apical-basolateral polarity of epithelial cells contributes to
directional transport. Correspondingly, the polarity pro-
teins Dlg1 (in concert with MTMR2, Sec8, and the kinesin
Kif13b) and Pals1 (required for the polarized distribution
of Sec8 and syntaxin4) affect myelin formation by regulat-
ing polarized vesicle trafficking and thus myelin membrane
formation (Pereira et al. 2012).
Also, basal lamina components, such as laminins, are
required for normal Schwann cell polarization (Feltri and
Wrabetz 2005) (see section 9). Schwann cells express various
types of laminin receptors exerting distinct roles (Previtali
et al. 2003). Integrin signaling often converges onto the regu-
lation of small Rho GTPases, such as Rac1, Rho, and Cdc42.
Indeed, Cdc42 and Rac1 are crucial for early stages of myelin
formation in the CNS (Thurnherr et al. 2006). A critical role
for Rac-dependent actin regulation has also been suggested
for Schwann cells during axonal sorting and myelination (Park
and Feltri 2011). Recently it was further demonstrated that
another actin regulator, neuronal Wiskott–Aldrich syndrome
protein (N-WASP), a downstream effector of RhoGTPases
including Rac1, is similarly required for axonal sorting and
myelination (Park and Feltri 2011). Together, these findings
strongly indicate that extracellular signals affecting the actin
cytoskeleton are required for myelination.
6 D I R E C T AXO N – G L I A I N T E R AC T I O N S
T H AT P R O M OT E M Y E L I N AT I O N A N D
MYELIN M AINTENANCE
Compared with Schwann cells, oligodendrocyte lineage
cells respond to a much larger number of stimulatory signals
(Taveggia et al. 2010), many of which also affect myelination,
but they are not as well studied in vivo. Moreover, the cellular
source of soluble factors in the CNS is not always clear and
can include astrocytes. Some of the myelin structural pro-
teins are not essential for myelin assembly, but for maintain-
ing long-term function and stability of the axon–myelin unit
(e.g., PLP/DM20 or MAG, Fig. 44.2), which is discussed in
more detail in chapter 62. Only few myelination-promoting
factors are clearly axonal in nature, and these have been
mostly studied in the PNS (as detailed for NRG1 above (see
section 4)).
FAC TO R S C O N T R O L L I N G M Y E L I N F O R M AT I O N
N EC T I N-L I K E P ROT E I NS
Nectin-like proteins (NECL1–NECL4) comprise a family of
immunoglobulin-like adherens junction proteins, localized at
both sides of the internodal axon–glia interface, which con-
tribute to Schwann cell–axon adhesion during myelin forma-
tion (Maurel et al. 2007; Spiegel et al. 2007) (see Fig. 44.2).
They predominantly undergo heterophilic binding, for exam-
ple, NECL1 (on axons) with NECL4 (on Schwann cells). In
myelinating DRG cocultures, NECL4 expression in Schwann
cells increases upon axonal contact, and is required for normal
Schwann cell differentiation and in vitro myelination (Maurel
et al. 2007; Spiegel et al. 2007). However, NECL function is
mechanistically distinct from that of signaling proteins. When
axonal NECL1 was ablated in mice, mutants were still nor-
mally myelinated in sciatic nerves, and only a delayed myeli-
nation of optic nerve and spinal cord was noted (Park et al.
2008). These discrepancies of in vitro and in vivo findings are
not well explained, but point to functional redundancy of
NECL isoforms, or differences between acute and chronic
perturbation experiments.
AXO NA L P R I O N P ROT E I N
The widely expressed prion protein, a membrane-anchored
protein known for its detrimental role in transmissible
Creutzfeldt-Jakob disease, plays an unexpected role in the long-
term maintenance of peripheral myelin (see Fig. 44.2). Mice
lacking prion protein cellular (PrPc) develop an adult-onset
demyelinating neuropathy, predominantly affecting large-cal-
iber fibers. Surprisingly, it is the transgenic ablation of axonal
(not glial) PrPc that can prevent that demyelinating pheno-
type in PrPc mutant mice (Bremer et al. 2010). The underlying
molecular mechanisms are not understood, and CNS myelin
appears unaffected by the absence of axonal PrPc.
7 AXO N A L E L E C T R I C A L AC T I VI T Y
That myelination is influenced by action potentials of the
axon was first suggested in the 1960s, when it was noted that
optic nerve myelination is decreased in animals raised in the
dark (Gyllensten and Malmfors 1963) (see also chapter 45).
With the help of neurotoxins, electrical activity was shown
to promote oligodendrocyte survival (Barres and Raff 1993)
and enhance myelination (Demerens et al. 1996). In CNS
preparations, the release of adenosine, adenosine triphos-
phate, and glutamate by electrically active axons promotes
oligodendrocyte differentiation (Stevens et al. 2002; Wake
et al. 2011). Furthermore, in vitro, Adenosine inhibits OPC
proliferation via adenosine receptors (Stevens et al. 2002;
Wake et al. 2011). Glutamate, released from vesicular sources
along the axonal membrane, was shown to induce myelina-
tion in DRG neuron/oligodendrocyte cocultures, mediated
by N-methyl-d-aspartate (NMDA) receptors and metabotro-
pic glutamate receptors (Wake et al. 2011). Here, glutamate
induces the phosphorylation of Fyn, leading to the local trans-
lation of transcripts for myelin basic protein (MBP), that is,
• 561
 Axon NRG1 type III
COOH

TACE/ NH2 Jagged

NECL1 ADAM22 ADAM19 ADAM17 Bace1

EGF
PrPc
Lgi4

EGF
? ATP

PIP3 PIP2 P P MAG

GPR 126 P2Y
P P p75NTR
NECL4 PI3K Myelin
PTEN P P maintenace PAR3
DIg1 Notch
ErbB2 - ErbB3
DIg1
Initiation of
P P myelination/
cAMP ? ? AKT PLCγ MEK SHP2
polarity

?
Ca2+ Calcineurin ?

SREBPs
P P P
mTOR NFATc4 NFATc4 Erk1/2 (+ cAMP)

Lipid/ P
Choresterol NFATc4 SOX10 YY1
biosynthesis

EGR2

Schwann cell MYELINATION

Figure 44.2 Multiple Factors Controlling Peripheral Nervous System Myelin Formation. In the peripheral nervous system, myelination by Schwann
cells depends on instructive cues from the underlying axon. The schema lists different ligand–receptor systems of axon–glia interaction and their
downstream signaling cascades that have been implicated in peripheral myelination. Question marks denote interactions that are still poorly under-
stood. Black lines mark positive myelination-promoting effects, orange lines inhibitory effects. At present, the best-studied axonal growth factor is
NRG1 type III. After proteolytic activation by BACE1 and other secretases, this NRG isoform remains membrane-tethered (via a second transmem-
brane domain), with the epidermal growth factor domain (EGF)–like domain at the new C-terminus becoming a juxtracrine signal. However, the
release of this EGF-like domain following a secondary proteolytic cleavage step is also possible and could lead to paracrine signaling underneath the
myelin sheath (not shown). In contrast to BACE1, protease TACE/ADAM17 cleaves NRG1 type III within the EGF-like domain, thereby inactivat-
ing it. Binding of NRG1 to ErbB3 induces ErbB2-ErbB3 heterodimerization, autophosphorylation and transphosphorylation of specific tyrosine
residues, and recruitment of adaptor proteins. This results in the activation of several signaling cascades, including the AKT/mTOR, RAS/ERK1/2,
and Calcineurin/NFAT pathways, which collectively control myelination at the transcriptional and posttranslational level (downstream transcription
factors are highlighted in light brown). For details and further references see text Modified from Pereira et al. 2012.

in compartments adjacent to electrical active axons (Wake
et al. 2011). The in vivo requirement for this type of regula-
tion is unclear, however, as NMDA receptors are dispensable
for oligodendrocyte development and myelination (De Biase
et al. 2011). In vitro, astrocytes respond to axonal electrical
activity (and the associated release of ATP) with secretion of
leukemia inhibitory factor (LIF), a cytokine that promotes
myelination by oligodendrocytes.
Interestingly, neuronal activity inhibits Schwann cell
proliferation and differentiation in vitro (Stevens and Fields
2000) (see Fig. 44.2). In Schwann cell/DRG neuron cocul-
tures, action potentials increase the ATP concentration and
activate Ca2+/calmodulin and MAPK signaling pathways via
purinergic P2 receptors. Subsequently, phosphorylation of the
transcription factor CREB and expression of c-fos and Krox24
are induced (Stevens and Fields 2000). Whether these imme-
diate early genes antagonize maturation of Schwann cells in
response to ATP remains to be established. Different puri-
nergic receptors in Schwann cells and oligodendrocytes may
account for different effects of electrically active axons.

562 • NEUROGLIA
 8 I N H I B I TO R S O F M Y E L I N G R OW T H
In the CNS, factors that negatively regulate myelin forma-
tion exceed myelination-promoting factors. This may reflect
a strong intrinsic drive of oligodendrocyte to myelinate target
structures at least in part by default. Thus, negative signals may
be required to control the spatiotemporal development of oli-
godendrocytes and inappropriate ensheathment of neuronal
and glial processes. Indeed, axon-derived inhibitory signals,
including Lingo1 or Jagged, can prevent precocious oligo-
dendrocyte differentiation and myelination. Clearly, the dys-
regulation of such inhibitory signals might contribute to the
failure of efficient myelin repair in diseases, such as multiple
sclerosis (see section 9.2).
L I N G O1
The leucine rich repeat and Ig domain containing NOGO
receptor interacting protein 1 (Lingo1) is a transmembrane
protein, originally described as a neuronal coreceptor for
Nogo-66 and other myelin-associated neurite outgrowth
inhibitors (Mi et al. 2004). Ablation of the Lingo1 gene in
mice results in an enhanced maturation of oligodendrocytes
and a premature onset of myelination (Mi et al. 2005). Lingo1
is expressed on axons and oligodendrocytes, and is thought to
exert its function via cis interactions with Nogo Receptor 1
(NgR1) and p75NTR . Lingo1 induces a downregulation of Fyn
kinase and subsequent activation of RhoA kinase in oligoden-
drocytes, at least in vitro. Likewise, disruption of Fyn inhibits
oligodendrocyte differentiation and myelination in vitro (Mi
et al. 2004, 2005). Also, axonal Lingo1 emerged as an inhibi-
tor of oligodendrocyte differentiation and myelination in vitro
(Lee et al. 2007). Transgenic mice that overexpress Lingo1 in
neurons show an impairment of CNS myelination, as demon-
strated by a reduced number of myelinated axons in the spinal
cord. Expression of axonal Lingo1 is regulated, at least in vitro,
by neurotrophins and the TrkA receptor (Lee et al. 2007). The
expression of Lingo1 has no obvious effect on Schwann cell
myelination (Lee et al. 2007; Mi et al. 2005).
N OTC H1
Different effects on PNS and CNS myelination have also been
reported for the receptor protein Notch1 expressed by glial
cells. At early developmental stages, the canonical Notch1
ligands Delta and Jagged are highly expressed in neurons of
the CNS, whereas in the PNS both Schwann cells and axons
only express the ligand Jagged1. Upon ligand binding, Notch
is cleaved by gamma-secretase, and the notch intracellular
domain (NICD) translocates to the cell nucleus and activates
gene transcription (Taveggia et al. 2010). Notch1 signaling
inhibits oligodendrocyte differentiation and myelination in
vitro (Givogri et al. 2002; Taveggia et al. 2010). In vivo, abla-
tion of Notch1 induces premature expression of myelin pro-
teins (Genoud et al. 2002). Interestingly, some myelinated
axons were also observed in the molecular layer of the cerebel-
lum, which normally remains unmyelinated. Thus, Jagged1
expression on axons may also prevent aberrant myelination
FAC TO R S C O N T R O L L I N G M Y E L I N F O R M AT I O N
(Givogri et al. 2002). Significantly, the function of Notch1 in
oligodendrocytes may depend on its interaction with different
ligands and downstream signaling pathways. For example, the
GPI-anchored neural cell recognition protein F3/Contactin1,
localized at the paranodal region, is a functional, noncanonical
ligand of Notch1. Interaction of Notch1 with F3/Contactin1
promotes oligodendrocyte differentiation, although in vivo data
are still lacking. In the PNS, inverse functions of Notch have
been observed. The canonical Notch1 pathway induces Schwann
cell proliferation and promotes the differentiation from precur-
sors into mature Schwann cells, possibly via Notch1-mediated
upregulation of ErbB2 receptors (Woodhoo et al. 2009). Before
myelination, the transcription factor Egr2 induces a decrease of
the NICD protein. This repression of Notch signaling during
myelination is required because Notch1 turns into an inhibitory
factor that delays myelin formation at later developmental stages
(see Fig. 44.2), most probably via noncanonical Notch signal-
ing and posttranslationally targeting Wnt/E-catenin (Woodhoo
et al. 2009).
P S A-N C A M
The polysialylated form of the neural cell adhesion molecule
NCAM (PSA-NCAM) is highly expressed on the axonal mem-
brane before myelination and later downregulated (Charles
et al. 2000). PSA-NCAM acts as a negative regulator of myeli-
nation in cell culture, possibly by preventing myelin-forming
cells to attach the axonal surface, and removal of PSA-NCAM
from axons is required for the initiation of myelination in vitro
(Charles et al. 2000). A transgenic mouse line lacking the normal
downregulation of PSA-NCAM exhibits myelin abnormalities
and axonal degeneration (Fewou et al. 2007). However, an effi-
cient inhibition of myelination by PSA-NCAM, as observed in
cell culture, has not yet been observed in vivo.
In summary, the “positive” regulation of myelination
by direct axo–glial signaling is most important in the PNS,
whereas the correct timing of myelination in the CNS involves
inhibitory signals that may also prevent aberrant myelination
of cellular processes.
9 R O L E O F T H E E X T R AC E L LU L A R
M AT R I X I N M Y E L I N AT I O N
The contact of a myelinating glial cell to its extracellular matrix
constitutes an important prerequisite for the correct forma-
tion of a myelin sheath. The extracellular matrix mediates
the integration of other signaling molecules, such as growth
factors, and the embedding of cytoskeletal dynamics (Pereira
et al. 2012; Taveggia et al. 2010). Among the extracellular
matrix molecules, laminins, integrins, and dystroglycans are
functionally best characterized.
L A M I N I NS
Laminins are heterotrimeric proteins comprising at least 15
different isoforms. They are essential components of the basal
• 563
lamina (Chernousov et al. 2008), which is a specific feature
of Schwann cells. The natural dystrophic mouse, defined by a
mutation in the gene for the laminin alpha 2 subunit, exhib-
its a peripheral neuropathy (Helbling-Leclerc et al. 1995). A
conditional null mutant of the laminin gamma-1 gene (lack-
ing all laminin expression) shows a severe defect of the radial
sorting of axons and an impaired formation of the basal
lamina, with mutant Schwann cells arrested at the premy-
elinating stage (Chernousov et al. 2008). Laminins are the
ligands for integrin and dystroglycan receptors that activate
different downstream signaling pathways. Laminin-deficient
Schwann cells display a reduction of ErbB2 phosphoryla-
tion and impaired PI3K signaling (Yang et al. 2005; Yu et al.
2005), suggesting that ErbB and laminin/integrin signaling
are coupled (see the following).
I N T EG R I NS A N D DY S T RO G LYC A N
Laminin binds to the E1 integrin and the dystroglycan recep-
tor. Ablation of either receptor in Schwann cells results in a
severe defect of axonal segregation, leading to hypomyelina-
tion and defective myelin compaction (Berti et al. 2011).
Importantly, E1 integrin and dystroglycan have sequential,
nonredundant roles for myelination. E1 Integrin is required
early in development for Schwann cell proliferation and sur-
vival (Berti et al. 2011). In contrast, dystroglycan mutant mice
develop extensive foldings of the myelin sheath, disorganized
nodal structures, and an abnormal distribution of sodium
channels at the axon surface (Berti et al. 2011). Laminin 2 and
dystroglycan have also been implicated in the compartmental-
ization and the elongation of the myelin sheath, presumably
by promoting Schwann cell polarization (Court et al. 2009).
Indeed, dystroglycan forms a complex with other proteins of
the dystrophin family in specific subcellular domains (Court
et al. 2009). The cleavage by matrix metalloproteinases 2 and
9 modulates the composition of this complex and the size of
cytoplasmic Schwann cell compartements (Court et al. 2011).
Additionally, E4 integrin is polarized in Schwann cells, with
predominant expression at the abaxonal site. This protein is
important for myelin stability since abnormal myelin infold-
ings have been observed in aged E4 integrin knockout mice
(Feltri et al. 1994; Nodari et al. 2008).
In the CNS, the laminin mutation of dystrophic mice
causes a regional hypomyelination, involving abnormal PI3K
and integrin-linked kinase (ILK) signaling (Chun et al.
2003). Laminin/integrin signaling induces oligodendrocyte
differentiation, at least in part, by modulating Fyn kinase
(Buttery and ffrench-Constant 1999; Relucio et al. 2009).
Also the integrin/contactin complex promotes myelination
by stimulating Fyn activity in vitro (Laursen et al. 2009).
Whether E1 integrin itself is required for CNS myelina-
tion is controversial. In two studies, mouse mutants with
targeted ablation of E1 integrin expression in oligoden-
drocytes exhibited an impaired initiation of myelination,
whereas in a third study E1 integrin was required for oligo-
dendrocyte survival but not for CNS myelination or remy-
elination (Barros et al. 2009; Benninger et al. 2006; Camara
et al. 2009).
564 • NEUROGLIA
Integrin interacts with NRG1/ErbB signaling in the reg-
ulation of oligodendrocyte differentiation, at least in vitro
(Barros et al. 2009; Colognato et al. 2002). Here, the binding
of laminin to E1 integrin switches NRG1 from a proliferation
signal to a differentiation signal of oligodendrocytes, which is
mediated by shifting the downstream response from PI3K to
MAPK signaling (Barros et al. 2009; Colognato et al. 2002).
Since laminin is localized on the axonal surface, laminin can
be considered an axonal signal that promotes oligodendrocyte
differentiation upon axonal contact (Colognato et al. 2002).
The dystroglycan receptor of laminin contributes to the
terminal steps of oligodendrocyte differentiation and myelina-
tion. Blocking dystroglycan in vitro impairs myelin formation
without affecting oligodendrocyte survival (Colognato et al.
2007). Because dystroglycan influences the ability of insulin-
like growth factor 1 (IGF1) to promote oligodendrocyte dif-
ferentiation, also laminin may act via a modulation of IGF1
signaling (Galvin et al. 2010).
10 E X T R AC E L LU L A R FAC TO R S A N D
O R P H A N R E C E P TO R S
I NS U L I N-L I K E G ROW T H FAC TO R 1
Insulin-like growth factor 1 (IGF1) acts mainly through the
type I IGF receptor tyrosine kinase (IGFR1), and both IGF1
and IGFR1 are expressed in neurons and glial cells (Anlar et al.
1999). Insulin-like growth factor signaling is also thought to
mediate the effect of growth hormone (GH) on myelination.
A positive role of IGF1 in myelination was convincingly dem-
onstrated in IGF1-overexpressing transgenic mice that are
hypermyelinated throughout the CNS (Carson et al. 1993;
Ye et al. 1995). In turn, ablation of the IGF1 gene impairs
oligodendrocyte proliferation and myelination. However,
no persistant myelin abnormalities can be observed in adult
mutants, suggesting that the upregulation of IGF2 compen-
sates functionally (Beck et al. 1995; Ye et al. 2002). The con-
ditional deletion of the receptor IGFR1 in oligodendrocytes
confirmed the role of IGF1 in myelin formation (Zeger et al.
2007). In vitro, IGF1 also promotes Schwann cell myelina-
tion (Russell et al. 2000) and induces fatty acid biosynthesis
by activating the PI3K/AKT pathway (Liang et al. 2007).
FI B RO B L A S T G ROW T H FAC TO R S
Fibroblast growth factors (FGF) and their receptor tyrosine
kinases (FGFR1–4) comprise complex protein families with
widespread functions in proliferation, differentiation, and
organogenesis (Mason 2007). Neurons and astrocytes express
FGF1 and FGF2 at the time of myelination, and FGFRs are
developmentally regulated in the oligodendrocyte lineage
(Fortin et al. 2005). Because FGF2 stimulates the prolifera-
tion of OPCs (in vitro), it appears inhibitory to differentia-
tion (Bansal, 2002). FGFR1 is expressed in both OPCs and
differentiated oligodendrocytes, whereas FGFR2 is more spe-
cific to the latter, enriched in membrane lipid-rafts, and associ-
ated with paranodes (Bryant et al. 2009). Fibroblast growth
factor receptor activation of mature oligodendrocytes stimu-
lates process-outgrowth (Fortin et al. 2005). A mild CNS
hypomyelination phenotype in mice with targeted disruption
of both FGFR1 and FGFR2 in oligodendrocytes suggests that
FGF signaling regulates myelin membrane growth and myelin
sheath thickness, perhaps similar to NRG1/ErbB signaling
in the PNS (Furusho et al. 2012). However, the responsible
ligand(s) are not well defined and their association with the
axonal membrane would only be indirect, for example, by
binding to extracellular matrix proteins.
WN T S I G NA L I N G
The canonical Wnt/E-catenin pathway regulates glia cell dif-
ferentiation and myelination via a complex transcriptional and
epigenetic modulation of gene expression (Fancy et al. 2009;
Feigenson et al. 2009; Tawk et al. 2011; Ye et al. 2009) (see
chapter 43). However, the cell type that provides Wnt during
development is not known. Classically, the binding of Wnt
to its receptor frizzled at the cell membrane stabilizes cytoso-
lic E-catenin, which subsequently forms a complex with the
transcription factors TCFs (T-cell factor/lymphoid enhancer
factor or TCF/LEF) to activate (or repress) target genes. In
transgenic mice, constitutive overexpression of E-catenin in
oligodendrocytes inhibits oligodendrocyte differentiation and
delays myelination (Fancy et al. 2009). That continuous Wnt
signaling has only a transient effect may be caused by its bind-
ing partner TCF4 (TCF7L2). Since TCF4 is normally down-
regulated in development, its absence at later stages is likely to
prevent efficient Wnt signaling, and thereby to allow myelin
gene transcription to resume (Rosenberg and Chan 2009). The
histone deacetylases HDAC1 and 2 antagonize Wnt signal-
ing and stimulate oligodendrocyte differentiation by prevent-
ing the complex formation of TCF4 with E-catenin (Ye et al.
2009). However, exogeneous Wnt has been shown to promote
myelin gene expression in Schwann cells and oligodendrocytes
in vitro, and ablation of Tcf3 in zebrafish inhibits myelinogen-
esis by Schwann cells (Tawk et al. 2011). These discrepancies
may be explained by a change of Wnt/E catenin function in
different developmental stages. In addition to its role during
myelination, Wnt signaling is also required for remyelination,
and a dysregulation of the Wnt pathway has been implicated in
remyelination failure of MS (Fancy et al. 2009).
G P ROT E I N– C O U P L E D R E C E P TO R S
G protein–coupled receptors (GPCR) have been identified
as crucial for glial cell differentiation and myelination. In a
mutant screen of zebrafish, GPR126 was identified as essen-
tial for Schwann cell differentiation and myelination (Monk
et al. 2009) (see Fig. 44.2). Mutant Schwann cells are arrested
at the promyelin stage and fail to express the transcription
factors Oct6/SCIP and Egr2, which are essential for myeli-
nation (Monk et al. 2009). However, GPR126 has remained
an “orphan receptor” because its natural ligand is unknown.
Because Schwann cells normally respond to elevated levels of
cAMP, it has been suggested that GPR126 promotes myelina-
tion via the activation of adenylate cyclase (Arthur-Farraj et al.
FAC TO R S C O N T R O L L I N G M Y E L I N F O R M AT I O N
2011; Monk et al. 2009). Indeed, pharmacological elevation
of cAMP levels in zebrafish GPR126 mutants can rescue the
Schwann cell differentiation arrest (Monk et al. 2009). The
mouse mutant of GPR126 develops a severe dysmyelination
and peripheral neuropathy, with defects of axonal sorting and
altered Remak bundles, leading to premature death at 16 days
(Monk et al. 2011).
GPR17 is a P2Y purinergic receptor of nucleotides as well as
a receptor for cysteinyl-leukotrienes (Ciana et al. 2006), and is
transiently expressed during oligodendrocyte differentiation.
GPR17 has been implicated in CNS myelination, because it
inhibits oligodendrocyte differentiation when transgenically
overexpressed (Chen et al. 2009). In turn, lack of this receptor
causes premature differentiation of oligodendrocytes. These
effects are in part mediated by the inhibitory transcription fac-
tors Id2 and Id4 that translocate to the nucleus upon GPR17
activation (see chapter 43) (Chen et al. 2009). Since Id2 and
Id4 physically interact and sequester the differentiation factors
Olig1 and Olig2, it is thought that GPR17 counteracts their
myelination-promoting functions (Chen et al. 2009; Emery
2010b).
11 N E U R OT R O P H I N S
Neurotrophins are growth and survival factors that are widely
expressed by neurons and glial cells, comprising NGF, BDNF,
NT-3, and NT-4/5. The vital function of neurotrophins in
early development has hampered their in vivo analysis during
postnatal myelination, but insight came from in vitro systems
(Rosenberg et al. 2006; Xiao et al. 2009). All neurotrophins are
ligands for the low-affinity receptor p75NTR and one or more
high-affinity Trk (tropomyosin-related kinase) receptors.
N E RVE G ROW T H FAC TO R
Nerve growth factor (NGF) promotes myelination in vitro by
activating TrkA on the axonal surface, potentially mimicking
glia-to-axon signaling (Chan et al. 2004). However, in vivo
only a subpopulation of small-caliber nociceptive axons that
remain largely unmyelinated expresses TrkA receptors (Xiao
et al. 2009). How axonal TrkA receptors influence myelina-
tion has not been clarified, but an interaction with NRG1
signaling has been suggested. For example, soluble NRG1 iso-
forms have been suggested to be locally released in response to
Schwann cell–derived NGF, BDNF, and GDNF (Esper and
Loeb 2004, 2009). In CNS-derived cultures, binding of NGF
to axonal TrkA receptors does not promote but rather inhibits
oligodendrocyte differentiation and myelination (Chan et al.
2004), potentially by inducing the inhibitor Lingo on the
axonal surface (Lee et al. 2007; Mi et al. 2005).
B R A I N-D E R I VE D N EU ROT RO P H I C FAC TO R
Brain-derived neurotrophic factor (BDNF) has been impli-
cated in axon-to-glia and glia-to-axon signaling. A neuronal
increase of BDNF expression enhances myelin formation in
DRG neuron/Schwann cell cocultures. This is presumably
• 565
by interaction with p75NTR and p75NTR mutant mice display
impaired peripheral myelination (Cosgaya et al. 2002). One
suggested mechanism is the interaction of p75NTR with the
polarity protein Par3, regulating the initial polarization of
Schwann cells that is required for myelination (Fig. 44.2)
(Chan et al. 2006). Whereas BDNF-mediated activation of
p75NTR on axons enhances myelin formation by Schwann cells
in vitro (Xiao et al. 2009), BDNF binding to the axonally
bound TrkB inhibits myelination in vivo, and the expres-
sion level of this neurotrophin varies widely, for example,
between different subpopulations of DRG neurons (Xiao
et al. 2009).
In oligodendrocyte cultures (in contrast to Schwann cells),
BDNF exerts a promyelinating effect by stimulating glial
TrkB receptors (Xiao et al. 2010). Different loss-of-function
mutants have been investigated. Heterozygous BDNF mutant
A sion in mice is associated with mildly impaired myelination,
control PTEN mutant
B
control AKT mutant
Figure 44.3 Elevation of PI3K Signaling in Oligodendrocytes Induces
Hypermyelination. A. Increasing the signaling lipid PIP3 in oligodendro-
cytes by cell-specific ablation of the lipid phosphatase PTEN. When ana-
lyzed by magnetic resonance imaging (T2 weighted images; top panel) the
subcortical white matter (arrow: corpus callosum) has a larger volume in
PTEN mutant mice compared with controls. This phenotype is associated
with increased myelin sheath thickness. B. Transgenic overexpression of a
constitutively active isoform of AKT kinase in oligodendrocytes also causes
a similar pattern of mTOR-dependent hypermyelination (Flores et al. 2008;
Goebbels et al. 2010; Narayanan et al. 2009). (A) Taken from Goebbels
et al. 2010. (B) and Flores et al. 2008.
566 • NEUROGLIA
mice revealed a mild hypomyelination in several CNS regions
(Vondran et al. 2010; Xiao et al. 2010). However, whether this
myelination defect is transient or persists in adulthood is less
clear and may be regionally different (Du et al. 2003; Vondran
et al. 2010; Xiao et al. 2010). When BDNF expression is con-
ditionally ablated selectively in postmitotic neurons, mutant
mice show normal oligodendrocyte differentiation and unal-
tered myelination in the optic nerve (Rauskolb et al. 2010).
Thus, other cellular sources of BDNF (possibly astrocytes)
may play a role and again myelination needs to be analyzed in
different brain regions.
N EU ROT RO P H I N-3
Neurotrophin-3 (NT3) signaling to Schwann cells for myelin
formation is still debated, with most in vitro data suggesting
that NT3 acts as an inhibitory factor of myelination by acti-
vating TrkC receptors. In contrast, ablation of NT3 expres-
but this myelination defect may be secondary to a massive
neuronal loss (Xiao et al. 2009).
In conclusion, neurotrophins act as modulators of myelin
formation. Their role in myelination can be both inhibitory
and promoting, depending on the receptor composition pre-
sented on glial cells and axons. Finally, Glial cell line–derived
neurotrophic factor (GDNF), is not a “neurotrophin,” but a
member of the TGF-E family of growth factors. GDNF has
been shown to trigger myelination of normally unmyelinated
axons in the sciatic nerve, which is most probably secondary
to a massive increase of Schwann cell proliferation (Höke
et al. 2003).
12 D I VE R S I F I C AT I O N O F
G R OW T H FAC TO R F U N C T I O N BY
P O S T T R A N S L AT I O N A L P R O C E S S I N G
Proteolytic processing is a posttranslational mechanism by
which growth factor signaling is modified, for example, by
determining whether the growth factor or its receptor is pre-
sented as a soluble or membrane-bound protein. This deci-
sion may alter downstream signaling pathways in axons and
myelinating glial cells. “Secretases,” such as ADAM proteins,
BACE1, and the J-secretase complex have all been implicated
in myelination. ADAM17, a D-secretase also known as TACE,
and BACE1 both cleave NRG1 type III at distinct but closely
spaced sites, leading to opposite effects on myelination (see
Figs. 44.1B and 44.2). While BACE1 activates NRG1 type
III and promotes the myelination by Schwann cells (Hu et al.
2006; Velanac et al. 2012; Willem et al. 2006) and also remy-
elination after nerve injury (Hu et al. 2008), TACE process-
ing negatively regulates peripheral myelination, most likely by
cutting within the EGF domain and thus reducing the level
of functional NRG1 type III (La Marca et al. 2011). It is yet
to be determined in which subcellular neuronal/axonal com-
partment these processing steps occur. That BACE1 activity
exerts a similar effect on CNS myelination (Hu et al. 2006)
has been debated (Treiber et al. 2012). Nardilysin (NRD1,
N-arginine dibasic convertase) is a metalloendopeptidase and
enhancer of protein ectodomain shedding that has been asso-
ciated with NRG1 cleavage (Ohno et al. 2009). Whether its
myelination-promoting function in PNS and CNS is related
to the activation of TACE, BACE1, or other proteases is not
known. Two other ADAMs have been involved in periph-
eral myelination (see Fig. 44.2): ADAM19/Meltrin-beta
deficient mice exhibit a delayed remyelination upon injury
(Wakatsuki et al. 2009). Mice devoid of the catalytically
inactive ADAM22 are hypomyelinated, most likely because
this neuronal membrane protein is the axonal receptor for
the secreted soluble Schwann cell protein Lgi4 (leucine-rich
repeat LGI family, member 4) (Ozkaynak et al. 2010; Sagane
et al. 2005). Lgi4 itself is an essential regulator of myelination
in the PNS, because a mutation of this gene causes a severe
congenital dysmyelination in the spontanous mouse mutant
claw paw (Bermingham et al. 2005). The broadly expressed
J-secretase complex cleaves and codetermines the turnover of
a range of transmembrane protein substrates, including those
that regulate glial development and myelination, i.e., NRG1
(Bao et al. 2003), ErbB4 (Ni et al. 2001), p75NTR (Zampieri
et al. 2005), and Notch1 (Fortini 2002). Oligodendroglial
J-secretase activity is important for the ensheathment of
retinal ganglion cell axons when maintained in a CNS cocul-
ture system (Watkins et al. 2008). In summary, proteolysis
comprises posttranslational and probably rapid regulation
of axonal myelination signals, and some of the enzymes are
potential targets in pharmacological attempts to boost myelin
repair in demyelinating diseases.
13 G L I A L R E C E P TO R AC T I VAT I O N A N D
D OW N S T R E A M S I G N A L I N G C A S C A D E S
Despite striking differences of axon–glia signaling in CNS
and PNS at the level of axonal ligands and their glial receptors,
essential downstream mechanisms in oligodendrocytes and
Schwann cells appear to be conserved. For example, activated
by glial receptors, the enzymes phosphatidylinositol 3-kinase
(PI3K) and AKT1 (v-Akt murine thymoma viral oncogene
homolog 1) are critical for myelination in the entire nervous
system. PI3K phosphorylates the membrane lipid PIP2 to
produce the key second messenger PIP3. This in turn leads to
the local recruitment and activation of various effector pro-
teins that can bind to PIP3 at the cell membrane with their
pleckstrin homology (PH) domain. Among those AKT1,
a serine-threonine protein kinase, has been studied in most
detail; however, mostly in the context of CNS myelination. For
example, transgenic expression of a constitutively active AKT1
isoform in oligodendrocytes enhances myelin growth by acti-
vating the serine-threonin protein kinase mTOR (mammalian
target of rapamycin) and its further downstream substrates
(e.g., S6 kinase and the ribosomal protein S6) (see Fig. 44.3)
(Flores et al. 2008; Narayanan et al. 2009). In addition, mTOR
regulates mRNA expression (specifically of MBP, PLP, and
enzymes for lipid synthesis) by forming one of two complexes,
mTORC1 or mTORC2, which are defined by the adaptor
FAC TO R S C O N T R O L L I N G M Y E L I N F O R M AT I O N
proteins raptor and rictor, respectively (Tyler et al. 2009).
Independent evidence for the role of PI3K signaling in CNS
myelination comes from mice lacking PTEN (phosphatase and
tensin homolog) in oligodendrocytes. This enzyme antagonizes
PI3K signaling by dephosphorylating PIP3 at the D3 position
of the inositol and recreating PIP2. Consequently, PTEN
ablation in oligodendrocytes leads to elevated PIP3 levels and
activation of mTOR and its downstream kinases, resulting in
enhanced myelin gene expression and widespread hypermyeli-
nation (see Fig. 44. 3) (Goebbels et al. 2010).
In Schwann cells, PI3K is activated when axonal NRG1
type III binds to ErbB2/ErbB3 receptor heterodimers
(Taveggia et al. 2005), but PI3K is also coupled to receptors
of other ligands, such as IGF-1 (insulin-like growth factor 1)
that stimulate myelination (Liang et al. 2007). Activation of
PI3K promotes myelination also in an AKT-dependent fash-
ion, at least in vitro. In cultured Schwann cells, the activation
of AKT after IGF-1 stimulation is thought to mediate the
EGR2-dependent upregulation of P0/MPZ gene expression
(Wakatsuki et al. 2009). Phosphorylated AKT also promotes
the expression of SREBPs (sterol responsive element binding
proteins), the key transcription factors that regulate the biosyn-
thesis of cholesterol, which is by itself crucial for myelination
(Porstmann et al. 2005; Saher et al. 2009). Similar to the CNS,
mTOR downstream signaling is suggested to play a central role
in regulating the growth of the myelin sheath in the PNS in
vivo (Sherman et al. 2012). However, transgenic mice that
overexpress a constitutively active AKT isoform in Schwann
cells reportedly show normal peripheral myelination (Flores
et al. 2008), in marked difference to the CNS phenotype (see
the preceding). Moreover, the elevation of PIP3 in Schwann
cells following the ablation of PTEN does not cause the same
hypermyelination phenotype in mice as the neuronal (axonal)
overexpression of NRG1 type III. Instead, isolated elevation of
PIP3 triggers progressive myelin pathology with focal myelin
outfoldings and tomacula, similar to findings in some patients
with demyelinating peripheral neuropathy (Goebbels et al.
2012). Although abnormal mTOR signaling may be causative
of abnormal myelin growth, the specific pathology is probably
the loss of PTEN as a local “brake,” which limits the insertion
of new membranes into the myelin sheath (Goebbels et al.
2012). This brake is formed by PTEN in conjunction with
the mammalian Disk-large homolog Dlg1 and stabilized by
NRG1 stimulation, likely to prevent ‘overmyelination’ during
myelin sheath assembly (Cotter et al. 2010).
In addition to PI3K/AKT/mTOR signaling, myelina-
tion control also involves other second messenger pathways
that transduce axonal signaling to Schwann cells, for exam-
ple, downstream of the activation of ErbB2/ErbB3 het-
erodimers by NRG1 type III. For example, the extracellular
signal regulated kinase 1/2 (Erk1/2) and the non–recep-
tor tyrosine phosphatase SHP2/PTPM11 are critical for
Schwann cells to fully respond to NRG1. In the absence of
ERK1/2 from cultured Schwann cells, NRG1 no longer has
a survival-promoting effect (Newbern et al. 2011). Likewise,
normal proliferative and migratory responses to NRG1 can-
not be triggered in SHP2-mutant cultured Schwann cells
(Grossmann et al. 2009). Conditional inactivation of SHP2
• 567
in murine Schwann cells causes marked hypomyelination of
peripheral nerves associated with a persistent impairment of
ERK1/2 signaling, whereas PI3K/AKT activation is unaf-
fected (Grossmann et al. 2009). Moreover, Schwann cell–
specific inactivation of ERK1/2 inhibits peripheral myelina-
tion (Newbern et al. 2011).
Finally, NFATc3/4 (nuclear factor of activated T cells) is
a transcription factor that is critical for NRG1 signaling to
Schwann cells (Kao et al. 2009). NRG1 binding to ErbB2/
ErbB3 receptors induces a phospholipase C (PLC-J)–
dependent increase in cytosolic calcium levels, which activates
calcineurin, a phosphatase that dephosphorylates this tran-
scription factor for subsequent nuclear translocation (Kao
et al. 2009). Mouse mutants that lack an essential regulatory
subunit of calcineurin (CnB1) in Schwann cells display severe
defects in Schwann cell differentiation (Kao et al. 2009).
Interestingly, calcineurin mutant mice also show decreased
expression of the transcription factor EGR2, indicating that
EGR2 requires calcineurin for its activity. In addition, NFAT
facilitates the binding of Sox10 to the regulatory region of
the Egr2 gene. Thus, a synergistic function NFAT and Sox10
upregulates myelin gene expression (Kao et al. 2009).
For oligodendrocyte maturation, Fyn kinase plays a cen-
tral role as an integrator and mediator of axon–glia signaling.
Mice lacking functional Fyn kinase depict a marked hypomy-
elination in the forebrain and have fewer myelinated fibers in
optic nerve and corpus callosum (Krämer-Albers and White
2011). Fyn kinase is activated by various axonal signals and
their respective oligodendroglial surface receptors, includ-
ing D6E1-integrin, Dcc (deleted in colorectal carcinoma),
Lingo-1, and the GPI-anchored protein F3/contactin. The
latter binds the axonal cell adhesion molecule L1 and activates
Fyn specifically in lipid-raft microdomains. Fyn itself relays
into three major downstream signaling pathways, all of which
affect the spatiotemporal control of oligodendrocyte differ-
entiation and myelination (Krämer-Albers and White 2011).
First, activated Fyn alters oligodendrocyte morphology by
regulating the Rho-family GTPases RhoA, Cdc42, and Rac1,
known modulators of actin cytoskeleton dynamics and oli-
godendrocyte process formation (Liang et al. 2004). Second,
active Fyn recruits microtubuli (via its SH2 and SH3 protein
binding domains) and the microtubule-associated protein
Tau (Klein et al. 2002), which stabilizes the glial cytoskeleton,
contributes to oligodendrocyte polarization and facilitates
(microtubule-dependent) transport of myelin-cargo toward
the axon–glia contact site. Finally, Fyn affects transport and
stability of MBP mRNA and activates translation of MBP, the
only known protein that is rate-limiting of CNS myelination
(Popko et al. 1987; Readhead et al. 1987). Specifically, MBP
mRNA is transported in RNA granules (mediated by hnRNP
A2) from the nucleus to distal oligodendroglial processes in
a translationally silenced state. Release of this repression is
thought to occur on contact between axonal L1 with the oli-
godendroglial cell process and subsequent Fyn activation and
phosphorylation of hnRNPA2 (White et al. 2008). Hereby,
the local dissociation of the transport granule allows for local-
ized MBP synthesis at the site of axon–glia contact where
myelin deposition is required. Further, it has been proposed
568 • NEUROGLIA
that axonal electrical activity and the release of glutamate
stimulates myelination by locally increasing Fyn-dependent
MBP synthesis (Wake et al. 2011).
14 S U M M M A RY A N D P E R S P E C T I VE S
The widely held model that neuronal signaling molecules trig-
ger the proliferation of axon-associated glial cells and control
the synthesis of myelin, originally derived from classical cell
culture and nerve graft experiments, has been largely confirmed
at the molecular level and by in vivo experiments. However,
in its simplest form the model only applies to Schwann cells
responding to the axonal growth factor NRG1 type III. In
the peripheral nervous system, this axonal membrane protein
has emerged with an unexpected broad range of develop-
mental functions, from promoting Schwann cell survival in
embryonic development to regulating myelin sheath thickness
according to axon size in postnatal life. In the CNS, oligoden-
drocytes and their precursor cells respond to a large number of
signals, derived mostly as soluble factors from different cellular
sources, including astrocytes, and including factors associated
with electrical activity of axons. However, oligodendrocytes
appear not to depend on NRG1 or other single growth fac-
tors to become myelination competent, and the ensheathment
of their axons appears to be triggered by a battery of signals
received by oligodendrocytes and a transcriptional program,
and ultimately to occur synchronously “by default.” Thus,
inhibitory cues that help restrict myelination to appropriate
axonal targets become developmentally equally important.
On the down side, these negative factors of CNS myelina-
tion appear to also limit myelin repair in neurological diseases,
which is required to protect axonal integrity. Therefore, recent
research activities have concentrated also on the intracellular
second messenger systems that appear to be largely shared by
my elinating glial cells in PNS and CNS, and that serve to inte-
grate the plethora of non–cell autonomous signals received.
It is anticipated that a better understanding of bidirectional
axon–glia interactions, and the even more complex neuron–
glia signaling in the CNS, will allow to identify rate limiting
steps of remyelination, which can then be pharmacologically
targeted for the future treatment of demyelinating diseases.
AC K N OW L E D G M E N T S
The authors thank members of the Nave lab for helpful com-
ments. Work in the authors’ lab was supported by the Max
Planck Society and grants from the DFG (CMPB, SFB/
TR43), BMBF (Leukonet), EU-FP7 (Ngidd, Leukotreat),
ELA, and the MS Society (USA). KAN holds an ERC
Advanced Grant (Axoglia).
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 45.
REGULATION OF MYELINATION BY
FUNCTIONAL ACTIVIT Y
R. Douglas Fields

A B B R E VI AT I O N S of neurons, producing a quantum leap in cognitive ability of

vertebrates.
ATP adenosine triphosphate When myelin is damaged through disease or injury, loss
CNS central nervous system of sensory and motor function causes severe debilitation.
DRG dorsal root ganglion Relegation of myelin to within the context of disease was
DTI diffusion tensor imaging responsible for the longstanding concept that myelin was
ERK/MAPK extracellular signal–regulated kinase/ static insulation and irrelevant to nervous system plasticity,
mitogen-activated protein kinase except after injury. This view has changed rapidly (Fields 2005,
Fyn Fyn kinase 2010). New cellular and molecular information on modes of
GABA gamma aminobutyric acid axon–glial communication regulated by electrical activity has
GFP green fluorescent protein emerged from the development of new methods of studying
L1-CAM L1 cell adhesion molecule myelination in cell culture. Specialized magnetic resonance
LIF leukemia inhibitory factor imaging (MRI) brain imaging techniques enable visualizing
MAG myelin-associated glycoprotein microstructural changes in white matter in the human brain in
MBP myelin basic protein response to environmental experience (Fields 2011a; Zatorre
mGluR metabotropic glutamate receptor et al. 2012). Lineage tracing in transgenic animals shows that
MRI magnetic resonance imaging immature oligodendrocytes (OPCs) persist in abundance in
NCAM neural cell adhesion molecule the adult brain (Paschoula et al. 2009; Rivers et al. 2008).
NMDA N-methyl-d-aspartate Myelination is no longer regarded strictly as an early develop-
OPC oligodendrocyte progenitor cell mental process; myelination continues well into adult life.
PDGF platelet-derived growth factor Myelination is now known to be influenced by functional
PNS peripheral nervous system activity in axons. Several mechanisms have been identified for
TTX tetrodotoxin activity-dependent effects on myelination and many more are
certain to be discovered. The consequences are wide-ranging
and profound, from illuminating how environmental experi-
1 INTRODUCTION ence modifies brain development through early life; to how
cognitive abilities decline with age; to understanding a new
A compelling argument can be made that more than any other aspect of mental illnesses and developmental disorders; and
development, the evolution of myelin was responsible for the expanding the cellular mechanisms of learning beyond neuro-
success of vertebrates. The multilayered wrapping of insulation transmitter signaling at synapses.
around axons and the formation of nodes of Ranvier funda-
mentally changed the way electrical impulses are conducted
1.1 H I S TO R I C A L P E R S P EC T I VE O N MY E L I N
through nerve axons, increasing impulse propagation speed by
A N D F U N C T I O NA L AC T I VIT Y
at least 50 times faster than in unmyelinated axons (see also
chapter 42). The ability to transmit impulses rapidly over long The concept that myelination is associated with development
distances permitted increased body size, rapid sensation and of functional activity in nerve fibers originated with the earli-
movement, and the ability to concentrate neurons into a single est histological studies of the brain, but much confusion and
organ, the brain, rather than the necessity of having isolated uncertainty persisted because the true nature and function of
ganglia distributed throughout the body and situated close to myelin was mysterious from the time it was first seen in the late
the structures they control, as they are in invertebrates, which 19th century. Electron microscopy in the mid-1950s to early
lack myelin (Bullock et al. 1984). The concentration of neurons 1960s finally revealed myelin to be a multi-laminar wrapping
into one organ (cephalization) and the miniaturization that of compacted membrane around an axon (Geren 1954), which
myelin enabled by allowing extremely slender axons to con- is formed by Schwann cells in the peripheral nervous system
duct impulses as rapidly as the largest axons in invertebrates, (PNS) and oligodendrocytes in the central nervous system
enabled complex integration among many different types (CNS) (Fig. 45.1). Originally, myelin was believed to be a fatty

573
 A C

Figure 45.1 Myelinating Glia A. Spindle-shaped Schwann cells form myelin in the peripheral nervous system. B. Multipolar oligodendrocytes form
myelin in the central nervous system. Individual processes of the oligodendrocyte (green) form contacts with axons (purple) to begin wrapping myelin
membrane around them. Electrical activity promotes myelination by increasing survival of oligodendrocytes, promoting differentiation of oligoden-
drocytes, and stimulating the local synthesis of myelin basic protein in oligodendrocyte processes associated with electrically active axons.
C. Cross-section of axons in optic nerve of an adult mouse showing the multilaminar compacted membranes of myelin wrapped around the axons.
(A,B) are from cell culture. Bar = 10 um (A,B), 100 nm (C).

substance secreted by the axon. In the CNS this was the most
reasonable explanation because the slender processes of oligo-
dendrocytes forming myelin on axons were not revealed by the
histological stains used by early anatomists. Thus, there was no
evidence linking the thick white coating on axons to any cel-
lular source other than the axon. In the PNS, Louis-Antoine
Ranvier suggested that myelin resided inside the Schwann cell
like a fat droplet clinging to the axon (Ranvier 1878). The func-
tion of myelin as an electrical insulator arose naturally with the
understanding that communication in the nervous system is
electrical, but it was also clear that conduction could proceed
perfectly well in the absence of myelin, as many fibers in the
brain and peripheral nerves are unmyelinated. Thus myelin
was not regarded as essential or as having any unique prop-
erties in serving as an electrical insulator, as expressed by the
renowned neuroanatomist Ramón y Cajal in the early twenti-
eth century:
Myelin does not enjoy a monopoly on insulation, even
if one acknowledges that it does indeed insulate, which
has not yet been demonstrated conclusively. Because
axons in invertebrates, the autonomic system of ver-
tebrates, the olfactory nerve, and most parts of the
cerebrum, cerebellum, and optic lobe of fish, reptiles,
and amphibians lack myelin sheaths entirely, their
only protection against the lateral escape of current, or
short circuit, appears to be a glial plexus [of astrocytes]
between fibers . . . because of its late appearance in the
animal series, it would seem to us that myelin repre-
sents nothing more than a refinement of the insulating
function assumed for so long by neuroepithelial (i.e.,
ependymal) cells and glia (i.e., astrocytes). S. Ramón
y Cajal Histology of the Nervous System of Man and
Vertebrates, p. 206. 1995.
Although the function of the nodes of Ranvier in salta-
tory conduction was suggested by Lillie in 1925 and shown
experimentally by Tasaki in 1939, the function in saltatory
conduction was not universally appreciated. “The function of
the nodes [of Ranvier] remains obscure,” wrote JZ Young in an
article published in Nature in 1944. “It may be that they serve
to prevent movement of the fluid within the fibres. Fibres of
the central nervous system are usually without nodes, and lack
the definite tubes which are present in peripheral nerve, no
doubt to meet the stresses imposed by movement” (Young
1944, p. 522).
This uncertainty accounts for the slow progress in under-
standing how functional activity could affect myelination.
Synapses are understandably the focus of studies on functional
plasticity in the nervous system. Nevertheless, there has always
been considerable interest in studying myelination in correla-
tion with development of various nervous system functions,
because unlike synapses, which were invisible through light
microscopes, myelination of fibers was readily apparent to his-
tologists. Although the correlation between development of
functional activity in fibers with the onset of myelination was

574 • NEUROGLIA
grasped by most early anatomists, the correlation was usually
perceived as providing an index of neuronal maturity, not that
myelination was necessarily stimulated by electrical activity in
the axon.
Paul Flechsig (1876), first observed that tracts become
myelinated in a definite sequence so that fibers belonging to
related functional systems become myelinated at the same time
(see Langworthy 1932). Birth was seen as a great stimulus to
myelination in human development. “Probably it is the result
of increased stimulation of the sensory organs” (Langworthy
1932, p. 1379). The vestibular portion of the acoustic nerve
is myelinated early in development, but the olfactory nerve is
unmyelinated at birth and the optic nerve is just beginning to
myelinate at birth. Motor roots are myelinated before sensory
roots, and sensory pathways begin to myelinate at the time
when sensory endings become functional. Sensory endings
in the skin, muscle, and vestibular and cochlear organs would
be stimulated in utero, but optic and olfactory nerves are not
strongly activated until after birth.
The suggestion that axon pathways become myelinated
when they become functional was also provided by animal
studies. The right eyelid of the rabbit opens slightly earlier
than the left and myelination begins in the retina of the right
eye of the rabbit before the left eye (Narang 1977), suggest-
ing that the onset of vision stimulates myelination of visual
circuits. In the minority of cases in which the left eye of rab-
bits opened first, that eye developed more myelinated fibers.
Similar asymmetry in myelination was seen in the optic
nerves.
Typically myelination begins after the axon has reached its
appropriate target, formed synapses, and begun firing action
potentials, even though the glial cells have been closely associ-
ated with the axons and fully matured from much earlier in
development. This suggests that myelination is influenced by
axonal factors, not simply determined by maturation of oli-
godendrocyte progenitor cells to a mature myelinating stage.
Migration and differentiation of oligodendrocytes in the optic
nerve of rat and most species progresses in a gradient from the
brain (optic chiasm) to the retina, but myelination in most spe-
cies proceeds in the reverse manner, from the cell body (near
the retina) to the distal axon (chiasma). In human cerebral
white matter, oligodendrocytes are present in some regions
for at least 3 months before the cells commit to myelino-
genesis, supporting dissociation between events regulating
oligodendrocyte maturation and their commitment to
myelinogenesis (Back et al. 2002).
Myelination of appropriate brain regions coincides with
the development of specific behaviors and cognitive func-
tions (Mabbott et al. 2006; Nagy et al. 2004; Yakolev and
Lecours 1967), such as reading (Kraft et al. 1980), develop-
ment of vocabulary (Pujol et al. 2006) and proficiency in
executive decision making (Giedd 2004; Liston et al. 2006).
Incomplete myelination of the forebrain until the early twen-
ties has been suggested as a neurological basis for weaker
decision-making skills in adolescence (Beckman 2004).
Whether these correlations reflect developmental limitations
on behavior or behavioral influences on development is the
intriguing question.
R E GU L AT I O N O F M Y E L I N AT I O N BY F U N C T I O N A L AC T I VI T Y
2 C O N T R O VE R SY A N D E A R LY E VI D E N C E
O N T H E R O L E O F I M P U L S E AC T I VI T Y
O N M Y E L I N AT I O N
Experimental studies on the effects of functional activity on
myelination have yielded a confusing and contradictory set
of data. Environmental experience during the period when
myelination is taking place has been shown to affect oligoden-
drocyte development and myelination (Fields 2008; Zalc and
Fields 2000). The number of oligodendrocytes increases 27%
to 33% in the visual cortex of rats raised in environments that
are enriched by additional play objects and social interaction
(Bennett et al. 1964; Sirevaag and Greenough 1987; Szeligo
and Leblond 1997). Enriched environments increase the num-
ber of myelinated axons in the corpus callosum connecting the
two cerebral hemispheres of rats ( Juraska and Kopocik 1988;
Markham and Greenough 2004), and the corpus callosum
increases in size in rhesus monkeys raised in enriched envi-
ronments in parallel with improved performance in cognitive
tests (Sanchez et al. 1998).
Sensory input to the visual system has been used to address
activity-dependent effects on myelination in several studies,
but results differ among studies. Mice reared in dark develop
fewer myelinated axons in the optic nerve compared with nor-
mally reared mice (Gyllensten and Malmfors 1964), and pre-
mature eye opening increases the level of myelin protein in the
optic nerve of rabbit (Tauber et al. 1980).
These findings from experimental manipulation of visual
experience are consistent with effects of disrupting impulse
activity by severing axons. There is marked reduction in myeli-
nation of the chick optic tectum following deafferentation
(Bondy and Madsen 1972), and sciatic neurectomy in the rat
inhibits myelination in the spinal cord (Kingsley et al. 1970),
suggesting myelination is related to physiological activity. The
axons are also slightly smaller in diameter after deafferenta-
tion, however, which could in turn affect myelination, because
there is a minimum threshold of axon diameter for myelina-
tion, and the thickness of the myelin sheath is proportional to
axon diameter.
In contrast, other studies have reported that intraocu-
lar injections of the sodium channel blocker tetrodotoxin
(TTX), which blocks action potential activity, had no effect
on the number of myelinated fibers or the time of myelination
onset in optic nerves of rat (Colello and Pott 1997; Colello
et al. 1995; Crespo et al. 1995). In experiments on goldfish,
action potential blockade by intraocular injection of TTX
during optic nerve regeneration had no effect on myelination
of regenerated fibers (Hayes and Meyer 1989). Dark-rearing
of kittens (Moor et al. 1976) and rats (Fukui et al. 1991)
has been reported to have no effect on initiation of myeli-
nation of the visual pathway during early postnatal develop-
ment. Mouse spinal cord explants in vitro undergo normal
oligodendrocyte development and myelination in the pres-
ence of TTX (Shrager and Novakovic 1995); however,
blocking potassium channels with tetraethylammonium ion
eliminated myelination in these experiments, suggesting pos-
sible involvement of ion channels in axons or oligodendro-
cytes in myelination.
• 575
 In apparent contradiction to the studies using TTX in spi-
nal cord explant cultures, in which no effects on myelination
were evident, dissociated cultures of embryonic mouse brain
hemispheres treated 2 to 4 days with TTX starting 5 to 7 days
before myelination normally begins, decreased the number
of myelinated fibers by 80% at 18 to 21 days in vitro (DIV)
(Demerens et al. 1996). The number of oligodendrocytes and
neurons was not affected. Results consistent with this were
also obtained after TTX injection into the eye to block action
potentials in the optic nerve, and increasing action potential
firing with the sodium channel activator alpha-scorpion toxin
increased myelination by 2.4 times without affecting the num-
ber of oligodendrocytes (Demerens et al. 1996). However, in
both in vitro and in vivo studies the effects were seen only when
treatments were given at narrowly defined points in develop-
ment. These results suggest that electrical activity affects the
myelination process only within a narrow time frame, and
around the time of eye opening when electrical activity in reti-
nal ganglion cells changes from a transient to a repetitive pat-
tern of firing (Rorig and Grantyn 1993; Skaliora et al. 1993).
Much of this apparent discrepancy in different studies,
and the sharp developmental windows for the effects, may be
explained in part by an expectation that functional activity in
axons could affect multiple processes involved in oligoden-
drocyte development and myelination (Fig. 45.2). This could
include regulating cell proliferation, survival, migration, dif-
ferentiation, and myelination. Depending on when functional
activity is modified in different experimental studies and when

A Early
development Cell survival?
Cell proliferation?
Cell migration?
OPC

Axon

B Differentiation Adenosine
Promotes differentiation of
OPCs to myelinating
ATP oligodendrocytes

ATP

Acts on promyelinating
oligodendrocytes to increase
LIF myelination
Astrocyte

C Myelination
Regulation of cell adhesion Formation of cholesterol-rich
molecules on axons signalling domain
Stimulates local translation
of MBP
CAMs

Oligodendrocyte

Glu

Myelinated axon

Figure 45.2 Electrical Activity in Axons Can Affect Multiple Steps in the Myelination Process. A. Activity-dependent release of growth factors, ions,
other diffusible cell–cell signaling molecules, or changes in cell adhesion molecules on the surface of axons can affect cell survival, proliferation, and
migration of myelinating glia. B. Differentiation of oligodendrocyte progenitor cells and myelination by promyelinating oligodendrocytes can be
stimulated by release of diffusible substances, including neurotransmitters (adenosine triphosphate and adenosine) released from axons, and cytokines
released from astrocytes responding to the neurotransmitter ATP liberated from electrically active axons. C. Initiation of myelination can be affected
by activity-dependent regulation of cell adhesion molecules on axons, and the vesicular release of glutamate from axons firing action potentials.

576 • NEUROGLIA
in development the results are evaluated, myelination could be
differentially affected or unaffected by the confounding pro-
cesses that could be involved. In addition, the effects on oligo-
dendrocytes and myelination could be secondary to a primary
effect on neurons. A major confound is that oligodendrocytes
and their precursors have many neurotransmitter receptors
and ion channels that could be directly affected by experi-
ments using neurotoxins applied with the intention of modu-
lating electrical activity in axons. Moreover, both positive and
negative factors may participate in controlling myelination,
and the pattern of impulse activity may be an important cri-
terion that would not be reproduced by using neurotoxins or
pharmacological treatments. These problems were eliminated
by cell culture studies in dishes equipped with electrodes for
electrical stimulation of axons in coculture with Schwann cells
or oligodendrocytes, as described in section 4.
2.1 HUM A N B R A I N I M AG I N G O F WH IT E
M AT T E R P L A S T I C I T Y
Magnetic resonance imaging, and in particular diffusion ten-
sor imaging (DTI), which is highly sensitive to microstructural
changes in brain tissue, has revealed many examples of differ-
ences in white matter structure that correlate with specific cog-
nitive abilities (Fields 2011a). This includes difference in white
matter structure associated with differences in performance
within the normal range of variation, not simply dysfunction
resulting from white matter defects. Individual differences in
normal cognitive development (Casey et al. 2000; Liston et al.
2006; Walhovd and Fjell 2007), IQ (Miller 1994; Schmithorst
et al. 2005), normal variation in reading skill (Gold et al. 2007;
Klingberg et al. 2000; Niogi and McCandliss 2006), work-
ing memory (Nestor et al. 2007), and musical proficiency
(Bengtsson et al. 2005; Hyde et al. 2006) are correlated with
differences in white matter structure in specific brain regions
mediating these tasks.
Recently, multiple MRI studies have revealed experience-
or learning-dependent changes in white matter in the human
brain. These studies indicate changes in white matter struc-
ture, rather than differences in structure that could predispose
proficiency in certain cognitive tasks. Complex skills, such as
playing the piano, are accompanied by increased organization
of white matter structure in appropriate brain tracts involved
in musical performance (Bengtsson et al. 2005). Significantly,
the level of white matter structure increased proportionately
to the number of hours each subject had practiced the instru-
ment, indicating white matter changes in acquiring the skill
rather than performance being predetermined by a limitation
on white matter development. Learning to read (Carreiras
et al. 2009), juggle (Scholz et al. 2009), and many other skills
(Fields 2011a), alters white matter structure in the human
brain.
Magnetic resonance imaging does not provide cellular reso-
lution, however, and the effects seen by MRI could result from
various cellular changes, including effects on axon branching,
sprouting, crossing, or diameter, and responses in astrocytes,
neurons, or vascular tissue, in addition to the possibility of
effects on myelination (Zatorre et al. 2012). In some studies,
R E GU L AT I O N O F M Y E L I N AT I O N BY F U N C T I O N A L AC T I VI T Y
diffusion imaging often shows increased resistance to diffu-
sion radially from axon tracts, which would be consistent with
an increase in myelin. Rats trained in the Morris water maze
showed changes in diffusivity or anisotropy in several brain
regions, including the corpus callosum (Blumenfeld-Katzir
et al. 2011). Histological analysis confirmed that the increased
fractional anisotropy was associated with increased staining
for myelin basic protein, suggesting that the learning task had
stimulated myelination.
Independent evidence is supportive of brain imaging
data, suggesting that white matter structure can change.
Oligodendrocyte progenitor cells remain resident in substan-
tial numbers in the adult brain (Psachoulia et al. 2009). These
cells could participate in repair after myelin damage, but they
could in theory participate in learning-dependent changes in
white matter if myelination of unmyelinated axons is stimu-
lated by functional activity. Internodal lengths decrease in
visual cortex of rhesus monkeys during normal aging (Peters
et al. 2008), suggesting active remyelination throughout life.
Changes in physical activity, such as bed rest or outer space
missions, temporarily reduce conduction velocities (Ruegg
et al. 2003), and hind-limb unweighting to reduce activity in
muscle fibers causes activity-dependent changes in myelin in
the PNS of adult rat (Canu et al. 2009).
3 AC T I VI T Y-D E P E N D E N T AXO N – G L I A L
C O M MU N I C AT I O N
A broad range of signaling molecules and transcription factors
are involved in regulating glial development and myelination,
many of which could be influenced by electrical activity in
axons. Glia express a wide variety of neurotransmitter-gated and
voltage-sensitive ion channels (Karadottir et al. 2005; Ransom
and Orkand 1996; Sontheimer et al. 1996). Modulation of ion
channel activity in oligodendrocytes can influence their pro-
liferation (Chiu and Wilson, 1989; Gallo et al. 1996; Knutson
et al. 1997; MacFarlane and Sontheimer 1997; Pappas et al.
1994). Inhibition of glial potassium channel function by tet-
raethylammonium ion eliminated myelination in spinal cord
explants (Shrager and Novakovic 1995). Blocking impulse
activity in optic nerve using TTX can reduce the proliferation
rate of oligodendrocyte progenitor cells, but exposure to the
mitogen PDGF (platelet-derived growth factor) can restore
the cell numbers to normal (Barres and Raff 1993).
3.1 VE S I CU L A R A N D N O N VE S I CU L A R R E L E A S E
O F N EU ROT R A NS M IT T E R FRO M AXO N S
It is not immediately obvious how myelinating glia, far
removed from synaptic endings, could monitor functional
activity in axons. However, vesicular release of neurotransmit-
ters from axons can occur at focal regions and from ectopic
sites along the axon (Fields 2011b; Kriegler and Chiu 1993;
Wake et al. 2011). In addition, synapses can form transiently on
some OPCs in white matter (DeBase et al. 2010; Kukley et al.
2007; Ziskin et al. 2007). Both glutamatergic and GABAergic
currents have been recorded in OPCs (see also chapter 21).
• 577
 Neurotransmitters also can be released from axons in an
activity-dependent manner in the absence of functional syn-
apses and SNARE-dependent release of synaptic vesicles
(Fields and Ni 2010). Activity-dependent release of the neu-
rotransmitter ATP from axons has been shown to take place
through volume-regulated anion channels in axons that
become activated by trains of action potentials (Fields and Ni
2010), and these channels are permeable to small amino acids,
including glutamate. Inhibiting the activation of these chan-
nels disrupts calcium responses in astrocytes seen in response
to action potential stimulation in axons. Thus, myelinating
glia along axons can have access to activity-dependent release
of neurotransmitters through nonvesicular release from axons
as well as vesicular release.
3.2 OT H E R P O S S I B L E M E C H A N I S M S O F AXO N
C O M MU N I C AT I O N WI T H S C H WA N N C E L L S
A N D O L I G O D E N D RO C Y T E S
Myelin formation requires cell recognition to myelinate the
appropriate axons, the formation of adhesive contacts, elabo-
ration of vast areas of cell membrane to form myelin sheets,
wrapping many layers of membrane around axons, and the
removal of cytoplasm between the wraps of myelin to form
compact stacks of lipid membrane, all of which might be
influenced by signaling from electrical activity in axons.
A large number of signaling molecules are involved in regu-
lating development of myelinating glia and myelination,
including growth factors, cytokines, ions (K+, pH, Ca2+), neu-
rotransmitters, cell adhesion, and extracellular matrix mol-
ecules, for reviews see (Emery 2010; Nave 2010; Taveggia et al.
2010), but few of these have been investigated in the context
of activity-dependent regulation of myelination.
4 C E L LU L A R A N D M O L E C U L A R
M E C H A N I S M S O F AC T I VI T Y-D E P E N D E N T
M Y E L I N AT I O N
Five cellular–molecular mechanisms for activity-dependent
regulation of myelination have been identified; all of them
from studies in cell cultures in which electrodes were used to
stimulate axons in specific patterns. These mechanisms can
be categorized as involving: (1) cell surface molecules, (2)
diffusible signals from axons, and (3) signals from astrocytes
responding to action potentials in axons.
4.1 C E L L SU R FAC E MO L ECU L E S I N
AC T I VI T Y-D E P E N D E N T MY E L I NAT I O N
The first mechanism to be identified for effects of impulse
activity on myelination involved regulation of a cell adhe-
sion molecule on axons (Stevens et al. 1998). Neural impulse
activity was found to lower L1-CAM expression on dorsal
root ganglion (DRG) axons in cell culture (Itoh et al. 1995).
Interestingly, specific frequencies of stimulation were required
to downregulate the gene, and other cell adhesion molecules
showed different responses to different frequencies of action
578 • NEUROGLIA
potentials. L1-CAM is downregulated by 0.1-Hz stimulation
for 5 days, but not by 0.3- or 1-Hz stimulation in the same
experiments (Itoh et al. 1997). Neural cell adhesion molecule
(NCAM) was not downregulated by any stimulus frequency
tested, and N-cadherin was downregulated in proportion to
stimulus frequency between 0.1 and 10 Hz.
L1-CAM is known to be essential for the initial wrapping
of Schwann cell membrane around the axon, after which the
expression of L1-CAM decreases in both the axon and Schwann
cell (Seilheimer et al. 1989; Wood et al. 1990). After stimulat-
ing neurons at different frequencies for 5 days, Schwann cells
in coculture were stimulated to form myelin by addition of
ascorbic acid. One-third the normal number of myelin seg-
ments formed on axons stimulated at 0.1 Hz compared with
unstimulated controls. Stimulation at other frequencies had
no effect on myelination in these experiments, and myelina-
tion was not reduced when the activity-dependent downregu-
lation in L1-CAM mRNA was prevented by addition of NGF
during stimulation. No effects on the number of Schwann
cells were detected in these experiments, consistent with the
involvement of L1-CAM in the initial stages of myelination.
In this case, 0.1 Hz electrical impulse activity acts as a negative
factor on myelin initiation.
Myelination in the CNS is known to be influenced by
L1-CAM (Barbin et al. 2004), but whether this cell adhe-
sion molecule contributes to activity-dependent regulation
of myelination in the CNS has not been reported. Electrical
stimulation of mixed cortical cultures of oligodendrocytes
and neurons increases myelination through an unknown
mechanism increasing oligodendrocyte survival (Gary et al.
2012). The effects depended on the frequency of electrical
stimulation and require contact between axons and OPCs,
suggesting possible activity-dependent regulation of unidenti-
fied cell adhesion molecules on axons that promote survival of
oligodendrocytes.
4.2 D I FF US I B L E S I G NA L I N G MO L ECU L E S I N
AC T I VIT Y-D E P E N D E N T MY E L I NAT I O N
Both glutamate and ATP released from axons have been
shown to regulate myelination in an activity-dependent man-
ner. Adenosine triphosphate released from axons firing action
potentials acts on Schwann cells to inhibit their proliferation
and development to a myelinating stage (Stevens and Fields
2000). Adenosine, generated by dephosphorylation of ATP
released from axons firing action potentials, inhibits prolif-
eration of Schwann cells via a different intracellular signaling
pathway involving ERK/MAPK activation through stimula-
tion of cAMP-linked A2A adenosine receptors, but unlike
ATP, adenosine does not inhibit differentiation of Schwann
cells (Stevens et al. 2004).
In the CNS, adenosine generated by electrically active
axons stimulates myelination by inhibiting OPC prolifera-
tion and stimulating differentiation. Nonhydrolyzable P2
receptor agonist 2MeS-ATP is not effective (Stevens et al.
2002), indicating involvement of P1 adenosine receptors in
the activity-dependent increase in myelination (Stevens et al.
2002).
 Initiation of myelination is regulated by axonal signals
beyond those that affect cell proliferation or differentiation.
Experiments using botulinum toxin to block vesicular release
from axons show that electrical stimulation promotes differ-
entiation of OPCs to a myelinating stage, even in the absence
of SNARE-dependent vesicular release from axons (Wake et
al. 2011). However, myelination is strongly inhibited on axons
in which vesicular release has been blocked by prior treatment
with botulinum or tetanus toxin, indicating that vesicular
release of substances from axons promotes myelination in a
manner distinct from signals promoting differentiation of
OPCs to a myelinating stage (Fig. 45.3). Calcium imaging
shows that nonvesicular release of ATP from axons induces
calcium responses in the cell soma of OPCs, but responses
in the slender cell processes of OPCs closely associated with
axons are inhibited when vesicular release from axons is
blocked by botulinum toxin (Wake et al. 2011). This is consis-
tent with vesicular release of glutamate from axons controlling
initiation of myelination locally in individual cell processes of
oligodendrocytes.
Glutamate release from synaptic vesicles along axons of
mouse DRG neurons in culture promotes myelin induction
by stimulating the formation of cholesterol-rich signaling
domains between oligodendrocytes and axons, and increasing
the local synthesis of the major protein in the myelin sheath,
myelin basic protein, through Fyn kinase-dependent signal-
ing (Wake et al. 2011) (Fig. 45.4). This axon–oligodendrocyte
signaling would promote myelination of electrically active
axons to regulate neural development and function accord-
ing to environmental experience. Pharmacological studies
show that these effects depend on activation of mGluR and
NMDA receptors on OPCs, but not AMPA or purinergic
receptors. Block of SNARE-dependent vesicular release from
axons could also impede activity-dependent delivery of other
proteins to the axonal membrane that may be involved in
myelination.
Even though the newly synthesized myelin basic protein
is generated locally in the OCP cell process in contact with
electrically active axons, if the myelin protein were free to
migrate within the lipid membrane, the effects of electrical
activity would not be confined specifically to the axon firing
action potentials. Studies using photoactivated GFP-labeled
myelin basic protein show that the diffusion of MBP in the
OPC membrane becomes highly restricted to discrete regions
adjacent to axons firing action potentials unless vesicular
release is blocked by botulinum toxin. This suggests that vesic-
ular release of glutamate from axons promotes formation of
a cytoskeletal membrane complex in oligodendrocytes that
restrict MBP to appropriate membrane regions adjacent to
the axon being myelinated.
4.3 A S T RO C Y T E S I N AC T I VIT Y-D E P E N D E N T
MY E L I NAT I O N
The fourth mechanism known for activity-dependent myeli-
nation involves activation of astrocytes by ATP released from
electrically active axons (Ishibashi et al. 2006). This stimu-
lates release of the cytokine LIF from astrocytes, which acts
to promote myelination by mature oligodendrocytes. This
activity-dependent pathway acts on mature oligodendrocytes,
in contrast with the effects of adenosine, which inhibits pro-
liferation at the progenitor stage and promotes differentiation
(Stevens et al. 2002).
5 SIGNIFICANCE OF
AC T I VI T Y-D E P E N D E N T M Y E L I N AT I O N
In theory, activity-dependent effects on myelination
would have profound effects on information process-
ing in the nervous system, but this is only beginning to be
explored. Environmental experience, physical therapy, and

A B

Figure 45.3 Vesicular Release from Axons Promotes Myelination. A. Compact myelin (green) forms on mouse dorsal root ganglion axons (purple)
stimulated to fire action potentials in cell culture. B. When oligodendrocyte progenitor cells are plated onto axons that have been previously treated
with botulinum toxin to inhibit vesicular release from axons, electrical stimulation promotes differentiation of oligodendrocytes, but the formation of
myelin is greatly inhibited. Myelin basic protein (green), neurofilament (purple), nuclei (blue). Adapted from Wake et al. 2011.

R E GU L AT I O N O F M Y E L I N AT I O N BY F U N C T I O N A L AC T I VI T Y • 579
 GluR +
axon

lam
+

ini
n
L1
integrin
F3

NMDA

mGluR

M
+ +

BP
cholesterol rich micro domain Fyn
P P translation

M
A2

+ BP

AAAAAA
mic

A2 MBP mRNA
rot
ubl
e

Figure 45.4 Vesicular release of glutamate from axons firing action potentials stimulates the formation of cholesterol-rich signaling domains between oligo-
dendrocytes and axons, and induces local protein synthesis of myelin basic protein from mRNA in OPC cell processes in contact with electrically active axons.
This signaling is dependent upon NMDA and mGluR receptor activation and Fyn kinase (Fyn) intracellular signaling. From Wake et al. 2011.

electrical stimulation can promote recovery from injury, and
activity-dependent myelination could contribute. Electrical
stimulation in vivo has been used to promote recovery from
spinal cord injury by increasing the pool of oligodendrocytes
in adult rats (Becker et al. 2010), for example. Myelination of
regenerated axons is required to restore function.
5.1 EFFEC T O F MY EL I N P L A S T I C I T Y O N
I N F O R M AT I O N P RO C E S S I N G
Activity-dependent myelination could contribute to nervous
system plasticity by optimizing the speed of impulse conduc-
tion. The speed and synchrony of impulse traffic between
distant cortical regions is critical for optimal mental perfor-
mance and learning, and myelin is one of the most important
factors affecting impulse conduction velocity. Millisecond
precision is necessary for the coincident arrival and summa-
tion of synaptic signals, because the postsynaptic potential
change is only 2 to 4 milliseconds in duration. Thus, axonal
conduction time is a critical variable in information pro-
cessing and synaptic function. The human corpus callosum
is largely unmyelinated at birth, for example, and in adults
30% of the fibers remain unmyelinated. The conduction time
between the left and right hemispheres is 30 milliseconds
through myelinated callosal fibers and 150 to 300 millisec-
onds through unmyelinated fibers (Swadlow et al. 1978).
Synaptic integration is affected profoundly by whether an
intercallosal axon becomes myelinated or remains unmyeli-
nated. Considering the many variables affecting conduction
delays, genetic instruction alone would seem inadequate to
specify the optimal conduction velocity in every axon, sug-
gesting the possibility of regulating impulse conduction
velocity and myelin by functional activity. Consistent with
this theory, conduction velocity varies widely among differ-
ent axons (over 100-fold) and many axons are slowly con-
ducting and ummyelinated.

580 • NEUROGLIA
 Rushton (1951) noted that along peripheral nerve trunks,
myelin thickness, and intermodal distance are adjusted so as
to maximize conduction velocity and thereby optimize func-
tion. Interestingly, many cases are known in which myelin
is structured not to maximize the speed of conduction of
action potentials, but instead to ensure simultaneous arrival
of impulses from multiple points that are separated by large
differences in distance (Waxman 1997). The olivocerebel-
lar projections carrying information to Purkinje cells in the
cerebellum (Sugihara et al. 1993), the electromotor axons of
certain electric fish (Waxman et al. 1972), and the axons of
retinal ganglion cells located at different eccentricities within
the retina (Stanford 1987) show differences in conduction
times that are adjusted by the structure of myelin to provide
simultaneous arrival of impulses within millisecond preci-
sion. There is an optimal spacing of nodes of Ranvier in the
auditory brain stem circuits of the barn owl to adjust conduc-
tion velocities appropriately to detect interaural time differ-
ences (Carr and Konishi 1990). These examples demonstrate
that the relationship between axons and myelinating glia are
in some way regulated to meet the precise functional require-
ments of individual axons.
Long-term electrophysiological recording shows that con-
duction velocity through axons can change. The conduction
velocity of one-third of callosal axons changes (increases or
decreases) over 1 year of chronic recordings in rabbit (Swadlow
1985), and conduction velocity and neural synchrony increase
in auditory circuits 1 year after congenitally deaf children
receive cochlear implants to restore hearing (Gordon et al.
2003). One of the largest categories of genes in which expres-
sion changes during sleep are genes that control oligodendro-
cyte development and myelination (Cirelli et al. 2004). The
reason for this is unclear, but sleep is linked to consolidat-
ing memory. As the human body grows, somatosensory and
motor conduction delays in CNS pathways remain constant
after 2 years of age despite substantial increases in axon length
with body growth, reflecting a compensatory increase in con-
duction velocity in CNS axons (Eyre et al. 1991). Interestingly,
this compensation does not occur in the peripheral nervous
system, and conduction delays increase in proportion to the
length of a growing limb. Conduction delays in the PNS are
relatively minor relative to the time required for integration in
the CNS, perhaps lessening the need for activity-dependent
regulation of PNS myelination.
5.2 E A R LY C H I L D H O O D E X P E R I E N C E
The human brain continues to undergo myelination until at
least the third decade of age, and the frontal regions of the
cerebral cortex, which carry out higher-level executive func-
tions, are the last to become myelinated. This prolonged
period of postnatal brain development may allow myelination
to be influenced by the specific environment that an indi-
vidual experiences during rearing rather than being specified
exclusively by genetic instruction. Early experience increases
white mater structure in the internal capsule and frontal
lobes in newborn human infants in parallel with improved
performance in behavioral tests (Als et al. 2007). Conversely,
R E GU L AT I O N O F M Y E L I N AT I O N BY F U N C T I O N A L AC T I VI T Y
children suffering severe childhood neglect have a 17% reduc-
tion in corpus callosum area (Teicher et al. 2004). A recent
study shows that environmental influences during the period
when the corpus callosum is undergoing myelination can have
lasting effects on white matter structure and behavior. In this
MRI study, individuals who reported experiencing verbal
abuse as middle school children had abnormal white mat-
ter development in the corpus callosum and they exhibited
increased psychiatric problems as adults (Teicher et al. 2010).
5.3 WH IT E M AT T E R P L A S T I C IT Y A N D
P SYC H I AT R I C I L L N E S S
Although synaptic dysfunction is the cellular basis for most
mental illnesses, disruptions in functional connectivity between
distant brain regions can impair information processing in
association with a range of neurological processes. Defects
in myelin insulation can lead to impaired cognitive function
in 40% of multiple sclerosis patients, for example (Kujala et al.
1997). Cognitive decline in aging also parallels subtle changes
in the integrity of white matter (Gootjes et al. 2004). This sug-
gests that impaired cognitive ability, disorganized thinking,
mood disorders or hallucinations, and accompanying psychiat-
ric illness might result from slowed or desynchronized impulse
conduction between distant cortical regions.
A diverse range of psychiatric disorders are accompa-
nied by changes in white matter structure evident by MRI
or abnormalities in myelin genes. Polymorphisms for sev-
eral myelin genes have emerged as unexpected risk factors
for schizophrenia (Hakak et al. 2001; Kubicki et al. 2005;
Tkachev et al. 2003), depression (Tkachev et al. 2003),
and obsessive-compulsive disorder (Stewart et al. 2007).
Post-mortem examination of brain tissue from patients suf-
fering schizophrenia (Georgieva et al. 2006; Steingard et al.
2002; Tkachev et al. 2003), major depression (Aston et al.
2005), and bipolar disorder (Tkachev et al. 2003) reveals
reduced abundance of several mRNA transcripts of myelin
genes or genes regulating differentiation and survival of
myelin-forming cells. Disrupting Neuregulin 1 signaling in
transgenic mice that express a dominant negative erbB recep-
tor in oligodendrocytes, results in white matter defects and
behavioral changes resembling schizophrenia in humans (Roy
et al. 2007).
6 FUTURE DIRECTIONS
The cellular mechanisms that have been identified thus far
for activity-dependent myelination all affect myelination
of unmyelinated axons, but it is unclear whether functional
activity can influence myelin or nodal structure once formed.
Because many axons remain unmyelinated throughout life, it
is possible that functional activity may promote myelination of
unmyelinated axons throughout life, but this is not adequately
verified experimentally. Histological analysis after brain imag-
ing in experiments studying environmentally induced white
matter changes should provide answers to these critical ques-
tions in forthcoming years.
• 581
 In theory there are many structural modifications of mye-
linated fibers that could affect impulse conduction, and there
is some circumstantial evidence in support of this possibility.
Myelin can influence conduction velocity by regulating axon
diameter, thickness of the myelin sheath, the number and
spacing of nodes of Ranvier, and nodal structure and molec-
ular composition of ion channels in the node and paranodal
regions.
Large-caliber fibers conduct impulses at higher speeds
because the resistance to electrical current is reduced as the cal-
iber of the fibers increases. However, myelin can also regulate
axon diameter (Sanchez et al. 1996). This is obvious at nodes
of Ranvier, in which the axon often shows marked changes in
diameter in the unmyelinated nodal regions. Axon diameter
is reduced in myelin-deficient mutants (Cole et al. 1994), and
signaling from myelin proteins, such as myelin-associated
glycoprotein (MAG) has been implicated in the signaling
cascade controlling neurofilament phosphorylation which
in turn affects axon caliber and axoplasmic transport (Hsieh
et al. 1994; Lunn et al. 2002; Yin et al. 1998).
The thickness of the myelin sheath can have a dominant
influence on conduction velocity. Myelin sheaths thicker or
thinner than the theoretical optimal g-ratio of 0.65 will reduce
conduction velocity (Rushton 1951). (The g-ratio is the ratio
of axon diameter divided by total fiber diameter, including the
myelin sheath.) There is, however, large variation in g-ratio
around this optimal mean among different myelinated fibers.
Even along the same fiber the conduction velocity is not always
constant along the length of an axon (Baker et al. 1990; Traub
and Mendell 1988).
The number of wraps of myelin increases as animals grow
and their axons lengthen, showing that myelin production is
a perpetual process and that the g-ratio can change (Berthold
et al. 1983). Increased myelin thickness can compensate for
increased internodal distance during body growth, which
would otherwise reduce conduction velocity (Friede, et al.
1985; Hara et al. 2003; Schro¨der et al. 1978). Experimentally
increasing nodal distance, by lengthening the femur of a rat,
increases myelin protein expression 160% (Hara et al. 2003),
suggesting a feedback signaling between changes in internodal
distance and myelin thickness to regulate conduction velocity.
Dimensions of the node or the morphology of the para-
nodal loops of myelin flanking the node may affect con-
duction velocity, but little is known about whether these
change. Swelling of paranodal region after intense repetitive
activation has been observed (Wurtz and Ellisman 1986).
Myelinating glia have a major influence on localization
of ion channels into concentrated domains at the node
during development through contact and diffusible fac-
tors (Dupree et al. 2004). The types of ion channels and the
spatial localization of channels in the axon, most prominently
concentration of sodium channels in the node and potas-
sium channels clustered in the paranodal region, affect the
excitability, frequency of impulse firing, refractory period,
and velocity of impulse conduction. In addition to affect-
ing conduction velocity, myelin also decreases the refractory
period, thus enabling transmission of spike trains at a higher
frequency.
582 • NEUROGLIA
7 S U M M A RY A N D P E R S P E C T I VE S
White matter is essential for proper impulse conduction, and
connectivity between distant cortical regions with precise
timing of transmission is critical for higher-level cognitive
functions. The new evidence showing white matter plas-
ticity in response to electrical activity widens the scope of
investigation into learning and functional plasticity beyond
the synapse to include the transmission of information through
neural networks. Changes in white matter have been seen
in humans in response to environmental experience and
learning, and extrapolating from cellular and animal stud-
ies, myelination could have a role. White matter differences
that correlate with the scoring on intelligence quotient
tests (Schmithorst et al. 2005) and certain psychiatric
conditions (Fields 2008) may be attributed in part to a direct
role for white matter plasticity in learning and cognitive
function.
AC K N OW L E D G M E N T S
This work is supported by funds for intramural research from
NICHD.
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 46.
IRON AND GLIA
James R. Connor

A B B R E VI AT I O N S transport and uptake proteins in the context of their specific

contributions to glial cell biology.
ATP Adenosine triphosphate
BBB blood-brain barrier
CSF cerebrospinal fluid 2 IRON AND GLIA
DAB 3-3´diaminobenzidine
DMT1 divalent metal transporter 1 Iron and Glia has both a distinctive regional and cellular distri-
HFE human hemochromatosis bution. Iron is quantifiable using atomic absorption spectros-
HMG-CoA 3-hydroxy-3-methylglutaryl-coenzyme A copy or can be visualized using a histochemical stain known as
IFN interferon the Perl reaction. At the regional level, iron is most prominent
IL interleukin in areas associated with motor functions such as basal ganglia,
IRE Iron Responsive Element substantia nigra (pars reticulata), and deep cerebellar nuclei.
LPS lipopolysaccharid Indeed the iron concentration in the globus pallidus is report-
MAG myelin-associated glycoprotein edly similar to that found in the liver (Hallgren and Sourander
MBP myelin basic protein 1958). The liver is considered the main iron storage organ in
MS multiple sclerosis the body. This proclivity for iron in regions associated with
NF- κB nuclear factor kappa-light-chain-enhancer the motor system is consistent among all animal species inves-
of activated B cells tigated (Guizzetti 1915; Spatz 1922) from rodents to monkeys
OPC oligodendrocyte precursor cell to humans. Moreover, there is also a high concentration of
PND postnatal day iron in white matter (Connor and Menzies 1990; Hallgren
RBC red blood cell and Sourander 1958; Rajan et al. 1976).
sla sex-linked anemia At the cellular level, the cells that most prominently stain
SVZ subventricular zone for iron are oligodendrocytes. Iron enriched oligodendro-
Tf transferrin cytes are found in white matter and even in the gray matter
TfR transferrin receptor regions where iron is enriched in select brain nuclei such as
Tim-2 T-cell immunoglobulin mucin domain the substantia nigra (Fig. 46.1A) and striatum (Fig. 46.1B).
2 protein Iron-positive oligodendrocytes are also found in lower animal
TNF tumor necrosis factor forms such as chickens but not frogs or fish (Erb et al. 1996),
WM white matter suggesting that the function of iron in oligodendrocytes is
WML white matter lesion not limited to myelin. The latter comment is perhaps consis-
tent with reports of perivascular and perineuronal oligoden-
drocytes staining for iron in rodents and humans (Fig. 46.2).
1 INTRODUCTION Nonetheless, peak uptake of iron in the rodent brain occurs
in concert with peak times of myelination (Taylor et al. 1991;
Iron is an essential metal in biological systems. It is necessary Taylor and Morgan 1990) and is consistent with oligodendro-
for the consumption of oxygen and production of adenos- cytes in white matter staining robustly for iron. Iron is report-
ine triphosphate (ATP). It is also an essential cofactor for edly associated with myelin sheaths (Francois 1981). Not all
many enzymes including those involved in cholesterol and oligodendrocytes in the white matter can be seen following an
neurotransmitter synthesis, and in degradation of proteins iron stain.1 In the human brain it is especially obvious that the
that require ubiquitination. Because of the ability of iron to iron-positive oligodendrocytes in white matter are organized
interact with oxygen, it can also be a potent generator of free
radicals. Thus, cells have developed an elegant system for keep- 1
There was confusion and controversy when iron staining in the brain was first
ing iron bound to proteins during delivery to cells and stor- reported in the white matter. Iron staining in the brain is significantly affected by
age within cells. The discovery of iron regulatory proteins fixation, storage in formalin where the formalin breaks down into formic acid and
including those involved in export of iron is an evolving story iron leeches out of the tissue. Moreover, iron staining in paraffin and frozen sec-
tions can be difficult to detect. The modification of 3-3’diaminobenzidine (DAB)
and the data to date are summarized in Table 46.1. Thus, this intensification to iron staining first described in 1980 was a significant advance to
chapter will focus on iron but will include discussions of iron the study of brain iron histology. Nguyen-Legros et al. 1980.

586
Table 46.1 PROTEINS INVOLVED IN IRON HOMEOSTASIS FOR GLIA
CELL TYPE PROTEIN FUNCTION REFERENCE

Oligodendrocytes Tf Nonsecreted form transports Fe within cell and is Bloch et al. 1985, Espinosa de los Monteros
required for maturation and myelinogenesis; taken et al. 1994, Bartlett, Li and Connor 1991,
up by OPCs de Arriba Zerpa et al. 2000, Møllgård et al.
1988

Tf R1 Expressed only by OPCs to take up iron via Tf Moos et al. 2007

endocytosis

Tim2 Ferritin receptor that may allow for iron to be Todorich et al. 2008, Todorich, Zhang and
taken up and incorporated in endogenous Tf Connor 2011

CXCR4 Interacts with ferritin; is important for Patel et al. 2010, Dziembowska et al. 2005,
oligodendrocyte migration and maturation Carbajal et al. 2011, Li et al. 2006

Hephastin Ferroxidase that mediates iron efflux in mature Schulz et al. 2011
oligodendrocytes
No ceruloplasmin Schulz et al. 2011
2+ +
DMT1 Fe and H symporter into cytosol; low expression Burdo et al. 2001
in subcortical white matter
Ferroportin Fe2+ efflux into cytosol; associated with hephaestin Burdo et al. 2001
Ferritin Iron storage and ferroxidase Connor and Menzies 1996, Blissman et al.
1996
IRP1 Iron sensor that modulates the translation of Hentze et al. 1987, Casey et al. 1989
ferritin and Tf R by binding to an IRE; cytoplasmic
aconitase activity
IRP2 Iron sensor that modulates the translation of ferritin Magaki et al. 2007
and Tf R by binding to an IRE

Microglia No Tf Benkovic and Connor 1993

No Tf R1 Lin and Connor 1989

Ferritin Iron storage and ferroxidase; secreted ferritin can Todorich et al. 2008, Todorich et al. 2011,
associate with receptors on oligodendrocytes Zhang et al. 2006, Cheepsunthorn, Palmer
and Connor 1998

CXCR4 Interacts with ferritin but iron related function is Lavi et al. 1997
unknown

HFE Low expression in CD68+ activated microglia in Gebril et al. 2011, Roy et al. 2004
white matter; associates with Tf R1 to reduce iron
uptake and is involved in innate immunity

IRP1 Iron sensor that modulates the translation of Hentze et al. 1987, Casey et al. 1989,
ferritin and Tf R by binding to an IRE; cytoplasmic Rathnasamy, Ling and Kaur 2011
aconitase activity

IRP2 Iron sensor that modulates the translation of ferritin Rathnasamy et al. 2011
and Tf R by binding to an IRE

Astrocytes Ceruloplasmin Ferroxidase that converts Fe2+ to Fe3+ so that it Patel and David 1997, Patel, Dunn and
can be exported by ferroportin David 2000

APP Ferroxidase that converts Fe2+ to Fe3+ so that it Rogers et al. 1999, Rogers et al. 2002, Duce
can be exported by ferroportin; increased protein et al. 2010
expression in response to IL-1

Ferroportin Fe2+ efflux into cytosol; associated with Wu et al. 2004

ceruloplasmin
Hepcidin Modulates iron export by binding to ferroportin Nemeth and Ganz 2006, Zechel,
and inducing internalization Huber-Wittmer and von Bohlen und
Halbach 2006

(continued)

IRON AND GLIA • 587

Table 46.1 (Cont’d)
CELL TYPE PROTEIN FUNCTION REFERENCE

DMT1 Fe2+ and H+ symporter into cytosol Burdo et al. 2001

Ferritin Iron storage and ferroxidase Connor et al. 2003

No Tf Connor and Benkovic 1992

No Tf R1 Moos 1996

HFE Low expression in white matter; associates with Gebril et al. 2011
Tf R1 to reduce iron uptake

IRP1 Iron sensor that modulates the translation of ferritin Hentze et al. 1987, Casey et al. 1989
and Tf R by binding to an IRE; cytoplasmic aconitase
activity
IRP2 Iron sensor that modulates the translation of ferritin Magaki et al. 2007
and Tf R by binding to an IRE

into patches (Todorich et al. 2009) (Fig. 46.3) but the patches
of iron-positive oligodendrocytes are also seen in rodents; to
visualize the patches in the rodents usually requires thicker
histological sections than standard paraffin or cryostat sec-
tions. The patches oligodendrocytes are only seen following
staining for iron. Those oligodendrocytes in the white mat-
ter that stain for transferrin, the iron mobilization protein, or
ferritin (iron storage protein) are not organized in discern-
ible patches (Connor and Menzies 1996a; Dwork et al. 1988)
(Fig. 46.4A,B). It appears, then, that some oligodendrocytes
contain much more stainable iron than others; this could be a
clinically meaningful observation because iron enriched oligo-
dendrocytes are more vulnerable to oxidative stress (Thorburne
and Juurlink 1996) and thus could be more susceptible to
inflammatory responses in diseases such as multiple sclerosis
(MS) ( see chapter 61). The relationship between the patches
of iron-positive oligodendrocytes and plaque formation in
MS or white matter (WM) lesions in the aging brain in which
iron distribution is altered (Bagnato et al. 2011; Gebril 2011)
has not been investigated and would be difficult to do so, but
the idea that patches of iron enriched oligodendrocytes are the
preferred site for initiation of MS or WM lesions with aging
is intriguing. Perhaps, as resolution with neuroimaging tech-
niques improves, the relationship between iron and WM his-
topathology can be interrogated (Bagnato et al. 2011).
The patches of iron-positive oligodendrocytes appear to
form early in development and are located around blood vessels.
The circumference of the patch increases with age and reaches

A B

Figure 46.1 Iron in the Brain. A. Visualization of iron in the substantia nigra of the mouse. Oligodendrocytes of the substantia nigra reticulata
contain most of the iron of the nigra and appear as small, dark, round cells. The substantia nigra pars compacta (bounded by the dashed line), in which
dopaminergic neurons that require iron for catecholamine production are located, contains comparatively less iron than the reticulata. The inset
provides higher magnification of oligodendrocytes in the reticulata. B. In the mouse striatum, iron-positive oligodendrocytes are robustly stained in
the white matter striations. These cells have few processes and appear “capped” at ends because of the eccentric position of the nucleus within the cell.
Other oligodendrocytes with the same capped morphology appear outside of the white matter patches.

588 • NEUROGLIA

bv
50 μm
Figure 46.2 DAB enhanced Perl reaction was used to stain iron in the stria-
tum of mouse brain. Iron-positive oligodendrocytes were located in the white
matter tract (arrows) and near blood vessels (rectangular box). The insert is a
representative of perivascular oligodendrocytes. bv, Blood vessel.
adult patterns by postnatal day (PND) 28 in the rat (Connor
et al. 1988). The appearance of transferrin in oligodendrocytes,
an early marker of oligodendrocyte differentiation (Espinosa
de los Monteros et al. 1988), in the developing optic nerve
has a similar relationship to blood vessels (Lin and Connor
1989b). This early relationship between iron accumulation by
oligodendrocytes and blood vessels is consistent with histori-
cal studies on the initiation sites of myelinogenesis in white
matter tracts, which were thought to occur near blood vessels.
The concept of iron accumulation by oligodendrocytes occur-
ring coincident with onset of myelin is consistent with numer-
ous reports of hypomyelination in iron deficiency. For example,
in studies on iron deficient children there is increased latency
100 μm
Figure 46.3 Iron visualization in the brain using the DAB-Perl stain. This
image from the human white matter tract demonstrates the “patchy” nature
of iron staining in white matter. The inset shows oligodendrocytes aligning
in a classic row-like pattern, which is a hallmark characteristic of these
cells.
IRON AND GLIA
of auditory and visual evoked potentials, which is interpreted
as insufficient myelination (Lozoff et al. 2006). In rodents,
in which myelin can be directly measured, restricting iron in
the diet during development results in decreased expression
of myelin proteins, lipids, and cholesterol (Beard et al. 2003;
Ortiz et al. 2004a; Yu et al. 1986). In contrast, it has recently
been reported that a mutation in the human hemochromato-
sis (HFE) protein that normally would limit iron uptake into
cells is associated with higher myelination ( Jahanshad et al.
2012). Because loss of myelin with age has been linked to cog-
nitive decline with aging (Bartzokis 2004), this mutation in
the HFE protein is argued to preserve cognitive function with
age; an intriguing concept that should be explored.
The accumulation of iron by oligodendrocytes is not depen-
dent of the structural integrity of myelin. In rodent models
with abnormal myelin, as long as oligodendrocytes develop
normally, they accumulate iron. (Connor and Menzies 1990;
LeVine 1991). On the other hand, if oligodendrocyte develop-
ment is compromised, the iron accumulates in astrocytes and
microglia in the white matter (Connor and Menzies 1990).
2.1 D EV E L O PM E N T
As mentioned, peak iron uptake into the brain coincides
with peak myelinogenesis in the rat brain (Taylor and
Morgan 1990). However, iron-positive cells in the rat brain
can be detected as early as PND 3 (Connor et al. 1995a). The
iron-positive cells at this time period are primarily found in
the subventricular zone (SVZ) and subcortical white matter
tracts. The morphological appearance and apparent migra-
tion of the iron-positive cells from SVZ to subcortical white
matter mirrors that of GD3+ oligodendrocyte precursor
cells (LeVine and Goldman 1988). The iron-positive cells
in the SVZ are large and round and non–process-bearing
(Connor et al. 1995a; Rathnasamy et al. 2011), whereas those
farther away from the SVZ have more processes and a smaller
soma. Over time, the cellular processes become shorter and
thinner until the cells become aligned with other iron-posi-
tive cells in the white matter and lose their processes. Many
iron-positive oligodendrocytes are seen in clusters around
blood vessels. It is not known if the oligodendrocytes around
the blood vessels migrated to that site with iron or began to
accumulate iron when they came in proximity to the blood
vessels. Oligodendrocytes from the SVZ do migrate to sites
near blood vessels (Levison et al. 1993; Levison et al. 1993).
The circumference of the patch expands from the blood vessel
with age in developing animals and in the Belgrade rat model
in which iron uptake is compromised, the circumference of
the patch of oligodendrocytes around the blood vessel is
smaller (Burdo et al. 1999). These data strongly suggest that
the oligodendrocytes are accumulating iron in situ as opposed
to migrating into the subcoritical white matter with iron.
The functional significance of the iron accumulation near
blood vessels is not clear but could relate to myelinogenic
foci near blood vessels (Skoff et al. 1980). The contribu-
tion to iron management in the brain by the different glial
cell types and oligodendrocyte development is shown in
Schematic 46.1
• 589
 A B

LV

50 μm 100 μm
10 μm

Figure 46.4 Transferrin and Ferritin in Oligodendrocytes. A. Immunohistochemical staining for Transferrin in the corpus callosum of mouse brain.
Transferrin-positive oligodendrocytes appear in a row in the corpus callosum. Very few of the cells are process bearing. The inset shows a representative
image of two transferrin-positive oligodendrocytes. The ependymal cells lining the lateral ventricles are also stained for transferrin (arrows). LV, Lateral
ventricle. B. Ferritin immunostaining of oligodendrocytes in human putamen. In contrast to iron distribution in white matter, ferritin immunostained
cells are homogeneously spread throughout the tissue. Arrowheads denote examples of oligodendrocytes aligned in rows; the inset provides higher
magnification of these cells.

The need for iron for oligodendrocytic maturation and
function is established. Although no direct regulatory roles
for iron in oligodendrocytes have been identified (i.e., there
are no known iron regulatory elements on the mRNAs of
myelin proteins) the list of enzymes and metabolic processes
that are unique to or highly expressed in oligodendrocytes
is extensive (Table 46.2). For example, glucose-6-phosphate
dehydrogenase is a key enzyme in the pentose-phosphate
shunt pathway used by oligodendrocytes for metabolism of
glucose (Cammer et al. 1982; Kugler 1994; Sanchez-Abarca
et al. 2001; Warringa et al. 1987). There are two steps in the
synthesis of cholesterol, a key component of myelin, that

OPC Oligodendrocyte Microglia

Key

Fe2+ Tim2

Fe3+
Endolysosome
Astrocyte DMT1
Ferritin/iron

TF
Ceruloplasmin

TFR Ascorbate

Schematic 46.1 Oligodendrocyte progenitor cells (OPCs) express transferrin receptors (TfR) and the ferritin receptor Tim2, which allow for iron uptake
from transferrin and ferritin. The iron is released from these two proteins while they are within an endolysosome. As the OPC matures into an oligo-
dendrocyte, TfR expression is reduced and Tim2 expression remains. Microglia secrete ferritin, which can bind to Tim2 that is expressed by OPCs
and mature oligodendrocytes, allowing for continuous iron uptake. Indeed this expression of Tim2 and the amount of iron deliverable via ferritin
may be a necessary step for oligodendrocyte maturation. It has been proposed that astrocytes release ascorbate, which can associate with ferrous iron.
Ferrous iron can then be taken up by the astrocyte and be used within the cell. Astrocytes also express ceruloplasmin, which is a ferroxidase that con-
verts ferrous iron into ferric iron. Ferric iron binds to transferrin, which can then bind to TfR and be taken up by a cell.

590 • NEUROGLIA
Table 46.2 IRON DEPENDENT ENZYMES IN GLIA
ENZYME FUNCTION REFERENCES
Ribonucleotide DNA synthesis that Wrigglesworth and Baum
reductase catalyzes rate-limiting 1988
step
Cytochrome Electron transport chain Wrigglesworth and Baum
oxidases 1988
Succinate Citric acid cycle and Friede 1962
dehydrogenase electron transport chain
NADH Electron transport chain Wrigglesworth and Baum
dehydrogenase 1988
Aconitase Citric acid cycle Wrigglesworth and Baum
1988
HMG-CoA Cholesterol syn- Cammer 1984
reductase thesis: enriched in
oligodendrocytes
Lipooxygenases Lipid synthesis: Shimizu and Wolfe 1990
enriched in
oligodendrocytes
requires iron including 3-hydroxy-3-methylglutaryl-coenzyme
A (HMG-CoA) reductase, which is the rate limiting step in
cholesterol synthesis and this enzyme is enriched in oligo-
dendrocytes (Nelissen et al. 2012; Pleasure et al. 1977). Lipid
synthesis and breakdown is also iron dependent (Bourre et al.
1984;). Iron is essential for cytochrome oxidase activity and
the production of ATP. Thus, in addition to a direct impact
on myelin synthesis, iron is also essential to support the meta-
bolic activity of oligodendrocytes. The relatively high content
of iron in the oligodendrocytes may reflect their reported
relatively high metabolic rate compared to other cells (Hyden
1960; Sanchez-Abarca et al. 2001). In support of the concept
that iron is metabolically essential to oligodendrocytes is the
consistent observation in studies that there is a general loss
of myelin and myelin proteins as opposed to loss of a specific
myelin component following iron deficiency.
That iron is required for myelin production was clev-
erly shown in a study using gastrostomy-reared rats that had
only the amount of iron changed in the milk substitutes that
the animals were fed. This approach eliminated concerns of
undernutrition that may accompany studies on dietary iron
deficiency. In this study, there were not extensive growth defi-
cits in the body or brain but the iron deficiency was associated
with a dramatic delay in the development of myelination (de
los Monteros et al. 2000).
The question of delay in myelination associated with
dietary iron deficiency is significant from a standpoint of
clinical intervention. The World Health Organization has
identified iron deficiency as the leading nutritional problem
in the world based on the cognitive and behavioral deficits
that are sustained into adulthood in children who were iron
deficient. Recent data has suggested that attention deficit dis-
order could be associated with iron deficiency during develop-
ment (Cortese et al. 2009) and myelin impairment (Luders
IRON AND GLIA
et al. 2009). The association between myelinogenesis and iron
and the cells involved are discussed later in this chapter, but
we have proposed that there are windows of opportunity that
need to be identified for rescuing a myelin deficit in the brain.
Moreover, a key question is whether iron deficiency impacts
the development and migration of oligodendrocytes or just
their ability to make myelin. This remains a key biological and
clinically important question—is there a sufficient number
of oligodendrocytes available in the white matter to rescue a
developmental hypomyelination caused by iron deficiency?
2.2 T R A NS F E R R I N
The early studies reporting on transferrin in oligodendrocytes
considered that iron could be released from oligodendro-
cytes via transferrin because transferrin is an iron mobiliza-
tion protein and is secreted by the liver and by the choroid
plexus (Watanabe et al. 1990; Zakin et al. 2002). Thus it was
logical to think that transferrin should also be secreted by
oligodendroyctes until a study by Zakin and colleagues (de
Arriba Zerpa et al. 2000) demonstrated that the mRNA for
transferrin lacks a secretory signal which distinguishes it from
liver transferrin. Thus, transferrin in oligodendrocytes was
not secreted, making them the only cells that synthesize but
do not secrete transferrin. The lack of secretion of transfer-
rin emphasizes the importance of iron in oligodendrocytes.
Moreover, the transferrin receptor is found primarily on neu-
rons in gray matter thus transferrin bound iron would have to
leave the oligodendrocytes in the white matter and make its
way to neurons in the gray matter, which is not the optimal
design for iron delivery to neurons. The current paradigm is
that the transferrin in the extracellular space in the brain is
likely from cerebrospinal fluid (CSF) and transport across the
blood-brain barrier (BBB).
What is the role of transferrin in oligodendrocytes?
Transferrin is expressed very early in oligodendrocyte matu-
ration in culture, colocalizing with myelin basic protein but
appearing before the expression of galactocerebroside in cul-
ture (Connor et al. 1993; Espinosa de los Monteros et al. 1988;
Espinosa de los Monteros and de Vellis 1988; Espinosa de los
Monteros et al. 1999). In vivo, transferrin (Tf ) expression
in oligodendrocytes preceded both galactocerebroside and
myelin basic protein (Lin and Connor 1989). Overexpression
of transferrin in oligodendrocytes will accelerate their differ-
entiation and is associated with more myelin in vivo (Saleh
et al. 2003; Sow et al. 2006). The expression of transferrin in
oligodendrocytes occurs even when myelin integrity is com-
promised as in the shiverer mouse mutant (Connor et al. 1993)
and the myelin deficient rat model (Lin and Connor 1989). If
oligodendrocyte development is compromised, however, the
expression of transferrin at both the protein and mRNA is
decreased (Bartlett et al. 1991). Studies on transferrin mobi-
lization within oligodendrocytes suggest that it is transported
in clathrin coated vesicles from cell bodies and into the oligo-
dendrocytic processes wherein it promotes stabilization of the
processes (Ortiz et al. 2005). Indeed, in transgenic mice over-
expressing transferrin, there is an increase in several microtu-
bule-associated proteins in the white matter particularly in
• 591
the stable tubule only peptide supporting the argument that
transferrin stabilizes processes in oligodendrocytes (Marta
et al. 2002). Thus, it appears that there is trafficking of endo-
somes in oligodendrocytes for iron delivery throughout the
cell and that trafficking into the oligodendrocyte processes
may be directly related to formation of myelin membranes and
perhaps consistent with the observations of iron in the myelin
processes that was noted by electron microscopy.
2.3 I RO N AC Q U I S I T I O N
Iron acquisition by developing oligodendrocytes has received
considerable attention in both cell culture and in vivo studies.
Transferrin was considered an obligatory protein for oligo-
dendrocytes in culture until recently when we demonstrated
that transferrin could be replaced by H-ferritin (Todorich
et al. 2011). The requirement of transferrin for oligodendro-
cyte survival in culture was incongruent with studies on the
distribution of transferrin receptors in the brain that consis-
tently failed to detect transferrin receptor expression in white
matter (Hill et al. 1985; Hulet et al. 1999a,b; Mash et al. 1990)
except during very early developmental periods (Lin and
Connor 1989b). Although one could argue that the lack of Tf
receptor expression on oligodendrocytes was a result of down-
regulation of the receptor on these cells because they had high
intracellular iron stores, there was no detectable expression of
Tf receptors even under conditions of dietary iron deficiency
that were sufficient to cause hypomyelination (Han et al.
2003). The data strongly argue that transferrin receptors
are not expressed on mature oligodendrocytes in vivo. We
demonstrated that receptors for H-ferritin were selectively
expressed in the white matter (Hulet et al. 1999a) and thus
proposed that H-ferritin was the major iron delivery protein
for oligodendrocytes.
The first suggestion that H-ferritin may have a trophic
relationship to oligodendrocytes, came from a developmen-
tal study in rats (Cheepsunthorn et al. 1998). In this study, it
was reported that ferritin was found predominantly in micro-
glia in white matter at PND 5 and by PND 30 the ferritin
staining was predominantly in oligodendrocytes. The salient
finding in this report was the temporal and spatial relation-
ship between the ferritin staining of microglia and the onset
of ferritin expression in oligodendrocytes. At PND 5, the
ferritin-positive microglia are numerous and robust in subcor-
tical white matter and corpus callosum. Very few oligodendro-
cytes are present and they are not ferritin positive. With age,
the number of ferritin-positive oligodendrocytes increase and
the number of ferritin-positive microglia decrease. Moreover,
this negative correlation occurs in microglia and oligoden-
drocytes that are within close proximity in specific regions of
the subcortical white matter. By PND 30, the ferritin posi-
tive oligodendrocytes almost completely replaced the micro-
glia and myelination of the callosum is homogeneous. This
developmental pattern of ferritin in microglia giving way to
ferritin-positive oligodendrocytes in the white matter is simi-
lar to that reported for cellular staining of iron in white mat-
ter during development (Connor et al. 1995). It is possible
that the ferritin in the oligodendrocytes was synthesized by
592 • NEUROGLIA
these cells rather than internalized from extracellular ferritin.
Indeed in cell cultures, H-ferritin mRNA is increased in oligo-
dendrocytes when they first adhere to culture plates (Sanyal
et al. 1996), indicating ferritin synthesis is an early event in
oligodendrocyte maturation. An alternative, but not mutually
exclusive, interpretation is that ferritin could be released by
the microglia and taken up by nearby oligodendrocytes. This
idea is consistent with our finding that ferritin could be taken
up by oligodendrocytes (Hulet et al. 2000). To test this idea,
we took conditioned media from microglial cells in culture
and demonstrated that the media has a trophic effect on oligo-
dendrocytes and that the trophic effect can be eliminated by
reducing the expression of H-ferritin in the microglia (Zhang
et al. 2006a). This indirect evidence that ferritin was a trophic
factor for oligodendrocytes was subsequently confirmed using
direct exposure of ferritin to oligodendrocytes in cultures
(Todorich et al. 2011). Moreover, a receptor for H-ferritin
was discovered on oligodendrocytes (Todorich et al. 2008)
(Fig. 46.5), which is consistent with our reports of ferritin bind-
ing to white matter tracts in rodent and human brains (Hulet
et al. 1999a). The receptor for H-ferritin is Tim-2 (T-cell immu-
noglobulin mucin domain 2) protein (Chen et al. 2005).
An iron-dependent relationship between macrophage acti-
vation and oligodendrocyte was further shown in a spinal cord
injury model (Schonberg and McTigue 2009). This study was
based on two findings: (1) lipopolysaccharide (LPS) injections
into the spinal cord results initially in a loss of oligodendrocytes
followed by significant oligodendrogenesis (Schonberg et al.
2007) and (2) iron-positive microglia are upregulated in the
brain following LPS injection (Zhang et al. 2005a). Following
the LPS injection there were numerous ferritin-positive
macrophages suggesting these cells were accumulating iron.
Around the lesion, NG2-positive cells were detected and these
cells contain ferritin. One week after the LPS injection, there
Figure 46.5 Tim-2 in Rodent Oligodendrocytes in Cell Culture. In this
confocal image, Tim-2 immunostaining appears in the green channel and
DAPI, a nuclear stain, is in the blue channel.
was a significant increase in ferritin-positive oligodendroyctes
in the lesions. Similar to the reports in the developmental
study in the white matter, there was a shift from iron-positive
macrophages to iron/ferritin-positive oligodendrocytes. The
oligodendrocyte response to the injury can be blocked by
treating the animals with an iron chelator. This effect of iron
chelation on oligodendrocytes is consistent with cell culture
studies that showed the trophic potential of the macrophages
on oligodendrocytes is lost when the cells are treated with
an iron chelator (Zhang et al. 2006a). The iron in these acti-
vated macrophages could be from a number of sources, but
one intriguing possibility is that the injured oligodendrocytes
released iron that was taken up by the macrophages and subse-
quently returned to oligodendrocyte progenitor cells once the
insult had subsided to allow oligodendrogenesis and myelina-
tion to proceed.
In the spinal cord injury model, there was very little demy-
elination (Schonberg et al. 2007). Thus, although this injury
model provides compelling evidence for an interrelation-
ship among macrophages, iron, and oligodendrogenesis, the
importance of this relationship for myelin production could
not be demonstrated. In the cuprizone-induced demyelina-
tion model, macrophages in the lesions expressed ferritin, but
because myelination proceeded when cuprizone was stopped,
the ferritin shifted to oligodendrocytes, which was followed
by the appearance of myelin basic protein (MBP) (Adamo
et al. 2006). These data strongly support a converse functional
and temporal relationship between ferritin in macrophages
and ferritin in oligodendrocytes and myelination. Why such
a relationship could not exist and support remyelination in a
demyelinating disease such as MS is not known but perhaps
supports the argument for an immune response that destroys
new oligodendrocytes before they can remyelinate. One
hypothesis under investigation is that the Tim-2 receptor, the
receptor for H-ferritin on oligodendrocytes, may interact with
semaphorin A from the immune system. Before the discovery
that Tim-2 binds H-ferritin, Sema4A was the only reported
ligand for Tim-2 (Chen et al. 2005; Kumanogoh et al.
2002). Semaphorins, such as class IV semaphorins (Sema4A
and Sema4D), are an example of shared signaling molecules
between the immune and nervous systems (Kikutani and
Kumanogoh 2003; Kumanogoh and Kikutani 2003). The
hypothesis is that when oligodendrocytes reach a stage of
maturation that they express Tim-2 for ferritin uptake, they
become targets for Semaphorin A. This notion is supported by
our observations that immature oligodendrocytes surround-
ing MS lesions are transferrin receptor-positive but there is no
ferritin binding in the same region (Hulet et al. 1999b).
Much more investigation is needed into the question of res-
cuing myelin following injury or subsequent to developmental
iron deficiency. These investigations must focus on both the
timing of the rescue attempt as well as the mode of delivery
of the iron. For example, in a cell culture study, providing iron
(ferric citrate) to glial-restricted precursor cells can increase the
differentiation of these cells into galactocerebroside-expressing
oligodendrocytes but providing iron to O2A progenitor cells
resulted in increased proliferation but not differentiation
(Morath and Mayer-Proschel 2001). The timing of delivery
IRON AND GLIA
and mechanism of delivery has been demonstrated in vivo as
well. In studies that inject apo-transferrin (iron poor trans-
ferrin) into the cerebral cortex as a mechanism to improve
myelination an effect is only seen between 2 and 7 days of age
(Marta et al. 2000). The data indicate that there is a window
when transferrin can enhance myelination, which is concur-
rent with the presence of prooligodendrocytes. The uptake of
transferrin into the oligodendrocytes could be mediated by
a transferrin receptor at this age because there are reports of
transferrin binding to or being expressed by developing oligo-
dendrocytes (Espinosa de los Monteros and Foucaud 1987;
Giometto et al. 1990; Lin and Connor 1989b).
Whether the effect of the apo-transferrin is iron indepen-
dent or if the apo-transferrin bound iron once it was injected
into the brain has not been resolved. There are cell culture
studies that show oligodendrocytes exposed to apo-transferrin
develop a multipolar morphology and increase expres-
sion of MBP and myelin-associated glycoprotein (MAG).
Apo-transferrin exposure also inhibits the migration of oli-
godendrocyte precursor cells and this effect is blocked by the
transferrin receptor (Paez et al. 2002). Thus these data suggest
that apo-transferrin supports maturation of oligodendrocyte
precursor cells. The data would also be consistent with the
notion that transferrin can promote stabilization of oligoden-
drocytic processes (Ortiz et al. 2005). In the cuprizone model
of demyelination, rats treated with apo-transferrin at the time
of cuprizone withdrawal had a significant improvement in
remyelination compared to spontaneous recovery (Adamo et
al. 2006). That apo-transferrin injections could be effective in
a disease such as multiple sclerosis is supported by the observa-
tions of transferrin receptor-positive oligodendrocytes in the
periplaque regions (Hulet et al. 1999b). Overall, the data sug-
gest that transferrin is an effective nutrient in oligodendrocyte
differentiation and maturation.
A major question, however, regarding the role of transferrin
in the development of oligodendrocytes is whether or not the
trophic influence of this protein is dependent or independent
of iron. The injections of apo-transferrin into the brain paren-
chyma would surely encounter iron and given that the associa-
tion constant of transferrin for iron is 1022M-1 apo-transferrin
would acquire iron even if the iron were bound to many other
proteins. Indeed when apo-transferrin was injected into the
cerebrospinal fluid, 52% of a 2-mg injection of apo-transferrin
was bound to iron within 4.5 hours (Ueda et al. 1993). That the
effect of apo-transferrin is iron dependent is supported by the
iron deficiency model where apo-transferrin injections were
not able to completely rescue the myelin deficit suggesting the
iron was needed for the apo-transferrin effect (Badaracco et al.
2008). It cannot be ruled out, however, that there were insuf-
ficient numbers of oligodendrocytes to completely rescue the
myelin.
The relationship between transferrin and oligodendro-
cytes was also investigated using hypotransferrinemic mice.
These mice have a splicing defect in the transferrin gene and
do not make transferrin. Hypotransferrinemic mice require
transferrin injections to survive and the injected transferrin
is detected in oligodendrocytes of these mice (Dickinson and
Connor 1995). Moreover, the hypotransferrinemic mice have
• 593
increased expression of integral myelin proteins and higher
than normal iron concentration in the white matter (Ortiz
et al. 2004b). These data clearly argue for a transferrin uptake
mechanism in the oligodendrocytes, yet transferrin receptors
were not detectable on the oligodendrocytes (Dickinson and
Connor 1998). We cannot rule out that iron had accumulated
in these cells early in development; but the injected transfer-
rin from those early time points would have been degraded
over time. Assuming the uptake of transferrin into the oligo-
dendrocytes was a receptor-mediated endocytotic process, the
injected transferrin in the oligodendrocytes of the hypotrans-
ferrinemic mice should not have entered the cytosol because
there is no endocytic export mechanism known for transfer-
rin, only iron. Thus, the transferrin in these cells should not
have had an intracellular effect (e.g., stabilization of processes)
beyond that of iron delivery. The hypotransferrinemic mice
are an underutilized model for intracellular effects of transfer-
rin in oligodendrocytes.
2.4 I RO N E F F LUX
Although much effort has been placed on understanding iron
uptake by oligodendrocytes, recent data have shown that there
is an iron export mechanism in place as well; and this mecha-
nism may differ between gray and white matter oligodendro-
cytes. The proteins of interest for iron export are hephastin
and ferroportin. Hephastin is a copper-dependent ferroxidase
initially identified in the duodenum wherein it was shown
to be involved in iron efflux by enterocytes. In a cell culture
model from the cerebellum, all of the oligodendrocytes but
none of the astrocytes expressed hephastin (Schulz et al. 2011).
In the sex-linked anemia (sla) mouse, there is a partial dele-
tion of the hephastin protein resulting in limited ferroxidase
activity (Chen et al. 2004; Vulpe et al. 1999). Ferroportin is
a membrane protein that couples with ferroxidases to export
iron from cells (Vulpe et al. 1999). Ferroportin is more ubiq-
uitously expressed in astrocytes and oligodendrocytes and
microglia. The expression of hephastin or ferroportin does
not appear in culture until the oligodendrocytes have matured
to express galactocerbroside. The appearance of hephastin and
ferroportin on oligodendrocytes has also been demonstrated
in vivo (Schulz et al. 2011).
The role of hephastin in iron efflux by oligodendrocytes
could be demonstrated using cells from that sla mice in cul-
ture. In this study, in addition to the novel observations around
hephastin, it is important to note the almost two-thirds of the
iron that is taken up by oligodendrocytes is released within
24 hours; a novel observation that there is iron release and
thus a dynamic iron concentration in oligodendrocytes. In
the oligodendrocytes from the sla mice almost no iron was
released, indicating the role of hephastin in the dynamics of
regulation of cellular iron content in oligodendrocytes. The
lack of iron release in the absence of hephastin by oligoden-
drocytes was also shown in vivo, wherein more intense iron
staining was also seen in vivo in the oligodendrocytes from
the sla mice compared with control (Schulz et al. 2011). The
increased iron-positive oligodendrocytes were more apparent
in the gray matter than the white matter. These findings are
594 • NEUROGLIA
surprising and inconsistent with some of the other findings in
this study; for example there was a dramatic downregulation
of Tim-2, the receptor for ferritin in the white matter oligo-
dendrocytes in the sla mice, clearly suggesting significant iron
accumulation by these cells. Nonetheless, these studies dem-
onstrate a dynamic system for iron uptake and release from
oligodendrocytes.
3 THE ROLE OF IRON IN
OLIGODENDROCY TIC VULNER ABILIT Y
TO OX I DAT I VE S T R E S S
Because oligodendrocytes are enriched in iron, have a high
rate of oxidative metabolism, and reside in the rich lipid envi-
ronment of myelin, these cells can be expected to be highly
vulnerable to oxidative damage. In addition, oligodendrocytes
have lower levels of endogenous antioxidant protection sys-
tems such as glutathione (Back et al. 1998). Oxidative damage
results from the interaction of iron with oxygen, which is nec-
essary for the transport of electrons in the production of ATP.
The presence of a relatively high concentration of catalase-rich
peroxisomes in oligodendrocytes (Kim and Kim 1991) is tes-
timony to both the high rate of oxygen consumption in the
oligodendrocytes and the need to detoxify H2O2, a normal
by-product of oxygen metabolism that can interact with iron
to generate the free hydroxyl radical, a reaction known as the
Fenton Reaction.
The vulnerability of oligodendrocytes to oxidative stress
has been clearly and consistently demonstrated (for two recent
reviews see (Benarroch 2009; McTigue and Tripathi 2008;
chapter 52). In vivo, two particular mechanisms that involve
oligodendrocyte cell death and oxidative stress are hypoxia
and exposure to proinflammatory cytokines. Overexpression
of tumor necrosis factor (TNF)-D or interferon (INF)-J in
the brains of transgenic mice results in severe central nervous
system (CNS) hypomyelination during development (Corbin
et al. 1996; Probert et al. 1995). Both direct and indirect
mechanisms can be identified for oligodendrocyte cell death
following exposure to cytokines. An example of the direct
mechanism is TNFD binding to p55 TNF receptor on oli-
godendrocytes followed by induction of apoptotsis ( Jurewicz
et al. 2005). Although this mechanism is direct, it can also
involve iron because TNFD is secreted from activated micro-
glia. Production of this cytokine and others is influenced by the
iron content of the microglia and the degree of vulnerability
of the oligodendrocytes is influenced by their iron content.
In cell culture studies, TNFD and IFN-J are toxic to oli-
godendrocytes in a dose-dependent manner but not interleu-
kin (IL)-1E (Zhang et al. 2005b). The toxic concentration of
TNFD and IFN-J can be decreased by exposing the cells to
iron. Iron status of the oligodendrocytes had no impact on the
lack of susceptibility to IL-1E. Interestingly, treatment with
deferoxamine (an iron chelator) did not protect the oligoden-
drocytes from the cytokines, perhaps supporting the evidence
that there are direct as well as indirect effects of the cytok-
ines on oligodendrocytes. The data also support the relative
vulnerability of oligodendrocytes to oxidative stress because
astrocytes and microglia actually accumulate iron in response
to activation through various injurious conditions including
inflammation (see later). The mechanism of action for toxicity
of the cytokines to oligodendrocytes appeared to be through
loss of mitochondrial integrity because the anti-oxidant TPPB
that targets mitochondria was protective. This observation is
consistent with in vivo studies in MS wherein mitochondrial
dysfunction is thought to underlie oligodendrocyte cell death
(Kalman 2006).
A curious observation in the cytokine exposure studies is
that the cytokines did not increase the expression of ferritin in
the oligodendrocytes (Zhang et al. 2005b). Ferritin is typically
increased following cytokine exposure, including in astrocytes
and microglia. The lack of ferritin increase is puzzling and
potentially important because it would be expected that such
an increase would be part of the cellular protective mechanism.
The lack of ferritin increase may couple with the relatively low
glutathione expression as part of the vulnerability profile of
the oligodendrocytes. The possible deleterious relationship
between microglia and oligodendrocytes involving iron and
inflammation was investigated in a cell culture model (Zhang
et al. 2006a). As discussed earlier, when conditioned media
from iron-loaded microglia is placed on oligodendrocytes the
media has a trophic effect and that trophic effect can be elimi-
nated by treating the microglia with siRNA for H-ferritin
(Zhang et al. 2006a). However, when the iron-loaded micro-
glia are treated by LPS, there is a decrease in H-ferritin release
from the microglia and an increase in pro-inflammatory
cytokines. The conditioned-media from the activated micro-
glial cells is toxic to the oligodendrocytes. Treatment of the
microglia with an iron chelator blocks the LPS-induced acti-
vation of nuclear factor kappa-light-chain-enhancer of acti-
vated B cells (NF-κB) in microglia and decreases the amount
of cytokines that are released.
Another physiological event that has been associated with
damage to the white matter, in particular the periventricular
white matter, is hypoxia. The hypoxic insult is associated with
increased levels of iron in serum and CSF (Shouman et al.
2008) and elevated iron staining in the periventricular white
matter (Kaur and Ling 1999; Palmer et al. 1999b; Qi et al.
1995; Rathnasamy et al. 2011). The iron in the periventricular
white matter is found in both oligodendrocytes and micro-
glia. Hypoxia leads to ferritin synthesis in oligodendrocytes in
culture but the ferritin synthesis appears secondary to a release
of iron in the oligodendrocytes (Qi et al. 1995). The release
of iron in the hypoxic oligodendrocytes is associated with a
decrease in intracellular pH. Thus, it appears that an increase
in iron in the cytosol is an early event following hypoxia in
oligodendrocytes. Ferritin in the oligodendrocytes may serve
to sequester the iron and limit damage from hypoxia but this
potentially protective ferritin reaction may be limited in vivo
because of the interaction of iron and microglia and hypoxia.
Ferritin expression in oligodendrocytes and microglia in
the periventricular white matter occurs in a spatiotemporal
relationship similar to that reported for iron (Cheepsunthorn
et al. 1998). The switch from ferritin-positive microglia to
ferritin-positive oligodendrocytes correlates temporally and
spatially with the appearance of myelin. Exposure to hypoxic/
IRON AND GLIA
ischemic insult during the first week of life in a developing
rat pup results in a marked increase in iron-positive micro-
glia (Palmer et al. 1999a; Rathnasamy et al. 2011). Likewise
hypoxia/ischemia in the PND 7 rat pup results in an increase
in ferritin-positive microglia and a delay in expression of
myelin markers. As myelin markers begin to appear, they are
accompanied by the appearance of ferritin-positive oligoden-
drocytes and the disappearance of ferritin-positive microglia
recapitulating the normal developmental pattern.
The trophic relationship between the iron-enriched micro-
glia in development and following injury or in demyelinating
diseases also presents potential problems. Microglia are able
to accumulate higher levels of iron and withstand higher
amounts of oxidative stress than most cells (Robb and Connor
1998; Zhang et al. 2006b). Indeed, microglia use iron to gen-
erate free radicals as part of a microbicidal defense mecha-
nism (Fang 2011) but the generation of these free radicals
in the vicinity of the lipid rich myelin environment and the
iron rich oligodendrocytes could result in oxidative stress and
death of the oligodendrocytes. We have discussed earlier the
relationship between microglia, iron and oligodendrocytes
in the developing brain. The periventricular white matter, a
region particularly susceptible to injury in preterm infants, is
enriched in iron-positive oligodendrocytes and microglia and
it has been argued that the vulnerability of this region is owing
to oxidative stress (French et al. 2009). Similarly, a potential
mechanism for oligodendrocyte death in multiple sclerosis
could be through production of free radicals by macrophages
in the vicinity of the MS lesions (Fisher et al. 1988; Haider
et al. 2012) (see chapter 61). Indeed, there is substantial histo-
logical evidence of oxidative stress in MS (Haider et al. 2012).
In support of a role for iron and oxidative stress in promotion
of demyelination is the finding of iron deposits in the white
matter in MS (LeVine 1997) and that treatment with an iron
chelator or an antioxidant can limit the amount of demyeli-
nation and behavioral abnormalities that are associated with
experimental allergic encephalomyelitis (Bowern et al. 1984;
Hartung et al. 1988; LeVine 1997). The relationship among
inflammation, hypoxia, iron, and microglia and oligodendro-
cytes has been studied in cell culture models. In response to
hypoxia, microglia become activated and accumulate iron. In
cell culture, exposure of microglia to hypoxia was coupled with
increased cellular iron consistent with the in vivo findings but
also production of reactive oxygen species, reactive nitrogen
species, and pro-inflammatory cytokines, all of which could
be reversed by treating the microglia with an iron chelator.
When medium from the hypoxic microglia was transferred
to oligodendrocyte cultures, there was a decrease in expres-
sion of glutathione in the oligodendrocytes. Glutathione
was increased in the oligodendrocytes, however, only if the
microglia-conditioned hypoxic medium contained deferox-
amine, an iron chelator. Deferoxamine alone had no effect on
glutathione expression by oligodendrocytes. Lipid peroxida-
tion in oligodendrocytes was elevated following exposure to
the medium conditioned by hypoxic microglia and this effect
is reduced if the microglia were also treated with deferoxam-
ine in the media at the time of hypoxia. Exposure to TNFD
or exposure to hypoxic-conditioned medium from microglia
• 595
will increase caspase 3 activation in oligodendrocytes and this
activation is reversed by deferoxamine or antiserum to TNFD
(Rathnasamy et al. 2011). These data strongly implicate iron in
the oligodendrocyte damage seen in hypoxia.
Another area that involves oxidative stress damage to
oligodendrocytes is radiation used to treat brain tumors.
Oligodendrocytes are the most radiosensitive of the glial cells
(Kim et al. 2008). No direct evidence could be found in the
literature for the contribution of iron to the radiosensitivity
of oligodendrocytes. However, exposing oligodendrocytes
to blue light increases oxidative stress sixfold in these cells
(Thorburne and Juurlink 1996) and the increase in oxidative
stress can be prevented by iron chelation.
4 ASTROCY TES
The role of astrocytes in iron neurobiology has mostly focused
on the accumulation of iron in these cells following damage
or alterations in development of myelin associated with muta-
tions in myelin proteins that altered the health of oligoden-
drocytes (Connor and Menzies 1996b). Normally there is no
detectable iron staining in astrocytes and rarely can ferritin-
positive astrocytes be found (Fig. 46.6). Recently, however,
compelling evidence has begun to emerge that astrocytes
have a role in maintaining iron homeostasis in the normal
brain. Specifically, astrocytes may regulate extracellular iron
availability in the brain and may be important for signaling
10 μm
Figure 46.6 Ferritin Immunostaining of Glia in Human Putamen.
Multiple glial cell-types express ferritin. The arrows indicate astrocytes
with multiple emanating projections protruding from the cell body.
The single arrowhead denotes a round oligodendrocyte, which is typical
morphology for this cell-type.
596
the blood-brain-barrier regarding brain iron status (Dringen
et al. 2007).
The mechanism of iron uptake into astrocytes does not
appear to involve transferrin receptors because these are
rarely seen on astrocytes in vivo, although they are expressed
on these cells in culture (Dringen et al. 2007). An interest-
ing model has been proposed that suggests astrocytes release
ascorbate that binds the ferrous (soluble) form of iron in the
extracellular space and then this ascorbate-iron complex is
imported into the astrocytes (Lane et al. 2011). Once inside
the cells the iron could be released from endosomes via diva-
lent metal transporter 1 (DMT1) protein (Burdo et al. 2001).
This protein is required to move iron from the endosomes
into the cytosol. Astrocytes express amyloid precursor pro-
tein, which has recently been described as having ferroxidase
activity (Duce et al. 2010), which will likely result in evi-
dence that astrocytes and their iron status play a direct role
in plaque formation and possibly Alzheimer disease (Zhao
et al. 2011). As mentioned earlier, ferritin-positive astrocytes
are rare, which would indicate that little iron is being stored in
astrocytes. There is a unique release mechanism for iron from
astrocytes, ceruloplasmin (GPI-linked). It has been shown
that ceruloplasmin-defective astrocytes will load iron but
not release it (Patel and David 1997). Moreover, in individu-
als or mouse models of aceruloplasminemia, astrocytes stain
strongly for iron. This last comment deserves a caveat because
the images of iron staining in these samples have the morpho-
logical appearance of oligodendrocytes but there is no question
that there are also ferritin-positive astrocytes in the acerulo-
plasmic brains (Oide et al. 2006; Texel et al. 2008). Astrocytes
release hepcidin, which is a protein that decreases iron release
from cells by binding to ferroportin. Thus, astrocytes through
secretion of hepcidin can regulate iron release from the BBB,
microglia (Du et al. 2012), and oligodendrocytes. Astrocytic
uptake of iron may be accelerated by hypoxia providing a
neuroprotective role (Yang et al. 2012). Thus, astrocytes may
have a critical role in brain iron homeostasis not only during
damage by accumulating iron but also by regulating brain iron
uptake from the BBB and release from other cells.
One additional possible source of iron to astrocytes, most
likely occurring only during damage, is hemin (Dang et al.
2012). Following injury, astrocytes strongly express heme-
oxygenase-1, which will degrade heme and release the iron.
Depending on the availability of ferritin, this iron could either
be stored or be a source of oxidative stress. The iron status of
the astrocytes will enhance their sensitivity to oxidative stres-
sors and treatment of astrocytes with an iron chelator will
minimize oxidative stress (Robb and Connor 1998). Iron
accumulation appears to cause damage to the mitochondria of
the astrocytes (Robb et al. 1999). Peroxide-induced death of
astrocytes involves generation of superoxide radicals and loss
of mitochondrial membrane potential as well as depletion of
ATP (Robb et al. 1999; Song et al. 2006). Iron chelation will
enable astrocytes to maintain membrane potential and pre-
serve production of ATP. However, because ATP production
requires iron, longer-term exposure of astrocytes to an iron
chelator is associated with a decline in ATP production. These
cell culture observations are directly relevant to histological
• NEUROGLIA
observations of increased peroxidase inclusions in astrocytes
of the substantia nigra with age and evidence of iron accu-
mulation in mitochondria of astrocytes in Parkinson Disease
(Dringen et al. 2007; Schipper et al. 2009) (also see chapters
51 and 65).
5 MICROGLIA
A common observation in activated microglia is that these
cells accumulate iron (Berg et al. 2001; Gorter et al. 2005).
Microglia can accumulate twice the amount of iron that an
oligodendrocyte can (Zhang et al. 2005c, 2006a).
Iron enriched microglia are seen in demyelinating disor-
ders such as MS (Adams 1988; Craelius et al. 1982; LeVine
1997) and in demyelination associated with Human immuno-
deficiency virus (HIV) (Gelman et al. 1992), and excess iron
in the MS white matter is supported by neuroimaging studies
(Haacke et al. 2009, 2010; Williams et al. 2012). For an excel-
lent review on iron and MS, see Williams et al. (2012) and
chapter 61. When the microglia are iron laden, there are few
to no iron-enriched oligodendrocytes seen in the white mat-
ter tracts consistent with our observations in development.
Iron-laden microglia are not limited to degenerative processes
in the white matter but are also seen in many neurodegen-
erative diseases such as Alzheimer disease in the vicinity of
neuritic plaques (Connor et al. 1992a) and in Parkinson dis-
ease ( Jellinger et al. 1993; Kienzl et al. 1995) including animal
models of Parkinson disease (Goto et al. 1996), demyelination
(Forge et al. 1998; Pedchenko and LeVine 1998), and stroke
(Kondo et al. 1995).
We have already mentioned the animal models of hypoxia/
ischemia, where iron-enriched microglia are present in the
damaged area (Bidmon et al. 2001; Cheepsunthorn et al.
2001; Rathnasamy et al. 2011). Moreover, when oligodendro-
cyte development is compromised, iron is found predomi-
nantly in microglia (Connor and Menzies 1990). Therefore,
microglia appear to play an important role in iron homoeosta-
sis in the injured brain. It is not known at this time if the iron
taken up by the microglia is part of the protective function
of the microglia or if the iron is also used by the microglia.
Certainly, the metabolic activity of the activated microglial
cells is increased. The amount of proinflammatory cytokines
released by microglia is increased by iron loading (Sindrilaru
et al. 2011; Zhang et al. 2006a) as well as increased release of
matrix metalloproteinase-9 (Mairuae et al. 2011) and activa-
tion of NF-NB (Crichton et al. 2002). One way to minimize
the impact of iron on damage to the brain following insult
may be to limit iron in the diet. Rats fed an iron-deficient diet
in the kainite model of epilepsy suffered less damage through-
out the brain and had less microgliosis when compared with
those on a control diet. In the same model, rats fed an iron
supplement diet had increased brain damage and microgliosis
(Shoham and Youdim 2000).
Iron acquisition by macrophages occurs primarily through
phagocytosis of damaged cells, myelin, and even red blood
cells (RBC)s. Although one group has reported expression
of transferrin receptors on microglia, these studies use whole
IRON AND GLIA
IgG secondary antibodies and thus the immunostaining may
have resulted from the interaction of the secondary antibody
to the Fc receptor on microglia (Kaur and Ling 1995). To date
we have not seen transferrin receptors on microglia, resting or
activated.
6 S U M M A RY A N D P E R S P E C T I VE S
Iron management in the brain may start with regulation of
iron transport and release from the BBB that is regulated by
astrocytes through their ability to release hepcidin. The iron
entering the brain, although not obligated to pass through
glial endfeet, seems to require a ceruloplasmin-mediated pro-
cess that is also associated with astrocytes. Astrocytes, under
normal conditions, appear to have little stored iron. The glial
cells most in need of iron for normal function appear to be
oligodendrocytes. These cells may acquire iron via transferrin
in earliest stages of development but appear to have their own
acquisition system: uptake of H-ferritin. One source of ferri-
tin in the brain is microglia although it is also possible for fer-
ritin to be transported across the BBB. Microglia accumulate
iron in both early stages of normal development and following
injury and thus iron appears critical to functions associated
with these cells as well. Moreover, the iron in microglia can
be released to oligodendrocytes bound in ferritin and be used
by the oligodendrocytes for maturation and myelin produc-
tion (see Schematic 46.1). Oligodendrocytes are also the most
sensitive to low iron because hypomyelination is a consistent
finding of decreased iron availability both in development
and adults. There is some suggestion in the literature that iron
changes in white matter tracts may be a biomarker for demy-
elination, which could be clinically meaningful information
to guide timing of treatment interventions. An exciting new
area is the role of genetic variations that impact brain iron sta-
tus and hence myelination—leading to alterations in rates of
cognitive decline with age or degree of damage with disease.
The balance of iron, however, is clearly critical to glial func-
tion because proinflammatory cytokines, hypoxia, and other
means of damage to the brain will use the ability of iron to
generate oxidative stress to promote cell death in all of the glial
subtypes but the most sensitive are oligodendrocytes followed
by astrocytes and then microglia. The ability of iron chelation
to protect oligodendrocytes not only from direct exposure to
proinflammatory cytokines but also from the toxic effects of
activated microglia suggest that attempts to treat hypoxic/
ischemic injury (Sorond and Ratan 2000) and demyelinating
diseases (Pedchenko and LeVine 1998) with iron chelators are
well founded. There is clear evidence that iron chelation will
decrease inflammatory reactions. A challenge will be in the
delivery of the iron chelator and the timing of delivery. The
chelating compound or the mechanism of delivery must be
able to traverse the BBB and in adequate amounts. Moreover,
the chelating compound has to discern between “good and
bad” iron so as not to limit energy production that may be
needed for remyelination. For example, we have shown (Robb
and Connor, 1998) that deferoxamine will protect astrocytes,
specifically maintaining ATP production, from oxidative
• 597
stressors but after a few hours of exposure ATP production
declines presumably because iron needed for cytochrome oxi-
dase activity is diminishing. Thus, iron chelation is a viable
clinical option to limit damage from hypoxia, ischemia, and
traumatic brain injury but mechanisms to regulate the chelat-
ing compound both in terms of amount of iron being chelated
and the distribution of the chelator within the brain (perhaps
limiting it to the injury site) may be critical to its function.
Lowering dietary iron may be one way to limit iron available
to exacerbate damage associated with neurological trauma,
but again, iron must be reintroduced for remyelination and
return to normal function.
AC K N OW L E D G M E N T S
I am grateful to the many outstanding students and postdoc-
toral fellows who worked with me in generating the data over
25 years from our first observation of transferrin in oligo-
dendrocytes into an exciting field of research involving brain
iron regulation and its importance in glial function. I am par-
ticularly grateful to current students Dominique Leitner and
Wint Nandar and postdoctoral fellow Dr. Amanda Snyder
who helped generate the micrographs, schematics, and tables
in this chapter. Dominique and Dr. Snyder also provided
valuable suggestions and editing of this manuscript. I am also
grateful to the funding agencies that allowed us to pursue
this work, primarily the National Institutes of Health, and
also other funding agencies including the National Multiple
Sclerosis Society.
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 47.
ROLE OF MICROGLIA IN THE NORMAL BRAIN
Frank Kirchhoff

A B B R E VI AT I O N S In this chapter the dynamic behavior of microglia and their

role for normal brain function is discussed and the following
2P-LSM two-photon laser-scanning microscopy points are addressed: the properties of microglial motility, its
5c-Nase 5c-nucleotidase mechanism and regulation as well as the role of microglia dur-
AMPA 2-amino-3-(5-methyl-3-oxo-1,2- oxazol- ing brain development in neuronal elimination and synaptic
4-yl)propanoic acid wiring (Fig. 47.1).
BDNF brain-derived neurotrophic factor
bFGF basic fibroblast growth factor
CNS central nervous system
EGFP enhanced green fluorescent protein 2 S U RVE I L L A N C E O F B R A I N F U N C T I O N
GFAP glial fibrillary acidic protein BY R A M I F I E D M I C R O G L I A
KCC K+-Cl– cotransporter
NBC1 Na+/bicarbonate cotransporter 1 Soon after entering the brain from the periphery, the amoe-
NDPase nucleoside diphosphatase boid precursor cells of microglia acquire a typical morphol-
NGF nerve growth factor ogy characterized by small rod-shaped somata with numerous
NO nitric oxide thin and highly ramified processes that extend radially. Their
NTPase nucleoside triphosphatase three-dimensional distribution in vivo appears rather homo-
PI3K phosphatidylinositol 3c-kinase geneous with a territorial organization and a typical cell-to-
PLOSL polycystic lipomembranous osteodysplasia cell distance of 50 to 60 μm. This kind of microglial cell has
with sclerosing leukoencephalopathy originally been described as resting. Indeed, if one monitors
PNPase purine nucleoside phosphorylase the position of microglial somata for several hours in vivo
SIRP signal regulatory protein using two-photon laser-scanning microscopy (2P-LSM)
TGF-E transforming growth factor-E (Helmchen and Denk 2005), the cell bodies generally remain
TNF-D tumor necrosis factor D stationary with only few signs of migration. Only 5% of
TSP thrombospondin microglial cells move their position minimally, by 1 to 2 μm/
trk tropomyosin-related kinase hour (Nimmerjahn et al. 2005). Since 2005 in vivo time-lapse
recordings of microglia became one of the most frequently
1 INTRODUCTION used experimental approaches to study their functional role.
In a transgenic mouse line in which the fractalkine receptor
Microglial glial cells represent a unique and abundant con- gene (cx3cr1) had been replaced by the open reading frame of
stituent of the central nervous system (CNS). Originating the enhanced green fluorescent protein (EGFP) all microglial
from primitive myeloid precursor cells of the embryonic yolk cells are fluorescently tagged and can be brightly and at high
sac, microglial cells populate the brain before developmental spatial resolution visualized by 2P-LSM ( Jung et al. 2000;
closure of the blood-brain barrier (Ginhoux et al. 2010). In Nimmerjahn et al. 2005).
the adult nervous system, they are almost evenly distributed In contrast to the somata microglial processes appear
throughout the various brain regions at a density of about highly motile with two different types of morphology
6,000 to 8,000 cells/mm3. Thereby, they roughly form 5% to changes: (1) growth and retraction of the cell processes them-
15% of the cellular elements with some variations in different selves, as well as (2) filopodia-like membrane protrusions that
brain regions and species (Lawson et al. 1990; Mittelbronn transiently appear at random locations along processes. These
et al. 2001). In general, they are described to comprise the structural changes of the processes as well as of the filopodia
main component of the innate immune system in the brain occur on a timescale of minutes (Fig. 47.2). Extensions and
by responding to any pathological insult. More recently, func- retractions display similar velocities of 1 to 3 μm/minute for
tions of microglia have been discovered that appear to be more processes and up to 4 μm/minute for filopodia. In general,
important for normal brain functions and that—in a strict thicker branches show slower extensions than thinner pro-
sense—are not related to immunological responses. However, cesses of higher order. The filopodia-like protrusions are of
our understanding of microglial function in the healthy ner- variable shape, but typically they form bulbous endings. These
vous system is only emerging and still limited. protrusions appear transiently for a few minutes, sometimes

605
 ~5mm
Motility Synaptic interactions Phagocytosis

Figure 47.1 Overview of Microglial Behavior in the Healthy Brain. Highly motile microglial processes continuously remodel their local environment
(left), structurally and functionally interact with synaptic elements (middle; dendritic branch and spines, green) through direct contacts and exchanges
of molecular signals, and contribute to restructuring of neuronal circuits by phagocytosing synaptic elements and newborn cells (right; cellular
inclusions, blue and green). Microglial morphology and behavior display variability across central nervous system regions and stages of the
lifespan. Figure from Tremblay and Majewska 2011.

microglia can continuously monitor the activities of neigh-

boring cells and effectively control the microenvironment.
In addition, with its phagocytic potential it can directly act
like a communal waste collection service and clear the paren-
chyma of accumulated metabolic products and deteriorated
tissue components. Indeed, membrane protrusions spontane-
ously engulf tissue components that can be further processed
in the microglial cell body.

Figure 47.2 Dynamic Motility of Microglial Processes in the Normal
Brain. Ten minutes of microglial movements in the mouse cortex (A)
and spinal cord (B). (Red) Process retractions at the start of recording.
(Green) Processes that extended and retracted during the recording.
(Blue) Process extensions.
even repeatedly, along main processes and at their terminal
endings. Frequently, the protrusive activity is interrupted for
several minutes before further extensions or retractions occur.
Despite this dynamic change of motility, the number of tran-
sient protrusions per cell (about 20 in the cortical gray matter)
and the total process length remains constant. Although the
morphological changes appear randomly, the overall motility
is as fast and abundant that each point of the extracellular ter-
ritory adjacent to a single microglia cell is encountered three to
four times during a day. Extended areas of extracellular space
that appear as translucent pockets in electron micrographs fre-
quently surround microglial processes (Tremblay et al. 2010).
Probably, microglial cells themselves generate these regions by
secreting proteases such as matrix-metalloproteinases to facili-
tate the process movement (Crocker et al. 2008).
Although the resident microglia of the healthy, unper-
turbed brain have previously been called “resting,” these cells
are structurally the most dynamic cells of the CNS. It is not
too tempting to speculate that the dynamic but persistent
motility fulfills housekeeping functions such as the removal
of cellular debris or toxic metabolites. The processes seem to
sample their three-dimensional territories. Interestingly, bor-
der zones between neighboring microglial cells are nonover-
lapping. When microglial processes encounter one of another
cell, endings mutually repelled each other. Equipped with
numerous transmitters, growth factor and cytokine receptor
3 M E C H A N I S M S A N D R E GU L AT I O N O F
M I C R O G L I A L M OT I L I T Y, S W E L L I N G ,
A N D C Y TO S K E L ETO N R E A R R A N G E M E N T
By addressing microglial motility, one has to distinguish mainly
three forms of cell movement and migration: (1) the baseline
activity of microglial motility that is generated by everlast-
ing extensions and retractions of microglial lamellipodia and
filopodia as well as being an indicator of their tissue survey-
ing function, (2) directed growth of single processes along the
gradient of a locally released chemoattractant, and (3) cellular
migration encompassing translocation of cell bodies. The lat-
ter two can be regarded as graded forms of cell activation.
Identifying the mechanisms of microglial motility and
migration is still an intense area of research. Currently, swell-
ing with the involvement of ion transporters and channels as
well as actin-mediated processes are considered. In particular,
the surveying motility of filopodia and lamellipodia seems
to be mediated by regulatory volume increases and decreases
(Zierler et al. 2008). Process extension would then require the
controlled influx of osmolytes and water. And indeed, asym-
metrical distributions of transporters and ion channels have
been observed. K+-Cl– cotransporter (KCC) mediate the
osmolyte influx and induce process swelling. Cl– influx persists
because of activation of swelling-activated Cl– channels. In the
opposite direction, K+-efflux is evoked by intracellular Ca2+
oscillations triggering Ca2+-activated K+ channels. Also, coop-
eration of Na+/H+ exchangers and Na+-K+/2Cl– cotransport-
ers have been implicated in local swelling. For the NaHCO3
cotransporter NBC1 and the KCC transporters a polarized
distribution has been suggested. The cytokine CCL21 that is
expressed after neuronal injury affects microglial motility. Its

606 • NEUROGLIA
action as a chemoattractant is accompanied by induction of a
distinct Cl– conductance (Rappert et al. 2002).
Part of the microglial motility is also mediated by the
actin-based cytoskeleton because it can be blocked by cytoch-
alasin B. Interestingly, the rapid depolymerization and repo-
lymerization, as well as the cytoplasmic redistribution of actin
filaments, is induced by microglial 2-amino-3-(5-methyl-
3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) type glutamate
receptors (Noda et al. 2000). Thereby, microglial motility is at
least partially modulated by excitatory neuronal activity.
Another important regulator of microglial motility is rep-
resented by components of the complement system. Short
application of C5a induces a ruffling of microglial membranes
within seconds (Nolte et al. 1996). Subsequently, a con-
comitant extension of lamellipodia and a change of the actin
cytoskeleton are observed. Although the activation of C5a
receptors induces intracellular rises of Ca2+, the microglial
motility appears to be independent from intracellular Ca2+.
However, the motility is effectively blocked by the G protein
inhibitor pertussis toxin. Although C5a receptors are also
found to be coupled to K+ channels, and therefore might be
involved in swelling-mediated process extensions, these chan-
nels are not linked to regulation of microglial motility.
4 MICROGLIA AND PURINERGIC
SIGNALING
Microglia not only recognize a wide range of signals, but also
display a rather broad plasticity in their response (Kettenmann
et al., 2011). Given the rather homogenous distribution of
microglia within various CNS regions, they contribute to
the structural and functional integrity of the CNS. Some
authors attribute a very low threshold of activation to micro-
glial cells. However, this is only partially true if one compares
cell responses evoked by triggers of normal brain function
with those induced during acute or chronic brain injuries.
The primordial transmitter ATP, for example, activates neu-
rons, astrocytes or microglia at similar concentrations (see also
chapter 25).
Interestingly, microglia are probably the cell-type with the
broadest response pattern to purinergic signals. In the CNS,
ATP is not only an energy metabolite itself and its derivatives
are released as important primary transmitter and cotransmit-
ter by a range of release mechanisms, which include vesicular
release, diffusion through gap-junction hemi-channels of the
pannexin-type, or transporters. Highest concentrations of
ATP are released during programmed or necrotic cell death.
Therefore, ATP is regarded as a universal “danger” signal.
Indeed, the excess of ATP in the brain parenchyma is neu-
rotoxic. On release, ATP is rapidly degraded by extracellular
ectonucleotidases to ADP, AMP, and adenosine, thereby gen-
erating a complex “soup” of purinergic signaling molecules.
A complete set of various ectonucleotidases, namely nucleo-
side triphosphatase (NTPase), nucleoside diphosphatase
(NDPase), 5c-nucleotidase (5c-Nase), and purine nucleoside
phosphorylase (PNPase) is directly expressed at the surface
of microglia. After hydrolysis, the purine metabolites can
ROLE OF MICROGLIA IN THE NORMAL BR AIN
effectively activate the numerous purinoceptors of P1 and
P2-type in an autocrine and/or paracrine manner. Microglia
express all P1 receptors, that is, the adenosine receptors A1,
A2A, A2B, and A3. In addition, they express the ionotropic
P2 receptors P2X4 and P2X7 as well as the metabotropic P2
receptors P2Y6 and P2Y12. Thereby, microglia are not only
the cell type expressing the most diverse set of purinergic sig-
naling molecules, it is perfectly equipped to be the best sentinel
of the evolutionary oldest transmitter system. Although ATP
is regarded as an alert signal, adenosine has been attributed
a more neuroprotective function. The distinct spatially and
temporally defined composition of the extracellular milieu in
purines such as ATP, ADP, and adenosine represents a sophis-
ticated trigger signal for the motility and migratory behavior
of microglia.
Equipped with this diversity of nucleotide sensing mol-
ecules, these receptors are used in distinct pathways. In the
context of motility the ADP-preferring P2Y12 receptor is
particularly important (Haynes et al. 2006). In the brain this
receptor is predominantly expressed on microglia on the pro-
cesses as well as on the somata. As soon as the receptors evenly
distributed along the processes sense a gradient of ADP/ATP
(e.g., elicited by a local brain injury or release of ATP from a
glass micropipette), the microglial processes extend and elon-
gate toward the source within minutes. In vivo this process out-
growth is not accompanied by cell migration. As soon as the
process reaches its target region, the P2Y12 receptor is down-
regulated to almost nondetectable levels. In P2Y12 receptor
knock-out mice the outgrowth is significantly delayed, but not
completely abolished, suggesting that additional mechanisms
are involved (Haynes et al. 2006). In acutely isolated brain
slices, release of ATP associated with astroglial Ca2+ waves is
sensed by microglia (Schipke et al. 2002). Subsequently, an
outwardly rectifying K+ conductance is induced. In platelets
a functional and structural interaction of the adenosine recep-
tor A2A and the P2Y1 receptor with P2Y12 has been found
(Suzuki et al. 2011). The role of adenosine for microglial motil-
ity or migration has also been strengthened by the analysis of
mice deficient of the ectonucleotidase ENTPDase1 or CD39,
an enzyme that very rapidly converts ATP into AMP that can
be readily hydrolyzed by 5c-nucleotidase, broadly expressed
in the brain. In ENTPDase1 knock-out mice ATP is inactive
to stimulate microglial motility and has to be substituted by
exogenous soluble ectonucleotidase or adenosine to observe
microglial outgrowth (Farber et al. 2008).
Analysis of the second messenger cascade revealed the
activation of phosphatidylinositol 3c-kinase (PI3K) and
Akt kinase (Irino et al. 2008). Subsequently, induction of
integrin-E1-extracellular matrix interactions has been sug-
gested to control the extension of microglial processes
(Ohsawa et al. 2010). Required rises of intracellular Ca2+ could
be provided by influx through the ionotropic P2X4 receptor
(Ohsawa et al. 2007).
Although ATP/ADP represents a strong chemoattrac-
tant for microglial processes, the volatile transmitter nitric
oxide (NO) induces migratory behavior of whole cells (Dibaj
et al. 2010). When the NO-donor spermine-NONOate was
injected into the mouse dorsal white matter of the spinal cord
• 607
with a fine glass micropipette, microglial cells retracted their
fine lamellipodia, converted into cells with a simpler, more
elongated shape, and started to migrate toward the local NO
source within minutes (Dibaj et al. 2010). A detailed phar-
macological analysis revealed that NO triggers the guanylate
cyclase pathway of microglia. For astrocytes it has already been
shown that dibutyryl-cGMP and atrial natriuretic peptide (a
ligand for a particulate guanylate cyclase) induce process elon-
gation and redistribution of the intermediate filament pro-
tein glial fibrillary acidic protein (GFAP) and actin filaments
(Boran and Garcia 2007). Expression of the small GTPase
RhoA, which is involved in cellular migration and contrac-
tion, is regulated by protein kinase G, another component of
the NO/cGMP signaling pathway (Sauzeau et al. 2003).
The baseline motility is effectively modulated by ambient
levels of purines such as ATP, uridine triphosphate, and their
diphosphate metabolites. So far, three different mechanisms
have been reported that affect extracellular purine levels.
Although a block of GABAergic inhibition enhances motil-
ity by about 10% (Nimmerjahn et al. 2005), hydrolyzing ATP
to AMP by the hydrolyzing enzyme apyrase as well as block-
ing gap junction channels reduces the motility (Davalos et al.
2005). Because ATP is often coreleased during neurotrans-
mission, the block of inhibition results in increased ATP.
Similarly, ATP can be released from astroglial pannexin-type
gap junction hemichannels (Iglesias et al. 2009).
5 M I C R O G L I A L C O N T R I B U T I O N TO
SY N A P S E F O R M AT I O N A N D N E U R O N A L
C O N N E C T I VI T Y
During development, and during synaptic plasticity as well,
microglia contribute to the formation of synapses as well in
determining their synaptic efficacy. Thrombospondins (TSPs)
belong to extracellular matrix proteins, are mainly produced
by astrocytes and induce synapse formation (Christopherson
et al. 2005). In the early nervous system also microglia secrete
TSPs (Chamak et al. 1995; Moller et al. 1996). TSP interacts
with the integrin-associated protein CD47 that can form a
link to the signal regulatory protein (SIRP) D SIRP D is a
transmembrane protein expressed by neurons and myeloid
lineage cells (Matozaki et al. 2009). The protein CD47 is a
widely expressed glycoprotein. In the CNS it is found on vas-
cular endothelial cells. The system is involved in the regulation
of migration and phagocytosis as well as immune homeostasis.
Via the release of TSPs by microglia SIRPD/CD47 also affects
neuronal networks by modulating synaptogenesis.
Discovering the dynamic extensions and retractions of
microglial processes in the normal brain led to a significant
change in our understanding of microglia. After the first in
vivo imaging studies, the term resting microglia characterizing
the nonactivated state in the healthy brain was changed to sur-
veying microglia to indicate its constantly active role in moni-
toring its environment (Hanisch and Kettenmann 2007).
These early observations revealed repeated contacts with the
surfaces of all adjacent cell types, such as neurons, astrocytes,
or the endothelial cells of the brain capillaries. However, it
608 • NEUROGLIA
remained unclear whether the apparently random pattern
of microglia motility was controlled by distinct mechanisms
other than the purinergic and NO signals released by local
microinjuries.
Although the first in vivo imaging experiments of micro-
glia revealed only a small influence of neuronal activity onto
the global microglial motility (Nimmerjahn et al. 2005), more
recent studies demonstrate a very selective interaction of micro-
glial processes with synaptic terminals (Paolicelli et al. 2011;
Tremblay et al. 2010; Wake et al. 2009). In transgenic mice
in which both cell types, neurons and microglia, were labeled
by expression of fluorescent proteins discrete morphological
interaction steps could be discerned by two-photon laser-scan-
ning microscopy (2P-LSM)(Wake et al. 2009; Tremblay et al.
2010). In the two studies different transgenic mice were used:
Thy1-EGFP (Feng et al. 2000) and Iba1-EGFP (Hirasawa et
al. 2005) by Wake and colleagues; Thy1-EYFP and CX3CR1-
EGFP by Tremblay and colleagues. During their tissue sur-
veying, microglial processes stop at different synapses with
variable duration ranging from a few minutes to 1 hour to
establish direct contacts with neuronal synapses, which could
be recognized by the fluorescent protein–expression in the
postsynaptic spine compartment. On serial electron micro-
graphs of imaged areas, it can be seen that microglial processes
touch also other synaptic elements, the presynaptic terminal,
perisynaptic process, and synaptic cleft (see Fig. 47.3). These
novel ultrastructural observations define microglia as a sec-
ond, additional glial element next to astrocytes (Perea et al.
2009) with important influence on synaptogenesis and neu-
rotransmission (Tremblay and Majewska 2011). Interestingly,
these ultrastructural inspections revealed that the dendritic
shaft was not contacted by microglia, thereby strengthening
the specificity of microglia–synapse interaction. The contacts
appeared rather regularly with a frequency of about once per
hour. In contrast to the observation that microglial motility is
slightly enhanced when neuronal inhibition is blocked, reduc-
ing neuronal activity resulted in a reduced frequency of syn-
aptic contacts. Permanent block of the visual input (either by
eye enucleation or tetrodotoxin injection into the retina) lead
to a reduction of neuronal activity in the observed layer two
to three regions of the visual cortex and concomitant retrac-
tion of microglial processes from the synapses (Wake et al.
2009). Under the condition of energy failure as observed in
the ischemic penumbra after transient occlusion of the middle
cerebral artery, the duration of microglia–synapse contacts is
prolonged to more than 1 hour. Because numerous presyn-
aptic terminals disappear during that period, it is likely that
microglia contribute to the increased structural reorganiza-
tion in penumbra by sensing the functional status of individual
synapses. In an injury model (axotomy of vagal motoneurons)
it was shown that synaptic activity decreased before synapse
elimination (Yamada et al. 2008).
Microglia–neuron interaction also affects synapse forma-
tion under less aggressive experimental conditions. Under
normal sensory stimulation dendritic spines with microglial
contacts display a much larger size distribution (Tremblay
et al. 2010). A reversible change of synaptic activity results
in a corresponding morphological modification. Although
 E Without microglia F
1000 With microglia 4000
Extracellular space area (nm2)
Exlracelluar space area (nm2)

800
3000
600
2000
400

1000
200

0 0
0 1000 2000 3000 4000 5000 6000 7000
Microglial process area (nm2)

Figure 47.3 Ultrastructural Interactions Between Microglia and Synapses During Normal Sensory Experience. A–C. electron microscopy images
showing IBA1-immunostained microglial (m+) cell bodies (A), as well as large (B) and small (C) processes, surrounded by extended extracellular space
(asterisks) and contacting axon terminals (blue), dendritic spines (pink), perisynaptic astrocytes (green), and synaptic clefts (arrowheads). d, dentrite; N,
nucleus; p, perikaryon. Scale bars = 250 nm. D. electron microscopy image showing extended microglia-associated extracellular spaces (asterisks) after
glutaraldehyde instead of acrolein fixation. The unlabeled microglial process (m) makes direct contacts with dendritic spines (s) and axon terminals (t),
and displays an inclusion (in), as well as a clathrin-coated pit (black arrow) at the site of contact with a spine. Scale bar = 250 nm. E. Extracellular space
areas with or without contact with IBA1-positive microglial process. F. Correlation between the areas of microglial processes and associated extracel-
lular space. From Tremblay et al. 2010.

ROLE OF MICROGLIA IN THE NORMAL BR AIN • 609

dark-adapted mice display more microglial processes and more
contacts to spines and axon terminals, the extent of processes
and contact sites return to control levels after novel light expo-
sure. Interestingly, the visual experience also influences the
size of the extracellular perisynaptic space around the micro-
glial processes, thereby suggesting an important bidirectional
signaling pathway from neurons to microglia that actively
involves the modulation of the extracellular matrix.
Surprisingly, although the in vivo imaging and the ultra-
structural studies convincingly demonstrated intense contacts
and engulfments of synaptic structures by microglia, they
have not yet provided clear evidences for microglial phago-
cytosis of synaptic components. It is, therefore, very likely
that membrane-bound ligand-receptor–mediated signaling
between the microglial process and the synaptic elements
exist. Several signaling pairs have already been identified. A
common feature is the activity of the ligand as a calming signal
for microglia. In the presence of the ligand microglia remain in
its surveying state, whereas its reduction or the corresponding
inactivation of the receptor lead to strong microglial responses
(Biber et al. 2007).
One of such signaling pairs is formed by fractalkine and
CX3CR1. Although CX3CR1 is a heptahelical, G protein–
coupled receptor, fractalkine (CX3CL1) is a transmem-
brane glycoprotein expressed by neurons that ectodomain
can be proteolytically cleaved and released into the extracel-
lular space. Fractalkine is the exclusive ligand for CX3CR1,
in the CNS expressed only by microglia (Cook et al. 2001;
Harrison et al. 1998). In CX3CR1-deficient mice the calm-
ing effect of fractalkine onto microglia is no longer exerted,
resulting in increased neuronal cell death after stimulation of
inflammatory responses by injection of lipopolysaccharide,
by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
intoxication leading to symptoms of Parkinson disease or in
genetically modified mice modeling amyotrophic lateral scle-
rosis (ALS) (Cardona et al. 2006). More information on the
pathology of microglia in neurodegeneration is provided in
chapters 61, 63 and 65.
To prevent microglial activation fractalkine is tonically
released throughout the CNS at high concentrations (>300
pg of soluble fractalkine per mg of aqueous extract of an adult
mouse brain). However, the interaction of fractalkine and
CX3CR1 is not only a static mechanism to silence microglia.
In the uninjured brain of CX3CR1-deficient mice significantly
less microglial cells were found during the first postnatal month
(Paolicelli et al. 2011). Although in these mice synapses were
also engulfed by microglial processes, almost twice as many
dendritic spines as well as immature synapses were detected.
This was accompanied by a decreased frequency of spontane-
ous excitatory postsynaptic currents recorded from hippocam-
pal pyramidal neurons in acutely isolated slices from 2- to
3-week-old-mice. These two observations indicate insufficient
maturation of synapses and, hence of hippocampal connec-
tivity. The fractalkine/CX3CR1 pair is a pivotal component
of synaptic pruning (Paolicelli et al. 2011). Subsequently, the
synaptic elimination occurs via a classical complement cascade
involving synaptic or perisynaptic localization of C1q and C3
(Stevens et al. 2007) as well as the complement receptor CR3
610 • NEUROGLIA
(CD11b/CD18) on microglia (Linnartz et al. 2012). In vitro
an additional mechanism of cell debris removal in the absence
of inflammation has been shown that involves the triggering
receptor expressed on myeloid cells-2 (TREM2)–mediated
signaling in microglia (Takahashi et al. 2005). Patients with
loss-of-function mutations of TREM2 develop a chronic
neurodegenerative disease, named Nasu-Hakola disease or
polycystic lipomembranous osteodysplasia with sclerosing leu-
koencephalopathy (PLOSL). PLOSL develops as a neurode-
generative disease with only latent inflammation and patients
die at the fourth or fifth decade of life. Because TREM2 is
required for tissue debris clearance by microglia, the disease
highlights the essential function of microglia for CNS tissue
homoeostasis (Neumann and Takahashi 2007).
Still intriguing is the possibility of direct sensing of the
synaptic status by recognizing the respective neurotransmit-
ter. Microglia are well equipped with a plethora of transmit-
ter and neurohormone receptors such as glutamate, GABA,
or substance P (Hanisch and Kettenmann 2007; Pocock
and Kettenmann 2007). However, the functional impact of
these receptors for synaptic regulation has not yet been dem-
onstrated conclusively. So far, in the healthy brain, the cor-
responding receptors and electrophysiological responses to
synaptic events could not be detected on microglia. Therefore
more indirect ways of regulating neuronal activity, for exam-
ple, via astrocytes, have to be considered (Fontainhas et al.
2011; Wu et al. 2008). Activation of microglia induces a rapid
production and release of ATP. Microglial ATP then triggers
astrocytes to amplify ATP production and release glutamate.
Subsequently, astroglial glutamate increases EPSC frequency
by modulation of the neuronal presynaptic metabotropic glu-
tamate receptor mGluR5.
Thus, microglia might act as a genuine regulator of neu-
rotransmission and upstream partner of astrocytes.
6 FAC TO R S R E L E A S E D BY M I C R O G L I A
Numerous studies have shown that microglial cells can release
a multitude of growth factors such as interleukin-1E, trans-
forming growth factor-E (TGF-E), basic fibroblast growth
factor (bFGF), hepatocyte growth factor, brain-derived neu-
rotrophic factor (BDNF), and nerve growth factor (NGF).
Therefore, the supernatant of microglial cell cultures display
a powerful neurotrophic action (Morgan et al. 2004) (see also
chapters 22 and 23). Although we can hypothesize that micro-
glia contribute to higher-order brain functions, we are still
missing not only a systematic approach to reveal the growth
factor production by microglia of different brain regions, we
also have to develop experimental paradigms for subsequent
functional evaluation in vivo.
Microglial-derived growth factors may also affect synap-
tic transmission directly, as it has been shown in the spinal
cord by investigating the molecular mechanisms of neuro-
pathic pain after peripheral nerve injury. The injury-mediated
release of ATP stimulates microglia to release BDNF, which
in turn changes the Cl–-equilibrium potential in lamina
I neurons. Subsequently, GABA- and glycine-mediated
Cl– currents reverse and the corresponding postsynaptic
responses become excitatory, instead of inhibitory (Coull
et al. 2005). BDNF, however, may act directly on micro-
glia. It induced a sustained increase in [Ca2+]i through bind-
ing with the truncated tropomyosin-related kinase (trk)
B receptor, resulting in activation of the phospholipase C
pathway and store-operated Ca2+ entry in rodent microglial
cells (Mizoguchi et al. 2009).
Recently, it has been shown in tissue culture and acutely
isolated brain slices that the proinflammatory cytokine tumor
necrosis factor alpha (TNF-D)–regulated synaptic scaling
depending on neuronal activity (Stellwagen et al. 2006).
Although it was not directly proven in that study, it is highly
likely that the TNF-D was produced by microglia present in
the culture.
These data highlight the important role of microglia in
the effective regulation of distinct synaptic transmission path-
ways by releasing various peptides. However, more studies are
required to reveal the molecular complexity and diversity of
microglial, astroglial, and neuronal interactions in common
cell circuits.
7 M I C R O G L I A D U R I N G D E VE L O PM E N T
Microglial cell do not only determine the correct number of
synapses during development by a mechanism called synap-
tic pruning, microglia may determine the correct number of
neurons as well. However, during the development of the ner-
vous system, microglia display Janus-like properties. In some
instances they positively influence neurogenesis, whereas in
other they directly induce programmed cell death and elimi-
nate developing neurons. In tissue culture, for example, the
astroglial differentiation of neural precursors isolated from
the largest neurogenic niche of the brain, the supraventricu-
lar zone, required the presence of microglia and their released
interleukin-6 and leukemia-inhibitory factor (Nakanishi et al.
2007; Walton et al. 2006). Differentiated astrocytes, in turn,
seem to determine the final steps of microglia ramification
that occurs later during development (Navascues et al. 2000).
In other regions of the brain microglia regulate cell death. In
the chick retina microglia release NGF that activates the trkB
receptor to eliminate developing neurons by inducing apopto-
sis (Frade and Barde 1998). Similarly, in the developing mouse
cerebellum, microglia induced the cell death of Purkinje neu-
rons produced in excess. In contrast with the retina, here the
cell death was mediated by microglial release of superoxide
anion (O2–) generated during respiratory bursts (Marin-Teva
et al. 2004). The translucent and developing zebrafish brain
provides an excellent model system: With a width of 400 μm
the brain can be completely imaged. Because it contains only
about 30 microglial cells, all of them can be visualized almost
simultaneously (Peri and Nusslein-Volhard 2008). Time-lapse
in vivo imaging of microglia revealed that all apoptotic, but
still alive neurons were engulfed by microglial cells. Dead
neurons, however, were found directly inside the microglia,
outlining the phagocytic function of microglia during neu-
ronal removal. This study also suggests that the molecular
ROLE OF MICROGLIA IN THE NORMAL BR AIN
mechanisms of neuron–microglia interactions responsible for
synapse detection, recognition, and engulfment present dur-
ing development might be similar with those required for
removal of dying neurons by phagocytosis (Peri and Nusslein-
Volhard 2008).
8 S U M M A RY A N D P E R S P E C T I VE S
In the CNS microglia are pivotal cells that permanently con-
trol the status of brain activity. For that purpose they show the
following features: (1) They are evenly distributed throughout
all brain regions. (2) They are equipped with a broad range
of transmitter, growth factor, and cytokine receptors to effec-
tively monitor the diversity of neural activity during devel-
opment and in the adult individual. (3) Receptor activation
occurs rapidly and results in drastic functional changes.
From these points one can extrapolate the following puta-
tive functions for the healthy brain:
1. During development, microglia can contribute to the
structural reorganization, for example, by removal of
excess and dispensable neurons that die by apoptosis or by
fine tuning and shaping synaptic structures as well as the
adjacent extracellular space.
2. Microglia fulfill a housekeeping function by removing
cellular waste products.
3. Microglia help to reshape brain connectivity during
sensory input.
4. Microglia cure microinjuries, for example, by removal of
cell debris after the death of single cells.
5. Microglia are always in an alert state to sense acute or
chronic injuries.
Current research has not only identified the dynamic
activity of microglia in the brain, but also characterized sev-
eral molecular mechanisms of bidirectional neuron–microglia
interactions during development, learning, and experience.
Present data also demonstrate that additional yet unknown
mechanisms exist. In particular, we are still missing a com-
prehensive description how microglia interact with other cell
types at the circuit level, for example, the communication with
excitatory or inhibitory neurons via perisynaptic astrocytes,
the interactions with astrocytes and the brain vasculature and
the influence on the oligodendrocyte lineage cells and NG2
glia as well as axons and myelin. Furthermore, future work has
not only to evaluate and quantify the relative importance of
already known pathways, but also to address region-depen-
dent variations in microglia function.
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 48.
FACTOR S CONTROLLING MICROGLIAL ACTIVATION
Uwe-Karsten Hanisch

A B B R E VI AT I O N S 8). Also later on, notions of their pathological and physiological

importance were inevitably linked to the process of activation.
AE amyloid-E However, the underlying mechanisms triggering a transforma-
AD Alzheimer disease tion and causing consequences for the central nervous system
APC antigen-presenting cell (CNS) tissue remained elusive, as was the nature of signals that
ALS amyotrophic lateral sclerosis would initiate the response. Now it is known that microglial
CNS central nervous system activation is complex regarding causes, courses, and outcomes.
COX cyclooxygenase Even the term activation may not adequately reflect shifts from
DAMP damage-associated molecular pattern the state in the healthy CNS to the behavior upon an insult
EAE experimental autoimmune encephalomyelitis because it implies nonactivity under normal conditions and
ECM extracellular matrix fails to acknowledge diversity of reactive phenotypes. Seminal
FcR Fc receptor discoveries over the recent years unraveled housekeeping func-
IFN interferon tions of the “resting” microglia (see chapter 47). Meanwhile, the
IGF insulin-like growth factor concept of reactive phenotype diversity is accepted and becom-
IL interleukin ing further refined. Common to all of these aspects, there are
iNOS inducible nitric oxide synthase molecular factors that govern a departure from the surveilling
LPS lipopolysaccharide mode, that shape executive performance to protect the CNS
M1 classically activated macrophages against threats, but that may occasionally lead to an inappro-
M2 alternatively activated macrophages priate reactivity by scale and properties. There is no shortage
mGluR metabotrobic glutamate receptor of literature reports on those factors, receptors, and signaling
MHC major histocompatibility complex cascades, conditions under which they may unfold an influence
MMP matrixmetalloproteinase and the proposed impact they leave. Here, a focus is given to
MS multiple sclerosis principles of actions along with a brief survey of the main classes
MyD88 myeloid differentiation primary response of factors controlling microglial activation and activities.
gene 88
NO nitric oxide
PAMP pathogen-associated molecular pattern 2 CONCEPTS OF MICROGLIAL
PD Parkinson disease AC T I VAT I O N A N D AC T I VAT I N G
PG prostaglandin FAC TO R S
PRR pattern recognition receptor
RNI reactive nitrogen intermediates Several roles of microglia in the CNS surveillance as well as
ROS reactive oxygen species preservation of its integrity and functionality have been unrav-
TGF transforming growth factor eled over the last decade (Davalos et al. 2005; Nimmerjahn
Th T helper (cell) et al. 2005; Paolicelli et al. 2011; Wake et al. 2009). Concepts
TLR Toll-like receptor and fundamental discoveries that boosted their formulation are
TNF tumor necrosis factor outlined in chapters 19 and 47. The process of activation—as
TRIF TIR-domain containing adaptor protein inducible shifts in activity states were traditionally termed—
inducing IFNE includes a dramatic rebuilding of the cytoskeleton and mem-
brane organization, induction of whole sets of genes and
release of direct and indirect gene products (also considering
1 INTRODUCTION synthesis by induced enzymes) as well as unfolding of func-
tional portfolios ranging from migration and phagocytosis
The conscious description of microglia by del Rio-Hortega (see chapter 49) to communication with the adaptive immu-
came already with the observation that they undergo morpho- nity, for example, contributions as antigen-presentating cells
logical changes in response to pathological events (see chapter (APC, see chapter 50). The present section stresses principles

614
of alert and activation as triggered and governed by molecu-
larly identifiable signals.
2.1 L I P O P O LYS AC C H A R I D E A N D T H E FI R S T
E X P E R I M E N TA L D E M O N S T R AT I O N O F
INDUCIBLE RESPONSES
Much of the knowledge about the molecular and cellular prin-
ciples as well as consequences of microglial activation has been
derived from experimental stimulations under controlled in
vitro conditions (Kettenmann et al. 2011). Even though a loss
of tissue context comes with unavoidable limitations (absence
of environmental cues with activity-controlling influence), the
steps and features of activation through many of the key recep-
tor systems can be essentially recapitulated. Certain manipula-
tion and monitoring options are still not equally accessible in
vivo, giving value to work with cell preparations as a comple-
mentary approach. If particular compounds should be nomi-
nated for delivering basic insights into microglia activation,
lipopolysaccharide (LPS) would lead the list.
As a cell wall component of gram-negative strains, LPS is
responsible for many of the initial responses to such infections
(Beutler and Rietschel 2003). Production of cytokines, such
as the pluripotent and proinflammatory tumor necrosis fac-
tor (TNF) alpha, interleukin 1 (IL-1), IL-6, or IL-12, and a
range of chemokines, like CCL2, CCL3, CCL5, CXCL1, and
CXCL2 with chemoattractive activity for monocytes, T-cell
populations, and neutrophils, organizes not only immune cell
infiltration and activities, but influences the functions and
vitality of neurons and glial cells, including microglia them-
selves (Hanisch, 2002; Kettenmann et al. 2011) (Fig. 48.1A).
Similarly, release of nitric oxide (NO) and reactive oxygen
species (ROS), of proteases and other mediators and expres-
sion of an array of surface molecules for cellular communi-
cation, including major histocompatibility complex (MHC)
structures (Fig. 48.1B), are in support of defense and protective
reactions, but some of these factors and their associated func-
tions can also cause substantial damage (Block et al. 2007).
Such activated microglia can exhibit massive neurotoxicity.
Structurally, LPS varies by the type and number of acy-
lations, by phosphorylation as well as by the size and the
composition of the carbohydrate moieties, giving rise to a
variety of chemotypes. Common to all is recognition by cell
surface–expressed Toll-like receptor (TLR) 4, triggering ini-
tial host defense responses of innate immune cells, including
microglia (Regen et al. 2011). Yet even long before the dis-
covery of TLRs, LPS was known and used for the profound
changes it triggers in macrophages and microglia (Schwartz
et al. 2006).
Dramatic shape changes, including granular appearance,
already indicate a response. However, it is more the massive
induction of release activities that would affect the CNS.
Morphological changes under LPS resembled the appearance
of microglia in the diseased CNS—and activation could not
be tolerated by its vulnerable neur(on)al structures. In con-
clusion, microglia earned the reputation of a harmful cell,
almost like a risk factor (Schwartz et al. 2006). This notion
dominated the view of microglia for years. Although it is
now known to be a poor reflection of the true physiological
spectrum, the early experiments employing LPS (and other
microbial agents) set the basis for experimental studies on fac-
tor-initiated microglial responses. Responses can be versatile,
depending on the driving stimulus and contextual signaling
of additional factors. Even for TLR4, functional implications
are diverse. Notably, microglia primarily serve supportive and

Figure 48.1 Microglial Responses to Stimulation of TLR4 with LPS. A. Mouse microglial cells were stimulated in vitro with S-LPS and Re-LPS at
various concentrations. The released cytokines and chemokines were determined in the culture supernatants after 18 hours. Absolute amounts were
then normalized to amounts obtained from stimulation at maximal agonist concentration. B. Microglia were challenged with Re-LPS (10–8 g/mL, 48
hours), stained with fluorescence-conjugated anti-MHCI and anti-CD11b antibodies and processed for flow cytometry. Analysis considered CD11b+
cells for MHCI expression. C. Microglia deficient in functional MyD88 or TRIF (myd88–/–, trif lps2) were stimulated with 10–7 g/mL of either LPS
chemotype for 18 hours. Cytokines and chemokines were analyzed as in (A), normalized to the release obtained with wild-type cells. Data are from
Regen et al. 2011.

FAC TO R S C O N T R O L L I N G M I C R O G L I A L AC T I VAT I O N • 615

protective functions under normal healthy conditions as well
as in many (if not most) situations in which the CNS has to
cope with exogenous and endogenous threats. Yet most of the
“daily” actions of microglia in solving minor and localized
abnormalities probably remain unnoticed because they never
surface with signs (Hanisch and Kettenmann 2007).
2.2 G E N E R A L FE AT U R E S A N D P H A S E S O F
T H E M I C RO G L I A L AC T I VAT I O N P RO C E S S
Until recently, microglia of the healthy adult CNS were con-
sidered quiescent, that is, functionally dormant, because of
low (absent) expression of activation-associated molecules and
their ramified morphology. This changed with in vivo imaging
studies demonstrating high motility of the cellular processes
for a scanning of the environment (Nimmerjahn et al. 2005;
Paolicelli et al. 2011; Wake et al. 2009).
Infection, trauma, ischemia, neurodegenerative diseases,
or altered neuronal activity, that is any disturbance or loss of
homeostasis, real or potential danger to the CNS can then
evoke rapid and profound changes in the microglial cell shape,
gene expression patterns, and functional behavior (Block et al.
2007; Davoust et al. 2008; Kettenmann et al. 2011) (refer also
to chapter 49). Pioneering work exploiting facial nerve transec-
tion set the fundaments of targeting microglial reactions in vivo
(Kreutzberg 1996). The process of microglial activation can
unfold the reactive repertoire of an innate immune cell. Stages
were defined by morphological, molecular, and functional
characteristics, with full-blown reactive microglia presenting
like (other) macrophages (Davoust et al. 2008; Graeber 2010).
Yet microglia are embedded in a specialized tissue (brain paren-
chyma), which has to protect itself from potentially damaging
consequences of an immune reaction. This special condition
has been described as “immune privilege” (Galea et al. 2007).
Microglia activation is thus a highly regulated process.
Traditional concept oversimplified and generalized the
adjustments to changes in their environment. The binary con-
cept of activity states (“resting” versus “activated”) did not
acknowledge default activities under normal conditions, nor
the diversity of response options. Microglial activation is not an
“all-or-none” process, like an action potential. It does not follow
a linear path to a uniform outcome. Instead, activated microg-
lia acquire distinct functional profiles (David and Kroner 2011;
Saijo and Glass 2011; Schwartz et al. 2006) (Fig. 48.2).
Features and consequences of activation are largely (yet
not entirely) determined by the initial cause. This cause comes

Figure 48.2 States of Microglia Activity and Steps of the Activation Process. In the normal healthy (mature) CNS tissues, microglia present with a
ramified morphology previously considered a “resting” state, but recent investigations proved that they are active in terms of surveillance. This includes
scanning of their environment with motile processes and intimate contacts with synapses as well as permanent integration of signaling inputs through
an array of receptors. Appearance of molecular cues indicating disturbed homeostasis can instruct rapid changes in morphology, movement, expres-
sion, and functional profiles. Infections, ischemia, trauma, or cell impairment along with neurodegenerative and autoimmune processes deliver a
range of activating signals that are sensed by already expressed receptors. Transformations to alerted and finally fully activated states are, however, also
controlled by calming signals that inform microglia about the well-being of neurons. Loss of such inputs may set off an alert or lower thresholds for
activating signs. Similar to other macrophages, but in a CNS-adapted fashion, microglia will then commit to distinct reactive phenotypes. Programs
come with particular transcriptional profiles as well as nontranscriptional adjustments. Their choice is dictated by the initial stimuli and their context.
Initial programs may also undergo changes while the activation proceeds, governed by a disappearance of driving forces and influences from other resi-
dent CNS as well as invading immune cells. Activation may resolve thereafter. Deescalation may come with a redistribution of microglia, their decline
by cell death, or even a departure from the CNS. Microglia may reacquire a nonactivated state—or remain altered by the previous experience. Their
behavior on another confrontation may differ from that of completely naïve cells. Scheme adapted from Hanisch and Kettenmann 2007.

616 • NEUROGLIA
with prominent molecular signals that are indicative of the
pathophysiological meaning for the CNS, such as an infec-
tious threat or cell impairment by intrinsic complications.
Microglia are prepared to understand many of these signals
and call up a variety of programs to cope with the challenge.
Common to all activating scenarios is that microglia have to
give up the surveilling mode for a situation-adapted response
program, based on the balance of incoming signs (Hanisch
and Kettenmann 2007).
2.3 I N I T I AT I O N O F A R E S P O NS E
Multiple signals converge on microglia under normal con-
ditions as well as on impairment of the CNS. Each of these
conditions comes with an ensemble of (molecular) factors
that will decide on whether microglia engage with a response
at all and which features are expressed. Sorting of activating
signals could be based on exogenous or endogenous sources
(infectious agents, self-derived molecules). Signals vary by
cellular origin and physiological implication (neurotransmit-
ters, inflammatory messengers). Delivery by autocrine or para-
crine routes or “leakage” differentiates between professionally
secreted factors and material delivered by damage. Factors can
be grouped by their biochemical or chemical nature or related
receptors, action as a soluble ligand, or as anchored in a mem-
brane or the extracellular matrix (ECM), such as integrins.
Signals can also be sorted by principles setting off an alarm.
In one case, the signal has to appear in the extracellular space
surrounding microglia, because it is usually not there at all
(bacteria), or close by, but not in physical contact (plasma
proteins), or usually not present in signaling-relevant format
(redox state, monomeric/aggregated, on necrotic cell death or

Table 48.1 LIGAND-RECEPTOR SYSTEMS FOR ACTIVATION AND MODULATION OF MICROGLIAL RESPONSES
FACTOR/COMPOUND CLASS LIGANDS AND RESPECTIVE RECEPTORS

Cell wall components, surface structures, Pam3CSK4, MALP-2, lipoteichoic acid, proteoglycans, various LPS chemotypes (S- and R-LPS),
DNA and RNA of viral, bacterial or zymosan, poly(I:C), poly(A:U), flagellin, ssRNA, CpG ODN as binding to PRR families, especially
fungal origin (PAMPs) TLR1/2, TLR3, TLR4, TLR5, TLR6/2, TLR7/8, and TLR9

Endogenous molecules (DAMPs)
HMGB1, fibronectin, fibrinogen, tenascin, versican, hsp60, hsp72, S100A8/9, neutrophil elastase,
lactoferrin, serum amyloid A, AE, AE25–35, AE40, AE42, prion protein (PrP), oxidized low density
lipoprotein, fatty acids, hyaluronic acid, heparan sulfate (fragments), IgG/chromatin complexes,
endogenous RNA/DNA as binding to TLR2, TLR3, TLR4, TLR7/8, and TLR9 as well as other
(scavenger) receptors, RAGE

Cytokines IFNJ and IL-4/IL-13 with crucial instruction of phenotypes, IL-10 as an immunosuppressive factor,
TNFD, IL-1E, IL-18, IL-33 with activating contributions, IFNE with modulating influences, IL-2,
IL-15, TGFE, colony stimulating factors (M-CSF, GM-CSF), activities via their respective receptors
and receptor subtypes

Chemokines CCL2/CCR2 pair with key role in monocyte/microglia recruitment, CX3CL1/CX3CR1 with calming
control, both systems also with distinct roles in monocyte/microglia subset identification, ligands for
CCR1, CCR3, CCR5, CXCR1, CXCR3, CXCR4, IL-8R with diverse functional effects

Complement Complement factor C1q, C5a via complement receptors

Antibodies Immunoglobulins of diverse sub/classes (IgA, IgG, IgM), largely active as immune complexes

Neurotrophins/neurotrophic factors Brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), nerve growth
(NF) factor (NGF), neurotrophin 3 (NT-3), NT-4

Coagulation factors Thrombin via proteinase-activated receptors (PAR) 1, 3, 4

Neurotransmitters/cotransmitters Adenosine, ADP, ATP via adenosine (A) and purinoreceptors (P2X, P2Y) of most diverse subtypes,
glutamate via AMPA and mGluR types, GABA via GABAB receptors, acetylcholine via nicotinic
receptors, nor/adrenaline via D and E subtypes of adrenergic receptors, dopamine via D1 and D2 recep-
tors, often with inhibitory and modulating influences on other receptor stimulations

Neurohormones/neuromodulators Bradykinin, neurokinin, substance P, VIP, endothelin, opioids, endorphins, angiotensin, somatostatin,
melatonin, platelet-activating factor (PAF), histamine, cannabinoids via their respective receptors and
receptor subtypes

Steroids, vitamins Gluco/corticoids via glucocorticoid as well as mineralocorticoid receptors, vitamin D3, importance for
attenuation of responses to other receptor activations (e.g., TLRs), own activities

Lipids and others Ceramides, gangliosides, prostaglandins, leukotrienes, lysophosphatidic acids

Systems with calming steady-state Ligand/receptor pairs with reciprocal expression by neurons and microglia, SIRP1D/CD47, CD200/
influences CD200R, CD22/CD45, and CX3CL1/CX3CR1

Examples are given as based on Chen and Nunez (2010), Hanisch and Kettenmann (2007), Hanisch et al. (2008), Kettenmann et al. (2011), and Piccinini and
Midwood (2010).

FAC TO R S C O N T R O L L I N G M I C R O G L I A L AC T I VAT I O N • 617

in neurodegenerative processes with protein deposition). All
of them alert microglia by (sudden, increased) presence as “on
signals.” Typical on signaling is mediated via TLRs or recep-
tors for TNFD or interferon (IFN) J (Table 48.1). In contrast,
certain ligand–receptor pairs keep microglia in a steady state
because their signaling imposes a calming influence, with an
“off signaling” effect on disruption (Biber et al. 2007; Hanisch
and Kettenmann 2007; Kettenmann et al. 2011; Saijo and Glass
2011; van Rossum and Hanisch 2004). CD200/CD200R,
CD47/SIRPD, CX3CL1/CX3CR1, or CD22/CD45 are
representative systems. Integration of both signal types deter-
mines whether surveilling microglia transform to other (exec-
utive) activity states (see Fig. 48.2). A distinction by “on” and
“off ” signals offers even more understanding of activation. On
signals require the pre-existence of receptors—TLRs cover-
ing multiple signs of infection. Blockage of constitutive off
signaling can increase basal activities and enhance stimulated
responses in situations of neuronal impairment of any kind,
for example. Interruption of the current in wires of a shop-
ping window turns on an alarm. Microglia get alerted when
off signals fade. As a consequence, microglia can sense danger
by “known” and “unknown” causes.
2.4 FAC TO R S C O N T RO L L I N G M I C RO G L I A L
AC T I VAT I O N
Pattern recognition receptors (PRRs) are germ line-encoded
sensors for microorganisms and enable the immune system
to fight infectious threats. They bind an assortment of evolu-
tionary conserved structural motifs within glycolipids, glyco-
peptides, lipoproteins, and proteoglycans as well as RNA and
DNA of diverse microbial origins that are collectively termed
pathogen-associated molecular patterns (PAMPs) (Kawai
and Akira 2010; Takeuchi and Akira 2010). Pattern recogni-
tion receptors come in families as to structural and functional
relations: NOD-like receptors (NLRs), RIG-1-like receptors
(RLRs), C-type lectin receptors (CLRs), absence in mela-
noma 2-like receptors, and TLRs (Chen and Nunez 2010).
Significantly, PRRs also observe the inner world because some
(notably TLRs) respond to molecules generated, released, or
modified in the context of tissue injury. Being of diverse origin
and physiological importance, as damage-associated molecular
patterns (DAMPs) they gain a role as danger indicators (alarm-
ins) in situations that are not (yet) linked to infection and that
(ab)use (innate) immune cells in sterile inflammation (Chen
and Nunez 2010). Microglia express PRRs, including TLRs,
and involve their obvious and cryptic signaling in probably all
scenarios of CNS diseases (Hanisch and Kettenmann 2007;
Hanisch et al. 2008; Kettenmann et al. 2011). Recognizing
PAMPs and DAMPs, TLRs prototypically fulfill roles of
omnipotent sensors of disturbed homeostasis (Hanisch and
Kettenmann 2007; Matzinger 2007).
Recognizing patterns rather than individual ligands, a
small number of TLRs (about 13 in mouse and humans)
cover a wide range of relevant pathogens. Expressed on the
cell surface or in endosomal compartments, TLRs signal via
two major pathways that are characterized by the signaling
adapter proteins myeloid differentiation primary response
618 • NEUROGLIA
gene 88 (MyD88) or TIR-domain containing adaptor protein
inducing IFNE (TRIF) (Lin et al. 2010; O’Neill and Bowie
2007; Park et al. 2009). MyD88 and TRIF, in turn, organize
a downstream activation of kinase cascades and transcription
factors controlling interferons, cytokines, and chemokines
with importance for immune cell recruitment and activa-
tion (Kawai and Akira 2010). Although most TLRs depend
on MyD88, and only TLR3 solely relies on TRIF, TLR4 is
in the unique situation to be linked to both adapters, which
organizes the various response elements (Fig. 48.1C). Toll-like
receptor expression in cells of the innate and adaptive immu-
nity as well as in nonimmune cells has been associated with a
wide range of functions, including responses to PAMPs and
DAMPs (Glezer et al. 2006; Hanisch et al. 2008; Miller et al.
2005; Rivest 2009; Rubartelli and Lotze 2007).
The unleashing and propagation of microglial activation
in (apparently noninfectious) CNS diseases has been attrib-
uted to some CNS-intrinsic signals that would be delivered
as a consequence of cell impairment. Now, more and more
of those factors are actually identified. DAMPs may not only
play roles in responses to trauma and cell injury, vascular dis-
ruption, and ECM breakdown, and contribute to damaging
cascades following CNS complications, such as ischemia and
stroke. DAMP signaling likely participates also in neurodegen-
erative processes, such as Alzheimer disease (AD), Parkinson
disease (PD), or amyotrophic lateral sclerosis (ALS), and to
some extent in autoimmune diseases such as multiple sclerosis
(MS) (Chen and Nunez 2010).
For microglia, especially (yet not only) TLR1, 2, 4, and 6
in their combinations or association with non-TLR receptors
would play a role not only in the response to bacterial infec-
tions but also for detection of endogenous factors, including
several heat-shock proteins (hsp), fibronectin, lactoferrin,
tenascin, versican, heparan sulfate, hyaluronic acid, HMGB1,
S100A8/S100A9, oxidized phopsholipids and lipoprotein
A, fatty acids, eosinophil-derived neurotoxin, serum amy-
loid, and amyloid-E (AE) (Hanisch and Kettenmann 2007;
Hanisch et al. 2008; Heneka et al. 2010; Lehnardt et al.
2008; Piccinini and Midwood 2010; Stewart et al. 2010).
Thus, DAMPs may prepare and assist reactions to PAMPs,
such as in meningitis, or independently drive inflammatory
scenarios. Disturbed protein expression, altered turnover,
and processing as resulting in abnormal (poly)peptide struc-
ture, aggregation, and deposition are assumed to be key ele-
ments of microglia activation and reaction in diseases such as
AD. Factors such as AE in AD, which could misuse DAMP
principles, are becoming increasingly better defined also as
to their receptors and signaling outcomes. This is illustrated
by a study reporting on an assembly of a TLR4/6 heterodi-
mer with CD36 that can promote sterile inflammation to
AE (Stewart et al. 2010). Of course, TLRs are not the only
class of receptors sensing danger and damage, and micro-
glial responses are not only orchestrated by DAMPs (see
Table 48.1). Microglia are a source and target of a plethora
of cytokines, which control diverse genes and functions by
themselves but also exert remarkable modulatory influences
on other receptor-signaling systems (Hanisch 2002; Häusler
et al. 2002; Regen et al. 2011).
 Among the cytokines/interferons, TNFD, IL-1, IL-4,
and IL-6 play essential roles as to their induction by diverse
insults, multiple implications, profound effects, and key
roles for governing microglial functions (Block et al.
2007; Hanisch 2002; Hanisch and Kettenmann 2007;
Kettenmann et al. 2011; van Rossum et al. 2008). IL-1 and
its relatives are essential to CNS responses as (mainly but
not only) mediated by microglia based on a deep involve-
ment in a regulation of (innate) immune function (Liew
et al. 2010; Prinz and Hanisch 1999; Smith 2011). Beneficial
outcomes of manipulating IL-1–like signaling, for example,
via IL-1 receptor antagonist, point to an implementation
in CNS-harming processes, but IL-1 also has functions in
development or endocrine axis (Bilbo and Schwarz 2009;
Hanisch 2002). IL-1 can claim special relations to situations
with TLR-agonistic PAMP/DAMP involvement owing to
common signaling pathways.
For IL-6, ambiguous impacts on microglia result from its
wide array of proinflammatory as well as antiinflammatory
consequences. It is the induction by tissue damage that may
determine the diverse importance for the CNS. Similarly as
for IL-1, IL-6 functions are modulated at both the level of the
ligand as well as its receptor activity (Hanisch 2002).
IFNγ and IL-4 are key determinants for instruction of
general orientations seen with activated macrophages, includ-
ing microglia (Hanisch and Kettenmann 2007; Kettenmann
et al. 2011). Their roles are further discussed in the follow-
ing. Importantly, production largely depends on other cells,
namely Th1 and Th2 T-cell subsets, as well as natural killer
(NK) cells, indicating a prominent control of infiltrating
immune cells on microglia (Hanisch, 2002; Hanisch and
Kettenmann 2007; Häusler et al. 2002; Kettenmann et al.
2011).
Chemokines comprise a family of chemoattractive cytok-
ines exerting functions in cell migration under both physi-
ological and pathophysiological conditions, but also beyond.
Their structures, receptors, signaling, and (glial) physiology
are subjects of chapter 22. Receptors are expressed abun-
dantly in microglia. Combined with a promiscuous ligand
acceptance, induced chemokine cocktails exert a sophisti-
cated control. Special mention is made regarding members
with exposed roles in the management of microglial func-
tions (Kettenmann et al. 2011). CCL2 is of key importance
as to its monocyte-attractive properties (Mildner et al. 2007).
CX3CL1, also known as fractalkine, organizes a complex
control over microglia activities by calming effects and other
involvement (Cardona et al. 2006; Prinz et al. 2011). CXCL10
signaling via CXCR3 plays roles in diverse neuropathologies.
Involvement in NMDA-induced neurotoxicity unraveled
cooperation with astrocytes for a complex function of micro-
glia in diminished or enhanced cell death, depending on CNS
regions (Kettenmann et al. 2011).
If a class of compounds is typical for the CNS, it is the range
of neurotransmitters. As it is for astrocytes, some can be sensed
by microglia. Interactions with neuronal signaling derive from
both their production and the expression of their receptors.
Although activating contributions are found largely for ATP
and related purines, other transmitters and cotransmitters
FAC TO R S C O N T R O L L I N G M I C R O G L I A L AC T I VAT I O N
may mainly exert modulating influences (Boucsein et al. 2003;
Kettenmann et al. 2011).
The list of microglia-activating factors comprises an
increasing number and diversity of additional molecules, con-
taining also lipid mediators or proteases with unusual receptor
mechanisms, as surveyed in detail by Kettenmann et al. (2011).
Examples are given in Table 48.1.
2.5 C O M M IT M E N T TO R E AC T I VE P H E N OT Y P E S
A global impact on understanding microglial functions derived
from research on macrophage phenotypes, that is, their abil-
ity to mount tailored responses (Gordon and Taylor 2005;
Martinez et al. 2006; Mosser and Edwards 2008; Murray and
Wynn 2011).
Microbial agents such as LPS, or cytokines such as TNFD
or IFNJ, trigger the “classical” and “innate” (immune) activa-
tion. Cells develop a phenotype with proinflammatory, APC,
and microbicidal activities in support of Th1-type adaptive
immune responses, which can also result in tissue damage,
however. Macrophages can “alternatively” commit to more
antiinflammatory (inflammation-resolving) profiles under the
influence of the Th2 cytokines IL-4 and IL-13, with endocy-
totic, parasite-killing, and tissue repair activities and fostering
Th2 immune responses (Murray and Wynn 2011). Following
the Th1/Th2 paradigm, classically and alternatively activated
macrophages were also termed M1/M2 types. The principle
appears to be evolutionarily conserved as it is seen in fishes.
Highlighted by the prominent expression of either the proin-
flammatory IL-12 (IL-12hi) or the immunosuppressive IL-10
(IL-10hi), M1 and M2 macrophages reveal characteristic (dis-
crete, partially reciprocal, and overlapping) patterns of induced
cytokines/chemokines, enzymes, and substrates for ECM
breakdown or rebuilding (Martinez et al. 2009) (Fig. 48.3).
The M1 phenotype is accompanied by the release of
TNFD, IL-1, IL-6, IL-23, CCL2, CCL3, CCL5, ROS,
reactive nitrogen intermediates (RNI), and diverse matrix
metalloproteinases (e.g., MMP-1, -2, -7, -9, or -12) as well as
(enhanced) expression of MHCII, CD16, CD32, CD64,
CD80, CD86, and TLR family members—to name a few.
M2 macrophages rather induce IL-1 receptor antagonist
(IL-1ra), CCL17, CCL22, coagulation factor XIII, decoy
IL-1RII, CD163, mannose/scavenger receptors, fibronectin,
and ECM-cross-linking enzymes (David and Kroner 2011;
Mantovani et al. 2004; Mosser and Edwards 2008; Murray
and Wynn 2011; Ransohoff and Perry 2009). The IL-10lo/
IL-12hi versus IL-10hi/IL-12lo bifurcation is thus mirrored by
preferential, selective, or even opposite induction of many
additional genes. M1 macrophages express cyclooxygenase
(COX) 2, whereas M2 orientation is associated with COX1
(Martinez et al. 2006). Expression of inducible nitric oxide
synthase (iNOS) or arginase directs the use of arginine for
production of NO or synthesis of polyamines, the former
being used for defense purposes, the latter being required for
tissue repair. Cells may switch between these pathways, M2
being the homeostatic default setting (Martinez et al. 2006).
This binary classification only temporarily served profile
descriptions (Mantovani et al. 2004; Mosser and Edwards
• 619
Figure 48.3 Reactive Phenotypes of Macrophages/Microglia. Exposure to different stimuli leads to distinct functional consequences. Certain cytok-
ines drive macrophage reactions in support of inflammation and Th1 type of (adaptive) immune responses, highlighted by IL-12 release, but consisting
also of numerous other gene activations. An array of induced cytokines and chemokines, enzymes for ROS and NO synthesis, proteases (matrix metal-
loproteinases), as well as expression of surface molecules for cell and matrix interactions is thereby serving largely in defense reactions, partially with
the risk of tissue impairment. A macrophage of this orientation is commonly known as classically or M1 activated. In contrast, cytokines, namely IL-4
or IL-13, rather induce an alternative or M2 activation. This is characterized by prominent induction of IL-10 and a more or less complementary set of
genes, for cytokines, chemokines, enzymes, and substrates that together promote antiinflammatory and inflammation-resolving processes, Th2 types of
immune responses and tissue repair. As exemplified by mannose receptor and MHCII expression, there can be substantial overlaps in genes and their
functional aspects, suggesting that reactive phenotypes are not sharply separated entities. Indeed, phenotypes cannot easily be categorized by a M1/M2
bifurcation. Subtypes were defined by triggering stimuli and outcomes, such as M2a, M2b (type II) or M2c on infections, tissue damage, and exposure
to apoptotic cells and (myelin) debris, in association with tumors and metabolic disorders. The concept of phenotypes considers major orientations
relating to homeostasis, defense, and repair, but also that actual transcriptional and functional profiles are versatile and fine tuned, and probably even
flexible. Microglia are thought (and already shown) to commit to similar phenotypes within a M1/M2 spectrum, but respond in a fashion that is
adapted to CNS conditions. For details, see a recent survey (Kettenmann et al. 2011).

2008) (see Fig. 48.3). Exposure to immune complexes (IC)
can reverse LPS-induced toxicity and IL-12 production,
switching macrophages into an IL-10hi phenotype termed type
II (M2b) (Anderson and Mosser 2002). FcJRI (CD64) seems
to be a critical FcR. M2b cells do not easily fit into a M1/M2
sorting. They produce TNFD, and all three subtypes express
MHCII. Dampening of inflammatory activity by FcJR liga-
tion was also observed with other proinflammatory stimuli,
such as lipoteichoic acid, CD40 ligand, or hyaluronic acid
(Gerber and Mosser 2001). The physiological impact of M2b
macrophages is tremendous. Transfer of 106 cells/mouse res-
cued LPS-treated animals, whereas animals receiving control
macrophages succumbed to lethal endotoxemia (Anderson
et al. 2002). In contrast to M1, M2b macrophages also induced
naïve CD4+ T cells to produce high IL-4 levels, even when
they were subsequently stimulated under nonbiasing condi-
tions. M2b macrophage transfer to ovalbumin-immunized
mice led to higher antibody titers and IgG1 preference, com-
pared with M1 delivery. Obviously, the macrophages instruct
T- and B-cell functions.
It turns out that macrophages, including CNS micro-
glia, take major orientations by the physiological context,
although actual sets of genes and functions allow for rather
flexible tuning. Accordingly, rigid phenotype classifications
cannot match biological diversity. In addition to homeo-
static control and maintenance, cells act in immunoregula-
tion and defense or healing and repair—with profiles of genes
and functions as to situations and location (Hanisch and
Kettenmann 2007; Mosser and Edwards 2008). Appropriate
phenotype initiation, propagation, maturation, and ter-
mination are essential for success. Excessive acute, chronic,
or maladapted responses can have detrimental outcomes
ranging from hyperinflammation to immunosuppression in
infection, autoimmunity, cancer, and neurodegenerative and
metabolic diseases (Hanisch and Kettenmann 2007; Pollard
2009).
For microglia, a similar array of phenotypes can be
assumed, and first studies report on the similarities and dif-
ferences, although detailed experimental proof is pending
(David and Kroner 2011; Hanisch and Kettenmann 2007;
Kettenmann et al. 2011; Olah et al. 2012; Schwartz et al.
2006). Phenotypic switches can occur without overt changes
in morphology, rendering subtle functional reorganizations
likely to escape detection. Orientations for M1 and M2 (e.g.,
under IFNJ, LPS, IL-4/IL-13, or IL-10) reveal essential over-
lap with responses of macrophages (van Rossum et al. 2008).
Nevertheless, side-by-side comparisons also reveal differences,
such as in the preferential induction of IL-10 versus TGFE, as

620 • NEUROGLIA
to the sensitivity to stimuli, organization of receptor/signal-
ing, and functional consequences (Regen et al. 2011).
The role of IFNJ and IL-4 for instruction of micro-
glial phenotypes has been addressed by a number of studies
(Butovsky et al. 2006a,b), also in relation to allowing, sup-
porting, or impeding regenerative processes (Kempermann
and Neumann 2003). In one of them, microglia were forced
toxic by treatment with aggregated AE or LPS and trans-
ferred to hippocampal slice preparations. Marked tissue
damage was observed. On the other hand, microglia treated
with IL-4 exerted neuroprotection. The effect was accom-
panied by induction of MHCII, upregulated insulin-like
growth factor (IGF) I, and altered TNFD production. IFNγ
had some protective effect, but conferred at high levels a
phenotype that interfered with oligodendrogenesis, which
could be overcome by IL-4. Injection of IL-4–educated
microglia into the cerebrospinal fluid of rodents with experi-
mental autoimmune encephalomyelitis (EAE) resulted
in increased oligodendrogenesis and ameliorated disease
symptoms. IL-4–induced oligodendrogenesis-promoting
orientation was further investigated, revealing that a
CD11b+CD11c+MHCII+TNFD– phenotype capable of
IGF-I delivery rescues cells, CNS tissue, and animals from
the adverse effects of toxic compounds, which drive a
CD11b+CD11c–MHCII–TNFD– microglia. Dendritic cell
(DC) orientation with APC potential (CD11c, MHCII)
of “protective” microglia, their plasticity and neurogenesis
support indicate that engagement with innate and adaptive
immunity (and even proinflammatory cytokines) may not
necessarily and always impair the CNS (Hanisch et al. 2008;
Rivest 2009; Simard and Rivest 2006).
Thus, harm might not only result from an overshoot-
ing (acute) reaction, but also from maladapted phenotypes.
Misinterpretation of DAMPs as signs of an exogenous threat
may trigger defense programs at a cost of neurons and glia
(Popovich et al. 1999; Schwartz et al. 2006). On the other
hand, failure to fight off brain tumors or metastasis—or even
providing assistance—would be equally devastating (Pollard
2009; Pukrop et al. 2010).
2.6 C O N T RO L OVE R T H E AC T I VAT I O N C O U R S E
Microglial activation is likely not a monophasic event. For a
population, or even a single cell, induced genes and functions
may change throughout the course (Hanisch and Kettenmann
2007) (see Fig. 48.2). In vivo evidence derives from injuries,
ischemia, or autoimmune challenges (Kettenmann et al. 2011).
Profile shifts were studied for macrophages in more detail,
but can also be shown for microglia. Microglial challenges by
PAMPs trigger CCL2, CCL3, CCL5, CXCL1, and CXCL2.
IFNγ, driving M1 polarization, was found to completely reor-
ganize the profile. It enhanced monocyte-attracting CCL2,
suppressed signals for neutrophils and Th1 cells, but spared
those for Th2 attraction (Häusler et al. 2002). Using IFNJ,
Th1 cells could thus self-limit their own recruitment. In turn,
Th2 cytokines, like IL-4, could then direct a M2-like pheno-
type in support of Th2 responses and repair. Microglia may
start with an emergency response to fight an infection or limit
FAC TO R S C O N T R O L L I N G M I C R O G L I A L AC T I VAT I O N
it after injury, but eventually convert to profile for deescala-
tion and restoration.
Experimental instructions in vitro enforce orientations
which in vivo may follow (and build on) each other (Hanisch
and Kettenmann 2007). Defects in such transitions could be
detrimental. Chronic activation may result from the persist-
ing presence of an activating stimulus that cannot be prop-
erly cleared or is constantly renewed. This might be the case
for AE plaques in AD. Vicious cycles can (further) drive
microglia owing to neuronal decline, fueling damage by self-
propelling recruiting of microglia (Lehnardt et al. 2008).
This may apply to various degenerative diseases (Block and
Hong 2007). Failure to progress through a sequence of
phenotypes could arrest microglia in a functional state that
can only transiently be tolerated by the CNS. Interestingly, cer-
tain elements of a microglial response can continue, whereas
others run down with time. Repetitive challenges through
TLRs can result in progressively declining responses, prob-
ably involving desensitization, as shown for LPS-triggered
NO and TNFD production. In contrast, cells continue
releasing prostaglandin E2 (PGE2). Accordingly, iNOS expres-
sion decreases, whereas COX2 and PGE synthase remain ele-
vated. The enzymes thus demonstrate a dissociated regulation
with time. A “continuum” of microglial phenotypes (Town
et al. 2005) or heterogeneity of reactive macrophages found
in a tissue (Kigerl et al. 2009; Mantovani et al. 2004) may,
therefore, not just reflect a variety of fixed options chosen a
priori. Cells may, indeed, switch phenotypes based on feed-
backs. Still it has to be verified whether phenotypes prevail-
ing at stages of a CNS lesion, as in MS or AD, are based on
distinct subpopulations with specialized duties or whether
individual cells adapt performances. Especially little is known
about human microglia and how reactive phenotypes are
installed and modulated—and how they compete in ambigu-
ous situations.
2.7 R E S P O NS E T E R M I NAT I O N A N D T H E FAT E
O F P O S TAC T I VAT E D M I C RO G L I A
The simplest feedback on activation could be based on dis-
appearance or clearance of the stimulus. Desensitization of
signaling cascades, as another mechanism, is frequently based
on receptor internalization. In some cases, receptor endo-
cytosis is rather a principle for triggering intracellular path-
ways. TLR4 initiates MyD88 signaling from the surface and
continues with TRIF activation in endosomes (Zanoni et al.
2011). In still other cases, signaling arrest is caused by block
of receptor domains, as for G protein–coupled receptors.
As to microglia, involved principles are as many-sided as
the multitude of expressed receptors. Failure of termi-
nating the activation can fuel smoldering inflammation.
However, the neuronal circuitry cannot tolerate such an envi-
ronment for long (Schwartz et al. 2006). Not only a tight con-
trol of the initiation of activation, but also of its termination,
is required.
Besides autonomous mechanisms, such as timed produc-
tion of IL-10 or induction of intracellular regulators of sig-
naling, feedbacks from other cells may impose de-escalation.
• 621
Whether and how microglia redistribute after local prolif-
eration and migratory congregation or whether coordinated
decline takes place by cell death or departure from the CNS
has not yet been properly addressed. Nor are the factors known
that would govern such processes (Hanisch and Kettenmann
2007) (see Fig. 48.2).
Postactivated microglia—cells that had mounted a tran-
sient reaction—may resume to the surveillance modus and
reacquire a naïve state. Alternatively, some microglia may also
remain altered, by receptor expression, signaling efficacy or
transcriptional regulation. Such effects were shown for extra-
neural macrophages (DeSanta et al. 2007). Experience from
a former activation, such as through TLR4 (DeSanta et al.
2009), could influence properties on re-challenge (e.g., by epi-
genetic modification).
Infectious complications especially in development do
not only affect maturing CNS structures, such as white mat-
ter, and causing lasting impairment, but also affect microglia
responses later in life (Bilbo and Schwarz 2009; Bilbo et al.
2005). Signaling systems with importance for defense as well
as developmental processes may perturb critical maturation
events when being induced by early infection. IL-1 serves as an
example because it shares MyD88 signaling with TLRs. The
original identification of the name-giving Toll, the Drosophila
homolog, was based on morphogenetic functions. Recent
studies addressed the TLR potential in mammalian develop-
ment (Rolls et al. 2007). Immune activation may thus conflict
with ontogenetic IL-1R/TLR/MyD88 signaling.
In combination with an assumed (life-)long tissue resi-
dence of the microglial cell pool, imprints of earlier activation
episodes should also be considered for CNS diseases that do
not have overt infectious associations, including brain tumors,
AD, or neuropsychiatric disorders. Provocative resemblance
to lymphocyte memory is “foisted” on purpose and has
already been attributed to natural killer cells (Ugolini and
Vivier 2009).
Practical implications of modulating (probably sustained)
microglial activity on an insult also relate to tactile allodynia.
Neuropathic pain can develop on nerve injury. A role for
microglia has been demonstrated, followed by an elegant dis-
section of a chain of molecular events that involve CCL21 and
P2X4 (Biber et al. 2011). Not only does such work improve
the understanding of how microglia engages with neuronal
functions, but it also nominates candidates for therapeutic
interference.
3 C E N T R A L N E RVO U S SYS T E M–S P E C I F I C
CONDITIONS IN CONTROL
OF MICROGLIAL FUNCTIONS
Tissue macrophages differ by their environment and func-
tional requirements (Davoust et al. 2008; Murray and Wynn
2011; Prinz et al. 2011). Alveolar macrophages are exposed
to airborne germs. Peritoneal macrophages may occasionally
detect trace amounts of agents spilling from the commensal
flora. The CNS harbors various types of macrophages, in addi-
tion to microglia proper. Parenchymal microglia may rarely
622 • NEUROGLIA
see microbes, and if so, only in truly threatening situations.
However, the CNS is a heterogeneous tissue, probably more
than any other organ accommodating macrophage-like cells.
Microglia in the various CNS divisions cannot ignore their
distinctions.
Microglia distribute throughout the CNS, but the den-
sity varies (Lawson et al. 1990). Regions with a high micro-
glia index, such as the substantia nigra (twice as high as other
areas), could be especially affected by inflammatory factors. In
combination with properties of the intrinsic neuronal popu-
lation (pathways for neurotransmitter synthesis), oxidative
stress can develop a strong impact (Block and Hong 2007).
Yet microglial density may not be the only determinant of
regional differences (Hanisch and Kettenmann 2007).
Microglia may themselves present with diversity in recep-
tor expression and signaling organization, underlying some
heterogeneity in responses. Several studies revealed distinct
expression patterns for CD11c, CD34, CD40, CD45, CD86,
CXCL14, FcJRII, IGF-I, IL-6, iNOS, integrins, MHCII,
neurotrophins, Tim-3, TNFD, Trem2, or 5D4+ keratan sul-
fate (de Haas et al. 2008; Kettenmann et al. 2011). Central
nervous system regions vary by cell communities as well as
micro milieus defined by prevalent neurotransmitters, ECM
components, and cues associated with myelinated or vascu-
lar structures. Expression profiles may reflect adaptations in
housekeeping duties and reactive options. This is indicated,
for example, by the CXCL10/CXCR3 system and its role in
neuronal death and glial activation in the hippocampus (van
Weering et al. 2010). These differences are disguised when
studying bulk responses of entire populations without cellular
or anatomical resolution.
Response heterogeneity could yet also apply to micro-
glia within a local population, as it is indicated by discrete
basal or regulated expression patterns for cells in the imme-
diate vicinity (Fitzner et al. 2011; Schmid et al. 2002, 2009).
Subsets are distinguished by the clearance of oligodendrocytic
exosomes laden with myelin, a contribution to physiological
myelin turnover (Fitzner et al. 2011). In turn, upregulation
of MHCII appears to be assigned to a complementary sub-
population. Compartmentalization of endogenous material
clearance and antigen presentation potential could segregate
conflicting functions. In the case of myelin, failure in seques-
tration could cause (auto)immune responses. A split by per-
formance is also seen when microglia are exposed to myelin
debris (Regen et al. 2011; van Rossum et al. 2008). Its removal
is a prerequisite for remyelination and may lower the risk for
autoimmune rechallenge. On the contrary, material taken up
by microglia could be presented to T cells. In both cases, the
physiological disposal and removal of damaged myelin, micro-
glial subtypes behave differently, probably based on receptor
sets for recognition, binding, and regulation, like Fc, comple-
ment, scavenger, phosphatidyl, and purine receptors or PRRs
(Gitik et al. 2011; Makranz et al. 2006; Neumann et al. 2009;
Rotshenker 2003, 2009; Town et al. 2005).
Evidence also points to subpopulations with distinct
electrophysiological properties (Kettenmann et al. 2011).
Privileged production of key cytokines, such as TNFD, by
subsets would grant them a master control among “the”
microglial response. Although the concept of monocyte/mac-
rophage diversity owing to origin and location has a long his-
tory (Treves 1984), studies now trace precursors and routes
of settlement, turnover rates under normal conditions, and
replenishment under disease conditions (Davoust et al. 2006;
Mildner et al. 2007; Prinz et al. 2011) (see chapter 15). First
investigations deal with microglia/subset-specific defects,
such as in Hoxb8, which correlate with dysfunctions even at
the behavioral level (Chen et al. 2010). A conscious consider-
ation of microglial heterogeneity could also open new avenues
for selective manipulation.
4 S U M M A RY A N D P E R S P E C T I VE S
Microglia are resident myeloid cells and serve as principal
innate immune cells of the CNS. Functions in the healthy
mature CNS relate to a surveillance of tissue homeostasis and
appear to include previously unnoticed contributions to the
maintenance of structural integrity, neurogenesis, neuronal
plasticity, and signaling. Developmental roles include but also
exceed phagocytosis. All these functions are tightly regulated
by signaling inputs as mediated through a broad array of con-
stitutively expressed receptors, including those that prepare
microglia for responses to infections, tumors, cell impairment,
and tissue injury of the most diverse cause and nature. PAMPs
and DAMPs, cytokines and chemokines, immunoglobulins
and complements lipid mediators and neurotransmitters exert
profound influences on reactive phenotypes. Shifts from the
surveillance (resting) mode to executive states are controlled by
the net influence of activating and calming factors. “Activation”
may induce further upregulation of receptors, ion channels, and
other cognate molecules to install, enhance, or modify sensitiv-
ity to additional signals or change expression to render cells
unresponsive—as part of adapted reactions. Cell contact and
messengers of resident CNS and invading immune cells may
subsequently further shape functional profiles and eventually
organize de-escalation and installation of a postactivation status.
Although the list of reported factors in microglia control is ever
growing, future challenges include the need for understand-
ing their integrative actions. Practical use shall be made based
on factors that decide on protective versus harmful actions of
microglia to develop strategies of therapeutic moderation.
AC K N OW L E D G M E N T S
This work was supported by grants from the German Research
Council (DFG, SFB/TRR43 and FOR1336). Figures have
been based on published work with respective permission
(Hanisch and Kettenmann 2007; Kettenmann et al. 2011;
Regen et al. 2011).
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• 625
 49.
ROLES OF ACTIVATED MICROGLIA
Kelly R. Miller, Stefan Prokop, and Frank L. Heppner

A B B R E VI AT I O N S “resting” microglia in the healthy brain are highly dynamic

cells (Davalos et al. 2005; Nimmerjahn et al. 2005) (see
AA arachidonic acid chapter 47); therefore, microglia activation refers specifically
AD Alzheimer’s disease to a distinct repertoire of cellular behavior that is restricted to
ATP adenosine triphosphate situations in which there is a disturbance in CNS homeostasis.
BMEC brain microvascular endothelial cell Careful consideration of this definition highlights at least
[Ca2+]i intracellular calcium activity three critical aspects of microglia reactivity: (1) The range of
CNS central nervous system responses that microglia exhibit upon activation are widely
GM-CSF granulocyte-macrophage colony varying and highly context-dependent; (2) in the absence
stimulating factor of primary “microgliopathies” (Bianchin et al.), microglia
IL-1β interleukin-1 beta activation constitutes a reaction to an already present CNS
IL-3 interleukin-3 disturbance; and (3) microglia activation refers to an in vivo
IL-6 interleukin-6 cellular response. By nature, the role of microglia is to detect
JAK janus kinase and respond to homeostatic perturbations in their microen-
LPS lipopolysaccharide vironment; therefore, cells in vitro are likely to exhibit behav-
MAPK mitogen–activated protein kinase iors that do not translate to the in vivo situation. This chapter
M-CSF macrophage colony stimulating factor describes how microglia respond to CNS insult with a focus
MCP1 monocyte chemotactic protein-1 on the in vivo setting.
NADPH nicotinamide adenine dinucleotide phosphate The functional significance of microgliosis varies depend-
NgR Nogo receptor ing on the pathological circumstances. Reactive microglia are
NO nitric oxide capable of responding to brain insult with a wide repertoire of
NPY neuropeptide Y specialized activity (Fig. 49.1). One prominent role of activated
PMA phorbol myristate acetate microglia in instances of neuronal degeneration is phagocyto-
PNS peripheral nervous system sis of tissue debris. Microglia also serve as antigen-presenting
ROS reactive oxygen species cells (for details, see chapter 50). Moreover, upon activation,
STAT signal transducer and activator of microglia migrate to sites of damage and proliferate. Finally,
transcription activated microglia upregulate or produce de novo immu-
TNFα tumor necrosis factor alpha nomodulatory factors, including cytokines and chemokines,
that mediate both autocrine and paracrine signaling. With the
exception of antigen presentation (see chapter 50), each of the
1 INTRODUCTION aforementioned aspects of microglia reactivity is elaborated in
the sections to follow.
The term microglia activation is a somewhat ambiguous
and problematic phrase that is often dangerously overused.
Historically, this term has been introduced as a mainly descrip- 2 M I C R O G L I A P R O L I F E R AT I O N
tive idiom derived from the morphological appearance of
microglia in pathological conditions, which implies a uniform The mitotic potential of microglia in the adult brain was
cellular response to a variety of stimuli, while misleadingly also first demonstrated in the late 1960s using autoradiographic
assuming that microglial cells are quiescent bystanders in the studies employing [3H] thymidine incorporation in the con-
normal, nondiseased central nervous system (CNS) that only text of brain injury (Friede and Johnstone 1967; Kreutzberg
awaken when called on. Of course, the microglia cosmos is not 1966). Although microglial cells have been shown to divide
black and white, and this is why we need to adjust our view in the absence of injury, this phenomenon occurs to a much
on, and even the terms we use to describe, microglia activa- lesser degree in the healthy brain and is thought to reflect nor-
tion. So, what is microglia activation? To activate is “to make mal cell turnover (Dalton et al. 1968; Tonchev et al. 2003).
something active or more active” (Merriam-Webster Online Population expansion of microglia in response to CNS insult
Dictionary 2012). In regard to microglia biology, more active is allows a more robust cellular response than might otherwise
indeed the appropriate description. It is now well known that be possible by the existing pool of cells in the vicinity of brain

626
 protein aggregates resting microglial cell injury/trauma

CNS Insult
infection ischemia

activated microglia cell

IL-1βra
NPY
cannabinoids
Nogo-A
anti-inflammatory drugs Apoptosis of excess
Stages of Microglia Activation

cells

1. Migration C5a
ATP
IL-1β 2. Proliferation
M-CSF
GM-CSF
IL-6
TNF-α
IL-1β
ATP
NADPH oxidase/H2O2

Ab
bacteria
phosphatidylsering
asialoglycoprotein
vitronectin
4. Phagocytosis 3. Cytokine production
IL-10
TGFb

IL-1β
IL-6
TNF-α
IFNγ
IL-2
M-CSF
Resolution of

resting microglial cell senescent microglial cell

Activation
Microglia

Figure 49.1 Functions of Activated Microglia. Initial CNS insult leading to microglia activation. Photomicrographs depict: (Upper left panel) Protein
aggregates (β-amyloid plaques [4G8 immunohistochemistry] left; and neurofibrillary tangles [AT-8 immunohistochemistry] right). (Lower left panel)
Infection (cerebral fungal abscess [PAS staining] left; and cerebral toxoplasmosis [H&E staining] right). (Upper right panel) Trauma (severe CNS
trauma with intracerebral hematoma). (Lower right panel) Ischemia (hypoxic neuronal damage [H&E staining] right; acute cerebral infarction [H&E
staining] left]. Microglia activation. Photomicrographs depict: (1) Migration—activated microglia [Iba-1 immunohistochemistry, brown] gathering
around cerebral β-amyloid plaques (4G8 immunohistochemistry, red) in a mouse model of AD. (2) Proliferation—proliferating microglia in the spinal
cord after peripheral nerve lesion (Iba-1 green; BrdU, blue). (3) Cytokine secretion—activated murine microglia after intracerebral LPS challenge
(Iba-1 immunohistochemistry, brown) signaling to astrocytes (GFAP immunohistochemistry, lower right) and neurons (H&E staining, lower left).
(4) Phagocytosis—in vitro phagocytosis assay using acute slice cultures (Iba-1 immunohistochemistry, red; Latex beads, green).

R O L E S O F AC T I VAT E D M I C R O G L I A • 627
damage; however, it is important to note that inhibition of
microglia proliferation does not necessarily prevent cellular
reactivity and effector functions (Raivich et al. 1994). Despite
the numerous contexts in which microglial activation has
been observed, mitosis proves to be a prominent and consist-
ent component of the cellular response to injury. Notably, the
proliferative activity of microglia contrasts other CNS cells,
including astrocytes, which undergo cell division in a much
more limited context (see chapter 51).
Much of our insight about microglia proliferation has been
gained by studying regenerating nerve models (Cammermeyer
1965; Kreutzberg 1966; Miller and Streit 2007; Streit and
Kreutzberg 1988). Data from these and other acute and
chronic neural injury models reveal that microglial prolifera-
tion begins as early as 12 hours post-lesion, peaks between 3
and 15 days after insult depending on the nature of the injury,
and declines thereafter (Ladeby et al. 2005; Sjostrand 1971;
Streit and Kreutzberg 1988; Tonchev et al. 2003). Following
a proliferative burst, homeostasis of the microglia popula-
tion is regulated through apoptotic death of excess cells, a
process that can be detected as early as 4 days postinjury and
persists for up to 21 days (Gehrmann and Banati 1995; Jones
et al. 1997). Although microglial mitotic activity typically
occurs during early stages of insult and subsequently resolves,
repeated stimulation in the form of lipopolysaccharide (LPS)
administration has been shown to induce continued prolifera-
tion for up to 45 days (Shankaran et al. 2007). Nevertheless,
the exact proliferative capacity of microglia is not known, and
data from in vivo experiments wherein microglia were sub-
jected to repeated activation over the course of a year suggests
that the mitotic potential of microglia may be finite (Miller
and Streit 2007), despite their telomerase enzyme expression
(Flanary and Streit 2004), which serves as a compensatory
mechanism for telomere attrition.
In contrast to the well-characterized proliferative response
of microglia, the mechanisms underlying its regulation prove
more complex and challenging to delineate.
Although microglia proliferation can be readily induced
by countless substances in vitro, the physiological relevance of
such manipulations is not clear, and the highly reductionistic
nature of the setting ensures that critical regulatory elements
of microglial activity are lacking. Nonetheless, careful consid-
eration of both in vivo and in vitro data together reveals some
converging mechanisms governing microglial mitosis.
In principle, nearly any stimulus that induces microg-
lia activation will also serve to incite proliferation, whether
directly or indirectly. Therefore, it is beyond the scope of this
chapter to detail every condition associated with microglia
proliferation (see chapter 48 for more details), but discussion
will rather focus on the most commonly identified signal-
ing pathways mediating microglia mitosis. The best studied
models wherein microglia activation and subsequent cell divi-
sion has been described include nerve injury models, stroke,
infection models, including LPS administration and models
of (degenerative) CNS disease, especially Alzheimer’s disease
(AD). These varying stimuli often elicit production of com-
mon promitotic factors, including, but not limited to, growth
factors, especially macrophage colony stimulating factor
628 • NEUROGLIA
(M-CSF) and granulocyte-macrophage colony stimulating
factor (GM-CSF) (Kloss et al. 1997; Raivich et al. 1994);
cytokines, such as interleukin-1 beta (IL-1β) (Mander et al.
2006), tumor necrosis factor alpha (TNFα) (Ganter et al.
1992; Merrill 1991), interleukin-6 (IL-6) (Kiefer et al. 1993;
Streit et al. 1998, 2000), and interleukin-3 (IL-3) (Kloss et al.
1997); chemokines, prominently including monocyte chemo-
tactic protein-1 (MCP1/CCL2) (Hinojosa et al. 2011) and
purines, specifically ATP (Baricordi et al. 1999).
Binding of these aforementioned factors to their respec-
tive receptors serves to activate many of the same signaling
pathways that ultimately converge to induce gene transcrip-
tion of, among other transcripts, cell cycle regulators such
as the cell cycle–associated proteins cyclin D1, E, A, and
cyclin-dependent kinase inhibitor p21. Specifically, the
janus kinase ( JAK)-signal transducer and activator of tran-
scription (STAT) pathway ( JAK-STAT) plays a prominent
role in signal transduction of a wide array of cytokines and
growth factors leading to changes in cellular activity, includ-
ing proliferation (Aaronson and Horvath 2002). Among the
proliferation-inducing molecules described in the preced-
ing, the CSFs and interleukins have been shown to initiate
microglia proliferation by signaling through the JAK-STAT
pathway (Liva et al. 1999; Nosaka et al. 1999). Although
common, the JAK-STAT pathway is certainly not exclusive
in the mediation of promitotic signaling cascades, and the
Ras- mitogen–activated protein kinase (Ras-MAPK) path-
way has also been described to act downstream of the men-
tioned proliferation-inducing factors, including the CSFs
and MCP-1, for example ( Jimenez-Sainz et al. 2003; Nicola
1990). Similarly, ATP binding to the ionotropic P2X7 recep-
tor on microglia has been shown to activate MAPK pathway
proteins, ERK 1/2 and MEK (Skaper et al. 2010) and leads
to transient Ca2+ currents in microglia (Eichhoff et al. 2011;
Oshimi et al. 1999).
An important regulator of microglia proliferation upon
stimulation by many of the discussed factors, specifically
including IL-1β, TNFα, ATP, and GM-CSF as well as arachi-
donic acid (AA), phorbol myristate acetate (PMA) and fibrillar
Aβ 1–40, is hydrogen peroxide (H2O2) generated from nico-
tinamide adenine dinucleotide phosphate (NADPH) oxidase
(Nox) by microglia ( Jekabsone et al. 2006). H2O2 produced
by the degeneration of superoxide can induce cell death at high
concentrations, but at low concentrations can affect cell signal-
ing pathways, including those previously mentioned, through
the oxidation of target molecules (Groeger et al. 2009). In this
way, H2O2 produced by the phagocyte-expressed Nox enzyme
is capable of inhibiting enzymes, including phosphatases,
thereby influencing signal transduction pathways such as the
JAK-STAT and Ras-MAPK pathways ultimately resulting in
microglia mitosis.
3 M I C R O G L I A M I G R AT I O N
Although resting microglia engage in continual extension and
retraction of their processes to survey the surrounding milieu,
they are believed to exist as nonmigratory cells in the absence
of activation. However, in vitro and in vivo studies have dem-
onstrated the migratory capacity of activated microglia in
response to a multitude of chemoattractants and brain insult,
respectively. The ability of microglia to migrate to sites of
brain damage and disease is crucial for fulfillment of their role
as CNS resident immunocompetent cells. Almost without
exception, microglia can be found perilesionally where they
secrete signaling molecules, including chemokines that serve
to alert other microglia and astrocytes and recruit more cells
to the injury site, phagocytose tissue debris and pathogens,
and present antigen to peripherally derived immune cells.
Other important activities of activated microglia that require
migration to injured neurons involves synaptic stripping (see
section 5). This phenomenon has now been observed in
numerous contexts, and in some instances is thought to
protect neurons from excitotoxic inputs (Kreutzberg and
Hager 1966; Streit and Kreutzberg 1988; Trapp et al. 2007).
Additionally, in AD for example, microglia migrate to and
cluster around amyloid plaques (Fig. 49.2), but their function
in this setting remains unresolved. Although microglia are
invariably present at sites of CNS pathology, the mechanisms
regulating migration, like all aspects of microglia reactivity,
are complex and tightly regulated and also difficult to study
in living organisms.
Most knowledge gained about the specific molecules
that induce microglia mobilization and the signaling path-
ways involved has been obtained from in vitro studies using
transwell migration assays involving the Boyden chamber and
time-lapse imaging of cells in culture or ex vivo brain slices.
One elegant study that provides great insight into the charac-
teristics of long-distance microglia migration was conducted
using time lapse confocal imaging of in vivo–injured, acutely
isolated living brain slices (Carbonell et al. 2005). This study
revealed that extensive migration of microglia occurs as early
as 24 hours after injury and peaks at 3 days. Furthermore, micro-
glia reached a maximum speed of greater than 10 μm/minute.
Interestingly, data from this study demonstrate that the speed of
50 μm
Figure 49.2 Activated microglia gathering around a cerebral
β-amyloid plaque in a mouse model of Alzheimer’s disease (Iba-1
immunohistochemistry, brown; 4G8 immunohistochemistry, red).
R O L E S O F AC T I VAT E D M I C R O G L I A
microglia migration varies depending on the inciting stimulus,
again highlighting the very specialized and heterogeneous
responses of microglia to different types of CNS insult.
Despite the multitude of activating stimuli so far shown
to induce microglia migration, some prevailing signaling
molecules have been identified. The initial phase of chemot-
axis involves binding of a chemoattractant to its receptor.
Perhaps the most well-defined factor known to induce migra-
tory activity in microglia (and leukocytes) is complement
5a (C5a). Complement proteins are known to be produced
at sites of CNS insult, and thus serve as ideal candidates for
mediation of microglia chemotaxis. The major C5a receptor,
CD88, is expressed on microglia and has even been found to
be upregulated on microglia in the vicinity of amyloid plaques
in AD mouse models (Cudaback et al. 2011). Binding of C5a
to its receptor has been shown to induce almost immediate
changes in microglia behavior, including ruffling of cellular
membranes followed by lamellipodia extensions (Nolte et al.
1996). This activity was accompanied by a rearrangement of
the actin cytoskeleton and increased intracellular calcium
activity [Ca2]i. Similarly, the neuronally derived signaling mol-
ecule fractalkine also elicits microglia chemotaxis in response
to injury that is accompanied by increased [Ca2]i (Harrison
et al. 1998). As mentioned, microglia are sensitive to extracel-
lular ATP levels, and data reveals that binding of ATP to puri-
nergic receptors also serves to induce microglia migration. In
fact, in contrast to the great deal of in vitro data gathered on
this topic, ATP-induced microglia motility has also been dem-
onstrated in the living neocortex of mice using two-photon
imaging (Davalos et al. 2005). Several lines of evidence sug-
gest that P2X4 and P2Y12 receptors may be the primary media-
tors of ATP-induced microglia motility, but involvement of
other purinergic receptors is not precluded (Honda et al.
2001; Horvath and DeLeo 2009). Like C5a, ATP is present at
foci of brain damage or disease through release from distressed
cells.
To balance pro-migratory signals, several factors have also
been identified that serve to inhibit microglia motility. In
general, antiinflammatory molecules that act to dampen the
overall microglia activation response can also inhibit migra-
tory activities. For example, exogenous agents ranging from the
tetracycline antibiotic minocycline to the antiinflammatory
spice curcumin inhibit numerous aspects of microglia reactiv-
ity, including migration (Karlstetter et al. 2011). Similarly, the
endogenously derived IL-1β receptor antagonist (IL-1βra)
and neuropeptide Y (NPY) inhibit IL-1β–induced microglia
migration (Ferreira et al. 2012). In addition, cannabinoids have
also been shown to hinder microglia chemotaxis toward the
microglia-derived virus-specified trans-activating protein, Tat
(Fraga et al. 2011). Alternatively, an example of a more spe-
cialized anti-migratory signaling axis involves Nogo-A and
Nogo receptor (NgR). In addition to neurons, microglia have
been found to express NgR, which when bound by its ligand,
Nogo-66 (a component of the myelin associated inhibitory
molecule, Nogo-A) mediates inhibition of microglia migration
by affecting cell adhesion, polarization, and membrane protru-
sion formation (Yan et al. 2011). Moreover, expression of NgR
and its coreceptor, Troy/Lingo-1, were found to be expressed
• 629
by microglia in demyelinating lesions of multiple sclerosis, sug-
gesting that this molecule may have a distinct role in regulation
of the microglial inflammatory response (Satoh et al. 2007).
Finally, in vitro findings point out that many CNS-resident
cells can and likely do participate in the complex and fine-tuned
orchestration of the brain’s intrinsic inflammatory response.
Specifically, evidence supports a role for soluble factors pro-
duced by brain microvascular endothelial cells (BMECs) in
inhibition of microglia migration (Wang et al. 2011). Although
this was not an exhaustive enumeration of all factors that may
influence the motility of activated microglia, and there are sure
to be many more interesting and important regulators identi-
fied in the future, the information known to date clearly indi-
cates that like all aspects of microglia reactivity, perilesional
migration is a well-controlled process within the CNS.
4 C Y TO K I N E A N D C H E M O K I N E
PRODUCTION
The production of cytokines and chemokines following activa-
tion is a prominent feature of reactive microgliosis and may per-
haps be the best-studied aspect of microglia function. Microglia
are capable of producing a great variety of cytokines and
chemokines, and new evidence for the impact of these signaling
molecules on the CNS, be it in disease or physiological settings,
is constantly added. Basic principles of cytokine and chemokine
production by activated microglia and important signaling mol-
ecules and interactions are discussed herein (Table 49.1). The
interested reader is referred elsewhere for a detailed overview
of factors produced by activated microglia (Hanisch 2002)
and to chapter 22 for a description of the receptors mediating
cytokine and chemokine signaling. Most evidence concerning
cytokine/chemokine production by microglia has been derived
from in vitro studies using isolated cells, mainly from perinatal
tissue, mixed glial cultures, and organotypic slice culture sys-
tems. Data extracted from these analyses have broadened our
understanding of microglia function, but must be interpreted
with care in light of the fact that primary cell cultures derived
from perinatal tissue may not reflect the resting state of micro-
glia, which is only achieved later in development (Draheim
et al. 1999). Furthermore, a fact that should not be underes-
timated is that single microglia or mixed-glial culture systems
lack other cell types that serve to form important signaling net-
works in the in vivo setting. Finally, acute tissue damage occur-
ring during preparation of organotypic slice cultures can lead to
inflammatory changes that may confound experimental results.
These may be some of the very reasons why we still do not have
a complete and accurate picture of “microglia activation” as it
occurs in reality, that is, in the living mature brain upon distur-
bance of CNS homeostasis.
Microglial elaboration of cytokines/chemokines is a tightly
regulated and gradually escalating process. The production of
these factors by activated microglia occurs in a graded fashion
in relation to the severity and nature of the insult. Although
microglia cytokine/chemokine secretion is generally aimed at
restoring homeostasis after an insult and is therefore locally and
temporally restricted, excess production of effector molecules
630 • NEUROGLIA
resulting from cell dysfunction or chronic stimulation may be
harmful to the surrounding brain tissue. Many factors secreted
by microglia act in a paracrine fashion on neighboring cells,
such as neurons and astrocytes, but microglia also engage in
critical autocrine signaling that can serve to self-regulate other
aspects of cellular activation, such as morphology (see chapter 8
for details), migration (discussed in the preceding) and fur-
ther cytokine elaboration. This section describes the modes of
cytokine signaling that microglia engage in and highlights the
varying and specific profiles of microglia cytokine elaboration
that occur under different pathological circumstances.
4.1 AU TO C R I N E S I G NA L I N G
Autocrine signaling refers to the secretion of a substance by a
cell that acts on surface receptors of the same cell. Microglia
express myriad cytokine and chemokine receptors that bind
molecules synthesized by the cells themselves (see chapters 19
and 22 for more details).
4.2 PA R AC R I N E S I G NA L I N G
Signaling molecules produced by activated microglia have a pro-
found impact on neighboring glial cells, neurons, and endothe-
lial cells. The cytokine/chemokine signaling cascade initiated
by activated microglia usually only affects tissue within the
local microenvironment. This mechanism is thought to act as
a safeguard to contain potentially harmful effects of overactive
cytokine production. However, long-range effects of signaling
molecules secreted by activated microglia include cross-talk
with the peripheral immune system, the recruitment of periph-
eral cells to the brain, and influences on the endocrine system.
Among the effects of microglia-mediated paracrine signaling,
it has been shown that cytokines derived from activated micro-
glia are important for astroglial responses to occur (Balasingam
et al. 1996). An excellent example is the instance of neuro-
pathic pain, in which it is hypothesized that early microglia
activation serves to instigate astrocyte reactivity and promote
an inflammatory process that culminates in central sensitiza-
tion (see chapter 68). This theory highlights the inhibition
of microglia activation as a therapeutic target for this condi-
tion. Among the numerous factors produced by microglia that
act on astrocytes throughout the brain are IL-1β, IL-2, IL-6,
TNFα, and M-CSF (for more detail, see chapters 47 and 53).
Furthermore, IL-1, IL-2, and IL-6 secreted by activated micro-
glia can impact growth of oligodendrocytes (Hanisch 2002),
and several microglia-derived cytokines have been implicated
in the pathogenesis of demyelinating diseases, such as multiple
sclerosis (for more detail, see chapter 61). Lastly, microglia can
influence neuronal activity via the elaboration of cytokines,
including IL-1, IL-2, interferons, and TNFα, among others
(Rothwell et al. 1996) (see chapters 53 and 68).
4.3 C O N T E X T-D E P E N D E N T C Y TO K I N E
P RO D U C T I O N
As mentioned, microglia activation and the resulting cytokine/
chemokine response is a process that is tightly regulated and
Table 49.1 CYTOKINES AND CHEMOKINES PRODUCED BY ACTIVATED MICROGLIA
CYTOKINE/CHEMOKINE ABBREVIATION TYPE OF INJURY MAIN TARGETS REFS.

Growth regulated oncogene α GROα Inflammation Leucocytes; (Filipovic et al. 2003;

oligodendrocytes Janabi et al. 1999)

Interleukin-1α/β Il-1α/β Infection, ischemia, stroke, T and B cells, monocytes, (Hanisch 2002; Raivich
excitotoxicity, mechanic microglia; neurons; et al. 1999)
injury pituitary cells
Interleukin-1 receptor antagonist Il-1ra Infection, ischemia, stroke, T and B cells, monocytes, (Simi et al. 2007)
excitotoxicity, mechanic microglia; neurons;
injury pituitary cells
Interleukin-2 Il-2 Infection T and B cells, NK cells, (Hanisch 2002)
macrophages, microglia
Interleukin-3 Il-3 Infection, neurodegeneration hematopoietic progeni- (Lee et al. 1993)
tor cells, macrophages,
microglia
Interleukin-4 Il-4 Infection, neurodegeneration T and B cells, mac- (Hanisch 2002)
rophages, microglia
Interleukin-6 Il-6 Infection, trauma T and B cells, plasma cells, (Hanisch 2002; Raivich
liver cells, microglia et al. 1999)
Interleukin-10 Il-10 Infection, neurodegeneration T and B cells, mac- (Hanisch 2002;
rophages, microglia Kettenmann et al. 2011)
Interleukin-12 Il-12 Infection T and B cells, mac- (Hanisch et al. 2001;
rophages, microglia Taoufik et al. 2001)
Interleukin-13 Il-13 Infection T and B cells, mac- (Hanisch 2002;
rophages, microglia Kettenmann et al. 2011)
Interleukin-15 Il-15 Infection, mechanic injury T and B cells, NK cells, (Gomez-Nicola et al.
macrophages, microglia 2008; Hanisch 2002)
Interleukin-18 Il-18 Infection T and B cells, mac- (Alboni et al. 2010)
rophages, microglia
Gamma interferon inducible IP-10 (CXCL10) Infection, T cells, macrophages, ( Jack et al. 2005; Xia and
protein-10 neurodegeneration microglia, dendritic cells Hyman 1999)
Monocyte chemoattractant MCP-1 (CCL2) Neurodegeneration, isch- T cells, macrophages, (Xia and Hyman 1999)
protein-1 emia, epilepsy microglia
Macrophage colony stimulating M-CSF Ischemia, injury Hematopoietic stem cells, (Kettenmann et al. 2011)
factor Neurodegeneration macrophages, microglia,
neurons
Macrophage-derived chemokine MDC ?? Macrophages, microglia, (Godiska et al. 1997)
NK cells
Macrophage inflammatory MIP-1α Infection, ischemia, Macrophages, microglia (Diab et al. 1999)
protein-1α/-1β (CCL3)/1β(CCL4) neurodegeneration
Macrophage inflammatory MIP-2 (CXCL2) Infection Neutrophils, hematopoi- (Diab et al. 1999)
protein-2 etic stem cells
Macrophage inflammatory MIP-3β (CCL19) Neuroinflammation Dendritic cells, (Columba-Cabezas et al.
protein-3β lymphocytes 2003)
Transforming growth factor TGFβ Infection, tissue damage, Lymphocytes, monocytes, (Streit et al. 1998;
neurodegeneration microglia Wyss-Coray et al. 2001)
Tumor necrosis factor α TNFα Infection, injury, Neurons, astrocytes, (Hanisch 2002; Raivich
neurodegeneration microglia, lymphocytes et al. 1999)
Regulated on activation, normal RANTES (CCL5) Infection, neurodegeneration T cells, eosinophils, baso- (Hanisch 2002)
T cell expressed and secreted phils, leukocytes
Fractalkine CX3CL1 Neuronal injury Microglia (Hanisch 2002; Zujovic
et al. 2005)

R O L E S O F AC T I VAT E D M I C R O G L I A • 631
adapted to the respective pathological stimulus. Not only
the specific factors themselves, but also the amounts that are
released are modulated to suit the severity and nature of the
tissue lesion. Low levels of factors, including M-CSF and
TGFβ1, can be detected in unchallenged adult brains (Kiefer
et al. 1993; Streit et al. 1998). However, on activation, micro-
glia cells upregulate and secrete a panel of cytokines/chemok-
ines that are common to many different pathological stimuli,
yet which are specifically adjusted to the particular injurious
setting at hand. Besides M-CSF and TGFβ1, the proinflam-
matory factors TNFα, IL-6, IL-1β, and nitric oxide (NO)
belong to the basic set of signaling molecules secreted by
activated microglia (Raivich et al. 1999).
4.3.1 Brain Injury
Traumatic spinal cord injury leads to a rapid upregulation of
IL-1β, IL-6, and TNFα by activated microglia (Bartholdi
and Schwab 1997; Streit et al. 1998; Yang et al. 2005).
Alternatively, the regenerating facial nerve transection model,
one of the most widely used animal models for studying injury-
induced microglia activation, is associated with only low lev-
els of TNFα and IL-1β and a sustained secretion of IL-6. In
contrast, IL-6 is rapidly downregulated after an initial spike
following traumatic spinal cord injury (Streit et al. 1998).
Likewise, TNFα is strongly induced by severe neuronal dam-
age following traumatic brain injury or ischemia (Bruce et al.
1996; Seilhean et al. 1997), but is almost absent in the facial
nerve axotomy paradigm (Streit et al. 1998). High levels of
TNFα have been associated with direct toxic effects on neu-
rons (Kraft et al. 2009) and myelin (Hartung 1993); however,
animal studies demonstrate enhanced neuronal death and
poorer prognosis on deficiency of TNFα signaling (Yang et al.
1998), indicating a neuroprotective effect of TNFα, especially
at low levels. Notably, this example serves to illustrate the dual
effects that cytokines produced by activated microglia can
have. Besides the mentioned factors that are primarily known
for their proinflammatory effects, microglia also produce the
antiinflammatory molecule TGFβ1 on activation (Kiefer
et al. 1995), which serves to subdue the proinflammatory
response occurring after injury and prevent harmful effects on
CNS tissue.
4.3.2 Infection
Infection of the CNS or meninges also leads to prominent
upregulation of the aforementioned cytokines in microglia.
Besides robust increases in TNFα expression, secretion of
IFN-γ is also observed in association with specific infectious
conditions (Suzuki et al. 2005; Wang and Suzuki 2007).
Although microglia are not a primary source of IFN-γ under
most pathological circumstances, IFN-γ is a strong regulator
of subsequent cytokine production by microglia and induces
the release of molecules important for phagocytosis, antigen
presentation, and cytotoxic functions of microglia (Raivich
et al. 1999). This activation-linked signaling cascade is simi-
lar to what can be observed with other tissue macrophages,
and which is known to aid in controlling and clearing the
632 • NEUROGLIA
infection. On the other hand, an uncontrolled production
of cytotoxic molecules can also harm the surrounding CNS
tissue. For example, high levels of IFN-γ can directly induce
apoptosis by causing upregulation of Fas and Fas ligand (Badie
et al. 2000). Again, the simultaneous production of TGFβ1
can counteract some effects of IFN-γ and helps to control the
inflammatory reaction.
4.3.3 Neurodegenerative Disease
Chronic activation of microglia and subsequent cytokine and
chemokine secretion has been described extensively in several
neurodegenerative diseases (McGeer and McGeer 1997; Wyss-
Coray 2006) (see chapters 63, 64 and 65). This protracted
reactivity is characterized by upregulation of the mentioned
cytokines in activated microglia, especially IL-1β and TNFα.
It has been proposed that microglia-mediated neuroinflam-
mation serves to exacerbate neurodegenerative processes,
thereby significantly contributing to disease progression. This
theory has been called the neuroinflammatory cascade hypothe-
sis (Streit 2004) in the context of AD and has fueled clini-
cal trials on the use of antiinflammatory medications for AD
treatment, with equivocal success. Notably, chronic microg-
lia activation in response to neurodegenerative conditions is
associated with a “senescent” phenotype that does not support
classical functions of activated microglia (Miller and Streit
2007). In this context, microglia-mediated bystander damage
may occur as a result of a dysregulation of otherwise tightly
controlled microglia activity. Taken together, this suggests
that fine-tuned manipulation of specific microglial signaling
pathways known to be altered in AD may be a successful ther-
apeutic strategy. At present, the impact of microglia activation
and subsequent cytokine/chemokine secretion on disease pro-
gression is a matter of hot debate and the interested reader is
referred to chapters 63, 64 and 65 and elsewhere (Wyss-Coray
2006) for further reading.
4.3.4 Classical versus Alternative Activation
In analogy to peripheral macrophages, activated microglia can
adopt two main functional phenotypes. Microglia may exhibit
a classically activated phenotype that is induced by IFN-γ
or LPS and generally promotes Th1 responses and neuronal
injury. This “classical” response better mimics the activation
profile well-known from ex vivo studies. Under these circum-
stances, microglia stereotypically secrete the proinflammatory
cytokines TNFα, IL-1β, and reactive oxygen species (ROS)
and NO. Alternatively, IL-4 or IL-10 can induce an antiin-
flammatory or alternatively activated phenotype in microglia
(Ebert et al. 2008; Ponomarev et al. 2007; Town et al. 2005).
Microglia that exhibit the latter activation profile promote
resolution of inflammation and tissue repair, support Th2
functions, and primarily secrete IL-4, IL-10, and IGF-1. This
concept highlights the functional plasticity of microglia and
is very helpful in explaining the differential impact of micro-
glia cells in various disease contexts or during progression
of one and the same disease. Significantly, one must keep in
mind that these two distinct phenotypes describe the state of a
single, highly specialized and adaptive cell type under specific
conditions rather than differing, that is, fixed subtypes within
the microglia population.
5 P H AG O C Y TO S I S BY AC T I VAT E D M I C RO G L I A
As the “cousins” of peripheral monocytes, microglia serve
as the brain’s intrinsic macrophages. In addition to the
ever-more-recognized role of microglia-mediated phagocy-
tosis in the developing brain and homoeostatic maintenance
of the adult CNS, phagocytic microglia play a pivotal part
in CNS pathology and repair. Microglia engage in multiple
types of ingestion of extracellular substances, including classi-
cal phagocytosis, pinocytosis, and uptake of particles through
their processes.
Phagocytosis describes a process in which a cell engulfs
a solid object with its cytoplasm, forming a phagosome
that is internalized via a specialized form of endocytosis.
The solid object is subsequently degraded in the lysosome
in either an oxygen-dependent or -independent fashion.
Oxygen-dependent degradation depends on the generation
of ROS, wherein myeloperoxidase generates hypochlorite
that ultimately aids in the destruction of ingested bacteria. The
oxygen-independent system relies on the release of effector mol-
ecules that are stored in cytosolic granules. Proteolytic enzymes,
such as defensins and lysozymes as well as lactoferrin, which
sequesters iron and leads to unfavorable growth conditions, also
help to eliminate ingested bacteria (Flannagan et al. 2011).
In contrast to instances of sublethal CNS injury, neuronal
degeneration incites microglia to adopt a phagocytic pheno-
type that represents an escalated form of activation not char-
acteristic of all reactive microglial cells. Phagocytic microglia
take on an ameboid morphology (which does not necessarily
mean that all ameboid microglia per definition are phagocytic)
characterized by shortened or absent processes, cytoplasmic
hypertrophy, and surface expression of macrophage mark-
ers such as CD68 (ED1) (Streit et al. 1999). The purpose of
microglia phagocytosis is to clear apoptotic neurons, necrotic
tissue or microbes, and this process is finely attuned to suit the
initiating trigger.
Infectious agents, in particular cell-wall components of
bacteria, activate microglia and stimulate phagocytosis via
Toll-like receptor signaling. This promotes a proinflamma-
tory response characterized by secretion of IL-1, TNFα, and
NO production (Ravichandran 2003). This inflammatory
reaction, in particular the production of ROS, is important
to kill and clear the infectious agent, but can also be harm-
ful to the surrounding CNS tissue when exaggerated. Besides
direct degradation of invading microbes, antigens of phago-
cytosed and degraded parasites are subsequently presented on
the cell surface via MHC class II molecules to fuel activation
of peripheral immune cells that further aid in clearance of the
CNS infection.
Apoptotic neurons trigger phagocytosis by microglia on
exposure of internal cell wall components on the outer cell
surface. Exposed entities of asialoglycoprotein, vitronectin
or phosphatidylserine on apoptotic cells bind to respective
receptors on microglia (Witting et al. 2000) and initiate a
R O L E S O F AC T I VAT E D M I C R O G L I A
phagocytosis program that is not associated with a proinflam-
matory reaction, but rather with secretion of the antiinflam-
matory cytokines TGFβ and IL-10 (Ravichandran 2003). This
process highlights the fact that although microglial phago-
cytosis coincides with neuronal death, dying neurons signal
their demise and microglial cells act as responders, rather than
instigators of cell death. Notably, in contrast to peripheral ner-
vous system (PNS) lesions, the assumption that synapses and
dendrites from injured CNS neurons are not automatically
retracted is substantiated by findings attained using the facial
nerve axotomy model, in which it was demonstrated that
microglia actively remove synapses from the perikaryon and
dendrites of injured neurons (Streit 2005). Migratory activity
positions microglia in close proximity to damaged cells, allow-
ing them to strip residual synapses and dendrites (Schiefer et al.
1999). Similar results have been garnered from experimental
entorhinal cortex lesion models in which it was observed that
the signaling cascade initiated by injured neurons is important
for removal of denervated dendrites (Rappert et al. 2004). In
response to neuronal necrosis, cell debris remains at the lesion
site, especially myelin. Also here, microglia take on the task
of clearing the remains. Finally, pinocytosis refers to uptake
of small molecules or fluid into endosomal vesicles that can
fuse with the lysosome and lead to degradation of its contents.
In contrast to phagocytosis, the process of pinocytosis is not
specific to the substance being transported.
Although microglia are the first line of defense in the
CNS, their phagocytic capacity relative to that of periph-
eral macrophages may be limited (Mosley and Cuzner 1996;
Popovich et al. 1999), and newly recruited blood-borne mac-
rophages within the injured or diseased brain may signifi-
cantly contribute to efficient clearance of cell debris (Stoll and
Jander 1999). Importantly, the removal of myelin debris by
phagocytes is crucial for subsequent regeneration via axonal
outgrowth and remyelination, both of which have been shown
to be inhibited by the persistent presence of myelin molecules
at the injury site (Kotter et al. 2005). This process was shown
to be more effective in young as compared with aged animals
(Dubois-Dalcq et al. 2005; Shields et al. 1999), suggesting an
age-related decline in macrophage/microglia function. Myelin
degrading phagocytes have been extensively described in mul-
tiple sclerosis lesions (Bruck et al. 1995), but the functional
impact of these cells on disease progression and pathogenesis
is still far from clear. The role of microglia in multiple sclerosis
is detailed in chapter 61.
Besides microbes, apoptotic cells and cellular debris, extra-
cellular aggregates of proteins are powerful activators of micro-
glia phagocytosis. In that respect, the relationship between the
accumulation of Aβ in AD and associated phagocytic activity,
or lack thereof, by microglia has been extensively studied and
is still a matter of vehement debate (see also chapter 64). There
is abundant evidence that microglia are activated and recruited
to Aβ-plaques via a proinflammatory cascade. Moreover, is
has been shown that microglia are capable of ingesting Aβ
into their cytoplasm (Wisniewski et al. 1991). This finding
has fueled numerous experimental studies indicating that
microglia-mediated clearance of Aβ is crucial for ameliora-
tion of AD pathology (Tan et al. 2002; Town et al. 2008;
• 633
Wyss-Coray et al. 2001). However, despite the fact that Aβ
can be demonstrated inside the cytoplasm of microglia, con-
clusive experimental proof of lysosomal degradation of Aβ by
microglia is lacking. In fact, in vitro studies show that although
Aβ is efficiently taken up by microglia, it is later found in the
medium again, indicating that microglia sample these extra-
cellular aggregates, but are apparently unable to degrade them,
resulting in re-release back into the extracellular space (Njie
et al. 2012). This activity may also contribute to the dynamic
nature of Aβ plaques. In accordance with these data, evidence
from analysis of post-mortem human AD tissue suggests that
microglia attempting to phagocytose Aβ may undergo a pro-
cess termed frustrated phagocytosis that could lead to produc-
tion and secretion of toxic molecules because the cell is not
capable of degrading the ingested agent (Streit 2004). Finally,
chronic Aβ exposure has been postulated to induce premature
aging of microglia cells in a process called microglia senes-
cence, and it has been speculated that a subsequent partial loss
of neuroprotective function may contribute to AD progres-
sion. In support of this theory, data from experimental studies
reveal that conditional depletion of microglia from the brains
of AD transgenic mice does not affect the number of amy-
loid plaques in the brain (Grathwohl et al. 2009). This find-
ing does not necessarily mean that microglia are redundant in
AD pathogenesis, but rather suggests that microglia in the AD
environment are, in fact, unable to efficiently clear Aβ. Thus,
removal of these cells from the system (at least for up to 1
month) does not result in the increase in plaques that might be
expected in the case of sudden removal of efficient mediators
of Aβ clearance, whereas functional parameters such as behav-
ioral changes on microglia deficiency in the aforementioned
experimental AD setting have not been assessed. In addition
to dying neurons, microglia have also been shown to engage
in phagocytosis of other microglia cells (Falsig et al. 2008).
A transgenic mouse model allowing for the conditional and
selective ablation of CD11b+ myeloid cells in the brain causes
widespread death of microglia on pharmacological treatment,
reaching nearly 100%, depending on the treatment protocol
(Heppner et al. 2005). Interestingly, although some cell debris
can be found throughout the microglia-free parenchyma,
nearly all dead cells are cleared. Although this phenomenon
warrants further investigation, it is most likely that the cells do
not all die at once, but are eliminated by a domino effect, leav-
ing behind neighboring microglia to clear the remains of dead
cells. In support of this hypothesis, highly “alerted” microglia
displaying an uncharacteristically large, bushy appearance can
be seen near the borders of microglia-depleted brain regions
(Fig. 49.3).
It is important to note that not all phagocytic activities
performed by microglia are carried out by activated cells under
conditions of duress. In the developing brain, microglia play a
critical role in removal of surplus neurons that have undergone
apoptosis via a caspase-3–mediated mechanism (Marin-Teva
et al. 2004). Furthermore, microglia are important for removal
of synapses during development, a process also termed synap-
tic pruning, which involves the classical complement cascade
(Stevens et al. 2007) and fractalkine signaling (Paolicelli et al.
2011). Last, it has recently been demonstrated that microglia
634 • NEUROGLIA
50 μm
Figure 49.3 Alerted microglia at the border of microglia depleted cortical
area in ganciclovir treated CD11b-Herpes simplex virus thymidine kinase
mice (Iba-1 immunohistochemistry, brown).
are important for maintenance of the neuronal stem cell pool
in the hippocampus through apoptosis-mediated phagocyto-
sis (Sierra et al. 2010). These physiological phagocytic activi-
ties of microglia are primarily triggered by neuronal signaling
and are performed by nonactivated, ramified microglia and
associated with little or no inflammatory cytokine reaction.
6 S U M M A RY A N D P E R S P E C T I VE S
Microglia are capable of mounting a rapid and robust response
to homeostatic disturbances in the CNS. Although basic prin-
ciples of microglia activation are stereotypic for all types of
insults, discriminating aspects of the reactive profile are highly
specialized and context dependent. Moreover, microgliosis
is a tightly regulated process balanced by incoming signals
from surrounding cells and through autocrine signaling. In
this regard, it is important to note that microglia activation
occurs as a response to signals from distressed CNS cells who
themselves serve as instigators of reactivity. Therefore, it must
be assumed that in the absence of primary microglia dysfunc-
tion, reactive microgliosis detected in early disease stages, even
before signs of obvious pathology, likely occurs in response to
signals from already afflicted neurons, and is not a primary
event in pathogenesis. In fact, the activation of microglia
preceding other pathogenic signs may serve as a warning of
impending illness that could prove clinically valuable, and may
also be useful for studying the region and cell-specific origin of
disease. On the other hand, new data are more and more sug-
gestive of the fact that microglial function deteriorates with
advanced age, and this decline may be exacerbated by chronic
CNS pathology. More research is needed to understand the
ways in which aging, repeated injury, such as head trauma, and
chronic neurodegenerative disease affect microglia viability.
In the event that microglia lose the ability to properly self-
regulate their activational profile or respond to exogenous
regulatory signals, exaggerated and detrimental inflammatory
processes may ensue. In this way, dysfunctional microglia may
also contribute to disease pathogenesis. An understanding of
the specific functional impediments exhibited by “senescent”
microglia may provide clues regarding strategies to maintain
or boost microglia health or substitute for lost CNS support.
Technological advances, including live in vivo microscopy that
allows for in situ visualization of behaving microglia, sophis-
ticated transgenic mouse models that facilitate microglia-
specific manipulation, and ever-advancing high-throughput
genetic and proteomic screening capabilities hold promise for
an era of new discoveries in the field of microglia biology that
may facilitate therapeutic strategies aimed at regulating micro-
glial responses to brain disease.
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• 637
 50.
IMMUNE FUNCTIONS OF MICROGLIA
Trevor Owens

A B B R E VI AT I O N S responses are induced. However, subsequent effector response

must occur in tissues, wherever pathogens are found.
APC antigen-presenting cell
BBB blood-brain barrier
1.2 M A J O R H I S TO C O M PAT I B I L IT Y C O M P L E X I
CNS central nervous system
A N D C D8+ T C E L L S
DAMP danger/damage-associated molecule pattern
DC dendritic cell The antiviral major histocompatibility complex (MHC
EAE experimental autoimmune encephalomyelitis I)–restricted effector CD8+ T cell response is necessarily less
GFP green fluorescent protein dependent on professional APC, so that virus-infected cells
IFN interferon can be susceptible to immune attack, whatever their cell type
IGF1 insulin-like growth factor-1 or wherever located. Therefore, it is consistent that MHC I is
IL18bp IL18 binding protein widely expressed, essentially by all nucleated cells of the body,
LPS lipopolysaccharide and this includes all cells within the CNS. (The preceding
MS multiple sclerosis statements represent “general knowledge” and are difficult to
MyD88 myeloid differentiation primary response precisely reference. Entry points to an extensive literature can
gene-88 be found in Goverman, 2009).
NFkB nuclear factor kappa B
PAMP pathogen-associated molecular pattern
1.3 M A J O R H I S TO C O M PAT I B I L IT Y C O M P L E X I I
PD-1 programmed cell death-1
A N D C D4 + T C E L L S
PRR pattern recognition receptor
RIG-I retinoic acid inducing gene-1 Requirements for response of CD4+ T cells are more com-
RLH RIG-I–like helicase plex. These T cells are required for cellular immunity such
TGFE transforming growth factor-beta as directed against intracellular bacteria and the induction
TLR Toll-like receptor of inflammatory responses, as well as being required as help-
TNFD tumor necrosis factor-alpha ers for antibody responses. CD4+ T cells recognize antigenic
TRAF TNF receptor–associated factor; peptides in association with MHC II and this is not widely
TRIF Toll/IL1R-containing adapter inducing expressed. Hence, there is great interest in cells within tis-
IFNE sues that can express MHC II and whether they play a role in
directing immune responses in those tissues.

1 INTRODUCTION
1.1 A N T I G E N P R E S E N TAT I O N:
GENERAL CONCEPTS
The concept of antigen presentation is a cornerstone of immu-
nology. T lymphocytes are, with B lymphocytes, the only two
cell types that express clonally restricted receptors encoded
by rearranged genes. Unlike the B cell receptor or cell surface
immunoglobulin, which can directly recognize conforma-
tional epitopes of antigenic moieties, the majority of T-cell
receptors (i.e., alpha-beta receptors) are restricted to recogni-
tion of peptides that are presented by association with MHC
molecules. Immune dogma holds that antigen is primarily pre-
sented to T cells by so-called professional antigen presenting
cells (APC), within lymphoid tissue, which is where immune
1.4 C O S T I MU L AT I O N
Although essential for presentation of antigen to CD4+
T cells, expression of MHC II is not the only requirement for a
cell to function as an APC. Additional signals from costimula-
tory ligands on APCs are required for optimal T-cell response
(Goverman 2009). It should be considered here whether
the immune response being induced is primary or secondary.
The costimulatory requirements for induction of a primary
T-cell response are stricter than for a secondary response. It
makes sense that activated antiviral CD8+ T cells should be
able to exert their effector function without too many con-
straints, for optimal protection against viruses. In the same
way, effector CD4+ T-cell responses in tissues should be less
restrained than primary responses, because this enables pro-
tection against pathogens and has obvious advantage for the

638
host. Naïve T-cell responses that may be induced in the con-
text of established inflammation, outside lymphoid tissue,
would equally be expected to be costimulation-dependent.
Nevertheless, even for a secondary effector response, MHC
II expression per se is not sufficient and costimulation is
required.
Not only does recognition of MHC II+peptide per se
not necessarily lead to a productive T-cell response, but in
the absence of any other signal, MHC II+peptide alone can
induce T-cell anergy, or antigen-specific nonresponsiveness.
Simultaneous engagement of costimulator receptor-ligand
pairs delivers signals that result in a proliferative and cytokine
effector T-cell response. The paradigmatic costimulator mol-
ecules that are upregulated on microglia are B7.1/CD80 and
B7.2/CD86 (Goverman 2009). Costimulatory signals are
delivered by these molecules via CD28 on the T cell. CD40
is a cell surface receptor expressed by B cells and myeloid cells
(including microglia) that is implicated in costimulation of
T-cell responses, through engagement of CD40L/CD154 on
activated CD4+ T cells. Opposing signals via CTLA4 or via
the programmed cell death-1 (PD-1)/PD-L1 pathway keep
T-cell responses in check (Goverman 2009).
2 A N T I G E N P R E S E N TAT I O N I N T H E
C E N T R A L N E RVO U S SYS T E M
The central nervous system (CNS), like other organs of the
body, is protected from infection and tumor development by
the immune system. Unlike most other organs, there is a lack
of lymphatic drainage from the CNS and transit of molecules
from blood to the local circulation (cerebrospinal fluid) is
tightly restricted by the blood-brain barrier (BBB) (Owens
et al. 2008). These differences, although important, are often
overemphasized, because although immune cellular traffic
across the BBB is dependent on specific and well-regulated
molecular interactions, it nevertheless occurs. This is evidenced
by the deleterious consequences of its blockade as well as by
diseases such as multiple sclerosis, in which immune infiltra-
tion is a pathological characteristic (Engelhardt and Ransohoff
2005). Therefore, there is a need for immune-competent cells
in the CNS that can interact with, support, and regulate
blood-derived immune responses.
2.1 M I C RO G L I A A S C E N T R A L N E RVO US SYS T E M
A N T I G E N-P R E S E N T I N G C E L L S
The requirements for a cell to function as an APC for a CD4+
T-cell response broadly include the ability to take up anti-
gen, process it into peptides that can associate with MHC II,
express MHC II on the cell surface, and express costimula-
tory ligands and mediators. These considerations direct atten-
tion to the molecular requirements for antigen presentation
and whether microglia fulfill them. Microglia are implicated
in phagocytosis, inflammation, and cytotoxicity in the CNS
(Hanisch and Kettenmann 2007), and express all of the capa-
bilities required for antigen presentation. Microglia are the
only glial cells that normally express MHC II at any level,
I M MU N E F U N C T I O N S O F M I C R O G L I A
therefore are candidate APCs for T-cell responses in the CNS
(Hanisch and Kettenmann 2007). Consequently, we need to
understand the role of microglia as antigen presenting cells in
the CNS. It is important to consider the circumstances under
which they would mediate such a role and their status relative
to non-glial APCs.
2.2 OT H E R C E N T R A L N E RVO US S Y S T E M–
R E S I D E N T A N T I G E N-P R E S E N T I N G C E L L S
Are there other cells within the CNS with potential to pres-
ent antigen to T cells? Astrocytes were shown to be capable
of presenting antigen for secondary T-cell activation in vitro,
especially after activation by inflammatory cytokines (Fontana
et al. 1984), but subsequent studies show that they cannot pres-
ent antigen for primary T-cell response (Williams et al. 1995),
they do not normally express costimulator ligands, even in vitro
(Williams et al. 1994), and their APC capability is strongly
influenced by co-cultured microglia, which must be taken into
consideration in any assay for astrocyte function (Holm et al.
2012; Saura 2007; Williams et al. 1995). Astrocytes are not
considered effective CNS APC for inflammatory responses,
although it has been reported that they might promote antiin-
flammatory Th2 cells (Aloisi et al. 1999).
2.3 D E N D R IT I C C E L L S
The most potent antigen-presenting cell type in the body is
the dendritic cell (DC) (Goverman 2009). Myeloid DCs are
implicated in induction of inflammatory immune responses
such as in experimental autoimmune encephalomyelitis
(EAE), whereas plasmacytoid DC, that do not express myel-
oid markers, are associated with Type I interferon (IFN) pro-
duction and immunoregulation (Goverman 2009). One of
the enduring paradigms in immunology is that of Langerhans
cells in skin, which after encounter with antigen migrate to
lymphoid tissue, where they become DC and present anti-
gen to T cells. The debate whether a similar concept of APC
migration applies to induction of immune responses against
CNS antigens has been given some impetus by a study show-
ing that monocytes can migrate from CNS to deep cervical
lymph nodes (Kaminski et al. 2012), which have been impli-
cated in CNS inflammation (van Zwam et al. 2009). It was
not demonstrated whether CNS-derived monocytes became
DC in lymph nodes.
Dendritic cells are defined by their morphology and a pro-
file of surface markers, no one of which is sufficient for their
identification. These include high levels of CD11c, MHC II,
and costimulator ligands, as well as absence of markers for other
lineages. Expression of MHC II and CD103/integrin-alphaE
(OX62) was used to distinguish populations of rat DC in dura
mater, leptomeninges, and choroid plexus from parenchymal
microglia and tissue macrophages, and this and other studies
demonstrated that DC are not normally found in the CNS
parenchyma (Greter et al. 2005; McMenamin 1999; Prodinger
et al. 2011). It has been proposed that DC in perivascular/sub-
arachnoid spaces act as gatekeepers for presentation of anti-
gen to infiltrating T cells (Becher et al. 2006; Prodinger et al.
• 639
2011). Once the CNS becomes inflamed as in multiple sclero-
sis (MS) or EAE, then DC may infiltrate the parenchyma, as
do activated macrophages.
Activated macrophages are also potent APC, and it has
been reported that macrophages and DC are much more effec-
tive APC, for secondary T-cell response, than microglia (Ford
et al. 1995). However, comparison to splenic or thymic APC
as reference controls may set the bar too high for identification
of functional APC in the CNS. As discussed in the following,
there are reasons to suspect that CNS DC may be less potent
APC than splenic DC, so that microglia may be equally effec-
tive to the CNS-resident DC that are normally found within
the CNS. Nevertheless, the case for microglia as APC must
always be considered in light of the fact that other APC are
available either within the CNS or immediately adjacent. The
physiological role of microglial APC may reflect their loca-
tion rather than their potency.
3 I S O L AT I O N A N D A S S AY O F M I C R O G L I A
Apparently simple questions such as whether a given cell type
can present antigen are complicated by the experimental dif-
ficulty of isolating cells in an unaltered state from the tissue.
Many studies have relied on in vitro culture for eventual purifi-
cation of glial cells, and this introduces possibilities for assay of
aberrant activation states, which in many cases may not reflect
even activation states in vivo. Furthermore, the APC capabil-
ity of a given cell type may include contribution of cocultured
contaminant cells, as has been mentioned for astrocytes and
microglia (Holm et al. 2012; Saura 2007). Likewise, cocul-
tured DC may need to be considered in interpretation of in
vitro assays of microglial function.
3.1 C D45 L EVE L S A S A M A R K E R F O R M I C RO G L I A
Microglia, as cells of the myeloid lineage, are phenotypically
similar to macrophages, and they are usually distinguished
in situ by morphology and location, but this is not useful for
functional assays. Seminal studies by Sedgwick and Hickey
led to understanding that parenchymal microglia can be dis-
tinguished from other myeloid lineage cells in CNS by their
relatively low expression of CD45 (Ford et al. 1995; Sedgwick
et al. 1991). This allows them to be analyzed separately from
blood-derived cells, and to be isolated using flow cytometric
methods (see Fig. 50.1). This approach is most commonly
used for rodent microglia given difficulties of accessing nor-
mal human tissue in sufficient quantities for isolation proce-
dures. Reduced CD45 expression (CD45low or CD45dim) is
an experimentally useful characteristic that has been exploited
in numerous studies (Babcock et al. 2006). Myeloid cells in
perivascular space, meninges, or subarachnoid space express
CD45 at levels similar to those in blood (CD45high).
Notwithstanding persistent residual concerns that elevated
levels of CD45 on activated microglia may obscure distinc-
tions, there is consensus that the CD45dim or CD45low cells
in CNS isolates do not include any astrocytes, macrophages
or DC.
640 • NEUROGLIA
Flow cytometric discrimination of microglia from
macrophages and DC in CNS
Macrophages, DC,
T cells granulocytes
Microglia
CD45
Non-bone marrow
derived cells
CDIIb
Figure 50.1 Flow Cytometric Discrimination of Microglia from Central
Nervous System–Infiltrating Myeloid Cells. The schematic illustrates
the principle whereby microglia and blood-derived myeloid cells, all of
which express CD11b/Mac1, can be distinguished by the relatively low
level of expression of CD45 by microglia, using flow cytometry. The red
population oval defines CD45dimCD11b+ microglia, which are drawn to
not overlap with the CD45highCD11b+ macrophages DC and granulo-
cytes. Microglia can increase their expression of CD45 when activated,
but there is general agreement that this does not move them into the
CD45high category. Even if it did, the CD45dim population remains dis-
tinct from blood-derived cells, which are always CD45high. Non-myeloid
CNS-infiltrating cells, which include T cells, do not express CD11b and
are identifiable using lineage-specific markers.
3.2 C O M P L E M E N T R EC E P TO R S
Additional receptors that are used in analysis and isolation of
antigen-presenting microglia are the complement receptors.
There are two prominent integrin beta2 family D/E heterodi-
meric receptors for complement that define myeloid cells, so
including microglia, as well as antigen-presenting function.
These are the complement receptor-3 or Mac-1/CD11b, and
complement receptor-4 or CD11c. They share expression of
the beta2/CD18 chain, and are defined by their alpha chain
expression (alphaM/ITGAM for CD11b, versus alphaX/
ITGAX for CD11c).
3.3 C D11B
CD11b is routinely used as a marker for myeloid cells in mice.
CD11b-deficient mice showed delayed onset and attenuated
EAE (Bullard et al. 2005). Transfer experiments reveal a role
for CD11b in T-cell priming, generating altered cytokine pro-
files and attenuated ability to transfer EAE, and as expected
a role for host–resident myeloid cells in transfer recipients—
these would include microglia.
3.4 C D11C
Complement receptor-4 CD11c is upregulated by microglia
under similar circumstances to B7 molecules and is implicated
in their antigen-presenting capability. CD11c is composed of a
heterodimer of CD18 and CD11c. This molecule is implicated
in phagocytosis and is associated with antigen presentation.
CD11c is frequently used as a “shorthand” marker for DC,
although always with recognition that it is per se insufficient
for their identification in absence of other DC markers. Mice
that lack CD11c are resistant to EAE, although systemic defi-
ciency also affects the peripheral immune response (Bullard
et al. 2007). Adoptive transfer of wild-type encephalitogenic
T cells to CD11c-deficient mice resulted in markedly attenu-
ated EAE, indicating that a non–T-cell source of CD11c is
also critical. CD11c-deficient T cells were also compromised
as inducers of EAE, showing altered cytokine profiles, reflect-
ing the impact of CD11c deficiency in priming; therefore,
inevitably, identification of the CD11c-expressing cell(s) that
are critical for EAE is going to be complex (Bullard et al.
2007). For example, lack of CD11c+ DC has also been shown
to attenuate EAE (Greter et al. 2005).
4 D I R E C T D E M O N S T R AT I O N O F
M I C R O G L I A L A N T I G E N -P R E S E N T I N G
CELL FUNCTION
The first unequivocal assay of APC function by directly isolated
ex vivo microglia used flow cytometry to isolate them from rat
CNS, using CD11b/c+ CD45dim as markers (Ford et al. 1995).
Their ability to present antigen to myelin basic protein-specific
T cells for a secondary response was compared with that of
CD11b/c+ CD45high macrophages/DC from CNS, using
thymic APC as a reference control. Microglia induced 20-fold
increases in thymidine incorporation by T cells, equivalent to
the fold increases induced by CD45high cells isolated from the
same CNS. However, the same microglia were relatively poor
inducers of interleukin (IL) production (Ford et al. 1995). The
conclusion drawn by the authors was that microglia were infe-
rior as APC to macrophages/DC in the CNS, and it remains a
commonly stated opinion today that despite the fact that micro-
glia are the only MHC II-expressing cells resident within the
CNS, their ability to present antigen to T cells is questionable
(Ford et al. 1995; Hanisch and Kettenmann 2007). Alternative
interpretations of those data are possible.
In a complementary study, Aloisi and colleagues (1999) iso-
lated microglia from neonatal mice and compared their ability
to act as APC with that of lymph node DC, for the activation of
naïve and secondary Th1- or Th2-biased T-cell response. The key
finding with relevance to this chapter was that IFNJ-activated
microglia were as effective as DC in induction of naïve or Th1
CD4+ T cells. Given that IFNJ is itself largely produced by Th1
T cells this of course represents a chicken-versus-egg argument
for any role for microglia in the initiation of a T cell response in
the CNS, but nevertheless reinforces the core findings of Ford
et al. (1995) that microglia are effective APC under appropriate
circumstances (Aloisi et al. 1999).
4.1 MO D U L AT I O N O F A N T I G E N-P R E S E N T I N G
C E L L F U N C T I O N BY T H E C E N T R A L N E RVO US
SYSTEM ENVIRONMENT
The CNS environment may also influence function of APC.
DC were shown to become microglia-like and inhibit CD4
I M MU N E F U N C T I O N S O F M I C R O G L I A
T cells after culture with cerebral endothelial cells (Bai et al.
2009). Central nervous system–resident dendritic cells (DC,
CD45highCD11c+) may not be as effective as APC as splenic
DC (Løbner and Owens, unpublished). The finding that a
subset of CD11b+ CD11c+ CD45dim microglia were equivalent
as APC to DC (Remington et al. 2007) in fact involved com-
parison to splenic DC, so if CNS DC should be modulated by
the local milieu, then this would if anything be an understated
finding.
5 A N T I G E N U P TA K E BY M I C R O G L I A
This occurs by phagocytosis or by pinocytocis. Both intro-
duce cell-extrinsic proteins to the lysosomal compartment,
where subsequent fusion in proteolysomes generates peptides
that associate to MHC II (Goverman 2009). These pathways
intersect with those involved in autophagy, although the pre-
cise role of cell-intrinsic autophagic mechanisms to presenta-
tion of cell-extrinsic molecules remains unclear (Mizushima
and Komatsu 2011). Human and rodent microglia have
been shown to be capable of phagocytosing cell debris, and
infection- and degeneration-related material, in vitro and
in vivo (Bechmann and Nitsch 1997; Kielian et al. 2002;
Koenigsknecht and Landreth 2004; Ulvestad et al. 1994),
albeit with some selectivity (Hughes et al. 2010). An alternate
mechanism for antigen uptake involves incorporation of exo-
somes released by oligodendrocytes (Fitzner et al. 2011).
What is clear is that microglia have the ability to ingest
particulate matter from their milieu and generate from this
peptides that can associate with MHC II on the microglial cell
surface. As direct demonstration that this normally occurs,
isolated microglia from CNS without any addition of antigen
could act as APCs in vitro for activation of myelin-specific T
cells (Ford et al. 1995). The fact that the responses induced
were suboptimal compared with those induced by APC that
had been antigen-loaded emphasizes the need for further
development of microglial APC capability. Part of this may
involve MHC II induction.
5.1 A N T I G E N P RO C E S S I N G/P ROT E A S O M E
An important component of a cell’s capacity for antigen pre-
sentation is the generation of peptides that can associate with
MHC. The 20S proteasome is a multicatalytic complex of
protease subunits that achieves this goal in APCs. Microglia
have been shown to selectively substitute immunoproteasome
for constitutive subunits in response to the bacterial Toll-like
receptor-4 (TLR4)-ligand lipopolysaccharide (LPS) as well as
in response to (IFNJ) (Stohwasser et al. 2000). Thus activated
microglia adapt their proteasome structure toward an MHC I
antigen-processing capability. IFNJ has separately been shown
to induce both mRNA for MHC I and antigen processing com-
ponents, with rapid kinetics, in mouse microglia. Importantly,
these mRNAs were expressed at relatively high levels in naïve
unstimulated microglia, and then upregulated in response to
coronoavirus infection, suggesting “innate immune prepared-
ness” (Malone et al. 2008).
• 641
5.2 M A J O R H I S TO C O M PAT I B I L I T Y C O M P L E X I I
E X P R E S S I O N A N D P E P T I D E A S S O C I AT I O N
Although microglia, as members of the myeloid cell lineage,
can express MHC II, this is not always detectable in situ with
conventional staining techniques. It was described that only
5% of ex vivo isolated CD45low rat microglia expressed MHC
II, compared with 15% of CD45high cells (Ford et al. 1995).
It should be noted that neither population expressed high
endogenous levels of MHC II. Once microglia are activated,
however, MHC II is upregulated and readily detectable. One
of the best known stimuli for induction of MHC II is the T-
and natural killer (NK)-cell cytokine IFNJ (Goverman 2009).
As has already been discussed, this may not be so helpful for
consideration of innate antigen-presenting ability because pro-
duction of IFNJ implies that antigen presentation has already
occurred, although of course it has relevance to induction of
secondary responses, an important aspect of MS. Stimuli that
may be more meaningful in terms of induction of protective
immune responses would include pathogen-associated molec-
ular pattern (PAMP), or damage-associated molecular pattern
(DAMP) antigens, both of which signal via innate receptors
(Mills 2011). A number of candidate CNS DAMPs have been
identified (Amor et al. 2010).
6 I N N AT E R E C E P TO R S I G N A L I N G F O R
MICROGLIAL RESPONSE
Innate receptors recognize conserved epitopes whose pre-
sentation in a tissue reflects either infection or damage. Such
“molecular pattern” epitopes do not therefore signal non-self
so much as loss of homeostasis. This is a very important con-
cept for the CNS where microglia (as described in chapters 47
to 49) are constantly surveying their surroundings. Encounter
with innate ligands triggers microglia to upregulate MHC
and costimulatory molecules, that is to say, to become a “pro-
fessional APC” (Takeda and Akira 2001). This concept is
illustrated in Figure 50.2. The innate or pattern recognition
receptors (PRR) that play the most well understood role are
the TLRs ( Janeway and Medzhitov 2002). Although there
PAMPs, DAMPs
phagocytosis
PRRs
Immature APC
Figure 50.2 Principles of Antigen Presentation. The figure shows that stimulation of immature APC by PAMPs or DAMPs, acting through PRRs,
induces expression of costimulatory ligands and maturation of the APC. Whether the PAMP/DAMP is a component of pathogen or tissue antigen, or
whether antigen is separately phagocytosed, the result is a mature APC expressing MHC+ peptide that is equipped to costimulate a T cell by virtue of
costimulator ligand and cytokine expression (IL12 and IL23 are shown). The result of this interaction is an effector T cell that secretes cytokines such
as IFNJ (Th1) and IL17 (Th17).
642
are numerous other innate receptor families, the concept of
innate signaling pathways that induce costimulation and anti-
gen presentation is exemplified by TLRs, and for the sake of
this discussion it is assumed that similar principles apply to
other receptors. The concept is important because it links
antigen presentation to infection or loss of homeostasis and
therefore satisfies a teleological consideration of Need driving
Function. Such principles, originally devised for DC, resonate
particularly strongly for processes that might lead to immune
response within the CNS.
6.1 TO L L-L I K E A N D OT H E R I N NAT E R EC E P TO R S
The TLRs are a family of Type I proteins almost all of which
signal via IL1-receptor homologous domain interactions with
the cytoplasmic adaptor molecule myeloid differentiation pri-
mary response gene-88 (MyD88) and downstream interme-
diates to induce transcriptional activation of genes encoding
costimulator receptors, cytokines, and chemokines (Fig. 50.3).
Toll-like receptor 3 and under some circumstances TLR4 do
not signal via MyD88, but use an alternative pathway to engage
with nuclear machinery (Kawai and Akira 2009). Microglia
stand out from other CNS-resident cells in detectably express-
ing all members of the TLR family, and can upregulate their
expression in response to infection and inflammation (Kielian
2006). Cooperation between TLRs in microglial signaling has
also been shown (Holley et al. 2012), which underscores that
the microglial response to local milieux is likely to be com-
plex. Notably some of the TLRs are predominantly expressed
intracellularly, in phagosomes, in which case ligand access is
dependent on cellular uptake. Another class of intracellular
innate receptors expressed by microglia, whose role in anti-
gen presentation remains less well worked out, are the retinoic
acid inducing gene-I (RIG-I)-like helicases, or RLH receptors
(Dann et al. 2011; Kawai and Akira 2009). These bind viral
RNA, similar to TLR3, 7, and 9 and so they act as primary
signals of cellular infection. However, release of RNA and
conformationally similar entities are also a consequence of cell
damage and their uptake may also trigger microglial antigen
presenting capability. Of particular interest is the ability of
IL12, IL23
Antigen Naive
T cell
Th1,
Costimulatory Th17
molecules
Mature APC
• NEUROGLIA
 TLR7, TLR9 TLR3 RLH TLR4 TLR2, TLR4
TIRAP TRAM TIRAP
MyD88
TRIF TRADD
TRIF MyD88
TRAF6
TRAF6
TRAF3
FADD
Type I IFN NFκB
Figure 50.3 Signaling Pathways Through Pattern Recognition Receptors
for Type I Interferon Production. TLR3,4, 7, 9, and RLH receptors signal
for Type I IFN production. Type I IFN inducing pathways are shown
in red. The TLR7 and TLR9 (receptors for virus PAMPs) and TLR2
and TLR4 (receptors for bacterial PAMPs) all engage the intracellular
signaling intermediate MyD88 via Toll/IL1 receptor domain containing
adapter protein TIRAP, and this leads to activation of TRAF6. In the
case of TLR7 and TLR9, TRAF6 then engages TRAF3, which results
in type I IFN transcription and production (red arrows), whereas for
TLR2 and TLR4 (gray arrow), TRAF6 leads to nuclear factor kappa B
(NFkB) activation and production of inflammatory cytokines. TLR4
also activates TRIF via TRIF-related adapter molecule TRAM, and this
too can activate TRAF6 and NFkB (gray half arrow), but can also lead
to TRAF3 activation and type I IFN (red half arrow). The viral PAMP
receptor TLR3 and the RLH receptors also activate TRAF3, either via
TRIF (TLR3) or the TNF receptor-associated death domain TRADD
(RLH), respectively, both inducing type I IFN. Signaling through all of
these innate receptors can also activate NFkB, shown by dotted arrows in
the background. The pathway to NFkB from RLH receptors involves the
Fas-associated death domain FADD.
PRRs to trigger production of Type I IFN. This is signaled via
TNF receptor-associated factor-3 (TRAF3) accessed either via
the MyD88 or Toll/IL1R-containing adapter inducing IFNE
(TRIF) pathways (see Fig. 50.3) (Kawai and Akira 2009).
Innate receptors may also regulate cytokine responses by
microglia. Microglia are known to express TLR2 in models of
CNS infection in which exogenous PAMPs are a likely trigger
(Nichols et al. 2009). In experimental brain abscesses induced
by Staphylococcus aureus, levels of IL17 and infiltrating Th17 as
well as gamma-delta IL17 producing T cells were significantly
enhanced in TLR2-knock-out mice (Nichols et al. 2009).
These findings potentially implicate TLR2 signals in microglial
regulation of the Th17 response in the CNS, as discussed in a
later section. In the brain abscess model, microglia were shown
to express the IL17RA/CD217 receptor and so were also com-
petent to respond to IL17, although the predominant effect
of IL17 in this system was to synergize with tumor necrosis
factor-alpha (TNFD) for induction of chemokine production
by macrophages (Nichols et al. 2009). Interestingly, microglia
also upregulate TLR2 in response to noninfectious stimuli
such as axonal lesion-induced synaptic degeneration (Babcock
et al. 2006) and spinal cord injury (Kigerl et al. 2007), and in
mice that lack TLR2, the CNS response to axonal lesion was
marked by reduced production of TNFD, including by micro-
glia (Babcock et al. 2006). This speaks to the innate immune
preparedness of microglia, as mentioned.
I M MU N E F U N C T I O N S O F M I C R O G L I A
7 C O S T I MU L AT I O N O F M I C R O G L I A L
RESPONSES
Although the molecular details remain elusive it is assumed
that expression of microglial components of both costimula-
tory and regulatory pathways is controlled by their milieu, via
innate receptors. Microglia have been shown to upregulate
B7.1/CD80 and B7.2/CD86 in EAE and in MS (Goverman
2009; Issazadeh et al. 1998; Windhagen et al. 1995). Functional
relevance of B7 molecule expression was evidenced by resis-
tance to EAE in mice that lacked their expression (Chang et al.
1999), although this did not directly implicate B7 in micro-
glial antigen presentation. Interestingly, in response to synap-
tic degeneration, an experimental system that does not have
a destructive autoimmune component, microglia upregulated
B7.2/CD86 but not B7.1/CD80 (Bechmann et al. 2001). This
was interpreted to indicate that failure to upregulate B7.1/
CD80 might account for the lack of a destructive autoimmune
T-cell response. However, a transgenic mouse in which micro-
glia expressed B7.2/CD86 developed spontaneous EAE-like
symptoms with demyelination and a marked infiltration of
CD8+ T cells to spinal cord (Zehntner et al. 2003). Although
transgenic systems can sometimes have idiosyncratic mecha-
nistic aspects; this exemplifies the fundamental costimulatory
paradigm. Induction of costimulation via for example infec-
tion-related stimuli results in populations of primed or mature
APC that can most effectively present pathogenic epitopes to
host-protective T cells (Fig. 50.4). Unfortunately, this results
in their also being primed for presentation of myelin or other
CNS-derived self-epitopes.
1).
2).
Microglia MHC/TCR
CD28
No costimulation-no response
Infection or other stimuli
(PAMPs, DAMPs)
3).
B7.2
Demyelination
Figure 50.4 How Costimulation Regulates Antigen Presentation in
the Central Nervous System. Microglia that have not been induced to
express costimulator ligands do not activate T cells that may enter the
CNS (1), even though they may express antigenic peptides associ-
ated with MHC. Recognition of such peptide+MHC by T cells in
the absence of a costimulatory signal (CD28 is shown) may anergize
T cells (2), thus contributing to the noninflammatory CNS environ-
ment. Microglia that have encountered PAMP or DAMP signals as
a consequence of CNS infection or damage upregulate costimulator
ligands (B7.2/CD86 is shown) (3), and can present antigen to T cells
for induction of effector function, which can result in demyelinat-
ing disease (4). The figure is modified from an original drawing by
Zehntner et al. 2003.
• 643
 Additional tuning influences on the T-cell response
induced by antigen-presenting microglia are provided by
cytokines, which in turn direct cytokine production by T cells.
T cells that induce inflammation are characterized by cytokine
profiles, which include IFNJ as well as IL17, IL21, and gran-
ulocyte-macrophage colony-stimulating factor. Induction of
these proinflammatory cytokines is driven by synergistic or in
some cases opposing actions of IL12, IL18, and IL23, with par-
ticipation of IL1, IL6, and transforming growth factor-beta
(TGFβ) (Gutcher and Becher 2007). All of these cytokines
can be produced by microglia and as for costimulatory ligand/
receptors, it either has been shown or can be assumed that
PAMP and DAMP signals control such glial response.
8 C Y TO K I N E A N D C H E M O K I N E
P R O D U C T I O N BY M I C R O G L I A
Microglia are a source of as wide a range of cytokines and
chemokines as other myeloid cells without distinguishing
characteristics from macrophages, for example. Under appro-
priate stimulus they can produce cytokines considered both
pro inflammatory and antiinflammatory, as well as chemok-
ines attractant for multiple T-cell subsets, macrophages, DC,
and granulocytes (Hanisch and Kettenmann 2007). This
broad capacity for cytokine and chemokine production sug-
gests that it is not only the cell but also its context that will
define the microglial contribution to any particular response.
8.1 I M MU N O R E GU L ATO RY RO L E
F O R M I C RO G L I A L C Y TO K I N E S
The possibility that microglia might subserve an immuno-
regulatory function is of particular interest. They are impli-
cated in inflammatory responses via “guilt by association,”
but their actual role remains unclear. Butovsky et al. showed
that microglia could be directed by the cytokine IL4 to a
CD11b+ CD11c+ phenotype associated with production of
insulin-like growth factor-1 (IGF1) and neurogenic poten-
tial (Butovsky et al. 2006). Two recent genomewide array
screens of microglia isolated by cell sorting from mice have
shown that microglia display proregenerative gene expres-
sion profiles. In one case CD11b+ CD45dim microglia from
the corpus callosum of mice undergoing cuprizone-induced
demyelination and subsequent remyelination showed upreg-
ulation of the CD11c-encoding gene itgax as well as chemok-
ines and cytokines that would support oligodendrocyte
precursor cell recruitment (Olah et al. 2012). Expression of
IGF1 and other proneurogenic genes was shown for CD11b+
CD45dim microglia isolated from the subventricular zone of
mice with EAE (Starossom et al. 2011). The authors called
attention to expression of IL18-binding protein (IL18bp), a
soluble factor that neutralizes an inhibitor of neuronal dif-
ferentiation, by these microglia (Starossom et al. 2011). It
has been reported that CD11b+ CD45dim microglia express
IL18bp in EAE, and shown that IL18bp can inhibit Th17 in
the CNS when overexpressed using a viral vector. This was
likely caused by suppression of APC-derived IL6, IL23, and
644 • NEUROGLIA
TGFβ (Millward et al. 2010). Such Th17 T cells are impli-
cated in EAE and MS, even though IL17 itself may not be
essential (Goverman 2009; Haak et al. 2009). Microglial
functional and regional heterogeneity are likely to play a role
in this context. The fact that intrathecal delivery of adenovi-
ral IL18bp inhibited Th17 and EAE was interpreted to show
that IL18bp production by cells at the BBB controls T cell
inflammatory potential as they enter the CNS (Millward
et al. 2010). Whether the critical APC were DC or microglia
was not determined, but proximity to blood vessels and the
BBB are a likely influence on microglial phenotype (Hanisch
and Kettenmann 2007).
8.2 T Y P E I I N T E R FE RO N
The type I IFNβ (used as a therapy for MS) has been shown
to inhibit Th17 responses (Guo et al. 2008), and whether
IFNβ inhibits EAE or MS correlated inversely with IL17 lev-
els (Axtell et al. 2010). Microglia express interferon regulatory
factor-7 in EAE (Salem et al. 2011) or in response to synaptic
degeneration (Khorooshi and Owens 2010), and they are con-
sidered a likely source of type I IFN. It has been shown that
the critical type I IFN-responding population for regulation
of EAE are of the myeloid lineage and so might include micro-
glia (Prinz et al. 2008). This reinforces that microglia may act
to dampen inflammation in the CNS.
9 C D 11 C + M I C R O G L I A
CD11c is upregulated under inflammatory conditions by a sub-
population of microglia that have preferential ability to pres-
ent antigen. It was reported that microglia become activated
early in EAE and that these activated microglia upregulate
MHC II, CD40, and CD86, as well as CD11c (Ponomarev
et al. 2005). The use of radiation bone marrow chimeras in that
study, to distinguish microglia from peripheral myeloid cells,
introduces a caution about the possibility of aberrant cell acti-
vation (see Mildner et al. 2007). However, analogous findings
were made in mice undergoing cuprizone-induced demyelina-
tion, wherein there is a pronounced activation of microglia in
demyelinating corpus callosum. These were identified as being
greater than 90% CD45dim microglia and not macrophages by
flow cytometry (Remington et al. 2007). Among the CD45dim
cells was a subpopulation that upregulated CD11c, which is
not normally easily detectable on microglia (Remington et al.
2007). Similar CD11c+ microglia were identified in the den-
tate gyrus of mice that had undergone experimental denerva-
tion of the hippocampus (Babcock et al. 2008). Populations
of microglia from cuprizone-treated mice, which included
CD11c-positive microglia, were shown to be highly potent
antigen presenting cells for T-cell response induction in vitro
(Remington et al. 2007). Correspondingly, one of the genes
strongly upregulated by CD11b+ CD45dim microglia sorted
from such tissues was the CD11c-encoding itgax gene (Olah
et al. 2012). Unexpectedly, in that same study, neither CD40
nor CD80 was detected as expressed and CD86 was not
regulated.
 9 L O C AT I O N O F A N T I G E N -P R E S E N T I N G
C E L L S I N T H E C E N T R A L N E RVO U S
SYSTEM
By now the reader may be wondering whether microglia are
just immature CNS DC, given that they appear to present
antigen equivalently and share at least some surface markers
once activated. Relative CD45 levels distinguish parenchymal
microglia from other myeloid cells in CNS, including DC,
but once they are sufficiently activated, overlap in phenotype,
and function cannot be formally excluded (see Fig. 50.1). It
is not easily resolved whether some of the CD45high CD11b+
CD11c+ cells in inflamed CNS started out as CD45low micro-
glia. There are technical limitations to the extent to which
individual cells originating within a tissue can be tracked to
other locations and phenotypes. Consensus at present is that
DC are a distinct subset of myeloid cells, but one should keep
an open mind as to the extent to which this distinction is
function- and context-dependent.
It is of interest that CD11c serves as a marker for APCs
in the CNS, as elsewhere. It has been suggested that DC are
primarily situated in perivascular locations appropriate to a
“gatekeeper” function, whereas microglia are parenchymally
located and so may play more of a role in progression of EAE
and MS than in initiation (Becher et al. 2006; Greter et al.
2005) (Fig. 50.5). Such compartmentalization (both physi-
cal and intellectual) helps to resolve continuing debate over
whether microglia can present antigen as effectively as DC, but
may also be an oversimplification. The CD11c+ DC is clearly
an effective APC, and it may be speculated that the primary
difference between them and microglia may turn out to be not
Parenchymal
microglia Neuropil
Perivascular Glia limitans
space
DC
Blood Perivascular
vessel microglia
Endothelium
DC
Figure 50.5 Importance of Location for Antigen Presentation in the
Central Nervous System. T cells that enter the CNS in MS and EAE
do so in postcapillary venules in which the vascular endothelial and glia
limitans basement membranes are separated by a perivascular space.
Dendritic cells in perivascular space are considered to play a role as
gatekeeper APC. Presentation of antigen by DC to T cells permits their
further activation and facilitates their transit across the glia limitans into
the CNS parenchyma. Juxtavascular DC and (it is speculated) microglia
extend processes across the glia limitans, which can also present antigen
to trafficking T cells. By contrast, parenchymal microglia, deeper in
the neuropil, only encounter T cells that make it that far. This imposes
restrictions on their capacity for T cell interaction, especially with naïve
T cells. The figure incorporates concepts described in Becher et al.
(2006), Owens et al. (2008), and Prodinger et al. (2011).
I M MU N E F U N C T I O N S O F M I C R O G L I A
so much their innate capability as APC as their location in the
tissue. A recent study tracked CD11c+ cells in mouse CNS via
itgax promoter-driven GFP expression. It is possible that some
of the cells that were visualized were microglia, because flow
cytometric analysis of relative CD45 levels was not employed
(Prodinger et al. 2011). It is of interest that CD11c promoter-
driven GFP+ cells in that study were localized juxtavascularly
at the glia limitans, which would place this subset of microglia
in communication with cells in perivascular space, a role that
has been conventionally assigned to DC (Becher et al. 2006).
Identification of CD45low CD11c+ microglia helps to rec-
oncile studies that reported parenchymal DC in mouse mod-
els of ischemia and brain infection (Fischer and Reichmann
2001). Discrimination of microglia from blood-derived cells
by measurement of relative CD45 expression was not done
in those studies, and it may be speculated that the CD11b+
CD11c+ cells that were described may have at least included
CD11c+ microglia. Curiously, neither itgax or the CD11b-
encoding itgam genes, nor CD80 or CD86 were among those
upregulated in CD45dimCD11b+ microglia isolated from the
subventricular zone of mice with EAE, although a plethora
of other genes associated with antigen presentation (includ-
ing CD40) were among the signature genes in that study
(Starossom et al. 2011).
10 R E S P O N S E TO C Y TO K I N E S
Dialog between microglia and inflammatory cells is mediated
at least in part via cytokines. This can result either in progres-
sion or remission of the inflammatory response. For example,
microglia have been shown to express the IL-17RA/CD217
receptor for the inflammatory cytokine IL17A/F, which
defines Th17 (Das Sarma et al. 2009). In vitro studies show
that IL17 induces microglial as well as astroglial production
of chemokines such as MCP-1/CCL2, MCP-5/CCL12, and
KC/MIP-2/CXCL1 (Das Sarma et al. 2009). This would
predict amplification of the inflammatory response via glial-
derived chemokines recruiting macrophages and granulo-
cytes. Because monocytes are also a potential source of IL17
(Moseley et al. 2003), the microglial response may not only
be directed by Th17, but also by infiltrating macrophages, and
microglia themselves are also reported to be a source of IL17
(Kawanokuchi et al. 2008; Lv et al. 2011). On the other hand
the IL17 response may itself be inhibited by mediators pro-
duced by microglia in the CNS. These include type I IFN and
IL18bp (Millward et al. 2010; Salem et al. 2011). Microglia
also respond to the immunomodulatory cytokine TGF-β
which can derive from T-cell subsets as well as from other glial
cells and neurons and is implicated in preserving the relatively
noninflammatory state of the CNS (Hanisch and Kettenmann
2007; Liu et al. 2006). Another well-known modulatory com-
ponent of the microglial environmental milieu is ATP acting
via purinergic receptors, which has been shown to reduce the
in vitro induction by LPS of IL12 as well as other cytokines and
chemokines (Boucsein et al. 2003). It should be kept in mind
that demonstration of microglial capability to respond to or
produce mediators that modulate production of cytokines
• 645
in vitro is not always a predictor of microglial response in
vivo, and there may also be issues of microglial heterogeneity
(Hanisch and Kettenmann 2007).
11 M I C R O G L I A L R E GU L AT I O N O F
T CELLS
Regulation of inflammatory immune response also involves
direct cellular ligand–receptor interactions. The PD-1 recep-
tor on CD4+ T cells signals for reduced Th1 response when
engaged by PD-L1/B7-H1/CD274 that is expressed by myel-
oid cells, including microglia (Schreiner et al. 2008). This
has been implicated in regulating relapses in EAE, as well as
in modulating inflammatory T cells in Theiler virus demy-
elination (Duncan and Miller 2011). Clearly, the stimuli for
induction of PD-L1/CD274 on microglia, as well as whether
there is heterogeneity of PD-L1 expression among microglia,
need to be defined, to more fully understand this microglial
regulation of Th1 response in the CNS. Of interest is that
PD-L1 was found to be strongly upregulated by microglia
responding to cuprizone demyelination, an experimental sys-
tem that is T-cell–independent (Arnett et al. 2001; Olah et al.
2012). Again one sees evidence of innate programs of micro-
glial response that predispose to interaction with cells of the
immune system, in this case to modulate response.
12 S U M M A RY A N D P E R S P E C T I VE S
Microglia secrete cytokines and chemokines and express sur-
face molecules consistent with antigen-presenting function.
Microglia have been directly demonstrated to present antigen
to T cells and they are the best-equipped parenchymal CNS-
resident cell to do this. Ongoing studies of the influence of the
CNS environment on the antigen-presenting ability of other
cells such as DC suggest that microglia may be equally effec-
tive APC as DC in situ. The challenge now is to devise ways
to address this. Recent cell isolation and microarray studies
point the way toward more precise gene and proteomic analy-
ses of microglial subpopulations and the immune cells whose
responses they control. Key questions for the future will be,
where are the antigen-presenting microglia located in the
CNS? And how heterogeneous are microglia both with regard
to tissue localization and in the context of response? It will be
of interest to determine the extent to which the local environ-
ment determines microglial phenotype, including antigen-
presenting ability, versus whether predetermined microglial
phenotypes define the local antigen-presenting environment.
Transplantation of microglial subpopulations is currently not
technically feasible and would seem challenging, so a more
plausible approach would be to control local production of
selected candidate mediators. A further challenge will be to
selectively activate or disable subpopulations of microglia in
otherwise physiologically normal circumstances, to assess their
relative contribution to CNS immunity. The environmental
cues for microglial participation in immune responses need to
be defined—unequivocal identification of CNS DAMPs and
their receptors will be necessary. Finally, to what extent can
646 • NEUROGLIA
microglia subserve DC functions, including the ability to traf-
fic out of the CNS for antigen presentation in lymph nodes A
number of groups are engaged in addressing these issues, and
description of progress and advances can be found in chapters
15, 19, and 47 to 49.
AC K N OW L E D G M E N T S
This work was supported by grants from the Danish Agency
for Research and Innovation, the Danish Multiple Sclerosis
Society, the Lundbeck Foundation, and the Novo Nordisk
Foundation.
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 SECTION 3
ROLE OF GLIAL CELLS IN DISEASE
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MECHANISMS OF GLIAL INJURY
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 51.
ASTROCY TE RESPONSES TO CENTRAL
NERVOUS SYSTEM INJURY AND DISEASE
Michael V. Sofroniew

A B B R E VI AT I O N S diseases. These findings have revealed that astrocyte reactivity is a

context-dependent, complex, and multivariate processes that has
ALS amyotrophic lateral sclerosis the potential to impact substantively on neural function in a vari-
APC adenomatous poliposis coli ety of ways. This chapter reviews current information about (1)
AQP4 aquaporin 4 cellular and molecular aspects of astrocyte changes in response to
ATP adenosine triphosphate injury and disease, (2) mechanisms that trigger and regulate these
BBB blood-brain barrier changes, and (3) functional consequences.
Ca2+ calcium
[Ca2+]i intracellular calcium activity
CNS central nervous system 2 T E R M I N O L O GY
CSPG chondroitin sulfate proteoglycans
EAAT2 excitatory amino acid transporter 2 The terminology used to describe astrocyte responses to injury
EAE experimental autoimmune encephalomyelitis can vary considerably among authors. There are no widely
EGF epidermal growth factor accepted categories that define different types of astrocyte
FGF fibroblast growth factor responses and the same terminology can be used to describe
GABA gamma-aminobutyric acid different things by different authors. In the interest of clarity it
GFAP glial fibrillary acid protein is useful to begin with a brief consideration of terminology and
Glt1 glutamate transporter 1 definitions of terms as used in this chapter (Table 51.1). Various
GP130 receptor for interleukin 6 terms that are in common use to describe astrocyte responses
GSNO S-nitrosoglutathione to injury or disease include: astrocyte reactivity, astrocyte acti-
INF-J interferon gamma vation, reactive astrogliosis, reactive astrocytosis, and glial scar
LPS lipopolysaccharide formation. As a general all-inclusive descriptor of astrocyte
NFκB nuclear factor kappa-light-chain-enhancer responses to stimuli associated with injury or disease, we pre-
of activated B cells fer the term reactive, as used in “reactive astrocytes” or “astro-
NGF nerve growth factor cyte reactivity” (see Table 51.1). Although the term astrocyte
NMO neuromyelitis optica activation is sometimes used to describe astrocyte responses to
NO nitric oxide injury or disease, we find this term confusing in light of the
SOC3 suppressor of cytokine signaling 3 now considerable evidence that astrocytes become activated
SOD superoxide dismutase in healthy tissue by exhibiting transient, ligand-evoked eleva-
STAT3 signal transducer and activator of tions in intracellular calcium activity ([Ca2+]i). This form of
transcription 3 healthy astrocyte activation is under intense investigation as
TGF-β transforming growth factor beta a potential means of mediating dynamic astrocyte functions,
VEGF-A vascular endothelial growth factor-A including interactions with synapses and regulation of blood
flow (Attwell et al. 2010; Barres 2008; Halassa and Haydon
2010) (see chapters 16, 17, 26, 37, 38, and 39). It seems appro-
1 INTRODUCTION priate at this time to differentiate this form of astrocyte activa-
tion in healthy physiological contexts from astrocyte reactivity
For well over 100 years, astrocytes have been known to undergo to stimuli associated with injury or disease.
anatomical changes in response to injury and disease in the mam- The terms reactive astrogliosis, reactive astrocytosis, and glial
malian central nervous system (CNS), and histopathological scar also deserve clarification (see Table 51.1). In this chapter we
changes in astrocytes are routinely used as hallmarks of disease or will use the term reactive astrogliosis to denote any astrocyte
damage in human CNS tissue. Until recently, however, the func- response, however mild or severe, undergone by astrocytes to any
tional implications of such astrocyte responses have been poorly form of CNS insult. Because the suffix “cytosis” is widely used
understood. Over the past 20 years, considerable information has in histopathology to denote an increase in cell number over that
accrued regarding molecular and functional changes associated normally observed, we will use the term reactive astrocytosis only
with astrocyte responses to many different types of injuries and when there is true evidence of astrocyte proliferation. Lastly, it

653
Table 51.1 TERMINOLOGY RELATED TO ASTROCYTE RESPONSES TO CNS INSULTS
TERM DEFINITION AS USED IN THIS ARTICLE

Reactive astrogliosis Umbrella term referring to all forms of astrocyte responses to all forms of CNS insults, ranging from very
mild reversible responses such as small changes in gene expression, to moderate responses involving large
changes in gene expression and structural changes such as hypertrophy, to severe responses that include
cell proliferation and permanent scar formation

Reactive astrocytosis Reactive proliferation of astrocytes that results from newly occurring cell division in direct response to a
CNS insult

Reactive astrocyte Umbrella term referring to an astrocyte that has responded in any way (mild to severe) to any form of
CNS insult

Scar forming astrocyte Reactive astrocyte that derives from newly proliferated cells and that lines the borders to lesions of
severely damaged tissue

Glial scar Multicellular structure that forms borders around severely damaged or inflamed CNS tissue, consisting of
newly proliferated, scar-forming astrocytes that abut and interact with fibromeningeal cells and fibro-
cytes (see Fig. 51.1D,E)

Mild to moderate astrogliosis
Severe astrogliosis
Astrogliosis in which astrocytes hypertrophy but the individual domains of their processes are preserved
and tissue cytoarchitecture is preserved, and in which there is potential for resolution if the insult resolves
(see Fig. 51.1C)
Astrogliosis in which there is astrocyte proliferation and substantial overlap of astrocyte processes with
loss of individual astrocyte domains; and in which there is permanent rearrangement of cytoarchitecture,
even if the insult resolves (see Fig. 51.1D,E)

deserves emphasis that the terms glial scar and reactive astrogliosis
are not synonymous (see Table 51.1), as discussed in detail in the
next section.
3 R E AC T I VE A S T R O G L I O S I S A S A
C O N T I N U U M O F C E L LU L A R C H A N G E S
Contrary to commonly held beliefs, reactive astrogliosis is not
a simple all-or-none stereotypic phenomenon. Instead, there
is now ample experimental evidence that reactive astrogliosis
is a finely gradated continuum of changes that occur in a
context-dependent manner and that can be independently
regulated by a multitude of different specific molecular signaling
events that mediate different specific responses (Table 51.2).
The changes that can be associated with astrocyte reactivity
range from reversible alterations in gene expression and cell
hypertrophy with preservation of cellular domains and tissue
structure, to long lasting scar formation that involves cell
proliferation and rearrangement of tissue structure (see Fig. 51.1,
Table 51.3) (Sofroniew 2009; Sofroniew and Vinters 2010).
Based on a large body of observations in experimental animals
and human pathological specimens, we have recently proposed
a definition of reactive astrogliosis that includes several grades
of severity that may be commonly encountered in experimental
and clinical histopathological examinations (Sofroniew 2009;

Table 51.2 EXAMPLES OF MOLECULAR REGULATORS AND MODULATORS OF REACTIVE ASTROGLIOSIS

FUNCTIONAL CATEGORIES IMPLICATED FACTORS

A. Extracellular mediators

Cytokines and growth factors IL6, IL1β, TNFα, INFγ, CNTF, LIF, TGF β, IL10,
EGF, FGF2, endothelin

Neurotransmitters and modulators glutamate, noradrenalin

Small molecules released by cell injury ATP

Molecules of oxidative stress nitric oxide, reactive oxygen species

Ischemia-associated hypoxia and glucose deprivation

Infection-associated lipopolysaccharide (LPS)

Neurodegeneration associated β-amyloid

Systemic metabolic toxicity ammonium (NH4+)

B. Intracellular signaling molecules STAT3, NFκB, SOC3, MAP-kinase, SOX9, Nrf2, Olig2, cAMP, Nurr1, SMAD, mTOR

654 • NEUROGLIA
Sofroniew and Vinters 2010). It is important to emphasize
that the different severities of reactive astrogliosis transition
seamlessly along a continuum. Nevertheless, it is convenient
for purposes of description, classification, and discussion to
recognize several broad categories as defined in the next three
sections and as summarized in structural terms in Figure 51.1
and Table 51.1. Representative examples of the continuous
gradations of reactive astrogliosis from mild to moderate to
scar formation can be seen adjacent to essentially all lesions of
tissue damage formed by stroke, trauma, or infection (Fig. 51.2).
Although differences in degree of reactive astrogliosis are readily
apparent in different degrees of hypertrophy, upregulation of
glial fibrillary acid protein (GFAP) and proliferation, much work
remains to be done in characterizing differences in transcriptome
and proteome. Early studies in this direction show that stroke
and lipopolysaccharide (LPS) mediate significantly different

A Healthy tissue
Cytoplasm &
cell membrane

Cytoskeleton

Figure 51.1 Changes Characteristic of Different Gradations of Reactive

B Mild to moderate reactive astrogliosis Astrogliosis. A. In healthy CNS tissue, the territories of astrocyte
Cytoplasm & processes have little or no overlap and many astrocytes do not express
cell membrane detectable levels of the prototypical astrocyte cytoskeletal protein, GFAP.
B. In mild to moderate reactive astrogliosis most (if not all) astrocytes
have upregulated expression of GFAP and exhibit hypertrophy of cyto-
Domain skeleton and stem processes, but with preservation of individual astrocyte
preservation domains and without pronounced overlap of astrocyte processes and
without cell proliferation. In addition there are variable changes in
Cytoskeletal
hypertrophy
molecular expression and associated functional activities. These changes
vary with insult severity and exhibit the potential for structural resolu-
tion if the triggering insult is removed or resolves. Such changes occur
C Severe diffuse reactive astrogliosis after mild metabolic or molecular insults, or mild infections or inflam-
matory activation, or after mild trauma, or at sites distant from a more
severe injury. C. Severe diffuse reactive astrogliosis is characterized by the
Process overlap appearance of newly proliferated astrocytes (with red nuclei in figure) in
with loss of addition to pronounced upregulation of GFAP expression with hypertro-
domains phy of cytoskeleton and stem processes. As a result there is considerable
intermingling of astrocyte processes and disruption of astrocyte domains,
Proliferated causing long lasting reorganization of tissue architecture. There are also
astrocytes substantial changes in molecular expression. Such changes are found in
areas surrounding severe focal lesions, infections or areas responding to
chronic neurodegenerative triggers. D. Severe reactive astrogliosis with
D Severe reactive astrogliosis with compact scar compact glial scar formation occurs along borders to areas of overt tissue
damage and inflammation after CNS trauma, stroke or infection. It is
Inflammatory characterized by newly proliferated astrocytes (with red nuclei in figure),
cell infiltrate particularly along the immediate borders to lesions, where scar forming
in tissue lesions astrocytes interact with other cell types such as fibroblasts and fibrocytes
to form a multicellular glial scar that also includes deposition of dense
collagenous extracellular matrix. In the immediate scar border, astrocytes
Fibroblasts
have densely overlapping processes. These astroglial scars function as
fibrocytes important barriers to inflammatory cells, infectious agents and non-CNS
etc cells in a manner that protects healthy tissue from nearby areas of intense
inflammation. In addition these scars act as barriers to axon regenera-
tion. Glial scar formation results in long lasting reorganization of tissue
Multicellular compact scar with barrier function architecture that appears to be long lasting or permanent. E. Perivascular
astrogliosis with scar formation is characterized by dense accumula-
E Perivascular astrogliosis with scar formation tions of the endfeet and processes of reactive astrocytes that align and
Perivascular scar intermingle around perivascular cuffs or clusters of inflammatory cell
with barrier function infiltrates. There are newly proliferated astrocytes (with red nuclei in
figure) as well as pronounced upregulation of GFAP expression with
Blood vessel hypertrophy of cytoskeleton and stem processes. These perivascular scars
form during certain CNS infections, in the vicinity of CNS traumatic
Perivascular cuff lesions and in autoimmune disorders. In a manner similar to astrocyte
of inflammatory scars that form around lesions after CNS trauma or stroke in (D), these
cell infiltrate perivascular astrocyte scars form important barriers that protect healthy
tissue by restricting the invasion of inflammatory cells from perivascular
clusters into CNS parenchyma.

A S T R O C Y T E R E S P O N S E S TO C E N T R A L N E RVO U S SYS T E M I N J U RY A N D D I S E A S E • 655

Table 51.3 EXAMPLES OF ASTROCYTE GENES POTENTIALLY INDUCED DURING REACTIVE ASTROGLIOSIS
FUNCTIONAL CATEGORIES EXAMPLES OF GENES

Cytokines IL6, IL1β, TNFα, INFγ, CNTF, LIF, TGF β, IL11, IL15,

Chemokines CXCL1, CXCL9, CXCL10, CCL2, CCL5, CCL7

Extracellular matrix MMP9, SEMA4A, TIMP1, chondroitin sulfate proteoglycans (various)

Growth factors and related BDNF, NGF, GDNF, Thsp1, GAP43, FGF2, PDGFb, BMP1, VEGFA

Astrocyte structural GFAP, VIM, NES

Small molecule producing enzymes NOS2, PTGES, SOD3

Intracellular signaling molecules STAT3, NFκB, SOC3, MAP-kinase, SOX9, Nrf2, Olig2, cAMP, Nurr1, SMAD, mTOR

For more details see references: Sofroniew 2009; Zamanian et al. 2012; Hamby et al. 2012.
Abbreviations as per nomenclature of Human Gene Compendium, GeneCards® www.genecards.org

changes in the astrocyte transcriptome in vivo (Zamanian et al.
2012) and that different molecular stimulators of astrogliosis
mediate significantly different changes in the astrocyte
transcriptome in vitro (Hamby et al. 2012), as discussed in more
detail as folllows.
3.1 M I L D TO MO D E R AT E R E AC T I V E
A S T RO G L I O S I S
Mild to moderate reactive astrogliosis consists of changes (up
or down) in gene expression that occur together with variable
degrees of hypertrophy of cell body and stem processes, with-
out loss of individual astrocyte domains and without astrocyte
proliferation (see Fig. 51.1B) (Sofroniew 2009; Sofroniew and
Vinters 2010). There is increased expression of astrocyte struc-
tural proteins such as the canonical astrocyte marker protein,
GFAP. In healthy tissue, not all astrocytes express detectable
levels of GFAP (see Fig. 51.1A), and so this increased GFAP
expression can lead to increased detection of astrocytes in mild
to moderate reactive astrogliosis (see Fig. 51.1B) that should
not be confused with the cell proliferation (astrocytosis) that
occurs in the more severe forms of reactive astrogliosis describe
below. Mild to moderate reactive astrogliosis is generally asso-
ciated with mild nonpenetrating and noncontusive trauma, or
with diffuse innate immune activation (viral infections, sys-
temic bacterial infections), or with areas that are some distance
to focal CNS lesions. It is noteworthy that in healthy tissue,
the extensive network of finely branched processes of individ-
ual astrocytes occupy contiguous, essentially nonoverlapping
domains (see Fig. 51.1A) (Bushong et al. 2002) (see chapter 4),
and that in mild to moderate reactive astrogliosis, there is
preservation of the individual nonoverlapping domains of
reactive astrocytes in spite of the hypertrophy of the cell body
and stem processes (see Fig. 51.1B) (Wilhelmsson et al. 2006).
Experimental evidence suggests if the triggering mechanism
is able to resolve, then mild or moderate reactive astrogliosis
exhibits the potential for resolution in which the astrocytes
return to a morphological appearance similar to that in healthy
tissue, suggesting that there is little or no long-lasting reor-
ganization of tissue architecture (Sofroniew 2009; Sofroniew
and Vinters 2010). The extent to which various molecular and
potential functional changes might resolve or persist is not well
known and this question warrants further investigation.

Figure 51.2 Continuous Gradient of Reactive Astrogliosis from Mild to
Moderate to Scar Formation Adjacent to Tissue Lesions. Areas of severe
tissue damage after stroke, trauma, infection, or autoimmune inflamma-
tory lesions become surrounded by compact glia scar. Spreading away
from the scar is a gradient of reactive astrogliosis that diminishes in
severity with increasing distance from the lesion until there is a seamless
transition to healthy tissue.
3.2 S EVE R E D I F F US E R E AC T I VE
A S T RO G L I O S I S
Severe diffuse reactive astrogliosis consists of changes (up or
down) in gene expression with pronounced up regulation of
GFAP expression, together with hypertrophy of cell body and
stem processes, as well as intermittent astrocyte proliferation
and loss of individual astrocyte domains such that there is sub-
stantive intermingling and overlapping of neighboring astro-
cyte processes (see Fig. 51.1C) (Oberheim et al. 2008; Sofroniew
2009; Sofroniew and Vinters 2010). These changes can result
in long lasting or permanent reorganization of tissue architec-
ture that can extend diffusely over substantive areas. This type
of response is generally found in areas that extend for some
distance away from severe focal lesions, or in areas responding
to chronic neurodegenerative triggers, or in response to certain

656 • NEUROGLIA
types of infection or seizures (Oberheim et al. 2008; Sofroniew
and Vinters 2010). Because there can be considerable tissue
reorganization, the potential for resolution and return to nor-
mal structure is reduced (see Fig. 51.1C) (Sofroniew 2009).
3.3 S EVE R E R E AC T I V E A S T RO G L I O S I S WI T H
C O M PAC T G L I A L S C A R F O R M AT I O N
Severe reactive astrogliosis with compact glial scar forma-
tion consists of changes (up or down) in gene expression
with pronounced upregulation of GFAP expression, together
with hypertrophy of cell body and stem processes, as well as
pronounced astrocyte proliferation and the pronounced
overlapping of astrocyte processes that interdigitate to form
compact borders that surround and demarcate areas of severe
tissue damage, necrosis, infection or autoimmune-triggered
inflammatory infiltration (see Fig. 51.1D) (Bush et al. 1999;
Drogemuller et al. 2008; Faulkner et al. 2004; Herrmann et al.
2008; Oberheim et al. 2008; Voskuhl et al. 2009). The borders
formed by compact glial scars include other cell types, in partic-
ular meningeal and perivascular derived fibroblasts, fibrocytes,
and other glial cells (Aldrich and Kielian 2011; Bundesen et al.
2003; Herrmann et al. 2008; Reilkoff et al. 2011; Sofroniew
2009; Sofroniew and Vinters 2010). These multicellular glial
scars also deposit collagenous extracellular matrix that con-
tains many molecular cues that inhibit axonal and cellular
migration (Silver and Miller 2004). It is important to note that
although other cells are present in and around the multicellu-
lar glial scar, proliferating scar-forming astrocytes exert essen-
tial scar organizing functions, such that scar formation does
not occur when astrocytes are dysfunctional (Bush et al. 1999;
Faulkner et al. 2004; Herrmann et al. 2008). Triggering insults
include penetrating trauma, severe contusive trauma, invasive
infections or abscess formation, neoplasm, chronic neurode-
generation, systemically triggered inflammatory challenges. It
is noteworthy that glial scar formation is associated with sub-
stantive tissue reorganization and structural changes that are
essentially permanent and persist even when triggering insults
may have resolved. An interesting and understudied variant
of astroglia scar formation is found surrounding perivascu-
lar cuffs of leukocytes in CNS autoimmune disease and viral
infections, in which proliferated reactive astrocytes tightly
surround infiltrating leukocytes that occupy the perivascular
space (see Fig. 51.1E) (Sofroniew and Vinters 2010; Voskuhl
et al. 2009). Transgenic loss of function studies show that these
reactive astrocytes exert barrier functions that limit the spread
of leukocytes and restrict them to the perivascular space, such
that disruption of this barrier results in increased spread of
leukocytes and tissue damage in autoimmune conditions such
as experimental autoimmune encephalitis (EAE) (Voskuhl et
al. 2009). Thus, there appear to be interesting similarities in
both structure and function between the glial scars that form
around focal traumatic injuries and perivascular autoimmune
lesions. In both cases, disruption of the astrocyte scar leads to
widespread invasion of inflammatory cells into CNS paren-
chyma, indicative of essential barrier functions.
Astrocyte contributions to glial scar formation can be
regarded as the most extreme form of reactive astrogliosis. The
A S T R O C Y T E R E S P O N S E S TO C E N T R A L N E RVO U S SYS T E M I N J U RY A N D D I S E A S E
striking differences in astrocyte changes along the continuum
of reactive astrogliosis, ranging from small modulations in
gene expression to compact scar formation, in response to
insults of different kinds, are likely to be of consequence when
considering the functions and impact of reactive astrogliosis
on CNS functions, as discussed further.
4 S I G N A L I N G C A S C A D E S T H AT
I N I T I AT E A N D R E GU L AT E R E AC T I VE
ASTROGLIOSIS
In agreement with the observations just described, which indi-
cate that reactive astrogliosis is not a single all-or-none phe-
nomenon, available evidence also indicates that there is not a
single genetic program that is simply turned on by different
stimuli and elicits all aspects of a stereotypic “reactive astroglio-
sis” response. Instead, current evidence indicates that different
aspects of reactive astrogliosis can be regulated separately and
individually as called for in different situations by a wide vari-
ety of different molecular mediators and signaling pathways
(see Table 51.2) (Sofroniew 2009). Different molecular media-
tors that can influence astrocytes can be released in response
to essentially all forms of CNS insults, ranging from subtle cel-
lular perturbations to intense tissue injury and cell death. Many
cell types can release molecular mediators of reactive astro-
gliosis including cells intrinsic to CNS tissue such as neurons,
microglia, oligodendrocyte lineage cells, endothelia, pericytes,
fibromeningeal cells and other astrocytes, as well as nonneu-
ral cells that gain entry into the CNS, such as bone marrow
derived leukocytes, fibrocytes, and microbial infectious agents,
and in some cases cells that remain outside the CNS but release
cytokines or toxins that can affect astrocytes. There is a broad
range of types of molecules that can trigger reactive astrogliosis
including small molecules such as purines and transmitters, to
large polypeptide growth factors and cytokines (see Table 51.2)
as reviewed recently (Sofroniew 2009; Sofroniew and Vinters
2010). Some of these molecular mediators are released via spe-
cific signaling mechanisms, while others are released by simply
cell damage or cell death. Most of the extracellular molecu-
lar triggers of reactive astrogliosis have well defined recep-
tor targets that have the potential to initiate a wide variety of
different intracellular signaling cascades that involve many dif-
ferent second messenger systems as reviewed (Sofroniew 2009;
Sofroniew and Vinters 2010). It is beyond the scope of this
chapter to recapitulate the voluminous amount of information
available, but it is useful to consider certain basic examples that
point toward broad principles that appear to be emerging.
Although we are in the early stages of dissecting the precise
nature of molecular signaling routes between specific forms of
CNS damage or disease and specific aspects of reactive astroglio-
sis, some evidence in this regard is available. A number of studies
from different laboratories have used transgenic mice with astro-
cyte specific gene deletions to examine roles of different compo-
nents of signaling pathways involving STAT3, SOC3, or NFκB
in regulating reactive astrogliosis and its functions in differ-
ent models of CNS disorders. Briefly, deletion of STAT3 or its
associated membrane receptor, GP130, markedly attenuates
• 657
certain aspects of reactive astrogliosis such as cell hypertrophy,
upregulation of GFAP, and scar formation, and was associated
with increased spread of inflammation and infection, increased
lesion size and demyelination, and impaired functional recov-
ery after spinal cord injury, autoimmune disease, or infection
(Drogemuller et al. 2008; Haroon et al. 2011; Herrmann et al.
2008; Okada et al. 2006). Deletion of SOC3 or NFκB also
attenuated certain aspects of reactive astrogliosis such as cell
hypertrophy and up regulation of GFAP, but was associated
with reduced inflammation and lesion size after spinal cord
injury or autoimmune disease (Brambilla et al. 2005, 2009;
Okada et al. 2006). Thus, different signaling pathways that can
trigger visible reactive astrogliosis in the form of cellular hyper-
trophy and increased GFAP expression are involved in mediat-
ing either pro- or antiinflammatory effects that are associated
with that reactive astrogliosis.
Findings such as those just described, combined with other
available evidence, support a model in which there are many dif-
ferent specific signaling mechanisms that trigger different specific
molecular, morphological, and functional changes in reactive
astrocytes. According to this model, not only will the responses
of reactive astrocytes vary in a context-dependent manner as
determined by different signals, but also the potential effects of
reactive astrogliosis on astrocyte functions will vary with different
types of stimuli. In agreement with this model are recent findings
that different types of CNS insults, such as stroke or exposure to
innate inflammatory mediators, have markedly different effects
on the genomic profiles of reactive astrocytes in vivo (Zamanian
et al. 2012), and that stimulation of astrocytes in vitro with dif-
ferent inflammatory mediators also leads to broadly different
changes in astrocyte genomic profiles (Hamby et al. 2012). This
model also predicts that the impact of reactive astrogliosis on
its host neural tissue and associated neural functions will not be
uniform or stereotypic, but will vary in a context-specific manner
as determined by specific signaling mechanisms.
Specific molecular triggers of astrocyte proliferation have also
been identified. As discussed above, not all reactive astroglio-
sis induces cell proliferation, but in its most severe forms, reac-
tive astrogliosis can lead to the appearance of newly proliferated
astrocytes, particularly in scars that form borders around or along
inflammatory cell infiltrates or tissue necrosis (see Fig. 51.1D,E).
Such astrocyte proliferation generally occurs in response to severe
tissue damage or inflammation after severe trauma, stroke, infec-
tion, autoimmune responses, or degenerative disease (Drogemuller
et al. 2008; Faulkner et al. 2004; Voskuhl et al. 2009; White et
al.). The molecular triggers that lead to proliferation of reactive
astrocytes in vivo are incompletely characterized but include epi-
dermal growth factor (EGF), fibroblast growth factor (FGF),
endothelin 1, and ATP (Gadea et al. 2008; Levison et al. 2000;
Neary and Zimmermann 2009). The source of newly divided
scar-forming astrocytes is not well established, and there are sev-
eral potential possibilities. Perhaps the strongest evidence thus far
is that mature astrocytes can reenter the cell cycle and proliferate
during scar formation (Buffo et al. 2008; Bush et al. 1999; Gadea
et al. 2008). Evidence has also been presented that some prolifer-
ating reactive astrocytes may derive from NG2 progenitor cells in
the local parenchyma (Magnus et al. 2008) or from ependymal
cell progenitors after injury or stroke (Carlen et al. 2009; Meletis
658 • NEUROGLIA
et al. 2008). Another potential source might be the multipotent
progenitors in subependymal tissue that express GFAP (Garcia et
al. 2004) and generate progeny cells that migrate toward sites of
injury after trauma or stroke (Ohab and Carmichael 2008), but
thus far no evidence has been presented that these particular pro-
genitors give rise to newly generated scar-forming reactive astro-
cytes. Additional studies will be required to establish the relative
contributions of these potential sources.
5 E F F E C TO R M O L E C U L E S
U P R E GU L AT E D A N D R E L E A S E D BY
R E AC T I VE A S T R O C Y T E S
Whole genome expression profiling is enabling the identifica-
tion of molecular changes associated with reactive astrogliosis
in vivo (Zamanian et al. 2012) or in response to stimulation
with specific molecular mediators of reactive astrogliosis in vitro
(Hamby et al. 2012). Such studies are identifying a wide variety of
potential effector molecules that may be produced and released
by reactive astrocytes to influence other cell types, including
many different cytokines, chemokines, growth factors, extra-
cellular matrix molecules, and small signaling molecules, some
examples of which are given in Figure 51.3 and Table 51.3. It is
also noteworthy that in vivo, different causes of reactive astro-
gliosis such as stroke and peripheral inoculation with LPS led
Microglia
Synapse LIF
NO LCN2 FGF2
TNFα IL6
IL1β
THBS1
Reactive Astrocyte
IL11
BDNF CCL7
CXCL10
GDNF
CCL2
NGF CXCL1
IGF1 VEGF-A
SEMA4A
GSNO wbc
NO PGE
MMPs
Neuron CSPGs bv
Figure 51.3 Examples of Effector Molecules Potentially Produced and
Released by Reactive Astrocytes in Response to CNS Insults. Reactive
astrocytes can be stimulated by different mediators to upregulate the
production of a wide variety of potential effector molecules that can
influence different local cells in a paracrine fashion. Only a cross-section of
molecular examples can be shown. The effector molecules include factors
that can regulate blood flow or blood-brain barrier (NO, prostaglandin
E (PGE), VEGF-A); chemokines, cytokines, and growth factors that
can regulate white blood cells or microglia (CXCL1, CXCL10, CCL2,
CCL7, interleukin (IL)-1β, IL6, IL11, LIF, FGF2, tumor necrosis factor
alpha (TNFα); growth factors and small molecules that can influence
neurons and synapses, neurotrophic factor, glial cell-derived brain-derived
neurotrophic factor (BDNF), nerve growth factor (NGF), insulin growth
factor (IGF)1, TNFα, thrombospondin (Thsp) 1, NO); as well as molecules
that contribute to extracellular matrix (chondroitin sulfate proteoglycans
(CSPG), matrix metallopeptidase 9 (MMP9), Semaphorin-4A (Sema4a)).
to significantly and markedly different changes in the astrocyte
transcriptome (Zamanian et al. 2012). In vitro, different media-
tors of reactive astrogliosis such as transforming growth factor
beta (TGF-β) or LPS plus interferon gamma (INF-γ) caused
substantially and significantly different changes in the astrocyte
transcriptome, and combinatorial interactions among these dif-
ferent mediators led to synergistic changes in gene expression
that could not be predicted simply by summing the effects of
the individual mediators (Hamby et al. 2012). These findings
clearly demonstrate the heterogeneity and specificity of reactive
astrogliosis under different conditions and as mediated by dif-
ferent molecular stimuli.
6 E F F E C T S O F R E AC T I VE A S T R O G L I O S I S
O N A S T R O C Y T E P H YS I O L O GY A N D O N
F U N C T I O N S E X E RT E D BY A S T R O C Y T E S
I N T H E H E A LT H Y C E N T R A L N E RVO U S
SYSTEM
In comparison with the voluminous information accumulating
about morphological and genetic changes associated with reactive
astrogliosis, far less is known about the impact of reactive astro-
gliosis on astrocyte physiology or on functions normally exerted
by astrocytes in the healthy CNS. As described elsewhere in this
volume, astrocytes exert numerous essential functions in healthy
tissue including maintenance of extracellular ion homeostasis,
clearance of transmitters, provision of energy metabolites, regula-
tion of blood flow, and interactions with synapses, and astrocytes
exhibit ligand-evoked intracellular calcium ([Ca2+]i) increases that
are under intense investigation as potential means of mediating
dynamic astrocyte functions, including interactions with synapses
and regulation of blood flow. The effects of reactive astrogliosis
on these activities are not well defined and in some cases are only
beginning to be studied. One recent study reports that virally
infected reactive astrocytes downregulated glutamine synthetase
and that local neurons exhibited reduced inhibitory synaptic cur-
rents (Ortinski et al. 2010). Other recent findings indicate that
exposure to inflammatory mediators alters astrocyte expression of
a large number of G-protein coupled receptors and alters astro-
cyte changes in [Ca2+]i evoked by specific ligands of these recep-
tors (Hamby et al. 2012). Together such findings point toward the
possibility of important effects of reactive astrogliosis on astrocyte
physiology in ways that may impact on neural function, and sug-
gest that this area warrants more extensive investigation.
7 F U N C T I O N S O F R E AC T I VE
ASTROCY TES
Glial scar formation is well known as an impediment to axon
regeneration migration (see chapter 56). Perhaps for this reason,
reactive astrogliosis per se has sometimes been regarded as a purely
maladaptive obstacle to functional recovery after CNS damage
and it has even been proposed that wholesale inhibition of reac-
tive astrogliosis might be therapeutically beneficial. It is important
to emphasize that this is not the case. There are now many differ-
ent kinds of experimental evidence that reactive astrocytes exert
A S T R O C Y T E R E S P O N S E S TO C E N T R A L N E RVO U S SYS T E M I N J U RY A N D D I S E A S E
various kinds of essential beneficial effects after CNS insults. It is
also becoming clear that reactive astrocytes are not only hetero-
geneous in their morphology as discussed above (see Fig. 51.1),
but also in the functions and effects that they exert in response
to different molecular triggers released by different kinds of CNS
insults.
7.1 R E AC T I VE A S T RO G L I O S I S ,
N EU RO P ROT EC T I O N A N D EFFEC TS O N
N EU RO NA L F U N C T I O N
Various different types of transgenic loss of function models
from multiple laboratories show that either ablation or attenua-
tion of reactive astrogliosis causes increased lesion size, increased
neuronal loss, demyelination, and exacerbated loss of function
after traumatic injury, stroke, autoimmune attack or infec-
tion (Bush et al. 1999; Drogemuller et al. 2008; Faulkner et al.
2004; Herrmann et al. 2008; Li et al. 2008; Myer et al. 2006;
Okada et al. 2006; Voskuhl et al. 2009). A large number of
studies provide different kinds of evidence in vivo and in vitro
that reactive astrocytes can protect CNS cells and tissue in
various ways, including by (1) uptake of potentially excitotoxic
glutamate (Bush et al. 1999; Rothstein et al. 1996; Swanson
et al. 2004), (2) protection from oxidative stress via glutathione
production (Chen et al. 2001; Shih et al. 2003; Swanson et al.
2004; Vargas et al. 2008), (3) protection via adenosine release
(Lin et al. 2008), (4) protection from NH4+ toxicity (Rao
et al. 2005), (5) protection by degradation of amyloid-beta pep-
tides (Koistinaho et al. 2004), and (6) stabilizing extracellular
fluid and ion balance and reducing seizure threshold (Zador
et al. 2009). In addition, recent findings indicate that reactive
astrogliosis can influence astrocyte functions in such a manner
as to have effects on synaptic transmission. For example, down-
regulation of gamma-aminobutyric acid (GABA) transporter
function by reactive astrocytes in periinfarct cortex was asso-
ciated with reduced cortical neuronal synaptic plasticity that
could be reversed by inhibition of extrasynaptic GABAA recep-
tor mediated currents (Clarkson et al. 2010). It can be speculated
that increasing GABA tone may initially be neuroprotective, but
later reduces capacity for plasticity and recovery. Together, these
findings indicate that reactive astrocytes can exert a range of
functions that impact on neuroprotection, repair, and recovery
in response to CNS insults of various kinds, including trauma,
infection, stroke, and degenerative disease (Sofroniew 2005,
2009; Sofroniew and Vinters 2010).
7.2 R E AC T I VE A S T RO C Y T E RO L E S I N B L O O D
B R A I N BA R R I E R B R E A K D OWN A N D R E PA I R ,
A N D R EGU L AT I O N O F E D E M A
Many CNS insults result in leakiness of the blood-brain bar-
rier, including traumatic injury, stroke, inflammation and
certain forms of neurodegeneration, and in many cases, the
blood-brain barrier (BBB) will repair spontaneously if the
damaging insults resolve, such as in acute traumatic injury or
stroke. Reactive astrocytes appear able to play critical roles
in both breakdown and repair of BBB. Recent findings indi-
cate that vascular endothelial growth factor-A (VEGF-A)
• 659
derived from astrocytes mediates BBB breakdown and lympho-
cyte infiltration during autoimmune CNS inflammation (Argaw
et al. 2009, 2012). On the other hand, experimental loss of func-
tion studies demonstrate that astrocytes are essential for sponta-
neous posttraumatic repair of the blood-brain barrier (Bush et
al. 1999; Faulkner et al. 2004) even though astrocytes are not
essential for normal development of the blood- brain barrier
(Daneman et al. 2010; Saunders et al. 2008) (see chapter 33). A
candidate molecule for astrocyte contribution to BBB repair is
S-nitrosoglutathione (GSNO), which is produced by astrocyte-
like enteric glia and stimulates barrier properties in gut epithelia
(Savidge et al. 2007). Astrocytes also play essential roles in reduc-
ing vasogenic edema after trauma, stroke or obstructive hydro-
cephalus (Bush et al. 1999; Zador et al. 2009).
7.3 R E AC T I V E A S T RO G L I O S I S , S C A R
F O R M AT I O N, A N D R E GU L AT I O N O F
I N FL A M M AT I O N
One of the essential functions exerted by reactive astrocytes
appears to be that of formation of scars that create migration
barriers around collections of highly active inflammatory cell
infiltrates that collect in areas of focal CNS lesions after trau-
matic injury, stroke, or suppurative infection, or in perivas-
cular clusters or cuffs during viral infections or autoimmune
disease (see Fig. 51.1D,E) (Sofroniew and Vinters 2010). As
described in this section, loss of function studies clearly show
increased spread of inflammation and increased tissue damage
when astrocyte scar formation is experimentally attenuated.
It has been recognized for some time that astrocytes in vitro
have the capacity to make many pro- or antiinflammatory mole-
cules in response to different kinds of stimulation (Eddleston and
Mucke 1993; John et al. 2003; Sofroniew 2009), and can exert
both pro- and antiinflammatory effects on microglia (Farina et al.
2007; Min et al. 2006). Recent studies from multiple laborato-
ries using transgenic loss of function models have extended such
observations and shown that reactive astrocytes in vivo can exert
both pro- and antiinflammatory regulatory functions. For exam-
ple, ablation or attenuation of reactive scar-forming astrocytes can
exacerbate the spread of inflammatory cells during (1) locally ini-
tiated innate inflammatory responses to traumatic injury (Bush
et al. 1999; Faulkner et al. 2004; Herrmann et al. 2008; Li
et al. 2008; Okada et al. 2006), or (2) during peripherally ini-
tiated adaptive immune responses such as EAE (Liedtke et al.
1998; Voskuhl et al. 2009). In this regard it is also noteworthy
that attenuation of reactive astrocytes also leads to increased
spread of infection (Drogemuller et al. 2008). Nevertheless,
reactive astrocytes also have proinflammatory potential, and
other studies provide evidence that deletion or knockdown of
certain molecules in astrocytes is associated with a reduction
in inflammation after traumatic injury or experimental autoim-
mune disease (Brambilla et al. 2005, 2009; Okada et al. 2006).
Together these findings indicate that astrocyte involvement in
the regulation of CNS inflammation is complex and likely to
be context dependent and regulated by multimodal extracellu-
lar signaling events that may differ at different time points after
insults.
660 • NEUROGLIA
Although it may initially seem counterintuitive that astro-
cytes could exert both pro- and antiinflammatory effects, we
have recently proposed a functional model that reconciles this
seeming paradox (Sofroniew 2009; Sofroniew and Vinters
2010). In this model, reactive scar-forming astrocytes function
not only to attract and instruct inflammatory cells, in particular
at early times after insults, but also over time these astrocytes
form cell migration barriers that surround and demarcate areas
where intense inflammation is needed and that restrict the
spread of inflammatory cells and infectious agents in order to
preserve nearby healthy tissue (Sofroniew 2005; Sofroniew
2009; Sofroniew and Vinters 2010) (see Fig 51.1D,E). This
model is congruent with the notion that evolutionary pressures
shaping CNS injury responses are likely to have favored mecha-
nisms to keep small injuries small and uninfected, and that the
inhibition of the migration of inflammatory cells and infectious
agents and other cells might have led to the accidental byprod-
uct of inhibiting axon regeneration owing to the redundancy
between migration cues across cell types (Bush et al. 1999;
Sofroniew 2005). In this context it is also interesting to consider
the variant of astroglia scar found surrounding perivascular col-
lections of leukocytes in CNS autoimmune disease and viral
infections as discussed previously (see Fig. 51.1E) (Sofroniew
and Vinters 2010; Voskuhl et al. 2009). Recent findings show
that the formation of such perivascular collections of leukocytes
in the form of ectopic lymphoid follicles can be induced by the
Th17 subtype of T cells (Peters et al. 2011). It is interesting to
consider that interactions between T cells and astrocytes may
be involved in the formation of such ectopic lymphoid follicles.
These follicles are surrounded by reactive scar-forming astro-
cytes (see Fig. 51.1E) in both experimental animals and human
pathology (Sofroniew and Vinters 2010; Voskuhl et al. 2009)
and fail to form when scar-forming astrocytes are experimentally
ablated in transgenic mice, resulting in the widespread spread
of T cells in the neural parenchyma (Voskuhl et al. 2009). The
nature and consequences of astrocyte–leukocyte interactions in
such follicles requires further study, but it is noteworthy that
astrocytes produce high levels of diverse chemokines and cytok-
ines in vivo and in vitro in response to innate inflammatory stim-
uli associated with injury and microbial infection (Hamby et al.
2012; John et al. 2003; Zamanian et al. 2012). It is interesting to
speculate that such ectopic lymphoid follicles within the CNS
may represent sites where astrocytes help to recruit and con-
strain immune cells in sites (see Fig. 51.1E) where they may be
instructed within the blood brain barrier by factors produced by
astrocytes and other cells, in such a manner that they may play
important roles in both adaptive situations such as viral infec-
tion and in maladaptive situations such as autoimmune disease.
8 R E AC T I VE A S T R O C Y T E S A N D
N O N - C E L L -AU TO N O M O U S
N E U R O D E G E N E R AT I O N
The work summarized thus far in this chapter provides com-
pelling evidence that reactive astrogliosis is a ubiquitous,
complex, essential and beneficial part of the repair response
to all CNS insults. Nevertheless, there is also evidence
that dysfunctions of astrocytes or reactive astrocytes can
contribute to, or to be a primary source of, CNS disease
mechanisms (Sofroniew and Vinters 2010). In the healthy
CNS, astrocytes exert many different essential functions,
as presented in other chapters of this volume. It is therefore
not surprising that there is a rapidly growing body of both
experimental and clinical evidence showing that genetic
defects or molecular abnormalities within astrocytes or
reactive astrocytes can precipitate non-cell-autonomous
neuronal dysfunction or degeneration. These effects can
occur as a result of either gain of abnormal effects, or loss
of normal functions, by astrocytes or reactive astrocytes.
8.1 G A I N O F D ET R I M EN TA L EFFEC TS BY
A S T RO C Y T E S O R R E AC T I V E A S T RO C Y T E S
There is evidence from both clinical and experimental stud-
ies that under specific circumstances astrocytes or reactive
astrocytes have the potential to exert detrimental effects. In
some cases, there is a clear association with a genetic defect
or molecular dysfunction. Clinical studies have identified at
least two single gene mutations that cause gain of detrimental
effects by astrocytes and/or reactive astrocytes. In Alexander
disease, a dominant, gain-of-function mutation of the gene
encoding GFAP is associated with changes in astrocytes that
resemble reactive astrogliosis, leukoencephalopathy, macroen-
cephalopathy, seizures, psychomotor disturbances, and prema-
ture death (Brenner et al. 2001) (see chapter 69). In a familial
form of amyotrophic lateral sclerosis (ALS) or motor neuron
disease, a dominant gain-of-function mutation of the gene
encoding superoxide dismutase (SOD) leads to production by
reactive astrocytes of molecules that are toxic to motor neurons
(Di Giorgio et al. 2007; Nagai et al. 2007) (see chapter 63). In
addition, gain of function transgenic mouse models indicate
that selective targeting to astrocytes of a mutant form of the
SOD associated with ALS leads to neuronal degeneration
(Lobsiger and Cleveland 2007; Nagai et al. 2007; Yamanaka
et al. 2008). It is important to emphasize that in these examples
of Alexander disease and familial ALS, the neuronal toxicity
and damage to neural tissue is caused by genetically mutated
and abnormal astrocytes rather than to the generic process of
reactive astrogliosis per se. In addition, there is also some evi-
dence that the process of reactive astrogliosis might in some situ-
ations exert detrimental effects, perhaps in an analogous fashion
to the manner by which the process of inflammation can in
some situations exert detrimental effects. In this regard, there is
evidence from transgenic animals and other experimental mod-
els that reactive astrocytes may be stimulated by specific signal-
ing cascades to gain detrimental effects such as (1) exacerbation
of inflammation via cytokine production (Brambilla et al. 2005,
2009), (2) production and release neurotoxic levels of reactive
oxygen species (Hamby et al. 2006; Swanson et al. 2004), (3)
release of potentially excitotoxic glutamate (Orellana et al.
2009; Takano et al. 2005), (4) contribution to seizure genesis
(Jansen et al. 2005; Tian et al. 2005), (5) compromise of blood-
brain barrier function owing to VEGF-production (Argaw et al.
2009), (6) cytotoxic edema during trauma and stroke through
aquaporin 4 (AQP4) overactivity (Zador et al. 2009), and
A S T R O C Y T E R E S P O N S E S TO C E N T R A L N E RVO U S SYS T E M I N J U RY A N D D I S E A S E
(7) contribution to chronic pain (Milligan and Watkins
2009). In these cases in which there is no obvious genetic
mutation, the gain of detrimental effects by reactive astrocytes
is incompletely understood but may result from specific sig-
naling cascades that result from complex interactions with
other cell types such as microglia and other inflammatory cells
(Farina et al. 2007), or for example by simultaneous exposure
to microbial antigens such as lipopolysaccharide, which can
markedly increase the levels of potentially toxic nitric oxide
produced by astrocytes in response to cytokines (Hamby
et al. 2006). Context-dependent acute or chronic combinato-
rial signaling events involving different cell types might thus
stimulate reactive astrocytes to produce potentially cytotoxic
levels of molecules or might lead to chronic inflammation or
neuropathic pain. A greater understanding of such combina-
torial signaling could facilitate targeted therapeutic strategies
that preserve the beneficial and attenuate the potentially detri-
mental aspects of reactive astrogliosis (Sofroniew 2009).
8.2 L O S S O F E S S E N T I A L F U N C T I O NS BY
A S T RO C Y T E S O R R E AC T I VE A S T RO C Y T E S
There are many potential ways in which loss of essential
functions by astrocytes or reactive astrocytes might lead to
non-cell-autonomous neuronal dysfunction or degeneration.
As summarized in many chapters in this volume, in healthy
neural tissue, astrocytes play critical roles in many functions
essential for neuronal health and function including homeo-
stasis of extracellular fluid, ions and transmitters, regulation of
blood flow, energy provision and interactions with synapses.
In addition, as summarized in this chapter, reactive astrocytes
play critical roles in additional functions including neuropro-
tection during various CNS insults, repair of the blood-brain
barrier, and regulation of inflammation. Although we are only
beginning to appreciate the specific mechanisms whereby
astrocyte dysfunction might impact on neural function, spe-
cific examples are becoming available from both experimental
studies and clinical observations.
From an experimental perspective, there is now clear
evidence that various single gene mutations that are tar-
geted exclusively to astrocytes in transgenic mice can lead
to loss of astrocyte functions that spontaneously precipitate
non-cell-autonomous neuronal dysfunction and degeneration
in the absence of additional CNS insults. For example, selec-
tive deletion from astrocytes of the endoribonuclease, Dicer,
leads to cell-non-autonomous neuronal degeneration of both
cerebellar granule neurons and Purkinje neurons (Tao et al.
2011). Dicer-deficient mice exhibit normal motor develop-
ment and neurological morphology until mid-adolescence and
then invariably develop a fulminant neurological decline char-
acterized by ataxia, severe progressive cerebellar degeneration,
seizures, uncontrollable movements, and premature death.
Concomitant with onset of symptoms, essential astrocytic
functions including glutamate uptake and antioxidant path-
ways are substantially impaired, leading to massive apoptosis
of cerebellar granule cells and degeneration of Purkinje cells
(Tao et al. 2011). Another example is that of the Wnt-signaling
pathway gene, adenomatous poliposis coli (APC), wherein
• 661
deletion of APC selectively from astroglia leads to the grad-
ual but relentlessly progressive cell-non-autonomous neu-
ronal degeneration of cerebellar Purkinje neurons (Wang et al.
2011). APC-deficient mice develop morphologically normal
Bergmann astroglia during the juvenile period, but then their
radial fibers become shortened, their cell bodies translocate to
the molecular layer, and they lose their pial contact and trans-
form into stellate-shaped cells reminiscent of reactive astro-
cytes. Purkinje neurons are normal in appearance throughout
the juvenile period and then begin gradually to degenerate
with a gradual diminution of the dendritic tree and eventual
cell death such that there is significant loss of Purkinje neurons
and cerebellar atrophy by middle age. Granule neurons are not
visibly affected. It is also well established that genetic deletion
of the astrocyte glutamate uptake transporter, Glt1 (EAAT2)
will lead to seizures and excitotoxic neuronal death (Rothstein
et al. 1996) (see chapter 63). As regards reactive astrocytes,
there is evidence that single gene mutations targeted exclu-
sively to astrocytes can lead to loss of essential functions by
reactive astrocytes that result in increased neuronal dysfunc-
tion and degeneration in the context of experimental models
of CNS injury or disease. For example, deletion of the signaling
molecule STAT3 or the cytokine receptor GP130 can impair
function and increase tissue damage and neurodegeneration
in experimental models of CNS trauma, infection, and auto-
immune disease (Drogemuller et al. 2008; Haroon et al. 2011;
Herrmann et al. 2008; Myer et al. 2006; Okada et al. 2006). In
addition, as discussed above, the transgenically targeted selec-
tive ablation of proliferating reactive astrocytes will cause the
failure of blood-brain barrier repair, increase inflammation,
and exacerbate loss of function and neurodegeneration in var-
ious models of CNS trauma or autoimmune disease (Bush et
al. 1999; Faulkner et al. 2004; Myer et al. 2006; Voskuhl et al.
2009), indicating that the consequence of loss of astrocytes
because of injury or disease leads to devastating consequences
and the comprehensive degeneration of neural tissue.
Another important and interesting question is the degree
to which the process of reactive astrogliosis might in itself in
some situations lead to a reduction or loss of essential func-
tions exerted by astrocytes in healthy tissue. Some experimen-
tal evidence is already available in this regard. For example,
recent findings indicate that reactive astrogliosis can influence
astrocyte functions that may impact on synaptic transmission.
Downregulation of GABA transporter function by reactive
astrocytes in periinfarct cortex was found to be associated
with reduced cortical neuronal synaptic plasticity and inhibi-
tion of extrasynaptic GABAA currents was found to benefit
recovery (Clarkson et al. 2010). Reactive astrogliosis associ-
ated with viral infection reduced astrocyte glutamine synthe-
sis and altered hippocampal neuronal excitability (Ortinski et
al. 2010). In addition, after traumatic spinal cord injury, the
majority of proliferating astrocytes were found to express low
or no detectable levels of a transgene reporting expression lev-
els of the major astrocyte glutamate transporter, Glt1, which
may influence the ability of the injured tissue to cope with
potentially excitotoxic glutamate load (Lepore et al. 2011). As
discussed above, proliferating astrocytes are characteristic of
scar formation and are intermingled with non-proliferating
662 • NEUROGLIA
astrocytes. Differences in the molecular expression of dif-
ferent forms of reactive astrocytes, and how such changes in
expression impact on essential functions normally exerted by
astrocytes in healthy tissue, may have important ramifications
for outcome after different types of CNS insults. Additional
work is needed to address these interesting issues.
From a clinical perspective, the disorder neuromyelitis
optica (NMO), a relapsing inflammatory demyelinating dis-
ease (see chapter 61), provides evidence that dysfunction or
loss of astrocytes can exacerbate CNS inflammation in a clini-
cally relevant context. Neuromyelitis optica is causally associ-
ated with autoantibodies that bind selectively to AQP4 on
astrocytes (Lennon et al. 2005) and precipitate compliment-
mediated astrocyte lysis that is associated with pronounced
inflammation and tissue destruction (Lucchinetti et al. 2002;
Roemer et al. 2007) and severe symptoms such as vision loss
and paralysis (Wingerchuk et al. 1999). The neuropathologi-
cal changes found in NMO appear in many ways to be simi-
lar to changes found in transgenic mouse models in which
ablation of reactive scar-forming astrocytes results in greatly
exacerbated inflammation and tissue destruction during CNS
autoimmune inflammation (Voskuhl et al. 2009).
9 S U M M A RY A N D P E R S P E C T I VE S
Astrocytes have been known to respond to CNS injury and
disease for well over 100 years. This response has often been
regarded as a stereotypic all-or-none phenomenon that uni-
formly results in glial scar formation. Recent progress dem-
onstrates that this is not the case and that instead, reactive
astrogliosis is a finely gradated continuum of changes that
range from subtle alterations in gene expression and cell
hypertrophy that are reversible, to scar formation with per-
manent tissue rearrangement. It has become clear that there
is not a single program for reactive astrogliosis with a simple
on/off switch, but that the different potential structural and
functional changes associated with reactive astrogliosis that
can occur are regulated separately by many different poten-
tial signaling mechanisms. For example, different signaling
events have been identified that separately regulate expression
of structural proteins, cell hypertrophy or cell division. Other
signaling mechanisms separately regulate expression of pro- or
antiinflammatory molecules. In addition, although it is clear
that reactive scar forming astrocytes are associated with the
inhibition of axon regrowth, a number of beneficial functions
have been identified that are mediated by reactive astrocytes,
including neuroprotection, blood brain barrier repair, and
the restriction of inflammation. Dysfunctions of astrocytes
and reactive astrocytes can also occur and contribute to CNS
disorders, either through gain of abnormal effects or loss of
normal functions. Combinations of mouse models of trans-
genic astrocyte manipulations with experimental models of
CNS injury or disease show that genetic modulations of reac-
tive astrogliosis and scar formation can markedly alter tissue
repair, disease progression and functional outcome. Ablation
of astrocytes and attenuation of certain astrocyte functions
exacerbate disease progression and tissue degeneration and
worsen functional outcome, whereas deletion of certain astro-
cyte genes appears to improve outcome in some situations.
Collectively such findings point toward an enormous, yet
incompletely understood, potential for astrocytes to contrib-
ute to, or play primary roles in, disease processes, tissue repair,
and functional outcome in a wide variety of clinical condi-
tions including stroke, epilepsy, and neurodegenerative disease
(Sofroniew and Vinters 2010). In this context it is noteworthy
that genetic polymorphisms that impact selectively on astro-
cyte functions are likely to become of interest in clinical neu-
roscience. Future studies are needed to identify specific factors
that might alter or perturb the normal process of reactive
astrogliosis through different means such as (1) genetic muta-
tions or polymorphisms, (2) autoimmune events, or (3) simul-
taneous exposure to multiple stimulators that might interact
synergistically.
AC K N OW L E D G M E N T S
Work in the author’s laboratory is supported by grants from
the National Institutes of Health (RO1 NS057624), Roman
Reed Spinal Cord Injury Research Fund, Adelson Medical
Research Foundation, National Multiple Sclerosis Society,
and Wings for Life.
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 52.
METAB OLIC INJURY OF
OLIGODENDROCY TES AND MYELIN
Peter K. Stys

A B B R E VI AT I O N S therefore possibly of limited effectiveness. This chapter will

summarize current knowledge of the molecular pathways,
AMPA α-amino-3-hydroxy-5-methyl-isoxazoIe-4- with an emphasis on Ca2+ dependent mechanisms, that may
propionate be involved in injury to the OL, its processes, and the vital
ADP adenosine diphosphate myelin sheath that plays such an important role in supporting
ASIC acid-sensing ion channel information transmission in the mammalian nervous system.
ATP adenosine triphosphate
CNS central nervous system
EAAT excitatory amino acid transporter 2 O L I G O D E N D R O C Y T E S T RU C T U R E ,
ER endoplasmic reticulum F U N C T I O N, A N D E N E R GY M ETA B O L I S M :
GluT glutamate transporter A B R I E F O VE RVI EW
GlyT glycine transporter
MS multiple sclerosis A detailed review of the development, structure, and function
NMDA N-methyl-d-aspartate of OLs, as well as metabolic pathways of energy production,
NO nitric oxide is presented elsewhere in this volume (see chapters 13, 20,
OL oligodendrocyte and 27). This section serves as a brief overview to put into
PNS peripheral nervous system context the demands placed on these glia, and as a result, their
TNFα tumor necrosis factor α predilection to injury. As OLs mature from precursors in the
developing CNS, their main role is to synthesize myelin, and
ensheath axons, thus tremendously improving conduction
1 INTRODUCTION velocity. This in turn allows axons to retain a much smaller
diameter, and thus greatly increases the packing density in
Oligodendrocytes (OLs) are one of the major glia of the cen- the CNS. This appears to be the main objective of myelina-
tral nervous system (CNS), subserving the important function tion, because the actual net energy saving may not be as great
of synthesizing myelin for ensheathment of axons in the brain as once assumed (Harris & Attwell 2012). Indeed, the syn-
and spinal cord. It is estimated that more than 95% of cal- thetic demands placed on myelinating OLs seem extreme:
losal axons in the human are myelinated (Aboitiz et al. 1992); each OL may synthesize myelin membrane whose total area
therefore, injury and death of oligodendrocytes have the pro- exceeds that of the parent soma by 10,000-fold, generating
pensity to cause major disruption both to the function and as much as 5,000 μm2 of membrane per day during the active
even long-term survival of vital axonal connections within the myelinating phase (Pfeiffer et al. 1993), placing an extreme
CNS. Numerous clinical disorders result in injury and death energy demand on these glia (Harris & Attwell 2012).
of OLs, and ultimately demyelination of axons. These include To fuel its energy needs, the OL possesses all the standard
trauma (brain and spinal cord injury), ischemia (acute stroke metabolic machinery and organelles (i.e., mitochondria) nec-
and more chronic vascular insufficiency leading to dementia), essary for glycolytic and oxidative adenosine triphosphate
infections, toxins, metabolic abnormalities, genetic diseases (ATP) synthesis. Oligodendrocytes express glucose transport-
resulting in leukodystrophy, and degenerative/inflammatory ers (Arluison et al. 2004), indicating that these glia can utilize
disorders whose causes are unknown such as Alzheimer’s glucose directly. However, glucose is not the only substrate that
disease and multiple sclerosis (for a more extensive list, see can support OL energy requirement: lactate can also be taken
Schmahmann et al. 2008). These conditions share, to a greater up by these cells, which they use for energy, lipid metabolism,
or lesser extent, pathology centered on the oligodendrocyte and myelin synthesis (Rinholm et al. 2011). Oligodendrocytes
and the myelin sheath, in addition to involving other elements are electrically polarized, with an estimated resting potential of
of the CNS. Yet we know comparatively little about the cellu- about −65 mV (Karadottir et al. 2005), and as with most elec-
lar and molecular pathophysiology of oligodendrocyte injury trically polarized cells, OLs must expend energy to maintain
and death, and as a result, any attempts at devising effective physiological transmembrane ionic gradients. They express
therapeutic interventions will be perforce incomplete, and all the major cation and anion transporters found in other

665
excitable and electrically polarized cells, including Na+/K+-
ATPase, Ca2+-ATPase, Na+ Ca2+ exchanger, a variety of anion
transport proteins, and intracellular Ca2+ release channels
(Ruiz et al. 2010; Mata & Fink 1989; reviewed in chapter 20).
All of these consume energy directly or indirectly, and many are
directly or indirectly involved in maintaining control over the
most important ion involved in cell signaling and death: cal-
cium. Thus, an imbalance between Ca2+ entry into the cytosol
(from extra- and/or intracellular sources) versus the cell’s abil-
ity to buffer this key cation, most often because of impairment
of energy supply, leads to injury and death. This “final common
pathway” of cell injury (Schanne et al. 1979) applies equally to
OLs, and much of the remainder of this chapter will focus on
mechanisms and conditions that lead to Ca2+ dyshomeostasis
in OLs, and equally importantly, in the myelin sheath, that
responds uniquely to pathological stimuli independently of its
parent cell body.
3 C A L C I U M O VE R L OA D I N J U R E S
OLIGODENDROCY TES AND MYELIN
Perhaps the earliest compelling data on the importance of Ca2+
ions in degeneration of myelin and injury of OLs stemmed
from experiments where Ca2+ ionophores were applied to
myelinated fibers in the PNS and CNS, or to cultured OLs.
The latter suffered injury at ionophore concentrations that did
not affect other cell types (e.g., astrocytes), and myelin under-
went severe vesiculation (Scolding et al. 1992; Smith et al. 1985;
Smith & Hall 1988). Also, OLs suffer injury upon release of
Ca2+ from internal stores (Benjamins & Nedelkoska 1996).
Together these observations underscore the key role played by
an excess accumulation of intracellular Ca2+ levels, regardless
of the source, as a fundamental mediator of demyelination and
OL injury (Deng et al. 2003; Follett et al. 2004; Karadottir
et al. 2005; Matute 2010; Micu et al. 2006; Salter & Fern 2005).
Clearly such experiments are deliberately contrived to directly
raise intracellular Ca2+ levels in order to study the consequences,
and of course such chemical ionophore-induced Ca2+ overload
would not occur in disease states. However, Ca2+ ionophores
mimic both complement and perforin attack, inducing lysis of
OLs (Jones et al. 1991). This suggests that even natural, albeit
pathological, conditions lead to direct OL/myelin Ca2+ excess
and cell damage. In addition to nonspecific ionophore- (artificial
or natural) induced Ca2+ entry, OLs and perhaps surprisingly,
myelin itself, express channels and receptors that permeate Ca2+.
This likely represents a physiological signaling arrangement, but
one that also promotes injury and demyelination if overdriven
under pathological conditions.
In fact, there exist many factors and stimuli capable of induc-
ing Ca2+ fluctuations in OLs, likely playing a role in development
and if excessive, in pathology. T-cell perforins trigger Ca2+ signals
as noted above (Jones et al. 1991), as well as a variety of inflamma-
tory mediators including histamine and bradykinin (Bernstein
et al. 1996; He et al. 1996), arachidonic acid and its by-products
(Soliven et al. 1993), and lysophosphatidic acid (Moller et al.
1999). The mechanisms involved in OL and myelin damage
by immune effectors are complex and beyond the scope of this
666 • NEUROGLIA
chapter; the reader is referred elsewhere for details (Buntinx et al.
2002) (see chapter 50). Voltage- and store-operated Ca2+ chan-
nels have also been detected on OLs, and are likely involved in
development and directional process growth (Butt 2006); their
role in OL injury is less established. Perhaps the best studied are
neurotransmitter-gated channels, many of which are capable of
permeating damaging amounts of Ca2+ ions in a wide variety of
pathological conditions.
The effectors of cell destruction in the face of a pathologi-
cal intracellular Ca2+ rise are likely a collection of Ca2+ activated
enzyme systems normally tightly regulated and essential for
growth, repair and remodeling. The calpains are a family of Ca2+
activated proteases that have as substrates numerous OL and
myelin proteins, ideally positioning these enzymes to wreak major
havoc if excessively activated (Vosler et al. 2008). Several phos-
pholipases, both Ca2+-dependent and independent, will in turn
degrade critical myelin lipids, which make up 70% of the content
of myelin. Peptidylarginine deiminase 2 is another Ca2+-activated
enzyme present in myelin that converts arginine residues in myelin
basic protein to citrulline. The alteration in net charge on this key
structural protein may lead to destabilization of myelin, poten-
tially rendering it more susceptible to breakdown or immune
attack (Harauz & Musse 2007). Taken together, abnormal Ca2+
accumulation inside the OL soma, its processes, and myelin can
in turn unleash a wide variety of nefarious biochemical pathways
that can severely damage or destroy the myelinating unit.
4 OLIGODENDROCYTES EXPRESS
C A L C I U M-P E R M E A B L E G LU TA M AT E
R E C E P TO R S
Myelin and OLs are inexcitable, although they are electrically
polarized (Karadottir et al. 2005) (this is speculative for myelin,
which is a rather unusual structure and difficult to study
physiologically; for further discussion see Stys 2011). Therefore it
was somewhat surprising to find voltage-gated ion channels and
transmitter-gated receptors on these cells. In fact, OLs and their
progenitors express a surprisingly rich variety of voltage- and
chemically-activated channels, including many Na+, K+ and Ca2+
channels, together with ionotropic and metabotropic glutamate
receptors, purinergic receptors, and GABA-sensitive channels.
The precise roles of these channels are unknown, but recent
data indicate they likely contribute importantly to development,
proliferation, and migration (Domingues et al. 2010; see chapter
20). The common physiological effect of activation of most of
these channels is cell depolarization and cytosolic Ca2+ increase,
both through transmembrane flux and release from internal
Ca2+ stores. Taken together, short of being able to generate
full-fledged action potentials, OLs appear to exhibit most of
the active signaling behavior of excitable neurons. It is therefore
not surprising that like neurons, which are famous for their
sensitivity to overactivation and suffering excitotoxicity in a wide
variety of pathological conditions, OLs also suffer damage from
overactivation of their surface channels and receptors and suffer
“excitotoxicity”; indeed, of all glia, those of the oligodendroglial
lineage are the most susceptible to this type of damage (Matute
et al. 2007a).
 A series of reports from the 1990s unequivocally included
OLs into the family of cells that suffer damage upon excess
exposure to glutamate (Matute et al. 1997; Oka et al. 1993;
Yoshioka et al. 1996). The receptors most responsible are the
ionotropic AMPA and kainate receptors. Notably, OL AMPA
receptors lack the GluA2 (formerly GluR2) subunit, and the
GluK2 (formerly GluR6) kainate subunit is minimally edited
(Matute et al. 2001), conferring substantial Ca2+ permeability
to both. Exposure of OLs to AMPA and kainate receptor ago-
nists results in OL apoptosis, or after more chronic exposure,
OL necrosis, demyelination, and axonal damage (Matute 1998).
Given the documented vulnerability of OLs to Ca2+ overload
and the highly Ca2+-permeable AMPA and kainate receptor
varieties expressed by these cells, it is highly likely that OLs are
directly damaged by receptor overactivation and Ca2+ influx
from outside the cell. Interestingly, AMPA receptor-induced
Ca2+ entry can be further amplified by a Ca2+-induced Ca2+
release phenomenon in OLs (akin to cardiac Ca2+-induced
Ca2+ release), leading to further endoplasmic reticulum (ER)
stress, mitochondrial damage, and cell injury (Ruiz et al.
2010). This phenomenon in OLs is curiously similar to what is
described in white matter axons that are myelinated by these
same cells; here submyelinic axonal AMPA receptors, orga-
nized into discrete signaling “nanocomplexes” (Stirling & Stys
2010), also trigger exaggerated Ca2+ release from the axoplas-
mic reticulum, the axonal equivalent of the ER (Ouardouz et
al. 2009). Depending on the intensity and chronicity of recep-
tor activation, glutamate receptor-mediated OL Ca2+ overload
may result not only in necrotic death, but may cause apopto-
sis. Interestingly, the apoptotic cascades triggered by AMPA
and kainate receptors are distinct, indicating selective signal-
ing pathways. Kainate receptors activate caspases 9 and 3,
whereas AMPA receptors induce caspase 8, truncation of the
Bid protein, followed by activation of caspase 3 and PARP-1
polymerase (Matute et al. 2007a). The presence of AMPA and
kainate receptors on OLs, and their importance in meditating
injury to this critical cell, have been confirmed and extended
by many groups in a variety of models (reviewed in Karadottir
& Attwell 2007). Glutamate receptor overactivation may also
result in indirect injury to OLs given that other glia such as
microglia, also express these receptors. The latter have been
shown to release tumor necrosis factor α (TNFα) in response
to glutamate receptor activation, and the generation of reac-
tive oxygen and nitrogen species which could further exacer-
bate damage to OLs (Matute et al. 2001).
The third major ionotropic glutamate receptor is the
N-methyl-d-aspartate (NMDA) receptor, which is ubiqui-
tous in the mammalian CNS, subserving a key role in learning
and memory. This receptor differs from the AMPA/kainate
classes being highly permeable to Ca2+, and thus implicated in
many acute and chronic degenerative CNS disorders (Lau &
Tymianski 2010). The presence of functional NMDA recep-
tors on OLs has been more controversial. A number of earlier
studies failed to conclusively demonstrate these (Karadottir
& Attwell 2007). A report by Sykova and coworkers demon-
strated NMDA-evoked currents in oligodendrocytes from
spinal gray matter, which diminished substantially during the
first 14 postnatal days, with only very weak currents detected
M ETA B O L I C I N J U RY O F O L I G O D E N D R O C Y T E S A N D M Y E L I N
mainly in gray matter OLs (Ziak et al. 1998). It wasn’t until
almost a decade later that Attwell and colleagues reported
functional NMDA receptors in OLs at all stages of matura-
tion (Karadottir et al. 2005; Karadottir & Attwell 2007;
Burzomato et al. 2010). These receptors are somewhat unique,
exhibiting weak Mg2+ block, which would imply that they
may generate a significant current even at the nominal rest-
ing potential of −60 to −65 mV. From knowledge of molecular
physiology of NMDA receptors, together with immunochem-
ical studies, it was suggested that the subunit composition may
also be unusual, composed of GluN1 (NR1), GluN2C/D
(NR2C/D) and/or GluN3 (NR3) subunits. Probably not
coincidentally, mature myelin itself also expresses NMDA
receptors with similar subunit compositions (see next sec-
tion) (Burzomato et al. 2010; Micu et al. 2006; Pin ῀ a-Crespo
et al. 2010). At variance with the above findings, more recent
electrophysiological experiments using acute brain slices from
transgenic mice revealed that OLs substantially downregulate
somatic AMPA and NMDA receptor densities and glutamate
receptor-coding mRNAs as they mature; although progenitor
NG2+ cells exhibit substantial NMDA receptor-dependent
currents, in this study pre-myelinating and mature OLs did
not (De Biase et al. 2010). In line with these observations,
Salter and Fern reported that OL somata preferentially express
AMPA/kainate receptor subunits, whereas the OL processes
contain NMDA receptors (Salter & Fern 2005); because
processes will be geographically and electrically distant from
the soma, they would tend not to be detected electrophysi-
ologically during patch clamp recordings, consistent with the
absence of detectable NMDA currents noted above. Moreover,
ischemic injury of OL somata was prevented by AMPA/kain-
ate receptor antagonists, but processes were only protected
when NMDA receptors were blocked (Salter & Fern 2005).
Similarly, Micu and coworkers found that ischemic Ca2+
increase in the OL cell body was prevented by the AMPA/
kainate antagonist NBQX, whereas myelin Ca2+ accumula-
tion was mainly dependent on NMDA receptors (Micu et al.
2006). Taken together, although the issue of NMDA recep-
tors on OLs is still not completely settled, current evidence
suggests the following: (1) Immature OLs express all ionotro-
pic receptors on their soma, likely explaining their vulnerabil-
ity in pre- and early postnatal life (Fern & Moller 2000; Deng
et al. 2003). (2) as OLs mature, somatic density of NMDA
receptors diminishes, perhaps not to zero, leaving AMPA/
kainate receptors as the predominant forms on their cell bod-
ies. (3) Instead, NMDA receptor expression seems to migrate
centrifugally, occupying processes and the myelin sheath.
This framework implies that cytoprotection of OLs is com-
plicated, and will require a combination approach, because
both the soma, processes, and sheaths are essential for survival
and function of the entire myelinating unit, which will likely
suffer damage by overactivation of distinct receptor subtypes.
Another important point pertaining to NMDA receptors is
the fact that they require two obligatory agonists for activa-
tion: glutamate and glycine (or D-serine). The latter has not
been studied much in the context of OLs, and will be briefly
discussed below. Although the previous discussion is focused
on pathophysiology, the obvious question that arises is, what
• 667
is the physiological purpose of these receptors on OLs?
Unmyelinated CNS axons have been shown to have vesicular
release apparatus, forming synaptic specializations with OL
precursor cells, and supporting AMPA receptor-mediated
activity-dependent glutamatergic transmission from axon to
this glial precursor (Kukley et al. 2007; Ziskin et al, 2007).
Moreover, demyelinated axons in the adult mouse corpus cal-
losum form functional AMPA/kainate receptor-dependent
synapses onto adult-born OL precursor cells migrating from
the subventricular zone after focal demyelination. This “syn-
apse” is downregulated as the OL precursors mature and com-
plete their remyelination (Etxeberria et al. 2010). Therefore it
is tempting to speculate that AMPA/kainate receptors play a
role in differentiation and migration of immature OLs during
(re)myelination. As for NMDA receptors, their role in this
process is less clear. Fields and colleagues showed that NMDA
receptors, together with metabotropic glutamate receptors,
stimulated through vesicular release of glutamate induced
by electrical activity from axons undergoing myelination, are
involved in promoting local protein synthesis in myelinating
processes (Wake et al. 2011). On the other hand, other reports
indicate that these receptors are not involved in postnatal OL
development or myelination (De Biase et al. 2011; Guo et al.
2012). Given the predilection of “centrifugal” expression of
OL NMDA receptors as alluded to above, perhaps their role
has more to do with physiology of OL processes and axomy-
elinic signaling (Stys 2011), possibly relevant to “metabolic”
support of the mature axomyelinic unit, but this is purely
speculative at this time.
Although OLs appear to express all three classes of iono-
tropic glutamate receptors that are poised to cause damage, the
other key part of the excitotoxic equation is clearance of this
transmitter. This is effected by Na+-dependent glutamate trans-
porters (gluTs), of which 5 distinct types have been identified
and are expressed in the mammalian CNS, denoted EAAT1
through 5 (excitatory amino acid transporter) (Danbolt
2001). Glutamate transporters may contribute to OL excito-
toxicity passively, by reducing their capacity for extracellular
glutamate clearance, or actively, by releasing glutamate from
the cytoplasm of cells during conditions of intracellular Na+
loading and depolarization, both of which will tend to drive
gluTs in the glutamate release mode. The importance of con-
tinuous and competent glutamate uptake is underscored by
reports that inhibition of these transporters, either pharma-
cologically or by antisense oligonucleotides, causes OL death,
demyelination, and axon damage in animal models (Domercq
et al. 2005). Such experiments are very instructive because
they tell us that the basal cycling (i.e., release and reuptake)
of glutamate is substantial, even in white matter; impairment
of uptake, or excess release alone, is sufficient to cause severe
damage to white mater in general, and OLs in particular.
Impaired gluT expression and function has been implicated
in exacerbating excitotoxicty in human disorders such as mul-
tiple sclerosis (Werner et al. 2001), where an inflammatory
milieu with generation of proinflammatory cytokines such as
TNFα and oxidative stress promotes downregulation of gluTs
(Domercq et al. 2007). To add additional insult to inflam-
matory injury, infiltrating activated microglia increase their
668 • NEUROGLIA
glutamate–cystine exchange activity, driving uptake of cystine
in exchange for release of cytosolic glutamate, further increas-
ing toxic extracellular glutamate levels (Domercq et al. 2007).
GluTs contribute to OL damage, and excitotoxicty in
general, not only by reducing transmitter uptake, but also by
releasing glutamate from the cytosol. Because of the sensitivity
of glutamate receptors (especially NMDA receptors) to this
transmitter, extracellular glutamate is maintained in the low
micromolar range. In contrast, cytoplasm of most cells con-
tains millimolar glutamate, and the intracellular space repre-
sents the major fraction of brain volume. Therefore, there is
potentially a large lethal source or glutamate lurking just on
the other side of every membrane of every cell in the CNS.
Under pathological conditions such as trauma or ischemia,
cells accumulate Na+ and depolarize, two ideal stimuli that
synergistically conspire to promote reverse operation of gluTs,
which are electrogenic and driven by the prevailing Na+ (and
K+) gradient (Danbolt 2001). Thus, reverse operation of glutTs
and release of glutamate is a major source of pathological glu-
tamate rise in the extracellular space, and causes injury to OLs,
neurons, and axons (Fern & Moller 2000). Indeed, any cell
that is depolarized and loaded with Na+ under pathological
conditions, and that expresses gluTs (which includes all major
cell types in white matter), will release abnormal amounts
of glutamate. This is particularly true of white matter axons,
which contain substantial quantities of glutamate in the axo-
plasm that is readily released during injury (Li et al. 1999). As
mentioned in the previous section, NMDA receptors require
not only glutamate, but also glycine, for activation. As with
glutamate, the CNS is equipped with Na+-dependent glycine
transporters (glyT1 and 2) (Aragon and Lopez-Corcuera
2003). We have shown that in CNS white matter, glyTs con-
tribute importantly to Ca2+ overload in both OLs and myelin
during chemical ischemia (Micu et al. 2007), indicating that
this transporter family may also play a major role in OL injury,
and white matter damage in general, by virtue of its ability
to source the other essential NMDA receptor coagonist. It is
interesting to note that inborn errors of metabolism leading
to abnormally high or low levels of glycine and/or D-serine
result in severe myelination defects (de Koning et al. 2000;
Press et al. 1989), underscoring the potential importance of
this less-studied, but essential, NMDA receptor coagonist in
myelination and white matter integrity.
5 OT H E R C A L C I U M-P E R M E A B L E
CHANNELS AND OLIGODENDROCYTE
I N J U RY
While glutamate, its receptor family and its transporters, are
the most thoroughly studied in the context of OLs, these
cells express additional receptors that are emerging as playing
important roles in OL pathophysiology. As their name implies,
purinergic receptors are activated by purines and pyrimidines,
and are divided into two main classes, P1 and P2, activated
by adenosine and ATP/ADP, respectively. The P2 family is
further subdivided into P2X and P2Y. The P1 and P2Y recep-
tors are G protein-coupled metabotropic, whereas P2X are
ionotropic receptors (Burnstock et al. 2011; Matute 2011).
Each of these main categories is itself further subdivided into
many subtypes. All four types of P1 receptor are expressed by
OLs, most of the Ca2+-permeable ionotropic P2X subtypes
(with the P2X7 receptor being particularly prominent) and
several metabotropic P2Y subtypes (for review, see Matute
& Ransom 2012). Activation of these purinergic receptors by
adenosine and ATP results in elevation of cytosolic [Ca] in
OLs, via release from internal Ca2+ stores or influx from the
extracellular space across the plasma membrane ( James & Butt
2002; Matute et al. 2007b). Under physiological conditions,
these receptors may function to transduce signals from axons
and astrocytes to control myelination (Butt 2011). Importantly,
given their propensity to raise OL Ca2+ levels via stores release
or permeation across the cell membrane, purinergic receptors
are well positioned to effect injury to OLs under pathologi-
cal conditions. The lifeblood of cellular energy supply, ATP, is
paradoxically highly toxic when it accumulates outside the cell.
Under pathological conditions, ATP can be released by several
mechanisms, including Ca2+-dependent exocytosis of synap-
tic vesicles, connexin- and pannexin-containing gap junction
hemichannels, and other ion channels, including P2X7 recep-
tors themselves, setting the stage for a potentially dangerous
positive feedback loop. Although P2X7 receptors have a rela-
tively low affinity for ATP, pathological conditions result in
substantial release of this nucleotide in sufficient amounts to
activate these highly Ca2+-permeable receptors (Burnstock et
al. 2011). An important source of ATP release may be from
astrocytes (Coco et al. 2003). This precipitates severe OL
injury (Matute et al. 2007b), and as will be discussed in the
next section, probably direct damage to myelin as well.
Acid-sensing ion channels (ASICs) are plasmalemmal
channels that are activated by low (<7) extracellular pH. These
cation channels are mainly permeable to Na+ but some isoforms
exhibit substantial Ca2+ permeability (reviewed in Krishtal
2003). Cells of the OL lineage express ASICs, and in particular,
the ASIC1a splice variant (Feldman et al. 2008), which is Ca2+
permeable. This would implicate these channels as potentially
important mediators of OL injury under acidotic conditions
as occurs during ischemia or inflammation (Friese et al. 2007).
These channels can flux damaging amounts of Ca2+ directly,
and can contribute additional indirect damage by promoting
Na+ entry and depolarization, in turn activating voltage-gated
Ca2+ channels, Na+ Ca2+ exchanger (Chen et al., 2007), and
glutamate transporters (the latter two driven to import Ca2+
and release glutamate respectively), all known to be expressed
by OLs. The importance of these channels is underscored by
reports of reduced tissue damage and improved clinical scores
after experimental autoimmune encephalomyelitis in animals
genetically lacking ASIC1 or where this channel was pharma-
cologically inhibited by amiloride (Friese et al. 2007). In con-
trast, activation of these channels promoted OL injury in an
ASIC-dependent manner (Vergo et al. 2011).
As more detailed experiments are focused on OLs and
myelin, additional channels, receptors and transporters are
being identified, any of which can participate in parallel to
mediate OL injury and demyelination. For instance, recently
nicotinic and adrenoreceptors have been implicated in white
M ETA B O L I C I N J U RY O F O L I G O D E N D R O C Y T E S A N D M Y E L I N
matter injury (Constantinou and Fern 2009; Nikolaeva et al.
2009). The list is already large (for a review, see Domingues et
al. 2010) and is likely to grow larger as time goes on, empha-
sizing the remarkable molecular complexity of nonsynaptic
white matter and its constituent cells.
6 MYELIN ALSO EXPRESSES CHANNELS
A N D R E C E P TO R S
Myelin is a very unusual structure, composed of multiple wraps
of lipid-rich membranes emanating from OL processes in the
CNS (see chapter 44). Its main role is to increase the resistance
and reduce the capacitance of the axon membrane allowing
rapid saltatory conduction along fibers that are one or two orders
of magnitude thinner than their unmyelinated equivalents,
an essential requirement in a high-density mammalian CNS.
Without an obvious intracellular compartment, electrical
polarization or source of signaling molecules, it was assumed
that myelin is an inert structure, playing its part only by virtue
of its passive biophysical properties. A major stumbling block
was a lack of techniques capable of reporting physiological
responses from this structure, in contrast to neurons and glia
where quantitative and sophisticated electrical and optical
recordings have been applied for decades. Given biochemical,
immunohistochemical, proteomics-based, and cytochemical
evidence that myelin per se expresses functional ion channels
and pumps, transporters and chemical receptors (Ishii et al.
2009; Jahn et al. 2009), we posited that it too can respond
to stimuli, both physiological and pathological, much like its
parent OL and possibly even like neurons. We developed a
technique based on 2-photon laser scanning microscopy that is
able to report Ca2+ fluctuations from the major dense line (the
thin cytosolic spiral) of mature CNS myelin (Micu et al. 2007).
Using this method, we showed that mature CNS myelin suffers
a substantial increase in Ca2+ levels following chemical ischemia
(Micu et al. 2006). Molecular dissection revealed a surprising
mechanism: while the ischemic Ca2+ rise in the OL cell body
is mainly mediated by AMPA/kainate receptors, myelinic Ca2+
overload does not passively follow the fate of the parent soma.
AMPA/kainate receptor blockers have only a modest effect on
myelinic Ca2+ increase, (nonetheless, the effect was significant,
consistent with earlier immunohistochemical demonstration
of GluA4 AMPA subunits in myelin [Li and Stys 2000], an
observation that was subsequently confirmed and extended to
include the GluK5 [KA2] kainate receptor subunit [Brand-
Schieber and Werner 2003]). Somewhat surprisingly, NMDA
receptor inhibitors completely block the ischemic Ca2+ rise in
myelin (but not in OL soma) (Micu et al. 2006), emphasizing
the independent and molecularly unique response of the
sheath compared to its parent OL cell body. Using a variety
of specific pharmacological agents, it was determined that
myelinic receptors are likely composed of the obligatory
GluN1 (NR1) subunit and GluN2C-D (NR2C-D) subunits,
with no evidence for GluN2A or B, which are prominent
synaptic and extrasynaptic (respectively) receptors in neurons.
Immunohistochemistry provided a direct demonstration of
the presence of all the major NMDA receptor subunits on
• 669
CNS myelin, located in clusters, mainly along the inner and
outer myelin loops (Micu et al. 2006) (Fig. 52.1). At the same
time, Attwell and colleagues independently corroborated the
presence of NMDA receptor subunits in myelin (Karadottir
et al. 2005), which was further confirmed by Fern and coworkers
(Alix and Fern 2009), lending credence to this unexpected
finding. A follow-up study revealed an even more unexpected
result. Using the same technique, but without chemical ischemia,
we subsequently showed that D-serine alone (a GluN1 glycine
site agonist) elicits a substantial rise in myelinic Ca2+ levels
(Pin῀ a-Crespo et al. 2010). Pharmacological dissection revealed
that these responses are likely mediated by an unusual “glycine-
only” NMDA receptor, activated by glycine or D-serine alone
(Chatterton et al. 2002), without the need for NMDA or
glutamate which bind to the GluN2 subunit. These “glycine-
only” NMDA receptors are instead composed of glycine/D-
serine-binding subunits, GluN1 and GluN3, without GluN2.
Figure 52.1 CNS Myelin Expresses Transmitter Receptors. A. Clusters
of NMDA receptor subunits (green) are seen surrounding axon cylinders
(neurofilament, red) of adult rat optic nerve axons that are fully myeli-
nated at this age. Scale bars = 2 μm. Immunogold labeling indicates that
most receptors are located at the inner or outer lops of myelin. Scale bars:
50 nm. B. Compact CNS myelin also expresses clusters of P2X7 puriner-
gic receptors with a similar distribution. Scale bars 300 nm. My: myelin,
Ax: axon. (A) Modified with permission from Micu et al.
2006. (B) Modified with permission from Matute et al. 2007b).
670 • NEUROGLIA
It may be no coincidence that myelin prominently expresses this
unusual GluN1/GluN3 receptor, as it has been shown to be
weakly susceptible to voltage-dependent Mg block, and exhibits
low Ca2+ permeability (reviewed in Henson et al. 2010). Unlike
conventional synapses, where AMPA/kainate receptors are
present to depolarize the postsynaptic membrane and relieve
NMDA receptors from their Mg block, perhaps myelin cannot
undergo similar depolarizations, and therefore must contain
a receptor that is not strongly blocked at hyperpolarized
potentials. Once activated however, the myelinic NMDA
receptor may need to show restraint with regards to how
much Ca2+ it permeates; the cytoplasmic volume of the inner
mesaxon (the uncompacted inner tongue of myelin adjacent to
the axon) and the myelin spiral is tiny compared with a neurons
cytosolic volume; therefore, an NMDA receptor with limited
Ca2+ permeability could suffice for whatever local signaling is
required, and in fact may be essential to avoid injuring the inner
loops. In summary, to date evidence indicates the inner myelin
loops contain GluA4 AMPA, and mainly GluN1/GluN2C-D
(or perhaps triheteromeric GluN1/GluN2C/GluN3A-
containing receptors [Burzomato et al. 2010]) and GluN1/
GluN3 “glycine-only” NMDA receptors, and that these
contribute importantly to myelin damage. The physiological
role of these receptors is unknown, but they may represent
the “postsynaptic” component of the proposed axomyelinic
synapse (Stys 2011). The potentially unique pharmacological
properties of the GluN1/GluN3 receptors raise intriguing
possibilities for rational design of selective therapeutic agents
for disorders where demyelination is prominent.
The rather unexpected finding of functional neurotrans-
mitter receptors on myelin was bolstered soon thereafter by
data from Matute and colleagues who reported the pres-
ence of Ca2+ permeable P2X7 receptors not only on OLs,
but also in myelin (Matute et al. 2007b). They found that
activation of P2X7 damaged OLs and promoted demyelina-
tion, similar to activation of glutamate receptors. Moreover,
P2X7 receptors are expressed on compact myelin, and like
NMDA receptors, appear to show a predilection for the
inner and outer myelin loops. Although these investigators
did not directly demonstrate that myelinic P2X7 receptors
cause Ca2+ overload in this structure (promotion or reduc-
tion of demyelination by modulating P2X7 may have been
due to injury or survival of the parent OLs in their study),
given the high Ca2+ permeability of P2X7 and their expres-
sion in myelin, it is highly likely that like NMDA receptors,
these purinergic receptors directly contribute to myelin
Ca2+ overload and demyelination. Although highly specu-
lative at this time, the emergence of various neurotrans-
mitter receptors, transporters and various ion exchangers/
cotransporters in myelin further raises the prospect that the
mature sheath is a far more active structure than previously
thought, capable of specific signaling possibly originating
from its ensheathed axon. Unfortunately for myelin, like
with glia and excitable neurons, when excessively activated
under pathological conditions, these same signaling path-
ways likely contribute to myelin damage and may represent
the most proximal events eventually leading to frank histo-
pathological demyelination.
 GluT
(EAAT1) Ca AMPAR
K
Na KAR
glu mGluR
cystine/
glutamate glu
RyR
antiporter P2YR
Ca IP3R G
cystine ROS ER
-
...glutathione Na
Ca NKCC
K
Ca
NCX Cl

Na
- Ca
ATPase
Na Na-K NO
ATPase -
ONOO
Ca
ROS K
ASIC1
NO P2X7
ONOO
- NMDAR H+
Ca
Ca K Cl Ca Na, Ca
Ca K Na
ATPase
Na-K GluA4 GluK5 NMDAR P2X7 calpain
KCC
ATPase Ca
PAD2
MYELIN
NET (GluN1/2C,D/3A)
GluT (EAAT1)
Na NE

AXON

Figure 52.2 Schematic Illustrating Some Major Pathways That Normally Subserve Important Physiological Functions, but May be recruited to Operate
Inappropriately Contributing to Injury to Ols and Myelin. The OL soma expresses most major ion channels, transporters, and AMPA/kainate and
metabotropic glutamate receptors. Ca2+ homeostasis in maintained by the plasmalemmal Na+-Ca2+ exchanger (NCX) and Ca2+-ATPase, as well as the
ER Ca2+-ATPase (not shown). Pathological Ca2+ accumulation can be mediated by many systems, including reverse NCX, AMPA/kainate receptor
overactivation, acid-sensing ion channels (ASIC1a), and ionoptropic (P2X7) and metabotropic (P2Y) purinergic receptors. Additional Ca2+ can be
released from the ER via ryanodine (RyR) receptors in a Ca2+-induced Ca2+ release manner or via IP3 receptors. The Na+ K+-Cl– cotransporter (NKCC)
can load the OL soma with Na+ leading to further injury by reverse NCX or promoting glutamate release via reverse glutamate transport (gluT). OL
processes are preferentially damaged by NMDA receptors. Myelin itself expresses a variety of receptors (GluA4 AMPA, NMDA, KA2 kainate, and
P2X7 purinergic) all of which are Ca2+-permeable. Reactive oxygen and nitrogen species (ROS, NO, peroxynitrite [ONOO-]), generated locally or by
nearby cells, can diffuse and promote membrane and myelin damage through lipid peroxidation and can exacerbate mitochondrial dysfunction (see text
for more detail).

7 N O N –R E C E P TO R -M E D I AT E D I N J U RY mechanisms (Back et al. 1998). This powerful intracellular

Neuroinflammatory lesions are characterized by a very toxic
milieu with elevated levels of nitric oxide (NO) (or reactive
nitrogen species in general), cytokines, glutamate, and oxygen
free radicals. Nitric oxide has both beneficial and deleterious
effects (reviewed in Smith and Lassmann 2002); OLs appear
particularly vulnerable to this free radical (Mitrovic et al.
1994), with cell death proceeding either via necrosis or apop-
tosis depending on the length and intensity of NO exposure
(Garthwaite et al. 2002). The particularly toxic metabolite of
NO, peroxynitrite, damages cell membranes through lipid
peroxidation, permanently altering their chemical and physi-
cal properties (van der Veen & Roberts 1999). This may be
particularly important for demyelination, because the altered
membrane composition promotes recognition and phagocy-
tosis by macrophages (Graham et al. 1993). Reactive oxygen
species are also toxic to OLs and likely myelin as well. Like
NO, these radicals are also generated in abundance by acti-
vated macrophages and microglia, resulting in damage to
white matter elements in neuroinflammatory conditions such
as multiple sclerosis (MS) (Haider et al. 2011; reviewed in
van Horssen et al. 2011). Indeed, cells of the OL lineage have
developed powerful strategies to cope with free radical stress,
with the glutathione system being one of the more important
antioxidant molecule depends on a steady supply of cystine,
supplied by uptake by the cystine/glutamate antiporter, which
is then converted to cysteine, essential for glutathione synthe-
sis (Conrad and Sato 2012). Interfering with this system by
cystine deprivation results in toxicity to OLs, especially imma-
ture OLs (Back et al. 1998). Upregulation of this antiporter,
under conditions that require a response to increased oxida-
tive stress, will release glutamate, potentially exacerbating
excitotoxicity (Conrad and Sato 2012) (see the preceding),
reflecting the complex interdependence of many pathways,
the net result of which is not always apparent.
Regulation of cellular volume is a complex and essential
function of all cells, and depends on a complex collection
of channels and anion transporters coupled to various other
ions (reviewed in Okada 2004). Given that the essential func-
tion of myelin depends on its structural integrity, and on a
tight apposition to its axon, swelling of this structure could
adversely affect its function in a significant way, by altering its
electrical characteristics and promoting current leaks if vol-
ume changes disrupt the intimate axomyelinic relationship.
The K+-Cl– cotransporter, one of the main anion cotransport-
ers involved in volume regulation, is expressed in OLs and
also in myelin (Malek et al., 2003). Experiments on anoxic
ex vivo white matter tracts revealed that while reducing Na+

M ETA B O L I C I N J U RY O F O L I G O D E N D R O C Y T E S A N D M Y E L I N • 671
influx through voltage-gated Na+ channels by TTX is highly
protective at restoring the total magnitude of evoked electrical
responses (Stys et al. 1992), the waveshapes of the compound
action potentials, governed by velocities of constituents axons,
remain significantly distorted (Malek et al. 2003). This suggests
that Na+ channel blockade rescues axons from injury, but the
axomyelinic integrity continues to be disrupted, causing slow-
ing and desynchronization of action potential propagation.
Interestingly, addition of the K+ Cl– cotransporter inhibitor
furosemide significantly improves the shape, but not the mag-
nitude, of the postanoxic compound action potential, suggest-
ing that myelin swelling mediated by aberrant Cl– movement
(and in turn water) across the myelin lamellae, mediated by
the K+ Cl– co-transporter, was mainly responsible for disrupt-
ing the essential physical association between myelin sheath
and axon (Malek et al. 2003). The Na+-K+-Cl- cotransporter
has also been shown to promote pathological Na+ entry into
OLs, which then triggers reverse Na+ Ca2+ exchange resulting
in Ca2+ entry across the OL plasmalemma and additional Ca2+
dependent injury (Chen et al. 2007).
8 I N J U RY TO O L I G O D E N D R O C Y T E S A N D
MYELIN IN SPECIFIC DISORDER S
Many prevalent and devastating disorders of the brain and
spine adversely affect the white matter, with OLs and myelin
being prominent targets that are rarely if ever spared injury.
Indeed, it is likely that OLs and myelin are probably affected,
directly or indirectly, in virtually every disorder of the CNS,
therefore it is not possible to cover every one in this section.
Instead, a few prominent and better understood conditions, at
least from the viewpoint of OL/myelin pathophysiology, will
be mentioned. Moreover, there are many overlaps in pathways
pathologically overdriven among various disorders (e.g., gluta-
mate excitotoxicity is prominent in ischemia, inflammation,
and trauma), therefore no clear lines should be drawn in the
discussion that follows. One of the commonest diseases is isch-
emia, either with an acute presentation as in ischemic stroke, or
a more chronic scenario of longstanding (mainly subcortical)
small vessel insufficiency and microinfarcts. Ischemia affects
not only older subjects, but also the very young, in the form
of perinatal ischemia resulting in the well-known pathology
of periventricular leukomalacia (Back et al. 2007). Ischemia is
particularly damaging because the general reduction of energy
supply in cells promotes multiple points of failure, with an
intracellular increase of [Ca2+]i being the final common path-
way leading to OL and myelin injury. Indeed, after neurons,
OLs are the most sensitive cells to hypoxia/ischemia (Fern &
Moller 2000; reviewed in Matute et al. 2006), with immature
OLs in the perinatal brain being even more sensitive probably
because of their collection of surface glutamate receptors. Influx
of extracellular Ca2+ through AMPA/kainate receptors is prob-
ably the most important route of Ca2+ overload in OLs (Matute
2011). As noted in previous sections, OL processes and myelin
are preferentially sensitive to NMDA receptor overactivation
(Micu et al. 2006; Salter & Fern 2005), though this distinction
between soma and processes/myelin is certainly not absolute
672 • NEUROGLIA
(Karadottir et al. 2005). To date, reperfusion strategies are the
only effective treatment for acute stroke. The burst of reactive
oxygen species generated by mitochondria when tissue is reox-
ygenated (Piantadosi and Zhang 1996) causes additional dam-
age, and OLs may be particularly vulnerable because of their
high iron content (LeVine and Macklin 1990), which catalyzes
the formation of free radicals and promotes lipid peroxidation.
This may be particularly important for lipid-rich myelin, and
is consistent with the frequent demyelination seen after brain
ischemia. Interestingly, detailed pathological studies indicate
that the inner myelin loops (those adjacent to the axon) are
preferentially damaged by ischemia as evidenced by loss of
myelin-associated glycoprotein, together with apoptotic OLs,
and often with still-preserved, though frequently spheroidized
axons (Aboul-Enein et al. 2003). This attests to the high vul-
nerability of OLs and myelin to ischemia. Moreover, the fact
that Ca2+-permeable myelinic NMDA receptors are preferen-
tially located on the inner loops, precisely where the earliest
myelin damage is observed, may not be a coincidence. In addi-
tion, expression of Ca2+-permeable P2X7 purinergic receptors
on OLs and inner myelin loops also contributes to ischemic
injury to these elements (Domercq et al. 2010).
Demyelinating neuroinflammatory conditions such as
multiple sclerosis are another prominent example of disorders
exhibiting prominent damage to OLs and myelin. Mechanisms
of damage by the immune system have been extensively studied
and are complex, the details being beyond the scope of this chap-
ter. The reader is instead referred to chapter 61 and other reviews
(Bruck 2005). In addition to the complex immunopathogenesis
of the MS plaque, inflammatory lesions represent a very toxic
milieu with high levels of excitotoxins (glutamate, ATP), NO,
and a plethora of inflammatory effectors. The detailed pathways
of glutamatergic and purinergic injury to OLs and myelin have
been described above. More relevant to MS and neuroinflam-
matory disease, however, are observations from animal and
human studies clearly indicating increased levels of glutamate
in the MS brain (Srinivasan et al. 2005), impaired homeostatic
mechanisms for glutamate (Werner et al., 2001), robust pro-
tective effects of AMPA/kainate (Pitt et al. 2000; Smith et al.
2000), and NMDA receptor (Wallstrom et al. 1996; but see Guo
et al. 2012; Matute 2010) antagonists in rodent models of experi-
mental autoimmune encephalomyelitis (an experimental model
mirroring the inflammatory lesions of MS) and genome-wide
association studies showing strong links with genes involved
in glutamate homeostasis (Baranzini et al. 2010). Interestingly,
this latter study indicated the strongest correlation in patients
with the greatest degree of progressive degeneration, suggest-
ing a direct link to MS pathogenesis independent of immune
or inflammatory effects. Another very important mediator of
cellular injury during inflammation is NO generation (Smith
& Lassmann 2002). Oligodendrocytes are far more sensitive
to damage by NO (or more likely peroxynitrite, its highly toxic
by-product (Jack et al. 2007)), than other glia (Mitrovic et al.
1994). Mechanisms include lipid peroxidation, with the large
area of myelin membrane conferring particular vulnerability.
Peroxynitrite might indirectly damage OLs (and most other
cells) by inhibiting mitochondrial metabolism, thus promoting
a state of “virtual hypoxia” (Stys 2004).
 Trauma to the brain and spinal cord is an unfortunately
common occurrence. The pathophysiology of secondary
degenerative events triggered by the initial injury is diverse
(Farkas and Povlishoc 2007), but OL injury and demyelina-
tion are a nearly universal feature that may progress for weeks
or months following the initial trauma (Totoiu and Keirstead
2005). Extracellular glutamate has been shown to rise signifi-
cantly after both brain and spinal trauma (McAdoo and Wu
2008; Chamoun et al. 2010), triggering secondary excitotox-
icty as discussed above. Another major excitotoxin, ATP, also
increases substantially after traumatic brain and spinal cord
injury, resulting in further injury to nearby OLs and neu-
rons via P2X7 receptors (reviewed in Burnstock et al. 2011).
Inflammation and relative ischemia are also prominent after
trauma, in turn entraining all the deleterious cascades associ-
ated with these conditions as detailed previously. The unfolded
protein response in response to ER stress has been shown to
be directly involved in OL damage, and by extension in sec-
ondary demyelination, following spinal cord injury (Ohri
et al. 2011). Finally, neurodegenerative conditions, in particu-
lar Alzheimer’s disease, also involve degeneration of white mat-
ter, including injury to OLs and demyelination. Traditionally
white matter pathology in Alzheimer’s has been ascribed to
small vessel ischemia and Wallerian degeneration secondary to
cortical atrophy. Recent evidence shows that Aβ alters NMDA
receptor kinetics leading to receptor overactivation, excessive
Ca2+ influx, and neuronal injury (You et al. 2012). Given that
myelin also expresses NMDA receptors, it is conceivable that
a similar mechanism may promote direct injury to myelin.
Traumatic brain injury and chronic traumatic encephalopa-
thy also exhibit β-amyloid deposition ( Johnson et al. 2010),
suggesting a potential similar mechanism of myelin and white
matter damage after trauma. These proposed new mechanisms
will require experimental confirmation however.
9 S U M M A RY A N D P E R S P E C T I VE S
Oligodendrocytes and the myelin they produce play a vital
role for the proper operation of the CNS. Numerous disorders
result in damage to OLs and demyelination, causing major
morbidity and mortality. As in many other cells, Ca2+ overload
plays a major role in the death of OLs and damage to myelin.
Recent studies have revealed a remarkably complex collection
of channels and receptors expressed on OL cell bodies, pro-
cesses, and myelin itself (Fig. 52.2), that contribute to Ca2+
accumulation and cell injury in a variety of conditions ranging
from ischemia, trauma, neuroinflammation, and neurodegen-
erative disorders. It is highly likely that there exist many more
chemically-gated receptors on OL cell bodies, processes, and
myelin itself, that may provide unique therapeutic opportuni-
ties and desired selectivity, both for protecting the myelinat-
ing unit from injury, and conceivably for boosting repair and
remyelination after an insult. Experimental approaches aimed
at understanding mechanisms of neurological disease have
been heavily “neurocentric,” that is, biased toward elucidating
cellular and molecular mechanisms of injury to neurons and
synapses. This is understandable because the neuronal element
M ETA B O L I C I N J U RY O F O L I G O D E N D R O C Y T E S A N D M Y E L I N
is the most vulnerable in most disorders of the CNS. However,
glia play critical roles as well, and in particular, the OL and
myelin, without which the neuron’s raison d’être is largely
extinguished: if the neuron cannot transmit its output to its
target, the neuron may as well not exist. Recent years have seen
a growing interest in the fundamental mechanisms of OL and
myelin injury, and this area is starting to catch up to the vast
knowledge base that has accumulated regarding the neuron
and the interneuronal synapse. The emerging complexity per-
taining to the OL and myelin is at the same time fascinating,
and humbling, as even this relatively simple cell is turning out
to be far more complex than once imagined. Perhaps the most
interesting aspect of OL/myelin biology is the discovery of
an ever richer collection of neurotransmitter-based signaling
mechanisms on the OL and even the mature myelin sheath
itself. Together with emerging data on transmitter release
machinery in axons, these observations are painting a fas-
cinating novel framework of an “axomyelinic” synapse (Stys
2011). This synapse may play a fundamentally important role
in myelination during development and repair, and may also
control white matter plasticity in the mature CNS. Equally
important, dysfunction of this synapse may participate in the
pathogenesis of a broad range of neurological disorders where
white matter is adversely affected. The challenge for the next
decade will be to expand our knowledge of axoglial, and in
particular, axomyelinic signaling, which in turn will pave the
road for developing potent and selective therapeutic agents
designed to mitigate damage to the white matter of the CNS.
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 53.
INTERACTION OF MICROGLIA WITH NEURONS
AND ASTROCY TES UNDER LESIONED NEURONAL
CONDITIONS
Kazuyuki Nakajima and Shinichi Kohsaka

A B B R E VI AT I O N S and pathological conditions. However, it is not always entirely

clear how the glial cells and neurons interact, or for what rea-
BDNF brain-derived neurotrophic factor son. A good model for examining the cellular interactions
CD11b cluster of differentiation 11b between glial cells and neurons is the peripheral nerve injury
Cdk cyclin-dependent protein kinase model, which often involves injuries from the transection of
CdkI cyclin-dependent protein kinase inhibitor facial nerves (Kreutzberg 1996) or hypoglossal nerves (Morita
ChAT choline acetyltransferase et al. 1996). Since this injury system maintains the blood brain
CM conditioned medium barrier under normal conditions, the reactions between neu-
CNS central nervous system rons and glial cells can be observed in the parenchyma without
CNTF ciliary neurotrophic factor considering the effects of blood-derived cells and blood con-
CSF colony stimulating factor stituents. Initially, the facial nerve injury system results in an
ERK extracellular signal-regulated kinase alteration of motoneurons, followed by glial responses includ-
GM-CSF granulocyte, macrophage-CSF ing microglial activation/proliferation and astroglial activation
GDNF glial cell line-derived neurotrophic factor (Kreutzberg 1996; Streit et al. 1988). This chapter summarizes
GFAP glial fibrillary acidic protein current knowledge on the events in injured neurons, microglia,
GLAST glutamate aspartate transporter and astrocytes, as well as the interaction between microglia and
GLT-1 glutamate transporter-1 neurons/astrocytes, in the peripheral nervous injury system.
Iba1 ionized Ca2+ binding adapter molecule
IL interleukin
JNK c-jun N-terminal protein kinase 2 CHANGES IN INJURED NEURONS
MAPK mitogen-activated protein kinase
M-CSF macrophage-CSF Transection of the facial nerve in adult rats leads to a retro-
MPF mitosis promoting factor grade injury in the motoneuron cell bodies. In this case, the
NGF nerve growth factor injured motoneurons are viable and regenerate, unlike in
NO nitric oxide neonatal rats. The injured neurons begin to change their gene
NT neurotrophin expressions, morphology and functional state.
PA plasminogen activator In the early stages after transection, immediate early genes
PAI-1 plasminogen activator inhibitor-1 including c-jun, Jun-B, and TIS11 are induced in the injured
PCNA proliferating cell nuclear antigen motoneurons (Haas et al. 1993). Subsequently, these injured
PLGn plasminogen cells undergo changes in their expressions of molecules such
TGFβ transforming growth factor beta as cytoskeleton components, metabolic enzymes, neuropep-
TNFα tumor necrosis factor alpha tides, and cytokines (Moran and Graeber 2004). For example,
tPA tissue-type PA the transcriptions of actin and tubulin are downregulated in
uPA urokinase-type PA the injured motoneurons (McNamara et al. 2000). Stearoyl-
VAchT vesicular acetylcholine transporter CoA desaturase, which mainly plays a role in energy storage, is
strongly promoted (Schmitt et al. 2003). Neuron-specific eno-
lase levels are increased in the motoneurons following injury,
1 INTRODUCTION and may contribute to their survival (Angelov et al. 1994).
Damage-induced neuronal endopeptidase is enhanced in the
The nervous system is constructed of heterogeneous cell types motoneurons of the axotomized hypoglossal nucleus, possibly
(i.e., neurons and glial cells), and these cell types are thought to to participate in a self-protection mechanism (Kiryu-Seo et al.
interact closely with each other. These processes are mediated 2000). Calcitonin gene-related protein (Haas et al., 1990),
by many molecules acting as signaling molecules under normal GAP-43 (Schwaiger et al. 1998) (see chapters 48 and 51)

677
and interferon-gamma (Olsson et al. 1989) are elevated in
response to neuronal injury. Accompanying these changes, the
functioning of the injured motoneurons declines along with
the downregulated levels of choline acetyltransferase (ChAT)
and the vesicular acetylcholine transporter (VAchT) (Tetzlaff
and Kreutzberg 1984). In fact the facial motoneurons reduce
the levels of ChAT and VAchT after the injury (Fig. 53.1).
3 R E S P O N S E S O F M I C R O G L I A TO
M OTO N E U R O N I N S U LT
3.1 AC T I VAT I O N
The morphological change from a ramified form to a short
process-bearing form is a characteristic feature of microglia
observed after motoneuronal insult. Accompanying or after
the transformation/morphological change, microglia start
to increase the expression of various kinds of proteins. The
classically established proteins in activated microglia include
immune-related proteins such as the cluster of differentiation
11b (CD11b), CD4, and Fc receptor (Kreutzberg 1989). Later
studies showed that ionized Ca2+ binding adapter protein 1
(Iba1) (Ito et al. 1998) and annexin III (Konishi et al. 2006)
are also induced in activated microglia. CD11b and Iba1 are
well-defined microglia-specific markers in the parenchyma of
the nervous system (Fig. 53.2). For a list of other molecules
upregulated in microglia in the axotomized facial nucleus, see
the review by Moran and Graeber (2004).
In the axotomized facial nucleus (Blinzinger and
Kreutzberg 1968) and in the axotomized hypoglossal nucleus
(Gesase and Kiyama 2000), microglial processes are observed
to squeeze into the synaptic cleft, leading to the concept of
A proliferation. After the injury-induced morphological trans-
ChAT
VAchT
L R L R L R L R L R
1d 3d 5d 7d 14d
B ChAT VAchT
C T C T
Figure 53.1 Injured Motoneurons in the Transected Facial Nucleus.
A. ChAT and VAchT levels in the injured nucleus. The right (R) facial
nerve of adult rats was cut, and after 1, 3, 5, 7 and 14 days the left (L) and
right (R) facial nuclei of each rat were recovered. The tissue homogenate
of each nucleus was immunoblotted for ChAT and VAchT.
B. Immunohistochemistry for ChAT and VAchT. Tissue sections of the
brainstem of an adult rat whose right facial nerve was transected 5 days
earlier was stained for ChAT (left side) and for VAchT (right side).
C, control side; T, transected side.
678
A
Iba1
GFAP
L R L R L R L R L R
1d 3d 5d 7d 14d
B CD11b
Figure 53.2 Activated Microglia in the Transected Facial Nucleus.
A. Iba1 and GFAP levels in the time course. The same samples
used in Figure 53.1A were immunoblotted for Iba1 and GFAP.
B. Immunohistochemistry for CD11b. The same tissue sections used in
Figure 53.1B were stained for CD11b. C, control side; T, transected side.
“synaptic stripping.” Although the localization of the microglia
would seem to suggest that they are involved in the “stripping”
of synaptic contact, later evidence indicates that synaptic with-
drawal from the motoneuron cell body following peripheral
nerve injury is likely a neuron-autonomous event, and not one
induced by activated microglia (Perry and O’Connor 2010).
3.2 P RO L I FE R AT I O N
One of the responses of microglia to motoneuronal injury is
formation, microglia begin to increase their numbers in the
ipsilateral facial nucleus (Ito et al. 1998).
3.2.1 Proliferation Factors and the Receptors
The factors controlling microglial proliferation have been
assumed as colony stimulating factors (CSFs) in in vitro
studies, in which macrophage-CSF (M-CSF); granulocyte,
macrophage-CSF (GM-CSF); and multi-CSF (interleukin 3;
IL-3) exert almost equivalent proliferative effects on microglia
(Guilian and Ingleman 1988). However, among the growth fac-
tors potentially involved in microglial proliferation, M-CSF is
perhaps the most likely candidate based on the finding that
osteopetrotic mice (Wiktor-Jedrzejczak et al. 1990), which
cannot produce biologically active M-CSF, exhibit almost no
increase in microglial number in the transected facial nucleus
(Raivich et al. 1994). In fact, the amount of M-CSF increases
in the ipsilateral facial nucleus at the time of microglial prolif-
eration (Yamamoto et al. 2010). At the same time, the recep-
tor for M-CSF, cFms (Ross 2006), is also remarkably induced
in the ipsilateral facial nucleus (Fig. 53.3A). These results are
essentially in good agreement with those from 125I-M-CSF-
binding experiments (Raivich et al. 1991).
• NEUROGLIA
 A suggesting that the induced CdkI brakes the progression of
M-CSF
cFms
PCNA
Cyclin A
L R L R L R L R L R
1d 3d 5d 7d 14d
B CFms Iba1 Cyclin A Iba1
Figure 53.3 Proliferation of Microglia in the Transected Facial Nucleus.
A. Time course of M-CSF, cFms, PCNA, and cyclin A levels. The same
samples used in Figure 53.1A were immunoblotted for M-CSF, cFms,
PCNA, and cyclin A. B. Immunohistochemistry for cFms and cyclin
A. The same tissue sections used in Figure 53.1B were dually stained for
cFms/Iba1 (left side) and for cyclin A/Iba1 (right side).
Apart from the facial nerve transection model, other CSFs
are also possible candidates for microglial mitogen. Human
fetal and adult microglia in vitro respond to M-CSF and
GM-CSF, and proliferate, but GM-CSF shows a much stron-
ger effect than M-CSF (Lee et al. 1994). Immunohistochemical
analyses have shown that GM-CSF is produced by reactive
astrocytes in the lesion sites of degenerative diseases. In addi-
tion, in a study by Bartolini et al. (2011), granulocyte-CSF
enhanced the proliferation of microglia in the normal cen-
tral nervous system (CNS). Thus, plural CSFs may serve as
microglial mitogens in normal and pathological states of the
CNS. Furthermore, particularly in inflammatory sites, cytok-
ines such as tumor necrosis factor alpha (TNFα) and IL-1β
(Ganter et al., 1992) may act as the mitogens.
3.2.2 Cell Cycle-Related Proteins
It is well known that cyclin pairs with a suitable cyclin-dependent
protein kinase (Cdk) and the resultant cyclin/Cdk complex
serves as a mitosis-promoting factor (MPF) that progresses
the cell cycle and leads to cell proliferation (Sherr 1993). More
recently, it was revealed that cyclinA/Cdk2 and cyclinD/
Cdk4 serve as the MPFs in microglia of the transected facial
nucleus (Yamamoto et al. 2012). After axotomy of the facial
nerve, microglia begin to express the cell cycle-genes cyclin A
and proliferating cell nuclear antigen (PCNA) (Fig. 53.3B), the
Cdk inhibitor (CdkI) p21, which serves in cell division arrest
(Fischer et al., 2002), is also induced in the injured nucleus,
I N T E R AC T I O N O F M I C R O G L I A W IT H N EU R O N S A N D A ST R O C Y T E S U N D E R L E S I O N E D N EU R O NA L C O N D IT I O N S
the microglial cell cycle advanced by MPF.
The participation of cyclin A and cyclin D in microglial
proliferation was reported in a GM-CSF-stimulated micro-
glial cell line (Koguchi et al. 2003). Zhang et al. (2009) dem-
onstrated that the microglial proliferation that occurs in focal
cerebral ischemia is inhibited by a reduction of cyclin A, B,
and E by applying a cell cycle inhibitor to the brain prior to
middle carotid artery occlusion.
3.2.3 Signaling Mechanism for Inducing
Microglial Proliferation
There is accumulating evidence that, after stimulation with a
mitogen, a specific signaling cascade for proliferation is acti-
vated in microglia. Liva et al. (1999) reported that, following
GM-CSF stimulation, extracellular signal regulated kinase
(ERK)1/2, Jak2, and STAT5A/5B were phosphorylated in
microglia in vitro. In human microglial cultures in which
microglia proliferate in a GM-CSF-dependent manner, Suh
et al. (2005) demonstrated that Hck tyrosine kinase and
the phophatidylinositol 3-kinase/AKt signaling cascade are
associated with microglial proliferation. The importance of
p38 mitogen-activated protein kinases (p38MAPKs) in the
microglial proliferation/activation induced by neuregulin-1
has been reported in the rat dorsal horn (Calvo et al. 2010).
In the facial nerve transection system, cFms stimulated by
M-CSF is activated and transduces the signals to downstream
MAPKs (Yamamoto et al., 2012). The stimulation of micro-
glia with M-CSF resulted in the activation of ERK, c-Jun
N-terminal kinase ( JNK), and p38MAPK, and JNK and
p38MAPK played a role in the induction of cyclins/PCNA
and cFms, respectively.
Mander et al. (2006) reported that microglial proliferation
in response to TNFα or IL-1β was mediated by hydrogen perox-
ide from nicotinamide adenine dinucleotide phosphate oxidase.
This finding suggests that reactive oxygen species may be associ-
ated with a signaling cascade leading to microglial proliferation.
3.2.4. Inhibition of Microglial Proliferation
Microglial proliferation occurring in the axotomized motor
nucleus can be blocked. In the transected facial nucleus, adminis-
tration of adriamycin inhibits microglial proliferation (Graeber
et al. 1989). Similarly, in hypoglossal nuclei treated with cytosine
arabinoside, microglial proliferation is inhibited (Svensson and
Aldskogius 1993). In the studies described above, these reagents
were thought to act as blockers of DNA replication.
3.2.5 Features of Proliferating Microglia
It would be valuable to analyze the properties of proliferat-
ing microglia from the viewpoint of neurotrophic cells/cyto-
toxic cells. From the observations reported so far, harmful
molecules including TNFα, IL-1β, IL-6, nitric oxide (NO)
and superoxide anion are not significantly promoted in the
transected facial nucleus. On the other hand, in the injured
facial nucleus, certain neurotrophic factors for motoneurons
• 679
are detected. One of these factors, transforming growth factor
b1 (TGFβ1), is expressed in activated microglia (Kiefer et al.
1993). Enhanced levels of brain-derived neurotrophic fac-
tor (BDNF) are localized in motoneurons (Kobayashi et al.
1996). Apart from the neurotrophic ligands, receptors for
glial cell line-derived neurotrophic factor (GDNF) (Burazin
et al. 1998), BDNF (Kobayashi et al. 1996), leukemia inhibi-
tory factor, and ciliary neurotrophic factor (CNTF) (Haas
et al. 1999) are also enhanced in motoneurons. These results
predict that these receptors will attempt to bind the ligands
released from surrounding glial cells in the injured facial
nucleus. Microglia and astrocytes in vitro exhibit the ability
to produce a variety of neurotrophic factors. Accordingly, it is
possible that proliferating microglia play a role as neurotrophic
cells in the lesioned site.
The observation that rubrospinal neurons degenerate in
the absence of microglial proliferation in the rubrospinal tract
transection model indicates that proliferating microglia serve as
neurosupportive cells in vivo (Streit 2002). Lalancette-Hebert
et al. (2007) indicated that selective ablation of proliferating
microglia leads to the exacerbation of ischemic injury, and
conversely, stimulation of microglial proliferation by M-CSF
results in an increase of insulin-like growth factor-1 levels. The
results suggest that proliferating microglia serve as neuropro-
tective cells in cerebral ischemia.
Another proof that proliferating microglia serve as neuro-
supportive cells in vivo was provided by Lopez-Redondo et al.
(2000), who observed that these cells express high levels of glial
type glutamate transporter-1 (GLT-1) in the area surrounding
the injured motoneuron cell body in the axotomized facial
nucleus. The location of GLT-1–expressing microglia seems
to suggest that the microglia protect injured motoneurons
from the abnormal glutamate-induced excitation that would
be otherwise induced.
4 R E S P O N S E O F M I C R O G L I A TO
N E U R O N A L S T I MU L AT I O N S I N V I T RO
As described above, an insult to the facial nerve may send a
signal to the motoneuron cell bodies that induces them to
change their morphology and metabolic and functional state.
Simultaneously, such injured neurons are presumed to out-
put various signaling molecules to trigger the activation of
glial cells, including an electrical signal (Rishal and Fainzilber
2010), CD200/CD200R interaction-derived signal (Lyons
et al. 2007), chemokines (Nishiyori et al. 1998), cytokines
(Tsuda et al. 2009), nucleotides, and transmitters. In response
to the injury signals, microglia are thought to be activated
and/or proliferated. However, how the microglial properties
are modulated by the neuronal stimulus is poorly understood.
To clarify this matter, it is necessary to analyze the influence of
neuronal stimuli on microglial properties by using an in vitro
system.
4.1 N EU ROT RO P H I C A B I L IT Y
Microglia in vitro produce various kinds of neurotrophic
factors (Nakajima and Kohsaka 2004). Whether or not
the microglial properties are altered by neuronal stimulus
is an interesting question that remains to be answered. The
stimulation of microglia with neuronal conditioned medium
(CM) enhanced the production of neurotrophic factors
including nerve growth factor (NGF), neurotrophin (NT)-
4/5, TGFβ1, GDNF, fibroblast growth factor, and IL-3
(Nakajima et al. 2007). These results suggest that the neuronal
stimulation changes microglia into more neurotrophic cells,
which contribute to neuronal survival and regeneration in
vivo (Table 53.1).
4.2 E XC ITOTOX I N-S C AVE N G I N G A B I L I T Y
Microglia cells in vitro express the glutamate transporter
GLT-1 (EAAT2) (Lopez-Redondo et al. 2000), and exhibit
a specific ability to uptake glutamate (Nakajima et al., 2001).
The glutamate uptake by microglia was enhanced by neu-
ronal CM, and its stimulation could be blocked by a GLT-1
inhibitor (Nakajima et al. 2008). Neuronal CM triggers the
upregulation of GLT-1, but not another glial-type glutamate
aspartate transporter (GLAST) (EAAT1). This implies that
proliferating microglial cells at injured motoneurons reduce
glutamate levels, and thereby reduce glutamate toxicity.

Table 53.1 RESPONSE OF MICROGLIA TO NEURONAL STIMULUS (NEURONAL CONDITIONED MEDIUM)

NONSTIMULATION NEURONAL CM-STIMULATION LPS-STIMULATION

mRNA PRODUCTION RELEASE mRNA PRODUCTION RELEASE mRNA PRODUCTION RELEASE

NGF + + − ++ ++ ++ + ++ ++

BDNF ++ ++ ++ ++ ++ ++ +++ +++ +++

NT-3 ++ − − + − − ++ +/− +/−

NT-4/5 ++ + + ++ +++ +++ ++ ++ ++

TGFb1 + + + +++ +++ +++ ++ ++ ++

GDNF + ++ ++ +++ +++ +++ ++ ++ −

The levels of mRNA expression and the protein production/secretion of NGF, BDNF, NT3, NT4/5, TGFb1, GDNF in nonstimulated microglia, lipopolysaccharide
(LPS)-stimulated microglia, and neuronal CM-stimulated microglia are summarized.

680 • NEUROGLIA
4.3 E X T R AC EL LU L A R P ROT EO LY T I C AC T I VIT Y
In the axotomized facial nucleus, microglia change their mor-
phology, proliferate, and migrate to the motoneuron cell bod-
ies, and astrocytes also become hypertrophic and extend their
processes to form lamellar sheets (Kreutzberg 1996). These
events represent the occurrence of significant tissue remodel-
ing in the axotomized facial nucleus, and involve an associa-
tion of proteinases which stimulate tissue rearrangement by
degrading extracellular proteins. One of the proteases shown
to play an important role in the tissue remodeling is plasmin
(Dano et al. 1985). Inactive proenzyme plasminogen (PLGn)
is changed into an active proteinase plasmin by plasminogen
activator (PA). PA is of two types: urokinase-type PA (uPA)
and tissue-type PA (tPA).
Tissue zymography of brainstem sections reveals that uPA
activity is induced in the axotomized facial nucleus (Nakajima
et al. 1996). The uPA activity increases consistent with the
time of microglial proliferation (Nakajima et al. 2004). This
phenomenon suggests the presence of an interaction between
injured motoneurons and microglia. An in vitro study revealed
that the amounts of uPA released from microglia are enhanced
by coculture with neurons or by neuronal CM (Nakajima et al.
2005). Thus, uPA induced in the axotomized facial nucleus
is considered to be produced in microglia stimulated with
injured motoneurons. A substrate for uPA and tPA, PLGn,
was demonstrated to be produced in microglia (Nakajima
et al. 1992). Thus, microglia are important cells for generating
plasmin in the CNS.
A plasmin-generating system (PLGn/PAs system) par-
ticipates in remodeling of the nervous system, including in
processes such as degradation of the extracellular matrix, and
transformation, activation, proliferation, and migration of
glial cells (Nakajima and Kohsaka 1996). At the same time,
plasmin activates inactive proforms such as procollagenase
(He et al. 1989), pro-uPA (Blasi et al. 1987), and pro-IL-8
(Nakagawa et al. 1991). Furthermore, plasmin is prerequisite
for activating latent TGFβ (Annes et al. 2003) and proNGF/
proBDNF (Lu et al. 2005). Thus, through the plasmin-gen-
erating ability, activated and proliferating microglia are sug-
gested to contribute to tissue remodeling in the injured facial
nucleus.
5 R E S P O N S E S O F M I C R O G L I A TO
M OTO N E U R O N C E L L D E AT H
The response of microglia to motoneuron cell death is dif-
ferent from the response to injured but living motoneurons.
The motoneuron cell death can be seen in the transected facial
nucleus in newborn rats (Aldskogius and Thomander 1986;
Graeber et al. 1998), in the ricin-administered facial nucleus
in adult rats (Streit and Kreutzberg 1988), and in the Cholera
toxin-applied nucleus (Llewellyn-Smith et al. 2000). In these
situations, the microglia were transformed into phagocytes.
This response is an essential process to remove needless struc-
tures in advance of the reconstruction of cellular connections
in the facial nucleus. An indicator of the phagocytes is CD68
(ED-1), which is a lysosomal protein (Damoiseaux 1994).
I N T E R AC T I O N O F M I C R O G L I A W IT H N EU R O N S A N D A ST R O C Y T E S U N D E R L E S I O N E D N EU R O NA L C O N D IT I O N S
The expression in the cells tells us that the cells are function-
ally in a phagocytic state.
5.1 N EWB O R N R ATS
The transection of newborn rat facial nerve leads to the
appearance of ED1-positive cells in the ipsilateral nucleus
(Graeber et al. 1998). In the same study, the number of ED1-
positive cells increased. Because the induction of the ED1
antigen is associated with the formation of phagosomes, this
finding demonstrated that most of the motoneurons had died.
The ED1-positive cells are microglia, which are identified by
Iba1 staining. In addition, some of the phagocytic microglia
were proliferating and major histocompatibility complex
class II-positive. Similar to the facial nerve transection model,
motoneuronal cell death is observed in the transected newborn
rat hypoglossal nucleus (Snider, 1989) and in the newborn rat
sciatic nerve transection paradigm (Iwasaki et al. 1995).
5.2 A D U LT R ATS
Motoneurons in the adult rat facial nucleus undergo cell death,
and microglia transform to phagocytes when the facial nerve
is transected and ricin is administered to the transection site or
injected into the facial nerve (Streit and Kreutzberg 1988). In
addition to these models, cycloheximide induces phagocytes
in the ipsilateral facial nucleus (Fig. 53.4). In the transected
A
ED-1
Actin
L R L R L R
Rat 1 Rat 2 Rat 3
B ED-1
Figure 53.4 Phagocytes Induced by Cycloheximide. A. Expression of
the ED-1 protein. The first rat did not receive an operation (Rat-1). In
the second adult rat, the right facial nerve was transected (Rat-2). In
the third adult rat, the right facial nerve was cut, and a gelform soaked
in cycloheximide (CH) solution was administered at the injury site
(Rat 3). After 5 days, each facial nucleus of the three rats was dissected,
and the tissue homogenate was immunoblotted for ED-1 and actin.
B. Immunohistochemistry for ED-1. Tissue sections of brainstem of
Rat-3 in A were stained with ED-1 antibody. C, control side; T+CH,
transected and CH-treated side.
• 681
facial nucleus without treatment, ED1-positive cells are not
observed. However, the ED1-positive cells appeared in the
transected and cycloheximide-treated facial nucleus. These
cells were PCNA-positive and cFms-positive, suggesting a
state of proliferation.
Dying neurons would be expected to express the “eat me”
signal (Elward and Gasque 2003) on the neuronal membrane.
Receiving the signals by scavenger receptor and T cell immu-
noglobin mucin 4, microglia internalize the dead neurons
by phagocytosis. The microglia are ED1-positive phagocytic
cells (Kinchen and Ravichandran 2008). As a matter of fact,
the phagocytic activity is regulated by chemokines and neu-
rotransmitters, like ATP (Koizumi et al. 2007).
6 R E S P O N S E S O F A S T R O C Y T E S TO
M OTO N E U R O N I N S U LT
6.1 R E S P O N S I V E N E S S O F A S T RO C Y T E S
Microglia are not the only cell type that responds to neuronal
insult. In the facial nerve transection model, astrocytes also
undergo morphological changes and functional transforma-
tions. One of the responses is morphological change from a
protoplasmic form to a fibrous form (Graeber and Kreutzberg
1986). One of the cellular constituents upregulated in astro-
cytes is glial fibrillary acidic protein (GFAP), which is an
astrocyte-specific intermediate protein and never expressed
in neurons and microglia. Although the level of the inter-
mediate protein is low in astrocytes under a normal state,
the level increases in astrocytes in the facial nucleus after the
transection, and the elevated levels continue for a long time
(Graeber and Kreutzberg 1988). Astrocytes in this state are
known as reactive astrocytes or activated astrocytes. The func-
tional significance of GFAP in activated astrocytes is associ-
ated with their hypertrophy and lamella formation. Analysis
of GFAP-knockout mice suggested that GFAP is neces-
sary to physically support the cellular construction in astro-
cytes (Liedtke et al. 1996). In addition, a role of GFAP as an
inhibitory barrier against axonal regeneration was reported in
GFAP/vimentin-deficient mice (Wilhelmsson et al. 2004).
In the CNS, astrocytes respond to neuronal damage
including trauma, ischemia, and chemicals, and undergo pro-
liferation leading to astrogliosis (Zhang et al. 2010). However,
astrocytes do not proliferate in the transected adult rat facial
nucleus. A similar phenomenon is observed in the adult hypo-
glossal nucleus (Svensson et al. 1994). In contrast to the adult,
in the neonatal rats astrocytes proliferate in response to facial
nerve lesions (Graeber et al. 1998).
Some molecules are known to be promoted in astrocytes in
response to neuronal injury. Plasminogen activator inhibitor-1
(PAI-1), which regulates PA activity, is induced in astrocytes
in the transected facial nucleus (Reddington et al. 1994).
Protein tyrosine phosphatase SHP1 is enhanced in astrocytes
as well as microglia in the injured facial nucleus (Horvat et al.
2001). In the transected hypoglossal nucleus, apolipoprotein J
is augmented in astrocytes (Svensson et al. 1995). Sciatic nerve
transection results in the elevation of N-methyl D-aspartate
682 • NEUROGLIA
receptor in astrocytes (Popratiloff et al. 1996). These upregu-
lated molecules may be associated with the cellular and bio-
chemical events necessary for tissue remodeling in the injured
facial nucleus.
The influence of these activated astrocytes on neurons is
important for the survival and restoration of neuronal func-
tion. Generally, the activated astrocytes have been viewed as
neuroprotective cells for supporting injured neurons by the
production of neurotrophic factors (Dong and Benveniste
2001). The capacity has been ascertained in many in vitro stud-
ies. Overexpression of GDNF by astrocytes prevents the pro-
grammed cell death of motoneurons (Oppenheim et al. 2000)
and the axotomy-induced motoneuron cell death (Zhao et al.
2004). These results strongly suggest that the neurotrophic
factors produced in astrocytes are actually delivered to the
region surrounding the neurons in the injured peripheral ner-
vous system.
From the viewpoint of glutamate toxicity, astrocytes can be
regarded as a glutamate eliminator. Activated astrocytes at the
lesion site express highly active uptake system for glutamate,
namely glutamate transporters (GLAST, GLT-1) (Mcnaugt
and Jenner 2000). The glutamate taken up into astrocytes is
converted to glutamine by glutamine synthetase (Brusilow
et al. 2010). Thus, astrocytes upon their activation are thought
to act as glutamate eliminators. In addition to the glutamate-
uptake capacity, astrocytes have an ability to protect against
the neuronal cell death induced by oxidative stress (Oshiro
et al. 2008). Accordingly, activated astrocytes in the injured
facial nucleus are suggested to play a major role in neuropro-
tection by releasing neurotrophic factors, and by eliminating
glutamate toxicity and oxidative stress.
6.2 I N T E R AC T I O N B ET WE E N M I C RO G L I A
A N D A S T RO C Y T E S
As seen above, both astrocytes and microglia respond to neu-
ronal injury, and they interact for the functional restoration of
neurons and tissue remodeling. The finding that the number
of astrocytes in the transected facial nucleus of osteopetrotic
mice was less than that of control mice suggests the presence
of an interaction between astrocytes and microglia (Raivich
et al. 1994). Other relevant phenomena include the associa-
tion of the microglial cell number with astroglial prolifera-
tion in an animal model (Miller and McAllister 2007), and
that the astrogliosis caused by microglia in vitro (Rohl et al.
2007) leads to an intercellular interaction between astrocytes
and microglia. Therefore, astrocytes and microglia appear to
form an activation loop in which one cell type–derived sig-
nal further stimulates the other cell type. The mediators in
the pathological state are predicted to be soluble molecules
including proinflammatory cytokines, chemokines, proteases,
nucleotides, and radicals, although many of the active materi-
als are not accurately identified yet.
There are some reports that astrocytes activate or regu-
late microglial properties/functions. In a study by Rohl and
Sievers (2005), astrocytes stimulated microglia to enhance
NO production in a coculture system with trimethyltin, sug-
gesting that astrocytes potentiate the trimethyltin-triggered
microglial activation. A certain soluble molecule(s) is specu-
lated to be the effective stimulator. Shih et al. (2006) reported
that astrocytes attenuate microglial activation by stimulating
their Nrf2-dependent antioxidant gene expressions, includ-
ing heme oxygenase-1, glutathione synthesis enzymes, and
superoxide dismutase expression, indicating that astrocytes
contribute to the regulation of microglia-triggered inflam-
mation. Astrocytes in vitro suppressed the phagocytic activ-
ity of microglia in a coculture system containing senile plaque
cores. The responsible molecules are thought to be diffusible
factor(s) (DeWitt et al. 1998).
On the other hand, in some cases microglia were shown
to be able to regulate astrocytic properties by releasing sol-
uble molecules. Microglia-derived PLGn induced PAI-1 in
astrocytes in vitro (Liu et al, 2000). Because PLGn and uPA
are produced in microglia and PAI-1 is produced in astro-
cytes (Reddington et al. 1994), a plasmin-generating system
could be regulated through the cellular interaction between
microglia and astrocytes. Similarly, microglia-derived PLGn
enhanced the production of TGFβ3, but not TGFβ1 or
TGFβ2, in astrocytes (Maeda et al. 2009). The TGFβs are
known to regulate the survival of neurons (Makwana et al.
2007). The neuroprotective function of astrocytes might be
regulated by microglial stimulus in vivo.
7 S U M M A RY A N D P E R S P E C T I VE S
Peripheral nerve injury leads to motoneuron downregulation,
microglial activation/proliferation, and astroglial activation
in the ipsilateral nucleus. In this chapter, the events occurring
in the injured motor nucleus were summarized (Fig. 53.5).
Activation
Proliferation
Astrocytes
tPA Microglia
PAI-1 uPA
PLGn
proTGFβ3
Plasmin Migration
Extracellular
proteolysis
Figure 53.5 Cellular Interaction Between Injured Motoneurons and Glial Cells. Activation, proliferation, enhanced neurotrophic action, association
with tissue remodeling of microglia, and astrocyte activation in the injured motor nucleus are depicted. The transformation of microglia into phago-
cytes in response to neuronal cell death is also shown.
I N T E R AC T I O N O F M I C R O G L I A W IT H N EU R O N S A N D A ST R O C Y T E S U N D E R L E S I O N E D N EU R O NA L C O N D IT I O N S
At an early time point following injury, the injured motoneu-
rons downregulated function-related molecules such as ChAT
and VAchT. Subsequently, the microglia were activated and pro-
liferated. In the facial nerve transection model, enhanced levels
of M-CSF were suggested to trigger the induction of cFms and
cyclinA/D in microglia to advance the cell cycle. The putative
roles of the proliferating microglia were suggested to include
serving as neurotrophic/neurosupportive cells, as glutamate
scavengers to prevent glutamate toxicity, and as a regulator of
tissue remodeling. On the other hand, in response to motoneu-
ronal death, microglia are activated, proliferate, and become
phagocytes. These cells were not essentially inflammatory.
In response to neuronal injury, astrocytes also become
activated and are suggested to act as neuroprotective cells.
Microglia and astrocytes are suggested to interact intimately
with each other by using certain mediators, and through their
crosstalk, they participate in neurosupportive actions and in
the tissue remodeling.
There are some points to be elucidated in the future. The
mechanism by which M-CSF is upregulated in microglia
stimulated with injured neurons is still uncertain. The signifi-
cance of proliferating microglia in the injured nervous system
should be clarified in vivo. Furthermore, the mechanism by
which microglia change into phagocytes in vivo, and the prop-
erties of phagocytic microglia remain to be elucidated. And
finally, the astrocytic response to motoneuron insult, and the
interaction between astrocytes and microglia in injured ner-
vous system will need to be analyzed in detail. These studies
will lead to a better understanding of the role of activated/
proliferating microglia, phagocytic microglia, and activated
astrocytes, and of the cellular interaction between astrocytes
and microglia in the lesioned nervous system.
Activation
Astrocytes
Microglia
Cell death
Microglia
Injured motoneuron Phagocytes
Ach↓
ChAT↓
VAchT↓ Microglia
Neurotrophic
NGF action
BNDF
NT-4/5
Glutamate TGFβ1
Microglia scavenger GDNF
• 683
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 54.
SCHWANN CELLS AND INJURY
Violetta Zujovic and Alexandros A. Lavdas

A B B R E VI AT I O N S of immune response, the support of axonal regeneration, and an

important actor in peripheral and central regeneration processes.
ADAM a disintegrin and metalloprotease Furthermore, we will also give an overview of all the pathologies
BACE β-site amyloid precursor protein cleaving that result from SC dysfunction.
enzyme
BDNF brain-derived neurotrophic factor
BM bone marrow 2 D E VE L O PM E N T
BMP bone morphogenic protein
Ccnd1 cyclinD1 Because a specific chapter (chapter 14) is dedicated to SC devel-
CMT Charcot-Marie-Tooth disease opment, we will give a general brief overview of all the features
CNS central nervous system of SC development that will be useful in understanding the pro-
DRG dorsal root ganglia cesses SCs go through during nerve degeneration-regeneration.
EAE experimental allergic encephalomyelitis
FGF fibroblast growth factor 2.1 T R A NS C R I P T I O N FAC TO R S
GFP green fluorescent protein
IFN interferon The embryonic phase of SC development involves three major
IL interleukin transitions. The transition from migrating neural crest cells to
LPS lipopolysaccharide SC precursor, the differentiation of SC precursors into imma-
MHC major histocompatibility complex ture SCs, and their final maturation to either myelinating or
miRNA micro RNA nonmyelinating SCs.
MS multiple sclerosis The time and space regulation of cell progression through SC
MOG myelin oligodendrocyte glycoprotein lineage is based on the balance of expression of transcription fac-
NGF nerve growth factor tors that can be divided in two groups, the negative and the posi-
NRG neuregulin tive regulators of myelination. Transcription factors such as Sox
NT neurotrophin (SRY-related HMG-box) 2, c-jun, Pax 3, Id2, Id 4, and Notch
OL oligodendrocytes signaling are designated as negative regulators, and Sox 10, Oct 6,
P0 peripheral myelin protein 0 and Krox 20 as positive regulators (see Fig. 54.1; chapter 14; and
PI-BMDC proinsulin-producing bone marrow derived Jessen and Mirsky 2008).
cells Recent data report that SC differentiation is also regulated
PLA2 phospholipase A2 at the epigenetic level. When micro RNA (miRNA), small non-
PNS peripheral nervous system coding RNA that bind to target RNA and inhibit their tran-
SC Schwann cell scription, are deleted in SCs by the specific ablation of DICER,
TACE TNFα-converting enzyme the SCs are “stuck” at the promyelinating stage (Yun et al. 2010).
TGF tumor growth factor Negative regulators of myelination, such as Sox 2, c-jun, and
TLR Toll-like receptors cyclin D1 are upregulated in SC-dicer mutants, whereas Krox 20
TNF tumor necrosis factor is dramatically downregulated (Yun et al. 2010). Thus, miRNA-
YFP yellow fluorescent protein 138 silencing results in an increase of c-jun and Sox 2. It appears
that miRNA are involved in promoting and stabilizing the dif-
ferentiated SC phenotype (Wienholds and Plasterk 2005).
1 INTRODUCTION
2.2 AXO NA L S I G NA L I N G
Schwann cells, the myelinating cells of the peripheral nervous
system (PNS), have attracted intense interest in the last decades, During development, the control of PNS myelination is
both because of their role in various pathological conditions and instructed by axonal signals, the most prominent being the neu-
because of their contribution to nervous system regeneration. regulins (NRGs), which are crucial at several stages of SC dif-
In this chapter, we document the numerous pleiotropic roles of ferentiation process (Jessen and Mirsky 2005). Neuregulins are
Schwann cells (SCs) particularly in repair processes such as being a family of growth factors with four different isoforms that act
a sensor of axonal integrity and injury, the activator and regulator through erbB tyrosine kinase receptors (Birchmeier and Nave
687
Figure 54.1 The balance between positive (red) and negative (green) regulators of SC program directs SC fate toward a myelinated or un/dedifferenti-
ated state. Sox 10 and Oct6 act in synergy to induce the expression of Krox 20 (purple arrow). High Krox 20 expression results in the triggering of the
myelination process along with the inhibition of negative regulators (yellow arrow). However after an injury, the reexpression of negative regulator
redirects SCs to an undifferentiated state. Micro RNA(miR)s stabilize the differentiated phenotype by inhibiting the expression of several negative
regulators and controlling the levels of Krox20.

2008). Axonally derived NRG1 signals via erbB2 and erbB3
receptor heterodimers present on cells of the SC lineage and is
required for SC precursor emergence, migration into the periph-
eral nerve, and survival before differentiation into immature SCs
(see chapter 14).
phagocytose extracellular debris resulting from dying or dam-
aged cells by activating the phospholipase A2 family of enzymes
(Martini et al. 2008). Another protease, the matrix metallopro-
tease 9, is also expressed by SCs after peripheral nerve injury and
is involved in both leukocyte migration and myelin breakdown
(Kobayashi et al. 2008).

3 S C H WA N N C E L L S ’ R O L E I N P N S I N J U RY
3.1 T H E MU LT ITA S K I N G AC T I VIT Y O F
S C H WA N N C E L L S I N P NS I N JU RY
Schwann cells are the first sensors of axonal damage and
react quickly to peripheral nerve injury by orchestrating the
immune response, clearing myelin debris, supporting axonal
regeneration, and finally remyelinating regenerated axons (for
overview see Fig. 54.2).
3.1.1 Phagocytic Activity/Immune Mediation
Schwann cell reaction to PNS injury is mediated through the
activation of Toll-like receptors (TLRs). The family of TLRs
belongs to the group of pattern-recognition receptors that recog-
nize specific conserved components of microorganisms (Takeda
et al. 2003) and they play a critical role in various inflammatory
disorders (Cook et al. 2004). Indeed, SCs express a variety of
TLRs such as TLR2, TLR3, TLR4, TLR7 constitutively, and
TLR1, which is upregulated after nerve axotomy (Goethals et
al. 2010). Axonal injury results in the liberation of TLR ligand
that will in turn activate the production by SCs of cytokines
and chemokines that will amplify and fine tune the inflamma-
tory response (Pineau et al. 2009). Indeed, the release of tumor
necrosis factor (TNF)α, interleukin (IL)α, ILβ, and CCL2
and CCL3 (Perrin et al. 2005) by SCs will trigger macrophage
recruitment to the site of injury, macrophages that will support
SC action in clearing myelin debris.
Denervated SCs are the major phagocytic cells for the first
days after injury. They can degrade their own myelin but also
3.1.2 Dedifferentiation/Peripheral
Nervous System Stem Cells
As in development, SC dedifferentiation is dependent on the
balance between positive and negative regulators of myelina-
tion (see Fig. 54.1). Upon nerve injury, negative regulators of
myelination such as Notch1, Ccnd1, c-jun, Sox2, Id 4, and Id2
are upregulated and drive SC dedifferentiation to a phenotype
similar to, but distinct from, that of immature SCs (Mirsky
et al. 2008; also chapter 14). One of the first negative regula-
tors of myelination to have been described is c-jun. The specific
deletion of c-jun in SCs leads to delay in SC dedifferentiation,
phagocytic activity, and a loss of axon regenerative ability.
Sharing the same expression pattern as c-jun is sox2, which is
also upregulated after injury. In a very elegant study, Woodhoo
et al. (2009) successfully deciphered the role of Notch signal-
ing in the SC dedifferentiation process. This study notably
provided evidence that when Notch is ectopically activated in
noninjured nerve, it is sufficient to induce myelin breakdown
and provoke SC dedifferentiation. It is the first study to dem-
onstrate that overexpression of a negative regulator is sufficient
to overcome Krox 20 inhibition and control SC plasticity.
The injury related changes that occur in dedifferentiated
SCs are also regulated at the epigenetic level. Indeed, immedi-
ately after injury most SC miRNAs are downregulated (Viader
et al. 2011). This allows the transcription of negative regula-
tors of myelination and the engagement of the dedifferentia-
tion process. When Dicer expression is specifically deleted in
SCs, there is a delay in the transition from myelinating SCs
to dedifferentiated SCs and from dedifferentiated SCs to

688 • ROLE OF GLIAL CELLS IN DISEASE

Figure 54.2 Overview of SC Participation in PNS Regeneration. After peripheral injury, SCs dedifferentiate, exert phagocytic activity, and regulate the
immune response. Once myelin and axonal debris are cleared, they form bands of Büngner, columns of cells that recreate a microenvironment permis-
sive for axonal regeneration. It is only when the axons have reinnervated their targets that SCs reacquire a remyelinating phenotype.

remyelinating SCs; indicating that SCs respond less efficiently
to injury when miRNA are absent. Among the miRNA high-
lighted in this study, miR-34a and miR-140 represent interest-
ing targets. Note that miR-34 interacts and inhibits cyclinD1
(Ccnd1) transcription and interferes with Notch signaling,
whereas miR-140 maintains Krox 20 expression to a specific
functional level.
While the dedifferentiation process of myelinating SCs
has been thoroughly studied, one should consider also the
participation of PNS stem cells in the regenerative process.
In recent years, two studies provided evidence for the per-
sistence of neural crest–derived stem cells in adult dorsal
root ganglia (DRG) (Li et al. 2007; Nagoshi et al. 2008).
Using lineage tracing with peripheral myelin protein 0 (P0)
and Wnt1-Cre/Floxed EGFP mice, Nagoshi and colleagues
demonstrated that the DRG-derived neural crest stem cells
showed a great ability to form secondary spheres and a high
multipotency, giving rise to both neuronal and glial cells.
However their participation in the PNS repair process
remains to be assessed.
3.1.3 Axonal Growth Support: Extracellular
Matrix/Neurotrophins
Next, the dedifferentiated SCs form Büngner bands and
express surface molecules that guide regenerating fibers. The
ability of SCs to support axonal regeneration is because of mul-
tiple factors. First, SCs produce extracellular matrix molecules
such as laminin and fibronectin that promote axonal growth
(Baron-Van Evercooren et al. 1982a). These molecules provide
a molecular link between the extracellular matrix (ECM), SCs,
and neuronal cytoskeleton through their interaction with their
receptors (integrins, dystroglycan) present at the growth cone.
Fibronectin is expressed at low levels in the SC basement mem-
brane, but is rapidly upregulated following PNS injury (Lefcort
et al. 1992). Fibronectin acts as a substratum for migrating
SCs (Baron-Van Evercooren et al. 1982b) and provides also
a direct substrate for adhesion and outgrowth of regenerat-
ing axons (Gardiner 2011). Schwann cells produce a specific
embryonic isoform of fibronectin, that is best fitted to interact
with α4β1 integrins present in the growth cone of regenerating
axons (Baron-Van Evercooren et al. 1982b; Vogelezang et al.
2001). Other ECM molecules that are upregulated after injury
include the laminins. Two types of laminins are expressed in
the intact nerve, laminin 2 composed of the α2β1γ1 subunits
and laminin 8 composed of the α4β1 γ1 subunits (Patton et
al. 1997); but after peripheral injury an increase of the α2, α4,
β1, and γ1 chains is observed (Wallquist et al. 2002). Their
essential role in nerve regeneration was shown by a series of
blocking experiments using function-blocking antibodies for
α2 laminin (Agius and Cochard 1998) and conditional knock
out for the γ1 laminin subunit (Chen and Strickland 2003),
both resulting in the inhibition of axonal growth (Gardiner
2011). But successful axonal regeneration is also regulated by
the finely tuned timely expression of laminin receptors on
growth cone and SCs. Thus, following injury, α6β4 integrin
is down-regulated on SCs (Feltri et al. 1994) while α1β1 and
α5β1 are induced in growth cone and SCs during the axonal
regeneration phase (Lefcort et al. 1992; Siironen et al. 1992);
conversely the reexpression of α6β4 integrin is correlated to
the remyelination process and full assembly of SC basal lamina
(Feltri et al. 1994).

S C H WA N N C E L L S A N D I N J U RY • 689
 Furthermore, SCs support axonal growth also by expressing
multiple neurotrophins including nerve growth factor (NGF),
neurotrophin-3 (NT3), brain-derived neurotrophic factor
(BDNF), fibroblast growth factor (FGF), glial cell line-derived
neurotrophic factor and ciliary neurotrophic factor (Lavdas
et al. 2008), all known to be essential for axonal growth dur-
ing development. After nerve injury, dedifferentiated SCs of the
distal nerve stump upregulate the synthesis of NGF, BDNF, and
NT-4 but not NT-3 (Funakoshi et al. 1993). Neurotrophins are
able to promote axonal regrowth from proximal nerve stumps
individually as well as in synergistic combinations (Cao and
Shoichet 2003). These molecules act through two different
types of receptors: high affinity Trk receptor tyrosine kinases,
expressed more prominently on axons, and low affinity p75
neurotrophin receptors, which are also present on SCs (Chao
2003). Although dedifferentiated SCs produce these growth
factors, they also reexpress p75 after nerve injury (Funakoshi
et al. 1993), bind some of this protein, and in this way control
their concentration and availability to the axons. To act prop-
erly, the effect of neurotrophins has to be finely regulated both
in time and in quantity. For example, at low doses BDNF medi-
ates its effects via both p75 and Trk receptor, but when high
doses of BDNF are present the neurotrophin will preferentially
signal through the p75 receptor, whose activation will inhibit
axonal growth (Gordon et al. 2011) (see chapter 55).
3.1.4 Remyelination
Neuregulin-1 plays an essential role in the neuroregenerative
process. After peripheral nerve injury, there is dysregulation of
the NRG1 isoform and erbB receptors (Nicolino et al. 2009) that
might be one of the signals that instruct myelinating SCs to revert
to a more immature phenotype (Guertin et al. 2005). An elegant
recent study (Fricker et al. 2011) demonstrated that the targeted
deletion of axonal NRG1 results in slower axonal regeneration,
defects in remyelination, and incorrect reinnervation. The
amount of functional NRG1 present at the axonal surface
regulates the myelination and remyelination process. Interestingly,
two members of a disintegrin and metalloprotease (ADAM)
secretase family have been implicated in the regulation of NRG1
type III activity notably by regulating its processing. The levels
of active NRG1 are regulated by proteolysis by β secretase β-site
amyloid precursor protein cleaving enzyme (BACE) 1 and the
α-secretase TNFα-converting enzyme (TACE), which both
cleave NRG1type III. These two secretases have different cleavage
sites and have an opposite effect on myelination because BACE1
is a positive regulator of myelination and remyelination (Hu
et al. 2006; Shirakabe et al. 2001), whereas TACE is a negative
regulator; these enzymes represent an interesting therapeutic
target because reducing TACE activity might increase the
remyelination process (La Marca et al. 2011).
To engage the remyelination process, the induction of
myelin genes has to be coordinated with high levels of lipid
synthesis required for the production of multi-layered choles-
terol rich membranes. Gene expression profiling revealed that
various lipid biosynthesis genes are upregulated after peripheral
nerve injury (Verheijen et al. 2003). One of the key regulators
of fatty acid and cholesterol biosynthesis in SCs is the sterol
690 • ROLE OF GLIAL CELLS IN DISEASE
regulatory element binding protein (SREBP). Leblanc and col-
leagues demonstrated that in SCs, Krox20 and SREBP trans-
activators control several target gene promoters such as HMG
CoA reductase, a gene essential for cholesterol biosynthesis
(Leblanc et al. 2005). Its role in myelination is further con-
firmed by SC-specific deletion of SREBP activation (Verheijen
et al. 2009) and mutations perturbing cholesterol biosynthesis
result in severe hypomyelination (Saher et al. 2009).
3.2 S C H WA N N C E L L S I N P E R I P H E R A L N E RVO US
S YS T E M D E MY E L I NAT I N G D I S O R D E R S
3.2.1 Autoimmunity Related: Inflammation
and Cytokines
Human inflammatory neuropathies are the results of an
immune attack against PNS autoantigens. According to the
currently accepted model, even under normal conditions
there may be autoreactive T lymphocytes recognizing
epitopes on the peripheral nerve, but their continuous
suppression ensures self-tolerance. During an infection,
these lymphocytes become activated upon encountering
microbial epitopes. These epitopes can resemble endogenous
peripheral nerve antigens, a situation termed molecular
mimicry (Yuki et al. 1993). For instance, in patients with
Guillain–Barré syndrome, one of the most serious forms
of autoimmune neuropathies, epitopes shared between
Campylobacter jejuni, cytomegalovirus, or Haemophilus
influenzae and nerve fibers have been identified (Kieseier
et al. 2004). In this situation, autoreactive T lymphocytes
stimulate B cells to produce autoantibodies, which in turn
block nerve conduction, activate complement, and trigger
macrophage attack against peripheral nerve constituents
(Sawant-Mane et al. 1996; Takigawa et al. 2000). Activated
T cells cross the blood-nerve barrier and provoke local
inflammation by proinflammatory cytokines (Kieseier et al.
2006). Attracted macrophages act as antigen-presenting cells,
release toxic mediators, and directly damage myelinating
SCs and axons (Kiefer et al. 2001). Functional consequences
are mainly determined by the degree of axonal degeneration.
The immune response may eventually be terminated by
increased T-cell apoptosis, as shown in animal models of
inflammatory neuropathies (Gold et al. 1997). Why has the
problem of molecular mimicry not been resolved through
evolutionary selective processes? Having to defend the
body from as many invaders as possible, the immune system
works like a double-edged sword. It may be that the current
situation represents the optimum balance between response
against invading pathogens and autoimmunity.
Toll-like receptors are usually found on antigen-presenting
cells, such as dendritic cells, but have also been found on SCs
(see 3.1.1). One of these molecules, TLR-2, expressed on pri-
mary human and rat SCs, is considered to be a target recep-
tor for Mycobacterium leprae (Harboe et al. 2005). Expression
of various TLRs is inducible on rat SCs in vitro. For example,
stimulation of TLR-4 on SCs with lipopolysaccharides (LPS)
elicits the production of inflammatory mediators such as pro-
tease inhibitors, chemokines, and growth factors (Qin et al.
2012; Shamash et al. 2002), suggesting that SCs can detect LPS
fragments and may act as a link between innate and acquired
immunity via TLR-4 activation in the inflamed PNS.
The presentation of antigens of cell-associated microorgan-
isms for recognition by T lymphocytes is performed by mol-
ecules of the major histocompatibility complex (MHC). Two
types of MHC gene products can be distinguished, class I and
class II MHC molecules, which differ both functionally and
in terms of cell expression, with all nucleated cells exhibiting
MHC class I molecules and only macrophages, dendritic cells,
and B lymphocytes expressing MHC class II molecules. The
levels of both class I and II molecules can be upregulated by
cytokines, particularly interferon (IFN)-γ and TNF-α. Other
cell types may acquire the ability to process and present anti-
gens in inflammatory conditions, thus representing facultative
antigen-presenting cells. Human and rat SCs in vitro have been
shown to constitutively express low levels of MHC class I; in
the presence of activated T lymphocytes and upon stimulation
with IFN-γ and TNF-α, significant amounts of MHC class II
molecules can also be detected (Gold et al. 1995). Also, SCs
stain positive for MHC class II in human nerve biopsies from
patients with Guillain–Barré syndrome, suggesting that these
cells may indeed act as facultative antigen-presenting cells
(Pollard et al. 1986). Schwann cells in vitro have been shown
to present antigens to antigen-specific syngeneic T-cell lines
(Wekerle et al. 1986). Mycobacterium leprae readily invades and
proliferates in nonmyelinating SCs (Rambukkana et al. 2002);
myelinated SCs, on the other hand, are resistant to M. leprae
invasion but undergo demyelination and axonal damage upon
attachment to SC axon units, thus creating more fertile ground
for the microbe expansion (Rambukkana 2004). Schwann cells
have been shown to present M. leprae-derived antigens to lym-
phocytes in an MHC II-dependent manner (Ford et al. 1993).
A large body of evidence has accumulated over the last two
decades implying that SCs can also produce and secrete a wide
variety of cytokines, which could act as immunomodulators.
Cultured SCs have been shown to be able to produce IL-1, a
cytokine involved in the initiation of an immune response.
Other proinflammatory cytokines, such as IL-6, TNF-α, and
TGF-β are generated and released by SCs under certain con-
ditions, either in vitro or in vivo (Lisak et al. 1997).
Proinflammatory cytokine production by SCs has also been
shown to be regulated in an autocrine fashion (Qin et al. 2008).
The specific receptors for some of the cytokines, such as TNF
receptor, are constitutively expressed on SCs, whereas other
proinflammatory and immunoregulatory mediators, such as
prostaglandin E2, thromboxane A2, and leukotriene C4, are syn-
thesized in large amounts by SCs, and may regulate the immune
cascade within the inflamed PNS (Bonetti et al. 2000).
Schwann cells can also modulate immune reactivity by
humoral mechanisms. One such pathway is through the produc-
tion and release of nitric oxide. Schwann cells possess inducible
nitrite oxide synthase, which is upregulated after stimulation
with proinflammatory cytokines (Wohlleben et al. 1999).
Finally, SCs may be involved in the termination of auto-
immune response in the PNS. The survival of lymphocytes
depends on a delicate balance between proapoptotic and
antiapoptotic factors. Lymphocyte apoptosis can be regu-
lated through the interaction of the cell surface receptor
S C H WA N N C E L L S A N D I N J U RY
Fas on T cells with its ligand, FasL, a member of the TNF
family (Krammer 2000). This ligand has been shown to be
expressed on SC surface after stimulation with proinflamma-
tory cytokines in vitro, and functional analysis has suggested
that the interaction between Fas on T cells and FasL on SCs
can promote apoptosis of T lymphocytes. Interestingly, FasL
can also act as a signal-transducing molecule in SCs, leading to
the secretion of NGF, and may thus contribute to peripheral
nerve regeneration (Mimouni-Rongy et al. 2011).
3.2.2 Myelin Malformation-Related: Genetic Causes
Charcot-Marie-Tooth (CMT) disease is a term used for
inherited peripheral neuropathies, after the names of the three
investigators who described them in the late 19th century.
In most cases, CMT is associated with autosomal dominant
inheritance, although a small proportion of X-linked domi-
nant and autosomal recessive forms also exist. In addition,
apparent sporadic cases occur, which may lead to new domi-
nantly inherited mutations (see chapter 62).
There is functional and anatomical evidence that axonal
degeneration is likely to be the result of abnormalities in
SC–axon interactions (Krajewski et al. 2000)). Trembler mice,
which have a dysmyelinating peripheral neuropathy caused by
a point mutation in pmp22 similar to CMT1A, have signifi-
cant changes in both axonal structure and function (Sahenk
et al. 1999).
Mutations of P0 myelin protein (see chapter 7) cause
another form of CMT, CMT1B (Li et al. 2006). There exist
many other, less common forms of CMT associated with dif-
ferent types of mutations. Further analysis of these will be pre-
sented in chapter 62.
Hereditary disorders of the PNS also trigger immune
responses in the peripheral nerve. These responses are consid-
ered to be important determinants of neuropathy. In CMT,
infiltration of lymphocytes and macrophages has been demon-
strated in peripheral nerves both in human patients (Vital et
al. 1992) and animal models (Schmid et al. 2000). However,
in addition to these secondary immune responses, a series
of studies have suggested an immunological involvement as
a primary cause for disease in some genetically determined
neuropathies. Mice with a heterozygous null mutation in P0
develop late-onset clinical paralysis associated with inflamma-
tory pathology in the peripheral nerves. Young P0(+/-) mice,
without behavioral symptoms, have a defect in central toler-
ance to P0 and are more prone to induction of experimental
autoimmune neuritis by sensitization against a P0 (180–199)
peptide (Miyamoto et al. 2003). The P0 gene was found to
be transcribed in the thymus of wild-type and the P0 (+/-)
mice in an amount proportional to the gene dosage. Thus, a
heterozygous mutation in an autoantigen could cause defective
central tolerance to this autoantigen. This finding may be rel-
evant to CMT1B, in which P0 is affected. Through this mech-
anism, autoimmune T cells may play some role in “genetic”
diseases caused by a heterozygous gene defect. Increased
immunogenicity owing to altered protein composition or
mechanical instability of the myelin sheath are also possible
explanations for the immunoreactive nature of the defective
• 691
myelin sheath. Molecules required for antigen presentation
could communicate this information from SCs to immune
cells, and it has been proposed that a myelin protein mutant
Schwann cell would not demyelinate if it lacked the molecules
required for antigen processing and presentation (Meyer zu
Horste et al. 2008)).
4 S C H WA N N C E L L S ’ R O L E I N C N S R E PA I R
In some pathological cases, SCs can participate in CNS
repair, in particular in the spinal cord (for an overview see
Table 54.1).
4.1 E N D O G E N O US S C H WA N N C E L L R E PA I R I N
C N S M E C H A N I C A L I N JU RY
Numerous studies have demonstrated the capacity of SCs to
remyelinate CNS axons and promote axonal regeneration in
the injured CNS (Hagg and Oudega 2006) (see also chap-
ter 55). Schwann cells’ myelinating central axons have been
detected at the edge of the lesion cavity after photochemical
thrombosis lesions in the rat (Bunge et al. 1994) and also after
contusion spinal injuries in the monkey (Bresnahan 1978),
whereas large numbers of SCs and axons have been reported
in long-term spinal injury sites in human studies (Bunge
1994). In contusion spinal injuries of the rat, significant axonal
sprouting and regeneration occur and the amount of this
regeneration is related, among other factors, to the amount of
SC invasion (Beattie et al. 1997). A recent study demonstrated
the peripheral origin of invading SCs using genetic-labeling
techniques for neural crest lineage cells (Nagoshi et al. 2011).
Furthermore, the study showed that spinal cord injury induces
the dedifferentiation of dorsal root SCs before their invasion
of the lesion.
However, endogenous SCs that invade the CNS after
injury are found mainly in areas lacking astrocytes, and
migration of SCs transplanted in rodent CNS white matter
is inhibited by astrocytes (Iwashita et al. 2000). When SCs
are grafted in the newborn CNS, in the presence of compet-
ing endogenous oligodendrocytes, astrocytes are able to limit
their migration and prevent myelination by the transplanted
SCs. In addition, astrocytes are able to form basement
membranes around the area of the graft, effectively exclud-
ing the grafted cells from the host parenchyma altogether
(Baron-Van Evercooren et al. 1992). In vitro studies with
cocultures of SCs and astrocytes have confirmed the mutual
exclusivity of the two cell types and attempted to interpret it
based on the presence of extracellular matrix molecules, with
no consensus reached yet (Ghirnikar and Eng 1995; Wilby
et al. 1999).
Schwann-cell–conditioned medium induces astrocyte
proliferation and production of chondroitin sulfate proteo-
glycan in a fibroblast growth factor receptor 1 (FGFR1)–
independent manner (Santos-Silva et al. 2007). In contrast,
stimulation of boundary formation, astrocytic hypertrophy
and induction of GFAP expression in astrocytes by SCs is
mediated in an FGF-dependent manner and is modulated
by another extracellular matrix proteoglycan, heparin sulfate
proteoglycan. Identification of the factors secreted by SCs
that induce these astrocytic responses will give us a better
understanding of the nature of astrocyte/SC interactions and
will point us in the right direction in our quest to manipulate
the inhibitory environment induced after injury to promote
regeneration (also see chapter 55).
Interestingly, another inhibitor of axonal regeneration,
semaphorin 3A, is repulsive for SCs in vitro and in vivo in a
model of spinal trauma. Treatment with a semaphorin 3A inhib-
itor was able to reverse this effect and increase SC-mediated
myelination and axonal regeneration (Kaneko et al. 2006).

Table 54.1 ENDOGENOUS SCHWANN CELL PARTICIPATION IN CENTRAL NERVOUS SYSTEM REPAIR

Humans Multiple sclerosis:

Feigin and Ogata 1971; Itoyama et al. 1983; Prineas and Connell 1979; Yamamoto et al. 1991
Neuromyelitis Optica:
Ikota et al. 2010
Spinal cord injury:
Buss et al. 2007
Animal models Development:
Focal irradiatiation of developing mouse spinal cord (Gilmore et al. 1983; Heard and Gilmore 1980)
Neuregulin 1 type III up-regulation in zebrafish CNS (Perlin et al. 2011)
Myelin Mutants:
Myelin deficient rat, taiep rat, canine shaking pup (Duncan and Hoffman 1997)
Demyelination:
Experimental allergic encephalomyelitis (Pender 1989)
Viral encephalomyelitis (Dal Canto and Lipton 1980; Miller et al. 1995)
Chemically induced demyelinating lesions in rodents (Blakemore et al. 1985) and macaques (Bachelin
et al. 2005; Lachapelle et al. 2005)
Spinal Cord Injury Models:
Photochemical thrombosis (Bunge et al. 1994)
Spinal cord injury (Bresnahan 1978; Nagoshi et al. 2011)

692 • ROLE OF GLIAL CELLS IN DISEASE

 4 .2 E N D O G E N O US S C H WA N N C E L L
R E MY E L I NAT I O N I N D E MY E L I NAT I N G
DISORDERS
4.2.1 Schwann Cell Participation in
the Remyelination Process in MS and
Demyelinating Animal Models
The dorsal root entry zone and motor axons exit represent
the transitional zone between the CNS and the PNS and
is composed of the glia limitans, dense astrocytic processes
covered by a basal lamina.
This boundary can be transgressed in demyelinating con-
ditions and the peripheral “invasion” of the CNS has been
observed in multiple sclerosis (MS) patients. Multiple scle-
rosis is a demyelinating disease in which successive inflam-
matory attacks result in the specific destruction of central
oligodendrocytes (OL) (see chapter 61). An interesting fea-
ture of MS is that during the development of the disease a
concomitant endogenous remyelination process is triggered.
In the CNS, this repair process is mainly conducted by OL
that form new myelin sheaths around the axons, but the
presence of SC remyelination of CNS axons is also detected
in MS lesions (Itoyama et al. 1983; Yamamoto et al. 1991).
Schwann cells invade the CNS mainly after the rupture of the
blood-brain barrier and their entry occurs in the optic nerve,
spinal cord, brainstem, and cerebellum (reviewed in (Duncan
and Hoffman 1997). Intraspinal expansion of SC remyelina-
tion was also detected in patients with Devic disease, a demy-
elinating disease affecting specifically the optic nerve and the
spinal cord (Ikota et al. 2010).
New insights into the demyelination-remyelination mech-
anisms of MS have been made by the development of a vari-
ety of toxin-induced animal lesion models (see Baron-Van
Evercooren and Blakemore 2004) (see also chapter 57). All
these lesions are totally remyelinated and present the same
pattern, with SC remyelination at the center of the lesion in
an astrocytic free area and a oligodendrocytic remyelination at
the border where astrocytes are present. Schwann-cell–derived
myelin restores axonal conduction (Felts and Smith 1992) and
severe neurological deficits can be reversed when massive SC
entry occurs in response to spinal cord demyelination ( Jasmin
et al. 2000). The recovery involves the establishment and main-
tenance of nodes of Ranvier by endogenous SCs for more than
a 1-year period, with nodes displaying normal distribution of
sodium and potassium channels (Black et al. 2006).
Although extremely useful, these models do not reflect the
complex environment of MS lesions. A preferred MS model is
experimental allergic encephalomyelitis (EAE), an inflamma-
tory demyelinating model induced by sensitizing the animals
with myelin-related proteins. As in MS lesions, SC-derived
myelin was detected in EAE models (Pender 1989) even after
longstanding disease duration.
4.2.2 Origin of Remyelinating Schwann Cells
Although the origin of remyelinating oligodendrocytes is well
described, the origin of remyelinating SCs remains a matter
of debate. Two major trends emerge from the literature, one
S C H WA N N C E L L S A N D I N J U RY
arguing that the SCs derive from central stem/precursor cells,
and the other considering that SC remyelination is the conse-
quence of SC invasion from the PNS.
A few years ago, the possibility of the transdifferentiation
of central precursors to peripheral myelinating cells arose with
a series of experiments. In vivo experiments using X-irradiated
ethidium bromide lesions, demonstrated that transplantation of
FGF-2 expanded polysialic acid neural cell adhesion molecule
(PSA-NCAM) glial restricted precursors (Keirstead et al. 1999),
myelin oligodendrocyte glycoprotein-expressing (MOG) oli-
godendrocyte precursors (Crang et al. 2004), or cloned human
neural precursors resulted in both SC and oligodendrocyte
remyelination (Akiyama et al. 2001). Recent data identified a
role for bone morphogenic protein (BMP) in instructing CNS
precursor into the SC fate. In vitro, BMP and FGF2 were found
to cooperate to induce a neural crest-like fate including SC dif-
ferentiation from fetal and adult neural stem cells (Sailer et al.
2005). In addition grafted OPCs were shown to differentiate
preferentially into SCs when in the presence of BMP (Talbott
et al. 2006). Depending on the environment they are trans-
planted in, neural stem/progenitor cells derived from the spinal
cord give rise to different cells (Mothe and Tator 2008). In the
adult shiverer mouse spinal cord, the neural stem/progenitor
cells generate oligodendrocytes, whereas in focal demyelinated
lesion of the spinal cord they differentiate in both oligodendro-
cytes and SCs. A recent study explored the origin of remyelinat-
ing SCs using transgenic Pdgfra-creERT2: Rosa26-YFP mice
that enabled the tracing of OL precursor cells (Zawadzka et al.
2010). They observed that after a demyelinating lesion of the
spinal cord, 30% of new peripheral myelin colocalizes with the
yellow fluorescent protein (YFP) reporter gene; providing evi-
dence that some SCs might have a central origin.
However the most favored hypothesis is that SCs come
from peripheral nerve roots in the spinal cord and small nerves
that innervate blood vessels in the brain, because SC remyeli-
nation typically occurs in proximity to spinal/cranial nerves
or around blood vessels (Duncan and Hoffman 1997). This is
likely to occur because injection of lentiviral vectors encoding
green fluorescent protein (GFP) in the dorsal roots near a spi-
nal cord contusion injury lead to the presence of GFP labeled
SCs in the lesion (Oudega and Xu 2006). This invasion of the
CNS by SCs might be the result of a transgression of the fron-
tier between the CNS and PNS, with possible permissive con-
ditions that might be induced by the demyelinated lesion. So,
the high SC remyelination observed in spinal cord of Devic
syndrome patients might be explained by the disturbance of
the astrocytes present at the PNS/CNS frontier (Ikota et al.
2010).
5 M ETA B O L I S M-I N D U C E D S C H WA N N
C E L L DA M AG E : F O C U S O N D I A B ET E S
MELLITUS
Neuropathy is the most common complication of diabetes,
occurring in over 50% of all diabetic patients (Feldman et al.
1997). A multitude of biochemical changes are associated with
diabetic neuropathy: oxidative stress (Vincent et al. 2004),
• 693
activation of the polyol pathway (Oates 2002), hypoxia and
ischemia (Low et al. 1989), activation of protein kinase C (Xia
et al. 1995), mitogen-activated protein kinases (Tomlinson
1999), and inducible nitric oxide synthase (Vareniuk et al.
2008), increased advanced glycation end products and their
receptors (Toth et al. 2008), and elevated cytokines such as
TNF (Yamakawa et al. 2011).
The participation of SCs in diabetic neuropathy is char-
acterized by degeneration of SCs and myelinated axons as
well as loss of a population of dorsal root ganglia neurons
(Schmeichel et al. 2003). The pathology of SCs in diabetes
is bad news for two reasons: it not only induces degenerative
phenomena, but it also makes regenerative processes more
difficult because these cells are key players during nerve
regeneration.
Biochemically, SCs are key players in hyperglycemia-
induced damage, because they express the enzyme aldose
reductase, which may be responsible for some of the harm-
ful effects of high glucose (Mizisin and Powell 1993). In
addition to the hyperglycemia-related mechanisms of nerve
injury, experimental and clinical studies have suggested that
dyslipidemia related to obesity and type 2 diabetes can also
play a role in the development and progression of peripheral
neuropathy (Obrosova et al. 2007).
Growth-factor–related regulation of SC function is also
affected. An enhanced expression of p75 neurotrophin recep-
tor is found on diabetic SCs and, because this receptor is
involved in an autocrine apoptotic loop, its increased expres-
sion may lead to increased cell loss (Eckersley et al. 2001).
An interesting twist to the story of diabetic neuropathy
has been added recently (Chan et al. 2011). These investigators
describe the appearance of proinsulin-producing cells in multi-
ple organs and tissues of streptozotocin-induced diabetic mice.
Close examination of these cells revealed that they first appear
in the bone marrow (BM) and eventually travel to different
peripheral organs and tissues through the circulation. The pro-
insulin-producing bone marrow-derived cells (PI-BMDCs)
appeared within one to three days of intermittent hyperglyce-
mia induced by repeated glucose injections. The initial conjec-
ture was that one could use such PI-BMDCs for diabetes cell
therapy, a thought reinforced by a report (Oh et al. 2004) that
incubation in high-glucose medium induces insulin-gene tran-
scription in BM cells in vitro and that these cells could be trans-
ferred to the kidney capsule of diabetic rodents and partially
ameliorate the hyperglycemia. However, it was found in vitro
that the tiny amounts of PI produced by the PI-BMDCs were
not detectable in the culture medium and thus PI-BMDCs
induced by exposure to a high glucose medium had no thera-
peutic potential. Quite the contrary, these cells played a nega-
tive role. In fact, PI-BMDCs are a subpopulation of highly
proinflammatory BMDCs specifically induced by diabetes
that are capable of fusing with cells in the nerves. They seem
to play a central role in the pathogenesis of diabetic neuropa-
thy and mediate much of the inflammation-related pathology
of diabetic complications. Importantly, these same cells have
great fusogenic potential that can be demonstrated under in
vitro conditions. They directly cause tissue damage by fusing
694 • ROLE OF GLIAL CELLS IN DISEASE
with both neurons and SCs are thus a major culprit in diabetic
neuropathy (Terashima et al. 2012).
6 S U M M A RY A N D P E R S P E C T I VE S
In this chapter, we discussed the various properties of SCs
that implicate them in pathological states; we saw that these
cells participate in the causative processes of a variety of con-
ditions that affect the PNS, but also constitute a most impor-
tant player in PNS regenerative processes. Last but not least,
we discussed their participation in CNS repair. The informa-
tion on SCs we have amassed over the last few decades allows
us not only to answer basic questions about their biology,
but also to plan realistic strategies for using SCs for cell ther-
apy. Their regeneration-supporting properties can be fully
exploited in the PNS in the construction of bioartificial nerve
grafts to bridge peripheral nerve gaps resulting from injury,
and their ability to support the regrowth and myelination of
central axons makes them prime candidates for cell replace-
ment therapy in the CNS, as will be discussed in chapter 57.
AC K N OW L E D G M E N T S
The authors would like to acknowledge all the former and
present members of their labs who contributed directly
or indirectly to the elaboration of the manuscript and in
particular Corinne Bachelin for her illustration skills. The
authors are also grateful to Anne Baron-Van Evercooren and
Rhona Mirsky for their helpful suggestions and comments
that greatly improved the quality of the manuscript. Also,
the FP7 EU project grant Neurosign/Regpot for financial
support.
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RECOVERY OF NEURAL FUNCTION

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 55.
NERVE REGENERATION IN THE PERIPHERAL
NERVOUS SYSTEM
Tessa Gordon and Olawale AR Sulaiman

A B B R E VI AT I O N S despite the regenerative capacity of the peripheral nervous system

and provide the rationale for experimental approaches to improve
BDNF brain-derived neurotrophic factor axonal regeneration and in turn, functional recovery.
cAMP cyclic adenosine monophosphate
ES electrical stimulation
2 P E R I P H E R A L N E RVE I N J U RY
FGF fibroblast growth factor
GDNF glial-derived neurotrophic factor
IGF insulin-like growth factor 2.1 T H E C E L L B O DY R E S P O NS E A N D T H E
N-CAM neural cell adhesion molecule G ROW T H M O D E O F I N J U R E D N EU RO N S
NGF nerve growth factor
NT3 neurotrophin 3
2.1.1 Chromatolysis and Gene Expression:
NT4/5 neurotrophin 4/5
PMP22 peripheral myelin protein 22 kDa Neurons that incur axonal disruption or axotomy with loss of
RAG regeneration-associated gene contact with target connection undergo characteristic morpho-
SC Schwann cell logical or “chromatolytic” changes. The morphological changes
TGF-β transforming growth factor beta include swelling, movement of the nucleus and nucleolus to an
VEGF vascular endothelial growth factor eccentric position in the cell body, and dissolution of Nissl bod-
ies that comprise the rough endoplasmic reticulum. They reflect
a marked increase in mRNA synthesis and the change in gene
1 INTRODUCTION expression that converts the neurons from the normally “trans-
mitting” to the “growth” mode (Fu and Gordon 1997). They
The capacity for injured neurons to regenerate their axons and include the upregulation of growth associated genes such as
remake functional connections with target organs distinguishes actin, tubulin, and GAP43 and downregulation of genes such as
the peripheral from the central nervous system. This unique neurofilament that is normally responsible for sustaining axon
characteristic of the peripheral nervous system stems from the diameter (Fig. 55.1).
ability of the Schwann cells (SCs) as opposed to the inability of
the oligodendrocytes in the central nervous system, to support 2.1.2 Altered Gene Expression Is Transient
axonal regeneration (see chapters 54 and 56). In consequence,
The conversion of the neuron from a transmitting to a growth
return of function after peripheral nerve injuries contrasts with
mode involves the downregulation of transmitter-encoding
permanent deficits associated with central nerve injuries. Yet,
genes that include choline acetyltransferase in motoneurons
functional recovery after nerve injuries in humans is generally
and the upregulation of neurotrophic factors and their receptors
poor despite the capacity of injured peripheral nerves to regenerate
(Boyd and Gordon 2003b; Fu and Gordon 1997). The altered
their axons. Minimal or no recovery may be the outcome for
gene expression occurs within days to reach a maximum within
injuries that sever the peripheral nerve far from the target and
weeks, the time course varying according to the particular genes
that incur considerable delays before target reinnervation. This is
that are expressed. However, the expression is not maintained
regardless of considerable advances made in microsurgical repair
with mRNA levels returning to baseline levels (Sulaiman et al.
of the injured nerves (Sunderland 1978). The processes of axonal
2011) (see Fig. 55.1).
regeneration and target reinnervation involve many factors that
pertain to the injured neuron, the growth environment of the
distal nerve stumps, and denervated targets. The neurons must 2.2 C O N VE R S I O N O F T H E D I S TA L N E RVE
survive the injuries to mount a regenerative response, regenerate S T UM P TO A G ROW T H P E R M I S S I VE
their axons within the growth environment of the denervated S TAT E A F T E R I N JU RY
distal nerve stump, and reinnervate the appropriate targets to
restore function. This chapter reviews the response of SCs to 2.2.1 Wallerian Degeneration
peripheral nerve injury in animal models. This information will Axons that are separated from the cell body by nerve injury
provide insight into why functional recovery is often so poor undergo Wallerian degeneration with ultimate phagocytosis of
701
 A GAP-43 ChAT

Tubulin AChE

Actin Neurofilament

Chromatolysis Axon Outgrowth Wallerian Degeneration

Neurotrophic Adhesion Myelination

Neurotrophic Neurotrophic factor & molecules, genes
factors factor receptors receptors mitogens/
receptors,
cytokines

B C
1.0 1.0
Gene expression

Gene expression
0.5 Neuron 0.5 Schwann cell
RAGs RAGs

0.0 0.0
07 100 200 07 100 200
Time after nerve injury (days) Time after nerve injury (days)
D E
1.2
Tubulin mRNA relative to 7d = 1

1600
*
% Change in GDNF mRNA

1.0 1400
1200 *
expression

0.8 1000
800 *
0.6 *
600
0.4 Intact 400
7 days 200
0.2 0
0 30 60 90 120 150 180 2d 7d 28d 90d 180d
Days after axotomy Days after chronic denervation

Figure 55.1 After nerve injury, regeneration-associated genes (RAGs) are upregulated transiently in the neurons (A,B) in concert with downregulation of
genes associated with normal synaptic transmission (A). Schwann cells in the denervated nerve stumps undergo proliferation during Wallerian degenera-
tion and express many RAGs in concert with downregulation of myelin-associated genes (A,C). The gene profiles support outgrowth of axons, but
their altered expression being transient, axonal growth declines with time as the growth response wanes and the growth support by Schwann cells
declines as the cells go into a senescent state. mRNA levels are plotted for tubulin in neurons (D) and GDNF in Schwann cells (E).

axonal and myelin debris by resident SCs and the macrophages and the ubiquitin-proteasome system (Zhai et al. 2003). The
that infiltrate the distal stump (Brushart 2011; Stoll and Muller axon debris releases mitogens that promote mitotic SC divi-
1999; Waller 1850) (see chapters 14 and 54) (Fig. 55.1). A sion and initiates a network of cytokines and transcription fac-
recent review breaks the events into three morphologically tors that stimulates myelin breakdown, macrophage invasion,
discernible stages: acute axonal degeneration, a latency period, and targets the myelin for phagocytosis by the SCs and mac-
and an abrupt granular degenerative stage (Wang et al. 2012). rophages (Brushart 2011; Fu and Gordon 1997). Schwann cell–
Within an hour of injury, “die back” in both the proximal derived neuregulins are not essential for this proliferative phase
and distal stumps is mediated by channel-mediated calcium (Atanasoski et al. 2006; Wang et al. 2012).
influx and activation of calpains that cleave neurofilament and The dividing SCs secrete many chemoattractive factors
microtubular-associated components such as spectrin and that include the cytokines, interleukin-1α, leukemia inhibi-
tubulin. In the peripheral nerve, this die back is to the first tory factor, and monocyte chemoattractant protein-1 (Fu and
node of Ranvier. The next stage of approximately 24 hours, Gordon 1997). They recruit macrophages into and through-
during which time action potentials continue to be conducted out the distal nerve stump, where they enhance SC prolifera-
(Miledi and Slater 1970) is followed by a longer period of dis- tion by releasing growth factors and assume the major role of
assembly of cytoskeletal proteins by calcium-activated calpains phagocytosis of axon and myelin debris (Avellino et al. 1995).

702 •
The growth factors include nerve growth factor (NGF), plate-
let-derived growth factor, epithelial growth factor, and acidic
and basic fibroblast growth factors (FGF) (Fu and Gordon
1997). Schwann cells and macrophage cytokines have been
implicated in both the enhancement (e.g., interleukin-1β and
tumor necrosis factor-α) and the curtailment (transforming
growth factor-β [TGF-β]) of phagocytosis and hence, the
regulation of the month-long process of Wallerian degenera-
tion in the rat (Shamash et al. 2002; You et al. 1997).
2.2.2 Conversion of Schwann Cells
to a Growth-Supportive State
2.2.2.1 Expression of Growth-Associated Genes
Schwann cells and macrophage-derived growth factors contrib-
ute to the conversion of SCs from the myelinating phenotype
associated with intact axons to the “growth-supportive” den-
ervated phenotype in the absence of axon contact. The dener-
vated SCs downregulate myelin-associated glycoproteins and
proteins including P0, myelin basic protein, myelin-associated
glycoprotein, peripheral myelin protein 22 kDa (PMP22),
connexin 32, periaxin, and transcription factors such as
Krox-20, Krox-24, and Oct-6. They upregulate genes for cell
adhesion molecules such as the immunoglobulins neural cell
adhesion molecule (N-CAM) and L1 and the cadherin super-
family. They also upregulate the genes for the large glycopro-
teins such as laminin that form the basal lamina substrate for
axonal regeneration, as well as many neurotrophins, growth
factors, and their receptors that play a role in regeneration of
the axons from the proximal nerve stump (Boyd and Gordon
2003b; Fu and Gordon 1997) (see Fig. 55.1) (see chapters 43
and 54).
The interleukins released in the distal nerve stumps after
nerve injury have been implicated in the production of growth
factors by the denervated SCs, including NGF in fibroblasts
and SCs (Boyd and Gordon 2003b; Fu and Gordon 1997).
Transforming growth factor beta (TGF-β) has also been
shown to be essential for the neurotrophic effect of several
neurotrophic factors, including glial-derived neurotrophic fac-
tor (GDNF), but the effects of TGF-β on SCs are phenotype-
dependent as they are in other cells (Einheber et al. 1995).
2.2.2.2 Motor and Sensory Specific Expression of
Neurotrophic Factors
The several growth factors expressed by denervated SCs
include the neurotrophins [NGF, brain-derived neurotrophic
factor (BDNF), neurotrophin-3 (NT-3) and neurotro-
phin 4/5 (NT4/5)], members of the TGF-β superfamily
including TGF-β and GDNF, vascular endothelial growth
factor (VEGF), pleiotrophin, the insulin-like growth
factors (IGF1 and 11), and FGF-1,2 (Brushart 2011). The
gene profile of neurotrophic factors differs in denervated SCs
within endoneurial tubes that surround motor and sensory
axons: BDNF is the predominant neurotrophin expressed by
motor SCs and the sensory SCs express a wider range of neu-
rotrophins that include NGF and NT4/5 (Hoke et al. 2006).
The profiles have been implicated in the preference shown for
motor and sensory neurons to regenerate their axons within
N E RVE R E G E N E R AT I O N I N T H E P E R I P H E R A L N E RVO U S SYS T E M
appropriate distal pathways, although the process is not
precise (Al-Majed et al. 2000; Brushart 1993; Eberhardt
et al. 2006).
2.2.3 Transient Expression of Growth-Associated Genes
The growth supportive state of denervated SCs is transient
like that of the growth state of axotomized neurons (Boyd
and Gordon 2003b; Fu and Gordon 1997) (see Fig. 55.1).
Neurotrophic factors such as GDNF that are upregulated
within weeks, are progressively downregulated to low lev-
els within months after denervation (Hoke et al. 2002). The
low affinity p75 receptor expression and protein progres-
sively decline to undetectable levels within the same time
frame (Li et al. 1997; You et al. 1997). Interestingly, the
declining expression of erb2/3 receptors for neuregulin is
reversed when the chronically denervated nerve stump is
placed into tissue culture (Li et al. 1997). The reduced expres-
sion of growth associated genes is accompanied by a transi-
tion from the growth supportive state to a dormant state in
which the SCs atrophy and decline in number (Li et al. 1997;
You et al. 1997).
2.3 R EG E N E R AT I N G AXO NS A N D T H E I R
I N T E R AC T I O NS WIT H S C H WA N N C E L L S
2.3.1 The “Bands of Büngner”
Schwann cells are normally anchored in intact nerve via
attachments to the large glycoproteins of the basal lamina
in the endoneurial tubes; these include laminin, fibronectin,
and collagen VI (Chernousov and Carey 2000). After injury,
the denervated SCs upregulate genes for these glycoproteins
and other adhesion molecules, including NCAM and L1,
and N-cadherin that are members of the immunoglobulin
and cadherin superfamilies, respectively (Brushart 2011; Ide
1996; Martini and Schachner 1988). Thereby, they sustain the
basal lamina and they line the endoneurial tubes as the “bands
of Büngner” (Büngner 1891) to support the regeneration of
axons through the endoneurial tubes (Chernousov and Carey
2000; Fu and Gordon 1997). Both the basal lamina and the
SCs are essential for axon regeneration, axon outgrowth, and
elongation failing in their absence (Hall 1986).
Schwann cell–derived neuregulin likely plays a role in
the secondary proliferation of SCs as the regenerating axons
contact the SCs and in the remyelination of the axons once
their processes are withdrawn (Fricker et al. 2011). There is
pharmacological evidence that the neurotransmitters, acetyl-
choline, and ATP that are released from growth cones act via
receptors on the SCs in the distal nerve stump to promote the
withdrawal of their long processes and in turn, convert them
from the nonmyelinating growth permissive state to a myeli-
nating one (Vrbova et al. 2009). Neurotransmitter mediated
calcium influx has been demonstrated to initiate retraction of
long processes of denervated perisynaptic SCs (Georgiou et al.
1999). Nogo receptors on macrophages in the lesioned nerves
mediate their efflux out of the SC basal lamina during axon
extension (Fry et al. 2007).
• 703
 Diameters of the regenerating axons progressively increase
in proportion to the proximal axons with concomitant increase
in the myelin rings around the axons (Devor and Govrin-
Lippmann 1979). Full recovery of nerve fiber diameters, how-
ever, depends on the reformation of functional contacts with
denervated targets (Fu and Gordon 1997).
2.3.2 Staggered Axonal Regeneration
The initial axon outgrowth within hours of nerve injury
is aborted before transport of elevated levels of actin and
tubulin for axonal extension (Geraldo and Gordon-Weeks
2009). Outgrowth from the proximal nerve stump into the
distal stump is a protracted process especially after transec-
tion injuries that disrupt the continuity of the basal lamina.
The accepted 1- to 3-mm/day regeneration rate in mammals
was calculated from rate at which regenerating axons covers
a distance. This was evaluated by the “pinch test” for sensory
neurons and the rate of recovery of a given motor function,
such as toe spreading for motoneuron regeneration (Gutmann
et al. 1942; Sunderland 1947). When this plot was extended
to the x-axis, the latent period of axon outgrowth was cal-
culated as a few days. However, the counts of backlabeled
motoneurons that regenerated their axons across a suture site
of a transected nerve demonstrated that only a small fraction
of motoneurons regenerate their axons into the distal stump
within 4 days, with the remainder progressively crossing the
suture site over a 30-day period (Al-Majed et al. 2002) (Fig.
55.2). This unexpected “staggering” of regenerating axons
across the suture line accounted for our earlier finding of an
8- to 10-week period during which all motoneurons progres-
sively regenerate their axons over a 25-mm distance rather
than an expected shorter period of 2 to 3 weeks if regenera-
tion of axons was synchronous rather than staggered across
the suture site (Al-Majed et al. 2000). Dorsal root ganglion
neurons demonstrate the same staggered outgrowth of axons
(Witzel et al. 2005). Cajal had described how, despite surgi-
cal apposition of transected nerve stumps, the axons that grow
out from the proximal nerve stump take tortuous routes to
navigate between surgically apposed proximal and distal nerve
stumps. In some cases the axons spiraled and some grew back
into the proximal nerve stump (Cajal 1928). In transgenic
mice whose neurons express yellow fluorescent protein, the
same tortuous pathways of axon growth were described after
nerve transection and surgical repair (Witzel et al. 2005).
Moreover, immunohistochemical visualization of events at
the suture site showed the same tortuous patterns of axon out-
growth in the context of the slow migration of misaligned SCs
into the disorganized laminin of the disrupted basal lamina
(Witzel et al. 2005).
Growth inhibitory molecules such as chondroitin
sulfate proteoglycans that are expressed in the endoneu-
rium and the surrounding nerve sheaths, may serve to con-
fine regenerating axons to the growth supportive SC-basal
lamina conduits (Udina et al. 2006). Their breakdown by
chondroitinase ABC promotes axon outgrowth by a sepa-
rate signaling pathway to that of the neurotrophins (Zhou
et al. 2006).
704 • NEUROGLIA
2.3.3 Sprouting, Axon Elongation, and
Reformation of Target Connections
Parent axons in the proximal stump of injured peripheral
nerve elaborate multiple growth cones and produce as many
as five daughter axons each that regenerate across the injury
site and into the distal nerve stumps (see Fig. 55.1). The fate of
these branches depends on the availability of a neural “guid-
ing” structure distal to the injury site. In its absence, regenerat-
ing axons form a neuroma that is a mixture of immature nerve
fibers and connective tissue (Fu and Gordon 1997; Sunderland
1978). Axonal sprouts that advance distally from an axon
within the proximal nerve stump comprise a “regenerating
unit” (Fu and Gordon 1997). The multiple axonal sprouts
remain in the distal nerve stumps unless axons make target
connections when all but one daughter axon are withdrawn
over months and even years (Aitken et al. 1947; Mackinnon
et al. 1991). The final diameter of the daughter axon depends
on reformation of target contacts (Fu and Gordon 1997).
At the neuromuscular junction, the specialized nonmyeli-
nating perisynaptic SCs normally respond to released trans-
mitters from the intact presynaptic terminals; they become
reactive, extend processes after denervation, and guide regen-
erating axons back to the denervated endplates to reinnervate
muscle (Son and Thompson 1995). These reactive perisynaptic
SCs resemble denervated SCs in the distal nerve stumps with
respect to their process formation. The perisynaptic Schwann
cells and the basal lamina of the adult endplate are sufficient
to guide regenerating axons back to the endplate region,
even after ablation of the denervated muscle (Dunaevsky and
Connor 1998).
2.4 T E M P O R A L D EC L I N E I N R EG E N E R AT I V E
C A PAC IT Y A N D S U P P O RT
2.4.1 Poor Functional Recovery After Nerve
Injury in Humans
Unfortunately, functional recovery in humans is frequently
poor after peripheral nerve injuries, especially when nerve
trunks close to the spinal cord and far from the target organs
are injured (Fig. 55.3). Under these conditions, the regenerat-
ing axons remain without target connections for months and
even years, a condition we refer to as chronic axotomy (Boyd
and Gordon 2002, 2003b; Fu and Gordon 1995a, 1997).
Schwann cells in the distal nerve stumps and the target organs
are chronically denervated for long periods, conditions of
chronic SC denervation (Fu and Gordon 1995b; Gordon et al.
2003, 2011; Sulaiman and Gordon 2000, 2002; Sulaiman
et al. 2002) and chronic muscle denervation (Gordon et al.
2011a), respectively. These periods may be months and years
for neurons to regenerate their axons and reinnervate targets
at 1 mm/day. The failure of functional return in the hand,
for example, is generally attributed to irreversible atrophy
of the denervated muscles and sense organs and fatty tissue
replacement (Sulaiman et al. 2011) (see Fig. 55.3). However,
our experiments over the past approximately 15 years provide
strong evidence that it is the progressive failure of chroni-
cally axotomized neurons to regenerate their axons and the
 A B

1.5 mm

25mm

Fluoro Fluoro
gold ruby

C 350 D 400
350
Labeled motoneurons

300 300
250
250 200
150
200 100
50
150 0
0 2 4 6 8 10 4d 1 wk 2 wk 3 wk 4 wk

E 350 F 400

350
300
Labeled motoneurons

All 300
250 250
200 200
150
150 Muscle
100 Stimulation
100 Sham
50
Cutaneous
50 0
0 2 4 6 8 10 4d 1 wk 2 wk 3 wk 4 wk

G H
350 180
160
300
Labeled motoneurons

All 140
250 120
100
200
80
Muscle
150 60
40
100
Cutaneous 20
50 0
0 2 4 6 8 10 0–4 4d– 1 wk– 2 wks– 3 wks–
Weeks after femoral ne
rve repair days 1 wk 2 wks 3 wks 4 wks
Weeks after femoral ne
rve repair

Figure 55.2 Numbers of retrogradely labelled femoral motoneurons that regenerated their axons 25 mm into muscle and cutaneous branches (A) and 1.5 mm (B) from
the site of transection and microsurgical repair are plotted as a function of time after repair with 1 hour sham or 20-Hz electrical stimulation. “Staggered motor axonal
regeneration” into the femoral nerve branches (C) and across the suture site (D) results in “preferential motor reinnervation” of the appropriate motor branch
(E). Electrical stimulation accelerates regeneration of axons across the suture site (F,H) and into the appropriate motor branch (G) with the arrows denoting a signifi-
cant increase (P < .01). The number of motoneurons in (H) represents new crossing events with each value being the mean motoneuron number labeled at the end of
the interval minus the mean labeled at the beginning of the interval. The rapid increase in crossing event that peaks between 1 and 2 weeks reflects the recruitment
of many motoneurons to regenerate across the suture line and penetrate the distal stump earlier with electrical stimulation than without stimulation. Adapted from
Al-Majed et al. 2000; Brushart et al. 2002.
progressive failure of chronically denervated SCs to support demonstrated in rat experiments in which the period of
the regenerating axons that play the predominant role in the motoneuronal axotomy was experimentally prolonged for
clinical failures of functional recovery after surgical repair up to a year before evaluating regenerative success (Boyd
(Boyd and Gordon 2002, 2003a,b; Fu and Gordon 1995a,b; and Gordon 2001, 2002, 2003a; Fu and Gordon 1995a). By
Gordon and Fu 1997; Gordon et al. 2011; Midha et al. 2005; counting backlabeled chronically axotomized motoneurons
Sulaiman and Gordon 2000, 2002, 2003, 2009; Sulaiman that regenerated their axons into a freshly denervated nerve
et al. 2002). The poor functional recovery is exacerbated by and counting reinnervated motor units to determine how
delayed nerve surgery. many motoneurons reinnervate denervated muscles, we
reported that these numbers progressively declined to
approximately 33% of those motoneurons that regenerated
2.4.2 Prolonged Axotomy of Neurons Reduces
after immediate axotomy (see Fig. 55.3). The exponential
Regenerative Capacity and Expression of
decline in regenerative capacity of the chronically
Regeneration Associated Genes
axotomized motoneurons was paralleled by a decline in the
The inherent ability of axotomized neurons to regenerate upregulated (RAGs Tetzlaff et al. 1996) (see Fig. 55.1).
axons is not sustained over long periods of time. This was

A 140
MN number
Percent of regenerating axons (%)

120 MU number

100

80

60

40

20
B 140
0 MN number
0 20 40 60 80 100 120 140 160 180 200
Percent of regenerating axons (%)

120 MU number
Days of chronic axotomy
100

C 80

60

40

20

Brachial plexus 0
injuries 0 20 40 60 80 100 120 140 160 180 200
Days of chronic denervation

800
mm
= 800
day
s

Median and
ulnar injuries
at the wrist
100 mm = Denervated
100 days hand muscle

Figure 55.3 Schematic illustration of proximal nerve injuries that require prolonged regeneration times for the regenerating axons to traverse lengthy
distances to reinnervate distant targets such as the muscles in the hand (C). Over these long periods, numbers of motoneurons that regenerate axons
(MN number) and reinnervate muscles (motor unit [MU] number) decline with time reflecting the reduced regenerative capacity of the chroni-
cally axotomized neurons (A) and the regenerative support by the chronically denervated Schwann cells (C). These combined account for the poor
functional outcomes of nerve surgeries that require regeneration over long distances. Chronic denervation of target organs does contribute a small
component to poor outcomes but irreversible atrophy of target organs and muscle in particular, is not the cause of these poor outcomes.

706 • NEUROGLIA
 The dramatic negative effect of reduced regenerative capac-
ity of the chronically axotomized motoneurons did not affect
the ability of the neurons to reinnervate up to three times as
many muscle fibers as normal, the fewer motoneurons enlarg-
ing their motor units and the number of muscle fibers supplied
by each reinnervating motoneuron (Fu and Gordon 1995b).
Thus, chronic axotomy impairs the regenerative capacity of
motoneurons, but does not impair the capacity of the smaller
number of regenerated motor axons to make functional con-
nections with denervated muscle fibers.
2.4.3 Prolonged Denervation Reduces the
Capacity of Denervated Schwann Cells to Support
Axonal Regeneration and Their Expression of
Regeneration-Associated Genes
The counts of axons regenerating into a chronically denervated
nerve stump after 100 days in early experiments conducted during
World War II, indicated that the rate of outgrowth and the axon
numbers were not affected by delayed nerve repair (Gutmann
and Young 1944; Holmes and Young 1942). These findings with
evidence of extreme atrophy of the denervated muscles was the
basis for the enduring view that it is the irreversible atrophy of
denervated targets and their replacement by connective tissue
and fat that is the prime basis for poor functional recovery after
nerve repair (Gutmann and Young 1944; Holmes and Young
1942). However, counts of axons that regenerated into the distal
nerve stumps severely overestimate the number of neurons that
regenerate their axons, especially when regenerated axons are
counted before target reinnervation. This is because each parent
axon gives rise to an average of five daughter sprouts (see section
2.3.3). Moreover, the axonal counts made just distal to the repair
site are unlikely to represent the number of axons that regener-
ate as far as the denervated muscle targets because of the progres-
sive loss of the capacity of chronically denervated SCs to support
axonal regeneration (Fig. 55.3B). We tested the latter possibility
in experiments in which we enumerated both the number of
freshly axotomized motoneurons that regenerated their axons
into chronically denervated nerve stumps and the number of rein-
nervated motor units in the target muscle after cross-suture of a
freshly axotomized nerve to a chronically denervated distal nerve
stump (see Fig. 55.3A) (Fu and Gordon 1995b; Sulaiman and
Gordon 2000). Under conditions in which the effect of chronic
SC denervation was isolated from the effect of chronic muscle
denervation, we noted that the permissive environment provided
by denervated SCs is short-lived with the numbers of motoneu-
rons regenerating their axons through chronically denervated SCs
declining rapidly (Gordon et al. 2011a). The numbers of freshly
axotomized motoneurons that regenerated their axons and rein-
nervated denervated muscles via the chronically denervated nerve
stumps, declined in parallel to a value of approximately 5% (see
Fig. 55.3) (Fu and Gordon 1995b; Sulaiman and Gordon 2000).
These findings provide strong evidence that poor axonal
regeneration is owing to the progressive failure of axons to regen-
erate in the atrophic nerve stumps and not an inability of chroni-
cally denervated muscle fibers to accept reinnervation after long
delays. In fact, wet weights of reinnervated muscles, which are
directly related to their isometric forces, demonstrated a very
N E RVE R E G E N E R AT I O N I N T H E P E R I P H E R A L N E RVO U S SYS T E M
sluggish rate of muscle atrophy compared with a drastic loss of the
trophic environment of the distal nerve stumps after long-term
denervation (Sulaiman and Gordon 2000). The declining num-
ber of motoneurons that did regenerate their axons to make tar-
get connections formed maximally enlarged motor units. These
enlarged units could not compensate for the approximately
95% reduction in numbers of functional motoneurons (Fu and
Gordon 1995b). The sustained capacity of denervated muscle
to accept reinnervation was also demonstrated by the parallel
recovery of both muscle weight and isometric force. Nonetheless,
isolation of the effects of SC denervation from muscle denerva-
tion identified that chronically denervated muscle fibers did not
fully recover their former size, arguing that there are limited
numbers of satellite cells that are incorporated into each atro-
phic muscle fiber to recover muscle fiber cross-sectional area
(Gordon et al. 2011a). Hence, progressive deterioration of the
growth-supportive capacity of SCs in the distal nerve stumps
plays a primary role in poor functional recovery after nerve injury
and the role of muscle atrophy is secondary.
It was striking that the long-term chronically denervated SCs
maintained their capacity to remyelinate the fewer axons that
regenerated (see Fig. 55.3) (Sulaiman and Gordon 2000), partic-
ularly in view of the progressive regression of the capacity of the
denervated SCs to sustain their growth permissive phenotype
(see Fig. 55.1) and the progressive decline in numbers of growth
supportive SCs in the chronically denervated distal nerve stumps
(Li et al. 1997; You et al. 1997).
2.5 M ISDIR E CT I ON OF R E G E N E R AT IN G AXONS
2.5.1 Appropriate Reinnervation After
Crush Injuries:
Functional outcomes after axonal injuries are best after nerve
crush injuries in which the endoneurium remains anatomically
intact all the way to the target and axonal sprouts of the regener-
ating unit are contained within the original endoneurium such
that the regenerating axons are “led” back to their original tar-
gets (Sunderland 1978). With time, reinnervated muscle fibers
display the normal mosaic distribution of muscle fiber types,
motor unit and muscle isometric forces recover fully, the original
number of functional motor units is restored, and the numbers
and diameters of the myelinated nerve fibers return to normal
(Gordon and Pattullo 1993).
2.5.2 Misdirection of Axons After Transection Injuries
The number of axons that regenerate into distal nerve stumps
and successfully remake target connection is far more variable
for transection injuries that disrupt endoneurial tubes. Axonal
sprouts emanating from the proximal nerve stump may enter
several different endoneurial tubes with target destinations that
they did not formerly supply as in the case of motor axons that
regenerate within pathways leading to the skin rather than mus-
cle (Brushart 1993). Regenerating axons fail to find their origi-
nal targets, especially when large nerve trunks are damaged and
require surgical repair.
• 707
 After microsurgical repair of severed nerves to the human
hand, voluntary recruitment of the reinnervated motor units
revealed the random reinnervation of several muscles across
the hand by the motoneurons that had previously supplied
only one muscle. The random distribution of regenerating
axons to different muscles essentially negated any fine con-
trol of hand movement (Thomas et al. 1987). Animal studies
of motor axonal regeneration also revealed random reinner-
vation of muscles after surgical repair of transected nerves.
Gillespie et al. (1987) demonstrated random distribution of
slow and fast motor units in reinnervated soleus and lateral
gastrocnemius rat muscles after surgical repair of the common
lateral gastrocnemius-soleus (LGS) nerve to the two muscles.
These studies demonstrate the inability of transected motor
axons to “find” their original endoneurial tubes, leading to the
appropriate muscles. Experiments that applied retrograde dyes
to regenerated axons of transected sciatic and facial nerves in
rats directly demonstrated misdirection of regenerating motor
axons from several different motoneuron pools to muscles
that the motoneurons did not formerly supply (Brushart and
Mesulam 1980). In cases in which the nerves supplied muscles
with antagonistic actions, axonal misdirection was associated
with inappropriate movements, quite consistent with findings
that flexor and extensor motoneurons sustain their normal but
inappropriate pattern of firing when they are directed to inap-
propriately reinnervate extensor and flexor muscles, respec-
tively (Gordon et al. 1986).
2.5.3 Preferential Motor Reinnervation
There are several peripheral nerve trunks whose branches
innervate skeletal muscles and sense organs in the skin and the
joints. Examples include the femoral nerve with two branches,
the muscle branch containing motor and sensory nerves to the
quadriceps muscle, and the other pure sensory saphenous nerve
branch to the skin (Brushart 1993). Although reinnervation
of motor and sensory nerve branches is initially random, the
motoneurons that progressively regenerate their axons across the
suture site send their axons into the appropriate motor branch
(Al-Majed et al. 2000; Brushart 1993). This “preferential motor
reinnervation” emerged in parallel with the progressive or stag-
gered regeneration of motor axons into the distal nerve stumps
(see Fig. 55.2E,F) (Al-Majed et al. 2000).
An unusual acidic glycan associated with myelin profiles
of motor but not sensory mouse axons that is recognized by a
monoclonal antibody L2/HNK-1 may be one mechanism of the
preferential reinnervation of appropriate pathways (Eberhardt
et al. 2006; Martini et al. 1994). Another is the differential neu-
rotrophic factor profile in SCs derived from sensory and motor
axons with the BDNF/NT4/5 profile likely to play an impor-
tant role in preferential reinnervation of motor pathways (Hoke
et al. 2006).
3 S T R AT E G I E S TO P R O M OT E AXO N A L
R E G E N E R AT I O N
Long delays incurred by staggered and slow rate of axonal regen-
eration frequently result in chronic axotomy of the injured
708 • NEUROGLIA
neurons that regenerate in chronically denervated distal nerve
stumps (see Fig. 55.1). The misdirection of regenerating axons to
inappropriate targets exacerbates the reduced numbers of neu-
rons that regenerate their axons and reinnervate denervated tar-
gets. Strategies developed to accelerate axonal regeneration and
counter the deleterious effects of long delays are presented here.
3.1 AC C E L E R AT I N G AXO N O U TG ROW T H
3.1.1 Conditioning Lesions Are Effective but
Not Feasible :
Several attempts have been made to accelerate the relatively slow
rate of axonal regeneration to optimize the recovery of function
after nerve injuries. Of the many attempts made, only the condi-
tioning crush lesion of the intact nerve before a more proximal
test crush lesion, significantly increases rate of axonal regenera-
tion (reviewed by Grafstein and McQuarrie 1978). The lesion ele-
vates mRNA expression of actin and tubulin and reduces levels of
neurofilament expression even further than normal in association
with more rapid transport of these cytoskeletal proteins (Tetzlaff
et al. 1996). The parallel increase in the rates of slow component b
of axonal transport of the cytoskeletal proteins and axonal regen-
eration in the injured neurons after the conditioning lesion pro-
vided further evidence for a direct causal link between these rates
(Hoffman and Lasek 1980; Jacob and McQuarrie 1991). The
more rapid transport of the cytoskeletal proteins into the growth
cones prevents their collapse (Jacob and McQuarrie 1991).
The conditioning lesion accelerates both outgrowth of
regenerating axons that normally begins within hours of the
injury and the later SC migration from the proximal stump after
approximately 2 days (Torigoe et al. 1999). These findings argue
for a role of both of the neurons and the SCs in the accelerated
axonal regeneration rate after a conditioning lesion. It has not yet
been ascertained as to whether the conditioning lesion, which so
effectively accelerates axonal outgrowth and regeneration rate, is
also effective in synchronizing the staggered axonal outgrowth
across the suture site. This staggered outgrowth normally delays
the process of axonal regeneration by many weeks (Al-Majed
et al. 2000; Brushart et al. 2002) (see Fig. 55.2). The condition-
ing lesion is nevertheless the only technique that has been dem-
onstrated to accelerate the rate of regeneration. The important
contraindication for the practical use of the conditioning lesion
to promote axon regeneration is that it must precede the test
injury to be effective (Jacob and McQuarrie 1993). This delay
obviously negates the conditioning lesion as a clinically viable
technique to promote axonal regeneration in human patients.
3.1.2 Brief Electrical Stimulation Accelerates
Axon Outgrowth
Early promising studies reported that continuous low fre-
quency electrical stimulation (ES) accelerates muscle rein-
nervation (Nix and Hopf 1983; Pockett and Gavin 1985).
The data were not necessarily explained by more rapid nerve
regeneration. Using backlabeling to count motoneurons that
regenerated their axons, we demonstrated that superthres-
hold low frequency ES immediately after the surgical repair
of transected femoral nerve accelerated axon regeneration of
axons from all the axotomized motoneurons. The duration of
the stimulation could be reduced to as short as 1 hour and the
effect required that action potentials be conducted to the cell
bodies (see Fig. 55.2G) (Al-Majed et al. 2000). Electrical stimu-
lation accelerated axon outgrowth across the suture site (see Fig.
55.2F), but did not accelerate regeneration rate (see Fig. 55.4H)
(Brushart et al. 2002). The ES effect is to synchronize the
growth of axons across the surgical suture line by accelerating
the axon outgrowth from the proximal nerve stump and their
passage across the suture line (Brushart et al. 2002).
The accelerating effect of the 1-hour period of 20 Hz ES was
also demonstrated in sensory neurons where it promotes the
appropriate reinnervation of sensory nerve pathways (Brushart
et al. 2005). Only a 1-hour period stimulation was effective in
promoting sensory nerve regeneration, longer periods of ES
being ineffective, coincident with downregulation of neuronal
BNDF (Geremia et al. 2007). Several laboratories have con-
firmed the positive effect of ES in accelerating regeneration in
rat and mouse models of transected nerves (Asensio-Pinilla et al.
2009; Eberhardt et al. 2006; English et al. 2007; Franz et al.
2008; Huang et al. 2010; Sharma et al. 2009; Yeh et al. 2010).
3.1.3 Electrical Stimulation Accelerates
Target Reinnervation
Electrical stimulation accelerates functional return in rats
(Eberhardt et al. 2006). In a proof of principle study of nerve
regeneration and target reinnervation in humans, we counted
reinnervated motor units after carpal tunnel release surgery
with and without immediate 20-Hz 1-hour electrical stimula-
tion (Gordon et al. 2010) (Fig. 55.4). Choosing only patients

Carpal Median nerve is released.

Incisition site tunnel

B 20Hz/
1hr ES

Median EMG
− nerve recording

Carpel
+ tunnel
release
surgery

C Control D Stimulation

400 * *
Number of innervated

300 Normal
motor units

300
Pre-op
x±SE
100

0
3m 6-8 m 12 m 3m 6-8 m 12 m

Figure 55.4 Immediately following open carpal tunnel release surgery (A) in patients with severe carpal tunnel syndrome (loss of >50% of functional motor
units in the thenar eminence muscle), the median nerve was stimulated at 20 Hz for 1 hour via implanted electrodes and action potentials generated were
monitored with electromyography. Mean (+SE) of the number of innervated motor units did not change from preoperative levels in the control unstim-
ulated group of patients. In contrast, there was already a significant increase by 6 months after the 1-hour stimulation with the number of functional
motor units at 1 year being equal to the normal levels.

N E RVE R E G E N E R AT I O N I N T H E P E R I P H E R A L N E RVO U S SYS T E M • 709

with severe carpal tunnel syndrome with electrophysiological
evidence of loss of more than 50% of their functional motor
units in the median eminence, the effects of the ES were strik-
ing. Especially as the number of reinnervated motor units did
not increase significantly even after 1 year without the stimu-
lation, the finding that the numbers had increased within 3
months in the stimulated group and reached normal levels 6
months after the release and ES was striking (see Fig. 55.4).
3.1.4 Role of Neurotrophic Factors and cAMP
The effect of ES on accelerating axonal growth across a suture
site is associated with an accelerated and augmented upreg-
ulation of endogenous neuronal BDNF and trkB followed
shortly after by elevated expression of RAGs (Al-Majed
et al. 2000, 2004; Geremia et al. 2007). Knockout experi-
ments in transgenic mice demonstrated that positive ES
effect on regeneration is dependent on neuronal expression
of NT4/5 (English et al. 2007).
Strong links have also been made between activity-induced
elevations in BDNF, intracellular calcium and intracellular
cyclic adenosine monophosphate (cAMP) that in turn,
promote neurite outgrowth and axonal regeneration on both
permissive and nonpermissive substrates in vitro and in vivo
(Qiu et al. 2002). The positive effects of ES in promoting axon
outgrowth is associated with elevation of cAMP, suggesting that
the ES mediates its effect via its upregulation of neurotrophins
and elevated cAMP, the effects likely converging on cAMP
response element-binding (CREB) to promote synthesis of
cytoskeletal proteins and/or rearrangement of these proteins
to accelerate axon outgrowth (Aglah et al. 2008). Rolipram, a
phosphodiesterase inhibitor that increases cAMP, mimicked
ES in accelerating axon outgrowth (Udina et al. 2010).
Neurotrophic factors
and receptors
Motoneuron pool
Time (d)
Figure 55.5 The time course of a month for all axons to regenerate across the suture site into the denervated distal nerve stump (Brushart et al. 2002)
corresponds with the time during which neurotrophic factors and their receptors in the distal nerve stump peak and, in most cases, decline. As a result
neurons regenerate their axons through the distal nerve stump as the Schwann cells progressively lose their growth support and become atro-
phic. Adapted from Furey et al. 2007.
3.2 C O U N T E R AC T I N G T H E N EG AT I VE
EFFEC TS O F C H RO N I C AXOTO MY O F
I N JU R E D N EU RO NS
3.2.1 By Neurotrophic Factors
3.2.1.1 Exogenous Sources of Neurotrophic Factors
The expression of neurotrophic factors by axotomized neu-
rons and denervated SCs peaks within weeks and declines
thereafter, the time course of the decline varying according
to the factor (Boyd and Gordon 2003b). In general, this time
course parallels the course of decline in capacity of chroni-
cally axotomized neurons to regenerate their axons (see sec-
tion 4.2). This suggested that exogenous application of these
factors might reverse the deleterious effects of chronic axo-
tomy on nerve regeneration (see Fig. 55.1). Indeed this proved
to be the case. Exogenous supply of BDNF and/or GDNF
significantly increased regenerating motoneuron numbers,
application of appropriate doses for a month during regen-
eration being effective in restoring regenerative competence
to that seen after immediate nerve repair (Boyd and Gordon,
2001, 2002, 2003a,b; Gordon et al. 2003). Not surprisingly,
these neurotrophic factors had no effect on regeneration
after immediate nerve suture. This is consistent with there
being sufficient endogenous supplies of neurotrophic fac-
tors by the axotomized neurons and SCs in the denervated
distal nerve stumps after immediate nerve repair (Fig. 55.5).
GDNF acting on its receptors was effective at lower doses
than BDNF and always facilitated axon regeneration (Boyd
and Gordon 2003a). BDNF had a bimodal dose-response
effect with its trkB receptors mediating a positive effect on
axon regeneration and p75 receptors a negative effect that
acts to negate the positive effects of trkB activation (Boyd
and Gordon 2001).
Distal stump
BDNF
Distal
stump
MN GDNF
BDNF
Ret
TrkB
GFR-α
0 10 20 30 40 50 60
10 mm
Site of
axotomy Schwann cell - growth supportive
Schwann cell - Atrophied
710 • NEUROGLIA
 3.2.1.2 Endogenous Sources of Neurotrophic Factors
Low frequency ES upregulates BDNF and GDNF and its
receptors in axotomized motoneurons with NT4/5 appearing
to be a critical factor that mediates the neurotrophic effect of
accelerated axon outgrowth (Al-Majed et al. 2000; English
et al. 2007). This is likely the reason for ES being effective in
promoting the regeneration of axons from chronically axo-
tomized neurons (Gordon et al. 2011b). The upregulation of
neurotrophic factors in freshly axotomized motoneurons was
effective in promoting axon regeneration even through chron-
ically denervated nerves (Gordon et al. 2011b).
3.2.2 FK506 Application
The immunophilin ligand FK506 that enhances neurite out-
growth in vitro and accelerates regeneration rate after crush
injury and after section and immediate resuture (Gold et al.
1995), was very effective in counteracting the negative effects
of chronic axotomy to promote axonal regeneration. Twice as
many chronically axotomized motoneurons regenerated their
axons after 3-week daily subcutaneous injection in association
with a corresponding increase in the number of regenerated
axons (Sulaiman et al. 2002). The effects are mediated via
FKBP-52, a chaperone component of mature steroid recep-
tor complexes, independent of the FK506-binding protein-12,
that mediates its immunosuppressive effects (Gold et al. 1999).
Evidence of increased numbers of regenerated axons and the
more rapid progression of axon remyelination indicated that the
FK-506 also accelerated the rate of axon outgrowth, the effect
being observed in both motor and sensory neurons (Sulaiman
et al. 2002).
3.3 CO U N T E R AC T I N G T H E EFFEC TS O F
P RO L O N G E D D E N E RVAT I O N O F T H E
D I S TA L N E RV E S T U M P S
3.3.1 Electrical Stimulation
Brief ES significantly increased the number of motor and sen-
sory neurons that regenerated their axons into a chronically
denervated nerve stump (Gordon et al. 2010). This finding
indicated that ES may raise neurotrophic factor levels in the
neurons sufficiently to “jump start” axon outgrowth into less
permissive growth environments provided by atrophic SCs
in the chronically denervated nerve stumps. It is also possible
that the ES may release sufficient neuregulins from the proxi-
mal nerve stump to “reawaken” dormant SCs to support axon
regeneration. Indeed, the effective myelination of those axons
that do regenerate through chronically denervated nerve
stumps provides evidence that the reawakening is possible
(Sulaiman and Gordon 2000).
3.3.2 TGF-β to Promote Permissive Growth
Phenotype in Schwann Cells
A possible role of the cytokine TGF-β in counteracting the
deleterious effects of chronic SC denervation was suggested
by the results of several in vitro studies that TGF-β plays a
role in maintaining the growth-promoting denervated SC
N E RVE R E G E N E R AT I O N I N T H E P E R I P H E R A L N E RVO U S SYS T E M
phenotype in addition to its mitogenic effects on the SCs
(Unsicker and Strelau 2000) (see section 2.2.2.1). TGF-β
increased SC proliferation within 2 days in vitro and forsko-
lin augmented the effects of TGF-β presumably by increas-
ing intracellular levels of cAMP. To assess the capacity of
reactivated SCs to support axonal growth, we reactivated
chronically denervated SCs in isolated nerve stumps with
TGF-β and forskolin for 2 days in vitro prior to placing
these reactivated SCs in a silastic cuff between proximal
and distal nerve stumps in vivo. The effect was dramatic.
The TGF-β- and forskolin-treated SCs increased the regen-
eration of axons through the silastic cuff fourfold as com-
pared with number of motoneurons that regenerated axons
into chronically denervated distal nerve stumps without
pretreatment. Moreover, the regenerated nerve fibers were
larger and better myelinated than those fibers that regener-
ated through the untreated nerve explants (Sulaiman and
Gordon 2002). These findings are consistent with previ-
ous reports that chronically denervated SCs sustain their
capacity to undergo proliferation and remyelinate regen-
erated axons (Sulaiman and Gordon 2000). They provide
promise that the reduced numbers of SCs that remain in
chronically denervated nerve stumps can be reactivated by
exposure to cytokines that are normally released during the
process of Wallerian degeneration. The reinstated growth-
supportive phenotype of the SCs is sufficient to support
axonal regeneration.
3.3.3 Side-to-Side Transfers to “Protect”
Chronically Denervated Schwann Cells
Nerve autografts have been placed between a donor nerve and a
recipient denervated distal nerve stump to promote axon crossing
and growth within the denervated stump (Viterbo et al. 2009).
Using this technique, we investigated whether these side-to-side
transfers could be used to “protect” a chronically denervated
nerve stump before the ingrowth of axons after delayed nerve
repair or from a more proximal nerve injury and surgical repair
(Ladak et al. 2011). Using a model of side-to-side bridging in rat
hindlimb nerves, we reported that the small number of donor
nerves that regenerate their axons into the denervated nerve stump
do provide “protection” of the SCs, significantly increasing the
number of neurons that regenerate their axons successfully into
the chronically denervated and protected distal nerve stumps.
This technique is particularly attractive for the protection of
long denervated distal nerve stumps such as the ulnar nerve after
a brachial plexus injury because donor nerves that cross inserted
side-to-side bridges regenerate in both proximal and distal
directions (Gordon and Borschel, unpublished findings). Hence
these donor nerves may protect the distal nerve sufficiently to
allow for more effective regeneration of axons over large distances
and in turn, to reinnervate denervated targets.
4 S U M M A RY A N D P E R S P E C T I VE S
The contributions of the injured neurons, SCs and mac-
rophages to the regenerative process in the peripheral nervous
• 711
system appear to be interlinked such that there is a window of
opportunity during which the growth potential of the neurons
translates into outgrowth and extension of regenerating axons.
This window is closely linked to the period during which there
is active Wallerian degeneration when macrophages interact
with SCs to provide the permissive growth environment. The
progressive but slow advancement of regenerating axons from
the proximal nerve across the site of nerve transection and
repair severely slows the progress of axonal growth into the dis-
tal nerve stumps. The delay exacerbates the lengthy time peri-
ods during which the neurons remain chronically axotomized
and the SCs remain chronically denervated. As we begin to
understand the limitations to peripheral nerve regeneration,
we can begin to devise effective interventions to prolong the
window of opportunity for axonal regeneration and promote
recovery of function after nerve injuries. Although exogenous
sources of neurotrophic factors for example, may be effective,
we should continue to pursue methods that are not only read-
ily applied clinically, as in electrical stimulation, but also that
effectively upregulate endogenous sources of molecules that
are needed for effective nerve regeneration.
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 56.
NERVE FIBER REGENERATION IN THE CENTRAL
NERVOUS SYSTEM OF HIGHER VERTEBRATES
Anita D. Buchli and Martin E. Schwab

A B B R E VI AT I O N S and structural plasticity as well as functional recovery take

place.
ATP adenosine triphosphate In the past 25 years, molecules and factors responsible for
BBB blood-brain barrier the nonpermissive environment of the adult CNS and cellular
ChABC chondroitinase ABC mechanisms involved have been elucidated. In parallel, novel
CNS central nervous system therapeutic approaches are being developed to overcome the
CSPG chondroitin sulfate proteoglycan impediment of nerve fiber outgrowth leading to functional
CST corticospinal tract recovery.
DCC deleted in colorectal cancer
ECM extracellular matrix
IGF-1 insulin-like growth factor 1 2 T H E PAT H O P H YS I O L O GY O F S P I N A L
GAP-43 growth-associated protein 43 C O R D I N J U RY A N D S T R O K E
GDNF glial cell line–derived neurotrophic factor
GFAP glial fibrillary acidic protein Limited functionally relevant regeneration takes place sponta-
GPI glycosyl-phosphate-inositol neously following CNS injury. Animals that underwent experi-
HSPG heparin sulfate proteoglycan mental CNS injury or patients who suffered a stroke or spinal
HA hyaluronic acid cord injury (SCI) often show recovery of initial impairments
LAR leukocyte common antigen-related with time. The extent of recovery largely depends on the degree
LIF leukemia inhibitory factor of CNS damage; that is, after small lesions often near to full
MAG myelin-associated glycoprotein recovery can be achieved, whereas very large CNS lesions are
mTOR mammalian target of rapamycin accompanied by poor functional outcome. After large lesions
NgR Nogo-66 receptor the spontaneous recovery curve plateaus after approximately 8
OMpG oligodendrocyte-myelin glycoprotein to 12 weeks in rodents and several months in humans, leaving
PIP3 phosphatidylinositol (3,4,5)-triphosphate most functional improvements to the first months postinjury in
PI3K phosphoinositide-3-kinase humans.
PKA protein kinase A In animal models as well as in humans the final tissue dam-
PKC protein kinase C age after SCI is much larger than that of the first mechanical
PNS peripheral nervous system insult. Within the first few hours a variety of reactive processes
PTEN phosphatase and tensin homolog take place that are commonly termed secondary injury (Beattie
RGMA repulsive guidance molecule A 2004; Beattie et al. 2002a,b). In this second phase, vascular
SCI spinal cord injury changes, excitotoxic events, inflammation, and scarring further
unc-5c unc-5-homolog C contribute to the loss of tissue (Dumont et al. 2001; Schwab
wt wild-type and Bartholdi 1996). Starting hours to days after injury, plastic
changes take place at different CNS levels—from the cortex to
subcortical regions such as the brainstem and spinal cord. They
1 INTRODUCTION include biochemical, molecular, and synaptic changes, but also
gross anatomical adaptations resulting in the reorganization of
Although nerve fiber injury in the peripheral nervous sys- neuronal networks with a functionally relevant outcome.
tem (PNS) often leads to successful regeneration over long Although small lesions of the CNS can lead to strong and
distances, in the adult central nervous system (CNS) of often complete functional recovery, very large lesions have a
higher vertebrates spontaneous regeneration is almost absent. poor functional outcome. Currently available data mainly
Therefore, central nervous system trauma such as large spinal from animal experiments, but also from human patients indi-
cord or brain injuries or stroke frequently results in perma- cate that functional recovery probably depends mainly on
nent and severe deficits. During development, while circuits the amount of spared CNS tissue and plastic processes taking
are formed and fine tuned, the CNS is more adaptive to injury, place at different CNS levels.

715
 Therapeutic interventions aim at increasing neuropro-
tection levels leading to a decrease of secondary damage.
Rehabilitation and training vigorously applied in the first
months after injury focus on enhancing spontaneous plastic
adaptations which result in functional recovery after CNS
injury.
Neuroplasticity and axonal regeneration in the adult
CNS are restricted by growth inhibitory molecules of myelin
(Gonzenbach and Schwab 2008; Liu et al. 2006; Maier and
Schwab 2006; Yiu and He 2006) and inhibitory factors pres-
ent in the glial scar and extracellular matrix (Carulli et al.
2005; Kwok et al. 2011; Liu et al. 2006; Yiu and He 2006).
Furthermore, complex processes of inflammation and second-
ary damage are being initiated after CNS injury (Barreto et al.
2011; Norenberg et al. 2004).
This chapter discusses the current state of knowledge on
the cellular and molecular processes taking place after CNS
injury and their influence on regeneration.
3 G L I A L A N D N E U R A L R E AC T I O N S TO
C E N T R A L N E RVO U S SYS T E M DA M AG E
3.1 T H E R EG E N E R AT I V E A B I L I T Y O F A D U LT
M A M M A L I A N N EU RO N S C A N B E A LT E R E D BY
T H E I R M I C RO E N V I RO N M E N T
First reported by Ramon y Cajal nearly a century ago (1928),
the intrinsic regenerative ability of the adult CNS and the
molecular components involved have only been unraveled
in the last few decades (David and Aguayo 1981; Richardson
et al. 1984). Early studies by Ramon y Cajal were followed
in the late 1970s by classic transplantation experiments by
David and Aguayo (1981). After injury to spinal cord neurons
a peripheral nerve graft linking spinal cord areas rostral and
caudal to the injury site allowed long-distance regeneration
of damaged CNS axons into the peripheral nerve graft. This
finding suggested that the glial environment of the periph-
eral nerve was permissive to CNS axon regrowth and that the
regeneration failure in the CNS might result from a nonper-
missive environment.
Myelin and the glial scar are currently the main inhibi-
tory structures described for nerve fiber regeneration in the
adult CNS. In vitro, oligodendrocytes, CNS white matter,
and myelin of adult rats were shown to be nonpermissive sub-
strates for growing neurites and to induce growth cone col-
lapse (Bandtlow et al. 1993; Schwab and Caroni 1988).
Several molecules, mainly expressed in myelin, have been
described based on their ability to inhibit neurite outgrowth
and induce growth cone collapse in primary neuron cultures:
the myelin-associated inhibitors Nogo-A (Chen et al. 2000;
GrandPre et al. 2000; Prinhja et al. 2000), myelin-associated
glycoprotein (MAG) (McKerracher et al. 1994a), and oligo-
dendrocyte glycoprotein (OMgp) (Wang et al. 2002a), axon
guidance molecules ephrin B3 (Benson et al. 2005), Sema4D
(Moreau-Fauvarque et al. 2003), Sema 5A (Goldberg et al.
2004; Kantor et al. 2004), netrin-1 (Low et al. 2008), and
tenascin-R ( Joester and Faissner 2001).
716 • NEUROGLIA
3.2 C E N T R A L N E RVO US SYS T E M MY E L I N
C O N TA I NS N E RVE C E L L G ROW T H
I N H I B ITO RY M O L EC U L E S
3.2.1 Nogo-A
Nogo-A, OMgp, and MAG are synthesized by oligodendro-
cytes and are enriched in myelin sheaths. Nogo-A was extracted
from a neurite growth inhibitory protein fraction of rat, bovine,
and human spinal cord, respectively, and was shown to be a
highly nonpermissive substrate in cell culture assays (Caroni
and Schwab 1988b; Spillmann et al. 1998). A monoclonal anti-
body raised against the rat neurite growth inhibitory fraction
enabled fibroblast spreading and neurite outgrowth of CNS
neurons in vitro (Caroni and Schwab 1988a). In rat models of
spinal cord injury/SCI and stroke application of these anti-
bodies promote regeneration of injured nerve fibers leading
to improvements in functional recovery (Buchli et al. 2007;
Buchli and Schwab 2005; Markus et al. 2005; Papadopoulos
et al. 2002; Schnell and Schwab 1990; Seymour et al. 2005;
Tsai et al. 2007; Tsai et al. 2011; Zorner and Schwab 2010).
Neutralization of Nogo-A or suppression of its downstream
effectors is accompanied by increased long-distance regen-
eration of lesioned axons as well as compensatory sprouting
of unlesioned fiber tracts in rodents and monkeys (Freund
et al. 2006; Gonzenbach and Schwab 2008; Maier and Schwab
2006; Schwab 2004; Yiu and He 2006).
Next to being expressed on myelinating oligodendrocytes,
Nogo-A is also found on neurons where it modulates growth
cone motility and the dynamics of neurite formation (Montani
et al. 2009; Petrinovic et al. 2010) both during development
and after injury (Cheatwood et al. 2008).
Nogo-A signaling is complex with different functions dur-
ing development and in the adult organism (Schwab 2010).
In the mature CNS Nogo-A takes on the role of a negative
regulator of neuronal growth—as described—and that of a
modulator of LTP and synaptic stability (Aloy et al. 2006;
Delekate et al. 2011; Lee et al. 2008; Liu et al. 2003; Raiker
et al. 2010; Wang et al. 2002c), implying that it negatively
regulates both functional and structural plasticity in mature
neuronal networks.
3.2.2 MAG
MAG (Cao et al. 2010; Filbin 2008) is present in both
CNS and PNS myelin and constitutes a member of sialic
acid binding proteins as well as of the immunoglobulin
superfamily. Early during development MAG functions
as a neurite growth–promoting molecule ( Johnson et al.
1989), whereas in the mature CNS it takes on inhibitory
characteristics toward neurite outgrowth (McKerracher et al.
1994b; Mukhopadhyay et al. 1994). Cultures of dorsal root
ganglia growing on myelin of mice lacking MAG expression
made long extensions, whereas they barely grew on wild-
type myelin (Shen et al. 1998). However, MAG knock-out
mice did not show major effects on axonal regeneration after
optic nerve or spinal cord lesion (Bartsch et al. 1995), indi-
cating that MAG may not be involved in robust regeneration
after SCI.
 3.2.3 OMgp
OMgp (Cao et al. 2010; Filbin 2008) is a GPI-linked, highly
glycosylated membrane protein expressed in CNS myelin and
different types of neurons. It is a potent inhibitor of neurite
outgrowth in vitro (Wang et al. 2002b) and inhibits axon
regeneration in vivo as shown in OMgp knock-out mouse
studies (Huang et al. 2005). The same OMgp-deficient mice
exhibited elevated collateral sprouting at the node of Ranvier,
whereas OMgp mice with a mixed genetic background showed
increased functional and anatomical regeneration after SCI
( Ji et al. 2008).
Nogo-A, MAG, and OMgp, although very different struc-
turally, interact with the same receptor, NgR1, leading to
growth cone collapse and inhibition of neurite outgrowth in
vitro. However, the absence of NgR has no effect on inhibi-
tion of neurite outgrowth in culture, and there is no improve-
ment in corticospinal tract regeneration in vivo, suggesting the
existence of another, more specific Nogo-A receptor (Schwab
2010). On the other hand, axons become insensitive to MAG
or OMgp on cleavage of NgR and introduction of exogenous
NgR to otherwise insensitive neurons renders them respon-
sive to MAG or OMgp (Liu et al. 2002; Wang et al. 2002a).
Although Nogo-A (Schwab 2010), MAG (McKerracher and
Winton 2002), and OMgp (Dou et al. 2009) may be localized
on the surface of either oligodendrocytes or neurons, NgR and
a proposed unknown, Nogo-A specific receptor are expressed
on oligodendrocytes, where they assemble with different
membrane proteins to form a multimeric signaling complex.
Several signaling cascades that are not conclusively under-
stood seem to be involved in Nogo-A signaling (Gonzenbach
and Schwab 2008; Hannila and Filbin 2008; Joset et al. 2010;
Schwab 2010; Walmsley and Mir 2007).
3.3 O L I G O D E N D RO C Y T E I N JU RY C AUS E S C E L L
D E AT H, MY E L I N DA M AG E , A N D MY E L I N L O S S
Oligodendrocytes are particularly susceptible to toxic sub-
stances released to the close environment at the site of CNS
injury. They acutely undergo both necrosis and apopto-
sis, with apoptosis continuing until chronic time points.
Oligodendrocyte loss leads to demyelination (see chapter 52),
as well as impaired axon function and survival. In parallel, a
rapid and prolonged oligodendrocyte progenitor cell (OPC)
proliferative response occurs, especially at the lesion borders.
Proliferation of OPCs after injury is induced and controlled
by factors present at the site of injury, some of which are dis-
cussed in the following. Proliferating and migrating OPCs
differentiate into myelinating oligodendrocytes, which remy-
elinate demyelinated axons starting at 2 weeks post injury.
3.4 MO L ECU L A R C H A N G E S R E L EVA N T TO
N EU R IT E G ROW T H A N D R EG E N E R AT I O N
In addition to the neurite growth inhibitory factors present
in myelin, proteins regulating axonal guidance during devel-
opment contribute to the failure of mature CNS neurons to
regenerate after injury (reviewed by Bashaw and Klein 2010).
N E RVE F I B E R R E G E N E R AT I O N I N T H E C E N T R A L N E RVO U S SYS T E M O F H I G H E R VE RT E B R AT E S
Among these are growth factors, cytokines, trophic factors,
semaphorins, ephrins, Tenascin-R, Wnt, neugenin, neuronal
receptor protein tyrosine phosphatide sigma/Leukocyte
common antigen-related (RPTPsigma/LAR) and chondroi-
tin and heparan sulfate proteoglycans (CSPGs/HSPGs) on
astrocytes, which are described in the following. Through spe-
cific membrane receptors some of these guidance cues eventu-
ally converge onto the cytoskeleton, where they interact with
cytoskeletal and cytoskeletal-associated molecules within the
axon, whereas others influence protein synthesis in the soma
(Fig. 56.1).
3.4.1 Neurotrophin Signaling
The mechanisms controlling the intrinsic axon growth abili-
ties during regeneration are still poorly understood. The
tumor suppressor and tyrosine phosphatase PTEN regulates
the activity of several molecular pathways, such as mTOR
driven protein translation in the neuronal soma and cytoskel-
etal assembly in axons, which are required for successful axon
regeneration. PTEN inactivation results in the accumulation
of PIP3 and the activation of AKT (Song et al. 2006) acting
on different downstream effectors in the soma and axon ter-
minal, which in turn control axon growth and injury-induced
axon regeneration. Neurotrophin signaling converges on the
PI3K/AKT pathway. The phenotypes of complete or condi-
tional knock-out of involved genes of this pathway indicate
that the pathway plays an important role for axon growth
(Eickholt et al. 2007; Zhong et al. 2007). PIP3/AKT signal-
ing leads to mTOR activation, the eventual outcome of which
is an increase in protein synthesis, cell growth and nerve fiber
regeneration (Guertin and Sabatini 2007; Liu et al 2010; Sun
et al. 2011).
3.4.2 Insulin Growth Factor 1, Glial
Cell Line–Derived Neurotrophic Factor, and
Leukemia Inhibitory Factor
In vivo growth factors do not exist in isolation and are modu-
lated by other factors, making it difficult to pin down certain
biological effects to a single growth factor.
Insulin-like growth factor I (IGF-I) exhibits neuropro-
tective effects and promotes neuronal differentiation and
survival as well as myelin integrity and function (Carson
et al. 1993; Russo et al. 2005). After ischemia, expression of
IGF-1 is induced in reactive microglia, oligodendrocytes,
astrocytes, and surviving neurons of the periinfarcted area
with other factors transporting IGF-1 from the site of synthe-
sis to the sites of action (Russo et al. 2005) (see also chapters
18 and 58).
Glial cell line–derived neurotrophic factor (GDNF) has
potent neuroprotective and neurotrophic effects on differ-
ent neuronal cell types of the CNS. GDNF neurotrophic
effects are mediated by a multisubunit receptor consisting
of the GPI linked GFRalpha1 and the transmembrane pro-
tooncogene c-Ret. Neuroprotective effects of GDNF have
been shown after treatment of SCI rats with recombinant
GDNF (Iannotti et al. 2004). GDNF also enhances axonal
• 717

Retrograde transport to the nucleus along microtubules
nucleus
Neuronal growth machinery:
GAP-43, cytoskeletal proteins
Jak/Sat3 signalling (inhibited by SOCS3)
PI3K/AKT/mTOR signalling (inhib. by PTEN)
RhoA signalling
Figure 56.1 Mechanisms Regulating Neurite Growth in the Adult Central Nervous System. Different growth inhibitory and promoting factors are present
in the ECM and expressed on neuronal and glial cells opposing the growth cone of neurons. The interaction of such guidance cues with specific receptor
components on the membrane of the growth cone eventually leads to rearrangements of the cytoskeleton or activation of gene transcription in
the nucleus.
regeneration and myelination after spinal cord injury (Zhang
et al. 2009).
Axons of electrically active neurons release ATP, which in
turn stimulates the production and release of leukemia inhibi-
tory factor (LIF) from astrocytes enhancing myelination by
oligodendrocytes (Ishibashi et al. 2006) (see also chapters 20,
25 and 45).
3.4.3 Semaphorins
Semaphorins constitute a large family of secreted, membrane-
associated proteins the receptors, of which plexins and neuro-
pilins, are found on myelinating oligodendrocytes (Bannerman
et al. 2008; Spassky et al. 2002). Several members of class 3
semaphorins have been shown to act as axon guidance cues
during development. In the mature CNS the predominant
neural response to semaphorins (and ephrins) is repulsive.
Their presence suggests a role in network stabilization by nar-
rowing down neuronal growth and survival. A proposed role
in remyelination builds on the findings that Sema3A expres-
sion is induced after injury in the mature rat CNS (De Winter
et al. 2002). Sema3s are also expressed by meningeal cells
718 • NEUROGLIA
Attractors/growth promotors:
Neurotrophic factors
ECM, cell adhesion molecules (CAM)
wnt4
Glial or neuronal cell
Local
protein
interactions
Repulsors/growth inhibitors:
Nogo-A, MAG, Omgp
Proteoglycans, ECM (HA, CSPGs)
Ephrins, semaphorins
Netrin
RGMA/neogenin
Wnt, wnt5a
Tenascin-R
associated with CSPGs in the glial scar; in vitro blocking of
the Sema3-CSPG interaction inhibits Sema3A repulsion, sug-
gesting a role in enhancing the inhibitory function of the glial
scar (Pasterkamp and Verhaagen 2006). Members of other
classes of membrane-bound semaphorins have only recently
been implicated in the failure of regeneration of injured CNS
axons. Sema4D is expressed on myelinating oligodendrocytes
after CNS injury where it inhibits axonal growth (Moreau-
Fauvarque et al. 2003) and seems to regulate the survival of
neuronal cells (Giraudon et al. 2004). Similarly, after injury
Sema6B and Sema7A expression is upregulated in white
matter of the brain or spinal cord, respectively (Küry et al.
2004; Pasterkamp et al. 2007). The repulsive effect of sema-
phorins is mediated through interaction with plexin cell
surface receptors on neurons, leading to collapse of the actin
cytoskeleton and retraction of outgrowing nerve fibers (Hung
and Terman 2011).
3.4.4 Ephrins and Eph Receptors
Ephrins and their Eph receptors are membrane molecules
which—similar to semaphorins—play a role in axonal
pathfinding and target recognition during development
(Klein 2001) and some of which are constitutively expressed
in the mature CNS of rodents and humans (Sobel 2005).
EphrinAs are anchored to the membrane through a GPI tail,
whereas ephrinBs are transmembrane proteins with an intra-
cellular cytosolic tail. The tyrosine kinase receptors EphAs and
EphBs transmit ephrin-A and ephrin-B signaling, respectively
(Pitulescu and Adams 2010). Their role in the formation of the
glial scar, in neurite outgrowth inhibition and in apoptosis has
been studied in vitro, after spinal cord, brain, or optic nerve
injury and in transgenic animals (Yaron and Sprinzak 2012).
Of interest for regeneration after CNS injury is the
enhanced expression of ephrinB3 in CNS myelin, the upregu-
lation of ephrinB2 in reactive astrocytes, and the increase in
ephrinA5 expression around ischemic cortical lesions (Benson
et al. 2005). Next to their role as growth inhibitory cues, Eph-
ephrin signaling influences the formation of the glial scar
and apoptosis: After SCI in the rat EphA3 is upregulated in
reactive astrocytes (Irizarry-Ramirez et al. 2005) and EphA7
regulates apoptosis of astrocytes (Figueroa et al. 2006).
Functional recovery has been described after blocking differ-
ent Ephrin-Eph interactions (Yaron and Sprinzak 2012).
3.4.5 Netrins
A third family of axonal guidance molecules involved in axon
regeneration are the secreted netrins. In the adult CNS, netrins
are expressed by neurons and myelinating oligodendrocytes
(Manitt et al. 2001). The response to netrin-1 is regulated by
the repertoire of netrin receptors expressed and their presenta-
tion on the cell surface as well as protein kinases PKA and PKC
within the neuron (Bouchard et al. 2004; Hong et al. 1999;
Williams et al. 2003). In the visual system, netrin-1 and its
receptors DCC and Unc5c are expressed by nonlesioned adult
retinal ganglion cells. After SCI, expression of the attraction-
mediating netrin-1 receptor DCC decreases, whereas that of
the repulsive receptor Unc5c returns to normal levels (Manitt
et al. 2006). In areas surrounding the lesion site levels of
netrin-1 remain unchanged in neurons and oligodendrocytes.
Taken together, netrin-1 acts as an inhibitor of axon regenera-
tion in the mature CNS and may play a role in myelination.
3.4.6 RGMA/Neogenin
The repulsive guidance molecule A (RGMA) accumulates in
the glial scar after SCI and induces growth cone collapse in
vitro. Under pathological conditions, RGMA accumulates
at the lesion site (Schwab et al. 2005). Inhibition of RGMA
with a function blocking antibody after SCI in the rat model
enhances growth of injured axons, promotes functional recov-
ery (Hata et al. 2006), and induces plastic changes such as
synaptic rearrangements of spared axonal projections (Kyoto
et al. 2007). This suggests that re-expression of embryonic
repulsive cues in adult CNS contributes to the failure of axon
regeneration in the CNS (Yamashita et al. 2007).
Repulsive guidance molecule A signaling converges on
the Rho/ROCK pathway in neurons through binding to
neogenin on the surface of neurons. Activation of RhoA is
N E RVE F I B E R R E G E N E R AT I O N I N T H E C E N T R A L N E RVO U S SYS T E M O F H I G H E R VE RT E B R AT E S
important for neurite growth inhibition and growth cone
collapse mediated also by other inhibitory proteins such as
Nogo-A, MAG, OMgp (via NgR signaling), Wnts, ephrin-A5,
or Semaphorin-3A.
3.4.7 Wnt Signaling
Wnts are axon guidance molecules particularly well described
for their role in developmental processes but which are also
involved in regeneration. Wnts bind to membrane bound
receptors that converge on downstream beta-catenin signal-
ing, activating gene expression, and resulting in the regulation
of a variety of processes.
Three Wnts have been shown to be expressed after CNS
damage. Wnt1, Wnt5a (both repel descending CST axons
during development), and the repulsive Wnt receptor Ryk are
expressed in spinal cord gray matter after SCI, whereas Wnt4
(attracts ascending sensory neurons during development) is
induced close to the lesion site (Liu et al. 2008). After SCI,
function blocking antibodies against Ryk promote axonal
sprouting beyond the lesion, implying that Wnt signaling
plays a role in inhibiting axon sprouting after injury.
3.4.8 Hyaluronic Acid and Chondroitinsulfate
Proteoglycans
Degradation of the ECM component hyaluronic acid (HA)
induces activation and proliferation of astrocytes (Struve et al.
2005). In vitro, HA decreases cell proliferation and reduces
CSPG production (Khaing et al. 2011). The CSPGs neurocan,
versican, aggrecan, and brevican can be anchored within the
ECM through binding to HA glycosaminoglycan, and treat-
ment with hyaluronidase leads to degradation of HA, result-
ing in the release of these CSPGs from the ECM. Indeed,
lesion of the rat nigrostriatal tract followed by treatment with
hyaluronidase led to partial degradation of HA and chondroi-
tin sulfate as well as depletion of HA-binding CSPGs, which
in turn enhanced local sprouting at the lesion site (Moon et al.
2003). This indicates that these three ECM components—HA,
chondroitin sulfate, and HA-binding CSPGs—contribute to
the lack of spontaneous axon regeneration after CNS injury.
3.4.9 Tenascins
Tenascins constitute a family of extracellular matrix (ECM)
glycoproteins known to regulate axonal guidance during
development (Joester and Faissner 2001), but also involved in
myelination and cell adhesion to fibronectin. Tenascin-R is also
expressed in the mature CNS, where its expression is restricted
mainly to oligodendrocytes and their precursors. It is secreted
into the ECM and is upregulated at lesion sites after injury. In
vitro it is inhibitory for retinal ganglion cell outgrowth and its
expression in the optic nerve persists after injury (Becker et al.
2000). Tenascin-R exerts its inhibitory function through bind-
ing to different membrane-bound molecules (Sandvig et al.
2004). ChABC treatment of tenascin-R neutralizes binding to
the heparin-binding fibronectin fragment which in turn pro-
motes cell adhesion (Probstmeier et al. 2000) (Fig. 56.2).
• 719
 Activated astroglia

Activated microglia
Oligodendrocytes
Differentiating OPC
Myelin debris Lesion cavity

Demyelinated axons Remyelination Sprouting axons

Figure 56.2 The Central Nervous System Environment Is Hostile for Axon Regeneration and Repair. After CNS injury nerve fibers are transected at the
site of injury and the surrounding tissue is damaged (red). During the early phase of injury, axon regneration is inhibited by myelin-associated inhibi-
tors (e.g., Nogo-A, MAG, EphA3, Sema3A) expressed on intact and damaged oligodendrocytes. Recruitment of inflammatory microglia and reactive
astrocytes to the site of lesion eventually leads to the formation of a glial scar, associated with an increased release of growth inhibitory CSPGs.
Spontaneously occurring processes supporting regeneration and repair (green) include the early neuroprotective effect of astrocytes, the astrocytic
production of antioxidants, the differentiation of OPCs into myelinating oligodendrocyes, and short-range regeneration and sprouting of axons.

3.5 A S T RO C Y T E R E S P O N S E TO I N JU RY well as the damaged axon. An attribute of the cellular response

Reactive astrocytes play a key role in neurotrauma, with
different roles during the course of the damage (see also
chapters 32, 51 and 57).
At an early stage they show neuroprotective functions,
whereas at later stages they facilitate the formation of a glial
scar and inhibit CNS regeneration. Formation of a glial scar
results from the proliferation of astrocytes, oligodendrocyte
progenitor cells, meningeal fibroblasts, and pericytes migrat-
ing into the lesion (Chen et al. 2002; Göritz et al. 2011).
In response to CNS injury, reactive gliosis takes place,
which is characterized by hypertrophy of the cell bodies and
processes, altered gene expression, an increase in the expression
of the glial intermediate filament marker GFAP, of nestin and
vimentin, and proliferation bordering the wound that occurs
in a graded fashion in relation to the severity of the injury.
Glial scar formation is influenced by interleukins, trophic fac-
tors, and cell adhesion molecules. The astrocyte-rich glial scar
has physical and chemical inhibitory properties: The processes
of astrocytes become hypertrophied and interdigitated, form-
ing a physical barrier for outgrowing neurites, and they express
inhibitory chondroitin sulfate proteoglycans (CSPGs). In
addition, injury astrocytic gap junctions remain open for sub-
stances such as proapoptotic factors, contributing to further
worsening of the cell injury (Giaume et al. 2010).
Next to enhancing glial scar formation in response to CNS
injury, astrocytes are important contributors to neuroprotec-
tion and repair (Ridet et al. 1997). The regulation of astro-
cytic production of growth factors and cytokines is complex
and involves the cross-talk between different types of glia as
to CNS injury is a rapid increase in damaging free radicals in
both astrocytes and neurons, in response to which astrocytes
produce beneficial antioxidants to enhance neuronal survival.
Studies with GFAP knockout mice suggest that modulat-
ing the astrocytic response can be detrimental as well as bene-
ficial. These mice exhibit larger lesions than wt mice following
brain injury, whereas mice lacking both GFAP and vimentin
have impaired astrocyte activation and decreased glutamate
uptake abilities but at the same time reveal posttraumatic
regeneration (Wilhelmsson et al. 2004).
3.6 M I C RO G L I A A F T E R C E N T R A L
N E RVO US SYS T E M I N JU RY
The roles of microglia in the developing and adult CNS are
covered by several chapters within this book (e.g., chapters
47–49). Here, we briefly summarize their roles in CNS trauma
and regeneration.
Microglial cells react within minutes to traumatic tissue
destruction by increased motility, phagocytosis, and release of
different factors such as pro-inflammatory and anti-inflamma-
tory cytokines, free radicals, antioxidants, and neurotrophic
and angiogenic factors. Depending on the microenvironment
and their state of activation, they can exacerbate neurodegen-
eration or in contrast take on an important role in tissue repair
and regeneration (Singh et al. 2011).
Activated microglia can promote axon regeneration after
CNS injury (Wake et al. 2009) and contribute to the plasticity
of neuronal circuits. Resting microglia contact neuronal syn-
apses through their processes in a neuron activity–dependent

720 • NEUROGLIA
manner. After cerebral ischemia, these contacts are markedly
prolonged and can lead to the disappearance of synapses, sug-
gesting that microglia monitor and react to the functional sta-
tus of synapses (Wake et al. 2009).
Next to the glial–neuronal interactions the cross-talk
between microglia and astrocytes seems to be pivotal for
microglia activation. Intercellular astrocyte communication
is typically mediated by gap junctions. Proinflammatory
cytokines secreted by activated microglia inhibit astrocyte gap
junction communication influencing the role of astrocytes in
providing neuronal support (Retamal et al. 2007).
4 R E G E N E R AT I VE A N D C O M P E N S ATO RY
F I B E R G R OW T H
After damage or injury to the CNS, for example, after SCI or
stroke, different types of recovery mechanisms can take place:
(1) regeneration of damaged axons involving elongation of the
damaged axon end leading to reconnection with deafferentated
targets, and (2) compensatory sprouting of undamaged spared
axons to form new axonal connections with deafferentated tar-
gets. The latter process is also termed compensatory plasticity.
Interestingly, these two different recovery processes share similar
molecular mechanisms, leading to regrowth and regeneration.
Several neuronal intrinsic mechanisms are involved in
axon growth and regeneration (Liu et al. 2011). MAPkinase,
Jak/Stat, and Akt/mTOR signaling play important roles for
the activation of the growth program in adult neurons (Gupta
et al. 2011; Watanabe et al. 2011). Activation of these pathways
externally (e.g., by neurotrophic factors) or internally, by dele-
tion of crucial pathway inhibitors such as SOCS3 or PTEN
were recently shown to induce massive enhancement of fiber
sprouting and regeneration after optic nerve or spinal cord
lesion (Liu et al. 2010; Sun et al. 2011). Cytoskeletal mRNAs
and proteins, regulatory proteins such as GAP-43, tau, and
MAPs, or the actin regulators cofilin/profilin, as well as cell
adhesion molecules are upregulated, allowing the formation
of growth cones, sprouts, and elongating axons.
A real gap of knowledge currently exists with regard to mech-
anisms of guidance and target finding for regenerating axons in
the adult CNS. The tissue composition, size, and architecture
are radically different from the developing CNS when these
fiber tracts originally grew. Information on detailed patterns of
guidance and growth factor expression in the adult, injured, or
denervated CNS is rare. Still, in the spinal cord, regenerating
corticospinal, red nucleus or serotonergic axons follow a rostral
to caudal direction, and the reverse is true for ascending sensory
fibers, suggesting the presence of rostrocaudal cues. The posi-
tion of the fibers is often abnormal, however. Detailed stud-
ies on regulation of branch formation, target recognition, and
specificity of synapse formation are lacking at present.
4.1 T R E AT M E N T S T R AT E G I E S
Multiple attempts have been undertaken to enhance the lim-
ited spontaneous recovery occurring after damage to the CNS
in experimental animal models and some are on their way to
N E RVE F I B E R R E G E N E R AT I O N I N T H E C E N T R A L N E RVO U S SYS T E M O F H I G H E R VE RT E B R AT E S
translation for human patients. The main treatment strategies
include neurorehabilitative training—the only treatment that is
routine in clinical settings today. Neurorehabilitation results in
structural rearrangements, upregulation of growth factors, adhe-
sion molecules, and components of the synapse in spinal cord
injured rats. Experimental and up-coming treatments include,
neuroprotective and antiinflammatory treatments, promotion
of fiber regeneration and compensatory sprouting, transplanta-
tion of bridges or stem cells to allow for growth over the lesion
and replacement of lost cell populations, and pharmacological or
electrophysiological manipulation of residual networks. Several
of these treatment strategies disclose beneficial effects in animals
on the anatomical and/or functional level (Giger et al. 2010;
Kwon et al. 2010, 2011; Noble et al. 2011; Tetzlaff et al. 2011;
Zorner and Schwab 2010). Recent reviews on clinical trials in
spinal cord injury include Zorner and Schwab (2010) and Kwon
et al. (2010, 2011).
4.1.1 Promotion of Fiber Regeneration
and Compensatory Sprouting
Initial molecular screens revealed the enhanced expression of
neurotrophic factors, axonal guidance molecules, and ECM
proteins in denervated spinal cord tissue, along with enhanced
expression of growth-associated and cytoskeletal proteins in
neurons (Bareyre and Schwab 2003; Bareyre et al. 2002), mak-
ing them key targets for regenerative treatment (Fig. 56.3).
Inactivation of Nogo-A, NgR, CSPGs and their down-
stream signaling molecules leads to long-distance regeneration
of transected axons, and enhanced compensatory fiber growth
(Carulli et al. 2005; Fournier et al. 2001; Schwab 2010). These
processes are paralleled by functional recovery pointing to the
formation of new networks and functional reorganization
taking place at different levels (Bradbury et al. 2002a; Li et al.
2004; Merkler et al. 2001). In a rat model of SCI sprouting of
axons into deafferentated areas of the spinal cord is paralleled by
improved functional outcome (Raineteau and Schwab 2001).
Similar effects occur in monkeys subjected to high cervical
lesions. Anti-Nogo-A antibody treatment specifically enhances
recovery of manual dexterity, sprouting and regeneration of the
corticospinal tract at the level of the spinal cord (Freund et al.
2006, 2007). Based on these preclinical data a clinical trial apply-
ing human anti-Nogo-A antibodies to paraplegic and tetraple-
gic subjects is currently ongoing (http://clinicaltrials.gov/ct2/
show/NCT00406016).
Prevention of the scar formation is not yet possible but
the inhibitory effects of CSPGs can be counteracted with
experimental ChABC treatment. After SCI, treatment with
ChABC enhances axonal regeneration (Moon et al. 2001;
Zuo et al. 1998), the restoration of postsynaptic activity and
functional recovery (Bradbury et al. 2002b).
Because of their ability to enhance the intrinsic cell
response of damaged neurons after injury, different trophic
factors have been applied after experimental SCI. Various
modes of delivery have been applied such as viral systems, and
grafts of genetically modified cells that secrete these factors,
pumps, or injections (Lacroix and Tuszynski 2000).
Application of neurotrophic factors results in neurite growth
• 721
 A

Figure 56.3 Nerve Fiber Regeneration Can Be Enhanced After Central Nervous System Injury. After spinal cord injury in the rodent, axon regeneration
and sprouting can be encouraged around the lesion site by blocking neurite growth inhibiting ECM molecules (e.g., CSPGs), membrane-associated
proteins (e.g., Nogo-A), or downstream signaling molecules (e.g., PTEN, RhoA). A. In PTEN knock-out mice some axons grow to the distal spinal
cord regeneration e.g. 5mm caudal of the injury. B. In rats, after spinal cord lesion and ChABC treatment regenerating fibers transverse the lesioned
tissue. C. Spinal cord injured rats treated with anti-Nogo-A antibodies show regenerative sprouting (left) and fiber regeneration. D. Treatment with
C3 transferase to inactivate Rho stimulates axon regeneration after SCI. (A) From Liu et al. 2010. (B) From Bradbury et al. 2002. (C) From Liebscher
et al. 2005. (D) From Dergham et al. 2002. All are reprinted with permission.

722 • NEUROGLIA
promotion and long-distance regeneration as well as functional
recovery in some models (Bregman et al. 2002; Lacroix and
Tuszynski 2000).
4.1.2 Transplantation of Bridges or Stem Cells
The topic of cell-based therapies is discussed in chapter 57.
A major obstacle for all cell replacement approaches—
including transplanted cells, tissues, or synthetic bridges—is
that after passing the bridge regenerating axons must re-enter
the CNS tissue to find their targets. This process is severely
hampered by scar- and myelin-associated inhibitory factors.
Different cell-based transplantation strategies attract and
support growing axons in the experimental animal model
(Rossi and Keirstead 2009). Some transplanted cells provide
cell replacement (e.g., for oligodendrocytes or neurons), which
assume relay functions (Cummings et al. 2006; Hooshmand
et al. 2009; Sharp et al. 2011), whereas others seem to confer
trophic or immunomodulatory effects.
4.1.3 Neuroprotective and Antiinflammatory
Treatments
Treatment with high doses of methylprednisolone, a cor-
ticosteroid with antioxidant neuroprotective effects in
the animal, within the first hours after injury had modest
beneficial effects also in acute SCI patients (Bracken et al.
1998). Because of the small size of the effects and the sub-
stantial side effects, the clinical use of methylprednisolone
therapy is being discontinued in many places at present.
Another experimental approaches for neuroprotection
in CNS-injured animals are the blockade of Rho GTPase
(Dubreuil et al. 2003). Rho blockade can also dampen the
regeneration inhibitory effects of Nogo-A and other growth
inhibitors. A Rho-blocking drug, applied as a depot epidu-
rally on the acutely injured spinal cord, is currently under
clinical investigation (http://clinicaltrials.gov/ct2/show/
NCT00610337).
5 M Y E L I N R E PA I R A F T E R C E N T R A L
N E RVO U S SYS T E M I N J U RY
In the CNS, neurons and glia share a mutual dependence
that becomes particularly evident in the process by which glia
forms myelin around axons. Signals regulating myelination are
discussed in chapters 43–45.
Oligodendrocytes are vulnerable by many influences that
occur at sites of CNS injury and inflammation. Therefore,
demyelination has been considered a major problem for
incomplete lesions in which spared axons can be nonfunc-
tional because of local loss of myelin (Arvanian et al. 2009;
Totoiu and Keirstead 2005). Oligodendrocyte proliferation
does happen, but the numbers of new oligodendrocytes
around spinal cord lesion sites is modest and high num-
bers of demyelinated axons have been reported in human
post-mortem material (Guest et al. 2005). In contrast with
N E RVE F I B E R R E G E N E R AT I O N I N T H E C E N T R A L N E RVO U S SYS T E M O F H I G H E R VE RT E B R AT E S
humans, myelin repair in the mouse was much more suc-
cessful (Lasiene et al. 2008). Oligodendrocyte replacement
by transplantation of precursor cells is a therapeutic option
that is currently considered for clinical trials. The use of oli-
godendrocyte-targeted mitogens or differentiation factors
to enhance myelin repair at sites of CNS trauma has not
been sufficiently tested experimentally so far.
6 S U M M A RY A N D P E R S P E C T I VE S
Much insight has been gained into the complex cross-talk
between neurons and different glial cells following CNS
injury. Cellular, molecular, and secreted factors have been
described, some of which influence nerve fiber sprouting,
regeneration, and functional recovery in experimental animal
models. Treatments to counteract some of the cues that nega-
tively control neurite outgrowth, to bridge the growth imped-
ing glial scar or boost plasticity are on their way to translation
for future therapies of spinal cord injury, brain injury, and
stroke.
AC K N OW L E D G M E N T S
The authors thanks the Journal of Neuroscience, Annals of
Neurology, Nature, and Nature Neuroscience for their permis-
sion to reproduce published material in this chapter.
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• 727
 57.
GLIAL CELL TRANSPLANTATION FOR CENTRAL
NERVOUS SYSTEM REPAIR
Anne Baron-Van Evercooren and Rebecca Matsas

A B B R E VI AT I O N S entered the segments of a peripheral nerve implanted into the

lesioned brain, and were myelinated by Schwann cells (SCs), the
BC boundary cap peripheral myelin-forming cells (Cajal 1928; Tello 1911). More
BMSC bone marrow mesenchymal cell than half a century later, William Blakemore demonstrated that
cAMP cyclic adenosine monophosphate SC remyelination occurred following placement of peripheral
CNS central nervous system nerve fragments over the areas of demyelination in the spinal
EAE experimental autoimmune encephalomyelitis cord (Blakemore 1977). Soon afterward Richardson et al. (1980)
ES embryonic stem cell published a landmark study that greatly influenced subsequent
FGF fibroblast growth factor research, showing that it was possible to bridge a complete
iPS induced pluripotent stem cell transection gap in the spinal cord with a peripheral nerve
LPC lysophosphatidylcholine implant. It was obvious that if the environment is permissive,
MBP myelin basic protein axons from central neurons are able to respond and regrow, and
MLD metachromatic leukodystrophy peripheral type myelination of CNS axons soon was described
MSC mesenchymal stem cell following engraftment of purified SCs (Duncan et al. 1981).
NCC Neural crest cell Madeleine Gumpel et al. then demonstrated for the first time
NC-SC Neural crest-derived stem cell that CNS fragment implantation could also lead to CNS-type
PS-NCAM poly-sialated-neural cell adhesion molecule myelination in myelin mutants (Lachapelle et al. 1983). Further
NPC neural stem/precursor cell experiments showed that graft-mediated oligodendrocyte
OEC olfactory ensheathing cell remyelination could be achieved in situations in which
OEp Olfactory ensheathing cell progenitor endogenous myelination/remyelination fails (Blakemore and
OPC oligodendrocyte progenitor cell Crang 1988). In the 1990s, Franklin et al. (1996) demonstrated
PDGF platelet-derived growth factor that transplantation of olfactory ensheathing cells (OECs)
PNS peripheral nervous system could also lead to robust remyelination of demyelinated CNS
PSA polysialic acid axons. Shortly afterward, stem cells derived from both neural
SC Schwann cell tissues (Hammang et al. 1997) and embryonic tissues (Brustle
SCI spinal cord injury et al. 1999) were identified as a promising source of myelin
SCP Schwann cell precursors following transplantation into myelin-deficient animals,
opening the field of cell therapy approaches based on ES- and
iPS-derived myelin forming cells.
1 INTRODUCTION
The complex changes occurring in spinal cord tissue
following injury have led to the idea of using combinatorial
This chapter provides an overview of the current knowledge
strategies. To repair the injured spinal cord it is essential to
relating to the use of transplanted glial cells and stem cells in the
limit the spread of secondary tissue damage, provide trophic
treatment of myelin diseases and spinal trauma. The chapter covers
support for neuroprotection, manipulate inflammation and
anatomical repair, including neuroregeneration and myelin repair,
scar formation, neutralize inhibitory molecules, promote
as well as bystander effects mediated by activation of endogenous
axonal growth and remyelination, and restore damaged
glia (oligodendrocyte progenitors, astrocytes) and modulation of
connectivity. Such combinatorial interventions include cellular
neuroinflammation.
transplants coupled with administration of neuroprotective
agents and growth factors, guidance channels to support
2 H I S TO RY O F C E L L T R A N S P L A N TAT I O N regrowing axons, addition of the enzyme chondroitinase to
F O R C E N T R A L N E RVO U S SYS T E M reduce inhibitory molecules or elevation of cyclic adenosine
M Y E L I N R E G E N E R AT I O N monophosphate (cAMP) to enhance axonal regeneration and
remyelination (Fouad et al. 2005; Pearse et al. 2004; Xu et al.
Santiago Ramon y Cajal and Francisco Tello were the first 1995). Although much of the work reported here is driven by
to indicate that central nervous system (CNS) axons can be exploration of the therapeutic potential of transplanted cells,
myelinated by exogenous cells. They observed that CNS axons these studies also stress the power of cell transplantation to
728
unravel the biology of glial cells during developmental and
pathological conditions.
3 TA R G ET D I S E A S E S
Transplantation of myelin-forming cells is considered as a
potential treatment for leukodystrophies, multiple sclerosis,
and spinal cord injury.
The wide family of leukodystrophies are congenital myelin dis-
orders characterized by genetic abnormalities resulting in a failure
of myelin sheath formation and/or maintenance (see chapter 62).
The mutations target genes encoding either proteins or enzymes
involved directly or indirectly in the development or maturation
of oligodendrocytes. They can lead to hypomyelination (lack
of myelin), dysmyelination (abnormal myelin), or demyelina-
tion (myelin breakdown) and are characterized by severe motor,
sensory, and cognitive neurological deficits. One could there-
fore speculate that in some of these diseases such as Pelizaeus
Merzbacher disease (PMD) introduction of genetically normal
myelinating cells could correct the genetic deficit in myelina-
tion. Transplanted cells may also serve as vehicles for the delivery
of normal enzymes to the endogenous cells, thus improving the
functional deficit (Goldman and Windrem 2006).
Multiple sclerosis (MS) is an inflammatory CNS disorder
characterized by disseminated demyelination and axonal loss
(see chapter 61). Even though remyelination occurs during
the early disease stages, it is progressively impaired, resulting
ultimately in lesions consisting of chronically demyelinated
axons, axonal loss, and reactive gliosis. Given that axonal loss is
the leading cause of neurological disability characterizing MS
patients and that normal myelin is required for axonal integ-
rity (Kornek et al. 2000), remyelination therapies are neces-
sary to ensure restoration of axonal function. In addition to
myelin reconstruction, further benefits of cell therapy for MS
have been suggested because transplanted cells may contribute
to axonal protection by suppressing ongoing inflammation
and activating endogenous repair.
Spinal cord injury (SCI) is one of the most devastating condi-
tions that affect the CNS and can lead to permanent disability.
It is characterized by lesions of CNS axons and a primary loss
of cells at the site of injury. This results in posttraumatic inflam-
matory responses, including activation of resident microglial
cells and infiltration of circulating immune cells, which together
with cytotoxic events, lead to secondary damages. These include
formation of fluid-filled cysts, demyelination, and degeneration
of axons. As a response to injury, a glial scar is formed primarily
by activated proliferating hypertrophic astrocytes or pericytes
(Goritz et al. 2011) generating a physical and chemical barrier
that seals off the injured tissue and restricts inflammation and cell
death. Although beneficial for tissue preservation during the early
stages after injury, eventually it creates a serious obstacle to regen-
eration. Inhibitory molecules, such as chondroitin sulfate proteo-
glycans (Dou and Levine 1994), are upregulated within the glial
scar, whereas myelin and myelin debris also constitute a major
source of factors preventing axonal growth and regeneration
(Caroni and Schwab 1988). Even after severe injuries some sur-
viving axons persist in the subpial rim of white matter, but exhibit
demyelination and consequently dysfunctional conduction
G L I A L C E L L T R A N S P L A N TAT I O N F O R C E N T R A L N E RVO U S SYS T E M R E PA I R
properties (Nashmi and Fehlings 2001). Because differentiation
of endogenous oligodendrocyte progenitors (OPC) into oligo-
dendrocytes following SCI is not sufficient to achieve remyelina-
tion, myelinogenic cell engraftment has been proposed as a means
to promote axonal regeneration, restore myelination, and protect
denuded axons against subsequent degeneration. Engineering the
transplanted cells to deliver growth factors associated with neural
protection and regeneration to the lesion site is also attempted, so
as to optimize the functional benefits.
4 ANIMAL MODELS
Animal models of myelin diseases in which either myelin is
the primary target or demyelination is secondary to axon dam-
age are distinguished here.
4.1 A N I M A L M O D E L S O F MY E L I N D I S E A S E S
Two groups of models are used to study remyelination by grafted
cells. These are models of hypomyelination/dysmyelination, and
models of induced demyelination of the adult CNS. Each of these
models has advantages and disadvantages. The primary character-
istic is that a substantial proportion of axons is available for the
transplanted cells to myelinate and therefore serves to assay the
biology and therapeutic potential of various cell preparations.
4.1.1 Animal Models of Hypomyelination/
Dysmyelination
This group includes animals characterized by (1) aberrant myelin
sheath production or maintenance owing to a defect in myelin
protein–related genes (myelin mutants), or (2) progressive myelin
loss resulting from abnormal accumulation of metabolites owing
to enzymatic deficiencies (models of Krabbe disease and metach-
romatic leukodystrophy). Because most myelin mutants are short
lived, transplantation is usually performed in developing animals.
Neonate recipients have a highly permissive environment for
myelination because transplanted cells respond to developmen-
tal cues instead of reflecting chronic disease situation. In the case
of mutants with an extended lifespan (e.g., canine shaking pup),
transplantation has been performed into adult animals (Archer
et al. 1997). One of the advantages of these models is that myelin
produced by the transplanted cells can be readily recognized
because of its normal composition. For example, MBP posi-
tive normal myelin is easily recognized in the shiverer mouse by
immunohistochemistry and electron microscopy (Lachapelle
et al. 1983). Myelin mutants are classified into groups according to
the myelin protein–related gene affected (for review see Duncan
et al. 2011. Major characteristics are listed in Table 57.1.
4.1.2 Induction of Demyelination in the Adult
Central Nervous System
Models of induced demyelination include toxin-induced
demyelination and induction of inflammatory pathologies of
the adult CNS. In toxin-induced models, cell grafting is gener-
ally performed directly in the parenchyma, whereas in inflam-
matory models cells are introduced in the venous or ventricular
• 729
Table 57.1 MYELIN MUTANTS
SURVIVAL/CLINICAL
MUTANTS SPECIES DEFECT SIGNS PATHOLOGY DISEASE

Shiverer Mouse Deletion of 7 out of 9 2–3 Months Dysmyelination : lack of —

Mikoshiba et al. 1991 exons in MBP gene shivering MBP protein leading to
seizures uncompacted myelin

Long Evans Shaker (les) Rat Disruption of MBP 6–9 Months Dysmyelination/ —
Kwiecien et al. 1998 gene: insertion of demyelination lack of
retrotransposon in MBP protein leading to
noncoding region uncompacted myelin

Jimpy Mouse Base pair substitution 1-Month Oligodendrocyte death Severe form of
in 4 introns of seizures Hypomyelination PMD
PLP1 gene on X and mild inflammation
chromosomes Dysmyelination: remaining
myelin poorly compacted
Lack of PLP protein

Myelin deficient Rat X-linked mutants 23–25 Days Oligodendrocyte death Severe form of
Duncan and Milward 1995 with effected PLP1 Severe hypomyelination PMD
gene Remaining myelin lacks PLP
protein

PLP overexpressor Mouse PLP1 gene 23 Days (homozygous) Hypomyelination/ Severe form of
(PLOA) dysmyelination PMD
Griffiths et al. 1995 and major inflammation
2 Months Hypomyelination after 3–4
(heterozygous) months

Bradl et al. 1999 Rat PLP1 gene 23–24 Days Dysmyelination

Shaking pup Dog Point mutation in >2 Years Dysmyelination/ PMD

Archer et al. 1997 PLP1 gene hypomyelination

Mag-fyn KO Mouse Deficient for MAG Normal Hypomyelination

Biffiger et al. 2000 and fyn

Pex5 Mouse PEX5 12 Months Demyelination, ALD

Kassmann et al. 2007 axon loss, inflammation

Taiep Rat 17 Months

Duncan et al. 1992

Twitcher Mouse Galactocerebrosidase 34–45 Days Demyelination CNS Krabbe Globoïd

Duchen et al. 1980 and PNSecurreloboïd Leukodystrophy
macrophages

Primate

Canine

Arylsulfatase A (ASA) KO Mouse ASA 2 Years Sulfatide storage MLD

Hess et al. 1996 No obvious demyelination
Aspartoacylase KO Mouse Aspartoacylase 2–9 Months Myelin vacuolation, no Canavan disease
Matalon et al. 2000 demyelination

MBP, Myelin basic protein; MLD, metachromatic leukodystrophy; PLP, proteolipid protein; PMD, Pelizaeus-Merzbacher disease.

systems (reviewed in Tepavcevic and Baron-Van Evercooren
2008). Focal models offer the possibility to graft cells either
directly in the lesion to assay their repair potential or remotely
form the lesion, to assay their ability to migrate. Their char-
acteristics are summarized in Tables 57.2 and 57.3. Several
models are characterized by more complex pathophysiologi-
cal features than gliotoxin-induced models of demyelination,
including clinical deficits. These include experimental auto-
immune encephalomyelitis (EAE), demyelination induced
by intraparenchymal zymogen injection and various virus-
induced demyelinating diseases (see Table 57.3). These models
offer an environment required to address more complex issues
for cell therapy such as the ability of cells to gain access to
lesions dispersed throughout the CNS, their survival within
an inflammatory environment characterized by recurrent epi-
sodes of demyelination, as well as the ability of transplanted
preparations to sustain clinical recovery via neuroprotective
and immunomodulatory properties.

730 • NEUROGLIA
Table 57.2 INDUCTION OF NONIMMUNE DEMYELINATION
AGENT/MODE SPECIES EXTENT CHRONICITY MODE PATHOLOGY

Lysolecithin
intra-CNS injection
Hall et al. 1972
Ethidium bromide (EB)
Intra-CNS injection
Mouse, rat, rabbit, Focal Acute Membrane Focal loss of oligodendrocytes
cat, primate solubilizing agent and myelin, axon sparing, little
inflammation, followed by
remyelination.
Remyelination slower in
rabbits and nonhuman
primates than rodents
Rodents Focal but more DNA-intercalating Focal loss of oligodendrocyte
extended agent and astrocytes leading to
than LPC demyelination, axon sparing,
lesions little inflammation

EB alone Acute Remyelination

Blakemore et al. 1982

EB combined with Chronic No remyelination

X-irradiation
Blakemore and Paterson,
1978
Cuprizone Rodents More diffuse Cuper chelator Widespread loss of
Diet oligodendrocytes, axon loss
Matsushima and
Morell 2001

6 wk Acute Remyelination
12 wk Chronic No remyelination

Table 57.3 INDUCTION OF IMMUNE-MEDIATED DEMYELINATION

INDUCTION MODE SPECIES EXTENT CHRONICITY MODE PATHOLOGICAL FEATURES DISEASE

Theilers’ virus (TMEV) Mouse Diffuse Persistent Viral- mediated Infection of neurons spreading MS
Lipton et al. 2005 to glial cells. including
oligodendrocytes, neuronal
degeneration, astrogliosis
Immune infiltrates
Mouse hepatitis virus Mouse Diffuse Acute Viral-mediated Transient replication in MS
(MHV) oligodendrocytes, leading to
Kristensson 1986 demyelination followed by slow
remyelination
EAE, peripheral Mouse, rat, Diffuse Monophasic, Immune-mediated Rupture of BBB, immune According to
injections of CNS guinea pig, relapse-remitting infiltrates, astrogliosis, axone loss, models :
homogenates, myelin, monkey (RR), oligodendrocyte ecurrentcurent RR, SP, or
myelin proteins, or primary (PP), events of demyelination followed PP MS
synthetic peptides and secondary by events of remyelination for
Gold et al. 2006 progressive (SP) RR, PP, and SP

Peripheral immunization Mouse, Rat Focal No prominent Immune-mediated Similar signs but localized in the Chronic MS
with myelin peptides clinical signs area of cytokine injections
followed by intra CNS
injections of cytokines
Kerschensteiner et al.
2004
Zymozan ip injection Rat Focal — Toll receptor-2 Focal inflammation, MS-like
Popovich et al. 2002 agonist demyelination, and axon loss

4.2 S P I NA L C O R D I N J U RY MO D E L S
As mentioned, remyelination of demyelinated axons follow-
ing spinal cord injury appears fundamental to achieve func-
tional recovery. However, in addition to remyelinating the
axons in the lesion, transplanted cells should ideally also sup-
port CNS axonal elongation and outgrowth. Available models
reflect various degrees of traumatic injury and include partial
or complete spinal cord transection, compression, or contu-
sion injury. Most cell transplantations are delivered directly at

G L I A L C E L L T R A N S P L A N TAT I O N F O R C E N T R A L N E RVO U S SYS T E M R E PA I R • 731

the site of injury or adjacent to it by injection of a cellular sus-
pension via fine needles or capillaries. Transplantation is per-
formed either immediately after injury (acute phase) or after 1
to 2 weeks following injury (subacute phase); unfortunately,
only a few studies have dealt with chronic injuries (Tom et al.
2009). The therapeutic potential of grafted cells is assessed by
demonstration of functional and histological recovery follow-
ing neuropathological analysis.
4.2.1 Transection or Hemisection Models
The complete spinal cord transection injury model reflects symp-
toms of complete SCI in patients. Following laminectomy, spinal
cord transection is performed with spring scissors. Occasionally,
to attach the two ends for regeneration, a sterile gel or polymer
bridge is placed between the two severed spinal cord ends. The
advantage of this model is that the question of spared axons or
sprouting fibers is eliminated, allowing a clear identification of
regenerated axons. The partial spinal transection injury model or
hemisection induces a less severe injury with motor dysfunction
appearing only in the ipsilateral injured side.
4.2.2 Contusion or Hemicontusion Models
Spinal contusion is the oldest and most widely used animal
model (Gruner 1992). In addition to motor dysfunction,
this injury elicits sensory dysfunction, including neuropathic
pain, tactile allodynia, and thermal hyperalgesia. Cervical
contusion is rarely reported, because death can often occur
(Fehlings and Robins-Steele 2012). Therefore, cervical hemi-
contusion, following hemilaminectomy, is used instead. In
cervical contusion models motor dysfunction appears in the
forelimbs (Gensel et al. 2006). The thoracic spinal cord con-
tusion model causing motor deficits in the hind limbs is more
popular and is induced with impactors, such as the controlled
weight-drop impactor.
4.2.3 Compression Injury Models
Following laminectomy, compression injury is induced with
clips calibrated to exert a force inducing moderate or severe
injury (Fehlings and Tator 1995). In either case the clip is
pressed dorsoventrally over the entire cord for 1 minute.
Figure 57.1 Human Schwann Cells Isolated from Peripheral Nerve Biopsy. The culture was expanded in the presence of heregulin and Schwann cells
were purified from contaminating fibroblasts by immunopanning using anti-p75 neurotrophin receptor antibody. A. Purified Schwann cells were
transduced with a lentiviral vector for expression of the green fluorescent protein (green). B. Their purity (>95%) was assessed by immunofluorescence
staining for p75 (red). C. The merged picture is shown.
732
The spinal cord becomes ischemic and the model mimics
common clinical injuries and outcomes. An alternative to clip
compression is balloon compression in which a deflated
balloon is inserted in the spinal cord at the desired level and is
then inflated to cause tissue injury.
5 R O L E O F E XO G E N O U S G L I A L C E L L S I N
R E G E N E R AT I O N A N D M Y E L I N R E PA I R
5.1 A NATO M I C A L R E PA I R
Glial cell types used for transplantation in CNS dysmyelina-
tion, demyelination, and SCI include Schwann cells, oligoden-
droglial cells, and olfactory ensheathing cells (OECs). More
recently and rather unexpectedly, astrocytes have been used
in SCI, which when primed with certain factors appear to be
transformed from a growth-inhibitory to a growth-supporting
phenotype (White et al. 2011). Recent data also indicate that
reactive astrocytes harvested from the lesion site after brain
injury acquire stem cell potential, suggesting that they may
be harnessed for repair strategies (Robel et al. 2011). Here, we
stress the role of these cells in regeneration and remyelination,
and refer to the repair potential of stem cells after transplanta-
tion in models of dysmyelination, demyelination, and SCI.
5.1.1 Committed Cells
5.1.1.1 Schwann Cells
Schwann cells (SCs) are the myelin-forming cells of the
peripheral nervous system. They are in intimate association
and dynamic communication with neuronal axons regulat-
ing the development, maintenance, and function of peripheral
nerves (see chapter 14). The well-established property of SCs
to myelinate CNS demyelinated areas (Zujovic et al. 2007) has
fostered the wide use of these cells as an attractive cell source
to drive exogenous remyelination. Schwann cells are appealing
candidates for autologous transplantation because they can be
grown and expanded from human biopsies (Fig. 57.1). Thus
the main advantage of using these peripheral nervous system
(PNS) myelin-forming cells is that SCs can be obtained from
neonate and adult sural nerve biopsies, cultured and expanded
• NEUROGLIA
extensively in vitro, cryopreserved, and autotransplanted into
demyelinated CNS areas (Lavdas et al. 2008). Moreover, these
cells and their myelin are most likely not a target in MS. Rodent,
monkey, and human SCs have been successfully used to recon-
struct myelin sheaths and restore axonal conduction in dysmy-
elination, demyelination, and SCI. Moreover, remyelination by
exogenous SCs improves neurological functions in focally demy-
elinated areas of the CNS in rodents as well as in SCI (Honmou
et al. 1996). Finally, preliminary data indicate that SCs grafted
in a diffuse model of EAE, survive, migrate, and differentiate in
myelin-forming SCs (Zujovic et al. 2012). Importantly, autolo-
gous grafts of SCs in the primate demyelinated spinal cord have
been proved feasible (Bachelin et al. 2005).
Because of their inherent properties to sustain axonal
regeneration in the PNS, SCs were the first type of cells to
be transplanted into the injured spinal cord (Duncan et al.
1981). A number of studies have since demonstrated the abil-
ity of SCs or SC precursors to promote regeneration of the
lesioned spinal cord in rodent and primate models (for review
see Lavdas et al. 2008) (Fig. 57.2). Schwann cells have been
used for grafting either alone or in combination with other
cell types, such as olfactory ensheathing cells, with or without
growth factor administration (Fouad et al. 2005; Pearse et al.
2007). An alternative strategy has been to combine SC grafts
with sustained cAMP signaling to promote axonal regenera-
tion after injury (Pearse et al. 2004). Additionally, SCs have
been genetically engineered to secrete higher levels of growth
factors than they normally do, or other neuroprotective or
regeneration-promoting molecules (Girard et al. 2005; Lavdas
et al. 2010a; Papastefanaki et al. 2007).
One limitation in using SC grafts stems from their inabil-
ity to be efficiently integrated in an astrocyte-rich environ-
ment (Iwashita et al. 2000). To facilitate their incorporation
and at the same time introduce a cellular vector that would
render the CNS more permissive to regeneration, SCs were
engineered to express the polysialylated form of the neural cell
adhesion molecule NCAM (PSA-NCAM) on their surface
(Lavdas et al. 2006). Expression of PSA on NCAM results in
attenuation of cell-to-cell interactions and allows for structural
changes necessary for CNS repair (El Maarouf et al. 2006).
Transplantation of PSA-NCAM expressing rodent SCs in a
mouse model of spinal cord compression injury proved supe-
rior to using control SCs and resulted in a marked restoration
of hindlimb locomotor function (Papastefanaki et al. 2007).
Enhanced SC-astrocyte mixing, migration, and remyelina-
tion was also noted with transplantation of macaque SCs,
engineered to express PSA-NCAM, in a focal demyelination
model in nude mice (Bachelin et al. 2010).
Figure 57.2 Parasagittal Section of the Lesioned Mouse Spinal Cord
5.1.1.2 Olfactory Ensheathing Cells Transplanted with Schwann Cells, 4 Weeks After Compression Injury.
Olfactory ensheathing cells (OECs) are a distinct popula- A. Double labeling for fibronectin to illustrate the lesion site (blue).
tion of glial cells that wrap around—but do not myelinate— B. Glial fibrillary acid protein immunoreactivity (red). C. Transplanted
the axons of olfactory receptor neurons in their entire length, Schwann cells were isolated from transgenic mice expressing the green
as they extend from the olfactory epithelium in the PNS to fluorescence protein green fluorescent protein under the actin promoter
and appear in green. D. The merged picture. Orientation is shown in
the olfactory bulb in the CNS (Doucette 1990). Unlike SCs, (A). C, caudal; D, dorsal; R, rostral; V, ventral. Scale bar: 100 μm. From
OECs intermingle well with astrocytes (Lakatos et al. 2000) Lavdas et al. 2010b.
and have been shown to support the development and regen-
eration of the olfactory nervous system (Su and He 2010).

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Although OECs do not produce a myelin sheath around the
axons of olfactory receptor neurons in situ, they can myelinate
when presented with large-diameter axons in vitro (Babiarz
et al. 2011). Moreover, rodent, canine, human, or porcine
OECs isolated from the adult olfactory bulb are capable of
extensive functional remyelination following transplantation
into the demyelinated rat CNS (Deng et al. 2006). Because of
their biological properties and relative accessibility in humans
from the olfactory mucosa, OECs have also been considered
as candidate cells for autologous transplantation and have
been tested in numerous animal models of SCI (Barnett and
Riddell 2007). Although promising, OEC transplantation
has not yielded sufficient anatomical or functional recovery
and, as with SCs, has led to implementation of combinatorial
approaches, such as cotransplantation with other cell types,
genetic modification, or additional interventions, for example
(Cao et al. 2004).
5.1.2 Stem Cells and Progenitor Cells
5.1.2.1 Neural Crest–Derived Stem and
Progenitor Cells
During development of the neural tube, neural crest cells
(NCC) colonize various embryonic tissues and differenti-
ate into a wide range of cells, including neurons and glia in
the PNS, pigment cells in the skin, and endocrine cells in the
adrenal and thyroid gland. They also contribute to the mesen-
chymal lineage, giving rise to cartilage, bone, dermis, adipose
tissue from the craniofacial area, and vascular smooth muscle
cells (pericytes). Within the PNS, SC go through different
transitional stages before becoming myelin competent cells
(Zujovic et al. 2007). Neural crest cells (NCC) differenti-
ate in SC precursors before becoming immature SC, and a
subpopulation of NCC arrest transiently at the PNS-CNS
BC
boundary to form boundary cap (BC) cells (Fig. 57.3). More
recently, embryonic Schwann cell precursors (SCp) or BC are
the subject of growing interest for cell-based therapy. These
immature cells appear to be less inhibited by CNS cues than
neonatal and adult SCs and are therefore able to colonize
and remyelinate more efficiently the demyelinated or injured
CNS (Zujovic et al. 2010). Although embryonic BC and SCp
differentiate in Schwann cells when transplanted in demyeli-
nated lesions (Zujovic et al. 2010), they differentiate in oligo-
dendrocytes when confronted with CNS developmental cues
(Binder et al. 2011; Zujovic et al. 2011) (Fig. 57.4). Finally, SCp
promote extensive axon regeneration after transplantation in
SCI (Agudo et al. 2008).
Neural crest–derived stem cells (NCSC) were also identified
in embryonic tissue, such as embryonic sciatic nerve (Morrison
et al. 1999) and various adult tissues such as gut (Kruger et al.
2002), skin (McKenzie et al. 2006; Sieber-Blum et al. 2004),
heart (Tomita et al. 2005), DRG (Li et al. 2007; Nagoshi
et al. 2008), and bone marrow (Nagoshi et al. 2008; Wislet-
Gendebien et al. 2012). Although the regenerative potential of
bone marrow mesenchymal cells (BMSCs) after transplantation
in the injured spinal cord (Kocsis et al. 2002) or injured nerve
(Keilhoff et al. 2006) suggested transdifferentiation of these cells
into remyelinating SC, the identification of NC-SC in bone
marrow suggest a possible NC origin of the remyelinating SC
in the mentioned studies. Stemlike cells can also be derived from
adult peripheral nerves in response to injury (Takagi et al. 2011)
or adult palatal ridges (Widera et al. 2011). However, so far, it
is not clear whether these cells arise from de-differentiation of
Schwann cells or a discrete population of unidentified stem cells.
Although the persistence of stem cell reservoirs in adult tissues
opens new perspectives for cell therapy in demyelinating disor-
ders and SCI, future work should further characterize the stem-
ness of these adult NC-SC compared to embryonic NC-SC.

nmSC

NCC iSC
SCp

Pro-mSC mSC
Sox-9
Sox-2
Sox-10
Krox-20

Pluripotene Mullipotent Unipotent (SC)

Unipotent (SC)
(mesenchymal and (N,SC,SM, O) Poory migratory
Non migratory
neuroepithelial cells) Migratory
Migratory

Figure 57.3 Schematic Drawing Illustrating the Migration and Differentiation Potential of Different Stages (Defined by Stage-Specific Transcription
Factors) of the Schwann Cell Lineage After Engraftment in Different Situations in the Central Nervous System. NCCs migrate extensively and gener-
ate a wide variety of mesenchymal and neuroepithelial derivatives. Early stages of the SC lineage such as BC and SCP still migrate efficiently and can
generate multiple PNS and CNS derivatives (in developmental conditions). By contrast, more mature cells (immature SC and mature SC) do not
migrate efficiently and differentiate essentially in SC (myelinating or nonmyelinating) when grafted in the demyelinated CNS. BC, boundary cap cells;
iSC, immature Schwann cells; mSC, myelinating Schwann cells; N, neurons; NCC, neural crest cells; nmSC, nonmyelinating Schwann cells; SCP,
Schwann cell precursors; SM, smooth muscle cells.

734 • NEUROGLIA
Figure 57.4 Mouse Boundary Cap Cells Give Rise to Myelinating Oligodendrocytes After Transplantation into the Forebrain of the Newborn
Shiverer Brain. (A1–A3) YFP-positive cells (green) give rise to mature myelin basic protein (MBP) expressing oligodendrocytes with features of
myelin-forming cells extending processes to internodes (arrows). B1–3. Illustrated at higher magnification. C. Immunodetection for MBP indicated
widespread of BC-derived myelin patches in the subcortical white matter. D,E. Ultrastructural analysis confirms the presence of compacted donor
myelin (E, inset) compared with uncompacted shiverer myelin (D, inset).

Olfactory ensheathing cell progenitors OEp were found in
the human and rodent olfactory epithelium (Winstead et al.
2005). Transplantation into the demyelinated spinal cord lesion
indicated that exogenous rat OEp provide extensive remyelina-
tion (Markakis et al. 2009). Moreover, human OEp were trans-
planted in the rodent traumatized spinal cord and shown to
rescue axotomized rubrospinal neurons and promote functional
recovery (Xiao et al. 2005). Yet their use in disease models of
trauma or demyelination deserves further attention.
Future studies should document the resistance to neuroin-
flammation and the neuroregenerative or neuroprotective
potential of these various adult NC-derived stem cells.
5.1.2.2 Oligodendrocyte Progenitors
Until recently it was considered that exogenous cells that
remyelinate axons in the adult CNS could be derived both
from immature OPC and mature oligodendrocytes (Chari
and Blakemore 2002) (see chapter 13). In agreement, some
transplantation studies suggested that rodent or human oli-
godendrocytes were capable of myelin formation (Baron-Van
Evercooren and Blakemore 2004.). However, this possibility
was not fully demonstrated mainly because of the difficulty
of obtaining cell preparations entirely devoid of OPC or the
insufficient specificity of markers allowing the selection of the
mature oligodendrocyte stage. Instead, OPC were the most
extensively studied cells of the lineage (see Fig. 57.5). Rodent
and human OPC elicit vigorous remyelination when trans-
planted in the developing brain of myelin mutants or focal
lesions of demyelination (Groves et al. 1993; Lachapelle et al.
1983; Windrem et al. 2008). Interestingly, multiple injections
of these cells in the developing CNS parenchyma led to impres-
sive colonization of the entire neuraxis by donor-derived myelin
(Archer et al. 1997). Moreover, remyelination by transplanted
OPC is associated with functional recovery (Utzschneider

G L I A L C E L L T R A N S P L A N TAT I O N F O R C E N T R A L N E RVO U S SYS T E M R E PA I R • 735

 Mature Olg
Pre-Olg Olg
NPC OPC

Olig1+
Olig2+/- NG2+ NG2+ O4+ Olig2+/–
Ollg 1, 2+ O4+ GalC+ Sox10+
Sox 10+ Ollg 1,2+ CNPase+ GalC+
PDGF-aR+ Sox 10+ Olig 1,2+ MOG+
A2B5+ PDGF-aR+ Sox10+ MBP+

Pluripotent Parenchymal OPC

Unipotent (O) Unipotent (O)
(N,O,A, SC) Multiotent (N,O,A, SC)
Migratory (+/–) Non mlgratory
Migratory Migratory (+)
SVZ OPC
Multiotent (N,O,A)
Migratory (++)

Figure 57.5 Schematic Drawing Illustrating the Migration and Differentiation Potential of Different Stages (Defined by Stage-Specific Markers) of
the Oligodendroglial Lineage After Engraftment in Different Situations in the Central Nervous System. Neural precursor cells migrate extensively and
generate neurons, astrocytes, oligodendrocytes and sometimes SC (demyelinating conditions). According to their origin, OPC migrate efficiently and
generate multiple CNS derivatives, including SC (demyelinating conditions). By contrast more mature cells (pre-Olg, Olg, and mature Olg) are non
migratory and generate solely and in limited numbers oligodendrocytes. A, astrocytes; N, neurons; NPC, neural precursor cells; Olg, oligodendro-
cytes; OPC, oligodendrocyte progenitor cells; Pre-Olg, pre-oligodendrocytes; SC, Schwann cells.

et al. 1994). Although OPCs were found to migrate poorly
in the adult normal CNS white matter, grafting CG4 cells in
a model of diffuse EAE (Tourbah et al. 1997) or at a distance
of a focal inflammatory lesion (Wang et al. 2011b) indicated
that inflammation sustains OPC migration through normal
white matter and subsequent remyelination, a major issue for a
disease such as MS, which is characterized by randomly spread
lesions.
Most of the mentioned studies were performed isolat-
ing OPC from whole forebrain or spinal cord and did not
discriminate between parenchymal OPC and those that are
localized in germinative niches such as the dentate gyrus
(DG) and subventricular zone (SVZ) (see chapters 5 and
40). Transplantation of cells selected on the basis of the OPC
marker NG2 in the newborn brain indicated that postnatal
and adult SVZ NG2+ cells generate functional neurons in
addition to CNS glia, and migrate more efficiently in the
CNS compared with parenchymal NG2+ cells, which gen-
erate essentially glial cells and migrate poorly in the CNS
parenchyma (Aguirre and Gallo 2004). These studies clearly
underline region-specific differences among NG2+ cells
(see chapters 5, 10 and 30).
5.1.2.3 Central Nervous System Neural Stem/
Precursor Cells
Embryonic or adult neural stem/precursor cells (NPCs)
harvested from germinal areas of the CNS are typically cul-
tured and expanded as neurospheres in the presence of EGF
and/or FGF (Reynolds and Weiss 1996). They contain pre-
cursors for neurons, astrocytes, and oligodendrocytes and
also some self-renewing cells. In view of their self-renewability
and multipotency, NPCs are regarded as cells of high thera-
peutic interest for a variety of neurological diseases, includ-
ing those affecting myelin, as well as CNS injuries. Numerous
studies have characterized the behavior of these cells after
intra-CNS transplantation. Of concern were the regulation
of their proliferation to avoid tumor formation, migration
capacity, differentiation in neural cell types other than oli-
godendrocytes, and consequently their reduced ability to
reconstruct myelin in their host. Preclinical studies in a vari-
ety of disease models established that tumor formation was
not an issue because these cells integrated efficiently within
the CNS parenchyma. Moreover, rodent and human NPCs
migrate extensively (and thus more efficiently than OPC)
leading to massive colonization of the neuraxis even in the
adult CNS (Buchet et al. 2011).
Transplantation of NPCs in the developing forebrain
leads to the generation of multiple neural cell types, includ-
ing astrocytes, neurons, and oligodendrocytes (Yandava
et al. 1999). By contrast they generate astrocytes and oligo-
dendrocytes (Buchet et al. 2011) (Fig. 57.6), and sometimes
SCs (Keirstead et al. 1999), when grafted in the demyeli-
nated spinal cord. Nevertheless, their capacity to generate
oligodendrocytes seems to be region and species depen-
dent. However, it is limited when grafted in conditions of
focal inflammatory demyelination (Walczak et al. 2011).
Adult SVZ fragments containing neural stem cells and
immature neural precursors and neurospheres form myelin
when grafted into the neonate brain (Lachapelle et al. 2002;
Zhang et al. 1999). This potential can be enhanced by prim-
ing SVZ donors with FGF (Lachapelle et al. 2002) (see
chapter 40). Most studies with NPC transplantation in SCI
have also reported that these cells integrate well and dif-
ferentiate robustly in the host spinal cord, thus conferring
some degree of improvement (Karimi-Abdolrezaee et al.
2006). To increase the yield in neuronal or oligodendrocyte
progeny, rodent and human NPCs have been genetically
modified with the use of viral vectors to express neuronal
or glial fate determinants (Hwang et al. 2009; Maire et al.
2009; Makri et al. 2010).

736 • NEUROGLIA
Figure 57.6 Human Fetal Neural Progenitor Cells Give Rise to Myelinating Oligodendrocytes After Transplantation into the lysophosphatidylcholine
(LPC)-Induced Demyelination of the Shiverer Mouse Spinal Cord. A–D. Immunohistochemical labeling against myelin basic protein (MBP) (green)
illustrating human-derived myelin in the shiverer spinal cord at different rostro (A) caudal levels (D). E–H. Immunohistochemical detection of MBP
(red), human NOGO (green), and NF-200 (blue), illustrating multipolar human neural stem/precursor cell–derived oligodendrocytes extending
processes and wrapping axons in dorsal and ventral white matter. E,F. Cross-sections. F. An orthogonal view of (E). H. A higher magnification of (G)
illustrating MBP+ internodes and typical nodes of Ranvier. I–L. Ultrastructural analysis of myelin compaction in (I) shiverer mice without lesion, (J)
shiverer mice with LPC lesion, (K), shiverer mice with LPC lesion, and hNPC graft, and (L) higher magnification illustrating compaction of hNPC–
derived myelin. Scale bars (A–D), 200 μm; (E), 20 μm; (F,H) 5 μm; (G) 10 μm; (I–K) 250 nm; (L) 20 nm.

5.1.2.4 Embryonic Stem Cells and Induced
Pluripotent Stem Cells
OPCs and NPCs can also be derived from embryonic
stem cells (ES). A few studies reported the first evidence
of myelination achieved by grafting ES-derived glial pro-
genitors, for example (McDonald et al. 1999; Perez-Bouza
et al. 2005). Protocols were established to derive OPC from
human-ES cells (Hu et al. 2009). However, the number of
newly generated glial progenitors in vitro and in vivo is weak.
Treatment of ES cells with all-trans retinoic acid alone or in
combination with morphogens generated OPC-enriched
populations, which generated myelin after grafting into
the neonate shiverer brain (Izrael et al. 2007) and a model
of SCI (Keirstead et al. 2005). Treatment with interleukin
6 (Zhang et al. 2006) or overexpression of Olig2 (Du et al.
2006) are other means to favor oligodendrogenesis from
mouse ES cells.
The future for regenerative medicine may reside in
induced pluripotent stem cells (iPS) as in theory they can be
derived from the patients’ own cells, expanded and used for
autologous transplantation (Fig. 57.7). Although endogenous
cells, such as Schwann cells, can also serve for autologous
transplantation, their use necessitates sacrificing a peripheral
nerve. Although the significance of such an approach may
be negligible in a completely paralyzed individual suffering
from SCI, the implications on other types of patients have to
be appreciated. Notably, the use of iPS meets the needs for
noninvasive isolation procedures. Rodent and human OPC
were generated via differentiation of mouse iPS into NPCs,
and transplanted in demyelinating lesions, where they differ-
entiated into a few myelin-forming oligodendrocytes (Czepiel
et al. 2011; Pouya et al. 2011)). In addition, NPCs were derived
from mouse embryonic iPS and human adult iPS and used in
transplantation paradigms for treatment of SCI (Nori et al.
2011; Sakai et al. 2012; Tsuji et al. 2010)). Rodent and human
NC-PC can also be derived from ES cells and iPS (Lee et al.
2010; Menendez et al. 2011). Wang et al. (2011a) exploited the
repair potential of hiPS-NC by seeding them in nerve con-
duits, and observed their differentiation in myelin-forming
SCs, correlating with improved regeneration of the sciatic
nerve. However, the role of these PNS cells in CNS remyeli-
nation and regeneration has not been addressed.
Although these pioneer studies remain to be confirmed and
their transplantation in various animal models of dysmyelination,
demyelination, and SCI are required to demonstrate the
safety and efficacy of these novel cellular sources, these
developments reported so far and the efforts made to improve
the differentiation of iPS cells into myelin-forming candidates

G L I A L C E L L T R A N S P L A N TAT I O N F O R C E N T R A L N E RVO U S SYS T E M R E PA I R • 737

Figure 57.7 Schematic Representation of a Paradigm of Autologous Transplantation. The future for regenerative medicine may reside in cells harvested
from accessible human tissues, such as peripheral nerves (Schwann cells), olfactory mucosa (olfactory ensheathing cells), or the skin (fibroblasts for
derivation of induced pluripotent stem cells). Schwann cells and olfactory ensheathing cells may be cultured and expanded in vitro for autologous
transplantation, whereas induced pluripotent stem cells may be differentiated to neural precursors before being grafted into the injured central nervous
system.

open the perspective for autologous cell-based therapy for
demyelinating disorders and CNS trauma.
5.2 BYS TA N D E R E F F E C T S
A series of experimental studies suggest that both mesenchy-
mal stem cells (MSC) and NPCs on transplantation exert
other therapeutic effects than anatomical repair. Indeed,
NPCs or BMSC transplanted either by intrathecal or intrave-
nous cell injection ameliorate the clinicopathological features
of rodents with acute, chronic, and relapsing EAE (Martino
et al. 2010; Uccelli 2008). This phenomenon is caused by the
remarkable neuroprotective and immunomodulatory capaci-
ties that NPC and BMSC exert within the CNS. These effects
occur most likely via the release of soluble molecules (e.g.,
cytokines, chemokines), which leads to downregulation of the
encephalitogenic inflammatory T cells, antigen-presenting
dendritic cells, and microglia/macrophages. Recent evidence
shows that intravenously or subcutaneously transplanted
NPCs might also exert immunomodulatory functions at the
level of peripheral lymphoid organs (Pluchino et al. 2005).
Although these could be features shared by immature cell
types, similar bystander effects or immunomodulatory prop-
erties of OPC or NC-SC were not reported. The bystander
effects of exogenous cells could also result from a trophic
effect on endogenous cells. Such trophic effect has been
reported for BMSC, but also more recently for NPCs after
their intracerebroventricular delivery in a cuprizone-induced
model of demyelination (Einstein et al. 2009). Although
NPC remained at an immature state, enhanced remyelination
resulted from increased endogenous OPC proliferation, an
effect mediated by platelet-derived growth factor (PDGF) A
and fibroblast growth factor (FGF) 2, and their subsequent
differentiation. In a recent study (Cusimano et al. 2012), it
was shown that NPCs transplanted in a model of SCI also
survived mostly undifferentiated within the host by finding
their way toward a perilesional inflammatory-like vascular
niche, in close proximity to macrophages and microglial cells.
The authors argue that from their homing site, undifferenti-
ated NPCs were able to control innate and adaptive immune
responses to induce tissue healing and functional recovery.
This and other studies highlight the remarkable potential of
transplanted NPCs, MSCs, and other cell types to influence
bystander events, such as myelin repair and axonal rescue or
regeneration after SCI.
The bystander effect of more committed cells is also the
subject of recent studies. Olfactory ensheathing cells release
diffusible factors that regulate the proliferation and differ-
entiation of NPCs and may be involved in neurogenesis,
which takes place throughout life in the olfactory system
(Zhang et al. 2008). Additionally, SC transplants facilitate
the emergence of host SCs in the lesioned spinal cord, which
alongside with grafted SCs contribute to axon regenera-
tion and protection. Yet their role in immunomodulation

738 • NEUROGLIA
has not been investigated in SCI or inflammatory CNS
demyelination.
6 S U M M A RY A N D P E R S P E C T I VE S
Over the years, the idea to treat demyelinating disorders or
SCI with cells capable of populating the lesion site and cre-
ate a regeneration permissive environment within the CNS
has attracted considerable attention. From the early days of
peripheral nerve grafts to recent paradigms of cellular trans-
plants, the idea has emerged that stem cells and progenitor
cells are more promising, than committed cells. Moreover,
combinatorial approaches hold promise as a means for-
ward. New knowledge has been acquired as to the fate of
transplanted cells in vivo and, most importantly, how donor
and host cells respectively interact. Thus, the notion of cell
transplantation as a cell replacement strategy has shifted
toward the concept that it represents a multifaceted thera-
peutic approach conferring neuroprotection, trophic support,
immunomodulation, and manipulation of reactive gliosis,
triggering endogenous regenerative processes in addition to
anatomical repair.
AC K N OW L E D G M E N T S
The authors thank Florentia Papastefanaki (RM’s lab) and
Delphine Buchet and Violetta Zujovic (ABVE’s lab) for pro-
viding original figures; Alexandros A. Lavdas and Violetta
Zujovic for help with editing the manuscript, and all mem-
bers of the Matsas and Baron-Van Evercooren laboratories for
their useful input. The authors acknowledge financial support
to RM from FP7 REGPOT NEUROSIGN Project 264083
and Fondation BNP Paribas; to ABVE from ELA, ARSEP
Foundation and FP7 LEUKOTREAT Project.
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