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HORMONES, BRAIN,
AND BEHAVIOR

THIRD EDITION
This page intentionally left blank
 HORMONES, BRAIN,
AND BEHAVIOR

THIRD EDITION

EDITORS-IN-CHIEF
Donald W. Pfaff
The Rockefeller University, New York, NY, USA

Marian Joëls
University Medical Center Utrecht, Utrecht, The Netherlands;
and University Medical Center Groningen, Groningen, The Netherlands

VOLUME 1
MAMMALIAN HORMONE-BEHAVIOR SYSTEMS

VOLUME EDITOR
Gabriela González-Mariscal
CINVESTAV-Universidad Autónoma de Tlaxcala, Tlaxcala, Mexico

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EDITORIAL BOARD

EDITORS-IN-CHIEF
Donald W. Pfaff
The Rockefeller University, New York, NY, USA

Marian Joëls
University Medical Center Utrecht, Utrecht, The Netherlands;
and University Medical Center Groningen, Groningen, The Netherlands

VOLUME EDITORS
Gabriela González-Mariscal (Volume 1: Mammalian Hormone-Behavior Systems)
CINVESTAV-Universidad Autónoma de Tlaxcala, Tlaxcala, Mexico

Jacques Balthazart (Volume 2: Non-Mammalian Hormone-Behavior Systems)

University of Liège, Liège, Belgium

E. Ronald de Kloet (Volume 3: Molecular and Cellular Mechanisms)

Leiden University Medical Center, Leiden, The Netherlands

Stafford Lightman (Volume 4: Clinically Important Hormone Effects on Brain and Behavior)
University of Bristol, Bristol, UK

Anthony P. Auger (Volume 5: Development of Hormone-Behavior Relationships)

University of Wisconsin, Madison, WI, USA

Catherine J. Auger (Volume 5: Development of Hormone-Behavior Relationships)

University of Wisconsin, Madison, WI, USA

v
This page intentionally left blank
CONTRIBUTORS TO VOLUME 1

Raúl Aguilar-Roblero M H Ferkin

Instituto de Fisiología Celular, Ciudad Universitaria, University of Memphis, Memphis, TN, USA
UNAM, México, Mexico
Jeannie M Fiber
Osborne F X Almeida Contract Scientist, Cambridge, MA, USA
Max Planck Institute of Psychiatry, Munich, Germany
Nicole J Gervais
Elvira Arellanes-Licea University of Massachusetts, Amherst, MA, USA
Instituto Potosino de Investigación Científica y
Tecnológica, San Luis Potosí, Mexico Oscar González-Flores
Centro de Investigación en Reproducción Animal,
Wayne G Brake Universidad Autónoma de Tlaxcala-CINVESTAV,
Center for Studies in Behavioral Neurobiology (CSBN), Tlaxcala, México
Concordia University, Montreal, QC, Canada
Gabriela González-Mariscal
Anne Sisto Burt Centro de Investigación en Reproducción Animal,
Facultad de Medicina Veterinaria y Zootecnia, CINVESTAV-Universidad Autónoma de Tlaxcala,
Universidad Nacional Autónoma de México, Mexico Tlaxcala, Mexico
City, Mexico
Erno J Hermans
Matthew P Butler Radboud University Medical Center, Donders Institute
Oregon Health & Science University, Portland, OR, for Brain, Cognition and Behaviour, Radboud University
USA Nijmegen, Nijmegen, The Netherlands
M Caba
A Y Herrera
Centro de Investigaciones Biomédicas, Universidad
University of Southern California, Los Angeles, CA, USA
Veracruzana, Xalapa, Veracruz, México
Kurt L Hoffman
C S Carter
Centro de Investigación en Reproducción Animal,
The Kinsey Institute, Indiana University, Bloomington,
Universidad Autónoma de Tlaxcala-CINVESTAV,
IN, USA
Tlaxcala, México
Annette D de Kloet
Elaine M Hull
College of Medicine, University of Florida, Gainesville,
Florida State University, Tallahassee, FL, USA
FL, USA
Ilia Karatsoreos
J delBarco-Trillo
Washington State University, Pullman, WA, USA
Liverpool John Moores University, Liverpool, UK
Matthieu Keller
José A Delgadillo
Behavioral & Reproductive Physiology, INRA/CNRS/
Centro de Investigación en Reproducción Caprina
University of Tours, Nouzilly, France
(CIRCA), Universidad Autónoma Agraria Antonio
Narro, Torreón, México E B Keverne
University of Cambridge, Cambridge, UK
Mauricio Díaz-Muñoz
Instituto de Neurobiología, Campus UNAM-Juriquilla, Eric G Krause
Querétaro, Mexico College of Pharmacy, University of Florida, Gainesville,
FL, USA

vii
viii Contributors to Volume 1
Lance J Kriegsfeld
University of California, Berkeley, CA, USA
Agnès Lacreuse
University of Massachusetts, Amherst, MA, USA
Yazmín Macotela
Instituto de Neurobiología, Campus UNAM-Juriquilla,
Querétaro, Mexico
A I Melo
Centro de Investigación en Reproducción Animal,
CINVESTAV-Universidad Autónoma de Tlaxcala,
Tlaxcala, México
Sally P Mendoza
University of California, Davis, Davis, CA, USA
Randy J Nelson
Neuroscience Research Institute, Ohio State University
Wexner Medical Center, Columbus, OH, USA
S E Nielsen
University of Southern California, Los Angeles, CA,
USA
Raymond Nowak
INRA, UMR85 Physiologie de la Reproduction et des
Comportements, Nouzilly, France; CNRS, UMR7247
Physiologie de la Reproduction et des Comportements,
Nouzilly, France; Université François Rabelais de Tours,
Tours, France; and Institut Français du cheval et de
l’Equitation, Nouzilly, France
Raúl G Paredes
Instituto de Neurobiologia, Universidad Nacional
Autónoma de México, Querétaro, México
Aras Petrulis
Georgia State University, Atlanta, GA, USA
Brian J Prendergast
University of Chicago, Chicago, IL, USA
Ryszard Przewlocki
Institute of Pharmacology, Polish Academy of Sciences,
Krakow, Poland
Gabriela Rodríguez-Manzo
Cinvestav, Mexico City, México
Benno Roozendaal
Radboud University Medical Center, Donders Institute
for Brain, Cognition and Behaviour, Radboud University
Nijmegen, Nijmegen, The Netherlands
Rae Silver
Columbia University, New York, NY, USA; Barnard
College, New York, NY, USA; and College of Physicians
and Surgeons, New York, NY, USA
Cheryl L Sisk
Michigan State University, East Lansing, MI, USA
Tyler J Stevenson
Institute of Biological and Environmental Sciences,
University of Aberdeen, Aberdeen, UK
Jennifer M Swann
Lehigh University, Bethlehem, PA, USA
Brian C Trainor
University of California, Davis, CA, USA
Francisco Vázquez-Cuevas
Instituto de Neurobiología, Campus UNAM-Juriquilla,
Querétaro, Mexico
Olivia Vázquez-Martínez
Instituto de Neurobiología, Campus UNAM-Juriquilla,
Querétaro, Mexico
Iván Villanueva
Escuela Nacional de Ciencias Biológicas, IPN, México,
Mexico
CONTENTS OF ALL VOLUMES

Editorial Board v
Contributors to Volume 1 vii
Preface of the First Edition xvii
Hormonal Effects on Specific Behaviors, on Global CNS States, and on Human Disease.
An Introduction to the Second Edition xix
Hormonal Effects on Specific Behaviors, on Global CNS States, and on Human Disease.
An Introduction to the Third Edition xxi

VOLUME 1: MAMMALIAN HORMONE-BEHAVIOR SYSTEMS

1.01 Male Sexual Behavior 1

Elaine M Hull and Gabriela Rodríguez-Manzo
1.02 Female Sexual Behavior in Rodents, Lagomorphs, and Goats 59
Oscar González-Flores, Kurt L Hoffman, José A Delgadillo, Matthieu Keller, and Raúl G Paredes
1.03 Parental Behavior 83
G González-Mariscal, M Caba, K L Hoffman, and A I Melo
1.04 The Neurobiology of Social Affiliation and Pair Bonding 117
C S Carter and E B Keverne
1.05 Hormones and the Development and Expression of Aggressive Behavior 145
Brian C Trainor, Cheryl L Sisk, and Randy J Nelson
1.06 Food Intake and Its Control by Signaling Molecules 175
Francisco Vázquez-Cuevas, Raúl Aguilar-Roblero, Elvira Arellanes-Licea, Yazmín Macotela,
Olivia Vázquez-Martínez, Iván Villanueva, and Mauricio Díaz-Muñoz
1.07 Body Fluid Homeostasis 211
Eric G Krause and Annette D de Kloet
1.08 Stress and Opioid Systems 225
Ryszard Przewlocki and Osborne F X Almeida
1.09 Social Stress: Concepts, Assumptions, and Animal Models 261
Sally P Mendoza

ix
x Contents of All Volumes

1.10 Communication by Chemical Signals: Physiological Mechanisms, Ontogeny and Learning,

Function, Evolution, and Cognition 285
M H Ferkin, J delBarco-Trillo, and A Petrulis
1.11 The Medial Amygdala, Hormones, Pheromones, Social Behavior Network, and Mating Behavior 329
Aras Petrulis, Jeannie M Fiber, and Jennifer M Swann
1.12 Circadian Regulation of Endocrine Functions 345
Matthew P Butler, Ilia Karatsoreos, Lance J Kriegsfeld, and Rae Silver
1.13 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates 371
Tyler J Stevenson, Brian J Prendergast, and Randy J Nelson
1.14 Sex Steroids, Learning and Memory 399
S E Nielsen and A Y Herrera
1.15 Adrenal Stress Hormone Effects on Memory 423
Benno Roozendaal and Erno J Hermans
1.16 Ovarian Hormones and Prefrontal Cortex-Related Cognition 439
Nicole J Gervais, Wayne G Brake, and Agnès Lacreuse
1.17 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals 453
Gabriela González-Mariscal, Anne Sisto Burt, and Raymond Nowak

VOLUME 2: NON-MAMMALIAN HORMONE-BEHAVIOR SYSTEMS

Non-mammalian Vertebrates: Fishes

2.01 Neuroendocrinology of Social Behavior in Teleost Fish 3
David Gonçalves, Ana S Félix, and Rui F Oliveira
2.02 Social Regulation of Sex: How the Brain Controls Reproductive Circuits 19
Russell D Fernald
2.03 Socially Controlled Sex Change in Fishes 31
John Godwin and Melissa Lamm
2.04 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish
as Model Systems 47
Ni Y Feng and Andrew H Bass
2.05 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology 69
Kent D Dunlap, Ana C Silva, G Troy Smith, and Harold H Zakon

Non-mammalian Vertebrates: Amphibians

2.06 Neuroendocrine Control of Social Behavior in Frogs 101
Walter Wilczynski
2.07 The Vertebrate Brain Stem as the Primary Site of Behavior Command:
Insights from an Amphibian 117
Emma Coddington
2.08 Hormones and Vocal Systems: Insights from Xenopus 131
Erik Zornik and Darcy B Kelley
2.09 Endocrinology of Complex Life Cycles: Amphibians 145
Robert J Denver
 Contents of All Volumes xi

Non-mammalian Vertebrates: Reptiles

2.10 Hormones, Brain, and Behavior in Reptiles 171
David Kabelik and David Crews

Non-mammalian Vertebrates: Birds

2.11 Neuroendocrine Regulation of Reproductive Behavior in Birds 217
Gregory F Ball and Jacques Balthazart
2.12 Neural and Hormonal Control of Birdsong 255
Barney A Schlinger and Eliot A Brenowitz
2.13 Rapid Effects of Estrogens on Avian Brain and Social Behavior 291
L Remage-Healey, S A Heimovics, K K Soma, and C A Cornil
2.14 The Function of Behavior as Assessed by Phenotypic Engineering with Testosterone 305
Nicole M Gerlach and Ellen D Ketterson
2.15 Ecophysiological Studies of HormoneeBehavior Relations in Birds: Future Challenges
in a Changing World 321
John C Wingfield

Non-mammalian Invertebrates
2.16 Actions of Developmental Hormones in Adult Social Insects 349
Susan E Fahrbach, Ashton M Trawinski, and Rodrigo A Velarde
2.17 Diverse Functions of Insect Biogenic Amines as Neurotransmitters, Neuromodulators, and
Neurohormones 367
Wendi S Neckameyer and Sandra M Leal
2.18 Regulation of Honeybee Worker (Apis mellifera) Life Histories by Vitellogenin 403
Gyan P Harwood, Kate E Ihle, Heli Salmela (nee Havukainen), and Gro V Amdam
2.19 Endocrine Influences on Insect Societies 421
A R Hamilton, H Shpigler, G Bloch, D E Wheeler, and G E Robinson
2.20 Hormonal Regulation of Behavioral and Phenotypic Plasticity in Bumblebees 453
Abraham Hefetz and Christina M Grozinger
2.21 Stressed Out Insects II. Physiology, Behavior, and Neuroendocrine Circuits Mediating
Stress Responses 465
Erik C Johnson

VOLUME 3: MOLECULAR AND CELLULAR MECHANISMS

3.01 Membrane-Initiated Effects of Estradiol in the Central Nervous System 1

Oline K Rønnekleiv and Martin J Kelly
3.02 Estrogen Regulation of Neurotransmitter and Growth Factor Signaling in the Brain 23
A M Etgen and L M Garcia-Segura
3.03 Genetic and Epigenetic Mechanisms in Neural and Hormonal Controls over Female Reproductive
Behaviors 55
Lee-Ming Kow, A W Lee, Carolyn Klinge, Margaret Warner, Jan-Ake Gustafsson, and D W Pfaff
3.04 Epigenetics of Glucocorticoid Action 83
Karen R Mifsud, Sylvia D Carter, Astrid C E Linthorst, and Johannes M H M Reul
xii Contents of All Volumes

3.05 Androgen Regulation of Neural Circuit Activity: Molecules and Mechanisms 101
Jaroslava Durdiaková and Peter Celec
3.06 Molecular Aspects of Thyroid Hormone-Regulated Behavior 111
Grant W Anderson and Cary N Mariash
3.07 Corticosteroid Actions on Electrical Activity in the Limbic Brain 131
M Joëls, R A Sarabdjitsingh, and H Karst
3.08 Genomic Aspects of Corticosteroid Action in the Brain 149
Lisa T C M van Weert and Onno C Meijer
3.09 Aldosterone Action on Brain and Behavior 159
Harald Murck
3.10 Mechanism of Progesterone Action in the Brain 181
Ignacio Camacho-Arroyo, Valeria Hansberg-Pastor, Edgar Ricardo Vázquez-Martínez, and Marco Cerbón
3.11 Progesterone: Synthesis, Metabolism, Mechanism of Action, and Effects in the Nervous System 215
Michael Schumacher, Xiaoyan Zhu, and Rachida Guennoun
3.12 Neuroactive Steroids and the GABAA Receptor 245
S S Smith and H Shen
3.13 Oxytocin 259
Larry J Young and Hans H Zingg
3.14 Vasopressin: Roles in Modulating Social Behaviors 279
Sarah K Williams Avram and Adi Cymerblit-Sabba
3.15 The Cell Biology of Oxytocin and Vasopressin Cells 305
Jeffrey G Tasker, Daniel L Voisin, and William E Armstrong
3.16 Electrophysiological and Molecular Properties of the Oxytocin- and Vasopressin-Secreting
Systems in Mammals 337
Gary S Bhumbra, Richard E J Dyball, Yoichi Ueta, and Hiroshi Yamashita
3.17 The Gonadotropin-Releasing Hormone and Its Receptor 363
Lothar Jennes, Alfredo Ulloa-Aguirre, Jo Ann Janovick, and P Michael Conn
3.18 Neurophysiology of Gonadotropin-Releasing Hormone Neurons 379
Catherine A Christian
3.19 Action of CRF/Urocortin Peptides 401
Jan M Deussing, Andrey E Ryabinin, Alfredo Zuniga, and Tamas Kozicz
3.20 The Brain Renin Angiotensin System 417
Annette D de Kloet and Eric G Krause

VOLUME 4: CLINICALLY IMPORTANT HORMONE EFFECTS ON BRAIN AND BEHAVIOR

Disorders of Development
4.01 The Gonadal Axis: A Life Perspective 3
Ilpo T Huhtaniemi, Sasha Howard, Leo Dunkel, and Richard A Anderson
4.02 Disorders of Sex Development in Males: Molecular Genetics, Epigenetics, Gender Identity,
and Cognition 59
Yuan-Shan Zhu and Julianne L Imperato-McGinley
 Contents of All Volumes xiii

4.03 Genetic Defects of Female Sexual Differentiation 105

Martine Cools, Hedi L Claahsen-van der Grinten, Elfride De Baere, Nina Callens, and Arianne B Dessens
4.04 HypothalamicePituitaryeAdrenal Axis: Congenital Adrenal Hyperplasia 135
Vickie Pasterski and Ieuan Hughes
4.05 Sexual Orientation in Men and Women 151
L J Gooren and W Byne
4.06 Gender Identity Variants 169
Heino F L Meyer-Bahlburg and Wylie C Hembree
4.07 HypothalamicePituitaryeThyroid Axis 179
Russell T Joffe
4.08 Glucocorticoids and Programming of the Fetal Brain 189
Rebecca M Reynolds and Jonathan R Seckl

Disorders in the Adult

4.09 Premenstrual Dysphoric Disorder 197
Henry J Orff and Barbara L Parry
4.10 Mood and Anxiety Disorders in the Context of Fertility Treatments 217
I Fried-Zaig, Y Joskowicz, and M Bloch
4.11 Thyroid Hormone Replacement 229
V Eligar, P N Taylor, O E Okosieme, and C M Dayan
4.12 Glucocorticoid Replacement in Man 241
Georgina M Russell, Kristian Løvås, and Stafford Lightman
4.13 Stress and Anxiety Disorders 251
C V Chen, S A George, and I Liberzon
4.14 Neuroendocrine Aspects of Posttraumatic Stress Disorder 275
Heather N Bader and Rachel Yehuda
4.15 Growth Hormone and Insulin-Like Growth Factor-I: Effects on the Brain 289
Zvi Laron
4.16 Aging and Alzheimer’s Disease 311
Shireen Sindi, Anna Rosenberg, Robert-Paul Juster, and Sonia J Lupien

Specific Disease Entities

4.17 Health-Related Quality of Life and Behavior in Patients with Both Pituitary and Hypothalamic
Diseases 343
Iris Crespo and Susan M Webb
4.18 Eating and Feeding Disorders 355
Madhusmita Misra and Kamryn T Eddy
4.19 Obesity and Appetite: Central Control Mechanisms 369
Tamas L Horvath
4.20 Alcohol Abuse: Endocrine Concomitants 377
P Bortoletto, E W Rosenthal, and E S Ginsburg
4.21 Endocrinological Effects of Drugs of Abuse 403
Brian Reed and Mary Jeanne Kreek
xiv Contents of All Volumes

4.22 Cognitive Dysfunction in Diabetes Mellitus 421

G J Biessels and C M Ryan
4.23 Sex Differences in Autoimmune Diseases 445
R Voskuhl and Taia T Wang

VOLUME 5: DEVELOPMENT OF HORMONE-BEHAVIOR RELATIONSHIPS

Early Life Stages

5.01 Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning 3
Margaret M McCarthy, Geert J De Vries, and Nancy G Forger
5.02 Sex Differences in the Age of Genetics 33
Arthur P Arnold
5.03 Epigenetic Contribution to Sex Differences in Brain and Behavior 49
Anthony P Auger and Margaret M McCarthy
5.04 Environmental Endocrine Disruption of Brain and Behavior 63
H B Patisaul, A C Gore, and D Crews
5.05 Sex Differences in and Hormone Effects on Mammalian Pheromonal Communication 89
James A Cherry and Michael J Baum
5.06 Parental and Early Developmental Stress Impact on Neurodevelopmental and Neuropsychiatric
Disorders 117
B M Nugent, J C Chan, E Jasarevic, and T L Bale
5.07 Early-Life Experiences: Enduring Behavioral, Neurological, and Endocrinological Consequences 133
Russell D Romeo, Akaysha C Tang, and Regina M Sullivan
5.08 Thyroid Hormones and Brain Development 159
Juan Bernal
5.09 Sexual Differentiation of Brain and Behavior in Birds 185
Jacques Balthazart, Arthur P Arnold, and Elizabeth Adkins-Regan
5.10 Sexual Differentiation of Behavior in Nonhuman Primates 225
Kim Wallen
5.11 Gonadal Hormones and Sexual Differentiation of Human Brain and Behavior 247
M Hines
5.12 Sexual Identity and Sexual Orientation 279
Laura Castellanos-Cruz, Ai-Min Bao, and Dick F Swaab

Later Life Stages

5.13 Gonadal Hormonal Influences on the Adolescent Brain and Trajectories of Behavioral
Development 293
Kalynn M Schulz and Cheryl L Sisk
5.14 Neuroendocrine Regulation of Puberty 309
Ei Terasawa and Jon E Levine
5.15 Human Puberty: Physiology, Progression, and Genetic Regulation of Variation in Onset 357
B A Kaminski and M R Palmert
 Contents of All Volumes xv

5.16 Hormone Regulation of Neurogenesis Across the Lifespan 373

P Duarte-Guterman, S Yagi, J R Epp, S Brummelte, S R Wainwright, C K Barha,
C Chow, J M Barker, and Liisa A M Galea
5.17 Effects of Sex Steroids on Damaged Neural Systems 411
Donald G Stein
5.18 Effects of Stress Throughout the Lifespan on the Brain and Behavior 443
C Nasca, E Davis, B Bigio, C Sandi, and B S McEwen
5.19 Reproductive Aging of Neuroendocrine Systems 465
Roberta Diaz Brinton

Index 477
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PREFACE OF THE FIRST EDITION

Because of the large number of neuroactive substances discovered by chemical endocrinologists and the
prominent roles of nuclear hormone receptors as ligand-activated transcription factors, scientists studying
hormoneebrainebehavior relations have made discoveries and achieved explanations of behavior that place
their field foremost in neurobiology. The purpose of these volumes is to review the current state of this
knowledge inclusively. That means covering true molecular genetic approaches as well as neuroanatomical,
electrophysiological, zoological, neurochemical, developmental, and behavioral studies. The medical impor-
tance of this work is clear from the last section of this treatise. The editors intend these reviews to be
comprehensive; if there are any gaps in the coverage, a second edition will correct them.

Acknowledgments

Lucy Frank and Carol Oliver, at The Rockefeller University, organized all the volumes. Noelle Gracy and Mica
Haley handled things efficiently at Academic Press. Jasna Markovac at Academic Press presided over all stages of
this project. The editors thank all of these folks for their intelligent and gracious efforts.

Donald W. Pfaff
The Rockefeller University, New York, NY, USA

xvii
This page intentionally left blank
HORMONAL EFFECTS ON SPECIFIC BEHAVIORS, ON GLOBAL
CNS STATES, AND ON HUMAN DISEASE. AN INTRODUCTION
TO THE SECOND EDITION

In 2002, there appeared the first edition of Hormones, Brain, and Behavior, dedicated to three generations of
productive scientists in our field, as it had developed at that point. With five volumes, totaling about 4100
pages, it represented a massive effort by over 200 scientists. The volumes were well received.
At least four developments have taken place since 2002 and justify a second edition. Two of them are
technical: the increasingly sophisticated application of molecular biological techniques and biophysical tech-
niques to the understanding of mechanisms for hormone/behavior relations. Third, we have entered an era of
translational research. Laboratory scientists are under increasing pressure to show how their work might lessen
the burden of disease upon the citizenry. The fourth development is the most subtle one and can be explained as
follows.
Some contemporary approaches to the hormonal controls of various forms of behavior can be distin-
guished sharply from the major theme in neuroscientific research in the past century. I characterize that
classical theme as a hunt for specificity. In the spinal cord, for example, the work of neurophysiologists such as
Sir Charles Sherrington (1906) and Sir John Eccles (1957) sought to explain why a particular reflex response
occurred specifically as a result of one particular stimulus and not another. At the level of the cerebral cortex,
work typified by that of David Hubel and Wiesel (1977) in the visual system and Vernon Mountcastle (1957)
in the somatosensory system described cells whose responses required stimuli of a specific type in a well-
defined receptive field. My own work, explaining mechanisms for lordosis behavior (Pfaff, 1999), fell into that
tradition.
In contrast, new approaches to changes of global states of the central nervous system involve the
regulation of large classes of behavioral responses to large sets of adequate stimuli. Thus, hormonal
influences on mood, exploratory tendencies, arousal, alertness, and attention would not be specific in the
manner displayed by neurons in the visual cortex, but they would be incredibly important for main-
taining mental and physical health. Our field of hormones and behavior increasingly embraces these
concepts.
In order to add new material, discovered since 2002, with special emphasis on clinical research, we had
to adopt the following strategy. We asked authors who had contributed to the first edition to reduce
markedly the length of their treatments of findings already covered in 2002. Therefore, the reader of this
current (second) edition will want to refer to the first edition as well, for the kind of comprehensive review
we intend.
Taking the first and the second editions of Hormones, Brain, and Behavior together will afford the reader
a tremendous number of facts, ranging from molecular genomics of hormone action, through biophysics,
neuroanatomy, and endocrinology to animal behavior and human disease. But this bulky coverage of primary
research findings is not, across the scope of several volumes, intended to be logically systematic. A parallel effort
in the text Principles of Hormone Behavior Relations takes a step in that direction.

References
Eccles, J., 1957. The Physiology of Nerve Cells. Johns Hopkins University Press, Baltimore, MD.
Hubel, D., Wiesel, T., 1977. Functional architecture of macaque monkey visual cortex. Proc. R. Soc. Lond. 198, 1e59.

xix
xx Hormonal Effects on Specific Behaviors, on Global CNS States, and on Human Disease

Mountcastle, V., 1957. Modality and topographic properties of single neurons of cat’s somatic sensory cortex. J. Neurophysiol. 20, 408e434.
Pfaff, D., 1999. Drive: Neural and Molecular Mechanisms of a Motivated Behavior. MIT Press, Cambridge, MA.
Sherrington, C., 1906. The Integrative Action of the Nervous System. Yale University Press, New Haven, CT (Reprinted 1947).

Further Reading
Pfaff, D., Phillips, M.I., Rubin, R.T., 2004. Principles of Hormone/Behavior Relations. Academic Press/Elsevier, San Diego.

Donald W. Pfaff
The Rockefeller University, New York, NY, USA
HORMONAL EFFECTS ON SPECIFIC BEHAVIORS, ON GLOBAL CNS
STATES, AND ON HUMAN DISEASE. AN INTRODUCTION TO THE
THIRD EDITION

In 2002, the first edition of Hormones, Brain, and Behavior appeared, dedicated to three generations of productive
scientists in our field, as it had developed at that point. With five volumes, totaling about 4100 pages, it rep-
resented a massive effort by over 200 scientists. In 2009 the volumes were updated, in the second edition. Both
sets of five volumes were well received.
At least four developments have taken place since 2009 which justify a third edition. Two are technical: the
application of increasingly sophisticated molecular biological techniques and biophysical techniques to the
understanding of mechanisms for hormone/behavior relations. Third, we have entered an era of translational
research. Laboratory scientists are under increasing pressure to show how their work might lessen the burden of
disease upon the citizenry. The fourth development is the most subtle one and can be explained as follows.
Some contemporary approaches to the hormonal controls of various forms of behavior can be distinguished
sharply from the major theme in neuroscientific research in the past century. We characterize that classical theme
as a ‘hunt for specificity.’ In the spinal cord, for example, the work of neurophysiologists such as Sir Charles
Sherrington (1906) and Sir John Eccles (1957) sought to explain why a particular reflex response occurred as
a result of one particular stimulus and not another. At the level of the cerebral cortex, work typified by that of
David Hubel and TorstenWiesel (1977) in the visual system and Vernon Mountcastle (1957) in the somato-
sensory system described cells whose responses required stimuli of a specific type in a well-defined receptive field.
Pfaff’s own work, explaining mechanisms for lordosis behavior (Pfaff, 1999), fell into that tradition.
In contrast, new approaches to changes of global states of the central nervous system involve the regulation of
large classes of behavioral responses to large sets of adequate stimuli. Experimental work on steroid hormone
regulation of responses to stress provide an example of this type of brain research (reviewed, Joels et al., 2013). Thus,
hormonal influences on mood, exploratory tendencies, arousal, alertness, and attention would not be specific in the
manner displayed by neurons in the visual cortex, but they would be incredibly important for maintaining mental
and physical health. Our field of hormones and behavior increasingly embraces these concepts.
In order to add new material, discovered since 2009, with special emphasis on clinical research, we had to
adopt the following strategy. We asked authors who had contributed to the first and second editions to reduce
markedly the length of their treatments of findings already covered in 2009; therefore, the reader of this current
(third) edition may want to refer to the earlier editions as well, for the kind of comprehensive review we intend.
In lieu of these earlier sections, the authors expanded information on more recent concepts and experiments.
Taking all editions of Hormones, Brain, and Behavior together will afford the reader a tremendous number of
facts, ranging from molecular genomics of hormone action, through biophysics, neuroanatomy, and endo-
crinology to animal behavior and human disease. But this bulky coverage of primary research findings is not,
across the scope of several volumes, intended to be logically systematic.
A parallel effort in the text Principles of Hormone Behavior Relations (the second edition of which will appear
soon) takes a step in that direction.

References
Eccles, J., 1957. The Physiology of Nerve Cells. Johns Hopkins University Press, Baltimore, MD.
Hubel, D., Wiesel, T., 1977. Functional architecture of macaque monkey visual cortex. Proc. R. Soc. Lond. 198, 1e59.

xxi
xxii Hormonal Effects on Specific Behaviors, on Global CNS States, and on Human Disease

Joels, M., Pasricha, N., Karst, H., 2013. The interplay between rapid and slow corticosteroid actions in brain. Eur. J. Pharmacol. 719, 44e52.
Mountcastle, V., 1957. Modality and topographic properties of single neurons of cat’s somatic sensory cortex. J. Neurophysiol. 20, 408e434.
Pfaff, D., 1999. Drive: Neural and Molecular Mechanisms of a Motivated Behavior. MIT Press, Cambridge, MA.
Sherrington, C., 1906. The Integrative Action of the Nervous System. Yale University Press, New Haven, CT (Reprinted 1947).

Further Reading
Pfaff, D., Phillips, M.I., Rubin, R.T., 2004. Principles of Hormone/Behavior Relations. Academic Press/Elsevier, San Diego.

Donald W. Pfaff
The Rockefeller University, New York, NY, USA
Marian Joëls
University Medical Center Utrecht, Utrecht, The Netherlands
PERMISSION ACKNOWLEDGMENT

The following material is reproduced with kind permission of Oxford University Press
Figure 1 of Regulation of Honeybee Worker (Apis mellifera) Life Histories by Vitellogenin
www.oup.com

i
 1.01 Male Sexual Behavior
Elaine M Hull, Florida State University, Tallahassee, FL, USA
Gabriela Rodrı́guez-Manzo, Cinvestav, Mexico City, México
Ó 2017 Elsevier Inc. All rights reserved.

1.01.1 Patterns of Male Sexual Behavior 2

1.01.1.1 Description of Behavioral Elements 2
1.01.1.1.1 Precopulatory Behaviors 2
1.01.1.1.2 Copulatory Behavior 2
1.01.1.2 Sexual Satiety 3
1.01.1.3 Motivation and Performance 4
1.01.1.4 Sexual Experience 5
1.01.1.5 Sexual Behavior during Puberty and Aging 5
1.01.1.5.1 Puberty 5
1.01.1.5.2 Aging 6
1.01.2 Sexual Reflexes 7
1.01.2.1 Observations during Copulation 7
1.01.2.2 Ex Copula Sexual Reflexes 7
1.01.2.2.1 Spontaneous or Drug-Induced Erections 7
1.01.2.2.2 Noncontact Erections 7
1.01.2.2.3 Reflexive Erections, Anteroflexions, and Seminal Emissions 7
1.01.2.2.4 The Urethrogenital Reflex 8
1.01.2.3 Erection 8
1.01.2.3.1 Anatomy of the Penis and Mechanisms of Erection 8
1.01.2.3.2 Neural Innervation 8
1.01.2.3.3 Cellular Mediators of Erection 9
1.01.2.4 Ejaculation 10
1.01.3 Role of Gonadal Steroids in the Control of Male Sexual Behavior 12
1.01.3.1 Testosterone and Its Metabolites 12
1.01.3.1.1 Time Course of Changes in Copulation Following Castration and T Restoration 12
1.01.3.1.2 Role of T Metabolites in Maintaining and Restoring Copulation 12
1.01.3.1.3 Effects of Castration and Hormone Replacement on Ex Copula Penile Responses 13
1.01.3.2 Steroid Action on Steriod Hormone Receptors 14
1.01.3.3 The Role of Progesterone in Male Sexual Behavior 14
1.01.4 Effects of Systemically and Intraventricularly Injected Drugs 14
1.01.4.1 Dopamine 14
1.01.4.2 Norepinephrine 16
1.01.4.3 Serotonin 16
1.01.4.4 Acetylcholine 18
1.01.4.5 Gamma Aminobutyric Acid 18
1.01.4.6 Glutamate 18
1.01.4.7 Nitric Oxide 18
1.01.4.8 Endocannabinoids 19
1.01.4.9 Endogenous Opioids 19
1.01.4.10 Oxytocin 20
1.01.4.11 Prolactin 20
1.01.4.12 Gonadotropin-Releasing Hormone 21
1.01.4.13 Orexin/Hypocretin 21
1.01.5 Brain Areas and Circuitry Implicated in the Control of Masculine Sexual Behavior 21
1.01.5.1 Sensory Inputs 21
1.01.5.1.1 Olfactory Bulbs 21
1.01.5.1.2 Amygdala 22
1.01.5.1.3 Bed Nucleus of the Stria Terminalis 24
1.01.5.1.4 Central Tegmental Field/Subparafascicular Nucleus of the Thalamus 25
1.01.5.2 Major Integrative Sites 26
1.01.5.2.1 Medial Preoptic Area 26
1.01.5.2.2 Mesocorticolimbic DA Tract 31
1.01.5.2.3 Nigrostriatal DA Tract 33

Hormones, Brain, and Behavior, 3rd edition, Volume 1 http://dx.doi.org/10.1016/B978-0-12-803592-4.00001-8 1

2 Male Sexual Behavior

1.01.5.2.4 Paraventricular Nucleus of the Hypothalamus 33

1.01.5.2.5 Lateral Hypothalamus 35
1.01.5.2.6 Ventromedial Hypothalamus 35
1.01.5.3 Major Motor Outputs 36
1.01.5.3.1 Ventral Premammillary Nucleus 36
1.01.5.3.2 Midbrain Periaqueductal Gray 36
1.01.5.3.3 Nucleus Paragigantocellularis of the Medulla 36
1.01.5.3.4 Other Brain Areas 37
1.01.5.3.5 Spinal Cord 37
1.01.6 Circuitry and Anatomical Interconnections 38
1.01.6.1 Sensory Inputs 38
1.01.6.2 An Ejaculation-Related Circuit 38
1.01.6.3 Efferents from the MPOA 40
1.01.6.4 Sexual Behavior in the Context of Mammalian Social Behavior 40
References 40

1.01.1 Patterns of Male Sexual Behavior
1.01.1.1 Description of Behavioral Elements
Male sexual behavior comprises a complex pattern of genital
and somatomotor responses, elicited, directed, and maintained
by external and internal signals. It includes copulation as well
as precopulatory behaviors that allow the male to detect and
locate a mate, assess her potential mating appropriateness,
and stimulate a receptive response.
1.01.1.1.1 Precopulatory Behaviors
Precopulatory behavior may consist simply of anogenital inves-
tigation or may include highly elaborate behavioral patterns,
depending on the species. Male and female rodents show an
initial period of mutual anogenital investigation; they also
emit ultrasonic vocalizations (USV) of 50 kHz, which have
been considered mutually arousing (Geyer and Barfield,
1978; Pomerantz and Clemens, 1981), although recent data
suggest that these USV lack incentive value for both male and
female rats (Snoeren and Ågmo, 2013, 2014). Males engage
in urine marking (Meisel and Sachs, 1994), the odor of which
possesses incentive value for female rats (Snoeren and Ågmo,
2014). Receptive females will solicit mating from the male by
characteristic proceptive behaviors, and the male will pursue
and mount her.
1.01.1.1.2 Copulatory Behavior
Male rodents and rabbits exhibit a highly stereotyped copula-
tory pattern, shaped by three distinct behavioral motor
patterns: mount (M), intromission (I), and ejaculation (E).
1.01.1.1.2.1 Mounts
Almost all male mammals mount females dorsally and from
the rear, posing his forelegs over the female’s back and with
his hind feet on the ground. The female may assume
a lordosis posture, a reflexive dorsoflexion of the spine,
accompanied by deflection of the tail to the side. The male
then begins anteroposterior pelvic thrusts (19–23 Hz) (Beyer
et al., 1981) that induce or intensify the female’s receptive
posture. During a nonintromissive mount, the male does
not achieve penile insertion and dismounts the female
slowly.
1.01.1.1.2.2 Intromissions
Intromission, the defining event of copulation, refers to
intravaginal penile insertion. The male rat mounts the
female, performs pelvic thrusting, and suddenly displays
a deeper thrust with a mean duration of 200–300 ms (Beyer
et al., 1981) that coincides with vaginal penetration, fol-
lowed by an abrupt backward dismount and grooming of
his genitalia. Male hamsters show a similar intromission
pattern, but after repeated ejaculations (9–10) a motor intro-
mission pattern called ‘long intromissions’ is displayed,
during which penile insertion is longer than during typical
preejaculatory intromissions and than during ejaculation.
Long intromissions include intravaginal thrusting, and this
motor response precedes sexual satiety (reviewed in Hull
and Dominguez, 2007). Male rabbits show rhythmic thrusts
for several seconds, which may or may not result in intromis-
sion; however, if intromission does occur, ejaculation begins
almost immediately (Morali et al., 2003).
1.01.1.1.2.3 Ejaculation
Most mammals achieve the ejaculatory threshold after
multiple intromissions. Ejaculatory behavior in male rats
starts with an intromission but includes a deeper, longer
thrust (750–2000 ms) (Beyer et al., 1981) that coincides
with seminal ejection. The male then raises his forelegs and
dismounts slowly, then typically grooms himself. Mice
show essentially the same ejaculatory pattern, except that
during ejaculation, they may freeze before dismounting.
Male canids begin ejaculating soon after penile insertion
and develop a swelling at the base of the penis, which results
in a ‘lock’ of the male to the female (Beach, 1969). Male
ungulates (Lott, 1981) and rabbits (Morali et al., 2003)
also ejaculate immediately upon intromitting. Rabbits’
mounts that result in ejaculation are shorter, more regular,
and of higher frequency (Morali et al., 2003). Rhythmic
contractions of skeletal and striated perineal muscles usually

accompany ejaculation and are associated with orgasm in
humans.
1.01.1.1.2.4 Postejaculatory Behavior
The most patent effect of ejaculation is a period of sexual
quiescence: the postejaculatory interval (PEI). In rats, refracto-
riness lasts 5–8 min after a first ejaculation and increases with
each successive ejaculation. During the initial 75% of the PEI,
the male emits 22 kHz USVs and will not copulate in response
to any stimulus; for that reason, it is called the absolute refrac-
tory period (Barfield and Geyer, 1975). The remaining 25% of
the PEI, the relative refractory period, is no longer accompa-
nied by vocalizations, and its duration can be reduced if the
male is subjected to nonspecific arousing stimuli, such as
a mild electrical shock (Barfield and Sachs, 1968) or a novel
female partner (Zucker and Wade, 1968). After the PEI,
male rats resume pursuance and mounting of the female.
Mice resume sexual activity 1–24 h after an ejaculation,
depending on the mouse strain, while hamsters have much
shorter PEIs, from a few seconds to 1.5 min (reviewed in
Burns-Cusato et al., 2004; Hull and Dominguez, 2007; Meisel
and Sachs, 1994). During successive ejaculatory series, the
duration of the PEI increases and the number of
preejaculatory intromissions decreases (Rodríguez-Manzo
and Fernández-Guasti, 1994).
The mechanisms involved in the postejaculatory refractory
period are poorly understood. In rats, there are data suggesting
the involvement of serotonergic (McIntosh and Barfield,
1984a) and GABAergic (Fernández-Guasti et al., 1986a) trans-
mission in the induction and maintenance of the PEI, while
both dopaminergic (McIntosh and Barfield, 1984b) and norad-
renergic (McIntosh and Barfield, 1984c) transmission are
thought to counteract it, but the evidence is limited. Data on
human PEI are scarce and therefore do not contribute to
a significant advance in its understanding (Levin, 2009).
1.01.1.2 Sexual Satiety
When allowed to copulate without restriction, male rats will
show approximately seven ejaculations before reaching sexual
satiety (Beach and Jordan, 1956; Rodríguez-Manzo and
Fernández-Guasti 1994). The main feature of this phenom-
enon is the instatement of a long-lasting sexual behavior inhi-
bition which may last up to 3 days, from which males gradually
recover (Rodriguez-Manzo et al., 2011). Twenty-four hours
after reaching satiety, male rats may show a complete absence
of sexual activity or may execute one ejaculatory series without
recovery (Rodríguez-Manzo and Fernández-Guasti, 1994).
In rabbits, sexual activity was studied over periods up to
15 days to determine when the male would become exhausted,
i.e., fail to copulate with a new female for 24 h (Jiménez et al.,
2012). Males copulated to satiety on one day, but would
resume the next day. To attain sexual exhaustion, rabbits copu-
lated ad libitum during several successive days (the number
varying from 2 to 15). During those tests, the number of
ejaculations decreased from 22 on the first day to 6 on the
last day, and the number of ‘misses’ (mounts without
ejaculation) increased. Thus, rabbits require more days to
become exhausted than do rats. Sexual satiety has also been
described in hamsters (Beach and Rabedeau, 1959), mice
Male Sexual Behavior 3
(Dewsbury, 1983), mandarin voles (He et al., 2013), and
rhesus monkeys (Bielert and Goy, 1973).
Copulation to satiety in rats increases dopamine (DA)
metabolite levels in the medial preoptic area (MPOA), which
remain elevated during the first 48-h period of sexual inhibi-
tion (Mas et al., 1995a), while nucleus accumbens (NAc) DA
levels also increase during copulation to exhaustion (Fiorino
et al., 1997). The hypothalamic content of enkephalins is
also increased in sexually satiated rats for at least 48 h after
sexual exhaustion (Rodríguez-Manzo et al., 2002a). A reduc-
tion in androgen receptor (AR) density within the MPOA,
NAc, and ventromedial hypothalamic nucleus (VMH), but
not in the bed nucleus of the stria terminalis (BST), of satiated
rats (Fernández-Guasti et al., 2003) and in the lateral septal
nucleus, medial amygdala (MeA), MPOA, and VMH of satiated
male mandarin voles (He et al., 2013) has been detected. Fos-
immunoreactivity (Fos-ir, a marker of neural activation) was
increased in regions of the BST, MPOA, and MeA following
sexual satiety in male hamsters (Parfitt and Newman, 1998),
while in rats the relevant brain structures were the MeA and
septum (Phillips-Farfán and Fernández-Guasti, 2007). A
contribution of the nucleus paragigantocellularis (nPGi) in
satiety can be inferred from the fact that nPGi lesions increased
the latency to and the number of ejaculations that preceded
sexual satiation (Yells et al., 1992).
Sexual satiety is regulated by diverse neurotransmitters as
indicated by drug-induced reversal of the sexual inhibitory
state. Among them, norepinephrine (NE) (Rodríguez-Manzo,
1999b; Rodríguez-Manzo and Fernández-Guasti, 1994,
1995b), serotonin (5-HT) (Arnone et al., 1995; Fernández-
Guasti and Rodríguez-Manzo, 1997; Rodríguez-Manzo and
Fernández-Guasti, 1994), endogenous opioids (Garduno-
Gutierrez et al., 2013b; Miller and Baum, 1987; Pfaus and
Gorzalka, 1987; Rodríguez-Manzo and Fernández-Guasti,
1995a), endocannabinoids (Canseco-Alba and Rodriguez-
Manzo, 2014; Rodriguez-Manzo and Canseco-Alba, 2015b),
glutamate (Rodriguez-Manzo, 2015), and DA (Mas et al.,
1995b; Rodríguez-Manzo, 1999b; Guadarrama-Bazante et al.,
2014), but not MPOA gamma-aminobutyric acid (GABA)
(Rodríguez-Manzo et al., 2000), all seem to play a role. Most
of the drugs that reverse satiety appear to do so via the NE
system (Rodríguez-Manzo and Fernández-Guasti, 1995b),
coupled, in turn, to the DA system, which would be a final
pathway for the reversal (Rodríguez-Manzo, 1999b). Both
endogenous opioids (Garduno-Gutierrez et al., 2013a) and
endocannabinoids (Canseco-Alba and Rodriguez-Manzo,
2014) are released during copulation to satiety and contribute
to the establishment of the long-lasting sexual inhibitory state.
The mesolimbic DA system is the target of the actions of the
released endogenous opioids, through the activation of mu
opioid receptors within the ventral tegmental area (VTA)
(Garduno-Gutierrez et al., 2013b,c). In male hamsters, the
cholinergic system in the MPOA was found to play a role in
the development of sexual exhaustion. Infusion of the musca-
rinic receptor agonist oxotremorine delayed, while the musca-
rinic antagonist scopolamine accelerated, the attainment of
sexual exhaustion (Floody, 2013).
The so-called Coolidge effect (the renewed sexual respon-
siveness of male rats that ceased to copulate after reaching
exhaustion that is produced by changing the stimulus female
4 Male Sexual Behavior
(Fisher, 1962)) interferes with the establishment of the sexual
inhibition characteristic of sexual exhaustion, 24 h after satiety;
neurotoxic lesions of NE neurons did not interfere with
this effect (Rodríguez-Manzo, 1999a). Bromocriptine, a DA
agonist, was recently found to renew sexual responsiveness of
sexually exhausted male rats, to the same sexual mate, immedi-
ately after sexual satiety, mimicking the Coolidge effect but
without changing the female partner (Rojas-Hernandez and
Juarez, 2015). Together these data suggest that a decreased
DA transmission may account for cessation of sexual activity
at satiety.
In addition to the inhibition of copulation, other responses
are also modified by sexual exhaustion. Thus, sexually satiated
rats show a general increase in sensitivity to drug actions,
including those of yohimbine, 8-hydroxy-2-(di-n propyla-
mino)tetralin (8-OH-DPAT), naloxone, and desipramine
(Martínez-Mota et al., 2005; Rodríguez-Manzo and
Fernández-Guasti, 1994, 1995a). Modifications in the neural
control of copulation appear to occur after sexual satiation in
rats. Hence, prior to sexual exhaustion, electrical stimulation
of the MPOA (Rodríguez-Manzo et al., 2000), the dorsal region
of the VTA (Rodríguez-Manzo and Pellicer, 2007), or the NAc
(Rodriguez-Manzo and Pellicer, 2010) markedly facilitated
sexual behavior, but after sexual satiety the same animals no
longer responded to this stimulation. Also, the anxiolytic-like
properties of ejaculation, seen in nonexhausted rats, disap-
peared in the population of sexually satiated males that can
ejaculate 24 h after satiation (Rodríguez-Manzo et al., 1999).
Thus, copulation to satiation promotes long-lasting changes
in male rats’ physiology.
1.01.1.3 Motivation and Performance
Motivation for sexual activity may be distinguished from actual
sexual performance. Sexual motivation may be compared to an
engine that may drive several goal-directed behaviors, whereas
hormone-sensitized factors may steer the behavior down
specific paths. However, experimental procedures designed to
test sexual motivation may have effects that are confounded
with sensory, motor, or learning abilities. Therefore, tests that
separate effects on motivation from those on motor, sensory,
or learning abilities are preferred.
Several tests do meet at least some of those criteria. Place pref-
erence tests assess whether previous copulation in one compart-
ment leads to greater time spent in that compartment, even
without a stimulus female. An advantage of this test is that little
motor ability is required to show a preference; another advan-
tage is that any drugs administered during conditioning would
be metabolized before the test day. However, it does require
learning ability for the male to associate the external stimuli
with the positive stimuli of copulation. A similar procedure
uses naïve males, which are allowed to spend time near either
an estrous or nonestrous female (Ågmo, 2003; Amstislavskaya
and Popova, 2004). Motor ability to approach the two stimulus
females is the same; however, sensory processing may be affected
by drugs administered before the test. Another test is the obstruc-
tion apparatus, in which a male is required to cross a barrier or
an electrified grid to access a female. However, this procedure
does confound motivation with motor ability. Another test
uses a bilevel apparatus in which a male and female can
copulate; later, the male is placed into the apparatus alone,
and the number of times he changes levels, presumably in search
of the female, is the measure of motivation (Mendelson and
Pfaus, 1989). However, this test also confounds motivation
and motor ability. Another test is lever pressing for a secondary
reinforcer that was previously associated with copulation
(Everitt, 1990). However, this also confounds motivation with
motor ability and with the ability to associate the secondary
stimuli with copulation. The X-maze or cross-maze uses the
percentage of trials on which the male chooses the
compartment containing an estrous female, with which he can
copulate, compared to empty compartments or those that
contain other goal objects (Hull et al., 1991; Warner et al.,
1991). Motor ability is measured as the running speed to all
goal boxes and the number of trials on which the male does
not leave the central start area. Motivation is then scored as
the percentage of trials on which he chooses the female, out of
all trials on which he leaves the start area. Copulatory
measures can also be assessed. However, drugs may affect
sensory processes or memory of cues marking the female’s
goal box. A similar test uses a runway and a goal compartment
with a one-way door, through which a male can enter
a female’s chamber, copulate, and then leave through a second
one-way door to the original start box (Beck et al., 2002).
Motivation is measured as time in the female’s compartment,
whereas run latency and duration and the number of
copulatory behaviors reflect both motivation and motor
ability. However, drugs on the test day may affect sensory
processes. Therefore, there are no ‘pure’ tests of motivation
that are free from potential confounds. Without additional
tests of sensory, motor, and learning abilities, it is not possible
to differentiate those factors from sexual motivation.
Therefore, reports of effects on sexual motivation should
include a caveat that those measures of motivation may be
influenced by changes in sensory, motor, or learning ability.
The term sexual arousal has also been discussed as a compo-
nent of motivation, although there is no general agreement
about its definition. For example, Ågmo (2008) suggested
that sexual arousal is nothing more than enhanced genital
blood flow, while others may regard it as more of a species-
specific first step in a series of appetitive behaviors leading
toward copulation (discussed in Heiman and Pfaff, 2011).
Ågmo (2011) later proposed that arousal should “refer to the
general state of activity of the organism and of its nervous sys-
tem.and cannot be specific to a particular class of stimuli or
reponses.” Furthermore, he proposed that “the execution of
copulatory acts leads to increased general arousal.” Similarly,
Schober et al. (2011) argue that a heightened generalized
CNS arousal energizes sexual arousal and vice versa. They
propose that a network of ascending excitatory neurons to
the hypothalamus and basal forebrain promote general arousal
and that descending axons from those areas activate brain stem
areas that control the specific behaviors. They report having
successfully bred, over six generations, mice that show high
versus low general arousal. They then found that males bred
for high general arousal had more mounts before vaginal intro-
mission, fewer intromissions before ejaculation, and ejaculated
more quickly after the first intromission, which the authors
took as evidence for high sexual arousal. Thus, high general
arousal can increase sexual arousal, which in turn may increase

sexual motivation and influence copulation. Conversely, sexual
activity can enhance general motivation.
1.01.1.4 Sexual Experience
Experience plays an important role in the full development and
efficiency of sexual behavior. Thus, sexual learning reduces the
time to initiate sexual contact and to achieve ejaculation, as
well as the amount of stimulation required to ejaculate (Pfeiffer
and Johnston, 1994). Sexual experience also improves copula-
tory ability (Domjan 1992; Pfaus et al., 2001; Woodson, 2002)
and sharpens the olfactory interest of male rodents in female
sexually related chemosignals (Hayashi and Kimura, 1974;
Lydell and Doty, 1972; Nielsen et al., 2013; Pfeiffer and John-
ston, 1994; Swaney et al., 2007). Some of the learning-induced
changes in male sexual ability can be interpreted as experience-
dependent increases in sexual motivation related to the
rewarding nature of male sexual activity (Pfaus and Phillips,
1991), since males engage in sexual behavior more rapidly,
ejaculate more often, and display shorter PEIs (Rodriguez-
Manzo and Canseco-Alba, 2014). Furthermore, sexual experi-
ence diminishes or eliminates the disruptive effect of a novel
environment, seen in sexually naïve males (Pfaus and Wilkins,
1995). Increased investigation of female chemosignals has also
been suggested to result from the increased sexual interest
exhibited by sexually experienced male mice (Swaney et al.,
2007), although a recent study in hamsters revealed that, in
this species, sexual experience did not affect male preference
for a receptive female (Ballard and Wood, 2007). Interestingly,
sexually experienced male cats (Rosenblatt and Aronson,
1958), mice (Manning and Thompson, 1976), and hamsters
(Constantini et al., 2007), but not rats (Bloch and Davidson,
1968), are less susceptible to the disruptive effects of castration.
Sexual experience also attenuates the negative effects of several
brain lesions on sexual performance, such as bilateral olfactory
bulbectomy in rats (Bermant and Taylor, 1969), ablation of the
vomeronasal organ (VNO) in hamsters (Meredith, 1986) and
rats (Saito and Moltz, 1986), but not those resulting from
zinc sulfate lesions of the main olfactory epithelium in mice
(Keller et al., 2006). The effects of lesions in the medial poste-
rior BST (Claro et al., 1995) on copulation were also greater in
sexually naïve than in sexually experienced male rats, while in
Syrian hamsters this lesion decreased opposite-sex odor prefer-
ence and delayed the whole copulatory sequence in sexually
naïve, but not in sexually experienced animals (Been and
Petrulis, 2010a). Lesion of the sexually dimorphic nucleus
(SDN) of the MPOA disrupted copulatory behavior in sexually
inexperienced male rats (de Jonge et al., 1989) but lacked an
effect in sexually experienced animals (Arendash and Gorski,
1983). In hamsters, discrete lesions of the MPOA caused severe
copulatory deficits only in sexually naïve males and not in
males with sexual experience (Been and Petrulis, 2010b).
Enhanced neuronal responses as a result of sexual experience
have also been reported. Thus, ejaculation activated more cells,
determined by the number of Fos-ir neurons, within the MPOA
(Lumley and Hull, 1999) and the NAc (Lopez and Ettenberg,
2002a) of sexually experienced male rats, compared with sexu-
ally naïve animals. By contrast, in male Japanese quail, sexual
experience decreased the expression of another immediate
early gene, egr-1, in brain areas involved in male sexual
Male Sexual Behavior 5
behavior, compared to that exhibited by first-time copulators
(Can et al., 2007). The MPOA, in particular, seems to be the
target of several sexual experience-induced changes in diverse
neurotransmitter systems. Thus, higher levels of nitric oxide
synthase (NOS) (Dominguez et al., 2006b), increased expres-
sion of oxytocin (OT) receptors (Gil et al., 2013), and an
augmented expression of phosphorylated DARPP-32 (DA-
and cAMP-regulated phosphoprotein, a downstream marker
of D1 receptor signaling) were detected in the MPOA of
sexually experienced rats (McHenry et al., 2012), as well as
an increased number of AR-positive neurons in the MPOA of
sexually experienced mice (Swaney et al., 2012), when
compared with sexually naïve animals. By contrast, mating-
induced Fos expression in NOS-containing neurons of this
brain region is higher in sexually naïve rats undergoing their
first sexual encounter than in sexually experienced males
(Nutsch et al., 2014). Testosterone (T) plasma and
hippocampal levels are also greater in sexually experienced
(Edinger and Frye, 2007) than in sexually naïve males, and
long-term alterations in glutamate receptor expression in rat
NAc neurons occur as a result of sexual experience (Pitchers
et al., 2012). Sexual experience increased precontact USVs
(50 kHz) (Bialy et al., 2000) and decreased anxiety-like
behavior of male rats (Edinger and Frye, 2007), while
promoting neurogenesis in the hippocampus of sexually
naïve adult male rats (Leuner et al., 2010).
In summary, sexual experience induces brain plasticity that
allows males to establish associations that strengthen sexual
responding. The rewarding component of male sexual activity
appears central to this strengthening, and it can even elicit
sexual arousal in response to nonsexual cues that were paired
with the early sexual experience, such as specific scents or
somatosensory cues (Pfaus et al., 2012). It has been proposed
that natural reinforcers, such as sexual reward, lead to brain
plasticity as a way to accomplish required behavioral adapta-
tions (Olsen, 2011).
In spite of this, two specific subpopulations can be found in
all strains of rats that do not respond to sexual experience: sexu-
ally sluggish and noncopulating animals. Sexually sluggish rats
are males that exhibit long latencies to initiate copulation and
to achieve ejaculation which are not reduced by repeated sexual
experience (Portillo et al., 2006; Rodriguez-Manzo and
Canseco-Alba, 2014), whereas noncopulating rats are appar-
ently normal, otherwise healthy animals that will not copulate
despite repeated exposure to sexually receptive females (Stefa-
nick and Davidson, 1987). The ‘lack’ of experience-driven
improvement in copulation of sluggish rats or of induction
of sexual behavior display in noncopulating males has no clear
explanation. However, the endocannabinoid anandamide has
been found to transiently reduce the ejaculatory threshold of
sluggish rats (Rodriguez-Manzo and Canseco-Alba, 2015a)
and to transform noncopulating rats into sexually active males
in a long-lasting manner (Canseco-Alba and Rodriguez-Manzo,
2013).
1.01.1.5 Sexual Behavior during Puberty and Aging
1.01.1.5.1 Puberty
Puberty is a developmental period during which the hypotha-
lamic–pituitary–gonadal axis is activated and reproductive
6 Male Sexual Behavior
capacity matures (Navarro et al., 2007; Suter et al., 1998). The
development of copulatory behavior has been studied in male
rats mainly by testing prepubertal animals with young estrous
females (Sachs and Meisel, 1979). The data from such studies
established that mounting behavior appears between 40
and 50 days of age; intromission, between 44 and 75 days;
and the behavioral pattern of ejaculation, between 48 and
75 days (reviewed in Meisel and Sachs, 1994). The develop-
ment of copulatory behavior in rats clearly depends on gonadal
hormones, since prepubertal castration prevents its appearance
(Larsson, 1967), while exogenous T or estrogen (E) hastens its
onset (Södersten et al., 1977). By contrast, in male Syrian
hamsters adult-typical reproductive behavior cannot be acti-
vated by gonadal steroids prior to puberty (Sisk et al., 2003).
In male rats reflexive erections appeared at 40 days of age,
penile flips at 44 days, and penile cups at 48 days (Sachs and
Meisel, 1979).
In male rats, T levels begin to rise about day 40, with the so-
called pubertal T surge occurring around day 50. However, the T
surge occurs after the onset of mount, intromission, and ejacu-
latory behaviors and the appearance of sexual reflexes (Sachs
and Meisel, 1979). Male hamsters begin to copulate after the
increase in pubertal T begins, but before the T surge (Romeo
et al., 2002). In this species, the lack of pubertal T impaired T-
induced mating in adulthood, produced in hamsters that were
castrated after puberty (Schulz et al., 2004). A series of recent
studies in rodents concluded that during puberty sex steroids
exert a second episode of organizational actions inducing
permanent changes in brain and behavior (Schulz et al., 2009;
Sisk and Zehr, 2005). Thus, the effects of pubertal hormones
on the adolescent brain are important for the maturation of
adult social behaviors such as copulation (Schulz and Sisk,
2006). During adolescence the interpretation of social cues
must change to enable adult responses. To illustrate this, it
has been reported that pheromones contained in female
hamster vaginal secretions (VSs) are rewarding for adult, but
not for juvenile male hamsters (Bell et al., 2013a). The
rewarding value of VS is T- and DA-dependent; however, T is
not sufficient to activate adult-like mesolimbic Fos expression
in response to this female sexual cue, but brain maturation is
also required (Bell et al., 2013b). Puberty and adolescence are
periods of particular vulnerability to stress (Holder and
Blaustein, 2014; Schneider, 2013), and perturbations during
this phase may result in important physiological and
behavioral alterations in adulthood (Romeo, 2005, 2010).
Indeed, chronic consumption of ethanol beginning at puberty
impaired sexual behavior and fertility in adult male rats
(Oliva et al., 2006), and consumption of anabolic androgenic
steroids (AAS), alone or combined, during puberty increased
or decreased sexual and aggressive behaviors in adulthood,
depending on the drug or drug combination used (Wesson
and McGinnis, 2006).
Neural changes have been associated with pubertal matura-
tion of sexual behavior. Pubertal maturation of the brain
includes remodeling of synaptic connections. Accordingly, at
the onset of puberty in male rats, dendritic arborization of
the sexually dimorphic spinal nucleus of the bulbocavernosus
(SNB) increases, followed by a decrease during puberty
(Goldstein and Sengelaub, 1994). Pubertal hormones have
been found to modulate the addition of new cells to sexually
dimorphic brain regions, such as the SDN of the MPOA and
the MeA (Ahmed et al., 2008). Actually, it has been suggested
that structural reorganization at the amygdala might be central
for the emergence of novel behaviors during adolescence in
humans (Scherf et al., 2013). Conversely, in male hamsters
the number of dendrites and spines in the posterodorsal MeA
(MeApd) decreases during puberty, which coincides with
sexual behavior maturation (Zehr et al., 2006), but in this
species pubertally newborn neurons and glia were found to
integrate into limbic and hypothalamic circuits (Mohr and
Sisk, 2013).
1.01.1.5.2 Aging
Aging is associated with changes in male sexual function in
humans, monkeys, and rodents. The probability of initiating
copulation decreases, and once initiated, the latencies to
mount (ML), intromit (IL), and ejaculate (EL) increase in old
male rodents and monkeys (Meisel and Sachs, 1994). A rela-
tionship between these deficits and lower T levels has not
been clearly established. In male rats, the decline in sexual
behavior is accompanied by a decrease in circulating T (Cham-
bers et al., 1991; Smith et al., 1992), but exogenous T only
partially restores copulatory behaviors (Chambers et al.,
1991). Maintenance of T levels by long-term replacement pre-
vented the decline in intromission frequency (IF) in old rats,
but did not prevent the age-related loss of ejaculatory response
(Hsu et al., 1986). By contrast, the decrease in sexual activity of
old male rhesus monkeys is not accompanied by a decline in
gonadal hormones or changes in the diurnal pattern of
androgen plasma levels, and exogenous T does not increase
their sexual activity; however, in old long-term castrated
monkeys T increased sexual behavior (Phoenix and Chambers,
1986). In men, the decline in T levels during aging has been
correlated with sexual impairment, as in young men with hypo-
gonadism; however, it is not clear whether T treatment
improves sexual performance (Moncada, 2006). A direct rela-
tionship between low T levels and decreased libido in aging
men has also been difficult to establish (Travison et al.,
2006). However, a role for sexual motivation in male rats’
age-related sexual behavior decline has been inferred from
the fact that treatment with yohimbine, a drug that stimulates
sexual arousal (Viitamaa et al., 2006), improved copulatory
behavior of old rats and increased mounting in old males
with penile anesthesia to levels of untreated young animals
(Smith and Davidson, 1990). Interestingly, it was documented
that a larger number of hypothalamic cells are recruited in aged
rats (30–36 months) to elicit a sexual response to chemosen-
sory stimulation as compared to adult males (12 months),
a phenomenon suggested to represent a compensatory mecha-
nism in the aged animal (Tobiansky et al., 2012).
A decline in estrogen receptors (ERs) (Roselli et al., 1993),
but not in ARs (Chambers et al., 1991), might underlie the ejac-
ulatory deficit of old male rats, since it was found that old intact
rats had fewer nuclear estrogen receptors (ERn) in the amygdala
than did young animals, with no difference in circulating E
levels. Castration reduced ERn in young and old males, but
exogenous T restored copulation and ERn in the amygdala
only in young males; T in old males restored copulation and
ERn only to precastration levels, not to that of young males
(Roselli et al., 1993). However, middle-aged (12 months)

and young (3 months) rats responded to castration and T
replacement with similar hypothalamic preoptic upregulation
of AR and downregulation of ERa densities, suggesting that
middle-aged rat brains retain the capacity to respond to exoge-
nous T (Wu and Gore, 2010). In men there was an age-related
loss of motoneurons in the lumbar spinal cord controlling
penile reflexes (Cruz-Sánchez et al., 1998). In a study in rats,
no age-related decrease in motoneuron number in the spinal
nuclei involved in rats’ sexual reflexes was found, but an age-
related atrophy of these motoneurons and the muscles they
innervate was detected (Fargo et al., 2007). Exogenous T
reversed the decreases in penile muscle weight and motoneu-
rons’ soma size and dendritic length, suggesting that T might
play a role in maintaining neuronal connectivity.
Various levels of sexual performance are observed in
middle-aged rats (18–19 months), with some able to ejaculate,
others showing only mounts and intromissions, and some
failing to exhibit any sexual activity. The loss of these specific
components of male sexual behavior has been related with
an age-related decrease in gonadotropin-releasing hormone
(GnRH) neurons in hypothalamic and septal regions (Tsai
et al., 2014). Incomplete sexual behavior has been associated
with decreased DA and NE, and increased 5-HT, in the NAc
(Tsai et al., 2006) and with decreased DA in the MPOA and
arcuate nucleus, as well as decreased NE in the MPOA (Chen
et al., 2007). Impaired sexual performance, on the other side,
was found to be related with decreased DA and NE in the
BST (Chen et al., 2008). Aged males showing complete copula-
tory behavior had monoamine levels similar to those of young
sexually active rats. These data suggest that changes in mono-
amine contents in diverse brain regions could play a role in
the sexual behavior decline of aged male rats. Recently, taurine
supplementation to aged rats (20 months) has been found to
enhance their sexual response by increasing T and nitric oxide
(NO) levels (Yang et al., 2013).
1.01.2 Sexual Reflexes
1.01.2.1 Observations during Copulation
Although the penis can be observed during copulation in many
species, erections in rodents are usually brief and obscured
from view. Characteristic behaviors are usually used to infer
the presence of erection, intromission, and ejaculation. The
female’s vagina can be examined for the presence of sperm after
a suspected ejaculation, and direct observation of the penis can
be made with a slanted mirror under the floor of a clear cage.
Electrodes may be implanted in the striated perineal muscles
to detect electrical activity during copulation (Holmes et al.,
1991), and penile pressure has also been measured (Bernabé
et al., 1999; Giuliano et al., 1994; reviewed in McMurray
et al., 2006). However, these techniques are technically diffi-
cult. Therefore, ex copula measures of sexual reflexes have
been developed. However, in copula and ex copula erections
may differ in their physiological, neurochemical, and
hormonal regulation (Sachs, 2000). Furthermore, experimental
manipulations may affect sexual reflexes without altering the
behavioral pattern. For example, male rats with a neurochem-
ical lesion of the NE system repeatedly showed the behavioral
pattern of ejaculation without seminal ejection, evidenced by
Male Sexual Behavior 7
the absence of a seminal plug in the female’s vagina, in
response to the a2-adrenoceptor antagonist yohimbine
(Rodríguez-Manzo and Fernández-Guasti, 1995b). Thus, we
must be cautious in extrapolating from ex copula tests to copu-
lation or vice versa.
1.01.2.2 Ex Copula Sexual Reflexes
1.01.2.2.1 Spontaneous or Drug-Induced Erections
Male rodents sometimes have erections in their home cage or in
a neutral test arena without any obvious sexual stimulus. Such
erections can be increased by certain drugs. These spontaneous
or drug-induced erections usually consist of extension of the
engorged glans outside of the penile sheath and are often
accompanied by genital grooming. The main advantage of
this model is its simplicity. However, drug-induced erections
can be modified by previous copulation (Sachs et al., 1994).
1.01.2.2.2 Noncontact Erections
Male rodents often have erections in the presence of an inacces-
sible receptive female or even the odors of an estrous female
(Kondo et al., 1999; Sachs, 1997). These erections are similar
to spontaneous or drug-induced erections in rodents and are
considered to be a model of psychogenic erection in men.
They are elicited by central, rather than peripheral stimuli.
However, the primary stimuli to elicit such erections in rodents
are olfactory, whereas the primary cues for psychogenic erec-
tions in men are visual and auditory; thus, the central pathways
controlling these erections may differ across species. Also, there
is evidence that different brain areas may regulate noncontact
versus in copula erections (Liu et al., 1997b). In addition,
noncontact erections may be elicited only in pigmented rats
and not in albinos (Sachs, 1996).
1.01.2.2.3 Reflexive Erections, Anteroflexions, and Seminal
Emissions
Reflexive erections, sometimes referred to as touch-based erec-
tions, can be elicited by manual stimulation of the penis in
numerous species. However, tactile stimulation of the rat penis
actually inhibits erection (Hart, 1968). To overcome this diffi-
culty, male rats (Hart, 1968) or mice (Sachs, 1980) can be
restrained on their backs and the penile sheath retracted,
thereby eliciting genital reflexes. Pressure around the base of
the penis elicits erections of the glans, due to vasodilation in
the corpus spongiosum, and also anteroflexions (‘flips’), which
are elevations of the penis caused by engorgement of the
corpora cavernosa and contraction of the ischiocavernosus
muscles. Three gradations of erections have been noted: (1)
elongation and rising of the body of the penis; (2) engorge-
ment and flaring of the glans; and (3) intense flaring of the
glans into a cup. The cup deposits the ejaculate around the
female’s cervix, where it coagulates to form a copulatory plug.
This plug prevents the semen from seeping out of the vagina;
without it, pregnancy rarely occurs (Sachs, 1982). Ex copula
seminal emission may also occur. Both reflexive and in copula
erections are mediated by parasympathetic vasodilation and
striated penile muscle contractions (Hart, 1968; Holmes
et al., 1991). However, as with noncontact erections, the neural
mechanisms of erection differ in the two contexts (Sachs,
1983).
8 Male Sexual Behavior
1.01.2.2.4 The Urethrogenital Reflex
A model for both erection and ejaculation has been devel-
oped in anesthetized, acutely spinalized male rats (McKenna
et al., 1991). The urethra is first distended with saline, and
then the pressure is released, resulting in clonic contractions
of the perineal muscles, rhythmic firing of the cavernous
nerve, erection, and ejaculation. The simultaneous firing in
all the perineal muscles is similar to bursts observed in
human climax (Gerstenberg et al., 1990) and in rats during
ejaculation (Holmes et al., 1991; Miura et al., 2001).
Synchronized activity in both the pelvic (parasympathetic)
and hypogastric (sympathetic) nerves drives the bursts in
the cavernous nerve, which are synchronized with somatic
muscle contractions. This reflex is usually evoked only after
spinal transection or lesions of certain brain nuclei,
evidencing the presence of a tonic descending inhibition
(Borgdorff et al., 2008; Carro-Juárez et al., 2003; Gravitt
and Marson, 2007; Stafford et al., 2006a,b). (However, see
Section 1.01.5.2.1.) The urethrogenital (UG) reflex has
been used as a model that provided important information
on the physiological, pharmacological, and neuroanatomical
control of ejaculation. By using this model the existence of
a spinal pattern generator for ejaculation (SGE) gained
support (Carro-Juárez et al., 2003; McKenna et al., 1991)
(see Section 1.01.2.4).
1.01.2.3 Erection
1.01.2.3.1 Anatomy of the Penis and Mechanisms of Erection
An erect penis is needed to deliver sperm into the female’s
reproductive tract. Some mammalian penes are highly vascular,
whereas others are more fibroelastic. The vascular penes of
humans, monkeys, dogs, cats, and rodents become engorged
due to vascular relaxation, coordinated with striated muscle
contraction. The fibroelastic penes of ungulates, including
sheep and goats, are extruded by penile muscles and rely very
little on engorgement. There is much variability across species
in the relative importance of these two factors.
The basic structure of the penis is similar across mammalian
species. Most of the penile shaft is occupied by the two corpora
cavernosa, while the corpus spongiosum surrounds the urethra
and enlarges into the glans at the end of the penis. The two
corpora cavernosa are fused in most species, including humans,
so that a drug injected anywhere in the structure can diffuse
throughout. Similarly, intracavernous pressure can be moni-
tored from any site. The corpora cavernosa are large cavernous
sinuses (trabeculae) that are supplied by the helicine arteries,
which in turn receive blood from the cavernosal artery
(reviewed in Anderson, 2011; Hull and Dominguez, 2015).
The corpora cavernosa are enclosed in a tough capsule (the
tunica albuginea), so when they fill with blood, the pressure
against the venous outflow traps blood in the penis, which
enlarges and becomes rigid. The proximal ends of the corpora
cavernosa taper into crura (‘legs’) that are attached to the
ischium (hip bone) and are surrounded by the ischiocaverno-
sus muscle. The proximal end of the corpus spongiosum
enlarges into the urethral bulb and is surrounded by
the bulbospongiosus (BS) muscle. Contraction of the
ischiocavernosus muscle increases cavernosal pressure, and
contraction of the BS muscle increases pressure in the glans
(Gerstenberg et al., 1990). The relative contribution of vascular
and muscular factors varies across species, with the rat relying
more on the striated penile muscles, and humans relying
more on vascular engorgement (Gerstenberg et al., 1990;
Schmidt and Schmidt, 1993).
Reflexive erections in the rat are due mostly to steady
increases in pressure in the corpora cavernosa (Bernabé et al.,
1999) and corpus spongiosum (Schmidt et al., 1995). These
increases are below systolic pressure, but are interrupted by

1 s peaks of suprasystolic pressure, elicited by contractions
of the ischiocavernosus and BS muscles. The bursts occur
during glans erections, anteroflexions, and cups. Excision
(Sachs, 1982) or denervation (Monaghan and Breedlove,
1992) of the BS muscle eliminated cups, whereas excision of
the ischiocavernosus muscle eliminated anteroflexions (Sachs,
1982). Stimulation of the pelvic and cavernous nerves
produced plateau increases in penile pressure, whereas
pudendal nerve stimulation elicited suprasystolic increases
(Giuliano et al., 1995; Lue et al., 1984). Therefore, erections
require the coordination of parasympathetically controlled
blood flow to the penis and pudendal stimulation of the stri-
ated perineal muscles.
1.01.2.3.2 Neural Innervation
The three major pathways that control penile erection are the
pelvic nerves (primarily parasympathetic and proerectile),
the hypogastric nerves (sympathetic and antierectile), and the
pudendal nerves (somatosensory and motor). The pelvic nerve
provides the major proerectile innervation (Lue et al., 1995; Tai
et al., 1998); it originates in the lumbosacral spinal cord and
travels via the pelvic plexus (pelvic ganglion) and cavernous
nerve to the penile corpora and vasculature. In addition to
the parasympathetic fibers, it also carries some sympathetic
postganglionic axons (Dail et al., 1986). The SNB and the
dorsolateral nuclei of the lumbosacral spinal cord provide
somatomotor control (Schrøder, 1980). Their axons travel in
the pudendal nerve, which splits into motor and sensory
branches, both of which also carry sympathetic efferents
(McKenna and Nadelhaft, 1986). Stimulation of the ischioca-
vernosus and BS muscles is not sufficient to cause an erection
when the penis is flaccid, but if the penis is erect, stimulation
dramatically increases its rigidity, and intracavernosal pressure
increases to suprasystolic levels (Schmidt and Schmidt, 1993).
The third major pathway provides mostly antierectile
sympathetic influence (Diederichs et al., 1991; Giuliano
et al., 1995). Preganglionic axons travel in the lumbar
splanchnic nerves or the paravertebral sympathetic chain of
ganglia (Jänig and McLachlan, 1987). They synapse in the
hypogastric plexus or the paravertebral sympathetic chain
ganglia, respectively. Postganglionic fibers travel via two major
routes. First, the hypogastric nerve sends input to the pelvic
plexus, and then via the cavernous nerve to the penis. Second,
paravertebral sympathetic chain axons travel via the pelvic
nerve to the pelvic plexus, which then sends output through
the cavernous nerve to the penis. This route provides most of
the noradrenergic input to the penis (reviewed in Anderson,
2011; Giuliano and Rampin, 2000). The penis is maintained
in a relaxed, nonerect state by tonic sympathetic input, and
injection of noradrenergic antagonists into the corpora caver-
nosa can elicit erections in men (Brindley, 1986).

Although the main influence of the sympathetic input is
antierectile, a proerectile effect has been observed in anesthe-
tized rats (Giuliano et al., 1997; Figure 1). Electrical stimula-
tion of the MPOA, a critical brain area for male sexual
behavior, increased cavernosal pressure. This increase was abol-
ished by bilateral section of the pelvic or cavernous nerves,
indicating that the major proerectile effect of MPOA stimula-
tion is through the parasympathetic system. However, bilateral
section of the paravertebral sympathetic chain at the level of L4-
L5 also significantly decreased the effect of MPOA stimulation,
as did lesion of sympathetic fibers by the catecholamine neuro-
toxin 6-hydroxydopamine (6-OHDA). Bilateral section of the
hypogastric nerves produced a nonsignificant decrease in the
Figure 1 Diagrammatic representation of peripheral autonomic path-
ways potentially involved in erectile response elicited by medial preoptic
area (MPOA) stimulation. Sites of neural lesions are represented by
hatched bars. No direct projections from MPOA to spinal autonomic
nuclei have been reported. CE, cauda equina; CN, cavernous nerve; HN,
hypogastric nerve; L4-L5, fourth and fifth lumbar levels of the para-
vertebral sympathetic chain; L6-S1, sixth lumbar and first sacral level of
the spinal cord; MPG, major pelvic ganglion; PN, pelvic nerve; PSC,
paravertebral sympathetic chain; PudN, pudendal nerve; T12-L2, 12th
thoracic to 2nd lumbar level of the spinal cord. Figure is reprinted from
Giuliano, F., Bernabé, J., Brown, K., Droupy, S., Benoit, G., Rampin, O.,
1997. Erectile response to hypothalamic stimulation in rats: role of
peripheral nerves. Am. J. Physiol. 273, R1990–R1997, with permission
from the American Physiological Association.
Male Sexual Behavior 9
penile response. The authors suggested that the sympathetic
system may produce vasoconstriction in nonpenile areas,
thereby diverting blood to the penis and enhancing erection.
Therefore, the MPOA can elicit erection by a coordinated acti-
vation of both the parasympathetic and sympathetic systems.
1.01.2.3.3 Cellular Mediators of Erection
Penile erection results from both increases in blood flow into
the penis, due to relaxation of arterial smooth muscle, and
opening sinusoids in the erectile tissue due to relaxation of
trabecular smooth muscle. Trabecular smooth muscle cells
are linked by gap junctions, which allow current to spread elec-
trotonically and second messengers to pass from cell to cell.
Therefore, autonomic input can influence the whole network
by innervating relatively few cells (Christ et al., 1999). The
penis is maintained in a flaccid state largely by NE from sympa-
thetic nerves, acting on postsynaptic a1 and, to a lesser extent,
a2 adrenergic receptors (Traish et al., 2000; reviewed in
Andersson, 2011). Drugs that block these receptors can elicit
erection or induce priapism (prolonged erection) (Abber
et al., 1987). Stimulation of a1 and a2 adrenergic receptors acti-
vates phospholipase C and thereby increases intracellular Ca2þ,
which ultimately leads to contraction of arterioles and trabec-
ulae, producing detumescence (Andersson and Stief, 1997;
Christ, 1995; Traish et al., 2000). However, soon after the
initial contraction, Ca2þ levels return to near basal levels, while
the contractile tone remains. The continuing contraction results
from Ca2þ sensitization, mediated by the GTP-binding protein
RhoA and its major effector, Rho-kinase (reviewed in Jin and
Burnett, 2006; Somlyo and Somlyo, 2000; Sopko et al.,
2014). A Rho-kinase antagonist (Y-27632) inhibited
contraction of human or rabbit cavernosal tissue strips that
had been elicited by an a-receptor agonist or electrical
stimulation in vitro (Rees et al., 2001). Y-27632 also
stimulated erection in rats (Dai et al., 2004; Rajasekaran
et al., 2005).
The main mediator of erection is NO, a short-lived gaseous
messenger produced by NOS (reviewed in Burnett, 2006).
Neuronal NOS (nNOS, or NOS I) is present in parasympathetic
nonadrenergic noncholinergic nerves that innervate the erectile
tissue. Endothelial NOS (eNOS, or NOS III) is in the endothe-
lium of erectile tissue. The initial stimulus for erection is NO
produced by nNOS in the parasympathetic nerves. It diffuses
into the smooth muscle cells and activates soluble guanylyl
cyclase, which produces cyclic guanosine monophosphate
(cGMP), which then activates protein kinase G (PKG), and to
a lesser extent protein kinase A (PKA). These enzymes phor-
phorylate regulatory proteins, which result in sequestration of
Ca2þ and the resultant relaxation of smooth muscles and erec-
tion (reviewed in Burnett, 2006). The activity of cGMP is termi-
nated by phosphodiesterase 5 (PDE5). Several drugs used to
treat erectile dysfunction, including sildenafil citrate (Viagra),
tadalafil (Cialis), and vardenafil (Levitra), act by inhibiting
PDE5 and thereby prolonging the effects of cGMP.
Although the initial stimulus for erection is NO from the
parasympathetic nerves, a longer-lasting mediator of erection
is NO from eNOS in the endothelium of blood vessels and
sinusoidal spaces. The initial increase in blood flow induces
shear stress in those tissues, which activates several enzymes,
which phosphorylate eNOS at Ser 1177 and sustain the
10 Male Sexual Behavior
production of NO (reviewed in Musicki and Burnett, 2006).
Phosphorylation at other sites by other enzymes may increase
or decrease eNOS activity. However, nNOS can also contribute
to sustained erection by PKA-induced phosphorylation at Ser
1412, which closely resembles the sequence surrounding
eNOS-Ser 1179 (Hurt et al., 2012). Thus, both neuronal and
endothelial sources of NO contribute to sustained erection.
In addition to its direct effects on vasodilation, NO also
inhibits the antierectile RhoA/Rho-kinase pathway (Mills
et al., 2002). Conversely, the RhoA/Rho-kinase pathway may
inhibit phosphorylation of eNOS at Ser 1177 and thereby
inhibit erection (Ming et al., 2002). It may also suppress
eNOS gene expression and enzyme activity (Bivalacqua et al.,
2004). Therefore, there is an inverse relationship between the
proerectile NO/cGMP/PKG pathway and the antierectile
RhoA/Rho-kinase pathway (reviewed in Sopko et al., 2014).
RhoA activates Rho-associated protein kinase (ROCK), which
contracts the cavernous sinusoidal and arteriolar smooth
muscle cells, keeping the penile smooth muscle contracted
during the flaccid state of the penis (Kimura et al., 1996).
The ROCK inhibitor Y-27632, initially studied for antihyper-
tensive properties, acts independently of NO-mediated
relaxation (Chitaley et al., 2001; Wang et al., 2002).
Inhibition of Rho-kinase not only improves erectile function,
but also decreases penile apoptosis after cavernous nerve
injury in rats (Hannan et al., 2014). Therefore, Rho-kinase/
ROCK inhibition may provide a new target for treatment of
erectile dysfunction.
Cavernous nerve damage resulted in increased ROCK and
decreased erectile ability in rats; erectile ability was restored
with Y-27632, which also increased NOS and decreased
apoptotic cells (Hannan et al., 2013). Castration also increased
RhoA and Rho-kinase protein levels, but intracavernosal injec-
tion of Y-27632 increased cavernosal pressure in the castrates,
as well as in intact and T-replaced rats (Wingard et al., 2003).
Therefore, upregulation of the RhoA/Rho-kinase pathway
may be one factor in the loss of erectile ability after
castration. Thus, inhibition of this pathway could provide
a potential treatment for erectile dysfunction. However, the
decrease in blood pressure that would accompany systemic
administration could require that these inhibitors be applied
locally or that tissue-specific isoforms that target RhoA
regulatory proteins be developed (Jin and Burnett, 2006).
In addition to NO, there has been recent investigation of the
roles of carbon monoxide (CO) and of hydrogen sulfide (H2S)
as gaseous mediators of erection (reviewed in Yetik-Anacak
et al., 2015). CO is produced by heme oxygenase and, like
NO, activates guanylyl cyclase. H2S is mainly produced by
the smooth muscle in human corpora cavernosa and also
targets the guanylyl cyclase pathway. These pathways may
complement and increase NO signaling. These other gaseous
transmitters may provide additional treatment targets for erec-
tile dysfunction.
Erection can also be stimulated by activation of adenylyl
cyclase by vasoactive intestinal peptide, calcitonin gene-
related peptide, and prostaglandin E1 (PGE1) (reviewed in
Bivalacqua et al., 2000). The resultant activation of PKA seques-
ters intracellular Ca2þ, leading to smooth muscle relaxation
and increased vasodilation. PGE1 also increases production of
NO, and repeated treatments increase both nNOS and eNOS
in the penis, resulting in greater erectile response to nerve stim-
ulation (Escrig et al., 1999). Additional erectile mechanisms
include activation of melanocortin (MC)4 receptors (Martin
et al., 2002). Systemic administration of the MC(4) agonist
THIQ increased intracavernous pressure and ex copula erections.
Intracerebroventricular administration also increased reflexive
erections. The erections elicited by THIQ were blocked by the
MC(4) antagonists, agouti-related protein and MPB10, and
were also attenuated by an OT antagonist (L-368899). Thus,
MC(4)-mediated erection may involve OT pathways.
1.01.2.4 Ejaculation
Ejaculation is the physiological process that describes the
expulsion of the semen from the urethra. This process involves
several organs including the distal epididymis, the vas deferens,
the seminal vesicles, the prostate, the prostatic urethra, and the
bladder neck (Giuliano and Clement, 2005) and consists of
two stages: an emissive phase and an ejective phase (Mitsuya
et al., 1976; Newman et al., 1982). The emissive phase involves
an autonomic component that elicits synchronized activation
of visceral accessory structures such as the prostate and the
tunica albuginea (Exintaris et al., 2006; Shafik et al., 2005),
while the ejective phase involves rhythmic contraction of peri-
neal and pelvic floor striated muscles and the maintenance of
penile rigidity. During the emissive phase, the bladder neck
sphincters close, and the seminal vesicles, prostate, vas defer-
ens, and coagulant glands contract, involving parasympathetic
and sympathetic mechanisms that promote deposition of
seminal secretions into the prostatic urethra. The expulsive
phase consists of the forceful ejection of semen from the
urethral meatus, caused by the rhythmic and coordinated
contraction of all genital muscles surrounding the genital tract
(Carro-Juárez and Rodríguez-Manzo, 2000; Gerstenberg et al.,
1990; Kollberg et al., 1972; Shafik, 1997) that coincides with
the rhythmic contraction of muscles from the abdominal
wall, i.e., cremasteric, iliopsoas, and pubococcygeus muscles
(Carro-Juárez et al., 2014). Thus, successful ejaculation
depends on coordinated responses involving autonomic and
somatic events. It has recently been proposed that during the
emissive phase the sympathetic effect on seminal vesicle’s
contraction is dominant in order to squeeze fluid into the
posterior urethra, but that during the ejective phase parasympa-
thetic seminal vesicle contractions dominate providing antire-
flux resistance to the pressure generated by BS rhythmic
contractions (Hsieh et al., 2014).
In copulating animals ejaculation requires the pelvic
thrusting pattern that results in vaginal penetration and
seminal deposition, and the genital motor pattern of forceful
seminal ejection (Moralí et al., 2003). Although these patterns
can be separately studied (Moralí et al., 2003), only the genital
motor pattern of ejaculation can be observed in experimental
models with lesions where penile rigidity, penile movements,
and ejaculatory expulsions are not disturbed (Carro-Juárez
and Rodríguez-Manzo, 2000; Carro-Juárez et al., 2003;
McKenna et al., 1991).
The stimulus that initiates ejaculation is at present
unknown; however, it has been proposed that genital sensory
stimulation that comprises friction of penile skin and intravagi-
nal pressure, acting against the penis during penetration,
 Male Sexual Behavior 11

commences seminal drain into the posterior portion of the (a)

urethra, and when the ejaculatory threshold is reached, due
to the concomitant mechanical stimulation of the urethra by
continuous seminal deposition (de Jong et al., 2006; Sachs
and Barfield, 1976) and penile afferent activation, ejaculation
takes place. The fluids that enter the urethra during emission
elicit single bursts of activity of the BS muscles via the pelvic (b′)
and pudendal nerves, but to elicit rhythmic bursting activity (b)
of perineal muscles during expulsion of seminal fluids, the
concomitant activation of penile afferents (via the pudendal
nerve) is necessary (Tanahashi et al., 2012). The synchronous
contractions of perineal and pelvic floor striated muscles, under
the control of spinal and brain centers are responsible for (c′)
(c)
semen expulsion (Dobberfuhl et al., 2014).
Individual differences in the ejaculatory threshold of rats
were described as part of the biological variability in ejacula-
tory behavior, suggesting the existence of ejaculatory endophe-
notypes in this species, and males have been classified into
(d)
praecox, intermediate, and retarded ejaculators (Pattij et al.,
2005). The physiological control of the ejaculatory threshold
remains one of the biggest riddles in the neurobiology of ejac-
ulatory function. Tonic descending inhibition from the
8-OH-DPAT
nPGi prevents untimely release of the reflex (see Sections
(e)
1.01.2.2.4, 1.01.5.3.3, 1.01.5.3.5, and 1.01.6.2), and supraspi-
nal excitatory influences originate in the MPOA and PVN (see
Sections 1.01.5.2.1 and 1.01.5.2.4).
The neural commands for ejaculation are organized at the Figure 2 Summary figure showing the evidence that the ejaculatory
motor pattern follows the general principles of rhythmic motor patterns
spinal level. It has been demonstrated that a central pattern
produced by a central pattern generator (CPG): (1) a rhythmic muscular
generator, located in the lumbosacral cord, is involved in the
response, the genital motor pattern, is registered during the ejaculatory
control of ejaculation (Figure 2; Carro-Juárez and Rodríguez- event (a); (2) this ejaculatory motor response has similar EMG charac-
Manzo, 2000, 2008; Carro-Juárez et al., 2003). A group of teristics in intact and in spinal urethane-anaesthetized male rats (a and
galanin-containing neurons in this portion of the cord is part b, respectively); (3) deafferentation does not disrupt the expression of
of this ejaculation generator (Truitt and Coolen, 2002), but the ejaculatory motor train (c); (4) a change in the stimulation interval
does not participate in emission in spinally intact male rats does not alter the intrinsic pacing of the ejaculatory-like response (d);
(Staudt et al., 2012). The pattern generator is positioned to and (5) fictive ejaculation can be pharmacologically induced (e). Cali-
relay and integrate all genital sensory and motor signals related bration bar 50 mV, 5 s. Tracing inserts (b0 and c0 ) illustrate two consec-
to ejaculation, and its activation is able to turn on and off all utive ejaculatory motor responses obtained in a 4-min period in a spinal
rat with intact afferents and in a deafferented spinal rat. Figure is adapt-
somatic and autonomic events associated with ejaculation,
ed from Carro-Juárez, M., Cruz, S.L., Rodriguez-Manzo, G., 2003.
including penile erection and movements of the penis driving
Evidence for the involvement of a spinal pattern generator in the control
seminal ejection (Carro-Juárez and Rodríguez-Manzo, 2000, of the genital motor pattern of ejaculation. Brain Res. 975, 222–228,
2003, 2005b, 2006; Carro-Juárez et al., 2003). The SGE can with permission from Elsevier.
modulate genitosensory-induced excitatory and/or inhibitory
mechanisms regulating the facilitation or inhibition of the ejac-
ulatory response at a spinal level (see Carro-Juárez and Rodrí- (Marson and McKenna, 1992), there are data suggesting an
guez-Manzo, 2008, for an extensive review). Thus, repeated intraspinal 5-HT excitatory mechanism, via activation of
activation of ejaculation-related afferent signals modifies the 5-HT1A (Carro-Juárez and Rodríguez-Manzo, 2001; Carro-
activity of the SGE, inducing a short-lasting inhibition that Juárez et al., 2003) and 5-HT2C receptors (Stafford et al.,
could be related to the ejaculation delay phenomenon attained 2006b), in addition to the descending inhibitory influence.
by the ‘pause squeeze method’ as well as long-lasting inhibitory A stimulating role for DA has also been postulated (Peeters
states (Carro-Juárez and Rodríguez-Manzo, 2005b) with char- and Giuliano, 2008; Stafford et al., 2006a), since activation of
acteristics similar to those observed in sexually exhausted DA receptors by apomorphine induces ejaculation-like
animals after repeated ejaculation (Carro-Juárez and Rodrí- responses in anesthetized rats (Stafford and Coote, 2006). Simi-
guez-Manzo, 2000, 2005b). Thus, the SGE can be modulated larly, NO in the lumbar spinal cord participates in the induction
through the activation of spinal sensory feedback mechanisms of a synchronized bursting pattern of sympathetic and somato-
(Carro-Juárez and Rodríguez-Manzo, 2000, 2001, 2005b). motor activity associated with ejaculation (Brack et al., 2007).
Spinal circuits in the lumbosacral spinal cord are modulated The spinal NE system also facilitates ejaculation. Ejaculatory
by supraspinal structures, since the SGE can be activated autonomic and somatic rhythmic patterns, including the
only after lesion of the nPGi or spinal cord transection expulsion of urethral contents and penile erections, are
(Marson and McKenna, 1990). Although the nPGi is thought obtained after increasing NE tone by systemic yohimbine
to exert a 5-HT-mediated tonic inhibition on ejaculation (Carro-Juárez and Rodríguez-Manzo, 2003, 2006). Stimulation
12 Male Sexual Behavior
of a1-adrenoceptors (Carro-Juárez and Rodríguez-Manzo, 2006,
2003) also activates the SGE. Moreover, inhibited ejaculation
due to repeated genital mechanical stimulation may be over-
come by blockade of a2-adrenoceptors with yohimbine
(Carro-Juárez and Rodríguez-Manzo, 2003). Cholinergic stimu-
lation of the SGE, mediated by the M2, M3, and M4 muscarinic
receptor subtypes, has also been suggested (Gomez et al., 2005).
Systemic injection of OT in male rats elicits ejaculatory
sequences similar to those obtained by genital-sensory stimula-
tion in adult (Carro-Juárez and Rodríguez-Manzo, 2005b) or
neonatal rats (Carro-Juárez and Rodríguez-Manzo, 2005a).
Also gastrin-releasing peptide (Sakamoto, 2011; Takanami
and Sakamoto, 2014) and NMDA receptor-mediated glutama-
tergic (Staudt et al., 2011) mechanisms are involved in the excit-
atory modulation of the SGE. Characterization of fast synaptic
responses of the motor neurons controlling BS muscles’ contrac-
tions evidenced the participation of glutamate, GABA, and
glycine receptors in their mediation (Best et al., 2013).
Endogenous opioids are the only intraspinal inhibitory
influence on SGE functioning so far described (Carro-Juárez
and Rodríguez-Manzo, 2009). Besides its central descending
inhibitory role, 5-HT has recently been postulated to modulate
the ejaculatory reponse through the activation of peripheral
receptors. Thus, a facilitative action, mediated by peripheral 5-
HT2 receptors, was recorded after intraurethral application of
5-HT (Ishigami et al., 2013), while activation of peripheral 5-
HT1A receptors was reported to inhibit seminal vesicle’s contrac-
tion (Hsieh et al., 2011), and selective serotonin reuptake inhib-
itors (SSRIs) were found to antagonize sympathetic human
seminal vesicle muscle contractions in vitro (Birowo et al.,
2009). Interestingly, chronic treatment with antidepressants
that modify the ejaculatory response, i.e., desipramine, fluoxe-
tine, and bupropion, also affect the functioning of the SGE
(Hueletl-Soto et al., 2010, 2012, 2014) (see Section 1.01.6.2).
1.01.3 Role of Gonadal Steroids in the Control
of Male Sexual Behavior
1.01.3.1 Testosterone and Its Metabolites
Male sexual behavior depends heavily on T and its metabolites.
T is secreted by the Leydig cells of the testes and is carried to its
nontesticular targets in the blood. In all mammals studied,
sexual behavior by adult males is promoted by circulating T
and/or its metabolites, estradiol (E2) and dihydrotestosterone
(DHT). A pubertal increase in T is essential for the increased
sexual activity of maturing males. However, there is interspecies
variation in the importance of gonadal steroids for sexual
activity. Testicular steroids are essential for mating in most
rodents, but they play a more modulatory role in humans
(Heim and Hursch, 1979).
The stimulatory effects of T in adulthood are referred to as
activational effects, as opposed to the organizational effects
of T during sex differentiation. In adult males T has primarily
slow, genomically mediated permissive effects that prepare
the male to respond to a receptive female. T is usually present
in higher concentrations than necessary to activate sexual
behavior, and small fluctuations in levels usually do not affect
behavior (reviewed in Ågmo and Ellingsen, 2003). The higher
levels are necessary for sperm production in the testes.
1.01.3.1.1 Time Course of Changes in Copulation Following
Castration and T Restoration
Levels of T in plasma decline to unmeasurable levels within
24 h after castration (Krey and McGinnis, 1990), but male
rats may continue to copulate for days or weeks, although
the latency to intromit begins to increase within days. The
number of intromissions required to elicit ejaculation actually
decreases for a few days after castration. Therefore, one function
of T may be to increase the number of intromissions preceding
ejaculation, which would increase the number of sperm in the
ejaculate, facilitate sperm transport, and trigger a progestional
state in the female (Toner et al., 1987). There is disagreement
about the effects of castration on sexual desire and copulation
in men. Kinsey et al. (1948) used anecdotal reports to conclude
that castration may not seriously impair sexual function in
most men. However, more detailed prospective studies of
men who were castrated as ‘treatment’ for sexual offenses found
that half to two-thirds of the men reported a rapid loss of sexual
interest (Heim and Hursch, 1979). The remaining men had
gradual decreases in interest, with 10% still able to copulate
20 years later. Men who were older at the time of castration
were most affected. Imaging studies have reported that visual
sexual stimuli activated brain areas that control autonomic
and neuroendocrine functions and that activation was corre-
lated with plasma levels of T (Stoléru et al., 1999). Hypogona-
dal men showed less activation of some of those areas,
compared to eugonadal men, and hormone replacement
therapy partially restored the stimulus-activated brain activa-
tion (Redoute et al., 2005). Testosterone treatment also led to
a recovery of erectile function in hypogonadal patients, as indi-
cated by nocturnal erections (Foresta et al., 2004).
Five to ten days of exposure to T is necessary to reinstate
copulation in long-term castrated rats (McGinnis et al., 1989;
Putnam et al., 2001), and 5–7 weeks are required for hamsters
(Ballard and Wood, 2007). However, in castrated rats T expo-
sure increased firing in the MPOA in response to female odors
within minutes (Pfaff and Pfaffmann, 1969). Castrated rats
started mounting within 35 min after E administration (Cross
and Roselli, 1999), and castrated mice started mounting within
60 min of T exposure (James and Nyby, 2002). Therefore,
steroids activate certain brain areas within minutes but require
longer-term genomic effects to restore copulation fully.
Furthermore, the amount of T affects the degree of recovery.
Castrated rats that received a high dose of T, which restored
normal levels, had the greatest recovery, compared to those
that received medium and low doses (Harding and Velotta,
2011). After 16 days of T exposure, 100% of those that received
the high dose copulated to ejaculation, whereas 80% and 39%
of those receiving the medium and low doses could ejaculate.
However, there are individual differences in responsiveness to
androgen treatment (Larsson, 1966).
1.01.3.1.2 Role of T Metabolites in Maintaining and Restoring
Copulation
T is the principal hormone produced by the testes and present in
systemic circulation. However, T is primarily a prohormone,
being converted in target organs either to E2 by aromatase or
to DHT by 5a-reductase. There are at least two E receptors,
ERa and ERb. Although both T and DHT bind to the
AR, DHT binds with approximately fivefold greater affinity

(Wilbert et al., 1983). DHT cannot be aromatized to E2, and there-
fore is considered to have only androgenic action. Some target
cells may produce both E2 and DHT and have both ERs and ARs.
The relative importance of estrogenic and androgenic stim-
ulation for male sexual behavior is complex and species-
specific. In castrated male rats E2 is sufficient to reinstate
most aspects of copulation (Cooke et al., 2003). Similarly,
synthetic androgens that can be aromatized to E2, but not 5a
reduced to DHT, restored copulation in castrated rats (Moralí
et al., 1993) or mice (Ogawa et al., 1996). In contrast, copula-
tion was not restored by either DHT or the nonaromatizable
androgen methyltrienolone (R1881) (reviewed in Hull et al.,
2006). The ability of T to restore copulation in castrated rats
was inhibited by aromatase inhibitors (Bonsall et al., 1992;
Roselli et al., 2003; Vagell and McGinnis, 1997) and ER antag-
onists (Beyer et al., 1976). These results support the ‘aromatiza-
tion hypothesis,’ which states that aromatization of T to E is
critical for maintaining or restoring copulation in male rats.
Furthermore, E is also important for sexual behavior in
macaques. Plasma concentrations of E2 and of the 5-HT
precursor tryptophan, but not of T or DHT, were correlated
with male macaques’ ability to copulate to ejaculation,
although androgen concentrations were more important for
sexual motivation (Barrett et al., 2006). The ER antagonist
(RU 58668) inhibited scent marking and 50 kHz vocalizations
in male rats, but did not diminish T’s ability to restore copula-
tion, although an AR antagonist (hydroxyflutamide) did
inhibit restoration of copulation as well as scent marking and
vocalizations (Vagell and McGinnis, 1998). Therefore, ARs
appear to play a role, at least in some experimental conditions,
in restoring copulation in male rats. In support of this hypoth-
esis, E is usually insufficient to fully maintain or restore copu-
lation after castration (Kaplan and McGinnis, 1989; Putnam
et al., 2003, 2005), and antiandrogens also reduce T’s ability
to restore copulation (Vagell and McGinnis, 1998). In another
experiment, male rats were implanted with either E or choles-
terol 35 days after castration and then tested for sexual incen-
tive motivation every fifth day for 3 weeks (Attila et al.,
2010). E implants failed to restore incentive motivation. In
other animals, T was administered alone or together with the
aromatase inhibitor fadrozole. T did restore behaviors, but
fadrozole significantly inhibited those effects. In a third group,
a smaller amount of E was administered with DHT, or E and
DHT were administered alone. Only the combination restored
sexual incentive motivation. Therefore, sexual incentive moti-
vation requires simultaneous stimulation of both ARs and
ERs (see Hull et al., 2006 for a more extensive review).
Sexually inexperienced gonad-inact male hamsters showed
a preference for a receptive female versus a stimulus male, which
was abolished by castration (Ballard and Wood, 2007). Other
castrates received a T implant and were tested for 6 weeks.
Both the loss of sexual preference in castrates and its restoration
by a T implant occurred after changes in mating behavior.
Studies of genetically altered mice that lack ERa or ERb have
provided new insights into the roles of E in male sexual activity.
Gonadally intact males that lacked the ERa (ERa ‘knockout’
(ERaKO) mice) mounted, but had fewer intromissions than
wild-type males, and very few ejaculations (Ogawa et al.,
1997, 1998). These males actually had higher levels of T than
wild-type mice, because of decreased negative feedback by E
Male Sexual Behavior 13
(Wersinger et al., 1997). Castrated ERaKO males with normal
levels of replacement T (Wersinger et al., 1997) or higher
than normal levels of DHT (Ogawa et al., 1998) showed
increased mounting, but few or no ejaculations. However,
treatment with T and a DA agonist restored the ability to ejac-
ulate (Wersinger and Rissman, 2000). Aromatase knockout
(ArKO) mice also showed mating deficits (Bakker et al.,
2002a; Matsumoto et al., 2003). In contrast, bERKO males
mated normally (Ogawa et al., 1999), although the pubertal
onset of ejaculation was delayed (Temple et al., 2003). There-
fore, ERa and aromatase, perhaps together with androgens,
are important for male mouse sexual behavior.
However, aromatization of T to E is not required in
numerous other species, including guinea pigs, hamsters, deer
mice, monkeys, and mice (reviewed in Hull et al., 2006).
Although T acting through ARs is most potent, E does contribute
to the ability of male copulation to ejaculation in macaques,
though androgens are important for sexual motivation (Barrett
et al., 2006). Also, aromatase inhibitors impaired copulation
in castrated T-treated monkeys (Zumpe et al., 1996). In
addition, plasma concentrations of E2 or the 5-HT precursor
tryptophan, but not of T or DHT, were correlated with male
macaques’ ability to copulate to ejaculation, although sexual
motivation was more closely related to androgen
concentrations (Barrett et al., 2006). In castrated hamsters the
aromatizable androgens androstenedione (A) and T restored
all sexual behaviors; E promoted anogenital investigation and
some mounting, but not ejaculation; and DHT was ineffective
(Arteaga-Silva et al., 2005). In that study the combination of E
plus DHT was less effective than A or T, suggesting that
aromatization, perhaps local, may be a factor. Although the
relative importance of E and A varies across species, males
normally have both classes of hormone, which together
promote appetitive and consummatory aspects of mating.
1.01.3.1.3 Effects of Castration and Hormone Replacement
on Ex Copula Penile Responses
1.01.3.1.3.1 Animal Studies
Ex copula reflexes are more sensitive to the presence or absence
of gonadal steroids than is copulation. Reflexes are lost more
rapidly after castration and restored more rapidly by T replace-
ment. Reflexive erections were diminished 24 h after castration
in spinally transected animals (Hart et al., 1983). In spinally
intact males the number of cups was decreased by day 4 after
castration, the earliest time tested, anteroflexions were
decreased by 7 days, and glans erections were decreased by
11 days (Meisel et al., 1984). Reflexive erections were restored
within 6 h of T replacement in spinally transected males,
with maximal increases by 24 h (Hart et al., 1983). In spinally
intact males, increases were observed 24 h after T replacement,
with maximal increases at 48 h (Gray et al., 1980). The longer
intervals required in spinally intact males may be due to the
time required to reduce supraspinal inhibition, so that the
already primed spinal effectors can function. Noncontact erec-
tions are also lost more rapidly after castration (3 days) and
restored more rapidly after T replacement (3 days) (Manzo
et al., 1999). Lower levels of T are effective in restoration
than are present in gonadally intact males (Davidson et al.,
1978), so, as with copulation, plasma T in adult males is higher
than required for penile reflexes.
14 Male Sexual Behavior
Although copulation in male rats is more dependent on E,
reflexes rely more heavily on androgenic stimulation. DHT is
both necessary and sufficient for maintaining and restoring
reflexive (Gray et al., 1980; Meisel et al., 1984) or NO-mediated
(Lugg et al., 1995, see below) erections in male rats. The DHT
regimens that maintained or restored ex copula reflexes were not
effective in activating mounting (Gray et al., 1980; Meisel et al.,
1984). Noncontact erections were also maintained by DHT,
but not by E (Cooke et al., 2003; Manzo et al., 1999). However,
erections during copulation can be maintained by E. E-treated
castrates achieved vaginal insertion on as high a percentage of
intromissions as did control males (O’Hanlon et al., 1981). E
was also as effective as T in maintaining the duration,
frequency, and amplitude of EMG bursts in the BS muscles
during intromissions (Holmes and Sachs, 1992). Sachs (1983)
proposed that E can activate a ‘behavioral cascade,’ organized
in the brain that can activate reflexes during copulation, but
cannot disinhibit those reflexes ex copula.
1.01.3.1.3.2 Studies on Human Males
Levels of T in men with erectile dysfunction are not significantly
lower than those in normally functioning men (Becker et al.,
2001; Rhoden et al., 2002). However, in aging men with
moderate decreases in T levels, exogenous T did improve erec-
tile function and nocturnal penile tumescence (Cavallini et al.,
2004; Schultheiss et al., 2000). Furthermore, T treatment of
hypogonadal men increased the number of erections in diary
reports (O’Carroll et al., 1985; Salmimies et al., 1982). Further-
more, exogenous T in hypogonadal men (which resulted in
normal levels) or in eugonadal men (which produced supra-
physiological levels) did increase sexual arousal in response
to sexual audiotapes (Alexander et al., 1997). T and DHT
were equally effective in stimulating sexual activity in agonadal
men, and administration of either an ER antagonist or an aro-
matase inhibitor did not inhibit sexual function. Thus, andro-
genic stimulation in men appears to facilitate sexual interest
and ability (reviewed in Traish and Guay, 2006).
1.01.3.2 Steroid Action on Steriod Hormone Receptors
ARs and ERs, distributed widely but selectively throughout the
brain, are thought to mediate most hormonal effects on sexual
behavior. These receptors are concentrated in areas that are
important for male sexual behavior, including the MPOA
(see Simerly, 1995). When bound to the appropriate ligand,
classical steroid receptors act as transcription factors to increase
synthesis of new proteins. Such actions are consistent with the
lengthy time course by which steroids promote male sexual
behavior (McGinnis and Kahn, 1997; Yahr and Ulibarri, 1987).
However, hormones may also have more rapid effects,
mediated by membrane receptors that affect ion channel
activity or second-messenger systems (Kelly et al., 2002; Mer-
melstein et al., 1996; Shakil et al., 2002; Xiao and Becker,
1998; reviewed in Frye, 2001; in Moore and Evans, 1999; in
Toran-Allerand, 2004). Either E or T in the perfusion medium
rapidly affected firing rates of neurons in MPOA slices; E- and T-
responsive cells were differentially distributed (Silva and
Boulant, 1986). In tests of reflexive erections, BS muscle
activity was increased 5 min after T injection, although no
erections were produced at that time; furthermore, the
protein synthesis inhibitor anisomycin did not interfere with
the ability of T to activate reflexive erections 24 h after T
injection, suggesting that protein synthesis was not necessary
for T’s effect (Sachs and Leipheimer, 1988).
Several studies have emphasized the role of local aromatiza-
tion of T to E and suggested that E should be considered to be
a neuromodulator or neurotransmitter (Balthazart and Ball,
2006; Balthazart et al., 2006; Cornil et al., 2006a; Voigt et al.,
2007). Aromatase is present in presynaptic boutons of several
species, including birds, rats, monkeys, and humans (Hojo
et al., 2004; Naftolin et al., 1996). In mice, systemic injection
of several aromatase inhibitors inhibited copulation in tests
starting 10 min after the injection; this inhibition was blocked
by coadministration of a large dose of E2 (Taziaux et al., 2007).
The inhibition was not seen in ArKO mice whose behavior was
maintained by daily injections of E2; furthermore, ArKO mice
responded within 15 min to a single dose of E2. Similar inhib-
itory effects of aromatase inhibitors were observed in male
quail (Cornil et al., 2006b). Therefore, locally produced E
may facilitate mating. However, questions remain concerning
the behavioral significance of local regulation of aromatase
(see Section 1.01.5.2.1).
1.01.3.3 The Role of Progesterone in Male Sexual Behavior
Progesterone (P), secreted from the testes and adrenals, is
a precursor to T. However, there are apparently contradictory
reports as to whether P facilitates or inhibits male sexual
behavior. P partially restored intromission, but not ejaculation,
in castrated rats (Witt et al., 1994, 1995). The P receptor antag-
onist RU486 decreased the number of erections elicited by para-
doxical sleep deprivation (Andersen and Tufik, 2005). Sexually
naïve P receptor knockout (PRKO) mice had fewer mounts than
did wild-type males, but PRKO mice mated normally on subse-
quent tests (Phelps et al., 1998). Sexually experienced PRKO
males showed more dramatic declines in copulation after castra-
tion than did wild-type males. In contrast to these findings,
which support a facilitative role of P, a later study of PRKO
mice found that they had shorter mount latencies and increased
likelihood of achieving ejaculation, compared to wild-type mice
(Schneider et al., 2005). In the same report, the P receptor antag-
onist RU486 also decreased intromission latencies and
increased mounts and intromissions. The PRKO mice in that
study had more ARs in the medial preoptic nucleus (MPN)
and BST, which may have contributed to their better perfor-
mance. The reason for the contradictory results obtained with
PRKO mice is not clear. However, in agreement with an inhib-
itory role of P, chronic administration of synthetic progestins
(medroxyprogesterone acetate and cyproterone acetate) in
men has been used to inhibit deviant sexual behavior (reviewed
in Andersen and Tufik, 2006). These compounds block ARs,
which may, at least partially, account for their inhibitory effects.
1.01.4 Effects of Systemically
and Intraventricularly Injected Drugs
1.01.4.1 Dopamine
The mesolimbic, nigrostriatal, and hypothalamic dopami-
nergic systems are all involved in the integration of male rodent

sexual response (Hull et al., 1999). DA has long been known to
facilitate male sexual function. L-DOPA, the precursor of DA,
administered to Parkinsonian patients, increases libido and
sexual potency (Barbeau, 1969; Bowers et al., 1971), and
some DA replacement therapies produce hypersexuality in
these patients (Politis et al., 2013). Besides, the nonspecific
DA agonist apomorphine has been used to treat sexual dysfunc-
tion (Dula et al., 2000; Giuliano and Allard, 2002; Lal et al.,
1987).
In rats, systemically administered DA agonists facilitate
male sexual behavior (reviewed in Bitran and Hull, 1987;
Giuliano and Allard, 2001; Hull et al., 2006; Melis and
Argiolas, 1995), induce sexually sluggish males to copulate
(Tagliamonte et al., 1974), elicit copulation in sexually
exhausted males (Mas et al., 1995b; Rodríguez-Manzo,
1999b; Guadarrama-Bazante et al., 2014), and partially restore
copulation in castrates (Malmnäs, 1976; Scaletta and Hull,
1990). In mice lacking the ERa, which usually show little sexual
behavior, apomorphine induces normal copulation (Wersinger
and Rissman, 2000). It also elicits penile erections and genital
grooming in mice (Rampin et al., 2003).
Systemically administered DA antagonists impair sexual
behavior in both sexually experienced (Ahlenius and Larsson,
1990; Pfaus and Phillips, 1989) and naïve rats (Ågmo and
Picker, 1990). The negative effects of DA antagonists range
from increased IL and EL to failure to copulate (reviewed in
Bitran and Hull, 1987; Giuliano and Allard, 2001; Melis and
Argiolas, 1995). Haloperidol, a nonspecific DA antagonist,
inhibits measures of sexual motivation as well as copulation
itself both after acute (Lopez and Ettenberg, 2000, 2001,
2002b) and chronic treatment (Zhang et al., 2011). In human
males, DA antagonists reduce sexual desire, arousal, and
orgasm (Baldwin and Mayers, 2003).
Differences in male sexual motivation and copulatory
patterns of two outbred rat lines (Roman high- and low-
avoidance rats) have been related to differences in the central
dopaminergic tone, evidenced by a differential sensitivity to
apomorphine and haloperidol between strains (Sanna et al.,
2014). In line with these data, both apomorphine and the
D1-like receptor agonist, SKF38393, facilitated sexual behavior
display in male rats with a diminished motivational tone due
to sexual exhaustion, but not in sexually experienced animals
with an intact sexual motivation (Guadarrama-Bazante et al.,
2014).
DA receptors are classified into two families; the D1-like
family includes receptors positively coupled to adenylyl cyclase
and comprises the D1 and D5 receptor subtypes, while the D2-
like family consists of D2, D3, and D4 receptor subtypes, which
are negatively coupled to adenylyl cyclase (Missale et al., 1998).
At the supraspinal level DA has a facilitative effect on ejacula-
tion, mediated especially in the MPOA and PVN by D2-like
receptors (reviewed in Hull et al., 2004). Systemic activation
of D2-like receptors with either D3 (7-OH-DPAT) or D2/D3
receptor agonists (SND 919 and BHT 920) facilitated ejacula-
tion by decreasing both IF and EL in sexually experienced
rats, but not in sexually inactive ones (Ferrari and Giuliani,
1995, 1996a; Giuliani and Ferrari, 1996). Selective D4 receptor
agonists (PD-168,077 and ABT-724) improved copulatory
behavior of sexually potent rats by decreasing MF, EL, and
PEI and increasing ejaculation frequency (EF), while the D4
Male Sexual Behavior 15
receptor antagonist L-745870 impaired copulatory behavior
(Sanna et al., 2015). Polymorphism of the dopamine trans-
porter gene (DAT1) was associated with differences in human
ejaculatory threshold that might be related to premature ejacu-
lation (Santtila et al., 2010).
Male rats can be conditioned to have a same-sex partner
preference if they undergo cohabitation with other males under
the effects of D2 receptor activation with quinpirole (Triana-Del
Rio et al., 2011), while D1-like receptors mediate ejaculation-
induced conditioned place preference (Dominguez-Salazar
et al., 2014), further evidencing the role of DA in the rewarding
effects of copulation. Sexual arousal has also been shown to be
conditionable in men (Brom et al., 2014), and there is evidence
for DA mediating the processing of subconsciously perceived
sexual stimuli (Oei et al., 2012).
In anesthetized rats, icv and intra-MPOA injection of a D3
agonist induced BS rhythmic contractions and ejaculation,
effects blocked by a D2/D3 (raclopride) and a preferential D3
antagonist (nafadotride) (Clement et al., 2007a; Kitrey et al.,
2007). Thus, supraspinal command of ejaculation appears to
be mediated by D2-like receptors, probably of the D3 subtype,
in the MPOA. D1-like receptor agonists increased sexual moti-
vation in rats (Beck et al., 2002) and facilitated copulation in
DA-deficient mice (Szczypka et al., 1998). Besides, D1-like
receptors mediate the apomorphine-induced reversal of
sexual satiation (Guadarrama-Bazante et al., 2014) and play
a role in the experience-induced enhancement of male rat
sexual behavior (McHenry et al., 2012). D1-like receptor
facilitation of male sexual behavior appears to be conserved
across phyla, since it has been detected in lizards (Woolley
et al., 2001), geckos (Woolley et al., 2004), Japanese quail
(Balthazart et al., 1997), and European starlings (Schroeder
and Riters, 2006).
The effects of DA agonists are dose-dependent, with low
doses facilitating and high doses inhibiting copulation, the
latter effect possibly due to the induction of stereotypic
behavior (Foreman and Hall, 1987; Szczypka et al., 1998).
Contradictory effects of D1- and D2-like receptor agonists on
ex copula genital reflexes have been reported. On one hand,
a D2 selective agonist was reported to elicit erections, while
a D1 receptor agonist inhibited them (Zarrindast et al.,
1992). However, the D2/D3 receptor agonist quinelorane
decreased the number of reflexive erections in restrained rats
(Bitran et al., 1989b), but promoted penile erection in rhesus
monkeys in the presence of sexually receptive females, which
they could see, hear, and smell, but could not contact (Pomer-
antz, 1991). Other D2/D3 selective receptor agonists elicited
erections in rats in a neutral arena (Ferrari et al., 2002).
However, a D2/D3 antagonist (eticlopride) actually increased
erections elicited by a presumed-selective D2 agonist or cocaine
in rats (Ferrari and Giuliani, 1996b). Cocaine would have
increased extracellular DA, which would have stimulated
both D1 and D2 receptors. It is possible that the presumed-
selective D2 agonist actually stimulated some D1 receptors, as
did cocaine, and that inhibition of D2 receptors disinhibited
the D1-like effect. With the use of dopaminergic agonists and
antagonists it was established that the proerectile effects
produced by D2-like receptor agonists, administered either
systemically or intra-PVN, are mediated by the D2 receptor
subtype, with D4 receptors playing only a modest role
16 Male Sexual Behavior
(Depoortere et al., 2009; Sanna et al., 2011). DA is released in
the MPOA (Hull et al., 1995; reviewed in Hull, 2011) and the
NAc (Fiorino et al., 1997) of male rats in response to an estrous
female and during copulation. The DA release patterns during
ejaculation in these two brain regions are virtually identical
(Pfaus, 2009).
1.01.4.2 Norepinephrine
NE has both facilitative and inhibitory effects on male sexual
behavior. Inhibition of NE synthesis impaired copulation,
increasing ML, IL, EL, and PEI (McIntosh and Barfield,
1984c). However, lesions of the NE system have rendered
inconsistent results. Electrolytic lesion of the locus coeruleus
(LC), which contains the majority of NE cell bodies in the brain
(Kuhar et al., 1999), inhibited copulation (McIntosh and
Barfield, 1984c), but similar lesions of the dorsal NE bundle,
which connects the LC with the cerebral cortex and hippo-
campus (Kuhar et al., 1999), decreased the PEI and increased
the ejaculations in a 1-h test (Clark, 1975, 1980). Central NE
depletion by the neurotoxin DSP-4 increased the PEI (Hansen
et al., 1982), but in another study it had no effect on copulation
(Fernández-Guasti and Rodríguez-Manzo, 1997), neither did
the lesion of the dorsal NE bundle with the neurotoxin 6-
OHDA (Clark, 1980). DSP-4-induced lesion did not interfere
with the Coolidge effect, although it reduced the number of
ejaculations prior to satiation and in the ensuing Coolidge
effect copulatory period (Rodríguez-Manzo, 1999a).
NE exerts its effects by stimulating a- and b-adrenoceptors.
a-adrenoceptors have been shown to modulate sexual arousal
(Viitamaa et al., 2006), since a2-adrenoceptor antagonists
such as yohimbine, idazoxan, and imiloxan, which enhance
NE transmission by blocking autoreceptors, increased
mounting rates in rats with genital anaesthetization,
augmented copulatory behavior in sexually sluggish and cas-
trated males (Smith et al., 1987; Tallentire et al., 1996),
increased the percentage of sexually naïve rats that copulated
to ejaculation (Benelli et al., 1993; Tallentire et al., 1996),
reversed the sexual inhibition that follows male rat sexual
exhaustion in intact rats (Rodriguez-Manzo, 1999b) as well
as exhaustion of the ejaculatory reflex in spinal cord-
transected males (Carro-Juárez and Rodríguez-Manzo, 2003).
Yohimbine produced biphasic effects after its systemic and
icv injection; it dose-dependently decreased IL, EL, and PEI in
sexually experienced males (Clark et al., 1984; Sala et al.,
1990) and stimulated penile erection through autonomic acti-
vation (Allard and Giuliano, 2001). Conversely, a2-adrenocep-
tor agonists (clonidine, guanabenz) dose-dependently
suppressed ejaculation and erection when systemically (Clark
and Smith, 1990) and icv administered (Clark, 1991), or
increased ML, IL, and PEI in sexually vigorous male rats and
decreased the number of inexperienced males achieving ejacu-
lation (Benelli et al., 1993). Blockade of a1-adrenoceptors with
prazosin increased IL, EL, and PEI, while the a1-adrenoceptor
agonist methoxamine had the opposite effect (Clark et al.,
1985; reviewed in Meisel and Sachs, 1994).
The influence of b-adrenoceptors on copulation is not clear.
b-Adrenoceptor antagonists, used for the treatment of cardio-
vascular diseases (Borchard, 1998), cause sexual dysfunction
in men (Düsing, 2005). In rats, sc and icv injection of the
b1/b2-blockers propanolol and pindolol profoundly inhibited
sexual behavior, almost abolishing ejaculation (Smith et al.,
1990, 1996). In sexually vigorous male rats, the b2-adrenocep-
tor agonist clenbuterol reduced mount frequency (MF) and IF
and increased the PEI, but in sexually sluggish animals it
improved copulatory behavior by increasing the percentage
of males achieving ejaculation and reducing ML, IL, EL, and
PEI (Benelli et al., 1990). Chronic oral administration of prop-
anolol to male rabbits impaired sexual behavior, affecting
performance more than arousal aspects (Grotthus et al.,
2007). Besides, an increase in NE transmission produced by
acute or chronic (14 days) treatment with desipramine, an anti-
depressant that inhibits the NE transporter, reduced the ejacu-
latory threshold (IF and EL) of sexually vigorous male rats
(Hueletl-Soto et al., 2010), thus supporting a facilitative role
of NE on copulation.
The NE system regulates male sexual functions through
ascending pathways to the brain and descending pathways to
the spinal cord (Giuliano and Rampin, 2000), and adrenocep-
tors are found in the brain and spinal cord of animals and
humans (Roudet et al., 1994; Smith et al., 1995; Wada et al.,
1996). The activity of spinal preganglionic neurons is modu-
lated by NE, and postganglionic sympathetic nerve terminals
release NE in the penis, promoting penile detumescence
(Giuliano and Rampin, 2004). In contrast to the effects on
copulation, both agonists and antagonists at a2-adrenoceptors
inhibit reflexive erections as well as seminal emission in rats.
However, the effects of the a2-receptor antagonist yohimbine
appear to be dose-dependent, with low doses facilitating and
higher doses inhibiting the erectile response (reviewed in
Meisel and Sachs, 1994). A similar effect is seen on seminal
emission in dogs (Yonezawa et al., 1991). Activation of a1-
adrenoceptors inhibits reflexive erection in dogs, but stimulates
seminal emission in rats. At the spinal level, it has been shown
that enhancement of noradrenergic transmission by blockade
of a2 autoreceptors with yohimbine and direct a1-adrenoceptor
stimulation with methoxamine both facilitate male rat ejacula-
tory response (Carro-Juárez and Rodríguez-Manzo, 2006).
There is also evidence that the b-adrenergic antagonist
propranolol has a negative impact on rat reflexive erectile and
ejaculatory reflexes (Smith et al., 1995). Together, these data
suggest that increased NE activity, either by blockade of a2
autoreceptors or by stimulation of a1-adrenoceptors, increases
sexual arousal. b-adrenoceptors facilitate copulation, but NE
inhibits reflexive erections, probably at the penis.
1.01.4.3 Serotonin
5-HT is generally inhibitory to male sexual behavior (reviewed
in Bitran and Hull, 1987; Hull et al., 2004). A common side
effect of SSRI antidepressants, which increase extracellular
5-HT, is to impair male sexual function in humans and rats.
SSRI’s sexual side effects seem to be best explained by inhibi-
tory actions of 5-HT on dopaminergic activity (Bijlsma et al.,
2014).
Contrary to the general inhibitory role of 5-HT in copula-
tion, stimulation of the 5-HT1A receptor subtype markedly
facilitates male rat ejaculation (Ahlenius et al., 1981). Fourteen
5-HT receptor subtypes, belonging to one of seven receptor
families, have been identified (Hoyer et al., 2002); however,

only a few of them have been related to male sexual function.
The inhibitory actions of 5-HT appear to be mediated by 5-
HT1B receptors in rats and mice (Ahlenius and Larsson, 1998;
Hillegaart and Ahlenius, 1998; Rodríguez-Manzo et al.,
2002b) and by the 5-HT2 receptor in rats (Foreman et al.,
1989; Klint and Larsson, 1995), while the 5-HT1A receptor
mediates the facilitative actions of this neurotransmitter in
rats (Ahlenius et al., 1981). Thus, systemic and intrabrain injec-
tion of 5-HT1B agonists inhibit male rat sexual behavior
(Fernández-Guasti et al., 1989, 1992), and coadministration
of subeffective doses of 5-HT1B agonists and the 5-HT precursor
5-hydroxytrytophan (5-HTP) synergize to inhibit copulation
(Fernández-Guasti and Rodríguez-Manzo, 1992). 5-HT1B
receptor antagonists completely block the inhibitory actions
of 5-HTP on copulation, while antagonists to other subtypes
do not (Ahlenius and Larsson, 1998). In 5-HT1B receptor
knockout (KO1B) mice, males become interested earlier in
sexual behavior and are less sensitive to the inhibitory actions
of 5-HTP than the corresponding wild-type strain. However,
KO1B mice require more stimulation to achieve ejaculation
than wild-type males. Pharmacological manipulation in these
animals revealed that in mice, in contrast to rats, both the
5-HT1B and the 5-HT1A receptor subtypes contribute to the
inhibitory actions of 5-HT (Rodríguez-Manzo et al., 2002b).
The inhibitory role ascribed to the 5-HT2 receptor subtype in
male rat sexual behavior is mainly derived from results
showing that systemic, intraraphe, and intralateral hypothal-
amus (LH) administration of 5-HT2 receptor agonists dose-
dependently decreased its expression (Foreman et al., 1989;
Riolo et al., 1999; Watson and Gorzalka, 1991, 1992), while
a 5-HT2 antagonist facilitated copulation (Gonzalez et al.,
1994). Stimulation of 5-HT2C receptors increased erections
and inhibited ejaculation in monkeys (Pomerantz et al.,
1993) and facilitated erection in anesthetized (Steers and
DeGroat, 1989) and conscious rats (Millan et al., 1997). Facil-
itative effects of 5-HT2C receptor activation on ejaculation have
also been reported in rats (Yonezawa et al., 2008), and activa-
tion of spinal 5-HT2C receptors elicited burst pattern responses
and evoked emission associated with ejaculatory-like responses
(Stafford et al., 2006b).
Brain sites where microinjection of 5-HT has been reported to
impair male rat sexual behavior are the MPOA (Dominguez and
Hull, 2010) and the NAc (Fernández-Guasti et al., 1992).
However, accumulation of 5-HT in the MPOA induced by
SSRI local injection did not affect sexual behavior, although
SSRI injection into the anterior LH did inhibit copulation
(Lorrain et al., 1997; see Section 1.01.5.2.5). Besides, a tonic
inhibitory influence on sexual reflexes exerted by brain stem
5-HT descending pathways to the spinal cord has been described
(Marson and McKenna, 1992, 1994a; Marson et al., 1992), while
a facilitative action of 5-HT on the expulsion phase of ejacula-
tion, through the activation of peripheral 5-HT2 receptors, was
recently reported in male rats (Ishigami et al., 2013).
The facilitative effects on ejaculation resulting from stimula-
tion of the 5-HT1A receptor subtype were characterized with the
5-HT1A receptor agonist 8-OH-DPAT, which dramatically
decreased the IF and EL of sexually competent male rats, to
the point that some rats ejaculated on their first intromission
(Ahlenius et al., 1981, 1989; Coolen et al., 1997a; Schnur
et al., 1989). Similar facilitation has been reported in sexually
Male Sexual Behavior 17
satiated rats (Rodríguez-Manzo and Fernández-Guasti 1994;
Fernández-Guasti and Rodriguez-Manzo, 1997). Other 5-
HT1A agonists such as flesinoxan (Haensel and Slob, 1997),
buspirone (Mathes et al., 1990), or ipsapirone (Fernández-
Guasti et al., 1989) induced a behavioral profile similar to
that of 8-OH-DPAT, although animals did not ejaculate on
their first intromission. That 5-HT1A receptor stimulation yields
opposite effects to those produced by 5-HT itself, together with
the fact that somatodendritic autoreceptors in 5-HT neurons
belong to the 5-HT1A receptor subtype, led to the postulation
that 8-OH-DPAT acted as an agonist at somatodendritic recep-
tors to reduce 5-HT release (Ahlenius et al., 1981). However,
lesion of raphe nuclei did not alter the 8-OH-DPAT facilitative
actions on copulation, suggesting that they were exerted at
postsynaptic sites (Fernández-Guasti and Escalante, 1991). In
addition, facilitative effects of 8-OH-DPAT were obtained after
its infusion into the MPOA and the NAc, but not into the dorsal
raphe, where somatodendritic receptors are found (Fernández-
Guasti et al., 1992; Matuszewich et al., 1999). The anterior LH
(Riolo et al., 1999) and the MeApd (de Castilhos et al., 2006)
are two additional brain areas where injection of 8-OH-DPAT
facilitates ejaculation.
8-OH-DPAT’s facilitative effects on copulation require
normal T levels and sexual experience (Rowland and
Houtsmuller, 1998). Both NE and DA systems are involved
in 8-OH-DPAT’s facilitative effects, since neurotoxic lesions of
the central NE system blocked or attenuated them in sexually
exhausted and nonexhausted rats, respectively (Fernández-
Guasti and Rodríguez-Manzo, 1997; Rodríguez-Manzo and
Fernández-Guasti, 1995b). Besides, the D2 antagonist raclopr-
ide, but not the 5-HT1A antagonist p-MPPI, decreased 8-OH-
DPAT’s facilitative effects in the MPOA (Matuszewich et al.,
1999). Intrathecal administration of 8-OH-DPAT at the level
of the lumbosacral spinal cord also reduced the IF and EL
(Lee et al., 1990).
Chronic SSRI treatment is frequently associated with
delayed ejaculation. In the case of fluoxetine, this effect seems
to be exerted directly at the spinal level (Hueletl-Soto et al.,
2012). Acutely, SSRIs inhibit 5-HT reuptake, elevating extracel-
lular 5-HT levels (Malagie et al., 2000), and usually do not
affect male sexual behavior (de Jong et al., 2005a; Hueletl-
Soto et al., 2012), but when infused into the anterior LH,
they inhibit copulation (Lorrain et al., 1997). Interestingly,
reverse dialysis of 5-HT into this brain region decreases DA
release in the NAc (Lorrain et al., 1999). After chronic admin-
istration, SSRIs cause desensitization of 5-HT1A receptors (Le
Poul et al., 1995).
Apparently, this desensitization plays a role, not only in
SSRIs’ antidepressant properties (Artigas et al., 1996), but
also in the chronic treatment–induced delayed ejaculation
(de Jong et al., 2005b). Differences among individual SSRIs
in their ability to desensitize 5-HT1A receptors and to delay ejac-
ulation appear to exist, with paroxetine and fluoxetine being
more potent than citalopram and fluvoxamine in both actions
(de Jong et al., 2007).
The absence of serotonin transporter (SERT) produced
lower ejaculation frequencies and prolonged EL in SERT
knockout male rats (Chan et al., 2011). Both T and E2 have
been found to increase SERT and 5-HT2A receptor densities in
male rats’ brains (Barrett et al., 2006). Stimulation of spinal
18 Male Sexual Behavior
5-HT1A receptors inhibits rat penile erection (Pomerantz et al.,
1993; Rehman et al., 1999), but facilitates ejaculation in spinal
cord-transected rats (Carro-Juárez et al., 2003). The concept of
5-HT exerting a general inhibitory influence on sexual
responses is changing. It has recently been suggested that 5-
HT is not only involved in the inhibitory control of male sexual
behavior, but also in disinhibiting processes to induce sexual
behavior (Snoeren et al., 2014). Further, it was proposed that
5-HT released in high concentrations exerts an inhibitory influ-
ence upon male sexual responses, whereas when released at
low concentrations this neurotransmitter contributes to the
disinhibition of sexual responses (Rubio-Casillas et al., 2015).
1.01.4.4 Acetylcholine
There is little information on selective effects of acetylcholine
(ACh) on copulation in males. Early studies reported suppres-
sion of male sexual behavior by high doses of nicotine and of
physostigmine, an acetylcholinesterase inhibitor, as well as by
antagonists such as atropine and scopolamine. However, a rela-
tively low dose of nicotine increased EF and reduced IF, EL, and
PEI (for review, see Bitran and Hull, 1987), and the muscarinic
agonist oxotremorine reduced IF and EL (Ahlenius and
Larsson, 1985). Later, physostigmine-induced increases in
ACh level and stimulation of muscarinic receptors with pilocar-
pine increased EF and promoted erections (Maeda et al., 1990;
Zarrindast et al., 1994). In hamsters, MPOA cholinergic trans-
mission seems to play a role in the development of sexual
exhaustion (Floody, 2013) (see Sections 1.01.5.2.1 and
1.01.5.3.5).
1.01.4.5 Gamma Aminobutyric Acid
GABA is the main inhibitory neurotransmitter in the mamma-
lian brain (Paul, 1995). Systemic administration of GABAergic
drugs inhibited male rat sexual behavior (Ågmo and Paredes,
1985; Paredes et al., 1997). There are reports of inhibitory
effects of GABAergic drugs in the MPOA, PVN, and spinal
cord (see Sections 1.01.5.2.1, 1.01.5.2.4, and 1.01.5.3.5). In
male gerbils, a pathway originating in the ventral BST (vBST)
and MPN and projecting to the retrorubral field (RRF) that is
activated during mating was reported to be mostly GABAergic
(Simmons et al., 2011). An equivalent pathway also important
for mating was described in male rats (Finn and Yahr, 2005). In
addition, cerebrospinal fluid concentrations of GABA increased
dramatically during the PEI (Qureshi and Södersten, 1986).
1.01.4.6 Glutamate
Excitatory amino acids appear to facilitate copulation and
penile erection. Glutamate is the major excitatory neurotrans-
mitter in the CNS. Intraperitoneal injection of low doses of
kainic acid, an agonist of AMPA/kainate receptors, enhanced
copulatory behavior in sexually sluggish male rats, but did
not affect good copulators; however it impaired sexual
behavior at higher doses (Drago and Busa, 1997). Systemic
injection of the NMDA antagonist MK-801 impaired sexual
behavior in sexually experienced and sexually naïve male
rats (Powell et al., 2003), an effect also obtained in the
MPOA (Dominguez et al., 2007). Repeated exposure of
sexually naïve animals to inaccessible estrous females
improves copulatory behavior, and this improvement was
blocked by MK-801 (Powell et al., 2003). There is consider-
able evidence for glutamatergic facilitation of both copulation
and genital reflexes following microinjections into the MPOA,
PVN, and spinal cord (see Sections 1.01.5.2.1, 1.01.5.2.4, and
1.01.5.3.5). Thus, glutamate appears to be a major facilitator
of copulation at both brain and spinal levels. However, the
sexual condition of male rats and/or the dose range of gluta-
matergic drugs administered appear to influence the role
played by glutamatergic transmission on copulatory behavior.
Thus, systemic injection of low doses of the NMDA antagonist
ketamine, the AMPA receptor antagonist CNQX, and the
mGluR5 receptor antagonist MPEP all facilitated sexual
behavior expression of sexually exhausted male rats
(Rodriguez-Manzo, 2015), characterized by being sexually
inhibited. Besides, systemic administration of high doses of
MPEP and of the mGluR7 receptor antagonist AMN082 were
reported to inhibit sex seeking behavior as well as copulatory
performance (Li et al., 2013).
Glutamate is released in the MPOA during copulation
(Dominguez et al., 2006a), and increases in local extracellular
glutamate produced by glutamate reuptake inhibitors facili-
tated copulation (Dominguez and Hull, 2010). On the other
hand, activation of glutamatergic transmission in the mesocor-
ticolimbic tract has also been associated to male sexual activity
(Pitchers et al., 2012).
1.01.4.7 Nitric Oxide
NO is a soluble gas that acts both as a second messenger and
as a neurotransmitter that has been implicated in male sexual
functions that include penile erection (Andersson, 2001) and
brain control of sexual behavior (Bialy et al., 1996). NO is
produced by the action of NOS, which converts L-arginine
into citrulline. Systemically administered NOS inhibitors
impair copulation, decreasing the proportion of sexually
naïve (Benelli et al., 1995) and experienced (Bialy et al.,
1996; Hull et al., 1994) male rats that ejaculate, while aug-
menting MF and decreasing IF. NOS inhibitors also decrease
the number of noncontact erections (Melis et al., 1998), as
well as reflexive erections, and also increase seminal emis-
sion (Hull et al., 1994). These effects are consistent with
a facilitative role of NO in parasympathetic function and
an inhibitory one in sympathetic activity. A NOS inhibitor
did not affect sexual motivation, evaluated in a female choice
maze (Hull et al., 1994), although another report found
a diminution evaluated by precoital activity and the
percentage of rats mounting (Ratnasooriya et al., 2000).
nNOS KO mice have normal penile function, due to
a compensatory increase in eNOS (Burnett et al., 1996),
and achieve ejaculation with fewer mounts and intromis-
sions (Kriegsfeld et al., 1999). Ip injection of L-arginine
increased the percentage of sexually naïve rats that copulated
and improved sexual performance in experienced males,
whereas icv injection of the NOS inhibitor L-NAME impaired
copulatory parameters only in sexually naïve rats (Benelli
et al., 1995), and increases in NO production in the MPOA
were detected in response to the first sexual experience of
male rats (Nutsch et al., 2014). NO may control both

copulation and gential reflexes via actions in the MPOA,
PVN, and spinal cord (see Sections 1.01.5.2.1, 1.01.5.2.4,
and 1.01.5.3.5). In rodents the expression of NOS in regions
implicated in the control of male sexual behavior is under
the control of gonadal hormones (Panzica et al., 2006).
NO also plays a crucial role in the initiation and mainte-
nance of increased intracavernous pressure and penile erec-
tion by acting on smooth muscle cells (Toda et al., 2005).
1.01.4.8 Endocannabinoids
Endocannabinoids are retrograde messengers acting as neuro-
modulators in the CNS. They are released from postsynaptic
cell bodies, travel backward across the synaptic cleft, and
bind to their receptors mainly located presynaptically at nerve
endings (Wilson and Nicoll, 2002). The main endocannabi-
noids are small molecules derived from arachidonic acid: anan-
damide and 2-arachidonoylglycerol. Its neuronal receptor, the
cannabinoid CB1 receptor, is located almost exclusively at
axon terminals, where its stimulation inhibits neurotransmitter
release (Freund et al., 2003). A general inhibitory role on male
sexual behavior was ascribed to the endocannabinoid system
based on the observation that antagonism of CB1 receptors
accelerated ejaculation in rats (Gorzalka et al., 2008), while
both plant-derived (delta-9-THC) and synthetic (HU 210)
cannabinoid CB1 agonists impaired copulation in mice and
rats (Shrenker and Barke, 1985; Murphy et al., 1994; Ferrari
et al., 2000). Exogenous administration of anandamide, the
endogenous ligand for CB1 receptors, was also found to impair
male sexual behavior similarly to high doses of exogenous
agonists (Martínez-González et al., 2004); however, inhibition
of anandamide metabolism did not importantly affect male
sexual behavior (Gorzalka et al., 2008). Recently, it was shown
that low doses of anandamide actually facilitate male sexual
behavior of sexually experienced and sexually exhausted rats
(Canseco-Alba and Rodriguez-Manzo, 2014). Facilitative
actions were also obtained after low anandamide doses in sexu-
ally sluggish (Rodriguez-Manzo and Canseco-Alba, 2015a),
noncopulating (Canseco-Alba and Rodriguez-Manzo, 2013),
and sexually naïve animals (Rodriguez-Manzo and Canseco-
Alba, 2015b), leading to the conclusion that anandamide
exerts dose-based biphasic actions on male rat copulatory
behavior (Rodriguez-Manzo and Canseco-Alba, 2015b).
CB1 receptors are found at glutamatergic terminals in
hypothalamic nuclei related to reproductive function (Melis
et al., 2006), but also at glutamatergic and GABAergic termi-
nals of the mesolimbic system, where endocannabinoids
modulate the activity of this brain reward circuit (Lupica
et al., 2004), involved in the motivational aspects of copula-
tion (Kelley and Berridge, 2002). CB1 receptor antagonism
in the PVN of male rats has also been reported to induce
penile erection (Melis et al., 2004a; Succu et al., 2006b). In
this case, it has been hypothesized that CB1 receptor antago-
nists act at GABAergic and glutamatergic neurons to increase
oxytocinergic transmission which in turn would promote
penile erection and facilitate ejaculation (Castelli et al.,
2007; Gorzalka et al., 2008). There is also evidence that the
endocannabinoid system interacts with gonadal androgens
by acting on the hypothalamus and the anterior pituitary
(Gorzalka and Dang, 2012). Together, these data show that
Male Sexual Behavior 19
the endocannabinoid system exerts a complex influence on
copulatory responses.
1.01.4.9 Endogenous Opioids
Exogenous opiates such as morphine and heroin are known to
have negative effects on the sexuality of male addicts, reducing
sexual interest, impairing genital responses, and blocking ejac-
ulation and orgasm (Pfaus and Gorzalka, 1987). Endogenous
opioids belong to one of three major classes: endorphins,
enkephalins, or dynorphins (Mains and Eipper, 1999). These
peptides are generated by enzymatic processing from three
precursor molecules, proopiomelanocortin, proenkephalin,
and prodynorphin (Akil et al., 1984). An inhibitory role of
endogenous opioid peptides in the control of male sexual
behavior is commonly accepted, based on animal studies
showing that acute and chronically administered morphine,
heroin, and methadone inhibit copulatory behavior in males
of various species (Pfaus and Gorzalka, 1987). Similar results
have been obtained after injection of opioid peptides such as
b-endorphin, morphicepin, and a Met-enkephalin analog
into the MPOA (Hughes et al., 1987; Matuszewich and Dornan,
1992; Pellegrini-Quarantotti et al., 1978). When infused into
the amygdala, they retard sexual performance (McGregor and
Herbert, 1992b). Moreover, it has been reported that sexually
inactive male rats have a constitutively increased basal concen-
tration of the endogenous opioid octapeptide Met-Arg(6)-
Gly(7)-Leu(8) in the hypothalamus (Rodríguez-Manzo et al.,
2002a), as well as increased expression of proenkephalin (the
precursor of the endogenous octapeptide) and prodynorphin
mRNAs in the PVN (Arletti et al., 1997), compared with sexu-
ally active animals.
Endogenous opioids exert their effects by acting on m, d, and
k receptor subtypes. The inhibitory actions of these peptides on
copulation appear to be mainly mediated by m and d receptors,
although involvement of k opioid receptors has also been
found. Systemic injection of the k opioid receptor agonist
U-50,488H inhibited several sexual parameters, which were
differentially prevented by intra-VTA, intra-NAc, or intra-
MPOA injections of the k receptor antagonist norbinaltorphi-
mine (Leyton and Stewart, 1992). The inhibitory role of
endogenous opioids in copulation is also suggested by studies
showing that opioid receptor antagonists (e.g., naloxone and
naltrexone) induce sexual behavior expression in sexually inac-
tive rats (Gessa et al., 1979), facilitate the display of mounts
and intromissions in sexually naïve rats (Pfaus and Wilkins,
1995), reverse the sexual inhibition of sexually satiated rats
(Rodríguez-Manzo and Fernández-Guasti, 1995a), decrease
the ejaculatory threshold in sexually competent intact rats,
and increase the percentage of ejaculating animals (Myers
and Baum, 1979). Furthermore, the content of opioid octapep-
tide was higher in the hypothalamus of sexually inactive males,
compared to sexually active males (Rodríguez-Manzo et al.,
2002a).
However, there are inconsistent effects of opioid agonists
and antagonists on rodent male sexual behavior. Although
a large body of evidence points to the inhibitory nature of
opioid agonist actions, there are also data suggesting facilitative
effects of these peptides. Thus, increasing enkephalin levels by
icv administration of an enkephalinase inhibitor decreased IF
20 Male Sexual Behavior
and EL (Ågmo et al., 1994), and intra-VTA injection of
morphine and dynorphin facilitated male sexual behavior
and increased DA transmission in the NAc (Mitchell and
Stewart, 1990). Opioid facilitation may result from disinhibi-
tion of mesolimbic DA neurons by acting at m receptors on
VTA GABAergic neurons that exert a tonic inhibitory influence
on DA transmission (Balfour et al., 2004). On the other hand,
opioid antagonists also have had inhibitory effects, increasing
the duration of the PEI (McConnell et al., 1981; Sachs et al.,
1981). Thus, a complex picture of potentially excitatory and
inhibitory effects of endogenous opioids on male sexual
behavior emerges. This complexity appears to be related to
several factors, including dose-based, biphasic effects of
agonists and antagonists, the brain site where they were admin-
istered, the time of the day when rats were tested, and the
sexual activity level of the animals. Thus, the inhibitory effect
of b-endorphin is dose dependent (Argiolas, 1999) and is
believed to occur mainly through actions at the MPOA and
the amygdala. In the former it disrupts copulatory behavior,
while in the latter it disrupts precopulatory exploration
(Bancroft, 1999). Infusion of endomorphin-1 into the MPOA
and the MeA also impaired copulatory behavior, effects that
were blocked by naloxone (Parra-Gamez et al., 2013). System-
ically administered opioid receptor antagonists enhance sexual
performance under certain testing conditions; for example, low
doses of naloxone facilitated behavior when tested nocturnally,
but not during the light phase (Van Ree et al., 2000). As to
sexual activity level, naloxone in the MPOA facilitates sexual
performance in poor copulators, but impairs it in average cop-
ulators (Van Ree et al., 2000); low ip-administered doses of
both naloxone and naltrexone induce copulation in sexually
satiated rats (Rodríguez-Manzo and Fernández-Guasti,
1995a), as does naltrexone when directly infused into the
VTA. However, in sexually experienced males, intra-VTA
naltrexone impairs copulation (Garduno-Gutierrez et al.,
2013b). This last datum suggests that the motivational status
of the animals rather than the sexual activity level might be
the defining factor for opioid antagonist effects, since sexually
exhausted animals were sexually competent males that became
sexually inhibited as a result of copulation to satiation. Endog-
enous opioids acting at the VTA are involved in sexual motiva-
tion (van Furth and van Ree, 1996).
Evidence suggests that opioid peptides are released during
sexual activity, since the physiological mechanisms of analgesia
and reward are concurrently activated during sexual behavior
(Szechtman et al., 1981), and both phenomena are blocked
by naloxone (Ågmo and Berenfeld, 1990; Forsberg et al.,
1987). In addition, increases in Met-enkephalin and the opioid
octapeptide Met-Arg(6)-Gly(7)-Leu(8) were detected in the
hypothalamus of rats that ejaculated once or copulated to sati-
ation 24 or 48 h earlier (Rodríguez-Manzo et al., 2002a). Block-
ing the actions of the endogenous opioids released by multiple
ejaculations during the process of copulation to exhaustion
interferes with the establishment of the characteristic sexual
inhibition 24 h later (Garduno-Gutierrez et al., 2013a).
Mating-induced m opioid receptor internalization, a marker
for ligand-induced receptor activation, was detected in the
MPOA (Coolen et al., 2004b) and VTA (Balfour et al., 2004)
of rats after one ejaculation. Ejaculation also induced d opioid
receptor internalization in the VTA and repeated ejaculation
produced a degree of VTA m opioid receptor internalization
proportional to the amount of ejaculatory activity displayed
(Garduno-Gutierrez et al., 2013c).
Systemic morphine dose-dependently reduced the propor-
tion of animals showing reflexive erections and virtually elim-
inated seminal emission; naloxone antagonized the effects of
morphine, but the lowest dose, administered alone, also
inhibited erectile response, suggesting that some opioid activity
can facilitate sexual reflexes (Gomez-Marrero et al., 1988).
Stimulation of CNS m opioid receptors prevents penile erection
(reviewed in Andersson, 2001), and opioid mechanisms in the
spinal cord appear to raise ejaculation threshold, since intra-
thecal morphine increased the IF before ejaculation
(Wiesenfeld-Hallin and Södersten, 1984). Moreover, opioids
in the spinal cord have been found to play an inhibitory role
in the spinal control of ejaculation (Carro-Juárez and
Rodríguez-Manzo, 2009) (see Section 1.01.5.3.5).
1.01.4.10 Oxytocin
OT is expressed in neurons of the magnocellular PVN and
supraoptic nucleus (SON), both of which project to the poste-
rior pituitary, and in parvocellular PVN neurons that project to
several brain areas and the lumbosacral spinal cord, where erec-
tile and ejaculatory reflexes are controlled. Either icv or
systemic injections of OT facilitated copulation in male rats
(reduced EL and PEI); conversely, icv injections of an OT antag-
onist impaired or abolished copulation in sexually experienced
rats (reviewed in Argiolas and Melis, 2005). Facilitative effects
were also seen in older (
20 months) rats that were ‘sexually
sluggish’; OT treatment reduced MLs, ILs, ELs, and PEIs and
increased the number of animals able to resume copulation
after the first ejaculation (Arletti et al., 1990). Intra-MPOA
injection of OT facilitated mating in sexually experienced (Gil
et al., 2011) and sexually naïve male rats, and copulation
increased OT receptor gene and protein expression in this brain
region, suggesting a reciprocal interaction between central OT
and sexual behavior (Gil et al., 2013). Systemic OT also
reversed the inhibitory effects of chronic fluoxetine, an SSRI,
on the ability of male rats to ejaculate (Cantor et al., 1999).
SSRI-induced delayed ejaculation was related to
desensitization of 5-HT1A receptors on OT neurons (de Jong
et al., 2007). Plasma OT is increased during sexual activity in
humans (Carmichael et al., 1987) and is released in the
hypothalamus of male rats during mating (Waldherr and
Neumann, 2007). OT infusion into the PVN of male rats
stimulates penile erection (Kita et al., 2006) an effect that is
also obtained when OT is injected into brain areas that
receive projections from the PVN, e.g., VTA, hippocampus,
amygdala, and spinal cord (Melis and Argiolas, 2011) (see
Section 1.01.5.2.4).
1.01.4.11 Prolactin
Prolactin (PRL) is secreted by the anterior pituitary into the
general circulation. This peptide has a well-established role in
lactation; however PRL has been reported to have more than
300 functions across vertebrates, a majority of which relate to
reproduction (Bancroft, 2005). Hyperprolactinemic patients
have low sexual desire often associated with erectile problems

(Corona et al., 2007). Plasma PRL levels increase markedly
following ejaculation and orgasm in men (Bancroft, 2005).
This postorgasmic PRL increase may act as a feedback control
of the refractory period following orgasm (Krüger et al.,
2003). In line with this hypothesis, there is a case report of
a multiorgasmic healthy man that showed no PRL response
to three consecutive orgasms (Haake et al., 2002). However,
male rats engaged in constant sexual activity show a sustained
increase in serum PRL during mating (Rojas-Duran et al.,
2015). Animal experiments confirm that chronic exposure to
elevated PRL impairs male rat sexual response, increasing IL
and EL and reducing reflexive erections; short-term PRL
increase had either no effect or facilitated copulation (Melis
and Argiolas, 1995).
1.01.4.12 Gonadotropin-Releasing Hormone
GnRH is a peptide hormone produced by the mediobasal
hypothalamus, lamina terminalis, and the MPOA that acts on
the anterior pituitary where it stimulates gonadotropin release.
Earlier studies found that GnRH, administered systemically or
icv, facilitated male sexual behavior in castrated rats main-
tained with low doses of T, but not in intact animals (Moss
et al., 1975). By contrast, Myers and Baum (1980) found a facil-
itative effect of GnRH only in gonadally intact rats and not in
castrates replaced with T. A facilitative action of GnRH on
sexual motivation was also established in a mounting test in
rats with penile anesthesia, after its icv infusion (Dorsa and
Smith, 1980). GnRH is released naturally when male rodents
(mice and hamsters) encounter female vaginal fluid chemosig-
nals (Westberry and Meredith, 2003), and GnRH-containing
neurons are activated in male rats by estrous female odors
(Kippin et al., 2003). In sexually naïve hamsters, GnRH
infused icv restored mating behavior impaired by removal of
VNOs (Meredith and Fernandez-Fewell, 1994). Systemic
GnRH facilitates sexual behavior in hyperprolactinemic male
rats treated with T (Dennison et al., 1996), and exogenous
GnRH restores fertility and sexual activity in men with
hypogonadism associated with low levels of circulating T
(Mortimer et al., 1974). The loss of GnRH neurons in the
medial septum and MPOA has been associated with aging-
related diminished mating ability (Tsai et al., 2014).
However, adverse effects of GnRH treatment have also been
reported. Thus, GnRH in old rhesus monkeys increased the PEI
(Phoenix and Chambers, 1990). Chronic administration of
a synthetic GnRH analog [(6-D-(2-napthyl)-alanine)GnRH]
initially increased IL in intact rats and eliminated sexual
behavior 6–8 weeks later, along with a decrease in T (Dorsa
et al., 1981). The inhibitory effects probably resulted from
the continuous high doses of GnRH, which inhibit LH release
and gonadal function, whereas endogenous GnRH is released
in a pulsatile fashion. Studies in hypogonadal mice bearing
a deletion of the GnRH-encoding gene suggest that this
peptide is not essential for male sexual behavior (Gibson
et al., 1997).
1.01.4.13 Orexin/Hypocretin
The orexin/hypocretin (orx/hcrt) peptides (orexin A and
B/hypocretin 1 and 2) are expressed in the LH; orexinergic
Male Sexual Behavior 21
neurons project widely to monoaminergic nuclei in the
midbrain and brain stem and to a number of basal forebrain
areas, including brain areas relevant for sexual behavior like
the PVN, the MPOA, and the VTA (Peyron et al., 1998). These
peptides are primarily known for their ability to regulate
feeding and wakefulness; however, they can also enhance
male sexual behavior. Their expression is hormonally regu-
lated, being decreased after castration and restored by E2;
a slight increase by DHT was not statistically significant
(Muschamp et al., 2007). Both copulation and estrous female
odors increased Fos expression in orx/hcrt neurons, implying
their activation during copulation; in addition, systemic
administration of an orx/hcrt antagonist (SB334867) delayed
the onset of copulation and decreased the number of ejacula-
tions per 30-min test (Muschamp et al., 2007). However,
specific lesion of orexin neurons did not impair sexual
behavior, but prevented the formation of a conditioned place
preference for copulation (Di Sebastiano et al., 2011) suggest-
ing a role of this neuropeptide in sexual reward. In line with
this idea, intra-VTA injection of orexin increased DA release
in the NAc (Korotkova et al., 2003) as well as dopaminergic
cell firing (reviewed in Hull, 2011); however, it has been re-
ported that icv-administered orexin-A attenuated male sexual
motivation measured in a preference paradigm (Bai et al.,
2009).
1.01.5 Brain Areas and Circuitry Implicated
in the Control of Masculine Sexual Behavior
1.01.5.1 Sensory Inputs
1.01.5.1.1 Olfactory Bulbs
Chemosensory signals are transduced in the olfactory mucosa
and VNO and are transmitted to the olfactory bulbs. The
importance of chemosensory cues varies across species, but
these cues are especially important in rodents and other
nocturnal species. The olfactory system includes the anatomi-
cally and functionally distinct main and accessory olfactory
systems. Volatile odors, processed by the main olfactory
system, are transduced by receptors in the nasal mucosa, whose
axons project through the cribriform plate to the main olfactory
bulb (MOB). Both nonvolatile and volatile species-specific che-
mosensory cues, or pheromones, are detected in the VNO,
located at the base of the nasal cavity and projecting to the
accessory olfactory bulb (AOB). Male rats that experienced
three ejaculations had a greater density of new cells, and specif-
ically, new neurons, in the posterior part of the granular cell
layer of the AOB 45 days after copulating (Unda et al., 2016).
No significant differences were found in the MOB. Thus, copu-
lating to three ejaculations can induce lasting changes in the
AOB. While vomeronasal cues are important in rodents and
other macrosmotic animals, the VNO and AOB are regressed
in humans and may be nonfunctional.
1.01.5.1.1.1 Effects of Lesions
Mating by male Syrian hamsters is abolished by olfactory bul-
bectomy. (See Hull et al., 2006 for review of the older research
in this area.) However, the relative importance of the main and
accessory systems is not clear. Removal of the VNO impaired
copulation in sexually naïve males, but had little effect in
22 Male Sexual Behavior
experienced males (Ballard and Wood, 2007; Meredith, 1986).
However, vomeronasal nerve cuts, which also damaged part of
the MOB, produced greater deficits, especially in naïve males
(Meredith, 1986). Destruction of receptors in the nasal mucosa
with zinc sulfate produced minimal damage (O’Connell and
Meredith, 1984), but deafferentation of both the MOB and
AOB abolished copulation (Meredith et al., 1980).
Male rats are somewhat less dependent on chemosensory
input than are hamsters, although bilateral bulbectomy did
severely compromise mating in some males and increased IL
and EL in those that did copulate (Edwards et al., 1990,
1996; reviewed in Hull et al., 2006). Noncontact erections, eli-
cited by female rat urine, were also eliminated by olfactory bul-
bectomy or zinc sulfate lesions of the nasal mucosa, but not by
removal of the VNO (Kondo et al., 1999).
Chemosensory stimuli also promote sexual activity of male
mice (reviewed in Hull et al., 2006). Removal of the VNO
blocked the preference for urinary odors of estrous females
but did not affect olfactory sex discrimination (Pankevich
et al., 2004). Male mice with a null mutation of the transient
receptor potential 2 (trp2) cation channel, which disrupted
VNO function, had little electrophysiological response to pher-
omones, but mated normally with females and also mounted
other males (Leypold et al., 2002; Stowers et al., 2002). In
contrast, mice genetically lacking the cyclic nucleotide-gated
channel alpha 2, necessary for odor-evoked signaling in the
main olfactory epithelium, failed to mate (Mandiyan et al.,
2005). Thus, in mice, the main olfactory system is more critical
for sexual behavior than the vomeronasal system.
1.01.5.1.1.2 Activation of c-Fos or Other Immediate-Early Genes
Immediate-early genes are expressed transiently after a stim-
ulus. Their protein products are transcription factors that may
affect neural activity or responsiveness to certain stimuli. In
situ hybridization for mRNA from the c-fos gene or immunocy-
tochemistry for its protein product Fos is widely used to map
neural activation in responsive brain areas. Fos was increased
in the MOB and AOB of male hamsters following either copu-
lation or exposure to female odors (Fernandez-Fewell and Mer-
edith. 1994; Fiber et al., 1993; Kollack-Walker and Newman,
1997; Swann, 1997). Furthermore, female odors elicited Fos
expression in the olfactory bulb even in castrates, although
the castrates were unable to copulate (Swann, 1997). Therefore,
castration may result in loss of signal in downstream areas
without altering detection of female cues. In rats and mice,
too, either copulation or exposure to female odors elicited
Fos-ir in the MOB, AOB, and downstream structures (Baum
and Everitt, 1992; Coolen et al., 1997b; Halem et al., 1999;
Robertson et al., 1991), with mating being more effective
than odor alone (Kelliher et al., 1999). In male mice, Fos-ir eli-
cited by female odors was localized to the rostral portion of the
AOB and was dependent on an intact VNO (Dudley and Moss,
1999; Matsuoka et al., 1999). However, intranasal zinc sulfate
in mice, intended to disrupt the main olfactory epithelium,
blocked the Fos response to female urine in both the MOB
and AOB (Martel and Baum, 2007). In rats, exposure to female
bedding elicited Fos-ir in the AOB of all males, regardless of
whether they were sexually experienced, noncopulators, or
sexually naïve (Portillo and Paredes, 2004); however, only in
sexually experienced copulators was Fos expressed in all
downstream structures of the vomeronasal pathway
(Hosokawa and Chiba, 2005; Portillo and Paredes, 2004).
The authors suggest that alterations in the pathway from the
VNO render some males unable to process fully the chemosen-
sory cues of estrous females, resulting in reduced sexual
motivation.
Although both the main olfactory and vomeronasal systems
contribute to rodent sexual behavior, male ferrets, which are
carnivores, rely only on the main olfactory system (Kelliher
et al., 1998). Female odorants did not elicit Fos-ir in the
AOB, but did activate clusters of glomeruli in the ventral–
caudal MOB (Woodley and Baum, 2004). The ferret MOB
has ARs, which have the potential to enhance neuronal
responses to chemosensory stimuli from the female.
1.01.5.1.2 Amygdala
1.01.5.1.2.1 Anatomy
There is controversy about whether ‘the amygdala’ should be
regarded as a single entity or a collection of nuclei that
contribute to learning, motivation, and fear (central nucleus
and basolateral division) or to chemosensory processing and
social behaviors (corticomedial division) (Swanson and
Petrovich, 1998). Clearly, the corticomedial region serves as
an integration site for chemosensory, somatosensory, and
hormonal stimuli and projects to the MPOA and other central
regulatory areas. The AOB projects via the lateral olfactory tract
to the medial and posteromedial nuclei, while the MOB
projects diffusely to the anterior and posterolateral cortical
nuclei and to the ventral allocortex (Figure 3). The MeA also
receives somatosensory input from the genitals, relayed via
the subparafascicular nucleus (SPF), which also projects
directly to the MPOA. There are abundant ERs and ARs in the
MeA, especially in the posterior subnucleus, as well as in the
MPOA and other hypothalamic nuclei. The volume and soma
size of the MeApd are larger in male than in female rats (Cooke
et al., 1999; Hines et al., 1992) and are maintained by stimula-
tion of both ARs and ERs (Cooke et al., 2003). Males also have
more neurons and glial cells in that area; the number of glia is
influenced by circulating androgens; neuron number appears
to be ‘organized’ before adulthood (Morris et al., 2008).
1.01.5.1.2.2 Effects of Lesions
Lesions of the central nucleus and basolateral division have
little effect on copulation, although they do impair condi-
tioned responses during fear and reinforcement (reviewed in
Everitt, 1990). In contrast, corticomedial lesions do impair
copulation, with the severity dependent on the specific location
and the species. Anterior MeA lesions abolish copulation in
male hamsters, while more posterior lesions impair, but do
not eliminate mating (Lehman et al., 1980). A more recent
study reported that lesions of the MeApd in male hamsters
decreased attraction to female odors, but anterior MeA lesions
led to prolonged investigation of both male and female odors
(Maras and Petrulis, 2006). The authors suggested that the
MeApd may increase arousal toward relevant social stimuli,
whereas the anterior MeA may filter stimuli and direct the
arousal toward appropriate social cues. In rats (de Jonge
et al., 1992; Dominguez et al., 2001; Kondo, 1992; McGregor
and Herbert, 1992a) and gerbils (Heeb and Yahr, 2000), MeA
 Male Sexual Behavior 23

Figure 3 Diagram of the ventral surface of the hamster brain. Shaded areas indicate brain regions that receive efferent projections of the olfactory
system, via the main olfactory bulb (left), and of the vomeronasal system, via the accessory olfactory bulb (right). ACo, anterior cortical nucleus of
the amygdala; AOB, accessory olfactory bulb; AON, anterior olfactory nucleus; BNST, bed nucleus of the stria terminalis; EC, entorhinal cortex; HR,
hippocampal rudiment; Me, medial nucleus of the amygdala; MeA, medial nucleus of the amygdala, anterior division; MeP, medial nucleus of the
amygdala, posterior division; MOB, main olfactory bulb; nAOT, nucleus of the accessory olfactory tract; nLOT, nucleus of the lateral olfactory tract;
OLF EPITH, olfactory epithelium; OTu, olfactory tubercle; PC, piriform cortex; PLCo, posterolateral cortical nucleus of the amygdala; PMCo, poster-
omedial cortical nucleus of the amygdala; VNO, vomeronasal organ. Figure is reprinted from Wood, R.I., Newman, S.W., 1995. Hormonal influence
on neurons of the mating behavior pathway in male hamsters. In: Micevych, P.E., Hammer Jr., R.P. (Eds.), Neurobiological Effects of Sex Steroid
Hormones. Cambridge University Press, New York, pp. 3–39 (Chapter 1) with permission from Cambridge University Press.

lesions delay and slow copulation and increase the intromis-
sions required to elicit ejaculation.
Radiofrequency lesions of the posterior MeA of male rats
eliminated noncontact erections and decreased preference for
an estrous female, but did not affect copulation (Kondo and
Sachs, 2002; Kondo et al., 1998). A subregion of the MeApd
has been linked to sexual satiety. Lesions of the MeApd in
male hamsters delayed sexual satiety (Parfitt et al., 1996) (see
Section 1.01.5.1.2.3).
1.01.5.1.2.3 Activation of c-Fos or Other Immediate-Early Genes
or Other Measures of Activity
Chemosensory stimuli can induce Fos-ir in the corticomedial
amygdala in rats (Bressler and Baum, 1996; Coolen et al.,
1997b; Kelliher et al., 1999), hamsters (Fernandez-Fewell and
Meredith, 1994; Fiber et al., 1993; Kollack and Newman,
1992), and gerbils (Heeb and Yahr, 1996). Increasing amounts
of sexual behavior elicited increasing amounts of Fos-ir in
nuclei of the amygdala of male rats (Baum and Everitt, 1992;
Coolen et al., 1996; Veening and Coolen, 1998), with the
largest number of Fos-ir cells in the MeA, especially the poste-
rior subnucleus, and fewer in the anterior and posteromedial
cortical nuclei (Kollack and Newman, 1992). However, expo-
sure to an inaccessible estrous female rat elicited about as
much Fos-ir in arginine vasopressin-containing neurons in
the MeApd as did copulation in male rats (Hari Dass and
Vyas, 2014). Perhaps those neurons are more attuned to chem-
ical stimuli than to copulation. Mating also elicited Fos-ir in
amygdaloid nuclei of hamsters (Kollack and Newman, 1992,
1997), gerbils (Heeb and Yahr, 1996), prairie voles (Wang
et al., 1997), and musk shrews (Gill et al., 1998). In gerbils
the medial part of the MeApd was activated (Fos-ir) by sex-
related odors, whereas the lateral portion was activated only
following ejaculation (Heeb and Yahr, 1996). Similarly, in
hamsters Fos-ir in the MeApd was correlated with the onset
of satiety (Parfitt and Newman, 1998). Fos-ir in the MeA, but
24 Male Sexual Behavior
not in the MPOA, of male rats was positively correlated with
the length of the PEI (Lumley and Hull, 1999), suggesting
that some neurons in that area may contribute to postejacula-
tory quiescence. In rats (Gréco et al., 1998b) and hamsters
(Wood and Newman, 1993), AR-containing cells in the
posterior MeA are activated by mating; however, castration
did not reduce mating-induced Fos-ir in the MeA of rats
(Baum and Wersinger, 1993) or odor-induced Fos-ir in the
MeA of hamsters (Swann, 1997). Similarly, mice lacking the
gene for aromatase had similar response to odors as in wild-
type mice (Aste et al., 2003).
Men and women underwent functional magnetic resonance
imaging (fMRI) while watching sexual or nonsexual pictures
(Cyders et al., 2016). Only men showed increased brain activity
in response to the sexual stimuli. Areas activated included the
bilateral amygdala, anterior cingulate cortex/medial prefrontal
cortex, anterior insula/lateral orbitofrontal cortex, and occipital
regions.
1.01.5.1.2.4 Effects of Hormone Implants or Manipulations
Affecting Hormone Receptors or Enzymes
In castrated male rats, bilateral T implants in the MeA delayed
the loss of mating and of noncontact erections (Berendsen and
Broekkamp, 1987), and E implants stimulated mounting, but
not ejaculation (Huddleston et al., 2003). Similarly, in cas-
trated hamsters either T (Wood, 1996) or E (Coolen and
Wood, 1999; Wood and Coolen, 1997), but not DHT
(Wood, 1996), implants in the MeA restored copulation.
However, DHT implants were successful in male rats treated
with subthreshold systemic injections of E (Baum et al.,
1982). Either an ER antagonist in male hamsters (Wood and
Williams, 2001), or an aromatase (Huddleston et al., 2006)
or AR (McGinnis et al., 1996) antagonist in male rats impaired,
but did not block, copulation, suggesting that both androgenic
and estrogenic stimulation contributes to mating in male rats
and hamsters. Furthermore, MeA implants of E2 conjugated
to bovine serum albumin (which prevents E2 from crossing
the membrane) were ineffective in maintaining mating
behavior (Huddleston et al., 2006), suggesting an intracellular
mechanism of action. Because both hormonal and chemosen-
sory stimuli are critical for mating in male hamsters and are
processed in the MeA, their interactions were probed. Unilat-
eral olfactory bulbectomy, either ipsi- or contralateral to a T
implant in the posterior MeA of castrated hamsters, rendered
the implant ineffective (Wood and Coolen, 1997), suggesting
that bilateral chemosensory input is essential for T in this
nucleus to stimulate sexual behavior. However, with similar T
implants in both the MPOA and BST, only ipsilateral bulbec-
tomy blocked the facilitation by T (Wood and Newman,
1995c). Thus, communication between groups of neurons
that process hormonal and chemosensory stimuli is critical
for male hamster sexual behavior.
The P receptor antagonist RU-486, administered to male
rat pups during the first postnatal week, resulted in increased
AR-ir in the MeA and several other areas, including BST,
MPOA, and ventromedial nucleus (Forbes-Lorman et al.,
2014). Copulation was also enhanced by the RU-486
treatment. In contrast, Antaramian et al. (2015) reported
that sexually sluggish male rats actually had increased
mRNA for aromatase in the amygdala and increased mRNA
for both aromatase and AR in the MPOA. The authors did
not suggest an explanation for the apparent inverse
relationship between aromatase (and AR in the MPOA) and
copulatory ability.
1.01.5.1.2.5 Microinjections of Drugs
Microinjection of the serotonin 5-HT1A agonist 8-OH-DPAT
into the MeApd speeded the onset of copulation and decreased
the PEI in male rats (de Castilhos et al., 2006). Therefore, the
MeApd is one site at which 5-HT1A agonists facilitate copula-
tion. Angiotensin microinjections into the MeA impaired copu-
lation in male rats; however, it is not clear whether increased
thirst may have interfered with sexual behavior (Breigeiron
et al., 2002).
1.01.5.1.2.6 Amygdaloid Efferents
A major output of the MeA is to the MPOA. Unilateral lesions
of the MPOA impaired, but did not abolish copulation in male
rats; however, contralateral lesions of the MeA and MPOA
severely disrupted sexual behavior (Kondo and Arai, 1995).
Similar results were reported for gerbils (Heeb and Yahr,
2000). Chemical stimulation of the MeA increased DA release
in the MPOA (Dominguez and Hull, 2001), and microinjec-
tion of the DA agonist apomorphine into the MPOA restored
copulation that had been abolished by large excitotoxic lesions
of the MeA (Dominguez et al., 2001). Basal extracellular DA in
the MPOA was not affected by small radiofrequency MeA
lesions, but the DA release in response to a female and during
copulation was eliminated by the lesions, which also impaired
but did not abolish copulation (Dominguez et al., 2001).
Indeed, chemical stimulation of the MeA elicited DA release
in the MPOA of a magnitude similar to that observed during
copulation (Dominguez and Hull, 2001). Thus, it appears
that one result of MeA activity is to increase DA in the MPOA
in anticipation of and during mating. There are no dopami-
nergic neurons in the MeA of rats; therefore, efferents from
the MeA may activate, either directly or indirectly, dopami-
nergic cell bodies or terminals in the MPOA and thereby facil-
itate mating.
Chronic isolation of normally monogamous prairie voles
resulted in accelerated copulation when males were paired
with a female (Perry et al., 2016). Pups had been reared with
same-sex siblings until adulthood, when they were isolated
for 4 weeks. Males had increased ERa in the MeApd but
decreased tyrosine hysroxylase (TH), which is the rate-
limiting enzyme for catecholamine production. As noted
above, the MeA projects to the MPOA; however, it lacks
DOPA decarboxylase, which would produce DA; therefore,
those neurons could release either L-DOPA or other trace
amines into the MPOA. Trace amine receptors have been
shown to decrease DA release (Lindemann et al., 2008), which
is important for copulation. Therefore, a reduction in trace
hormone release in the MPOA could promote mating before
pair bond formation could occur, which would facilitate polyg-
amous strategies.
1.01.5.1.3 Bed Nucleus of the Stria Terminalis
1.01.5.1.3.1 Anatomy
Output from the MeA travels either directly to the MPOA or
synapses in the BST, which then relays the information to the

MPOA and other sites. The posteromedial division of the BST is
especially relevant to the control of male sexual behavior
(Alheid et al., 1995). As with the posterior MeA, the posterome-
dial BST has abundant steroid hormone receptors (Li et al.,
1993; Simerly et al., 1990; Wood and Newman, 1993).
1.01.5.1.3.2 Effects of Lesions
In male rats and hamsters the more dorsal part of the BST
appears to contribute more to preparation for mating than to
copulation per se (reviewed in Hull et al., 2006). In rats, lesions
decreased the number of noncontact erections and slowed the
rate of copulation, but did not affect the intromission ratio,
suggesting that in copula erections were unaffected; in contrast,
lesions of the MPOA had little effect on noncontact erections
but severely impaired copulation (Liu et al., 1997b). Finn
and Yahr (2005) suggested that previous studies that showed
little effect on copulation targeted the dorsal part of the BST.
In contrast, they made excitotoxic lesions of the vBST, which
projects to the retrorubral field (A8) of the midbrain and other
downstream sites. vBST lesions in rats resulted in severe copu-
latory deficits, although damage to the adjacent posterodorsal
preoptic nucleus (PdPN) may have contributed to the impair-
ment (Finn and Yahr, 2005). The authors suggested that the
vBST in rats may be the counterpart to the gerbil sexually
dimorphic area (SDA) of the preoptic area, which is critical
for male gerbil sexual behavior. In gerbils contralateral lesions
of the caudal medial BST and the SDA severely impaired copu-
lation, but contralateral lesions of the MeA and SDA produced
less of a deficit, suggesting that the caudal medial BST does
more than simply transmit information from the MeA to the
SDA (Sayag et al., 1994).
1.01.5.1.3.3 Activation of c-Fos or Other Immediate-Early Genes
In male rats, hamsters, and gerbils, copulation, or to a lesser
extent, exposure to female odors elicited Fos-ir in the BST, espe-
cially in the posterior and medial subdivisions (Coolen et al.,
1996; Fernandez-Fewell and Meredith, 1994; Heeb and Yahr,
1996; Kelliher et al., 1999; Kollack-Walker and Newman,
1995). In quail, both visual and physical contact increased
Fos-ir in the BST (Iyilikci et al., 2014). In contrast, mating
decreased Fos-ir in male macaques (Michael et al., 1999),
and female odors did not affect Fos-ir in male ferrets (Kelliher
et al., 1998).
1.01.5.1.3.4 Hormonal Effects
There are abundant steroid receptors in the posteromedial BST,
extending from the stria terminalis to the MPN (Li et al., 1993;
Simerly, 1995; Simerly et al., 1990; Wood and Newman,
1993). Castration decreased, and T restored, immunoreactivity
to CCK in the encapsulated portion of the BST (Simerly and
Swanson, 1987). Similar effects were seen in SDAs of the
MeA and MPN.
1.01.5.1.4 Central Tegmental Field/Subparafascicular Nucleus
of the Thalamus
1.01.5.1.4.1 Anatomy
There are reciprocal connections between the midbrain
tegmentum and the MPOA, MeA, and anterior hypothalamus
(Coolen et al., 1998, 2003; Gréco et al., 1999, 1998b; Murphy
et al., 1999a; Simerly, 1995; Simerly and Swanson, 1988).
Male Sexual Behavior 25
Subregions of the tegmentum have been referred to as the
central tegmental field (CTF, e.g., Baum and Everitt, 1992;
Gréco et al., 1999; Simerly and Swanson, 1986) or dorsolateral
tegmentum (DLT, e.g., Edwards and Einhorn, 1986; Giordano
et al., 1998; Maillard and Edwards, 1991). The CTF/DLT is
dorsal to the lateral half of the substantia nigra and includes
the SPF, part of the zona incerta, the peripeduncular nucleus,
the mesencephalic reticular nucleus, and the anterior pretectal
nucleus. The medial parvocellular division of the subparafascic-
ular nucleus (SPFp) relays somatosensory input from the geni-
tals to the MPOA and MeA. It is considered to be part of an
ejaculation circuit (see Section 1.01.6).
1.01.5.1.4.2 Effects of Lesions
Bilateral neurotoxic lesions of the CTF decreased the percent-
ages of male rats that could mount, intromit, and ejaculate
(Romero-Carbente et al., 2007). Partner preference and sexual
incentive motivation were not affected. Even misplaced lesions
in adjacent areas impaired behavior, suggesting that the effects
were not specific to the CTF. In tests 3 weeks after the lesion,
copulation had returned to normal. Earlier studies reported
more severe deficits (Brackett and Edwards, 1984; Giordano
et al., 1998), and contralateral lesions of the MPOA and CTF
impaired both copulation and pursuit and sniffing of the
female (Edwards and Einhorn, 1986). Combined ipsilateral
lesions of the CTF and MeA abolished mating-induced Fos-ir
in the MPOA, whereas single lesions of each did not (Baum
and Everitt, 1992). Bilateral SPFp lesions in gerbils did not
affect copulation, suggesting that the Fos-ir elicited in that
nucleus following ejaculation resulted from sensory input,
rather than motor control of ejaculation (Heeb and Yahr,
2000). However, in male rats the SPFp has been shown to
receive projections from lumbar spinothalamic neurons
(Coolen et al., 2003), which are essential for ejaculation (Truitt
and Coolen, 2002) and express ejaculation-related Fos-ir
(Truitt et al., 2003).
1.01.5.1.4.3 Activation of c-Fos or Other Immediate-Early Genes
Fos-ir was selectively increased in either the CTF or SPFp after
ejaculation in rats (Baum and Everitt, 1992; Coolen et al.,
1997a,b, 1996; Wersinger et al., 1993), gerbils (Heeb and
Yahr, 1996), hamsters (Kollack-Walker and Newman, 1997),
and musk shrews (Gill et al., 1998). Fos-ir was not increased
after chemosensory investigation, mounts, or intromissions.
In men, ejaculation stimulated blood flow in the SPFp, as
measured with positron emission tomography (Holstege
et al., 2003). Also, electrical stimulation of the CTF facilitated
mating in rats (Shimura and Shimokochi, 1991).
1.01.5.1.4.4 Presence of Steroid Receptors
SPFp neurons contain ARs (Gréco et al., 1996), and many AR-ir
neurons that project to the MPOA express ejaculation-induced
Fos-ir (Gréco et al., 1998b).
1.01.5.1.4.5 Connections
The medial SPFp contains many galanin-ir fibers, and neurons
containing mating-induced Fos-ir in the medial SPFp are
surrounded by galanin-ir fibers (Veening and Coolen, 1998).
Neurons in laminae 7 and 10 of the lumbosacral spinal
cord project to the medial SPFp (Coolen et al., 2003;
26 Male Sexual Behavior
Gréco et al., 1999; Truitt et al., 2003), which also connects recip-
rocally with forebrain nuclei that control copulation (Coolen
et al., 2003). Therefore, galanin may convey ejaculation-related
somatosensory input through the SPFp to higher brain areas.
1.01.5.2 Major Integrative Sites
1.01.5.2.1 Medial Preoptic Area
1.01.5.2.1.1 Anatomy
The MPOA is a critical integrative site for male sexual behavior
in all vertebrate species tested. This is remarkable, in that a wide
range of sensory stimuli elicit mating, and diverse motor
patterns comprise the species-specific behaviors. Every sensory
modality sends indirect input to the MPOA, and reciprocal
connections allow the MPOA to modify the processing of
sensory input (Simerly and Swanson, 1986). Furthermore,
steroid receptors in the MPOA and its major afferents can
bias input to favor sexually relevant stimuli. T can influence
the neuroanatomy of MPOA neurons. Gonadally intact and
T-treated castrates had more dendritic spines and greater
spine density than did castrates without T (Garelick and
Swann, 2014). However, dendrite length was not increased.
Therefore, existing dendrites produce more spines that are
available for synapses, but do not extend their length.
Efferents from the MPOA to hypothalamic, midbrain, and
brain stem nuclei regulate somatomotor or autonomic
patterns and motivational states (reviewed in Simerly and
Swanson, 1988; Yahr, 1995).
The MPOA comprises heavily interconnected subnuclei
with various functions, connections (Maragos et al., 1989; Sim-
erly et al., 1986; Wang and Swann, 2014), and neurotrans-
mitter content (Simerly et al., 1986). A medial periventricular
zone regulates neuroendocrine function, and a medial zone,
including MPN and PdPN, controls male sexual behavior and
maternal behavior. The magnocellular medial preoptic nucleus
in male Syrian hamsters receives input from chemosensory
pathways and steroid receptor-containing areas and sends its
efferents to several brain stem nuclei that regulate aspects of
male sexual behavior, including the periaqueductal gray
(PAG), deep mesencephalic nucleus, retrorubral field, VTA,
and lateral paragigantocellular nucleus (Wang and Swann,
2014). Other targets are steroid-containing nuclei in the
septum, preoptic area, and hypothalamus. Wang and Swann
suggest that its lack of efferents to chemosensory processing
areas implies that it integrates the chemosensory and hormonal
signals and then relays that information to brain stem areas
that control motor behavior.
1.01.5.2.1.2 Effects of Lesions
Large lesions of the MPOA abolish copulation in numerous
species ranging from monkeys to fish (reviewed in Hull and
Dominguez, 2015). Similar effects are obtained with electro-
lytic lesions, which destroy both cell bodies and axons passing
through, and axon-sparing neurotoxic lesions; thus neurons in
the MPOA are critical for activating copulation. Generally,
more severe deficits were found with more caudal lesions
that included portions of the anterior hypothalamus. Smaller
lesions have produced less severe deficits. In gerbils small
lesions of the PdPN, which is activated selectively by ejacula-
tion (Heeb and Yahr, 1996), decreased mounting and delayed
ejaculation (Heeb and Yahr, 2000). Unilateral lesions of the
medial SDA in gerbils, together with lesions of the contralateral
lateral SDA, impaired copulation as effectively as bilateral
lesions of each (Yahr and Gregory, 1993).
Large MPOA lesions disrupt the initiation of copulation as
well as its performance, suggesting that it contributes to sexual
motivation. However, male rats, cats, dogs, and monkeys
showed interest in sexual behavior even after MPOA lesions
abolished their ability to copulate (reviewed in Everitt, 1990;
Hull and Dominguez, 2015). Similarly, noncontact erections
were not affected by lesions that abolished copulation (Liu
et al., 1997b). This apparent dissociation of copulatory perfor-
mance and sexual motivation following MPOA lesions led
Everitt (1990) to suggest that the MPOA controls only copula-
tory performance and not sexual motivation.
However, MPOA lesions have decreased sexual motivation
in several contexts, including female partner preference in rats
(Edwards et al., 1996; Paredes et al., 1998) and ferrets (Kindon
et al., 1996; Paredes and Baum, 1995), pursuit of a female by
male rats (Paredes et al., 1993), or precopulatory behaviors
in marmosets (Lloyd and Dixson, 1988). Therefore, MPOA
lesions do not eliminate sexual motivation, but they clearly
diminish it. The evolutionary conservation of MPOA influence
on both sexual performance and motivation is seen in studies
of Japanese quail and starlings, in which small MPOA lesions
decreased time spent in front of a window through which the
male could see a female (Balthazart et al., 1998) or reduced
singing and gathering of nesting materials (Riters and Ball,
1999).
1.01.5.2.1.3 Effects of Electrical or Chemical Stimulation
Because MPOA lesions impair male sexual behavior, it was ex-
pected that stimulation would facilitate it, and, indeed,
numerous early studies of rats, guinea pigs, and opossums
showed such facilitative effects (reviewed in Hull and Domi-
nguez, 2015). Decreases in mounts and intromissions
preceding ejaculation and shortened ejaculation latency and
PEIs were reported. One such study found facilitation of copu-
lation following electrical stimulation of the MPOA of male
rats; however, such stimulation did not restore copulation
24 h after they had copulated to sexual satiety (Rodríguez-
Manzo et al., 2000). Furthermore, combining electrical stimu-
lation with subthreshold doses of the DA agonist apomorphine
or the a2-adrenoceptor antagonist yohimbine was also ineffec-
tive. Higher doses of both drugs had previously restored sexual
behavior in satiated males (Rodríguez-Manzo, 1999b). There-
fore, sexual satiety and postejaculatory quiescence may be
based on different neural mechanisms.
Repeated electrical stimulation of the MPOA in previously
noncopulating male rats resulted in ‘kindling’ (increased after-
discharges leading to a seizure) and the ability of most males to
copulate on subsequent stimulation-free tests (Paredes et al.,
1990). Males that could initially copulate without stimulation
were not further facilitated. In another study, kindlinglike stim-
ulation that did not produce a seizure nevertheless facilitated
copulation for at least 8 months in previously noncopulating
rats (Portillo et al., 2003). Even in T-treated female rats,
MPOA kindling resulted in male-like sexual behavior
(Dominguez-Salazar et al., 2003).

Either electrical or glutamatergic stimulation of the MPOA
of anesthetized rats increased intracavernous pressure
(Giuliano et al., 1996; Sato et al., 2001) and elicited the UG
reflex, even without urethral stimulation (Marson and
McKenna, 1994b). The effects of MPOA stimulation on intraca-
vernous pressure were enhanced by intrathecal injection of an
NO donor, a cGMP analog, and sildenafil, and were inhibited
by the NOS inhibitor L-NAME (Sato et al., 2001). Axons from
the MPOA do not project directly to the lumbosacral spinal
cord, where erection and ejaculatory reflexes are controlled;
thus, efferents from the MPOA must stimulate downstream
sites that then regulate the reflexes. However, the MPOA is
not necessary for genital reflexes, since MPOA lesions have
minimal effect on noncontact erections (Liu et al., 1997b),
reflexive erections (Szechtman et al., 1978), or spontaneous
seminal emission (Ågmo et al., 1977).
1.01.5.2.1.4 Effects of Direct Application of Steroids or Steroid
Antagonists
Numerous studies have shown facilitative effects of steroid
implants in the MPOA of castrated rats, ferrets, birds, and
lizards (reviewed in Hull and Dominguez, 2015). However,
these implants have not resulted in complete copulatory
behavior, as other central and peripheral targets also require
hormonal priming. In castrated male rabbits, T implants
restored territorial marking (chin rubbing), but not copulation
(Melo et al., 2008). Aromatization of T to E in the MPOA is
important for T’s facilitative effects. The aromatase inhibitor
fadrozole, microinjected into the MPOA of gonadally intact
male rats, inhibited copulation; only the MPOA contained
numerous unoccupied ERs as a result of the inhibitor, suggest-
ing that the inhibitor had not spread to other sites (Clancy
et al., 1995). Conversely, implantation of E into the MPOA
of intact males treated systemically with fadrozole increased
MF, IF, and EF, compared to males with systemic fadrozole
but no E in the MPOA (Clancy et al., 2000). Therefore, E in
the MPOA, together with androgens throughout the body,
could partially restore male sexual behavior. The importance
of aromatization for male sexual behavior is also seen in quail,
in which the effects of T implants in castrates correlated with
the induction of aromatase immunoreactivity (ARO-ir) in the
caudal POM (Balthazart and Ball, 2007). Similarly, lesions in
the caudal POM of quail produced the most marked
copulatory deficits (Balthazart et al., 1998), and there was the
highest Fos-ir in response to copulation in the caudal POM
(reviewed in Balthazart and Ball, 2007). On the other hand,
anterior POM lesions were associated with deficits in
appetitive measures (Balthazart et al., 1998). Sexual
preference in rams may also be affected by aromatization in
the MPOA. Rams that preferred to mate with other males had
lower levels of serum T and E, as well as decreased aromatase
activity in the MPOA, than did those that preferred to mate
with females (Resko et al., 1999). Also, sexually high-
performing rams with ewes had more ERs and more
occupied ERs in the MPOA than did low-performing rams
(Alexander et al., 1993).
In contrast to E, DHT in the MPOA of castrated male rats is
relatively ineffective, unless administered with subthreshold
systemic E or DHT (reviewed in Hull and Dominguez, 2015).
However, such implants in castrated guinea pigs restored
Male Sexual Behavior 27
copulation in almost all males (Butera and Czaja, 1989). While
E is the primary steroid for activating sexual behavior in male
rats, androgen in the MPOA does contribute to their sexual
behavior. The antiandrogen flutamide, administered into the
anterior MPOA, impaired copulation, but not partner preference,
whereas flutamide in the posterior MPOA impaired motivation,
but not copulatory performance (McGinnis et al., 2002).
Gonadally intact males that did not copulate when tested
repeatedly received either E or T implants in the MPOA
(Antonio-Cabrera and Paredes, 2014). All noncopulating males
receiving T ejaculated consistently from week 11 after the
implant until the end of testing at 5 months. All subjects
implanted with E ejaculated from week 16 until the end of
testing. The noncopulating rats had been shown to have normal
T and E plasma concentrations, but did have reduced
aromatase in the MPOA. Therefore, providing additional T or
E directly into the MPOA was sufficient to stimulate copulation.
1.01.5.2.1.5 Effects of Direct Application of Drugs Affecting
Specific Neurotransmitters
Drugs administered into the MPOA influence a number of
behavioral measures. Most of these effects resemble those of
the same drug administered systemically, though there are
some site-specific differences.
1.01.5.2.1.5.1 Dopamine. Neurons of the periventricular DA
system lie along the third ventricle and send branching axons
laterally into the MPOA and anterior hypothalamus (Moore
and Lookingland, 1995). The classic D1/D2 agonist apomor-
phine, microinjected into the MPOA, facilitated copulation in
both intact and castrated male rats and increased the number
of ex copula reflexes; conversely, the D1/D2 DA antagonist cis-
flupenthixol impaired copulation, reflexes, and sexual motiva-
tion, measured as choice of the female in an X-maze (reviewed
in Hull and Dominguez, 2015). There was both spatial and
behavioral specificity in the drug effects. MPOA injections of
a different antagonist, haloperidol, decreased the number of
anticipatory level changes in search of a female in a bilevel
apparatus and also impaired copulation (Pfaus and Phillips,
1991). Similarly, 6-OHDA lesions of the MPOA impaired
copulation for at least 4 h after the injection, although
increased synthesis in the remaining neurons restored both
DA levels and sexual behavior within 24 h (Bazzett et al.,
1992). The effect was specific to DA; levels of NE and
epinephrine were not affected. In addition, apomorphine in
the MPOA fully restored copulatory ability of males whose
large excitotoxic lesions of the amygdala had severely
impaired their ability to copulate (Dominguez et al., 2001).
D1- and D2-like receptors in the MPOA appear to have
different roles in the regulation of copulation. Stimulation of
D1-like receptors facilitated copulation and reflexive erections,
whereas stimulation of D2-like receptors produced dose-
dependent effects (reviewed in Hull and Dominguez, 2015).
A low dose of the D3/D2 agonist quinelorane decreased the
latency to the first ex copula reflex without affecting the numbers
of reflexes, suggesting a disinhibition of reflexes; however,
a high dose of quinelorane, or of the D1 antagonist SCH-
23390, decreased erections but increased sympathetically
mediated seminal emissions (Bazzett et al., 1991). The D1
agonist dihydroxyphenyl-tetrahydrothienopyridine (THP)
28 Male Sexual Behavior
increased reflexive erections and facilitated copulation but
inhibited ex copula seminal emissions (Hull et al., 1995;
Markowski et al., 1994). Experiments using low and high doses
of apomorphine with D1- and D2-like antagonists also support
the suggestion that stimulation of D1-like receptors promotes
erections, while intense stimulation of D2-like receptors facili-
tates ejaculation (Hull et al., 1992). Similarly, in tests of copu-
lation the D1-like agonist increased copulatory efficiency
(Markowski et al., 1994), and the D1-like antagonist increased
intromission latency but decreased the threshold for ejacula-
tion (Hull et al., 1989). The antagonist also decreased sexual
motivation (choice of the female in an X-maze) (Moses and
Hull, 1999). Similarly to the D1-like antagonist, a high dose
of the D3/D2 agonist quinelorane delayed the start of
copulation, but decreased ejaculation threshold (Hull et al.,
1989; Moses et al., 1995). It is not clear whether the dose-
dependent effects of quinelorane are mediated by different
subtypes of D2-like receptors or by different populations of
neurons that are under different levels of tonic inhibition.
However, the data consistently support the hypothesis that
stimulation of D1-like receptors increases parasympathetically
mediated erections and the early phase of copulation,
whereas stimulation of D2-like receptors shifts the autonomic
balance to favor sympathetically mediated ejaculation. This
hypothesis is supported by a report that the preferential D3
agonist 7-OH-DPAT, microinjected into the MPOA, elicited
ejaculation-related events in anesthetized male rats (Kitrey
et al., 2007). In addition, the facilitative role of D1-like
receptors was supported by a demonstration that a D1
antagonist (SCH-23390), microinjected into the MPOA
before each of seven exposures of a male rat to an estrous
female placed over his cage, blocked the facilitative effects of
those exposures on a drug-free copulation test (McHenry
et al., 2012). In that same report, other animals received
acute and/or chronic mating experience; those with both
acute and chronic mating showed increased phosphorylation
of DA- and cAMP-regulated phosphoprotein (DARPP-32) in
the MPOA, compared with all other groups. DARPP-32 is
a downstream marker of D1 receptor signaling and is
a substrate of cAMP-dependent protein kinase (PKA).
Therefore, D1 receptors in the MPOA appear to contribute to
experience-induced enhancement of male sexual behavior,
perhaps through a PKA-regulated mechanism.
In male European starlings microinjections of a D1 agonist
into the MPOA elicited sexually motivated, but not aggression-
motivated, song (Riters et al., 2014). However, there was an
inverted U curve, with either very low or very high doses elicit-
ing little singing. The effect of the D1 agonist was blocked by
the D1 antagonist SCH-23390. Therefore, an optimal level of
D1 receptor stimulation is needed for sexually motivated vocal
production.
1.01.5.2.1.5.2 Serotonin. Microinjection of large doses of
5-HT into the MPOA inhibited copulation (Fernández-Guasti
et al., 1992; Verma et al., 1989), and a 5-HT1B agonist delayed
ejaculation (Fernández-Guasti et al., 1992). As with systemic
injections of 5-HT1A agonists, reverse-dialysis of 8-OH-DPAT
into the MPOA facilitated copulation (Matuszewich et al.,
1999) and increased both DA and 5-HT levels (Lorrain et al.,
1998). The D2 antagonist raclopride partially blocked the
effects of 8-OH-DPAT, but the 5-HT1A antagonist did not, sug-
gesting that elevation of extracellular DA levels resulted in stim-
ulation of D2 receptors in the MPOA, which mediated much of
the facilitation (Matuszewich et al., 1999). Microinjection of an
SSRI (alaproclate) into the MPOA did not significantly affect
behavior, although it did inhibit copulation when microin-
jected into the anterior lateral hypothalamus (LHA) (Lorrain
et al., 1997).
1.01.5.2.1.5.3 Gamma-Aminobutyric Acid. High concentrations
of GABA have been reported in the MPOA of male rat brains
(Andersson, 2001) as well as in mating-activated neurons of
this brain area and of the MeA in male gerbils (Simmons and
Yahr, 2003). Enhancement of GABAergic transmission in the
MPOA by blocking GABA degradation or stimulating GABAA
receptors with muscimol reduced the proportion of copulating
rats (Fernández-Guasti et al., 1986a). Inhibition of GABA
synthesis or blockade of GABAA receptors within the MPOA
had a potent stimulatory effect on male rat sexual behavior,
expressed by a marked reduction of the PEI (Fernández-
Guasti et al., 1986a) and the virtual elimination of the USVs
that accompany the absolute refractory period (Fernández-
Guasti et al., 1986b). However, bicuculline did not restore
copulation in satiated male rats, suggesting that sexual satiety
is regulated by mechanisms different from those that control
the PEI (Rodríguez-Manzo et al., 2000).
1.01.5.2.1.5.4 Opioids. The lowest dose of the m agonist
morphine and of the k agonist dynorphin (1–13), microin-
jected into the MPOA, facilitated copulation, but the highest
dose of morphine led to the failure to resume mating after
the second ejaculation (Band and Hull, 1990). The m agonist
morphiceptin increased the latency to copulate, but did not
affect copulatory performance, motivation, or locomotion
(Matuszewich et al., 1995). However, b-endorphin delayed
the start of mating and inhibited its performance (Holmes
et al., 1997; van Furth et al., 1995). In support of facilitative
effects of low levels of opioids in the MPOA, the antagonist
naloxone in the MPOA prevented the induction of sexual rein-
forcement (Ågmo and Paredes, 1988). A review by Paredes
(2014) summarizes both indirect evidence of opioid activity
during copulation and conditioned place preference (CPP)
studies that directly test the importance of opioids in the
MPOA for sexual reward in both males and females.
1.01.5.2.1.5.5 Norepinephrine. Microinjection of NE into the
MPOA facilitated both sexual arousal and copulation, whereas
the a-adrenergic antagonist phenoxybenzamine and the
b-adrenergic antagonist propranolol inhibited mating (Mallick
et al., 1996). Stimulating autoreceptors with the a2-agonist
clonidine, which would decrease NE levels in the MPOA,
impaired copulation, and inhibiting autoreceptors with the
a2-antagonist yohimbine blocked the inhibitory effects of
systemic clonidine (Clark, 1991). Therefore, NE in the MPOA
may facilitate copulation.
1.01.5.2.1.5.6 Nitric Oxide. Reverse dialysis of the NO
precursor L-arginine into the MPOA increased, and the NOS
inhibitor L-NMMA decreased the rate of mounting by male
rats (Sato et al., 1998). L-NMMA also decreased the number

of sexually naïve males that copulated and increased the
number of ex copula seminal emissions (Moses and Hull,
1999; Moses et al., 1995). The NOS inhibitor L-NAME also pre-
vented copulation in sexually naïve male rats and decreased
numbers of intromissions and ejaculations in experienced
males (Lagoda et al., 2004). L-NAME microinjections before
each of seven exposures to an inaccessible estrous female
blocked the facilitative effects of those exposures, as seen in
saline-injected males, compared to males not exposed to
a female (Lagoda et al., 2004).
NOS in the MPOA is hormonally regulated. In both rats
(Du and Hull, 1999; Putnam et al., 2005) and hamsters
(Hadeishi and Wood, 1996), castration decreased NOS-ir in
the MPN. T replacement in castrated rats for 2, 5, or 10 days
increased NOS-ir and improved copulation; greater NOS-ir
density was correlated with shorter mount latencies (Sato
et al., 2005). There was also less NOS-ir in ERaKO mice,
compared to both wild-type mice and those lacking the AR
due to the testicular feminization mutation (tfm)
(Scordalakes et al., 2002). Therefore, ERs are more likely than
ARs to mediate the hormonal upregulation of NOS. nNOS is
colocalized with both ERa and AR in the MPOA of rats (Sato
et al., 2005), mice (Scordalakes et al., 2002), and hamsters
(Hadeishi and Wood, 1996). Furthermore, improvement in
copulation following T replacement correlated with density
of NOS-ir in the MPOA, but not the number of NOS-positive
neurons (Sato et al., 2005). Reverse dialysis of an NO donor
(sodium nitroprusside) into the MPOA of castrated rats that
were maintained on systemic DHT (to maintain genital and
sensory structures) fully restored copulation in half the
animals (Sato et al., 2007). NO’s effects are at least partially
mediated by cGMP. A guanylyl cyclase inhibitor decreased
the ability of the NO donor to improve copulation and
increased the MPOA DA release in gonadally intact male rats
(Sato and Hull, 2006). In contrast to the reports of
facilitative effects of hormones in mice, rats, and hamsters,
there is one report of increased NOS-ir after castration of rats
and a reduction following T or DHT replacement (Singh
et al., 2000). The reason for the seeming contradiction may
lie in the more posterior location examined by Singh et al. or
their use of an antibody that labeled very few cells. In
summary, NO in the MPOA facilitates copulation, and NOS
is upregulated by T, probably via aromatization to E, in the
MPN, but not in more posterior areas.
1.01.5.2.1.5.7 Glutamate. Microinjection of glutamate into
the MPOA of anesthetized male rats elicited erectile
responses (Giuliano et al., 1996) and also the UG reflex
without genital stimulation (Marson and McKenna,
1994b). Conversely, copulation was inhibited by microinjec-
tions of the NMDA antagonist MK-801 into the MPOA of
sexually naïve or experienced males (Dominguez et al.,
2007). Nearly all neurons in the MPOA that expressed Fos
after mating also contained the NR1 subunit of NMDA recep-
tors, and mating increased phosphorylation of NR1 in the
MPOA, thereby activating the receptor. In addition, blocking
NMDA receptors decreased mating-induced Fos-ir and
mating-induced phosphorylation of NMDA receptors. There-
fore, glutamate in the MPOA facilitates several measures of
sexual behavior, at least in part through NMDA receptors.
Male Sexual Behavior 29
1.01.5.2.1.5.8 Orx/Hcrt, OT, and Other Neurotransmitters. Orx/
hcrt is best known for its regulation of arousal and feeding.
However, microinjections of Orx/hcrt into the MPOA of male
rats also facilitated mating (Gulia et al., 2003). Microinjection
of OT into the MPOA also facilitated copulation, whereas
microinjection of an OT antagonist impaired copulation (Gil
et al., 2011). Furthermore, sexual experience increased both
OT receptor mRNA and protein levels in the MPOA (Gil
et al., 2013). Thus, a neuropeptide known for facilitating
parental behavior, mating, and social trust also acts in the
MPOA to facilitate mating in male rats. In addition, there is
evidence for facilitative effects of ACh (Hull et al., 1988a,b)
and PGE2 (Clemens and Gladue, 1977) in the MPOA
(reviewed in Hull et al., 2006).
1.01.5.2.1.6 Electrophysiological Recordings
Some neurons in the MPOA increased their firing rates only
before male rats began to mate, and others increased their firing
only during copulation (Shimura et al., 1994). MPOA neurons
in monkeys also responded differentially during lever pressing
to gain access to a female and during copulation; activity
decreased markedly after ejaculation (Oomura et al., 1988).
In MPOA slices from male quail, bath applications of DA
inhibited 52–80% of cells, but excited 10–25% (Cornil et al.,
2002). The inhibitory and excitatory effects were mediated by
a2 and a1 adrenoreceptors, respectively. Therefore, in the quail
MPOA DA affects neural activity via cross-talk with NE recep-
tors. In voltage-clamp recordings from dissociated neurons
from the MPOA of male rats, with presynaptic nerve endings
intact, 5-HT inhibited both GABAergic miniature inhibitory
postsynaptic currents (mIPSCs) and glutamatergic miniature
excitatory postsynaptic currents (mEPSCs) (Lee et al., 2008).
The inhibition of mIPSCs was mediated by 5-HT1A receptors,
whereas the inhibition of mEPSCs was mediated by 5-HT1B
receptors. The authors suggest that stimulation of adenylyl
cyclase, and the consequent activation of PKA, increase mIPSCs
and mEPSCs and counteract the effects of 5-HT. Therefore, even
though 5-HT did not increase in the MPOA at the time of ejac-
ulation (Lorrain et al., 1997; see Section 1.01.5.2.5.3), and an
SSRI microinjected into the MPOA did not significantly impair
behavior (Lorrain et al., 1997; see above), 5-HT may influence
sexual behavior by reducing the effects of GABAergic and gluta-
matergic transmission.
1.01.5.2.1.7 Chemical Changes Detected by Microdialysis or from
Tissue Punches
There is a close relationship between extracellular DA levels in
the MPOA and male rat sexual behavior. DA levels rose when
an inaccessible female was introduced and remained high or
increased further during copulation (Hull et al., 1995; Sato
et al., 1995). The recent presence of T was permissive for
both copulation and DA release, with two-thirds of 1-week
castrates able to copulate and to show a DA response to the
female, while no 2-week castrate could copulate or show
a DA response (Hull et al., 1995). There was both anatomical
and behavioral specificity for the DA response (Hull et al.,
1995, 1993). The fact that DA increased before copulation
began suggests that the increase was not caused by copulation,
but was likely associated with sexual motivation. Not only is
30 Male Sexual Behavior
the DA response to the female lost after castration, but basal
extracellular levels are also lower than in gonadally intact
males; however, intracellular levels were actually higher than
in intact males (Du et al., 1998). Therefore, synthesis and
storage of DA in the MPOA was at least as great in castrates
as in intact males; the deficiency in castrates was in their ability
to release their abundant stores. Restoration of T for 2, 5, or
10 days resulted in increasing sexual behavior and DA response
to the female (Putnam et al., 2001). No 2-day T-treated castrate
could copulate or show a DA response; eight of nine 5-day
T-treated castrates copulated and showed a DA response, with
five of the eight able to ejaculate. All of the 10-day T-treated
castrates copulated and all showed the DA response. There
were numerous correlations between DA levels and
copulatory measures. Therefore, both the loss of copulation
following castration and its restoration by T are closely
associated with the female-elicited DA response in the
MPOA. E and DHT were differentially effective in restoring
basal and female-stimulated DA release in long-term castrates
(Putnam et al., 2003). E2 resulted in high basal DA levels but
no increase in response to a female; E2-treated castrates
intromitted, but did not show an ejaculation pattern. DHT
was no more effective than the oil vehicle in restoring
copulation, basal, and female-stimulated DA levels. However,
the combination of E2 and DHT fully restored both basal and
female-stimulated DA levels, as well as copulation. In
contrast to the positive correlations between extracellular DA
and behavior, tissue (stored) DA levels were negatively
correlated with mating, suggesting that tissue DA levels were
high because it could not be released (Putnam et al., 2005).
A similar relation between apparent DA release and ability to
copulate was reported in male hamsters (Schulz et al., 2003).
Adult males had an increase in the major metabolite DOPAC
in punches from the MPOA in response to VSs from female
hamsters. The increase in DOPAC is taken as evidence that
DA was released, transported back into the axon terminal,
and metabolized. Juveniles showed no such increase and also
were unable to copulate.
DA release in the MPOA is regulated by NO. Both basal
(Lorrain and Hull, 1993) and copulation-induced (Lorrain
et al., 1996) extracellular DA levels were dependent on NOS
activity. As noted above, T and E positively regulate NOS-ir in
the MPOA. Thus, gonadal steroids may maintain DA levels in
the MPOA by upregulating NOS.
A major stimulus for the DA response to a female is input
from the MeA. Small radiofreqency lesions of the MeA impaired
copulation and abolished the MPOA DA response to the female
but did not affect basal DA levels in the MPOA (Dominguez
et al., 2001). Thus, as with E2 restoration of copulation in
castrates (Putnam et al., 2003), basal DA levels in the MPOA
were sufficient for suboptimal mating ability, but an additional
increase in response to a female is required for optimal copula-
tion. As noted above, microinjections of apomorphine into the
MPOA completely restored copulation in males with large exci-
totoxic lesions of the amygdala (Dominguez et al., 2001),
providing additional evidence that a major way in which the
MeA facilitates sexual behavior is by increasing extracellular
DA in the MPOA in response to a female. In agreement with
a facilitative effect of DA, chronic systemic administration of
an extract of Ginkgo biloba increased tissue DA levels in the
MPOA and arcuate nucleus, decreased serum PRL, and facili-
tated copulation (Yeh et al., 2008). Similarly, tissue levels of
DA in the MPOA and arcuate were higher in middle-aged rats
that could copulate to ejaculation, compared to middle-aged
males that could not copulate or that could mount and
intromit, but not ejaculate (Chen et al., 2007). Tissue levels of
NE were not different among the groups.
Chemosensory stimuli processed by the olfactory bulbs
provide the major signal relayed by the amygdala to the
MPOA of rodents. In male hamsters with sham bulbectomies,
or with unilateral bulbectomy contralateral to the MPOA micro-
dialysis probe, presentation of an estrous female increased DA
levels, and animals copulated to ejaculation (Triemstra et al.,
2005). Males with ipsilateral bulbectomy also copulated to ejac-
ulation, but there was no female-stimulated DA response. Bilat-
eral bulbectomy abolished both copulation and the DA
response to the female. Threfore, chemosensory stimuli are
essential for the MPOA DA response in male hamsters.
Glutamate is also released in the MPOA of male rats during
copulation (Dominguez et al., 2006a). Extracellular glutamate
increased
170% during copulation and
300% in the 2-min
sample during which the male ejaculated. Glutamate fell
dramatically after ejaculation, and the decrease was highly
correlated with the length of the PEI. Reverse dialysis of a gluta-
mate reuptake inhibitor increased levels to
280%, increased
the number of ejaculations, and decreased the latencies to ejac-
ulate and to resume copulation after the PEI. In a later study,
reverse dialysis of 5-HT into the MPOA decreased glutamate
levels, decreased the number of ejaculations, and increased
ejaculation latencies and PEIs (Dominguez and Hull, 2010).
The 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA)
was increased in the POA of male rats following ejaculation
(Fumero et al., 1994; Mas et al., 1995a), and 5-HT was
increased in POA tissue punches after ejaculation (Mas et al.,
1987). The authors suggested that increased 5-HT may have
contributed to the PEI. However, a more recent microdialysis
study that measured 5-HT itself found that 5-HT in the
MPOA and the more lateral POA remained constant during
copulation, ejaculation, and the PEI (Lorrain et al., 1997).
However, 5-HT did increase in the LHA in the sample during
which the male ejaculated. The 5-HIAA increase in the earlier
studies may have diffused from the LHA to the adjacent POA.
Thus, 5-HT in the POA may not contribute significantly to
the PEI. However, see below for a microdialysis study showing
that reverse dialysis of 5-HT into the MPOA decreases gluta-
mate release and number of ejaculations and increases ejacula-
tion latency and PEI (Dominguez and Hull, 2010). Two
resolutions to this apparent contradiction may be that 5-HT
levels during reverse dialysis are higher than during copulation
and the PEI or that a small increase in 5-HT during the PEI was
not detectable by microdialysis.
1.01.5.2.1.8 Activation of c-Fos or Other Measures of Neural
Activity
Copulation increases Fos expression in the MPOA of male
rats (Baum and Everitt, 1992; Robertson et al., 1991; Veening
and Coolen, 1998), gerbils (Heeb and Yahr, 1996), hamsters
(Fernandez-Fewell and Meredith, 1994; Kollack and
Newman, 1992; Kollack-Walker and Newman, 1995), and
mice (Halem et al., 1999). Sexual experience may enhance

future responsiveness to sexual stimuli; there were more Fos-
ir neurons in the MPN of sexually experienced male rats after
one ejaculation than in previously naïve males, even though
the naïve males required more intromissions to trigger
the ejaculation (Lumley and Hull, 1999). Similarly,
chemosensory cues in sexually experienced male hamsters
stimulated greater Fos expression than in sexually naïve
males (Fewell and Meredith, 2002). Fos expression was
also increased in the MPN and BST of Japanese quail by
both visual and physical contact with females (Iyilikci
et al., 2014).
Copulation-induced Fos-ir in the MPOA can be elicited by
input from either the MeA or the CTF (Baum and Everitt,
1992), and Fos-ir neurons in the MPOA project to the
PAG (Struthers, 2001) and to the BST (Maejima et al., 2015).
Sexual behavior was also associated with an increase in
2-deoxyglucose incorporation in the MPOA of birds (Dermon
et al., 1999) and increased cytochrome oxidase activity in lizards
(Sakata and Crews, 2004).
There is disagreement as to whether chemosensory cues are
sufficient to induce Fos expression in the MPOA. There have
been positive reports of such induction in rats (Bressler and
Baum, 1996; Kelliher et al., 1999), hamsters (Fiber et al.,
1993; Kollack-Walker and Newman, 1997), macaques
(Michael et al., 1999), gerbils (Heeb and Yahr, 1996), and
ArKO mice (Aste et al., 2003). It is not clear whether the
main or accessory olfactory system mediates the Fos responses
to chemosensory cues (Fewell and Meredith, 2002; Swann
et al., 2001). Similarly, fMRI indicated pheromone-induced
arousal of the MPOA in marmosets (Ferris et al., 2001), and
temperature fluctuations in the MPOA of rats occurred in
response to odor cues (Kiyatkin and Mitchum, 2003). Fos
was also elicited in the MPOA of male hamsters by electrical
stimulation of the VNO (Meredith and Fewell, 2001).
However, there have also been negative reports of Fos induc-
tion in rats (Baum and Everitt, 1992; Coolen et al., 1997b),
mice (Halem et al., 1999), hamsters (Fernandez-Fewell and
Meredith, 1994), and ferrets (Kelliher et al., 1998). Ejaculation
selectively activated the MPN of hamsters (Kollack-Walker
and Newman, 1997) and rats (Coolen et al., 1997b), and the
PdPN of gerbils (Heeb and Yahr, 1996).
The neurochemical identity of Fos-ir neurons has been
addressed. In gerbils half of the Fos-ir neurons in the medial
SDA and PdPN were GABAergic; almost one-fourth of the
neurons in the medial SDA were glutamatergic, but there
were no glutamatergic cells in the PdPN (Simmons and Yahr,
2003). In rats galanin-containing cells are selectively activated
by ejaculation (Bakker et al., 2002b). Ejaculation-induced
Fos-ir in the MPOA of male rats was decreased by administra-
tion of a D1 antagonist (Lumley and Hull, 1999), suggesting
that stimulation of D1 receptors mediated at least some of
the copulation-induced Fos-ir. Copulation increased Fos-ir in
AR-containing neurons in the MPOA of male rats (Greco,
1998a), but not in aromatase-containing neurons in male
quail (Foidart et al., 1999). Copulation also increased
expression of Fos in NOS-containing neurons in the MPOA
of male rats (Nutsch et al., 2014). As mentioned above, NO
is important for MPOA DA release and copulation. Previous
sexual experience also increased the coexpression of Fos and
D2-like receptors in the MPOA, and numbers of D2- and Fos-
Male Sexual Behavior 31
expressing neurons correlated with levels of sexual experience
and sexual activity (Nutsch et al., 2016).
Some B6D2F1 mice are able to copulate after castration
(McInnis et al., 2016). The ability to copulate without T is
passed from father to son and may be related to increased
production of amyloid precursor protein, tau, and synaptophy-
sin in the MPOA. The mechanisms by which these proteins may
affect mating behavior are not clear.
1.01.5.2.1.9 Effects of Intracerebral Grafts
Injection of fetal MPOA neurons into the MPOA of aging male
rats improved their ability to copulate between 21 and 45 days
after implantation; the benefits lasted until the end of the exper-
iment, 4.5 months later (Hung et al., 1997). Serum T was also
increased. Implants from other brain areas were ineffective.
1.01.5.2.1.10 Activation of Neurotransmitter Receptors
Endocytosis of m opioid receptors occurred within 30 min after
copulation and lasted for at least 6 h (Coolen et al., 2004b).
The opioid antagonist naloxone prevented receptor internaliza-
tion. Microinjection of a m agonist elicited similar internaliza-
tion, and mating elicited Fos-ir in m receptor-containing
neurons. However, naloxone did not prevent the mating-
induced Fos expression in m receptor-containing neurons, sug-
gesting that the mating-induced Fos response did not result
from stimulation of m receptors.
1.01.5.2.1.11 Summary of MPOA Functional Roles
The MPOA receives indirect sensory input from all senses and
has reciprocal connections with the sources of afferent input,
allowing hormone-concentrating neurons to bias the processing
of sexually relevant input. MPOA efferents to other hypotha-
lamic, midbrain, and brain stem nuclei regulate somatomotor
patterns and genital reflexes and contribute to sexual motivation.
1.01.5.2.2 Mesocorticolimbic DA Tract
1.01.5.2.2.1 Anatomy
The mesocorticolimbic circuit comprises DA cell bodies in
the VTA ascending to the NAc and the medial prefrontal
cortex (mPFC) and plays a central role in motivated behav-
iors (Alcaro et al., 2007). Electrical stimulation studies
have implicated this system in positive rewarding states
(Wise and Rompre, 1989) and appetitive-motivated behav-
iors (Berridge and Robinson, 1998). Sexual behavior is
a rewarding and reinforcing behavior that activates the mes-
ocorticolimbic circuit (Balfour et al., 2006). Hence, DA efflux
is increased in the NAc in response to the presence of an
estrous female behind a barrier, as well as during copulation
(Damsma et al., 1992; Fiorino et al., 1997; Pfaus et al., 1990;
Robinson et al., 2002). The VTA and the NAc are activated
during sexual arousal (Guevara et al., 2008). Recording of
male rat NAc neuronal activity during sexual behavior
showed that many NAc shell neurons exhibited firing rate
changes during sexual activity that were related to sexual
behavior performance, its reward-related processing, and
the sexual behavior inhibition that follows ejaculation. These
data suggest that the NAc shell is importantly involved in
encoding distinct aspects of male sexual behavior
(Matsumoto et al., 2012). In the VTA, sexual behavior acti-
vates both DA and non-DA neurons. VTA activation appears
32 Male Sexual Behavior

to be mediated by endogenous opioids released during
sexual activity, which inhibit VTA GABAergic interneurons,
thereby releasing DA cells from tonic inhibition (Balfour
et al., 2004). Actually, ejaculation induced m and d opioid
receptor internalization (an indicator of ligand-induced
receptor activation) in neurons of the VTA, and a correlation
between the amount of ejaculatory activity displayed and the
degree of in vivo m opioid receptor internalization could be
established (Garduno-Gutierrez et al., 2013c). The mPFC
receives DA inputs from the VTA and sends projections
back to this structure (Tzschentke, 2000). Projections from
the mPFC to the VTA are largely glutamatergic; thus, it is
possible that mPFC inputs contribute to excitation of VTA
neurons via glutamate release during copulation (Balfour
et al., 2006). Efferent projections from the mPFC also contact
other brain areas involved in sexual behavior and motiva-
tion, including the NAc shell and core, the MPOA, BST,
and SPF.
Functional neuroimaging studies in humans showed that
the NAc is likely to participate in the motivational component
of male sexual arousal and is activated during erection evoked
by visual erotic stimulation (Redoute et al., 2000), while gray
matter atrophy of the NAc was related to psychogenic erectile
dysfunction in men (Cera et al., 2012).
1.01.5.2.2.2 Effects of Lesions or Electrical Stimulation
Lesion of the mPFC disrupted male rat sexual behavior, partic-
ularly its initiation (Ågmo et al., 1995). VTA lesion increased
the duration of the PEI (Brackett et al., 1986), while NAc
lesions impaired both copulation and noncontact erections,
suggesting that the NAc plays an excitatory role in the regula-
tion of sexual arousal (Kippin et al., 2004). Electrical stimula-
tion of the VTA facilitated copulation (Markowski and Hull,
1995); however, the facilitative effect is restricted to its dorsal
portion, and stimulation of its ventral portion inhibited mating
(Rodríguez-Manzo and Pellicer, 2007). Electrical stimulation
of the NAc also facilitated sexual behavior display in sexually
experienced rats (Figure 4), but lacked an effect when the
same animals were sexually exhausted (Rodriguez-Manzo and
Pellicer, 2010).
1.01.5.2.2.3 Activation of c-Fos or Receptors
Fos expression is induced in the NAc and VTA of male rats in
response to mating (Robertson et al., 1991; Balfour et al.,
2004). Female odors and conditioned nonsexual odors paired
with mating-stimulated Fos expression in NAc (Kippin et al.,
2003). Moreover, sexual experience enhanced estrous female-
induced NAc Fos-ir (Lopez and Ettenberg, 2002a). mPFC is
also activated during sexual behavior and sends projections to

Figure 4 Percentage of sexually experienced male rats achieving one to four successive ejaculations (EJAC) within a 30-min period when electri-
cally stimulated with high- (100 Hz) or low-frequency (20 Hz) in the nucleus accumbens (dashed columns) or caudate-putamen nucleus (double
dashed columns). Fisher exact probability test, þ, p ¼ 0.054; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Ventral striatum stimulation facilitated, while
dorsal striatum stimulation inhibited, copulation, regardless of the stimulation frequency used. Reproduced from Rodriguez-Manzo, G., Pellicer, F.,
2010. Electrical stimulation of dorsal and ventral striatum differentially alters the copulatory behavior of male rats. Behav. Neurosci. 124, 686–694,
adapted with permission from APA.

sex-activated neurons in the VTA, as determined by Fos-ir (Balfour
et al., 2006). Mating increased Fos-ir in the dopaminergic neurons
of the VTA that were apposed to orexin fibers, suggesting that
orexin promotes sexual behavior by activating the mesocortico-
limbic circuit (Hull, 2011). Sexual behavior was shown to cause
DFosB (a stable transcription factor of the Fos family related to
enhanced motivation and reward induced by drugs of abuse)
accumulation in the NAc. Overexpression of DFosB in this brain
region facilitated sexual behavior in naïve male rats which
behaved more like experienced animals, but had no effect in
already experienced males (Wallace et al., 2008) DFosB accumu-
lation was also found to occur in the mPFC and VTA as a result of
sexual experience (Pitchers et al., 2010). Besides, sexual activity
causes long-term changes in glutamate NMDA and AMPA
receptor expression and function in the NAc (Pitchers et al.,
2012).
1.01.5.2.2.4 Microinjections of Drugs
Microinjection of D1- and D2-like receptor antagonists into the
NAc decreased sexual motivation (Pfaus and Phillips, 1991).
Apomorphine, a nonselective DA agonist, microinjected into
the VTA, delayed the onset of copulation and slowed its rate,
presumably by stimulating autoreceptors and thereby decreasing
dopaminergic activity (Hull et al., 1990). Conversely, the nonse-
lective DA antagonist cis-flupenthixol reduced IL for those rats
that copulated, presumably by blocking autoreceptors, but also
decreased the proportion of copulating rats, perhaps due to depo-
larization block. These effects appeared to be due to motoric slow-
ing, not a decrease in specifically sexual motivation or genital
reflexes (Hull et al., 1991). D1 agonist injection into the NAc
augmented the percentage of sexually naïve male rats copulating
during their first sexual encounter (Bialy et al., 2010). Infusion of
5-HT into the NAc impairs copulation in rats, while a facilitative
effect was obtained after 8-OH-DPAT (Fernández-Guasti et al.,
1992). Intra-VTA microinjection of morphine and dynorphin
facilitated male sexual behavior and increased DA transmission
in the NAc (Mitchell and Stewart, 1990), both effects probably
resulting from m opioid receptor-mediated release from
GABAergic tonic inhibition (Balfour et al., 2004). Conversely,
the opioid receptor antagonist naltrexone microinjected into
the VTA inhibited ejaculation in sexually experienced rats, but
facilitated sexual behavior expression in sexually satiated males.
This result suggests that endogenous opioids within the VTA
take part in the maintenance of the sexual inhibition that follows
sexual exhaustion, evidencing the participation of the mesocorti-
colimbic system in this phenomenon. The reversal of sexual
exhaustion was due to blockade of m opioid receptors, since
intra-VTA infusion of the d opioid receptor antagonist naltrindole
was ineffective (Garduno-Gutierrez et al., 2013b).
It was shown that direct administration of phosphodies-
terase type 5 inhibitors in the caudal VTA facilitates noncontact
penile erections and increases DA levels in the NAc, suggesting
that the increase in sexual arousal induced by these drugs
involves the mesolimbic circuit (Sanna et al., 2009). Similarly,
OT injected into the VTA and other brain regions induces
penile erection (Melis and Argiolas, 2011). In particular, penile
erection induced by OT infusion into the hippocampus is
mediated by an increase in glutamate levels in the VTA, which
in turn increase DA levels within the mesocorticolimbic system
(NAc and mPFC) (Succu et al., 2011). Intra-VTA injection of
Male Sexual Behavior 33
orexin-A and orexin-B was reported to increase DA release in
the NAc (Narita et al., 2006) and, at specific doses, to augment
dopaminergic cell firing (Muschamp et al., 2007). Using rat
midbrain slices, it was demonstrated that orexin-B enhances
glutamatergic transmission in the VTA (Borgland et al., 2008).
1.01.5.2.3 Nigrostriatal DA Tract
The nigrostriatal pathway originates in the zona compacta of
the substantia nigra and sends its projections to the caudate
and putamen nuclei of the striatum. The nigrostriatal DA
system has been implicated in the control of procedural
aspects of movement and motivated behaviors, as it reaches
dorsal areas of the basal ganglia (Alcaro et al., 2007). Mating
induces an increase in DA concentrations both in ventral and
dorsal striatum, but DA is released in the dorsal striatum only
after the male begins to copulate, suggesting that this release
reflects motor activation, rather than motivational aspects of
copulation (Damsma et al., 1992). Electrical stimulation of
the dorsal striatum (caudate putamen) had an inhibitory
influence on copulation of sexually competent male rats, con-
sisting of slowing its display and decreasing the ejaculatory
frequency, in contrast to ventral striatum stimulation, which
accelerated copulation (Rodriguez-Manzo and Pellicer,
2010; Figure 4). Bilateral lesions of the substantia nigra also
slowed the rate of copulation and decreased ejaculatory
capacity (Brackett et al., 1986). Copulation did not induce
Fos-ir in the dorsal striatum, in contrast to mesolimbic areas
such as the NAc, VTA, and mPFC (Coolen et al., 1996);
however, DFosB accumulated in the caudate putamen, as
well as in the NAc and VTA, as a result of sexual experience
(Pitchers et al., 2010). Apomorphine injected into the stria-
tum did not affect copulation in male rats (Hull et al.,
1986), and bilateral haloperidol infusions only increased EF
(Pfaus and Phillips, 1991).
It is important to call attention to the fact that anatomical
and neurochemical studies suggest that nigrostriatal and meso-
limbic dopaminergic systems are involved in the organization
of many if not all motivated behaviors including sexual
behavior (Alcaro et al., 2007). No clear boundaries can be
established between these two systems, since VTA and substan-
tia nigra dopaminergic neurons partly overlap in their projec-
tion fields (Nauta et al., 1978), while copulation-related cues
and mating simultaneously activate nigrostriatal and mesolim-
bic circuits (Robinson et al., 2002). Thus, motor, motivational,
and reward functions of these two dopaminergic systems func-
tion in a unified way (Nicola, 2007; Robbins and Everitt, 2007)
and might in the future be integrated in a single dopaminergic
circuit.
1.01.5.2.4 Paraventricular Nucleus of the Hypothalamus
1.01.5.2.4.1 Anatomy
The PVN is an important integrative site for endocrine and
autonomic functions. It comprises a parvocellular division,
which projects to several brain areas and the spinal cord, and
a magnocellular division, which releases OT and vasopressin
from the posterior pituitary (reviewed in Swanson and
Sawchenko, 1980). Axons that project to the spinal cord release
OT, vasopressin, somatostatin, DA, and other, undetermined
transmitters. Direct projections from the PVN to the motoneu-
rons of the dorsomedial nucleus of the lumbosacral cord,
34 Male Sexual Behavior
which innervate the levator ani and BS muscles mediating erec-
tion and ejaculation, have also been identified (Dobberfuhl
et al., 2014). Periventricular DA neurons and brain stem NE
and 5-HT nuclei provide input to the parvocellular PVN. The
PVN integrates afferent input of multiple sources as well as
efferent autonomic output, the latter with the participation of
both glutamate and GABA interneurons that play a role in
controlling the excitability of magnocellular and parvocellular
output neurons (Ferguson et al., 2008).
OT is present in neurons whose cell bodies are located in the
PVN and project to the neurohypophysis as well as in neurons
that project from the PVN to extrahypothalamic brain regions.
The PVN is the most sensitive brain area to OT -induced penile
erection in male rats. Actually, all other brain areas in which
local OT injection was reported to induce penile erection
receive oxytocinergic projections from the PVN. DA, glutamate,
and NO can facilitate penile erection and copulation by stimu-
lating OTergic neurons of the PVN, while GABA, opioid
peptides, and endocannabinoids inhibit those neurons
(Argiolas and Melis, 2013).
1.01.5.2.4.2 Effects of Electrolytic or Cell Body Lesions
Excitotoxic lesions of the parvocellular PVN decreased noncon-
tact erections without affecting copulation (Liu et al., 1997a).
Similar lesions decreased the volume of semen ejaculated
and decreased the OT-ir innervation of the lumbosacral
spinal cord, but again did not affect mating (Ackerman et al.,
1997). Larger lesions of both the parvo- and magnocellular
divisions inhibited both reflexive and noncontact erections
and led to some impairment of mating (Liu et al., 1997a).
However, electrolytic lesions actually speeded the first
reflexive erection (Monaghan et al., 1993). Thus, the PVN
contributes to both noncontact and reflexive erections and
seminal emission, but does not consistently affect copulation
or reflexive erections. Lateral parvocellular PVN lesions
destroyed neurophysin-ir axons to the sexually dimorphic
SNB (Wagner and Clemens, 1993). Neurophysin is a marker
for OT and vasopressin; thus, the PVN is the source of OT
projections to the SNB that promote erection, seminal
emission, and ejaculation.
1.01.5.2.4.3 Effects of Direct Applications of Drugs Affecting
Specific Transmitters
1.01.5.2.4.3.1 DA and OT. Microinjections of mixed and selec-
tive D2-like agonists or of OT elicited drug-induced erections
and increased reflexive erections and seminal emissions
(reviewed in Argiolas and Melis, 2005). The mixed DA agonist
apomorphine also increased intracavernous pressure in anes-
thetized rats (Allard et al., 2002). The D2-like receptor has
more recently been hypothesized to be the D4 receptor, which
apparently opens N-type calcium channels; the calcium then
activates NOS in the OT neurons (Melis et al., 2005, and
references therein). The noncontact erections elicited by DA
agonists were inhibited by an OT antagonist administered
icv, but not into the PVN, suggesting that DA excites neurons
that release OT elsewhere (Melis et al., 1999a). OT released
in the PVN itself apparently acts via OT receptors on OT
neurons by increasing calcium influx, thereby stimulating
NOS, which acts intracellularly to stimulate OT neurons
(reviewed in Argiolas and Melis, 2005). Penile erection is
stimulated by direct infusion of OT into the parvocellular
division of the PVN (Kita et al., 2006). D2-like receptors are
coexpressed by OT neurons of the PVN, and it has been
suggested that the OT neurons projecting to the spinal cord
respond to DA to induce penile erection (Baskerville et al.,
2009). Besides, PVN apomorphine acts via OT projections to
the VTA to increase DA release in the NAc and elicit erections
(Melis et al., 2007).
1.01.5.2.4.3.2 Nitric Oxide. Microinjection of NO donors or
a high dose of the NO precursor L-arginine into the PVN eli-
cited erections, whereas the NOS inhibitor L-NAME decreased
noncontact erections and impaired mating (Melis et al.,
1998). Reverse dialysis of another NOS inhibitor, L-NMMA,
decreased reflexive erections, and L-arginine increased erec-
tions; however, neither drug affected copulation (Sato et al.,
1999). Those authors reported that similar administration
of L-NMMA into the MPOA did impair copulation. Thus,
NO-related drugs have consistently affected noncontact and
reflexive erections, but have had inconsistent effects on
mating.
1.01.5.2.4.3.3 Amino Acids. Microinjections of the glutamate
agonist NMDA elicited erections, an effect blocked by an
NMDA antagonist and a NOS inhibitor in the PVN (Melis
et al., 1997) and also by an OT antagonist injected icv, but
not into the PVN (Melis et al., 2000b). The NMDA antagonist
impaired both noncontact erections and copulation when
administered alone (Melis et al., 2005). NMDA in the PVN
also increased intracavernosal pressure in awake and anesthe-
tized male rats (Chen and Chang, 2003; Zahran et al., 2000).
Conversely, a GABAA, but not a GABAB, agonist in the PVN
inhibited erections elicited by apomorphine, OT, or NMDA
(Melis et al., 2000a).
1.01.5.2.4.3.4 Other Transmitters. Morphine microinjections
before introduction of an estrous female prevented both
noncontact erections and the rise in NO that occurred in
control rats (see below) (Melis et al., 1999b). Hexarelin analog
peptides, which were originally known for their ability to
induce growth hormone release, activate specific receptors on
OT neurons in the PVN to admit calcium and thereby stimulate
NOS in the OT neurons and elicit erections (reviewed in
Argiolas and Melis, 2005). A cannabinoid CB1 antagonist,
microinjected into the PVN, elicited erections, an effect reduced
by CB1 agonists, an NMDA antagonist, and a NOS inhibitor in
the PVN, and by an icv OT antagonist (Melis et al., 2004b). The
CB1 receptors in the PVN are mostly on GABAergic terminals,
which in turn impinge on both OT and glutamatergic neurons
(Castelli et al., 2007). The authors note that one would expect
inhibition of the inhibitory CB1 receptors to increase GABA
release; however, they suggest that stimulation of CB1 receptors
may inhibit GABA reuptake in the PVN; therefore, blocking CB1
receptors would increase GABA reuptake and disinhibit gluta-
matergic and OT neurons. Thus, the mechanism of CB1 antag-
onists’ effects is not clear. Pro-VGF-derived peptides are
cleavage products of VGF, the product of the vgf gene, which
is selectively induced by nerve growth factor (see references
in Argiolas and Melis, 2005). They may activate OT neurons
in the PVN and thereby facilitate erections; their effects are

inhibited by both NOS and OT antagonists (reviewed in
Argiolas and Melis, 2005).
1.01.5.2.4.4 Chemical Changes Detected by Microdialysis
DA levels increased during noncontact erections and, to
a greater extent, copulation (Melis et al., 2003, 2004b). Both
noncontact erections and copulation were accompanied by
an increase in NO, inferred from increases in NO2 and NO3
(Melis et al., 1998). In the same study, L-NAME in the PVN
inhibited both the increase in NO2 and noncontact erections;
however, PVN injections of hemoglobin, an NO scavenger,
blocked the increase in NO2, but not noncontact erections, sug-
gesting that NO works only intracellularly in the PVN. Dopa-
mine agonists of the D2-like receptor family injected into the
PVN increase NO production, apparently with the D2 receptor
subtype playing a major role in this effect (Sanna et al., 2012).
Morphine microinjections into the PVN inhibited the
copulation-induced increase in NO and copulation (Melis
et al., 1999b). Reverse dialysis of L-arginine increased both
reflexive erections and the NO increase, and L-NMMA inhibited
both (Sato et al., 1999). Apomorphine or a D2 agonist, but not
a D1 agonist, increased NO production and erections (Melis
et al., 1996), as did NMDA (Melis et al., 1997). Omega cono-
toxin, an inhibitor of N-type calcium channels, prevented the
elicitation of erections and NO production by apomorphine
and OT; NO donors overcame the need for calcium channel
activation (Succu et al., 1998). Microinjection of a CB1
antagonist increased extracellular glutamate (Succu et al.,
2006b) and NO production (Melis et al., 2006), whereas
microinjection of morphine decreased both erections elicited
by a CB1 antagonist and the increases in glutamate and NO
production (Succu et al., 2006a).
1.01.5.2.4.5 Immunocytochemistry
30% of lateral parvocellular neurons in the PVN contain ER-ir,
and almost half of them project to the lumbar spinal cord
(Wagner et al., 1993). Therefore, some hormonal effects on
the SNB may be indirect, via steroid-sensitive afferents.
Ejaculation induced greater Fos expression in the parvocel-
lular and magnocellular PVN than did intromission, especially
in the caudal lateral parvocellular PVN, with one-third of the
Fos-ir neurons containing OT (Witt and Insel, 1994). Neurons
in that area project to the brain stem and lumbar spinal cord.
Furthermore, female odor elicited Fos-ir in OT-containing
neurons in the parvocellular PVN of sexually experienced, but
not naïve male rats; direct exposure to an anesthetized
estrous female elicited Fos-ir in both groups, compared to
nonexposed controls (Nishitani et al., 2004). However, in
gerbils neither mating nor exposure to a previous mating
arena elicited Fos-ir in the PVN (Heeb and Yahr, 1996). The
PVN has been implicated in the deterioration of male sexual
behavior produced by the alarm pheromone, since the
copulatory decline is accompanied by an increase in Fos
expression in the parvocellular division and a decrease in the
magnocellular division of the PVN. Naloxone pretreatment
blocked sexual behavior decline and magnocellular PVN Fos
expression changes. The authors suggest that pheromone
detection by the olfactory system activates the parvocellular
PVN, which in turn activates opioidergic neurons that
suppress magnocellular PVN activation, thus contributing to
Male Sexual Behavior 35
sexual behavior deterioration (Kobayashi et al., 2015).
Sexually competent male rats had more OT mRNA and less
mRNA for proenkephalin and prodynorphin in the PVN than
did impotent males (Arletti et al., 1997). Furthermore, NOS
and OT were colocalized (Yamada et al., 1996).
In addition to projections to the hippocampus, lumbosacral
spinal cord, and other brain areas, there is a direct projection to
the nPGi, where terminals form close appositions to seroto-
nergic neurons that project to the lumbosacral cord and inhibit
genital reflexes (Bancila et al., 2002).
1.01.5.2.5 Lateral Hypothalamus
1.01.5.2.5.1 Anatomy
The LH is reciprocally connected with brain regions that
process emotional information. It may control the autonomic
nervous system via connections to the nucleus of the solitary
tract and parabrachial nucleus, endocrine secretion via those
to the PVN, and emotional responses via connections to PAG
and midbrain extrapyramidal area. Activation of this system
leads to both motor arousal and emotional/cognitive arousal
(Ikemoto, 2007).
1.01.5.2.5.2 Effects of Electrical Stimulation and Lesions
LH electrical stimulation has rewarding effects and has induced
stimulation-bound copulation in male rats (Huston, 1971),
while males with lesions in the anterior LH intromitted repeat-
edly, but few copulated to ejaculation (Kippin et al., 2004).
During exposure to an inaccessible receptive female, anterior
LH-lesioned males had increased noncontact erections. Thus,
the anterior LH may inhibit sexual arousal but facilitate
ejaculation. On the other hand, orexin cell-specific lesion in
the LH did not affect sexual behavior expression, but
prevented formation of a CPP for sexual behavior, suggesting
that orexin is not essential for male sexual behavior display,
but is important for copulation-related reward processing
(Di Sebastiano et al., 2011).
1.01.5.2.5.3 Microinjections, Microdialysis, and Fos-ir
Microinjection of an SSRI into the anterior LH delayed the onset
of copulation and increased the EL (Lorrain et al., 1997), while
reverse dialysis of 5-HT decreased basal and female-elicited DA
release in the NAc (Lorrain et al., 1999). In addition, anterior
LH 5-HT levels were increased at the time of ejaculation, coin-
cident with the copulatory refractory period (Lorrain et al.,
1997). The LH contains orx/hcrt neurons, which appear to
play a critical role in arousal and reward (Harris and Aston-
Jones, 2006) and are activated following copulation
(Muschamp et al., 2007). It has been proposed that 5-HT in
the anterior LH might inhibit male sexual behavior by inhibit-
ing orx/hcrt neurons, which would eliminate their facilitative
influence on VTA DA cell firing (Muschamp et al., 2007).
Measurement of brain perfusion in healthy men during
sexual activity with the aid of fMRI showed increased activity-
related blood flow in the LH that correlated with penile erec-
tion (Georgiadis et al., 2010).
1.01.5.2.6 Ventromedial Hypothalamus
1.01.5.2.6.1 Anatomy
Although the VMH is primarily known for its role in female
receptive behavior, it may also influence male sexual behavior.
36 Male Sexual Behavior
It has numerous ERs and ARs (Simerly et al., 1990; Wood et al.,
1992) and receives both genitosensory (Coolen et al., 2003)
and chemosensory (Canteras et al., 1995; Coolen and Wood,
1998; Gomez and Newman, 1992) information. Copulation
to satiety reduces AR density in the VMH of male rats and
mandarin voles (Fernández-Guasti et al., 2003; He et al., 2013).
1.01.5.2.6.2 Effects of Lesions and Hormonal Manipulations
Electrolytic microlesions in the dorsomedial VMH impaired the
ability of T implants to restore USVs and scent marking, but
had relatively little effect on mating (Harding and McGinnis,
2005). T implants in VMH failed to restore copulation or
USVs in rats (Harding and McGinnis, 2003) or mice (Nyby
et al., 1992), but did restore partner preference in rats (Harding
and McGinnis, 2003) and increased urine marking in mice
(Nyby et al., 1992). Hydroxyflutamide (AR antagonist) in the
VMH of castrated, T-replaced rats inhibited both sexual
motivation (Harding and McGinnis, 2004) and mating
(Harding and McGinnis, 2004; McGinnis et al., 1996). Thus,
ARs in the VMH may contribute to male sexual behavior.
1.01.5.2.6.3 Expression of c-Fos
Copulation induced Fos-ir in the VMH of both rats (Coolen
et al., 1996) and gerbils (Heeb and Yahr, 1996). Chemosensory
signals induced Fos-ir in gerbils (Heeb and Yahr, 1996), but
not in rats (Bressler and Baum, 1996). Pelvic nerve transection
did not affect ejaculation-induced Fos-ir in rats, suggesting that
other sensory inputs, such as the pudendal nerve or chemosen-
sory input, contribute to mating-induced Fos-ir (Wersinger
et al., 1993). Copulation did not increase Fos-ir in the VMH
of musk shrews (Gill et al., 1998), hamsters (Kollack-Walker
and Newman, 1997), mice (Halem et al., 1999), or ferrets
(Kelliher et al., 1998; Lambert et al., 1992) and actually
decreased Fos-ir in macaques (Michael et al., 1999). The
ventrolateral portion of the VMH contains neuronal subpopu-
lations that are activated at the initiation of male–female
encounters in mice and become progressively inhibited as the
encounter proceeded to active copulation, intermingled with
neurons activated by intermale aggression (Lin et al., 2011).
1.01.5.3 Major Motor Outputs
1.01.5.3.1 Ventral Premammillary Nucleus
The MeA, the BST, and the MPN are the major sources of inputs
to the ventral premammillary nucleus (PMv), while the ventral
subiculum, the arcuate nucleus, and the ventrolateral subdivi-
sion of the VMH provide moderate-to-high-density afferents
to this nucleus (Cavalcante et al., 2014). The PMv contains
ARs in rats (Canteras et al., 1992; Simerly et al., 1990; Yokosuka
and Hayashi, 1996; Yokosuka et al., 1997) and hamsters
(Wood and Newman, 1995a). Mating increased Fos-ir in
AR-containing neurons in rats (Gréco et al., 1998b). Fos-ir
was elicited in the PMv of male mice by female-soiled
bedding (Yokosuka et al., 1999). The PMv contains
numerous neurons expressing receptors for leptin (LepRb),
the adipocyte-derived hormone signaling energy status and
contributing thereby to the metabolic control of
reproduction. LepRb neurons in the PMv of male mice are
activated by female-soiled bedding odors and directly
innervate hypothalamic GnRH neurons, suggesting that they
represent a unique site for the integration of sexual odorant
cues with leptin-mediated signals of energy balance to
regulate reproduction (Leshan et al., 2009).
1.01.5.3.2 Midbrain Periaqueductal Gray
The ventrolateral portion of the PAG (vlPAG) contains neurons
that receive input from the MPOA (Murphy and Hoffman,
2001) and project to the nPGi. These neurons also receive input
from the lumbosacral spinal cord (Normandin and Murphy,
2008). Sexual behavior induces extensive FOS expression in
the PAG; however, very little FOS is observed in the PAG output
neurons to the nPGi, suggesting that these neurons may be
inhibited during mating. Since the nPGi exerts a descending
inhibitory influence onto the spinal cord, this pathway must
be disinhibited to allow genital reflexes to occur. It has been
speculated that the PAG output to the nPGi might be the source
of disinhibition to the spinal cord during mating (Normandin
and Murphy, 2008). Neurons of different regions of the PAG
express 5-HT, including those projecting from the vlPAG to
the nPGi; this serotonergic input might be relevant for the
modulation of the tonic inhibition of spinal cord sexual
reflexes from the nPGi (Bago et al., 2002). Supporting this
idea, serotonergic lesion of the vlPAG decreases ejaculation
latency and increases the mean number of ejaculations
exhibited by male rats (Normandin and Murphy, 2011). Elec-
trolytic lesions of the PAG, including the vlPAG, blocked elici-
tation of the UG reflex by MPOA electrical stimulation
(Marson, 2004), but unilateral lesions of vlPAG and contralat-
eral SDA of gerbils did not affect copulation (Finn and Yahr,
1994).
There are numerous ERs and ARs in the caudal two-thirds of
the PAG (Murphy et al., 1999b), with afferents from the MPOA
in close apposition to ER- and AR-ir neurons, some of which
project to nPGi (Murphy and Hoffman, 2001). AR and ERa
distribution within the caudal PAG of male rats was dense,
with the majority of these receptors localized in the lateral/
ventrolateral PAG (Loyd and Murphy, 2008). Thus,
hormones can modulate transmission from the MPOA to the
nPGi, via the PAG. The PAG of monkeys projects to
a premotor area of the medulla, the nucleus retroambiguous
(Vanderhorst et al., 2000).
1.01.5.3.3 Nucleus Paragigantocellularis of the Medulla
In rats, the nPGi of the rostroventrolateral medulla is consid-
ered the site of origin of the descending inhibitory influence
to the spinal cord that controls genital reflexes (Marson and
McKenna, 1990). The nPGi receives direct projections from
the PVN, MPOA, and PAG and indirect projections from the
MPOA through a relay in the PAG. Direct efferents from the
MPOA are activated during mating, while indirect projections
are not (Normandin and Murphy, 2008). The nPGi projects
directly to pudendal motoneurons in the lumbosacral spinal
cord involved in sexual refelexes (Hermann et al., 2003;
Marson et al., 1992) (see Section 1.01.6.2).
1.01.5.3.3.1 Effects of Lesions
Spinal nuclei that control genital reflexes are under tonic
inhibitory control from the nPGi; spinal transection releases
fictive ejaculation (Carro-Juárez and Rodríguez-Manzo,
2008) and increases the number or intensity of erections

(reviewed in Hull et al., 2006). Bilateral nPGi lesions
increased the proportion of sexually naïve male rats that
ejaculated on their first exposure to an estrous female and
reduced the stimulation and time needed by sexually experi-
enced male rats to achieve ejaculation. This lesion also
increased the latency and number of ejaculations displayed
before reaching sexual exhaustion (Yells et al., 1992). Similar
lesions decreased the latency and increased the numbers of
reflexive erections (Holmes et al., 2002; Marson et al., 1992)
and allowed the UG reflex to be elicited without spinal tran-
section (Marson and McKenna, 1990).
1.01.5.3.3.2 Effects of Electrical Stimulation
Electrical stimulation of the nPGi elicited field potentials in the
lumbosacral spinal cord near the SNB (Tanaka and Arnold,
1993). nPGi stimulation specifically activated sympathetic
fibers in the pudendal nerve (Johnson and Hubscher, 2000),
and activation of pudendal neurons by electrical stimulation
of the dorsal nerve of the penis was inhibited by stimulation
of nPGi (Johnson and Hubscher, 1998).
1.01.5.3.3.3 Immunocytochemistry
Most nPGi neurons that project to the lumbosacral spinal cord
contain 5-HT (Marson and McKenna, 1992). The main 5-HT
source to the nPGi comes from the vlPAG neurons that project
directly to the nPGi (Normandin and Murphy, 2011). The
inhibitory effects of systemic SSRIs on ejaculation are reversed
(Clement et al., 2007b) or prevented (Yells et al., 1994) by
nPGi lesions. Serotonergic receptors of the 5-HT1A (Thor
et al., 1990), 5-HT1C (Hoffman and Mezey, 1989), 5-HT2A, 5-
HT2C, and 5-HT3 (Fonseca et al., 2001) subtypes are expressed
in this brain structure. Interestingly, the inhibitory effects of
systemic SSRIs on ejaculation are reversed or prevented by
nPGi lesions, leading to the suggestion that SSRIs might inhibit
male rats’ genital reflexes, not only at the spinal-cord level
(Marson and McKenna, 1992), but this could also involve
the nPGi via vlPAG 5-HT input (Normandin and Murphy,
2011).
1.01.5.3.4 Other Brain Areas
Several additional brain areas have been implicated in the regu-
lation of male sexual behavior, although there is little informa-
tion about their specific contributions. These include the lateral
septum (Kondo et al., 1993), hippocampus (Chen et al., 1992),
caudal zona incerta (Edwards and Isaacs, 1991; Maillard and
Edwards, 1991), and cortical regions in men: inferior temporal
cortex, right insula, right inferior frontal cortex, and left anterior
cingulate cortex (Stoleru et al., 1999).
1.01.5.3.5 Spinal Cord
The spinal cord contains the autonomic and somatic nuclei
controlling erection and ejaculation. The sympathetic and
parasympathetic tones involved in the control of these
reflexes are under the influence of sensory stimuli from the
genitalia and are integrated at the spinal-cord level, where
supraspinal information also converges. The spinal cord
contains thoracolumbar sympathetic, sacral parasympathetic,
and sacral pudendal motoneurons anatomically linked with
the penis. Erection is likely to occur when the spinal cord
reduces the activity of the thoracolumbar sympathetic
Male Sexual Behavior 37
antierectile pathway with a concomitant increase in the
activity of the proerectile parasympathetic sacral and
pudendal pathways (for review see Giuliano and Rampin,
2004). The neural commands controlling ejaculation are
also organized at the spinal level, and a central pattern gener-
ator, located at the lumbosacral-cord level, is involved in the
relay and integration of the genital sensory and motor signals
related to ejaculation (Carro-Juárez and Rodríguez-Manzo,
2008; Carro-Juárez et al., 2003). A group of lumbar spinotha-
lamic neurons, located in lamina VII and X of L3 and L4
segments in this portion of the cord, which express galanin,
cholecystokinin, and neurokinin receptors, form part of this
ejaculation generator (Truitt and Coolen, 2002). Selective
lesion of these neurons disrupted bulbocavernosous muscle
bursting characteristic of ejaculation in anesthetized, spinal-
ized male rats, but did not affect emission or erectile function
in spinally intact rats (Staudt et al., 2012).
A system of gastrin-releasing peptide (GRP) neurons in the
upper lumbar spinal cord (L3-L4) that innervates lower lumbar
(L5-L6) and sacral S1 regions controlling erection and ejacula-
tion has been described in male rats. This system is the first
neural pathway known to restore ejaculation in castrated
male rats (Sakamoto et al., 2008). It was recently demonstrated
that the perinatal androgen surge is essential for the develop-
ment of this GRP system, which is sexually dimorphic (Oti
et al., 2016).
In humans, innervation of anatomical structures involved
in ejaculation is organized similarly to rats, but the spinal
cord segments harboring the sympathetic, parasympathetic,
and somatic centers are slightly different due to differences in
metamerization between species. Thus, the parasympathetic
centers that innervate the prostate and seminal vesicles that
secrete seminal fluid are located in the L6-S1 segments of rats
but in the S2-S4 segments of men; the sympathetic centers
innervating smooth muscles of the tract, including epydidimus,
vas deferens, seminal vesicles, prostate, prostatic urethra, and
the bladder neck, are located in T13-L2 rat segments and
between T12 and L2 in humans, while the location of the
somatic centers that innervate the pelvic striated muscles is
between L6 and S1 in rats, and in the ventral horn of S2-S4
segments in humans (Onuf’s nucleus). Studies about ejacula-
tion in spinal cord-injured men support the existence of an
SGE in humans that appears to be located at L3-L5 spinal
segments (Chehensse et al., 2013).
Spinal cord transection releases sexual reflexes, indicating
that supraspinal centers exert a descending inhibitory influence
(McKenna et al., 1991), probably mediated by 5-HT (Marson
and McKenna, 1994a). Intrathecal administration of 5-HT
abolished the UG reflex (Marson and McKenna, 1992), and
chronic treatment with the SSRI fluoxetine diminished the
response capacity of the ejaculation generator (Hueletl-Soto
et al., 2012); however 5-HT1A (Carro-Juárez and Rodríguez-
Manzo, 2001; Carro-Juárez et al., 2003) and 5-HT2C receptor
stimulation (Stafford et al., 2006b) facilitated the UG reflex,
suggesting that 5-HT exerts a dual influence on male sexual
behavior at the spinal level.
Cerebrospinal fluid levels of GABA, glutamate, and aspar-
tate markedly increased after ejaculation, with the increase in
GABA concentrations almost fivefold that of excitatory amino
acids (Qureshi and Södersten, 1986). Lumbosacral injection
38 Male Sexual Behavior
of the GABAB agonist baclofen inhibited ex copula reflexive
penile erection (Bitran et al., 1989a), while that of a competitive
NMDA or AMPA-kainate receptor antagonist depressed both
dorsal penile nerve (DPN)-stimulated erection in
anesthetized rats and reflexive erection in conscious animals
(Rampin et al., 2004). Also, NMDA receptor-mediated
glutamatergic mechanisms are involved in the excitatory
modulation of the SGE (Staudt et al., 2011). Thus, GABA
may inhibit, and glutamate may facilitate, sexual reflexes at
the spinal level. The DA agonist apomorphine, injected
intrathecally at the lumbosacral level of conscious rats,
impaired reflexive erection, but evoked erection in normal
and spinal animals (Andersson, 2001) and induced
ejaculation-like responses in anesthetized rats (Stafford and
Coote, 2006). However, chronic treatment with bupropion,
a dual reuptake inhibitor increasing DA and NA availability,
disrupted the functioning of the SGE (Hueletl-Soto et al.,
2014).
Reflexive erections are inhibited by both a1- and a2-adreno-
ceptor agonists. a2-antagonists have dose-dependent effects,
with low doses facilitating, and higher doses inhibiting,
reflexive erections in rats (reviewed in Meisel and Sachs,
1994) and seminal emission in dogs (Yonezawa et al., 1991).
The spinal NE system facilitates ejaculation, since increasing
NE tone by systemic yohimbine and stimulation of a1- or a2-
adrenoceptors (Carro-Juárez and Rodríguez-Manzo, 2003,
2006) all activate the SGE.
The ACh agonist muscarine, intrathecally administered,
facilitated ejaculation, while intraspinal injection of different
muscarinic receptor antagonists decreased the percentage of
rats that ejaculated and increased the EL in those ejaculating
(Durán et al., 2000; Gomez-Martinez and Cueva-Rolon,
2009). Intrathecal injection of a NOS inhibitor abolished,
and an NO donor enhanced apomorphine-evoked bursting
activity in the vas deferens nerve that is associated with ejacula-
tory responses (Brack et al., 2007).
Opioids exert an inhibitory influence on the functioning of
the SGE, since morphine blocks the expression of the
rhythmic motor pattern that accompanies ejaculation in
spinal rats, and this effect is prevented by naloxone pretreat-
ment. By contrast, naloxone facilitated the SGE activity and
delayed the inhibition of the ejaculatory response resulting
from repeated urethral stimulation. So far, endogenous
opioids represent the only intraspinal inhibitory influence
on SGE functioning described (Carro-Juárez and Rodríguez-
Manzo, 2009).
Electrical microstimulation of the SGE in rapid ejaculating
rats revealed that the expulsion phase in this subpopulation
is accelerated, supporting the notion that the reduced ejacula-
tory latency in these rats is related to an altered SGE functioning
(Borgdorff et al., 2009).
These data show that the effects of neurotransmitters in the
spinal cord may vary from those exerted in the brain or periph-
eral nervous system. Thus, opposite results may be obtained
with the same drug on a given sexual reflex, depending on
the animal model used (i.e., in copula vs ex copula responses,
conscious vs anesthetized rats, spinally intact vs spinally trans-
ected animals). To integrate this knowledge to understand the
spinal cord-mediated sexual functioning of an intact copu-
lating animal is one of today’s challenges.
1.01.6 Circuitry and Anatomical Interconnections
Organisms in natural settings weigh, prioritize, and integrate
multiple internal and external signals in order to choose and
perform various motivated behaviors, including mating. Here
we summarize several functional circuits that analyze and inte-
grate sensory and hormonal stimuli and execute behavioral
outcomes.
1.01.6.1 Sensory Inputs
Rodents depend heavily on chemosensory signals for mating.
The MeA, BNST, and MPOA are critical nodes in this circuit,
and each contains numerous steroid receptors (Rasia-Filho
et al., 1991; Wood and Newman, 1995b). T or E, but not
DHT, in the MeA or MPOA enhances mating (Wood, 1996)
and responsiveness to odor cues (Sipos and Nyby, 1998;
Wood and Newman, 1995b). Hormones may permit or
enhance release of transmitters, promote the production or
placement of postsynaptic receptors, and increase growth and
branching of neurons.
The major source of afferent input from the penis is the
dorsal nerve of the penis (DNP, a branch of the pudendal nerve),
which carries signals from the penile skin, prepuce, and glans;
input from deeper structures is carried by the cavernous nerve
(Steers, 2000). Afferents end mostly in medial portions of the
dorsal horn and the medial central gray matter of the lumbosa-
cral cord (reviewed in Hull et al., 2002, 2006). Electrical
stimulation of the DNP elicited responses in the nPGi, PVN,
MPOA, and cortex (Hubscher and Johnson, 1996; Yanagimoto
et al., 1996). Mating elicits Fos-ir in the CTF of rats (Baum and
Everitt, 1992) and SPFp of rats (Coolen et al., 1996, 1997a,b),
gerbils (Heeb and Yahr, 1996), and hamsters (Kollack-Walker
and Newman, 1997); these areas all project to the MPOA.
Axons from the L3 and L4 spinal segments terminate adjacent
to SPFp neurons that project to the MPOA or BSTpm
(Wells and Coolen, 2001). Electrical stimulation of the DNP
in men elicited activity in the midline of somatosensory cortex
(Gerstenberg et al., 1991; Guerit and Opsomer, 1991).
1.01.6.2 An Ejaculation-Related Circuit
As mentioned above, ejaculation is controlled by a central
pattern generator, located in the lumbosacral spinal cord, named
the SGE (Carro-Juárez et al., 2003), coordinating genital inputs
and sympathetic, parasympathetic, and motor outputs to trigger
ejaculation (Carro-Juárez and Rodríguez-Manzo, 2008). The
activity the SGE depends on cerebral descending facilitatory
and inhibitory influences. A group of spinothalamic neurons
located in the lumbar spinal cord (LSt cells) form part of the ejac-
ulation generator and project to the SPFp (Truitt and Coolen,
2002). Ejaculation, but not intromissions without ejaculation,
elicited small areas of Fos-ir in MeApd, BSTpm, PdPN, and
SPFp of rats (for review see Coolen, 2005; Coolen et al.,
2004a), hamsters (Fernandez-Fewell and Meredith, 1994;
Kollack-Walker and Newman, 1997; Parfitt and Newman,
1998), and gerbils (Heeb and Yahr, 1996, 2001; Simmons and
Yahr, 2002, 2003; Figure 5). These regions, activated specifically
following ejaculation, are part of a broader ejaculation-related
network that includes the MPOA, the PVN, and the nPGi.
 Male Sexual Behavior 39

AOB
Chemosensory inputs

vl SPFp
st st
sm ot
fx
v3 fx
aq
v3 PD

fr ml

MPN
MPN

Rostral MeApd
Caudal
BNSTpm BNSTpm LSSC

Visceral and somatosensory inputs

Figure 5 Schematic overview of Fos immunoreactivity in medial preoptic nucleus (MPN), posteriomedial part of the bed nucleus of the stria termi-
nalis (BNSTpm), posterodorsal preoptic nucleus (PD), posterodorsal part of the medial amygdala (MeApd), parvocellular division of the sub-
parafascicular nucleus (SPFp), and accessory olfactory bulbs (AOB). Areas where Fos is induced following chemosensory cues or chemosensory
investigation are illustrated by diagonal stripes from upper left to lower right. Areas where Fos is induced only following ejaculation are illustrated in
gray. Areas where Fos is induced following all consummatory elements of sexual behavior are illustrated by diagonal stripes from lower left to upper
right. LSSC, lumbosacral spinal cord; v3, third ventricle; fx, fornix; vl; lateral ventricle; st, stria terminalis; s, stria medularis; ot, optic tract; aq, aque-
duct; fr, fasciculus retroflexus; ml, medial lemniscus. Figure is reprinted from Coolen, L.M., Allard, J., Truitt, W.A., McKenna, K.E., 2004. Central
regulation of ejaculation. Physiol. Behav. 83, 203–215.

The MPOA and PVN appear to mediate excitatory, and the nPGi,
inhibitory, influences on the spinal control of ejaculation. The
MPOA is a key structure for ejaculation, but it does not directly
project to the spinal cord. However, neurons in each of the
ejaculation-activated regions have reciprocal connections with
the MPOA (Coolen et al., 1998; Gréco et al., 1998a; Heeb and
Yahr, 2001), and the MPOA projects to the PVN and nPGi.
The parvocellular region of the PVN sends neural projections
to the spinal cord, including the lumbosacral segments
(Hallbeck and Blomqvist, 1999), to the vlPAG (Luiten et al.,
1985), and the nPGi (Kobayashi et al., 2013). This PVN descend-
ing pathway is considered to exert, directly or indirectly, a facili-
tatory influence on ejaculation (Veening and Coolen, 2014). The
MPOA projects also to the nPGi, the primary source of descend-
ing inhibition on the SGE, both directly (Normandin and
Murphy, 2008) and through a PAG relay (Murphy and Hoffman,
2001; Figure 6). It was recently postulated that the 5-HT
pathway that runs from the vlPAG to the nPGi may act as a gating
mechanism for descending inhibition of genital reflexes
(Normandin and Murphy, 2011).
The MeApd and SPFp of rats and gerbils contribute to acti-
vation of the MPOA, as unilateral lesions decreased mating-
induced Fos-ir there (Baum and Everitt, 1992; Heeb and
Yahr, 2000). It is not clear whether the Fos-ir neurons received
sensory input or activated motor patterns of ejaculation.
In gerbils, PdPN or MeApd lesions decreased mounting and
delayed ejaculation; thus, these areas contribute to mating, but
are not essential for ejaculation (Heeb and Yahr, 2000).
However, lesions that included the SPFp and zona incerta of
rats almost eliminated ejaculation (Maillard and Edwards,
1991). The BSTpm and MePD contribute to sexual satiety of
hamsters, as Fos-ir increased only after multiple ejaculations
(Parfitt and Newman, 1998), and small lesions increased the
number of ejaculations before satiety (Parfitt et al., 1996). LH
lesions in rats disrupt ejaculation, but not intromissions or
mounts (Kippin et al., 2004). 5-HT is released in the anterior
LH at the time of ejaculation (Lorrain et al., 1997); however,
it is not clear whether this release is related to triggering ejacu-
lation or promoting the quiescence of the PEI. However, brain-
descending pathways originating in the LH, caudal raphe
nuclei, and the ventrolateral division of the gigantocellular
nucleus were recently described which directly project onto
spinal neurons, including the LSt cells (Facchinetti et al., 2014).
In men, positron emission tomography showed increases in
regional cerebral blood flow during ejaculation in the meso-
diencephalic region, which includes the SPFp, as well as the
cerebellum, lateral putamen, claustrum, and several cortical
regions (Holstege et al., 2003). No increases were seen in the
MPOA or BNST, in agreement with a study in male macaques
(Michael et al., 1999). Neuroimaging techniques also showed
that the major effect of ejaculation in male human brain is
not the activation of certain regions, but deactivation of regions
involved in emotional processing. Activation following ejacula-
tion was detected in the right posterior insula, the dorsal poste-
rior temporal lobe, the central midbrain, the medial globus
pallidus, and major parts of the cerebellum, while deactivation
40 Male Sexual Behavior
Ejaculatory circuit
BNSTpm
mPOA
SPFp
PAG
Figure 6 Brain control of ejaculation. Scheme illustrating the principal brain regions that form part of the ejaculatory circuit influencing the
functioning of the spinal ejaculation generator. BNSTpm, posteriomedial part of the bed nucleus of the stria terminalis; MeApd, posterodorsal part of
the medial amygdala; mPOA, hypothalamic medial preoptic area; nPGi, nucleus paragigantocellularis; PAG, periaqueductal gray; PVN, hypothalamic
paraventricular nucleus; SGE, spinal generator for ejaculation; SPFp, parvocellular division of the subparafascicular nucleus (described in
Section 1.01.6.2).
was identified in the ventral part of the temporal lobe and
extended regions of the parietal, medial orbitofrontal, and
temporal cortices (Holstege and Huynh, 2011).
1.01.6.3 Efferents from the MPOA
Efferents from the MPOA target the periventricular and medial
zones of the hypothalamus; the LH; midbrain motivation/
somatomotor regions, including the VTA and pedunculopon-
tine nucleus; midbrain and brain stem areas that project to
the spinal cord, including the PAG, certain raphe nuclei,
and nPGi; the BNST; and part of the septal area (Simerly and
Swanson, 1988; reviewed in Hull et al., 2006). These connec-
tions are mostly reciprocal, allowing ‘downstream’ sites to
influence the input that they receive. They provide multiple
ways to influence autonomic, motivational, and somatomotor
patterns. Output to the nPGi, either directly or via the PAG,
may disinhibit erections and the UG reflex. However, it seems
unlikely that mere disinhibition could elicit the reflexes that
can be evoked by MPOA stimulation (Giuliano et al., 1996,
1997; Marson and McKenna, 1994b). Such excitatory inputs
may include parasympathetic efferents, via the pelvic and
cavernous nerves, and sympathetic outflow, via the paraverte-
bral sympathetic chain and possibly the hypogastric nerve
(Giuliano et al., 1997). A number of projection sites contain
abundant steroid hormone receptors, allowing hormones to
bias sexually relevant cues and responses.
1.01.6.4 Sexual Behavior in the Context of Mammalian Social
Behavior
The brain areas that regulate male sexual behavior also influ-
ence other social behaviors. Newman (1999) suggested that
MeApd
PVN
nPGi
SGE
these brain areas form a reciprocally interconnected circuit
that serves all mammalian social behaviors. Most of the areas,
except the midbrain, are richly endowed with steroid receptors,
and all influence more than one behavior. Therefore, perinatal,
adolescent, and adult hormones can provide a bias toward
sexually dimorphic responses to social stimuli. It is not clear
whether the same neurons within a structure contribute to
more than one behavior, or whether neurons specific for one
behavior mingle with those for other behaviors. However, there
may be common themes that underlie the various social behav-
iors and the neural mechanisms that control them.
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 1.02 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
Oscar González-Flores and Kurt L Hoffman, Centro de Investigación en Reproducción Animal, Universidad Autónoma de Tlaxcala-
CINVESTAV, Tlaxcala, México
José A Delgadillo, Centro de Investigación en Reproducción Caprina (CIRCA), Universidad Autónoma Agraria Antonio Narro,
Torreón, México
Matthieu Keller, Behavioral & Reproductive Physiology, INRA/CNRS/University of Tours, Nouzilly, France
Raúl G Paredes, Instituto de Neurobiologia, Universidad Nacional Autónoma de México, Querétaro, México
Ó 2017 Elsevier Inc. All rights reserved.

1.02.1 Introduction 59
1.02.1.2 Estrous Behavior in Rodents, Lagomorphs, and Ruminants 60
1.02.1.2.1 Behavioral Aspects of Estrous Behavior 60
1.02.1.2.2 Effect of Steroid Hormones in Rodents 62
1.02.1.2.3 Effect of Steroid Hormones in Lagomorphs (Rabbits) 64
1.02.1.2.4 Seasonality of Reproduction in Goats 65
1.02.1.2.5 Endocrine Control of Sexual Behavior in Goats 66
1.02.1.2.6 Nonsteroidal Agents on Estrous Behavior 66
1.02.1.3 Cellular Processes Related to the Action of Steroid Hormones on Estrous Behavior 67
1.02.1.3.1 ER and PR Expression in the Brain 67
1.02.1.3.2 Cellular Mechanisms Proposed to Explain the Regulation of Estrous Behavior by Different Agents 68
1.02.1.3.3 Protein Kinase A 68
1.02.1.3.4 Leptin and Protein Kinase 69
1.02.1.3.5 Src Tyrosine Kinase, MAPK Pathway, and Lordosis Behavior 69
1.02.1.4 Neuroendocrine Factors Participating in the Behavioral Transition across the Reproductive Cycle of Rats,
Rabbits, and Goats 71
1.02.1.4.1 Anestrous to Estrous in Rodents 71
1.02.1.4.2 Anestrous to Estrous in Rabbits 71
1.02.1.4.3 Postpartum and Lactation in Rats 73
1.02.1.4.4 Postpartum and Lactation in Rabbits 73
1.02.1.4.5 Postpartum Anestrous and Lactation in Goats 73
1.02.1.5 Female Sexual Motivation in Rats and Goats 73
1.02.1.5.1 Paced Mating and Female Sexual Motivation 74
1.02.1.5.2 The Importance of Testing Conditions in the Analysis of Sexual Motivation 74
1.02.1.5.3 The Male Effect 75
1.02.1.5.4 Neurogenesis and Sexual Behavior 76
1.02.1.6 Conclusions 76
Acknowledgments 77
References 77

1.02.1 Introduction
In general, female mammals show periods of sexual activity
(estrous) alternating with periods of sexual refractoriness (dies-
trous). Depending on the effect of coitus on ovulation and on
the presence of functional corpora lutea, three types of estrous
cycles have been distinguished (Beyer et al., 1976; Morali and
Beyer, 1979). In the first type, ovulation occurs spontaneously
establishing functional corpora lutea with sexual receptivity
taking place around ovulation. The estrous cycle is long, with
a variable duration among species; for example, 16 days in
guinea pigs and 28 days in primates. In the second type of
estrous cycle, ovulation is spontaneous but functional corpora
lutea are not formed unless the animal copulates and becomes
pseudopregnant or pregnant. As in the case of the first type of
cycle, maximal receptivity is displayed around ovulation.
However, the estrous cycle is much shorter in duration, lasting
only 4–5 days, as is the case for rats and hamsters. In both of
these cycles, ovulation occurs in response to a hormonal
change: follicular estrogen secretion, waxing, and waning at
regular intervals. In contrast, in the third type of estrous cycle,
ovulation occurs following coitus: it is reflexive or induced.
This type of cycle is displayed by female rabbits, voles, mink,
ferrets, cats, camels, to name a few. Mating is followed by
ovulation with cessation of estrous and formation of active
corpora lutea. In contrast to spontaneously ovulating species,
induced ovulators generally do not show steroid-induced
ovulation. For example, in rabbits (Sawyer and Markee,
1959), voles (Milligan, 1978, 1980), and ferrets (Baum,
1990), estradiol (E2) treatment, with or without concurrent
progesterone (P), failed to stimulate ovulation. The cat may
be one exception to this rule, as spontaneous ovulation has

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60 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
been occasionally observed in group-housed female cats
(Gudermuth et al., 1997; Lawler et al., 1993). However, as
for any biological phenomenon, the distinction between
induced and spontaneous ovulation is sometimes unclear.
There is evidence, for example, that spontaneously ovulating
species can also show reflexive ovulation in certain circum-
stances. Under laboratory conditions, during mating, female
rats must receive several mounts and intromissions from a sexu-
ally active male in order for the ovaries to develop fully func-
tional corpora lutea. Thus, mating stimulates the release of
prolactin from the anterior hypophysis, which is necessary for
luteal activity (Terkel, 1988). In this condition, the female
spontaneously exhibits persistent vaginal estrous (Everett,
1939).
In most mammals, female sexual behavior is stereotyped
and predictable over time, and occurs in organized patterns
that can be very different between species. However, in general,
a complete sequence of sexual behavior generally includes pre-
copulatory behavior, copulation, termination, and conse-
quences of copulation.
Precopulatory behaviors: These are species-specific behaviors
displayed by males or females in order to attract the opposite
sex. Thus, the genital contact that characterizes copulation
usually is preceded by sequences of precopulatory or courtship
behavior, the complexity and duration of which varies from
species to species. These behaviors are often associated with
morphological and physiological signals such as swelling of
the genital area and the production of odoriferous substances
in the case of the female and anogenital investigation, scent
marking, and urine investigation by the male. Thus, courtship
brings together a male and a female of the same species at
the time when they are likely to mate, enhancing the proba-
bility of successful reproduction.
Copulation: Completion of courtship activity results in a tran-
sition to copulatory behavior, by which fertilization of the ova
is accomplished internally. Behavioral patterns that comprise
copulation are often elaborate and highly stereotyped. Typi-
cally, mammals engage in repeated copulations that can be of
long duration, although there are some species in which
mating entails a single, brief genital contact.
Termination: When hormonal levels are optimal, copulatory
activity may persist for a considerable period of time, either
through a single copulation of a long duration, or many brief
series of copulations, the latter characterizing the copulatory
behavior of laboratory rats. Thus, in female mammals, the
duration of estrous is largely determined by adequate
hormonal levels, and copulatory activity often wanes when
circulating levels of these hormones decline. In some cases,
even when apparently sexually satiated, a change of mating
partners often can lead to a reinstatement of copulatory
behavior, the so-called ‘Coolidge effect’ (Tlachi-López et al.,
2012; Ventura-Aquino et al., 2016).
Consequences of copulation: Pregnancy is the major conse-
quence of copulatory activity. In the female, successful fertiliza-
tion of the ova and pregnancy depend on a multitude of
endocrinological and physiological processes, including ovula-
tion and formation of the corpora lutea, the vaginal deposition
of sperm and their transference to the site of fertilization, and
implantation of the fertilized ova in the uterus. For example
in rabbits, the appropriate copulatory stimulation induces
ovulation and the initiation of a functional luteal phase that
is essential in order to maintain pregnancy (Conaway, 1971).
In this species, copulation is followed by a decrease in female
estrous behaviors, including sexual receptivity and scent-
marking (González-Mariscal et al., 1997, 1990; Hoffman and
González-Mariscal, 2007).
Although sexual behavior is the result of an ongoing inter-
action between the male and the female, each sex displays
specific, quantifiable behaviors that reflect motivational or
consummatory aspects of sexual behavior. Among female
mammals, patterns of sexual behavior are generally stereotyped
within a species, but can be quite variable between species. In
the present chapter we first describe salient behavioral and
hormonal aspects of female sexual behavior in rodents, lago-
morphs, and ruminants. This section is followed by an over-
view of cellular processes underlying the action of sex
hormones on estrous behavior, as well as neuroendocrine
factors participating in behavioral transitions across the repro-
ductive cycle of rats, rabbits and goats. Finally, the last section
discusses studies of female sexual motivation in rats and goats,
along with a brief overview of long-lasting changes in neuro-
genesis induced by sexual behavior.
1.02.1.2 Estrous Behavior in Rodents, Lagomorphs,
and Ruminants
1.02.1.2.1 Behavioral Aspects of Estrous Behavior
1.02.1.2.1.1 Rodents
During estrous, female rodents display a series of stereotyped
behaviors in order to attract a male and copulate. Classical
studies by Beach in 1976 distinguished three typical components
of female sexual behavior: attractivity, proceptivity, and recep-
tivity. Attractivity was defined as the female’s “value as a sexual
stimulus to evoke a sexual response in the male, which is deter-
mined by a number of factors, including olfactory and visual
cues.” Proceptivity was defined as a reaction by the female
toward the male which constitutes her assumption of initiative
in establishing or maintaining sexual interaction. Receptivity
was defined as “the female responses necessary and sufficient
for the male’s success in achieving intravaginal ejaculation”
(Beach, 1976). Over the years, these three components of female
sexual behavior have been further characterized and analyzed in
detail by many different research groups, resulting in a more
detailed account of estrous behavior and female sexual motiva-
tion (Agmo, 1999). Sexual attractivity of the female rat is neces-
sarily inferred from certain appetitive behaviors that the male rat
displays in response to signals emitted by the female. These
behaviors include approaching and investigating the female in
response to olfactory and visual cues. Experimental procedures
that allow for the investigation of sexual incentive motivation
(appetitive behavior) independently of copulation (consumma-
tory behavior) have shown that the female rat is an uncondi-
tioned sexual stimulus for the male. In other words, cues
emitted by the receptive female rat evoke and maintain approach
behavior by the male, and the possibility of copulating with her
has no lasting effect on her incentive value (Agmo, 2003).
Proceptive behavior displayed by female rats has been
described as having different components including approach,
orientation, and runaway (Beach, 1976; McClintock and Adler,
1978). During the approach, which is often initiated from
 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
a distance of at least four body lengths away from the male, the
female brings herself into proximity with the male. The orienta-
tion component involves the female positioning herself so that
her head is near the male, sometimes nudging or grooming him,
crawling over or dragging herself under his chin, or positioning
herself so that her genital region is near his nose. The female
then suddenly turns and departs from the male while displaying
a hopping or darting (zigzagging) behavior – the runaway
component. This often provokes the male to pursue her and
attempt a mount or intromission. If the male does not pursue
her, the female reapproaches the male and then again with-
draws. The female also can display ‘ear wiggling,’ which is the
result of rapid head shaking (Beach, 1976; McClintock and
Adler, 1978). In some rodents, proceptive scent-marking behav-
iors are displayed; these behaviors comprise rubbing the surface
of objects in the environment with specialized scent glands or
by depositing urine (Reynolds, 1971). Another term that has
been introduced is ‘paracopulatory’ behavior, which includes
proceptive behaviors mentioned above, along with ultrasonic
vocalizations. These paracopulatory behaviors are species-
typical behaviors that are displayed by females, which arouse
males and stimulate them to mount (Blaustein, 2009).
Receptivity in the female is characterized by the display of
a posture that facilitates penile insertion and ejaculation. In
rodents, as in many other mammalian species, this is accom-
plished by the lordosis reflex, which comprises arching of the
back, elevation of the pelvis, and deviation of the tail
(Komisaruk and Diakow, 1973; Kuehn and Beach, 1963). In
this posture, the legs are extended from the initial crouching
position while the head is elevated above its usual resting posi-
tion. Usually, lordosis is triggered by the male’s mount,
although tactile stimulation of the perineal region alone can
elicit this reflex in many rodent species such as rats, guinea
pigs, hamsters, etc. (Komisaruk, 1972). The latency to reinitiate
proceptive behavior after having received sexual stimulation is
determined in large part by the intensity and characteristics of
that stimulation. If the stimulus comprised a mount, intromis-
sion, and ejaculation, proceptive behavior will be delayed for
a longer time than if a mount and intromission were received
without an ejaculation. Likewise, the latency to reinitiate pro-
ceptive behavior is longer when both a mount and intromis-
sion are received, compared to a mount alone (Beach, 1976).
Receptivity in the female rat decreases progressively as
a result of repeated stimulation from the male (Hardy and
DeBold, 1972); eventually, the female shows a refractory
period during which she will no longer mate with the male.
This inhibition depends on the activity of the corpora
lutea and was first successfully mimicked in ovariectomized
(OVX) guinea pigs. Thus, in OVX animals that had received P
during estrogen priming, the later administration of P was inef-
fective at promoting estrous behavior a phenomenon called
‘concurrent inhibition’ (Goy et al., 1968; Zucker, 1966). In
the guinea pig a ‘biphasic’ effect of P on sexual behavior was
also demonstrated. It was observed that, following sponta-
neous receptivity in cycling female guinea pigs, there was
a refractory period during which exogenous hormone adminis-
tration failed to induce receptivity (Goy et al., 1968; Zucker,
1968). This refractoriness to hormone stimulation was repli-
cated in OVX guinea pigs that were made receptive by exoge-
nous estrogen priming followed 36 h later by P. After
61
experimentally inducing receptivity in this manner, a second
series of estrogen and P injections was ineffective at stimulating
estrous behavior; this phenomenon was termed ‘sequential
inhibition’ (Zucker, 1966).
1.02.1.2.1.2 Lagomorphs (Rabbits)
Female sexual behavior in the European rabbit (Oryctolagus
cuniculus) appears long before the ability to ovulate. However,
such behavior is regarded as prepubertal play, rather than as
a sign of puberty. In contrast to the rat, the sexually mature
female rabbit does not display an estrous cycle with regular
periods of heat, and ovulation occurs only after mating. In
captivity (in the laboratory or on the farm), under controlled
long-daylight conditions, unmated does are considered to be
in a more or less constant state of estrous. However, observa-
tions of rabbits under natural or seminatural conditions have
suggested that does may display subtle cycles of waxing and
waning receptivity with a cycle length of approximately
7 days (Myers and Poole, 1962).
Proceptive behaviors in the female rabbit have been much
less studied compared to those of the rat and, as a result,
have not been as thoroughly described and characterized. In
the wild, on the farm, or under laboratory conditions, procep-
tive female rabbits are observed to display circling of the male
and ‘flagging’ of the tail (elevation and lateral displacements of
the tail). However, specific proceptive behaviors have proven
difficult to characterize in the rabbit (Stoufflet and Caillol,
1988). González-Mariscal et al. (1990) proposed that a scent-
marking behavior called ‘chinning’ (where the rabbit rubs the
undersurface of its chin over objects in its environment, thereby
depositing scent gland secretions) might function as a procep-
tive behavior, since its heightened expression closely corre-
sponds to periods of sexual receptivity and rapidly declines
following copulation (González-Mariscal et al., 1990, 1997;
Soares and Diamond, 1982).
Chinning is a frequently observed behavior, being
expressed by both females and males within a variety of envi-
ronmental, social, and reproductive contexts. The waxy secre-
tion of the submandibular gland is deposited by chinning
on vertical or irregular surfaces, such as trees, grass, branches,
burrow entrances, cages, feeders, waterers, and mates. When
a rabbit is placed in a new cage, for example, it will explore
and chin-mark this unfamiliar environment (Lebas et al.,
1986); similarly, when unfamiliar objects are encountered
within a familiar environment, both female and male rabbits
preferentially chin-mark these objects, compared to familiar
ones simultaneously encountered within the same environ-
ment (Hoffman and Basurto, 2013, 2014; Hoffman et al.,
2010). In males, chinning apparently serves to delineate
territory, to mark conspecifics within the social group, and
to promote territorial confidence (Mykytowy, 1965;
Mykytowycz, 1962; Mykytowycz et al., 1976). Chinning has
been positively associated with social dominance and, in the
male, elevated levels of circulating testosterone (T) (Arteaga
et al., 2008).
In the female rabbit, the expression of chinning is closely
tied to reproductive state. Across prepubertal development,
chinning frequency steadily increases until sexual maturity is
reached (González-Mariscal et al., 1992). Sexually mature
does display increased chinning behavior during estrous,
62 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
compared to during pregnancy (González-Mariscal et al., 1990;
Soares and Diamond, 1982), as this behavior is stimulated by
circulating E2 and inhibited by P (Hudson et al., 1990). Stimuli
associated with mating (e.g., the male’s mounts and intromis-
sions) engage physiological mechanisms that inhibit chinning
across immediate (within minutes after mating), early (across
the first days postmating), and long-term (from day 4 post-
mating through the rest of pregnancy) time frames. These
mechanisms will be described in more detail in a later section.
The estrous female rabbit displays a lordosis posture in
response to the male mount, with the back and the hindquar-
ters raised (Lebas et al., 1986). This posture allows intromis-
sion and the immediate ejaculation by the male (unlike rats,
the male rabbit ejaculates immediately after each intromission
(Beyer et al., 1975; Beyer and Mcdonald, 1973). The lordosis
reflex is stimulated by a characteristic male mounting pattern,
in which the male mounts the female from behind, clasping
her flank region with his forepaws while displaying vigorous
pelvic thrusting directed at the perineal region (Contreras and
Beyer, 1979; Morali and Beyer, 1992; Morali et al., 2003).
Like chinning, the lordosis reflex is inhibited after ovulation,
whether induced naturally by mating, or experimentally by
the administration of gonadotropin (Hoffman et al., 2010),
and remains repressed throughout gestation (Beyer and
Rivaud, 1969). Similarly, sexual receptivity in female rabbits
is reduced during lactation. Studies have shown that lactating
does consistently refuse the buck after the 12th day of lactation
(Hammond and Marshall, 1925) or show sexual behavior only
occasionally (Yaschine et al., 1967). In one study, only 50% of
does sporadically displayed sexual behavior between the third
day of lactation and weaning in both New Zealand white and
California rabbits (Beyer and Rivaud, 1969). Like chinning,
inhibition of sexual receptivity during lactation was found to
be related to litter size: suckling a litter of 10 pups resulted in
significantly reduced sexual receptivity on lactation day 15,
compared to mothers that suckled litters of only one or three
pups (García-Dalman and González-Mariscal, 2012). Thus,
the ‘estrous cycle’ of the doe consists only of alternating periods
of sexual receptivity and nonreceptivity associated with preg-
nancy, pseudopregnancy, and lactation.
1.02.1.2.1.3 Goats
In natural or seminatural conditions males and females of
ruminant species live most of the year in segregated unisex
flocks (Alexander et al., 1980). This sexual segregation between
males and females disappears around the time of reproduction
where males leave their group to look for females. As in other
species, estrous behavior is composed of proceptivity and
receptivity. Proceptivity consists of seeking out and stimulating
the male partner while receptivity consists in the active immo-
bilization in response to male nudges. At the beginning of
estrous, proceptivity always precedes receptivity, but later on
these behaviors are expressed simultaneously. The expression
of proceptivity and receptivity in ruminant species is quite
different from that of rodents. Although various sensory cues
play a role in the attraction of males, the most important cue
for attracting males is the females’ proceptive behavior. An
increase in motor activity is observed in most female ruminants
when estrous starts. Goats express vocalizations and male-type
courtship behavior and female–female reciprocal mounting.
This behavior attracts males and stimulates sexual activity in
male goats (Shearer and Katz, 2006).
Once in contact, the receptive behavior of the goat when in
estrous consists of an immobilization. However, this immobi-
lization is not a passive posture but is rather an active response.
Indeed, the female will actively resist any attempt of movement
by an external manipulator. This immobilization is rather
subtle and is only observed as a slight arching of the back
during immobilization (Fabre-Nys and Gelez, 2007). This
immobilization is a sign both of proceptivity and receptivity.
In the buck, ejaculation is rather rapid and there is no need
for several intromissions: ejaculation generally occurs after
the first intromission and lasts a few seconds (Alexander
et al., 1980). Therefore, the amount of vaginal stimulation
received by females is rather limited in these species, contrary
to what can be observed in rodents (Rodríguez-Sierra et al.,
1977).
1.02.1.2.2 Effect of Steroid Hormones in Rodents
1.02.1.2.2.1 Estrogens
In females of all subprimate species that have been studied,
estrogens are essential for the expression of lordosis behavior
(for a review, see Young, 1961). While some species require
only estrogen for the display of sexual behavior, others need
the sequential action of estrogen and P. Rodent species such
as rat and guinea pig, which require both estrogen and P,
have short periods of estrous or heat and are sexually nonrecep-
tive throughout the major part of the cycle.
The estrous cycle of the rat is 4–5 days, comprising the
following stages (identified by cellular and histological changes
of the vaginal walls): diestrous I (or metestrus), diestrous II,
proestrous, and estrous. Proestrous is associated with a peak
in circulating E2, occurring approximately at midday, followed
by a sharp peak in P across the afternoon and evening, which
coincides with the release of luteinizing hormone (LH) from
the pituitary. Levels of all three hormones decline to low levels
by the morning of estrous. Female rat sexual behavior – both
proceptive and receptive components – is triggered by overlap-
ping peaks of circulating E2 and P that occur across the after-
noon and evening of proestrous. Likewise, in OVX female
rats, the administration of estradiol benzoate (EB; a synthetic
estrogen with an extended biological half-life), followed 48 h
later by the administration of P, promotes the full expression
of female sexual behavior. E2 is conceptualized to prime the
neural circuitry that underlies sexual behavior, for its later
responsiveness to P. Thus, the full expression of sexual
behavior – proceptive as well as receptive components – in
the female rodent has generally been considered to require P
in addition to a priming dose of EB (Glaser et al., 1983).
In physiological and behavioral studies of estrous behavior
that involve exogenous hormone administration, esterified
forms of 17b-E2 are often used, because the circulating levels
of 17b-E2 do not remain elevated for a long time after systemic
administration. A variety of esters and other modifications have
been used in this regard, such as ethynylestradiol (Patisaul
et al., 2003), E2 valerate (García-Segura et al., 1986), or
E2 dipropionate (Hlinak, 1993). However, the most
common form used is EB, which is 17b-E2 with a benzoic
acid esterified in the third carbon position. This form is hydro-
lyzed in vivo to the physiologically active 17b-E2, and it is more
 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
potent than free steroids for inducing estrous behavior. Thus, in
OVX rats, EB is 7 times more potent than E2 for inducing
estrous behavior (Powers, 1975). Similarly, exogenously
administered catecholestrogens, which are natural estrogen
metabolites, vary in their ability to induce estrous behavior in
OVX female rats (Luttge and Jasper, 1977; Naish and Ball,
1981). This variability depends on the route of administration
or the presence of other estrogens. For example, when
2-hydroxyestradiol and E2 were implanted together in the
hypothalamus, the level of receptivity was higher than when
E2 was administered alone (Marrone et al., 1977). Similarly,
Luttge and Jasper (1977) found that intracerebral implants of
2-hydroxyestradiol are ineffective in stimulating lordosis
behavior in female rats but, when combined with intravenous
administration of E2, such implants induced receptivity.
Although estrous behavior in the intact female rat is nor-
mally triggered by the combined action of E2 and P, in some
experimental conditions, the administration of E2 or EB can
substitute for P in the induction of female sexual receptivity,
in OVX or OVX-adrenalectomized rats (the adrenal gland is
another significant source of P) (Beach, 1942). Thus, a single
high-dose injection of EB can induce sexual behavior, as can
the combination of a single low dose of EB followed by
a second high dose of EB or E2. Moreover, OVX or OVX-
adrenalectomized rats displayed sexual behavior after having
received low doses of EB across several successive days (Beyer
and Komisaruk, 1971; Blaustein et al., 1987; Kow and Pfaff,
1975). Classic early studies demonstrated that various
estrogenic compounds showed different potencies with
respect to their ability to induce estrous (lordosis), after
subchronic treatment (daily injections, across 10 days) in
OVX rats. Thus, E2 was the most potent estrogen in this
regard, followed by estrone (E1), estriol (E3), and estrone
sulfate (review in Beyer et al., 1976). The intensity and
frequency of proceptive behavior in the rat (e.g., ear wiggling
and hopping) and some characteristics of the lordosis
response are directly correlated with the dosages of estrogen
administered. Thus, by increasing the estrogen dose in OVX
rats, the degree of arching of the back during lordosis
increases, the latency to respond with lordosis to male
mounting shortens, and the duration of the lordosis posture
is extended (Zemlan and Adler, 1977). Likewise, Domínguez-
Ordoñez et al. (2015) showed that lordosis was facilitated by
subcutaneous or intracerebral administration of several doses
of free E2, in OVX Sprague Dawley female rats primed with
EB (Domínguez-Ordonez et al., 2015). Taken together, these
observations indicate that, in the induction of lordosis in
OVX female rats, exogenously administered E2 can act both
to prime the later responsiveness to P and to facilitate the
expression of sexual behavior in rats that had previously been
primed with an estrogen.
More recently, it has been shown that chronic, repeated
administration of EB (2 or 10 mg) in sexually experienced
OVX Long-Evans and Wistar rats induced ‘sensitization’ of
receptivity (Jones et al., 2012; Jones and Pfaus, 2014). Thus,
rats administered EB every 4 days, across 32 days, displayed
significantly higher levels of proceptive behaviors and sexual
receptivity in response to EB administration, compared to
rats that had not received chronic EB treatment. EB-induced
sensitization did not involve the release of adrenal steroid
63
hormones (such as progestins) because adrenalectomy did
not block the sensitization of sexual behavior. Moreover, EB-
induced sensitization occurred independently of whether or
not they had interacted with a sexually vigorous male during
the course of hormone treatment, indicating that hormonal
treatment alone was sufficient to induce behavioral
sensitization (Jones and Pfaus, 2014).
1.02.1.2.2.2 Progestins
P, either alone or in combination with other hormones, modu-
lates several reproductive processes such as sexual behavior,
ovulation, gonadotropin secretion, pregnancy, maternal
behavior, among others. In intact rodents, estrous behavior is
normally induced by the combined action of estrogen and P.
During the 48 h period preceding the onset of sexual receptivity
in the rat, estrogen levels gradually rise and reach a peak late in
the afternoon of proestrous. At about this time, P levels begin
to rise rapidly, reaching a peak during the early evening of pro-
estrous. The onset of sexual receptivity is temporally associated
with this preovulatory peak in circulating P (Freeman, 1994;
Ichikawa et al., 1974). Early experiments carried out in rats
and hamsters demonstrated that lordosis was abolished if the
ovaries were removed shortly before the preovulatory peak of
P, whereas lordosis was consistently displayed by animals
that had been ovariectomized after the preovulatory peak in
P had already occurred (Moreines and Powers, 1977; Powers
and Valenstein, 1972). Other studies showed that P greatly
facilitated the effect of estrogen on estrous behavior in OVX
females, but only if the female had been previously primed
with estrogen for a minimal period of time, independently of
the amount of estrogen given (Boling and Blandau, 1939;
Dempsey et al., 1936; Quadagno et al., 1972). On the other
hand, the duration of the estrogen priming effect does depend
on the dose injected: if large dosages of E2 are given, sexual
behavior can be facilitated by P administration even 96 h
after estrogen administration (Boling and Blandau, 1939;
Quadagno et al., 1972). Thus, in normal cycling female rats,
estrogen acts to prime the subsequent responsiveness to P.
Rodent species differ widely with respect to the amount of
systemically administered P that is required to induce lordosis
in OVX, estrogen primed females. For example, after being
primed with suprathreshold doses of estrogen, the minimum
dose of P per 100 g body weight was 4 mg for guinea pigs
(Wade and Feder, 1972), 50 mg for the golden hamster (Frank
and Fraps, 1945), and 100 mg for the rat (Powers and
Valenstein, 1972). These differences in potency appear to be
related to variations in the brain’s capacity to incorporate and
retain P. Thus, the guinea pig takes up more P than the hamster,
which in turn retains more than the rat brain (Wade et al.,
1973). In estrogen primed rats, P promotes sexual attractivity,
as well as the display of proceptive behaviors such as ear
wiggling and darting (Hlinak and Madlafou, 1969; Hlinak
and Madlafousek, 1972; Whalen and Gorzalka, 1972). P has
been particularly associated with the display of proceptive
behavior in the OVX female rat: in one study, ear wiggling
was highly correlated with the dose of P administered, whereas
no significant relationship was observed between proceptive
behavior and the amount of E2 administered (Tennent et al.,
1980).
64 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
In the brain, P is rapidly reduced in its ring A to
5a-pregnanedione (a-DHP), which subsequently can be
reduced in C3 to 3a- or 3b-pregnanolones by the action of
3a- or 3b-hydroxysteroid oxoreductases, respectively (Beyer
and González-Mariscal, 1991; Cheng and Karavolas, 1973;
Karavolas et al., 1984; Whalen, 1985). A review of the litera-
ture reveals large variations between studies in the reported
capacity of ring A-reduced P metabolites to stimulate estrous
behavior. In some cases, these differences among studies can
be ascribed to differences in the solvents used and in the
manner in which the hormone was administered (Gorzalka
and Whalen, 1977; Karavolas et al., 1984; Wade and
Feder, 1972). Thus, a-DHP or 5b-pregnanedione, 3a- or 3b-
pregnanolones were much less effective than P for inducing
lordosis when dissolved in oil and administered subcutane-
ously to OVX rodents primed with estrogen (Czaja et al.,
1974; Johnson et al., 1976; Pleim and DeBold, 1984; Whalen
and Gorzalka, 1972). However, when a-DHP was dissolved in
Tween-80 and administered to OVX rats, it was just as effective
as P at inducing estrous behavior (Gorzalka and Whalen,
1977). On the other hand, additional studies showed that
ring A-reduced progestins were less potent than P when
administered intravenously (Meyerson, 1972). Finally, some
ring A-reduced progestins, such as 5b,3b-pregnanolone
(5b,3b-Pgl) and 5a,3b-pregnanolone (5a,3b-Pgl), reported
to be ineffective when systemically injected (Kubli-Garfias
and Whalen, 1977), were as effective as P to facilitate lordosis
when implanted directly into the ventromedial hypothalamus
(VMH) or the medial preoptic area (MPOA) of OVX, estrogen-
primed rats (Beyer et al., 1988; González-Mariscal et al., 1989;
Rodríguez-Manzo et al., 1986). Therefore, the low potency of
these P metabolites to induce estrous when dissolved in oil
solutions and administered SC is most likely due to the low
bioavailability of these compounds when that route of
administration is used (Gorzalka and Whalen, 1977;
Karavolas et al., 1984).
1.02.1.2.2.3 Androgens
In female mammals, androgens are secreted by the ovaries
and adrenals and can promote female sexual behavior
(Dupon and Kim, 1973). In OVX rodents, the most effective
androgens in this regard are those that possess the delta 4–3
keto structure, such as T. Such steroids fluctuate across the
female’s ovarian cycle and show maximal levels during
the afternoon of proestrous, temporally corresponding to
the preovulatory peak in P (Belanger et al., 1981). Early
studies showed that testosterone propionate (TP) had
a weak, but clear stimulatory effect on the lordosis reflex
in OVX rats when administered in large dosages
(6 mg day
1, across l0 days) (Beyer and Komisaruk, 1971).
Similar results had previously been described by Ball and
Beach (Ball, 1940; Beach, 1942). However, other studies
in which lower doses of TP were administered (200 mg daily
for 9–11 days) failed to observe a stimulatory effect of TP
on lordosis (Pfaff, 1970). Other androgens, including 5a-
dihydrotestosterone (DHT) and androsterone, also failed
to stimulate the lordosis reflex in the OVX female rat (Beyer
and Komisaruk, 1971). These results were concordant with
studies carried out in the OVX female rabbit, discussed in
the next sections.
1.02.1.2.3 Effect of Steroid Hormones in Lagomorphs
(Rabbits)
1.02.1.2.3.1 Estrogen
In the female European rabbit, E2 is the principal gonadal
hormone that promotes estrous behavior: in contrast to the
rat, estrous behavior in the rabbit is not associated with
a peak in circulating P levels (Beyer and Mcdonald, 1973). In
unmated female rabbits, under controlled laboratory condi-
tions, ovarian follicles develop in overlapping waves,
producing a relatively constant plasma level of E2. Under these
conditions, does display a state of relatively persistent sexual
receptivity. However, in more natural conditions, does were
observed to display waxing and waning receptivity, with a cycle
length of approximately 7 days, and a heightened attractiveness
to males across approximately 1–3 days. The receptivity cycle
length corresponds closely to that of follicular development
(reviewed in Beyer and Mcdonald, 1973). Ovulation is induced
in receptive females by a combination of diverse stimuli asso-
ciated with mating, arising from tactile (flank, perineal,
vaginal), olfactory, visual, and auditory channels (Staples,
1967). Immediately following coitus, ovarian output and/or
plasma levels of E2, P, 20a-hydroxyprogesterone (20a-OHP),
and T increase, reaching a peak at approximately 2 h post-
mating, and then return to baseline. Ovulation occurs approx-
imately 10 h postmating (Bakker and Baum, 2000; Hilliard and
Eaton, 1971; Mills et al., 1981). As a result of ovulation,
a number of distinct mechanisms become engaged that inhibit
receptivity during the immediate and early postmating time
frames, across pregnancy (or pseudopregnancy), and during
lactation.
Contrary to what is observed in rodents, some sexual recep-
tivity is retained in a small proportion of adult female rabbits
for at least 2 months after ovariectomy (Beyer et al., 1969).
Moreover, sexual activity was displayed by a small proportion
of adult female rabbits that had been ovariectomized prepuber-
tally; that is, 30 days after birth (for review, see Beyer and
Mcdonald, 1973). Taken together, these results indicate that,
in some individuals, the ovaries are not indispensable for initi-
ating and maintaining sexual behavior.
In OVX rabbits, E2 administration potently promotes sexual
receptivity (Beyer et al., 1975). Thus, in OVX New Zealand
white rabbits, 3 mg of EB was sufficient to induce receptivity
within 24 h (Palka and Sawyer, 1966). Later, a series of studies
found that the threshold dose for inducing lordosis behavior in
OVX rabbits was between 250 ng day
1 (40% response) and
1 mg day
1 (100% response). In these studies, EB was found
to be much more potent than estrone in eliciting lordosis
(McDonald et al., 1970).
Chin-marking behavior is also increased by E2 in OVX
rabbits. Hudson et al. (1990) studied the effects of daily injec-
tions of 1 or 10 mg on chin-marking and sexual receptivity.
They found that both doses of EB significantly increased both
behaviors, over baseline values, relative to the control,
vehicle-treated group (Hudson et al., 1990). Likewise, EB
implants into the VMH or MPOA reliably stimulated chinning
in OVX females. In this study, most of the does implanted in
the VMH and around half of those that had received EB in
the MPOA or diagonal band of Broca (DBB) showed lordosis
(Melo et al., 2008). These observations suggest that the VMH
is an estrogen-sensitive brain area that stimulates both
 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
chinning and lordosis while the MPOA seems to contain
subpopulations of neurons involved in both behaviors.
1.02.1.2.3.2 Progestins
In contrast to the rat, progestins (e.g., P and 20a-OHP, a P
metabolite secreted by ovarian interstitial tissue) have no
known stimulatory effects on rabbit female sexual behavior.
Circulating progestin levels are practically nonexistent during
estrous and do not increase until immediately after ovulation
(in which a brief, sharp peak in 20a-OHP release is observed)
and, later, as a result of the formation of the corpora lutea,
beginning around day 4 of pregnancy or pseudopregnancy
(Beyer and Mcdonald, 1973; Hilliard, 1973; Hilliard et al.,
1968). In the case of fertile matings, plasma P levels rise
steadily from day 4 postmating, reach a peak at approximately
day 16, and then steadily decline back to baseline (premating)
levels by day 31 (parturition) (Hilliard et al., 1968). Sexual
behavior, scent marking as well as sexual receptivity, is abruptly
inhibited after mating. Activation of the progesterone receptor
(PR) appears to participate in the ‘early’ postcoital inhibition of
sexual behavior observed prior to the formation of the corpora
lutea, when circulating P levels are negligible, as well as across
pregnancy, when plasma P levels are high (Beyer and Rivaud,
1969; Hoffman and González-Mariscal, 2006, 2007). Likewise,
in OVX, E2-primed female rabbits, P administration inhibits
sexual receptivity and scent-marking behavior (Beyer et al.,
1969; Hudson et al., 1990). Beyer and his group studied the
effect of 0.5, 1.0, and 2.0 mg of P on sexual receptivity in
OVX rabbits that had been treated with twice daily injections
of 0.5 mg of EB for 4 days. In these experiments, 2 mg of P
inhibited receptivity in all females, while 0.5 mg inhibited
this behavior in half of the subjects. This amount of P is compa-
rable to the daily output of P secreted by the ovaries of mid-
pregnant rabbits (Hilliard et al., 1968). Similarly, P inhibited
chinning behavior and sexual receptivity in OVX, EB-primed
does (Hudson et al., 1990), an effect that is primarily
mediated by activation of the PR (Hoffman and González-
Mariscal, 2006).
Possible facilitatory effects of ring A-reduced progestins on
female sexual behavior have not been investigated in the
rabbit, but since P itself has no stimulatory effects, it seems
unlikely that its ring A-reduced metabolites would have such
effects. However, it remains possible that 20a-OHP might
participate in the early postcoital inhibition of sexual behavior.
Thus, 20a-OHP is secreted by the interstitial tissue of the ovary
in very large amounts just prior to ovulation, while at the same
time P levels are very low or nondetectable (Hilliard et al.,
1963). These endocrine changes correlate with an abrupt
drop in sexual behavior (Beyer et al., 1969), suggesting that
20a-OHP, which weakly activates the PR, could be involved
in the early postcoital inhibition of sexual behavior. Consistent
with this idea, the administration of a PR antagonist combined
with a small dose of EB prevented the early postmating decline
in sexual receptivity and scent marking in intact female rabbits,
while neither treatment by itself was able to do so (Hoffman
et al., 2010).
1.02.1.2.3.3 Androgens
Some androgens are known to stimulate estrous behavior in
intact and OVX female rabbits. Beyer et al. (1970a,b) evaluated
65
the effects of several types of androgens, both aromatizable
(capable of being converted to estrogen) and nonaromatizable
(unable to be converted to estrogen), on lordosis behavior in
OVX rabbits. They reported that aromatizable androgens
(T, androstenedione, 19-hydroxy-androstenedione, and dehy-
droepiandrosterone) administered during a 10-day period,
stimulated lordosis and mounting behavior, while nonaroma-
tizable androgens (DHT, androsterone, and 4-chloro-testos-
terone) did not. Based on these results, these authors
proposed that androgens primarily stimulate lordosis behavior
in the rabbit through their aromatization, i.e., conversion to E2
(reviewed in Morali and Beyer, 1979). These studies were
among the first to suggest that some biological effects of T –
in this case, stimulatory effects on female sexual behavior –
might be mediated by its aromatization to E2. This idea was
supported by studies showing that an estrogen receptor (ER)
antagonist, ethamoxytriphetol (MER-25), blocked the ability
of TP to induce estrous behavior in the rabbit (Beyer and Vidal,
1971).
1.02.1.2.4 Seasonality of Reproduction in Goats
Reproductive seasonality is a common characteristic of many
mammalian species, allowing females to produce offspring at
the optimal time of year for the survival of the neonate.
Thus, female goats originating from temperate or subtropical
latitudes display reproductive seasonality. In several breeds of
goats from the north hemisphere, the breeding season, charac-
terized by the display of estrous behavior and ovulation, starts
in autumn (August–September) and finishes in winter
(February–March). In these females, the anestrous season, char-
acterized by the absence of estrous behavior and ovulation,
occurs during spring and summer (Chemineau et al., 1992;
Duarte et al., 2008).
In goats from temperate or subtropical latitudes, the repro-
ductive seasonality is mainly controlled by photoperiod.
Indeed, in experimental conditions where the duration of
photoperiod is controlled, short days, i.e., 10 or 8 h of light
per day, stimulate ovulation, whereas long days, i.e., 16
or 14 h of light per day, inhibit the ovulatory response
(Chemineau et al., 1992; Duarte et al., 2008). In goats exposed
to natural light–dark conditions, photoperiod synchronizes an
endogenous rhythm of reproduction allowing the alternation
of the sexual and anestrous seasons (Gebbie et al., 1999;
Gomez-Brunet et al., 2008). During the sexual season,
nonpregnant females show several successive estrous cycles of
a mean duration of 21 days. Nevertheless, short (around
8 days), or long (around 39 days) estrous cycles are commonly
observed. Most estrous cycles are associated with ovulation;
however, ovulation without estrous or estrous without ovula-
tion can also occur (Chemineau et al., 1992).
Photoperiod modifies the E2 negative feedback on gonado-
tropin secretion between the breeding and anestrous seasons.
During increasing or long days, the feedback of E2 reduces
dramatically the secretion of gonadotropins (LH and follicular
stimulating hormone (FSH)), inhibiting ovulation. By contrast,
during decreasing or short days, this feedback decreases, stimu-
lating the secretion of gonadotropins, which allow ovulations
to occur (Chemineau, 1987; Mori et al., 1987). The seasonal
changes in E2 negative feedback were described in OVX goats
treated with subcutaneous implants constantly releasing
66 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
estradiol-17b (OVX þ E2). In these OVX þ E2 females, high
and low plasma LH concentrations coincide with ovulatory
and anovulatory periods of intact goats, respectively (Duarte
et al., 2008; Henniawati et al., 1995). Therefore, the modifica-
tion of the E2 negative feedback on gonadotropin secretion
induced by photoperiod constitutes the neuroendocrine mech-
anisms determining the breeding and seasonal anestrous
seasons.
1.02.1.2.5 Endocrine Control of Sexual Behavior in Goats
There is evidence showing the importance of steroids in the
control of female sexual behavior in goats. First, as in many
other mammalian species, hormonal changes in female goats
include an increase in E2 concentration that is observed around
24–48h before the expression of sexual behavior (Thorburn
and Schneider, 1972). This change suggests that E2 may be
a good candidate for the control of sexual behavior. In this
regard, it is well known that E2 alone is sufficient to induce
estrous behavior in French-Alpine goats (Billings and Katz,
1998; Kalivas et al., 2006), even if the amount of E2 used is
in the pharmacological range (around 30 mg), while E2 concen-
trations on the day of estrous were reported to be around
15 pg ml
1 in intact Surti and Mahwari goats (Pathak et al.,
1990), or in Shiba goats (Mori and Kano, 1984). In addition,
the proportion of OVX females displaying estrous behavior as
well as the intensity of expression of proceptivity and recep-
tivity increase according to the dose of E2 given to the animals
(Billings and Katz, 1998). Finally, the adequate display of
female sexual behavior appears to be dependent on the dura-
tion of E2 treatment rather than on the doses or the maximal
concentration achieved (Okada, 1998).
The increase in E2 concentration appears only after the
regression of the corpus luteum, which leads to a decline in P
concentration around 48 h before the expression of sexual
behavior. During the breeding season, expression of sexual
behavior is preceded by a 12- to 15-day luteal phase during
which plasma concentrations of P are high (between 2 and
5 ng ml
1). In addition, it is known that the first estrous of
the breeding season may be behaviorally ‘silent’ in some breeds
of goats (Jainudeen, 1993). As the concentration of P is very
low during the nonbreeding season, this suggests that exposure
to P may be necessary for the full display of sexual behavior at
the onset of the breeding season as is the case in sheep.
However, in OVX goats, P priming did not increase the display
of estrous at the end of the anestrous period (Sutherland and
Lindsay, 1991).
These results should not point to a lack of a role of P in the
display of female sexual behavior. Indeed, it has been also
shown that the timing between the secretion of P and the rise
in E2 is important for the expression of female sexual behavior
(Billings and Katz, 1998). In fact, P facilitated E2-induced
attractivity and receptivity in the anestrous period if the P treat-
ment occurred 48 or 72 h prior to the treatment with E2. During
the breeding season, P facilitated attractivity when treatment
occurred 72 h prior to treatment with E2. By contrast, P has
an inhibitory effect on all sexual behaviors when the treatment
occurred 12 or 24 h prior to injection of E2. Finally, P did not
facilitate E2-induced proceptivity in either sexual or anestrous
seasons. Therefore, the decline in circulating P concentrations
prior to the rise in E2 is more important than just the presence
of P itself. These results confirm that the temporal sequence of
P and E2 administration affects sexual behavior in OVX goats.
This sequence of secretion of both E2 and P is quite different
from that seen in many rodent species in which the increase
in P follows the increase in E2. This suggests that the role of
these two hormones differs according to the species.
Regarding androgens, it has been shown that aromatizable
androgens, such as T and androstenedione, stimulate the
expression of sexual behavior in OVX goats (Katz, 2007). In
contrast, a nonaromatizable androgen, such as dihydrotestos-
terone is not effective (Lindia and Katz, 2003). One study
tested the effect of the androgen receptor antagonist flutamide,
a potent androgen receptor antagonist. It was suggested that
while estrogens facilitate proceptive and maybe attractive
behaviors, an androgen-dependent pathway modulates recep-
tivity in female goats (Imwalle and Katz, 2004).
1.02.1.2.6 Nonsteroidal Agents on Estrous Behavior
A number of studies, many of which have been carried out in
rodent species, demonstrate that nonsteroidal hormones
participate along with ovarian hormones in the regulation of
female sexual behavior. Beginning in the early 1970s, pharma-
cological studies showed that gonadotropin-releasing
hormone (GnRH) (Moss and McCann, 1973) and prosta-
glandin E2 (PGE2) (Rodríguez-Sierra and Komisaruk,
1977) facilitated lordosis behavior. Since then, a surprising
variety of nonsteroidal compounds have been shown to stimu-
late lordosis behavior. These compounds include certain small
neuropeptides (e.g., oxytocin (OT), melanocyte-stimulating
hormone), proteins (prolactin, ACTH), and neurotransmitters
(noradrenaline, dopamine, GABA, acetylcholine) (for review,
see Beyer and González-Mariscal, 1986). The diversity of
compounds that promote lordosis in the female rat speaks to
the complex nature of the brain networks that underlie female
sexual behavior. In the following paragraphs, we discuss some
of these compounds: GnRH, OT, leptin, and nitric oxide (NO).
1.02.1.2.6.1 Gonadotropin-Releasing Hormone
In addition to triggering the release of LH from the anterior
pituitary, ultimately resulting in ovulation, GnRH also facili-
tates lordosis in the female rat after either systemic or intracere-
bral administration (Beyer et al., 1997; González-Mariscal
et al., 1993; Mahesh, 1985; McCann, 1977; Moss and McCann,
1973; Ramirez-Orduna et al., 2007). In OVX, estrogen-primed
rats, infusions of this peptide directly into the MPOA or the
VMH facilitated lordosis behavior, while infusion of a GnRH
antibody reduced the frequency of the lordosis response (Pfaff,
1993). In these studies, the latency to induce the maximal
lordosis response after GnRH infusion was relatively long
(1 h 45 min). However, the effect of GnRH was independent
of pituitary hormones since it persisted in hypophysectomized
E2-primed rats, and the combined administration of LH, FSH,
and E2 did not elicit lordosis (Moss and Mccann, 1975).
Ramirez-Orduña et al. (2007) showed that GnRH (0.005 and
0.05 mg doses) infused into the right lateral ventricle (intracer-
ebroventricular (icv)), induced proceptivity and lordosis
behavior in OVX, EB-primed rats (Ramirez-Orduna et al.,
2007). These results are in agreement with previous studies
that showed that GnRH induced female sexual behavior
when administered either intracerebrally or systemically (Beyer
 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
et al., 1997; González-Mariscal et al., 1993; Hall and Luttge,
1977; Hall et al., 1975; Moss and Foreman, 1976; Moss and
McCann, 1973; Rodríguez-Sierra et al., 1977; Rodríguez-Sierra
and Komisaruk, 1982).
1.02.1.2.6.2 Oxytocin
Administration of OT directly into the hypothalamus or MPOA
facilitated lordosis behavior in E2-primed female rats (Caldwell
et al., 1989, 1994; Schulze and Gorzalka, 1991), whereas treat-
ment with E2 increased OT binding in the VMH (Caldwell et al.,
1994). Likewise, in the rat, OT injected into the cerebral ventri-
cles stimulated proceptive behavior, while an OT antagonist
inhibited female sexual behavior (Caldwell et al., 1989,
1994; Schulze and Gorzalka, 1991). The stimulatory effects of
OT on lordosis depended on the time of administration rela-
tive to the light/dark schedule as well as the exact hypothalamic
location of the OT infusion (Caldwell et al., 1989). These vari-
ables appeared to influence measures of lordosis frequency and
duration independently (Schulze and Gorzalka, 1991). For
recent reviews of the effects of OT circulating in the CSF, see
Veening et al. (2010) and Veening and Olivier (2013).
1.02.1.2.6.3 Leptin
Leptin is produced primarily in adipose tissue, and it is an
important hormonal regulator of food intake, weight gain,
and energy balance (Campfield et al., 1996). Moreover, leptin
has been reported to influence estrous behavior in rodents in
a manner that appears to depend on the animal’s metabolic
or nutritional state (Fox and Olster, 2000; García-Juarez
et al., 2013, 2012, 2011; Wade et al., 1997). Thus, leptin facil-
itated sexual behavior in female hamsters fed ad libitum, but
intensified the fasting-induced inhibition of lordosis in food-
deprived animals (Wade et al., 1997). By contrast, in geneti-
cally obese Zucker rats treated with E2 and P, intracerebrally
administered leptin did not augment lordosis and inhibited
proceptivity (Fox and Olster, 2000). Taken together, these
studies suggest that this hormone can have either facilitatory
or inhibitory effects on rodent sexual behavior, apparently
depending at least in part on the animal’s metabolic or nutri-
tional state. Only a few studies have been carried out on the
modulatory effects of leptin on lordosis in the ad libitum fed
rat. It has been shown that doses of 1 and 3 mg produced signif-
icant lordosis quotient at 60 min postinjection, with maximal
response being induced at 120 min, without inducing procep-
tive behavior at any dose (García-Juarez et al., 2011). The
observation that leptin can facilitate lordosis while inhibiting
proceptive behavior indicates that this hormone can directly
or indirectly dissociate neural mechanisms underlying female
rodent sexual behavior.
1.02.1.2.6.4 Nitric Oxide
NO is a diffusible gas neurochemical transmitter that is
produced from L-arginine, via the action of NO synthase
(NOS) (Knowles et al., 1989), and that participates in major
aspects of brain function related to reproduction (Dawson
and Snyder, 1994; García-Juarez et al., 2012). Studies have
shown that NOS activity is induced by E2 and is present in
neurons of the VMH of female rats (Rachman et al., 1996).
In the intact cycling female rat, NO levels in the hypothalamus
doubled between the afternoon and evening of proestrous,
67
concomitant with the LH surge and the period of behavioral
receptivity (Calka, 2006). In OVX female rats primed with E2
plus P, intracerebroventricular infusion of NOS inhibitors or
NO scavengers attenuated lordosis (Mani et al., 1994).
Conversely, an NO donor facilitated lordosis in E2-primed
OVX rats in the absence of P. Given these observations, Chu
and Etgen (1997) proposed that cGMP, which is synthesized
subsequent to the activation of soluble guanylyl cyclase by
NO, could be an important cellular second messenger to stim-
ulate lordosis in hormonally primed female rats. In order to
test this idea, they used 1H-[1,2,4]oxadiazolo[4,3-a]quinoxa-
lin-1-one (ODQ), a selective inhibitor of NO-stimulated
soluble guanylyl cyclase. As predicted, ODQ infused into the
lateral or third ventricle suppressed lordosis in a dose-
dependent manner in OVX female rats treated with E2 plus P
(Chu and Etgen, 1999; Chu et al., 1999).
1.02.1.3 Cellular Processes Related to the Action of Steroid
Hormones on Estrous Behavior
Intracellular receptors for steroid hormones (e.g., estrogens, P’s,
glucocorticoids, androgens, mineralocorticoids) are members
of the nuclear receptor superfamily of ligand-dependent tran-
scription factors, which also include receptors for certain other
nonsteroid lipophilic hormones and ligands (such as thyroid
hormone, retinoic acid, 9-cis retinoic acid, vitamin D3 and
eicosanoids, fatty acids, and lipids), as well as orphan receptors
that have no known ligand (Beato, 1987; Brinton et al., 2008).
Steroid receptors, cloned in various species (Faber et al., 1976;
Horwitz et al., 1986; Schrader et al., 1981), comprise four main
domains: (1) the DNA-binding domain (DBD), which contains
two Zn fingers that can interact with the DNA hormone
response element; (2) the ligand-binding domain (LBD) at
the C-terminal of the molecule, which is responsible for
hormone binding and the interaction with the 90 and 70 kd
heat shock proteins; (3) the hinge region, between the DBD
and LBD; and (4) the region located at the N-terminus of the
molecule, whose specific functions are unknown, but with
transactivation properties that can increase gene transcription
regardless of hormone binding (Beato, 1987; Brinton et al.,
2008; Horwitz et al., 1986; Schrader et al., 1981).
Additionally, steroid receptors contain at least two domains
necessary to mediate transcriptional activation, designated
AF-1 and AF-2. AF-1 is located within the N-terminal
domain, while AF-2 is found in the C-terminal domain.
1.02.1.3.1 ER and PR Expression in the Brain
1.02.1.3.1.1 Rodents
The classic intracellular ER and PR are expressed in several brain
regions associated with reproductive behavior. Two subtypes of
ERs are synthesized in distinct cell types: ER-a (Greene et al.,
1986) and ER-b (Kuiper et al., 1996). These isoforms are the
product of separate genes, the ER-b protein being somewhat
smaller than ER-a (530 and 595 amino acids, respectively).
Similarly, in rodents, PR has two isoforms, designated PR-A
and PR-B, and which are coded by a single gene through alter-
nate promoter usage. The PR-A isoform is an N-terminal
truncated version of the PR-B isoform (minus 130 amino
acids).
68 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
ER-a and ER-b have distinct patterns of expression in the
rodent brain (Greco et al., 2001; Osterlund et al., 1998;
Shughrue et al., 1997, 1998; Simerly et al., 1990). For example,
in the female rat brain, ER-a mRNA expression was found to be
most intense in the anterolateral VMH, arcuate, and the ventral
premammillary nucleus. By contrast, the expression of ER-
b mRNA was greatest in the paraventricular hypothalamic
nucleus, supraoptic nucleus, and the ventral premammillary
nucleus. Both isoforms were widely expressed in the amygdala,
with ER-a expression being highest in the medial posterodorsal
nucleus, while ER-b expression was greatest in the medial post-
erodorsal nucleus (Osterlund et al., 1998).
In the intact cycling rat, PR is present in the anteroventral
periventricular nucleus, the MPOA, the arcuate nucleus, and
the VMH during diestrous and across proestrous–estrous
(Numan et al., 1999). During mid-pregnancy, PR expression
decreases in the arcuate nucleus and VMH, most likely due to
the high levels of circulating P that are present at this time. Like-
wise, the expression of the individual PR-A and PR-B isoforms
is regulated by ovarian steroids, in a brain region-specific
manner, across the ovarian cycle. In OVX female rats, E2
administration increased the expression of both PR isoforms
in the hypothalamus and preoptic area, whereas the
subsequent administration of P decreased the expression of
both isoforms only in the hypothalamus. In intact cycling
rats, PR-A and PR-B were highly expressed in the preoptic
area during diestrous, while their expression in the
hypothalamus was low (Guerra-Araiza et al., 2003).
Activation of the PR appears to be necessary for the normal
stimulation of estrous behavior in the rodent species that are
most commonly studied in the laboratory. In OVX, estrogen-
primed female rodents, RU486, a PR antagonist, blocks the
P-mediated facilitation of sexual behavior. Likewise, P fails to
stimulate lordosis in knockout mice lacking the A isoform of
the PR (Mani and Oyola, 2012). González-Flores and
colleagues investigated the participation of the PR-A and PR-
B isoforms in stimulating lordosis in estrogen-primed OVX
female rats. These studies, which used antisense
oligonucleotides to selectively inhibit the activity of both
isoforms or of PR-B alone, indicated that PR-B activation was
necessary for lordosis induced by P, 5a-DHP, and
5b,3b-pregnanolone in OVX, estrogen-primed female rats
(Guerra-Araiza et al., 2009). Using the same experimental
strategy, Carrillo-Martínez et al. (2011) showed that P
induced the expression of tryptophan hydroxylase and
glutamic acid decarboxylase in the hypothalamus of OVX rats
via PR-B activation, whereas P reduced the expression of
tyrosine hydroxylase through the activation of both PR
isoforms (Carrillo-Martínez et al., 2011). Taken together,
these studies indicate that the PR-B isoform is essential for
the display of lordosis behavior in rats, and that, considering
the findings of studies in the mouse, the role of specific PR
isoforms in female sexual behavior may be species specific
(Mani and Oyola, 2012; Mani et al., 2009).
1.02.1.3.1.2 Rabbits
Clear cytoplasmic ER-a immunoreactivity was observed in
distinct forebrain areas of the female rabbit (Caba et al.,
2003a). Thus the nucleus X, located in the ventrolateral part
of the VMH, was very rich in cytoplasmic immunostaining.
Dense aggregations of ERa-immunoreactive (IR) neurons
were also found in the infundibular nucleus (IN), the MPOA,
the bed nucleus of the stria terminalis (BNST), and in the amyg-
dala. Numerous ERa-IR neurons were observed in the paraven-
tricular hypothalamic nucleus, but not in the supraoptic or
suprachiasmatic nuclei. The hippocampus lacked ERa-IR
neurons, but the ventral subiculum in the hippocampal forma-
tion had a dense group of such cells (Caba et al., 2003a).
In OVX, E2-treated female rabbits, PR mRNA was also highly
expressed in the hypothalamus and preoptic area. The subse-
quent administration of P resulted in a dramatic reduction of
PR mRNA in these brain regions (Camacho-Arroyo et al.,
2007, 1994). Likewise, in OVX, EB-treated does, a high
density of PR-IR cells was observed in the nucleus X, preoptic
region, paraventricular nucleus, and IN. Injections of P
provoked an intense downregulation of PR immunoreactivity
(Caba et al., 2003b). Thus, similar to the rat and guinea pig,
estrous in the rabbit is associated with increased expression
of PR in the MPOA and in the VMH.
1.02.1.3.2 Cellular Mechanisms Proposed to Explain
the Regulation of Estrous Behavior by Different Agents
In this section we describe findings regarding the cellular mech-
anisms involved in the regulation of rat female sexual behavior
by a variety of agents. They include specific intracellular path-
ways, such as those that involve protein kinase A (PKA), NO/
protein kinase G (PKG), and the Src/PR/MAPK system. As
described below, these pathways participate in estrous behavior
induced by progestins, GnRH, and leptin, and by vaginal-
cervical stimulation (VCS).
1.02.1.3.3 Protein Kinase A
The molecular mechanisms that regulate cell function often
involve changes in the specific conformation of proteins (allo-
steric processes) or in their chemical composition (e.g., methyl-
ation, acetylation, glycosylation, and phosphorylation).
Protein phosphorylation is a major mechanism through which
many hormones regulate intracellular processes (Nestler et al.,
1994). The enzymes responsible for protein phosphorylation
are called protein kinases and are specifically activated by
a number of second messengers, including cyclic AMP
(cAMP), cyclic GMP (cGMP), peptides, calcium, and phospho-
lipids (Beyer et al., 2003; Nestler et al., 1994).
cAMP is a major intracellular mediator of hormone
responses, through its role as a stimulator of a specific protein
kinase, called PKA. Early studies by Beyer et al. (1981) evaluated
the involvement of the cAMP-PKA signaling pathway for the
display of estrous behavior of OVX, E2-primed rats, finding
that subcutaneous or intracerebral administration of
dibutyryl cAMP facilitated lordosis in such animals (Beyer
et al., 1981). These seminal results were the foundation of
a unitary model to explain how, in estrogen-primed rats,
lordosis could be facilitated by P as well as by a variety of
other hormones and neurotransmitters (Beyer et al., 1981,
2003, 1997; Beyer and González-Mariscal, 1986; Blaustein,
2009; Blaustein and Mani, 2007).
In the proposed model, estrogen binds to its intracellular
receptor, and this ‘hormone-receptor’ complex is translocated
to the nucleus. There, it induces the expression of certain
proteins, most of which are in an inactive condition (inactive
 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
estrogen-induced proteins, or iEIPs). The subsequent activation
of these iEIPs occurs as a result of their phosphorylation by
cAMP-dependent PKA. cAMP can be produced from the
activation of membrane receptors linked to adenylate cyclase,
thus allowing for ‘cross-talk’ between intracellular hormone
receptors and membrane receptors. The first such ‘cross-talk’
model proposed that cAMP – generated from the activation
of membrane NE receptors linked to adenylate cyclase –
activates intracellular iEIPs. There is evidence suggesting that
one such iEIP could be the PR. This possibility is supported
by a variety of different experimental strategies, including
blocking the PR with the antiprogestin RU486, the
administration of PR antisense oligonucleotides, as well as
studies in knockout mice lacking the PR (Beyer et al., 2003,
1995, 1997; Blaustein et al., 1987). Studies suggest that
kinases (e.g., PKA) that are activated by agents that induce
lordosis do not directly phosphorylate the PR (Bai et al.,
1997), but work indirectly through the activation of MAPK,
which in turn phosphorylates serine residues on the PR
(Faivre et al., 2008; Lange et al., 2000; Qiu et al., 2003).
Nevertheless, it is important to note that diverse agents that
induce lordosis may act through different protein kinase
systems. For example, in OVX, estrogen-primed rats,
pharmacological inhibition of PKC prevented the facilitation
of lordosis by several ring A-reduced progestins, but had no
effect on lordosis induced by P (González-Flores et al.,
2006). On the other hand, lordosis induced by P, GnRH,
prostaglandin (PG), E2, and db-cAMP were blocked by
RpAMPC, an inhibitor of PKA (Ramirez-Orduna et al., 2007).
Therefore, it appears that lordosis is positively modulated by
the coordinated action of multiple intracellular signaling
systems that converge on the PR, which may be a common
effector for the facilitation of lordosis by both steroidal and
nonsteroidal agents.
1.02.1.3.3.1 NO Pathway
As described above, several lines of evidence indicate that NO is
a modulator of lordosis behavior. Studies show that the stimu-
latory effects of NO on lordosis behavior might involve the
NO-cGMP pathway. Beyer et al. (1982) found that, in OVX-
adrenalectomized female rats primed with E2, administration
of cGMP, dpc-GMP, and 50 GMP stimulated lordosis
behavior. Later, Chu and Etgen (1999) proposed that the
coupling of hypothalamic a1-adrenoreceptors to the NO-
cGMP pathway is steroid hormone-dependent and that this
pathway mediates the facilitation of lordosis by NE in OVX
rats treated with E2 and P (Chu and Etgen, 1999; Chu et al.,
1999). This idea was supported by experiments showing
that (1) icv infusion of ODQ, a selective inhibitor of NO-
stimulated cGMP, suppressed lordosis in OVX rats treated
with E2 and P, and (2) lordosis was also inhibited by icv
infusion of KT5823, a cell-permeable, highly specific
inhibitor of PKG. Moreover, the PR apparently also
participates in this pathway, as RU486, a PR antagonist,
inhibited lordosis induced by the administration of
8-br-cGMP (Chu et al., 1999).
Experiments carried out by González-Flores and colleagues
(González-Flores et al., 2007, 2009; González-Flores and
Etgen, 2004) explored whether the NO/cGMP/PKG pathway
participates in the induction of estrous behavior by diverse
69
agents. They found that icv administration of LNAME (an
NOS inhibitor) and ODQ (a specific inhibitor of soluble gua-
nylyl cyclase) suppressed lordosis behavior induced by P and
by 5a-reduced P metabolites, as well as lordosis facilitated by
VCS. Lordosis was suppressed at 2 h after drug administration
but returned to control values by 4 h. Likewise, icv infusion of
the PKG inhibitor KT5823 significantly inhibited lordosis
induced by all three progestins at 2 h after drug administration.
Moreover, they found that LNAME and ODQ also blocked
estrous behavior induced by GnRH, PGE2, and db-cAMP
(González-Flores et al., 2009). By contrast, KT5823 reduced
PGE2- and db-cAMP-induced estrous behavior, but did not
affect the behavioral response to GnRH. Thus, the NO/
cGMP/PKG pathway appears to be one important mechanism
by which estrous behavior in estrogen primed rodents can be
facilitated by a variety of progestins, neurotransmitters, and
neuromodulators, including GnRH and PGE2.
1.02.1.3.4 Leptin and Protein Kinase
As described above, leptin is also a modulator of female sexual
behavior. The possible interaction of this hormone with the
various signaling pathways implicated in regulating estrous
behavior in the rat has been investigated by González-Flores
et al. (2011). Several doses of leptin, infused icv, significantly
enhanced lordosis behavior in OVX, E2-primed female rats.
Additional experiments explored the participation of specific
receptors, such as the PR and GnRH-1 receptors, on leptin-
facilitated lordosis behavior. These experiments revealed that
the facilitatory effects of leptin on lordosis are mediated
through these receptors, as the administration of RU486 or
antide (an antagonist of the GnRH-1 receptor) prior to leptin
administration inhibited the ability of this hormone to facili-
tate lordosis (García-Juarez et al., 2011).
In a follow-up study, the same group investigated the partic-
ipation of the NO-cGMP-PKG pathway in the leptin-induced
facilitation of lordosis (García-Juarez et al., 2012). They
found that LNAME, ODQ, and KT5823 significantly reduced
the lordosis behavior induced by the icv administration of
leptin. Additionally, these authors explored the participation
of several other protein kinases, including JAK2, Src tyrosine
kinase, and MAPK, in leptin-induced lordosis behavior in
OVX, E2-primed rats fed ad libitum. Inhibitors of JAK2
(AG490), Src tyrosine kinase (PP2), and MAPK (PD98059)
caused a significant decrease in leptin-facilitated lordosis.
These results demonstrate that the stimulation of lordosis
behavior in estrogen-primed rats by leptin requires the
simultaneous or sequential activation of several signaling
pathways (see Figure 1), which most likely converge on
hypothalamic neurons that regulate the expression of lordosis
behavior (García-Juarez et al., 2012). The mechanism
through which the PR participates in the facilitation of
lordosis by leptin is unclear, but it may involve the
phosphorylation of this receptor.
1.02.1.3.5 Src Tyrosine Kinase, MAPK Pathway, and Lordosis
Behavior
The cellular tyrosine kinase c-Src may be one of the most funda-
mental components for the intracellular integration of steroid
and growth factor signaling. This kinase is expressed by virtu-
ally all eukaryotic cells, where its activation is critical for
70 Female Sexual Behavior in Rodents, Lagomorphs, and Goats

Leptin
GnRH neuron (b)

GnRH

(a)
GnRH-1

Antide
JAK2

X
PR PP2
C-Src
RU486

X
X
MAPK

P Gene
PR
Promoter

Lordosis behavior

Figure 1 Direct (a) and indirect (b) effects of leptin potentially involved in lordosis facilitation in OVX, estrogen-primed rats. A direct activation of
the long leptin receptor directly activates JAK2 system that in turn stimulates the Src-MAPK pathway to facilitate lordosis. A potential indirect effect
of leptin is depicted in (b). In this case, leptin releases GnRH through indirect activation of GnRHergic neurons. GnRH, in turn, would activate the Src
kinase (c-Src) pathway, which has been shown to activate lordosis behavior in OVX, estrogen-primed rats. As shown in the figure, specific inhibitors
of PR, RU486, GnRH-1 receptor, antide or PP2, Src inhibitor, interfere with the behavioral action of leptin. The effect of Src could be direct at the
genome or mediated by MAPK. OVX, ovariectomized; GnRH, gonadotropin-releasing hormone; PR, progesterone receptor.

generating the appropriate cellular responses to a variety of
extracellular signals, including growth factors, cytokines, adhe-
sion molecules, and antigen receptors (Brown and Cooper,
1996; Thomas and Brugge, 1997). All members of the Src
kinase family comprise a conserved tyrosine kinase (SH1)
domain, SH2, SH3 domains, and an SH4 domain at the
N-terminus that targets the protein to the cell membrane. Src
kinase activates the MAPK pathway, which is a prominent
intracellular signaling pathway that is present in virtually all
eukaryotic cells and that can modulate the activity of
a variety of effector molecules, including transcription factors,
cytoskeletal proteins, and protein kinases.
Importantly, the PR has the potential to interact with cyto-
plasmic signaling molecules as well as with nuclear elements,
as it is actively translocated between the cell cytoplasm and
the nucleus by import and export mechanisms. In cell culture
systems, ligand-activated PR has been shown to interact with
Src kinase, resulting in the stimulation of the Src/Ras/Raf/
MAPK signaling pathway (Boonyaratanakornkit and
Edwards, 2004; Leonhardt et al., 2003; Migliaccio et al.,
1996, 1998). Thus, Migliaccio et al. (1998), demonstrated
that P induced the rapid activation of MAPK through an
Src-dependent mechanism. Subsequent studies indicated
that the ER was required for the interaction between PR and
Src, as well as for the PR-induced activation of the MAPK
pathway (Migliaccio et al., 1996, 1998). On the other hand,
studies using the synthetic progestin R5020 as a ligand
indicated that, alternatively, PR can activate the Src/MAPK
pathway independently of the ER (Boonyaratanakornkit
et al., 2008).
 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
The MAPK pathway appears to be critical for the hormonal
facilitation of estrous behavior. Etgen and Acosta-Martinez
(2003) demonstrated that, in OVX female rats, if MAPK inhib-
itors (PD98059 and U0126) were infused icv during E2
priming, subsequent lordosis behavior was completely abol-
ished. Moreover, in E2-primed female rats, icv infusion of
PD98059 significantly diminished lordosis behavior induced
by a variety of agents, such as ring A-reduced progestins, the
delta opioid agonist DPDPE, GnRH, PGE2, cAMP, leptin, as
well as by VCS (García-Juarez et al., 2013; González-Flores
et al., 2009). Interestingly, lordosis induced by the cGMP
analog, 8-br-cGMP, was also inhibited by a MAPK inhibitor,
as well as by RU486, a PR antagonist. These results suggested
that cGMP-mediated facilitation of lordosis involves ligand-
independent activation of PR in the brain by MAPK
phosphorylation (González-Flores et al., 2004b).
A possible role for MAPK phosphorylation in the phenom-
enon of sequential inhibition was suggested based on the
finding that MAPK phosphorylation of the PR promotes
receptor downregulation via proteasomal degradation. This
possibility was tested in OVX, E2-primed female rats by
infusing a MAPK inhibitor (PD98059) icv, 30 min before the
first injection of P; 4 h later, these subjects did not display
lordosis. Then, 24 h later, these same animals received a second
injection of P. Normally, this second P injection would be inef-
fective at inducing lordosis (sequential inhibition); however, in
the females that had received the MAPK inhibitor along with
the first P injection showed high levels of lordosis, indicating
that sequential inhibition had been blocked by inhibiting
MAPK (González-Flores et al., 2004b). Downregulation of
hypothalamic PRs was also inhibited by PD98059 (González-
Flores and Etgen, 2004). From these findings, we suggest that
MAPK-dependent phosphorylation of PRs has a dual role in
regulating the actions of P and the PR in female rat sexual
behavior: initially, MAPK phosphorylation contributes to the
facilitatory actions of P, but subsequently it targets PR for
degradation by the 26S proteasome pathway. This
mechanism appears to underlie the phenomenon of
sequential inhibition. Further support for this idea comes
from the observation that sequential inhibition is likewise
prevented by a proteasome inhibitor (González-Flores and
Etgen, 2004).
Based on these studies, González-Flores and colleagues
proposed the novel idea that the Src/PR/MAPK pathway is an
essential component in the facilitation of estrous behavior
(see Figure 2). This model is supported by the finding that
the specific Src kinase inhibitor PP2 reduced lordosis behavior
stimulated by progestins (P and its ring A-reduced
metabolites), peptides, and VCS (González-Flores et al.,
2010; Lima-Hernández et al., 2012). Moreover, these results
suggest that several agents, including GnRH and PGE2, as
well as VCS, act on membrane GPCRs that then activate
serine/threonine kinases (PKs) such as PKA, PKC, and PKG.
Stimulation of these kinases might also activate Src kinase
through several mechanisms. For example, ring A-reduced
progestins may stimulate Src activity either by acting directly
on a membrane-associated PR or indirectly by stimulating the
release of GnRH. It appears that PR acts as a scaffold within
a multiprotein Src/MAPK signaling complex, in order
to recruit a variety of enzymatic effectors. Additionally,
71
Src–MAPK signaling also appears to interact with the classical
pathways of PR action by phosphorylating Ser-294 in this
protein, thereby enhancing its transcriptional activity (Lange
et al., 2000; Migliaccio et al., 1996, 1998).
1.02.1.4 Neuroendocrine Factors Participating in the
Behavioral Transition across the Reproductive Cycle of Rats,
Rabbits, and Goats
1.02.1.4.1 Anestrous to Estrous in Rodents
Anestrous in the female rat is associated with low serum
FSH levels, compared to those observed in animals that are
in constant estrous, pseudopregnant, or young cycling animals.
Serum LH levels are undetectable in anestrous females (Huang
et al., 1976, 1978). Likewise, estrogen and progestin levels are
also lower during anestrous, relative to those observed in any
other reproductive state. By contrast, serum prolactin levels
are sixfold higher among aging anestrous rats, compared to
those measured in young (4- to 6-month-old) animals (Huang
and Meites, 1975). Thus, the transition to the anestrous condi-
tion coincides with the decrease in plasma gonadotropins and
an increase in plasma prolactin. It has been suggested that the
progressive reduction in hypothalamic catecholamines that
occurs during aging might be associated with the anestrous
state (Huang et al., 1978). Senescent, anestrous animals display
atrophic ovaries with few primary follicles as well as infantile
uteri, suggesting diminished ovarian steroid production
(Huang and Meites, 1975).
The transition from anestrous to estrous behavior in the
cycling female rat occurs on the afternoon of proestrous,
approximately 10 h before ovulation. This transition is tempo-
rally associated with a rise in P and high circulating levels of E2.
This latter hormone is secreted in increasing amounts across
day 2 of diestrous, ultimately stimulating a surge of LH, which
in turn elicits a surge of P, produced by the granulosa cells, in
the early afternoon of proestrous (Freeman, 1994). The P surge
wanes across the morning of estrous, concomitant with
a decline in estrous behavior. During this phase, hypothalamic
PR is also downregulated, a process that participates in the
termination of estrous behavior (Blaustein and Erskine,
2002). In the experimental paradigm of sequential inhibition,
a second administration of P, given 24 h after administering
E2 and P, does not induce sexual behavior in female rats.
Sequential inhibition relies on the P-mediated induction of
the 26S proteasome, a protease that degrades PR (among
other proteins) in the brain (González-Flores et al., 2004a).
Across pregnancy, female rats fail to display estrous behavior
(Hardy, 1970), an effect that is most likely due to high
circulating levels of P.
1.02.1.4.2 Anestrous to Estrous in Rabbits
In the prepubertal or seasonally anestrous rabbit, sexual recep-
tivity increases gradually as ovarian follicles mature and begin
to secrete E2. In the adult female rabbit, under favorable envi-
ronmental conditions, estrous behavior generally persists until
after mating occurs. Thus, the proportion of females that
remain receptive decreases dramatically during the first 24 h
after mating (Beyer and Rivaud, 1969). Sexual receptivity
remains low across pregnancy, most likely due to P secreted
by the corpora lutea, which likely counteracts the stimulatory
72 Female Sexual Behavior in Rodents, Lagomorphs, and Goats

ProgesƟns VCS

GnRH PGE2
P

Gα Gα Gα
P

PKs

Cytoplasm
P
c-Src
PR

PP2
P

P MAPK
PR

Nucleus Ser-294 P
CREB STAT

? ?

Estrous behavior
havio
Figure 2 Diagram of the possible mechanisms of action through which several progestins, GnRH, PGE2, and vagino-cervical stimulation (VCS) acti-
vates the PR-Src system to elicit estrous behaviors in OVX, EB-primed rats. As shown in the upper right section of the diagram, VCS stimulates
release of GnRH and PGE2, which act on G protein-coupled membrane receptors (GPCRs). A direct action on the PR–Src neurons is shown on the
left side of the diagram by progestins. GnRH and PGE2 on membrane GPCRs to activate second messenger-regulated several kinases. Progestins
also stimulate Src activity by either acting at the membrane or indirectly through releasing GnRH or directly activating PR. Src signaling through the
MAPK system occurs in the context of a multiprotein complex in which the PR acts as a scaffold to recruit an assortment of enzymatic effectors.
Activation of the Src–MAPK system results in rapid and transient membrane effects on ion channels and membrane receptors and delayed genomic
effects mediated through proteins such as CREB or STATs modulating gene transcription. Src–MAPK signaling also interacts with the classical path-
ways of PR action by phosphorylating Ser-294 in this protein, a reaction enhancing its transcriptional activity. The cross on the arrow indicates the
site of PP2 inhibitory effect on Src action. GnRH, gonadotropin-releasing hormone; PR, progesterone receptor; PGE2, prostaglandin estradiol; PK,
protein kinase.

effects of E2 and T secreted by the ovaries. During the last week
of pregnancy, sexual receptivity was reported to increase, as
plasma P levels decrease and levels of E2 and T increase (Beyer
and Rivaud, 1969; González-Mariscal et al., 1994). After a brief
postpartum estrous, sexual receptivity continues to be inhibited
during lactation (Beyer and Rivaud, 1969).
The proceptive scent-marking behavior called ‘chinning’ has
been intensively studied with respect to changes in its expres-
sion across the female reproductive cycle. Thus, immediately
after mating, chinning is inhibited proportionally to the inten-
sity of sexual stimulation that the doe received (González-
Mariscal et al., 1997). This immediate (or ‘acute’) postmating
inhibition of chinning, which is accompanied by an overall
decrease in ambulatory activity, occurs independently of ovula-
tion and any other contribution from the ovaries and is most
likely related to changes in electroencephalographic (EEG)
brain activity (Hoffman et al., 2010; Kawakami and Sawyer,
1959; Sawyer and Kawakami, 1959). The ‘early’ inhibition of
chinning (observed across the first 4 days following mating)
depends on ovulation and endocrine signals from the ovaries,
and it is proposed to involve the simultaneous activation of the
PR and a decline in E2 receptor activation, the latter possibly
being mediated by a temporary postovulatory decline in circu-
lating E2 (Hoffman et al., 2010; Hoffman and González-
Mariscal, 2007). The long-term ‘luteal’ inhibition of chinning
that occurs across pregnancy is associated with the formation
of the corpora lutea and the high circulating levels of P
(González-Mariscal et al., 1990). This inhibition is clearly
mediated by PR activation (Hoffman and González-Mariscal,
2006). Notably, in all of the aforementioned studies, low chin-
ning frequency was associated with periods of reduced sexual
receptivity.
 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
1.02.1.4.3 Postpartum and Lactation in Rats
Intact cycling rats normally display a postpartum estrous
between 12 and 14 h after parturition (Connor and Davis,
1980a,b). Proceptivity and lordosis are displayed during this
time, and mating can result in a new pregnancy (Gilbert,
1984). Thus, at about 3 h postpartum, around the time of
ovulation, a weak lordosis response is observed; this response
becomes more intense and is accompanied by proceptive
behaviors by about 10 h postpartum (Connor and Davis,
1980a). A pregnancy resulting from mating during the post-
partum estrous normally lasts about 1 week longer than if the
fertile mating had occurred during proestrous of a typical cycle
(Woodside et al., 1981). This is due to a delay in implantation
(Lataste, 1981) that can be induced at any moment by removal
of the suckling litter for 24 h (Zeilmaker, 1964). When the
female is prevented from nursing on postpartum day 4 and
then mated, the resulting pregnancy lasts 22 or 23 days, only
1 day longer than the duration of a normal pregnancy. In this
case, there is an overlap of lactation with the last week of the
postpartum pregnancy. P sharply declines to very low levels 2
or 3 days before parturition, while E2 production is maintained
or continuously rises. Thus, at parturition, plasma levels of E2
and T are high while P levels are negligible. The initial stimula-
tory effect of E2 may be enhanced by a subsequent rise in P
plasma levels which peak around 12 h postpartum (Connor
and Davis, 1980b).
1.02.1.4.4 Postpartum and Lactation in Rabbits
In the case of the rabbit, a brief period of estrous is observed
during postpartum day 1. In one study, the proportion of
females that were sexually receptive declined to approximately
40% on lactation day 8, and then to 10% on day 12
(Hammond and Marshall, 1925). Beyer and Rivaud (1969)
studied female rabbits daily during their first and second lacta-
tions. Hammond and Marshall (1925) also observed an
intense postpartum heat in the first days of lactation. Following
this postpartum estrous, sexual receptivity showed an overall
decrease, but was still observed in about 50% of the females
at some point (Beyer and Rivaud, 1969). In this study,
a possible correlation between the display of receptivity and
the number of young in the litter was not explored. Yet, the
fact that some lactating rabbits displayed lordosis behavior
suggests that significant plasma levels of E2 and T are main-
tained across days 10 and 25 of lactation. Withdrawal of suck-
ling induces a surge of LH and FSH stimulating both follicular
growth and E2 secretion. This latter observation might explain
why the omission of one suckling episode (amounting to the
doe’s separation from the pups for 44 h, as female rabbits nor-
mally nurse only once per day) allows the recovery of sexual
receptivity in 24 h. This phenomenon, called ‘biostimulation’
(preventing the doe from nursing for at least 44 h), was
observed even after nursing was subsequently resumed
(Bonanno, 1999). In fact, biostimulation is a strategy used by
rabbit breeders to improve the fertility of lactating does
(Theau-Clément, 1998).
Lactation is also marked by a dramatic reduction in chin-
ning behavior (García-Dalman and González-Mariscal, 2012;
González-Mariscal et al., 1990). This behavior was found to
be inhibited on two levels during lactation: a transient,
dramatic, and immediate inhibition that followed each
73
nursing, and a chronic, persistent inhibition of chinning that
was observed across lactation. Thus, chinning was immediately
inhibited after each daily nursing bout, in a manner qualita-
tively similar to the immediate postmating inhibition of this
behavior (González-Mariscal et al., 1997). The immediate post-
suckling inhibition of chinning was observed regardless of the
total number of pups nursed; that is, independently of the
intensity of suckling stimulation. In contrast to the immediate
postnursing inhibition of chinning, baseline chinning levels
across lactation were inversely related to the total number of
pups that were nursed (García-Dalman and González-
Mariscal, 2012).
1.02.1.4.5 Postpartum Anestrous and Lactation in Goats
In goats displaying reproductive seasonality, the length of the
postpartum anestrous depends on several factors, including
nursing of the young and season of parturitions (Delgadillo
et al., 1998). The influence of these two factors on the duration
of postpartum anestrous was clearly demonstrated in goats
kidding on different seasons of the year, and nursing their
young for 2, 30, or 90 days. In seasonal breeds of goats, partu-
ritions generally occur during spring, and the postpartum anes-
trous can coincide with the seasonal anestrous, prolonging its
duration. This prolongation occurred because the onset of the
ovulatory activity after parturitions can only be observed
when the animals enter the breeding season. As a consequence,
in goats kidding in winter (January) or spring (May), the dura-
tion of postpartum anestrous last about 200 and 100 days,
respectively, and it is not modified by the duration of nursing,
i.e., 2, 30, or 90 days. By contrast, in females kidding in autumn
(October), the duration of postpartum anestrous is shorter
than in females kidding in January and May. Moreover, in these
cases the duration of nursing modifies the length of the post-
partum anestrous. Therefore, the postpartum anestrous lasts
around 46 days in goats kidding in autumn, and this duration
is not modified by the nursing the young for 2 or 30 days. On
the contrary, this postpartum anestrous lasted 82 days in
goats nursing their young for 90 days (Delgadillo et al.,
1998). Interestingly, in nonnursing goats, it seems that the level
of milk production does not modify the length of postpartum
anestrous. Indeed, females of the Saanen breed produce more
milk than Anglo-Nubian ones, but the length of postpartum
anestrous does not differ between breeds (Freitas et al.,
2004). These results show that in goats displaying a reproduc-
tive seasonality, the season of parturition has an important
influence on the time of onset of ovulatory activity following
delivery. The effect of nursing is observed only in females
kidding during their breeding season.
1.02.1.5 Female Sexual Motivation in Rats and Goats
In this section we focus mostly on rats, a species in which the
study of sexual motivation has been approached by different
methodologies that reflects the difficulty in defining this
concept (for reviews, see Agmo, 1999; Pfaus, 1999). We will
also include a description of the male effect in goats which
clearly demonstrates the plastic capacity of the brain to rapidly
respond to hormonal release with fast behavioral changes.
As described above early studies, Beach, (1976) identified
the motivational or appetitive components of female sexual
74 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
behavior as those proceptive or soliciting behaviors (also called
paracopulatory behaviors) displayed by the estrous female, to
attract and facilitate the sexual interaction with the male
(Blaustein, 2009). These behaviors will be displayed, when
hormonal levels are adequate, because the male rat is an incen-
tive stimulus for the female (Agmo, 2003). That is, the female
does not need to associate the male with a previous learning
experience or a rewarding sexual encounter to approach the
male and display solicitation and proceptive behaviors because
the male is an unconditional incentive. When tested under
seminatural or natural conditions, it is the female that usually
initiates contact with the male by approaching and running
away from him (McClintock and Adler, 1978). This type of
solicitation pattern appears to be the most effective way to acti-
vate pursuit and mating by the male because, under seminat-
ural or natural conditions, 90% of the intromissions
followed a female solicitation (McClintock and Adler, 1978).
This ability to control the rate of the sexual interaction has
also been observed in laboratory conditions using different
methodologies including pressing a lever to copulate with
a male rat (Bermant, 1961), mating with tethered males
(Edwards and Pfeifle, 1983), copulating in bilevel chambers
(Pfaus et al., 1999), or mating in cages with one hole (Erskine,
1989; Paredes and Alonso, 1997) or four holes (Ismail et al.,
2009; Yang and Clemens, 1997) in which the female is able
to pace the sexual interaction.
1.02.1.5.1 Paced Mating and Female Sexual Motivation
When females pace the sexual interaction, fewer intromissions
are needed to induce the progestational state of pregnancy; rats
also show a higher release of prolactin and have a higher
number of pups than females not pacing the sexual interaction.
The behavioral and physiological differences between paced
and nonpaced mating have been extensively described before
(Blaustein and Erskine, 2002; Erskine, 1989). A series of studies
by different groups have shown that the ability to control or
pace the sexual interaction induces a reward state as evaluated
by conditioned place preference (CPP) (Ismail et al., 2009;
Paredes, 2009; Paredes and Fernández-Guasti, 2008). The
combination of paced mating and CPP reduces the influence
of confounding variables and enhances the rewarding aspects
of mating. The different factors that contribute to the reward
state induced by paced mating have been described before
(Paredes, 2009; Paredes and Fernández-Guasti, 2008) and
will not be discussed in the present chapter. One key aspect
is that only when males or females control the sexual interac-
tion they develop a reward state. When males and females
mate without controlling the sexual interaction, no CPP is
observed (Martínez and Paredes, 2001) indicative that under
this conditions the sexual interaction does not induce a reward
state. There is one study showing that females that mated
without pacing the sexual interaction with only one male in
three different conditioning sessions showed CPP (Meerts
and Clark, 2007). In our laboratory, we directly tested whether
females that mated with the same male along the conditioning
sessions developed CPP and no changes were observed, that is,
no CPP was induced. Possible explanations for these discrep-
ancies have been presented before (Camacho et al., 2009).
It is also well established that sexual behavior induces
a reward state as evaluated by CPP in male rats, mice, and
hamsters (see Paredes, 2009 and references therein). There
are also several indirect lines of evidence indicating that
opioids are released during sexual behavior in both male
and female rats (see Paredes, 2014; Pfaus and Gorzalka,
1987 for reviews). Therefore, it is not surprising that the
reward state induced by mating in male and female rats is
mediated by opioids. Systemic administration of the opioid
antagonist naloxone blocks the CPP induced by mating in
males (Agmo and Berenfeld, 1990; Miller and Baum, 1987)
and females (Paredes and Martínez, 2001). Together with
the data on the effects of administration of naloxone in
different brain regions before mating and CPP, it has been
suggested that in males and females there is a common opioid
system mediating sexual reward in which the MPOA plays
a crucial role (review in Paredes, 2014). The release of opioids
during sexual behavior, which would induce a CPP, ensures
that the behavior will be repeated for the simple reason that
it is rewarding, favoring the survival of the species. This can
be achieved through the social decision-making network
that regulates adaptive behaviors. This network is formed by
two circuits important in behavioral regulation. One is the
mesolimbic reward system and the other is the social behav-
ioral network composed of several brain regions, including
the MPOA, that regulate different forms of social behaviors
which were already present in early vertebrates (O’Connell
and Hofmann, 2011).
1.02.1.5.2 The Importance of Testing Conditions in the
Analysis of Sexual Motivation
In natural and seminatural conditions, males and females
have a different temporal pattern of copulation. They mate
in groups when estrous is synchronized in females that live
together interacting with several males (McClintock and
Adler, 1978; McClintock and Anisko, 1982; McClintock
et al., 1982). Males and females mate with several partners,
changing them in the middle of copulation without an
ordered sequence of behavioral events (as is characteristic of
laboratory testing). A female can receive an ejaculation after
few intromissions if she encounters a male that has already
experienced intromissions with other females and a male
can achieve intromissions with one or several females. What
is clear is that both sexes experience a similar amount of copu-
lation (McClintock and Anisko, 1982; McClintock et al.,
1982), controlling the sexual interaction and assuring that
the rewarding state induced by mating outlasts the execution
of the behavior, making sex rewarding for both sexes
(Martínez and Paredes, 2001).
A recent series of studies have evaluated rat sexual
behavior in a seminatural environment where groups of three
males and four females are housed together for 8 days and
evaluating sociosexual interactions during the entire period
of behavioral estrous. Contrary to what has been shown in
laboratory conditions female proceptive behaviors were not
reduced and rejection behavior did not increase at the end
of behavioral estrous. In the same study, it was shown that
females showed a preference for a male with whom they dis-
played the higher number of lordosis. The preferred male also
had more intromissions and ejaculated more frequently than
the avoided male, the one with which the female displayed
the lowest number of lordosis. Yet, even from the avoided
 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
male, the female received mounts, intromissions, and ejacula-
tions (Chu and Agmo, 2014). Using the same seminatural
environment, it was shown that male reproductive success is
related to the intensity of copulatory behavior, for example,
the higher the number of intromissions and ejaculations the
higher the likelihood of producing offspring (Chu et al.,
2015). A surprising observation in these series of studies is
that females responded with lordosis to almost every mount
by the male from the beginning of estrous until the end of
it (Chu and Agmo, 2014) indicating that the female suddenly
changes from a state of complete nonreceptivity to full recep-
tivity and then abruptly changes back to nonreceptivity with
males exceptionally mounting the female before the mount
was able to induce lordosis (Chu and Agmo, 2015). Accord-
ing to Agmo and colleagues, their results indicate that initia-
tion and termination of copulatory behavior are tightly
associated with sudden modifications of the amount of
female paracopulatory behaviors and male pursuit of the
female. Thus, from the beginning to the end of behavioral
estrous, the female is fully receptive displaying lordosis in
response to every mount by the male (Chu and Agmo,
2015). These observations are at odds with what has tradi-
tionally been observed in laboratory conditions in which
only one male and one female are tested together and where
the transition from nonreceptivity to receptivity (and vice
versa) have been reported to be gradual. These studies remind
us that the context in which the sexual interaction is observed
can determine how the behavior is expressed. They also exem-
plify the complexity of the sexual behavior response, which
can have different expressions depending on the context in
which it is displayed.
1.02.1.5.3 The Male Effect
In goats, introduction of males into a group of seasonally
anovulatory goats stimulated and synchronizes their sexual
activity during the anestrous season (Chemineau, 1983; Ott
et al., 1980; Shelton, 1960). This phenomenon, where socio-
sexual relationships between males and females impact the
occurrence of ovulation, is known as the ‘male effect’
(Figure 3). The exposure of females to males induces a rapid
increase of LH secretion followed by estrous behavior and
ovulation (Bedos et al., 2014; Hawken and Martin, 2012;
Vielma et al., 2009). A variable proportion of goats exposed
to males show estrous behavior at the first ovulation induced
by males, between 2 and 5 days after the first contact with
them. Most of these goats display a short ovulatory cycle of
5–7 days of duration, followed by a second ovulation
7–10 days after the introduction of males. These second
ovulations are associated with estrous behavior and an
estrous cycle of normal duration (Chemineau, 1987;
Delgadillo et al., 2002; Walkden-Brown et al., 1999).
The endocrine and sexual responses of female goats to the
male effect depend on various sensory cues emitted by males,
including their odor but also the level of male sexual behavior
(Delgadillo et al., 2009). In fact, the exposure of anovulatory
goats to hair of males obtained during their breeding season
stimulates the activity of GnRH neurons, LH secretion, and
ovulation (Claus et al., 1990; Hawken and Martin, 2012;
Okamura et al., 2010). Thus, the olfactory cues are considered
the primary signal involved in the response of female goats to
75
50
Goats showing estrous (%)
40
30
20
10
0
0 5 10 15
Days after buck introduction
Figure 3 Daily proportions of goats displaying estrous behavior. One
group of females was exposed to male goats subjected to natural
changes in day length ( ), and were therefore in sexual rest. The other
group of females was exposed to males goats subjected to 2.5 months
of artificially long days (16 h of light per day) from November 1 fol-
lowed by natural day length ( ), and were therefore sexually active.
Females and males were put in contact during the seasonal anestrous.
Adapted from Delgadillo, J.A., Flores, J.A., Veliz, F.G., Hernandez, H.F.,
Duarte, G., Vielma, J., Poindron, P., Chemineau, P., Malpaux, B., 2002.
Induction of sexual activity in lactating anovulatory female goats using
male goats treated only with artificially long days. J. Anim. Sci. 80 (11),
2780–2786.
the male effect. Indeed, 4-ethyloctanal is the olfactory molecule
that activates the GnRH pulse generator (Murata et al., 2014).
Nonetheless, the proportion of goats that ovulate is lower
when exposed to olfactory cues alone than after exposure to
whole males (Claus et al., 1990; Walkden et al., 1993). This
difference may be related to the intensity of the sexual behavior
of males. In fact, exposure of males to 2.5 months of long days,
starting on November 1, followed by natural photoperiod,
stimulates the secretion of T (Figure 4) and increases their
20
LD
15
10
5
0
Nov Jan Mar May Jul Sep
Figure 4 Plasma testosterone concentrations (mean  SEM) in male
goats subjected to natural changes in day length ( ), or to 2.5 months
of artificially long days (LD: 16 h of light per day) from November 1 fol-
lowed by natural day length ( ). Blood samples were taken once
a week. Stars indicate significant differences between groups. Adapted
from Delgadillo, J.A., Flores, J.A., Veliz, F.G., Hernandez, H.F., Duarte,
G., Vielma, J., Poindron, P., Chemineau, P., Malpaux, B., 2002. Induc-
tion of sexual activity in lactating anovulatory female goats using male
goats treated only with artificially long days. J. Anim. Sci. 80 (11),
2780–2786.
76 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
odor; and sexual behavior during the nonbreeding season, i.e.,
in March and April. These photo-stimulated, sexually active
males are more efficient than untreated, sexually inactive
ones, to induce ovulation in anovulatory goats. In fact, the
photo-stimulated males induce ovulation in 80–100 % of
goats, whereas only around 10% of females ovulated when
exposed to the untreated males. The higher ovulatory response
of goats exposed to the photo-stimulated males seems to be
related with the intensity of the sexual behavior, rather than
with more intense male olfactory cues. Indeed, goats exposed
to photo-stimulated males that also displayed an intense sexual
behavior, displayed the preovulatory LH surge, and ovulated in
>95% of cases. By contrast, goats exposed to the sedated,
photo-stimulated males to prevent the display of sexual
behavior showed neither a preovulatory LH surge nor ovula-
tion, even though the males were emitting a strong odor
(Martínez-Alfaro et al., 2014). Interestingly, the continuous
presence of the photo-stimulated males prevents the display
of seasonal anestrous (Delgadillo et al., 2015). Altogether,
these results strongly suggest that in goats, the intensity of
sexual behavior displayed by males is a critical factor in the
responsiveness of females to the male effect. Finally, it is impor-
tant to mention that the use of photo-stimulated males to
control the sexual activity of females is a sustainable breeding
technique for goat’s reproduction.
1.02.1.5.4 Neurogenesis and Sexual Behavior
The incorporation of new neurons that are produced in the
adult brain (neurogenesis) is well documented in several
species including humans. Neurogenesis in the adult mostly
occurs within two brain regions: the subventicular zone
(SVZ) of the lateral ventricles, where the new cells and neurons
migrate to the olfactory bulb system, and the subgranular zone
(SGZ) of the dentate gyrus of the hippocampus. Several studies
have clearly demonstrated that social and reproductive behav-
iors modulate adult neurogenesis (see Peretto and Paredes,
2014 and references therein). For example, in female mice,
where recognition of the sexual partner is essential for the
display of sexual behavior and maintenance of pregnancy,
the simple exposure to male pheromones increases the prolif-
eration in the SVZ, the number of new cells in the MOB
(Mak et al., 2007) and in the AOB (Oboti et al., 2011). Chronic
exposure (28 days) to male mice soiled bedding produces
a significant increase in the number of new neurons in the
AOB of adult females that is dependent on vomeronasal organ
activity (Oboti et al., 2009). The newborn granule cells that
reach the AOB are functional, activated by male pheromones,
and are important in mate recognition processes such as in
the Bruce effect (reviewed in Oboti et al., 2011; Peretto and
Paredes, 2014).
Few studies have evaluated the effect of mating in females
on neurogenesis. For example, in hamsters, new cells generated
in the adult are activated by Fos expression after mating
(Huang and Bittman, 2002). Since the olfactory bulbs and
the processing of chemosensory relevant cues are crucial for
a successful reproduction, we investigated if mating itself can
increase the number of new cells or neurons along the
SVZ-OB system. To that aim we allowed female rats to pace
the sexual interaction or to mate in a condition where they
were not able to control the sexual stimulation received. We
added groups exposed to banana scent or to a sexually
experienced male but where physical contact was not
possible. The proliferation marker BrdU was administered 1 h
before the behavioral test, at the end of the behavioral test
(which lasts 1 h), and the third injection was administered
1 h later. Females were then sacrificed at different time
intervals to evaluate the number of new cells and the number
of new neurons. We found that 15 days after mating there is
an increase in the number of new cells in the granular cell
layer of the AOB in females that paced the sexual interaction
in comparison to the other groups, indicating that pacing
behavior promotes an increase in cell proliferation that
induces a higher density of new cells in the AOB (Corona
et al., 2011). Indeed when subjects were sacrificed 2 days
after the behavioral test, an increase in the number of cells in
the rostral migratory stream was found in females that
mated, either pacing or not the sexual interaction, suggesting
that sexual behavior induces cell proliferation in females
independently of the possibility of pacing the sexual
interaction. When females were sacrificed 45 days after the
behavioral test no differences in the number of cells were
found in the OB, but a significantly higher number of
neurons were observed in the AOB in the groups that mated
(Corona et al., 2016). In the studies described above, females
mated only once. Yet, if the stimulus is repeated, that is, if
rats are injected with BrdU on the day of the first behavioral
test and then mate in three more sessions, a higher number
of cells are observed in the granular layer of the AOB in
comparison to those females that mated only once.
Moreover, an increase in the number of cells is observed in
other layers of the AOB and MOB in females that paced the
sexual interaction. When the number of neurons was
analyzed, a significant increase was observed in both the AOB
and MOB suggesting that repeated paced mating promotes
the arrival of more newborn neurons to the OB (Arzate et al.,
2013). Together, these results indicate that sexual behavior in
females can induce long-lasting plastic changes in the
olfactory bulbs. Future studies will need to determine what
the function of the new neurons is.
Adult neurogenesis has also been found in sheep (Brus
et al., 2013, 2014). However, the contribution of this phenom-
enon within the context of sexual behavior in ruminants is not
known. The only result reported so far in sheep is that the intro-
duction of the ram into a group of anestrous ewes induces an
increase in cell proliferation in the hippocampus (Hawken
et al., 2009). However, the functional significance of this effect
is unclear.
1.02.1.6 Conclusions
Most of the research analyzing the interaction between
hormones, receptors, and cellular mechanisms that trigger
female sexual behavior has been done in rodents, rabbits,
and goats. There are obvious differences, for example, in the
length of the estrous cycle. In the case of the rat, ovulation
occurs in response to hormonal changes with E2 and P secre-
tion at regular intervals. In the rabbit, ovulation occurs
following coitus, being reflexive or induced, while in
goats the reproductive seasonality is mainly controlled by
 Female Sexual Behavior in Rodents, Lagomorphs, and Goats
photoperiod. There are also similarities between species
regarding the physiological control, as well as differences in
behavioral patterns. For example, during estrous, female
rodents display a series of stereotyped behaviors including
attractivity, proceptivity, and receptivity in order to attract
a male and copulate. Proceptive female rabbits display circling
of the male, elevation, and lateral displacements of the tail
(flagging), and a scent-marking behavior called ‘chinning.’ In
the female goat (and in most ruminants), motor activity
increases when estrous starts. Goats express vocalizations and
male-type courtship behavior and female–female reciprocal
mounting to attract males and stimulate sexual activity. It is
evident then that although the behavioral patterns are different
among the three species, they are directed to attract the male.
On the other hand, the receptive response in rats and rabbits
is similar. Both display a clear lordosis posture in response to
male mounts, while female goats remain immobile when in
estrous to facilitate mounting by the male.
With respect to effects of hormones on estrous behavior, E2
is important for the three species but the role of P is different.
Estrous behavior in female rats is triggered by the combined
action of E2 and P while in rabbit, E2 is the principal gonadal
hormone that promotes estrous behavior. In female goats, an
increase in E2 concentration is observed before the expression
of sexual behavior, and exposure to P may be necessary for the
full display of sexual behavior at the onset of the breeding
season. Intact cycling rats normally display a postpartum
estrous between 12 and 14 h after parturition while in the
case of the rabbit, a brief period of estrous is observed during
postpartum day 1. In goats displaying reproductive seasonal-
ity, the length of postpartum anestrous depends on several
factors, including nursing of the young and season of
parturition.
It is also evident that much research is needed in the field. A
number of studies, many of which have been carried out in
rodent species, demonstrate that nonsteroidal hormones
participate in the regulation of female sexual behavior. These
include GnRH, OT, leptin, NO, etc. As well, the cellular mech-
anisms involved in the regulation of female sexual behavior by
a variety of intracellular pathways including PKA, PKC, PKG,
and the Src/PR/MAPK system have been studied with detailed
in the rat. Future studies need to address the importance of
these systems in the control of female sexual behavior in other
species.
Sexual motivation has mostly been studied in rats where it
has been shown that the context in which the animals are tested
can modify the expression of the behavior. A clear example of
the interaction between the context and behavior is also
observed in the ‘male effect’ in goats. This is mediated by olfac-
tion but clearly shows the capacity of the brain to respond to
external stimuli modifying the interaction between hormones,
receptors, and cellular mechanism in different brain regions to
modify behavior. The analysis of these interactions requires,
without doubt, a comparative approach.
Acknowledgments
Research from Paredes supported by CONACYT: Fronteras 374;
DGAPA: IN210215.
77
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 1.03 Parental Behavior
G González-Mariscal, Centro de Investigación en Reproducción Animal, CINVESTAV-Universidad Autónoma de Tlaxcala, Tlaxcala,
México
M Caba, Centro de Investigaciones Biomédicas, Universidad Veracruzana, Xalapa, Veracruz, México
KL Hoffman and AI Melo, Centro de Investigación en Reproducción Animal, CINVESTAV-Universidad Autónoma de Tlaxcala,
Tlaxcala, México
Ó 2017 Elsevier Inc. All rights reserved.
This chapter is a revision of the previous edition chapter by G. González-Mariscal, C.H. Kinsley, volume 1, pp. 109–138, Ó 2009, Elsevier Inc.

1.03.1 Introduction 84
1.03.2 Behaviors at Parturition: Combined Actions of Hormones and Somatosensory Stimuli 84
1.03.3 Postpartum Activities 85
1.03.3.1 Nursing: Variability Rules 85
1.03.3.2 Maternal Behavior in Nonlactating Females: What Does It Reveal? 85
1.03.4 Neural Control 86
1.03.4.1 Cortex, Trigeminal Complex, and Olfactory Circuit 86
1.03.4.2 Septum and BNST 86
1.03.4.3 MPOA and Its Connections 86
1.03.4.3.1 Studies Using Lesion, Inactivation, or Deafferentation Techniques 86
1.03.4.3.2 Studies Using Implantation of Hormones and Detection of Their Receptors 87
1.03.4.4 Midbrain Tegmentum, PVN, and Habenular Complex 87
1.03.4.5 Sampling the Active Parental Brain 88
1.03.4.5.1 The Expression of Immediate Early Genes 88
1.03.4.5.2 Other Methods 88
1.03.5 Impact of Maternal Behavior on the Mother: Effects on Neuroanatomy, Cognition, and Emotional Reactivity 89
1.03.5.1 Theoretical Context 89
1.03.5.2 Fear and Anxiety Regulation 89
1.03.5.2.1 Behaviors Affected 89
1.03.5.2.2 Brain Mechanisms 89
1.03.5.3 Regulation of Cognitive Effects 89
1.03.5.3.1 Learning and Memory 89
1.03.5.3.2 Neuroanatomical Changes Observed in the Maternal Brain 89
1.03.6 Timing: A Little-Explored Dimension of Maternal Care 89
1.03.6.1 A ‘Healthy Clock’ Is Necessary for Ovulation and Pregnancy 99
1.03.6.2 Circadian Timing of Parturition 101
1.03.6.3 Circadian Rhythms in Breast and Breast Milk 101
1.03.6.4 Daily Rhythms in the Mother during Lactation 102
1.03.6.4.1 Locomotor Behavior 102
1.03.6.4.2 Core Body Temperature 102
1.03.6.4.3 Rhythms of PER1 Protein in Neuroendocrine Cells 102
1.03.6.4.4 Rhythms of PER1 Protein in Areas Related to Maternal Behavior 103
1.03.7 Beyond Motherhood: Maternal Behavior as an Experimental Model for the Study of Some Neuropsychiatric
Disorders 103
1.03.7.1 Emotional Systems 104
1.03.7.1.1 Motivation: Concepts and Definitions 104
1.03.7.2 Maternal Behavior in the Context of SEEKING 105
1.03.7.3 Maternal Behavior in the Context of the CARE Emotional System 106
1.03.7.3.1 Relationship of the CARE Emotional System to Neuropsychiatric Disorders 106
1.03.7.3.2 Early Parental Preoccupations and Their Relationship to Obsessive–Compulsive Disorder 106
1.03.8 Conclusions and Perspectives 107
Acknowledgments 108
References 108

Hormones, Brain, and Behavior, 3rd edition, Volume 1 http://dx.doi.org/10.1016/B978-0-12-803592-4.00003-1 83

84 Parental Behavior
1.03.1 Introduction
The chapters written for the first and second editions of this
book presented a thorough review of the many factors that
participate in the onset, maintenance, and decline of parental
behavior in mammals (González-Mariscal and Poindron,
2002; González-Mariscal and Kinsley, 2009). In addition, two
books (Numan, 2006; Numan and Insel, 2003), several review
chapters in books (González-Mariscal and Melo, 2013; Numan
et al., 2006) and journals (Olazábal et al., 2013a, b), plus
a special issue of the journal Hormones and Behavior (January
2016), all devoted to the topic of parental behavior, have
appeared within the last 15 years. This vast literature indicates
that new lines of research have emerged, both from the area of
parental behavior itself and from the finding that the display of
this activity modifies the mother’s brain itself as well as several
behavioral and physiological functions. Moreover, maternal
behavior (MB) is no longer viewed as a ‘reflexive’ activity but,
rather, as a complex function involving the coordinated activity
between neural circuits regulating other functions (e.g., social-
ization, emotionality, fear) but ‘recruited’ to subserve MB, and
those mediating ‘strictly maternal’ activities (e.g., nursing, nest
building). From this background, for this third edition chapter
we chose to (1) Highlight topics that have emerged recently,
such as (a) the effects of mother–young contact on the
maternal brain; (b) the time dimension in reproductive physi-
ology and maternal care; (c) maternal behavior as a model for
studying neuropsychiatric disorders. (2) Reflect on what new
questions can be asked regarding parental care and where the
field stands now.
1.03.2 Behaviors at Parturition: Combined Actions
of Hormones and Somatosensory Stimuli
Placentophagia and amniotic fluid ingestion are consistently
observed following delivery in most mammals studied
(Poindron et al., 2007a; Gregg and Wynne-Edwards, 2006;
Kristal, 1991; Melo and González-Mariscal, 2003). Moreover,
this activity has also been reported in males from some bipa-
rental species, e.g., dwarf hamsters (Phodopus campbelli; Gregg
and Wynne-Edwards, 2005; Jones and Wynne-Edwards, 2000),
California mice (Peromyscus californicus; Lee and Brown, 2002;
Perea-Rodríguez and Saltzman, 2014). Despite its prevalence
in mothers and in fathers of some species, the biological func-
tion or advantage of placentophagia has not been determined.
The interaction between mother and offspring at parturi-
tion is critical to ensure a continuous, ‘unfailing’ maternal
responsiveness across lactation, especially in primiparous
females. Clear evidence in rabbits (González-Mariscal et al.,
1998b), rats (Bridges, 1975), sheep (Poindron et al., 2007a),
and goats (Poindron et al., 2007b) has shown that interfering
with mother/young contact at this time disrupts or seriously
alters maternal behavior. A similar interference in early- or
midlactation provokes milder effects. These results suggest
that the particular conditions that exist at parturition – in
terms of changes in the concentration of several hormones
in blood (e.g., oxytocin, OT; prostaglandin F2-alpha, PGF2a;
corticosteroids; prolactine, PRL; progesterone; estradiol; for
review, see González-Mariscal and Poindron, 2002),
contractions of the uterus, changes in the distribution, and/
or number of receptors for specific hormones and neurotrans-
mitters in the brain, simultaneously with the multisensorial
perception of stimuli from the newborn – favor the consolida-
tion and direct the responsiveness of the mother to the young.
In the case of ungulates an additional process takes place at
this time: the recognition of the mother’s own offspring which
determines the exclusive nursing of a particular lamb or kid
across lactation (see the following section). Parturition has
long been recognized as a critical period for the consolidation
of maternal behavior, and recent evidence is beginning to
unveil the factors underlying this process. Results obtained
in rats suggest that a pathway similar to learning mediates
this consolidation (Fleming et al., 1996; Graham et al.,
2006; Li and Fleming, 2003). Dopaminergic neurotransmis-
sion (involving D1 and D2 receptors; Byrnes et al., 2002)
and OT action in the nucleus accumbens (NAcc) shell (D’Cu-
nha et al., 2011) seem to be critical in this regard.
Vaginocervical stimulation (VCS), received at parturition,
plays a major role in the establishment of maternal behavior.
Peridural anesthesia results in the failure to accept the suckling
attempts of the newborn in primiparous sheep and goats
(Poindron et al., 2007a,b). Experimental evidence suggests
that these effects may be mediated by the release of OT occur-
ring at parturition: in rats, icv injections of an OT antagonist at
this time prevent the onset of maternal behavior (Van Leen-
goed et al., 1987) and in sheep, the disruptive effect of peri-
dural anesthesia is partly overcome by icv injection of OT
(Krehbiel et al., 1987). Similar results have been obtained in
nonpregnant animals: VCS (Kendrick et al., 1991) or icv OT
injections (Kendrick et al., 1987) promote maternal behavior
in multiparous ewes primed with ovarian steroids; conversely,
an OT antiserum reduces the facilitation of maternal behavior
normally seen in virgin rats primed with ovarian steroids
(Pedersen et al., 1985).
OT has been shown to regulate many forms of social
behavior, such as maternal nurturing and bonding (Rilling
and Young, 2014), social recognition (Ferguson et al., 2001;
Skuse et al., 2014), and pair-bonding (Johnson and Young,
2015; Young and Wang, 2004; Numan and Young, 2016).
There are large variations in the topography of OT receptors
(OTRs) in the mammalian brain, for instance, in rats, OTR
binding and mRNA have been detected in the medial preoptic
area (MPOA; Numan and Insel, 2003) and the prelimbic cortex
(Krémarik et al., 1993; Young et al., 1997). OTR binding
increases on postpartum day 1 – relative to pregnancy day
15 – in the bed nucleus of the stria terminalis (BNST) and
ventromedial nucleus of the rat hypothalamus (Insel, 1990),
and over the course of pregnancy and lactation, changes in
OTR mRNA have been documented (Young et al., 1997). In
the socially monogamous and biparental prairie voles a high
abundance of OTR has been observed in the prelimbic cortex,
BNST, NAcc, and lateral amygdala. Moreover, in biparental
prairie voles there is a correlation between individual variation
in OTR expression in the NAcc and spontaneous parental
behavior (Olazábal and Young, 2006a, b). In contrast, the
polygamous, uniparental montane and meadow voles have
few OTR in the NAcc and lateral amygdala but high densities
in the lateral septum (LS; Insel and Shapiro, 1992). Further-
more, manipulating the density of OTR in the NAcc provokes

alterations in pair-bonding (Keebaugh et al., 2015; Keebaugh
and Young, 2011; Ross et al., 2009).
In rabbits, also polygamous and uniparental (González-
Mariscal et al., 2016), a high density of OTR has been found
in the prefrontal cortex (PFC), preoptic area (POA), LS, hippo-
campus, and medial amygdala (Jiménez et al., 2015). OT is
released at parturition (Fuchs and Dawood, 1980) and at each
nursing bout (Fuchs et al., 1984). Despite their restricted contact
with the young throughout lactation (a single nursing bout per
day, lasting around 3 min), indirect evidence suggests a role of
OT for the regulation of specific aspects of rabbit maternal
behavior. A minimal number of suckling kits (four to five) is
essential for (1) maintaining maternal responsiveness across
lactation (González-Mariscal et al., 1998, 2013b); (2) express-
ing nursing behavior with circadian periodicity (González-
Mariscal et al., 2013a); and (3) allowing a normal duration of
the nursing bout (González-Mariscal et al., 2013b). Moreover,
suckling increases the number of c-FOS-IR cells in the paraven-
tricular nucleus (PVN; González-Mariscal et al., 2009), and the
amount of OT released at each nursing bout is dependent on the
number of suckling kits (Fuchs et al., 1984).
1.03.3 Postpartum Activities
1.03.3.1 Nursing: Variability Rules
This is undoubtedly the main behavior displayed by all
mammalian mothers toward their progeny, from parturition
until weaning. Yet, the specific characteristics of nursing and
the display of activities that surround it vary greatly among
species. For instance, in rodents, nursing is preceded by
retrieving the pups that have strayed away and grouping
them in the nest. Rabbits and sows also nurse within a nest,
but they do not retrieve their young (despite their small size);
ewes and goats do not retrieve their large, well-developed
offspring, and they nurse in the absence of a nest. The predict-
able, unfailing display of nursing and its associated behaviors
depends on several hormones that act in late pregnancy (e.g.,
PRL; Andrews and Grattan, 2002; González-Mariscal et al.,
2000, 2004b) and throughout lactation (e.g., OT and vaso-
pressin; Landgraf and Neumann, 2004; Bosch et al., 2005).
The fine-tuning of maternal behavior, that is, the display of
nursing with its associated species-typical ethogram
throughout lactation, depends largely on somatosensory
factors. Thus, in species that give birth to multiple young
(such as rodents, rabbits, and pigs), recognition of the mother’s
own progeny is irrelevant as they effectively nurse young other
than their own (Algers and Uvnäs-Moberg, 2007; González-
Mariscal and Gallegos, 2007; Rosenblatt and Lehrman,
1963). In these animals stimuli coming from the progeny regu-
late other aspects of maternal behavior. Specifically, in rats the
adoption of a high-crouch nursing posture critically depends
on the quantity and quality of the ventral stimulation provided
by the suckling pups (Stern and Johnson, 1990; Stern et al.,
1992). Moreover, in rats the number and duration of nursing
bouts per day declines from midlactation onward (as does
the amount of milk produced) in relation to the intensity of
suckling, which becomes more vigorous as the pups grow
(Mena and Grosvenor, 1972). In rabbits, nipple stimulation
plays a major role in regulating the duration of each nursing
Parental Behavior 85
bout: the normal c.3 min day
1 (González-Mariscal et al.,
1994; Zarrow et al., 1965) requires a minimum litter size of
five. When a single pup is provided, the time inside the nest
box (during which nursing occurs) is greatly increased, regard-
less of the day of lactation (González-Mariscal et al., 2013b). A
similar effect is observed when the mother’s nipples are covered
or removed (thelectomy) or when the kits’ mouths are covered
(González-Mariscal, 2007). In sows, suckling stimulation from
the piglets modulates a specific component of the maternal
ethogram: the mother’s grunting (Algers and Uvnäs-Moberg,
2007). As the sow lies down to initiate nursing, she emits
a type of grunting that signals the piglets to her teats. Their
suckling promotes the initial release of OT which, in turn, trig-
gers a change in the type of maternal grunting: the piglets
respond to it by suckling more vigorously, thus initiating
milk letdown.
In contrast to the above species, ewes and goats show selec-
tive nursing throughout lactation; that is, they will accept at the
udder only their own lamb or kid and actively reject alien ones
(Poindron et al., 2007a,b). The recognition of the mother’s
own offspring in these species depends on the perception of
the olfactory signature of the newborn at parturition and its
consolidation as an olfactory memory (Lévy et al., 2004). As
lactation progresses, visual and acoustic cues begin to play an
important role in the mother’s recognition of her progeny
(Nowak et al., 2011; Poindron et al., 2007b) and also in the
reciprocal process, that is, in the capacity of the young to recog-
nize their own mother (Poindron et al., 2007a,b; Nowak et al.,
2007). This mutual recognition is essential to adjust the phys-
iology and behavior of both parties to each other across lacta-
tion. Proximity with the offspring is essential to maintain such
maternal memory because ewe–lamb separation for 3 days
leads to a loss of maternal responsiveness and rejection of
the young (Keller et al., 2005). Remarkably, goat mothers
continue to recognize the vocalization of their own kids for
up to 13 months postweaning (Briefer et al., 2012).
1.03.3.2 Maternal Behavior in Nonlactating Females: What
Does It Reveal?
Although the hormones of pregnancy prime the maternal brain
to effectively respond to the stimuli she receives from the young
starting at parturition, nonpregnant, nonlactating rodents can
behave maternally under specific conditions. Thus, rats,
hamsters, and gerbils gradually behave maternally to foster
young after several days of cohabitation with them (for review,
see González-Mariscal and Poindron, 2002). Virgin female
mice of several strains are spontaneously maternal upon first
exposure to foster pups with a latency of a few minutes
(Gandelman, 1973; Noirot, 1972). This supports the idea
that, in these rodents, the hormones of pregnancy and lactation
are irrelevant for the onset of maternal behavior. However,
important differences exist between the care exhibited by truly
lactating versus sensitized mice (Stolzenberg and Rissman,
2011). Specifically, (1) the latency to retrieve pups and adopt
crouching over the litter is significantly smaller in lactating
animals and (2) the number of pups retrieved is larger in
lactating mice. By contrast, intact doe rabbits do not become
maternal even after 2 weeks of daily exposure to kits
(González-Mariscal et al., 2004a, 2015). Yet, in both rats
86 Parental Behavior
(Fleming and Rosenblatt, 1974a,b) and rabbits (González-
Mariscal et al., 2004b, 2015; Chirino et al., 2007), lesions to
the main or the accessory olfactory systems (MOS, AOS) stim-
ulate the display of maternal behavior. In rats this effect is facil-
itated by estrogens (reduced latency to become maternal;
Mayer and Rosenblatt, 1979), whereas in rabbits estrogens
are essential (absent in OVX animals; González-Mariscal
et al., 2004a; Chirino et al., 2007).
The above findings indicate that the neural circuits under-
lying the motivation to interact with the progeny and the regu-
lation of species-typical behaviors are present in all female rats,
mice, and rabbits, regardless of their reproductive status, but
are tonically inhibited by the MOS and AOS. The way(s)
through which that inhibition is overcome at parturition –
such that the olfactory cues from the young are perceived as
attractive rather than as repulsive – has been investigated in
sheep (by means of electrophysiology) and in rabbits (through
a behavioral assay). Olfactory bulb mitral cells, when recorded
during pregnancy in ewes, respond mainly to food odors. After
birth, however, a dramatic increase occurs in the number of
cells that respond preferentially to lamb odors (Kendrick
et al., 1992). These shifts in the specificity of relevant stimuli
are associated with changes in the release of gamma aminobu-
tyric acid (GABA), glutamate, and OT within the olfactory bulb
and with the activation of the noradrenergic system that
projects to it (Lévy et al., 1995). In rabbits, virgin animals
show no sniffing preference for a box containing neutral odors
versus one containing pup olfactory cues. However, already on
pregnancy day 7, doe rabbits sniff the ‘pup box’ significantly
more than the neutral one. Interestingly, these differences
disappear close to parturition (Chirino and González-
Mariscal, 2015).
1.03.4 Neural Control
The investigation of the brain structures, fiber pathways, and
neurotransmitters/neuromodulators involved in regulating
the expression of the many behavioral patterns characteristic
of parental care has required the use of diverse experimental
strategies. As will be described below, brain lesions attempt-
ing to obliterate in the mother the perception of specific
cues (olfactory, visual, auditory, and tactile) from the young
assessed the role of the different senses on the display of
maternal behavior. More refined methods (e.g., chemical
lesions, deafferentation of individual brain regions, intracere-
broventricular infusion and intracranial implantation of
hormones, localization of receptors and neuroactive agents
in specific brain areas, and electrical stimulation of fiber path-
ways) have provided detailed information on the ways
through which a complex network of nuclei and tracts may
be activated by hormones and organized to coordinate the
perception of stimuli from the young with the expression of
the appropriate behavioral patterns. The ‘active’ parental
brain has been sampled by the use of methods (e.g., micro-
dialysis, immunostaining of immediate early gene proteins,
uptake of 2-deoxy-glucose, immunostaining of ‘clock gene’
proteins) that allow an association between the activity of
particular brain regions and the display of specific behavioral
patterns.
1.03.4.1 Cortex, Trigeminal Complex, and Olfactory Circuit
Beach and Jaynes (1956) determined that no single sensory
modality is essential for the performance of retrieving behavior
in postpartum rats. Rather, they proposed that this behavior is
under a multisensorial control because the elimination of
vision, olfaction, or tactile sensitivity of the snout and perioral
region did not interfere with pup retrieval. Later work (Benuck
and Rowe, 1975; Herrenkohl and Rosenberg, 1972; Herrenkohl
and Sachs, 1972) confirmed the lack of effect of anosmia,
blindness, and deafness of lactating rats on maternal behavior.
However, desensitization of the perioral region – by either local
anesthesia or section of the infraorbital branch of the trigem-
inal nerve – does interfere with pup retrieval (Kenyon et al.,
1981, 1983; Stern and Kolunie, 1989).
Lesions of the amygdala (a structure that receives major
input from the MOS and AOS) do not disrupt maternal
behavior in lactating mice (Slotnick and Nigrosh, 1975), and
lesions to the stria terminalis (the major efferent pathway of
the amygdala) have no effect on the maternal care of lactating
rats (Numan, 1974). By contrast, lesions of the corticomedial
amygdala (DelCerro et al., 1991; Fleming et al., 1980; Numan
et al., 1993), bed nucleus of the accessory olfactory tract (Del
Cerro et al., 1991), or stria terminalis in virgin rats stimulate
maternal behavior (Fleming et al., 1980), while kindling-type
electrical stimulation of the medial amygdala delays the onset
of maternal responsiveness to foster pups in experienced
mother rats (Morgan et al., 1999).
1.03.4.2 Septum and BNST
The septal area has been lesioned in mother rats (Fleischer and
Slotnick, 1978; Terlecki and Sainsbury, 1978), mice (Carlson
and Thomas, 1968; Slotnick and Nigrosh, 1975), and rabbits
(Cruz and Beyer, 1972). In rodents, these lesions do not
abolish maternal motivation but provoke a spatial disorganiza-
tion of maternal behavior: mothers pick up pups, carry them
around, drop them at random (thus altering crouching and
nursing), and may also build several small nests where they
retrieve young (Terlecki and Sainsbury, 1978). Excitotoxic
amino acid lesions of the BNST disrupt retrieval behavior in
postpartum rats (Numan, 1996). Rats lesioned in the septal
region also show increased defensiveness, an alteration
proposed to explain the disorganization of maternal behavior
(Sheehan et al., 2000). In contrast, the behavior of rabbits is
severely altered in all its aspects following septal lesions;
mothers do not build nests and refuse to enter the nest box
for nursing (Cruz and Beyer, 1972).
1.03.4.3 MPOA and Its Connections
1.03.4.3.1 Studies Using Lesion, Inactivation,
or Deafferentation Techniques
Much evidence has been accumulated to identify the MPOA as
a critical site for the expression of maternal behavior. Electro-
lytic or radiofrequency lesions of this region disrupt retrieving,
nest building, and nursing in lactating rats (Gray and Brooks,
1984; Jacobson et al., 1980; Numan, 1974; Numan et al.,
1977) and hamsters (Marques et al., 1979; Miceli and
Malsbury, 1982). A similar effect is provoked by injecting

N-methyl-D-aspartate (NMDA), which selectively destroys cell
bodies but spares fibers of passage, into the MPOA of lactating
rats (Numan et al., 1988). Moreover, virgin females (ovariecto-
mized or intact) lesioned in the MPOA do not show maternal
behavior despite many days of exposure to pups (Gray and
Brooks, 1984; Miceli et al., 1983; Numan et al., 1977). Further-
more, the facilitation of maternal behavior provoked in virgin
rats by lesioning the corticomedial amygdala does not occur if
such females have also been lesioned in the MPOA (Fleming
et al., 1983). In contrast, by applying kindling-type electrical
stimulation to the MPOA, maternal responsiveness to foster
pups is promoted in both experienced mothers and virgins
(Morgan et al., 1999), a finding supporting the idea that
activity of this brain structure is critical for the performance
of maternal behavior.
In sheep, a transitory inactivation of the MPOA at parturi-
tion (provoked by a local injection of lidocaine) severely
disrupts the display of MB 2 h later in primiparous animals
(Perrin et al., 2007).
To investigate which connections of the MPOA were
involved in regulating maternal behavior, the rostral, caudal,
lateral, or dorsal connections of this region were severed and
the effects were assessed in lactating rats. Several studies (Franz
et al., 1986; Miceli et al., 1983; Numan, 1974; Numan and
Callahan, 1980; Numan and Corodimas, 1985; Terkel et al.,
1979) revealed that the lateral projections of the MPOA exert
the most important influence. Some of these projections travel
to the ventral tegmental area (VTA; Barone et al., 1981; Conrad
and Pfaff, 1976; Swanson, 1976) a region that, according to
Numan, plays a major role in the regulation of rat maternal
behavior (Numan and Stolzenberg, 2009; Stack et al., 2002).
Specifically, (1) bilateral electrolytic (though not NMDA)
lesions of the VTA severely disrupt maternal behavior in post-
partum rats (Gaffori and Le Moal, 1979; Numan and Smith,
1984) and (2) bilateral knife cuts through the lateral hypothal-
amus disrupt maternal behavior only when performed at a level
(dorsal) in which fibers coming from neurons in the lateral
hypothalamus and going to the VTA are severed (Numan
et al., 1985). Efferent dopaminergic fibers from the VTA to
the NAcc promote behavioral reactivity to a variety of ‘signifi-
cant’ biological stimuli, among them, pup cues. The relevance
of a circuit involving dopaminergic neurotransmission within
the MPOA-VTA-NAcc circuit is supported by the finding that
the infusion of flupenthixol (a D1–D2 antagonist) into the
NAcc shell antagonizes maternal memory in primiparous
rats; that is, the latency to reengage in maternal behavior
following a 10-day separation is increased relative to vehicle-
injected dams (Parada et al., 2008).
1.03.4.3.2 Studies Using Implantation of Hormones
and Detection of Their Receptors
Diluted estradiol, implanted into the MPOA of rats that were
ovariectomized–hysterectomized on pregnancy day 16, effec-
tively stimulates maternal behavior (Fahrbach and Pfaff, 1986;
Numan et al., 1977). Similarly, estradiol benzoate implants
into the MPOA of ovariectomized rabbits promote nest building
when combined with peripheral injections (followed by with-
drawal) of progesterone (González-Mariscal et al., 2005). These
findings coincide with the presence of abundant estradiol
receptor alpha in the MPOA of rabbits and rats, as determined
Parental Behavior 87
by several methodologies (Caba et al., 2003a; Fahrbach and
Pfaff, 1986; Giordano et al., 1989, 1990; Wagner and Morrell,
1996; Wagner et al., 1998). Progesterone receptors (PRs) are
also abundant in the MPOA in rats (Numan et al., 1999), guinea
pigs (Blaustein and Turcotte, 1989; Blaustein et al., 1988; Ware-
mbourg et al., 1986, 1989), sheep (Scott et al., 2000), and
rabbits (Caba et al., 2003b), but their possible role in the control
of maternal behavior has been little explored (see below).
Regarding PRL, implants of this hormone, placental
lactogen-I, or growth hormone into the MPOA of ovariecto-
mized, bromocriptine-treated rats, given progesterone and
then estradiol, readily facilitate maternal behavior (Bridges
and Freemark, 1995; Bridges and Mann, 1994; Bridges et al.,
1990, 1997b). 25I-ovine PRL binding sites have been detected
in the hypothalamus (Muccioli and Di Carlo, 1994), and
estrogen-induced PRL receptors were found in the MPOA
using immunocytochemistry (Pi and Grattan, 1998) and in
situ hybridization (Pi and Grattan, 1999a), especially at the
end of pregnancy (Bakowska and Morrell, 1997) and during
lactation (Pi and Grattan, 1999a,b,c). In late pregnant rabbits
the intracerebroventricular injection of rabbit PRL counteracts
the inhibitory effects of s.c. bromocriptine on nest building
and crouching over the litter (González-Mariscal et al.,
2004b). These findings agree with the detection of PRL
binding sites in the hypothalamus of nonpregnant rabbits
through affinity labeling of 125I-ovine PRL (Di Carlo and
Muccioli, 1981) and by autoradiography of 125I-ovine PRL
(Walsh et al., 1990).
1.03.4.4 Midbrain Tegmentum, PVN, and Habenular Complex
The lateral midbrain tegmentum, which includes the peripe-
duncular nucleus, receives input from descending preoptic
efferents and also from trigeminal sensory pathways that pass
through the VTA and carry input from the perioral region
(Nadaud et al., 1984; Smith, 1973). Lesions in this area abolish
maternal aggression toward males, block milk ejection, but do
not interfere with nursing behavior (Hansen and Ferreira,
1986; Hansen and Gummesson, 1982; Hansen and Kohler,
1984). However, knife cuts made caudal to the VTA (thus dis-
rupting ascending and descending pathways traveling through
the mesencephalon) abolish maternal behavior (Numan and
Numan, 1991). These results indicate that fibers passing
through the VTA and relevant for maternal behavior terminate
at levels lower than the mesencephalon (e.g., caudal periaque-
ductal gray (PAG) and central tegmental field). The PAG has
also been implicated in behavioral switching, that is, as part
of a decision-making apparatus that allows the maternal female
to decide whether to hunt or to act maternal (Felicio and
Canteras, 2008). Apparently, the PAG ‘weighs’ the relative
merits of chasing prey versus caring for the offspring, based
on concurrent internal states.
The PVN seems to participate in the onset of maternal
behavior in several species through the release of OT and its
parvocellular extrahypothalamic projections. Thus, in rats, (1)
OT is released in the supraoptic nucleus (SON) and the PVN
at parturition (Neumann et al., 1993); (2) isolation of the
mediobasal hypothalamus, a procedure that leaves the PVN
and its extrahypothalamic connections intact but severes path-
ways between the PVN and the medial basal hypothalamus,
88 Parental Behavior
abolishes milk output but does not affect maternal behavior
(Herrenkohl and Rosenberg, 1974); (3) lesions to the PVN
disrupt rat maternal behavior, although only if performed
before its onset (Insel and Harbaugh, 1989; Numan and
Corodimas, 1985); if performed after parturition PVN lesions
significantly reduce maternal aggression (Consiglio and
Lucion, 1996); and (4) kainic acid-induced lesions of the
PVN on postpartum day 2 affect retrieving behavior but not
other maternal behavior components (Olazábal and Ferreira,
1997), while lesions performed on day 5 postpartum with ibo-
tenic acid (a procedure that preferentially lesions parvocellular
neurons) or infusion of antisense oligonucleotides against OT
increase maternal aggression (Giovenardi et al., 1997, 1998).
Moreover, OT is released within the PVN during nest defense
(Bosch et al., 2004), particularly in rats bred for high-anxiety-
related behavior (Bosch et al., 2005).
In sheep there is strong evidence for the participation of OT
from the PVN in the activation of maternal behavior. Thus, (1)
OT is released in this structure during parturition, and retro-
dialysis of this peptide induces maternal behavior in nonpreg-
nant steroid-primed females (Da Costa et al., 1996a); (2) birth
or VCS activates the PVN (as determined by expression of the
FOS protein), where oxytocinergic neurons and fibers as well
as OTRs have been found (Kendrick et al., 1997); (3) the
number of OT immunoreactive (IR) neurons and mRNA tran-
scripts for the OTR are increased in the PVN at parturition and
following progesterone/estradiol priming (Broad et al., 1993);
and (4) previous maternal experience further enhances the
expression of OTR mRNA in the PVN (Broad et al., 1999).
From this evidence, it has been proposed that, at parturition,
OT facilitates its own release (by acting on autoreceptors),
not only in the PVN itself but also in the brain regions it
projects to, for example, MPOA and olfactory bulbs (Kendrick
et al., 1997).
1.03.4.5 Sampling the Active Parental Brain
1.03.4.5.1 The Expression of Immediate Early Genes
A large number of studies have used the detection of the c-FOS
protein (product of the c-fos gene) by means of immunocyto-
chemistry to explore the brain regions activated during the
display of maternal behavior. Thus, Numan and Numan
(1994) found that lactating rats exposed to pups (following
a 3-day separation) showed significantly higher numbers of
FOS-IR cells in the MPOA, ventral (though not dorsal) BNST,
anterior cortical, and posterodorsal medial amygdaloid
nuclei, compared to lactating rats that were given candy
instead of pups on the day of testing. Similarly, Fleming et al.
(1994) found that postpartum rats exposed to pups showed
many more FOS-IR cells in MPOA, piriform cortex, and
medial and cortical amygdala than mothers exposed to
a familiar adult female or to food. In addition, sensitized
adult virgin rats also show an increased number of c-FOS IR
cells in the MPOA and ventral BNST following a 2-h
interaction with pups (Kalinichev et al., 2000).
To further understand the meaning of changes in the
number of c-FOS-IR cells associated with the display of
maternal behavior, Lonstein and De Vries (2000) analyzed
brain sections costained with FOS and an antiserum against
GAD67 (an isoform of the rate-limiting synthesizing enzyme
for GABA, found in the cytoplasm; Martin and Rimvall,
1993). They found that, in lactating rats, interaction with
pups increased the number of colabeled cells in the MPOA,
ventral BNST, and caudal PAG (ventrolateral region), a result
suggesting that GABAergic neurons may promote specific
aspects of maternal behavior by removing tonic inhibitory
influences. Similarly, Lonstein et al. (2000) reported that
lactating rats allowed to interact with pups showed an
increased number of cells colabeled to c-FOS and ERa than
did mothers not given young. These differences were particu-
larly evident in the MPOA, lateral habenula, and ventral BNST.
In virgin Balb/c mice a single brief (30 min) exposure to
pups increased the number of FOS-IR cells in the MPOA,
corticomedial amygdala, entorhinal and piriform cortex, and
anterior olfactory nucleus, regardless of whether females were
intact or ovariectomized (Calamandrei and Keverne, 1994).
Interestingly, the presentation of pups inside a dark,
perforated box (that allowed females to perceive only
olfactory and auditory signals from the young) stimulated
FOS expression in the olfactory areas but not in the MPOA.
In contrast, the depletion of noradrenaline in the olfactory
bulb (provoked by injecting 6-hydroxy-dopamine into the
medial olfactory stria) reduced FOS expression only in the
anterior olfactory nucleus and the piriform cortex. These
results coincide with those obtained in rats and support the
notion that FOS expression in the MPOA occurs as a result of
the active display of maternal behavior.
In multiparous sheep, the interaction with the lamb for the
first 30 min after parturition stimulated c-fos mRNA transcrip-
tion (detected by in situ hybridization) in the cerebral cortex
(e.g., entorhinal, piriform, and somatosensory), habenula,
hippocampus (CA3 region, dentate gyrus), limbic system
(e.g., BNST, LS, and olfactory bulb), MPOA, and several hypo-
thalamic nuclei (e.g., PVN and SON). Very similar changes
were observed in nonpregnant multiparous ewes treated with
estrogen and progesterone and given 5 min of VCS (Da Costa
et al., 1997).
In primiparous doe rabbits a single brief (c.3 min) nursing
bout significantly increased the number of c-FOS-IR cells in
the PVN, SON, and LS on lactation days 1 and 7 (González-
Mariscal et al., 2009). By contrast, in the POA the number of
c-FOS-IR cells did not differ between does given a litter to nurse
on the day of sacrifice (lactation days 1 or 7) and those not
given kits on that day. However, both groups of mothers
showed many more c-FOS-IR cells than did virgin animals.
These results suggest that the POA, though not activated by
suckling itself, is part of a ‘circuit’ underlying the regulation
of maternal behavior which, in rabbits, starts in midpregnancy
with the construction of an elaborate maternal nest (see
above).
1.03.4.5.2 Other Methods
The uptake of 14C-2-deoxy-glucose (2DG) has been used as an
index of activity in neuronal terminals but not in cell bodies
(Nudo and Masterton, 1986; Sharp et al., 1988). At parturition,
mother rats showed an increased uptake of 2DG in the MPOA
and VTA, while in sensitized virgins displaying maternal
behavior a decreased 2DG uptake was observed in several struc-
tures of the AOS (i.e., accessory olfactory bulb, medial amygda-
loid nucleus, and bed nucleus of the accessory olfactory tract;

Del Cerro et al., 1995). These results coincide with the idea that
the facilitation of maternal behavior in virgin rats occurs as
a consequence of reducing the impact of aversive olfactory
stimulation coming from the pups. To distinguish between
excitatory and disinhibitory synaptic relations in specific brain
areas, associated with the display of maternal behavior, the
same group of investigators combined the 2DG method with
the immunocytochemical detection of c-FOS (Komisaruk
et al., 2000). In the three models of maternal behavior explored
(parturient, sensitized virgins, and rats hysterectomized on
pregnancy day 16), the MPOA showed increased uptake of
2DG and increased expression of c-FOS. This indicates that
the expression of maternal behavior (regardless of how it is
induced) involves excitatory input to the MPOA and excitatory
output from it. A different pattern was observed in structures of
the olfactory system (e.g., bed nucleus of the accessory olfactory
tract, BAOT and medial amygdala): while parturient rats
showed increased 2DG uptake and increased FOS expression
(i.e., excitatory/excitatory relations), in sensitized virgins 2DG
uptake decreased and c-FOS increased. This observation further
supports the notion that the expression of maternal behavior
through sensitization in virgin rats involves a reduction in the
activity of the olfactory system.
1.03.5 Impact of Maternal Behavior on the Mother:
Effects on Neuroanatomy, Cognition, and Emotional
Reactivity
1.03.5.1 Theoretical Context
Any new mammalian mother is, on the one hand, the same
female she was before the birth of her offspring. Yet, the tran-
sition from nulliparous to parous female involves substantial,
likely permanent, alterations to both brain and behavior. These
changes are related to the simple recognition that a new set of
activities emerge in the new mother, that include both behav-
iors directly related to pup care and maintenance (the so-
called maternal behavior), and others that are ancillary to
maternal behavior and allow the mother to be efficient in her
search for resources (Kinsley et al., 1999; Lambert et al., 2005).
In uniparental rodents the mother is confronted with a stark
lose–lose situation. She can stay on her nest, safe with her
offspring, and face eventual starvation and the loss of her litter.
Or she can leave the nest and her vulnerable brood in search of
food and water, in which case she places both herself and the
young in danger. Departing the safe confines of the nest for
environments and dangers unknown requires overcoming the
fear inherent to doing so and successfully confronting any risks
encountered (predators, rogue males, impassable obstacles,
etc.). Then, any foraging costs (increased time and effort) could
be reduced by enhancements to spatial and reference memory.
These latter changes would ensure a rapid return to the nest and
the defenseless offspring. Therefore, reductions in fear and
anxiety and improvements to memory systems should arise
as a consequence of pregnancy and/or lactation. As summa-
rized in Tables 1 and 2, abundant evidence indicates that
maternal experience modifies brain neuroanatomy, the respon-
siveness to several hormones, the functioning of some neuro-
transmitter systems, several forms of memory, and emotional
reactivity.
Parental Behavior 89
1.03.5.2 Fear and Anxiety Regulation
1.03.5.2.1 Behaviors Affected
In the realm of stress regulation and the anxiolytic properties of
reproduction, much research demonstrates altered emotional
reactivity in parous females. Many works report a reduction
in stress and fear responses of both primiparous and multipa-
rous rats, compared with virgins (see specific references in
Tables 1 and 2). Multiparity reduces anxiety, relative to a single
maternal experience, but primiparous rats show much attenu-
ated anxiety responses compared with virgins (see Tables 1A
and 2).
1.03.5.2.2 Brain Mechanisms
The changes in hormonal and neurotransmitter systems that
may underlie the reductions in fear and anxiety associated
with maternal experience are multiple and involve, mainly,
(1) modifications in the responses to estradiol and prolactin
in the anterior pituitary, hypothalamus, POA, striatum, and
amygdala; (2) changes in opiate receptors; (3) enhanced gluta-
matergic and dopaminergic neurotransmission; and (4) other
(e.g., kisspeptin neurons). These effects (and the corresponding
references) are detailed in Table 1B.
1.03.5.3 Regulation of Cognitive Effects
1.03.5.3.1 Learning and Memory
Reproductive experience also exerts strong and long-lasting
effects on various forms of learning and memory. As shown
in Table 2, spatial, reference, prospective, working, and
maternal memory are better in rats that have experienced
motherhood. In addition, functions such as foraging and atten-
tion are more finely tuned in mothers. Not surprisingly,
maternal behavior per se is more efficient in multiparous
dams. Moreover, the possibility that such changes, induced
by maternal experience, are permanent is supported by reports
showing that aging-related declines in brain function are
reduced in multiparous dams, followed by primiparous and
virgin rats (Table 2). The specific tests used to determine the
above (as well as the corresponding bibliography) are detailed
in Table 1C and 1D.
1.03.5.3.2 Neuroanatomical Changes Observed in the
Maternal Brain
The behavioral responses described above involve the activity
of several neurotransmitter systems in various diencephalic
and telencephalic regions that regulate enhanced attention
toward and care of the young (see Section 1.03.3). Table 1E
presents a detailed description (and bibliography) of the
changes in dendritic spine length and density, cell proliferation
and survival, characteristics, and abundance of glia found in the
brains of multiparous, primiparous, and virgin rats.
1.03.6 Timing: A Little-Explored Dimension
of Maternal Care
The suprachiasmatic nucleus (SCN), a small-paired nucleus in
the hypothalamus, is considered the master circadian clock that
control circadian rhythms in the body (see specific chapter on
Table 1 Effects of maternal experience on fear and anxiety, neuroendocrinology and neurotransmitters, learning and memory, maternal behavior, and neuroanatomya

90
Primiparousb Multiparousb References

Parental Behavior
A. Fear and anxiety
No [ plasma OT following immobilization (PP 6) Carter and Lightman (1987)
< OT content posterior pituitary Higuchi et al. (1991)
Y OT release by electrical stimulation in isolated posterior pituitary
[ % time and # entries in open-arm EPM (only with pups into the maze) Pereira et al. (2005)
Pups in the maze overrode the Y % time in open arm by haloperidol
\ young (proestrus) Byrnes and Bridges (2006a)
[ time in open-arm EPM
[ travel distance open arms
[ % time center open field
\ middle aged
Opposite effects found in young females
OVX eliminated effect of reproductive experience
>> \ at 18 and 22 months \ 10, 14 months old Love et al. (2005)
[ time in open arms in EPM [ % time in open arms in EPM
[ time contact novel stimuli
After EPM and FS: Neumann et al. (1998)
No [ plasma ACTH and CORT (PP 7)
No [ plasma OT (late pregnancy and lactation)
> CORT plasma (early lactation) < CBG Pawluski et al. (2009)
 CBG
[ time in open arms of EPM Byrnes et al. (2012)
(PPT; ERa agonist treatment) Larsen and Grattan (2010)
[ time open arms of EPM
Y latency to full maternal behavior in a novel cage (PP 2)
\ Pregnancy days 15–18 and lactation days 2–6 Neumann et al. (2000)
OT antagonist induces:
Y entries and time in the open arm (EPM)
No [ CORT and ACTH release
Basal and after EPM and FS tasks
[ OT secretion by stress (only pregnant)
< Corticosterone-binding globulin Shanks et al. (1999)
No [ c-fos mRNA in PVN
No [ AVP mRNA in PVN after LPS endotoxin (lactation days 9–12)
\ PP 7–10 Windle et al. (1997)
No [ plasma CORT or ACTH by white noise stress but, 72 h after weaning,
[ plasma CORT or ACTH by 2 h noise stress
< basal CRF mRNA in PVN but noise stress had no further effect
6 weeks postweaning diazepam vs veh EPM, novel cage tasks: Byrnes and Bridges (2006b)
[ locomotor activity
No anxiolytic effects
< CORT release
> % time
> % distance traveled
\ late pregnancy/early lactation: Da Costa et al. (1996b)
Y plasma CORT by stress
< c-fos mRNA in PVN, lateral septum, cingulate cortex, and MeA
< After weaning: Wartella et al. (2003)
[ < c-fos in CA3 and BLA after 60 min in restraint tube
Y [ # number of center blocks crossed (open field test)
and rearing
Y # freezing (similar results for primigravid and
multigravid \s)
\ OVX Walf and Frye (2008)
[ time spent in open arms in EPM
Y immobility time in FST only in \ pretreated with
17-b E2
\ lactation days 7–8 Miller et al. (2011)
> time light chamber
Y GABA-A receptor activity (by pentylenetetrazol)
B. Neuroendocrinology and neurotransmitters
<  plasma PRL (vs OVX) Anderson et al. (2006)
< estradiol < plasma PRL in mating-induced diurnal/nocturnal peak Bridges et al. (1993)
> pituitary PRL Bridges and Hammer (1992)
(proestrus, diestrus) Bridges and Byrnes (2006)
Byrnes and Bridges (2005, 2007)
Byrnes et al. (2001)
Carvalho-Freitas et al. (2007)
Sider et al. (2003)
\ in proestrus: After EB (125 mg kg
1): Bridges and Byrnes (2006)
< estradiol-17b and PRL [ plasma PRL and estradiol
> ERa in anterior pituitary After EB (1 mg kg
1):
Y plasma PRL and estradiol
After oPRL (250 mg): Anderson et al. (2006)
< serum PRL and estradiol
[ long-form PRL-R mRNA in MPOA and ARC

Parental Behavior
[ SOCS-1 and SOCS-3 mRNA in MPOA and ARC
(diestrous \)
\ ovx Bridges et al. (1997)
< responsive to HAL (sc) to: Hucke et al. (1998)
Induce PRL release in plasma
Induce DOPAC and HVA
(Continued)

91
Table 1 Effects of maternal experience on fear and anxiety, neuroendocrinology and neurotransmitters, learning and memory, maternal behavior, and neuroanatomyadcont'd

92
Primiparousb Multiparousb References

Parental Behavior
\ proestrus Byrnes and Bridges (2007)
After HAL:
[ D2 long mRNA
[ D2 short mRNA Anterior pituitary
Y D2 long/D2 short mRNA
[ response to apomorphine-induced stereotypy and PPI Byrnes et al. (2001)
Hucke et al. (2001)
(diestrus):
[ DA and DOPAC
Y PRL
[ distance and time moving, total velocity
[ level DA and DOPAC in STR, but not in NAcc (shell)
\ primigravid (day 7) \ multigravid (day 7) Felicio et al. (1996)
[ j DA in STR and hypothalamus and tendency of DOPAC in STR
[ After HAL:
[ PRL in serum j 5-HT in STR
[ PRL in serum
In hypothalamus: Carvalho-Freitas et al. (2007)
Y [ ] DOPAC
Y DOPAC/DA ratio
Y HVA þ DOPAC/DA
(2–3 weeks after weaning)
\ primigravid (day 7–8) \ multigravid (day 7–8) Sider et al. (2003)
<  plasma PRL
[ j DA and NE [ ]
Y k DOPAC/DA
Y k HVA/DA Ratio (light-dark shift)
Y k 5-HIAA/5HT
[[[ Y disrupted effect of b-endorphin (sc and MPOA infusion) on MB Kinsley and Bridges (1988)
[ Y analgesic after morphine injection Mann and Bridges (1992)
[[[ MPOA opiate receptor density (PP 5) Bridges and Hammer (1992)
[[[ Oprm1, Oprk1 gene expression in PAG Teodorov et al. (2011)
[[[ m and k opiate receptors in PAG
[ DA release NAcc (first 8 min pup-exposure) Afonso et al. (2008)
[ response of glutamatergic neurons exposed to pup- Cohen and Mizrahi (2015)
odor (PP 4; mice)
[ kisspeptin neurons colocalized with pSTAT5 in ARC Sjöeholm et al. (2011)
(after icv rat PRL)
[ pSTAT5 IR-cells MPOA, PVN, ARC after PRL (icv)
\ young proestrus Byrnes et al. (2009)
[ # ERa-cells in anterior-dorsal MPOA
Y # ERa-cells in anterior-medial and cortical AMG
\ middle-aged (13 months)
[ ERa-IR cells in dorsal STR
PPT (ERa agonist) treatment: Byrnes et al. (2012)
[ CRH mRNA in PVN
Y in lateral AMG (after EPM task)
Pregnant days 7 and 16: Macbeth et al. (2008a)
> exploration ratio (novel environment)
Y DA, DOPAC, HVA, NE, MHPG, 5-HT, 5-HIAA in PFC
[ NE, HVA, MHPG (PP 9)
Y DOPAC, HVA (PP 18), MHPG (PP 16), 5-HT (PP 16)
in CA1
\ 13 months old Macbeth et al. (2008b)
[ [ ] DA and DOPAC, NE, MHPG, 5HT and 5HIAA in olfactory bulb.
[ [ ] BDNF in CA1 and septum
Pup exposure Akbari et al. (2013)
[ DRD1 and DAT gene expression in MPOA
[ cort-related genes in MeA
[ OPRM1
[ 5HT-R2A in MPOA
C. Learning and memory
< < errors in eight-arm radial Pawluski et al. (2006b)
maze (working memory) Pawluski et al. (2006a)
< errors on spatial learning and memory performance task
Y latency to find baited food (PP 21) > time in closed baited well Franssen et al. (2012)
< time with familiar object Kinsley et al. (1999)
> correct choices to find baited food Lambert et al. (2005)
Pawluski et al. (2006a,b)
Pregnant: WMT task Bodensteiner et al. (2006)
Y latency and distance to find platform, and find new
location of platform
[ time in quadrant of previous platform
Learning sessions: PP 1–4 Darnaudery et al. (2007)
[ latency to reach platform (WMT)
> distance traveled
Recall session: PP 14

Parental Behavior
> % time exploring
Y proliferation cells
< cell decreased over time
(Continued)

93
Table 1 Effects of maternal experience on fear and anxiety, neuroendocrinology and neurotransmitters, learning and memory, maternal behavior, and neuroanatomyadcont'd

94
Primiparousb Multiparousb References

Parental Behavior
2 weeks and 16 months after weaning Lemaire et al. (2006)
Y distance traveled to find hidden platform
[ AMPA/Kainate fEPSP slope
[ NMDAr-mediated (LTP in Hipp)
However
Whether \ was stressed during pregnancy,
No effects on WMT
No effects on LTP
Y \ 6,12, 28, and 24 months old Gatewood et al. (2005)
Y k Latency to find baited food
k Latency to new location of food
(DLM and WMT tasks)
[ \ 5, 9, and 13 months old Love et al. (2005)
Y j # retrieve fruit loops
Y latency to previously baited food
\ 17 and 21 months old vs 9 months
Y latency to reach baited food
Y latency to food reward (DLM; visual and no visual cues, reversal task)
Y predation latency in lactation, mid- and late-pregnancy Kinsley et al. (2014)
[ phosphorylation of CREB and LTP in Hipp slices (mice) by OT Tomizawa et al. (2003)
OT antagonist (icv):
Y # of errors: reference memory task
[ # discrimination ratios of object-in-place memory (foraging) (from lactation Cost et al. (2014)
day 14–42 days postweaning)
\ 13 months old Macbeth et al. (2008b)
> exploration ratio time to new object (object recognition)
> exploration ratio time to object placement
D. Maternal behavior
¼ After cesarean section: Moltz et al. (1966)
¼ ¼ % nest-building
¼ Pup retrieval
Maternal retention test (pup exposure at partum for 2– Bridges (1977)
24 h) Fleming and Korsmit (1996)
Y latency to retrieve pups (test 10, 25–35 days later) Fleming and Sarker (1990)
Stern and Mackinnon (1976)
AVP vs veh (icv) Nephew and Bridges (2008)
Y maternal aggression (PP 5)
Y maternal behavior (PP 15)
Antagonist AVP vs veh
[ maternal aggression (PP 5 and PP 15)
Y maternal behavior (PP 15)
Y grooming (PP 5)
 AVP (icv) vs veh Nephew et al. (2010)
k maternal aggression (PP 5)
V1a receptor antagonist vs veh
j aggression (PP 15)
Pregnant days 8, 9, 10, and 20 Serafim and Felicio (2002)
[[[ mammary gland grooming
Y latency to retrieve pups Lopatina et al. (2011)
[ plasma OT
[ hypothalamus
[ pituitary
(CD38
/
knockout and wild-type mice)
55 and 80 postweaning days: Scanlan et al. (2006)
Y latency to full maternal behavior
[ exploration and distant travel in an open arm (by pup
exposure)
[ C-fos in cortical AMG, NAcc (shell), BLA
Y prefrontal cortex (4–10 days later)
Y k latency to attacks _s Nephew et al. (2009)
[ j # of attacks
[ j CRH mRNA in lateral septum
Y k OT-Rec mRNA in PVN
Y k OT mRNA in PVN
Y PP 5
k AVP mRNA in PVN
Y k V1a mRNA SO
[ j OT mRNA in lateral septum and BNST
j CRH mRNA in PVN PP 15
k OT-Rec mRNA in central AMG
j time fighting (mice) (6 lactations) Svare and Gandelman (1976)
Nest building (lactating mice > pregnant > nulliparous) Bond et al. (2002)
E. Neuroanatomy
[ Thickness of frontal, somesthetic, and occipital cortex Diamond et al. (1971)
[ Basal and apical spine density in PFC, DG, CA1 Hipp (PP Leuner and Gould (2010)
21)
24–36 h PP þ pups – test: PP 8 Shams et al. (2012)
Y total dendritic length and dendritic intersection in CN
[ body and brain weight
[ dendritic branch in NAcc (shell)
[ # dendrites per branch order in MPOA

Parental Behavior
PP 2 and PP 8 Leuner et al. (2007)
Y BrdU-labeled cells in DG (cell proliferation)
[ CORT
But, pups’s removal for 3 days prevents these effects.
PP 2, 1 week post-BrdU shot
Y BrdU-labeled cells in DG (cell survival)
(Continued)

95
Table 1 Effects of maternal experience on fear and anxiety, neuroendocrinology and neurotransmitters, learning and memory, maternal behavior, and neuroanatomyadcont'd

96
Primiparousb Multiparousb References

Parental Behavior
Pregnancy day 7, lactation days 7 and 14 (mice) Gregg et al. (2007)
[ OPC proliferation and generation of new oligodendrocytes in CC and SC
[ MBP expression
[ myelinated axons (genu of CC)
[ oligodendrocytes, < demyelinated axons, > remyelinated axons, and Y
volume of lesion on pregnancy day 14
[ dendritic spines in CA1 Hipp (late pregnancy, PP 5, and Kinsley et al. (2006)
OVX þ P þ E2 null)
Y cell proliferation and % cell survival in DG of Hipp Y cell proliferation in DG Pawluski and Galea (2007)
(lactation day 2) [ % cell survival than primiparous in DG (PP 2)
OVX \ þ estrogen; 13–14 months old Barha and Galea (2011)
[ cell proliferation in DG
17 a-E2 > estrone > 17 bE2
[ dendritic spine in MeA (anterodorsal) Rasia-Filho et al. (2004)
Y dendritic spine in posterodorsal MeA (after weaning)
[ [[[ # GFAP-IR cell in MPOA Featherstone et al. (2000)
Y YYY # GFAP-IR cells; MeA and habenula (PP 5)
[ [ basic GFAP-IR Cg2 Salmaso and Woodside (2008)
(3 h postpartum and PP 24)
\ Late pregnancy and lactation day 16 Salmaso et al. (2005, 2009)
[ bFGF-IR in PVN, SON, MPOA Salmaso and Woodside (2006, 2008)
[ GFAP-IR in Cg2
[ GFAP-ir and bFGF-ir Cg2 at 3 h and PP days 1, 4,
16, 24
Y vimentin (marker GS) in MPOA (PP 1, PP 4, PP 16) Salmaso et al. (2011)
[ GS-IR (PP 1), glutamate/protein, and glutamine/protein
(3 h, PP 1, PP 16) in Cg2 but not in MPOA
Cell body size: number and length of basal dendritic Keyser-Marcus et al. (2001)
branches in MPOA
Late pregnant and OVX þ P þ E2 > lactating \
[ j apical and basal dendritic length and apical branch points in CA3 and CA1 Pawluski and Galea (2006)
[ of Hipp
j # spine density basal region CA1
Y \ 24 months old Gatewood et al. (2005)
kAPP in CA1
kAPP in DG
Positive correlations: latencies in the mazes (WMT, reversal task) and # APP ir
neurons in CA1 and DG
48 h after KA Franssen et al. (2012)
Y % GFAP-IR cells in HFm, HFI, and CA3 of Hipp
[ cell death Hipp CA3 (16 postshot)
No effects on spatial memory and memory of familiar object
24 h after KA lesion (ip) vs veh, diestrus, OVX:PP 10–14 Monasterio et al. (2008)
< Fos-ir cells in PVN
24 h after KA (ip): PP 18 Vanoye- Carlo et al. (2008)
Dorsal Hipp
< loss of cells (>cellular density) in CA1, CA3, CA4
< # IR-cells positive for caspase-3 in CA1, CA3, CA4
< # positive tunel nuclei in CA1, CA3, CA4
[ Bcl-2 ir (antiapoptotic protein) in CA1, cerebral
cortex, and piriform cortex
24 h after KA (ip): PP 19 vs metestrus \ Vanoye- Carlo et al. (2009)
[ ERb in CA1, CA3 (dorsal Hipp)
Y ERa in CA3
KA lesion (icv): PP 12–14 Cabrera et al. (2009)
24 h postlesion: < Fluoro–Jade C staining (marker of cell
degeneration) in CA3, CA4
72 h postlesion: < Fluoro–Jade C in CA1, CA3, CA4
a
Studies done in rats unless otherwise specified.
b
Relative to virgins unless otherwise specified.
ACTH, adrenocorticotropic hormone; AMG, amygdala; AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (agonist for glutamate); APP, amyloid precursor protein; ARC, arcuate nuclear; AVP, arginine vasopressin;BDNF, brain-
derived neurotropic factor; bFGF, astrocytic basic fibroblast growth factor-2; BLA, basolateral amygdala; BNST, bed nucleus of stria terminalis; CA1, cornu ammonis (region of hippocampus); CBG, corticosteroid binding globulin; CC, corpus
callosum; Cg2, cingulate cortex (area 2); CN, caudate nucleus; CREB, cAMP response element-binding; CORT, corticosterone; CRH, corticotropin-releasing hormones; DA, dopamine; DAT, dopamine transporter; DG, dentate gyrus; DLM, dry
land maze; DOPAC, 3,4-dihydroxyphenylacetic acid; DRD1, DRD4, dopamine-related genes; E2, estradiol; EB, estradiol benzoate; ERs, estrogen receptors; EPM, elevated plus maze; fEPSPs, field excitatory postsynaptic potential; FS, force
swimming; GABA, gamma-aminobutyric acid; GFAP, glial fibrillary acid protein; GS, glutamine synthetase; 5-HIAA, 5-hydroxyindolacetic acid; 5-HT, 5-hydroxytryptamine (serotonin); 5HT-R2A, 5-HT biosynthesis gene; Hipp, hippocampus;
HVA, homovanillic acid (DA metabolite); KA, kainate acid; L-LTP, long-lasting, long-term potentiation; LPS, lipopolysaccharide (endotoxin); MBP, myelin basic protein; MeA, medial amygdala; MHPG, 3-methoxy-4-hydroxyphenylglycol; MPOA,
medial preoptic area; MWM, Morris water maze; NAcc, nucleus accumbens; NE, norepinephrine; OT, oxytocin; OPC, oligodendrocyte precursor cells; Oprm1, opiod receptor m-1 gene; OVX, ovariectomized; PAG, periaqueductal gray; PFC,
prefrontal cortex; PP, postpartum; PVN, paraventricular nucleus; PRL, prolactine; PPI, prepulse inhibition; PPT, 4,40 ,400 -(4-propyl-[1H]-pyrazole-1,3,5-tryl) trisphenol; PSTAT5, phosphorylated signal transducer and activator of transcription
five; SC, spinal cord; SON, supraoptic nuclear; STR, striatum; SUZ, subventricular zone; V, vasopressin; WMT, water maze task.

Parental Behavior
97
98 Parental Behavior

Table 2 Changes in responses of the whole organism as a consequence of motherhood

Responses affected Comparison References

Stress and fear Multip ¼ primip < null Byrnes et al. (2012)
Carter and Lightman (1987)
Da Costa et al. (1996b)
Neumann et al. (1998, 2000)
Pawluski et al. (2009)
Shanks et al. (1999)
Wartella et al. (2003)
Windle et al. (1997)
Anxiety Multip < primip < null Byrnes and Bridges (2006a)
Byrnes et al. (2009, 2013, 2012)
Love et al. (2005)
Neumann et al. (1998, 2000)
Pereira et al. (2005)
Larsen and Grattan (2010)
Scanlan et al. (2006)
Walf and Frye (2008)
Macbeth and Luine (2010)
Spatial memory Multip > null (>16–24 months: better and faster) Cost et al. (2014)
Gatewoodet al. (2005)
Kinsley et al. (1999)
Lemaire et al. (2006)
Love et al. (2005)
Macbeth et al. (2008a)
Pawluski et al. (2006a,b)
Tomizawa et al. (2003)
Reference memory Primip > null and _s Pawluski et al. (2006a,b)
Working memory Pregnant > virgin Tomizawa et al. (2003)
Bodensteiner et al. (2006)
Maternal memory Primip > null Bridges (1977)
Fleming and Korsmit (1996)
Fleming and Sarker (1990)
Fleming et al. (1999)
Orpen and Fleming (1987)
Lopatina et al. (2011)
Scanlan et al. (2006)
Attention Primip > null Leuner and Gold (2010)
Foraging Multip > primip > null Kinsley et al. (2014)
Love et al. (2005)
Brain plasticity Multip ¼ primip > null Barha and Galea (2011)
Diamond et al. (1971)
Featherstone et al. (2000)
Furuta and Bridges (2005)
Gatewoord et al. (2005)
Gregg et al. (2007)
Kinsley et al. (2006)
Kinsley and Lambert (2006)
Leuner and Gould (2010)
Pawluski and Galea (2006)
Pawluski et al. (2016)
Rasia-Filho et al. (2004)
Shingo et al. (2004)
Salmaso and Woodside (2006, 2008)
Salmaso et al. (2005, 2009, 2011)
Shams et al. (2012)
Sensitivity to E2 Multip < null Bridges et al. (1993)
Bridges and Byrnes (2006)
Byrnes et al. (2009, 2012, 2013)
 Parental Behavior 99

Table 2 Changes in responses of the whole organism as a consequence of motherhooddcont'd

Responses affected Comparison References

Brain responsiveness to hormones and peptides (e.g., Primip > null Anderson et al. (2006)
PRL, OT, Glu) Brown et al. (2011)
Carter and Lightman (1987)
Cohen and Mizrahi (2015)
Fleming et al. (1999)
Grattan and Kokay (2008)
Gregg et al. (2007)
Higuchi et al. (1991)
Larsen and Grattan (2010),
Nephew et al. (2009)
Neumann et al. (2000)
Pereira et al. (2005)
Salmaso et al. (2011)
Sjöeholm et al. (2011)
Tomizawa et al. (2003)
Enhanced activity of dopaminergic system Primip > null Afonso et al. (2008)
Bridges et al. (1997a)
Bridges (2016)
Byrnes et al. (2001)
Carvalho-Freitas et al. (2007)
Felicio et al. (1996)
Hucke et al. (2001)
Macbeth et al. (2008a,b)
Sider et al. (2003)
Sensitivity to opioids and benzodiazepines Multip < primip Bridges and Hammer (1992)
Kinsley and Bridges (1988)
Mann and Bridges (1992)
Teodorov et al. (2011)
Byrnes and Bridges (2006b)
Reduced aging-related decline in brain function Multip > primip > null Barha and Galea (2011)
Byrnes et al. (2009)
Gatewood et al. (2005)
Love et al. (2005)
Macbeth et al. (2008b)
Protection from neurotoxics Primip > null Franssen et al. (2012)
Morales (2011)
Shanks et al. (1999)
Vanoye-Carlo et al. (2008, 2009)
Monasterio et al. (2008)
Cabrera et al. (2009)

E2, estradiol; Glu, glutamate; Multip, multiparous mother; Primip, primiparous mother; Null, nulliparous female; OT, oxytocin; PRL, prolactine.

this topic in this book). At the core of these rhythms are a group
of genes, the clock genes, such as Per, Clock, and Bmal1, and
their proteins that form an autoregulatory negative transcrip-
tion–translation feedback loop with a periodicity of around
24 h. This molecular mechanism is found in many cells of
the body and regulates several clock-controlled genes. As
a result there is a timing mechanism perhaps in all tissues
and organs, but the master circadian clock is at the top of the
hierarchy in the control of circadian oscillations. In this section
we will focus on clock genes and circadian rhythms in relation
to reproductive process. Long ago it was established that the
SCN plays a key role in the timing of ovulation. Besides that,
very little is known about the circadian control of other repro-
ductive process, namely pregnancy and lactation and the
outcome of these processes: maternal behavior. As expected,
these genes are also present in reproductive organs and also
in brain areas related to reproductive processes. Yet, only
recently has the issue of circadian rhythmicity on reproductive
function been explored in laboratory animals and in humans,
e.g., in relation to shift work. Thus, the study of circadian
rhythms in reproductive processes is a topic of growing
importance.
1.03.6.1 A ‘Healthy Clock’ Is Necessary for Ovulation
and Pregnancy
Everett and Sawyer (1950) in a seminal publication demon-
strated that the preovulatory release of luteinizing hormone
(LH) in the rat could be blocked by barbiturate sedation at
‘certain critical hours during proestrus,’ usually between 2
p.m. and 4 p.m. (Everett and Sawyer, 1950). Exogenous admin-
istration of estradiol induces an LH surge, but always in the late
100 Parental Behavior
afternoon, even on multiple successive days (Caligaris et al.,
1971). After mating the copulatory stimulus induces the initia-
tion of a pattern of biphasic circadian prolactin surges: one
diurnal and one nocturnal of low and high amplitude, respec-
tively (Freeman and Neill, 1972). As expected, the timing of LH
and PRL release is controlled by the SCN (Kawakami et al.,
1980; Poletini et al., 2007).
Rhythmic oscillation of clock genes had been observed in
the ovary, uterus, and oviduct, and it is thought that such
oscillations contribute to the timing of several reproductive
events (reviewed in Sellix, 2013). By using genetic mouse
models with disruption of the core molecular clock, our
knowledge about the importance of a ‘healthy clock’ on repro-
ductive events has significantly advanced. Clock/Clock mutant
mice have a reduced pregnancy rate and even those animals
that do become pregnant have an increased rate of midgesta-
tion fetal reabsorption and pregnancy failure at full term. This
failure was indicated by severe dystocia, and after 24 h of
unsuccessful labor females were sacrificed. In all cases the
fetuses were fully developed but were dead and showed
morphological abnormalities (Miller et al., 2004; Miller and
Takahashi, 2014). Similarly, knockout mice of Per1 and Per2
show deficiencies in implantation, maintenance of pregnancy,
and/or parturition (Pilorz and Steinlechner, 2008). Moreover,
Bmal1 knockout mice show a complete lack of embryonic
implantation (Boden et al., 2010). It has been proposed
that these abnormalities are due mainly to an inappropriate
circadian daily timing to coordinate hypothalamic control
of the specific hormonal milieu of pregnancy (Miller and
Takahashi, 2014). Indeed, Clock mutant mice have a shortened
pseudopregnancy, indicative of a disruption of PRL release
(Miller et al., 2004). Previously it was demonstrated that
blocking of the circadian clock by antisense deoxyoligonu-
cleotides for clock genes in the SCN disrupts the circadian
rhythm of PRL release (Poletini et al., 2007). In humans
studies that analyzed polymorphisms in four circadian clock
genes found that a single-nucleotide polymorphism of the
gene ARNTL is associated with an increase in miscarriages
(Kovanen et al., 2010). This is the first study to establish a rela-
tionship between a polymorphism of one clock gene and
fertility in humans, although the reason for this infertility
remains unclear.
The importance of the circadian clock for reproductive
processes in humans had been established primarily through
abundant evidence related to the effects provoked by exposure
to lighting conditions during pregnancy. This procedure imme-
diately affects the normal rhythm of the hormone melatonin
(MEL). This hormone is produced in the pineal gland and
secreted to the blood during the night. Its concentration reaches
a peak after midnight, then decreases to reach low, almost
undetectable levels during the day. The duration of MEL secre-
tion is affected by the length of the night period and by expo-
sure to a bright light during the night (Pevet, 2003). Lewy et al.
(1980) were the first to demonstrate that the exposure to bright
light (2500 lux) during the night inhibits the normal pattern of
MEL secretion. Now it is known that very low light intensity
(<500 lux), equivalent to the normal illumination of a room
during the night, is sufficient to inhibit the onset of MEL during
the night (Boivin et al., 1996). Specifically, wavelengths in the
range of 446–477 nm, in the blue spectrum of intensities, and
as low as 1 lux are enough to drastically decrease MEL concen-
tration in the blood (Zeitzer et al., 2000).
It has been calculated that 15–20% of the working popula-
tion in Europe and the United States is engaged in shift work
(PLoS One Medicine Editors, 2011). Shift work is defined by
the International Labor Organization as “a method of organiza-
tion of working time in which workers succeed one another at
the workplace so that the establishment can operate longer
than the hours of work of individual workers” (PLoS One
Medicine Editors, 2011). Epidemiological studies of working
hours during the night or rotating work schedules have been
associated with a higher risk of developing cardiovascular/
metabolic/gastrointestinal disorders, some types of cancer,
and mental disorders (Gamble et al., 2013).
Due to the different styles of altered working hours and
individual preferences of sleep patterns, it is difficult to have
a clear correlation between exposure to abnormal lighting
conditions and reproductive processes. However, studies in
different populations in general agree that altered working
hours greatly increase the risk of endometriosis, painful and
irregular menstrual cycles and menstrual flow, dysmenorrhea
duration of menstrual bleeding, a general delay in ovulation,
miscarriage, low birth weight, and preterm delivery (reviewed
in Gamble et al., 2013; Lohstroh et al., 2003). After birth and
upon return to their predelivery work schedule, mothers
show a low percentage of breastfeeding and early introduction
to complementary foods, which increase the risk of infections
and poor nutrition in the newborn (Lakati et al., 2002).
Recent studies in pregnant rats with controlled chronic
phase shifts have found specific alterations in hormonal and
metabolic rhythmic parameters (Varcoe et al., 2013). These
authors found that timing of corticosterone, leptin, glucose,
insulin, free fatty acids, triglycerides, and cholesterol concentra-
tions were profoundly disrupted. Moreover, gluconeogenic and
circadian clock genes in the maternal and fetal liver became
either arrhythmic or were in antiphase relative to control
animals. This result in the liver is most probably related to
altered hours of food consumption as a consequence of phase
shifts. As already stated, peripheral organs also have functional
clock genes, which can be entrained by specific cues others than
light. Among them, timing of food consumption is a powerful
zeitgeber (entraining cue) for the circadian clock machinery of
the liver and other peripheral organs (Damiola et al., 2000).
Similarly, in women an increased risk of Type 2 diabetes melli-
tus has been reported as a consequence of rotating periods of
night shift work (Pan et al., 2011). These alterations in the
hormonal and metabolic parameters of the mother during
pregnancy have detrimental effects on their offspring, which
are glucose intolerant due to whole-body insulin resistance
(Varcoe et al., 2011). MEL seems to play a central role in this
effect because (1) rats born from pinealectomized mothers
were glucose intolerant due to hepatic insulin resistance and
(2) offspring from pinealectomized mothers that received
exogenous MEL were not glucose intolerant (Ferreira et al.,
2012). These results agree with previous reports that pinealec-
tomy induces glucose intolerance and increased glucose
production in adult rats (Nogueira et al., 2011). From this
evidence it is clear that disruption of the normal pattern of
MEL during pregnancy has profound consequences on the
fetus. Indeed, exposure to constant light during the night

suppresses the rhythmic secretion of MEL in pregnant dams
(Mendez et al., 2012). This has important consequences for
the developing fetus, which is exposed to rhythmic varying
concentrations of MEL, as this hormone crosses the placenta
(Okatani et al., 1998). Because the SCN is not a functional
master clock in the fetus, it has been considered that rhythmic
maternal MEL is necessary to drive the fetal circadian system
(Mendez et al., 2012). Consequently, blocking the MEL rhythm
in the pregnant mother by exposure to constant light induced
intrauterine growth retardation and markedly affected mRNA
expression of clock genes and clock-controlled genes as well
as the rhythm of corticosterone secretion (Mendez et al.,
2012). This, in turn, affects normal development of other
organs because in the fetus corticosterone acts as a peripheral
circadian clock, whose rhythm sustains peripheral clocks in
many tissues of the fetus (Torres-Farfan et al., 2011). In general
these studies support the proposal that maternal MEL acts as
a zeitgeber for different fetal peripheral oscillators (Serón-
Ferré et al., 2016).
1.03.6.2 Circadian Timing of Parturition
Parturition in nocturnal rodents usually occurs across their
resting period, i.e., during the day or subjective day in animals
kept in constant darkness (Olcese et al., 2013; Takayama et al.,
2003). In humans, data of labor onset cluster statistically in the
late nighttime and early morning hours, and parturition occurs
12–24 h later, at least in nulliparous women (reviewed in
Olcese et al., 2013). Experiments in the rat revealed that partu-
rition is controlled by circadian signals as destruction of the
maternal SCN disrupts this temporal gating of birth (Reppert
et al., 1987). In an elegant experiment, Takayama et al.
(2003) demonstrated that the circadian signal is MEL from
the pineal gland. Pinealectomy eliminated the circadian timing
of parturition, which then occurred randomly across the 24-h
light/dark cycle. Administration of MEL during the night, but
not during the day, restored the temporal daytime phasing of
parturition. Yet, in primiparous rabbits kept under a 14:10
photoperiod, parturitions occurred throughout the day
(González-Mariscal et al., 2013a).
Evidently, the control of pregnancy maintenance and the
necessary physiological events for timing parturition requires
a complex balance of other hormones. In women, plasma
progesterone has a significant diurnal rhythm before week 35
of pregnancy (Challis et al., 1981; Junkermann et al., 1982),
but this rhythm is not longer detected by week 38 of gestation
(Challis et al., 1983) coincident with an increase of inhibitory
progesterone receptor isoforms (Mendelson, 2009) and pro-
contractile mechanisms necessary for parturition (Olcese
et al., 2013).
If MEL stimulates nocturnal uterine contractions at parturi-
tion, then inhibiting its secretion must affect such contractions.
A recent experiment in humans confirmed this proposal
(Olcese et al., 2013). Uterine contractions of pregnant women
>38 weeks of gestation were monitored continuously from 7
p.m. to 7 a.m. under dim light. At 11 p.m. they were exposed
to a 10 000 lux full-spectrum lamp. Plasma MEL concentration
immediately decreased by 45% as did uterine contractions.
This evidence strongly supported the proposal of a circadian
control of parturition through the differential secretion of
Parental Behavior 101
MEL, which, in turn, is controlled by the master clock. Thus,
MEL seems to be a zeitgeber for the timing of parturition
(Olcese et al., 2013).
1.03.6.3 Circadian Rhythms in Breast and Breast Milk
Levels of MEL have a circadian rhythm with higher levels
during the night, as already mentioned. Concurrently, in breast
milk too, there is a rhythm of MEL. During the day these
concentrations are very low; they start to increase during the
early night, reach a peak after midnight, and then decrease
(Engler, 2012). MEL itself has important benefits for the
newborn, for instance, regarding infantile colic. This is
a common pathology worldwide in infants younger than
3 months of age, and the episodes of frequent crying occur in
the evening at about the same hour every day (Savino et al.,
2014). Colic follows a relationship with the sleep–wake cycle,
and its alteration seems to be a trigger for colic episodes
(Epstein et al., 2013). As mentioned, in utero the fetus is
exposed to a circadian release of MEL coming from the mother
but, after birth, a clear rhythm of the infant’s own MEL is
detected at around 3 months of age (Kennaway et al., 1996),
coincident with the consolidation of nighttime sleep
(Henderson et al., 2010). Consequently, it has been proposed
that MEL, received through breast milk, improves nocturnal
sleep and reduces infantile colic (Engler et al., 2012). This
datum is important as sales of milk formula substitutes are
growing yearly by around 10%, mostly in low- and middle-
income countries (Piwoz and Huffman, 2015), and some of
them are promoted to increase night sleep, which is not true
(Montgomery-Downs et al., 2010). In cows (as in humans),
exposure to artificial light during the night significantly reduces
MEL content in milk, a result that is being considered to
produce ‘chronofunctional milk’ from cows (Asher et al.,
2015).
Besides MEL, other components of breast milk show
temporal fluctuations. Cytokines, important for the develop-
ment of the immune, nervous, and gastrointestinal systems,
show chronobiological variations in breast milk (Morais
et al., 2015). Additionally, the profile of antibodies and
complement proteins of the immune system are higher during
the day than at night in human breast milk (Franca et al.,
2010). The chronobiological aspect of breast milk is an impor-
tant and necessary area of research that needs urgent attention,
especially considering that suboptimal breastfeeding accounts
for around 800 000 child deaths annually, and one of the
causes is the use of breast-milk substitutes (Piwoz and Huff-
man, 2015). Some attempts have been made to reproduce
circadian variations of selected carbohydrates, aminoacids,
and proteins (though not MEL) claiming that they help to
consolidate the sleep/wake rhythm in bottle-fed infants from
4 to 20 weeks of age (Cubero et al., 2007). At 3 months of
age, when the infant’s pineal starts to produce MEL, the addi-
tion of the amino acid tryptophan (necessary for MEL
synthesis) significantly improves the development of the
sleep/wake rhythms (Aparicio et al., 2007).
In agreement with the daily and circadian variations in the
components of breast milk, the ductal epithelium of the
mammary gland shows robust circadian expression of PER1
protein in lactating, but not, virgin mice (Metz et al., 2006).
102 Parental Behavior
Detailed genetic analysis in rat dams indicates that from day 20
of pregnancy through day 1 of lactation there are significant
changes in several genes, including clock genes, in the liver
and mammary gland which the authors suggest are orches-
trated by the master clock in order to support the increased
metabolic demands of milk synthesis at the onset of lactation
(Casey et al., 2009). In humans a microarray study of RNA ob-
tained from breast milk fat globules revealed more than 14 000
transcripts, 7% of which, including clock genes, changed signif-
icantly throughout the day; some were more highly expressed
during the day and others during the night (Maningat et al.,
2009).
The above evidence points to the importance of MEL (and
perhaps other components in mother’s milk that show
a rhythmic secretion pattern) for the development of circadian
rhythms in the newborn, when it is no longer exposed to the
circadian environment in utero. This deficiency of environ-
mental circadian maternal influence after birth is more evident
in preterm infants. Usually these infants are exposed to
constant bright light in the neonatal intensive care units. This
condition negatively affects the normal growth and develop-
ment of infants (Blackburn, 1998). By placing a removable
helmet over the premature infant’s head, the exposure to the
bright light during the night was drastically reduced. The idea
was to create an artificial light–dark cycle instead of the 24-h
constant illumination. As a result, infants gained weight faster
and had a shorter hospital stay than newborns kept in constant
illumination. Importantly, they developed higher concentra-
tions of MEL during the night than during the day, as opposed
to infants under constant illumination in which this difference
was not observed (Vásquez-Ruiz et al., 2014).
1.03.6.4 Daily Rhythms in the Mother during Lactation
During the transition from pregnancy to lactation, there are
tissue-specific changes in molecular clocks. In mice, the ampli-
tude and robustness of rhythms change between the SCN, liver,
and mammary gland, as well as the stoichiometric relationship
between positive (CLOCK) and negative (PER2) component
proteins of the core molecular clock. Thus, this relationship
remained 1:1 in the liver but was increased to 4:1 in the
mammary gland during this transition (Casey et al., 2014).
Following parturition, all mammals engage in lactation and
display various forms of maternal behavior. Yet, little is known
about the daily or circadian control of this behavior, except in
one species. The rabbit is unique among mammals as the
mother nurses the kits once a day at around the same hour
throughout the 30-day lactation. This predictive behavior has
important consequences both for the mother and the kits
(reviewed in González-Mariscal et al., 2016). In this section
we will review behavioral and physiological parameters, activa-
tion of some neuroendocrine cells, and rhythmic patterns of
clock gene machinery in lactating rabbits. Whenever possible,
literature in other species will also be included.
1.03.6.4.1 Locomotor Behavior
Intact female rabbits kept in nonisolated conditions under
a light/dark cycle have a circadian rhythm of locomotor
behavior with a significant increase in activity before and after
the first hours of lights on. The activity steadily decreases from
then onward, reaching lowest levels at around the time of lights
off (Meza et al., 2008). However, the timing of nursing in
lactating does is a powerful stimulus that drastically affects
this rhythm. Lactating does spontaneously nurse their kits
during the night (Caba and González-Mariscal, 2009; Gonzá-
lez-Mariscal et al., 2013a), but timing of once-a-day nursing
can be scheduled either during the day or during the night
(Caba et al., 2008). By using this strategy it was demonstrated
that the rhythm of locomotor behavior shifts in parallel to the
timing of nursing (Meza et al., 2008); that is, locomotor activity
is maximal at the time of scheduled nursing. Moreover, there is
a clear increase in locomotor behavior even before nursing
time, which suggests an anticipatory activity. This is very inter-
esting because in other animal models before access to a timed
stimulus, such as food or other rewards, animals show an
increase in locomotor behavior, a finding that has been inter-
preted as the behavioral output of an endogenous clock
(Caba et al., 2008; Antle and Silver, 2009). Coincident with
this interpretation, a rhythmic expressing of PER1 protein has
been reported in several brain areas of the lactating rabbit brain
(see below).
In other species, such as the Djungarian (P. campbelli) and
Siberian (Phodopus sungorus) hamsters (Scribner and Wynne-
Edwards, 1994) and in the rat (Rosenwasser et al., 1987), there
is a decrease in wheel running or locomotor activity rhythms
during pregnancy and lactation, coincident with an increased
time spent in the nest. The locomotor activity gradually
recovers to levels seen before pregnancy at the end of lactation.
1.03.6.4.2 Core Body Temperature
Pregnant rabbits have a circadian rhythm of temperature with
a peak that free-runs with a period length >24 h (Jilge et al.,
2001). In contrast, after parturition the peak of temperature
is shifted to the time of once-a-day nursing. Similar to the loco-
motor rhythm, body temperature starts to increase in the hours
preceding nursing, which also supports the proposal of an
endogenous clock in the control of anticipatory activity.
In lactating rodents the dam shows hyperthermia perhaps
because of the frequent contact bouts with her pups (Woodside
and Leon, 1980; Scribner and Wynne-Edwards, 1994). Nursing
bouts in the rat occur every 24–54 min without an apparent
circadian periodicity (Lincoln et al., 1973). Temperature
rhythm is not associated with these nursing events because it
reaches a peak exclusively during the night (Leon et al.,
1984). Yet, it shows an advance in its rising phase and a reduc-
tion in rhythm amplitude during gestation and an increase
during lactation (Krittrell and Satinoff, 1988). These changes
during lactation are associated with the presence of pups.
When the litter is removed from the nest, these changes in
temperature disappear immediately.
1.03.6.4.3 Rhythms of PER1 Protein in Neuroendocrine Cells
In rabbits (Caba et al., 1996) and rats (Jirikowski et al., 1989),
changes in the immunoreactivity of oxytocinergic cells occur in
association with specific reproductive conditions. In doe
rabbits the number of OT cells increases in the lateral hypotha-
lamic area and the PVN after parturition in comparison to
estrous or pregnant subjects. In the rat there is an increase in
immunostaining for OT cells in several regions of the forebrain,
associated with the onset of maternal behavior and the

physiological demands of lactation. By analyzing the expres-
sion of PER1 protein in OT cells in the rabbit, it was demon-
strated that the timing of nursing entrains rhythmic activity
of OT cells. In estrous does, PER1 protein has a rhythmic
expression in the supraoptic and paraventricular nuclei, but,
in lactating rabbits, the timing of nursing entrains the rhythm
of this protein. Specifically, at the time of nursing, PER1 cell
numbers are low in both nuclei, but, 4–8 h after nursing,
they increase significantly and reach a peak. From these results
it was proposed that periodic nursing is a time signal for the
synthesis and/or release of OT, both at central and peripheral
levels (Meza et al., 2008). No studies had explored clock genes
or their proteins in OT cells during lactation in other species.
Besides OT, dopaminergic cells are also entrained by the
timing of nursing in the rabbit. Dopaminergic cells of TIDA
(tuberoinfundibular dopaminergic) and PHDA (periventricu-
lar hypophyseal dopaminergic) cell groups in the hypothal-
amus have a rhythmic expression of PER1 protein that peaks
3 h after lights-off in estrous subjects. The timing of lactation
entrains both dopaminergic populations, and now higher
levels of PER1 are attained 4–8 h after the time of scheduled
nursing (Meza et al., 2011). This shift in the rhythmic activity
of dopaminergic cells is probably associated with the daily
release of prolactin, which is released massively to the plasma
in response to the suckling stimulus of the kits (Fuchs et al.,
1984). In contrast, no change in the number of PER1 cells
was found in the incertohypothalamic dopaminergic cell group
in the diencephalon, which does not project to the anterior
pituitary and has not been associated with the control of
prolactin secretion, as TIDA and PHDA cell groups (Cheung
et al., 1998).
It had been suggested that suckling stimulus could be the
entraining signal for the observed rhythms in OT and dopami-
nergic cells during lactation. When lactating females were pre-
vented from nursing for 24 or 48 h, there was a significant
decrease of PER1 at peak time in those cells (Meza et al.,
2008, 2011). Indeed, the intensity of suckling, determined by
the number of suckling pups, is essential to maintain the circa-
dian periodicity of nursing: does with litters smaller than four
kits show a disruption in the normal pattern of daily periodic
nursing. From this finding it was proposed that suckling could
be an entraining signal for periodicity of nursing (González-
Mariscal et al., 2013a) and for neuroendocrine cells (Meza
et al., 2008, 2011). Moreover, virgin rabbits – that do not
produce milk – induced to behave maternally by lesioning their
olfactory epithelium show nursing behavior (toward foster kits)
with a periodicity of c.24 h in 55% of cases (González-Mariscal
et al., 2015). This finding suggests that exposure to the
hormones of pregnancy and lactation is not essential for
the operation of a system that determines the occurrence of
the doe–litter interaction with a periodicity of c.24 h.
In contrast to the effects of timing of nursing on OT and
dopaminergic neurons, no change in the rhythm of PER1 in
the SCN was observed. However, the number of PER1 cells
significantly decreases at peak time in this nucleus (Meza
et al., 2008, 2011), which suggest that perhaps suckling is
a nonphotic stimulus for the SCN (Maywood et al., 1999),
but this has not been explored properly. The studies in lactating
rabbits clearly illustrate the differential control of clock gene
rhythms in the brain, with some cells locked to the light/dark
Parental Behavior 103
rhythm (SCN) and the uncoupling of other cells (OT and dopa-
minergic) to a different stimulus. This flexibility in the circa-
dian machinery is very useful to sustain specific physiological
demands, such as the synthesis and/or release of hormones
necessary to sustain milk production and output.
1.03.6.4.4 Rhythms of PER1 Protein in Areas Related
to Maternal Behavior
Extensive studies, mainly in rodents, have provided informa-
tion about several brain areas important for the control of
maternal behavior (reviewed in Olazábal et al., 2013a,b). The
POA and the LS, among others, have been explored in the
rabbit (reviewed in González-Mariscal et al., 2016). As timing
of nursing entrains neuroendocrine cells (see above), it was
hypothesized that areas related to the control of maternal
behavior could also be entrained. In fact, timing of nursing
induces a robust rhythm of PER1 protein in the POA and in
the dorsal and ventral regions of the septum in comparison
to estrous does. The effect in the POAs was most dramatic as
no rhythm at all was detected in controls (Meza et al., 2015).
Similar to neuroendocrine cells this effect seems to be main-
tained by suckling particularly in the LS as the number of
PER1 cells significantly decreases after 24–48 h of nursing
deprivation (Meza et al., 2015). The authors suggested that
the POA and LS are part of an ‘entrainable maternal circuit’
that supports maternal behavior in the rabbit. Future studies
are warranted to determine the phenotypical identity of these
PER1 rhythmic cells, which could help to understand the
almost unexplored control of the daily nursing of the rabbit.
Besides these studies in the rabbit, there is little evidence in
other species about changes in clock genes in relation to the
neural control of behavioral and physiological changes during
pregnancy or lactation. Changes in the rhythms of PER2 and c-
FOS proteins were found in subregions of the SCN as well as in
the ventral subparaventricular zone in early pregnancy
(Schrader et al., 2010). Additionally c-FOS expression was
reduced and its daily rhythm was lost or attenuated in other
regions such as the medial septum, diagonal band of Broca,
lateral hypothalamus, and MPOA (Schrader et al., 2013). The
authors suggest that these changes may support the above re-
ported adaptations of locomotor behavior and temperature
during pregnancy and parturition and also perhaps the rhythm
of sleep/wake cycle.
1.03.7 Beyond Motherhood: Maternal Behavior
as an Experimental Model for the Study of Some
Neuropsychiatric Disorders
Scientific investigations are often grounded on the use of exper-
imental models. Models are experimentally accessible represen-
tations of a natural phenomenon that is being investigated, and
information obtained by observing and manipulating such
models can be used to generate hypotheses about its nature.
A model’s validity refers to the extent to which information ob-
tained from the model is useful for generating accurate hypoth-
eses. One important application of experimental models is to
understand the biological underpinnings of pathological
conditions in which core mental or behavioral processes are
disrupted. In humans, such disruptions can result in a wide
104 Parental Behavior
range of neuropsychiatric symptoms including (1) psycho-
motor dysfunction, e.g., hyperactivity, tics, and compulsive
behavior; (2) emotional disturbances such as depression and
anxiety; (3) cognitive alterations including psychosis, obses-
sional thinking and alterations in attentional processes; and
(4) altered motivational states ranging from compulsive drug
seeking in the case of addiction, to avolition and anhedonia
characteristic of depressive disorders and schizophrenia.
Although there is some reason to believe that humans are
uniquely capable of consciously reflecting on and experiencing
these behavioral and mental states as psychopathological
symptoms (e.g., Craig, 2002), the core brain processes that
underlie these states also operate in nonhuman animals and
are similarly subject to pathological disruption. In this context,
valid animal models would include those in which informa-
tion can be obtained, and useful hypotheses can be generated,
about these core processes and their disruption (Hoffman,
2015, 2016).
Most investigators that use animal models in order to learn
about human psychopathologies recognize the difficulties – or
impossibility – of reproducing a human mental disorder in
a nonhuman animal. Given these limitations, some investiga-
tors have emphasized modeling strategies in which core symp-
toms or endophenotypes of a given neuropsychiatric disorder
are convincingly replicated in the animal: for example, anhe-
donia and alterations in sleep patterns in the case of models
of depression (Gottesman and Gould, 2003; Uys et al., 2003;
Hoffman, 2011, 2013). Many emphasize pharmacological
responsiveness of the animal model to drugs that successfully
treat the human disorder as an important validating criteria
(Willner, 1991; Wolmarans et al., 2013). Others have proposed
that important information can be obtained from considering
human neuropsychiatric symptoms as the consequence of
alterations in fundamental, evolutionarily adaptive psycho-
physiological processes that are conserved between human
and nonhuman animals. According to this view, studying the
biological underpinnings of these core processes in controlled
experimental conditions, and using a comparative approach in
which a variety of animal species are studied, can give impor-
tant insights into the genetic, environmental, and neurobiolog-
ical processes that underlie human psychopathology
(Hoffman, 2015, 2016; Matthysse, 1986; Kas et al., 2007,
2011; Uys et al., 2003; Insel, 2007; Panksepp, 2006, 2011).
1.03.7.1 Emotional Systems
The concept of evolutionarily conserved psychophysiological
processes has been applied to understanding the roots of
human emotions. Panksepp (2006) proposes that the brains
of all mammals possess conserved neural substrates for seven
fundamental, adaptive, and evolutionarily ancient ‘emotional
systems.’ The names of these systems, by convention written
in upper case in order to distinguish them from the common,
everyday meaning of these words, are LUST, CARE, PANIC,
PLAY, FEAR, RAGE, and SEEKING. These brain emotional
systems are proposed to have arisen concomittantly with brain
systems that underlie basic adaptive behavioral and physiolog-
ical responses: in other words, an adaptive behavior and its
associated affective experience are ‘two sides of the same
coin.’ For example, the subjective emotional experience of
fear is proposed to have arisen together with the adaptive
behavioral responses of freezing or flight, and autonomic
responses such as increases in blood pressure, heart rate, and
reactivity to startling stimuli. Importantly, neurobiological
studies overwhelmingly indicate that the fundamental brain
processes that underlie these emotional systems are primarily
subcortical and are well conserved across mammalian species,
and perhaps in some cases (FEAR and PANIC, for example)
across vertebrate species in general (Davis et al., 2010; Stewart
et al., 2012; Sufka et al., 2009). In the context of animal
modeling, this theoretical framework provides a foundation
for the use of specific behaviors in nonhuman animals as
proxies, or ‘endo-psycho-phenotypes’ for human emotional
states, both normal and pathological (Panksepp, 2006).
Maternal care involving direct interactions with the young,
such as feeding, grooming, carrying, and defending, is ubiqui-
tous among mammals, as described in this chapter. According
to the theoretical framework proposed by Panksepp (2006),
mammalian maternal behavior is the core of the CARE
emotional system, while the SEEKING emotional system –
encompassing general motivational processes – is suggested
to be a fundamental component of all emotional systems.
There are many converging lines of evidence indicating that
the brain mechanisms associated with these emotional systems
are conserved across mammalian species and comprise path-
ways that interconnect the anterior cingulate, orbitofrontal,
and medial prefrontal cortices with the basal ganglia, medial
thalamus, hypothalamus, and midbrain (PAG and VTA)
(Panksepp, 2006, 2011). In the following sections, we will
discuss mammalian maternal behavior in the context of the
SEEKING and CARE emotional systems.
1.03.7.1.1 Motivation: Concepts and Definitions
The term motivation encompasses behavioral and psychological
processes by which an animal regulates the proximity and avail-
ability of a certain motivating stimulus. Such stimuli include
external ones that are important for survival and which are
approached, as in the case of food or a mate, as well as those
that signal danger and which are avoided, such as a predator.
Interacting with motivating stimuli provokes species-typical
consummatory behaviors, or unconditioned responses, that are impor-
tant for survival and adaptation to the environment. The expres-
sion of an unconditioned response is associated with a particular
affective state. These affective states are suggested to be generated
from the basolateral amygdala (BLA) and the central nucleus of
the amygdala (CeA), which modulate hypothalamic and brain
stem mechanisms that control unconditioned autonomic and
behavioral responses (Cardinal et al., 2002).
Motivated behavior (also referred to as goal-directed or instru-
mental behavior) is described as having appetitive and consum-
matory phases. During the appetitive phase, the animal
actively regulates the proximity of the motivating stimulus.
The consummatory phase is when the animal interacts with
the motivating stimulus, displaying behaviors that are often
stereotypical and species-specific. The psychobiological
construct of motivation is considered to have components of
‘wanting’ and ‘liking,’ each arising from separate brain
processes (Berridge, 2004). Wanting is manifested by the
animal’s behavior directed toward the motivating stimulus,
while liking comprises the subjective experience of pleasure

in response to interacting with the motivating stimulus. Inter-
fering with dopamine neurotransmission in the NAcc markedly
reduces the animal’s willingness to work to gain access to
a motivating stimulus (wanting), while liking responses are
mediated by endogenous opioid neurotransmission in the
NAcc and ventral pallidum and are independent of dopamine
(Smith et al., 2011; Castro et al., 2015).
Importantly, goal-directed behavior must be flexible and
adaptable to the animal’s internal physiological state and to
external contingencies. This flexibility is determined in large
part by brain processes that represent the incentive value, or moti-
vational value of a given stimulus. The incentive value of a stim-
ulus refers to the relative capacity of a given stimulus to elicit
goal-directed behavior. The incentive value of a given stimulus
varies according to the animal’s internal state, prior experiences
with that stimulus, and current external contingencies. Most
laboratory studies of goal-directed behavior utilize operant
conditioning paradigms, in which the animal (most often,
a rodent) is trained to perform a specific arbitrary behavior
(e.g., pressing a bar) in order to receive a motivating stimulus
(e.g., a food or sweet ‘reward’) (Berridge, 2004). In these test
paradigms, the amount of work that the animal is required to
do in order to receive the reward can be experimentally varied,
as can the incentive value of the reward. The former is experi-
mentally varied by increasing or decreasing the number of
bar presses that the animal is required to do in order to receive
the reward (thereby increasing or decreasing the amount of
‘work’ it is required to do), and the latter is varied by prior
deprivation of the reward (increasing its incentive value), or
by satiating the animal to the reward (decreasing its incentive
value by allowing the animal to eat it ad libitum just prior to
the test). These measures assess the ‘wanting’ component of
motivation. In the specific case of sweet rewards, ‘liking’ is
outwardly manifested by the animal’s unconditioned response
of tongue protrusion and smacking of the lips (Berridge, 2000).
To condense the findings of a substantial body of literature
into a pair of sentences: the incentive value of a particular
reward is associated with activity of the orbitofrontal cortex
(OFC) and is determined by interactions between this region
and the BLA; while the vigor and persistence to work in order
to obtain the reward (‘wanting’) is augmented by dopamine
release in the NuAcc core (Baxter et al., 2000; Berridge, 2000,
2004; Cardinal et al., 2002; Salamone and Correa, 2002;
Kringelbach et al., 2003; Kringelbach and Rolls, 2004;
Schoenbaum et al., 2011; Balleine, 2011). The unconditioned
affective response to the goal (‘liking’) appears to be modulated
by endogenous opioid neurotransmission in the NuAcc and
ventral pallidum (Castro et al., 2015).
1.03.7.2 Maternal Behavior in the Context of SEEKING
Studies of maternal behavior can offer a complementary
perspective on motivational processes and the neural under-
pinnings of goal-directed behavior. Let us consider one
frequently used measure to assess maternal behavior in the
rat: pup retrieval. In the laboratory, when a dam’s pups are
removed from the nest site and scattered around on the floor
of the cage, she quickly begins to pick them up and carry
them, one by one, back to the nest site. This behavior is goal-
oriented in that it is aimed toward a specific end: to return all
Parental Behavior 105
of the pups to the nest. However, unlike most commonly
studied operant conditioning paradigms, pup retrieval itself is
an innate, unconditioned response. The experimental variables
that are measured are the latency to begin collecting, propor-
tion of pups returned to the nest site, and duration of pup-
carrying behavior. These measures clearly reflect the ‘wanting’
component of motivated behavior, as they involve behavioral
activation and ‘working’ to achieve a specific outcome. More-
over, similar to conditioned instrumental behavior, dopamine
in the NuAcc augments the vigor and persistence of this uncon-
ditioned behavior (Hansen, 1994; Keer and Stern, 1999).
In other experiments, rat or mouse dams were conditioned
to display bar-pressing in order to gain access to a live pup
(Hauser and Gandelman, 1985; Lee et al., 2000; Van Hemel,
1973). These studies demonstrated that, for rodents, contact
with a live pup can serve as a rewarding stimulus that can
support behavioral conditioning. Similarly, maternal rats
preferred pup contact over cocaine during the early postnatal
period – but no longer during the late postnatal period – in
conditioned place preference paradigms where the female
chose between spending time in a chamber previously associ-
ated with pups and an alternative chamber previously associ-
ated with cocaine (reviewed in Pereira and Morrell, 2011).
In other words, similar to food or cocaine reward, contact
with a pup apparently elicits an unconditioned pleasure
response, or ‘liking.’ Likewise, the incentive value of the pup
changes across the reproductive cycle, and the behavioral
response of the female changes accordingly. Changes in the
incentive value of maternally relevant stimuli are most likely
related to changing circulating levels of certain hormones of
pregnancy. One particularly clear demonstration of this
phenomenon is the maternal behavior of the female domestic
rabbit.
In nature, the pregnant female European rabbit (Oryctolagus
cuniculus) digs a burrow, ending in a nest chamber, approxi-
mately 1–2 weeks before parturition (gestation in the rabbit
is 31 days). When finished, she exits the burrow and conceals
its entrance by covering it with soil. Then, a few days before
parturition, she reopens the burrow and begins to collect dry
grass in her mouth and carries it into the chamber, where she
constructs a nest. About 20–40 trips are necessary in order to
collect enough grass to finish the nest (Hoffman and Rueda-
Morales, 2009). When finished, she leaves the burrow, again
concealing its entrance. Later, on the night of parturition, she
reopens the burrow, pulls out hair from her ventrum and lines
the inside of the nest with it, and then gives birth to the kits
inside the maternal nest. She eats the placentae (Melo and Gon-
zález-Mariscal, 2003), cleans the newborn and nurses them,
and then leaves the nest burrow and conceals its entrance.
One time per day thereafter, she returns to the nest to nurse
her kits for a very brief time (approximately 5 min), each
time reopening and concealing the burrow’s entrance (Myers
and Poole, 1961).
This behavioral pattern is faithfully replicated in the labora-
tory. A series of studies have shown that the rise in circulating
progesterone (against a background of constant estradiol
levels) during pregnancy stimulates the motivation to dig while
the subsequent fall in the levels of this hormone, which occurs
a few days before parturition, triggers the drive to collect straw,
carry it into the nest box, and construct a straw nest. A surge in
106 Parental Behavior
prolactin levels just before parturition is associated with the
onset of hair pulling and the incorporation of this hair into
the straw nest (González-Mariscal et al., 1994, 1996, 2004b).
Controlled experiments strongly suggest that these behavioral
changes are associated with changes in the incentive value of
specific external stimuli. Thus, several days before parturition
the pregnant rabbit displays a clear preference to collect and
carry straw over collecting real rabbit hair or synthetic material
that resembles rabbit hair, when both materials are freely avail-
able. By contrast, just before parturition, she preferentially
collects and carries real or synthetic hair (González-Mariscal
et al., 1998). Moreover, the motivation to collect and carry
straw is also modulated by external cues associated with the
nest: whether constructed by the rabbit herself, or by the exper-
imenter, interacting with a finished nest inside the nest box
quenches her motivation to collect and carry straw. Conversely,
straw carrying motivation is prolonged during experimental
manipulations in which the rabbit is able to collect and carry
straw but unable to interact with a completed nest. In other
words, the incentive value of straw is adjusted according to
external cues associated with the status of nest completion
(Hoffman and Rueda Morales, 2009). Perhaps most strikingly,
the female rabbit will eat meat only during a very brief period
just after parturition, when she would normally consume the
placentae. During the rest of her life, the rabbit is a very strict
herbivore (Melo and González-Mariscal, 2003). The theme
running through this body of work is that the incentive value
of specific stimuli (straw, hair, meat) is determined by a combi-
nation of internal physiological cues (e.g., hormone levels) and
external cues (e.g., the presence or absence of a completed nest
inside the nest box). What is the neural substrate underlying
such dramatic changes in the incentive value of these stimuli?
Studies using operant conditioning paradigms, in which the
incentive value of the reinforcer (most often, a food reward) is
experimentally controlled, point to OFC-BLA connections as
the substrate for determining the incentive value of food
reward (Baxter et al., 2000; Kringelbach et al., 2003;
Kringelbach and Rolls, 2004). Are these areas similarly
involved in determining the incentive value of unconditioned
maternally related stimuli? A current model for the
neurocircuitry of maternal behavior implicates the BLA and
PFC as regions that process and relay information about
maternally relevant stimuli to the NuAcc and ventral
pallidum, which gate the motor response to these stimuli
(Olazábal et al., 2013a,b). In support of this circuit model,
lesions to the BLA severely disrupted bar-pressing for pups in
maternal rats (Lee et al., 2000) and temporary inactivation of
the basomedial nucleus of the amygdala (BMA) disrupted
pup retrieval (Numan et al., 2010), suggesting that these
manipulations reduced the incentive value of pup contact.
Likewise, temporary inactivation of the medial prefrontal
cortex (mPFC, infralimbic or paralimbic regions) reduced the
preference for pup contact over cocaine administration in
a conditioned place preference paradigm (Perreira and
Morell, 2011) and greatly increased the latency to retrieve
pups (Febo et al., 2010). However, the specific involvement
of OFC-BLA connections in determining the incentive value
of maternally relevant stimuli apparently has not been
investigated in laboratory animals. On the other hand, there
is a growing body of work in human mothers that implicates
the OFC in the positive affective response to stimuli
associated with their own infant (discussed later).
1.03.7.3 Maternal Behavior in the Context of the CARE
Emotional System
1.03.7.3.1 Relationship of the CARE Emotional System
to Neuropsychiatric Disorders
Some evolutionary theories suggest that mother–infant attach-
ment is the most basic and evolutionarily ancient example of
affiliative behavior. A reasonable hypothesis is that the
mammalian CARE emotional system arose along with lacta-
tion, around 200 million years ago. Comparative investiga-
tions of mammalian maternal behavior in placental
mammals, as well as in marsupials and monotremes, can illu-
minate the inner workings of the CARE emotional system.
Components of this fundamental system are most likely dis-
rupted in neuropsychiatric conditions such as autism, which
is characterized by difficulties in forming social attachments,
and schizophrenia, which often involves symptoms of disin-
terest in social interactions and difficulties in processing social
stimuli. In both disorders, some studies suggest that pharmaco-
logical manipulations of oxytocinergic neurotransmission
might alleviate these symptoms (Hofman et al., 2015). Brain
processes associated with the sudden dissolution of a strong
social bond (e.g., death of a loved one or the termination of
a romantic relationship) are proposed to be similar to those
associated with depressive disorders as well as drug withdrawal
symptoms in opioid addiction (Panksepp, 2006).
1.03.7.3.2 Early Parental Preoccupations and Their
Relationship to Obsessive–Compulsive Disorder
In nonhuman animals, the CARE emotional system is activated
by a combination of internal endocrine and neurochemical
signals and external stimuli. For example, in the case of the
female rabbit, the interactions between internal hormonal
milieu and specific external stimuli, and their affects on moti-
vation and behavior, have been elegantly demonstrated. In
the case of humans the situation is much more complex, but
it is apparent that becoming a new parent is associated with
significant emotional, cognitive, and behavioral changes.
Leckman et al. (1999) and Abramowitz et al. (2007) described
thought and behavior patterns in new parents that strikingly
resemble the symptoms of obsessive–compulsive disorder
(OCD). Persons with OCD suffer from uncontrollable intrusive
thoughts (obsessions) that provoke the performance of specific
repetitive behaviors or behavioral rituals (compulsions).
Approximately 80–90% of the new mothers and fathers re-
ported experiencing recurrent and intrusive thoughts related
to some harm occurring to the baby. Moreover, similar propor-
tions of new parents in both studies (approximately 30–40%)
reported having intrusive thoughts about intentionally harm-
ing the baby. Other intrusive thoughts included ‘misplacing’
the baby (for example, leaving it at the supermarket checkout;
experienced by 50% of new parents), illness (30%),
contamination (50%), and sexual thoughts about the baby
(10%) (Leckman et al., 1999; Abramowitz et al., 2007).
Leckman et al. (1999) propose that such ‘early parental preoc-
cupations and behavior,’ or EPPB, are triggered by enhanced
activity of highly conserved neural pathways that link

heightened error detection and risk assessment to harm avoi-
dant behaviors. They also propose that OCD involves similar
alterations in these same systems.
Functional magnetic resonance imaging (fMRI) studies in
human mothers have implicated certain cortical and subcor-
tical regions in maternal responsiveness to stimuli associated
with their child (Hipwell et al., 2015; Kim et al., 2011;
Lorderbaum et al., 1999, 2002; Swain et al., 2008; Leibenluft
et al., 2004; Nitchke et al., 2003; Noriuchi et al., 2008; Ranote
et al., 2004). Baby-associated cues almost invariably activate
the OFC and/or anterior cingulate cortices. Activation in the
anterior cingulate cortex is frequently associated with hearing
a baby’s cries, compared to hearing white noise (the control
sound), along with activations in motor-associated regions
(premotor, supplementary motor, primary motor). Interest-
ingly, activations in the anterior cingulate do not seem to
distinguish between cries of the mother’s own baby and those
of a different child. On the other hand, activation of the OFC
was also associated with hearing a baby’s cry, but in this case
activations were greater for the mother’s own baby compared
to cries from an unfamiliar baby. Similarly, when mothers
were shown pictures or video clips of their own child or an
unfamiliar child, seeing her own child elicited greater activa-
tions in the OFC and posterior cingulate cortex. In one study,
OFC activation correlated positively with the mother’s self-
reported ratings of pleasant mood evoked by the photograph
of her child (Nitchke et al., 2003). Additionally, specific subcor-
tical regions were also activated in response to baby-associated
cues. Subcortical areas that were activated in this study
included many components of the cortico-striatal-thalamo-
cortical looped circuits: the caudate and NuAcc, globus pal-
lidus, thalamus, VTA, and substantia nigra, as well as the amyg-
dala and hypothalamus.
It is striking many of these very same regions have been
associated with the symptoms of OCD. The results of many
fMRI studies concur: obsessive–compulsive symptoms are
associated with increased activity in the OFC, anterior cingu-
late and insular cortices, caudate and putamen, globus pal-
lidus, and medial thalamus. Some studies have also pointed
to increased activity in the amygdala and ventral striatum
(NuAcc) (Rotge et al., 2008; Menzies et al., 2008). Together,
these regions form looped circuits that are proposed to serve
as an interface between mechanisms that perceive and inter-
pret emotionally salient stimuli and those that initiate and
maintain the appropriate motor response to these stimuli
(Burquière et al., 2015; Haber, 2016). In this context it is
compelling that, although the specific characteristics of obses-
sions and compulsions in an individual can be quite ideosyn-
cratic, they virtually always have at their core adaptive
processes of harm avoidance (to self, loved ones, or one’s
home and belongings), contamination and uncleanliness,
and symmetry and order in one’s personal space (Hoffman,
2011, 2015). The hypothesized CARE emotional system,
which is proposed to have arisen as a mechanism to allow
the formation and maintenance of the mother–infant bond
as well as to promote behaviors in the mother that increase
the chances that her offspring will survive, might participate
at some level in each of these processes.
In further support of a relationship between the CARE
emotional system and OCD, pregnancy and childbirth
Parental Behavior 107
appear to be times of increased vulnerability to the onset
of clinical OCD in women. Several studies have reported
that a considerable proportion of women that suffer from
OCD retrospectively associate the onset or worsening of their
symptoms with pregnancy or childbirth (Forray et al., 2010;
Neziroglu et al., 1996; Maina et al., 1999; Labad et al., 2005;
Guglielmi et al., 2014; reviewed in Abramowitz et al., 2004;
McGuinness et al., 2011). Likewise, the results of two
prospective studies were consistent with the proposal that
pregnancy and the postpartum period are times of increased
risk for the onset or exacerbation of obsessive–compulsive
symptoms (Chaudron and Nirodi, 2011; Abramowitz et al.,
2007). Moreover, women who reported OCD symptom
worsening during pregnancy or the postpartum period were
more likely to also have suffered from premenstrual wors-
ening of OCD symptoms, suggesting that reproductive
hormones might play a role in OCD symptom exacerbation
(Forray et al., 2010). Consistent with this idea, some studies
have reported an increase in subclinical obsessive–
compulsive-like thoughts and behaviors – typically having
to do with contamination and cleaning – during the premen-
struum. These obsessive–compulsive-like experiences were
positively correlated with salivary progesterone concentra-
tions (Fleischman and Fessler, 2011; Dillon and Brooks,
1992). Fleischman and Fessler (2011) propose that such
cognitive and behavioral responses to elevated progesterone
might be evolutionarily adaptive responses to progesterone’s
inhibitory effects on the immune system: behavioral
compensations to minimize contact with pathogens. There-
fore, a detailed understanding of the fundamental brain
mechanisms that underlie how the hormones of pregnancy
alter the female’s perception of specific stimuli should shed
light on possible pathological mechanisms of OCD (Hoff-
man and Rueda-Morales, 2009, 2012).
1.03.8 Conclusions and Perspectives
The study of mammalian maternal behavior has expanded
much since the last edition of this book. New areas of research
have flourished, e.g., the modifications to the female’s brain
that occur as a consequence of her engaging in maternal
behavior. An abundance of data have revealed that the
offspring (via their combination of sensory load) can perma-
nently change the mother’s emotional reactivity, her capacity
for learning and memory, and her subsequent maternal
behavior. These findings agree with the notion that plasticity
is an inherent property of the mammalian brain that is not
restricted to the infantile–juvenile stage.
A new dimension has been added to the study of maternal
behavior: time. In both animals and humans many works are
exploring the relationship between the circadian system and
particular aspects of reproductive physiology (e.g., parturition,
milk production) and reproductive behavior. The underlying
subcellular mechanisms are beginning to be revealed by
showing the activation of several ‘clock genes’ in particular
brain regions that participate in the regulation of maternal
behavior and nursing. The rabbit is emerging as a particularly
good species for exploring this topic given its natural once-a-
day nursing pattern.
108 Parental Behavior
The richness of the behavioral repertoire displayed by
mothers (human and animal), plus the accompanying changes
in emotionality, has emerged as a useful model for exploring
some aspects of particular neuropsychiatric diseases. As the
key processes that underlie symptoms such as hyperactivity,
deficits in attention, and anxiety also operate in animals,
specific models have been generated in rodents and rabbits to
obtain objective information for generating hypothesis about
the disruption of particular neuronal networks in specific
neuropsychiatric diseases.
In summary, the investigation of maternal behavior has
spilled over its traditional borders. The varied repertoire of activ-
ities (and associated physiological changes) shown by mamma-
lian mothers as they care for their offspring is now viewed from
the perspectives of developmental psychobiology, chronobi-
ology, biological psychiatry, and, of course, behavioral neuroen-
docrinology. The authors of this chapter hope that, from reading
our text, young investigators will be recruited to continue
studying maternal behavior and thus broaden our under-
standing of this most complex, essential mammalian activity.
Acknowledgments
The authors recognize the work of the late Craig Kinsley, who coau-
thored this chapter for the second edition of this book. His valuable
contributions to the field will forever remain a testimony to the crea-
tivity and originality of his academic thinking. We thank Francisco
Torres Quintanar for his generous help in the preparation of the
bibliography.
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 1.04 The Neurobiology of Social Affiliation and Pair Bonding
CS Carter, The Kinsey Institute, Indiana University, Bloomington, IN, USA
EB Keverne, University of Cambridge, Cambridge, UK
Ó 2017 Elsevier Inc. All rights reserved.

1.04.1 Introduction 117

1.04.2 The Natural History of Pair Bond Formation 118
1.04.2.1 Experiences Associated with Pair Bonding 118
1.04.2.2 Measurement of Social Bonding 119
1.04.3 Oxytocin and Vasopressin – Classical Neuroendocrine Peptides 119
1.04.3.1 Oxytocin and Vasopressin Receptors 120
1.04.3.2 Opioid, Oxytocin Interactions in the Brain 120
1.04.4 Mother–Infant Bonding 121
1.04.4.1 Mechanisms Underlying Maternal State 121
1.04.4.2 Oxytocin, Infant Recognition, and Bonding 122
1.04.4.3 Frontal Cortex: Role in Social and Parental Reward 122
1.04.4.4 Social and Maternal Bonding in Primates 124
1.04.5 Adult Pair Bonds 125
1.04.5.1 Mating Systems and Pair Bonds 125
1.04.5.2 Social Systems 125
1.04.5.3 Sexual versus Social Monogamy 125
1.04.5.4 Social Monogamy in Prairie Voles 126
1.04.5.5 Experimental Analysis of Partner Preferences 126
1.04.5.6 Selective Aggression as a Component of Pair Bonding 126
1.04.5.7 Affiliative Behaviors and Positive Sociality 127
1.04.5.8 Selective Social Behavior, a Role for Oxytocin and Vasopressin 128
1.04.6 Stress and Pair Bond Formation 128
1.04.6.1 Dopamine and Pair Bonding 130
1.04.7 Responses to Separation 130
1.04.7.1 Consequences of Isolation May Be Mediated through Oxytocin 131
1.04.7.2 Social Behavior, the Polyvagal Theory and Oxytocin 132
1.04.8 Phylogenetic and Ontogenetic Factors Influencing Pair Bonding 132
1.04.9 Peptides and Human Behavior 133
1.04.10 Clinical Implications of a Peptidergic Theory of Social Attachment 134
1.04.11 Attachment and Bonding in Primates: An Evolutionary Perspective for Humans 135
1.04.11.1 The Evolutionary Basis of Bonding 136
1.04.11.2 Genomic Imprinting, Social Behavior, and Cortical Growth 137
1.04.11.3 Oxytocin and Love 137
1.04.11.4 Oxytocin and Development 138
1.04.12 Summary 138
Acknowledgments 138
References 138

1.04.1 Introduction either positive, including affiliations and social bonds, or

Positive social interactions, including affiliations and social
bonds, dominate the behavioral repertoire of humans and
many higher vertebrates. Social behaviors are the product of
evolution and result from phytogenetic, ontogenetic, and
epigenetic processes. Social behaviors provide a context for
reproduction and can facilitate survival. Social behaviors also
are inherently species-typical and variable across individuals.
Social behaviors by definition involve two or more individuals
and require the willingness of individuals to aggregate and
remain together. Social behaviors are typically identified as
agonistic, including aggressive or defensive behaviors. Selective
social behaviors and social bonds are critical components of
long-lasting social interactions.
The purpose of this review is to examine neuroendocrine
mechanisms underlying social affiliation and social bonds in
mammals. The biological substrates of social behaviors are
shared with those necessary for birth and lactation. As with
reproduction, sociality and social bond formation rely on
neural systems that incorporate neuropeptides, including
oxytocin, vasopressin, opioids, corticotropin-releasing factor
(CRF), and related hormones. These peptides also serve to

Hormones, Brain, and Behavior, 3rd edition, Volume 1 http://dx.doi.org/10.1016/B978-0-12-803592-4.00004-3 117

118 The Neurobiology of Social Affiliation and Pair Bonding
coordinate the physiological and behavioral consequences of
positive social experiences, thus implicating reward systems
and especially dopamine. These molecules will be the focus
of this chapter.
1.04.2 The Natural History of Pair Bond Formation
Social bonds may form between a parent and infant, between
two adults, and among other members of a social group
(Table 1). These apparently very different bonding relation-
ships have a common function – namely to enhance reproduc-
tion and ensure reproductive success. This is achieved in pair
bonding and social group bonding by providing a readily avail-
able mate and predator defense, while mother–infant bonding
and social group bonding secure the nurture of infants and
their protection. Pair bonding is especially favored in those
mammalian species where offspring survival cannot be assured
without the help of mates.
Clear examples of pair bonding and the physiological basis
for social bond formation are provided from studies on voles,
marmosets, and other species that are identified as “monoga-
mous” (Kleiman, 1977). In domesticated species, social bonds
may cross species barriers (e.g., dogs and humans) where
a combination of tameness, docility, and dependence, selected
for by human intervention, has secured their survival and
enhanced reproductive success.
In promiscuous mammalian species, where parental males
are frequently absent, mother–infant bonding may help to
guarantee infant survival. This often involves individual infant
recognition, and examples of filial bonding are found among
ungulate species. Research on maternal bonding has taken
advantage of the fact that precocial ungulates, including sheep,
are excellent subjects for neurobiological investigation and do
develop selective mother–infant bonding.
In social living mammals and especially in primate socie-
ties, an alternative strategy for promoting reproductive success
through infant survival is seen in the assistance given by other
females (Hrdy, 2009). Serving as surrogate parents may involve
a self-sacrifice for reproductive potential and often is under-
taken by females that are genetically related to the offspring.
Table 1 Animal models for studying the biological bases of pair
bonding
Maternal–infant bonding:
Most commonly studied in animals in which the mother is selective.
Mother usually cares for only her own young.
Example: Sheep.
Maternal behavior:
Not selective, but may share physiological substrates with pair
bonding.
Examples: Rats and other rodents, rabbits, pigs.
Adult pair bonding:
Most commonly studied in adult monogamous mammals. Most
commonly studied in adult monogamous mammals.
Examples: Prairie voles and titi monkeys.
Reproduced from Carter, C.S., Keverne, E.B., 2002. The neurobiology of social
affiliation and pair bonding. In: Pfaff, D., Arnold, A., Etgen, A., Fahrbach, S., Rubin,
R. (Eds.), Hormones, Brain, and Behavior. Academic Press, New York, pp. 299–337,
with permission from Elsevier.
Old World primate societies are frequently referred to as being
‘female-bonded.’ Such primate matrilines not only provide for
group cohesion and social continuity, but kin also benefit from
the learning experience gained when helping mother. The
neurobiology of same-sex social bonds remains largely unex-
plored, but may rely on the underlying neural systems that
regulate other forms of social bonding.
Alloparenting and male parental behavior are traits of
species, ranging from birds to rodents, canids, and New World
primates, which are defined as socially monogamous (reviewed
in Solomon and French, 1997). Although both related and
unrelated animals may aid in the care of young, alloparenting
is most common among genetically related individuals. Selec-
tive social behaviors and social bonds are defining characteris-
tics of species that engage in alloparenting or paternal behavior,
suggesting that common underlying mechanisms may regulate
social bonding and other social traits in species that tend to
form communal families.
Social bonds also may provide direct benefits to the
members of a pair or group, by serving to buffer the indi-
vidual from stress. The physiological mechanisms for stress
buffering are related to those responsible for the formation
of pair bonds.
1.04.2.1 Experiences Associated with Pair Bonding
The essential first steps in social bond formation involve prox-
imity and social engagement (Porges, 2011). Potential part-
ners must interact in a species- and sex-appropriate manner.
The choice of a social partner, the tendency to engage in social
behaviors, and patterns of social behavior differ among
species and individuals, and between males and females
(Carter et al., 1995; DeVries et al., 1996). In addition, because
the outcome of social interactions can be positive or negative,
the emotional experience of a particular social encounter may
determine whether that encounter leads to pair bond
formation.
Both positive social interactions and social bonds may func-
tion to provide a sense of safety and reduce anxiety or the nega-
tive feelings associated with stressful experience. In species that
do tend to form pair bonds, stressful experiences (such as preg-
nancy and parturition), anxiety, neophobia, and isolation
often precede the formation of long-lasting social attachments
(Carter, 1998). Threatening situations may encourage return to
a ‘secure base’ or otherwise strengthen social bonds (Bowlby,
1969). These circumstances may increase social ‘drive’ or ‘moti-
vation,’ and subsequent social interactions, and thus increase
the tendency to form a social attachment.
The association of pair bonding with overcoming stressful
experiences also offers clues to the physiology of pair bond
formation. Hormones can reduce fear or behavioral inhibition
and permit the subsequent expression of selective social behav-
iors, such as those necessary for pair bonding. The neurochem-
ical processes that are capable of overcoming neophobia also
may be needed to permit the formation of new social attach-
ments. Although sharing some common substrates, the neural
substrates for social engagement and selective social bonding
are probably not identical (Cho et al., 1999), and it is likely
that the processes leading to social interactions differ from
those necessary for a bond to form.
 The Neurobiology of Social Affiliation and Pair Bonding
Table 2 Measurements of pair bonding
Selective contact or physical proximity in the presence of an attachment
object
Aggression in defense of the attachment object
Autonomic and/or endocrine responses
Distress after separation
Crying or vocalization
Increased release of stress hormones (HPA activation)
Reproduced from Carter, C.S., Keverne, E.B., 2002. The neurobiology of social
affiliation and pair bonding. In: Pfaff, D., Arnold, A., Etgen, A., Fahrbach, S.,
Rubin, R. (Eds.), Hormones, Brain, and Behavior. Academic Press, New York,
pp. 299–337, with permission from Elsevier.
1.04.2.2 Measurement of Social Bonding
Social bonds are hypothetical constructs, and thus measure-
ments require analysis of a complex assortment of behaviors
over long periods of time. However, selective behavioral,
hormonal, or autonomic responses to social stimuli, or their
absence, may be used to infer social bonds (Table 2). Labora-
tory analyses of social bonding often involve preference tests
in which experimental animals are allowed to choose between
or express differential responses to social stimuli.
Differential responses to the entire animal, visual cues,
vocalizations, or odors can be used to assess preferences.
The most compelling evidence for positive social preference
is usually obtained in tests in which an experimental animal
is allowed to simultaneously or sequentially make a choice
between a familiar animal or an otherwise similar unfamiliar
conspecific (or stimuli associated with those individuals).
Physical proximity and positive social interactions are
commonly used to index preference. Testing conditions in
which the experimental animal is allowed elective physical
contact with the stimulus animals are especially sensitive
(Williams et al., 1992). In addition, conditions that allow
the experimental animal to remain alone are desirable,
because voluntary social preferences may differ from forced
choices.
Responses to separation or reunion can also be evidence
for social bonding. In mammals and birds, ‘distress’ vocaliza-
tions may increase following separation and decline
following reunion. Secretion of hormones from the hypotha-
lamic–pituitary–adrenal (HPA) axis, usually cortisol or corti-
costerone or adrenocorticotrophic hormone (ACTH), also
follows separation from the ‘attachment figure’ and HPA
activity tends to return to normal upon reunion (DeVries
et al., 1996). However, vocalizations, changes in the HPA
axis, and autonomic responses may be triggered by either
social or nonsocial stressors. The most compelling evidence
for social bonding is obtained when various measures
converge and when those measures can be interpreted in light
of the biology of the entire organism and especially the neuro-
endocrine system.
1.04.3 Oxytocin and Vasopressin – Classical
Neuroendocrine Peptides
Because oxytocin and vasopressin are released during social
and sexual interactions, and under conditions that result in
119
pair bonding, the neural systems that incorporate these neuro-
peptides are potential candidates for a role in the mediation
of social behavior and especially social bonding (Table 3).
Oxytocin and vasopressin are neuropeptides composed of
nine amino acids, including a six amino acid ring with a three
amino acid tail. Oxytocin and vasopressin differ from each
other in two amino acids, one in the ring and one in the tail,
and may have evolved from a common ancestral peptide
(Van Kesteren et al., 1992; Acher et al., 1997). The gene for
each of these two peptides is located on the same chromosome.
The stereochemical similarities between these peptides allow
functional interactions with each other’s receptors. Oxytocin
and vasopressin are the predominant neurohypophyseal
hormones in mammals, although structurally similar peptides,
such as vasotocin, mesotocin, and isotocin, have been identi-
fied in other vertebrates (Acher et al., 1997). In addition, struc-
turally related peptides with consequences for reproductive
behavior are found in invertebrates, suggesting that variations
on these molecules appeared prior to the evolution of verte-
brates (Van Kesteren et al., 1992; Garrison et al., 2012).
Oxytocin is released in a coordinated fashion within the
brain and at the posterior pituitary, into general circulation
(Neumann and Landgraf, 2012; Gainer, 2012). It is likely
that the ability of oxytocin to have exceptionally broad behav-
ioral and physiological consequences is related to this capacity
for movement throughout the brain and body (Stoop, 2012;
Grinevich et al., 2016).
Oxytocin is produced tonically. In typical humans, basal
levels of oxytocin in plasma are notably consistent across
time (Gouin et al., 2010; Dai et al., 2012; Weisman et al.,
2013). However, oxytocin also can be released as pulses, thus
promoting muscle contractions in the uterus and mammary
gland. The pulsatile release of oxytocin neurons may be related
to the notable plasticity of these cells. In adult rats, oxytocin
neurons undergo physical transformations in response to
hormonal and social stimulation. During pregnancy and lacta-
tion, and perhaps under other conditions such as dehydration
or sexual stimulation (Carter, 1992), glial processes that nor-
mally separate the oxytocin-containing neurons are retracted
allowing electrical coupling and then the pulsatile release of
oxytocin. Vasopressin-containing neurons typically do not
Table 3 Endocrine mediators of pair bonding
Steroids
Gonadal steroids – not essential
But may modulate effects of peptides
Adrenal steroids – not essential
May modulate effects of peptides
Effects may be sexually dimorphic
Tends to be elevated then decline
Peptides
Oxytocin – may be especially important in females
Vasopressin – may be especially important in males
Corticotropin-releasing hormone – may modulate effects of stress
Opioids – modulation of separation responses
Reproduced from Carter, C.S., Keverne, E.B., 2002. The neurobiology of social
affiliation and pair bonding. In: Pfaff, D., Arnold, A., Etgen, A., Fahrbach, S., Rubin,
R. (Eds.), Hormones, Brain, and Behavior. Academic Press, New York, pp. 299–337,
with permission from Elsevier.
120 The Neurobiology of Social Affiliation and Pair Bonding
show this form of plasticity and pulsatile release. Furthermore,
oxytocin-producing cells are sensitive to oxytocin itself; thus
a form of autocrine feedback regulates the functions of
oxytocin-producing cells. Stimulation of the oxytocin system
may ‘feed forward’ to release more oxytocin, and in some cases,
administration of oxytocin appears to enhance the synthesis of
endogenous oxytocin (Grippo et al., 2012).
Oxytocin exposure is of particular significance during
development. Specifically oxytocin can influence cellular
growth, death or motility, inflammation, or differentiation
(Gutkowska and Jankowski, 2012). Oxytocin treatments in
early life can have dose-dependent effects on physiology and
behavior (Carter et al., 2009). Low doses of exogenous
hormone facilitate pair bonding, as well as the expression of
the endogenous oxytocin, and inhibit the expression of the
vasopressin (V1a) receptor. Furthermore, the oxytocin receptor
is susceptible to epigenetic regulation, for example, by
silencing genes via methylation. Changes in the expression
of the oxytocin receptor as a function of early experiences
could help to account for species and individual differences
in sociality and emotionality. As just one example, hyperme-
thylation of the oxytocin receptor gene has been detected in
autism (Gregory et al., 2009). Through mechanisms such as
these, the developmental effects of oxytocin may fine-tune
the nervous system, including the capacity for pair bonding
and attachment in later life.
The major sources of systemic oxytocin and vasopressin in
mammals are the magnocellular neurons of the supraoptic
nucleus (SON) and paraventricular nucleus of the hypothal-
amus (PVN) (reviewed in Russell and Leng, 1998). Oxytocin
and vasopressin, in conjunction with neurophysin carrier
proteins, are transported from magnocellular neurons in the
SON and PVN to the posterior pituitary, where they are secreted
into the blood stream. The release of oxytocin into the systemic
circulation is typically pulsatile. In addition, oxytocin and vaso-
pressin are released within the central nervous system (CNS)
from smaller, parvocellular neurons, located in the PVN and
other brain areas. The release of peptides within the CNS and
posterior pituitary can occur independently, although central
and peripheral release patterns also may be coordinated
(Kendrick et al., 1986; Landgraf and Neumann, 2004;
Grinevich et al., 2016).
In addition to the oxytocin that is produced in the SON and
PVN, cell bodies and fibers staining for oxytocin also have been
identified in the bed nucleus of the stria terminalis (bNST), the
anterior commissural nucleus, and the spinal cord (Sofroniew,
1983). The latter fibers terminate within the CNS or release
oxytocin into the cerebrospinal fluid (CSF). Oxytocin gene
expression increases in the PVN and SON during lactation
and around birth or under hormonal conditions that mimic
birth. Oxytocin also is produced in other tissues including
the ovary, uterus, placenta, and thymus. The multiple actions
of this hormone may integrate a variety of peripheral and
central processes, including those involved in reproduction
and social behavior.
As with oxytocin, the most abundant sources of vasopressin
are found in the PVN and SON. Vasopressin is synthesized and
released within the CNS by parvocellular neurons in the PVN.
Vasopressinergic cell bodies also have been identified in the
suprachiasmatic nucleus, medial amygdala (mAMY), bNST,
and areas of the caudal brain stem. Androgens facilitate the
synthesis and release of vasopressin, particularly in the
mAMY and lateral septum (LS) (DeVries and Panzica, 2006),
accounting for clear sex differences in the abundance of vaso-
pressin in the CNS.
Although vasopressin plays an important role in social
behavior, many of the functions of stimulation of the vaso-
pressin receptor differ from those of oxytocin (Neumann
and Landgraf, 2012; Stoop, 2012; Albers, 2015). For example,
in maternal behavior, oxytocin is critical to nursing and
important to nurture, while vasopressin has been implicated
in maternal (Bosch and Neumann, 2012) and paternal
defense of the young (Kenkel et al., 2012, 2013). Some
aspects of vasopressin’s functions within the nervous system
are sexually dimorphic, with possible implications for sex
differences in the tendency to show defensive behaviors and
for disorders, such as autism, that are male-biased (reviewed
Carter, 2007).
1.04.3.1 Oxytocin and Vasopressin Receptors
The oxytocin receptor is a member of the seven transmembrane
G-protein coupled receptors and is distinguished by differences
in its anatomical distribution across different mammalian
species. There is at present only evidence for one oxytocin
receptor, which is similar across species. However, species
differences do exist in the regulation of the oxytocin receptor
(Freeman and Young, 2016), allowing a variety of other
neuroendocrine factors, including steroids, to determine
species-typical patterns of receptor distribution (Witt et al.,
1991; Insel and Shapiro, 1992; Jiménez et al., 2015).
Oxytocin receptors have been identified by receptor autora-
diography and receptor mRNA expression in several brain
areas. Among the areas that contain high concentrations of
the oxytocin receptor in rodents are the olfactory bulbs, ante-
rior olfactory nucleus (AON), olfactory tubercle, nucleus
accumbens (NAcc), prelimbic cortex, ventral subiculum,
bNST, central nucleus of the amygdala, ventromedial nucleus
of the hypothalamus (VMH), LS, cingulate cortex (CC), dorsal
motor nucleus of the vagus (DMX), and the nucleus tractus sol-
itarius (NTS) (Barberis and Tribollet, 1996). These areas have
been implicated in both social behavior and the regulation of
the autonomic nervous system.
Although three isoforms of the vasopressin receptor have
been identified, most of the behavioral effects have been attrib-
uted to the V1a receptor, which is expressed in the brain. Vaso-
pressin V1a binding has been identified in the granule cell layer
of olfactory bulbs, accessory olfactory bulbs, the diagonal band
of Broca, CC, central and lateral aspects of the amygdala, the
dorsolateral thalamus, and superior colliculus (Insel, 1997).
Like oxytocin, the distribution of V1a receptors is highly
species-specific, and follows a pattern that implicates this
receptor in social behavior and pair bonding (Hammock and
Young, 2005).
1.04.3.2 Opioid, Oxytocin Interactions in the Brain
The interactions between opioids and oxytocin in the brain are
complex. During pregnancy, it is important that oxytocin is not
released from the magnocellular neurons into peripheral
 The Neurobiology of Social Affiliation and Pair Bonding
circulation as this produces a risk for preterm labor. Release of
oxytocin from neurohypophysial terminals is sensitive to inhi-
bition by opioid receptor agonists (Ortiz-Miranda et al., 2005).
Thus, in pregnancy, a central inhibitory opioid mechanism
restrains magnocellular oxytocin neurons from responding to
stress and cytokine release, minimizing the risk of preterm
labor (Brunton et al., 2006).
A number of findings indicate that endogenous opioids as
well as oxytocin are active during parturition. Plasma concen-
trations of b-endorphin increase during labor and birth in
humans (Goland et al., 1981), and in the rat brain, b-endor-
phin concentrations are elevated in the hypothalamus,
midbrain, and amygdala during pregnancy and parturition
(Wardlaw and Frantz, 1983). The terminal areas of distribu-
tion for endorphin neurons have much in common with
oxytocin neurons, and in certain areas the release of oxytocin
is strongly regulated by b-endorphin. However, considerable
species differences have been demonstrated with respect to
the receptor concentrations in different areas of the brain
(Mansour et al., 1988). Synthesis of proopiomelanocortin,
the precursor peptide for b-endorphin, increases in ewes
following estradiol or progesterone replacement therapy
which mimics the effects of pregnancy hormones. In sheep,
but not in rats, expression of proopiomelanocortin mRNA
during lactation increases significantly. Also in ewes, levels
of preproenkephalin messenger RNA expression are signifi-
cantly higher during lactation compared to late pregnancy
and parturition (Kendrick and Keverne, 1992). Taken together,
these findings provide an anatomical basis for opioid–
oxytocin interactions during pregnancy and lactation. A func-
tional relationship between these peptides has also been
demonstrated in the context of maternal behavior (Keverne
and Kendrick, 1991) and also in pair bond formation in voles
(Burkett et al., 2011).
1.04.4 Mother–Infant Bonding
The most notable form of an enduring social bond is that
between a mother and her infants. Bonding, although integral
to maternal behavior, can be distinguished from this. This
distinction is most obvious in a postpartum mother that is
highly maternal but rejects her offspring through a failure to
bond. This dissociation of maternal behavior and infant rejec-
tion occurs following failure in the recognition process.
Recognition is prerequisite for bonding, and in ungulate
species a sensitive period following birth provides the
window of opportunity for this recognition bond to occur.
A failure to make this recognition within the sensitive period,
as sometimes occurs in primiparous mothers, or multiparous
mothers if their infant is experimentally removed, results in
a rejection of the offspring (Poindron et al., 2007). It might
seem counterintuitive that a maternally motivated female,
having experienced the hormones of pregnancy and parturi-
tion, would reject her genetically related infant. Indeed, this
only makes sense when we view maternal rejection behavior
in its appropriate biological context. Within minutes after
birth in ungulates, the mother starts to lick the newborn,
which can usually stand and suckle within 2 h of birth
(Poindron and Le Neindre, 1980). It is around this same
121
time period that maternal care becomes exclusive. Thus in
sheep, the ewe, having ‘bonded,’ accepts only her own lamb
at suckling and rejects, often violently, any strange lamb
that attempts to suckle. Because sheep are seasonal breeders
with synchronized multiple births occurring throughout the
flock, all within a few weeks, it is important that the mother
recognizes and only permits her own lamb to suckle.
Nurturing is energetically costly to the mother, and ‘bonding’
ensures that this energetic sacrifice is only made to her own
genetically related offspring.
The mechanisms which underlie the formation of the
mother–infant bond involve the female being in a maternal
state, being able to recognize her offspring dependent on
unique sensory cues, and activation of the sensitive window
for recognition contingent upon the appearance of the
offspring. The recognition window is activated by parturition
and delivery of the neonate.
1.04.4.1 Mechanisms Underlying Maternal State
The ovarian hormones of importance in maternal behavior are
estrogens and progestins, and because there is no restriction to
the passage of steroid hormones from the vascular to the cere-
bral compartment, high-affinity binding neurons for these
hormones will be activated in all parts of the brain simulta-
neously (Kendrick and Keverne, 1991). Although studies have
tended to focus attention on the hypothalamus as a site of
action for steroid hormones, it should be remembered that
steroid binding neurons have a widespread distribution
throughout the limbic and olfactory brain. The pattern of secre-
tion of steroid hormones during pregnancy is remarkably
similar among all nonprimate mammals and is characterized
by high levels of progesterone in the postimplantation period,
which decreases prior to parturition with a concomitant
increase in estradiol. This prolonged priming of the brain by
exposure to high progesterone and low estrogens is important
for the suppression of sexual behavior in mammals and for
genomic activation promoting the synthesis of hypothalamic
peptides (oxytocin, CRF, prolactin, and b-endorphin). Proges-
terone decline followed by the sequential increase in estradiol
is important for synthesis of receptors for these peptides in
many parts of the limbic brain. In other words, steroid
hormone receptors have one of their actions in the brain by
binding to DNA motifs on the promoter regions of certain
genes, many of which code for neuropeptides and their recep-
tors (Table 4).
The steroid hormones of pregnancy are integral to the
induction of maternal responding but do not exclusively
code for these events; they also influence sexual behavior,
feeding behavior, and exploratory behavior, and thus have
the recruiting capacity for a wide range of neural systems. The
ovarian steroids are therefore perhaps best viewed as part of
a widely distributed addressing system, provided with a degree
of specificity in the context of maternal behavior by the events
which occur at parturition (Keverne and Kendrick, 1992). The
prolonged action of progesterone throughout pregnancy
suppresses estrous cycles and behavior, while parturition itself
recruits other neural systems for the initiation of maternal
behavior, thereby synchronizing maternal care with the birth
of infants.
122 The Neurobiology of Social Affiliation and Pair Bonding
Table 4 Mechanisms through which steroids and/or peptides may
influence social bonding
Developmental regulation of species-typical traits (Often including sexual
dimorphism in nervous system and endocrine function)
Alterations in peptide sensitivity or reactivity by altering:
Hormones or neurotransmitters and/or their receptors
In adulthood:
Peptide synthesis and/or release
Peptide receptors and/or binding
Altering behaviors that release peptides
Peptide–peptide interactions
Steroid–steroid interactions
Steroid–peptide–neurotransmitter interactions
Hormonal effects on the autonomic nervous system
Reproduced from Carter, C.S., Keverne, E.B., 2002. The neurobiology of social
affiliation and pair bonding. In: Pfaff, D., Arnold, A., Etgen, A., Fahrbach, S., Rubin,
R. (Eds.), Hormones, Brain, and Behavior. Academic Press, New York, pp. 299–337,
with permission from Elsevier.
1.04.4.2 Oxytocin, Infant Recognition, and Bonding
Pedersen and Prange (1979) first tested the hypothesis that
central oxytocin has a role in maternal behavior. It is an attrac-
tive idea that both the central and peripheral components of
a neuroendocrine system may be activated simultaneously as
part of a coordinated behavioral and neuroendocrine response.
Infusions of oxytocin and its analogue, tocinoic acid, had
a significant effect in promoting maternal behavior within
a 2 h period (Pedersen et al., 1982). Subsequent studies have
shown that the onset of maternal behavior can be delayed by
intracerebroventricular (ICV) treatment with oxytocin antisera
or a synthetic oxytocin antagonist (Fahrbach et al., 1985),
while olfactory stimuli stimulate maternal care through
increased expression of oxytocin receptors in specific brain
areas (Francis et al., 2002). In contrast, long periods of
mother–infant separation inhibit maternal care, again through
modulation of oxytocin receptors (Pedersen and Boccia, 2002).
In the sheep, vaginocervical stimulation and parturition
increase the levels of oxytocin within the brain, even though
the blood–brain barrier is relatively impermeable to oxytocin
(Keverne and Kendrick, 1992). Vaginocervical stimulation
can itself promote a rapid onset of maternal behavior in non-
gestant ewes (Keverne et al., 1983), while ewes that deliver
under epidural anesthesia show no interest in lambs until
such ewes are subsequently given intracerebral oxytocin
(Levy et al., 1992). Also in sheep, administration of oxytocin
directly to the brain’s ventricular system stimulates a rapid
onset of maternal behavior and induces the ewe to maintain
proximity with lambs in an open field (Kendrick et al.,
1987).
Microdialysis studies provide direct evidence for the release
of oxytocin at sites in the brain known to be involved in
maternal behavior. In ewes, oxytocin is released during labor,
birth, and suckling in the bNST, medial preoptic area
(mPOA) and olfactory bulbs (Kendrick et al., 1992). For
some time, anatomists have been confused by the mismatch
between neurotransmitter terminals and their receptors in the
brain. Indeed, under basal conditions, oxytocin may only be
effective at terminal areas. During critical life events such as
mating and parturition, however, it has been proposed that
the whole brain may be flooded with the peptide, activating
those receptors where axon terminals fail to project; however,
there is increasing evidence that oxytocin is neuronally secreted
more widely than was previously recognized (Grinevich et al.,
2016).
Whether secreted locally or through wide perfusion of the
nervous system, it may be the case that the exceptionally high
levels of oxytocin released into CSF at parturition act both as
transmitter and neurohumoral factors. This may permit
oxytocin to orchestrate maternal responses that require activity
throughout widespread areas of the brain, including the olfac-
tory bulbs, for integration of complex sensorimotor programs.
Administration of oxytocin to CSF in nonparturient ewes
evokes the complete complement of sensorimotor patterns
that make up full maternal responsiveness (Kendrick et al.,
1987).
The main source of intracerebral oxytocin is the parvocellu-
lar neurons in the PVN, which project widely throughout the
brain. Not only is the PVN the main source of intracerebral
oxytocin, but it also seems to be one of the targets for oxytocin.
The relative importance of this oxytocin feedback for the induc-
tion of maternal behavior has been shown following low doses
of oxytocin infused directly to the PVN (Da Costa et al., 1996).
The consequent induction of behavior is comparable to the
induction of maternal behavior by vaginal–cervical stimulation
and by ICV infusions of high doses of oxytocin. Oxytocin
release in the PVN at parturition is probably facilitating a posi-
tive feedback on both parvocellular and magnocellular neurons
to coordinate high levels of oxytocin release that are important
for the induction of maternal behavior, infant recognition, and
bonding.
The half-life of peptides such as oxytocin in CSF is consider-
ably longer than that found in peripheral plasma. Nevertheless,
the duration of its behavioral effectiveness is approximately
1 h, a finding in parallel with a rapid waning of maternal
behavior in parturient ewes that fail to bond because their
lambs are removed (Poindron and Le Neindre, 1980). In this
context, it is interesting to note that not only parturition but
also suckling increases CSF oxytocin in sheep (Keverne and
Kendrick, 1992), while chemosensory cues from anogenital
licking of pups maintain the efficiency of oxytocin release in
rats in response to suckling. Likewise, a baby’s crying can
induce oxytocin release and milk ‘let-down’ in lactating
women. Hence the initiation and maintenance of maternal
care appear to differ in important ways. For initiation, oxytocin
and its receptors depend largely on steroid hormone priming,
while sustained release of oxytocin is evoked by somatosensory
stimulation at parturition. During the maintenance of maternal
behavior, steroid levels are low and oxytocin sustains its own
receptors, while its release can be evoked by suckling and other
sensory stimuli from the offspring. The duration of the mainte-
nance phase is therefore dependent on the frequency of sensory
stimulation from the offspring, which diminishes in both type
and frequency as the infant becomes self-sufficient.
1.04.4.3 Frontal Cortex: Role in Social and Parental Reward
The prefrontal cortex has shown progressive enlargement
throughout mammalian evolution (Carter, 2014). In small-
brained rodents, the medial frontal cortex (mPFC) matures
 The Neurobiology of Social Affiliation and Pair Bonding
around day 13–15 postpartum (Diergaade et al., 2005), while
in the human, this development continues late into adoles-
cence or later (Sowell et al., 2001). In rats, this area of the cortex
mainly receives a strong olfactory input via the mediodorsal
thalamic nucleus, and lesions to this system decrease the
frequency with which the mother protects her pups by attack-
ing intruders who threaten to cannibalize her infants (Ferreira
et al., 1987). Unlike males, females rarely initiate aggression
to others unless they are nursing offspring (Bosch and
Neumann, 2012). The mPFC synchronizes this response to
odor cues via the release of CRF from the bNST and PVN where
the majority of CRF neurons are found (Spencer et al., 2005). A
second major output from mPFC is to the ventral striatum
(NAcc), an area of the brain concerned with reward. Hence,
in the context of emotionally rewarding behaviors associated
with maternal care, olfactory signals relayed in the mPFC are
endowed with incentive salience. The reward value provided
by the NAcc requires a biological context signaled by the
hormonal state of the female and activated by release of
opioids and dopamine in the NAcc. It is well established that
estrogens increase synthesis of preproenkephalin in the NAcc
(LeSaux and DiPaolo, 2005) and that the dopaminergic projec-
tions from the mesolimbic neurons of the midbrain are also
immunoreactive for estrogen receptors (Creutz and Kritzer,
2004).
In rats, low levels of maternal care are associated with
reduced dopamine release in the NAcc in response to pup
cues (Champagne et al., 2004). In contrast, drugs which act
in the frontal cortex and NAcc that are associated with reduced
dopamine show reduced PFC response to pup suckling as
revealed by fMRI (Ferris et al., 2005). Oxytocin has also been
shown to be active in brain regions associated with mother–
pup bonding during suckling (Febo et al., 2005; Ferris et al.,
2015).
In sheep that have already given birth, exposure to lamb
odors provokes intense c-fos activity in the mPFC as well as
in the pyriform and cortical amygdala, central relays for main
olfactory inputs known to be important in lamb recognition
and selective bonding. However, in sheep, reversible inactiva-
tion of the mPFC (medial limbic area) using tetracaine infu-
sions demonstrates this region of the brain is not required for
the performance of proactive maternal behavior or for own
lamb recognition. It is, however, required to enable the ewe
to execute an aggressive motor action in response to the odors
of strange lambs. This reversible inactivation does not interfere
with other forms of motor activity related to maternal care
including emotionally generated rejection vocalizations,
walking, approaching, licking and suckling lambs, and/or
feeding (Broad et al., 2002). Rejection behavior directed at
strange lambs is associated with increases in c-fos mRNA
expression in the large output cells of layer V of the mPFC
(which project to the ventral striatum). This suggests that in
order to execute the aggressive motor response underlying
rejection, novel, anxiety provoking olfactory information needs
to be routed by the projections from the frontal cortex to the
ventral striatum (Lidow et al., 1998).
Aggressive responses to the odors of strange lambs are also
associated with increased levels of dopamine, serotonin, gluta-
mate, and gamma-aminobutyric acid (GABA) in this cortical
region, increases that do not occur in the absence of rejection
123
behavior (Broad et al., 2002). It seems likely that the primary
action of the local anesthetic under these circumstances is to
cause a shutdown of neuronal activity in the mPFC, which
promotes local large increases in inhibitory transmitter concen-
trations (notably GABA and serotonin). Tetracaine has similar
effects to localized cooling on inhibiting membrane conduc-
tance (Luzzati et al., 1999) and so it is likely that behavioral
and neurochemical actions observed with tetracaine infusions
result from inhibition of these mPFC neurones, disabling their
communication with the ventral striatum. Infusions of tetra-
caine in the mPFC, which prevent overt motor rejection of
strange lambs, do not interfere with recognition memory nor
prevent memory retrieval for generating emotional vocal
responses, but this treatment does prevent downloading the
information necessary for initiating an aggressive motor
response via the striatum. Hence the process of maternal
bonding in the sheep brain is strongly driven by olfactory
cues processed in the mPFC. Ungulates are intrinsically averse
to strangers, and mothers are especially aggressive to the
approach of strange lambs. Therefore proactive maternal care
and subsequent bonding require an active suppression of the
aggressive rejection driven by unfamiliar olfactory signals.
Interestingly, the frontal cortex projection to the ventral stria-
tum is known not only to activate reward, but also to be acti-
vated in the context of punishment which also involves
dopamine release in the ventral stratum (Schultz, 2007). Hence
an integral component of reward-based bonding, initially
dependent upon olfaction and subsequently expanded to other
social cues (visual and auditory), employs the frontal–striatal
reward pathways, but first requires the aversive intrinsic
circuitry to be gated and silenced.
The evolutionary enlargement of the frontal cortex in
primates plays a notable role in social interactions and activa-
tion of social reward associated with bonding. Nursery-reared
rhesus monkeys deprived of a maternal upbringing demon-
strate reduced oxytocin secretion and reduced social interac-
tions, but show increased aggressive and stereotypical
behaviors. Increased stereotypical behaviors are often associ-
ated with disruption of frontal cortical function and often
result from an inability to suppress inappropriate behavioral
responses (Winslow et al., 2003). Squirrel monkeys, 4 years
after experiencing separations from their mothers early in
development, exhibit differences in emotional behavior, stress
physiology, and development of the brain, notably in the
mPFC. Behavior tests have subsequently shown these monkeys
to be impaired in reward-related memory tasks (Lyons and
Schatzberg, 2003). Electrophysiological recordings from
normal adult monkeys have shown the mPFC mediates the
achievement of goals (Matsumoto and Tanaka, 2004). The
precise involvement of the PFC in reward-related behavior
has also been examined in humans using imaging techniques.
Interestingly, the detection of unfavorable outcomes, response
conflict, and decision uncertainty elicit overlapping clusters of
activation foci in the mPFC (Ridderinkhof et al., 2004).
Choosing between actions associated with uncertain rewards
and punishments in humans is mediated by neural circuitry
involving frontal cortex, anterior cingulate, and striatum
(Rogers et al., 2004). Showing mothers alternating videos of
their own child, versus that of a stranger, provokes the greatest
signal contrasts in the mPFC and orbitofrontal cortex. These
124 The Neurobiology of Social Affiliation and Pair Bonding
distinctions require face recognition and emotional processing,
which correlate with activation in the visual cortex, temporal
pole, and amygdala (Ranote et al., 2004).
Recent human neuroimaging research documents the extent
to which social cognition, the ability to make inferences about
psychological aspects of other people, relies on distinct frontal
cortical areas. For example, regions of the mPFC appear to
contribute to the recognition of different emotions (Hornak
et al., 1996; Shamay-Tsoory et al., 2005) and to the task of
forming a ‘person impression.’ This area is activated specifically
when a subject is asked to form an impression of a person, but
not when they are asked to form an impression of an inanimate
object, suggesting that this region specifically indexes social
cognition (Mitchell et al., 2005). Studies of the perception of
animacy also implicate activation in the medial mPFC and
related brain regions. In one recent study, individual differ-
ences in plasma levels of oxytocin were strongly related to acti-
vation in these areas critical for social cognitive processes
including the mPFC, suggesting that oxytocin may help to
explain individual differences in social perception (Lancaster
et al., 2015).
1.04.4.4 Social and Maternal Bonding in Primates
The evolutionarily conserved biology that underpins mother–
infant bonding in mammals raises the question as to what
neural changes have occurred in the maternal behavior of
humans and certain nonhuman primates that enables
female–infant bonding to occur outside the context of preg-
nancy and parturition and in the absence of lactation. In Old
World monkeys, apes and humans, the hormones of preg-
nancy, parturition, and lactation are not necessary for maternal
or alloparental care, as females of these species can be motherly
toward infants without ever being pregnant. Nevertheless, of
major significance in primate and human maternal care is the
endogenous opioid system. Indeed, it has been suggested that
the activation of this system at parturition and during suckling
promotes the positive affect arising from maternal behavior
(Broad et al., 1993; Martel et al., 1993, 1995; Franceschini
et al., 1989). Studies on naloxone (an opioid receptor antago-
nist) treatment of postpartum rhesus monkey mothers living in
social groups have addressed the importance of opioids in
maternal bonding.
In the early postpartum period, a mother’s social interac-
tions are almost exclusively with her new infant, and opioid
receptor blockade in the mother has marked effects on this
mother–infant relationship. This treatment reduces the
mother’s caregiving and protective behavior shown toward
their infants. In the early weeks following birth when infant
retrieval is normally very high, mothers treated with an opioid
receptor blocker neglect their infants and show little retrieval
even when the infant moves a distance away. As the infants
approach 8 weeks of age, when a bonded grooming relation-
ship normally develops between mother and infant, mothers
again treated with an opioid receptor blocker fail to groom
their infant. Moreover, they permit other females to groom
their infants, while saline-treated control females are very
possessive and protect their infants from contact with others
at this stage. Mothers do not reject from suckling, but the
mother’s usual possessive preoccupation with the infant
declines with opioid receptor blockade. Mother is not the
normal attentive caregiver, and mother–infant interactions
are invariably infant-initiated (Keverne et al., 1997; Martel
et al., 1993, 1995).
Primates and other mammals have in common opioid
involvement in maternal care, but the consequences of opioid
blockade in small-brained mammals are much greater for the
biological aspects of maternal behavior. In sheep, interference
with the endogenous opioid system severely impairs maternal
behavior, including suckling, whereas monkeys neglect to show
a focused preoccupation with infant care but still permit suck-
ling (Kendrick and Keverne, 1989; Martel et al., 1993). These
differences may reflect the degree of emancipation from endo-
crine determinants that maternal behavior has undergone in
primates, and the increased importance of ‘emotional reward’
for the bonding process.
If the endogenous opioid system in monkeys is positively
linked to mother–infant bonding, then heroin addiction,
which acts at the same opioid receptor, would be predicted
to have severe consequences for human maternal bonding.
Women who are heroin addicts have many aspects of their
social and economic life disrupted, making the data difficult
to disentangle. Nevertheless, by 1 year of age, nearly 50% of
children are living away from their biological mothers, and
by school age, only 12% remain with their biological mothers
(Mayes, 1995). These infants have been abandoned for adop-
tion or are taken into the care of their grandparents and other
female kin. Moreover, methadone-maintained mothers, when
compared with controls matched for ethnicity, socioeconomic
status, infant birth weight, and gestational age, are less likely to
have remained the child’s primary parent. Their children are
significantly more likely to have been referred to child protec-
tive care or special service agencies for neglect, abandonment,
or abuse.
Integral to the bonding process in large brained primates is
the action of the endogenous opioid system on receptors local-
ized to the ventral striatum (Koob and Le Moal, 1997). This
area of the brain is involved in ‘reward’ and also requires the
mesolimbic dopamine projection, which detects rewarding
stimuli and also when they occur at unpredicted times to
enable ‘updating’ of the stimulus (Schultz and Dickinson,
2000). In primates, the oxytocin system is also important in
bonding, and in humans, oxytocin release is increased at birth,
following female orgasm and following exposure to neonatal
images or sounds (Carmichael et al., 1987; McNeilly et al.,
1983). Indeed, as reviewed earlier in this chapter, oxytocin,
the endogenous opioids, and their receptors are intimately
coordinated by the hormones of pregnancy and interlinked
in the context of mother–infant bonding. The mother–infant
bonding process in monkeys entails obsessive grooming espe-
cially to hands, face, and genitalia by mothers, and these are
the morphological traits of infant monkeys that show the
greatest changes during development. Because primates
show extended postpartum care, offspring recognition
requires the continual updating of any changes in these
morphological features and in behavioral development. This
updating of infant recognition involves visual cues and
prefrontal–ventral striatal pathways, which are also intimately
linked to the emotional brain via the amygdala. The positive
emotional responses that infants generate in females enable
 The Neurobiology of Social Affiliation and Pair Bonding
parental care to occur without the continual priming by preg-
nancy and parturition.
Human mothers also experience preoccupations and rituals
in the context of maternal care, and even before the birth of
their baby, they are obsessive with cleaning and creating
a safe environment. After birth, safety is the major concern,
and mothers frequently check on their baby even at times
when they know the baby is fine. The evolution of these obses-
sive psychological and behavioral states can be seen as a devel-
opmental extension of the preoccupations all primates show
for their infants. Thus it is notable that areas of the human
brain that MRI findings shown to be responsive to babies’
crying include the brain’s reward structures (mesolimbic dopa-
mine inputs from the ventral tegmental area to the ventral stria-
tum and amygdala) (Lorberbaum et al., 2002). These same
regions of the brain are also active in the context of romantic
love (Fisher et al., 2015).
The evolutionary progression away from hormonal centric
determinants of maternal behavior to emotional, reward-ful-
filling activation, probably involves dopaminergic and opioi-
dergic activity in the ventral striatum. The enhanced role of
this circuitry for regulating behavior in humans may also
provide vulnerability to various forms of psychopathology
such as obsessive compulsive disorder (OCD) and substance
abuse. Mild forms of addictive behavior (gambling, video
games, internet use, and consumption of caffeine and choco-
late) may be such indicators of this neurological predisposition
for obsessive behavior seen in humans (Greenberg et al., 1999).
In vivo neuroimagining studies identify the orbital frontal
cortex, head of the caudate, and closely associated ventral stria-
tum and anterior cingulate as being involved in OCD (Rauch,
2000), while acquired OCD occurs later in life in patients
with striatal lesions (Chacko et al., 2000). There is also
evidence that CSF levels of the neuropeptide, oxytocin, are
elevated in OCD, and this peptide also plays a fundamental
role in many obsessive aspects of maternalism (Leckman
et al., 1994). Not surprisingly, therefore, OCD is more
common in women. The influence of gonadal hormones on
periodicity of OCD (Weiss et al., 1995) and the postpartum
exacerbation of OCD symptoms in women suggest that the
course of this disorder may be influenced by the hormones
of pregnancy (Williams and Koran, 1997; Labad et al., 2005).
Hence some components of human behavior that occur in
the postpartum maternal period are influenced by hormones,
but interestingly they relate to areas of the brain concerned
with reward and not with the direct execution of maternal
behavior per se.
1.04.5 Adult Pair Bonds
1.04.5.1 Mating Systems and Pair Bonds
One common method for categorizing species is based on
mating systems, characterized by the presumed choice or
number of sexual partners, and often focused on male
behavior. The most common mating system in mammals is
called polygamy (many mates) or polygyny (many wives).
The less common alternative, monogamy (one mate) is esti-
mated to occur in about 3% of mammalian species (Kleiman,
1977), while polyandry (many husbands) is even rarer.
125
In mammals, female reproduction is constrained by gesta-
tion, birth, and lactation. In contrast, males have the option
to invest less than females in their offspring, with the minimal
investment being a contribution of sperm and genetic material.
It has been conventional to describe mating systems in terms of
male fitness or reproductive success. However, defining mating
systems in terms of male behavior tends to ignore female
fitness, female–female interactions, and the importance of the
larger family unit. In addition, DNA fingerprinting has revealed
that at least some of the offspring raised by seemingly monog-
amous partners of most species are in fact due to ‘extra-pair
copulations’ or EPCs (Okhovat et al., 2015). The high inci-
dence of EPCs and extra-pair paternity creates a problem for
theories that focus on monogamy purely as a mating system-
and have encouraged scientists to shift their attention to social
systems.
1.04.5.2 Social Systems
A vast array of social systems have been described, many of
which are based directly on mating systems (such as polygamy
or monogamy), while others may be defined by the living
arrangements or genetic relationship among the members of
a group. In comparison to mating systems, definitions of social
systems are more likely to take into account the role of females
and offspring. For example, as described above, many mamma-
lian social systems can be described as matrilinear, and a variety
of mammalian species live in groups in which young animals
may remain in the natal family. The tendency to form extended
families, often, but not always, based around a monogamous
pair, has been termed communal or cooperative breeding
(reviewed Solomon and French, 1997).
1.04.5.3 Sexual versus Social Monogamy
Most of what has been written regarding monogamy focuses on
sexual monogamy. The awareness that – at the species level –
sexual exclusivity is rarely created a challenge for the concept
of sexual monogamy (Carter et al., 1995). Nonetheless, selec-
tive social behaviors and pair bonds between adults do provide
a social matrix for reproductive behaviors, including sexual
behavior and care of offspring, supporting the welfare of indi-
viduals, groups, and species.
The traits of mammalian social monogamy typically occur
together, forming what has been called a ‘syndrome’ (Kleiman,
1977). The cardinal characteristic of social monogamy is
a male–female pair bond, characterized by selective partner
preferences and social interactions. Selective aggression is
another feature of social monogamy. Monogamous mammals
are highly social within their families, although they can show
lethal aggression toward strangers (Carter et al., 1995; Bowler
et al., 2002). Aggression is most common toward members
of the same sex and usually occurs during or after pair bond
formation. The capacity of socially monogamous partners to
defend their mates, family, or territory may influence reproduc-
tive fitness and their own survival. Incest avoidance, a tendency
for reproductive suppression of the young, who remain as allo-
parents in communal families, and a relative absence of phys-
ical sexual dimorphism also characterize monogamous
mammals.
126 The Neurobiology of Social Affiliation and Pair Bonding
1.04.5.4 Social Monogamy in Prairie Voles
Prairie voles (Microtus ochrogaster) exhibit all of the features of
social monogamy including the formation of pair bonds
between adult conspecifics. Prairie voles, which are small
rodents found in grasslands throughout midwestern North
America, have attracted particular interest because they can be
studied under both field and laboratory conditions. Field
studies begun in the 1960s provided the first evidence that
prairie voles form long-lasting pair bonds (Carter et al.,
1995). Male and female pairs of this species maintain
a common nest and territory and tend to enter live traps
together as long as both members of the pair are alive. By
contrast, less social, polygamous meadow voles (Microtus pen-
nysylvanicus) and montane voles (Microtus montanus) show
few indications of pair bonding; males and females of these
species have separate nests and territories and are usually trap-
ped alone. In the laboratory, nonmongamous species are less
likely to engage in social contact and less likely to exhibit selec-
tive partner preferences.
Prairie voles live in nature in communal family groups
comprised primarily of a male and female breeding pair and
their offspring. About 70–75% of young voles do not leave
their natal family (Getz et al., 1993). The original breeding
pair within a family has a reproductive advantage, while
most other members of the communal groups are reproduc-
tively inactive offspring. These offspring serve as helpers at
the nest or alloparents, presumably gaining reproductive
advantages through inclusive fitness. Familiarity inhibits repro-
duction in young prairie voles, and incest is avoided through
several mechanisms, including reluctance to mate with a family
member. Young males remain sexually suppressed within the
family nest and must leave the family group to reproduce
(Carter et al., 1995; Okhovat et al., 2015). New pairs are
most likely to form when previously naïve males and females
leave their group, meet an unfamiliar member of the opposite
sex, develop new pair bonds, and mate and generate their own
families. Pair bond formation in prairie voles commences
within hours, or sooner, after meeting. In addition, when
young males and females meet strangers of the opposite sex,
a cascade of events is initiated which leads to reproductive acti-
vation in both sexes and eventual estrus induction in the
female (Carter et al., 1995). Female prairie voles do not exhibit
ovarian cycles, and spontaneous estrus is rare. Mating usually
follows within a day or two of male–female pairing. In newly
formed pairs, intermittent mating bouts continue for about
24–48 h followed by a 21-day gestation. After an initial mating
and pregnancy, prairie vole females experience a postpartum
estrus, allowing them to mate again and nurse one litter while
they are pregnant with a second litter (Witt et al., 1989).
Attempts to use sexual preferences as an index of pair
bonding have not been successful. However, significant social
preferences for physical contact with a new partner can be
measured within hours following an initial pairing (Carter
et al., 1995). The onset of social preferences is facilitated by
mating, but can occur in the absence of mating and even in
gonadectomized animals, albeit less readily (Williams et al.,
1992).
Females in postpartum estrus that have remained with
a male for one or more litters may exhibit a sexual preference
for the familiar male. However, even in postpartum estrus, if
given an opportunity, a significant proportion of females will
mate with an unfamiliar male. Once females are in estrus,
sexual postures allowing mating are reflexive and may be less
selective than social contact (Carter et al., 1995).
Pair-bonded males become highly aggressive following
mating and probably patrol the runways that lead to their
nest. If successful in defending his partner, then the male
may father the offspring that his partner delivers. In nature,
when one member of the pair dies or abandons the nest, fewer
than 20% of the remaining partners form a new pair bond
(Getz et al., 1993); thus for most prairie voles, pair bonds
last until the pair is separated by death.
1.04.5.5 Experimental Analysis of Partner Preferences
Prairie voles adapt easily to the laboratory, and therefore it has
been possible to investigate various aspects of the behavioral
and neuroendocrine control of pair bonding. Voles may be
placed in a large three-chambered apparatus that allows the
experimental animal to spend time with either a stimulus
animal designated by familiarity as the ‘partner,’ or with
a comparable ‘stranger.’ or elect to be alone in a neutral cage
(Williams et al., 1992). Female stimulus animals are tethered
loosely, allowing them to move only within their own
chamber, whereas the experimental male is free to explore the
entire test apparatus. This testing paradigm yields reliable social
preferences that can be studied as a function of physiological
and experimental manipulations. In female prairie voles, social
preferences for the familiar partner are established more quickly
if mating occurs prior to testing. Nonsexual cohabitation also is
eventually followed by a significant partner preference (DeVries
and Carter, 1999). As mentioned above, however, social prefer-
ence may bias reproduction to favor the familiar or resident
male; laboratory studies do not indicate that newly paired
females form a strong sexual preference for familiar or unfa-
miliar males (Carter et al., 1995). In fact during the early stages
of pair bonding, females are equally likely to mate with familiar
and unfamiliar males. For this reason, social contact is more
selective and probably more useful as an index of pair bonding
than is sexual preference (Carter and Getz, 1993).
1.04.5.6 Selective Aggression as a Component of Pair
Bonding
Sexually naïve prairie voles of either sex are rarely aggressive
toward conspecifics. By contrast, after approximately
12–24 h of sexual experience, male prairie voles become
extremely aggressive toward unfamiliar males (Winslow
et al., 1993). Sexually experienced ‘breeder’ males are
socially dominant and may drive away or even kill male
strangers. However, they are usually not aggressive to
familiar animals, and breeder males will allow their own
offspring to remain in the family, at least as long as these
offspring are sexually naïve. Cohabitation with a nonrepro-
ductive female, even for several days, usually does not
produce a reliable increase in male aggression; however,
nonsexual cohabitation does cause hormonal changes that
are associated with reproductive activation. Females also
 The Neurobiology of Social Affiliation and Pair Bonding
become aggressive after mating, but postcopulatory aggres-
sion develops more slowly in females than in males. In
females the onset and intensity of aggression is variable
and does not require sexual interactions (Bowler et al.,
2002). If two unrelated females are placed with a male,
both may mate with the male (Witt et al., 1989). Under
these conditions, one female becomes socially dominant,
and the nondominant female is blocked from social contact
with the male (Firestone et al., 1991).
In both sexes, postpartum aggression is common and may
be directed toward both unfamiliar females and unfamiliar
males (Getz et al., 1981). However, female prairie voles do
not express aggression toward their own offspring. Female–
female aggression, sometimes leading to death of one female,
is also a feature of the behavior of paired tamarins and other
monogamous mammals (Kleiman, 1977).
Partner preferences and selective aggression characterize
established pair bonding in prairie voles while sexual and
social experiences regulate the onset of the pair bond (Carter
et al., 1995). However, partner preferences can develop even
in the absence of gonadal hormones. The initial development
of a partner preference usually occurs prior to mating during
a time when gonadal hormones are comparatively low. Subse-
quent sexual experience may consolidate the pair bonding
process (Williams et al., 1992). Hormonal events associated
with social interactions therefore are likely contributors to
pair bond formation. Prior to these studies, it was known
that neuropeptides, including oxytocin and vasopressin, are
released by both sexual (reviewed Carter, 1992) and nonsexual
contact (Uvnas-Moberg, 1998). In addition, as described
above, oxytocin is implicated in both nonselective maternal
behavior and maternal bonding.
Research on pair bonding has focused on factors capable of
affecting the formation of a pair bond and the induction of
selective aggression. Relatively, little is known regarding the
mechanisms involved in the maintenance of these behaviors,
although there is reason to believe that the factors responsible
for the induction of pair bonding can differ from those
involved in its maintenance. For example, compounds that
block vasopressin prevent the development, but not the expres-
sion, of selective aggression (Winslow et al., 1993). In addition,
as detailed below, the induction and maintenance of pair
bonds may rely on different dopamine receptors subtypes
(D2 versus D1) (Aragona et al., 2006).
1.04.5.7 Affiliative Behaviors and Positive Sociality
Most socially monogamous mammals show high levels of
social contact within the family. Sociality can be either selec-
tive, and thus in some cases indicative of social bonds, or
nonselective. Oxytocin and vasopressin can increase the
tendency to show both selective and nonselective social
behaviors. For example, treatment (ICV) with either oxytocin
or vasopressin approximately doubles nonselective social
contact in male and female prairie voles (Cho et al., 1999).
Pretreatment with a selective antagonist for either peptide
(oxytocin plus an OT antagonist (OTA), or vasopressin
plus an AVP V1a antagonist (AVPV1aA)) reduces social
contact. High levels of social behavior are still seen if either
of these peptides is combined with an antagonist for the
127
heterologous receptor (oxytocin plus AVPV1aA or vaso-
pressin plus OTA). However, when animals are treated with
either an OTA or an AVPV1aA, they no longer show a selec-
tive partner preference. These results indicate that either
oxytocin or vasopressin, at least when applied at high doses,
can stimulate nonselective social contact in prairie voles of
either sex; however, it is possible that both types of receptors
must be accessible and stimulated to allow these peptides to
facilitate the formation of a selective social preference. Cross-
reactivity between oxytocin and vasopressin and their recep-
tors probably occurs in most mammals and under many
conditions that regulate dynamic social interactions (Albers,
2015).
Vasopressin facilitates partner preference formation, espe-
cially in male prairie voles. ICV infusion of an AVPV1aA
prevents mating-induced partner preference formation as well
as the selective aggression associated with mating in male
prairie voles (Winslow et al., 1993). Prairie voles have higher
levels of V1a receptors in several brain regions including the
ventral pallidum, medial dorsal thalamus, and mAMY, espe-
cially in comparison to nonmonogamous montane or meadow
voles.
In male prairie voles, infusing the AVPV1aA into the ventral
pallidum, but not other regions, prevents partner preference
formation (Lim and Young, 2004). In addition, upregulating
the V1a receptor in this region facilitates the formation of
partner preferences. Species differences in pair bonding may
arise from species differences in the expression pattern of the
V1a receptor in males. In male meadow voles, which do not
normally form partner preferences, overexpression by viral
vector of the prairie vole V1a receptor in the ventral pallidum
resulted in the development of partner preferences (Lim
et al., 2004). A separate study found that infusing AVPV1aA
into the LS inhibits partner preference (Liu et al., 2001). Septal
V1a receptors also regulate selective social recognition in male
mice (Bielsky et al., 2005). The LS projects heavily to the NAcc
and may allow social signals to activate reward pathways and
facilitate pair bonding.
Based on the behavioral effects of vasopressin and species
differences in the vasopressin receptor, Young and his associ-
ates (1999) have created transgenic mice overexpressing the
gene for the vasopressin receptor. These transgenic animals
express the vasopressin receptor (V1a) in a pattern that was
different from wild-type mice and similar in some brain
regions to prairie voles. For example, V1a receptor binding
is elevated in transgenic mice in the CC, claustrum, laterodor-
sal and ventroposterior thalamic nuclei, and especially the
olfactory bulbs. Oxytocin receptor distribution is not altered
in the V1a transgenic mice. When the V1a transgenic male
animals are treated with exogenous vasopressin, they also
show high levels of social contact with female stimulus
animals.
A highly repetitive microsatellite DNA element is found in
the promoter of the prairie vole; this gene promoter is virtually
absent in the montane and meadow voles. Variations in this
microsatellite can alter gene expression in a cell-specific
manner (Hammock and Young, 2004). In prairie voles, V1a
receptor abundance varies among individuals (Okhovat et al.,
2015). The microsatellite in the gene promoter varies signifi-
cantly among individuals (Hammock and Young, 2002,
128 The Neurobiology of Social Affiliation and Pair Bonding
2005). Prairie voles have been bred based on the length of their
microsatellites. Those with a long microsatellite have higher
levels of V1a receptor expression in several brain regions,
including the olfactory bulb and LS, and are more likely to
display partner preferences than males with short microsatel-
lites (Hammock and Young, 2005). The repeat sequences
found in microsatellites often recruit methylation and gene
silencing, which is opposite to the findings demonstrated in
these studies. Moreover, the generality of this finding remains
uncertain. A survey of other nonmonogamous vole species sug-
gested that the microsatellite is also present in several other
nonmonogamous rodent species, and therefore is not solely
responsible for differences in social organization across other
vole species (Fink et al., 2006).
One study reports that an OTA, but not an AVPV1aA,
prevents mating-induced pair bonding in females (Insel and
Hulihan, 1995). A separate study found that an AVPV1aA,
but not an OTA, significantly inhibits partner preference in
males (Winslow et al., 1993). These data suggest a sex differ-
ence in the relative role of endogenous oxytocin and vaso-
pressin in pair bond formation. However, in male prairie
voles, infusions of either oxytocin or vasopressin can facilitate
partner preferences after a brief cohabitation (Cho et al.,
1999). In addition, infusion of an OTA into the LS blocks
partner preference formation in male prairie voles (Liu
et al., 2001). Taken together, these studies suggest that activa-
tion of both the oxytocin receptor and V1a receptor may be
necessary in selective partner preference formation in prairie
voles.
In general, sex differences have not been reported in
oxytocin or vasopressin V1a receptor binding in the prairie
vole brain (Insel and Shapiro, 1992; Wang et al., 1996). Quan-
tification of oxytocin synthesis in the PVN and SON does not
reveal sex differences (Yamamoto et al., 2004), although the
release of oxytocin may, under some conditions, be sexually
dimorphic (Kramer et al., 2004). However, in various species
including voles, vasopressin synthesis and release is higher in
males in the mAMY, BNST, and LS, brain regions that may be
important in a variety of social and defensive behaviors (De
Vries and Panzica, 2006). It is also possible that, under some
conditions, oxytocin is preferentially released in the female
(Kramer et al., 2004), which might be especially relevant
during the vaginocervical stimulation associated with copula-
tion. In addition, oxytocin is essential for social recognition,
which is implicit in pair bonding in both sexes.
Additional information regarding the neural basis of social
behavior comes from studies of ‘knockout’ mice, created with
selective genetic lesions. Deletions of the exon that encodes
the oxytocin peptide in mice produce an animal (OTKO)
capable of all aspects of reproduction except milk ejection
(Nishimori et al., 1996). However, the social behavior of
OTKO mice also is not normal. OTKO mice tend to be more
defensively aggressive and reactive to stressors than normal
mice (Winslow et al., 2000; Amico et al., 2004). OTKO mice
also have deficiencies in ‘social memory,’ which are restored
by injections of oxytocin (Ferguson et al., 2000). Social recog-
nition is necessary for pair bond formation, and other studies
suggest that both oxytocin and vasopressin play a role in social
recognition in male rodents (Bielsky and Young, 2004; Bielsky
et al., 2005).
1.04.5.8 Selective Social Behavior, a Role for Oxytocin
and Vasopressin
Taken together, these data suggest that neural systems respon-
sible for the expression of sociality can be stimulated by either
vasopressin or oxytocin. However, the formation of social
bonds may require both peptides and/or the activation of addi-
tional neural systems, beyond those required to facilitate
nonselective social behaviors (Cho et al., 1999).
In prairie voles, sexual experience can hasten the onset of
pair bonding (Williams et al., 1992) and, in males, induces
aggression toward strangers (Winslow et al., 1993). It is also
known that oxytocin and vasopressin can be released during
mating (reviewed in Carter, 1992). These observations sug-
gested the hypothesis that oxytocin and/or vasopressin could
promote social bonding. Oxytocin infusions, when centrally
administered, do indeed facilitate the onset of partner prefer-
ences in sexually naïve female and male prairie voles (Williams
et al., 1994; Cho et al., 1999). Moreover, oxytocin antagonists
reduce the behavioral effects of exogenous oxytocin and also
block partner preference formation during prolonged cohabita-
tion. In addition, in male prairie voles, vasopressin is necessary
to the onset of stranger-directed aggression, which in nature
would promote mate guarding, another essential aspect of
pair bonding.
Experimental evidence for a role of vasopressin in males
comes from the fact that pretreatment with a V1a vaso-
pressin antagonist inhibits pair bonding that normally
results from sexual experience (Winslow et al., 1993). In
addition, exogenous vasopressin administration facilitated
aggression induction in this species. When tested under
comparable conditions, infusions of vasopressin were not
effective in facilitating female aggression. Thus, the physi-
ology of female aggression in pair bonding species remains
to be understood.
1.04.6 Stress and Pair Bond Formation
Oxytocin and social behaviors are critical components of the
capacity of the mammalian body to manage and downregulate
reactivity to stressful experiences. Oxytocin has effects on the
regulation of emotion, the mammalian autonomic nervous
system, homeostasis, coping, and healing, helping to explain
the consequences of the presence or absence of social engage-
ment and attachment. For example, oxytocin has been impli-
cated in human wound healing (Gouin et al., 2010) and is
protective against cardiovascular dysfunction, acting to repair
tissue (Karelina and DeVries, 2011). Oxytocin also has antiox-
idant and anti-inflammatory properties across the life span
(Jankowski et al., 2016). Acute stressors, especially of high
intensity, can release oxytocin in both sexes (Neumann and
Landgraf, 2012), possibly helping the organism to adapt in
the face of a challenge. These adaptive properties of oxytocin
help to explain the capacity of social bonds to protect and
heal in the face of stress and adversity. Concurrently, stressors
seem to play a major role in the formation of social bonds.
Evidence for a role for hormones of the HPA axis in pair
bond formation comes from studies which reveal the repeated
association between stressful experiences and social bonding
 The Neurobiology of Social Affiliation and Pair Bonding
(Carter, 1998; Sachser et al., 1998). More direct evidence can be
found in laboratory studies of the effects of stressful stimuli or
administration of hormones of the HPA axis on the formation
of partner preferences.
Evidence for the role of biochemistry in the formation of an
attachment comes from behavioral changes associated with
mammalian birth, lactation, and sexual interactions. In addi-
tion, novel or stressful experiences may encourage increased
social behaviors and attachment. Comparatively high levels
of HPA axis activity or indications of sympathetic arousal,
and the subsequent release of oxytocin, have been measured
under conditions that commonly precede or are associated
with the formation of social bonds.
Mammalian birth is a uniquely stressful experience. In the
mother, the physiological events preceding and during parturi-
tion involve exceptionally high levels of adrenal activity (both
glucocorticoids and catecholamines) and the subsequent
release of peptides, including endogenous opioids, oxytocin,
and vasopressin (Keverne and Kendrick, 1992; Kendrick,
2000; Landgraf et al., 1991). Infants also experience parturi-
tional stress or ‘birth trauma’ and probably experience
increased exposure to maternal oxytocin during labor.
Oxytocin has a large profile of neural consequences which
may help to reduce the vulnerability of infants during the birth
process, although whether this applies to exogenous oxytocin,
administered, for example, to induce birth, requires further
study. Hormonal experiences associated with birth may affect
the tendency of young animals to form social bonds, although
such effects remain to be demonstrated.
In species that form heterosexual pair bonds, including
prairie voles, sexual interactions are associated with the forma-
tion of social attachments (Carter et al., 1995). Sexual behavior
also can be physiologically stressful for both sexes. Adrenal
steroids, vasopressin, oxytocin, and endogenous opioids are
released during sexual behavior (reviewed in Carter, 1992).
Stress or corticosterone facilitates pair bond formation in
male prairie voles (DeVries et al., 1996). Although female
prairie voles do not form pair bonds with familiar males
following stressful treatments, stress does encourage the devel-
opment of preferences for other females, consistent with the
communal breeding pattern of this species (DeVries, George
and Carter, unpublished data). Steroid hormones, including
glucocorticoids, can influence the synthesis of, release of,
and/or receptors for neuropeptides, including oxytocin. In
addition, steroid and peptide hormones may regulate or
interact with each other to influence behavior.
Hormones can reduce fear or behavioral inhibition and
permit the expression of social behaviors, such as those
necessary for pair bonding, maternal behavior (Chirino and
González-Mariscal, 2015; Fleming et al., 1989; McCarthy
et al., 1992), or sexual behavior (Carter, 1992). The neuro-
chemical processes that overcome neophobia also may be
needed to permit the formation of new social attachments. Pro-
social behavior or social contact is facilitated, and aggression is
diminished following central oxytocin treatments in estrogen-
treated female prairie voles (Witt et al., 1990). Increases in
social contact also follow oxytocin treatment in both male
and female rats (Witt et al., 1992). In addition, in sexually naïve
male rats, a brief (15 min) heterosexual interaction is followed
by an approximate doubling of serum oxytocin levels; this
129
change was not detected in sexually experienced males for
which this situation may have been less novel (Hillegaart
et al., 1998). Oxytocin treatments may reduce anxiety as
measured by exploration of a novel environment in rats
(McCarthy et al., 1992; Uvnas-Moberg, 1997). In humans
(Chiodera et al., 1991) and prairie voles (DeVries et al.,
1997), oxytocin inhibits the secretion of glucocorticoids. Social
contact also can inhibit HPA axis activity in prairie voles
(DeVries et al., 1995, 1996). In reproductively naïve prairie
voles, either oxytocin (ICV) or social contact produce a 50%
decline in corticosterone, which occurs within 30–60 min. In
male prairie voles, either mating (Insel et al., 1995) or vaso-
pressin treatment (Dharmadhikari et al., 1997) is associated
with increased exploration in the open arm of a plus maze,
a measure often considered indicative of reduced anxiety.
The unanticipated finding that socially naïve prairie voles
respond to exposure to a novel stranger of the opposite sex
with a decline in corticosterone suggests that pair bonding in
prairie voles might be inhibited by hormones of the HPA axis
(DeVries et al., 1995). This has been shown to be the case
when, in naïve female prairie voles, removal of the adrenal
glands facilitates the development of a partner preference.
Adrenalectomized females form a significant preference for
the familiar male within 1 h or less of cohabitation compared
with the usual requirement of in excess of 3 h. The facilitatory
effect of adrenalectomy is reversed by corticosterone replace-
ment prior to female exposure. In addition, increasing cortico-
sterone levels in females, by exogenous injection or pellets of
corticosterone, produce a dose-dependent inhibition of the
preference formation.
In contrast, in socially naïve male prairie voles, the behav-
ioral effects of corticosterone, when examined in experiments
that paralleled those in females, produce strikingly different
results (DeVries et al., 1996). Injections of corticosterone, or
the stress of swimming, facilitated the development of partner
preferences in males. By contrast, removal of the adrenal
gland was followed by failure to form a partner preference,
which was subsequently restored by corticosterone
replacement.
Sex differences in the behavioral effects of oxytocin and
vasopressin are consistent with a larger literature implicating
oxytocin in various aspects of positive sociality and female
reproduction and vasopressin in territoriality and defensive
behaviors. As described above, vasopressin is more abundant
in males, and vasopressin production, especially within the
mAMY and bNST, is increased by androgens. Sex differences
also exist in pair bonding (DeVries et al., 1996), and it has
been suggested that pair bonding in female prairie voles
depends primarily on oxytocin, while males rely on vaso-
pressin (Winslow et al., 1993; Insel and Hulihan, 1995).
However, there are no dramatic sex differences in oxytocin
or vasopressin receptors in prairie voles (Insel, 1997). As
mentioned above, steroid-dependent increases in hypotha-
lamic oxytocin receptors have been reported in rats
(Schumacher et al., 1990). In female prairie voles, estrogen
treatments do not change hypothalamic oxytocin receptors;
only the AON shows an obvious estrogen induction of
oxytocin receptor expression (Witt et al., 1991). Furthermore,
when given as an exogenous treatment, either oxytocin or vaso-
pressin can facilitate pair bonding in both male and female
130 The Neurobiology of Social Affiliation and Pair Bonding
prairie voles, and receptors for both peptides may need to be
available to allow pair bond formation (Cho et al., 1999).
The finding, described elsewhere, that the effects of stressful
experiences on prairie vole pair bonding are sexually dimorphic
(DeVries et al., 1996) is most easily interpreted in the context of
natural history. Male prairie voles must leave the family to
breed and must be capable of forming new pair bonds and
mating under stressful conditions. In contrast, females of this
species may respond to stressful conditions by remaining
within the natal family (Getz et al., 1993). Thus, sexually
different behavioral strategies and the hormonal response to
stress from the HPA axis may both reflect and support a sex
difference in reproductive strategies.
CRF regulates the release of ACTH and also is known to
have a number of behavioral effects including increased
arousal. In addition, in rats, glucocorticoids are capable of
enhancing the behavioral effects of CRF (Schulkin, 1999).
CRF, administered ICV and in moderate doses, is capable of
facilitating pair bond formation in male prairie voles (DeVries
et al., 2002). There is recent evidence from male voles that loss
of a partner disrupts the capacity to cope with stress through
pathways that involve the NAcc; this disruption can be restored
by application of oxytocin into the shell of the NAcc in which
oxytocin receptors are colocalized with receptors for CRF
(Bosch et al., 2016).
These findings are consistent with the observation in sheep
that CRF can facilitate the maternal acceptance of a lamb
(Keverne and Kendrick, 1991). In addition, there is evidence
in rats that CRF is colocalized with oxytocin (Levin and Saw-
chenko, 1993), suggesting that functional interactions between
these peptides are likely.
1.04.6.1 Dopamine and Pair Bonding
Dopamine modulates pair bonding behavior in prairie voles
(Gingrich et al., 2000; Aragona et al., 2003, 2006; Liu and
Wang, 2003). The effects of dopamine may be due to effects
on a wide variety of events that are important for partner pref-
erences, such as sensory processing, reward, memory forma-
tion/consolidation, and behavioral expression. Additionally,
dopamine is released during mating (Gingrich et al., 2000),
which in turn facilitates pair bonding (Williams et al., 1992).
Reward associated with the formation of social bonds is regu-
lated by the dopamine transmission in the NAcc. Intra-NAcc
administration of a general dopamine receptor antagonist
blocks mating-induced partner preferences, indicating that
dopamine transmission within the NAcc is necessary for pair
bonding. In addition, intra-NAcc administration of a general
dopamine receptor agonist dose-dependently induces this
behavior without mating, showing that dopamine receptor
activation is sufficient to produce pair bond formation
(Aragona et al., 2003, 2006).
Dopamine acts on two distinct groups of receptors, D1 and
D2. Dopamine D1-type and D2-type receptors have different
effects on pair bonding in both male and female prairie voles.
Blockade of D2-type receptors in NAcc blocks mating-induced
partner preferences. Conversely, activation of D2-type receptors
in NAcc induces partner preferences without mating (Gingrich
et al., 2000). Such effects are not found following a similar
manipulation of D1-type receptors in NAcc. In addition,
activation of D1-type receptors in NAcc block partner prefer-
ence formation induced either by mating or by D2-type
receptor activation (Aragona et al., 2003, 2006), whereas
blockade of D1-type receptors in the NAcc of pair-bonded
males (paired with a female for 2 weeks; a manipulation that
upregulates D1-type receptors within the NAcc) diminished
aggressive behavior toward a novel conspecific female
(Aragona et al., 2006). This selective aggression is considered
as an additional behavioral index of monogamy and pair
bonding (Winslow et al., 1993). Together, these data demon-
strate that in NAcc, D2-type activation is important for pair
bond formation. In contrast, D1-type activation prevents
partner preference formation and is related to high levels of
selective aggression toward a conspecific stranger and thus
may play a role in the maintenance of an established pair
bond (Aragona et al., 2006).
It is likely that dopamine also plays an important role in
human social bonding. For example, dopamine receptor-rich
areas of brain have been implicated in romantic love in
humans (Fisher et al., 2015).
1.04.7 Responses to Separation
One indication of pair bond formation is the degree to which
animals show behavioral or autonomic responses to the
absence of a familiar or preferred partner. Because responses
to separation are species-typical, they are best understood in
the context of social organization and ecology.
Separation from an attachment figure may be associated
with various behavioral and physiological changes (Reite and
Bocca, 1994; Hennessy, 1997; Sachser et al., 1998; Mendoza
and Mason, 1997; Mason and Mendoza, 1998). In young
animals, vocalizations, in either the audible or ultrasonic range,
often increase following separation. Measurements of these
vocalizations have been used as indices of ‘distress’ and may
be indicative of attachment (Nelson and Panksepp, 1998).
Physiological changes, including increased secretion of gluco-
corticoids and/or ACTH, cardiovascular measures, and
immune system parameters, also have been described
following social separation in primates (Reite and Boccia,
1994). Cortisol responses are sometimes used to assess the
intensity of separation ‘distress’ and/or to examine the hypoth-
esis that the presence of a partner may provide a form of social
buffering (Hennessy, 1997; Mendoza and Mason, 1997; Mason
and Mendoza, 1998).
Social separation in both male and female prairie voles is
followed by an increase in glucocorticoid (corticosterone)
levels (DeVries et al., 1996). When reunited with a partner,
corticosterone levels drop to below baseline in previously
paired males and females. However, if previously paired
animals and separated animals are placed with an unfamiliar
partner of the opposite sex, corticosterone levels remain
elevated.
Social separation for a period of a few days in both male
and female prairie voles is followed by an increase in corticoste-
rone levels, as well as CRF (Ruscio et al., 2007). When reunited
with a partner, corticosterone levels drop to below baseline in
previously paired males and females. However, if previously
paired and then separated animals are placed with an
 The Neurobiology of Social Affiliation and Pair Bonding
unfamiliar partner of the opposite sex, corticosterone levels
remain elevated (DeVries et al., 1996).
Prolonged separation or chronic isolation over a period of
weeks has many consequences including increases in heart
rate, reductions in parasympathetic activity, and increases in
behavioral responses, such as immobility in a forced swim
test, anhedonia, and reduced exploration in an elevated plus
maze. These behavior measures are considered to index depres-
sion and anxiety (Grippo et al., 2007a, 2008, 2009; Bosch et al.,
2016). In experiments subjecting voles to chronic isolation,
corticosterone is not elevated. However, chronic isolation,
especially in female prairie voles, is associated with elevations
in endogenous oxytocin and oxytocin synthesis, which may
help to downregulate the HPA axis (Grippo et al., 2007b).
Support for the latter hypothesis comes from experiments in
voles showing that chronic treatment with exogenous oxytocin
(administered peripherally) prevents or reverses several of the
detrimental cardiac and behavioral effects of isolation (Grippo
et al., 2009; Bosch et al., 2016).
Social experiences also may release oxytocin. In adult,
nonreproductive prairie voles, pup exposure can facilitate pair
bond formation, possibly through the release of oxytocin
(Kenkel et al., 2012; 2013). Social stimuli from a pup exposure
also can increase cell proliferation including neurogenesis as
measured by BrdU; these effects are most pronounced in the
dentate gyrus of the hippocampus (Ruscio et al., 2008). Inter-
estingly, in male voles, those that attack pups have the highest
level of neurogenesis and also have more OT-ir neurons and
fibers in and around the PVN.
Experiments on separation ‘distress’ have tested the capacity
of peptides to prevent behavioral changes during separation.
The intense calling behavior of isolated domestic chicks
declines following various treatments including, injections of
oxytocin/vasotocin, opioids that stimulate m receptors, and
prolactin (Panksepp et al., 1997). Opiate injections also
diminish distress vocalizations in guinea pigs (Herman and
Panksepp, 1978), but separation cries of infant rats do not
show the predicted decline following opiate treatments
(Winslow and Insel, 1991). However, in rat pups, separation
cries are inhibited by central treatments with oxytocin or vaso-
pressin (Insel and Winslow, 1991; Winslow and Insel, 1993).
In squirrel monkeys, there is evidence that both vasopressin
and oxytocin can reduce isolation calling, although the effects
are dependent on social status and high doses of the peptides
were necessary to obtain behavioral effects (Winslow and Insel,
1991).
1.04.7.1 Consequences of Isolation May Be Mediated
through Oxytocin
In the context of the shared physiology among social and
emotional behaviors, it is not surprising that social interactions
and isolation have powerful physiological consequences. Indi-
viduals with a perceived sense of social support are more likely
to avoid or survive illness and have longer lives than otherwise
similar people who live alone, especially those who experience
a sense of loneliness (Cacioppo et al., 2006).
Experiments in animals provide an opportunity to examine
in more depth the physiological consequences of the absence
of a social partner. Highly social mammals, including prairie
131
voles, offer useful models for examining the biology of isola-
tion because they share with humans the capacity to form
long-lasting social relationships. For example, prairie voles
exhibit social monogamy (Carter et al., 1995). They also have
a humanlike autonomic nervous system, with high levels of
parasympathetic–vagal activity and a dependence on social
behavior for emotion regulation (Kenkel et al., 2013). Because
the parasympathetic nervous system mediates many of the
consequences of social interactions, this aspect of prairie vole
biology helps us understand the high levels of sensitivity to
the presence or absence of social experiences in this species.
In prairie voles, isolation from a partner for a few weeks
produced significant increases in several behavioral measures
of depression and anxiety (Sun et al., 2014). Isolated animals
were less exploratory, showed increases in anhedonia (indexed
by a loss of preference for sweet liquids), and were more likely
to show immobility in response to a stressor. In prairie voles,
separation from a partner, followed by prolonged isolation, is
associated with increases in heart rate, decreases in parasympa-
thetic function, and increased behavioral reactivity to stressors,
such as the presence of a social intruder. As one example,
following a 5-min social stressor (intruder), isolated prairie
voles required an average of more than 15 h for heart rate to
return to baseline. In contrast, animals living in sibling pairs
required about 2.5 h for their heart rate to recover. In the
absence of a social partner, the neuropeptide hormone oxytocin
increased in blood in female prairie voles (Grippo et al., 2007c)
and centrally in male prairie voles (Sun et al., 2014). Elevated
oxytocin may be protective against the negative consequences
of isolation, which include reductions in the expression of the
oxytocin receptor (Pournajafi-Nazarloo et al., 2013) and
increases in both vasopressin and CRF (Sun et al., 2014). In
the context of personal safety, the release of oxytocin could
encourage social interactions including those associated with
detecting and responding to the emotions or experiences of
others. We also tested this hypothesis by treating isolated or
paired prairie voles with exogenous oxytocin. Oxytocin injec-
tions over a period of weeks were capable of reversing the
cardiac and behavioral effects of isolation, including protecting
against the increases in heart rate and reductions in vagal tone
that typically accompany isolation (Grippo et al., 2009).
In postmenopausal women, increases in oxytocin also have
been associated with ‘gaps in social relationships’ (Taylor et al.,
2006). Releasing oxytocin may be a component of a self-
regulatory process that helps mammals deal with isolation or
other stressful experiences. Such responses also might facilitate
seeking out social engagement or relationships, functions that
could be especially adaptive in females who may be less able
than males to live and maintain a family alone (Taylor et al.,
2000).
Monogamous monkeys, such as titi monkeys (Mendoza
and Mason, 1997; Mason and Mendoza, 1998), form social
bonds that can be observed in the laboratory. In fact, in this
species, pairs typically sit in physical contact with their long
tails braided together. In titi monkeys, increases in cortisol in
response to disturbances are attenuated by the presence of
a familiar companion. Titi monkeys also respond to the
absence of adult partners. In contrast, there is little evidence
that titi monkeys differentiate between familiar and unfamiliar
infants of comparable ages, and it is reported that this species
132 The Neurobiology of Social Affiliation and Pair Bonding
typically will select their adult mate over their infant. The pres-
ence of an adult partner could have survival consequences in
this monogamous species, since maintenance of adult social
bonds permits subsequent reproduction.
Squirrel monkeys are small New World primates that
usually live in unisex groups. Behavioral changes in squirrel
monkeys do occur following the removal of companions,
although these responses may reflect general arousal or physi-
ological adjustments to being alone, rather than the loss of
a particular companion. Although squirrel monkeys appear
highly social, physiological measures do not suggest selective
pair bonding or stress buffering by companions (reviewed
Mendoza and Mason, 1997; Hennessy, 1997).
1.04.7.2 Social Behavior, the Polyvagal Theory and Oxytocin
The evolutionary origins of human behavior are especially
informed by awareness of the ontogeny and phylogeny of
the physical structures that are necessary for social communi-
cation (Porges, 2011). Ancient gill arches and associated
muscles and nerves gave rise to human face, larynx and
pharynx, and middle ear. The detachment from the skull of
the middle-ear bones occurred in the transition from reptiles
to mammals, possibly serving to permit a higher level of
acoustic social communication in mammals. Of particular
importance to social behavior was the evolved expansion of
the human face and skull, formation of the detached
middle-ear bones, larynx, pharynx, and eventually the expan-
sion of the neocortex. Together, these structures and their
neural control systems allowed the emergence of human
cognition, speech, and language.
Components of this complex system are under voluntary,
corticomotor control. However, these same structures may be
influenced by the autonomic nervous system. The social
engagement system, critical to social communication and
cognition, is innervated in part by the parasympathetic compo-
nent of the autonomic nervous system.
The cranial part of the parasympathetic nervous
system consists of the vagus nerve, which exits the skull as
the 10th cranial nerve. The vagus is functionally a coaxial cable
with two source nuclei, containing both motor and sensory
fibers. The myelinated motor component of the vagus arises
in the ventral vagal complex (VVC) and supports the metabolic
demands of the upper part of the body, including the face and
brain. Actions of the VVC allow the reduction in fear and the
concurrent emergence of calm states necessary for social
engagement and bonding. Also originating in the VVC, the
special visceral efferent (SVE) pathways regulate the striated
muscles of the face and head. The myelinated SVE system
allows dynamic and rapid autonomic input in social interac-
tions, including eye movements and auditory communication,
which are necessary for mammalian social engagement.
Primary features of emotion and affect (i.e., neural regulation
of facial muscles and the heart) are regulated by this system.
The more ancient unmyelinated vagus arises from the dorsal
vagal complex (including the DMX). The unmyelinated vagus
primarily serves visceral organs below the diaphragm.
However, some organs including the heart and immune system
have dual or ‘polyvagal’ – myelinated and unmyelinated –
innervation.
In addition, the afferent or sensory vagus provides a major
source of input to the brain from visceral organs, via the NTS.
At least some of this feedback via the autonomic nervous
system, and especially the visceral, unmyelinated vagus, is
experienced by the cortex as vague but powerful emotions.
Although difficult to cognitively control or interpret, visceral
experiences modulate arousal and play a major role in state
regulation and social cognition (Porges, 2011), which in turn
are important to social behaviors such as attachment and pair
bonding.
Oxytocin appears to regulate both sympathetic and vagal
branches of the autonomic nervous system. The central cells
of origin of the autonomic nervous system and autonomic inte-
grative centers, such as the PVN, synthesize and respond to
oxytocin. Exposure to exogenous oxytocin, especially in a social
context, neurally coupled activity in the PVN to brain stem
nuclei implicated in the regulation of the autonomic nervous
system (Yee et al., 2016). In addition most, if not all, of the
peripheral target organs of the autonomic nervous system
contain receptors for oxytocin. Behavioral, physiological and
anatomic data from rodents and humans (Grewen and Light,
2011) suggest that the ‘antistress’ effects of chronic oxytocin
may include downregulating the sympathetic nervous system,
while supporting the protective and behavioral functions of
the vagal systems. As one behavioral example, individuals
with higher levels of parasympathetic activity showed more
rapid increases in self-described positive emotions and a sense
of connectedness (Koh and Fredrickson, 2010).
1.04.8 Phylogenetic and Ontogenetic Factors
Influencing Pair Bonding
The expression of attachment behaviors varies widely among
species, and the mechanisms responsible for these behaviors
also must have species-specific components (Sachser et al.,
1998). Peptide hormones, including oxytocin and vasopressin,
with species-typical patterns of peptide production, receptor
distributions, and functions (Insel and Shapiro, 1992; Insel
et al., 1997; Wang, 1995; Wang et al., 1996, 1997; Witt et al.,
1991; Young et al., 1996, 1999), are particularly well posi-
tioned to influence behaviors, such as pair bonding, that vary
among different species (Carter et al., 1995, 1997). Both
general patterns of oxytocin receptor expression and binding
and receptor responses to steroids differ among species (Insel
and Shapiro, 1992; Insel et al., 1994, 1997; Jiménez et al.,
2015; Tribollet et al., 1992; Witt, 1997; Young et al., 1996).
For example, estrogen increases oxytocin binding within the
VMH in rats, but not in prairie voles (Witt et al., 1991). In
rabbits (polygamous, monoparental), ovariectomy increases
oxytocin binding in the prefrontal cortex but has the opposite
effect in the POA (Jiménez et al., 2015).
Species differences in peptide receptor activity are presum-
ably an important source of interspecific variation in the behav-
ioral effects of oxytocin and vasopressin including the capacity
to form social bonds. Species-typical variations in peptide
receptors are apparent in early development. For example,
vasopressin receptor binding increased rapidly in the second
week of life in the LS of nonmonogamous montane voles,
but not prairie voles (Wang et al., 1997). Insel, Young, and
 The Neurobiology of Social Affiliation and Pair Bonding
their colleagues compared the genes for oxytocin receptors in
prairie voles and montane voles and found that these receptors
were ‘virtually identical’ in genetic structure (Insel, 1997;
Young et al., 1996, 1998). However, promoter elements can
regulate the expression of these receptor genes in particular
tissues; subtle, but potentially important, species differences
in base sequences may be responsible for the interspecific vari-
ations in peptide receptor distributions. Based on rodent work,
especially in voles, Wang et al. (1996) suggest that “neuroendo-
crine systems may evolve by changes in receptor distribution
rather than by restructuring the presynaptic pathway.” Compar-
isons among related vole species with very different patterns of
social behavior offer insights into the role of peptides and their
receptors in species-typical social behaviors (Insel, 1997;
Hammock and Young, 2005).
Among the traits of monogamous mammals is a relative
absence of physical sexual dimorphism in appearance
(Kleiman, 1977). It is well known that steroid hormones,
including testosterone and dihydrotestosterone, play an impor-
tant role in the development of masculine genitalia. Thus, the
absence of physical sexual dimorphism in monogamous
species may reflect a comparative absence of, or insensitivity
to sex steroids, especially in early life (Carter and Roberts,
1997). It is possible that social behaviors, including the
tendency to form pair bonds, which characterize monogamous
mammals, also emerge in the relative absence of develop-
mental exposure to high levels of androgens. For example,
male parental care is a trait of some, but not all, pair bonding
species. There is evidence from a variety of species that exposure
to androgens in early life and in some cases in adulthood can
inhibit the tendency to be parental.
In the relative absence of high levels of androgens, some
species may rely more directly on neuropeptides, including
vasopressin, to determine ‘masculine’ patterns of physiology
and behavior – for example, selective aggression. Neonatal
exposure to vasopressin also can facilitate aggression especially
in male prairie voles (Stribley and Carter, 1999). Peptide treat-
ments either in development (Boer, 1993; Swaab and Boer,
1994) or in adulthood (Poulain and Pittman, 1993) may alter
the sensitivity of the nervous system to subsequent hormonal
experiences (reviewed in Carter, 2003, 2007). For example, in
rats, treatment with vasopressin during the first week of life
reduced gene expression for the oxytocin receptor in the PVN
during adulthood (Ostrowski, 1998). Because vasopressin is
part of the HPA axis and is sensitive to androgens, this finding
suggests that developmental changes associated with perinatal
stress or sex-dependent androgenization could alter the subse-
quent sensitivity of the oxytocinergic system.
It is possible that the presence or absence of androgens at
various times in the life span plays a role in the development
of the capacity to form pair bonds. It is known that exposure
to steroids, including testosterone or corticosterone during
perinatal life, can alter the tendency of young prairie voles to
prefer a sibling versus a novel stranger (Roberts et al., 1997).
In addition, even in prairie voles, there is a sex difference in
the initial preference for male or female stimulus animals
(DeVries and Carter, 1999), which may reflect the develop-
mental effects of steroids in this species. During development,
varying exposure to peptides and steroids may ‘retune’ the
nervous system, altering thresholds for sociality and aggression;
133
these changes in turn may affect the subsequent tendency of
a species or individual to develop social bonds.
Ontogenetic experiences, including levels of perinatal stress
and varying amounts of parent–young interaction, can
contribute to the development of species-typical patterns of
social behavior (Carter, 2003, 2007). As one example, reduc-
tions in early handling during the first week of life, probably
mediated by differential maternal stimulation, were associated
with a reduced capacity later in life to form social bonds in
female prairie voles (Bales et al., 2007b). This form of differen-
tial handling is also associated with lower levels of central
oxytocin (Bales et al., 2007a). Behavioral flexibility, such as
that seen in prairie voles, and possibly mediated by peptide–
steroid interactions during development, allows animals to
individually adapt their social systems to accommodate early
experiences and environmental demands.
In the transition from reptiles to mammals, and especially
to primates, high levels of encephalization and cognition
appeared (Somel et al., 2013). Sophisticated autonomic
systems also emerged in mammals capable of supporting the
physiological demands of the expanding cortex for oxygen
(Porges, 2011). The neuroendocrine systems that regulate
social interactions evolved in conjunction with the autonomic
nervous system include processes necessary for homeostasis.
These in turn facilitate adaptation to ever-changing social and
physical environments (Dunbar, 2009).
In humans the capacity to adapt to environmental demands
often relies on the neocortex and cognition, in ways that exceed
that of other mammals. However, the neocortex rests physically
and functionally upon an ancient brain stem, relying for suste-
nance on autonomic mechanisms (Porges, 2011). The neural
substrates for social behavior and emotion involve not only
the cortex, but also subcortical brain structures, below
conscious awareness. Voluntary and conscious aspects of social
behavior and social engagement behaviors may be directly
influenced by the same neural systems, including the central
and autonomic nervous systems, which regulate survival and
homeostasis. For example, neonatally decorticated rats appear
remarkably normal and continue to engage in social contact
(Carter et al., 1982). In contrast, lower brain stem lesions
generally are fatal since they interrupt the autonomic supply
lines for oxygen and nutrients to the cortex.
1.04.9 Peptides and Human Behavior
Two aspects of mammalian life, birth and lactation, are clearly
associated with the release of oxytocin. Among the neuroendo-
crine adaptations that accompany both birth and subsequent
lactation are hormonal changes that may promote selective
social interactions, including high levels of physical contact
between parent and offspring and maternal–infant bonding.
Although, it is likely that the experiences associated with birth
facilitate maternal bonding in humans as they do in other
animals, studies of the physiology of mother–infant interac-
tions are at present primarily correlational.
Birth is a hormonally complex event that is difficult to
study. In addition, it is well known that strong social attach-
ments between adults and children can occur in the absence
of parturition. Thus, the hormonal events of birth are not
134 The Neurobiology of Social Affiliation and Pair Bonding
essential for social bonding. However, as described above,
research on bonding in sheep provides strong evidence for a
role for vaginocervical stimulation and birth-related hormonal
events (Kendrick, 2000). Recent studies in women reveal
a stability of individual differences in plasma oxytocin; in
this study, oxytocin is positively correlated with several indices
of maternal interactions including gaze, positive affect, affec-
tionate touch, and attachment-related thoughts (Feldman
et al., 2007).
Lactation is a defining property of mammals and, until
modern times, was necessary for mammalian reproduction.
Oxytocin plays an essential role in milk let-down, but not
maternal behavior (Nishimori et al., 1996; Russell and
Leng, 1998). However, lactating women interact more posi-
tively with their babies, directing more touching and smiling
toward their infants than do bottle-feeding mothers. It is also
reported that nursing mothers are more likely to describe
positive mood states and be less reactive to stressful experi-
ences than bottle-feeding mothers (reviewed in Carter and
Altemus, 1997).
Oxytocin injections, given peripherally to the mother, can
facilitate nipple attachment by young rat pups, suggesting
that oxytocin may change the response of a young animal to
its mother (Singh and Hofer, 1978). Rat pups also show pref-
erences for specific odors that are associated with exposure to
their mothers. Preferences for the mother do not develop in
animals that are pretreated with oxytocin antagonists (Nelson
and Panksepp, 1996). Thus, oxytocin may act on both the
mother and infant to influence the response of young animals,
including humans, to their mother.
Interacting with an infant may release oxytocin in adult
humans, including human males (Feldman, 2012), as well as
in male prairie voles (Kenkel et al., 2012). Oxytocin has been
shown to enhance the processing of social information and
in some cases to facilitate a sense of trust (Kosfeld et al.,
2005) or empathy. Oxytocin may mediate the buffering effects
of social support and modulate reactivity to stressful experi-
ences, playing a role in what humans experience as ‘love’
(Carter, 1998; Carter and Porges, 2013). In general, oxytocin
tends to support a sense of safety and social behaviors charac-
terized by immobility without fear (Porges, 2011).
Understanding the actions of peptides in humans will
require greater knowledge of the capacity of the peptide to
affect the peptide receptors. Individuals vary in the sensi-
tivity of their receptors to oxytocin and vasopressin.
Research on pair bonding species, such as prairie voles,
suggest that these differences are likely the result of selec-
tion pressures acting on receptors, including those for
oxytocin and vasopressin. Receptor variation, in turn, may
permit adaptive individual or species differences in the
sensitivity to a given peptide, and hence in the capacity to
exhibit pair bonding or other characteristics of social
monogamy (Okhovat et al., 2015).
The effects of oxytocin on behaviors such as pair bonding
are due to actions on both the oxytocin and the related vaso-
pressin receptor (Cho et al., 1999; Albers, 2015). The relative
importance of these across species, between males and females,
and among individuals is increasing being examined in a search
for the source of differences in the capacity for sociality and pair
bonding. For example, single nucleotide polymorphisms
(SNPs) in the gene for the oxytocin receptor have been linked
to individual differences in human behavior (Jacob et al.,
2007) and in vagal functions relevant to social behavior
(Kanthak et al., 2016). The strength of these relationships varies
among SNPs, among ethnic groups, and among individuals.
However, genetic patterns are emerging suggesting that some
individuals are highly susceptible to both peptides and social
factors, while others are relatively insensitive to the impact of
the social environment and perhaps also relatively insensitive
to the effects of peptides.
1.04.10 Clinical Implications of a Peptidergic
Theory of Social Attachment
The presence or absence of attachments has broad conse-
quences across the life span. Like other mammals, humans
rely on positive social interactions for both safety and reproduc-
tion. It has been argued that the tendency to form pair bonds or
social attachments is a universal human characteristic (Money,
1980; Fisher, 2016; Hazan and Shaver, 1987). Social support
has documented health benefits, and the absence of positive
social interactions or social bonds typically is associated with
both physical and mental illness (Ryff and Singer, 1998).
Forced social separations or the absence of social attach-
ments can trigger stress, anxiety, fear, and even depression
(Sachser et al., 1998). The behaviors and physiological changes
associated with bereavement or grief are similar to those used
to define depression (Reite and Boccia, 1994). Understanding
the nature of physiological processes that regulate social attach-
ment also could be of value in the treatment or prevention of
disorders, such as autism, depression, or schizophrenia, which
can involve dysfunctional social attachment (Kirkpatrick, 1997;
Henry and Wang, 1998).
In primate infants the absence of adequate maternal care
and opportunities to form attachments has been associated
with growth retardation, social withdrawal, inadequate inter-
personal relationships, and inhibited verbal communication
(Harlow, 1971). This complex of symptoms has even been
recognized in human development as a medical syndrome,
termed ‘Reactive Attachment Disorder of Infancy.’ Although
the concept of attachment disorder has begun to generate treat-
ment strategies, the relationship between this ‘disorder’ and
normal human attachment remains to be described.
It has been proposed that autism, which can be character-
ized by atypical social behavior and a failure to form social
attachments, may involve abnormal activity in endogenous
peptidergic systems. For example, a variety of clinical studies
implicate opioids in autism (Bouvard et al., 1995). Treatment
with naltrexone produces some clinical benefits and alters
biochemical profiles in a subset of autistic children. Studies
also have begun to explore the role of oxytocin in autism (Insel,
1997). In autistic children, deficits in oxytocin (Modahl et al.,
1998; Guastella and Hickie, 2016), genetic variation in (Jacob
et al., 2007) or epigenetic silencing of the oxytocin receptor
(Gregory et al., 2009), have been correlated with some symp-
toms of autism. However, the possible role for oxytocin, vaso-
pressin, endogenous opioids (or receptors for these) in
selective human social attachments, more analogous to pair
bonding, remains less well understood.
 The Neurobiology of Social Affiliation and Pair Bonding
Other neurochemicals, such as catecholamines and sero-
tonin, have effects on behavior and influence the release and
actions of oxytocin and vasopressin. For example, selective
serotonin reuptake inhibitors can influence peptidergic systems
(Li et al., 1993; Uvnas-Moberg et al., 1999). The clinical effects
of these chemicals in the context of attachment are largely
unknown, although drugs that affect these systems could influ-
ence social bonding.
Forced isolation, anxiety, fear, and other forms of stressful
conditions are associated with increased levels of HPA activity.
Such conditions or experiences normally tend to encourage
social interactions. Both the human and animal literature
(Carter, 1998) suggest an association between HPA activation
and stressful experiences and the development of social attach-
ments. The role of hormones of the HPA axis in attachment is
probably not linear, because both animals and humans under
extreme conditions may become either self-protective or
immobilized – conditions which are at odds with the forma-
tion of social bonds (Porges, 1998, 2011). Excessively stressful
conditions, such as those that compromise survival or intense
grief, may lead to a breakdown of social relationships (Reite
and Boccia, 1994). Thus, chronic or extreme stress could inhibit
subsequent attachment. However, research with rodents
suggests that within a homeostatic range, stress-related physio-
logical processes, including hormones of the HPA axis, can
promote the subsequent development of social bonds, espe-
cially in males (DeVries et al., 1996). In addition, positive
social interactions, including social bonds, may help to create
physiological states that are anxiolytic or stress reducing.
Oxytocin, perhaps released by positive social interactions,
has the capacity to produce both acute and chronic reductions
in the activity of the HPA axis (Carter, 1998; Petersson et al.,
1999). Studies of lactating women support this hypothesis in
humans (Altemus et al., 1995). Thus, oxytocin, with both
central and peripheral processes, is part of an endogenous
homeostatic system. This system has the concurrent capacity
to increase social attachment and other positive social behav-
iors, providing the additional indirect benefits of sociality.
The role of vasopressin in human social behavior is more
difficult to characterize. Vasopressin is believed to be similar
to the ancestral peptide from which oxytocin and vasopressin
were derived. Vasopressin binds to several types of receptors,
including the oxytocin receptor. Vasopressin has been impli-
cated in territoriality, agonistic behaviors, and HPA
arousal and may be part of a more primitive adaptive system
for mobilization and self-defense (Carter, 1998; Porges, 1998,
2007; 2011). In some cases the functions of vasopressin are
apparently similar to those of oxytocin. However, under other
conditions, oxytocin and vasopressin may have antagonistic
actions (Engelmann et al., 1996). Dynamic and complex inter-
actions between oxytocin and vasopressin (Albers, 2015),
working in the presence of a more slowly changing steroid
background, could help to regulate human visceral states and
emotions.
Awareness of the importance of peptides, including oxytocin
and vasopressin, in human behavior is comparatively recent,
but of considerable importance. Many aspects of daily life can
affect the release of the peptides. Social or sexual contact, food
intake (Uvnas-Moberg, 1994), the use of steroid hormones, or
drugs of abuse or alcohol (Kovacs et al., 1998) are only a few
135
examples of experiences that have been shown to influence
the endogenous production, release, or actions of these
peptides.
Many components of human behavior, such as motor
patterns, are regulated at least in part, by cognitive processes.
However, the decision to engage in a given behavior and
various mood states are strongly determined by visceral
processes or autonomic states, which may in turn motivate
the occurrence of specific behaviors (Porges, 2011). Peptides,
including oxytocin and vasopressin, regulate these visceral
states or emotional feelings. Steroids, opioids, oxytocin, and/
or vasopressin may induce a physiological process or ‘social
motivation’ that increases the probability of social interactions
and the formation of social bonds. In addition, oxytocinergic
processes, possibly due to their unique central position in the
nervous system, may help to modulate autonomic and behav-
ioral reactivity. Thus, by understanding the neurobiology of
peptides, such as oxytocin and vasopressin, we may gain
a better understanding of the processes through which social
bonds promote physical and emotional health.
Hormones, including oxytocin and vasopressin, are directly
and indirectly manipulated by various medical practices. For
example, large doses of ‘Pitocin,’ a synthetic version of
oxytocin, are routinely used to hasten childbirth, with unex-
plored effects on the social behavior or propensity to attach-
ment of both the mother and child (Boer, 1993). Long
labors, caesarian sections, and the decision to breast or bottle
feed are indirectly peptidergic manipulations (Kenkel et al.,
2014). Only recently has attention begun to be directed to
the behavioral or hormonal consequences of these peptide-
related events that can have profound effects on behavioral,
homeostatic, and emotional systems for both the parent and
the child.
1.04.11 Attachment and Bonding in Primates: An
Evolutionary Perspective for Humans
Human infants form attachment bonds with their caregiver
who is usually the mother, for it is she who provides the secure
base for fostering exploration, play, and other social behaviors
(Klaus et al., 1995). The nature of this attachment figure
becomes more visible under conditions that are fearful or novel
and threatening to the infant. By seeking protection through
attachment, the immature offspring are believed to increase
their likelihood of survival. Infant attachment is not indepen-
dent of caregiving and therefore it is important that mothers
should find ‘satisfaction with the parental role,’ and across
many cultures this appears to be the case. To the extent that
such ‘satisfaction’ may help to maintain investment in rearing
offspring to reproductive age, maternal caregiving behavior
must have evolved with infant attachment behavior. Also
common to all cultures are the changes in hormonal status
during pregnancy and parturition that predispose women to
respond to an infant’s signals.
The quality of mothering, particularly in times of fear and
stress, contributes substantially to the infant’s security of
attachment. Bowlby (1969) argued that infant attachment
and maternal bonding provided the foundation for human
attachment behavior from the cradle to the grave. Although
136 The Neurobiology of Social Affiliation and Pair Bonding
the ‘internalized’ bond is secured by the various forms of
attachment behavior that contribute to it, touching and
huddling are features of attachment behavior that persist into
adult life, particularly in times of distress, fear, and illness.
A question of some importance is how this relationship,
engendered by maternal bonding and reciprocated by infant
attachment, and which extends into adult relationships and
bonds, is subserved at the neural level. If, as Bowlby suggests,
all meaningful relationships are structured on the foundations
of early attachment, are there common underlying mecha-
nisms, and if so how have they evolved? The biology of
mammalian bonding, as exemplified by monogamous prairie
voles and sheep with their lambs, focuses heavily on the impor-
tance of peripheral steroid hormones (estradiol, progesterone,
testosterone, and corticosterone) and their effects on central
release of peptides including vasopressin, oxytocin, CRF, and
opioids, and the synthesis of receptors for these peptides. A
primary site of action for these peptides, although not exclu-
sive, may be on olfactory recognition that is important in the
bonding process. It is likely that attachments or bonding in
humans are more complex and probably more dependent on
cognitive processes and mental representations rather than
the olfactory processes that occur in rodents or sheep (Kim
et al., 2016). However, some insight regarding the processes
involved in social bonding in humans may come from studies
in nonhuman primates.
Many of the basic features in human infant behavior that
helped Bowlby formulate his attachment theory can be
observed in the mother directed behavior of infant monkeys.
This sharing of behavioral propensities and emotional labilities
of monkey and human infants (sucking, clinging, crying, and
following) is consistent with their close evolutionary histories.
The first detailed studies of attachment relationships were
undertaken in the 1960s on rhesus monkeys and provided
descriptions of infant behavioral development that generalize
to many primate species (Harlow, 1971; Kraemer, 1992).
Primate mothers provide their infants with nourishment,
protection, and warmth; protection from predators; and
a secure base to which they can retreat when disruptions occur
in the social group. The attachment that a rhesus monkey infant
develops with its mother is especially enduring in females,
lasting a lifetime, while in infant males, it rarely lasts beyond
puberty.
1.04.11.1 The Evolutionary Basis of Bonding
What are the evolutionary developments that have enabled
expansion of the affectional system deployed in mother–infant
bonding to incorporate other contexts of social/affiliative rela-
tionships? In considering primates, strong interrelations are
beginning to emerge between the two important features noted
earlier, neocortical expansion, and matrilineal inheritance.
These are common to all mammals that exhibit complex social
organizations. The development of a larger neocortex has
enabled motivated behavior to occur at will, such that maternal
affiliation may take place without pregnancy and parturition
(Keverne, 2013; Carter, 2014). This unique development in
primate evolution has matched parturient females with non-
parturient females in sustaining the behavioral potential for
infant caregiving (Hrdy, 2009). Such an emancipation of
behavior from endocrine determinants has only been evolu-
tionarily possible with the development of a bigger brain,
because decision-making processes, especially in the context
of maternal behavior, are complex and need to be strategically
correct. Progression away from the synchronization of maternal
behavior with the hormones of pregnancy and replacement
with a system of cognitive control require exceptional cognitive
abilities. These abilities are not inherited, but a larger brain
enables social factors to take over from hormonal factors in pre-
dicting reproductive success. This would explain the overriding
importance of social and maternal experience in order to
achieve successful maternal care, experience that is acquired
during early social development under the watchful eye of
mothers, but outside the immediate context of pregnancy
and parturition (Kim et al., 2016).
From the available fossil records, it appears that many
mammalian lineages have evolved increased cranial capacity,
but because it is claimed that the push for an exceptionally
larger neocortex in primates has developed from complex
social living, then differences in maternal and paternal lifestyles
may have subjected brain evolution to differential selection
pressures. In Old World monkeys, females provide social
stability and group cohesion, are more affiliative than males,
and maintain the continuity of the group over successive gener-
ations. Females are the primary caregivers with social rank of
daughter, but not sons, being related to the matriline. This
kind of matrilineal inheritance is compatible with genomic
imprinting, which not only results in the development of
a larger forebrain from maternally expressed alleles (Keverne
et al., 1996a), but also the advantages of genomically
imprinted inheritance are transmitted to both sons and daugh-
ters. Hence, the differential selection pressures operating
through the matriline could have resulted in a larger neocortex
giving greater cognitive control over behavior (Keverne et al.,
1996b). For such neocortical control over behavior to be
successful, the process of attachment and early learning is
essential for the subsequent development of normal affiliative
relationships.
The development of a larger brain has been particularly
important to enable the lifetime attachments such as those
that occur in humans. For the operation of ‘attachments’ to
sustain relationships, and endure a human lifetime, a brain
that can develop growing insights into self as well as the
maternal attachment figure is clearly an advantage. As young
children develop, their knowledge base expands especially in
regard to the changing ways in which their mother responds
to them and, more importantly, how each is likely to respond
to the other. This knowledge steadily becomes organized in the
form of internal working models of self and mother, encom-
passing an understanding of both her moods and intentions.
Building on this early knowledge provides the infant with an
ability to simulate happenings in an expanding world of rela-
tionships. Forward planning may occur with the advantages
of foresight and security. Because these working models are
in constant daily use, their influence on thought, feeling, and
behavior becomes routine and all pervasive.
Deploying our brains in the construction of internalized
working models of self and others would be difficult, if not
impossible, without an attachment figure, and we would
further argue, that attachment figures would be extremely
 The Neurobiology of Social Affiliation and Pair Bonding
scarce without maternal bonding. Those parts of the brain that
are characteristically enlarged in humans (neocortex and stria-
tum) develop in the postnatal period (neotany) when infants
are forming their internal working models. Important in this
context is the availability of exclusive access to mother and
her undivided attention. Biology has assisted this possibility
by extending the interbirth interval, aided by the contraceptive
effects of infant suckling. There can surely be no better way of
increasing maternal investment than by reducing the number
of infants in which the mother invests.
1.04.11.2 Genomic Imprinting, Social Behavior, and Cortical
Growth
Oxytocin may have both direct and indirect effects on neocor-
tical development. Live birth puts restrictions on the physical
size of an infant and especially the head. A large baby with
an expanded neocortex is a physical burden for the mother.
In primates, infants are gestated, nursed, and carried for
months or years. Reproductive restrictions are further increased
in bipedal primates, since mothers must give birth through
a pelvic girdle adapted for upright locomotion. Thus, the
capacity of the mother to regulate offspring development and
especially the size of the neocortex may be critical to both
her survival and reproductive success (Keverne, 2013). At the
same time the father’s genome may be better served by larger
offspring.
Through a process known as genomic imprinting, the
expression of a subset of genes, of particularly relevant impor-
tance to growth and development, can be determined by one
parent versus the other. This is accomplished by selective
silencing of one of a pair of alleles for a given gene, allowing
the other allele to dominate. In theory the mother seeks to
produce a smaller infant with a smaller brain. Research in
mice (Keverne, 2013) showed that the matrilinear germ line
contains cells that will become the neocortex, while progenitor
cells that will become the hypothalamus originate in the
father’s genome. The primary source of oxytocin is from the
hypothalamus, in neurons that are regulated by paternally
expressed genes that are susceptible to genomic imprinting.
One paternally expressed gene, known as Peg3, plays a critical
role in the development of the hypothalamus, as well as the
placenta (Broad et al., 2009; Champagne et al., 2009). Evidence
for the importance of Peg3 comes from experiments in which
the Peg3 gene was inactivated. In the absence of Peg3 expres-
sion, females had a reduced number of hypothalamic oxytocin
neuron, lower reproductive success, and specific reductions in
the growth of the offspring that survive. Peg3 mutant mothers
also were less attentive to their young, showing increased indi-
cations of anxiety and aggression, all suggestive of reductions in
the functions of oxytocin. Genomic imprinting or silencing of
the paternal allele of the Peg3 gene allows the maternal
genome to dominate the development of her fetus.
Thus, although the genes that produce oxytocin may origi-
nate in the father, it is the mother who determines the expres-
sion of these genes, and thus the size of the infants. By
genomically silencing genes from the father and allowing her
own to dominate, the mother’s genome can regulate the growth
of the brain and body of her offspring (Keverne, 2013). The
capacity of the mother to regulate cortical development also
137
may help to explain the exceptionally long period of time
necessary to produce a fully developed human nervous system.
The slow maturation of the nervous system also increases the
developmental significance of social interactions and long-
term attachments.
It has been shown that apoptosis can be inhibited by
oxytocin, and especially by the ‘fetal,’ extended form of oxytocin
(Jankowski et al., 2016). Oxytocin also has been shown to
reduce apoptosis in adult animals and to promote adult neuro-
genesis (Leuner et al., 2012). High levels of oxytocin might
facilitate neocortical growth, by inhibiting the programmed
destruction of brain cells, thus sculpting the cortex, and
synchronizing neocortical development to the physical
demands of mammalian reproduction (Carter, 2014).
It is also important to note that in these early years, when
the neocortex is forming and making its connections and asso-
ciations with other parts of the brain, the limbic emotional
brain is well developed. The large emotional repertoire of
young infants bears testimony to this. Understanding
emotions, curbing these emotions, and channeling these
emotions for beneficial purposes must represent an important
phase in brain development. These cognitive abilities have
surely prospered from experiencing the dynamics of reciprocity
in the interplay between mother and child, while the subse-
quent expansion of social relationships, based on secure attach-
ment, provides a second cushion for the emotional turmoil of
puberty. The child’s need for an attachment figure and the
mother’s predisposition to bond provides an optimal social
environment in which the human brain can develop. Weaning
and expansion of the child’s social world have not only coin-
cided, but are made easier by having a secure base from which
to explore.
1.04.11.3 Oxytocin and Love
Among the most interesting behaviors and emotional states in
which oxytocin has been implicated are those that humans call
‘love’ (Carter, 1998; Carter and Porges, 2013; Fisher, 2016). Key
to loving relationships are selective social behaviors and recip-
rocal social bonds. Studies originally conducted in socially
monogamous prairie voles revealed that oxytocin was capable
of facilitating social contact, as well as selective social prefer-
ences in both sexes. Research in prairie voles showed that
mating facilitated the onset of pair bonding (Williams et al.,
1992), a behavior that was later shown to be dependent on
oxytocin (Williams et al., 1994). In this context, it is important
to note that in the prairie vole model, access to both oxytocin
and vasopressin receptors is necessary for pair bonding to
emerge, while either oxytocin or vasopressin alone can facilitate
nonselective sociality (Cho et al., 1999). However, as explained
here, it is likely that the quality of the biological experiences
that are influenced by oxytocin and vasopressin differ (Carter,
1998).
Research in animals, including humans, has confirmed and
extended many of the general conclusions drawn from that
research (Carter, 1998), leaving no doubt that oxytocin plays
a central role in the social behavior that lie at the heart of the
human experience of love. Oxytocin is released in response
to a variety of circumstances, both positive and negative
(Carter, 1992; Feldman, 2012; Dai et al., 2012). However,
138 The Neurobiology of Social Affiliation and Pair Bonding
whether human social behavior and attachments can be
formed in the absence of oxytocin is not known.
1.04.11.4 Oxytocin and Development
The importance of oxytocin is especially appreciated in the
context of the development of the human nervous system.
The human brain is two to three times larger than that of other
related primates (Somel et al., 2013). This difference is due
primarily to increases in cortical tissue, especially neurons,
located in association areas such as the prefrontal cortex. The
elaboration of the neocortex was critical to the evolution of
novel features of human behavior, including cognition and
speech. The origins of the neocortex are traced to the reptilian
ancestor of early mammals. These reptiles are believed to
have had a thin sheet of cortical cells, which by the time of
the evolution of the earliest mammals has been estimated to
be only about six cell layers in thickness (Rakic, 2009). During
development, the cells of the neocortex differentiate, migrate,
enlarge, and in some cases undergo cell death (including
apoptosis). Initially subtle developmental events may have
allowed the evolution of the human neocortex. For example,
it has been estimated by Rakic (2009) that “fewer than 7 extra
rounds of cell division in the progenitor cells at an early embry-
onic stage would be sufficient to create the 1000 fold difference
in total cortical surface area that differentiates the brains of
mice from those of humans.”
In rats, maternal oxytocin can act as a signaling mechanism
between the mother and fetus (Kenkel, 2014). Of particular
importance to cortical and hippocampal functioning is
GABA. Maternal oxytocin released during birth triggers a switch
in GABA signaling in the fetal brain from excitatory to inhibi-
tory. In vivo administration of an oxytocin antagonist before
delivery prevented this switch of GABA activity in fetal neurons
and aggravated the severity of hypoxic episodes. Maternal
oxytocin apparently inhibits fetal neurons and concurrently
increases their resistance to hypoxia, which can serve to protect
cortical tissue during birth. The birth-related surge in oxytocin
also helps to regulate the synchronization of the fetal hippo-
campal neurons, possibly allowing the transition from prenatal
to postnatal life (Tyzio et al., 2006; Mazzuca et al., 2011). Such
changes have long-term consequences for both emotional and
cognitive functions, including the capacity to exhibit positive
social behaviors and form social bonds.
1.04.12 Summary
Pair bonding is an evolved trait and may play a critical role
in reproduction, as well as in individual and species
survival. The neurobiology of pair bonding or other forms
of social affiliation are most readily understood in this
context. The proximate mechanisms underlying various
forms of positive social behaviors, including pair bonding
and maternal–infant behavior, rely on common neural and
endocrine systems. At the heart of pair bonding are neural
systems dependent on peptides, including oxytocin, vaso-
pressin, opioids, CRF, and related hormones. Steroid
hormones, although not directly essential for pair bonding,
may facilitate and modulate these behaviors. Neuropeptides
also serve to coordinate the autonomic and endocrine
consequences of positive social experiences with behavioral
states that support the formation and maintenance of social
bonds.
Unique actions of social behavior and of oxytocin may
have been necessary for encephalization, by facilitating birth,
lactation, maternal behavior, genetic regulation of the
growth of the neocortex, and the maintenance of the blood
supply to the cortex (Keverne, 2013; Carter, 2014).
Peptide-facilitated attachment also allows the extended
periods of nurture necessary for the emergence and optimiza-
tion of human intellectual development. In general, oxytocin
acts to encourage a sense of safety, and the high levels of
social sensitivity and attunement necessary for human soci-
ality and for rearing a human child. Oxytocin dynamically
moderates the autonomic nervous system, and effects of
oxytocin on vagal pathways, as well as the antioxidant and
anti-inflammatory effects of this peptide, help to explain
the pervasive adaptive consequences of social behavior for
emotional and physical health.
Acknowledgments
We gratefully acknowledge many scientists, including our collaborators,
whose work has contributed to the understanding of this subject.
Studies on maternal bonding in sheep and primate behavior were
sponsored by grants to EBK from the BBSRC and the Medical Research
Council of the United Kingdom. Studies on pair bonding in prairie
voles were sponsored by grants to CSC from the United States National
Institutes of Health (NICHD and NIMH) and National Science Foun-
dation, and National Alliance for Autism Research. Studies of human
breast feeding were sponsored by a grant from the United States
Department of Defense. We are particularly grateful for conceptual
guidance regarding the role of the autonomic nervous system in social
behavior from Stephen Porges.
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 1.05 Hormones and the Development and Expression of Aggressive Behavior
Brian C Trainor, University of California, Davis, CA, USA
Cheryl L Sisk, Michigan State University, East Lansing, MI, USA
Randy J Nelson, Neuroscience Research Institute, Ohio State University Wexner Medical Center, Columbus, OH, USA
Ó 2017 Elsevier Inc. All rights reserved.

1.05.1 Introduction 146

1.05.1.1 Categories of Aggression 146
1.05.2 Animal Models and Tests of Aggression 147
1.05.3 Endocrine Signals’ and Receptors’ Contribution to Aggression 148
1.05.3.1 Steroid Hormones 148
1.05.3.1.1 Androgens 149
1.05.3.1.2 Estrogens 151
1.05.3.1.3 Glucocorticoids 151
1.05.3.2 Anabolic Steroid Abuse and Aggression 151
1.05.4 Brain Regions Contributing to Aggression 152
1.05.4.1 Studies in Rodents 153
1.05.4.2 Nonhuman Primates and Humans 154
1.05.5 Neurotransmitters, Hormones, and Aggression 155
1.05.5.1 Serotonin 155
1.05.5.2 Arginine Vasopressin 156
1.05.5.3 Monoamine Oxidase 156
1.05.5.4 Nitric Oxide 157
1.05.6 Development of Aggression 158
1.05.6.1 Rough-and-Tumble Play as an Antecedent to Aggressive Behavior 158
1.05.6.2 Endocrine Contributions to the Development of Aggressive Behavior: Perinatal Organizational Effects 159
1.05.6.3 Endocrine Contributions to the Development of Aggressive Behavior: Pubertal Organizational Effects 160
1.05.6.4 Endocrine Contributions to the Development of Aggressive Behavior: Pubertal Activational Effects 161
1.05.6.5 Social Experience and the Development of Aggressive Behavior 162
1.05.6.6 Conditioned Defeat 162
1.05.6.7 Aggression in Aged Individuals 162
1.05.7 Reciprocal Effects of Aggression on Steroid Hormones 163
1.05.7.1 Gene–Environment Interactions 163
1.05.8 Integration 165
References 166

Glossary
Aggression A form of social interaction that includes
threat, attack, and fighting.
Hormone response element Sequences of DNA in
promoter regions that are bound by hormone receptors.
Binding of the receptor complex promotes transcription.
Immediate early gene A gene that is expressed rapidly and
transiently in response to various cellular stimuli. Several of
these genes are used by neuroscientists as indirect markers
of neuronal activity because they are expressed when
neurons fire action potentials.
Intermittent explosive disorder A disorder characterized
by repeated episodes of aggressive, violent behavior that is
grossly out of proportion to the situation: thought to affect
as many as 7.3% of adults in the United States.
Lifetime History of Aggression (LHA) Scale An interview-
based scale that is used by mental health workers to assess
general aggressive tendencies in humans. Interviews can
be supplemented with other sources such as clinical
records.
Organizational/activational hypothesis The proposal that
early androgen exposure permanently organizes the
nervous system of mammals in a male-like manner. After
pre- or perinatal organization by androgens, these
hormones more readily activate male-typical postpubertal
behaviors by acting upon the organized structures.
Piloerection The erection of hair on the skin, used as
a threatening display by many animals.

Hormones, Brain, and Behavior, 3rd edition, Volume 1 http://dx.doi.org/10.1016/B978-0-12-803592-4.00005-5 145

146 Hormones and the Development and Expression of Aggressive Behavior
1.05.1 Introduction
Aggression may be defined as overt behavior with the intention
of inflicting harm or the threat of harm upon another indi-
vidual. Aggressive behaviors range from lethal to subtle. The
possibility for aggressive behavior exists whenever the interests
of two or more individuals conflict (Svare, 1983). Conflicts
typically arise over limited resources including territories,
mates, and food. Among social species, conflicts may arise
over social status and limited resources. A social interaction
decides which individual gains status or access to the contested
resource. In many cases, a submissive posture or gesture from
one individual avoids actual combat. Individuals may also
participate in threat displays or ritualized combat in which
dominance is determined, but no physical damage is inflicted.
Species- and situation-specific rules exist to regulate aggression.
When aggressive behaviors break these rules or when the
aggression is excessive to situational norms, then it may be
considered to be pathological or violent (Haller and Kruk,
2006; Miczek et al., 2007). Pathological or violent aggression
causes much personal and societal suffering and thus is an
important topic for study. Treatment for violent or pathological
aggressive behavior remains primitive. Essentially, no effective
interventions exist for violent humans – the most common
‘treatment’ for violent people is incarceration (Eastman and
Campbell, 2006). Remarkably, a death sentence is the sole
treatment for an aggressive dog that bites a person. Thus,
understanding the pathophysiology of aggression remains an
important, yet understudied endeavor.
Indeed, in common with categorizing human behavior, it is
often difficult for observers to draw the line between adaptive
aggression and pathological aggression in nonhuman animals
(Sluyter et al., 2003). An important strategy for improving our
ability to draw this line is to understand the mechanistic basis
for both species-specific aggression and pathological aggres-
sion. This review will examine the role of neural circuits, devel-
opment, and neuroendocrine mechanisms in the regulation of
aggression, primarily in species-specific aggression. Under
certain conditions, aggression can deviate from normal
patterns (Miczek et al., 2013). Although these models are
beginning to provide important insights into potential mecha-
nisms of violence, it is necessary to first understand the basis for
normative forms of aggression.
Mechanistic studies of aggression have historically focused
on aggression in nonpathological states, and in this state
gonadal hormones play an important role. Across most species,
males are more aggressive than females, and this difference
usually emerges at puberty, which coincides with important
sex differences in gonadal hormone secretion. These patterns
have contributed to a historic focus on the role of gonadal
hormones such as testosterone in the regulation of aggressive
behaviors. Initial studies using hormone ablation and replace-
ment approaches showed a clear role for androgens such as
testosterone in many species (Vandenbergh, 1971; Leshner
and Moyer, 1975; Soma, 2006). However, exceptional cases
appeared in which removal of testes had no obvious effect on
aggression (Demas et al., 1999; Trainor and Marler, 2001).
Furthermore, for many years the link between testosterone
and aggression in humans appeared very weak (Archer,
1991). These apparently inconsistent observations raised the
question of whether neuroendocrine mechanisms of aggres-
sion were evolutionarily conserved. However, improved exper-
imental design and methodologies have provided new insights.
For example, androgens can be synthesized de novo within the
brain (Pradhan et al., 2010), so removal of testes does not
necessarily remove all sources of testosterone. In addition,
rapid changes in testosterone (rather than baseline levels) are
now known to have important effects on aggression (Gleason
et al., 2009). Improved experimental designs have revealed
that pairing testosterone measurements with competitive tasks
are more effective at revealing hormone-behavior relationships
in humans (Section 1.05.4.2). Hormones affect aggression by
acting on neural circuits in the brain, and these circuits have
been identified using lesions, immediate early gene studies,
neuropharmacology studies, and more recently optical stimu-
lation. Within these neural circuits neurotransmitters such as
serotonin have important modulatory effects on aggression.
Finally, experience has important effects on how these neural
circuits respond in competitive contexts. These effects of expe-
rience occur across development, both early in life and in the
adult.
1.05.1.1 Categories of Aggression
Several categories of aggression are generally recognized, and
the different types of aggression have different neuroendocrine
bases. For example, there are important differences in the
neuroendocrine mechanisms of maternal aggression versus
territorial aggression, but there is also a significant degree of
overlap. Androgens (Bouissou, 1983) and estrogens (Laredo
et al., 2014b) have important modulatory effects on intermale
aggression, especially in territorial contexts. Hormonal changes
associated with the pregnancy, including high levels of estro-
gens, have an important role for inducing maternal aggression
(Gammie et al., 2007). In the most widely studied rodent
species (domestic mice and rats), maternal aggression is the
main form of aggression in females. However, in other rodent
species such as Syrian hamsters intrafemale aggression is more
prevalent (Gutzler et al., 2010; Staffend and Meisel, 2012),
which provides opportunities for examining neuroendocrine
mechanism of female aggression outside of the context of
reproduction. Another type of agonistic behavior commonly
studied in the laboratory has been called fear-induced aggres-
sion, but this is more correctly termed ‘defense’ and is not
strongly modulated by estrogens or androgens (Blanchard
and Blanchard, 1989). Recently there has been increased
interest in abnormal forms of aggression (or escalated aggres-
sion) that might resemble aspects of human violence.
Abnormal aggression has been characterized using quantitative
or qualitative criteria (Natarajan and Caramaschi, 2010; Miczek
et al., 2013). Aggression can differ quantitatively such as induc-
tion with low provocation or occur at higher intensity. Alterna-
tively, aggression can differ qualitatively such as through
attacks directed at vulnerable regions or through a lack of
responding to submissive displays that normally trigger cessa-
tion of aggressive behavior. Abnormal aggression can be
induced by very low glucocorticoid levels induced by adrenal-
ectomy (Haller et al., 2001). Intriguingly, in this context aggres-
sion occurs in the absence of autonomic activation, which is
observed in some forms of human violence.
 Hormones and the Development and Expression of Aggressive Behavior
Whereas specific aggressive behaviors in animals are typi-
cally highly stereotyped (wrestling, chasing, bites), aggression
behaviors in humans can take many forms (e.g., physical versus
verbal). Quantifying aggression in humans is a challenge. Early
efforts to study aggression relied primarily on self-reports of
aggression that estimated general aggressive tendencies. These
measures generally correlate poorly with neuroendocrine
measures such as testosterone (Archer, 2006). However,
a variety of tasks have been developed to induce aggressive
behavior under controlled conditions (e.g., point subtraction
task). Aggressive behavior in these tasks is more closely linked
to testosterone levels, which are rapidly modulated during
aggressive interactions (Carré et al., 2011). These tests also
induce sympathetic nervous system activation (Gerra et al.,
2007). Increased arousal and anger are main components of
what has been termed reactive aggression (Vitiello and Stoff,
1997), which is considered to be more impulsive yet can lead
to sustained aggressive responses. It has been thought that reac-
tive aggression accounts for most societal problems related to
aggression (Blair et al., 2006). Reactive aggression is usually
associated with impulse control and low serotonergic signaling
(Mehlman et al., 1994; Krakowski, 2003). In contrast instru-
mental aggression occurs in the absence of physiological
arousal and is considered to be a more goal-oriented behavior.
High-profile incidents (e.g., mass killings, genocides, or assassi-
nations) are likely to reflect instrumental mechanisms of
aggression. Attacking or otherwise bullying your neighbors to
intimidate them is another example of instrumental aggres-
sion. The controlled–instrumental subtype of aggression is
thought to be regulated by higher cortical systems and less
dependent on the hypothalamic and limbic systems that are
known to mediate impulsive aggression (Viding et al., 2007;
and see below), and likely less dependent on hormones than
other types of aggression.
Mental disorders such as intermittent explosive disorder
and posttraumatic stress disorder are associated with increased
autonomic arousal, which can contribute to sudden and
uncontrolled reactive aggression (Blair et al., 2006; Viding
et al., 2007). In contrast, individuals who are diagnosed with
conduct disorder or antisocial personality disorder show
unusually low autonomic responsiveness (Viding et al.,
2007), which can contribute to increased instrumental aggres-
sion by blunting the typical emotional responses (Raine,
2002). Thus, exaggerated aggressive responses can be observed
in both high- and low-arousal states, with different biochem-
ical, neuroanatomical, and neuroendocrine systems contrib-
uting to behavior in each context. These results illustrate that
more than one approach to studying neuroendocrine mecha-
nisms of aggression will be required. Animal model studies
in which testosterone has an important modulatory effect on
aggression will be more informative for reactive aggression
whereas approaches examining aggression under low-arousal
conditions may be more informative for understanding instru-
mental aggression.
Aggressive behavior is a motivated behavior. In common
with other motivated behaviors, four types of questions arise:
(1) What are the external factors that elicit aggressive behavior?
(2) What neural circuitry mediates aggressive behaviors?
(3) How does aggression develop across ontogeny? (4) What
are the internal signals that mediate aggressive behaviors?
147
Here we will focus on questions 2–4. First we will review
animal models and tests of aggression, because much of what
is known about neurobiological mechanisms and development
of aggression is based on animal models.
1.05.2 Animal Models and Tests of Aggression
The rigorous quantification of aggressive behaviors is an essen-
tial requirement for identifying underlying neuroendocrine
mechanisms. The most common behavior test for quantifying
overt aggressive behaviors is the resident–intruder test
(Koolhaas et al., 2013). In a typical test the focal animal is
housed in a home cage for at least 3 days. This allows the focal
animal to become familiar with home cage and adopt it as
a territory. Next an unfamiliar intruder is introduced into the
cage. The introduction of the intruder usually prompts the initi-
ation of anogenital sniffing by the resident, which results in the
detection of nonvolatile pheromones. Depending on the
species or strain, aggressive threats such as tail rattles or vocal-
izations may precede overt aggressive behaviors (Nelson and
Chiavegatto, 2000). In rats and mice, attacks are usually
directed toward the flanks which have thick skin that prevent
wounding. Each occurrence of these behavioral elements can
be measured in terms of frequency, as well as the onset and
termination of specific behaviors from both live observations
and video records. An advantage of the resident–intruder test
is that it can be used in several different contexts, which can
allow the study of several forms of aggression.
In many cases androgens either promote aggressive
behavior in the resident–intruder test (Vandenbergh, 1971;
Lima and Spinelli de Oliveira, 2014) or are secreted in response
to engaging in aggression (Marler et al., 2005). These contexts
appear to correspond to reactive aggression in humans, which
is more likely to be linked with increases in testosterone (Carré
et al., 2011). Under these conditions residents typically reduce
aggression in response to the intruder engaging in submissive
postures. However, when adrenalectomized rats are tested as
residents in this test, submissive displays by the intruder are
ignored resulting in escalated aggression (Haller et al., 2001).
It is thought that aggression under these conditions more
closely resembles the instrumental aggression associated with
conditions such as conduct disorder (Haller and Kruk, 2006).
Aggression can also be observed in females using the resi-
dent–intruder test. Most often this is studied in lactating
dams with male intruders. Maternal aggression under these
contexts is considered defensive aggression (Lonstein and
Gammie, 2002). It is characterized by short latency attacks of
high intensity, mostly directed toward the head/neck region
of the opponent and usually without the introductory threat-
ening behaviors typically displayed by male animals con-
fronted with an intruder. In laboratory rats and mice, females
are rarely aggressive toward other females. However, female–
female aggression is quite prevalent in other species. The Syrian
hamster is a solitary species, and females are very aggressive
toward other females (Solomon et al., 2007; Staffend and
Meisel, 2012). In the California mouse, males and females
form monogamous pairs, and females are also very aggressive
toward other females (Davis and Marler, 2004; Silva et al.,
2010; Trainor et al., 2010b). These species allow for the ability
148 Hormones and the Development and Expression of Aggressive Behavior
to examine whether neuroendocrine mechanisms of aggression
identified in males extend to females (Gutzler et al., 2010).
The resident–intruder test can be adapted to examine exag-
gerated aggressive behavior following provocation. In this
protocol an intruder is first placed into the resident’s home
cage behind a protective barrier, which is then removed
(Miczek et al., 2013). Elevated aggression using this protocol
can be observed in both males (Fish et al., 1999) and females
(Potegal, 1991). This approach may provide insights into
mechanisms contributing to exaggerated aggression following
perceived provocation.
Under normal conditions, it is rare for male domestic mice
and rats to direct aggression toward females. However among
humans, women are often targets of male violence (Crowell
and Burgess, 1996). Recently it was discovered that male rats
exposed to psychosocial stress during the peripubertal period
were more aggressive with female breeding partners compared
to controls (Figure 1; Cordero et al., 2012). Intriguingly this
effect was also observed in the male offspring of these pairs.
Female-directed aggression by males was reversed with mono-
amine oxidase inhibitor (Marquez et al., 2013). A recent
prospective study reported that individuals that were neglected
as children were more likely to injure an adult intimate partner
(Widom et al., 2014). However, the neurobiological mecha-
nisms contributing to this effect are unknown. Rodent peripu-
bertal stress models may facilitate the identification of
physiological mechanisms contributing to intersex aggression.
Clearly, disrupting generational transmission of this form of
aggression is an important public health problem.
The resident–intruder test can provide insights into mecha-
nisms of aggression in multiple contexts. However, several
factors need to be considered when interpreting the outcome
of this test. For example, the behavior of the stimulus animal,
or intruder, has an important impact on the behavior of the
focal animal or resident. Previous experiences of the intruder
can confound the results. Thus, a previously defeated or naïve
* circuits are also altered by aggressive experience in female
50
Aggressive behavior (%)
Control
40
Stress
30
20
10
0 Contribution to Aggression
0 10 20 30
Time (min)
Figure 1 Peripubertal stress–induced abnormal aggressive behaviors
in resident–intruder tests performed in adulthood. Aggressive behaviors
of resident rats when intruders were similar to the resident rat in body
weight (stress effect: F1, 29 ¼ 9.52, p ¼ 0.004; n ¼ 15–16 per group).
Reprinted from Marquez, C., Poirier, G.L., Cordero, M.I., Larsen, M.H.,
Groner, A., Marquis, J., Magistretti, J., Trono, D., Sander, C., 2013.
Peripuberty stress leads to abnormal aggression, altered amygdala and
orbitofrontal reactivity, and increased prefrontal MAOA gene expression.
Transl. Psychiatry 3, e216 with permission from Nature.
intruder can elicit different reactions from the resident. One
way that this potential problem can be reduced, however, is
to determine which group-housed intruders are not aggressive
before the onset of the behavioral tests. An additional source of
variability can come from dominance relationships among
cage mates. For example, in the widely used C57Bl6 strain,
each cage of males has a dominance hierarchy. There are impor-
tant differences in behavior and brain function between the
most dominant and most subordinate cage mate (Howerton
et al., 2008). For example, the lowest ranking cage mates had
less corticotropin-releasing hormone 2 mRNA and estrogen
receptor alpha in the bed nucleus of the stria terminalis than
the most dominant cage mate (Greenberg et al., 2014). An
easy solution for quantifying this variation is to run mice
through a ‘tube test,’ which provides an accurate estimate of
dominance status.
Experience is also important in the relationship between
hormones and aggressive behavior (Miczek and Fish, 2006).
Castration and hormone-replacement studies of males repre-
senting several species of reptiles, fish, and birds clearly demon-
strate reduced postcastration levels of aggression and
restoration of aggression after testosterone treatment (e.g.,
Crews and Moore, 1986; Wingfield et al., 1987). In mammals,
the effects of androgens in supporting aggressive behavior
depend largely on experience. Castrated mice and rats without
prior aggressive experience rarely fight when tested with
another male conspecific (Christie and Barfield, 1979). Individ-
uals that have won aggressive encounters before castration will
exhibit aggression long after the testes have been removed (e.g.,
Christie and Barfield, 1979; DeBold and Miczek, 1981, 1984).
Winning aggressive encounters usually induces a temporary
surge in testosterone referred to as the challenge effect. The
combination of winning experience and testosterone has
a powerful enhancing effect on aggressive behavior (Trainor
et al., 2004; Fuxjager et al., 2011). Winning also increases the
expression of androgen receptors in motivational circuits
including the nucleus accumbens and ventral tegmental
area (Fuxjager et al., 2010). Interestingly, these motivational
rodents. Female Syrian hamsters also become very aggressive
with repeated aggressive encounters (Staffend and Meisel,
2012), and this behavioral change is mediated in part by gluta-
matergic synaptic plasticity in the nucleus accumbens (Been
et al., 2016).
1.05.3 Endocrine Signals’ and Receptors’
1.05.3.1 Steroid Hormones
The role of steroid hormones has long been a focus of investi-
gators studying the neuroendocrine bases of aggressive
behavior. Although testosterone is generally a key hormone
regulating aggression, either directly or by serving as a prohor-
mone for DHT or estradiol, detailed experiments have demon-
strated that the relationships between steroid hormones such as
testosterone and aggression are complex. Other factors such as
steroid hormone synthesis in the brain, differential expression
of steroid receptors, and environmental context have important
influences on the behavioral effects of circulating hormones.
 Hormones and the Development and Expression of Aggressive Behavior
1.05.3.1.1 Androgens
The idea that hormones produced in the testes promote aggres-
sive behavior dates back to the mid-nineteenth century in the
classic experiments of Arnold Berthold (Quiring, 1944). In
these studies aggressive behavior in male chickens was abol-
ished by removal of testes and restored when donor testes
were implanted. Subsequent castration and hormone-replace-
ment experiments have identified androgens as a key class of
hormones produced by the testes that facilitates aggression.
Early studies focused on establishing correlations between
plasma concentrations of androgens and aggression. Androgen
levels are often increased during the breeding season when
males aggressively compete for breeding opportunities
(Lincoln et al., 1972; Wingfield, 1984; Moore, 1986; Bales
et al., 2006). Similarly, male aggressive behavior often increases
at the time of puberty (Wallen et al., 1991; Pellis et al., 1997;
Delville et al., 2005), when testes mature and begin to secrete
androgens. More definitive evidence that androgens facilitate
aggressive behavior comes from studies in which androgens
are manipulated. Castration reduces male aggressive behavior
in Syrian hamsters (Mesocricetus auratus) (Vandenbergh,
1971), mice (Mus musculus) (Leshner and Moyer, 1975), pigs
(Sus scrofa) (Zamaratskaia et al., 2008), bulls (Bos taurus)
(Huxsoll et al., 1998), rats (Rattus rattus) (Albert et al., 1987),
and red deer (Cervus elaphus) (Lincoln et al., 1972), whereas
testosterone replacement restores aggression in these species.
Similarly, elevated testosterone concentrations via implants
increase aggression in a variety of passerine birds (Wingfield
et al., 1987; Ketterson and & Nolan, 1992) and spiny lizards
(Marler and Moore, 1989).
Strain differences certainly exist among house mice in the
extent to which aggressive behaviors are expressed and in the
extent to which these aggressive behaviors are mediated by
androgens. The effects of castration on predatory, shock-
induced, maternal, and isolation-induced aggression were
studied in Swiss albino mice. Isolation-induced aggression
was generally reduced after castration; postgonadectomy treat-
ment with testosterone, 5a-dihydrotestosterone (DHT), or
estradiol restored this form of aggression (reviewed in Brain,
1983). Castration increased intruder aggression toward
lactating females, and treatment with testosterone, DHT, or
estradiol reversed the elevated rate of aggressive responses in
this situation (Brain, 1983). These results imply that steroid
hormones do not merely ‘trigger’ aggression, but act to affect
the animal’s perception of and response to aggression-
provoking stimuli (Haug et al., 1986).
In another series of experiments (Whalen and Johnson,
1987), male mice were pitted against either lactating females
or olfactory bulbectomized males (reviewed in Johnson and
Whalen, 1988). Gonad-intact males and castrated males
treated with testosterone attacked the olfactory bulbectomized
males, but did not attack lactating females. Untreated castrated
males tended to display tremendous individual differences in
aggressiveness, with some attacking either type of opponent,
others attacking only one type of opponent, and others display-
ing no attack behaviors (Johnson and Whalen, 1988). Because
castration was associated with large individual variation in
aggressive responding, and because androgen treatment
reduced that variation, Johnson and Whalen (1988) proposed
that testicular steroid hormones act to induce ‘behavioral
149
homogenization’ to reduce behavioral variability. This is an
intriguing hypothesis to account for the disparate aggressive
responses of males to different aggression-provoking stimuli,
although further experiments are necessary to evaluate it fully.
Although baseline testosterone concentrations regulate
aggression in many species, studies have identified several
species in which castration does not reduce male aggression
(Caldwell et al., 1984; Demas et al., 1999; Trainor and Marler,
2001; Trainor et al., 2006a). Furthermore, dominance in more
complex social organizations may not be related to blood
concentrations of testosterone, especially in stable groups. For
example, dominant dogs or squirrel monkeys can be castrated
without affecting their position in the hierarchy (Dixson,
1980). Also, treatment of low-ranking individuals with andro-
gens does not change their status. The intuitive conclusion
from these results is that testosterone does not affect aggression
in these species. However, there are several ways in which
aggressive behavior could be influenced by androgens indepen-
dent of baseline testosterone concentrations.
Acute hormonal responses to the environment can have
different effects on behavior than the baseline hormonal state
(Leshner, 1979). Winning aggressive encounters increases
male testosterone concentrations (‘challenge effect’) in birds
(Wingfield et al., 1990), fish (Oliveira et al., 2002), rodents
(Oyegible and Marler, 2005), nonhuman primates (Rose
et al., 1971), and humans (Mazur and Booth, 1998). Interest-
ingly, the increase in testosterone after a winning experience
is not observed unless the individual is in a familiar environ-
ment (Figure 2; Fuxjager et al., 2009). Initially, these rapid
and transient responses were puzzling because it was thought
that the effects of steroid hormones such as testosterone
required at least several hours for a behavioral effect to be
observed. It is now apparent, however, that these challenge
effects can influence behavior. Transient increases in testos-
terone may help crystallize the experience of winning an aggres-
sive encounter (Trainor et al., 2004). Several studies have
demonstrated that individuals that win aggressive encounters
are more likely to win future encounters (Parmigiani and Brain,
1983; Chase et al., 1994; Kudryavtseva, 2000), even when
variables such as intrinsic fighting ability are controlled
Home cage Unfamiliar cage
2000
testosterone (pg/ml)
1500
Post-encounter
1000
500
0
3 prior wins Handled controls
Figure 2 Postencounter levels of testosterone in focal individuals.
The bars represent mean hormone levels and the error bars represent
the standard error. The ) indicates significant differences between treat-
ment groups (p < 0.025). The data were log transformed for statistical
analysis. Reprinted from Fuxjager, M.J., Mast, G., Becker, E.A.,
Marler, C.A., 2009. The ‘home advantage’ is necessary for a full winner
effect and changes in post-encounter testosterone. Horm. Behav. 56,
214–219 with permission from Elsevier.
150 Hormones and the Development and Expression of Aggressive Behavior
(Oyegible and Marler, 2005). In addition, steroid hormones
are now known to exert nongenomic effects which can occur
within seconds or minutes (Vasudevan and Pfaff, 2006).
Injections of testosterone can act within minutes to reduce
anxiety-like behavior in mice (Aikey et al., 2002), and an acute
injection of estradiol (a testosterone metabolite) can increase
aggressive behavior in Peromyscus within 15 min (Nelson and
Trainor, 2007; Trainor et al., 2007a). Testosterone can be con-
verted to estradiol within the brain, a conversion mediated by
the aromatase enzyme. Environmental factors can rapidly
convert testosterone to estrogens in the brain and affect aggres-
sive behaviors in a number of contexts (reviewed in Laredo
et al., 2014b).
Steroid synthesis in the brain is not limited to the conver-
sion of androgens to estrogens by the aromatase enzyme.
Many of the enzymes required for de novo steroid synthesis
have been identified in the brain (Baulieu and Robel, 1990;
Young et al., 1996; Soma, 2006), raising the possibility that
the brain may be producing androgens independently of the
testes. Dehydroepiandrosterone (DHEA) is produced in the
adrenal gland but requires only two metabolic steps to convert
to testosterone (Demas et al., 2007). Studies in hamsters
(Demas et al., 2004; Figure 3) and song sparrows (Soma
et al., 2002) suggest that adrenal steroids may promote aggres-
sive behavior, especially under environmental conditions in
which gonadal testosterone secretion is low. Most studied
rodents have elevated aggression levels under short-day photo-
periods (Jasnow et al., 2000; Trainor et al., 2007b, 2008), an
effect that is mediated by extended periods of elevated
18
16
* behavior (for example, Matthews, 1979), the consensus is
14
Total duration of attacks (s)
12
10
8
6 activities and (2) androgens promote a constellation of traits,
4 seeking, that may be expressed either as antisocial or as proso-
2 background. To distinguish between these two possibilities,
0 beginning in 1985. Analyses of their psychological profiles
Sham ADx
Saline Melatonin
Figure 3 Mean (SEM) total duration of attacks (s) in hamsters that
received bilateral adrenalectomies (ADx) or sham operations (Sham)
and subsequently treated with either melatonin or control (Saline) injec-
tions. Significant differences between pairwise means are indicated by
an asterisk (*) if p < 0.05. Reprinted from Demas, G.E., Polacek, K.M.,
Durazzo, A., Jasnow, A.M., 2004. Adrenal hormones mediate
melatonin-induced increases in aggression in male Siberian hamsters
(Phodopus sungorus). Horm. Behav. 46:582–591 with permission from
Elsevier.
melatonin concentrations (Jasnow et al., 2002; Laredo et al.,
2014a). In hamsters, removal of the adrenal glands blocks
the aggression enhancing effects of melatonin (Figure 3).
This suggests that adrenal steroids are critical for sustaining
aggression outside of the breeding season when testosterone
concentrations are low. However, in female hamsters, mela-
tonin appears to work directly on adrenal function to elevate
DHEA. Short-day female hamsters are more aggressive than
their long-day counterparts; long-day hamsters provided with
short-day melatonin doses also displayed increased aggression
and elevated DHEA concentrations (Rendon et al., 2015).
Furthermore, melatonin increased DHEA secretion from
cultured adrenal glands. As noted above, testosterone some-
times exerts behavioral effects indirectly through its conversion
to estrogens.
Several studies of human aggression in which psychological
rating scales were used to quantify levels of aggressiveness or
hostility reported no relationship between blood or saliva
androgen concentrations and aggressiveness (Doering et al.,
1975; Monti et al., 1977; Persky et al., 1977). However, rela-
tionships between blood testosterone concentrations and
behavior have been reported among aggressive, violent, and
antisocial individuals, especially those incarcerated in prison
(Kreuz and Rose, 1972; Ehrenkranz et al., 1974). Prison
inmates with high circulating testosterone concentrations,
usually defined as the top 5% or 10% of the normal distribu-
tion, had committed violent crimes (Ehrenkranz et al., 1974;
Dabbs et al., 1987, 1988), were more unruly in prison, and
were judged more harshly by their parole boards (Dabbs
et al., 1987, 1988). High testosterone concentrations have
also been associated with male juvenile delinquency (Olweus,
1983). Although some studies of criminal populations show
no association between plasma testosterone and violent
that violence among prison inmates and blood androgen
concentrations are positively correlated. A similar relationship
was observed among female prison inmates (Dabbs and
Hargrove, 1997).
Two related hypotheses have been proposed to explain the
association between high androgen concentrations and human
antisocial behavior as observed in delinquent or criminal pop-
ulations: (1) androgens directly mediate the antisocial
including social dominance, competitiveness, and thrill-
cial behavior depending upon the individual’s resources and
a large sample of 4462 US military veterans was examined
and saliva concentrations of testosterone suggested that andro-
gens directly mediate antisocial behavior in human males,
although socioeconomic status has a small moderating effect
(Dabbs and Morris, 1990).
Few studies have addressed the role of androgens in aggres-
sive behavior in women; no consistent correlation between
androgen concentrations and aggressive behavior has been
reported for women (Persky et al., 1977; Dabbs et al., 1988;
Dabbs and Hargrove, 1997). However, subtle effects of andro-
gens may influence aggression in women. Saliva testosterone
concentrations did not differ between female prison inmates
 Hormones and the Development and Expression of Aggressive Behavior
and female college students. But further analyses discovered
that testosterone concentrations were highest in women pris-
oners convicted of unprovoked violent crimes and lowest in
women convicted of ‘defensive’ violent crimes, such as killing
abusive husbands (Dabbs et al., 1988).
1.05.3.1.2 Estrogens
Often considered to be primarily a female class of hormones,
estrogens have important effects on many male behaviors
including aggression. In most species that have been examined,
estrogens increase aggressive behavior. Blocking estrogen
production with an aromatase inhibitor reduces aggression in
Japanese quail (Schlinger and Callard, 1990) and song spar-
rows (Soma et al., 2000), whereas aromatase knockout mice
display low aggression levels in resident–intruder tests (Toda
et al., 2001; Matsumoto et al., 2003). In CFW and CF-1 strains
of mice, the negative effect of castration on aggression can be
reversed by treatment with estradiol (Simon and Whalen,
1986). The effects of estrogens on aggression can be rapid in
both mammals and birds (reviewed in Heimovics et al., 2015).
Estrogens can bind to one of at least two estrogen receptor
(ER) subtypes, a and b. Most of what is known about the effects
of these receptors on aggression comes from a series of studies
on knockout mice. Male mice with targeted disruption of ERa
display reduced aggression when tested with other males in
a number of testing situations (Ogawa et al., 1997; Scordalakes
and Rissman, 2003, 2004). Curiously, male ERa knockout
mice are more likely than wild-type mice to attack female
intruders. In male CD-1 mice, levels of aggression directed
toward other males are positively correlated with the number
of ERa immunopositive cells in the LAS, BNST, and AHA
(Trainor et al., 2006a). The deletion of ERb is generally associ-
ated with increased aggression (Ogawa et al., 1999; Nomura
et al., 2006), although this effect appears to be context depen-
dent (Nomura et al., 2002). Deletion of both receptors is asso-
ciated with increased male aggression (Ogawa et al., 2000). In
these knockout studies, the effects of ERa and ERb could be
organizational, activational, or both. Studies using ER-specific
ligands in adult animals have suggested that the directional
effects of these ERa and ERb may occur primarily during devel-
opment (see Section 1.05.7.1), although additional studies are
needed to test this hypothesis.
1.05.3.1.3 Glucocorticoids
The effects of glucocorticoids on aggression are also variable,
although the mechanistic basis for this is poorly understood.
Generally, chronic elevations in glucocorticoid concentrations
(usually associated with stress) inhibit aggressive behavior
(Leshner et al., 1980; Maestripieri et al., 1991; Summers
et al., 2005), whereas chronic deficiencies in glucocorticoid
secretion are associated with increased aggression (Haller
et al., 2001, 2004). Elevated baseline glucocorticoid concentra-
tions inhibit testosterone secretion (Viau, 2002), increase sensi-
tivity to serotonin (Meijer and de Kloet, 1998), and increase
glutamate neurotransmission in frontal cortex (Moghaddam,
2002). All of these physiological responses could contribute
to reduced aggressive behavior and appear to do so in animals
subjected to chronic social defeat (Haller, 2014). However, the
immediate effect of a transient increase in glucocorticoids on
aggression is quite different than a chronic increase in baseline
151
concentrations. Corticosterone acts rapidly to increase aggres-
sion in rats (Mikics et al., 2004), hamsters (Hayden-Hixson
and Ferris, 1991), and mice (Poole and Brain, 1974). The
increase in aggression due to acute elevated corticosterone is
particularly salient in challenge-situations, for example, when
confronted with unfamiliar opponents or other novel situa-
tions (Mikics et al., 2007). In rough-skinned newts (Taricha
granulosa) corticosterone acts rapidly to promote mating
behavior (Moore and Miller, 1984), an effect that has been
linked to nongenomic hormone action (Orchinik et al.,
1991). This raises the possibility that the effects of chronic
elevation of glucocorticoids on aggressive behavior are medi-
ated by changes in gene expression (via activation of mineralo-
corticoid and glucocorticoid receptors), whereas the effects of
a transient increases in glucocorticoids are mediated by nonge-
nomic responses of membrane receptors.
1.05.3.2 Anabolic Steroid Abuse and Aggression
The use of anabolic steroid hormones (such as testosterone) as
performance-enhancing drugs has become a high-profile topic
in the news media, especially as several elite athletes have been
disqualified or stripped of titles or medals. Anabolic steroids
are used because they stimulate the growth and development
of muscle tissue. Although anabolic steroids have certain ther-
apeutic uses (Ferrando and Wolfe, 2007), when used in excess
or abused they have many negative side effects such as infer-
tility, suppressed immune function, and increased risk of
cardiovascular, liver, and kidney disease (Bahrke and Yesalis,
2004; Bonetti et al., 2008). In addition there is accumulating
evidence that anabolic steroid abuse has adverse psychological
effects, including aggression. Surveys have indicated that
anabolic steroid abusers are more likely to engage in verbal
aggression, fighting, violence toward women, and risk-taking
behaviors (Choi and Pope, 1994; Pope and Katz, 1994;
Galligani et al., 1996). However, there are some inconsistencies
across studies, and study participants may not be forthcoming
about their usage, especially because anabolic steroids are out-
lawed in many countries (McGinnis, 2004). To address
whether many commonly abused anabolic steroids exert effects
on aggression, researchers have developed animal model
systems in which dosages and environmental variables are
controlled.
Studies in hamsters and rats have established that many
commonly abused anabolic steroids can influence aggressive
behaviors, although in some cases these effects depend on
testing conditions. Methyltestosterone, but not stanozolol
(a nonaromatizable androgen), increases male aggressive
behavior in castrated male rats compared to castrated rats
receiving oil injections (Clark and Barber, 1994). Other studies
in rats (Farrell and McGinnis, 2004) and mice (Martinez-
Sanchis et al., 1996) have also reported that stanozolol does
not increase aggression (Figure 4). Nandrolone is another
commonly abused androgen, but its effect on aggressive
behavior in rodents is variable. One study reported that nan-
drolone administration to male rats increased aggression
(Long et al., 1996), whereas two other studies observed that
nandrolone administration had no effect on aggressive
behavior in rats (McGinnis et al., 2002a,b). It has been hypoth-
esized that the effects of nandrolone on aggression may depend
152 Hormones and the Development and Expression of Aggressive Behavior
(a)
20
Intact opponent
Composite aggression score
15 Castrated opponent
10
5 over 3% of 12th-grade males in the United States report
* *
0
Test Nand Stan
Control
(b)
20
Home cage
Composite aggression score
Opponent cage
15 Neutral cage
* bites toward a male intruder compared with untreated males
10
5
* *
* ences by endogenous testosterone. Similarly, a mild provoca-
0
Test Nand Stan Control
Figure 4 Social discrimination is seen in aggression toward a gona-
dally intact versus a castrated male opponent (a). Environmental
discrimination is depicted in (b). Aggression is shown toward a gona-
dally intact male in the anabolic androgenic steroid (AAS) males’ home
cage, the opponents’ home cage, or a neutral cage. The rats were
treated with testosterone (Test), nandrolone (Nand), Stanozolol (Stan),
or vehicle (Control). )p < 0.05. Reprinted from Farrell, S.F., McGinnis,
M.Y., 2003. Effects of pubertal anabolic-androgenic steroid (AAS)
administration on reproductive and aggressive behaviors in male rats.
Behav. Neurosci. 117, 904–911, with permission from American
Psychological Association.
on experience or testing conditions (McGinnis, 2004). For
example, tail pinching can be used as a form of physical prov-
ocation (Miczek et al., 2004), and this can exaggerate the effects
of anabolic steroids on aggression (McGinnis et al., 2002a).
A further complicating factor is that many abusers of anabolic
androgens use more than one steroid simultaneously, a practice
also known as stacking (Trenton and Currier, 2005). Studies in
hamsters show that activation of 5-HT1B receptors blocks the
effects of an androgenic cocktail (testosterone cypionate, nor-
testosterone, and dihydroxytestosterone) on aggression in
a resident–intruder test (Grimes and Melloni, 2005). Although
stanozolol treatment alone reduced aggressive behavior in
male rats, stanozolol treatment with testosterone increased
aggression in aggression tests preceded by a tail pinch (Wesson
and McGinnis, 2006). Anabolic androgens appear to affect
aggressive behaviors by working at several biochemical and
neurobiological levels. In hamsters, anabolic androgen
administration increases arginine vasopressin (AVP) immuno-
reactivity (Grimes et al., 2007) and baseline c-fos immunoreac-
tivity (Ricci et al., 2007) in the anterior hypothalamus,
a forebrain region that facilitates male aggressive behavior.
There is also evidence that anabolic androgens can downregu-
late GABAergic neurotransmission, thereby facilitating aggres-
sive behavior (Henderson et al., 2006). The emerging picture
then is that anabolic androgens facilitate aggressive behaviors
by affecting several biochemical pathways and these neuro-
chemical changes are influenced by environmental factors.
Androgenic anabolic use is also a problem of adolescence.
According to the 2005 Monitoring the Future Survey,
having used anabolic androgens (http://www.nida.nih.gov/
ResearchReports/Steroids/AnabolicSteroids.html). Anecdot-
ally, anabolic steroid use by adolescents is associated with irri-
tability and heightened aggression, but a causal link has not
been established. On the other hand, animal studies have
provided compelling evidence for anabolic steroid-induced
aggression in adolescent males. Adolescent male hamsters
treated chronically with an anabolic steroid cocktail have
shorter attack latencies and a greater number of attacks and
(Melloni et al., 1997; Harrison et al., 2000), and anabolic
steroid-induced increases in aggression are more robust in
adolescent than in adults (Salas-Ramirez et al., 2008). These
effects are all the more striking considering that male–male
aggression in hamsters is not under strong activational influ-
tion (tail pinch) produces a persistent increase in aggression
in adolescent male rats treated with anabolic androgens,
including aggression toward females (Cunningham and
McGinnis, 2007). Importantly, adolescent exposure to
anabolic androgens causes long-lasting changes in agonistic
behavior (Grimes and Melloni, 2006), neurotransmitter
systems (Grimes and Melloni, 2006; Ricci et al., 2007), and
synaptic organization (Cunningham and McGinnis, 2007)
that persist even after the period of drug exposure. In light of
the evidence that endogenous testosterone organizes aggressive
behavior during puberty and adolescence, it seems likely that
anabolic steroid use during adolescence would result in larger
magnitude or more enduring effects on the brain and aggres-
sive behavior than use in adulthood. Taken together, the
evidence suggests significant aggression-promoting effects on
aggressive behaviors (e.g., Oberlander and Henderson, 2012).
1.05.4 Brain Regions Contributing to Aggression
A complicating factor in studying neural circuits affecting
aggression is that many brain nuclei that regulate aggression
also affect other social behaviors. For example, in rodents the
medial amygdala (MEA) is activated during both aggression
and reproductive behavior (Kollack-Walker and Newman,
1995; Choi et al., 2005). Although there are sometimes subtle
anatomical differences in activity as measured with immediate
early genes (Holt and Newman, 2004), more detailed analyses
will be necessary to sort out whether different cell types are acti-
vated in different contexts, or whether different cellular
responses result in different behavioral responses. Despite
this uncertainty, it is clear that a common set of hypothalamic
and limbic brain areas play a role in regulating some form of
aggressive behavior in a variety of species. Homologous brain
 Hormones and the Development and Expression of Aggressive Behavior 153

structures appear to regulate social behaviors, including aggres-
sion, in both mammalian and nonmammalian species (Gregg
and Siegel, 2001; Crews, 2003; Goodson, 2005). Aggression is
a primitive, yet highly conserved vertebrate behavior, and it is
reasonable to expect that the molecular mechanisms under-
lying aggression are also similar (and possibly ancient) among
vertebrates. Species-specific features of aggression are likely the
result of adaptive ‘co-opting’ of novel molecules as modulators
that are ‘added to’ the primary neural circuits. These findings
support the hypothesis that at least some neurobiological
and neurochemical mechanisms governing the motivation to
engage in aggressive behavior are evolutionarily conserved
(Scott, 1975). Thus, results from studies of aggression in
rodents and other nonprimate species should provide insight
on the motivational circuits regulating aggression in other
species, including humans.
1.05.4.1 Studies in Rodents
In rodents, sensory input from the olfactory bulbs (DaVanzo
et al., 1983) is sent to the MEA and then relayed to the bed
nucleus of the stria terminalis (BNST), medial preoptic area
(MPOA), lateral septum (LAS), anterior hypothalamus
(AHA), ventral medial hypothalamus (VMH), and the peria-
queductal gray region (PAG) (Wood and Newman, 1995;
Delville et al., 2000; Figure 5(a)). This pathway has been
referred to as a social behavior network (Newman, 1999;
Goodson, 2005), and it has also been hypothesized that
different subnuclei are more active in different contexts. For
example, the posteroventral MEA and dorsomedial VMH are
thought to be more important for regulating aggression in
defensive contexts, whereas the posterodorsal MEA and ventro-
lateral VMH are thought to be more important in offensive
contexts (Swanson, 2000). The components of this network
have been identified over the years through increasingly precise
experimental methods. The involvement of the LAS, BNST,
AHA, VMH, and MEA in aggression was first established
through lesion studies (Miczek et al., 1974; Annen and Fujita,
1985; Kruk, 1991; Figure 5(a)). Next, the use of electrical stim-
ulation of specific brain regions was found to induce aggressive
behavior in regions such as the AHA (Kruk et al., 1984; Kruk,
2014). Both lesions and electrical stimulation can activate
fibers of passage, and so local infusion of GABAergic or gluta-
matergic agonists and antagonists were used to examine local
cell bodies (Haller et al., 1989; Roeling et al., 1993). Optoge-
netic stimulation of the ventrolateral VMH was also found to
increase aggression in male mice (Lin et al., 2011). The applica-
tions of immunostaining for immediate early gene products
such as c-fos largely confirmed that engaging in aggressive
behavior increases the activity of neurons in the LAS, BNST,
AHA, and MEA in both males (Kollack-Walker and Newman,
1995; Delville et al., 2000) and females (Davis and Marler,
2004; Hasen and Gammie, 2005; Gammie et al., 2007).
More recently, immunostaining for phosphorylated proteins
such as the transcription factor CREB has proved to be a useful
complementary method for identifying changes in cellular
activity in the brain following aggressive encounters (Trainor
et al., 2010b; Heimovics et al., 2012). An important advantage
of immediate early gene studies is the ability to identify cell
types through multilabeling. For example subordinate male
mice had more AVP neurons in the caudal paraventricular
nucleus coexpressing fos compared to dominant mice (Ho
et al., 2010). Similarly both male and female California mice
had more AVP/c-fos-expressing cells in the caudal PVN
following a resident–intruder test if they had previously experi-
enced social defeat (Steinman et al., 2015). There is growing

(a) (b) Visual and auditory

social cues
Stress
Olfactory
social cues

OFC
Hippocampus
LAS
Olfactory OFC
bulb BNST BNST
PAG
AHA MeA
PVN
−
+
AHA PAG
MeA

Aggressive Aggressive
behavior behavior

Figure 5 Neuroanatomical pathways of aggression in rodents and nonhuman primates. In rodents (a), information from the olfactory bulb is pro-
cessed by the medial amygdala (MEA) and sent to the lateral septum (LAS), bed nucleus of the stria terminalis (BNST), and anterior hypothalamic
area (AHA). These brain areas are thought to prompt the periaqueductal gray (PAG) into promoting species-specific aggressive behaviors. Stress can
inhibit aggression via inhibitory inputs from the orbital frontal cortex (OFC), the hippocampus, and the paraventricular nucleus (PVN). In nonhuman
primates (b), aggression is typically evoked by vocal or visual signals. Activation of the MEA is thought to result in activation of the BNST and AHA,
which in turn activate the PAG. In general, the OFC appears to be important for the interpretation of social cues, and inhibitory inputs from the OFC
might inhibit aggression by reducing responsiveness in the amygdala. Thick arrows represent inputs and outputs to and from the brain; thin arrows
represent connections within the brain; dotted lines represent inhibitory connections. Reprinted from Nelson, R.J., Trainor, B.C., 2007 Neural mecha-
nisms of aggression. Nat. Rev. Neurosci. 8, 536–546 with permission from Nature.
154 Hormones and the Development and Expression of Aggressive Behavior
interest in understanding how specific cells types within the
social behavior network are activated under different social
contexts.
The development of viral gene transfer techniques has created
new approaches for studying neural circuits of aggressive
behavior. So far most studies have focused on the MEA and
VMH in mice. In one study aromatase-expressing neurons in
the MEA were inhibited using a designer receptor exclusively acti-
vated by designer drugs (DREADD) (Unger et al., 2015). Inhibi-
tion of MEA aromatase neurons in males increased the latency to
engage in aggression without affecting the overall frequency of
attacks or threats. In females inhibition of MEA aromatase
neurons increased the latency and decreased the frequency of
maternal aggression. The role of GABAergic and glutamatergic
neurons in the MEA has been examined using optical stimula-
tion. Activation of GABAergic neurons in the MEA was found
to induce male–male aggressive behaviors while activation of
glutamatergic neurons inhibited aggression (Hong et al., 2014).
In the ventrolateral VMH infusion of AAV expressing small
hairpin RNAs targeting ERa reduced male–male aggressive
behavior (Sano et al., 2013). Intriguingly optical stimulation of
ERa neurons increased male aggression directed toward both
male and female intruders (Lee et al., 2014). Overall the results
of experiments using modern genetic approaches are largely
consistent with original reports while adding a new layer of
understanding of how specific cell types control behavior.
1.05.4.2 Nonhuman Primates and Humans
As in rodents, the hypothalamus seems to play a key role in
regulating aggression in nonhuman primates (Figure 5(b)).
Electrical stimulation of the ventromedial hypothalamus
increases vocal threats and piloerection in male marmosets,
Callithrix jacchus (Lipp and Hunsperger, 1978). Similarly,
lesions of the AHA and POA reduce vocal threats toward an
intruder in male C. jacchus (Dixson and Lloyd, 1988). In rhesus
monkeys (Macaca mulatta), electrical stimulation of the AHA,
BNST, or POA increases the frequency of aggressive vocaliza-
tions (Robinson, 1967) and aggression toward subordinate
males (Alexander and Perachio, 1973).
Other studies have focused on the amygdala and orbital
frontal cortex (OFC). Lesions of the amygdala either increase
(Machado and Bachevalier, 2006) or decrease intermale aggres-
sion (Emery et al., 2001) in rhesus monkeys. One explanation
Right
1.2
Prefrontal/whole brain
1.15
1.1
1.05
1 1
0.95
0.9
0.85
Nml Vlt
Figure 6 PET scan indicating brain changes in violent people. Individual values for relative metabolism in right and left prefrontal and medial
temporal cortex of adult control volunteers (Nml) and violent patients (Vlt). Reprinted from Volkow, N.D., Tancredi, L.R., Grant, C., Gillespie, H.,
Valentine, A., Mullani, N., Wang, G.J., Hollister, L., 1995. Brain glucose metabolism in violent psychiatry patients: a preliminary study. Psychiatry
Res. 61, 243–253, with permission from Elsevier.
for these conflicting results is that studies that reported
increased aggression reintroduced lesioned monkeys into
groups, whereas studies that reported decreased aggression
tested monkeys in groups of two (Emery et al., 2001), which
might be less threatening. Lesions of the OFC are generally
associated with reduced affiliative behavior such as grooming
or close contact (Butter et al., 1970; Machado and Bachevalier,
2006), whereas their effects on aggressive behavior depend on
context. For example, OFC lesions produce increased aggres-
sion in dominant, but not subordinate males (Machado and
Bachevalier, 2006). In a different study, OFC lesions in domi-
nant animals led to an initial increase in aggression that disap-
peared after several months (Butter and Snyder, 1972). In
general, it seems that the OFC is important for the interpreta-
tion of social cues and contributes to appropriate behavioral
responses in complex social situations.
A creative study used positron emission tomographic
imaging to examine brain activity in rhesus macaques in the
context of mate competition (Rilling et al., 2004). Dominant
male monkeys witnessed a potential sexual interaction between
a female they had been previously paired with and a subordi-
nate male. This mate competition ‘challenge’ condition was
designed to model aspects of jealousy in humans. Males
exposed to this ‘challenge’ condition showed increased activa-
tion in the right amygdala and right superior temporal sulcus
compared to males exposed to the control condition in which
the subordinate male was absent. Interestingly, similar results
were observed in a functional MRI study on human partici-
pants. Brain activation was increased in the amygdala and
hypothalamus when men read sentences depicting sexual infi-
delity compared to neutral sentences, whereas women showed
increased activation in the posterior superior temporal sulcus
in the same comparison (Takahashi et al., 2006). Other studies
suggest that the superior temporal sulcus is activated when
assessing deception (Calarge et al., 2003), trustworthiness
(Winston et al., 2002), and violation of social norms (Greene
et al., 2001). Thus, it appears that there are at least qualitative
similarities between human and nonhuman primate circuitries
that function during mate competition.
A more direct link between brain activation in humans and
aggression was observed in imaging studies that reported an
inverse relationship between average baseline activity in the
frontal cortex and measures of reactive aggression (Raine
et al., 1994; Volkow et al., 1995; Soderstrom et al., 2000;
Left Right Left
1.2
Med-temp/whole brain
1.15
1.1
1.05
0.95
0.9
0.85
Nml Vlt Nml Vlt Nml Vlt
 Hormones and the Development and Expression of Aggressive Behavior 155

Soloff et al., 2003; Figure 6). The frontal cortex provides inhib-
itory inputs to circuits in the hypothalamus and amygdala that
might promote aggression (Davidson et al., 2000), although
the role of these brain areas remains less well-established in
humans than in other animals. In one study, individuals that
had been diagnosed with intermittent explosive disorder
increased activation in the amygdala in response to angry faces
when compared to control participants, and amygdala activa-
tion across both groups was positively correlated with scores
on the Lifetime History of Aggression (LHA) scale (Coccaro
et al., 2007). Insights into brain areas that affect human aggres-
sive behavior also come from observing the behavioral effects
of brain injuries. Many studies have reported a link between
brain damage to the frontal cortex and increased aggressive
behavior (Grafman et al., 1996; Anderson et al., 1999). Brain
injury rarely causes selective damage to the hypothalamus or
amygdala. However, during a grim period in the mid-twentieth
century, electrolytic lesions of these brain regions were used to
treat what was deemed ‘excessive aggression’ (Heimburger
et al., 1966; Sasano et al., 1998). Although lesions of the hypo- Figure 7 Inverse correlation between the binding potential in the
thalamus and amygdala were reported to inhibit aggression, orbital prefrontal cortex and lifetime aggression score as assessed by
these conclusions are limited. Measurements of behavior in the Brown–Goodwin Scale. Open circles are males, filled circles are
these studies were usually crude and fail to account for the females. R ¼ 0.53, p ¼ 0.007 for the combined data. Reprinted from
complexities of human behavior (Blair, 2004; Cherek et al., Parsey, R.V., Oquendo, M.A., Simpson, N.R., Ogden, R.T., Van Heer-
2006; Scarpa and Raine, 2006; Trainor et al., 2006b). Addition- tum, R., Arango, V., Mann, J.J., 2002. Effects of sex, age, and aggres-
ally, electrolytic lesions damage fibers of passage as well as the sive traits in men on brain serotonin 5-HT1A receptor binding potential
target nuclei, and damage to the hypothalamus and amygdala measured by PET using [C-11]WAY-100635. Brain Res. 954, 173–182
with permission from Elsevier.
affect general arousal (Tonkonogy and Geller, 1992), not just
aggression. An intriguing experimental approach was devel-
oped by Carré and colleagues to examine the effects of testos-
terone on neural activation in response to threat-related aggression (Winslow and Insel, 1991; DeVries et al., 1997;
stimuli (Goetz et al., 2014). Healthy men were given a gonado- Winslow et al., 2000) and that oxytocin receptors are abundant
tropin-releasing hormone antagonist, which normalized T in the amygdala (Insel, 1992; Jiménez et al., 2015).
concentrations to low baseline levels. Then men were Thus oxytocin might reduce human aggressive responses in
randomly assigned to be treated with T or placebo and tested some contexts, although this should be tested pharmacologi-
in a face-matching task while undergoing fMRI. Men treated cally. These studies show that combining biochemical manipu-
with T had greater increases in reactivity in the amygdala and lations with a realistic social context can allow investigators to
hypothalamus to angry faces compared to men treated with ethically test hypotheses that been developed using animal
placebo. Several studies have taken an integrative approach to models.
studying the neurobiological circuits that influence aggression.
Previous studies reported reduced activation of the prefrontal
cortex in patients who were rated highly for impulsive 1.05.5 Neurotransmitters, Hormones, and
aggression and also showed that selective serotonin reuptake Aggression
inhibitors (SSRIs) reduced ratings of aggression (Coccaro and
1.05.5.1 Serotonin
Kavoussi, 1997); the effect of SSRIs on prefrontal cortex
(PFC) activity was examined in patients who had been Serotonin is the neurotransmitter most closely associated with
diagnosed with borderline personality disorder (these patients regulation of aggressive behavior. Nonetheless, there is no
score highly on measures of impulsive aggression). Twelve simple relationship between serotonin levels and aggressive
weeks of SSRI treatment increased baseline activation in the behavior overall (Takahashi et al., 2011). More granular anal-
PFC, and PFC activation was negatively correlated with ratings yses have established both subtle and not so subtle relation-
of aggression (New et al., 2004). In addition, positron emission ships between serotonin and aggression. For example, it is
tomography (PET) imaging studies using a selective serotonin now generally accepted that 5-HT1A and 5-HT1B receptor inhi-
receptor type 1A (5-HT1A) antagonist showed that scores on bition can reduce aggression (Morrison and Melloni, 2014),
the Lifetime History of Aggression (LHA) test were negatively although sex differences in 5-HT1A receptor activation have
correlated with 5-HT1A binding in the amygdala and been observed (Joppa et al., 1997). Rats display rapidly elicited
PFC (Parsey et al., 2002; Figure 7). Intranasal administration aggressive responses when an intruder is introduced into the
of the neuropeptide hormone oxytocin to human participants home cage and treatment with the 5-HT1A receptor agonist
reduced activation of the amygdala in response to (8-OH-DPAT) decreases offensive aggressive behaviors in resi-
fear-inducing pictures (e.g., sharks, snakes) (Kirsch et al., dent males (De Boer et al., 1999; De Boer and Koolhaas,
2005). Studies in animals indicate that oxytocin can reduce 2005). However, these drugs have no effect on aggression
156 Hormones and the Development and Expression of Aggressive Behavior
when administered to intruders. Additionally, highly aggressive
male mice with depleted CNS 5-HT ameliorate agonistic
responses after 8-OH-DPAT treatment suggesting further that
the 5-HT1A receptors inhibit aggressive responses.
Generally, activation of the 5-HT system dampens aggres-
sion in animals and violent behavior in humans. Impulsivity
and high aggressiveness are correlated with low cerebrospinal
fluid concentrations of 5-HT metabolite, 5-HIAA, in humans
and nonhuman primates, and reduced 5-HT levels or turnover
in the brain of laboratory animals (reviewed in Lesch and
Merschdorf, 2000). Pharmacological strategies of increasing
5-HT levels such as the use of 5-HT precursors, 5-HT reuptake
inhibitors, as well as 5-HT1A and 5-HT1B receptor agonists are
able to reduce aggressive behavior in rodents (reviewed in
Manuck et al., 2006). Nonetheless, in models of excessive,
proactive aggression, 5-HT is unable to modulate the abnormal
behavioral responses (Haller, 2013). This suggests that seroto-
nergic mechanisms are more important for species-typical
aggressive behaviors rather than models of abnormal or violent
aggressive behavior.
Genetic evidence for a role of 5-HT in aggression comes
from mice missing specific genes that either directly or indi-
rectly affect 5-HT concentrations or metabolism. The 5-HT1B
receptor is expressed in a variety of brain regions, including
the basal ganglia, periaqueductal gray, hippocampus, lateral
septum, and raphe nuclei, either presynaptically inhibiting
5-HT release or as a heteroreceptor modulating the release of
other neurotransmitters (Bibancos et al., 2007). Male mice
that lack functional expression of the 5-HT1B receptor gene
(5-HT1B /) are more aggressive than wild-type mice (Saudou
et al., 1994). Lactating female 5-HT1B / mice also attack unfa-
miliar male mice more rapidly and violently than wild-type
females (Ramboz et al., 1996). Notably, administration of
the nonselective 5-HT1B agonist eltoprazine (one of the so-
called ‘serenics’) significantly reduces aggressive behavior in
both 5-HT1B knockout mice and WT mice, presumably by
affecting 5-HT1A receptors (Ramboz et al., 1996). Although
the 5-HT1B receptor contributes to aggression, these results
suggest that the 5-HT1B receptor subtype is not the sole 5-HT
receptor modulating aggressive behavior. Specifically, 5-HT1A
receptor activation, which is also induced by eltoprazine, can
also influence aggressive behaviors. Although both 5-HT1A
and 5-HT1B receptors control the tone of 5-HT system, it seems
likely that these two receptors contribute differently in partic-
ular brain areas modulating the postsynaptic 5-HT inhibitory
effects on aggression (Bibancos et al., 2007). The role of other
5-HT receptor subtypes on aggression remains unspecified, but
these receptors likely play a minor role (Montoya et al., 2012).
Long-term treatment with selective serotonin reuptake
inhibitors (SSRIs) reduces aggression in many species ranging
from fish to primates (reviewed in Morrison and Melloni,
2014). Acute SSRI treatment blocks the pro-aggressive effects
of several agents such as synthetic anabolic androgenic steroids
(AAS), testosterone, alcohol, and apomorphine (Morrison and
Melloni, 2014). Taken together, the evidence suggests that sero-
tonin in the CNS tends to decrease aggressive behaviors
(Takahashi and Miczek, 2014). However, a recent meta-
analysis of the serotonin–aggression relationship in humans
only revealed a small effect size between central 5-HT and
aggression, hostility, and anger (Duke et al., 2013).
Androgens, either acting directly or via estrogenic metabo-
lites, tend to facilitate aggression, whereas increased CNS
5-HT tends to inhibit aggression. Androgens interact with
5-HT in several ways to influence aggression. For example, peri-
natal exposure influences the expression and distribution of 5-
HT receptor subtypes (Simon et al., 1998; Sumner and Fink,
1998; Cologer-Clifford et al., 1999). Either testosterone or
estradiol elevates 5-HT2A receptor mRNA expression and
binding site densities in male rat brains (Ferrari et al., 1999).
Importantly, both androgens and estrogens modulate 5-HT1A
and 5-HT1B receptor agonist effects on murine aggression
(Simon, 2002). Thus, sex steroid hormones and 5-HT interact
on several levels to influence the likelihood of aggression.
Several other classical neurotransmitters have also been
linked to aggression. The most commonly related neurotrans-
mitters associated with aggressive behaviors are discussed
below.
1.05.5.2 Arginine Vasopressin
AVP is another hormone that plays a critical role in aggression
and other social behaviors (Goodson and Bass, 2001; Ferris,
2006). AVP produced in the anterior hypothalamus (AH) can
modulate aggression through its effects on AVP V1a receptors,
5-HT, and the serotonin receptors 5-HT1A and 5-HT1B. Treat-
ment of male hamsters with a V1A antagonist increases attack
latencies and decreases frequency of biting in a standard
resident–intruder test (Ferris et al., 2006). Interestingly, V1a
antagonist infusions into the AH of female hamsters increased
aggression (Gutzler et al., 2010). Somewhat consistent with
this effect, V1a antagonist infused into the lateral ventricle
also enhanced maternal aggression in rat dams (Nephew and
Bridges, 2008). These findings indicate that the V1a receptor
has very different effects on aggression in males and females.
Microinjections of AVP into the AH in combination with
5-HT1A or 5-HT1B receptor agonists revealed that only the
5-HT1A receptor activation inhibited AVP-facilitated
aggression (Ferris, 2006). 5-HT neurons project into the AH,
and 5-HT appears to inhibit AVP-facilitated offensive
aggression by activating 5-HT1A receptors (Ferris, 2006).
Several drugs that affect both AVP and the 5-HT systems
reportedly reduce aggression. For example, systemic treatment
of humans with nicotine increases blood AVP concentrations
and central 5-HT release while reducing aggression (reviewed
in Morrison and Melloni, 2014). It appears that development
can impact the effects of V1a receptors on aggression. V1A
knockout mice have normal levels of aggression after
isolation, whereas isolation of V1B knockout mice reduces
aggression (Wersinger et al., 2002; Morrison and Melloni,
2014). This suggests that genetic deletion of V1a results in
a compensatory developmental effect in aggression circuits,
possibly a greater role for V1b receptor.
1.05.5.3 Monoamine Oxidase
Metabolic enzymes such as monoamine oxidase A (MAOA)
also influence aggression because they function to alter neuro-
transmitter concentrations. MAOA is predominantly located in
catecholaminergic neurons in the brain, but MAOA catalyzes
with high affinity the oxidative deamination of 5-HT,
 Hormones and the Development and Expression of Aggressive Behavior
norepinephrine, and dopamine (Shih et al., 1999). Although
MAOA deficiency due to a point mutation in its coding gene
is correlated with impulsive aggression in several males from
a single Dutch family (Brunner et al., 1993), humans treated
with pharmacological MAO inhibitors for depression generally
display no change in impulsivity or aggression (Manuck et al.,
2006; Buckholtz and Meyer-Lindenberg, 2008). Substantial
work has related low MAOA expression with genetic factors
to influence aggressive behaviors in humans (reviewed in
Dorfman et al., 2014).
MAOA knockout mice display markedly increased aggres-
sive behaviors (Cases et al., 1995; Godar et al., 2011). Ablation
of the MAOA gene in mice leads to high levels of offensive
aggression despite elevated 5-HT concentrations (Cases et al.,
1995); the metabolic disturbances caused by MAOA deficiency
throughout life likely account for the effects on aggression.
Notably, the elevated aggression in humans and mice with
MAOA gene disruption mostly affects males (Manuck et al.,
2000; Buckholtz and Meyer-Lindenberg, 2008). MAOA activity
is directly regulated by estrogen (Chakravorty and Halbreich,
1997), which may be mediated by hormone receptor response
elements present in the MAOA promoter (Ou et al., 2006). In
humans, environmental risk factors interact with the MAOA
to produce elevated risk for aggression (Kim-Cohen et al.,
2006). Interestingly, low MAOA gene expression interacts
with prenatal cigarette exposure to result in elevated conduct
disorder diagnoses later in life (Wakschlag et al., 2010).
1.05.5.4 Nitric Oxide
Nitric oxide (NO) was initially identified as an endogenous
regulator of blood vessel tone, but is now recognized as
a neurotransmitter in both the central and the peripheral
nervous systems (Baranano and Snyder, 2001). Male mice
with targeted deletion of the gene encoding the neuronal
version of NOS (nNOS/ or NOS1/) displayed three to
four times more aggressive behaviors than wild-type mice in
the intruder–resident test (Nelson et al., 1995; Trainor et al.,
2007c; Bedrosian and Nelson, 2014). Nearly 90% of the aggres-
sive encounters were initiated by the nNOS/ animals. In all
test situations, male nNOS/ mice rarely displayed submissive
behaviors (Nelson et al., 1995). Behavioral studies of mice with
targeted deletion of specific genes suffer from the criticism that
the gene product is not only missing during the testing period,
but also missing throughout ontogeny when critical develop-
mental processes, including activation of compensatory mech-
anisms, could be affected (Nelson et al., 1997). Furthermore,
differences in genetic background might also contribute to
the observed changes in behavior of knockout mice (Wolfer
et al., 2002). To address these criticisms, mice were treated
with 7-nitroindazole (7-NI) to specifically inhibit nNOS
formation in vivo (Demas et al., 1997). Isolated mice treated
with 7-NI displayed substantially increased aggression in two
different tests of aggression compared to control animals
(Demas et al., 1997). The combination of the traditional phar-
macological approach and a targeted gene disruption approach
to studies of aggression enhances the strengths and minimizes
the weaknesses of each single approach.
Plasma androgen concentrations in mice do not differ
between wild types and nNOS/ either before or after
157
aggressive interactions (Nelson et al., 1995). However, cas-
trated nNOS/ mice indicate that testosterone is necessary, if
not sufficient, to maintain elevated aggression in these
knockout mice (Kriegsfeld et al., 1997). Androgen replacement
therapy restored the elevated levels of aggression to precastra-
tion levels in both nNOS/ and wild-type mice.
Serotonin function was hypothesized to be disrupted in the
aggressive nNOS/ mice because of the inverse relation of
5-HT system activity and aggression. Serotonin metabolism
was reduced in several brain regions involved in aggression
(Chiavegatto et al., 2001). Changes in 5-HT turnover were
due to increased concentrations of 5-HT with no changes in
its metabolite in most brain regions studied (Figure 8). The
disturbed neurochemical profile appears specific to the 5-HT
system, because norepinephrine, dopamine, and metabolites
were generally unaffected. As noted, monoamine oxidase has
been implicated in aggression; however, the relatively normal
norepinephrine and dopamine values suggest that it is unlikely
that alterations in monoamine oxidase account for the 5-HT
abnormalities in the nNOS knockout mice (Chiavegatto
et al., 2001).
Gonadal hormones directly influence the expression of
nNOS in many regions within the hypothalamus and limbic
system (Panzica et al., 2006). The effects of sex steroid
hormones have primarily been achieved after medium- or
long-term treatments. However, significant changes occur in
particular physiological conditions, for example, during the
estrous cycle. Changes are not uniform throughout the brain,
but vary in specific directions in different populations of
neurons (Panzica et al., 2006). Estradiol treatment seems
to increase nNOS activity in the ventrolateral nucleus of
guinea pigs (Warembourg et al., 1999) and in PVN (Sanchez
et al., 1998) and MPA (Okamura et al., 1994) of rats. These
discrepancies in the effects of gonadal hormones on nNOS
60 5-HT *
5-HIAA
Change from WT values (%)
5-HIAA/5-HT
40
*
* *
20 *
0
−20
* * *
†
−40
Cor Hyp Hipp Amy Mid Cer
Figure 8 Serotonin (5-HT), 5-HIAA, and 5-HT turnover (5-HIAA/5-HT)
ratios are reduced in mice lacking the gene for nNOS. Determination of
5-HIAA/5-HT ratio was made by HPLC in the cerebral cortex (Cor),
hypothalamus (Hyp), hippocampus (Hipp), amygdala (Amy), midbrain
(Mid), and cerebellum (Cer) of nNOS/ as compared to WT mice. Data
are percent change in relation to WT mice SEM; )p < 0.05. Reproduced
from Chiavegatto, S., Dawson, V.L., Mamounas, L.A., Koliatsos, V.E.,
Dawson, T.M., Nelson, R.J., 2001. Brain serotonin dysfunction
accounts for aggression in male mice lacking neuronal nitric oxide syn-
thase. Proc. Natl. Acad. Sci. U.S.A. 98, 1277–1281 with permission
from the National Academy of Sciences.
158 Hormones and the Development and Expression of Aggressive Behavior
could reflect a combination of factors including species
differences, methodology, regional specificity, or assays of
mRNA compared to protein (Panzica et al., 2006). Further
work is necessary to understand the relationship among sex
steroid hormones, 5-HT, and NO in mediating aggression.
Additional work is necessary to understand the relationship
among sex steroid hormones, 5-HT, and NO in mediating
aggression.
Interestingly the effects of nNOS on aggression are mediated
by the social environment. Initial studies examining effects of
nNOS inactivation on aggression were conducted on singly
housed males. Effects of nNOS inactivation through gene
knockout or inhibition with 3BrN were blunted in males that
were housed in pairs (Trainor et al., 2007c). Thus the effects
of nNOS on aggression are dependent on the social environ-
ment. Social isolation decreases 5-HT1A receptors (Rilke et al.,
1998) and increases V1a receptors (Albers et al., 2006), which
could potentially mediate the impact of nNOS on aggression.
Interestingly nNOS inactivation reduced motivation to
approach unfamiliar individuals or social odors. The combina-
tion of increased aggression and reduced social interaction has
also been observed in rhesus monkeys with the low activity
form of the serotonin transporter promoter and raised in
peer-only groups. In monkeys raised by their mothers, no
differences between serotonin transporter genotypes were
observed (Barr et al., 2003).
Two recent studies implicate a role of NO in human aggres-
sion as well. For example, a NOS1 promoter repeat length vari-
ation (NOS1 Ex1f variable number tandem repeat (VNTR))
was identified, and the short repeat variant is associated more
frequently with adult ADHD, personality disorder, and aggres-
sion. The short variants lead to reduced transcriptional activity
of the NOS1 exon 1f promoter (Reif et al., 2009). Adverse envi-
ronmental stressors can interact with the short variant to
promote elevated impulsive behaviors, including impulsive
aggressive behavior, in children aged 9–18 (Reif et al., 2011).
Taken together, these studies suggest a role of NO in mediation
of human impulsive aggression.
1.05.6 Development of Aggression
1.05.6.1 Rough-and-Tumble Play as an Antecedent
to Aggressive Behavior
Juveniles of most species engage in agonistic behaviors that at
least superficially resemble adult aggression. These behaviors
are referred to as rough-and-tumble play or play fighting. In
common with adult aggression, juvenile rough-and-tumble
play comprises both offensive and defensive maneuvers in
which animals attack, bite, pin, wrestle, roll over, and flee.
Unlike adult aggression, juvenile rough-and-tumble play does
not involve competition for resources, territory, or mates. In
most species, including humans, juvenile males engage in
more rough-and-tumble play than females. Rough-and-tumble
play predominates during social interactions in prepubertal
and juvenile animals and gradually declines over the course
of pubertal maturation. In general, overt aggression is relatively
uncommon prepubertally and increases concomitantly with
reproductive maturation and the associated rise in circulating
concentrations of gonadal steroids.
The gradual replacement of play fighting by ‘serious’
fighting over the course of ontogeny, coupled with the male
bias in rough-and-tumble play, invites the conclusion that
play fighting and adult fighting are a developmental
continuum in which play fighting is the immature form of
adult aggression. However, based on several lines of evidence
gleaned primarily from studies in rats and hamsters, Pellis
and colleagues contend that play fighting and adult aggression
are distinct behaviors, and that play fighting is not practice for
adult fighting skills, but instead is practice for general social
skills and competence (Pellis and Pellis, 1988, 1997, 1998,
2007). First, play fighting and adult fighting have different
topographies. In play fighting, attacks are initiated toward the
head and nape, whereas in serious fighting, attacks are initiated
toward the rump (Pellis and Pellis, 1988; Wommack et al.,
2003; Taravosh-Lahn and Delville, 2004). Defensive maneu-
vers during play and adult fighting differ as well. To evade an
attack, juvenile male rats rotate their bodies fully to a supine
position, but adults rotate only partially so that their hind
feet remain on the ground (Pellis, 2002). Play fighting is also
characterized by frequent role reversals; for example, the chasee
may suddenly turn around and become the chaser, or a larger
animal may voluntarily self-handicap, letting a smaller animal
pin or chase the larger one (reviewed in Pellis and Pellis, 2007).
Such role reversals are uncommon in adult aggression. Further-
more, infant (preweaning) rats display adultlike defensive
tactics, which are then replaced by the juvenile tactics (Pellis
and Pellis, 1997). Thus, it does not appear that the specific
motor patterns of juvenile play fighting are immature or
simpler forms of adult fighting. Second, although the frequency
of play fighting decreases over the course of pubertal matura-
tion, play fighting is not unique to the juvenile period, and
both play fighting and adult fighting can and do occur in adult-
hood (Pellis and Pellis, 1988). When play fighting occurs
among adult animals, however, it is more likely to escalate to
adult fighting, presumably because the adults have decreased
tolerance for one another (Pellis and Pellis, 1988). Third,
play fighting and adult fighting appear to have opposite
valences. In anticipation of play and during play, rats emit
50 kHz ultrasonic vocalizations, which are associated with
rewarding stimuli and positive social affect. In contrast, during
threatening situations, including intermale fighting, rats emit
22 kHz vocalizations, which are associated with aversive
stimuli and negative social affect (Knutson et al., 1998;
reviewed in Knutson et al., 2002). Thus, play fighting and adult
aggression appear to involve different psychological states.
Fourth, neurochemical correlates of male rat juvenile play
and adult aggression are not identical. Specifically, juvenile
play is associated with a decrease in hypothalamic levels of
cholecystokinin (CCK), whereas submission during adult
aggressive encounters is not (Burgdorf et al., 2006). This
finding supports the notion that juvenile play has positive
valence in light of the fact that elevated levels of CCK in cortex
are associated with submissive behavior during adult aggres-
sion and negative affective states (Knutson et al., 2002;
Panksepp et al., 2004). Finally, opportunities to engage in
rough-and-tumble play during the juvenile period appear to
facilitate development of social skills and competency and, if
anything, lead to reduced overt aggressive encounters in adult-
hood (Pellis and Pellis, 2007; Cooke and Shukla, 2011;
 Hormones and the Development and Expression of Aggressive Behavior
Panksepp and Scott, 2012). For example, play-deprived adult
rats do not appear to have learned the behavioral strategies
that signal submission to a dominant rat, and this lack of social
competency only invites more aggression (Von Frijtag et al.,
2002). Together, these lines of evidence do not support the
idea that play fighting is a practice for adult aggression.
The relationship between play fighting and adult aggression
is viewed differently by Delville and colleagues, who maintain
that they are the same behaviors expressed during different
stages of development (Delville et al., 2003; Cervantes et al.,
2007; Wommack and Delville, 2007). Based on their extensive
studies on the development of aggression in Syrian hamsters,
they argue that play fighting attacks are similar to adult attacks
in intent, even though the body part that is the target of the
attack is different at the two ages (head vs rear), because both
juveniles and adults flank mark during agonistic interactions
as a means of communicating dominant/subordinate status.
Furthermore, because the selective serotonin reuptake inhibitor
fluoxetine inhibits both juvenile play fighting and adult aggres-
sion, there appears to be a common underlying neurobiology
(Delville et al., 2003). The key to resolving these opposing
viewpoints about whether play fighting is an immature form
of adult aggression may lie in the different methodologies
used to evaluate agonistic interactions. Pellis and colleagues
have studied play fighting almost exclusively among group-
housed siblings or familiar males in familiar environments,
whereas Delville and colleagues have studied play fighting
almost exclusively using a resident–intruder paradigm in which
the resident has been socially isolated since weaning and the
intruder is a younger and smaller animal. The latter conditions
create competition and favor aggressive responses by an advan-
taged resident. Therefore, agonistic interactions between juve-
nile males under these circumstances may in fact be adultlike
aggression in defense of territory, despite the animal’s young
age and immature reproductive status, and different topog-
raphy of aggressive behavior. Thus, the distinction between
play fighting and adult fighting may not rest so much on the
age of the animal as it does on whether or not stakes are
involved.
As a case in point, sibling rivalry between spotted hyena cub
twins involves overt aggression and can result in siblicide
(Frank et al., 1991; Wahaj et al., 2007). Sibling aggression
within the first year of life in hyenas establishes a rank relation-
ship within the litter and is primarily over competition for milk
and food. Sibling aggression is more intense when local prey is
scarce and tends to be higher within litters of low-ranking
females, who are disadvantaged for access to resources within
the clan (Wahaj and Holekamp, 2006). Thus, siblicide in
hyenas is not obligate, as once proposed, but instead is rela-
tively uncommon and facultative, occurring when maternal
resources are insufficient to sustain two cubs (Smale et al.,
1999). This example reinforces the idea that the distinction
between play fighting and aggression is not age per se, but
rather whether competition for resources is involved.
1.05.6.2 Endocrine Contributions to the Development of
Aggressive Behavior: Perinatal Organizational Effects
Given that levels of both play fighting and adult aggression are
higher in males than in females of most species, numerous
159
investigations have examined the role of gonadal steroids in
the sexual differentiation of these behaviors. Overall, sexual
differentiation of play fighting conforms to the classical model
in which the presence of testosterone prenatally (nonhuman
primates) or during the first few days after birth (rodents)
masculinizes play fighting, and in the absence of testosterone
or in the presence or absence of the ovaries, a female-typical
level of play fighting is observed (reviewed in Wallen, 1996;
Pellis, 2002; Cooke and Shukla, 2011). In addition, human
females with congenital adrenal hyperplasia, who experience
relatively high levels of adrenal androgens in utero, display
higher levels of rough-and-tumble play and ‘tomboyism’ relative
to unaffected siblings (reviewed in Collaer and Hines, 1995).
Perinatal masculinization of play behavior most likely
involves activation of both androgen and estrogen receptors
in the nervous system. The androgen receptor blocker fluta-
mide disrupts masculinization of play behavior, either when
given to rat dams during the last half of pregnancy (Casto
et al., 2003) or when given to male pups over the first
10 days of life (Meaney et al., 1983). Other experiments
provide evidence that perinatal estrogen receptor activation
also contributes to the masculinization of play behavior. Two
of these experiments investigated play fighting in tfm rats, in
which a mutation in the androgen receptor gene renders the
receptor protein nonfunctional and the rats androgen-
insensitive. Therefore, effects of testosterone, which is synthe-
sized and secreted by Tfm rats, are presumably due to estrogen
receptor activation after aromatization of testosterone to estra-
diol. The two investigations of play fighting in tfm rats are
somewhat contradictory. One of them reports similar levels
of play fighting in tfm and wild-type male rats, suggesting
that estrogen receptor-mediated mechanisms are sufficient to
masculinize the behavior (Field et al., 2006). The other one
reports that levels of play fighting are less in tfm than in wild-
type males, supporting a role for androgen receptor-mediated
mechanisms (Meaney et al., 1983). However, play behavior
was measured in different social contexts in the two studies,
and while this may make the results not directly comparable,
together they implicate both androgenic and estrogenic action
in the sexual differentiation of play behavior.
Hormone-mediated masculinization of play behavior may
involve pathways apart from the classical mechanism of
hormone activation of its cognate receptor. The first of these
is ligand-independent activation of the estrogen receptor.
Administration of a dopamine D1 receptor agonist to neonatal
female rats masculinizes their play fighting behavior, and this
effect can be blocked by prior treatment with the estrogen
receptor antagonist tamoxifen (Olesen et al., 2005). The second
of these involves epigenetic regulation of gene transcription,
specifically DNA methylation and repression of gene expres-
sion in the amygdala. Testosterone delivered directly to the
amygdala of neonatal female rats is sufficient to induce male-
typical levels of play fighting (Meaney and McEwen, 1986),
suggesting that testosterone or a biologically active metabolite
acts within the amygdala to organize sex differences in play
behavior. If the methyl-binding protein MeCP2 is silenced
within the amygdala of newborn male rats, juvenile play
fighting is reduced to female-typical levels (Kurian et al.,
2008); this effect on behavior is similar to what one would
predict if testosterone action within the amygdala of newborn
160 Hormones and the Development and Expression of Aggressive Behavior

males were blocked. Overall, it appears that sexual differentia-
tion of play fighting involves multiple hormones and multiple
mechanisms.
Similar principles apply to the perinatal sexual differentia-
tion of adult aggression. That is, the transient elevation in
testosterone in male neonates leads to higher levels of aggres-
sion in adulthood (compared with females), and surgical or
pharmacological castration of neonatal males leads to reduced
levels of aggression in adulthood (Bronson and Desjardins,
1969). Conversely, treatment of neonatal females with testos-
terone masculinizes their levels of adult aggression (Bronson
and Desjardins, 1970). Prenatal androgens also appear to
masculinize aggression in humans, as a study found that girls
with CAH are not only rated as more aggressive than unaffected
girls, but also are as aggressive as boys. A fascinating variation
on this theme occurs in spotted hyenas, in which higher social
rank of females within the clan is associated with higher
maternal androgens during late gestation (Dloniak et al.,
2006). These higher gestational concentrations of androgen
lead to higher levels of aggression in the offspring. Thus,
maternal androgens not only organize aggressive behavior,
but they also are a mechanism through which social status
traits are epigenetically transferred from mother to daughter.
At least some of the masculinizing effects of perinatal testos-
terone on adult aggression are due to estrogenic action
(Martinez-Sanchis et al., 1996). As described elsewhere in this
chapter, males have both an androgen and an estrogen-sensi-
tive circuitry that underlies hormone-facilitated aggression.
Work by Simon and colleagues in mice has demonstrated
that estradiol, presumably derived from aromatized testos-
terone, masculinizes the estrogen-sensitive circuit, while
masculinization of the androgen-sensitive circuit is due to
direct androgenic action during early postnatal development
(Martinez-Sanchis et al., 1996).
decline. These behaviors are replaced by flank marking as
a means to more peacefully maintain the relationship. If male
hamsters are castrated prepubertally, then testosterone replace-
ment in adulthood fails to activate flank marking behavior, as
it normally does if hormone replacement is given to hamsters
that are castrated in adulthood (Schulz et al., 2006). Further-
more, male hamsters deprived of testosterone during puberty
fail to replace overt aggression with flank marking and resort
to fighting again when reintroduced to each other after having
established a dominant–subordinate relationship during earlier
encounters (De Lorme and Sisk, 2013). Similarly, territorial
scent marking in tree shrews is organized by the pubertal rise
in testosterone, since castration prior to puberty prevents activa-
tion of this behavior by testosterone in adulthood (Eichmann
and Holst, 1999).
Testicular hormones during puberty also program the level
of aggression displayed by adult hamsters. In one study (Schulz
et al., 2006), males were castrated either before or after puberty,
and then 6 weeks later were treated with either vehicle or testos-
terone. One week after hormone replacement, agonistic behav-
iors were assessed in a resident–intruder test (Figure 9).

1.05.6.3 Endocrine Contributions to the Development of

Aggressive Behavior: Pubertal Organizational Effects
Another period of hormone-dependent organization of social
behaviors, including aggression, occurs during puberty, when
testicular hormone concentrations are once again elevated in
males and when ovarian hormone cycles commence in females
(reviewed in Schulz et al., 2009). An organizational role for
pubertal hormones has been demonstrated by experiments in
which gonadectomy performed after the perinatal period of
sexual differentiation but before the onset of puberty results
in long-lasting alterations in agonistic interactions. Prepubertal Figure 9 Aggressive and submissive behaviors expressed by male
castration prevents the normal transition from complete to Syrian hamsters in a 10-min resident–intruder test. Subjects were cas-
partial rotations in male play fighting defensive behaviors. trated either prepubertally or in adulthood, and 6 weeks later treated for
Interestingly, ovarian hormones appear to suppress in females 1 week with either placebo or testosterone. When endogenous testos-
the pubertal emergence of a male-typical increase in roughness terone was absent during adolescent development (prepubertal castra-
of play fighting (Pellis, 2002). tion group), behavior in adulthood was characterized by fewer attacks
Testicular hormones during puberty program agonistic and more escapes compared to when endogenous testosterone was
present during adolescent development. Thus, testicular hormones,
behaviors in adult hamsters. One agonistic behavior commonly
acting during puberty, program higher levels of aggression in adult-
observed in male–male encounters is flank marking, in which
hood, even though testosterone does not exert activational effects on
flank gland secretions are rubbed onto objects in the environ- these behaviors in adulthood in this species. Reprinted from Schulz,
ment as a means of communicating dominant/subordinate K.M., Sisk, C.L., 2006. Pubertal hormones, the adolescent brain, and
status. Male hamsters initially use overt aggression and submis- the maturation of social behaviors: lessons from the Syrian hamster.
sion to quickly establish a dominant–subordinate relationship. Mol. Cellular Endocrinol. 254–255, 120–126, with permission from
In subsequent encounters, aggressive and submissive behaviors Elsevier.
 Hormones and the Development and Expression of Aggressive Behavior 161

Figure 10 Aggressive and submissive behaviors expressed by male Syrian hamsters in a 10-min resident–intruder test. Subjects were castrated
either prepubertally or in adulthood, and 6 weeks later treated for 1 week with either placebo or testosterone. When endogenous testosterone was
absent during adolescent development (prepubertal castration group), behavior in adulthood was characterized by fewer attacks and more escapes
compared to when endogenous testosterone was present during adolescent development. Thus, testicular hormones, acting during puberty, program
higher levels of aggression in adulthood, even though testosterone does not exert activational effects on these behaviors in adulthood in this species.
Reproduced from Schulz, K.M., Sisk, C.L., 2006. Pubertal hormones, the adolescent brain, and the maturation of social behaviors: lessons from the
Syrian hamster. Mol. Cellular Endocrinol. 254–255, 120–126, with permission from Elsevier

Irrespective of testosterone or vehicle treatment in adulthood,
males castrated prior to puberty did not attack the intruder
and displayed high levels of submissive behaviors. In contrast,
males that were castrated after puberty attacked the intruder
and rarely displayed submissive behaviors. Organizational
effects of adolescent hormones on male aggression have also
been reported in other species, as evidenced by long-lasting
changes in aggressive behavior when hormones are manipu-
lated during the pubertal period. Male DBA/1Bg mice are nor-
mally very aggressive, but the absence of gonadal hormones
during adolescence prevents activation of aggressive behavior
by testosterone in adulthood (Shrenker et al., 1985). Similarly,
adult testosterone treatment only partially restores aggressive
behavior in prepubertally castrated male gerbils (Lumia et al.,
1977), indicating that pubertal hormones program behavioral
responses to hormones in adulthood.
Agonistic behaviors in female rodents may also be orga-
nized during adolescence. If female mice are ovariectomized
at the onset of puberty (30 days of age), treated with testos-
terone for 3 weeks during adolescent development, and then
tested 6 weeks after discontinuation of testosterone treatment,
the levels of aggressive behavior toward another female in
a neutral arena are much higher than in females treated with
vehicle (Edwards, 1970). Thus, adolescent exposure to
androgen has long-term effects on aggression in female mice,
and the nervous system remains sensitive to organizing influ-
ences of gonadal steroid hormones well into postnatal life.
However, the adolescent brain appears to be less sensitive
than the neonatal brain to organizational effects because
more testosterone and longer duration of treatment are
required to masculinize aggression during puberty than
on PND1.
Overall, sexual differentiation of play fighting and adult
aggression is a two-stage process involving gonadal hormone
action in the nervous system during perinatal and pubertal
periods of development. Perinatally, testicular hormones, via
both androgenic and estrogenic action, drive the initial mascu-
linization and defeminization of circuits underlying juvenile
play and adult aggression. Pubertally, both testicular and
ovarian hormones reinforce and refine the sexual
differentiation of neural circuits to result in sex-typical expres-
sion of aggressive behavior in adulthood (Figure 10).
1.05.6.4 Endocrine Contributions to the Development of
Aggressive Behavior: Pubertal Activational Effects
Generally speaking, levels of aggression increase over the
course of puberty as an animal achieves reproductive fertility
and faces the responsibility of obtaining its own food and
shelter, fending for itself, finding a mate, and potentially caring
for offspring. Because the pubertal increase in aggression
temporally coincides with the pubertal rise in gonadal and
adrenal steroid hormones, it is logical to conclude that pubertal
hormones activate the behavior in particular social contexts.
Indeed, as detailed above, there is strong evidence for andro-
genic and estrogenic activation of adult aggressive behavior in
many rodent species, with the caveats that the causal relation-
ship between hormones and aggression is often a two-way
street, and that effects of hormones on aggression are modu-
lated by genetic background, experience, and complex interac-
tions between the two. Evidence for activational effects of
hormones on aggression in adulthood notwithstanding, the
expression of agonistic behaviors over ontogeny is not gov-
erned by hormones in all cases. Play fighting during the prepu-
bertal and juvenile periods of life is clearly not under gonadal
hormone control, because hormone levels are at their nadir at
this point in development. However, during early puberty,
initiation of play is activated by testicular hormones, as evi-
denced by a reduction in play in male rats gonadectomized
just prior to puberty (Cooke and Woolley, 2009). In Syrian
hamsters, pubertal increases in intermale aggression proceed
similarly in both gonad-intact males and males castrated
a few days after weaning (Whitsett and Vanderbergh, 1975).
Thus, the pubertal increase in testosterone is not an absolute
requirement for the expression of adult aggression.
Normal pubertal changes in play fighting involve both
androgen and estrogen receptor-mediated processes as revealed
by an examination of play fighting in tfm rats. Tfm rats do not
show the typical decrease in play fighting with age and are more
likely than wild-type rats to show the juvenile-typical defensive
162 Hormones and the Development and Expression of Aggressive Behavior
complete rotation in adulthood (Field et al., 2006). On the
other hand, tfm males do show normal age-related changes in
the use of partial rotations and upright postures. Thus, func-
tional androgen receptors appear to be necessary for some,
but not all, developmental changes in the quantity and quality
of play fighting. The transition from play fighting (attacks
toward head) to adult aggression (attacks toward rear) in
hamsters appears to be due to increasing corticosteroid concen-
trations during puberty (Wommack and Delville, 2007).
1.05.6.5 Social Experience and the Development of
Aggressive Behavior
In both humans and animals, early life social experience and
opportunities for juvenile play fighting influence the expression
of aggression in adulthood (reviewed in Cooke and Shukla,
2011; Veenema, 2012; Haller et al., 2014). For example, male
rats that are singly housed from 3 to 5 weeks of age (during
the prepubertal/adolescent period) show less submissive
behavior during territorial aggression by a resident male
compared with rats that are group-housed during adolescence
(van den Berg et al., 1999). Similarly, isolation rearing from
3 to 7 weeks of age leads to increased shock-induced defensive
aggression, an effect that is ameliorated by daily play fighting
experience during the period of social isolation (Potegal and
Einon, 1989). Thus, social interactions in the form of juvenile
play appear to buffer against heightened aggression in response
to provocation.
Research using the Syrian hamster to examine the effects of
social subjugation on subsequent expression of aggression illus-
trates the importance of two types of interaction that influence
the development of aggressive behavior. First is the interaction
between social experience and context. If prepubertal male
hamsters are socially subjugated by experiencing repeated defeat
in male–male social encounters, then they subsequently show
enhanced aggression toward a smaller and younger intruder,
whereas they show reduced offensive responses toward an
intruder of similar age and size (Delville et al., 1998). Thus,
subjugation can lead to heightened or reduced aggression,
depending on social context. Prepubertal subjugation also accel-
erates the transition from play fighting to adult aggression and
increases aggression in adulthood, which may be mediated by
an increase in adrenal glucocorticoid secretion brought about
by the stress of defeat (Wommack et al., 2003). Second, the
effect of subjugation on aggression depends on the age at which
subjugation occurs. In contrast to prepubertal subjugation,
subjugation in adulthood leads to complete suppression of
aggressive behavior in male hamsters, a phenomenon known
as conditioned defeat (see below Huhman et al., 2003). Subju-
gation of hamsters after mid-puberty leads to an adult-typical
response to subjugation, i.e., decreased aggression toward an
intruder (Delville et al., 2003), suggesting that the develop-
mental switch responsible for the different responses to subju-
gation observed in juvenile and adult hamsters occurs shortly
after the pubertal rise in testosterone. However, it is not clear
that testosterone either triggers the switch or is part of the
switch, because as described above, the absence of testicular
hormones during adolescent brain development renders male
hamsters less aggressive and more submissive during male–
male encounters (Schulz et al., 2006). This finding does not
easily lead to the prediction that the presence of testicular
hormones during adolescence would result in conditioned
defeat responses to subjugation.
1.05.6.6 Conditioned Defeat
After defeat in the home cage of an aggressive conspecific, male
hamsters (Mesocricetus auratus) will subsequently fail to defend
their home territory even if the intruder is a smaller, nonaggres-
sive male (Huhman et al., 2003). This phenomenon has been
called conditioned defeat and appears to evoke a stress
response via fear conditioning (Huhman and Jasnow, 2005).
The physiological effects of defeat include elevated HPA axis
activity such as increased plasma ACTH, b-endorphin, cortisol,
and corticosterone concentrations, as well as decreased plasma
testosterone and prolactin concentrations (Huhman et al.,
1990, 1991). This endocrine profile is observed among previ-
ously defeated hamsters upon reexposure to another animal
– even when the new opponent is blocked by a physical barrier
(Huhman et al., 1992). This latter response suggests that this
change in endocrine profile is in response to a psychological
stressor and not to the pain or anxiety of the combat itself.
Social defeat also affects immune responses (Fleshner et al.,
1989; Jasnow et al., 2001). The physiological and behavioral
consequences of conditioned social defeat persist for at least
33 days (Huhman et al., 2003), and perhaps throughout adult-
hood (Delville et al., 1998). Few female hamsters exhibit
conditioned social defeat, although ACTH concentrations
were reduced in those females that displayed low levels of
submissive/defensive behavior (Huhman et al., 2003). In
contrast to males, the conditioned defeat response did not
persist beyond the first test among female hamsters. These
results suggest that in male hamsters conditioned defeat is
a profound, persistent behavioral change characterized by
a total absence of territorial aggression and by the frequent
display of submissive and defensive behaviors (Huhman and
Jasnow, 2005).
1.05.6.7 Aggression in Aged Individuals
At the other end of the life span, heightened aggressive and
sexually offending behaviors by aged individuals can pose diffi-
culties for themselves, their caretakers, family members, and
fellow elderly residents living in assisted care facilities (Pulsford
and Duxbury, 2006). Some clinical studies have assessed
hostility as a ‘proxy’ for human aggression. Despite its impreci-
sion, hostility has proven to be a useful construct in studies of
the influences of hormones among aggressive elderly people.
For example, postmenopausal women using HRT scored lower
on hostility scales than women that did not use HRT (Steffen
et al., 1999; Olson et al., 2004). These reports suggest estrogens
can influence the expression of aggressive behavior. Studies of
men and women who have been diagnosed with dementia and
display physical or verbal aggression suggest a positive correla-
tion with circulating testosterone and a negative correlation
with circulating estradiol (Orengo et al., 2002). Treatment of
patients with dementia with estrogens reduced aggression
and sexually offending behaviors (Kyomen et al., 1991,
1999). Despite the significant problems associated with height-
ened aggression among some elderly patients, especially those
 Hormones and the Development and Expression of Aggressive Behavior
with moderate-to-severe dementia, there has been remarkably
little animal research on this topic. Mice with mutated human
amyloid precursor protein (APP) and presenilin (PS1) genes
display shorter latencies to attack and increased numbers of
attacks, suggesting that the plaques and tangles associated
with dementia may contribute to aggression (Minkeviciene
et al., 2004; Alexander et al., 2011).
1.05.7 Reciprocal Effects of Aggression on Steroid
Hormones
Hormones influence aggressive behaviors, but it should be
emphasized that aggressive behavior can feed back and affect
hormone concentrations. Male mice and Syrian hamsters
reduce circulating androgen concentrations if they lost a fight
in paired aggressive encounters (Lloyd, 1971; Huhman and
Jasnow, 2005). This endocrine suppression lasted for several
days postdefeat. Similarly, rhesus monkeys that were defeated
by a higher-ranking male had dramatically reduced testos-
terone concentrations for several weeks postdefeat. In contrast,
winning males’ circulating testosterone concentrations quadru-
pled within 24 h of victory (Bernstein et al., 1977).
1.05.7.1 Gene–Environment Interactions
Studies of aggression are typically conducted under a single set
of environmental conditions. However, mechanisms of aggres-
sive behavior have evolved in fluctuating physical and social
environments. Perhaps not surprisingly, several neurochemical
pathways of aggression function differently depending on the
environment.
One of the most repeatable observations in studies of
human aggression is the interaction between early life experi-
ence and genotype for the human MAOA gene. Caspi et al.
(2003) reported that the effect of child abuse on behavior
was significantly stronger if the child carried alleles associated
with low MAOA activity (Figure 11(a)). Abused children
with low MAOA activity had increased antisocial behavior,
greater prevalence of conduct disorder, and a higher likelihood
of convictions of violent offenses than abused children with
high MAOA activity. In children who were not abused, the
polymorphism had no effect on these measures of behavior.
This gene–environment interaction has been replicated in
some studies (Foley et al., 2004; Kim-Cohen et al., 2006),
but not others (Huizinga et al., 2006; Young et al., 2006). An
initial meta-analysis indicated that on average, children with
genotypes for low MAOA activity have elevated rates of antiso-
cial behavior when exposed to parental maltreatment (Kim-
Cohen et al., 2006). A more recent meta-analysis of 31 studies
(including both community-based and clinically referred
samples) also found that the allele for low MAOA activity
was significantly associated with antisocial behavior and
aggression (Ficks and Waldman, 2014). However, variability
in effect size was quite large across studies and this variability
was not associated with sample or study characteristic. Thus
it is still unclear how the environment interacts with MAOA
function to modulate antisocial behavior and aggression.
There is also growing evidence that genetic variation in sero-
tonin transporter (5-HTT) is linked to aggression and antisocial
163
behavior. The short allele of the 5-HTT gene is associated with
reduced expression of 5-HTT in the brain and inefficient reup-
take of 5-HT from the synapse (Greenberg et al., 1999). The
interaction between stress and 5-HTT genotype was examined
in men and women who were instructed to administer shocks
to a confederate as punishment for incorrect responses in
a memory task (no shocks were actually delivered) (Verona
et al., 2006). Half of the participants were subjected to a phys-
ical stressor (unpredictable air blasts to the throat) whereas the
other half were not. Men, but not women, who were homozy-
gous for the short allele were more likely to administer shocks
under the stressed condition, whereas there were no genotype
differences in the control condition. This interaction could be
mediated by differences in threat perception, as individuals
carrying the short allele have increased activation in the amyg-
dala in response to fear-inducing pictures (Hariri et al., 2002).
A recent meta-analysis of 18 studies also found that the short
allele was significantly associated with increased antisocial
behavior (Ficks and Waldman, 2014). Interestingly, there was
some evidence for publication bias such that studies with
smaller sample size tended to report large effect sizes. In addi-
tion, as with MAOA, there are many unanswered questions on
how the environment interacts with 5-HTT function to modu-
late aggression.
In rodents several studies have demonstrated that parental
behaviors can influence the effects of genes on aggression.
Males of the NZB strain are more aggressive the CBA/H strain
(Roubertoux and Carlier, 1988). If male NZB mice are crossed
with females of the CBA/H strain, then the resulting male
offspring are more aggressive, but only if raised by CBA/H
dams. However, if hybrid pups are cross-fostered to hybrid
mothers, then the pups are no more aggressive as adults
than male CBA/H strains (Carlier et al., 1991). In these studies
the specific differences in maternal care were not identified.
More detailed studies have observed the effects of parental
care on aggression in Peromyscus. Male Peromyscus californicus
are more aggressive than male Peromyscus leucopus (Bester-
Meredith et al., 1999), but if male P. californicus are cross-
fostered to P. leucopus parents, then this species difference in
aggression disappears (Bester-Meredith and Marler, 2001).
Correlational analyses suggested that parental retrieving
behavior was a critical factor (Bester-Meredith and Marler,
2003; Marler et al., 2003), and a subsequent study showed that
experimentally increasing retrieval behavior in P. californicus
increased aggression in male and female offspring (Frazier
et al., 2006), possibly by inducing increased T secretion in
pups (Becker et al., 2010). Interestingly, the absence of
a California mouse father has a major impact on the develop-
ment of aggression. Female but not male California mice
raised without the father were significantly more aggressive
and had reduced the sensitivity of the frontal cortex to dopa-
mine (Bambico et al., 2015).
The context in which mice are tested can also have impor-
tant effects on behavior. Male mice are usually more aggressive
in resident–intruder tests, when intruders are introduced into
a residents’ home cage compared to neutral tests when two
mice are introduced into a neutral arena. Patterns of aggression
in one context do not necessarily transfer to a different context.
For example, the correlation between mossy fibers in the
hippocampus and aggression is positive if male mice are tested
164 Hormones and the Development and Expression of Aggressive Behavior

Figure 11 Gene–environment interactions in humans and mice. The interaction between the monoamine oxidase A (MAOA) genotype and the rear-
ing environment affects aggressive behavior (a). Although they have not been replicated in every study, most data suggest that children carrying the
short form of the MAOA promoter gene, which confers decreased MAOA activity, are more likely to develop conduct disorders and increased antiso-
cial behavior when exposed to abusive home environments. This environmental effect is less prevalent in individuals carrying the long form of the
promoter. Photoperiod determines the directional effects of estrogens on aggressive behavior in beach mice (Peromyscus polionotus) (b). Peromyscus
polionotus are more aggressive when exposed to short days (shown in the left graph) than when exposed to long days (shown in the right graph).
Treatment with the estrogen synthesis inhibitor fadrozole (fad) decreases aggression if beach mice are tested in short days, but increases aggression
if tested in long days. The effects of fad are reversed with cotreatment with estradiol (E2). This does not appear to be mediated by differences in
receptor expression, because the drugs PPT (propylpyrazole-triol, an estrogen receptor (ER)-a agonist) and DPN (diarylpropionitrile, an ERb agonist)
both increase aggression on short days and decrease aggression on long days. Photoperiod apparently regulates the molecular actions of estrogens,
acting rapidly on short days (presumably nongenomically) and more slowly on long days (presumably genomically). Reprinted from Nelson, R.J.,
Trainor, B.C., 2007. Neural mechanisms of aggression. Nat. Rev. Neurosci. 8, 536–546 with permission from Nature.

in a resident–intruder test (Guillot et al., 1994), but is absent if
mice are tested in a neutral arena (Roubertoux et al., 1999). It
has also become clear that social status may be a critically over-
looked variable in studies using domestic mice. For example,
male C57 mice are typically housed four to five mice per
cage. However, each cage typically has one dominant mouse
that directs aggression at its cage mates (Howerton et al.,
2008). This alters not only behavior, but brain circuits that
modulate aggression (Greenberg et al., 2014). This is likely
an important factor contributing to increased variability in
neuroscience studies using male rodents compared to studies
using females, even when estrous cycle is not controlled for
(Prendergast et al., 2014). Finally, the outcomes of resident–
intruder tests also depend on whether the intruders used are
a different genotype than the test mice (Maxson et al., 1989).
For example, genetic variation in the steroid sulfatase gene
(Sts) affects male aggressive behavior when there is no risk of
the opponent retaliating (such as when males are olfactory bul-
bectomized), but has no effect when there is a risk of injury
from the opponent (Maxson et al., 2001).
 Hormones and the Development and Expression of Aggressive Behavior
In mice of the genus Peromyscus, photoperiod determines
the mechanisms through which estrogens control male aggres-
sive behavior (Laredo et al., 2014b; Figure 11(b)). Similar to
hamsters, three species of Peromyscus are more aggressive
when exposed to short days than when exposed to long days
(Peromyscus maniculatus and Peromyscus polionotus (Trainor
et al., 2007b); P. californicus (Nelson and Trainor, 2007; Trainor
et al., 2007b)). In P. polionotus, estrogens decrease aggression
when mice are housed in long days, but increase aggression if
mice are housed in short days (Trainor et al., 2007a). Hormone
manipulation studies showed that the ERa agonist PPT and the
ERb agonist DPN increased aggression in ‘short-day’ mice and
decreased aggression in ‘long-day’ mice. These data suggested
that photoperiod regulates processes that occur after estrogens
bind their cognate receptors. Steroids can affect physiological
and behavioral processes via genomic or nongenomic path-
ways (Vasudevan and Pfaff, 2006). Classical genomic action
occurs when ligand-bound receptors bind to hormone
response elements that facilitate transcription. This process
typically takes hours or days. Nongenomic action can occur
through several pathways including phosphorylation of
cellular signaling pathways and changes in intracellular
calcium. Nongenomic effects can occur within seconds of estro-
gens binding receptors. Gene chip analyses of P. polionotus indi-
cated that estrogen-dependent gene expression was increased in
the BNST of long-day mice compared to short-day mice, sug-
gesting estrogens might act via nongenomic pathways in mice
exposed to short days. In P. polionotus estradiol injections acted
rapidly (15 min) to increase aggression in short-, but not long-
day mice, suggesting that estradiol increases aggression via
nongenomic action (Trainor et al., 2007a). This same result
was also observed in P. californicus (Trainor et al., 2008). The
rapid effects of estradiol on aggression in short-day mice were
not blocked by pretreatment with a protein synthesis inhibitor,
consistent with nongenomic action (Laredo et al., 2013).
However, these follow-up studies identified an unexpected
twist. Whether estradiol increased or decreased aggression
was dependent on type of bedding used to line the cages.
In the initial studies, cages were lined with corncob bedding
and estrogens increased aggression under short days (Trainor
et al., 2007a, 2008). However, when a cardboard-based
bedding was used, estradiol decreased aggression under short
days (Laredo et al., 2013). Importantly, the rapid effects of
estradiol on aggression were absent under long days. When
the two beddings were directly compared, the aromatase inhib-
itor fadrozole increased aggression in mice housed with card-
board-based bedding and decreased aggression in mice
housed with corncob bedding (Figure 12; Villalon Landeros
et al., 2012). Mice housed on corncob bedding had elevated
blood levels of tetrahydrofuran diols (THF-diols), which are
estrogen-like compounds that can alter estrogen-dependent
behavior such as lordosis (Markaverich et al., 2002). Corncob
bedding has been found to reduce anxiety-like behavior and
decrease sex differences in stress responses. Although the
exact mechanism through which corncob bedding alters
estrogen-dependent behaviors is unknown, one possibility is
the regulation of extracellular signal regulated kinase (ERK).
California mice housed on corncob bedding had greatly
reduced pERK immunoreactivity in the BNST, MPOA, MEA,
and VMH. The effects of bedding are significant because
165
Saline Fadrozole
20
**
15
Bites (freq)
10
5
0
Carefresh Corncob
Figure 12 The effects of estrogens on aggression depend on cage
bedding. Aromatase inhibition with fadrozole increased the number of
bites on Carefresh bedding but not on corncob bedding. **, P < 0.01,
planned comparison for effect of fadrozole; n ¼ 10–14 per group.
Reprinted from Villalon Landeros, R., Yoo, H.J., Morisseau, C., Fu, S.,
Hammock, B.D., Trainor, B.C., 2012. Corncob bedding reverses the
effects of estrogens on aggressive behavior and reduces estrogen
receptor alpha expression in the brain. Endocrinology 153, 949–953
with permission from the Endocrine Society.
pERK immunoreactivity in BNST and MEA is increased
during resident–intruder tests when aggression is induced
(Trainor et al., 2010a,b). Further study is needed to
determine how environmental estrogens modulate the rapid
effects of endogenous estrogens on aggression.
1.05.8 Integration
Neurochemical and neuroanatomical pathways of aggression
have been investigated in various species, and it is apparent
that some pathways are common to humans and nonhuman
animals. Increasing serotonergic activity decreases reactive
aggression in humans and also reduces aggression in a mouse
resident–intruder test, probably by decreasing impulsivity.
A more challenging task is determining how murine behavior
in a resident–intruder test relates to reactive or instrumental
aggression in humans. Aggression researchers have been strug-
gling with this question, and a comprehensible answer has not
yet emerged. This may be because there is no unambiguous
answer. In humans, reactive aggression appears to be governed
more by serotonergic pathways, whereas the motivated charac-
teristics of instrumental aggression suggest a role for dopami-
nergic pathways. Given the enormous differences in biology
and social structure, it is unlikely that mouse and human
aggression can be classified into homologous categories.
However, it is clear that many neurochemical systems (such
as the serotonergic system) have coevolved in mice and
humans to regulate species-specific aggressive behaviors.
Thus, although aggressive behavior is expressed in different
contexts with different behavioral outputs in mice and
humans, similar neurochemical and neuroanatomical path-
ways are activated. Difficult questions remain to be answered.
For example, to what extent does an impoverished background
influence the development of these neurochemical and neuro-
anatomical pathways, and to what extent are they activated by
observing aggression? Considerable debate ensues on the
166 Hormones and the Development and Expression of Aggressive Behavior
effects of violence in the media on aggression, and myriad con-
founding factors make it difficult to study these putative effects.
Aggression is increased in animals that observe conflicts among
other individuals (Earley and Dugatkin, 2002; Peake et al.,
2002). Generally overlooked by mental health researchers,
these data show that vicarious experiences have important bio-
logical effects. Sports fans respond to watching their team win
or lose with corresponding increases or decreases in testos-
terone levels (Bernhardt et al., 1998; Figure 13). Children play-
ing violent video games show reduced activation of brain areas
involved in affect, such as the amygdala and the anterior cingu-
late cortex (Mathiak and Weber, 2006). Reduced brain activity
in frontal areas has also been reported in children with high
exposure to violent video games and television programs
(Mathews et al., 2005). Although it is not clear whether these
experiences have long-term behavioral effects, it is clear that
vicarious experiences have consistent short-term influences
on brain activity. It is perhaps unsettling that these patterns
resemble those identified in individuals with dysregulated
aggression (Volkow et al., 1995; Soloff et al., 2003). Biology-
based approaches to examine the effects of observing violence
on aggressive behavior, if they are conducted in realistic social
contexts (in addition to questionnaires and other pencil-and-
paper approaches), have potential because they allow more
precise measurements of the neural circuits that influence
aggressive behaviors. Another issue of concern to clinicians is
how to treat uncontrolled aggression. This is a complicated
issue because, although it is agreed that unchecked aggression
has negative consequences, a certain amount of human aggres-
sion is probably necessary to succeed in life. Clinical trials have
investigated many treatments aimed at reducing elevated
aggression that is associated with mental disorders, but treat-
ments that can ameliorate excessive aggression have unwanted
side effects on processes such as arousal (Cherek et al., 2006).
Although further advances in drug development may lead to
additional improvements in the treatment of pathological
aggression, the complexity of aggressive behavior suggests
that it might not be possible to control aggression. A more
9 Bedrosian, T.A., Nelson, R.J., 2014. Nitric oxide and serotonin interactions and
Salivary testosterone (ng dl−1)
8 Winning
fans
7 vicarious experiences of winning and losing among fans at sporting events. Physiol.
6 influencing primate testosterone levels. J. Hum. Evol. 6, 517–525.
Losing
5 fans
4
Before After
College basketball game
Figure 13 Basketball fans’ testosterone levels before and after their
team has won or lost. Reprinted from Bernhardt, P.C., Dabbs, J.M.,
Fielden, J.A., 1998. Testosterone changes during vicarious experiences
of winning and losing among fans at sporting events. Physiol. Behavior.
65, 59–62, with permission from Elsevier.
effective strategy for dealing with uncontrolled aggressive
behavior may lie in a combination of biological and behavioral
approaches, especially for instrumental aggression.
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 1.06 Food Intake and Its Control by Signaling Molecules
Francisco Vázquez-Cuevas, Instituto de Neurobiología, Campus UNAM-Juriquilla, Querétaro, Mexico
Raúl Aguilar-Roblero, Instituto de Fisiología Celular, Ciudad Universitaria, UNAM, México, Mexico
Elvira Arellanes-Licea, Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, Mexico
Yazmı́n Macotela and Olivia Vázquez-Martı́nez, Instituto de Neurobiología, Campus UNAM-Juriquilla, Querétaro, Mexico
Iván Villanueva, Escuela Nacional de Ciencias Biológicas, IPN, México, Mexico
Mauricio Dı́az-Muñoz, Instituto de Neurobiología, Campus UNAM-Juriquilla, Querétaro, Mexico
Ó 2017 Elsevier Inc. All rights reserved.

1.06.1 Preface 177

1.06.2 Hunger–Satiety Cycle–Historical View and Modern Perspective 177
1.06.2.1 Energy Input: Food Intake and Its Physiological Control 178
1.06.2.2 Not All Feeding Is Related to Energy 178
1.06.2.3 Study of the Physiological Control of Food Intake 179
1.06.2.4 Main Concepts in the Study of Food Intake Control and Their Historical Development 179
1.06.2.5 Energy: A Compulsory Element 180
1.06.2.6 Quest for the Signals 180
1.06.3 Bioenergetics of the Fasting–Feeding Cycle 183
1.06.4 Hypothalamus and Feeding Control 184
1.06.4.1 General Perspective 184
1.06.4.2 Neuronal Circuits 184
1.06.4.3 Feeding Circuits in the Brain 185
1.06.4.3.1 Homeostatic Circuits 185
1.06.5 Chronostasis: Physiological Regulation across Time 187
1.06.6 Neuropeptides 188
1.06.6.1 Neuropeptides Regulating Feeding Behavior 188
1.06.6.1.1 Orexins 188
1.06.6.1.2 Orexins and Feeding Control 189
1.06.6.1.3 Orexins and Energy Regulation 189
1.06.6.2 Endocannabinoid System 190
1.06.6.2.1 Endocannabinoids and Feeding Control 190
1.06.6.3 Melanocortin System 190
1.06.6.3.1 POMC-Derived Peptide Family 190
1.06.6.3.2 Aguoti-Related Peptide (AgRP), a Natural Antagonist of the Melanocortin System 191
1.06.6.3.3 Receptors of Melanocortin 191
1.06.6.3.4 Melanocortins and Feeding Control 191
1.06.6.4 Neuropeptide Y 192
1.06.6.4.1 NPY and Feeding Control 192
1.06.7 Endocrine Systems: Hormones in Fasting and in the Feeding Response 193
1.06.7.1 Ghrelin and Leptin Biology and Their Role in Food-Intake Regulation 193
1.06.7.1.1 Biology: Synthesis, Secretion, Physiology, and Signaling 193
1.06.7.1.2 Leptin 194
1.06.7.1.3 Food-Intake Regulation by Ghrelin and Leptin 195
1.06.8 Other Adipokines Regulating Food Intake 196
1.06.8.1 Adiponectin 196
1.06.8.2 Resistin 197
1.06.8.3 Nesfatin 197
1.06.8.4 Apelin 198
1.06.9 Pancreas and Feeding Control 198
1.06.10 Glucocorticoids and the Control of Food Intake 199
1.06.10.1 Glucocorticoids and the Stress System 200
1.06.11 Concluding Remarks 200
Acknowledgments 201
References 201

Hormones, Brain, and Behavior, 3rd edition, Volume 1 http://dx.doi.org/10.1016/B978-0-12-803592-4.00006-7 175

176 Food Intake and Its Control by Signaling Molecules

Abbreviations
11-b HSD 11-b-hydroxysteroid dehydrogenase LH Luteinizing hormone
ACTH Adrenocorticotropic hormone LHA Lateral hypothalamic area
AG Acylated ghrelin b-LPH b-Lipotropin
AGRP Agouti-related peptide LR Leptin receptor
AMPK AMP-dependent kinase MAPK Mitogen-activated protein kinase
ANS Autonomic nervous system MCR Melanocortin receptor
APJ Apelin receptor MR Mineralocorticoid receptors
ARC Arcuate nucleus mRNA Messenger RNA
ATP Adenosine triphosphate a-MSH a-Melanocyte-stimulating hormone
BBB Blood–brain barrier N-POC N-terminal peptide proopiomelanocortin
cAMP/PKA Protein kinase A activated by cAMP NADPD Nicotinamide adenine dinucleotide phosphate,
CART Cocaine- and amphetamine-regulated transcript oxidized form
CB1 Cannabinoid receptor one NADPH Nicotinamide adenine dinucleotide phosphate,
CCK Cholecystokinin reduced form
cDNA Complementary DNA NE Norepinephrine
CLIP Corticotropin-like intermediate lobe peptide NEFA/NUCB2 Nesfatin or nonesterified fatty acid/
CNR1 Endocanabinoids receptor 1 nucleobindin2
CNR2 Endocanabinoids receptor 2 NPY Neuropeptide Y
CPE Carboxypeptidase E NS Nervous system
CRF Corticotropin-releasing factor NTS Nucleus of the solitary tract
CRH Corticotropin-releasing hormone OXR Orexin receptor
CSF Cerebrospinal fluid PAM Peptidyl a-amidating mono-oxygenase
DAG Diacylglycerol PBMC Blood mononuclear cells
DMH Dorsomedial hypothalamic nucleus PBN Parabrachial nucleus
DREADD Designer receptors exclusively activated by PC Posterior commissure
designer drugs PEPCK Phosphoenol pyruvate carboxykinase
DVC Dorsal vagal complex PI3K Phosphatidylinositol 3-kinase
b-end b-Endorphin PKA Protein kinase A
FAS Fatty acid synthase PLC Phospholipase C
GABA a-Aminobutyric acid POMC Proopiomelanocortin
GABAA GABA receptor type A PP Pancreatic polypeptide
GH Growth hormone PPARs Peroxisome proliferator-activated receptors
GHRL Ghrelin/obestatin prepropeptide PPOX Prepro-orexin
GHRS Ghrelin receptors PVH Paraventricular nucleus of the hypothalamus
GHSR Growth hormone secretagogue receptor PVN Paraventricular thalamic nucleus
GLP-1 Glucagon-like peptide-1 PYY Peptide tyrosine tyrosine
GOAT Ghrelin O-acyltransferase RSTN Resistin
GPCR G protein-coupled receptor SF1 Steroidogenic factor 1
GR Glucocorticoid receptors SOC3 Suppressor of cytokine signaling
H6PDH Hexose-6-phosphate dehydrogenase SON Supraoptic nucleus
HCl Clorhydric acid STAT3/5 Signal transducers and activators of transcription
HCRTR Hypocretin (orexin) receptor TNF-a Tumor necrosis factor-a
HPA Hypothalamic–pituitary–adrenal axis TRH Thyrotropin-releasing hormone
IML Intermediolateral column of the spinal cord TZDs Thiazolidinediones
InsR Insulin receptors UAG Unacylated ghrelin
IP3 1,4,5-trisphosphate VLDL Very low density lipoproteins
Jak2 Janus kinase family VMH Ventromedial hypothalamus
JP Joining peptide WAT White adipose tissue
LC Locus caeruleus

1.06.1 Preface
Frequently, feeding implies moving within a subject’s habitat
in search for food, perhaps engaged in intense motor activity
to suppress the sensation of hunger. When the animal has
secured its food it continues to the actual process of feeding,
which will eventually finish as hunger vanishes or the food
has been consumed. This oversimplified view of feeding
already lets us grasp the complexity of the physiological
processes involved in the regulation and execution of feeding
behavior, such as interoception involving neuronal networks,
behavioral state control, motivation, selective attention, spatial
and temporal memory, motor coordination, secretions from
exocrine and endocrine glands (associated with the gastrointes-
tinal tract) involved in digestion and absorption of nutrients,
and biochemical processes of intermediate metabolism inte-
grated mainly, but not exclusively, at the hepatic level. In the
case of humans, beyond the already complex system outlined
above, there is an additional level of regulatory interactions
arising from the fact that we can disregard satiety signals and
continue eating when we are not hungry. In the present chapter,
we will review some aspects of the system regulating feeding
behavior.
The term feeding is linked to the concept of nourishment,
which is the process by which nutrients are obtained. As chem-
ical entities, nutrients are a set of molecules with an energetic
value for living beings. Living entities, from single cells to
multicellular fungi, plants, and animals, are thermodynami-
cally open systems formed by dissipative structures specialized
in exchanging matter and energy with the environment. All of
these systems are complex, dynamic, nonlinear, and far from
being at equilibrium. Indeed, living beings fulfill the Second
Law of Thermodynamics of increasing global entropy while
presenting emerging properties that are characteristic of biolog-
ical organization (Mrosovsky, 1990).
According to physics, energy is defined as the ability to
execute work. In animals energy is assimilated through the
degradation of dietary carbohydrates, lipids, and proteins.
Within the cellular milieu, the principal forms of energy are
those with the capacity to allow chemical (biochemical reac-
tions within metabolic networks), electrical (maintaining
membrane potential in excitable and nonexcitable tissues),
and mechanical work (assembly and disassembly of cytoskel-
etal components). Energy is, therefore, essential to maintain
life processes such as metabolism, growth, reproduction, and
ambulatory activity. In particular, life on our planet is depen-
dent on radiant solar energy and its subsequent fixation by
photosynthetic mechanisms and conversion by green plants
and algae into biochemical energy (i.e., carbohydrates). Carbo-
hydrates and other biomolecules are an energy source for
animals that have the ability to oxidize them through the
process of metabolic respiration. For example, when an animal
releases the potential energy of glucose during mitochondrial
respiration, approximately two-thirds of it is converted into
chemical and mechanical energy to be used for work (metabo-
lism and growth), and one-third is dissipated as heat (Jequier
et al., 1987).
Energy is usually expressed in terms of heat units, since all
forms of energy are convertible and eventually turn into heat
energy. The basic heat unit normally used is the calorie. One
Food Intake and Its Control by Signaling Molecules 177
calorie is defined as the amount of heat required to raise the
temperature of 1 g of water by 1
C. Since for many purposes,
calorie (cal) is too small, kilocalorie (kcal) is often used;
1 kcal ¼ 1000 cal. In many scientific studies, calorie is replaced
by joule (J) as the unit of energy; 4.184 J ¼ 1 cal and
4184 J ¼ 1 kcal (Franklin et al., 2010).
This chapter is focused on the underlying mechanisms that
enable the physiological activity of food intake in animals,
endothermic organisms with heterotrophic nutrition. The first
section explains feeding as a complex function with regulatory
principles that can be studied using homeostatic, rheostatic,
and chronostatic criteria. This section will illustrate the concept
that eating should be visualized as a rhythmic function (with
ultradian or circadian periods) within the chronobiological
regulation of the organism’s physiology.
In animals, food intake is a behavior comprising volitional
and reflexive activities of more than 30 nerves and muscles.
However, feeding involves several steps, such as searching for
food, food ingestion, assimilation of nutrients, the restoration
of energetic deposits (hepatic glycogen and adipose lipids), and
eventually the energy consumption that empties those
deposits and returns the system to the first step. Underlying
this fasting–feeding cycle is the equilibrium between satiety
and hunger, the physiological responses to the energetic state
of the organism. The second section of this chapter will review
the principal theories that have been postulated to explain the
hunger–satiety cycle. Some of them have historical significance,
but others continue to provide the foundation for an active
field of biomedical research.
Eating involves a display of behavioral patterns that vary
with environmental conditions. Most of the time food intake
is driven by sensory inputs, such as tastes and odors, as well
as by affective or emotional states. Indeed, it has been postu-
lated that eating behavior is based on genetics, but recently
this behavior has also been postulated to be partially attribut-
able to an environmental parameter called behavioral suscepti-
bility (Carnell and Wardle, 2008).
The chapter also contains a section dealing with signaling
molecules that enable coordination among organs and tissues
during feeding activity. We will discuss the principal hypotha-
lamic peptides and factors (purines and endocannabinoids
(ECs)) associated with hunger and satiety as well as peripheral
endocrine molecules with regard to their site of synthesis,
modulation of secretory events, types of receptors, and mecha-
nisms of signal transduction. Each messenger molecule will be
dealt with in the context of its role under fasted or fed condi-
tions. Finally the chapter will end with some concluding
remarks that highlight the most recent concepts in the control
of feeding activity.
1.06.2 Hunger–Satiety Cycle–Historical View
and Modern Perspective
Energy is acquired by organisms in a number of ways. Prokary-
otic and plant cells employ diverse strategies to obtain energy
from their environment. Virtually all animal cells are heterotro-
phic, which means that they depend on the energy captured
and stored by other living species to capture and store the
energy they need in the form of carbon present in specific
178 Food Intake and Its Control by Signaling Molecules

organic molecules. Consequently, animals must feed on these
energy-rich molecules synthesized by other living organisms.
Energy availability often becomes an environmental
constraint for the perpetuation and growth of animal popula-
tions. Animals have developed a series of mechanisms to
improve the balance between energy acquisition and expendi-
ture, so that proportionally more energy is available for their
internal processes. This explains the exquisite specialization
and efficiency of the physiological mechanisms dedicated to
food acquisition, processing, and storing, along with the
notable strategies for optimizing internal energy use. Birds
and mammals must also face the energetic cost of endothermy,
which implies a several-fold increase of the continuous energy
requirements of their body mass and thus, a still greater ener-
getic constraint. As a whole, these particular conditions result
in a set of physiological mechanisms that finely control the
acquisition and incorporation of energetic molecules, the
internal management of metabolic fuels, and the distribution
of the energy-consuming processes.
1.06.2.1 Energy Input: Food Intake and Its Physiological
Control
The ingestion of food is the only way to fulfill the elevated
energy demands derived from the high metabolic rate in
mammals. As such, food intake has the primary function of
providing metabolic fuels in accord with the energy require-
ments of the organism. The recognition of this basic relation-
ship results in two logical principles:
1. Energy availability is a critical variable that requires physi-
ological mechanisms devoted to its maintenance. These
mechanisms (involving a diversity of sensory cells, nerve
pathways, hormones, and other messengers, brain centers,
and subcellular enzymatic processes in a number of effector
tissues) have an enormously complex organization and are
grouped together under the concept of ‘physiological
control of food intake.’
2. In order to assure energy availability, food intake should be
physiologically adjusted to ensure energy balance. A nega-
tive energy balance (i.e., expenses larger than input) would
elicit a strong physiological response promoting food search
and consumption, which we call ‘hunger’; a positive balance
(i.e., more energy available than needed) would cause the
weakening of this drive and the cessation of feeding, which
we call ‘satiety.’ A cybernetic model of this relationship is
presented in Figure 1.
1.06.2.2 Not All Feeding Is Related to Energy
The formation and maintenance of animal tissues also
requires a series of exogenous elements whose primary role
is structural and their functions go beyond providing only
energy; instead, they have specific roles as biochemical constit-
uents and catalytic factors. Some of these elements are so crit-
ical that their availability is maintained by specialized
physiological mechanisms specifically controlling their acqui-
sition and elimination. Though independent from energy,
these special requirements are met primarily by the same route
as energy, i.e., by ingestion. This creates additional drives that
are combined together in a single food-intake response. In
some cases, such as water, the particular requirements generate
a special repertoire of physiological and behavioral traits
(thirst/drinking) that is clearly distinguishable from that
devoted to energy (hunger/feeding). In most cases, however,

Figure 1 The control of hunger in the maintenance of energy availability. Energy is consumed continually in biological work and must be provided
from the environment. The energy availability to tissues results from the balance between energy acquisition and expenditure. The only significant
form of energy acquisition in mammals is food intake. The energy availability is critical and it requires a constant measurement of the variable by the
sensor and the processing of this information in an integration center. If too low or too high, the integration center ‘corrects’ the energy availability
by adjusting the processes determining it (feedback loops). In mammals, the correction is made essentially on the energy intake (i.e., feeding) while
expenditure is only modestly modified, so that feeding becomes a major determinant of the energy status of the organism. A positive energy balance
(increased input relative to expenditure; downward dotted arrow) results in excessive accumulation and obesity. A negative balance (increased expen-
diture; upward dotted arrow) implies fat reserve consumption. The physiological control of food intake refers to the mechanisms of adjustment of
food intake according to the energy status of the organism (dark frame in the figure). Based on Cabanac, M., 2001. Regulation and the ponderostat.
Int. J. Obesity Relat. Metab. Disord. 25 (Suppl. 5), S7–S12.

Figure 2 Feeding behavior is the resultant of a decision-making process aimed at maintaining energy availability. The process recruits information
from diverse sources, integrates it, and generates the pertinent outputs. The outputs include corrective adjustments in both energy income and
expenditure. Metabolic energy expenditure does not change greatly, so that most adjustments are made on the energy input by food intake.
the special requirements become manifest as subtle prefer-
ences for a certain flavor or food type, and they are called
‘specific appetites.’ These specific drives can produce clearly
directed preferences such as for sodium (salt appetite), but
in many cases (as for specific amino acids) an expected prefer-
ence behavior does not manifest itself clearly, probably
because it is overridden by another preference or because
such a mechanism simply does not exist. Either way, the turn-
over rate of energy is much larger than that of the nonenergetic
nutriments, which means that the energetic molecules are
depleted and need to be replenished at a much higher rate
than the nonenergetic ones. Consequently, the molecules
potentially yielding metabolic energy are consumed in larger
quantities, for which they are called ‘macronutrients.’ In
contrast, nonenergetic molecules, except water, generally
have long residence times within the organism and low elim-
ination rates, making their need quantitatively low (hence
‘micronutrients’). The consequence is that the physiological
control of food intake is centered on energy availability, while
the contribution of the specific appetites to feeding behavior is
controversial.
1.06.2.3 Study of the Physiological Control of Food Intake
Food intake can be envisaged as an array of behavioral mecha-
nisms devoted to the acquisition of energy from the environ-
ment in accord with the particular requirements of the
organism. The behavioral events that make up the food-
intake pattern are typically divided in two categories: the
appetitive (related to craving and search of food) and the
consummatory (related to actual ingestion: biting, chewing,
and swallowing). Each behavioral trait can be considered as
the execution of a series of operative commands (i.e., an
output) produced by a physiological decision-making process.
In order to match the actual body requirements, the decision-
making process recruits and integrates a vast amount of infor-
mation from different sources, either directly related or not to
the energy needs. The decision-making process consists of
composing a comprehensive picture of the energetic state of
the organism and determining the necessary actions, if any,
Food Intake and Its Control by Signaling Molecules 179
to recover a certain ideal state. Ingestive behavior (and the
resulting food intake) is a main mechanism to meet that
purpose. A schematic representation of the integrative
decision-making process and its behavioral output is presented
in Figure 2. From this perspective it emerges that feeding
behavior results from the integration of physiological signals
related mostly, but not exclusively, to energy availability.
Over a time span of more than 100 years, diverse research
groups have tried to extract a logical principle describing the
physiological control of food intake. The motivation could
have been an interest to manipulate food intake for therapeutic
(such as obesity treatment) or economical (such as cattle
growing) purposes, but in most cases the motivation seems
to be rooted in the human wish to understand biological
complexity. The study of the physiological control of food
intake is represented by a succession of theories, enriched
over time by previous experience, so that a progression is noted
toward more accurate models. Let us make a brief review of this
process.
1.06.2.4 Main Concepts in the Study of Food Intake Control
and Their Historical Development
As noted before, a negative energy balance is the main motiva-
tion for food intake. Energy deficit generates a series of physio-
logical signals that result in enhanced feeding behavior. This is
called hunger. The inhibition of these signals by the ingestion
of food, with the consequent cessation of feeding, is called
satiety. In simple terms, feeding behavior can be visualized as
the alternation of periods of hunger and satiety. According to
this, the first approach to understanding food-intake control
is to elucidate the physiological origin of hunger.
In everyone’s personal experience, the most evident and
first-hand manifestation of hunger is a characteristic sensation
similar to a painful contraction of the stomach, known as an
epigastric pang. The relation between this pang and the physi-
ological process of hunger is intuitive, as the epigastric pang
originates after a certain time of food deprivation, it increases
in intensity with continued deprivation, ceases shortly after
food is ingested, and is clearly distinguishable from any other
180 Food Intake and Its Control by Signaling Molecules
gastric ache. The first attempt to establish a physiological mech-
anism of hunger was the demonstration by Cannon and
Washburn (1912) that the epigastric pang coincides with the
periodic contractions of the empty stomach. On this basis,
Carlson (1916) explained hunger as a set of sensations that
originated from contractions of the stomach when empty and
ceased as food distended the gastric wall. This can be consid-
ered the first model proposed to explain the physiological
origin of hunger and satiety, and it is remarkable that it is
centered in the stomach as a major determinant of the feeding
response. In fact, experimental evidence demonstrates that
gastric stimulation strongly influences feeding behavior. For
instance, permanent gastric filling by means of a balloon
suppresses feeding in rats to the point of causing death by star-
vation, but many aspects of feeding behavior cannot be
explained in terms of gastric processes. In particular, this
‘gastric theory’ of hunger disregards the theoretical relationship
between food intake and metabolic energy. Over the following
years the perceived role of the stomach in the control of food
intake has changed and it is currently visualized as a major
element of a feeding-control submechanism committed specif-
ically to the digestive process.
1.06.2.5 Energy: A Compulsory Element
In parallel with the motivational analysis of feeding behavior,
the problem of the physiological control of body weight was
examined experimentally. For several decades, it was debated
whether body weight is a regulated variable or a passive result
of the processes on which it depends, i.e., energy intake and
expenditure. On the one hand, strong evidence indicated
that body weight variability over time is much less than would
be expected from a passive outcome; yet, human obesity sug-
gested a lack of tight physiological control of fat accumula-
tion. The controversy motivated a number of studies
focused on the physiology of body weight control, indirectly
including food intake as a factor. In this context a body
weight-centered hypothesis emerged that guided much of
the subsequent research in the field: the lipostatic theory.
Proposed by Gordon Kennedy (1953), the lipostatic theory
establishes that a certain ‘desirable’ amount of fat stores is
to be kept by adjusting the underlying processes. This mecha-
nism assumes that fat stores are constantly being
measured and that the information is sent to a regulatory
center that ‘monitors’ the stores and determines the appro-
priate corrective actions if needed. This center must have
outputs to effector structures that execute the corrective
actions, such as adjustments in food intake.
The lipostatic theory was originally proposed as a predictive
model meaning that although some specific mechanisms,
structures, and messengers were unknown at the time, their
discovery was expected. This was particularly the case for the
mechanism of fat measurement, central to the theory, for which
a number of hypotheses were issued but not convincingly
solved until leptin was discovered in 1994 (see below). The
lipostatic theory is centered on the control of adipose mass
and not directly on feeding; however, it must be taken as
a crucial antecedent for the study of food intake as it offered
the first comprehensive model linking the ingestive response
to the broad energy status of the organism.
1.06.2.6 Quest for the Signals
Based on the concept that the main purpose of feeding is to
provide energy-rich substrates, the study of food intake
encountered a very complex set of facts to disentangle. Meta-
bolic energy has multiple forms that acquire different configu-
rations in different tissues; it is sensitive to different signals, it
changes over time, and it has different functional meanings.
For instance, of the three main metabolic substrates, glucose
enters and exits the blood pool at a high rate but, unlike fatty
acids and amino acids, it is subjected to a strict regulatory
mechanism that severely opposes change. Fatty acids provide
c.2.4 times more energy per gram than carbohydrates, but
energy extraction in this case requires an additional metabolic
apparatus based on mitochondrial oxidation, so that this
energy is inaccessible for some tissues. The estimated size of
the energy stores varies strikingly between the different forms;
fats exceed by several hundred-fold the stores of glucose. This
implies that the utilization of a certain quantity of stored
energy impacts very differently the size of each deposit. Lastly,
certain organs (e.g., the brain) are very sensitive to changes in
energy availability, for which they make use of vigorous
compensatory responses. Other organs (e.g., the liver) easily
handle marked reductions in metabolic energy eliciting,
instead, moderate routine responses. As the understanding of
food-intake control developed, it took the form of different
hypotheses that focused on certain processes or variables
considered to be indicative of the animal’s general energy
status. Still, the subject was hunger, and the question was how?
Glucose is the only substrate from which energy can be
extracted in the absence of mitochondria and/or oxygen.
Tissues in hypoxia or lacking mitochondria (e.g., erythrocytes)
obtain energy exclusively from glucose or certain closely related
compounds. Moreover, the enormous operating cost of
nervous tissue (c.20% of the daily energy expenditure of the
whole body for a tissue that represents only 2% of the body
mass) is supplied almost exclusively by glucose, because
nervous tissue does not oxidize fatty acids. Glucose reserves
are relatively small (hepatic glycogen lasts for less than 24 h
of fasting), and when they have been exhausted the availability
is maintained by hepatic de novo glucose synthesis at the
expense of body proteins. It is evident that glucose is a critical
energy substrate that also impacts more than any other energy
source on brain metabolism. These premises point to glucose
as a reliable index of energy availability and thus, it can act
as a feasible indicator of the need for food. The glucostatic
theory proposed by Jean Mayer in 1953 states that blood
glucose is the signal determining hunger: the blood glucose
pool is replenished after feeding and the subsequent extraction
gradually reduces the size of the pool. The demonstration of
brain neurons sensitive to high and to low glucose concentra-
tions completed the scheme. Sensed by brain glucoreceptors,
the reduction in blood glucose would be interpreted as an
energy deficit, triggering the motivation to feed, i.e., provoking
hunger.
The glucostatic theory was amply accepted as an explana-
tion of hunger, probably because of its logical basis. However,
there are some practical observations that conflict with it. First,
glycemia is a regulated variable, which means that changes in
glucose availability are physiologically compensated for, so

that blood glucose is expected to vary minimally. In fact,
a significant hunger-triggering drop in blood glucose has
been hard to demonstrate. Modern measuring techniques
have revealed modest drops of 10%, at best, preceding sponta-
neous food intake (Campbell, 1991). Also, brain tissue is rela-
tively protected from variations in glucose availability because
of the blood–brain barrier, which allows the cerebrospinal
fluid to buffer the variations in plasma composition. However,
the strongest argument against the glucostatic theory is feeding
behavior in the diabetic state. Both types of diabetes mellitus
reduce insulin functionality and elevate blood glucose levels.
Glucose entry into neural tissue is constitutive and not
insulin-dependent, so that diabetic hyperglycemia implies
excessive glucose provision to the brain. The ingestive response
predicted by the glucostatic theory would be permanent satiety,
but instead, the diabetic state is characterized by overfeeding.
The glucostatic theory was too simple to encompass all the
complexity involved in the physiology of hunger. Blood
glucose is a signal to be taken into account, but evidently it
does not suffice per se to explain feeding behavior. The gluco-
static theory was not discarded, but in subsequent years
a number of research groups sought a model that could
complement or substitute satisfactorily for the glucostatic
idea. In fact, the quest was for a signal that could simulta-
neously indicate metabolic state of the organism and match
the known relationships with feeding behavior. The different
approaches shared the premise that food intake was part of
a mechanism intended to keep constant some other critical,
still undetermined, variable which then became the signal
determining the feeding responses. Since the models were
based on maintaining a certain variable, the suffix -static
(implying steadiness) was used to name the concepts. Hence,
glucostatic theory implies the control of feeding according to
the glucose stability requirements.
Over the following decades a series of hypotheses were
proposed to explain hunger and satiety in the terms described
above. The aminostatic theory proposed by Sherman
Mellinkoff et al. (1956) was based on the satiating power of
dietary protein, which is much stronger than that of carbohy-
drates. In this case a supposed regulation of amino acid avail-
ability would be the main drive for feeding. Yet, this theory
did not resolve well the difficulties already posed against the
glucose-centered model and it did not gain further acceptance
as a general model for the control of feeding.
The thermostatic theory (Brobeck, 1948) had a different
rationale. Feeding is accompanied by an increase in body
temperature (prandial thermogenesis) originated mainly in
visceral tissues involved in the digestion and absorption of
food. This warming is a consequence of both an increase in
the rate of metabolic reactions and the reflex activation of the
sympathetic nervous system. Artificial warming of some
visceral organs, particularly the liver, has been shown to reduce
spontaneous feeding in rats, presumably to avoid excessive
warming. From this, it was proposed that food intake is
controlled by a visceral temperature-centered mechanism
based on the capacity of food to stimulate thermogenesis and
on the impact of this effect on the thermal balance of homeo-
thermic animals. Again, this thermostatic principle does not
seem to operate consistently in all situations and cannot, there-
fore, be considered a general mechanism of feeding control.
Food Intake and Its Control by Signaling Molecules 181
The theoretical models of hunger became gradually more
inclusive as they were enriched with the concepts of previous
theories and with new experimental evidence. In 1963
a comprehensive model was issued that explained hunger in
terms of the glucose reserves instead of the glucose concentra-
tion in blood. Glucose is a critical energy source, and metabo-
lism is clearly tuned to preserve glucose availability. Glucose
cannot be stored in large quantities because glycogen retains
water osmotically, and this effect multiplies the volume and
the weight of the reserve. Virtually all cell types keep a glycogen
reserve under these conditions to be used by the same cell.
Only the liver maintains a glucose reserve (the hepatic
glycogen) intended to provide glucose to other tissues during
the nonfeeding periods. This reserve is replenished from the
intestinal blood after feeding, and the glucose is gradually
returned to the circulation as the fasting period progresses. In
fact, the liver is irrigated by a special arm of the circulation,
the hepatic-portal system that conveys the intestinal blood
directly through the hepatic tissue before draining into the
general circulation. Hepatocytes express diverse enzymatic
mechanisms that can destroy, store, or transform metabolites
during their transit through the liver. Thus, the portal system
funnels all absorbed nutriments from the intestine first to the
liver, which can sense and modify blood composition before
it reaches other tissues. The liver is also the main site of glucose
synthesis and export when glycogen reserves are exhausted. It is
strategically situated to be a key element in energy metabolism
controlling the availability not only of glucose, but of all major
energy substrates in the blood.
The hepatostatic theory, proposed by Mauricio Russek in
1981, states that the control of hunger relies on the size of
the hepatic glycogen store, sensed by glucosensitive cells in
hepatic tissue. This information is conveyed to the brain
mainly by the hepatic branch of the vagus nerve and it is inte-
grated at specific brain regions to produce the proper response,
hunger or satiety. Satiety is maintained as a function of the
hepatic glycogen, and when this reserve is consumed beyond
a certain threshold, hunger begins to develop as a stimulus to
replenish it. At first the hunger sensation is mild but gradually
becomes more intense as glycogen wanes. Hepatic glycogen
lasts for several hours; its consumption is initially rapid but it
gradually slows down as glucose is spared at the expense of
other fuels, mainly fat. The glycogen reserve is completely
depleted after c.18 h of fast. The time frame of these events
roughly matches the chronology of spontaneous feeding.
The hepatostatic theory includes the central elements of the
glucostatic theory while it solves the theoretical difficulties.
Glycogen varies in a consistent way over time and it is
a much better indicator of glucose availability than blood
glucose itself. Most importantly, in the diabetic state, glycemia
is elevated at the expense of glycogen and amino acid reserves;
hepatic glycogen is persistently low in this condition, which is
consistent with a persistent craving for food. Finally, the liver is
a key factor for the dynamics of other metabolites such as fats
and amino acids. Information on the supply of other fuels can
thus be incorporated into the feeding response by the same
nerves from the liver to the brain; this possibility is excluded
if the brain itself acquires the information from blood glucose.
The most controversial aspect of the hepatostatic theory has
been the central role it attributes to the liver and its innervation.
182 Food Intake and Its Control by Signaling Molecules
Previous observations had established the importance of other
organs besides the liver in determining feeding behavior, for
example the stomach, the adipose mass, and the brain. It was
even debated how patients who suffered liver transplants
showed hunger–satiety cycles if the liver innervation had
been completely sectioned. Subsequent experiments favored
the view that the liver is a major controller of metabolism
and a strong determinant of feeding behavior, but it also sup-
ported the integration of information from other tissues to
form a comprehensive picture of the metabolic needs of the
body. The prevailing view was that the metabolic information
controlling food intake should come from different sources,
involving structures both inside (central) and outside (periph-
eral) the central nervous system.
In the 1950s stereotaxic surgery, an experimental technique
developed 10 years earlier, began to be applied to explore
experimentally the role of the brain in controlling feeding in
laboratory animals. Stereotaxic preparations make it possible
to manipulate a specific brain region while sparing neighboring
zones. The selective destruction of a certain cell group creates
a permanent condition allowing the effects of such lesions to
be thoroughly studied. Stereotaxic destruction of different
brain regions demonstrated that a group of hypothalamic
neurons, the so-called ventromedial hypothalamus (VMH)
was implicated in the production of satiety, for animals lacking
it ate beyond normal limits and developed obesity. The loss of
appetite, even leading to death, was observed after the destruc-
tion of a nearby region known as the lateral hypothalamic area
(LHA) (Anand and Brobeck, 1951). These results were inter-
preted as evidence that the brain contains a group of neurons
dedicated to causing hunger (a ‘hunger center’) and another
one to causing satiety (the ‘satiety center’). In the following
years a great deal of work was dedicated to collecting additional
data reinforcing the concept. A model was then proposed in
which hunger and satiety originate by the alternating inhibition
of these two antagonistic centers by each other. Moreover, the
model proposes that the centers themselves collect information
about the general metabolic state by means of glucosensitive
neurons measuring glucose concentration directly from blood
or cerebrospinal fluid, in much the same way proposed by
the glucostatic theory. This model is known as the dual-center
hypothesis. Understandably, the points of conflict previously
noted in the glucostatic theory were also present in the dual-
center hypothesis. In particular, this hypothesis again
conceived the control of feeding as based mainly, if not exclu-
sively, on glucose availability to the brain, thus neglecting all
the possible contributions made by the peripheral organs.
This conflicted with the main views at that time and initiated
a debate that motivated research in the field. The dual-center
hypothesis soon became widespread in spite of its various
weaknesses, probably because it offered a simple solution to
the problem of the origin of hunger and because it assumed
a brain-centralized control of the physiological functions of
the body in the way a man-designed mechanism would be
organized.
The controversy extended for years and derived basically
from two opposite perspectives: one asserting brain self-
sufficiency for collecting all pertinent information and gener-
ating the corresponding responses (a kind of ‘centralism’)
and another supporting the critical involvement of peripheral
organs in providing information for central integration and
even in coordinating general responses for which the brain
was only one of many effectors (a kind of ‘peripheralism’). In
this latter view, a major peripheral administrator was the liver,
much in the way proposed in Russek’s hepatostatic theory.
The subsequent incorporation of experimental evidence
and the corresponding evolution of ideas led to the ischymetric
theory, proposed by Stylianos Nicolaïdis in 1987. This theory
proposes that the essential factor in the control of food intake
is the potential contribution that the different fuel forms in the
body can make to the general energy budget. Conversion of the
chemical energy contained in the fuel molecule to cell-usable
ATP (i.e., the ‘power’ of the fuel) is then the critical variable
to be measured and maintained with little regard for the loca-
tion of the fuel. This theory still holds the liver as a prime
element, but it broadens the scope of the model to include
other reserve structures, particularly the adipose tissue. As can
be seen, the ischymetric theory did not resolve the central
versus peripheral controversy, but it incorporated and
condensed the main ideas at that time about the different
aspects involved in the physiology of hunger.
The central versus peripheral controversy was never
resolved. The debate was abandoned in 1994 when the newly
available molecular biology tools facilitated the discovery of
leptin. The next step was to find the main site of leptin action,
i.e., the location of leptin receptors, which turned out to be the
brain. From that time on, a series of studies gradually unveiled
the details of the mechanism by which the brain receives
peripheral information carried by hormones. A number of
previously unknown hormones have been described that
constitute an elaborate network of metabolic signals involved
not only in the control of food intake but also in the long-
term maintenance of energy balance and body weight and
how they are coordinated with massive, energy-consuming
processes such as growth and reproduction. The discovery of
leptin brought about the key element missing in Kennedy’s
lipostatic theory, i.e., a signal indicating the size of the fat
deposit. The discovery of the hunger hormone ghrelin, secreted
by the stomach, paradoxically reintroduced gastric fullness as
an important determinant of hunger in a way similar to that
conceived by Cannon’s gastrostatic concept.
The new data have been incorporated into a modern model
of the control of food intake. This model relies on the activity of
two neuron groups inside the hypothalamic arcuate nucleus
(ARC) at the base of the brain. These neurons receive hormonal
information from the blood and nervous information from
other areas of the central nervous system. One of the neuron
groups initiates a nerve pathway that has the overall effect of
promoting food intake (hence orexigenic group), and the other
reduces food intake by a similar route (hence anorexigenic
group). The orexigenic group inhibits the anorexigenic one.
The output of this integration is conveyed to other hypotha-
lamic areas, including the VMH and the LHA as second-order
centers. Integration successively involves other brain areas
and terminates in hormonal, nervous, and behavioral outputs
that determine the compensatory responses executed by the
effectors. These responses include the appetitive and the
consummatory phases of food intake. This new model has
been extensively adopted as an appropriate substitute for all
previous ones.

In some aspects, the modern model can be envisaged as a re-
adaptation of the dual-center hypothesis. The antagonistic
hunger and satiety centers have been replaced by the orexigenic
and anorexigenic neuron groups, though with the addition of
a sensory mechanism that gathers diverse peripheral informa-
tion. However, the participation of the peripheral organs is
again underestimated. The contributions made by the vagal
afferents and the brainstem integration are barely considered,
and the possibility of extra-brain control of feeding is simply
beyond the scope of the model. Clearly, the current model
for the control of food intake still needs to be improved.
The implicit purpose in constructing a model for the phys-
iology of hunger is to create a comprehensive structure that
includes all known factors affecting feeding behavior, describes
their interrelations, and predicts their effects (Figure 2). In the
past, the evolution of ideas progressed from theories based on
specific structures or signals to complex models encompassing
many elements. Involvement and interrelationship seem to be
fundamental features of physiological mechanisms, so there is
no reason to expect that the mechanism of food-intake control
should reproduce the straightforward design of a man-made
machine. The way we now explain feeding is predicted to
grow in complexity and inclusiveness over time.
1.06.3 Bioenergetics of the Fasting–Feeding Cycle
All heterotrophic organisms are subject to a metabolic rhyth-
micity dictated by the fasting–feeding cycle. Even in particular
circumstances, such as animals in a vivarium or on the farm
with nourishment available all the time, or many humans in
modern societies with food constantly accessible, the organ-
isms display a clear dichotomy in their behavior: a period of
resting/fasting and a period of activity/feeding. This rhyth-
micity is more pronounced in wild animals and in nonindustri-
alized human cultures where meal availability is uncertain on
a daily basis. From a metabolic and endocrine point of view,
these two periods are characterized by specific adaptations
that have important consequences for the bioenergetic status
of the biological systems. Recognizing that the fasting–feeding
cycle is continuous, we will start by describing the adaptations
associated with food ingestion.
Feeding triggers a complex set of coordinated responses that
recruit different organs as the digestive process progresses.
Digestion allows the complex nutrients ingested to be broken
down into biomolecules (carbohydrates, lipids, and amino
acids). The steps involved in this process take place in different
sections of the digestive track: (1) in the mouth the food is
macerated, and a set of amylase enzymes start to hydrolyze
polysaccharides, mainly starches; (2) from the mouth the
food is swallowed and transits to the gastric chamber through
the esophagus; (3) digestive juice secreted into the stomach
contains zymogens that are activated by the acidic environ-
ment, unmasking their hydrolytic activity toward proteins
and peptides; (4) the assimilative part of the digestive process
occurs in the first section of the small intestine called the
duodenum. In this segment the pancreatic and bile ducts
release pancreatic juice and bile via the ampulla of Vater in
an action controlled by the sphincter of Oddi. It is in the
duodenum where nutrients are simplified by the coordinated
Food Intake and Its Control by Signaling Molecules 183
action of bile acids and lipases, peptidases, and amylases,
producing mono- and disaccharides, free fatty acids, and glyc-
erol, as well as free amino acids and short peptides.
Assimilation of nutrients into the ‘internal medium’ of the
body occurs in two phases: triacylglycerides are packed by intes-
tinal cells into lipoproteins called chylomicrons which are
directed to lymphatic vessels. Eventually, chylomicrons reach
the blood circulation via the subclavian vein. All the other
nutrients are directed from duodenum to the liver by the portal
artery. In the liver, nutrients are first in contact with a special-
ized population of hepatocytes called ‘periportal’ hepatocytes.
This part of the liver is rich in metabolic pathways that need
a high concentration of O2, such as b-oxidation of fatty acids,
gluconeogenesis, and ureagenesis. Periportal hepatocytes also
show more numerous mitochondria and higher xenobiotic
processing as well as glutamine synthesis activity. Some meta-
bolic pathways such as glycogen synthesis do not show hepatic
‘zonation.’
When food is available, the anabolic reactions are favored
and hence, pathways such as those for the synthesis of
glycogen, fatty acids, triacylglycerols, cholesterol, and the
pentose phosphate pathway, are activated. This anabolic
‘wave’ is coordinated by a set of endocrine messengers, among
which insulin plays a prominent, but not unique, role. Other
hormones that are secreted in response to feeding are the intes-
tinal peptides tyrosine tyrosine (PYY) and cholecystokinin
(CCK), adipose tissue leptin, pancreatic polypeptide (PP),
gastric obestatin, etc. Overall, the endocrine response to
feeding promotes the synthesis of biomolecules and their poly-
mers, cellular growth and maturation, storage of caloric fuels,
and the message of satiety for the hypothalamus. Levels of gly-
cemia are increased.
Within the liver, triacylglycerides combine with other
components to form lipoproteins, in this case, very low density
lipoproteins (VLDL) which contain a high proportion of lipidic
material (more than 90%), and are exported to the circulation
for transport to other peripheral tissues and organs.
The definition of fasting varies. For some authors, fasting
starts as soon as the organism stops eating; for others, fasting
exists until the anabolic response associated with feeding
culminates and the metabolic fuels, glycogen and triacylglycer-
ols, are replenished. Most organisms are subject to a period of
fasting when they are sleeping/resting. This natural or physio-
logical fasting is usually enough to deplete the carbohydrate
reserves that form the hepatic glycogen deposits, releasing
glucose units to keep blood glucose at normal levels. Coordi-
nated with the depletion of glycogen, the liver responds by
activating the expression of gluconeogenic enzymes (phosphe-
nolpyruvate kinase, PEPCK, glucose 6-phosphatase, G6Pase,
pyruvate carboxylase, PC, and others) to allow the conversion
of nonglycosidic molecules (glycerol, alanine, lactate) to
glucose that is mainly released into the blood but can also be
incorporated into glycogen depots. These hepatic adaptations
are coordinated by endocrine regulators, mainly pancreatic
glucagon and the glucocorticoids produced in the suprarenal
gland cortex.
After the glycogen reserve is depleted, the next fasting adap-
tation that occurs is the mobilization of lipid deposits in the
adipose tissue. Activation of hormone-sensitive lipase activity
promotes the hydrolysis of triacylglycerides into fatty acids
184 Food Intake and Its Control by Signaling Molecules
and glycerol. Both molecules leave the adipocytes, enter the
circulation, and are captured by gluconeogenic tissues (mainly
liver) and cardiac muscle. In the liver, fatty acids are oxidized by
the coordinated action of mitochondria and peroxisomes to
yield energy substrates that fuel the Krebs cycle. In addition,
modified forms of fatty acids act as ligands of transcriptional
factors such as peroxisome proliferator-activated receptors
(PPARs) to orchestrate an adaptive fasting response. In the
liver, the excess oxidized fatty acids are channeled to the gener-
ation of ketone bodies (acetoacetate and b-hydroxybutyrate;
dimethyl ketone is formed by spontaneous decarboxylation
of acetoacetate). As a consequence, ketonemia levels increase
in the fasting condition. Ketone bodies serve as oxidizable
substrates, being incorporated into the Krebs cycle in the brain,
heart, kidney, skeletal and cardiac muscles, and the lactating
mammary gland.
Peripheral mechanisms for regulating appetite include the
motor functions of the stomach, such as the rate of emptying
and accommodation, which convey symptoms of satiation to
the brain. The rich repertoire of peripherally released peptides
and hormones provides feedback from the arrival of nutrients
in different regions of the gut from where they are released to
exert effects on satiation or to regulate metabolism through
their incretin effects. Ultimately, these peripheral factors
provide input to the highly organized hypothalamic circuitry
and vagal complex of nuclei to bring about cessation of energy
intake during a meal and the return of appetite and hunger after
fasting. Understanding these mechanisms is key to the physio-
logical control of feeding and the derangements that occur in
obesity and other pathological conditions related to feeding.
1.06.4 Hypothalamus and Feeding Control
1.06.4.1 General Perspective
The hypothalamus is a diencephalic structure formed by
a variety of nuclei (around 15 according to neuroanatomy text-
books) which play a dominant role in maintaining the internal
milieu. It is the anatomical substrate that controls important
physiological activities such as body temperature, thirst, sleep,
and circadian rhythmicity. Moreover, the hypothalamus is
a key element in regulating hunger and the energetic state of
the body (Lechan and Toni, 2013). Located in the tuberal
region, the ARC plays a preeminent role in the driving of
feeding and the ventromedial nucleus is one of the major brain
regions controlling satiety. The tuberomammillary nucleus
(TMN), situated in the posterior hypothalamus, also partici-
pates in feeding and the sensing of energy balance. Nuclei in
the anterior region that participate in the control of food intake
are the lateral nucleus, which contains orexigenic neurons that
project throughout the brain and spinal cord, and the supra-
chiasmatic nucleus (SCN), which is the major pacemaker that
controls circadian physiology (Buijs et al., 2006).
Classical ablation experiments demonstrated that bilateral
lesions of the extreme lateral part of the ventromedial nucleus
impaired food intake. In contrast, stimulation of this zone of
the nucleus promoted increased feeding. The medial part of
the ventromedial nucleus was also shown to be important in
feeding since its bilateral lesion was associated with hyper-
phagia, eventually leading to obesity (Swaab et al., 1993).
The ARC has a major role in the control of feeding. In this
hypothalamic region there are two populations of neurons
that integrate nutritional information: one expresses neuropep-
tide Y (NPY) and the Agouti-related peptide (AgRP), both
orexigenic factors; the other population expresses anorexigenic
peptides such as proopiomelanocortin (POMC) and the
cocaine- and amphetamine-regulated transcript (CART). All
these neurons are differentially sensitive to circulating
hormones such as leptin, insulin, peptide tyrosine tyrosine
(PYY), and ghrelin, and to metabolites such as glucose, fatty
acids, and amino acids (Blouet and Schwartz, 2010).
1.06.4.2 Neuronal Circuits
Anatomical regions participating in feeding behavior can be
viewed from different degrees of complexity, for example: as
a set of organs forming the digestive system; as individual
organs like the liver, with all its diverse and coordinated cellular
types; or as a set of specialized cells communicating with each
other, sending and receiving chemical messages. Additionally,
we can introduce the concept of assemblies of neuronal cells,
i.e., neuronal circuits that establish dynamic connections to
regulate diverse physiological responses such as feeding.
Considered simplistically, such neuronal circuits seem
linear, but in reality they form overlapping and interconnected
populations of neurons. Importantly, the neuronal resting
membrane potential and the action potential firing frequency
of the active circuits controlling feeding behavior are under
a dual regulation: (1) by the intrinsic biophysical properties
of each neuron and (2) by the integrated synaptic activity in
response to inputs from other neuronal circuits and from
peripheral signaling molecules such as leptin, insulin, CCK,
and others. Overall, the set of these communicating networks
integrate neuronal and endocrine stimuli, relevant to the
eating response, such as (1) satiety hormones from the gut,
(2) homeostatic sensors of energetic demands, (3) hedonic
and motivational drives, (4) taste and smell clues, and
(5) retrieved memory of previous experiences (Hirayama and
Gillette, 2012).
The principal circuits that regulate feeding and energy
metabolism are located in the hypothalamus and the
midbrain. Among the best-characterized chemical messengers
in these neuronal circuits are the orexigenic peptides NPY/
AgRP, which are secreted by neurons in the ARC to target nuclei
in other brain areas and the spinal cord. The anorexigenic factor
a-MSH (a-melanocyte stimulating hormone) is synthesized in
POMC neurons (within the arcute nucleus) and secreted
into the paraventricular nucleus of the hypothalamus (PVH),
parabrachial nucleus (PBN), dorsal vagal complex (DVC),
and intermediolateral column of the spinal cord (IML) (Valassi
et al., 2008).
Recent studies have begun to demonstrate a role for non-
neuronal cell types in the regulation of energy homeostasis.
In particular, the potential importance of different glial cell
types is increasingly being recognized. A number of studies
have described changes in the activity of hypothalamic macro-
glia (principally astrocytes and tanycytes) in response to states
of positive and negative energy balance, such as obesity and
fasting (Buckman and Ellacott, 2014). For example, endoge-
nous proteins with ‘benzodiazepine-like’ activity known as
 Food Intake and Its Control by Signaling Molecules 185

endozepines are downregulated in fasting conditions, whereas
cerebral administration of benzodiazepines reduces food
intake in rodents (Lanfray et al., 2013). Receptors for endoze-
pines are not well characterized. They have been reported to
bind with high affinity to the a-aminobutyric acid receptor
type A (GABAA) receptor as well as to recognize a mitochondrial
protein with translocation activity (Christian and Huguenard,
2013). Further studies are necessary to clarify the physiological
role of this pathway in the regulation of energy homeostasis.
1.06.4.3 Feeding Circuits in the Brain
Neuronal circuits involved in sensing peripheral signals of
energy balance from the body, and the regulation of feeding
itself are mainly located in the hypothalamus and the hind-
brain (Drazen and Woods, 2003; Blouet and Schwartz,
2010). Neurons from these areas are characterized by the
expression of receptors for adiposity signals such as leptin
and insulin, hunger signals such as ghrelin or hypoglycemia,
or to respond to postprandial increase of circulating levels of
nutrients or release of gastrointestinal peptides such as CCK
or NPY (Drazen and Woods, 2003; Wynne et al., 2005;
Kleinridders et al., 2009; Berthoud et al., 2012). Additionally,
other neuronal systems, like the corticolimbic and the reward
systems of the brain, participate in a nonhomeostatic regula-
tion of feeding (Woods, 1991; Berthoud, 2004, 2011). These
circuits are shown in Figure 3.
1.06.4.3.1 Homeostatic Circuits
Primary relay nuclei that receive peripheral signals for hunger
and satiation are located in the hindbrain and the ARC in the
mediobasal hypothalamus (Leibowitz and Wortley, 2004;
Sanchez-Lasheras et al., 2010). Neurons responding to gluco-
privation after intraventricular administration of 5-thioglucose
were localized in the hindbrain (Ritter et al., 1981), in catechol-
aminergic nuclei A1/C1 C2–C3, and in surrounding nuclei
such as the reuniens (Ritter et al., 1998). In addition, the area
postrema (AP) and the solitary tract nucleus (NTS) vagal sensi-
tive neurons are necessary to integrate the glycoprivic-induced
feeding (Ritter and Taylor, 1990). NTS/PVT projects to the
PBN in the pons and the paraventricular nucleus (PVN), the
ARC, and other areas of the hypothalamus (Juárez and
Tongju-Koh, 1978). The PB also projects to the PVN, ARC,
VMH, and dorsomedial hypothalamic nucleus (DMH) (Saper
and Loewy, 1980). These connections indicate that hindbrain
primary relay nuclei connect to hypothalamic primary relay
neurons and also to some of the secondary relay hypothalamic
neurons.
In the ARC a set of neurons coexpress AgRP/NPY, which
initiate feeding (Ollmann et al., 1997; Hahn et al., 1998; Gropp
et al., 2005; Luquet et al., 2005; Aponte et al., 2011). Another
neuronal group coexpresses POMC/CART which stops feeding
(Koylu et al., 1997; Schwartz et al., 1997; Kristensen et al.,
1998; Elias et al., 1999; Chen et al., 2015). AGRP/NPY neurons
in the ARC also contain GABA (Ovesjo et al., 2001; Meister,
2007), while GABAA receptors have been located both in
AGRP/NPY neurons in the ventromedial ARC and POMC/
CART neurons in the ventrolateral ARC. It has been proposed
that GABA release from ARGP/NPY neurons into POMC/
CART neurons in the ARC is part of the regulatory mechanism
of feeding (Cowley et al., 2001).
ARC neuronal populations containing AgRP/NPY or
POMC/CART express the growth hormone secretagogue
receptor (GHSR) (Guan et al., 1997; Willesen et al., 1999)
and the orexin A receptor (OXI1-R) (Guan et al., 2002; Suzuki
et al., 2002). In response to gherlin or orexin, AgRP/NPY
neurons show pacemaker bursts of action potentials driven
by low-threshold calcium conductances and a transient

Figure 3 Schematic representation of a sagittal view of rodent neural networks involved in the control and regulation of feeding behavior. Inputs or
primary relay nuclei are shown in red; integration or secondary relay nuclei are shown in light blue; efferent or third order relay nuclei are shown in
green. Modulatory neural networks involved in nonhomeostatic regulation of food intake are shown in brown. Arrows extending beyond the brain
represent peripheral inputs (red) and outputs (green), thin arrows represent neural projections while very thick arrows indicate hormonal signaling.
Abbreviations: Amy, amygdala; AP, area postrema; ARC, arcuate nucleus; DMH, dorsomedial hypothalamus; E, endocrine inputs/outputs; Hip, hippo-
campus; LA, lateral hypothalamus; NAc, nucleus accumbens; NTS, nucleus of the solitary tract; PBN, parabrachial nucleus; PFA, perifornical area;
PFC, prefrontal cortex; PS, parasympathetic system; PVN, paraventricular nucleus of the hypothalamus; PVT, thalamic paraventricular nucleus;
S, sympathetic system; SCN, suprachiasmatic nuclei; VMH, ventromedial hypothalamus; VTA, ventrotegmental area.
186 Food Intake and Its Control by Signaling Molecules
outwardly rectifying potassium conductance (van den Top
et al., 2004). ARC neurons also express leptin receptor (OB-
R) in both populations AgRP/NPY (Mercer et al., 1996;
Schwartz et al., 1996a) and POMC/CART (Cheung et al.,
1997; Elias et al., 1998a; Cowley et al., 2001). Leptin
administration inhibits electrical activity and mRNA
expression in AgRP/NPY arcuate neurons (Elias et al., 1999;
Higuchi et al., 2005) but increases electrical activity and
mRNA expression of POMC/CART neurons (Wilson et al.,
1999; Cowley et al., 2001; Cone, 2005). Since leptin inhibits
NPY/AgRP neurons in the ARC, the concomitant decrease in
GABAergic input to the POMC/CART neurons would
synergize leptin-induced activation in the latter.
Secondary relay nuclei integrating hunger and satiety receive
projections from both NPY/AgRP and POMC/CART neuronal
populations in the ARC. These secondary relays include the
lateral hypothalamus (LH), the perifornical area (PFA), the
VMH, the DMH, and the PVN. The LH was considered
the neural ‘center’ controlling hunger but nowadays neurons
synthesizing melanin-concentrating hormone (MCH) have
been described in the LH. Within the LH and PFA, neurons syn-
thetizing orexin (OXI) have been described. ICV administra-
tion of either peptide induces spontaneous feeding in
satiated animals (Qu et al., 1996; Sakurai et al., 1998). Both
MCH and OXI neurons in the LH and PFA express melanocor-
tin receptors MC3R-MC4R, stimulated by a-MSH and b-MSH
(peptides produced by posttranslational processing of
POMC), and inhibited by AgRP (Sánchez-Lasheras et al.,
2010). NPY receptors Y1 (NPY1-R) and Y5 (NPY5-R) are
present in all secondary feeding relay nuclei (Eva et al., 2006;
Higuchi, 2012). On the other hand, OXI neurons from the
PFN project to the ARC, PVN, DMH (Elias et al., 1998b;
Horvath et al., 1999; Suzuki et al., 2002), locus coeruleus
(LC), pedunculo pontine and laterodorsal tegmental pons
(PPT/LDT), and the TMN to regulate arousal (Siegel, 2004;
Saper et al., 2005; Harris and Aston-Jones, 2006) and its
relation to energy balance (Yamanaka et al., 2003). LH
neurons expressing orexin also project to the mesencephalic
ventral tegmental area (VTA) and to the shell region of the
nucleus accumbens (NAc), which participate in the brain
reward system, possibly involved in the effects of food as
a behavioral reinforcer.
The VMH has been implicated in the regulation of food
intake since the early 1940s; after a revision of its role on meta-
bolic regulation recent new evidence also supports its role in
food intake. Similarly, DMH neurons have been implicated
in food intake and body mass regulation as its lesion produces
hypophagia and hypodipsia and decreases body weight in indi-
viduals with normal body composition. It has been suggested
that the DMH functions as an interface between the LH and
the VMH which projects to the PVN, thus integrating informa-
tion related to feeding behavior with the autonomic and endo-
crine regulation of metabolism (Bernardis and Bellinger,
1998). The PVN has also a role in the regulation of ingestive
behaviors; the results of early experiments in which VMH
lesions deeply affected feeding have been reinterpreted and it
is now known that such VMH lesions were disrupting the
ventral noradrenergic bundle that connects the hindbrain
noradrenergic nuclei with the PVN (Gold, 1973). Furthermore,
injection of low doses of noradrenaline (less than 5 ng) in the
PVN induced feeding and drinking behavior (Leibowitz, 1978).
Similar but more potent effects were also found after PVN injec-
tions of NPY at picogram doses (Stanley and Leibowitz, 1985).
The current view points to a role of the PVN as part of the
output pathway that regulates the autonomic and endocrine
influence on the control of feeding (Leibowitz and Wortley,
2004).
Efferent or third-order relay nuclei that participate in the
output pathways for regulation of food intake and metabolic
energy balance involve mainly the dorsal and ventral medulla
and AP/NTS nuclei (Adan et al., 2008), the hypothalamic
projections from PVN to the median eminence, as well as its
projections to the autonomic sympathetic system in the spinal
cord (Jobst et al., 2004; Leibowitz and Wortley, 2004; Cone,
2005). Dorsal and ventral medulla and AP/NTS nuclei are
capable of integrating feeding responses. This was shown in
decerebrated animals, which discriminate taste (bitter from
sweet), increase sucrose intake in response to 24 h food depri-
vation or glucoprivation induced by 2-deoxyglucose or
5-thioglucose administration, and show a reduced intake after
direct sucrose injection into the stomach (Norgren and Grill,
1982; Flynn and Grill, 1983; Ritter and Taylor, 1990; Ritter
et al., 1994; Andrew et al., 2007; Darling and Ritter, 2009). It
has been shown that GLUT2-expressing neurons from NTS
comprise a distinct population of hypoglycemia-activated
neurons via increased AMP-activated protein kinase
activity and closure of leak Kþ channels (Lamy et al., 2014).
These receptors seem to be involved in the parasympathetic
response leading to glucagon secretion. On the hand, the PVN
projects to the median eminence from CRH- and thyrotropin-
releasing factor (TRF)-containing neurons (Palkovits, 1987;
Fliers et al., 1994) to control adrenocorticotropin (ACTH) and
TSH secretion from the pituitary in relation to energy balance
(Smith and Ferguson, 2008). On the other hand, a PVN
projection to the intermediolateral column of the spinal cord,
the NTS, and X nucleus in the medulla (Saper et al., 1976;
Luiten et al., 1985) is involved in the parasympathetic and
sympathetic regulation promoted by insulin and glucagon in
relation to feeding (McCabe et al., 1984; Weiss and Leibowitz,
1985; Thorens, 2015).
Other neural circuits involved in the regulation of feeding
are the reward system of the brain, which assigns a hedonic
value to food (Alonso-Alonso et al., 2015) and the corticolim-
bic regions, which regulate feeding in relation to food choice
and preferences (Berthoud, 2007). These areas include the orbi-
tofrontal, insular, and cingulate cortices, as well as the amyg-
dala and hippocampus (Berthoud, 2011; Parent et al., 2014).
The reward system lies in the mesolimbic dopaminergic projec-
tion from the VTA to the NAc and the prefrontal cortex. In turn,
these nuclei form a circuit feeding-back among them and
receiving inputs from the LH (Harris and Aston-Jones, 2006;
Adan et al., 2008). Furthermore, VTA neurons have insulin
receptors (InsR) that assign a reward value to food intake by
the release of dopamine into the NAc (Karatsoreos et al.,
2013). EC and opioid signaling at LH also participate in the
assignment of hedonic value to food (Di Marzo and Matias,
2005; Di Marzo et al., 2009; Alonso-Alonso et al., 2015; Liu
and Borgland, 2015). Corticolimbic areas are involved in the
cognitive and learning processes related to food recognition
and preferences which are particularly relevant for human

feeding behavior (Berthoud, 2004; Zheng et al., 2009). Thus
previous experience with food is represented in the orbitofron-
tal and insular cortices, while food- or feeding-related affective
information is processed and stored in the amygdala and the
hippocampus (Zheng and Berthoud, 2007). A neural circuit
which may integrate homeostatic information along with the
hedonic and affective value assigned to food includes the para-
ventricular thalamic nucleus (PVT), which is reciprocally con-
nected with the feeding areas of the brain stem and
hypothalamus and the NAc and amygdala (Li and Kirouac,
2008; Kirouac, 2015; Lee et al., 2015).
Finally, the SCN is the circadian clock controlling the
expression of behavioral and physiological circadian
rhythms in mammals (Morgado et al., 2015). The SCN
projects to hypothalamic nuclei involved in the regulation
of food intake and metabolism mainly the DMH, the subpar-
aventricular zone (SPvZ) and to the PVN (Watts and Swan-
son, 1987; Watts et al., 1987; Chou et al., 2003;
Abrahamson and Moore, 2006; Kalsbeek et al., 2010). The
SCN also projects to the sympathetic system via the ventro-
lateral medulla A5 which, in turn, projects to the intermedio-
lateral column of the spinal cord and then to the adrenal
medulla and other sympathetic outputs (Nagai et al., 1996;
Buijs et al., 1999). SCN regulation of hypothalamic nuclei
allows chronostatic regulation of feeding and metabolism,
as discussed below.
1.06.5 Chronostasis: Physiological Regulation
across Time
Feeding behavior starts when we feel hungry and ends when we
feel satiated. If only it were as simple as that! But when do we
feel hungry? The answer is quite complex, particularly in the
case of humans and other domesticated animals, but when
food is easily available – even in the wilderness – feeding
behavior starts because it is time. Such time or, rather, times
to hunt or collect and then to eat are initially selected consid-
ering the status of energy deposits and circulating levels of
nutrients (homeostatic process) and adjusted on the basis
of the effort involved in gathering food, the risk involved in
the process, and the preferences for a particular diet (nonho-
meostatic processes) (Lenard and Berthoud, 2008; Shin et al.,
2009; Berthoud et al., 2012). The latter involve cognitive
processes such as motivation, learning, and memory related
to previous experience (Alonso-Alonso et al., 2015). But
besides cognitive processes, the animal must be able to
measure the time of day. Circadian rhythms are fluctuations
in physiological and behavioral parameters which are repeated
almost every 24 h; these rhythms are generated by endogenous
biological clocks in living organisms (Menaker et al., 1978).
Circadian rhythms and their underlying clocks have evolved
in relation to a cyclic environment and provide adaptive rele-
vance by synchronizing the individual to daily environmental
cycles such as light–dark, temperature, and food availability
(Albrecht, 2012).
Circadian rhythms manifest themselves in physiological
regulation across time or chronostasis (Aguilar-Roblero and
Díaz-Muñoz, 2010; Aguilar-Roblero, 2015). Thus each physio-
logical system adjusts itself to different but optimal values
Food Intake and Its Control by Signaling Molecules 187
throughout the day, in a coordinated manner within the whole
organism. Chronostasis operates upon ongoing homeostatic
processes by updating the set point of physiological systems
according to a timetable determined by the circadian clocks.
In mammals the SCN is the only neural structure shown to
function as a biological clock, although the molecular oscilla-
tors driving the clock are present in most (if not all) cells in
the organism and are known as peripheral circadian oscillators
(Albrecht, 2012; Aguilar-Roblero et al., 2015). The set point has
been defined as the information used to compare the instant
value of a variable by a regulatory system in order to generate
an error signal to correct deviations from this value (Russek
and Cabanac, 1982). The coding of this information in the
system could result from the structure and dynamics of the
feedback loops in the regulatory system, such as endocrine or
neural networks. Alternatively, it could be represented in the
concentration of chemical signals (hormones or neurotrans-
mitters), the density of specific receptors or effectors, such as
ionic channels, or even the level of gene expression related to
all of the above.
The value of the set point for each physiological variable,
along with its daily variations, has resulted from natural
selection throughout thousands of years of evolution.
However, it is possible that nowadays such set point values
may be outdated, since during at least the last 1000 years
we have dramatically altered the environment, and adapta-
tion through natural selection occurs in a much larger time
scale. Frequent transoceanic flights, night work, and shift
work schedules are good examples of altered chronostatic
regulation. These alterations are due to outdated and time-
misaligned set points in relation to habits and demands of
modern lifestyle and they induce physiological changes
leading to obesity and metabolic diseases (Bass and
Takahashi, 2010; Buxton et al., 2012).
Chronostatic regulation of feeding involves SCN neural
outputs to the hypothalamic and autonomic neural networks
related to feeding and metabolism, which are responsible for
circadian rhythms in food intake, activity of digestive enzymes,
gastrointestinal motility and absorption, and hormonal secre-
tion of cortisol, insulin, and leptin (Kalsbeek et al., 2006;
Garaulet and Madrid, 2010; Kalsbeek et al., 2011). Further-
more, restriction of food availability to 2–3 h each day
entrains circadian rhythmicity of feeding behavior, endocrine
secretion, and liver metabolism (Escobar et al., 1998; Díaz-
Muñoz et al., 2000; Escobar et al., 2000; Aceves et al., 2003;
Ángeles-Castellanos et al., 2004; Martínez-Merlos et al.,
2004; Báez-Ruiz et al., 2005), even in SCN-lesion animals
(Stephan, 1983, 1989; Marchant and Mistlberger, 1997).
Such evidence has supported the idea of a food entrainable
oscillator whose location has been so far elusive
(Rosenwasser and Adler, 1986). This has lead us to propose
a distributed oscillator involving neural, endocrine, and
gastrointestinal elements (Aguilar-Roblero and Díaz-Muñoz,
2010) where ghrelin inputs to the DMH may play a major
role as an interphase between peripheral and neural
components (Mieda et al., 2004).
Recent advances in molecular genomics have shown that
chronostatic regulation of feeding may occur by direct interac-
tion among ‘clock genes’ and genes related to metabolism
which include per1, 2 and 3, cry1 and 2, dec1 and 2, rev-erb-a,
188 Food Intake and Its Control by Signaling Molecules
rora, bmal1 (also named arntl), and clock (Green et al., 2008).
Thus, mice with a mutation in the gen clock increase their food
intake during the day which decreases the amplitude of
feeding circadian rhythms. They also present increases in
blood levels of glucose, triacylglycerides, cholesterol, and lep-
tin and decreases in insulin; plus, they become obese (Turek
et al., 2005). On the other hand, mice with a mutation of
bmal-1 show a decreased insulin sensitivity throughout the
day and also become obese (Shi et al., 2013). These data
clearly indicate a chronostatic regulation of metabolism
which, if absent, leads to metabolic dysfunctions. Other exam-
ples of chronostatic regulation by interaction among clock and
metabolic genes include (1) SIRT, a deacetylase which senses
NADþ, and thus functions as a metabolic sensor; it binds to
Clock/Bmal1 to promote Per2 deacetylation and degradation
which, in turn, may amplify the oscillation in the transcription
of other clock-controlled genes (which contain an E-BOX in
their promoter region) (Nakahata et al., 2008; Grimaldi
et al., 2009); (2) cyclic AMP-activated protein kinase
(AMPK) also functions as a nutrient sensor due to its
sensitivity to the AMP/ADP/ATP ratio; by promoting
phosphorylation and dephosphorylation of different
proteins including CRY, it could function as an energy-
charge transducer to the circadian oscillator (Lamia et al.,
2009); and (3) the PPAR-1a (and also PPAR-b/d and PPAR-
g) a nuclear receptor functioning as a transcription factor
that regulates metabolism. It has been recently shown that
PPAR-1a binds PER while REV-ERBa is a target of PPAR-g
thus promoting bmal-1 expression via the PPAR gamma
coactivator 1-alpha (PGC-1a) (Liu et al., 2007; Eckel-Mahan
and Sassone-Corsi, 2013).
Figure 4 Alignment (a) and percent identity (b) of pre-pro-orexin (PPOX) peptides from different species of mammals by the Clustal Wallis
algorithm.
1.06.6 Neuropeptides
1.06.6.1 Neuropeptides Regulating Feeding Behavior
1.06.6.1.1 Orexins
The orexins, also named hypocretins are neuropeptides
described in the late 1990s. The family comprises two peptides:
orexin A with 33 amino acids and orexin B with 28 amino
acids. The sequence of orexin A contains four cysteines, and it
has been suggested that it forms two different interchain disul-
fide bonds (Sakurai, 1998). Both orexins originate from the
same precursor named prepro-orexin (PPOX), a peptide of
130 amino acids in rodents and 131 in humans. The gene
coding for this precursor is on chromosome 17 in humans
and on chromosome 10 in mice (De Lecea et al., 1998; Sakurai
et al., 1998). The PPOX sequence is highly conserved among
vertebrates; the human sequence shows 82% identity with
that of mice, 95% with macaca, and 39% with zebrafish
(Figure 4). Orexin function as a regulator of feeding has been
maintained since the origin of vertebrates.
The orexins were first described as the endogenous ligands
of an orphan G protein-coupled receptor (GPCR) named
OX1R (Sakurai et al., 1998). It is now known that orexins
have two specific receptors, OX1R and OX2R, also named
HCRTR1 and HCRTR2, both of which are preferentially
coupled to Gaq proteins and consequently to the PLC-IP3-
Ca2þ pathway (Voisin et al., 2003). Human OX1R and OX2R
display 64% amino acid identity (Sakurai et al., 1998).
The distribution of the mRNA encoding for OX1R and
OX2R in the rat brain showed marked differences; OX1R is
most abundant in the VMH nucleus, but it is also present in
the tenia tecta, dorsal raphe, and nucleus coeuruleus. OX2R

is expressed most abundantly in the paraventricular nucleus,
and it is also expressed in the cerebral cortex, NAc, subthala-
mic and paraventricular thalamic nucleus, and anterior pre-
tectal nucleus (Trivedi et al., 1998). Interestingly, dogs and
mice without a functional OX2R gene display a phenotype
similar to humans with narcolepsy (Lin et al., 1999; Chemelli
et al., 1999).
Orexinergic neurons in the LHA project their axons to
several regions of the cerebral cortex, limbic system, and brain
stem (Nambu et al., 1999). The distribution of orexinergic
neurons had been analyzed in detail by immunohistochem-
istry in slices of the rat brain. The main immunoreactive
neurons were detected in the hypothalamus, specifically in
the LHA, DMH, perifornical nucleus and posterior hypotha-
lamic area. The axons of these neurons are widely distributed
in the brain. The orexinergic terminals have been identified
in several regions of the hypothalamus, two of which, the
ARC and the paraventricular hypothalamic nucleus, are
strongly related to feeding control. The extrahypothalamic
nuclei showing orexinergic terminals include the cerebral
cortex, hippocampus, amygdala, brain stem, locus coeuruleus,
and raphe nuclei (Nambu et al., 1999).
1.06.6.1.2 Orexins and Feeding Control
The location of the orexinergic neurons in the LHA, a region
classically related to feeding that also contains other peptides
associated with appetite regulation (Bernardis and Bellinger,
1993), suggests that the orexinergic neurons have a role in
this function. Other seminal observations support an essential
role for these peptides in feeding control: (1) the intracerebro-
ventricular injection of orexin A or B in the light phase induces
an increase in food consumption (Sakurai et al., 1998) and
water intake (Kunii et al., 1999); (2) fasting for 48 h or acute
hypoglycemic manipulation induces an increase in the tran-
script of PPOX (Cai et al., 1999); and (3) the intraperitoneal
injection of the selective antagonist for OX1R, SB-334867-A,
reduces food intake in rats (Haynes et al., 2000). Feeding regu-
lation by orexins has also been documented in bullfrog larvae
(Shimizu et al., 2014) and zebrafish (Yokobori et al., 2011).
Genetic approaches have been applied to the study of
orexin; a knockout (KO) mouse for PPOX has been developed;
its most conspicuous phenotype was a severe sleep alteration
very similar to narcolepsy in humans (Chemelli et al., 1999;
Hara et al., 2001), revealing that orexinergic neurons display
complex functions including feeding behavior and sleep
control. The ataxin-3 gene product was isolated from
a Machado-Joseph disease patient under the control of the
PPOX gene promoter; using gene ablation techniques it was
possible to reduce the number of orexinergic cells selectively.
Like the PPOX/ mice, this transgenic mouse has sleep alter-
ations and narcolepsy, as well as metabolic alterations.
Although the transgenic mice show a decrease in food intake
in the first weeks after birth, adult mice are obese. The authors
explain that this apparent contradiction is the result of low
motor activity and/or a low metabolic rate and that the loss
of other secreted peptides regulating feeding might be partici-
pating in the induction of this phenotype in the transgenic
mice (Hara et al., 2001).
A new model with conditional genetic ablation employing
the tet-off system, where the transgene is expressed when
Food Intake and Its Control by Signaling Molecules 189
tetracycline is removed from the diet was recently applied to
study the orexinergic system. These mice conditionally express
diptheria toxin A under the control of the PPOX locus. When
the toxic transgene is expressed after puberty, a rapid loss of
orexinergic neurons was observed (80% cell loss in 7 days).
In addition to the marked sleep alterations and narcolepsy,
these mice were also obese and had a low rate of activity; their
levels of leptin and insulin were higher than those of control
animals but the levels of free fatty acids and glucose were not
affected (Tabuchi et al., 2014).
1.06.6.1.3 Orexins and Energy Regulation
The actions of orexinergic neurons involve an intricate web of
interactions that are only partially known. The orexinergic
terminals contact neurons relevant for feeding regulation.
Important interactions are those established with NPY- and
POMC-positive neurons of the ARC nucleus; thus, injection
of orexins into the ARC nucleus induces food intake. Moreover,
in isolated NPY neurons, orexins induce a rise in intracellular
Ca2þ ([Ca2þ]i), and in POMC neurons they induce the oppo-
site effect (Muroya et al., 2004). In agreement with this result,
food intake induced by intracerebroventricular injection of
orexin-A (10 nmol) is partially blocked by the administration
of BIBO3304 (60 mg), an antagonist of the Y1 receptor for
NPY (Yamanaka et al., 2000). These pieces of evidence suggest
that NPY- and POMC-expressing neurons are effectors of
orexins.
Orexinergic neurons are sensitive to nutritional status, since
fasting induces an increase in PPOX (Sakurai, 1998). Orexins
can also induce increases in glucose levels by interacting with
the sympathetic system (Yi et al., 2009). Indeed, orexinergic
neurons are able to sense glucose levels since, in isolated orex-
inergic neurons, glucose application induces hyperpolarization
and abolishes action potentials; conversely, glucose depriva-
tion induces depolarization and increases the frequency of
action potentials (Yamanaka et al., 2003).
Leptin, a hormone secreted by adipocytes that negatively
regulates feeding, also interacts with the orexinergic system.
The intracerebroventricular administration of leptin reduces
the hypothalamic expression of PPOX and of the OX1 receptor
and also inhibits the fasting-dependent increase in PPOX
mRNA (López et al., 2000). In agreement, leptin stimulation
of isolated orexinergic neurons, which express the leptin
receptor (Håkansson et al., 1999), has the direct effect of
reducing the electrical activity of these neurons (Yamanake
et al., 2003). Orexins can also regulate the activity of glucose-
responsive neurons, with effects opposite to those of leptin,
particularly in neurons from the VMH (Muroya et al., 2004).
Experimental evidence indicates that the orexinergic system is
part of a complex circuitry modulating feeding in an opposite
way to leptin.
Recent studies of orexinergic neurons with a pharmacoge-
netic technique named designer receptors exclusively activated by
designer drugs (DREADD), a method that makes possible the
selective activation or ablation of orexinergic neurons, have
reinforced a role for orexins in feeding behavior. The pharma-
cogenetic stimulation of orexinergic neurons induced an
increase in locomotor activity, food and water intake, and the
blood glucose level. When ablation was induced, it was
observed that the elimination of 73% of the orexinergic
190 Food Intake and Its Control by Signaling Molecules
neurons did not induce any changes; however, elimination of
83% of these same neurons resulted in decreased food and
water intake (Inutsuka et al., 2014). This study demonstrates
that the selective activation or elimination of orexinergic
neurons has clear effects on feeding.
1.06.6.2 Endocannabinoid System
The ECs are chemical messengers derived from arachidonic acid
(AA). All are eicosanoids produced by the conjugation of AA
with ethanolamide to form fatty acid amides (FAA), i.e.,
N-arachidonoylethanolamide (anandamide), or with glycerol
to form monoacylglycerols (MAGS), the best known of which
is 2-arachidonoyl-glycerol (2-AG) (Devane et al., 1992;
Mechoulam et al., 1995; D’Addario et al., 2014).
EC exert their action through binding to specific receptors
that belong to the GPCR superfamily. Two subtypes of EC
receptors have been described in vertebrates: CNR1 and
CNR2 (originally named CB1 and CB2); they were cloned in
the early 1990s from mouse, rat, and human (Matsuda et al.,
1990; Gerard et al., 1991; Munro et al., 1993). Both are coupled
to proteins Gi/o, and consequently to the negative regulation of
adenylyl cyclase; their activation modulates the activity of
mitogen-activated protein kinase (MAPK), inward-rectifying
potassium channels (Kir), A and D outward-rectifying potas-
sium channels, and N- and P/Q-type calcium channels (Mu
et al., 1999; Pertwee, 2005). In addition, CNR1 can also acti-
vate the Gs protein, thereby activating adenylyl cyclase (Chen
et al., 2010).
CNR1 is the main cannabinoid receptor expressed in the
nervous system. It has been reported to be present in neuron
terminals of the central and peripheral nervous system
(Egertová et al., 2003), but it is also expressed in some periph-
eral organs (reviewed in Maccarrone et al., 2014); CNR2
is mainly expressed in the immune system (Pertwee, 2005).
In situ binding studies in brain slices of several mammalian
species (rat, guinea pig, dog, rhesus monkey, and human)
using a high-affinity ligand named [H3]CP55,940 showed
specific binding mainly in the basal ganglia (the sustantia nigra
pairs reticulata and globus palidus), the hippocampus, and the
cerebellum (Herkenham et al., 1990). By in situ hybridization,
mRNA coding for CB1 was detected in neuronal populations of
the rat dorsal ganglia (Hohmann and Herkenham, 1999).
1.06.6.2.1 Endocannabinoids and Feeding Control
Early evidence demonstrated that D9-tetrahydrocannabinol
(Williams et al., 1998) or anandamide induces hyperphagia
(Williams and Kirkham, 1999). This role for cannabinoids is
conserved in chicken (Alizadeh et al., 2015).
There has been growing clinical interest in the EC system
since the introduction of the CB antagonist SR141716A (rimo-
nabant or AcompliaÒ) by the pharmaceutical company Sanofi-
Aventis for the treatment of obesity.
The study of the CNR1 KO mouse (CNR1/) highlighted
the role of components of the cannabinoid system in feeding.
Thus, the CNR1/ mice exhibit marker hypophagia, probably
induced by the disruption of the central control of appetite;
these mice also showed decreased body weight and reduced
fat mass, as well as adipocyte dysfunction. In addition, CNR1
KO mice exhibit lower levels of plasma leptin, but insulin
and glucose levels were normal. CNR1/ mice also display
important changes in hypothalamic neuropeptides, for
example, high levels of corticotropin-releasing hormone
(CRH) and the CART, which is also related to feeding control
(Cota et al., 2003).
Studies analyzing a second CB1/ mouse (Di Marzo et al.,
2001) indicate that, in response to 18 h of fasting, CB/
animals reduced their food consumption compared with
normal littermates, in agreement with a role for the CB1
receptor in this response. The administration of the CB1 antag-
onist SR1416A reduced food intake in the wild type but not in
the KO mice, also indicating a role for CB1 in the control of
feeding. This study outlined a functional interaction between
leptin, the main signal regulating the anorexigenic response,
and the EC system; thus, the administration of leptin in normal
rats induced a 50% reduction in the hypothalamic concentra-
tion of anandamide and 2-AG; in agreement, obese Zucker
rats deficient in leptin signaling displayed high levels of hypo-
thalamic ECs.
On the other hand, an interaction of the EC system with
ghrelin has also been shown; thus, blocking the CB1 receptor
in the hypothalamic paraventricular nucleus (PVN) induces
an increase in the fasting-induced hyperphagia and potentiates
the orexigenic effect of ghrelin. These observations suggested
that the PVN participates in the integration of orexigenic–
anorexigenic signals (Soria-Gómez et al., 2014).
A functional interaction between the cannabinoid and orex-
inergic systems has emerged from evidence demonstrating that
these two systems act synergistically to modulate feeding (for
detailed review see Flores et al., 2013). This notion arose
from the fact that CB1, OX1R, and OX2R have similar expres-
sion patterns in the central nervous system (Hervieu et al.,
2001; Mackie, 2005), and it is reinforced by the fact that CB1
and OX1R can form dimers (Hilairet et al., 2003) that,
regarding some functions, e.g., regulating energy balance,
constitute a functional unit (Crespo et al., 2008; Verty et al.,
2009; Cristino et al., 2013).
It has long been established that POMC neurons promote
satiety (Yang et al., 2011); however, recent findings, obtained
by applying DREADD to regulate the activity of specific
POMC neurons, suggested that POMC neural activity is neces-
sary for the CB1-dependent activation of food intake in the
satiety state (Koch et al., 2015). These observations reveal
that the new experimental approaches open novel ways to
study the details of feeding control circuits.
1.06.6.3 Melanocortin System
1.06.6.3.1 POMC-Derived Peptide Family
Existing evidence shows that the melanocortin system is
strongly associated with feeding; the melanocortins (a-, b-
and g-MSH), the ACTH, and b-endorphin (b-end) are a family
of peptides derived from one precursor named
POMC (Figure 5; Switonski et al., 2013). The POMC gene is
located on chromosome 2p23; it consists of three exons and
two introns, and its organization and processing have
been analyzed (Chang et al., 1980; Drouin and Goodman,
1980). In humans, POMC is expressed in corticotrophs of
the hypophysis; however, in other vertebrates it is also
expressed in melanotrophs, cells that are vestigial in humans

Figure 5 Posttranslational processing of proopiomelanocortin (POMC) peptide. Modified from Coll, A.P., Tung, Y.C.L., 2009. Pro-opiomelanocortin
(POMC)-derived peptides and the regulation of energy homeostasis. Mol. Cell. Endocrinol. 300, 147–151.
(Raffin-Sanson et al., 2003). POMC is also produced by a well-
defined neuronal cell population in the ARC nucleus (Civelli
et al., 1982). Other tissues expressing POMC are dermal micro-
vascular endothelial cells (Scholzen et al., 2000) and keratino-
cytes (Schauer et al., 1994).
POMC by itself is inactive; it is posttranslationally processed
by subtilisin-related proprotein convertases PC1-3 and the
expression of these proteins in a specific tissue determines the
presence of a specific, POMC-derived peptide. The processing
of POMC potentially generates various peptides of
physiological importance, for example ACTH, a-MSH,
b-MSH, and b-end. Thus, in the anterior pituitary where PC1
is expressed, the cleavage of POMC produces mainly ACTH;
in lower vertebrates the intermediate lobule of the pituitary
expresses PC2 and cleaves ACTH to produce a-MSH (Seidah
and Chretien, 1992).
In the hypothalamus, the most important structure for
feeding regulation, the 32-kDa precursor POMC is cleaved by
PC1 to generate pro-ACTH and b-lipotropin (b-LPH), and
pro-ACTH is cleaved again by PC1 to form ACTH, joining
peptide (JP), and N-POC. After that, PC2 cleaves ACTH to
generate ACTH1–17 and corticotropin-like intermediate lobe
peptide (CLIP), and it cleaves b-LPH to form a-LPH and
b-end; the processing of POMC in the hypothalamus is
specific, because the hypophysis does not express PC2.
Carboxypeptidase E (CPE), peptidyl a-amidating mono-
oxygenase (PAM), and N-acetyltransferase catalyze the
conversion of ACTH1-17 into mature a-MSH (Zhou et al.,
1993; reviewed in Pritchard et al., 2002).
1.06.6.3.2 Aguoti-Related Peptide (AgRP), a Natural
Antagonist of the Melanocortin System
An outstanding characteristic of melanocortin system is the
existence of natural antagonistic peptides; the most relevant
of these for food-intake control is AgRP. This peptide of 132
amino acids was originally cloned by two groups (Ollman
et al., 1997; Shutter et al., 1997). It displays a high similarity
with the Agouti protein present in the melanocytes that
produce the agouti-colored coat in mice; AgRP inhibits the
actions of a-MHS on MCR1 in skin cells.
AgRP is mainly expressed in the hypothalamus, and its
main function is to act as an antagonist of the MC3R and
MC4R melanocortin receptors, thereby inhibiting the actions
of a-MSH (Ollman et al., 1997; Yang et al., 1999). The
Food Intake and Its Control by Signaling Molecules 191
hypothalamic neurons expressing AgRP are located specifically
in the ARC nucleus, an essential structure for feeding regula-
tion. These neurons are also positive for NPY and they inner-
vate hypothalamic and extra-hypothalamic structures
(Broberger et al., 1998; Hahn et al., 1998).
1.06.6.3.3 Receptors of Melanocortin
The melanocortin family of peptides exerts its action by acting
on specific receptors. The first melanocortin receptor (MCR)
was cloned in the early 1990s and was designated the a-MSH
receptor (Mountjoy et al., 1992; Gantz et al., 1993). Currently,
the family of MCR contains five members (MC1R–MC5R) that
are encoded in autosomes by single-exon genes. All of them
belong to the GPCR super-family and they display differential
affinity for the natural agonist and antagonists, giving speci-
ficity to the system (Rodrigues et al., 2015).
In general, MCRs transduce their signals via the cAMP/PKA
system because they are coupled to Gs proteins. In addition,
MC3R is able to couple Go proteins and MC4R to Go and Gq
proteins (Newman et al., 2006); signaling through MC4R
also implicates activation of the PLC-IP3-Ca2þ pathway.
The expression of melanocortin receptors is tissue specific:
MC1R is mainly expressed in melanocytes and leukocytes,
MC2R in the adrenal gland, MC3R and MC4R in the nervous
system and MC5R in diverse tissues (Yang et al., 2011).
MC3R and MC4R are strongly related to feeding and energy
balance and mutations in MC3R are associated with type 2 dia-
betes and pediatric obesity (Mountjoy, 2010; Feng et al., 2005).
MC4R mutations are linked to morbid obesity in humans
(Loos, 2011).
1.06.6.3.4 Melanocortins and Feeding Control
The first insight that POMC participated in feeding regulation
was provided by the report of children lacking Pomc products.
These individuals developed different degrees of metabolic
deficiencies including hypocortisolemia, hyperphagia, and
obesity (Krude et al., 1998).
Animal models to study this deficiency were developed: KO
mice lacking all the POMC peptides have characteristics resem-
bling those observed in humans: hyperphagia, obesity, altered
pigmentation, and adrenal insufficiency (Yaswen et al., 1999;
Challis et al., 2004). Pomc/ mice also showed reduced
activity, low metabolic rate, and reduced activity of the
hypothalamus–hypophysis–thyroid axis (Challis et al., 2004).
192 Food Intake and Its Control by Signaling Molecules
Taking into account that deletion of the Pomc gene resulted in
the lack of several peptides, these mice were used to define
the importance of each one of the POMC-derived peptides
in the metabolic phenotype; thus, Pomc/ mice were treated
with intracerebroventricular injections of the different POMC-
derived peptides at the onset of the dark period, and the
anorexigenic effect was evaluated. a-MSH had the most
potent effect, reducing food intake by 65%; this peptide also
induced a decrease in body weight after 3 days of
administration (Tung et al., 2006). These observations
showed that a-MSH is the main POMC-derived anorexigenic
peptide. On the other hand, a potent orexigenic effect has
been described for AgRP when it was administered by
intracerebroventricular injection into the paraventricular
nucleus (PVN) (Wirth and Giraudo, 2000) or ARC nucleus
(Aponte et al., 2011; Krashes et al., 2011).
Feeding control is the result of the integration of several
signals; importantly, the actions of hormones regulating
diverse aspects of feeding and energetic state by acting on hypo-
thalamic neurons are now outlined. The functional interactions
between POMC and AgRP hypothalamic neurons and leptin,
ghrelin, and insulin have already been described.
Starvation and inhibition of leptin signaling induce a greater
increase in hypothalamic AgRP cDNA than in POMC mRNA
(Thornton et al., 1997; Mizuno et al., 1998). In fact, POMC
and AgRP neurons in the ARC nucleus express leptin receptors
and are the main target for the anorexinergic actions of this
hormone, since leptin induces depolarization of POMC
neurons (Cowley et al., 2001) and hyperpolarization of AgRP
neurons (van den Top et al., 2004).
Recent genetic restitution experiments in mice lacking the
expression of leptin (Lepob/ob) or its receptor (Leprdb/db)
showed that the recovery of leptin signaling specifically in
hypothalamic AgRP neurons, but not in POMC or SF1 neurons,
is sufficient to restore the regulatory actions of leptin on glucose
concentration (Gonçalves et al., 2014). This evidence demon-
strates that the hypothalamic melanocortin system, specifically
AgRP neurons, is essential to regulate the glucose levels in mice.
Another important element in the machinery of feeding
control is ghrelin, an orexigenic hormone secreted by a popula-
tion of enteroendocrine cells (Cummings et al., 2001). Impor-
tantly, the injection of ghrelin into the ARC nucleus induces
a strong food-intake response (Nakazato et al., 2001; Wren
et al., 2001). This hypothalamic structure expresses ghrelin
receptors (GHRS), mainly in AgRP-positive neurons
(Kamegai et al., 2000). These observations led to the
proposal that ghrelin actions are mediated, at least in part, by
AgRP hypothalamic neurons; this hypothesis was supported
by the fact that ghrelin modulates the electrical activity of
AgRP neurons (Cowley et al., 2003). Recently, using directed
genetic restitution in mice lacking GHRS expression, it was
shown that AgRP neurons are sensitive to ghrelin signaling
(Wang et al., 2013).
On the other hand, deletion of InsR in the brain was found
to induce obesity and increase food intake of female mice
(Brüning et al., 2000), raising the possibility that insulin acts
directly on the CNS. This idea was tested by the development
of transgenic mice with selective inhibition of the InsR in
POMC or AgRP hypothalamic neurons. Although no direct
effects on food intake or body weight were observed, the
mice lacking InsR expression in AgRP neurons had altered
hepatic glucose production (Könner et al., 2007), revealing
that these neurons are elements of a long chain that participates
in integrating information from the whole organism.
1.06.6.4 Neuropeptide Y
The hypothalamic peptide named NPY was discovered in the
early 1980s, in extracts of porcine brain (Tatemoto et al.,
1982). NPY, peptide YY (PYY), and PP belong to the same
family; NPY is most abundant in the central and peripheral
nervous system (Pedragosa-Badia et al., 2013) and is highly
expressed in the brain stem, in the hypothalamus, and in the
adenohypophysis. Hypothalamic neurons expressing NPY
have been detected in the ARC and DMH nuclei (Chronwall
et al., 1985; Bi et al., 2001).
NPY receptors (NPYR) belong to class A (rhodopsin-like) of
the GPCR superfamily; in mammals five subtypes are currently
known: Y1, Y2, Y4, Y5, and Y6; in humans Y6 have not been
reported. All subtypes are coupled to Gi/o heterotrimeric
G-proteins and, in consequence, their activity induces
a decrease in the concentration of cAMP; through a Gbg
dimer they regulate Ca2þ and Kþ ion channels (Pedragosa-
Badia et al., 2013).
The NPYR are expressed in a tissue-specific pattern, and the
location and receptor subtype in the CNS determines their
physiological function. In accord with their widespread expres-
sion, they participate in diverse processes, e.g., learning and
memory, anxiety, and circadian rhythms (Mercer et al.,
2011). The Y4 and Y5 receptors are noteworthy for the orexi-
genic actions of NPY since their expression has been described
in the ARC and VMN hypothalamic nuclei. Thus, Y5 transcript
was identified in the hippocampal dentate gyrus, in the CA1
area, and in the ARC nucleus (Naveilhan et al., 1998); Y4 tran-
script was found in the brain stem, solitary tract nucleus (NTS),
the vagal complex, and the ARC and VMN nuclei (Parker and
Herzog, 1999).
The projections of NPY-ergic neurons connect some
hypothalamic nuclei such as the PVN, VMN, LHA, and DMN
(Mercer et al., 2011) to establish an informational web.
1.06.6.4.1 NPY and Feeding Control
A role for NPY in food intake has been demonstrated by diverse
experiments. Thus, the acute intracerebroventricular injection
of NPY induces an increase in food intake (Clark et al.,
1985), and chronic administration of the peptide leads to
obesity (Zarjevski et al., 1993). In agreement, NPY levels are
increased in the hypothalamus of obese rats (Dryden et al.,
1995), and conditions of low energy, such as food restriction
or fasting, induce elevated hypothalamic NPY levels (Brady
et al., 1990).
NPY exerts its orexigenic action by directly regulating the
anorexigenic POMC neurons which express NPYR. Neurons
expressing NPY in the hypothalamus also express AgRP and
GABA, and these neurons release NPY in the postsynaptic
POMC neurons, negatively regulating their electrical activity
(Hahn et al., 1998; Cowley et al., 2001; Roseberry et al.,
2004; Acuña-Goycolea et al., 2005).
Although the first mice lacking NPY expression (NPY/)
did not manifest altered feeding phenotypes such as obesity

or fat accumulation (Erickson et al., 1996), this genetic back-
ground sensitized the pathway related to feeding. Thus, the
cross between NPY/ with the leptin-deficient mice ob/ob
attenuated notably the obesity syndrome that resulted from
the deletion of leptin expression (Erickson et al., 1996).
It is well known that the blood–brain barrier in the ARC
nucleus is semipermeable, allowing actions of the feeding-
control hormones leptin and insulin. Leptin inhibits NPY/
AgRP neurons of the ARC nucleus and stimulates POMC
neurons. The result is a potent anorexigenic response (Ahima
et al., 1996); similar findings had been described for insulin,
a hormone produced by b-pancreatic cells (Schwartz et al.,
1992). However, it is not possible to generalize the actions of
hypothalamus NPY and AgRP neurons, since a study applying
genetic ablation techniques, using transgenic mice expressing
the diphtheria toxin receptor in the brain under the regulation
of the promoter of AgRP showed that the populations of AgRP
and NPY neurons in the hypothalamus are not identical
(Luquet et al., 2005), suggesting differential roles in feeding-
behavior regulation.
To define the contribution of the NPYR subtypes in feeding
regulation, some studies have used KO animals. Y4 KO mice
were crossed with ob/ob mice, but the obese phenotype of ob/
ob was not affected (Sainsbury et al., 2002). Meanwhile, Y5
null mice develop mild late-obesity characterized by elevated
food intake, adiposity, and body weight, unchanged re-feeding
after fasting or sensitivity to leptin, and the absence of a response
to intracerebroventricular injection of NPY (Marsh et al., 1998).
These results demonstrated that the Y5, but not the Y4, receptor
mediates the actions of NPY on feeding.
A recent study showed that germline ablation of the
expression of both the Y1 and Y5 receptors (Y1Y5/) results
in hypophagia. When the ablation was directed to the hypo-
thalamus (Y1Y5 (hyp/hyp)), despite hypophagia, the mice
showed increased body weight and fat accumulation possibly
due to compensatory mechanisms; however, these results did
reveal a role for Y1 and Y5 in food-intake regulation (Nguyen
et al., 2012).
1.06.7 Endocrine Systems: Hormones in Fasting
and in the Feeding Response
1.06.7.1 Ghrelin and Leptin Biology and Their Role in Food-
Intake Regulation
1.06.7.1.1 Biology: Synthesis, Secretion, Physiology, and
Signaling
Ghrelin from the Proto-Indo-European language root ghre,
meaning grow, was purified in 1999 from rat and human stomach
extracts with activity as a growth hormone- (GH-) releasing
peptide. Moreover, it proved to be the endogenous ligand for
the GH secretagogue receptor (Kojima et al., 1999). Ghrelin is
a peptide hormone of 28 amino acids (molecular weight of
3.37 kDa) with a lipid moiety, an n-octanoyl group, on serine
three; it is designated acylated ghrelin (AG). This serine acylation
is catalyzed by the ghrelin O-acyltransferase (GOAT) enzyme,
a membrane-bound acyltransferase of the endoplasmic
reticulum (Yang et al., 2008).
Ghrelin is encoded by the GHRL gene; in humans it is
located on the short arm of chromosome 3 (3p25–26). The
Food Intake and Its Control by Signaling Molecules 193
GHRL gene encodes six exons, four of which code for preprogh-
relin mRNA products of 117 amino acids, which include the
peptides: AG, UAG (unacylated ghrelin), and obestatin (Seim
et al., 2010; Wajnrajch et al., 2000); however, it also contains
transcriptional variants. The promotor region of the ghrelin
gene has several consensus sequences for a number of transac-
tivating regulatory proteins (e.g., upstream stimulatory factor
1/2) and includes an E-box site (Kanamoto et al., 2004).
Finally, ghrelin gene expression in human tissue is
widespread in endocrine and nonendocrine tissues, with the
highest ghrelin mRNA levels found in the stomach fundus
(Gnanapavan et al., 2002).
Ghrelin is also present in the unacylated isoform or de-
octanoylated (UAG). UAG comprises 70–90% of total plasma
ghrelin (AG plus UAG), which is partially explained by the
short half-life (<8 min) of AG (Hosoda and Kangawa, 2004;
Kojima et al., 1999). Ghrelin biosynthesis and secretion occur
primarily in the stomach, specifically in endocrine cells of the
oxyntic gland; however, it is also produced by other cells and
organs such as gut, alpha and epsilon cells of pancreatic islets,
placenta, kidney, testis, lung, and the hypothalamic
ARC (Ghelardoni et al., 2006; Granata et al., 2010; Kanamoto
et al., 2004; Kojima et al., 1999). In rats, ghrelin-containing
cells are localized in the large curvature of the pars glandularis
and also in the small stomach curvature, while ghrelin-
containing cells are distributed throughout the human
stomach, although mainly in the fundus (Li et al., 2011).
The regulation of ghrelin secretion has not been completely
elucidated. Fasting, CCK-octapeptide, and acetylcholine stimu-
late total ghrelin release (Shrestha et al., 2009); in contrast,
inhibitory stimuli are feeding, hyperglycemia, insulin, and
somatostatin (Sakata et al., 2012; Shrestha et al., 2009). In
pathological conditions such as obesity, ghrelin circulating
levels are reduced, whereas they are increased by malnutrition
or cachexy (Veldhuis and Bowers, 2010). Basal plasma ghrelin
levels are usually lower in obesity and higher in individuals
suffering from anorexia nervosa (Germain et al., 2007; Yildiz
et al., 2004).
Ghrelin is secreted in a pusatile pattern, and it is released in
larger quantities before meals, in agreement with its important
role for meal initiation (Cummings et al., 2001; Kojima and
Kangawa, 2005). In rodents, the daily profile of circulating
AG or UAG is distinctly modulated in response to feeding para-
digms (Arellanes-Licea et al., 2014; Bodosi et al., 2004) and the
AG receptor participates in regulating food anticipatory activity
under conditions of regularly scheduled feeding (Blum et al.,
2009; LeSauter et al., 2009).
Beyond their metabolic functions, both ghrelin isoforms
have pleiotropic ones, ranging from cell proliferation to cogni-
tive processes (Müller et al., 2015; Seim et al., 2010). Even
though the UAG receptor(s) has not yet been characterized,
UAG downregulates lipogenic gene expression in the absence
of the GHSR in muscle and white adipose tissue (WAT) of
mice (Delhanty et al., 2010). In the digestive system, some of
the functions of AG and UAG are antagonistic. AG controls
gastric motility, augmenting its tone, while UAG decreases it;
HCl secretion is stimulated by AG, but UAG, does the opposite.
In pancreatic islets, AG modulates glucose metabolism and the
sensitivity to insulin secretion depending on its concentration
and metabolic state. AG, but not UAG, inhibits insulin
194 Food Intake and Its Control by Signaling Molecules
secretion and the latter reverts AG insulin-desensitizing effects
(Delporte, 2013; Gauna et al., 2007; Salehi et al., 2004). In
the cardiovascular system, the two ghrelin isoforms regulate
cell proliferation and blood pressure (Delporte, 2013; Li
et al., 2006). The third gene product of the ghrelin gene, obesta-
tin, might have effects opposite to those of AG, reducing food
intake; however, debate exists as it might not affect food-intake
regulation (Murphy and Bloom, 2006).
AG stimulates hypophyseal GH secretion directly or by
modulating GH-releasing hormone (Feng et al., 2011; Kojima
et al., 1999; LeRoith et al., 2001). Further, AG causes a positive
energy balance, i.e., a surplus of energy stores in the organism,
that mechanistically could be explained by the adipogenic
effects of AG, as it promotes the proliferation of adipocytes
from WAT (Lanfranco et al., 2010; Veldhuis and Bowers,
2010; Wells, 2009). Another explanation is based on the
relevant orexigenic role of AG in central feeding regulation
(Murphy and Bloom, 2006; Tschöp et al., 2001), explained in
the following sections.
Ghrelin was originally discovered as the natural ligand for
the GHSR, an orphan receptor known to bind drugs that
increased the release of GH from the pituitary gland (Howard
et al., 1996; Kojima et al., 1999). GHSR belongs to the G
protein-coupled receptor family, and it occurs as isoforms 1a
and 1b. GHSR-1a is functional, possesses seven transmem-
brane domains; it is highly expressed in somototrophs, the
ARC nucleus, and WAT and, to a lesser extent, in thyroid,
pancreas, spleen, myocardium, adrenal gland, testis, ovary,
and liver. It has a Kd of 0.1 nM; however, tissue distribution
in humans of mRNA for isoform 1b is broader than for 1a,
although the former is a truncated, nonfunctional isoform
(Gnanapavan et al., 2002; Davenport et al., 2005; Wells, 2009).
GHSR-1a has several signaling mechanisms, including the
activation of distinct kinases and constitutive activity (Yin
et al., 2014). In pituitary and other targets, this receptor acti-
vates phospholipase C (PLC) through the G protein G11a,
producing inositol 1,4,5-trisphosphate (IP3) and diacylglycerol
(DAG). IP3 binding to the IP3 receptor causes Ca2þ release from
intracellular Ca2þ stores, while DAG activates protein kinase C
(Yin et al., 2014). In NPY-positive ARC neurons, the IP3
receptor activates cAMP/protein kinase A (PKA) through G
protein Gas, leading to ion channel modulation that increases
the intracellular Ca2þ concentration (Kohno et al., 2003). In
the VMH nucleus region, AG regulates energy balance through
AMPK (AMP-dependent kinase), inhibiting fatty acid
biosynthesis (López et al., 2008).
Finally, a number of genetically altered mouse models have
been developed that lack or overexpress either GOAT, GHSR, or
ghrelin isoforms, and upon diverse feeding protocols, they
have yielded useful information about the ghrelin system
(Kirchner et al., 2009; Sun et al., 2003; Zigman et al., 2005).
Additionally, ghrelin mimetic drugs show promise as treat-
ments for GH deficiency or gastrointestinal disorders (Müller
et al., 2015).
1.06.7.1.2 Leptin
1.06.7.1.2.1 Biology: Biosynthesis, Secretion, Physiology, and
Signaling
Leptin, from Greek leptos meaning thin, has anorexigenic and
weight-diminishing effects; it was discovered and cloned in
1994 as a protein hormone secreted by adipocytes (Halaas
et al., 1995; Zhang et al., 1994). The discovery of leptin came
in part from the existence of mutant obese rodents. One was
a mouse colony with an obese phenotype at the Jackson Labo-
ratory in 1950, designated ob/ob. Another strain had a pheno-
type similar to ob/ob but was also markedly diabetic and it was
termed db/db; both mouse strains were infertile and lethargic
(Coleman, 1978; Friedman and Halaas, 1998). It was later
found that the genes that were mutated in these animals were
the ligand leptin (ob) and the long leptin receptor isoform
(db) genes (Friedman and Mantzoros, 2015).
The genes ob (obese) and Lep (leptin) are localized on chro-
mosome 7 in humans and chromosome 6 in mice (Green et al.,
1995). Leptin gene expression has been detected in WAT,
stomach, liver, heart, placenta, pituitary gland, and brain
(Denver et al., 2011; Green et al., 1995). The profile of leptin
gene expression in rat WAT is circadian, with a peak during
the dark phase (Saladin et al., 1995).
Leptin is a protein with 167 amino acids, a molecular
weight of 16 kDa, and a crystal structure homologous to the
members of the long-chain helical cytokine family with a 4-
helix bundle (Zhang et al., 1997), proteins with a high degree
of conformational conservation among mammals. Leptin
expression is broad; its main site of biosynthesis is WAT,
although it is also present in brown adipose tissue, skeletal
muscle, placenta, ovaries, bone marrow, gastric and mammary
epithelium, and brain (Ahima and Flier, 2000; Münzber and
Morrison, 2015). In epididymal WAT of rats, leptin is continu-
ously synthesized and secreted from secretory vesicles;
however, de novo synthesis is required for a long-term leptin
secretion (Cammisotto et al., 2006). In this sense, constitutive
leptin secretion is the main secretory pathway (Bradley et al.,
2001). Leptin synthesis is regulated by several factors, in a posi-
tive fashion by insulin, glucocorticoids, tumor necrosis factor-
a (TNF-a), and estrogens, and negatively by catecholamines,
androgens, and b-adrenergic agonists (Bradley et al., 2001;
Cammisotto et al., 2006; Saladin et al., 1995). Moreover, leptin
production is sensitive to the size of the adipose depot in obese
subjects; subcutaneous adipocytes are more responsive to
insulin than to glucocorticoids, but the reverse is true for
omental adipocytes (Russell et al., 1998), which also present
gender differences. Male adipose tissue responds only weakly
to steroid hormones, as compared to female adipose tissue
(Casanueva and Diéguez, 1999).
Leptin circulates as a free form or bound to the soluble lep-
tin receptor; in lean subjects up to 98% of total leptin is in the
bound form while in obese ones the majority of leptin is in the
free form (Sinha et al., 1996). The half-life of leptin in humans
is approximately 75 min (Hill et al., 1998). Leptin is expressed
and secreted by mice or human adipocytes, regardless of their
location (retroperitoneal, omental, or subcutaneous); however,
there is more leptin synthesis in subcutaneous than in visceral
WAT (Casanueva and Diéguez, 1999; Ahima and Flier, 2000).
The level of plasma leptin is positively correlated with body
mass index in lean and obese humans and in rodents (Maffei
et al., 1995), i.e., levels of this hormone are related to body
fat, and its circulating levels are higher in obesity (Considine
et al., 1996). The circulating concentration of leptin is sexually
dimorphic; it is higher in females than in males with a similar
body fat mass, an effect attributed to regulation by sexual

hormones or to different proportions of subcutaneous fat
(Rosenbaum and Leibel, 1999; Ahima and Flier, 2000).
Leptin expression and circulating levels show circadian fluc-
tuations, and they also change with nutritional state; in fact,
leptin circulating levels decrease in mice during short-term star-
vation (Saladin et al., 1995; Ahima et al., 1996). Plasma leptin
has a diurnal rhythm, with the highest concentrations at
midnight and entrained to meal time (Schoeller et al., 1997).
Leptin is an important player in energy homeostasis; the
mechanism for its essential role in decreasing appetite and aug-
menting energy expenditure will be explained in the following
section. In addition, leptin has numerous physiological roles in
metabolism as an adiposity signal, affecting reproductive and
immune function, hematopoiesis, and brain and bone devel-
opment (Ahima and Flier, 2000; Friedman and Mantzoros,
2015). In fact, leptin forms a linkage between nutritional status
and neuroendocrine and immune functions (La Cava and
Matarese, 2004). The anorexigenic role of leptin has been
evolutionarily conserved in vertebrates, suggesting that its
effects on energy balance regulation are ancient (Denver
et al., 2011).
Leptin participates in fertility regulation and it is a permis-
sive factor for puberty (Casanova and Dieguez, 1999). Leptin
also functions as an essential factor for brain development,
promoting formation of hypothalamic pathways that later
convey leptin signals to brain regions regulating food intake
and energy consumption (Bouret et al., 2004). In the immune
system, leptin is an immune mediator and is considered
a hormone/cytokine, affecting a number of immune functions;
for instance, this hormone modulates experimental autoim-
mune diseases and regulates secretion of interleukin-1 and
tumor necrosis factor (La Cava and Matarese, 2004).
Further, the central action of leptin and insulin on hypotha-
lamic ARC POMC neurons in mice promotes the development
of beige adipocytes in WAT and weight loss (Dodd et al., 2015).
Recently reviewed (Stieg et al., 2015), leptin has been associ-
ated with neuropathologies due to its neuroendocrine interac-
tions. It has antidepressant effects and the decrease of leptin
levels in dementia facilitates neurodegeneration.
The leptin receptor (LR) is encoded by the lepr gene, with 17
exons. In mice there are six LR isoforms, products of alternative
splicing, named LRa to LRf, that belong to the categories:
secreted, short and long, all sharing the leptin-binding domain.
The secreted forms may regulate the concentration of free leptin
and the transport of short leptin across blood–brain barrier
(BBB). It is the long form, LRb (or LepRb), that is crucial for lep-
tin signaling in db/db mice, as they only lack this isoform
(Myers et al., 2008; Münzberg and Morrison, 2015).
The db gene for the leptin receptor was cloned in 1996 (Chen
et al., 1996; Leibel, 2008). Human leptin long receptor has 1165
amino acids, and its expression is highest in neurons within
nuclei of the basomedial hypothalamus, i.e., the ARC, dorsome-
dial hypothalamic and VMH nuclei, and LHA. It is also present
in other nervous system regions such as the VTA and the nucleus
of the solitary tract (NTS) and, to a lesser extent, in the liver,
kidney, lung, stomach, pancreatic beta, and immune cells (Myers
et al., 2008; Denver et al., 2011). LRb belongs to the class I family
of cytokine receptors and it possesses three functional domains:
an extracellular ligand-binding, a single transmembrane, and
a cytoplasmic-signaling domain without intrinsic kinase activity.
Food Intake and Its Control by Signaling Molecules 195
Intracellularly, it signals through the Janus kinase family (Jak2)-
signal transducers and activators of transcription (STAT3/5)
signaling pathway, among others (Myers et al., 2008; Münzberg
and Morrison, 2015).
Ligand binding allows transphosphorylation and activation
of Jak2 which, in turn, phosphorylates Tyr residues in a complex
signal transduction phosphorylation pattern. In fact, mutations
of specific signaling sites on the leptin receptor in mice have
provided information about its in vivo phenotype and functions
and also suggested a targeted therapy for obesity and type 2 dia-
betes (Myers, 2010). LRb activates a number of downstream
mechanisms, such as the ERK (extracellular regulated kinase)
signaling pathway; STAT3 activation and nuclear translocation
for regulation of transcription, including a feedback inhibitor
of this pathway, the SOC3 (suppressor of cytokine signaling
3); STAT5 or PI3K (phosphatidylinositol 3-kinase) (Bates
et al., 2003; Myers et al., 2008; Münzberg and Morrison,
2015). Hypothalamic neuropeptides that participate in food-
intake regulation and are modulated by LRb include POMC,
CART, AgRP, and NPY (Korner et al., 2003; Kristensen et al.,
1998; Schwartz et al., 1996b, 1997).
Leptin resistance is a condition in which hyperleptinemia
in obese subjects is unable to activate the leptin receptor,
suppress feeding, and prevent or mitigate obesity (Myers
et al., 2008). Several mechanisms have been suggested to
explain this failure, such as the involvement of leptin transport
by the brain–blood barrier (BBB), alterations in the cellular
LRb signal, neurodevelopmental hypothalamic programming
by leptin or even a population of ARC/LRb neurons with
increased sensitivity to circulating leptin prone to develop
cellular leptin resistance (Bouret and Simerly, 2006;
El-Haschimi et al., 2000; Faouzi et al., 2007; Myers et al.,
2008). Yet, this is still an unsolved issue.
1.06.7.1.3 Food-Intake Regulation by Ghrelin and Leptin
Although both ghrelin and leptin have been linked to the
cephalic-phase response in the anticipatory regulation of
feeding (Power and Schulkin, 2008), one of the main actions
of AG and leptin in the counterregulation of food intake, is
via the central NS, particularly through the modulation of
ARC subpopulations of neurons with marker neuropeptides.
On one side, a ventromedial population that coexpresses the
36-amino acid peptide, NPY (Muroya et al., 1999; Tatemoto
et al., 1982), and the 112-amino acid protein, AgRP (Gropp
et al., 2005; Luquet et al., 2005) that coexists with a GABAergic
system (Meister, 2007); on the other side, a ventrolateral pop-
ulation that colocalizes with the 241-amino acid precursor
POMC that is cleaved to produce and secrete the melanocortin
a-melanocyte-stimulating hormone (a-MSH) and the CART,
that coexists with a glutamatergic system (Meister, 2007;
Millington, 2007; Figure 6).
Anatomically, the ARC nucleus is outside of the BBB, and it
expresses receptors for both leptin and AG. Moreover, AG is
locally produced in neurons of the ARC nucleus, which central
leptin can access by several potential mechanisms (Cowley
et al., 2003; Kojima et al., 1999; Münzberg and Morrison,
2015). Central administration of AG or in vitro treatment of
ARC or ARC slices with AG increases the activity and stimulates
the synthesis and release of NPY/AgRP neurons, exerting orexi-
genic effects by the stimulation of feeding (Cowley et al., 2003;
196 Food Intake and Its Control by Signaling Molecules

Figure 6 Schematic overview of acylated ghrelin (AG) and leptin action on the nervous system to regulate food intake. Abbreviations: PVN, para-
ventricular nucleus; ARC, arcuate nucleus; LHA, lateral hypothalamic area; NTS, nucleus of the solitary tract; GHSR-1a, growth hormone secreta-
gogue receptor-1a; LepRb, leptin long receptor; NPY, neuropeptide Y; AgRP, agouti-related protein; MCR, melanocortin receptors; POMC,
proopiomelanocortin; a-MSH, a-melanocyte-stimulating hormone; CART, cocaine and amphetamine regulated transcript.

Nakazato et al., 2001). In contrast, leptin has dual mechanisms
on ARC; first it activates POMC/CART neurons which, in turn,
produce and release a-MSH, activating MC3 and MC4 melano-
cortin receptors on second-order neurons. Importantly, orexi-
genic AgRP is an endogenous antagonist of MC3 and MC4
receptors, further blocking this anorexigenic pathway
(Ollmann et al., 1997). Secondly, leptin inhibits the expression
and secretion of NPY/AgRP neurons by blocking NPY gene
expression, and it lowers GABA release (Cowley et al., 2001;
Morton et al., 2006; Murphy and Bloom, 2006; Schwartz
et al., 1996b; Woods et al., 1998). In this way, NPY/AgRP orexi-
genic neurons activated by AG and inhibited by leptin, stimu-
late feeding and inhibit satiety, while anorexigenic POMC/
CART neurons, activated by leptin, stimulate satiety, block
feeding, and increase energy expenditure.
The ARC nucleus is innervated bidirectionally by reciprocal
connections to second-order neurons in hypothalamic areas
such as the PVN (anorexigenic signaling) with catabolic effects,
and in the LHA (orexigenic signaling) with anabolic effects. It is
also connected to other brain regions (e.g., the NTS), together
with which the ARC participates in the energy homeostasis
regulatory circuit (Meister, 2007; Münzberg and Morrison,
2015; Murphy and Bloom, 2006; Schwartz et al., 2000).
Although initial reports of murine models that lack either
ghrelin or its receptor showed that, under normal feeding,
this hormone had a redundant or masked role in the regulation
of appetite, under diet-induced obesity AG participates in the
selection of the metabolic substrate for energy, favoring fat
ingestion. Consequently, the lack of AG protects against obesity
(Wortley et al., 2004, 2005; Sun et al., 2003; Zigman et al.,
2005). Moreover, leptin regulates food intake by reducing the
hypothalamic content of ECs with an orexigenic effect
(Di Marzo et al., 2001) and by modulating the levels of neuro-
transmitters such as noradrenaline, dopamine, and serotonin
that also contribute to decrease food intake (Schwartz et al.,
2000). Additionally, the number of LRb in the ARC nucleus
constitutes 15–20% of the total number of LRb within the
CNS, so leptin signaling participates in a number of homeo-
static mechanisms, such as glucose homeostasis and hedonic
feeding (Myers et al., 2008; Münzberg and Morrison, 2015).
Both AG and leptin interact with GLP-1 signaling via vagal
afferent neurons; AG blocks translocation of GLP-1R to the
plasma membrane, whereas leptin interacts with GLP-1 to
induce satiation (Ronveaux et al., 2015).
1.06.8 Other Adipokines Regulating Food Intake
Adipose tissue is an endocrine organ that plays a central role in
the regulation of whole-body metabolic homeostasis. As previ-
ously discussed, the most important adipocyte product regu-
lating food intake is leptin; however, adipose tissue produces
a myriad of proteins that regulate many aspects of metabolism.
To date, additional adipokines with well-documented roles in
food intake are adiponectin, resistin, nesfatin, and apelin.
1.06.8.1 Adiponectin
Adiponectin was discovered 20 years ago by Scherer and
colleagues (Scherer et al., 1995), and 1 year later by three other
groups (Hu et al., 1996; Maeda et al., 1996; Nakano et al., 1996)
as a highly adipocyte-specific secreted protein. It has a structure
similar to the complement C1q and the tumor necrosis factor
superfamilies of proteins (Shapiro and Scherer, 1998).
 Food Intake and Its Control by Signaling Molecules 197

Adiponectin is a highly abundant circulating protein present in 1.06.8.2 Resistin

blood in micrograms per milliliter, and it is estimated that it
Resistin (RSTN) was first described in 2001 as an adipocyte-
accounts for 0.01% percent of total plasma proteins (Turer
derived protein downregulated by the insulin-sensitizing thia-
and Scherer, 2012). It is a pleiotropic adipokine exerting
zolidinediones (TZDs) drugs (Steppan et al., 2001). Its levels
a wide variety of actions in different tissues; it acts as an insulin
increase in obesity in humans (Savage et al., 2001; Degawa-
sensitizer, promotes healthy adipose tissue expansion, and is
Yamauchi et al., 2003) and in rodents, in which its neutraliza-
anti-inflammatory. Accordingly, adiponectin-deficient mice are
tion improves insulin sensitivity (Steppan et al., 2001). In
glucose intolerant and insulin resistant (Kubota et al., 2002;
agreement, lean mice treated with resistin become insulin resis-
Nawrocki et al., 2006). Adiponectin has received great attention
tant and glucose intolerant (Steppan et al., 2001). However, in
as a therapeutic factor due to its roles as an antidiabetic, antia-
humans, RSTN is not expressed in adipocytes but in blood
therogenic, and possibly anticarcinogenic factor (Ye and
mononuclear cells (PBMC), mainly macrophages (Fain et al.,
Scherer, 2013; Yamauchi and Kadowaki, 2013). Adiponectin
2003; Tomaru et al., 2009). Human RSTN is upregulated by
levels correlate inversely with obesity and insulin resistance
proinflamatory stimuli (TNFa, IL1b, IL6, LPS) (Kaser et al.,
(Arita et al., 1999; Turer et al., 2011; Gao et al., 2013) and
2003; Lehrke et al., 2004) and, as in rodents, it is downregu-
also with several types of cancer (Dalamaga et al., 2012). Adipo-
lated by TZDs (Patel et al., 2003). RSTN promotes insulin resis-
nectin actions initiate when the protein binds to its receptor
tance, inflammation, atherosclerosis, and heart failure, acting
species AdipoR1 and AdipoR2, which are seven transmembrane
on several cell types such as endothelium, smooth muscle,
domain proteins linked to different signaling molecules such as
and cardiomyocytes (Park and Ahima, 2013). The receptor
AMPK, Ca2þ, PPARa, PPARg, SIRT1, and ceramide/sphingoli-
mediating resistin actions is still unknown.
pids. Independently of the classical 1 and 2 receptors, adiponec-
Interestingly, RSTN is expressed in the mouse brain, partic-
tin binds to T-cadherin, and this interaction mediates its
ularly in the hypothalamus and in the pituitary gland (Morash
cardioprotective effects (Denzel et al., 2010) and also activates
et al., 2002). In rats, i.c.v. injection of resistin transiently
the NFkB pathway in many cell types by activating a still
reduces food intake in fasted and fed animals (Tovar et al.,
unidentified receptor entity (Yamauchi et al., 2014). AdipoR1
2005; Vázquez et al., 2008; Cifani et al., 2009). Although it
is expressed ubiquitously, whereas AdipoR2 is mostly
has no long-term effect (Park et al., 2008), resistin abolishes
expressed in the liver (Yamauchi and Kadowaki, 2013).
the anorectic effect of leptin when they are administered
Regarding adiponectin effects on the central nervous
together, in both control and diabetic animals (Park et al.,
system, its receptors are present in the brain (Yamauchi
2008). In fasted rats, central RSTN administration prevents
et al., 2003; Kubota et al., 2007), and the peptide is present
the normal increase in mRNA expression of the orexigenic
in the cerebrospinal fluid of rodents and humans (Kusminski
peptides NPY and AgRP and the decrease in the anorexigenic
et al., 2007; Kos et al., 2007). In lean animals, systemic treat-
CART, without effects on POMC in the ARC (Vázquez et al.,
ment with adiponectin increases energy expenditure, reduces
2008). In fed rats, RSTN reduced FAS mRNA expression in
body weight and fat mass, but does not alter food intake
the VMH (Vázquez et al., 2008), a mechanism that contributes
(Pajvani et al., 2003; Yamauchi et al., 2001; Berg et al.,
to decreased feeding (Chakravarthy et al., 2007). Consistent
2001). In obese animals it also improves insulin sensitivity
with this, RSTN reduces the orexigenic effect of NPY when
and glucose and lipid metabolism. These results are recapitu-
both are administered centrally (Cifani et al., 2009). Some
lated upon i.c.v. adiponectin injection (Qi et al., 2004). Intra-
contradictory results exist, such as the fact that in mice, central
venous injection of adiponectin increases cerebrospinal fluid
resistin increased the levels of NPY in the ARC nucleus, suggest-
levels of the hormone, suggesting serum to brain transport
ing possible species differences in the RSTN effects.
(Qi et al., 2004). Conversely, i.c.v. adiponectin injection
Additionally, central RSTN treatment promotes liver insulin
does not alter its circulating levels, suggesting adiponectin
resistance (Muse et al., 2007; Singhal et al., 2007), stimulates
receptor-mediated actions on the brain (Qi et al., 2004).
fatty acid synthesis, and pro-inflammatory cytokine expression
In contrast, others have shown that adiponectin stimulates
in liver and in adipose tissue (Vázquez et al., 2008).
food intake and decreases energy expenditure through activa-
tion of AdipoR1 and AMPK in the ARC of the hypothalamus
(ARH), without changes in body weight (Kubota et al.,
1.06.8.3 Nesfatin
2007). In agreement, adiponectin-deficient mice fed a high-
fat diet exhibited reduced food intake (Kubota et al., 2007). Nesfatin or nonesterified fatty acid/nucleobindin2 (NEFA/
Levels of adiponectin in serum and cerebrospinal fluid (CSF) NUCB2) was identified in 2006 as a satiety factor produced
and of its receptor in the ARH increase during fasting condi- throughout the hypothalamus (Oh et al., 2006) and later found
tions and decrease upon refeeding (Kubota et al., 2007). Adi- to be expressed in many different areas of the brain such as the
ponectin is proposed as a satiety signal, promoting food intake insular cortex, amygdala, periventricular nucleus, raphei nuclei,
and fat storage during fasting. These contradictory results may ventrolateral medulla, LC, cerebellum, DMV, NTS, etc. (Stengel
reflect different adiponectin preparations or molecular forms, et al., 2013b). Despite its recent discovery, there is a vast litera-
as it exists in trimers (main form in human CSF) (Kusminski ture describing the effects of NUCB2 in reducing food intake
et al., 2007), hexamers, and high molecular weight adiponec- (recently reviewed by Stengel et al., 2013b). NUCB2 is cleaved
tin containing 18–36 subunits. Thus, further studies are by prohormone convertases into an N-terminal fragment
needed to clarify the effects of the various adiponectin multi- named Nesfatin-1 that exerts potent anorexigenic activity (Oh
mers on food intake in different physiological and patholog- et al., 2006); however, it is controversial whether Nesfatin-1 is
ical conditions. produced in vivo (Stengel and Tache, 2013). Nevertheless, the
198 Food Intake and Its Control by Signaling Molecules
full-length NUCB2 molecule (50 KDa, 396 aa) exerts the same
anorexigenic activity as the cleaved product (9.7 kDa, aa
1–82), so it may well be the endogenous active molecule
(Stengel and Tache, 2013). Immunoneutralization or
knockdown of NUCB2/Nesfatin-1 increases food intake (Oh
et al., 2006; Sedbazar et al., 2013) and conversely, i.c.v. or i.p.
administration of this molecule reduces feeding (Oh et al.,
2006; Shimizu et al., 2009). The anorexigenic effects of central
nesfatin treatment lead to decreased body weight and adiposity
in rats. Nesfatin-1 expression in the PVN and SON is regulated
by the nutritional status: it is reduced upon fasting and
increases after re-feeding in rats (Oh et al., 2006; Kohno et al.,
2008). Also, supporting its physiological role in food-intake
regulation, NUCB2 expression has a circadian expression
pattern, decreasing during the light phase, when rats reduce
their feeding behavior, and increasing in the dark phase
coincidentally with increased feeding (Sedbazar et al., 2013).
The anorexigenic effects of NUB2/Nesfatin-1 are indepen-
dent of leptin, as Zucker rats are still responsive to this protein
and in the same fashion; nesfatin-1 neutralization does not
alter the anorexigenic effects of leptin (Oh et al., 2006; Maejima
et al., 2009). Nesfatin actions involve oxytocin and fix a-MSH
signaling, as intraventricular administration of oxytocin and of
a-MSH receptor antagonists blunts the anorexigenic effect of
Nesfatin-1 (Maejima et al., 2009; Yosten and Samson, 2010,
2009; Oh et al., 2006). Nesfatin-1 also induces oxytocin secre-
tion from PVN neurons in vitro (Maejima et al., 2009). NUCB2
is increased by central administration of a-MSH, but it fails to
alter the expression of either orexigenic or anorexigenic
peptides in the ARC or PVN (Oh et al., 2006). However,
nesfatin-1 treatment increases expression of the mRNAs for
POMC and CART in the nucleus tractus solitarius (NTS)
(Shimizu et al., 2009). Other mediators of Nesfatin-1 effects
reducing food intake are CRF, histamine, serotonin, TRH
(Stengel et al., 2013b), and dopamine (Chen et al., 2015).
The molecular mechanisms that mediate NUNCB2/Nesfatin-
1 effects await the discovery of its receptor.
Just 5 years ago, NUNCB2 was found to be produced by
adipose tissue from mice and humans, adding to the list of adi-
pokines with effects on feeding behavior. NUCB2/Nesfatin-1
protein levels are reduced by fasting and increase in mice given
a high fat diet (Ramanjaneya et al., 2010). In adipose tissue and
adipocytes, Nesfatin-1 levels are induced by proinflammatory
cytokine treatment. Interestingly, Nesfatin-1 is expressed by
subcutaneous adipose tissue more than by other fat depots,
and its levels are positively correlated with body mass index
in humans (Ramanjaneya et al., 2010).
Besides its expression throughout the brain, NUCB2/
nesfatin-1 is also expressed in the pituitary gland, pancreas,
testis (González et al., 2009; Stengel et al., 2013b), and the
gastric mucosa of rodents and humans, in the same cells that
produce ghrelin (Stengel et al., 2009, 2013a). Given the wide-
spread tissue localization of NUNCB2/Nesfatin-1, further
studies are needed to determine the role of adipose tissue-
derived NUNCB2 in the regulation of food intake.
1.06.8.4 Apelin
Apelin was first described and isolated from bovine stomach in
1998 by the group of K. Tatemoto and colleagues, as an
endogenous ligand for the orphan G protein-coupled receptor
APJ (Tatemoto et al., 1998). Apelin is expressed by many cell types
including the hypothalamus (Brailoiu et al., 2002; Reaux et al.,
2002), and it was described as an adipokine in 2005 because it
is expressed by mature adipocytes in both rodents and humans
(Boucher et al., 2005). Apelin expression in adipocytes is
inhibited by fasting and increased by refeeding conditions, and
its circulating levels are increased in obese rodents and humans
(Boucher et al., 2005). Apelin is synthesized as a propeptide
and processed to different peptides, from which apelin-13 is the
main isoform found in the circulation and has the greatest activity
upon receptor binding (Hosoya et al., 2000). Apelin receptors are
present in the hypothalamus and the molecule regulates fluid
homeostasis (De Mota et al., 2004; Mitra et al., 2006), pituitary
hormone release (Taheri et al., 2002), cardiovascular function
(Lee et al., 2005; Tatemoto et al., 2001), neuroprotective effects
(O’donnell et al., 2007; Zeng et al., 2010), gastrointestinal
motility (Lv et al., 2011; Yang et al., 2010), etc.
Regarding effects on food intake, i.c.v. injection of apelin-13
inhibits food intake in fed and fasted rats and mice during the
light and dark phase, respectively (Sunter et al., 2003; Lv et al.,
2012). However, apelin-13 has no effect on food intake in
mice during the light period (Lv et al., 2012). Apelin-13 effects
reducing food intake are dependent on the APJ receptor, as
demonstrated by the use of an APJ antagonist that blocks
apelin-13 effects (Lv et al., 2012). Apelin-13 effects are mediated
by corticotropin-releasing factor (CRF); in vitro, apelin-13 stimu-
lated CRF and AVP release from hypothalamic explants (Taheri
et al., 2002), and a CRF receptor antagonist blocks the apelin-
13 inhibitory effect on food intake (Lv et al., 2012). Apelin
also increases a-MSH release from POMC neurons, and both
apelin and its receptors are expressed by these neurons, suggest-
ing autocrine effects (Reaux-Le Goazigo et al., 2011). a-MSH
stimulates CRF release from PVN neurons resulting in inhibition
of food intake. Thus, apelin inhibits food intake by stimulating
both a-MSH and CRF release (Reaux-Le Goazigo et al., 2011).
Interestingly, apelin receptors are reduced in the hypothalamus
of obese rats in parallel with reduced apelin effects on food
intake (Clarke et al., 2009). However, elevated apelin levels in
obese rats and humans are suggested to occur as an attempt to
maintain metabolic homeostasis (Carpéné et al., 2007).
1.06.9 Pancreas and Feeding Control
In vertebrates the pancreas is an exocrine/endocrine gland that
plays a crucial role in the metabolic status of the organism as
well as in the digestive processing of complex nutrients. Of
endodermal origin, the pancreas communicates to the
duodenum through the ampulla of Vater to release a variety
of zymogens and enzymes that allow the digestion of proteins,
polysaccharides, and fats. Within the pancreatic tissue several
specialized endocrine cells form aggregates known as the islets
of Langerhans. The number of islets varies from hundreds to
thousands depending on the species; the diameter of each islet
is about 200 mm and overall, the islets constitute nearly 1.5% of
the pancreatic mass (Bockman, 2008).
The exocrine pancreas is formed by ducts that are arranged
in clusters known as acini. There are two principal types of cells:
centroacinar cells that secrete bicarbonate (HCO 3 ) ions that

serve to neutralize acid coming from the stomach to the
duodenum, and basophilic cells that synthesize a set of diges-
tive enzymes (trypsinogen, chymotrypsinogen, and pancreatic
lipase and amylase). Both types of cells are regulated by endo-
crine cues. HCO 3 secretion is mainly stimulated by the peptide
hormone secretin which is produced in the S cells of the
duodenal glands, whereas the basophilic cells are activated by
CCK, another peptide hormone synthesized in the mucosal
epithelium of the small intestine (Bockman, 2008).
As to the endocrine pancreas, most of the cells within the
islets of Langerhans are b, which synthesize and secrete insulin
and amylin, also known as islet amyloid PP (in ratio of approx-
imately 100:1). Cells producing and secreting glucagon are
named a, and they are less abundant. Other cellular types
present, but in a much smaller proportion, are d, ε, and PP cells.
They produce and secrete somatostatin, ghrelin, and PP, respec-
tively. Endocrine pancreatic signals are key elements in the
digestive process, prior to (glucagon) and after mealtime
(insulin and amylin) (Adler and Beglinger, 1990).
The pancreas is under the modulation of the autonomic
nervous system (ANS): the sympathetic branch is active during
stressful situations and physical exercise. ANS neurons promote
the secretion of glucagon from the a cells of the islets of Lang-
erhans; the parasympathetic neurons become active during the
digestive process, stimulating the secretion of insulin from the
b cells of the islets of Langerhans, as well as the digestive zymo-
gens from the exocrine ducts (Cerf, 2011).
1.06.10 Glucocorticoids and the Control of Food
Intake
Glucocorticoids are hormones produced and secreted from the
zona fasciculata in the adrenal cortex. They were named for their
ability to increase serum glucose. In primates, the main glucocor-
ticoid is cortisol, whereas in rodents it is corticosterone. These
hormones modulate the metabolism of carbohydrates, proteins,
and fats, and they are also important for their role in the fasting
response as well as their action as immunosuppressors. Access of
the active ligand to the mineralocorticoid and glucocorticoid
receptors is controlled by the activity of an enzyme called 11-
b hydroxysteroid dehydrogenase (11-b HSD, types 1 and 2).
The enzymatic activity is a NADP(H) redox reaction that
converts the active ligand to its corresponding inactive 11-keto
metabolite (cortisol to cortisone in humans, and corticosterone
to 11-dehydrocorticosterone in rats) (Ricketts et al., 1998).
The glucocorticoids are produced by activation of the
hypothalamic–pituitary–adrenal axis (HPA) by some stressor
(internal or environmental); thus, the release of cortisol activates
the mobilization of energy reserves. In fasting or starvation,
glucocorticoids induce skeletal muscle activation and the release
into the circulation of amino acids that are substrates for hepatic
gluconeogenesis (Braun and Marks, 2015).
Alterations in glucocorticoid homeostasis are associated
with severe diseases such as osteoporosis, metabolic syndrome,
cardiovascular complications, immune deficiencies, and even
psychiatric disorders. Glucocorticoids are the most prescribed
drugs around the world due to their anti-inflammatory effects.
Glucocorticoids are recognized by both cytoplasmic and
membrane receptors, and they are part of a complex
Food Intake and Its Control by Signaling Molecules 199
signaling process with physiological and pathological conse-
quences. Within the target cells two transcription factors are
activated by the presence of cortisol/corticosterone, gluco-
corticoid receptors (GR), and mineralocorticoid receptors
(MR). Paradoxically, the MR has a higher affinity for gluco-
corticoids than the GR. This observation has been explained
by postulating that glucocorticoids attach to GR only in
stress conditions, when they are present at high circulating
levels after activation of the HPA. Nongenomic effects eli-
cited by membrane receptors are faster than transcriptional
actions, and they modulate the activity of signaling kinases
as well as the permeability of ion channels (Prager and
Johnson, 2009).
Overproduction of glucocorticoids due to adrenal tumors or
to prolonged administration of high therapeutic doses can
cause Cushing’s syndrome. This disease shows metabolic
disturbances similar to those observed in patients with meta-
bolic syndrome. Interestingly, these pathologies may have in
common the enzyme 11b-HSD1, which in diverse organs and
tissues converts inactive keto-steroids to active glucocorticoids.
For example, experimental overexpression of 11b-HSD1 in
adipose tissue produces abdominal obesity, hyperglycemia,
insulin resistance, dyslipidaemia, and hypertension; in
contrast, overexpression of 11b-HSD1 in the liver yields an
attenuated metabolic syndrome with mild insulin resistance,
dyslipidaemia, hypertension, and fatty liver, but not obesity
or glucose intolerance. These data corroborate the notion that
the enzyme may be a good therapeutic target in the treatment
of metabolic syndrome (Morton and Seckel, 2008).
The enzyme 11b-HSD is also interesting from a biochemical
perspective, since it catalyzes a redox reaction that depends on
NADPH. This coenzyme is produced by the activity of hexose-
6-phosphate dehydrogenase (H6PDH), a microsomal protein
that fulfills a role similar to that of glucose-6-phosphate dehy-
drogenase, the marker enzyme of the pentose phosphate
pathway. Figure 7 illustrates the coordinated activity of these
Figure 7 Coordinated action of 11b-hydroxy-steroid dehydrogenase
(11b-HSD) and hexose-6-phosphate dehydrogenase (H6PDH). G6P,
glucose-6-phosphate; 6 PG, 6-phosphogluconate; NADPþ/NADPH, nico-
tinamide adenine dinucleotide phosphate, oxidized and reduced form.
Modified from Odermatt, A., Klusonova, P., 2015. 11b-Hydroxysteroid
dehydrogenase 1: regeneration of active glucocorticoids is only part of
the story. J. Steroid Biochem. 151 (3), 85–92.
200 Food Intake and Its Control by Signaling Molecules

Figure 8 Glucocorticoid response to endogenous and exogenous stress regulates the HPA axis and influences the satiety-related hormones (leptin,
ghrelin, and insulin) to alter feeding behavior long term. Stress reduces glucocorticoids receptor (GR) expression in hypothalamus and hippocampus
and increases PVN arginine vasopressin (AVP) and corticotropin-releasing hormone (CRH) by epigenetic mechanisms. Circulating glucocorticoids
(GC) and 11-bHSD activity within the hippocampus promote synaptic pruning. The circuit is closed when PVN is affected by hippocampal regulation.
Stress can also induce increased release of satiety-related hormones influencing appetite, feeding behavior, and metabolism throughout life. Modified
from Sominsky, L., Spencer, S.J., 2014. Eating behavior and stress: a pathway to obesity. Front. Psychol. 5 (434), 1–8.

two enzymes, their dependence on the NADPþ/NADPH ratio,
as well as some alternative substrates besides glucocorticoids,
which can act as inhibitors of the enzyme (Odermatt and
Klusonova, 2015).
1.06.10.1 Glucocorticoids and the Stress System
It has been reported that glucocorticoids have orexigenic effects
and can cause obesity in dogs. Besides, in humans, the admin-
istration of glucocorticoids increases energy intake by 20–59%
and also increases body weight in patients to 59%. The reports
suggest that these effects relate to the ability of glucocorticoids
to act on the CNS.
As described in earlier sections leptin regulates body weight
by controlling energy balance and food intake; glucocorticoid
administration in humans increases circulating leptin by acti-
vating insulin release (Nishii et al., 2006). In addition, some
reports indicate that orexins regulate the HPA axis in response
to stressful signals, for example, orexin A directly promotes the
secretion of ACTH and, eventually, corticosterone. This obser-
vation has been confirmed in in vitro experiments, using human
and rat adrenal gland slices (Srinivasan et al., 2013).
All living organisms must respond to many extrinsic and
intrinsic stressful stimuli that constantly challenge the equilib-
rium of the internal homeostasis. The term stress is used to
define a state of being threatened or perceived as being threat-
ening. To give a response, organisms have developed
a sophisticated system that provides appropriate central periph-
eral neuroendocrine responses (Figure 8).
The system responsive to stress functions through the activa-
tion of the HPA axis and the locus caeruleus (LC)/norepineph-
rine (NE)-autonomic nervous system. The end result of
activating this axis is the secretion of circulating glucocorti-
coids. These hormones regulate the basal activity of the HPA
axis and terminate the stress response by acting mainly at the
hypothalamus and the pituitary gland, forming negative feed-
back loops on the secretion of CRH and adrenocorticotropic
hormone (ACTH), respectively. CRH is released in response
to acute fasting from the hypothalamic PVN. Consequently,
ACTH release from the pituitary follows and promotes the
cascade of events that allows the secretion of glucocorticoids
(Sominsky and Spencer, 2014).
1.06.11 Concluding Remarks
After decades of intensive research, our knowledge of the phys-
iological events that underlie the regulation of food intake,
nutrient assimilation, and metabolic performance has
advanced considerably. Nowadays it is common to read in
scientific journals reports about the control of food intake
that involve the removal or insertion of diverse genes in diverse
tissues and organs. Indeed, conclusions are reached from
studying, e.g., the type of receptors, the cellular signaling

systems involved, and the resultant transcriptional responses.
Yet, impressive as it is, we must not forget the original contribu-
tions and the first concepts elicited by a set of renowned phys-
iologists that set the basis for the contemporary advances in the
physiology of food intake. Overall, the understanding of the
basic mechanisms underlying the physiology of food intake
will help us to address how to treat the pathologies of eating
disorders. Effective therapies for obesity and anorexia nervosa,
just to mention two of the more prevalent diseases related to
feeding, will benefit from the integration of the findings
emerging from many biomedical research laboratories with
the experience of clinicians and nutriologists.
Acknowledgments
M.D.-M. and F.V.-C. are researchers from the Cellular and Molecular
Department of the Neurobiology Institute, UNAM. Their research is
funded by the following grants: M.D.-M., DGAPA, PAPIIT, project
IN200815 and F.V.-C., DGAPA, PAPIIT, IN205114, and CONACyT,
166725. We thank Dr Dorothy Pless for her professional and diligent
review of the English grammar. We also thank Nutriologist Fernando
López-Barrera for his support in the elaboration of the figures.
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 1.07 Body Fluid Homeostasis
Eric G Krause, College of Pharmacy, University of Florida, Gainesville, FL, USA
Annette D de Kloet, College of Medicine, University of Florida, Gainesville, FL, USA
Ó 2017 Elsevier Inc. All rights reserved.
This chapter is a revision of the previous edition chapter by D. Daniels, S.J. Fluharty, volume 1, pp. 259–289, Ó 2009, Elsevier Inc.

1.07.1 Introduction 211

1.07.2 In Defense of Osmolality 211
1.07.2.1 Vasopressin and OT 211
1.07.2.2 Thirst 212
1.07.2.3 Peripheral Osmoreceptors 213
1.07.2.4 Central Osmoreceptors 213
1.07.2.5 Sodium Deficit/Reduced Osmolality 213
1.07.3 In Defense of Volume 214
1.07.3.1 The Renin–Angiotensin–Aldosterone System 214
1.07.3.2 Hypovolemic Thirst 215
1.07.3.3 Salt Appetite 216
1.07.3.3.1 Aldosterone and Salt Appetite 217
1.07.3.3.2 Angiotensin-II and Salt Appetite 217
1.07.3.3.3 Angiotensin/Aldosterone Interactions 218
1.07.3.3.4 Baroreceptors, Osmoreceptors, and Salt Appetite 219
1.07.3.3.5 Taste and Salt Appetite 219
1.07.3.4 Blood Pressure 219
1.07.3.5 Hypervolemia 220
1.07.4 Body Fluid Homeostasis and Psychological Stress 220
1.07.5 Summary and Conclusion 221
References 221

1.07.1 Introduction 1.07.2 In Defense of Osmolality

Maintaining body fluid homeostasis requires neural and endo-
crine control of behavioral, cardiovascular, and renal responses
that adjust the osmolality and volume of body fluid to levels
that are optimal for physiological function. Perturbations to
osmolality and volume are detected by sensors that relay
imbalance to the appropriate effector organs, which in turn,
initiate compensatory responses that maintain or restore blood
pressure and hydromineral balance. Ultimately, these compen-
satory responses are orchestrated by the brain, which utilizes
endocrine signals that exert effects on target organs expressing
receptors that promote precise imposition of appropriate
responding.
Challenges to body fluid homeostasis can result in concom-
itant changes in plasma osmolality and effective circulating
volume; however, plasma osmolality and volume are regulated
independently by distinct behavioral and endocrine responses.
Plasma osmolality is maintained by changes in water balance,
accomplished by thirst, water intake, and activation of the argi-
nine vasopressin (AVP)/oxytocin (OT) renal axes. Conversely,
blood volume and pressure is largely maintained by activation
of the renin–angiotensin–aldosterone system (RAAS) which
promotes vasoconstriction as well as sodium conservation
and consumption. Here, we review the neurohumoral systems
that control behavioral, cardiovascular, and renal compensa-
tory mechanisms that maintain body fluid homeostasis.
In most mammals, extracellular fluid osmolality is tightly regu-
lated near 290 mOsm, and this maintains optimal water distri-
bution between vascular, interstitial, and intracellular fluid
compartments. Solutes that elicit the movement of water across
the phospholipid bilayer that comprises the semipermeable
membrane separating the intracellular and extracellular fluid
are capable of generating ‘osmotic pressure’ that activates
osmoreceptors located within the brain and periphery. Osmor-
eceptors are thought to be mechanoreceptors that detect the
swelling or shrinkage of specialized neurons during osmotic
challenges. These ‘osmosensing neurons’ detect changes in
osmolality and, subsequently, transduce this information
into action potentials relayed to effector brain nuclei that
initiate responses alleviating such perturbations and restore
osmolality to homeostatic levels (Figure 1).
1.07.2.1 Vasopressin and OT
In humans and rodents, a 1–2% increase in systemic plasma
osmolality causes the release of AVP and OT into the systemic
circulation and also causes the sensation of thirst (Rennke and
Denker, 2010). Magnocellular neurons within the supraoptic
(SON) and paraventricular (PVN) nuclei of the hypothalamus
send their axons to the median eminence and posterior pitui-
tary and release AVP and OT into the systemic circulation.

Hormones, Brain, and Behavior, 3rd edition, Volume 1 http://dx.doi.org/10.1016/B978-0-12-803592-4.00007-9 211

212 Body Fluid Homeostasis
Figure 1 Response to hyperosmotic (intracellular) dehydration. ACC, anterior cingulate cortex; MnPO, median preoptic nucleus; PVN, paraventricular
nucleus of the hypothalamus; SON, supraoptic nucleus.
Elevated circulating AVP binds to V2 receptors in the kidney
that initiate a Gs-mediated intracellular signaling cascade that
results in the insertion of aquaporin 2 channels into the apical
membrane of cells within the collecting tubule. These aqua-
porin channels allow the reabsorption of water, which
decreases urine output but increases urine osmolality. The
release of OT into the systemic circulation elicits activation of
OT receptors that promote natriuresis or renal sodium excre-
tion (Verbalis et al., 1991; Sjoquist et al., 1999). In the end,
the renal actions of AVP and OT allow the conservation of
body water and the excretion of solutes in urine, which buffer
against further elevations in plasma osmolality.
The AVP and OT secretion that follows increased plasma
osmolality is believed to be controlled by osmosensing
neurons within the organum vasculosum of the lamina termi-
nalis (OVLT). The OVLT is a circumventricular organ residing
near the ventral surface of the forebrain. Circumventricular
organs lack a blood–brain barrier, and this allows the move-
ment of water in or out of OVLT neurons during decreases or
increases in plasma osmolality, respectively (McKinley et al.,
2003; Ciura et al., 2011). The activity of OVLT neurons is
tightly coupled to the osmolality of the extracellular fluid,
and increasing concentrations elicit depolarization and
increased action potential frequency (Ciura and Bourque,
2006). Neurons in the OVLT have direct axonal projections
to the SON, PVN, and median preoptic nucleus (MnPO)
(Johnson and Gross, 1993; Bourque, 2008), and these connec-
tions are believed to constitute a neurosecretory circuit that
controls the systemic release of AVP and OT. Evidence support-
ing this idea comes from neuroanatomical and lesion studies
demonstrating functional connections between these brain
nuclei that are sensitive to changes in osmolality. For example,
hyperosmotic stimulation elicits activation of OVLT neurons
with monosynaptic connections to the PVN, suggesting that
increases in plasma osmolality elicit activation of OVLT
neurons that likely synapse onto PVN neurons to evoke their
excitation and the release of AVP and OT into the systemic
circulation (Shi et al., 2008). Consistent with this idea, lesion
of the OVLT attenuates the AVP and OT secretion that occurs
with increased plasma osmolality in dogs, rats, or sheep
(Thrasher and Keil, 1987; Negoro et al., 1988; McKinley
et al., 2003). In addition to the SON and PVN, the OVLT is con-
nected to the MnPO, which has robust efferent projections to
the SON that synapse onto AVP neurons (Oldfield et al.,
1991). Interestingly, brain lesions that encompass both the
OVLT and MnPO result in greater impairments to AVP secre-
tion relative to lesions confined to the OVLT (Johnson et al.,
1978; Thrasher and Keil, 1987), suggesting that the MnPO
also contributes to the defense of body fluid homeostasis
that occurs subsequent to osmotic challenges.
1.07.2.2 Thirst
While activation of neuroendocrine axes buffer against further
elevations in plasma osmolality, it is the sensation of thirst and
the resulting water consumption that ultimately dilutes the
plasma osmolality and restores its concentration to homeo-
static levels. In this regard, the OVLT also plays a prominent
role in the thirst and water intake that results from elevated
plasma osmolality. Lesion of the OVLT decreases water
consumption subsequent to a hyperosmotic challenge
(Thrasher and Keil, 1987; McKinley et al., 2003). Similar to
neuroendocrine secretion, lesions including the MnPO reduce
osmotically induced water intake to a greater extent than those
specifically targeting the OVLT (Johnson et al., 1978), indi-
cating that the MnPO may serve as a relay center within brain
circuits controlling drinking behavior. However, the OVLT
also has projections to thalamic and cortical nuclei, and these

brain regions likely control the perception of thirst (Bourque,
2008; Hollis et al., 2008). In addition to the SON, PVN, and
MnPO, the OVLT sends direct efferent projections to various
thalamic nuclei, the anterior cingulate cortex (ACC), and
insular cortex (Hollis et al., 2008). In particular, the ACC has
been implicated in the generation of thirst because electrical
stimulation of the ACC in conscious monkeys elicits drinking
behavior within seconds of onset (Robinson and Mishkin,
1968) though ablation of the ACC does not completely abolish
water intake (Hollis et al., 2008). Functional magnetic reso-
nance imaging studies show that the lamina terminalis and
ACC are robustly activated in humans rendered thirsty via
delivery of intravenous hypertonic saline (Egan et al., 2003).
Remarkably, when these subjects drank water to satiation, the
perception of thirst quickly subsided as did the intensity of
ACC activation; however, the plasma osmolality and activation
of the lamina terminalis persisted (Egan et al., 2003). Taken
together, these results implicate the OVLT and MnPO as sensors
that detect osmotic stimuli that initiate thirst, the perception of
which arises within cortical brain nuclei, like the ACC.
1.07.2.3 Peripheral Osmoreceptors
It is interesting that drinking water relieves the sensation of
thirst and eliminates the activation of the ACC but not the
OVLT, and this occurs prior to resolving the elevated plasma
osmolality (Egan et al., 2003). In other words, drinking water
causes the perception of thirst to subside before plasma osmo-
lality is lowered and brain osmoreceptors are deactivated. This
phenomenon suggests the existence of peripheral osmorecep-
tors that anticipate the effects of fluid consumption and, subse-
quently, elicit compensatory changes that prepare the organism
for subsequent increases or decreases in plasma osmolality.
Indeed, in the periphery, osmoreceptors are expressed in the
oropharyngeal cavity (Kuramochi and Kobayashi, 2000),
gastrointestinal tract (Dooley and Valenzuela, 1984; Carlson
et al., 1997; Andersen et al., 2000), splanchnic mesentery
(Choi-Kwon and Baertschi, 1991), and hepatic portal vein
(Baertschi and Vallet, 1981). In regard to the hepatic portal
vein, delivering intragastric concentrated saline to rats signifi-
cantly increases portal venous osmolality several minutes
before circulating plasma osmolality is elevated, and this effect
is associated with increased AVP secretion and neuronal activa-
tion of brain networks implicated in the regulation of body
fluid osmolality (Carlson et al., 1997). Thus, peripheral osmor-
eceptors can prime the brain to respond to an osmotic chal-
lenge prior to actual elevations in circulating plasma
osmolality. Inversely, in both osmotically dehydrated humans
and rats, consuming water or gastric infusion of water rapidly
lowers plasma levels of AVP and OT prior to restoring plasma
osmolality (Geelen et al., 1984; Baertschi and Pence, 1995;
Huang et al., 2000). Taken together, these studies indicate the
existence of peripheral osmoreceptors that detect the concen-
tration of fluids within specific viscera and, subsequently,
convey this information to brain circuits controlling body fluid
homeostasis through ascending afferent signals. In this regard,
several lines of evidence suggest the vagus, cranial nerve X, is
the afferent connection that relays signals from peripheral
osmoreceptors to the CNS. For example, the hepatic branch
of the vagus nerve increases or decreases firing rate in response
Body Fluid Homeostasis 213
to changes in osmolality (Adachi et al., 1976; Adachi, 1984),
and transection of the hepatic vagus nerve suppresses AVP
release when osmolality is elevated via infusion of hypertonic
saline into the hepatic portal vein (Chwalbinska-Moneta,
1979). Similar to AVP secretion, water intake stimulated by
intragastric infusion of hypertonic saline is reduced by
vagotomy, indicating that the vagal afferent signals are capable
of influencing both behavioral and endocrine responses to
increased osmolality (Starbuck et al., 2002). Thus, peripheral
osmoreceptors monitor the concentration of fluid within
discrete vascular beds and use ascending vagal signals to prime
the brain for impending alterations in systemic osmolality.
1.07.2.4 Central Osmoreceptors
The molecular identity of the peripheral osmoreceptor is
largely unexplored. In contrast, the molecular mechanism
underlying central osmosensing is an active area of research,
and investigations have centered on ion channels gated by
changes in osmolality. Specifically, the transient receptor
potential vanilloid 1 receptors (TRPV1) are cation channels
that have been implicated in osmosensing because TRPV1
knockout mice have impaired thirst and AVP responses subse-
quent to elevated plasma osmolality (Ciura and Bourque,
2006; Sharif Naeini et al., 2006). The increased action poten-
tial discharge that occurs in OVLT neurons subjected to hyper-
osmolality is absent in TRPV1 knockout mice, further
substantiating that TRPV1 is the molecular mechanism under-
lying central osmosensing (Ciura and Bourque, 2006).
However, subsequent studies reported that TRPV1 knockout
mice and wild-type mice have similar increases in plasma
osmolality, water intake, and activation of neurons residing
in the OVLT and MnPO subsequent to cellular dehydration
elicited by administration of hypertonic saline or mannitol
(Taylor et al., 2008; Kinsman et al., 2014). Moreover, TRPV1
reporter mice that have had the TRPV1 genetic locus modified
to accurately report the functional expression of TRPV1 gene
products do not demonstrate the presence of these channels
in brain nuclei implicated in osmosensing (Cavanaugh
et al., 2011). Collectively, these studies indicate that the iden-
tity of the central osmosensor is controversial and additional
research is required to resolve underlying mechanisms.
1.07.2.5 Sodium Deficit/Reduced Osmolality
As may be expected, decreases in plasma osmolality also stim-
ulate osmoreceptors residing in the brain and periphery, but
such stimulation inhibits rather than activates renal and
behavioral mechanisms that promote thirst and water reten-
tion. It is important to note that decreased fluid osmolality
is often the result of lowering the plasma sodium concentra-
tion, which is a strong determinant of blood volume.
Lowering of the plasma sodium concentration is often
coupled to a reduction in blood volume and decreased perfu-
sion pressure, which increases the activity of the RAAS, which
in turn, initiates physiological and behavioral compensatory
responses that restore blood volume and perfusion pressure
to homeostatic levels. These compensatory responses and
their underlying neural and humoral mechanisms are dis-
cussed in the subsequent sections.
214 Body Fluid Homeostasis
1.07.3 In Defense of Volume
Much like the maintenance of plasma osmolality, complex
humoral, neural, and behavioral mechanisms exist that main-
tain blood volume (Figure 2). In contrast to increases in
plasma osmolality, however, decreased blood volume requires
the conservation and replacement of both water and solute
(i.e., sodium) to correct the extracellular volume loss that
occurs. Therefore, hypovolemia triggers physiological changes
that involve renal and other peripheral mechanisms that mini-
mize further loss of sodium and water and behavioral changes
that maximize their intake. For example, aldosterone and AVP
act at the level of the kidney to maximize the reabsorption of
sodium and water, thereby minimizing their loss in urine.
Furthermore, angiotensin-II (Ang-II) and aldosterone are
largely responsible for the consumption of water and sodium
that occur as a result of volume loss, and collectively, these
physiological and behavioral changes act to restore volume to
both the extra- and intracellular compartments.
Another important point is that, at least in a clinical setting,
a loss in blood volume is most often also associated with
a decrease in blood pressure, and this decrease in pressure is
responsible for the initiation of many of the physiological
mechanisms that allow one to overcome hypovolemia. In
this regard, the carotid sinus and aortic baroreceptors respond
to this decrease in pressure by reducing their tonic firing
frequency, thereby activating neurons within the hindbrain
nucleus of the solitary tract (NTS) that elevate sympathetic
and decrease parasympathetic tone (Fisher and Paton, 2012).
If the magnitude of the reduced pressure is considerable, this
then prompts the release of AVP that acts systemically to
Figure 2 Response to hypovolemic (extracellular) dehydration. IML, intermediolateral cell column of the spinal cord; NTS, nucleus of the solitary
tract; PVN, paraventricular nucleus of the hypothalamus; RVLM, rostral ventrolateral medulla.
promote vasoconstriction and water reabsorption (Aoyagi
et al., 2009; Sampey et al., 1999). Furthermore, this reduced
pressure is sensed by baroreceptors in the renal vasculature to
initiate activation of the RAAS (Hainsworth, 2014; Schweda
and Kurtz, 2011).
1.07.3.1 The Renin–Angiotensin–Aldosterone System
The RAAS is a critical regulator of hydromineral balance, that
impacts both peripheral mechanisms (e.g., systemic blood
pressure, urinary sodium excretion, and renal hemodynamics)
and central mechanisms (e.g., sympathetic outflow, thirst, and
sodium appetite) that aim to restore blood volume and pres-
sure during hypovolemic threats. Stimuli such as hemorrhage
(Yamaguchi and Hama, 2011), pharmacologically induced
vasodilation (Stocker et al., 2000), sodium loss (Badauê-Passos
et al., 2007), or hypovolemia induced by extravascular admin-
istration of polyethylene glycol (Stricker and Verbalis, 1986)
potently trigger the activation of the RAAS, the consequent
synthesis of the effector peptide, Ang-II, and its stimulation
of aldosterone secretion. While there is some evidence for
tissue-specific formation and actions of Ang-II, as discussed
in Chapter 3.20, The Brain Renin–Angiotensin System, it has
long-been established that the formation of this octapeptide
occurs predominantly within the systemic circulation and
requires the coordination of many organ systems (Figure 3).
Initially, liver-generated angiotensinogen is cleaved into
angiotensin-I by renin that arises predominantly from the
juxtaglomerular apparatus of the kidney. Renin is the rate-
limiting enzyme for Ang-II synthesis, and consequently, hypo-
volemia leads to elevations in Ang-II by increasing the synthesis

Figure 3 The classical endocrine renin–angiotensin system. Hypo-
volemia leads to the synthesis of Ang-II by stimulating the secretion of
renin from the kidney. Reduced renal arterial perfusion, reduced sodium
delivery to the macula densa, increased sympathetic nerve activity to
the b1 receptors of the juxtaglomerular apparatus, and increased circu-
lating catecholamines all occur in response to hypovolemic threats and
lead to increased secretion of renin.
and secretion of renin from the kidney. Reduced renal arterial
perfusion, reduced sodium delivery to the macula densa,
increased sympathetic nerve activity to the b1 receptors of the
juxtaglomerular apparatus, and increased circulating catechol-
amines all occur in response to hypovolemic threats and lead
to increased secretion of renin. Angiotensin-converting enzyme
(ACE; found in the lungs) then removes two amino acids from
angiotensin-I to form Ang-II, which acts at its receptors in
various areas of the body to maintain blood pressure and
volume via effects on vasoconstriction, renal sodium reabsorp-
tion, sympathetic nervous system activity, aldosterone secre-
tion, and water and sodium consumption.
The receptors to which Ang-II binds are G-protein-coupled
receptors that, in humans, can be subdivided into two types:
the angiotensin type-1 receptor (AT1R) and the angiotensin
type-2 receptor (AT2R) (Mendelsohn et al., 1984; Tsutsumi
and Saavedra, 1991; Johren et al., 1995, 1996; Johren and
Saavedra, 1996; Lenkei et al., 1997; Hauser et al., 1998;
Gonzalez et al., 2012; de Kloet et al., 2016). Activation of
these receptors triggers a number of intracellular signal
transduction pathways, such as G-protein-mediated (Gq and
Gi), JAK/STAT, and MAPK or ERK intracellular signaling
cascades which have been implicated in Ang-II’s impact on
physiology and behavior (Sadoshima et al., 1995; Velloso
et al., 1996; Daniels et al., 2005, 2009; Daniels, 2014).
Importantly, AT1Rs are the target of most of the known
physiologic actions of Ang-II, such as vasoconstriction and
water consumption. Conversely, AT2R activation is thought
to antagonize AT1R activation in many regards (Yayama and
Okamoto, 2008; Sumners et al., 2015). It is also relevant to
note here that in rodents, the AT1R subtype is further
subdivided into the angiotensin type-1a receptor (AT1aR)
and type-1b receptor (AT1bR) whose genes are present
on separate chromosomes, but are highly homologous
Body Fluid Homeostasis 215
(Sasamura et al., 1992; MacTaggart et al., 1997). Within the
brain of rodents, AT1R is primarily present in its AT1aR form;
however, in some cases, AT1bR has been detected in the
brain, and it is also abundant in the anterior pituitary and
adrenal cortex (Kakar et al., 1992; Burson et al., 1994; Lenkei
et al., 1997). For the purposes of this chapter, however, it is
only important to distinguish between the AT1R (which in
rodents refers to both AT1aR and AT1bR) and the AT2R.
At the level of the brain, blood-borne Ang-II acts at CVOs
which, as discussed above, are brain regions with a ‘leaky’
blood–brain barrier that serve as an interface between signaling
factors within the systemic circulation and the brain. As a result
of this positioning, CVOs are ideally suited to relay hydromin-
eral imbalance and cardiovascular function to the brain, and
one way that they do this is by communicating the level of
Ang-II in circulation. In particular, the subfornical organ
(SFO) and the OVLT are known to express AT1R and to initiate
central responses to hypotension and hypovolemia. Ang-II
receptors are also expressed on neurons residing on the
protected side of the blood–brain barrier and are heavily
implicated in the brain circuits that control responses to hypo-
volemia and hypotension. The modulation of Ang-II levels
and/or actions at its receptors within these blood–brain
barrier-protected areas is known to impact physiology and
behavior, and it is hypothesized that a brain-specific renin–
angiotensin system generates Ang-II that acts on these recep-
tors. This notion is discussed in detail in Chapter 3.20,
The Brain Renin–Angiotensin System.
1.07.3.2 Hypovolemic Thirst
Much like an increase in plasma osmolality, a reduction in
blood volume is also a potent stimulus of thirst and water
consumption. In contrast to osmotically driven thirst, however,
which is stimulated by a minimal, 1–2 % increase in plasma
osmolality, a decrease in plasma volume by
10% is necessary
to stimulate hypovolemic thirst. Clinically, such pronounced
reductions in plasma volume occur with hemorrhage or with
excessive vomiting, diarrhea, or sweating. Some experimental
methods by which hypovolemia is provoked include bleeding
(Fitzsimons, 1961; Oatley, 1964), surgical methods to reduce
venous return to the heart (Fitzsimons, 1969; Fitzsimons and
Moore-Gillon, 1980), induced sodium deficiency (Cizek
et al., 1951), and the extravascular administration of colloidal
solutions (Fitzsimons, 1961; Stricker, 1966). The latter leads to
the sequestration, in particular, of extracellular fluid allowing
for a reduction in plasma volume without impacting plasma
osmolality and pressure (Fitzsimons, 1961; Stricker, 1966),
thereby allowing for the determination of physiological and
behavioral responses to volume loss, per se.
Among the hormones involved in the physiological
responses to volume loss is the above-described AVP, which
causes vasoconstriction and water retention not only during
increased plasma osmolality, but also in response to severe
hypovolemia (Fitzsimons, 1961; Stricker, 1966; Dunn et al.,
1973). That is, AVP secretion is stimulated in the absence of
any changes in plasma osmolality, if the volume loss is
substantial enough (Fitzsimons, 1961; Stricker, 1966; Dunn
et al., 1973). Again, this response is advantageous because
AVP acts at the vasculature to promote vasoconstriction as
216 Body Fluid Homeostasis
well as at the kidney to conserve urinary water, and these
combined effects allow maintenance of perfusion pressure
and fluid conservation subsequent to severe volume depletion.
Similar to AVP, Ang-II promotes vasoconstriction and renal
fluid reabsorption; however, it is also a potently acting dipso-
gen. As discussed, hypovolemia stimulates the secretion of
renin resulting in elevated circulating Ang-II that acts centrally
to restore blood volume and pressure. In support of a role for
Ang-II in thirst and drinking behavior, administration of
this peptide systemically or intracranially causes prompt
and intense drinking responses in water-replete animals
(Fitzsimons, 1969; Fitzsimons and Simons, 1969; Simpson
and Routtenberg, 1978; Vanhouten et al., 1980; Mendelsohn
et al., 1984). This phenomenon is true for many species,
including mammals (Epstein et al., 1970; Lotter et al., 1980;
Weisinger et al., 1997), birds (Schwob and Johnson, 1977),
reptiles (Fitzsimons and Kaufman, 1977), and fish (Beasley
et al., 1986). The most sensitive region in the generation of
Ang-II-induced drinking responses is the SFO (Simpson and
Routtenberg, 1973, 1978; McKinley et al., 1983), which is a cir-
cumventricular organ that binds circulating factors and
communicates their levels to the brain. Specific inhibition of
AT1R in the SFO decreases water intake elicited by increasing
the circulating concentration of Ang-II but has no effect on
drinking induced by increasing the plasma sodium concentra-
tion (Krause et al., 2008). This study suggests that the water
intake that occurs as a consequence of increased circulating
Ang-II is dependent, in part, on activation of AT1R residing
in the SFO. That being said, the ability of Ang-II to stimulate
water intake requires the coordination of numerous brain
structures, including not only CVOs but also areas that are pro-
tected by the blood–brain barrier (e.g., MnPO, BNST, lateral
hypothalamus, and portions of the amygdala). The brain
nuclei and neural circuits that are involved in the central
actions of Ang-II are discussed in detail in Chapter 3.20,
The Brain Renin–Angiotensin System. In regard to Ang-II’s
role in the brain’s control of water balance, circulating Ang-II
stimulates water consumption by activating neurons in SFO
that express AT1R, and the efferent projections of these neurons
terminate within brain nuclei (e.g., preoptic, anterior hypotha-
lamic, and limbic regions) that coordinate the perception of
thirst with drinking behavior (Fitzsimons, 1998; McKinley
et al., 2003). Furthermore, there is evidence for Ang-II actions
within blood–brain barrier-protected regions in the stimula-
tion of thirst and water consumption. For example, injection
of Ang-II into the preoptic area also induces drinking (Richard-
son and Mogenson, 1981).
It is important to note that, in spite of the hypovolemic drive
to consume water, the powerful inhibitory effect of osmotic dilu-
tion on water intake is able to override hypovolemic thirst. That
is, a 2–3 % decrease in plasma osmolality will abolish water
intake and AVP secretion in volume-depleted rats (Stricker,
1969). This phenomenon may be due to the fact that although
increased water intake is a compensatory response to hypovole-
mia, consuming only water will not alleviate deficits in vascular
volume because water is stored mostly within intracellular
compartments. A more advantageous response would be to
ingest sodium and water to produce a mixture near the concen-
tration of isotonic saline, which would more effectively replace
the volume lost, thereby allowing normal water distribution
among fluid compartments. Perhaps consequently, several
RAAS mechanisms are centered on promoting salt appetite or
the ingestion of sodium that follows volume depletion.
1.07.3.3 Salt Appetite
From a historical perspective, much of our understanding of
salt appetite stems from the pioneering work of Curt Richter,
which focused largely on how changes in behavior compensate
for perturbations in the internal environment (Krause and
Sakai, 2007). In his experiments, Richter manipulated diet,
interfered with the nervous system, and removed or replaced
glandular secretions in attempt to understand the neural and
endocrine signals that contribute to behavior. One of the
most striking behaviors that Richter observed was that of rats
voluntarily ingesting salt when faced with sodium deficit.
Sodium chloride was removed from the rats’ food and they
were given access to water and 3% NaCl solution. The intake
of the NaCl solution was
1% of the total diet, which is the
amount of salt that was calculated as necessary for healthy
development of the rat (Richter, 1956). These results suggested
that the rats were consuming NaCl, which effectively compen-
sated for the loss of salt in their food.
Subsequent studies examined the behavioral responses to
sodium deficiency when physiological mechanisms to conserve
sodium were surgically eliminated. While it was previously
known that adrenalectomy produced sodium insufficiency
(Rubin and Krick, 1933), Richter found that immediately after
adrenalectomy the consumption of NaCl solution was
augmented while water intake was reduced (Richter, 1936).
The increased consumption of NaCl was robust and occurred
the first time the rats were given access to the solutions, suggest-
ing that the behavior was innate because there was little time to
learn of the postingestive consequences. Importantly, adrenal-
ectomized rats did not increase intake of chloride supplied as
magnesium chloride or aluminum chloride, but did readily
ingest different sodium compounds indicating that the appetite
was specific for sodium (Richter, 1956). As a result of having
access to saline, the survival rate of the adrenalectomized rats
was greatly increased, and except for the propensity to consume
large volumes of salt solutions, there were no indications of
deficiency. However, when the salt solutions were removed,
almost 90% of the rats died within 5 days, indicating that the
increased appetite for salt was keeping these animals alive.
This is also of clinical relevance, as described in a case study
by Wilkins and Richter (1940) that concerned a 3 1/2-year-old
boy who was admitted to the hospital with symptoms of early-
onset puberty. Shortly after admittance the child died, and it
was only after his death that it was determined that he was
unable to retain sodium due to adrenal pathology. Interviews
with the child’s parents revealed that at an early age the boy
had an insatiable appetite for salt, which likely kept him alive
until the relatively low-sodium diet at the hospital resulted in
his death.
While Richter’s initial studies demonstrated that sodium
deficiency was accompanied by an increased salt appetite that
promotes vitality, the signals relating to the arousal of the appe-
tite were poorly understood. Richter found that grafting adrenal
tissue into the eye of adrenalectomized rats decreased sodium
intake to that of intact rats within 10 days, suggesting that

the increased intake is due to the loss of some adrenal secretion
(Richter, 1956). Consequently, it was hypothesized that salt
appetite was dependent on the presence of the salt-retaining
hormone, aldosterone, which is produced in the adrenal glo-
merulosa. Richter treated adrenalectomized rats with the aldo-
sterone precursor, deoxycorticosterone (DOC), and NaCl
intake returned to normal, presumably, because excessive
amounts of sodium were no longer lost in urine (Richter and
Eckert, 1938). However, subsequent studies revealed that treat-
ing intact rats with DOC elicited a salt appetite similar to that of
adrenalectomized rats, which at the time seemed paradoxical
(Rice and Richter, 1943). Adrenalectomized rats lacking endog-
enous mineralocorticoids had a robust salt appetite that could
be attenuated by DOC, but administration of this agonist in
normal rats increased NaCl intake with increasing doses. Subse-
quent research would attempt to resolve this conundrum by
investigating the mechanisms underlying DOC-induced NaCl
intake and evaluating other factors contributing to salt appetite.
1.07.3.3.1 Aldosterone and Salt Appetite
Wolf (1965) expanded on Richter’s initial observations by
examining the NaCl intake of intact and adrenalectomized
rats after administration of various doses of DOC. As expected,
intact rats increased NaCl intake with elevating doses of DOC.
In contrast, the dose–response curve of the adrenalectomized
rats resembled an inverted U-shape, with DOC diminishing
intakes at low doses, but increasing ingestion at higher
doses. Intriguingly, pretreating adrenalectomized rats with
corticosterone, an endogenous adrenal steroid, decreased
the doses of DOC required to produce NaCl intake. In
addition, Wolf and others found that aldosterone, another
endogenous adrenal steroid, specifically augmented the
intake of sodium salts (Wolf and Handal, 1966; Weisinger
and Woods, 1971). Taken together, these studies suggested
that endogenous adrenal steroids contributed to the arousal
of salt appetite; however, the mechanism underlying the
natrorexigenic effects of these hormones remained unknown.
Early investigation of adrenal steroid regulation of salt appe-
tite focused on the central actions of corticosterone and aldoste-
rone because certain brain lesions inhibited the salt intake that
follows DOC administration (Wolf, 1964; Nitabach et al.,
1989). Brain regions that readily took up these hormones were
identified with the use of receptor autoradiography. Specifically,
corticosterone and aldosterone were found to compete for
binding sites in the hippocampus, septum, and amygdala
(Gerlach and McEwen, 1972; Birmingham et al., 1979; McEwen
et al., 1986). However, the relative contribution of adrenal
steroids to salt appetite in specific brain areas was uncertain until
specific antagonists for glucocorticoid (GR) and mineralocorti-
coid (MR) receptors were developed. In this regard, McEwen
et al. (1986) administered RU-28318, an MR-specific
antagonist, to adrenalectomized rats and observed that the
same dose of RU-28318 that inhibited aldosterone binding in
the hippocampus, septum, and amygdala also blunted the
hormone’s effects on salt intake. These results provided
indirect evidence that aldosterone regulation of salt appetite
involves, in part, the actions of aldosterone in the brain.
Aldosterone activates central pathways mediating salt appe-
tite via two mechanisms (Fluharty et al., 1995; Karst et al.,
2005; see Chapter 3.09, Aldosterone Action on Brain and
Body Fluid Homeostasis 217
Behavior). First of all, aldosterone is known to bind to cytosolic
receptors that act as transcription factors and initiate changes in
gene expression. This mode of action takes hours to days and is
likely a factor in the long-term regulation of sodium intake.
Second, aldosterone can also induce rapid changes in sodium
consumption through actions at the cell surface (Fluharty
et al., 1995; Sakai et al., 2000). Specifically, when converted
into its tetrahydro metabolites, aldosterone interacts with
ligand-gated ion channels to influence neuronal firing. This
nongenomic mechanism was thought to contribute to the
rapid ingestion of sodium that accompanies DOC administra-
tion (Fluharty et al., 1995; Sakai et al., 2000). Thus, it was
hypothesized that aldosterone regulates short- and long-term
consumption of sodium through genomic as well as nonge-
nomic actions within the brain.
Sakai et al. (2000) provided evidence supporting this
hypothesis by using antisense oligodeoxynucleotides (ASDNs),
single-stranded DNA molecules that inhibit the expression of
targeted genes, to evaluate the role of MR in DOC-induced
salt intake. Antisense oligodeoxynucleotides directed against
the GR or MR were injected into the amygdala, thereby
inhibiting the expression of these receptors locally within this
brain region. Subsequently, DOC was administered and salt
intake was measured. Administration of ASDNs directed
against MR significantly reduced DOC-induced sodium
intake, whereas ASDNs directed against the GR had no effect.
Interestingly, the same study reported that salt intake could
be elicited 15 min after direct injection of tetrahydro-
aldosterone into the amygdala, and this effect was not
blocked by administration of MR antagonists that inhibit the
genomic actions of this receptor. Collectively, these results
suggested that salt appetite could be aroused through both
genomic and nongenomic effects of mineralocorticoids.
However, the fact that adrenalectomized rats lacking
endogenous adrenal steroids demonstrated a robust salt
appetite made it unlikely that aldosterone was the only signal
capable of eliciting sodium intake.
1.07.3.3.2 Angiotensin-II and Salt Appetite
It has long-been appreciated that Ang-II contributes to the elic-
itation of salt appetite indirectly by stimulating the release of
aldosterone (Davis and Spielman, 1974), while the direct
involvement of Ang-II as a stimulator of sodium intake was
often disregarded (Stricker, 1971). However, Buggy and Fisher
(1974) found that intracranial administration of Ang-II
increased water and sodium intake. Importantly, the elevated
consumption of NaCl was specific to Ang-II because adminis-
tration of carbachol, another centrally acting dipsogen, elicited
only water intake. Subsequent studies further implicated the
peptide in the arousal of salt appetite and provided potential
sites of actions for this effect. For example, salt appetite result-
ing from the natriuretic–diuretic furosemide was attenuated by
pharmacological reduction of circulating Ang-II and was
restored by intravenous administration of this peptide
(Weisinger et al., 1987a, 1987b, 1997).
As with Ang-II-induced water intake, investigators considered
the SFO and OVLT potential sites of action for Ang-II arousal of
salt appetite. In support of a role for these CVOs in this process,
ablation of the SFO decreased sodium intake elicited by
administration of the furosemide (Weisinger et al., 1990;
218 Body Fluid Homeostasis
Thunhorst et al., 1999) and lesion of the OVLT attenuated
sodium-depletion-induced intake (Chiaraviglio and Perez
Guaita, 1984), while the site-specific injection of Ang-II into
the OVLT stimulated NaCl consumption (Fitts and Masson,
1990). Although these studies suggested that Ang-II acted cen-
trally to elicit salt appetite, the biological relevance of this
phenomenon was called into question because supraphysiolog-
ical doses of Ang-II were required to produce the behavior. Large
doses of Ang-II administered centrally or intravenously elevate
blood pressure and can cause natriuresis (Findlay and Epstein,
1980; Fluharty and Manaker, 1983). This complicated interpre-
tation of studies examining Ang-II-induced salt appetite because
it was possible that the NaCl intake caused by Ang-II treatment
was the consequence of renal sodium excretion. However, the
recognition that circulating levels of Ang-II and aldosterone
increased under normal conditions of sodium depletion Stricker
et al. (1979) led to a new understanding of hormonal regulation
of salt appetite.
1.07.3.3.3 Angiotensin/Aldosterone Interactions
Because sodium depletion leads to the concurrent elevations in
aldosterone and Ang-II, it has been hypothesized that these two
hormones work in concert, or synergistically, to arouse sodium
appetite (Epstein, 1982). Consistent with this, Fluharty and
Epstein (1983) determined that simultaneous AT1R and MR
activation via the coadministration of Ang-II and DOC
produced a salt intake that was greater than the sum of the
amounts consumed when the same doses of Ang-II or DOC
were separately administered. Furthermore, low doses of Ang-
II and DOC that were ineffective at evoking salt appetite
when given separately became potent at inducing sodium
intake when given concurrently. This enhanced salt intake
was not accompanied by pressure-induced natriuresis, obvi-
ating the possibility that renal sodium excretion was respon-
sible for the augmented ingestion of NaCl. Additionally,
central antagonism of MR using RU-28318 reduced, but did
not eliminate, the salt appetite elicited by sodium depletion;
however, when both Ang-II and MR actions were pharmacolog-
ically inhibited, depletion-induced sodium intake was
completely abolished (Sakai et al., 1986). Again, these effects
were likely not due to altered renal sodium handling or general
inhibition of ingestive behaviors, as they occurred independent
of alterations in urinary sodium excretion or food intake (Sakai
et al., 1986). Thus, it was concluded that AT1R and MRs did
indeed work synergistically to arouse salt appetite.
The synergistic interactions between Ang-II were also
quickly appreciated to be brain-mediated, as blocking the
effects of Ang-II and aldosterone in the brain, but not in the
periphery, eliminates the salt appetite of furosemide-treated
rats (Sakai et al., 1986, 1990). Furthermore, chronic adminis-
tration of DOC increases Ang-II receptor binding in brain tissue
homogenates that included the hypothalamus, thalamus, and
septum (Wilson et al., 1986), and drinking stimulated by
central injection of Ang-II is augmented after DOC administra-
tion, demonstrating the behavioral consequences of these
increased Ang-II binding sites (Wilson et al., 1986). Subsequent
studies identified increased Ang-II receptor binding in specific
brain regions following DOC administration (Gutkind et al.,
1988; De Nicola et al., 1993) as well as within cultured neurons
(Sumners and Fregly, 1989).
Because DOC is a precursor for both aldosterone and corti-
costerone and its administration leads to elevations in both of
these adrenal steroids, it was not clear whether the observed
upregulation of Ang-II receptors was due to the actions of
MRs, GRs, or a combination of both. Nonetheless, a role for
GRs was proposed because DOC increased Ang-II receptor
binding to a greater degree than aldosterone (Wilson et al.,
1986) and because pretreatment with dexamethasone, a GR
agonist, also potentiated drinking stimulated by Ang-II
(Ganesan and Sumners, 1989). Moreover, intravenous infu-
sion of dexamethasone, corticosterone, or the GR receptor
agonist RU 28362 increased aldosterone-induced sodium
consumption, further suggesting that GRs contribute to the
arousal of salt appetite (Ma et al., 1993).
The strongest support for the regulation of salt appetite by
glucocorticoids comes from studies examining NaCl intake
and AT1R expression subsequent to DOC and dexamethasone
administration (Shelat et al., 1999b). Rats treated with both
DOC and dexamethasone ingested more NaCl than DOC given
by itself. Additionally, coadministration of DOC and dexa-
methasone augmented Ang-II receptor binding in the PVN,
SFO and area postrema; DOC alone evoked no such effect
(Shelat et al., 1999a). Consequently, some models of the
hormonal regulation of salt appetite combine the contribu-
tions of MRs as well as GRs.
It is worth noting that more recent studies have implicated
hindbrain neurons that are uniquely sensitive to aldosterone in
the control of sodium appetite. The MR has affinity for both
aldosterone and corticosterone with the latter circulating at
concentration orders of magnitude greater than the former
(Funder et al., 1988). Consequently, MR-expressing cells
that are uniquely responsive to aldosterone also express
11-b-hydroxysteriod dehydrogenase type 2 (11-bHSD2) which
inactivates glucocorticoids that under normal conditions
occupy MRs, which in turn, permits their binding of aldoste-
rone (Funder et al., 1988). Anatomical studies conducted by
Geerling and Loewy (2006) identified neurons residing in the
NTS that expressed both MRs and 11-bHSD2. These ‘HSD2’
neurons were found to send direct axonal projections to the
ventral portion of the bed nucleus of the stria terminalis, the
pre-locus coeruleus, the external lateral parabrachial nucleus,
the ventral tegmental area, the lateral and paraventricular
nuclei of the hypothalamus, and the central nucleus of the
amygdala (Geerling and Loewy, 2006). Given the hypothesized
role of brain MRs in salt appetite, the authors proposed that the
connectivity of HSD2 neurons comprised a neuronal circuit
controlling the drive to consume sodium when challenged
with such deficit. Indeed, their subsequent study determined
that HSD2 neurons in the NTS were mostly sensitive to
aldosterone and were activated by DOC or sodium depletion,
and this activation was inhibited by voluntary consumption
of NaCl (Geerling et al., 2006). Consistent with these results,
studies conducted by the Mifflin laboratory found that salt
appetite elicited by exogenous administration of aldosterone
is greatly reduced in rats delivered viral constructs that knock-
down MR expression in the NTS (Koneru et al., 2014).
However, it is interesting to note that adrenalectomized rats
that cannot synthesize aldosterone or corticosterone have
robust activation of HSD2 neurons that is abrogated by NaCl
consumption, indicating that adrenal steroids are not necessary

for the activation of HSD2 neurons or the emergence of salt
appetite (Geerling et al., 2006). These results suggest that
HSD2 neurons in the NTS are activated by aldosterone to drive
salt appetite, but in the absence of adrenal steroids sodium
deficiency still engages these HSD2 neurons to drive sodium
consumption.
1.07.3.3.4 Baroreceptors, Osmoreceptors, and Salt Appetite
Baroreceptors are mechanoreceptors located in blood vessels
near the heart that provide the brain with information pertain-
ing to blood volume and pressure, by detecting the level of
stretch on vascular walls. As blood volume increases, vessels
are stretched and the firing rate of baroreceptors increases. In
contrast, as vessels contract in response to reduced blood
volume, baroreceptors decrease firing. Since sodium insuffi-
ciency is often accompanied by changes in blood volume, it
was reasoned that baroreceptors were involved in the regula-
tion of sodium appetite. Consistent with this, surgical manipu-
lation of baroreceptors (Thunhorst et al., 1994) or the
sinoaortic nerves that communicate their status to the brain
(Thunhorst et al., 1994) alters sodium consumption. For
example, Toth et al. (1987) implanted small balloons into
the superior vena caval–right atrial junction of rats in order
to model volume expansion and determine its impact on
sodium appetite. Inflation of these balloons led to stretching
of the baroreceptors and signaled volume expansion to the
brain. This manipulation also attenuated NaCl intake elicited
either by peritoneal dialysis or by administration of DOC,
and intakes returned to normal when the balloons were
deflated. Similarly, NaCl intake elicited by furosemide is signif-
icantly reduced when baroreceptor input is eliminated by
sinoaortic denervation (Thunhorst et al., 1994). Collectively,
these studies suggest that baroreceptors provide the brain
with neural signals pertaining to blood volume that are critical
for the normal expression of sodium appetite.
While cardiac baroreceptors regulate salt appetite by detect-
ing changes in blood volume, the above-described cerebral
osmoreceptors influence sodium consumption by monitoring
the concentration of sodium within cerebrospinal fluid
(CSF). Along these lines, increasing the sodium concentration
within the brain via the intraventricular injection of hyperos-
motic CSF [Naþ, 500 mM] leads to an attenuation of
deprivation-induced sodium intake (Weisinger et al., 1979).
On the other hand, when osmolality of the CSF is increased
by intraventricular injection of mannitol, which decreases the
sodium concentration, sodium intake is augmented (Weisinger
et al., 1979). These results suggest that in addition to regulating
thirst, AVP, and OT secretion (as described above) by detecting
central changes in osmolality cerebral osmoreceptors may also
regulate the ingestion of sodium.
1.07.3.3.5 Taste and Salt Appetite
The sensory modality that is most important for the detection
of sodium is taste, and early studies conducted by Richter
and others revealed that adrenalectomy and the resulting
sodium insufficiency creates a clear preference for sodium solu-
tions that were previously indistinguishable from water
(Richter, 1939). Therefore, it was hypothesized that sodium
depletion led to changes in the oral cavity that contributed to
the increased salt intake, and experiments were conducted to
Body Fluid Homeostasis 219
investigate whether sodium deficiency enhanced the detection
threshold for NaCl solutions. Using a two-bottle preference
test, Richter determined that intact rats preferred a NaCl solu-
tion over water when the concentration was about 0.055%;
however, immediately after adrenalectomy rats drank more of
the NaCl solution when it was at a nearly 15-fold lesser concen-
tration of 0.0037% (Richter, 1939). That is, these results indi-
cated that sodium insufficiency decreases the threshold at
which rats prefer NaCl solutions over water, possibly by
increasing the sensitivity for the taste of sodium. Also along
these lines, examination of the ingestion of saline solutions
at various concentrations revealed that while intact rats have
a preference curve that resembles an inverted U-shape with
maximum consumption occurring near the concentration of
isotonic saline (Richter, 1956), sodium deficiency dramatically
changes this preference curve by elevating intake of NaCl at low
and high concentrations (Epstein and Stellar, 1955; Nachman,
1962; Curtis et al., 2001).
Of particular relevance, sodium-deficient rats develop this
preference for sodium within 15 s after they are given a choice
between sodium and nonsodium solutions (Nachman, 1962).
This short latency to develop a preference is also consistent
with the effects being taste-mediated as there is little time for
the animal to learn of the consequences of the ingestion.
Furthermore, sodium depletion alters orofacial patterns of
rats in response to intraoral infusions of high concentrations
of NaCl in a manner suggestive of the depletion rendering
the taste of sodium more palatable (Flynn and Grill, 1988).
High concentrations of NaCl that elicit aversive orofacial
behaviors in normal rats elicit ingestive behaviors when rats
are salt deficient (Berridge et al., 1984). In sum, sodium deficit
enhances the gustatory processing of sodium, thereby facili-
tating the likelihood that sodium will be detected and ingested.
1.07.3.4 Blood Pressure
Other important aspects of overcoming hypovolemia and the
concomitant hypotension include factors that are involved in
elevating blood pressure. To this end, hypotension is remedied,
at least in part, by the above-described mechanisms to mini-
mize sodium and water excretion and maximize their intake.
That is, an important outcome of increasing blood volume is
to also increase blood pressure. Also important, however, are
Ang-II’s (and AVP’s) more direct roles in elevating blood pres-
sure by activating receptors in the peripheral vasculature and
within certain brain nuclei. In regard to central effects of Ang-
II, the sensing of elevated Ang-II levels by CVOs, such as the
SFO also communicates to the brain the need to elevate blood
pressure; the goal being to restore blood pressure to normal
levels (Ferguson, 1988, 2009; Ferguson and Bains, 1997;
Krause et al., 2011b; Osborn et al., 2012). This ultimately leads
to an increase in blood pressure that, again, is partly an indirect
consequence of the behavioral effects discussed above.
However, it is also due to its ability to impact blood pressure
by other means, such as activation of the sympathetic nervous
system (SNS).
Much like with Ang-II’s behavioral actions, in order to
exert these influences over the SNS, CVO neurons send projec-
tions to and influence the activity of numerous cardiovascular
control centers that are generally protected by the BBB,
220 Body Fluid Homeostasis
including the PVN (Ferguson, 1988; Krause et al., 2011b).
Upon activation of these CVO projections, the release of tradi-
tional neurotransmitters (e.g., glutamate) from their nerve
terminals and onto neurons situated in downstream cardio-
vascular control nuclei is modulated, and sympathetic
outflow and blood pressure become elevated (Tanaka et al.,
2001; Saxena et al., 2014). That being said, much like the
positioning and influence of the AT1R within the BBB-
protected nuclei involved in the behavioral responses to
Ang-II, many of the downstream brain nuclei involved in
sympathetic outflow also densely express AT1R and Ang-II’s
actions at its AT1R within these nuclei impact cardiovascular
function (see Chapter 3.20, The Brain Renin–Angiotensin
System).
1.07.3.5 Hypervolemia
An increase in blood volume, hypervolemia, is also sensed by
the stretch-sensitive atrial baroreceptors to trigger the secretion
of atrial natriuretic peptide (ANP), from the cardiac atria (Chiu
et al., 1987; Palkovits et al., 1995). This volume-regulatory
hormone inhibits the secretion of renin and aldosterone
(Atlas and Maack, 1987), promotes natriuresis (Soares et al.,
1999), and opposes the stimulatory actions of Ang-II on water
intake and vasoconstriction (Antunes-Rodrigues et al., 1985;
Saavedra, 1990; McCann et al., 2003). There is also evidence
that ANP reduces sodium appetite and consumption (Ehrlich
and Fitts, 1990; Stellar and Epstein, 1991; McCann et al.,
1994). Collectively, ANP acts at its receptors throughout the
body to facilitate restoration of blood volume and pressure
to normal levels subsequent to volume expansion.
1.07.4 Body Fluid Homeostasis and Psychological
Stress
Stressors can be classified as either real (i.e., physiological) or
perceived (i.e., psychological) in nature and lead to behavioral,
endocrine, and autonomic adjustments that allow one to
attempt to cope with the stressor. Some examples of physiolog-
ical stressors include the threats to hydromineral balance dis-
cussed above, such as hypovolemia, while examples of
psychogenic stressors may include the loss of a loved one or
social isolation (Jankord and Herman, 2008). The origin and
initiation of the responses to these types of stressors are distinct
from one another; however, they are similar in that they
both activate the hypothalamic–pituitary–adrenal (HPA) axis
(see Chapter 4.07, Hypothalamic–Pituitary–Thyroid Axis).
Activation of the HPA axis is initiated by stimulation of
corticotrophin-releasing hormone (CRH) neurons within the
PVN that trigger the release of adrenocorticotrophin hormone
(ACTH) from the anterior pituitary and glucocorticoid
synthesis and release from the adrenal cortex into the systemic
circulation. In addition to the fact that physiological stress
(such as threats to body fluid homeostasis) and psychological
stress both activate the HPA axis, it is also evident that many
brain regions and neural circuits that are involved in the regu-
lation of body fluid homeostasis are also essential for the
neural, humoral, and behavioral responses to psychological
stress. It is perhaps not surprising, therefore, that challenges
to body fluid homeostasis can have a profound impact on
the responses to psychological stress.
For example, sodium depletion is anxiogenic in rats
(Leshem, 2011), and several factors that are traditionally
known to regulate hydromineral balance have also been impli-
cated in the responses to psychological stress. The RAAS, which
again, is activated during hypovolemic threats, is thought to
facilitate physiological and behavioral responses to psycholog-
ical stress (Saavedra et al., 2006; Krause et al., 2011b; Saavedra,
2011). Psychological stress activates the RAAS and upregulates
certain populations of AT1R in the absence of threats to hydro-
mineral balance (Yang et al., 1993; Aguilera et al., 1995; Krause
et al., 2011b) and inhibition of Ang-II’s actions at the SFO also
inhibits anxiety-like behavior and stress-induced activation of
the HPA axis (Krause et al., 2008, 2011b). Along these lines,
the RAAS has gained considerable attention over the past
decade as a potential therapeutic target for treating stress-
related disorders (Baghai et al., 2002, 2006; Saavedra et al.,
2006; Krause et al., 2011b; Khoury et al., 2012; Hurt et al.,
2015). Angiotensin receptor blockers administered either
peripherally or centrally improve mood and cognition as well
as attenuate HPA axis and sympathetic responses to stressors
having strong psychogenic components (Barnes et al., 1990;
Kaiser et al., 1992; Jezova et al., 1998; Kubo et al., 2001;
Saavedra et al., 2006).
On the other hand, challenges to body fluid homeostasis
that manifest as increases in osmolality oppose or dampen
the physiological and behavioral responses to psychological
stress (Krause et al., 2011a; Frazier et al., 2013; Smith et al.,
2014). As discussed above, excess body sodium or hypernatre-
mia creates a distinct neural and humoral milieu that alleviates
elevations in body sodium levels. One important aspect of this
milieu is the inhibition of the RAAS, and it is possible that this
reduction contributes to the stress-dampening effects of hyper-
osmotic conditions by removing or reducing the facilitating
influence of Ang-II on stress responses. That being said, there
is also evidence for a prominent role of OT in these stress-
dampening actions. This is supported by rodent studies
demonstrating that increased plasma osmolality induced by
the systemic administration of hypertonic (2.0 M) saline fol-
lowed by 60 min water deprivation significantly increases
plasma OT concentrations that correspond to a reduced HPA
axis response to an acute psychological stressor (Krause et al.,
2011a). In addition to blunting the HPA axis response to the
psychological stressor, this paradigm also results in a blunted
cardiovascular response to stress and decreased anxiety-like
behavior in the elevated plus maze and social interaction para-
digm (Krause et al., 2011a). Subsequent anatomical studies
revealed that acute hypernatremia elicits robust activation of
OT neurons in the PVN but attenuates stress-induced activation
of CRH neurons (Smith et al., 2014). Furthermore, whole-cell
patch-clamp recordings found that acute hypernatremia
causes an inhibition of CRH neurons in the PVN that is
dependent on activation of OT receptors; in other words, the
increased plasma osmolality is associated with an elevated
inhibitory oxytocinergic tone that is exerted onto CRH neurons
(Frazier et al., 2013). The implication is that this oxytocinergic
tone on CRH neurons observed in electrophysiological
experiments suppresses stress-induced activation of CRH
neurons and consequent elevations in ACTH and

glucocorticoids in vivo. Therefore, while disturbances to body
fluid homeostasis that manifest in reduced blood volume
may heighten responses to psychological stress, those that
manifest in increased plasma osmolality may act to dampen
responses to psychological stress.
1.07.5 Summary and Conclusion
As discussed throughout this chapter, alterations in body fluid
homeostasis resulting from changes in the osmolality,
volume, and/or pressure of the blood are detected by sensors
in the periphery and brain that relay the imbalance to effector
organs. Consequently, neural, humoral, and behavioral
compensatory responses are initiated and maintain or restore
body fluid homeostasis. Although changes in plasma osmo-
lality and changes in effective circulating volume and blood
pressure are similarly considered threats to body fluid homeo-
stasis, the behavioral and endocrine mechanisms by which
osmolality, volume, and pressure are regulated are distinct
and these mechanisms are summarized in Figures 1 and 2.
That is, plasma osmolality is maintained largely by changes
in water balance and activation of the AVP/OT renal axes.
On the other hand, the RAAS is largely responsible for the
mechanisms by which blood volume and pressure are main-
tained. That being said, AVP is involved both in the restora-
tion of osmolality and in the restoration of blood volume
and pressure subsequent to hyperosmotic or hypovolemic
threats, respectively. That is, AVP secretion is stimulated in
response to increased plasma osmolality and also in response
to profound hypovolemia. In both conditions, it acts to regu-
late body fluid homeostasis by activating its receptors in the
kidney and vasculature, to promote water reabsorption and
vasoconstriction.
In regard to the behavioral responses that act to preserve
body fluid homeostasis, it is particularly important to note
that although both hypovolemic and hyperosmotic stimuli
both induce the sensation of thirst and the subsequent act
of drinking, hypovolemic thirst differs in that it is also accom-
panied by an increase in sodium conservation, appetite, and
consumption; the goal being to restore the extracellular deficit
in fluid which requires the restoration of both solute
and water.
The regulation of body fluid homeostasis is critical for all
living organisms and requires coordinated responses with
cardiovascular, renal, neuroendocrine, and behavioral nodes.
The humoral and neural mechanisms that orchestrate these
responses are highly conserved and in some instances are
hardwired. Consequently, investigation of such mechanisms
reveals fundamental brain circuits controlling the compensa-
tory responses and motivated behaviors that promote the
homeostatic concentrations of water and solute that are
essential for normal physiological function. Given their
importance to the vitality of an organism, it is likely that
such mechanisms were first established to maintain hydro-
mineral balance, but subsequently, evolved to promote other
types of adaptive physiological and behavioral responses. In
this way, studying the neuroendocrine control of thirst and
salt appetite, as well as those mediating blood pressure and
renal function, provides insight toward an infrastructure
Body Fluid Homeostasis 221
that also supports neuronal networks implicated in a variety
of behaviors ranging from stress and anxiety to sociality and
affiliation.
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 1.08 Stress and Opioid Systems
Ryszard Przewlocki, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland
Osborne FX Almeida, Max Planck Institute of Psychiatry, Munich, Germany
Ó 2017 Elsevier Inc. All rights reserved.
This chapter is a revision of the previous edition chapter by R. Przewlocki, volume 1, pp. 289–332, Ó 2009, Elsevier Inc.

1.08.1 Introduction: The Concept of Stress 226

1.08.2 Opioid Systems 227
1.08.2.1 Multiple Opioid Peptides 227
1.08.2.2 Opioid Receptor Subtypes and Their Ligands 227
1.08.3 Distribution of Opioid Peptides and Receptors in the Stress Network 232
1.08.3.1 Proopiomelanocortin System 232
1.08.3.2 Proenkephalin System 232
1.08.3.3 Prodynorphin System 232
1.08.3.4 Endomorphins 232
1.08.3.5 Opioid Receptors 232
1.08.4 Stress-Induced Alterations in Endogenous Opioidergic Systems 233
1.08.4.1 Proopiomelanocortin System 233
1.08.4.2 Proenkephalin System 234
1.08.4.3 Prodynorphin System 234
1.08.4.4 Opioid Receptors 235
1.08.5 Multiple Neurochemical Interactions with Opioid System as a Consequence of Stress 236
1.08.5.1 Dopaminergic System 236
1.08.5.2 Adrenergic System 236
1.08.6 Involvement of Opioids in Modulation of the HPA Axis 237
1.08.6.1 Corticotropin Releasing Factor 237
1.08.6.2 Vasopressin System 238
1.08.7 Involvement of Opioids in the Behavioral and Physiological Response to Stress 238
1.08.7.1 Locomotor Activity 238
1.08.7.2 Reward 239
1.08.7.3 Nociception: SIA 240
1.08.7.4 Thermoregulation: Stress-Induced Hyperthermia 243
1.08.7.5 Feeding Behavior 243
1.08.7.6 Cardiovascular Effects 244
1.08.7.7 Respiration 245
1.08.7.8 Reproduction 245
1.08.7.9 Autonomic Nervous System 245
1.08.7.10 Immune System 246
1.08.8 Therapeutic Potential of Opioid Agonists and Antagonists in Stress-Related Psychiatric Disorders 247
1.08.9 Opioidergic Control of Stress Responses – Conclusions 248
Acknowledgments 248
References 249

Nomenclature
ACTH adrenocorticotropic hormone GAS general adaptation syndrome
CRF corticotrophin-releasing factor HPA hypothalamo–pituitary–adrenal axis
CTOP D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH 2 (MOP ICI 174,864 [allyl]2-Tyr-alpha-amino-isobutyric acid
receptor selective agonist) (Aib)-Aib-Phe-Leu-OH (DOP receptor antagonist)
DAMGO [D-Ala2, MePhe4, Gly-ol]-enkephalin (selective m- ICSS intracranial self-stimulation
receptor agonist) IFN-a interferon-a
DAT dopamine transporter KOP k-opioid peptide
DOP d-opioid peptide LH lateral hypothalamus
EOPs endogenous opioid peptides LPS lipopolysaccharide
b-FNA beta-funaltrexamine (MOP receptor antagonist) MOP m-opioid peptide

Hormones, Brain, and Behavior, 3rd edition, Volume 1 http://dx.doi.org/10.1016/B978-0-12-803592-4.00008-0 225

226 Stress and Opioid Systems
Mr2266 (1R,5R,9R)-5,9-diethyl-2-(3-furylmethyl)-20 -
hydroxy-6,7-benzomorphan (opioid antagonist)
MSH melanocyte-stimulating hormone
NTS nucleus tractus solitarii
PDYN prodynorphin
PENK proenkephalin
1.08.1 Introduction: The Concept of Stress
The term ‘stress’ refers to the mental or bodily tension that
occurs upon experiencing a stressful stimulus (‘stressor’) that
challenges the organism’s homeostatic mechanisms and poten-
tially threatening its health and well-being. The stress response
is the compensatory reaction of the body to the disturbance
imposed by the stressor. Generally, the stress response evolves
as an adaptive mechanism, but in extreme cases, depending on
the quality, intensity, or duration of the noxious stimulus, the
response can produce maladaptive responses (McEwen and
Wingfield, 2003; de Kloet et al., 2005). Importantly, the indi-
vidual’s state at the moment the stressor arrives, is also an
important, albeit often neglected, consideration; it is important
because the way the individual perceives the stressor will deter-
mine its subsequent psychological as well as physiological reac-
tion (Lovallo, 1997; Koolhaas et al., 2011). It is worthwhile to
briefly mention some historical landmarks that helped make
stress the intensely studied subject it is today.
The idea of the critical need to maintain constancy of the
milieu de l’intérieur (internal environment) was introduced by
Claude Bernard in the mid-nineteenth century and later extended
by Walter Cannon (1939) who introduced the term homeostasis to
describe physiological reactions which maintain the steady state
of the organism in the face of external stimuli. Cannon noted
that the brain plays an important role in maintaining homeo-
stasis as it communicates with the rest of the body via specialized
sensory nerves to recognize the external threats and to detect the
internal state of the body. He also suggested that the brain can
activate multiple mechanisms to compensate for any disequilib-
rium caused, and importantly, he observed that psychological
as well as physiological disturbances could elicit responses
from the sympathetic nervous system and adrenals.
Today’s concept of stress was framed first by Selye (1936). He
observed that, irrespective of the nature (or identity) of the
stressor(s), the physiological changes were relatively similar
and were primarily mediated by the hypothalamo–pituitary–
adrenal (HPA) axis. He used the term ‘stress’ to refer to the phys-
iological response to ‘stressor’ such as heat, cold, toxic, and infec-
tious agents. He discovered that exposure to stressors caused
enlargement of the adrenal cortex, reduction of thymus and
lymph gland weights, and appearance of stomach ulcers. Selye
described the process in terms of a three-stage model called the
General Adaptation Syndrome (GAS). In the first, alarm stage,
there is an adaptive reaction to the external challenge that is likely
to be flight or fight; this involves increased heart rate, pupillary
dilatation, a stop to digestion, muscle tension, and activation
of the adrenals. If this reaction is unsuccessful, the organism
enters the state of arousal or resistance for which it uses existing
POMC proopiomelanocortin
RU (11b,17b)-11-[4-(dimethylamino)phenyl]-17-
hydroxy-17-(1-propynyl)estra-4,9-dien-3-one
SIA stress-induced analgesia
U50,488H trans-()-3,4-dichloro-N-methyl-N-[7-(1-
pyrrolidinyl)cyclohexyl] (selective k-receptor agonist)
reserves (e.g., energy) to help it cope. Selye referred to the third
phase of the stress response as the exhaustion stage when the
organism has failed to overcome or adapt successfully to the
environmental challenge, thereby increasing its chances of death.
While earlier theories of stress tended to focus on physiolog-
ical responses only, it is now common to also consider the
emotional and psychological aspects of stress. Importantly,
stressors that are not accompanied by an emotional component
elicit only minor physiological responses (Mason, 1971). Munck
et al. (1984) suggested that the initial response was specific to
a particular stressor and targeted toward maintaining homeo-
stasis. Accordingly, the subsequent and slower hormonal
response of the HPA axis is a response to the initial behavioral
response rather than to the stressor per se. In an important
conceptual advance, Kopin et al. (1988) defined stress as a state
in which expectations did not match current or anticipated
perceptions of the internal or external environment and that
the maintenance of homeostasis involves (usually) unconscious
adjustments in multiple systems; failure in any of these would
lead to a state of distress associated with emotional components
such as fear or anxiety. All physical stressors have a psychological
component (Lovallo, 1997), and as alluded to above, physiolog-
ical and psychological factors are equally important determi-
nants of the stress response.
An activated HPA axis is the most overt and easily measure-
able physiological response to stress. Stress stimulates the secre-
tion of proopiomelanocortin (POMC)-derived peptides such
as adrenocorticotropic hormone (ACTH) and opioid peptide
b-endorphin as well as corticotropin releasing factor (CRF)
and glucocorticoids (GCs). This reaction is accompanied by
autonomic responses, namely, activation of the sympathetic
nervous system and the release of noradrenaline from sympa-
thetic nerve endings and adrenaline from the adrenal medulla.
These changes trigger rapid alterations in cardiovascular and
immune system functioning.
The discovery of the endogenous opioid peptides (EOPs),
and research into their physiology, pharmacology, and molec-
ular biology, provided new insights into our understanding of
stress. The EOPs modulate many behaviors and affect physio-
logical functions in both the brain and periphery, ranging
from motor activity and nociceptive threshold to feeding and
sexual behavior; all of these functions are sensitive to the dis-
rupting effects of stress and interestingly encompass interac-
tions between central opioidergic pathways, neuroendocrine
systems, the sympathetic nervous system, and the immune
system. The many nodes at which EOP can act to influence
physiology and behavior suggest a potentially important role
for them in facilitating the organism’s interaction with, and
response to, the ever-changing external environment.

In this chapter, we will discuss the possible involvement of
opioids in mediating physiological as well as behavioral reac-
tions to stress. Further, we will focus on the neurobiological
mechanisms that underlie interactions between EOP systems
and other stress-processing pathways. We will also discuss alter-
ations in EOP systems and multiple opioid receptors which
occur as a result of the organism’s reaction and adaptation to
stressful stimuli. Lastly, we will consider current research on
the use of opioid receptor agonists and antagonists for the treat-
ment of stress-related disorders in psychiatry.
1.08.2 Opioid Systems
1.08.2.1 Multiple Opioid Peptides
Over the last two decades, our understanding of the biogenesis
of various EOPs, their anatomical distribution, and characteris-
tics of the multiple receptors with which they interact, has
advanced considerably. The EOPs derive from three different
precursor proteins: POMC, prodynorphin (PDYN), and proen-
kephalin (PENK), which were cloned in the late 1970s and early
1980s (Nakanishi et al., 1979; Kakidani et al., 1982; Noda et al.,
1982). Details concerning EOP discoveries, their body and brain
distribution, and properties have been reviewed elsewhere
(Höllt, 1990). The main groups derived from PENK, PDYN,
and POMC are enkephalins, dynorphins, and b-endorphin,
respectively. PENK is the source of Met- and Leu-enkephalins
and several longer peptides. EOPs such as dynorphin A, dynor-
phin B and a- and b-neoendorphin and several larger molecules
are generated from PDYN. POMC is the precursor of b-endor-
phin, a-endorphin, and several nonopioid peptides. Recently,
a novel group of brain peptides, the endomorphins,
endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2
(Tyr-Pro-Phe-Phe-NH2), have been discovered. These unique
EOPs have a characteristic structure and anatomical distribution
and show high selectivity for the m-opioid peptide (MOP)
receptor (Zadina et al., 1997).
EOP-containing neurons are commonly found in tissues that
make up the HPA axis, namely the hypothalamus, pituitary, and
adrenal glands and all of which are involved in mounting the
neuroendocrine response to stress. In addition, central and
peripheral opioidergic neurons innervate the autonomic
nervous system centers. Further, EOPs are widely distributed in
brain areas (Figure 1(a) and 1(b)), also in structures associated
with emotional components of stress. It is important to note
that, most EOPs are usually not tonically active, and hence,
opioid antagonists have little or no effect under homeostatic
states. However, they are activated by stressful stimuli and act
to influence the physiological responses to stress. Interestingly,
b-endorphin derives from the same precursor (POMC) as that
from which adrenocorticotropin (ACTH) is cleaved, and both
molecules are cosecreted by hypothalamic neurons and
pituitary corticotropes. Colocalization and corelease also
occurs in the case of adrenaline and the enkephalins in the
adrenals, and the dynorphins and CRF in hypothalamic neurons.
1.08.2.2 Opioid Receptor Subtypes and Their Ligands
Three members of opioid receptor family were cloned in the
early 1990s, first, the mouse d-opioid peptide (DOP) receptor
Stress and Opioid Systems 227
(Evans et al., 1992; Kieffer et al., 1992), then the MOP receptor
(Chen et al., 1993a; Fukuda et al., 1993; Thompson et al.,
1993), and lastly, the k-opioid peptide (KOP) receptor (Chen
et al., 1993b; Li et al., 1993; Meng et al., 1993; Minami et al.,
1993; Nishi et al., 1993). All three receptors share extensive
structural homologies and belong to the family of seven trans-
membrane G-protein-coupled receptors (GPCR). In some
cases, certain EOPs bind to more than a single opioid
receptor, albeit with differing affinities; the binding properties
of EOPs and of related opioidomimetics are reviewed
elsewhere (Okada et al., 2002, 2003). Generally, the
enkephalins and dynorphins are considered to be high-
affinity agonists of the DOP and KOP receptors, respectively.
However, b-endorphin binds similarly to both MOP and
DOP receptors.
Functional studies led to postulation that different subtypes
of main opioid receptors exist (Cox et al., 2015). That postulate
was confirmed by molecular analysis which also showed the
existence of several variants of opioid receptor subtypes (Pas-
ternak and Pan, 2013; Dietis et al., 2011). In addition, oligo-
merization of various opioid receptors has been shown to
generate receptors with unique functional properties; for
example, an association between DOP receptors and MOP
receptors was recently demonstrated, with the occupancy of
DOP receptors (by DOP receptor antagonists) enhancing
MOP receptor binding and signaling activity (Alfaras-Melainis
et al., 2009).
Pharmacological studies have led to the description of
several opioid receptor subtypes (MOP1, MOP2; DOP1,
DOP2; KOP1, KOP2, KOP3) (Dietis et al., 2011). While
such categorization has not been confirmed by molecular
biology to date, the existence of different variants of the
known opioid receptors has been suggested (Koch et al.,
1998; Uhl et al., 1999; Abbadie et al., 2000; Pasternak and
Pan, 2000). Alternatively spliced MOP receptor subtypes
have in fact been described in mice (Pan et al., 1999, 2000,
2001, 2003), rats (Zimprich et al., 1995), and humans (Bare
et al., 1994). The particular MOP receptor domains encoded
by various exons of MOP-1 mediate different mechanisms
of MOP receptor activation (Schuller et al., 1999), and various
MOP receptor ligands, acting at different MOP-1 splice vari-
ants, can be ranked differentially according to efficacy
(Pasternak, 2004). Although it is not known how the alterna-
tive variants correspond to the pharmacologically defined
receptors types, their existence may help explain some of
various phenomena observed in clinical settings, e.g., incom-
plete cross-tolerance to different MOP receptor agonists
(Pasternak, 2004). Interestingly, genetic studies suggest that
humans carrying one or two copies of the 118G MOP receptor
allele may be at increased risk for opiate and alcohol addic-
tion (Kreek and LaForge, 2007), and of pertinence to the
present chapter, Lovallo (2006) suggested individuals with
an altered MOP receptor allele coding for the high-affinity
MOP receptor may be more vulnerable to stress and more
prone to develop addiction as a consequence of stressful situ-
ation. The pharmacologically defined subtypes of KOP
receptor have also been proposed to have functional signifi-
cance; for example, dopamine and acetylcholine release in
rat neostriatum was shown to be modulated by KOP1 and
KOP receptors, respectively (Schoffelmeer et al., 1997).
228 Stress and Opioid Systems

Figure 1 Anatomical distribution of opioid peptides (a) their place of gene expression (b) and opioid receptors and (c) and sites of their expression
(d) in the rat brain. Colors correspond to each of the three opioid receptor or peptide precursor. Densities are represented by symbols from low
(L) medium (M) to high (D). AD, anterodorsal thalamus; AL, anterior lobe, pituitary; AMB, nucleus ambiguus; AON, anterior olfactory nucleus; ARC,
arcuate nucleus, hypothalamus; BLA, basolateral nucleus, amygdala; BNST, bed nucleus of the stria terminalis; CEA, central nucleus, amygdala; Cl,
claustrum; CL, centrolateral thalamus; CM, centromedial thalamus; COA, cortical nucleus, amygdala; CPU, caudate putamen; C, cerebellum; DMH, dor-
somedial hypothalamus; DMR, dorsal and medial raphe; DR, dorsal raphe; DTN, dorsal tegmental nucleus; EN, endopiriform cortex; ENT, entorhinal
cortex; FRCX, frontal cortex; G, nucleus gelatinosus, thalamus; GP, globus pallidus; HbL, lateral habenula; HbM, medial habenula; HPC, hippocampus;
IL, intermediate lobe, pituitary; IP, interpeduncular nucleus; LC, locus coeruleus; LD, laterodorsal thalamus; LG, lateral geniculate, thalamus; LH, lateral
hypothalamus; LRN, lateral reticular nucleus; MD, mediodorsal thalamus; ME, median eminence; MEA, median nucleus, amygdala; MG, medial genicu-
late; MM, medial mammillary nucleus; NAc, nucleus accumbens; NL, neuronal lobe, pituitary; NRGC, nucleus reticularis gigantocellularis; NST, nucleus
tractus solitarius; OB, olfactory bulb; OCX, occipital cortex; PAG, periaqueductal gray; PCX, parietal cortex; PIR, piriform cortex; PN, pontine nucleus;
PNR, pontine reticular; PO, posterior thalamus; POA, preoptic area; PV, paraventricular thalamus; PVN, paraventricular hypothalamus; RE, reuniens
 Stress and Opioid Systems 229

Figure 1 (continued).

=thalamus; RM, raphe magnus; S, septum; SON, supraoptic nucleus; SN, substantia nigra; STN, spinal trigeminal nucleus; TCx, temporal cortex; Tu,
olfactory tubercle; VL, ventrolateral thalamus; VM, ventromedial thalamus; VMH, ventromedial hypothalamus; VP, ventral pallidum; VPL, ventropostero-
lateral thalamus; VR, ventral raphe; VTA, ventral tegmental area; ZI, zona incerta. Adapted from Le Merrer, J., Becker, J.A., Befort, K., Kieffer, B.L.,
2009. Reward processing by the opioid system in the brain. Physiol. Rev. 89 (4), 1379–1412; Erbs, E., Faget, L., Veinante, P., Kieffer, B.L., Massotte,
D., 2014. In vivo neuronal co-expression of mu and delta opioid receptors uncovers new therapeutic perspectives. Recept. Clin. Investig. 1 (5), 210.
230 Stress and Opioid Systems

Figure 1 (continued).
 Stress and Opioid Systems 231

Figure 1 (continued).
232 Stress and Opioid Systems
1.08.3 Distribution of Opioid Peptides and
Receptors in the Stress Network
1.08.3.1 Proopiomelanocortin System
b-Endorphin and related peptides are present in the nucleus
arcuatus of the mediobasal hypothalamus (Sofroniew, 1979;
Bugnon et al., 1979; Khachaturian et al., 1985). An extensive
nerve fiber system originating in the arcuate nucleus terminates
in many areas of the brain which have been implicated in the
stress response, e.g., various hypothalamic nuclei, limbic and
raphe nuclei, and some pontine nuclei. Moreover, POMC
mRNA is also found in the reward system, specifically in the
medial prefrontal cortex, nucleus accumbens, and ventral
tegmental area (Leriche et al., 2007). Some of these structures
may also be innervated by laterally projecting POMC neurons
located in the nucleus tractus solitarii (NTS) of the caudal
medulla; POMC projections from the NTS are also found in
the spinal cord (Bronstein et al., 1992; Maley, 1996). Endocrine
cells of the intermediate lobe of the pituitary, cells of some
peripheral tissues, and immunocytes also synthesize and
release POMC peptides (Bloom et al., 1978; Blalock et al.,
1985) and may contribute to the peripheral responses to stress;
further, POMC peptides secreted from mast cells in the skin
have been implicated in the regulation of stress response to
exogenous stressful stimuli (Slugg et al., 2000; Arck et al.,
2006; Slominski et al., 2006).
1.08.3.2 Proenkephalin System
PENK neurons are widely distributed throughout the central
and peripheral nervous systems, primarily within interneu-
rons, some of which form longer local tract projections.
They are especially abundant in the paraventricular and arcua-
tus nuclei of the hypothalamus and are relatively well repre-
sented in limbic structures such as the hippocampus,
septum, and bed nucleus of the stria terminalis. Septal
PENK neurons project directly to the amygdala, extend
throughout the bed nucleus of the stria terminalis, and from
there, project to the hypothalamic paraventricular nucleus
and median eminence. PENK neurons have also been mapped
in the spinal cord, cranial sensory systems, and in the major
pain-signaling network. Further a variety of PENK-
containing cells are present in the adrenal medulla (Viveros
et al., 1979).
1.08.3.3 Prodynorphin System
PDYN neurons are widely distributed in brain areas associated
with stress (Watson et al., 1981; Khachaturian et al., 1985).
Dynorphin and related peptides are coexpressed with arginine
vasopressin in the magnocellular neurons of the paraventricu-
lar nucleus of the hypothalamus and also present in the NTS,
an area closely associated with the regulation of vagal and other
autonomic functions. Further, PDYN neurons occur in the
limbic system and in areas of the spinal cord involved in the
transmission of nociceptive stimuli. Cells expressing PDYN
mRNA are also present in a subpopulation of anterior pituitary
lobe gonadotrophs (Khachaturian et al., 1985), in POMC-
expressing melanotrophs of the intermediate pituitary lobe
(Day et al., 1993) as well as in neuronal terminals of
posterior pituitary pituicytes, the adrenal glands; in the latter
PDYN mRNA is strongly expressed in the adrenal cortex (Day
et al., 1991).
1.08.3.4 Endomorphins
Endomorphins are endogenous peptides that were relatively
recently isolated from bovine and human brain (Zadina
et al., 1997; Hackler et al., 1997). These peptides differ in their
amino acid sequences from other known EOPs; rather than
having a Tyr residue followed by Gly, endomorphins resemble
members of another related family of peptides (e.g., morphi-
ceptin, hemorphin, and casomorphins) that instead contain
Tyr–Pro residues. Endomorphins are localized in neuronal
circuits involved in processing nociceptive information and
also in many stress-responsive areas of the brain and auto-
nomic nervous system where MOP receptors are present
(Zadina et al., 1999). Endomorphins act as endogenous ligands
of MOP receptors and thus are important in modulating
responses to pain and stress by acting on autonomic and neuro-
endocrine systems (Zadina, 2002; Wang et al., 2003; Glatzer
and Smith, 2005; Silverman et al., 2005; Greenwell et al.,
2007). In addition, the common distribution of endomorphins
and other neurotransmitters suggests their modulatory role in
neurotransmission (Greenwell et al., 2007).
1.08.3.5 Opioid Receptors
Opioid receptors are differentially distributed in brain areas
(Figure 1( c) and 1(d)), also in structures involved in the regu-
lation of the stress response. Dense MOP and KOP receptor
binding and high expression is present in many hypothalamic
nuclei in the monkey (Mansour et al., 1988) and human
(Peckys and Landwehrmeyer, 1999). However, equivalent areas
of the rat brain display few or no MOP-binding sites (Mansour
et al., 1987). DOP receptors are also very poorly distributed in
hypothalamic nuclei, e.g., weakly detectable in the
ventromedial nucleus in the rat. All three opioid receptors
(MOP, DOP, and KOP) are present in the monkey median
eminence, with KOP receptors being most densely expressed
in this area; KOP receptors are also richly expressed in the
monkey and rat posterior pituitary lobe (Mansour et al.,
1988). This pattern of opioid receptor distribution is
consistent with the view that opioids are important regulators
of neuroendocrine secretion. In addition, a moderate density
of MOP and KOP receptor binding has been found in the
periaqueductal gray, locus coeruleus, substania nigra, ventral
tegmental area, raphe nuclei, and NTS, while low DOP
receptor binding has been reported in the substantia nigra
and NTS. Further, opioid receptor-containing neurons have
also been described in the limbic system where they may
modulate the emotional component of stress and in areas of
the spinal cord involved in the transmission of nociceptive
stimuli. Opioid receptors have also been found in the
peripheral nervous system (Bechara and van der Kooy, 1985;
Hedner and Cassuto, 1987; Wittert et al., 1996) as well as in
various immune cells (Wybran et al., 1979; Blalock et al.,
1985; Sibinga and Goldstein, 1988; Carr et al., 1988, 1989;
Stein et al., 1990; Gaveriaux et al., 1995; Chuang, 1995;
Peterson et al., 1998).

1.08.4 Stress-Induced Alterations in Endogenous
Opioidergic Systems
Most studies on the interrelationship between stress and the
endogenous opioid system have been on male laboratory
rodents. The main reason for this is that male physiology is
not subject to cyclical changes in the secretion of gonadal
hormones; the short duration of the rodent ovarian cycle in
commonly used experimental species (4–5 days) makes study
design and results interpretation in rodents particularly diffi-
cult. Greater effort should be placed on overcoming these diffi-
culties, however, because of well-known sex differences in the
stress response (Bale and Epperson, 2015; Ferin, 1993; Kokras
and Dalla, 2014; Patchev and Almeida, 1999). Readers inter-
ested in the modulation of opioidergic activity by sex
hormones are referred to studies by Almeida and Pfeiffer
(1991), Chartoff and Mavrikaki (2015), Ferin et al. (1984),
and Piva et al. (1995).
1.08.4.1 Proopiomelanocortin System
Acute and chronic stress appear to influence the activity of
EOP systems in the brain. Evidence indicates that releasable
pools of b-endorphin exist within the hypothalamus
(Osborne et al., 1979), and early in vivo studies showed
that short-term footshock stress depletes b-endorphin in
the hypothalamus, septum, and periaqueductal gray of rats
(Rossier et al., 1977; Millan et al., 1981a; Przewlocki et al.,
1982). Interestingly, conditioned fear-induced stress mark-
edly decreased hypothalamic content of b-endorphin (Przew-
locka et al., 1990), a finding in line with an earlier
observation that there is a measurable decrease of b-endor-
phin levels in the midbrain when chronically stressed rats
are acutely restressed (Akil et al., 1986). On the other
hand, cold swim stress was found to increase hypothalamic
b-endorphin levels (Vaswani et al., 1988), although a more
severe stress (acute prolonged intermittent footshock) did
not alter hypothalamic and midbrain levels of the peptide
(Akil et al., 1986; Przewlocki et al., 1987). Thus, the stress-
induced decrease in hypothalamic b-endorphin levels might
be detectable only after mild, short-term stress. Various kinds
of chronic stress (repeated footshock, repeated electroconvul-
sive shock) reportedly do not induce substantial alterations
in brain levels of b-endorphin (Lason et al., 1987; Przewlocki
et al., 1987). b-Endorphin levels were found to increase in
the nucleus accumbens septi following acute intermittent
footshock and after extinction of opiate self-administration
(Zangen and Shalev, 2003). On the other hand, hypotha-
lamic and amygda levels of b-endorphin were found to be
increased by social isolation of adult gerbils (Raab et al.,
1985), probably pointing to the intricate control of tissue
neuropeptide levels that reflect peptide release, biosynthesis,
and axonal transport. It is also worth noting that the POMC
gene is under the negative control of adrenal steroids
(Beaulieu et al., 1988).
Studies examining how POMC mRNA levels are modulated
by various stressors have produced divergent results. An acute
(1 h) restraint stress resulted in increased levels of POMC
mRNA in the arcuate nucleus of the hypothalamus (Larsen
and Mau, 1994), whereas arcuate POMC mRNA levels were
Stress and Opioid Systems 233
decreased following repeated immobilization (Makino et al.,
1999) and food deprivation (Gayle et al., 1999). In contrast,
water restriction had no effect on hypothalamic levels of
POMC mRNA (Zhou et al., 1999).
Stress leads to a substantial release of b-endorphin into the
blood of animals and humans (Molina, 2006), most likely
originating from corticotrophs in the anterior pituitary lobe.
Increased blood levels of b-endorphin are seen following
different acute stressors, such as electrical footshock (Guille-
min et al., 1977; Rossier et al., 1977; Millan et al., 1981a;
Przewlocki et al., 1982), swim stress (Przewlocka et al.,
1988; Vaswani et al., 1988; Young et al., 1993a), burn injury
(Osgood et al., 1987), immobilization (Forman and Estilow,
1988; Kjaer et al., 1993b; Larsen and Mau, 1994), cold stress
(Forman et al., 1988), hemorrhagic shock, muscle crush
injury, administration of LPS (Molina, 2002), food (Majeed
et al., 1986) and water deprivation (Zhou et al., 1999), labor
(Pancheri et al., 1985; McLean et al., 1994), electroacupunc-
ture (Pan et al., 1996), surgical stress (Roth-Isigkeit et al.,
2000), septic shock and sepsis (Legakis et al., 1998), and
simulated combat military flight (Leino et al., 1998). These
changes in peptide release are paralleled by increases in
the rate of POMC synthesis and processing in the anterior
pituitary (Akil et al., 1982; Höllt et al., 1986; Harbuz and
Lightman, 1992; Zhou et al., 1999).
The intermediate lobe of pituitary responds to stress in
a similar way to the anterior lobe (Przewlocki et al., 1982;
Berkenbosch et al., 1983; Akil et al., 1985; Young et al.,
1993b). Acute exposure to 30-min swim stress at room temper-
ature increased plasma b-endorphin levels (derived from the
intermediate lobe) several fold (Young, 1990; Young et al.,
1993b); the effect was abolished by apomorphine, indicating
the involvement of dopaminergic receptors. Acute ether stress
followed by 1-h restraint stress also increased POMC mRNA
in the intermediate lobe cells, which appeared to be mediated
by tonic activity of the serotoninergic system (Garcia-Garcia
et al., 1998).
POMC-containing cell types in the anterior and
intermediate pituitary lobes are derived from a single
embryonic rudiment and synthesize the same hormone
precursor, POMC, but differ in the pattern of precursor
processing and regulation of peptide secretion. Whereas
b-endorphin is the predominant EOP produced and secreted
by anterior pituitary (Young and Akil, 1985), the
intermediate lobe shows a slight increase in its content of
N-acetyl-b-endorphin following stress; this peptide is devoid
of opioidergic activity but contributes to the changes in the
circulating b-endorphin levels (Akil et al., 1985; Young,
1990). The secretion of POMC-derived peptides from the
anterior lobe and intermediate lobe is differentially
regulated, and the relative contributions of each lobe may
vary with the stimulus. For example, in response to restraint
stress, b-endorphin and ACTH are secreted in equal
amounts from the anterior lobe and intermediate lobe,
whereas the intermediate lobe is the most important source
of b-endorphin and a-MSH when ether stress is used (Kjaer
et al., 1995).
Chronic stress leads to a substantial increase in b-endorphin
levels in both, the anterior and intermediate lobes of the pitui-
tary (Akil et al., 1985; Höllt et al., 1986; Przewlocki et al., 1987).
234 Stress and Opioid Systems
Interestingly, prolonged stress decreases the proportion of
b-endorphin in the releasable pool of anterior lobe cortico-
tropes. Chronic footshock releases proportionally more
b-lipotropin than b-endorphin in the rat (Young et al.,
1993b). Repeated immobilization (Lopez-Calderon et al.,
1991; Marti et al., 1999; Slominski, 2006), water restriction
(Zhou et al., 1999), repeated injection of hypertonic saline
(Kiss and Aguilera, 1993), and adjuvant-induced inflammation
(Harbuz and Lightman, 1992; Aguilera et al., 1997) increase
POMC mRNA in the anterior pituitary. Interestingly, the level
of POMC mRNA increases selectively in the anterior, but not
in the intermediate, lobe after repeated footshock (Höllt et al.,
1986). This finding suggests selective activation of POMC
synthesis in the anterior lobe only in response to chronic stress.
In contrast, other studies have indicated that rats subjected to
chronic footshock or forced swim stress display an increase in
POMC mRNA levels in the intermediate pituitary (Kelsey
et al., 1984). Therefore, it is likely that POMC biosynthesis in
the intermediate lobe is regulated differentially, depending on
the type of stressor.
1.08.4.2 Proenkephalin System
Stressful stimuli strongly activate the PENK system within the
hypothalamus. Early studies demonstrated that footshock
stress (Rossier et al., 1978; McGivern et al., 1983) and social
isolation (Raab et al., 1985) induced a decrease in the hypo-
thalamic content of PENK-derived peptides. In contrast,
other studies reported no obvious effect of immobilization,
footshock, or cold swim stress upon PENK peptide levels and
its mRNA in the rat hypothalamus (Millan et al., 1981a;
Takayama et al., 1986; Przewlocki et al., 1987; Vaswani et al.,
1988; Vellucci and Parrott, 1997). However, a plethora of
other studies clearly indicated that stressful stimuli enhance
PENK gene expression in cells localized in the parvocellular
part of the hypothalamic paraventricular nucleus (Harbuz
and Lightman, 1989; Lightman and Young, 1989; Larsen and
Mau, 1994; Priest et al., 1997; Garcia-Garcia et al., 1998).
Stress induced by the injection of hypertonic saline increased
PENK mRNA levels in the paraventricular nucleus, an effect
blocked by the administration of the GC receptor antagonist,
RU-38486 (Garcia-Garcia et al., 1998). Levels of PENK
mRNA were upregulated in the medial parvocellular
subdivision of the hypothalamic paraventricular nucleus
following 1 h restraint stress (Young and Lightman, 1992;
Larsen and Mau, 1994); novelty and hypertonic saline
injections induced a similar response (Yukhananov and
Handa, 1997). Social deprivation (14–20 days) in rats was
also associated with an activation of the central PENK opioid
system. This paradigm increased the levels of PENK mRNA
and Met-enkephalin immunoreactivity in the hypothalamus.
Interesting studies were performed in transgenic mice with
human PENK-b-galactosidase fusion transgene; the transgene
resulted in correct phenotypic expression and was regulated
by osmotic and hypovolemia stress in the hypothalamic
paraventricular and supraoptic nuclei (Borsook et al.,
1994a,b,c). In the forebrain, proenkephalin mRNA levels
were found to be transiently decreased in the anterior and
medial aspects of the caudate–putamen and the nucleus
accumbens after 7 or 14 days of isolation stress, but the
levels returned to control levels after 28 days of isolation
(Angulo et al., 1991).
A few data are available on the effects of PENK in the pitu-
itary. Zhu and Desiderio (1994) observed that the space flight
stress diminished the Met-enkephalin level in the posterior
pituitary of rats and suggested that posterior pituitary enkepha-
linergic system may respond to this type of unique stress.
Further the same authors demonstrated that the level of Met-
enkephalin significantly increased 10 days after head injury in
rats (Grigoriants et al., 1995).
Lewis et al. (1982) demonstrated the role of adrenal medul-
lary PENK opioids in some behavioral responses to stress. The
exposure of rats to short, intermittent footshock caused
a decrease in the adrenal medullary content of PENK-derived
peptides (Lewis et al., 1982), pointing to an enhanced release
of these opioids. Levels of PENK peptides in the adrenal
medulla were decreased after acute stress, but did not differ
from control levels in chronically stressed rats. The PENK
mRNA levels increased several fold over control after
hypoglycemic stress (Kanamatsu et al., 1986).
One study in humans demonstrated biphasic changes in the
plasma levels of peptide F, the peptide derived from PENK, in
response to heavy exercise. Initially the level of the peptide F
decreased but significantly increased during recovery period
(Bush et al., 1999). This result suggests that the biosynthetic
activity of adrenal PENK cells increases upon prolonged stress,
thus compensating for the enhanced peptide release into the
blood. However, Van Loon et al. (1990) suggested that plasma
Met-enkephalin in rats derived from sympathetic peripheral
neurons and not only from the adrenals. Repeated daily expo-
sure to restraint stress resulted in an adaptive loss of the plasma
Met-enkephalin response. Repeated immobilization stress,
however, increased PENK mRNA in sympathetic neurons of
the rat cervical and stellate ganglia (Nankova et al., 1996).
Thus, it seems that, in the periphery, adrenal opioidergic cells
and sympathetic neurons contribute to the stress response.
Additionally, it is likely that different types of stress selectively
activate different pools of PENK peptides.
1.08.4.3 Prodynorphin System
Hypothalamic levels of PDYN-derived peptides were
significantly increased after acute swim stress and starvation
(Przewlocki et al., 1983a, 1988a), probably indicating an
inhibition of their release. In contrast, a pronounced fall in
hypothalamic dynorphin levels was observed in rats subjected
to electroconvulsive shocks, cold swim stress, or 2-h exposure
to 4  C (Morley et al., 1982; Lason et al., 1987; Vaswani et al.,
1988). This finding may indicate the enhancement of PDYN-
derived peptide release during seizures and extreme stress. In
contrast, repeated electroconvulsive shock markedly increased
dynorphin content and PDYN mRNA levels in the
hypothalamus (Hong et al., 1985; Lason et al., 1987). Thus,
hypothalamic PDYN neurons appear to be particularly
sensitive to seizures. There is no evidence that chronic stress,
e.g., recurrent footshock (Przewlocki et al., 1987), or
conditioned fear-induced stress (Przewlocka et al., 1990)
influences the hypothalamic PDYN system. Also no evidence
of the changes in PDYN biosynthesis has been found in
hippocampal dentate neurons after chronic restraint stress

(Watanabe et al., 1995). However, isolation induced
a significant accumulation of PDYN mRNA selectively in the
paraventricular nucleus, but no changes were noted in mRNA
content within the supraoptic nucleus (Matthews et al., 1993).
No significant differences were observed in the level of PDYN
mRNA in the nucleus accumbens between low and high
responders to novelty stress in rats (Hooks et al., 1994). On
the contrary, a profound increase in PDYN mRNA levels was
found in the brain, in particular in the nucleus accumbens,
during intake and withdrawal from various drugs of abuse
(Trujillo et al., 1995; Przewlocka et al., 1996; Wang et al., 1999).
The hypothalamic PDYN system clearly responded to dehy-
dration and water deprivation, evidenced by an increase in
hypothalamic PDYN-derived peptides (Przewlocki et al.,
1983b; Majeed et al., 1986; Höllt, 1990) and PDYN mRNA
levels (Sherman et al., 1986). Dehydration significantly
increased PDYN mRNA in the magnocellular neurons of the
paraventricular and supraoptic nucleus (Matthews et al., 1993).
Intermittent footshock and swim stress did not alter PDYN-
derived peptide content in the anterior pituitary (Przewlocki
et al., 1987; Vaswani et al., 1988). Another study showed that
dynorphin levels in the posterior pituitary (containing
dynorphin neuronal terminals) were unchanged after acute
footshock (Przewlocki et al., 1987). Thus, the
abovementioned observations do not provide a clear
indication as to whether stress activates PDYN peptide
biosynthesis and release in the pituitary.
1.08.4.4 Opioid Receptors
Several studies have assessed stress-induced alterations in
opioid receptors. The presumptive mild stress of handling
was sufficient to decrease MOP or DOP receptor binding in
several investigated brain regions, including the frontal cortex
and olfactory tubercle when compared to unhandled control
animals (Stein et al., 1992). Fear-induced stress decreased
[3H]Leu-enkephalin binding to the rat brain (Chance et al.,
1978; Sumova and Jakoubek, 1989). Similar results were ob-
tained in rats after exposure to forced swimming (Christie
et al., 1981). Low-affinity [3H]Leu-enkephalin binding to brain
homogenates at low temperature was significantly reduced in
mice exposed to acute and chronic forced swimming. It is likely
that the reduced binding reflects increased in vivo occupation of
opioid binding sites by EOPs (Christie and Chesher, 1983).
Seeger et al. (1984) reported that either prolonged intermittent
footshock or forced swimming caused a significant reduction in
[3H]diprenorphine binding in the hypothalamus and other
brain structures, as measured by autoradiography. A single
20-min footshock resulted in diminished [3H]DAMGO
binding in the septum (Stein et al., 1992). A decrease in
high-affinity [3H]etorphine binding after restraint stress in rat
brain membranes (Hnatowich et al., 1986) and a drop in the
number of MOP receptors in the midbrain after inescapable
shock (Stuckey et al., 1989) and acute noise stress (Vitale
et al., 2005) have also been documented. Thus, acute stress
appears to decrease the binding of opioid receptor ligands, sug-
gesting a persistent activation of opioid receptors due to an
enhanced release of EOPs.
Repeated restraint stress for 40 days reduced opioid
receptor binding in several CNS structures in the rat
Stress and Opioid Systems 235
(Dantas et al., 2005). Chronic, recurrent stress induced by
repetitive electroconvulsive shocks was shown to downregu-
late both DOP and MOP receptors in the rat brain (Nakata
et al., 1985). Sleep deprivation-induced stress decreased the
Bmax of DOP and MOP receptors in the limbic system (Fadda
et al., 1991, 1992). It is reasonable to assume that these effects
reflect an adaptive response to the enhanced release of EOPs
during stress. Rank-related stress has been shown to change
responsiveness of the DOP system, and social dominance
was linked to increased excitatory effects of DOP receptor
agonists, indicating enhanced sensitivity of this receptor in
dominant versus nondominant rats (Pohorecky et al.,
1999). In line with these findings, Holaday et al. (1982) re-
ported an upregulation of DOP receptors in response to
repeated electroconvulsive shock treatments. Further, an
increase in the number of DOP in the corpus striatum was re-
ported after repeated immobilization stress (Zeman et al.,
1988). Moreover, another study showed that stressors such
as intermittent footshock and 4 days of water deprivation
induced an increase in DOP- and MOP-binding sites in the
rat limbic system. A 90-h water deprivation induced
increases in [3H]DAMGO binding in the septum as well as
increases in[3H]DSTLE binding in the caudate and
accumbens nuclei (Stein et al., 1992). Offspring from
prenatally stressed female rats showed increases in MOP
receptors in brain areas such as the striatum, lateral
amygdala, and pyriform nucleus (Insel et al., 1990). Further,
mice selectively bred for high swim stress-induced analgesia
(SIA) were found to have significantly higher m receptor
density in the whole brain than those with low analgesic
response to stress (Mogil et al., 1994). In contrast, defeat
stress was without effect on the expression of MOP receptor-
encoding mRNA in the substantia nigra (Nikulina et al.,
1999). However, Lewis et al. (1987) were unsuccessful in
demonstrating any changes in the number or affinity of
DOP, KOP and MOP receptor sites in various brain and
spinal cord regions of rats exposed to chronic stress.
A number of studies have analyzed the changes in the
biosynthesis of opioid receptors as a result of stress. Novelty
stress increased the levels of KOP receptor mRNA in the
ventral zone of the medial parvocellular part but not in the
lateral parvocellular part of the paraventricular nucleus, claus-
trum, nucleus accumbens, or the nucleus of the lateral olfac-
tory tract (Yukhananov and Handa, 1996). Within 30 min
(and for up to 6 h) after social defeat stress, the levels of
MOP-encoding mRNA increased in the lateral ventral
tegmental area, as detected and quantified by in situ
hybridization histochemistry. These data suggest that stress-
induced alteration of MOP receptor-encoding mRNA
expression in the ventral tegmental area may be involved in
the consequences of social defeat stress (Nikulina et al.,
1999, 2005). On the other hand, KOP receptor mRNA but
not MOP receptor mRNA levels were decreased in the ventral
tegmental area of mice that were repeatedly winners in
aggressive episodes (Goloshchapov et al., 2005). Evidently,
there are discrepancies between the results reported by
different labs, and some results are contradictory. Clearly,
more studies are needed before a conclusive picture
regarding the influence of stress on the regulation of specific
opioid receptors can be obtained.
236 Stress and Opioid Systems
1.08.5 Multiple Neurochemical Interactions with
Opioid System as a Consequence of Stress
1.08.5.1 Dopaminergic System
Stress has been shown to selectively activate the mesolimbic
dopaminergic system located in the nucleus accumbens, the
prefrontal cortex, and the ventral tegmental area (VTA) (Deutch
et al., 1985; Scatton et al., 1988; Fadda et al., 1993; Cuadra
et al., 1999; George et al., 2000), while the nigrostriatal dopa-
minergic neurons seem not to be affected by stressful stimuli
(Kalivas and Abhold, 1987). Further, dopamine turnover in
the mesolimbic system is accelerated by cues associated with
a previously applied stressor (Deutch et al., 1985). Thus, fear
and anxiety that are evoked by stress appear to be related to
the changes in dopaminergic transmission.
Stress-induced alterations in the activity of the central dopa-
minergic system can be modulated by opioids. Opioid admin-
istration into the VTA has been shown to enhance dopamine
turnover in both the nucleus accumbens septi and the
prefrontal cortex (Leone et al., 1991; Spanagel et al., 1992;
Noel and Gratton, 1995) and to influence the motor stimulant
effect of footschock stress (Kalivas and Abhold, 1987). On the
other hand, the administration of opioid antagonists prevented
stress-induced variations in dopamine turnover (Kalivas
and Abhold, 1987). Further, repeated immobilization stress
enhanced sensitivity to dopamine agonists as a result of hyper-
activity of opioidergic systems. In fact, Kalivas et al. (1988)
showed that daily exposure to mild footshock stress enhanced
the motor stimulatory effect of DAMGO, a DOP receptor
agonist, injections into the VTA, whereas daily intra-VTA
administration of this enkephalin analog potentiated the
capacity of acute footshock to elevate dopamine metabolism
in the nucleus accumbens. Furthermore, it was shown that
daily pretreatment with naltrexone prior to stress prevented
augmentation of the effects of DAMGO when both substances
were administered into the VTA, indicating the involvement of
EOPs released locally into the structure during stress. Microin-
jection of enkephalin analogs into the VTA produced an
increase in spontaneous motor activity, and this effect was
antagonized by intra-accumbens or peripheral administration
of dopamine receptor antagonists (Broekkamp et al., 1979;
Kelley et al., 1980). Systemic administration or microinjection
of morphine into dopamine cells into the VTA excited dopami-
nergic neurons in the rat (Gysling and Wang, 1983; Matthews
and German, 1984). The enkephalin-evoked increase in motor
activity was associated with an increase in dopamine metabo-
lism in the mesolimbic system and appeared to be mediated
by MOP receptor (Latimer et al., 1987) most likely localized
on local GABAergic interneurons in the VTA since the
DAMGO-induced effects could be antagonized by GABA
antagonists (Kalivas et al., 1990).
Both, DOP and KOP receptors appear to be involved in the
modulation of dopaminergic neuron activity in the VTA. Acti-
vation of DOP (but not KOP) receptors in the VTA were found
to facilitate the brain reward system (Jenck et al., 1987) and
dopamine release therein (Devine et al., 1993). On the other
hand, administration of the KOP receptor agonist U50,488H
and the stable dynorphin analog E 2078 into the VTA induced
dose-dependent aversion in the place-conditioning procedure
(Bals-Kubik et al., 1993). Thus interestingly, the aversive effects
of KOP receptor agonists observed following their peripheral or
intraventricular administration may be mediated at least partly
by the modulation of dopaminergic cell activity. The effect of
exogenous or endogenous opioids appears to be mediated
through local GABA interneurons, e.g., via disinhibition of
GABA neurons, thereby releasing dopamine neurons from
tonic GABA inhibition.
Under basal (stress-free) conditions, EOP tonic activity is
minimal or absent, allowing a dominant role for GABAergic
inhibition; however, during stress, EOPs inhibit GABA interneu-
rons, resulting in an activation of dopaminergic transmission.
On the other hand, EOP-containing terminals provide direct
synaptic input to dopaminergic neurons in the VTA and may
directly modulate the activity of dopaminergic neurons during
stress. EOPs may also influence the activity of the mesolimbic
dopaminergic system in the nucleus accumbens by altering
activity at dopaminergic terminals. Interestingly, a single
immobilization stress produced an increase in striatal PENK
mRNA levels, while repeated immobilization stress resulted in
reduction of PENK mRNA (Lucas et al., 2007). It is known
that the release of dopamine from the nucleus accumbens is
enhanced by some opioids (Di Chiara and Imperato, 1988;
Spanagel et al., 1990). This effect appears to be mediated by
MOP as well as by DOP receptors (Spanagel et al., 1990). In
contrast, KOP receptor agonists inhibit dopamine release from
this structure (Di Chiara and Imperato, 1988; Spanagel et al.,
1990).
Restraint stress triggers dopamine release in the frontal
cortex, an effect that becomes further sensitized when the stress
is applied chronically. The sensitization is preventable by
naloxone pretreatment, implicating an endogenous opioider-
gic mechanism in the frontal cortex in the development of
stress-induced sensitization (Cuadra et al., 1999).
The interaction of hypothalamic tuberoinfundibular dopa-
minergic neurons with EOPs may be of some relevance in
stress. The hypothalamic tuberoinfundibular dopaminergic
neurons localized in the nucleus arcuatus are inhibited by
opiates (Haskins et al., 1981). The inhibition of dopamine
release into the portal vessels during stress, as a consequence
of enhanced release of EOPs (most likely b-endorphin), may
promote the release of POMC peptides and vasopressin from
the intermediate lobe and posterior lobe of pituitary, respec-
tively. On the other hand, an intrinsic dopaminergic system
exerts an inhibitory control on POMC peptide release via D2
dopaminergic receptors (Tiligada and Wilson, 1990; Tong
and Pelletier, 1992; Yamaguchi et al., 1996). It is apparent
from the preceding paragraph that the opioidergic network in
both the VTA and nucleus accumbens is critically involved in
the regulation of dopaminergic neuron activity during stress.
Opioids modulate activity of the mesocorticolimbic dopa-
minergic system, and this interaction appears to underlie major
aspects of stress-coping behaviors, motor response to stress,
and reward and drug-seeking.
1.08.5.2 Adrenergic System
It is well established that stress activates the adrenergic system
in several brain structures (Tsuda et al., 1986; Tanaka, 1999).
Psychological stress and conditioned fear cause increases in
noradrenaline release in the hypothalamus, amygdala, and

locus coeruleus (Tanaka, 1999). Noradrenergic neurons in the
locus coeruleus neurons have been suggested to regulate states
of attention and vigilance as well as activity of the sympathetic
nervous system. These neurons have also been implicated not
only in the actions of stress but also as a critical target for
opioid action. Some data indicate that CRF release upon stress
stimulates the brain systems by triggering the activity of cate-
cholaminergic neurons (Emoto et al., 1993; Otagiri et al.,
2000) and opioidergic systems. On the other hand, opioids
influence catecholaminergic neurons. Several in vitro studies
have documented that the activation of MOP (but not KOP
and DOP) receptors inhibits [3H]noradrenaline release from
rat brain cortical slices (Illes, 1989). In hypothalamic and neo-
striatal slices of rats, morphine also depresses the evoked secre-
tion of [3H]noradrenaline, again suggesting the involvement of
presynaptic inhibitory MOP receptors (Diez-Guerra et al.,
1986; Schoffelmeer et al., 1988), although other studies suggest
that the activation of MOP and KOP receptors does not modu-
late noradrenaline release from the basal hypothalamus
(Heijna et al., 1990). MOP receptors also seem to activate
noradrenaline release from the rat hippocampus (Jackisch
et al., 1988). On the contrary, presynaptic KOP receptors
appear to inhibit noradrenergic transmission in the rabbit
hippocampus (Jackisch et al., 1988).
Microinjections of morphine suppress the activity of norad-
renergic neurons in the locus coeruleus of freely moving cats
(Abercombie et al., 1988), and iontophoretic application of
MOP (but not KOP and DOP) receptor agonists cause a marked
and naloxone-reversible inhibition of locus coeruleus cell
activity in vitro due to an increase in Kþ conductance and subse-
quent hyperpolarization. This suggests that MOP receptors
influence excitatory transmission to the locus coeruleus. Thus,
the vast majority of current data support the view that the
noradrenergic system is under tonic inhibitory control of an
opioidergic network in several structures of the central nervous
system. The enhanced opioidergic activity occurring during
stress seems to modulate the function of the noradrenergic
system.
How do EOPs communicate with catecholaminergic
neurons upon stress, and what is the evidence for such interac-
tions? An early study reported that naloxone administration
enhanced stress-induced increases in noradrenaline turnover
in the rat brain (Tanaka et al., 1983), and it was later demon-
strated that stressor-induced conditioning of the locus coeru-
leus unit activity was profoundly potentiated by systemic
naloxone administration in freely moving cats (Abercombie
et al., 1988). Consistent with these reports is the observation
that immobilization stress increased noradrenaline release
from the hypothalamus, amygdala, and thalamus, an effect
attenuated by morphine and enhanced by naloxone (Tanaka
et al., 1983, 1988, 1991). The intraventricular administration
of Met-enkephalin during the early phase of stress attenuated
the stress-induced increase in noradrenaline turnover in several
brain structures (Tanaka et al., 1985, 1989).
These studies suggest that various kinds of stress activate
noradrenergic neurons in the brain, and EOPs counteract this
enhanced activity. CRF neurons and enkephalin neuronal fibers
impinge on locus coerelus cell dendrites and may have oppo-
site actions in controlling their activity and homeostasis
(Valentino and Van Bockstaele, 2015). Prolonged stress and
Stress and Opioid Systems 237
chronic opiates may alter the homeostatic balance and lead
to hyperactivity of the adrenergic system (Valentino and Van
Bockstaele, 2001).
Together, these findings indicate that coactivation of the
EOP system, which inhibits the activity of locus coeruleus
and lateral tegmental noradrenergic neurons, would favor
adaptive behavioral coping in response to the emotional
elements of stress.
1.08.6 Involvement of Opioids in Modulation of the
HPA Axis
1.08.6.1 Corticotropin Releasing Factor
Stress of various origins induces the secretion of CRF and
enhances its synthesis in neurons of the paraventricular nucleus
of the hypothalamus (Haas and George, 1988; de Goeij et al.,
1991; Bartanusz et al., 1993; Imaki et al., 1996). CRF is a potent
secretagogue of the three major endogenous opioid peptides
(b-endorphin, Met-enkephalin, and dynorphin) acting via
specific CRF receptors (Nikolarakis et al., 1986) and stimu-
lating opioidergic neurons in the hypothalamus (Almeida
et al., 1993). Interestingly, CRF neurons appear to control
EOPs release tonically, since the application of the CRF
receptor antagonist, a-helical CRF9-41, lowered the rate of the
basal release of both b-endorphin and Met-enkephalin from
the hypothalamus. On the other hand, EOPs may act on CRF
neurons to modulate CRF secretion, although there is no clear
evidence for this. Enkephalins as well as low doses of b-endor-
phin were shown to stimulate CRF release from the hypothal-
amus in vitro in a naloxone-reversible fashion (Buckingham,
1986). An increase in CRF mRNA levels in the paraventricular
nucleus and increased plasma ACTH concentration were
observed in vivo after the intraventricular injection of moderate
doses of b-endorphin in rats (Wang et al., 1996). Both effects
were mediated via opioid receptors. The effect of b-endorphin
on ACTH release was inhibited by intravenous injection of anti-
CRF antiserum. These results suggest that the injection of
b-endorphin increases the neuronal activity and the biosyn-
thesis of CRF in the paraventricular nucleus, and a rise in
CRF secretion stimulates the secretion of ACTH. On the other
hand, higher doses of b-endorphin administered intraventricu-
larly inhibited both basal and acetylcholine- and serotonin-
stimulated CRF release (Buckingham, 1986). Furthermore,
intraventricular administration of both b-endorphin and
dynorphin induced a dose-related inhibition of CRF secretion
into the hypophysial portal circulation of rats (Plotsky,
1986), and the effect was antagonized by naltrexone, suggest-
ing the involvement of opioid receptors. Centrally adminis-
tered EOPs inhibited hypoglycemia-induced CRF gene
expression in the hypothalamus and suppressed CRF release
which, in turn, led to a decrease in ACTH secretion and
POMC mRNA levels in the anterior pituitary (Suda et al.,
1992). Further, both KOP and MOP (but not DOP) agonists
inhibited the stimulated release of CRF from the rat hypothal-
amus in vitro (Tsagarakis et al., 1990), the effects being specifi-
cally reversed by opioid antagonists. Moreover, it was shown
that chronic morphine administration changes the anxiogenic
response to CRF intracerebroventricular administration, which
suggests that alteration in central CRF transmission may evoke
238 Stress and Opioid Systems
the psychological disturbances after chronic morphine admin-
istration (Blatchford et al., 2006). Interestingly, naltrindole,
a selective antagonist of DOP receptors, clearly enhanced basal
and stimulated CRF release. Thus, a complex relationship
between the EOP and CRF systems appears to exist. It is
possible that depending on circumstances, EOP systems act
directly on CRF neurons, or indirectly via other neuronal
systems. Several pieces of evidence indicate that the EOP and
CRF systems interact in opposing manners to regulate the adap-
tive response to stress (Valentino and Van Bockstaele, 2015).
1.08.6.2 Vasopressin System
Stress activates the neurohormone vasopressin in animals
(Knepel et al., 1985; Vellucci and Parrott, 1997). In humans,
anticipation of novelty seems to be a human-specific stress
stimulus for sustained elevation of plasma vasopressin in
men (Ehrenreich et al., 1996). Vasopressin, released upon
stress, appears to affect EOP systems. Subcutaneous administra-
tion of arginine vasopressin to conscious rats induces a dose-
dependent increase in plasma b-endorphin levels (Mormede
et al., 1986). This effect seems to be mediated via both arginine
vasopressin V1- and V2-receptors (Kjaer et al., 1993a). Further,
passive immunization with antivasopressin antibodies
inhibited stress-induced POMC-derived peptides secretion
(Linton et al., 1985). Vasopressin system may directly
contribute to the regulation of POMC-derived peptide release
from the pituitary. In vitro, vasopressin was shown to release
both b-endorphin and ACTH from the anterior pituitary
(Arimura et al., 1969; Vale et al., 1978; Przewlocki et al.,
1979). In fact, vasopressin acts synergistically with CRF both
in vitro and in vivo (Gillies et al., 1982; Rivier et al., 1984).
Studies have shown that vasopressin is a potent secretogogue
of hypothalamic b-endorphin in vitro and in vivo (Burns et al.,
1989; Barna et al., 1990). Interestingly, vasopressin receptor
blockade reduced CRF-induced b-endorphin release in vitro.
Therefore, vasopressin appears to participate in the effects of
CRF. While the significance of this control of the mechanism
of b-endorphin release remains to be established, an
interesting possibility is that CRF tonically activates some
central b-endorphin neurons (as well as PENK and, to some
extent, PDYN cells) and controls basal release of these
peptides. On the other hand, in stress, vasopressin may
participate in the mediation of opioid release. Hypothalamic
vasopressinergic neurons appear to be an essential mediator
of CRF effect, suggesting the occurrence of CRF synapses on
or in the vicinity of vasopressinergic neurons (Almeida et al.,
1993). Thus, it appears that vasopressin might be an
important modulator of POMC-derived peptide secretion,
making it a potentially important factor in the mediation of
stress reactions.
On the other hand, EOPs appear to participate in the
control of vasopressinergic neurons in the hypothalamus
and posterior pituitary. In rats, EOPs were shown to inhibit
vasopressin release in response to stressors such as footshock
and immobilization (Knepel et al., 1985). Electrophysiolog-
ical evidence obtained in hypothalamic slices in vitro
confirmed a role for EOP in the regulation of magnocellular
vasopressinergic (and oxytocinergic) neuronal activity (Kne-
pel and Reimann, 1982). These results suggest lack of tonic
inhibition of vasopressin release by EOPs. EOPs appear to
inhibit excitatory input to vasopressinergic and oxytocinergic
neurons via the MOP receptors (Liu et al., 1999a).
Knepel and Reimann (1982) showed that morphine and
b-endorphin inhibited the electrical field-stimulated release
of vasopressin from the mediobasal hypothalamus in vitro.
The inhibition of secretion can occur both at the level of the
terminals and cell bodies of magnocellular cells. In fact, their
activity is modulated by presynaptic inhibition of afferent
inputs to magnocellular cells by opioids as well as direct effects
of afferent input cells on the cell bodies (Brown et al., 2000).
Systemic administration of morphine to rats reduced vaso-
pressin release into the pituitary stalk blood. This effect was
antagonized by naloxone, which itself was without any effect.
On the other hand, the inhibition of vasopressin release from
posterior lobe terminals appears to be mediated rather through
KOP than through MOP or DOP receptors. KOP selective
agonists as well as dynorphin A1-13 inhibited secretion of vaso-
pressin, and this effect was antagonized by naloxone and selec-
tive KOP receptor antagonist nor-binaltorphimine (Bondy
et al., 1988; Zhao et al., 1988). KOP receptors may also directly
modulate the activity and release of vasopressin (but not
oxytocin) in the hypothalamic magnocellular neurosecretory
neurons (Brown and Leng, 2000). Furthermore, the KOP
receptor might be an autoreceptor in this system because of
a high degree of colocalization of KOP receptor, dynorphin,
and vasopressin in magnocellular nerve terminals (Shuster
et al., 2000).
1.08.7 Involvement of Opioids in the Behavioral and
Physiological Response to Stress
The behavioral and physiological responses to stress are modu-
lated by EOPs. The responses are varied, and they include
motor activity, nociception, reward, learning, memory, regula-
tion of emotion, and mood. The EOPs are also involved in the
modulation of endocrine, cardiovascular, respiratory, gastroin-
testinal, autonomic, and immune functions.
1.08.7.1 Locomotor Activity
Stressors in the form of restraint (Zurita and Molina, 1999), tail-
pinch pressure (Amir, 1986), footshock (Van den Berg et al.,
1998), and forced swimming (Walker et al., 1981; Overstreet
et al., 1986; Abel, 1993) affect motor and investigatory behavior.
The motor responses are modified by opioids, as demonstrated
by experiments in which opioid receptor antagonists decreased
motor activity (Arnsten and Segal, 1979; Katz, 1979; Walker
et al., 1981; Roth et al., 1981; Kavaliers and Innes, 1987). The
opioid receptor antagonist naloxone was found to decrease loco-
motor activity and rearing in rats exposed to an open field arena
(Rodgers and Deacon, 1979; Walker et al., 1981) and potenti-
ated the effect of immobilization stress (acute and chronic) on
amphetamine-induced hyperlocomotion (Holtzman, 1974;
Diez-Otanez et al., 1997). Likewise, naloxone reverses the
enhanced immobility time in the forced swimming test
following restraint stress (Zurita and Molina, 1999). Emotional
stimulus (forced perception of another rat receiving footshocks)
induces a transient decrease in ambulation and rearing

immediately after the last session, but an increase in ambulation,
rearing, and sniffing for at least 15 days; this emotion-driven
motor effect is blocked by naloxone (Van den Berg et al.,
1998). The results suggest that activation of EOP systems under-
lies some of the changes in motor behavior evoked by exposure
to stress. In contrast, however, the decrease in ambulation, rear-
ing, and sniffing and an increase in immobility in open field
observed after footshock stress are not antagonizable by
naloxone (Van den Berg et al., 1998). Thus, EOPs appear to be
involved in the changes in motor activity that are induced by
very selective stressors. Interestingly, naloxone potentiated
stress-evoked freezing in male, but not female, rats (Klein
et al., 1998), pointing to sex-specific regulation of EOPs during
the emotional response to stress. Notably, stress-induced
increases in locomotor activity in the mainland deer mice can
be blocked by the DOP receptor antagonist, ICI 154,129,
whereas naloxone had the opposite effect in a related, island-
residing, species. These findings suggest the involvement of
different opioid receptor types (DOP and possibly MOP) in
the behavioral responses to stress (Kavaliers and Innes, 1987);
they also suggest that EOPs and their receptors differentially
modulate the stress response in different species.
Exposure to stressful situations has been also shown to alter
the effects of opiates. Both exogenous opioids and EOPs have
been observed to exert bidirectional (depending on the dose)
effects on motor activity. Moreover, there are contradictory
reports regarding the influence of opioids and EOPs on loco-
motor activity, with increases and decreases, being reported.
Given this, one may assume that stress elicits various biochem-
ical changes that then differentially intervene and modulate the
effects of opiate administration (Stohr et al., 1999). In general,
low opiate doses produce arousal and increase activity, while
higher doses decrease motor activity, leading to sedation,
stupor, or catalepsy. Further, stress may alter the locomotor
stimulatory effects of morphine in rats, a phenomenon called
stress-induced behavioral sensitization to morphine (Frances
et al., 2000). Thus, the locomotor effects of morphine admin-
istration are augmented by low, but not high, doses of
morphine administered after repeated stressors (restraint,
handling, and social defeat) (Stohr et al., 1999). Similar effects
were observed after food deprivation, a condition which poten-
tiates sensitivity to opioids. Interestingly, the latter effects were
inhibited by blockade of corticosterone secretion (Deroche
et al., 1993). It would therefore appear that stress-evoked corti-
costerone release underlies stress-induced behavioral sensitiza-
tion to morphine (Stohr et al., 1999; Deroche et al., 1992).
Interestingly, morphine elicits hyperactivity in hamsters
habituated to handling, whereas it produces hypoactivity in
nonhabituated animals. The effects of handling diminished
across test days, and morphine eventually elicited hyperactivity
in both habituated and nonhabituated animals (Schnur et al.,
1988). These results are in line with the suggestion that stress
induces the release of EOPs which summate with exogenous
opiates to exert the final effect.
Several authors have sought to identify the specific EOP
mechanisms activated by stress. For example, the immobility
induced in mice subjected to a conditioned footshock (fear-
conditioning) paradigm was associated with decreased Met-
enkephalinergic activity in the striatum (Nabeshima et al.,
1986). The conditioned suppression of mobility was prevented
Stress and Opioid Systems 239
by administration of inhibitors (thiorphan and bestatin) of
Met-enkephalin degradation as well as by naloxone. The results
from the experiments with thiorphan and bestatin suggest that
the evoked attenuation of the conditioned suppression of
mobility may be directly proportional to increased striatal levels
of Met-enkephalin (Nabeshima et al., 1988). Confirming a role
for Met-enkephalin is the observation that mice lacking the pro-
enkephalin gene display increased freezing (immobility) in
response to fear (Ragnauth et al., 2001). On the other hand,
overexpression of proenkephalin in the olfactory tubercle
decreased freezing behavior in response to mild footshock and
produced behavioral activation in the open field arena
(Primeaux et al., 2003). Administration of enkephalin analog
D-Ala2-Met5-enkephalinamide into the ventral tegmental area
of rats increased spontaneous motor activity (Kalivas and
Abhold, 1987). Furthermore, rats receiving daily footshock
stress for few days exhibited a significantly greater motor
stimulant response to intraventral tegmental area injection
with the peptide than control rats. These results indicate that
the endogenous enkephalin system in the ventral tegmental
area may participate in stress-induced motor sensitization.
The KOP receptor agonists tifluadom, bremazocine, and
U50,488H reduced locomotor activity in C57BL/6 and DB/2
mice, and the effect of the agonists was enhanced by immobi-
lization stress (Castellano et al., 1988). This finding is consis-
tent with suggestions that PDYN and KOP receptor may
contribute to stress-induced behavioral immobilization
(Pliakas et al., 2001; Newton et al., 2002; Mague et al., 2003;
McLaughlin et al., 2003; Shirayama et al., 2004). Indeed,
immobilization stress in rats increases dynorphin A and dynor-
phin B immunoreactivity in the hippocampus and nucleus
accumbens, although it should also be noted that forced
swim stress transiently increases dynorphin A levels in the
hippocampus (Shirayama et al., 2004).
In summary, various opioid receptors appear to be differen-
tially involved in the modulation of stress-induced alterations
of motor activity. Stress-induced motor suppression may be
mediated or potentiated by PDYN and KOP receptors, while
PENK and DOP receptors (Hebb et al., 2005) may act to atten-
uate the locomotor response to stress.
1.08.7.2 Reward
Morphine (Katz and Gormezano, 1979; Van der Kooy et al.,
1982; Mucha and Iversen, 1984; Mucha and Herz, 1986),
EOPs (Bals-Kubik et al., 1993; Zangen et al., 2002; Terashvili
et al., 2004; Dong et al., 2006; Der-Avakian et al., 2007), as
well as various MOP and DOP receptor agonists (Spanagel
et al., 1992) elicit conditioned place preference; for example,
animals receiving an opioid are inclined to choose an initially
nonpreferred environment. In contrast, the opioid antagonist
naloxone (Mucha and Iversen, 1984; Mucha and Herz, 1986)
and KOP receptor agonists (Iwamoto, 1985; Mucha and
Herz, 1985) evoke place aversion. EOP systems play a key
role in modulating reward and mood and in regulating neural
hedonic homeostasis. Opioid-induced place preference is abol-
ished in m receptor KO mice (Marquez et al., 2007) but is unaf-
fected when the genes encoding the KOP receptor or
prodynorphin are deleted (Simonin et al., 1998; Mizoguchi
et al., 2010; Zimmer et al., 2001). On the other hand, higher
240 Stress and Opioid Systems

level of prodynorphin expression in the nucleus accumbens of 1.08.7.3 Nociception: SIA

DBA/2J and SWR/J inbred mice correlated with lower
A year after the discovery of enkephalins in 1975, Akil et al.
morphine place preference, and pretreatment of these mice
(1976) made the pioneering observation that footshock stress
with KOP receptor antagonist nor-BNI enhanced the place pref-
produces potent antinociception in rats. These authors also
erence (Gieryk et al., 2010).
found that naloxone partially reversed this analgesia, suggest-
Other work suggests that enkephalins, but not b-endor-
ing an involvement of EOPs. A number of later studies showed
phin, may be responsible for opioid-mediated reward since
that various kinds of stress such as exposure to novel stimuli
PENK knockout mice failed to show aversion to naloxone,
(Kavaliers and Innes, 1988), inescapable footshocks (DeVries
while b-endorphin-deficient mice displayed conditioned
et al., 1979; Lewis et al., 1980; Terman et al., 1984; Rosecrans
place aversion to the opioid antagonist (Skultetyova and
et al., 1986; Hemingway and Reigle, 1987), restraint (Kelly
Jezova, 1999). Briefly, the current consensus is that both
and Franklin, 1987; Kurumaji et al., 1987), food deprivation
PENK- and POMC-containing neurons and MOP and DOP
(Konecka et al., 1985; Wideman et al., 1996), acute noise stress
opioid receptors mediate reward mechanisms while the
(Vitale et al., 2005), forced swimming (Cooper and Carmody,
PDYN/KOP receptor system is involved in dysphoria
1982; Terman et al., 1986a; Suaudeau and Costentin, 2000),
(Chavkin, 2013); importantly, it is thought that PDYN may
cold water stress (Bodnar et al., 1979; Girardot and Holloway,
limit drug-produced reward (Shippenberg et al., 2007;
1985b), burn injury (Osgood et al., 1987), learned helplessness
Gieryk et al., 2010). Stress appears to influence opioid-
(Maier et al., 1983; Hemingway and Reigle, 1987), social isola-
conditioned place preference, albeit through poorly
tion (Konecka and Sroczynska, 1990), food deprivation
understood mechanisms; however, GC receptors in the
(Konecka et al., 1985; Hodgson and Bond, 1996), pregnancy
hippocampus and nucleus accumbens are apparently
(Baron and Gintzler, 1984, 1987; Iwasaki and Namiki,
essential for place preference for morphine (Dong et al.,
1997), conditioned fear in animals and man (Levine et al.,
2006). There are now numerous examples of how stress
1984; Fanselow, 1986; Przewlocka et al., 1990; Pitman et al.,
influences the rewarding effects of opiates and opioids.
1990), handling stress (Fanselow and Sigmundi, 1986),
Inescapable shock enhances morphine-conditioned place
mild social stress (Kulling et al., 1988; Pohorecky et al.,
preference for several days thereafter (Will et al., 1998), and
1999), defeat stress (Miczek et al., 1982), stressful odors
an acute emotional stress or mild intermittent stress,
(Fanselow and Sigmundi, 1986), and even exposure of
respectively, resulted in an increase in the sensitivity to the
domestic and wild animals to fight-inflicted wounds (Colwell
rewarding effects of morphine (Kuzmin et al., 1996) and
and Kavaliers, 1992) elicit antinociception, in a naloxone-
enhanced the reinforcing efficacy of heroin in rats (Shaham
sensitive manner (Akil et al., 1984, 1986; Girardot and
and Stewart, 1994; Spanagel et al., 1998; Shaham et al.,
Holloway, 1985a; Terman et al., 1985). The analgesic effects
2000). Interestingly, footshock reliably reinstated
of opioids were comparable to the stress-like responses of
extinguished heroin-taking behavior, suggesting that stress
rodents given analgesic doses of morphine (5–10 mg kg1),
may be a critical factor leading to relapse (Spanagel et al.,
albeit with a faster onset and of shorter duration.
1998). Remarkably, the controllability of the stress is
Interestingly, experiments demonstrated cross-tolerance
important since escapable tail shock does not enhance
between morphine and swim SIA (Sadowski and Panocka,
morphine CPP in the rat (Der-Avakian et al., 2007).
1993; Sadowski and Konarzewski, 1999). In rodents, mild
Recent findings suggest that chronic social defeat stress acti-
stressors usually produce opioid-dependent analgesia while
vates the KOP system resulting in potentiation of the acute
severe stressors induced analgesia independent of opioids
rewarding properties of cocaine (McLaughlin et al., 2003).
(Izumi et al., 1983; Hamm and Knisely, 1987; Hawranko
On the other hand Bruchas et al. (2007) showed that repeated
et al., 1994; Mogil et al., 1996; Sadowski and Konarzewski,
swim stress caused activation of both KOP receptors and p38
1999). Stress severity plays an important role in determining
mitogen-activated protein kinase coexpressed in GABAergic
the neurochemical basis of SIA; increasing severity (duration
neurons in the nucleus accumbens, cortex, and hippocampus,
or intensity) of stress causes a shift from opioidergic to nonop-
indicating that the endogenous PDYN-KOP receptor system
ioidergic mechanisms. For example, the severe conditions of
constitutes a key component of the molecular mechanisms
swimming (longer duration or lower water temperature)
mediating the aversive properties of stress (Bilkei-Gorzo
produce SIA that is insensitive to the opiate antagonist,
et al., 2012). Furthermore, administration of ethanol under
naltrexone, whereas naloxone significantly attenuates analgesia
conditional fear stress leads to development of place
induced by less severe swimming conditions (Terman et al.,
preference, and KOP receptors modulate the development of
1986b). Similar findings have been reported with other
the rewarding effects of ethanol under psychological stress
stressors such as footshock (Rodgers and Deacon, 1981;
(Matsuzawa et al., 1999). Stress also appears to influence
Cannon et al., 1984). Interestingly, SIA is followed by a period
opioid self-administration (Piazza and Le Moal, 1998). Thus,
of hyperalgesia with a series of symptoms characteristic of the
mild footshock applied before each self-administration
exogenous opioid abstinence syndrome (Cristea et al., 1993).
session was seen to evoke higher rates of lever pressing for
Some studies have indicated that there are substantial sex and
heroin in rats (Shaham et al., 1997). However, a previous
population/genetic differences in the novelty-induced anal-
study reported that acute footshock stress did not
gesia displayed by various populations of deer mice (Kavaliers
significantly affect opioid self-administration (Kuzmin et al.,
and Innes, 1987, 1988; Marek et al., 1989; Mogil et al., 1997).
1996). Stress has been shown to reduce self-stimulation from
Some findings suggest an early evolutionary development
ventral tegmental area that was attenuated by intracerebral
and phylogenetic continuity of stress responses to aversive
administration of opioid peptides (Zacharko et al., 1998).

stimuli mediated by opioidergic and nonopioidergic mecha-
nisms (Kavaliers, 1987). Exposure to either cold or warm stress
increased the thermal nociceptive thresholds of the terrestrial
snail, Cepaea nemoralis. Warm SIA was blocked by opiate antag-
onists while cold SIA was unaffected. Exposure to tail-pinch
stress increased the thermal nociceptive thresholds (which
was antagonized by naloxone) of the slug, Limax maximus.
These results indicate that this mollusk displays both opioider-
gic and nonopioidergic forms of SIA in a manner analogous to
that reported in mammals (Kavaliers, 1987). Tolerance to the
analgesia induced by repeated forced intermittent cold water
swim has been reported (Girardot and Holloway, 1985b).
Naltrexone antagonized the adaptive aspect of all those forms
of analgesia. Thus, EOPs may play a functional role in the
behavioral adaptation to aversive stressful environmental
situations.
A series of studies in humans have shown that physical exer-
cise leads to temporary hypoalgesia. Reduced sensitivity to pain
is demonstrable after long-distance exercise (such as marathon
run) but also after intensive physical exercise. Pain threshold
elevation is most pronounced during maximal exertion, but
hypoalgesia persists after exercise has been stopped, demon-
strating that a systemic analgesic effect is induced by the exer-
cise process (Droste, 1992).
Exposure to acute stress potentiates the magnitude and dura-
tion of analgesia following both the peripheral and intracerebro-
ventricular administration of several opioid agonists as
compared to nonstressed controls (Hassen et al., 1982). Both
supraspinal and spinal analgesic opioidergic mechanisms signif-
icantly contribute to the enhanced analgesic potency of opioids
in subjects exposed to various types of stress. Opioid-treated rats
exposed to restraint stress showed potentiation of the magnitude
and duration of opioid analgesia (Calcagnetti et al., 1990;
Calcagnetti and Holtzman, 1992; Woolfolk and Holtzman,
1995). However, rats given agonists with high intrinsic activity
at the MOP displayed the most potent and consistent potentia-
tion of analgesia compared to unrestrained controls. Thus, the
results suggest that the activation of MOP is of primary impor-
tance for restraint to potentiate analgesia (Calcagnetti et al.,
1990, 1992). Naloxone or naltrexone at low doses antagonized
certain kinds of SIA. However, neither antagonist is selective for
the MOP, and this experiment demonstrates only the involve-
ment of an opioid receptor in the phenomenon. Few studies
authenticated the above conclusion using more specific MOP
receptor antagonist b-FNA and CTOP (Fanselow et al., 1989).
The octapeptide CTOP dose-dependently reversed fear-induced
conditional analgesia in rats when administered intraventricu-
larly in rats (Fanselow et al., 1989). More recently, other authors
have demonstrated that CTOP suppressed SIA, when injected
into the periaqueductal gray (Wiedenmayer and Barr, 2000).
Thus, MOP localized in the midbrain periaqueductal gray appear
to be involved in opioid SIA. Fields (2000) has described the
neuronal pain-modulating circuit, which includes the periaque-
ductal gray, amygdala, and raphe nuclei in the brain stem.
Through descending projections, this circuit controls both the
spinal and trigeminal dorsal horn pain-transmitting neurons.
This system may be activated by acute stress and mediates stress
analgesia in animals and humans. Interestingly, prolonged
chronic stress such as repeated cold swim, in contrast to acute
stress, decreases sensitivity to the antinociceptive effects of
Stress and Opioid Systems 241
morphine. Chronically stressed mice were hyposensitive to
MOP-mediated antinociception (Omiya et al., 2000).
Interestingly, rats exposed to the stress of repeated exposure to
noxious heat exhibited SIA, but antinociception was reduced
(detected using the tail-flick test) after the administration of
b-endorphin into the periaqueductal gray region of the brain
(Hawranko et al., 1999).
Several studies demonstrated the potential involvement of
DOP in SIA (Hart et al., 1983, 1985; Kitchen and Pinker,
1990; Killian et al., 1995). Those studies showed that naltrindole
and ICI 174874 inhibited analgesia induced by warm water
(20  C) swim stress in adult and young rats (Kitchen and Pinker,
1990). Also cold water swim stress (5  C) was antagonized by
the DOP antagonist ICI 174,864. This antinociceptive response
was antagonized by prior administration of the DOP2-opioid
receptor antagonist, naltriben, but not by the DOP1 receptor
antagonist, 7-benzylidenenaltrexone in mice (Killian et al.,
1995). Another study showed that the cold water swim stress
response was selectively antagonized by the naltrindole, but
not by the DOP1 antagonist, [D-Ala2-Leu5- Cys6]enkephalin,
the MOP antagonist, b-funaltrexamine, or the KOP antagonist,
nor-binaltorphimine (Vanderah et al., 1993). These studies
suggest that cold water swim stress analgesia is mediated
by DOP2-opioid receptors. On the other hand, the selective
DOP receptor antagonist naltrindole had no influence on
psychological SIA (Takahashi et al., 1990), indicating the
specific involvement of DOP in certain kinds of stress, such as
swim stress.
The involvement of KOP receptor in SIA has also been
demonstrated. An early study suggested that KOP receptor
antagonists reversed nonopioid SIA while MOP receptor might
mediate opioid analgesia (Panerai et al., 1984, 1987). It was
subsequently demonstrated that nor-binaltorphimine, a selec-
tive KOP receptor antagonist, blocked footshock SIA when
administered systemically or intrathecally in rats (Menendez
et al., 1993). Further, the stress analgesia induced by exposure
to psychological stress was also antagonized by pretreatment
with nor-binaltorphimine and Mr2266 (Takahashi et al.,
1990). Furthermore, chronic cold swim stress increased the
antinociceptive response via KOP receptor (Omiya et al.,
2000); thus, the antinociceptive activity of U50,488H was
demonstrated in mice subjected to the repeated cold stress.
Interestingly, the antinociceptive activity of the MOP receptor
agonist DAMGO was attenuated by stress. These results suggest
that spinal KOP receptors may be responsible for endogenous
analgesia induced by some types of stress.
The potential involvement of the brain POMC and b-endor-
phin systems in SIA was indicated by early evidence that
showed that hypothalamic and midbrain b-endorphin levels
were changed upon footshock stress, possibly due to the
enhanced release of this peptide (Przewlocki et al., 1987,
1991). Conditioned fear stress evoked naloxone-sensitive anal-
gesia and a marked decrease in b-endorphin levels in the hypo-
thalamus and the anterior and intermediate lobes of the
pituitary, together with an increase in the peptide level in
plasma (Przewlocka et al., 1990). On the other hand, b-endor-
phin levels were increased in the periaqueductal gray, where
terminals of b-endorphinergic neurons are located (Kulling
et al., 1989; Nakagawasai et al., 1999), as well as in the arcuate
nucleus of the medial basal hypothalamus, after exposure of
242 Stress and Opioid Systems
rats to mild social stress (aggressive confrontation) and forced
walking stress (Nakagawasai et al., 1999), respectively. Lesions
of the arcuate nucleus reduced opioid-mediated SIA, but inter-
estingly, they enhanced a form of footshock SIA that was not
blocked by injections of the opiate receptor blocker, naltrexone
(Kelsey et al., 1986). Thus, the arcuate lesions led to compen-
satory changes in the nonopioid analgesic system, resulting
in the enhanced nonopiate-mediated stress-induced analgesia.
Further, naloxone injections into the periaqueductal gray and
arcuate nucleus blocked analgesia in defeated mice (Miczek
et al., 1985). In addition, microinjections of antibodies against
b-endorphin into the midbrain periaqueductal gray attenuated
antinociception elicited by electroacupuncture (Xie et al.,
1983). Another study suggested the potential role of b-endor-
phinergic cells within the NTS in SIA. Electrical stimulation
of this structure evoked opioid-mediated analgesia in the rat
(Lewis et al., 1987). Such results suggest that b-endorphin-
containing neuronal cells may, at least partly, be involved in
this phenomenon.
There are some suggestions that SIA may also depend on
peripheral pools of b-endorphin. It is not clear whether pitui-
tary pools of b-endorphin play a role in the phenomenon since
some kinds of stress induce the b-endorphin release into the
blood without affecting nociception. Other studies have sug-
gested that although the integrity of the adenohypophysis is
essential for the manifestation of SIA, probably involving
neither ACTH nor b-endorphin is essential for the development
of the analgesia which accompanies stress (Millan et al., 1980,
1981a). Lim et al. (1982) have suggested that plasma levels of
b-endorphin do not reflect changes in pain threshold, and
b-endorphin levels in the anterior pituitary, neurointermediate
lobe, and plasma are probably not causally related to stress
effects such as footshock-induced analgesia. Some results
suggest that the stress of birth causes an increase in maternal
secretion of b-endorphin which is not related to the degree of
pain itself (Jouppila et al., 1983). Mean levels of b-endorphin
in cerebrospinal fluid was significantly greater in the patients
with PTSD compared with normal subjects. Thus, the increased
endorphinergic activity in the central nervous system may exist
in PTSD patients, and that hypersecretion of opioids might
constitute an adaptive response to traumatic experience (Baker
et al., 1997). However, there is poor correlation between cere-
brospinal fluid and plasma b-endorphin levels in combat
veterans with PTSD (Baker et al., 1997). The study on mice
with a selective deficiency of b-endorphin clearly demonstrated
the critical role of b-endorphin in SIA. In these mice, the lack of
opioid-dependent naloxone-reversible analgesia was observed
in response to mild swim stress (Rubinstein et al., 1996). The
mice exhibited normal analgesia in response to morphine,
indicating the presence of functional MOP receptors. Mutant
mice also displayed significantly greater nonopioid analgesia
in response to cold water swim stress compared with the
controls and displayed paradoxical naloxone-induced anal-
gesia. These changes may reflect compensatory upregulation
of alternative opioid-independent pain inhibitory mecha-
nisms. More recently, SIA was further studied in b-endorphin
knockout mice; results demonstrated attenuated opioid-
mediated SIA (Skoubis et al., 2005).
There is some evidence suggesting a role of the brain PENK-
derived peptides in SIA (Kurumaji et al., 1987). Exposure of
mice to forced swim stress in cold water produced opioid
analgesia that was blocked by intrathecal pretreatment with
antiserum to Met-enkephalin, but not to Leu-enkephalin,
b-endorphin, or dynorphin (Mizoguchi et al., 1997). The
results suggest that swim SIA is mediated by spinal Met-
enkephalin. Inhibition of PENK peptide degradation has
been reported to potentiate this phenomena, when applied
intraventricularly in mice (Chipkin et al., 1982; Christie and
Chesher, 1983). The enkephalinase inhibitor thiorphan
evoked a dose-related potentiation of both the peak effect
and the duration of the SIA after exposure of rats to
inescapable footshock (Chipkin et al., 1982). Moreover, it
was shown that adrenal demedullation abolished the
analgesic response (Lewis et al., 1982), suggesting the
participation of circulating PENK peptides. Unexpectedly,
however, enkephalin-deficient knockout mice exhibited
normal SIA (Konig et al., 1990; Bilkei-Gorzo et al., 2004).
The involvement of the PDYN system in the mediation of SIA
remains less clear. A recent study in mice demonstrated that
psychological SIA was fully antagonized by the selective KOP
antagonist nor-binaltorphimine, while this compound was
without any effect on footshock- and swim-induced antinocicep-
tion (Takahashi et al., 1990). Further, Starec et al. (1997)
demonstrated that dynorphin exerted an analgesic effect in
mice when combined with a stressor. On the other hand, an
intermittent fasting diet in mice produced analgesia that was
blocked by nor-binaltorphimine, accompanied by increased
spinal PDYN mRNA expression (de los Santos-Arteaga et al.,
2003). Furthermore, the KOP receptor antagonist blocks SIA eli-
cited by the repeated forced swim test in mice, and PDYN-
knockout mice fail to display SIA following the forced swim
test (McLaughlin et al., 2003).
Such results indicate that the PDYN system, as well as KOP
receptors, may be involved in the mechanisms underlying the
analgesia effects of certain kinds of stress. Notably, however,
certain stressors may inhibit, rather than activate, the brain
PDYN system. For example, it has been shown that the
in vitro hypothalamic release of a-neoendorphin, a peptide
derived from PDYN, is lowered in rats subjected to conditioned
fear-induced stress, which itself is accompanied by naloxone-
reversible analgesia (Przewlocka et al., 1990). Further, a rise
in the hypothalamic content of dynorphin after footshock
stress has been reported, suggesting a suppression of dynorphin
release (Millan et al., 1981b).
It has been demonstrated that SIA is abolished in prodynor-
phin knockout mice (McLaughlin et al., 2003). Furthermore,
MOP receptor-deficient CXBK mice displayed no opioid SIA
(Moskowitz and Goodman, 1985), and the late component
of opioid SIA was reduced in MOP knockout mice (LaBuda
et al., 2000). A recent study demonstrated that mild SIA (3-
min swim at 32  C) was reversed by the triple mutation of
the MOP, KOP, and DOP receptors and decreased in MOP
and DOP receptor knockout female mice (Contet et al.,
2006). Interestingly, the stress-induced opioid-mediated
responses were modified in CB1 cannabinoid receptor knock-
outs. Indeed, these mutants did not exhibit antinociception
following a forced swim in warm water. However, absence of
the CB1 cannabinoid receptor did not modify the antinocicep-
tive effects induced by different opioid agonists. These results
indicate that a physiological interaction between the opioid

and cannabinoid systems is necessary to allow the develop-
ment of opioid-mediated responses to stress (Valverde et al.,
2000).
Experimental data clearly demonstrate that opioids are able
to inhibit nociception arising in inflamed tissue by a local
peripheral action, presumably via the terminal region of the
sensory nerves. Similar effects are observed when endogenous
opioid peptides are released under stress conditions from
immune cells present in the inflamed tissue (Stein et al.,
1990; Przewlocki et al., 1992; Herz, 1995, 1996). Immunoreac-
tive b-endorphin and enkephalins, processed in these cells,
seem to be the relevant peptides in this respect. Although the
mechanism of stress-induced release of opioid peptides from
the immunocytes is presently not clear, there is an indication
that this process involves cytokines and CRF.
EOPs appear to be involved in the mechanisms of placebo-
analgesic effects in human (Bendetti and Amanzio, 1997; Sher,
2004). In fact, positron-emission tomography with [(11)C]car-
fentanil has revealed that placebo effects were accompanied by
release of EOPs, as measured by enhanced MOP receptor-
dependent neurotransmission in the rostral anterior cingulate,
dorsolateral prefrontal cortex, insular cortex, nucleus accum-
bens, amygdala, and ventral pallidum (Zubieta et al., 2005).
These studies suggest that one of the possible mechanisms of
placebo analgesia results from an enhanced EOP response to
noxious stimuli, and the results help explain how expectancy
of noxious stimuli can influence affective and nociceptive
components of pain (Gracely et al., 1983; Wager et al., 2007).
Thus, whether or not EOPs play a role in the antinocicep-
tion depends on the kind of stress and variables studied.
Some stressors activate the EOP systems, whereas others do
not. Moreover, when EOPs are involved, they frequently
interact with other neuronal systems. Therefore, the analgesic
effects of stress are complex and difficult to characterize. Never-
theless, the reactions to certain kinds of stress have been shown
to be mediated by specific neuronal opioid systems, since they
are known to cause release of these opioid peptides and may be
modulated by opiates.
1.08.7.4 Thermoregulation: Stress-Induced Hyperthermia
Several forms of stress have been shown to cause hyperthermia
in the rat. Footshock stress produced hyperthermia, the degree
of which was found to be a function of current intensity
(Pechnick and Morgan, 1987). Restraint stress and novelty
stress also produced hyperthermia (Vidal et al., 1984).
Naloxone (Vidal et al., 1984; Kapas et al., 1989) slightly
reduced restraint and ‘novelty’ hyperthermias (Vidal et al.,
1984) or emotional hyperthermia (Blasig et al., 1978). These
effects suggest that endogenous opioids are not significantly
involved in the thermal effects of the stressors. They may play
only a minor role in the regulation of basal temperature (Vidal
et al., 1984). Peak rise in temperature following footshock
stress was not affected by naltrexone or chronic morphine
administration; however, the rate of return to baseline temper-
ature was slowed by these treatments. Thus, the endogenous
opioidergic system appears to be involved in the return to
normal body temperature following footshock, but not in the
footshock-induced rise in temperature (Pechnick and Morgan,
1987). On the other hand, a recent study demonstrated that
Stress and Opioid Systems 243
repeated swim stress developed thermal hyperalgesia that was
blocked by naloxone and naloxonazine, but not by naltrindole,
DOP receptor antagonist, and nor-binaltophimine, a KOP
receptor antagonist (Suarez-Roca et al., 2006) suggesting
involvement of MOP receptors in development of SIA.
Restraint stress affects the changes in body temperature
induced by morphine and opioid peptides administered
either systemically or intracerebroventricularly (ICV) in rats.
The unstressed group of rats responded to all doses of
morphine, DAMGO, DADLE, and D-Met2-Pro5-enkephalin,
with an increase in core temperature. In contrast, restrained
rats showed a decrease in core temperature following the
injection of opioids (Szikszay et al., 1983; Spencer et al.,
1985; Appelbaum and Holtzman, 1986). Interestingly, the
administration of cholecystokinin reduced the hypothermic
response to systemic morphine in restrained rats, while
hyperthermia elicited by an administration of morphine to
freely moving rats was not diminished by cholecystokinin
pretreatment. These results support the hypothesis that
cholecystokinin may contribute to the regulation of the
endogenous opioid system (Kapas et al., 1989).
There is indirect evidence for involvement of the opioid
peptide b-endorphin in the hypothalamic regulation of the
development of fever and stress-induced hyperthermia. In
the unanesthetized rabbits, microinjection of b-endorphin in
the preoptic/anterior hypothalamus resulted in the elevation
of body temperature. It has been suggested that b-endorphin
reduces sensitivity of hypothalamic neurons to high ambient
temperature and that this reduction leads to the increased
peripheral vasoconstriction, inhibition of evaporative heat
loss, and modification of behavioral thermoregulation, result-
ing in the elevation of body temperature (Gordon et al., 1984).
Plasma b-endorphin response under three exercise-
thermoregulatory stress conditions was measured in humans
during stationary upright cycling. The b-endorphin response
pattern closely paralleled rectal temperature changes in all
conditions. These data suggest that conditions of increasing
thermoregulatory stress caused by exercise are associated with
rising peripheral b-endorphin concentration (Kelso et al.,
1984).
It has been previously reported that sauna-induced fevers
result in a rise in b-endorphin level in normal human volun-
teers (Jezova et al., 1985). Another report described similar
changes in plasma b-endorphin in cancer patients undergoing
whole body hyperthermia (Robins et al., 1987). The presented
results show that there is a relationship between thermal stress,
defined in terms of core temperature and/or duration of
hyperthermia, and the quantitative rise in plasma b-endorphin
levels.
1.08.7.5 Feeding Behavior
Stress is known to influence feeding. Some kinds of stress
augment feeding in animals, and opioid antagonists appear
to abolish it (Antelman and Rowland, 1981; Ferin and Vande
Wiele, 1984; Teskey and Kavaliers, 1988). Stress produced by
pinching the tail has been shown to compel satiated animals
to eat and display oral stereotypies. Eating induced by the tail
pinch was reduced by microinjections of naloxone and the
MOP-selective antagonist CTOP into the substantia nigra,
244 Stress and Opioid Systems
indicating the selective involvement of MOP. In fact, stress-
induced eating did occur after treatment with KOP and DOP
receptor antagonists (Hawkins et al., 1994). Interestingly,
opioids are thought to influence the palatability of food and
its relative reward value (Taha et al., 2006; Olszewski and
Levine, 2007). MOP in the nucleus accumbens shell are
postulated to mediate this influence (Ward et al., 2006;
Woolley et al., 2007a,b). Secretion of enkephalin and
dynorphin in the hypothalamus is also modulated by the
nutritional value of food (Chang et al., 2007; Naleid et al.,
2007). Additionally, injection of morphine into the rostral
portion of lateral hypothalamic area increases food intake in
the rat (Li et al., 2006).
Opioid peptides may partly determine the rewarding
aspects of eating. Mercer and Holder (1997) argue that altered
EOP activity may elicit food cravings which, in turn, may influ-
ence food consumption. Support for this opioidergic theory of
food cravings is provided by various clinical conditions
(bulimia nervosa, anorexia nervosa, Prader–Willi syndrome,
and eating-induced obesity) which are associated with the
altered EOP levels, intensified food cravings, and increased or
decreased food intake (Johnson, 1995; Morley et al., 1982,
1983). Furthermore, food deprivation results in alterations of
EOP levels in the brain and pituitary of the rat (Majeed et al.,
1986; Tsuji et al., 1987), similar to those induced by chronic
stress. The data support the notion that EOPs, in particular,
dynorphin and the MOP receptors, play a role in the modula-
tion of food intake by stress. Stress-induced alterations in EOP
activity may elicit food craving, which may then influence food
consumption (Mercer and Holder, 1997).
1.08.7.6 Cardiovascular Effects
Cardiovascular responses to stress include increased catechol-
amine secretion, tachycardia due to elevated cardiac sympa-
thetic and reduced vagal efferent activity, peripheral
vasoconstriction in certain vascular beds, arrhythmia, and
hypertension. Some data suggest the involvement of partic-
ular EOP systems in these processes. EOPs are present in the
cerebrospinal fluid and the cerebrovascular bed, and opioid
receptors have been found in cerebral perivascular nerves.
Their activation may modulate the function of vasoregulatory
mechanisms that are involved in the control of the cerebrovas-
cular tone. Furthermore, EOPs have been found in cardiac
tissue and EOPs of myocardial origin have also recently
been shown to play a role in regulation of the heart functions
(van den Brink et al., 2003). PENK synthesis have been
demonstrated in isolated rat heart (Younes et al., 2000), while
PDYN expression have been found in cultured rat myocytes
(Ventura et al., 1994).
Under resting conditions, EOPs do not appear to play an
important role in the regulation of the cardiovascular system,
but they become important under stress (Benyo and Wahl,
1996). Restraint stress evoked an increase in heart rate, blood
pressure, and plasma catecholamine levels in rats. Pretreatment
with b-funaltrexamine partially attenuated the increase in heart
rate in response to stress (Houdi et al., 1996). During MOP
receptor activation by DAMGO, restraint stress resulted in
bradycardia. Psychosocial stress appears to elevate blood pres-
sure via an opioid-dependent mechanism in normotensive
rats since naloxone has been demonstrated to attenuate or
reverse the elevation in blood pressure in both renovascular
and spontaneous hypertension (Szilagyi, 1991).
During periods of severe stress, opioid blockade increased
ambulatory blood pressure in humans. These observations
suggest that opioidergic mechanisms inhibit ambulatory blood
pressure responses during naturally occurring stress (Nordin
et al., 1987; McCubbin et al., 1998). The results of another
study indicated that relaxation training reduced the diastolic
pressure response to mental arithmetic stress. Opioid receptor
blockade with naltrexone antagonized the effects of relaxation
training. The study suggests that some of the physiological
effects of relaxation training are mediated by augmentation
of inhibitory opioid mechanisms (McCubbin et al., 1998).
Thus, endogenous opioid mechanism appears to inhibit the
cardiovascular response to stress.
In the brain, the POMC neurons are involved in the
control of the function of the NTS, the structure known to
participate in the control of cardiac function. Intravenous or
intracerebroventricular administration, as well as the injection
into the NTS of b-endorphin, has been shown to decrease
blood pressure (Hassen et al., 1982; Sitsen et al., 1982). The
localization of enkephalins in the paraventricular nucleus of
the hypothalamus, a brain region important for the regulation
of the stress response, further substantiates the involvement of
the opioid system in the modulation of stress-induced hyper-
tension. Studies on the cardiovascular responses to centrally
administered PENK products are contradictory so far and
provide little insight into the physiological role of central
PENK in cardiovascular functions. However, adrenal PENK
peptides, released by the stimulation of the splanchnic nerve,
may induce bradycardia and hypotension as shown in reserpi-
nized dogs (Hanbauer et al., 1982).
Dynorphin decreases blood pressure and produces brady-
cardia when applied intravenously or into the cisterna magna
(Laurent and Schmitt, 1983). In contrast, application of dynor-
phin into the NTS or cerebral ventricles does not alter cardio-
vascular function (Hassen et al., 1982; Glatt et al., 1987).
Prodynorphin peptides appear to modulate the release of
vasopressin from the posterior lobe and may regulate diuresis
in this way. KOP receptor agonists are powerful diuretics
(Leander, 1982). Therefore, prodynorphin peptides and KOP
receptors may influence the cardiovascular system. In humans,
mental stress affects blood pressure and increases various
opioid peptides in the plasma. Subjects responding to stress
with a low increase in blood pressure had high levels of
b-endorphin, while those who reacted with a high stress-
induced blood pressure had elevated levels of dynorphin and
Met-enkephalin. Pretreatment with naloxone enhanced blood
pressure in low responders but not in high blood pressure
responders (Fontana et al., 1997). Interestingly, naloxone
decreased blood pressure response in hypertensive subjects
with acute stress-induced increase in blood pressure suggesting
the pressor effects of some EOPs, possibly dynorphins or
PENK-derived peptides, in hypertensive patients (Fontana
et al., 1997).
In summary, it seems likely that certain EOPs may, under
certain circumstances, counteract the cardiovascular effect of
moderate stress, e.g., tachycardia and increased blood pressure,
and mediate cardioprotection (Tanaka et al., 2014). On the

other hand, some EOPs appear to also mediate cardiovascular
depression which occurs in response to severe stress. In fact,
a number of studies have demonstrated that naloxone reverses
the hypotension induced by most cardiovascular shock states
(Reynolds et al., 1980; Vargish et al., 1980; Feuerstein et al.,
1981; Boeuf et al., 2003).
1.08.7.7 Respiration
It is well documented that opioids and EOPs influence
respiration (McQueen, 1983) and that stress modulates
respiration. Rats exposed to inescapable footshock displayed
an increase in respiratory rate and naloxone potentiated
the footshock-induced increase in ventilation (Isom and
Elshowihy, 1982). Chronic footshock stress attenuated both
the respiratory stimulation produced by acute footshock
and the potentiation induced by naloxone. These results
strongly suggest that stress can influence respiratory function
through the activation of endogenous opioid systems and
release of the endogenous opioids as a compensatory reac-
tion which prevents excessive stimulation of respiration
(Isom and Elshowihy, 1982).
1.08.7.8 Reproduction
Stress is frequently accompanied by an impairment of reproduc-
tive functions (Calogero et al., 1998). Stressors generally induce
a depression of the HPA axis, seen as a fall in plasma luteinizing
hormone (LH) and testosterone levels (Petralgia et al., 1986).
There is accumulating evidence that CRF is a critical stress factor
which exerts inhibitory actions upon sexual behavior and secre-
tion of gonadotropins. However, CRF may inhibit hypothalamic
neurons producing gonadotropin-releasing hormone most
likely via endogenous opioidergic pathways although direct
effects of CRF on gonadotrophin-releasing hormone neurons
are also likely to occur (Almeida et al., 1989). Endogenous
opioids, acting through CRF-independent mechanisms in the
brain or pituitary, may also influence gonadotropin
production (Chatterton, 1990). Acute opioid administration
decreases plasma LH levels which is due to an inhibitory
modulation of gonadotrophin-releasing hormone discharge
from the hypothalamic neurons (Briski et al., 1984; Ferin and
Vande Wiele, 1984; Petralgia et al., 1986; Doma nski et al.,
1989; Genazzani et al., 1993). It is indicated that EOPs
released from the hypothalamus inhibit LH secretion, which
results in an inhibition of ovulation and termination of
pregnancy. Data suggesting the involvement of EOPs in the
direct control of the neuroendocrine mechanism modulating
gonadotrophin secretion have been reported.
In stress, the presence of low plasma LH levels and
abnormal LH pulsatile secretion has been related to the
increased opioidergic activity thus supporting the role of
opioids in the integration of hormonal and neuronal systems
of the brain (Genazzani et al., 1993). Inhibitory effects of
footshock stress on LH release were found to be antagonized
by MOP and KOP (but not DOP) receptor antagonists as well
as by antibodies against both b-endorphin and dynorphin
(but not Met-enkephalin) (Petralgia et al., 1986). Therefore,
these results suggest the involvement of POMC and PDYN
(but not PENK) neurons in the control of LH release during
Stress and Opioid Systems 245
stress. Restraint stress reduced plasma testosterone levels in
control rats in a naloxone-sensitive manner (Akibami and
Mann, 1996). Testicular steroidogenesis may also be locally
inhibited by opioid peptides via peripheral opioid receptors
(Kostic et al., 1998), in particular, b-endorphin secreted into
plasma and secreted locally by the Leydig cells (Fabbri et al.,
1988; Eskeland et al., 1992, 1989). On the other hand,
estrogen treatment enhanced PENK gene expression in the
ventromedial hypothalamus (Quinones-Jenab et al., 1996),
and PENK expression therein can be associated with estrogen
as well as progesterone concentration during the estrous cycle
(Funabashi et al., 1995). Although stress and estrogen appear
to have specific effects on PENK expression in the hypotha-
lamic neurons (Priest et al., 1997), it has also been demon-
strated that the administration of 17-b-estradiol to
ovariectomized females upregulates MOP receptor mRNA
expression in the ventromedial nucleus and arcuate nucleus
of the hypothalamus, indicating estrogenic regulation of
MOP receptors in the hypothalamus (Quinones-Jenab et al.,
1997). The involvement of MOP receptors in sexual function
was recently observed in MOP receptor knockout mice. Inter-
estingly, male homozygotes showed unexpected changes in
sexual function as shown by reduced mating activity,
a decrease in sperm count and motility, and smaller litter
size (Tian et al., 1997).
1.08.7.9 Autonomic Nervous System
Stress results in a broad spectrum of autonomic effects. They
include (1) elevation of plasma catecholamine levels due to
the enhanced adrenomedullary and sympathetic outflow; (2)
an increase in mean arterial pressure and tachycardia (Fisher,
1989); (3) stress-induced intestinal effects (Tache et al.,
1990); and (4) immunosuppression (Blazar et al., 1986;
Glaser et al., 1987; Fujiwara et al., 1999). Further, stress mobi-
lizes the release of b-endorphin from the pituitary gland and
PENK peptides from the adrenomedullary cells and sympa-
thetic nerves (Farrell et al., 1983; Jarry et al., 1985; Matthews
and Challis, 1995; Nankova et al., 1996). Little is known
about the effect of stress on peripheral PDYN-derived
peptides. A source of these peptides in the periphery could
also be the gastrointestinal tract since it has been shown
that they are released from the duodenum in vitro (Majeed
et al., 1986).
Circulating EOPs released from pituitary and adrenals, as
well as those released from the postganglionic nerves, appear
to regulate functions of the autonomic nervous system and
may modulate the effects of stress. These EOPs may inhibit
sympathetic outflow via the action on peripheral sympa-
thetic nerves. This suggestion is supported by several obser-
vations. It has been shown that tachycardia induced by the
stimulation of the accelerans nerve was reduced by ethylketo-
cyclazocine, a preferential KOP receptor agonist. The chrono-
tropic effect of noradrenaline was not changed by this
compound, and MOP and DOP receptor agonists were
without effect. Therefore, Starke et al. (1985) have concluded
that postganglionic sympathetic neurons innervating the
sinus node of the rabbit heart may express presynaptic
KOP receptors. Further, it has been shown that KOP and
DOP receptor types (but not MOP) are localized on
246 Stress and Opioid Systems
sympathetic nerves in the isolated guinea pig atria (Ledda
et al., 1985). In isolated arteries, KOP and DOP receptor
agonists depressed the response to sympathetic stimulation.
Agonists of the KOP receptors decreased noradrenaline
release from postganglionic neurons into the blood. On
the other hand, EOPs and opiates may stimulate sympathetic
nervous system. ICV administration of b-endorphin to rats
increased plasma noradrenaline and adrenaline (Yamauchi
et al., 1997) via opioid receptors. On the other hand, the
increase in catecholamine levels by restraint stress was not
inhibited by anti-b-endorphin antisera but was clearly
diminished by naloxone. The results suggest that some
EOPs other than b-endorphin are involved in restrain
stress-induced activation of autonomic nervous system. In
fact, immobilization stress activated enkephalin neurons in
the ventral medulla paragigantocellularis and lateral reticular
nuclei which might be involved in autonomic response to
stress (Mansi et al., 2000). Exposure to stress increased
sympathetic nervous system activity inducing an elevation
of plasma noradrenaline and might disturb glucose homeo-
stasis. Liu et al. (1999b) found that a hypoglycemic effect
was produced in the rats with streptozotocin-induced dia-
betes after cold-exposure, and the effect was reversed by
naloxone. It was suggested that the hypoglycemia was medi-
ated by b-endorphin since the increase in the plasma concen-
tration was observed upon stress. Moreover, intravenous
injection of b-endorphin in the rats with streptozotocin-
induced diabetes produced a lowering of plasma glucose
level. Therefore, b-endorphin appears to be responsible for
the induction of hypoglycemic effects in diabetic rats after
cold stress. In rats, postexercise peak of insulin in response
to glucose was markedly reduced when compared to resting
controls. Interestingly, the administration of naloxone
further decreased the insulin response. These results suggest
that EOPs may participate in the physiological adaptation
to exercise stress, maintaining postexercise insulin response
to glucose (Bouix et al., 1996).
Studies in humans provided evidence that DOP receptors
and possibly enkephalins might influence autonomic sympa-
thetic reactivity. The selective DOP receptor agonist deltorphin
failed to modify basal plasma levels of noradrenaline in the
control rats, but completely suppressed the insulin-evoked
elevation of noradrenaline and the release of both noradrena-
line and adrenaline elicited by cold stress. These findings
provide evidence that DOP receptors and possibly enkephalins
may modify autonomic sympathetic output (degli Uberti et al.,
1993).
Lymphoid organs, like many others, are innervated by the
autonomic nervous system, and there is a growing body of
evidence that this system can have immunomodulatory effects.
Noradrenergic postganglionic nerve fibers are found in the
thymus, spleen, lymph nodes, and gut-associated lymphoid
tissue, where they can make direct contact with immunocytes
(Ader et al., 1990). It would appear that opioids may influence
peripheral noradrenergic nerves and noradrenergic innervation
of lymphoid organs. On the other hand, there is evidence that
cells of the immune system produce EOPs (Blalock et al.,
1985). Therefore, it is likely that in situations such as inflam-
mation, immunocytes may release EOPs under stress and
through paracrine or direct synaptic-like communication
mediate the peripheral effects of stress (Stein et al., 1990;
Przewlocki et al., 1992; Herz, 1995).
1.08.7.10 Immune System
A variety of stressors have been found to alter immune func-
tions. EOPs released in response to stress from endocrine and
neuronal systems may interact with the immune system by
modulating immune responses to various factors (Shavit
et al., 1985; Jodar et al., 1994; Sacerdote et al., 1994, 2003;
Sacerdote, 2003; Moynihan et al., 2000). On the other hand,
EOPs produced and released by immune cells display paracrine
and autocrine action and demonstrate functional activities
that are in part similar to cytokines.
Several in vitro studies suggested that EOPs enhanced
immune responses (Plotnikoff and Miller, 1983; Wybran,
1985; Gatti et al., 1993), while others led to the opposite
conclusion (Greenberg et al., 1984; Shavit et al., 1986;
Ben-Eliyahu et al., 1990). In fact, stress suppresses immune
function. Greenberg et al. (1984) have found that stress
induced by tail shock suppressed natural killer cell cytotox-
icity and that this effect was blocked by naltrexone. Subse-
quent studies showed that inescapable footshock stress
(Shavit et al., 1986) and forced swimming (Ben-Eliyahu
et al., 1990) decreased natural killer cell activity and finally
reduced the resistance of rats to a mammary ascites tumor
(Shavit et al., 1984). Other studies demonstrated that heat
stress-induced immunosuppression during pregnancy was
mediated by opioid system most likely by placental b-endor-
phin release into blood (Nakamura et al., 1998). The effect
was inhibited by naloxone indicating the involvement of
opioid receptors. Overnight restraint stress of mice decreased
concanavalin A-driven lymphocyte proliferation, plaque-
forming cell response to sheep red blood cells, and natural
killer cell activity in the spleen, but the phagocytic activity
was enhanced (Marotti et al., 1996). Interestingly, injection
of Met-enkephalin before restraint stress abolished these
changes (except for the natural killer cell activity) and
attenuated stress-induced elevation of GCs, although Met-
enkephalin itself affected the immune responses to stress: it
decreased natural killer cell activity and the plaque-forming
cell response and enhanced phagocytic activity (Brown and
Van Epps, 1985). Further studies revealed that the
concentrations of b-endorphin in splenocytes, peripheral
blood mononuclear cells, and lymph node cells were
significantly increased after the exposure to inescapable
intermittent footshock for 20 min (Sacerdote et al., 1994).
In contrast, the exposure to a continuous footshock for
3 min did not affect the b-endorphin concentrations
(Sacerdote et al., 1994). Recent studies have indicated that
the brain produces interferon-a (IFN-a) in response to
inflammatory stress and that the effect of IFN-a was
inhibited by naloxone. Central administration of IFN-
a inhibited natural killer cytotoxicity. Further study revealed
that IFN-a decreased the activity of hypothalamic neurons
via opioid receptors which, in turn, resulted in the
activation of CRF neurons, thereby suppressing natural killer
cytotoxicity through the activation of the splenic sympathetic
nerves in splenocytes (Hori et al., 1998). Interestingly, EOPs are
synthesized and released under stress conditions from immune

cells present in the inflamed tissue (Stein et al., 1990, 2003;
Przewlocki et al., 1992; Herz, 1995). The majority of studies
indicated that the effects of stress on immune response were
blocked by opioid receptor antagonists, indicating that they
were mediated by endogenous EOPs mobilized during stress.
1.08.8 Therapeutic Potential of Opioid Agonists and
Antagonists in Stress-Related Psychiatric Disorders
Chronic stress is associated with several mental disorders; it is
strongly implicated in anxiety and depression in humans
(Holsboer and Ising, 2010; Lucassen et al., 2014; Patchev
et al., 2014) and thought to play a role in the development of
PTSD (Daskalakis et al., 2013), eating disorders (both anorexia
and hyperphagia; Bazhan and Zelena, 2013), drug and
substance (Koob and Kreek, 2007; Koob and Le Moal, 1997;
Koob and Mason, 2015), stroke (Craft and Devries, 2009),
and more recently, neurodegenerative disorders such as Alz-
heimer’s (Machado et al., 2014; Sotiropoulos et al., 2008)
and Parkinson’s (Ros-Bernal et al., 2011) diseases. These effects
may be attributed to the hypersecretion of GCs that accom-
panies the stress response, which may continue for an extended
period well after the stressful stimulus has faded due to failure
in the mechanisms mediating GC negative feedback at the
central and pituitary levels (Holsboer and Ising, 2010; Lucassen
et al., 2014). Chronically elevated GC levels result in altered
neuronal structure, namely synaptic dysfunction and loss,
dendritic atrophy, and even death of neural precursor cells or
mature neurons. Such morphological mal(adaptations) –
primarily observed in the hippocampus and prefrontal cortex
– may extend to electrophysiological and functional disconnec-
tion between brain nuclei; all of them are linked with
emotional, affective, and cognitive impairments (Sousa and
Almeida, 2012; Lucassen et al., 2014). This, and the previously
discussed (1) distribution of EOPs and EOP receptors in fronto-
cortical, limbic, and hypothalamic areas of the brain and (2)
the reciprocal regulatory interactions between EOPs and the
HPA axis, has led to interest in the potential of opioid-based
treatments for stress-related disorders of the brain such as major
depression. However, the idea that opioid receptor ligands may
be useful to alleviate psychiatric symptoms has been around
and practiced for centuries (Weber and Emrich, 1988); in the
modern era of medicine, opium was widely used to treat
mood disorders until the early 1950s when tricyclic antidepres-
sants were introduced (Tenore, 2008). In the following para-
graphs, we will only review studies in which opioidergic
ligands were tested for their antidepressant efficacy. We will
not discuss such investigations in patients with addictive disor-
ders although depression and drug and alcohol addiction
often coexist; readers interested in those aspects should refer
to Nunes et al. (2004) and Lalanne et al. (2014).
Anhedonia, the inability to sense pleasure, is a cardinal
feature of depression (Nestler and Carlezon, 2006). Since
MOP receptor-activating opioids as well as the prototypic
opiate morphine, and DOP receptor agonists, induce feelings
of well-being, decreased anxiety, and tranquility (Jaffe and
Martin, 1990), it has been hypothesized that EOP deficiency
may contribute to depressive illness (Byck, 1976; Herz and
Emrich, 1983; Lutz and Kieffer, 2013). This hypothesis is
Stress and Opioid Systems 247
supported by reports of upregulated MOP receptors in the
brains of depressed suicide victims (Gabilondo et al., 1995;
Gross-Isseroff et al., 1998), as well as pioneering studies on
the antidepressant-like actions of b-endorphin (Kline et al.,
1977; Gerner et al., 1980), the partial opiate agonist cyclazo-
cine (Fink et al., 1970), and buprenorphine (Emrich et al.,
1982). Additional indirect evidence that opioids may be
involved in the regulation of mood comes from the observa-
tion that withdrawal from exogenous opiate usage causes
a decline in dysphoria and a PET-scan report of reduced
MOP receptors in the neural circuitry regulating emotions
(Kennedy et al., 2006). Buprenorphine is an approved
medication for chronic pain and addiction; the compound
has far fewer side effects (respiratory, immune, and cognitive)
and displays lower abuse potential (dependence) than most
other opiates (Shmygalev et al., 2011; Davis, 2012; Karp
et al., 2014). Buprenorphine acts rapidly and effectively to
ameliorate depressive symptoms in patients who were
otherwise resistant to other antidepressant medications
(Emrich et al., 1982; Bodkin et al., 1995); treatment-resistant
depression, which affects about 50% of depressed patients,
remains one of psychiatry’s greatest challenges (Rush et al.,
2006). Buprenorphine is a partial MOP agonist and KOP
antagonist (Cowan, 2007), a characteristic referred to again
later in this section. Lastly, mention should be made of
a compound (ALKS 5461) that combined buprenorphine
with a m opioid receptor antagonist (samidorphan); this drug
entered Phase III clinical trials on the basis of evidence from
a placebo-controlled study that it is efficacious and safe and
lacks abuse potential (Ehrich et al., 2015).
Considerable attention has been paid to the possible anti-
depressant actions of DOP receptors (Pradhan et al., 2012;
Saitoh and Yamada, 2012). The rationale for pursuing DOP
receptors as possible therapeutic targets included the demon-
stration that mice with a deletion of the DOP receptor-coding
gene display increased immobility in the Porsolt forced swim
test (Filliol et al., 2000) and that various DOP receptor agonists
have antidepressant properties (Tejedor-Real et al., 1998;
Torregrossa et al., 2006; Vergura et al., 2008; Jutkiewicz and
Roques, 2012). The latter effects may be attributed to increased
availability of the brain levels of serotonin and/or brain-
derived neurotrophic factor (BDNF) (Narita et al., 2006;
Torregrossa et al., 2005). As already mentioned, DOP receptor
agonists would appear to be candidate treatments because they
also have some euphorigenic properties, but possibly lower
abuse liability than MOP receptor-activating drugs (Jutkiewicz,
2006). Convulsions are a major undesired effects of many DOP
receptor agonists, but since similar side effects of conventional
antidepressant drugs can be avoided by appropriate dosage,
this would not necessarily be an insurmountable barrier. A
number of DOP agonists have entered clinical trials in recent
years (www.clinicaltrials.gov); while some trials were termi-
nated due to inconclusive evidence, published reports on
others are still unavailable.
Antagonism of KOP receptors as a means to prevent or treat
depression appears to be the most promising ‘opium cure’
(Harrison, 2013) as Emil Kraepelin would have put it (Weber,
1987). Recent PET studies suggest disturbed KOP receptor
activity in depression (Pietrzak et al., 2014). Moreover, whereas
MOP receptor agonists increase reward and produce euphoria,
248 Stress and Opioid Systems
KOP receptor-activating drugs are aversive and dysphoric in
humans and animals (Chefer et al., 2013; Pfeiffer et al., 1986;
Shippenberg and Herz, 1986; Roth et al., 2002; Chavkin,
2013). These KOP-mediated actions result from their
inhibitory effects on dopaminergic, serotoninergic, and
glutamatergic innervation of the corticolimbic reward pathway
(reviewed in Lalanne et al., 2014) and possibly from their
ability to modulate BDNF synthesis (Zhang et al., 2007) and
hippocampal neurogenesis (Drake et al., 2007). The latter
phenomena are implicated in the development of, and
recovery from, depression (Krishnan and Nestler, 2010;
Mateus-Pinheiro et al., 2013; Miller and Hen, 2015). Stress
results in dynorphin release and KOP receptor activation and
concomitantly induces KOP receptor antagonist-reversible
anxiolytic and depression-like behaviors in animals (del
Rosario et al., 2002; Mague et al., 2003; Shirayama et al., 2004;
McLaughlin et al., 2006; Carr et al., 2010; Van’t Veer and
Carlezon, 2013; Falcon et al., 2015). If stress-induced
hypersecretion of GC is indeed a key culprit in generating
depressive behavior (Holsboer and Ising, 2010), it is important
to note that mice with deletion of either the prodynorphin
(Bilkei-Gorzo et al., 2008) or KOP receptor genes (Wittmann
et al., 2009) fail to increase GC secretion upon exposure to
certain stressors. For a comprehensive and critical review of the
involvement of the prodynorphin and KOP receptor systems in
the relationship between stress and depression/depressive-like
behavior, readers are referred to a recent review by Van’t Veer
and Carlezon (2013). With respect to the potential of KOP
receptor-targeted drugs for the alleviation of depression, readers
are reminded that the efficacy of buprenorphine appears to
depend as much on its MOP receptor activity as on its KOP
receptor antagonism. Reference was also previously made to
ALKS 5461, a drug presently in Phase II clinical trials (Ehrich
et al., 2015). This compound combines buprenorphine with an
MOP receptor antagonist (samidorphan); presumably, the
added benefit of samidorphan is that it helps titrate down the
euphoric actions of buprenorphine, thus reducing abuse
potential of the new compound.
1.08.9 Opioidergic Control of Stress Responses –
Conclusions
EOP systems appear to play an important role in the interaction
of the organism with different stressors by limiting the harmful
effects of stress (Drolet et al., 2001; Molina, 2006). While rela-
tively quiescent in the resting state, EOPs are released during
intense stressful stimuli. Acute, mild, and short-lasting stressors
appear to mobilize EOPs, which may, in turn, act to oppose
stress-precipitated reactions and, in concert with other factors,
counteract the initial response and help restore homeostasis.
Systemic stressors act predominantly by interacting with
hypothalamic neuropeptides and catecholaminergic systems.
EOPs can limit the HPA axis response to stress by dampening
the adrenocortical system during stress and uncoupling the
adrenal gland from hypothalamo–pituitary stimulation. Interac-
tions of EOPs with brain catecholaminergic systems appear to
influence the ability of the CNS to cope with stress. Coactivation
of EOPs (especially b-endorphin) inhibits activity of the locus
coeruleus to promote adaptive behavioral responses. On the
other hand, EOPs derived from POMC and, possibly, from
PENK may enhance the activity of the mesolimbic dopaminergic
system during stress, resulting in the reinforcement of a positive
emotional state, a decrease in anxiety, and better adaptation. In
contrast, PDYN peptides may have an opposite effect on the
dopaminergic system under stress. Stress-induced activation of
the PDYN system produces dysphoria and depression.
It seems likely that the endogenous PDYN system may act
upon motor and emotional aspects of the stress response; the
PDYN system appears to act in a manner opposite to that of
the POMC and PENK systems.
Repeated or chronic stress leads to alterations in EOPs that
may, in turn, contribute to the onset of stress-related patholo-
gies. Several findings suggest that peripheral EOPs released
during stress may contribute to the production of gastric ulcers,
while the central pool of EOPs appears to have an opposite func-
tion (Hernandez et al., 1983). In addition, EOPs participate in
the regulation of the circulatory response to stress and the path-
ogenesis of hypertension. Activation of EOPs in response to
traumatic injury, ischemia, and infection has been observed in
both preclinical and clinical studies (Molina, 2006).
While the reactions of EOP systems to stress generally help
restore altered homeostasis, EOPs may also be, at least partly,
responsible for the pathological effects of some forms of shock
(e.g., circulatory shock and hypoxia) and may facilitate or even
accelerate death.
While the organism’s response to stress serves to maintain
or restore physiological and behavioral homeostasis in the
event of adverse challenges from the environment, the EOP
system is implicated in dissociative symptoms in patients
with personality disorder and PTSD. Specifically, hyperactivity
of the EOP system appears to contribute to flashbacks (a key
feature of PTSD) and borderline personality disorder (Bohus
et al., 1999).
The role of stress hormones in the development of drug
addiction and depression is significant but the underlying
mechanisms remain poorly understood. Interestingly, human
addicts and depressive patients display dysregulation of the
HPA axis and these subjects are hyporesponsive to stress
(Lovallo, 2006). This blunted response to stress, which is
considered an important risk factor for relapse into drug abuse
and depression (Sinha, 2007; Valentino and Van Bockstaele,
2015), may be, at least partly, caused by changes in the func-
tioning of EOP systems; a disturbed balance in the activity of
the PENK and PDYN systems may be especially important in
this respect.
Many stressors encountered by humans are chronic in
nature; therefore, elucidation of exactly how EOPs influence
the adaptation to stress can have potentially important thera-
peutic implications for a variety of systems that become disrup-
ted by stress. More research on the interactions between EOP
systems and stress therefore remain a high research priority.
Acknowledgments
This work was supported by research grant 2013/08/A/NZ3/00848
MAESTRO from National Science Foundation (NCN, Poland). We
express thanks to Lidia Radwan for invaluable help with the preparation
of the manuscript and Mikolaj Przewlocki for figure preparation.

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 1.09 Social Stress: Concepts, Assumptions, and Animal Models
Sally P Mendoza, University of California, Davis, Davis, CA, USA
Ó 2017 Elsevier Inc. All rights reserved.
This chapter is a revision of the previous edition chapter by C.R. McKittrick, D.C. Blanchard, M.P. Hardy, R.J. Blanchard, volume 1, pp. 333–366,
Ó 2009, Elsevier Inc.

1.09.1 Introduction 261

1.09.1.1 The Stress Response: What Is It and How Is It Measured? 262
1.09.1.2 Social Systems 263
1.09.1.2.1 Limitations of Inclusion 263
1.09.1.2.2 What Constitutes a Social Stressor? 264
1.09.2 Aggression, Intruders, Social Defeat 264
1.09.2.1 Resident/Intruder Models of Social Stress 264
1.09.2.2 Application and Limitation of Resident/Intruder Model 265
1.09.3 Aggression and Dominance in Social Groups 266
1.09.3.1 Case Study: Visible Burrow System in Rats 266
1.09.3.2 Mice in Groups 267
1.09.3.3 Social Dominance in Primates 268
1.09.3.3.1 Case Study: Physiological Processes Associated with Dominance in Baboons 269
1.09.3.3.2 Case Study: Social Influences on Stress Response Systems in Squirrel Monkeys 270
1.09.4 Social Instability 271
1.09.5 Disruption of Established Relationships: Separation, Bereavement, and Loneliness 273
1.09.5.1 Comparative Case Study: Polygynous Squirrel Monkeys versus Monogamous Titi Monkeys 274
1.09.5.2 Comparative Case Study: Voles 276
1.09.5.3 Comparative Case Study: Guinea Pigs 277
1.09.5.4 Social Isolation in Humans 278
1.09.6 Conclusions 279
References 281

1.09.1 Introduction
Social stress is by definition a hybrid field, combining concepts
in endocrinology and social processes into a single investigation.
Two contributions stand out as facilitating the shift from an
occasional mention of social stress to a field of inquiry in its
own right. The first was provided by Henry and Stephens
(1977) in their treatment of personality types expressed within
and created by the social environment. This work was based
on a long history of animal research organized to deal with
a problem posed by human research – differences between folks
with Type-A or Type-B personality. The second was an article by
House et al. (1988) that posited that social stress, particularly
social isolation, should be considered a risk factor of comparable
magnitude to cigarette smoking. This work combined expertise
in epidemiological studies in human populations with experi-
mental studies, largely conducted in animals.
From its inception social stress has been an interdisciplinary
effort, and most of the investigators look to studies in animals
to model aspects of human biology. Recent years have seen
a blossoming of studies of social stress into investigations of
neuroscience, immunology, health-care practices, and more.
Given the diversity of expertise that is currently required to
conduct studies of social stress and the rapidity within which
the field has developed, it is timely to examine the underlying
assumptions and concepts both with respect to the models
used and what they have contributed to our understanding of
social stress in animals and humans.
We will begin with examination of the stress concept and its
origins. In our everyday language, and in much of the literature,
stress refers both to the conditions that create discomfort in our
lives and to the physiological reactions to those conditions. The
most commonly evoked metaphor for understanding the phys-
iological reactions to stressful conditions is the encounter
between predator and prey – from the perspective of the
prey. Nothing is more important than escape from the certain
death that awaits. All physiological systems involved with
mobilizing resources are activated while others concerned
with immediately nonessential processes are shut down.
Collectively this concerted physiological reaction to an emer-
gency situation is referred to as the stress response. How apt
is this metaphor for explaining the kinds of reactions to the
social and psychological conditions that are referred to as
stressful in the modern condition? Our understanding of stress
responses has grown immensely in the time since studies of
social stress emerged. A particularly important distinction,
that was not considered important initially, is between acute
and chronic responses to stress.
Similarly, our understanding of social systems has
increased enormously. Relationships first became a target of
research with Bowlby (1969) focusing on mother–infant
relationships and elaborated to more diverse relationships

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262 Social Stress: Concepts, Assumptions, and Animal Models
with Hinde’s (1974) contribution to understanding social
dynamics in human and nonhuman primates. This was fol-
lowed by a flurry of studies investigating the proximate mech-
anisms that contribute to animal societies necessitating new
conceptual understanding of social systems (Mason, 1976).
From this work we know that social organization is a charac-
teristic of the species which can vary within limits for any
given species but changes dramatically between species.
Animal studies of social stress need to be evaluated with
a keen understanding of the social system. Moreover, studies
of social stress have helped to illuminate social systems
(Mendoza and Mason, 1989). For these reasons emphasis in
this chapter will be placed on systematic investigations of
particular research programs with particular species that
were influenced by and influence the field of social stress as
it has developed and emerged as a field of inquiry.
1.09.1.1 The Stress Response: What Is It and How Is It
Measured?
Walter B. Cannon (1871–1945) and Hans Selye (1907–82)
together provide the historical view of stress that we currently
employ. Both Cannon and Selye were influenced by another
physiologist – Claude Bernard (1813–78), a French physiolo-
gist, who introduced the concept of milieu interieur. Bernard
recognized the importance of maintaining a constant internal
environment – temperature, chemical balance, fluids, etc. –
in the face of a continually changing external environment.
He viewed physiology as compensating for even the smallest
deviations in the external environment in order to maintain
constancy in the internal environment. Regulation of the milieu
interieur, Bernard (1872) also recognized, was the purview of
the brain creating the possibility that mental imagery could
initiate physiological processes, such as those involved with
eating or thermogenesis.
Cannon (1932) elaborated Bernard’s notion that physiolog-
ical systems exist to regulate internal processes in relation to
external events, processes Cannon collectively referred to as
homeostasis. He too was concerned with anticipatory activa-
tion of metabolic processes and coined the phrase ‘fight or
flight’ to characterize the emergency metabolic state underlying
the preparation for sudden action (Cannon, 1929). Whether
responding fearfully or aggressively was determined by evalua-
tion of the circumstance, an appraisal process subsequently
elaborated by Lazarus (1999). Cannon’s focus in these studies
was the sympathetic nervous system (SNS).
The fight-or-flight response largely comprised the initial
stage (Alarm) of Selye’s (1956) General Adaptation Syndrome.
In addition to high levels of SNS activity, the Alarm stage,
according to Selye, is characterized by activation of the hypo-
thalamic–pituitary–adrenal (HPA) system. The second stage
of the General Adaptation Syndrome is Resistance during
which elevations in glucocorticoids reach their peak response.
Glucocorticoids during this stage serve multiple functions
including freeing stored energy in the form of glucose,
preserving use of energy for brain, and, most importantly,
serving as a negative feedback signal to inhibit further activa-
tion of the HPA and SNS, restoring homeostasis (Dallman,
2007). Responses to most stressors, according to Selye, are
effectively over at the second stage.
Most animal studies of stress focus on the alarm reaction
and the stage of resistance which together comprise the acute
stress response. Acute stress has a predicable temporal
sequence: corticotropin-releasing hormone (CRH) activates
SNS, including adrenomedullary release of epinephrine (Epi),
and stimulates adrenocorticotropin (ACTH) release; ACTH, in
turn, stimulates adrenocortical release of corticosteroid
(cortisol in primates; corticosterone in most rodents); finally,
corticosteroids acting through negative feedback mechanisms
inhibit further activation of the system. The temporal character-
istics of the stress cascade are predictable. With the onset of the
stressor, CRH release from the hypothalamus occurs within
milliseconds, and the sympathetic response follows within
seconds; conversion of norepinephrine (NE) to Epi occurs
rapidly, and Epi released into the bloodstream augments and
extends SNS effects; increased ACTH levels become detectable
within 1–2 min; cortisol takes longer time to increase appre-
ciably (5–10 min) in the general circulation and does not reach
peak values for at least 20 min after stressor onset. At this point
there are hypothetically two opposing processes acting within
the HPA and SNS system. Activational mechanisms are stimu-
lating release of CRH and negative feedback mechanisms are
operating to restore homeostasis. Corticosteroids are both the
signal that activational mechanisms are still operating and
the means by which they are shut off or rendered ineffective.
Negative feedback sites regulating the HPA system include
the hippocampus, amygdala, hypothalamus, pituitary, and
adrenal. The multiplicity of feedback sites and their efficacy
ensure that the HPA system is not activated continuously
even under circumstances when the stressor is sustained
(de Kloet, 2003; Dallman, 2007). In addition to inhibitory
effects from corticosteroids, the other branch of the autonomic
nervous system (ANS), the parasympathetic nervous system
(PNS), opposes many SNS effects at target organs, and it is
intimately involved in mediating social processes (Porges,
2007, 2009).
Because of the rapidity of response in SNS and CRH and
ACTH, technological sophistication is required to measure
early stages of the stress cascade especially in freely moving
and interacting animals. Measurement of corticosteroid
responses is relatively easy in comparison. Blood samples, for
example, can be collected within 5–10 min time delay from
activation of the stress response to initial detection of stress-
induced elevations in corticosteroid concentrations. Corticoste-
roid levels are often used to monitor the magnitude and
duration of the stress response, both of which are considered
to reflect the intensity of the stressor (Hennessy and Levine,
1978). Most procedures used to collect blood samples,
however, do constitute a stressor, and physiological events
subsequent to collection of an initial blood sample would
reflect the response to the experimental procedures as well as
antecedent conditions (Coe et al., 1978). This has led people
to turn to less invasive methods for inferring corticosteroid
dynamics. As steroid hormones corticosteroids can pass lipid
barriers unless prevented from doing so by binding to its carrier
protein, corticosteroid-binding globulin (CBG), or albumin.
Only a small percentage of the circulating corticosteroid is
not bound to large carrier proteins. The free component can
pass into saliva, urine, or feces directly or in metabolized
form. Of these salivary concentrations of corticosteroids mimic
 Social Stress: Concepts, Assumptions, and Animal Models
the temporal cascade most directly (Hellhammer et al., 2009).
Urine and feces are not instantly available and corticosteroid
concentrations in these biological specimens reflect all events
since last void.
Measurement of Epi and NE are possible in both plasma
and urine, but capturing the dynamics of early stress events is
not possible with either measure. This has led to the use of
known effects of the ANS system on heart rate (HR), blood
pressure, and the like. HR, in particular, has been a useful
measure of both SNS and PNS activity (Porges et al., 2007).
In summary, the SNS and the HPA system are often referred
to as the stress-response systems, and their end-product
hormones, Epi (adrenalin) and cortisol (or corticosterone), are
the stress hormones. So profound has been the influence of
Cannon and Selye that we often equate levels of these hormones
with the degree of stress so that higher hormone levels
mean greater stress therefore greater adverse health outcomes.
It is important to remember that these hormones are activated
for reasons other than stress. Cortisol, for example, displays
circadian activity which rises just before awakening. In humans
there is an additional activation of the HPA system upon awak-
ening, the magnitude and form of which is similar to an acute
stress response (Hellhammer et al., 2007). Similarly, as a primary
metabolic hormone, cortisol is also released in large quantities
surrounding meals (Warne et al., 2009). Exercise activates both
HPA system and SNS. We would not want to suggest that any
of these activities – awakening, exercise, and eating – are stressful
and detrimental to health. The principal way we have differenti-
ated the stress response from activation for other purposes is by
the nature of the stressor. It is our value judgment, if you will,
that the nature of the eliciting stimulus defines the response as
stressful or simply metabolic adjustment.
Selye incorrectly believed that under conditions of extreme
stress the homeostatic mechanisms would be overwhelmed
and the Stage of Exhaustion initiated. We now know that the
Stage of Exhaustion does not happen as Selye envisioned.
Negative feedback mechanisms are too potent to permit
continued activation of the HPA system even in the presence
of a persistent stressor (Dallman, 2007). Likewise, repeated
stressors lose their ability to activate the HPA system even
when habituation to the stressor does not occur (Ruys et al.,
2004). Increasingly it appears that chronic, persistent, or
repeated stressors lead to altered regulation and not activation
to the point of adrenal depletion (Mendoza et al., 2000; Munck
et al., 1984). Dysregulation of HPA system is increasingly being
considered a sign of chronic stress. Dysregulation can take the
form of excessive activation, lack of negative feedback regula-
tion, excessive feedback regulation, or flattened circadian regu-
lation. Importantly, dysregulation can take the form of an
overactive system (e.g., diminished negative feedback) or an
underactive one (e.g., enhanced negative feedback), and corti-
costeroid concentrations, likewise, can be either higher or lower
(Lupien et al., 1995).
1.09.1.2 Social Systems
The problems of evaluating social stress are further com-
pounded by the ongoing nature of social stress. For animals
living in simple social systems where the appearance of
a conspecific is either a potential mate or potential rival, the
263
principles governing the response to acute stress provide
adequate explanatory value. The initiation of response can be
equated with the perception and identification of the intruder
and the physiological response followed, at least theoretically,
from initiation to reestablishing homeostasis. That is, the acute
response to stress provides an adequate conceptual framework
to understand isolated incidences or contrived events. But in
most species, including humans, individuals are inextricably
embedded within the social system, and its constant demands,
potentials, benefits, and hazards are ongoing and difficult to
isolate from one another. Without being able to identify stress
onset or isolate one stressor from another, more complex
models of social stress are needed than those provided by
our understanding of the acute stress response.
To further complicate studies of social stress, the type of
social structure varies from species-to-species. While we can
recognize common features among many different species
not all types of relationships are equally relevant to each. For
example, dominance relations which primarily govern interac-
tions between same-sex individuals are not relevant for under-
standing social stress for monogamous species. Conversely, the
adult emotional bond which is characteristic of monogamous
species is not a feature of adult relations in polygynous species.
Moreover, social systems while species-typical are not invariant
and adaptations within and between generations are
commonly reported. No element of animal societies can be
said to follow a phylogenetic progression, yet rapid phyloge-
netic changes in form of society do occur (Mendoza and
Mason, 1989).
1.09.1.2.1 Limitations of Inclusion
In this chapter we will consider social stress to be any condition
emanating from the social environment that significantly alters
activity in the SNS and/or HPA system for normally socialized
mammalian individuals. This pursuit will include changes that
are both protective and detrimental to health. To truly fit the
definitions of stress intended by Cannon and Selye negative
health outcomes are expected to result from too much social
stress. Too often, however, the necessary steps to link stress
and disease have not been identified. Consequently, social situ-
ations may have been incorrectly categorized as beneficial or
detrimental based on incomplete information. Mistakes have
been made.
Behaviorally and physiologically our approach is too
limiting to capture the full essence of studies of social stress.
Many other physiological systems contribute to the outcome
of social stress or provide evidence for its effects. Oxytocin
and vasopressin, for example, are intimately involved in medi-
ating social relationships, and they impinge on other physio-
logical processes, including HPA and SNS, in ways that can
obscure or reveal instances of social stress (Insel et al., 1993;
Carter et al., 2008). These hormones receive some scant atten-
tion in this chapter, and they are limited to a discussion of
prairie voles. Focusing on normally socialized animals leaves
out a wealth of information on developmental consequences
of early social manipulations (Mason, 2008). Our focus will
be on mammals leaving aside birds and reptiles. Within
mammals, we are further limited by the typical choice of study
subjects. The most studied laboratory animals are mice, rats,
guinea pigs, and voles. Systematic studies of social stress have
264 Social Stress: Concepts, Assumptions, and Animal Models
also been conducted extensively in several primate species
including tree shrews, titi monkeys, squirrel monkeys,
macaques, baboons, and humans. In order to preserve our
focus on species-typical social systems, discussion of each
will be accomplished in succession and more or less separately.
1.09.1.2.2 What Constitutes a Social Stressor?
By virtue of their reproductive systems all mammals are at least
moderately social in order to breed and nurture offspring.
Perhaps the most typical social system among mammals is
one of overlapping territories. In these species females typically
have smaller territories which overlap with one or more larger
territories maintained by males. Other adults are not usually
encountered and when they are they are treated either as an
intruder or a potential mate. Only about 5% of mammals
live in permanent social groups. Some are reliant on interper-
sonal relationships; other species respond to the category
another represents (male/female, young/mature) rather than
differentiating among individuals within category. The forms
of relationships in each society and their importance vary
from loose associations in herds or colonies without individual
recognition; monogamous groups characterized by a single
male and a single female and their offspring; harems with
a single male and multiple females; to complex societies with
individuals of all ages and both sexes and within which all indi-
viduals are uniquely identified.
What constitutes a social stressor for mammals needs to be
filtered through the social system of the test subject. We would
expect isolation to be a stressor for a macaque that ordinarily
lives in very large social groups with complex and highly indi-
vidualized relationships, but not for orangutans which live an
isolated existence normally. Current circumstances are also
important. Consider the monogamous titi monkey, for
example. When living with a mate, a stranger is treated as an
intruder to be expelled or avoided regardless of sex of intruder;
when living without a mate, a stranger represents the potential
for a new mate if it is the right sex.
Studies of social stress can also inform our understanding
of a particular social system. For example, if presence of
a stranger but not a familiar social companion elicits a stress
response, then we can infer at least that the test subject can
recognize the difference and the stranger represents a need
to mobilize resources. If we further determine that separation
from one familiar animal induces a stress response and sepa-
ration from another does not, we can conclude that the test
subject perceives absence of one as more important socially
than the other.
1.09.2 Aggression, Intruders, Social Defeat
Aggression is typically the social behavior that immediately
comes to mind when thinking about a stressor emanating
from within the social environment (McKittrick et al., 2009).
Early studies of aggression noted that spontaneous aggression
occurs during mate competitions, territorial encounters,
defense of young particularly by mothers, resource competi-
tion, and so forth (Hinde, 1966). Competition for incentives
as the primary motivating source of aggression was questioned
when Southwick (1967) failed to find sustained increases in
interanimal aggression when food was restricted or space
severely reduced. Instead it was discovered that novel animals,
intruders, entering the living area defined physically or socially,
were the most effective aggression eliciting stimuli (Bernstein
and Gordon, 1974).
The HPA-axis response to an agonistic interaction appears
to depend, in part, on the outcome of the encounter. After
fighting between male rats, corticosterone levels are increased
in the losers (Koolhaas et al., 1983), while corticosterone
levels decline in the winner (Haller et al., 1996). The SNS is
likewise altered in defeat. For example, if a smaller rat (S/JR)
experiences defeat by a rat of a larger strain (Long-Evans), the
defeated, smaller rat exhibits decreases in systolic BP that
persists for up to 2 h (Adams et al., 1987).
Most animals show an affinity not only for particular indi-
viduals but also for place. Territoriality is most common in
animals with social structures defined by overlapping territories
or monogamy (Davis and Marler, 2003). For many animals
defense of territory is an important social task and, with the
possible exception of mating, the primary social interaction
especially for males. The resident/intruder test capitalizes on
that tendency. Miczek (1979) is credited with developing the
resident/intruder model used extensively in studies of aggres-
sion in rodents.
1.09.2.1 Resident/Intruder Models of Social Stress
Due to the sex difference in proclivity to fight upon encoun-
tering another like-sex animal, resident/intruder studies in
rodents are almost exclusively performed using male subjects.
Residents in this model are generally favored in outcomes. In
some cases the scales are further tipped by having the resident
or intruder preselected for size or aggressiveness (Davis and
Marler, 2003). If focus is on the winner of the encounter,
commonly referred to as the challenge hypothesis (Wingfield
et al., 1990), one typically finds that corticosteroids are not
altered. Repeated defeat, on the other hand, leads to reduction
in body weight and adrenal hypertrophy with elevations in
corticosteroids (Archer, 1970; Kudryavtseva and Avgustinovich,
1998). The importance of territory is underscored by compar-
ison of rates of aggression in the resident/intruder model
with aggression if the same two males meet in an environment
equally novel to both animals. When the encounter takes place
in the resident/intruder situation, latency to attack is faster,
more frequent, and more intense than are aggressive bouts
when the males meet in a place equally unfamiliar to both
(Takahashi et al., 2012).
Rats placed with a resident from a highly aggressive strain of
rats show increases in NE, Epi, and corticosterone to defeat
(Sgoifo et al., 1996). In this study males were fitted with an
indwelling cardiac catheter which permitted collection of
blood samples throughout the test period without additional
stress introduced with the collection of samples. Results from
social defeat were compared to introduction of a shock prod
to males while in the home cage. Within 1 min Epi and NE
were significantly elevated in response to social defeat and
shock. By 5 min Epi and NE had reached peak values and
by 15 min levels of Epi plateaued and NE decreased even
though the test was still ongoing. Following the 15-min test
period, both catecholamines declined to pretest basal levels
 Social Stress: Concepts, Assumptions, and Animal Models
15–45 min following removal from the test situation. Cortico-
sterone, as expected, was slower to react to the test situation
reaching significant elevations at 5 min, but not reaching
peak until 15–30 min following test initiation with peak levels
occurring after the cessation of the stressor. Even though the
time course of the response of each of the hormones to each
stressor was the same, the magnitude of response to social
defeat was much exaggerated in comparison to shock. Pretests
of latency to attack an intruder placed in the subject’s home
cage were used to determine trait aggressiveness. Aggressiveness
did not alter the corticosterone response to either stressor. In
contrast, males with shorter attack latencies in the pretest
were considered more aggressive, and these animals showed
a greater NE and Epi response to both shock and social defeat
that could not be explained by different behavioral reactions
to defeat.
These results illustrate how closely the response to defeat
mimics the classic stress response, but it is a substantially
greater response than another stressor, electric shock. At least
a possible explanation for this is that the two stress situations,
shock and social defeat, afford the mice differing degrees of
control. The shock was delivered by a shock prod which
afforded the rats an opportunity to avoid subsequent exposure
to the shock device. The mice spent considerable amounts of
time burying the shock prod (Sgoifo et al., 1996). Engagement
in this behavior could be considered a coping response as Weiss
(1972) noted providing a modicum of predictability and
control in the situation could reduce the stress response to elec-
tric shock. The notion that coping responses provide a total
explanation for the differential response to shock prod and
social defeat is somewhat qualified by the finding that those
animals that exhibited the most attempts to bury the shock
prod also exhibited the greatest response to social defeat. The
authors suggest that animals that are inclined to bury the shock
prod are generally more likely to respond to stressful circum-
stances by adopting an active coping style (Sgoifo et al.,
1996). The authors further note that if this is true it did not
lead to observed differences in the behavior of defeated rats.
The response to a single exposure to social defeat and the
attendant physiological changes can leave lasting changes
(Koolhaas et al., 1997). Long after the stressful experience,
these authors note that binding capacity of Type I and Type II
receptors for glucocorticoids is reduced in hippocampus
3 weeks later; circadian temperature rhythms take 8–10 days
to recover; and growth is suppressed for 5 days, but body
weight may be permanently altered. The consequences of
a severe attack leave the attacked individual altered.
Experiencing multiple defeat episodes does not produce
habituation to the procedure (Sgoifo et al., 2005). Victors in
a series of resident/intruder episodes (i.e., the residents from
those encounters) who are placed in the cage of another aggres-
sive resident display long-term changes to the circadian rhythm
of HR. In this circumstance the response of the defeated animal
is determined by his behavior rather than that of the attacker. If
the defeated animal responds to being placed in the resident’s
cage with environmental exploration and responds to being
attacked with counterattack (i.e., an actual fight occurs), then
the effect of being the intruder after having been the victorious
resident is minimized. In spite of the lack of habituation to
social defeat in the resident/intruder paradigm, repeated
265
exposure to the conditions of social defeat did not produce
evidence of HPA dysfunction (such as increased adrenal
weight, thymus involution, altered circadian rhythms, or basal
corticosterone outside the test situation) as has been found for
other repeated daily stressors (Bhatnagar and Vining, 2003).
Repeated exposure to the conditions of social defeat does sensi-
tize the response to a novel stressor, physical restraint, indi-
cating at least some long lasting consequences.
Studies pharmacologically manipulating HPA axis suggest
that corticosteroids promote aggressive behavior in the
residents during resident/intruder tests (Mikics et al., 2004).
Metyrapone blocks synthesis of cortisol, and administration 2
or 20 min prior to resident/intruder tests decreased initiation
of aggression by residents directed to smaller opponents.
Central or peripheral injections of corticosterone reinstated
aggressive reactions. Blocking protein synthesis by administra-
tion of cycloheximide prevents glucocorticoid effects if admin-
istered immediately (2 min) before the test but is ineffective in
modulating glucocorticoid effects if administered 20 min prior
to the test. These findings imply that cortisol acts centrally to
facilitate aggressive responses and that it acts through a rapid
mechanism – cell membrane changes perhaps – that does
not require the inevitable time delay for genomic-mediated
mechanisms.
1.09.2.2 Application and Limitation of Resident/Intruder
Model
Given the involvement of SNS and the long-term changes
produced by social defeat on cardiovascular function, the resi-
dent/intruder model has proven useful for study of early stage
cardiovascular disease, including hypertension, determinants
of arrhythmias, and cardiac myopathy (Sgoifo et al., 2012).
The resident/intruder model is extremely useful in identifying
characteristics of the acute stress response, its dynamics, and
its regulatory control (Koolhaas et al., 1997). Hamsters show
similar responses to the resident/intruder paradigm as
described for mice and rats, except the defeated animal is
equally likely to be the resident or intruder (Kollack-Walker
et al., 1997). Examination of neural activity, as measured by
expression of c-fos mRNA, reveals that defeated hamsters
exhibited enhanced activity in regions typically associated
with acute stress including, cingulate cortex, lateral septum,
bed nucleus of the stria terminalis, medial preoptic area, central
amygdala, and locus coeruleus. Moreover, most of these
regions have also been implicated in the behavior of stressed
animals. The resident/intruder model is not a good model, as
illustrated in the next section for spontaneous formation of
dominance–subordinate relationships in complex social
animals.
For a few, modestly social species, social behavior can be
modeled effectively using the resident/intruder model. Tree
shrews are one such species. Tree shrews, considered a rodent
by most taxonomies but a primate by some, exhibit a particu-
larly virulent response to an intruder. Resident males will
immediately attack another male and defeat them within
a few minutes. Shortly after the fight has ended the resident
(victor) shows no further signs of arousal and generally ignores
the defeated male. The defeated tree shrew typically withdraws
as completely as possible from the purview of dominant and
266 Social Stress: Concepts, Assumptions, and Animal Models
may even die within a few days (von Holst, 1972). Those
subordinates that survive the defeat, engage in active avoidance
of the dominant, quitting the field anytime the dominant
approaches. For these subordinates, high levels of SNS activity
have been reported (Fuchs, 1984) with concomitant loss of
body weight and reproductive function (Fischer et al., 1985).
For studies wanting to examine the consequences of unbri-
dled attack, perhaps the best animal model might be found in
the response of female rats, protecting their litter. Presenting
a male intruder to a lactating female rat elicited maternal aggres-
sion which rarely included elements of ritualized attack and
submission typical of male–male encounters. Instead females
responded to intruders with bites directed to the most vulnerable
body regions. Defeat in this circumstance induces increased
blood pressure, catecholamines, and corticosteroids (Koolhaas
and Bohus, 1991). For the female, engaging in maternal aggres-
sion (15 min) induced increased HR with evidence of higher
SNS activity. Defeat (of the intruder) induced elevations in Epi
and NE. If the intruder were presented behind a mesh barrier,
effectively eliminating direct fighting but perhaps enhancing
the threat of aggression, elevations in Epi and NE were evident
but not to the same extent as with direct aggression. Elevations
in HR were likewise noted in the intruder, but did not differ in
conditions in which direct contact was or was not permitted.
HR variability indicative of heightened PNS activity positively
correlated with passive behavior (immobility, on the back,
upright defense) but did not change during testing and instead
increased during recovery from aggressive encounters (Sgoifo
et al., 1998).
The resident/intruder model is an excellent means of
inducing and studying the acute stress response. The question
must be asked, however, to what extent the resident/intruder
situations model the natural behavior of the animals. It would
virtually never be the case that animals appear in the core of
another animal’s territory as an initial encounter. Instead
such initial encounters occur at the periphery of territories,
and a vigorous attempt to expel another is likely to be met
with flight on the part of the intruder (Barnett, 1975). More-
over, most studies utilize methods to exaggerate the incidence
of attack by using larger residents, selecting the most aggressive
animals to be residents, or by enhancing aggression in residents
by prior social isolation using various taunting methods to
induce aggression. We would not expect to witness the
complete resident/intruder consequences to occur in nature
even for the species typically used in these experiments. With
the possible exception of tree shrews, the resident/intruder
paradigm, therefore, is not a good model of stress emanating
spontaneously from the social environment.
1.09.3 Aggression and Dominance in Social Groups
While we might expect the physiological response to threat to
share some similar response elements with an actual bout of
injurious aggression, the stressor in the instance of threat is
purely psychological which, obviously, is not the case with
injurious aggression. We reason that animals respond to threat
by preparing for the possibility of injury. While possibly true
this assumes that we know the animals are processing a threat
as a situation that will inevitably lead to attack. This
assumption is safest to make in the common intruder para-
digms that prevent physical aggression by introducing animals
to one another in circumstances that preclude direct interaction
(Sgoifo et al., 1998). In these circumstances the response to
threat is attenuated in comparison to direct interactions. In
group-living situations threats to attack are more common
than attacks even when there are limiting circumstances –
such as confinement in small spaces that preclude escape.
Also in contrast to controlled dyadic encounters such as in
the resident/intruder paradigm, aggression or the threat of
aggression within social groups is part of an ongoing stream
of behavior which includes multiple agonistic encounters as
well as feeding, sexual, and affiliative behaviors which may
mitigate any effects of stress by social or nonsocial means.
In most group-living primates and rodents it is evident that
all-out fighting is rare. Within group aggression is generally
mild, ritualized, and also infrequent – most of the time, in
most circumstances, for most species. When aggression does
occur, it is usually unidirectional. Dominance relationships
are a particularly common and often prominent feature of
male–male relationships in large group-living species and in
some species can describe aspects of female–female relations
as well. It is important to note, however, that male–male rela-
tionships in many species, particularly primates, are more
complex than can be described only by the direction and
frequency of quasi-aggressive interactions. Nonetheless, it is
generally assumed that the subordinate member of the dyad
is inhibited and socially suppressed in a way that is largely
detrimental to well-being. “Subordination constitutes a natural
and often chronic stress condition for males of virtually all
species of social mammals” (Blanchard et al., 1998: p. 307).
1.09.3.1 Case Study: Visible Burrow System in Rats
Wild rats, including the progenitor of most laboratory strains
(Rattus norvegicus), live in colonies in which multiple adults
share nesting sites and feeding grounds (Barnett, 1975). Such
colonies can number more than 100 individuals. Most early
laboratory studies of dominance and aggression in group-
housed rats used an open arena in which previously unfamiliar
animals were introduced to one another and the novel environ-
ment simultaneously. In these circumstances rats are often in
proximity to each other and will typically huddle together in
groups during sleep. Attacks are usually reserved for noncolony
members and often results in death of the intruder or, if the
situation allows, expulsion of the stranger. Aggressive episodes
between adults within the colony, natural or artificial, are
restricted to ritualized male–male encounters and females pro-
tecting the young (Barnett, 1975).
Rats are normally burrowing animals and, since feeding
sites are above ground, they are removed from sleeping or nest-
ing sites. In order to study dominance in an environment that
permits the full range of behaviors generally employed by rats,
a visible burrow system was devised (Blanchard and Blanchard,
1990). This apparatus includes an open arena containing food
and water which is lighted for 12 h per day simulating daytime;
the arena during the dark phase and remainder of the apparatus
is lit by red light that permits observation but is perceived as
dark by the rats. The open arena is attached to burrows via
tunnels. Rats must move from the burrows to the open arena
 Social Stress: Concepts, Assumptions, and Animal Models
to forage. This is accomplished during the dark phase and
provides an opportunity to engage with others or presents
a challenge to obtain food and water while avoiding others.
Thus the visible burrow system provides an elegant solution
to problems posed by nocturnal animals which spend the
majority of their time below ground. Within the visible burrow
system rats can organize their daily activities in a species-typical
fashion closely approximating natural colonies while permit-
ting investigators to observe virtually all social interactions.
Small colonies formed with six rats (three males, three
females) quickly adapt daily schedules to the visible burrow
system remaining in the burrows during the light portion of
the day and emerging to the open arena during the dark phase
(Blanchard and Blanchard, 1990). Rats do not form linear
dominance hierarchies, but instead a single male will emerge
as dominant to the others within a few days. Dominant males
account for 75–100% bites within the colony and engage in
offensive behaviors and few if any defensive behaviors. Domi-
nant males also have freer movement within the burrow
system; typically emerging into the open arena first within
the first hour of the dark phase, they engage in more eating
and drinking bouts, and in more sexual behavior. Following
exposure to a predator (a docile cat) dominant males engage
in significant risk assessments – peeking out of the tunnel
and surveying the open arena, rapid forays to the corners,
and concomitant reductions in offensive behaviors directed
to subordinates and sexual overtures to females (Blanchard
and Blanchard, 1989). Thus, it may be supposed that dominant
rats provide an important role in risk assessment for the entire
colony. The tendency to emerge first into open areas is consis-
tent with this role, and the suppression of subordinate
behavior serves to limit the risk of predation for subordinate
males and females while increasing risk for dominant males.
Thus, dominance in rats develops from a series of aggressive
encounters which include at least minor injurious aggression
and may be likened to a contest of strength. High status also
confers a role of vigilance for the entire colony.
Studies of the physiological response of male rats to the
visible burrow system were conducted by placing four to
five males and two females into the burrow system (Blanchard
et al., 1995). In most animals this disparity in sex ratio would
result in heightened competition among the males for access
to females, thus aggression in these studies would be expected
to occur at a higher rate than would occur naturally. In each of
the groups a single male emerged as dominant. After 2 weeks
of colony living, subordinate males showed elevated cortico-
sterone as compared to dominant animals. If colonies were
formed using males preselected for aggressiveness, clear
dominance status was settled more quickly. Changes in defen-
sive behavior was evident on day 1, and status differences in
offensive behavior, quite variably expressed on day 1,
emerged by day 2. Wounding was largely restricted to subor-
dinate males and generally was found on the less vulnerable
regions of the body. Subordinate males experienced weight
loss by day 6, and somewhat surprisingly dominant males
experienced significant weight loss by day 13. Corticosterone
levels were also elevated in both dominant and subordinate
males, and subordinate males had significantly higher cortico-
sterone levels than dominant males. Thus preselection for
aggressiveness resulted in changes in dominant as well as
267
subordinate animals indicative of hypersecretion of cortico-
sterone. The high levels of cortisol were not accompanied by
significant changes in brain or pituitary glucocorticoid
receptor populations or organ weights (adrenal, thymus,
spleen) although directional shifts suggest that even longer
time in the burrow may have produced such effects. These
animals did show a large reduction in CBG in subordinate
(70%) and dominant (40%) males relative to nonsocial
control males (Spencer et al., 1996). The drastic reduction
in CBG combined with the finding that there were fewer
Type II glucocorticoid receptors in spleen suggests dysregula-
tion of HPA function, particularly in subordinate males.
This interpretation is further supported by the finding that
adrenalectomy, a necessary precondition for receptor studies,
led to the death of 9 of 24 subordinate animals. Dominant
and control animals did not suffer mortally from the surgical
intervention perhaps due to sustained presence of the pool of
cortisol preserved in circulation by CBG.
Comparison of groups that are selected for aggressiveness
and those that are formed by random assignment differ in
the response of the dominant. When groups are preselected
for aggression, the dominant male shows physiological
changes that are indicative of social stress, but not to the
same degree as subordinates. This group of investigators was
trying to establish animal models of human health conditions
that also show HPA dysregulation such as depression, anxiety,
and autism (Blanchard et al., 2013). Since the most profound
effects of dominance/subordination only occur in groups that
would not likely be found in nature – more males than females
in the colony and colonies composed of only the most aggres-
sive males – the translational value of studies focusing on
aggression or dominance in rat colonies is minimal.
1.09.3.2 Mice in Groups
Like rats, mice live in colonies that can number over 100 indi-
viduals and dominance relations best described as a single
dominant with all others assuming subordinate status. Early
studies of mice suggest that circumstances that lead to social
instability are likely to produce greater indices of physiological
stress reactions than dominance relations per se (Henry et al.,
1971). Formerly isolated animals have difficulty in meeting
demands of prolonged social interaction and failed to develop
a stable hierarchy. In a stable group, levels of wounding are
relatively low and the dominant male has higher blood pres-
sure than subordinate males. In poorly socialized groups
everyone has higher blood pressure.
Stable laboratory groups were formed by simultaneously
placing 5 males and 10 females (approximating the natural
sex bias) into a colony cage which consisted of eight cages
interconnected by tunnels (Ely and Henry, 1978). Stabiliza-
tion of dominance relations took 45 days and was signaled
by decrease in wounding rates and threat gestures replaced
biting or other forms of aggression. As with rats, then, domi-
nance status appears to be determined by a contest of strength
which is only partially ritualized. Behavior of the dominant
male included patrolling behavior characterized by rapid
movement between the interconnected boxes. In contrast to
the antipredator behavior described for rats, the patrolling
behavior of mice seems more concerned with intragroup
268 Social Stress: Concepts, Assumptions, and Animal Models
processes such as seeking and expelling competitors. This
behavior was very frequent early following group formation
but stabilized at a much lower level (but still higher than
subordinate males) around the time wounding decreased.
Tyrosine hydroxylase and PNMT (rate-limiting enzymes for
catecholamine synthesis) increased in all animals after
14 days, continued increasing until day 42, and then declined
precipitously by day 105. The increased concentrations of the
enzymes necessary for production of NE and Epi were most
pronounced in dominant animals and remained higher in
dominant animals even through day 105. Subordinate
animals while exhibiting substantially lower levels of tyrosine
hydroxylase and PNMT were still elevated above control
animals living in social isolation. Evaluation of HPA activity
revealed that corticosterone levels in subordinate animals
were elevated substantially at day 14, but declined precipi-
tously by day 42 and again slightly by day 105. Corticosterone
levels for dominant males also showed a slight increase that
was not observed until Day 42 and by day 105 corticosterone
levels of dominant and subordinate did not differ and both
were slightly elevated relative to control levels. Dominance
reversal resulted in a reversal in physiological profile as well
(Ely and Henry, 1978).
The mice in these studies did not show a pattern of physio-
logical activation that could simply be described as activation
of the acute stress response to differing degrees in dominant
and subordinate animals. Dominant animals responded to
group formation with an increased evidence of SNS activity
whereas subordinate animals responded with a greater HPA
activation. It was this distinction that led Henry and Stephens
(1977) to conclude that there are two different stress-
response profiles, one emphasizing adrenomedullary or SNS
activity and one emphasizing adrenocortical activity. Although
the generality of these findings beyond mice does not suggest
that this distinction is applicable to other species, it did spawn
a great deal of future work investigating individual differences
in stress responsiveness.
The studies in rats and mice support the notion that domi-
nance status is imposed on one animal by another. In either
case, fighting and wounding happens but in a kind of
controlled manner that is generally not life-threatening.
A reasonable question from these studies is whether the phys-
iological changes noted are due to dominance per se and how
much is due to an untenable social circumstance. The increase
in sympathetic activity in dominant animals early in the group
formation might be expected to be supportive of the hypervig-
ilant state and increased activity displayed by them. The
increased adrenocortical activity of the subordinate males is
a response similar to the intruder’s response in the resident/
intruder paradigm and may be a consequence of directed
aggression.
Traditionally, it has been assumed that the formation of
dominance relationships is a simple case of mutual shaping.
These aggressive encounters, typical of rats and mice during
initial stages of group formation, are likened to contests of
strength. The loser of fights learns that the strength and
fighting ability of the other supersedes its own. The subordi-
nate role, according to this view, is established when the
loser responds at the outset of each agonistic or competitive
encounter with submissive responses. Dominance relations
thus function to ritualize aggressive tendencies between
potential rivals (Bernstein, 1981).
1.09.3.3 Social Dominance in Primates
Unlike mice and rats, formation of dominance relationships in
primates is not based on outcomes of contests of strength
(Mendoza, 1993). Studies of initial encounters in macaques
among previously unfamiliar, like-sex animals have shown
that while agonistic behavior is frequent initially it is highly
ritualized and orderly. Notably absent in studies of initial
encounters is attack followed by counterattack – or actual
fighting. Indeed, most contact aggression is mild – grabbing,
hitting, or comprised of ritualized threat gestures such as stare
or open-mouth threat. This is the case if dyadic encounters
occur in isolation of other social partners (Maxim, 1976) or
if they occur within the context of larger group formations
(Bernstein and Mason, 1963; Hawkes, 1970). It is also not
likely that animals assess fighting ability in their opponents
and concede dominance in the face of obviously superior part-
ners. Reducing the ability of animals to predict outcome by
carefully matching individuals for characteristics associated
with status such as age, sex, weight, and aggressiveness leads
to less, not more aggression (Mendoza, 1993). At first both
animals in the new dyad are equally likely to initiate agonistic
interactions, but very quickly the individuals limit themselves
to behaviors consistent with the dominant or the subordinate
role within the dyad – without anything closely approximating
a contest of strength.
Group level processes are also involved in formation of
dyadic status relationships in many animals (Chase, 1984)
including macaques (Barchas and Mendoza, 1984). In
contrast to rats and mice, linear hierarchies are a frequent
feature of primate societies. In triads of rhesus macaques,
for example, three dyadic relationships must be resolved.
The outcome of dyadic status resolution can be transitive
(A / B / C; A / C) or intransitive (A / B / C; C / A).
Intransitive or circular dominance relations would be inimical
to a linear hierarchy. A study of 13 like-sex triads of rhesus
monkeys revealed that in both male and female triads the
sequence and direction of dyadic agonistic interactions during
the first 45 min of initial encounters occur in an orderly
fashion which promotes linearity in the resultant hierarchy.
Moreover, some interaction sequences are suppressed in
newly formed groups (Barchas and Mendoza, 1984). Given
an initial encounter – A threatens B, there was a high proba-
bility (90.2%) the next aggressive overture conformed to
one of three sequences: A threatening B again (Repeat), A
threatening C (Double Attack), or C threatening B (Double
Receive). Each of these patterned sequences support the
formation of transitive relationships. Surprisingly, three
possible sequences were infrequent (9.8% of interactions): B
threatening A (Reverse), C threatening A (Attack the Attacker),
or B threatening C (Pass-on). These interaction sequences do
not inevitably lead to intransitive patterns of interaction, but
they have a 50% theoretical probability of doing so. The
predominance of Repeat, Double Attack, Double Receive
patterns of interaction held for all behaviors including those
that are not considered to be concerned with dominance
interactions such as sniffing or grooming. It should also be
 Social Stress: Concepts, Assumptions, and Animal Models
noted that the patterning of successive acts according to the
Repeat, Double Initiate, and Double Receive patterns need
not imply that dyadic interactions are unidirectional. For
dominance and submissive behaviors, directionality was not
immediately apparent, but emerged in most dyads by the
end of the 45-min observation period, and all triads presented
a transitive hierarchy.
As relationships in macaques stabilize, patterns of interac-
tions change somewhat and the frequency of redirected aggres-
sion (Pass-on pattern) and defending an ally (Attack the
Attacker) increase and become a part of the rich repertoire of
expressing and controlling aggression in groups (Chase,
1984; de Waal, 1993). For large groups it is worth noting
that dominance relations are most commonly formed in
within-sex dyads and not in male–female dyads and that
a middle ranking animal performs the dominant role with
some group members and the subordinate role with others.
Examination of physiological correlates of dominance
status reveals considerable species variability in studies of
nonhuman primates (Abbott et al., 2003). Nonetheless where
differences are found it is generally in the direction of domi-
nant animals exhibiting lower cortisol and lower indices of
SNS activity than subordinates.
1.09.3.3.1 Case Study: Physiological Processes Associated
with Dominance in Baboons
The most detailed studies regarding the physiological under-
pinning of dominance are provided by studies of free-ranging
baboons (Papio anubis) (Sapolsky, 1993). Baboons live in large
social groups, and as is the case with most species of primates
living in large groups, group members can be ranked within
a pecking order or dominance hierarchy. Using darting tech-
niques to anesthetize baboons and using elegant physiological
probes Sapolsky has demonstrated status includes physiolog-
ical differentiation of HPA and SNS systems. In his initial
studies, Sapolsky (1982, 1989) found that dominant males
have lowest initial concentrations of plasma cortisol and the
most rapid rise in circulating levels in response to the stress
of darting and anesthetization. Administration of labeled
cortisol (3H-cortisol) indicated that dominant and
subordinate were equally efficient in metabolizing and
removing cortisol from the circulation. The low HPA activity
characteristic of dominant individuals was due to a low
secretion rate of cortisol rather than enhanced clearance rates
(Sapolsky, 1983). The possibility that the adrenal of
subordinate animals is more sensitive was eliminated by the
comparable response of dominant and subordinate animals
to ACTH. Paradoxically, subordinate animals were found to
be less sensitive to CRH, suggesting that central mechanisms
are even more active in stimulating HPA activity in
subordinate than in dominant animals (Sapolsky, 1989,
1990). Dexamethasone, a synthetic glucocorticoid that can be
biochemically distinguished from cortisol, but presents a very
potent negative feedback signal to the HPA system, was more
effective in suppressing endogenous HPA activity in
dominant animals than in subordinate animals (Sapolsky,
1983). In short, dominant animals have an efficient HPA
system in which basal levels of HPA activity are relatively
low, and the response to acute stressors is rapid and rapidly
terminated.
269
With regard to the SNS similar differences were found.
Dominant males had the largest and fastest response to Epi,
greatest HR increase and peak systolic pressure, and the most
rapid recovery (Sapolsky and Share, 1994). Interestingly,
administration of an alpha blocker (proterenol) did not
equalize the differences in blood pressure nor did administra-
tion of a beta blocker eliminate status differences in HR.
Administration of metyrapone which blocks synthesis of
cortisol did eliminate status differences in both blood pressure
and HR. Thus it appears that status differences in HPA function
has an impact on the actions of the SNS and confers a physio-
logical advantage for dominant individuals.
Other glucocorticoid-related differences between dominant
and subordinate baboons include diminished high-density
lipoprotein cholesterol with elevated cortisol levels character-
istic of subordinate monkeys (Sapolsky and Mott, 1987).
Likewise, circulating lymphocytes are higher in dominant indi-
viduals but were reduced to the same level as seen in subordi-
nate males when cortisol levels were pharmacologically raised
in dominant males to that observed in subordinate males
(Sapolsky, 1993). One rank-related physiological effect that
was not attributable to differential glucocorticoid levels was
insulin-like growth factor which is suppressed in subordinate
monkeys (Sapolsky and Spencer, 1997).
All of the above studies with baboons were conducted
during periods of social stability when rates of aggression and
other indices of dominance status were relatively low. Sapolsky
also studied his troop of baboons during periods of instability
– following ousting of a dominant male by a cohort of young
males who themselves failed to establish a clear hierarchy once
the dominant animal was gone. With social instability the
dominant animals lost the physiological benefit of high status,
and their HPA system looked like that of subordinate males
(Sapolsky, 1993). When a highly aggressive male entered the
troop he became dominant, but unlike previous, less aggressive
dominant animals in the troop, the aggressive dominant
male had the highest cortisol and lowest lymphocyte levels
(Sapolsky, 1995).
Individual differences in sociability also altered status
differences in HPA regulation. A subset of subordinate males
had high rates of prolonged association with estrous females,
or consortships, comparable to dominant males. These subor-
dinates also had larger stress responses, similar to that
described for dominant males, but they also exhibited elevated
basal cortisol typical of a subordinate male. Similarly, furtive
males with high rates of stolen copulations did not show
elevated basal cortisol, and subordinate males who initiated
aggression or who exhibited redirected aggression (usually
threatening a bystander) also show lower cortisol typical of
a dominant individual (Virgin and Sapolsky, 1997). As noted
above redirected aggression is virtually nonexistent during rela-
tionship formation when all strangers meet for the first time.
The data from Sapolsky’s studies show that redirected aggres-
sion does occur in stable groups and that it also can serve
a protective function for the animals that engage in this
behavior pattern.
A particularly interesting set of changes in one troop of
baboons occurred when an outbreak of bovine tuberculosis
from spoiled meat at a garbage dump led to the death of all
adult males that frequented the site, but spared those that
270 Social Stress: Concepts, Assumptions, and Animal Models
did not (Sapolsky and Share, 2004). Aggressiveness of animals
competing for food at the garbage site was much higher than
aggressive tendencies in the animals that did not visit. The
result was a baboon troop comprised of the least aggressive
males. When new males transferred to the troop, the lower
levels of aggression and higher levels of affiliation were main-
tained well after the troop consisted only of new males. This
altered pattern of interactions among troop members did not
alter the overall composition of the new troop nor the expres-
sion or stability of the hierarchy. The subordinate males in the
new troop, however, no longer exhibited hypercortisolemia.
These studies in baboons illustrate a few principles perti-
nent to the study of social stress. The social system is capable
of exerting an influence on the physiology of its members.
Whether the society is stable or unstable, more aggressive over-
all or more affiliative, all contribute to rank-related differences
in stress physiology. When cortisol levels are sustained at high
levels, there are strong reasons to suppose that they extract
a cost to the animals in terms of physical health and probably
psychological well-being (Sapolsky et al., 2000). We also know
from this body of research that heightened cortisol is not
simply the result of being subordinate even within the same
baboon troop. Highly aggressive animals, even though domi-
nant, exhibit heightened cortisol and highly affiliative animals
may not, even though subordinate. It is also important to
note that even in the troops with stratified cortisol related to
dominance status, high or low position in hierarchy is not
a life-long commitment. Male tenure in a given group, and
hence occupation in a given position in the hierarchy, is rela-
tively short, generally a year or two. Therefore, it is entirely
likely that adverse consequences of heightened cortisol levels
when they occur are not sustained long enough to cause irrep-
arable damage.
1.09.3.3.2 Case Study: Social Influences on Stress Response
Systems in Squirrel Monkeys
Studies of captive squirrel monkeys present a quite different
picture than Sapolsky describes for wild baboons. When
rank-related differences in HPA activity are found, they occur
in newly formed and relatively unstable social groups. Domi-
nant male squirrel monkeys in newly formed same-sex dyads
or triads exhibited the highest levels of cortisol and subordi-
nates exhibited the lowest (Coe et al., 1979; Mendoza et al.,
1979). Examination of hormonal change from living alone to
living in male triads to living in male–female groups (through
the addition of five females to each of three male triads) sug-
gested that each stage of group formation was responded to
by an increase in plasma cortisol that was greatest in the
most dominant male, but in this study dominance effects
were not significant until the females were added to the male
triads. It is not always the case that male squirrel monkeys
respond to group formation with elevated HPA activity. In
one study, formation of male triads did not result in an imme-
diate rank-related difference in cortisol, but simultaneous
introduction of males, all strangers to one another, in a novel
cage led to an overall reduction in cortisol in all group
members (Lyons et al., 1994); 3 months following triad forma-
tion cortisol levels did reflect dominance status, and once
again, the dominant male had significantly higher cortisol level
than the other two males and the most subordinate males had
the lowest cortisol level (Mendoza, 1991). Cortisol levels for all
males, including the alpha male, were still lower than individ-
ually housed males.
Although the methods used to perform group formations,
collect blood samples, and cage configurations were similar
between the studies, several differences in subjects and
methods could account for the discrepant results. One such
difference is subspecies variation. Male Bolivian squirrel
monkeys, the subject of the earlier studies (Coe et al., 1979;
Mendoza et al., 1979), are considerably larger than other
subspecies, and they are dominated by the much smaller
females acting in concert to displace males from preferred
resting sites, which is not the case for other subspecies
(Mendoza et al., 1978; Lyons et al., 1992). Cortisol concentra-
tions are overall higher for Bolivian males, and their HPA
system is more reactive than other subspecies of squirrel
monkeys (Coe et al., 1979, 1978). Perhaps the most influential
difference between the studies was timing relative to the
breeding season. In the Mendoza et al. (1979) study, group
formations were timed to occur 6 months out of phase with
the breeding season; group formation induced breeding in
each of the three groups. In contrast, the triad formations in
the Lyons et al. (1994) study occurred just prior to the breeding
season and by 3 months following formation of male triads
colony females were cycling (Mendoza, 1991). Perhaps differ-
ential HPA activity only occurs in new groups in which
breeding is also happening. Heterosexual groups were formed
5–7 months following triad formation. At this time most
colony females had ceased cycling. Male cortisol was not
altered by the addition of females to their groups and breeding
was not induced. Heterosexual group formation, however, did
affect the timing of the subsequent breeding season which
occurred 12 months following group formation, considerably
later than that predicted by timing of heightened gonadal
hormone activity in the previous year (Schiml et al., 1996).
Female squirrel monkeys also show a reduction in cortisol
upon formation of female triads (Mendoza and Mason,
1991). The reduction in cortisol did not happen right away.
Cortisol levels 24 h following triad formation were comparable
to cortisol titers when females were housed individually. In this
study a single male was added to each female triad, and
breeding was induced within the first week of heterosexual
group formation. Somewhat surprisingly, cortisol levels
remained low in the females in spite of all females undergoing
massive changes in gonadal hormone activity. Cortisol levels
do show circannual changes (Schiml et al., 1996). For females
living in established groups, elevations in cortisol coincide with
the onset of ovarian cyclicity typical of the breeding season. In
males the seasonal elevation in cortisol coincides with annual
weight gain – the so-called fatting response – both of which
peak in the month prior to seasonal elevations in testosterone.
Both males and females show clear and linear within-sex domi-
nance relations based on the direction and frequency of
agonistic interactions; neither males nor females exhibit rank-
related differences in cortisol levels or in the magnitude and
timing of circannual changes once the groups have stabilized.
The reduction in cortisol upon the formation of like-sex
groups of squirrel monkeys could suggest an alleviation of
stress associated with isolation. This seems unlikely for several
reasons. First, the reduction takes time to develop. In our
 Social Stress: Concepts, Assumptions, and Animal Models
studies, more than 24 h and less than 6 days is required for
cortisol levels to reach their socially induced low level. This
slowly developing time frame in response to separation and
pair formation is of the type delineated by Hofer (1987) for
removal or provision of homeostatic regulation possibly
induced by a shift in receptor populations. Once developed
the hypocortisolemia is apparent at all points in the circadian
rhythm (Mendoza et al., 2000). Secondly, the acute responses
to isolation and pair formation are distinctly different than
the prolonged effects (Mendoza et al., 1992). Female squirrel
monkeys, living in established female–female pairs, responded
to separation from their cage mate with reduced cortisol 1 h
later; by 6–8 days following separation cortisol levels were
significantly elevated. Subsequent formation of new female
dyads resulted in further cortisol elevations at 1 h followed
by significant reductions 6–8 days later. Thus, long-term conse-
quences of separation and pair formation are opposed to the
acute response to these social manipulations and cannot be
considered an extension or continuation of the acute response.
Finally, cortisol levels in established groups do not differ from
animals housed individually (Schiml et al., 1999). Following
the increase in cortisol associated with the first full breeding
season as cortisol declines to nonbreeding levels, the social
effect on HPA activity disappears and does not reemerge in
stable groups.
HR in male squirrel monkeys shows a similar reduction
upon formation of new relationships (Mendoza, 1991). Within
the first hour of pair formation HR was considerably reduced
compared to HR in the same testing situation while living
alone. Following 2–3 weeks of continuous cohabitation, HR
was further reduced. It is possible that the reduction in HR
was attributable partially to sympathetic withdrawal, but the
evidence suggests that the inhibitory influence of parasympa-
thetic activity was more influential. HR variability, a measure
of parasympathetic activity, increased with pair formation
and, like HR, was further increased after the males had lived
continuously together. Female squirrel monkeys, tested simi-
larly, also showed the lowest HR when living in established
pairs, but this was not significant nor was there any indication
of enhanced parasympathetic activity (Saltzman et al., 1991).
This finding of increased parasympathetic activity in male
squirrel monkeys is particularly interesting since squirrel
monkeys living without like-sex companions show very little
parasympathetic activity in the regulation of HR (Mendoza
and Mason, 1997).
In summary, the hypocortisolemia characteristic of newly
formed squirrel monkey groups is evident only during the
period following group formation and preceding the first
breeding season of the new group. At least the initial
response to formation of within-sex relationships is also
accompanied by an increase in parasympathetic activity in
males but not in females. The tendency to ascribe the socially
induced reduction in cortisol to something akin to stress
buffering does not conform to the data. Instead the group
formation studies in squirrel monkeys suggest a chronic
response to the stress of encountering strange animals for
the first time, but it is in the opposite direction from that
usually considered indicative of chronic stress. The question
of greatest import is whether the reduced cortisol is beneficial
or detrimental to health.
271
One notable absence in all of our studies of group forma-
tion in squirrel monkeys was any evidence of dominance
contests. Dominance relations were detectable early following
relationship formation in both males and females. In all of
the group formations conducted in squirrel monkeys only
one reports any instances of actual fighting (Gonzalez et al.,
1981). That fighting was severe is evidenced by the fact that
the most subordinate males in two of four groups in this study
died within a week of group formation. Why severe fighting
occurred in this study and fighting rarely, if at all, in other
studies is related to methods employed in forming groups. In
the Gonzalez et al. study heterosexual groups were formed by
combining subgroups that were familiar with one another,
some of which were living together and transferred as a group
to the new heterosexual group. These are the conditions
that lead to increased aggression during group formations
(Bernstein et al., 1974).
In combination the studies of dominance and stress-
response systems reveal some generalizations and lack thereof.
Fights are rare. Contrary to popular assumptions, even in
newly formed groups dominance relationships are established
quickly without contests of strength. Once established domi-
nance relationships are stable. Subordinate animals are not
continually striving for dominance. In contrast to mice and
rats, the dominance relations in most polygynous primates
are linear, implying some group level processes control the
behavior of the individuals therein. Early in primatological
research, KRL Hall (1964) suggested that aggression within
primate societies is most often the result of violating social
order either by acting at variance with their status and role
within the group or in response to attempts to gain access to
the group by outsiders. As Sapolsky’s work, reviewed above,
suggests there are occasions when the subordinate animal
displays a physiological profile consistent with a chronically
stressful state, which if sustained might result in health prob-
lems. It is also the case that dominant baboons can exhibit
the same stress-related pathologies during times of instability
or that all animals are saved from experiencing chronic stress
effects if the larger group culture is one characterized by higher
than usual affiliation and lower conflict. How dominance
affects stress response systems is species specific. Physiological
differentiation of status in squirrel monkeys when it appears
suggests the dominant male is most at risk, whereas in
baboons the subordinate male is most at risk. Rank-related
differences in stress physiology in squirrel monkeys are also
transitory, appearing only in situations in which social insta-
bility and breeding readiness coincide, and thus unlikely to
pose risks to health. Social instability, rather than rank, in
a well-ordered hierarchy appears to be key in understanding
social stress.
1.09.4 Social Instability
The impact of social instability on stress response systems and
health is provided by a study of rhesus macaques (Capitanio
et al., 1998). In this study male rhesus monkeys were allowed
social interaction for 100 min day 1 for 3–5 days week 1. All
other times the animals were housed individually, but within
sight and sound of other monkeys. During the social
272 Social Stress: Concepts, Assumptions, and Animal Models
interaction periods animals were either placed in triads with
the same other animals each day (Stable condition) or they
were placed in groups of two to four with the identity of the
other group members changing from session to session
(Unstable condition). The rationale for this study was that in
the Stable group, in spite of relatively limited daily interaction,
the animals would quickly learn the identity and proclivities of
one another and form stable relationships. The Unstable
groups would afford the same amount of interaction time for
each male but since formation of stable dyadic relationships
is facilitated by stability at the group level, it was reasoned
that males from the Unstable group would not be able to
form stable relationships. Instead, each time they met a new
social order, in particular new dominance relations, would
need to be established. After three interaction days half of the
animals were inoculated with simian immunodeficiency virus,
SIVmac251, and stress-related physiology and disease progres-
sion were monitored.
Aggression was rare in all groupings, but males from the
Unstable condition were more likely to be the recipients of
threats, contact aggression, and chasing; males in the Stable
condition were not observed to engage in either physical
aggression or chases. Animals in the Unstable condition
exhibited a more rapid disease course and a shorter survival
time than animals in the Stable condition. The ability to mount
an antibody response to the virus was significantly related to
survival. However, animals experiencing stable social encoun-
ters did not differ in formation of SIV-specific antibody from
males experiencing unstable social encounters. Therefore,
antibody formation could not account for social condition
effects on survival.
As with squirrel monkeys during the initial period following
triad formation, cortisol levels declined for male rhesus
monkeys in both the Unstable and Stable social conditions
during the first 12 weeks of social experience. After this point
cortisol levels for monkeys in the Stable condition recovered,
whereas cortisol levels in the monkeys experiencing unstable
social conditions continued to decline through 24 weeks of
testing. After this time the cortisol levels for males experiencing
Unstable social conditions rose once again, and cortisol no
longer differed between Stable and Unstable social experiences.
Thus monkeys in both conditions experienced an initial drop
followed by recovery in basal cortisol levels, but for animals
in the Unstable group the decline in cortisol was greater and
persisted for a longer period of time. Blood samples were
collected in this study on days when no social interaction
testing occurred so the changes in cortisol are in response to
long-term socially induced alterations in HPA regulation and
not an acute response to social experience. Perhaps because
of the intermittent nature of social encounters in the rhesus
or perhaps due to known species differences in HPA regulation
(Mendoza and Moberg, 1985), the time to fully develop hypo-
cortisolemia in the rhesus monkeys (weeks) was considerably
longer than in squirrel monkeys (days). Nonetheless, survival
differences between Stable and Unstable animals in the face
of a lethal virus implicates low cortisol as a health risk or at
the very least associated with a more lethal physiological state.
The reduction in cortisol during the early weeks of stable
and unstable conditions was accompanied by a dampened
HPA response to physical restraint and a greater suppression
of HPA activity following dexamethasone administration
(Capitanio et al., 1998). The lower cortisol levels exhibited
by squirrel monkeys and rhesus macaques during periods
of social instability are reminiscent of findings of patients
suffering from posttraumatic stress disorder (PTSD)
(Daskalakis et al., 2013). Also similar to PTSD (Yehuda
et al., 2004), there appears to be a heightened sensitivity of
GR receptors leading to increased negative feedback evident
both in the unmanipulated HPA system and in the HPA
response to dexamethasone suppression.
PTSD does not occur in all individuals exposed to a trau-
matic stressor, and work is beginning to suggest that ante-
cedent to a PTSD-inducing trauma, differences exist which
increase the risk of developing PTSD (Yehuda and Bierer,
2008; Yehuda et al., 2002). The possibility that individual
differences in genotype or personality contribute to stress-
induced susceptibility to SIV was investigated in macaques.
Early in the disease process as the immune system is
activated to contain the virus and the virus is replicating
rapidly, the two biological processes reach a set point in
which viral replication is maintained at a static level
throughout the remainder of the infection until the
development of full-blown AIDS immediately preceding
death. Viral set point is established 6 weeks postinfection,
is measured by viral RNA (vRNA), and is the single best
predictor of survival. In two studies, for example, survival
duration was correlated .79 and .73 with viral set point
(Capitanio et al., 1998; Capitanio et al., 2008). Immune
processes early in infection include interferons, produced by
infected cells within hours, and their effector molecules
whose effects on viral replication are processed via receptors
on other immune cells. Each of these processes influences
viral set point, and each is influenced by behavior and
behavioral tendencies created by personality and genetic
polymorphisms (Capitanio et al., 2008).
Specifically, animals were selected as subjects based on
a personality assessment conducted with animals in their natal
field cages. Males scoring in the highest or lowest third of the
colony on sociability, a trait associated with affiliative tenden-
cies (Capitanio, 2004), were tested for their allelic variation of
the promoter region for the serotonin transporter (5-HTTLPR).
A short allelic variant, is associated with aggressiveness, anxiety,
and neuroticism (Lesch et al., 1997; Barr et al., 2003; Caspi and
Moffett, 2006). Animals received socialization opportunities
for 100 min day 1, 3–5 days per week. Triads of animals in
the Stable social condition met with the same animals each
socialization period, whereas animals in the Unstable condi-
tion met with animals of varying number (2–4) and identity.
All groupings were performed within personality type (High
or Low Sociable) and all were inoculated with SIV following
three such pairings. None of the categorical variables in this
study (social condition, high or low sociability, or short versus
long 5-HTTLPR) directly influenced viral set point. However,
each was found to influence viral set point indirectly largely
through altering actions on IFN-a which is more directly
involved in establishing viral set point. Animals in the Unstable
group exhibited sustained aggression and reduced affiliation.
Sustained aggression performed predominantly by animals in
the Unstable group with the short 5-HTTLPR genotype was
associated with decreased expression of CXCR3 (receptor for
 Social Stress: Concepts, Assumptions, and Animal Models
both IFN-a and IFN-g effectors). Recipients of sustained aggres-
sion had reduced SIV antibody (anti-SIV IgG). Sustained
submission, displayed most prominently by Low Sociable
animals, was correlated reductions in cortisol and with
increased ISG (an IFN-a effector). Aggression initiated in Stable
groups, performed by animals with long 5-HTTLPR genotype,
was associated with increased ISG activity. Reduced anti-SIV
IgG, increased expression of ISG, and decreased expression of
CXCR3 were all related to increased viral set point and thus
point to a more rapid disease course. Interestingly the reduc-
tions in cortisol found earlier to be related to survival was
only found to be related to increased ISG levels and to sus-
tained submission. Whether hypocortisolemia is an inter-
vening variable between the association of submissive
expression and viral set point is not known. It is clear, however,
that HPA function is not clearly related to establishing viral set
point, the best predictor of disease course available during early
stage disease. Low levels of cortisol may, however, permit many
disease processes to go unchecked.
In addition to low cortisol, PTSD is also characterized by
high levels of catecholamines, CRH, and proinflammatory
cytokines suggestive that the low cortisol is insufficient to
inhibit these aspects of stress response (Daskalakis et al.,
2013). In keeping with this suggestion, inquiry into the role
of SNS in understanding differences in SIV disease progression
between animals in Stable and Unstable social conditions was
conducted. Examination of neural fibers enervating lymphoid
tissues suggest a role for SNS in altered disease progression. At
37 weeks postinoculation, lymph nodes for 20 animals were
biopsied. Replication of the SIV virus, as indicated by local
SIV RNA expression, was nearly four times higher in proximity
to axonal varicosities of sympathetic fibers (Sloan et al.,
2006). Although the number of sympathetic fibers was
reduced in SIV-infected animals, for surviving animals of the
Unstable social condition innervation was greater than for
animals in the Stable social condition and viral replication
was nearly 60% greater for Unstable males (Sloan et al.,
2007). The enhanced SNS innervation of lymph tissue was
associated with altered interferon activity – reduced INF-
b and increased INF-a. Both greater innervation and altered
INF expression account for enhanced SIV gene expression.
Social instability was also associated with increased expres-
sion of neurotrophic factors, BDNF and NGF, in lymph tissue
(Sloan et al., 2008b). NGF was particularly influential,
inducing more SNS innervation providing more varicosities
and greater local release of NE in Unstable animals, which
moderated the INF response ultimately increasing viral repli-
cation (Sloan et al., 2008a).
The role of the SNS in mediating the enhanced SIV disease
progression with social instability was examined in another set
of studies (Capitanio and Cole, 2015). Selection of animals for
this study balanced groups for sociability. Animals were social-
ized in stable groups (male triad with same animals each day)
for 5 weeks, inoculated with SIV, followed by 5–15 additional
weeks of stable groupings, then switched to 5 weeks of unstable
groups in which animals met in groups of two to four with
number and identity of interactants changed daily. Animals
were then replaced in their original stable groups and testing
continued for at least 5 weeks. When in Unstable groups, males
groomed less, spent more time alone, and aggressed more.
273
Urinary analysis of catecholamine metabolites indicated that
social instability was associated with higher catecholamine
output. Enhanced expression of proinflammatory genes
(IFN-g, IL-6, and TNF-a) was induced with social instability.
Behavioral, endocrine, and immune consequences of social
instability were reversed when social stability was reinstated.
Switching from Stable to Unstable social conditions did not
affect measures of disease progression – anti-SIV IgG increased
at each stage of the study and vRNA did not change.
In another study (Capitanio and Cole, 2015) administra-
tion of methamphetamine to animals socialized in Stable or
Unstable social conditions was used to enhance the apparent
SNS contribution to viral set point. Interestingly, administra-
tion of methamphetamine did not affect lymph architecture
even though clear elevations in plasma EPi and NE were
accomplished. Animals from the Unstable social condition
exhibited greater innervation of lymph tissue than animals in
the Stable condition and neither were different whether meth-
amphetamine was administered or not.
It is clear that the stress response systems, HPA and SNS, are
both altered by the same circumstances – social instability –
that alter disease processes and the body’s effort to combat
them. It is less clear that measures of global activity as repre-
sented by circulating levels of the hormones of each system
can be linked to specific disease processes. For the SNS system,
in particular, local changes in lymph tissue appear to be altered
by social conditions which, in turn, alter the ability to deal with
disease processes independently of circulating hormone levels.
It is also clear that the HPA response to social instability in
squirrel monkeys and rhesus macaques results in lower circu-
lating levels of cortisol than in social contexts that enable
animals to form stable social relationships. Thus in both
systems, looking for consequences of social stress can be
misleading, in the SNS because tissue effects do not mimic
circulating hormone levels and in the HPA system because
our tendency is to use high cortisol as a marker for what is
and what is not stressful.
1.09.5 Disruption of Established Relationships:
Separation, Bereavement, and Loneliness
Structured and recurrent interchanges between individuals
within relatively stable social units are a general feature of life
for social mammals. When such interchanges occur regularly
and predictably, it is customary to refer to them as relationships
(Hinde, 1976, 1983). Another aspect of social mammals is that
each species tends to display characteristic modal form of social
organization. At a gross and superficial level this is reflected in
the size and composition of the group. Social organization is
generally considered to reflect the combined nature and quality
of relationships among the members of the group (Hinde,
1976; Mendoza, 1984). This is not to say that relationships
create the existing social structure. The most obvious evidence
that relationships alone cannot account for group structure is
that for most social systems the group structure is defined in
part from the individuals excluded from the core of social inter-
actions. One-male groups or monogamous groups, for
example, are defined by the absence of potential relationships
as much as those which are actually present. Comparative
274 Social Stress: Concepts, Assumptions, and Animal Models
studies aid in understanding generic aspects as well as species-
typical variants in relationships.
As indicated in the previous section, formation of new
relationships is a prolonged process and is accompanied by
periods of vulnerability, behaviorally and physiologically.
Cooperation or protection is as much of a mainstay of social
life as competition. What happens when stable relationships
are disrupted? Some of the answer to this question depends
on the nature of the relationship and the society within which
the relationship is embedded. Removal of a bully might be
responded to positively by other group members. Removal
of a mother can have devastating consequences for the infant,
even for infants beyond the stage of nutritional dependency.
Friendships describe a tendency to affiliate with particular
others and to engage infrequently with others – a common
feature of large polygynous groups. Do these friendships
confer behavioral or physiological advantages? Virtually
all adult humans have experienced a friend moving to
another geographical location preventing regular interaction.
Is the response to loss of a friend of many years at all compa-
rable to separation from a lover even if the relationship is
relatively new?
1.09.5.1 Comparative Case Study: Polygynous Squirrel
Monkeys versus Monogamous Titi Monkeys
Titi monkeys live in family groups consisting of an adult male
and female and one to three offspring (Mason, 1966). The
adults are monogamous, and like-sex encounters are generally
restricted to highly ritualized territorial displays at the
boundary of adjacent territories (Van Belle et al., 2016). Fathers
provide the majority of infant care, typically infants transfer
from the father to the mother for brief periods surrounding
nursing (Mendoza and Mason, 1986b).
Squirrel monkeys, in contrast, live in large groups and,
with the exception of infants, primarily associate with
animals of the same age and sex. Females tend to cluster
into smaller social units or cliques and may act in concert
with clique members to actively exclude other group
members, including males (Leger et al., 1981), and are often
credited with maintaining the sexually segregated social
structure (Mendoza, 1991). Interactions among females
within a clique are generally affiliative, but agonistic interac-
tions are also more frequent within cliques. Membership in
cliques is transitory, and changes in clique size and member-
ship are frequent in laboratory populations and field data
indicate that females periodically transfer between groups
(Boinski, 1987). Male relationships are generally less affilia-
tive than female relationships but are more stable. Both
males and females form within-sex linear dominance hierar-
chies that are more clearly demarcated among males
(Mendoza et al., 1978). Infants are cared for almost exclu-
sively by mothers, although occasional ‘aunting’ by other
adult females occurs (Mendoza, 1991).
Social separation studies have helped to elucidate the rela-
tional dynamics in these species. The first studies of social sepa-
ration focused on the HPA response and the mother–infant
relationship (Mendoza et al., 1978). Mother and infant squirrel
monkeys react to the disruption of their relationship with
a substantial and rapid increase in HPA activity. Brief
involuntary separation of 3-month-old infants from their
mothers elicits an increase in plasma cortisol of up to 100%
of baseline values in both mother and infant within 30 min,
and cortisol levels return to baseline levels 1–6 h following
reunion (Mendoza et al., 1980). When infants are cared for
by a familiar adult female during separation from the mother,
they show as great an increase in cortisol as when they are left
alone. One of the features of the above separation studies
includes a disturbance control condition. Establishing this
condition includes capture of the pair, brief separation, and
immediate return to familiar living conditions. This procedure
was anticipated to elicit a modest stress response albeit not as
substantial as separation per se. Indeed, capture and handling
have been used as a stressor to elicit an acute stress response
(Mendoza et al., 1978). Surprisingly, neither mother nor infant
shows a response to the disturbance control procedures. This
finding has been used to argue that the counterpart to the sepa-
ration response is stress buffering.
The acute pituitary–adrenal response to disruption of the
mother–infant bond is remarkably resistant to habituation.
Hennessy (1986b) found that over the course of 80 mother–
infant separations conducted between 12 and 31 weeks of
age, each 2-h separation reliably elicited an increase in cortisol
levels. Inasmuch as basal cortisol levels in these repeated sepa-
rated infants remained unchanged and did not differ from
basal values in nonseparated control infants. These recurrent
reactions to mother–infant separation in squirrel monkeys
occur around a roughly stable physiological set point – regula-
tion of the HPA system was not altered. The HPA response to
separation for infant squirrel monkeys does not occur if infants
are within sight of their mother but unable to contact her (Vogt
and Levine, 1980). Behavioral agitation, however, is most
extreme under these circumstances. When visually isolated
from mother, the cortisol response to separation is sustained
for at least a week, in spite of a lack of sustained behavioral
agitation and an absence of depressive symptoms noted under
similar circumstances in macaques (Coe et al., 1985).
These findings, together with data from other species,
confirm the initial interpretation the effective social dimension
triggering the acute physiological response to separation is not
removal of maternal behaviors, but rather it symbolized the
disruption of a well-integrated and specific psychosocial attach-
ment between mother and infant (Hennessy, 1997). Many
investigators proceed as if all affinitive social relationships
resemble the attachment bond in form, structure, and conse-
quences, differing only in the intensity of participant involve-
ment. Surprisingly, this is not the case.
Our initial study designed to extend our findings to other
relationships focused on the response of female squirrel
monkeys to involuntary separation from like-age, same-sex
companions, relationships which might be expected to
conform most closely to mother–infant relationships
(Hennessy et al., 1982). To our surprise, the results indicated
that juvenile females did not show a specific response to sepa-
ration from isosexual companions, nor did familiar compan-
ions reduce the adrenocortical response to stressful events
such as capture, handling, or exposure to a novel environment.
These findings were extended to adult females removed from
large group cages and placed in novel cages with a preferred
or not preferred female (Hennessy, 1986a). The HPA response
 Social Stress: Concepts, Assumptions, and Animal Models
of adult females to a novel environment was not influenced by
the presence of a familiar like-sex cage mate, and the degree of
affiliation displayed by the companions in the home cage did
not influence the response to a novel environment. Similarly,
disruption of established heterosexual relationships does not
induce behavioral or physiological indications of stress
(Mendoza and Mason, 1986a).
Similar studies in titi monkeys, some of which were per-
formed in direct comparison with squirrel monkeys, show
quite different patterns of responses to separation from group
members. Adult males and females living in established hetero-
sexual pairs respond to separation from their mate with a rapid
and substantial increase in HPA activity (Mendoza and Mason,
1986a,b). When parental response to separation was tested,
neither titi monkey mothers nor fathers responded to removal
of their infant. Disturbance control procedures are not different
from basal values of cortisol and for parents not different than
when separated from their infant. Infants respond to separation
from their fathers even with their mother present; when sepa-
rated from both parents (remaining alone in the home cage),
cortisol levels for infant titi monkeys reach their highest levels
(Hoffman et al., 1996). For infants, removal of the mother does
not elicit a separation response as long as the father is present.
Titi monkeys continue to respond to separation from their
fathers into adulthood and do so with prolonged activation
of the HPA axis (Mendoza et al., 2000). The response to sepa-
ration was compared in infant (6–12 months of age), juvenile
(1–2 years of age), and adult offspring (2–3 years of age).
Cortisol concentrations were determined from blood samples
collected on day 1 and day 5 of separation from the parents.
The results from the separation condition were compared to
a separation–reunion condition in which the parents were
separated from their offspring for 1–2 h the day before the first
blood sample was collected. All offspring responded to separa-
tion from their parents with an increase in plasma cortisol
levels, even though all were tested in the familiar home cage
and 20 of 21 subjects were housed with siblings throughout
the separation period. The separation response was sustained
through the 5-day period without attenuation. In addition to
demonstrating that the response to parental separation
continues into adulthood, these data importantly show that
stress buffering for titi monkeys is provided only by parents,
and together with the results from the previous studies only
effectively by the father.
Further extensions of the separation response in adult titi
monkeys were performed in order to determine if longer time-
frames would lead to an attenuated response and if reduced
cortisol would be reinstated after a prolonged absence from
the family group (Mendoza et al., 2000). Adult daughters
were removed from their groups and placed in a new cage
(identical to the home cage) in the same colony room for
30 days and then reunited with their families and monitored
for the ensuing 30 days. Blood samples were collected at weekly
intervals in both phases beginning 24 h following separation
and 4 h (with the second sample collected at 48 h) following
reunion. Plasma cortisol was elevated 24 h following separa-
tion compared to basal samples collected in the previous
week and remained at high levels throughout the month long
separation period. Reunion with the parents led to return to
the preseparation levels within 48 h.
275
Surprisingly, the only time we have found the response to
parental separation in young adult titi monkeys truncated by
anything other than reunion with the father was if they were
provided a potential mate (Mendoza et al., 2000). In the
process of forming 10 new pairs, most of the new partners
(six males and seven females) were removed from their natal
groups and hence experienced separation from their fathers
(and other family members) immediately prior to pair forma-
tion. Blood samples were collected twice per week for 3 weeks
before and 4 weeks following new pair formation. Cortisol
levels were elevated 24 h following pair formation but there-
after returned to preseparation levels. The response to the
formation of new pairs was the same for monkeys who were
removed from their natal groups and for monkeys who were
not currently living with either a mate or their parents at the
time of pair formation. We do not know from these studies
when titi monkeys would begin to exhibit a separation
response from their new mate, but clearly a new mate, even
though a stranger, is capable of buffering the potent parental
separation response. Further underscoring the unique response
of the young adult titis to pair formation, is the finding that for
animals living in established pairs provision of a potential
mate does not ameliorate the response to separation from
the actual mate and may even augment it (Cubicciotti and
Mason, 1975).
There is no indication that the persistent elevations in
cortisol pose a health risk in titi monkeys. It is likely that the
sustained cortisol in response to separation facilitates the
motoric output deemed necessary to find the object of attach-
ment or a replacement. Titi monkeys are one of the truly
monogamous primate species. It is likely that the unique
response to social separation supports their equally unique
social system. Other species for which paternal behavior is
common occasionally have nonrelated adults or adult
offspring contribute to infant care, neither of which are avail-
able for titi monkeys (Fernandez-Duque et al., 2009; Van Belle
et al., 2016).
Separation studies have been a critical element in
categorizing relationships as attachment bonds or friendships
(Hennessy, 1997; Mendoza et al., 1980; Mendoza, 1991). In
addition to evidence of agitation upon separation, recognition
of and selective preference for the object of attachment, prox-
imity seeking, and stress buffering which is expressed behavior-
ally by using the object of attachment as a secure base are all
important elements of the attachment relationship in human
and nonhuman primates (Ainsworth, 1972). In squirrel and
titi monkeys several relationships meet the criteria for attach-
ment. These include the mother–infant relationship in squirrel
monkeys (bidirectional); the pair bond of adult titi monkeys
(bidirectional); and the relationship of young titi monkeys to
the father (unidirectional). Relationships that are affiliative
behaviorally but do not meet the criteria of an attachment
bond include female–female relationships in squirrel
monkeys; male–male relationships in squirrel monkeys;
sibling relationships in titi monkeys; and titi mother’s and
father’s relationship with their offspring of any age. All other
dyadic potential relationships in both species are not generally
affiliative and/or do not occur with any great frequency and
thus cannot be categorized as either friendships or attachment
bonds.
276 Social Stress: Concepts, Assumptions, and Animal Models
The separation studies of these species have helped to clarify
the relational aspects of social organization and their possible
functional consequences. Titi parents are never confronted
with a situation in which their baby can be mixed up with
another baby, whereas this is a daily occurrence for squirrel
monkey mothers particularly once the infants begin to leave
mother and interact in playgroups. The bidirectional attach-
ment relationship in squirrel monkeys may facilitate a mother’s
reunion with her own infant in sometimes chaotic group activ-
ities during travel and foraging. If visual contact is retained,
then agitated behavior on the part of either mother or infant
is sufficient to signal to the other that it is time for reunion.
If visual contact is broken, the activation of the stress response
would provide the necessary metabolic resources to initiate
a search and find operation in both mother and infant. Short
term, the equivalent response in both mother and infant would
be considered adaptive, but long term the mother should
consider cutting her losses, whereas the infant needs the
mother for survival for some time following nutritional inde-
pendence and must sustain efforts to reestablish contact.
For titi monkeys, all group members form an attachment
bond with the father. It is the seeking of proximity with the
father by all group members and the father’s attraction to the
mother that accounts for remarkable group unity characteristic
of titis. It is common to see all group members sitting in huddle
groups with tails entwined with the father at the center, or
maintaining close proximity during travel and foraging. The
infant is not a particularly cherished member of the group.
The father is tolerant of infant contact but other group
members are not. Except for short periods surrounding nursing
most titi mothers are intolerant of prolonged infant contact
and attempts by the infant to maintain contact with her or
even brief contact with siblings are met with active efforts to
dislodge the infant. Indeed, when the father is carrying the
infant all other family members stay a little further away
from him (Van Belle et al., 2016).
1.09.5.2 Comparative Case Study: Voles
Another monogamous species that has received a lot of atten-
tion in physiological studies of social stress is the prairie vole.
Many studies have been done comparing this species with its
very closely related cousin species the nonmonogamous
montane vole. In nature, a variety of behavioral mechanisms
facilitate monogamy in the prairie vole. Females are selectively
aggressive toward strange females, sexually naïve males are
nonaggressive unless they are met with aggression, and sexually
experienced males are highly aggressive to unfamiliar animals
(Carter et al., 1995). This means that females will not associate
readily in multifemale groups and experienced males actively
exclude any strangers. Forming new pairs is more likely to occur
with a naïve male since experienced males are generally too
aggressive even toward potential mates. In the laboratory,
studies of pair bond formation are tested by allowing a period
of cohabitation between a male and a female who are repro-
ductively naïve and without prior pairing experience. Testing
proceeds with the experimental animal allowed to choose
between three chambers – an empty middle chamber and
two side chambers with tethered stimulus animals. One
chamber contains their now familiar cohabitation partner
and the other chamber contains unfamiliar potential mate.
Pair bonding is operationally defined by spending more time
in contact and proximity to the familiar stimulus animal.
Females typically require 3 h of cohabitation to show a clear
partner preference, whereas males require longer (up to
24 h). In addition to partner preference, the pair bonding expe-
rience also leads to increased aggression directed to strange
animals (potential intruders) (Carter et al., 1995).
The main focus of this research has been the roles of
oxytocin and vasopressin in the formation of pair bonds, oper-
ationally defined as selective preference for partner over other
potential mates. Generally, vasopressin facilitates development
of partner preference in males and oxytocin in females (Devries
et al., 2007). Centrally, oxytocin and vasopressin are believed
to act via dopaminergic reward system to promote social inter-
action and recognition of mate as mate (DeVries et al., 2007).
The powerful role these peptides can play in partner preference
is illustrated by a study comparing the effects of centrally
administered oxytocin and vasopressin on partner preference
formation and social contact in male and female prairie voles
(Cho et al., 1999). After only 1 h of cohabitation and pretreat-
ment with either AVP or OT, both males and females exhibited
increased social contact and significant preference for the
familiar partner. If either oxytocin receptors or vasopressin
receptors were blocked, partner preferences were not evident
in either males or females and neither oxytocin nor vasopressin
was effective in eliciting partner preference. Thus, both receptor
populations must be available for pair preference to develop,
but either of these closely related peptides is effective in elicit-
ing pair formation. Sex differences in the roles of oxytocin and
vasopressin in pair bond formation are clear, and their interac-
tions with HPA may help to understand differing impact of
HPA activity in regulating the social system of prairie voles.
Vasopressin stimulates release of ACTH thus potentially aug-
menting release of corticosteroids, whereas oxytocin tends to
suppress ACTH and corticosteroids and these conditions
influence pair bonding in a sex-specific fashion (DeVries
et al., 2007).
Like titi monkeys, monogamous prairie voles exhibit
responses to separation and reunion that are indicative of an
emotional bond or attachment. Following 2 weeks of cohabita-
tion, ovariectomized adult females and intact males were sepa-
rated for 24 h and then either left alone, reunited with familiar
mate for 30 min, or placed with a stranger for 30 min. A sepa-
ration of 24 h leads to elevated corticosterone levels. Reunion
with the mate leads to reductions in corticosterone, below
basal values for females, and exposure to an unfamiliar partner
instead of the mate during the reunion period results in
elevated levels of corticosterone (Carter et al., 1995). Socially
inexperienced males exposed to a strange naïve female and
males that had been living in a pair bond had exactly the
same response to a novel female, suggesting that for males
the reduction in cortisol with pairing is not selective or at least
does not change the male’s subsequent response to another
female. Males exposed to another male in the same testing
paradigm failed to reduce adrenocortical responses, so the buff-
ering response is to a category – female rather than to a specific
female (DeVries et al., 1997). Similarly, females living with
a female sibling also respond to isolation with increased corti-
costerone and reunion with the cage mate leads to reduction of
 Social Stress: Concepts, Assumptions, and Animal Models
corticosterone. For female siblings the reduction in corticoste-
rone is also apparent when just the bedding the sibling has
used is returned. This may be a specific response to a specific
individual, but it certainly indicates that, at least for siblings,
contact is not required. Similar findings for peer effectiveness
in reducing the response to social isolation has been found
in sheep with exposure to simply the face of a familiar partner
(da Costa et al., 2004).
Exogenous administration of corticosteroids to females
immediately before the cohabitation period leads to an
inability to bond with the familiar partner and is reported to
actually induce preference for the stranger (Carter et al.,
1995; DeVries et al., 1996). Similarly, if females are exposed
to a stressor, such as forced swim test, immediately before
cohabitation with a naïve male, pair bonding does not happen.
Adrenalectomy leads to greater efficiency in forming partner
preference and, without corticosterone, females require much
less time in the cohabitation phase to induce partner prefer-
ence. Timing of corticosterone effects is critical. Administration
of corticosterone following cohabitation but before testing is
not disruptive of partner preference formation in females. In
males, the effects of corticosterone treatment and exposure to
a stressor were opposite to that in females. Artificially elevating
corticosterone prior to the cohabitation phase facilitates forma-
tion of partner preference, whereas adrenalectomy abolished
partner preference formation even with extended periods of
cohabitation.
Autonomic activity using HR as the signal also shows signif-
icant change with social isolation. In the first study of auto-
nomic balance, female prairie voles living with like-sex
siblings exhibited a predominant vagal tone (Grippo et al.,
2007a). Pharmacologically blocking the PNS significantly
increased HR, reduced HR variability, and reduced the ampli-
tude of respiratory sinus arrhythmia. Pharmacologically block-
ing the sympathetic input to the heart significantly reduced HR.
Combined PNS and SNS blockade significantly increased HR,
and reduced HR variability and respiratory sinus arrhythmia
amplitude. Placing young naïve females in social isolation for
4 weeks reverses autonomic tone from predominantly para-
sympathetic to predominantly sympathetic; HR increases and
HR variability decreases (Grippo et al., 2007b). In another
study, adult female prairie voles were exposed to social isola-
tion or continued pairing with a female sibling for 4 weeks.
During weeks 3 and 4 of this period, animals were adminis-
tered oxytocin daily for a total of 14 days. As in the previous
experiment, isolation significantly increased basal HR and
reduced HR variability and vagal regulation of the heart; these
changes in isolated animals were prevented with oxytocin
administration (Grippo et al., 2009).
Disruption of an established bond can also lead to altered
regulation of HR in male prairie voles (McNeal et al., 2014).
This study monitored males living initially with siblings then
formed into new pairs for 5 days followed by removal of the
female and the males subjected to at least 8 days of social isola-
tion. Moving from living with siblings to formation of new
pairs did not produce a change in HR. Isolation resulted in
increased HR largely due to enhanced sympathetic input to
the heart as previously shown for females. Recently, social
isolation has also been shown to result in impaired immune
functions as indicated by reduced hemolytic complement and
277
bacteria killing ability in both males and females (Scotti
et al., 2015).
There are many parallels in the titi monkey and prairie voles
related to their monogamous lifestyle. Research with the prairie
voles focuses on pair bonding, and research with the titi
monkey has focused more on stable family units. Nonetheless
parallels exist including suggestions that prairie voles, like titi
monkeys, develop an attachment bond between adults.
Imaging studies in titi monkeys and more invasive studies of
brain circuits important to pair bond formation in prairie voles
indicate that a neural circuit beginning with olfactory input and
involving the dopaminergic reward circuit (ventral pallidum,
nucleus accumbens, ventral tegmental area) and the social
recognition circuits in the medial amygdala and lateral septum
are critically involved in pair bonding (Bales et al., 2007; Young
et al., 2005).
1.09.5.3 Comparative Case Study: Guinea Pigs
Guinea pigs are perhaps the most studied rodent from the
perspective of social separation and stress buffering for any
polygynous rodent. When housed in large heterosexual groups
with multiple animals of each age and sex categories, guinea
pigs form harem groups consisting of one male and several
females. Adult males exposed to a novel environment exhibit
reduced cortisol response to the procedure if also placed with
an adult female from his harem, but not a familiar female
outside the harem or an unfamiliar female (Sachser et al.,
1998). Similarly, the male buffers the female’s response to
novelty. For females, however, the response to novelty is best
attenuated by the harem male, but other familiar males and
familiar females also provide a modicum of buffering the
HPA response (Hennessy et al., 2008; Kaiser et al., 2003).
Guinea pig mothers left in the home cage when their litters
were removed showed an increase in cortisol (Ritchey and Hen-
nessy, 1987); however, like squirrel monkeys (see above), if
pups were within auditory range, mothers do not exhibit an
HPA response. In a novel environment mothers are capable
of buffering the response to novelty for their infants, but
another adult female and siblings are not effective in reducing
HPA response to stressful circumstances (Graves and Hennessy,
2000; Ritchey and Hennessy, 1987; Sachser et al., 1998). Infant
guinea pigs allowed to stay in their familiar environment (in
this case a large mixed sex group), unlike squirrel monkeys,
show activity and vocal behaviors indicative of distress, but
do not show heightened pituitary–adrenal activity. Infant
guinea pigs do not show the full stress buffering effect if the
mother and infant are separated by wire mesh (and cannot
make full contact), and HPA responses are greatest for the
infant when confronting the novel environment completely
alone (Hennessy, 1988).
For young guinea pigs raised with their mother and siblings,
the mother continues to be a source of stress buffering for her
offspring still living in the natal nest into adulthood. However,
the ability to provide stress buffering is no longer selective, and
for offspring beyond the infancy period other adult females are
also capable of buffering the response to novelty (Graves and
Hennessy, 2000; Hennessy et al., 2000). This picture is some-
what more complicated if young guinea pigs are colony raised
and, hence, have a multitude of social partners to select or
278 Social Stress: Concepts, Assumptions, and Animal Models
avoid in interactions during development. One obvious differ-
ence is that males can now choose to interact with females
other than the mother and may even begin to prefer to do so.
Studies of colony-raised guinea pigs reveal interesting
changes that occur surrounding achievement of sexual and
social maturity which in guinea pigs, like humans, occurs
somewhat later than reproductive maturity (Hennessy et al.,
2006). Animals in this study were tested with their favored
female social partner, a strange female, or alone during a 4 h
exposure to a novel environment. Infant guinea pigs’ favored
adult female was always the mother and, when with her, they
showed reduced adrenocortical response novelty. When alone
or with an unfamiliar female, cortisol levels were higher than
with the mother and were sustained for the full 4 h, whereas,
when with the mother, cortisol levels returned to basal values
by 4 h. Beyond the period of infancy none of the males showed
an HPA response that persisted through the 4-h period. This is
not likely to reflect maturation of negative feedback mecha-
nisms, since infants with mothers are capable of terminating
the HPA response within the 4-h period. As juveniles, some
males (1/3) still preferred their mothers, while others prefer
to interact with another adult female. The preferred females,
however, are not effective in buffering juvenile males’ response
to a novel environment; instead, unfamiliar females provide
a modicum of stress buffering. At sexual maturity all males
shifted preference from mother to other females. Sexually
mature (but not socially mature) males appear to go through
a stress hyporesponsive period. For these males, cortisol levels
remained low throughout the testing period, rising only
slightly above basal levels at 1 h; by 4 h levels were below basal
values. Cortisol levels for sexually mature males were not influ-
enced by either favored or unfamiliar adult females. With social
maturity the reduced response to novelty was retained when
novelty was encountered with a favored female. When alone
response to novelty was as high at 60 min as for juvenile males,
but for socially mature males cortisol levels returned to base
more quickly. The response to unfamiliar females for socially
mature males was intermediate and not clearly different
from the response to novelty when either alone or with
a favored female.
The distinctive response profile shown by sexually, but not
yet socially, mature guinea pigs comes at a time in the male
guinea pig’s life during which it might leave the natal colony
altogether (Hennessy et al., 2006). The lack of HPA responsive-
ness to novel environments may induce a tendency to range
further from the nest in sexually mature guinea pigs than would
be the case for younger or even older guinea pigs. Adolescence
is also a critical period in determining how a male guinea pig
will behave and react to social challenge later in life (Sachser
et al., 2013). In laboratory studies of guinea pigs, males that
experience adolescence in large colonies apparently learn social
skills associated with formation of stable hierarchies and can
integrate themselves in new social groups with relatively little
aggression and in the absence of stress profiles sometimes asso-
ciated with subordination. For males that live with only a single
female from early adolescence, interactions with other males
become increasingly aggressive and long-term associations
difficult. Stress responsiveness to novelty in males living in
pairs is also greater than that in colony-raised males. Moreover,
these profiles can be shifted in the expected direction by
providing pair-living males with extra-social interactions
during the early stage of adolescence. In wild cavies, the
ancestor species for the guinea pig, comparisons of adolescent
males from high and low population densities show similar
behavioral differences between adolescents reared in pairs
and those reared in large colonies. These findings suggest that
the shifting male responsiveness, both with respect to behavior
and the organization of physiological processes, occurs in
response to the density of population and the shifting resources
populations rely upon.
1.09.5.4 Social Isolation in Humans
Separation and stress buffering studies in animals have
provided important insights in human stress and vulnerability
to disease. The most striking example is bereavement (Stroebe
et al., 2007). Although details vary from study to study, the
overwhelming result of the research to date suggests that
following bereavement the surviving spouse is at greater risk
of mortality. Excess mortality is greatest in early months
following bereavement and decreases with duration of bereave-
ment. Bereaved individuals are more likely to have new or
worsened illness, increased pain, and weight loss and corre-
spondingly higher rates of disability, medication use, and
hospitalization. Heightened ACTH levels and resistance to
dexamethasone suppression indicating impaired feedback
mechanisms have been found for bereaved individuals,
although cortisol did not differ except following treatment
with dexamethasone (Gerra et al., 2003).
There are many changes that occur in the immediate after-
math of bereavement, and it is no surprise that physical and
psychological processes need time to fully adapt. For our
purposes it is difficult to compare the human response to
sudden separation from spouse by death with the findings
from animals simply because of the number of changes and
the extent of differences in the extended social circumstances
within which the bereaved individual is embedded. The clarity
of animal studies is suggestive that mechanisms involved with
isolation are similar to those involved in bereavement
(Karelina and DeVries, 2011), but we cannot with certainty
suggest the mechanisms of social isolation and bereavement
are the same. It is almost certainly the case, however, that
studies of humans during bereavement or otherwise are not
studying the complete social isolation typical of animal studies.
In research on social isolation in humans the social network
size is operationalized as indicating the degree of social isola-
tion. This, in turn, is associated with greater awakening
response to cortisol and higher cortisol at the trough of the
cycle which is accompanied by insensitivity of glucocorticoid
receptor to cortisol if isolation is prolonged. Together with
enhanced SNS activity, the physiological changes associated
with relative social isolation in humans create a proinflamma-
tory immune profile with attendant risks for inflammatory
disease (Hawkley et al., 2012).
As Cacioppo et al. (2015) note a given type of relationship
may be supportive or dismissive, protective or threatening, and
the perception of the relationship by its participants is likely
a determinant of the physiological consequences of engaging
or disrupting the relationship. Moreover, they argue that the
number of others in the network or frequency of interactions
 Social Stress: Concepts, Assumptions, and Animal Models
with others is not as important as is the perception of loneli-
ness. Indeed, individual differences in the degree to which indi-
viduals seek social stimulation are not considered a risk factor,
but if an individual’s social environment or interaction
network does not provide the level of stimulation desired,
loneliness ensues with attendant risks to health. Health risks
associated with loneliness cannot be explained by simple avail-
ability of family and friends or by changes imposed by others in
one’s network to improve healthful behaviors. Thus, loneliness
is a risk factor for morbidity and mortality beyond that which
can be ascribed to isolation, which in humans is virtually never
complete in any event.
Studies of the physiological processes attendant to loneli-
ness are just beginning. Early results indicate that both SNS
and HPA systems are apparently responsive to perceived lone-
liness (Cacioppo et al., 2015). Enhanced sympathetic activity
particularly in tissue, elevated daily cortisol output, and
decreased glucocorticoid receptor sensitivity have all been
found to be associated with loneliness. Moreover, loneliness
may increase negative evaluations of social potential – others
are perceived as more dangerous and less supportive. These
authors argue that the psychological dampening of perceived
benefits of social interaction prevents stress buffering mecha-
nisms. These findings have led to the conclusion that animal
studies of repeated social threat are a better model for the
neuroendocrine changes and the immunological consequences
than are animal studies of separation or isolation.
1.09.6 Conclusions
In this chapter we have concentrated on presenting several
influential attempts to study social stress largely using animal
models. To evaluate the efficacy of the models two major
criteria should be considered. First is the extent to which the
research illuminates aspects of the social biology of the species
investigated. Does the paradigm permit natural expression of
social proclivities? As a result of the research do we come to
a better understanding about the animals being investigated?
Second, the justification for conducting most social stress
research is its translation potential. How aptly do the studies
capture aspects of human biology? How might understanding
sociophysiological processes in animals contribute to under-
standing vulnerability to disease processes in humans and
inform medical practice?
Starting with the notion that aggression is the key behavior
mediating negative consequences of sociality, studies were
initiated, largely with rodents to explore the physiological
underpinnings of aggression. The resident/intruder paradigm
was central to early explorations. This approach has the virtue
of being able to describe stress onset and follow physiological
changes throughout the ensuing response. The defeated animal
experiences a substantial acute stress response that is compa-
rable to the response to other stressors in form and temporal
sequence and does not habituate (Bhatnagar and Vining,
2003; Sgoifo et al., 2005). As a translational tool to study the
acute stress response the resident/intruder model has much
to recommend. However, the resident/intruder model does
not fulfill its promise as a means of studying social stress.
With the possible exception of maternal aggression displayed
279
by mothers protecting their nest, the resident/intruder test
does not model an aspect of the natural biology of most
rodents. Aggressive encounters in colonies of mice and rats
generally occur at the periphery rather than the core of the terri-
tory, or in neutral foraging areas. Therefore, the advantage of
the resident is likely to result in flight of the intruder rather
than inescapable conflict. Moreover, even in the circumstances
of the resident/intruder tests, considerable care must be taken
to ensure expression of aggression in the test situation. Resi-
dents are often selected to be larger, more aggressive, spend
prolonged periods in isolation, and even teased to elicit the
attack behavior of the resident. Social defeat might happen in
nature, but it would be rare. Thus, rather than modeling sources
of normative social stress, the resident/intruder model may be
more likened to an unusual traumatic experience outside the
purview of ordinary social experience. Modification of the resi-
dent/intruder model has proven useful to identify a shift in
aggressiveness or attraction directed to an unfamiliar animal.
This is illustrated, for example, in the shift of voles’ response
to unfamiliar animals from nonaggressive to more aggressive
as a consequence of mate formation.
Prolonged periods of aggressive and more ritualized forms
of agonistic behavior are seen in newly formed colonies of
mice and rats. Particularly informative of behavior in natural
social situations are models such as the visible burrow system
(Blanchard and Blanchard, 1990) or other means of providing
a laboratory simulation of burrowing and foraging typical of
mice and rats. In these circumstances, normally socialized
rats and mice form groups in which one male is dominant
and the others submissive. Submissive animals are vulnerable
and typically experience greater and earlier mortality than
dominants. Altered regulation of SNS and HPA can accompany
formation of dominance relations perhaps contributing to
mortality. When colonies are allowed to stabilize, the physio-
logical differences are minimal, but believed to reemerge
when stressors are encountered (Henry and Stephens, 1977).
It is important to note that the dominant animal can also be
viewed as more vulnerable to predation by virtue of his status.
Dominant mice and rats tend to be hypervigilant and more
often exposed to the risk of predation (Blanchard and
Blanchard, 1989). While dominance/subordination in groups
of mice and rats have great potential for understanding social
influences on physiological regulation, their generality to
processes in common with human and nonhuman primates
must be treated cautiously. Establishing dominance/submis-
sive relations in mice and rats are generally dyadic in contrast
to most primates for which linear dominance hierarchies are
formed implying at a minimum a triadic process (Chase,
1984). Also in contrast to large group-living primates, agonistic
encounters are prolonged when new colonies of mice and rats
are formed and include a fair amount of contact aggression and
wounding. Status relations in mice and rats take days and
weeks to be evident, whereas, for primates formation of domi-
nance relations in comparable circumstances does not include
prolonged periods of agonistic interactions; dominance rela-
tions are settled within minutes and typically without fighting.
Sapolsky’s studies of feral groups of baboons provide a truly
excellent model for engaging in studies of social stress. His
combination of behavioral observation of a natural population
and physiological probes is unique in animal studies of social
280 Social Stress: Concepts, Assumptions, and Animal Models
stress. Clearly his work has enriched our understanding of both
social processes in baboons and endocrine physiology.
A particularly important outcome of his research is the clear
finding that physiological responses to social dominance can
be altered by group level processes. A concern with this research
is the extent to which generalizations beyond baboons is advis-
able. The nature of physiological processes associated with
dominance is not the same in baboons as in other species
and often not even in the same direction. Squirrel monkeys,
for example, show status differentiation of stress response
systems only when instability and breeding coincide; when it
does appear, status differentiation is opposite to that found
in baboons. Thus, while the approach Sapolsky used is an
ideal approach, the results are at present best considered
species specific.
The focus on aggression and dominance relations as key to
understanding social stress must also be questioned. The
studies of group formations in primates indicate that domi-
nance relationships are an effective means of limiting aggres-
sion and not a manifestation of unregulated aggression. Is
the subordinate’s lot in life as bad as often depicted? Almost
certainly not. Moreover, in most primate societies, duration
in a particular status is delimited by processes of emigration
and immigration. Dominance relations provide an easy means
of role differentiation and enhanced predictability particularly
during periods of instability, but a subordinate role is not a life-
time sentence. One of the problems with the research on domi-
nance and aggression is that it is only one aspect of social life,
particularly in primates. Clear testament to the myopia in this
research is that females, often considered the cohesive force in
primate societies, are completely neglected in research on
aggression and dominance.
Studies of social instability provide promising avenues for
examining the impact of group level processes. Work by Capi-
tanio and colleagues have shown clear linkages between stress-
response systems, immune processes, and disease progression.
In addition to providing another excellent example of pursuit
of understanding social processes, behavior, and physiology
in a disease context, these studies offer caution in the assump-
tions that we bring to studies of social stress. SNS processes play
an important role in disease progression but seem to do so
independently of circulating levels of Epi and NE. This means
that correlational studies of social processes and activity in
SNS are in danger of being misleading rather than informative.
Similarly, rhesus monkeys and squirrel monkeys respond to
formation of new social groups with an initial reduction in
cortisol and evidence of dysregulation of HPA by excessive
negative feedback. Rather than conferring advantage, reduced
cortisol is associated with more rapid disease course through
as yet unidentified mechanisms. The clear message from these
findings is cortisol levels cannot be equated with stress as is
so often the case. Lower than optimal levels of cortisol can be
disadvantageous and even on occasion lethal (Heim et al.,
2000). A more general cautionary note these studies provide
is that assumptions regarding disease processes and vulnerabil-
ities based on correlational studies should be considered a start-
ing point rather than a conclusion in studies of social stress.
In fairness, it should be noted that group formation studies
are also a laboratory artifact. It is only in the lab that new groups
are ever formed de novo with all animals unfamiliar to one
another initially. Whether the instability induced by the forma-
tion of new laboratory groups, models spontaneous occurrence
of periods of instability in established or feral groups is debat-
able. Unfortunately, we do not have the methods to describe
relative degrees of stability or instability in large groups other
than spontaneous outbreaks of aggression which may or may
not be sensitive measures of cohesion or the lack thereof.
If dominance relations are one aspect of sociality important
to understanding social stress, availability of emotional bonds
is another that has received a great deal of research attention.
Some relationships can be characterized by an attachment
bond. Filial bonds are the most commonly studied relation-
ship of this type and dependent offspring usually, but not
always, form a strong and abiding emotional bond with the
mother. Disruption of the bond induces an acute stress
response and engagement in the bond can be stress buffering
(Hennessy, 1997). Considerable insight into the characteristic
social structure can be provided by studies that explore the
extent to which relationships conform to the requirements
of an attachment bond as delineated by Bowlby (1969),
Ainsworth (1972), and others. Our understanding of the social
systems of voles, guinea pigs, and titi monkeys have benefitted
from application of concepts developed to understand the
filial bond in humans – a valuable instance where transla-
tional research occurs in the reverse direction.
In the last decade we have improved our technical ability to
pursue the physiological and neurobiological mechanisms
necessary to link processes originating in the social environ-
ment to health and disease. If we are to improve our
approaches to social stress, better behavioral tools are required.
In particular, objective measures of cohesion and stability in
groups, the rules that govern individual behavior within
society, and the degree of embeddedness of an individual
within its society are needed. Recent studies of loneliness
suggest the perception of loneliness is a risk factor but the
number of family and friends is not (Cacioppo et al., 2015).
Modeling loneliness or other aspects of human sociality is
not currently possible, but promising methods are being devel-
oped (McCowan et al., 2011) which link individual proclivities
and experience with group processes.
To appropriately model human social stress with animal
studies, we also need to be clear that the processes we study
in animals are relevant to human societies. As we have seen
social structure is species specific. More concern needs to be
paid to what human society means to humans. The attach-
ment processes are probably the human relationship best
modeled in animal research, and animal studies have helped
to generalize processes involved in filial attachment to other
primate relationships including adult–adult emotional bonds
(Mason and Mendoza, 1998). The extension of attachment
processes in humans to health issues currently includes adoles-
cent and adult romantic attachment and parental behavior in
addition to the more commonly considered filial attachment
as a unique relational system (Mikulincer and Shaver, 2012).
This work parallels work on monogamous species, such as titis
or voles, which also show adult emotional bonds and similar
neurobiological mechanisms. This suggests humans are
monogamous. But unlike titi monkeys or voles, human
monogamy is embedded in a social nexus that more closely
resembles the complex societies exhibited by baboons or
 Social Stress: Concepts, Assumptions, and Animal Models
macaques. At present we do not have adequate and similar
tools for comparing human and nonhuman societies and
these need to be developed if truly translational research on
social stress is to be implemented.
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 1.10 Communication by Chemical Signals: Physiological Mechanisms,
Ontogeny and Learning, Function, Evolution, and Cognition
MH Ferkin, University of Memphis, Memphis, TN, USA
J delBarco-Trillo, Liverpool John Moores University, Liverpool, UK
A Petrulis, Georgia State University, Atlanta, GA, USA
Ó 2017 Elsevier Inc. All rights reserved.
This chapter is a revision of the previous edition chapter by R.E. Johnston, J. delBarco-Trillo, volume 1, pp. 395–441, Ó 2009, Elsevier Inc.

1.10.1 Introduction 286

1.10.1.1 Brief Overview of Odor Cues and Scent Marking 286
1.10.1.1.1 Sources of Scents and Odors 287
1.10.1.1.2 The Attractiveness of Scent Marks and Conspecific Odor Preferences 287
1.10.2 Physiological Mechanisms 289
1.10.2.1 Neural Substrates 289
1.10.2.2 Mechanisms of Communication 289
1.10.2.2.1 Scent Marking 290
1.10.2.2.2 Ultrasonic Vocalizations 292
1.10.3 Development and Learning 293
1.10.3.1 Reproductive States and Odors 293
1.10.3.2 Individual Discrimination 294
1.10.3.2.1 Habituation
Dishabituation 295
1.10.3.2.2 Two Primary Polymorphic and Multigenic Complexes 296
1.10.3.3 Kin Recognition 297
1.10.3.3.1 Mechanisms of Kin Recognition 297
1.10.3.4 Group Discrimination 298
1.10.3.5 Species Discrimination: Competitors and Predators 298
1.10.3.6 Sex Identification 299
1.10.3.6.1 Endocrine Effects 300
1.10.3.7 Social Effects 300
1.10.3.7.1 Role of Odors in Social Dominance and Aggression 300
1.10.3.7.2 Food and Odors: Diet and Food Deprivation 302
1.10.3.7.3 Effect of Health and Environmental Stressors on Odors 303
1.10.3.7.4 Age and Odors: From Infancy to Senescence 303
1.10.4 Functions of Odor Communication 304
1.10.4.1 Scent Marking 304
1.10.4.1.1 Hypotheses about Function 304
1.10.4.2 Over-marking 306
1.10.4.2.1 Top- and Bottom-Scent Donors Are Same-Sex Conspecifics 306
1.10.4.2.2 Mixed-Sex Over-marks and the Effects of Familiarity 307
1.10.4.2.3 Ten Hypotheses about the Function of Over-marks 309
1.10.4.3 An Over-mark versus a Single Scent Mark 310
1.10.4.4 Self-Grooming as a Form of Scent Marking 311
1.10.4.4.1 Mate Attraction 311
1.10.4.5 Scent Marks Provide Public Information 311
1.10.5 Evolution 312
1.10.5.1 Basic Concepts 312
1.10.5.2 Comparative Approaches to the Study of Olfactory Communication 312
1.10.5.3 Genetic Variation, Natural Selection, and Odors 313
1.10.5.4 Odors and Sexual Selection 313
1.10.6 Cognition and the Assessment of Scent Marks 314
1.10.6.1 Processing Scent Marks 315
1.10.6.2 Cognitive Ability, Numerosity, and Scent Marks 315
1.10.7 Concluding Thoughts 316
Acknowledgments 316
References 316

Hormones, Brain, and Behavior, 3rd edition, Volume 1 http://dx.doi.org/10.1016/B978-0-12-803592-4.00010-9 285

286 Communication by Chemical Signals
1.10.1 Introduction
This chapter focuses on scent marking and odor communica-
tion. We provide examples of odor communication in terres-
trial vertebrates but focus most of our efforts on mammals,
the class of vertebrates most familiar to the authors. Such an
endeavor was previously carried out in a text by Brown and
Macdonald (1985) and by Wyatt (2014). Because of space limi-
tations in this chapter our focus will be less comprehensive.
However, much of the chapter will focus on scent marking
and, to a lesser extent, on other forms of olfactory communica-
tion such as self-grooming in terrestrial mammals, which are
excellent subjects for the study of such topics. This is because
olfaction is a major sensory modality for terrestrial mammals,
most of which display scent marking (Biben, 1980; Brashares
and Arcese, 1999; Drea, 2015; Ferkin, 2015; Hurst, 1990a,b,c;
Heymann, 1998; Johnson, 1973; Johnston and delBarco-
Trillo, 2009; Klailova and Lee, 2014; Lewis, 2005; Macdonald,
1980; McClintock, 2002; Sliwa and Richardson, 1998).
To understand odor communication in terrestrial
mammals, it is necessary to differentiate between studies
that examine proximate causation and ultimate consequences
of scent marking and then integrate the findings of these
disparate studies into a comprehensive picture of this
behavior. In this chapter, our approach is to discuss advances
and controversies in odor communication among terrestrial
mammals. Our approach is based on Tinbergen’s (1963)
four levels of behavioral analysis. Specifically, we categorized
and discussed studies that deal with proximate causation,
such as the physiological mechanisms that underlie and
mediate odor communication and the role of ontogeny and
learning in the development of odor communication. We
also identify and discuss studies that pertain to ultimate
consequences, such as the function and adaptive significance
of odor communication, the benefits and costs of scent
marking in terms of survival and fitness, and its evolution in
terrestrial mammals. We also discuss the role of cognition in
odor communication. We selected papers that are recent
and provide new information and detail and also ‘classics,’
many of which provided the initial hypotheses and theoretical
framework for current researchers studying odor communica-
tion in terrestrial mammals.
1.10.1.1 Brief Overview of Odor Cues and Scent Marking
In most terrestrial mammals scent marks are typically depos-
ited on prominent objects or along paths that are shared with
conspecifics (Brown and Macdonald, 1985; Clapham et al.,
2013; Sharpe, 2015). Therefore, individuals will enter areas
that contain single scent marks and overlapping scent marks
(over-marks) which are comprised of scent marks from two
or more conspecifics (Ferkin et al., 2011a,b). These scent
marks may be a signal that provides particular information
about the donors to individuals that encounter such marks.
These, in turn, may affect how the receivers respond to the
donors (Wyatt, 2014). Typically, individuals spend more
time investigating the scent marks of opposite-sex conspe-
cifics than of same-sex conspecifics (Ferkin and Seamon,
1987). The response of individuals to scent marks as well
as the location where individuals deposit their own scent
marks may affect subsequent interactions with conspecifics.
Presumably, individuals may spend more time investigating
the scent marks of more attractive than of less attractive
opposite-sex conspecifics. Similarly, individuals in better
physical condition may scent mark and over-mark more
often than would individuals in worse condition (Ferkin,
2011, 2015; Hurst and Beynon, 2004; Johnston and
delBarco-Trillo, 2009).
For many terrestrial mammals, scent marks convey informa-
tion about the donor to nearby conspecifics and heterospecifics
(Johnston and delBarco-Trillo, 2009; Kaur et al., 2014; Roberts
et al., 2014; Wyatt, 2014). These scent marks are viewed as
honest signals of the donor’s quality or condition (Ferkin,
2011; Roberts, 2007; Thonhauser et al., 2013a) because
many of the substances used as scent marks are digestive
exudates (Albone, 1984). Thus, scent marks from sources
such as the urine (Drickamer, 1995; Zala et al., 2004; Charlton,
2015), saliva (Block et al., 1981), feces (Tinnesand et al., 2015),
and those from specialized glands, such as the preorbital gland
(Ceacero et al., 2015; Pluhácek et al., 2015), anal gland (Bills
et al., 2013), foot pads (Owen et al., 2015), submandibular
glands (Camacho-Arroyo et al., 1999; Mykytowycz, 1965),
and the integument (Martin et al., 2014; McLean, 2014) accu-
rately reflect the condition and phenotype (Sabau and Ferkin,
2013a), height (Sharpe, 2015) or genotype of the sender
(Green et al., 2015). Scent marks from these multiple sources
likely provide unique and overlapping information about the
sender (Ferkin and Johnston, 1995a; Hurst and Beynon,
2004; Johnston, 2003, 2009; Kaur et al., 2014). As a result,
the response of terrestrial mammals to scent marks and odors
of conspecifics has remained of interest to researchers in
anthropology, biology, chemistry, psychology, neuroscience,
and medicine (Apps, 2013; Drea, 2015; Ferkin, 2015; Grimm,
2014; Johnston and delBarco-Trillo, 2009; Logan, 2014;
Petrulis, 2013a,b, 2015; Roberts, 2007; Roberts et al., 2014;
Schulte et al., 2013; Wyatt, 2014).
The fact that researchers from different fields are interested
in olfactory communication has led to confusion in the termi-
nology to describe chemical signals. Johnston and delBarco-
Trillo (2009) created a means to identify different types of
signals involved in odor communication. The researchers
argued that chemical signals may be categorized as being
a pheromone, a pheromone blend, or a mosaic. Accordingly,
a pheromone would be a single chemical compound, which
would be sufficient to induce one or more responses in
the receiver. A pheromone blend would be comprised of
several compounds in relatively specific proportions. These
compounds, in the proper amount, would be necessary to stim-
ulate responses in the receiver. A mosaic signal contains a large
number of chemical compounds, which are necessary to evoke
responses in the receiver. It is not known if a mosaic signal
creates an individually distinctive odor, sexually distinctive
odor, or group (species) odors based on differences in the
proportions of these compounds across individuals. It is
possible that most odor signals produced by terrestrial
mammals are mosaic signals as they often involve many
different compounds from multiple sources of scent. The ratio-
nale for this conjecture is that odor signals contain many chem-
ical compounds (Apps, 2013; Schulte et al., 2013) and are
derived from multiple sources of scent, including by-products

of hormone breakdown, excreta, and specialized tissues and
glands in the integument (Johnston and delBarco-Trillo,
2009; Petrulis, 2013).
1.10.1.1.1 Sources of Scents and Odors
Some sources of scent produce marks that convey sexual infor-
mation about the donor.
For example, meadow voles, Microtus pennsylvanicus, have
a highly localized pattern of sexual information on their bodies
during the breeding season (Ferkin and Johnston, 1995a). The
feces, anogenital area, and urine scent marks of meadow voles
are attractive to opposite-sex conspecifics but not to same-sex
conspecifics. However, saliva/mouth secretions of female voles
were attractive to male but not to female conspecifics, whereas
the posterolateral region scent marks of male voles were attrac-
tive to both male and female conspecifics. For meadow voles,
the secretions from the mouth and posterolateral region likely
provide different information than that from the feces, anogen-
ital area, and urine (Ferkin and Johnston, 1995a).
Similarly, the chemical signals for discrimination and recog-
nition of individuals can come from many different specific
sources, such as a variety of specialized scent glands (e.g., seba-
ceous glands, apocrine and eccrine sweat glands) as well as
urine and feces. In Syrian hamsters, Mesocricetus auratus, for
example, there are five different sources of individually distinc-
tive scents, namely flank gland, vaginal secretions, ear glands
(inside the pinna), urine, and feces (Johnston et al., 1991).
Six other potential sources of odors in Syrian hamsters were
tested but were not found to be individually distinctive, as
measured by habituation
dishabituation tests (fur from the
midline ventral surface, fur from the dorsal surface between
the shoulders, saliva, feet, fur behind the ears, and the flank-
gland area from flank-glandectomized males) (Johnston
et al., 1991). Similar results were found with Djungarian
hamsters, Phodopus campbelli. Male hamsters were able to
discriminate individual differences between other males using
scents from the midventral gland, urine, feces, mouth, and
the sacculi from the corner of the mouth (Lai and Johnston,
1994). These different odor sources that provide information
about individual identity may contain some redundant infor-
mation, but the combined information from of all of these
sources may facilitate the creation of stronger memories about
the identity of a given conspecific.
1.10.1.1.2 The Attractiveness of Scent Marks and Conspecific
Odor Preferences
The attractiveness of scent marks from the above sources can
vary according to, for example, the diet and reproductive condi-
tion of senders (Boonstra, 1994; Wade et al., 1996). This can be
reflected in the attractiveness of its scent marks to receivers
(Pierce et al., 2005, 2007; Sabau and Ferkin, 2013a; Sabau
et al., 2014). Scent marks also provide honest signals of health
(Zala et al., 2004) and nutritional status of the sender to the
receivers (Hobbs and Ferkin, 2011a,b; Pierce et al., 2005).
Nutritional stresses such as food deprivation and food restric-
tion are an ecological challenge faced by small herbivores
that live in transitional grasslands, where food sources are
patchy and vary in quality across the territories of female
conspecifics (Batzli, 1985; Bronson, 1989; Getz, 1985). Food
availability affected the amount of time male and female
Communication by Chemical Signals 287
meadow voles spent self-grooming in response to the odors
of opposite-sex conspecifics. Specifically, male and female
meadow voles that were food deprived for either 6 or 24 h
spent less time self-grooming compared to voles that had
continuous access to food (Hobbs and Ferkin, 2012b). It is
not clear at this time why food deprivation and restriction
had different effects on self-grooming and scent marking in
voles. Perhaps, the difference lies in the fact that voles self-
groom when they encounter the scent marks of conspecifics
that are in close proximity, whereas voles scent mark when
they encounter the scent marks of conspecifics that may be
nearby and when they are distant (Hobbs and Ferkin, 2012b;
Hobbs et al., 2008).
Given the importance of finding potential mates of the
opposite sex and competing with rivals of the same sex, identi-
fication of males and females is probably universal in all
species that reproduce sexually. Chemical signals often provide
crucial information about the sex of conspecifics; a few exam-
ples of species in which individuals have been shown to be
more attracted to odors of opposite-sex conspecifics than those
of same-sex conspecifics include many species of terrestrial
mammals (Eisenberg and Kleiman, 1972; Ferkin and Johnston,
1995a,b; Johnston, 1983). Male mice (Mus musculus) respond
with a surge in luteinizing hormone when exposed to female
mouse odors but not when exposed to male mouse odors or
female hamster odors (Maruniak and Bronson, 1976). Such
hormonal responses indicate that males discriminate sex
from odors even if a behavioral preference cannot be detected.
In most species, there may be several distinct sources of odor
that provide information about sex and/or reproductive state,
although not all odors necessarily contain such information.
For example, in Djungarian hamsters, males showed a prefer-
ence for the odors of receptive females over the odors of males
when the odor source was urine, anogenital secretions, saliva,
or midventral gland secretion but not when the odor source
was feces or secretions from the feet (Lai et al., 1996).
Several studies have shown that the attractiveness of the
scent marks of spontaneous ovulators also varies temporally
(Brown and Macdonald, 1985). The changes in the attractive-
ness of a sender’s scent mark appear to be concomitant with
changes in the responses of receivers to such marks (Ferkin,
2011; Johnston, 2009; Roberts, 2007; Sabau and Ferkin,
2013a). The scent marks of female house mice and Syrian
hamsters are attractive to male conspecifics only when the
female is in estrus (delBarco-Trillo et al., 2009c; Johnston,
1983; Kavaliers et al., 1994; Figure 1). Postpartum estrus
female mammals produced odors and scent marks that were
more attractive to males than were those produced by females
not in postpartum estrus (Ferkin and delBarco-Trillo, 2014;
Ferkin and Johnston, 1995b; Lai et al., 1996; Witt et al.,
1990; Zeigler et al., 1993). Postpartum estrus female voles, rela-
tive to females not in postpartum estrus, also spent more time
and showed increases in scent marking, over-marking, and self-
grooming when they encountered the scent marks of males
(Ferkin and Leonard, 2010; Ferkin, 2011).
In addition to showing a preference for odors of opposite-
sex individuals, male mammals usually show a preference for
odors of females that are sexually receptive over those of
females that are in other reproductive states (Brown, 1985).
For example, male meadow voles prefer the odors of females
288 Communication by Chemical Signals

Figure 1 Habituation
dishabituation test. A male Syrian hamster was exposed to vaginal secretions of a donor female during four habituation trials
(‘Hab 1’ to ‘Hab 4’) and consequently to the vaginal secretions of a second donor female during the dishabituation trial (‘Test’). The four panels
represent four scenarios in which: (a) both female donors were in estrus; (b) both female donors were in diestrus-2 (i.e., not in estrus); (c) the first
female was in diestrus-2 and the second female in estrus; (d) the first female was in estrus and the second female in diestrus-2. A dishabituation
response (a significant increase in investigation time in the dishabituation trial compared to the last habituation trial) was observed only when the
second donor female was in estrus. E, estrus; Di, diestrus-2; *p < 0.05; **p < 0.01; ***p < 0.001. Reproduced from delBarco-Trillo, J., LaVenture,
A.B., Johnston, R.E., 2009. Male hamsters discriminate estrous state from vaginal secretions and individuals from flank marks. Behav. Process. 82,
18–24, with permission from Elsevier.

in postpartum estrus (when females are in a state of heightened
sexual receptivity) than those of females in induced estrus or
undergoing pregnancy (Ferkin and delBarco-Trillo, 2014).
Sexually experienced female rats, Rattus norvegicus, prefer odors
of normal males over the odors of castrated males (Carr et al.,
1965). Similarly, male rats prefer odors of receptive females
over odors of nonreceptive females. These preferences,
however, may depend on knowledge that males have learned
during previous interactions with females. For example, sexu-
ally experienced male rats prefer odors of receptive females
over odors of nonreceptive females but sexually naïve males
do not show this preference (Carr et al., 1965). Yet, the ability
of males to discriminate between male and female and the
male preference for female odors may be two distinct mecha-
nisms. Experiments with house mice showed that males with
the vomeronasal organ removed did not show a preference
for the odors of estrous females over odors of males (Pankevich
et al., 2004). However, these males were still able to discrimi-
nate between odors of males and estrous females when tested
in a habituation
dishabituation method. These results make
the important point that the ability to discriminate between
odors of males and females does not necessarily lead to func-
tionally relevant responses.
The preference for opposite-sex conspecifics is also often
dependent on the reproductive state of the receiver. In domestic
rats, males prefer the odors of estrous females to those of
females in other reproductive states but castrated males do
not show such preference (Carr et al., 1965; Randall, 1986).
Similarly, the preference that diestrous, estrous, and lactating
female Syrian hamsters show for the odors of intact male
hamsters over castrated males is not shown by pregnant
females (Johnston, 1979).
Familiarity of a female with a male and/or his odors can
also be a determining factor in her odor preference. A female
repeatedly exposed to odors of a male may show a preference
for that male over a male that is unfamiliar, possibly because
familiarity indicates the ability to defend a territory or living
area for some period of time. Female pygmy loris, Nycticebus
pygmaeus, in captivity were first exposed to urine marks from
one male over 14–20 weeks. When the female was close to
 Communication by Chemical Signals 289

ovulation, males confined in small cages were placed in the

much larger living areas of females. Using three different behav-
ioral measures, females showed strong preferences for the
familiar male (Fisher et al., 2003a). In another example, female
collared lemmings were first housed with a male for 30 days
and were then separated for 1, 12, or 24 days. Females showed
a strong preference for their partners’ odors over the odors of an
unfamiliar male 12 days after separation, but they did not
show such a preference after 24 days of separation (Huck and
Banks, 1979). Female rats also show a preference for the odors
of a previous, familiar mate over the odors of a novel male. In
contrast, male rats show a preference for the odors of a novel
female over the odors of a previous mate (Carr et al., 1980).
In socially monogamous species one predicts that mated indi-
viduals will not only be able to recognize their partner from
other conspecifics but also that they will show a preference
for the partner’s odors compared to those of unfamiliar,
opposite-sex individuals (Duarte et al., 2015).

1.10.2 Physiological Mechanisms

1.10.2.1 Neural Substrates
Most social odors are processed by two complimentary chemo-
sensory systems, the main (MOS) and accessory olfactory
(AOS) system (Baum and Kelliher, 2009) as represented in
Figure 2. AOS sensory neurons are located in the vomeronasal
organ (VNO), a blind-ended tube that primarily takes up con-
tacted, nonvolatile, chemosignals (Chamero et al., 2012;
Halpern and Martinez-Marcos, 2003; Meredith, 1994; Zufall
and Leinders-Zufall, 2007). In contrast, sensory neurons of
the MOS (main olfactory epithelium: MOE) are located within
the nasal turbinates and respond primarily to volatile chemo-
signals via sniffing (Murthy, 2011; Wachowiak, 2011). These
systems also have separate projection zones within the olfac-
tory bulbs (OB): MOE neurons project to main olfactory bulbs
(MOB) and the VNO neurons project to the accessory olfactory
bulbs (AOB) (Scalia and Winans, 1975). The separation of AOS
and MOS is extended further into the brain systems such that
there are few areas of overlap, primarily in the anterior part
of the medial amygdala (MA) (Fan and Luo, 2009; Kang
et al., 2009; Mohedano-Moriano et al., 2012) or through indi-
rect connections in the cortical amygdala (MOB input, projects
to MA) and posterior bed nucleus of the stria terminalis (BNST;
AOB input, projects to MA) (Maras and Petrulis, 2010a;
Martinez-Marcos, 2009). Indeed, the MOS can alter how the
AOS processes social odors. For example, MOB inputs to the
anterior MA alter how the AOB responds to volatile phero-
mones (Martel and Baum, 2009) and the detection of volatile
odors by the MOS triggers VNO sampling, thereby increasing
chemosignal access to the VNO (Meredith, 1998).
The MA is not only the primary area of chemosensory inte-
gration, it is also a steroid-sensitive and sexually dimorphic
structure (Cooke, 2006) as are its connected structures, such
as the BNST, medial preoptic area (MPOA), anterior hypothal-
amus (AH), and ventromedial hypothalamus (VMH) (Segovia
and Guillamon, 1993; Simerly, 1990; Wood, 1997). Detailed
analysis of MA and its connected structures shows that steroid
and chemosensory information are represented by partially
separate and parallel systems (Maras and Petrulis, 2010a,b;
Figure 2 Abbreviated schematic of main olfactory (MOS; gray) and
accessory olfactory or vomeronasal (AOS; white) systems along with
integrative areas (black). For clarity, only unidirectional olfactory bulb
connections are presented and several areas without known relevance
to social odor processing are omitted. AOB, accessory olfactory bulb;
ACo, anterior cortical amygdala; BNST, bed nucleus of stria terminalis;
END, endopiriform nucleus; ENT, entorhinal cortex; HIPP, hippo-
campus; MA, medial amygdala; MDth, mediodorsal thalamus; MOB,
main olfactory bulb; MOE, main olfactory epithelium; MPOA, medial
preoptic area; OFC, orbitofrontal cortex; PIR, piriform cortex; PMCo,
posteromedial cortical amygdala; VMH, ventromedial hypothalamus;
VNO, vomeronasal organ; VPH, ventral premammilary hypothalamus.
Reproduced from Petrulis, A., 2013. Chemosignals, hormones and
mammalian reproduction. Horm. Behav. 63, 723–741, with permission
from Elsevier.
Newman, 1999; Wood, 1997) that provide a substrate where
steroid hormones and chemosensory systems may interact
(Been and Petrulis, 2011; Maras and Petrulis, 2010a). Impor-
tantly, these interconnected brain regions form the core of
the “social behavior neural network” (SBNN), a highly
conserved system of interconnected, steroid-sensitive and sexu-
ally dimorphic limbic structures that control multiple social
behaviors (Newman, 1999; O’Connell and Hofmann, 2012).
1.10.2.2 Mechanisms of Communication
Chemosensory cues from conspecifics guide almost every kind
of mammalian social behavior studied to date, including
aggressive, sexual, and parental behaviors, and the physiolog-
ical control of these behaviors is described elsewhere (Petrulis,
2013). Here we focus on the hormonal and neural control of
behaviors representing productive (scent marking, vocaliza-
tions) aspects of odor communication. The vast majority of
290 Communication by Chemical Signals
detailed information on this topic comes from the study of
only a few species: the Syrian hamster, the Mongolian gerbil,
the house mouse, the Norway rat, and the rabbit. Conse-
quently, our coverage will be constrained to these species, but
it should be noted that neural studies with similar findings
have been reported in other species, such as domestic cats
(Hart and Voith, 1978; Lischka et al., 2008) and dogs (Hart,
1974; Gadbois and Reeve, 2014; Jia et al., 2014).
When rodents of either sex investigate conspecific odor cues,
they often deposit their own odor cues through stereotyped
scent-marking responses and/or vocalize, often in the ultra-
sonic range (Brown and Macdonald, 1985; Petrulis, 2013;
Pierce et al., 1989; Wyatt, 2014). Both of these behaviors serve
to facilitate localization of mates over different temporal and
spatial ranges (Pfaff et al., 2008). Although the details may
vary as to exact dominant vocal frequencies or the postural
and chemical nature of deposited scent marks, there is remark-
able consistency in the chemosensory, hormonal, and neural
substrates of these behaviors across model species. Indeed,
the aggregate evidence, described below, suggests that both
scent marking and vocalizations are driven by a steroid-
dependent neural network composed of the chemosensory
inputs to the extended medial amygdala (MA, BNST), its
outputs to basal forebrain (MPOA, AH, VMH) and connected
midbrain elements (periaqueductal gray, PAG).
1.10.2.2.1 Scent Marking
1.10.2.2.1.1 Syrian Hamster: Flank Marking
Unlike species discussed below, both male and female
hamsters deposit products of dorsal flank sebaceous scent
glands on vertical surfaces, a behavior termed flank marking
(Johnston, 1975a, 1977). Flank marking is thought to reflect
territorial or competitive motivation as it occurs primarily in
response to same-sex odors and following establishment of
dominant
subordinate relationships (Johnston, 1975b,
1977). This behavior has been extensively investigated at the
neural level following the accidental discovery that MPOA
injections of arginine-vasopressin (AVP) injections elicit excep-
tional levels of flank marking in both males and females
(Albers, 2015; Ferris et al., 1984). Both AVP- and odor-
induced flank marking is blocked by MPOA injections of AVP
V1a receptor antagonists (Albers et al., 1986). The key locations
within MPOA that trigger AVP-induced marking are primarily
in the ventral parts of the anterior hypothalamus (AH) (Ferris
et al., 1986a) and the source of the AVP appears to be from
nonpituitary projecting nucleus circularis (NC) cells within
AH and the medial supraoptic nucleus (mSON) (Delville
et al., 1998; Ferris et al., 1990b, 1992; Mahoney et al., 1990).
Indeed, subordinate hamsters (that do not mark) have lower
levels of AVP in NC (Ferris et al., 1989) and these cells (and
mSON) show increased activity during aggressive interactions
(Delville et al., 2000). Further studies have indicated that
AVP action within the AH requires excitatory glutamatergic
neurotransmission (Bamshad et al., 1996) and that it is
antagonized by serotonin action (Albers et al., 2002). Input
to the AH from the broader AVP-sensitive brain also regulates
flank marking as LS/BNST lesions reduce marking and AVP
injections in lateral septum (LS), or in nearby BNST, trigger it
(Ferris et al., 1990a). Lesions of AH block the induction of
flank marking by AVP injections in LS/BNST. In contrast, LS
lesions have no effect on the ability of AVP injections in AH
to drive flank marking (Ferris et al., 1994). This strongly
suggests that AH is downstream of LS control and is the
primary regulator of AVP-driven flank marking, perhaps via
midbrain structures (Hennessey et al., 1992).
Initial reports demonstrated that both flank marking and
V1a receptors (but not AVP itself) in MPOA/AH are dependent
on gonadal steroids, especially androgens (Albers and
Rowland, 1989; Albers and Prishkolnik, 1992; Albers et al.,
1988, 1996; Huhman and Albers, 1993; Young et al., 2000).
However, more recent work suggests a more complicated
picture. Although male hamsters in a nonbreeding photope-
riod have regressed testes and low levels of circulating testos-
terone, flank marking levels are testes-independent and not
reduced during social encounters, despite steroid-dependent
reductions in V1a receptors (Caldwell et al., 2008).
The AVP manipulations within AH alter flank marking irre-
spective of social status, but they do not have lasting effects on
dominance interactions (Ferris et al., 1986b), suggesting that
AH AVP primarily regulates behavioral output rather than stim-
ulus evaluation or dominance/subordination itself. So, what
then is the source of social odor information to the AH?
Initially, this information comes from both the MOS and
AOS representations in the olfactory bulb as olfactory bulb
removal eliminates flank marking (Kairys et al., 1980). Periph-
eral lesions of the MOS reduce male and female flank marking
in arenas scented with the odors of both male and female
conspecifics (Johnston, 1992; Johnston and Mueller, 1990).
Although VNO removal does not reduce flank marking in
this context, excising the VNO in females reduces preferential
targeting of this behavior toward other females, without
altering their low baseline response to male odors (Petrulis
et al., 1999). As the MA serves as the main point of convergence
between MOS and AOS, it is perhaps unsurprising that
complete lesions of MA virtually eliminate flank marking in
females (Petrulis and Johnston, 1999); smaller lesions of either
anterior or posterodorsal MA result in reductions rather than
outright elimination of this behavior (A.P., unpublished obser-
vations). As the MA has substantial inputs to MPOA/AH, it is
likely that odor modulation of flank marking is mediated by
MA (and perhaps cortical amygdala; (Petrulis et al., 2000) effer-
ents to AVP-sensitive regions of AH; this awaits further
investigation.
1.10.2.2.1.2 Syrian Hamster: Vaginal Marking
Female hamsters scent mark (vaginal mark) to advertise
impending sexual receptivity (Johnston, 1977) similar to rats,
house mice, and rabbits (Birke, 1984; Chang et al., 2000;
González-Mariscal et al., 1990; Rich and Hurst, 1999). Vaginal
marking is a stereotyped behavior that deposits sexually attrac-
tive vaginal fluid on the surface (Been and Petrulis, 2008; Been
et al., 2012) and is specifically targeted toward male odors
(Johnston and Brenner, 1982; Johnston, 1977; Petrulis and
Johnston, 1997). This behavior changes substantially across
the female’s estrous cycle (Figure 3) such that females mark
most on the night prior to sexual receptivity (during high circu-
lating estrogen levels) and then show no vaginal marking
during sexual receptivity (during high progesterone levels)
(Johnston, 1977; Lisk and Nachtigall, 1988). This remarkable
hormone-dependent shift in vaginal marking is likely regulated

(a) Diestrous day 2
30
Number of vaginal marks
25
20
15 *
10
0
SHAM BNST-X
Figure 3 Mean (SEM) number of vaginal marks to male and female odors. SHAM, but not BNST-X, females preferentially marked at higher levels
to male odors compared to female odors on both (a) diestrous day 2, and (b) proestrus. *p < 0.01, male versus female odor condition.
by the steroid action on MPOA and VMH: E implants in these
areas restore vaginal marking in OVX females (Takahashi et al.,
1985). However, the VMH may not be critical for vaginal
marking, as VMH lesions do not eliminate the behavior
(Floody, 2002). The role of the MPOA is presently unclear as
large, fiber-damaging MPOA lesions disrupt vaginal marking
(Malsbury et al., 1977), whereas smaller, fiber-sparing lesions
limited to MPOA do not (Martinez and Petrulis, 2013). This
discrepancy is likely due to the concomitant damage to BNST
present in the earlier study, as fiber-sparing lesions confined
to the posterior BNST impair vaginal marking (Martinez and
Petrulis, 2011) without altering the cyclicity of this behavior
(Figure 3).
As is the case for flank marking, olfactory bulbectomy
reduces vaginal marking (Kairys et al., 1980) and the disruptive
effects of MOS peripheral lesions on vaginal marking are much
more pronounced than those following VNO removal (John-
ston, 1992; Petrulis et al., 1999). MOS inputs to the MA, rather
than to other MOS targets (Petrulis et al., 1998, 2000), appears
to regulate vaginal marking as MA lesions lead to significant
reductions in vaginal marking (Petrulis and Johnston, 1999;
Takahashi and Gladstone, 1988). Interestingly, females with
MA lesions still show differentially high marking rates to
male odors and still retain normal cyclic variation in vaginal
marking. This suggests that MA is not critical for hormonal
control of marking (likely mediated by hypothalamic struc-
tures) or differential marking responses. In contrast, the BNST
is critical for differential marking to male odors (Martinez
and Petrulis, 2011). This effect is likely mediated by oxytocin
action because blockade of oxytocin receptors in BNST and
MPOA reduces male-directed vaginal marking (Martinez
et al., 2010, 2013).
1.10.2.2.1.3 House Mouse: Urine Marking
Urine marking by socially dominant male mice is generally
used in the context of territorial defense (Arakawa et al.,
2008a,b; Desjardins et al., 1973; Lumley et al., 1999). However,
male urine marking does not appear to be differentially tar-
geted toward males versus females (Hurst, 1990a,b,c; Labov
and Wysocki, 1989), suggesting a more general advertising
function. Nevertheless, males do urine mark at much greater
levels than do females, an effect largely driven by sex differ-
ences in adult and developmental exposure to testosterone
Communication by Chemical Signals 291
(b) Proestrus
Male
*
Female
SHAM BNST-X
and its estrogenic metabolites (Juntti et al., 2010; Kimura and
Hagiwara, 1985; Wu et al., 2009). These steroid effects appear
to be exerted on MPOA/AH, BNST, or VMH neurons as testos-
terone or estradiol implants near or in these structures can
maintain urine marking in gonadectomized males (Matochik
et al., 1994; Nyby et al., 1992), perhaps through interactions
with midbrain dopamine systems (Sipos and Nyby, 1996).
The available evidence suggests that the MA is not a substrate
for steroid effects on urine marking (Sipos and Nyby, 1998;
Unger et al., 2015). Recent evidence suggests that central
oxytocin decreases urine marking and thereby provides
a possible chemical mechanism through which marking can
be decreased in subordinate animals (Arakawa et al., 2015).
Surprisingly, little is known as to how chemosensory informa-
tion is transmitted to steroid-sensitive structures. What
evidence exists suggests that urine marking is partially medi-
ated by the AOS (MOS contribution not yet assessed) as evi-
denced by decreased marking in males following VNO
removal (Labov and Wysocki, 1989; Maruniak et al., 1986).
As in many other social contexts, removal of VNO has a greater
effect in sexually naïve animals than in males with sexual expe-
rience (Clancy et al., 1984; Labov and Wysocki, 1989; Maru-
niak et al., 1986).
1.10.2.2.1.4 Mongolian Gerbil: Ventral Marking
Dominant male gerbils engage in more scent marking than do
subordinate male gerbils. Much of this scent marking involves
the ventral gland. Briefly, individuals rub odors from this
ventral sebaceous scent gland onto the substrate (Thiessen
et al., 1968, 1970; Whitsett and Thiessen, 1972; Yahr, 1977).
Adult gonadectomy reduces both male and female marking
in gerbils and can be reinstated with administration of testos-
terone or its metabolites in males (Yahr and Stephens, 1987;
Yahr et al., 1979) or estradiol plus progesterone in females
(Owen and Thiessen, 1974; Yahr and Thiessen, 1975). The
sex difference in overall levels of marking is due to organiza-
tional effects of gonadal steroids as neonatal testosterone and
subsequent adult T in females will increase female marking
to male levels (Ulibarri and Yahr, 1988, 1996). Testosterone
appears to exert its effect via the MPOA; implants near the
most rostral or caudal limits of MPOA are most effective in
restoring marking in gonadectomized males (Thiessen and
Yahr, 1970; Thiessen et al., 1973; Yahr et al., 1982). Indeed,
292 Communication by Chemical Signals
large MPOA lesions that include these areas reduce ventral
marking in males (Yahr et al., 1982) particularly if these lesions
remove the sexually dimorphic cell groups in caudal MPOA
and BNST (Commins and Yahr, 1984). However, small fiber-
sparing lesions of these cell groups had no effect on ventral
marking (Yahr and Gregory, 1993) indicating that previous
effects were due to damage to passing axon fibers. Further
attempts at identifying brain regions that regulate ventral
marking have not been successful: small, fiber-sparing lesions
of posterodorsal MA, medial preoptic nucleus, or posterome-
dial BNST were without effect on ventral marking (Heeb and
Yahr, 2000; Sayag et al., 1994).
1.10.2.2.1.5 Rabbits: Chinning
Studies have examined the mechanisms underlying chinning,
a type of scent marking in rabbits. For example, González-
Mariscal et al. (1990) found that the frequency of chinning
varied with the female’s reproductive cycle. Moreover, during
estrous there were more acini/field than during pregnancy
and lactation (Cerbón et al., 1996). Gonadectomy reduced
the number of acini/field and increased their diameter in
females but had the opposite effect in males (Cerbón et al.,
1996). Hudson et al. (1990) found that estradiol treatment
was necessary to increase chinning in ovariectomized rabbits.
Interestingly, progesterone treatment given to estradiol-
primed, ovariectomized females induced an immediate
suppression of chin marking and lordosis, but progesterone
therapy alone had no effect on these measures in ovariecto-
mized female rabbits (Hudson et al., 1990). González-
Mariscal et al. (1993) found that chinning in males depends
on testicular steroids, as castration decreased the frequency of
chinning. This behavior was nearly restored to precastration
levels by treatment with testosterone propionate, whereas the
administration of estradiol and dihydrotestosterone increased
chinning above precastration levels (Gonzalez-Mariscal et al.,
1993). Melo et al. (2008) examined the brain sites where
steroids act to stimulate chinning. They did so by implanting
testosterone propionate or estradiol benzoate into the ventro-
medial hypothalamus (VMH) or the medial preoptic area
(MPOA) of gonadectomized male and female rabbits, respec-
tively. Melo et al. (2008) found that, for female rabbits,
chinning was stimulated by EB implants into the VMH or
MPOA. In contrast, for males, chinning was stimulated by
testosterone implants into the MPOA, but not into the VMH
(Melo et al., 2008). Camacho-Arroyo et al. (1999) discovered
progesterone and estrogen receptors in the nucleus of acini
cells of the submandibular (chin) glands of rabbits of both
sexes. Intact males had a smaller number of estrogen
receptor-immunoreactive cells and progesterone receptor-
immunoreactive cells than did estrous females. Progesterone
receptor-immunoreactive cells were more numerous in
estrous than they were in ovariectomized rabbits. Estradiol
therapy increased the number of progesterone receptor-
immunoreactive cells in ovariectomized female rabbits
(Camacho-Arroyo et al., 1999).
1.10.2.2.2 Ultrasonic Vocalizations
1.10.2.2.2.1 House Mouse
In mice, reproductively active and territorial/dominant adult
males produce high amounts of ultrasonic vocalizations
(USV) specifically during investigation of novel females or their
urine; males or their urine do not elicit USVs from other males
or females (Guo and Holy, 2007; Nyby et al., 1977; Nyby,
1983b; Wang et al., 2008; Warburton et al., 1989; White
et al., 1998) and so these USVs are often considered to be
male-typical promoters of opposite-sex interactions (Chabout
et al., 2015; Musolf et al., 2010; Pomerantz et al., 1983; Sugi-
moto et al., 2011). Female mice do produce a modest amount
of USVs during female–female mutual investigation (Moles
et al., 2007) but, unlike female-directed calling by males, this
female-directed calling habituates rapidly (Maggio and
Whitney, 1985; Warburton et al., 1989).
Removal of olfactory bulbs in males eliminates USVs to
female odors and similar or lesser effects are also observed after
AOS damage, especially in sexually naïve animals (Bean, 1982;
Wysocki et al., 1982). Females with genetically induced VNO
dysfunction display increased USVs to males suggesting that
the VNO may normally suppress male-typical behavior in
female mice (Kimchi et al., 2007; Stowers et al., 2002, but
see Pankevich et al., 2004). Data on the role of the MOS in
USV generation is more equivocal: peripheral lesions of MOS
are reported to either have no effect (Bean, 1982) or to reduce
USVs (Sipos et al., 1995).
Given the sex difference in USV production, it is perhaps not
surprising that male USVs are strongly steroid-dependent. Male
USVs are eliminated by adult gonadectomy and restored, and
even induced in females, by circulating testosterone or its estro-
genic/androgenic metabolites (Bean et al., 1986; Juntti et al.,
2010; Matochik and Barfield, 1991; Nunez et al., 1978;
Pierman et al., 2008; Wu et al., 2009). The male-biased pattern
of USV production does not appear to require organizational
hormone action, as neonatal castration does not alter male-
typical patterns of calling (Warburton et al., 1989). The identi-
fied brain sites of gonadal steroid action on USVs (MPOA/AH,
BNST, or VMH) are the same ones identified for urine marking
as testosterone or estradiol implants around these structures
maintains USVs in gonadectomized males (Matochik et al.,
1994; Nyby et al., 1992), perhaps interacting with midbrain
dopamine systems (Sipos and Nyby, 1996). However, small
lesions to MPOA do not change USVs (Bean et al., 1981) and
so suggests that MPOA steroid implants are effective because
of steroid spread to nearby areas, such as BNST. The available
evidence suggests that the MA is not a substrate for steroid
effects on USVs (Sipos and Nyby, 1998; Unger et al., 2015),
but MA’s broader role in regulating USVs has not been assessed.
1.10.2.2.2.2 Syrian Hamster
Both estrous female and male hamsters (Floody et al., 1977,
1979a; Johnston and Kwan, 1984) produce USVs in response
to opposite-sex conspecifics or their odors; this response is
steroid-dependent in both sexes (Floody and Petropoulos,
1987; Floody et al., 1979a,b, 1987). Hamster USVs to odors
requires intact olfactory bulbs (Kairys et al., 1980) and either
a functioning MOS or AOS (Johnston, 1992). Lesions of the
MA leads to deficits in USV production by female hamsters,
suggesting that MOS/AOS chemosensory information
converge in this structure (Kirn and Floody, 1985). It is likely
that MA projections to MPOA/AH or BNST are critical for this
behavior as damage to MPOA/AH impairs USVs (Floody,
1989) as do knife cuts that damage BNST connections

(Miceli and Malsbury, 1982). Similar to studies in other
species (Jürgens, 2009), hamster vocalizations require
processing by the periaqueductal gray (PAG) region of
the midbrain (Floody and DeBold, 2004; Floody and
O’Donohue, 1980) via a steroid-sensitive mechanism (Floody
et al., 1986).
1.10.2.2.2.3 Norway Rat
Both male and estrous female rats emit USVs (50 kHz) during
interactions with opposite-sex individuals or their odors (Geyer
and Barfield, 1978; White et al., 1991). Male USVs are depen-
dent on both androgen- and estrogen-receptor activity
(Matochik and Barfield, 1991; Vagell and McGinnis, 1998).
Testosterone action within the AH and/or VMH appears to be
necessary for USVs in males (Harding and McGinnis, 2004)
even though it is not sufficient (Harding and McGinnis,
2003). Indeed, small lesions in VMH inhibit the ability of testos-
terone to restore USVs in gonadectomized male rats (Harding
and McGinnis, 2005) suggesting that the VMH is one of the crit-
ical steroid-sensitive brain regions for USVs. Other structures
may also be involved in regulating USVs: pharmacological stim-
ulation of the BNST or MPOA/AH increase 50 kHz USVs in
males (Burgdorf et al., 2007; Fu and Brudzynski, 1994; Wintink
and Brudzynski, 2001), likely via their direct connections with
midbrain vocal centers (Kyuhou and Gemba, 1998).
1.10.3 Development and Learning
1.10.3.1 Reproductive States and Odors
In many mammalian species females approaching or in estrus
may release odors or increase their scent marking to advertise
their sexual receptivity and thus attract mates. For example,
female Syrian hamsters increase their vaginal and flank
marking immediately prior to sexual receptivity, concentrating
those scent marks around the dominant male territories and
also creating a trail of scent marks between the burrow of
a dominant male and her own burrow (Huck et al., 1985a).
Several mechanisms may be used for advertising sexual recep-
tivity. The quantity of secretions produced and available for
scent marking may be increased, the frequency of scent
marking may increase, the composition of odors may change
with the female’s reproductive state, or a combination of these
mechanisms may be employed. With the first two mechanisms
there need not be a change in the quality of the odor, but in
most cases both the quality and the quantity of odors may
change. Indeed, given that metabolic products of sexual
hormones are likely to be contained in the female’s odors,
such odors can be a direct reflection of the reproductive state
of the female and the attractiveness of female odors.
As a further example of how females advertise their sexual
receptivity through odors, we can consider those species in
which females undergo postpartum estrus. Postpartum estrus
is a relatively short period of heightened sexual receptivity
that occurs immediately after parturition. Females that mate
and become pregnant during postpartum estrus gestate a set
of offspring while they are nursing the previous litter. As
a consequence, postpartum estrus allows females to be simul-
taneously pregnant and lactating, maximizing the number of
litters that they can produce during the breeding season.
Communication by Chemical Signals 293
Postpartum estrus occurs in many species but it is especially
prevalent in small-sized rodents (Ferkin and delBarco-Trillo,
2014) and in lagomorphs (i.e., rabbits, hares, pikas). Female
meadow voles in postpartum estrus are more sexually receptive
and likely to become pregnant than females in other estrous
states (Ferkin and delBarco-Trillo, 2014). These females in
postpartum estrus also deposit more scent marks, advertising
their heightened sexual state to males, who show a preference
for the odors of postpartum-estrus females compared to those
of females in other estrous states (Ferkin and delBarco-Trillo,
2014). In contrast, in mice and prairie voles (Microtus
ochrogaster), females do not increase scent marking when in
postpartum estrus (Coquelin, 1992; Ferkin and delBarco-
Trillo, 2014), possibly because females in these species nor-
mally mate with the same partner, who may be familiar with
the female’s state of sexual receptivity.
Different sources of female odor may contain information
about her sexual receptivity. Male Syrian hamsters show high
levels of copulatory behavior when all of a female’s odors are
present but they show declining levels of copulatory behavior
as specific odor sources are removed (Johnston, 1986). Elimi-
nation of vaginal secretions significantly reduced male copula-
tory behavior and removal of flank, ear, and Harderian
secretions caused additional decreases in copulatory behavior.
Therefore, these four different odor sources carry information
about the attractivity of females, influencing male sexual
arousal and performance (Johnston, 1986). Similarly, male
Djungarian hamsters show a preference for the urine, saliva,
and vaginal secretion of estrous females over similar odors of
diestrous females, whereas a preference is not shown when
males investigate other types of body odor, such as feces and
midventral gland secretions, even though these odors are
important in other aspects of communication (Lai et al.,
1996). Also in Djungarian hamsters, the attractiveness of three
different odors of females varied across the estrous cycle, each
odor being maximally attractive at a different stage of the cycle
(Lai and Johnston, 1994). That is, different odors from the
same female may be most attractive at different times during
the estrous cycle. This type of pattern is especially interesting
because it might allow males to accurately track and predict
the timing of estrous.
It has already been mentioned that males show a preference
for the odors of sexually receptive females. Such preference for
odors of receptive females may be universal and has indeed
been shown to exist in many species, including the house
mice (Hayashi and Kimura, 1974), brown lemmings, Lemmus
trimucronatus, and collared lemmings, Dicrostonyx groenlandicus
(Huck and Banks, 1984), meadow voles (Ferkin and Johnston,
1995a), Indian desert gerbils, Meriones hurrianae (Kumari and
Prakash, 1984), Mongolian gerbils (Block et al., 1981), wood-
rats, Neotoma lepida (Fleming et al., 1981), Columbian ground
squirrels, Spermophilus columbianus (Harris and Murie, 1984),
dogs, Canis lupus familiaris (Beach and Gilmore, 1949; Dunbar,
1977), rams, Ovis aries (Lindsay, 1965), and pygmy marmosets,
Cebuella pygmaea (Converse et al., 1995). A lack of a preference
for odors of estrous females over odors of nonestrous females is
difficult to interpret and may be due to methodological short-
comings, but it has been reported in some species such as
deer mice, Peromyscus maniculatus (Dewsbury et al., 1986), and
guinea pigs, Cavia porcellus (Nyby, 1983a). Indeed, in species
294 Communication by Chemical Signals
that have been studied by many investigators, such as mice, rats,
and Syrian hamsters, some studies report male preferences for
estrous females (Carr et al., 1965; Johnston, 1980; Lydell and
Doty, 1972), whereas other studies report a lack of such a pref-
erence (Brown, 1977; Kwan and Johnston, 1980; Landauer
et al., 1978; Taylor and Dewsbury, 1990). Such differing results
are probably due to differences in methodology (delBarco-Trillo
et al., 2009c; Taylor and Dewsbury, 1990). For example, in one
set of experiments with female Syrian hamsters, six different
methods were compared using measures of investigation of
a single stimulus and five similar methods were used to measure
preference for one of two odor stimuli. Results showed that tests
in the subject’s home cage or in a moving air stream were rela-
tively poor, whereas tests in which the stimulus (or stimuli)
were in the center of a relatively large arena or were outside
the arena yielded data that often demonstrated differences in
attraction (Johnston, 1981).
In addition to showing a preference for the odors of recep-
tive females, males may increase their scent-marking rates when
in areas marked by sexually receptive females. For example,
male meadow voles over-mark more the scents of females in
a heightened state of sexual receptivity, i.e., females in post-
partum estrus, than those of females in other estrous states
(Ferkin and delBarco-Trillo, 2014). Males can also respond
physiologically to odors of receptive females; for example,
more erections were elicited by exposing males to odors of
receptive females than to odors of unreceptive females (Sachs,
1999). Males can also use female odors to discriminate
between mated and unmated females (Thomas, 2011).
In several species, sexually experienced males show a prefer-
ence for odors of receptive females but sexually naïve males do
not (Carr et al., 1965; Hayashi and Kimura, 1974; Huck and
Banks, 1984; Johnston, 1980; Lydell and Doty, 1972). In other
studies, however, both sexually naïve and sexually experienced
males show a similar preference for odors of estrous females
over odors of nonestrous females, e.g., in male mice (Rose
and Drickamer, 1975).
Changes in seasonality may result in changes in the compo-
sition of odors and the scent-marking rates of individuals,
which may affect the preference of individuals for the odors
of conspecifics. When meadow voles were tested in short-day
conditions, females showed a preference for the odors of other
short-photoperiod females over those of males, probably asso-
ciated with the habit of often nesting with other females in
winter to maximize warmth (Ferkin and Johnston, 1995a).
Short-photoperiod males did not show a preference for the
odors of short-period females over those of other short-
period males. Furthermore, short-photoperiod voles (both
sexes) did not show sex-specific preferences for long-
photoperiod conspecifics, and vice versa (Ferkin and Johnston,
1995a). Some of these seasonal changes in odor preferences
may depend on the endocrine state of individuals. For
example, during long photoperiods, the attractiveness of
male scent to females depends on high titers of both testos-
terone and prolactin (Ferkin et al., 1997a).
Mate attraction is not universally driven by females. There
are species in which males produce odors to attract females.
For example, female frogs of the genus Hymenochirus are
attracted to secretions produced by male ‘breeding glands’
(Pearl et al., 2000). Females are attracted to water containing
males, but not to water containing females or males without
breeding glands. Males are not attracted to any of these odors,
indicating that the attractant is specific to females and not
a general signal that promotes aggregation (Pearl et al.,
2000). Sexually mature, terrestrial female toadlets, Pseudo-
phryne bibronii, tested in a two-choice Y-maze, were attracted
to the odors deposited by males compared to the clean arm
of the Y-maze (Byrne and Keogh, 2007). Similarly, male red-
bellied newts, Cynops pyrrhogaster, produce a peptide
pheromone in a cloacal gland that has a female-attracting
function (Kikuyama et al., 1995). In contrast, female palmate
newts, Triturus helveticus, are equally attracted to male and
female chemical cues (Secondi et al., 2005). In this species,
males and females aggregate on breeding sites at high
densities. Attraction to odors of conspecifics, irrespective of
sex, may serve to locate a breeding site and then short-range
chemical or visual signals may be used to locate mates
(Secondi et al., 2005). In terrestrial salamanders, Plethodon
jordani, the mental gland of males produces a proteinaceous
secretion that operates as a ‘courtship pheromone,’
increasing female receptivity and easing insemination
(Feldhoff et al., 1999). There are also several mammalian
examples, including mice, Syrian hamsters, dogs, and pigs,
Sus scrofa, in which females show a preference for the odors
of intact males over those of castrated males, indicating that
male attractivity may be important during sexual interactions
across many species (Brown, 1977; Johnston, 1981; Lisberg
and Snowdon, 2009; Signoret, 1976).
1.10.3.2 Individual Discrimination
Individual discrimination involves the ability of a subject to
detect the odors of different donors as being different. Recogni-
tion does not only involve the ability to discriminate between
the cues/signals from one individual and another, but it also
implies that the subject has some previous knowledge about
the stimulus animal (Halpin, 1986). At the simplest level,
this knowledge may just involve familiarity with one or more
signals from another individual and the ability to categorize
a stimulus into one of two categories (familiar or unfamiliar).
For example, using the knowledge about the odors of one’s
own group may be sufficient to accept a given individual into
the nest or not. True individual recognition, however, implies
more thorough knowledge of the characteristics of other
individuals.
Individual recognition requires the existence of distinct
signals and the ability to detect and discriminate between these
signals. Most animals may combine a broad spectrum of
signals to communicate individual identity, including a combi-
nation of chemical, visual, and auditory cues, producing multi-
modal signals (Higham and Hebets, 2013). Individuals are the
fundamental units of social interaction and social organiza-
tion. Thus discrimination and recognition of individuals, or
classes of individuals, are fundamental to the understanding
of animal behavior. Even though individual recognition medi-
ated by odors is not restricted to vertebrates (Caldwell, 1985),
most of such research has been conducted on vertebrates,
including many mammalian species (Kent and Tang-
Martínez, 2014).

Studies on rodents such as Mongolian gerbils, Meriones
unguiculatus, ground squirrels, Syrian hamsters, meadow voles,
otters (Lontra canadensis), koalas (Phascolarctos cinereus),
raccoons (Procyon lotor), and house mice have also provided
evidence that these animals can distinguish between the scent
marks of different individuals (Charlton, 2015; Ferkin et al.,
2016; Halpin, 1986; Kean et al., 2015; Kent and Tang-
Martínez, 2014; Thom and Hurst, 2004), responding preferen-
tially or displaying a selective memory for the scent mark of
a familiar conspecific compared to that of an unfamiliar
conspecific (Heth et al., 1998; Johnston, 1993; Kaur et al.,
2014; Mateo and Johnston, 2000a,b). Recent work on house
mice has shown that females can learn the individual identity
of males from their scent marks and females can use this infor-
mation to select potential mates (Roberts et al., 2014).
In a series of elegant experiments, Johnston and colleagues
(Heth et al., 1999; Johnston, 1993, 2003; Johnston and
Bullock, 2001; Mayeaux and Johnston, 2002; Todrank et al.,
1998) used a habituation/dishabituation paradigm to show
that Syrian hamsters could distinguish between scent marks
of two same-sex conspecifics that were similar in condition,
treating each scent mark as if it were individually distinct. John-
ston and coworkers found that the amount of time that Syrian
hamsters spent sniffing the scent mark of an individual donor
decreased with each successive exposure to that individual’s
marks, indicating that the hamsters habituated to the scent
marks, and viewed them as being familiar. The hamsters were
then exposed to a scent mark of a different conspecific and
that of a familiar donor. During this exposure, the hamsters
spent more time sniffing the novel scent mark than the familiar
scent mark, suggesting that scent marks convey individually
distinct information to conspecifics (Johnston, 2009).
1.10.3.2.1 Habituation
Dishabituation
Different methodological approaches can be used to demon-
strate individual discrimination and recognition. The first
studies to demonstrate discrimination of odors in mammalian
species used training procedures with food reinforcement to
demonstrate such abilities (Bowers and Alexander, 1967;
Rasa, 1973). During positive reinforcement the subject is pre-
sented with odors from two different donors but rewarded
only when responding to only one of those two odors. If
over a series of trials the subject animal learns to discriminate
between the two odors we can assume that the animal has
the ability to distinguish between them. Positive reinforcement
is a useful approach to determine the discriminating potential
of animals but it has a limited use to understand individual
recognition under normal, nontaught conditions.
The most commonly used approach to study individual
discrimination involves variations of the habituation
dishabi-
tuation technique (Halpin, 1986). A subject animal is first pre-
sented with the odor of one donor (odor A) over several trials
(this is the habituation phase), and then the same subject is
presented with the odor of a second donor (odor B; this is
the discrimination phase). In each trial, the time the subject
spends investigating the odor is recorded. During the habitua-
tion phase, with repeated presentations of new samples of the
same odor stimulus, the behavioral response declines signifi-
cantly, indicating that habituation has occurred. In this case,
individuals habituated to odor A at the end of the habituation
Communication by Chemical Signals 295
phase should investigate odor B longer than odor A during the
last dishabituation trial. This result would suggest that the indi-
vidual views odor A and odor B as being different from one
another. The habituation
dishabituation technique is much
more naturalistic and much easier to implement than reinforc-
ing techniques. A variation on this method is to present two
stimuli on the test trial after the habituation phase, namely,
the stimulus that the subject has been habituated to and a novel
stimulus. Simultaneous presentation of the familiar and novel
stimuli is an easier task and may allow the visualization of
differences in response to the novel stimulus when a single-
stimulus test does not (Brown et al., 1987). The optimal dura-
tion of the interval between trials will depend on the species. In
hamsters, varying the intertrial interval from 1 s to 2 days led to
similar results (Johnston, 1993). In contrast, dogs and captive
wolves would not even approach the stimulus on the second
trial after an interval of 15–30 min, apparently because they
could determine from a distance that the odor presented on
the second trial was the same as that on the first trial; when
the interval between trials was shifted to 24 h, both dogs and
wolves showed the typical habituation
dishabituation
response (Brown and Johnston, 1983).
Some studies have used across-odor habituation approaches
to determine if animals can remember familiar individuals
using several different distinctive signals and integrate these
separate memories into integrated representations of others.
For example, male hamsters first had a series of brief interac-
tions with females on four successive days. During these
4 days males had the opportunity to investigate several odor
sources from those females. After habituation to one individu-
ally distinctive odor of a stimulus animal (e.g., that from vaginal
secretions), males were also habituated to other odors from the
same individual (e.g., sebaceous flank glands). That is, subjects
showed an across-odor habituation (Johnston and Jernigan,
1994). Since the odors themselves are composed of very
different chemical compounds, it is not likely that this effect
was due to chemical similarities in the two odors. Additional
evidence for this conclusion comes from results of control
experiments in which subjects were not familiar with the stim-
ulus animals. If there was similarity in odor quality across
different odor sources, subjects should show across-odor habit-
uation without familiarity with the scent donor. However, if
subjects had not previously interacted with the scent donors,
no cross-odor habituation was observed (Johnston and Bullock,
2001; Johnston and Jernigan, 1994). Subsequent experiments
showed that the same effects were observed with other pairs
of odors (Johnston and Bullock, 2001).
In virtually all laboratory studies aimed at individual recog-
nition, it has proven difficult to provide evidence for ‘true’
individual recognition. That is, the unique significance of an
individual, not just the significance of categories of individuals
such as familiar versus unfamiliar or dominant versus subordi-
nate (Halpin, 1986). In particular, it has been difficult to
demonstrate the emotional significance of another individual
without possible confounding interpretations, such as
differences in familiarity or dominance status (Martin and
Beauchamp, 1982). In one attempt to disentangle the effect
of familiarity during individual discrimination, male hamsters
were exposed to two different males. One stimulus male beat
the subject in a series of three brief fights, whereas the subject
296 Communication by Chemical Signals
became familiar with a second male by interacting across
a wire-mesh screen. This latter type of exposure is sufficient
for males to develop multicomponent memories of the stim-
ulus animal (Johnston and Peng, 2008). After both of these
experiences, male subjects were then tested for their responses
in a Y-maze with odors and other cues from the two stimulus
animals. Subjects were attracted to the odors and other cues
from a familiar neutral male but were hesitant to approach
the odors of the familiar winner or stay near him (Lai et al.,
2004, 2005). These results indicate specific types of response
to two different individuals that have different significance to
the subject but have equivalent (or at least very similar)
levels of familiarity. These results are based on a relatively
simple set of procedures and experiences compared to the
number and potential complexity of experiences in the wild.
One disadvantage of habituation tasks is that a failure to
show dishabituation to a novel stimulus may have two
different interpretations: (1) the animal did not discriminate,
or (2) the animal did not investigate the stimulus because of
other motivational/emotional reasons, for example, if the
novel stimulus came from an animal that had beaten the
subject in a fight the subject might be reluctant to investigate
an odor from the familiar winner. Thus, failure to discriminate
in habituation
dishabituation tasks may be difficult to
interpret.
Both trained discrimination and the basic habitua-
tion
dishabituation tests are primarily useful for determining
whether animals discriminate between and remember indi-
vidual signatures. If one wants to determine other types of
information, such as the content of the memory or the
emotional salience of a memory for an individual, other
methods of testing are necessary. After individuals interact
with one another, what do they remember about each other?
Two types of information may be valuable to remember: (1)
memories that incorporate several different characteristics of
the individual, such as several separate odors or other cues
(sound of voice, visual features) and (2) the emotional or func-
tional significance of the individual to the subject.
1.10.3.2.2 Two Primary Polymorphic and Multigenic
Complexes
There are two primary polymorphic and multigenic complexes
that are important in studies of olfactory communication that
contribute to or determine individual differences in odors.
These complexes are the major histocompatibility complex
(MHC) and the major urinary proteins (MUPs). MHC genes
produce highly polymorphic glycoproteins involved in immune
system function (Yamazaki et al., 1980). MUPs are mostly
produced in the liver and become concentrated in the urine in
mice. MUPs are nonvolatile molecules, but they bind smaller,
volatile molecules and release them slowly (Hurst and Beynon,
2004), thus prolonging the effectiveness of volatile compounds
contained in scent marks (Hurst et al., 1998). Without such
binding the volatile compounds might be lost from the scent
mark relatively quickly (in minutes), which would decrease
the information contained in the scent mark (Hurst and
Beynon, 2004). By being bound to MUPs, these signaling vola-
tiles can emanate from the scent mark for up to 24 h
(Humphries et al., 1999). MUPS also contain individually
distinctive information on their own (Hurst et al., 2001).
In fact, the components involved in individual discrimination
either are the MUPs themselves or the MUP
ligand complexes,
rather than the volatiles emanating from a scent mark (Nevison
et al., 2003).
Studies have also shown that some subsets of MUPs
found in scent marks of male house mice and rats provide
information about the identity of the sender, essentially
a ‘bar code’ (Gómez-Baena et al., 2014; Hurst et al., 2001).
This information is stable over time and individual as well
as species-specific and appears to be based on the individ-
ual’s genotype and not its phenotype (Cheetham et al.,
2007; Green et al., 2015; Hurst et al., 2001). Recently, Stow-
ers and colleagues discovered that wild male house mice
expressed their own unique combination and ratio of
MUPs in their urine and that the subsets of MUPs were indi-
vidually distinct (Kaur et al., 2014). For example, Kaur et al.
(2014) discovered that wild male house mice learn their own
subset of MUPs that provide individually distinct informa-
tion and distinguish it from those found in the urine of other
male house mice. Male house mice used this information to
avoid countermarking their own scent marks and those of
male conspecifics whose subset of MUPs is similar to their
own. In contrast, male house mice countermarked the scent
marks of conspecifics whose composition of MUPs was
unlike their own (Kaur et al., 2014). Male house mice,
however, did not countermark the scent marks of male
conspecifics that they had learned to be socially dominant
to them (Kaur et al., 2014). In addition, in a recent study
Green et al. (2015) found that MUPs, which act as a genetic
marker, can also be used by female house mice to select
closely related females as nest partners to help look after
their offspring and that they can do so without being familiar
with those close relatives.
Mice were shown to spontaneously notice differences in the
odors of conspecific mice with different MHC types (Penn and
Potts, 1998b). Norway rats also discriminated body odors
based on slight genetic differences in the MHC (Brown et al.,
1987, 1989, 1990). These results demonstrate that genes in
the MHC complex do influence body odors and mice and
rats can discriminate between the odors of individuals with
different MHC types. However, later studies that specifically
tested whether mice can use MHC-related odors to recognize
the owner of a given scent found negative results (Cheetham
et al., 2007; Hurst et al., 2005). In these studies, MUPs were
involved in individual discrimination but the MHC was not
(Cheetham et al., 2007). It is important to notice that MHC-
related odors are affected by such factors as status and diet
(Schellinck et al., 1997) and thus are not a good candidate to
offer a stable individual chemical fingerprint (Hurst et al.,
2005). Therefore, there seems to be no clear evidence that the
MHC of an animal offers an individual signature that is
perceived by conspecifics. In addition, Green et al. (2015)
found that the vertebrate-wide MHC was not involved in kin
recognition in house mice.
MUPs appear to be a better candidate for individually
distinctive information, at least in mice (Hurst and Beynon,
2004), because the pattern of MUPs expressed by an individual
is unchanged and thus provides a constant individual signature
not affected by factors such as diet or social status (Nevison
et al., 2003). For example, when two individuals share the

same pattern of MUPs, odors of one mouse do not trigger
competitive behaviors in the other (Hurst et al., 2001). MUPs
from other conspecifics have also been shown to elicit more
countermarking than self-MUPs (Kaur et al., 2014). Also,
when a purified MUP is added to the urine of a male, he treats
his own modified urine as if it belonged to an intruder male
(Hurst et al., 2001).
A recent study challenges the fingerprint properties of MUPs
(Thoß et al., 2015). In this study the MUP profiles were more
similar between brothers than between unrelated males.
However, the MUP profile of a given individual changed over
the course of several weeks, supporting more a ‘dynamic
expression’ hypothesis than a ‘barcode’ hypothesis. Further
studies will need to determine if mice in natural conditions,
i.e., having the ability to investigate the scent marks of neigh-
boring conspecifics in a daily basis, adjust their memories of
individual neighbors according to the slowly changing compo-
sition of their scent marks. That is, it is possible that animals
recognize individuals by investigating relatively dynamic
signals instead of fixed ones.
Although MUPs have been described and thoroughly
studied in the house mice, similar proteins can also have equiv-
alent semiochemical functions in other species (Gómez-Baena
et al., 2014). A particular MUP, Darcin, also found in the urine
of male house, plays a particular role in chemical communica-
tion. Roberts et al. (2010) found that Darcin stimulated very
rapid learning of spatial cues associated with its location in
male and female house mice. Female mice recalled the location
where they encountered Darcin and later returned to that area,
presumably to locate that male donor. Similarly, male mice
also rapidly learn and remember the location of rival male
scents and drive off these rivals or over-mark their scent marks
(Roberts et al., 2010). Mice that encountered Darcin scent only
once would return to that location 2 weeks later even if the Dar-
cin scent was absent (Roberts et al., 2010).
Some species of male mammals do not produce large quan-
tities of MUPs. Instead, some animals such as Syrian hamsters
produce a proteinaceous compound referred to as Aphrodisin
(Singer et al., 1986). Exposure to Aphrodisin from the vaginal
secretions of female hamsters was sufficient to increase copula-
tory behavior among male hamsters (Macrides et al., 1986). In
a recent paper, Stopková et al. (2010) found that Aphrodisin
like proteins are produced by prostate, preputial, and salivary
glands, and liver and uterus in male and female bank voles,
Myodes glareolus. Stopková et al. (2010) also identified three
novel odorant-binding proteins, Obp1, Obp2, and Obp3, in
urine and saliva. Stopková et al. (2010) suggested that due to
their high sequence homology (i.e., Obp1, Obp2, and Obp3
with Aphrodisin) Obps may have capacity to bind ligands
similar to those bound by Aphrodisin like proteins. If so,
Aphrodisin-like proteins and their ligands binding proteins
are likely to play a role in chemical communication similar
to that of MUPs in attracting mates (Humphries et al., 1999;
Kaur et al., 2014; Roberts et al., 2010).
1.10.3.3 Kin Recognition
The main functions of kin recognition involve nepotism and
inbreeding avoidance. Kin recognition allows an individual
to focus its cooperative behavior toward extended kin and to
Communication by Chemical Signals 297
focus aggressive behavior toward nonkin as a means of
increasing its inclusive fitness. Many animals use chemical
signals from conspecifics to differentiate between kin and non-
kin. For example, odors of kin elicit lower levels of agonistic
scent marking (flank marking) in hamsters than did odors of
nonkin (Heth et al., 1998). Recognizing kin also allows an indi-
vidual to avoid mating with closely related individuals,
achieving optimal outbreeding, and thus reducing inbreeding
depression in its offspring (Nunes, 2007).
A special aspect of kin recognition is that of parent

offspring recognition. Mutual recognition between a mother
and her offspring is likely to be beneficial to both parties under
most scenarios. Most if not all senses may be involved in paren-
t
offspring communication, but olfaction is especially impor-
tant, particularly in rodents (Porter, 1983) but also in other
mammalian groups such as ungulates (Grau, 1976) and carni-
vores (Pitcher et al., 2011). In altricial species, neonates remain
blind and deaf during the first days, whereas they can already
process scents from the mother and from other pups (Porter,
1983). Rodent pups may rely on olfaction to locate the mother’s
nipples as shown by olfactory bulbectomy of pups impairing
nipple location (Porter, 1983). In rabbits, a pheromone
emanating from the mother’s ventrum triggers a stereotyped
behavior in the pups that guides them toward the mother’s
nipples and suckling (Hudson and Distel, 1983). This volatile
pheromone is sensed by the main olfactory system of the young
(Hudson and Distel, 1986) and it is also involved in reinforced
learning (Coureaud et al., 2006; Schaal et al., 2003). The emis-
sion of such a pheromone is hormonally controlled (González-
Mariscal et al., 1994; Hudson et al., 1990). The source of the
pheromone 2-methylbut-2-enal is milk (Schaal et al., 2003).
Yet, even females that are not producing milk, i.e., estrous
or pregnant, can still produce an olfactory cue that triggers
the same behaviors as those seen following exposure to
2-methylbut-2-enal (González-Mariscal et al., 2016).
Mothers can identify the odors produced by their offspring.
For example, human mothers can distinguish the odors on T-
shirts of their children from those of unfamiliar children of
the same age and sex (Porter and Moore, 1981). Using
a similar methodological approach mothers, but not fathers,
were able to recognize the odor of their children better than
by chance (Ferdenzi et al., 2010). Many studies on goats and
sheep, two precocial species, have established that mothers
use olfactory information to discriminate between their own
newborns and those of other females, although olfaction is
not the only sensory modality involved in such discrimination
(Lévy et al., 2004; Romeyer et al., 1994; Viviers et al., 2014).
Mothers may also mark their offspring for later recognition.
Female Mongolian gerbils use their ventral gland to scent
mark their own pups. Females will scent mark their pups if
they have been licked and groomed, and females will
preferentially retrieve pups marked with their own secretion
compared to unmarked pups (Wallace et al., 1973).
1.10.3.3.1 Mechanisms of Kin Recognition
There are at least three mechanisms by which an individual can
use odors to recognize kin: familiarity-based recognition,
phenotype matching, and self-referent phenotype matching.
In familiarity-based recognition, animals learn the characteris-
tics of others that they grow up with and later in life treat only
298 Communication by Chemical Signals
these individuals as kin. This type of learned recognition can
only be useful for actual kin recognition in species in which
litters of one female’s pups are physically separated from the
litters of other females so that developing pups are only
exposed to kin, and in which future kin interactions are
restricted to the mother and siblings. This type of recognition
can be demonstrated by using a cross-fostering design in which
pups grow up with siblings or foster siblings from another
litter. When these individuals are adult, results showing that
individuals treat nonsiblings that shared their nest like kin indi-
cate that the mechanism underlying such recognition is the
association in the nest.
In phenotype matching, animals learn the phenotype of the
mother and siblings and later compare those phenotypes with
the phenotypes of unfamiliar individuals, treating unfamiliar
individuals as kin if there is a close match between phenotypes.
Phenotype matching in which features of mothers can be used
as a template occurs in Belding’s ground squirrels, Spermophilus
beldingi (Mateo, 2009). Young were exposed to odors from an
unrelated mother during early stages of development and
before natal emergence. These young chose as play partners
not only their littermates but also juveniles from the unrelated
mother, indicating that the odors of the unrelated mother were
incorporated into the kin template of the subject young.
A third mechanism is self-referent phenotype matching, in
which an individual compares its own phenotype (e.g., odors)
to that of other conspecifics. If there is a high correlation
between these characteristics, other individuals are treated as
kin. This method of kin recognition may be found in species
with multiple paternity or maternity, where full-siblings and
half-siblings may share a nest. It also may occur in species in
which pups of unrelated females can mix early in life. In this
type of situation, recognition by association may not be a reli-
able method to establish kinship. Self-referent phenotype
matching can also facilitate optimal inbreeding and
outbreeding. An example of self-referent phenotype matching
has been shown in female Syrian hamsters. Estrous females
that had been cross-fostered shortly after birth were more sexu-
ally attracted to unfamiliar nonkin than to unfamiliar kin
(Mateo and Johnston, 2000a).
Unless using a complex experimental design, it may be
impossible to determine whether kin recognition is based on
phenotype matching or self-referent phenotype matching. For
example, female meerkats, Suricata suricatta, exposed to odors
of unfamiliar individuals discriminated between the scent of
unfamiliar kin and unfamiliar nonkin. However, it could not
be determined whether meerkats used information from known
kin or from themselves as templates (Leclaire et al., 2013).
The basis for chemical recognition of kinship must be
provided by odor similarities based on genetic similarities
between close kin (Heth and Todrank, 2000; Heth et al.,
1998, 1999, 2001; Todrank and Heth, 2003; Todrank et al.,
1998). In ring-tailed lemurs, Lemur catta, for example, there is
a positive association between chemical and genetic differences
between individuals (Boulet et al., 2009), and males respond
more to the odor of less-related females than to those of
more-related females, although only during the breeding
season (Charpentier et al., 2010). Both the MHC and the
MUPs seem to be involved in kin discrimination (Thoß et al.,
2015; Yamazaki et al., 2000). Female mice are more likely to
retrieve pups with a similar MHC (Yamazaki et al., 2000).
Pups also show a preference for the odors of the mother and
siblings, which supposedly share a similar MHC, over the odors
of individuals with a dissimilar MHC (Yamazaki et al., 2000).
Little is known about the specific mechanisms beyond the role
of MUPs and MHC genes. Nonetheless, it is clear that genetic
similarity does translate into odor similarity. Many genes
must have roles in producing odors, since many different meta-
bolic processes can be reflected in the output of scent glands
and other secretory and excretory products.
1.10.3.4 Group Discrimination
Recognition of group members is important in social species in
which agonistic behaviors are directed preferentially to alien-
group individuals. Group discrimination may occur by individ-
ually recognizing each member of the own group and/or by
means of a shared group odor. Not in all social species,
however, there may be a need to discriminate between the
scents of other groups. For example, in banded mongooses,
Mungos mungo, there is no difference in the composition of
chemical signals from different groups, and individuals do
not discriminate between the scents of different groups of
similar familiarity, i.e., either two familiar neighboring groups
or two unfamiliar groups (Jordan et al., 2010).
The scent of a group can be conferred by a high degree of
genetic similarity, either due to kin relationships (e.g., family
group) or to high levels of inbreeding. If odors are diet-
related and all individuals from a group share the same food
source, this can also create a shared group odor (Halpin,
1986). A group odor can also be produced by mixing the scent
of all individuals together on each individual by scent marking
one another (allomarking), or merely by being in contact with
one another or by scent marking on the same sites. For
example, European badgers, Meles meles, produce a secretion
in the subcaudal pouch which contains distinctive group
membership information (Buesching et al., 2003). This secre-
tion is partly produced by bacterial flora. Group members
transfer such flora between one another by pressing the subcau-
dal pouch against the body of another group member. In some
cases, two individuals press both pouches together, so that the
bacterial flora and/or secretions are transferred between the
subcaudal pouches of the two individuals. The authors
observed thousands of instances of allomarking between 40
individuals in natural conditions, indicating the regularity
and importance of this behavior (Buesching et al., 2003).
Another example is the European rabbit, which lives in close-
knit social groups (Mykytowycz, 1968, 1970). Dominant males
mark all members of the group with their chin glands. If a rabbit
from one group is experimentally scented with scent from
a dominant male of another group, and then introduced
back into its natal group, it is attacked (Mykytowycz, 1968).
Dominant males even attack females of other groups and
females of his own group that are scented with the odors of
a male or female from another group (Mykytowycz, 1968).
1.10.3.5 Species Discrimination: Competitors and Predators
Discriminating between individuals of one’s own species and
individuals of a closely related species is common across taxa

since individuals who mate with other species usually do not
produce young or may produce infertile young. Female prefer-
ence for odors of conspecific males and male attraction only to
odors of conspecific females may serve as a precopulatory
isolating mechanism resulting in the avoidance of interspecific
mating. Indeed, there are many species that show female pref-
erences for odors of conspecific males over odors of closely
related heterospecific males (Johnston, 1983), including deer
mice (Doty, 1973; Moore, 1965), collared and brown
lemmings (Huck and Banks, 1980), the fossorial mole rat, Spa-
lax ehrenbergi (Nevo et al., 1976), and two species of sac-winged
bats, Saccopteryx bilineata and Saccopteryx leptura (Caspers et al.,
2009).
In pairs of closely related species in which hybridization is
possible, individuals may not only show a preference for
conspecific over heterospecific odors, they may even show
a preference for conspecific odors over those of hybrids. Such
preferences has been shown in the well-studied hybrid zone
between Mus musculus musculus and Mus musculus domesticus
across Europe (Latour et al., 2014). Male musculus mice around
the hybrid zone showed a preference for the urine of conspe-
cific females over the urine from hybrid females (Latour
et al., 2014). Interestingly, such preference did not occur
when subject males were from allopatric populations (where
hybridization does not take place), suggesting that in the
sympatric populations there has been a process of character
displacement involving signal perception or processing, or
preference response (Latour et al., 2014).
Further support for the notion that female preferences for
odors of conspecific males may have the ultimate function of
avoiding interspecific mating comes from several studies
showing that preference for conspecific odors occurs only
when females are sexually receptive. This effect has been
shown, for example, in fossorial mole rats (Nevo et al.,
1976), and deer mice (Doty, 1972). In the striped mouse,
Rhabdomys pumilio, females of three populations showed a pref-
erence for the odors of males of the same population over those
of other populations, but only when females were in estrus
(Pillay, 2000).
Closely related species may not only pose the risk of intra-
specific mating. Given that closely related species may still
share many similarities in their ecology (e.g., feeding habits),
they may be direct competitors between closely related species.
For example, two sympatric species of Acomys spiny mice have
similar foraging needs. As a result, one species (A. russatus)
shifts its foraging activity from nocturnal to a less optimal
diurnal foraging activity when A. cahirinus is present in the
same area. Odors of A. cahirinus in a particular area also reduce
the time that A. russatus will spend in that area (Baudoin et al.,
2013).
Prey
predator relationships can also promote the need for
the recognition of odors of different heterospecific species.
Both predators and prey can benefit by recognizing the odors
from each other. Predators will benefit by recognizing and
tracking down the odors of prey, whereas prey may benefit
by recognizing the odors of predators and avoiding areas
recently marked by predators.
From the prey perspective, scent marking may suppose
a trade-off between the benefits accrued by intraspecific
communication and the cost of advertising its presence to
Communication by Chemical Signals 299
predators. A flexible solution to this trade-off by many prey
species is to decrease scent-marking rates under high predation
risk (Hughes et al., 2010; Roberts et al., 2001; Rosell and Sanda,
2006), or to avoid the areas or the paths containing those pred-
ator odors (Gorman, 1984; Je˛ drzejewski et al., 1993; Roberts
et al., 2001; Rosell and Sanda, 2006; Vlautin and Ferkin,
2012). Mice do not change their scent-marking rate in the pres-
ence of predator cues, but they tend to cluster their scent marks
when there are predator cues (Hughes and Banks, 2010).
However, some prey species that are heavily preyed upon
may not change their behavior in response to the odors of pred-
ators. The reason for this is that the risk of predation may
always be high. It may be impossible for these prey species to
avoid areas marked by predators and counterproductive to
decrease scent marking. Instead, prey species may change
foraging locations (Orrock et al., 2004; Wolff, 2004a) and
increase vigilance (Buchanan-Smith et al., 1993; Monclús
et al., 2005). Finally, males and females may respond differ-
ently to the odors of predators in a way that reflects their
different mating strategies and the level of risk that they are
prepared to undertake. Normally, it is likely that males will
take riskier approaches if their reproductive fitness would be
negatively affected otherwise. For example, female field voles,
Microtus agrestis, avoid areas containing the scent marks of pred-
ators (Ronkainen and Ylönen, 1994), whereas males do not
avoid such areas if they also contain the scent marks of females
(Norrdahl and Korpimäki, 1998).
Recognition of odors from heterospecific species can be
innate, learned during the first stages of development, or
learned during adulthood (Griffin et al., 2000). Male brown
and collared lemmings that were raised by their mothers
preferred odors of conspecific females in adulthood. However,
when pups of these two species were cross-fostered, males
showed a preference for the female odors of the cross-
fostered species, i.e., heterospecific odors (Huck and Banks,
1980). Females that were not cross-fostered also showed
a strong preference for conspecific male odors, but cross-
fostered females did not show any longer a preference between
conspecific and heterospecific male odors (Huck and Banks,
1980). It is however important to notice that any preference
between the odors of conspecific and heterospecific opposite-
sex individuals may not necessarily relate to mating decisions.
Syrian hamster females showed an innate preference for the
odors of conspecific males over those of heterospecific males
(delBarco-Trillo et al., 2009a). However, female hamsters that
have never interacted with heterospecific males will respond
sexually in a similar way to both conspecific and heterospecific
males (delBarco-Trillo et al., 2009b). Interestingly, when adult
females were exposed to a heterospecific male during 4 or
8 days (one or two estrous cycles) behind a wire-mesh barrier,
they stopped mating with heterospecific males. These female
hamsters started to behave in a way that reflects their innate
odor preference for conspecific males (delBarco-Trillo and
Johnston, 2011; delBarco-Trillo et al., 2010).
1.10.3.6 Sex Identification
Sex identification based on chemical signals must rely on differ-
ences in the composition of specific scents from male and
female donors. However, information about the chemical basis
300 Communication by Chemical Signals
of such differences is surprisingly limited. There are, however,
a few cases in which active compounds involved in sex discrim-
ination have been characterized. In mice, two farnesene
compounds (E,E-a-farnesene and E-b-farnesene) are found in
male preputial glands, and sexually experienced female mice
are attracted to these farnesenes when added to bladder urine
or water (Jemiolo et al., 1991). Another compound (methyl-
thio) methanethiol is found in male urine but not in female
urine. This compound is attractive to females and may be
important in the attraction of estrous females to adult males
(Lin et al., 2005). The MUP Darcin is also only found in
male urine and females are attracted to it. Darcin is also
involved in odor learning, reinforcing the attraction of females
to male volatile compounds that might not elicit otherwise an
innate attractive response in females (Roberts et al., 2010).
Several studies have shown that females show a preference
for odors of males with dissimilar MHC types compared to
those of males with their own MHC type (Penn and Potts,
1999). By mating with males that have a different MHC type,
females may produce MHC-heterozygous offspring and such
offspring should be able to respond more effectively to
a wider range of pathogens (Penn and Potts, 1998a). Also,
a preference for a male with a different MHC type reduces
inbreeding and produces offspring that are more
heterozygous across all of the genome, not just in the MHC
(Brown and Eklund, 1994; Potts and Wakeland, 1993). Most
of the studies on the role of the MHC in mate choice have
been conducted using inbred laboratory mice that are
genetically identical except at the MHC locus (Brown et al.,
1990; Yamazaki et al., 1980). In these studies, male mice
show the ability to discriminate between the odors of two
conspecific females that have a dissimilar MHC (Yamazaki
et al., 1980). Studies with laboratory rats show similar results
(Brown et al., 1990). Female mice in estrus also show
a preference for the odors of males with a dissimilar MHC
when tested in a Y-maze (Egid and Brown, 1989).
Interestingly, females that were not in estrus did not show
such a preference (Egid and Brown, 1989). Women in the
fertile phase of their menstrual cycle also find the odors of
men that are the most dissimilar in their MHC the most
attractive and pleasant (Wedekind et al., 1995). When
women are taking oral contraceptives, such preferences are
reversed, i.e., women then prefer the odors of men with
a similar MHC type. Contradicting some of the previous
literature on mice, studies using wild mice living in large
enclosures have shown that MHC is not a relevant marker
that animals use for inbreeding avoidance, whereas MUPs
were sufficient to explain inbreeding avoidance (Sherborne
et al., 2007).
1.10.3.6.1 Endocrine Effects
Hormones such as testosterone and estradiol influence the
attractiveness of odors of the opposite sex. Gonadectomy
usually reduces the attractiveness of opposite-sex odors and
the attractive properties of such odors can be restored by
replacement therapy. Similarly, ovariectomy often reduces
responses of males to female odors and replacement treatment
with estradiol often restores the attractiveness of these odors
(Ferkin and Johnston, 1993). When several body odors contain
information about the sex of an individual, the degree to which
each odor is affected by steroid hormones can vary. In meadow
voles, odors from feces, mouth, and the posterolateral region
were clearly affected by testosterone concentrations in the
blood of males and by estradiol in females. Gonadectomy of
scent donors eliminated preferences for these odors by
opposite-sex subjects and these preferences were restored by
replacement therapy with testosterone for male donors and
estradiol for female donors. In comparison, the attractiveness
of urine and anogenital odors were reduced but not eliminated
by gonadectomy (Ferkin and Johnston, 1993).
1.10.3.7 Social Effects
1.10.3.7.1 Role of Odors in Social Dominance and Aggression
Mammals can also learn the identity of dominant males, which
affects the location and frequency of scent marking by subordi-
nate males. Dominant male rodents mark more often, along
the boundaries of territories and on prominent objects that
serve as sign posts. Subordinate male house mice and snow
voles, Chionomys nivalis, however, scent mark at a much lower
rate, and diffusely through the territory and usually not at
sign posts (Drickamer, 1995; Luque-Larena et al., 2001,
2002). Similarly, socially dominant female primates deposited
more scent marks than did socially subordinate female
primates (Kappeler, 1998; Pochron et al., 2005). Several studies
on house mice have shown that their response to the scent
marks of conspecifics depends on their ability to distinguish
between dominant and subordinate individuals. For example,
Hurst et al. (1993) tested the hypothesis that the scent marks
of subordinate mice maintain social tolerance between familiar
mice living in the same group. Hurst et al. (1993) found that
after investigating the scent mark of a familiar subordinate,
the resident male mouse would attack that individual. If the
scent mark was from an unfamiliar, subordinate male, the resi-
dent dominant male would investigate the scent marks of all
the subordinates in his territory. If, however, the scent mark
was from an unfamiliar, dominant male, the resident over-
marked it and would flee if it encountered that male. If the
scent mark was from a familiar, dominant male that was
a neighbor, the resident male would ignore the scent mark
(Hurst et al., 1993). This suggests a ‘dear enemy’ effect, in
which residents ignore the cues and signals of neighbors to
reduce confrontations with them (Rosell and Bjorkoyli, 2002;
Wyatt, 2014). In addition, male house mice that deposited
more scent marks had higher reproductive success than other
males (Thonhauser et al., 2013b). This observation indicates
that scent marking is maintained by sexual selection, as it
enhanced males’ reproductive success. Studies on a variety of
terrestrial mammals suggest that scent marks appear to be
a secondary sexual trait, which conspecifics use to assess the
quality of a potential mate and competitors (Ferkin, 2015;
Roberts et al., 2014; Thonhauser et al., 2013a,b; Tinnesand
et al., 2015).
In species that live in permanent social groups, there are
often well-established dominance relationships between males
in a local area and dominance rank is often related to the ability
of one male to monopolize or have preferential access to
resources. Females should prefer dominant males as mates
because at least some of the qualities of dominant males may
be based on genetic characteristics that may be passed on to

offspring. If odor parameters (e.g., testosterone metabolites)
are related to male dominance, females may be able to distin-
guish between dominant and subordinate males just by inves-
tigating their odors. For example, female brown lemmings
showed a preference for odors of dominant males over odors
of subordinate males, even when the females were not familiar
with the two male stimulus animals before testing (Huck and
Banks, 1982; Huck et al., 1981). Female brown lemmings
also preferred odors of socially naïve males that later became
dominant over the odors of socially naïve males that later
became subordinate, suggesting that the differences between
males were at least partially determined by genetic differences
(Huck et al., 1981). Female European rabbits also show a pref-
erence for the odors of dominant males (Engel, 1990). These
types of results all indicate that there are some constituents
of male odors that correlate with social status, possibly related
to levels of testosterone, cortisol, or other stress-related
hormones (Engel, 1990; Huck et al., 1981).
Subordinate individuals will also benefit by discriminating
between subordinate and dominant individuals and respond-
ing accordingly in order to reduce aggression from dominant
individuals. In many species, including Syrian hamsters, mice,
rats, lagomorphs, and canids, individuals avoid areas marked
by another individual that defeated them in a dyadic encounter,
whereas winners do not normally avoid areas marked by indi-
viduals they defeated in such encounters (Drickamer, 2001;
Petrulis et al., 2004). Reactions to odors of dominant or subor-
dinate males may be determined by information in the odor
that identifies an animal as having a dominant status or not.
Before the establishment of a polarized status relationship
between two male snow voles, both males investigated areas
scent marked by the other male similarly. However, after the
establishment of a dominant
subordinate relationship, the
subordinate male investigated the area scented by the dominant
male much less than the dominant male investigated the area
scented by the subordinate male (Luque-Larena et al., 2002).
These results can be explained either by individual recognition
or by a reaction to information in the odors about status. Other
results show that cues to status do influence the behavior of
animals. For example, group-housed male mice avoid areas con-
taining urine of a dominant male, whereas urine of a subordi-
nate male or water in the area results in much more time
spent in a similar test chamber (Jones and Nowell, 1973).
Also, urine of a dominant male spread on a castrated male trig-
gers aggressive behavior in other conspecifics, whereas subordi-
nate urine spread on a castrated male does not promote
aggression in other conspecifics (Jones and Nowell, 1973).
After a subordinate
dominant relationship is established,
odors can be used to advertise dominant status. In many
species, dominant individuals scent mark more than subordi-
nate individuals (Ralls, 1971). A larger number of marks or
a greater density of marks by one male may signal dominant
status within the area that was marked (Gosling, 1990; Hurst,
1987; Rich and Hurst, 1999). Even in the absence of qualitative
differences in the chemical composition of their odors between
dominant and subordinate individuals, quantitative differ-
ences in marking may be used by conspecifics to determine if
a particular individual has a dominant status or not. Some
examples of species in which dominant individuals scent
mark more than subordinates include the house mice
Communication by Chemical Signals 301
(Desjardins et al., 1973), Syrian hamsters (Ferris et al., 1987;
Johnston, 1975a,b), Mongolian gerbils (Thiessen et al.,
1971), stoats, Mustela erminea (Erlinge et al., 1982), European
rabbits (Mykytowycz, 1965), sugar gliders, Petaurus breviceps
(Schultze-Westrum, 1969), marmosets, Callithrix jacchus (Ralls,
1971), and ring-tailed lemurs (Kappeler, 1990).
Instead of or in addition to differential marking rates, the
dominant status of an individual may be coded by odors
having a chemical composition that differs from that of subor-
dinate individuals. For example, the chemical profiles of anal
scent gland secretions from female spotted hyenas differ
depending on whether females have a low social ranking or
a high ranking (Burgener et al., 2009). The functional impor-
tance of these chemical differences is suggested by the fact
that adult females respond to secretions of other females faster
and more often and are more likely to anoint themselves with
those secretions if they were produced by a high-ranking female
(Burgener et al., 2009). In beavers the chemical composition of
anal gland secretions also differs between dominant and subor-
dinate individuals, and beavers can use those chemical differ-
ences to distinguish between dominant and subordinate
unfamiliar intruders (Tinnesand et al., 2013). Similarly, in
house mice, the more aggressive females excreted a higher
amount of MUPs in their urine (Stockley et al., 2013).
Normally, larger males are dominant to smaller males
because size gives them an advantage during competitive
displays and fights. However, other factors may also influence
which male becomes dominant. Smaller male mice, for
example, may initially become dominant over larger males
by investing more in preputial glands and in scent marking
than larger males (Gosling et al., 2000). However, this strategy
is energetically very costly – a high investment in scent marking
leads to slower growth and lower adult body weight, so these
small males are vulnerable to reversal of dominance status later
in life (Gosling et al., 2000).
The odors that a male normally deposits in his territory,
home range or in the vicinity of a burrow can serve as a confi-
dence-building context in aggressive interactions. For example,
male Syrian hamsters took more time to enter an enclosure
when another male’s odors were present compared to a clean
area or an area scented by a flank-glandectomized male; subjects
also spent less time in an arena scented by an intact male
compared to a flank-glandectomized male (Alderson and
Johnston, 1975). The effect of an ‘own-odor’ or ‘home-odor’
context should be maximal when the odor in one location is
consistent with other information about location (familiar
landmarks, etc.). In experiments in which agonistic interactions
take place in an unfamiliar, neutral area, however, odors may
take on an especially important role and give an advantage to
the male whose odors are present. For example, when two
European rabbits were placed in a neutral arena containing feces
from one of the two contestants, the owner of the feces was
more aggressive, initiated more interactions and was more likely
to win the contest (Mykytowycz, 1973). Thus, the factor deter-
mining dominance was not any intrinsic feature of the winner,
but the fact of being in an environment containing the odors of
one of the males. When two female Syrian hamsters that are
unfamiliar with each other are placed in a neutral arena scented
with vaginal secretion of one of the females, the female whose
odor is in the arena is more likely to become dominant;
302 Communication by Chemical Signals
subsequently the winner does all of the vaginal marking and
most of the flank marking (Fischer and McQuiston, 1991).
When two females with an established dominant–-subordinate
relationship are placed in a neutral arena scented with vaginal
secretion from the subordinate female, a reversal in the domi-
nance relationship was observed in 8 of 12 pairs (Fischer and
McQuiston, 1991). Similarly, when the encounter between
two male Syrian hamsters takes place in the territory or home
cage of male A or in a neutral arena that contains odors of
male A, that male is more likely to win the initial encounter
and become dominant. The results of the initial encounter
have a strong effect on later encounters between the same two
individuals, even if a later encounter is in a neutral arena (Lai
and Johnston, 2002; Lai et al., 2004). In house mice, an intruder
is more submissive when there is a match between the odors on
the substrate and the other individual (e.g., a resident), than
when there is not a match between the substrate odors and
the other male (Gosling and McKay, 1990).
After the establishment of a status relationship, there are
usually differences between the dominant and subordinate
individuals in how they respond to the odors of the other indi-
vidual. For example, subordinate males may avoid odors of
dominant males, whereas dominant males usually do not
avoid odors of subordinate males. Male cavies, Cavia aperea,
that were dominant to one male and subordinate to another
male spent more time in the side of an arena containing odors
of the subordinate male than in the side containing odors of
the dominant male (Martin and Beauchamp, 1982). A male
Syrian hamster avoids odors of the male that beat him in a fight,
but this subject does not avoid the odors of either a familiar
male with whom he did not fight or an unfamiliar dominant
male (Lai et al., 2004, 2005; Lai and Johnston, 2002; Petrulis
et al., 2004). These latter results indicate that the identity of
the individual and the memories associated with a specific indi-
vidual are crucial factors that determine the type of response
observed toward an individual’s odor.
The response of dominant and subordinate individuals to
the same unfamiliar odors may also differ. For example, domi-
nant male meerkats had a stronger response than did subordi-
nate males to intruder scent marks (Mares et al., 2011).
1.10.3.7.2 Food and Odors: Diet and Food Deprivation
Across a population, different individuals may differ in their
particular diet. This may be the case even in species with
a very specialized diet, given that a particular food source
may vary in quantity and quality across an area. If the distribu-
tion of food is heterogeneous, dominant individuals are likely
to defend a territory that contains better higher-quality food
resources and thus will consume a higher-quality diet than
subordinate males. A higher-quality diet should lead to better
condition and odors that reflect such a higher condition. In
several species, subjects have been shown to prefer the odors
of opposite-sex conspecifics that have been feeding on
a high-quality diet compared to those on a lower-quality diet.
For example, both male and female meadow voles are more
attracted to odors of opposite-sex conspecifics that were fed
a high-protein diet compared to those fed a diet containing
the standard level of protein in commercial rodent food or
a low level of protein in the food (Ferkin et al., 1997b). Simi-
larly, gravid female salamanders, Plethodon cinereus, preferred
to associate with males that fed on a high-quality diet
(termites) compared to a low-quality diet (ants) and were
attracted to the feces of males on the high-quality diet (Jaeger
and Wise, 1991; Walls et al., 1989). To the extent that a male
can defend or obtain high-quality food resources and to the
extent that this ability is heritable, females who choose such
males should have higher quality offspring and greater repro-
ductive success than females that do not discriminate between
males on the basis of the diet they consume.
Phytoestrogens are nonsteroidal plant compounds that can
cause estrogenic or antiestrogenic effects on the animals that
consumed phytoestrogen-rich plants (Pierson and Ferkin,
2015). Despite the fact that such estrogenic and antiestrogenic
effects could include changes in scent marking, over-marking,
and the response of individuals to the odors of other conspe-
cifics, the impact of phytoestrogens on olfactory communica-
tion requires further investigation (Pierson and Ferkin, 2015).
Food restriction and food deprivation can have dramatic
effects on the information contained in the odors of affected
individuals. For example, long-term dietary restriction leads
to a reduction in the amount of MUPs in male mouse urine
(Giller et al., 2013). Female swordtail fish, Xiphophorus birch-
manni, show a preference for odors of males that are well fed
over the odors of males that were food deprived for 5 days
(Fisher and Rosenthal, 2006). Food restriction also affects the
highly sexually attractive odors of females in postpartum
estrus. Food deprivation or restriction prior to the beginning
of postpartum estrus renders the postpartum-estrous odors of
those females less attractive than the odors of postpartum-
estrous females that were not food deprived or food restricted
(Sabau and Ferkin, 2013a). In contrast, the odors of male
meadow voles that were food deprived for 24 h were still attrac-
tive to females (Pierce et al., 2005). Such sex differences in
meadow voles can be attributed to the different energetic
demands placed on female and male meadow voles during
the breeding season (Pierce et al., 2005, 2007).
The loss of attractivity of odors may not only result from
food restriction during adulthood but also from food restric-
tion during development. Female rat-like hamsters, Cricetulus
triton, that were food restricted during pregnancy produced
male offspring whose odors during adulthood were less attrac-
tive to female conspecifics than those of males reared by
untreated dams (Liang et al., 2004; Meikle et al., 1995). A
similar experimental approach has been used in meadow voles,
comparing the offspring reared by either untreated dams or
dams that were food restricted during early, middle, or late
lactation (Sabau and Ferkin, 2013b, 2014; Sabau et al.,
2014). Male offspring from dams food-restricted during mid-
lactation produced scent marks that were less attractive to
opposite-sex conspecifics than those of male offspring reared
by control dams or dams food-restricted during early or late
lactation (Sabau and Ferkin, 2014; Sabau et al., 2014). In
contrast, female offspring from dams that were food-
restricted during early lactation produced odors that were not
as attractive as those produced by female offspring from
control dams or dams that were food restricted during middle
or late lactation (Sabau et al., 2014). These differences between
males and females indicate that there may be different sensitive
periods for the development of scent-producing tissues in male
and female meadow voles (Sabau et al., 2014).

Food-restricted and food-deprived animals can also modify
their normal responses to conspecific odors. For example,
female meadow voles that were food deprived for 6 h or longer
stopped showing a preference for the odors of a male over
those of a female (Pierce et al., 2005).
1.10.3.7.3 Effect of Health and Environmental Stressors
on Odors
Individuals may use odors as honest signals to assess the health
status of conspecifics and choose mates accordingly (Kavaliers
et al., 2005; Penn and Potts, 1998a). A high parasite load or
active disease processes can affect the information contained
in odors, due to changes in the composition of commensal
microbes, the production of metabolites related to immuno-
logical responses and a decrease in hormonal metabolites
due to a reduction in androgen levels during infection (Penn
and Potts, 1998a). Infected individuals can also reduce their
scent-marking rates, which may reduce their attractiveness to
opposite-sex conspecifics (Zala et al., 2004). In several species,
females have been shown to be more attracted to the odors of
unparasitized males over the odors of parasitized males. For
example, female mice avoid the urine of males infected with
lice, compared to the urine of males that do not have lice
(Kavaliers et al., 2003). Female mice and rats also show a pref-
erence for urine of males without endoparasites over the urine
of males infected with diverse species of such parasites
(Kavaliers et al., 1998; Willis and Poulin, 2000). In addition,
female mice are as attracted to water as to urine of males
infected with the influenza virus, whereas the same females
are more attracted to urine of uninfected males than to urine
of infected males, which indicates that viral infection can
reduce the attractiveness of male odors (Penn et al., 1998).
Sick animals can also be more prone to predation or para-
sites. For example, mice and rats parasitized by Toxoplasma
will suffer a behavioral change induced by the parasite so
that instead of avoiding feline odors will be attracted to
them, increasing their risk of predation and the transfer of
the parasite to the predator gut where it can complete its repro-
ductive cycle (Vyas et al., 2007). Ticks show a preference for the
odors of sick hedgehogs, Erinaceus europaeus, possibly because
of the compound indole being produced in higher proportions
in the scents of sick hedgehogs (Bunnell et al., 2011).
Many species are exposed to anthropogenic substances that
can affect the synthesis of chemical signals, signal detection,
and the responses of conspecifics to those signals (Blocker and
Ophir, 2013; Lürling and Scheffer, 2007; Tuomainen and
Candolin, 2011). For example, female palmate newts, Lissotriton
helveticus, preferred odors of unexposed males to those of males
exposed to nitrates (Secondi et al., 2009). Similarly, pesticides
disrupt sexual olfactory communication and mate-choice
behaviors in red spotted newts, Notophthalmus viridescens
(Lürling and Scheffer, 2007). Agricultural runoff and sewage
effluents can alter mate choice in the swordtail fish, Xiphophorus
birchmanni, leading to interspecies mating and hybridization
with the closely related species Xiphophorus malinche (Fisher
et al., 2006). Chemicals can also interfere with the perception
of chemical signals; male Atlantic salmon exposed to anti-
androgenic insecticides suffer a reduced response to female
pheromones (Tierney et al., 2010). In regards to predation,
tadpoles of the Columbia spotted frog, Rana luteiventris, exposed
Communication by Chemical Signals 303
to heavy metals decreased their fright response to the odors of
predators (Lefcort et al., 1998). The endocrine system can be
involved in the production and perception of scents. Therefore,
exposure to compounds affecting the endocrine system can have
a direct effect on olfactory communication. This includes
substances that unintentionally enter in the animals, but it
also includes hormone-based contraceptives. In ring-tailed
lemurs, a contraceptive altered the composition of odors, elim-
inating individual-specific olfactory information, and rendered
these odors less interesting for males than were odors from
control females (Crawford et al., 2011).
1.10.3.7.4 Age and Odors: From Infancy to Senescence
Fell et al. (2006) studied social integration in European badger
cubs from their time of emergence to full independence. They
discovered that as cubs grew older they interacted more with
adults, initiating a greater number of social interactions with
adults. These cubs would mark themselves with the subcaudal
gland secretions of adult group members. Fell et al. (2006)
referred to this behavior as scent-theft and concluded that
cubs do so to carry a group scent. Such a group scent would
allow the cubs to become integrated into the social group
and be recognized by adults.
Due to their particular endocrine state, juvenile individuals
may produce odors that are not yet attractive to adult conspe-
cifics. Some scent glands may not even have developed and
started to produce attracting compounds. On the other hand,
a lack of adult, aggression-eliciting odors may provide juveniles
with protection against aggression from adults. In Syrian
hamsters, adult males paired with a juvenile male in a neutral
arena spent most of their time investigating the anogenital area
of the juvenile and were not aggressive against that juvenile,
while the juvenile male raised his tail and assumed
a lordosis-like position (two behaviors that are typical in
estrous females). Adult males in this species spend little time
investigating the anogenital area of other adult males, so it is
possible that the anogenital odors of juvenile males share
some similarity with those of adult females or at least contain
some compounds that elicit a nonaggressive response from
adult males (delBarco-Trillo et al., 2011b; Figure 4).
Females in many species show a preference for the odors of
adult males over those of males around puberty. For example,
the chemical composition of urine from prepubescent and
adult male rats differs in the quantities of three volatile
compounds, and female rats are more attracted to the urine
of adult males than the urine of prepubescent males (Osada
et al., 2009). Female meadow voles also prefer odors of older
males, possibly because such males tend to be heavier, have
larger testes, and be in better condition (Ferkin, 1999).
However, as males start to senesce and their condition starts
to decrease, the attractivity of their odors may also decrease.
This may be the case because a decrease in general condition
gets reflected in the composition of their odors, or because sen-
esced males cannot invest as many resources into the produc-
tion of costly sexual signals as younger males can. In mice,
urine of senescent males contains a lower concentration of
MUPs and of the androgen-dependent volatile compounds
brevicomin and thiazole compared to middle-aged males
(Garratt et al., 2011). When urine samples of senesced males
and middle-age males are freshly deposited, females do not
304 Communication by Chemical Signals

Figure 4 Effect of juvenile age on the time that adult males investigate anogenital and flank areas in Syrian hamsters. Data represent the percentage
of sniffing time during which the adult male sniffed the anogenital area of the young male. Sniffing time is an aggregate measure of anogenital area
sniffing and flank sniffing. The 50% line represents equal anogenital and flank sniffing. Values over the line represent a major percentage of anogen-
ital area sniffing, whereas values below the line represent a major percentage of flank area sniffing. Black circles indicate trials without aggression
and white circles indicate trials with adult aggression. Juveniles did not show any aggression against adult males. Reproduced from delBarco-Trillo,
J., McPhee, M.E., Johnston, R.E., 2011. Syrian hamster males below an age threshold do not elicit aggression from unfamiliar adult males. Aggres-
sive Behav. 37, 91–97, with permission from Wiley.

show a preference for the urine of middle-age males. However,
when the urine samples had been deposited for 12 h, females
were less attracted to the urine of senesced males than to the
urine of middle-age males, indicating that the reduced content
of MUPs in the urine of senesced males affects the longevity
and attractivity of the scent signals (Garratt et al., 2011).
The sensitivity to odors and the ability to discriminate
between odors of conspecifics can also decrease in aged
animals (Doty and Kamath, 2014). Such decrease in olfactory
sensitivity can be due to changes in several structures, including
the olfactory epithelium, the olfactory bulb, and central brain
regions, and several processes, including loss of selectivity of
receptor cells, changes in neurotransmitters, and neuronal mal-
function due to neurodegenerative disease (Doty and Kamath,
2014; Mobley et al., 2014). In a recent study in meadow voles,
individuals of up to 13 months of age were able to distinguish
among odors of different conspecifics and they could also
distinguish between the odors of males and females (Ferkin
et al., 2016). However, 15- to 18-month-old meadow voles
could not differentiate between two same-sex individuals,
although these older voles were still able to distinguish
between male and female odors, showing a preference for the
odors of opposite-sex conspecifics (Ferkin et al., 2016).
1.10.4 Functions of Odor Communication
1.10.4.1 Scent Marking
Individual recognition is functionally relevant across different
social and mating systems. In asocial species, recognition and
avoidance of agonistic neighboring individuals may reduce
the potential danger of physical conflicts (delBarco-Trillo
et al., 2009c). In social species, individual recognition is neces-
sary for the establishment and maintenance of social interac-
tions (Doty, 1986). In pair-bonding species, recognition of
the partner is obviously important to maintain close and
long-term relationships. In promiscuous species, an individual
must discriminate between a recent mate and other opposite-
sex individuals, in order to mate preferentially with new indi-
viduals (Coolidge effect) (Carr et al., 1970). Most terrestrial
mammals must be able to distinguish between the myriad of
scent marks they encounter as they move their habitat to find
mates, secure resources, and avoid predation. How animals
use such information has been a topic of discussion for many
years. The adaptive significance of scent marking has been pre-
sented in a multitude of papers (Ferkin, 2015; Hurst and
Beynon, 2004; Johnston and delBarco-Trillo, 2009; Roberts,
2007; Roberts et al., 2014). Several hypotheses have been sug-
gested by authors (Brown and Macdonald, 1985; Ralls, 1971;
Thiessen and Rice, 1976; Roberts, 2007).
1.10.4.1.1 Hypotheses about Function
Here, we list 15 putative functions of scent marking and its role
in communication. We provide representative citations of
recent work which relate directly to the functions (Table 1).
The first and most popular hypothesis is that scent marking
serves a role in spacing behavior, particularly territoriality.
Several studies on a variety of species, particularly involving
males, have suggested that scent marks provide a chemical
fence or deterrent that prevents intrusions in a territory by
 Communication by Chemical Signals 305

Table 1 Fifteen hypotheses that explain the role scent marking

Hypothesis Support/Species Recent References

1. Scent marks function in territoriality
2. Scent marks function in mate attraction
3. Scent marks function in providing social
status information
4. Scent marks function in providing
individually distinct information
5. Scent marks function to indicate a food
source
6. Scent marks function during interspecies
competition
7. Scent marks allow for scent matching
8. Scent marks provide a chemical bulletin
board
9. Scent marks provide an information center
10. Scent marks allow group cohesion
11. Scent marks provide information about
a social bond
12. Scent marks provide information that affects
aggression in social encounters
13. Scent marks of prey associated with those of
predators reduces prey activity in that area
14. Scent marks provide information about
species identity
15. Scent marks are used to facilitate parent

offspring bond
Eurasian beaver (Castor fiber); wolf (Canis lupus);
European badger (Meles meles)
House mice (Mus musculus); lynx (Lynx lynx);
sifaka (Propithecus candidus)
House mice; snow voles (Chionomys nivalis)
Meadow voles (Microtus pennsylvanicus); otters
(Lontra canadensis); koalas (Phascolarctos
cinereus); house mice
Hog badger (Arctonyx collaris); canids (Canis
spp.); wildcats (Felis silvestris)
Black-tailed prairie dogs (Cynomys ludovicianus)
Meriones unguiculatus, Meriones tamariscincus,
Meriones meridianus
Lynx; puma (Felis concolor); white-footed
sportive lemurs (Lepilemur leucopus)
Ocelots (Leopardus pardalis)
Red foxes (Vulpes vulpes)
African wild dogs (Lycaon pictus)
C57BL/6J mice (Mus domesticus); Rusa deer
(Rusa timorensis); barasingha (Rucervus
duvaucelii)
Cabrera vole (Microtus cabrerae)
Strepsirrhine primates
House mice; humans (Homo sapiens); sheep
(Ovis aires); rats (Rattus norvegicus)
Cross et al. (2014), Llanenza et al. (2014), and
Tinnesand et al. (2015)
Roberts et al. (2014), Thonhauser et al.,
2013a,b, Vogt et al. (2014), Drea (2015), and
Greene and Drea (2014)
Hurst et al. (1993), Stockley et al. (2013), and
Luque-Larena et al. (2001, 2002)
Ferkin et al. (2016), Kean et al. (2015), Charlton
(2015), and Kaur et al. (2014)
Zhou et al. (2015), Potts et al. (2012), and
Piñeiro and Barja (2015)
Eads et al. (2016)
Gromov (2015)
Vogt et al. (2014), Allen et al. (2014), and
Dröscher and Kappeler (2014)
Rodgers et al. (2015)
Soulsbury and Fawcett (2015)
Jordan et al. (2014)
Arakawa et al. (2015), Ceacero et al. (2015), and
Pluhácek et al. (2015)
Gomes et al. (2013)
delBarco-Trillo and Drea (2014)
Al Aïn et al. (2013), Corona and Lèvy (2015), and
Lonstein et al. (2015)

conspecifics (Brown and Macdonald, 1985; Thiessen and Rice,
1976). Many recent studies have shown partial support for this
hypothesis, in that scent marks reduce intrusions by conspe-
cifics (Cross et al., 2014; Llaneza et al., 2014; Tinnesand
et al., 2015). Another frequently proposed function is that scent
marking is used to attract mates (Johnston, 1983; Thiessen and
Rice, 1976). Many studies provide data that support this
hypothesis, particularly from those on house mice (Roberts
et al., 2014; Thonhauser et al., 2013a,b), lynx, Lynx lynx
(Vogt et al., 2014) and sifaka, Propithecus candidus (Drea,
2015; Greene and Drea, 2014). A third hypothesis is that scent
marks provide information about the social status of the sender
(Hurst et al., 1993; Luque-Larena et al., 2001, 2002; Stockley
et al., 2013). A fourth hypothesis is that scent marks provide
individually distinct information about the sender (Halpin,
1986; Thom and Hurst, 2004) in a variety of animals, including
meadow voles (Ferkin et al., 2016), otters (Kean et al., 2015),
koalas (Charlton, 2015), and house mice (Kaur et al., 2014).
A fifth function is related to defending or indicating a food
resource among hog badgers, Arctonyx collaris (Zhou et al.,
2015), canids (Potts et al., 2012), and wildcats, Felis silvestris
(Piñeiro and Barja, 2015).
It has also been hypothesized that scent marks are involved
in competition between species (Eads et al., 2016). A seventh
function is related to scent matching of conspecifics
(Gromov, 2015; Gosling, 1982; Gosling and Roberts, 2001).
Another function for scent marking is to provide a chemical
bulletin to indicate one’s presence in an area among lynx
(Vogt et al., 2014), puma, Felis concolor (Allen et al., 2014),
and lemurs (Dröscher and Kappeler, 2014). A ninth function
suggests that scent marks act as an information center (Rodgers
et al., 2015). A tenth function of scent marking is to provide
cohesion among group members (Soulsbury and Fawcett,
2015). Scent marks may also provide signals of a social bond
between conspecifics to group members (Jordan et al., 2014).
A twelfth hypothesis is that scent marks provide information
that affects the aggression displayed during paired encounters
between conspecifics (Arakawa et al., 2015; Ceacero et al.,
2015; Pluhácek et al., 2015). A thirteenth function suggests
that scent marks from prey that are associated with those depos-
ited by a predator serve as alarm signals that decrease activity in
nearby conspecifics of the prey (Gomes et al., 2013). The 14th
hypothesis is that scent marks provide information about species
identity (delBarco-Trillo and Drea, 2014). The last hypothesis is
that scent marks and odors produced by the parent and the
offspring are used to facilitate a bond or attachment (Al Aïn
et al., 2013; Corona and Lévy, 2015; Lonstein et al., 2015).
It is important to note that the 15 hypotheses listed above
may not be mutually exclusive. Some hypotheses may overlap
and may be necessary for other functions to be possible. For
306 Communication by Chemical Signals
example, it is difficult to imagine that a terrestrial mammal can
identify the scent marks of conspecifics and learn features about
the donor’s phenotype, genotype, space use, and social biology
(Ferkin, 2011; Green et al., 2015; Halpin, 1986; Johnston,
2009; Kaur et al., 2014).
1.10.4.2 Over-marking
Over-marks are typically formed when the scent marks of two
or more donors overlap (Ferkin and Pierce, 2007; Johnston
et al., 1994; Macdonald, 1980). Our knowledge about how
animals respond to over-marks, how they distinguish between
the top- and bottom-scent donors of such marks, and what
kinds of information individuals can glean about the top-
and bottom-scent donors has been discussed over the past
two decades and still continues in the present (Jezierski et al.,
2015; Jordan et al., 2014; Kaur et al., 2014; Tinnesand et al.,
2015; Vlautin and Ferkin, 2014). The pioneering work about
over-marking was carried out by Robert Johnston and co-
workers, who were interested in the perceptual mechanisms
that an animal may use to assess and differentiate between
the top-scent mark and bottom-scent mark of an over-mark.
Johnston (1994) provided essentially two hypotheses to
explain how an individual may perceive an over-mark and its
constituent components, the top-scent mark and the bottom-
scent mark. One hypothesis stated that the over-mark became
a mixture of two or more scent marks that were no longer
distinct from one another. Johnston (1994) considered this
mixture of two or more over-marks could be characterized as
new or group scent which was made up of features that re-
flected the identity of the group members rather than the iden-
tity of the individual members themselves. The other
hypothesis was that the scent marks in the over-mark did not
mix. Rather, the over-mark retains features of the individuals
that contribute to it (Johnston, 1993). In a series of elegant
experiments Johnston et al. (1994, 1995, 1997a,b) determined
whether Syrian hamsters and meadow voles treated the over-
lapping scent marks of conspecifics as if the top- and bottom-
scent mark mixed or remained distinct. To do so, Johnston
and colleagues used a habituation/dishabituation paradigm
to show that Syrian hamsters exposed to an over-mark of two
same-sex conspecifics later behaved as if they had a selective
memory for the top-scent mark but not for the bottom-scent
marks (Johnston et al., 1994, 1995). Similar results were ob-
tained when meadow voles were exposed to the over-mark of
two same-sex conspecifics (Johnston et al., 1997a,b). These
early papers suggested indicated that the two scent marks did
not mix but remained distinct. These data led to the hypothesis
that by over-marking a scent mark the actor is attempting to
physically cover the bottom-scent mark or behaviorally block
its information to subsequent receivers.
This hypothesis was tested in studies on Syrian hamsters
(Johnston and Bullock, 2001) and meadow voles (Ferkin
et al., 1999; Woodward et al., 1999). The results of these studies
were striking in that both meadow voles and Syrian hamsters
were able to also discriminate the top-scent mark from a novel
scent mark that was not part of an over-mark and the bottom-
scent mark from a novel scent mark. This suggested that the
top-scent does not block the information contained in the
bottom-scent mark but also that the voles and hamsters viewed
the top- and bottom-scent marks as being distinct from one
another but also from a novel scent mark. These findings sug-
gested that over-marks provide receivers with a chemical
bulletin board and on this bulletin board the top-scent mark
may have more value attached to it or salience compared to
the bottom-scent mark or to a scent mark that was not part
of the over-mark (Johnston et al., 1995; Woodward et al.,
1999, 2000). This finding has been repeated in subsequent
studies on these rodents (Ferkin et al., 2010, 2011a,b;
Johnston, 2003, 2009) as well as in some primates (Fisher
et al., 2003b; Heymann et al., 1998; Kappeler, 1998). The liter-
ature is also replete with observations of other rodents, insecti-
vores, ungulates, carnivores, and primates depositing their
scent marks on or near the scent marks of conspecifics (Biben,
1980; Brashares and Arcese, 1999; Heymann et al., 1998; Hurst,
1990a,b,c; Johnson, 1973; Johnston et al., 1994, 1997a; Lewis,
2005; Lisberg and Snowdon, 2009; Macdonald, 1980; Sliwa
and Richardson, 1998).
1.10.4.2.1 Top- and Bottom-Scent Donors Are Same-Sex
Conspecifics
Our knowledge about how individuals respond to same-sex
over-marks is growing. The first wave of studies examined
how conspecifics responded to same-sex over-marks in which
the top- and bottom-scent donors were similar in features of
their quality and were unfamiliar to the investigating conspe-
cific. These studies on pygmy lorises, Syrian hamsters, prairie
voles, and meadow voles showed that after individuals were
exposed to same-sex over-marks, they later displayed a selective
memory for the mark of the top-scent donor (Johnston et al.,
1994, 1995) or they preferred that mark to the mark of the
bottom-scent donor (Ferkin and Pierce, 2007; Fisher et al.,
2003a; Woodward et al., 1999).
Ferkin (2011, 2015) has extended the three original hypoth-
eses of Johnston (1994) to explain how individuals may
perceive over-marks and respond to the top- and bottom-
scent marks of two donors that are the same sex. One
hypothesis is that individuals do not necessarily discriminate
the top-scent mark from the bottom-scent mark. Instead, the
individuals respond selectively to the top-scent mark because
it was the last mark to be deposited. In essence, the preferences
for the top-scent mark would be based on how recently it was
deposited. This simple rule of thumb would work if individuals
encounter an over-mark in which the top- and bottom-scent
marks do not differ in features of their phenotype (Johnston
et al., 1994, 1995; Woodward et al., 1999) or if the top-scent
donor is socially dominant to the bottom-scent donor (Ferkin
and Pierce, 2007; Hurst and Rich, 1999; Rozenfeld et al., 1987).
The preference for the scent mark of the top-scent donor over
that of the bottom-scent donor suggests that many rodents
and some primates exposed to same-sex over-mark behave as
if they have a placed a higher value on the top-scent mark rela-
tive to that of the bottom-scent mark (Ferkin and Pierce, 2007;
Fisher et al., 2003a; Johnston, 2009). This top-scent preference
may be rules of thumb for these animals. The first rule appears
to be to prefer or have a selective memory for the top-scent
donor if the overlapping scent marks come from donors that
do not differ sufficiently in some feature of their phenotype,
such as their age, reproductive condition, social status, sex,
and diet (Ferkin and Pierce, 2007). Thus, we would expect to

see a preference for the top-scent donor of a same-sex over-
mark if higher quality donor is the top-scent donor, the resi-
dent in an area rather than an intruder, or socially dominant
to the bottom-scent donor. However, research on a variety of
rodents suggests that discriminating between and responding
to the top-scent and the bottom-scent mark of an over-mark
may be more complex than simply preferring or having a selec-
tive memory for the top-scent mark. It appears the response
chosen by the individual depends on features of its genotype
and phenotype, those of the top- and bottom-scent mark
donors, the association between the investigating individual
and the donors, and context. And, most importantly, the
response chosen represents a trade-off in the benefits and costs
associated with over-marking and fitness and survival (Ferkin,
2015).
Because over-marking may occur in high-traffic areas
(Brown and Macdonald, 1985; Ferkin et al., 2004a,b; Hurst,
1990a,b,c; Hurst et al., 1993), individuals may encounter
over-marks in which the top- and bottom-scent donors differ
in features of their phenotype and genotype. For example,
they may encounter an over-mark in which a subordinate
conspecific or an individual that is in poor nutritional health
over-marks the scent mark of a socially dominant conspecifics
or one that is in good nutritional health. However, it is possible
that lower quality individuals may over-mark the scent marks
of higher quality individuals. If this occurs, do animals still
maintain their preference for the top-scent donor over the
bottom-scent donor? If so, over-marking may not be an honest
signal but an alternative tactic that allows all top-scent donors
to gain an edge in the transfer of their olfactory information to
conspecifics, increase the value of their scent mark, or lower the
value of the bottom-scent mark. Interestingly, in those situa-
tions when the top-scent donor is lower in quality relative to
the bottom-scent donor of a same-sex over-mark, investigating
individuals no longer follow the rule of thumb to prefer the
top-scent donor to that of the bottom-scent donor or the alter-
native rule of thumb to prefer the marks of the higher quality
scent donor to those of the lower quality scent donor.
Individuals may alter their response depending on features
of the top- and bottom-scent donor. For example, meadow
voles exposed to same-sex over-marks in which the top- and
bottom-scent donors differed in gonadal steroid hormone
titers, male voles preferred the marks of females that had higher
titers of estradiol than those that had lower titers of estradiol;
independently of whether they were the top- or the bottom-
scent donor. Female voles, however, preferred the scent marks
of males with higher testosterone titers than those of males
with lower testosterone titers but only when the former males
were the top-scent donors of the same-sex over-mark (Leonard
et al., 2001). Similar results were obtained when meadow voles
preferred the top-scent mark if the donor was fed a diet high in
protein content but not if top-scent donor was fed a diet low in
protein content. Voles preferred the bottom-scent mark if the
donor was fed a diet with a higher protein-content (Hobbs
and Ferkin, 2011a, 2012a).
1.10.4.2.2 Mixed-Sex Over-marks and the Effects
of Familiarity
Ferkin et al. (2010) determined whether the amount of
time individuals investigate the scent marks of opposite-sex
Communication by Chemical Signals 307
conspecifics is affected by 4 days of olfactory experience with
those conspecifics. They found that female meadow voles spent
more time investigating the scent mark of the novel male
conspecific than that of the familiar male donor, whereas
male voles spent similar amounts of time investigating the
scent mark of the familiar female and a novel female conspe-
cific (Figure 5). Ferkin et al. (2010) also found that voles
exposed to a mixed-sex over-mark in which subjects did not
have 4 days of olfactory experience with either the top-scent
donor or the bottom-scent donor spent more time investi-
gating the scent mark of the opposite-sex conspecific that
provided the top-scent mark than that of a novel, opposite-
sex conspecific. Male and female voles spent similar amounts
of time investigating the scent mark of the bottom-scent donor
and that of a novel opposite-sex conspecific (Figure 6). They
also discovered that male voles exposed to a mixed-sex over-
mark that contained the scent mark of a female conspecific
with which they had 4 days of olfactory experience spent
more time investigating the mark of the familiar, top-scent
female than the scent mark of a novel female donor but spent
similar amounts of time investigating the mark of the familiar,
bottom-scent female and that of a novel female donor. In
contrast, female voles spent more time investigating the mark
of a novel male donor than that of either the familiar, top-
scent male or that of the familiar, bottom-scent male
(Figure 7).
Olf exp
Novel 1
Novel 2
*
30
25
Investigation time (s)
20
15
10
5
0
Males Females
Figure 5 The amount of time (mean  SEM) that male and female
meadow voles spent investigating the scent mark of an opposite-sex
conspecific with which they had 4 days of olfactory experience (olf exp)
and that of a novel, opposite-sex conspecific as well as the time they
spent investigating the scent marks of two different novel, opposite-sex
conspecifics. The scent marks used in the pairings were encountered
separately and were not part of a mixed-sex over-mark. * indicates
a statistical difference in investigation time between scent-mark pairings
(p < 0.05; multiple paired comparisons, Holm-Sidák method). Repro-
duced from Ferkin, M.H., Ferkin, D.A., Ferkin, B.D., Vlautin, C.T., 2010.
Olfactory experience affects the response of meadow voles to the
opposite-sex scent donor of mixed-sex over-marks. Ethology 116, 821–
831, with permission from Elsevier.
308 Communication by Chemical Signals
Top scent
Novel scent
Bottom scent
* *
30
25
Investigation time (s)
20
15
10
5
0
Males Females
Figure 6 The amount of time (mean  SEM) that male and female
meadow voles spent investigating the scent mark of the top-scent,
opposite-sex donor of the mixed-sex over-mark and that of a novel,
opposite-sex conspecific as well as the time they spent investigating the
bottom-scent, opposite-sex donor of a mixed-sex over-mark and that of
a novel, opposite-sex conspecific. * indicates a statistical difference in
investigation time between scent-mark pairings (p < 0.05; multiple
paired comparisons, Holm-Sidák method). Reproduced from Ferkin,
M.H., Ferkin, D.A., Ferkin, B.D., Vlautin, C.T., 2010. Olfactory experience
affects the response of meadow voles to the opposite-sex scent donor
of mixed-sex over-marks. Ethology 116, 821–831, with permission
from Elsevier.
Collectively, these observations suggest that familiarity with
an opposite-sex conspecific has different effects on the behavior
of male and female meadow voles. Male voles may be spending
more time with the familiar female donor because she was the
top-scent donor in the mixed-sex over-mark and not because of
any value that she may have gained by being familiar to that
male. Being the top-scent female of a mixed-sex over-mark
may provide a signal to males that she is seeking mates
(Woodward et al., 2000). Thus, she may somehow be more
attractive to investigating males because of her prior associa-
tion with the bottom-scent male (Ferkin, 2011). However,
male voles behave as if the scent marks of females that are
bottom-scent donors are similar to those of novel female
donors in their attractivity. Thus, the bottom position in the
over-mark may signal to males that she has already found
a mate because of her association with the top-scent male
(Woodward et al., 2000). Alternatively, by not being part of
a mixed-sex over-mark, and as a result not being associated
with the scent mark of a male, the novel female donor’s scent
mark could indicate that she may or may not have mated
with another male. In any case, male voles may not respond
favorably to the scent marks of the bottom-scent female
because he is attempting to reduce competition with the top-
scent male, who may have mated the bottom-scent female.
Similar conclusions about the role of familiarity and responses
to scent marks have been adduced for house mice (Arakawa
et al., 2015; Kaur et al., 2014) and the white-footed sportive
lemur, Lepilemur leucopus (Dröscher and Kappeler, 2014).
Top scent
Novel scent
Bottom scent
*
30 * *
25
Investigation time (s)
20
15
10
5
0
Males Females
Figure 7 The amount of time (mean  SEM) that male and female
meadow voles spent investigating the scent mark of the opposite-sex,
top-scent donor of a mixed-sex over-mark with which they had 4 days
of olfactory experience and that of a novel, opposite-sex conspecific as
well as the time they spent investigating the scent mark of the
opposite-sex, bottom-scent donor of a mixed-sex over-mark and that of
a novel, opposite-sex conspecific. * indicates a statistical difference in
investigation time between scent mark pairings (p < 0.05; multiple
paired comparisons, Holm-Sidák method). Reproduced from Ferkin,
M.H., Ferkin, D.A., Ferkin, B.D., Vlautin, C.T. 2010. Olfactory experience
affects the response of meadow voles to the opposite-sex scent donor
of mixed-sex over-marks. Ethology 116, 821–831, with permission
from Elsevier.
These results augment those obtained by Woodward et al.
(2000), who found that after encountering a mixed-sex over-
mark, prairie voles, and meadow voles later preferred the scent
mark of the opposite-sex donor to that of the same-sex donor,
independent of whether the opposite-sex donor was encoun-
tered first as the top-scent donor or the bottom-scent mark in
the over-mark. Interestingly, male and female voles showed
no preference between the mark of the bottom-scent donor
and the mark of an opposite-sex conspecific donor that was
not part of the over-mark. Thus, the position of the opposite-
sex donor’s mark in the mixed-sex over-mark determines how
conspecifics will respond to its scent mark relative to the scent
mark of a novel, opposite-sex conspecific.
Unlike males, female voles spent more time during the test
phase investigating the mark of the novel male donor than the
scent mark of the familiar male conspecific, independent of
whether he was the top- or bottom-scent donor. Female voles
may not respond preferentially to a familiar male if his scent
mark was part of an over-mark with the scent mark of another
female (Woodward et al., 2000), which may make that male
less attractive to a female seeking a mate. The preference of
a female meadow vole for a novel male is similar to the partner
preference found among female house mice (Cheetham et al.,
2007). By preferring novel males to familiar males, female
voles may be encouraging sperm competition between males,
increasing paternity confusion, or avoiding repeated copula-
tions with the same partners.
 Communication by Chemical Signals 309

1.10.4.2.3 Ten Hypotheses about the Function of Over-marks
The literature is replete with observations of mammals over-
marking the scent marks of conspecifics and at least 10 hypoth-
eses have been attributed to its role in odor communication
(Ferkin and Pierce, 2007; Table 2). Many of these hypotheses
suggest functions that are similar to those we presented for
scent marks earlier in this chapter. Briefly, the 10 hypotheses
state that over-marking may be a form of competitive marking,
a bulletin board, a form of self-advertisement, a territorial
behavior, a form of mate attraction, a means to increase
foraging efficiency, a guide for navigation, the formation of
a group/colony scent, a threat behavior, a means to identify
conspecifics, and an artifact of laboratory studies (Table 2).
Some of these hypotheses are not mutually exclusive or may
occur under very special conditions (Ferkin and Pierce, 2007).
The first hypothesis is that over-marking is a type of counter-
marking that provides the top-scent donor with some advan-
tage in information transfer over the bottom-scent donor.
The advantage may be as simple as physically masking the pres-
ence of the bottom-scent mark, as a means to incite competi-
tion, as an indication of social rank or as a threat. Support
for the competition hypothesis comes from studies on a variety
of terrestrial mammals (Ferkin et al., 2004a,b; Hurst, 1990a,b;
Lewis, 2005; Mertl-Millhollen et al., 1986; Palagi et al., 2004;
Smith, 2006).
The second hypothesis is that over-marking provides
a bulletin board that allows individuals to place their mark
along paths traversed by conspecifics or on a large feature in
the area, such as a rock, tree, or mound (Johnston et al.,
1994). In this way, individuals may indicate their presence in
an area (Ferkin, 2015). Direct and indirect evidence for this
hypothesis can be drawn from studies on prairie voles, red-
bellied tamarins (Saguinus labiatus), marmosets, and meadow
voles (Lazaro-Perea et al., 1999; Smith and Gordon, 2002;
Thomas and Wolff, 2002; Woodward et al., 1999).
The third hypothesis is that an individual over-marks the
scent marks of intruders but is less likely to do so when it is
an intruder. Support for this hypothesis can be inferred from
studies on hyena (Crocuta crocuta), Eurasian beavers (Castor
fiber), Ethiopian wolves (Canis simensis), rabbits, Alpine
marmots (Marmota marmota), aardwolves (Proteles cristata),
konik stallions (Konik polski), and klipspringers (Oreotragus
oreotragus) (Drea et al., 2002; Jezierski et al., 2015; Roberts
and Dunbar, 2000; Rosell and Sanda, 2006; Rosell et al.,
2000; Sillero-Zubiri and Macdonald, 1998; Sliwa and
Richardson, 1998; Theis et al., 2008).

Table 2 Ten hypotheses to explain the role of over-marking

Hypothesis Support/Species Recent citations

1. Over-marking is a type of counter-marking
that provides the top-scent donor with
some advantage in information transfer
over the bottom-scent donor
2. Over-marking provides a chemical bulletin
board
3. An individual over-marks the scent marks of
intruders but is less likely to do so when it is
an intruder
4. Over-marking is a form of mate attraction
5. Over-marking is an indication of a social or
sexual bond between the top- and bottom-
scent donors
6. Over-marking allows scent marks to mix,
thereby providing group members with
a unique group scent
7. Over-marking the scent marks of
conspecifics increases foraging efficiency
by the individual and/or family members for
locating food sources
8. Over-marking allows aids in navigating
between familiar and unfamiliar areas
9. Over-marking serves no function
10. Over-marking serves multiple functions
Meadow voles; house mice; sifaka; wolf;
lemurs; common marmosets (Callithrix
jacchus)
Prairie voles; red-bellied tamarins (Saguinus
labiatus); common marmosets; meadow
voles
Hyena (Crocuta crocuta); Eurasian beavers
(Castor fiber); Ethiopian wolves (Canis
simensis); rabbits; Alpine marmots (Marmota
marmota); aardwolves (Proteles cristata);
konik stallions (Konik polski); klipspringers
(Oreotragus oreotragus)
Meadow voles; zebras; hyenas; wolf; ring-tailed
lemurs; moustached tamarins; house mice
Hyena; prairie voles; wolf; klipspringers;
antelope; moustached tamarins; ring-tailed
lemurs
Sifaka
Honey badgers (Mellivora capensis); otters
Not tested directly
Prairie voles
Not tested directly
Ferkin et al. (2004a,b), Hurst (1990a,b,c), Lewis
(2005), Palagi et al. (2004), and Smith
(2006)
Lazaro-Perea et al. (1999), Thomas and Wolff
(2002), Smith (2006), and Woodward et al.
(1999)
Theis et al. (2008), Drea et al. (2002), Jezierski
et al. (2015), Roberts and Dunbar (2000),
Rosell et al. (2000), Sillero-Zubiri and
Macdonald (1998), and Sliwa and
Richardson (1998)
Ferkin et al. (2004a,b), Heymann (1998), Hurst
(1990c), Jezierski et al. (2015), Kappeler
(1998), Mertl-Millhollen et al. (1986),
Penzhorn (1984), and Tinnesand et al.
(2015)
Brashares and Arcese 1999, Drea et al. (2002),
Jordan et al. (2014), Heymann, 1998, Mertl-
Millhollen et al. (1986), Woodward et al.
(1999, 2000), and Palagi et al. (2004)
Greene and Drea (2014)
Begg et al. (2003) and Kruuk (1995)
Peterson (1988)
Thomas and Wolff (2002) and Wolff (2004b)
Ferkin (2011) and Ferkin and Pierce (2007)
310 Communication by Chemical Signals
The fourth hypothesis is that over-marking is a form of mate
attraction. Thus, the top-scent mark may serve as a greeting or
invitation to the donor whose mark was over-marked, and
facilitate interactions between the two scent donors. The mate
attraction hypothesis has gained some support from studies
on ungulates, carnivores, rodents, and primates (Ferkin et al.,
2004a,b; Heymann, 1998; Hurst, 1990c; Jezierski et al., 2015;
Kappeler, 1998; Mertl-Millhollen et al., 1986; Penzhorn,
1984; Tinnesand et al., 2015).
The fifth hypothesis is that over-marking may be an
indication of a social or sexual bond between the top- and
bottom-scent donors. If the over-mark is between opposite-
sex conspecifics it may be a form of mate guarding (Hurst
and Beynon, 2004; Woodward et al., 1999). For example, Jor-
dan et al. (2014) found that dominant African wild dogs
(Lycaon pictus) over-mark the scent marks of their mates more
often than their own marks and more likely to be part of
tandem marks with those of their siblings than those of a non-
siblings. Presumably, matched scents and tandem scent
marking may be a way to combine territory defenses or to
advertise the pair’s relationship status to their group.
The sixth hypothesis is that over-marking allows scent
marks to mix, thereby providing group members with a unique
group scent. This group scent may provide cohesion among
group members and allow individuals the means to distinguish
between group and nongroup members. Greene and Drea
(2014) discovered that mating pairs of sifakas copied each
other’s scent-marking behavior and produced similar odors
after they reproduced, whereas unmated pairs failed to do so.
The seventh hypothesis states that over-marking the scent
marks of conspecifics increases foraging efficiency by the indi-
vidual and/or family members for locating food sources.
Thus, individuals will over-mark the scent marks of conspe-
cifics and their own scent marks to create a path that allows
them to locate a food source, and may be akin to trail marking
in ants (Ferkin and Pierce, 2007). The foraging efficiency
hypothesis has gained indirect support from studies on honey
badgers (Mellivora capensis) and otters (Begg et al., 2003; Kruuk,
1995).
The eighth hypothesis is that over-marking allows aids in
navigating between familiar and unfamiliar areas (Peterson,
1988). This hypothesis has not been tested directly.
The ninth hypothesis states that over-marking serves no
function. This is essentially a null hypothesis, which is based
on reports of some mammals that rarely over-mark or over-
mark a very small proportion of the scent marks of conspecifics
(Thomas and Wolff, 2002; Wolff, 2004b). Although this
hypothesis raises some interesting issues with regard to how
and where some over-marking studies are conducted, there is
little empirical evidence that shows that over-marking serves
no function.
The tenth hypothesis states that over-marking serves
multiple functions. While it is tempting to assign all such tasks
to over-marking, it may be premature to suggest over-marking
is an all-purpose type of odor communication. The manner in
which individuals respond to scent marks and whether they
over-mark the scent marks of conspecifics may be context-
dependent (Ferkin, 2011). Differences in over-marking
behavior may be concomitant with changes in their life history
patterns, season, space use and social tolerance, reproductive
condition, social status, age, and health (Ferkin and Pierce,
2007).
1.10.4.3 An Over-mark versus a Single Scent Mark
It had been suggested that over-marks provide individuals with
the opportunity to directly assess features of two same-sex
conspecifics that may not be available if individuals encoun-
tered the scent marks of these two conspecifics separately
(Hurst and Beynon, 2004; Johnston, 2003). A study by Ferkin
et al. (2011a) tested the hypothesis that after meadow voles
enter areas containing different proportions of over-marks
and single scent marks of two same-sex scent donors, voles
will differ in the amount of time they later spend investigating
the marks of these two donors. Males and females were allowed
to enter an arena that contained 10 over-marks and 0 single
marks of the conspecific that provided the bottom-scent mark
of the over-mark, nine over-marks and one single scent mark
of the bottom-scent donor, eight over-marks, and two single
scent mark of the bottom-scent donor, and so on. Ferkin
et al. (2011a) discovered that after male voles entered an arena
containing at least six over-marks and four single scent marks of
the bottom-scent female donor of the over-mark, they spent
more time investigating the mark of the top-scent female donor
than that of the bottom-scent female donor. When the ratio of
over-marks to single scent marks was less than 6:4, the males
did not discriminate between the top- and bottom-scent female
donors. After female voles entered an arena containing at least
four over-marks and six single marks, they spent more time
investigating the mark of the top-scent male donor than that
of the bottom-scent male donor. When the ratio of over-
marks to single scent marks was less than 4:6, the females did
not discriminate between the top- and bottom-scent male
donors. Female voles also performed better than male voles
did in others tests involving discriminating between larger
and smaller sets of scent marks (Ferkin and Hobbs, 2014; Fer-
kin et al., 2005; Vaughn and Ferkin, 2011). It is also worth
pointing out that the proportions of over-marks that male
and female voles needed to encounter to display a preference
for the mark of the second scent donor over that of the second
scent donor were similar to the proportion of scent marks
males and females use to over-mark the scent marks of
a same-sex conspecific (Ferkin et al., 2004b).
Collectively, these results support and extend the view that
over-marks provide individuals with the opportunity to
directly assess features of two same-sex conspecifics that may
not be available if individuals encountered the scent marks
of these two conspecifics separately (Ferkin et al., 2011a).
In this way, over-marks may also provide information about
a possible association between the top- and bottom-scent
donors (Ferkin and Pierce, 2007; Hurst and Beynon, 2004;
Johnston, 2009). For example, over-marks provide a temporal
component in which the position of a donor’s scent mark as
being either the top-scent donor or bottom-scent donor
conveys information to a receiver about the relative time frame
in which both donors deposited their marks (Ferkin et al.,
2011b). An over-mark may also contain information about
the two scent donors that led to the establishment of the
over-mark itself. Thus, it may represent an olfactory snap
shot of a past ‘olfactory-based’ encounter between two scent

donors (Hurst and Beynon, 2004). However, single marks may
be easier for individuals to learn about features of the pheno-
type and genotype of individual scent donors (Ferkin, 2011,
2015; Green et al., 2015; Kaur et al., 2014) and recall them
later (Roberts et al., 2014).
1.10.4.4 Self-Grooming as a Form of Scent Marking
Thiessen (1977) suggested that self-grooming may spread
odoriferous substances found in the integument, which may
affect the responses of the groomer and the receiver. For
example, prairie voles, meadow voles (Ferkin et al., 1996),
and male Mongolian gerbils (Harriman and Thiessen, 1986;
Thiessen and Harriman, 1986) that recently self-groomed
produced odors than those of males that did not self-groom
to females. These results suggest that self-grooming may be
a form of olfactory communication, akin to scent marking,
which allows groomers to transmit information about features
of their identity and sex to nearby conspecifics (Ferkin et al.,
1996; Thiessen, 1977; Wiepkema, 1979). Self-grooming may
enhance features of this information, which may make nearby
conspecifics that encounter these odors more responsive to the
groomer (Ferkin and Leonard, 2010; Oliveira et al., 2013;
Thiessen and Harriman, 1986; Vaughn et al., 2010).
1.10.4.4.1 Mate Attraction
Several studies have also provided indirect and direct support
for the role of self-grooming in mate attraction. Ground squir-
rels, deer (Odocoileus virginianus), Mongolian gerbils, hedge-
hogs, blind mole rats (Spalax spp.), and voles self-groom
more when they are exposed to odors of opposite-sex conspe-
cifics than when they are exposed to those of same-sex conspe-
cifics (Brockie, 1976; Ferkin and Leonard, 2010; Ferkin et al.,
1996; Shanas and Terkel, 1995; Steiner, 1973, 1974; Thiessen
and Harriman, 1986; Witt et al., 1988, 1990). Next, male and
female voles spent more time self-grooming in response to
odors of reproductively active opposite-sex conspecifics than
those that are reproductively quiescent (Ferkin and Leonard,
2010). Male voles also spend more time self-grooming in
response to odors of a female vole in postpartum estrus than
they did in response to odors of females not in postpartum
estrus (Ferkin, 2006; Ferkin and delBarco-Trillo, 2014). Male
house mice spent more time self-grooming in response to urine
of estrus female mice compared to that of female mice not in
estrus (Achiraman et al., 2014). However, male root voles,
Microtus oeconomus, spent more time self-grooming in response
to urinary odors of lactating females than to urinary odors of
nonlactating females (Yu et al., 2010). Thus, the sexual condi-
tion of the scent donor affects the amount of time that rodents
self-groom.
1.10.4.4.1.1 Condition and Familiarity of the Scent Donor
The condition and familiarity of the scent donor affects the
amount of time that rodents self-groom. Gonadectomized
male voles, house mice, and rats spent less time self-
grooming in response to odors of reproductively active
opposite-sex females than did their intact counterparts
(Achiraman et al., 2014; Ferkin, 2006; Moore, 1986). In house
mice, self-grooming behavior was partially restored by testos-
terone treatment, whereas self-grooming rates were restored
Communication by Chemical Signals 311
to levels comparable to intact male voles by testosterone
therapy (Ferkin and Leonard, 2010; Leonard et al., 2001,
2005). Additionally, food-deprived voles spent less time than
those that were not food deprived self-grooming in response
to the odors of a sexually receptive, opposite-sex conspecific
(Hobbs et al., 2008). Lastly, meadow voles and prairie voles
also spent more time self-grooming in response to odors of
unfamiliar and unrelated females than in response to those
of their reproductively active sisters (Paz-y-Miño et al., 2002).
1.10.4.4.1.2 Seasonal Differences
Seasonal differences also exist in self-grooming in meadow
voles. Short-photoperiod voles spent similar amounts of time
self-grooming in response to the odors of both opposite and
same-sex long photoperiod and short-photoperiod scent
donors (Leonard and Ferkin, 2005). In contrast, long-
photoperiod voles spent more time self-grooming in response
to the odors of long-photoperiod, opposite-sex conspecifics
compared to that in response to the odors of short-
photoperiod, opposite-sex conspecifics (Ferkin et al., 1996;
Leonard and Ferkin, 2005).
These observations and findings indicate a communicative
role for self-grooming (Ferkin and Leonard, 2010; Ferkin
et al., 1996). During the breeding season, self-grooming
behavior may be an indicator of detection and discrimination
of sexually receptive females by males and vice-versa and serve
to attract opposite-sex conspecifics to the groomer. In male
rodents, this response is mediated by testosterone (Achiraman
et al., 2014; Ferkin and Leonard, 2010). Thus, seasonal differ-
ences in self-grooming in voles may be attributed to photoperi-
odically induced changes in gonadal steroids, prolactin, and
melatonin titers (Ferkin et al., 2007; Leonard et al., 2005),
which are needed to support reproduction (Ferkin and Leonard,
2010; Leonard and Ferkin, 2005). In contrast to its role during
the breeding season, self-grooming during the nonbreeding
may have a function outside of mate attraction. Perhaps, during
the nonbreeding seasons self-grooming is a communicative
behavior directed at both male and female conspecifics, serving
to announce the groomer’s presence in an area (Ferkin and
Leonard, 2010; Leonard and Ferkin, 2005) or to reduce agonism
between conspecifics (Simeonovska-Nikolova and Dekov,
2013). These latter two hypotheses remain untested.
1.10.4.5 Scent Marks Provide Public Information
Because scent marks and over-marks provide individually
distinct information about senders (Halpin, 1986; Johnston
and delBarco-Trillo, 2009; Kaur et al., 2014), receivers gain
specific information about the scent marks of individuals that
they encounter (Charlton, 2015; Ferkin, 2015; Roberts, 2007;
Sharpe, 2015). Indeed, this information may be viewed as
public information. Public information as defined by Valone
(2007) is the “indirect information used by individuals to esti-
mate the quality of environmental parameters.” Public infor-
mation may be gleaned by individuals noting the behavior of
others or by the overt decisions made by them (Danchin
et al., 2004). This social information provided may not be
intentional. For example, scent marks may contain inadvertent
social information. To examine this possibility Ferkin and Fer-
kin (unpublished data) first exposed male voles to the scented
312 Communication by Chemical Signals
bedding of a sexually receptive female and the bedding or
a varying number of male conspecifics that were placed next
to the bedding of that female. After the exposure, male voles
were tested to assess if they spend different amounts of time
investigating the fresh scent mark of the female from their
exposure phase and the scent mark of a novel female. The tests
revealed that male voles preferred the scent mark of a female
whose bedding was associated with the bedding of three or
five males compared to that of a novel female. Male voles
showed no preferences for the scent mark of a novel female
or that of the female who was associated with the bedding of
zero, one, or two males. These preliminary findings suggest
that male voles may use social information provided by the
scent marks of both rival males and that of a female conspecific
to influence their odor preferences for a particular female. Such
preferences for the ‘popular’ female may facilitate mate
copying.
Mate copying by males has been reported among species of
fish. Male guppies (Poecilia reticulata), sail fin mollies (Poecilia
latipinna), and mosquito fish (Gambusia affinis) preferred to
associate and copulate with female conspecifics that they
have observed to have had more suitors compared to females
that had no suitors (Wagner and Danchin, 2003; Witte and
Ryan, 2002). It appears that male meadow voles, akin to
male guppies and mosquito fish (Bierbach and Somer-
Trembo, 2015; Callender et al., 2012; Mautz and Jennions,
2011), make facultative adjustments in their choice of a female
if male conspecifics are present and the number of male
competitors increases. This speculation is intriguing in that
male meadow voles are promiscuous (Berteaux et al., 1999;
Boonstra et al., 1993), and mating with a female that may
have mated with three other males would lower that male’s
paternity assurance (Birkhead, 2000), and result in an increase
in the risk and intensity of sperm competition (Parker and
Pizzari, 2010), which result in him increasing the amount of
sperm in his ejaculate and lowering the amount of sperm in
future ejaculations (delBarco-Trillo and Ferkin, 2004; Vaughn
et al., 2008). Thus, it is likely that individuals may have to
balance the costs of sending and receiving potentially public
information, which may reduce their survival or attract compet-
itors, with the benefits of attempting to mate with a ‘popular’
female or that of the female who was associated with the
bedding of zero, one, or two males (Morand-Ferron et al.,
2010).
1.10.5 Evolution
1.10.5.1 Basic Concepts
Odor-related behaviors may have evolved from behaviors asso-
ciated with the excretion of by-products of digestion (Steiger
et al., 2011). Scent marking likely evolved from the excretion
of metabolic wastes. Because the digestive exudates provide
information about the phenotype and genotype of the sender
it is likely to provide accurate information to receivers. Another
route that may have been involved in the evolution of odor
communication may be attributed to the specialization and
elaboration of scent-producing tissues in the integument.
Perhaps, early terrestrial mammals were able to draw secretions
from the integument by scratching themselves with an
appendage or rubbing the anogenital area against the surface
of a substrate. These behaviors may have been initially involved
in thermoregulation, maintenance of the pelage, or removal of
ectoparasites that may also have concomitantly released
substances in the environment. These secretions likely con-
tained odiferous substances that affected the behavior of
receivers (releasing effect) or modified the behavior of the
sender (priming effect) (Bossert and Wilson, 1963). If the net
result of these behaviors increased an individual’s survival
and fitness and if variation existed among individuals, selection
pressures may have led to formalization and stereotyping of
these behaviors, such that over time senders would be able to
make this signal more easily detected (Rozhnov, 2004). Such
a process would lead to the ubiquity of scent marking in terres-
trial mammals and its role in odor communication (Brown and
Macdonald, 1985; Ferkin. 2015; Johnston and delBarco-Trillo,
2009). For the sender, the specific behaviors involved in odor
communication would involve behaviors like scent marking,
over-marking, and self-grooming, which have been reported
in nearly all terrestrial mammals (Ferkin, 2011), as well as
the production of odiferous substances that have primer and
releasing-like properties (Bossert and Wilson, 1963; Müller-
Schwarze, 1984) and may be species-specific (delBarco-Trillo
and Drea, 2014; Setchell et al., 2011). For the receiver, they
would need to have the functional morphology to detect chem-
ical signals that allows them to attend to those of conspecifics
more than those of heterospecifics. For odor communication to
evolve, the sender must gain a benefit that outweighs the
survival or fitness cost of signaling this to occur. Likewise, the
receiver that encounters the scent mark must not incur in costs
that are greater than what is gained in terms of survival and
fitness by attending to and responding to the message (Roberts,
2007; Roberts et al., 2014). In addition, because male and
female terrestrial mammals differ in some aspects of the
frequency, duration, and timing of displaying odor-related
behavior suggests that odor communication may also have
been influenced by sexual selection (Ferkin, 2011, 2015; Petty
and Drea, 2015; Stockley et al., 2013).
1.10.5.2 Comparative Approaches to the Study of Olfactory
Communication
As pointed out in a recent paper by delBarco-Trillo and Drea
(2014), there is a paucity of data on the evolution of odor
communication in terrestrial mammals. This is partly due to
the difficulty of combining chemical data from different studies
to perform comparative studies, the only solution to this
problem being the chemical characterization of odors in the
several species to be compared, which is quite a complex and
time-consuming task (delBarco-Trillo et al., 2011a). This type
of de novo approach has been used in strepsirrhine primates
(delBarco-Trillo et al., 2011a, 2012; delBarco-Trillo and Drea,
2014). These studies have shown, for example, that (1) species
that rely on urine as a signal for chemical communication have
more chemical compounds in their urine than species that rely
more on glandular secretions; (2) such differences between
urine markers and nonurine markers suggest urine marking being
an ancestral behavior in solitary and nocturnal species,
whereas glandular marking would have evolved in diurnal
and social species; (3) chemical richness is higher in
 Communication by Chemical Signals 313

multimale
multifemale species (in which more interindi-
vidual interactions and thus transfer of information occur)
than in pair-bonded species; (4) male odors are more chemi-
cally complex in codominant species than in female-
dominant species; (5) the chemical composition of odors is
very species-specific, even in species that belong to the same
genus, providing reliable species scent signatures; and (6) chem-
ical signals in strepsirrhines follow a gradual mode of evolution
(i.e., there is a linear and positive relationship between chem-
ical differences and phylogenetic distances), rather than
a saltational mode of evolution, both when we consider strep-
sirrhines as a whole or a series of species from the same genus
(Figure 8).
1.10.5.3 Genetic Variation, Natural Selection, and Odors
Olfactory communication can be a very important component
during speciation processes (Smadja and Butlin, 2009). In the
Skyros wall lizard, Podarcis gaigeae, islet and mainland popula-
tions differ in the chemical composition of their odors, and
there is a relationship between pheromonal and genetic diver-
gence among populations (Runemark et al., 2011). In two
subspecies of mice, Mus musculus musculus and Mus musculus
domesticus, hybrids in the sympatric area produce an odor
phenotype that is distinct from the two parental species, which
facilitates the avoidance of intraspecific mating (Ganem et al.,
2014). The genetic basis for odor divergence between these
two Mus subspecies involves autosomal genes and genes on
the X chromosome (Ganem et al., 2014). Particular proteina-
ceous pheromone families, like androgen-binding proteins,
are also shaped by selection in mice (Karn and Laukaitis,
2014). In several mammalian species a direct relationship has
been reported between chemical odor profiles and MHC geno-
types (Drea, 2015; Setchell et al., 2011). There is also a genetic
underpinning for olfactory reception (Logan, 2014):
Olfactory-receptor genes vary greatly among individuals, and
this genetic variation may result in individualized receptor
repertoires that provide different individuals with a differential
perception of the environment (Logan, 2014).
1.10.5.4 Odors and Sexual Selection
Sexual selection is divided in four subprocesses, two of them
occurring before copulation (male–male competition and
female choice) and the other two occurring after copulation
(sperm competition and cryptic female choice) (Kuijper et al.,
2012). The role of odors in female choice has already been
described earlier in this chapter, whereas the relationship
between olfactory communication and cryptic female choice
remains to be investigated. Consequently, in this section we
will address how odors can mediate behaviors involved in
male–male competition and sperm competition.
Some degree of male–male competition can be found in all
vertebrate species, and it includes behaviors in which males
compete for sexual access to females and/or prevent other
males from gaining access to their mates. Scent marks are often
used in such competitions. For example, ring-tailed lemurs
spread odors on their tails and direct the odors toward
competing individuals by waving their tails in the direction
of their opponent, a behavioral interaction called a ‘stink fight’
(Jolly, 1966). Many ungulates mark or urinate as part of terri-
torial and/or aggressive contests for dominance. Some species,
such as the hartebeest, Alcelaphus buselaphus, perform ‘scent-
marking fights’ at territorial boundaries, in which two males
scent mark alternately and also attempt to remove the scent
marks of their opponents (Gosling, 1982). In addition to scent
marking in his territory, a male may also rub his scent gland
secretions, urine, or other scents onto his own body; other
males can then match the scent marks deposited in the terri-
tory with the male that deposited them. For example,

Figure 8 Types of information obtained by comparing the chemical composition of urine in several strepsirrhine primates. (a) Mean  SEM number
of volatile compounds (including either all compounds detected or only those with a purported semiochemical function) in the urine of six species of
urine markers and six species of nonurine markers. (b) Relationship between the urinary chemical distance between all possible pairs of species and
their phylogenetic distance. ***p < 0.0005. Reproduced from delBarco-Trillo, J., Burkert, B.A., Goodwin, T.E., Drea, C.M., 2011. Night and day: the
comparative study of strepsirrhine primates reveals socioecological and phylogenetic patterns in olfactory signals. J. Evol. Biol. 24, 82–98, with
permission from Wiley.
314 Communication by Chemical Signals
territorial male hartebeest rub their antorbital gland onto their
side, and also rub their body in their own feces (Gosling and
Roberts, 2001). Many other ungulate species produce wallows
in which they urinate and then roll around in the wallow
(Grau, 1976), e.g., the American bison, Bison bison (Coppedge
and Shaw, 2000). Capuchin monkeys (Cebus spp.) ‘urine
wash’ their hands and then rub their hands up their flanks
(Zeller, 1987).
An advantage of assessing rivals via their odors is that
dangerous physical interactions may be reduced or avoided.
Males may recognize each other’s scent, and either withdraw
or continue competing depending on the information ob-
tained. For example, after male snow voles investigated an
arena containing the odors of another male, they behaved
less aggressively toward that male (i.e., the resident male)
than toward an unfamiliar male (Luque-Larena et al., 2001).
The level of response to the odors of other males may also
differ between monogamous and promiscuous species. In the
monogamous California mouse, Peromyscus californicus, males
scent mark and over-mark more in response to the odors of
a conspecific male that is the case in the promiscuous white-
footed mouse, Peromyscus leucopus (Becker et al., 2012).
Sperm competition occurs when two or more males copu-
late with the same female and their sperm ‘compete’ within
the female’s reproductive tract for the fertilization of her ova
(Parker, 1970). Males may change their sexual behavior
(Stockley and Preston, 2004) or the number of sperm ejacu-
lated (delBarco-Trillo and Ferkin, 2006) depending on the
risk of sperm competition (the probability that a female will
mate with other males) or the intensity of sperm competition
(number of males mating with a female). Some studies have
shown that males assess the risk and intensity of sperm compe-
tition by paying attention to the odors of other males on
females or in the immediate environment of a female. For
example, a ram will copulate faster when semen of another
male is spread on the female’s vulva (Lezama et al., 2001).
The ram’s semen does not trigger the same response, suggesting
that a male is able to discriminate the odor of his own semen
from the semen of other males.
A high risk of sperm competition can also be signaled by the
presence of the odors of a conspecific male in the environment.
A series of experiments investigated this possibility in meadow
voles (delBarco-Trillo and Ferkin, 2004; delBarco-Trillo and
Ferkin, 2006). Male voles mated either in a context with high
risk of sperm competition (conveyed solely by the presence
of odors of another male) or in a context with low risk of sperm
competition (no odors of conspecifics present during the trial).
When another male’s odors were present, male voles increased
the number of sperm that they delivered to their mate
(delBarco-Trillo and Ferkin, 2004). This increase was not due
to any change in copulatory behavior (delBarco-Trillo and
Ferkin, 2004), which suggests that the odors of another male
trigger a physiological response that ultimately produces an
increase in the contractility of the cauda epididymis and vas
deferens, and thus a larger number of sperm being ejaculated
(delBarco-Trillo and Ferkin, 2007a). In contrast to what has
been found in voles, male house mice do not increase the
number of sperm in their ejaculates in response to a high level
of sperm competition conveyed by odors of other males
(Ramm and Stockley, 2009).
When a male vole mates while being exposed to odors of
many other males, he decreases the number of sperm ejacu-
lated in comparison to when he is exposed to odors of only
one competing male (delBarco-Trillo and Ferkin, 2006). This
result is in agreement with sperm competition theory. That is,
as the number of competing males increase, the advantage of
increasing the number of sperm decreases.
The response of a male may depend on the status or condi-
tion of competing males. If the odors of surrounding males
come primarily from subordinate males that are not likely to
mate, a subject male may respond differently than if those
odors belong to a highly competitive male. Similarly, when
a male meadow vole mates with a female in the presence of
odors from an ad libitum fed male, he increases the number
of sperm ejaculated, but he will not do this in the presence of
odors from a food-deprived male, indicating that subject males
assign a lower competitive threat to odors of food-deprived
males than to odors of ad libitum fed males (Vaughn et al.,
2008).
Exposure during sexual development to conspecific odors
that convey a context with a high level of sperm competition
can have developmental effects. For example, in bank voles,
Myodes glareolus, males raised in a context in which odors of
adult males resembled a high level of sperm competition devel-
oped larger seminal vesicles (Lemaître et al., 2011).
The increase in sperm allocation outline above in response
to contexts of sperm competition can be used for conservation
purposes. In the giant pandas, males exposed to the odors of
a conspecific male became more interested in investigating
females (Bian et al., 2013). In this and other species that repro-
duce poorly in captivity, such enhancement in sexual motiva-
tion could lead to important increases in sexual performance.
Given that the risk of sperm competition is lower in
unmated females than in recently mated females, males are
generally expected to show a preference for unmated females.
Indeed, in collared lemmings, sexually experienced males
prefer the odors of an estrous unmated female than the odors
of a female recently mated with another male, whereas sexually
inexperienced males do not (Huck et al., 1984). In some
species, however, as in Syrian hamsters, males mate indiscrim-
inately and do not show a preference when exposed to
unmated and mated estrous females (Johnston and
Rasmussen, 1984).
In promiscuous species, males that mate with several females
may become sperm-depleted, and females mating with such
males may incur a reproductive cost (Wedell et al., 2002). If
females are able to discriminate between recently mated and
unmated males, they may reduce the cost of mating with
a sperm-depleted male by avoiding mating with recently mated
males. Female rats, for example, prefer the odors of males that
have not copulated than those of males that have recently mated
with another female (Krames and Mastromatteo, 1973).
1.10.6 Cognition and the Assessment
of Scent Marks
Chemical signals from scent marks and odor-producing tissues
provide a great deal of information about the donor to receivers
(Ferkin, 2011; Roberts, 2007; Wyatt, 2014). The hypotheses

that best explain the functions of scent marking are likely to be
several and dependent on the species and the variety of envi-
ronmental and biological challenges it must face. Nevertheless,
the manner in which an animal uses this information to make
decisions will depend on the context and manner in which it
encounters these scent marks (Franklin and Ferkin, 2006,
2008; Roberts et al., 2014). It is unlikely that a learned a rule
of thumb that will allow the animal to respond appropriately
when they encounter different scent marks of different donors.
To select the appropriate response, an individual will need to
identify the phenotype, genotype of the donor, recall any inter-
actions with that donor, be aware of its own condition and
respond in a manner that will presumably increase the individ-
ual’s survival and fitness (Ferkin, 2015; Green et al., 2015;
Tinnesand et al., 2015).
Receivers can use the information found in the scent marks,
over-marks, and scent plumes from self-grooming to locate the
donor, learn its identity, and determine the donor’s phenotype
or genotype. This will involve the receivers learning spatial and
temporal associations among scent donors and their marks
(Ferkin and Pierce, 2007; Roberts et al., 2014), storing them,
and later recalling these associations (Franklin and Ferkin,
2006, 2008). Receivers will also need to learn other features
associated with the scent donor such as when the scent mark
was deposited, how long it has been since the receiver encoun-
tered the scent donor, and where that encounter occurred
(Franklin and Ferkin, 2006, 2008). Higher level cognitive pro-
cessing involving procedural memory, episodic memory, auto-
biographical memory, and making judgments of numerical
discrimination would be required for receivers to make poten-
tially informed decisions, when they encounter the scent marks
of particular conspecifics (Ferkin, 2011; Ferkin and Hobbs,
2014; Ferkin et al., 2005, 2008).
1.10.6.1 Processing Scent Marks
Gosling and Roberts (2001) and Roberts (2007) provided us
with an excellent flowchart for the processing of scent marks in
house mice. Ferkin (2015) extended this flow chart to incorpo-
rate key cognitive elements that would be used by voles and
potentially other mammals when they encounter scent marks,
which was developed by Franklin and Ferkin (2006, 2008),
and illustrated in Figure 9. Briefly, the schema is as follows.
The first step in the schema involves the detection of the scent
mark, which involves sensory processes (environment, Figure 9).
After the scent mark is detected, it must be attended to (percep-
tion, Figure 9). This involves cognitive processes that underlie
or mediate the interpretation of the scent mark by the individual.
Interpreting the scent mark will involve discrimination, percep-
tual, procedural, spatial memory, and episodic memory, catego-
rization, and the weighting or assessment of valence that may be
attached to the sender (donor) of the scent mark by the receiver
(Franklin and Ferkin, 2006, 2008; Roberts et al., 2014; Figure 9).
The number of scent donors, their identity, age, and sex and
eavesdroppers may affect the value of the scent mark to the
receiver (Ferkin and Hobbs, 2014; Kaur et al., 2014; Roberts
et al., 2014). At this point, the receiver may choose whether to
respond to the scent mark (action selection, Figure 9). If the scent
mark induces a response, the simplest response may depend on
the receiver’s determination of whether the scent mark provides
Communication by Chemical Signals 315
Episodic
memory
Recall Encode
Percept
Perception Attention
Perceptual Reinforcement
learning
Action Procedural
selection memory
Environment
Senses Acts
Figure 9 The cognitive processes that underlie or mediate the inter-
pretation of the scent mark by the individual. Reproduced from
Franklin, S., Ferkin, M.H., 2006. An ontology for comparative cognition:
a functional approach. Comp. Cogn. Behav. Rev. 1, 36–52, with
permission from the authors.
information about the sender posing some type of threat or
a mating opportunity (Franklin and Ferkin, 2006, 2008). If the
scent mark is from a potential mate, the response would likely
be to self-groom, scent mark, or over-mark and seek out the
donor (Ferkin and Leonard, 2010; Ferkin and Pierce, 2007;
Gosling and Roberts, 2001). If the scent mark is from a familiar
same-sex conspecific, it may be ignored (Hurst et al., 1993). If,
however, the scent mark is from an unfamiliar same-sex conspe-
cific, it may be investigated (Gosling, 1982; Roberts, 2007). If
the scent mark is from a heterospecific, the response would
depend on whether or not the donor was perceived as a threat
(Eads et al., 2016; Vlautin et al., 2010). If the threat is such that
it is too costly for the individual to be detected, it would avoid
scent marking or seeking out interactions with the particular
donor (Apfelbach et al., 1991), or it may shift its nest or area
of activity away from that donor (Je˛ drzejewski et al., 1993).
If a receiver chooses, it may deposit its own scent mark and
become a sender (Ferkin, 2015; Johnston, 2003, 2009). This
action would return us to the first step of the schema (Figure 9).
1.10.6.2 Cognitive Ability, Numerosity, and Scent Marks
A variety of nonhuman animals in different taxa can discrimi-
nate between a group and entity containing more objects from
one containing fewer of the same objects (Beran, 2001; Pahl
et al., 2013; Perdue et al., 2012; Reznikova and, Ryabko,
2011). One way to assess if animals display numerousness is
to expose them simultaneously to a set with relatively large
numbers of stimuli for one response and a set with a relatively
small number of stimuli for another response, and then give
them a test to determine if they respond differently to either
set or if they respond selectively to the larger set (Boysen and
Capaldi, 1993; Hauser, 2000). If animals respond selectively to
the larger set over the smaller set, usually by attending to it or
investigating it (Boysen, 1997; Hauser et al., 2003), that would
be an indication that they may have the ability to spontaneously
discriminate between two different numerosities and have
a concept of relative numerousness (Beran, 2001; Beran et al.,
2012). This capacity for making judgments of numerousness
316 Communication by Chemical Signals
may allow individuals to discriminate between potential mates.
Carazo et al. (2009) found that male mealworm beetles (Tene-
brio molitor) discriminated between the odors of females if the
ratio between the female scent donors exceeded 1:2. By keeping
track of the number of females and their odors, males can maxi-
mize their mating opportunities. Likewise, mammals may keep
track of the odors and scent marks produced by conspecifics to
find mates. In that scent marks are cheat-resistant signals (Ferkin,
2011; Roberts, 2007) that provide accurate and current informa-
tion about the genotype and phenotype of a donor (Green et al.,
2015; Kaur et al., 2014) differences in the number of scent marks
or over-marks deposited by donors may be used by conspecifics
to select potential mates (Ferkin et al., 2005; Ferkin and Hobbs,
2014; Vaughn and Ferkin, 2011). For example, female voles were
offered the scent mark of both male donors; they spent more
time investigating the scent mark of the male donor that earlier
provided more scent marks to that of the male that had provided
fewer scent marks (Ferkin and Hobbs, 2014). In that study,
female meadow voles discriminated between the scent marks
of two males when the difference in scent marks was 2 and
the total number of scent marks was 6 (Ferkin and Hobbs,
2014). Spending more time investigating the scent marks of
a particular conspecific compared to that of another conspecific
has been interpreted as the subject anticipating an amicable
social interaction with the preferred individual if it was
a same-sex conspecific and a mating attempt with that individual
if it was an opposite-sex conspecific (Ferkin and Seamon, 1987).
Similarly, mealworm beetles were trained by using a sponta-
neous two-choice procedure in which males were exposed to
substrates bearing odors from different numbers of females in
increasing numerosity ratios (1:2, 1:3 and 1:4). Males discrimi-
nated sources of odors reflecting 1 versus 4 and 1 versus 3
females, but not 2 versus 4 or 1 versus 2 (Carazo et al., 2009).
So, the beetles demonstrated the ability to discriminate between
‘more or less smells’ within the limit of four with an operational
signature ratio of 1:2.
1.10.7 Concluding Thoughts
The manner in which many terrestrial mammals behave
depends on the information they receive from their environ-
ment (Ferkin, 2011). This information should serve some
function or functions involved in intraspecific and interspe-
cific communication (Johnston, 2009). For many terrestrial
mammals, scent marks convey information about the individ-
ual’s biotic and abiotic environment to nearby conspecifics
and heterospecifics (Hurst and Beynon, 2004). These scent
marks are typically deposited on prominent objects or along
paths that are shared with conspecifics (Brown and
Macdonald, 1985). Thus a variety of individuals will enter
areas that contain scent marks and overlapping scent marks,
over-marks of a variety of donors, including heterospecifics
and conspecifics. First, these signals must be detected. And,
as we have described in this chapter, terrestrial mammals
have exquisite sensory apparatus to detect these signals and
specialized areas in the brain to decode, integrate, store, recol-
lect this information (Petrulis, 2013). The response should be
tailored to maximize the fitness of the participants (Ferkin,
2015; Wyatt, 2014).
The information found in a scent mark, for example, should
therefore be individually distinct (Halpin, 1986; Johnston, 1993;
Kaur et al., 2014). The response when they encounter scent
marks from different donors or under different contexts should
be flexible, allowing receivers to adjust their responses accord-
ingly, depending on the identity of the sender, past associations,
and context (Ferkin, 2011). Thus, the response chosen will be
modulated by the receiver’s ability and capacity for discrimina-
tion, categorization, judgments of relative numerousness, and
recollection of the emotional valence attached to the event
(Ferkin, 2011; Franklin and Ferkin, 2006, 2008; Johnston and
delBarco-Trillo, 2009; Roberts et al., 2014). The particular
response should represent a balance of the costs and benefits
associated with that choice (Ferkin, 2015) and the likelihood
of eavesdropping or copying by conspecifics (Danchin et al.,
2004; Valone, 2007). The response in question would have
been modified over time through evolutionary pressures placed
on individuals in that species (Roberts, 2007). In this way,
receivers in species that face similar ecological and social chal-
lenges may display responses to scent marks that are more
similar compared to those facing different challenges (Wyatt,
2014), indicating some degree of convergence and stability
over time (Ferkin, 2015).
Acknowledgments
We dedicate this chapter to Dr Robert E. Johnston. Bob was our mentor,
colleague, and friend. He will be missed.
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 1.11 The Medial Amygdala, Hormones, Pheromones, Social Behavior Network,
and Mating Behavior
Aras Petrulis, Georgia State University, Atlanta, GA, USA
Jeannie M Fiber, Contract Scientist, Cambridge, MA, USA
Jennifer M Swann, Lehigh University, Bethlehem, PA, USA
Ó 2017 Elsevier Inc. All rights reserved.
This chapter is a revision of the previous edition chapter by J. Swann, C. Fabre-Nys, R. Barton, volume 1, pp. 441–474, Ó 2009, Elsevier Inc.

1.11.1 Introduction 329

1.11.2 Pheromones in Mammalian Reproduction: The Rodent Model 330
1.11.2.1 Impact of Mating-Related Pheromones on Males 330
1.11.2.2 Impact of Mating-Related Pheromones on Females 330
1.11.3 The Impact of Mating-Related Hormones on Social Behaviors 331
1.11.3.1 The Impact of Gonadal Hormones on Mating in Males 331
1.11.3.2 The Impact of Gonadal Hormones on Mating in Females 331
1.11.4 The Social Behavior Network 331
1.11.5 MA Subdivisions 333
1.11.6 Comparative Anatomy 334
1.11.7 Sex Differences 334
1.11.8 Role of the MA and Connected Structures in Social Odor Investigation and Sexual Behavior in Males 334
1.11.8.1 MA Connections with BST 335
1.11.8.2 MA Connections with MPOA 336
1.11.8.3 MA Connections with VMH 336
1.11.9 The Role of the MA in Females 336
1.11.9.1 MA Connections with BST and MPOA 336
1.11.9.2 MA Connections with VMH 337
1.11.10 Steroids May Act Centrally and Peripherally to Impact Responses to Pheromones 337
1.11.11 Future Directions 337
References 338

Abbreviations
AH Anterior hypothalamus MApv Posteroventral subdivision of the medial nucleus of
AOB Accessory olfactory bulb the amygdala
AR Androgen receptor MOB Main olfactory bulb
AVP Arginine-vasopressin MPOA Medial preoptic area
BST Bed nucleus of the stria terminalis OBX Olfactory bulbectomy
DHT Dihydrotestosterone OT Oxytocin
E Estrogen SBN Social behavior network
ER Estrogen receptor USV Ultrasonic vocalizations
IEG Immediate-early gene T Testosterone
MA Medial nucleus of the amygdala VMH Ventromedial hypothalamus
MAa Anterior subdivision of the medial nucleus of the VMHdm Dorsomedial subdivision of the ventromedial
amygdala hypothalamus
MApd Posterodorsal subdivision of the medial nucleus of VMHvl Ventrolateral subdivision of the ventromedial
the amygdala hypothalamus

1.11.1 Introduction pheromones coordinate between conspecifics by signaling their

Sexual reproduction is a complicated business requiring coor-
dination within and between individuals. Hormones and pher-
omones play key roles: hormones coordinate within the
organism by aligning the activity of organs and cells, while
location and internal state. In many vertebrates, these signals
come together to act on a network of interconnected neural
groups to affect appropriate social behavior. At the heart of
this circuit is the medial amygdala (MA), an almond-shaped
nucleus in the temporal lobe that serves as a gatekeeper,

Hormones, Brain, and Behavior, 3rd edition, Volume 1 http://dx.doi.org/10.1016/B978-0-12-803592-4.00011-0 329

330 The Medial Amygdala, Hormones, Pheromones, Social Behavior Network, and Mating Behavior
integrating hormonal and pheromonal signals and facilitating
social behavior. The MA is a bellwether for the individual’s
internal state as its morphology and function are modified
and supported by gender and hormonal milieu. The MA is
but a few synapses away from the external environment,
receiving direct and indirect projections from the main and
accessory olfactory bulbs. Hormonal sensitivity and sex differ-
ences in structure, neurochemistry and perhaps connectivity,
impact the perception of pheromonal cues that regulate
behavior. While this ensures appropriate reproductive behav-
iors, it also has larger implications for how (sometimes
competing) social cues are filtered and perceived by males
and females and by individuals, to achieve specific, appropriate
behavioral outcomes. Thus, the MA, as a hub in several neural
networks involved in social behaviors, plays a critical role in
a wide variety of social interactions including aggression, kin
recognition, parenting, and copulation. Our review explores
the complexity of that role in mating. It concludes that what
we have learned over the past 40 years lays a solid foundation
for new questions to be addressed with new technologies
that further our understanding of pheromones and mating
behavior and more broadly, neural networks and behavioral
outcomes. This chapter represents a new version of a previously
published review on this topic (Swann et al., 2009).
1.11.2 Pheromones in Mammalian Reproduction:
The Rodent Model
Although chemical communication is involved in all social
contexts, it is most evident in reproduction. Chemosignals
impact reproductive development and physiology and, impor-
tantly, drive the appetitive components of reproductive behav-
iors such as investigation and approach toward opposite-sex
conspecifics, precopulatory behaviors that advertise the pres-
ence/absence of appropriate partners, as well as behaviors
such as anogenital investigation (AGI) that initiate copulation
(Ball and Balthazart, 2008; Petrulis, 2013). However, phero-
mones are generally less important for the maintenance of
sexual behavior once copulation has commenced. For example,
the display of lordosis in rats and hamsters is primarily stimu-
lated by somatosensory input (reviewed by Pfaff, 1980), and
the role of chemosignals is limited to reinitiation of interest
in the male (Rajendren et al., 1990). Removal of the olfactory
bulbs of female rats or hamsters does not inhibit and may
even enhance (Williams et al., 1992) the expression of lordosis
(Carter, 1973; Kelche and Aron, 1984). This, however, does not
occur in mice (Keller et al., 2006a,b). In addition, in male
hamsters (a species that requires chemosensory input for
expression of male-typical copulatory behavior (Murphy and
Schneider, 1970)), blocking chemosignal processing during
copulation has no effect on sexual behavior, whereas blocking
chemosensory input before copulation or during long intervals
between copulatory bouts eliminates male sexual behavior
(Devor and Murphy, 1973).
1.11.2.1 Impact of Mating-Related Pheromones on Males
Males may use mnemonic (anticipated location of females),
temporal (freshness), and spatial (location, arrangement)
aspects of odor cues, to orient toward mating partners (Ferkin
et al., 2008; Johnston, 2008; Pankevich et al., 2006), and will
readily approach and investigate female odors as part of their
mate-location strategy (Beauchamp and Beruter, 1973; Brown,
1978; Johnston, 1974). Upon detecting female odors, males of
several rodent species, including rats (Geyer and Barfield,
1978), mice (Nyby et al., 1977), hamsters (Johnston and
Kwan, 1984), and guinea pigs (Eisthen et al., 1987), will
emit ultrasonic vocalizations (USVs), which appear to facilitate
the location of the female (Pfaff et al., 2008). These responses
are eliminated by olfactory bulbectomy (OBX) (Bean, 1982;
Eisthen et al., 1987; Wysocki et al., 1982).
Once a male has located a female, he will usually engage in
substantial amounts of chemoinvestigation targeting the ano-
genital region of the female, prior to mounting (Dewsbury,
1975). OBX eliminates AGI and subsequent mounting,
and therefore copulation, in Syrian hamsters (Murphy and
Schneider, 1970) and mice (Rowe and Edwards, 1972), and
it impairs AGI/copulation in rats and guinea pigs (Beauchamp
et al., 1977; Edwards et al., 1990, 1996). In some species, such
as rats, robust investigation of female odors depends on socio-
sexual experience with the opposite sex, and exposure to vola-
tile chemosignals from female urine/feces alone can elicit
noncontact erections (NCEs) (Kondo et al., 1999; Sachs et al.,
1994). In contrast, both sexually naïve and sexually experi-
enced male Syrian hamsters are strongly attracted to female
odors (reviewed in Petrulis, 2013), and nonvolatile compo-
nents of female vaginal secretion (O’Connell and Meredith,
1984; Singer et al., 1984) are sufficient, but not necessary
(Johnston, 1986), for eliciting mounting behavior (Johnston,
1975; Murphy, 1973).
1.11.2.2 Impact of Mating-Related Pheromones on Females
Female Syrian hamsters (Johnston, 1979), rats (Carr et al.,
1965), mice (Hurst, 1990), meadow voles (Ferkin and Zucker,
1991), and ferrets (Kelliher and Baum, 2002) are preferentially
attracted to chemical cues of adult male conspecifics. Moreover,
females scent-mark to advertise their current or impending
receptivity in mice (Rich and Hurst, 1999), Syrian hamsters
(Johnston, 1977), rabbits (Gonzalez-Mariscal et al., 1990),
ferrets (Chang et al., 2000), and rats (Birke, 1984). For
example, female Syrian hamsters engage in vaginal marking,
a stereotyped behavior that places a vaginal fluid on the
substrate (Been and Petrulis, 2008, 2012; Been et al., 2012)
and is specifically targeted toward the location of male odors
(Johnston and Brenner, 1982; Johnston, 1977; Petrulis and
Johnston, 1997). Vaginal marking increases substantially on
the night before behavioral receptivity but is absent during
sexual receptivity, consistent with its role in attracting dispersed
males for mating (Gattermann et al., 2001; Johnston, 1977).
Similarly, estrous female rats (White et al., 1991) and hamsters
(Floody et al., 1977) also produce USVs in response to males or
their odors, a behavior that aids in short-range localization of
mates (Pfaff et al., 2008). Both types of precopulatory behavior,
marking and USVs, critically require chemosensory input as
OBX female hamsters strongly reduce both their USVs and
vaginal marking toward males (Kairys et al., 1980).
Even though chemosignals are not required for the expres-
sion of the sexually receptive posture (lordosis) in some
 The Medial Amygdala, Hormones, Pheromones, Social Behavior Network, and Mating Behavior
mammalian species (Pfaff, 1980), chemosignals can facilitate
lordosis, e.g., in pigs (Melrose et al., 1971; Reed et al., 1974),
rats (Rajendren and Moss, 1994), musk shrews (Rissman,
1989), and prairie voles (Curtis et al., 2001). In contrast, recep-
tivity is much more dependent of chemosignals in female mice
(Haga et al., 2010; Keller et al., 2006a,b) although it is not clear
if this chemosensory requirement is limited to the initiation
of sex behavior, or the maintenance of the receptive posture.
1.11.3 The Impact of Mating-Related Hormones on
Social Behaviors
Hormones exert a powerful impact on social behaviors. For
example, the presence or absence of gonadal steroids dictates
whether mating takes place or not. Information about the
internal steroid milieu and external chemosensory signals are
integrated within brain regions involved in social behaviors.
This section provides a brief overview. Extensive reviews of
this topic are available in other chapters within this volume.
1.11.3.1 The Impact of Gonadal Hormones on Mating in
Males
Gonadectomy, which removes the greatest source of gonadal
steroids, gradually and permanently eliminates male mating
behavior (Clark et al., 1995; Ballard and Wood, 2007).
Systemic treatment with testosterone (T) via injections or
timed-release capsules restores mating (Hull and Dominguez,
2007). T competes with dihydrotestosterone (DHT) to bind
to androgen receptors (ARs) (Pihlajamaa et al., 2015) and to
exert its actions. While ARs are found throughout the central
nervous system (Sarkey et al., 2008; Simerly et al., 1990) they
are densely concentrated in the MA and bed nucleus of the stria
terminalis (BST) (Wood et al., 1992). An interesting experi-
ment by Sheridan suggests the possibility of more than one
AR: one that binds unmetabolized T, and another one that
binds DHT (with greater affinity for DHT than T). The study
utilized radioactive T that would lose its label if metabolized
or aromatized (Sheridan, 1979). The study found the labeled,
unmetabolized T in only two brain areas – the BST and the
MA suggesting that the MA and BST alone may be directly influ-
enced by T in addition to its metabolites (Sheridan, 1983,
1991). Therefore, the BST and MA are unique in that they are
responsive to actions by unmetabolized T in addition to its
metabolites.
Circulating androgen is essential for male sex behavior, and
treatment with T restores mating in castrate male hamsters
over several weeks (Ballard and Wood, 2007). Gonadal
hormones in males are released in pulses or bursts that reflect
the pulsatile nature of the pituitary hormones that regulate
steroid hormone release (Gan and Quinton, 2010). However,
pulsatile release of pituitary hormone is not critical for T produc-
tion (Chase et al., 1988) or steroidal regulation of male
behavior, as copulation is restored in male hamsters, rats, and
mice given injections (Piekarski et al., 2009) or timed-release
capsules (Matochik et al., 1994; Antonio-Cabrera and Paredes,
2012).
T is metabolized to DHT and estradiol (E) by 5-alpha
reductase and aromatase, respectively. Both metabolites are
331
formed in the brain and peripheral tissues (Degtiar et al.,
1981). Moreover T has been shown to upregulate the produc-
tion of both enzymes in the mammalian brain (Wagner
and Morrell, 1997; Aste et al., 1998), suggesting a role for
DHT and E in the regulation of mating by T.
Neither hormone alone is sufficient for the expression of
mating; simultaneous treatment with both steroids restores the
full expression of male sexual behavior in castrated rats (Baum
and Vreeburg, 1973; Larsson et al., 1976, 1973b,a; Beyer et al.,
1973) and rabbits (Beyer et al., 1975; Beyer and Rivaud, 1973).
1.11.3.2 The Impact of Gonadal Hormones on Mating in
Females
Females also rely on gonadal steroids for the expression of
mating behavior. In spontaneous (Erb et al., 1971) – though
not in induced (Kauffman and Rissman, 2006) – ovulators,
gonadal hormones are released in a cyclical manner with
estrogen rising before progesterone rises. Estrogen recruits
progesterone receptors (Blaustein et al., 1980) which must be
activated for mating to occur (Mani and Oyola, 2012). Ovarec-
tomized females can be made sexually receptive if treated with
estrogen and progesterone in a pattern that mimics their
normal release. For example, most rodents have 4-day estrous
cycles. Treatment with estradiol for 2 days followed by proges-
terone is sufficient to induce motivational and consumatory
aspects of female mating behavior. Both steroids appear to
have central sites of action (Maggi and Perez, 1985), as estrogen
and progesterone placed in the hypothalamus restore mating
(Barfield et al., 1984). Ovarian steroids act peripherally as
well as centrally to coordinate responses to somatosensory
stimuli and act within the hypothalamus to optimize and coor-
dinate hypothalamic neurotransmitter (noradrenergic and
others) release, cellular responses to neurotransmitter release,
and second messenger systems (i.e., cAMP, cGMP) to facilitate
expression of lordosis at the appropriate time (Etgen et al.,
1999; González-Flores et al., 2010).
1.11.4 The Social Behavior Network
Early research outlined a series of pathways, collectively
described as the limbic system, connecting the amygdala with
the hippocampus for learning and memory and the cortex for
subsequent decision-making (MacLean, 1990; Papez, 1937).
More recent work with more sophisticated techniques has ques-
tioned the designation of these groups as a single system and
suggested, instead, that it operates as two or three circuits with
different, specific roles (Rolls, 2015; Catani et al., 2013).
Research using rodent models has identified a subset of limbic
system nuclei that play major roles in the regulation of social
behaviors (Newman, 1999). Described as the ‘social behavior
network’ (SBN), it is comprised of six reciprocally connected
regions: the extended MA (central-medial amygdala and BST),
anterior hypothalamus, midbrain, lateral septum, medial pre-
optic area (MPOA), and the ventromedial hypothalamus
(VMH) (Newman, 1999; O’Connell and Hofmann, 2011;
Figure 1). Together, these regions are nodes within a network
that regulates sexual, aggressive, and parental behavior in males
and females. Newman’s model broke new ground in suggesting
332 The Medial Amygdala, Hormones, Pheromones, Social Behavior Network, and Mating Behavior

Mesolimbic
reward system
Social behavior
network Str

POA NAcc

AH LS VP

VMH BNST/ blAMY

meAMY

PAG/CG HIP

VTA

Figure 1 The social behavior network and social decision-making network: the left panel shows the six reciprocally interconnected regions of the
social behavior network as described by Newman (1999). The lateral septum and BST/MA are also involved in the social decision-making network
(O’Connell and Hofmann, 2011). Arrows indicate projection neurons from one region to the other, and double-headed arrows indicate that the
connection is reciprocal. AH, anterior hypothalamus; blAMY, basolateral amygdala; BNST/meAMY, bed nucleus of the stria terminalis/medial amyg-
dala; HIP, hippocampus; LS, lateral septum; NAcc, nucleus accumbens; PAG/CG, periaqueductal gray/central gray; POA, preoptic area; Str, striatum;
VMH, ventromedial hypothalamus; VP, ventral pallidum; VTA, ventral tegmental area. Reprinted with permission from O’Connell, L.A., Hofmann, H.A.,
2011. The vertebrate mesolimbic reward system and social behavior network: a comparative synthesis. J. Comp. Neurol. 519, 3599–3639.

that it is the collective, rather than the individual, elements of the
group that drives behavior. Exactly which behavior is expressed
is determined by the degree to which relevant environmental
and internal parameters activate a given area.
The critical node of this network for processing social che-
mosensory cues related to male mating behavior is the medial
nucleus of the amygdala (MA). The nucleus forms a small
almond-shaped collection of cells in the ventromedial extent
of the temporal lobe in vertebrates (Medina et al., 2011;
Martinez-Garcia et al., 2002). It is in the MA that chemosensory
signals from both the accessory and the main olfactory bulbs
are integrated with hormonal signals to initiate and regulate
reproductive behavior. The MA also processes chemosensory
cues salient to other social behaviors, including parental
behavior, aggression, and conditioned defeat (Newman,
1999; Markham and Huhman, 2008). The MA has also been
implicated as an extension of the mesolimbic dopamine-
based reward system, in a ‘social decision-making network’
(O’Connell and Hofmann, 2011; Figure 1). In the expanded
group or social decision-making network the rewarding proper-
ties of dopamine impart salience to stimuli and associated
behaviors (O’Connell and Hofmann, 2011). However, it
appears that the reward component of sexual pheromones is
mediated via the vomeronasal amygdala (medial and cortical
posteromedial nuclei) projections to the ventral striatum and
not via the dopaminergic tegmento-striatal pathway (Lanuza
et al., 2008). Two nuclei, the septum and the BST (the exten-
sion of the MA), belong to both the SBN and social decision-
making network, supporting the role of the MA as a central
organizing hub for social behaviors.
In addition, the MA, MPOA, and parts of the hypothalamus
involved in the SBN overlap as part of a ‘survival circuit’ where
cues related to arousal, reinforcement, motivation, and
emotion surrounding survival are processed (LeDoux, 2012;
Sternson, 2013). For example, as part of this circuit, chemosen-
sory cues related to responses to unconditioned threats are
mediated via the MA (LeDoux, 2012). In addition to inte-
grating chemosensory and hormonal signals to allow mating
behavior or other behaviors to occur, the MA appears to serve
as a filter or sorting center to inhibit certain behaviors or choose
between competing chemosensory-driven behaviors (e.g.,
defensive response versus mating behavior if both cues are
present).
There is evidence for this type of MA-initiated inhibitory
chemosensory circuit regulating maternal behavior. Lesions of
the MA (especially the posteroventral subdivision), anterior
hypothalamus, or VMN decrease the latency for nonmaternal
rats to exhibit maternal behavior, suggesting that these regions
normally act to inhibit the behavior (Sheehan et al., 2001).
In addition, pup-induced Fos stimulation in the anterior
hypothalamus and VMN as well as other regions of the SBN
are reduced with ipsilateral lesions of the MA (Sheehan et al.,
2001). Taken together, these data suggest that the MA, through
its projection to the hypothalamus, and acting together with
other parts of the SBN, is involved in both facilitating and
inhibiting chemosensory-induced behaviors depending on
the internal hormonal milieu and external signals received.
It should be noted that more recent models describe a more
complex brain circuitry involved in chemosensory-driven
expression of the initiation of maternal behavior (Olazabal
et al., 2013). For example, stimulation from pup chemosensory
cues can differentially impact specific subdivisions of the lateral
septum or MPOA to inhibit or facilitate maternal behavior
(reviewed by Olazabal et al., 2013). The nucleus accumbens
 The Medial Amygdala, Hormones, Pheromones, Social Behavior Network, and Mating Behavior 333

and ventral pallidum are also involved in processing the reward
salience of pup olfactory cues; and the central and basolateral
amygdala are involved in processing the emotional salience of
pup cues to facilitate or inhibit behavior (see Olazabal et al.,
2013 for review). While the MA is one of the first regions to
integrate external chemosensory signals and internal hormonal
milieu to help guide appropriate social behavior, other regions
are involved in an extraordinarily complex circuitry of decision-
making and expression of social behaviors, concomitantly
processing different aspects of chemosensory salience for the
individual.
1.11.5 MA Subdivisions
The MA can be divided into three main subdivisions based on
their connections within the SBN. Two of these – the anterior
(MAa) and the posterodorsal (MApd) – maintain connections
with nuclei involved in reproduction (Coolen and Wood,
1998; Maras and Petrulis, 2010a; Gomez and Newman, 1992;
Canteras et al., 1995) while the third – the posterior ventral
(pv) subdivision – is also connected to nuclei that mediate
defensive behaviors (Canteras, 2002; Figure 2). Using axon
tracers genetically encoded for different LIM homeodomain
transcription factors, Choi et al. (2005) identified GABAergic
cells within the MApd of the male rat that respond to mating-
related olfactory cues (female urine). These cells project to the
ventrolateral (vl) VMH, and the authors suggest that this allows
reproductive behavior to occur, likely by disinhibiting cells
within the VMHvl, or indirectly through the BST. These
GABAergic cells in MApd that respond to mating-related chemo-
sensory cues and project to regions that facilitate mating
behavior also contain the transcription factor Lhx6 (Choi et al.,
2005). They are distinct from cells within the MApv that contain
other Lhx transcription factors, respond to olfactory defensive
cues (cat odor), and are glutamatergic. Choi et al. (2005) have
proposed that the MApv glutamatergic cells that project to the
dorsomedial (dm) part of the VMH are allowing or facilitating
defensive behaviors in response to odor cues and may serve to
inhibit mating behavior when a concomitant threatening cue is
present via projection to the VMHvl. This elegant study suggests
that the subdivisions of the MA respond to different types of

MA posterodorsal (pd)
Receives input from AOB and MOB
Projects to VMH(vl) and posteromedial BST
Contains androgen receptors (++++) and
estrogen receptors (+++(+))
Regulates mul ple aspects of reproduc ve
MApd
behavior; regulates female odor preference
via androgen receptors in pd
Contains GABAergic cells that respond to
ma ng-related chemosignals

MAa

MA anterior (a)
Receives input from AOB and MOB
Projects strongly to to VMH(vl), posterointermediate
BST and laterodorsal BST MApv
Contains androgen receptors (+++) and estrogen MA posteroventral (pv)
receptors (+) Receives input from AOB and MOB
Regulates interest in male and female odors (males) Projects to VMH(dm) (and vl to inhibit ma ng
and responses to odor in BST/MPOA behavior when threat is present), and the
May act as social filter for salience of cues via posteromedial BST
interac on with MApd Contains androgen receptors (+++) and
Oxytocin and arginine-vasopressin implicated in social estrogen receptors (+)
chemoinves ga on in MAa Regulates defensive behavior via glutaminergic
cells that respond to threat/defense-related
chemosignals

Figure 2 Subdivisions of the medial nucleus of the amygdala (MA) and their roles in chemosensory-driven mating behaviors. The double arrows in
this schematic illustrate the reciprocally connected subdivisions of the MA. All three subdivisions, anterior (a), posteroventral (pv), and posterodorsal
(pd), receive input from the AOB, and pd and anterior receive input from the MOB (Choi et al., 2005; Kang et al., 2009; Cadiz-Moretti et al., 2016).
The AOB projects to deeper layer Ia laminae of the amygdala versus MOB (Kang et al., 2009). All three subdivisions contain steroid receptors, to
different degrees as indicated by number of þ symbols (Simerly et al., 1990), and play differential roles in regulating aspects of chemosignal-driven
mating behaviors. All MA subdivisions project to the medial preoptic area, with the strongest projection from pd to the medial preoptic nucleus
(based on a mouse study) (Pardo-Bellver et al., 2012). Differential projections to the BST are described in the schematic. Despite some differences,
the three main subdivisions appear to interact to cooperatively regulate expression of (sometimes competing) chemosensory-driven behaviors. The
drawings of MA subdivisions are adapted from “A Stereotaxic Atlas of the Golden Hamster Brain” (Morin and Wood, 2001). This figure and legend
are also based on the work of Wood et al. (1992), Pardo-Bellver et al. (2012), Portillo et al. (2006), Greco et al. (1996), Ferguson et al. (2001),
Arakawa et al. (2010), Maras and Petrulis (2010a), and Keshavarzi et al. (2014).
334 The Medial Amygdala, Hormones, Pheromones, Social Behavior Network, and Mating Behavior
cues, but there is some prioritization and filtering occurring at
this hub of the SBN to ensure that responding to threats takes
precedence over mating behavior. In social mammals, this mech-
anism may be akin to social judgment. Details of the pathways
and mechanisms of this type of filtering or gating require further
study. A cell fate mapping study showed a differential pattern of
expression of Sonic hedgehog (Shh)- and Nkx2-1-derived inhib-
itory output cells in the MApv and MApd, and that these cell
groups possess different neurotransmitter profiles (Carney et
al., 2010). These findings support the idea that the two subdivi-
sions of MA, which have differing cell lineages, play different
integral roles within the SBN. The findings suggest that the MA
subdivisions are programmed by cell migration early in develop-
ment to manage chemosensory input and regulate appropriate
behavioral responses. The MApd is anatomically sculpted by
internal processes during puberty that determine axon place-
ment and pruning of dendrites and spines (Zehr et al., 2006).
In addition, social signals and experiences after weaning impact
MApd volume and neuronal soma size, as well as male mating
behavior (Cooke et al., 2000). Thus, both pubertal anatomical
developments and social experiences wire the MA for processing
the array of social olfactory cues that allow appropriate behav-
ioral responses to those cues via the SBN.
1.11.6 Comparative Anatomy
Beyond mammals, key elements of the SBN appear to be
similar in the nonmammalian vertebrate brain (Goodson,
2005; O’Connell and Hofmann, 2011, 2012) suggesting that
the SBN is an evolutionarily conserved, key feature of the verte-
brate brain (Goodson, 2005) and that diversity in social
behavior is likely due to variation in an otherwise conserved,
homologous neural network and the array of gene expression
within that network (O’Connell and Hofmann, 2011, 2012).
It should be noted that, while not listed as part of the SBN,
the olfactory bulbs also play an obvious and critical role in the
organization and expression of olfactory-mediated social behav-
iors. The pioneering work of Keverne (Lloyd-Thomas and
Keverne, 1982; Kaba et al., 1989) showed that memory of
a mate may not be encoded in the amygdala or the SBN. Rather,
the site of the phenomenon known as the Bruce effect (preg-
nancy termination after exposure to chemosignals from an unfa-
miliar male) lies in the accessory olfactory bulb (Kaba et al.,
1989, 1994; Brennan et al., 1990). Moreover, research suggests
that the main olfactory system may play a larger role in process-
ing reproductively relevant chemosignals than initially consid-
ered (Baum and Cherry, 2015; Woodley and Baum, 2004;
Dhungel et al., 2011a; Swann et al., 2001; Kang et al., 2009)
and that the piriform cortex may play a significant role in the
processing of socially relevant cues (Veyrac et al., 2011; Petrulis,
2009). Thus, the basic networks and circuitry do not function
as solitary units, but as part of an interconnected system of
networks that also includes afferent and efferent brain regions.
1.11.7 Sex Differences
The MA is sexually dimorphic in a number of species for
a variety of parameters. The volume of both the neurons and
the nucleus is greater in males and controlled by circulating T
(Morris et al., 2008; Rasia-Filho et al., 1991). Male rats have
a greater number of cells – neuronal and supporting (Johnson
et al., 2008) – than females in the MApd and MApv (Rasia
et al., 2002). The morphology of the cells is also dimorphic.
Both glia (Johnson et al., 2008) and neurons (Rasia et al.,
1999) are more complex in males than females. Moreover,
neuronal morphology is sex specific so that dendrites are differ-
entially oriented in both the MApd and MApv (Dall’Oglio
et al., 2008) of male and female rats. While most of these differ-
ences are in the steroid-responsive MApd, sex differences in
dendritic spines are also found in the anterior versus posterior
divisions of MA (Rasia et al., 1999). Male rats have greater
concentrations of opioid receptors (Wilson et al., 2002),
substance P (Malsbury and McKay, 1989), and CCK (Micevych
et al., 1994) in the MApd than females. There are species-
specific neurochemistry differences as well. For example, while
the MApd of male prairie voles has more tyrosine hydroxylase
than that of females (Cavanaugh and Lonstein, 2010), this sex
difference is not found in rats or hamsters (Northcutt et al.,
2007). Taken together the data suggest that the MA of males
and females are differentially organized, and this may play
a role in differential responses to chemosignals. This hypoth-
esis is further explored below.
1.11.8 Role of the MA and Connected Structures in
Social Odor Investigation and Sexual Behavior in
Males
The MA plays a critical role in the attraction of males to the
odors of females. Bilateral ablation of either the MAa or
MApd eliminates preferences for female odors in Syrian
hamsters and rats, albeit in different ways. Specifically, in
hamsters, MAa damage causes unusually high levels of interest
in male odors but unaltered (high) levels of interest in female
odors, whereas males with damage to MApd reduce their inves-
tigation of female odors (Dhungel et al., 2011a,b; Maras and
Petrulis, 2006). This may suggest that the rostral aspect of the
nucleus, with its strong chemosensory input, provides a ‘social
filter’ that evaluates the significance of a social odor. This infor-
mation then interacts with a steroid-dependent mechanism in
the caudal aspect of MA that adjusts the ‘gain’ driving increased
investigation. Further evidence for this distinction between
MAa and MApd function comes from (1) the ability of MAa,
but not MApd, lesions to eliminate immediate-early gene
(IEG) responses throughout MA, BST, and MPOA following
exposure to social odors (Maras and Petrulis, 2010b,c; Wang
et al., 2013; Maras and Petrulis, 2010b), (2) highly specific
IEG and electrophysiological responses to social odors in
MApd, but not MAa (Bergan et al., 2014; Meredith and
Westberry, 2004; Samuelsen and Meredith, 2009), (3) finding
that olfactory bulb stimulation only increases IEG expression
in MAa, but not MApd (Blake and Meredith, 2010; Meredith
and Westberry, 2004; Nolte and Meredith, 2005), and (4) the
ability of AR blockade to impair female odor preference in
MApd, but not MAa (Hosokawa and Chiba, 2010). Indeed,
the MAa and MApd must interact to drive attraction to female
odors, either through reciprocal MAa–MApd connections
(Maras and Petrulis, 2010a,b,c) or through downstream
 The Medial Amygdala, Hormones, Pheromones, Social Behavior Network, and Mating Behavior
structures, as functional disconnection of MAa and MApd elim-
inates odor preference (Maras and Petrulis, 2010c; Figure 2).
Although the MA is critical for male attraction and interest
in female odors, the particular neurochemical mechanisms
within MA that regulate this process are not well understood.
There is evidence, however, that the oxytocin (OT) and
arginine-vasopressin (AVP) neuropeptide systems within the
MA are implicated in social investigation. This relationship
appears bidirectional because, in mice, OT receptor blockade
impairs IEG responses in MA (and connected structures) to
females (Ferguson et al., 2001) or their odors (Samuelsen
and Meredith, 2011). Additionally, MA lesions reduce social
IEG responses in hypothalamic OT/AVP cell groups (Wang
et al., 2013) and central/peripheral OT release (Wang et al.,
2015).
The typical reductions in male chemoinvestigation of
familiar females (‘social memory’) appear to require OT action
in the MA: OT receptor antagonist injected into MApd of male
mice and rats blocks social memory whereas OT injected into
MApd rescues social memory in OT knockout mice (Ferguson
et al., 2001; Lukas et al., 2013). This effect may be due to OT
facilitation of long-term depression of AOB, but not MOB,
inputs to MA (Gur et al., 2014). However, OT effects on MA
are not limited to opposite-sex investigation as OT receptor
antagonist infusions in MA increase approach toward predator
odors (Samuelsen and Meredith, 2011) and decrease approach
toward odors of healthy same-sex conspecifics (Arakawa et al.,
2010). This pattern of results would suggest that OT in MA nor-
mally promotes interest in complex odors except that avoid-
ance of odors from sick males is unaffected by OT receptor
blockade (Arakawa et al., 2010); this discrepancy remains to
be explained. A similar discrepancy is apparent with AVP action
in MA: blockade of AVP receptors (V1a type) here inhibits
avoidance of sick conspecifics (Arakawa et al., 2010), but upre-
gulation of AVP within MA by Toxoplasma gondii infection leads
to an abnormal attraction to predator (cat) odors in rats (Hari
Dass and Vyas, 2014). This difference in results may reflect
different loci of AVP action. That is, local AVP release and
action within MA promotes avoidance of unhealthy or
dangerous odors whereas increased AVP produced in MA
may be released at distal targets to drive attraction.
The MA initiates male sexual behavior through its integra-
tion of chemosensory and sex steroid signals (Wood and
Coolen, 1997). MA ablation in hamsters, like olfactory bulb
removal (Murphy and Schneider, 1970), eliminates sexual
behavior and strongly inhibits AGI of females; this deficit is
much more evident following MAa lesions than after MApd
damage (Lehman et al., 1980). In contrast, in male rats it is
damage to MApd, and to a lesser extent MAa, which impairs
initiation of sexual behavior (McGregor and Herbert, 1992a)
and sexual arousal (NCEs) toward female odors (Kondo
et al., 1998; Kondo and Sachs, 2002).
Sex steroids acting within MApd appears to be necessary and
sufficient for initiating mounting and female investigation.
However, the precise nature of this relationship may vary across
species, and the evidence is, in some cases, contradictory. For
example, implanting T or estradiol, but not dihydrotestoster-
one, into the MApd restores mounting and female investiga-
tion in castrated hamsters (Coolen and Wood, 1999; Wood
and Newman, 1995c; Wood, 1996), and estrogen receptor
335
(ER) blockade here reduces mounting in hormonally intact
males (Wood and Williams, 2001). Although these results
suggest that estrogen action in MApd is critical for sexual
behavior, systemic aromatase blockade has no effect on female
investigation or mounting behavior in hamsters (Cooper et al.,
2000). In rats, implants of estradiol within MApd restore
mounting behavior in castrates (Huddleston et al., 2003;
Rasia-Filho et al., 1991), and blocking aromatase action here
(Huddleston et al., 2006) reduces mounting in intact male
rats. However, blocking ER-alpha in MApd does not impair
mounting (Paisley et al., 2012) nor is mounting maintained
by specific ER-alpha or ER-beta agonist infusions in MApd
(Russell et al., 2012). This suggests that combined ER-alpha
and ER-beta activity is required in the MApd for sexual
initiation and/or AR activation is also needed. Indeed,
blocking AR within MApd reduces mounting in rats
(McGinnis et al., 1996) and is necessary and sufficient for
sexual arousal to distal female cues in rats (Bialy and Sachs,
2002; Bialy et al., 2011). Similarly, the initiation of sexual
behavior in mice survives deletion of virtually all aromatase
neurons in MApd (Unger et al., 2015).
Perhaps the strongest argument against DHT acting through
the MAa is in studies of MA morphology. Systemic T, E, and
DHT þ E affect changes in neuronal morphology of the MAa,
but DHT alone has no effect in either the hamster (Gomez
and Newman, 1991), or the rat (Cooke et al., 2003), suggesting
that DHT acts on other sites to affect behavior or affects
behavior without affecting morphology of the MA.
The neurochemical mechanisms by which the MA initiates
investigation and sexual behavior are becoming somewhat
clearer. Even though there is little overlap between the MApd
cells that respond during copulation or during aggression
(Lin et al., 2011), the initiation of copulation and aggressive
behavior both require GABA neuron activation in MApd
(Hong et al., 2014). A low-intensity increase in activity of
GABAergic neurons in MApd stimulates mounting behavior
or social grooming whereas attack behavior is initiated at
higher levels of GABA cell activity/recruitment. In contrast,
MApd glutamatergic neurons appear to normally inhibit
ongoing social behavior. Perhaps glutamatergic MApd cells
produce these effects by coreleasing beta-endorphin as injec-
tions of this peptide into MApd decrease investigation
of females and subsequent transition to mounting in rats
(McGregor and Herbert, 1992b,c). There is also evidence that
AVP cells within MA play a role in decreasing hippocampal
activity during initial investigation and mounting in male
rats, but the precise functional role of this connection has not
been established (Smock et al., 1998).
1.11.8.1 MA Connections with BST
The massive interconnections between MA and BST are a key
neural substrate for attraction to, and interest in, opposite-sex
odors. In male hamsters, damage to just the BST eliminates the
high levels of interest in volatile female odors and strongly
decreases interest in contacting female scents (Been and Petrulis,
2010a; Powers et al., 1987). Similarly, male rats with damage to
BST also show a lack of preferential interest in sexually active
females (Edwards et al., 1996). The preference to investigate
volatile (but not contacted) components of female odors
336 The Medial Amygdala, Hormones, Pheromones, Social Behavior Network, and Mating Behavior
requires functional interactions between BST and the MA (Been
and Petrulis, 2012). This pattern of results suggests that MA-BST
interactions are critical for attraction to distal female cues,
but that preference for odors when contact is allowed is
mediated by the limited, but direct, AOB projections to BST.
Although the BST, or its connections with MA, is not critical
for copulatory behavior (Been and Petrulis, 2010a, 2012), BST
lesions do reduce the AGI that precedes copulation (Claro
et al., 1995; Powers et al., 1987) as well as penile erections in
response to female odors (Liu et al., 1997). T or E implants
in BST/MPOA can restore female investigation and mounting
in hamsters (Wood and Newman, 1995b) and mice (Matochik
et al., 1994; Nyby et al., 1992), but no studies have investigated
how hormonal manipulations restricted to BST influence
sexual behavior.
1.11.8.2 MA Connections with MPOA
The MPOA may be part of the circuit that links interest in
opposite-sex cues to motivated approach behavior (Everitt,
1990), but this relationship is complicated by the oft-
observed concomitant damage to BST following MPOA lesions
and interactions with the sexual experience of the subject.
Damage that includes the MPOA reverses the preference for
female odors in sexually naïve ferrets (Alekseyenko et al.,
2007; Hurtazo and Paredes, 2005) and eliminates preference
for estrous female odors in sex-experienced rats (Dhungel
et al., 2011b; Hurtazo et al., 2008; Hurtazo and Paredes,
2005). However, the ventral/caudalmost part of the BST was
also lesioned in these studies and so any deficits in investiga-
tion could be due to ventral or caudal BST damage and not
to MPOA damage itself. Indeed, in hamsters, only damage to
both the MPOA and the ventral/caudal BST was sufficient to
eliminate interest in volatile female odors by sexually experi-
enced male hamsters (Been and Petrulis, 2010b). Specific
MPOA lesions only reduce volatile odor preference in sexually
naïve males, not in sex-experienced animals (Been and Petrulis,
2010b). In fact, this caudal/ventral aspect of BST is already
known to be critical for sexual behavior in both rats (Finn
and Yahr, 2005) and gerbils (Yahr and Gregory, 1993). Simi-
larly, within MPOA, AR blockade only impairs opposite-sex
preference when targeted to caudal and dorsal MPOA, an
area contiguous with caudal/ventral BST (Harding and
McGinnis, 2004; McGinnis et al., 2002).
The MPOA has long been known to be critical for male
sexual behavior (Hull and Dominguez, 2007). Indeed, lesions
limited to the MPOA eliminate copulation in Syrian hamsters
(Been and Petrulis, 2010b; Powers et al., 1987), gerbils
(Heeb and Yahr, 2000; Yahr and Gregory, 1993; Yahr et al.,
1994), mice (Bean et al., 1981), and rats (Dorries et al.,
1997). Connections from MA to MPOA are critical for the initi-
ation of copulation in hamsters (Been and Petrulis, 2012), rats
(Kondo and Arai, 1995), and gerbils (Heeb and Yahr, 2000),
indicating that this pathway is the likely circuit for chemosen-
sory control of male copulatory behavior (Triemstra et al.,
2005). Downstream of MA, the MPOA is also a site of conver-
gence of steroid and chemosensory signals for regulating copu-
lation (Greco et al., 1998; Wood and Newman, 1995b) as
implanting T or E into MPOA restores mounting and female
investigation in castrated hamsters (Lisk and Bezier, 1980;
Wood and Newman, 1995a,b), rats (Lisk, 1967), and mice
(Nyby et al., 1992) (also see Sipos and Nyby, 1996, 1998).
Conversely, blocking aromatase or ER-alpha action in MPOA
reduces mounting in rats (Clancy et al., 2000; Huddleston
et al., 2007; Paisley et al., 2012; Russell et al., 2012; Sano
et al., 2013). However, AR action in the MPOA also appears
important for initiating mounting as blocking AR in MPOA
reduces mount behavior in rats (Harding and McGinnis,
2004; McGinnis et al., 1996, 2002).
1.11.8.3 MA Connections with VMH
In the context of sexual behavior, there has been a strong focus
on the VMH as a key brain substrate for hormonal control of
female, rather than male, sexual behavior (Pfaff, 1980).
However, several lines of evidence now indicate that the VMH
actually plays a very important role in initiating male investiga-
tion and mounting of females. First, lesions limited to the MAa
projection zone of the VMH impair precopulatory behavior in
male rats (Harding and McGinnis, 2005). Second, within the
VMH, T implants restore, and AR blockade impairs, preference
for investigating sexually active females (McGinnis et al., 1996;
Harding and McGinnis, 2003, 2004). Third, deletion of proges-
terone receptor-bearing neurons in VMH decreases male
mounting behavior as well as female sexual behavior (Yang
et al., 2013). Lastly, low-level stimulation of ER-alpha neurons
within VMH, much like MApd stimulation (Hong et al.,
2014), induces prolonged investigation of females and
subsequent mounting (Lee et al., 2014), suggesting that MA-
VMH connections are critical for managing and filtering
conflicting social states (i.e., aggression vs copulation).
1.11.9 The Role of the MA in Females
The MA plays a critical role in chemosensory-driven sexual
behavior in females. Damage to MA eliminates differential
investigation by females of male odors in Syrian hamsters
(Petrulis and Johnston, 1999), mice (DiBenedictis et al.,
2012), and rats (Kondo and Sakuma, 2005) as well as the
heightened receptivity following repeated mating in rats (Rajen-
dren and Moss, 1993). However, ER-alpha knockdown within
MApd has no effect on preference and proceptive behavior in
female rats (Spiteri et al., 2012) and so the behavioral role
that gonadal steroids play within MA of females is currently
unclear.
1.11.9.1 MA Connections with BST and MPOA
The BST is well known as a brain region that is sexually differ-
entiated in size and cell phenotype (de Vries and Sodersten,
2009) which may explain the relative lack of data on the role
of BST in female sexual attraction or behavior. What evidence
exists suggests that the female BST is involved in appropriate
targeting of sexual solicitation, via an OT mechanism, and is
not involved in directing interest toward male odors (Martinez
et al., 2010; Martinez and Petrulis, 2011). Although the MPOA
is more typically associated with male copulatory behavior,
this brain area does play a role in regulating both interest in
males and initiation of female copulatory behavior. For
 The Medial Amygdala, Hormones, Pheromones, Social Behavior Network, and Mating Behavior
example, when females are allowed to pace mate, MPOA
lesions impair female copulatory behavior (Yang and Clem-
ents, 2000; Whitney, 1986) and eliminate preferences for
male scents in rats (Xiao et al., 2005) and hamsters (Martinez
and Petrulis, 2013) but not in ferrets (Robarts and Baum,
2007). Moreover, knockdown of ER-alpha within rat MPOA
impairs preferential investigation of reproductively active
males (Spiteri et al., 2012).
1.11.9.2 MA Connections with VMH
The VMH has long been implicated in the control of female
sexual receptivity (Pfaff et al., 2008), but it also regulates
female precopulatory behavior. For example, lesions of the
VMH in ferrets eliminate preference for investigating male
cues (Robarts and Baum, 2007) whereas blocking ER-alpha
in VMH reduces preference for reproductively active males in
rats (Spiteri et al., 2010) as well as proceptive behaviors in
mice (Musatov et al., 2006). Similarly, estrogenic stimulation
of VMH reinstates sexual solicitation in ovariectomized
hamsters (Takahashi et al., 1985).
1.11.10 Steroids May Act Centrally and Peripherally
to Impact Responses to Pheromones
Both sex and circulating hormones impact neural responses to
chemosensory cues (Fiber and Swann, 1996; Brock et al., 2012;
Kang et al., 2011; Bodo and Rissman, 2007; Swann and Fiber,
1997). Circulating steroids not only influence behavioral
responses to pheromones, but may also impact the sensitivity
to reception or perception of mating-related chemosignals.
Steroids likely act on both central regions mediating behavioral
responses as well as peripheral structures that process incoming
sensory information (Gandelman, 1983). Studies using Fos as
a marker for neural stimulation show that T increases phero-
mone (female vaginal secretion)-stimulated Fos in medial pre-
optic nucleus magnocellular (MPNmag) of male hamsters
(Fiber and Swann, 1996; Kollack-Walker and Newman,
1997) and fails to affect pheromone-stimulated Fos in the post-
eromedial BST and the posterior MA of either sex (Fiber and
Swann, 1996). In a recent study using calcium imaging of vom-
eronasal sensory neurons to directly visualize cellular response
to male pheromone ligands, it was demonstrated that the
pheromone-responsive sensory neurons respond to the phero-
mone ligands during estrus, but not during diestrus when
progesterone is present (Dey et al., 2015). Taken together, these
studies appear to confirm that circulating steroids act at both
peripheral sites and central sites to govern the reception or
perception of sensory cues and the behavioral responses to che-
mosensory cues.
1.11.11 Future Directions
What next for this complex nucleus? While the posterior dorsal
and posterior ventral subdivisions seem committed to specific
behavioral roles, the anterior subdivision does not, and may
play a role in filtering social information. The MA also medi-
ates aggression and parenting, behaviors that, such as mating,
337
are modified by steroids and sex. How can one area be respon-
sible for multiple behaviors? To answer that question, one
need only look to the vast research on the VMH. Decades of
research have implicated this tiny group of cells in feeding,
copulation, and lordosis. Clues to the mechanisms that
underlie its complex roles have been addressed using optoge-
netics coupled with genetic cell markers and other genetic
approaches. Stimulation and ablation of specific cells within
the VMH reveal that the functional subdivisions of the VMH
defy conventional approaches. Neurons participating in
specific behaviors are not segregated to different regions –
they are distributed throughout (Lin et al., 2011). Moreover,
the study supports earlier findings, described above, that the
VMH is also involved in male sexual behavior (Lin et al.,
2011). Similar work in the MA could begin with optogenetic
approaches, building on previous LIM homeodomain data
(Choi et al., 2005).
More comparative work also needs to be done. The large
body of work presented here is focused on mammals primarily
because work in birds and reptiles is in short supply. Homolog-
ical studies identifying the amygdala in birds and fishes suggest
functional differences in the rostral and caudal regions of the
tenia (homologous to the MA) (Absil et al., 2002; Thompson
et al., 1998). Additional work would broaden our
understanding of the relationship between the development of
the amygdala and the evolution of the behaviors it subserves.
Similarly, studies of the primate MA are few and far
between. With recognition of the powerful work of LeDoux
(2007), researchers have focused on fear as mediated by the
lateral amygdaloid nucleus, virtually ignoring the contribu-
tions of the MA. This may be an oversight in the search for
neural correlates of cognition. MA mediates aggression which
is an important hallmark of some cognitive diseases (Van den
Stock et al., 2015). Olfaction has been implicated in cognitive
diseases such as Parkinson’s, schizophrenia, and Alzheimer’s
(Good and Sullivan, 2015; Yildirim and Derksen, 2012;
Markham, 2012). Plaques and Lewy bodies’ characteristic of
these diseases are present in the olfactory bulbs of patients
with disease (Attems et al., 2014; Wilson et al., 2011). Olfac-
tory dysfunction is also associated with combat-related trau-
matic brain injury (Xydakis et al., 2015). Anosmia
determined by smell tests has become a useful part of diag-
nostic confirmation and to inform medical decisions (Otta-
viano et al., 2015; Xydakis et al., 2015). Steroids have also
been implicated in the prognosis for cognitive diseases (Yil-
dirim and Derksen, 2012; Markham, 2012). Facial expression
or emotion recognition are common deficits among certain
cognitive disorders (Torres et al., 2015; Yoshimura et al.,
2005; Rangel et al., 2015), and one study found the amygdala
function was compromised in individuals with Parkinson’s
disease (Yoshimura et al., 2005). The combination of olfac-
tory dysfunction and impairments of social cue perception
in individuals with cognitive disorders suggests the possible
involvement of the MA.
As we seek to know more about these devastating cognitive
diseases, we ignore this area at our own peril. As a central hub
of several important social networks within the connectome,
the amygdala with its ability for internal and external refer-
ence may ultimately be where we find the answer to Pee
Wee Herman’s question “I know you are, but what am I?”
338 The Medial Amygdala, Hormones, Pheromones, Social Behavior Network, and Mating Behavior
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 1.12 Circadian Regulation of Endocrine Functions
Matthew P Butler, Oregon Health & Science University, Portland, OR, USA
Ilia Karatsoreos, Washington State University, Pullman, WA, USA
Lance J Kriegsfeld, University of California, Berkeley, CA, USA
Rae Silver, Columbia University, New York, NY, USA; Barnard College, New York, NY, USA; and College of Physicians and
Surgeons, New York, NY, USA
Ó 2017 Elsevier Inc. All rights reserved.
This chapter is a revision of the previous edition chapter by M.P. Butler, L.J. Kriegsfeld, R. Silver, volume 1, pp. 473–507, Ó 2009, Elsevier Inc.
1.12.1 Introduction to Circadian Aspects of Endocrine Function
1.12.2 The Circadian Timing System
1.12.2.1 Entrainment through a Hierarchy of Clocks
1.12.3 Circadian Regulation of the Endocrine System
1.12.4 Circadian Regulation of Hypothalamo–Pituitary–Adrenal Axis
1.12.5 Circadian Rhythmicity in the Hypothalamic–Pituitary–Gonadal Axis
1.12.6 Circadian Regulation of Melatonin
1.12.7 Circadian Dysfunction and Chronotherapy
Acknowledgments
References
1.12.1 Introduction to Circadian Aspects of
Endocrine Function
Overview of circadian timing. The significance of circadian
rhythms to hormones and behavior is twofold. First, time of
day must be taken into account in considering ‘normal’
hormone levels as most hormones are secreted with circadian
periodicity. These rhythms are determined by the interaction
between the brain’s master clock which is typically synchro-
nized to the external light–dark cycle and hormone secreting
tissues that are also rhythmic. Second, the physiological effects
of a hormone depend on the timing of its secretion and sensi-
tivity of the target tissue to its effects. Cellular actions are deter-
mined by the concentration of a hormone relative to affinity for
its specific receptor, receptor availability and/or abundance,
and postreceptor signaling factors. Time of day, acting through
the circadian timing system, modulates each of these processes.
The evidence of time of day effects has long been available. Our
understanding of the causal mechanisms continues to emerge.
The importance of time of day in the regulation of endo-
crine processes was known well before the biological basis of
the circadian timing system was understood. As an interesting
example, Everett and Sawyer’s (1950) pioneering work on the
timing of ovulation was motivated by the question of whether
there was any role for the central nervous system in the regula-
tion of hormone release. Everett and Sawyer knew that the
luteinizing hormone (LH) (Grundschober et al., 2001) surge
in rats occurs at a particular time of day – termed the critical
period – on the afternoon of proestrus. They demonstrated
that when hypothalamic activity is blocked in proestrus
females by transient barbiturate anesthesia, the LH surge is
delayed for a full 24 h rather than for the 2 h of anesthetic seda-
tion, pointing to the participation of a circadian timing system
in generating the LH surge. It is now known that a daily signal
from the brain’s master clock times the preovulatory LH surge.
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While this signal occurs every day, it is effective at stimulating
gonadotropin-releasing hormone (GnRH) neurons to produce
the LH surge only when estrogen levels reach a critical
threshold. The occurrence of this daily stimulatory suprachias-
matic nucleus (SCN) signal can be demonstrated by implant-
ing female rodents with capsules that produce high
concentrations of circulating estrogens, in which case an LH
surge occurs daily (Legan et al., 1975; Christian et al., 2005).
Clearly, this work points to a role for neural mechanisms in
timing daily surges in hormone secretion. The principle that
time of day of exposure to a hormone has marked effects on
the physiological response has also been recognized (Figure 1).
For example, glucocorticoid secretion by the adrenal gland
in vitro responds differentially to a fixed dose of ACTH depend-
ing on time of day (Ungar and Halberg, 1962).
Since the time of these initial studies, the phenomenology
of circadian rhythmicity has exploded, with ubiquitous exam-
ples of circadian regulation. We now understand more fully
how the circadian clocks gate and modulate endocrine
rhythms. Developments in molecular biology have led to the
identification of genes that are gated or modulated by the
circadian system and canonical circadian ‘clock’ genes that
are part of the cellular timing mechanism. While questions
of how the circadian system regulates endocrine secretions
remain, the nature of the unknowns and the kind of questions
asked and answered has changed dramatically. This is due, in
part, to the ability to more easily and accurately measure circa-
dian variables over long time intervals making the work of col-
lecting time series data less grueling. Technical breakthroughs
now allow real-time measurement of clock function in vivo and
in vitro making high-throughput screening of gene expression
possible.
Functions of rhythms: entrainment, internal synchronization,
and anticipation. The fundamental functions of the circadian
timing system are to ensure the correct temporal staging of
345
346 Circadian Regulation of Endocrine Functions
12
Direct extraction
Incubation without ACTH
Incubation with 0.04 I.U.ACTH
Incubation with 0.40 I.U.ACTH
Incubation with 4.00 I.U.ACTH
10
Mean of 2 pools
Corticosterone (μg/IO adrenals/2 h)
Individual mean
8
6
4
2 from early days and play increasingly important roles in
0
24
08 16
Time (clock hours)
Figure 1 Time of hormone administration determines the amplitude
of the rhythmic response. In phenomenological studies done
throughout the world, Franz Halberg and his associates monitored
physiological rhythms in numerous variables as part of the ‘neuro-
endocrinologic armamentarium.’ In experimental laboratory work, he
showed a circadian influence on the response of mouse adrenals to
fixed doses of adrenocorticotropic hormone (ACTH) administered
in vitro. Corticosterone was extracted either directly from adrenal
glands or after incubation with several doses of ACTH. Figure from
Ungar, F., Halberg, F., 1962. Circadian rhythm in the in vitro response
of mouse adrenal to adrenocorticotropic hormone. Science 137,
1058–1060.
numerous processes internal to the body and to enable the
entrainment of these processes to the local environment.
This coordination is necessary for optimal regulation of phys-
iology and behavior. Coordination is achieved by a master
clock, located in the SCN, which is synchronized each day to
the local photoperiod. The SCN, in turn, sets the phase of
the cellular clocks throughout the body. The output of some
of these downstream systems, such as hormones produced
by the major glands of the body, in turn, feeds back to provide
temporal information to the brain clock. Together the brain
and bodywide circadian clocks enable the anticipation of regu-
larly recurring daily events and the optimal temporal phasing
of key events within the body, relative to each other. Notably,
conditions such as shift work can disrupt the delicate balance
of oscillators across the body and impair this coordination
leading to pathology.
Intense interest in circadian chronobiology derives from
a convergence of practical and medical needs with technolog-
ical and theoretical developments. In the practical domain,
the range of medical phenomena that are modulated by the
circadian system has expanded to include every organ of the
body, numerous disrupted states such as alcoholism, cardio-
vascular crises, cancer and cell division, and phenomena
such as stress, jet lag, shift work, and aging. The goal of this
chapter is to delineate the basic phenomena important in
the field of circadian rhythmicity and to highlight ways in
which circadian timing impacts the understanding of
hormone effects on behavior and physiology. Terminological
notes and definitions esoteric to circadian studies are
provided in Box 1.
1.12.2 The Circadian Timing System
Overview. The trajectory of chronobiological research has been
‘top-down’ from macroscopic studies of rhythms in animals
and humans to more reductionist levels. An excellent review
of the history of research is available for work up to the initial
discovery of clock genes in mammals (Weaver, 1998). Biolog-
ically relevant mathematical models have guided research
revealing how oscillators are coupled from the neuron to
the tissue level (Bechtel, 2016; Pauls et al., 2016). The enigma
of how isolated and interacting cells measure the passage of
08
time has been fruitfully addressed at the biochemical,
network, and whole organism levels. The role of transcrip-
tion–translation feedback loops is very well documented
(Albrecht, 2012). More recently, the contribution of
nontranscriptional mechanisms has drawn attention to the
multitude of oscillatory mechanisms (Hoyle and O’Neill,
2015). Importantly, it is now well established that virtually
all tissues in the body exhibit circadian rhythms that are
based on a cellular clock and a robustly cycling transcriptome
(Zhang et al., 2014). This literature lays the groundwork for
understanding circadian rhythms in endocrine and neuroen-
docrine systems. In the following section, we highlight key
aspects of circadian timing in a ‘bottom-up’ manner, starting
from the cellular basis to the hierarchical control of clocks
throughout the body.
Core clock genes and transcription/translation feedback loops.
Cell-based circadian oscillators had initially been construed
as composed of a small number of gene families linked in tran-
scription–translation feedback loops that complete one cycle in
approximately 24 h (Figure 2). In the primary loop, two basic
helix-loop-helix transcription factors, CLOCK and BMAL1,
dimerize and drive transcription of the Period (Per1, Per2, and
Per3) and the Cryptochrome (Cry1 and Cry2) genes by binding
to the E-box motif (CACGTG) in the promoter regions of
these genes. The resultant PER and CRY proteins form hetero-
and homodimers in the cytoplasm of the cell and are then
translocated back to the nucleus where they inhibit their own
transcription via direct interactions with the CLOCK:BMAL1
protein complex (Zhang and Kay, 2010; Partch et al., 2014;
Dibner and Schibler, 2015).
In addition to rhythmic negative feedback via PER and CRY,
there is a rhythm in CLOCK:BMAL1 transactivation based on
cycling abundance of BMAL1. These rhythms are generated by
a second loop: CLOCK:BMAL1 acts as a positive regulator, driving
transcription of two Rev-erb genes and four Ror genes. The protein
products compete to bind retinoic acid–related orphan receptor
response elements (RORE) in the promoter region of Bmal1,
with REV-ERBa-inhibiting and RORa-stimulating transcription,
ultimately leading to rhythms in BMAL1 abundance
 Circadian Regulation of Endocrine Functions 347

Box 1 Terminological notes

Amplitude: The strength or magnitude of an oscillation.
Arrhythmia: When a system that is normally rhythmic loses rhythmicity. For example, lesions of the SCN lead to arrhythmia in mice.
Circadian time (CT): Unit of time based on an organism’s free-running period. Typically CT0 is the onset of activity in diurnal organisms and CT12 is the onset of
activity for nocturnal organisms.
Circadian: A cycle or oscillation with a period close to 24h, from circa ¼ about and diem ¼ day. This term is generally used to describe processes that are
endogenously driven and that will persist in the absence of any external cues, such as light.
Clock gene: A gene involved in the basic genetic negative feedback loops underlying circadian rhythms.
Desynchronization: When a rhythm becomes uncoupled from a synchronizing cue or when two or more internal rhythms are no longer coupled.
Diurnal: Used to describe rhythmic phenomena that are not endogenous but rather driven by the external light–dark cycle. Also used to refer to day-active animals.
Endogenous rhythm: Self-sustained rhythm that persists in constant conditions, such as constant darkness.
Entrainment: Synchronization of the circadian system to external cues. The light–dark cycle is the dominant entraining signal for most organisms, but other cues, such
as food, can also entrain organisms.
Free-running: The state of an oscillator running at its own endogenous period, without external perturbations, such as entrainment to a light–dark cycle. The free-
running period of an animal is usually measured in constant darkness.
Nocturnal: Night active.
Pacemaker: Anything that can generate robust, endogenous rhythms and can synchronize other rhythmic entities to its own rhythm. The SCN is the master
pacemaker, responsible for synchronizing all of the body’s rhythms to its own.
Period: The duration of a cycle or oscillation, typically close to 24 h for circadian processes.
Phase: The time at which a cyclic event occurs as measured against a reference time frame, such as time of day. A phase advance causes the event to occur earlier in
the day, while a phase delay causes it to occur later.
Subjective day/night: Under free-running conditions subjective day is the period from CT0 to CT12, when diurnal organisms would typically be active. Subjective night
is the period from CT12 to CT0 when nocturnal organisms would typically be active.
Zeitgeber: Any cue or signal that can synchronize or entrain a rhythm. For example light is a very powerful zeitgeber capable of entraining the body clock to
environmental light/dark cycles. Zeitgeber time (ZT) is a unit of time measured from the onset of a zeitgeber. For example, ZT0 is the moment when the cue appears
and ZT12 is 12 hours after the cue is presented.

Figure 2 The cartoon depicts the fundamental core clock elements in mammalian cells. At the genetic level, circadian rhythms are generated in
single cells by interlocked positive and negative feedback loops. BMAL1 and CLOCK (or its homolog NPAS2 depending on the tissue) dimerize and
transactivate several clock genes, including those of the Period (Per) and Cryptochrome (Cry) families and Rev-erba and other clock-controlled genes
(Ccg). The negative regulators, Per and Cry, are translated and then translocated as a single complex to the nucleus where they inhibit CLOCK:BMAL1
transactivation. Casein Kinase 1-epsilon (CK1ε) and other kinases then alter the phosphorylation states of core clock components, thereby regulating
their retention in the cytoplasm or degradation, which determines the period and amplitude of the cellular rhythms. Outputs of the clock set the
phase of expression of numerous additional genes, regulating myriad cellular functions.
348 Circadian Regulation of Endocrine Functions

(Guillaumond et al., 2005). The Bmal1 gene is necessary for
circadian function – indeed it is the only single gene whose
knockout produces arrhythmic behavior. Numerous other
integrated feedback loops, often with elements that are
themselves regulated by the core clock loop, mediate clock
output to clock-controlled genes (Mirsky et al., 2009; Albrecht,
2012; Takahashi, 2015).
Posttranslational and metabolic modulation of oscillation: Post-
translational effects have a strong influence on clock speed, espe-
cially regulatory kinases that determine phosphorylation state of
clock proteins (Albrecht, 2012). For example, the circadian
mutation named tau encodes a dominant negative allele of
casein kinase I epsilon (CK1ε) that shortens the period of loco-
motor activity rhythms (Ralph and Menaker, 1988; Lowrey et al.,
2000). The wild-type forms of CK1ε and CK1d shape endoge-
nous period by determining the phosphorylation state of core
clock genes (Etchegaray et al., 2009; Isojima et al., 2009; Mong
et al., 2011).
Phosphorylation can modulate rhythm speed, but may
also represent a timekeeping mechanism. Phosphorylation
state drives the protein-based circadian timer in cyanobacte-
ria (Nakajima et al., 2005), and kinase–phosphatase
coupling alone can generate endogenous rhythms (Jolley
et al., 2012). Circadian timekeeping by the transcriptional/
translational feedback loop is also linked to nontranscrip-
tional metabolic and redox oscillations (Rey and Reddy,
2015). Peroxiredoxins are members of an antioxidant
protein family. On a circadian timescale, they display circa-
dian rhythms in oxidation, detectable in red blood cells,
which lack a transcriptional mechanism. The interconnec-
tions between the circadian transcription/translation feed-
back loop and redox pathways point to a substantial
contribution of metabolism to circadian timekeeping (Asher
and Sassone-Corsi, 2015; Perelis et al., 2015; Brown, 2016).
This is important in the present context as, to date, the endo-
crine systems have been considered primarily as outputs of
the circadian timing system. The discovery of circadian meta-
bolic clocks points to new models of circadian rhythmicity
and new roles for endocrine factors, as these have major
effects on metabolism (Figure 3).
Clock-controlled genes. The core clock genes are the same across
tissues, but the eventual set of rhythmic genes is large and tissue
specific. There is a small subset of canonical clock-controlled
genes, defined as those that exhibit E or D boxes and are rhythmi-
cally transactivated by CLOCK:BMAL1, exemplified by albumin
D-element-binding protein (Dbp) (Ripperger and Schibler,
2006). Nevertheless, the circadian clock may have a much
longer reach via its transcriptional and epigenetic effects.
Across the genome, the core clock genes bind to thousands of
sites suggesting that the clock genes have large spheres of
influence (from 4600 CLOCK sites to 16 000 CRY1 sites)
(Koike et al., 2012). Microarray studies have identified
many cycling transcripts in several tissues, including the
SCN, liver, heart, skeletal muscle, adrenals, and fibroblasts

Figure 3 Within a cell, rhythms depend on an intact molecular clock based on transcription and translation of core clock proteins (molecular
clock). This clock is intimately associated with nontranscriptional oscillations, including self-sustaining redox rhythms that depend on the redox state
of peroxiredoxins (nontranscriptional clocks) as well as metabolic flux rhythms in ATP and NADH (metabolic rhythms). There are several key interme-
diate molecules that link the molecular and metabolic clocks, including the sirtuins, PPAR, and PARP-1 (Brown, 2016).
 Circadian Regulation of Endocrine Functions 349

# circadian genes detected

0 1,000 2,000 3,000

Liver 16%
Kidney 13%
Lung 12%
Brown fat 8%
Heart 6%
Adrenal gland 5%
Aorta 4%
White fat 4%
Skeletal muscle 4% 1+ spliceforms present
Cerebellum 4% 2+ spliceforms present
Brainstem 4%
Hypothalamus 3%

Figure 4 The graph depicts the number of protein-coding genes that express circadian rhythms in various organs (Zhang et al., 2014).

(Figure 4; Grundschober et al., 2001; Akhtar et al., 2002; Duffield
et al., 2002; Panda et al., 2002a; Storch et al., 2002; Ueda et al.,
2002; Oster et al., 2006a; McCarthy et al., 2007). Zhang et al.
(2014) used RNA-seq and DNA arrays to quantify the
transcriptomes of 12 mouse organs (adrenal gland, aorta, brain
stem, brown fat, cerebellum, heart, hypothalamus, kidney,
liver, lung, skeletal muscle, and white fat) and produced an
atlas of changes over time in these tissues. What is most
surprising is that 43% of all protein-coding genes showed
circadian rhythms in transcription somewhere in the body,
largely in an organ-specific manner with little overlap between
organs. Only 10 genes, including the core clock genes, Per1,
Per2, and Per3, oscillated in all organs. This maps well to prior
data indicating that there is only 5% overlap between the
rhythmic genes in the SCN and in the liver (Panda et al.,
2002a), or between the heart and liver (Storch et al., 2002).
This finding immediately suggests that cycling transcripts are
associated with specific tissue functions. Interestingly, in most
organs, the expression of many oscillating genes peak just
before dawn and/or after dusk (Zhang et al., 2014). Finally, the
majority of the top-selling drugs target genes that exhibit
circadian rhythms (Zhang et al., 2014).
Rhythmic transcription within a tissue is intimately related
to rhythms in RNA polymerase function and epigenetic regu-
lation of the chromatin (Koike et al., 2012; Masri et al., 2015).
Indeed, the gene CLOCK is a histone acetyltransferase (Doi
et al., 2006), and numerous bodily tissues exhibit daily
rhythm in patterns of chromatin modifications (Koike et al.,
2012). There is a strong dawn peak in genome binding by
BMAL1 and CLOCK coincident with increased histone acetyla-
tion (H3K9ac) and methylation (H3K4me1), and there is
a strong evening peak in PER and CRY binding, also with
distinct acetylation and methylation changes (H3K27ac and
K3K4me3) (Figure 5). This suggests that systems biology

Figure 5 Circadian chromatin rhythms in the liver. Profiles show the timing and degree of genome binding by core clock genes, the times of major
histone modifications including acetylation and methylation, and the time of peak binding by RNA polymerase. Reprinted with permission from AAAS,
Koike, N., Yoo, S.H., Huang, H.C., Kumar, V., Lee C., Kim, T.K., Takahashi, J.S., 2012. Transcriptional architecture and chromatin landscape of the
core circadian clock in mammals. Science 338 (6105), 349–354.
350 Circadian Regulation of Endocrine Functions
approaches will be valuable in the field of circadian rhythms
(Hogenesch and Ueda, 2011).
1.12.2.1 Entrainment through a Hierarchy of Clocks
Hierarchy of circadian oscillators. The many clocks within the
body are synchronized in a hierarchical system in which
a master circadian clock in the SCN entrains downstream
rhythms via an array of signaling mechanisms (Figure 6). The
SCN receives environmental time information via direct retinal
projections and also receives information as to the internal
state of the body, from hormonal signals that act on receptors
in the SCN, or indirectly via afferent input from other sites.
SCN outputs entrain rhythms in target sites in the CNS and
periphery. The oscillatory behavior of these extra-SCN sites
can communicate phase information back to the SCN, creating
a closed-loop feedback system. The stability of this hierarchical
arrangement is necessary for normal body functioning. Disrup-
tion of coordinated timing is a hallmark of numerous subop-
timal and disease states. We now characterize the hierarchical
organization of the circadian timing system, from input to
output.
Input to the brain clock. Circadian rhythms are entrained (or
synchronized) to local environmental time primarily via light
information conveyed to the SCN by the retinohypothalamic
tract (Moore and Klein, 1974; Klein and Moore, 1979).
SCN
Pineal gland
Pituitary gland
Thyroid
Adrenal
gland
Liver
Ovary
Testis
Figure 6 The circadian system is hierarchically organized. The master clock of the suprachiasmatic nucleus (SCN) provides timing cues to periph-
eral clocks in the rest of the body, including those in endocrine organs, via both direct (e.g., neural control) and indirect (e.g., humoral outputs,
feeding rhythms, body temperature) signals. These peripheral clocks in turn participate in coordinate local endocrine function and feed back at many
sites (not shown). Each hormone rhythm cycles with its own characteristic waveform and phase, shown here for a typical diurnal mammal. On the
right panel, the shaded area indicates night.
This non-image-forming pathway is necessary and sufficient
for photic entrainment. If the primary visual pathway is trans-
ected at the level of the optic tract beyond the optic chiasm (i.e.,
caudal to the SCN), then the animal is visually blind, but the
circadian system continues to respond to photic cues and the
animal remains entrained (Klein and Moore, 1979; Johnson
et al., 1988).
The threshold of entrainment and the relative contributions
of the retinal photoreceptors-rods, cones, and intrinsically
photosensitive retinal ganglion cells (ipRGCs) have been exam-
ined (Butler and Silver, 2011). The entrainment threshold is
determined by rods and is more sensitive by 1–2 log units
than other measures of the non-image-forming visual system.
That is, dim light that entrained mice did not elicit either mask-
ing or the pupillary light response.
Although the dim threshold of entrainment is determined
by rod function, all three major photoreceptor classes (rods,
cones, and melanopsin-expressing intrinsically sensitive retinal
ganglion cells) are capable of driving entrainment (Panda et al.,
2002b, 2003; Ruby et al., 2002; Hattar et al., 2003; Lucas et al.,
2003). All three photoreceptors must be knocked out to
abolish entrainment (Hattar et al., 2003). Anatomically,
ipRGCs make up 2% of the total RGC population and serve
as the principal conduit to the SCN: ablation of these cells
alone prevents entrainment even while preserving pattern
vision (Guler et al., 2008; Figure 7). The axon collaterals of
Melatonin
Prolactin
Cortisol
Testosterone
 Circadian Regulation of Endocrine Functions 351

(a) Retinal input (b) SCN (c) Output

hν hν Actogram Endocrinogram

Neural Intact
and DD
diffusible
Rod Cone output
Shell SCN-x
hν Core

SCN
RGC ipRGC
Optic chiasm transplant
Anterior 0 24 0 24
Posterior Hours Hours

Figure 7 Inputs and outputs from the suprachiasmatic nucleus (SCN) master clock. (a) A small fraction of retinal ganglion cells contain the photo-
pigment melanopsin and are photosensitive (termed intrinsically photosensitive retinal ganglion cells: ipRGCs). The ipRGCs also receive photic infor-
mation from rods and cones, and they project via the retinohypothalamic tract (RHT) to the SCN. Elimination of ipRGCs eliminates circadian
responses to light (entrainment and phase shifting) but does not impact vision (Guler et al., 2008). (b) The core region of the SCN receives a dense
retinal input. Both core and shell send efferents to target sites in the brain. The SCN communicates timing information to the body by means of both
neural and diffusible signals. (c) The schematic shows locomotor activity in constant conditions in an animal before and after an SCN lesion. Each
consecutive day is plotted on lines from the top, and the black bars indicate the periods of activity. The animal becomes arrhythmic following the
lesion. In support of an SCN diffusible output signal, transplantation of fetal SCN tissue sealed in a semipermeable membrane into the lesioned host
restores locomotor rhythmicity. In contrast, the graft does not restore endocrine rhythms, indicating that neural outputs are required for these
responses.

these ipRGCs are extensive (Fernandez et al., 2016) and are in
a position to directly or indirectly regulate reproductive func-
tion and a variety of other endocrine-mediated behaviors and
physiological processes (Fernandez et al., 2016). Note that
there is no evidence for extraretinal photoreception in rodents
(Nelson and Zucker, 1981). Extraocular photoreception also
appears to be absent in humans (Lockley et al., 1998; Barinaga,
2002; Wright and Czeisler, 2002), despite one report to the
contrary (Campbell and Murphy, 1998).
The SCN and its oscillator neurons. Many lines of evidence
confirm that the SCN is a light-entrainable master pacemaker.
It receives direct projections from the retina (Moore and Lenn,
1972), ablation of this nucleus abolishes circadian rhythms
(Moore and Lenn, 1972; Stephan and Zucker, 1972), the
SCN exhibits rhythms of electrical activity and gene expres-
sion both in vivo and in vitro (Inouye and Kawamura, 1979;
Green and Gillette, 1982; Groos and Hendriks, 1982; Shibata
et al., 1982; Yamazaki et al., 2000; Ono et al., 2015), and
transplants of SCN tissue restore rhythmicity in arrhythmic
hosts (Lehman et al., 1987; Ralph et al., 1990). In the last,
the donor SCN, rather than the host, determines period,
showing that the transplanted tissue does not act by restoring
host-brain function but that the ‘clock’ is contained in the
transplanted tissue. The fact that SCN rhythms persist for
many cycles in vitro (longest reported is 478 days!) (Yamazaki
and Takahashi, 2005) shows that input from other brain areas
is not necessary.
The cells of the SCN are heterogeneous in neurotransmitter
and peptide content, in their dendritic and axonal morphol-
ogies, and in their afferent and efferent connections. There
are distinct components of the clock and important emergent
properties of its interacting elements. In the intact animal, the
SCN oscillation generates a coordinated circadian rhythm,
but dissociated individual SCN neurons exhibit different free-
running periods (Welsh et al., 1995). Neurons communicate
among themselves, and there is a functionally important
underlying circuitry. The SCN has a core (also termed the
ventral or ventromedial subregion) and shell (dorsal or dorso-
medial subregion), based on anatomical, neurochemical, and
functional studies (reviewed in Antle and Silver, 2005). In
the core, a light pulse induces the expression of clock genes,
including Per1 and Per2, but these genes do not oscillate detect-
ably. In the shell compartment, Per1 and Per2 oscillate with
a circadian rhythm, but are not induced by light (Hamada
et al., 2001). The question of how light-induced cells and oscil-
lating cells of the SCN interact to communicate their phasic and
rhythmic information to the rest of the brain/body has been
examined both in terms of effects on molecular components
and its overall circuitry, evident in the spatial and temporal
patterns of clock gene expression (Hamada et al., 2001; Herzog,
2007; Yan et al., 2007; Pauls et al., 2014). The network exhibits
a spatiotemporal pattern of oscillation that adopts different
conformations in response to the environment (e.g., long or
short day lengths) (Evans et al., 2013). While multiple mecha-
nisms have been proposed for coupling among SCN neurons
(Antle et al., 2003, 2007), to date, relatively little is known of
the precise nature of its anatomical circuitry (Drouyer et al.,
2010). Furthermore, while the SCN oscillates independent of
photic input from the retina, some cells within the SCN act
as ‘slave oscillators’ whose rhythm depends on the presence
of connections from the retina (Lee et al., 2003).
SCN output is diffusible and neural. The SCN signals the body
by several mechanisms. Among them are both diffusible and
neural output signals. Some aspects of rhythmicity, including
locomotor behavior, are sustained in the presence of diffusible
signals from the master clock. On the other hand, the circadian
352 Circadian Regulation of Endocrine Functions
control of the endocrine system requires neural connections
from the SCN to sustain oscillation.
SCN tissue from a fetal donor, when implanted into the third
ventricle of an adult, SCN-lesioned host, restores circadian
patterns in activity-related behaviors such as locomotor,
drinking, and gnawing rhythms (Lehman et al., 1987; Ralph
et al., 1990; Silver et al., 1990). While some neural
connections are established between the donor tissue and the
host brain, the transplant also produces diffusible factors. That
a diffusible signal is sufficient to restore locomotor
rhythmicity in SCN-lesioned hosts was demonstrated
definitively by encapsulating donor SCN tissue in a membrane
that prevented neural outgrowth while allowing the diffusion
of signals between graft and host (Silver et al., 1996). The
efficacy of paracrine signaling in circadian timing was
demonstrated with an in vitro coculture technique using wild-
type SCN atop a membrane that communicated by shared
culture medium enabling it to drive oscillation in a target
‘host’ SCN deficient either in elements of neuropeptidergic
signaling or in elements of the core feedback loop (Maywood
et al., 2011). Paracrine signaling restored pacemaking in SCN
circuits lacking vasoactive intestinal peptide (VIP) and
maintained rhythmicity in arrhythmic Cry-null SCN. More
specifically, this work suggested a role for VIP, arginine
vasopressin (AVP), and gastrin-releasing peptide (GRP) in
synchronizing SCN neurons. Thus, a hierarchy of paracrine
neuropeptidergic signals determines cell- and circuit-level
circadian pacemaking in the SCN.
SCN output: Neural efferents. In contrast to behavioral
rhythms, endocrine rhythms require neural projections from
the SCN to neurosecretory cells in the brain. Endocrine rhythms
are abolished after knife cuts severing SCN efferents (Nunez and
Stephan, 1977; Hakim et al., 1991) and are not restored in SCN-
lesioned animals with SCN transplants, presumably due to
inadequate neural innervation of the host brain by the graft
(Nunez and Stephan, 1977; Silver et al., 1996; Meyer-Bernstein
et al., 1999). Further evidence for a neural SCN output signal
regulating hormone secretion is seen in studies of hamsters.
When housed in constant light, the locomotor activity of
hamsters ‘splits’ into two separate activity bouts within a 24-h
interval (as though they have 12-h days). Split females display
two daily preovulatory LH surges, 12 h apart and each
approximately half the concentration of a single surge in
a nonsplit female (Swann and Turek, 1985). While both halves
of the bilaterally symmetrical SCN are active in synchrony
under normal conditions, in split hamsters, rhythms of SCN
activity on each side of the brain are 12 h out of phase (de la
Iglesia et al., 2000). Remarkably, FOS expression in GnRH
neurons is only observed ipsilaterally to the FOS-expressing
side of the SCN (de la Iglesia et al., 2003). These findings
support the conclusion that the precise timing of the LH surge
is derived from a neural signal originating in the SCN and
communicated to ipsilateral GnRH neurons (likely via
kisspeptin neurons, see below), as a diffusible output signal
would reach both sides of the brain. Further analysis of the
split hamster indicates that within each nucleus, the SCN splits
into two parts, oscillating in antiphase (Nakajima et al., 2005;
Tavakoli-Nezhad and Schwartz, 2005). Surprisingly, in most
brain areas, SCN targets, including the preoptic area,
paraventricular nucleus (PVN), and dorsomedial nucleus of the
hypothalamus (DMH), and orexin neurons of the lateral
hypothalamus all exhibit 12-h rhythms. These data suggest
each SCN (left and right) may signal each monosynaptic target
once daily, thereby producing 12-h days in the animal (Butler
et al., 2012).
Neural output from the SCN has been extensively investi-
gated in mice, rats, and hamsters using tract-tracing techniques
(Morin, 2013). Projections arise from both the core and the
shell of the SCN. Importantly, many of these monosynaptic
projections target brain regions containing neuroendocrine
cells producing hypothalamic releasing hormones. Direct
projections have been traced from the SCN to the medial pre-
optic area (MPOA), supraoptic nucleus, anteroventral periven-
tricular nucleus (AVPV), the PVN, DMH, lateral septum, and
the arcuate nucleus (Arc). The SCN also projects to the pineal
through a multisynaptic pathway (Klein et al., 1983; Klein,
1985). There is abundant evidence for direct neural SCN
control of neuroendocrine cell populations (van der Beek
et al., 1993, 1997; Vrang et al., 1995; Kalsbeek et al., 1996,
2000; Buijs et al., 1998, 2003; Horvath et al., 1998; Gerhold
et al., 2001; Kalsbeek and Buijs, 2002; Kriegsfeld et al., 2002;
Egli et al., 2004). Because these cell populations can regulate
neurochemicals that are secreted into the cerebrospinal fluid,
pituitary portal system, and general circulation, SCN-derived
signals can control widespread systems in the brain and body
(Skinner and Malpaux, 1999; Reiter and Tan, 2002; Skinner
and Caraty, 2002; Tricoire et al., 2003).
Extra-SCN Oscillators. Unlike the long-lasting and self-
sustained rhythms exhibited by the SCN in culture, most
gene expression rhythms damp out after several cycles in vitro
(Balsalobre et al., 1998; Yamazaki et al., 2000). This damping
of the rhythms reflects desynchronization of self-sustained
and cell-autonomous oscillators (Nagoshi et al., 2004; Welsh
et al., 2004). In vivo, similar desynchronization among organs
is observed in the absence of the SCN (Yoo et al., 2004).
The coordination of peripheral oscillations, and therefore
the expression of overt rhythms in physiology and behavior,
depends primarily on the SCN, but may be modulated by other
mechanisms. The SCN projects via multisynaptic pathways to
peripheral endocrine organs, including the pineal gland, adre-
nals, thyroid, and pancreas (Moore, 1996; Buijs et al., 1999;
Kalsbeek et al., 2000; Bartness et al., 2001; Buijs and Kalsbeek,
2001). The SCN can also transmit timing cues indirectly via
SCN-dependent behaviors, such as eating, that can alter
physiological signals. The presence of oscillators throughout
the body confers flexibility in entraining to cues. For
example, when the feeding schedule and the light–dark
schedule are dissociated, rhythms in SCN gene expression
remain entrained to the light–dark schedule, but rhythms in
the liver entrain to the feeding schedule (Damiola et al.,
2000; Stokkan et al., 2001). By examining SCN-lesioned mice
parabiotically paired with animals bearing an intact SCN
(sharing 1–4% of their blood supply), it was revealed that
nonneural signals are adequate to maintain circadian
rhythms of clock gene expression of Per1, Per2, or Bmal1 in
the liver and kidney (but not in the heart, spleen, or skeletal
muscle) (Guo et al., 2005). This implicates a blood-borne
cue, perhaps endocrine or metabolic, that can provide phase
information to some but not all peripheral oscillators.
Alternatively, the intact animal may communicate timing

information to the SCN-lesioned mouse via activity and/or
body temperature fluctuations. One interesting implication of
this work is that an SCN-derived cue may synchronize each
individual liver hepatocyte every single day in order to
sustain phase coherence in peripheral tissues. A similar
principle may apply to the process of synchronizing
individual cells of glandular organs.
Interactions among central and peripheral oscillators: Peripheral
organs are entrained by the SCN, but there are many potential
intermediate pathways, and their relative importance is still not
well described. In addition to direct effects of SCN neural output
or SCN-derived diffusible cues, SCN-controlled behaviors and
secondary messengers are critical zeitgebers. One of the most
important of these mechanisms is the feeding–fasting rhythm.
Rhythmic presentation of food is sufficient to entrain the
locomotor activity rhythms of animals (food anticipatory
activity) even in otherwise arrhythmic SCN-lesioned animals
(Mistlberger, 2006). The endogenous nature of this rhythm is
revealed during subsequent total food deprivation. At these
times, the food-deprived animals continue to display free-
running anticipatory behavior, and this behavior demonstrates
the existence of a food-entrained oscillator (FEO). There is
considerable interest in understanding whether this FEO is
localized in a specific site or constitutes a network – a topic of
heated debate (Gooley and Saper, 2007; Landry and
Mistlberger, 2007; Landry et al., 2007). The phenomenon of
food-entrained oscillation is important because food-derived
signals constitute cues that can influence rhythmicity of
downstream oscillators in both the brain and in endocrine
organs (discussed below). In turn, changes in the phase
hormone secretion may influence other timed physiological
and behavioral responses.
In addition to food-related entraining cues, peripheral
tissues may also be entrained by glucocorticoids (Balsalobre
et al., 2000; Le Minh et al., 2001). Body temperature rhythms
as well, governed directly and indirectly through rest–activity
cycles, are also cues for peripheral oscillators (Buhr et al.,
2010). Impressively, while the molecular makeup of circadian
oscillators is nearly identical in all cells, they may not be
equally sensitive or similarly responsive to entraining cues.
Though tempting to consider a peripheral tissue clock phase
as the endpoint of entrainment, this is simplistic. Each cell
exhibits a multilayered rhythm from epigenetic regulation of
the chromatin, to transcription, to cellular function. These
may differ in their sensitivity to the potential internal and
external entraining cues. For example, of 2000 genes that
are rhythmic in whole brain extracts, only 400 remain
rhythmic in sleep-deprived mice, indicating a strong entraining
role for sleep and its correlates (Maret et al., 2007). Similarly, of
3000 genes that cycle in the liver during ad-lib feeding condi-
tions, only 400 genes remain rhythmic during fasting condi-
tions (Vollmers et al., 2009).
1.12.3 Circadian Regulation of the Endocrine
System
Circadian patterns of hormone secretion occur in both diurnal
and nocturnal species (Kriegsfeld et al., 2002a). Although the
timing of expression of clock genes in the SCN is not different
Circadian Regulation of Endocrine Functions 353
between nocturnal and diurnal species (Ramanathan et al.,
2010), the precise pattern of variation in plasma hormone
levels does differ and results from interactions between regu-
larly recurring periodic cues (such as eating) and nonrhythmic
factors such as homeostatic mechanisms (e.g., rest/activity
cycles) (Kriegsfeld and Silver, 2006; Morin, 2013; Dibner and
Schibler, 2015). This is shown elegantly in studies of humans
where cycles of fasting–feeding, sleep–wake, and rest–activity
are easily dissociated (Morris et al., 2012).
1.12.4 Circadian Regulation of Hypothalamo–
Pituitary–Adrenal Axis
Rhythms of the hypothalamo–pituitary–adrenal (HPA) axis.
Rhythms in glucocorticoid secretion and serum concentration
are well established (Dickmeis, 2009); indeed, elimination of
corticosterone rhythms after SCN lesions were among the first
data implicating the SCN as a brain clock (Moore and Eichler,
1972). Humans, nonhuman primates, and rodents all display
circadian changes in glucocorticoid secretion that continue in
constant conditions (Weitzman et al., 1971; Moore and Eichler,
1972; Gallagher et al., 1973; Dubey et al., 1983; Van Cauter and
Refetoff, 1985; Czeisler and Klerman, 1999). The phase of the
rhythm differs between nocturnal and diurnal species, with
corticoid secretion beginning to rise before waking in both
but then peaking during the day in diurnal species and during
the night in nocturnal species (Wong et al., 1983; Albers et al.,
1985; Ottenweller et al., 1987). In humans, cortisol levels have
a robust daily rhythm, peaking around the morning wake-up
time. The adrenal gland itself manifests rhythms in clock
gene expression and these underlie rhythmic responsiveness
to pituitary adrenocorticotropic hormone (Gallagher et al.,
1973), and to physical and physiological stressors (Ungar
and Halberg, 1962; Dunn et al., 1972; Buijs et al., 1997;
Bittman et al., 2003; Kalsbeek et al., 2003; Oishi et al., 2003;
Guo et al., 2006; Oster et al., 2006b; Fahrenkrug et al., 2008).
The robust circadian pattern of circulating corticoids is
generated by changes in pulsatile secretion that occurs on an
ultradian basis, with a periodicity of about 1 h (Walker et al.,
2012; Spiga et al., 2014; Russell et al., 2015). Both modeling
and empirical data show that this ultradian pattern of secretion
is driven by biological delays and feed-forward mechanisms at
the level of the pituitary and adrenal gland, without any
requirement for a pulse generator in the hypothalamus (Walker
et al., 2010, 2012). These ultradian pulses are important for
adrenal function as it has been demonstrated that the adrenal
gland responds optimally to an oscillatory profile of ACTH,
but that constant ACTH infusion results in a decrease in corti-
coid output (Spiga et al., 2011).
Role of SCN in HPA axis rhythmicity. Rhythms in circulating
glucocorticoids are eliminated by SCN lesions and are not
restored by SCN transplants (Moore and Eichler, 1972;
Meyer-Bernstein et al., 1999). Similarly, rhythms in clock
gene expression in the adrenal depend on the SCN (Guo
et al., 2006). Two SCN efferent pathways are implicated in
the regulation of the adrenal axis (Figure 8). First, monosyn-
aptic projections to the corticotropin releasing hormone
(CRH) neurons in the PVN are thought to directly regulate
ACTH release (Vrang et al., 1995; Kalsbeek et al., 1996;
354 Circadian Regulation of Endocrine Functions

PVN

CRH cells

IML
SCN

Eye Pituitary
H ion
ACT nervat
mic in
Circadian rhythm ono
Aut
of ACTH sensitivity

Rhythmic
glucocorticoid
Adrenal release

Figure 8 Schematic depiction of the hypothalamo–pituitary–adrenal (HPA) axis and its connections. The suprachiasmatic nucleus (SCN) projects to
the adrenal gland via synapses in the autonomic portion of the paraventricular nucleus (PVN) and the intermediolateral column (IML). In addition,
SCN neurons make contacts with corticotropin releasing hormone (CRH)-expressing cells of the PVN, which in turn promote the release of ACTH
from the pituitary. The combination of signals synchronizes the cycling transcriptome of the adrenal which includes genes associated with adrenal
function. The local cycling of gene expression within the adrenal gland may also contribute to gating the response to ACTH.

Buijs et al., 1998). In addition, the SCN may directly control
adrenal rhythms via multisynaptic autonomic innervation of
the adrenal cortex (Buijs et al., 1999). This second pathway
may be critical to establishing proper adrenal function, as
hypophysectomy does not alter adrenal clock gene expression
rhythms, indicating that rhythms in ACTH do not by them-
selves induce rhythms in the adrenal glands (Fahrenkrug
et al., 2008).
Regulation of HPA axis by peripheral oscillators. Transcription
profiles of the mouse adrenal reveal a large number of cycling
genes (5% transcripts on an Affymetrix MG430v2.0 chip),
among which are those involved in such adrenal functions
as steroid biosynthesis and catecholamine metabolism (Oster
et al., 2006a). The majority of the cycling transcriptome peaks
at the sleep to wake transition; notably, 10 of 14 rhythmic
genes associated with steroid biosynthesis were synchronous,
peaking in early subjective night in parallel with the
increasing plasma corticoid levels at this time (Oster et al.,
2006a). The gating of adrenal ACTH sensitivity depends on
this local cycling, as rhythms in sensitivity are eliminated in
cultured adrenal slices from arrhythmic Per2/Cry1 knockout
mice (Oster et al., 2006b). Synchronization of the adrenal
oscillators by other cues allows for rhythmic function in the
absence of the SCN. For example, SCN lesions do not prevent
the corticosterone peak associated with restricted feeding
(Krieger et al., 1977).
In an elegant study, Oster et al. (2006b) tested the role of
a functioning adrenal clock by cross-implanting adrenals
between wild-type and Per2/Cry1 mutant mice: the mutant
mice lack rhythmic ACTH, corticosterone, and adrenal clock
gene expression. They found that the rhythm of ACTH respon-
siveness observed in vivo was also observed in vitro in wild-type
but not in Per2/Cry1 knockout adrenals. This implicated the
local, intra-adrenal clock in responses to ACTH. They then
showed that corticoid excretion rhythms of wild-type mice
with wild-type adrenal implants were nearly double the ampli-
tude of wild-type mice that had mutant adrenal implants. These
results indicate that functioning peripheral clocks are necessary
for optimal glucocorticoid production by the adrenal gland.
Consequences of adrenal hormone rhythms in brain and
periphery. As noted, glucocorticoid hormones produced by the
adrenal gland cortex are rhythmically released under the
control of the SCN. The SCN itself does not express glucocorti-
coid receptors (GRs) (Rosenfeld et al., 1988; Balsalobre et al.,
2000). Glucocorticoids, nevertheless, may have indirect effects
on the SCN: glucocorticoid treatment in humans disrupts sleep
and in the SCN, reduces AVP (Liu et al., 2006). In rats, gluco-
corticoids upregulate glial fibrillary acidic protein in the SCN,
possibly through effects on serotonergic neurons (Maurel
et al., 2000). For the most part, the specific neural targets by
which corticoids influence SCN function remain to be
determined.
In extra-SCN brain regions, it is quite clear that glucocorti-
coid rhythms can communicate phase information in a region-
specific manner. In rats, PER2 is rhythmic in several brain
areas, including the oval nucleus of the bed nucleus of the stria
terminalis (BNST-OV), and the central (CEA) and basolateral
nuclei of the amygdala (BLA) (Amir et al., 2004; Lamont
et al., 2005). The PER2 rhythm in the BLA is not affected by
adrenalectomy. In contrast, rhythms in the BNST-OV and
CEA depend on peripheral glucocorticoid cues for
entrainment, for in these two structures, the rhythm in PER2
is abolished by adrenalectomy. Rhythms are restored in
animals provided with corticosterone in the drinking water
(producing a rhythm in circulating corticosterone), but not
by implanted constant-release corticosterone pellets (Segall
et al., 2006). While these effects have been well characterized
in subcortical brain areas, there are similar effects observed
in the cortex. A direct role for diurnal changes in corticoids
has been shown, with diurnal changes in circulating
corticosterone important for spine turnover in motor cortex.
This has implications for performance, showing that
glucocorticoid rhythms promote learning of motor tasks
(Liston and Gan, 2011).

Glucocorticoids also communicate timing information to
peripheral organs (Balsalobre et al., 2000). Dexamethasone
(DEX), a potent agonist of the GR, is effective in synchronizing
circadian gene expression in cultured cells and in activating
transcription of Per1, a core clock gene. Intraperitoneal injec-
tion of DEX in mice during the day results in phase shifts in
the liver, suggesting that glucocorticoid signaling synchronizes
liver clocks. Using gene chip microarrays, Oishi et al. (2005)
described 169 genes that differed in expression level between
night and day in the livers of normal mice. The day–night
difference in 100 of these genes was abolished by
adrenalectomy. Reddy et al. (2007) further identified 366
transcripts whose rhythmic expression were abolished by
SCN lesions; of these, rhythms in 57% were reinstated by
DEX treatment. Multiple pathways drive rhythmicity across
the whole cycling transcriptome. For example, only two-
thirds of the genes whose rhythms were induced by DEX had
glucocorticoid response elements (GRE) in their promoters
(Reddy et al., 2007). Rhythms in some of the remaining genes
may have been induced by clock genes, themselves with DEX-
induced rhythms. Finally, other rhythmic genes must act as
intermediaries between the clock- or DEX-responsive genes
and remaining genes of the cycling transcriptome that lack
both glucocorticoid and clock responsive promoter elements.
1.12.5 Circadian Rhythmicity in the Hypothalamic–
Pituitary–Gonadal Axis
Overview. As noted in the introduction to this chapter, the
importance of the brain in regulating the hypothalamic–pitui-
tary–gonadal (HPG) axis was first established in the 1950s. It
was in this era that the existence of neurosecretory hormones
was first described in the classical studies of Harris where he
showed that the hypothalamohypophysial portal vasculature
carries neurosecretions from hypothalamic nerve fibers to the
anterior pituitary, unlike the direct neural control of the poste-
rior pituitary. The first hypothalamic releasing hormone,
GnRH, was isolated in 1969, and its importance was acknowl-
edged in a Nobel Prize in 1977 for its seekers, Guillemin and
Schally (Initially GnRH was called luteinizing hormone
releasing hormone as it was thought to regulate only LH
(Grundschober et al., 2001)). Following that work, other hypo-
thalamic releasing hormones were soon isolated and synthe-
sized, leaving one with the impression that the most
important neural elements of the HPG axis were known.
Thus, it came as a surprise to find major new neuroendocrine
elements in the brain in the twenty-first century, namely kiss-
peptin and gonadotropin-inhibitory hormone (GnIH), funda-
mental stimulatory and inhibitory regulators of the HPG axis,
respectively. The search for brain hormones has been dramatic
(Loriaux, 2016), and the tale of the more recent discoveries is
an interesting (but perhaps not unusual) example of the jagged
course of discovery in research.
Rhythms of the HPG axis. Circadian rhythms are evident at all
levels of the HPG axis. The importance of the SCN is under-
scored by the observation that disruptions of circadian rhythms,
either by surgical lesions or by mutations in the underlying
clock genes, interfere with or abolish female reproductive
rhythms and maintenance of pregnancy (Gray et al., 1978;
Circadian Regulation of Endocrine Functions 355
Miller et al., 2004; Chu et al., 2013) Two aspects of gonado-
tropin regulation have been described. First, a circadian rhythm
in gonadotropin secretion is seen around the time of puberty
due to increases in nighttime secretion of LH and follicle-
stimulating hormone (FSH) (Dunkel et al., 1992; Apter et al.,
1993). As peripubertal children enter adulthood, daytime secre-
tion of the gonadotropins increases; consequently, adults lack
circadian patterns of gonadotropin secretion (Krieger et al.,
1972; Veldhuis et al., 1986). Second, as already noted, circadian
rhythms are striking in the regulation of the preovulatory LH
surge. The high estradiol (E) concentrations during proestrus
are permissive in this regard, and constant E treatment in ovari-
ectomized (OVX) females reveals a circadian rhythm in daily LH
surges at the same time each day (Legan et al., 1975). In adult
males, there is a robust circadian rhythm in circulating testos-
terone (T), with a trough in late evening and a peak in the early
morning (Spratt et al., 1988). T rhythms persist in constant
conditions (Dubey et al., 1983). The disparity between a lack
of adult male gonadotropin rhythms and a robust rhythm in
T suggests some regulation of the T rhythm downstream from
LH release, potentially through autonomous clock function in
the testes (see below). In adult women, plasma levels of ovarian
hormones vary across the menstrual cycle, but their circadian
rhythmicity is less clear. Nonhuman primates show diurnal vari-
ation in estrogen and progesterone (P) during the luteal, but not
during the follicular, stage of the menstrual cycle (Spies et al.,
1974). In humans, results are mixed, with some studies finding
no diurnal variation in P or E, and others finding rhythms
during some phases of the menstrual cycle (Aedo et al., 1981;
Carandente et al., 1989; Rossmanith et al., 1990).
SCN regulation of the HPG axis rhythms. Work in rodents indi-
cates, at the whole-animal level, that the circadian system deter-
mines the duration of the female reproductive cycle. In Syrian
hamsters entrained to a 24-h day, the duration of the estrous
cycle is normally 4 days. When the animals free-run, their
estrous cycle is a quadruple-multiple of the free-running
rhythm period (Fitzgerald and Zucker, 1976). This is a general
pattern: in treatments that lengthen the circadian period (e.g.,
with deuterium oxide in the drinking water), or in non-24-h
days (e.g., 11 h light:11 h dark), the estrous cycle duration is
reliably a fourfold function of the circadian day. Similarly,
the preovulatory LH surge occurs at a specific time of day on
proestrus (Colombo et al., 1974). Notably, surgical lesions of
the SCN abolish both the LH surge and estrous cycling
(Brown-Grant and Raisman, 1977; Gray et al., 1978). Work
with mutant mice confirms a role for circadian clocks at the
cellular level. Mice with mutated Clock or Bmal1 have an
impaired LH surge and abnormal estrous cyclicity (van der
Horst et al., 1999; Miller et al., 2004; Chu et al., 2013). Women
bearing single-nucleotide polymorphisms in ARNTL (Bmal1)
have more miscarriages than those lacking this mutation
(Kovanen et al., 2010).
SCN efferents from core (VIP) and shell (AVP) cells. The finding
that the SCN underlies the unilateral activation of neurosecre-
tory cells in split hamsters (de la Iglesia et al., 2003; Gibson
et al., 2008; Butler et al., 2012) provides strong evidence that
the SCN controls neuroendocrine secretion through neural
output signaling. Neuroanatomical and physiological studies
indicate two sets of neural SCN efferents that signal daily
temporal information to the reproductive axis: VIP in the
356 Circadian Regulation of Endocrine Functions
ventrolateral core part of the SCN and AVP containing neurons
in the dorsomedial shell part. The VIP peptide does not vary
rhythmically in constant darkness, but under light–dark condi-
tions, VIP content decreases over the course of the light period
and then gradually recovers during the dark period, and thus
may transmit lighting information (Shinohara et al., 1993).
While VIP decreases, the content of GRP, another less well
studied peptide in the core, increases.
VIP: Efferents of VIP SCN neurons contact GnRH neurons
which express the VIP receptor (VPAC2) (van der Beek et al.,
1993, 1997; Smith et al., 2000). Interestingly, female rats
have greater VIPergic innervation than males (Horvath et al.,
1998) and VIP contacts increase following puberty (Kriegsfeld
et al., 2002). Pharmacological studies indicate that blocking
the VIP receptor, VPAC2, attenuates GnRH cell firing on the
proestrous afternoon surge in female mice. In brain slices taken
from OVX E-primed mice, acute VIP treatment increases firing
rates in GnRH neurons from mice sacrificed around the time
of LH surge onset, but generally had little effect on neurons
before the surge (Christian and Moenter, 2008). Central
administration of VIP antiserum reduces the LH surge in
female rats (van der Beek et al., 1999). By contrast, infusion
of VIP into the third ventricle inhibits LH surges triggered by
progesterone injection in OVX E-primed rats (Weick and
Stobie, 1995). The reasons for this discrepancy are unclear.
Finally, VIP might contribute to the LH surge through
interactions with neuroglia that envelop GnRH cells.
Astrocytes express VIP receptors, and their envelopment of
GnRH neurons varies with time of day (Gerhold and Wise,
2006). Suppression of the SCN VIP rhythm with antisense
nucleotides prevents these rhythms in astrocyte coverage.
Such glial–neuronal signaling is an important aspect of the
regulation of neuroendocrine secretion (Garcia-Segura and
McCarthy, 2004) and is a key element of SCN oscillation
(Marpegan et al., 2009).
AVP neurons of the SCN: Another important SCN peptide,
AVP, has been implicated in the LH surge. The observation
that vasopressin peaks coincident with the onset of the preovu-
latory LH surge provided the initial impetus for further investi-
gating the role of the SCN peptide in surge generation
(Schwartz et al., 1983). However, unlike VIP, there is no
evidence of direct SCN to GnRH connections by AVPergic
neurons, and GnRH cells very rarely express the 1a subtype of
AVP receptor (V1a) (Kalamatianos et al., 2004). Despite not
directly acting on the GnRH system, pharmacological work
established that AVP was critical for surge generation. For
example, administration of AVP into the MPOA induces an
LH surge in SCN-lesioned, OVX rats treated with E2, whereas
treatment with an AVP receptor, V1a, antagonist reduced the
E2-induced LH surge (Palm et al., 1999; Funabashi et al.,
2000). Likewise, in cocultures of POA and SCN tissue, the
rhythm of GnRH release is in phase with the rhythm of AVP
release, but not VIP (Funabashi et al., 2000). AVP gene
transcription in the SCN is directly controlled by the CLOCK/
BMAL1 dimer (Jin et al., 1999; Silver et al., 1999). The SCN
content of AVP and its release in the cerebrospinal fluid
displays a daily rhythm that is maintained in constant
darkness (Schwartz and Reppert, 1985; Gillette and Reppert,
1987; Takahashi et al., 1989; Kalsbeek et al., 1995; Krajnak
et al., 1998). As one might expect given that AVP is clock-
controlled in the SCN, Clock mutant mice (which have
reduced AVP mRNA expression in the SCN) lack an LH surge,
a response that is rescued by central injections of AVP, further
linking this peptide to the circadian control of ovulation
(Miller et al., 2006). How does AVP have such a pronounced
impact on the LH surge without communication directly to
GnRH cells? As discussed further below, AVP appears to
mediate the LH surge via indirect projections to kisspeptin
cells in the preoptic area that, in turn, project to GnRH cells.
FMRFamide. The cardioexcitatory neuropeptide containing
the C-terminal Phe-Met-Arg-Phe-NH2 (FMRFamide) was first
identified in the ganglia of the clam, Macrocallista nimbosa
(Price and Greenberg, 1977). Following this discovery,
antibodies to FMRFamide peptides were applied as a tool for
labeling structurally similar peptides across taxa, although the
identity of labeled peptides remained unknown. Subsequently,
a host of vertebrate peptides sharing the RF motif (Arg-Phe-
NH2: RFamide) have emerged as prominent regulators of
neuroendocrine activity. Two RFamide peptides, kisspeptin
and GnIH, have marked direct, but opposing, actions on the
reproductive axis. Kisspeptin provides positive drive to the
GnRH system, whereas GnIH acts to suppress GnRH output.
Given the location of these peptides in the rodent brain, both
are in a position to relay circadian information to the
reproductive axis (Figure 9).
Gonadotropin-inhibitory hormone. GnIH was first identified in
quail brain by probing the proteome using competitive
enzyme-linked immunosorbent assays (ELISA) with antibodies
Figure 9 Cartoon depicts the circadian control of two key RFamide
mediators of the reproductive axis. Kisspeptin (Kp) and gonadotropin-
inhibitory hormone (GnIH) neurons project directly to the gonadotropin-
releasing hormone (GnRH) system to positively and negatively regulate
GnRH secretion, respectively. GnIH cells also communicate directly with
the anterior pituitary portal system to regulate gonadotropin secretion.
The suprachiasmatic nucleus (SCN) controls the daily and seasonal
activity of the kisspeptin and GnIH systems. There are prominent direct
projections from the SCN to anteroventral periventricular nucleus
(AVPV). Whether these projections specifically make synapses on kiss-
peptin cells is not known. Finally, the SCN projects directly to GnIH and
GnRH neurons (Williams and Kriegsfeld, 2012).

directed against the RF motif. Using this approach, a novel RFa-
mide peptide was identified that rapidly and dose-dependently
inhibits gonadotropin release from cultured quail pituitaries
(Tsutsui et al., 2000) and was named GnIH. In rodents,
GnIH cells are confined to the dorsomedial hypothalamus
(DMH), with widespread projections to hypothalamic and
limbic structures (Kriegsfeld, 2006). GnIH cells project upon
GnRH perikarya and to the median eminence. Injections of
GnIH lead to rapid suppression of LH in rats, mice, and Syrian
hamsters. Importantly, GnIH cells express estrogen receptors
and subserve the steroid negative feedback on the reproductive
axis (Kriegsfeld et al., 2006). The SCN sends pronounced
projections to the DMH (Leak and Moore, 2001; Kriegsfeld
et al., 2004), and in Syrian hamsters, over 60% of GnIH cells
are contacted by SCN terminals (Gibson et al., 2008; Russo
et al., 2015). Important in the present context, the SCN acts
to suppress GnIH cell activity at the time of the LH surge,
thereby allowing the coordinated release of the GnRH system
from estrogenic negative feedback at this time via VIPergic
projections (Gibson et al., 2008; Russo et al., 2015). In
summary, GnIH is regulated by the circadian timing system
and serves as a key player regulating reproduction in verte-
brates, acting on the brain and pituitary to modulate reproduc-
tive physiology and behavior (Smith and Clarke, 2010;
Kriegsfeld, 2013; Tsutsui et al., 2015).
Kisspeptin. The Kiss1 gene was originally discovered in a screen
for tumor metastasis suppressors, and its protein was named
metastin for this property when it was identified in 2001 as
the endogenous ligand for the orphan G-protein-coupled
receptor, GPR54 (Kriegsfeld, 2006; Kauffman et al., 2007). A
role for this RFamide, now designated kisspeptin, in
regulating reproductive function emerged in 2003 when
individuals with hypogonadism were shown to have
a mutation in GPR54 (de Roux et al., 2003; Seminara et al.,
2003). The effects of kisspeptin are mediated through its
actions on the GnRH system; administration of kisspeptin
leads to immediate early gene expression (i.e., FOS) in GnRH
cells (Irwig et al., 2004; Matsui et al., 2004). Additionally,
kisspeptin-induced gonadotropin secretion is blocked across
species by the GnRH receptor antagonist, acyline (Gottsch
et al., 2004; Irwig et al., 2004; Shahab et al., 2005). Finally,
a majority of GnRH cells coexpress GPR54 mRNA across
species (Irwig et al., 2004; Han et al., 2005; Messager et al.,
2005), and kisspeptin acts directly on GnRH cells to increase
their neural firing in brain slices (Liu et al., 2008). In rodents,
kisspeptin cell bodies are concentrated in the AVPV and Arc
nuclei. As with the GnIH system in rodents, kisspeptin cells
colocalize sex-steroid receptors, making them a direct target
for sex steroid actions (Smith, 2008). Several lines of evidence
led researchers to suspect that kisspeptin might act as a critical
node in the circadian network stimulating the LH surge. First,
the AVPV was known to be a critical brain region mediating
the positive feedback effects of estrogen, crucial to the
initiation of the preovulatory LH surge and ovulation
(Wintermantel et al., 2006). The AVPV expresses both
estrogen receptor subtypes (ERa and ERb) and V1a (Shughrue
et al., 1997; Kalamatianos et al., 2004). In the AVPV, Kiss1
mRNA expression is at a maximum at the time of the LH
surge, and these neurons show peak activity, as measured by
FOS expression, on the afternoon of proestrous (Robertson
Circadian Regulation of Endocrine Functions 357
et al., 2009; Williams et al., 2011). These findings, combined
with the fact that the SCN projects extensively to the AVPV
(Leak and Moore, 2001; Kriegsfeld et al., 2004), suggested
that the circadian system may stimulate the LH surge via
projections to the kisspeptin cells in the AVPV. Indeed,
AVPergic SCN cells project directly to GnRH cells in mice and
hamsters (Sarvari et al., 2010; Williams et al., 2011) and AVP
increases kisspeptin cellular activity as measured by FOS
(Williams et al., 2011) and intracellular calcium signaling (Piet
et al., 2015). A substantial amount of work has been done on
the neuroanatomy and function of the kisspeptin signaling
system, its receptors (Kiss1r), and its projections across
mammalian species (Lehman et al., 2013). Across species, the
majority of Arc kisspeptin cells coexpress the positive and
negative regulators of the reproductive axis, neurokinin B
(NKB) and dynorphin, respectively (Goodman et al., 2007).
These triple-phenotype neurons are now commonly
referred to as KNDy (pronounced like ‘candy’) and
(K ¼ kisspeptin, N ¼ NKB, and Dy ¼ dynorphin) (Lehman
et al., 2010). KNDy neurons are reciprocally connected and
express receptors for NKB and dynorphin, providing the ability
to propagate stimulatory or inhibitory signaling (Lehman et al.,
2010). KNDy neurons also express progesterone as well as
estrogen receptors, providing a mechanism for mediating
steroid negative feedback (Akhtar et al., 2002; Foradori et al.,
2002). This Arc population of kisspeptin neurons is thought
to participate in the regulation of GnRH pulses and sex steroid
negative feedback. Additional kisspeptin neurons, fibers, and
Kiss1R can be detected in areas outside the hypothalamus,
but their physiological role(s) in these regions is not
understood.
Intrinsic oscillation in GnRH neurons. Circadian oscillators in
GnRH neurons or rhythmic changes in the properties of these
neurons may participate in regulating GnRH secretion. In hypo-
thalamic slice cultures in the absence of the SCN, circadian
rhythms in neural firing rate of GnRH neurons persist for several
days in vitro when the slices are prepared from ovariectomized,
E-treated mice but not from those of ovariectomized, untreated
mice (Christian et al., 2005). Clock gene and GnRH mRNA
expression are rhythmic in GnRH-secreting GT1-7 cell lines
(Gillespie et al., 2003). Moreover, transfection of the
dominant negative mutant Clock-D19 gene into GT1-7 cells
decreases GnRH pulse frequency, indicating that the circadian
clock genes could participate in regulating pulsatility
(Chappell et al., 2003). The evidence for a functional clock in
GnRH cells in vivo is less clear, however. GnRH mRNA is
rhythmic in both OVX and intact female rats, but the GnRH
neurons in vivo do not themselves seem to exhibit rhythms in
clock gene expression (Kriegsfeld et al., 2006; Schirman-
Hildesheim et al., 2006). The GnRH system exhibits time-
dependent sensitivity to stimulation directly by VIP and
indirectly by AVP (Zhao and Kriegsfeld, 2009; Williams et al.,
2011), suggesting that this timekeeping mechanism might
subserve such daily changes. In GT1-7 cells, Kiss1R is
expressed rhythmically when cells are exposed to estradiol
(Tonsfeldt et al., 2011) suggesting that autonomous GnRH
cell clocks might alter relevant receptor expression to mediate
these daily changes in sensitivity to upstream neurochemicals.
Ovarian rhythms. The ovary exhibits clock gene rhythms
in vivo and these rhythms are self-sustained for several days in
358 Circadian Regulation of Endocrine Functions
culture (Nakamura et al., 2010). Both Per1 and Per2 message
and protein are rhythmically expressed in follicles, corpora
lutea, and the interstitium; furthermore, distinct rhythms of
cytoplasmic and nuclear localization are observed (Fahrenkrug
et al., 2006). These circadian oscillations may be synchronized
directly by rhythmic release of LH and FSH, both of which
induce Per1 and Per2 rhythms in cultured granulosa cells (He
et al., 2007). Research in birds suggests a connection between
ovarian rhythms and ovulation. Clock genes are rhythmic in
preovulatory follicles in Japanese quail, but not in smaller folli-
cles (Nakao et al., 2007). This follicular clock in turn may
control other physiological processes: the steroidogenic acute
regulatory protein (StAR), a component of the progesterone
synthesis pathway, is expressed rhythmically, again in preovu-
latory but not in smaller follicles. The StAR promoter region
includes several E-box elements: the authors used a luciferase
assay to show that Clock and Bmal1 induce promoter activity
and that mutation of the E-boxes abolishes this response.
In rodents, granulosa cells cultured from mouse or rat
ovaries of transgenic reporter strains show many cycles of clock
gene expression (Sellix, 2015). Circadian oscillations in the
corpus luteum, which contains both thecal and granulosa cells,
appear to be stronger than in the follicle (Chen et al., 2013).
These data support the notion that clock-driven events in the
gonad are necessary for critical local processes and the timing
of hormone secretion. The clock/StAR relationship has been
explored in the mammalian ovary. Levels of StAR were reduced
in ovary, as in testis and adrenal, of Bmal1-deficient mice
(Alvarez et al., 2008). In female Bmal1 knockouts, suppression
of gonadal steroidogenesis leads to prevention of implantation
(Liu et al., 2014).
Testicular rhythms. Unlike data for the ovary, the evidence for
a functional role of clock genes in the testis is complex; period-
icity of testicular function has been described along multiple
timescales, from ultradian to infradian to circadian and
seasonal (Bittman, 2016). Evidence for circadian rhythms in
T secretion is well established (Van Reeth et al., 1994), but its
origins are not well understood (Neill, 2005). There is some
evidence of circadian variation in Per gene expression (Zylka
et al., 1998; Guo et al., 2005), but other studies have not
detected such rhythms (Miyamoto and Sancar, 1999; Bittman
et al., 2003; Morse et al., 2003; Yamamoto et al., 2004; Alvarez
and Sehgal, 2005; Liu et al., 2007; Bittman, 2016). Additional
findings have led to further confusion: although Bmal1 message
is not expressed rhythmically, BMAL1 protein oscillates in Ley-
dig cells, and knockout of Bmal1 reduces T production and
fertility in mice (Alvarez and Sehgal, 2005; Alvarez et al.,
2008). Although much work appears contradictory, real-time
reporting of bioluminescence of testis slices from Per2::luc
mice confirms that at least ex vivo, circadian rhythmicity occurs
in testes (Nishide et al., 2014). This method, however, does not
clarify the cell type that produces the oscillation. The resolution
of apparently contradictory evidence likely lies in the heteroge-
neity of testicular tissue, with different temporal patterns of
change unique to various cell types (Bittman, 2016).
Circadian rhythms and differentiation of endocrine tissue.
Alvarez et al. have suggested that circadian rhythms are sus-
pended during differentiation based on data from testis and
thymus (Alvarez et al., 2003; Alvarez and Sehgal, 2005). This
contention is supported by in vitro data using a destabilized
luciferase driven by the Per2 promoter (Per2-dluc) in rats (He
et al., 2007). Several 24 h cycles of Per2-dluc biolumines-
cence, although gradually decreasing in amplitude, could be
induced by medium changes or by DEX in nondifferentiating
ovarian luteal cells, proliferative uterine stromal cells, and
testicular interstitial cells. In contrast, no rhythms were detected
in decidualizing uterine stromal cells, differentiating Leydig
cells, or thymocytes. Notably, ovarian granulosa cells displayed
a single circadian cycle of bioluminescence; loss of Per2-dluc
rhythmicity was concomitant with increased LH receptor
expression, a marker of granulose cell differentiation (He
et al., 2007). Whether circadian rhythms interfere with differen-
tiation if not inactivated is an interesting possibility.
Estrogenic feedback to the SCN. Ovarian hormones have
profound effects on circadian locomotor activity rhythms. Nor-
mally cycling hamsters display a phase advance every fourth
day on the day of estrus when E levels are highest (Morin
et al., 1977). Continuous administration of E shortens the
free-running rhythm compared to OVX controls (Morin et al.,
1977), and when such animals are held in constant light that
normally reduces consolidation of the activity bout and can
lead to splitting of activity, continuous E prevents these changes
(Morin, 1980). Finally, E may act either directly on the SCN,
where ER are sparsely expressed (Shughrue et al., 1997;
Hileman et al., 1999; Gundlah et al., 2000; Vida et al., 2008),
or indirectly (Figure 10). Cells that express ERa in the preoptic
area, amygdala, BNST, and Arc provide input to the SCN (de la
Iglesia et al., 1999). Estrogens may act by altering gap junction
connectivity within the SCN. In female rats, estrogen increased
expression of connexin-36, a subunit that makes up interneu-
ronal gap junction channels in the SCN (Shinohara et al.,
2001; Rash et al., 2007). Functional gap junctions are revealed
in cultured SCN cells by dye and electrical coupling (Jiang et al.,
1997; Colwell, 2000; Long et al., 2005), and gap junction
blockers reversibly alter rhythms in electrical activity and
hormone release in vitro (Prosser et al., 1994; Shinohara
et al., 2000). Locomotor activity rhythms are dampened in
male connexin-36 knockout mice, suggesting that gap junc-
tions may be important to normal behavioral rhythms in vivo
(Long et al., 2005).
Interactions of clock genes with ERa and ERb. Per2, a core clock
gene, is regulated both transcriptionally and posttranslationally
by multiple cues (Albrecht, 2012). Relevant in the present
context is a feedback loop comprising ovarian hormones, the
ER signaling network, and Per2. ERb expression is rhythmic
in several peripheral tissues in wild-type but not in arrhythmic
Bmal1 knockout mice. Its promoter region includes an E-box,
a target for the CLOCK:BMAL1 complex, suggesting direct
regulation by the circadian clock (Cai et al., 2008). Per2
enhances ERa protein degradation, and Per2 suppression
leads to ERa stabilization. Estrogen can in turn feedback
induce Per2 expression (Gery et al., 2007). Such feedback
mechanisms point to a critical role for peripheral circadian
regulation in tissue homeostasis. While this work was done
in a cancer cell model, it is likely to apply to other ERa
containing cells.
Androgenic feedback to the SCN. Castration of mice lengthens
the free-running period, decreases the precision of activity
onset, and reduces consolidation of activity (Daan et al.,
1975; Karatsoreos et al., 2007). Both T and dihydrotestosterone

Figure 10 Schematic representation of differences between the sites of action of estrogenic and androgenic hormones on the suprachiasmatic
nucleus (SCN). Estrogen receptor (ER)-rich nuclei (pink) including the retina, the intergeniculate leaflet (IGL), and the dorsal raphe (DR), via the
median raphe project to the SCN. The DR also contains androgen receptors (ARs, blue) and projects to the SCN. ERs are largely constrained to the
shell SCN region. Unlike ERs, ARs are densely localized in the core SCN. The SCN regulates circadian timing in physiology and behavior by sending
outputs to the neuroendocrine systems, including the gonads.
(DHT: a nonaromatizable androgen) restore the gonadally
intact phenotype, suggesting that the effects of T are mediated
by androgen receptors (ARs) rather than by ERs. While it is
likely that androgens modulate locomotor behavior at multiple
sites, the AR is concentrated in the core region of the SCN of
mice, and local administration of T to the SCN restores
intact-typical circadian periods, strongly indicating a direct
effect of androgens, mediated by the AR, in the SCN (Figure 8;
Karatsoreos and Silver, 2007; Model et al., 2015). More diffuse
AR expression has also been detected in other species (see
Karatsoreos et al., 2007 for review). Androgenic effects on
period in males are mediated by an increased sensitivity of
the SCN to light cues from the retina (Butler et al., 2012).
Although the mechanism is not known, this may be associated
with an increased response to NMDA as observed in the hippo-
campus (Pouliot et al., 1996).
Comparison of AR expression indicates sex differences in
the SCN in humans (Fernandez-Guasti et al., 2000) and in
rodents (Iwahana et al., 2008). Western blots and immuno-
chemistry indicate that ARs are more highly expressed in
male than in female mice; gonadectomy eliminates and
androgen treatment restores these sex differences. At the behav-
ioral level, gonadectomy produces a dramatic loss of the
evening activity onset bout in males, but has no such effect
in females. Consistent with the implications of these findings,
treatment with T or DHT restores male locomotor activity and
eliminates sex differences in the behavioral response (Iwahana
et al., 2008).
1.12.6 Circadian Regulation of Melatonin
Overview. The study of melatonin and the clock grew from
a strong literature on the mechanisms of seasonality, and mela-
tonin’s important role as an endocrine representation of day
Circadian Regulation of Endocrine Functions 359
length (Arendt, 2006; Johnston and Skene, 2015). Melatonin
is also a chronotherapeutic and can phase shift circadian
rhythms in both rodents and humans, although only at times
when melatonin would not be secreted endogenously
(Armstrong, 1989; Lewy et al., 1998). Here we consider the
regulation of melatonin and its potential effects on clocks
within the body.
SCN regulation of melatonin secretion. Melatonin is secreted at
night by the pineal gland; its synthesis is governed rhythmically
by cues from the SCN via a multisynaptic pathway with
synapses in the PVN, the medial forebrain bundle, and the
superior cervical ganglion of the spinal cord (Moore and Klein,
1974). From the superior cervical ganglion, b-adrenergic
neurons drive pineal melatonin secretion during the dark but
not during the light when their activity is inhibited (Cassone
et al., 1990; Larsen et al., 1998; Larsen, 1999; Teclemariam-
Mesbah et al., 1999; Card, 2000). Both stimulatory and inhib-
itory outputs of the SCN are implicated in controlling the final
noradrenergic stimulation of the pineal gland at night. During
the day, GABAergic projections to the PVN inhibit excitatory
output to the pineal and therefore suppress melatonin
(Kalsbeek et al., 2000). During the night, stimulatory glutama-
tergic outputs from the SCN to the PVN stimulate melatonin
synthesis (Perreau-Lenz et al., 2004). As with other hormonal
systems, lesions of the SCN abolish circadian rhythms in
melatonin production and secretion (Scott et al., 1995;
Tessonneaud et al., 1995). Given its high-amplitude rhythm,
strong dependence on circadian phase, and rapid rise from
basal levels, melatonin secretion patterns are the gold standard
for measuring clock phase in humans, usually defined as the
time of melatonin secretion onset under dim light conditions
(Lewy and Sack, 1989).
Melatonin as an entraining agent (endogenous and exogenous).
Stimuli such as light and melatonin can shift the phase of the
circadian clock, but only when they are administered at
360 Circadian Regulation of Endocrine Functions
Figure 11 Melatonin is most effective in phase shifting the circadian
clock at times when endogenous melatonin secretion is low. Here, the
phase response curve to 3.0 mg of oral melatonin is shown, with
maximum advances in the afternoon and delays in the morning. Endog-
enous melatonin secretion (gray), in contrast, is high during the night-
time hours. Modified from Burgess, H.J., Revell, V.L., Molina, T.A.,
Eastman, C.I., 2010. Human phase response curves to three days of
daily melatonin: 0.5 mg versus 3.0 mg. J. Clin. Endocrinol. Metab.
95 (7), 3325–3331.
particular times of day. The relationship of time of treatment to
the magnitude of response is described by a phase response
curve. Melatonin can cause phase advances, phase delays, or
have no effect depending on its dose and time of administra-
tion (Figure 11). It is noteworthy, however, that the most effec-
tive phase advancing effect occurs in the evening, well before
bedtime, at a time when melatonin suppression is minimal.
Therefore, endogenous melatonin secretion is unlikely to play
a role in phase resetting the central clock.
Whether endogenous melatonin secretion plays a role in
entraining peripheral clocks is not well studied, although this
remains an intriguing mechanism for clock coordination (Pevet
and Challet, 2011). Melatonin receptors are described in
several endocrine tissues, including the pituitary gland, adrenal
gland, pancreas, and ovaries (Slominski et al., 2012). In the
pituitary gland, long and short duration endogenous mela-
tonin secretion differentially entrain clock genes in the pars
tuberalis; the altered relationships among clock genes are
thought to be involved in day-length encoding in seasonally
breeding species (Lincoln et al., 2002). Until melatonin phase
response curves are defined for other specific peripheral tissues,
it will not be possible to determine the role that melatonin
plays in coordinating peripheral rhythms. The one area in
which endogenous melatonin has a strong documented
entraining effect is in maternal–fetal communication. Maternal
melatonin synchronizes the clocks of rat pups (Bellavia et al.,
2006).
Exogenous melatonin, when administered at the proper
times, can have strong phase resetting properties that are worth
highlighting. Melatonin is an effective treatment to synchronize
the free-running rhythms of blind subjects (Sack et al., 2000).
In sighted individuals, evening melatonin can phase advance
rhythms and its effect is additive to that of light (Burke et al.,
2013). Some affective disorders, such as seasonal affective
disorder and major depression, are associated with altered
clock phase (Lewy et al., 2006; Emens et al., 2009). In the
former, melatonin timed to normalize clock phase improves
mood (Lewy et al., 2006).
1.12.7 Circadian Dysfunction and Chronotherapy
The universal experience of our sleep–wake cycles and feelings
associated with their disruption lend immediate salience to this
realm of basic research. It is easy to intuit how physiological
functioning suffers without an internal circadian clock synchro-
nized to the environment. Most of us have experienced this
state, manifested by general feelings of malaise after a long
flight to a new time zone. While we can recover from acute
jet lag within a few days, millions of frequent flyers, shift
workers, and those with sleep disorders are exposed repeatedly
to temporal disruptions. However, it has also been demon-
strated that the nearly weekly changes in sleep patterns caused
by workdays and days off also cause significant temporal disor-
ganization, a phenomenon known as ‘social jet lag’ (Wittmann
et al., 2006).
To return to points made in the first paragraphs of this
chapter, the function of the SCN brain clock is to synchronize
the many cellular clocks in the rest of the body. The function
of individual cellular clocks is to generate oscillations in the
production of clock-controlled output genes, which in turn
drive overt rhythms in physiology and behavior. This is
achieved, in part, by oscillating hormones of the adrenal and
gonadal axes, which have been discussed here.
Under normal conditions, signals from the SCN to clocks in
peripheral tissues produce coherent and stable phase relation-
ships among different organs. Of course, the precise phase of
tissue clocks is specific to each organ due to tissue-specific func-
tions. When perturbed, such as occurs following alterations in
lighting cycle subsequent to long distance travel or shift work,
the SCN shifts in phase much more quickly (within a day or
two) than do peripheral clocks (which can take a week or
more), creating desynchrony (Lowrey et al., 2000). Similarly,
systematic manipulation of external cues such as unusual
feeding times can disturb the usual relationship between
peripheral clocks and the SCN clock (Damiola et al., 2000),
and these effects of food are independent of the SCN and
activity rhythms (Pitts et al., 2003).
How might mistimed feeding-related signals contribute to
circadian desynchrony? The HPA axis activity is modulated
by leptin, a hormone involved in regulating feeding behavior
and energy homeostasis. Leptin binds to its receptors in the
hypothalamus and midbrain and signals the availability of
peripheral energy stores (Stieg et al., 2015). Leptin is released
rhythmically and in antiphase to the release of cortisol
(Aschbacher et al., 2014). Elevated leptin inhibits glucocorti-
coid activity in the medial hypothalamus (Grosbellet et al.,
2015) and thus suppresses appetite and stimulates energy
expenditure. Reduction in leptin levels increases glucocorticoid
release, activating circuits that stimulate eating (Ahima et al.,
1996). Thus the interaction between rhythms in leptin and
cortisol provide a pathway by which neuroendocrine stimuli
and the brain circuits on which they act can influence circadian
rhythmicity and the production of clock-controlled genes by
altering normal phase relationships among organs.
Chronic circadian dysregulation has significant negative
consequences for health and has been associated with
increased cancer susceptibility, inflammation, and altered
metabolism (Karatsoreos et al., 2011; Golombek et al.,
2013). Experimental evidence indicates that individuals with
 Circadian Regulation of Endocrine Functions 361

chronic circadian disruptions often present with clinical

pathologies, pointing to the importance of synchrony between
brain and environmental rhythms. Elderly mice subjected to
temporal disruptions equivalent to a flight from Washington
to Paris, once a week for 8 weeks, suffered high mortality rates
due to these phase shifts (Davidson et al., 2006). Flight atten-
dants who frequently travel across time zones exhibit cognitive
deficits associated with reductions in temporal lobe structures,
have slower reaction times, and make more errors on cognitive
tasks (Cho et al., 2000; Cho, 2001). Shift workers have a higher
incidence of cancer (Hansen, 2006; Kubo et al., 2006; O’Leary
et al., 2006; Patel, 2006; Conlon et al., 2007), diabetes
(Sanborn et al., 1982; Robinson et al., 1990; Poole et al.,
1992; Karlsson et al., 2005; Morikawa et al., 2005), ulcers
(Kolmodin-Hedman and Swensson, 1975; Segawa et al.,
1987; Costa, 1996; Koda et al., 2000), hypertension and cardio-
vascular disease (Costa, 1996; Alstadhaug et al., 2005; Hwang
and Lee, 2005; Wolk et al., 2005; Kivimaki et al., 2006),
psychological disorders (Skipper et al., 1990; De Koninck,
1991; Leonard et al., 1998; Venuta et al., 1999; Munakata
et al., 2001; Bildt and Michelsen, 2002), and are a host of other
clinical issues. Social jet lag has also been associated with alter-
ations in metabolism and increases in obesity (Roenneberg
et al., 2012).
One important aspect of the circadian system is individual
differences – and these appear to be characteristic of individ-
uals. In one study, eight hormones were measured over two
different nights (Schulz et al., 1996). The temporal organiza- Figure 12 Circadian genes, genes associated with disease and drug
tion of hormone secretion into the blood was highly indi- targets. There is substantial overlap between circadian genes, known
vidual, and the intraindividual patterns were conserved. disease-associated genes, and drug targets (Zhang et al., 2014).
Colloquially, we have similar intuitive experiences and can
often categorize ourselves as larks or owls. A major break- Brown, 2008). These cell-based clock models are ideal for
through for circadian biologists interested in humans has target discovery and chemical biology and some of the
been the use of cells of peripheral tissues as proxies for the screening principles and measuring technologies may be
oscillators found in the SCN. Interindividual genetic differ- useful in the study of relationships between hormones, clock
ences appear to be manifest similarly in central and periph- genes, and clock-controlled genes at various levels of the neu-
eral oscillators (Yagita et al., 2001; Pando et al., 2002). raxis (Figure 12).
Several strategies for assessing individual differences in We have emphasized in this chapter the significance of the
timing, with the potential for optimizing medical treatment, circadian system in the brain and periphery in studies of
are promising. Molecular gene cycling is useful for deter- endocrine physiology and behavior. Circadian regulation
mining body time with important applications in personal- and dysregulation have profound consequences in health
ized medicine, including cardiovascular disease and cancer, and disease. It is clear that precise temporal organization
our leading causes of death (Liu et al., 2007). Detection of helps to promote optimal function of many endocrine and
an individual’s body time via a single-time-point assay can neuroendocrine systems. Disruption of this organization is
be achieved by a ‘molecular timetable’ composed of also linked to disease, even at the molecular level. Clock genes
hundreds of ‘time-indicating genes’ (Ueda et al., 2004). link estrogen receptors and breast cancer, and their dysregula-
Diurnal protein cycling in blood using high-throughput pro- tion has been implicated in cancers of other endocrine tissues
teomics is also an attractive possibility; blood draws are mini- (Gery et al., 2007; Suzuki et al., 2008). Understanding circa-
mally invasive, and the proteomic profile can indicate what is dian rhythms can increase experimental control in empirical
happening elsewhere in the body in health and disease studies and improved understanding of tissue-specific
(Martino et al., 2007). Rhythms in fibroblasts also vary rhythms should help to refine clinical diagnosis and hasten
among individuals, and the correlation between activity the development of individualized, timed courses of
patterns and fibroblast rhythms suggest that these cells treatment.
accurately indicate circadian properties (Brown et al.,
2005). Because peripheral cells oscillate and are easily acces-
sible, it has also been possible to introduce genetic materials Acknowledgments
into such cells by transfection or transduction; this holds
promise for the analysis of biochemical mechanisms of oscil- We thank Erica Mezias and Malini Riddle for invaluable assistance in
lation, quantitative trait mapping, and the search for indi- preparing this chapter and figures. Our work has been supported by
vidual chronotypes (Brown et al., 2008; Cuninkova and NIH grants MH075045, NS37919, and by NSF grant 125605.
362 Circadian Regulation of Endocrine Functions
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 1.13 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates
Tyler J Stevenson, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, UK
Brian J Prendergast, University of Chicago, Chicago, IL, USA
Randy J Nelson, The Ohio State University Wexner Medical Center, Columbus, OH, USA
Ó 2017 Elsevier Inc. All rights reserved.
This chapter is a revision of the previous edition chapter by B.J. Prendergast, R.J. Nelson, I. Zucker, volume 1, pp. 507–540, Ó 2009, Elsevier Inc.

1.13.1 Introduction 371

1.13.2 Evolution of Seasonal Rhythms 372
1.13.3 Formal Classifications of Seasonal Rhythms 373
1.13.3.1 Type I Rhythms 373
1.13.3.1.1 Induction and Termination of Photorefractoriness 373
1.13.3.1.2 Critical Day Lengths 374
1.13.3.1.3 Puberty 375
1.13.3.1.4 Sexual Behavior and Physiology 375
1.13.3.2 Type II Rhythms 376
1.13.3.2.1 Entrainment of Type II Rhythms 377
1.13.3.2.2 Circannual Modulation of Circadian Organization 377
1.13.4 Endocrine Transduction of Photoperiod Signals 377
1.13.4.1 Duration versus Phase of the Melatonin Signal 377
1.13.4.2 Decoding Melatonin Signals 378
1.13.5 Neural Bases for Photoperiodism 378
1.13.5.1 Pathways for Melatonin Secretion and Hypothalamic-Binding Sites 378
1.13.5.2 Localized Thyroid Hormone Synthesis Induces Morphological Changes in the CNS 380
1.13.5.3 Photoperiod and Hormone Regulation of GnRH, Kisspeptin, and RFRP-3 380
1.13.6 Maternal–Fetal Communication of Day Length 381
1.13.7 Seasonal Rhythms in Primates 382
1.13.7.1 Nonhuman Primates 382
1.13.7.2 Humans 383
1.13.7.3 Evolution and Human Seasonality 383
1.13.7.4 Human Seasonal Reproductive Rhythms 383
1.13.7.5 Melatonin and Human Reproduction 384
1.13.7.6 What Can Animal Studies Tell Us about Human Seasonality? 384
1.13.8 Seasonal Rhythms in Nonreproductive Traits 384
1.13.8.1 Immune Function 384
1.13.8.2 Energetics and Immune Function 385
1.13.8.3 Social Organization: Affiliation and Aggression 387
1.13.8.4 Brain Development 388
References 391

Glossary
Circannual rhythm An endogenous self-sustained Photoperiodism The ability of an organism to measure
oscillation with a period of approximately 12 months. environmental day length.
Melatonin An indoleamine hormone secreted by the
pineal gland.

1.13.1 Introduction
Seasonal phenotypes in behavior and physiology are pervasive;
many reflect adaptations to matching environmental periodic-
ities and presumably have their highest fitness in the seasons
in which they occur (Brakefield, 1996). Seasonality is appropri-
ately conferred upon traits that recur during a relatively limited
time in the same months on successive years. Seasonality can
be inferred or distinguished from mere synchrony when more
than a single cycle has been tracked. Strong seasonality generally
implies interanimal synchrony within a population, but the
converse is not necessarily true (Di Bitetti and Janson, 2000).
Pronounced seasonal variations in day length, temperature,
and humidity derive from the yearly orbit of Earth about the

Hormones, Brain, and Behavior, 3rd edition, Volume 1 http://dx.doi.org/10.1016/B978-0-12-803592-4.00013-4 371

372 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates

Sun. The amount and intensity of solar radiation varies with
latitude; the greater the distance from the equator, the more
pronounced interseasonal differences in ambient temperature
and solar radiation. Plant growth, which is also affected by
other abiotic factors such as seasonal winds and patterns of
rain and snowfall, determines food availability and reproduc-
tion in herbivores and, consequently, in carnivores too.
Climatological shaping of animal structure and function is
a neglected aspect of mammalian neuroendocrinology research;
this is a major limitation, considering that the annual variation
in mean temperature in North America exceeds the glacial–
interglacial changes in mean annual temperature during the
Pleistocene (Potts, 1998). Nonclimatological factors also
contribute to the evolution of seasonality, not least in humans,
where social customs, religious practices, and legal regulations
account for some seasonal rhythms (Farrell and Pease, 1994;
Brewis et al., 1996; Dickert-Conlin and Chandra, 1999). This
chapter updates the article published in the second edition of
this series (Prendergast et al., 2009). Our aim is to review recent
seasonality research in the context of established frameworks.
The selective approach taken in this chapter does not
provide an in-depth treatment of formal models of photoperi-
odism (Goldman, 2001), nor does it address literatures on
nonmammalian vertebrates or invertebrates, each of which
informs analyses of mammalian seasonality (see Nelson
et al., 2010). The extensive literature on endocrine changes
unrelated to behavior is not considered. The emphasis on
reproductive, immunological, and select behavioral and cogni-
tive rhythms, largely in rodent models, reflects our own
research backgrounds and is not intended to diminish the
importance of nonreproductive rhythms in the overall
economy of mammals, nor the significance of other mamma-
lian and nonmammalian orders.
1.13.2 Evolution of Seasonal Rhythms
Successful reproduction is profoundly influenced by food avail-
ability. Foraging effort, ambient temperatures, presence of
predators, and quality and quantity of food located during
foraging determine whether or not reproduction will succeed
(Bronson, 1989). Two different modes of responding to food
abundance have been identified. At the time of mating, food
and energy reserves may be relatively low for income breeders,
and natural selection favors individual females that produce
offspring coincident with an abundance of high-quality food
during lactation and at the time of weaning (income breeders;
Figure 1(a)). The latter stage of lactation is an extreme energetic
bottleneck for small mammals (Bronson, 1985). An alternative
mating strategy is capital breeding; mating is triggered by an
abundance of food and/or prior energy storage; lactation and
weaning of young occur during declines in food availability
(Figure 1(a)). Animals resident in highly seasonal environ-
ments with short growing seasons are more likely to evolve

Figure 1 (a) Two distinct modes of responding to seasonality in the abundance of preferred foods. In A1, females are selected to give birth before
the annual food peak; they have evolved responsiveness to environmental cues that lead to conception one gestation period before this optimal
timing (income breeding). In A2, females respond directly to increases in food abundance as cues for conception, leading to births one gestation
period after that event, often coinciding with the decline in food availability (capital breeding). Line arrows indicate timing of conceptions; outlined
arrows indicate timing of births. (b) Timing of births among Soay sheep on the island of St Kilda in relation to availability and digestibility of food.
Births are restricted to a period early enough for the ewe and lambs to capitalize on abundant food of the spring pasture. Data from Lincoln, G.A.,
Short, R.V., 1980. Seasonal breeding: nature’s contraceptive. Recent Prog. Horm. Res. 36, 1–52.
 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates
seasonal phenotypes than conspecifics whose niches provide
more evenly distributed food resources throughout the year.
Food generalists, such as baboons, monitor and exploit many
plant and fruit species, switching to less preferred items in
times of scarcity (Hemingway and Bynum, 2005) and are less
subject to seasonal pressures than specialists reliant on a few
seasonally variable foods (e.g., a local population of vervets
went extinct when their main food source, fever trees, died
off (Alberts et al., 2005).
Small mammals have higher metabolic rates, higher costs
of thermoregulation, reduced fat stores with which to bridge
intervals of food scarcity, and shorter life spans than do larger
animals. They tend to breed more opportunistically than large
species, with greater year-to-year and locale-to-locale vari-
ability. Although seasonal timing of food availability is
usually generally constant from year to year, animals that
rigidly commit to reproduction at a specified calendar date
in disregard of local temperature variations, time of the first
frost, or snow melt are disadvantaged (Lee and Gorman,
2000). Day length, by far the most accurate predictor of phase
within the geophysical cycle, is sufficient to synchronize most
seasonal rhythms, and in many environments it is an accurate
predictor of food availability (Figure 1(b)), but mammalian
reproduction in the wild is typically affected by multiple
cues (Bronson, 1989). The neglect of interactions among the
several proximate determinants of seasonal rhythms is
a feature of all but a handful of laboratory investigations.
Also typically missing is the distinction between ‘strategic’
(photoperiod-dependent) versus ‘tactical’ (supplemental)
cues in seasonality (Follett, 2015) and differential neural
circuits that may be involved in these two approaches
(Migaud et al., 2015). Seasonal breeding may be abandoned
or relaxed in animals approaching the end of their lives; their
residual reproductive value is reduced (Horton and Yellon,
2001), and presumably the cost–benefit ratio of reproduction
versus survival favors out-of-season reproduction.
The above views of seasonality emphasize post-hoc adap-
tive scenarios of considerable surface plausibility. This adaptive
hypothesis approach has been criticized because it stops short
of direct field validation (Huey and Berrigan, 1996), and
assumes that physiological adjustments to the immediate envi-
ronment must increase fitness. A given seasonal phenotype can
reflect random drift, factors other than natural selection, and
adaptation to past rather than present environmental condi-
tions (Huey and Berrigan, 1996).
The mediation of mammalian seasonal breeding by day
length and photoperiodic changes in melatonin production is
well established. Goldman et al. (2003) noted that the similar
actions of pineal melatonin in various mammals support
a single ancestral origin for photoperiodism early in the history
of the mammalian lineage.
1.13.3 Formal Classifications of Seasonal Rhythms
Two principal forms of seasonal rhythms have been distin-
guished (Zucker et al., 1991). Type I rhythms are based on an
endogenous interval timer and do not persist for more than
a single cycle in the absence of environmental resetting
(Figure 2(a)). Common among many short-lived temperate
373
and boreal mammals, this has been argued to reflect an ances-
tral form of mammalian seasonality from which type II
rhythms evolved (Farner, 1985). Type II rhythms are endoge-
nous but persist for two or more cycles even when day length,
temperature, humidity, and food availability are held constant
throughout the year (Figure 2(b); Pengelley and Asmundson,
1974; Gwinner, 1986). Also referred to as ‘circannual’ rhythms,
type II rhythms are characteristic of longer-lived mammals
from several orders (primates, bats, carnivores, ungulates,
rodents); their period is usually <12 months, generated by bio-
logical oscillators that have eluded localization (Zucker, 2001),
although recent molecular evidence points to coordinated
seasonal activation of molecular events in the pars tuberalis
(PT) that may constitute output signals from the circannual
oscillator(s) (Sáenz de Miera et al., 2014). Seasonal changes
in day length entrain both type I and II rhythms to a period
of 12 months. Syrian (Mesocricetus auratus) and Siberian (Pho-
dopus sungorus) hamsters and golden-mantled ground squirrels
(Spermophilus lateralis) are the most thoroughly studied species
with type I and II rhythms, respectively.
Type III rhythms, which have been neglected by chronobio-
logists, are based on a strict stimulus-response system. Typi-
cally, environmental signals present at a particular time of
year control effector systems with little or no modulation
from time-keeping mechanisms (Figure 2(c)).
Below we provide an overview of type I and II rhythms, with
an emphasis on hormone–behavior interactions and recent
work in the field. The version of this chapter in the first edition
of this series addressed these issues in greater detail.
1.13.3.1 Type I Rhythms
Many North Temperate Zone female rodents restrict reproduc-
tion to the months of April–August, during which they
produce one or more litters. The seasonal rhythm for such
long-day breeders can be replicated by exposing animals to
natural photocycles, even if all other factors are held constant.
For sexually mature individuals, the decreasing day lengths of
mid- to late summer trigger the onset of gonadal involution,
eventually eliminating spermatogenesis, ovulation, and
mating behavior. Reproductive quiescence endures for
5 months, yielding in mid-winter to gonadal regrowth
(recrudescence); approximately 6 weeks later animals mate
(Butler et al., 2007a,b).
1.13.3.1.1 Induction and Termination of Photorefractoriness
Late-winter initiation of gonadal recrudescence reflects devel-
opment of refractoriness to short day lengths and is indepen-
dent from the small increases in day length after the winter
solstice (Gorman and Zucker, 1995). Mid-winter gonadal reju-
venation is termed ‘spontaneous recrudescence’ because it
occurs in the absence of long day lengths; it defines the onset
of the refractory state in which reproduction is disinhibited
and no longer depends on the presence of long days
(Figure 2(a)), and it is triggered by an interval timer set during
the first few weeks of short days (Prendergast et al., 2000a; Park
et al., 2006). The interval timer exhibits considerable inter-
sexual and interindividual variability; males typically initiate
recrudescence 4–6 weeks in advance of females (Prendergast
et al., 2004b; Butler et al., 2007a,b). Refractoriness to short
374 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates

(a) Type I rhythm

Mixed: Driven by endogenous and exogenous components
(e.g., Syrian hamster reproduction)

Reproductively Reproductively
active active

e al
ce o n ad
Te
sti

nc
cu

rud us g
es
Ce us c

lar
tro
ss

es
rec neo
Termination of

reg
ati yclic
photorefractoriness

ta
on

res

on
of y

sio

Sp
it

n
Reproductive
quiescence
Photorefractory Photosensitive

M A M J J A S O N D J F M A M J J A S O N

(b) Type II rhythm

Endogenous: Product of circannual clock(s)
(e.g., ground squirrel body weight)

Events analogous to type I rhythms,

but no phase transition is dependent
on photoperiod.

M A M J J A S O N D J F M A M J J A S O N

(c) Type III rhythm

Exogenous: Environmentally triggered
(e.g., hay fever)

Events analogous to type I & type II rhythms,

but all phase transitions are dependent
on stimuli in the environment.

M A M J J A S O N D J F M A M J J A S O N

Figure 2 Schematic representation of seasonal rhythms. (a) Type I (mixed) rhythms involve exogenous (photoperiod) and endogenous (interval
timer) components. Decreasing day lengths (1) initiate gonadal regression in the late summer and (2) trigger an interval timer that eventually renders
animals refractory to short days 20 weeks later leading to gonadal recrudescence. Exposure to long day lengths in the spring and summer ‘breaks’
refractoriness and resensitizes the neuroendocrine axis to short days. (b) Type II (circannual) rhythms are entirely endogenous and typically have
a period less than 12 months. Seasonal changes in day length synchronize type II rhythms to a period of exactly 1 year. (c) Type III rhythms are
entirely exogenous and driven by environmental stimuli.

days persists until animals are exposed for several weeks to the
relatively longer days of spring and summer (Kauffman et al.,
2003); this allows overwintered animals to undergo a second
gonadal regression when day lengths again decrease in late
summer. Although sufficient in this regard, recent work indi-
cated that melatonin is not mandatory for the ‘breaking’ of
refractoriness. Under a simulated natural photoperiod,
hamsters that were pinealectomized at the spring equinox
(prior to the appearance of categorically long photoperiods)
nevertheless exhibited gonadal regression in response to
long-duration melatonin signals delivered weeks later, suggest-
ing an endogenous component to the seasonal cycle that is
entrained by natural photoperiods but not evident under static
exposure to short days (Butler et al., 2010). The foregoing
description applies to several reproductive and nonreproduc-
tive type I photoperiodic traits.
1.13.3.1.2 Critical Day Lengths
The critical day length was initially proposed as a value above
and below which long-day breeders exhibit the summer and
winter phenotypes, respectively (Elliott, 1976). Critical day
length values were originally established with a protocol that
transferred animals between invariant long or short photope-
riods (e.g., 16–10 L) in a single day, an event that bears no
 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates
resemblance to the gradual changes in day length experienced
in nature. The critical day length concept generally refers to
a fixed value at which a trait reverts between seasonal pheno-
types under conditions of thermoneutrality, access to unlim-
ited free food, inescapable exposure to light, absence of
conspecifics, and other conditions that likewise seldom prevail
in nature. Several lines of evidence have compelled
modification of the strict critical day length framework,
including photoperiod history effects (Duncan et al., 1985;
Darrow and Goldman, 1986), graded effects of photoperiods
below the critical day length (Powers et al., 1997), and robust
effects of simulated natural photoperiods (Gorman and
Zucker, 1995, 1998). The pattern of change in day length codes
important information, and in some instances changes in day
length are more salient than the absolute length of the day
(Nicholls et al., 1988; Gorman and Zucker, 1995). Although
useful as an analytic tool, its value in the exegesis of seasonality
in photoperiodic rodents has likely been overemphasized.
1.13.3.1.3 Puberty
Rates of somatic and reproductive development differ mark-
edly in beginning cohorts versus end-of-season cohorts; rapid
somatic growth and puberty occur in early-born offspring of
several species (Worth et al., 1973), whereas those born several
weeks after the solstice forego reproduction in the season of
birth, overwinter, and first attain sexual competence and adult
body mass at 6 months of age or greater. Variations in day
length directly control whether puberty occurs in the season
of birth. Hamsters housed in simulated natural photoperiods
and born 8 or more weeks after the summer solstice fails to
undergo sexual maturation in the year of their birth; rather,
they initiate synchronous puberty in the following spring, irre-
spective of birth date (Butler et al., 2007a,b). The interval timer
underlying vernal spontaneous development is set by the pre-
vailing photoperiod shortly after weaning (3–9 weeks of
age) and is dependent on melatonin signals generated during
this interval (Gorman, 2003). The subset of individuals
born 8 weeks after the summer solstice likely forms the
main breeding nucleus the following spring (Butler et al.,
2007a,b). Depending on species, individuals born before the
summer solstice may not survive for more than a single
breeding season (Sadleir, 1969).
1.13.3.1.4 Sexual Behavior and Physiology
Changes in photoperiod are potent regulators of male and
female sex behaviors in the laboratory. In principle, only one
sex needs to terminate reproductive activity to prevent out-of-
season breeding. In Syrian hamsters, the inhibitory effect of
short days on mating behavior occurs several weeks earlier in
females relative to males; in both sexes, a seasonal decline in
fertility is evident prior to a measurable decrease in sex
behavior (Beery et al., 2007). Because copulation in most
mammals is strictly tied to gonadal hormones, photoperiodic
changes in sex behavior could be achieved by a reduction in
hormone secretion several weeks before conditions become
unfavorable for offspring survival. Alternatively, substrates
that mediate mating activity could become unresponsive
to steroid hormones several weeks before the decline of
gonadal steroidogenesis eliminates copulatory behavior. Lastly,
375
steroid-independent mechanisms may also participate in the
termination of the breeding season.
1.13.3.1.4.1 Males
Mating behavior declines in Syrian hamsters transferred from
long to short days (Morin and Zucker, 1978; Campbell et al.,
1978). Male hamsters undergo gonadal regression after 6–
10 weeks of exposure to short photoperiods and circulating
testosterone (T) declines over this interval (Reiter, 1980). Defi-
cits in male sexual behavior are apparent after several weeks of
exposure to short days (Powers et al., 1989). Castrated
hamsters in short days copulated less than their long-day coun-
terparts during replacement therapy with testosterone (Morin
and Zucker, 1978). In agreement with other studies (Powers
et al., 1989), short days apparently reduce responsiveness to
hormones in neural substrates that activate copulatory
behavior and thus may elevate thresholds for hormonal activa-
tion of copulation. However, conclusions derived from
hormone replacement studies can be questioned on grounds
that hormone implants fail to replicate the episodic pattern
of testicular androgen secretion. For example, the presence of
T in the circulation during the circadian interpulse interval is
not required to maintain male hamster sexual behavior (Park
et al., 2007).
In Syrian hamsters, there is little indication that a steroid-
independent decrease in motivation to mate contributes to
the seasonal suppression of reproductive behavior. In Siberian
hamsters, however, copulatory behavior is sustained for
months following castration but can be inhibited during this
interval by exposure to short days, implicating gonadal
hormone-independent effects of photoperiod in this species
(Park et al., 2004).
Part of the decline in mating among short-day male
hamsters may be caused by altered responses to vaginal chemo-
attractants, although the data are inconsistent across species
(Miernicki et al., 1990). In some reports, chemosensory stimu-
lation of luteinizing hormone (LH) by heterosexual conspe-
cifics was categorically abolished by exposure to short days
(Anand et al., 2002); in others, investigation of estrous females
was greater in short-day males than in long-day males
(Honrado et al., 1991).
Social stimuli interact with seasonal changes in day length
to influence gonadal status. In short-day housed deer mice
(Peromyscus maniculatus), growth of the testes and seminal
vesicles, indicators of increased androgen secretion, was stim-
ulated by the presence of a long-day female in each male’s
cage (Whitsett and Lawton, 1982). Recovery of sexual
behavior in short days was also accelerated in photorefractory
male Syrian hamsters provided with opportunities to interact
with long-day females (Honrado and Fleming, 1996). Cohab-
itation with heterosexual conspecifics inhibited gonadal size
in Siberian hamsters housed in intermediate-duration day
lengths (Paul et al., 2009). Testicular regression was blocked
in Siberian hamsters transferred to short days in tandem
with a gonad-intact summer-phenotype female (Hegstrom
and Breedlove, 1999b) but was unaffected by cohabitation
with an ovariectomized female (Weil et al., 2007). In this
species, maintenance of LH production in males requires
pheromones contained in the female feces (Anand et al.,
2004). Females do not elicit LH surges in males adapted to
376 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates
short days, indicating that photoperiod gates LH responsive-
ness to pheromones (Anand et al., 2002). The extent to which
interactions with females prevent males in the field from
undergoing gonadal involution as the end of the breeding
season approaches is unknown.
An intact pineal gland is essential for inhibition of sexual
behavior in short day lengths (Miernicki et al., 1990). Pinealec-
tomized male–female pairs of Syrian hamsters, unlike intact
controls, produced offspring during the winter when housed
in a natural photocycle (Reiter, 1974), again emphasizing the
essential role of the pineal gland in transducing effects of short
day lengths on the reproductive axis.
Activation of male sex behavior in Syrian hamsters involves
conversion of T to estradiol via aromatization in neural tissue
(Wood, 1996). Aromatase activity in the preoptic area(POA)
was not affected by day length or castration but was decreased
in the anterior hypothalamus of short-day males and may
contribute to behavioral deficits (Hutchison et al., 1991).
Callard et al. (1986) documented decreased aromatase activity
in whole hypothalamus of both intact and castrated males
housed in short days; androstenedione and T replacement in
castrated hamsters lowered aromatase activity and induced
fewer estradiol receptors in short than long days. The sugges-
tion that aromatase activity is an important rate limiting step
that determines the number of estrogen receptors in dience-
phalic tissues is a plausible mechanism by which day length
may control androgen-dependent processes.
Low ambient temperature does not by itself induce gonadal
regression in some long-day breeding rodents (e.g., Desjardins
and Lopez, 1980), but it is effective in Turkish hamsters (Hall
et al., 1982). Maintenance at 5  C in conjunction with short
day lengths markedly accelerated gonadal regression in several
rodent species (Steinlechner et al., 1991; Ruf et al., 1997; Larkin
et al., 2002). Conversely, higher ambient temperatures delayed
appearance of short-day gonadal responses (Larkin et al.,
2002). The day length at which testicular regression occurs in
Siberian hamsters is 2 h longer at 5 than 22  C (15 vs
13 L; Steinlechner et al., 1991). At 50 N latitude, well within
the home range of this species, this would accelerate gonadal
regression by 5 weeks (Steinlechner et al., 1991). We are
unaware of studies of mating behavior of photoperiodic
rodents under conditions of elevated or reduced ambient
temperature.
1.13.3.1.4.2 Females
Syrian hamsters are rendered anovulatory within 6 weeks of
transfer from long to short days (Seegal and Goldman,
1975). They cease to display lordosis following transfer to short
days (Badura and Nunez, 1989). In Siberian hamsters, estrous
cyclicity is eliminated for 20 weeks beginning 7 weeks into
short-day exposure (Schlatt et al., 1993).
Short day lengths reduced behavioral responsiveness to
estradiol and progesterone in female Syrian hamsters (Karp
and Powers, 1993; Bittman et al., 1990; Honrado et al., 1991;
Badura and Nunez, 1989; Mangels et al., 1998). This effect
was only observed at relatively low doses of estradiol (Badura
et al., 1987; Karp and Powers, 1993). In one instance, sexual
receptivity was attenuated in response to treatment with estra-
diol but not to estradiol followed by progesterone (Badura
et al., 1987). Behavioral responsiveness to steroid hormones
is reduced but not eliminated in short day lengths and, as in
males, may reflect an elevation of the threshold for activation
of specific behavioral components (cf. Morin and Zucker,
1978).
There is disagreement about whether photoperiodic inhibi-
tion of female sex behavior in Syrian hamsters is mediated by
the pineal gland. Karp and Powers (1993) implicated the pineal
gland, whereas Badura and Nunez (1989) did not. Differences
between the two studies in the interval between pinealectomy
and behavioral assessments may explain the discrepant claims,
as residual effects of pineal hormones can persist for as long as
a month (Ruby et al., 1989), perhaps accounting for the dimin-
ished responsiveness to steroid hormones in recently pinealec-
tomized females. Pinealectomy accelerated resumption of
estrous cycles in anestrous Siberian hamsters previously housed
in short days (Schlatt et al., 1993). The pineal gland mediates
effects of day length on intrasexual aggressive behavior of
female hamsters (Fleming et al., 1989).
Reduced behavioral responsiveness to estradiol in short-day
female hamsters is accompanied by reduced estrogen receptor
immunoreactivity (ER-ir) in the medial POA and by increases
in ER-ir positive cells in the amygdala. Induction of
progesterone receptors by estradiol is significantly reduced in
the hypothalamic ventromedial nucleus (Mangels et al.,
1998), a target tissue for hormonal induction of lordosis
(Pleim et al., 1990).
In some rodents, day length may affect female fecundity
indirectly, via changes in male physiology and behavior. Wean-
ling female Peromyscus caged together with adult males in short
days sustained greater uterine weights than social isolates
(Garcia and Whitsett, 1983). Pheromones or the behavior of
one sex may override inhibitory effects of short days on the
other sex (Walkden-Brown et al., 1999).
Almost all female meadow voles are pregnant during the
summer but fewer than 50% are pregnant during the winter
(Meek and Lee, 1993a). Complex behavioral changes
contribute to the decreased incidence of winter pregnancy.
Virgin female voles maintained in short days for 8 weeks all
mated when paired with long-day males, but fewer short-day
females produced litters (Meek and Lee, 1993a). Female
responsiveness to male pheromones and male responsiveness
to the female interact to affect winter pregnancy rates in this
species. Additional complexity is indicated by the finding that
prior sexual experience inhibited reproductive responsiveness
to short days (Meek and Lee, 1993b).
1.13.3.2 Type II Rhythms
Endogenous annual rhythms have been documented in a wide
variety of animals (Paul et al., 2008). In the golden-mantled
ground squirrel, these rhythms persist for many cycles irrespec-
tive of environmental lighting conditions (Zucker, 2001); in
others, only certain photoperiodic or temperature conditions
are compatible with rhythm expression (Gwinner, 1986), and
in still others the rhythm fades after several years.
The annual sequence of gonadal growth, a restricted
breeding season, and gonadal involution are superficially
similar in type I and II cycles. Closer examination reveals signif-
icant differences: reproduction in type II species is not main-
tained indefinitely during exposure to any sequence of
 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates
constant or variable day lengths; either gonadal recrudescence
is not accompanied by refractoriness or, if refractoriness occurs,
it terminates spontaneously (Paul et al., 2008). This feature
permits a self-sustaining annual rhythm. Recent high-
throughput screening in the 13-lined ground squirrel identified
scores of plasma metabolites that change significantly over the
hibernation cycle, indicating widespread changes in metabo-
lism that accompany the profound heterothermy exhibited
by hibernating rodents (Epperson et al., 2011).
An alternative view is that type II rhythms emerge from
sequential refractoriness to short and long days, respectively.
Ferrets and sheep develop refractoriness to both long and short
day lengths (Herbert and Klinowska, 1978; Almeida and
Lincoln, 1984; Robinson et al., 1985). This feature may be char-
acteristic of many type II rhythms but is not universally present;
ground squirrels make repeated, spontaneous transitions from
winter to summer phenotypes even when housed continuously
in an unvarying day length (Zucker, 2001).
1.13.3.2.1 Entrainment of Type II Rhythms
The period of Type II rhythms deviates from 12 months in the
absence of entraining signals; endogenous periods of body
mass, hibernation and reproductive rhythms of squirrels are
10.5 months, necessitating a phase delay of 1.5 months
each year to synchronize with the annual geophysical cycle.
Simulated annual variations in day length effect this correction
(Lee and Zucker, 1991). Pinealectomy eliminates melatonin
from the circulation and obliterates most type I rhythms, which
invariably revert to the summer or long-day phenotype (Reiter,
1980). Type II rhythms, in contrast, persist after pinealectomy
(Zucker, 1985; Woodfill et al., 1991), but the ability to entrain
to a simulated natural photoperiod is lost (Hiebert et al.,
2000). Infusions of melatonin that mimic durations of
nocturnal melatonin during the interval from the summer
solstice to the autumnal equinox entrain the LH rhythm of
pinealectomized ewes to a period of 12 months (Woodfill
et al., 1994). In ground squirrels, melatonin treatments initi-
ated in late summer produced durable phase shifts of body
mass and reproductive rhythms; identical treatments in the
spring were ineffective (Zucker, 2001). Circannual oscillators
in ground squirrels and sheep evidently respond differently
to melatonin at discrete phases of the annual cycle.
1.13.3.2.2 Circannual Modulation of Circadian Organization
Home range size and the amount of wheel-running activity
vary seasonally in several rodents. In type I species (e.g., voles,
hamsters), these transitions in activity are driven by seasonal
changes in day length, temperature, and food availability; in
type II species, however, seasonal variations in locomotor
activity are evident in the absence of environmental change.
The period of the squirrel free-running circadian locomotor
rhythm is less than 24 h during the subjective summer and
greater than 24 h during the subjective winter (Zucker et al.,
1983); locomotor activity begins earlier and ends later in the
day during subjective spring and summer than in autumn
and winter (Lee et al., 1990; Lee and Zucker, 1995). The interval
between successive recurrences of summer or winter patterns of
activity is 10.5 months and is driven by an endogenous cir-
cannual mechanism. Although increases in the duration and
distribution of activity are correlated with the reproductive
377
cycle and with high-amplitude seasonal changes in body
temperature, circannual rhythms in circadian organization
persist independent of reproductive (Lee and Zucker, 1995)
and thermoregulatory rhythms (Freeman and Zucker, 2000).
1.13.4 Endocrine Transduction of Photoperiod
Signals
Initial clues that the pineal gland played a role in seasonality
came out of the work of Czyba et al. (1964): excision of the
pineal gland reduced the rate and magnitude of seasonal
gonadal regression in male Syrian hamsters. This outcome
anticipated results obtained under more controlled laboratory
conditions by Hoffman and Reiter (1965) who reported that
gonadal regression in response to blinding or exposure to short
photoperiods was absent in pinealectomized hamsters. Mela-
tonin is secreted by the pineal gland in a circadian pattern,
with peak gland and blood concentrations occurring during
subjective night. The light-entrainable circadian clock in the
suprachiasmatic nuclei (SCN) drives the melatonin secretory
rhythm; separate circadian oscillators, entrained to lights-off
and lights-on, regulate the onset and offset of nocturnal
hormone secretion, respectively (Illnerova, 1991). Over the
annual cycle, the duration of nocturnal melatonin varies
inversely with day length (Darrow and Goldman, 1986).
1.13.4.1 Duration versus Phase of the Melatonin Signal
The circadian phase during which melatonin is elevated and
the duration of nocturnal elevation are two features which
could account for its gonadotropic effects. Syrian hamsters
housed in 14 L exhibited gonadal regression when injected
with melatonin during late afternoon (shortly before the onset
of darkness) but not when treated earlier in the light phase
(Reiter et al., 1974; Tamarkin et al., 1976b). This initially sug-
gested a circadian rhythm in reproductive responsiveness to
melatonin (Tamarkin et al., 1977), but subsequent experi-
ments indicated that, for regulation of seasonal traits, the dura-
tion of the nocturnal melatonin peak is paramount (Carter and
Goldman, 1983; Bittman and Karsch, 1984). Timed melatonin
infusions induced gonadal regression in pinealectomized
hamsters irrespective of the stage of the circadian cycle of its
administration; only the duration of the signal was relevant
to its antigonadotropic effects (Goldman et al., 1984; Gorman,
2003; but see Gunduz and Stetson (2001) for a contrary view
that emphasizes coincidence of melatonin secretion with
a circadian rhythm of sensitivity to the hormone). Melatonin
infusions 8 h per day suppressed serum gonadotropin and
prolactin (PRL) concentrations and provoked gonadal regres-
sion, whereas infusions 6 h per day stimulated the reproduc-
tive axis. As few as 4 weeks of daily long-duration melatonin
signals induced gonadal regression in adult Siberian hamsters,
and 6 weeks of such treatments sufficed in Syrian hamsters
(Maywood and Hastings, 1995; Prendergast et al., 2000a).
From this and other work, the ‘duration hypothesis’ has
emerged. The apparent circadian rhythm in responsiveness to
melatonin injections in pineal-intact hamsters reflected the
summation of exogenous and endogenous melatonin to result
in a longer daily signal. A version of the circadian sensitivity
378 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates
hypothesis was resurrected to account for the observation that
Syrian hamsters exhibited gonadal regression when provided
with two 2.5-h melatonin infusions per day separated by a 5-
h melatonin-free interval (Pitrosky et al., 1995); however, this
interpretation has been challenged (Hastings, 1996). The
viability of the duration hypothesis has been established in
several rodent and ungulate species and at present best
accounts for the way in which melatonin mediates photoperi-
odism (Arendt, 1995).
1.13.4.2 Decoding Melatonin Signals
Over the course of a year, the duration of nightly melatonin
secretion neither varies markedly nor remains static from
night-to-night; rather, melatonin signals either gradually
increase or decrease as days get shorter and longer, respectively.
Just as photoperiod information is contained in the direction
of change in day length, independently of absolute day length
(Gorman and Zucker, 1995; Butler et al., 2007a,b), the direc-
tion of change in melatonin secretion conveys seasonal infor-
mation to the neuroendocrine system. Melatonin infusions
gradually decreasing in duration from 10 to 7.5 h per night eli-
cited gonadal growth in Siberian hamsters, whereas those grad-
ually increasing from 5 to 7.5 h per night induced regression
(Gorman and Zucker, 1997).
Delivery of ‘unnatural’ melatonin signals has unveiled
formal properties of the melatonin signal-processing system.
Short-duration melatonin signals that occur every other night
fail to induce gonadal growth; likewise, long-duration mela-
tonin infusions at 48 h intervals do not trigger regression
(Elliott et al., 1989; Prendergast and Hugenberger, 1999).
Neuroendocrine responsiveness to melatonin is frequency-
tuned: only signals recurring at frequencies approximating
the organism’s endogenous frequency (in most cases 24 h)
elicit duration-appropriate responses. When challenged with
a series of melatonin signals that vary in duration by several
hours from night-to-night, hamster reproductive responses sug-
gested that a signal-averaging algorithm is used to abstract
seasonal information (Piatkowska et al., 2003).
Just as prior photoperiod history modifies the critical day
length for reproductive stimulation and inhibition, photope-
riod history likewise determines whether melatonin signals of
intermediate duration are regarded as long- or short-day cues.
For example, 7-h melatonin infusions elicited gonadal growth
in hamsters previously housed in short days but caused
gonadal regression in hamsters from long days (Prendergast
et al., 2000a). Photoperiodic mammals encode a representation
of recent prior day lengths which may provide a continuously
changing context for the interpretation of intermediate day
lengths. In nature, the encoding and eventual decay of such
photoperiodic history information may ensure that hamsters’
responses to ambiguous photoperiod cues are influenced
only by relatively recent day lengths.
1.13.5 Neural Bases for Photoperiodism
The neuroendocrine control of photoperiodism involves
an evolutionarily conserved cluster of hypothalamic brain
regions that includes the PT, ependymal layer along the third
ventricle (3rdV) and a series of discrete, interconnected nuclei
(Paul et al., 2008; Wood and Loudon, 2014; see Figure 3 for
current working model). The primary hypothalamic nuclei
include the arcuate nucleus (Arc), anteroventricular periventicu-
lar nucleus (AvPv), dorsomedial nucleus (DMH), and the POA.
These core structures are critical for the generation and mainte-
nance of the constellation of genomic, molecular, and neuroen-
docrine changes that govern the photoperiodic regulation of
seasonal rhythms (Weems et al., 2015). As described in the prior
section, the pineal hormone melatonin is a key internal signal
that encodes day length; thus the duration of melatonin secre-
tion provides the physiological mechanism for photoperiodic
time measurement. Seasonal changes in melatonin binding
within core hypothalamic structures are essential to trigger
genomic and morphological plasticity that underlies the photo-
period information essential for generation (type I) and/or
entrainment (types I and II) of seasonal rhythms. The role of
melatonin on seasonal rhythms has been most extensively
investigated using reproductive endpoints, and this section
will focus primarily on this literature.
1.13.5.1 Pathways for Melatonin Secretion
and Hypothalamic-Binding Sites
Seasonal changes in day length impinge on the neuroendocrine
substrates via a retinal–hypothalamic–pineal pathway (Morin
and Allen, 2006). Light entrains circadian oscillators and alters
gene expression in the SCN, which in turn initiate and termi-
nate nightly melatonin synthesis (Illnerova et al., 1984; Elliott
and Tamarkin, 1994). Melatonin is secreted at night, in propor-
tion to the duration of the scotophase. Consistent with the
essential role of melatonin in mammalian photoperiodism,
lesions along the retina–SCN–pineal pathway disrupt mela-
tonin secretion and reproductive responses to day length
(Rusak and Morin, 1976; Pickard and Turek, 1983; Bittman
and Lehman, 1987).
Two classes of melatonin receptors have been identified in
the mammalian CNS: membrane-bound, G protein-coupled
high-affinity receptors (MT1 and MT2; Reppert et al., 1994)
and isoforms of the RZR/ROR family of orphan nuclear recep-
tors, which bind melatonin with nanomolar affinity (Becker-
Andre et al., 1994). There is no evidence that nuclear receptors
participate in control of seasonal changes by melatonin
(Hazlerigg et al., 1996); the consensus is that these actions
are mediated by MT1 receptors (Weaver et al., 1996; Prender-
gast, 2010). Although inbred laboratory mice are not reproduc-
tively photoperiodic in the laboratory, Yasuo et al. (2009)
reported that such mice exhibit molecular hypothalamic
responses to melatonin treatment but failed to do so following
targeted deletion of the MT1 melatonin receptor. 125I mela-
tonin binds with high affinity in the SCN of several rodents
(Weaver et al., 1989). In Siberian hamsters, the thalamic para-
ventricular nucleus (PVN) and reunions nuclei (NRe) also
exhibit high-density melatonin binding (Duncan et al.,
1989). Among photoperiodic mammals, there is considerable
interspecific variability in the neural locations of high-affinity
melatonin-binding sites (Bittman et al., 1994; Bittman and
Weaver, 1990), ranging from diffuse (Peromyscus species: Kor-
ytko et al., 1995; Heideman et al., 1999) to nonexistent (ferrets:
Weaver and Reppert, 1990).
 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates 379

(a) (b)
Long day Short day

S
Caudal
lH
GnRH

ME
Tanycyte
Light
DIO2/3

↓
Rostral PT TSHβ
NSt Thyrotropes

T3 MEL
PVN PT
RN ME
DM

SCN
↓ Gonadotropes

LH/FSH

Figure 3 Light-dependent regulation of seasonal rhythms. The nocturnal secretion of the pineal gland hormone melatonin (MEL) codes night length
in all known vertebrate species. Annual changes in day length lead to short (long days in summer) or long (short days in winter) durations in MEL
secretion. MEL acts both centrally and peripheral by binding to MEL receptors. (a) MEL receptor is widely distributed in the brain; this schematic
provides the general distribution using a rodent brain. (b) The pars tuberalis (PT) and median eminence (ME) in the ventral hypothalamus are the key
brain regions for the seasonal regulation of physiology and behavior in many species. Long day lengths (>12 h; left side) stimulate thyrotropes in
the PT to produce thyrotropin-stimulating hormone subunit-b (TSHb). Increased TSHb alters the ratio of thyroid hormone enzymes (deiodinase type
II and III, DIO2, and DIO3) leading to the increased local synthesis in triiodothyronine (T3). T3 then permits the release of gonadotropin-releasing
hormone1 (GnRH) which in turn stimulates gonadotropes to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH
induce gametogenesis and steroidogenesis facilitating reproductive physiology and behavior. In short days (right side), increased MEL duration
inhibits TSHb expression, reversing the ratio of DIO2/3, reducing local T3 concentrations and inducing gonadal involution. AvPv, anteroventral peri-
ventricular nucleus; DM, dorsomedial nucleus; lH, lateral habenula; NSt, nucleus of the stria terminalus; PVN, paraventricular nucleus; RN, reunions
nucleus; S, subiculum; SCN, suprachiasmatic nucleus. Adapted from Stevenson, T.J., Visser, M.E., Arnold, W., Parrett, P., Biello, S., Dawson, A.,
Denlinger, D.L., Dominoni, D., Ebling, F.J., Elton, S., Evans, N., Ferguson, H.M., Foster, R.G., Hau, M., Haydon, D.T., Hazlerigg, D.G., Heideman, P.,
Hopcraft, J.G., Jonsson, N.N., Kronfeld-Schor, N., Kumar, V., Lincoln, G.A., MacLeod, R., Martin, S.A., Martinez-Bakker, M., Nelson, R.J., Reed, T.,
Robinson, J.E., Rock, D., Schwartz, W.J., Steffan-Dewenter, I., Tauber, E., Thackeray, S.J., Umstatter, C., Yoshimura, T., Helm, B., 2015. Disrupted
seasonal biology impacts health, food security and ecosystems. Proc. R. Soc. B 282, 1–10.

The PT is the predominant neuroendocrine substrate
located within the core evolutionarily conserved hypothalamic
nuclei implicated in seasonal rhythms that binds melatonin in
mammals. MT1 receptors are highly expressed in the PT and the
absence of melatonin binding facilitates the secretion of
thyrotropin-stimulating hormone subunit-b (TSHb) from PT
thyrotropes (reviewed below). Increased TSHb synthesis in
long-day conditions triggers a cascade of events that include
thyroid hormone-mediated neuroendocrine plasticity that
permits gonadotropin secretion from the pituitary gland
(Wood and Loudon, 2014).
Lesions of hypothalamic substrates enabled the necessity of
melatonin receptors in anatomically localized regions to be
examined. PT lesions are very difficult due to the small and
elongated structure of the tissue; however, lesions of several
other hypothalamic nuclei involved in seasonal rhythmicity
(e.g., DMH, VMH) reveal these regions are critical for the main-
tenance of annual reproductive cycles. In male Syrian hamsters,
ablation of melatonin targets in the DMH eliminated the
gonadal response to long-duration melatonin infusions
(Maywood and Hastings, 1995; Maywood et al., 1996); in
female hamsters, similar lesions centered on the junction of
the DMH and the ventromedial nuclei (VMH) abolished
the suppressive effect of short days on estrous cycles
(Lewis et al., 2002). Lesions of the SCN eliminated the antigo-
nadal effects of long-duration melatonin signals in Siberian
(Bartness et al., 1991) but were without effect in Syrian
hamsters (Bittman et al., 1979, 1989). DMH and VMH lesions
in Syrian hamsters (Maywood and Hastings, 1995; Bae et al.,
1999; Lewis et al., 2002) and SCN lesions in Siberian hamsters
(Bartness et al., 1991) spared the lactotropic (PRL) response to
short-day melatonin signals. Melatonin signals are transduced
into the hypothalamo–pituitary axis via multiple pathways
that may be trait specific (Lincoln, 1990; Bartness et al.,
1991; Maywood and Hastings, 1995). In hypothalamic–pitui-
tary disconnected sheep, melatonin implants in the PT-
inhibited PRL secretion in a manner that mimicked the cycle
of responsiveness and refractoriness to short days, even as
gonadal responses to melatonin were absent (Lincoln and
Clarke, 1997). In common with sheep, the hamster PT contains
substrates sufficient to decode melatonin signals that control
PRL and gonadotropin secretion.
There is interspecific variability and intraspecific redun-
dancy in the neural substrates that decode the nightly duration
of melatonin signals. In pinealectomized sheep, Siberian
hamsters, and white-footed mice, melatonin implants or infu-
sions in the anterior hypothalamus, SCN, MBH, NRe or PVT
are sufficient to engage seasonal reproductive responses
380 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates
(Glass and Lynch, 1982; Badura and Goldman, 1992; Lincoln
and Maeda, 1992). In pinealectomized Siberian hamsters,
melatonin implants localized to the SCN, NRe, or PVT were
each sufficient to induce gonadal regression. The eventual
loss of gonadal responsiveness to melatonin in any one of these
nuclei was indicative of tissue-specific development of
refractoriness and did not preclude responsiveness to
melatonin by other tissues (Freeman and Zucker, 2001).
Refractoriness appears to occur independently at several mela-
tonin target tissues. Other melatonin-mediated effects of
photoperiod in hamsters occur in a more target-specific
manner: elimination of melatonin signaling in the NRe, but
not the SCN or PVT interfered with long-day stimulation of
the testes, whereas obscuring melatonin signals to the SCN
and NRe but not the PVT disrupted acquisition of photoperi-
odic histories (Teubner and Freeman, 2007).
1.13.5.2 Localized Thyroid Hormone Synthesis Induces
Morphological Changes in the CNS
During the 1990s, thyroid hormones were clearly demon-
strated to play an essential role for mammalian photoperi-
odism (Parkinson and Follett, 1994, 1995; Moenter et al.,
1991). Given the consistent observation of thyroid-
dependent regulation of seasonal reproductive rhythms across
vertebrates, thyroid hormone signaling may reflect an evolu-
tionarily conserved system (Paul et al., 2008). Photoperiod
itself has little effect on circulating T3 or T4 concentrations in
Siberian hamsters (O’Jile and Bartness, 1992; Prendergast
et al., 2002a), although short days inhibited secretion of these
hormones in Syrian hamsters (Vriend, 1983). Hypothyroidism
does not abolish gonadal regression (Vriend and Wasserman,
1986; Prendergast et al., 2002a) in short-day exposed hamsters.
The recent identification of enzymes involved in thyroid
hormone catabolism has led to a significant advancement in
our understanding of the molecular mechanisms that govern
the internal representation of seasonal time information. Two
key enzymes called deiodinase type II and III (DIO2 and
DIO3, respectively) are expressed in cells along the ependymal
layer in the third ventricle (Wood and Loudon, 2014). DIO2
functions to convert the prohormone thyroxine (T4) into the
bioactive triiodothyronine (T3). Conversely, DIO3 reduces
the bioactive forms of thyroid hormone by converting T4 into
reverse T3, and T3 into diiodothyronine (T2). Adaptation to
short days or treatment with melatonin yields decreases in
the expression of DIO2 in Syrian hamsters (Watanabe et al.,
2004, 2007), and a single afternoon injection of melatonin
inhibits DIO2 production (Yasuo et al., 2007). In contrast,
DIO3 increases under short photoperiod (Watanabe et al.,
2007; Barrett et al., 2007), an effect that is paralleled by
decreases in methylation of the promoter region of the gene
that encodes DIO3 (Stevenson and Prendergast, 2013).
Changes in T3 concentrations induce tanycyte end-feet along
the ependymal layer to retract (Yamamura et al., 2004); these
morphological changes facilitate exposure of gonadotropin-
releasing hormone (GnRH) neurosecretory terminals to the
basal membrane of the median eminence. This response likely
facilitates the release of gonadotropins from the pituitary gland
and, ultimately, drives photoperiodic changes in reproductive
physiology. In support of this conjecture, exogenous T3
provokes gonadal growth in photoinhibited hamsters and
blocks short day-induced gonadal regression (Freeman et al.,
2007; Barrett et al., 2007). In addition to a T3-dependent facil-
itation of gonadotropin release from the pituitary, T3 has also
been implicated in the direct regulation of neuropeptides
located in many hypothalamic nuclei (i.e., Arc, DMH, AvPv;
Henson et al., 2013).
The eventual induction of photorefractoriness and loss of
responsiveness to short days may also depend on hypotha-
lamic regulation of thyroid hormone signaling. Expression of
thyroxine binding proteins, along with hypothalamic T4
uptake, was each lower in photorefractory relative to responsive
hamsters (Prendergast et al., 2002a). DIO2 expression
remained low after the development of refractoriness, but
DIO3 returned to long day-like levels (Watanabe et al.,
2007). Thus in recent years, it has become apparent that T3
can affect reproductive physiology in seasonal breeders via
morphological changes in tanycytes and via the expression of
key neuropeptides involved in the neuroendocrine control of
reproduction.
1.13.5.3 Photoperiod and Hormone Regulation of GnRH,
Kisspeptin, and RFRP-3
As described in Section 1.13.4, a series of long-duration mela-
tonin signals suppresses anterior pituitary gonadotropin secre-
tion (Bartness et al., 1993; Goldman and Nelson, 1993).
Melatonin may act in the hypothalamus to control GnRH
secretion, the pituitary to influence follicle-stimulating
hormone (FSH) and LH release, the gonads to affect respon-
siveness to gonadotropins, or at a combination of these sites.
In hamsters, negative-feedback sensitivity of LH to testosterone
is enhanced by exposure to short days (Tamarkin et al., 1976a;
Ellis and Turek, 1980) and attenuated by the development of
photorefractoriness (Ellis et al., 1979; Matt and Stetson,
1979; Prendergast et al., 2006); the neural sites and mecha-
nisms by which these effects occur are not known. There is
no consistent evidence that day length or melatonin alters
direct pituitary responsiveness to GnRH independent of steroid
feedback effects (e.g., Martin et al., 1977; Jetton et al., 1994). In
ruminants a significant modulatory role for photoperiod
signals in this regard has been documented (Xu et al., 1992).
In some studies of Syrian and Siberian hamsters, the
number of hypothalamic GnRH-immunoreactive (GnRH-ir)
neurons was increased in short day lengths (Ronchi et al.,
1992; Bernard et al., 1999). This effect was not found in
other studies (Urbanski et al., 1991; Yellon, 1994; Bittman
et al., 1996). Interpretation of these data is further
complicated by the fact that decreased GnRH-ir may reflect
less peptide production or more peptide release. It is not
known whether melatonin affects GnRH neurons directly or
indirectly, although in some species there is overlap between
brain nuclei that exhibit high-density melatonin binding and
those that contain GnRH-ir neurons (Glass, 1986; Korytko
et al., 1995).
In contrast to GnRH, two recently discovered neuropep-
tides: kisspeptin (KiSS) and RFamide-related peptide-3
(RFRP3, also referred to as gonadotropin-inhibitory hormone;
GnIH) exhibit marked seasonal variation in hypothalamic
expression (Weems et al., 2015). KiSS, is expressed in two
 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates
hypothalamic nuclei (Arc, AvPv), and stimulates GnRH secre-
tion via GnRH neurons expressing the GPR54 receptor
(Dungan et al., 2006). Adaptation to short days in Syrian
hamsters inhibited kisspeptin-ir and mRNA expression in the
Arc (Revel et al., 2006). This effect was not a consequence of
changes in circulating testosterone but was abolished by pine-
alectomy, thereby implicating photoperiod-driven changes in
melatonin secretion, as the mediator of seasonal kisspeptin
rhythms (Revel et al., 2006). In ovariectomized ewes, seasonal
anestrus is accompanied by lower ARC kisspeptin mRNA
expression (Smith et al., 2007). By contrast, kisspeptin-ir was
increased in the ARC and decreased in the anteroventral peri-
ventricular nucleus following adaptation to short days in
male and female Siberian hamsters (Greives et al., 2007; Mason
et al., 2007). Exogenous kisspeptin is sufficient to stimulate
gonadal hormone secretion in both hamster species (Revel
et al., 2006; Greives et al., 2007). Reports that kisspeptin also
responds to changes in energy balance (Castellano et al.,
2005) suggest it may transduce masking effects of food avail-
ability on seasonal reproductive rhythms. KiSS neurons in the
Arc and AvPv do not appear to coexpress with melatonin recep-
tors (Clarke and Caraty, 2013) suggesting that melatonin does
not directly regulate seasonal variation in KiSS expression. In
Siberian hamsters, daily peripheral injections of T3 in short
day significantly increased the number of cells expressing
KiSS-ir in the AvPv, and a reduction was observed in the Arc
(Henson et al., 2013). These findings indicate a potential
dual role for T3: in addition to the regulating tanycyte
morphology (and thus GnRH neurosecretory terminal
exposure), the local synthesis of T3 in the hypothalamus
regulates KiSS neuronal function leading to enhanced GnRH
activity.
RFRP3 was originally identified in birds and shown in vivo
and in vitro to inhibit gonadotropin release in a dose-
dependent manner (reviewed in Bentley et al., 2006). RFRP3
is localized to the DMH and directly interacts with GnRH to
regulate GnRH release. RFRP3 exhibits robust seasonal varia-
tion in expression with greater mRNA and protein expression
in long-day conditions (Revel et al., 2008; Mason et al.,
2010; Ubuka, 2012). Daily melatonin injections are sufficient
to inhibit RFRP3 expression and pinealectomized hamsters
maintain high DMH content after transfer to short-day photo-
periods (Revel et al., 2008). Moreover, T3 has also been shown
to stimulate RFRP3 expression, as 2 weeks of afternoon injec-
tions to short day-regressed hamsters stimulated more immu-
noreactive RFRP3 (Henson et al., 2013). These data indicate
that photoperiodic regulation of RFRP3 plasticity may lie
downstream of melatonin-driven changes in PT function (via
T3 mechanisms) as well as the potential for direct effects on
expression.
This report has primarily focused on the role of melatonin,
thyroid hormones, and neuropeptides (i.e., GnRH, KiSS, and
RFRP3) for seasonal rhythms in reproductive physiology. It is
important to note that several other modulatory systems are
involved, and these have been covered in detail elsewhere. In
brief, transcription of several hypothalamic genes is affected
by photoperiod and the induction of photorefractoriness
(GPR50, VGF, histamine H3 receptor, CRBP1 and CRABP2,
RXR/RAR nuclear receptors; Barrett et al., 2005; Ross et al.,
2005; Watanabe et al., 2004, 2007; Bechtold et al., 2012).
381
Some of these genes change in a manner that cannot be dis-
counted as masking effects of steroid feedback or entrainment
(Ross et al., 2005; Watanabe et al., 2007), leaving viable the
hypothesis that their protein products may constitute critical
intermediates between melatonin-mediated day length infor-
mation and the HPG axis or are output signals of an endoge-
nous circannual interval timer.
Cyclic expression of ‘clock genes’ in the central daily pace-
maker, the SCN and extra-SCN tissues are a key component
of an autoregulatory feedback loop that confers circadian peri-
odicity on these cells (Takahashi, 2004). Because several of
these genes are phase-locked to dusk and dawn, their expres-
sion provides a neural representation of ambient photoperiod,
which may complement the endocrine representation of day
length contained in the nightly melatonin signal (Lincoln,
2002; Hastings and Herzog, 2004). Expression of at least
some clock genes rises and falls in response to melatonin
(Morgan et al., 1998; Messager et al., 1999, 2000; Johnston
et al., 2003, 2005). Melatonin-proficient Per2 mutant mice
exhibited photoperiod-driven changes in Tshb, Dio2, and
Dio3 gene expression, suggesting that Per2 is not mandatory
for initial molecular responses to photoperiod change (Ikegami
et al., 2013). The role of clock gene expression in the induction
of photorefractoriness has also been investigated. Clock gene
expression in the short-day refractory hamster brain (Johnston
et al., 2003) and the long-day refractory sheep PT (Lincoln
et al., 2005) continued to track dusk and dawn, and the
onset/offset of a melatonin signal to which the tissues were
no longer responsive.
Lesion and histological analyses over annual time scales
suggest that the SCN and circadian clock genes are not neces-
sary for the generation and maintenance of some seasonal
rhythms. Annual oscillations in body mass persist in many
golden-mantled ground squirrels subjected to SCN lesions
(Dark et al., 1985). Moreover, Arctic reindeer exhibit robust
seasonal variation in melatonin duration that does not appear
to be entrained by circadian clock genes (Lu et al., 2010). These
findings indicate the integrity of the SCN as well as cellular
oscillations in circadian clock genes are not critical components
of the proximate mechanisms for seasonal rhythms in some
species. This raises the tantalizing conjecture for endogenous
circannual clock timers for the expression of seasonal rhythms.
Indeed, annual patterns in the coastal dinoflagellate, Alexan-
drium tamarense, suggest that even unicellular organisms
contain endogenous circannual molecular machinery for the
timing of seasonal rhythms (Andersen and Keafer, 1987).
With the advent of ‘omics’ technologies, the ability to sequence
entire genomes, delineate cellular and tissue transcriptomes,
and examine the biological effectors contained within proteo-
mic analyses offers opportunities to generate meaningful new
insights into the neural mechanisms that govern seasonal
rhythms.
1.13.6 Maternal–Fetal Communication of Day
Length
An earlier version of this chapter reviewed the literature
documenting the ability of rodent dams to communicate to
their fetuses’ photoperiods prevailing during gestation. In
382 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates
a laboratory setting, prevailing day lengths around the time of
weaning arrest or accelerate somatic and reproductive growth
in several photoperiodic rodents (Johnston and Zucker, 1980;
Horton, 1984; Stetson et al., 1989). When weanlings encounter
intermediate day lengths (12–14 h light per day) at weaning
(intended to simulate early spring or late summer), reproduc-
tive development is markedly affected by day length during
gestation. Comparison of pre- and postnatal day lengths
provides pups with directional information regarding changes
in photoperiod duration, and thereby either promotes or
retards growth and development.
The view that photoperiodic rodents are obliged to compare
prenatal with postnatal photoperiods in order to accurately
trigger or delay puberty has been modified by recent experi-
ments on Siberian hamsters maintained during gestation and
postnatally on simulated natural photoperiods. These incre-
mentally changing photoperiods exert a strong organizing
effect on seasonal rhythms by providing hamsters with a richer
source of environmental timing cues than are available in
simple static day lengths. Butler et al. (2007b) found no
evidence that prenatal photoperiod history influenced post-
natal gonadal development of female hamsters in the presence
of simulated natural photoperiods, and concluded that the first
animals to delay puberty may do so predominantly on the
basis of postnatal decreases in day length, whereas in later
cohorts, a comparison of postnatal day length to gestational
day length may contribute to arrested development.
1.13.7 Seasonal Rhythms in Primates
1.13.7.1 Nonhuman Primates
Discrete birth seasons have been conclusively established or are
probable for some two dozen species of nonhuman primates
(Lindburg, 1987). Other primates, though they have birth
peaks, produce some young at all times of year. The volume
edited by Brockman and van Schaik (2005) provides a compre-
hensive overview of primate seasonality. The editors note that
90% of extant primates live in tropical forests, with limited
seasonal variation in ambient temperature and day length
but often striking seasonal variations in rainfall and sunshine
(van Schaik and Brockman, 2005). Most primates have diverse
diets composed of over 50 species of plants and generally vary
food types ingested over the course of the year (Hemingway
and Bynum, 2005). Dietary diversity, reliance on specific
food types, and readiness with which animals switch between
food items may contribute to the evolution of seasonal
rhythms in reproduction (e.g., Bercovitch and Harding, 1993;
Jablonski et al., 2000). Dietary diversity may be a necessary
but not sufficient condition for nonseasonal reproduction
(Bercovitch and Harding, 1993; van Schaik et al., 1993).
Birth seasonality is lowest for foliovores and more
pronounced among omnivores or frugivore-insectivores of
equivalent body mass (Janson and Verdolin, 2005). There is
little effect of latitude on birth seasonality between 0 and
15 . Among new world primates, squirrel monkeys (Saimiri
sciureus) are highly seasonal, with births restricted to a 3- to
5-month interval each year. In a captive colony exposed to
natural day length variations in California, 87% of births
were recorded between May and September (Kaplan, 1977).
Squirrel monkeys housed indoors for 3 years in a 14-L photo-
period sustained marked annual rhythms; full-term pregnan-
cies usually terminated in the months of August to October.
Striking annual body mass rhythms and variations in testicular
size also persisted under constant conditions (Clewe, 1969).
These rhythms suggest a type II circannual organization.
The rhesus macaque (Macaca mulatta) is perhaps the best
exemplar of seasonality among Old World monkeys. At lati-
tudes between 24 and 31 N, approximately 80% of rhesus
births occur between March and May and none between
November and March (reviewed in Lindburg, 1987). Clear
seasonal rhythms in plasma testosterone secretion persisted
in males housed in a 14-L photoperiod for up to 3 years (Plant
et al., 1974; Michael and Bonsall, 1977). Individually housed
males maintained in a 13-h day length for 14 months gener-
ated clear rhythms in serum concentrations of PRL, LH, and
testosterone but not FSH (Beck and Wuttke, 1979). Marked cir-
cannual variations in spermatogenesis, testicular volume, spon-
taneous and provoked ejaculations, and sperm output, with
maximum values in the autumn and winter months that
encompass the mating season in the natural habitat, were
documented in males (Wickings and Nieschlag, 1980). Female
rhesus monkeys sustain seasonal anovulatory rhythms when
housed in a fixed 13-h day length (Riesen et al., 1971).
Changes in day length are sufficient to entrain annual
rhythms of rhesus monkeys. Males exposed to alternating
16-week cycles of 16 and 8 L manifested reproductive rhythms
with a period of 31  1.3 weeks, without evidence of 52-week
periodicity. Day length variation is also posited as the proxi-
mate synchronizer of rhythms of testosterone, dihydrotestos-
terone, frequencies of mounts, ejaculations, number of
female partners, courtship displays, and aggression in Japanese
macaques (Macaca fuscata; Rostal et al., 1986).
Ovulation was restricted to the months of August to March
in a group of rhesus females housed outdoors in Atlanta, GA,
USA; estradiol failed to induce gonadotropin surges during
the summer months at a time when endogenous concentra-
tions of gonadotropins and ovarian steroids are low (Riesen
et al., 1971). Estradiol lowered basal LH concentrations to
a greater extent in ovariectomized females treated during the
summer anovulatory season than during the fall-winter season
during which ovulation ordinarily occurs. As in hamsters and
sheep, negative feedback sensitivity of gonadotropin secretion
to steroid hormones increases during the nonbreeding season
and steroid-independent seasonal regulation of gonadotropin
secretion is documented for untreated ovariectomized females
who generate LH pulses with a lower frequency in summer
than fall (Wilson et al., 1987).
Behavioral responsiveness to estradiol varies seasonally in
rhesus macaques. Ovariectomized females treated with estra-
diol engaged in heterosexual behavior during the breeding
season but not during the nonbreeding season (Pope et al.,
1987). The duration of female proximity to males was influ-
enced by season but not by estradiol and the percentage of
time spent with nonkin was increased by estradiol, regardless
of time-of-year. Females were not attracted to males during
the nonbreeding season, nor were males interested in females
during this time of year, despite the induction of high out-of
season testosterone secretion. Males as well as females may
be refractory to behavioral actions of steroids during the
 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates
nonbreeding season, as previously established for Syrian
hamsters (see Section 1.13.3).
There is little evidence for seasonality of reproduction
among the apes (Lindburg, 1987; Watts, 1998). Savanna
baboons at the equator in Kenya experience two rainy seasons
with two intervening dry seasons each year but display no
significant birth seasonality; nor is the number of births per
year significantly correlated with rainfall (Bercovitch and
Harding, 1993). No single pattern of seasonality is discernable
among the many nonhuman primates. In Nepal, ovulatory
cycles and estrous behavior were restricted to the period from
July to October, during which most conceptions occurred
(Ziegler et al., 2000). In contrast, Struhsaker (1997) found no
evidence of a restricted birth season in five monkey species in
a tropical rain forest in Kimbale. In some species, conception
is associated with the presence of abundant food, and at the
time of parturition both food availability and quality are at
their worst (Ziegler et al., 2000; capital breeders). In other
species the opposite relation obtains (Struhsaker, 1997).
1.13.7.2 Humans
Given humans’ ability to buffer themselves from the environ-
ment, the number and diversity of human traits that vary
seasonally are impressive. Depression, stroke, suicide, sudden
infant death, cardiac arrest, infections, homicides, hip fractures
among the newborn, multiple sclerosis, intracerebral hemor-
rhage, and autism, as well as nonpathological functions such
as breast feeding, hormone secretion, food consumption, and
energy expenditure are a subset of human traits that vary
seasonally (Stevenson et al., 2015). The underlying mecha-
nisms are essentially unknown. The following discussion
focuses on historical and evolutionary trends that contribute
to the blunting of human seasonality.
1.13.7.3 Evolution and Human Seasonality
Early hominids originated in East Africa, with current evidence
localized to the Rift Valley (Maslin et al., 2014). The develop-
ment of humanlike attributes coincided with a decline in
global temperatures during the middle Pliocene and the emer-
gence of mosaic habitats far more seasonal than previously
thought (Foley, 1993). The ability to contend with drought
and associated nutritional stress is considered a key feature in
hominid evolution (Foley, 1993; Jablonski, 2005; Maslin
et al., 2014). The savanna occupied by our early ancestors is
marked by striking seasonal variation in plant and animal
resources that have experienced marked changes in climate
over the past 4 million years (Maslin et al., 2014). For extended
periods, each year hunting and scavenging were likely insuffi-
cient to provide adequate sustenance, in part because deteriora-
tion of grazing and browsing matter during drought renders the
meat of game animals marginally nutritious (Stewart, 1994).
Hominids from the late Pleistocene may have relied heavily
on fish when other foods were scarce or of low quality (Stewart,
1994). They may also have built up fat reserves to ameliorate
seasonal food shortages (Speth, 1987).
Thermoregulatory stress from extreme winter conditions is
thought to have limited expansion of African hominids from
tropical and warmer mid-latitudes to more temperate and
383
boreal regions (Foley, 1993). Modern humans, dating from
40 000 to 50 000 years ago, colonized the entire Earth not by
habitat-specific adaptations but by relying on increased cogni-
tive and behavioral capacity. The use of fire, improved weapons
for hunting, language for communication, and deployment of
animal skins for warmth, all contributed to this development
(Foley, 1993; Potts, 1998) which presumably involved selec-
tion for expanded, more complex neural machinery.
The past 18 000 years have seen some of the largest climatic
changes in the Earth’s history (Webb et al., 1993) with large
temperature changes in mid- to high latitudes accompanied
by significant alterations in atmospheric circulation and mois-
ture balance. The heightened seasonality of northern hemi-
sphere solar radiation from 12 000 to 6000 years ago
increased seasonality of northern hemisphere climates,
bringing warmer summers and cooler winters (Kutzback and
Webb, 1993). The development of agriculture some
12 000 years ago and domestication of animals led to the even-
tual worldwide settlement of agrarian societies 8000–
3000 years before the present. During this time the climate of
the Earth was highly variable relative to preceding and succeed-
ing millennia (Sandweiss et al., 1999). Potts (1998) has
proposed that human traits were shaped by a succession of
habitat changes over the course of the late Pliocene and early
Pleistocene epochs, with strong selection for genes that permit
adaptation to multiple habitats (discussion in Alberts et al.,
2005).
1.13.7.4 Human Seasonal Reproductive Rhythms
Environmental factors contribute to seasonal variations in
human births (Roenneberg and Aschoff, 1990a,b; Becker,
1991; Bronson, 2004). The amplitude of these rhythms pales
in comparison to those of most other mammals; peak to trough
variations usually are in the range of 10–20%. Reproduction in
many populations occurs throughout the year and typically is
not markedly seasonal, unless driven by food shortages or reli-
gious and cultural customs. With the advent of ‘big-data,’ the
assessment of seasonal rhythms in human reproduction can
easily be examined with impressive statistical power. Recent
work assessing historical and modern month-of-birth estab-
lishes a significant reduction in seasonal amplitudes from
pre-Baby Boom (1931–45) through Baby Boom Era
(1946–64) to Modern Era (1965–2008) (Martinez-Bakker
et al., 2013). A strong latitudinal gradient in seasonal human
birth was observed: countries located at higher latitudes
(35–65 ) were found to exhibit peak births in spring–
summer months, while lower latitude countries (0–35 )
have peaks in the fall–winter periods (Martinez-Bakker et al.,
2013). Modern assessments of large survey data sets of approx-
imately 450 000 individuals indicated marked seasonality of
birth on weight, age at menarche, and height (Day et al.,
2015). For example, individuals born in summer experienced
later pubertal development. Given such effects of birth month
on measures such as reproduction and immunity (Stevenson
et al., 2015), there likely exists a fetal programming effect
that varies by season. One consistently reported finding is the
seasonal variation in human semen parameters, such as
motility and morphology (Proctor et al., 2004; Levitas et al.,
2013). Sperm motility is significantly increased seasonally,
384 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates

with greater ‘normal’ morphology during fall and winter
periods compared to samples analyzed in spring and summer
(Levitas et al., 2013; Proctor et al., 2004). However, in vitro
fertilization–embryo transfer procedures do not have seasonal
outcomes; indicating that in the ovary, fertilization and
implantation processes may not be affected by seasonal
patterns in sperm (Revelli et al., 2005).
Identifying the factors (i.e., environmental/internal) that
mediate effects of birth season has been challenging. Evidence
suggests that shortages in metabolic fuels suppress reproduc-
tion in female mammals, with puberty as the most sensitive
phase of the life cycle to such resources (Bronson, 1989; Bron-
son and Heideman, 1994). Shortages in energy impact the
GnRH pulse generator, which stops functioning when energy
supplies are restricted or expenditures are very great, as in
anorectic women or athletes (Bronson, 1995, 2000). Studies
of subsistence foragers, agriculturists and pastoralists suggest
this mechanism impacts human reproduction (Bronson,
1995, 2000; Bentley, 1999). Among the Ache people, contem-
porary hunter-gatherers in eastern Paraguay, the low food
period is associated with lowest conception rates (Hill and
Hurtado, 1996). In a far ranging review, Bronson (2004)
contends there is no evidence that photoperiod plays an adap-
tive role in modern humans, a view shared by Ellison et al.
(2005). The evidence supporting the hypothesis that human
reproduction is sensitive to seasonal variation in day length is
inconsistent (but see Roenneberg, 2004). The suggestion that
individual humans are to different degrees seasonally photores-
ponsive (Bronson, 2004) has parallels in an extensive rodent
literature.
1.13.7.5 Melatonin and Human Reproduction
Humans, in common with all other mammals, secrete mela-
tonin nearly exclusively at night and exposure to light at night
interrupts melatonin secretion (Wehr, 1997). In urban environ-
ments with artificial lighting, people differ in the extent to
which duration of nocturnal melatonin secretion corresponds
to seasonal changes in day length (Wehr, 1997; Vondrasova
et al., 1997). Vondrasova and colleagues suggest that during
the middle ages, when people worked the fields from dawn
to dusk, summer versus winter melatonin durations may
have differed by as many as 4 h at 50 N. If so, then uncoupling
of melatonin secretion from gonadotropin regulatory mecha-
nisms must have been in place for many centuries in Homo
sapiens. Otherwise seasonal rhythms in human reproduction
would be far more pronounced in preindustrial societies than
indicated by the historical record (Lummaa et al., 1998).
Furthermore, molecular evidence indicates that the disconnect
of pituitary responsiveness from melatonin signaling preceded
the hominidae lineage. Female baboon pituitary cells cultured
with melatonin did not impact long-day, FSH or TSH expres-
sion nor release (Ibanez-Costa et al., 2015). Melatonin only
stimulated increased growth hormone and PRL expression in
a dose-dependent manner. These data indicate that mela-
tonin-driven seasonal physiology in primate physiology is
localized to the pars distalis and regulates GH and PRL, but
not components of steroidogenesis or gametogenesis. There is
presently no convincing evidence for a functional relation of
human reproduction and melatonin (Reiter, 1998).
1.13.7.6 What Can Animal Studies Tell Us about Human
Seasonality?
Indeed humans exhibit seasonal rhythms over multiple levels
of analyses, how these rhythms are generated and maintained
at a mechanistic level remains poorly defined. Animal research
provides a conceptual framework for the exegesis and manipu-
lation of human seasonal rhythms. Lessons learned from
research on rodents and monkeys raise the possibility that
hormone treatments will be effective in modifying behavior
at one but not another stage of the annual cycle. The demon-
stration that drugs and hormones phase shift annual rhythms
at some but not other stages of the annual cycle can open the
way for rational drug treatments for human rhythm dysfunc-
tion. Over the past few years it has become clear that disrup-
tions to daily cycles, via jet-lag or shift work, has a significant
and negative impact on many traits, at the genetic, physiolog-
ical, behavioral, and cognitive levels. One particularly good
example is the impact of dim light at night on immunity
(Scheer et al., 2009; Fonken et al., 2010). Animal models
have been extremely valuable for directing research efforts to
understand the effects of daily disruptions on humans.
However, we have a rudimental understanding of the impact
of seasonal disruption, such as climate change, on wild and
laboratory animal species (Stevenson et al., 2015). There are
simply no data available in humans. Rectifying this lack of
knowledge is particularly important given the hypotheses of
climate change driven evolution of hominidae (Trauth et al.,
2005). Advances in ‘omics’ research and the availability of
‘big-data’ resources will undoubtedly yield significant insights
into the effects of disruption in daily and seasonal rhythms.
Limits to the usefulness of animal models (Zucker, 1988) are
apparent, however, when we consider that the many social, reli-
gious and cultural customs that influence human seasonality
have no parallels in the animal kingdom.
1.13.8 Seasonal Rhythms in Nonreproductive Traits
Several nonreproductive traits that fluctuate seasonally are
mediated by reproductive steroid hormones, whereas others
are modulated by melatonin or other seasonal events.
Many seasonal changes conserve energy during the fall and
winter. Some species accomplish this goal by migrating to
better conditions; others cope with winter by undergoing
torpor or hibernation. Individuals that brave winter use
changing photoperiod signals and decreases in steroid
hormone secretion to inhibit food intake, increase or
decrease body mass, increase pelage density (Park et al.,
2007), or transition from nocturnal to diurnal locomotor
activity to conserve energy. These topics were reviewed exten-
sively in the previous version of this chapter (Prendergast
et al., 2002b) and are not addressed here. We focus on
immune function, social interactions, and brain size as key
nonreproductive adaptations.
1.13.8.1 Immune Function
Reduced food availability, in common with many other ener-
getic challenges, is a potent stressor. The number and types of
 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates
stressors vary seasonally (Breuner et al., 1999) and are corre-
lated with seasonal changes in immune function (Nelson,
2004). The role of temporal fluctuations in response to stress
has been well reviewed elsewhere (e.g., Chrousos, 1998;
Dallman et al., 1993).
Seasonal cycles of illness and death, as well as birth, are
common among many mammals (e.g., Bolinger et al., 1996;
Bradley et al., 1980; Lochmiller et al., 1994). Mammals may
have evolved mechanisms to combat seasonal stress-induced
reductions in immune function (Nelson and Drazen, 1999;
Yellon et al., 1999; Sinclair and Lochmiller, 2000; Nelson,
2004). It is plausible that fitness is increased in individuals
that bolster immune function in advance of seasonally recur-
ring immunosuppression.
Numerous studies have now documented marked changes
in immune function in response to photoperiod change in
the laboratory. Here we review broad concepts related to the
adaptive significance of seasonal immunomodulation and
highlight classic studies along with select, recent developments.
The previous version of this chapter reviewed this material
extensively, as have several recent review papers (Prendergast
et al., 2010; Stevenson and Prendergast, 2015; Weil et al.,
2015).
Consistent with laboratory analyses, many field studies
reveal enhanced immune function and decreased disease
prevalence during winter as compared to summer. Several
reports, however, document the opposite pattern of results
(Nelson and Demas, 1996; Nelson et al., 1995, 1998); i.e.,
immune function is compromised during the short days of
winter. Additional environmental factors, not usually manip-
ulated in laboratory studies, may account for the discrepancy.
For example, winter-associated stressors such as restricted
food availability and low ambient temperatures counteract
short-day enhancement of immune function in the laboratory
(reviewed in Demas and Nelson, 1996). Thus, immune func-
tion may be enhanced and compromised, respectively, during
mild and severe winters. Long-term field studies are required
to test this hypothesis (see, e.g., Sinclair and Lochmiller,
2000). Although the effects of melatonin on immunity are
well-established (see Nelson, 2004 for review), an under-
standing of how the ecological context affects immune func-
tion is necessary to appreciate that these actions of
melatonin may be adaptive, rather than merely physiological
oddities.
The cascade of cellular events during the acute phase
immune response and inflammation, and the elevation of
body temperature in response to cytokine activation, presum-
ably requires substantial energy, although precise quantifica-
tion is lacking (Henken and Brandsma, 1982; Maier et al.,
1994). Cytokine activation elevates body temperature, and
the energy requirements of inflammation and acute phase
immune responses may increase metabolic rate by more than
10% for each degree of increase in body temperature (reviewed
in Grimble, 1994). In house mice (Mus musculus) injection of
keyhole limpet hemocyanin, a specific antigen that evokes an
antibody response without inducing fever or symptoms of
illness resulted in increased oxygen consumption and meta-
bolic heat production. Animals with energy deficits that impair
the ability to sustain immune function display elevated risk of
infection and mortality.
385
In response to a simulated bacterial infection, Siberian
hamsters maintained in long day lengths that mimic summer
conditions have significantly higher levels of interleukin
(IL)-6 in circulation and produce higher fevers compared to
animals maintained in short days that mimic winter condi-
tions (Bilbo et al., 2002a, Figure 4(a)). Both IL-6 production
and febrile responses are mediated in part by macrophages
(Medzhitov, 2007). In addition, long-day, more than short-
day, hamsters increase macrophage-mediated phagocytosis
activity and increase oxidative burst activity (Yellon et al.,
1999). These photoperiod differences may be mediated by
activation of Toll-like receptors, which during infection stim-
ulate macrophages to produce the proinflammatory cytokine,
tumor necrosis factor a (TNFa) (Aderem and Ulevitch, 2000)
among other acute-phase cytokines. Elicited peritoneal
macrophages cultured from long-day hamsters produced
significantly more TNFa after a challenge with a bacterial
component known to stimulate specific Toll-like receptors,
lipopolysaccharide (LPS) compared to macrophages cultured
from short-day hamsters (Prendergast et al., 2003; Navara
et al., 2007, Figure 4(a)). However, TLR2 and TLR4 expres-
sion was similar between photoperiods and unrelated to
macrophage responsiveness (Navara et al., 2007); other
downstream intracellular mechanisms may be responsible
for photoperiod-evoked variation in macrophage responsive-
ness. Photoperiodic changes in immune function in, e.g.,
Siberian hamsters occur in parallel with photoperiodic
changes in reproductive physiology. Several lines of evidence
suggest that the suite of changes evident in the hamster
immune system during adaptation to short days is both
gonadal hormone (i.e., sex steroid)-dependent and gonadal
hormone-independent, to varying degrees, according to the
trait (reviewed in Stevenson and Prendergast, 2015).
Gonadal hormone-independent effects of photoperiod on
immune function in hamsters may be mediated by changes
in thyroid hormone availability to immune tissues. Exoge-
nous T3 enhanced several aspects of hamster immune func-
tion, and photoperiod-driven changes in deiodinase
expression in immune cells may mediate these effects
in vivo (Stevenson et al., 2014). However, other hormones,
including glucocorticoids (Bilbo et al., 2002a), PRL (Weil
et al., 2015), and leptin (Carlton and Demas, 2014) appear
to mediate other aspects of photoperiod-induced changes
in immunity in photoperiodic rodents.
1.13.8.2 Energetics and Immune Function
Maintaining optimal immune function is energetically expen-
sive; the cascades of dividing immune cells, the onset and
maintenance of inflammation and fever, and the production
of humoral immune factors require substantial energy
(Demas et al., 1997; Spurlock, 1997). Mounting an immune
response likely requires resources that could otherwise be
allocated to other functions (Sheldon and Verhulst, 1996;
Demas, 2004). Thus, it is reasonable to consider immune
function from the perspective of energetic trade-offs (Martin
et al., 2008). Individuals may ‘optimize’ immune
function and allocate energy resources to immune function,
other maintenance functions, or reproduction (Demas,
2004; Martin et al., 2008). Seasonal changes in immune
386 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates

(a)

20 80
10 *
70

TNFα (pg ml–1)

16 * 60

IL-1β (pg ml–1)

IL-6 (pg ml–1)

*
50
12
1 40
8 30
20
4
10
0.1 0 0
LD SD LD SD LD SD

(b)
0–24 h 24–48 h 48–72 h 0–24 h 24–48 h 48–72 h

0 0

Decrease in food intake (%)

Decrease in milk intake (%)

* *
−20
*
−20
*
−40 −40

−60 −60

−80 −80

Figure 4 Exposure to short photoperiods for 10–12 weeks attenuates lipopolysaccharide (LPS)-induced cytokine production of Siberian hamsters
(top panel; TNFa production from peritoneal macrophages; interleukin (IL)-1b and IL-6 production from lymphocytes), and LPS-induced sickness
behaviors (bottom panel). Milk and food intake are commonly used dependent measures for sickness symptoms. Sweetened-condensed milk is
highly palatable and reflects both a hedonic and an energetic measure. The impact of LPS on milk intake is attenuated, indicating that these sickness
symptoms (anorexia and/or anhedonia) are resolved faster in short days. Data from Bilbo, S.D., Drazen, D.L., Quan, N., He, L., Nelson, R.J., 2002b.
Short day lengths attenuate the symptoms of infection in Siberian hamsters. Proc. R. Soc. B 269, 447–454.

function are likely driven by seasonal changes in energy
requirements and availability (Nelson and Demas, 1996).
According to this hypothesis, in an environment where energy
needs and demands fluctuate, individuals maintain the high-
est level of immune function that is energetically possible
(without evoking autoimmune diseases) subject to
constraints of other survival needs. A complete understanding
of seasonal cycles in human immune function requires
consideration of how energetic and other stressors impinge
on immunocompetence. Animal models illustrate these
relations.
Among domesticated mammals, reduced food intake is
associated with compromised immune function (reviewed in
Spurlock, 1997). For example, proinflammatory cytokines,
particularly IL-1, IL-6, and TNFa, interrupt anabolic processes
and alter nutrient uptake and utilization (Grimble, 1994;
Johnson, 1997). These cytokines also cause the release of
glucocorticoids, catecholamines, and prostaglandins, which
in turn modulate metabolism and immune function. Although
proinflammatory cytokines are critical in the responses to
infection, an excess of cytokines can damage tissue and
compromise survival. Excess or inappropriate secretion of cyto-
kines has been linked to morbidity in malaria, sepsis, menin-
gitis, inflammatory bowel disease, and rheumatoid arthritis
(Beutler and Cerami, 1988; Waage et al., 1989; Kelley and
Dantzer, 1990; Kwiatkowski et al., 1990). Consequently,
a balanced release of cytokines must be achieved for maximal
effectiveness in protecting the host from infection.
Day length and ambient temperature interact to affect
immune function. In deer mice maintained at 20  C, serum
immunoglobulin G (IgG) concentrations were elevated in
short-day mice as compared to long-day mice (Demas and
Nelson, 1996). At 8  C, long-day deer mice had reduced IgG
concentrations and short-day animals had IgG concentrations
comparable to those of long-day mice at 20  C; i.e., IgG
concentrations were higher in short than long days, and low
temperatures significantly reduced IgG concentrations. Short
day lengths and low temperatures thus appear to produce
opposite effects on IgG concentrations, in essence restoring
values to those of animals in long days, which may help
animals cope with seasonal stress of winter and ultimately
increase reproductive fitness (Demas and Nelson, 1996).
2-deoxy-D-glucose (2-DG) induces glucoprivation and is
a metabolic stressor that increases serum corticosterone
concentrations in male rats (Weidenfeld et al., 1984) and
inhibits murine splenic T lymphocyte proliferation in a dose-
dependent fashion in laboratory mice (Miller et al., 1994). In
long days, corticosterone concentrations were elevated in
 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates
mice injected with 2-DG versus saline; an effect that was absent
in short days (Demas et al., 1997a), again underscoring impor-
tance of understanding interactions between stressors and
photoperiod in the effect on immune responses. 2-DG was
associated with reduced mitogen-stimulated splenocyte prolif-
eration in long-day mice, but short-day mice, regardless of
experimental treatment, exhibited enhanced immune function
in response to 2-DG treatment (Demas et al., 1997a). 2-DG
treatment of long-day hamsters decreased antibody response
compared with control animals but did not affect antibody
responses in short-day hamsters (Zysling and Demas, 2007).
These data suggest that short day lengths buffer animals
against metabolic stress. Reduced corticosterone concentra-
tions in animals maintained on short days or treated with
melatonin likely reflect improved metabolic function (Nelson
and Demas, 1996; Sinclair and Lochmiller, 2000). Accordingly,
enhancements in several aspects of immune function in short
days are likely one component of the winter adaptation medi-
ated by melatonin. The expectation is that natural perturba-
tions of energy associated with pregnancy, lactation, torpor,
and hibernation will affect immune function in predictable
directions (Lochmiller and Deerenberg, 2000; Nelson et al.,
2002).
Exposure to pathogens or to inflammatory stimuli such as
LPS activates the immune system and initiates physiological
and behavioral responses collectively termed the acute phase
response. These so-called sickness responses, including fever,
may be constrained by energy availability; if so, then cytokine
production, fever, and anorexia should be attenuated in
infected Siberian hamsters housed under short days (simulated
winter). Indeed, short days attenuated the response to LPS by
diminishing the duration of fever and anorexia (Bilbo et al.,
2002b). Remarkably, decreased ingestion of dietary iron,
a nutrient vital to bacterial replication, was reduced in short-
day hamsters. These changes in sickness responses corre-
sponded with decreasing production of IL-6 and IL-1b (Bilbo
et al., 2002b; Figure 4(a)). Taken together, short days attenu-
ated the symptoms of infection, presumably to optimize energy
expenditure and facilitate survival (Figure 4(b)). Similar results
were attained with melatonin treatment (Bilbo and Nelson,
2002), suggesting a therapeutic use of melatonin or its receptor
agonists.
Although much of the research on photoperiodic effects on
immune responses has been conducted in adult animals, early-
life photoperiods are also important sources of seasonal infor-
mation that establish a developmental trajectory and have
profound consequences for the immune system. Early-life
photoperiods appear to program adult immune responses, as
Siberian hamsters enhance immune responses as adults if
they were housed in short days both pre- and postnatally
(simulating early winter births) relative to hamsters that were
in long days during any developmental period (Weil et al.,
2006). Perinatal and adult photoperiods interact to determine
the adult immunological phenotype in developing animals
(Prendergast et al., 2004a,b).
Lastly, recent evidence is emerging indicating that exposure
to dim light at night, a phenomenon that is becoming more
prevalent in modern times, can impair multiple aspects of
immune function (Bedrosian et al., 2011). The aforementioned
enhancements in select aspects of immunity conferred by
387
changes in photoperiod may be antagonized by such artificial
stimuli (see Weil et al., 2015 for discussion).
1.13.8.3 Social Organization: Affiliation and Aggression
Many rodent species shift from highly territorial asocial
behavior during the breeding season to a social, highly interac-
tive existence during winter. These species typically undergo
reproductive quiescence at the end of the breeding season in
response to short days; the resulting decrease in androgen secre-
tion may be necessary for and permissive of the seasonal shift
in sociality, although in wood rats (Neotoma fuscipes) nonste-
roidal mechanisms mediate the seasonal transition in social
behavior (Caldwell et al., 1984). During the breeding season,
rodents control resources that promote their own survival
and that of their offspring and often aggressively exclude non-
kin from access to resources. During the winter, however, this
strategy is abandoned in favor of group living which conserves
energy and enhances survival in the face of low temperatures
and reduced food availability. Many species of rodents
conserve energy during the winter by forming aggregations of
huddling animals (West and Dublin, 1984). In these aggrega-
tions, different sexes and even different species commingle
(Madison et al., 1984).
Even in the absence of huddling behavior, animals may
tolerate one another better in close quarters during the winter
than during the breeding season. For example, male meadow
voles are highly territorial in the spring and summer and
occupy open meadows whereas red-backed voles (Clethriono-
mys gapperii) breed in forest habitats. During the winter
months, meadow voles move into the spruce forest habitats
occupied by the red-backed voles, presumably to take advan-
tage of the protective cover provided by trees. In some cases
they share nests with other rodent species (Madison et al.,
1984). Individual meadow voles trapped during the winter
tested in paired encounters in a small neutral arena exhibit
less interspecific aggression than voles trapped in summer
(Turner et al., 1975). The winter reduction in aggressiveness
permits energy-saving habitat sharing. As the animals enter
breeding condition in the spring, they reestablish mutually
exclusive territories.
Some males do not undergo reproductive regression and
maintain testicular function and produce sperm and androgens
during simulated winter conditions (Nelson, 1987; Prendergast
et al., 2001). The advantages of continuous breeding evidently
incur substantial hidden costs, because only a minority of each
population adopts this strategy. Reproductively competent
males, because of unusual aggressiveness during the winter,
are unable to participate in communal huddling and thus
may incur greater energetic costs in overwintering. High behav-
ioral and energetic costs associated with maintenance of the
reproductive system in winter may explain why nonregressive
types do not normally predominate in temperate or boreal
zone populations of rodents (Nelson, 1987; Nelson et al.,
1989). This contention is supported by a field study of winter
nesting behavior of prairie voles (McShea, 1990). Most voles
in the population studied were reproductively inactive during
the winter and formed groups of huddling individuals. Two
males, however, remained in breeding condition and were
never observed to huddle with other animals. In pair-wise tests
388 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates
of aggression, these two males were much more aggressive than
reproductively quiescent individuals. In another study, repro-
ductive status also influenced odor preferences of meadow
voles maintained in simulated winter day lengths (Gorman
et al., 1993). Males that retained reproductive capability in
winter day lengths preferred the odors of females that also
failed to inhibit reproduction during short days. This prefer-
ence may facilitate the sporadic occurrences of winter breeding
frequently reported for this species (reviewed in Nelson, 1987).
The decrease in circulating androgens associated with cessa-
tion of reproduction may mediate the increase in male affilia-
tive interactions during winter; males that maintain ‘summer’
reproductive status may thereby also maintain ‘summer’
patterns of aggression. This hypothesis is supported by the
observation that castration results in decreased aggressiveness.
Although the relation between castration and reduced male
aggression is well established, anecdotal observations of male
prairie voles suggest that low circulating testosterone concen-
trations are not sufficient to reduce aggressive behavior. Castra-
tion did not reduce the frequency of male aggression in tests
that involved resident–intruder, grouped aggression, or aggres-
sion against a lactating female (Demas et al., 1999). Similarly,
postpubertal castration of male dusky-footed wood rats also
did not eliminate the marked vernal increase in aggression in
laboratory encounters with castrated or gonadally intact oppo-
nents (Caldwell et al., 1984). Aggressive behavior may be inde-
pendent of gonadal steroid hormones in some adult male
rodents; further studies will be needed to establish the gener-
ality of the notion that aggression is maintained by testicular
androgens.
Individuals of many, if not most, species reduce aggression
outside of the breeding season. For some species, however,
aggression must be maintained throughout the year indepen-
dent of breeding season. In such cases, aggression would likely
be modulated by mechanisms unrelated to reproduction. In
hamsters, short days increase male resident–intruder aggression
(P. sungorus, Demas et al., 2004; M. auratus, Garrett and
Campbell, 1980). This effect is paradoxical because increased
aggression occurs when testosterone concentrations are often
undetectable. Despite the lack of plasma androgens, estrogens
may still be important (see below). Adrenalectomy prevents
increased aggression in short-day Siberian hamsters (Demas
et al., 2004).
Adrenal steroid hormones appear to be involved in the
short-day aggressive phenotype in several species of rodents
and birds (Soma et al., 2015). Both male and female Siberian
hamsters housed in short day lengths undergo gonadal regres-
sion and reductions in circulating gonadal steroids, but
substantial increases in aggression, compared with animals in
long days (Jasnow et al., 2000; Scotti et al., 2007). Replacement
of gonadal steroid hormones such as 17b-estradiol in short-day
females (to mimic long-day concentrations) does not reduce
aggression to summer levels (Scotti et al., 2007). Thus, another
neuroendocrine mediator was sought. Recently, it was reported
that melatonin bolsters DHEA secretion from adrenal glands in
culture (Rendon et al., 2015; Figure 5). Furthermore, long-day
hamsters provided with short-day melatonin doses displayed
increased aggression and elevated DHEA concentrations
(Rendon et al., 2015). These results suggest that DHEA is
a key peripheral regulator of aggression and that melatonin
coordinates a ‘seasonal switch’ from gonadal to adrenal regula-
tion of aggression by direct action on the adrenal glands.
In common with hamsters, short day lengths increase
aggression in beach mice (Peromyscus polionotus) (Trainor
et al., 2007). This effect is apparently mediated by estrogens.
Remarkably, estrogens have the opposite effects depending
on day length (Figure 6). The ERa agonist PPT
(propylpyrazole-triol) and the ERb agonist DPN (diarylpropio-
nitrile) increased aggression in ‘short-day’ P. polionotus and
decreased aggression in ‘long-day’ mice (Trainor et al., 2007).
These results indicate that photoperiod controls processes
that occur after estrogens bind to their appropriate receptors.
Steroid hormones, including estrogens, can affect physiological
and behavioral processes via slow genomic (hours to days) or
rapid nongenomic (seconds to minutes) pathways (Vasudevan
and Pfaff, 2007). Estradiol injections of beach mice evoked
rapid (<15 min) increases in aggression in short-day, but not
long-day mice (Trainor et al., 2007). These data suggest that
estradiol increases aggression via nongenomic actions in
short-day, but not long day mice. Estrogen-dependent expres-
sion of genes with estrogen response elements in their
promoters was decreased in the bed nucleus of the stria termi-
nalis (BNST) of short-day mice compared with that of long-day
mice. Environmental factors may regulate the effects of steroid
hormones on aggression in P. polionotus by determining the
molecular pathways activated by steroid receptors (Nelson
and Trainor, 2007).
Male song sparrows (Melospiza melodia) display estrogen-
dependent territorial aggression throughout virtually the entire
year (Wingfield and Soma, 2002). Although territorial aggres-
sion in response to a territorial intrusion appears similar
throughout the year, the neuroendocrine responses after terri-
torial aggression vary seasonally. After a territorial intrusion
during the breeding season, territorial residents continue
patrolling their territories and display spontaneous song for
hours, whereas during the nonbreeding season, resident birds
stop behaving aggressively within minutes (Wingfield, 1994).
Thus, breeding territorial aggression is persistent, whereas
nonbreeding territorial aggression is transient. It appears that
estrogen varies in function by season as in Peromyscus described
above. To test this hypothesis, captive song sparrows in either
breeding or nonbreeding conditions were provided with wax
moth larvae dosed with either estradiol or the vehicle. Birds
provided with estradiol-laced larvae increased aggressive
behaviors within 20 min of treatment indicating that estrogens
increase aggression during the nonbreeding season via a rapid
nongenomic mechanism of action (Heimovics et al., 2015).
Birds in breeding condition did not show this rapid onset of
aggression in response to estradiol. In song sparrows and other
small animals, transient expression of aggressive behavior
during the nonbreeding season is presumably highly adaptive
as it minimizes energy expenditure and maximizes the amount
of time available for foraging (Heimovics et al., 2015).
1.13.8.4 Brain Development
Seasonal changes in brain weight have been documented in
rodents and shrews (e.g., Clethrionomys glareolus, Clethrionomys
rutilus, Microtus oeconomus, Microtus gregalis, Sorex araneus, Sorex
minutus; Bielak and Pucek, 1960, 1965; Yaskin, 1984). Brain
 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates 389

(a) 30 (b) 2.0

25 ∗ ∗

Serum DHEA (ng ml–1)

1.5
20

No. of attacks
15 1.0

10
0.5
5

0 0
Control Melatonin Control Melatonin

(c) 1.0 Control

Long days
Melatonin-induced in vitro DHEA

b Short days
0.8 b

0.6
(ng ml–1)

d
0.4

a a
0.2
c

0
Adrenal Ovarian

Figure 5 Melatonin-induced changes in aggression and DHEA. In vivo-induced (a) number of attacks, (b) serum DHEA concentrations in melatonin
and control-treated female Siberian hamsters. (c) In vitro DHEA response to melatonin or control treatment. Data from Rendon, N.M., Rudolph, L.M.,
Sengelaub, D.R., Demas, G.E., 2015. The agonistic adrenal: melatonin elicits female aggression via regulation of adrenal androgens. Proc. R. Soc. B
282, 20152080.

weights tend to be higher in summer than winter (Yaskin,

1984). Seasonal variations in brain weight may decrease energy
Saline Estradiol expenditure (Jacobs, 1996). Although the brain constitutes
20
only 2–3% of the total body mass in rodents, it consumes
*
Offensive attacks (freq)

a somewhat higher percentage of total energy expenditure

10
0
Long day
Figure 6 Photoperiod alters nongenomic actions of estradiol on
aggressive behavior. Beach mice (Peromyscus polionotus) were housed
in long or short photoperiods for 8 weeks. One week prior to behavioral
testing, mice were injected daily with the aromatase inhibitor fadrozole,
mice were then injected with saline or cyclodextrin-conjugated estradiol
(cE2), a water-soluble, rapid-acting estrogen, and tested in resident–
intruder tests 15 min later. Data from Trainor, B.C., Lin, S., Finy, M.S.,
Rowland, M.R., Nelson, R.J., 2007. Photoperiod reverses the effects of
estrogens on male aggression via genomic and nongenomic pathways.
Proc. Natl. Acad. Sci. U.S.A. 104, 9840–9845. Copyright (2007)
National Academy of Sciences, U.S.A.
(Mink et al., 1981). It has been suggested that minor reductions
in brain mass could result in substantial energetic savings
(Jacobs, 1996). A significant part of the seasonal change in
brain weight may be attributable to differences in water
content; however, the neocortex and the basal portion of the
brains (i.e., the corpus striatum) of rodents and shrews show
seasonal cytoarchitectural changes. The relative weight of the
forebrain declines during the winter, the relative weight of
Short day the hippocampus increases from winter to summer, and the
relative weight of the olfactory bulbs, myelencephalon, and
cerebellum increases during the winter (Yaskin, 1984). A sex
difference in brain weight is observed among bank voles
(C. glareolus) only during the winter months when male brains
are heavier than those of females. The absolute and relative
weight of the hippocampus is significantly higher in males
than in females throughout the year, but the difference is
most pronounced during the winter (Yaskin, 1984). Meadow
voles also show seasonal changes in brain weight that are
controlled by photoperiod (Dark et al., 1987); males kept
under short-day conditions have lighter brains than long-day
390 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates

animals. Meadow voles born into long day lengths have greater
brain growth (i.e., more cells) than cohorts born in short days
(Dark et al., 1990). Furthermore, long days enhance myelina-
tion of both midbrain and hindbrain regions of developing
male meadow voles (Spears et al., 1990).
Photoperiodic effects on cell division in the dentate gyrus
and subependymal zone of adult mammals were noted by
Huang et al. (1998). Long-term exposure to short days in Syrian
hamsters doubled the number of new neurons produced in
these brain regions, as well as in the hypothalamus and cingu-
late/retrosplenial cortex. There were no appreciable photoperi-
odic differences in the brain volume of either the granule cell
layer of the hippocampus or the dentate gyrus. And in white-
footed mice, exposure to short days increased progenitor cell
survival in the posterior plexiform and granulae cell layers of
the olfactory bulb (Walton et al., 2012); a potential functional
consequence of this difference was demonstrated in photoperi-
odic changes in habituation to odor cues. Effects of photope-
riod on adult mammalian neurogenesis have been reported
in other species as well (e.g., sheep) and have been extensively
reviewed by Migaud et al. (2015).
Sex differences in hippocampal size were evident in male
polygamous meadow voles (Microtus pennsylvanicus), but not
in monogamous pine voles (Microtus pinetorum; Jacobs et al.,
1990). Similar sex differences, but not seasonal changes, were
also reported for eastern gray squirrels (Sciurus carolinensis;
Lavenex et al., 2000). In both cases, it was suggested that the
larger hippocampal complex in males was related to their
increased reliance on spatial memory in foraging and mainte-
nance of territories (Jacobs, 1996). These results suggest that

Figure 7 Photoperiod alters spatial learning and hippocampal activity. (a) Male white-footed mice (Peromyscus leucopus) were housed in long or
short photoperiods for 13 weeks and trained on a Morris water maze. Latency to reach the hidden platform over successive blocks of training. (b)
Impairment of long-term potentiation of fEPSPs in the Schaffer collateral–CA1 pathway of white-footed mouse hippocampal slices after 10 weeks of
exposure to short days. Upper left and middle: representative traces of fEPSPs (1) pretetanus and (2) 50 min posttetanus. Calibration bar: 0.2 mV;
10 ms. Upper right: location of stimulating (black arrow) and recording (white arrow) electrodes. Lower panel: comparison of LTP between long-day
(open box) and short-day (black box) mice. Tetanus stimulation was delivered at the time indicated by two arrows. Data from Pyter, L.M., Reader,
B.F., Nelson, R.J., 2005. Short photoperiods impair spatial learning and alter hippocampal dendritic morphology in adult male white-footed mice
(Peromyscus leucopus). J. Neurosci. 25, 4521–4526; Walton, J.C., Chen, Z., Weil, Z.M., Pyter, L.M., Travers, J.B., Nelson, R.J., 2011. Photoperiod-
mediated impairment of long-term potention and learning and memory in male white-footed mice. Neuroscience 175, 127–132.
 Mammalian Seasonal Rhythms: Behavior and Neuroendocrine Substrates
developmental effects of steroids might mediate changes in
hippocampal size. There was, however, no overall sex differ-
ence in hippocampal volume or dentate gyrus width in wild-
captured male and female meadow voles (Galea et al., 1999).
A sex difference in hippocampal volume emerged if only the
males with relatively high circulating testosterone concentra-
tions (top 50th percentile) were compared to females with rela-
tively low circulating estradiol concentrations (bottom 50th
percentile; Galea et al., 1999).
Despite the evidence for seasonal changes in brain weight in
rodents, there has been relatively little research investigating
seasonal changes in learning among mammalian species (but
see Jacobs, 1996). Voles trapped in winter make more errors
and require longer to learn mazes than do summer-captured
voles (Jacobs, 1996).
Similarly, short-day white-footed mice (Peromyscus leucopus)
require longer to learn the Morris spatial water maze (Pyter
et al., 2005; Figure 7(a)). Short days also decreased brain
mass and hippocampal volume compared to mice housed in
long days (Pyter et al., 2005). Despite impairments in spatial
learning and memory, there were no photoperiodic effects on
sensory discrimination or other types of memory. Short days
decreased apical (stratum lacunosum-moleculare) CA1 spine
density, as well as increased basilar (stratum oriens) CA3 spine
density (Pyter et al., 2005). Short days also decreased LTP in
the Schaffer collateral–CA1 pathway and impaired spatial
learning and memory in the Barnes maze (Walton et al.,
2011; Figure 7(b)).
The motoneurons of the spinal nucleus of the bulbocaver-
nosus and their target muscles, the bulbocavernosus and
levator ani are sexually dimorphic and control penile erection
in rodents (Breedlove, 1992). Short days reduce the size and
neuronal numbers in these regions in white-footed mice and
Siberian hamsters (Forger and Breedlove, 1987; Hegstrom
and Breedlove, 1999a). These neuronal changes may help
conserve energy during winter.
The interaction among sex and seasonal differences is
complex but may be important in typical development. For
example, androgenic induction of sexual dimorphism in
meadow voles proceeds under long, but not short, days (Kelly,
1993). The slight, but significant, increases in neurodevelop-
mental disorders associated with autumn conceptions may
involve subtle interactions among sex steroid hormones,
photoperiod, and brain development in humans (Liederman
and Flannery, 1994).
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 1.14 Sex Steroids, Learning and Memory
SE Nielsen and AY Herrera, University of Southern California, Los Angeles, CA, USA
Ó 2017 Elsevier Inc. All rights reserved.
This chapter is a revision of the previous edition chapter by G. Dohanich, D. Korol, T. Shors, volume 1, pp. 539–578, Ó 2009, Elsevier Inc.

1.14.1 Gonadal Steroids, Learning and Memory 400

1.14.1.1 History 400
1.14.1.2 Brief Introduction into Ovarian Hormones and the Hypothalamic–Pituitary–Gonadal Axis 400
1.14.1.3 Ovarian Hormone Cycling 401
1.14.1.3.1 Rodents and the Estrous Cycle 401
1.14.1.3.2 Humans and the Menstrual Cycle 401
1.14.1.3.3 Birth Control and the HPG Axis 401
1.14.1.3.4 Summary 402
1.14.1.4 Ovarian Steroids and Working Memory 402
1.14.1.4.1 Ovarian Hormones and Working Memory Paradigms in Animals 402
1.14.1.4.2 Ovarian Hormones and Working Memory in Humans 403
1.14.1.4.3 Ovarian Hormones and Working Memory in Postmenopausal Women 403
1.14.1.4 Summary 404
1.14.1.5 Ovarian Steroids and Reference Memory in Animals 404
1.14.1.5.1 Water and Radial Arm Mazes 404
1.14.1.6 Ovarian Steroids and Spatial Learning in Humans 406
1.14.1.6.1 Influence of Ovarian Steroids on Spatial Learning in Women 406
1.14.1.6.2 Sex Differences in Spatial Learning in Humans 406
1.14.1.7 Ovarian Steroids and Associated Learning in Animals 406
1.14.1.7.1 Avoidance Learning and Ovarian Steroids 406
1.14.1.7.2 Stress and Conditioning Paradigms 407
1.14.1.8 Summary 407
1.14.2 Mechanisms of Steroid Action 407
1.14.2.1 Cellular Mechanisms of Steroid Action 407
1.14.2.2 Effects of Ovarian Steroids on Electrical Properties 407
1.14.2.3 A Model of Estrogen Action 408
1.14.2.3.1 Estrogen Receptor Subtypes 408
1.14.2.3.2 Estrogen as a Neuroprotective Steroid 409
1.14.2.4 A Model of Progesterone Action 409
1.14.2.4.1 Progesterone and the Stress Response 409
1.14.2.4.2 Progesterone Action and Cognition 410
1.14.3 Interaction between Steroid Hormones, Learning and Memory 410
1.14.3.1 Overview 410
1.14.3.2 Glucocorticoids, Learning and Memory 410
1.14.3.2.1 Factors Influencing the Relationship between Stress and Learning 410
1.14.3.2.2 Impairing Effects of Stress on Learning and Memory in Animals 410
1.14.3.2.3 Impairing Effects of Stress on Learning and Memory in Humans 411
1.14.3.2.4 Sex Steroid Hormones and Stress Response Interactions 411
1.14.3.3 Summary 412
1.14.3.4 Steroid Hormone Interactions in Learning and Memory of Arousing Information 412
1.14.3.4.1 Ovarian Steroids, Stress and Emotional Memory in Animals 412
1.14.3.4.2 Ovarian Steroids and Emotional Memory in Humans 412
1.14.3.4.3 Sex Differences in Emotional Memory Paradigms 413
1.14.3.4.4 Shifts in Emotional Memory across the Menstrual Cycle 413
1.14.3.4.5 Hormonal Contraceptive Use and Emotional Memory 415
1.14.4 Ovarian Steroids and Clinical Implications for Human Neuropathologies 415
1.14.4.1 Overview 415
1.14.4.2 Gonadal Steroids and AD 415
1.14.4.3 Gonadal Steroids and Disorders of Emotional Memory 416
References 416

Hormones, Brain, and Behavior, 3rd edition, Volume 1 http://dx.doi.org/10.1016/B978-0-12-803592-4.00014-6 399

400 Sex Steroids, Learning and Memory
Glossary
Alzheimer’s disease The most common form of dementia
that leads to issues with memory, cognition, and behavior.
The disease worsens over time, and although the greatest
risk factor is increasing age, up to 5% of people with the
disease have early onset Alzheimer’s.
Amygdala Almond-shaped structure located in the medial
temporal lobe of the brain and has been shown to be critical
for processing emotions, fear responses, and pleasure.
Emotional memory Storage and recall of events and details
that are coupled with physiological responses present when
the event occurred.
1.14.1 Gonadal Steroids, Learning and Memory
1.14.1.1 History
Evidence collected over the past 25 years has shown there are
profound sex differences in learning and memory. Research
with both animals and humans indicates that these sex
differences are mediated in large part by ovarian steroid
hormones, namely estradiol and progesterone. Work demon-
strating these effects of sex steroids on learning and memory
was largely prompted by reports showing that estradiol
promoted neurogenesis in the dentate gyrus of the hippo-
campus and subventricular zone (Fowler et al., 2008). In
these early studies, the highest number of new cells in the
dentate gyrus of female rats was identified during proestrus,
when there is a significant surge in estradiol levels (Tanapat
et al., 1999). Moreover, ovariectomy decreases neurogenesis
while estradiol replacement restores it (Tanapat et al.,
1999). Similarly, when female meadow voles receive acute
treatment with estradiol, they also show a transient increase
in the number of new neurons in the dentate gyrus
(Ormerod and Galea, 2001), suggesting that estradiol may
facilitate neurogenesis. These early studies of hormones
and neurogenesis promoted future research on the relation-
ship between ovarian hormones and learning and memory
processes in both animal and human models. Additionally,
the early ovarian hormone work encouraged the exploration
of sex differences in the underlying neurobiology of learning
and memory, particularly with regard to brain structures such
as the amygdala and hippocampus (for review, see Andreano
and Cahill, 2009).
Research has also focused on how ovarian hormones may
play a role in the onset of different neurological disorders,
such as Alzheimer’s disease (AD), a common type of dementia
in older adults (Paganini-Hill and Henderson, 1994) and
disorders of emotional memory, such as anxiety, depression,
and posttraumatic stress disorder (PTSD). For example, early
studies in the 1990s provided evidence that hormone replace-
ment therapy (HT) in older women may offset the risk of devel-
oping AD (Paganini-Hill and Henderson, 1994). However,
follow-up studies have had mixed findings, indicating that
more work is still needed for researchers to truly elucidate the
relationship between ovarian hormones and neurological
disorders.
Hippocampus Located in the medial temporal lobe of the
brain and plays an important role in consolidation of
information from short-term to long-term memory.
Hormonal contraception A form of birth control
composed of synthetic steroid hormones that acts on the
endocrine system.
Reference memory Knowledge for aspects of a task,
typically spatial, that remain constant between trials.
Working memory Short-term memory that allows for
keeping information ‘online’ and readily available for
access.
To better understand how ovarian hormones might affect
the onset and/or treatment of neurological disorders, many
learning and memory researchers have focused their work on
how these hormones might alter the underlying neurobiology
of different memory processes. The multiple memory systems
approach (Sherry and Schacter, 1987) accounts for differential
effects of ovarian hormones on certain forms of memory,
including working memory, reference memory, and even
emotional memory. As we explore here, ovarian hormones
sometimes have impairing or enhancing effects on learning
and memory, or even no effect at all, depending on the type
of memory being tested.
In this chapter, we will explore the physiological mecha-
nisms underlying steroid hormone production and how steroid
hormones influence different types of learning and memory in
both animals and humans. We will also provide information
about how hormone manipulations, such as hormonal contra-
ception in humans, can alter learning and memory functions in
adults. Finally, we will address the implications of steroid
hormone effects on learning and memory in the clinical setting.
1.14.1.2 Brief Introduction into Ovarian Hormones and the
Hypothalamic–Pituitary–Gonadal Axis
The hypothalamic–pituitary–gonadal (HPG) axis is primarily
responsible for regulating reproductive activity and the release
of ovarian hormones in animals and humans (Couse et al.,
2003; Meethal and Atwood, 2005). In this way, the HPG axis
also plays a key role in promoting healthy brain function, as
ovarian hormones exert neuroprotective effects and facilitate
neurogenesis, neuronal differentiation and survival, and cogni-
tive function (Blair et al., 2015).
The HPG axis is responsible for orchestrating the release
of both centrally and peripherally produced ovarian
hormones. Centrally produced regulatory hormones include
gonadotropin-releasing hormone (GnRH) from the hypothal-
amus and gonadotropins from the pituitary, specifically lutei-
nizing hormone (LH) and follicle-stimulating hormone
(FSH) (Meethal and Atwood, 2005). Recently, researchers iden-
tified a central regulator of GnRH; kisspeptin was identified as
a neuromodulator that modulates GnRH release and subse-
quently, controls activity of the gonadotropins and the HPG
axis (Skorupskaite et al., 2014). The other gonadotropins,

LH and FSH, are instrumental in modulating behavioral and
neuronal changes (Meethal and Atwood, 2005).
Receptors for estradiol, progesterone, and testosterone are
widely distributed throughout the brain and are highly concen-
trated in regions outside of the hypothalamus and pituitary,
such as the amygdala and cerebral cortex (Meethal and
Atwood, 2005). Additionally, estradiol is locally generated
within the hippocampus (Mukai et al., 2006), and levels of
estradiol in this region can fluctuate based on circulating levels
(Barker and Galea, 2009). Since estradiol is generated in the
hippocampus, it not only has an effect on the negative-
feedback loop regulating HPG axis activity, but estradiol, as
well as progesterone and testosterone, can also have effects
on different neuronal pathways (McEwen, 2002). In addition
to ovarian hormones, there are peripherally produced protein
complexes that are responsible for regulating the activity of
the HPG axis. For example, activins and inhibins are dimeric
paracrine protein complexes with opposing physiological
effects on FSH secretion; activins are produced in all tissues,
whereas inhibins are generated in the gonads (Meethal and
Atwood, 2005). Activins are critical for stimulating FSH secre-
tion, activating the HPG axis, and facilitating neuronal plas-
ticity, whereas inhibins are responsible for inhibiting FSH
secretion and inactivating and modulating the effects of acti-
vins. A third protein complex, follistatin, also works with
inhibin to regulate the activin activity. Together, these protein
complexes help regulate the onset and offset of HPG axis
activity (Meethal and Atwood, 2005) in coordination with
circulating levels of the ovarian hormones.
1.14.1.3 Ovarian Hormone Cycling
Given that this chapter will focus on the influences of ovarian
hormones on learning and memory in rodents and humans,
only the ovarian hormone cycling in these species will be dis-
cussed. The following section addresses ovarian hormone
cycling in females of both the rodent and human species.
1.14.1.3.1 Rodents and the Estrous Cycle
In rodents, there are four phases in the estrous cycle, and the
average cycle length is 4–5 days. In metestrus (day 1), proges-
terone and estradiol circulate at relatively low levels. The
following day (day 2), diestrus ensues, and early on in this
postovulation phase, progesterone increases sharply and then
drops quickly by the end of day (Butcher et al., 1974). At

1200 h on day 3, proestrus is initiated by a significant surge
in estradiol levels. This marked increase in estradiol causes
a rapid peak of LH and FSH between 1600 and 1800 h as
well as an increased progesterone secretion. Finally, in estrus,
ovulation occurs and all hormones return to baseline, save
for a temporary peak in estradiol in the evening of estrus on
day 4 (Butcher et al., 1974).
1.14.1.3.2 Humans and the Menstrual Cycle
The average menstrual cycle in women lasts 28 days, though it
can vary between 23 and 38 days depending on the individual.
Most women have monthly menstrual cycling starting at
menarche around the age of 11–13 years old until the onset
of menopause (Reilly, 2000). Usually, the menstrual cycle
consists of three phases: the follicular phase, ovulation, and
Sex Steroids, Learning and Memory 401
the luteal phase. These phases can be further subdivided into
early follicular, late follicular and midluteal based on notable
changes in the hormonal milieu.
The onset of menstruation marks the first day of the cycle
and this period lasts 4–5 days on average. The menstrual cycle
begins with the follicular phase, which lasts from the first day
of menstruation until ovulation (days 1–13) (Reilly, 2000).
Throughout menstruation, estradiol, progesterone, and LH
circulate at relatively low levels while FSH levels are slightly
elevated relative to baseline in order to stimulate the develop-
ment of follicles. After menstruation, the generated follicles
begin to secrete estradiol, gradually increasing the levels of
this hormone during the early follicular phase (days 1–6).
Simultaneously, one follicle develops into the dominant
follicle destined for ovulation; this follicle typically has
a high concentration of FSH receptors.
In the latter half of the follicular phase (days 7–13),
increasing levels of estradiol facilitate a gradual decrease of
FSH levels as estradiol inhibits FSH production. Simulta-
neously, the dominant follicle outcompetes other follicles for
the remaining FSH, thereby maturing and secreting increasing
amounts of estradiol. Between days 10 and 13, there is
a preovulatory surge of estradiol. Concurrently, the pituitary
initiates positive feedback, resulting in a preovulatory surge
of FSH and LH and subsequently, ovulation. Ovulation usually
occurs on day 14 of the menstrual cycle, and at this point, the
mature dominant follicle forces out the oocyte into the perito-
neal cavity. During ovulation, LH, FSH, and estradiol decrease
to levels slightly above those observed during menstruation,
and FSH and LH circulate at low levels for the remainder of
the menstrual cycle.
The second half of the menstrual cycle is known as the luteal
phase (days 15–28), which is initiated by the LH surge and is
characterized by the luteinization of follicle elements that
were not ovulated. Different cells and tissues, namely the gran-
ulosa cells, theca cells, and connective tissue, are transformed
into the corpus luteum, which is responsible for secreting
progesterone and estradiol. Progesterone and estradiol secre-
tion by the corpus luteum typically peaks between days 22
and 24 of the menstrual cycle. Notably, progesterone levels
in the menstrual cycle reach their highest peak during this mid-
luteal phase. Estradiol levels, while elevated, are lower than the
levels observed during the preovulatory surge. At day 24 of the
cycle, the corpus luteum starts to evolve into the corpus albi-
cans and no longer secretes estradiol and progesterone. The
decrease in estradiol and progesterone levels facilitates an
increase in FSH levels and subsequently, the initiation of the
next menstrual cycle.
1.14.1.3.3 Birth Control and the HPG Axis
The menstrual cycle can be manipulated and regulated by
synthetic analogs of estradiol and progesterone. To better
understand how hormone manipulations such as hormonal
contraception can alter learning and memory processes in
women, it is important to know how hormonal contraception
affects HPG axis activity. These synthetic hormone formula-
tions have become a widely used and well-known form of
reversible contraception for women. Since its introduction in
1960, hormonal contraception has undergone many changes
to its synthetic hormone elements and dosage as well as the
402 Sex Steroids, Learning and Memory
method of administration. In spite of these alterations,
however, the original utility of the drugs has been preserved.
The most common method of hormonal contraception is
known as a combination formulation; here, ethinyl estradiol
and a synthetic progestin work synergistically to prevent ovula-
tion by inhibiting different components of the HPG axis (Lobo
and Stanczyk, 1994). Although interference at either the hypo-
thalamus or the pituitary is thought to be sufficient to prevent
ovulation (Lobo and Stanczyk, 1994; Rapkin et al., 2006),
combined contraceptive formulations target both regions to
ensure maximum effectiveness. It is well established that
ethinyl estradiol/progestin contraceptives utilize negative feed-
back to the hypothalamus to inhibit the midcycle GnRH surge
(Lobo and Stanczyk, 1994). In addition, these synthetic
hormones suppress the release of LH and FSH from the pitui-
tary to inhibit the gonadal production of endogenous estrogen
and progesterone. Thus, hormonal contraception significantly
suppresses endogenous sex hormone levels in women, and
this hormone suppression has been shown to be involved in
different forms of learning and memory processes in women
(Warren et al., 2014).
1.14.1.3.4 Summary
The HPG axis is responsible for the production of ovarian and
testicular hormones. Additionally, these steroids are locally
produced in brain regions that are critical for learning and
memory, such as the hippocampus. Circulating levels of
ovarian hormone levels fluctuate throughout ovarian monthly
cycling in female rodents and humans, and importantly, these
levels can be further modified in humans by the use of
hormonal contraceptives. In induced ovulators, that do not
have an estrous cycle (e.g., rabbits, musk shrews), ovulation
is induced by mating-related sensory stimulation (Bakker and
Baum, 2000).
(a) 6.6
6.4
Correct choices in first 8 visits
Correct choices in first 8 visits
6.2 *
6.0
5.8
5.6
E CH
Figure 1 Effect of estradiol administered prior to and during training on a radial arm maze. Ovariectomized rats were implanted with 5-mm silastic
capsules containing 25% estradiol (E, n ¼ 14) or 100% cholesterol (CH, n ¼ 16) for 30 days prior to and continuing throughout 24 days of maze
training. (a) Mean number of correct arm choices in first eight visits (SEM) averaged over 24 days of training (*p < 0.01; ANOVA). (b) Mean
number of correct arm choices in the first eight visits (SEM) presented as 4-day blocks over 24 days of training. Reproduced from Daniel, J.M.,
Fader, A.J., Spencer, A.L., Dohanich, G.P., 1997. Estrogen enhances performance of female rats during acquisition of a radial arm maze. Horm.
Behav. 32, 215–225, with permission from Elsevier.
1.14.1.4 Ovarian Steroids and Working Memory
Working memory is considered a critical cognitive function
(Janowsky et al., 2000) and is classified as a form of short-
term memory in which information is stored for the period
of time that it remains useful and relevant. Retention intervals
for working memory experiments vary from seconds to hours,
depending on the parameters of the task as defined by the
experimenter; working memory load can be further intensified
by increasing the quantity or complexity of information. This
section will cover how steroid hormones modulate working
memory in rodents and humans.
1.14.1.4.1 Ovarian Hormones and Working Memory Paradigms
in Animals
1.14.1.4.1.1 Land Mazes in Animals
In animals, estradiol has been shown to facilitate learning and
memory processes. For example, if ovariectomized (OVX)
mammals were treated with estradiol, they made fewer working
memory errors during a radial arm maze than OVX-females not
treated with estradiol (Daniel et al., 1997; Bimonte and
Denenberg, 1999; Fader et al., 1999). Likewise, females
receiving only a sham surgery exhibited a greater number of
consecutive correct choices on a radial arm maze, compared
with OVX-alone females (Wilson et al., 1999).
17b-estradiol also modulates working memory perfor-
mance on radial arm mazes, even when maintained at low
levels. For example, subcutaneous implantation of continuous
release 17b-estradiol capsules decreased errors during the
acquisition of the radial arm maze in OVX-females (Daniel
et al., 1997; Luine et al., 1998). Please refer to Figure 1.
This improvement in working memory performance was
also observed in a food-reinforced T-maze; OVX-females
systemically treated with estradiol benzoate over days or
(b) 7.4
7.2
7.0
6.8 Estradiol
6.6
6.4
6.2
6.0
5.8 Cholesterol
5.6
1 2 3 4 5 6
4-day blocks

weeks improved performance on a nonmatching-to-position
maze (Fader et al., 1998), and those treated with
17b-estradiol improved on a matching-to-position task
(Gibbs et al., 1998).
1.14.1.4.1.2 Water Mazes in Animals
Benefits of estradiol also have been observed in other tests of
working memory, such as water mazes. In a delayed match-
to-place task, ovariectomized animals treated with estradiol
benzoate displayed better retention for the location of a hidden
platform compared to OVX-alone females; this performance
enhancement was stable across 10-, 30-, and 100-min delay
intervals (Sandstrom and Williams, 2001). Likewise,
estradiol-treated animals also required less time to reach
learning criteria of a delayed-match-to-position task than
OVX-alone animals (Gibbs, 1999).
1.14.1.4.1.3 Object Recognition and Placement Tasks in Animals
The same pattern of estrogens improving working memory has
also been observed in an object recognition task; here, animals
were exposed to a total of four novel objects. They were first
allowed to explore a pair of two novel objects, and after
some delay they were exposed to another two objects (one
object from pair #1 and the third novel object); after a second
delay period the animals were exposed to two additional
objects (one being the second object from pair #1 and the
fourth novel object). Correct recognition was scored when
the animal spent less time exploring the familiar object
compared with time spent exploring the novel object. Ovariec-
tomy followed by 21 days of estradiol treatment enhanced
retention for the first-tested object after a 3-h delay and of
the second-tested object after a 6-h delay, compared with
nonestradiol-treated mice (Vaucher et al., 2002). These data
suggest that in female animals, estradiol enhances working
memory performance on a variety of different paradigms.
1.14.1.4.2 Ovarian Hormones and Working Memory in Humans
1.14.1.4.2.1 Young Adults
Research on working memory in humans largely supports the
findings from the animal literature. Several studies have found
a positive relationship between estradiol and working memory
in young, healthy women. One study tested how performance
on a verbal span task varied over the course of the menstrual
cycle. Naturally cycling women were tested four times
throughout the menstrual cycle, and researchers found that
performance was enhanced at days 7 and 14 (high-estradiol
periods in a regular cycle) (Rosenberg and Park, 2002). Another
study indicated that women tested at menstruation (low estra-
diol period) made significantly more working memory errors
on the test of spatial working memory than age- and
education-matched women tested at other phases of the cycle
(Hampson and Moffat, 2004). These studies suggest that estra-
diol has a positive effect on working memory processes;
however, each of these studies relied on self-report and failed
to measure hormone levels in the women (Rosenberg and
Park, 2002; Hampson and Moffat, 2004).
Later studies focused on assessing the correlational relation-
ship between hormone levels and working memory perfor-
mance and women. For example, when women were treated
with a GnRH analog for 4 weeks, estradiol levels were reduced
Sex Steroids, Learning and Memory 403
to those of an older woman postmenopause (Grigorova et al.,
2006), and working memory performance also decreased
significantly as estradiol decreased.
Other studies explored the effect of endogenous hormone
levels on working memory in young women. In a working
memory task with visual stimuli (i.e., faces), women performed
better on the task during high-estradiol (late follicular and mid-
luteal) compared to the low-estradiol (early follicular) phases
(Vranic and Hromatko, 2008). However, this study was unable
to determine a direct effect of estradiol since the high-estradiol
group was comprised of two cycle phases with different
hormone profiles. In particular, the high-estradiol group con-
sisted of the late follicular phase, where progesterone levels
are still very low but estradiol peaks to initiate ovulation, and
the midluteal phase, where estradiol is moderately high but
progesterone is also reaching its peak levels. A more recent
study in naturally cycling women focused explicitly on estra-
diol; when salivary estradiol levels were elevated, women
committed fewer working memory errors on a spatial working
memory task compared to when their estradiol levels were low
(Hampson and Morley, 2013). Additionally, estradiol nega-
tively correlated with a number of working memory errors.
Although most studies have focused on the relationship
between estradiol and working memory, some studies have
explored how progesterone may affect working memory perfor-
mance in women. These studies indicate that progesterone may
have a negative effect on working memory performance in
women (Man et al., 1999; Schmitt et al., 2005) given that
performance on working memory and short-term memory
tasks were worse in the midluteal relative to the follicular
phase. However, effects of progesterone on working memory
in women remain relatively understudied and unclear at this
point.
In summary, progesterone and estradiol both appear to
modulate working memory performance in young women;
however, they may have opposing effects. A number of studies
support a positive influence of estradiol on working memory,
but more work is needed to determine the true role of proges-
terone. As research continues in this domain, researchers
should focus on how hormone levels and activity in different
brain regions relate to different kinds of working memory in
women.
1.14.1.4.3 Ovarian Hormones and Working Memory
in Postmenopausal Women
1.14.1.4.3.1 Endogenous Hormones
Few studies have examined the cognitive effects of naturally
declining estradiol levels during menopause in the absence of
hormone replacement. One study showed that postmeno-
pausal women performed significantly worse than pre- and
perimenopausal women on delayed verbal memory tasks;
additionally, these postmenopausal women performed signifi-
cantly worse than perimenopausal women on phonemic verbal
fluency tasks (Weber et al., 2013).
Another study showed no differences between women
within 5 years of menopause and beyond 5 years of meno-
pause on measures of sustained attention, category generation,
or episodic memory. However, higher estradiol levels in
women within 5 years of menopause were associated with
better performance in measures of mental flexibility and
404 Sex Steroids, Learning and Memory
planning (i.e., executive function) compared with the steady
low-estradiol levels in the women beyond 5 years of meno-
pause (Elsabagh et al., 2007; refer to Figure 2), an effect main-
tained after controlling for age and IQ of women within and
beyond 5 years of menopause. Endogenous levels of estradiol
in postmenopausal women not taking hormone supplements
also were positively correlated with performance on the
Stroop task, another measure of executive function (Wolf and
Kirschbaum, 2002).
1.14.1.4.3.2 Exogenous Hormones and the Women’s Health
Initiative Studies
Postmenopausal women exhibit improved working memory
performance with exogenous estradiol treatment. For example,
when women received 2 weeks of transdermal estradiol treat-
ment, those experiencing higher endogenous estradiol levels
in response to the dosage performed better on the delayed
recall portion of a verbal memory test than women experi-
encing lower dose-induced endogenous estradiol levels (Wolf
et al., 1999). Additionally, women randomly assigned to trans-
dermal hormone replacement experienced improved perfor-
mance on the Stroop task, spatial working memory (Krug
et al., 2006; Baker et al., 2012), digit ordering (Krug et al.,
2006), and delayed recall (Baker et al., 2012). Similarly,
women already using hormone replacement also made fewer
errors on nonspatial working memory (sequential memory of
an unfamiliar story, digit ordering, and digit span backward)
and spatial working memory tasks (Duff and Hampson,
2000), performed better on measures of verbal fluency and
working memory (Miller et al., 2002), and displayed better
performance on a nonverbal memory task (Smith et al.,
2001) compared with women not taking any hormone
supplements.
However, others report no effect (Maki et al., 2001; Smith
and Zubieta, 2001; Yaffe et al., 2006; Pefanco et al., 2007) or
negative effects of estradiol replacement (Mulnard et al.,
2000; Rapp et al., 2003; Shumaker et al., 2003; Espeland
et al., 2004; Resnick et al., 2006). The most widely publicized
negative findings come from the Women’s Health Initiative
(WHI) studies. At the time of inception, the Women’s Health
Initiative Memory Study (WHIMS) and Women’s Health Initia-
tive Study of Cognitive Aging (WHISCA) were the largest
randomized placebo-controlled trials testing claims that
hormone replacement after menopause maintained cognitive
function otherwise observed to decline after menopause.
However, the WHIMS and WHISCA studies found that conju-
gated equine estrogens, a form of synthetic estrogen used in
some HTs, negatively affected performance on various cogni-
tive measures, including the California Verbal Learning Test
(test of verbal retention and recall; Resnick et al., 2006) and
the Modified Mini Mental State Exam (a measure of global
cognitive function; Rapp et al., 2003; Espeland et al., 2004),
as well as increased risk of developing dementia (Shumaker
et al., 2003).
In order to reconcile these reports of estradiol-induced
cognitive impairment and basic science reports of estradiol-
related protection of the central nervous system (Pike, 1999;
Brinton et al., 2000; Hosoda et al., 2001; Chen et al., 2006),
some have proposed a critical window for estradiol treatment
initiation (for review, see Resnick and Henderson, 2002;
Maki, 2013). In line with the critical window hypothesis,
Brinton (2005) proposed the healthy cell bias of estradiol
action as a mechanism for why such disparities exist in the
research canon (Brinton, 2005). Work in the Brinton lab sup-
ported the healthy cell bias theory, showing that introduction
of estradiol after 5 days of beta-amyloid exposure exacerbated
beta-amyloid-induced damage, but estradiol pretreatment
before beta-amyloid exposure protected the cells from damage
and death (Chen et al., 2006).
1.14.1.4 Summary
Estradiol treatment after menopause appears to facilitate cogni-
tion, although certain limitations exist. The primary of these
limitations appears to be the timing of treatment initiation.
When considering this point, treatment should be initiated as
close to the final menses as possible and the patient should
be in good health prior to beginning any treatment regimen.
1.14.1.5 Ovarian Steroids and Reference Memory in Animals
Reference memory refers to knowledge for aspects of a task that
remain constant between trials. Rodent researchers often test
reference memory in paradigms similar to those used to test
working memory (i.e., radial arm maze and Morris water
maze), but reference memory is distinguishable by its memory
consolidation processes. It is difficult to map reference memory
processes onto humans; however, it appears to resemble
knowledge that is consistent across episodes rather than
specific to one event (Nadel and Hardt, 2011). Thus, the
following section will focus on how ovarian hormones modu-
late reference memory specifically in rodents.
1.14.1.5.1 Water and Radial Arm Mazes
Reference memory is typically assessed using either water or
radial arm maze. For the radial arm maze, studies will utilize
eight arms, four of which are baited on all trials while the other
four arms are never baited (Hodges, 1996). When rodents enter
a maze arm that is never baited, this is considered a reference
memory error (RME). In the case of the water maze, rodents
learn that the submerged platform which represents an escape
is always in the same location (Hodges, 1996); here, an RME
occurs when a rodent does not acquire the escape response.
Studies have found that ovarian hormones modulate refer-
ence memory differently at acquisition, consolidation, and
retrieval. In the case of acquisition, high levels of estradiol
appear to be impairing. Studies have shown that elevated levels
of estradiol impair acquisition of the escape response in the
Morris water maze (Galea et al., 1994, 1995, 2000; Frye,
1995; Galea et al., 1995; Warren and Juraska, 1997). Proges-
terone also seems to have an impairing effect on acquisition
as the combination of estrogen and progesterone replacement
in OVX-females impaired acquisition of the place and cued
version of the Morris water maze (Chesler and Juraska, 2000).
Although gonadal hormones appear to have detrimental
effects on acquisition in reference memory, there are mixed
findings in how they affect consolidation and retrieval.
For example, researchers found that posttraining injections
of estrogen enhanced the consolidation and retention of
the Morris water maze (Packard and Teather, 1997a,b).
 Sex Steroids, Learning and Memory 405

Figure 2 Executive function (scores are mean  SEM) in early and late postmenopausal stages. (a) In the IDED task: stages completed; extra-
dimensional shift (EDS) errors; trials to criterion; errors to criterion. (b) In the stockings of Cambridge planning task: number of moves; subse-
quent thinking time (ms). ***p < 0.001, **p < 0.01, *p < 0.05 significant difference between postmenopausal stages. Reproduced from
Elsabagh, S., Hartley, D.E., File, S.E., 2007. Cognitive function in late versus early postmenopause stage. Maturitas 56, 84–93, with permission
from Elsevier.
406 Sex Steroids, Learning and Memory
Other studies have found the complete opposite with both
endogenous and exogenous sex steroid hormones. For
example, intact rats exhibited worse performance on a spatial
reference memory version of the Morris water maze during
proestrus (high hormone) compared to estrus (low hormone)
(Warren and Juraska, 1997) or simply when levels of ovarian
hormones were high (Markham et al., 2002). Other work using
exogenous estradiol treatment found that estradiol-treated
females made significantly more RMEs on a radial arm maze
compared to the vehicle-treated animals (Galea et al., 2001).
Thus, although there is some evidence to support enhancing
effects of ovarian hormones on reference memory perfor-
mance, most of the findings indicate that estradiol, and likely
progesterone, has impairing effects on acquisition and retrieval
of reference memory.
Biological sex can also be a mediating factor on reference
memory performance. Across a number of studies, male
rodents are far superior to females in acquiring the reference
memory component of the water-escape radial arm maze
(Gresack and Frick, 2003). Males tended to make fewer initial
RMEs and repeated RMEs compared to females (Williams
et al., 1990; Williams and Meck, 1991; LaBuda et al., 2002).
This sex difference in reference memory performance is likely
linked to elevated levels of estradiol and progesterone in female
rodents compared with males.
1.14.1.6 Ovarian Steroids and Spatial Learning in Humans
Spatial learning and memory pertains to the ability to encode
and subsequently recall the spatial layout of a particular envi-
ronment (Postma et al., 1999). As with other memory systems,
the hippocampus is a critical brain region for the encoding and
subsequent storage of relevant spatial information, evidenced
by the structure’s role in the construction of cognitive maps
of space (O’keefe and Nadel, 1978). Given that this region is
modulated by ovarian hormones, the following section will
review the influence of ovarian hormones on the processing
of spatial information and sex differences in spatial learning.
1.14.1.6.1 Influence of Ovarian Steroids on Spatial Learning in
Women
Although some studies have found no relation between
menstrual cycle phase in women and performance on spatial
tasks (Gordon et al., 1986; Ho et al., 1986; McBurney et al.,
1997), some studies have found a relationship between cycle
phase and spatial abilities that may be estradiol-mediated
(Silverman and Phillips, 1993). For example, on a spatial test
battery (Rod and Frame, Space Relations, Hidden Figures),
Hampson (1990a) found that women in the menstrual phase
of the cycle (low estradiol and progesterone) performed better
than women in the midluteal phase (higher estradiol and
progesterone). A within-subjects study using this paradigm
found similar effects of phase in that women performed best
on the spatial tasks when they were in their menstrual phase
compared to the preovulatory phase (high estradiol and low
progesterone) (Hampson, 1990b). Interestingly, women on
oral contraceptives did not differ in their performance on the
tasks from menstrual women. Subsequent studies with
different spatial tests found similar effects of estrogen;
menstrual women with lower levels of estradiol performed
best (Hampson and Kimura, 1988), particularly when
compared to midluteal women (Phillips and Silverman, 1997).
1.14.1.6.2 Sex Differences in Spatial Learning in Humans
The impairing effect of estradiol on spatial learning in humans
may account for the prevalent sex differences in this type of
learning. Early on, studies used two-dimensional maps to
assess spatial learning differences between men and women.
One study used a two-dimensional map of a fictional town
and found that males required fewer trials and made fewer
errors when learning a new route compared to females;
researchers also noted that men retained more information
about Euclidean geometric properties of the map, while
females recalled more landmarks (Galea and Kimura, 1993).
Other work supported these findings by showing a significant
male advantage for spatial route learning through an unfa-
miliar environment (Moffat et al., 1998). In this study, males
learned a novel route through a simulated maze more quickly
and accurately than females (Moffat et al., 1998).
Subsequent studies expanded upon this work by using
virtual mazes. Some studies used a virtual water maze to assess
sex differences in spatial learning. In these tests, men navigate
to a hidden platform better than females (Astur et al., 1998,
2004), and when availability of landmarks and room geometry
were manipulated, men were able to use either landmarks or
room geometry to find the platform, but women were impaired
when landmarks were removed (Sandstrom et al., 1998). Other
work supported this notion that women rely much more
heavily on landmarks to navigate through a maze compared
to men (Andersen et al., 2012). Additionally, men tend to
explore more space initially in the virtual water maze compared
to females, whereas females spend a greater amount of time
fixating on one location (Mueller et al., 2008).
These data suggest that men utilize multiple strategies to
navigate new spaces and acquire knowledge about their space
more quickly than women. Sex differences in spatial learning
may be related to differences in estradiol, and possibly proges-
terone and testosterone, levels at the time of learning. Given
that women perform better on spatial tasks when their ovarian
hormone levels are lower, it may be the case that low levels of
circulating estradiol and progesterone in males facilitate the
encoding and memory of spatial information.
1.14.1.7 Ovarian Steroids and Associated Learning
in Animals
The following section will address ovarian hormone effects on
avoidance learning and how these hormones interact with
stress to alter learning processes in animals. Specifically, we
will focus on classical conditioning paradigms.
1.14.1.7.1 Avoidance Learning and Ovarian Steroids
There are two types of tasks that assess avoidance learning. In
active avoidance tasks, rodents learn over multiple trials to
avoid a mild presignaled shock by actively moving into a safe
location within the testing space. In a test of two-way active
avoidance, intact female rats in proestrus showed facilitation
of avoidance acquisition compared to OVX-females (Sfikakis
et al., 1978); compared to OVX-alone females, estradiol
benzoate-treated OVX-females showed the same facilitation,

particularly after long-term, chronic estradiol-benzoate
treatment (Singh et al., 1994). Interestingly, however, when
OVX-females received acute estradiol benzoate treatment,
they showed impaired acquisition of two-way avoidance
behavior compared to untreated OVX-females (Diaz-Veliz
et al., 1989). These data suggest that long-term exposure to
estrogens facilitates active avoidance learning, but acute
exposure to the hormone may have an impairing effect or no
effect on acquisition at all (Farr et al., 1995).
Estrogen is not the only ovarian hormone that modulates
active avoidance learning. Progesterone may also play a critical
role in this type of learning. For example, Farr et al. (1995)
showed that mice in estrus, with elevated levels of proges-
terone, needed more trials to reach acquisition and retention
criteria on a T-maze avoidance task compared to diestrous
mice, when progesterone increases sharply and then drops
quickly; similar impairing effects of exogenous progesterone
were observed in OVX-female mice (Farr et al., 1995).
Other studies of ovarian hormone effects on avoidance
learning focus on passive avoidance, where rodents learn to
avoid a mild shock in a particular location in one training trial.
Research indicates that elevated levels of ovarian hormones,
particularly estrogen, may have an impairing effect on passive
avoidance learning. For example, in intact female rats, passive
avoidance was inhibited during proestrus, when there is a surge
in estradiol levels; this phenomenon was also observed in
OVX-females treated with 17b-estradiol and/or progesterone
(Rivas-Arancibia and Vazquez-Pereyra, 1994; Mora et al.,
1996; Foster et al., 2003).
1.14.1.7.2 Stress and Conditioning Paradigms
In the absence of stress, ovarian hormones can alter perfor-
mance on a hippocampal-dependent learning task. For
example, when OVX-females are trained on an eye-blink
conditioning task, acute exposure can enhance performance
(Leuner et al., 2004). Furthermore, results indicated that
removal of estrogen does not necessarily prevent female
rodents from acquiring trace memory or extinguishing
a learned association, rather the exposure to highly elevated
levels of estradiol actually enhance trace conditioning, and
subsequently, memory performance (Leuner et al., 2004;
Jasnow et al., 2006).
Ovarian hormones also interact with stress to further modu-
late performance on conditioning tasks. In the case of fear
conditioning, estradiol benzoate-treated mice froze signifi-
cantly more to a conditioned tone 24 h after training compared
to vehicle-treated mice (Morgan and Pfaff, 2001). Other work
showed that when intact female rats were trained and tested
in proestrus, when estradiol levels are surging, they acquired
the classically conditioned eye-blink response faster than
females in other stages of the estrous cycle (Shors et al.,
1998), and the most prominent sex differences in the acquisi-
tion of the eye-blink conditioning response were observed
when female rats commenced training in proestrus (Wood
and Shors, 1998; Shors, 2001; Hodes and Shors, 2005;
Bangasser and Shors, 2007). Figure 3 illustrates this point.
Thus, estrogen appears to facilitate both fear and conditioning
response learning.
Exposure to external stressors also changes how condi-
tioned responses are acquired in male and female rats. For
Sex Steroids, Learning and Memory 407
example, exposure to stress facilitated the acquisition of
a conditioned response in males, but impaired acquisition in
females (Wood and Shors, 1998). Females’ performance under
stress was further affected by ovariectomy. When sham-OVX-
females were exposed to stress, they exhibited a significant
impairment in their conditioning relative to unstressed control
animals; this impairment was not observed in stressed OVX
animals (Wood and Shors, 1998). Thus, these data suggest
that estrogen mediates the stress-induced impairment observed
in female acquisition.
1.14.1.8 Summary
Ovarian hormones exert a robust influence on cognitive
processes in female mammals and may account for sex differ-
ences observed in spatial and associative learning although
the extent and direction of such influence appears to depend
on the duration of hormone exposure and the cognitive para-
digm used.
1.14.2 Mechanisms of Steroid Action
1.14.2.1 Cellular Mechanisms of Steroid Action
Neuronal plasticity and synaptic remodeling appear to be inte-
gral to learning and memory processes (Garcia-Segura et al.,
1994), and recent theories of memory formation are often
based on the concept of synaptic plasticity, particularly in the
hippocampus. Ovarian hormones play a key role in modu-
lating synaptic plasticity in the hippocampus, particularly the
density and number of synaptic inputs to the hippocampal
pyramidal cells (Woolley, 1998). The following section will
review the effects of ovarian hormones on cellular mechanisms
underlying learning and memory processes, specifically electri-
cal properties within the hippocampus and estrogen and
progesterone effects at the receptor level.
1.14.2.2 Effects of Ovarian Steroids on Electrical Properties
Given that ovarian hormones modulate synaptic plasticity in
the hippocampus (e.g., Woolley, 1998), a subset of research
has focused on how estrogens alter the morphology of neurons.
For example, research indicates that estradiol-induced changes
in the density of dendritic spines occur without changes in
overall dendritic length or the number of dendritic branches
(Woolley and McEwen, 1994), suggesting that the hormone-
induced alterations in spine density are due to changes in the
number of spines rather than an expansion and shrinkage of
the dendritic tree (Woolley and McEwen, 1994). Studies with
fetal primary hippocampal neurons exposed to conjugated-
equine estrogens (Brinton et al., 2000) or estradiol (Chen
et al., 2006) showed similar effects of estrogens on neurites;
here, studies found that both forms of estrogen increased the
number and length of neurites, as well as secondary branching
and branch length when compared to estrogen-free cultures.
Other studies tested how exogenous estradiol in OVX-female
rats modified dendritic spines. Gould et al. (1990) found that
6 days after female rats were ovariectomized, there was
a significant decrease in the density of dendritic spines on
lateral dendritic branches on CA1 pyramidal cells; however,
408 Sex Steroids, Learning and Memory

Figure 3 Effects of stress of trace eyeblink conditioning before, during, and after puberty. For (a)–(c), the bars represent the mean (SEM)
percentage of conditioned responses (CRs) across 300 trails of training. For (d)–(f), the bars represent the mean (SEM) percentage of CRs on
conditioned stimulus (CS)-alone trails. (a) Prior to puberty, there were no differences in conditioning between males and females and no effect of
stress on trace conditioning as measured 24 h after exposure to an acute stressor of brief, periodic tailshocks. (b) During puberty, there were no sex
differences in trace conditioning, although exposure to the stressful event enhanced levels of conditioning in males and females. (c) In adulthood,
females emitted more CRs than did males and exposure to the stressful event enhanced conditioned responding in males and impaired responding in
females. (d) Prior to puberty, there are no sex differences or effects of stress on the responses to the CS on CS-alone trials. (e) During puberty,
stress enhanced these responses regardless of sex. (f) In adulthood, females emitted more CRs than did males but in response to stressor emitted
fewer. In contrast, males emitted more CRs on CS-alone trials if they had been exposed to the acute stressor. Reproduced from Hodes, G.E., Shors,
T.J., 2005. Distinctive effects on learning during puberty. Horm. Behav. 48, 163–171, with permission from Elsevier.

estradiol administration prevented the OVX-induced decrease
in spine density (Gould et al., 1990). A subsequent study
found that estradiol administration actually reversed the
OVX-induced reduction in spine density (Woolley and
McEwen, 1993). Thus, estradiol seems to enhance spine
density and branching in neurons within the hippocampus,
a critical region for learning and memory processing.
Given that hippocampal dendrite spine density (Gould
et al., 1990; Woolley et al., 1990; Woolley and McEwen,
1993) and synapse density (Woolley and McEwen, 1992) are
modulated by estradiol levels, as a function of NMDA receptor
activation (Woolley and McEwen, 1994), estradiol should be
able to modulate the ability of the hippocampus to exhibit
long-term potentiation (LTP). LTP of synaptic transmission in
this region is considered the primary candidate for major
synaptic changes underlying learning and memory processes
(Malenka and Nicoll, 1999), and estradiol decreases the
threshold for LTP induction (Cordoba Montoya and Carrer,
1997). For example, studies showed that electrophysiological
sensitivity of CA1 pyramidal cells is modified by exogenous
estradiol treatment (Montoya and Carrer, 1997) and changes
in ovarian levels across the estrous cycle (Warren et al.,
1995); in both cases, elevated levels of estradiol are related to
enhanced LTP. A more recent study assessed whether estradiol
affected LTP differently in male and female rats (Vierk et al.,
2014). In females, synaptic loss emerged after inhibiting estra-
diol synthesis in hippocampal neurons. This synaptic loss was
attributed to impaired LTP. However, this effect was not
observed in males. Thus, it is likely that estradiol synthesis in
the hippocampus occurs exclusively in females, and this local
estradiol synthesis assists in the maintenance of LTP and
synapses; therefore, inhibition of estradiol synthesis results in
impaired LTP and eventually synapse loss in females only.
1.14.2.3 A Model of Estrogen Action
1.14.2.3.1 Estrogen Receptor Subtypes
Estrogens are a class of steroid hormones comprised of estrone,
estriol, and estradiol; each estrogen binds to a series of
receptors including ERɑ, ERb, and G-protein-coupled ER 1
(GPER1) (Almey et al., 2015). ERɑ and ERb are both found
in the cell membrane, nuclei, and cytoplasm. These receptors
are expressed in different tissues and organs throughout the
body, including the uterus, breast tissue, testicles, prostate,
the cardiovascular system (Rollerova and Urbancikova,
2000), and importantly for learning and memory, the brain.

ERɑ and ERb have been identified in brain regions critical for
learning and memory, such as the hippocampus, prefrontal
cortex, and amygdala (Almey et al., 2015).
GPER1 was discovered more recently and was first identified
in breast tissue (Carmeci et al., 1997). This G-protein-coupled
receptor is found at the endoplasmic reticulum in neurons and
plasma membrane in cells of the hippocampus and
hypothalamus (Almey et al., 2015); binding at GPER1 is
thought to modulate effects of estrogens at ERɑ and ERb
(Revankar et al., 2005; Sakamoto et al., 2007) and have
direct effects on neuronal transmission in the hippocampus
and hypothalamus (Funkoshi et al., 2006; Prossnitz et al.,
2008; Waters et al., 2015).
1.14.2.3.2 Estrogen as a Neuroprotective Steroid
Of the class of estrogens, much of the work on estrogen action
in learning and memory has focused on estradiol since, in addi-
tion to promoting neurogenesis and maintaining neural integ-
rity, estradiol is also neuroprotective. For example, in vitro
studies found that when hippocampal fetal neurons were
pretreated with either estradiol (Chen et al., 2006) or
conjugated-equine estrogens (Brinton et al., 2000), neurode-
generation caused by beta-amyloid exposure was blocked, the
degree of apoptosis decreased, and beta-amyloid-induced
decrease in ATP levels was attenuated. In vivo studies also report
patterns of neuroprotection by estradiol in hippocampus and
entorhinal cortex, as well as neurogenesis in prefrontal cortex.
For example, when the entorhinal cortex and the hippocampus
were pretreated with estradiol in OVX-alone females, the
marked dose-dependent neuron loss in response to kainic
acid was blocked (Hoffman et al., 2003). Estradiol may also
protect the brain from age-related declines in females. In
young-adult female rhesus monkeys, ovariectomy followed
by cyclic estradiol replacement increased dendrite spine
density in layer III of dorsolateral prefrontal cortex; estradiol
also restored density in aged female monkeys, compared
with female rhesus monkeys not treated with estradiol (Hao
et al., 2007).
Neuroimaging studies in women support findings of
estrogenic neuroprotection (Smith and Zubieta, 2001). For
example, in a structural magnetic resonance imaging (MRI)
study, postmenopausal women using unopposed estradiol
for over 20 years experienced less ventricular enlargement
and white matter loss than women who did not use estradiol
(Ha et al., 2007), possibly resulting from estrogenic protection
of oligodendrocytes and subsequent promotion of myelination
(Gerstner et al., 2007).
Functional magnetic resonance imaging (fMRI) studies
have also reported greater activity in brain regions associated
with the task of interest. In one study, women who started
a combination estradiol/progestin treatment during perimeno-
pause performed better on a verbal recognition task; perfor-
mance on the task was subsequently associated with greater
activation in the hippocampus compared with women not
using the hormone treatment (Maki et al., 2011). When
women started combination hormone treatment within 2 years
of menopause and continued uninterrupted for 10 years, they
exhibited greater frontal cortex and hippocampal activation
during a visual working memory task performed during an
fMRI scan. This increased activity positively correlated with
Sex Steroids, Learning and Memory 409
performance on the task; a pattern not observed in women
who had never used hormone treatment (Berent-Spillson
et al., 2010). Women using combination hormone treatment
also exhibited higher cholinergic activity in the hippocampus
and posterior cingulate at rest, compared with never-users
(Smith et al., 2011), which may contribute to the positive asso-
ciation between hormone use and cognition, as the neurotrans-
mitter is intimately involved in attention, learning, and
memory (for reviews, see Klinkenberg et al., 2011; Micheau
and Marighetto, 2011).
Research also indicates that estradiol can act as a neuropro-
tective steroid in males. For example, intracerebroventricular
injection of estradiol 10 min prior to quinolinic acid injection
protected the CA1 region of the hippocampus in males (Kuroki
et al., 2001). Male mice pretreated with estradiol were also pro-
tected from stereotypical motor deficits associated with methyl-
mercury exposure and exhibited reduced lipid peroxidation in
the cerebellum (Malagutti et al., 2009); these effects were
thought to be the result of estradiol’s protection of the antiox-
idant glutathione (Malagutti et al., 2009).
The aforementioned findings indicate that estradiol protects
the brain via regulation of excitatory ion influx, antioxidant
systems, and maintaining proper energy metabolism despite
facing trauma. On a molecular level, estradiol utilizes subme-
chanisms to modulate these systemic neuroprotective effects,
including upregulation of extracellular signal-regulated kinase
(ERK) phosphorylation in the face of quinolinic acid exposure
(Kuroki et al., 2001), reduction of oxidative stress and mainte-
nance of respiratory capacity of mitochondria faced with
various mitochondrial-specific toxins (Yao et al., 2011), and
blockade of glutamate-induced and hydrogen peroxide-
induced cell loss by increasing nitric oxide synthesis (Mannella
et al., 2009; Andozia et al., 2010).
1.14.2.4 A Model of Progesterone Action
Progesterone is synthesized by different regions, including the
adrenal cortex, the ovaries, and Leydig’s cells, and binds to
progesterone receptors (Barros et al., 2015) throughout the
uterus, ovaries, brain, and bones (Graham and Clarke,
1997). Progesterone can have both excitatory and inhibitory
effects (Mahesh et al., 1996) and is considered a neurosteroid
with protective effects on the central and peripheral nervous
system (Barros et al., 2015). Research indicates that release
of progesterone is related to a myriad of biological functions,
including reproduction and stress; the sex steroid hormone
is also important for modulating cognition (McEwen and
Alves, 1999).
1.14.2.4.1 Progesterone and the Stress Response
Work with animals and humans has shown that progesterone
secreted by the ovaries and adrenals may relate to stress-
related cortisol increases. The effect of stress on adrenal output
in animals and men has shown that the adrenal glands secrete
not only cortisol, but also progesterone, in response to stress
(Fajer et al., 1971; Brown et al., 1976; Deis et al., 1989; Breier
and Buchanan, 1992; Cooper et al., 1995; Elman and Breier,
1997; Duncan et al., 1998; Romeo et al., 2004, 2006) with
limited work examining the effect in women (Childs et al.,
2010; Gaffey and Wirth, 2014; Herrera et al., 2016).
410 Sex Steroids, Learning and Memory
In addition to stress-induced adrenal release of proges-
terone, baseline levels of the hormone have been associated
with greater salivary cortisol response to stress. For instance,
the high-progesterone luteal phase of menstrual cycle is asso-
ciated with greater cortisol responses to social stress,
compared with the response observed during the low-
progesterone follicular phase (Kirschbaum et al., 1999).
More recent studies show that hormonal contraceptives also
attenuate the salivary cortisol response to physical stress
compared to naturally cycling women (Nielsen et al.,
2013b) and more specifically, luteal women (Nielsen et al.,
2014), suggesting the inhibition of ovarian hormone release
exerted by birth control pills contributes to the reduced
cortisol response to stress. This reduction with ovarian inhibi-
tion is likely a result of reduced ovarian progesterone, as
women with induced hypogonadism (via administration of
the GnRH agonist Lupron) exhibited amplified cortisol
responses to exercise stress when also administered proges-
terone but not estradiol (Roca et al., 2003).
In spite of these compelling data, the interpretation that
progesterone amplifies the cortisol response to stress fails to
acknowledge that the adrenal glands also secrete progesterone.
Importantly, the purported influence of progesterone fluctua-
tions during the menstrual cycle on cortisol response to stress
may be masking whether and how adrenal progesterone may
be responding to stress. If women also experience adrenal release
of progesterone in response to stress, then this may contribute to
the pattern of greater bioavailability of cortisol in response to
stress during high-progesterone phases of the menstrual cycle
(Kirschbaum et al., 1999; Kudielka and Kirschbaum, 2005).
There are multiple purposes stress-induced progesterone
release may serve, such as recruitment of the progesterone
metabolite allopregnanolone in the shutting down the HPA
axis (Patchev et al., 1994), reducing feelings of anxiety
(Dennerstein et al., 1985), and/or exerting a slight sedative
effect (Freeman et al., 1992, 1993; Söderpalm et al., 2004).
Additionally, social closeness has been shown to raise proges-
terone levels (Brown et al., 2009). This effect may prove bene-
ficial during times of stress, as feelings of social support
increase well-being in the face of stress (Cohen and Wills,
1985), and might promote individuals to seek out beneficial
social support systems (Wirth, 2011).
1.14.2.4.2 Progesterone Action and Cognition
In relation to cognition, recent studies have focused on the rela-
tionship between progesterone levels and cognitive domains
such as information processing and memory. In studies exam-
ining the relationship between progesterone and the processing
of emotional or biologically salient stimuli, higher proges-
terone levels during the luteal phase are negatively correlated
with recognition of emotional facial expressions (Derntl
et al., 2008), while oral progesterone administration reduced
recognition of faces and brain activation associated with
successful performance of the task (Van Wingen et al., 2007).
Other studies found that progesterone levels during the
menstrual cycle are associated with enhanced memory for
emotional items (Ertman et al., 2011; Felmingham et al.,
2012a) with heightened memory for peripheral details of
a scene rather than its general theme in a nonstressful context
(Nielsen et al., 2013a), and with enhanced memory for
peripheral details of a scene under stress (Nielsen et al.,
2014). Thus, although progesterone can have different effects
on cognition, research does clearly indicate that progesterone
is responsible for modulating cognitive processes related to
information processing and memory.
1.14.3 Interaction between Steroid Hormones,
Learning and Memory
1.14.3.1 Overview
Steroid hormones have been shown to exert robust effects on
cognition. Depending on the hormone under investigation,
the course of exposure, and the cognitive domain of interest,
these steroid hormones can influence cognitive processes in
a negative or positive fashion. The following sections cover
the effects of glucocorticoids and ovarian hormones on various
cognitive domains.
1.14.3.2 Glucocorticoids, Learning and Memory
Glucocorticoids, a class of stress hormone, are necessary for
proper neuronal and cognitive functioning. Much focus is
placed on the detrimental effects of stress on cognition since
long-term or extreme exposure to glucocorticoids or stress
can impair learning and memory. However, stress can also
have enhancing effects on learning and memory as well. The
following section will address impairing and enhancing effects
of glucocorticoids and stress on learning and memory in
animals and humans.
1.14.3.2.1 Factors Influencing the Relationship between
Stress and Learning
Different factors mediate these potential effects of stress. For
example, the magnitude of the stress applied is extremely
important in determining how stress affects memory. The rela-
tionship typically follows an inverted-U-shaped curve, with too
little or too much being related to poor performance. However,
a moderate amount of stress tends to be related to peak perfor-
mance on a variety of tasks, including shock avoidance, spatial
learning, and working memory (Yerkes and Dodson, 1908;
Mateo, 2008; Salehi et al., 2010). It is important to note
that there is no defined ‘optimal’ level of stress; this can
shift depending on the difficulty of the task (Dodson, 1915;
Anderson, 1994; Shors, 2004; Salehi et al., 2010).
1.14.3.2.2 Impairing Effects of Stress on Learning
and Memory in Animals
There are numerous reports of glucocorticoid- or stress-
induced cognitive impairment related to chronic or excessive
stress (for review, see Holmes and Wellman, 2009). Some
studies have used exogenous administration of corticosterone
to test the effects of stress on working memory and reference
memory. For example, 8 weeks of daily corticosterone injec-
tions resulted in male rodents committing significantly
more working memory errors during a Y-maze task
compared to control animals (Coburn-Litvak et al., 2003).
Similarly, 9 weeks of corticosterone treatment induced more
working memory errors during a radial-arm-maze task
(Arbel et al., 1994), and 3 weeks of daily corticosterone

injections impaired male rodents’ reference memory on the
Barnes maze (Coburn-Litvak et al., 2003).
To test the effects of stress on learning in animals,
researchers also use psychological and physical stressors. For
instance, male rats exposed to a combination of predator and
social stress exhibited significant working memory impair-
ments on a radial arm water maze (Park et al., 2001). Compa-
rably, males exposed to an unfamiliar environment during
a testing delay period for 21 days made more working memory
errors on a radial arm maze compared with animals housed in
their home cages during the delay period (Diamond et al.,
1996). Males exposed to the physical and psychological stress
of restraint also displayed working memory impairments on
a Y-maze (Kleen et al., 2006) and a delayed alternation task
in a T-maze (Lee and Goto, 2015) compared with rats not
exposed to restraint stress.
Exposure to brief periods of stress, rather than long-term
exposure, can also cause cognitive impairments in animals.
For example, while 4 weeks of unpredictable stress resulted in
impaired acquisition, working, and reference memory and
impaired reversal learning in male rodents, shorter regimens
of 6 days and 3 days of unpredictable stress also negatively
impacted reference memory and reversal learning or just refer-
ence memory, respectively (Cerqueira et al., 2007). Even
shorter exposure can lead to decrements in performance. For
example, 15 min of tail pinch stress increased error rates during
a delayed win-shift task in a radial arm maze, an effect blocked
in animals given a glucocorticoid antagonist (Butts et al.,
2011). Such short-term exposure also applies to exogenous
application, such that corticosterone 30 min prior to testing
impaired performance on a delayed alternation task in T-
maze in male rodents (Roozendaal, 2004). Thus, even short-
term and acute exposures to stress can negatively impact
learning and memory in animals.
1.14.3.2.3 Impairing Effects of Stress on Learning and
Memory in Humans
Although much of the animal literature focuses on how chronic
glucocorticoid or stress exposure affects cognitive processes,
research with humans focuses on how acute stress modulates
cognitive processes. Nevertheless, the impairing effects of stress
observed in animals are also seen in humans using acute phar-
macological, psychological, and physical stress manipulations.
For example, men treated with acute intravenous hydrocorti-
sone showed greater working memory errors during a visuospa-
tial sketchpad task, as well as higher error rates in a paired
associate task (Young et al., 1999). Comparably, chronic oral
hydrocortisone administration in men caused similar impair-
ments on a Sternberg item recognition task (Lupien et al.,
1999), suggesting that elevated cortisol levels impaired
working memory processes in men.
Acute psychological stressors, such as the Trier Social Stress
Test (TSST), can also have impairing effects on learning. When
men completed the TSST, which reliably elevates participants’
cortisol levels by requiring them to give a speech and perform
mental arithmetic in front of an audience (Elzinga and Roelofs,
2005; Oei et al., 2006; Schoofs et al., 2008; Wolf et al., 2009),
they displayed increased reaction times and other impairments
on tests of working memory given 10–45 min later, such as the
reading span task (Luethi et al., 2009), the Sternberg item
Sex Steroids, Learning and Memory 411
recognition task (Oei et al., 2006), and the N-back task
(Schoofs et al., 2008). Similarly, when tested during the
social stressor, men and women made fewer correct responses
during the compound remote associates test and took
significantly longer to complete anagrams (Alexander et al.,
2007). The TSST was also related to impaired performance
on a go/no-go task in men (Scholz et al., 2009). These
impairments are likely related to stressor-induced elevated
cortisol levels, as men also showed impairments on other
tasks (i.e., a mental arithmetic task) when they exhibited the
highest cortisol responses during the task or after giving
a surprise speech (Al’Absi et al., 2002).
Other acute laboratory stressors, such as the cold pressor
task (CPT), are more physical in nature. The CPT reliably
induces a stress response by requiring participants to hold
one of their hands in ice water for an extended period of
time (Bullinger et al., 1984; Edelson and Robertson, 1986;
Lighthall et al., 2009, 2011; Mather et al., 2010). Like the
TSST, stress associated with the CPT modulates learning and
memory. For example, following exposure to the CPT, men
had impaired performance on working memory measures
such as the operation span and digit span backward tasks
(Schoofs et al., 2009); both men and women exhibited
impaired performance on the Sternberg item recognition task
(Duncko et al., 2009).
Although stress effects on learning and memory are quite
consistent in the laboratory, the ecological validity of labora-
tory studies is sometimes difficult to determine. However,
research indicates that stressors encountered outside of the
laboratory can interfere with cognition in both men and
women. For example, a meta-analysis revealed that interme-
diate intensity exercise led to declines in performance on
a variety of working memory tasks (McMorris et al., 2011).
In other instances, attention-shifting was impaired in
students preparing for a major academic exam; those
students’ scores of chronic stress on the Perceived Stress Scale
predicted their performance on the attention-shift task
(Liston et al., 2009). Decision making on the game of dice
task was also impaired in participants experiencing anticipa-
tory stress after being told they would need to give a surprise
speech (Starcke et al., 2008). Additionally, another cohort
with higher perceived chronic stress showed poorer working
memory than those rating lower perceived chronic stress
(Öhman et al., 2007). Even within a population of patients
suffering from unipolar major depression, morning cortisol
levels were negatively correlated with performance on the
Wisconsin Card Sorting Task, a measure of executive function
(Egeland et al., 2005).
1.14.3.2.4 Sex Steroid Hormones and Stress Response
Interactions
Thus far, we have addressed how stress alone affects learning;
however, there is also research showing how stress interacts
with ovarian hormones to modulate learning and memory
processes in animals and humans. Although comparisons
have been made between the animal and human literature
thus far, the following discussion will be limited to studies in
humans. This limitation is because the physiological response
to stress differs between female rodents and women. For
example, female rodents exhibit larger glucocorticoid responses
412 Sex Steroids, Learning and Memory
to stressors than males (Handa et al., 1994; Suzuki et al., 2001;
McCormick et al., 2002; Seale et al., 2004a,b), likely due to
excitatory actions of estradiol on the female stress response
system (Burgess and Handa, 1992; Suzuki et al., 2001; Weiser
and Handa, 2009) and inhibitory actions of testosterone on
the male stress response system (Bingaman et al., 1994; Suzuki
et al., 2001).
In humans, however, women display smaller glucocorticoid
responses than men (Kirschbaum et al., 1992, 1999; Kudielka
et al., 1998, 2004; Dixon et al., 2004; Kudielka and Kirsch-
baum, 2005). The contradicting glucocorticoid response
profiles of rodents and humans limit the translational general-
ization of stress response profiles and suggest the way in which
the two systems may differ across species. Thus, the way
ovarian hormones influence the stress response and cognition,
and implications for peri- and postmenopausal women, will be
limited to studies with humans.
1.14.3.2.4.1 Estradiol and Stress Response Interactions
in Humans
In humans, there is evidence of estrogenic blunting of the
hypothalamic–pituitary–adrenal (HPA) axis activation, as
women display dampened HPA responses to stressors. Natu-
rally cycling young-adult women show lower adrenocortico-
tropic hormone (ACTH) (Kudielka et al., 1998; Kirschbaum
et al., 1999; Faraday et al., 2005) and biologically active free
cortisol (Davis and Emory, 1995; Kudielka et al., 1998, 2004;
Kirschbaum et al., 1999) responses compared with young-
adult men. Likewise, women tested during the high-estradiol
phase of the menstrual cycle showed reduced stress-related acti-
vation of different brain regions compared to men, including
the anterior cingulate gyrus, orbitofrontal cortex, medial and
ventromedial prefrontal cortex, amygdala, and hippocampus
(Goldstein et al., 2010), with similar activational differences
in women tested during the low-estradiol versus higher-
estradiol phases of the menstrual cycle (Goldstein et al.,
2005). Furthermore, women using combined formulations of
hormonal contraception show blunted cortisol responses
compared with naturally cycling women, possibly as a result
of the ethinyl estradiol component (Kirschbaum et al., 1999).
Evidence in young-adult men also suggests that estradiol
dampens sympathetic responses to a stressor, with just 1 day
of estradiol treatment resulting in blunted systolic blood pres-
sure, pulse rate, epinephrine, and norepinephrine responses to
a mental stressor (Del Rio et al., 1994).
Similar dampening of the stress response has been
observed in postmenopausal women on estradiol treatment.
Transdermal or oral estradiol interventions spanning 1 day
(Del Rio et al., 1998), 3 weeks (Ceresini et al., 2000), 1 month
(Puder et al., 2001), 6 weeks (Lindheim et al., 1992), and
8 weeks (Komesaroff et al., 1999) reduced the epinephrine
(Del Rio et al., 1998; Ceresini et al., 2000), norepinephrine
(Ceresini et al., 2000), diastolic blood pressure (Lindheim
et al., 1992; Komesaroff et al., 1999; Ceresini et al., 2000),
systolic blood pressure (Lindheim et al., 1992; Komesaroff
et al., 1999), ACTH and cortisol (Puder et al., 2001) responses
to various stressors, such as mental stressors (Lindheim et al.,
1992; Del Rio et al., 1998; Ceresini et al., 2000), the CPT
(Lindheim et al., 1992), and an endotoxin challenge (Puder
et al., 2001).
1.14.3.3 Summary
Pharmacological, physical, psychological, and nonlaboratory
stressors can interfere with normal, or optimal, cognitive func-
tion in animal models and humans. In particular, the evidence
suggests that chronic or excessive glucocorticoid levels impair
cognition in tasks requiring executive resources, such as
working memory. The following section will address how
ovarian steroids interact with stress to modulate memory for
arousing material and also how these hormones generally
affect emotional memory.
1.14.3.4 Steroid Hormone Interactions in Learning and
Memory of Arousing Information
1.14.3.4.1 Ovarian Steroids, Stress and Emotional Memory
in Animals
Rodent studies have explored how ovarian hormones modu-
late emotional memory using a series of acutely arousing tasks.
For example, when cycling female rats were stressed prior to
training, eye-blink conditioning was impaired; the impairment
was prevented if females were ovariectomized or treated with
the estrogen antagonist, tamoxifen (Wood and Shors, 1998).
Similarly, Gupta et al. (2001) found that in a fear conditioning
paradigm, ovariectomized female rodents significantly
increased their freezing behavior, an enhanced response similar
to males (Gupta et al., 2001). Please refer to Figure 4 for graph-
ical representation of this behavior.
Another study examined differences between male and
female rats in the effects of restraint stress on spatial recogni-
tion memory, and results showed that 1 h of restraint impaired
spatial memory in males, but had the opposite effect in female
animals (Conrad et al., 2004). A second experiment revealed
that acute stress facilitated spatial memory in females regardless
of the estrous cycle phase. These aforementioned studies indi-
cate that sex differences in emotional memory may be
estrogen-mediated.
Other work has shown that ovarian hormone effects on
memory for arousing material may depend on the phase of
the estrous cycle. For example, one study assessed effects of
the estrous cycle on memory for a spatial water maze task
when the water temperature was varied (Rubinow et al.,
2004). Results indicated that ovarian hormones enhanced or
impaired performance on the task depending on the water
temperature, and plasma corticosterone levels were higher in
proestrous, when there is surging estradiol levels, rats
compared to estrous rats. Shors et al. (2000) found similar
results in that the impairing effect of stress on delay and trace
conditioning was more pronounced in female rats in proestrus.
Other work also supports these findings (Shansky et al., 2006;
Dalla et al., 2009), which strongly implicate ovarian hormones
in the mediation of stress effects on emotional memory in
animals.
1.14.3.4.2 Ovarian Steroids and Emotional Memory in Humans
Emotionally arousing stimuli tend to be better remembered than
neutral stimuli (McGaugh, 2000). Studies have shown that
although men and women exhibit emotional memory enhance-
ments, their memory patterns can differ. In a number of studies,
sex has been shown to modulate the encoding, consolidation,

Figure 4 (a) Mean (SEM) percentage of freezing on the conditioning
day in female rats that received sham surgery (filled), female rats that
were ovariectomized (open), and male rats (hatched). The values were
normalized to preshock freezing and averages across the three 1-min
postshock periods. (b) Mean (SEM) percentage of freezing collapsed
across the 8-min of the context extinction test over the four consecutive
days of testing for groups described in (a). Reproduced from Gupta,
R.R., Sen, S., Diepenhorst, L.L., Rudick, C.N., Mare, S., 2001. Estrogen
modulates sexually dimorphic contextual fear conditioning and hippo-
campal long-term potentiation (LTP) in rats. Brain Res. 888, 356–365,
with permission from Elsevier.
and/or retrieval of emotional material (Andreano and Cahill,
2009). The following section will address sex differences in
emotional memory, shifts in emotional memory across the
menstrual cycle, and how emotional memory is influenced by
hormonal contraceptive use.
1.14.3.4.3 Sex Differences in Emotional Memory Paradigms
The study of sex differences in memory has received increasing
interest over the last 20 years. Researchers identified a female
advantage in autobiographical memory; women are more accu-
rate in their recall (Pohl et al., 2005; Bloise and Johnson, 2007)
and provide more details (Seidlitz and Diener, 1998). The
female advantage in autobiographical memory may be linked
to a female advantage in recall of emotional experiences
(Andreano and Cahill, 2009). For example, some studies
Sex Steroids, Learning and Memory 413
have shown that women recall more emotional information
than men (Davis, 1999) and that women outperform men
on emotional memory assessments (Fujita et al., 1991; Bloise
and Johnson, 2007). These behavioral studies indicate that
women recall emotionally arousing material better than men;
however, these studies do not provide evidence for how sex
modulates the underlying neurobiology of emotional memory.
Additional neuroimaging studies have explored sex effects
on brain regions implicated in the processing of emotional
memory, namely the amygdala (McGaugh, 2000). Several
studies have reported a sex-related hemispheric lateralization
of amygdala activity in relation to long-term emotional
memory (Andreano and Cahill, 2009). Cahill et al. (2001)
found that activity of the right amygdala in men viewing
emotional stimuli was significantly correlated with long-term
recall of the stimuli; however, there was no relationship with
the left amygdala. However, activity in the amygdala of the
left hemisphere of the brain while viewing emotional stimuli
was significantly related to long-term recall in women; this rela-
tionship was not observed between activity in the right hemi-
sphere amygdala and memory. This relationship was
replicated in different paradigms investigating sex influences
on emotional memory (Canli et al., 2002; Cahill et al., 2004;
Mackiewicz et al., 2006). A subsequent study demonstrated
that a sex-related hemispheric lateralization of amygdala func-
tion exists even when men and women are resting (Kilpatrick
et al., 2006); these results have been strongly confirmed (Savic
and Lindström, 2008).
Several studies expanded upon this sex-related hemisphere
laterality of amygdala function to examine sex influences on
emotional information processing. There is evidence for
a hemispheric specialization in the processing of global (gist)
versus local (detail) information from an event; the right hemi-
sphere is associated with the processing of gist, whereas the left
hemisphere is associated with detail processing (Fink et al.,
1996, 1999). One study integrated these hemispheric laterality
findings to test the hypothesis that a b-adrenergic blocker
should impair the amygdala’s modulatory effects on memory.
That is, the drug would impair memory for gist of an emotion-
ally arousing story in men (right amygdala/hemisphere bias)
but memory for details of the same story in women (left amyg-
dala/hemisphere bias) (Cahill and van Stegeren, 2003). Results
from the study supported the hypothesis – emotional arousal
appears to enhance memory for gist in men via activation of
the right amygdala/hemisphere and memory for detail in
women via activation of the left amygdala/hemisphere. This
point is illustrated in Figure 5.
1.14.3.4.4 Shifts in Emotional Memory across the Menstrual
Cycle
Sex differences in emotional memory appear to be driven by
sex steroid hormone influences on emotional memory
processes, particularly the ovarian hormones estradiol and
progesterone (e.g., Andreano et al., 2008). In women, memory
for emotional material can shift with changes in sex hormone
levels, particularly across the menstrual cycle. Some studies of
emotional memory shifts across the menstrual cycle have inves-
tigated interactions between sex and stress hormones
(Andreano et al., 2008). For example, when female participants
read a mildly arousing story and were then administered
414 Sex Steroids, Learning and Memory

Figure 5 Multiple-choice test scores do each experimental group and story phase. (a) Values for questions defined as pertaining to central informa-
tion. (b) Values for questions defined as pertaining to peripheral detail. Values represent means percent correct (SEM) on the multiple-choice test
in each experimental group. P1, P2, and P3 indicate story phases 1, 2, and 3, respectively. Emotional story elements were introduced in P2.
*p < 0.01 placebo compared with corresponding P2 propranolol group. Reproduced from Cahill, L., van Stegeren, A., 2003. Sex-related impairment
of memory for emotional events with b-adrenergic blockade. Neurobiol. Learn. Mem. 79, 81–88, with permission from Elsevier.

a physical stressor (cold pressor stress), stress-induced cortisol
increases positively correlated with story recall for women in
the midluteal phase of the menstrual cycle (Andreano et al.,
2008). In the late follicular phase, cortisol responses tended
to negatively correlate with story recall, and women in the early
follicular phase exhibited no relationship between cortisol and
memory performance. A more recent study showed similar
effect of cortisol on memory in women with high and low
progesterone; high-progesterone women displayed greater
recall of negative images under stress compared to low-
progesterone women under stress (Felmingham et al.,
2012b). Other research with arousing and nonarousing words
found that when women were administered a posttraining cold
pressor stress after learning these words, those in the follicular
phase had enhanced long-term memory for arousing words
relative to nonarousing words (Zoladz et al., 2015). The find-
ings from these studies indicate that the relationship between
cortisol and memory changes depending upon levels of circu-
lating sex hormones in naturally cycling women.
Other studies have focused on how the menstrual cycle and
sex hormones modulate emotional memory in the absence of
a posttraining stressor. For example, Ertman et al. (2011)
examined whether naturally cycling women in hormonally
distinct phases of the menstrual cycle differentially recalled
emotional images 1 week after encoding. Enhanced recall of
emotional images was only observed in women in the luteal
(high hormone) phase of the menstrual cycle; furthermore,
regression analyses revealed a positive correlation between
memory and progesterone levels at encoding (Ertman et al.,
2011). Nielsen et al. (2013a) found similar results using an
emotional story paradigm. Women in the luteal phase recalled
significantly more details, but not gist, from the negatively
valenced emotional story 1 week after encoding, whereas
women in the follicular phase did not exhibit enhanced reten-
tion for gist or detail in the emotional story.
Although these behavioral studies provide evidence for
shifts in emotional memory across the menstrual cycle, they
do not speak to the underlying neurobiological mechanisms.
However, over the past two decades, human neuroimaging
studies have revealed potential mechanisms modulating sex
hormone influences on emotional memory. For example,
Goldstein et al. (2005) demonstrated that in women viewing
negative, emotionally arousing material, activity in several
limbic, frontal, and hypothalamic regions was related to

menstrual cycle phase; women in the late follicular phase
showed significantly decreased responses in these areas as
compared to those in the early follicular phase. In another
study, researchers administered synthetic progesterone to
young women and found that high levels of synthetic proges-
terone increased amygdala responses to emotional images
relative to neutral images (Van Wingen et al., 2008). These
findings were further supported by a study where women in
the midluteal phase exhibited an enhanced response to
emotional images in the hippocampus and amygdala as
compared to women in the early follicular phase (Andreano
and Cahill, 2010). These findings suggest that sex hormones,
particularly progesterone, modulate the responsiveness of
a key node in the brain’s emotional memory circuitry – the
amygdala.
1.14.3.4.5 Hormonal Contraceptive Use and Emotional
Memory
There are limited studies on how hormonal contraception
influences learning and memory of emotional stimuli. Some
of these studies have focused on how stress and hormonal
contraceptives interact to modulate emotional memory
processes. For example, Nielsen et al. (2013b) found that post-
training glucocorticoid release enhanced memory for positive
images in hormonal contraceptive users, but not in naturally
cycling women. In a follow-up study, posttraining stress
enhanced memory for gist and detail from a negative
emotional story in luteal women, but did not affect retention
for the emotional story in hormonal contraceptive users
(Nielsen et al., 2014). These studies demonstrate that stress
and hormonal contraception interact to modulate emotional
memory in ways not observed in naturally cycling women.
Other studies have utilized pharmacological manipulations
to explore the relationship between hormonal contraceptive
use, stress, and memory. Kuhlmann and Wolf (2005) tested
the influence of cortisol on verbal retrieval in oral contraceptive
users and nonusers; results showed that exogenous cortisol
administration impaired verbal retrieval in naturally cycling
women, but not in those using oral contraception (Kuhlmann
and Wolf, 2005). More recent studies with exogenous cortisol
administration found that cortisol enhanced fear learning in
oral contraceptive users compared to men and naturally cycling
women (Merz et al., 2012).
Other studies have used classical conditioning paradigms to
explore learning and memory of arousing stimuli in hormonal
contraceptive users. Several studies using classical conditioning
found that hormonal contraceptive use was associated with
facilitated acquisition of classical conditioning responses
(Beck et al., 2008; Holloway et al., 2011), and this facilitated
learning persisted even under stress conditions (Merz et al.,
2013a). Imaging work with a classical fear conditioning para-
digm also revealed that in oral contraceptive users and men,
but not in women in the luteal phase, there was a positive
correlation between basal cortisol levels and differential activa-
tion in the amygdala (Merz et al., 2013b). Together, these
studies suggest that fear learning, even under conditions of
stress, is significantly altered in women using hormonal
contraception.
Finally, other work has explored emotional memory in
hormonal contraceptive users in the absence of external
Sex Steroids, Learning and Memory 415
stressors using long-term memory paradigms. A recent study
showed that the use of hormonal contraception altered
memory for information from an emotional story (Nielsen
et al., 2011). Hormonal contraceptive users exhibited enhanced
memory for gist, but not details of an emotional story, whereas
naturally cycling women exhibited enhanced emotional
memory for details, but not gist (Nielsen et al., 2011).
1.14.4 Ovarian Steroids and Clinical Implications
for Human Neuropathologies
1.14.4.1 Overview
The topics discussed throughout this chapter demonstrate
that ovarian steroids have a profound effect on learning and
memory processes in both animal and human models. In
addition, a subset of research has identified interactions
between ovarian and stress hormones to modulate these
cognitive processes. The final section of the chapter will
discuss implications of ovarian hormones in the clinical
setting, particularly with respect to AD and disorders of
emotional memory.
1.14.4.2 Gonadal Steroids and AD
Women are at greater risk to develop AD compared to men due
to the substantial loss of estrogens and progesterone at meno-
pause (for review, see Pike et al., 2009). If the decrease of
ovarian hormones at menopause contributes to women’s
increased risk of AD, then it should follow that treatment
with HT would be effective at preventing or treating the disease.
However, results to date are inconclusive. Some studies have
found that women who used HT had a reducing risk of AD
compared to nonusers (Henderson and Buckwalter, 1994;
Paganini-Hill and Henderson, 1994, 1996; Tang et al., 1996;
Kawas et al., 1997; Carlson et al., 2001). These findings support
estrogen as a protective agent against the development of AD in
women postmenopause.
However, other studies have found that estrogen supple-
mentation can negatively affect the development of AD. For
example, the AD Cooperative Study, a randomized, double-
blind, placebo-controlled, clinical trial, found that older
women with preexisting mild to moderate AD had a faster
progression to AD when they were on estradiol supplementa-
tion (Mulnard et al., 2000). Other studies found that HT use
was not associated with a reduced risk of AD or that HT use
did not yield any significant benefits on cognition (Wang
et al., 2000; Almeida et al., 2006).
Although studies have found opposing effects of HT on AD
development in postmenopausal women, there is a growing
consensus that estrogen-based HT can positively affect AD
development depending on the time of initiation and length
of time that women are on HT relative to the onset of meno-
pause. If women initiate HT in the perimenopausal period,
research indicates that HT is more likely to reduce the risk of
AD and improve age-related deficits in cognition (for review,
Pike et al., 2009). This effect may be due to the healthy cell
bias (Brinton, 2005) discussed above in section Exogenous
Hormones, Working Memory, and the Women’s Health
Initiative Studies.
416 Sex Steroids, Learning and Memory
1.14.4.3 Gonadal Steroids and Disorders of Emotional
Memory
Over the course of their lifetime, women suffer from disorders
of emotional memory at a significantly higher rate than men
(Risbrough and Stein, 2006; Toufexis et al., 2006). For
example, compared to men, women are more likely to suffer
from depressive disorders such as dysthymia, depression,
atypical depression, and seasonal depression (Risbrough
and Stein, 2006; Toufexis et al., 2006). Not only are women
more likely to suffer from depressive disorders, but their
depressive symptoms can last longer, be more severe, tend
to recur (Nolen-Hoeksema, 1987).
Research has also shown that women significantly
outnumber men in anxiety disorders. For example, women
are twice as likely as men to suffer from generalized anxiety
disorder, PTSD, and panic disorder. Additionally, they are 1.5
times as likely to develop obsessive compulsive disorder and
social anxiety disorder (Pigott, 2003).
The increased prevalence of anxiety and depressive disorders
in women is likely related to ovarian hormone levels. Some
research indicates that lower levels of ovarian steroids facilitate
the onset of these disorders. For instance, in humans, reduced
estrogen levels have been associated with the onset of depres-
sion, sleep disturbance, irritability, anxiety, panic disorders,
and cognitive dysfunction (Campbell and Whitehead, 1977;
Arpels, 1996; Sherwin, 1998). Additionally, estrogen supple-
mentation can improve symptoms of these emotional memory
disorders. Research found that in postmenopausal women,
estrogen replacement therapy was associated with improved
mood, increased energy levels, feelings of general well-being,
and improved learning (Halbreich, 1997; Sherwin, 1998).
Other work suggests that increased levels of gonadal
hormones actually perpetuate disorders of emotional memory.
Some studies have linked levels of estrogens and progestins to
increased vulnerability to anxiety disorders in women (Pigott,
2003), and other work indicates that hypo- and hyperestrogen-
ism and the pre-ovulatory estrogen surge are linked to anxiety
and increased nervous energy in women (Arpels, 1996).
A subset of research has focused on how major hormonal
changes throughout a woman’s lifetime affect the onset of
emotional memory disorders. Sex differences in anxiety tend
to emerge only after puberty and may only be due in part to
the activational effects of gonadal hormones on processes
such as fear extinction (Graham and Milad, 2013). Also, sex
differences in depression incidence decrease after women go
through menopause and their monthly hormonal changes
are no longer a factor (Sonnenberg et al., 2013).
When assessing depression, it is most prevalent in women
during the reproductive years and periods of major hormonal
fluctuations, such as puberty, postchildbirth, and after meno-
pause (Rapkin et al., 2002; Parker and Brotchie, 2010). Women
are also susceptible to postpartum depression (Shors and
Leuner, 2003), which begins after childbirth. During the time
of childbirth, estrogen levels are extremely high and following
the birth of the child, they decline rapidly. This suggests that
depression may be related to a decrease, or at least a dramatic
change in ovarian steroid levels (Shors and Leuner, 2003);
therefore, estrogen levels may be necessary, but not sufficient
for inducing depression in women.
Although there are mixed findings with ovarian hormones
and disorders of emotional memory in women, all studies indi-
cate that these steroids affect depression and anxiety in some
manner.
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 1.15 Adrenal Stress Hormone Effects on Memory
Benno Roozendaal and Erno J Hermans, Radboud University Medical Center, Donders Institute for Brain, Cognition and Behaviour,
Radboud University Nijmegen, Nijmegen, The Netherlands
Ó 2017 Elsevier Inc. All rights reserved.
1.15.1 Introduction
1.15.2 Effects of Adrenal Stress Hormones on Memory Formation
1.15.2.1 Epinephrine
1.15.2.2 Glucocorticoids
1.15.2.3 Adrenergic–Glucocorticoid Interactions
1.15.3 Role of Noradrenergic Activity within the Basolateral Amygdala in Mediating Adrenal Stress Hormone
Effects on Memory Consolidation
1.15.3.1 Noradrenergic Activity of the Basolateral Amygdala Mediates Epinephrine Effects on Memory
Consolidation
1.15.3.2 Glucocorticoids Interact with Noradrenergic Mechanisms within the Basolateral Amygdala
1.15.4 Adrenal Stress Hormone Effects on Other Memory Functions
1.15.4.1 Memory Retrieval
1.15.4.2 Working Memory
1.15.5 Concluding Remarks
References
1.15.1 Introduction
Every day, we experience stress in varying forms and degrees.
When we are exposed to potential threats (stressors), our
brain initiates a course of action that releases numerous trans-
mitters, peptides, and hormones throughout our body
(Joëls and Baram, 2009). In particular, two systems become
activated during stress: the fast-acting sympathetic nervous
system and the slow hypothalamus–pituitary–adrenal
(HPA) axis. Sympathetic nervous system responses include
the release of the catecholamines epinephrine (adrenaline)
and norepinephrine (noradrenaline) from the adrenal
medulla and presynaptic nerve terminals (McCarty and
Gold, 1981; Smith and Vale, 2006), which induces, for
example, an increase in heart rate, energy metabolism, and
enhanced blood flow to skeletal muscles to prepare the
organism for the ‘fight-or-flight’ response. Activation of the
HPA axis triggers a cascade of events that induces the release
of glucocorticoids (mainly cortisol in humans, corticosterone
in rodents) from the adrenal cortex (Ulrich-Lai and Herman,
2009). First, corticotropin-releasing factor (CRF) is released
by the hypothalamic paraventricular nucleus into the portal
system. CRF then induces the release of adrenocorticotropin
from the adrenal pituitary gland, which subsequently stimu-
lates the release of glucocorticoids from the adrenal cortex
into the bloodstream (Axelrod and Reisine, 1984). In the
periphery, glucocorticoids exert, for example, immunosup-
pressive actions and increases in blood glucose levels
(McEwen, 1998). In addition to preparing an individual for
the acute consequences of dangerous or threatening situations
and the return to homeostasis, an important function of these
stress hormones and other stress mediators is to induce long-
term adaptive responses (de Kloet et al., 2005; McEwen,
1998).
Here, we focus on how adrenal stress hormones affect
learning and memory processes. Stressful and emotionally
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arousing experiences typically leave lasting and vivid memo-
ries (Sandi and Pinelo-Nava, 2007) and, in most cases, it
certainly seems highly adaptive to have such (neuro)biolog-
ical processes that enable the significance of events to regulate
their remembrance. Extensive evidence indicates that
epinephrine or glucocorticoids administered shortly after
a learning experience enhance the consolidation of memory
of such an experience (Roozendaal and McGaugh, 2011).
Experimental studies in humans have shown that this
memory retention benefit is observed also when stress is
induced after learning (Abercrombie et al., 2006; Andreano
and Cahill, 2006; Cahill et al., 2003; Preuss and Wolf, 2009;
Smeets et al., 2008), demonstrating that the effects of stress
and emotional arousal on memory cannot be explained
simply by immediate effects on attention or memory encod-
ing. In the first part of this chapter, we will summarize animal
and human studies on the acute effects of epinephrine and
glucocorticoids on the consolidation of memory processing.
It is now well established that adrenal stress hormones prefer-
entially strengthen the consolidation of memory of emotion-
ally arousing, and not emotionally neutral, training
experiences (de Quervain et al., 2009; Okuda et al., 2004).
In the second part, we will address how adrenal stress
hormones interact with arousal-induced noradrenergic
activity within the basolateral complex of the amygdala
(BLA), and several other brain regions, to selectively modulate
memory of emotionally arousing information or during
emotionally arousing test situations. Moreover, we will
present some recent developments indicating that such gluco-
corticoid interactions with the noradrenergic system do not
appear to be mediated through the classic genomic action of
glucocorticoids but, rather, to involve rapid effects through
an activation of membrane-associated steroid receptors and
the recruitment of downstream endocannabinoid signaling
(Atsak et al., 2012a; Tasker et al., 2006). Finally, we will
briefly describe the modulatory influences of adrenal stress
423
424 Adrenal Stress Hormone Effects on Memory
hormones on other memory functions such as memory
retrieval and working memory.
1.15.2 Effects of Adrenal Stress Hormones
on Memory Formation
1.15.2.1 Epinephrine
Ralph Gerard (1961) may have been the first to suggest that the
adrenomedullary stress hormone epinephrine might have
enhancing effects on memory consolidation. Memory consoli-
dation is the process by which a fragile short-term memory
trace is transferred into stable long-term memory. However,
not all information is equally well transferred into long-term
storage. In fact, it is well recognized that especially emotionally
arousing experiences are well remembered, even after a long
time (McGaugh, 2003). Gerard proposed that as, “. epineph-
rine . is released in vivid emotional experiences, such an
intense adventure should be highly memorable.” Early support
for this suggestion came from experiments using systemic
injections of amphetamines. In addition to the well-known
effects of amphetamine on dopaminergic mechanisms,
amphetamine increases the release of adrenergic catechol-
amines from peripheral and central storage sites (Rothman
et al., 2001). Numerous studies have shown that amphetamine
enhances memory when administered peripherally shortly
after a training experience. This finding provides strong
evidence that the drug enhanced memory by affecting time-
dependent memory consolidation processes, as the subjects
are drug-free during both acquisition and retention testing
processes (McGaugh, 1973; McGaugh and Petrinovich,
1965). Enhancing effects of amphetamine in rats and mice
have been observed in a variety of tasks such as inhibitory
avoidance, active avoidance, and appetitive discrimination
(McGaugh, 1989). Moreover, amphetamine administered to
human subjects either shortly before or immediately after
learning lists of unrelated words gradually enhanced free recall
of this information (Soetens et al., 1995, 1993). As amphet-
amine readily passes the blood–brain barrier, other experi-
ments investigated whether the memory-enhancing effects of
amphetamine involve influences on peripheral or central cate-
cholamines. Posttraining systemic injections of 4-OH amphet-
amine, a derivative of amphetamine that does not pass the
blood–brain barrier, also enhanced inhibitory avoidance
memory (Martinez et al., 1980). Moreover, adrenal demedulla-
tion, i.e., elimination of peripheral epinephrine, blocked the
effects of both amphetamine and 4-OH amphetamine on
memory, whereas removal of peripheral norepinephrine by
sympathetic denervation did not attenuate the memory-
enhancing effects of either drug (Martinez et al., 1980). These
findings thus provide support for the view that amphetamine
enhances memory consolidation by stimulating the release
of epinephrine from the adrenal medulla (Williams and
McGaugh, 1994).
Systemic administration of epinephrine after a training expe-
rience also enhances memory consolidation in a wide variety of
tasks, including inhibitory avoidance, multitrial avoidance,
a one-trial appetitive task, an aversively motivated discrimina-
tion task, and object recognition (Costa-Miserachs et al., 1994;
Dornelles et al., 2007; Gold and van Buskirk, 1976, 1975;
Introini-Collison and McGaugh, 1986; Izquierdo and Dias,
1985; Liang et al., 1986; Sternberg et al., 1985). Epinephrine
is most effective when given immediately after training, and
its memory-enhancing capacity rapidly decays with an
increasing delay between training and posttraining epinephrine
administration (Gold and Van Buskirk, 1975). Moreover,
moderate doses produce greatest enhancement, whereas larger
doses are less effective or even impair retention. Highly
comparable to the findings of these studies in animals, epineph-
rine administered to human subjects after they viewed emotion-
ally arousing slides also enhanced the subjects’ long-term
memory of the slides (Cahill and Alkire, 2003). Systemic
administration of the b-adrenoceptor antagonist propranolol
blocks epinephrine effects on memory consolidation
(Introini-Collison et al., 1992). As epinephrine does not readily
cross the blood–brain barrier (Weil-Malherbe et al., 1959), its
effects on memory consolidation appear to be initiated, at least
in part, by activation of b-adrenoceptors located in the
periphery. This conclusion is supported by the finding that sota-
lol, a b-adrenoceptor antagonist that does not readily enter the
brain, also blocks the effects of systemically administered
epinephrine on memory enhancement (Introini-Collison
et al., 1992).
Epinephrine effects on memory consolidation are most
likely mediated by activation of b-adrenoceptors located on
vagal afferents in the periphery that project to noradrenergic
cell groups within the nucleus of the solitary tract (NTS) in
the brainstem (Schreurs et al., 1986; Williams and Clayton,
2001). These noradrenergic cells directly innervate brain
regions involved in memory consolidation, including the
amygdala. The NTS may also influence noradrenergic activa-
tion via its projection to the locus coeruleus (LC), which has
noradrenergic cells with more widespread projections to
forebrain regions, including the hippocampus, cortex, and
amygdala (Ricardo and Koh, 1978; Valentino and Aston-
Jones, 1995; Williams and Jensen, 1991; Williams and
McGaugh, 1993). The key role of the NTS in mediating
epinephrine effects on memory is indicated by the finding
that temporary inactivation of the NTS with lidocaine
prevents epinephrine-induced release of norepinephrine in
other brain regions and blocks epinephrine influences on
memory consolidation. Further, stimulating either the vagus
(Clark et al., 1999) or the NTS immediately after training
enhances memory consolidation (Williams and Jensen,
1991). Thus, the NTS plays a critical role in mediating the
transmission of adrenergic signals from the periphery to
brain systems that process memory for emotionally signifi-
cant experiences.
Epinephrine may also influence memory consolidation by
enhancing glycogenolysis in the liver (Gold, 1995; Messier
and White, 1984, 1987). Glucose administered peripherally
immediately after a training experience produces dose- and
time-dependent effects on memory comparable to those
produced by epinephrine (Gold, 1986). Further, doses of
epinephrine and glucose that are optimal for enhancing
memory induce comparable levels of plasma glucose (Hall
and Gold, 1986). In contrast to their effects on epinephrine,
b-adrenoceptor antagonists such as propranolol do not block
glucose effects on memory (Gold, 1986). Glucose readily
enters the brain and, thus, can directly influence brain

mechanisms involved in memory consolidation (Oomura
et al., 1988). However, the finding that memory is also influ-
enced by peripherally administered fructose, a sugar that has
little effect on the brain, suggests that this sugar, as well as
glucose, also acts, at least in part, at peripheral sites in influ-
encing memory (Messier and White, 1987). In support of this
view, Talley et al. (2002) reported that vagotomy blocks the
memory-enhancing effects of peripherally administered
L-glucose, an enantiomer of glucose that does not cross the
blood–brain barrier.
1.15.2.2 Glucocorticoids
There is compelling evidence from studies in both animals and
humans that glucocorticoids are also involved in regulating
the consolidation of memory processes (de Kloet, 2000; de
Quervain et al., 2009; Flood et al., 1978; Het et al., 2005;
McGaugh and Roozendaal, 2002; Roozendaal, 2000, 2002,
2009a; Sandi and Pinelo-Nava, 2007; Schwabe et al., 2012).
Blockade of glucocorticoid production with the synthesis
inhibitor metyrapone impairs memory consolidation in both
animals and humans (Cordero et al., 2002; Maheu et al.,
2004). In contrast, acute systemic administration of glucocorti-
coids or synthetic glucocorticoid ligands such as dexametha-
sone enhances long-term memory when given before
(Abercrombie et al., 2003; Buchanan and Lovallo, 2001; Sandi
and Rose, 1994) or immediately after a training experience
(Flood et al., 1978; Roozendaal and McGaugh, 1996;
Roozendaal et al., 1999; Sandi and Rose, 1994; Wilhelm
et al., 2011). Glucocorticoid manipulations applied before
learning may affect both encoding and consolidation
processes. Human work has shown that administration of
glucocorticoids affects sensory (Miller et al., 2015) and atten-
tional processes (Hermans et al., 2014b; Putman and Roelofs,
2011). Research on the effects of glucocorticoid administration
on medial temporal lobe activity during memory encoding
remains inconclusive, with rapid deactivations (Lovallo et al.,
2010), delayed deactivations (Henckens et al., 2012), and
null findings (van Stegeren et al., 2010). Glucocorticoids also
play a role in the consolidation of memory of extinction
training (Barrett and Gonzalez-Lima, 2004; Bohus and Lissák,
1968; Cai et al., 2006; Yang et al., 2006). Like epinephrine
effects, such glucocorticoid influences on memory consolida-
tion follow an inverted U-shaped dose–response relationship:
moderate doses enhance memory, whereas lower or higher
doses are typically less effective or impair memory
consolidation (Lupien et al., 2007; Roozendaal, 2000).
Glucocorticoid hormones are highly lipophilic (McEwen
et al., 1979) and can bind directly to mineralocorticoid recep-
tors (MRs) and glucocorticoid receptors (GR) (de Kloet,
2000; McEwen et al., 1968; Reul and de Kloet, 1985). MRs
have a high affinity for the natural steroids corticosterone,
cortisol, and aldosterone. GRs have an approximately 10 times
lower affinity for corticosterone and cortisol but show a high
affinity for the synthetic ligands dexamethasone and RU
28362 (Reul et al., 1987; Sutanto and de Kloet, 1987). The
memory-enhancing effects of glucocorticoids appear to involve
the selective activation of the low-affinity GR (Oitzl and de
Kloet, 1992; Roozendaal and McGaugh, 1997) as a blockade
of GRs, but not MRs, shortly before or immediately after
Adrenal Stress Hormone Effects on Memory 425
training impairs long-term memory (but see Atucha et al.,
2015). Corticosterone or a GR agonist administered directly
into a variety of brain regions enhances memory consolidation
of training on many different kinds of learning tasks, including
inhibitory avoidance, contextual and cued fear conditioning,
water-maze spatial and cued training, object recognition,
conditioned taste aversion, and appetitive taste learning
(Roozendaal et al., 2006a). Glucocorticoids are known to act
through intracellular and intranuclear receptors and can affect
gene transcription by direct binding of receptor homodimers
to glucocorticoid response elements on the DNA (Beato
et al., 1995; Datson et al., 2001) or via protein–protein interac-
tions with other transcription factors such as Jun or Fos (Heck
et al., 1994). However, as discussed below, glucocorticoids may
also act more rapidly by interacting with membrane receptors
and/or potentiating the efficacy of the norepinephrine
signaling cascade via an interaction with G protein-mediated
actions and downstream molecular events, including activation
of the transcription factor cAMP response-element binding
(CREB) protein and associated epigenetic (e.g., histone acetyla-
tion) mechanisms (Barsegyan et al., 2010; Dallman, 2005;
Joëls et al., 2011; Karst et al., 2010; Roozendaal, 2002, 2010;
Tasker et al., 2006). These genomic and nongenomic glucocor-
ticoid actions may ultimately, and collectively, result in
neuroplasticity and structural changes, e.g., via modifications
of cell-adhesion molecules and strengthening of cell–cell inter-
actions (Sandi, 2011).
MR-induced effects have been primarily associated with
stress appraisal and responsiveness to stressful stimuli (Joëls
et al., 2008). Some studies, however, indicated that MR
function, either alone or in conjunction with GRs, might also
be implicated in mnemonic functions, but in most cases
evidence for a direct influence on consolidation processes is
lacking. In healthy individuals it has been shown that oral
administration of the MR agonist fludrocortisone improves
performance in a wide range of neuropsychological tests,
including visuospatial memory, working memory, and verbal
memory (Hinkelmann et al., 2015), whereas blocking MR
function typically leads to impaired cognitive performance
(Cornelisse et al., 2011; Otte et al., 2007; Rimmele et al.,
2012) and, for instance, prevents a stress-induced shift
away from hippocampal-dependent memory functions
(Vogel et al., 2015). There is also some evidence from animal
studies that enhancing MR function improves memory
performance (Harris et al., 2013) but mainly so in transgenic
models (Ferguson and Sapolsky, 2007; Lai et al.,
2007). In one study, the MR antagonist spironolactone
administered systemically to rats after inhibitory avoidance
training impaired later retention (Atucha et al., 2015), but
earlier studies in mice found no effect of posttraining
spironolactone treatment on context- and cue-dependent
conditioning (Zhou et al., 2010). It is currently not known
whether the effects of peripherally administered MR agonists
and antagonists on cognitive performance in animals and
humans are caused by a direct influence on MR function in
the brain (Ferguson and Sapolsky, 2007; Karst et al., 2005) or
whether the effects are mediated via peripheral pathways by
modifying MR-induced influences on, for example, HPA-axis
activity (Lembke et al., 2013) or autonomic outflow (Borrell
et al., 1984; Rahmouni et al., 2001). The cognitive changes
426 Adrenal Stress Hormone Effects on Memory

may also involve an MR-dependent influence on GR function
(Liu et al., 1995).
1.15.2.3 Adrenergic–Glucocorticoid Interactions
Evidence from several kinds of studies indicates that although
epinephrine and glucocorticoid effects are initiated by different
brain mechanisms, these adrenal stress hormones interact to
influence memory consolidation. For example, glucocorticoid
administration alters the efficacy of epinephrine for influencing
memory consolidation in adrenalectomized rats (Borrell et al.,
1984, 1983). Moreover, the corticosterone-synthesis inhibitor
metyrapone attenuates the memory-enhancing effects of
amphetamine and epinephrine (Roozendaal et al., 1996).
Thus, synergistic actions of epinephrine and corticosterone
may be essential in mediating stress effects on memory
enhancement (Joëls et al., 2011).
Animal experiments investigating stress hormone effects on
memory consolidation typically use highly arousing training
conditions, such as inhibitory avoidance or contextual and
cued fear conditioning, that are known to induce the release
of both corticosterone and epinephrine. Studies using an object
recognition task investigated whether adrenergic activation
induced by emotional arousal is essential in enabling cortico-
sterone effects on memory consolidation (Okuda et al.,
2004). Rats were given either extensive habituation to an appa-
ratus, or no prior habituation, and were then allowed to
explore objects in the apparatus. Placing rats in a novel testing
apparatus evokes novelty-induced arousal and habituation of
rats to the apparatus is known to reduce this arousal response
(De Boer et al., 1990). Corticosterone administered immedi-
ately posttraining to nonhabituated (i.e., emotionally aroused)
rats enhanced their 24-h retention performance. In contrast,
posttraining corticosterone did not enhance retention of object
recognition in habituated rats (Okuda et al., 2004), providing
evidence that training-associated emotional arousal is essential
for enabling glucocorticoid effects on memory consolidation.
Other findings indicated that training-induced (nor)adren-
ergic activation is a critical component of emotional arousal
in enabling glucocorticoid effects on memory consolidation.
As shown in Figure 1(a), the b-adrenoceptor antagonist
propranolol administered systemically together with the corti-
costerone immediately after object recognition training
blocked the corticosterone-induced memory enhancement of
rats without prior habituation to the training context
(Roozendaal et al., 2006b). Additionally, and shown in
Figure 1(b), in habituated rats, pharmacological reinstatement
of adrenergic activity by the administration of the noradren-
ergic stimulant yohimbine (an a2-adrenoceptor antagonist)
after object recognition training enabled corticosterone to
induce dose-dependent enhancement of memory (Roozendaal
et al., 2006b). These findings strongly suggest that emotional
arousal-induced adrenergic activation is essential for enabling
glucocorticoid enhancement of memory consolidation.
Human studies generally support the conclusion of animal
experiments that glucocorticoids can enhance memory consol-
idation only when their activity is paralleled by emotional
arousal (i.e., noradrenergic activity). Cortisol administered
shortly before or after training selectively enhances long-term
memory of emotionally arousing, but not of emotionally
neutral, items (Buchanan and Lovallo, 2001; Kuhlmann and
Wolf, 2006). Moreover, a cold pressor stress in humans (i.e.,
placing the arm in ice water), a procedure that significantly
elevates endogenous cortisol levels, enhances memory of
emotionally arousing slides but does not affect memory of
emotionally neutral slides (Cahill et al., 2003; but see Preuss
and Wolf, 2009). Consistent with these findings, it has been re-
ported that levels of endogenous cortisol at the time of learning
correlated with enhanced memory consolidation only in indi-
viduals who were emotionally aroused (Abercrombie et al.,

(a) (b)

Figure 1 Glucocorticoid effects on memory consolidation for object recognition training require arousal-induced noradrenergic activation. Rats were
either habituated to the training context for 7 days (prior habituation) or not habituated (no prior habituation). On day 8, they were given a 3-min
training trial during which they could freely explore two identical objects, followed by systemic drug administration. Retention was tested 24 h later
by placing the rats back into the apparatus for 3 min. On the retention trial, one object was similar to the training objects, whereas the other was
novel. Data represent discrimination index (%) on the 24-h retention trial, expressed as mean
SEM. The discrimination index was calculated as the
difference in time spent exploring the novel and the familiar object, expressed as the ratio of the total time spent exploring both objects. (a) Effect of
immediate posttraining administration of the b-adrenoceptor antagonist propranolol (3.0 mg kg1, s.c.) on corticosterone-induced enhancement of
object recognition memory in nonhabituated (emotionally aroused) rats. (b) Effect of coadministration of the a2-adrenoceptor antagonist yohimbine
(0.3 mg kg1, s.c.) with corticosterone on object recognition memory in habituated (emotionally nonaroused) rats. **, p < 0.0001 vs vehicle. Adapt-
ed from Roozendaal, B., Okuda, S., van der Zee, E.A., McGaugh, J.L., 2006. Glucocorticoid enhancement of memory requires arousal-induced norad-
renergic activation in the basolateral amygdala. Proc. Natl. Acad. Sci. U.S.A. 103 (17), 6741–6746.

2006). Thus, these findings from animal and human studies
indicate that training-associated endogenous emotional
arousal is essential for enabling glucocorticoid effects on
memory consolidation.
1.15.3 Role of Noradrenergic Activity within
the Basolateral Amygdala in Mediating Adrenal
Stress Hormone Effects on Memory Consolidation
As summarized up to this point, both epinephrine and gluco-
corticoids enhance memory consolidation. The memory-
enhancing effects of epinephrine are initiated by activating
b-adrenoceptors on vagal afferents in the periphery, which
will then activate noradrenergic mechanisms in the brain.
Glucocorticoid hormones readily enter the brain, but also their
effects on memory consolidation depend critically on interac-
tions with emotional arousal-induced noradrenergic activity.
In this section we will go deeper into the neurobiological mech-
anisms of how epinephrine and glucocorticoids interact with
central noradrenergic activation in enhancing memory consol-
idation of emotionally arousing experiences. Furthermore, we
will present some recent evidence indicating that glucocorti-
coid interactions with the noradrenergic arousal system involve
fast, nongenomically mediated actions via a membrane-
associated GR and downstream endocannabinoid signaling.
1.15.3.1 Noradrenergic Activity of the Basolateral Amygdala
Mediates Epinephrine Effects on Memory Consolidation
Extensive evidence indicates that norepinephrine, originating
from cell groups in the NTS and LC, affects memory consolida-
tion by influences involving the amygdala and that the effects
of peripheral epinephrine on memory involve noradrenergic
activation of the amygdala. Early findings have shown that
adrenal demedullation or posttraining administration of
epinephrine alters the memory-modulating effects of electrical
stimulation of the amygdala (Liang et al., 1985) and that
lesions of either the amygdala or stria terminalis, an important
amygdala input–output pathway, block epinephrine effects on
memory consolidation (Cahill and McGaugh, 1991; Liang and
McGaugh, 1983). Experiments using the expression of imme-
diate early genes as markers for activated neurons reported
that stimulation of the vagus nerve, the pathway that mediates
epinephrine effects on the brain, increases the number of amyg-
dala neurons expressing c-fos. Electrophysiological findings
indicate that the firing rate of amygdala neurons is increased
following electrical stimulation of either the vagus nerve
(Radna and MacLean, 1981) or the NTS (Rogers and Fryman,
1988). In contrast, depletion of amygdala norepinephrine
with local injections of the norepinephrine-specific neurotoxin
DSP-4 blocks systemic epinephrine effects on memory consol-
idation (Liang, 1998). Posttraining intra-amygdala infusions of
the b-adrenoceptor antagonist propranolol also block the
memory enhancement induced by systemic administration of
epinephrine (Liang et al., 1986, 1995), whereas posttraining
infusions of norepinephrine into the amygdala attenuate reten-
tion impairment induced by adrenal demedullation (Liang
et al., 1986).
Adrenal Stress Hormone Effects on Memory 427
Extensive evidence now indicates that the BLA is the critical
region of the amygdala involved in mediating noradrenergic
influences on memory consolidation. The BLA is a cortical-
like structure that has many reciprocal connections with other
higher brain regions, including the hippocampus and
prefrontal cortex (Petrovich et al., 2001; Pitkänen, 2000).
Many studies found that posttraining infusions of norepineph-
rine or the b-adrenoceptor agonist clenbuterol into the amyg-
dala (or selectively into the BLA) induce a dose-dependent
enhancement of retention (Barsegyan et al., 2014; Bianchin
et al., 1999; Hatfield and McGaugh, 1999; Huff et al., 2005;
Izquierdo et al., 1992; Liang et al., 1986, 1995). In contrast,
b-adrenoceptor agonists or antagonists infused into other
amygdala regions such as the central amygdala are ineffective
(Hatfield and McGaugh, 1999). In addition to b-adrenoceptor
effects, a-adrenoceptor influences in the BLA also modulate
memory consolidation. Intra-BLA administration of the
a1-adrenoceptor antagonist prazosin impairs inhibitory avoid-
ance memory, whereas an activation of a1-adrenoceptors
enhances retention (Ferry et al., 1999a). The a1-adrenoceptor-
induced memory enhancement most likely involves an interac-
tion with b-adrenoceptors, as posttraining intra-BLA infusions
of the b-adrenoceptor antagonist atenolol block the memory
enhancement produced by activation of a1-adrenoceptors
(Ferry et al., 1999b). Considered together, these findings
strongly suggest that noradrenergic activation in the BLA plays
a critical role in enhancing memory consolidation and medi-
ating the effects of peripheral epinephrine on memory
consolidation.
Gold and van Buskirk (1978) reported evidence suggesting
that noradrenergic activity in the brain is increased in rats
trained on an inhibitory avoidance task or given memory-
enhancing doses of epinephrine immediately after training.
Subsequent studies using in vivo microdialysis and high-
performance liquid chromatography (HPLC) to measure
ongoing changes in norepinephrine levels indicated that
norepinephrine is normally released within the amygdala
during training on aversively motivated tasks. A single, brief
footshock stimulation of the kind typically used in fear-based
training induced norepinephrine release within the amygdala
that varied directly with the stimulus intensity (Galvez et al.,
1996; Quirarte et al., 1998). Moreover, epinephrine adminis-
tered systemically in a dose known to enhance memory
consolidation potentiated footshock-induced increases in
norepinephrine levels in the amygdala (Williams et al.,
1998). Further, stimulation of the vagus nerve or the NTS
increases norepinephrine levels in the amygdala and enhances
memory consolidation (Clayton and Williams, 2000; Hassert
et al., 2004). McIntyre et al. (2002) found that norepinephrine
levels in the amygdala increased significantly after inhibitory
avoidance training. Additionally, and importantly, the magni-
tude of training-induced increases in amygdala norepinephrine
levels assessed shortly after training correlated highly with the
rats’ subsequent long-term retention performance, providing
additional evidence suggesting that noradrenergic activity of
the amygdala regulates memory consolidation.
Human research corroborates these findings: administra-
tion of yohimbine either before (O’Carroll et al., 1999) or after
(Southwick et al., 2002) learning has been shown to enhance
memory. Carriers of a common functional deletion in the
428 Adrenal Stress Hormone Effects on Memory

gene coding for the presynaptic a2b-adrenoceptor (ADRA2B), (a)

exhibit a stronger enhancement of emotional memory (de
Quervain et al., 2007b). Conversely, blocking the effects of
norepinephrine using the b-adrenoceptor antagonist propran-
olol results in impaired memory for emotionally arousing
events (Cahill et al., 1994). Such noradrenergic effects are cen-
trally mediated: nadolol, a b-adrenoceptor blocker that does
not readily cross the blood–brain barrier, does not mimic the
effects of propranolol (van Stegeren et al., 1998). Human neu-
roimaging work furthermore supports the notion that such
noradrenergic effects are mediated by the amygdala. For
instance, increased activity in the amygdala was observed
shortly after experimental induction of stress in healthy volun-
teers (Cousijn et al., 2010; Oei et al., 2012; van Marle et al., (b)
2009) or after symptom provocation in anxiety-disordered
patients (Osuch et al., 2008). Pharmacological elevation of
levels of norepinephrine using reboxetine yields similar results
(Onur et al., 2009). Furthermore, carriers of the functional
deletion in the ADRA2B gene mentioned above show stronger
amygdala responses to aversive stimuli (Rasch et al., 2009) and
stronger stress-induced enhancements of amygdala activation
(Cousijn et al., 2010).
There is now compelling evidence that this amygdala (or
more specifically, BLA) activation induced by stress hormones
and norepinephrine during emotionally arousing stimulation
acts via efferent projections to enhance memory storage and
neuroplasticity mechanisms in other brain regions (McGaugh, Figure 2 Corticosterone treatment after inhibitory avoidance training
2004; McIntyre et al., 2005). These regions include the hippo- increases norepinephrine levels in the BLA. Microdialysis samples were
campus, entorhinal cortex, striatum, medial prefrontal cortex, collected every 15 min. Norepinephrine levels (mean
SEM) are
anterior cingulate cortex, and insula, and this list is likely to expressed as a percentage change from average baseline levels.
expand in the future (Hermans et al., 2014a). Notably, the (a) Corticosterone treatment (3.0 mg kg1, i.p.) significantly increased
norepinephrine release in the amygdala of animals trained on an inhibi-
same regions have been shown to exhibit strong intrinsic func-
tory avoidance task compared with vehicle-injected animals. *, p < 0.05
tional connectivity within distinct large-scale neural systems in
vs vehicle. (b) No changes in norepinephrine levels were observed in
the human brain. For instance, the hippocampal complex and corticosterone- or vehicle-injected animals that were not trained on the
medial prefrontal cortex are part of the midline-centered task. Adapted from McReynolds, J.R., Donowho, K., Abdi, A.,
‘default mode network,’ while insular, anterior cingulate, and McGaugh, J.L., Roozendaal, B., McIntyre, C.K., 2010. Memory-
striatal structures are part of the cingulo-opercular ‘salience’ enhancing corticosterone treatment increases amygdala norepinephrine
network (Hermans et al., 2014b; Menon, 2011). Functional and Arc protein expression in hippocampal synaptic fractions. Neuro-
connectivity, in particular within the latter network, is regulated biol. Learn. Mem. 93 (3), 312–321.
by norepinephrine (Hermans et al., 2011). Our understanding
of these network interactions and their contributions to
memory functions is likely to develop rapidly in the coming the b-adrenoceptor antagonists propranolol or atenolol into
years due to novel techniques such as multiunit recording the BLA, but not into the neighboring central amygdala,
and optogenetics. blocked the memory enhancement induced by a glucocorticoid
administered systemically (Quirarte et al., 1997). Atenolol or
the protein kinase A (PKA) inhibitor Rp-cAMPS administered
1.15.3.2 Glucocorticoids Interact with Noradrenergic
into the BLA also blocks memory enhancement induced by
Mechanisms within the Basolateral Amygdala
infusions of a GR agonist into the BLA (Roozendaal et al.,
Considerable evidence indicates that glucocorticoids also 2002) or dorsal hippocampus (Roozendaal et al., 1999). These
interact with training-associated noradrenergic activation findings, indicating that glucocorticoid effects on memory
within the BLA in enhancing the consolidation of memory of consolidation require concurrent activation of the b-adreno-
emotionally arousing training experiences (Roozendaal et al., ceptor-cAMP/PKA pathway in the BLA, are thus consistent
2009a). As shown in Figure 2(a), an in vivo microdialysis study with the notion that glucocorticoid administration selectively
reported that systemic administration of a memory-enhancing modulates memory consolidation of emotionally arousing
dose of corticosterone after inhibitory avoidance training information that induces the release of norepinephrine within
rapidly augmented norepinephrine levels within the amygdala the BLA. Other findings suggested that glucocorticoids
(McReynolds et al., 2010). In contrast, the same dose of corti- enhance memory consolidation, in a permissive fashion, by
costerone administered to nontrained control rats did not potentiating b-adrenoceptor-cAMP/PKA efficacy in the BLA
increase amygdala norepinephrine levels (Figure 2(b)). More- (Roozendaal et al., 2002). Posttraining intra-BLA infusions of
over, attenuation of noradrenergic signaling with infusions of the b-adrenoceptor agonist clenbuterol or the cAMP analog

8-bromo-cAMP are known to dose-dependently enhance
memory consolidation. Importantly, suppression of glucocorti-
coid signaling with a GR antagonist infused into the BLA
shortly before training reduced the sensitivity of BLA neurons
to the memory-enhancing effects of clenbuterol such that
a much higher dose of clenbuterol was required to induce
memory enhancement (Roozendaal et al., 2002). In contrast,
the GR antagonist did not modify the dose–response effects
of 8-bromo-cAMP, indicating that cAMP acts in the BLA down-
stream from the locus of interaction of glucocorticoids with the
b-adrenoceptor-cAMP/PKA pathway. In subsequent studies we
reported that such glucocorticoid effects on potentiating
b-adrenoceptor-cAMP activity results in increased phosphoryla-
tion of CREB protein (Roozendaal et al., 2006b, 2010). Collec-
tively, these findings indicate that glucocorticoids interact with
the noradrenergic system in modulating the molecular
machinery involved in memory consolidation to selectively
strengthen the consolidation of long-term memory of emotion-
ally significant events.
An interaction between glucocorticoids and noradrenergic
activity within the amygdala in emotionally influenced
memory has also been investigated in studies using functional
MRI (fMRI) in healthy humans. van Stegeren et al. (2007) re-
ported that the relationship between amygdala activity during
encoding and subsequent long-term memory was greatest for
the most emotionally arousing stimuli and for participants
with higher endogenous levels of cortisol. Importantly, b-adre-
noceptor antagonists blocked both the increase in amygdala
activity and the enhanced retention induced by emotional
stimuli. Kukolja et al. (2008) furthermore showed increased
activity in the amygdala in response to negatively arousing
images after combined administration of reboxetine and
hydrocortisone but not with either drug alone. Highlighting
again the fact that the effects of hydrocortisone depend on
concurrent noradrenergic activity, a study with administration
of only hydrocortisone showed suppression of amygdala
activity (Henckens et al., 2010).
Importantly, glucocorticoid interactions with the noradren-
ergic system are not limited to the BLA. For example,
a b-adrenoceptor antagonist administered into the nucleus
accumbens shell prevented glucocorticoid-induced memory
enhancement on both an appetitive and an aversive version
of taste learning (Wichmann et al., 2012). Posttraining infusion
of the GR agonist RU 28362 into the medial prefrontal cortex
also enhances memory consolidation of inhibitory avoidance
training (Roozendaal et al., 2009b), and a b-adrenoceptor
antagonist or PKA inhibitor coinfused into the medial
prefrontal cortex prevented this memory enhancement
(Barsegyan et al., 2010). Moreover, corticosterone adminis-
tered systemically immediately after inhibitory avoidance
training increased PKA activity in the medial prefrontal cortex
within 30 min.
Several experimental findings further suggested that gluco-
corticoid effects on increasing noradrenergic signaling might
have an onset that is too fast to be mediated via transcriptional
regulation in the nucleus and likely involve a rapid, nonge-
nomic mode of action (Dallman, 2005; de Kloet, 2000; Popoli
et al., 2012). Findings of electrophysiological experiments in
brain slices are consistent with the view that glucocorticoids
rapidly interact with the noradrenergic system to influence
Adrenal Stress Hormone Effects on Memory 429
neural plasticity and cellular activity in BLA neurons (Joëls
et al., 2011). Corticosterone applied simultaneously with
a b-adrenoceptor agonist rapidly facilitates AMPA receptor-
mediated responses in BLA neurons (Liebmann et al., 2009).
Nongenomic glucocorticoid actions likely involve the activa-
tion of a membrane-associated variant(s) of the steroid
receptor (Dallman, 2005; Johnson et al., 2005; Losel et al.,
2003; Riedemann et al., 2010). In support of this view, recent
findings indicate that the administration of the membrane-
impermeable glucocorticoid ligand cort:BSA, corticosterone
conjugated to a large bovine-serum albumin molecule, into
a variety of brain regions of the rat is sufficient to enhance
the consolidation of long-term memory of emotionally
arousing training experiences (Lee et al., 2011; Roozendaal
et al., 2010). As these cort:BSA effects are blocked by coadmin-
istration of a GR antagonist (Barsegyan et al., 2010;
Roozendaal et al., 2010), these findings suggest a role for
a membrane-associated GR in mediating rapid glucocorticoid
effects on memory.
Recent findings indicate that such rapid actions of glucocorti-
coid hormones on memory consolidation involve the endocan-
nabinoid system. The endocannabinoid system is a fast lipid
system in the brain and recently emerged as an important
stress–response system (Atsak et al., 2012b; Campolongo
et al., 2009; Evanson et al., 2010; Hill and McEwen, 2009; Hill
and Tasker, 2012; Morena et al., 2016). The first evidence for
a role of the endocannabinoid system in regulating glucocorti-
coid effects on the brain originated from an elegant series of
in vitro studies by Tasker and colleagues. They demonstrated
that corticosterone rapidly induced the release of endocannabi-
noids in the hypothalamus. Endocannabinoids then act in
a retrograde fashion to inhibit the release of glutamate in the par-
aventricular nucleus and suppress HPA-axis activity (Di et al.,
2003, 2005). More recently, an in vivo study by Hill et al.
(2010) corroborated these findings and showed that
a single injection of corticosterone rapidly (within 10 min)
elevated levels of the endocannabinoid anandamide in the
hypothalamus but also in the amygdala and hippocampus.
Endocannabinoid transmission appears to be essentially
involved in mediating glucocorticoid effects on memory
consolidation as the cannabinoid type 1 (CB1) receptor
antagonist AM251 administered into the BLA blocked the
ability of posttraining systemic corticosterone to facilitate
memory consolidation of inhibitory avoidance training
(Campolongo et al., 2009). Similarly, a CB1 receptor
antagonist infused into the hippocampus blocked memory
enhancement induced by the synthetic glucocorticoid
dexamethasone (de Oliveira Alvares et al., 2010). As shown in
Figure 3(a), training rats on an emotionally arousing
inhibitory avoidance task with high footshock intensity rapidly
increased anandamide levels in the BLA, whereas training rats
on this task with a lower footshock intensity did not increase
anandamide levels (Morena et al., 2014). These findings thus
indicate that endocannabinoids are released within the BLA
during emotionally arousing training conditions that are
associated with glucocorticoid release. To investigate whether
glucocorticoid–endocannabinoid interactions on memory
consolidation are dependent upon an adrenal steroid receptor
on the cell surface, we performed an additional experiment. As
shown in Figure 3(b) and 3(c), the CB1 receptor antagonist
430 Adrenal Stress Hormone Effects on Memory

(a) (b)

(c) (d)

Figure 3 Glucocorticoids interact with the endocannabinoid system of the BLA in enhancing memory consolidation of inhibitory avoidance training.
(a) Training rats on an inhibitory avoidance task with a high footshock (high FS; 0.45 mA, 1 s), but not low footshock (Low FS; 0.35 mA, 1 s), inten-
sity increased anandamide (AEA) levels in the amygdala 10, 30, and 60 min after the training. *, p < 0.05; **, p < 0.01 vs the no footshock (No FS)
group. Results represent mean
SEM. (b) Forty-eight-hour retention latencies (in seconds, mean
SEM) of rats given immediate posttraining intra-
BLA infusions of the GR agonist RU 28362 (1, 3, or 10 ng in 0.2 mL) alone or together with the CB1 receptor antagonist AM251 (0.14 ng). **,
p < 0.01 vs vehicle; , p < 0.01 vs RU 28362 alone. (c) Forty-eight-hour retention latencies of rats given immediate posttraining intra-BLA infu-
sions of the membrane-impermeable glucocorticoid cort:BSA (1, 3, or 10 ng in 0.2 mL) alone or together with AM251 (0.14 ng). *, p < 0.05; **,
p < 0.01 vs vehicle; , p < 0.05 vs cort:BSA alone. (d) Forty-eight-hour retention latencies of rats given immediate posttraining intra-BLA infusions
of the cannabinoid agonist WIN55,212-2 (10, 30, or 100 ng in 0.2 mL) alone or together with the GR antagonist RU 38486 (1 ng). **, p < 0.01 vs
vehicle. Adapted from Morena, M., Roozendaal, B., Trezza, V., Ratano, P., Peloso, A., Hauer, D., Atsak, P., et al., 2014. Endogenous cannabinoid
release within prefrontal-limbic pathways affects memory consolidation of emotional training. Proc. Natl. Acad. Sci. U.S.A. 111 (51), 18333–18338;
Atsak, P., Hauer, D., Campolongo, P., Schelling, G., Fornari, R.V., Roozendaal, B., 2015. Endocannabinoid signaling within the basolateral amygdala
integrates multiple stress hormone effects on memory consolidation. Neuropsychopharmacology 40 (6), 1485–1494.

AM251 infused into the BLA blocked the memory-enhancing
effects induced by concurrent infusions of either a specific GR
agonist or the membrane-impermeable ligand cort:BSA
(Atsak et al., 2015). In contrast, the GR antagonist RU 38486
infused into the BLA did not alter the memory-enhancing
effects of a CB1 receptor agonist (Figure 3(d)). Therefore, these
findings indicate that endocannabinoid transmission is
required for mediating glucocorticoid effects on memory
consolidation, presumably involving the activation of a GR on
the cell surface. Subsequent experiments indicated that such
glucocorticoid interactions with the endocannabinoid system
also mediate the rapid effects of glucocorticoids onto the
noradrenergic system (Atsak et al., 2015). These findings are in
line with prior evidence that systemic or local administration
of a CB1 receptor agonist increases norepinephrine levels in
limbic and cortical brain regions (Oropeza et al., 2005; Page
et al., 2007).
Such glucocorticoid-induced increases in endocannabi-
noid signaling might either directly influence noradrenergic
activity or, alternatively, alter noradrenergic function indi-
rectly via a modulation of GABAergic or glutamatergic
activity. Within the BLA, CB1 receptors are in particular
abundantly expressed on GABAergic interneurons (Katona
et al., 2001) and activation of CB1 receptors has consistently
been shown to suppress the release of GABA (Katona et al.,
2001, 1999; Ohno-Shosaku et al., 2001) via a rapid inhibi-
tion of calcium entry into the terminals (Hoffman and Lup-
ica, 2000; Wilson and Nicoll, 2001). Interestingly, inhibition
of GABAergic transmission with GABA receptor antagonists
is known to enhance memory consolidation by stimulating
norepinephrine release (Hatfield and McGaugh, 1999). As
illustrated in Figure 4, these findings thus suggest that corti-
costerone might bind to a GR on the cell surface and rapidly
induce the release of endocannabinoids. The released endo-
cannabinoids then bind to CB1 receptors on GABAergic
interneurons and inhibit the release of GABA that can then
result in an increased noradrenergic transmission in BLA
neurons.

Figure 4 A model illustrating the role of the endocannabinoid system
in integrating the effects of glucocorticoids and norepinephrine within
the BLA on memory consolidation. Glucocorticoids, released during
emotionally arousing situations, bind to a membrane-bound GR and
activate the intracellular cAMP/PKA signaling cascade. This triggers the
release of endocannabinoids, particularly anandamide (AEA). Ananda-
mide then activates CB1 receptors on GABAergic interneurons and
thereby inhibits GABA release. This subsequently disinhibits norepi-
nephrine (NE) release and increases the excitability of pyramidal
neurons within the BLA. This overall increases the sensitivity of BLA
neurons to the effects of norepinephrine and results in an increased
activation of the cAMP/PKA pathway and phosphorylation of CREB
protein. These stress hormone effects in the BLA are required for the
optimal enhancement of memory for emotionally arousing experiences
by influencing information storage processes in other brain regions.
Adapted from Atsak, P., Hauer, D., Campolongo, P., Schelling, G.,
Fornari, R.V., Roozendaal, B., 2015. Endocannabinoid signaling within
the basolateral amygdala integrates multiple stress hormone effects on
memory consolidation. Neuropsychopharmacology 40 (6), 1485–1494.
1.15.4 Adrenal Stress Hormone Effects on Other
Memory Functions
Most studies investigating the effects of adrenal stress hormones
on memory have focused on the neurobiological processes
underlying the consolidation of recent experiences. There is,
however, considerable evidence that adrenal stress hormones
also influence memory retrieval and working memory. In
contrast to the enhancing effects of stress hormones on memory
consolidation, adrenal stress hormones, and in particular gluco-
corticoids, have been shown to impair memory retrieval and
working memory. As will be discussed below, these stress
hormone effects also depend on rapidly mediated interactions
with both the noradrenergic and endocannabinoid systems.
1.15.4.1 Memory Retrieval
Studies showing that stress induces subsequent memory
impairments when retention is tested soon after learning,
Adrenal Stress Hormone Effects on Memory 431
before memory consolidation took place and when glucocorti-
coid levels are still elevated (Diamond et al., 1996; Kirschbaum
et al., 1996), provided the first indication that glucocorticoids
might influence memory retrieval processes. Subsequent
studies specifically investigated this issue by exposing rats to
stress or by administering glucocorticoids shortly before reten-
tion testing. Stress exposure or systemic corticosterone admin-
istration to rats shortly before testing for memory of training on
inhibitory avoidance, contextual fear conditioning, or water-
maze spatial tasks (24 h earlier) produces temporary impair-
ment of retention performance (Bohus, 1973; Cai et al.,
2006; de Quervain et al., 1998; Pakdel and Rashidy-Pour,
2006; Roozendaal et al., 2004a; Sajadi et al., 2006; Yang
et al., 2003). As the same treatments administered shortly
before training do not affect either acquisition or retention
performance assessed immediately after acquisition, such find-
ings indicate that glucocorticoids impair retention by influ-
encing memory retrieval. Several further studies from
different laboratories have indicated that glucocorticoids
impair memory retrieval of spatial or contextual memory in
rats and declarative (mostly episodic) memory in humans
(Buss et al., 2004; Coluccia et al., 2008; de Quervain et al.,
2000, 2003; Het et al., 2005; Kuhlmann and Wolf, 2005;
Rashidy-Pour et al., 2004; Roozendaal et al., 2003, 2004b;
Sajadi et al., 2007; Wolf, 2008; Wolf et al., 2001). Moreover,
increased cortisol levels due to psychological stress have also
been shown to impair declarative memory retrieval (Buchanan
et al., 2006; Domes et al., 2004; Kuhlmann et al., 2005b).
Although the vast majority of studies have investigated the
effects of stress and glucocorticoids on the retrieval of
hippocampus-dependent forms of memory, some animal and
human studies have shown that stress exposure or glucocorti-
coid administration before retention testing also impairs the
retrieval of cortex-dependent recognition memory (Barsegyan
et al., 2015) and striatal-dependent stimulus–response associa-
tions (Atsak et al., 2016; Guenzel et al., 2013).
Glucocorticoid effects on memory retrieval are highly
comparable to those seen in studies investigating memory
consolidation in that the effects depend on emotional arousal.
Specifically, it has been shown in studies in humans that
emotionally arousing information is especially sensitive to
the retrieval-impairing effects of glucocorticoids (Buchanan
et al., 2006; Buchanan and Adolphs, 2004; Buss et al., 2004;
de Quervain et al., 2000, 2007a; Het et al., 2005; Kuhlmann
et al., 2005a,b; Kuhlmann and Wolf, 2006; Schwabe and
Wolf, 2009; Smeets et al., 2008, 2009; Tollenaar et al., 2009;
Wolf et al., 2001). Studies of rats investigating the neural mech-
anisms underlying this selectivity have indicated that glucocor-
ticoid effects on memory retrieval depend critically on
noradrenergic activity within the brain. The b-adrenoceptor
antagonist propranolol administered systemically 30 min
before inhibitory avoidance retention testing blocks the
memory retrieval impairment induced by concurrent injections
of corticosterone (Roozendaal et al., 2004a). A b-adrenoceptor
antagonist infused into the hippocampus also prevents the
retrieval-impairing effect of a GR agonist administered concur-
rently (Roozendaal et al., 2004b). Together with the finding
that norepinephrine is activated by emotional arousal, this
could explain why emotionally arousing information or an
emotionally arousing test situation is a prerequisite for
432 Adrenal Stress Hormone Effects on Memory

enabling glucocorticoid effects on memory retrieval. In line
with this idea and illustrated in Figure 5, it has been reported
that the b-adrenoceptor antagonist propranolol blocked the
impairing effect of cortisone administration on the retrieval
of emotionally arousing material in healthy humans (de
Quervain et al., 2007a). Another study indicated that propran-
olol also blocked the impairing effect of stress induction on
memory retrieval (Schwabe et al., 2009). Further studies have
indicated that arousal-induced BLA activation interacts with
the hippocampus in mediating glucocorticoid effects on the
retrieval of emotionally arousing information. Lesions of the
BLA or infusions of a b-adrenoceptor antagonist into the BLA
block the impairing effect of a GR agonist infused into the
hippocampus on memory retrieval of spatial information
(Roozendaal et al., 2003, 2004b). In support of the importance
of an interaction between the amygdala and the hippocampus
during retrieval of emotionally arousing information, human
imaging studies have indicated that the degree of interaction
between these two brain regions is greater during the retrieval
of emotionally arousing declarative information as compared
to neutral information (Dolcos et al., 2005; Smith et al., 2006).
The temporary effects of glucocorticoids on memory
retrieval impairment are fast and do not seem to depend on
gene transcription (Sajadi et al., 2006). Recent findings indicate
that endocannabinoid signaling is also involved in mediating
glucocorticoid-induced impairment of memory retrieval. For
this experiment, rats were trained on a contextual fear condi-
tioning task and tested 24 h later for fear memory retention
(Atsak et al., 2012a). Blockade of hippocampal CB1 receptors
by local infusions of AM251, 1 h before retention testing pre-
vented the impairing effects of systemically coadministered
glucocorticoids on retrieval of contextual fear memory. More-
over, a retrieval-impairing dose of corticosterone elevated
hippocampal levels of 2-arachidonoyl glycerol (2-AG), but
not anandamide. As mentioned before, glucocorticoid effects
on memory retrieval highly depend on noradrenergic activity.
Thus, in order to determine whether endocannabinoids
mediate the effects of glucocorticoids on the noradrenergic
system, possible interactions between the endocannabinoid
and noradrenergic systems during retrieval processing of
contextual fear memory were examined. The CB receptor
agonist WIN55,212-2 infused into the hippocampus 1 h before
retention testing impaired the retrieval of contextual fear
memory; however, the b-adrenoceptor antagonist propranolol
blocked the impairing effect of WIN55,212-2 on memory
retrieval. Conversely, the CB1 receptor antagonist AM251
infused into hippocampus together with an impairing dose
of norepinephrine failed to abolish the impairing effect of
norepinephrine on memory retrieval.
1.15.4.2 Working Memory
Stress exposure is also known to impair working memory
(Arnsten and Goldman-Rakic, 1998; Schoofs et al., 2008).
Working memory is a dynamic process whereby information
is updated continuously, providing a temporary storage of
information (Baddeley, 1992; Jones, 2002). Evidence from
lesion, pharmacological, imaging, and clinical studies indicates
that working memory depends on the integrity of the medial
prefrontal cortex (Brito et al., 1982; Fuster, 1991; Lee and
Kesner, 2003; Levy and Farrow, 2001; Rowe et al., 2000; Stern
et al., 2001; Taylor et al., 1999). Basal levels of endogenous
glucocorticoids are required to maintain prefrontal cortical
function (Mizoguchi et al., 2004), but systemic injections of
stress doses of corticosterone or a GR agonist administered
into the prelimbic region of the medial prefrontal cortex
impair delayed alternation performance in rats, a task
commonly used to assess working memory performance in
rodents (Roozendaal et al., 2004c). As similar GR agonist infu-
sions into the medial prefrontal cortex do not impair delayed
alternation performance on nonmnemonic control tasks that
have similar motivational and motor demands (Barsegyan
et al., 2010), these findings indicate that stress levels of gluco-
corticoids, via GR activation, impair working memory. Addi-
tionally, stress-level cortisol treatment impairs prefrontal
cortex-dependent inhibitory control of behaviors in squirrel
monkeys (Lyons et al., 2000), as well as working memory
performance in human subjects during demanding tasks which
require a high level of arousal (Lupien et al., 1999; Wolf et al.,
2001; Young et al., 1999).

(a) (b)

Figure 5 Glucocorticoid effects on memory retrieval depend on emotional arousal and noradrenergic activity. (a) Cortisone (25 mg) administered to
humans 1 h before recall testing impaired retrieval of high-arousal words, but not of low-arousal words. (b) Concurrent administration of propranolol
(40 mg) prevented the impairing effect of cortisone on the recall of high-arousal words. Data are presented as mean þ SEM. **, p < 0.0001 vs the
corresponding placebo condition. Adapted from de Quervain, D.J.-F., Aerni, A., Roozendaal, B., 2007. Preventive effect of beta-adrenoceptor blockade
on glucocorticoid-induced memory retrieval deficits. Am. J. Psychiatry 164 (6), 967–969.

Importantly, glucocorticoid effects on working memory
impairment also depend on interactions with arousal-
associated noradrenergic mechanisms. In humans, negative
effects of stress induction on working memory (Elzinga and
Roelofs, 2005; Luethi et al., 2008; Oei et al., 2006; Taverniers
et al., 2010; Qin et al., 2012; Schoofs et al., 2008, 2009) and
prefrontal cortex activity (Ossewaarde et al., 2011; Qin et al.,
2009) are usually observed within the time window in
which both glucocorticoid and noradrenergic activity are
elevated. In rats, a b-adrenoceptor antagonist administered
systemically blocks the impairing effect of corticosterone on
working memory (Roozendaal et al., 2004c). Furthermore,
a b-adrenoceptor antagonist or cAMP blocker infused into the
medial prefrontal cortex blocks working memory impairment
induced by a GR agonist administered concurrently (Barsegyan
et al., 2010). Animal studies have shown that glucocorticoid
effects on working memory also depend on functional interac-
tions between the BLA and the medial prefrontal cortex. The
BLA both sends projections to and receives projections from
the medial prefrontal cortex (Likhtik et al., 2005; McDonald,
1991; Pérez-Jaranay and Vives, 1991; Rosenkranz and Grace,
2002). Drug administration into the BLA does not appear to
affect working memory (Bianchin et al., 1999; Wan et al.,
1994), but disruption of BLA activity blocks the impairment
induced by either systemic administration of corticosterone
or infusions of a GR agonist into the medial prefrontal cortex
(Roozendaal et al., 2004b).
1.15.5 Concluding Remarks
The past decades have witnessed a steady increase in our under-
standing of how adrenal stress hormones affect different
aspects of memory function. This knowledge is increasingly
used to gain a mechanistic understanding of the etiology and
treatment of intense emotionally arousing or traumatic
memory in psychopathology. For instance, the finding that
glucocorticoids impair memory retrieval of emotionally
arousing experiences has been successfully translated to patient
studies to alleviate symptoms of chronic stress after a highly
traumatic experience (Schelling et al., 2001) and in patients
with phobic fear (Soravia et al., 2006). A recent study further
showed that administration of hydrocortisone can enhance
the efficacy of extinction-based psychotherapy such as exposure
therapy in phobic patients (de Quervain et al., 2011). A critical
future challenge for this field is therefore to apply the detailed
knowledge acquired in experimental work to improve treat-
ment and prevention of memory-related and stress-related
disorders such as posttraumatic stress disorder and phobias.
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 1.16 Ovarian Hormones and Prefrontal Cortex-Related Cognition
Nicole J Gervais, University of Massachusetts, Amherst, MA, USA
Wayne G Brake, Center for Studies in Behavioral Neurobiology (CSBN), Concordia University, Montreal, QC, Canada
Agnès Lacreuse, University of Massachusetts, Amherst, MA, USA
Ó 2017 Elsevier Inc. All rights reserved.

1.16.1 Introduction 439

1.16.2 Anatomy of the PFC 439
1.16.3 Effects of Ovarian Hormones on PFC-Mediated Cognition 440
1.16.3.1 Working Memory 440
1.16.3.1.1 Humans 440
1.16.3.1.2 Nonhuman Primates 440
1.16.3.1.3 Rodents 441
1.16.3.1.4 Summary of E2 Effects on WM 442
1.16.3.2 Executive Function 442
1.16.3.2.1 Updating 442
1.16.3.2.2 Selective Attention 443
1.16.3.2.3 Response Inhibition 444
1.16.3.2.4 Task Switching 444
1.16.3.2.5 Decision Making 445
1.16.3.3 Multiple Memory System Bias 446
1.16.4 Effects of Ovarian Hormones on the PFC 447
1.16.4.1 Receptor Expression 447
1.16.4.2 Local Synthesis 447
1.16.4.3 Effects on Synaptic Plasticity and Signaling in the PFC 447
1.16.4.4 Correlations between Changes in Neural Substrates and Cognitive Performance 447
1.16.5 Conclusion 448
References 448

1.16.1 Introduction
This chapter focuses on the effects of ovarian hormones on
prefrontal cortex (PFC)-mediated cognitive function. These
cognitive processes encompass several distinct, yet overlap-
ping, constructs that together form executive function.
Executive function includes working memory (WM) and
processes that manipulate WM, such as updating, selective
attention, response inhibition, task switching, planning,
and decision making (Chudasama and Robbins, 2006).
Although there is some disagreement as to whether executive
function is a functionally homogeneous or heterogeneous
ability (Dalley et al., 2004), for the purpose of the present
chapter, each construct will be described separately. We
will address how ovarian hormones modulate each of these
processes, by reviewing data from the human, nonhuman
primate (NHP), and rodent literature. Much work has been
conducted on the effects of estrogens on PFC-dependent
function in females, primarily in the context of hormone
replacement therapy (HRT) in postmenopausal women and
related animal models. Less is known about the effects of
estrogens in younger women, and there is a remarkable
paucity of studies devoted to the effects of progestins. The
later sections of the chapter will summarize what is
currently known about the neural circuitry underlying the
effects of ovarian hormones on PFC-mediated function.
Finally, we will offer some suggestions for future studies
on this topic.
1.16.2 Anatomy of the PFC
The PFC in primates is located in the anterior portion of the
frontal lobe in both humans and NHPs. Although the subdivi-
sions are not entirely clear, and vary across studies (Wilson
et al., 2010), there is general agreement that the primate PFC
is composed of three main subregions, the dorsolateral
(DLPFC, Brodmann’s areas 9, 46, and 9/46; Petrides et al.,
2012), ventrolateral (VLPFC, areas 45 and 47/12; Petrides
and Pandya, 2002), and orbitofrontal cortices (OFC, areas
10, 11, 14, and 25, Elliott et al., 2000).
The DLPFC (area 9, 46, and 9/46 in primates) receives input
from the multimodal superior temporal sulcal cortex, the
rostral superior temporal gyrus, the anterior and posterior
cingulate cortex, and the retrosplenial cortex (Petrides and Pan-
dya, 1999; Petrides et al., 2005). This connectivity between
multimodal temporal areas and paralimbic cortical areas
(cingulate, retrosplenial, and rostral temporal cortex) reflects
its role in cognitive control and WM (Petrides et al., 1999).
The majority of the VLPFC of primates lies on the inferior
frontal gyrus. Area 45 receives input from the superior temporal
gyrus (auditory system) and multimodal areas of the superior

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440 Ovarian Hormones and Prefrontal Cortex-Related Cognition
temporal sulcus (Petrides et al., 2005). Area 47/12 has strong
connections with the rostral inferotemporal visual association
cortex and ventral limbic areas (i.e., perirhinal cortex, rostral
parahippocampal gyrus; Petrides and Pandya, 2002). The
OFC lies on the orbital surface of the frontal lobe (Elliott
et al., 2000) and receives input from visual association areas,
somatosensory, inferior temporal cortex and temporal pole,
amygdala, and mediodorsal thalamus (Elliott et al., 2000).
The OFC projects to the inferior temporal and entorhinal
cortices, anterior cingulate, hypothalamus, ventral tegmental
area, and caudate nucleus (Elliott et al., 2000).
There is some disagreement as to whether rodents are suit-
able models for understanding the functions of the PFC (see
Wise, 2009). While rats do not have a frontal lobe, they do
possess a PFC (Uylings et al., 2003) with three subregions,
including the medial PFC (mPFC). The mPFC can be further
subdivided into the prelimbic cortex (PL), infralimbic cortex
(IL), frontal cortical area (area Fr2), and dorsal anterior cingu-
late area (Heidbreder and Groenewegen, 2003; Uylings et al.,
2003). The PL is located in the dorsal portion of the mPFC,
while the IL is located below it. These two subregions show
functional similarities with the DLPFC of primates (Uylings
et al., 2003). While the IL/PL are not as anatomically special-
ized as the primate DLPFC, as will be discussed below, these
regions are associated with abilities in rodents that overlap
with PFC-dependent abilities in primates. Therefore, while
rodents may not demonstrate all the PFC-dependent abilities
observed in primates, they are useful models to study the
functional role of the PFC and neuronal mechanisms in less
complex cognitive abilities.
1.16.3 Effects of Ovarian Hormones
on PFC-Mediated Cognition
1.16.3.1 Working Memory
WM in humans is defined as “a system for temporary storage
and a mechanism for online manipulation of information
that occurs during a wide variety of cognitive activities”
(Baddeley and Hitch, 1974). For nonhuman animals, WM is
defined instead as “short-term memory for an object, stimulus,
or location that is used within a trial, but not typically across
trials” (Dudchenko, 2004). This type of memory is often distin-
guished from reference memory, which is memory for informa-
tion that is relevant across trials, such as rules of a particular
task (Dudchenko, 2004). In contrast, WM involves a representa-
tion of stimuli that is delay-dependent, subject to decay over
time, and sensitive to attentional processes.
1.16.3.1.1 Humans
Many tests are available for assessing WM in humans, most of
which involve the manipulation of WM through executive
control, rather than simple maintenance of information in
memory. The Digit Span subtest of the Wechsler Adult Intelli-
gence Scale (Wechsler, 2008), which assesses verbal WM, is
one exception. In this test, participants are required to recall
the order of digits either in the same (Digit Span forward) or
opposite order (Digit Span backward) in which they are pre-
sented. The total number of correctly listed items is taken to
reflect the strength of WM. Few studies in humans examined
the effects of estrogens on WM per se, as opposed to more
complex tasks of executive function. Estrogen replacement
therapy (ERT) administered cyclically or continuously to post-
menopausal women has no effect on the Digit Span-forward
subtest (Duff and Hampson, 2000; Keenan et al., 2001), but
improves performance on the backward subtest relative to the
non-ERT group (Duff and Hampson, 2000). Since the Digit
Span-backward subtest involves some manipulation of infor-
mation in WM, the observed effect of ERT on this subtest, in
the absence of an effect on the Digit Span-forward subtest,
suggests a potential effect of estrogens on the manipulation
of WM.
1.16.3.1.2 Nonhuman Primates
One commonly used WM test in NHPs is the delayed response
(DR) task, initially implemented by Jacobsen (1936). In this
task, the monkey watches as food reward is placed into one
of two food wells. Two identical tokens are then placed over
the wells, and then an opaque door is closed for a brief reten-
tion delay blocking the subject’s view of the wells. Following
the delay, the door is lifted and the monkey is allowed to
displace one of the two tokens. The monkey must remember
the location of the food reward during the retention delay in
order to perform well on this task. Several lines of research
demonstrate a role of the DLPFC in DR performance. For
example, single unit recordings in monkeys demonstrate
persistent firing of DLPFC neurons during the delay phase of
the DR task (Fuster and Alexander, 1971). An imaging study
conducted in humans reported sustained activation of the
lateral PFC during the delay phase of a spatial WM test
(Courtney et al., 1998). It is thought that this persistent firing
in the DLPFC is necessary to maintain task-relevant informa-
tion in WM (Goldman-Rakic, 1996). Several studies demon-
strate that damage to the DLPFC results in impaired
performance on the DR task (for a review, see Chudasama
and Robbins, 2006). Altogether, these studies convincingly
show a crucial role of the DLPFC for DR task performance.
Another commonly used test of WM in NHP is the delayed
matching/nonmatching-to-sample (DMS/DNMS) task, first
described by Mishkin and Delacour (1975). Similar to the
DR task, the DMS/DNMS task involves two phases, separated
by a retention delay that varies from a few seconds to minutes.
During the first phase, the monkey is presented with a sample
object and is required to displace it in order to obtain a food
reward hidden underneath it. An opaque door is then closed
and following a delay, the door is lifted, and the monkey
must now choose between the now familiar (i.e., sample
object), or a novel object. For the DMS task, the sample object
is paired with the reward, and during the DNMS task, it is the
novel object that is paired with reward. While this task, like
many other WM tasks, requires maintaining information
acquired during the first phase of each trial, it also requires
recognition of the sample object, which is an ability associated
with medial temporal lobe structures, such as the perirhinal
cortex (as reviewed by Winters et al., 2008; Warburton and
Brown, 2010). There is also evidence that the DLPFC (Moore
et al., 2012) and the VMPFC play a role, as damage to these
regions impairs performance on this task (Bachevalier and
Mishkin, 1986; Meunier et al., 1997). However, Meunier
et al. (1997) suggest that the role of the PFC in recognition
 Ovarian Hormones and Prefrontal Cortex-Related Cognition
tasks is likely to maintain associative memory, rather than
recognition per se.
Studies focusing on DR and DNMS performance in NHPs
have provided mixed evidence for beneficial effects of E2.
Roberts et al. (1997) compared performance of peri-/postmen-
opausal macaques to age-matched premenopausal females,
and young control monkeys on the DR task. While the two
older groups performed worse than the young control
group, the greatest WM deficit was observed in the peri-/
postmenopausal group. In addition, accuracy scores were posi-
tively correlated with urinary levels of estrogen metabolites
(i.e., estrone conjugate). These data are consistent with the
idea that estrogen loss during menopause accelerates the effect
of normal aging on WM. A subsequent study (Rapp et al.,
2003) tested aged OVX macaques (
22 years) on the DR and
DNMS, following replacement with low cyclic E2 (100 mg estra-
diol cypionate, i.m.) or vehicle every 3 weeks. Relative to the
vehicle-treated group, E2 improved performance on the DR
and on two delays of the DNMS. While these findings suggest
that E2 can reverse age-related cognitive decline on WM, not
all studies report consistent effects. For example, Voytko et al.
(2008) observed modest improvements on the DMS and no
effect on DR performance in OVX aged macaques
(19.7  0.5 years) given a cyclic hormonal regimen that
included continuous low E2 replacement (40–80 pg ml1,
serum, via silastic implants) and acute injections of E2 valerate
(0.10–0.20 mg ml1). Another study comparing long-term
OVX (>12 years) monkeys to gonadally intact age-matched
only revealed small impairments on the DNMS task in OVX
animals (Lacreuse et al., 2000). Furthermore, administration
of continuous ethinyl estradiol (EE2, 450 ng kg1 per day,
p.o.) or vehicle for a month did not affect performance on
the DR or DNMS task (Lacreuse et al., 2002). So, while ERT
appears to attenuate age-related cognitive decline in WM, this
appears to be influenced by both the hormone regimen (cyclic
versus continuous) and the duration of estrogen deprivation.
While studies using aged macaques (either peri-/postmeno-
pausal, or OVX with ERT within a critical window) suggest that
estrogens improve WM, this is not observed in younger
monkeys. For example, two studies found that endogenous
fluctuations in ovarian hormones do not influence WM perfor-
mance, as assessed by the DMS (Lacreuse et al., 2001; Kromney,
2015). In addition, OVX and various regimens of E2 replace-
ment have no effect on DR performance (Voytko, 2000; Hao
et al., 2007) or DMS tasks in young OVX macaques (Lacreuse
and Herndon, 2003, 2009).
1.16.3.1.3 Rodents
The most basic WM test used with rodents is the delayed spatial
alternation (DSA) test (Tolman, 1925). This test is often
administered in a T-shaped maze. The starting location is the
end of one arm. At the end of the starting arm is a junction,
with a goal arm to the left and right. Food rewards are placed
in wells at the end of each goal arm. Like WM tasks in NHPs,
the DSA test involves two phases, which are separated by
a retention delay. During the first phase, one of the goal arms
is closed and so the animal is forced to turn either left or
right to retrieve a reward from the accessible arm. The animal
is returned to the start arm for a retention delay, after which
the test phase of the trial begins. During this phase, both goal
441
arms are accessible, and the rodent must enter the previously
unentered goal arm to retrieve the reward. Like WM tests for
NHPs, longer delays can be introduced between the two
phases of each trial to increase WM load. Lesion studies
demonstrate that the mPFC, and in particular the PL, is
necessary for accurate performance on this test (Larsen et al.,
1978; Thomas and Brito, 1980; Wolf et al., 1987).
Studies in aged rats provide support for the idea that
estrogen loss during midlife accelerates age-related cognitive
decline. For example, Markowska and Savonenko (2002)
tested the effects of long-term estrogen loss on the DSA in
middle-aged-to-old rats (13–23 months). Performance deficits
emerged beginning 4 months post-OVX. WM ability was
restored among females given a cyclic regimen of E2 replace-
ment involving continuous low E2 in addition to three daily
subcutaneous injections of E2 (
25 pg ml1, serum) followed
by 1 day of no injection. A similar pattern of results was
observed in a study by Gibbs (2000) using the delayed
matching-to-place (DMP) task, a task similar to the DSA test
except that the reward appears in the same arm during both
phases of each trial: continuous low E2 (15–25 pg ml1,
serum, via silastic implants) or cyclic E2 (10 mg, s.c., once
a week) and progesterone (500 mg, s.c. 2 days after E2) replace-
ment prevented impairments in the middle-aged rats when
administered within 3 months of OVX.
Several lines of evidence suggest that DSA and DMP
performance is also improved by estrogens in young rats.
Impaired DSA performance was observed during estrous (low
E2) relative to the other phases of the estrous cycle in one study
(Korol et al., 2004). In another study, DMP performance was
improved by acute E2 replacement. Velázquez-Zamora et al.
(2012) showed that young rats (75–90 days) given estradiol
benzoate (EB, 10 mg, s.c.) or vehicle 6 days post-OVX
performed better on the DMP than vehicle-treated rats. Interest-
ingly, performance was also improved following administra-
tion of two selective estrogen receptor modulators, tamoxifen
(1 mg kg1) and raloxifene (1 mg kg1). Unlike acute adminis-
tration, continuous E2 replacement does not necessarily
improve WM in young rats. For example, continuous E2
replacement (86.2  8.2 pg ml1 serum, via silastic implants)
given immediately following OVX was found to reduce
accuracy on the DSA test (Wang et al., 2008, 2009). While these
data suggest that acute and continuous E2 regimens have
differential effects on WM, they might also reflect dose-
dependent effects, as opposing effects of low and high
E2 replacement are observed on the DSA task when reliance
on extramaze cues is minimized (Wide et al., 2004).
In that study, low continuous E2 replacement
(27.6  6.3 pg ml1, serum) improved, whereas higher doses
(medium: 103.3  8.5 pg ml1; high: 197.0  28.1 pg ml1,
serum) impaired, DSA performance in adult rats. These studies
provide evidence that low E2 replacement given within
3–4 months of OVX enhances performance on WM tasks in
both young and older rats. It is important to note that the tests
used (DMP and DSA) rely on the integrity of both the mPFC
and HPC (Dudchenko, 2004; Yoon et al., 2008). With the
exception of the study by Wide et al. (2004), which reduced
reliance on the HPC by limiting the availability of extramaze
cues, the reported improvement of WM performance by E2 in
rats may reflect enhanced function of either brain region.
442 Ovarian Hormones and Prefrontal Cortex-Related Cognition
1.16.3.1.4 Summary of E2 Effects on WM
Limited support exists for the enhancement of WM by estro-
gens in postmenopausal women. Stronger evidence for the
enhancement of WM following estrogen administration is
provided by studies in NHP and rats. However, a few factors
influence whether the effects are detected. Age, in particular,
appears to be an important factor in NHP studies. In aged
macaques, OVX impairs while E2 replacement enhances WM
ability, but the effects are more reliable following cyclic than
continuous replacement, and only occurs within a critical
window following OVX. While E2 is shown to enhance WM
performance in both young and older rats, the effect appears
to be dependent on the dose of E2 replacement, with low
replacement improving while moderate to high replacement
impairing WM ability. However, the majority of studies in
rats use tests that rely on both the PFC and HPC, and so these
effects might not solely reflect an influence of E2 on PFC func-
tion. Studies incorporating intra-PFC infusions of E2 are
needed to confirm whether E2 influences performance on these
tests via effects on this structure.
1.16.3.2 Executive Function
WM involves maintaining information during a delay often to
perform higher-order cognitive operations, such as updating,
selective attention, response inhibition, task switching, plan-
ning, and decision making. Together, these abilities are referred
to as executive function.
1.16.3.2.1 Updating
1.16.3.2.1.1 Humans
This ability is typically assessed in humans using the N-back
test (Cohen et al., 1997). In this test, participants are given
a random list of items, usually numbers, and when a target
item is identified, he/she must identify the item that appeared
N items previously. For example, in the two-back test, partici-
pants must identify the number that preceded the target by
two items. To perform well on this task, participants must
keep track of the last two numbers presented to them and
update these items as new numbers are presented. Both the
DLPFC and VLPFC are activated during this test (Owen et al.,
2005). Similar tests include the Digit Ordering task (Petrides
et al., 1993), the Self-Ordered Pointing test (SOPT; Petrides
and Milner, 1982; Petrides, 2000), and the spatial WM task
(SPWM; Duff and Hampson, 2000), which involve selecting
items among an array without repeating any selections.
Positive effects of estrogens have often been reported in
tasks that require updating WM. For example, menopausal
women (
52 years) on ERT (either oral CEE, transdermal E2,
or vaginal CEE cream) had stronger performance on the
N-back test relative to menopausal women not on ERT (Keenan
et al., 2001). ERT given briefly (100 mg, transdermally over
3 days; Krug et al., 2006), either alone or combined with
progesterone for at least 70 months (Duff and Hampson,
2000), is also associated with improved performance on the
Digit Ordering test. In addition to the Digit Ordering test,
HRT also improved performance on the SPWM (Duff and
Hampson, 2000). ERT may not have the same beneficial effects
in older women, however. Janowsky et al. (2000) reported no
effect of CEE (0.625 mg day1 for 1 month) on the Self-
Ordered Search test in older hysterectomized women
(61–74 years). Similarly, 12 weeks of ERT (transdermal E2,
50 mg day1) had no effect on spatial WM in older postmeno-
pausal sample (mean age 71; Schiff et al., 2005). These results
are consistent with the critical period hypothesis in that estro-
gens may improve performance in WM tasks that require
updating, only if given within a short period of time following
estrogen loss.
In women of reproductive age, elevated circulating levels of
E2 are associated with better performance on the SPWM task
(Hampson and Morley, 2013).
1.16.3.2.1.2 Nonhuman Primates
In NHPs, several tests have been developed to closely model
the updating of WM, such as the monkey version of the
SOPT (Petrides, 2000), the Self-Ordered Spatial Search task
(Weed, 1999), or the Delayed Recognition Span tests (DRST;
Moss et al., 1997). All require the animals to monitor their
selection in order to avoid retouching a previously selected
stimulus. The PFC has been shown to be involved in the perfor-
mance of these tasks (Beason-Held et al., 1999; Lamar and
Driscoll, 2007; Collins et al., 1998).
Seemingly contrasting the results in human participants,
Lacreuse et al. (2000) reported positive effects of long-term
ovariectomy (12 years) on the spatial-DRST in aged macaques,
when compared to gonadally intact age-matched controls.
However, a subsequent study (Lacreuse et al., 2002) found
beneficial effects of EE2 (450 ng kg1 per day, p.o. for
1 month) on the same task. Later work in rodents provided
convincing evidence that the loss of progesterone in long-
term OVX animals benefits spatial WM (assessed with a water
radial-arm maze task (RAM); Bimonte-Nelson et al., 2003,
2004). Thus, both the lack of progesterone and the presence
of E2 can benefit WM tasks requiring updating in aged NHP.
In young, gonadally intact macaques (5–7 years old),
impaired performance on the spatial-DRST was observed
during the periovulatory period (high E2; Lacreuse et al.,
2001). No effect was found on DMS, suggesting that the
impairing effect of E2 is specific to updating, without affecting
the simple maintenance of information in WM. In OVX young
macaques, E2 administration (EB, 2 mg kg1, s.c. given for
2 weeks) had negligible effects on three versions of the DRST,
the spatial-, object-, and face-DRST (Lacreuse et al., 2009).
However, a previous study with continuous EE2
(450 ng kg1 day, p.o.) given for 1 month reported negative
effects of EE2 on the face version of the DRST (Lacreuse and
Herndon, 2003), possibly due to the interactions between
estrogens and the processing of emotional material (i.e.,
conspecific faces).
1.16.3.2.1.3 Rodents
A rodent task that requires the updating of WM is the
RAM (Olton and Samuelson, 1976). The more common
configuration of the maze involves eight goal arms radiating
from a center platform. A food well is located at the end of
each arm, and doors can be lifted or lowered by the experi-
menter to control access to each arm. During the first phase
of each trial, doors to a number of arms (e.g., three or four)
are lifted allowing the rodent to enter each arm to retrieve
the rewards. Once rewards from these arms are consumed,
 Ovarian Hormones and Prefrontal Cortex-Related Cognition
the rodent reenters the center compartment for a retention
delay, whereby access to all arms is blocked. All doors are
then lifted allowing the rodent to collect remaining rewards.
WM is inferred from the number of errors, i.e., the number of
times the rodent reenters each arm. In addition to maintaining
spatial information about the previously entered arms, this test
also requires monitoring arm entries to avoid reentering an
arm. The PL (Seamans et al., 1995) and the HPC (Dudchenko,
2004) appear to play a role in this task.
Similar to effects seen on WM tests, E2 replacement
enhances RAM performance in middle-aged and older rats. In
one study (Daniel et al., 2006), 12- and 17-month-old female
rats were OVX, then given continuous low E2 replacement (20–
25 pg ml1, serum) or vehicle. E2 replacement began either
immediately following OVX, or 5 months later. While E2
replacement was associated with fewer WM errors (arm reen-
tries), this was only true for those that received replacement
within 5 months of OVX. These data provide support for the
critical period hypothesis in that higher E2 levels benefit updat-
ing only if administered around the time of estrogen loss.
RAM performance is also influenced by E2 in younger rats.
During proestrus, fewer WM errors are observed relative to the
estrous phase in gonadally intact female rats (Pompili et al.,
2010). Similar to the DSA test, there appears to be a dose-
dependent effect of E2 replacement on RAM in young OVX
rats, as low EB replacement (0.3 mg day1, s.c.) is associated
with more WM errors, whereas high EB replacement
(5 mg day1, s.c.) is associated with fewer WM errors relative
to controls (Sinopoli et al., 2006). Reduced WM errors is also
observed when E2 (0.9 mg/0.5 ml, but not 0.1 mg/0.5 ml) is
infused directly into the PL prior to testing on the RAM.
Another study (Holmes et al., 2002) reported opposite dose-
dependent effects of EB replacement on RAM performance,
where low replacement (0.3 mg day1, s.c) enhanced perfor-
mance, whereas medium (1 mg day1, s.c.) and high
(5 mg day1, s.c.) replacement impaired performance. Whereas
Sinopoli et al. (2006) used the win-shift version of this task,
which is dependent on both PFC and HPC, Holmes et al.
(2002) kept the same pattern of baited and unbaited arms
across sessions, decreasing reliance on the PFC. Therefore, the
majority of the results suggest that E2 enhances performance
on the RAM, with some evidence that it acts directly in the PL
to promote the updating of WM.
1.16.3.2.1.4 Summary of E2 Effects on Updating WM
There is reasonable evidence that updating information in WM
is improved by HRT or ERT in women. This is observed in
younger women and in postmenopausal women who begin
therapy during a critical window. In NHPs, the effect of E2
on updating is less clear, as higher levels are associated with
improved updating in older, but impaired updating in
younger, monkeys. A study reporting contrasting results
(Lacreuse and Herndon, 2003) used emotional stimuli
(conspecific faces) that may have reflected an effect of E2 on
emotional reactivity rather than cognitive performance per se.
Studies in rats used the RAM, which is a task that involves
both the PFC and HPC. Low continuous E2 replacement is
associated with improved RAM performance in older rats, but
impaired RAM performance in younger rats. Higher EB
replacement or intra-PL infusion of E2 enhances performance
443
on a win-shift version of the RAM in young rats. The extent
to which the tests used involve the HPC may contribute to
the inconsistent findings.
1.16.3.2.2 Selective Attention
The PFC, and in particular, the DLPFC, is also implicated in
visual attention, which is the ability to process important
sensory information while ignoring irrelevant information
(Squire et al., 2013). Damage to this region results in impaired
performance on tasks that require selective attention in both
macaques (Wardak et al., 2006) and humans (Knight et al.,
1995; Rueckert and Grafman, 1996). Impaired attention is
also observed in both rats (Muir et al., 1996; Chudasama and
Muir, 2001; Passetti et al., 2002) and mice (Kahn et al.,
2012) following damage to the mPFC. In addition, enhanced
neuronal responses have been observed in the DLPFC when
attention is directed at stimuli within the visual receptive field
(Lebedev et al., 2004; Buschman and Miller, 2007; Armstrong
et al., 2009; Gregoriou et al., 2009).
1.16.3.2.2.1 Humans
A test of selective visual attention is the continuous perfor-
mance test (Rosvold et al., 1956). Although there are several
versions of this test, they all involve presentation of stimuli
on a screen, and the participant must respond by pressing
a key when the target stimulus is presented. Other tests include
the Digit Symbol subtest of the WAIS. This test involves pre-
senting a set of digit–symbol pairs, and participants are shown
a list of digits and are asked to identify the symbols that are
paired with each symbol as quickly as possible. Studies report
inconsistent results, with some studies reporting no effect of
HRT on attention (Keenan et al., 2001; Wolf et al., 2005), while
other studies report improved attention following HRT (Smith
et al., 2001; Schmidt et al., 1996).
1.16.3.2.2.2 Nonhuman Primates
Visual attention can be assessed in NHPs using a variety of cued
search tasks. These tests require monkeys to identify a target
stimulus among an array of distractors or to attend to a cue
that indicates the probable location of a target (Baxter and Voy-
tko, 1996). Similar to the results in humans, studies using
NHPs report inconsistent effects of E2 replacement on atten-
tion. Voytko et al. (2009) trained aged macaques (
20 years
old) to criterion before ovariectomies were performed.
Some animals received immediate cyclic E2 replacement
(E2
70 pg ml1 via silastic implants) plus acute E2
(0.10–0.20 mg ml1, s.c. given for 12 days every 4 weeks).
Other monkeys received continuous E2 replacement (via
implants) and cyclic P replacement (0.2 mg kg1 given for
12 days month1). A third group received no replacement.
All macaques were tested on the cued search task 2, 12, and
24 weeks post-OVX. While hormone replacement had no effect
on overall performance, it increased release times on valid
(when cue is presented on same side as target) and invalid trials
(when cue is presented on opposite side as target), suggesting
a specific impairment in the processing of cues. The opposite
pattern of results was reported in a study with young monkeys
(6–11 years old). Decreased release times were observed
following E2 replacement (
130 pg ml1 via silastic implants)
relative to a no replacement group (Voytko, 2002). In another
444 Ovarian Hormones and Prefrontal Cortex-Related Cognition
study, no effect of EB replacement (2 mg kg1, s.c. given for
2 weeks) in young monkeys (7–16 years old) was observed
(Lacreuse et al., 2009).
1.16.3.2.2.3 Rodents
In rodents, selective attention is typically assessed by using the
5-choice serial reaction time test (5-CSRT; Robbins, 2002). The
rodent responds to a brief visual stimulus presented randomly
in one of five locations in order to obtain a food reward. The
number of correct responses over incorrect ones is taken as
a measure of attention. Performance on the 5-CSRT is
enhanced by continuous E2 replacement (
37 pg ml1,
serum) relative to a no replacement condition (Bohacek and
Daniel, 2010) in both young and older rats. In older rats, the
enhanced effect on selective attention is only observed when
given immediately, but not 5 months following OVX.
Latent inhibition (LI) is another way to assess selective
attention (Escobar et al., 2002). LI occurs when preexposure
to the conditioned stimulus results in a reduction in the
subsequent association with the unconditioned stimulus
(Cassaday et al., 2014). In contrast to the 5-CSRT test, LI is
impaired by both continuous (17–37 pg ml1, serum via
silastic implant) and cyclic (implant: 28 pg ml1, serum,
and acute E2:10 mg kg1, s.c. every fourth day) E2 replacement
(Almey et al., 2013). LI is also reduced when conditioning
occurs during proestrus, when circulating levels of E2 are
highest (Quinlan et al., 2010). These data provide paradoxical
effects on two different measures of selective attention in
OVX rats. Research is needed to identify potential mecha-
nisms by which E2 impairs LI while enhancing attention on
the 5-CRST test.
1.16.3.2.2.4 Summary of E2 Effects on Selective Attention
Studies on selective attention have provided mixed evidence for
an effect of E2, with several studies in humans and NHPs
reporting no effect. Among those that do report an effect,
some find enhanced attention following E2 replacement, while
others report impaired attention. In rats, E2 replacement
appears to enhance performance on the 5-CSRT test, while
impairing LI. More research is needed to clarify the role of
ovarian hormones in attention.
1.16.3.2.3 Response Inhibition
Response inhibition in humans is typically assessed using the
Stroop test (Stroop, 1935), the Go/No-Go test (Donders,
1969), or the Stop-Signal task (Logan et al., 1984). These tasks
involve inhibiting a prepotent response. For example, the
Stroop test involves presenting a list of words describing colors.
The words themselves are written in different colors. Partici-
pants are required to report the color each word is written in,
while avoiding reading the word. The Go/No-Go test and
Stop-Signal task require participants to make a response
when certain stimuli are presented and withhold a response
when other stimuli are presented.
One study (Krug et al., 2006) reported beneficial effects of
ERT (100 mg, transdermally over 3 days) in postmenopausal
women (50–65 years old) on the incongruent component of
the Stroop test. This effect was not replicated in an older sample
(58–75 years old) of women who were hysterectomized

20 years prior, then given either E2 alone (2 mg day1, p.o.),
or combined with P (daily E2: 2 mg day1; P: 100 mg day1,
p.o.), or placebo (Wolf et al., 2005). These contrasting results
are consistent with the critical period hypothesis. Findings in
younger women suggest a different pattern of results. For
example, high E2 during the menstrual cycle impairs response
inhibition, as assessed by a Stop-Signal task (Colzato et al.,
2010) and a Go/No-Go task (Reimers et al., 2014).
There is a paucity of studies investigating the effects of
ovarian hormones on response inhibition in NHPs. A recent
study in baboons (Lacreuse et al., 2016) found that Stop-
Signal performance was unaffected by the menstrual cycle in
females, although large sex differences in favor of females
were observed in response inhibition and overall response
times. Additional studies are needed to understand how
gonadal hormones may influence these sexually differentiated
patterns of performance.
The effect of E2 replacement on response inhibition has also
been assessed in young, middle-aged, and older OVX rats
(Wang et al., 2011). OVX rats receiving continuous E2 replace-
ment were trained on the differential reinforcement of low rates
of responding task, which requires the rats to suppress a lever-
pressing response for a fixed period of time in order to obtain
a food reward. In the young and middle-aged groups, contin-
uous E2 replacement (20 and 40 pg ml1, serum) was associ-
ated with reduced ratio of reinforced to nonreinforced
responses, suggesting impaired response inhibition in these
animals.
1.16.3.2.3.1 Summary of E2 Effects on Response Inhibition
Few studies have investigated the effects of ovarian hormones
on response inhibition. Studies in postmenopausal women
suggest improved response inhibition following ERT. However,
higher E2 levels appear to impair response inhibition in young
cycling women and in rats regardless of age. More research is
necessary to better understand the role of E2 in response
inhibition.
1.16.3.2.4 Task Switching
Task switching is defined as the ability to switch attention from
an initially important feature to another that subsequently
conveys information about where to respond correctly to
obtain a reward (Butts et al., 2013). Task switching ability
involves the DLPFC in humans and NHPs (Heekeren et al.,
2006; Seo et al., 2007; Nakahara, 2002) and the mPFC in
rodents (Floresco et al., 2009; Ragozzino et al., 1999). One
basic form of task switching is reversal learning, which is the
ability to shift responses to stimuli following a shift in stim-
ulus/reward contingencies (Butts et al., 2013). This ability is
dependent on the OFC (Butts et al., 2013; Kehagia et al.,
2010). Damage to the OFC is often associated with persevera-
tive responding, the selection of the previously rewarded stim-
ulus, despite a change in stimulus/reward contingency (Jones
and Mishkin, 1972; Rolls et al., 2004; Dias et al., 1996a,b;
Fellows and Farah, 2003).
1.16.3.2.4.1 Humans
Task switching is typically assessed in humans using the Wis-
consin Card Sorting Test (WCST, Heaton, 1981). In this test,
participants are presented with cards displaying a varying
number of shapes of different colors. The participant is
 Ovarian Hormones and Prefrontal Cortex-Related Cognition 445

required to figure out, by using the experimenter’s feedback, one possibility is that E2 has opposing effects on the OFC
whether to sort the cards by shape, number, or color. Once and PFC. More research is needed to address this potential
the rule is acquired, the experimenter switches the rule and explanation.
records the number of trials required to acquire the new
rule. There is conflicting evidence that ERT improves this
1.16.3.2.5 Decision Making
ability. For example, Schmidt et al. (1996) compared WCST
Decision making is often assessed in humans via the Iowa
performance between postmenopausal women (
60 years
Gambling task (IGT; Bechara et al., 1994). The IGT is a comput-
old) taking ERT and those who never took ERT. While the
erized task that involves presenting four deck of cards on
number of category shifts was higher for the ERT group, the
a screen. Decks vary in the number of cards that result in lost
overall number of errors, and number of perseverative errors,
money, with two decks being considered advantageous, and
was equal across the two groups. Using a cross-over design,
two decks disadvantageous. Performing well on the IGT
Berman et al. (1997) reported no effect of ERT or P on
involves learning from as few trials as possible the payoffs asso-
WCST performance in younger women (age: 27–49 years
ciated with each deck and selecting cards from the advanta-
old) whose ovarian function was suppressed via injection of
geous decks, while minimizing selections from the
a gonadotropin-releasing hormone agonist (GnRH-a).
disadvantageous ones. The task involves the VMPFC/OFC
However, the activity patterns in the PFC were clearly
and the DLPFC (Bechara et al., 1998; Christakou et al., 2009;
reduced by the GnRH-a and restored following treatment
Lawrence et al., 2009). Another decision-making task for use
with E2 or P.
in humans is the Delayed Discounting (DD) task (Altamirano
et al., 2011; Smith and Boettiger, 2012). This task requires
1.16.3.2.4.2 Nonhuman Primates
participants to make choices between smaller, immediate
NHP studies provide evidence for a modulatory effect of E2 on
rewards and larger, eventual ones. This task taps into the ability
task switching. Voytko et al. (2009) reported that aged
to choose delayed larger rewards over smaller immediate ones.
macaques (
20 years old) treated with cyclic E2 or E þ P
A related decision-making task used in rodents is called the
performed better than the no replacement group in a monkey
Effort Discounting (ED) task. Subjects are trained in an operant
version of the WCST, as indicated by fewer trials to learn the
chamber containing two levers. One lever is paired with the
set-shift and fewer perseverative errors. However, E2 replace-
immediate delivery of a small reward (two pellets), and the
ment (EE2, 450 ng kg1 per day) failed to enhance perfor-
second reward is paired with a delayed delivery of a large
mance on the primate-WCST in aged (23–26 years) long-term
reward (four pellets). The rodent must choose between the
OVX macaques (Lacreuse et al., 2004).
two levers. Upon choosing the lever paired with the large
E2 replacement also modulates reversal learning in marmo-
reward, the subject must press that lever an additional 5–20
sets. Adult OVX marmosets (2.5–5 years) given immediate
times, with the number of additional level presses increasing
continuous E2 replacement (360  24.21 pg ml1, serum) per-
as the test sessions progress. Therefore, choosing the lever
formed worse on one of three reversals and showed an increase
paired with a large reward becomes increasingly costly as the
in perseverative responding across the three reversals compared
session progresses.
to the control group (Lacreuse et al., 2014).
E2 does not appear to influence performance on the IGT
in both young and postmenopausal women (Reavis and
1.16.3.2.4.3 Rodents
Overman, 2001). However, there is some evidence that perfor-
Impaired reversal learning was also found in aged OVX rats
mance on the DD task varies across the menstrual cycle. Smith
given continuous E2 replacement (39 pg ml1; Gibbs et al.,
et al. (2014) tested naturally cycling women (age 18–40 years)
2011). Another study in young OVX rats used the extradimen-
and reported a decrease in the tendency to prefer smaller
sional set-shifting task to assess task switching (ED task;
immediate rewards from the menstrual (low E2) to the follic-
Lipatova et al., 2016). This task was administered in a plus
ular phase (high E2). In addition, this decrease was negatively
maze, with arms that differed in terms of brightness (light or
correlated with E2 levels, suggesting that higher levels of E2 are
dark) and texture (smooth or rough), and the rat had to attend
associated with a decreased bias for immediate reward (Smith
one or the other feature. Three groups were tested, an acute E2
et al., 2014).
group (two injections, 10 mg kg1, s.c.; 48 h and 24 h before
The effects of E2 on decision making has also been assessed
training), a continuous E2 (
40 pg ml1, serum via silastic
in rats (Uban et al., 2012) but yielded opposite results, as high
implants), and a no replacement group. Both E2 groups
EB replacement (10 mg, s.c.) resulted in reduced preference for
required less trials than the no replacement group to acquire
the more costly/higher reward lever on the ED task.
the ED shift.

1.16.3.2.4.4 Summary of E2 Effects on Task Switching
Mixed findings are reported across species, and the direction
of the effect appears to depend on the task. E2 impairs
reversal learning in both NHPs and aged rats and enhances
set-shifting in postmenopausal women, aged monkeys, and
young rats. The enhanced effect of E2 on set-shifting is
dependent on the timing of replacement, as it has no effect
when administered following a critical period. Since
different PFC structures are implicated in these two tasks,
1.16.3.2.5.1 Summary of E2 Effects on Decision Making
Few studies have investigated the effects of ovarian
hormones on decision making. While ovarian hormones
do not appear to influence performance on the IGT,
elevated E2 levels during the menstrual cycle in young
women are associated with improved performance on the
DD task. However, high EB replacement in young rats is
associated with impaired ED task performance. While these
results provide some support for the idea that E2 modulates
446 Ovarian Hormones and Prefrontal Cortex-Related Cognition
cost/benefit analysis, more research is necessary to clarify
the direction of the effect.
1.16.3.3 Multiple Memory System Bias
Rats learning to complete a maze can use one of several
memory systems (Tolman et al., 1946). Firstly, they may use
several cues from the environment, or allocentric cues, to
develop a cognitive map and use this approach to find the
reward. This approach is referred to as ‘place’ learning and
memory and is mediated predominantly by the hippocampus
(Packard and McGaugh, 1996). Secondly, rats may use egocen-
tric cues to learn where to go to find the reward. In this case, the
rats would learn a series of habitual motor responses such as
turn left or turn right. This approach is referred to as ‘response’
learning and memory and is mediated predominately by the
dorsal striatum, also known as the caudate/putamen (Packard
and McGaugh, 1996). Thirdly, rats may also rely on the
memory of a single, specific extramaze cue such as go toward
the door in the room, or an intramaze cue such as go down
the arm that has a scratch on it. As would be expected, rats
use a combination of these memory systems under normal
circumstances when they are allowed to use any memory
system to learn where to go in a maze to find a reward (Chang
and Gold, 2003).
It was Tolman and colleagues in the 1940s (Tolman et al.,
1946) who first proposed that rats can use either place or
response memory to solve a maze. They showed that initially,
male rats more readily use place memory over response
memory but are capable of using either one. We now know
that male rats use a combination of both memory systems
when both can be used to solve the maze (Chang and Gold,
2003) switching from an initial place memory system to a later
response memory system upon repetition of testing in the same
maze. It was later shown by Norman White and colleagues
(e.g., McDonald and White, 1993, 1994) that selective inactiva-
tion of the hippocampus impairs the use of a place memory,
while inactivation of the dorsal striatum results in deficient
response memory (Packard and McGaugh, 1996). Spatial
WM is impaired in rats with hippocampal damage but not
dorsal striatal damage (Kesner, 1990). Response memory of
a directional turn, on the other hand, is attenuated in rats
with dorsal striatal lesions but not hippocampal lesions
(Cook and Kesner, 1988). During response tasks in which
dorsal striatal lesions significantly impair performance, rats
with damaged hippocampi, but intact dorsal striatal function,
exhibit an enhanced performance compared to normal rats
(Matthews et al., 1995). This suggests a dissociation of the
neural bases for particular memory systems in which the effec-
tiveness of a particular strategy in solving a task is enhanced if
relevant information from the alternative structure is made
unavailable.
These studies, notably all using male rats, strongly suggest
the existence of structurally and functionally distinct
multiple learning and memory systems in the brain. It is
also important to note that it is not the mere loss of function
of one structure that allows the other to take over, but that
when one structure is impaired, the learning and memory
system subserved by the other brain structure appears to be
enhanced. That is, lesion of the hippocampus, for example,
not only biases animals to use response memory, but it
also enhances response learning and memory and vice versa.
Under normal circumstances, male rats will begin to solve
a maze by using place memory, but as they become highly
trained on the task they switch to using response memory
(Chang and Gold, 2003).
A seminal paper by Donna Korol et al. (2004) showed
that when naturally cycling female rats were allowed to
solve a modified plus maze using any learning and memory
system, the majority of proestrous rats used place memory
while most estrous rats used response memory. This finding
has been repeatedly confirmed (for review see Hussain
et al., 2014). Female rats in the modified plus maze para-
digm are considered overtrained; that is, upon reaching
criterion they have found the reward in the same arm for
at least 40 trials. This led to the question, why do high E
female rats persist in using a hippocampus-based place/
spatial memory system? It is well established that E
improves hippocampus-based behaviors, but this does not
explain why these rats do not eventually switch to using
response memory like the males do.
E interacts with DA to influence memory bias in female
rats. Systemically administered DA D1 and D2 receptor antag-
onists are able to differentially shift the memory system used
by female rats administered low but not high E (Quinlan
et al., 2008). However, there is a limitation to the interpreta-
tion of those findings because DA antagonists were adminis-
tered systemically. At that point, it could not be concluded
that E is acting via DA specifically in the striatum to shift
memory system bias. Another experiment correlated striatal
dopamine levels, measured via in vivo microdialysis, with
the amount of bias in using either place or response memory
(Hussain et al., 2016a). In that study it was found that while E
levels were a significant predictor of whether a female rat
would use place or response memory, dorsal striatal dopa-
mine levels themselves were not a significant predictor of
memory system bias. Could estrogen be acting on dopamine
elsewhere in the brain to affect which memory system is used
in females?
Another study infused E directly into the PFC and tested
memory bias 15 min later. A dramatic shift in memory system
bias was observed. When E was infused into the PFC of low E
rats, they all switched to using place memory but did not switch
when vehicle was infused (Almey et al., 2014). The fact that E
has a rapid effect on memory system bias in the PFC indicated
that estrogen must be acting on membrane-associated E recep-
tors in the PFC and not intracellular receptors which would
take hours.
There has only been one study on women and multiple
memory system bias published to date. Women also show
a bias on which memory system they use that is dependent
upon phase of the menstrual cycle. Women tested in the
mid/late luteal phase, when progesterone is high, predomi-
nantly use spatial/place memory, whereas the opposite
pattern is observed in women in the early follicular and ovula-
tory phases (Hussain et al., 2016b). Surprisingly, it appears
that progesterone is most important for memory system
bias in women. The actions of progesterone in the PFC in
women are unknown. We are not aware of any study investi-
gating multiple memory system bias in NHPs.
 Ovarian Hormones and Prefrontal Cortex-Related Cognition
1.16.4 Effects of Ovarian Hormones on the PFC
1.16.4.1 Receptor Expression
All three estrogen receptors (ERa, ERb, GPER1) are expressed in
the PFC of rats and monkeys (Almey et al., 2014; Crimins et al.,
2016; Pau et al., 1998). Among female rats, these receptors are
localized almost exclusively at extranuclear sites, including
axons and axon terminals (Almey et al., 2014), suggesting
that estrogens alter presynaptic transmission in the PFC.
GPER1 in the monkey PFC is also localized predominantly
on presynaptic membranes (Crimins et al., 2016). ERa and
ERb mRNA are found in human PFC, although their distribu-
tion varies, with ERb found in layers V–VI, whereas ERa is
restricted to layer V (Osterlund et al., 2000a; Osterlund et al.,
2000b).
Both progesterone receptor isoforms (PR-A and PR-B) are
expressed in the frontal cortex of rats (Guerra-Araiza et al.,
2003), although the PR-B isoform appears to be more
abundant than PR-A in this area (Guerra-Araiza et al., 2000).
1.16.4.2 Local Synthesis
While E2 is synthesized in the ovaries of females, there is also
evidence that it is produced locally in the brain of both sexes,
including in the PFC. Aromatase, which is the enzyme that
converts testosterone to E2, is expressed in astrocytes in this
region (Luchetti et al., 2011). Aromatase expression in the
PFC appears to be higher in female than male rats (Wei et al.,
2014). While E2 concentrations are detectable in the PFC,
levels are comparable between gonadally intact and OVX
female rats (Barker and Galea, 2009), further supporting the
idea that E2 is synthesized locally in the PFC. Given that E2
levels were not detectable in males (intact and GDX), it seems
plausible that sex differences exist in the local synthesis of E2 in
this region.
1.16.4.3 Effects on Synaptic Plasticity and Signaling
in the PFC
While cyclic E2 replacement (100 mg estradiol cypionate, i.m.
given every 3 weeks) improves WM in aged OVX macaques,
no effect is observed in young monkeys (Hao et al., 2007;
Rapp et al., 2003). However, dendritic spine density in layer
III pyramidal neurons of the DLPFC (area 46) is enhanced by
this E2 regimen in both age groups (Hao et al., 2007). OVX
is associated with reduced dendritic spine density in the
mPFC of female rats (Wallace et al., 2006). In addition to spine
density, cyclic E2 replacement (100 mg every 3 weeks, i.m.) is
also associated with increased expression of spinophilin, a post-
synaptic protein, in young macaques (Tang et al., 2004).
Spinophilin expression is also enhanced by continuous E2
replacement (via time-release pellets) in OVX rats (Khan
et al., 2013). Taken together, these data suggest that E2
increases synaptic density in the PFC.
In addition to increasing synaptic density, E2 replacement
also appears to increase excitatory glutamatergic synapses, but
has no effect on GABAergic synapses in the PFC (Khan et al.,
2013). In the same study E2 replacement was shown to rapidly
enhance ERK (2–7 min) and Akt (10–30 min) signaling in this
structure (Khan et al., 2013). Synaptic potentiation that is
447
GluN2B-dependent is also influenced by endogenous
fluctuations of E2 (Galvin and Ninan, 2014). While E2
rapidly affects signaling in the PFC, suggesting nongenomic
mechanisms, it is currently unknown whether the effect on
PFC-dependent cognition is also nongenomic in nature.
Studies are needed to determine the mechanisms through
which E2 influences cognition in this region.
A limited number of studies have examined the effect of
progesterone treatment on synaptic plasticity in the PFC, and
among these studies none have examined the independent
effects of this hormone. One study examined the effect of
combined E2 replacement (p.o., 25–30 pg ml1, serum) with
either P (via implant) or medroxyprogesterone acetate (MPA;
via implant) on synapse number in middle-aged OVX rats.
MPA is a synthetic progestin that is most commonly prescribed
to women (Chisholm and Juraska, 2012). The number of
synapses was inferred from the number of boutons expressing
synaptophysin, a synaptic protein, which was measured in both
the PL and IL of the mPFC. While E2 alone had no effect on
synapse number, E2 combined with MPA treatment increased
the number of synaptophysin-expressing boutons. E2
combined with P had no effect on synapse number. These
results provide some support for the idea that certain proges-
terone treatments may regulate synaptic number, although it
is not clear if the results reflect independent or combined effects
of E2 and MPA. While MPA when combined with E2 may
increase synapse number, it does not appear to influence
dendritic spine density. Progesterone given either cyclically or
continuously (cyclic: 100 mg, given for 10 days after acute
injection of E2, then every 28 days; continuous: 2.79–
3.40 ng ml1, serum via daily capsules) combined with E2
(cyclic: 100 mg, i.m, given once every 28 days; or continuous:
91.62–449.28 pg ml1, serum via silastic capsules) had no
effect on spine density in the DLPFC in aged OVX macaques
(Ohm et al., 2012; Young et al., 2013). However, since synap-
tophysin is a presynaptic marker, it is possible that the effects of
P are restricted to presynaptic neurons.
1.16.4.4 Correlations between Changes in Neural Substrates
and Cognitive Performance
Wang et al. (2010) provide some evidence for a role of ERa in
PFC-dependent cognition by correlating receptor expression
with DR task performance. In young OVX monkeys, DR task
performance was shown to correlate with presynaptic
expression of ERa (Wang et al., 2010). In aged OVX given
cyclic E2 replacement (100 mg ml1 estradiol cypionate, i.m.
every 3 weeks), DR task performance was correlated with
postsynaptic expression of ERa within the postsynaptic
density. A decrease in the expression of GPER1-containing
nonperforated axospinous synapse density was also observed
in aged OVX macaque, which was partially restored following
cyclic E2 replacement. However, this marker was not
correlated with DR task performance.
Multisynaptic boutons (MSBs), which are presynaptic
protrusions that form multiple synapses with other neurons,
are sensitive to OVX in aged monkeys in area 46 of the DLPFC.
While OVX reduces the frequency of these boutons, cyclic E2
replacement (100 mg ml1 estradiol cypionate, i.m. every
3 weeks) restores MSBs to levels comparable to young and
448 Ovarian Hormones and Prefrontal Cortex-Related Cognition
aged premenopausal monkeys. MSBs in this region are also
positively correlated with DR task performance (Hara et al.,
2016), suggesting a potential role in E2 mediated improve-
ments in WM ability in aged OVX monkeys.
1.16.5 Conclusion
This chapter was organized around a set of cognitive constructs
that form executive function, the mental processes orchestrated
by the PFC that are necessary for the cognitive control of
behavior. These cognitive processes, which manipulate WM
through updating, selective attention, response inhibition, task
switching, and decision making, all show some degree of sensi-
tivity to estrogens in females, but the direction and magnitude
of these effects are far from being uniform. Differential effects
of estrogens on PFC-dependent cognition are observed
according to a variety of task-, drug-, and population-related
factors such as specific task demands, dose, duration and type
of E2, duration of estrogen loss, and age of the subjects. There
are also species differences that are not clearly understood. For
example, most rodent studies report significant effects of
estrogens on PFC-dependent function in young as well as
older animals. In contrast, NHP studies rarely find significant
effects in younger subjects. As most human studies have been
conducted in older women, it is not clear how age affects the
E2 modulation of PFC function in women. Another
important factor to consider is how E2 interacts with
neurotransmitters such as dopamine, acetylcholine, and
serotonin to influence PFC-dependent cognition. For example,
it has been demonstrated that E2 can have enhancing or
impairing effects on WM depending on dopamine levels
(Jacobs and D’Esposito, 2011), and dopaminergic drugs
can influence the effect of E2 on LI (Almey et al., 2013).
While some studies examine how E2 interacts with
neurotransmitters to modulate cognition, more attention is
needed to understand the discrepant results reported in the
literature.
Another fruitful avenue of research is to further our under-
standing of the impact of neuroestrogens on cognition. Indeed,
as suggested by others in the context of hippocampal function
(Prange-Kiel et al., 2013), estrogens synthesized in the brain
may be more important for cognitive function than peripheral
estrogens. Interestingly, we still do not know whether E2
synthesized in the brain declines with age in any species.
Furthering our knowledge in this area would likely provide
crucial cues to understanding the complex effects of age and
ovarian function loss on cognitive function. Recent data in
the macaque HPC (Higaki et al., 2012) revealed that aromatase
expression was upregulated in premenopausal OVX monkeys
without changes in ERb expression, while ERb expression was
markedly upregulated in menopausal monkeys, with little
change in aromatase expression. These data suggest that neuro-
estrogens are likely to play a crucial part in the modulation of
cognition throughout the life span. Another important issue is
to further our knowledge of the effects of progestins on PFC-
dependent function. With a few exceptions, the role of
progesterone in PFC-dependent processes has been largely
overlooked. Finally, there is a paucity of data across species
on the effects of androgens (or estrogens) on PFC-mediated
cognitive function in males, despite evidence that androgens
alter synaptic plasticity in PFC in male rats (Hajszan et al.,
2007) and that some PFC function is modulated by
androgens in humans (Janowsky et al., 2000), and rodents
(Kritzer et al., 2007; Wallin and Wood, 2015). A better
understanding of the effects of gonadal hormones and
neuroestrogens throughout the life span in males and
females will allow the design of new interventions for
successful cognitive aging in men and women.
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 1.17 Behavioral and Neuroendocrine Indicators of Well-being in Farm
and Laboratory Mammals
Gabriela González-Mariscal, Centro de Investigación en Reproducción Animal, CINVESTAV-Universidad Autónoma de Tlaxcala,
Tlaxcala, Mexico
Anne Sisto Burt, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Mexico City, Mexico
Raymond Nowak, INRA, UMR85 Physiologie de la Reproduction et des Comportements, Nouzilly, France; CNRS, UMR7247
Physiologie de la Reproduction et des Comportements, Nouzilly, France; Université François Rabelais de Tours, Tours, France; and
Institut Français du cheval et de l’Equitation, Nouzilly, France
Ó 2017 Elsevier Inc. All rights reserved.

1.17.1 Introduction 454

1.17.2 On Rodents 454
1.17.2.1 Regarding Lodging (Cages, Floors, Enrichment) 454
1.17.2.2 Regarding Environment and Procedures (Room Temperature, Noise, Cage Change, Blood Sampling) 455
1.17.2.3 Regarding Reactivity to Stress, as Modulated by Environment (Cages, Enrichment, Handling, Music) 455
1.17.2.4 Regarding Euthanasia Methods 456
1.17.3 On Rabbits 456
1.17.3.1 Regarding Lodging (Cages, Floors, Enrichment) 456
1.17.3.2 Regarding Environment (Temperature, Noise, Vibration) 458
1.17.3.3 Regarding Reduction in Reactivity to Stress by Neonatal Manipulations or Supplements in Adulthood 459
1.17.4 On Sheep and Goats 460
1.17.4.1 Regarding Behavioral Needs 460
1.17.4.1.1 Sexual Behavior 460
1.17.4.1.2 Welfare Issues Specific to Sexual Behavior 461
1.17.4.1.3 Maternity 462
1.17.4.1.4 Welfare Issues Specific to Maternal Behavior 463
1.17.4.1.5 Infancy 464
1.17.4.1.6 ‘Followers’ versus ‘Hiders’ 465
1.17.4.1.7 Welfare Issues Specific to the Young 465
1.17.4.2 Regarding Lodging (Cages, Floors, Nest Boxes, ‘Enrichment’) 467
1.17.4.2.1 Outdoor Facilities 467
1.17.4.2.2 Floor 467
1.17.4.2.3 Social Enrichment 467
1.17.4.2.4 Physical Enrichment 468
1.17.4.2.5 Human Contact 468
1.17.4.3 Regarding Environment (Light, Sound, Vibration, Temperature, Density of Animals) 469
1.17.4.3.1 Flocking 469
1.17.4.3.2 Dominance and Agonistic Behaviors 469
1.17.4.3.3 Social Isolation 469
1.17.4.3.4 Density of Animals 470
1.17.4.3.5 Heat and Cold Stress 471
1.17.4.3.6 Lighting and Noise 471
1.17.4.4 Regarding Modifications of Reactivity to Stress by Neonatal Manipulations 471
1.17.4.4.1 Prenatal Stress in Small Ruminants 471
1.17.4.4.2 Neonatal Stress in Small Ruminants 472
1.17.4.5 Regarding Methods to Quantify Parameters Presumably Indicative of Stress 472
1.17.4.6 Regarding Sacrifice Methods 473
1.17.5 On Dairy Cattle 473
1.17.5.1 Lodging and Handling during the Peripartum Period: Relevance for the Establishment of Maternal
Behavior and Nursing 473
1.17.5.1.1 Calf Lodging 474
1.17.5.2 Lodging of Dairy Cows 475
1.17.5.3 Regarding Environment 477
1.17.5.4 Behavioral Indicators of Well-being in Bovines 477
1.17.5.4.1 Handling of Adult Bovines 478
1.17.6 Conclusions and Discussion 479
Acknowledgment 480
References 480

Hormones, Brain, and Behavior, 3rd edition, Volume 1 http://dx.doi.org/10.1016/B978-0-12-803592-4.00017-1 453

454 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
1.17.1 Introduction
Animal husbandry and experimentation in the laboratory
have created artificial environments where a variety of
mammalian species spend their entire lives. The question of
whether such living conditions (and the procedures associ-
ated with them) are adequate for the well-being of such
animals is, therefore, a valid one and constitutes a research
topic in itself. What do we mean by well-being? What biolog-
ical indicators can we use to measure it? An approach used to
investigate these questions has been to ask whether farm and/
or laboratory environments generate stress in the animals
therein confined (e.g., see Moberg, 2000; Yehuda and
McEwen, 2004). The advantage of such an approximation is
that the stress response has been formally investigated since
Hans Selye (1956) coined the term ‘General Adaptation
Syndrome.’ Consequently, a vast scientific literature exists
on the behavioral, hormonal, immunological, and autonomic
responses observed following the exposure of animals to
a variety of presumably ‘stressful’ stimuli. Although abundant
information has, indeed, shown that some housing condi-
tions, handling procedures, noise, lighting schedules, etc. do
change specific biological indicators in particular species,
the key question of whether such changes alter the well-
being of those animals requires a theoretical framework
within which such results can be interpreted.
Most researchers distinguish between ‘acute’ (i.e., short
lasting) and ‘chronic’ (i.e., lasting for several days) stress
because the consequences of each one are different and the bio-
logical ‘meaning’ of these two types of stress responses varies
widely. Acute stress is generally considered to be adaptive;
that is, it is a generalized response that allows animals to react
‘as a whole’ to sudden changes in the environment. In healthy
animals once the perceived threat is over, hormonal and auto-
nomic responses return to baseline. Chronic stress, by contrast,
can provoke distress, a state of (semi) permanent alteration in
multiple systems that, in turn, leads to disease vulnerability,
infertility, depression, and even death. This apparently simple
dichotomy, however, is complicated by the observation that
repeated exposures to the same acute stressor or successive
contact with several different acute stressors can ‘add up’ and
lead to distress.
As we will illustrate below there is great variability among
farm and laboratory mammals regarding what they perceive
as ‘stressful.’ This should not be surprising in view of the fact
that a given stimulus per se is not stressful; it becomes such
depending on its interpretation by the animal. This, of course,
is a function of the central nervous system whose responsive-
ness is modified (even within a species) by factors such as
age, sex, reproductive condition, early rearing, familiarity
with the environment, etc. Moreover, a number of studies
have shown that using a single biological indicator to deter-
mine if a given situation or procedure is ‘stressful’ may not
be appropriate as, for instance, the concentration of cortisol
in blood may rise as a consequence of restraint, exercise,
mating, etc. Finally, we must emphasize that, in both the
farm and the laboratory, we are dealing with domesticated
mammals selected across many years for specific traits, a process
that may (or not) have also influenced their responsiveness to
specific environmental conditions and handling by humans.
From the above considerations in this chapter, we will
present studies in which specific biological indicators have
been measured in farm and laboratory mammals (i.e., bovines,
sheep, goats, rabbits, and rodents) under a variety of housing
conditions and husbandry practices. We hope that putting
together this information will reveal the consistency of findings
within and across species and will also allow a solid interpreta-
tion of results within a broad scientific framework.
1.17.2 On Rodents
Unlike the rest of mammals dealt with in this chapter, rodents
have been largely bred and used in laboratory settings. None-
theless, similar concerns as those expressed for rabbits (raised
and used both in the lab and on the farm; see the next section)
have been addressed in the studies described below. Specifi-
cally, the impact of housing conditions (in terms of animal
density, floor type, temperature, environmental enrichment)
and ‘standard’ manipulations of the animals (e.g., cage change,
injections) on corticosteroid secretion, physiological responses,
and particular behaviors (presumably indicative of stress) have
been the most explored topics. Two growing areas of study
relate to (1) the effect of manipulating young rodents on their
responsiveness to stressful situations as adults and (2) the
possibility that stress is ‘communicable,’ i.e., that observing
(presumably) stressful procedures performed on other rodents
generates stress in the witnesses.
1.17.2.1 Regarding Lodging (Cages, Floors, Enrichment)
An enriched environment (provided with, e.g., ladders, balls,
hiding places) plus a partner increases sleep, exploratory
behavior, body weight, and weight of thymus and spleen,
relative to (male) rats housed singly in barren cages
(Abou-Ismail and Mahboub, 2011). In pair-housed (female)
mice a high clustering of the elements added for enrichment
increased aggression, stereotypic behavior, and displacement
of ‘the other’ mouse from resources, but it had no effect on
fecal corticosterone (CORT) metabolites (Akre et al., 2011).
Are the effects of environmental enrichment due to novelty
or ‘complexity of the environment’? Do they exert long-
lasting consequences? Housing young mice in a variety of
enriched cages and transferring them back to standard cages
after several weeks did not modify the incidence of stereoty-
pies or the performance in the elevated-plus maze (EPM) or
the open-field tests, relative to animals always housed in stan-
dard cages provided only with cotton wool (Gross et al.,
2011). The effects provoked by an enriched environment are
critically dependent on the strain used and the responses
measured. Adding different types of items to standard cages
effectively reduced mean arterial pressure in male rats from
the F344 but not the BN strain. Moreover, enrichment did
not affect fecal CORT, heart rate, or immunoglobulin A excre-
tion (Kemppinen et al., 2010).
The extent to which stereotypies reflect stress was assessed
by comparing the coincidence of such behaviors with CORT
secretion (following a stress reactivity test) in three lines of
mice: a commercial outbred stock (CD-1) and two lines
selected for high (HR) or low (LR) stress reactivity. Although
 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
acute CORT secretion followed the expected outcome (i.e.,
HR > commercial stock > LR), only CD-1 mice displayed
stereotypies (Engel et al., 2011). These findings challenge
the assumption that the display of such behaviors necessarily
indicates stress. Moreover, in mice raised in either standard
or enriched cages there was no evidence of a causal relation-
ship between the incidence of stereotypies (in the home
cage) and perseverance (quantified in an extinction task;
Gross et al., 2012). Yet, both rats and striped mice raised
in an enriched environment are less likely to develop stereo-
typies when transferred to standard cages (Gross et al., 2012;
Jones et al., 2011). These findings would suggest a ‘protective’
effect of early enrichment on the emergence of stereotyped
behaviors.
Male rats housed in cages with wire-bottom floors showed
a higher heart rate, more body temperature fluctuations, and
a reduction in locomotion and body weight than their coun-
terparts housed in cages with bedding floors (Giral et al.,
2011). The impact of type of bedding (aspen shaving vs chips)
on breeding, nest-building, and anxiety-related behaviors
was compared in four strains of mice. Aspen shaving favored
nest building (thereby increasing the number of kits weaned/
litter) but had no effects on other behaviors (Jackson et al.,
2015). If given the choice mice, show a preference for specific
kinds of bedding by spending more time on softwood
shavings > coarse-grained chips > medium chips ¼ fine chips
(Kirchner et al., 2012). Similarly, rats prefer wood shavings
and paper bedding over sawdust and wire mesh (Van de
Weerd et al., 1996).
The stress generated by transport (as determined by fecal
CORT) can be reduced in guinea pigs by providing huts (to
hide in), but only if animals are pair-housed (Walters et al.,
2012).
Single-level versus two-level cages for rat housing improved
their ‘affective state’ but only if animals were transferred from
the former to the latter. No differences were seen in several
behaviors, immune function, or metabolism in animals that
remained in either type of cage (Wheeler et al., 2015).
1.17.2.2 Regarding Environment and Procedures (Room
Temperature, Noise, Cage Change, Blood Sampling)
Cage change is perhaps the most common ‘laboratory proce-
dure’ to which rodents are routinely subjected to. This practice,
although essential to maintain hygiene for both animals and
humans, is not trivial as it entails a major change in the
animals’ olfactory environment. Thus, the effect of the type of
cage rats were transferred into was assessed in Sprague-Dawley
and Wistar adult males. Heart rate and arterial blood pressure
were increased for up to 5 h following cage change, but the
largest effects were seen when rats were transferred into
a new, clean barren cage with a new lid. Interestingly, adding
an enrichment object had practically no effect but keeping
the old lid reduced ‘stress reactivity’ in both strains (Meller
et al., 2011). Pregnant rodents can be subject to an additional
cage change, i.e., maternity cages where they will give birth and
nurse the litter. The gestational stage when this is performed
can have a major impact on maternal behavior, pup survival,
and stress reactivity. Minks transferred in early pregnancy built
better nests and weaned more pups than did mothers
455
transferred in midpregnancy (highest CORT levels) or late
gestation (poor nest building; Malmkvist and Palme, 2015).
In addition to cage change individually ventilated cage
systems are increasingly being used in rodent colonies. The
preference of mice for different levels of air speeds and air
changes inside the cage has been explored. Animals do react to
drafts but they do not seem to be affected by a high number
of air changes delivered without draft (Krohn and Hansen,
2010).
Blood sampling is one of the most common experimental
procedures performed in rodents. Several methods have been
devised to minimize stress, especially when the procedure is
performed repeatedly on the same animal. Automated blood
sampling (through a catheter implanted in the carotid artery)
provoked the smallest effects on all parameters measured while
mice subjected to cheek blood sampling lost more body
weight, had higher levels of CORT, and expressed more anxious
behavior. Mice sampled from repeated puncturing in the tail
vein showed intermediate values (Teilmann et al., 2014).
1.17.2.3 Regarding Reactivity to Stress, as Modulated by
Environment (Cages, Enrichment, Handling, Music)
There is an ample literature showing that, in rats, the early rear-
ing environment permanently modifies their stress reactivity in
adulthood (for reviews see Champagne et al., 2003; Fleming
et al., 1999; Lomanowska and Melo, 2016). However, the
effects vary depending on the method used to assess stress reac-
tivity and the type of manipulation young animals were sub-
jected to. Thus, four different types of handling treatments
provided to male rats across PP days 57–74 were compared:
(1) minimal (i.e., just cage cleaning); (2) passive hand
(2 min); (3) tickling (2 min); or (4) restrained on back
(2 min). Responses of rats were assessed across days 77–87
using the following tests: EPM and reactivity to human
approach and to cat odor. Ease of weighing and injection was
also determined. Tickled rats showed less fear in the human
approach test, but even the passive hand and restraint treat-
ments provoked this effect. No treatment facilitated handling
during weighing and no effects were seen in the EPM test or
Cat Odor Test (CAT) (Cloutier et al., 2012). The effect of tick-
ling young male rats (across days 32–41) was further explored
by quantifying the emission of ultrasonic vocalizations (USV)
(50 kHz, positive effect; 22 kHz, negative effect) and audible
squeals. Tickling rapidly induced a positive effect (i.e., it
increased 50 kHz USV) when applied before and even immedi-
ately after injections (Cloutier et al., 2015).
In an attempt to reduce stress reactivity during an experi-
mental procedure adult ovariectomized rats were exposed to
silence, ‘white’ noise (with all the frequencies), or a piano
sonata. In the EPM and the light–dark box transition tests noise
was found to be anxiogenic and music anxiolytic. Interestingly,
the anxiolytic effect of music was enhanced by injecting proges-
terone or allopregnanolone (Escribano et al., 2014).
Experimental procedures are commonly done in rooms
where ‘other’ rodents are present. Consequently, those animals
are ‘unwilling witnesses’ to the fate of their peers. To determine
if observing, hearing, or smelling the reactions of experimental
animals (across 4 weeks) has an impact on the witnesses, the
thymus, spleen, adrenal gland, and body weight was
456 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
determined in adult rats, housed four per cage. Sleeping,
grooming, and agonistic behavior were also quantified. Rats
which witnessed managerial procedures during the light phase
showed more indicators of stress than those exposed during the
dark phase (Abou-Ismail et al., 2015). Do these results indicate
that the (presumed) stress of other animals is communicable
(Makowska and Weary, 2013)?
1.17.2.4 Regarding Euthanasia Methods
Concerns about animal welfare in the laboratory have to
include the ways by which animals are sacrificed at the end
of an experiment. A recently introduced method involves the
use of CO2 inhalation. A combination of CO2 concentration
and flow rate that minimizes dyspnea before the onset of insen-
sitivity and, ultimately, death was explored in mice. Higher
CO2 flow rates reduced the duration of dyspnea but, even
under such conditions, mice do experience it, for about 30 s
before they become insensitive (Moody et al., 2014).
1.17.3 On Rabbits
A growing concern for the well-being of rabbits kept in inten-
sive production units or in small farms has promoted the study
of how specific variables impact the health, productivity,
growth, and behavior of animals of different ages and repro-
ductive conditions. Other works have investigated how specific
manipulations or nutritional supplements ameliorate
unwanted consequences of environmental conditions or facil-
itate animal handling. In the following subsections we will
review such studies, highlighting their common findings as
well as the heterogeneity of the models explored.
1.17.3.1 Regarding Lodging (Cages, Floors, Enrichment)
The type of cage used to lodge rabbits (e.g., size, animal density
therein, floor material) has been, by far, the most investigated
variable. ‘Alternative’ cages (usually larger and provided or not
with a platform) have been compared to the conventional ones
in which rabbits are kept individually, either for fattening after
weaning or where lactating does nurse their kits (for review see
Szendrö et al., 2016). Both pregnant and lactating does spend
about a quarter of their time on the provided platform, but
this device creates hygienic problems inside the cage
(Alfonso-Carrillo et al., 2014). Young broilers, housed in
collective cages with a platform for several weeks, stand on it
at night and under it during the daytime, but the device does
not impact daily weight gain, mortality, morbidity, or body
lesions (Lang and Hoy, 2011).
In an attempt to mimic the ‘natural’ conditions where
rabbits live, i.e., underground colonies usually consisting of
males and females cohabiting according to a hierarchical order
(Mykytowycyz, 1958a,b), various types of social arrangements
have also been explored. Rather than individual housing,
groups of rabbits varying in age or reproductive condition are
kept together in large pens. The development of young females
reared in either same-sex or mixed-sex groups was not different
regarding weight gain, food intake, mortality, gonadotropin
concentrations, or weight of ovaries, uterus, adrenals, kidneys,
or perirenal fat. However, evidence of dominance hierarchies
emerged under both conditions, as evidenced by scarring (Bell
and Bray, 1984). Young bucks housed contiguously in two-
floor cages (that allowed looking at each other closely) ‘social-
ized’ more by looking at the animal in the immediate vicinity
(Negretti et al., 2004). A different impact of bicellular cages
versus wired pens was seen in Leprino versus New Zealand
White (NZW) young bucks: mortality was highest in bicellular
cages for Leprinos but in pens for the NZW breed (Mugnai
et al., 2014).
The effect of animal density within a cage was explored by
housing unfamiliar young bucks and does in cages of different
sizes (eight animals per cage). Hardly any differences were seen
in the animals’ behavior: only sternal lying increased with
decreasing space. Placing a platform decreased social interac-
tions, and enriching the environment with a wooden stick
decreased cage manipulation (Buijs et al., 2001). Food intake
and weight gain were compared between fattening rabbits
housed in either of two cages: ‘low-density and enriched’
(with platform, hiding box, and gnawing material) or ‘high-
density and barren.’ Food intake was higher in enriched cages
only across the first 2 weeks, and aggression did not occur
under any condition. Average weight gain was not significantly
different between the two cage types (Maertens et al., 2004).
The combined effects of animal density and introduction of
a gnawing stick were tested on productive parameters (live
weight, daily weight gain) and ‘spontaneous’ behaviors.
Although productivity was not affected, more varied behaviors
were observed in animals living two per cage. Addition of
gnawing stick reduced incidence of stereotypies, alertness,
and aggression (Verga et al., 2004; Figure 1). Single-housed
versus group-housed NZW adult females were monitored for
12 weeks regarding immune response (humoral and delayed-
type hypersensitivity response), lymphocyte counts, circulating
corticosterone, food intake, growth rate, adrenal gland weight,
cross-sectional area of adrenal cortex, and social interactions.
The only differences between both groups were that group-
housed animals engaged in more behaviors (social,
9
8 Enriched
7 Control
6
Incidence (%)
5
4
3
2
1
0
Hops Sniffs Grooming Aggression Alert Cage bi ng
Figure 1 Effect of housing rabbits, from 35 to 75 days of age, in
either ‘control’ or ‘enriched’ cages on the incidence of specific behav-
iors. Modified from Verga, M., Zingarelli, I., Ferrante, V., et al., 2004.
Effect of housing and environmental enrichment on performance and
behaviour in fattening rabbits. In: Proceedings of the 8th World Rabbit
Congress, pp. 1283–1288.
 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals 457

exploratory, play) that were not possible in the single-housed the nest boxes before kindling. Does kept in collective cages
rabbits. The former preferred small social groups (Whary showed a more varied behavioral repertoire relative to those
et al., 1993). The impact of individual cages versus collective housed individually. However, the interactions among the
pens on productivity was assessed in nulliparous NZW does former were not always ‘friendly’: a high incidence of aggres-
that were inseminated either artificially (AI) or by natural siveness, attack, and dominance was observed, especially in
mount. In the latter condition, the type of housing did not does ‘unfamiliar’ with the nest boxes. Reproductive perfor-
influence litter size, kit mortality, or kit weight at 14 days of mance and productivity were also lowest in such group as
age. However, when AI was used, a lower kindling rate and a low sexual receptivity was found in nearly 50% of animals
lower kit weight at weaning were found in animals housed in and fertility rates were 41%. Moreover, due to severe
collective pens. This was due largely to a high incidence of injury 8.3% of does in that group had to be replaced (Mugnai
pseudopregnancies (23% vs 0% in females housed individu- et al., 2009).
ally). Additionally, evidence of aggression (i.e., skin injuries) Other authors compared the productivity and behavior of
was found in 16–20% of does housed collectively (Rommers multiparous pregnant rabbits housed at a density of four
et al., 2006). does per cage, in standard versus in large cages. Reproductive
The reactions of lactating does kept in large communal pens performance (i.e., number of live/dead pups born, milk
with their litters differ from the ones observed in young or production, milk intake/kit, number of weaned kits, kit weight
adult animals housed together. In nature, in the laboratory, at weaning) was not affected by the type of cage (Dal Bosco
and on the farm lactating does nurse their kits inside a nest et al., 2004; Table 1). Rabbits housed in large cages showed
they construct with straw and their own body hair (for reviews more episodes of specific behaviors, e.g., moving, licking/
see González-Mariscal et al., 2007, 2016). Nursing usually scratching, lying down, sitting up, standing on hind legs.
occurs only once a day, with circadian periodicity, and lasts Females housed in standard cages showed more sniffing,
for 3–5 min (González-Mariscal et al., 2013a,b). In nature crouching, sitting hunched, standing alert, and biting bars
the site selected for building the maternal nest (and nursing (Dal Bosco et al., 2004; Table 2). In contrast, recently weaned
the kits) is determined by each individual doe, and it is located kits huddle spontaneously; that is, they prefer to be housed at
away from the communal burrow (Mykytowycz, 1958a,b). a high density (Matics et al., 2004). This condition resembles
Despite this natural pattern of ‘isolated nursing,’ attempts the one they experience in the maternal nest across lactation
have been made to house lactating does (and their litters) in and which is critical for conserving body heat and allowing
large communal pens. For instance, pregnant does were trans- a normal development (Bautista et al., 2003, 2008).
ferred from the communal pen they had shared with other
pregnant does to individual cages on prepartum day 1 and
kept there until lactation day 12. They were then placed back Table 1 Effect of housing rabbits in individual or collective
in communal pens together with the same (S) group of cages on specific reproductive parameters
(familiar) females or a group in which two or three of the
Type of cage
females were alien and mixed (M group) among the ‘familiar’
ones. Fecal corticosterone metabolites (FCM), lesions, and Parameter measured Individual Collective
agonistic behaviors were quantified before and after regroup-
ing. Lesions across days increased more in the M groups than Receptivity 81% 80%
Fertility 74% 70%
in the S groups. FCM raised after regrouping only in the M
Number of live-born kits 7.5  2.4 6.9  2.4
groups. These results indicate that familiarity with conspecifics
(mean  se)
provokes a more attenuated stress response after regrouping Milk intake per kit 19.3  3.5 20.5  3.5
than mixing with a few unfamiliar animals (Andrist et al., (g day1; mean  se)
2012). In another attempt to minimize agonistic interactions
among lactating does, they were transferred from the Modified from Dal Bosco, A., Mugnai, C., Castellini, C., et al., 2004.
A prototype of colony cage for improving the welfare of rabbit does:
communal pens they occupied throughout pregnancy to indi- preliminary results. In: Proceedings of the 8th World Rabbit Congress,
vidual cages across lactation days 1–12 and back to communal pp. 1229–1234.
pens. Does were individually sprayed (30 min before moving
them back to the communal pens) with water, 4.5% vinegar,
or fruit-scented alcohol. The presence of lesions, agonistic inter- Table 2 Effect of housing pregnant or lactating rabbits in individual
or collective cages on the display of specific behaviors
actions, glucose levels, and body temperature were measured.
On regrouping day 2, 43% of does had lesions; within days Type of cage
1–5 new lesions appeared in 60% of does; 32% were serious
lesions. Blood glucose concentrations and body temperature Percent time displaying Individual Collective
increased relative to baseline (i.e., before regrouping). Only General movement 22 26
a minor effect of spraying was observed: on regrouping day Eating–drinking 4.9 3.5
1, body temperature increased less in vinegar-scented does, Biting cage 8.2 1.4
compared to the other two groups. These results indicate that Sniffing 15.4 9.1
‘masking’ with alternative odors does not counteract agonistic Rearing 1.9 0.6
interactions among lactating does housed together (Andrist
Modified from Dal Bosco, A., Mugnai, C., Castellini, C., et al., 2004. A prototype of
et al., 2014). Another strategy used to reduce aggression was colony cage for improving the welfare of rabbit does: preliminary results. In:
to familiarize pregnant does, housed in collective pens, with Proceedings of the 8th World Rabbit Congress, pp. 1229–1234.
458 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
Enriching the animals’ housing environment has been
attempted as a means to reduce ‘boredom’ and counteract
anxiety-related behaviors, presumably provoked by barren cages.
Three different types of gnawing blocks were added to the cages
of pregnant rabbits, housed in conventional wire cages, as an
enrichment and dietary supplement. Litter weight or individual
kit weights were not significantly modified by the type of gnaw-
ing block provided, although a high variability in consumption
of the blocks occurred among females. Locomotion and intake
behavior in late pregnancy and at two time points in lactation
were significantly increased when a gnawing block was available,
but this did not modify the total number of behavioral transi-
tions (Maertens et al., 2013). In multiparous lactating hybrid
rabbits the effect of introducing a pinewood stick, straw in
a plastic bin, a compressed plywood block, or straw plus pine-
wood stick was compared. Across lactation more does were
engaged for more time in the group provided with straw þ ply-
wood than with the pinewood stick (Rommers et al., 2014).
An important consideration has been given to the type of
cage floor, to avoid footpad injuries. Young rabbits (kept at three
different animal densities) spent more time over a wire net floor
than over deep litter (Orova et al., 2008). Similarly, at low,
medium, or high ambient temperatures the lowest preference
was for deep litter. Plastic floor was preferred at low temperature
and wire floor at high. No differences among groups were seen
on food intake, feed conversion ratio, or mortality (Gerencsér
et al., 2014). Broilers (8–12 weeks old) kept on bedding spent
less time resting and grooming and more time eating than did
animals kept on wire floors (Ribikauskas et al., 2010). Providing
plastic floor mats to does housed individually in wire cages
across five reproductive cycles significantly reduced the incidence
of injured footpads (Rommers and de Jong, 2011).
In addition to determining the effects of housing conditions
on productivity a few studies have explored the impact of such
parameters on the behavior of rabbits in tests presumably
measuring anxiety or fear. Specifically, the effects of group
density (12 or 16 rabbits/m2) and floor type (wire or slat)
were compared in the tonic immobility test (no differences
related to group density or floor type), the efficiency of feed
conversion (higher under high density), and the open-field
test (rabbits under high density or wire net floor spent more
time exploring). The same work also found that growth param-
eters in young rabbits were not affected by animal density
(Table 3; Trocino et al., 2004). The fearfulness of young female
offspring raised under a semigroup housing system was assessed
Table 3 Effect of varying animal density in collective cages
on growth parameters in young rabbits
2
Animal density (rabbits/m )
Parameter measureda 12 16
Initial weight (g) 616  39 620  39
Final weight (g) 2646  102 2664  102
Weight increase (g day1) 48.3  2.0 48.7  2.0
Food intake (g day1) 149  6.4 148  6.4
a
mean  residual standard error.
Modified from Trocino, A., Xiccato, G., Queaque, P.I., et al., 2004.
Group housing of growing rabbits: effect of stocking density and cage
floor on performance, welfare, and meat quality. In: Proceedings of the
8th World Rabbit Congress, pp. 1277–1282.
using the following tests: open field, novel object, and social
runway. Semigroup housing consisted of rearing the kits in
a litter with their siblings and mother in an individual cage
across lactation days 1–18, when they were transferred to collec-
tive cages (four lactating does þ their own litters/pen) until
weaning. Kits reared under semigroup housing traveled less
distance in the open-field test and had a longer latency to leave
the ‘start’ corner than control kits. In the novel-object test they
reared less than control kits, but their latency to approach the
object was not different. No differences were found in the social
runway test either (Buijs and Tuyttens, 2015). The impact of
varying rabbit density in the cage and introducing a wooden
stick on the tonic immobility and emergence tests was studied
in young, hybrid, growing rabbits. Neither the stick nor the
animal density/cage had any impact on those tests or on weight
gain. Such factors, however, did increase locomotor activity and
social interactions (Zucca et al., 2012a).
1.17.3.2 Regarding Environment (Temperature, Noise,
Vibration)
Rabbits are raised worldwide in farms of different sizes and in
different latitudes/altitudes; they are transported from the
farms to the abattoirs; they are subjected to cage changes and
to a variety of husbandry procedures. Thus, the impact of
factors such as temperature, noise, vibration, and handling
on productivity and some indicators of stress have been
explored. For instance, Spanish Giant rabbits of both sexes
(55–65 days old) were exposed for 4.5 h before slaughter to
heat (42  C), cold (5  C), noise (96 dB), or mixing with other
rabbits. At slaughter, blood samples were taken to determine
the concentration of cortisol, creatinine kinase, and lactate
dehydrogenase; packed cell volume; and osmolarity. Cortisol
was significantly elevated (over values in the control group)
in animals exposed to heat or noise while cold or mixing had
no effect on this parameter (de la Fuente et al., 2007; Figure 2).
Young bucks (12 weeks old) placed in plastic transport cages
1.8 Control
1.6 Heat
Cold
1.4
Noise
Cor sol, log 10 μg/dl
1.2 Social mix
1
0.8
0.6
0.4
0.2
0
Type of stressor
Figure 2 Effect of five different types of potential stressors, applied
for 4.5 h prior to slaughter, to young adult male and female rabbits, on
the concentration of cortisol in blood (mean  standard error). Modi-
fied from de la Fuente, J., Díaz, M.T., Ibáñez, M., et al., 2007. Physio-
logical response of rabbits to heat, cold, noise and mixing in the
context of transport. Anim. Welf. 16, 41–47.
 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
traveled by road for 2 h at 80 km h1 without air-conditioning
(ambient temperature ¼ 20  C; humidity 69%; Group A).
Control groups were placed in transport cages (Group B) or
in standard battery-style cages (Group C). Rectal temperature,
glucose, lactate, packed cell volume, and total proteins were
determined before (baseline), immediately, and at 6 and
24 h after transport. All parameters showed significant
increases in Group A, relative to baseline and to both control
groups. Baseline values of Group A were restored at 24 h (Fazio
et al., 2015). A temperature–humidity index (THI) of 30 or
more severely altered male and female rabbit physiology,
including reproduction parameters and the resistance to stress.
In females, conception rate, embryonic development, litter size,
litter weight, and milk production declined while age at
puberty onset and periweaning mortality increased as a conse-
quence of heat stress. In males, the factors affected are sperm
count and motility, abnormal and dead sperm, testosterone
concentration, fertility, sexual libido, and ejaculate volume.
In both sexes, feed conversion, feed intake, water metabolism,
mineral balance, and energy balance were altered by a high THI
(Marai et al., 2002). Similarly, Rex rabbits placed under heat
stress (or control temperature) for 9 weeks, followed by a return
to control conditions, showed reductions in libido, sperm
count, and semen volume. Heat stress also elevated apoptosis
in germ cells in seminiferous tubules. All parameters were
restored to normal values upon return to control temperature
(Pei et al., 2012).
To compare the effects provoked by high temperature versus
intense sound, young male hybrid rabbits aged ca. 42 days
were exposed to continuous heat (30  C) or intermittently to
noise (90 dB; from 8:30 to 12:30 h and again from 15:30 to
19:30 h) for 1 week. Sound-exposed rabbits showed significant
increases in corticosterone across the whole trial while in
animals exposed to heat such an increase was evident across
the first 3 days, after which CORT values declined (Verde and
Piquer, 1986).
To determine the consequences of transport on pregnant
does, they were placed daily for 2 h, across gestation days
5–29, in or near a machine that generated vibration stress
and sound, thus simulating transport. This procedure did not
affect maintenance of pregnancy, gestation length, litter size,
perinatal mortality, or condition of the kits at birth (relative
to unexposed controls). Respiration rate increased from 65
to 120 min1 by moving animals from their cages and to
160/min1 by placing them in the machine. Noise alone
provoked intermediate responses. Values returned to baseline
after 20 min to 4 h (Stephens and Adams, 1982).
The consequences of confinement within a small space were
explored by placing rabbits for 15 min inside a restriction box
and measuring blood corticosterone concentration and temper-
ature (skin and rectal). Corticosterone increased from 17.7
(baseline) to 85.2 mg ml1 immediately after the restriction.
Skin temperature decreased from 35.3 to 34.8  C while rectal
temperature rose from 39.2 to 39.7  C (Luzi et al., 2007). The
effects provoked by exposing animals to a new environment
were explored in adult NZW rabbits of both sexes. The concen-
trations of corticosterone, epinephrine, and norepinephrine,
determined 10 min later, increased (relative to baseline) by
two- to threefold and two- to fourfold, respectively, while
norepinephrine was hardly modified (Fenske et al., 1982).
459
In an attempt to optimize the use of artificial daylight
without compromising fertility the effect of photoperiod dura-
tion on rabbit does’ productivity was determined. Starting at
11 weeks of age does were housed under a long (16 h light:8 h
dark; control group) or a short (8 h light:16 h dark) photope-
riod. The latter was ‘supplemented,’ 8 days prior to insemina-
tion, by an extra hour of light applied in the middle of the
dark phase (experimental group). The annual performance
per doe, across five consecutive reproductive cycles, was supe-
rior in the control group for number of kits born alive, and
number and weight of kits weaned (Gerencsér et al., 2011).
1.17.3.3 Regarding Reduction in Reactivity to Stress by
Neonatal Manipulations or Supplements in Adulthood
Two main strategies have been used to minimize the conse-
quences of stress provoked by environmental factors or
husbandry practices (see previous discussion): neonatal
manipulations of kits and administration of specific nutritional
supplements to young or adult rabbits of both sexes. Early
studies handled kits twice per day, at fixed times between 09:00
and 16:00 h, from days 1–10 or 11–20 postpartum. At
98 days of age handled and nonhandled animals were tested
in the open-field test. No significant differences were found
between the groups, a finding the authors attributed to a large
interlitter variability (Kersten et al., 1989). Later studies,
however, have shown that the kits’ brain is especially suscep-
tible to permanent changes in reactivity to stress when partic-
ular manipulations are given around the time of nursing. This is
due to the fact that a state of arousal is generated spontaneously
in the kits shortly before the mother’s arrival for the single daily
nursing bout (Hudson and Distel, 1982). Indeed, a light
manipulation of the kits, performed 15 min before or up to
30 min after nursing (across the first postnatal week), reduced
the latency to approach humans and increased the frequency of
contacts in tests performed in adulthood. Manipulating the kits
at other times relative to nursing was ineffective at modifying
their reactivity to stress later on (Bilkó and Altbäcker, 2000;
Pongrácz and Altbäcker, 1999; Figure 3). The effectiveness of
this strategy was further substantiated by the finding that just
introducing a human hand into the nest box for 5 min
(without touching the kits), across postpartum days 1–7 within
30 min after nursing reduced, in 28-day-old kits, their latency to
approach humans and increased the number of approaches to
the same extent as did the actual handling of the litter (Dúcs
et al., 2009). Moreover, such procedure also reduced the
latency to emerge from a dark box and the duration in the
supine position (‘tonic immobility test’; Zucca et al., 2012b).
A different type of manipulation that modifies the kits’ reac-
tivity to stress in adulthood is a 48 h mother–young separation
in early lactation. This so-called ‘biostimulation’ method is
used worldwide to restore estrus in lactating does (Alvariño
et al., 1998; Castellini et al., 1998; Theau-Clément and Mercier,
1999). It consists of increasing the interval between two succes-
sive nursing bouts from the usual 24 to 48 h. This increased
time period without suckling provokes, in lactating does, an
increased expression of estrous behaviors (scent-marking and
lordosis; García-Dalmán and González-Mariscal, 2012), the
release of estradiol (Ubilla et al., 2000), and an increased
responsiveness of specific forebrain regions to mating stimuli
460 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals

240 had a lower mortality rate and were significantly heavier than
220 the control group (Jezierski and Konecka, 1996). In a similar
200 paradigm NZW kits were handled six times per week, from
Latency to approach human (s)

birth until weaning (at 4 weeks of age), for 5 min in the after-
180
noon, i.e., not around nursing time. After weaning bucks
160
(housed in communal cages) were handled every other day
140 for 5 min. Across the next 12 weeks several tests were per-
120 formed to determine the effect of handling on the animals’
100 reactivity: human-approach latency, novel object, tonic immo-
80 bility, and open field. In general, results showed a marked effect
60 of handling in reducing emotional reactivity; that is, bucks were
more social, less sensitive to tonic immobility, made more and
40
faster contact with novel objects and humans, and moved more
20
in the open field (Verwer et al., 2009). Taken together, the
0
Control 30 min pre 15 min pre 30 min post 1 h post above results indicate that ‘windows of opportunity’ exist in
Manipula on me rela ve to nursing kits and in young adult rabbits at which the performance of
specific manipulations can permanently reduce the animal’s
Figure 3 Neonatal manipulation of kits at specific times before (pre-) reactivity to potentially stressful stimuli.
or after (post-) nursing, across postnatal days 1–7, reduces their In addition to such ‘prevention strategies’ some studies have
latency to approach humans, when tested at 28 days of age (mean - explored if providing particular nutritional supplements to
 standard error). Modified from Pongrácz, P., Altbäcker, V., 1999. The
adult animals can reduce the consequences of specific environ-
effect of early handling is dependent upon the state of the rabbit (Oryc-
tolagus cuniculus) pups around nursing. Dev. Psychobiol. 35, 241–251.
mental stressors. For instance, adult males housed under heat
conditions were supplemented with royal jelly (RJ) twice per
week for 20 weeks. RJ significantly increased: sperm count, total
(González-Mariscal et al., 2015). As a consequence of the doe’s sperm output, sperm motility, and the percentage of live and
‘biostimulation,’ the kits that missed a nursing bout (usually normal sperm, relative to control (i.e., nonsupplemented)
on lactation day 10) show a reduced release of corticosterone bucks. RJ also increased testosterone concentration, total
in response to a saline injection in adulthood (Brecchia et al., protein, albumin, globulin, glucose, and high-density lipopro-
2006; Figure 4). teins in plasma. RJ reduced total plasma lipids, triglycerides,
More prolonged manipulations (e.g., petting the kits daily cholesterol, and low-density lipoproteins (El-Hanoun et al.,
for 10 min from the age of 10 days up to 3 weeks) can provoke 2014). Similarly, the capacity of vitamin E or propolis to coun-
similar reductions in reactivity to stress in adulthood even if teract the impact of heat was explored in adult NZW bucks.
they are provided outside the perinursing period. In the ‘tonic Both supplements improved (relative to the control, nonsup-
immobility test’ the proportion of ‘bold’ (i.e., less time in plemented group) libido, sperm count, sperm viability, and
‘freezing’ posture) animals was 69% in the handled group testosterone concentration. They also increased number of
and 37% in control animals. Additionally, handled animals red blood cells, hematocrit value, and hemoglobin concentra-
tion. High-density lipoprotein and blood plasma glucose
were higher while cholesterol and triglycerides were lower in
40 both supplemented groups (Hashem et al., 2013). Another
supplement tried to ameliorate some of the adverse effects of
35 Control heat consequences is betaine. This agent, which has methyl
30 Separated donor and osmoprotective properties, decreased rectal temper-
Cor costerone (ug/dl)

ature and respiration rate; it also increased the humoral and

25 cell-mediated immune response and the serum concentrations
of T3, T4, total protein, globulin, and total lipids and enhanced
20
growth performance (Hassan et al., 2011). Polysaccharides
15 extracted from alfalfa and added to the regular diet of heat-
exposed NZW young males (40 days old) had a positive effect
10 on average daily weight gain, food intake, and food conversion
rate. Supplemented bucks also had lower cortisol concentra-
5
tions (Wei Liu et al., 2010).
0
0 30 60 90 120
Minutes postsaline, i.m.
1.17.4 On Sheep and Goats
Figure 4 Rabbit kits that skipped a single nursing episode in early 1.17.4.1 Regarding Behavioral Needs
lactation show a reduced release of corticosterone following an injec-
tion of saline (IM) in adulthood (mean  standard error). Modified from 1.17.4.1.1 Sexual Behavior
Brecchia, G., Bonanno, A., Dall’Aglio, C., et al., 2009. Neuroendocrine In wild and domesticated species of sheep and goats, reproduc-
responses in neonatal mother-deprived rabbits. Brain Res. 1304, tion is characterized by short breeding periods and long
105–112. periods of sexual segregation and inactivity, when females are
 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
in anestrus. Both species are short-light breeders which means
that they usually reunite and breed when the light phase of the
photoperiod decreases (Chemineau et al., 1992). As the season
advances, males will begin to fight with one another. The fight
consists of head clashing and nudging behavior similar to that
seen in courtship. Periodic reunion has two major conse-
quences: initiating breeding activity, by triggering the transition
from anestrus to estrus, and synchronizing the sexual cycle in
the females (Ungerfeld, 2007; Walkden-Brown and Martin,
1999). As a secondary consequence, this will also enable
synchronous parturition, a defense mechanism that under
natural conditions buffers the impact of predation on
offspring. The induction of sexual activity in the females is
called the ‘male effect.’ In responsive ewes and does the primary
reaction to the male is an immediate increase of LH pulse
frequency, which leads to ovulation within 24 h (Fabre-Nys
and Gelez, 2007). Similarly, in both rams and bucks, exposure
to estrous females may stimulate an immediate secretion of LH,
a phenomenon that has been termed the ‘female effect.’
Although its functional significance remains unclear, it may
lead to behavioral changes that improve the success of mating
(Walkden-Brown and Martin, 1999). Female sexual behavior is
characterized by three components: (1) attractiveness: physical
changes to attract the attention of the male; (2) proceptivity:
active search for and attraction toward the male; and (3) recep-
tivity: acceptance of mating attempts (Fabre-Nys and Gelez,
2007). Although attractiveness is not very explicit, rams find
ewes more attractive around the time when they are receptive
and when they are not shorn. During estrus, does appear rest-
less, show increased motor and vocal activity, and display
male-type behavior and reciprocal mounting. Ewes are less
demonstrative, but they do search actively the proximity of
males. The expression of such behavior is important when
males are not highly motivated, or when a large number of
ewes are in estrus in which case they compete for access to
the rams. Around the time of ovulation, ewes and does present
a well-defined response to the courtship of the males
(Fabre-Nys and Gelez, 2007). There is no ritualized sequence
of events leading to mating, but the repertoire of behaviors
expressed by both rams and bucks is very standardized and
similar. The male approaches the proceptive female which
remains motionless, kicking her occasionally with its front
leg. Alternatively, the two partners walk together in circles,
the female following the male and the male trying to place
himself behind the female. There is considerable evidence
that olfactory cues from the female and the male are important
for sexual communication; however, both in sheep and in goats
the sexual partner is a multisensory stimulus, olfactory cues
being only part of it (Walkden-Brown and Martin, 1999).
1.17.4.1.2 Welfare Issues Specific to Sexual Behavior
1.17.4.1.2.1 Rearing Conditions in Early Age
Contact with the mother in early life can dramatically influence
sexual preferences. Sheep and goats cross-fostered at birth, but
raised in mixed-species groups, display play and grooming
behavior resembling that of their foster rather than their genetic
species (Kendrick et al., 2001). In adulthood, cross-fostered
juveniles prefer to socialize and mate with partners of their
foster mother’s species, even if raised with a conspecific of their
own species. This effect is weaker in females than in males.
461
Even after 3 years of life in a flock made of members of only
their own species, cross-fostered males maintain their social
and mating preferences for females of their mother’s species.
Females shift their preference to the genetic species within
1–2 years even though the social interest for female members
of their foster species does not vanish. Although this phenom-
enon resembles ‘sexual imprinting,’ described in birds, it is
more likely to be a social learning determined by the parental
species. While the effect in males is very striking and looks irre-
versible, the development of a strong social and mating prefer-
ence for individuals of the foster species is never exclusive.
Social learning is also important for males to develop hetero-
sexual experience in order to prefer females over males as
sexual partners. While isolated rams show some aberration in
behavior when first exposed to estrous females, most are even-
tually able to mate. When reared in monosexual groups, some
rams and bucks are sexually inhibited or show homosexual
preferences and do so for long periods or permanently after
maturity (Zenchak et al., 1981; Figure 5(a)). Homosexual
behavior may be overcome by short exposure to ewes before
puberty (Tilbrook and Cameron, 1990). Nonetheless, rams
who do eventually mate with estrous ewes continue to exhibit
subnormal sexual activity. Bucks reared in groups of males
develop homosexual behavior as well and display fewer
agonistic interactions with other bucks at puberty (Price and
Smith, 1984; Figure 5(b)). The dominant male initiates homo-
sexual behavior over the subordinate ones (Price et al., 1994).
Homosexual behavior does not interfere with the ability to
ejaculate in an artificial vagina (Orgeur et al., 1990) showing
that breeding failure relates more to the formation of
individual-specific sexual attachment with other males rather
than with failure to develop normal mating skills. Conse-
quently, early social experience must be viewed as critical for
the behavioral development of the individual, and rearing juve-
nile males in same-sex groups must be avoided.
Under laboratory conditions, the duration and time of the
mating season is controlled by humans and will not neces-
sarily follow the natural breeding season of the animals. The
impossibility to fulfill the sexual drive may be seen in some
species as a potential source of frustration and consequently
as a decrease in welfare (Webster, 1995). While in most
species, the sexual drive of males is considered stronger than
that of females, in sheep and goats, it could be argued that
the females’ sexual drive is fairly strong as well because they
search actively the proximity of males. Therefore, the frustra-
tion of sexual restriction during the breeding season is a real
welfare issue. Because males and females are aroused in
response to the sight and the smell of females in estrus, the
segregation of sexes during the breeding season should not
be a source of excessive frustration. On the other hand, the
confinement of males in a single sex group during the
breeding season is likely to increase the amount of aggressive
behavior and, therefore, injuries. Care should be taken to
minimize fighting by rearing males (especially bucks) in indi-
vidual pens while also allowing social contact with conspe-
cifics, an important issue due to the gregarious nature of
these ungulates. During the nonbreeding season the separa-
tion of sexes should not be a problem because sheep and
goats, like most ungulates, naturally segregate outside of the
mating period.
462 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals

(a) Rams
First approach Inves gate Preference
15 15 15
Number of responses
10 10 10

5 5 5

0 0
0
Normal Low response Normal Low response Normal Low response
Ewe Ram Ewe only Ram and ewe Ram only Ewe Ram

(b) Bucks
Subjects that mounted a male Subjects that did not mount a
Mean sexual responses

Mean sexual responses

12
male
10 6
8
6 4
4
2
2
0 0
Nudges Mounts Nudges Mounts
Estrous goat Diestrous goat Buck Estrous goat Diestrous goat Buck

Figure 5 Sexual behavior displayed by rams and bucks reared in unisexual groups. (a) First approach, olfactory investigation and preference
expressed by rams when given the choice between a ram and a ewe (Normal: rams displaying normal levels of sexual performance toward ewes; low
response: rams showing little interest in the ewes). (b) Responses of sexually active bucks when given the choice between an estrous goat, a dies-
trous goat, and a buck. Subjects are divided into two groups: whether they had been seen mounting a male or not in their home enclosure. Adapted
from Price, E.O., Smith, V.M., 1984. The relationship of male-male mounting to mate choice and sexual performance in male dairy goats. Appl. Anim.
Behav. Sci. 13, 71–82; Zenchak, J.J., Anderson, G.C., Schein, M.W., 1981. Sexual partner preference of adult rams (Ovis aries) as affected by social
experiences during rearing. Appl. Anim. Ethol. 7, 157–167.

1.17.4.1.3 Maternity conditions, females show restlessness, frequent vocalization,

1.17.4.1.3.1 Maternal Responsiveness and increased intolerance of conspecifics, and ewes isolate
Despite their strong social needs, as parturition approaches themselves if cubicles are provided in the group (Gonyou
ewes and goats will easily withdraw from their flock (Nowak and Stookey, 1985). As parturition approaches, females
et al., 2000; Poindron et al., 1993, 2007b). Isolation-seeking vocalize, paw the ground, circle, and try to lick their back.
behavior persists in most breeds of sheep even if domestication Parturition is most likely to occur during the day in goats, while
is associated with reduced time spent on the birth site (Dwyer ewes do not show any diurnal pattern. The amniotic fluids
and Lawrence, 2005; Figure 6). Even under farm or laboratory dripping from the vagina become suddenly attractive and
trigger intense licking of the contaminated bedding. Attraction
8 to these fluids incites females to stay on the birth site, and once
7 the newborn is expelled, it becomes the core of the mother’s
Days a er parturi on

6
attention. Removal of such fluids greatly disrupts the onset of
maternal care especially in primiparous animals. In sheep
5
and goats, like in many other mammals, it is the rapid variation
4 in progesterone and estrogens that takes place in the days or
3 hours preceding parturition that are responsible for the peripar-
2 turient behavioral changes. In addition, the mechanical stimu-
1 lation caused by the expulsion of the fetus, which leads to the
0 release of oxytocin, reinforces the expression of maternal
responsiveness (Dwyer, 2014; Poindron et al., 1993). Does
defend the area where they gave birth and their kid against
other females. This is rarely the case in sheep, and the strong
Figure 6 Maximum time spent on the birth site by various species maternal responsiveness of nearby periparturient ewes may
and genotypes of sheep. Adapted from Dwyer, C.M., Lawrence, A.B., result in lamb stealing, especially under indoor conditions.
2005. A review of the behavioural and physiological adaptations of hill Licking of the neonate usually starts from the head down to
and lowland breeds of sheep that favour lamb survival. Appl. Anim. the anal region and has important consequences. Firstly, it
Behav. Sci. 92, 235–260. removes much of the fluid present on the coat and helps reduce
 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
heat loss, as young lambs and kids are both susceptible to chill-
ing (Dwyer et al., 2016). Secondly, it has a dual impact on the
newborn’s activity: licking of the head is soothing and triggers
head movement oral activity (teat-seeking) while grooming the
back and anogenital area is associated with leg movement and
may stimulate standing (Poindron et al., 1993). Finally, licking
of the neonate contributes to the establishment of the mother–
offspring bond, since this intimate relationship will make ewes
and does able to learn their offspring’s identity by smell.
Usually, the fetal membranes are licked off and consumed
but the placenta is exceptionally eaten. Mothers emit simulta-
neously a very-low-pitched bleat or rumble, a call that is
specific to the periparturient period or when they are in close
contact with their young. If mother and young are left undis-
turbed, maternal behavior develops normally and lasts for
several months. However, the maintenance of maternal
behavior depends very strongly on the events occurring imme-
diately after parturition. Should mother and young be sepa-
rated at birth, maternal behavior vanishes within hours. The
ability of ewes and goats to remain responsive to a neonate is
therefore limited to a sensitive period starting around parturi-
tion and ending a few hours later and is under the direct control
of the neuroendocrinological changes that take place at partu-
rition (Dwyer, 2014; Poindron et al., 1993, 2007b).
1.17.4.1.3.2 Maternal Selectivity
Maternal responsiveness is soon followed by selective maternal
care. It is characterized by the acceptance of suckling attempts
by their own young along with the rejection of alien ones.
Many ewes and does show selective nursing after only
30 min of contact with their young but most are selective after
2–4 h postpartum (Nowak et al., 2011; Poindron et al.,
2007b). There is some evidence that selectivity in goats may
result from olfactory labeling of the kid though maternal
licking and nursing (Gubernick, 1981), while in sheep, recogni-
tion of the lamb relies on its distinctive individual odor. None-
theless, selective nursing in goats reflects learning of some
individual olfactory characteristics: even twins have different
odors (Poindron et al., 2007b). Selective nursing depends
primarily on the main olfactory system since when ewes and
goats are made anosmic, affecting only the main olfactory
system, they subsequently nurse their own as well as alien
young. If separated from their young, mothers express strong
distress reactions with intense vocal (high-pitched bleats) and
motor activity (walking, circling). They stop vocalizing and
moving when reunited with their own lamb while they keep
being agitated in the presence of an alien young. Mothers
also identify their neonate from a distance by using auditory
and visual cues (Nowak et al., 2011; Poindron et al., 2007b).
Nonolfactory recognition develops slightly later than olfactory
recognition, but it is fully effective by 6 h postpartum. The
neural basis involved in these two types of recognition
processes seems to be independent since anosmic ewes can
discriminate between their own and an alien young from
a distance even though they are willing to nurse any lamb
(Ferreira et al., 2000). Olfaction alone as a means of recogni-
tion declines in importance with time, and after several days
it is probably used only for close contact discrimination. The
respective roles of hearing and vision have not been clarified
yet, although ewes do recognize the voice of their young
463
(Searby and Jouventin, 2003) and cues from the lambs’ face
seem important for visual recognition (Alexander and Shillito,
1977). As lambs and kids grow older, recognition improves
and it seems very likely that mothers learn a composite image
of their young, including appearance, behavior, and voice.
Once initiated, maternal behavior lasts for at least 3 months
after which lambs and kids are usually weaned by humans.
However, if ewes and goats are left undisturbed, natural wean-
ing occurs when their young are between 4 and 6 months of age
(Poindron et al., 1993; Miranda-de la Lama and Mattiello,
2010). Maternal memory and motivation seem to be rather
labile if mothers do not maintain close proximity with their
young. Seven days after parturition, if lambs are temporarily
removed, maternal selectivity remains following 36 h of sepa-
ration, but after 3 days, most ewes lose their maternal drive
and reject their young (Keller et al., 2005). Yet, vocal recogni-
tion of kids by their mothers persists for up to 13 months after
weaning, at a time when goats are already nursing a second
generation of young (Briefer et al., 2012). The fact that mothers
remembered the vocalizations of their weaned kids recorded
1.1 years earlier is quite remarkable and demonstrates the exis-
tence of long-term memory.
1.17.4.1.4 Welfare Issues Specific to Maternal Behavior
1.17.4.1.4.1 Management of Pregnant and Parturient Ewes
and Goats
The neonatal period is always of special concern for lambs and
goats as it is a period of high vulnerability (Dwyer, 2008a,
2014; Dwyer et al., 2016; Nowak et al., 2000). Several aspects
have to be taken into account to ensure maximum chance of
survival and optimum development of the young. First of all,
proper nutrition during gestation is a key element since under-
nutrition leads to reduced udder weight, delayed onset of lacta-
tion, reduced colostrum yield, and impaired maternal behavior
(Dwyer, 2014; Dwyer et al., 2016; Terrazas et al., 2009).
Improper nourishment may also lead to metabolic imbalance
(e.g., pregnancy toxemia) and death. Nutrient requirements
for sheep and goats are provided in detail in the document
Nutrient Requirements of Small Ruminants: Sheep, Goats, Cervids
and New World Camelids (NRC, 2007). Secondly, both ewes
and goats tend to separate themselves from their flocks,
a process that facilitates the development of the mother–young
bond. Even under indoor conditions sheep display such
a behavior if cubicles are provided (Nowak et al., 2008). Any
disturbance during that period, especially with highly reactive
females, may jeopardize the onset of maternal behavior, and
primiparous mothers are more prone to react. Prolonged labor,
associated with pain and stress, increases the risk of hypoxia
and brain trauma; it impairs maternal and infantile behavior
and it also diminishes the chance of survival of the newborn.
A swift parturition is, therefore, important and should it be pro-
longed, it is important to intervene and pull the fetus out.
Under crowded situations, care should also be taken to facili-
tate the development of a bond and to avoid mother–young
separation due to excessive human intervention or to interfer-
ence by other females. In some cases, when several ewes give
birth simultaneously, lamb stealing by preparturient ewes
occurs (Dwyer, 2014; Nowak et al., 2000). Unfortunately, these
lambs may be rejected once the ‘alien’ ewe has given birth
to her own lamb and they are not taken back by their own
464 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
mother: a fatal rejection. It is often advised in highly stocked
areas to isolate postparturient females with their neonates in
individual pens to favor early mother–young interactions,
especially in case of multiple births. However, restricting the
movements of postparturient females increases the risk that
they lie on their offspring and suffocate them to death. There-
fore, the pen should be wide enough to allow the animals to
move freely (1.8–2.0 m2). It is also recommended to isolate
females only if parturition is well advanced or terminated
and just for the time needed to establish the mother–young
bond (24–48 h). Then, mothers and newborn can be released
and subsequent separations are no longer risky.
1.17.4.1.4.2 Fostering
There may be a number of occasions when neonates cannot be
reared by their own mother (no expression of maternal
behavior, rejection, insufficient milk yield, death of mother).
While under unsupervised conditions such young are likely
to die from starvation, close supervision at lambing may often
save them. A way to save them is to force another maternal
female to accept these orphans by triggering the development
of another bond. Fostering has been more or less successful
in sheep using three methods: (1) bonding an alien lamb to
a ewe during the period of maternal responsiveness, i.e., before
maternal selectivity takes place; (2) transferring the mother’s
own lamb characteristics to the one to be fostered; and
(3) allowing a second bond to form progressively with the alien
lamb by cohabitation (Nowak et al., 2008). In all cases the
procedure consists of penning a periparturient ewe (or goat)
with an additional young, helping it have access to the udder,
and preventing excessive expression of selective maternal
behavior until it is fully accepted. This may take a few days.
Should this method not be successful, lambs can be artificially
fed (see the later discussion)
1.17.4.1.5 Infancy
1.17.4.1.5.1 Attachment with the Mother
Lambs and kids are born in a well-advanced stage of physical
and behavioral development which allows them to interact
with their mother on a multisensory basis. The activity of
the newborn, which has to be able to stand steadily before ori-
enting toward the body of the mother, needs to be coordi-
nated with that of the mother. Most neonates stand within
30 min after birth. As their coordination improves, they
nose the side of their mother’s body until a teat is found,
usually within the first hour. They are guided by various
cues emanating from the mother’s body, and in sheep many
ewes may also help the young by lowering their back and
bending a hind leg to facilitate access to the teat (Vince,
1993). Both kids and lambs learn the position and shape of
the udder very quickly, a learning process that is probably sus-
tained by the dynamics of mother–young interactions and the
reinforcing properties of suckling. This spontaneous attrac-
tiveness to the mother’s udder, reinforced by the fact that
suckling is displayed at hourly intervals, brings the newborn
in contact with maternal cues and behaviors required for the
establishment of individual recognition. Most lambs and
kids develop a clear preference for their dam by 12–24 h of
age, and their ability to discriminate their mother from an
alien ewe improves markedly over the first few days of life
(Nowak et al., 2011; Poindron et al., 2007a). While at
12–24 h identification of the mother is mostly based on
cues that lambs can perceive at close quarters, lambs can
clearly locate their mother from a distance of several meters
when they are 3 days old. Olfactory cues are not necessary
for newborn lambs to discriminate between two ewes at close
quarters even though they do recognize maternal odors at
birth (Vince, 1993). Instead, both auditory and visual cues
are used (Nowak et al., 2011). This reflects the fact that initial
orientation toward the mother is based on general maternal
signals since they are the differences between acceptance
behaviors (low-pitched bleats signal acceptance) and rejec-
tion behaviors (high-pitched bleats indicate aggression) that
make lambs and kids choose a specific adult (Poindron
et al., 2007a; Terrazas et al., 2002). Step by step neonates learn
various individual features from their mother which results in
an improved ability to recognize her from a distance. The role
of hearing seems to be as important in lambs as in kids once
they are several days old (Briefer and McElligott, 2011; Searby
and Jouventin, 2003), but there is no doubt that infants form
a multisensory image of their mother. Bit by bit this specific
responsiveness to individual maternal cues leads to a specific
attachment process that consolidates into an enduring
emotional bond, characterized by a tendency to seek and
maintain closeness, especially during times of stress (Nowak
et al., 2011; Gaudin et al., 2015). This bond with the mother
is based on the infant’s need for safety, security, and
protection.
1.17.4.1.5.2 Relation with Humans
Farm animals develop an important social network with inter-
specific partners, such as humans. Unlike other forms of social
attachment, the relationship sheep and goats can develop with
humans is not well understood. It seems that both species
develop intra- and interspecific relationships but not in
a concomitant manner since the presence of the mother
reduces strongly the lamb’s motivation to interact with
a human. However, when newborns are separated from their
mother and reared under artificial feeding conditions, the
human becomes a salient figure and this may facilitate the
management of lambs and kids under laboratory conditions
as well as on the farm (Nowak and Boivin, 2015; Miranda-de
la Lama and Mattiello, 2010). Usually, human–animal rela-
tionships are facilitated by positive social contacts (stroking,
bottle-feeding) provided by a specific caregiver in the first few
weeks after birth. In both species, stroking alone reinforces
the motivation to interact with the stockperson (Boivin and
Braastad, 1996; Tallet et al., 2005). Stroking is appeasing, it
induces relaxing postures in lambs, and it decreases heart rate
due to an activation of the parasympathetic system. Behavioral
tests comparing responses directed toward a familiar caregiver
and unfamiliar or familiar humans show that lambs do search
specifically the proximity of their human caregiver. Differential
emotional responses in the presence (calmness) or the absence
of the partner (agitation) are also expressed (Figure 7). The
clear and durable signs of appeasement shown by lambs in
the presence of their caregiver suggest that the human can be
perceived as an affective substitute in artificially reared animals
(Nowak and Boivin, 2015). The same phenomenon may apply
to young goats (Miranda-de la Lama and Mattiello, 2010).
 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals 465

Number or zones crossed Number of zones crossed

30 40
*
25 35
30
20 25
15 20
10 15
10
5 5
0 0
Isola on Reunion Separa on Isola n Reunion Separa

Number of vocaliza ons Number of vocaliza ons

40 40
35 * * 35
30 30 *
25 25
20 20
15 15
10 10
5 5
0 0
Isola on Reunion Separa on Isola n Reunion Separa

Familiar caregiver Unfamiliar person Familiar person

Figure 7 Affinity of lambs to their caregiver. Lambs were tested alone (isolation), then with a person (reunion), and alone again once the person
left the pen (separation). The subjects’ reactions were observed in two comparative situations: familiar caregiver versus unfamiliar person (left histo-
gram) or familiar caregiver versus familiar person (right histogram). The number of zones crossed and the number of vocalizations emitted indicate
that lambs are soothed during reunion and agitated after separation. Appeasement during reunion and distress after separation is more important
when the test is performed with the familiar caregiver.

1.17.4.1.6 ‘Followers’ versus ‘Hiders’
One of the main differences between sheep and goats is that
lambs follow their mother within hours after birth and, there-
fore, are classified as ‘followers,’ while goat kids remain tempo-
rarily hidden from their mother while she forages. Timing and
duration of the hiding phase varies from a few days to several
weeks, but it is a general trend that remains even when animals
are reared indoors (Miranda-de la Lama and Mattiello, 2010;
Poindron et al., 2007b). The mother leaves her kid for periods
of up to 8 h before returning to the precise location where the
kid is lying. During that stage, mother–young separation does
not trigger any distress response. In contrast to goats, lambs
stay close to their mother for the first few days after birth and
display distress behavior should they be separated. Both types
of mother–young relationships are believed to be antipredator
strategies. In one case hiding allows neonates to remain out of
sight; in the other, the young imitate the flight reaction of their
mother (or other adults) as the flock runs away from a potential
source of danger. Over time, kids and lambs tend to associate
and form bonds with age mates and to engage in play behavior.
1.17.4.1.7 Welfare Issues Specific to the Young
1.17.4.1.7.1 Thermal Discomfort
At birth, lambs and kids lose heat because their coat is covered
with amniotic fluid. A direct consequence may be hypothermia
as the ambient temperature can fall from 39  C in the uterus to
10  C or less outdoors, if parturition takes place in winter
(Dwyer, 2008a; Dwyer et al., 2016). Once dried and properly
fed, the neonates are resistant to cold weather and can maintain
homeothermia even below freezing temperature. It is the
combination of humidity, wind, and inadequate colostrum
intake that weakens them (Obst and Day, 1968; Figure 8(a)).
Newborn lambs and kids seek shelter more readily than their
mothers, which are insulated by their full fleece or hair.
Providing sheltered areas may, therefore, protect the young
from the deleterious effects of wind and rain and contribute
to their thermal comfort. Under indoor conditions, weak hypo-
thermic lambs should be put temporarily under a heating lamp
or a warm air blower that will help them maintain an adequate
body temperature, after which they can be put back with their
mother. While cold stress is a common source of thermal
discomfort, it should be borne in mind that young ruminants
are also vulnerable to heat stress. Lambs respond to high
temperature by increased respiratory rate, irregular breathing,
and periods of apnea (Riesenfeld et al., 1996). Indeed, severe
heat waves can lead to death after several days. Therefore,
should lambs and kids be kept in buildings, effective ventila-
tion is essential to allow free air circulation and decrease
ambient temperature.
1.17.4.1.7.2 Colostrum Needs
Ruminants are born markedly hypogammaglobulinemic, the
mother providing passive immunological protection through
the immunoglobulins contained in the colostrum. Passive
immunity must be acquired during a very narrow time window
since in newborn lambs, gut closure to immunoglobulin
absorption occurs between 24 and 36 h of birth (Dwyer
et al., 2016; Nowak and Poindron, 2006). Consequently,
lambs and kids must find the teat and suckle as soon after birth
as possible in order to receive an adequate amount of
466 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
(a)
% mortality 0–48 h a er birth
80
60
40
10–16 −1 )
20
1–8
d (km
0 0
ee
> 1.5 0.5–0 0 sp
ind
W
Rainfall (mm day−1)
Figure 8 (a) Lamb mortality during the first 48 h of life according to rainfall and wind speed. (b) Average periods for which carbohydrates (lactose
from colostrum, glycogen from the liver and the skeletal muscle) available during the first day after birth can sustain heat production in lambs at
different ambient temperatures and colostrum intakes. Adapted from Obst, J.M., Day, H.R., 1968. The effect of inclement weather on mortality of
Merino and Corriedale lambs on Kangaroo Island. Proc. Aust. Soc. Anim. Prod. 8, 239–242 and Mellor, D.J., Cockburn, F., 1986. A comparison of
energy metabolism in the new-born infant, piglet and lamb. Q. J. Exp. Physiol. 71, 361–379.
colostrum. Lambs born outdoors need 280 ml kg1 body
weight during the first day, while 210 ml kg1 should be
adequate for lambs born indoors (Mellor, 1990). Insufficient
colostrum intake may lead to exhaustion of energy reserves
and hypothermia, which in severe cases is fatal (Mellor and
Cockburn, 1986; Figure 8(b)). Care should be taken that
mothers have sufficient colostrum yield and that neonates
display proper suckling activity. If this is not the case, lambs
and kids should be supplemented with an external source of
colostrum. Usually, cow, sheep, and goat colostrum are equally
efficient and have interspecific positive actions; however,
commercial colostrum is not as satisfying to ensure proper
immunological protection (Argüelloa et al., 2004).
1.17.4.1.7.3 Behavioral Needs
Sheep and goats are social animals. Immediately after birth, the
core of their attention is the mother. Care should be taken that
the mother–young bond is not disrupted, especially with twins
and triplets which are more likely to lose contact with their
mother. Concerning newborn kids, because of their natural
tendency to hide even under housing management (Miranda-
de la Lama and Mattiello, 2010; Poindron et al., 2007b),
providing shelter will satisfy their behavioral need. If animals
have to be penned at parturition, it is important not to isolate
the mother–infant dyad beyond the first few days after parturi-
tion. With time, lambs and kids interact socially with peers and
adults, a process that plays an important role in shaping the
behavioral development of juveniles: the mother facilitates
the transmission of acquired social behaviors, environmental
preferences, and consumption of solid food while sexual play
with peers has a strong impact on later sexual preferences
(Dwyer, 2008a; Nowak et al., 2008).
1.17.4.1.7.4 Mother–Young Separation
Separation of mother and young occurs fairly frequently in
small ruminant husbandry either temporarily during manage-
ment procedures or permanently at birth, to rear lambs and
kids artificially, and at weaning when it is time to raise adults
and juveniles in separate flocks. Artificially reared lambs
(b)
90 18–26 °C
80 0–10 °C
70
60
50
Hours
40
30
20
s
10
0
0 150 210 290
Colostrum intake (ml)
experience a marked emotional stress represented by the loss
of their attachment figure, the mother, and a nutritional stress
provoked by the abrupt transition from maternal milk to
a commercial milk substitute. Artificial rearing of lambs and
kids requires close attention and a high standard of supervi-
sion. First of all, it is essential that all animals receive an
adequate supply of colostrum. Their body temperature should
be monitored to avoid hypothermia and, if needed, a heating
lamp should be provided. Where automatic feeding equipment
is used, lambs and kids must be trained to suck from the artifi-
cial rubber teats several times per day until fully independent.
Thereafter, an adequate amount of milk substitute must be
provided, and from the second week onward, palatable and
nutritious solid food should be given. Despite close attention
and human care, the welfare of artificially fed lambs is now
questioned. Compared to young reared by their mother, those
lambs display abnormal behaviors (cross-sucking activity) and
have higher basal cortisol levels. When tested in an open field,
artificially fed lambs emit less distress vocalization and their
cortisol response increases more sharply than that in lambs
reared with their mother. They also have decreased immuno-
logical responses and a high incidence of diarrhea (Napolitano
et al., 2008). Attempts to reduce the detrimental effects of early
separation focus on the emotional and nutritional aspects of
artificial rearing. It is not recommended to leave a lamb alone,
except for specific experimental reasons and only for short
periods of time. However, even in a group, peers do not
compensate for the loss of the attachment figure. In this case,
a human can provide a ‘maternal substitute’ (Boivin and
Braastad, 1996; Boivin et al., 1997). The necessary condition
is for the human to perform gentle stroking and feeding, two
forms of physical attention that trigger the affinity for the care-
giver. Regarding nutrition it is known that mixing ewe milk and
a milk substitute during the first week and then moving gradu-
ally to only milk substitute reduces some problems. Ewe–lamb
separation at weaning on the farm occurs generally at 3 months
of age, which is considered early weaning compared to natural
conditions (Poindron et al., 1993). It results in a temporary
increase in cortisol levels, distressed behavior, altered social
 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
interaction, and disrupted circadian rhythmicity (Dwyer,
2008a). Care should be taken not to wean the lambs too early;
otherwise, growth rate is retarded and lambs have an increased
susceptibility to disease. Nonetheless, maternal deprivation
appears to be worse when performed at 2 days (artificial rear-
ing) than at 3 months of age (early weaning).
1.17.4.2 Regarding Lodging (Cages, Floors, Nest Boxes,
‘Enrichment’)
1.17.4.2.1 Outdoor Facilities
Among large domestic animals, sheep and goats present the
widest diversity of breeds, and they live in the largest variety
of habitats. They are found from tropical to temperate regions,
and they can face very hot or very cold climatic constraints.
They are reared in sheds, in fenced meadows, in open moun-
tain pastures, in arid steps driven by nomadic people, and
even in remote rangelands with hardly any human supervi-
sion (for sheep see Dwyer, 2008b; Kilgour et al., 2008).
From such diversity recommendations for ‘standard’ manage-
ment practices or housing cannot be derived. The general
guideline is to take into consideration local conditions and
to ensure that animals are properly cared for. Sheep and goats
well adapted to local conditions usually take advantage of
natural shelter, and the use of habitat is strongly influenced
by season or weather conditions (Dwyer, 2008b). Nonethe-
less, under outdoor conditions local climate and extreme
temperatures should be incentive for providing shelter. Too
often animals are fenced off in barren paddocks with nothing
to protect them from adverse weather conditions. Various
types of shelter may be used, from artificial settings to more
natural windbreakers like shrubs, gramineous plants (e.g.,
Phalaris) which are not ingested by sheep, or tall deciduous
trees. Such shelter reduces wind velocity, protects adult
animals from rain and snowstorms, and provides valuable
shade during hot sunny days (Gregory, 1995). With shade,
sheep can support ambient temperatures of up to 50  C
(Dwyer, 2008b). Because sheep give birth normally in winter,
wind shelter can reduce mortality rates by up to 3% in single
lambs and 37% in twins. It is recommended that, to be effec-
tive against lamb mortality, shelter should be (1) very dense,
Figure 9 Preference for lying areas expressed by ewes before and after shearing. Adapted from Færevik, G., Andersen, I.L., Bøe, K.E., 2005. Prefer-
ences of sheep for different types of pen flooring. Appl. Anim. Behav. Sci. 90, 265–276.
467
in order to limit heat loss, (2) designed to allow neonates to
take full advantage of the sun, and (3) sufficiently familiar to
encourage ewes to isolate from other sheep at lambing
while minimizing the risk of mother–young separation
(Pollard, 2006).
1.17.4.2.2 Floor
Sheep and goats are reared under various floor surfaces including
compacted soil, straw bedding, concrete, concrete- or wood-
slatted floors, not naming all the types of surfaces they can be
in contact with when reared outdoors. All of them are considered
to be suitable as long as they are kept dry and clean and they are
nonskid surfaces. However, some studies show that while
unsheared ewes show no clear flooring preferences at low
ambient temperatures (1.4–8.6  C), sheared ewes choose softer
floors with low thermal conductivity (Figure 9). Wooden floors
are preferred over expanded metal ones, straw to wooden floor,
and straw to expanded metal floor (Færevik et al., 2005). Goats
kept under moderate temperatures (10–12  C) prefer expanded
metal to solid wood, while solid wood and mattress are equiva-
lent, and straw the least preferred flooring (Figure 10). During
cold periods (8–12  C), mattress and solid wood are the most
preferred types of flooring, expanded metal intermediate, and
surprisingly, straw is not perceived as attractive by goats (Bøe
et al., 2007). Thus both species do express a need for flooring
with lower thermal conductivity under low temperature
constraints. While the welfare of sheared ewes is improved if
straw is provided during the first weeks after shearing, softness
does not have any effect on the choices of goats. The fact that
in the wild goats are adapted to rest on rocky hills may explain
their lack of interest in soft surfaces.
1.17.4.2.3 Social Enrichment
Sheep and goats are highly gregarious species; they live in
flocks of various sizes, and therefore, they should never be
reared in social isolation. There is surprisingly no evidence
of what could be an optimal group size that satisfies the social
needs of these mammals. Under natural conditions, the
preferred group size in sheep is influenced by the type of breed
(Dwyer, 2008b). In stable flocks, social positive relationships
improve group affinity, and when reared in paddocks, sheep
468 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
Figure 10 Preference for lying areas expressed by dairy goats under moderate (12–12  C) and cold temperature periods (8–12  C). Adapted from
Bøe, K.E., Andersen, I.L., Buisson, L., 2007. Flooring preferences in dairy goats at moderate and low ambient temperature. Appl. Anim. Behav. Sci.
108, 45–57.
do form subgroups. While sheep display little affiliative
behavior with each other, goats groom, sniff, and lick each
other (Miranda-de la Lama and Mattiello, 2010). It has been
empirically suggested that the minimal group size is four or
five individuals for adult sheep, but the familiarity with the
environment may influence this number. One study reports
that when 1-month-old lambs are put in a novel environ-
ment, the minimal group size for suppressing nearly all the
distress vocalizations is four individuals (Rault et al., 2011).
A second study reveals that while separation from mates in
the familiar pen increases respiration rate, cortisol concentra-
tion, number of steps, vocalizations, and urinations, these
reactions decline to basal levels once a single companion
ewe is present. There is no additional advantage to having
two companion ewes (Carbajal and Orihuela, 2001). No
equivalent data are available for goats, but if adults need to
be housed in groups, care must be taken to minimize harmful
social behavior such as aggression.
1.17.4.2.4 Physical Enrichment
Because ewes show a strong tendency to isolate from the flock
when they are about to give birth outdoors, providing such
a possibility in an enclosed building is beneficial for the early
mother–young interactions and bonding. Also ewes that had
been previously exposed to 1.2  1.8 m cubicles use them
more readily than naïve animals (Figure 11(a)). After giving
birth, the ewes occupying 1.2  1.8 m cubicles remain longer
on the birth site and closer to their lambs, are rarely disturbed
by other ewes, and have fewer lambs separated or stolen from
them (Gonyou and Stookey, 1985; Figure 11(b)). In goats
living within a confined group, the strong hiding drive of the
newborn needs to be taken into account. Thus, small hiding
boxes have been provided (50  15  25 cm) and newborn
kids use them for 4–7 days (Lickliter, 1984). Moreover, because
goats are known to climb on a variety of objects, providing such
devices may contribute to their well-being. Environmental
enrichment consisting of old tires and wooden railway sleepers
to climb on, and PVC piping to mouth and butt, increased
weight gain in a feedlot of castrated goats by reducing aggres-
sive behavior at the feed trough (Flint and Murray, 2001). It
is recommended that ample climbing space is provided to
avoid dominant animals from displacing subordinates.
1.17.4.2.5 Human Contact
The development of a positive human–animal relationship in
small ruminants has obvious beneficial effects by lowering
their level of fear or increasing their level of confidence in
people. Gentle stroking by a familiar caregiver is a strategy
that helps sheep and goats calm down in potentially stressful
situations (e.g., isolation: Boivin and Braastad, 1996; Boivin
et al., 1997). Additional benefits of gentle stroking have been
observed on milk production in goats and on lambs’ immune
system and cortisol response to handling (Caroprese et al.,
2009b; Miranda-de la Lama and Mattiello, 2010). However,
a relationship with a human takes place more easily when
lambs are reared without their mother. The bond with the care-
giver can be equivalent to the filial attachment developed by
mother-reared lambs, and the underlying mechanisms medi-
ating these processes may be similar (Nowak and Boivin,
2015). Although the existence of a sensitive period is still
debated, affinity with the caregiver develops more rapidly if
positive interactions take place immediately after birth.
 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals 469

Figure 11 (a) Importance of previous experience on the percentage of parturient ewes using a 1.2  1.8 m cubicle to isolate themselves. Ewes had
never experienced such cubicles before, or had been exposed and did or did not use them. (b) Percentage of parturient ewes that were interfered
with by other ewes whether they had no hiding place (control), had access to cubicles and lambed outside, or had access to cubicles and lambed in
them. Adapted from Gonyou H., Stookey, J.M., 1985. Behavior of parturient ewes in group-lambing pens with and without cubicles. Appl. Anim.
Behav. Sci. 14, 163–171.

1.17.4.3 Regarding Environment (Light, Sound, Vibration, 1.17.4.3.2 Dominance and Agonistic Behaviors
Temperature, Density of Animals) Within an established flock of sheep, many expressions of
dominance are not necessarily associated with overtly aggres-
1.17.4.3.1 Flocking
sive behavior. Sheep maintain hierarchy through subtle activi-
Both goats and sheep show a strong desire to remain with
ties, like head movements and eye contact, rarely head-butting.
their group mates, and they become very vocal when sepa-
Subordinate sheep usually move away and do not retaliate after
rated from the flock. They may separate themselves into
receiving aggressions (Nowak et al., 2008). Dominance is more
subgroups consisting of family units or of animals from
obvious in a flock of goats, which are a much more aggressive
different origins when mixed together to form bigger flocks
species, and the presence of horns is an important determinant
(Miranda-de la Lama and Mattiello, 2010; Nowak et al.,
of dominance. Accordingly, most agonistic interactions are
2008). Unlike the mother–infant dyad, little is still known
brief feints or rushes in which the dominant animal lowers
about recognition between adult individuals. Sheep, and in
its head and points its horns at the subordinate. However,
all likelihood goats as well, are predominantly visual
long fights may occur in which horns and head are clashed
animals. Cohesion of the group is maintained by each indi-
together repeatedly when two animals are of equal or undeter-
vidual adjusting its behavior or position relative to the
mined rank. If the conflict escalates, aggressive behavior
postures or activities of other members. This leads to
involves one goat standing up on its hind legs, lowering its
synchronized feeding activity, resting, spreading of alertness
head, and striking it against its opponent’s head. When space
states, and tendency to follow a leader. But above all, sheep
is limited and butting cannot be performed, biting is a more
are known to recognize individuals on the basis of visual
frequent means to maintain individual distances (Miranda-de
cues: up to 50 different sheep faces can be discriminated in
la Lama and Mattiello, 2010). Usually, the level of aggression
an operant conditioning task at one time, and such recogni-
is higher among animals kept indoors than among individuals
tion may last for up to 2 years (Kendrick, 2008). Sheep can
living outdoors because of the difference in the amount of
also discriminate conspecifics, based on a similar test para-
space available and the impossibility for subordinates to avoid
digm, using olfactory cues contained in urine, wool, feces,
dominants. There are a number of consequences for subordi-
saliva, and secretions from the interdigital, inguinal, and
nate individuals. When access to food is limited, subordinate
infraorbital pouches. How these senses are used in group
sheep are likely to have access to poorer-quality forage or
cohesion and interindividual communication remains
they may be displaced from the feeding-trough if access is
unknown. The tendency to aggregate varies between breeds,
limited. Subordinates may also be displaced from shaded
and mountain breeds usually tolerate greater individual
areas, males may mate the less preferred females, and females
distances than sheep from lowland breeds. Leadership is an
may have their lambs stolen by dominant subjects.
additional component of sheep and goat social behavior,
and it is expressed by animals that initiate a movement in
the group. Leadership is never associated with dominance. 1.17.4.3.3 Social Isolation
Rather, it is negatively correlated with the tendency to join Sheep and goats are highly gregarious, and separation from the
the flock; thus, it is generally the most independent indi- flock is perceived as very stressful. Immediately after being
vidual that initiates movement. isolated, they show increased behavioral and vocal activity
470 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals

(Price and Thos, 1980). Various physiological responses are
also observed after sheep are isolated in a pen, specifically,
increases in heart rate, number of neutrophils, concentration
of catecholamines, cortisol and prolactin, plus a decreased
number of lymphocytes (Cockram et al., 1994; Minton et al.,
1992; Parrott et al., 1994). The physiological responses vanish
with repetitive testing, but the behavioral changes are still
visible when sheep are isolated for the seventh time. Goats
do not habituate to social isolation either (Sieberta et al.,
2011; Figure 12(a)). While paradigms used to investigate the
effect of social isolation usually confound the direct effects of
isolation with other factors (e.g., being moved to a novel of
environment, e.g., Cockram et al., 1994), removing group
mates from a room also triggers behavioral distress and eleva-
tion of cortisol in the ewe left alone (Guesdon et al., 2012;
Figure 12(b)). If sheep have to be put in individual pens, social
contact must be maintained by the presence of peers in adja-
cent pens. It has also been suggested that mirrors may serve
as a social surrogate under total social isolation; however,
they do not completely abolish the physiological stress
response (Parrott et al., 1988).
1.17.4.3.4 Density of Animals
Crowding, which implies an excessive density of animals in
a restricted space, is typically found indoors, but its potential
deleterious effects are difficult to evaluate. Nonetheless, it is
a fact that sheep confinement within enclosures activates the
hypothalamic–pituitary–adrenal (HPA) axis with an increase
of cortisol and the display of stereotypic behaviors (Caroprese,
2008). Reduction of the lying area (1.0, 0.75, and 0.5 m2 per
ewe) alters the resting patterns of pregnant ewes (Bøe et al.,
2006). Reduction in space allowance from 3 to 1.5 m2 per
individual in dairy ewes is associated with a decreased propor-
tion of animals walking and an enhanced incidence of aggres-
sions (Caroprese et al., 2009a). In addition, immune response
and milk production are increased in dairy ewes when space
allowance increases from 1 to 2 m2 per ewe illustrating an
improvement in health condition and well-being (Sevi et al.,
1999). Compared to sheep, goats are reported to have a signif-
icantly higher rate of aggressive interactions; they display biting
to maintain individual distances if space allowance does not
allow butting (Miranda-de la Lama and Mattiello, 2010).
Increased interindividual competition, which results in denied
access to the feeding-trough, leads to longer time spent queuing
and shorter time spent feeding, and an increased number of
displacements and aggressive interactions (Jørgensen et al.,
2007; Loretz et al., 2004). Reduced available space impairs
the growing performance in young lambs as well (Gonyou
and Stookey, 1985; Horton et al., 1991). In animals reared in
pens, space allowance should allow them to lie down, stand
up, circle, and feed without restriction. For instance,
a maximum of three adult ewes per square meter of feeding-
trough is usually recommended to ensure proper access to
food; however, this should be adjusted according to the size
of the breed and whether ewes are pregnant or not. In Europe
ewes are allowed 1–1.4 m2 per head according to their size and
physiological status, rams between 0.5 and 2 m2, and lambs
0.25–0.90 m2 according to their size. Similar housing condi-
tions may apply to goats (Sevi et al., 2009). However, it is
uncertain if these figures are chosen on objective grounds and
truly satisfy the needs of sheep. In Europe recommendations
for animals used for scientific purposes are provided by the
Directive, 2010/63/EU (Directive, 2010), and they differ from
those concerning animals reared on farms and given by the

(a) Goats (b) Ewes

Vocaliza on
Vocaliza on 70
Number of high bleats
Number of high bleats

120 60
100 50
80 40
60 30
40 20
20 10
0 0
1 2 3 4 5 6 Session 1 Session 2 Session 3
Isola on sessions
Rearing on back legs Pawing with foreleg
Number of occurrences

20
Number of occurrences

8
15
6
10
4
2 5

0 0
1 2 3 4 5 6 Session 1 Session 2 Session 3
Isola on sessions Before A er

Figure 12 (a) Distress behavior in goats submitted to social isolation on six consecutive days. (b) Distress behavior before and after isolation in
ewes submitted to three consecutive testing sessions. Vocal and locomotor activities show that subjects do not habituate. Adapted from Sieberta, K.,
Langbein, J., Schön, P.C., et al., 2011. Degree of social isolation affects behavioural and vocal response patterns in dwarf goats (Capra hircus). Appl.
Anim. Behav. Sci. 131, 53–62 and Guesdon, V., Ligout, S., Delagrange, P., et al., 2012. Multiple exposures to familiar conspecific withdrawal is
a novel robust stress paradigm in ewes. Physiol. Behav. 105, 203–208.
 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
Department for Environment, Food and Rural Affairs in the UK
(DEFRA, 2000) and by the Guide des Bonnes Pratiques Ovines
in France (GBPO, 2011). Apparently these space allowance
ranges do not raise major practical problems for farmers in
terms of production nor any ethical concerns in terms of
research. Nonetheless, as sheep and goats cannot escape from
their pen to seek shelter or avoid unpleasant environmental
conditions, care must be taken not to expose them to excessive
heat, cold, noise, or dampness.
1.17.4.3.5 Heat and Cold Stress
Sheep and goats are rather resistant to climatic extremes, and
they are found in tropical regions as well as in mountain
pastures. However, under very hot conditions they reduce
their locomotion and food consumption, seek shade, and
feed preferentially at nighttime. In bare areas they congregate
together side by side (Silanikove, 2000). Such a peculiar
behavior occurs when the heat being taken by the body
exceeds the ability to dissipate it. Thus, by huddling together,
individuals reduce the intake of direct solar energy per body
surface and thermoregulate in a better way than when they
stand alone. There is little information considering specifi-
cally the effects of heat stress on recognized welfare indicators
compared to the impact on productivity (Caroprese, 2008;
Silanikove, 2000; Marai et al., 2007; Sevi and Caroprese,
2012). Goats seem to be less affected by heat than sheep,
which reflects their adaptation to harsh environments: 88%
of goats are found in Asia and Africa, mostly in tropical and
subtropical areas. In addition to reducing feed intake in sheep,
heat stress exposure (above 30  C) disturbs water metabo-
lism, energy and mineral balances, enzymatic reactions,
hormonal secretion (cortisol increase and decrease in thyroid
hormone activity), and blood metabolites and increases pulse
rate, respiration rate, skin and rectal temperature. However,
breeds native to tropical regions are better adapted to heat
than temperate breeds (Dwyer, 2008b). In pregnant ewes
a reduction in total embryo cell number, placentome size,
and lamb birth weight is observed under extreme heat.
Lactating ewes exposed to direct solar radiation (maximum
range: 33–37  C) exhibit high concentrations of nonesterified
fatty acids in blood (due to enhanced mobilization of body
reserves), depressed lymphocyte proliferation, and reduced
mammary defense capacity. Sheep are extremely cold-hardy
and rarely experience environmental conditions below their
lower critical temperature (the minimum body temperature
before bodily functions are altered). By contrast, because of
their short and wet fleece, and their small size, lambs and
kids are particularly vulnerable to hypothermia (Dwyer
et al., 2016). Soon after birth, their body temperature falls
by 1–2  C before stabilizing and then rising progressively to
reach an hour later the initial value (39  C). Their physiolog-
ical adaptive mechanisms to increase heat production are
limited and include nonshivering thermogenesis involving
brown adipose tissue metabolism and shivering. The second
mechanism usually occurs in older animals under cold and
wet conditions and metabolizes muscle glycogen. In case of
unfavorable environmental conditions, shelter should be
provided when animals are kept outside, and heating lamps
are provided indoors for small hypothermic lambs. Under
dry and no wind conditions thermal well-being in sheep is
471
considered to be optimal in the range of 6–19  C for adults
and 14–20  C for lambs. Natural physiological adaptation
occurs between 9 and 31  C in adults and between 2 and
25  C in lambs. Beyond these limits animals are considered
to be under thermal stress.
1.17.4.3.6 Lighting and Noise
Sheep and goats are seasonal breeders, and as such they both
are very sensitive to changes in the duration of light. It is the
circadian rhythmicity that controls the secretion of sexual
hormones via the release of melatonin. As a consequence, arti-
ficial control of light–dark ratio results in the control of repro-
duction in lightproof buildings (Chemineau et al., 1992). In
a more general sense, light intensity and color affect sheep
behavior, but data in that domain are scarce. Hypoactivity
with a decrease in the time spent feeding is observed in lambs
exposed to low light intensity (10 lx); conversely, they are
hyperactive at high light intensity (1000 lx). Green light
appears to increase the time spent standing and feeding
compared to white, red, and blue lights, and body weight
gain is improved (Caroprese, 2008). We also know very little
about the effect of noise on sheep and goats. Cortisol may
increase moderately in lambs under some conditions (2 kHz
and 75, 85, 95 dB) but not under others (100–6300 Hz and
85 dB) even though walking activities increase in the latter
condition. In pregnant ewes, exposure to loud noise (2 kHz
and 95 dB) results in shorter active behavior including eating,
but without change in cortisol levels or immune responses.
Despite the scarcity of information, available data suggest
that lambs and ewes are not particularly affected by intense
illumination nor by loud noises (Caroprese, 2008). In any
case, it is recommended that loud noise be avoided and that
sheep housed indoors must be exposed to natural daylight
cycle using either artificial or natural light, except for breeding
animals under a controlled light regime. Should supplemented
light be added, illumination intensity should be set to provide
75–100 lx (Caroprese, 2008).
1.17.4.4 Regarding Modifications of Reactivity to Stress
by Neonatal Manipulations
1.17.4.4.1 Prenatal Stress in Small Ruminants
Aversive events taking place in early life can alter the ability to
cope with subsequent stressful circumstances. Compared to the
vast amount of research on prenatal and neonatal stress in
rodents, only a limited number of experiments have been per-
formed on small ruminants. Work on sheep and goats has
shown that aversive prenatal experiences may influence post-
natal physiological and behavioral responses. Kids exposed to
repeated prenatal stress develop heavier adrenal medullas
and higher HPA axis activity at 1 month, and they are more
reactive in open-field tests; females seem to be more sensitive
than males (Duvaux-Ponter et al., 2003; Roussel et al., 2005).
Similarly, lambs born to mothers stressed during pregnancy
show increased exploratory behavior and locomotion at
8 months of age compared to control lambs (Roussel et al.,
2004). While no behavioral differences are observed at an
earlier age, basal cortisol concentrations are nonetheless higher
in 25-day-old prenatally stressed lambs compared to controls.
Some authors report a longer-term effect on cognitive and
472 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
emotional aspects since at the age of 1.5–2.5 months lambs
subjected to prenatal stress are more fearful, do not perform
as well in a T-maze, and show pessimistic-like judgment in
a cognitive bias test (Coulon et al., 2015). The effects are not
necessarily comparable between studies and species since the
types of stressors vary (e.g., pregnant mothers exposed to
social isolation, transport, or dog) and postnatal evaluation
involves a diversity of testing situations (e.g., isolation tests,
surprise tests, dark–light test, novel environment). For
instance, social instability – which triggers aggression among
pregnant goats – has minor effect in terms of the behavioral
development of the kids. Compared to young born into
stable social groups, they seem to be less fearful at 5 weeks of
age and have lower basal cortisol levels (Andersen et al.,
2008). Whether these effects persist beyond infancy is
unknown.
1.17.4.4.2 Neonatal Stress in Small Ruminants
In dairy goats, kids are taken away from their mothers the
moment they are born and reared artificially. In sheep, many
lambs follow the same fate for various reasons: nonmaternal
ewe, insufficient milk yield, large litter size. Artificial rearing
of lambs is not without consequences (Napolitano et al.,
2008). It is associated with immediate behavioral disturbances,
such as increased vocalization and redirected sucking on pen
mates, as well as increased basal neonatal cortisol and oxytocin
levels indicative of stress. Such lambs also have a lower anti-
body titer to keyhole limpet hemocyanin in the following
weeks after injection, illustrating a marked depression of
immunological responses and suggesting reduced level of cyto-
kines stimulating B cell activity. Moreover, when tested in isola-
tion at 3 weeks of age they show reduced locomotion and
vocalization and an increased cortisol response (Napolitano
et al., 2008). Yet, an increased cortisol response is not system-
atically observed if lambs are tested at an older age (Moberg
and Wood, 1982). The behavioral consequences of early social
experience are more consistent and can still be observed at
42 days; they are even more accentuated if lambs had been
reared in total isolation. In an open-field test, artificially reared
lambs kept in isolation express a nonfunctional stereotyped
behavior called flank touch and described as ‘lamb reaching
back and touching its flank with its muzzle’ (Moberg and
Wood, 1982). This self-directed behavior may be displayed in
substitution of proximity searching and suckling that is nor-
mally directed toward the mother. The long-term effect of
maternal depravation has never been investigated in small
ruminants.
1.17.4.5 Regarding Methods to Quantify Parameters
Presumably Indicative of Stress
The physiological responses to stress relate to the functioning
of the HPA system, the sympathetic-adrenal medullary system
(SAM), and some assessment of the immune system. The HPA
axis is a neuroendocrine response triggered by external or
internal factors that act on the hypothalamus. Specific neurons
of the paraventricular nucleus are involved, and in sheep,
isolation, restraint, or exposure to a dog activates neurons
producing CRH, AVP, and enkephalin (Cook, 2004; Minton,
1994; Rivalland et al., 2007). This provokes an increase in
circulating cortisol. The autonomic SAM is also activated at
detection of some threat and causes the release of catechol-
amines from the adrenal medulla. However, the release of
adrenaline and noradrenaline is far more difficult to detect
as the dynamics of secretion is more rapid than for cortisol
(Parrott et al., 1994, 1998). Because catecholamines influence
peripheral systems, they can be monitored by measurement of
heat rate, respiration rate, body temperature, or blood pres-
sure. Temporary rise in cardiac rate and cortisol indicates
a normal response to a threat, and if the stressor is short
lasting, the physiological responses eventually decline to base-
line. However, standard stress responses are difficult to charac-
terize in sheep as they may be influenced by breed, age,
experience, physiological state, gender, and considerable inter-
individual variation. Prolonged or repeated exposure to
a stressor causes a specific form of adaptation with reduced
activation of the HPA axis (e.g., Cockram et al., 1994), which
is believed to help the individual cope with the actual situation
since excessive amounts of cortisol can be detrimental. As
a consequence, lower levels of cortisol may be indicative of
chronic stress. However, cortisol by itself cannot be always
interpreted as indicative of a state of stress since it can increase
in times of excitement or positive emotional states (e.g.,
mating). Consequently, its concentration in blood must be
associated with other physiological or behavioral evidence to
support an indication of stress. By and large, small ruminants
avoid the stressor and display agitation and increased vocaliza-
tion. Behavior should, nonetheless, be analyzed cautiously as
it varies according to the context and does not always correlate
with physiological responses. For instance, when tested in an
open-field arena, behavioral and physiological responses
expressed by lambs reared with or without their mother may
differ or not according to the context (Napolitano et al.,
2008). Another well-described response of sheep and goats
exposed to stressors is the alteration of the immune function
(Cockram et al., 1994; Minton et al., 1992; Sejian and
Srivastava, 2010). The main characteristic is an alteration in
leukocyte population, usually an increase in the number of
neutrophils and a decrease in the number of lymphocytes
suggestive of immunosuppression. Under chronic stress, alter-
ation in immune system has an effect on disease resistance. For
instance, stress associated with weaning or transport and
indoor housing reduces resistance to parasite infestation
(Sotiraki et al., 1999). Small ruminants are less likely to
perform stereotyped behavior, but for animals kept indoors,
a number of abnormal behaviors were reported such as
ground searching, rail-standing, bar-chewing, or pen-butting
(Marsden and Wood-Gush, 1986a,b). Sheep may also show
other forms of abnormal behavior such as cross-sucking and
wool-pulling. Cross-sucking occurs in artificially reared lambs
where some individuals suck the navels or scrotum of conge-
ners. This activity is common in the initial days, but it may
last until weaning and it is not without consequences. The
lamb being sucked may be disturbed and develop navel infec-
tions, while the animal performing sucking, poorly adapted to
artificial feeding, may reduce the ingested amounts of reconsti-
tuted milk (Napolitano et al., 2008). Wool-pulling is exclu-
sively observed in indoor-housed sheep. It is frequent at
high stocking density and reduced by increased provision of
roughage (Vasseur et al., 2006).
 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
1.17.4.6 Regarding Sacrifice Methods
Euthanasia may be necessary for experimental reasons or
because of natural causes (traumatic injury, incurable disease).
There are no specific regulations concerning this issue but there
are guidelines which are accepted worldwide, e.g., the American
Veterinary Medical Association in the USA (Leary et al., 2013), the
Euthanasia of Experimental Animals published by the European
Commission (Close et al., 1997). Only two methods are
considered to be suitable for sheep and goats: chemical
methods with the use of injectable agents such as barbiturates
and physical methods such as captive bolt or electric stunning
followed by exsanguination. Other methods may be used
under specific conditions. Barbiturates (pentobarbitone,
sodium pentobarbitone) act by depressing the central nervous
system in three steps: from consciousness to unconsciousness,
then deep anesthesia, and finally death. The jugular vein is the
normal intravenous route of injection; shearing the neck prior
the injection facilitates the procedure. It is important to ensure
that the animal is dead before disposing of the body. The use of
captive bolt is an acceptable physical method for small rumi-
nants. Projection of the shot should be on a line running
between the bases of the ears and aiming toward the throat.
Electric stunning is an acceptable form of euthanasia as long
as the research facility has a slaughter room. In both cases
adjunctive methods such as exsanguination should be applied
immediately following the loss of consciousness of the animal.
Operation training, experience, and confidence are required,
but shooting may only be suitable if the operator has an appro-
priate firearms license. Potassium chloride and magnesium
sulfate are effective methods to ensure death in sheep and goats
previously rendered unconscious by mechanical methods or
administration of anesthetics. Because lambs and kids are
born in an advanced stage of development, the same methods
of euthanasia should be applied on them.
1.17.5 On Dairy Cattle
Dairy cows have had the opportunity to live in a friendlier envi-
ronment than some other animals in intensive productions,
having spaces to lie down, eat, drink, and walk and having
the freedom to decide when to do it. Unfortunately, this does
not mean that they are free from welfare concerns. Calves are
separated from their mothers at birth or a few days later,
housed individually, lacking the opportunity for social contact,
and artificially fed. Cows also lack the opportunity to graze;
they can suffer from metabolic problems, lameness, and other
infectious diseases.
1.17.5.1 Lodging and Handling during the Peripartum Period:
Relevance for the Establishment of Maternal Behavior and
Nursing
Parturition is a difficult process. The calf’s development and
survival is dependent upon being strong and vigorous at birth
and receiving appropriate maternal care. However, difficulty at
delivery can result in less vigorous offspring and maternal care
can be altered, probably as a consequence of exhaustion, pain,
and human intervention. Barrier et al. (2012) have found that
assisted cows at parturition do not take longer to lick the calf
473
and perform as much licking as unassisted cows; this indicates
no delayed onset or impaired expression of maternal behavior
in dams given assistance at delivery. However, although maternal
behavior was unaffected by a difficult delivery, dairy calves born
following difficult calvings have lower vigor in the first 3 h after
birth than unassisted calves. Moreover, Barrier et al. (2013)
found that calves that survive a dystocia experience have lower
passive immunity transfer, higher mortality, and higher indica-
tors of physiological stress (salivary cortisol). This might have
longer-term effects on the health and survival of the calves.
Reproductive disorders such as lack of estrus, embryonic
loss, or early abortion can reflect prolonged or short-term
poor welfare (Algers et al., 2009). Stress during parturition or
in early lactation can lead directly to poor well-being, particu-
larly dystocia and genital infections, associated with pain or
inflammatory reactions. The weeks immediately after parturi-
tion are important since cows are at a high risk for both meta-
bolic and infectious diseases (Ingvartsen et al., 2003). Both are
related to inadequate nutrition and are one of the most serious
welfare issues affecting dairy cows (von Keyserlingk et al.,
2009). Several behavioral changes are observed in sick animals,
such as reduced activity, feeding, grooming, and rumination,
which are used as behavioral indicators of a lack of well-
being (Borderas et al., 2008; von Keyserlingk et al., 2009).
Cows are ‘hiders’ and they maintain their calves separated
from the rest of the group for the first days after parturition
(Kilgour and Dalton, 1984). Calves reared under natural condi-
tions develop a bond with their mothers through grooming,
lactation, and vocalizations. Most dairy cattle productions
discourage maternal behavior, with the exception of milk
production. When domestic cattle are permitted to raise their
calves, their behavior is similar to that of wild ungulates. These
behaviors permit the cow to bond with her calf, protect it,
and nourish until the breakup of this bond at weaning
(von Keyserlingk and Weary, 2007). Colostrum is essential
for the calf, and several studies indicate that many calves do
not ingest sufficient quantities. Management practices that
maximize the calf’s opportunities to ingest colostrum and
minimize the contact with pathogens will have beneficial
effects on their welfare. Albright (1983) demonstrated that
colostrum (even when bottle fed) has a better chance to be
absorbed when the calf is next to its mother and that grooming
helps the calf to drink with ease (Figure 13).
Unfortunately, the calf’s separation from the cow in the first
days after parturition has behavioral, health, and production
effects, on both the mother and the calf. There is evidence
that 5-min contact with a calf immediately postpartum is suffi-
cient for the formation of a strong, specific maternal bond with
that calf. Hudson and Mullord (1977) found that this bond
lasts, even if the calf is removed for 12 h and then returned.
After 24 h of separation, the cow still shows signs of distress,
but can no longer recognize her own calf. If no contact is
allowed between mother and young for periods of up to 5 h
postpartum, this maternal bond is not formed in half of the
animals. These results suggest that there is a sensitive period
for the development of maternal responsiveness in dairy cattle.
Hopstee et al. (1995) found only mild effects immediately after
the calves were removed. After separation cows vocalized
initially and moved to the feeding rack and started feeding
shortly after that. Heart rate effects were restricted to the first
474 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals

30 70
1 day
Primiparous 60 2 weeks
25
Mul parous

20 50

# vocaliza ons
Minutes

40
15

30
10
20
5
10
0
1 2 3 4 5 6 0
Days 1h 18 h 24 h

Figure 13 Time licking their calves in primiparous and multiparous Figure 14 Calf vocalization after 1, 18, or 24 h of separation, when
cows. Redrawn from von Keyserlingk, M.A.G., Weary, D.M., 2007. calves were separated at 1 day or 2 weeks of age. Data obtained from
Maternal behavior in cattle. Horm. Behav. 52, 106–113. Flower, F.C., Weary, D.M., 2003. The effects of early separation on the
dairy cow and calf. Anim. Welf. 12, 339–348.

minutes after separation and cortisol effects were not found.
They concluded that multiparous dairy cows respond only
mildly immediately after their calves had been removed.
Lidfors (1996) found that separation after 4 days has an
effect on activity, vocalization, and rumination in the cows
and on the activity and oral behavior of the calves. Stehulova
et al. (2008) concluded, from the study of specific behavioral
reactions, that separating dairy cows from their calves is more
stressful for both parties if it is performed on days 4 and 7
than on day 1. The behavioral response to separation was
more intense and lasted longer in both of the animals when
they had visual and auditory contact after separation. The heart
rate response of cows was not influenced by the age of the
calves at separation or postseparation contact, but the heart
rate increase in the separated calves lasted longer the more
they had been with their mothers. Indeed, calves that stayed
with their mothers had less incidence of diarrhea, better weight
gain, and better maternal ability than calves separated a few
days after parturition (Flower and Weary, 2003), findings sug-
gesting that production and welfare benefits are greater when
calves stay longer with the cows. Even though late separation
would be a challenge for the milk industry, this study explains
how a natural imprinting between the mother and the calf can
be used to improve production and welfare (Figure 14).
1.17.5.1.1 Calf Lodging
After the second week, calves, under natural conditions, are left
in ‘crèches’ or ‘kindergartens,’ where one or two cows take care
of all the calves. This facilitates social behavior (Phillips, 2008).
Play can be used as a welfare indicator, since calves play longer
when they are well fed and healthy (Houpt, 2005). Calves
reared in intensive farms live under very different conditions
to those encountered in nature, and this can be an important
source of welfare problems. Calves under these conditions
can be housed in groups or individually, with different space
allowance and levels of climatic control. Milk production
systems have undergone several changes in the last 50 years.
In the 1960s, when farms grew and became more specialized,
calves went from living in small groups near their mothers to
living in elevated crates in a closed building. In the last 20 years
hutches have become more popular.
Friend et al. (1985a,b) worked on different tests to measure
the well-being of calves under different housing systems
(Table 4). Chronic deprivation of movement that does not
allow natural behaviors such as locomotion, rest, grooming,
exploration, and social interaction can be accompanied by
behavioral and physiological changes indicative of stress
(Friend et al., 1985a,b; Sisto and Friend, 2001). Calves housed
in crates and hutches tend to stumble more frequently than
those housed in groups, with more space and allowed to exer-
cise (Friend et al., 1985a,b; Figure 15).
Individually confined calves do not show normal social
interaction patterns and are, therefore, unable to compete for
food with calves reared in groups (Broom and Leaver, 1978).
This is reflected in a lower weight gain and higher cortisol levels
at transport (Trunkfield, 1990). Calves reared in groups show
fewer fear reactions when confronted with new stimuli at
3 months of age, suggesting that individual management prac-
tices compromise calf welfare by inhibiting their normal
behavior patterns such as grouping in crèches (Donovan,
1992). The European Union has legislated that calves older
than 8 weeks have to be housed in groups, and in organic farms
they must be housed in groups since the first week of age
(Jensen, 2003). Socialization in calves is considered a priority.
Table 4 Physiological changes in calves under different housing
systems
Treatment
Elevated crate Hutch Group pen
Basal cortisol 5.4 4.8 3.4
ACTH challenge 103 81 75
Lymphocytes (%) 61 69 71
Neutrophil/lymphocyte 0.69 0.45 0.36
Based on data from Friend, T.H., Dellmeier, G.R., Gbur, E.E., 1985a. Comparison of
four methods of calf confinement. I. Physiology. J. Anim. Sci. 60, 1095–1101 and
Friend, T.H., Dellmeier, G.R., Gbur, E.E., 1985b. Comparison of four methods of calf
confinement. II. Behavior. J. Anim. Sci. 60, 1102–1109.
 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
12
Galloping
10
8 site effect (de Pasillé and Rushen, 1998), suggesting that the
Incidence
6 and not lactose per se. Rushen and de Pasillé (1995) examined
4 the calf drank its milk and found that there was an inhibitory
2 vation to suckle diminished after 10 min. de Pasillé and Caza
0 preferred them over their group mates, reducing cross-suckling
Elevated crate Hutch Group pen
Figure 15 Behavioral changes during an open-field test in different
housing systems. Based on data from Friend, T.H., Dellmeier, G.R.,
Gbur, E.E., 1985a. Comparison of four methods of calf confinement.
I. Physiology. J. Anim. Sci. 60, 1095–1101 and Friend, T.H., Dellmeier,
G.R., Gbur, E.E., 1985b. Comparison of four methods of calf confine-
ment. II. Behavior. J. Anim. Sci. 60, 1102–1109.
1.17.5.1.1.1 Calf Feeding
Feeding behavior of artificially reared calves is very different
from that of naturally reared calves. Besides the lack of the
natural stimulus associated with the cow’s udder and maternal
behaviors, calves are usually fed only twice a day and they
consume, at each bout, considerably more milk than what
they would in any of the 4–10 feedings seen under natural
conditions (bouts lasting 8–12 min; Wilt, 1985; Jensen,
2003). The ways by which milk is offered to artificially reared
calves promote the development of crossed suckling (i.e., suck-
ling of ears, tail, prepuce, and other parts of the body). Some
researchers have compared the behavior of calves fed using
different methods, while others have investigated the motiva-
tional basis of nonnutritive suckling by manipulating different
aspects of milk feeding and the opportunity to suckle. Milk
producers hesitate at the possibility of maintaining calves in
groups due to the problem of crossed suckling as they believe
it could lead to milk stealing in adult cows (Spinka and
Illmann, 1992; Keil et al., 2000). Rings and metal plates are
placed on their noses when these animals are identified in
order to control the behavior, but this practice raises concerns
regarding the animal’s welfare (Keil et al., 2000). To be able
to improve group management and control these unwanted
behaviors, we need to understand what makes calves want to
suckle.
de Pasillé and Rushen (1995) have extensively studied
calves’ motivation to suckle. Contrary to what most people
think nonnutritive suckling is greater after feeding and not
before, and therefore it is not motivated by hunger as long as
the calves are well fed. Even when their milk rations were cut
in half nonnutritive suckling did not increase. This suggests
that neither the quantity of milk in the stomach nor the oral
sensation of ingesting milk inhibits nonnutritive suckling.
When calves do not drink milk or water there is some nonnu-
tritive suckling, but much less than what is observed after
drinking milk (de Pasillé et al., 1992). Injecting small quanti-
ties of milk into the mouth of a calf produces a great amount
of nonnutritive suckling, as well as when milk replacement
475
concentration is elevated, suggesting that milk flavor is what
motivates suckling (de Pasillé et al., 1997; Figure 16). When
lactose levels are elevated, nonnutritive suckling is also
elevated, and when lactose levels are reduced, you get the oppo-
most important factor is the sugar concentration in the milk
if suckling reduced the motivation to suckle using dry teats after
effect on nonnutritive suckling. They also found that the moti-
(1997) found that calves which got dry teats after feeding
by 75%. Jung and Lidfors (1999) reported similar results.
Many have tried to extinguish the suckling instinct by trying
to teach the calves to drink from a bucket in the first days of life,
but this increases, rather than decreasing, the problems. The
same happens when bottles with big nipple holes are used.
Cross-suckling is less frequent when a teat or nipple is used
(bottles or buckets with nipples) compared to only using
buckets. When calves take longer to drink the milk through
the nipple, they engage in less nonnutritive suckling (Haley
et al., 1998). Offering water through a nipple or straw after
feeding reduces, but does not eliminate, nonnutritive suckling
(Haley et al., 1998; de Pasillé, 2001).
1.17.5.2 Lodging of Dairy Cows
Housing systems differ all over the world depending on climate,
economy, and personal preferences of the farmers. In many
countries dairy cows are kept on pastures year-round or during
good weather. In the United States, dairy cows are usually kept
in large lots with cement or dirt floors, some with free stalls
(cubicles) or tie stalls. Algers et al. (2009), following a request
from the European Commission, analyzed the most important
hazards regarding housing conditions and found that poor
cubicle design and lack of space in cubicle houses and tie stalls
had the highest estimated risk values. Inadequate ventilation,
160
140
120
% observa ons
100
80
60
40
20
0
Milk Water Milk×2
Figure 16 Percentage of nonnutritive suckling after a milk meal,
water, or increased concentration of milk replacer. Adapted from de
Pasillé, A.M., Rushen, J., Jensen, M., 1997. Some aspects of milk
that elicit non-nutritive sucking in the calf. Appl. Anim. Behav. Sci. 53,
167–173.
476 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
temperature, and humidity were the highest-ranked hazards in
straw yards. However, the risk estimate values in straw yards
were much lower than in cubicles and tie stalls.
Studying the normal behavior of cows helps to improve and
promote the well-being of the animals. To do so it is important
not only to see the size of the cows, but also to observe their
movement sequences when lying down. The problem with
cubicles is that some are poorly designed; this, together with
the recent increase in the size of the animals, makes them
poorly suited to fit the behavioral needs of the cows. They
need to lie down in a comfortable, dry, clean environment as
deprivation of rest can affect their behavior (Cooper et al.,
2007). Cows lie down for 7–12 h (or more) during a 24-h
period, and if they cannot find a comfortable lying space,
they stand or lie in dirty passageways which will lead to poor
health issues, specially foot and udder problems (Wierenga
and Hopster, 1990; Arney and Aland, 2012). Lying is an impor-
tant behavior for cows, as it enables them to rest and to avoid
confrontations. Moreover, a reduction in lying time affects their
welfare since the cow rests and ruminates when it is lying
down; the cow’s hoofs rest and dry off and blood circulation
to the udder increases by up to 30% (Bewley, 2011). Uncom-
fortable stalls result in less frequent or shorter-duration resting
periods, and consequently, there is an increased standing time
on concrete surfaces (Nordlund and Cook, 2003).
As the cow lies down, she puts about two-thirds of her
body weight on her front knees. These drop freely to the floor
from a height of 20–30 cm, so it is very important to have
adequate bedding to avoid injury. Wet knees mean wet
bedding, and cows do not like to lie down in dirty stalls
(Manteca, 2009). The time a cow takes to lie down is
a good indicator of welfare or lack thereof. For example, if it
takes her 5 min or less (on average), the welfare is good;
moderate, from 5 to 6.3 min, and bad if it is longer than
6.3 min (Manteca, 2009; Bewley, 2011).
It is important to consider lunge space to allow the cow to
lie down and stand up without difficulty. Cows that are not
using all of the stalls or enter them in reverse indicate poorly
180
160
140
120
100
80
60
40
20
0
Hock Hoof Teat injury Systemic
injury lesions
Figure 17 Effect of different diseases on cumulative reduction of feed intake and cumulative loss of milk production. Based on data presented in
Table 2.1 of Rushen, J., de Pasillé, A.M., von Keyserlingk, M.A.G., et al., 2008. The Welfare of Cattle. Springer, The Netherlands.
designed cubicles (Haley et al., 2000; Manteca, 2009). Errors
in stall design and maintenance have long been recognized as
significant risk factors for mastitis, hock abrasions, teat trauma,
and entrapment injuries of dairy cows. Moreover, if the stalls
are uncomfortable, the cow may choose to lie down in
a slurry-covered alleyway and put herself at risk for environ-
mental mastitis infection (Nordlund and Cook, 2003).
Floors also play an important role in foot health. Concrete
floors can be slippery for cows, and when they are rough, they
damage the hoof and animals have to alter their gait (Phillips
and Morris, 2000; Telezhenko and Bergsten, 2005). There is
increasing evidence for an association between standing time
on concrete and the induction of laminitis (Nordlund and
Cook, 2003; Espejo et al., 2006). Behavior also seems to play
a role in foot problems. For instance, half-standing in the
cubicle (perching) is performed more frequently by low-
ranking cows, as they use cubicles to hide from dominant indi-
viduals (Metz and Wierenga, 1987; Potter and Broom, 1987).
Galindo et al. (2000) found that low-ranking cows performed
this behavior for a longer time and had a greater reduction in
heel depth of the hind digits. This predisposed them to infec-
tion in the interdigital space and heel, causing clinical lame-
ness. Loberg et al. (2004) found that when given access to an
outdoor paddock, adult cows used the time to walk, trot, and
explore the environment, and these behaviors had a positive
effect on the claw conformation.
Regula et al. (2004) found that cows housed in free stalls
with access to a pen, yard, or pasture where they could regularly
exercise had lower odds of presenting lameness, hock joint
alterations, and teat injuries, therefore requiring fewer overall
medical treatments (Figure 17). Many cows have subclinical-
level claw disorders, and welfare is an important issue on
lameness in dairy cows. Together with mastitis, lameness is
recognized the most important health problem having a nega-
tive effect on the welfare of dairy cows (Somers et al., 2003).
‘Housing conditions affect animals throughout their lives’
(Rushen et al., 2008), and it is extremely important to be aware
of this and to take measures to correct any problem.
Reduced feed (Kg)
Lost milk (Kg)
Local Difficult Very
mas s mas s calving difficult
calving
 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals 477

1.17.5.3 Regarding Environment
Alleviating heat stress is critical to milk production. Heat
stress from high environmental temperatures can reduce
food intake and milk production by more than 25% and it
is estimated that for every 1-lb decrease in dry matter intake
2 lbs of milk production is lost (Garcia, 2006). When cows
have heat stress, they lie down less, and the risk for mastitis
is higher (Manteca, 2009).
Water, often overlooked as an important nutrient, is
extremely important to the modern, high-yield dairy cow, and
insufficient or poor-quality water is an obvious welfare concern
(Meyer et al., 2004). Water intake is closely related to feeding
intake and thus animal productivity. Cattle are sensitive to the
palatability of water and prefer to drink clean water without
contamination (Garcia, 2006; Manteca, 2009). Water trough
design can affect both preferences and consumption by lactating
and nonlactating dairy cattle. Dairy cows prefer and drink more
from larger troughs (Pinheiro Machado Filho et al., 2004).
1.17.5.4 Behavioral Indicators of Well-being in Bovines
The time spent performing normal behaviors, such as eating
and resting, versus abnormal behaviors, such as stereotypies,
can tell much about the welfare of the animals (Broom,
1991; Sherman and Serpell, 2008). Automated monitoring of
normal behaviors such as feeding, drinking, or walking can
help identify sick animals before clinical signs appear or cows
that are in heat to correctly time artificial insemination. Tech-
nology is advancing, but we must not rely on the automatic
measurement of behavioral indicators just because it is easy
(Rushen et al., 2012).
Stereotypies are important indicators of poor welfare, and
in dairy cattle we mainly find oral stereotypies such as
tongue-rolling (Sambraus, 1985; Bergeron et al., 2006) due
to diets that take too little time to find, chew, or ruminate.
This leaves animals with an unfulfilled motivation to perform
these natural foraging activities. Object-licking, which is
a repetitive licking or biting of fences, walls, or pen fixtures,
has also been reported in dairy cattle (Seo et al., 1998; Bashaw
et al., 2001).
When measuring welfare we usually focus on the absence of
stress rather than on positive behaviors. Adequate environ-
mental stimulation favors good welfare and positive indicators
such as play (Napolitano et al., 2009). Animals are highly moti-
vated to play only if their primary needs are satisfied, while
illness and injury are associated with the absence of play
(Friend et al., 1985a,b; de Pasillé and Rushen, 1995; Boissy
et al., 2007; Napolitano et al., 2009). In calves, play is mainly
expressed as locomotive play, that includes galloping, bucking,
and kicking, and social play, which includes play fighting and
nonreproductive mounting (Emmering, 2004; Napolitano
et al., 2009). Although also described in adult cattle, play is
more frequent in calves.
Cattle, under natural or extensive conditions, are highly
synchronized as a result of allomimetic motivation (Bouissou
et al., 2001). Therefore, a high degree of synchronization
within the group may indicate a positive welfare state (Galindo
and Broom, 1993). When cows have enough space and their
primary needs are satisfied, all animals can feed and rest
without competition, they usually synchronize, and it could
be said that their welfare is good. Their synchrony should
exceed 80% to consider a positive evaluation of the welfare
state of the herd (Napolitano et al., 2009; Figure 18).
Self-licking and scratching have been also proposed as indi-
cators of adequate living conditions. A reduced expression of
these behaviors has been related to slippery floors, thus reflect-
ing poor housing conditions (Winckler et al., 2002). Allog-
rooming plays a key role with regard to the so-called
sociopositive interactions. Allogrooming seems to serve both
hygienic (body care) and social functions as it is thought to
play a major role in reinforcing social bonds and in reducing

Behavior indoors
100 Behavior outdoors
90
80
70
60
% of cows

50
40
30
20
10
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31
Time of day

Figure 18 Feeding synchronization in the same group of cows when kept indoors (free stall) or outdoors (at pasture). Redrawn from data of
Galindo, F.A., Broom, D.M., 2000. The relationships between social behaviour of dairy cows and the occurrence of lameness in three herds. Res. Vet.
Sci. 69, 75–79.
478 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals

tension in groups of animals. Soothing effects regarding Table 5 Examples of physiological indicators of well-being used in
a reduction in heart rate have been demonstrated in cattle dairy cattle
(Sato et al., 1993; Boissy et al., 2007; Napolitano et al.,
Heart rate Broom (1991), Mohr et al. (2002), Hagen
2009). Sato et al. (1993) found that all animals were groomed
et al. (2005), and Stenhulova et al. (2008)
but that only 75% performed grooming. Plasma cortisol Friend et al. (1985a), Hopster et al. (1999),
and Sisto and Friend (2001)
1.17.5.4.1 Handling of Adult Bovines Hair cortisol Comin et al. (2012) and González de la Vara
The way animals are handled by people has a major effect on et al. (2011)
their welfare, and unfortunately, until recently this issue had Fecal cortisol Palme et al. (2000), Möstl and Palme (2002),
been neglected (Rushen et al., 2008; Waiblinger et al., 2006). and Morrow et al. (2002)
Salivary cortisol Barrier et al. (2013)
Dairy cattle that are roughly handled often become frightened
ACTH challenge Friend et al. (1985a,b) and González de la
of humans, and this may result in a substantial drop in milk
Vara et al. (2011)
production. Rushen et al. (1999) found that the mere presence Temperature Sisto (2002)
of an aversive handler during milking may be sufficient to cause Infrared thermography Stewart et al. (2007)
the cows to ‘hold back’ milk, through a stress-induced suppres-
sion of oxytocin secretion. Many people believe that cattle are
not sensitive and are difficult to move, and this leads to rough
handling. Through classical conditioning the cows learn to
associate rough handling with the handler and soon show Table 6 Examples of behavioral indicators of well-being used in
fear (Rushen et al., 1999). Munksgaard et al. (1999) studied dairy cattle
the role of the color of overalls as a cue used by cows to discrim-
inate between people. The cows kept a longer distance when Maternal (licking of calf) Flower and Weary (2003) and Barrier et al.
the handler wore the color worn during the aversive treatment (2012)
than when the handler wore the color worn during the gentle Vocalizations Lidford (1996) and Flower and weary (2003)
Play Friend et al. (1985a,b), de Pasille and Rushen
treatment. The cows also responded according to the color of
(1995), Sisto and Friend (2001), Emmering
the clothes when worn by an unfamiliar person, although (2004), Houpt (2005), Boissy et al. (2007),
they kept a shorter distance from the unfamiliar person than and Napolitano et al. (2009)
from the handler. Rushen et al. (1998) mention that one way Synchronization Galindo et al. (2000), Boissou et al. (2001),
to reduce the magnitude of the fear that cows develop toward and Napolitano et al. (2009)
their handlers as a result of aversive handling may be to apply Grooming and Sato et al. (1993), Winckler et al. (2003),
those procedures outside the cows’ home stalls. Flight distance allogrooming Boissy et al. (2007), and Napolitano et al.
is, therefore, a good indicator of animal welfare (Figure 19). (2009)
Good management consists of much more than handling the Fear (flight distance) Rushen et al. (1999, 2008), Rushen (1996),
animals properly. Everyday routine animal care tasks, such as Rushen et al. (1999, 2008), Munksgaard
et al. (1999), and Waiblinger et al. (2006)
cleaning, are very important in ensuring good animal welfare
Social interactions Broom and Leaver (1978), Friend and
(Rushen et al., 2008). Dellmeier (1988), Galindo et al. (2000),
Tables 5–8 summarize the main behavioral, neuroendo- and Phillips (2008)
crine, immunological, and physiological indicators that have Feeding Witt (1985), Sisto (2002), Jensen (2003),
been used in bovines to determine their well-being. Garcia (2006), Borderas (2008), and von
Keyserlingk (2009)
Drinking Sisto (2002), Meyer et al. (2004), Pinheiro
6 et al. (2004), Garcia (2006), and Manteca
(2009)
Gentle handler Feeding and drinking Rushen (2012)
5 Aversive handler automated measures
Cross-suckling Spinka (1992), Haley et al. (1998),
4 Rushen and de Pasillé (1992, 1995),
de Pasillé et al. (1997), de Pasillé and
Caza (1997), de Pasillé and Rushen
Meters

3
(1998), Jung and Lidfords (1999), and
de Pasillé (2001)
2 Lying, resting Haley et al. (2000), Sisto (2002), Manteca
(2009), Cooper et al. (2007), and Bewley
1 (2011)
Standing Wierenga and Hopster (1990), Sisto (2002),
Nordlund et al. (2003), and Arney and Alan
0 (2012)
Perching Metz and Wierenga (1987), Potter and Broom
Figure 19 Flight distance in the presence of a gentle or an aversive (1987), and Galindo et al. (2000)
handler after 19 times of handling. Adapted from Rushen, J., de Pasillé, Stereotypies Sambraus (1985), Seo et al. (1998), Bashaw
A.M., von Keyserlingk, M.A.G., et al., 2008. The Welfare of Cattle. et al. (2001), and Bergeron et al. (2006)
Springer, The Netherlands.
 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
Table 7 Examples of health indicators used in dairy cattle welfare
Passive immunity Barrier et al. (2013)
Lymphocytes Friend et al. (1985a,b) and Sisto and Friend (2001)
Lymphocyte/ Friend et al. (1985a,b)
neutrophil
Diarrhea Flower and Weary (2003)
Lameness Galindo et al. (2000), Phillips and Morris (2000),
Somers et al. (2003), Nordlund (2003), Bergsten
(2003), Loberg et al. (2004), Regula et al. (2004),
Telezhenko and Bergsten (2005), and Espejo
et al. (2006)
Mastitis Somers et al. (2003)
Injuries Nordlund et al. (2003)
Table 8 Examples of other indicators of well-being used in dairy
cattle
Weight gain Flower and Weary (2003)
Precollected registered data, such as Algiers (2009)
percentage of cows with repeated
inseminations, stillbirths, or mortality
1.17.6 Conclusions and Discussion
Throughout this chapter we have reviewed the ways by which
lodging, environmental conditions, and handling can impact
the well-being and stress reactivity of rodents, rabbits, goats,
sheep, and dairy cattle kept in the laboratory or on the farm.
Although the studies performed are numerous and varied,
when viewed as a whole the field seems to be lacking in unifor-
mity of the work performed; allowing a multiplicity of interpre-
tations in some findings; concentrating on a particular age, sex,
or reproductive condition; rarely including both behavioral
and neuroendocrine indicators in the same work. For instance,
what do the results of the open-field test mean.reduced fear-
fulness? Larger motivation to explore? How are these possible
interpretations related to welfare? How about the display of
stereotypies? Are they indicative of stress or boredom? They
are not always displayed in association with increased CORT.
Moreover, is boredom a psychological stressor (Panksepp,
2011)? Similar reflections can be made regarding studies in
which animals are ‘asked’ to choose between two conditions.
For instance, if an animal ‘prefers’ a certain type of bedding
or cage, does that indicate that ‘the other’ choices are not in
accord with the animal’s well-being?
The social organization of a mammalian species has
a profound impact on the type of lodging that is ‘optimal’ for
it in the lab and on the farm. Most species housed therein are
social, and one should rethink if ‘larger is better’; that is, space
allowance should consider the species’ habits which, in turn,
are modulated by factors such as sex and reproductive state.
Female rabbits, cows, and goats are very social, but when it
comes to ‘acting maternal,’ they retreat and they do not
welcome encounters with other males or females. Indeed,
maternal behavior is always ‘peculiar,’ in the sense that the
activities performed by mothers – and aimed at their offspring
– are unique to the lactation period (and late pregnancy). Yet,
the setups provided for lactating mothers and offspring do not
479
always consider each species’ needs, for example, isolation, nest
building. To what an extent is this a welfare concern? Recom-
mendations for ‘optimal’ lodging of mammals of different
reproductive states need to balance the impact of, e.g., type
of cage, animal density, ‘enriched’ environment versus its
consequences on other important issues, such as hygiene,
fertility, ‘peaceful cohabitation.’
An important finding that has consistently emerged across
species is the existence of the so-called ‘critical periods’ or
‘windows of opportunity,’ i.e., particular times in the early
postnatal period during which specific manipulations of
the young can permanently alter their reactivity to poten-
tially stressful stimuli in adulthood. In rodents this is a partic-
ularly intense area of research, and important advances have
been made on the subcellular mechanisms underlying this
phenomenon (Champagne et al., 2003; Fleming et al.,
1999; Lomanowska and Melo, 2016). By contrast, few
studies have been done on this issue in nonrodent mammals
(Jensen, 2014; Rodenburg, 2014). Much work is needed to
define the duration of ‘critical periods’ in particular species
and the type of early-life manipulations that effectively
reduce the stress generated by handling, transportation,
lodging, etc. On the other hand, practices like cross-
fostering in bovines, sheep, and goats provide evidence of
plasticity regarding social preferences in later life. From this
information management strategies can be implemented to
reduce stress responses to, e.g., moving flocks, mixing
animals in pens, transportation, etc. Moreover, bonding
with humans is well known to occur in several domesticated
mammals, and this phenomenon can also be used to facili-
tate management in the lab and on the farm. Furthermore,
animal–human bonding is a fascinating topic in itself and
the neurobiological mechanisms underlying it should be
investigated in their own right.
An important consideration should be given to the housing
and management of recently weaned and ‘adolescent’ animals.
Specific issues such as individual versus group housing, type of
feed, size of cages (or pens), mixing with adults, etc. have been
scarcely addressed in ‘classic’ Behavioral Neuroendocrinology.
Yet, we know that (peri) adolescent mammals are a category
in their own right and that experiences during such period
can modify a variety of behaviors and neuroendocrine
responses in adulthood (Schulz et al., 2009).
A puzzling, yet consistent, phenomenon emerging from
the need to help animals deal with environmental stress is
the effect of the so-called ‘nutritional supplements.’ A variety
of them have been tried, several of them do reduce
immunological and endocrine parameters indicative of heat
stress, but little is known regarding their mechanism of
action. Clearly, this can be a fruitful area of research in
Psychoneuroimmunoendocrinology.
It is essential to ‘bridge’ from lab to farm and back. The
experimental models, environmental conditions, and findings
are complementary to each other and mutually enriching.
Sadly, the ‘compartmentalization’ of science (i.e., ‘basic vs
applied’ research; ‘mainstream’ vs ‘local’ journals; evermore
specialized scientific meetings) does not favor such interaction.
Many issues related to the well-being of farm and laboratory
animals are rich topics for investigation in Behavioral Neuroen-
docrinology. The authors hope that, from reading our chapter,
480 Behavioral and Neuroendocrine Indicators of Well-being in Farm and Laboratory Mammals
students and researchers will become interested in exploring
new aspects of the animals they already work with or venture
into establishing connections with investigators that study
the same species, but from a different perspective.
Acknowledgment
The authors thank Francisco Torres Quintanar for his generous help in
the preparation of figures.
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HORMONES, BRAIN,
AND BEHAVIOR

THIRD EDITION
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 HORMONES, BRAIN,
AND BEHAVIOR

THIRD EDITION

EDITORS-IN-CHIEF
Donald W. Pfaff
The Rockefeller University, New York, NY, USA

Marian Joëls
University Medical Center Utrecht, Utrecht, The Netherlands;
and University Medical Center Groningen, Groningen, The Netherlands

VOLUME 2
NON-MAMMALIAN HORMONE-BEHAVIOR SYSTEMS

VOLUME EDITOR
Jacques Balthazart
University of Liège, Liège, Belgium

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CONTENTS OF VOLUME 2

Editorial Board vii

Contributors to Volume 2 ix
Preface of the First Edition xi
Hormonal Effects on Specific Behaviors, on Global CNS States, and on Human Disease.
An Introduction to the Second Edition xiii
Hormonal Effects on Specific Behaviors, on Global CNS States, and on Human Disease.
An Introduction to the Third Edition xv

Non-mammalian Vertebrates: Fishes

2.01 Neuroendocrinology of Social Behavior in Teleost Fish 3
David Gonçalves, Ana S Félix, and Rui F Oliveira
2.02 Social Regulation of Sex: How the Brain Controls Reproductive Circuits 19
Russell D Fernald
2.03 Socially Controlled Sex Change in Fishes 31
John Godwin and Melissa Lamm
2.04 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish
as Model Systems 47
Ni Y Feng and Andrew H Bass
2.05 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology 69
Kent D Dunlap, Ana C Silva, G Troy Smith, and Harold H Zakon

Non-mammalian Vertebrates: Amphibians

2.06 Neuroendocrine Control of Social Behavior in Frogs 101
Walter Wilczynski
2.07 The Vertebrate Brain Stem as the Primary Site of Behavior Command:
Insights from an Amphibian 117
Emma Coddington
2.08 Hormones and Vocal Systems: Insights from Xenopus 131
Erik Zornik and Darcy B Kelley
2.09 Endocrinology of Complex Life Cycles: Amphibians 145
Robert J Denver

v
vi Contents of Volume 2

Non-mammalian Vertebrates: Reptiles

2.10 Hormones, Brain, and Behavior in Reptiles 171
David Kabelik and David Crews

Non-mammalian Vertebrates: Birds

2.11 Neuroendocrine Regulation of Reproductive Behavior in Birds 217
Gregory F Ball and Jacques Balthazart
2.12 Neural and Hormonal Control of Birdsong 255
Barney A Schlinger and Eliot A Brenowitz
2.13 Rapid Effects of Estrogens on Avian Brain and Social Behavior 291
L Remage-Healey, S A Heimovics, K K Soma, and C A Cornil
2.14 The Function of Behavior as Assessed by Phenotypic Engineering with Testosterone 305
Nicole M Gerlach and Ellen D Ketterson
2.15 Ecophysiological Studies of HormoneeBehavior Relations in Birds: Future Challenges
in a Changing World 321
John C Wingfield

Non-mammalian Invertebrates
2.16 Actions of Developmental Hormones in Adult Social Insects 349
Susan E Fahrbach, Ashton M Trawinski, and Rodrigo A Velarde
2.17 Diverse Functions of Insect Biogenic Amines as Neurotransmitters, Neuromodulators, and
Neurohormones 367
Wendi S Neckameyer and Sandra M Leal
2.18 Regulation of Honeybee Worker (Apis mellifera) Life Histories by Vitellogenin 403
Gyan P Harwood, Kate E Ihle, Heli Salmela (nee Havukainen), and Gro V Amdam
2.19 Endocrine Influences on Insect Societies 421
A R Hamilton, H Shpigler, G Bloch, D E Wheeler, and G E Robinson
2.20 Hormonal Regulation of Behavioral and Phenotypic Plasticity in Bumblebees 453
Abraham Hefetz and Christina M Grozinger
2.21 Stressed Out Insects II. Physiology, Behavior, and Neuroendocrine Circuits Mediating
Stress Responses 465
Erik C Johnson
EDITORIAL BOARD

EDITORS-IN-CHIEF
Donald W. Pfaff
The Rockefeller University, New York, NY, USA

Marian Joëls
University Medical Center Utrecht, Utrecht, The Netherlands;
and University Medical Center Groningen, Groningen, The Netherlands

VOLUME EDITORS
Gabriela González-Mariscal (Volume 1: Mammalian Hormone-Behavior Systems)
CINVESTAV-Universidad Autónoma de Tlaxcala, Tlaxcala, Mexico

Jacques Balthazart (Volume 2: Non-Mammalian Hormone-Behavior Systems)

University of Liège, Liège, Belgium

E. Ronald de Kloet (Volume 3: Molecular and Cellular Mechanisms)

Leiden University Medical Center, Leiden, The Netherlands

Stafford Lightman (Volume 4: Clinically Important Hormone Effects on Brain and Behavior)
University of Bristol, Bristol, UK

Anthony P. Auger (Volume 5: Development of Hormone-Behavior Relationships)

University of Wisconsin, Madison, WI, USA

Catherine J. Auger (Volume 5: Development of Hormone-Behavior Relationships)

University of Wisconsin, Madison, WI, USA

vii
This page intentionally left blank
CONTRIBUTORS TO VOLUME 2

Gro V Amdam John Godwin

School of Life Sciences, Arizona State University, Tempe,
AZ, USA; and Norwegian University of Life Sciences,
Aas, Norway
Gregory F Ball
University of Maryland, College Park, MD, USA
Jacques Balthazart
University of Liège, Liège, Belgium
Andrew H Bass
Cornell University, Ithaca, NY, USA
G Bloch
The Hebrew University of Jerusalem, Jerusalem, Israel
Eliot A Brenowitz
University of Washington, Seattle, WA, USA
Emma Coddington
Willamette University, Salem, OR, USA
C A Cornil
GIGA Neurosciences, University of Liege, Liege, Belgium
David Crews
University of Texas at Austin, Austin, TX, USA
Robert J Denver
The University of Michigan, Ann Arbor, MI, USA
Kent D Dunlap
Trinity College, Hartford, CT, USA
Susan E Fahrbach
Wake Forest University, WinstoneSalem, NC, USA
Ana S Félix
Gulbenkian Institute of Science, Oeiras, Portugal; and
ISPA e University Institute, Lisbon, Portugal
Ni Y Feng
Cornell University, Ithaca, NY, USA
Russell D Fernald
Stanford University, Stanford, CA, USA
Nicole M Gerlach
University of Florida, Gainesville, FL, USA
North Carolina State University, Raleigh, NC, USA
David Gonçalves
University of St. Joseph, Macao, China
Christina M Grozinger
Center for Pollinator Research, Center for Chemical
Ecology, Huck Institutes of the Life Sciences, The
Pennsylvania State University, University Park, PA,
USA
A R Hamilton
University of Illinois, Urbana, IL, USA
Gyan P Harwood
School of Life Sciences, Arizona State University, Tempe,
AZ, USA
Abraham Hefetz
George S. Wise Faculty of Life Sciences, Tel Aviv
University, Tel Aviv, Israel
S A Heimovics
University of St. Thomas, St. Paul, MN, USA
Kate E Ihle
University of Zurich, Zurich, Switzerland
Erik C Johnson
Wake Forest University, Winston-Salem, NC, USA
David Kabelik
Rhodes College, Memphis, TN, USA
Darcy B Kelley
Columbia University, New York, NY, USA
Ellen D Ketterson
Indiana University, Bloomington, IN, USA
Melissa Lamm
North Carolina State University, Raleigh, NC, USA
Sandra M Leal
Harris-Stowe State University, St. Louis, MO, USA

ix
x Contributors to Volume 2

Wendi S Neckameyer G Troy Smith

Saint Louis University School of Medicine, St. Louis,
MO, USA
Rui F Oliveira
Gulbenkian Institute of Science, Oeiras, Portugal;
ISPA e University Institute, Lisbon, Portugal; and
Champalimaud Neuroscience Program, Lisbon, Portugal
Indiana University, Bloomington, IN, USA
K K Soma
University of British Columbia, Vancouver, BC, Canada
Ashton M Trawinski
Wake Forest University, WinstoneSalem, NC, USA

L Remage-Healey Rodrigo A Velarde

Center for Neuroendocrine Studies, University of
Massachusetts, Amherst, MA, USA
G E Robinson
University of Illinois, Urbana, IL, USA
Heli Salmela (nee Havukainen)
Centre of Excellence in Biological Interactions,
University of Helsinki, Helsinki, Finland
Barney A Schlinger
Brain Research Institute, University of California, Los
Angeles, CA, USA
H Shpigler
University of Illinois, Urbana, IL, USA
Ana C Silva
Unidad Bases Neurales de la Conducta, Instituto de
Investigaciones Biologicas Clemente Estable,
Montevideo, Uruguay; and Universidad de la Republica,
Montevideo, Uruguay
University of Buenos Aires, Buenos Aires, Argentina
D E Wheeler
University of Arizona, Tucson, AZ, USA
Walter Wilczynski
Neuroscience Institute and Center for Behavioral
Neuroscience, Georgia State University, Atlanta, GA,
USA
John C Wingfield
University of California, Davis, CA, USA
Harold H Zakon
University of Texas, Austin, TX, USA
Erik Zornik
Reed College, Portland, OR, USA
PREFACE OF THE FIRST EDITION

Because of the large number of neuroactive substances discovered by chemical endocrinologists and the
prominent roles of nuclear hormone receptors as ligand-activated transcription factors, scientists studying
hormoneebrainebehavior relations have made discoveries and achieved explanations of behavior that place
their field foremost in neurobiology. The purpose of these volumes is to review the current state of this
knowledge inclusively. That means covering true molecular genetic approaches as well as neuroanatomical,
electrophysiological, zoological, neurochemical, developmental, and behavioral studies. The medical impor-
tance of this work is clear from the last section of this treatise. The editors intend these reviews to be
comprehensive; if there are any gaps in the coverage, a second edition will correct them.

Acknowledgments

Lucy Frank and Carol Oliver, at The Rockefeller University, organized all the volumes. Noelle Gracy and Mica
Haley handled things efficiently at Academic Press. Jasna Markovac at Academic Press presided over all stages of
this project. The editors thank all of these folks for their intelligent and gracious efforts.

Donald W. Pfaff
The Rockefeller University, New York, NY, USA

xi
This page intentionally left blank
HORMONAL EFFECTS ON SPECIFIC BEHAVIORS, ON GLOBAL
CNS STATES, AND ON HUMAN DISEASE. AN INTRODUCTION
TO THE SECOND EDITION

In 2002, there appeared the first edition of Hormones, Brain, and Behavior, dedicated to three generations of
productive scientists in our field, as it had developed at that point. With five volumes, totaling about 4100
pages, it represented a massive effort by over 200 scientists. The volumes were well received.
At least four developments have taken place since 2002 and justify a second edition. Two of them are
technical: the increasingly sophisticated application of molecular biological techniques and biophysical tech-
niques to the understanding of mechanisms for hormone/behavior relations. Third, we have entered an era of
translational research. Laboratory scientists are under increasing pressure to show how their work might lessen
the burden of disease upon the citizenry. The fourth development is the most subtle one and can be explained as
follows.
Some contemporary approaches to the hormonal controls of various forms of behavior can be distin-
guished sharply from the major theme in neuroscientific research in the past century. I characterize that
classical theme as a hunt for specificity. In the spinal cord, for example, the work of neurophysiologists such as
Sir Charles Sherrington (1906) and Sir John Eccles (1957) sought to explain why a particular reflex response
occurred specifically as a result of one particular stimulus and not another. At the level of the cerebral cortex,
work typified by that of David Hubel and Wiesel (1977) in the visual system and Vernon Mountcastle (1957)
in the somatosensory system described cells whose responses required stimuli of a specific type in a well-
defined receptive field. My own work, explaining mechanisms for lordosis behavior (Pfaff, 1999), fell into that
tradition.
In contrast, new approaches to changes of global states of the central nervous system involve the
regulation of large classes of behavioral responses to large sets of adequate stimuli. Thus, hormonal
influences on mood, exploratory tendencies, arousal, alertness, and attention would not be specific in the
manner displayed by neurons in the visual cortex, but they would be incredibly important for main-
taining mental and physical health. Our field of hormones and behavior increasingly embraces these
concepts.
In order to add new material, discovered since 2002, with special emphasis on clinical research, we had
to adopt the following strategy. We asked authors who had contributed to the first edition to reduce
markedly the length of their treatments of findings already covered in 2002. Therefore, the reader of this
current (second) edition will want to refer to the first edition as well, for the kind of comprehensive review
we intend.
Taking the first and the second editions of Hormones, Brain, and Behavior together will afford the reader
a tremendous number of facts, ranging from molecular genomics of hormone action, through biophysics,
neuroanatomy, and endocrinology to animal behavior and human disease. But this bulky coverage of primary
research findings is not, across the scope of several volumes, intended to be logically systematic. A parallel effort
in the text Principles of Hormone Behavior Relations takes a step in that direction.

References
Eccles, J., 1957. The Physiology of Nerve Cells. Johns Hopkins University Press, Baltimore, MD.
Hubel, D., Wiesel, T., 1977. Functional architecture of macaque monkey visual cortex. Proc. R. Soc. Lond. 198, 1e59.

xiii
xiv Hormonal Effects on Specific Behaviors, on Global CNS States, and on Human Disease

Mountcastle, V., 1957. Modality and topographic properties of single neurons of cat’s somatic sensory cortex. J. Neurophysiol. 20, 408e434.
Pfaff, D., 1999. Drive: Neural and Molecular Mechanisms of a Motivated Behavior. MIT Press, Cambridge, MA.
Sherrington, C., 1906. The Integrative Action of the Nervous System. Yale University Press, New Haven, CT (Reprinted 1947).

Further Reading
Pfaff, D., Phillips, M.I., Rubin, R.T., 2004. Principles of Hormone/Behavior Relations. Academic Press/Elsevier, San Diego.

Donald W. Pfaff
The Rockefeller University, New York, NY, USA
HORMONAL EFFECTS ON SPECIFIC BEHAVIORS, ON GLOBAL CNS
STATES, AND ON HUMAN DISEASE. AN INTRODUCTION TO THE
THIRD EDITION

In 2002, the first edition of Hormones, Brain, and Behavior appeared, dedicated to three generations of productive
scientists in our field, as it had developed at that point. With five volumes, totaling about 4100 pages, it rep-
resented a massive effort by over 200 scientists. In 2009 the volumes were updated, in the second edition. Both
sets of five volumes were well received.
At least four developments have taken place since 2009 which justify a third edition. Two are technical: the
application of increasingly sophisticated molecular biological techniques and biophysical techniques to the
understanding of mechanisms for hormone/behavior relations. Third, we have entered an era of translational
research. Laboratory scientists are under increasing pressure to show how their work might lessen the burden of
disease upon the citizenry. The fourth development is the most subtle one and can be explained as follows.
Some contemporary approaches to the hormonal controls of various forms of behavior can be distinguished
sharply from the major theme in neuroscientific research in the past century. We characterize that classical theme
as a ‘hunt for specificity.’ In the spinal cord, for example, the work of neurophysiologists such as Sir Charles
Sherrington (1906) and Sir John Eccles (1957) sought to explain why a particular reflex response occurred as
a result of one particular stimulus and not another. At the level of the cerebral cortex, work typified by that of
David Hubel and TorstenWiesel (1977) in the visual system and Vernon Mountcastle (1957) in the somato-
sensory system described cells whose responses required stimuli of a specific type in a well-defined receptive field.
Pfaff’s own work, explaining mechanisms for lordosis behavior (Pfaff, 1999), fell into that tradition.
In contrast, new approaches to changes of global states of the central nervous system involve the regulation of
large classes of behavioral responses to large sets of adequate stimuli. Experimental work on steroid hormone
regulation of responses to stress provide an example of this type of brain research (reviewed, Joels et al., 2013). Thus,
hormonal influences on mood, exploratory tendencies, arousal, alertness, and attention would not be specific in the
manner displayed by neurons in the visual cortex, but they would be incredibly important for maintaining mental
and physical health. Our field of hormones and behavior increasingly embraces these concepts.
In order to add new material, discovered since 2009, with special emphasis on clinical research, we had to
adopt the following strategy. We asked authors who had contributed to the first and second editions to reduce
markedly the length of their treatments of findings already covered in 2009; therefore, the reader of this current
(third) edition may want to refer to the earlier editions as well, for the kind of comprehensive review we intend.
In lieu of these earlier sections, the authors expanded information on more recent concepts and experiments.
Taking all editions of Hormones, Brain, and Behavior together will afford the reader a tremendous number of
facts, ranging from molecular genomics of hormone action, through biophysics, neuroanatomy, and endo-
crinology to animal behavior and human disease. But this bulky coverage of primary research findings is not,
across the scope of several volumes, intended to be logically systematic.
A parallel effort in the text Principles of Hormone Behavior Relations (the second edition of which will appear
soon) takes a step in that direction.

References
Eccles, J., 1957. The Physiology of Nerve Cells. Johns Hopkins University Press, Baltimore, MD.
Hubel, D., Wiesel, T., 1977. Functional architecture of macaque monkey visual cortex. Proc. R. Soc. Lond. 198, 1e59.

xv
xvi Hormonal Effects on Specific Behaviors, on Global CNS States, and on Human Disease

Joels, M., Pasricha, N., Karst, H., 2013. The interplay between rapid and slow corticosteroid actions in brain. Eur. J. Pharmacol. 719, 44e52.
Mountcastle, V., 1957. Modality and topographic properties of single neurons of cat’s somatic sensory cortex. J. Neurophysiol. 20, 408e434.
Pfaff, D., 1999. Drive: Neural and Molecular Mechanisms of a Motivated Behavior. MIT Press, Cambridge, MA.
Sherrington, C., 1906. The Integrative Action of the Nervous System. Yale University Press, New Haven, CT (Reprinted 1947).

Further Reading
Pfaff, D., Phillips, M.I., Rubin, R.T., 2004. Principles of Hormone/Behavior Relations. Academic Press/Elsevier, San Diego.

Donald W. Pfaff
The Rockefeller University, New York, NY, USA
Marian Joëls
University Medical Center Utrecht, Utrecht, The Netherlands
PERMISSION ACKNOWLEDGMENT

The following material is reproduced with kind permission of Oxford University Press
Figure 1 of Regulation of Honeybee Worker (Apis mellifera) Life Histories by Vitellogenin
www.oup.com

i
 NON-MAMMALIAN
VERTEBRATES: FISHES
This page intentionally left blank
 2.01 Neuroendocrinology of Social Behavior in Teleost Fish
David Gonçalves, University of St. Joseph, Macao, China
Ana S Félix, Gulbenkian Institute of Science, Oeiras, Portugal; and ISPA – University Institute, Lisbon, Portugal
Rui F Oliveira, Gulbenkian Institute of Science, Oeiras, Portugal; ISPA – University Institute, Lisbon, Portugal; and Champalimaud
Neuroscience Program, Lisbon, Portugal
Ó 2017 Elsevier Inc. All rights reserved.
2.01.1 Introduction
2.01.2 The Social Decision-Making Network
2.01.2.1 Short History of the Concept
2.01.2.2 Homologies between Teleost Fish and Mammalian Brain Areas in the SDMN
2.01.2.3 Functional Analysis of the SDMN in Teleost Fish
2.01.3 Endocrine Regulation of Social Behavior in Teleost Fish
2.01.3.1 Aggressive Behavior
2.01.3.2 Mating Behavior
2.01.3.3 Parental Care
2.01.3.4 Prosocial Behavior
2.01.4 Hormones as Social Semiochemicals
2.01.5 Conclusions
Acknowledgments
References
2.01.1 Introduction
Animals need to interact with other members from the same
species in order to survive and reproduce successfully, and
the set of behaviors used in these social interactions are
labeled social behaviors. From a functional perspective, these
social behaviors can be classified into major groups accord-
ing to the type of interaction in which they are expressed,
namely, aggressive, mating, parental, and prosocial. Interest-
ingly, despite their different functions, all these types of
social behaviors seem to share a common underlying mech-
anism, composed of a neural network, recently named the
social decision-making network (SDMN) (O’Connell and
Hofmann, 2012a, 2011), whose overall state parallels the
expression of the specific type of social behavior expressed
in a given moment in time, such that the state of the network
is a better predictor of social behavior than the activity of
a specific brain region (Goodson and Kabelik, 2009; Teles
et al., 2015). Moreover, the fact that all nodes in this network
express receptors for steroid hormones and for neuropeptides
opens the possibility for its neuromodulation and concomi-
tantly for the regulation of its behavioral output by these
agents. Thus, by acting on the SDMN, hormones can regulate
the expression of social behavior and integrate it with the
organismal state of the individual. Hormones can also regu-
late the social behavior of animals other than the one in
which they are produced when they act as hormonal phero-
mones. In such cases, hormones are released into the envi-
ronment and act on receptors located in sensory tissues of
other individuals triggering changes on their behavior. In
this chapter we will use the conceptual framework sketched
above to illustrate how hormones regulate social behaviors
in teleost fish.
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2.01.2 The Social Decision-Making Network
2.01.2.1 Short History of the Concept
In 1999, Newman challenged the neuroscientific community
by proposing the existence of a core set of brain areas that
collectively regulate social behavior in mammals. Each one
of these areas is reciprocally connected with the others,
contains sex steroid hormone receptors, and it is involved
in the activation or regulation of several social behaviors. It
was designated as social behavior network (SBN), and it is
composed of six limbic areas: the lateral septum (LS), the
medial extended amygdala (medial amygdala, meAMY, and
bed nucleus of the stria terminalis, BNST), the medial pre-
optic area (POA), the anterior hypothalamus (AH), the
ventromedial and ventrolateral hypothalamus (VMH), all
localized in the forebrain, and the midbrain periaqueductal
gray and tegmentum (PAG/CG), lying in the midbrain. Her
model was based on a considerable amount of behavioral,
neuroanatomical, and neuroendocrine evidences from
diverse studies in rodents and other mammals, which used
electrical stimulation, neuropharmacological manipulations,
specific brain lesions, and detection of immediate early gene
(IEG) expression. Together, these data show that common
areas jointly influence sexual, parental, or even aggressive
behavior, counteracting the idea of one area (or even a sepa-
rate minicircuit) is determining a specific behavior. Instead,
all these areas represent the nodes of a neuroanatomical
network, whose dynamic activation patterns are responsible
for multiple behaviors. For instance, male sexual behavior
would be the result of successive behavioral responses such
as sniffing, mounting, ejaculation, or grooming, which alto-
gether are activated by this integrated circuit and modulated
by environmental stimuli and sex steroids. Newman also
3
4 Neuroendocrinology of Social Behavior in Teleost Fish
highlighted that species and sex differences in social behav-
iors are a consequence of brain organization and connec-
tivity divergences, influenced by variations in hormone
sensitivity along development, on this central network.
Later, Goodson (2005) expanded the same framework to
other nonmammalian vertebrates describing important
evidences for birds and teleost fish, and providing foundations
for the evolutionarily conservation of the SBN in vertebrates.
He also contributed to a better insight into this network by add-
ing the role of peptidergic neuromodulation such as arginine
vasotocin (AVT, homologue of mammalian arginine vaso-
pressin) or isotocin (IT, homologue of oxytocin) on social
behavior and specifically as an integrating component of SBN.
More recently, O’Connell and Hofmann (2012a, 2011)
proposed that social behavior would be regulated by an even
wider network. The SDMN would include SBN and the meso-
limbic reward system. The latter system is the brain circuit
responsible for the evaluation of the salience of a stimulus
(via dopaminergic signaling) prior to the behavioral response,
which in turn is elicited by the former network. Thus, the
reinforcing/rewarding component of social behavior as
a substantial feature of an individuals’ adaptive response to
the environment is the main argument of the authors. The
mammalian mesolimbic reward system is constituted by the
striatum (Str), the nucleus accumbens (NAcc), the ventral pal-
lidum (VP), the basolateral amygdala (blAMY), the hippo-
campus (Hyp), the ventral tegmental area (VTA), and the LS
and the meAMY/BNST, overlapping with nodes of the SBN.
The authors performed a comparative analysis of the two neural
circuits in five vertebrate lineages: mammals, birds, reptiles,
amphibians, and teleost fish. Putative brain homologies were
described based on neuronal connections, the presence of
steroid hormone receptors, gene expression, neurochemistry,
developmental and behavioral studies. Concurrently, they
provided a very useful resource to study the neural substrates
responsible for social behavior in vertebrates and a relevant
framework to make species comparisons.
Nevertheless, attention must be drawn to the fact that
some proposed homologies are not complete, instead they
are only partial. In that sense, in a recent review, Goodson
and Kingsbury (2013) proposed the inclusion of the paraven-
tricular nucleus (PVN) of the hypothalamus within the
mammalian POA node (i.e., POA/PVN), in order to compre-
hend vasopressin–oxytocin nonapeptide neurons crucial for
the regulation of social behavior. With this incorporation the
POA/PVN mammalian node would be similar to the anam-
niotes POA node.
On the other hand, for some nonmammalian species,
homologies of the SDMN nodes are not clear, and functional
studies are still missing, especially for the mesolimbic reward
system (Goodson and Kingsbury, 2013). So, despite being
strongly supported in mammals, for other taxa, the SDMN
must be cautiously evaluated and tested.
2.01.2.2 Homologies between Teleost Fish and Mammalian
Brain Areas in the SDMN
In teleosts, the SBN is assumed to be composed by the ventral
(Vv) and lateral (Vl) part of ventral telencephalon, supracom-
misural part of the ventral pallium (Vs), POA, ventral tuberal
region (vTn), anterior tuberal nucleus (aTn), all localized in
the forebrain, in addition to the PAG, lying in the midbrain
(O’Connell and Hofmann, 2011). On the other hand, the mes-
olimbic reward system is presumably constituted by the dorsal
(Vd) and central (Vc) part of the ventral telencephalon, the
medial part of the dorsal telencephalon (Dm), the lateral part
of the dorsal telencephalon (Dl), the posterior tuberculum
(TPp) on the midbrain, and also Vv/Vl and Vs, that are also
nodes of the SBN (O’Connell and Hofmann, 2011; Figure 1).
Both Vv and Vl seem to be homologous of mammalian LS
region. Cholinergic neurons were detected only in this telence-
phalic area. It is reciprocally connected to several important
nuclei, and it expresses sex steroid receptors (see Wullimann
and Mueller, 2004; O’Connell and Hofmann, 2011 for more
details). It is also involved in reproductive behavior since elec-
trical stimulation of Vv in Sockeye salmon, Oncorhynchus nerka,
elicits females’ digging and spawning while Vv and Vs ablation
in the goldfish Carassius auratus impairs male ejaculation (Kyle
and Peter, 1982; Satou et al., 1984). Vs is putatively homolo-
gous of meAMY/BNST based on developmental evidences,
as well as neurochemical and connectivity similarities (see
Wullimann and Mueller, 2004; O’Connell and Hofmann,
2011 for further details), even though some consider that
ventral telencephalon (Vp) should also be included within
this node (Goodson and Kingsbury, 2013). The teleost POA
homology is well established. This node that is imperative for
the regulation of sexual, parental, and aggressive behaviors
(Demski and Knigge, 1971; Macey et al., 1974; Satou et al.,
1984; Wong, 2000) is localized in the hypothalamus along
the third ventricle, just like in mammals. It is divided into three
subregions according to cell size: parvocellular, magnocellular,
and gigantocellular, and it is reciprocally connected with the
telencephalon and other hypothalamic regions (reviewed in
O’Connell and Hofmann, 2011). vTn is the putative homolo-
gous of AH because it is localized between the POA and the
ventral hypothalamus, it receives and sends projections to
several hypothalamic regions, and it expresses sex steroid recep-
tors (O’Connell and Hofmann, 2011 reviewed this informa-
tion). However, functional studies are yet not available to
confirm this homology. aTn was proposed to be the teleost
equivalent of VMH, although only a subset of aTn cells are
actually homologous (Goodson and Kingsbury, 2013). Lying
in the ventrocaudal region of the hypothalamus, it is recipro-
cally connected with several parts of the telencephalon and
contains sex steroid hormone receptors (reviewed in O’Connell
and Hofmann, 2011) but functional studies are as well
limited. PAG is also present in teleosts, located near the torus
semicircularis and receiving and sending projections to several
other nuclei (see O’Connell and Hofmann, 2011 for more
information). Functionally, it is associated with social commu-
nication, specifically, in sound production of the plainfin
midshipman Porichthys notatus (Kittelberger et al., 2006). Vc is
comparable to Str in mammals, while Vd seems homologous
to NAcc, but this is only supported by neurochemical studies
and some hodological evidences (consider Wullimann and
Mueller, 2004; O’Connell and Hofmann, 2011 for more
details). Unfortunately, a homologous for the mammalian
VP node has not yet been identified. Dm is the putative homol-
ogous of blAMY based on developmental, tract tracing, and
lesions studies, demonstrating its implication in emotional
 Neuroendocrinology of Social Behavior in Teleost Fish 5

Brain
SDMN
Dl Dm
POA

Sensory interface

Motor output
Input Vd Vs PAG Behavior

Vc Vv/Vl aTn

vTn
TPp VP?

IT AVT

Peripheral hormones

Sex steroids Glucocorcoids

Body

Figure 1 Representation of the interaction between hormones and the social decision-making network (SDMN) within teleosts’ social behavior:
putative nodes of the mesolimbic reward system in white – dorsal (Vd) and central (Vc) part of the ventral telencephalon, medial part of the dorsal
telencephalon (Dm), lateral part of the dorsal telencephalon (Dl), posterior tuberculum (TPp) – and the social behavior network in black – medial pre-
optic area (POA), ventral tuberal region (vTn), anterior tuberal nucleus (aTn), and midbrain periaqueductal gray (PAG). Ventral and lateral (Vv/Vl) part
of ventral telencephalon and supracommisural part of the ventral pallium (Vs), overlapping nodes of the social behavior network and the mesolimbic
reward system, are in gray. A homologous for the mammalian ventral pallidum (VP) node has not yet been identified. AVT, arginine vasotocin; IT,
isotocin.

learning, as shown in mammals (reviewed in O’Connell and
Hofmann, 2011; Portavella et al., 2002). The homologous of
Hyp is considered to be the Dl mainly because of its involve-
ment in spatial learning (Dl lesions of C. auratus impair
maplike memory representations, Rodríguez et al., 2002),
besides some of its hodological features (reviewed in
O’Connell and Hofmann, 2011). Finally, even though amphib-
ians and teleosts lack a midbrain dopaminergic population,
TPp, located in the ventral diencephalon, has been suggested
to present homologies to VTA, the A10 dopaminergic cell
group, because of its dopaminergic ascending projections to
Vd and conserved neurochemical patterns (see O’Connell and
Hofmann, 2011 for details). However, this seems to be refutable
as a recent study on the zebrafish, Danio rerio, ‘projectome’
underlines that posterior tuberculum cells (DC2 and DC4–6
cell groups) seem homologous to A11 mammalian dopamine
neurons based on transcription factor conservation and projec-
tion patterns (Tay et al., 2011). Actually ascending projections
to telencephalon are scarce (and only from DC2 and DC4
neuronal cells), while the most important dopaminergic
connections between the subpallium and the ventral dienceph-
alon are descending (Tay et al., 2011). Consequently, the exis-
tence of a mesolimbic reward system in fish is questionable
since the connection between the VTA and forebrain regions is
considered the core of the dopaminergic reward system
(Bromberg-Martin et al., 2010; Spanagel and Weiss, 1999).
2.01.2.3 Functional Analysis of the SDMN in Teleost Fish
A recent study in zebrafish functionally tested the SDMN
concept. In this study, IEG expression was determined along
selected nodes of the SDMN while animals were in different
behavioral states (Teles et al., 2015). The aim of this work
was to test for functional brain specialization or alternatively
for functional connectivity and, for example, determine
whether expression of social behavior is explained by the acti-
vation of a single node or by the overall combination of the
activity in the various nodes. They quantified IEG expression
in the brains of winners and losers of agonistic fights, as well
as in individuals who only experienced mirror fights and
compared them with a reference noninteracting group. IEG-
transcription patterns of c-fos and egr-1 as measured by qPCR
were used as markers of neuronal activity in the Vv, Vs, POA,
Dm, and Dl. The similarity of IEG activation between groups
and areas showed that there were no patterns of localized
activity in a specific nucleus. Instead socially driven behavioral
states demonstrated patterns of functional connectivity across
the nodes. The notion of an SDMN is therefore supported in
a teleost fish.
On the other hand, a considerable number of studies
centered on the behavioral responses of teleost fishes have
documented the activation of specific SDMN nodes, hence
establishing their involvement in the regulation of social
behavior. For instance, Desjardins et al. (2010) focused on
how mate information impacts female neural activity. In this
work, they measured IEG (c-fos and egr-1) expression levels
by qPCR in gravid females of the Burton’s mouthbrooder
cichlid, Astatotilapia burtoni, that saw their preferred males
winning or losing a fight. They looked specifically to the Vv
(LS), POA, vTn (AH), aTn (VMH), PAG, Dm, and Dl. Results
demonstrate that the POA and VMH, two nuclei known to be
involved in the control of reproduction, are highly activated
6 Neuroendocrinology of Social Behavior in Teleost Fish
when females see their preferred male winning, whereas IEG
response in the mammalian LS homologue region (a nucleus
associated with anxiety) is elicited when females see the male
losing.
O’Connell et al. (2013) directed their interest to how indi-
viduals integrate social information. These researchers pre-
sented A. burtoni males with different social stimuli and
discovered that visual information (seeing a female or
a male) is sufficient to elicit c-fos transcription in dopaminergic
neurons of Vc, and this transcription is significantly correlated
to aggressive behavior in the case of exposure to an intruder
male. These data suggest that Vc seems to be involved in assess-
ing stimulus visual valence. Another interesting survey was
also carried out in this species. Since A. burtoni males can revers-
ibly switch between dominant and subordinate status and
rapidly present distinct phenotypes, investigators examined
IEG levels in several brain areas of males ascending or descend-
ing in social status, as compared with control individuals
(Maruska et al., 2013a,b). In socially ascending males, both
c-fos and egr-1 levels were higher than in control males in all
the SDMN nuclei (Vv, Vs, POA, vTn, aTn, Dm, and Dl)
(Maruska et al., 2013b). Descending males presented different
activation patterns for c-fos and egr-1 across the same areas. c-fos
expression levels were increased in the Vs, POA, and aTn
by comparison with controls while egr-1 mRNA levels were
higher in the Vv, Vs, vTn, Dm, and Dl (Maruska et al.,
2013a). Another relevant study used the monogamous cichlid
Amatitlania nigrofasciata as a model to study the influence of IT
in parental care (O’Connell et al., 2012). The authors
compared males housed with their mate (control males), single
fathers who had the mate removed or lone males (mate and
offspring removed), and quantified c-fos expression in Vv,
POA, and the central part of the dorsal telencephalon (Dc).
At the behavioral level, only single fathers increased paternal
care immediately after removal of their mate, and they also
presented significantly higher IEG activity levels in Vv
compared to lone males, as well as increased c-fos expression
in the parvocellular POA IT neurons. Together these data
suggest that IT promotes paternal care after mate removal
and that Vv and POA are important brain areas in this process.
Finally, a very interesting study with P. notatus, where reproduc-
tive behavior is intimately associated with social acoustic
signals, measured c-fos activation in several brain nuclei
including vTn, aTn, and TPp (Petersen et al., 2013). The authors
report a significant increase of IEG expression in aTn and TPp
of males exposed to acoustic signals of other males compared
to control males, showing the importance of these nuclei in
social reproductive communication in this species.
2.01.3 Endocrine Regulation of Social Behavior in
Teleost Fish
The pioneering work of Arnold Berthold on the endocrine
regulation of sexual behavior in animals demonstrated the
influence of a ‘blood-borne product’ released in peripheral
glands on behavior (Berthold, 1849). Likewise, early studies
on the endocrine regulation of behavior in fish focused on
the role of hormones produced in the periphery, mainly
gonadal steroids, prostaglandins, and corticosteroids, and
there is now substantive information on the role of these
hormones as modulators of social behavior. However, in
some cases, the expression of social behavior seems to be inde-
pendent from hormones produced in the periphery, and
the role of brain-synthesized hormones, in particular neuroste-
roids and neuropeptides, has been receiving increasing atten-
tion (for a review see Gonçalves and Oliveira, 2010; Oliveira
and Gonçalves, 2008).
Regardless of the source, still little is known on how molec-
ular and cellular mechanisms of hormonal action in the brain
modify social behavior in fish. The recent concept of the
SDMN is useful as it allows studies on the neuroendocrine
modulation of social behavior to be focused in relevant brain
areas. As described above, recent work has addressed the inter-
play between social environment and activation of the nodes
of the SDMN using IEG as proxies of neuronal activity (e.g.,
Desjardins et al., 2010; Teles et al., 2015). However,
studies investigating the regulatory role of hormones in the func-
tionality of the SDMN are still scarce in spite of the fact that
major modulatory effects of hormones in the SDMN are ex-
pected, as evidenced by the widespread distribution of hormone
receptors in its nodes. In particular, estrogen (P. notatus, Forlano
et al., 2005; Micropogonias undulatus, Hawkins et al., 2005;
D. rerio, Menuet et al., 2002; A. burtoni, Munchrath and
Hofmann, 2010; Dicentrarchus labrax, Muriach et al., 2008),
progesterone (A. burtoni, Munchrath and Hofmann, 2010),
androgen (P. notatus, Forlano et al., 2010; C. auratus, Gelinas
and Callard, 1997; A. burtoni, Harbott et al., 2007; Munchrath
and Hofmann, 2010), vasotocin (A. burtoni, Huffman et al.,
2012; rock hind, Epinephelus adscensionis, Kline et al., 2011),
and IT (A. burtoni, Huffman et al., 2012) receptors are widely
distributed along the areas of the SDMN (see also Diotel et al.,
2011).
Below we review the evidence for the endocrine modulation
of different types of social behavior in fish, addressing their
possible mechanisms of action at the brain level.
2.01.3.1 Aggressive Behavior
Different categories of hormones, notoriously gonadal steroids,
corticosteroids, and nonapeptide hormones, have been associ-
ated with the regulation of aggressive behavior in fish. For
gonadal steroids, early evidence demonstrated that male domi-
nance correlated with circulating androgen levels, in particular
with the nonaromatizable androgen 11-ketotestosterone
(11KT; for a review, see Oliveira and Gonçalves, 2008; Oliveira
et al., 2002). Classical castration–androgen replacement experi-
ments suggested an effect of gonadal androgens in aggression,
and a meta-analysis confirmed that exogenous administration
of androgens promotes aggression in fish (Hirschenhauser and
Oliveira, 2006). In one of the best studied models in this
respect, the cichlid A. burtoni, a change from a submissive to
a dominant status in males increases the expression of the
GnRH1 gene and the concomitant production of its peptide in
neurons of the POA, inducing gonadal development and a sur-
ge in plasma 11KT levels within 30 min (Francis et al., 1993;
Maruska and Fernald, 2010; White et al., 2002). Interestingly,
in this species a submissive experience seems to have more rapid
and more profound physiological consequences than a domi-
nant one as aggressive behaviors were reduced more promptly

in males undergoing a descent in social status than they emerged
in animals ascending in social status (White et al., 2002). Simi-
larly, in zebrafish males, losing a fight induces a more
pronounced change in future aggressive behavioral displays
and in the neurogenomic state of the whole brain than winning
(Oliveira et al., 2011, 2016).
Aggression in females has been much less investigated
although, interestingly, the role of androgens in the modulation
of aggressive displays seems to be more consistent than
for males. In the Mozambique tilapia, plasma testosterone
(T) levels in females peak during a phase of the reproductive
cycle that matches increased aggression (Oliveira and Canário,
2000). In the cichlid Neolamprologus pulcher, females and males
jointly defend a territory all year round and females were shown
to be more aggressive than males in response to a territorial
intrusion and to experience a higher increase in androgen levels
(including 11KT, usually undetected in females, Desjardins et al.,
2006). Similarly, in the blue acara, Andinoacara pulcher, T admin-
istration increased aggressive behavior in females (Munro and
Pitcher, 1985) and daily injections of T to females of the Siamese
fighting fish Betta splendens for a period of 9 weeks increased
aggression directed toward males, although it decreased aggres-
sion directed toward females (Badura and Friedman, 1988).
Finally, in all-female groups of A. burtoni, it was observed
that females start to exhibit typical male behaviors, including
territorial and aggressive displays (Renn et al., 2012). In this
experiment, dominant females had higher T levels when
compared with subordinates and T levels correlated with aggres-
sive displays like chasing and threatening.
In birds and mammals, the central effects of androgens on
aggressive behavior seem to depend on the aromatization of
T into estradiol (E2) (Trainor et al., 2006). In fish, however,
in spite of aromatase being abundant in nuclei of the SDMN
(e.g., Forlano et al., 2001), there are conflicting results on the
role of aromatization in aggression. Several lines of evidence
suggest a direct action of androgens in the expression of aggres-
sive behavior in fish: (1) androgen receptors are widely distrib-
uted in nuclei of the telencephalon and diencephalon,
including in areas of the SDMN (e.g., Harbott et al., 2007);
(2) differences in plasma androgen levels between aggressive
and less aggressive phenotypes seem to be more evident for
the nonaromatizable androgen 11KT than for T (Oliveira,
2005) and the peripheral administration of 11KT has been
shown to promote aggression in teleosts (e.g., Rodgers et al.,
2013); (3) in some species, peripheral administration of
estrogens has an inhibitory effect on male aggression (e.g.,
three-spined stickleback, Gasterosteus aculeatus, Bell, 2001;
B. splendens, Clotfelter and Rodriguez, 2006; D. rerio, Colman
et al., 2009; Filby et al., 2012; peacock blenny, Salaria pavo,
Gonçalves et al., 2007; A. pulcher, Munro and Pitcher, 1985;
sand goby, Pomatoschistus minutus, Saaristo et al., 2010);
and (4) whole brain aromatase activity was shown to be
inversely correlated with aggression in females of the sex-
changing blue-banded goby, Lythrypnus dalli (Black et al.,
2005), suggesting that a higher availability of T (or a decrease
in E2 synthesis) promotes aggression. Contrarily, in the cichlid
A. burtoni, E2 administration increased male aggression
(O’Connell and Hofmann, 2012b) and pharmacologically
blocking aromatization with Fadrozole decreased these behav-
iors in the same species (Huffman et al., 2013) and also in the
Neuroendocrinology of Social Behavior in Teleost Fish 7
weakly electric fish Gymnotus omarorum (Jalabert et al., 2015).
This would suggest that aromatization of T into E2 is needed
to promote male aggressive displays in these species. Clearly,
more data are needed to interpret the divergent results across
species and understand what the general pattern in fish is.
The above data suggest that a dominance experience (e.g.,
winning fights) activates the hypothalamic–pituitary–gonadal
axis, increasing the secretion of gonadal androgens that in
turn act on different tissues to promote the expression of
a dominant phenotype, including increased aggression, via
effects on the brain (and the reverse for submissive experi-
ences). This feedback between the environment and behavior,
translated via neuroendocrine modulation by androgens,
would allow animals to dynamically adjust their behavior
and physiology to a particular social context (Oliveira, 2004).
However, the hypothesis that high levels of aggression are
maintained through a positive effect of gonadal androgens in
the brain is contradicted by other studies. The majority of
studies where males were gonadectomized during the breeding
season, thus reducing the circulating levels of androgens, failed
to find a significant effect on male aggression (reviewed in Gon-
çalves and Oliveira, 2010). As an example, in the Mozambique
tilapia gonadectomy impaired the expression of reproductive
behaviors in males, including nest building and courtship
displays, but did not affect aggressive behavior toward a
conspecific male (Almeida et al., 2014). Also, in the social
sex-changing bluehead wrasse Thalassoma bifasciatum, gonadec-
tomy did not prevent female-to-male behavioral change,
including an increase in the expression of aggressive behavior,
when females were given an opportunity to occupy a vacant
territory (Godwin et al., 1996). Finally, variation in aggressive
behavior has been shown to occur in immature individuals
and animals outside the breeding season, questioning the
hypothesis of aggression being modulated by gonadal andro-
gens in these contexts. For example, in the G. omarorum, high
levels of aggression were decoupled from 11KT levels outside
the reproductive season (Jalabert et al., 2015) and in the
damselfish Stegastes nigricans, also a year-round territorial
species, androgen levels did not increase when an aggressive
challenge was presented to males (Ros et al., 2014).
The contradictory results found for the effect of sex steroids
on aggressive behavior have driven the search for alternative
modulators of aggression. A pathway that has also been shown
to relate with the neuroendocrine modulation of aggression in
fishes is the hypothalamic–pituitary–interrenal axis. In the
abovementioned study in S. nigricans, where androgens failed
to respond to an aggressive challenge, cortisol levels in the
plasma increased after males were presented with intra- and
interspecific challenges, and its concentration was strongly
correlated with aggressive behaviors (Ros et al., 2014). Also,
in the cichlid A. pulcher, cortisol administration increased
aggression toward a model intruder (albeit not toward a mirror
image, Munro and Pitcher, 1985). In juvenile rainbow trout,
Oncorhynchus mykiss, cortisol administration failed to promote
aggression 1 h after being administered, but pharmacological
blockage of GR and MR receptors reduced aggression levels,
suggesting that basal levels of cortisol were contributing to
aggressive behavior via activation of intracellular GR and MR
receptors (Schjolden et al., 2009). Nevertheless, prolonged
(48 h) exposure to cortisol reduced aggression, suggesting
8 Neuroendocrinology of Social Behavior in Teleost Fish
a complex time-dependent effect of this hormone in aggressive
behavior (Øverli et al., 2002).
AVT and IT have also been implicated in the regulation of
aggression, and more generally social behavior, in fishes
(reviewed in Godwin and Thompson, 2012). AVT neurons
occur in the POA and project to the neurohypophysis, releasing
AVT into circulation when activated, but also project to many
other brain regions, including the ventral telencephalon, thal-
amus, and mesencephalon (for details on the neuroanatomy
of the AVT system in fish, see Godwin and Thompson, 2012;
Huffman et al., 2012; Thompson and Walton, 2013). Within
the POA, three subpopulations of AVT neurons can be identi-
fied; parvocellular, magnocellular, and gigantocellular, and
they have been suggested to play different roles in osmoregula-
tion and modulation of behavior (Greenwood et al., 2008).
Receptors for both AVT and IT have been found throughout
nuclei of the SDMN, suggesting a direct neuromodulatory
action of these neuropeptides in nodes of this brain network
(Huffman et al., 2012; Lema, 2010).
Following studies in mammals that associated AVP with
increased expression of aggressive behavior, AVT has also been
implicated in the regulation of aggression in fishes, although
with inconsistent results between species. A positive effect of
AVT on aggression has been described for some species, as,
for example, nonterritorial phase males of T. bifasciatum (Semsar
et al., 2001), males of the damselfish Stegastes leucostictus
(Santangelo and Bass, 2006), and males of A. nigrofasciata
(Oldfield and Hofmann, 2011) while a suppressive effect of
AVT on aggression has been described for other species, as, for
example, territorial phase males of T. bifasciatum (Semsar et al.,
2001), males of the brown ghost knifefish, Apteronotus leptorhyn-
chus (Bastian et al., 2001), males of Amargosa pupfish, Cyprino-
don nevadensis amargosae (Lema and Nevitt, 2004; but see Lema
et al., 2015), and males and females of D. rerio (Filby et al.,
2010). These contradictory results may have different, but not
necessarily exclusive, explanations. In studies with butterfly fish
of the genus Chaetodon, it was shown that a territorial species
had larger AVT-immunoreactive somata within the POA area
and higher AVT fiber densities within a number of
telencephalic nuclei than a nonterritorial species and that
aggression correlated positively with the number of POA
gigantocellular AVT cells and negatively with the number
and size of POA parvocellular AVT cells (Dewan and Tricas,
2011; Dewan et al., 2008). In the C. nevadensis amargosae,
telencephalic proAVT mRNA levels were found to be elevated
in subordinate males and to correlate with aggression. These
males also had higher V1a1 receptor transcript levels in the
telencephalon and hypothalamus, as compared with
dominant males. On the other hand, the levels of proAVT
mRNA were fourfold higher in the hypothalamus of
dominant males, which also had higher levels of
hypothalamic V1a2 receptor transcript abundance (Lema
et al., 2015). This may be interpreted as hypothalamic AVT
playing a role in the expression of aggressive behavior in
dominant males via the activation of the V1a2 receptor,
while AVT action in forebrain targets would promote
aggression only in subordinate animals. Greenwood et al.
(2008) showed an opposite pattern of AVT mRNA
expression in A. burtoni parvocellular and magnocellular
subpopulations of AVT cells, with territorial males having
higher levels of expression of AVT in the gigantocellular
layer but lower levels in the parvocellular layer, as compared
with nonterritorial males. The authors suggested that
gigantocellular neurons might be more related with the
modulation of dominance-related traits, including the
expression of aggressive behavior, while parvocellular cells
may relate to the activation of the stress axis or submissive
behaviors. Following this model, Godwin and Thompson
(2012) suggest that AVT projections from the POA may
regulate ‘sociosexual circuits,’ including those related with
aggression, by modulating neuronal action in central brain
regions, including nodes of the SDMN. On the other hand,
AVT would also be able to promote submissive and
escape behaviors by producing peripheral changes that
feedback to the brain. These effects could be induced either
directly, via modulation of hindbrain autonomic nuclei that
regulate peripheral states, or indirectly, as, for example,
through the demonstrated capacity of AVT to stimulate the
stress axis (Baker et al., 1996). As a consequence, peripheral,
or even central, administration of AVT may have variable
effects in behavior as it activates the targets of multiple
subpopulations of AVT neurons. In addition, and as
demonstrated in mammalian models, the effects of AVP/AVT
in behavior are greatly dependent on tissue sensitivity which
may vary across phenotypes, developmental stages, or seasons
due to variation in the abundance of its receptors (e.g., Walton
et al., 2010). Future studies manipulating the AVT system
using techniques such as optogenetics, transgenics, or
others may offer an opportunity to investigate the exact
function of the different subpopulations of AVT neurons and
subtypes of receptors in the modulation of aggression, and
other categories of social behavior, in fish.
The effect of IT on aggressive behavior in fish has been less
investigated. In the plainfin midshipman, IT administered to
the POA-AH elicited fictive aggressive vocalizations in a neuro-
physiological preparation of parasitic (sneaker) males but not
of territorial males (Goodson and Bass, 2000). On the other
hand, IT administered to males of the beaugregory damselfish
had no effect in aggressive displays (Santangelo and Bass,
2006), and similar results were found in females and males
of the cichlid N. pulcher (Reddon et al., 2012). Further studies
are needed before a role for IT in the modulation of fish aggres-
sion can be established.
Finally, hormones involved in somatic growth, in particular
the growth hormone (GH) and somatostatin (SS), have also
been proposed as modulators of aggressive behavior in fish.
These hormones are synthesized at the level of the hypothal-
amus projecting to somatotropes in the pituitary. In
A. burtoni, SS seems to inhibit the expression of aggressive
behavior in a dose-dependent fashion and independently of
any potential effect in gonadal androgen secretion (Trainor
and Hofmann, 2006). In the rainbow trout, peripheral admin-
istration of GH was shown to increase male aggression, but this
was interpreted as an indirect effect as GH also increased swim-
ming activity that promoted agonistic encounters (Jönsson
et al., 1998). Later, Jönsson et al. (2003) confirmed this
hypothesis by administering GH directly into the third ventricle
of juvenile rainbow trout and observing also an increase in
swimming activity. Interestingly, in the A. burtoni study, only
chasing behavior, and not threatening behavior, was affected

by SS. As SS is known to inhibit the release of GH, the inhibi-
tory effects of SS in aggressive displays could be explained by
a decrease in general locomotor activity induced by a reduction
in GH levels. These data are also contradictory to the findings
of Hofmann and Fernald (2000) showing that dominant
males have larger SS immunoreactive neurons in the POA as
compared with subordinate animals, suggesting that SS
administration should increase aggression if it is directly
related with the endocrine regulation of these behaviors.
2.01.3.2 Mating Behavior
The brain regions associated with the expression of sexual
behaviors have been extensively described in vertebrates,
including fish (for a related review, see Forlano and Bass,
2011). Notoriously, the POA and AH are known to be central
brain regions for the control of reproduction as they contain
the GnRH neurons that command the release of the gonadotro-
pins LH and FSH from the pituitary, regulating gonadal develop-
ment and secondarily gonadal steroid secretion. These regions
also synthesize neuropeptides relevant for reproduction and
are rich in sex steroid receptors. Early studies highlighted the
role of this area in reproduction by showing that electrical stim-
ulation of the POA induced reproductive behaviors in male
bluegill sunfish Lepomis macrochirus (Demski and Knigge,
1971), results later confirmed in the hime salmon O. nerka
(Satou et al., 1984). As mentioned above, females of the cichlid
A. burtoni, observing their preferred male winning fights acti-
vated the POA (as measured by IEG expression) and also the
Vm, another area implicated in female sexual displays in verte-
brates (Desjardins and Fernald, 2010). In a study in female
Medaka Oryzias latipes, mating induced widespread c-fos expres-
sion in the POA, telencephalon, optic tectum, and cerebellum
(Okuyama et al., 2011), suggesting the implication of a wide-
spread set of brain nuclei in female mating behavior. In addition
to these central brain regions, hormones can also modulate
reproductive displays by acting in sensory or effector systems.
As an example, in female midshipman, E2 acts in the inner
ear’s sacculus to increase the degree of temporal encoding of
the frequency content of male vocalizations (Sisneros et al.,
2004), thus synchronizing female phonotaxis and receptivity
with maturation of the ovaries (for a review, see Sisneros, 2009).
Both female and male reproductive behaviors are expected
to be coordinated with gonadal function, and thus hormones
of gonadal origin, in particular sex steroids for males and sex
steroids and prostaglandins for females, have been seen as
main candidates for endocrine regulation of reproductive
behaviors in fish.
The main androgens detected in fish plasma are T, 11KT,
and 11b-hydroxytestosterone (Borg, 1994). Males have usually
higher plasma levels of 11KT than females while T levels often
do not differ between sexes (Borg, 1994; Lokman et al., 2002).
The impact of manipulating androgen levels in male reproduc-
tive displays is highly variable (see Oliveira and Gonçalves,
2008 for a review). While gonadectomy is effective in reducing
plasma androgen levels (e.g., Almeida et al., 2014; Gonçalves
et al., 2007; Salek et al., 2001), in some cases a concomitant
reduction in reproductive behaviors occurs (e.g., Oreochromis
mossambicus, Almeida et al., 2014; G. aculeatus, Hoar, 1962;
Morone americana, Salek et al., 2001) while in others they are
Neuroendocrinology of Social Behavior in Teleost Fish 9
maintained (e.g., G. aculeatus, Páll et al., 2002; T. bifasciatum,
Semsar and Godwin, 2003). Also, exogenous administration
of androgens either to gonadectomized or to intact males has
variable effects on male mating displays (for a list of studies,
see Oliveira and Gonçalves, 2008). For example, androgen
administration to gonadectomized males of M. americana
restored sexual displays, with 11KT being more effective than
T (Salek et al., 2001), while 11KT administration to intact
males of the rock-pool blenny Parablennius parvicornis failed
to promote sexual behavior (Ros et al., 2004). Finally, pharma-
cologically blocking androgen receptors decreased male
nesting behavior in G. aculeatus (Sebire et al., 2008) and male
courtship displays in the guppy Poecilia reticulata (Baatrup
and Junge, 2001), in agreement with the hypothesis that
male mating behaviors are directly facilitated by androgens.
The administration of androgens to juveniles, parasitic
‘sneaker’ males or females, all phenotypes with lower plasma
androgen levels than males, likewise produced variable results.
In the peacock blenny, T implants inhibited female-like
displays in castrated parasitic males but failed to promote nest-
ing male behaviors (Gonçalves et al., 2007), while in C. auratus
T and 11KT implants given to intact females induced the full-
suite of male sexual behaviors (Stacey and Kobayashi, 1996).
The variable results obtained between studies of the effects of
androgens on male sexual behavior surely have multiple causes,
including differences in hormone concentrations, type of
androgens and antiandrogens used, hormone delivery mode,
species-specific differences, season when experiments were per-
formed or duration of exposure to the hormone, just to mention
a few. Nevertheless, the overall pattern suggests that androgens
have a positive effect on male sexual displays also in fish.
The central effects of androgens on male displays and in
particular their potential action in the nodes of the SDMN
are still poorly understood. In mammals, aromatization of
T into E2 in the brain plays a crucial role in the regulation
of male sexual behavior (reviewed in Ball and Balthazart,
2004; Baum, 2003). However, although aromatase is abun-
dant in the areas of the SDMN and partly colocalizes with
androgen receptors (Forlano et al., 2010; e.g., Gelinas and
Callard, 1997; Harbott et al., 2007), the evidence for aroma-
tization playing a role in the activation of male sexual
displays in fish via local conversion of T into E2 is less
obvious than in birds or mammals. In fact, in some studies
the nonaromatizable 11KT seems to have a more effective
role in the induction or recovery of male sexual displays
than the aromatizable T (e.g., Stacey and Kobayashi, 1996),
and pharmacologically blocking aromatization with Fadro-
zole was shown to inhibit male displays in P. reticulata
(Hallgren et al., 2006) but not in A. burtoni (Huffman
et al., 2013). Also, exposure to estrogens or xenoestrogens
generally reduces male sexual displays (e.g., P. reticulata,
Bayley et al., 1999; C. auratus, Bjerselius et al., 2001; Colman
et al., 2009; D. rerio, Pradhan and Olsson, 2015). Further-
more, while androgens masculinize the electric organ
discharge signal in ghost knifefishes, estrogens feminize it
(reviewed in Smith, 2013). In zebrafish, E2 seems to femi-
nize the male brain and 11KT to masculinize the female
brain, as assessed by gene transcriptomic profiling (Pradhan
and Olsson, 2015), further supporting a direct action of
androgens on male reproductive behavior.
10 Neuroendocrinology of Social Behavior in Teleost Fish
Male sexual displays are often more elaborated than female
displays, and consequentially there are more published studies
testing the effect of endocrine manipulations in sexual behavior
in males than in females. The regulation of female sexual
behavior was initially hypothesized to be controlled by gonadal
hormones and determined by the mode of reproduction
(Stacey, 1981). In internal fertilizers, sexual behavior and fertil-
ization are temporally dissociated, and sex steroids were
proposed to regulate female displays. By contrast, in external
fertilizers, female sexual behavior was considered to be mostly
restricted to oviposition, which may be regarded as homologous
to parturition. Thus ovarian prostaglandins, which induce
uterine contractions in mammals and oviposition in fishes
(Jalabert and Szöllösi, 1975), were proposed to modulate
female spawning behaviors. This idea was originally proposed
based mainly on data for P. reticulata, an internal fertilizer,
and C. auratus, an external fertilizer, but new evidence suggests
that the sex steroid and prostaglandin pathways may actually
be complementary. In a sex-role reversed population of the
peacock blenny, an external fertilizer, females court males with
very elaborate displays (Gonçalves et al., 1996), providing an
opportunity to test the effects of endocrine manipulations in
female sexual behavior. Ovariectomy was effective in quantita-
tively reducing the expression of female courtship displays and
nuptial coloration 2 weeks after the removal of the ovaries but
still a majority of ovariectomized females (9 out of 13) courte-
d at least once a nesting male, showing that removal of the
ovaries does not completely suppress the expression of sexual
behavior (Gonçalves et al., 2014). Interestingly, both the steroid
E2 and the prostaglandin PGF2a restored female sexual displays
(but not nuptial coloration), although PGF2a was more effec-
tive than E2 in this respect. In the same population, parasitic
males that mimic female displays occur and the removal of
the testes in these males has the paradoxical effect of promoting
even further the expression of female-like behaviors (Gonçalves
et al., 2007). This is probably explained by the fact that andro-
gens suppress female-like displays in sneaker males (Gonçalves
et al., 2007; Oliveira et al., 2001), and removing the testes
reduces circulating androgen levels, releasing this inhibition
(Gonçalves et al., 2007). Because E2 levels are very low in
sneaker males and E2 administration does not promote
female-like displays (Gonçalves et al., 2007), other neuroendo-
crine mechanisms are proposed to regulate these behaviors (see
below).
Prostaglandins have been shown to promote female
displays in other externally fertilizing species, including the
paradise fish, Macropodus opercularis (Villars et al., 1985), the
black acara Cichlasoma bimaculatum (Cole and Stacey, 1984),
the barb Puntius gonionotus (Liley and Tan, 1985), the cichlid
A. burtoni (Kidd et al., 2013) and, notoriously, the goldfish
C. auratus (reviewed in Kobayashi et al., 2002). Recently, Juntti
et al. (2016) confirmed this role of PGF2a in A. burtoni by
showing that the expression of a putative PGF2a receptor in
areas such as vagal lobe and POA increase during spawning
and that the activation of this receptor is needed for spawning
behavior to occur. This evidence suggests that ovarian prosta-
glandins act in external fertilizing teleost species, and probably
also in amphibians, as a short-duration endogenous messenger
to synchronize sexual behavior with the presence of mature
oocytes in the ovaries. However, alternative explanations exist
including the possibility of direct neural communication
between the gonads and the brain via the vagal nerve, which
would induce neural synthesis of PGF2a and the activation
of female reproductive displays (Juntti et al., 2016), or the acti-
vation of brain PGF2a synthesis by other ovarian hormones.
For example, in mammals, E2 has been found to promote
the synthesis of prostaglandins both in the uterus (PGF, Ham
et al., 1975) and in the POA (PGE2, Amateau and McCarthy,
2002). It seems possible that the above-described positive
effects of both E2 and PGF2a in the activation of sexual
displays in ovariectomized females of S. pavo could occur via
a direct effect of PGF2a in the brain and to a stimulatory
effect of E2 in the neural synthesis of PGF2a. Studies investi-
gating in further detail the interconnection between sex
steroids and prostaglandins in fish, in particular the effects of
gonadal steroids in brain PGF2a synthesis and receptor expres-
sion, and the modulation by PGF2a of SDMN nodes look
like promising venues for future research.
Nevertheless, evidence that the expression of sexual
behavior in fish does not rely on hormones of gonadal origin
was obtained by Godwin et al. (1996) who showed that, in
the sex-changing wrasse T. bifasciatum, females could rapidly
occupy a territory left vacant by the removal of the dominant
male and express male courtship and spawning displays in
the absence of gonads. Hypothalamic abundance of proAVT
mRNA in the brain of these females during sex change increases
fourfold when compared with nonchanging females (Godwin
et al., 2000) and is higher in ovariectomized dominant
females than in subordinate females (Semsar and Godwin,
2003), suggesting that AVT may be a key peptide regulating
the transition into male sexual displays in this species.
However, the effect of AVT seems to be context dependent as
AVT administration failed to induce male sexual behaviors
when a dominant male was present (Semsar and Godwin,
2004). Interestingly, 11KT administration also promoted
male displays in subordinate ovariectomized females but did
not change AVT hypothalamic levels (Semsar and Godwin,
2004, 2003), suggesting that gonadal androgens modulate
male reproductive displays via a pathway independent from
AVT. The positive effects of AVT on male sexual displays are
confirmed by studies in other species (e.g., A. leptorhynchus,
Bastian et al., 2001; white perch, M. americana, Salek et al.,
2001). For example, in the gymnotiform Brachyhypopomus gau-
derio, AVT stimulated the production of electric signals used for
courtship by direct action on the hindbrain pacemaker cells
(Perrone et al., 2010). However, in the cichlid A. burtoni, block-
ing the V1a receptor inhibited aggression and stimulated court-
ship displays in subordinate males that ascended to become
dominant, suggesting that AVT impairs male reproductive
displays in this context, although the same manipulation did
not produce any effects in stable dominant or subordinate
fish (Huffman et al., 2015).
AVT has also been shown to promote female displays. In
a sex-role reversed population of the peacock blenny S. pavo,
AVT mRNA levels were higher in the courting morphs, i.e., in
females and in female-mimicking parasitic males, than in
noncourting nesting males (Grober et al., 2002). Accordingly,
AVT administration promoted sexual displays in females and
in parasitic males but not in nesting males (Carneiro et al.,
2003).

Taken together, these results suggest that AVT promotes
sexual behavior in fishes and, following the model proposed
by Godwin and Thompson (2012), this is probably achieved
via modulation of central brain areas, including nodes of the
SDMN, by AVT projections from the POA.
2.01.3.3 Parental Care
The wide diversity of modes of reproduction in fishes extends
to the patterns of care to eggs or juveniles, and examples of
no care, paternal, maternal, biparental, or even alloparental
care can be found (Breder and Rosen, 1966). This offers an
excellent opportunity to study the proximal mechanisms of
parental care in vertebrates but not much is known on the brain
substrates of parental care in fish. In mammals, the POA, thal-
amus, BNST, and the LS have been associated with the expres-
sion of paternal care (for a review, see Dulac et al., 2014). In
fish, the putative homologue for the LS is the Vv, but an equiv-
alent area to the BNST is still ambiguous. Also, Dc, the central
part of the dorsal telencephalon, has been suggested to relate
with paternal care in bluegill sunfish (Demski and Knigge,
1971). In the only study so far investigating the neural
substrates of parental care in fish using IEG, it was shown in
the cichlid A. nigrofasciata that parental males have a higher
activation of the Vv, but not of the POA or Dc, as compared
with nonparental males (O’Connell et al., 2012).
Paternal care is more common than maternal care (Breder
and Rosen, 1966), and thus the endocrine regulation of
parental behavior has been mainly investigated in males. A
conceptual framework for the relationship between androgens
and parental care was proposed by Wingfield (1990), following
the observation in birds that male androgen levels decrease
during the parenting phase as compared with the mating phase,
even in animals that continue to reproduce after the initiation
of parental care (Wingfield, 1984). This hypothesis postulates
that androgen levels should be lower during the paternal phase,
when social instability is usually reduced, as compared to the
mating phase, when males need to compete for territories
and sexual partners. In fish, androgen levels drop during the
parental phase thus supporting this prediction of the challenge
hypothesis (Oliveira et al., 2002). However, there are many
exceptions to this pattern (e.g., Rodgers et al., 2006; Ros
et al., 2003), and exogenous administration of androgens to
parenting males failed to have the expected suppressive effect
in parental behavior in some species (T propionate implants
in L. macrochirus, Rodgers et al., 2012; e.g., 11KT implants in
Parablennius sanguinolentus parvircornis, Ros et al., 2004). In
the context of the challenge hypothesis, the regulation of
paternal behavior by androgens is interpreted as a trade-off
between parental behavior and androgen-induced aggression.
The decrease in androgen levels postulated to occur during
the parental phase would decrease the frequency of aggressive
behaviors, releasing more energy and time to parental duties.
Androgens may thus be better seen as secondary modulators
of parental behaviors, more related with aggressive displays,
while other hormones are expected to have a more direct regu-
latory action on specific parental care displays.
Prolactin (PRL) has been suggested as a prime candidate for
the endocrine modulation of both paternal and maternal
behavior across vertebrates (e.g., Bachelot and Binart, 2007;
Neuroendocrinology of Social Behavior in Teleost Fish 11
Schradin and Anzenberger, 1999), including fish (reviewed in
Whittington and Wilson, 2013). The first study on the effects
of this hormone in fish paternal behavior was conducted in
the ocellated wrasse Symphodus ocellatus where PRL administra-
tion was shown to promote egg fanning behavior in nesting
males (Fiedler, 1962). These results were corroborated by
studies in other species, both in males (e.g., blue discus, Sym-
physodon aequifasciata, Blüm and Fiedler, 1965; L. macrochirus,
Kindler et al., 1991; G. aculeatus, Páll et al., 2004) and females
(e.g., S. aequifasciata, Blüm and Fiedler, 1965). However, there
are also studies where the expected positive effect of PRL on
parental behavior was not observed. In the cooperatively
breeding cichlid N. pulcher, PRL mRNA levels in the pituitary
were not elevated in breeding females as compared with
nonbreeding females, and PRL administration to both males
and females did not promote parental behavior (Bender
et al., 2008). In the Nile tilapia Oreochromis niloticus, pituitary
and plasma levels of the two PRL isoforms described in fish
also did not differ between female mouthbrooding eggs and
nonincubating females (Tacon et al., 2000). Nevertheless,
PRL II showed high variation during the mouthbrooding
period, and the authors did not exclude a possible role of
PRL in the regulation of maternal behavior. In a comparative
study between a monogamous and a polygynous species of
cichlids of the genus Herichthys, gene expression levels of PRL
and of a PRL receptor in brain macroareas were not associated
with paternal care (Oldfield et al., 2013).
Interestingly, sex steroids have been found to interact with
PRL, raising the possibility of modulation of parental behavior
by sex steroids occurring indirectly via effects in PRL. Estrogens
have been found to promote the secretion of PRL from pitui-
tary glands (e.g., O. mossambicus Barry and Grau, 1986; rainbow
trout O. mykiss, Williams and Wigham, 1994). Onuma et al.
(2005) report variable effects of E2, T, and 11KT in PRL
mRNA expression levels in pituitary cell cultures of masu
salmon depending on gender and time in the reproductive
season. During the prespawning stage, E2, T, and 11KT
increased the expression of PRL mRNA while opposite effects
were detected during the spawning stage, suggesting that sex
steroids may regulate both positively and negatively PRL secre-
tion. This study also highlights the importance of integrating
variation in tissue sensitivity to modulatory agents to under-
stand their mode of action.
Similar to what has been described for oxytocin in
mammals, IT has also been found to regulate paternal care in
fish. In the monogamous cichlid, A. nigrofasciata, c-fos expres-
sion was higher in POA parvocellular IT neurons in fathers
than in nonfathers, and the administration of an IT receptor
antagonist blocked paternal care (O’Connell et al., 2012).
Interestingly, IT fibers and IT receptors are present in the Vv,
a brain area observed to be more activated in fathers than in
nonfathers, raising the possibility of stimulatory effects of IT
on parental behavior occurring via modulation of neuronal
signals in this brain region (O’Connell et al., 2012).
2.01.3.4 Prosocial Behavior
The idea that social bonding, or affiliative behavior, can be regu-
lated by evolutionary conserved brain and neurochemical
systems across vertebrates is relatively new. In fact, since the
12 Neuroendocrinology of Social Behavior in Teleost Fish
ground-breaking research on the role of AVP and oxytocin in
the regulation of social behavior in rodents (reviewed in Young
et al., 2011), evidence has been accumulating showing that
these nonapeptides, and their nonmammalian homologues,
play an important role in social bonding, affiliative behavior,
and attention to social stimuli also in other taxa, including
fish (reviewed in Godwin and Thompson, 2012). In goldfish,
intracerebroventricular administration of AVT and IT has oppo-
site effects in social behavior, with AVT-reducing approach
behavior toward a conspecific and IT increasing the duration
of this behavior (Thompson and Walton, 2004). The inhibitory
effects of AVT seemed to be mediated by AVT cell projections
from the POA to the hindbrain (Thompson and Walton,
2009). This was confirmed by showing that the behavioral
effects of AVT infusions into the fourth ventricle were more
potent than the effects of infusions into the third ventricle
(Thompson et al., 2008). Accordingly, it was demonstrated
that seasonal changes in behavioral responsiveness to AVT are
associated with changes in hindbrain sensitivity to AVT, as
measured by the expression of the V1a AVT receptor in this
brain region (Walton et al., 2010). In the cichlid N. pulcher,
peripheral administration of IT seemed to increase responsive-
ness to socially relevant information during aggressive contests
as IT-treated fish fought in accordance with the size of the
opponent while control animals fought according to their
intrinsic aggressive levels (Reddon et al., 2012). In the
monogamous cichlid A. nigrofasciata, the peripheral
administration of an AVP/IT receptor antagonist to males
reduced affiliative behavior although it did not prevent pair
bonding nor did it disrupt pair bonding after pairs had been
established (Oldfield and Hofmann, 2011). In the cleaner
wrasse, Labroides dimidiatus, AVT inhibited interspecific
cleaning activities while it did not affect other social
conspecific behaviors (Soares et al., 2012). In the same study,
IT administration failed to affect social behavior. In contrast
with previous studies, in N. pulcher, IT administered
intraperitoneally reduced affiliative behavior and, in a second
study, brain IT levels were found to be negatively correlated
with these behaviors (Reddon et al., 2015).
(a) Ancestral state (b)
No communicaƟon
Hormone released into water Hormone release unchanged
Chemical Cue
No mechanism
h for
f detecƟon/
d / response. EvoluƟon of detecƟon and response.
Figure 2 Stages in the evolution of chemical signaling in fish: (a) ancestral state, in which fish release a hormone that is not detected by conspe-
cifics; (b) spying state, where conspecifics are able to detect and respond adaptively to the hormone; and (c) communication state, characterized by
a response of the receiver which selects for signal specialization.
Information on the brain areas targeted by AVT or IT to
regulate social bonding is still very scarce but Godwin and
Thompson (2012) suggest forebrain nodes of the SDMN to
be likely candidates for AVT modulation; indeed motor output
pathways descending from these regions show dense AVT
innervation and project into multiple central targets. New
studies selectively manipulating subpopulations of AVT cells
will be able to test this hypothesis.
2.01.4 Hormones as Social Semiochemicals
When hormones are released into the environment, either
actively or passively, they convey information about the sender
that becomes potentially available to other conspecifics, and
thus can be seen as social semiochemicals (i.e., chemical cues
of conspecific origin). If released hormones have evolved to
convey information about the sender and trigger a specific
and adaptive response in the receivers, then they can be viewed
as pheromones (Sorensen, 2014; Wyatt, 2010). Thus, the
evolution of hormonal pheromones has been proposed to
follow a cue-signal continuum, where different evolutionary
states can be recognized (Sorensen and Scott, 1994; Sorensen
and Stacey, 1999; Wisenden, 2014; Figure 2). In the ancestral
state, senders release hormones into the environment, but
potential receivers are unable to detect them. In a second state,
receivers evolved the capacity to detect and respond adaptively
to the hormone; hence they are now spying on senders based
on the hormone that acts as a chemical cue for the internal
state of senders. Finally, a third evolutionary state may evolve
if the selective pressure imposed by spying on senders leads
to the evolution of specialized production and/or release of
the hormonal pheromones, which becomes a signal (i.e.,
true pheromone) according to animal communication termi-
nology (Wisenden, 2014).
An apparently easy way to discriminate between spying and
true signaling would be to look for the occurrence of special-
ized structures in the production and/or release of phero-
mones. Many such structures have been described among
Spying state (c) CommunicaƟon state
Change in receiver Change in sender
Change in hormone and/or its
release
Signal
Response
Response off the
h receiver selects
l ffor
signal specializaƟon
 Neuroendocrinology of Social Behavior in Teleost Fish 13

teleost fish, such as the seminal vesicles in catfish (Clarias garie-
pinus) that release female attractants (Resink et al., 1989), the
hypertrophied mesorchial glands in the testes of gobies that
secrete steroids that attract females (e.g., Colombo et al.,
1980; Murphy et al., 2001), or anal glands in blennies, which
consist of a transformation of the first rays of the anal fin
into a sex pheromone-producing gland (Serrano et al.,
2008a,b). Interestingly, in the case of both gobies and blennies,
i.e., in species in which male alternative reproductive tactics
occur and both territorial and sneaker males are present, only
territorial males develop the glands that produce female attrac-
tants (blennies: Gonçalves et al., 1996; gobies: Locatello et al.,
2002), suggesting a secondary loss of the pheromone-
producing tissue in sneakers. However, the absence of such
specialized structures cannot by itself rule out the ability to
release/store pheromones, and thus apparent cases of chemical
spying in fish may just reflect our failure to detect these more
subtle specializations. A good example of such scenario is the
goldfish (C. auratus), where, despite lacking any obvious
specialization for production and release of pheromones,
females release sequentially two hormonal pheromones: (1)
a preovulatory pheromone, consisting of progestins (17,20-b-
dihydroxy-4-pregen-3-one and its sulfated form) and androste-
nedione, that has a primer effect on sperm production in
males; and (2) a postovulatory pheromone, consisting of pros-
taglandins (prostaglandin F2-a and 15-keto-PGF2-a), that trig-
gers male courtship and makes ovulated females attractive to
the males (reviewed in Stacey and Sorensen, 2002). Given
that these female pheromones are the result of passive release
into the water of sex hormones and their metabolites involved
in female ovulation (progestins and androstenedione) and in
the regulation of female reproductive behavior (prostaglan-
dins), and there are no specialization for the production and/
or their release in the scope of chemical communication, this
system has initially been considered a classic example of males
spying on reproductive state of females in order to increase
their reproductive success (Stacey and Sorensen, 2002;
Wisenden and Stacey, 2005). However, subsequently it has
been found that these hormones are mainly released through
the urine and that female goldfish increase the frequency of
pulses of urine when in the presence of a male, in particular
if in the presence of oviposition substrate (Appelt and
Sorensen, 2007), indicating a specialization in the sender for
the release of the signal, compatible with a true signaling
scenario. This form of control of signal release is also present
in other species where hormonal pheromones are released
through the urine and males adjust the urination rate in
response to the presence of receptive/preovulatory females
(e.g., Mozambique tilapia, O. mossambicus, Almeida et al.,
2005; Burton’s mouthbrooder, A. burtoni, Maruska and Fernald,
2012; swordtail, Xiphophorus birchmanni, Rosenthal et al.,
2011). Thus, the role of hormones as pheromones may be
more common than initially thought.
Most of the examples of hormonal pheromones provided
above are related to reproduction and to their effect on the
behavior and physiology of the opposite sex. However, evidence
has accumulated indicating a role for chemical cues in other
aspects of fish social behavior such as intrasexual aggression,
parental care, and affiliative behaviors (Keller-Costa et al.,
2015; e.g., Sorensen and Baker, 2014). Unfortunately, for most
of these other cases of chemical communication, the chemical
identity of the cue/signal is not known, and thus hormones
cannot be directly implicated. One particular function that has
been the focus of recent research is the role of chemical commu-
nication in the regulation of male–male aggression in cichlids
(Keller-Costa et al., 2015). In the Mozambique tilapia, males
also release urine during agonistic interactions in pulses of short
duration and those that become subordinate stop releasing
urine (Figure 3; Barata et al., 2007). If urination is surgically
prevented, agonistic interactions escalate and more overt

Figure 3 Examples (1–3) of behavior of two territorial tilapia males (a) and (b) interacting for 45 min (submissive: white; not aggressive: light gray;
aggressive displays: dark gray; highly aggressive: black) and their corresponding release of urine pulses (urination). In (1), male (a) started aggres-
sive behavior and urination (time point around 25 min), subsequently male (b) initiated aggressive displays and then agonistic interaction escalated
to high symmetrical aggression. In (2), both males increased their urination frequency and aggressivity almost at the same time (within 10–15) and
maintained agonistic behavior until the end of experiment, although urine pulses decreased significantly. In (3), after the release of several urine pul-
ses from both males and a short period of symmetrical high aggression, male (a) became submissive whereas male (b) continued with agonistic
displays; both males stopped urine release at this time point (around 18 min). Adapted from Barata, E.N., Hubbard, P.C., Almeida, O.G., Miranda, A.,
Canário, A.V.M., 2007. Male urine signals social rank in the Mozambique tilapia (Oreochromis mossambicus). BMC Biol. 5, 1–11.
14 Neuroendocrinology of Social Behavior in Teleost Fish
aggressive behaviors are expressed (Keller-Costa et al., 2012).
Moreover, in stable dominance hierarchies the olfactory
potency of the urine is correlated with the male’s social rank
(Barata et al., 2007), and urine of dominant and subordinate
males triggers different patterns of gene expression in olfactory
brain regions of male receivers (Simões et al., 2015). Together
these results strongly suggest the presence of a chemical signal
in the urine used in social status assessment in this species.
Interestingly, dominant males also store large volumes of urine
(Barata et al., 2007), having hypertrophied urinary bladders
(Keller-Costa et al., 2012), which indicates a specialization for
urine release supportive of the evolution of a true dominance
pheromone rather than just a dominance cue. The chemical
identity of this putative dominance pheromone has not
been established yet, but it is known to have multiple compo-
nents, present both in polar and nonpolar urine fractions
(Keller-Costa et al., 2016) and to be distinct from the sex
pheromone that has primer physiological effects on females
(Keller-Costa et al., 2014). Similar evidence for the occurrence
of putative dominance pheromones has also been collected for
two other cichlid species (Burton’s mouthbrooder, Crapon de
Caprona, 1980; Maruska and Fernald, 2012; Nile tilapia,
Giaquinto and Volpato, 1997; Gonçalves-de-Freitas et al.,
2008), suggesting that pheromones may be also a widespread
phenomena in this teleost family.
2.01.5 Conclusions
To conclude, it is now clear that hormones exert powerful
modulatory effects on social behavior by acting on a core of
forebrain and midbrain areas that underlie the expression of
these behaviors in fishes. However, the exact mechanism
through which hormones change the functional connectivity
of the SDMN to affect behavior remains poorly understood,
and this is a promising area for research. Studies manipulating
hormone levels and investigating the effect in the neuroge-
nomic states and neuronal output of nodes of the SDMN
will help to elucidate how hormones modulate the expression
of social behaviors. Also, different hormonal systems are
known to interact with each other, and studies addressing the
cross-talk between endocrine systems are valuable. Finally,
hormones may also act on social behavior by acting as phero-
mones, and their role in the regulation of social interactions
has started to be unveiled.
Acknowledgments
During the writing of this review, RFO and ASF were supported by
Fundação para a Ciência e a Tecnologia (grants EXCL/BIA-ANM/0549/
2012 and SFRH/BD/102892/2014, respectively) and DG by Fundo para
o Desenvolvimento das Ciências e da Tecnologia de Macau (grant 012/
2012/A1).
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 2.02 Social Regulation of Sex: How the Brain Controls Reproductive Circuits
Russell D Fernald, Stanford University, Stanford, CA, USA
Ó 2017 Elsevier Inc. All rights reserved.
2.02.1 Social Dominance Influences Reproductive Physiology in Many Vertebrate Species
2.02.1.1 Social System of Astatotilapia burtoni: Reproductive Consequences of Status
2.02.2 How Does Social Status Regulate the HPG Axis?
2.02.3 How Is GnRH1 Released in a Pulsatile Fashion?
2.02.4 Can Males Be Deceptive about Their Reproductive State?
2.02.5 Genomic Consequences of Female Mate Choice
2.02.6 What Social Information Influences Changes in Status?
2.02.6.1 Attention Hierarchies in Male Astatotilapia burtoni
2.02.6.2 Transitive Inference in Astatotilapia burtoni
2.02.7 Conclusions
Acknowledgments
References
2.02.1 Social Dominance Influences Reproductive
Physiology in Many Vertebrate Species
Animals experience of life on the planet varies according to
their sensory systems that are differentially responsive to
unique perceptual worlds. Correspondingly, their responses
depend on their abilities to act in that private world. Von
Uexküll (1921) described the perceptual world as the ‘umwelt’
of a species, and since then, scientists have discovered new
dimensions of the umwelts for many species. While Von Uex-
küll conceptualized his idea of the umwelt idea in relation to
physical stimuli, Lorenz extended this concept, recognizing
that animals also have a social ‘umwelt’ (Lorenz, 1937)
through which signals from other individuals influence
behavior. Lorenz’ influential article, ‘Companions as factors
in the bird’s environment,’ (1937) showed that behavioral
scientists should include other individuals in considering the
umwelt and, importantly, the overall social context of the
behavior. Such interactions are particularly important for
sexual behavior in which multimodal signals orchestrate repro-
ductive activities. Since behavior is the ultimate arbiter of
animal survival, response of animals during their interactions
with others and with their environment shapes the phenotype.
Behavior, in turn, depends on intricate physiological, cellular,
and molecular adaptations forged during evolution and modi-
fied during development. Ultimate questions about the evolu-
tion and control of behavior and especially interactions that
sculpt behavior require understanding the causal mechanisms
responsible as animals interact with one another, preferably
in a natural setting. Yet, the vast majority of experiments are
performed on isolated individuals, often in domesticated
species. Clearly little can be learned about how evolution has
shaped social behavior by analyzing individual animals since
social interactions cannot occur.
Fish species have emerged as important models for studies
of how hormones influence social behavior and its neural
underpinnings because of the many examples of sophisticated
behavioral interactions regulating sex. For example, it has been
shown that fish might exploit Machiavellian strategies (e.g.,
Hormones, Brain, and Behavior, 3rd edition, Volume 2 http://dx.doi.org/10.1016/B978-0-12-803592-4.00021-3
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Bshary and Würth, 2001), cooperate (e.g., Pitcher et al.,
1986), learn in a social context (Laland et al., 2003), eavesdrop
on interactions among conspecifics to gain social insights
(McGregor, 1993), show transitive inference (Grosenick et al.,
2007), and generally display a wide range of social activities
comparable in some instances with those of primates (e.g.,
Bshary et al., 2002). In addition to their social sophistication,
fish species have proven important and quite useful for under-
standing mechanisms controlling social behavior. With several
fish genomes now sequenced, it has become much easier to
trace genetic and neurobiological mechanisms responsible for
sophisticated social interactions in fishes.
Why might fish social behavior provide general insights
about hormone and brain mechanisms of social interactions?
Stable social systems in any animal species require the individ-
uals to behave predictably. However, what an individual does
at any moment in time depends on its status relative to other
individuals, its reproductive state, and its recent behavioral
interactions. In addition, environmental factors (e.g., preda-
tors, prey, or resource competitors) also need to be incorpo-
rated into any plan for behavioral action. So, to be successful
in a social group, individuals must be aware of the immediate
behavior of others and use that information to regulate their
own activity. But what exactly does an individual need to
know and how do they acquire the knowledge that lets them
act appropriately? It is possible that the apparently subtle social
interactions can be explained and understood in terms of
contingencies. That is, a set of ‘if-then’ rules with associated
probabilities might suffice in many situations to explain the
behavior of animals in social groups. Since it has been argued
that species in the fish taxon have demonstrated many but not
all the social skills that arguably led to the evolution of
complex brain structures in primates (Bshary et al., 2002),
how could we proceed to exploit fish social skills in under-
standing neural mechanisms? Here I will describe results
from experiments using the relationship between social domi-
nance, hormones, and reproduction to understand how social
signals regulate the hypothalamic–pituitary–gonadal (HPG)
axis in Astatotilapia burtoni. I also explore the range of social
19
20 Social Regulation of Sex: How the Brain Controls Reproductive Circuits

requirements for A. burtoni to be reproductively successful and
describe evidence about how social behavior sculpts the brain
in ways that depend on the developmental stage, social circum-
stance, and environmental context.
2.02.1.1 Social System of Astatotilapia burtoni: Reproductive
Consequences of Status
In their natural habitat, the shallow shore pools and river estu-
aries of Lake Tanganyika (Fernald and Hirata, 1977a; Coulter,
1991), A. burtoni live in a leklike social system in which domi-
nant males vigorously defend contiguous territories (Fernald
and Hirata, 1977b). Social communication among these fish
appears to depend primarily on visual signals (Fernald, 1984;
see below). Astatotilapia burtoni males live as one of two revers-
ible, socially controlled phenotypes: reproductively competent
dominant (D) males and reproductively incompetent
nondominant (ND) males (see Figure 1). D males are brightly
colored, aggressively defend territories, and actively court
females (Fernald and Hirata, 1977b). In striking contrast, ND
males have a dull coloration, mimic female behavior, and
school with females and other ND males except when fleeing
from an attacking D male.
These obvious external differences are reflected in major
physiological and neural adaptations in response to differences
in social status. As animals transition from one phenotype to
the other, some changes including expression of the black bar
through the eye, brightening of the body color, and switching
behavioral repertoires are expressed in minutes while concom-
itant physiological and neural changes happen over days.
ND males attend closely to the unfolding social scene,
assessing when they might be able to gain a territory by defeat-
ing a resident male. When this happens, there is typically
a dramatic fight during which males engage in mouth-to-mouth
biting, hitting each other with their bodies, and nipping at each
other’s fins. If the ND male successfully defeats the resident, he
rapidly turns on his bright body colors (Fernald and Hirata,
1977b; Burmeister et al., 2005) and will quickly begin perform-
ing many of the 17 distinct behaviors characteristic of dominant
males upon his social ascent. Over a few days, the reproductive
system of the ascending male is remodeled rendering the male
reproductively competent, producing changes that are evident
at several levels along the HPG axis (Maruska and Fernald,
2014). In A. burtoni, as in all vertebrates, reproduction is
controlled by GnRH1-containing neurons in the hypothalamus
that deliver the eponymously named GnRH decapeptide to the
pituitary. When a male ascends (ND/D), delivery of this mole-
cule sets in motion a cascade of actions ultimately resulting in
reproductive competence and release of sex steroids from the
gonads. The GnRH neurons increase in volume by eightfold
(Davis and Fernald, 1990), extend their dendrites (Fernald,
2012), and rapidly increase production of GnRH mRNA
(Burmeister et al., 2005) and GnRH peptide (White et al.,
2002). However, when a D male is moved into a social system
with larger D males (>5% longer), it abruptly loses its color
(<1 min) and joins other ND males and females in a school.
Its GnRH-containing neurons in the preoptic area (POA)
shrink to one-eighth their volume and produce less GnRH
mRNA and peptide, causing hypogonadism and loss of
reproductive competence (
2 weeks) (Davis and Fernald,
1990; Francis et al., 1993). Similarly, androgen, estrogen, and
GnRH receptor mRNA expression levels depend on social
status (Au et al., 2006; Burmeister et al., 2007; Harbott et al.,
2007) as do electrical properties of the GnRH neurons
themselves (Greenwood and Fernald, 2004).
Astatotilapia burtoni D males perform 17 distinct behavioral
patterns during social interactions (Fernald, 1977). D males dig
a pit in their territory, exchange threat displays with neigh-
boring territorial males, chase ND animals from their territo-
ries, and solicit and court females. D males display bright
coloration patterns, particularly during courtship. A D male
will lead a female toward his territory, typically using large

Figure 1 Sketch of an observation site along the edge of a shore pool on the north end of Lake Tanganyika, near Bujumbura, Burundi, Africa. Solid
dots represent grid stakes spaced at
50 cm intervals that label grids (1–5; A–D) for identification. Dark circles represent spawning pit locations of
dominant males. Lighter-colored outlines circumscribe the approximate territories of individuals. Male territories are arrayed over the food source of
detritus. Nondominant males and females school together near the territorial area. Based on Fernald, R.D., Hirata, N.R., 1977a. Field study of Hap-
lochromis burtoni: habitats and co habitants. Environ. Biol. Fish. 2, 299–308.
 Social Regulation of Sex: How the Brain Controls Reproductive Circuits
movements of his tail and court by quivering his opened,
brightly colored anal fin in front of the female. When a D
male manages to lure a female into his territory, she will nor-
mally eat by sifting the substrate in the territory. ND males
will mimic female behavior sufficiently well so that the D
male allows NDs to enter the territories and feed before their
deception is discovered. This ND male behavior occurs because
only sites defended as territories contain food so that ND males
need to enter to eat. ND female impersonators are quickly
chased off, but for females it is different. The D male will
lead her to his pit and continue courtship movements. D males
swim vigorously in front of the female, quivering their entire
body with spread anal fins. If appropriately stimulated, the
female will lay her eggs in the pit and collect them in her mouth
immediately. After she has deposited several eggs, the male will
swim in front of her displaying the egglike spots on his anal fin
(ocelli). D males display this fin because the spots may seem to
the female like eggs not yet collected (Wickler, 1962). Thus,
while attempting to ‘collect’ the egg-spots, the female ingests
milt ejected near them by the male and ensures fertilization.
The spawning male may repeatedly interrupt his courtship
and mating to chase off intruders into his territory. After several
bouts of egg laying and fertilization, the female departs with
fertilized eggs that she broods in her mouth for
2 weeks
(Fernald, 1977).
Even this abbreviated description of the natural behavior of
A. burtoni shows the important role visual signals play in medi-
ating social behavior. As is typical for this kind of rapid social
interactions, each behavioral act influences the next, both in
the individual and in other animals involved in the encounter.
What do animals attend to during aggressive social interac-
tions? Using ethological methods, early workers identified
several fixed action patterns and key stimuli that mediate
social signaling in A. burtoni. Specifically, Leong (1969)
analyzed the role of the black eyebar by testing how T males
responded to A. burtoni dummies painted with various config-
urations of the distinctive body patterns. When the eyebar was
presented alone, T males increased their readiness to attack
targets while presentation of the orange-red patch of humeral
scales alone decreased attack readiness. Subsequent experi-
ments tested the importance of the orientation of the eyebar
relative to the body and other visual stimuli (Heiligenberg
and Kramer, 1972; Heiligenberg et al., 1972). All the work sup-
ported the hypothesis that the black eyebar and the red
humeral patch influence the aggressiveness of T males in
opposite directions.
2.02.2 How Does Social Status Regulate the HPG
Axis?
Being reared with broodmates suppresses early social and phys-
ical development, but in A. burtoni even more effective social
regulation occurs when older, larger animals are housed with
younger animals. Davis and Fernald (1990) raised animals
from hatching in the presence of adult males and showed these
fish have suppressed gonadal maturation relative to fish reared
without the presence of adults. Suppressed animals not only
had hypogonadal testes but also GnRH1-containing neurons
in the POA c.1/8 the volume of those in dominant males.
21
GnRH1 neurons control the brain–pituitary–gonadal axis
regulating reproduction in all vertebrates via a projection to
the pituitary (Bushnik and Fernald, 1995). GnRH1 is the
signaling peptide sent from the brain to the pituitary to trigger
release of gonadotropins and ultimately regulate testes growth.
Thus social control of maturation in A. burtoni is achieved by
changing the GnRH-containing cells in the brain.
Social status can regulate the physiology of the reproduc-
tive state, even in adult A. burtoni as shown by converting
males from D > ND or ND > D by moving them to new
communities. Specifically, when D males were moved to
communities containing larger D males, they rapidly became
ND (e.g., D > ND), and similarly when ND males were moved
to communities with smaller conspecifics, they became D (e.g.,
ND > D). Following 4 weeks in the altered social setting,
GnRH cell size was measured and showed that changing the
social status alone was sufficient to change GnRH neuron
size in the brain (Figure 2). As expected, the gonadosomatic
index (GSI) changed correspondingly (Francis et al., 1993).
Thus reproduction is socially controlled in adults, as well as
in juveniles, via changes induced in the GnRH neurons in
the brain.
Although causing a large change in brain structure by
changing social status is quite remarkable, the timescale of
this initial experiment did not reflect how rapidly behavioral
and neural changes could occur. Indeed, the 4-week interval
tested was substantially longer than any observed changes in
behavior following status switches, which can occur in
minutes. Analyzing socially induced changes in neural struc-
tures on a significantly shorter timescale revealed another
surprise.
Clearly social status sets both soma size of POA GnRH-
containing neurons and through this pathway, GSI, and these
effects are reversible. The relatively large testes and GnRH
neurons characteristic of D males are a consequence of their
social dominance, and when this dominance advantage is
lost, both neurons and testes shrink, although as shown by
White et al. (2002), there is striking asymmetry in the
physiological responses. Thus social information about status
causes the changes in the brain, but how this is achieved is
not known.
It is important to emphasize that the effect of social status
on GnRH1 cell size and GnRH1 mRNA expression is limited
to the GnRH1-containing neurons of the POA. As we have
previously shown, A. burtoni has three distinct genes that
code for three distinct GnRH-like molecules (White et al.,
1994; White and Fernald, 1998) expressed at three distinct
sites in the brain (White et al., 1995). The GnRH forms not
found in the POA are expressed in two other distinct cell
groups, one located in the terminal nerve region (GnRH3),
the other in the mesencephalon (GnRH2; see White et al.,
1995 for details). Neither of these other GnRH-containing
cells showed any changes in size or mRNA production as
a function of social status (Davis and Fernald, 1990; White
et al., 2002). Males and females share the brain–pituitary–
gonadal axis used to control reproduction, but female
A. burtoni have a strikingly different system that regulates
reproduction. GnRH1-containing cells in the POA of females
also change size, but size change depends on reproductive
status alone (White and Fernald, 1993).
22 Social Regulation of Sex: How the Brain Controls Reproductive Circuits

100
90
80
100
70
90
60
80 50
70 40
60 30
%
50 20
40 10
30 100
20
10
350
100 400
150 450
200
So

250
m

300
a
si

350 ND>D
ze

400
(µ

D>ND
m

450
2

D
)

ND

Figure 2 Three-dimensional plot of mean soma diameter of preoptic area (POA) gonadotropin-releasing hormone (GnRH1)-immunoreactive
neurons in males of different social status. There are significant differences between D and D/ND males and between ND and ND/D males.
Percentage of individuals with mean soma size in a given size bin are plotted for each treatment condition. Redrawn from Francis, R.C., Soma, K.,
Fernald, R.D., 1993. Social regulation of the brain-pituitary-gonadal axis. Proc. Natl. Acad. Sci. U.S.A. 90, 7794–7798.

White et al. (2002) also showed that the socially induced
changes in status resulted in significant changes in gene expres-
sion. Measuring changes in mRNA from all three forms of
GnRH found in A. burtoni, they found that only the POA
GnRH1 mRNA was regulated corresponding to a change in
social status. The change in mRNA in the POA form of GnRH1
was evident at 3 days after a change in social status. Such social
regulation demonstrates clearly that key social information is
used to control cellular and molecular processes in the brain.
Given the rapid macroscopic change in GnRH neurons
following a change in status, we devised a novel behavioral
paradigm to test the speed of this process. We placed a D
male in an aquarium with one ND male and three females,
with associated fish colonies on each side. The target D male
was removed before dawn, so when the lights came on, the
ND male was the sole male in the tank with three females.
We videotaped the response and subsequently recorded when
the ND male first exhibited an eyebar and the first D behaviors.
Twenty minutes after the first expression of D behavior,
we sacrificed the animal and subsequently mapped the
expression of an activity-dependent immediate-early gene
(egr-1; Burmeister et al., 2005). We found a rapid genomic
response in the GnRH1 neurons of the POA in response to
the perception of social opportunity 20 min after the NT
male adopted dominant coloration and behaviors. This
suggests that these animals are fully primed to ascend, and
based on their recognition of the new circumstances, they
respond at both the behavioral and molecular levels.
As expected, social control of the reproductive axis via
GnRH1 also influences important endocrine factors, including
androgen released from the gonads. Castrated A. burtoni T
males have hypertrophied GnRH neurons (Francis et al.,
1992a; Soma et al., 1996) showing that androgen has a feed-
back effect on GnRH cell size. The important discovery is that
the set point for this feedback loop is the social status since
D males have larger GnRH neurons despite having higher
androgen levels (Soma et al., 1996). D males that are castrated
are able to maintain their rank despite having lowered
androgen levels (Francis et al., 1992b). Possibly, prior domi-
nance experienced on the part of the D male and the size differ-
ence among animals contribute to this result.
Clearly, social status regulates the production and release of
GnRH1 into the pituitary. Another potential site for regulation
is the GnRH1 receptor in the pituitary. Our laboratory has
shown that A. burtoni has genes that encode two distinct
GnRH receptors (Robison et al., 2001; Flanagan et al., 2007).
Using real-time PCR, we have been able to show that the
mRNA of one of these receptor types is upregulated rapidly
and dramatically in the pituitary of D males as compared
with ND males (Au et al., 2006). It remains to be discovered
whether this receptor regulation results solely from a change
in social status or if other factors are also involved. In addition
to its primary role, analysis of the distribution of GnRH recep-
tors (Chen and Fernald, 2006) revealed that this peptide must
play a modulatory role in the animal as well as regulating
reproduction.
 Social Regulation of Sex: How the Brain Controls Reproductive Circuits 23

While the changes in GnRH neuron size and concomitant
changes in GnRH production and in hormone receptors are
part and parcel of a response to new reproductive opportunity,
other brain changes caused by social status are more subtle. As
is typical of many social hierarchies, D males have high levels
of testosterone and low levels of the stress hormone cortisol,
and these levels are reversed in ND males (Fox et al., 1997).
Among other changes caused by social subordination, the stress
response inhibits the reproductive axis in many species and
results in chronic elevation of stress hormones, which, if sus-
tained, is known to be detrimental (Webster et al., 2008;
Kaplan and Manuck, 2004; Kaplan, 2008). Thus, it is a puzzle
how socially suppressed ND A. burtoni cope with sustained
levels of elevated cortisol levels. One possible mechanism for
modulating the responsiveness of nondominant individuals
to stress would be to change the sensitivity of the cortisol recep-
tors that mediate its potentially damaging effects on the body
(Avitsur et al., 2001) and brain (Sapolsky, 1996).
We first showed that A. burtoni has three receptor genes for
cortisol: two glucocorticoid receptors (GR1 and GR2) and
a mineralocorticoid receptor (MR) (Greenwood et al., 2003).
Of these genes, GR1 has two splice variants (GR1a and
GR1b), which differ by a nine-amino acid insertion in the
DNA-binding domain in GR1b that greatly reduces binding
to cortisol, and has been shown to act as a dominant
negative inhibitor of transcription in zebrafish (Schaaf et al.,
2008). We have recently shown that when A. burtoni males
become ND, they replace the high sensitive receptor with this
less sensitive receptor in the hypothalamic/POA, site of the
GnRH neurons (Korzan et al., 2014). This modulation of
cortisol receptor subtype expression could mitigate the
consequences of socially induced increases in cortisol levels
in ND males. Just how external social information about the
change in social status is transduced into this molecular
change in the cortisol receptor remains a mystery.
2.02.3 How Is GnRH1 Released in a Pulsatile
Fashion?
The development and function of the reproductive system in
vertebrates ultimately depends on the timing and levels of
signaling by gonadal sex steroids (Phoenix et al., 1959). As
described above, production of these steroids is controlled by
neurons expressing GnRH1, which comprise the final output
of the brain to the HPG axis. Over 30 years ago, Belchetz
et al. (1978) showed that action of GnRH1 depends on coordi-
nated, pulsatile GnRH1 release, not simply elevated levels of
the decapeptide, requiring some kind of synchronization in
the output of these neurons. Such episodic activation of the
pituitary gonadotropes has been observed in multiple verte-
brate taxa, including mammals and fish (9–12); however,
specific mechanisms that underlie this required coordinated
activity of GnRH1 neurons are unknown in any system.
Synchrony could in principle derive from coincident input
from a ‘pacemaker’ neural population, from direct coupling
of GnRH1 neurons, or from some combination of
mechanisms.
Recording calcium signals from brain slices taken from
dominant and nondominant A. burtoni males, we found
a striking difference in the relationship among neuronal firing
patterns inferred from records of calcium signaling (Figure 3).
Specifically, cross-correlograms of the time of occurrence of
calcium signals recorded from nondominant males show no
correlation among GnRH1 neurons; that is, they are no
different from random firing. In contrast, in dominant males,

Figure 3 Cross-correlograms of calcium signals of gonadotropin-releasing hormone (GnRH1) neurons for nondominant (a) and dominant
(b) animals recorded from in vitro slices taken through the preoptic area of Astatotilapia burtoni males. Recordings from the nondominant males
(a) show no significant correlation in their expression of calcium signals representing neuronal firing, while those from the dominant males (b) are
highly correlated. Red bar indicates the expected value calculated from the data generated by randomly reordering the recordings. Kagawa,
unpublished.
24 Social Regulation of Sex: How the Brain Controls Reproductive Circuits

the occurrence of calcium signals is significantly correlated.
These data suggested that firing patterns of GnRH1 neurons
in A. burtoni, as in other vertebrates, are pulsatile in reproduc-
tively competent dominant males and not in reproductively
incompetent nondominant males.
To identify possible origins of this synchrony among
GnRH1 neurons, we used A. burtoni in which the GnRH1
neurons had been labeled transgenically (EGFP; Juntti et al.,
2013). We recorded from pairs of genetically labeled GnRH1
neurons in the brains of reproductively active fish and showed
that these cells are strongly and uniformly interconnected by
electrical synapses that can be reversibly blocked (Ma et al.,
2015, Figure 4). The electrical coupling we measure between
GnRH1 neurons predicts that these neurons express connexins,
the proteins that comprise gap junctions. We searched the
A. burtoni genome for likely connexin genes and focused on
the neuronal connexin gap junction delta 2 (Gjd2; also known
as Cx36 in mammals and Cx35 in fish). We found three homo-
logues most closely related to mammalian Gjd2, which we call
Gjd2a, Gjd2b, and Gjd2c (Ma et al., 2015). One of these,
Gjd2a, appears to be a true ortholog, as the genes to the 50
end of mouse Gjd2 (Aqr, Dph6, Meis2) are also found flanking
A. burtoni Gjd2a. We generated antisense RNA probes against
each to determine whether any was expressed in the POA. We
found that one of these, Gjd2a, was expressed in a population
of large-diameter cells of the parenchyma of the POA, in
a pattern similar to that of GnRH1 expression. To test whether
Gjd2a is expressed in GnRH1 neurons, we colabeled brain
sections and found that 96.3  1.8% of GnRH1þ neurons
also express Gjd2a mRNA (Figure 5; n ¼ 4 fish). These results
suggest that the electrical coupling we observe between
GnRH1 neurons is mediated by the gap junction Gjd2a.
Previously, gap junction-mediated coupling has been sus-
pected to play a role, as synchronous firing can be observed
in neurons mechanically isolated from brain slices, in cultures
of embryonic mouse and primate neurons, and immortalized
mouse GnRH1 neurons express the connexin proteins that
constitute gap junctions (13–15). These data are the first to
confirm that gap junctions connect GnRH1 neurons in any
vertebrate and that they very likely contribute to the coordi-
nated firing of GnRH1 neurons to produce the pulsatile output
needed for reproduction, the most important act of any
organism.
2.02.4 Can Males Be Deceptive about Their
Reproductive State?
Can A. burtoni males be deceptive about their reproductive
state? Deception in animals is an important question in animal
cognition because it is often taken as a hallmark of cognitive

Figure 4 Gonadotropin-releasing hormone (GnRH1) neurons are connected via electrical synapses. (a) Ventral view of the hypothalamus/preoptic
area (POA) of an Astatotilapia burtoni brain showing GnRH1þ cell bodies and fibers labeled with EGFP fluorescence at the top of the figure and termi-
nations of these neurons in the pituitary at the bottom of the figure (top: anterior; bottom: posterior). (b) Arrows indicate schematically patch clamp
electrophysiological recordings from pairs of GnRH1 neurons in the POA. Representative paired whole-cell recordings revealing postsynaptic spikelets
driven by full action potential in presynaptic GnRH1 cells V1 (red trace) in response to suprathreshold DC current injection to one cell (I1-red trace) or
the other cell (I2-blue trace). All paired recordings were similarly symmetric. Scale bar, 5 mV; time, 0.2 s. Modified from Ma, Y.Y., Juntti, S.A., Hu,
C.K., Huguenard, J.R., Fernald, R.D., 2015. Electrical synapses connect a network of gonadotropin releasing hormone neurons in a cichlid fish. Proc.
Natl. Acad. Sci. U.S.A. 112, 3805–3810.
 Social Regulation of Sex: How the Brain Controls Reproductive Circuits 25

(a) (b) (c) (d)

Figure 5 Gonadotropin-releasing hormone (GnRH1) neurons express mRNA for the connexin Gjd2a. Preoptic area (POA) sections were fluo-
rescently colabeled for mRNA encoding GnRH1 (a) and Gjd2a (b); merged channels are shown in (c). The red square in the Nissl-stained section
highlights region displayed (d). Scale bar, 30 mm in a–c. Modified from Ma, Y.Y., Juntti, S.A., Hu, C.K., Huguenard, J.R., Fernald, R.D., 2015.
Electrical synapses connect a network of gonadotropin releasing hormone neurons in a cichlid fish. Proc. Natl. Acad. Sci. U.S.A. 112, 3805–3810.

skill. To ask this question, we used a novel paradigm in which
two differently sized males share a tank, divided in half by
a clear, watertight divider and a black removable divider
(Chen and Fernald, 2011). The general idea is to see how
a small D male responds to a larger D male when he can
only see the larger fish. Experimentally, a half terra-cotta pot,
cut in half lengthwise, was placed so that it allowed the males
on each side to occupy a ‘shared’ shelter, though with the black
divider in place, neither animal knew the other animal was
present (Figure 6). On one side was a male that was about
four times larger than the male on the other side, and each
male had an appropriately sized female in their hemitank.
Both the small and large fish were habituated to the tank
with the opaque barrier in place for 2 days during which time
each behaved like a normal dominant male in its territory:
excavating gravel from the hemipot, courting the female in
their half of the tank, leading her back to the shelter, and per-
forming typical courtship and territorial male behaviors, all
of which were quantified and matched the behavior of normal
D males. On the third day, the opaque barrier was lifted, and
although there was no physical or chemical contact possible,
the larger male made several ‘attacks’ toward the small male
by swimming rapidly toward the smaller male behind the
barrier. The smaller male quickly lost his coloration, including
his eyebar in response upon seeing these ‘attacks.’ The behavior
of the smaller male was typical for an animal losing his territo-
rial status as confirmed by quantifying the behavior. Indeed,
the smaller males essentially abandoned their part of the
shelter, digging a new pit remote from the site of the flowerpot.
Interestingly, this suppression of dominance, based entirely on
visual signals, as reflected by the behavioral quantification
(Chen and Fernald, 2011), also reduced the expression of
androgen hormones, but only for the first 3 days. Seven days
after the black barrier was lifted, the smaller animals recovered
their hormone expression levels and other brain markers of
dominance while maintaining the coloration of an ND male.
Moreover, they could be seen courting their females when
out of view of the dominant male. So the effects of the visual
suppression resulted in changes in the expression of aggressive,
territorial behavioral responses by the smaller male but not in

Figure 6 Front view of the aquarium (45 l) divided in half with a watertight, clear divider (gray midline) and a removable opaque barrier (black
midline). There is a small male fish (left compartment) and the large male fish (about four times larger in the right compartment). The half terra-cotta
pot (red curved line) was cut so that both fish ‘shared’ the same shelter, though they were not aware of each other’s presence. This ‘shared’ shelter
was hemisected by both center dividers. A layer of gravel covered the bottom of the tank.
26 Social Regulation of Sex: How the Brain Controls Reproductive Circuits
sustained physiological changes. This suggests that the
smaller males uncoupled physiological changes in circulating
hormones from their effects on outward appearance, seemingly
presenting a false outward appearance not consonant with
internal changes. This appears to be an example of deceptive
behavior on the part of the male, allowing him to continue
his courtship but not be influenced by the larger male. One
can assume that the smaller animal had learned that the clear
barrier prevented the larger male from actual physical attacks,
and this recognition led the smaller animals to this novel
strategy. The smaller ND male attended carefully to the D
male when carrying out his behavior consistent with the atten-
tion hierarchy described in a later section.
2.02.5 Genomic Consequences of Female Mate
Choice
For females of many species, information about potential
mates can change their behavior dramatically, which makes
sense since female mate choice is extremely important for
the survival of their offspring. We wondered what cognitive
activity might accompany behavioral and physiological
changes in females in response to potential mates. Specifi-
cally, how does the female brain respond to social informa-
tion about potential mates? This experimental question
depends on deciding both how and where to look for a signal
in the brain in response to female mate choice. We chose to
measure brain sites that were marked by changes in gene
expression using immediate-early (IEG) genes, egr-1 and
c-fos, as a female chooses a mate. IEGs are inducible transcrip-
tion factors that comprise a part of the first wave of gene
expression induced in neurons being activated. Extensive
experimental work has shown that a range of natural experi-
ences including sensory stimuli can induce IEG expression,
and consequently, it has been used in mammals and birds
(e.g., Mello et al., 1992; Rusak et al., 1990) to identify social
responses. More recently, we showed that egr-1 is highly
conserved in A. burtoni and that functionally it responds
robustly within 30 min of stimulation (Burmeister and Fer-
nald, 2005) and that c-fos is also a valuable genetic signal indi-
cating brain responses in A. burtoni. As for the brain location,
we hypothesized that the conserved vertebrate social behavior
network (SBN) would be a logical place to look. The SBN was
originally described by Newman (1999) as a collection of
brain nuclei implicated in a variety of social behaviors
including male mating behavior, female sexual behavior,
parental behavior, and aggressive behavior. Anatomical
homologues to this network have subsequently been identi-
fied in birds and fish (Goodson, 2005; Goodson and Bass,
2002) and are likely to exist in other vertebrates as well. It
was unknown, however, whether they might also respond to
social information as well as to behavioral actions. In
previous experiments on female mate choice, we showed
that reproductively ready (i.e., gravid) females prefer to asso-
ciate with dominant, reproductively active males while non-
gravid females prefer nondominant, nonreproductive males
(Clement et al., 2005).
Using a similar paradigm, we placed females in a tank
with one male at each end, behind a clear, watertight
Plexiglas barrier so the female received only visual informa-
tion from the males. We quantified the female’s preference
based on her interactions and proximity to that male over
a 20-min period. Following this, we staged a fight between
the two males in one of the male compartments chosen at
random. Subsequently, the chosen male either won or lost
that fight. Our control condition was for the female to
choose between two males and not to see a subsequent fight.
We hypothesized that different IEG expression patterns
would be generated by the condition of females seeing their
chosen male win or lose a fight. We then mapped brain gene
expression patterns, comparing the mRNA levels of egr-1 and
c-fos in six brain nuclei comprising the SBN (Desjardins et al.,
2010).
Surprisingly, females seeing their selected male win or lose
a fight produced dramatically different brain IEG expression
patterns. Females who saw their preferred male win a fight
had activated brain nuclei associated with reproduction and
reproductive behavior. Specifically the anterior hypothalamus,
ventromedial hypothalamus, POA, and the periaqueductal gray
all had significantly more activation. In striking contrast,
females who saw their preferred male lose a fight had a much
higher expression of the IEGs that were in a part of the brain
associated with anxiety, the lateral septum (Desjardins et al.,
2010). A remarkable aspect of these results is that the females
were responding to visual information alone, since they did
not mate with the male, suggesting that the social information
had caused changes in activation of key brain areas. It is impor-
tant to remember that the IEG expression we measured is surely
only a very small part of the total brain response and hence is
just a glimpse of the genomic response to social information.
However, the differential responses in specific brain areas
show that females are activating their brains based on visual
information and may use this to guide decisions about what
to do.
One additional question is how this information might
inform the female’s choice of a mate. In a separate experiment,
we performed the same protocol but after the female had
chosen and seen the staged fight, she had to choose again. In
this second choice, if she had seen her preferred male lose,
she almost always switched her choice, and if he won, she rarely
switched her choice (Figure 7).
Figure 7 Tabulation of female Astatotilapia burtoni choices after she
saw her preferred male either win or lose a fight (top row) as compared
to her choices after not seeing a fight (bottom row). After seeing her
preferred male win a fight, the female rarely switches her choice
(p ¼ 0.0002; Fishers exact test), but after seeing her preferred male
lose, she nearly always switches her choice (p ¼ 0.0004; Fishers exact
test; Klausner et al., in preparation).
 Social Regulation of Sex: How the Brain Controls Reproductive Circuits 27

2.02.6 What Social Information Influences Changes D male cannot see them, another behavior not possible when
in Status? a D male is present. These behaviors reflect a sophisticated
social calculus in which ND males are doing the most they
2.02.6.1 Attention Hierarchies in Male Astatotilapia burtoni
can to increase their chances of becoming dominant. It is
Animals in social groups monitor the behavior of conspecifics possible that they are also learning about being dominant
and use their observations to guide their ongoing behavior. For through watching, a skill we have also reported elsewhere
example, individuals may attend to male fighting, females (Alcazar et al., 2014).
choosing mates, or many other ongoing social interactions. In an analysis of eye movements in A. burtoni, we observed
In all social hierarchies, subordinate animals attend very that when a D male was in the presence of ND males and he
closely to the behavior of dominant animals in what has oriented his eyes toward ND males, they would typically
been named an ‘attention hierarchy’ (Chance and Larsen, move out of his way (Figure 9; Fernald, 1985). This
1976). Animals watch higher-ranking animals carefully
presumably to calibrate their potential opportunities of social
ascent. Attention hierarchies have been identified in humans,
particularly in groups of children (Boulton and Smith, 1990)
where individuals modulate their behavior depending on their
own status relative to that of others. Within a hierarchy, when
a higher-ranking individual threatens or attacks a lower-
ranking individual, that individual often then subsequently
attacks an individual of still lower in rank (Vaughn and Waters,
1981). In addition to humans, attention hierarchies have been
described in baboons and mandrills (Emory, 1976) as well as
in reptiles (Summers et al., 2005).
To understand attention hierarchies in A. burtoni, we video-
taped groups of individually marked animals (n ¼ 20 per
group; four replicates) and quantified interactions between
dominant and nondominant males (Desjardins et al., 2012).
We found that D and ND males never behaved aggressively
at the same time. Even more interesting was that when the D
male was out of view in a shelter, the ND males that were larger
and attempting to ascend, behaved aggressively, and even
courted females, behaviors that never occurred when the D
male could see the ND male (Figure 8).
In this example, each time the dominant animal is out of
view, the intermediate male attacks another ND male until Figure 9 An example of eye movements during social interactions.
the dominant male reappears. When the dominant male Fish with eye positions have been drawn from film images at 160 ms
returns to the scene, he attacks within a few seconds but does intervals with the 1st through 11th frames labeled. Relative eye loca-
tions of the dominant animal are shown by lines extending stalks
not specifically target fish that have been aggressive to others
attached to the eyes at an arbitrary angle behind the central axis of the
in his absence (Desjardins et al., 2012).
eye. Note that the nondominant (ND) animals move out of the region
Our data show that the ND males are attending to the D being approached by the dominant male well before he arrives.
males and altering their behavior by acting aggressively which Redrawn from Fernald, R.D., 1985. Eye-movements in the African
is not possible when the D male is present. In addition, these cichlid fish, Haplochromis burtoni. J. Comp. Physiol. A Sens. Neural
males on occasion will approach and court females when the Behav. Physiol. 156, 199–208.

Figure 8 Schematic illustration of typical dominant male behavior in the presence of an intermediate male attempting to ascend to dominance.
Large gray rectangles represent the dominant male in his shelter and small black bars show when an individual chases or attacks another fish. Note
that intermediate males only attack other animals when they cannot be seen because the dominant male is in his shelter. From data presented in
Desjardins, J.K., Hofmann, H.A., Fernald, R.D., 2012. Social context influences aggressive and courtship behavior in a cichlid fish. PLoS One 7,
e32781.
28 Social Regulation of Sex: How the Brain Controls Reproductive Circuits
anticipation of the actions of the D male contributes to the
attention hierarchy of males within the social hierarchy and
suggests that the ND males attend to specific attributes of the
male behavior.
2.02.6.2 Transitive Inference in Astatotilapia burtoni
Social living requires more sophisticated cognitive abilities in
many vertebrates because the social environment offers indi-
viduals an opportunity to gather and use information about
the behavior of others (Brown and Laland, 2003). But how
do animals collect and use social information (for reviews
see McGregor, 2005; Brown et al., 2011)? Eavesdropping, the
collection of information via observation can provide benefits
to the bystander (McGregor, 2005). Eavesdropping behavior,
best documented in conflict situations, suggests that individ-
uals adjust their fighting behavior according to knowledge
gained by observing their opponent (Johnstone, 2001;
Johnstone and Bshary, 2004).
In A. burtoni, we have recently shown that males can infer
who will win fights solely from observing pairs of individuals
fighting. This ability, called ‘transitive inference,’ is tested
when a subject is given information about a specific relation-
ship among items based on a property of each item. In social
animals, the ability to make inferences about other individuals’
relative place in a dominance hierarchy and, therefore, predict
the outcome of competition should be a useful skill (Emery,
2006). Transitive inference has been demonstrated in a number
of avian species including pigeons, jays, and corvids (for
summary and review see Emery, 2006). However, we recently
showed that A. burtoni males show transitive inference about
dominance among other animals Grosenick et al. (2007).
Two groups of dominant bystander fish saw staged, pairwise
fights between five pairs of conspecifics. One group of five
had a dominance hierarchy of A > B > C > D > E while the
other had no dominance hierarchy (e.g., A ¼ B ¼ C ¼ D ¼ E).
Figure 10 Time spent near rival males during preference testing. Bar
plots of the difference in time spent near the higher-ranking versus the
lower-ranking rival dominant males in the AE (top) and BD (bottom)
preference tasks by context. Filled bars, familiar context (e.g.,
rearranged training aquarium); open bars, novel context (e.g., new
aquarium). One-sample permutation t-tests (50 000 replicates) showed
that in all cases bystanders spent significantly more time near the
lower-ranking rival, namely D or E (AE familiar context, P50.01; AE
novel context, P50.003; BD familiar context, P50.003; BD novel context,
P50.008; where P50.01 etc. refers to 50 000 replicates with a 0.01
probability of significance). Error bars indicate s.e.m.
Bystanders were then tested in a forced choice paradigm to
demonstrate their preference for either A and E, or B and D.
Both of these pairings were novel to the bystanders and differed
only in their position in the inferred dominance hierarchy. In
both tests (A vs E and B vs D) bystander fish spent more time
near rival males that were lower in the dominance ranking
(Figure 10). These results show that fish learned the implied
hierarchy as bystanders and, importantly, that A. burtoni can
use transitive inference to understand the likely outcome of
fights between dominant males.
2.02.7 Conclusions
Clearly, social information influences hormones, brain
action, and behavior. In the cichlid model system presented
here, social information via multiple sensory systems
impacts complex behavioral patterns and the entire HPG
axis from the expression and secretion of GnRH in the brain
to the development of sperm in the gonads. In this species,
we have shown that numerous cognitive processes play
a role in the regulation. Specifically, males pay close atten-
tion to one another, and they can predict who will be the
dominant male in a fight. Females also make choices that
reflect their understanding of social dominance among
males. It has been a mystery how GnRH neurons are con-
nected to produce the requisite pulsatile secretion. We
suggest that the gap junctions we describe between these
neurons are likely central to that key function. While we
now have a better understanding of the relationship between
social behavior, use of social information, and brain activa-
tion, we do not know how animals process and store social
information and then retrieve it in the service of complex
cognitive tasks. The cichlid fish, A. burtoni, because of its
complex social organization and the wealth of background
information into brain activation, is likely to be integral in
the study of dominance, social information, and their impact
on the brain.
Acknowledgments
I have been privileged to work with a superb collection of undergrad-
uates, graduate students, and postdoctoral fellows without whom this
work would not have been possible. Supported by NIH-NS 034950,
NIH MH101373, and NSF IOS-0923588 to R.D.F.
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 2.03 Socially Controlled Sex Change in Fishes
John Godwin and Melissa Lamm, North Carolina State University, Raleigh, NC, USA
Ó 2017 Elsevier Inc. All rights reserved.

2.03.1 Introduction 31
2.03.1.1 Hermaphroditism in Fishes 32
2.03.1.2 Social Regulation of Sex Change 33
2.03.1.3 Gonadal Change in Sex-Changing Fishes 33
2.03.1.4 Steroid Hormones and Sex Change 34
2.03.1.4.1 Sex Steroids and Behavioral Change 36
2.03.1.4.2 Corticosteroids and Sex Change 38
2.03.2 Neural Signaling Systems and Sex Change 39
2.03.2.1 Gonadotropin-Releasing Hormone and Gonadotropins 39
2.03.2.2 Kisspeptins and Gonadotropin-Inhibitory Hormone 39
2.03.2.3 Monoamine Neurotransmitters and Sex Change 40
2.03.2.4 Nonapeptides and Sex Change 41
2.03.3 Conclusions and Future Directions 42
References 43

2.03.1 Introduction gobies (Gobiidae), and others (see Godwin, 2009, 2010;
Kobayashi et al., 2013; Lamm et al., 2015 for recent reviews).
Teleost fishes are the oldest and, in many respects, the most While the reasons why sex change in general and socially
diverse of the vertebrate lineages. This diversity takes a variety controlled sex change in particular are observed in fishes and
of forms, but one that particularly stands out is the variety of not in other groups are not clear, several possibilities have
sexual expression seen across teleosts. Teleosts exhibit the been suggested. These include reduced evolutionary constraints
full range of sexual development patterns observed in in fishes with external fertilization since the ducts for release of
vertebrates more generally (see Devlin and Nagahama,
2002; de Mitcheson and Liu, 2008; Kobayashi et al., 2013
for detailed reviews; Figure 1). The majority of fish
Gonochorism Adult female
species display the familiar pattern of genotypic sex
determination and gonochorism, where individuals mature Adult male
as one sex or the other and remain that sex. However, there
is wide variation in genotypic sex-determining mechanisms Gonochorism with alternate male phenotypes
across fishes including male heterogamety, female hetero- Adult female
gamety, and polyfactorial sex determination. Some groups Fixed expression
of AMP Parasitic male
of fishes display gonochorism that is characterized instead
by environmental sex determination in two major forms. Bourgeois male
Temperature-dependent sex determination (TSD) is seen in Adult female
Plastic expression
several groups of fishes and shows some strong similarities of AMP Bourgeois
Parasitic male male
in patterns and mechanisms to those described especially for
reptiles. Fishes also exhibit social determination of sex, Protandry
a phenomenon that is not seen in other vertebrate groups. Juvenile (female) Adult male Adult female
This can occur either before or after sexual maturation
and has been documented from 27 families of fishes Monandric protogyny)
(de Mitcheson and Liu, 2008). The behavioral and neuroen- Juvenile (female) Adult female Adult male
docrine aspects of sex change that occur postmaturationally
in response to social cues are the focus of this chapter. Diandric protogyny Adult female
Terminal phase
Socially controlled sex change was first documented in the Juvenile (female)
(TP) male
early 1970s for two coral reef dwelling species, the fairy basslet Adult initial phase
Pseudanthias squamipinnis and the Indo-Pacific cleaner wrasse (IP) male
Labroides dimidiatus (Fishelson, 1970; Robertson, 1972, respec- Bidirectional sex change Adult female
tively; Pseudanthias was formerly classified as Anthias). This Juvenile (female)
sexual pattern has subsequently been documented for a variety Adult male
of other species including other members of the families that
include wrasses (Labridae), parrotfishes (Scaridae), damsel- Figure 1 Range of sexual patterns in fishes for both gonochoristic
fishes (Pomacentridae), groupers and basslets (Serranidae), and hermaphroditic species. ‘AMP’ refers to alternate male phenotypes.

Hormones, Brain, and Behavior, 3rd edition, Volume 2 http://dx.doi.org/10.1016/B978-0-12-803592-4.00022-5 31

32 Socially Controlled Sex Change in Fishes
gametes are relatively similar between males and females, and
less modification is therefore necessary with a change in sex
(Francis, 1992). There is also strong selection for sex change
in some species that can provide evolutionary advantages to
sex-changing individuals (Ghiselin, 1969; Warner et al., 1975;
Warner, 1984). Finally, it has been proposed that there is
a fundamental difference in the relative timing of differentia-
tion for the brain and gonadal tissues between tetrapod verte-
brates and at least many species of fishes (Francis, 1992).
Specifically, while the gonads develop relatively early and the
production of gonadal hormones plays an important role in
organizing the brain in well-studied mammalian models, the
reverse is generally true for fishes. Gonadal differentiation
occurs during a free-living and fully independent juvenile
period where developing individuals are essentially ‘miniature
adults’ fully able to sense and integrate information about their
abiotic and social environments. This creates at least the possi-
bility of adaptively adjusting sex determination in the direction
likely to be reproductively advantageous for the individual.
Consistent with this hypothesis, studies in cichlid and labrid
fishes show that social dominance can significantly influence
sexual differentiation with larger individuals disproportion-
ately becoming males (Francis and Barlow, 1993; Munday
et al., 2006). Social influences on sex determination in adult
functional sex change may act through similar physiological
mechanisms.
Sex-changing fishes provide attractive models for studying
the hormonal bases of sexual and aggressive behavior for
several reasons. The first is that, unlike most model species,
sexual developmental processes can be studied in an adult
animal in which behavioral changes take place as sexual devel-
opment is occurring. A second experimentally valuable feature
of sex change is that it often occurs relatively quickly, particu-
larly with protogynous (female to male) species. Both cleaner
wrasses and bluehead wrasses complete gonadal sex change
in approximately 1–2 weeks following removal of dominant
males from social groups in nature (Robertson, 1972; Warner
and Swearer, 1991). Some anemonefish and gobies also
show rapid behavioral changes during sex change (Godwin,
1994a; Black et al., 2005, 2011). Another attractive feature of
many sex-changing species is the presence of alternate male
mating phenotypes that exhibit within-sex dimorphisms in
morphology and behavior that are useful for exploring
hormonal mechanisms regulating these characteristics (Moore,
1991; Crews, 1993; Godwin, 2010). In these species, one of
these phenotypes is typically large, often colorful, and exhibits
strong courtship, territorial defense, and aggression. The second
male type typically exhibits what Taborsky termed ‘parasitic’
male tactics where reproductive success is obtained through
what are termed ‘sneaker’ or ‘streaker’ tactics (Gross, 1980;
Taborsky, 1998).
This chapter primarily focuses on the hormonal regula-
tion of socially regulated sex change and both within- and
between-sex differences in behavior in species that exhibit
this sexual pattern. We first introduce the different forms
of socially regulated sex change observed in teleost fishes.
Then, we review the roles of steroid hormones, neuropep-
tide hormones and other neural signaling systems in regu-
lating sex differences in morphology and behavior as well
as sex change. This will be followed by a discussion of
promising directions for the study of this phenomenon in
future studies.
2.03.1.1 Hermaphroditism in Fishes
The diversity of sexual patterns in fishes exceeds that observed
in any other group of vertebrates. These patterns have been
reviewed in detail elsewhere starting with Atz in 1964 and
with many excellent contributions in the years since (Rein-
both, 1980, 1988; Devlin and Nagahama, 2002; de Mitcheson
and Liu, 2008), so only a relatively broad overview is
provided here. The diversity in sexual patterns displayed by
teleosts includes several patterns that do not involve sex
change, and most groups of fishes are gonochoristic, maturing
as either males or females and remaining that sex through the
life history. However, gonochoristic species can nevertheless
exhibit sexual determination that is wholly or partly deter-
mined by environmental cues. While best characterized in
reptiles, TSD is seen in several groups of fishes with the best
studied examples being from the Atheriniforms (Conover,
2004) and flatfishes (Luckenbach et al., 2009). The key envi-
ronmental influence can also be social as demonstrated for
Midas cichlids (Francis and Barlow, 1993) and initial sex
differentiation in bluehead wrasses (Munday et al., 2006 –
discussed further below). Many species that are gonochoristic
nevertheless display lability in the development of secondary
sexual characteristics and behavior to produce within-sex
dimorphism and alternate mating phenotypes (reviewed in
Moore, 1991; Taborsky, 1998; Godwin, 2010). Alternate
male mating phenotypes are also seen in a number of sex-
changing species including especially the wrasses and
parrotfishes.
Forms of sexual lability seen in mature fishes include
sequential hermaphroditism, simultaneous hermaphroditism,
and some species that are simultaneous hermaphrodites before
losing ovarian tissue to become males. Sequential hermaphro-
ditism or sex change occurs in both protandrous (male-to-
female) and protogynous (female-to-male) directions. Protandry
(‘first male’) is characterized by male-to-female sex change.
Protandry may also be a functional description for develop-
mental patterns that are essentially protogynous. For example,
anemonefishes in the genera Amphiprion and Premnas mature as
males and can become females on becoming socially dominant
members of groups resident on large tropical sea anemones.
However, examining development in several of these species
shows that gonads are initially immature ovaries and only
develop testicular tissue if an individual assumes the male
breeding role (Fricke and Fricke, 1977; Fricke, 1983; Godwin,
1994b). Protogyny (‘first female’) is characterized by func-
tional, postmaturational female-to-male sex change. Monandric
protogynous species tend to first develop as immature females
that can then first sexually mature as females in what is termed
the initial phase (IP) and then later change sex to become males
in the terminal phase (TP males). Diandric protogynous
species, by contrast, can mature as either females or males in
the IP and then change to become TP males later. This develop-
ment of TP males involves sex change for females and role
change for IP males. The transition to TP male by either females
or IP males also often involves the development of TP-typical
morphology and behaviors including defense of a breeding

territory and increased display of aggressive and courtship
behaviors.
Many species in the family Serranidae, which contains the
groupers as well as basslets and hamlets, display simultaneous
hermaphroditism. This sexual pattern is characterized by the
presence of mature ovarian and testicular tissue within the
same gonad, and individuals can alternate between female
and male mating roles over the course of a brief spawning
period in an elaborate mating behavior termed ‘egg trading’
(Fischer, 1980). Other serranid fishes can display simultaneous
hermaphroditism when relatively small and then lose the
ovarian tissue to become males on attaining large size and
social dominance (Petersen, 1987).
Sex change is typically unidirectional, being either protogy-
nous or protandrous. However, a number of goby species
exhibit bidirectional sex change. This is hypothesized to be
an adaptation in at least some cases to strong predation pres-
sure in their coral reef habitats (Hobbs et al., 2004). Gobies
are typically relatively small, bottom-dwelling fishes that are
not strong swimmers, and it would therefore be dangerous to
move from their densely branching coral homes to find mates.
Bidirectional sex change allows any two conspecific gobies on
a coral head to form a female–male pair for breeding.
2.03.1.2 Social Regulation of Sex Change
A full understanding of socially controlled sex change will
require understanding the sequence of physiological events
that ‘transduce’ a social cue or cues into the neuroendocrine
changes that regulate gonadal transformation. While we have
clear evidence that social interactions can regulate sex change,
the sensory modalities that mediate these social influences
are not well understood.
The initial studies showing social control of sex change in
two coral reef fishes, the fairy basslet P. squamipinnis and cleaner
wrasse L. dimidiatus, showed that removal of large and socially
dominant males from social groups led to rapid sex change by
the largest resident female. Later demonstrations of similar
inhibitory influences on sex change by dominant secondary
sex individuals followed in a number of other species including
both protogynous and protandrous sex changers (e.g., Fricke,
1983; Ross et al., 1983; Warner and Swearer, 1991; Godwin,
1994b; Black et al., 2005). Precisely what are the critical inhib-
itory influences provided by these socially dominant individ-
uals remain poorly understood. There is evidence for both
visual and olfactory cues providing this inhibitory input, and
auditory/vibrational cues are possible as well.
One of the most in-depth studies of sensory cues regulating
sex change was conducted by Ross et al. (1983) in the Hawaiian
saddleback wrasse (Thalassoma duperrey). Working in experi-
mental pens, they were able to show that female-to-male sex
change in this species involved both stimulation from small
conspecifics and inhibition from larger conspecifics. Visual
cues are of critical importance as allowing sex-change candi-
dates to see, but not physically interact with a larger conspecific,
prevented sex change. Blocking visual contact with a porous
barrier did not allow females to undergo sex change despite still
presumably being exposed to olfactory and possibly auditory
cues. By contrast, olfactory cues appear to be important in other
species. Cole and Shapiro (1995) found that waterborne,
Socially Controlled Sex Change in Fishes 33
presumably olfactory, cues affected the incidence of sex change
in isolated females of the goby Coryphopterus glaucofraenum,
although group size was also important. Females in tanks
receiving water from tanks housing conspecific females were
significantly more likely to change sex than females in tanks
receiving water from tanks holding conspecific males. Fishes
can be extremely sensitive to pheromonal cues that affect the
reproductive axis (reviewed in Stacey, 1987). It is interesting
to note that increases in close ‘inspection’ of the cloacal region
of gravid females is one of the first clear behavioral changes
seen in sex-changing female bluehead wrasses (Godwin and
Lamm, pers. obs.). These ‘inspections’ could expose them to
important olfactory cues to help initiate sex change.
The pattern of exposure to key sensory cues also seems likely
to be very important in regulating sex change. This is primarily
seen in experiments that vary the ratio of males and females
that a sex change candidate is exposed to. Increasing the fema-
le:male ratio in experimental groups increases the likelihood of
sex change in the fairy basslet P. squamipinnis (Shapiro, 1980),
the saddleback wrasse T. duperrey (Ross et al., 1990), the bridled
goby C. glaucofraenum (Cole and Shapiro, 1995), and Potter’s
angelfish Centropyge potteri (Lutnesky, 1994). Lutnesky showed
quantitatively that the encounter rate of female C. potteri with
males was negatively associated with the occurrence of
female-to-male sex change. It is also noteworthy that removal
of dominant TP male bluehead wrasses from small reefs results
in an approximate one-to-one replacement by large females
who change sex, suggesting sex ratios are important in natural
social groups as well (Warner and Swearer, 1991). It is
intriguing to consider how encounter rates with larger and
smaller conspecifics might influence the neural systems regu-
lating behavioral and gonadal sex change, but no studies
have yet directly examined this question.
2.03.1.3 Gonadal Change in Sex-Changing Fishes
Sex change involves a dramatic restructuring of the gonad that
can occur very rapidly. For example, both bluehead and cleaner
wrasses can completely transform a functional and mature
ovary into a mature testes and become capable of fertilizing
eggs in as little as 7 to 10 days. A full review of gonadal changes
in sex-changing fishes is beyond the scope of this chapter, so
just a few examples are provided here.
Gonadal change has been well studied in protogynous Tha-
lassoma wrasses. Nakamura et al. (1989) described the process
of sex change in the saddleback wrasse T. duperrey at both the
light and electron microscopic level. Figure 2 illustrates the
sequence of changes in both gonadal structure and body color-
ation over the course of sex change in bluehead wrasses (Thalas-
soma bifasciatum). In this species, females induced to change sex
by removal of the large TP males from their social groups on
small reefs in the field exhibit very rapid changes in behavior,
gonadal structure, and body coloration. The ovaries exhibit
breakdown in the first 2–3 days of sex change, exhibiting
atresia of large, mature follicles. This is followed by the begin-
nings of spermatogenesis approximately 4–5 days after the
initiation of sex change (TP male removal). This new spermato-
genic tissue arises within the lamellae containing degenerating
ovarian tissue. Coloration changes depend on the teleost
androgen 11-ketotestosterone (11KT), analogous to the actions
34 Socially Controlled Sex Change in Fishes

(a) (b) (c)

(d) (e) (f)

Figure 2 Morphological and gonadal sex change in the bluehead wrasse. Left: color changes from female (top) to TP male (bottom) (image by J.
Godwin). Right: six gonadal sex change stages from ovary to functional testis (images by M. Lamm and J. Godwin). (a) Stage 1: mature ovary with
healthy vitellogenic oocytes (VO) and previtellogenic oocytes (PVO); (b) stage 2: atretic vitellogenic oocytes (AVO) with degraded zona pellucida;
(c) stage 3: atretic previtellogenic oocytes (APVO); (d) stage 4: proliferation of presumed spermatogonia (SG) and Leydig cells; (e) stage 5: onset of
spermatogenesis, indicated by spematocytes (SC) in spermatocysts; (f) stage 6: mature, tailed sperm (SP). Scale bar: 50 mm. Based on classification
by Nakamura, M., Hourigan, T.F., Yamauchi, K., Nagahama, Y., Grau, E.G., 1989. Histological and ultrastructural evidence for the role of gonadal
steroid hormones in sex change in the protogynous wrasse Thalassoma duperrey. Environ. Biol. Fishes 24 (2), 117–136.

of dihydrotestosterone in many tetrapods. The beginning of
permanent coloration changes in sex-changing bluehead
wrasses coincides approximately with the onset of spermato-
genesis (Lamm et al., 2015). These sex changers can then be
capable of fertilizing eggs from female spawning partners in
as few as 8 days after the initiation of sex change.
Anemonefishes exhibit protandrous sex change and a social
system characterized by mating pairs on host anemones con-
sisting of large, socially dominant females, mature males, and
often a variable number of subadults. Removal of the domi-
nant females stimulates male-to-female sex change in the
mature males (Fricke and Fricke, 1977; Godwin, 1994a,b).
The gonads of these mature males consist of an immature
ovarian portion and a distinct part of the gonad that is mature
spermatogenic tissue. In the anemonefish Amphiprion melano-
pus, this spermatogenic portion of the gonad rapidly matures
in the 10 days after onset of sex change, but immature oocytes
replace this tissue by 10 days later and go on to mature as the
entire gonad becomes a mature ovary.
Perhaps the most interesting examples of gonadal transfor-
mation are seen in several genera of bidirectionally sex-
changing gobies. In these species, the tissues that form the
ovarian and spermatogenic portions of the gonad are physi-
cally delimited, and sex change involves activation of one
portion and inactivation of the other. The goby Trimma okina-
wae exhibits serial bidirectional sex changes that can be
completed in as little as 4 days, and these involve rapid alter-
ations in gonadotropin receptor mRNA expression (Kobayashi
et al., 2009). We return to these rapid endocrine changes in
gobies in the next section.
2.03.1.4 Steroid Hormones and Sex Change
Sex steroid hormones play key roles in sex determination and
differentiation across vertebrate animals, and so attention has
focused on these mediators since the earliest physiological
studies of sex change. The first studies examined the effects
of exogenous hormone administration on the occurrence of
sex change, beginning with Stoll (1955) manipulating andro-
gens in the bluehead wrasse. However, as techniques for
measurement developed there has been an increasing focus
on examining patterns in circulating steroid hormone levels
and biosynthesis across sexual phenotypes and sex change.
Attention has primarily focused on gonadal steroid
hormones, but increasing evidence suggests a role for gluco-
corticoids in sex determination events both during develop-
ment in gonochoristic species and in adult sex change in
hermaphroditic species.
As with gonochoristic fishes, sex-changing species often
show strong sexual dimorphisms in circulating levels of sex
steroid hormones (Table 1). As in tetrapods, estradiol 17b
(E2) is the key circulating estrogen in fishes and tends to be
found at higher levels in females (Devlin and Nagahama,
2002). The key hormone responsible for inducing male
secondary sexual characteristics in fishes is the nonaromatiz-
able 11KT, which plays an analogous role in this process to
that played by dihydrotestosterone in tetrapods. Expression
of these male secondary sexual characteristics, primarily color
changes and often elaboration of fins, coincides with the onset
of spermatogenesis and rising 11KT levels (e.g., Nakamura
et al., 1989; Hourigan et al., 1991; Cardwell and Liley,
1991a,b; Warner and Swearer, 1991; Ohta et al., 2007;
Figure 3). These changes can also be induced by exogenous
androgens (Stoll, 1955; Cardwell and Liley, 1991a; Semsar
and Godwin, 2004). The role of testosterone in fishes is some-
what less clear (Borg, 1994; Frisch, 2005; Godwin, 2010) and
shows a less clear pattern in terms of female–male differences
including in sex-changing species where testosterone levels
may be higher in females (e.g., Godwin and Thomas, 1993;
Figure 3). Testosterone appears to be important primarily as
 Socially Controlled Sex Change in Fishes 35

Table 1 Circulating steroid levels in sex-changing fishes

Direction of
Species, common name Family sex change E2 Testosterone 11-Ketotestosterone References

Thalassoma duperrey, Labridae Diandric \ > (⚥ ¼ TP_) \ ¼ ⚥ ¼ TP_ (\ ¼ ⚥) < TP_ Nakamura et al. (1989)
saddleback wrasse protogyny IP_ ¼ TP_ IP_ ¼ TP_ IP_ < TP_ and Hourigan et al.
(1991)
Halichoeres trimaculatus, Labridae
three-spot wrasse
Pseudolabrus sieboldi, Labridae Protogyny \¼⚥¼_ NA \<⚥<_ Ohta et al. (2008)
bambooleaf wrasse
Sparisoma viride, stoplight Scaridae Diandric \ > (⚥ ¼ TP_) \ ¼ ⚥, \ < TP_ \ << (⚥ ¼ TP_) Cardwell and Liley
parrotfish protogyny IP_ < TP_ (1991a,b)
Amphiprion melanopus, Pomacentridae Protandry \ > (_ ¼ ⚥) \>(_ ¼ ⚥) _>(⚥ ¼ \) Godwin and Thomas
Cinnamon anemonefish (1993)
Lythrypnus dalli, Gobiidae Protogyny \>_ Mixed results \¼_ Lorenzi et al. (2008)
bluebanded goby and Lorenzi et al.
(2012)
Gobiodon histrio, coral Gobiidae Bidirectional \>_ \¼_ Kroon et al. (2003)
goby
G. erythrospilus, coral goby Gobiidae Bidirectional \¼_ Kroon et al. (2009)
Coryphopterus nicholsii, Gobiidae Protogyny \>_ \<_ \<_ Kroon and Liley (2000)
blackeye goby
Plectropomus leopardus, Serranidae Protogyny \>_ \¼_ \<_
coral trout

IP, initial phase; TP, terminal phase; E2, estradiol 17b.

a ‘prohormone’ in many species of fishes, serving as a biochem-
ical precursor for either E2 or 11KT production.
A critical role for sex steroid hormone signaling in gonadal
sex change is supported by two types of studies where exoge-
nous steroids are administered to IP individuals and where
steroid hormone synthesis is manipulated pharmacologically.
A first and continued focus of these studies has been androgen
signaling, stimulated at least in part by the responsiveness of
male sex determination to androgenic stimulation more gener-
ally in fishes (extensively reviewed in Devlin and Nagahama,
2002). Administration of androgen in the form of either the
testosterone-derivative methyltestosterone or 11KT successfully
induced female-to-male sex change in species from several
families of fishes that display socially controlled sex change
including wrasses (Labridae), parrotfishes (Scaridae), the
basses (Serranidae), gobies (Gobiidae), and a variety of species
where social control of sex change has not been demonstrated
(see Devlin and Nagahama, 2002; Frisch, 2005 for reviews).
Despite clear evidence that androgens can induce protogynous
sex change, it is not clear that an increase or alteration in
androgen production is the key initiating event in protogynous
sex change. In one of the most comprehensive studies of steroid
profiles during naturally occurring sex change in wild fish,
Nakamura et al. (1989) did document increases in circulating
11KT, but increases over female levels were not observed until
the animals were fully developed TP males – well after the initi-
ation of sex change.
Declines in estrogenic signaling are also reliably associated
with protogynous sex change. For example, E2 levels showed
immediate and significant declines with the initiation of sex
change in saddleback wrasses (Nakamura et al., 1989). This is
consistent with the observation that widespread atresia
(degeneration) of yolky oocytes is one of the first histologically
discernible signs of gonadal sex change in Thalassoma wrasses
and other species (Sadovy and Shapiro, 1987). Several lines
of evidence are consistent with a key role for changes in estro-
genic signaling in the initiation of sex change. These include
demonstrations that inhibiting estrogen production pharmaco-
logically using both steroidal and nonsteroidal aromatase
inhibitors is effective in inducing gonadal sex change in several
families of sex-changing fishes (Table 2). Estrogen synthesis
can decline very quickly at the initiation of sex change. The
California goby species Lythrypnus dalli shows rapid declines
in aromatase activity in both the gonad and brain at the initia-
tion of protogynous sex change (Black et al., 2005, 2011)
although reductions in estrogen production at least at the
whole-brain level lag behind increases in aggressive behavior
in sex changers. Several other species also show declines in
either estrogen production or expression of the gonadal form
of aromatase with sex change (fishes have two aromatase genes,
cyp191a1a and cyp19a1b, expressed predominantly in the
gonads and brain, respectively: Gelinas et al., 1998; Forlano
et al., 2006).
As with many gonochoristic fish species during develop-
ment, sex change can be manipulated effectively with exoge-
nous estradiol in species across several families (Table 2).
The bidirectionally sex-changing coral goby Gobiodon erythrospi-
lus was the subject of a study notable both because sex change
was manipulated in both directions and also because the study
was performed on corals in the field (Kroon et al., 2005). This
species typically lives in pairs that shelter in the branches of
corals and has limited movement ability across the reef as their
small size and relatively weak swimming abilities are thought
to contribute a high predation risk when away from shelter.
36 Socially Controlled Sex Change in Fishes
RelaƟve blood steroid levels
- were elevated in testes (11b-hydroxylase for both sea bream
RelaƟve blood steroid levels
-
Figure 3 Top panel: circulating steroid hormone levels in the anemo-
nefish Amphiprion melanopus for males and females in natural habitat as
well as males undergoing protandrous sex change induced by experi-
mental removals of dominant females from social groups. Bottom
panel: circulating steroid hormone levels in the saddleback wrasse
(Thalassoma duperrey) shown for wild-caught females and terminal
phase (TP) males and in females induced to undergo sex change in
experimental pen. Stages in blue boxes indicate progress in sex change
process when samples were taken. Relative levels of different hormones
are depicted to approximately the same scale except for cortisol in top
figure, where levels were substantially higher. Figures redrawn from
Godwin, J.R., Thomas, P., 1993. Sex change and steroid profiles in the
protandrous anemonefish Amphiprion melanopus (Pomacentridae, Tele-
ostei). Gen. Comp. Endocrinol. 91, 144–157; Nakamura, M., Hourigan,
T.F., Yamauchi, K., Nagahama, Y., Grau, E.G., 1989. Histological and
ultrastructural evidence for the role of gonadal steroid hormones in sex
change in the protogynous wrasse Thalassoma duperrey. Environ. Biol.
Fishes 24 (2), 117–136, respectively.
As noted above, bidirectional sex change is predicted to be
adaptive under these conditions as it allows any two adults
to form a breeding pair. Kroon and colleagues demonstrated
estrogenic control over this process by implanting male
G. erythrospilus with E2 to induce male-to-female change and
implanting females with the nonsteroidal aromatase inhibitor
fadrozole to induce female-to-male sex change. Additionally,
capitalizing on a pattern where unpaired individuals become
male, these investigators were able to show that estrogen could
block this transition in isolated females and induce male-
to-female change in isolated males.
Protogynous sex change can also be manipulated in other
gobies, wrasses, and serranids through manipulations of estro-
genic signaling (Table 2), and one report indicates male-
to-female sex change can be induced in the three-spot wrasse
with estrogen implants (Kojima et al., 2008). Differences in
plasma hormone levels and gonadal structure indicate sex
change involves significant changes in steroidogenic pathways
in the gonad. This is supported by recent transcriptomic studies
in both sea bream (Sparidae) and bluehead wrasse (Manousaki
et al., 2014; Liu et al., 2015). Gonadal aromatase mRNA
(cyp19a1a) is expressed at higher levels in ovaries in both
species while mRNAs for key androgen synthetic enzymes
and bluehead wrasses, a variety of other androgen synthetic
enzymes for wrasses as well). Both species showed a very large
number of differences in the ovarian transcriptome, but a rela-
tively modest number of differences overall when comparing
brain transcriptomes. This is consistent with other findings
indicating very modest sex differences in fish brain transcrip-
tomes, at least at an overall level (e.g., Wong et al., 2014). Of
note with respect to estrogenic regulation of sex change, the
brain form of aromatase showed a trend toward higher expres-
sion in the brains of female bluehead wrasses (Liu et al., 2015),
a result consistent with significant differences found in our
more recent quantitative PCR and in situ hybridization compar-
isons (Lamm et al., unpublished data).
2.03.1.4.1 Sex Steroids and Behavioral Change
The dramatic alterations in circulating sex steroid levels and
extensive literature supporting a role for these hormones in
mediating sexual and aggressive behavior in other vertebrates
suggest a critical role in regulating the behavioral changes
that occur with sex change. While there is certainly support
for this general hypothesis, there are also conflicting findings
and a lack of clarity about what the key initiating events are
in the behavioral sex change process. A large body of research
in tetrapods going back to Berthold’s studies with capons and
particularly in the modern era with work in Guinea pigs by Wil-
liam Young’s group (Phoenix et al., 1959) has implicated
androgens of testicular origin as critical for organizing the
neural substrates of male-typical sociosexual behaviors during
development and activating these behaviors in adulthood.
Fishes, however, may be fundamentally different from tetra-
pods in terms of the hormonal bases of neural sexual dimor-
phisms, with some studies suggesting these differences are
purely activational in nature (Hiraki et al., 2012). Although
there are good examples of critical roles for androgens in regu-
lating male-typical sociosexual behaviors in fishes (Borg, 1994;
Munakata and Kobayashi, 2010; Godwin, 2010), it has also
been postulated that aromatization and effects on brain
estrogen levels are the overriding steroid hormone influence
on sexual differentiation of neural function in fishes (Le Page
et al., 2010).
As noted above, circulating E2 levels are typically higher in
females and 11KT levels are higher in males, and this is true of
both socially controlled protogyny and protandry (Table 1).
However, relatively few studies have experimentally induced
sex change and followed the time course of both hormonal
 Socially Controlled Sex Change in Fishes 37

Table 2 Experiments involving sex steroid hormones, steroidogenesis inhibitors, and neuropeptide hormone manipulations

Direction of sex Color

Species, common name Family change Manipulation Gonad change? change? References

Thalassoma bifasciatum Labridae Diandric protogyny Methyltestosterone Yes: \ ➔ _ Yes Stoll (1955)
administered to females
Halichoeres trimaculatus, Labridae Protogyny E2 administered in feed to Yes: _ ➔ \ Kojima et al. (2008)
three-spot wrasse IP males
H. trimaculatus, three-spot Labridae Protogyny 11KT or AI administered in Yes: \ ➔ _ Higa et al. (2003)
wrasse feed to females
T. bifasciatum Labridae Diandric protogyny Testosterone administered Yes: \ ➔ _ Kramer et al. (1997)
to females
T. bifasciatum Labridae Diandric protogyny 11KT or AI administered to Yes: \ ➔ _ Yes Godwin (2009)
females
Sparisoma viride, stoplight Scaridae Diandric protogyny KT injections Yes: \ ➔ _ Yes Cardwell and Liley
parrotfish (1991b)
Coryphopterus nicholsii, Gobiidae Protogyny Implants of 11KT, KA, or Yes: \ ➔ _ Kroon and Liley (2000)
blackeye goby AI
Gobiodon erythospilus, coral Gobiidae Bidirectional E2 implants Yes: _ ➔ \ Kroon et al. (2005)
goby AI implants Yes: \ ➔ _

AI, aromatase inhibitor; 11KT, 11-ketotestosterone; KA, 11-ketoadrenosterone; IP, initial phase; E2, estradiol 17b.

and behavioral changes. One system that has been well
studied in this way is the bluebanded goby (L. dalli). This is
a small-bodied species and has facilitated studies in captive
conditions that nevertheless can allow naturalistic social
interactions. Bluebanded gobies form clear dominance hierar-
chies, and removal of dominant males leads to rapid sex
change in the largest and most dominant (alpha) female
(Rodgers et al., 2005, 2007). Female-to-male sex change is
associated with significant decreases in gonadal aromatase
activity, although significant reductions are not seen early
on in the sex change process (Black et al., 2005). By contrast,
reductions in brain aromatase activity coincide with and are
significantly correlated to increases in dominance-related
behaviors by alpha females undergoing sex change. These
findings are consistent with a reduction in neural estrogen
levels being necessary for male-typical dominance behavioral
profiles and sex change to proceed, but a later study in L. dalli
that profiled behavioral changes early in the initiation of sex
change found a lack of concordance between brain aromatase
activity and dominance-related behavior, leading the authors
to suggest behavioral changes were not caused by reductions
in neural aromatization (Black et al., 2011). These were
whole-brain measures of aromatase activity due to the small
size of this fish, and more neuroanatomically localized effects
cannot be excluded, but these authors have also suggested the
intriguing hypothesis that decreases in aromatization could
lead to behavioral effects by channeling more of the
hormonal substrate (testosterone) into 11KT synthesis (Black
et al., 2005, 2011; Lorenzi et al., 2012).
Bluehead wrasses also show significant increases in 11KT
with female-to-male sex change, but several observations are
inconsistent with these increases being the causal drivers of
increases in TP male sexual and aggressive behavior. Sex
change is very rapid in bluehead wrasses under natural field
conditions, with the transition from functional female to
the capability to produce sperm and fertilize eggs occurring
in as few as 8 days (Warner and Swearer, 1991). Interestingly,
behavioral sex change is much faster, with sex changers being
able to exhibit male-typical behaviors within minutes of
assuming social dominance and typically spawning in the
male role within 1–2 h during the spawning period (Warner
and Swearer, 1991; Godwin et al., 1996). Behavioral change
occurring very rapidly at the onset of sex change has been
observed in a variety of species now and is likely the usual
situation under field conditions (e.g., Robertson, 1972). The
time course of behavioral change is consistent with a model
where gonadal alterations and related changes in gonadal
steroid hormone profiles are not driving behavioral
changes (although steroid hormones can act rapidly through
nongenomic mechanisms). Also consistent with gonadal
change not driving behavioral change is the finding that
gonadectomy does not block behavioral sex change in blue-
head wrasses, although it does prevent the development of
TP male-typical color change (Godwin et al., 1996). Likewise,
castration of TP males does not reduce or discernibly alter the
expression of male-typical courtship and aggressive behavior
under field conditions (Semsar and Godwin, 2003). While
these findings do not support a role for gonadal steroids in
TP male-typical behavior and behavioral sex change, other
findings do indicate a role. Implanting ovariectomized
females with 11KT induces the development of both TP
male-typical body coloration and alters behavior over the
course of 10–12 days in the presence of dominant TP males
under field conditions (i.e., socially inhibitory conditions;
Semsar and Godwin, 2004). Importantly with respect to
behavior, this manipulation did not increase aggression or
induce territorial behavior generally but did increase the like-
lihood of showing courtship toward a gravid female. Also
noteworthy in this context is that territorial TP male stoplight
parrotfish (Sparisoma viride) had higher circulating 11KT than
‘bachelor’ TP males that did not hold territories on reefs
(Cardwell and Liley, 1991b).
As with bluebanded gobies, estrogenic influences appear
to play a role in sex change in bluehead wrasses. Estradiol
38 Socially Controlled Sex Change in Fishes
implants administered to females did prevent behavioral sex
change under socially permissive conditions (i.e., TP males
removed from social groups) and also increased aromatase
mRNA abundances in the preoptic area (POA) of the hypo-
thalamus (Marsh-Hunkin et al., 2013). These observations
along with higher aromatase mRNA levels in females relative
to TP males (Liu et al., 2015) and strong aromatase expression
in the POA (Marsh et al., 2006) suggest brain levels of E2 are
higher in females than TP males and likely drop with behav-
ioral sex change. However, consistent with the observation
that declines in aromatase activity lag behind behavioral
changes in the bluebanded gobies discussed above (Black
et al., 2011), preliminary evidence suggests that declines in
forebrain aromatase mRNA expression lag behind the initia-
tion of behavioral sex change in bluehead wrasses (Cabral
et al., unpublished results). However, aromatase activity can
be altered very rapidly through phosphorylation (reviewed
in Charlier et al., 2015), and this could be important in behav-
ioral change. It will be critical to assess estrogen signaling at
a detailed neuroanatomical level to more fully address
a potential role in behavioral sex change. It is noteworthy in
this context that brain aromatase in fishes is expressed in
radial glia rather than neurons and this glial expression is
strong in the POA (see Forlano et al., 2001; Forlano and
Bass, 2006). These aromatase-positive glia are found in very
close proximity to neurons of key neuroendocrine signaling
systems in bluehead wrasses, creating the likelihood of high
and potentially rapidly changing local estradiol levels (Marsh
et al., 2006).
2.03.1.4.2 Corticosteroids and Sex Change
Socially controlled sex change is mediated through social inter-
actions and typically involves changes in individual domi-
nance status within a social group. The behavioral
mechanisms mediating these social status effects appear to be
primarily related to being the initiator or recipient of aggressive
dominance-related behaviors, and these typically change
rapidly at the onset of sex change (Robertson, 1972; Warner
and Swearer, 1991; Godwin et al., 1996; Black et al., 2005,
2011). Differences in social status are often correlated with
differences in activity of the glucocorticoid endocrine stress
axis, and these hormones can produce rapid changes in
behavior and endocrine function. Therefore, this endocrine
system is a logical candidate for ‘transducing’ changes in
dominance status into changes in reproductive function.
Solomon-Lane et al. (2013) make a detailed case that the hypo-
thalamic–pituitary–interrenal (HPI) axis is a likely regulator of
the sex change process due to its involvement across vertebrates
in social status, agonistic behavior, and important life history
transitions. In support of this hypothesis, these authors cite
(1) studies linking social status to glucocorticoid levels in
a range of fishes, (2) links to agonistic behavior in a range of
vertebrates including some teleost models, and (3) the role
the primary glucocorticoid in fishes, cortisol, plays in sex
determination in distantly related species. This regulation of
sex determination is seen in both gonochoristic species
(medaka; Hayashi et al., 2010) and several species that
exhibit TSD (e.g., Japanese and southern flounders: Yamaguchi
et al., 2010; Mankiewicz et al., 2013; and South American
pejerrey: Hattori et al., 2009; Fernandino et al., 2012).
Nozu and Nakamura (2015) report a similar effect for the pro-
togynous three-spot wrasse where a high dose of cortisol
administered to females in feed for 6 weeks induced at least
the initiation of sex change in all individuals.
Perry and Grober (2003) developed a model that postu-
lated chronically elevated cortisol levels prevented sex change
in social subordinates (‘release of social stress’ hypothesis).
One experimental test involving implanting dominant
females of the protogynous sandperch (Parapercis cylindrica)
with cortisol did not support this model as this treatment
did not prevent sex change in a permissive environment
(Frisch et al., 2007), but effects elsewhere in the HPI axis
such as on corticotropin-releasing hormone activity remain
possible. Solomon-Lane et al. (2013) proposed an alternative
model they termed ‘classical facilitation of metamorphosis’
based on the evolutionarily conserved role of glucocorticoid
signaling in promoting metamorphic processes. This model
proposes that it is instead an increase in glucocorticoid
signaling that facilitates sex change at the onset of permissive
conditions. This model is consistent with patterns observed
following removal of dominant males from social groups
in bluebanded gobies, where dominant females (sex-
changing individuals) show elevations in excreted cortisol
at 1 and 3 days following removal of dominant males
(Figure 4). This may be a common pattern as sex change in
many species is correlated with significant increases in
activity including especially elevations in aggressive behavior
(e.g., bluehead wrasses show three- to fourfold increases in
aggression early on in sex change). The direction of sex
change may not be important for potential glucocorticoid
effects as the anemonefish A. melanopus exhibits protandry
with significant increases in both aggressive behavior and
circulating cortisol during the sex change process, although
males and females were not different (Godwin and Thomas,
1993; Godwin, 1994a). Likewise, social dominance in groups
of the protandrous anemonefish Amphiprion ocellaris is associ-
ated with elevated blood cortisol and expression of
Figure 4 Changes in cortisol released, waterborne cortisol, by sex-
changing bluebanded gobies (Lythrypnus dalli). ‘Pre’ refers to dominant
females before sex change is initiated and the 2 h to 15 days times are
sampling points following male removal, which produces the conditions
that induce sex change in females. Figure from Solomon-Lane, T.K.,
Crespi, E.J., Grober, M.S., 2013. Stress and serial adult meta-
morphosis: multiple roles for the stress axis in socially regulated sex
change. Front. Neurosci. 7, 210, http://dx.doi.org/10.3389/fnins.2013.
00210.

glucocorticoid receptor mRNA in the brain (Iwata et al.,
2012). Interestingly, given that dominance induces female
differentiation in this species, the dominant fish also showed
significantly higher neural expression of brain aromatase
mRNA.
2.03.2 Neural Signaling Systems and Sex Change
Social control of sex change implies the critical initiating events
of the process must involve neural signaling systems. These
events must induce both adaptive changes in sexual and aggres-
sive behavior and a reorganization of the reproductive system.
Studies have primarily focused on components of the hypotha-
lamic–pituitary–gonadal (HPG) axis, but other neural
signaling systems have also been implicated in regulating
behavioral and gonadal sex change.
2.03.2.1 Gonadotropin-Releasing Hormone and
Gonadotropins
Gonadotropin-releasing hormone (GnRH) is generally
considered the key ‘gatekeeper’ hormone in vertebrate
reproduction with activation of GnRH neurons being crit-
ical for such key life history events as puberty in mammals.
GnRH neurons are also critically involved in the activation
of the reproductive axis during assumption of social domi-
nance in males of the gonochoristic cichlid fish Astatotilapia
burtoni (Davis and Fernald, 1990; Burmeister et al., 2005;
reviewed in Maruska and Fernald, 2011). It is noteworthy
in this context that cichlid fishes are in the suborder of
fishes, the Labroidei, that also includes well-studied sex-
changing species in the wrasse, parrotfish, and damselfish
families (Streelman and Karl, 1997). This relationship
suggests that mechanisms by which social influences induce
reproductive activation in A. burtoni can likely inform our
understanding of this phenomenon in socially controlled
sex change.
A number of correlative and manipulative studies point to
a role for gonadotropic signaling in socially controlled sex
change, and wrasses and gobies are well studied in this
respect. Bluehead wrasses show differences in GnRH neuron
size in the POA across sexual phenotypes with TP males
having greater numbers than either females or IP males
(Grober and Bass, 1991). These numbers could be increased
to TP-male-typical levels by 11KT implants in females,
although this treatment also induced sex change so it is
possible that 11KT influences were not directly causal in this
change (Grober et al., 1991). Consistent with these findings
in bluehead wrasses, males of both Ballan wrasses and the
protandrous anemonefish A. melanopus also show greater
numbers of POA GnRH neurons than females (Eloffson
et al., 1999, 1997, respectively).
Although focusing on gonadal tissue rather than the brain,
a particularly intriguing finding regarding gonadotropin
signaling and sex change comes from a study of the bidirection-
ally sex-changing goby T. okinawae (Kobayashi et al., 2009).
The gonads in T. okinawae have both ovarian and testicular
portions, although only one of these is active at a given time.
Sex change involves regression of one tissue type and activation
Socially Controlled Sex Change in Fishes 39
of the other, and these changes take approximately 4 days.
Kobayashi and colleagues found that mRNA expression for
the two gonadotropin receptors (follicle-stimulating hormone
(FSH) receptor and luteinizing hormone (LH) receptor)
showed significant changes within 8–12 h of the initiation of
sex change, increasing in testicular tissue and declining in
ovarian tissue with female-to-male change while showing the
opposite pattern with male-to-female change. These changes
appeared to be functionally important as they conferred
responsiveness to gonadotropic stimulation as measured by
in vitro steroid hormone production.
Direct manipulative approaches also provide some support
for a GnRH role in sex change. Also working with bluehead
wrasses, a series of studies by Kramer and colleagues showed
that varying degrees of sex change could be induced in isolated
captive female bluehead wrasses through manipulations that
either directly altered gonadotropins or altered systems that
affect gonadotropin production and release. These experiments
involved injections of (1) human chorionic gonadotropin
(Koulish and Kramer, 1989), (2) a synthetic form of GnRH
with the dopamine receptor antagonist domperidone (Kramer
et al., 1993), and (3) neuropeptide Y which was hypothesized
to affect gonadotropin signaling (Kramer and Imbriano, 1997).
While these studies certainly do provide support for the
hypothesis that socially controlled sex change involves changes
in gonadotropic signaling and are generally consistent with
results in another protogynous wrasse (Coris julis, Reinboth
and Brusle-Sicard, 1997), a few points should be noted. These
studies involved female wrasses in captive conditions receiving
frequent injections (three injections/week). These conditions
could influence baseline activation of the HPA axis which, as
noted above (Section 2.03.1.2, glucocorticoids), could impor-
tantly influence sex change. Second, the degree of sex change
induced was relatively modest in terms of both progression
through the process in some cases and frequency, particularly
for the synthetic GnRH and NPY studies. Finally, the socially
isolated conditions of the experimental females provided
a high degree of experimental control but do not mimic the
inhibitory influence of larger individuals that is a key regulator
of sex change under natural conditions (Warner and Swearer,
1991; Ross et al., 1983 for the congeneric saddleback wrasse).
A convincing demonstration that the neuroendocrine system
of interest is part of the ‘final common pathway’ regulating
sex change would show that sex change could be induced by
this putative mediator in the presence of a more dominant
individual and, ideally, in the natural environment.
2.03.2.2 Kisspeptins and Gonadotropin-Inhibitory Hormone
The strong influences of kisspeptins and gonadotropin-inhibi-
tory hormone (GnIH) on the HPG axis in other fishes and
other vertebrates generally suggest these should be a productive
focus in studies of socially controlled sex change. Kisspeptins,
a group of RY- or RFamide peptides encoded by the kiss1
and/or kiss2 genes, typically stimulate expression and release
of GnRH in the POA and gonadotropins in the pituitary of
fishes and other vertebrates (reviewed by Elizur, 2009; Oakley
et al., 2009; Tena-Sempere et al., 2012). However, effects can
depend on the gonadal state of individuals and the partially
divergent activities of kiss1 and kiss2 in fishes that possess
40 Socially Controlled Sex Change in Fishes
both genes (Zmora et al., 2012, 2014). For example, intramus-
cular injections of kiss2 in gonochoristic female and male
hybrid bass (Morone saxatilis  Morone chrysops) induced release
of LH at both puberty and recrudescence, whereas kiss1 was
stimulatory only at recrudescence. Furthermore, kiss1 and
kiss2 increased gnrh1 expression at pre-prepuberty, but kiss2
downregulated gnrh1 expression during recrudescence (kiss1
had no effect) (Zmora et al., 2012). During the breeding
season, kiss1 and kiss2 had mostly stimulatory but partially
divergent effects on the genes of the HPG axis and gonadotro-
pins in male brain and pituitary cultures. The kisspeptin
receptor antagonists peptide 234, which blocks activation of
kissr2, and peptide 359, which blocks activation of kissr1 and
kissr2, reversed these effects (Zmora et al., 2015).
Interestingly, the same study by Zmora et al. (2015) found
that the kisspeptin receptor antagonists decreased avt mRNA
levels in the POA. In both this study and a study in medaka
(Kanda et al., 2013), kisspeptins were found to directly regulate
arginine vasotocin (AVT) neurons. Studies in fishes have also
found direct connections between the AVT and GnRH1
systems, e.g., in the sex-changing rock hind grouper (Kline
et al., 2016), and studies in mammals found AVP innervation
of kisspeptin neurons in mice (Vida et al., 2010) and Syrian
hamsters (Williams et al., 2011). Interactions among the AVT,
kisspeptin, and GnRH systems could be important connections
regulating behavioral and gonadal changes during sex change
in fishes.
In the protogynous orange-spotted grouper (Epinephelus
coioides), mature females had higher expression of hypotha-
lamic kiss2 than did males (Shi et al., 2010). During 4 weeks
of methyltestosterone treatment to induce sex change in
females, kiss2 and gnrh1 mRNA levels decreased during the first
3 weeks, but increased two- and eightfold, respectively, during
the fourth week. Shi et al. (2010) suggest that lower kiss2 and
gnrh1 expression during early sex change may be important
for ovarian atresia, and levels may increase during testicular
development. We found that implantation of kiss2 for
9–10 days in female bluehead wrasses with either active or
regressed ovaries at sampling was not sufficient to override
social inhibition and induce sex change in the presence of TP
males (kiss1 was not tested; Lamm et al., unpublished). Effects
of blocking kisspeptin signaling have not been described in any
sex-changing species.
GnIH (named in birds) and its related peptides, also known
as RFamide-related peptides (RFRP) in mammals and LPXRFa-
mides (LPXRFa) in fishes, are another recently discovered
group of RFamides that also interact with GnRH neurons and
gonadotropes to regulate the release of gonadotrophins in
vertebrates (reviewed by Tsutsui et al., 2010; Ogawa and
Parhar, 2014). The gnih precursor transcript encodes a prepro-
peptide that is cleaved into two to four GnIH-related
RFamides. One of the mature RFamides was first discovered
in quail and named GnIH due to its ability to inhibit LH
release from cultured pituitaries (Tsutsui et al., 2000). The
gnih gene and its receptor(s), part of the neuropeptide
FF receptor family, have been found in a variety of fishes, but
in vivo and in vitro studies in gonochoristic fishes have shown
complex and even conflicting results of GnIH on the
synthesis and release of gonadotrophins (Zhang et al., 2010;
Moussavi et al., 2012; Qi et al., 2013; Biran et al., 2014;
reviewed by Ogawa and Parhar, 2014). For example, goldfish
LPXRFa-1, -2, and -3 stimulated LH and FSH release from
pituitary cultures of precocious male sockeye salmon (Amano
et al., 2006), and goldfish LPXRFa-1 increased mRNA levels
for the beta subunits of both LH and FSH (lhb and fshb) in
pituitary cultures of sexually mature grass puffers of both
sexes (Shahjahan et al., 2011). Similarly, in Nile tilapia,
LPXRFa-2 treatment increased LH and FSH release in sexually
mature females injected intraperitoneally and in pituitary
cells in culture from sexually mature males (Biran et al.,
2014). However, studies in goldfish have also shown negative
effects of GnIH on gonadotropin synthesis and release
(Zhang et al., 2010; Qi et al., 2013), which has been shown
to be dependent on the spawning season, state of the gonads,
and circulating gonadal steroid levels (Moussavi et al., 2012),
and also suggested by Qi et al. (2013) to potentially be
socially dependent.
To our knowledge, the actions of GnIH on sex change or
sexual behavior in fishes have not yet been investigated. In
male quail, intracerebroventricular (ICV) injection of GnIH
inhibited male-typical sexual and aggressive behaviors and
increased estrogen synthesis in the POA via aromatase activa-
tion within 30 min of injection, whereas GnIH RNA interfer-
ence increased these behaviors 1 day after injection (Ubuka
et al., 2014). GnIH RNA interference also induced sexual and
aggressive behaviors 2 days after injection in male white-
crowned sparrows (Ubuka et al., 2012). Effects are also seen
in male rats where RFRP-3 ICV injection reduced sexual behav-
iors during the photophase (light period), but not scotophase
(dark period) (Johnson et al., 2007). In estradiol-implanted
female white-crowned sparrows, GnIH inhibited copulation
solicitation behavior upon male song playback within
30 min of ICV injection and decreased LH secretion (Bentley
et al., 2006). Though the actions of GnIH peptides on sexual
behavior in fishes are unknown, these studies nevertheless
suggest a potential for rapid modulation of behavior and
gonadal function by GnIH in fishes that are partially dependent
on social and abiotic cues.
2.03.2.3 Monoamine Neurotransmitters and Sex Change
Monoamine neurotransmitters are critical modulators of sexual
and aggressive behaviors across vertebrate animals including in
fishes (Summers and Winberg, 2006). To date, however, rela-
tively few studies have addressed a potential role for mono-
amine signaling in socially controlled sex change.
Larson and colleagues examined monoamine involvement
in protogynous sex change in saddleback wrasses using the
experimental pen approach that had previously established
social control of the process in this species (Larson et al.,
2003a,b). Measures of monoaminergic activity using HPLC
approaches demonstrated a complex set of changes in the
brains of sex-changing females including indications of
a decrease in serotonergic activity in the raphe nucleus during
the first week of social dominance. Importantly, these correla-
tive studies were followed by manipulative studies testing the
ability of pharmacological manipulations of monoamines to
influence sex change. Under social conditions that normally
inhibit sex change (i.e., a large TP male present in the group),
either putatively reducing serotonergic signaling with the

5-HT2 receptor antagonist ritanserin or increasing noradren-
ergic signaling with the drugs ephedrine or maprotiline
significantly increased the occurrence of ovary-to-testes differ-
entiation. Conversely, under socially permissive conditions
(i.e., experimental female was largest individual in the group),
ovary-to-testes transformation could be significantly inhibited
through either putatively increasing serotonergic signaling
using the serotonin-reuptake inhibitor sertraline or reducing
noradrenergic signaling with the a-receptor antagonist
phenoxybenzamine.
Support for a role of monoamines in regulating sex
change in other species is lacking overall. Manipulating sero-
tonergic activity did not affect the likelihood of sex change
in bluebanded gobies, and brain levels of serotonin and
the key serotonin metabolite 5-hydroxyindoleacetic acid
(5-HIAA) were not correlated with aggressive or courtship
behavior. These results were mirrored at a detailed neuroan-
atomical level with a similar lack of significant sex or social
status differences in serotonergic immunoreactive neurons in
bluebanded gobies (Lorenzi and Grober, 2012). Although
not directly related to sex change, the serotonin-reuptake
inhibitor fluoxetine can inhibit aggressive behavior of TP
male bluehead wrasses with both acute administration
under field conditions and chronic administration under
captive conditions (Perreault et al., 2003). Interestingly
and of potential relevance to behavioral sex change, chronic
fluoxetine treatment also reduced expression of AVT mRNA
in TP male bluehead wrasses (Semsar et al., 2004 and
Section 2.03.2.4 below).
2.03.2.4 Nonapeptides and Sex Change
Perhaps no class of neural signaling molecules has generated
more attention as modulators of social behavior than the
conserved nonapeptides in the arginine vasopressin and
oxytocin families (Goodson and Thompson, 2010). Fishes
express these peptides as AVT and isotocin (IST), respectively,
and provide intriguing models for studying nonapeptide
modulation of sociosexual behavior because of the diversity
of social systems and sexual differentiation patterns they
exhibit. The first demonstration of a nonapeptide effect on
sociosexual behavior was in a teleost (Wilhelmi et al., 1955;
Pickford and Strecker, 1977), and work in teleost models has
provided some critical advances in our understanding of these
hormones (e.g., Goodson and Bass, 2000; Godwin and
Thompson, 2012 for review). Most attention in sex-changing
fishes has focused on the AVT system, but it increasingly
appears IST actions may also be important.
Bluehead wrasses show rapid behavioral changes at the
onset of sex change (Warner and Swearer, 1991; Godwin
et al., 1996), and these changes are correlated with increases
in AVT mRNA abundances in the POA in field experiments
(Godwin et al., 2000). As noted above, behavioral sex change
does not depend on the presence of gonads in this species
and neither do dominance-related increases in AVT mRNA
abundances, occurring even in dominant ovariectomized
females exhibiting behavioral sex change (Semsar and Godwin,
2003; Figure 5). Evidence for a causal role of AVT in TP-male-
typical behavior and behavioral sex change comes from field
experiments where (1) administration of AVT could induce
Socially Controlled Sex Change in Fishes 41
Dominant & intact
Dominant & gonads removed
Subordinant & intact
Subordinant &
gonads removed
Relative levels of AVT mRNA
Figure 5 Social status influences AVT mRNA levels in the preoptic
area of the hypothalamus in bluehead wrasses while gonadal status
does not. Dominant individuals were the largest in their social groups
while subordinants were not. Intact animals underwent ‘sham’ opera-
tions to control for the surgical procedure. Inset shows AVT mRNA in
the preoptic area visualized by the technique of in situ hybridization.
AVT, arginine vasotocin. Figure depicts relative AVT mRNA levels in this
comparison from Semsar, K., Godwin, J., 2003. Social influences on
the arginine vasotocin system are independent of gonads in a sex-
changing fish. J. Neurosci. 23, 4386–4393.
territorial behavior in nonterritory holding TP males and the
AVP V1 receptor antagonist ‘Manning compound’ could
reduce it (Semsar et al., 2001) and (2) administration of
Manning compound could prevent establishment on open
territories by nonterritorial TP males and prevent behavioral
sex change by large females after removal of TP males from
their social groups on small reefs (Semsar and Godwin, 2004).
Bluehead wrasses also exhibit differences in AVT receptor
mRNA expression across sexual phenotypes (Lema et al.,
2012). As with many other gene families, a genome duplication
in the teleost lineage led to multiple forms of vasotocin recep-
tors (Lema, 2010), and bluehead wrasses express two forms of
the V1a receptor subtype: V1a1 and V1a2. Both receptor
subtypes are expressed in some key brain regions for socio-
sexual behavior including the ventral telencephalon, POA,
lateral recess, and periventricular portion of the posterior
tuberculum (Lema et al., 2012). These include some critical
regions of what has been called the ‘Social Behavior Network’
or ‘Social Decision-Making Network’ in vertebrate animals
(Newman, 1999; O’Connell and Hofmann, 2012). As with
AVT mRNA expression, sexual phenotype differences are seen
with AVT receptor mRNAs with higher V1a2 mRNA abundance
in the hypothalamus of TP males than females and evidence of
increases during sex change. Interestingly, these receptor
mRNAs are also expressed in the gonads where V1a1 mRNA
shows higher abundances in testes than ovaries and increases
during sex change while V1a2 mRNA shows significant eleva-
tions in sex-changing gonads relative to the ovary (Lema
et al., 2012).
42 Socially Controlled Sex Change in Fishes
Information on AVT signaling in sex-changing fishes is
otherwise relatively limited but does include a few intriguing
studies. The goby T. okinawae is a bidirectional sex changer,
and females that transformed into males under experimental
conditions showed larger AVT-immunoreactive neurons than
fish that transitioned from male to female (Grober and
Sunobe, 1996). The protogynous rock hind grouper
(Epinephelus adscensionis) exhibits a haremic social structure in
the wild, and sex change can be induced in captive groups of
females (Kline et al., 2011a). As with bluehead wrasses, rock
hinds express a V1a-type AVT receptor in a variety of brain
areas implicated in sociosexual behaviors and reproduction
including those described above for bluehead wrasses (Kline
et al., 2011b). AVT signaling may also be important in
protrandrous sex-changing species. Administration of
Manning compound reduced aggressive behaviors in both
juveniles and mature males in the protandrous anemonefish
A. ocellaris in staged laboratory encounters (Yaeger et al.,
2014). These investigators also examined neural activation
through use of c-Fos immunohistochemistry and showed that
mature males receiving Manning compound also showed
reduced c-Fos protein expression in the POA and the
posterior tuberculum (putative homolog of the ventral
tegmental area in tetrapods), although not the anterior
tuberal nucleus (putative homolog of the ventromedial
hypothalamus of tetrapods).
A potential role for IST in sex change and sex-changing
species is much less studied. Sex differences in IST expression
are observed in one very well-studied gonochoristic teleost
model, with female medaka exhibiting higher IST mRNA abun-
dances than males (Kawabata et al., 2012), and at least two sex-
changing species. Female bluebanded gobies showed greater
numbers of IST-immunoreactive cells than either control,
established males or fish in the late stages of female-to-male
sex change (Black et al., 2004). By contrast, RNA sequencing
showed a trend toward TP male bluehead wrasses having
higher levels of IST mRNA at the level of the forebrain and
midbrain transcriptome (Liu et al., 2015) and IST mRNA
levels were significantly higher in TP males in the POA when
measured by in situ hybridization (Thompson et al.,
unpublished).
While more studies are needed, the available information
is consistent with an important role for AVT signaling in
mediating important events in sex change. It is less clear
precisely what the role of this signaling may be. Behavioral
sex change is typically associated with the assumption of
social dominance and increases in aggressive behavior, and
these actions are consistent with known effects of AVT and
related peptides in vertebrates including fishes, although
the precise nature of AVT effects can vary across species
(Godwin and Thompson, 2012). There are also intriguing
possibilities for AVT signaling in the ‘transduction’ of social
cues into reproductive responses. Hamsters and mice show
direct projections of AVP neurons onto kisspeptin neurons
(Vida et al., 2010; Williams et al., 2011), and so AVT
signaling could act through kisspeptin neurons to influence
the HPG axis. Kline (2010) also found evidence of AVT
V1a receptor expression in POA GnRH neurons in the rock
hind grouper, providing evidence for potential direct interac-
tions of AVT signaling with the reproductive axis. Lastly, both
AVT and AVP are implicated in activation of the endocrine
stress axis (Engelmann et al., 2004; Huffman et al., 2015)
and, as noted above, there is evidence for activation of the
HPI axis during behavioral sex change in bluebanded gobies
and the anemonefish A. melanopus (Solomon-Lane et al.,
2013; Godwin and Thomas, 1993; fishes have interrenal
glands rather than adrenal glands, and the interrenals are
the key source of cortisol). Increases in AVT signaling leading
to activation of the HPI axis could be consistent with the
‘classical facilitation of metamorphosis’ model described by
Solomon-Lane and colleagues, but this remains to be rigor-
ously tested.
2.03.3 Conclusions and Future Directions
In his 1988 review of mechanisms by which the endocrine
system might direct the process of sex change in fishes, Rudolf
Reinboth regretfully noted that “It is hard to admit that till now
neither my own efforts nor those of other research groups have
succeeded in generating promising concepts.” Dr Reinboth
underestimated his early and sustained contributions to this
area of study that proved both informative and inspiring for
the researchers who followed. While many important ques-
tions remain, our understanding of this fascinating reproduc-
tive phenomenon has moved well ahead in the ensuing
decades and this progress appears likely to accelerate. This is
due in part to advances that allow matching sophisticated tech-
nical approaches with the heuristic advantages of nontradi-
tional model systems and the ‘experiments of nature’ that
these systems provide.
Gonadal steroid hormones play a prominent and important
role in control of sex change at the gonadal level. There is also
evidence for a role of these hormones in behavioral sex change
and effects on the behavior of sex-changing species more gener-
ally, but it is not yet clear whether changes in steroid hormone
signaling play a role in the key initiating events of sex change
because behavioral sex change can occur in at least one model
system in the absence of gonads (bluehead wrasses), and signif-
icant declines in estrogen production by brain aromatase in the
brain lag behind the beginnings of behavioral sex change in
another (bluebanded gobies). Important next steps in address-
ing the potential role of steroid signaling in behavioral sex
change will be more neuroanatomically focused to assess
changes in the key brain areas for sexual and aggressive
behavior, primarily those in the social behavior network.
Our understanding of the neural circuitry subserving social
behavior has advanced significantly in recent years. A major
advance in this area was the identification of what was termed
the ‘Social Behavior Network’ in mammals by Newman
(1999). This concept has more recently been extended to verte-
brates more generally and into a ‘Social Decision-Making
Network’ that includes both the Social Behavior Network and
the mesolimbic reward system (O’Connell and Hofmann,
2011, 2012). There have been no systematic studies of this
network in sex-changing fishes, but these animals should
provide valuable models. This is because the rapid behavioral
and neurodevelopmental events of sex change take place in
observable and manipulable adult animals. The immediate
early gene approaches that have been used so productively to

explore neural activity in mammals and birds are becoming
increasingly well established in fishes, making this a very acces-
sible approach (e.g., Burmeister et al., 2005; Yaeger et al.,
2014).
Lastly, rapid advances in genomics are making these
approaches feasible in a range of species beyond traditional
models, including sex-changing fishes (e.g., Manousaki et al.,
2014; Liu et al., 2015). Combining transcriptional approaches
such as RNA sequencing at the level of brain areas implicated in
rapid behavioral change should be particularly useful for
understanding the initiating events in the sex change process.
While transcriptional approaches such as RNAseq are inher-
ently descriptive and manipulative tests have until recently
been challenging in nonmodel species, recent methodological
advances including siRNA and vivo morpholinos (e.g., Notch
et al., 2011) should facilitate these important functional tests
of key gene expression events in sex change.
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 2.04 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive
Tactics: Vocal Fish as Model Systems
Ni Y Feng and Andrew H Bass, Cornell University, Ithaca, NY, USA
Ó 2017 Elsevier Inc. All rights reserved.
This chapter is a revision of the previous edition chapter by A.H. Bass, M.S. Grober, volume 1, pp. 579–610, Ó 2009, Elsevier Inc.

2.04.1 Introduction 47
2.04.1.1 Plasticity in a Suite of Characters 48
2.04.2 Alternative Male Reproductive Morphs in Midshipman Fish – A Case Study 48
2.04.2.1 Reproductive and Vocal–Acoustic Behaviors 48
2.04.2.2 Somatic, Gonadal, and Endocrine Traits 51
2.04.2.3 Central Vocal Network 51
2.04.2.3.1 Central Vocal Network, Structure and Function 51
2.04.2.3.2 Intra- and Intersexual Divergence in Vocal Network 53
2.04.3 Hormones, Vocal Behavior, and Vocal Central Pattern Generator 53
2.04.3.1 Divergent Steroid Hormone Profiles and Action between Sex and Male Morphs 54
2.04.3.1.1 Long-Term and Seasonal Steroid Action 54
2.04.3.1.2 Rapid Steroid Action 54
2.04.3.2 Sex- and Male Morph-Specific Patterns of Nonapeptide Actions on Vocal Neurophysiology 56
2.04.4 Hormonal Modulation of Hearing 56
2.04.4.1 Seasonal Auditory Plasticity 58
2.04.4.2 Steroid-Dependent Auditory Plasticity 59
2.04.5 Sites of Hormone Synthesis and Action 59
2.04.5.1 Neuropeptides 59
2.04.5.2 Aromatase 60
2.04.5.3 Androgen and ERs 61
2.04.5.4 Glucocorticoids 61
2.04.5.5 Catecholamines 62
2.04.6 Daily and Seasonal Rhythms in Vocal–Acoustic Mechanisms – Genetic Insights 62
2.04.6.1 VMN Transcriptome 63
2.04.6.2 Saccular Epithelium Transcriptome 65
2.04.7 Summary 65
Acknowledgments 65
References 65

Glossary
Advertisement calls Vocalizations made by males to attract Saccule Division of the vertebrate inner ear that
females. primarily functions as an auditory organ in many species
Arginine vasotocin Nonapeptide in nonmammalian of teleost fish.
vertebrates, including fish, that is homologue of Satellite-spawning A mating tactic whereby a male
vasopressin. attempts to steal fertilizations by spawning immediately
Aromatase (estrogen synthase) Converts testosterone to outside of the nests of other nest-guarding males when
estradiol. a female is present.
Gonosomatic index (GSI) Ratio of gonad mass/body Sneak-spawning A mating tactic whereby a male attempts
mass. to steal fertilizations by spawning inside the nests of other
Isotocin Nonapeptide homologue of mammalian oxytocin nest-guarding males when a female is present.
found in fish.

2.04.1 Introduction reproductive phenotypes that present the opportunity to inves-

tigate how natural variation in neural and hormonal mecha-
Teleost fish, which comprise nearly half of all living species nisms drive plasticity in behavior. In order to causally link
of vertebrates (Nelson, 2006), exhibit a wide range of neural and hormonal events to variation in behavioral output,

Hormones, Brain, and Behavior, 3rd edition, Volume 2 http://dx.doi.org/10.1016/B978-0-12-803592-4.00018-3 47

48 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems
it is beneficial to study behaviors that are readily quantifiable,
amenable to experimental manipulation, relatively simple
in their biomechanical basis, yet naturally variable. Vocal–
acoustic behaviors exhibit these characteristics, as they are vari-
able in several quantifiable characteristics such as duration,
frequency, and amplitude. Furthermore, vocalizations are often
reliable readouts of motor command signals from the central
nervous system (CNS) and reflect an animal’s internal state
and hormonal milieu (Bradbury and Vehrencamp, 2011).
For many species of vertebrates, including teleost fish, vocal-
ization is a key feature of social communication (Bradbury and
Vehrencamp, 2011; Fine and Parmentier, 2015). Fish vocal
behaviors exhibit natural variation across social contexts
(e.g., Brantley and Bass, 1994), daily and seasonal cycles
(e.g., Locascio and Mann, 2008; Forlano et al., 2015b), and
reproductive phenotypes (see below). Furthermore, compared
to other vertebrates such as birds that utilize complex neural
circuitry to control multiple groups of sonic muscles, fish
have a relatively simple and dedicated central pattern generator
(CPG) network that controls as few as one pair of sonic muscles
(see recent reviews Bass et al., 2015; Fine and Parmentier,
2015). Thus, the simple yet naturally variable vocalizations of
fishes are excellent models for understanding the neural and
hormonal basis of vertebrate behavior.
While most species of teleost fish exhibit a single reproduc-
tive phenotype within a sex, at least seven life history patterns
of sexual polymorphisms in behavioral and neurobiological
characters have arisen in teleosts. Specifically, these include
species exhibiting (1) alternative reproductive tactics (ARTs)
with two distinct phenotypes within a sex that utilize different
behavioral tactics to acquire mates; (2) singular, unidirectional
adult sex change and within-sex morph change; (3) singular,
unidirectional sex change; (4) singular, unidirectional intrasex-
ual (male) morph change; (5) serial, back-and-forth, sex
change; (6) bisexual, simultaneous hermaphrodite; and
(7) serial, back-and-forth shift by males between reproductive
and nonreproductive states (for further discussion, see Foran
and Bass, 1999). Elucidating how this staggering diversity in
teleost mating strategies is reflected in and determined by the
CNS is therefore a challenging task that requires a comparative
approach. Here, we review insights on the neural and
hormonal mechanisms driving inter- and intrasexual dimor-
phisms in vocal–acoustic behavior gained from fish models
for acoustic communication. Though we focus on species
with life history pattern 1, the reviewed studies show how
neurobiological characters that lead to the performance of
a social communication behavior, in this case vocalization,
can vary between reproductive morphs. Hence, investigations
of vocal teleosts can inform us about potential mechanisms
in other species (patterns 2–7) that underlie short- and long-
term phenotypic plasticity both within and between the sexes.
2.04.1.1 Plasticity in a Suite of Characters
A suite of characters can often be used to define reproductive
phenotypes in teleost fishes, including body size and colora-
tion, gonad anatomy and physiology, genital papilla shape,
sex-specific accessory gonadal structure, courtship display
behaviors and related structures (e.g., vocal organs), as well
as neuroendocrine and associated endocrine mechanisms
(Bass, 1992). Here, we largely focus on the last two character
sets with respect to acoustic communication in a family of
highly vocal fish, known as the toadfishes (Batrachoididae),
which includes the plainfin midshipman fish (Porichthys notatus
Girard) (e.g., Greenfield et al., 2008; Rice and Bass, 2009).
Section 2.04.2 provides an overview of investigations of vocal
and reproductive behaviors in the midshipman fish to demon-
strate that ARTs can be manifested at multiple levels of biolog-
ical organization. Sections 2.04.3 and 2.04.4 assess hormonal
modulation of neurophysiological mechanisms, namely vocal
(Section 2.04.3) and auditory (Section 2.04.4) that underlie
acoustic courtship behaviors crucial for toadfish reproduction.
Section 2.04.5 reviews sexual polymorphisms in neuroendo-
crine anatomical phenotypes. This includes the distribution
of neuropeptides that have been extensively studied in the
context of reproductive plasticity among species exhibiting
a wide range of life history patterns (see Section 2.04.1) –
gonadotropin-releasing hormone (GnRH) and the nonapepti-
des arginine vasotocin (AVT) and isotocin that are the fish
homologues of mammalian arginine vasopressin and oxytocin,
respectively (Bentley, 1998). Also reviewed are the expression
patterns of steroid receptors and steroid synthesizing enzymes
such as aromatase that catalyzes the conversion of testosterone
to estrogen. Section 2.04.6 reports on the most recent investiga-
tions using transcriptomic RNA sequencing methods to
identify genetic and molecular mechanisms of reproductive-
related plasticity in vocalization and audition. Section 2.04.7
presents summary comments on reproductive and behavioral
plasticity among teleost fishes.
2.04.2 Alternative Male Reproductive Morphs
in Midshipman Fish – A Case Study
The mechanisms underlying the expression of ARTs, from
behavioral ecology and life history strategies to neuroendocri-
nology and central motor control, have been extensively
studied in the plainfin midshipman fish (Figure 1; Table 1).
We first describe reproductive and vocal–acoustic behaviors
and then review the current understanding of the underlying
anatomical and physiological mechanisms.
2.04.2.1 Reproductive and Vocal–Acoustic Behaviors
The plainfin midshipman is a highly vocal species of fish that
mainly breeds at night during the late spring and summer in
the rocky intertidal zone along the western United States and
Canada (Figure 1(a); Bass, 1996). Successful reproduction in
midshipman and other toadfishes involves a suite of courtship
and spawning behaviors and relies on species and sex-typical
vocalizations (Figure 1(b) and 1(c); Brantley and Bass, 1994;
McIver et al., 2014). The plainfin midshipman has two male
reproductive morphs, types I and II, that diverge in courtship
and spawning behaviors as well as a range of morphological
and hormonal characters (Section 2.04.2.2; Figure 1, Table 1;
Bass, 1996). Comparable tactics and morphs have been
described in at least one other species of toadfish, the Lusita-
nian toadfish, Halobatrachus didactylus (e.g., Modesto and
Canário, 2003; Vasconcelos et al., 2010).
 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems 49

(a) Midshipman’s rocky intertidal habitat

(b) Midshipman spawning behavior

(c) Type I male vocal repertoire

Advertisement hum

Continous for mins to hrs

30 ms
Grunt train
Agonistic growl

100 ms

40 ms Also produced by type II males and females

(d) Somatic traits
I II

Figure 1 Midshipman ecology, spawning behavior, and alternative reproductive tactics. (a) Left: During the summer breeding season, type I males
defend nests under rocks along the rocky intertidal zone. White arrows in left panel point to the residential type I males whose rocks have been over-
turned in the picture. Right: Each midshipman nest can contain one or more resident type I males, visiting females, sneak/satellite-spawning type II
males, and newly fertilized eggs, embryos and hatched larvae attached to the underside of the rock. (b) Schematic drawings depicting spawning
scenarios, one where there is no sneaker type II male present (left) and one where a type II male satellite spawns by positioning his body adjacent to
the nest while fanning sperm into the nest of the spawning pair. (c) Midshipman vocal repertoire used under different social contexts. Type I males
produce long duration, ‘hum’ advertisement calls, and agonistic ‘growls’ and ‘grunt trains.’ Type II males and females have only been observed to
produce isolated grunts under agonistic contexts. (d) Somatic characters that diverge across and within sexes include the size and morphology of the
gonads and the vocal organ. Type I males have larger swim bladder and vocal muscles, while type II males and females have larger gonads relative
to body size. Photos by Margaret Marchaterre, line drawings by Margaret Nelson.
50 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems

Table 1 Inter- and intrasexual polymorphic characters in the plainfin midshipman fish

Type I male Type II male Female

Somatic traits
Body size Large Small Intermediate
Gonad/body size ratio Small Large Large (gravid); small (spent)
Ventral coloration Olive-gray Mottled yellow Bronze/golden (gravid);
mottled (spent)
Behavioral traits
Nest building Yes No No
Egg guarding Yes No No
Vocal repertoire Hums, grunts, Grunts Grunts
grunt trains, growls
Call duration Long Short Short
Call fundamental frequency High Low Low
Vocal motor traits
Vocal muscle mass, fiber number, Large Small Small
and diameter
Vocal motoneuron size Large Small Small
Fictive call discharge frequency High Low Low
Fictive call duration Long Short Short
Endocrine traits
Predominant circulating steroids 11-Ketotestosterone Testosterone Testosterone, estradiol
Neuroendocrine traits
Aromatase enzyme activity, mRNA, protein Low High High
GnRH-POA neuron size Large Small Small
GnRH-POA neuron number/body size ratio Low High Low
AVT-POA neuron size Large Small Large
AVT-POA neuron number/body size ratio Low High Low
Fictive call response to exogenous hormones
11-ketotestosterone Facilitation No effect No effect
Testosterone No effect AR-dependent facilitation ER-dependent facilitation
Estradiol Facilitation Facilitation Facilitation
Cortisol Facilitation Suppression Suppression
Arginine vasotocin Suppression No effect No effect
Isotocin No effect Suppression Suppression

AVT, arginine-vasotocin; GnRH, gondotropin-releasing hormone; POA, preoptic area.

Modified from previous version of chapter Bass, A.H., Grober, M.S., 2009. Reproductive plasticity in fish: evolutionary lability in the patterning of
neuroendocrine and behavioral traits underlying divergent sexual phenotypes. In: Pfaff, D.W., Arnold, A.P., Moss, R., Etgen, A.M., Fahrbach, S.E.,
Rubin, R.T., (Eds.), Hormones, Brain and Behavior. Academic Press, New York, pp. 579–610.

Type I males were those already known in the literature
to build nests under rocky shelters in the intertidal and sub-
tidal environments where they fertilize the eggs of females
that are deposited on the roof of the nest (Figure 1(a)
and 1(b)) (e.g., Greene, 1924; Arora, 1948). Females depart
after releasing all of their eggs and type I males remain to
care for the eggs. Midshipman are polygynous in that a single
nest may have several thousand eggs from different females;
a gravid female produces at most close to 200 eggs each
breeding season (DeMartini, 1988; Bass, 1996; McIver
et al., 2014). Juvenile males and females ranging in age
from 5 to 12 months are not found in nests, but rather in
eel grass beds in close proximity to nesting sites (Brantley
et al., 1993a).
Type II males do not acoustically court females, do not
build nests, and do not guard eggs. Rather, type II males
attempt to steal fertilizations from type I males by satellite-
spawning from positions immediately outside, or by sneak-
spawning inside, the nest of a type I male (Figure 1(b); Brantley
and Bass, 1994). Lee and Bass (2004) and Lee and Bass (2006)
later showed that small type I males also exhibit sneaklike
tactics, suggesting an additional level of plasticity within the
type I morph (Lee and Bass, 2004, 2006).
Ibara et al. (1983) first recognized that nesting males
produce an unusually long duration, tonelike acoustic signal
that they called ‘hums’ (Figure 1(c); Ibara et al., 1983),
perhaps the same ‘peculiar humming sounds’ reported even
earlier by Hubbs (1920). Brantley and Bass (1994) showed
that hums function to attract females to nests and character-
ized their spectral and temporal properties along with the
properties of agonistic grunt vocalizations (Figure 1(c); see
McIver et al., 2014 for extensive sound analysis). Individual
hums may continue without cessation for an hour or more
and then continue after a brief pause so that males will
produce them throughout an entire evening (McIver et al.,
2014; Feng et al., 2015).
Underwater playbacks show that females exhibit positive
phonotaxis to hums or tones that mimic the spectral and
temporal properties of hums (Ibara et al., 1983; McKibben
and Bass, 1998, 2001). Individual grunts, on average about
200 ms in duration, may be produced singly or repetitively
at intervals of about 400 ms (‘grunt trains’) during agonistic
 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems
interactions (Figure 1(c); Brantley and Bass, 1994; McIver
et al., 2014). The fundamental frequency (F0) of the multi-
harmonic hum and the pulse repetition rate (PRR) of the
grunt are temperature-dependent, but hover close to 90–
100 Hz in the midshipman’s natural habitat (F0 is equivalent
to the PRR of hums) (Brantley and Bass, 1994; McIver et al.,
2014). Type I males also produce growls with physical attri-
butes that are an amalgam between grunts and hums
(Figure 1(c); McIver et al., 2014). Grunts do not evoke posi-
tive phonotaxis (McKibben and Bass, 1998). Neither type II
males nor females are known to either growl or hum,
although they do generate low-amplitude, isolated grunts
in nonnesting contexts (Figure 1(c); Brantley and Bass,
1994).
2.04.2.2 Somatic, Gonadal, and Endocrine Traits
Like other species exhibiting multiple reproductive phenotypes
(see Section 2.04.1; Bass and Grober, 2009), type I and type II
male midshipman can be distinguished by a suite of gonadal,
somatic, and endocrine characters (Bass, 1996). The two male
morphs show a dramatic dimorphism in body, gonad and
vocal muscle size (Bass and Marchaterre, 1989a,b). On average,
type II males are about 50% smaller in body size than type I
males. The GSI is close to ninefold greater in type II males
who may invest close to 20% of their body mass in testes
(Figure 1(d)). Gravid females with mature eggs have a large
GSI like that of type II males, although the female GSI is greater
(Figure 1(d)).
Midshipman, such as other toadfishes, generate sound by
contracting a pair of superfast muscles attached to the wall of
the swim bladder (Figure 1(d); Cohen and Winn, 1967;
Rome, 2006). The vocal muscles are more massive in type I
males, due in part to dramatic dimorphisms ranging from
muscle fiber number to the dimensions of muscle fibers, sarco-
meres, and Z-lines (zone of overlap between thin, actin
filaments of adjacent sarcomeres), to the number of
mitochondria, all of which are greater in type I males
compared to type II males and females that are similar to each
other (Bass and Marchaterre, 1989a; Brantley et al., 1993a).
Both male morphs and females have olive-gray, dorsal body
coloration. The ventral body coloration of type I males in
reproductive condition is light to dark gray, while that of type
II males is mottled yellow, and of gravid females is bronze to
golden (Table 1). Females that have released their mature eggs
are more like type II males in their ventral coloration.
The two male midshipman morphs diverge from each
other, and from females, in plasma levels of androgens (testos-
terone and 11-ketotestosterone), estrogens (17b-estradiol), and
cortisol that is the main glucocorticoid present in teleost fish
(Table 1; see Bury and Sturm, 2007 for review of steroid
biosynthesis in teleosts). Type II males have two- to threefold
higher cortisol levels compared to type I males (Arterbery
et al., 2010a). Circulating testosterone levels are detectable in
both male and females, though found to be the highest in
type II males and the lowest in type I males, while intermediate
in females (Brantley et al., 1993b; Sisneros et al., 2004b).
17b-Estradiol is the predominant circulating steroid in females,
although some males also show detectable but much lower
levels than testosterone (Brantley et al., 1993b; Sisneros et al.,
51
2004b). 11-Ketotestosterone is a nonaromatizable (i.e., cannot
be converted to an estrogen by aromatase) form of testosterone
found among teleost fishes. Plasma 11-ketotestosterone levels
are several times greater than testosterone in reproductively
active type I males, but essentially nondetectable in type II
males and females (Brantley et al., 1993b; Knapp et al.,
1999b; Sisneros et al., 2004b). Within type I males, hormone
levels are correlated with courtship activity as actively
humming type I males have higher 11-ketotestosterone levels
than nonhumming males (Genova et al., 2012). Regardless
of sex, the levels of predominant steroids tend to peak in the
prenesting and early nesting season (Sisneros et al., 2004b).
The decline in 11-ketotestosterone levels over the breeding
season in nesting type I males also corresponds to increased
investment in parental care of fertilized eggs and larvae (Knapp
et al., 1999b). The circulating steroid profiles of type I male and
female midshipman resemble those in closely related toad-
fishes that exhibit two male morphs (Modesto and Canário,
2003) and, more generally, teleosts with ARTs (Brantley et al.,
1993b; Oliveira, 2006; Knapp and Neff, 2007). In summary,
seasonal profiles of circulating steroids diverge between sexes
and within male morphs, with type II males aligning with
females instead of type I males.
2.04.2.3 Central Vocal Network
We first present an overview of the organization of the vocal
motor network in the brain of midshipman fish and, more
generally, in toadfishes. This provides the framework for
a more complete review in Section 2.04.4 of the hormonal
modulation of the neurophysiological mechanisms of vocali-
zation. The vocal network of midshipman also provides an
exemplar of how a suite of neurobiological characters that
lead to the performance of a social communication behavior,
in this case vocalization, can vary between reproductive
morphs.
2.04.2.3.1 Central Vocal Network, Structure and Function
The initial demonstration that low molecular weight biotin
compounds (biocytin and neurobiotin) could be transneuro-
nally transported across multiple synapses in midshipman
fish allowed the visualization of the entire complement of
vocal hindbrain neurons (Figure 2(a); Bass et al., 1994). Subse-
quent studies revealed forebrain and midbrain vocal sites that
together with more focal studies of hindbrain sites delineated
a central vocal network that was highly interconnected with
both the auditory and neuroendocrine systems (e.g., Bass
et al., 2000; Goodson and Bass, 2002; Kittelberger, 2006;
Chagnaud et al., 2011; Kittelberger and Bass, 2013; Chagnaud
and Bass, 2013). Comparable patterns of connectivity were
observed in closely related toadfish (Chagnaud and Bass,
2014) and in more distantly related species of vocal teleosts
(Bass et al., 2015).
Intracellular recording and staining identified the func-
tional properties of a vocal CPG in the caudal hindbrain
and the rostral spinal cord that included three anatomically
and physiologically distinct neuronal populations: vocal
motor nucleus (VMN), vocal pacemaker nucleus (VPN),
and vocal prepacemaker nucleus (VPP) (Figure 2(a)–2(c);
Bass and Baker, 1990, 1991; Chagnaud et al., 2011, 2012;
52 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems

(a) (d) Natural call

VPP

VPP Vocal nerve fictive call

VPN

.1mV
1s

VMN VMN

vTr .1mV
25 ms
VPN Frequency
(b)
VOC duration
Spinal cord Hindbrain Midbrain
VMB Forebrain
VMN
VPN VPP POA
VN

(c)

Figure 2 Hindbrain central pattern generator (CPG) for vocalization in the plainfin midshipman fish, Porichthys notatus. (a) Applying biocytin to
the cut end of the nerve that innervates a single vocal muscle at the level of the swim bladder leads to bilateral, transneuronal labeling of the vocal
CPG that includes three topographically separate populations of neurons. Vocal muscles are innervated by a vocal motor nucleus (VMN) whose
axons make up the vocal tract (vTr) and vocal nerve (VN). The VMN is innervated by an adjacent column of vocal pacemaker nucleus (VPN) that
receives input from a more rostral vocal prepacemaker nucleus (VPP). Bar scale represents 200 mm in A and B (25 mm in the insets). (b) The
vocal CPG, mapped onto a lateral view of a midshipman brain, receives input from a vocal midbrain region (VMB) that is driven, in turn, by the
forebrain’s preoptic area (POA) and anterior hypothalamus. (c) Shown here are representative intracellular records from vocal motor, pacemaker,
and prepacemaker neurons (see text for more complete description). Vocal prepacemaker neurons project to the auditory system, thereby
providing a vocal corollary discharge that relays information from the vocal CPG to the auditory system about the temporal properties of natural
vocalizations. A descending forebrain–midbrain pathway drives the vocal CPG. (d) Spontaneous neurophysiological responses from the VN,
a fictive call, are aligned with a naturally occurring ‘grunt train,’ highlighting the correspondence between the temporal properties of the VN motor
volley and natural vocalizations. The lower trace is an expansion of a single vocal nerve response (VOC) that corresponds to a single grunt;
duration is the time between first and last pulse and frequency is the pulse repetition rate. Timescale and amplitude for all records are indicated.
Modified from Chagnaud, B.P., Baker, R., Bass, A.H., 2011. Vocalization frequency and duration are coded in separate hindbrain nuclei. Nat.
Commun. 2, 346–11; Chagnaud, B.P., Zee, M.C., Baker, R., Bass, A.H., 2012. Innovations in motoneuron synchrony drive rapid temporal modula-
tions in vertebrate acoustic signaling. J. Neurophysiol. 107, 3528–3542.
 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems
Chagnaud and Bass, 2013, 2014). Bilateral and contiguous
VMNs lie along the midline, extending from the caudal hind-
brain into the rostral spinal cord (Figure 2(a)–2(c)). Each
VMN gives rise to axons, coalescing into a vocal tract that
exits the brain via the paired occipital nerve roots to form
a vocal nerve that innervates the ipsilateral vocal muscle
(Figure 2(a)–2(c)). The VPN neurons form a column that
extends along the ventrolateral border of each VMN
(Figure 2(b) and 2(c)). Individual pacemaker neurons bilater-
ally innervate both VMNs. The more rostral VPP (originally
designated the ventral medullary nucleus (Bass et al., 1994))
innervates the paired VPN columns (Figure 2(a)–2(c)).
Each neuronal compartment of the vocal CPG (VMN, VPN,
VPP) has distinct neurophysiological attributes (Figure 2(c)).
The VMN is the last node that relays the output of the CPG
directly to vocal muscles. The highly synchronous output of
each VMN generates a vocal motor volley that is readily
recorded, intracranially, from each vocal nerve root as it exits
the brain (Figure 2(c) and 2(d)). Bilaterally, the vocal motor
volleys fire in phase, leading to the simultaneous contraction
of both vocal muscles (Cohen and Winn, 1967). VMN moto-
neurons fire action potentials with a highly stereotyped repeti-
tion rate/frequency that determines muscle contraction rate in
a 1:1 manner (Figure 2(d)). Furthermore, the differential size-
dependent recruitment of VMN neurons determines call ampli-
tude (Chagnaud et al., 2012).
Afferent input to the VMN arises from VPNs that generate
pacemaker-like (hence their name) action potentials that are
matched 1:1 in frequency with the highly synchronous output
of the VMN (Chagnaud et al., 2011). Finally, upstream to the
VPN, the VPP generates sustained depolarizations whose dura-
tion correlates with the duration of VPN-VMN activity
(Chagnaud et al., 2011). In summary, VMN, VPN, and VPP
separately code for distinct properties of natural vocalizations,
namely amplitude (VMN), PRR that sets the fundamental
frequency (VPN), and duration (VPP) (Figure 2(c); Bass and
Baker, 1990; Chagnaud et al., 2011, 2012; Chagnaud and Bass,
2014).
In the sections below, we refer to the neurophysiologically
recorded vocal motor volley (Figure 2(d)) as a fictive call because
it is a patterned motor command that directly determines the
properties of a natural behavior (see Marder and Calabrese,
1996), in this case vocalization. The ability to quantify changes
in fictive calls induced by pharmacological perturbations has
allowed us to elucidate causal roles of hormones and neuromo-
dulators in the patterning of vocalization (Section 2.04.3).
2.04.2.3.2 Intra- and Intersexual Divergence in Vocal Network
As summarized in Section 2.04.2.1, type I male midshipman fish
have a more extensive vocal repertoire than type II males and
females, which are similar to each other. The divergence in vocal
behavior is paralleled by a divergence in the anatomy of both the
sonic muscles (Section 2.04.2.2, Figure 1(d)) and of the vocal
network. Centrally, inter- and intrasexual polymorphisms in
midshipman fish range from the dimensions of motoneurons
(VMN) and premotoneurons (VPN, VPP) to hormone-specific
influences on the output of the vocal network (see Section
2.04.3; Table 1). Specifically, inter- and intrasexual dimorphisms
are found in the size of vocal motoneurons, such that type I
males have larger somata (Bass and Marchaterre, 1989b; Bass
53
and Baker, 1990; Bass et al., 1996), axon diameters (Bass and
Andersen, 1991), and neuromuscular junctions (Fluet and
Bass, 1990) than type II males and females. The volume of the
VMN follows a similar pattern (Bass et al., 1996). Unlike VMN
dimensions, there is no significant sexual dimorphism in the
number of vocal motoneurons as determined by the number
of myelinated axons in the vocal nerve, although type II males
have fewer myelinated fibers compared to type I males and
females (Bass and Andersen, 1991). Because type I males have
three- to fivefold more muscle fibers, but a similar number of
motoneurons compared to females and type IIs, it is predicted
that type I males have larger motor units, i.e., each motoneuron
innervates a larger number of muscle fibers (Bass and Baker,
1990; Bass and Andersen, 1991). Along with peripheral vocal
muscle morphology (see above), these central traits likely
contribute to the inter- and intrasexual differences in the vocal
repertoire (Brantley and Bass, 1994; McIver et al., 2014).
Developmental studies of the vocal motor system support
the hypothesis that the two male reproductive morphs in the
plainfin midshipman fish adopt nonsequential, mutually
exclusive growth trajectories during their first year of life
(Bass, 1996; Bass et al., 1996; Knapp et al., 1999a). Sexual
maturation of the type I male’s vocal CPG parallels that of
the vocal muscle (Brantley et al., 1993a; Bass et al., 1996).
Type II males and females retain a juvenile phenotype, whereas
a subset of juvenile males exhibits a growth trajectory that leads
to the type I male vocal CPG phenotype. Both type II male and
female midshipman reach sexual maturity at an earlier age and
smaller body size than type I males (Bass et al., 1996).
Together, the results indicate a life history trade-off between
investment in larger body size and an expansive vocal motor
system by type I males versus earlier reproduction and gonad
investment by type II males and females. The adoption of
one developmental trajectory versus the other involves trade-
offs between the age and size at sexual maturity and a suite
of secondary sex characters. Though both male morphs share
gonadal sex, they are highly divergent in a suite of other char-
acters that the type II male morph shares with females, reflect-
ing a common pattern of developmental trade-offs (Table 1).
2.04.3 Hormones, Vocal Behavior, and Vocal
Central Pattern Generator
Given that vertebrate vocalizations reflect an interaction between
internal states and external social/reproductive contexts, it is not
surprising that steroid hormones, known to regulate other
aspects of reproductive behavior and physiology, also directly
control vocalizations by acting upon central vocal control path-
ways (e.g., Remage-Healey and Bass, 2006; Bass and Remage-
Healey, 2008). Aside from early qualitative reports of testoster-
one’s potentiating effects on sound production in the satinfin
shiner, Cyprinella analostana (formerly Notropis analostanus)
(Winn and Stout, 1960), steroid regulation of fish vocalization
has not been well studied outside of the toadfish family. In the
last decade, comprehensive studies in toadfishes have established
rapid and robust steroid effects on vocal behavior and steroid
sensitivity of the underlying central vocal motor network
(Remage-Healey and Bass, 2004, 2005, 2006, 2007). Further-
more, inter- and intrasexual divergence in circulating hormone
54 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems
profiles (see Section 2.04.2.2) are paralleled by sex and male
morph patterns of neural sensitivity to specific steroid hormones
(Brantley et al., 1993b; Sisneros et al., 2004b; Remage-Healey
and Bass, 2007; Arterbery et al., 2010a; Fergus and Bass, 2013).
Steroid action is mediated by ligands binding with specific
intracellular cytosolic receptors, forming a steroid–receptor
complex that translocates into the nucleus and targets
steroid-specific DNA response elements to regulate gene expres-
sion (Lösel and Wehling, 2003). This mode of action initiates
a genomic response that could take 30 min to days. Steroids
can also exert rapid, nongenomic effects that occur on the order
of seconds to less than 30 min. Rapid steroid action may
involve activation of nonclassical membrane receptors in addi-
tion to classical intracellular receptors that lead to changes in
secondary messenger pathways (Lösel and Wehling, 2003). In
toadfish, both long-term and rapid modes of steroid action
have been observed, including seasonal changes in reproduc-
tive states and rapid changes in context- and stimulus-driven
vocalization (see Table 1 for summary). These steroid effects
on vocal behavior are likely mediated by steroid receptors
expressed throughout the vocal control network that patterns
vocalizations (see Section 2.04.5).
2.04.3.1 Divergent Steroid Hormone Profiles and Action
between Sex and Male Morphs
It is well established that steroid hormones act over a dynamic
range of timescales. During developmental critical periods
among mammals (e.g., perinatal and puberty), steroids exert
long-lasting organizational effects on peripheral and central
tissues that lay down the foundation for sex differences in
adulthood (see review by Arnold, 2009). In adulthood, steroids
exert reversible activational effects that control sexually dimor-
phic phenotypes (Arnold, 2009). In this section, we review the
current knowledge of steroid hormone regulation of toadfish
vocalization at vocal motor and behavioral levels over multiple
timescales. Because this topic has been recently extensively
reviewed (Bass et al., 2015), we focus discussions here on sex
and morph differences.
2.04.3.1.1 Long-Term and Seasonal Steroid Action
A prominent effect of steroid action over the developmental and
seasonal timescale is the growth of primary and secondary sexual
characteristics. In the context of midshipman courtship vocal
behavior, 11-ketotestosterone (11-KT) has been shown to act
peripherally to increase the size of the vocal muscle and
testosterone-propionate centrally to increase the size of vocal
motoneurons (Brantley et al., 1993a; Bass, 1995; Bass and
Forlano, 2008). As mentioned above, the marked sexual and
morph dimorphism in vocal muscle mass parallels their respec-
tive vocal repertoires: type I males who produce long duration
courtship hums have sixfold larger vocal muscle mass (normal-
ized to body weight) when compared to type II males or females
who can only produce isolated grunts (Figure 1(d); Brantley and
Bass, 1994). The vocal muscle hypertrophy in type I males is
androgen-dependent (Brantley et al., 1993a). Long-term
(8–9 weeks) treatment with androgen implants, including
11-KT and testosterone-propionate, increased the vocal muscle
weight and myofibril and mitochondrial-filled sarcoplasm ultra-
structure in juvenile type I males, type II males, and females,
toward levels seen in adult type I males (Brantley et al., 1993a).
Although circulating steroid levels were not measured by
Brantley et al. (1993a), a later study that induced 11-KT levels
in type II males to physiological levels found in type I males
also showed an expansion of the vocal muscle (Lee and Bass,
2005).
Long-term (8–9 weeks) testosterone-propionate treatment
also enlarged the soma size of VPP, VPN, and VMN neurons
in juvenile males compared to size-matched controls (Bass,
1995; Bass and Forlano, 2008). Neurophysiologically, this
treatment increased the PRR of the vocal motor volley, or fictive
calls (Section 2.04.2.3.1), evoked from juvenile males (Bass,
1995). In adult type II males, this treatment had no effects
on VMN soma size (Bass and Forlano, 2008). Furthermore,
increasing 11-KT plasma levels in type II males to those of
type I males did not induce an increase in overall VMN volume,
nor the expression of type I courtship behaviors (Lee and Bass,
2005). Instead, exogenous 11-KT intensified type II cuckholdry
behaviors (Lee and Bass, 2005). Thus, after committing to the
type II trajectory, the vocal CPG is no longer sensitive to the
masculinizing effects of androgens leading to a type I male
phenotype, suggesting that male morph differences in
midshipman are organized during an earlier critical period.
Finally, the rise in androgen levels at the beginning of the
breeding season likely activates and prepares the central and
peripheral nervous systems structurally and metabolically for
acoustic courtship activity, especially in type I males.
2.04.3.1.2 Rapid Steroid Action
In the midshipman and the closely related Gulf toadfish (Opsa-
nus beta), field and in vivo neurophysiology studies show that
steroids act rapidly on the order of minutes to modulate vocal
behavior and vocal CPG output (Table 1). Steroid hormones
are likely acting via specific receptors within neuroendocrine
and vocal pathways (Figure 3(a)). The neuroanatomical loca-
tion of steroid receptors is reviewed in more detail below in
Section 2.04.5. Rapid steroid modulation of the vocal CPG
was first demonstrated by in vivo neurophysiological investiga-
tions in type I male midshipman (Remage-Healey and Bass,
2004) and subsequently in male and female Gulf toadfish
(Remage-Healey and Bass, 2006) as well as type II male and
female midshipman (Remage-Healey and Bass, 2007).
Steroid action in the vocal neural network in midshipman is
both sex- and male morph-specific (Figure 3(b); Remage-
Healey and Bass, 2007). In both type I male midshipman
and male Gulf toadfish, which is only known to have one
male morph, systemic injections of 11-KT, but not testosterone,
increased the duration of electrically evoked fictive calls within
5 min (Figure 3(b)) (Remage-Healey and Bass, 2004, 2006). In
contrast, systemic injections of testosterone, but not 11-KT,
rapidly increased fictive call duration in type II male and female
midshipman (Figure 3(b)) (Remage-Healey and Bass, 2007).
These results are consistent with the divergent profiles of
predominant circulating androgens found between the male
morphs and sexes of midshipman (see Section 2.04.2.2).
Although testosterone potentiated vocal CPG output in both
type II male and females, its aromatization to 17b-estradiol
was necessary for potentiating the female vocal CPG, while
androgen receptor activation was necessary for potentiation
in type II males (Remage-Healey and Bass, 2007).
 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems 55

(a) Steroid receptor and aromatase distribution within vocal network

Cerebellum Midbrain
Forebrain
Hindbrain 1,2,4,5
Spinal cord PAG
1-5 OB
VMN PL 1-5
1 POA On
VPN VPP 1,5
1,2,4,5 AT Opn
vT
Vocal nerve 1 mm
POA-AH
Pituitary
α 3:ERβ1 4:ERβ2 5:ARO
1:AR 2:ERα

(b) Rapid steroid effects on vocal physiology

Type I male
200 11-KT 200 T
Type II male
180 180
Female
160 160
140 140
120 120
100 100
80 80
% Baseline duration

60 60
40 40
0 15 30 45 60 75 90 105120 0 15 30 45 60 75 90 105120

200 Cortisol 200 Estradiol

180 180
160 160
140 140
120 120
100 100
80 80
60 60
40 40
0 15 30 45 60 75 90 105120 0 15 30 45 60 75 90 105120
Time (min)

Figure 3 Rapid steroid action in the vocal network reveals sex and morph differences. (a) Neuroanatomical distribution of steroid receptors and
aromatase (estrogen synthase). Abbreviations: AR, androgen receptor; ER, estrogen receptor; ARO, aromatase; OB, olfactory bulb; On, olfactory nerve;
Opn, optic nerve; POA-AH, preoptic area–anterior hypothalamus; vT, ventral tuberal nucleus; AT, anterior tuberal nucleus; PL, paralemniscal nucleus;
PAG, periaqueductal gray; VPP, vocal prepacemaker nucleus; VPN, vocal pacemaker nucleus; VMN, vocal motor nucleus. (b) Summary of results from
in vivo neurophysiology studies demonstrating rapid (within 5 min) effects of peripheral steroid injection on the duration of electrically evoked fictive
calls (% change with respect to baseline). Plotted are means for each time point taken from Remage-Healey and Bass (2004, 2007). When multiple
doses were used, the means from the most effective dose were plotted. Abbreviations: 11-KT, 11-ketotestosterone; T, testosterone. Modified from
Bass, A.H., Chagnaud, B.P., Feng, N.Y., 2015. Comparative neurobiology of sound production in fishes. In: Ladich, F. (Ed.), Sound Communication in
Fishes, Animal Signals and Communication. Springer Vienna, Vienna, pp. 35–75, with kind permission from Springer Science þ Business Media.

The rapid effects of cortisol in midshipman also diverged
across phenotypic sex, as cortisol injections increased fictive
call duration in type I males but decreased duration in type II
males and females (Figure 3(b); Remage-Healey and Bass,
2007). However, no sex differences were observed in Gulf toad-
fish, as cortisol induced a rapid increase in fictive call duration
in both males and females (Remage-Healey and Bass, 2006).
Interestingly, when the hindbrain vocal CPG was isolated
from descending forebrain and midbrain input in type I male
midshipman and male Gulf toadfish, the effects of 11-KT and
cortisol on fictive call duration did not last more than
30 min, compared to 2 h for 11-KT and 45 min for cortisol in
intact preparations (Remage-Healey and Bass, 2004, 2006).
Thus, the results suggest that steroid action within upstream
vocal–acoustic centers mediates sustained stimulation of vocal
behavior, while steroid action within the hindbrain alone is
sufficient to elicit short-term increases in vocalization
(Remage-Healey and Bass, 2004).
Unlike 11-KT, testosterone, and cortisol, the rapid effects of
17b-estradiol did not differ dramatically between the male
56 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems
morphs or sexes (Figure 3(b)). In midshipman, 17b-estradiol
increased fictive call duration in type I males, type II males,
and females (Remage-Healey and Bass, 2004, 2007). Thus,
regardless of within-male comparisons, type II males always
aligned with females in their neurophysiological responses to
steroids, including 17b-estradiol (Figure 3(b)). In the Gulf
toadfish, 17b-estradiol had no effect in either males or females
(Remage-Healey and Bass, 2006), revealing an unexpected
divergence in hormone sensitivity between closely related toad-
fish species. In summary, in vivo neurophysiology studies have
uncovered sex, morph, and species differences of rapid steroid
action within the vocal CPG.
Rapid steroid effects on natural vocal behavior have also
been investigated in the Gulf toadfish by field experiments,
which largely support in vivo neurophysiology results summa-
rized above (Table 1) (Remage-Healey and Bass, 2005,
2006). First, it was observed that focal nesting males began
vocalizing within 48 h of exposure to an actively calling natural
population, and this exposure increased plasma 11-KT levels in
exposed males compared to males not exposed to calling
neighbors (Remage-Healey and Bass, 2005). Furthermore,
males not exposed to calling neighbors did not begin to
vocalize. Finally, nesting males exposed to acoustic playbacks
of conspecific calls with durations that approximated the pop-
ulation mean increased call rate and duration as well as plasma
levels of 11-KT within 20 min. These results suggested that
a social stimulation of 11-KT levels potentiates vocal behavior.
A causal relationship between 11-KT levels and expression
of natural vocal behaviors was established by a follow-up study
of Gulf toadfish showing that exogenous delivery of food con-
taining 11-KT increased calling rate in nesting males within
10–20 min (Remage-Healey and Bass, 2006). Interestingly,
there was no change in call duration, suggesting an influence
of sound playbacks and the sense of hearing on the results of
the earlier study showing increases in call duration as well
(Remage-Healey and Bass, 2005). Together, these field experi-
ments support the hypothesis that 11-KT rapidly facilitates
male vocal behavior, likely by acting via classical intracellular
receptors, which was supported by receptor-specific antagonist
treatments during fictive calling (Remage-Healey and Bass,
2004). 11-KT presumably acts within the vocal network
expressing androgen receptors (see Figure 3(a); Forlano et al.,
2010), though the effects of focal steroid injections directly
into vocal nuclei remain to be investigated.
In summary, complementary field behavior and laboratory
neurophysiological studies in toadfishes demonstrate steroid
regulation of dynamic social behaviors that fluctuate on the
minute-by-minute timescale. Neurophysiology revealed sex,
male morph, and species-specific steroid action in the vocal
network (Table 1; Figure 3(b)). The accessibility to monitor
and manipulate natural vocal behavior as well as the output
of the vocal network makes toadfishes excellent models for
studying hormone regulation of the neural substrates of a verte-
brate behavior.
2.04.3.2 Sex- and Male Morph-Specific Patterns
of Nonapeptide Actions on Vocal Neurophysiology
AVT and isotocin are nine amino acid peptides, known as non-
apeptides, that belong to the arginine vasopressin-oxytocin
family of peptides (Bentley, 1998) that modulates a range of
vertebrate social behaviors (reviewed in Goodson and
Bass, 2001; Godwin and Thompson, 2012; Goodson, 2013).
In teleosts, AVT (homologue to mammalian vasopressin)
and isotocin (homologue to mammalian oxytocin) are
produced exclusively within the POA (Figure 4(a); see Section
2.04.5.1 for anatomical distribution) (Goodson and Bass,
2001; Goodson, 2008). Although nonapeptide regulation of
vocal behavior is investigated in far less detail compared to
steroids, complementary in vivo neurophysiology studies have
demonstrated sex and male morph-specific actions within
the midshipman vocal network (Goodson and Bass,
2000a,b). Furthermore, in addition to fish (Goodson and
Bass, 2000a,b), nonapeptide modulation of vocalization is
widespread among vertebrates, including amphibians and
birds (reviewed in Goodson and Bass, 2001).
In type I male midshipman, AVT injection into the region
of the caudal POA and anterior hypothalamus (AH) induced
a dose-dependent inhibition of fictive call duration and the
number of calls elicited from the POA-AH (Figure 4(b);
Goodson and Bass, 2000a). Supporting vocal inhibition by
AVT, delivery of an arginine-vasopressin V1a receptor
antagonist significantly increased call duration (Figure 4(b);
Goodson and Bass, 2000a). Additionally, AVT injection into
a vocally active midbrain site in the tegmentum inhibited
the number of fictive calls elicited from the same area, as
well as increased the latency of fictive call initiation, but did
not affect call duration (Goodson and Bass, 2000b). These
results show that AVT inhibits vocal network excitability at
both forebrain and midbrain levels in type I males. In
contrast to AVT, isotocin was ineffective in changing vocal
output parameters in type I males, suggesting that despite
the high conservation in amino acid sequence and synthesis
within the POA, AVT and isotocin bind to receptors with
high specificity.
In type II males and females, however, AVT had no effects
on the output of the vocal network (Figure 4(b); Goodson
and Bass, 2000b). Instead, isotocin injection in the POA-
AH of type II males and females exerted dose-dependent
inhibition of vocal output, while an oxytocin receptor
antagonist facilitated vocal output (Figure 4(b)). These
results suggest that in species with ARTs such as the
midshipman, vasotocin-like peptides are dissociated from
gonadal sex and align instead with phenotypic sex. More
broadly, these results along with studies in other
vertebrates show that nonapeptides belonging to the
vasotocin family are evolutionarily labile in their sex-,
morph-, and species-dependent actions on reproductive
and courtship behaviors (Goodson, 2008; Bass and Grober,
2009).
2.04.4 Hormonal Modulation of Hearing
Sound-producing teleost fishes provide important model
systems for investigating peripheral and central mechanisms
encoding acoustic stimuli (Bass and McKibben, 2003; Edds-
Walton and Fay, 2008). Most studies of hormonal influ-
ences on audition in fishes have focused on plasticity at
the level of the saccule, the main auditory end organ of
 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems 57

(a)
Cerebellum Midbrain
Forebrain
Spinal cord Hindbrain
PAG OB
VMN PL
VPN POA On
VPP
AT Opn
vT
Vocal nerve 1 mm
POA-AH
Pituitary
(b)
Summed fictive call duration
150 Type I males
(% baseline)
100

50

0
1,000ng 0ng 0.1ng 10ng 1,000ng 1,000ng
Anti-AVP Control AVT IT

325
Females
Summed fictive call duration

300

100
(% baseline)

50

0
100ng 0ng 0.1ng 10ng 1,000ng 1,000ng
Anti-OXY Control IT AVT

300 Type II males

Summed fictive call duration
(% baseline)

100

50

0
100ng 0ng 0.1ng 10ng 1,000ng 1,000ng
Anti-OXY Control IT AVT

Figure 4 Arginine vasotocin neural expression and regulation of vocal central pattern generator output. (a) Simplified representation of arginine–
vasotocin (AVT) pathways (red arrows) mapped onto a sagittal view of the plainfin midshipman fish vocal network (black arrows) based on immuno-
cytochemistry (Goodson and Bass, 2000b). Arrow thickness denotes projection density. AVT-immunoreactive (AVT-ir) pathways originate almost
exclusively from the preoptic area (POA) and innervate vocally active brain sites in the POA, anterior tuberal nucleus (AT), ventral tuberal
hypothalamus (vT), paralemniscal midbrain tegmentum (PL), and midbrain periaqueductal gray (PAG). Other abbreviations: OB, olfactory bulb; On,
olfactory nerve; Opn, optic nerve; POA-AH, preoptic area–anterior hypothalamus; VMN, vocal motor nucleus; VPN, vocal pacemaker nucleus; VPP,
vocal prepacemaker nucleus. (b) Vocal motor output of the midshipman in response to POA-AH stimulation 10 min after delivery of AVT, isotocin
(IT), or their respective mammalian antagonists (Anti-AVP; Anti-OXY). Top panel, type I males; middle panel, females; bottom panel, type II males.
Total fictive call duration of type I males is inhibited in a dose-dependent manner by AVT, disinhibited by anti-AVP, and not affected by IT. By
contrast, fictive calls of females and type II males are not affected by AVT, but inhibited by IT and disinhibited by anti-OXY. From Bass, A.H.,
Chagnaud, B.P., Feng, N.Y., 2015. Comparative neurobiology of sound production in fishes. In: Ladich, F. (Ed.), Sound Communication in Fishes,
Animal Signals and Communication. Springer Vienna, Vienna, pp. 35–75, with kind permission from Springer Science þ Business Media.

the inner ear in toadfishes and many other teleost species distinct branches of the eighth (VIII) cranial nerve. We first
(Cohen and Winn, 1967; Bass and McKibben, 2003; discuss patterns of seasonal plasticity that provided the
Edds-Walton and Fay, 2008). The saccule’s hair cell epithe- foundation for subsequent studies looking at hormonal
lium is innervated by ganglion cell afferents that form influences. A recent and more detailed review of hormonal
58 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems

mechanisms and hearing in fishes is available elsewhere
(Forlano et al., 2016).
2.04.4.1 Seasonal Auditory Plasticity
Sisneros and Bass (2003) provided the first example of natu-
rally occurring, seasonal changes in peripheral frequency sensi-
tivity in a teleost fish, in this case by saccular, auditory afferents
in female midshipman fish (Figure 5(a); Sisneros and Bass,
2003). The saccular afferents of female midshipman that are
in nonreproductive condition show maximal frequency sensi-
tivity in the range of approximately 60–100 Hz, whereas
females in reproductive condition during the breeding season
show peak sensitivity extending from approximately 60 to
350 Hz. This broader range of sensitivity encompasses the
fundamental frequency and upper harmonics of male adver-
tisement calls that can have as much and sometimes even
more energy than the fundamental (Figure 5(a)). From
a neurophysiological perspective, the higher harmonics might
improve the neurophysiological encoding of a call’s funda-
mental frequency (McKibben and Bass, 1999). From a behav-
ioral perspective, one advantage of enhanced detection of the
higher harmonics for all members of the species would be an
improvement in the ability to detect the calls of nesting type
I males from a greater distance since the upper harmonics
would propagate farther in the shallow water environment
where midshipman spawn (Fine and Lenhardt, 1983; Bass
and Clark, 2003).
Adopting methods first used in goldfish to record a saccular
evoked potential (Furukawa and Ishii, 1967), the analog of
a cochlear microphonic, Sisneros (2009) showed that the
seasonal changes in frequency encoding in the midshipman
female saccule could be explained by plasticity at the level of
the hair cell epithelium (Sisneros, 2009). Subsequent studies
showed the same seasonal phenotype for type I males, with
pilot studies supporting this pattern for type II males
(Sisneros, 2009; Whitchurch and Sisneros, 2011; Rohmann
and Bass, 2011). Thus, seasonal changes in peripheral hearing
sensitivity show neither a sex- nor male morph-specific
pattern. Interestingly, seasonal shifts in peripheral frequency
sensitivity may not be shared with other toadfish species, at
least based on a study of the Lusitanian toadfish that has
ARTs (Section 2.04.2.1) but does not show the seasonal migra-
tion to deeper water habitats observed for midshipman
(Vasconcelos et al., 2011).
More recent studies have investigated the molecular basis
for peripheral auditory plasticity. The frequency tuning of
hair cells in reptiles and birds can be explained by electrical
resonance, with the abundance of large conductance,
calcium-activated potassium (BK) channels increasing as reso-
nant frequency increases (Fettiplace and Fuchs, 1999). Saccular
hair cells in teleosts also exhibit electrical resonance (Steinacker
and Romero, 1992; Sugihara and Furukawa, 1995). Rohmann
et al. (2013) showed that seasonal changes in saccular encod-
ing in midshipman could largely be explained by changes in
BK mRNA abundance (Rohmann et al., 2013). This study

(a) (b) Apical

Control Summer
Estradiol Testosterone
1.0 F1 0
F0
F2 -20
0.8
Power spectrum (dB/Hz)

-40

-60
0.6
VS

-80
Apical
0.4 -100

-120
0.2
-140

0.0 -160
0 100 200 300 400 500 600 700 800
Frequency (Hz)

Figure 5 Neurophysiological and anatomical evidence for steroid- and season-dependent auditory encoding of higher call frequencies. (a) The
power spectrum of frequency components of the multiharmonic hum is plotted in gray (right y-axis). The phase-locking precision of saccular affer-
ents to different frequencies is plotted as a function of the vector strength of synchronization (VS, left y-axis). In summer breeding females and
winter nonbreeding females implanted with estradiol or testosterone, the saccular afferents are better able to encode the upper harmonics of the hum
(F1 ¼ 200 Hz; F2 ¼ 300 Hz) that contain as much or more power than the fundamental frequency (F0 ¼ 100 Hz). (Adapted from Sisneros, J.A.,
Forlano, P.A., Deitcher, D.A., Bass, A.H., 2004a. Steroid-dependent auditory plasticity leads to adaptive coupling of sender and receiver. Science 305,
404–407.) (b) Localization of estrogen receptor (ER) hair cells of the saccular epithelium, ERb1 (green in top panel) is at apical end of hair cells
(white arrows), while ERb2 (magenta in bottom panel) is distributed throughout the body of the hair cells with varied levels of expression along the
epithelium. Hair cells are labeled with a hair cell-specific antibody (magenta in top panel; green in bottom panel). Scale bar represents 20 mm.
Adapted from Fergus, D.J., Bass, A.H., 2013. Localization and divergent profiles of estrogen receptors and aromatase in the vocal and auditory
networks of a fish with alternative mating tactics. J. Comp. Neurol. 521, 2850–2869.
 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems
also showed that BK channels were localized to the somata of
saccular hair cells and were not detectable in other cells within
the epithelium or in nearby auditory ganglion cells. Together,
the results strongly support the proposal that seasonal changes
in peripheral frequency encoding depend on BK channels in
saccular hair cells (Rohmann et al., 2013). Morphological
changes complementing these physiological and molecular
changes in saccular hair cell function include seasonal increases
in the numbers of saccular hair cells in reproductive compared
to nonreproductive females (Coffin et al., 2012).
2.04.4.2 Steroid-Dependent Auditory Plasticity
As reviewed in Section 2.04.2.2, midshipman fish, like other
seasonally breeding teleosts, show seasonal changes in
plasma levels of circulating steroid hormones (Brantley
et al., 1993b; Oliveira, 2009). In particular, levels of both
17b-estradiol and testosterone begin to peak during the
spring, just before males and females move into the intertidal
zone for spawning. Treatment of nonreproductive females
with an intraperitoneal implant of either 17b-estradiol or
testosterone for 3–4 weeks leads to plasma levels of these
steroids comparable to those of females in reproductive
condition and induces an improved auditory phenotype
nearly identical to that of the naturally occurring one of repro-
ductive females (Figure 5(a); Sisneros et al., 2004a). The
improvements are gradual, as nonreproductive females with
testosterone implants for up to 2 weeks still resemble controls
(Sisneros et al., 2004a). Supporting evidence for the periph-
eral effects of estrogen comes from the anatomical demonstra-
tion for estrogen receptors (ERs) in the hair cell epithelium
(Figure 5(b); Fergus and Bass, 2013), as well as aromatase
synthesis by saccular ganglion cells that could provide for
the local conversion of testosterone to estrogen (Forlano
et al., 2005; Fergus and Bass, 2013). The most recent studies
show two types of ERs, ERb1 and ERb2, localized to saccule
hair cells (Fergus and Bass, 2013). Consistent with the neuro-
physiological results showing shared patterns of seasonal
plasticity in frequency sensitivity between male morphs and
the sexes, quantitative PCR shows no morph- or sex-specific
patterns in the abundance of mRNA transcripts for both forms
of ERb and aromatase (Fergus and Bass, 2013). ERa, shown in
regions of the epithelium adjacent to hair cells (Forlano et al.,
2005), also shows similar levels of abundance across morphs
and the sexes (Fergus and Bass, 2013).
In sum, while the influence of circulating steroids on encod-
ing mechanisms by the hair cell epithelium of the auditory
saccule remains correlative (see Rohmann and Bass, 2011),
the available studies for the plainfin midshipman fish show
that steroid-dependent auditory plasticity leads to seasonal
improvements in the sense of hearing that inform both mate
identification and localization during the breeding season.
The findings of seasonal shifts in the sense of hearing in
midshipman fish are complemented by more recent studies
in songbirds showing seasonal changes in hearing (e.g., see
Caras, 2013; Vélez et al., 2015; and references therein). These
studies, together with those showing the influence of steroids
on seasonal peripheral auditory function in midshipman,
complement studies in other teleosts (e.g., Maruska et al.,
2012; Zeyl et al., 2013), birds (Gall et al., 2013) and mammals,
59
including humans (e.g., Chong and Liu, 2016; Frisina and
Frisina, 2016). Peripheral action of estradiol is likely mediated
by ERs located in auditory hair cells in midshipman (see
above), as well as in birds and mammals (Hultcrantz et al.,
2006; Noirot et al., 2009). There has been extensive neurophys-
iological study in midshipman and other teleost fishes of the
main auditory nucleus in the midbrain (Bass and McKibben,
2003; Edds-Walton and Fay, 2008) that expresses steroid recep-
tors (Forlano et al., 2010; Fergus and Bass, 2013). However, the
potential for steroid- and/or seasonal-dependent plasticity in
auditory physiology in the midbrain or other central auditory
sites that express steroid receptors (Forlano et al., 2010; Fergus
and Bass, 2013) has yet to be investigated as it has been in other
vertebrates (see Caras, 2013).
2.04.5 Sites of Hormone Synthesis and Action
Studies mapping the distribution and/or abundance of
neuropeptide-synthesizing neurons along with steroid recep-
tors provide support for the action of these hormones in vocal
and auditory systems. These investigations also provide a more
general roadmap for future studies investigating the neural
basis for the actions of these hormones on reproductive
behaviors.
2.04.5.1 Neuropeptides
The CNS distribution of neurons synthesizing GnRH and AVT
has been extensively studied in teleost fish. The POA is a major
site of synthesis for both of these peptides. Prior reviews have
extensively considered dimorphisms and polymorphisms in
the numbers and dimensions of GnRH and AVT neurons across
several teleost species exhibiting reproductive plasticity (e.g.,
Bass and Grober, 2009). Since the first studies that identified
polymorphisms in the POA-GnRH phenotype of a sex
changing fish (Grober et al., 1991; Grober and Bass, 1991),
comparable investigations of such species have largely
examined AVT mechanisms (see Godwin and Thompson,
2012; Almeida and Oliveira, 2015). The reader is referred to
other chapters in this volume for more detailed coverage of
these species and those that exhibit serial changes in social
status from reproductive to nonreproductive states (Godwin,
2016; Fernald, 2016). For the purposes of this chapter, more
detailed remarks are confined to species like midshipman fish
that exhibit ARTs.
GnRH containing cells in the POA were an early focus of
studies of reproductive plasticity in fishes because these
neurons directly innervate the teleost pituitary (Oka and
Ichikawa, 1990) and thus can potentially exert rapid changes
in gonadotropin release from the anterior pituitary and, in
turn, affect the secretion of gonadal steroids. The first such
studies were in a poeciliid, the southern platyfish (Xiphophorus
maculatus) (Halpern-Sebold et al., 1986). Though ARTs were
not described for poeciliids until later (Zimmerer and Kallman,
1989; Ryan et al., 1990), the investigators showed that the
number of GnRH neurons in the POA diverged between small
and large male morphs of platyfish. Later studies documenting
POA GnRH polymorphisms in the adult reproductive morphs
of midshipman fish, together with studies of several species
60 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems
showing sex and role change, provided neuroanatomical
support for the hypothesis that these neurons play a central
role in triggering events that lead to age- and male morph-
related changes in reproductive status (Grober et al., 1994;
Bass and Grober, 2009).
Like GnRH neurons in teleosts, AVT-containing neurons are
found in the POA and show both intra- and intersexual
dimorphisms in size and number in midshipman fish that
are comparable to polymorphisms found in teleosts
exhibiting adult sex and role change (Foran and Bass, 1998;
Bass and Grober, 2009). The actions of AVT on behavior, like
the neuroanatomical patterns, appear to be species-specific
(see Godwin and Thompson, 2012; also see discussion in
Santangelo and Bass, 2006). In the midshipman, AVT is
expressed in the parvocellular and magnocellular nuclei of
the POA (Goodson and Bass, 2000b). These AVT-containing
POA neurons project locally to the immediate surrounding
neuropil, as well as throughout diverse regions of the anterior
hypothalamus and midbrain (Figure 4(a)). Forebrain targets
include the pituitary and hypothalamic regions such as the
lateral hypothalamus and the anterior tuberal nucleus,
a vocal active region that is also a part of the ascending
auditory system, which showed a greater number of
AVT-positive somata in nonreproductive fish collected during
the winter compared to reproductive fish collected during the
summer (Goodson and Bass, 2000b; Goodson et al., 2003).
Dense terminal fields are also found in vocally active areas in
the midbrain tegmentum such as the paralemniscal nucleus
(Goodson and Bass, 2000b; Feng and Bass, 2014). Although
little AVT immunoreactivity was found in the hindbrain vocal
CPG, dense staining was observed in the area postrema
above the VMN (Goodson and Bass, 2000b).
The distribution of putative isotocin matched closely to that
observed for AVT, especially concerning the vocal circuitry
(Goodson et al., 2003). Briefly, isotocin-immunoreactive
somata were found in the POA and projected to vocal regions
in the hypothalamus and midbrain (Goodson et al., 2003). At
hindbrain levels, isotocin-immunoreactive terminals could be
found within the vicinity of the vocal CPG, but there was no
evidence of a direct projection within the vocal nuclei
(Goodson et al., 2003). However, isotocin showed expression
in many regions associated with auditory processing, such as
the central posterior nucleus of the thalamus (CP) that is
a major recipient of ascending input from the auditory
midbrain, as well as in CP’s telencephalic targets (Bass et al.,
2000; Goodson et al., 2003). Since no sex or morph differences
were observed in AVT or isotocin distributions, the differential
effects of these peptides on vocal motor output could be medi-
ated by sex- or morph-specific expression of AVT or isotocin
receptors within individual vocal nuclei, which remain to be
characterized in midshipman.
2.04.5.2 Aromatase
It has been well established that the brain itself is capable of de
novo steroid synthesis in addition to being sensitive to and
metabolizing circulating steroids derived from peripheral sour-
ces (Baulieu and Robel, 1990; Forlano et al., 2006; Saldanha
and Schlinger, 2008). Aromatase is an important steroidogenic
enzyme that converts aromatizable androgens such as
testosterone into estrogens (Simpson et al., 1994). Neural aro-
matase activity can therefore modulate steroid-dependent
behaviors by controlling the local ratio of testosterone and
estrogens (Bass and Remage-Healey, 2008; Adkins-Regan,
2013). Importantly, the more potent androgens, such as
11-KT in teleosts and 5a-dihydrotestosterone in tetrapods, are
not aromatizable and cannot be shunted by aromatase from
exerting androgen action (Bentley, 1998; Adkins-Regan, 2013).
Compared to other taxa, teleosts exhibit a remarkably high
level of neural aromatase activity (Callard et al., 1990; Forlano
et al., 2001, 2006). In the plainfin midshipman, the rate of aro-
matase activity, as measured by enzymatic conversion of radio-
active ligand, was higher in the forebrain compared to the
midbrain and hindbrain (Schlinger et al., 1999). Furthermore,
hindbrain aromatase activity was higher in females and type II
males than in type I males (Schlinger et al., 1999). This male
morph difference was likely a constitutive property of the
CNS and independent of circulating gonadal testosterone,
because type II male hindbrain had higher aromatase activity
than type I males even after castration (Schlinger et al.,
1999). During the nesting season, type II males had the highest
aromatase mRNA expression in the peripheral regions of the
VMN (Forlano and Bass, 2005a). Sex and morph differences
were also observed by quantitative PCR, which showed that
in hindbrain tissue that had the VMN removed, type II and
females had significantly higher aromatase expression than
type I males (Fergus and Bass, 2013). Furthermore, within
the isolated VMN, type II males had the highest levels of aroma-
tase expression, though this was not statistically significant
(Fergus and Bass, 2013). This constitutive high level of hind-
brain aromatase activity in females and type II males could
shunt testosterone from exerting androgenic and masculiniza-
tion effects on the vocal network.
Both females and type I males showed seasonal expression
of aromatase in several brain areas (Forlano and Bass, 2005a).
During the prenesting season in early spring, females exhibited
higher aromatase mRNA levels in the central and peripheral-
glial containing regions of VMN, the midbrain PAG, and the
magnocellular nucleus of the POA that includes the GnRH
and AVT synthesizing neurons described in Section 2.04.5.1
(type II males were not available for seasonal comparisons)
(Forlano and Bass, 2005a). These brain regions in type I males,
on the other hand, expressed the highest aromatase during the
nesting season (Forlano and Bass, 2005a). A companion study
of nonreproductive females showed that aromatase mRNA
expression was increased to prenesting levels after 20–30 days
of testosterone or estrogen implants (Forlano and Bass,
2005b). Thus, the seasonal changes in steroid levels could
modulate neural aromatase expression in a sex-dependent
manner.
Aromatase distribution within the midshipman vocal
network has been characterized using midshipman-specific
mRNA probes and antibodies (Figure 3(a); Forlano et al.,
2001). Aromatase transcript and protein expression were abun-
dant throughout critical nodes of the vocal network, including
the forebrain POA, midbrain PAG, and hindbrain VMN
(Forlano et al., 2001). Double labeling with glial and neuronal
specific markers revealed that aromatase is mostly expressed
within glial, but not neuronal, cell populations (Forlano
et al., 2001). For example, the highest density of aromatase
 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems
was found in telencephalic radial glia that surrounded the
surface of the telencephalon as well as the medial ventricular
surface (the telencephalon of teleosts has a single central
ventricle). Furthermore, dense aromatase-containing glial cells
were found in periventricular cell layers in the PAG and
surrounding the dorsolateral margin of VMN and extended
their processes throughout the VMN in a radial-like pattern.
Thus, aromatase is found within every major midbrain–hind-
brain node of the midshipman vocal control network, likely
providing sources of locally converted estradiol that vary in
levels across seasons and reproductive morphs, which was
shown to rapidly increase fictive call duration (Table 1;
Figure 3(b); Remage-Healey and Bass, 2004, 2007).
2.04.5.3 Androgen and ERs
Neuroanatomical studies in midshipman have localized tran-
script and protein expression of both androgen and ERs. These
receptors are expressed within neuroendocrine regions impor-
tant for reproductive behavior as well as within vocal regions
dedicated to controlling vocal behavior. Figure 3(a) summa-
rizes steroid receptor expression within the midshipman vocal
network.
Teleosts have two paralogous copies of androgen recep-
tors, ARa and ARb, that likely arose due to a teleost-specific
whole genome duplication (Ogino et al., 2009, 2016). Phylo-
genetic analyses indicate that ARb is closer to the ancestral AR
sequence, with ARa having accumulated novel substitutions
at a faster rate than ARb (Ogino et al., 2009, 2016). Character-
ization of ARa and ARb in medaka (Oryzias latipes) and the
western mosquitofish (Gambusia affinis) indicated that ARa
is a more potent transcription factor and is constitutively
expressed in the nucleus, whereas ARb exhibits cytosolic
expression with ligand-inducible nuclear translocation
(Ogino et al., 2009, 2016). For both ARs, 11-KT tends to be
a more potent ligand than testosterone (Bentley, 1998; Ogino
et al., 2009, 2016).
In type I male midshipman, relative expression of the two
AR subtypes in vocal muscle was dependent on recent
humming activity (Genova et al., 2012). Vocal muscle ARa
expression was significantly higher in humming males than
in nonhumming males, whereas ARb was higher in nonhum-
ming males (Genova et al., 2012). Perhaps the highly active,
type I vocal muscles require the more efficient functional
properties of ARa, such as its higher transcriptional activity
(Ogino et al., 2009, 2016).
In the CNS, only ARb expression has been characterized in
the midshipman (Forlano et al., 2010). ARb mRNA was highly
expressed in the POA, anterior and ventral tuberal nuclei of the
hypothalamus, vocal active sites from which fictive calls can be
evoked (Goodson and Bass, 2000a; Kittelberger, 2006; Forlano
et al., 2010). ARb mRNA was also found in midbrain vocal–
acoustic regions, such as the PAG, as well as in hindbrain vocal
pattern generator, including the VPP and VMN (Forlano et al.,
2010). An early report in the oyster toadfish (Opsanus tau) using
steroid autoradiography reported a sparse distribution of
androgen receptors in the medulla at the level of VMN,
including a region that likely corresponds to VPP (Fine et al.,
1996). It is unclear if ARa and ARb receptor subtypes exhibit
divergent localization or expression patterns in the
61
midshipman brain, as have been shown in other teleosts
(Harbott et al., 2007). Furthermore, it remains to be tested
whether divergent sex- or morph-specific AR subtype expres-
sion in vocal nuclei could account for differences in neural
androgen sensitivity.
Several ERs, including ERa, ERb1, and ERb2, have been
sequenced and localized in the midshipman brain
(Figure 3(a); Forlano et al., 2005; Fergus and Bass, 2013).
Similar to neuropeptides, AR, and aromatase, protein expres-
sion of all three ERs were found in the POA (Forlano et al.,
2001). In the hypothalamus, ERb2 showed robust expression
in the vocally active AT (Goodson and Bass, 2000a; Fergus
and Bass, 2013). Within midbrain vocal centers, the PAG
expressed both ERb1 and ERb2, though ERb2 showed a broader
distribution (Fergus and Bass, 2013). ERb2 was also expressed
in the periventricular cell layer of the main auditory nucleus in
the midbrain, the torus semicircularis (Fergus and Bass, 2013).
In the hindbrain vocal pattern generator, ERa, ERb1, and ERb2
were all strongly expressed within VMN neurons (Forlano et al.,
2005; Fergus and Bass, 2013). Interestingly, ERb2 staining in
VMN neurons was stronger in females and type II males
compared to type I males, which was confirmed by quantitative
PCR (qPCR) of transcript levels in the isolated VMN (Fergus
and Bass, 2013). These qPCR studies also revealed a significant
elevation of ERa within type II male VMN compared to female
VMN. These sex and morph differences in ER expression were
specific to the VMN, as qPCR of ER expression in surrounding
hindbrain tissue did not show any significant differences.
2.04.5.4 Glucocorticoids
As reviewed in Section 2.04.3.1.2, neurophysiological studies
in midshipman fish identified male morph-specific profiles
for the influences of cortisol, the main circulating glucocorti-
coid in teleost fish (Section 2.04.2.2), on vocal CPG activity.
Two enzymes are integral to the biosynthetic pathway
for cortisol, 11-beta(b)-hydroxysteroid dehydrogenase
(11bHSD), and 11b-hydroxylase (11bH) (Bury and Sturm,
2007). Especially interesting for teleosts is that 11bHSD and
11bH are also integral to the biosynthesis of 11-KT. Given
the duality of enzyme function, it is perhaps not surprising
that the mRNA expression patterns for 11bHSD and 11bH in
the brain of midshipman fish exhibit no single, morph-
typical pattern. For example, 11bHSD mRNA abundance was
highest in type II males in the vocal hindbrain, a region
extending between the cerebellum and rostral spinal cord
that houses the vocal CPG, but highest in type I males in the
forebrain (Arterbery et al., 2010b). Significant differences in
11bH mRNA levels were only found in the forebrain with
type II males having the highest levels. Given the absence of
detectable circulating levels of 11-KT in type II males (Brantley
et al., 1993b), we presume that regulation of the expression
levels of 11bHSD and 11bH in the CNS is related to shifts in
regional cortisol levels. A major caveat to these studies is
that the analysis included large brain regions, e.g., the fore-
brain included the olfactory bulb, telencephalon, and the
POA. This sampling method could have masked local varia-
tion in individual regions (e.g., POA) or nuclei (e.g., VMN).
The expression patterns for 11bHSD and 11bH mRNA in
the vocal muscle were less complex and thus more
62 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems
amenable to functional interpretations (Arterbery et al.,
2010b). Compared to type II males and females, type I
males had several fold higher transcript levels for both
enzymes that could provide a local source of 11-KT, which
has been linked to seasonal increases in vocal muscle
size (Brantley et al., 1993a; Walsh et al., 1995; Sisneros
et al., 2009).
The abovementioned transcript levels of 11bHSD and
11bH likely represented constitutive expression levels as
the tissues were collected from animals not engaged in vocal
activity at the time of sacrifice (Arterbery et al., 2010b).
A follow-up study of the vocal muscle in type I males showed
that the transcript expression levels of 11bHSD varied with
the behavioral states of humming versus nonhumming
(Genova et al., 2012). There was a strong trend for higher
11bHSD levels in nonhumming males that may help to
protect the vocal muscle from the potentially catabolic effects
of higher circulating cortisol levels by converting cortisol to
its inactive metabolite, cortisone (see Joëls et al., 2007;
Arterbery et al., 2010b). In this study, it was also shown
that the higher circulating 11-KT levels in humming males
were matched by higher levels of 11-KT within the testis
(Genova et al., 2012). There was also a positive correlation
between the levels of circulating 11-KT and of 11bHSD
mRNA in the testis, consistent with the testis being a major
site of synthesis for systemically available 11-KT (Genova
et al., 2012).
Teleosts also have two glucocorticoid receptors (GR), GR1
and GR2, that may differ in their sensitivity to corticosteroids
(see Arterbery et al., 2010a and references therein). In general,
the expression pattern of GR resembles that of 11bHSD with
one exception – GR1 was highest in all brain regions in type
II males and not just in the vocal hindbrain (Arterbery et al.,
2010a). In the vocal muscle of type I males, GR1 and GR2
mRNA levels were higher in nonhummers (Genova et al.,
2012). Perhaps together with elevated ARb (Section 2.04.5.3)
and 11bHSD (see above) transcript levels, the two GRs help
maintain the muscle in a state of preparedness for the onset
of long duration humming.
In sum, the complexity of the expression profiles for
mRNA transcript abundance of receptors and biosynthetic
enzymes involved in both glucocorticoid and androgen
signaling pathways likely relates to the duality in biosyn-
thetic enzyme function and of duplicate receptor subtypes.
It also remains important to assess the impacts of changing
enzyme transcript levels on protein expression and local
abundance of the steroids themselves in brain and
peripheral tissues, such as the vocal muscle (e.g., see Pradhan
et al., 2014).
2.04.5.5 Catecholamines
Recent studies in midshipman have characterized the CNS
distribution, seasonal and social regulation, as well as intrasex-
ual differences in expression of tyrosine hydroxylase (TH), the
rate-limiting enzyme for catecholamine synthesis (Petersen
et al., 2013; Forlano et al., 2014, 2015a; Ghahramani et al.,
2015). Like in other teleosts, dense clusters of catecholamin-
ergic (CA)-containing neurons were found in the olfactory
bulb, the telencephalon, the POA, the diencephalon, the locus
coeruleus, and the area postrema (Forlano et al., 2014).
Specific to midshipman was a dense population of CA neurons
along the rostral, dorsolateral margin of VMN that provided
dense CA input to the paired VMN. Catecholaminergic
neurons in the diencephalon had dense input to the central
and peripheral auditory systems. This included the hindbrain
octavolateralis efferent nucleus that innervates the hair cell
epithelium of the inner ear’s auditory saccule, as well as the
saccular epithelium itself. The pattern of CA input varied
between the breeding and nonbreeding seasons: input to the
efferent nucleus was denser in reproductive females compared
to nonreproductive females, whereas the saccular epithelium
showed the opposite pattern, namely denser input in nonre-
productive females (Forlano et al., 2015a). The CA input to
VMN was also greater in type II males compared to type I
males during the reproductive summer months (Ghahramani
et al., 2015). Thus, CA action in auditory and vocal areas of the
midshipman brain could underlie seasonal and morph-
dependent differences in auditory processing and vocal
function.
Support for the role of catecholamines in mediating social
stimulation of audition comes from behavioral studies that
quantified activation of CA neurons in response to underwater
playback of advertisement hums (Petersen et al., 2013). Activa-
tion of CA neurons was measured by quantifying the number
of CA neurons showing co-label with the immediate early
gene c-fos, a proxy for neural activation (Petersen et al.,
2013). Type I males who were exposed to hums had increased
activation of CA neurons in the dopaminergic periventricular
posterior tuberculum and the noradrenergic locus coeruleus
when compared to those exposed to ambient noise (Petersen
et al., 2013). Furthermore, the activation of CA neurons in
these two brain regions showed positive correlations with
neuronal activation of the ascending auditory pathway,
including auditory regions of the midbrain (torus semicircula-
ris) and thalamus (central posterior nucleus) (Petersen et al.,
2013). Together, this recent body of work suggests that CA
actions within auditory and vocal brain regions likely impact
seasonal and social regulation of midshipman acoustic
communication. The neurophysiological consequences of CA
inputs to the vocal and auditory systems remain to be
investigated.
2.04.6 Daily and Seasonal Rhythms in Vocal–
Acoustic Mechanisms – Genetic Insights
Thus far, as reviewed in the preceding sections, we have
employed a hypothesis-driven approach to study in detail
several hormonal pathways and their effects on vocal behavior
across multiple levels, including behavior, neurophysiology,
and gene expression. Recently, we used next-generation RNA
sequencing (RNA-seq) to characterize global gene expression
patterns in crucial nodes within vocal motor and acoustic
pathways: the VMN (Feng et al., 2015) and the hair cell
epithelium of the auditory saccule (Fergus et al., 2015).
From this approach, we not only gained insights into
the general molecular processes important for vocal
(Section 2.04.6.1) and auditory (Section 2.04.6.2) function,
but also expanded our list of functionally important
 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems 63

candidate genes, including specific ion channels and (Table 2), from the large data set generated from differential
hormone/neuromodulators, to be validated and studied in expression and downstream data analyses. Furthermore, the
future hypothesis-driven research. wealth of neurophysiological, anatomical, and behavioral
data in the midshipman model system provided validation
for transcriptome-identified candidate molecular mechanisms
2.04.6.1 VMN Transcriptome
underlying vocal characters such as duration and frequency
Within the broader aim of linking genes to behavior, we asked (Figure 2). Here, we focus our discussion on hormone-
whether the VMN, housing a homogeneous population of related insights gained from transcriptome analyses.
vocal motoneurons (alongside glia) (Figures 2 and 6), By comparing gene expression in all VMN samples to all H
expresses a unique molecular profile that supports the samples, we identified close to 3000 transcripts whose expres-
patterning of temporally precise vocal behavior. Furthermore, sion levels were upregulated in the VMN (Figure 6(a)). We first
we wondered whether VMN gene expression changes over subjected these VMN-upregulated transcripts to Gene Ontology
daily and seasonal cycles to drive variation in neural excit- (GO) term enrichment analysis, i.e., enrichment of number of
ability and in turn, natural vocal behavior. To answer these genes belonging to a certain GO term/molecular function.
questions, we compared gene expression in surgically isolated There were a large number of cellular respiration-related GO
VMN to the surrounding hindbrain tissue (H) at three time terms that were enriched in the VMN, consistent with the
points corresponding to minimal or maximal vocal activity: VMN’s ability to maintain firing at 100 Hz for minutes to
summer night (high vocal activity), summer morning (low hours repetitively throughout a single night of courtship
vocal activity), and winter night (low vocal activity) (Feng activity in type I males. There were also several hormone-
et al., 2015). Because we used brain tissue from type I males related GO terms that were enriched in the VMN, including
who were not vocally active at the time of tissue collection, ‘gonadotropin secretion,’ ‘endocrine hormone secretion,’ and
our results represent baseline changes in gene expression ‘sterol biosynthetic process.’ Additionally, many transcripts
with implications in changes in neuronal excitability, and upregulated in the VMN, but no transcripts upregulated in H,
not activity-induced changes. mapped to KEGG (Kyoto Encyclopedia of Genes and
Drawing from previously accumulated knowledge of the Genomes) pathways related to steroid hormone synthesis
midshipman vocal and auditory circuits as well as relevant liter- and degradation (Feng et al., 2015). Together, these results
ature, we were able to identify potentially functionally impor- indicate that VMN is both an energetically and hormonally
tant candidate genes, many of which were hormone related active nucleus, supporting previous studies.

(a) Differential transcript expression (b) VMN neuroanatomy and candidate genes
1e5 4
VMN VMN_summer
3
1e4
2
VMN mean

1e3 1
0
1e2
−1

1e1 −2
Centered log2(FPKM+1)

−3
1e0
−4
1

5
3
0

WNH SNH SMH WNV SNV SMV

1e

1e
1e
1e

1e

1e

H mean
4 4
All_summer Peak_SNV
Centered log2(FPKM+1)

3 3 VN
2 2
1 1
Motoneuron somata
0 0
Glial AR beta mRNA (ar-a)
−1 −1
Glial aromatase-ir and mRNA (cyp19a1a)
−2 −2
ER alpha mRNA & ER beta-ir (esr1, esr2)
−3 −3
GABA-ir (gabra3, gabra5)
−4 −4
Gap junction-coupled motoneurons (cx30, cx43)
WNH SNH SMH WNV SNV SMV WNH SNH SMH WNV SNV SMV

Figure 6 Differential expression identifies functionally important candidate genes. (a) Two methods were used to determine tissue (upper left) and
daily/seasonal (remaining panels) differential expression patterns (see Feng et al., 2015 for method details). Pattern names are shown in the upper
left of each panel and used in Table 2. Sample group names are acronyms indicating the tissue type and time of collection. W, winter; S, summer; N,
night; M, morning; V, VMN; H, surrounding hindbrain; FPKM, fragments per kilobase of transcript per million mapped reads. (b) Schematic of
a cross section of the VMN showing known anatomical features (also see Figure 2(a)). Transcriptome-identified candidate genes correlating to known
VMN characteristics are listed in parentheses. Adapted from Feng, N.Y., Fergus, D.J., Bass, A.H., 2015. Neural transcriptome reveals molecular mech-
anisms for temporal control of vocalization across multiple timescales. BMC Genomics 16, 417.
64 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems

Table 2 Select hormone-related candidate genes identified by
differential expression in the VMN (Feng et al., 2015) and saccular
epithelium (inner ear) transcriptomes (Fergus et al., 2015). Candidate
genes are categorized by function; expression patterns are listed in the
right column and correspond to Figure 6(a)
Table 2 Select hormone-related candidate genes identified by
differential expression in the VMN (Feng et al., 2015) and saccular
epithelium (inner ear) transcriptomes (Fergus et al., 2015). Candidate
genes are categorized by function; expression patterns are listed in the
right column and correspond to Figure 6(a)dcont'd

Transcript name Expression pattern Transcript name Expression pattern

Steroids Insulin receptor-like All-Summer

17b-Hydroxysteroid Ear-Summer Neuropeptide b precursor VMN
dehydrogenase 14 Opioid growth factor receptor-like VMN-Summer
3-Keto-steroid reductase-like VMN, VMN-Summer Parathyroid hormone/parathyroid VMN-Summer
Androgen receptor alpha VMN, All-Summer hormone-related peptide
Androgen-induced gene 1 VMN, VMN-Summer receptor-like
Cholesterol 25-hydroxylase-like All-Summer, Ear-Summer Peptide yy-like VMN
protein member 1-like Pituitary adenylate cyclase-activating VMN
Dihydroxyvitamin VMN polypeptide type i receptor-like
d 24-mitochondrial-like isoform 1 Vasoactive intestinal polypeptide VMN
Estrogen receptor alpha Peak-SNV receptor 1-like
Estrogen receptor beta 2 VMN Thyroid hormone
Estrogen-related receptor alpha VMN Thyroid hormone receptor alpha VMN and Ear
Estrogen-related receptor b type 1 Ear-Summer Thyroid hormone receptor- VMN
Estrogen-related receptor g Ear-Summer associated protein 3-like
Glucocorticoid receptor VMN-Summer Thyroid receptor-interacting protein VMN
Hydroxysteroid 11b-dehydrogenase VMN, Ear-Summer 6-like
1-like Other neuromodulators
Hydroxysteroid dehydrogenase-like VMN-Summer Adenosine receptor a1-like VMN
protein 2 D-like dopamine receptor-like VMN
Lanosterol 14a-demethylase-like VMN, Ear-Summer Melatonin receptor type 1a-like VMN
Liver x receptor VMN-Summer,a Ear-Summer Prostaglandin e synthase 2-like VMN
Neutral cholesterol ester hydrolase VMN-Summer Prostaglandin f2-alpha receptor-like VMN
1-like Acetylserotonin o-methyltransferase- All-Summer
Non-specific lipid-transfer Ear-Summer like
Non-specific lipid-transfer protein Ear-Summer
Nuclear receptor ror-a VMN,a Ear-Summer Note the subset of candidates shared by both VMN and inner ear.
Nuclear receptor subfamily 2 group f Ear-Summer
a
Indicates transcripts that were significantly upregulated in the VMN but not
reported in candidate lists reported in Feng et al. (2015).
member 6 Modified from Feng, N.Y., Fergus, D.J., Bass, A.H., 2015. Neural transcriptome
Ovarian aromatase VMN reveals molecular mechanisms for temporal control of vocalization across multiple
Oxysterol-binding protein 10 VMN-Summer timescales. BMC Genomics 16, 417; Fergus, D.J., Feng, N.Y., Bass, A.H., 2015. Gene
isoform 1 expression underlying enhanced, steroid-dependent auditory sensitivity of hair cell
epithelium in a vocal fish. BMC Genomics 16, 782.
Oxysterol-binding protein 8-like VMN-Summer
Oxysterol-binding protein 9-like Peak-SNV
isoform 3
Retinoid x receptor g Ear-Summer To further identify genes and pathways important for VMN
Sterol 26-mitochondrial-like Peak-SNV
function, we looked for genes whose expression levels were
Sterol regulatory element-binding VMN
increased during time points corresponding to natural levels of
protein 1
Sterol regulatory element-binding VMN high vocal activity. The focus was on candidate genes showing
protein 2 biologically relevant expression patterns, mainly those with
Sterol-c4-methyl oxidase Ear-Summer elevated expression during: the summer night only in the
Neuropeptides/peptide hormones VMN (‘Peak-SNV’); both the summer morning and night in
Atrial natriuretic peptide-converting VMN VMN (‘VMN-Summer’); the summer in both H and VMN
enzyme-like (‘All-Summer’) (Table 2; Figure 6(a)). These results suggest
Calcitonin gene-related peptide VMN, VMN-Summer, All-VMN that hormone synthesis and receptor levels within the VMN
precursor vary dynamically across behaviorally relevant timescales. The
Cholecystokinin type a receptor VMN
expression patterns of several candidate genes and gene
Growth hormone receptor VMN, Peak-SNV
products in the VMN, such as steroid receptors and aromatase,
Growth hormone-regulated tbc All-Summer
protein 1-a-like were consistent with results from prior studies that employed
Inhibin beta b chain-like VMN a variety of quantitative and anatomical methods (see Section
Insulin gene enhancer protein isl-1 VMN, Summer ear 2.04.5 and Figure 6(b) for summary). Although many
Insulin gene enhancer protein VMN-Summer candidates have been previously studied, thus providing
isl-2a-like corroborating evidence for the transcriptome results, most
Insulin-like growth factor binding VMN, VMN-Summer candidates were unstudied in the context of vocal behavior,
protein 1 such as those listed under ‘Neuropeptides/peptide hormones,’
Insulin-like growth factor i VMN ‘Thyroid hormone,’ and ‘Other neuromodulators’ (Table 2;
 Neural, Hormonal, and Genetic Mechanisms of Alternative Reproductive Tactics: Vocal Fish as Model Systems
see Feng et al., 2015 for more in-depth discussions). Many of
these candidates would be ideal candidates for future
behavioral, neurophysiological, and anatomical studies in the
midshipman vocal CPG.
2.04.6.2 Saccular Epithelium Transcriptome
We sequenced the transcriptome of the saccular epithelium
(SE) collected from summer reproductive and winter nonre-
productive type I males to identify candidate genes and path-
ways underlying the well-documented seasonal plasticity in
auditory sensitivity (Rohmann and Bass, 2011; Fergus et al.,
2015). Like in the VMN, several candidate genes were upregu-
lated in the summer reproductive SE related to steroid action,
including estrogen signaling (Table 2), previously shown to
enhance auditory sensitivity (see Section 2.04.4; Sisneros
et al., 2004a; Fergus et al., 2015). Because this experiment
was performed in parallel with the VMN study, and sequences
from both studies were pooled during de novo transcriptome
assembly, SE and VMN results were directly comparable.
Compared to the VMN and H, the SE contained the highest
number of unique transcripts. Furthermore, seasonal changes
in gene expression were more dramatic in the SE than in the
CNS tissues. Nevertheless, we identified a suite of candidate
genes that were shared between the SE and VMN (Table 2),
suggesting that audio-vocal coupling may have a genetic basis,
though further studies are needed to test this hypothesis
(Fergus et al., 2015).
2.04.7 Summary
The adaptive radiation of teleost fishes, which comprise nearly
half of all living species of vertebrates (Nelson, 2006), exhibits
a wide range of sexual phenotypes. Teleost models thus allow
us to link natural variation in brain and hormonal mechanisms
to patterns of adult reproductive plasticity. As discussed in this
chapter, behavioral phenotypes within a single species and sex
that follow different reproductive tactics can often exhibit
equally divergent characters ranging from body coloration
and signaling structures (e.g., those used in acoustic communi-
cation) to circulating hormone levels, body and gonad size,
and a range of neurobiological traits. Many characters are
expressed as a correlated suite, with shifts between reproductive
morphs involving divergence in a ‘covariant character set’ (after
West-Eberhard, 1989).
ARTs have been especially well studied at multiple levels of
biological organization in the highly vocal, plainfin
midshipman fish that has two male morphs with one of the
morphs resembling females in vocal behavior and underlying
vocal mechanisms (Table 1). Each of two male morphs, type
I courting and type II satellite/sneak-spawning, has a distinct
suite of phenotypic characters (Table 1). Like other closely
related toadfishes (family Batrachoididae), type I midshipman
exhibit vocal courtship, nest building and defense, and parental
care (Amorim et al., 2015; Bass et al., 2015). Type II males
neither build nests nor guard eggs (Brantley and Bass, 1994).
This led to the proposal that type II males are a derived char-
acter state (Bass, 1992). Small type I males also exhibit
sneak-spawning tactics (Lee and Bass, 2006), further consistent
65
with the hypothesis that the type II male phenotype is the more
recent innovation.
Although types I and II male midshipman express diver-
gent suites of morph-specific characters (Table 1), some
characters are shared between the two morphs such as the
sneak-spawning tactic (Lee and Bass, 2006). The uncoupling
of gonadal phenotype (male or female) from secondary sex
characters, like those witnessed for the vocal motor system,
leads to the evolution of conserved suites of behavioral
and neuroendocrine characters for each reproductive morph
between species and unique traits expressed within species,
e.g., vocal traits (Bass and Grober, 2009). Midshipman fish
and other teleosts with comparable ARTs (e.g., sunfish, see
Neff and Svensson, 2013) may represent an intermediate
condition between species with genetic morph determina-
tion, such as some poeciliids (Zimmerer and Kallman,
1989; Ryan et al., 1990) and those in which males and/or
females can change sex during their lifetime (see Godwin,
2016).
Taken together, the available evidence suggests that the
brain of toadfishes can be characterized as an exquisitely sensi-
tive endocrine organ that directs the expression of sexually
dimorphic and reproductive strategy-specific vocal behavior
under multiple social and environmental contexts. Recent
large-scale gene expression studies have enlarged the cast of
hormonal and molecular pathways that can regulate vocal
acoustic communication over daily and seasonal cycles (Fergus
et al., 2015; Feng et al., 2015). These candidates will inform
future experimental investigations in the toadfish model
system to validate and characterize their function. The
hormone sensitivity and relative simplicity of the vocal
behavior and its underlying neural circuit make toadfishes
excellent models for elucidating the hormonal and neural basis
of vocalization in vertebrates and how these mechanisms
diverge between and within sexes.
Acknowledgments
We offer a very special thanks to all of our collaborators for their input
into multiple aspects of the work reviewed here and to Margaret
Marchaterre and Margaret Nelson for the photographs and line draw-
ings in Figure 1, respectively. Thanks to the editors for the opportunity
to submit this review and especially to Jacques Balthazart for his helpful
edits. The authors’ research was supported by NSF (IOS 1457108)
during the preparation of this chapter.
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 2.05 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
Kent D Dunlap, Trinity College, Hartford, CT, USA
Ana C Silva, Unidad Bases Neurales de la Conducta, Instituto de Investigaciones Biologicas Clemente Estable, Montevideo, Uruguay;
and Universidad de la Republica, Montevideo, Uruguay
G Troy Smith, Indiana University, Bloomington, IN, USA
Harold H Zakon, University of Texas, Austin, TX, USA
Ó 2017 Elsevier Inc. All rights reserved.
This chapter is a revision of the previous edition chapter by H.H. Zakon, G.T. Smith, volume 1, pp. 611–639, Ó 2009, Elsevier Inc.

2.05.1 Introduction 69
2.05.1.1 Electric Fish as a Model in Behavioral Neuroendocrinology 70
2.05.1.2 The EOD as a Communication Signal 71
2.05.1.3 Breeding Cycle and Endogenous Levels of Hormones 71
2.05.2 EOD Waveform and Amplitude and Their Control by Electrocytes 72
2.05.2.1 Generation of the EOD Waveform 72
2.05.2.1.1 Pulse Fish 72
2.05.2.1.2 Wave Fish 73
2.05.2.2 EOD Waveform: Communication Function 73
2.05.2.3 EOD Waveform and Electrocytes: Sexual Dimorphism 74
2.05.2.4 EOD Waveform and EOD Amplitude: Hormonal Regulation 74
2.05.2.4.1 Effects of Exogenous Steroids on EOD Waveform and Electric Organ Morphology 74
2.05.2.4.2 Effects of Exogenous Steroids on Electric Organ Ion Currents 75
2.05.2.4.3 EOD Waveform: Seasonal Modulation and Its Hormonal Regulation 76
2.05.2.4.4 EOD Waveform: Diel, Social, and Stress-Induced Modulation and Its Hormonal Regulation 76
2.05.3 EOD Rate/Frequency and Its Regulation by the Pacemaker Nucleus 78
2.05.3.1 EOD Rate in Pulse Fish: Social and Diel Influences and Their Hormonal Regulation 79
2.05.3.1.1 Social Influences 79
2.05.3.1.2 Diel Influence 79
2.05.3.2 EOD Frequency in Wave Fish: Role in Social Behavior and Its Hormonal Regulation 80
2.05.3.2.1 Social Behavior 80
2.05.3.2.2 Hormonal Regulation 80
2.05.4 EOD Modulations and Their Regulation by Prepacemaker Nuclei 81
2.05.4.1 EOD Modulations: Communication Function 81
2.05.4.1.1 Pulse Fish 81
2.05.4.1.2 Wave Fish 83
2.05.4.2 EOD Modulations: Neural Control by Prepacemaker Nuclei 86
2.05.4.3 EOD Modulations: Species Diversity and Their Hormonal Control in Apteronotidae 87
2.05.4.3.1 Species Diversity 87
2.05.4.3.2 Neuroendocrine Regulation of Chirping in Apteronotus 87
2.05.4.4 EOD Modulations: Seasonality and Neuroendocrine Regulation in Brachyhypopomus 88
2.05.4.5 EOD Modulations: Steroidal Regulation of Nonsex-Biased Territorial Aggression in Gymnotus 89
2.05.5 EOD Reception through the Electrosensory System 89
2.05.5.1 Electroreceptor Types, Function, and Tuning 89
2.05.5.2 Electroreceptor Coding of EOD Waveforms 91
2.05.5.2.1 Pulse Fish 91
2.05.5.2.2 Wave Fish 91
2.05.5.3 Sensory Coding of Chirps 92
2.05.5.3.1 Electroreceptors 92
2.05.5.3.2 Central Nervous System 92
2.05.5.4 Electroreception: Sex Differences and Neuroendocrine Regulation 92
2.05.6 Conclusions 93
References 94

2.05.1 Introduction organ discharges (EODs) and those of other fish using special-
ized sensory receptors called electroreceptors. There are two
Weakly electric fish generate weak electric fields from modified clades of weakly electric fish: the Gymnotiformes from South
muscle cells in the electric organ. They sense their own electric America and the Mormyroidea from Africa. These groups are

Hormones, Brain, and Behavior, 3rd edition, Volume 2 http://dx.doi.org/10.1016/B978-0-12-803592-4.00019-5 69

70 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
so phylogenetically distant that electric organs and electrosen-
sory capabilities must have evolved independently in each
group. Electric fish use this extraordinary electric sense to locate
and identify nearby objects (electrolocation) and to communi-
cate with each other (electrocommunication) (Hopkins,
1974a,b). EODs enable fish to communicate about their
species identity, sex, social status, and motivational state.
2.05.1.1 Electric Fish as a Model in Behavioral
Neuroendocrinology
Electric fish are especially tractable experimental subjects for
studying the neural and hormonal mechanisms underlying
behavior. Electric signals are stereotyped and easily quantifi-
able, allowing detailed analysis of signal structure. They vary
by waveform, amplitude, rate (or frequency), and transient
modulations of rate and amplitude. Each of these signal
parameters can be traced to three cell types in the underlying
electrocommunication circuitry: (1) EOD waveform and
amplitude are determined by the electrocytes of the electric
organ located primarily in the tail, (2) baseline EOD rate or
frequency is determined by neurons in the hindbrain pace-
maker nucleus (PN), and (3) EOD modulations are deter-
mined by neurons in the thalamic and midbrain
prepacemaker nuclei (Figure 1). The detection of the EOD
occurs through a fourth cell type, the electroreceptors, distrib-
uted all over the body surface and concentrated near the head.
Diencephalon
Telencephalon Electromotor neurons
EO
SPPn PN
PPn
Figure 1 Pacemaker nucleus of a South American fish and its inputs.
Schematic illustration of the two inputs to the pacemaker nucleus: the
prepacemaker nucleus (PPn) and the sublemniscal prepacemaker
nucleus (SPPn). These inputs modulate the firing frequency of the
pacemaker neurons in the pacemaker nucleus (PN) that synapse on the
electromotor neurons in the spinal cord and ultimately drive the electric
organ (EO).
Figure 2 Pulse-type electric organ discharges (EODs) from the South American fish, Brachyhypopomus gauderio. Males (above) possess thicker
longer tails than females (below) and produce EOD pulses that are longer in duration, especially the second (P2, negative) phase. The EODs are from
representative mature fish of each sex, scaled in amplitude and overlain. Modified from Perrone, R., 2012. La vasotocina modula el comportamiento
social de dos especies de peces eléctricos con diferente socialidad. PEDECIBA, Universidad de la República, Montevideo.
EOD signal parameters and electroreception differ between
the sexes, participate in reproductive and agonistic communica-
tion, and respond to hormones. Because each process in electro-
communication is regulated by a discrete set of cells, it is
relatively easy to pinpoint the locus and cellular mechanisms
of hormone action. Most cells in the circuit are accessible for elec-
trophysiological recordings, so their biophysical properties can
be compared before and after hormone treatment. Moreover,
EODs are generated by current flow across ion channel proteins,
enabling us to identify candidate genes for molecular analysis of
sex differences in behavior and their hormonal regulation.
Another attractive aspect of these fish is the richness and
diversity of their behavior and ecology. For example, while
most species show a strong sexual dimorphism in the EOD
(Figures 2 and 3), a few show a ‘reverse’ sexual dimorphism,
and some seem to be monomorphic (Westby, 1988;
Alves-Gomes and Hopkins, 1997; Zhou and Smith, 2006; Ho
et al., 2007; Turner et al., 2007; Smith, 2013). Some species
lay eggs on vegetation or in the leaf litter and abandon them,
whereas others construct nests and tend to their young
(Hagedorn and Heiligenberg, 1985; Crampton and Hopkins,
2005). Species also differ in habitat choice and degrees of soci-
ality: in some, individuals are solitary and establish territories
in shallow streams; in others, fish school in open water or
even gather in large multispecies assemblages (Hopkins,
1981; Oestreich and Zakon, 2005; Tan et al., 2005). Because
phylogenies of both clades of weakly electric fish are available
(Alves-Gomes et al., 1995; Alves-Gomes, 1999; Lavoue and
Sullivan, 2004; Crampton and Albert, 2006; Tagliacollo et al.,
2016), it is possible to make reasonable speculations about
how sexual dimorphisms and the underlying neuroendocrine
mechanisms have evolved. Molecular phylogenetics has shown
that signals can evolve rapidly within clades to yield a remark-
able diversity of communication behaviors even within closely
related species (Arnegard et al., 2005, 2010; Feulner et al.,
2006, 2007; Turner et al., 2007).
In this chapter, we review the role of electrocommunication
in the social behavior of electric fish and its regulation by
gonadal steroid hormones and neuromodulators. After intro-
ducing the general function of the EOD in communication
and patterns of endogenous hormone secretion, we organize
the chapter into four sections according to EOD signal param-
eter and its cellular substrate: first, the EOD waveform and
amplitude and their control by electrocytes; second, EOD
rate/frequency and its control by the PN; third, EOD
 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
Brienomyrus brachyistius (t.p.)
Brienomyrus brachyistius (I.bp.)
Ivindomyrus opdenboschi
juv
ad
Stomathorhinus corneti
Mormyrus rume
1 ms
Figure 3 Pulse-type electric organ discharges (EODs) from five African
electric fish. Note the sexual dimorphism and/or age difference (Ivindomy-
rus opdenboschi) in EOD pulse shape. Courtesy of C.D. Hopkins.
modulations and their control by prepacemaker nuclei; and
finally, EOD detection and processing by electroreceptors and
central electrosensory circuits.
2.05.1.2 The EOD as a Communication Signal
Just as olfactory, visual, or acoustic signaling in other groups of
animals, electric signaling coordinates social interaction in
weakly electric fish. Electric signals are restricted to aquatic
environments, and they attenuate with distance more rapidly
than acoustic or visual stimuli (Hopkins, 1999). Consequently,
electric signals are used for communication only over short
distances of a few body-lengths. However, they can be used
at night or in light-restricted habitats such as silty streams or
the bottoms of large rivers, and they can be delivered from
within a protected location such as a clump of weeds. Impor-
tantly, the temporal fine structure of electric signals is not dis-
torted by the medium allowing slight variations of EOD
waveforms to carry important information (Hopkins and
Bass, 1981; McGregor and Westby, 1992; Hopkins, 1999).
Electric signals provide a private communication channel
since most predators cannot sense them. Nevertheless, some
predators are electroreceptive. For example catfish, which are
abundant in the rivers inhabited by electric fish, possess ampul-
lary electroreceptors (see 2.05.5.1) allowing them to detect
direct current (DC) and low-frequency components of EODs.
In streams with the highest density of catfish, as many as
45% of electric fish (Brachyhypopomus occidentalis) have injured
tails, mostly likely due to predation attempts by catfish
(Dunlap et al., 2016). Electric eels also hunt weakly electric
fish by tracking their EODs (Westby, 1988). Electroreceptive
predators have exerted a formidable selection pressure on
71
both groups of electric fish to evolve signals that are less detect-
able to these predators (i.e., minimize the DC and low-
frequency components of their EODs) (Stoddard, 1999;
Hanika and Kramer, 1999).
2.05.1.3 Breeding Cycle and Endogenous Levels of Hormones
Both mormyroid and gymnotiform electric fish breed primarily
during the rainy season (Kirschbaum, 1975, 1995), although
there are a few exceptions to this rule such as the electric eel
which reportedly breeds in the dry season (Assuncao and
Schwassman, 1995) and B. occidentalis, reported to be an acycl-
ical breeder (Hagedorn, 1992). Environmental cues that trigger
gonadal recrudescence and reproduction in these species
include the pH and conductivity of the water, water level, the
physical stimulation of rainfall, and food availability (reviewed
in Kirschbaum, 1995). In general, electric fish are transequato-
rial in their distribution so that day length and temperature are
not reliable zeitgebers for them. Some species of electric fish,
however, also inhabit the temperate zone. For example, in
the southern temperate zones of Uruguay, Brachyhypopomus
gauderio (formerly Brachyhypopomus pinnicaudatus) times its
spawning season based on seasonal temperature fluctuations
(Silva et al., 2002, 2003; Quintana et al., 2004).
Electric fish show a pattern of steroid secretion typical of
many teleost fish. Sex steroids are highest in mature adults,
with 11-ketotestosterone (11kT) as the predominant gonadal
steroid in males, 17b estradiol (E2) prominent in females,
and testosterone of similar values in males and females. For
both sexes, plasma steroids concentrations are greatest during
the breeding season.
In several cases, plasma steroid concentrations have been
measured directly from fish in their natural habitat. In male
B. gauderio in Uruguay, 11kT is higher in males during the
breeding season (January) than in the nonbreeding season
(June) (Dunlap et al., 2011b) and correlates positively with
testicular size, plasma levels of testosterone, and masculine
features of the EOD (Gavassa et al., 2011). E2 is higher in
females than in males and correlates positively with testos-
terone levels and feminine EOD features, but is unrelated to
gonadal size. In field-captured Sternopygus macrurus, androgen
levels of males correlate positively with the degree of testicular
development and body size, and masculine EOD parameters
(Zakon et al., 1991). However, while all large adult males
have high androgen levels and low EOD frequencies, immature
males, which have low androgen levels, have either high or low
EOD frequencies. Thus, some factor other than androgen level
influences EOD frequency in immature males (Zakon et al.,
1991). Female Sternopygus possess little or no 11kT and have
testosterone levels comparable to males. E2 levels in immature
females are similar to those of mature or immature males
(<0.5 ng ml
1), and gravid females with well-yolked eggs
have higher levels (2 ng ml
1) (Zakon et al., 1991).
In the laboratory, individual variation in androgens among
males is associated with body size, EODs, and social status.
Male brown ghosts (Apteronotus leptorhynchus) show a positive
correlation between plasma 11kT levels, body size, and two
EOD parameters (EOD frequency and chirp rate) (Dunlap,
2002). In the mormyrid Brienomyrus brachyistius, males housed
in captive social groups (three males and three females) in
72 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
which the males establish a dominance hierarchy, 11kT levels is
highest (16 ng ml
1) in the dominant male, next highest
(10 ng ml
1) in the next highest-ranked male, and least
(6 ng ml
1) in the subordinate male (Carlson et al., 2000).
2.05.2 EOD Waveform and Amplitude and Their
Control by Electrocytes
In most electric fish, the EOD is generated from muscle-derived
cells in the electric organ termed electrocytes. Like muscle cells,
they generate action potentials, but unlike muscle cells, they
fire in near-synchrony and do not contract. Because the electro-
cytes lie in columns in series, the voltage of the action poten-
tials sum to create a signal strong enough to form an electric
field that extends several body-lengths into the water.
The shape of the EOD is species-specific and is categorized
into two groups. Pulse-type EODs are generally short-
duration (0.2–4 ms and rarely up to 20ms) electrical waves
that are emitted by a fish at variable and relatively low rates
(5–50 Hz) (Figures 2 and 3). Wave-type EODs are generally
longer in duration but, within an individual fish, are emitted
at a very constant baseline rate (Figure 4). However, across
species with wave-type EODs, the baseline EOD frequency
varies broadly (60–1600 Hz). (For historical reasons, the
pace of the EOD in pulse fish is usually termed ‘EOD rate’ while
that of wave fish is termed ‘EOD frequency.’ We keep that
convention below, though these terms are essentially synony-
mous, given that both are controlled by the rhythmic firing
of the same hindbrain PN (see Section 2.05.3).)
Pulse and wave EODs each have their own advantages and
disadvantages. On one hand, wave-type EODs allow a more
rapid sampling rate – up to 1600 Hz in some apteronotids –
than pulse-type fish. On the other hand, pulse-type fish can
discharge at low baseline rates and rapidly increase EOD rate
when they need information faster. This also lets them regulate
energy expenditure on an as-needed basis and maintain a very
Sternopygus macrurus EOD range
50−200 Hz
Eigenmannia virescens
250−500 Hz
Apteronotus lephorhynchus
600−1100 Hz
Apteronotus albifrons
800−1250 Hz
10 ms
Figure 4 Wave-type electric organ discharges (EODs) from four
South American species. From Smith, G.T., 1999. Ionic currents that
contribute to a sexually dimorphic communication signal in weakly elec-
tric fish. J. Comp. Physiol. A 185, 379–387.
low resting rate during the day when they are inactive. Wave-
type EODs are continuously produced and, therefore, energeti-
cally demanding (Lewis et al., 2014). However, some wave-type
fish overcome this problem by dramatically decreasing EOD
amplitude in the daytime (see Section 2.05.2.4.4). With one
exception (Gymnarchus niloticus), all mormyroids produce
pulse-type EODs; about half of gymnotiform species have
pulse-type EODs and the other half have wave-type EODs.
The shape of the EOD waveform is determined by the
morphological and electrophysiological properties of the elec-
trocytes. The number of phases and their polarities depend on
fixed features of the electric organ such as the site(s) of innerva-
tion on the electrocytes, the number of electrically excitable
faces, the geometry of the electrocytes, the location of electro-
cytes across the body, and the coordination of neural inputs
along the length of the electric organ (Rodríguez-Cattáneo
et al., 2013). Variation within a species, such as sex differences
or individual differences, depends on more subtle variation in
electrophysiological properties of the action potentials gener-
ated by each electrocyte face (see Section 2.05.2.4.2).
2.05.2.1 Generation of the EOD Waveform
2.05.2.1.1 Pulse Fish
Among species with pulse-type EODs, the EOD differs in the
number and polarity of their phases and by the amplitude
and duration of each phase (Figures 2 and 3). The simplest
EOD is a monophasic pulse. This is generated by electrocytes
with one innervated face that generates an action potential
and another face that is electrically unexcitable (Bennett, 1961;
Figure 5(a)). Upon depolarization by synaptic input, the inner-
vated face fires an action potential during which positive current
(Naþ ions) enters the innervated face. This causes positive
(a) Monophasic EOD pulse
(b)
1 Diphasic EOD pulse 1
2
2
Figure 5 Generation of electric organ discharge (EOD) pulses.
(a) A monophasic EOD pulse is generated by current flow into the
innervated face of an electrocyte and out the opposite face. This
generates a pulse as in the right-hand side of the figure. (b) The first
phase of a diphasic EOD pulse is generated similarly. However, if the
uninnervated face is excitable, inward current may then flow into it
and out the innervated face. This results in the second phase of the
EOD as indicated on the right-hand side of the figure.
 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
current (probably Kþ ions) to exit the opposite face. If an electro-
cyte is innervated on its posterior face, then current flows in that
face and out the anterior face. The net positive current from all
the simultaneously active electrocytes flows toward the head
along the length of the electric organ into the water, and back
into the fish’s tail, resulting in a head-positive pulse.
In species that produce a biphasic discharge, both faces of
the electrocyte are excitable. First, the innervated face fires an
action potential during which current flows in one direction
(Figure 5(b)). The flow of current through the other membrane
depolarizes it, causing an action potential; current then flows in
the opposite direction in the organ. The alternate flow of
current headward and then tailward gives the EOD its biphasic
shape (Bennett, 1961).
The waveform of the pulse EOD can even be tri- or quadri-
phasic (Figure 3). Multiphasic EOD pulses occur in both gym-
notiform and mormyrid fishes, although the cellular
mechanisms responsible for their generation differ in the two
groups (Hopkins, 1999; Pedraja et al., 2014; Pereira et al.,
2007; Rodriguez-Cattaneo et al., 2008). Mormyrid electrocytes
are uniform, flattened, pancake-like discs restricted to and
tightly stacked in the tail peduncle (the part of the tail just ante-
rior to the anal fin). Distinct phases of a multiphasic EOD are
generated as current propagates along stalks that penetrate (tri-
phasic) and, in some species, repenetrate (quadriphasic) the
flattened electrocyte. Thus, the mormyrid electric organ acts
as a localized current source approximating a dipole producing
a simple field that is essentially the same close to and far away
from the fish (i.e., in near and far fields).
In gymnotiform pulse fish, the electrocytes are boxlike cells
surrounded by ample extracellular space that run in long tubes
of connective tissue along the whole length of the long knife-
like body. These tubes or groups of tubes span different lengths,
the electrocytes in them may vary locally in their properties
(e.g., size or innervation pattern), and the neural inputs to
them may arrive at different times to stagger the discharge of
different EOD components. These complexities cannot be
modeled as a simple dipole; they produce an EOD that differs
in its waveshape with time and along the body, and in the
near and far field (Pedraja et al., 2014).
2.05.2.1.2 Wave Fish
A wave-type EOD is essentially a pulse-type EOD with an
extremely regular discharge (Figure 4). If the duration of the
EOD pulses matches the interpulse interval, the EOD will be
a periodic and nearly sinusoidal wave. In wave-type fish that
possess myogenic electric organs, the EOD pulse is monopha-
sic, and the electrocytes have a single active face. The frequency
of a wave-type EOD is determined by the firing rate of the PN.
However, in order for the waveform to approximate a sine
wave, the electric organ pulse duration must also vary. So, for
example, the EOD frequency of Sternopygus sp. varies from 50
to 200 Hz while electric organ pulse duration varies inversely
from 12 to 3 ms (Figure 6). These two parameters covary so
that the fish with the lowest EOD frequency has the longest
pulse (Mills and Zakon, 1987, 1991). Both independent
parameters are sensitive to hormonal modulation (see Sections
2.05.2.4.1 and 2.05.2.4.2).
Fish in the family Apteronotidae are the only electric fish
with a neurogenic electric organ, and they all emit wave-type
73
(a) Pacemaker nucleus
Electric organ
(b)
Pacemaker
nucleus
Spinal electromotoneurons
Electrocytes
(c)
High EOD frequency
Pacemaker
EOD
3 ms
Low EOD frequency
Pacemaker
EOD
12 ms
Figure 6 Relationship between electric organ discharge (EOD)
frequency and electric organ (EO) pulse duration in gymnotiform wave
fish. (a) The location of the pacemaker nucleus and the electric organ.
(b) The output neurons of the pacemaker nucleus (the relay cells)
synapse on electromotor neurons and these synapse on the cells of the
electric organ, the electrocytes. (c) Pacemaker firing frequency and
electric organ pulse duration must be precisely correlated to produce
a sinusoidal EOD as in the gold-lined black knifefish (Sternopygus
macrurus). Note the difference between an individual with a high EOD
frequency (such as a female) and one with a low EOD frequency (such
as a male). From McAnelly, L.M., Zakon, H.H., 2000. Coregulation of
voltage-dependent kinetics of Naþ and Kþ currents in electric organ.
J. Neurosci. 20, 3408–3414.
discharges. Some apteronotids produce EOD frequencies in
excess of 1 K Hz. Their electromotor system is electrotonically
coupled from the PN to the electromotor neurons (Pappas
et al., 1975; Waxman et al., 1972). Apteronotus larvae begin
life with a myogenic electric organ, but it degenerates within
20 days of hatching, concurrent with a developmental increase
in EOD frequency (Kirschbaum, 1977; Kirschbaum and
Westby, 1975). The loss of the myogenic electric organ elimi-
nates the one obligatory chemical synapse in the electromotor
circuit that might fail at these high operating frequencies.
Although the EOD frequency ranges of some apteronotids
overlap, the waveform of each EOD cycle may differ among
species, with some having a number of phases (Kramer et al.,
1981; Turner et al., 2007).
2.05.2.2 EOD Waveform: Communication Function
One important communication function of the waveform is to
convey information about species identity. EOD waveforms are
species-specific, and behavioral tests show that they are suffi-
cient for species recognition (Hopkins and Bass, 1981;
74 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
Hopkins, 1976, 1981). Species-specific EODs are important for
reproductive isolation. Among the gymnotiformes, each wave
species may ‘broadcast’ over a specific range thereby enjoying
a ‘window’ with minimal interference from other species
(Hopkins, 1976, 1988). In areas of diverse species assemblages,
some species (especially apteronotids) may utilize similar EOD
frequency ranges. Nevertheless, the EODs are potentially
discriminable by the shape of the pulse of each cycle (Turner
et al., 2007). Pulse-type species easily discriminate waveform
differences between conspecific and heterospecific EODs.
In addition to these interspecific differences in waveform,
most electric fish species also show individual and sexual differ-
ences in EOD waveform within the species (Figures 2 and 3).
They have an exquisite capacity for detecting fine waveform
differences as shown by laboratory conditioning experiments
using real or synthesized EOD stimuli (Kramer and Otto,
1991; McGregor and Westby, 1992).
Intraspecific variation in waveform is used in the context of
social communication (McGregor and Westby, 1992; Carlson,
2000) and may be the basis for mate choice. When female
pintail knifefish (B. gauderio) are presented males of different
sizes, they preferentially associate with larger, sexually mature
males, whose EOD pulses are longer in duration than those
of smaller, immature males. This choice indicates a sexual
attraction since the females often spawn with them. Interest-
ingly, females spend less time near sexually immature males
than the empty chamber (Curtis and Stoddard, 2003). This is
not surprising given the propensity of gymnotiform pulse fish
to spread out by a few meters in their natural habitat (Hopkins,
1974a,b; Westby, 1988; Crampton, 1998).
2.05.2.3 EOD Waveform and Electrocytes: Sexual
Dimorphism
Most pulse fish possess sexually dimorphic EODs, although
some are monomorphic (Crampton et al., 2013). In a case of
apparent convergence, the males of species with myogenic
EODs (i.e., not apteronotidae) produce pulses that are longer
in duration than those of females. These patterns occur in
both Gymnotiformes and Mormyrids and likely arose through
evolutionary convergence (Figures 2 and 3; Carlson et al.,
2000; Hanika and Kramer, 2005; Hopkins et al., 1990;
Hopkins, 1974a,b; Mills and Zakon, 1987).
The ability of electric fish to perceive and respond to these
sex differences in the EOD waveform has been amply demon-
strated in numerous species. For example, male fish court
female, but not male, EODs or EOD mimics in playback exper-
iments (Hopkins and Bass, 1981). Conversely, females respond
preferentially to male versus female EOD pulses (Shumway
and Zelick, 1988).
Species differ in which components of the EOD are used for
sex recognition. For example, in many mormyroids, the EOD
pulse is longer in males than in females, and playback experi-
ments show that EOD pulse duration is the critical variable
for sex recognition (Hopkins and Bass, 1981; Machnik and
Kramer, 2008). By contrast, male Pollimyrus adspersus (formerly
classified as Pollimyrus isidori; Crawford, 1997), another mor-
myrid, ignore the EOD waveform, even though it possesses
a subtle sexual dimorphism in pulse amplitude (Crawford,
1992). Instead, it attends to sex differences in the regularity
of the pattern in which the discharges are emitted. The EOD
pulse is exceptionally brief in this species (85–90 ms in both
sexes), perhaps too brief to reliably resolve sex differences.
The correct pattern of interpulse intervals in a discharge may
be necessary for females to treat the pulse as a courtship signal
(Crawford, 1991, 1992; Machnik and Kramer, 2008; Wong and
Hopkins, 2007).
What is the function of sex differences in EOD waveform?
EODs from males are likely more ‘stimulatory’ to females
than those of females or juveniles. For example, the longer
duration of the second phase of the EOD pulse in male pintail
knifefish (B. gauderio) makes the signal waveform asymmet-
rical. This results in a brief DC component to the signal that
stimulates the ampullary electroreceptors of a female (Metzner
and Heiligenberg, 1991). Presumably the more dimorphic is
the second phase of the EOD, the more DC is generated, and
this may be attractive to females. Unfortunately, it also makes
the EOD pulse more detectable by electroreceptive predators
such as catfish. Indeed, male pintail knifefish are more likely
than females to have a wounded or regenerated tail, presum-
ably lost to predation (Hopkins et al., 1990). In addition,
sexual dimorphism of EOD waveform, as any other secondary
sexual character, likely acts as an honest proxy of male quality.
Indeed, in B. gauderio, pulse duration is a good indicator of
circulating androgen levels and gonad size in males (Gavassa
et al., 2011).
2.05.2.4 EOD Waveform and EOD Amplitude: Hormonal
Regulation
2.05.2.4.1 Effects of Exogenous Steroids on EOD Waveform
and Electric Organ Morphology
Many studies have demonstrated that exogenous sex steroids
modify the EOD waveform in ways that are consistent with
sexual dimorphism in waveform. In all species of pulse fish
tested to date, including both gymnotiforms and mormyrids,
androgen (testosterone or nonaromatizable androgen) treat-
ment to juveniles of both sexes and castrated males broadens
the pulse, mimicking the naturally occurring longer pulse dura-
tion in males (Bass and Hopkins, 1983; Freedman et al., 1989;
Hagedorn and Carr, 1985; Landsman and Moller, 1988; Mills
and Zakon, 1991; Silva et al., 1999; Figure 7). Similar results
occur when females are treated with the aromatizable androgen
testosterone, indicating that the effects are activational. Since
estrogens do not affect pulse-type EODs when given alone or
in combination with androgens, testosterone likely acts
through androgen receptors rather than binding to estrogen
receptors after aromatization (Hagedorn and Carr, 1985;
Landsman and Moller, 1988).
In some pulse fish, the long-term actions of sex steroids
affect only a portion of the EOD waveform. For example, in
B. gauderio and other species, androgen treatment broadens
only the second phase of the EOD pulse (Figure 7). This
suggests that changes in ion currents are restricted to the ante-
rior face of the electrocyte since this is the face that generates the
second phase of the EOD (Hagedorn and Carr, 1985; Goldina
et al., 2011; Markham and Stoddard, 2013).
In most wave-type gymnotiforms, males have a longer EOD
pulse duration than females, and pulse duration is closely tied
to EOD frequency. In these species (e.g., Eigenmannia and
 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
(a)
Pretreatment
Posttreatment
(b)
Juvenile/DHT
0d 10 d
Figure 7 Androgens broaden the electric organ discharge (EOD) pulse
of both gymnotiform and mormyroid pulse-type fish. (a) The EOD pulse
of a male pintail knifefish (Brachyhypopomus pinnicaudatus) before and
15 days after implantation with a capsule containing testosterone (Silva
et al., 1999). (b) Broadening of the EOD pulse of a juvenile mormyrid
Brienomyrus brachyistius 10 days after implantation with DHT. Adapted
from Bass, A.H., Hopkins, C.D., 1985. Hormonal control of sex differ-
ence in the electric organ discharge (EOD) of mormyrid fishes.
J. Comp. Physiol. A 156, 587–604.
Sternopygus), systemic androgen treatment broadens EOD dura-
tion over a 2-week period while simultaneously lowering EOD
frequency (Meyer, 1983; Mills and Zakon, 1987; Dunlap and
Zakon, 1998). However, in Sternopygus, localized androgen
treatment of the electric organ broadens pulse duration without
influencing EOD frequency (Few and Zakon, 2001). Thus,
androgens exert a concerted but independent influence on
the electric organ and PN. How androgens coordinate their
effects on two different target cell types of the electromotor
system raises the general question of how a hormone that
acts on multiple sites in a neural network exerts a coordinated
action.
In Sternopygus, estrogen implants in juvenile fish of both
sexes raise (feminize) EOD frequency (Meyer, 1983; Dunlap
et al., 1997). In addition, treatment of intact females or gonad-
ectomized fish of both sexes with human chorionic gonado-
tropin (hCG) powerfully increases EOD frequency (Zakon
et al., 1990). It is not known whether hCG acts directly on
the pacemaker and electrocytes or whether it acts indirectly
by stimulating the release of another hormone from a nongona-
dal source.
The size and shape of the electrocytes is sexually dimorphic
in some species of pulse fish in both orders (Hagedorn and
Carr, 1985; Bass et al., 1986; Bass, 1986b; Freedman et al.,
1989). In some cases (e.g., B. gauderio), this is so pronounced
that sexes differ in the length or thickness of the tail (Hopkins
et al., 1990). In mormyrids, electrocytes in males are thicker
and larger, although this does not result in a visible dimor-
phism in the tail. As expected, treatment of females or sexually
immature males with androgens induces these morphological
changes in electrocytes.
75
2.05.2.4.2 Effects of Exogenous Steroids on Electric Organ Ion
Currents
The cellular mechanisms underlying steroid action on EOD
waveform are best understood in the wave-type gymnotiform
Sternopygus macrurus, the gold-lined knifefish (Hopkins,
1974a,b). As discussed above, the electrocytes of Sternopygus
generate action potentials that vary from 3 ms, in a fish with
a high EOD frequency such as a sexually mature female, to
12 ms, in a fish with a low EOD frequency, such as a mature
male (Figure 6). Action potential duration is lengthened
(masculinized) over days to weeks by chronic androgen treat-
ment (Mills and Zakon, 1991; Figure 8).
At the cellular level, androgens broaden action potentials by
influencing the ionic currents of the electrocyte. Three different
ion currents are present in the Sternopygus electrocyte: a voltage-
dependent Naþ current, a delayed rectifying Kþ current, and an
inward rectifying Kþ current (Ferrari and Zakon, 1993). The
inward rectifying Kþ current sets the resting potential while
the Naþ current and delayed rectifying Kþ current shape the
EOD pulse. The rate at which the Naþ current turns off (inacti-
vates) and the delayed rectifying Kþ current turns on (activates)
varies systematically with EOD frequency and pulse duration.
Individuals with low EOD frequencies have currents that acti-
vate and inactivate slowly, while fish with the highest EOD
frequencies have currents with the fastest rates (Ferrari et al.,
1995; McAnelly and Zakon, 2000; Figure 8). In males, the
slow inactivation of the Naþ current and slow activation of
the Kþ current means that Naþ flows into the cell unopposed
by an efflux of Kþ thereby producing a long duration action
potential.
Treatment of fish with the nonaromatizable androgen 5-a
dihydrotestosterone (DHT) slows inactivation rate of the Naþ
current and the activation rate of the delayed rectifying Kþ
current (McAnelly and Zakon, 2007). As described above, these
changes lengthen electrocyte action potential duration and
broaden the EOD pulse. Conversely, estradiol speeds up the
inactivation of the Naþ current, which should shorten EOD
pulse duration (Dunlap et al., 1997). It is not known if estra-
diol also acts on Kþ currents. The actions of steroid hormones
on these currents are genomic, as they take days to manifest.
The most likely targets for these hormonal influences on
waveform are the ion channel genes themselves. Both Naþ
and Kþ channel genes have been cloned from the electric organ.
Two Naþ channel genes (Nav1.4a and Nav1.4b) and an acces-
sory Naþ channel subunit (b1) are expressed in the electric
organ (Liu et al., 2007, 2008). One gene, Nav1.4a, is expressed
uniformly across the range of electrocyte action potential dura-
tions. The other two genes, Nav1.4b and the b1 subunit, are
expressed at higher levels in fish with narrow EOD pulses
(females) than in those with long EOD pulses (males);
Nav1.4b and the b1 subunit are downregulated by androgens.
Compared to expression of Nav1.4b alone, coexpression of the
Nav1.4b Naþ channel with the b1 subunit in Xenopus oocytes
increases the inactivation rate of the Naþ current. (Nav1.4a
has not yet been expressed in oocytes.) In the current model
(Figure 9), Nav1.4a, which is expressed equally in all electro-
cytes and is the dominant Naþ channel gene expressed in
males, produces a Naþ current with a slow inactivation rate.
As the level of Nav1.4b increases, relatively more of the Naþ
current will be transmitted through a channel with a rapidly
76 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology

(a) (b) 140 Hz (c)

π = 0.87 ms
145 Hz Long spike
21%

Change in duration (ms)

2
15% pulse
60 nA 15%
1
105 Hz
π = 0.87 ms
101 Hz 0

−4% −3%
200 nA −1
DHT Control
65 Hz 55 Hz
π = 0.87 ms
20 mV
5 ms
60 nA
2 ms

þ
Figure 8 Electrocyte action potential duration and Na current are individually distinct and influenced by hormones. (a) Intracellular recordings
made in electrocytes of high, mid, and low EOD (electric organ discharge) frequency juvenile Sternopygus. Current was injected with one electrode,
and the voltage responses of the membrane were recorded with another. The number to the right of each trace gives each fish’s EOD frequency. Note
the systematic individual variation in action potential duration. (b) Voltage clamp recordings from electrocytes from fish with comparable EOD
frequencies as in ‘a’ show that the Naþ current activates and inactivates at different rates. EOD frequency and the exponential time constant of decay
of the current (its rate of inactivation) is given to the right of each family of current traces. (c) Androgens broaden the EOD pulse and the electrocyte
action potential (labeled ‘long spike’). In this experiment recordings of these two parameters are made before and after treatment with DHT or empty
capsules. Note that the control fish show no change in either parameter, but the hormone-treated fish show an increase in both. From Zakon, H.H.,
1999. Sex steroids and weakly electric fish: a model system for activational mechanisms of hormone action. In: Matsumoto, A. (Ed.), Sexual Differen-
tiation of the Brain. CRC Press, Boca Raton, FL, pp. 95–112.

inactivating current. As levels of the b1 subunit increase in
tandem with Nav1.4b, the b1 subunit interacts with and speeds
up the inactivation rate of both Naþ channels and further
increases the inactivation rate and narrows the duration of
each EOD pulse.
Less is known about the molecular control of Sternopygus Kþ
current. Levels of mRNA for certain members of the Kv1 family
(Kv1.1a, Kv1.2a) are abundant in the electric organ of fish with
short pulses and low in the electric organ of fish with long
pulses (Few and Zakon, 2007). The level of Kv1.2b mRNA
does not vary with EOD pulse duration. Furthermore, the tran-
script abundance of Kv1.1a and Kv1.2a is suppressed by DHT
while that of Kv1.2b is unaffected. These genes have not yet
been expressed in oocytes, so the biophysical properties of the
channel proteins are currently unknown. However, the current
model suggests that the androgen-dependent subunits have
faster activation rates, so Kþ currents will be slower in males.
2.05.2.4.3 EOD Waveform: Seasonal Modulation and Its
Hormonal Regulation
Sexual dimorphism of EOD waveform cycles seasonally
because it is regulated by seasonal changes in gonadal steroid
secretion. This was demonstrated by sampling a natural popu-
lation of B. gauderio at the subtropical edge of gymnotiform
distribution in South America, where electric fish and most
other freshwater teleosts breed during the austral spring to
summer (November to February) in association with warm
temperature and long photoperiod (Silva et al., 2003). In the
nonbreeding season, males and females are indistinguishable
externally. However, during the breeding seasons, males
exhibit large differences from females in morphological (broad
tails) and electrophysiological (longer EOD second phase)
traits that coincide with gonadal maturation (Silva et al.,
2002; Quintana et al., 2004) and elevated plasma 11kT
(Dunlap et al., 2011b). An experimental elevation in water
temperature causes gonadal recrudescence and increased
androgen levels in nonbreeding males, and experimental
androgen treatment enhances morphological and electrophys-
iological traits characteristic of males during the breeding
season (Silva et al., 1999; Quintana et al., 2004).
2.05.2.4.4 EOD Waveform: Diel, Social, and Stress-Induced
Modulation and Its Hormonal Regulation
The changes in EOD waveform induced by exogenous steroids
described above occur over days to weeks. However, changes in
EOD waveforms may also occur within minutes in freely
moving fish. Hagedorn and Heiligenberg (1985) first reported
that the EOD amplitude of the dominant male Eigenmannia in
a community tank increases in amplitude (i.e., gets very loud
on an audio monitor) on the night of breeding, so much so
that it drowns out the signals of other fish. This was a radical
suggestion at a time when it was believed that the characteris-
tics of the EOD were determined by fixed aspects of the
anatomy and physiology of the electromotor system.
Under conditions where accurate EOD recordings can be
made in swimming fish, the sexually dimorphic EOD phase of
male B. gauderio increases rapidly in duration and amplitude after
the lights turn off (Franchina and Stoddard, 1998; Figure 10).
Such nocturnal excursions occur in isolated males, but are further
enhanced in males exposed to conspecifics behind a mesh
barrier. Stimulus males elicit a greater effect on EOD parameters
than stimulus females (Franchina et al., 2001). The hormonal
 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology 77

(a) Total β1 (a)

Expression levels (low to high) smNav1.4bL

Day

smNav1.4a Night

1 ms
(b)
8

Amplitude (mV/cm@10 cm)

2 6

Duration (ms)
(b) EOD frequency (low to high)
Long Short 4
EOD pulse duration
1
Amplitude 2
Electrocyte Na+ current Duration
Light Dark Light
0 0
smNav1.4a smNav1.4bL
Noon Midnight Noon
Time of day
(c)

Figure 9 Model for androgen action in the electric organ of a wave-

type gymnotiform (Sternopygus macrurus). (a) Two sodium channels
(smNav1.4bL; smNav1.4a) and a sodium channel auxiliary subunit
(beta1) are expressed in the electric organ (EO). smNav1.4a is
expressed equally in all individuals and is hypothesized to inactivate
(i.e., close after opening) slowly (b). Nav1.4bL and b1 are expressed at
high levels in fish (females) with high electric organ discharge (EOD)
frequencies and short EOD pulses (right side of the graph); these fish
also have sodium currents that inactivate rapidly. Androgens suppress
expression of Nav1.4bL and beta1 (which act on sodium channels to
further speed inactivation). Thus, males have low levels of these genes
(left side of the graph) but relatively higher levels of smNav1.4a that
would generate a current that inactivates slowly and lengthens the EO
action potential.
action underlying this effect on waveform must be only on the
anterior membrane of the electrocyte since this is the membrane
that generates the second phase of the EOD pulse. Daily fluctua-
tions of EOD amplitude in this pulse species suggest that fish
optimize their energy expenditure by generating the largest
amplitude discharge only during periods of significant social
interaction. Two wave-type species, Sternopygus (Markham
et al., 2009) and Eigenmannia (Markham et al., 2013), also
produce rapid changes in EOD amplitude in response to social
or circadian stimuli, indicating that such modulation may be
widespread among gymnotiforms.
The hormonal link between environmental stimuli and
electrocyte excitability are the melanocortin peptides (alpha
melanocyte stimulating hormone (a-MSH) and/or adrenocor-
ticotropic hormone (ACTH)) that act via melanocortin recep-
tors on electrocytes. On the cellular level, the melanocortin
receptor obligatorily activates a G-protein-coupled pathway
that increases cAMP thereby activating protein kinase A.
The target of this kinase is not yet identified. However, the
200 nA
3 ms
8Br-cAM
Figure 10 Diurnal changes in the electric organ discharges (EODs) of
a pulse fish (Brachyhypopomus pinnicaudatus) and its possible biophys-
ical basis. (a) Traces of EOD waveform during the day and night illus-
trating the nocturnal increases in amplitude of positive and negative
phases of the EOD and duration of the negative phase of the EOD.
(b) Measurements of peak-to-peak amplitude and the duration of the
second phase of the EOD pulse of a male over 24 h. (c) Voltage clamp
recordings of the peak Naþ current from an electrocyte of a wave fish
(Sternopygus) before and after treatment with 8-bromo-cyclic AMP. Note
that the amplitude of the Naþ current is enhanced. Such an increase in
the Naþ current likely increases the current output of each electrocyte
thereby increasing the EOD amplitude. From (a) and (b) Franchina,
C.R., Stoddard, P., 1998. Plasticity of the electric organ discharge
waveform of the electric fish Brachyhypopomus pinnicaudatus: I. Quantifi-
cation of day-night changes. J. Comp. Physiol. A 183, 759–768;
(c) from McAnelly, L.M., Zakon, H.H., 1996. Protein kinase A activation
increases sodium current magnitude in the electric organ of Sternopy-
gus. J. Neurosci. 16, 4383–4388.
result of its activation is to increase the exocytosis of constitu-
tively trafficked Naþ channels, which increases electrocyte
Naþ current and, therefore, EOD amplitude (Markham and
Stoddard, 2005; Markham et al., 2009; McAnelly et al., 2003;
McAnelly and Zakon, 1996; Figure 11). In B. gauderio, females
78 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
Figure 11 Summary diagram of how activation of melanocortin receptors results in trafficking of Naþ channels into the electrocyte membrane to
increase electric organ discharge (EOD) amplitude. From Markham, M.R., McAnelly, L.M., Stoddard, P.K., Zakon, H.H., 2009. Circadian and social
cues regulate ion channel trafficking. PLoS Biol. 7, e1000203.
given androgen pretreatment show a stronger melanocortin-
induced EOD modulation (Allee et al., 2009; Goldina et al.,
2011). This is an example of a long-term steroidal process
enhancing a short-term process.
In addition to the rapid daily changes in EOD amplitude,
social and metabolic stress can modify EOD waveform or
amplitude. For example, socially stressing mature male B. occi-
dentalis causes the sexually dimorphic portion of the EOD pulse
to decrease in duration. When two males of comparable size
are placed together in a tank and compete for a single hiding
place, the winner shows a similar decrease of its EOD pulse
duration and its EOD amplitude falls. Both EOD changes occur
within 2 h of the interaction (Hagedorn and Zelick, 1989).
During the annual rain cycles of the tropics, fish are
commonly exposed to periods of low oxygen and food avail-
ability and thereby experience metabolic stress (Crampton,
1998). To conserve energy during hypoxic stress, electric fish
decrease EOD amplitude (Crampton, 1998; Reardon et al.,
2011). Presently, there is little information whether this is
hormonally driven or a direct effect of oxygen or ATP concen-
tration on the ion channels of the electrocytes (Markham
et al., 2013).
However, researchers have examined the hormonal basis
of the EOD response to another metabolic stress, food
shortage. One expected energy-conserving response to food
deprivation is also a decrease in EOD amplitude. Indeed,
this occurs in the wave fish Eigenmannia virescens (Sinnett
and Markham, 2015). Surprisingly, under similar condi-
tions, the pulse species B. gauderio increases EOD amplitude
(Gavassa and Stoddard, 2012). This species difference seems
paradoxical until the life histories of the two species are
compared. Eigenmannia virescens may live for a few years
and breed each year (iteroparous), whereas, under some
circumstances, it appears that some B. gauderio may live
less than a year and breed only once (semelparous). In
principle, iteroparous species save their energy ‘for a better
day’ while semelparous species ‘go for broke’; further
research is necessary to test this hypothesis broadly among
in electric fish.
These two species (E. virescens and S. macrurus) also have
different endocrine responses to metabolic and social stress.
In E. virescens, cortisol, an important stress hormone in many
fish, does not appear to play a role in the response to food
deprivation. Endogenous cortisol levels do not change with
food deprivation, and cortisol treatment does not influence
EOD amplitude. Instead, the response appears mediated by
the peptide leptin: systemic leptin treatment increases EOD
amplitude in starved, but not fed fish (Sinnett and Markham,
2015). By contrast, in B. gauderio, exogenous cortisol adminis-
tration reduces EOD amplitude and duration, but endogenous
cortisol levels do not rise under food limitation or social chal-
lenge. When starved, males respond to social challenge by
increasing androgen levels and enhancing EOD amplitude
and duration (Gavassa and Stoddard, 2012). Socially chal-
lenged males simultaneously elevate testosterone and cortisol
in proportion to signal amplitude. Thus, B. gauderio appears
to protect its cortisol-sensitive electric advertisement signal by
increasing food intake, limiting cortisol release and offsetting
signal reduction from cortisol with signal-enhancing andro-
gens. The role of leptin in this species is not yet known.
2.05.3 EOD Rate/Frequency and Its Regulation
by the Pacemaker Nucleus
(By convention, we refer to the pace of the EOD as ‘EOD rate’
in pulse fish and ‘EOD frequency’ in wave fish. However, these
terms refer to the same phenomenon and are essentially
synonymous.)
EOD rate/frequency conveys the full range of information
encoded by the electric channel of communication, including
species identity, sexual identity, arousal, social status, and
motivational state. In addition, a broad range of environ-
mental, social, and physiological factors impact EOD rate/
frequency (reviewed in Silva et al., 2007). For example, in all
gymnotiform species tested, EOD rate/frequency is temperature
dependent (Enger and Szabo, 1968; Boudinot, 1970; Dunlap
et al., 2000; Ardanaz et al., 2001). In many pulse-type species
 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
of both orders, EOD rate increases during the night when fish
become more active. In pulse type fish, sudden or novel stimuli
in the environment also cause changes in EOD rate of pulse-
type fish, a reaction known as the novelty response. Stimuli
activating Mauthner cells elicit an abrupt and prolonged
increase in EOD rate (Falconi et al., 1995). Finally, in the pres-
ence of a nearby interfering fish, most gymnotiform wave-type
species shift their EOD frequency (jamming avoidance
response; (Heiligenberg, 1986). Thus, given all these influences
on EOD rate, it is probably rare that a fish discharges at its EOD
at basal rate.
During breeding, complex and precise information is
encoded in the EOD rate, and different kinds of rate changes
occur in the context of reproduction. Several wave-type species
show sexually dimorphic EOD frequencies used for mate recog-
nition and selection (see Section 2.05.3.2.1). Furthermore, in
both wave- and pulse-type species, males and females modu-
late EOD rate to produce transient social signals that participate
in courtship, spawning, and aggression (see Section 2.05.4.1).
In all electric fish, EOD rate/frequency is controlled by the
medullary PN (Figure 1). Indeed, electric fish are one of the
few vertebrate groups in which the control of specific behav-
ioral displays can be traced reliably to a single conspicuous
hindbrain nucleus (Bennett, 1971; Ellis and Szabo, 1980).
In this section, we review examples of how EOD rate varies
by season and sex, how hormones modify EOD rate, and
how rate variations are generated at the cellular level in the
PN. These rate changes are particularly meaningful in species
with regular discharge rates; otherwise, the information they
carry could be masked if the background rate varied widely.
Since all mormyroids (except Gymnarchus) emit pulse-type
EODs with an irregular pattern of emission, we limit this
section to studies on Gymnotiformes. In addition, we focus
on variation in EOD basal rate, and not on the emission of
transient EOD rate modulations, which is covered in Section
2.05.4. The important conclusion emerging from these studies
is that the PN is an extremely plastic medullary central pattern
generator that responds to higher brain regions and a broad
set of neuromodulators to adapt its function to diverse
contexts.
2.05.3.1 EOD Rate in Pulse Fish: Social and Diel Influences
and Their Hormonal Regulation
2.05.3.1.1 Social Influences
In pulse fish, changes in EOD rate signal dominance status in
agonistic interactions. In B. gauderio dominant males show
higher EOD rates than subordinate males after reproductive
aggression, while subordinates of Gymnotus omarorum reduce
their EOD rate after nonbreeding inter- and intrasexual
agonistic encounters (Silva et al., 2013). Interestingly, the
hypothalamic neuropeptide arginine vasotocin (AVT), an inte-
grative mediator of social behavior, regulates this phenotype-
dependent variation in EOD rate. In B. gauderio, the electrical
dominance is abolished when a competitive antagonist of
AVT V1a receptors (Manning Compound) is administered to
the potential dominant fish prior to the encounter (Perrone,
2012). In G. omarorum, AVT treatment to potential subordi-
nates before the contest enhances their electric submission
(Perrone, 2012).
79
When tested in the most basal conditions (isolated and
immobile during daytime), AVT treatment increases EOD rate
in B. gauderio and G. omarorum (Perrone et al., 2010, 2014).
However, the effect of AVT on EOD rate differs between species,
depending on the degree of sociality. The effect is more
pronounced and persistent in the gregarious B. gauderio than
in the solitary G. omarorum. In vitro electrophysiological record-
ings of the PN demonstrate that these different sensitivities
result from differential effects of AVT on the PN itself (Perrone
et al., 2010, 2014). Furthermore, immunolabeling of preoptic
AVT neurons confirms the presence of hindbrain AVT projec-
tions close to the PN that might constitute the anatomical
substrate for AVT influences on PN activity. Differences in the
spatial distribution of AVT projections between species might
account for interspecific differences in AVT modulation
(Perrone et al., 2014).
2.05.3.1.2 Diel Influence
EOD rate increases during nocturnal arousal have been re-
ported in several species of the genera Brachyhypopomus and
Gymnotus (Silva et al., 2007). Although the circadian system
of weakly electric fish has not yet been fully characterized,
these daily variations in EOD rate probably constitute a circa-
dian rhythm of electric behavior. This is based on the
following evidence reported recently in both B. gauderio and
G. omarorum: strong daily variations of EOD rate (1) can be
replicated in isolated fish in laboratory controlled conditions
under a 12:12 light/dark photoperiod; (2) persist, albeit
shifted, when placed in constant darkness; (3) depend on
the timekeeping hormone melatonin; and (4) do not depend
on fish locomotion, which is more frequent during the
nocturnal active phase (Migliaro and Silva, 2016a,b). Interest-
ingly, this rhythm of EOD rate occurs in the wild despite the
fact that fish are in constant darkness underneath aquatic
plants during the 24-h cycle (Migliaro and Silva, 2016a,b).
This suggests that other environmental cues, e.g., temperature,
social interaction, might be crucial for entraining circadian
rhythmicity in weakly electric fish.
The control of circadian rhythms of electric behavior is
embedded in the complex integrative control of the brain.
In Brachyhypopomus, seasonal changes and social stimuli
modulate the nocturnal increase of EOD rate, which is
more pronounced in breeding than in nonbreeding fish (Silva
et al., 2007) and more in socially interacting fish than in iso-
lated fish (Silva et al., 2007; Stoddard et al., 2007). Also inter-
esting, species differ in the nocturnal increase of EOD rate in
parallel with their different social structure. The nocturnal
increase is mild and transient in the solitary G. omarorum,
whose individuals rarely interact, and strong and persistent
in the gregarious B. gauderio, in which social stimuli further
enhances the nocturnal increase (Migliaro and Silva,
2016a,b). AVT, known to support species-, phenotype- and
context-dependent actions, has been postulated to mediate
this species-specific effect. Although AVT acts directly on the
PN of both species to enhance EOD rate in a species-
specific manner (Perrone et al., 2010, 2014), it is not respon-
sible for the nocturnal increase of EOD rate in isolated
individuals (Migliaro and Silva, 2016a,b). However, AVT
mediates the social component of EOD rate nocturnal
increase in B. gauderio (Perrone et al., 2014), suggesting
80 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology

intricate relationships between the AVTergic and the melato- (a) 1100
nergic system in the coupling of timekeeping and social
behavior in electric fish.

1000

EOD frequency (Hz)

2.05.3.2 EOD Frequency in Wave Fish: Role in Social Behavior
and Its Hormonal Regulation
2.05.3.2.1 Social Behavior 900
EOD frequency signals sex among several gymnotiform wave-
type fish. In most species (e.g., Sternopygus macrurus, E. virescens,
and Apteronotus albifrons), EOD frequency is lower in males 800
than in females (Figures 6(c) and 12(b); Hopkins, 1972;
Dunlap et al., 1998). Some species have sexually monomor-
phic EOD frequencies (e.g., Adontosternarchus devenanzii, Para- 700
pteronotus hasemani, and Apteronotus bonapartii; Zhou and 0 10 20 30 40
Smith, 2006, Ho et al., 2010; Petzold and Smith, 2016), and Body mass (g)
in a few species (e.g., A. leptorhynchus and Apteronotus rostratus),
males have higher EOD frequencies than females (b)
(Figure 12(a); Hagedorn and Heiligenberg, 1985). The ances- 1200
tral and common pattern is for males to have lower EOD
frequencies than females, with the alternative (males with
higher EOD frequencies) evolving as a case of reversed sexual

EOD frequency (Hz)

dimorphism (Dunlap et al., 1998; Dunlap and Zakon, 1998).
In addition to signaling sexual identity, EOD frequency can
also convey information about social status. In a tank commu-
nity of Eigenmannia, the largest male possessed the lowest EOD
frequency among the males, and the largest female had the
highest EOD frequency among the females (Hagedorn and
Heiligenberg, 1985). The largest fish of both sexes became
dominant; they bred with each other and prevented other
fish from breeding. When a playback recording of the high-
frequency EOD of the dominant male was substituted for
him during courtship, the female continued to respond and
lay her eggs.
Similar relationships between EOD frequency and domi-
nance have been found in studies of brown ghost knifefish
(A. leptorhynchus). In small groups (three males, one female)
the largest male with the highest EOD frequency (this species
has a reversed sexual dimorphism) bred with the female
(Hagedorn and Heiligenberg, 1985). In larger groups
(12 males, 12 females) housed with a limited number of
shelter tubes, males with the highest EOD frequency controlled
the most desired shelter tubes (Dunlap and Oliveri, 2002).
Without playback experiments it is not possible to know
whether the EOD frequency was the basis for differences in
social status.
EOD frequency can also signal within-sex variation in
a species with different male morphs (Sternarchogiton nattereri;
Fernandes et al., 2010). In this species, toothed males,
compared to toothless males, have larger body size, testes
growth, and higher EOD frequencies in association with higher
levels of 11-kT, even though both male types are reproductively
mature. The association of all these factors not only impact
reproductive performance but also may vary in their expression
across seasons in concert with the highly variable environ-
mental conditions.
2.05.3.2.2 Hormonal Regulation
Sexual dimorphism in EOD frequency arises from the differen-
tial action of gonadal steroid hormones. In many species,
1100
1000
900
800
0 20 40 60 80 100 120 140
Body mass (g)
Figure 12 Electric organ discharge (EOD) frequencies of two related
gymnotiform wave-fish with sex differences in EOD frequency in
opposite directions. (a) Brown ghost females are lower in frequency
than males. (b) Black ghost females are higher in frequency than
males. From Zakon, H.H., Dunlap, K.D., 1999. Sex steroids and
communication signals in electric fish: a tale of two species. Brain
Behav. Evol. 54, 61–69.
treatment with exogenous steroids mimics the endogenous
sexual dimorphism. In Sternopygus and Eigenmannia, androgen
treatment lowers EOD frequency over a 2-week period (Meyer,
1983; Mills and Zakon, 1987; Dunlap and Zakon, 1998). While
decreasing EOD frequency, androgens also lengthen EOD dura-
tion, and with this combination, the waveform stays the same
shape. In the brown ghost, A. leptorhynchus, in which males
have higher EOD frequency than females (Figure 12(a)),
11kT raises EOD frequency, whereas E2 or T lowers it (Meyer
et al., 1987; Schafer and Zakon, 1996). Interestingly, in the
closely related species, A. albifrons, in which males have lower
EOD frequencies than females (Figure 12(b)), 11kT or dihy-
drotestosterone (DHT) treatment lowers EOD frequency
(Dunlap et al., 1998). The close phylogenetic relationships
between these two species with opposite androgenic actions
upon EOD frequency make them good subjects for studying
how sexual dimorphisms evolve.
 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
In many cases (e.g. Sternopygus and Eigenmannia), estrogen
implants in juvenile fish of both sexes raise (feminize) EOD
frequency (Meyer, 1983; Dunlap et al., 1997; Dunlap and
Zakon, 1998). However, Schaefer and Zakon (1996) found
that estradiol reduced EOD frequency in female A. leptorhynchus
which is consistent with the observation that females
have lower EOD frequency in this species. Treatment of intact
females or gonadectomized fish of either sex with
hCG potently increases EOD frequency in Sternopygus (Zakon
et al., 1990), in which females have higher EOD frequencies.
By contrast, male Sternopygus injected with hCG tandemly
increased 11kT plasma levels and decreased EOD frequency
(Zakon et al., 1990). It is not known whether hCG acts directly
on the pacemaker and electrocytes or whether it acts indirectly
by releasing another hormone from a nongonadal source.
In several studies, EOD frequency and plasma steroid
concentrations have been correlated in natural populations.
In the field, androgen levels of male S. macrurus correlate posi-
tively with the degree of testicular development and body
size and correlate negatively with EOD frequency (Zakon
et al., 1991). However, while all large adult males have high
androgen levels and low EOD frequencies, immature males,
which have low androgen levels, have either high or low
EOD frequencies. Thus, some factor other than androgen level
influences EOD frequency in immature males (Zakon et al.,
1991). Population differences in sexual dimorphism have
been reported in A. albifrons (Ho et al., 2013). Interestingly,
EOD frequency in a Colombian population is more sexually
dimorphic and more sensitive to androgen treatment than
the EOD frequency of a Brazilian population. Thus, the evolu-
tion of sexual dimorphism in electric behavior at the popula-
tion level appears related to changes in androgen sensitivity
across populations.
Male brown ghosts in the laboratory also show a positive
correlation between plasma 11kT levels, body size, and EOD
frequency; males with low levels (<2.5 ng ml
1) have low
EOD frequencies, whereas those with levels from 5 to
15 ng ml
1 have the highest EOD frequencies (Zucker, 1997;
Dunlap, 2002). The role of endogenous sex steroids in control-
ling EOD parameters is further supported by the fact that the
EOD changes in the expected direction following gonadectomy
(Meyer, 1983; Zucker, 1997). Furthermore, in A. bonapartii,
EOD frequency is not sexually dimorphic and is not correlated
with plasma levels of androgens in either sex (Ho et al., 2010).
Because EOD frequency is ultimately set by the activity of
the hindbrain PN, sexual dimorphism of EOD frequency
most likely depends on the action of steroid hormones on
the firing properties of PN neurons. Androgen receptors have
been identified in PN neurons (Pouso et al., 2010), and andro-
gens and estrogens change EOD frequency by directly
changing the rhythmic firing rates of neurons in the PN and/
or of the spinal electromotor neurons whose axons innervate
or form the electric organ. The rhythmic firing of the PN and
electromotor neurons is regulated primarily by transient
and persistent sodium and potassium currents (Schaefer and
Zakon, 1996; Smith and Zakon, 2000; Smith, 2006), but the
effects of gonadal steroids on these conductances have not
been clarified so far.
Glucocorticoids do not appear to influence EOD
frequency in Apteronotus (Dunlap et al., 2006). Endogenous
81
cortisol levels do not correlate with EOD frequency, and
exogenous cortisol treatment does not alter EOD frequency.
However, thyroxine treatment increases EOD frequency,
consistent with the role of thyroxine in many rate-
dependent processes in neurons (Dunlap and Ragazzi,
2015).
2.05.4 EOD Modulations and Their Regulation
by Prepacemaker Nuclei
As discussed above, features of the EOD that convey informa-
tion on species, sex, individual identity, or sexual maturity
are stable or change slowly. By contrast, information on moti-
vational states that change quickly, particularly during
agonistic and courtship interactions, are signaled by rapid
EOD changes termed EOD modulations. Both wave and pulse
fish transiently modify EOD frequency (wave fish) or pulse rate
(pulse fish) and amplitude (Black-Cleworth, 1970; Kramer,
1979; Zakon et al., 2002) during aggression and courtship.
Such EOD modulations occur at greatest rates during the
breeding season (however, see Gymnotus below).
In male–male interactions, aggression can be rather intense,
with fish often chasing and biting each other while they modu-
late their EOD to signal dominance or submission. The expres-
sion of EOD modulations commonly depends on the stage of
the fight, with modulations changing frequency or form
between the assessment, contest, and resolution phases of the
encounter (Hopkins, 1974a,b; Hupe and Lewis, 2008; Perrone
et al., 2009; Batista et al., 2012).
In all electric fish studied, courtship occurs over a period of
a few nights. Typically courtship activity intensifies each night
with the greatest activity on the night of spawning. Courtship
is accompanied by a variety of stereotyped body postures and
movements and variations in the rhythm of the EOD, with
each sex usually displaying a different pattern of EOD modula-
tion. Spawning occurs in a single bout or, more frequently, in
a series of bouts (Hagedorn and Heiligenberg, 1985;
Henninger, 2015). Below we give an overview of EOD modu-
lations in a broad range of electric fish, and then focus on the
hormonal control of EOD modulations in fish in the family
Apteronotidae, and in the genera Brachyhypopomus and
Gymnotus, where the neuroendocrine control has been studied
in depth.
2.05.4.1 EOD Modulations: Communication Function
2.05.4.1.1 Pulse Fish
During social interactions, the pulse-type gymnotiform B. gau-
derio displays three broad classes EOD modulations collectively
termed social electric signals (SESs) (Perrone et al., 2009; Silva
et al., 2008). Interruptions are near-complete cessations of the
EOD for approximately 100–200 ms. Accelerations are rela-
tively small increases in pulse rate with little change in EOD
amplitude that last 900ms. Chirps are brief periods (approx-
imately 50–300ms) when EOD pulses occur at much higher
rates and lower amplitudes than the basal EOD pulse rate.
Chirps can be classified into four chirp types (A, B, C, and M)
based on the duration, amplitude modulation, and degree of
symmetry (Figure 13).
82 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology

Figure 13 Context-dependent chirping behavior in Brachyhypopomus gauderio. (a) Dyadic male–female electric organ discharge (EOD) recordings
during courtship; the male emits chirps Types A, B, C, while the female maintains its regular discharge (indicated by dots). Chirps exhibited differ-
ences in duration, relative amplitude, time to minimal amplitude, and symmetry among chirp types. (b) Dyadic male–male EOD recordings during
agonistic interactions, the subordinate male turns off its discharge and emits short chirps Type S, while the dominant emits Type M chirps. Dots
indicate the occurrence of subordinates’ EODs. Modified from Perrone, R., 2012. La vasotocina modula el comportamiento social de dos especies de
peces eléctricos con diferente socialidad. PEDECIBA, Universidad de la República, Montevideo.

The production SESs varies by social context, sex, time of
day, and season (Perrone et al., 2009; Silva et al., 2008). The
overall rate of SES production is about four times greater in
male–male dyads than in male–female dyads. Virtually all
SESs occur at night, and some are produced differentially
according to the phase of the dark period. For example, type
A chirps are emitted early in the dark phase while types B
and C chirps are emitted just before sunrise. Finally, fish string
together long bouts and reciprocal exchanges of SESs that
might be analogous to the complex songs and duets of birds;
however, the communication value of these higher-order
patterns has not been examined.
Male–male aggressive interactions in Brachyhypopomus only
occur among breeding males; that is, only males that display
electric courtship toward females engage in agonistic competi-
tive encounters with other males. The larger resident male in
the courtship arena struggles aggressively toward the smaller
intruder male. Fish adopt an antiparallel position and tend to
bite each other’s tail. They emit primarily interruptions (prob-
ably signaling submission) and chirps (probably signaling
dominance), with one chirp type (M) emitted exclusively
during male–male interactions (Figure 13). On average, males
produce chirps every 2s, but these chirps are emitted in
‘bouts’ interspersed with periods without chirps. The contest
ends with the retreat of the intruder fish and the emergence
of a clear dominance–subordination status signaled by EOD
rate, in which the dominant fish discharges at a higher
frequency than the subordinate (Perrone et al., 2009).
In opposite-sex interactions, males emit chirp types A, B,
and C and accelerations while females produce interruptions
(Perrone et al., 2009). In this context, chirping likely conveys
information about male quality, while interruptions likely
signal acceptance by the female. During courtship, the male
swims near his potential nest site next to a plant and emits
pulses at rates of approximately 50–70 pulses/s. When a female
approaches, the pair may engage in duets in which their EOD
rates sometimes shift in parallel, and other times mirror each
other. The male prods the female with his snout or chases
her as his pulse rate increases dramatically in half-second bouts
of up to 200 pulses/s. Eventually the female moves toward
the nest plant, and the male begins making courtship chirps
toward her (these are distinct from the chirps given in aggres-
sive interactions, called decrement bursts by; (Hagedorn,
1988). A typical spawning chirp consists of approximately
25–200 low-amplitude pulses in rapid succession lasting
100–200 ms. Males give long trains of chirps during spawning.
The chirping male enters the plant and backs out. Then the
female enters, lays her eggs, and backs out. Finally, the male
reenters the plants, probably fertilizing the eggs. This cycle
may be repeated until the female leaves.
In Brachyhypopomus, aggression and courtship are linked;
both occur at greatest rates during the breeding season.
However, in G. omarorum, aggression also occurs outside the
breeding season when it serves mostly in territorial interactions
(Batista et al., 2012; Zubizarreta et al., 2015). Both male and
female Gymnotus are very aggressive, launching open mouth
attacks within seconds of the encounter. A clear sequence of
EOD modulations follows. In the contest phase, ‘offs’ or
complete interruptions of the EOD are emitted as submissive
signals. The fights themselves are very short (approximately
2–3 min). After the contest resolution, the submissive fish
increases the duration of the interruption and adds character-
istic chirps of 400ms in duration. Males and females display
indistinguishable EOD modulations.
Analogous modulations of the EOD have convergently
evolved in the pulse-type mormyrid fishes. Brienomyrus
 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
brachyistius produces three distinct types of EOD modulations
in different social contexts (Carlson and Hopkins, 2004).
Eleven different types of EOD modulations have been
described in this species and have been correlated with social
behaviors (Wong and Hopkins, 2007). Scallops are highly
stereotyped, short (0.5 s), abrupt increases in EOD rate that
are hypothesized to function as advertisement signals by domi-
nant fish. Accelerations are more variable, longer-duration
increases in EOD rate and are thought to be agonistic signals.
Rasps combine the abrupt increase in EOD rate of scallops
with the variable, longer-duration rate increase of an accelera-
tion. Rasps are produced primarily by males prior to spawning
and are likely courtship signals. Receptive females respond to
rasps by producing fast bursts, in which they increase their
EOD rate to 30–100 Hz for less than 1 s. Courting males and
females often ‘duet’ with alternating rasps and fast bursts
(Wong and Hopkins, 2007).
During aggressive encounters, the mormyrid P. isidori
increases pulse repetition rate from a baseline of tens of pulses
per second up to 125 pulses/s for hundreds of milliseconds
(Kramer, 1978). Each barrage is followed by a brief silent
period. The losing fish either discharges at a low and regular
rate of about 10 pulses/s or shuts off its EOD completely.
Both sexes behave similarly.
During courtship and spawning, P. isidori communicates
with both electric and acoustic signals (Crawford et al., 1986;
Bratton and Kramer, 1989; Crawford, 1991). Early in the
spawning cycle, a male establishes a territory and chases away
other fish, even females. Eventually he builds one or two nests
out of plant material and begins vocalizing. The resting EOD
pattern during this phase is irregular. On the night of spawning,
the female emits EODs very regularly at about 10 pulses/s,
starting on her own territory and continuing throughout the
night. During her visits, the male also changes his EOD pattern
to a similar regular low pulse rate and vocalizes profusely
(grunt-moan-growl calls); he is electrically silent when he
vocalizes. As courtship intensifies, both fish engage in a series
of stereotyped acrobatic swimming movements. In this phase,
they maintain the regular rhythm of EOD pulses except for
a brief moment when they are vent to vent and the male shuts
off his EOD. The male ceases vocalizing for the rest of the night,
and the pair undergoes a series of spawnings. Following each
spawning, the male picks up the eggs in his mouth and trans-
fers them to a nest. After a night of spawning, the male chases
the female away. In playback experiments, Crawford (1991)
showed that the regular EOD pulse rate by females during
the night of spawning elicits courtship vocalizations from the
males, whereas the normal irregular rhythm given by the
male does not.
2.05.4.1.2 Wave Fish
In wave-type fish, most EOD modulations fall into three two
categories based on the duration and degree of frequency excur-
sion (Zakon et al., 2002). Chirps are large frequency increases
(20–300 Hz) of short duration (10–200 ms) and rises are rela-
tively small increases (10–30 Hz) that last longer (1–10 s)
(Figures 14 and 15(a)). Chirps are further categorized by their
duration and magnitude of frequency and amplitude change.
In several species (e.g., A. leptorhynchus) chirping is sexually
dimorphic, with chirps emitted at greatest rates by males during
83
agonistic interactions, but also produced at lower rates by
females (Dunlap, 2002) and by both sexes during courtship
(Hagedorn and Heiligenberg, 1985). In many other apterono-
tids, there is no sex difference in chirp rate (Smith, 2013)
(Figures 14 and 16).
2.05.4.1.2.1 Agonistic Interactions
The best-studied example of agonistic chirping is in the brown
ghost (A. leptorhynchus). Most chirps during aggression have
relatively low-frequency excursion. These small chirps, which
have also been called ‘low-frequency’ or ‘type-II’ chirps, are
produced much more commonly by males than by females
(Zupanc and Maler, 1993, 1997; Dulka and Maler, 1994; Dun-
lap et al., 1998; Figures 14 and 16). This sex difference
depends, in part, on the circumstances under which the fish
are observed. Chirping is often studied by placing a fish in
a perforated plastic tube to constrain its movements – a chirp
chamber – and presenting an electrical sine wave stimulus
across the tube. This produces a well-controlled stimulus but
one with an unnatural geometry. Under these conditions,
males chirp robustly, whereas females seldom chirp when stim-
ulus presentations last a minute or less. Females chirp moder-
ately after a longer exposure to a playback stimulus
(Triefenbach and Zakon, 2008), and even more intensely in
real social encounters (Tallarovic and Zakon, 2002).
Chirp rate depends heavily on the specific parameters of the
stimulus. For example, when males are studied in a chirp
chamber with an EOD mimic near their own EOD frequency,
chirp rate is directly proportional to stimulus intensity
(Figure 16; Dunlap et al., 1998) and varies by stimulus
frequency. Stimulus frequencies near the fish’s own EOD
frequency, which would mimic a same-sex encounter, elicit
far more chirps than those mimicking an opposite-sex
encounter (Engler and Zupanc, 2001; Hupe et al., 2008; Smith,
2013). Similarly, in staged encounters between two fish sepa-
rated by a mesh barrier, males chirp at other males at twice
the rate as they do to females (Dunlap, 2002). In addition,
males respond at different rates toward stimulus frequencies
that might mimic males of different social status (Triefenbach
and Zakon, 2003). When freely swimming fish are presented
with EOD mimic stimuli, males with low EOD frequencies
(which tend to be the smallest and least mature) chirp more
at stimulus frequencies near or below their own EOD frequen-
cies but less at stimulus frequencies higher than their own
(which is the range of the larger dominant males). Conversely,
males with high EOD frequencies (larger dominant fish) chirp
at EOD frequencies across the male range.
Within conspecific male interactions, chirping by one fish
affects chirp rate of another fish. This influence also depends
on the stimulus context. While confined in a chirp chamber,
fish chirp less toward stimuli containing chirps than identical
stimuli without chirps (Dunlap and Larkins-Ford, 2003).
However, in dyadic interactions, the overall chirp rate by one
fish correlates positively with the other fish (Dunlap, 2002; Sal-
gado and Zupanc, 2011), and chirps commonly occur in an
“echo” pattern, with one fish producing a chirp with a latency
of 600ms after the other fish’s chirp (Salgado and Zupanc,
2011). This reciprocity can also change even within a particular
encounter, with positive correlation in some phases and nega-
tive correlation in other phases. During the actual attack phase,
 84
(a) (c) 1040

Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology

High-frequency chirp Gradual frequency rise
965
1300

EOD frequency (Hz)

1020
EOD frequency (Hz)

1200
1000 960

1100 980
955
1000 960

900 940
EOD EOD
(head–tail (head–tail
voltage) voltage)

0 20 40 60 80 100 0 200 400 600 800 1000

(b) Time (ms) (d) 500 Time (ms)

Low-frequency chirp 450

1300
EOD frequency (Hz)

400 High-frequency

Frequency modulation (Hz)

chirps
1200
350

1100 300
Male
250 Female
1000
200
900
150 Low-frequency
chirps
EOD
(head–tail 100
Gradual frequency
voltage) 50 rises

0 20 40 60 80 100 0
0.005 0.05 0.5 5 50
Time (ms)
Duration (s)

Figure 14 Types of electric organ discharge (EOD) modulations in brown ghost knifefish (Apteronotus leptorhynchus). (a–c) Blue traces show head–tail voltage and red traces illustrate EOD frequency during
chirps (a, b) and gradual frequency rises (GFRs, c). (a) During high-frequency chirps EOD frequency increases more than 200 Hz above its baseline, and there is a pronounced frequency undershoot at the
end of the chirp (red trace). EOD amplitude is also significantly reduced during high-frequency chirps (blue trace). (b) A low-frequency chirp plotted on the same frequency and timescale as in (a). Low-
frequency chirps are characterized by substantially less frequency modulation, the reduction or absence of a frequency undershoot at the end of the chirp, and the reduction or absence of amplitude modula-
tion. (c) A GFR. Note the different frequency and timescales compared to (a) and (b). GFRs typically have much less frequency modulation and last longer than chirps. Inset shows the GFR on an expanded
frequency scale. (d) Scatter plot illustrating frequency modulation and duration of >10 000 chirps and GFRs produced by male () and female (þ) Apteronotus leptorhynchus. Note that the duration scale is
logarithmic. High-frequency chirps, low-frequency chirps, and GFRs cluster in largely discrete parameter ranges. Both sexes produce low-frequency chirps and GFRs, but males produce most of the high-
frequency chirps.
 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology 85

(a) Chirps chirp rates decrease and rises are produced in greater rates
(Hupe and Lewis, 2008).
While chirp rates are very high during short-term (<20 min)
social interactions and stimulus presentation, the chirping
10 s
response to male stimuli habituates at longer timescales.
(b)
When presented with repeated artificial stimuli, chirp produc-
140 tion is almost extinguished after 90 min (Harvey-Girard et al.,
120 2010). This habituation occurs only when the stimulus
frequency is constant, and it can be reversed by presentation
Chirp rate (chirps min–1)

100 of a novel stimulus frequency. This may indicate a form of

80
60
40
20
0 et al., 2002).
0 5 10
Plasma 11KT (ng ml–1)
Figure 15 Agonistic chirping in brown ghost wave fish. (a) Males
maintain a steady electric organ discharge (EOD) frequency, but when
presented with an electrical sine wave mimicking an EOD, they make
a series of brief (20 ms) upward frequency modulations called chirps
(from Dulka and Maler, 1994). (b) Chirp rate in males is positively
correlated with their levels of 11-ketotestosterone From Dunlap, K.D.,
2002. Hormonal and body size correlates of electrocommunication
behavior during dyadic interactions in a weakly electric fish, Apteronotus
leptorhynchus. Horm. Behav. 41, 187–194.
long-term learning. When two males are housed together but
R2 = 0.7508 separated by a mesh barrier, chirp rate is high, 10 times back-
ground level, for the first 36h, but then decreases to about 3
times background level over the next 5 days (Dunlap et al.,
2011a). However, this long-term social interaction potentiates
the chirp response to artificial stimuli, indicating that fish
become sensitized to novel chirp-eliciting stimuli (Dunlap
15 20 The exact function of EOD modulations during male–male
interaction is not entirely clear (Smith, 2013; Zakon et al.,
2002). Because they are emitted at highest rates by the largest
and most dominant males, they have commonly been consid-
ered aggressive signals. However, in laboratory encounters,
chirp rates are actually lower during the attack phase than in
the periods just preceding and following the attack (Hupe
and Lewis, 2008). Moreover, chirp rates of dominant fish do
not change during aggression, but they increase in subordinate
fish. Similarly, recent field data show that when an intruding
male approaches a spawning pair, he is chased by the breeding

Apteronotus albifrons Apteronotus leptorhynchus

100 100

75 75
% Chirping
% Chirping

50 50

25 25

0 0
−60 −50 −40 −30 −20 −10 −60 −50 −40 −30 −20 −10

30
100
Chirps min–1

75
Chirps min–1

20

50
10 Males
25 Females

0 0
−60 −50 −40 −30 −20 −10 −60 −50 −40 −30 −20 −10
Stimulus strength (dB) Stimulus strength (dB)

Figure 16 Species differences in sex differences in chirping. Brown ghosts (Apteronotus leptorhynchus, right-hand side) show a large sex difference
in the percent of individuals that chirped in a minute long test period, as well as the number of chirps made. Black ghosts (Apteronotus albifrons, left-
hand side), on the other hand, show no sex difference in these parameters. From Dunlap, K.D., Thomas, P., Zakon, H.H., 1998. Diversity of sexual
dimorphism in electrocommunication signals and its androgen regulation in a genus of electric fish, Apteronotus. J. Comp. Physiol. A 183, 77–86.
86 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
male and emits chirps at high rates as he flees (Henninger,
2015). This suggests chirps serve as submissive signals or as
a deterrents to further aggression.
Rises are commonly considered submissive signals. In social
encounters, submissive fish produce long slow rises (Hopkins,
1974b; Hagedorn and Heiligenberg, 1985). When presented
with EOD mimic stimuli, females with the highest EOD
frequencies (the smallest, least mature females) make more
rises than females with the lowest EOD frequencies (which
are larger and more mature). Conversely, male brown ghost
with the lowest EOD frequencies (smallest, least mature males)
make more rises than those with higher EOD frequencies
(larger and more mature males) (Triefenbach and Zakon,
2008). However, in aggressive interactions, rises are emitted
at greatest rates during an attack by both the attacking fish
and the fish receiving the attack. This indicates that rises may
also serve as an aggressive signal (Cuddy et al., 2012; Hupe
et al., 2008).
2.05.4.1.2.2 Courtship Interactions
The courtship of wave fish (Eigenmannia, A. leptorhynchus) in
a laboratory setting was described thoroughly by Hagedorn
and Heiligenberg (1985). Males and females modulate their
EOD in different ways and at different phases. In the early
phase of Apteronotus courtship, the male chirps using small
chirps characteristic of aggressive encounters, and the female
responds with ‘long rises,’ which are typically submissive
signals. Courtship then intensifies over a few nights. On the
night of spawning, the male chirps incessantly (60–80
chirps/s), but then, as the chirping switches to big chirps
(also known as type-I or high-frequency chirps), chirp rate
diminishes. In the presence of a male, the female continues
making long rises, which now last tens of seconds. The male
often responds to the long rises of the female with renewed
bouts of courtship chirping. Within these episodes the female
repeatedly deposits her eggs. Females make a series of brief (5–
10 ms) chirps during ovulation. This might signal to the male,
who may be patrolling the territory, that ovulation has
occurred (Hagedorn and Heiligenberg, 1985). Such periovula-
tory chirping was also found in a naturally occurring spawn-
ing, but the chirps have a different character. In a field
population in Panama, Henninger (2015) observed that the
female produced long-duration and high-frequency chirps
just when she releases an egg. At that point, the male actually
stops chirping for approximately 10–60 s and then resumes
his high rate of chirping. Such EOD modulations produced
by the male may contribute to the induction of ovulation. In
Eigenmannia, playing signals of a courting male to gravid
females induces them to spawn. The question of how EOD
signals influence the neuroendocrine axis or, conversely, how
various hormones initiate the various components of court-
ship electrical displays, is ripe for future investigation.
The proportion of chirp types differs considerably between
opposite- and same-sex encounters. Males produce far more big
chirps (type I, III, and IV) when interacting with females or
when presented with stimuli in the female frequency range
than when presented with another male or its EOD. Males
occasionally also emit big chirps in aggressive contexts to stim-
ulus frequencies across the species range, but they tend to make
them more often to stimuli in the female EOD frequency range
(Triefenbach and Zakon, 2008). This suggests that these chirps
are courtship signals. However, subordinate males tend to
produce big chirps toward dominant males, indicating that
they may also serve as submissive signals (Cuddy et al., 2012;
Hupe et al., 2008).
2.05.4.1.2.3 Interspecific Interactions
In A. leptorhynchus, conspecific fish elicit chirp production at
higher rates than heterospecific fish (Sternopygus and Brachyhy-
popomus) that live sympatrically with Apteronotus. In addition,
artificial stimuli at frequencies within the species-specific range
cause more chirping than stimuli in the frequency ranges of
these heterospecifics (Dunlap et al., 2010). The fact that A. lep-
torhynchus chirps even at low rates to heterospecific stimuli
suggests that chirps may serve in interspecific interactions or
communicate to conspecifics about the presence of heterospe-
cifics. When EODs of closely related fish that have different
waveforms are presented all at the same frequency, male A. lep-
torhynchus show no differential response, indicating that EOD
waveform is not a salient feature of chirp-eliciting stimuli
(Fugere and Krahe, 2010).
2.05.4.2 EOD Modulations: Neural Control by Prepacemaker
Nuclei
The neural mechanisms underlying the generation of EOD
modulations are best understood in gymnotiform fish.
Inputs from two premotor nuclei called the prepacemaker
nucleus (PPn) and the sublemniscal prepacemaker nucleus
(SPPn) cause transient changes in the output of the
PN (Figures 1 and 17). Chirps are generated when neurons
from the PPn that synapse on relay cells in the PN release
glutamate, which binds to AMPA (kainate/quisqualate)
receptors to accelerate firing rates (Figure 17). Interruptions
occur when the glutamatergic input is so strong or long-
lasting that it depolarizes the relay cells enough to inactivate
their sodium channels and cause them to cease firing (Spiro,
1997). When this happens, no command signals are sent to
the spinal cord motor neurons, and the EOD transiently
ceases as well (Kawasaki and Heiligenberg, 1989). Different
chirp types (i.e., small vs big chirps) may result either from
activating more inputs from the PPn or by recruiting inputs
from the SPPn. Rises are likely regulated through inputs
from an additional midbrain nucleus, the PPn-G. These
inputs, which cause more gradual changes in EOD frequency,
use glutamate to activate NMDA receptors, which have much
slower activation kinetics than the AMPA receptors used in
chirp production.
While production of EOD modulations is regulated by
changes in prepacemaker nuclei, long-term habituation
appears regulated by the forebrain (Harvey-Girard et al.,
2010). Repeated chirp-eliciting stimuli cause a reduction in
chirp rate and decreases immediate early gene expression
(Egr-1) in a specific telencephalic region (DDmg). This expres-
sion then increases when novel stimuli reverse this habituation.
These observations suggest that Egr-1 expression in forebrain
contributes to learning electrocommunication signals in
a manner similar to that observed in bird song learning.
Steroids and neuromodulators modify neurotransmission
between the PPn and the PN (see Section 2.05.4.3), and such
 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
PPnC PPnG SPPn
NMDA
or Pacemaker
AMPA cells
Relay cells
AMPA NMDA
(a) To neurogenic electric organ
Relay cells
V
(b
Figure 17 (a) Social signals such as chirps and rises result from glu-
tamatergic modulation of the pacemaker neurons. This figure illustrates
the cell types (pacemaker and relay) on which the inputs from the SPPn
and the two divisions of the PPn synapse. NMDA and AMPA refer to
two classes of glutamate receptors. (b) Fish (in this case a pulse fish of
the genus Hypopomus) can be induced to make social signals in an
electrophysiological experiment. Relay cells (above) fire a barrage of
action potentials as glutamatergic input from the prepacemaker nucleus
depolarizes them. The electric organ discharge (EOD) (below) shows
a 1:1 relationship with each pacemaker action potential. During the
barrage of action potentials, the EOD show a burst of pulses of
decreasing amplitude (due to the inactivation of the Naþ channels in
the electrocytes). From Kawasaki, M., Heiligenberg, W., 1989. Distinct
mechanisms of modulation in a neuronal oscillator generate different
socialsignals in the electric fish Hypopomus. J. Comp. Physiol. A 165,
731–741.
changes underlie sex and seasonal differences in chirping.
Several studies also indicate that socially induced changes in
neurogenesis in the PPn contribute to long-term changes in
chirping behavior (Dunlap et al., 2006, 2013). Throughout
adulthood, the PPn receives cells that are born in the adjacent
periventricular region. Compared to other brain regions, the
PPn adds new cells at a particularly high rate (Zupanc and
Horschke, 1995), and about two-thirds of these cells differen-
tiate into neurons (Dunlap et al., 2008). Long-term social inter-
action increases the addition of new cells and the density of
radial glial fibers that guide the migration of new cells to the
PPn while also enhancing chirping behavior. Social manipula-
tions that increase chirping behavior, such as presentation with
EOD stimuli (Dunlap et al., 2008), novel social partners (Dun-
lap and Chung, 2012), or complex social groups (Dunlap et al.,
2011b), also increase cell proliferation in the periventricular
region that contributes cells to the PPn. These studies suggest
that social enhancement of neurogenesis contributes to
changes in chirping behavior; however, experimental
87
techniques are not yet available to verify whether this relation-
ship is causal (Dunlap et al., 2013).
2.05.4.3 EOD Modulations: Species Diversity and Their
Hormonal Control in Apteronotidae
2.05.4.3.1 Species Diversity
Fish in the largest family of gymnotiforms, Apteronotidae,
display a remarkable diversity of EOD modulations (Turner
et al., 2007; Smith, 2013). Species variation is much higher
for chirps than for rises. Rises are not sexually dimorphic,
appear less important in social interaction, and thus may
have evolved relatively slowly. By contrast, chirps are central
to courtship and aggression, which likely puts them under
strong diversifying selection. Interspecifically, chirps vary in
almost every possible way, including chirp rate, duration, the
magnitude of frequency and amplitude modulation, and the
degree of sexual dimorphism. Discriminant analysis indicates
that interspecific variation in chirp parameters could be used
by fish for species identification. This may be particularly
important in Apteronotidae, in which species’ EOD frequency
ranges overlap. Despite this broad diversification in chirps,
some chirp parameters appear to have evolved in a concerted
way. For example, species with the largest frequency modula-
tion during chirps also show the largest amplitude modulation,
suggesting biophysical constraints within the chirp-generating
neurons (Turner et al., 2007).
Most species show sex differences in chirping consistent
with their role in sexual and aggressive behavior. However,
species vary considerably in which specific chirp parameter
differs by sex. For example, in A. leptorhynchus, chirp rate is
highly sexually dimorphic (Figure 16) and dimorphic chirp
types vary primarily in frequency excursion. In the congeneric
A. albifrons, chirp rate is monomorphic and dimorphic chirp
types vary primarily in duration (Dunlap et al., 1998;
Kolodziejski et al., 2005). In yet another closely related species
(A. devenanzii and A. bonapartii), sexes differ only in chirp
complexity (single vs multipeaked chirps) and no other chirp
parameter (Ho et al., 2010; Zhou and Smith, 2006).
2.05.4.3.2 Neuroendocrine Regulation of Chirping
in Apteronotus
2.05.4.3.2.1 Steroids
In general, sex differences in chirping are regulated by activa-
tional effects of androgens. In dyadic male interactions, chirp
rate correlates positively with plasma 11kT levels
(Figure 15(b)). Gonadectomy in adult male A. leptorhynchus
eliminates sexual dimorphism in chirp rate (Dunlap et al.,
1998), and androgen receptor blockade with flutamide in
P. hasemani eliminates sexual dimorphism in chirp duration
(Petzold and Smith, 2016). Androgen treatment increases
the expression of sexually dimorphic chirping. Interestingly,
species differences in androgen sensitivity parallel the degree
of sexual dimorphism across species. For example, 11kT
treatment increases chirp rate in A. leptorhynchus, which is
dimorphic in chirp rate, but not in A. albifrons, which is mono-
morphic in chirp rate. This parallel between degree of sexual
dimorphism and androgen sensitivity is borne out at the pop-
ulation level as well. Apteronotus albifrons from the Orinoco
and Amazon, which differ in sexual dimorphism of EOD
88 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
frequency and its androgen sensitivity, do not differ in sexual
dimorphism of chirp rate, and their chirp rates are similarly
insensitive to androgen (Ho et al., 2013).
Many studies show clearly that androgens play a large role in
generating sexually dimorphic chirping, but several studies indi-
cate that other factors are involved as well. Females treated with
androgen (T or DHT) chirp at higher rates (10–20 chirps/min)
than females treated with empty capsules (2–4 chirps/min) but
less than normal males under the same conditions
(>40–60 chirps/min) (Dulka et al., 1995; Dunlap et al.,
1998). This partial effect of androgen in females may be due
to organizational differences in the brains of the two sexes
that make them differentially responsive to androgen. Alterna-
tively, some testicular compound other than androgen might
be necessary for full expression of chirping behavior or
a female-specific compound (e.g., estrogen) might inhibit chirp-
ing. Other studies indicate that within-sex variation in chirp rate
depends crucially on the sensory stimuli. In dyadic male interac-
tions, chirp rates correlate positively with plasma levels of 11kT
(Dunlap, 2002; Figure 15(b)), but when fish are presented with
synthetic EODs in a chirp chamber, 11kT levels (as inferred
from excretion rates) are unrelated to chirp production (Cuddy
et al., 2012).
In addition to steroidal effects on chirping responses to
acute presentation of social stimuli, steroids also modify chirp-
ing responses to long-term social interaction. The effect of
social housing on chirping is likely mediated by glucocorti-
coids (Dunlap et al., 2011a, 2013). Compared to isolated
males, males housed in pairs (but separated by mesh that pre-
vented physical contact) show an increase in plasma cortisol
associated with an elevation of chirp rate in their dyadic inter-
actions and potentiation of chirp rate to synthetic stimuli.
Plasma 11kT levels are not affected. Cortisol treatment to iso-
lated fish causes a similar potentiation of chirping, and gluco-
corticoid receptor blockade with RU486 suppresses chirp
production in dyadic interactions (Dunlap et al., 2011a).
Several lines of evidence show that this effect of long-term
social interaction on chirp production is mediated, in part, by
a cortisol-dependent effect on neurogenesis. Social interaction
simultaneously increases plasma cortisol and cell proliferation
in the periventricular zone that contributes new cells to the
PPn. Many of these cells then differentiate into neurons in
the PPn within 7 days, coinciding with the onset of changes
in chirping behavior. Cortisol treatment causes similar
increases in cell addition (Dunlap et al., 2006). However treat-
ment with a glucocorticoid receptor antagonist only partially
blocked this socially induced increase in cell addition, indi-
cating that cortisol may also act via mineralocorticoid receptors
or that some other pathway contributes to social effects on neu-
rogenesis (Dunlap et al., 2011a).
2.05.4.3.2.2 Neuromodulators: Peptides and Amines
Steroid hormones modulate EOD properties presumably at the
transcriptional level (see Section 2.05.2.4.2). To date, no rapid
nongenomic actions of steroids have been reported. Instead,
short-term modulation of the EOD is mediated by peptides
and amines. The PPn receives input from neurons containing
many neuromodulators, including dopamine, norepinephrine,
serotonin, AVT, somatostatin, galanin, substance P, and meten-
kephalin. Most research has focused on three of these
compounds, serotonin, substance P, and AVT, as modulators
of chirping behavior and its sexual dimorphism. In brown
ghosts (A. leptorhynchus) substance P-like immunoreactivity in
the PPn is stronger in males than in females, and the levels of
immunoreactivity in females can be brought almost to the
level of a sexually mature male by T implants (Weld and
Maler, 1992; Dulka and Maler, 1994; Dulka et al., 1995). The
effects of androgens on the substance P-like system is likely
entirely due to its upregulation of the neuropeptide since
there are no sex differences in the number of tachykinin
receptors in the PPn (Weld et al., 1994). Interestingly, the
expression of substance P in the PPn is also highly sexually
dimorphic in A. albifrons, a species in which chirp structure,
but not chirp rate, is sexually dimorphic (Kolodziejski et al.,
2005). This finding suggests that substance P might regulate
sex differences in the structure of chirps across species.
In contrast to the stimulatory effects of substance P, sero-
tonin inhibits chirping. Serotonin injections in the brain
suppress chirping (Maler and Ellis, 1987), and, consistent
with their lower chirp rates, females have more serotonin-
containing fibers in the PPn than males (Telgkamp et al.,
2007). Serotonin may also contribute to individual variation
in chirping behavior associated with social rank. However,
studies using pharmacological manipulation of serotonin
receptor subtypes show that seratonergic regulation of chirping
is complex and may involve both direct and indirect pathways.
While 5HT2 receptor agonists decrease chirp rate, 5HT1A
agonists increase chirp production, specifically of high-
frequency chirps (Smith and Combs, 2008).
Another neuromodulator, AVT, which modulates sexual
and agonistic behavior in a variety of other vertebrates, affects
chirping in males by altering the proportion of chirp types
(Bastian et al., 2001). It elevates the production of high-
frequency chirps but decreases production of low-frequency
chirps. This suggests that AVT may serve to shift male behavior
away from aggression to courtship. However, because AVT is
not present in the PPn, it likely acts via indirect inputs to
the PPn.
2.05.4.4 EOD Modulations: Seasonality and Neuroendocrine
Regulation in Brachyhypopomus
In Brachyhypopomus, the rate of EOD modulations (SESs)
increases dramatically (10-fold) during the breeding season,
with all three SESs increasing during male–female pairings
and just chirps showing a seasonal increase in male–male pair-
ings (Perrone et al., 2009). Sexual and seasonal variations in
the production of SESs are likely driven, at least in part, by regu-
lation of glutamatergic transmission and androgen binding in
the PN (Quintana et al., 2010). Glutamate injection into the
ventral PN causes interruptions in females and in nonbreeding
males but induces chirping in breeding males by acting on
AMPA receptors. These observations suggest that the default
state of the ventral PN is to generate interruptions, but that it
can be modified in breeding males. During the breeding
season, plasma 11kT levels rise, and androgen receptor expres-
sion increases in many parts of the brain, including in pace-
maker and relay cells of the PN (Pouso et al., 2010). The
seasonal upregulation in androgen receptor is itself likely regu-
lated by androgens, since androgen receptor expression in
 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
testosterone-treated, nonbreeding fish rise to levels found in
breeding fish. Together, these results suggest that androgens
cause seasonal activation of chirping in males by increasing
the expression of AMPA receptors in the PN.
Another possible mechanism of behavioral change in
B. gauderio is the socially induced and seasonal addition of
new cells to brain regions that contribute to electrocommunica-
tion (Dunlap et al., 2011b). Fish housed in mixed-sex groups
under seminatural captive conditions that promote gonadal
maturation and reproductive behavior show elevated rates of
cell proliferation in the PN, in the periventricular zone adjacent
to the PPn, and in electrosensory regions (electrosensory lateral
line lobe, ELL). This effect of social interaction on brain cell
proliferation occurs only in the breeding season, when fish
emit abundant SESs. This suggests that perception of social
stimuli promotes neurogenesis in the electrocommunication
circuitry and may contribute to seasonal changes in its output.
However, it is still unknown whether or how new neurons
modify the activity of the electrocommunication circuitry.
2.05.4.5 EOD Modulations: Steroidal Regulation of Nonsex-
Biased Territorial Aggression in Gymnotus
In most electric fish species, males are larger and exhibit more
aggression than females, particularly in the breeding season.
However, in Gymnotus, aggression is exhibited equally by males
and females both in and outside of the breeding seasons
(Batista et al., 2012; Zubizarreta et al., 2015). In such nonsex-
biased aggression, interruptions are emitted early in the contest
as a form of ‘electrical hiding.’ After the resolution of the fight,
individuals losing the contest produce chirps to signal
subordination.
Across many vertebrates, elevated male aggression during
the breeding season is regulated by testicular androgens.
However, Gymnotus displays aggression outside the breeding
season, and several experiments indicate that extragonadal
steroids regulate this aggression (Jalabert et al., 2015). Circu-
lating 11-kT levels in dominant (aggressive) and isolated
(nonaggressive) males are similar, and castration fails to affect
aggression in dominant fish. However, treating dominant fish
with an aromatase inhibitor decreased aggression, indicating
that aggression in the nonbreeding season depends on a nongo-
nadal, estrogenic pathway.
2.05.5 EOD Reception through the Electrosensory
System
2.05.5.1 Electroreceptor Types, Function, and Tuning
Weakly electric fish have two classes of electroreceptors: ampul-
lary receptors and tuberous receptors (Table 1). These receptors
are distributed over the fish’s body, although they are often
most dense in the skin of the head and in some species along
the dorsal margin of the fish (Szabó, 1974). In the South
American ghost knifefishes (Apteronotidae), a specialized
structure that can be extended dorsally above the tail (dorsal
filament) also contains a high concentration of tuberous recep-
tors (Franchina and Hopkins, 1996). Electroreceptors are
evolutionarily derived from the neuromasts of the lateral line
system and are innervated by the lateral line nerves.
89
Ampullary-type receptors are a relatively ancestral trait in
vertebrates and are found in many aquatic animals that lack
electric organs (e.g., elasmobranchs, lampreys, catfishes, some
amphibians, platypuses) as well as in both clades of weakly
electric fishes (Bodznick and Northcutt, 1981). Ampullary
receptors are similar in gymnotiform and mormyroid fish.
They are broadly tuned and behave as low-pass filters
(30–40 Hz to virtually DC). This low-frequency tuning
allows ampullary receptors to integrate inputs over time,
which makes them exquisitely sensitive to very weak electrical
fields (approximately 10–20 mV cm
1 in freshwater fish,
and <1 mV cm
1 in marine fish), but largely unable to detect
the high-frequency components present in most EODs
(Bodznick and Montgomery, 2005). Ampullary receptors func-
tion primarily in passive electrolocation – i.e., in the ability of
fish to passively detect the bioelectric fields generated even by
animals that do not have electric organs. However, in weakly
electric fishes, ampullary receptors may also contribute to the
detection of low-frequency components of electrocommunica-
tion signals (see below).
Tuberous receptors are much more variable across species,
and their filtering properties are often plastic. Unlike ampullary
receptors, tuberous receptors act as high-pass filters, and they
are typically two to four orders of magnitude less sensitive
than ampullary receptors. Consequently, tuberous receptors
cannot passively detect bioelectric fields, but are well adapted
to detect the stronger, high-frequency components of EODs
for active electrolocation and communication. Tuberous recep-
tors are usually tuned to the frequency components of each
fish’s EOD. The receptors of pulse fish are broadly tuned to
capture the wide frequency spectrum of an EOD pulse (Bass,
1986; Bastian, 1977; Hopkins and Heiligenberg, 1978;
Kawasaki, 1985; Watson and Bastian, 1979). The tuberous
receptors of wave fish are usually more sharply tuned to a fish’s
own EOD frequency (Hopkins, 1976). Since EOD frequency
differs between sexes, receptor tuning is sexually dimorphic
as well (see below; Zakon and Meyer, 1983).
There are two types of tuberous receptors in the pulse-type
mormyroids (Table 1). Mormyromasts are specialized to detect
a fish’s own EOD and are used in active electrolocation. Mor-
myromasts contain two types of electroreceptor cells. A-type
cells encode the local amplitude of the EOD by firing at
higher rates in response to stronger electric fields, but are
relatively insensitive to EOD phase or waveform; B-type cells
also encode EOD amplitude, but in addition are sensitive to
very small changes in EOD phase. Thus, they precisely
encode EOD timing and waveform (Kawasaki, 1985). There
are also differences across mormyrid species in the relative
sensitivities of A and B cells, which suggests mechanisms of
electroreceptive coding may vary across species (Bell, 1990).
A second type of electroreceptor organ, the knollenorgan, is
used exclusively for social communication and detects the
EOD pulses of other fish. Whenever a fish fires its own
electric organ, a corollary discharge from the electromotor
system strongly inhibits pathways from knollenorgans
through the nucleus of the ELL. This inhibition of responses
to the fish’s own EOD allows the knollenorgan inputs to
respond specifically to the EODs of other fish (Bell, 1986;
Bennett and Steinbach, 1969). The only mormyroid fish that
produces wave-type discharges, Gymnarchus niloticus, has two
90 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology

Table 1 Types, functions, and sex differences in electroreceptors

Sex differences and hormonal

Taxon and EOD type Receptor type Stimulus encoded Function regulation

All electroreceptive fish Ampullary receptors Weak low-frequency/DC Passive electrolocationa Female stingrays: more ampullary
electric fieldsa Chirp duration in species in afferentsc
which chirps have DC Male stingrays: ampullary
componentsb receptors tuned to lower
frequencies in breeding seasond
Androgens lower frequency tuning
of ampullary receptorsd
Mormyrid pulse Knollenorgans Timing and waveform Communication (detecting Male knollenorgans tuned to lower
(zero-crossings) of and discriminating other frequency (male-like) EOD
other fishes’ EODse EODs)e spectraf
Androgens indirectly masculinize
tuningf
Mormyromasts Active electrolocation No known sex differences
A cells Local EOD amplitudeg
B cells Local EOD amplitudeg
Phase/waveform distortion
caused by object
capacitanceg
Gymnotiform pulse Tuberous receptors:
Burst duration coders EOD amplitudeh Active electrolocationh No known sex differences
Communicationi
Pulse markers EOD phase/timingh Active electrolocationh No known sex differences
(capacitance detection)
Communicationg–i (EOD
timing and waveform)
Gymnotiform wave Tuberous receptors:
P (probability)-type EOD amplitudej Active electrolocationj Receptors tuned to fishes’ own
Communication (beat/ EOD frequency (i.e., tuning
chirp detection)k sexual dimorphism matches)
Jamming avoidancel EOD sexual dimorphism
Androgens directly masculinize
tuning
T (timing)-type EOD phase/timingj Active electrolocationj Receptors tuned to fishes’ own
Communication (beat EOD frequency
phase shifts during Androgens directly masculinize
chirps)m tuning
Jamming avoidancel
a
Kalmijn (1974).
b
Metzner and Viete (1996), Naruse and Kawasaki (1998), and Stockl et al. (2014).
c
Kempster et al. (2013).
d
Sisneros and Tricas (2000).
e
Hopkins and Bass (1981).
f
Bass (1986) and Bass and Hopkins (1984).
g
Bell (1990) and Kawasaki (1985).
h
Bastian (1976).
i
Shumway and Zelick (1988).
j
Scheich et al. (1973).
k
Benda et al. (2006) and Nelson et al. (1997).
l
Heiligenberg (1986).
m
Stockl et al. (2014). EOD, electric organ discharge.

tuberous receptor types, gymnarchomast types I and II that are
somewhat morphologically and perhaps functionally similar
to the mormyromasts and knollenorgans of mormyrids
(Szabó, 1974; Zakon, 1986). The physiology and function of
gymnarchomasts, however, has not been well studied and
will not be further considered here.
Unlike mormyrids, gymnotiforms do not have tuberous
receptors dedicated only to detecting communication signals.
Instead, the tuberous receptors of gymnotiforms must perform
the double duty of both detecting the fish’s own EOD for
electrolocation and EODs of other fishes for electrocommuni-
cation. Pulse-type gymnotiforms have two types of tuberous
receptor organs whose responses are analogous to those of
the A and B cells of the mormyrid mormyromasts. Pulse marker
electroreceptors fire at most a single spike to each EOD, but the
spike is phase-locked to the EOD and can thus serve to encode
EOD timing and waveform (Bastian, 1976). Burst duration
coding electroreceptors are more sensitive than pulse markers
and fire multiple spikes per EOD. Their firing rate encodes
EOD amplitude. In both pulse-type gymnotiforms and
 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
mormyrids, tuning of electroreceptors may vary across the pop-
ulation of electroreceptors on individual fish, but at least
a subset are narrowly tuned to the power spectrum of the fish’s
EOD (Bass and Hopkins, 1984; Bastian, 1976; Watson and
Bastian, 1979).
Wave-type gymnotiforms have two types of tuberous electro-
receptor organs used for electrolocation communication
(Table 1; Scheich et al., 1973). P-type electroreceptors, like the
burst duration coders in pulse gymnotiforms and the A cells
in mormyrid mormyromasts, encode EOD amplitude with
a rate code. P-type electroreceptor afferents fire once or not at
all to each EOD, and their firing probability depends on EOD
amplitude. T-type electroreceptors are one to two orders of
magnitude more sensitive than P-type electroreceptors and
typically fire once for every EOD cycle. T-type afferent firing to
each EOD is phase-locked, and the timing of T-type afferent
action potentials thus precisely encodes EOD timing/phase.
2.05.5.2 Electroreceptor Coding of EOD Waveforms
2.05.5.2.1 Pulse Fish
The waveform of pulse EODs varies substantially across species
and between sexes, and many pulse-type mormyrids and gym-
notiforms behaviorally discriminate among signals based on
pulse waveform (Hopkins, 1988; Hopkins and Bass, 1981;
Shumway and Zelick, 1988). Variation in EOD pulse waveform
could theoretically be encoded by the electrosensory system in
one of two ways: (1) electroreceptors could encode ‘landmarks’
in the EOD pulse waveform by precisely encoding the timing of
positive and negative zero-crossings of the EOD (i.e., when the
voltage of the EOD goes from negative to positive) and/or
(2) because the power spectrum of the EOD changes as its
waveform changes, electroreceptors could encode EOD wave-
form by using electroreceptor populations with different
frequency tuning to encode the spectral content of the EOD.
Pulse-type mormyrids use the first of these mechanisms
(Hopkins and Bass, 1981). In pulse-type mormyrids that
have sexually dimorphic EOD waveforms and that discriminate
EOD waveform, knollenorgan electroreceptors fire a single
action potential precisely on positive zero-crossings of the
EOD. This allows them to encode the EOD pulse waveform
of other fish. Knollenorgans on one side of the body fire during
positive to negative transitions of the EOD, and ones on the
other side fire during positive to negative transitions. This is
due to the geometry of current flow: current emanating from
a neighboring fish enters one side of the body and exits the
opposite side, depolarizing knollenorgans on the side it enters
and hyperpolarizing those on the side it exits. Neurons in the
anterior exterolateral nucleus (ELa) in the mormyrid midbrain
compare the timing of the EOD zero-crossings between knolle-
norgans on each side of the body. These neurons thus encode
EOD waveform and help identify the species and sex of the
sender’s EOD (Amagai, 1998; Xu-Friedman and Hopkins,
1999). Some mormyrid species do not have sexually dimorphic
EOD waveforms, and their knollenorgans do not encode EOD
waveform with a two-spike code (Baker et al., 2015). Instead,
the knollenorgans in these species spontaneously oscillate
and produce synchronized, time-locked spikes in response to
an EOD. This response optimizes the detection of other
EODs, but does not allow discrimination based on EOD
91
waveform. These species also lack the ELa circuitry to compare
knollenorgan spike timing across the body and encode EOD
waveform differences (Carlson et al., 2011).
Like the mormyrid knollenorgans, the pulse marker electro-
receptors of pulse-type gymnotiforms have the capacity to
generate a two-spike code to encode the timing of EOD zero-
crossings, and giant cells in the midbrain torus, like those in
the ELa of mormyrids, can compare timing of zero-crossings
across electroreceptors in different locations on the skin.
However, gymnotiforms may also compare the firing rates of
narrowband and broadband burst duration encoding electrore-
ceptors to encode the spectral content of the EOD and allow
fish to discriminate between male EODs that have more power
in lower frequencies and female EODs that have more power in
higher frequencies (Shumway and Zelick, 1988).
2.05.5.2.2 Wave Fish
Wave-type gymnotiforms detect and differentially respond to
EOD frequency, and thus may be able to use EOD frequency
to obtain information about the species, sex, and/or social
rank of other fish (Fugere and Krahe, 2010; Kolodziejski
et al., 2007; Triefenbach and Zakon, 2003). EOD waveform
also varies across and sometimes within species in wave-type
gymnotiforms, but the evidence that they use EOD waveform
as a communication signal is mixed. For example, Eigenmannia
can detect sex differences in EOD waveform, and both sexes
preferentially approach electrodes playing EODs with a female
waveform (Kramer and Otto, 1988). In contrast, despite the
fact that EOD waveform is more variable across species of ghost
knifefishes (Apteronotidae) than in any other clade of wave-
type gymnotiforms (Turner et al., 2007), a recent study did
not find EOD waveform discrimination in A. leptorhynchus.
Males did not approach or chirp more toward playbacks of
EODs with conspecific versus heterospecific waveforms, which
suggests that at least in these contexts, EOD waveform is not
a behaviorally relevant signal feature in this species (Fugere
and Krahe, 2010). It remains to be determined whether EOD
waveform is discriminated by A. leptorhynchus in other contexts
(e.g., courtship) or by other apteronotid species that have more
complex EOD waveforms.
Coding EOD waveform and/or frequency in wave-type fish
is more complicated than in pulse-type fish because wave-type
electric fish continuously produce a high-duty cycle EOD (i.e.,
the time between EODs is about equal to the duration of each
EOD). The tuberous receptors of wave-type fish thus simulta-
neously sense both the fish’s own EOD and that of neighboring
fish. This makes the EOD an unusual communication signal
because the receiver cannot perceive the signal directly. Instead,
wave-type electric fish detect the presence of other EODs based
on the interactions of the fish’s own EOD with those of nearby
conspecifics. Specifically, when two fish are near each other,
their combined EODs produce a signal that beats (oscillates
in amplitude and comes in and out of phase) at a frequency
equal to the difference between the two fishes’ EOD frequencies
(Heiligenberg, 1986). P-type tuberous electroreceptors (P-
units) in the fishes’ skin encode the amplitude modulation of
these beats, and T-type receptors in different parts of the
fishes’ skin encode the phase modulation. Because EOD
frequency is sex-specific in many species, the sex of a nearby
conspecific can be determined from the beat frequency. Fish
92 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
of the same sex have EODs of similar frequencies that produce
slow beats when combined, whereas fish of the opposite sex
have more distant EOD frequencies that produce higher beat
frequencies when their EODs interact.
Much less is known about whether and how EOD waveform
is encoded by electroreceptors in wave-type fish. Information
about EOD waveform is reflected in the structure of the beat
produced by interacting EODs; fish with more complex EOD
waveforms produce complex EOD beats when they interact
(Petzold and Smith, 2014). The complexity of the beat (and
thus EOD waveform) theoretically should be reflected in the
firing patterns of P-units, but because most studies of
electroreceptors in wave-type electric fish have used
sinusoidal stimuli, the relationship between EOD waveform/
beat complexity and electroreceptor coding has not been
empirically examined.
2.05.5.3 Sensory Coding of Chirps
2.05.5.3.1 Electroreceptors
The beat produced by the interaction of the EODs of two
nearby wave-type electric fish is a rhythmic amplitude modula-
tion whose frequency is determined by the difference between
the frequencies of the two EODs. When a fish chirps, its EOD
frequency increases abruptly, and in some cases the amplitude
of its EOD decreases. These transient frequency and amplitude
changes disrupt the regular beat of two interacting EODs, and
fish detect the chirps as the disruption of the beat.
Both P-type and T-type tuberous receptors and in some
species even ampullary receptors encode chirps. P-type
receptors encode the amplitude disruption in the EOD beat
by either increasing or decreasing the synchrony of their
firing (Benda et al., 2006; Stockl et al., 2014). T-type
electroreceptors encode the disruption of the phase
modulation in the beat by changing their interspike intervals.
In some electric fish species, the EOD is interrupted during
a chirp, which unmasks a head-negative DC component of
the EOD. This DC signal stimulates ampullary receptors and
allows them to encode chirp duration (Metzner and Viete,
1996; Naruse and Kawasaki, 1998; Stockl et al., 2014).
Chirp encoding by the electrosensory system has been most
extensively studied in brown ghost knifefish (A. leptorhynchus).
P-type electroreceptors in this species use distinct mechanisms
to encode different types of chirps. Small chirps, which are
often emitted during same-sex interactions, are most
detectable by P-units (i.e., produce the most dramatic change
in firing synchrony) when they occur on low-beat frequencies
created by the interaction of same-sex EODs. In contrast, big
chirps, which are produced during courtship, cause changes
in the synchrony of P-unit firing when they occur on
a background of the high beat frequencies produced when
male and female EODs interact (Benda et al., 2006). These
findings suggest a fascinating coevolution of the sensory
physiology of signal detection with signal structure and the
social context (i.e., courtship vs aggression) in which the
signals are used.
2.05.5.3.2 Central Nervous System
Each P-type tuberous electroreceptor afferent projects to
pyramidal cells in three somatotopically mapped subregions
of the ELL in the hindbrain (Carr and Maler, 1986). These
three ELL subregions process electrosensory input in parallel
for both electrolocation and communication and are
functionally specialized. Pyramidal cells in the centromedial
segment of the ELL have small spatial receptive fields,
respond best to low-frequency beats, and are necessary for
the jamming avoidance response. Pyramidal cells in the
lateral segment of the ELL have large receptive fields, respond
best to somewhat higher frequency beats, and are necessary
for fish to respond to conspecifics with chirping (Metzner,
1999; Metzner and Juranek, 1997).
In A. leptorhynchus, the distinct sensory coding mechanisms
for low-frequency (agonistic) chirps and high-frequency (court-
ship) chirps that are found in the periphery extends into the
ELL. The ELL has two types of pyramidal cells that receive input
from P-units. E-cells receive direct input from P-type afferents
and are excited by increases in EOD amplitude. I-cells receive
indirect input from P-type afferents via an inhibitory
interneuron and fire more in response to decreases in EOD
amplitude. E-cells respond to low-frequency chirps on low-
frequency beats (i.e., small chirps produced when same-sex
individuals interact) by producing stereotyped bursts that
facilitate the detection of these chirps, but do not allow
discrimination of subtle variation in the parameters of these
chirps (Marsat and Maler, 2012). In contrast, I-cells respond
to high-frequency chirps on high-frequency beats (i.e., big
chirps produced during opposite-sex encounters) with
a heterogeneous, graded firing pattern that enables
populations of them to encode the fine structure of the chirp
and thus enables fish to discriminate among different high-
frequency chirps. Thus, central processing mechanisms may
be adapted with signal structure in this species to allow
females to extract information about the quality of male
courtship chirps.
Less is known about how chirps are coded in the T-type
tuberous or ampullary pathways in the ELL or at higher levels
in the electrosensory system. ELL neurons project to the
dorsal torus semicircularis (TSd) in the midbrain. The TSd
contains populations of neurons that respond selectively to
chirps with a sparse code and that project to midbrain and
forebrain circuits mediating social behavior and diencephalic
circuits that mediate chirping (Metzner and Heiligenberg,
1991; Vonderschen and Chacron, 2011). However, how the
sparse code in the TSd arises and whether it allows merely
the detection of chirps or fine discrimination of chirp
properties are not yet clear.
2.05.5.4 Electroreception: Sex Differences and
Neuroendocrine Regulation
Much less is known about sex differences and hormonal regula-
tion of the neural mechanisms by which EOD and chirps are
perceived than is known about the mechanisms controlling sex
differences in the production of electrocommunication signals.
Most of what is known focuses on sex differences and hormonal
regulation of tuning in electroreceptors. Some sex differences in
ampullary receptors have been found in elasmobranchs. Female
stingrays have more ampullary afferents than males, which
might make it easier for them to distinguish between approach-
ing conspecifics versus predators (Kempster et al., 2013).
 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
The ampullary receptors of male stingrays are tuned to lower
frequencies in the breeding season than in the nonbreeding
season, and it is hypothesized that this tuning facilitates locating
mates buried in the substrate. The seasonal change in ampullary
tuning in males is mediated by androgens; treatment with DHT
lowers the best frequency of ampullary receptors (Sisneros and
Tricas, 2000).
In gymnotiform fish with wave-type discharges, tuning of
tuberous receptors is also sexually dimorphic and hormonally
regulated. Sex differences in EOD frequency are mediated
largely by activational effects of androgens and estrogens.
Because tuberous electroreceptors are precisely tuned in wave-
type fish, electroreceptors must shift their tuning when
hormones change EOD frequency to remain tuned to a fish’s
own EOD. Like EOD frequency, the tuning of tuberous electro-
receptors is sexually dimorphic in wave-type gymnotiforms,
and fish treated with androgens show parallel shifts of EOD
frequency and electroreceptor tuning (Meyer et al., 1987; Meyer
and Zakon, 1982; Zakon and Meyer, 1983).
A key question is whether the hormones act directly or indi-
rectly on electroreceptors to change their tuning. That is,
hormones might act directly on the PN to induce a change in
EOD frequency and this change in the frequency at which the
electroreceptors are driven might somehow reset electrorecep-
tor tuning to match. Alternatively, androgens might act inde-
pendently on electromotor circuits and on electroreceptors to
coordinate EOD frequency and receptor tuning. In wave-type
gymnotiforms, hormones appear to act independently on
motor circuits and electroreceptors. Androgens still shift elec-
troreceptor tuning in Sternopygus whose EOD has been abol-
ished by transecting the spinal cord or lesioning the PN
(Ferrari and Zakon, 1989; Keller et al., 1986). Furthermore,
androgen receptors have been localized immunohistochemi-
cally in electroreceptors, so they likely have the capacity to
respond directly to androgens (Gustavson et al., 1994). Thus,
the actions of androgens on the electroreceptors, the pacemaker
neurons, and the electric organ are coordinated to ensure
matched tuning, but this is accomplished via independent
hormone actions in each cell type.
In mormyrid knollenorgans, electroreceptor tuning is also
sexually dimorphic. Pulse-type EODs have a broad power spec-
trum, and electroreceptors are broadly tuned. However, male
EODs have more power in lower frequencies and their knolle-
norgans are more sensitive to lower frequencies than those of
females (Hopkins and Bass, 1981). This is interesting because
it implies that males are more sensitive to the EODs of other
males than they are to those of females. As in wave-type gym-
notiforms, the sex difference in receptor tuning in mormyrids is
regulated by androgens, which shift knollenorgan tuning
curves toward lower frequencies. Unlike in gymnotiform
tuberous receptors, however, the tuning shift in knollenorgans
depends on the presence of the fish’s EOD. Spinal transection
of Brienomyrus blocked the effects of androgens on knollenor-
gan tuning (Bass, 1986; Bass and Hopkins, 1984). Thus, in
mormyrids, sex differences in tuning likely result through indi-
rect actions of hormones.
Unfortunately, no more recent studies have examined the
hormonal regulation of sex differences in electroreceptor
tuning. In particular, the question of the biophysical mecha-
nisms by which receptor tuning is derived and shifted by
93
hormone treatment is a fertile one. Similarly, no studies have
examined the possibility of sex differences or effects of gonadal
hormones on the coding of EODs and chirps in the CNS.
However, recent studies have found that neuromodulators
may alter the excitability of pyramidal cells to influence sensory
processing of EODs and chirps in the ELL. In particular, sero-
tonin, acting on 5HT2-like receptors, enhances the excitability
of ELL pyramidal cells and selectively increases their responses
to stimuli created during same-sex encounters (low-frequency
EOD beats and small chirps), but not to signals produced
during opposite-sex encounters (Deemyad et al., 2013; Larson
et al., 2014). Combined with the ability of serotonin (via 5HT2
like receptors) to inhibit chirping (Maler and Ellis, 1987; Smith
and Combs, 2008), this finding led to the ‘shut up and listen’
hypothesis that serotonergic activation during aggressive
encounters may modulate signaling by reducing the produc-
tion of chirps and enhancing detection and responsiveness to
chirps (Deemyad et al., 2013).
2.05.6 Conclusions
What general lessons can be reached from studying hormones
and behavior in electric fish? First, although electrocommuni-
cation signals may be unique to only two orders of fish, they
are utilized in much the same ways as signals in other modal-
ities and are similarly influenced by natural and sexual selec-
tion. As with other sexually dimorphic communication
signals, the EOD conveys information on the species, age,
sex, and individual identity of the signaler and can also trans-
mit information about breeding status, dominance/subordi-
nance, and motivation. As with many other sexually
dimorphic communication signals, EODs are often regulated
by gonadal steroid hormones through actions on the
morphology and physiology of the neurons that control these
signals.
Other similarities between systems are apparent in the
development and interaction of natural and sexual selection
on the behavioral phenotypes. The EOD waveform of pulse
fish (and EOD frequency in many wave fish) is similar in juve-
nile and female gymnotiforms, reminiscent of the plumage
similarities of juvenile and female birds. For many sexually
dimorphic signals, particularly in species in which males
compete for reproductive access to females, male signals are
more conspicuous and/or ornate than those of females. These
signals may carry costs, including increased risk of predation
and/or energetic costs (Stoddard, 1999; Salazar and Stoddard,
2008). Similarly, in electric fish there is evidence that the elec-
trical signals produced by males are more conspicuous both to
conspecific females and to potential predators.
Another striking feature of the electrocommunication
system in electric fish is the wealth of species diversity in the
biology of these fishes. This diversity includes variation in the
magnitude and direction of sex differences in body size, EOD
properties, and other electrical behaviors (i.e., chirping); diver-
sity in habitat preferences, sociality, and associated diversity in
physiology and patterning of the EOD; and variation in court-
ship and parental behavior. This diversity is coupled with
numerous striking examples of convergence in the behavior
and physiology between the two different orders of fish that
94 Weakly Electric Fish: Behavior, Neurobiology, and Neuroendocrinology
independently evolved electrocommunication. These features
lend themselves well to comparative studies of the hormonal
control of reproductive and communication behavior. For
example, evolutionary gains, losses, and even reversals in the
direction of sex differences in EODs and chirps in apteronotid
fishes make these signals an excellent model system for
studying how evolutionary lability of neuroendocrine mecha-
nisms that regulate sex differences can lead to species diversity
in sexually dimorphic behavior.
A final highlight of the electrocommunication system is that
it has numerous features that allow us to study reproductive
communication behavior at multiple levels of analysis. These
features include a signal that can be quantitatively analyzed,
manipulated, and played back and a neural circuit that controls
the EOD behavior that is well characterized, simple, and acces-
sible enough to allow cellular analyses of the mechanisms
underlying species, sex, and individual differences in behavior.
The recent addition of genomic and bioinformatic tools in this
system (Gallant et al., 2014; Salisbury et al., 2015) will facili-
tate future studies investigating both how hormones regulate
gene expression to influence sex differences and how
hormone–gene networks evolve to yield species diversity in
sexually dimorphic behavior.
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 2.06 Neuroendocrine Control of Social Behavior in Frogs
Walter Wilczynski, Neuroscience Institute and Center for Behavioral Neuroscience, Georgia State University, Atlanta, GA, USA
Ó 2017 Elsevier Inc. All rights reserved.

2.06.1 Introduction 101

2.06.2 Seasonal Patterns of Hormones and Behavior 102
2.06.2.1 Seasonal Changes in Gonadal Steroids 102
2.06.2.2 Seasonal Changes in Social Behavior Match Changes in Gonadal Steroids 103
2.06.2.3 Seasonal Variation in Other Hormones: Adrenal Steroids 105
2.06.2.4 Seasonal Variation in Other Hormones: Melatonin 105
2.06.3 Steroid Hormone Receptors in the Brain 106
2.06.4 Hormone Effects on Hearing 108
2.06.4.1 Seasonal Changes in Hearing Track Seasonal Changes in Sex Steroids and Behavior 109
2.06.4.2 Experimental Manipulation of Circulating Gonadal Steroids Modifies the Auditory System 109
2.06.4.3 Implications for Social Behavior 110
2.06.5 Hearing Effects on Hormones 110
2.06.5.1 Anatomical Links between the Auditory Midbrain and Hypothalamic Areas Define an Audioendocrine
Circuit 110
2.06.5.2 Hearing Calls Elevates Levels of Circulating Steroid Hormones 111
2.06.5.3 Short-Term Exposure to Aggressive or Advertisement Calls Has Different Effects 111
2.06.5.4 Implications for Social Behavior 113
2.06.6 Summary and Conclusions 113
References 114

2.06.1 Introduction
Anuran amphibians (frogs and toads) occupy a greater diversity
of habitats and express a wider variety of reproductive social
behaviors than is generally appreciated outside of the field of
herpetology (Feder and Bruggren, 1992; Duellman and Trueb,
1994; Wells, 2007). Anuran species range from the arctic to
the tropical, and from dry desert regions to constantly wet habi-
tats; some species, such as the Pipidae, are even fully aquatic.
Anurans also vary in the degree to which they express different
kinds of social behavior. Most work on anuran social behavior
has focused on the male and female adult behaviors that lead
to mate choice and ultimately reproduction. These behavioral
patterns are more consistent across species than are other types
of social behavior. Patterns of aggression for example, to date
only described among males, vary in degree across species, and
parental care can range from none (the most common condi-
tion), to depositing eggs in foam nests, to guarding or feeding
eggs and tadpoles by either males, females, or both. The variety
of ecological and behavioral profiles, viewed within the context
of several largely conserved elements of social behavior that
define this taxonomic group, provides ample opportunities for
comparative and evolutionary studies in brain–hormone–
behavior relationships that have not been fully exploited.
The understanding of anuran social behavior, and the
brain–hormone interactions behind it, is based on relatively
few temperate and neotropical species that have long served
as models for neuroethology, acoustic communication, and
evolutionary animal behavior. With this caveat, there are
several features of anuran social behavior that define this group
of vertebrates.
Reproduction is seasonal. Like most vertebrates, frogs engage in
reproduction and its attendant social behavior during specific
times of the year and become reproductively quiescent during
others. Frogs need water, or at least high local humidity, for
successful egg deposition and larval development, and reproduc-
tion is restricted to periods providing that. What defines the
season can differ. In temperate zones, the usual circannual
patterns of temperature and day length define seasons. In other
areas, seasonally occurring environmental cues dictate reproduc-
tion. Tropical species breed during defined rainy seasons, for
example. The season can be very long, as in many North Amer-
ican species such as green treefrogs (Hyla cinerea) that are repro-
ductively active, weather permitting, from late spring to early fall,
or very short, sometimes being restricted to a matter of days in
very early spring, as in wood frogs (Rana sylvatica) which emerge
for a brief, frenetic breeding period in ephemeral ponds. In
extreme cases, breeding ‘seasons’ in challenging habits such as
the southwest American desert occupied by spade foot toads
in the family Scaphiopodidae consist of unpredictable rain
showers that bring out immediate, but very brief, bursts of repro-
ductive activity that can be over in a day. Regardless of the length
or trigger, something must gate reproduction and coordinate the
physiological, neural, and behavioral components of it. Gonadal
steroid hormones play this role throughout vertebrates. At least
for temperate zone species with defined annual breeding
seasons, seasonal variation in gonadal steroids coincides with
seasonal variation in reproductive behavior and physiology.
Acoustic communication is the foundation for anuran social
behavior. Except for the small number of species in the most
primitive frog families (the genera Ascaphus and Leiopelma) all
anurans studied to date base their social interaction on vocali-
zations. Most species have a lek-like mating system (Höglund
and Alatalo, 1995) in which males aggregate on communal
display sites (choruses) and produce advertisement, or mating,
calls to attract females (Wells, 1977, 2007). Females use the

Hormones, Brain, and Behavior, 3rd edition, Volume 2 http://dx.doi.org/10.1016/B978-0-12-803592-4.00026-2 101

102 Neuroendocrine Control of Social Behavior in Frogs
calls to find males and to compare them in order to make
a choice among them to initiate mating. Once a female has
picked a male, they enter into amplexus, in which the male
clasps her from the dorsal side, and eggs and sperm are
co-released for external fertilization. Amplexus can last for
hours in many species. This social system leads to intense selec-
tion pressure on males to call vigorously throughout the
breeding season with clearly defined species-specific call
features attractive to females. Females in turn are under the
pressure of their developing egg masses to find a mate within
the cacophony of vocally competing males before their eggs
must be released fertilized or not.
In addition to advertisement calls, other vocalizations may
be produced. In some species males produce an aggressive call
that may be a modification of the advertisement call or a sepa-
rate vocalization. In some species females produce recognition,
courtship, or release calls, the last signaling nonreceptivity. In
a few species, females have been found to produce a vocaliza-
tion that seems to function as some sort of advertisement call
(Emerson and Boyd, 1999), although this is relatively rare (at
least among commonly observed species).
Acoustic communication is a sexually differentiated behavior.
In the vast majority of anuran species, advertisement calling
is a male trait, and vocal behavior overall is strongly male
biased. The sex difference can be so great that females do
not vocalize at all, a feature of several commonly studied
North American and neotropical species. In other commonly
studied species such as Xenopus sp. both sexes produce
communication sounds, but the behavior and the features
of the underlying neural circuits and biomechanical mecha-
nisms of the larynx clearly show a male-biased sex difference
(reviewed in Kelley, Chapter 2.08, Hormones and Vocal
Systems: Insights from Xenopus). The great sex difference in
calling in many species provides a substrate for investigating
sex differences in the neural and biomechanical mechanisms
of this behavior. How these vary across species in which
females do and do not call has not been studied in a systematic
way.
Although the sexes diverge in their capacity to call, both
males and females hear and respond to the advertisement calls
that males produce. These responses, however, differ between
the sexes. Males respond by calling back to neighbors’ calls or
with aggressive responses that may lead to attacking nearby
calling neighbors. Females respond by orienting and displaying
phonotaxis behavior (that is, moving toward a sound) to
conspecific calls (which males may also do depending on the
situation Bernal et al., 2007). It is clear that females evaluate
the features of male calls, discriminate among them, and
base their mate choice on their assessment (Ryan, 1985). In
general, males are much less discriminating in terms of the
advertisement calls they will respond to, as predicted by theo-
retical considerations of sex differences in the fitness costs of
recognition errors (Trivers, 1972).
2.06.2 Seasonal Patterns of Hormones and
Behavior
The hormonal state of a seasonal organism varies greatly over
a year as physiological demands change and behaviors shift
along with them. In amphibians, these seasonal changes corre-
late most obviously with seasonal changes in the gonadal
steroid hormones, androgens and estrogens. Both the calling
and the responses to calls are the most obvious seasonal social
behaviors in male and female frogs. As such, one would expect
them to be gated by seasonal cycles of gonadal steroid
hormones, which in fact they are. Kelley (in Kelley, Chapter
2.08, Hormones and Vocal Systems: Insights from Xenopus)
discusses this for calling circuitry (see also Coddington,
Chapter 2.07, The Vertebrate Brain Stem as the Primary Site
of Behavior Command: Insights from an Amphibian, for
a similar analysis focusing on newts), and as such this chapter
will focus more on the auditory system providing the sensory
side of this communication. Hormones such as adrenal
steroids and melatonin change seasonally as well. Their contri-
butions to the control of social behavior remain less well
understood but an important avenue for further study.
2.06.2.1 Seasonal Changes in Gonadal Steroids
As expected for vertebrates with sexually dimorphic, seasonally
gated, reproductive social behavior, seasonal variation in circu-
lating gonadal steroid levels is present in anurans (Licht et al.,
1983; Pierantoni et al., 1984; Iela et al., 1986; Rastogi et al.,
1986; Itoh et al., 1990; Herman, 1992; Narayan et al., 2010;
Scaia et al., 2013). Studies that describe this pattern, coming
mainly from work in northern temperate species with pro-
longed breeding seasons, indicate both a basic seasonal pattern
of endocrine changes plus variation within a season pegged to
reproductive cycles or environmental and social context. As in
the yearly cycle seen in many seasonally breeding vertebrates,
gonadal steroid levels are higher during the breeding season,
when reproductive and aggressive social behavior is also
high. In several species investigated, anuran males have been
found to have the highest gonadal steroid (testosterone) levels
at the beginning of the breeding season; in others, androgens or
estrogens either remain constant or increase throughout the
breeding season. In either case, a rapid decline in gonadal
steroids marks the end of the breeding season, and the end of
reproductive social behavior. What the patterns look like in
frogs with extremely short breeding seasons, or with unpredict-
able behavior based on local climate conditions, is less well
understood, but Harvey et al. (1997) described a similar
pattern in the spadefoot toad, Scaphiopus couchii, an explosive
desert breeder that emerges and breeds quickly when rain
occurs. There, as in animals with a prolonged breeding season,
androgens in males, and estrogens and progesterone in
females, were highest when individuals had emerged to breed.
In addition to the seasonal hormone changes that gate the
breeding season, individual variation in androgens and estro-
gens occurs within the breeding season. Some of this is driven
by individual reproductive cycles. For example, in species
with long breeding seasons, females may cycle through
several clutches of eggs with estrogens and/or progesterone
levels cycling accordingly, peaking when eggs are ready to
be released, then dropping quickly once mating is over
(Iela et al., 1986; Harvey et al., 1997; Medina et al., 2004;
Lynch and Wilczynski, 2005; Gramapurohit and Radder,
2013; Gordon and Hellman, 2015). Interestingly, in one
species males were found to have a similar testosterone

pattern: highest immediately before and during mating, then
dropping quickly once mating was over (Gramapurohit and
Radder, 2013). More unpredictable context-dependent endo-
crine changes may also occur related to any number of envi-
ronmental cues. The immediate social environment is one
such cue. The effect of social interactions on endocrine state
will be discussed in Section 2.06.5.
2.06.2.2 Seasonal Changes in Social Behavior Match
Changes in Gonadal Steroids
Male and female reproductive social behavior waxes and wanes
with the seasonal changes in gonadal steroids, providing strong
correlative evidence that these hormones are important modu-
lators of male calling and female phonotaxis. On average,
seasonal changes in male calling track seasonal changes in
androgen levels (Polzonetti-Magni et al., 1984; Varriale et al.,
1986; Itoh and Ishii, 1990; Gobbetti et al., 1991; Harvey
et al., 1997). Male calling is often most vigorous at the point
of the breeding season when testosterone is highest. In green
treefrogs, this is at the beginning of the season. One laboratory
study (O’Bryant and Wilczynski, 2010) using this species held
environmental conditions constant throughout the summer
and found that both testosterone and spontaneous calling
declined together from June through September (Figure 1).
Experimental manipulation of gonadal steroid levels also
clearly shows the role these hormones play in modulating
frog social behavior. In males, vocal behavior is androgen
dependent (Wada et al., 1976; Wada and Gorbman, 1977;
Figure 1 Mean (þSE) plasma testosterone (a) and log spontaneous
calls per hour (b) in May, July, and September in green treefrogs (Hyla
cinerea) held over their normal summer breeding season in constant
summer environmental conditions. Data from O’Bryant, E.L., Wilczynski,
W., 2010. Changes in plasma testosterone levels and brain AVT cell
number during the breeding season in the green treefrog. Brain Behav.
Evol. 75 (4), 271–281.
Neuroendocrine Control of Social Behavior in Frogs 103
Wetzel and Kelley, 1983; Solís and Penna, 1997; Burmeister
and Wilczynski, 2001) and eliminated when males are cas-
trated (Schmidt, 1966; Palka and Gorbman, 1973; Kelley and
Pfaff, 1976; Burmeister and Wilczynski, 2001; Wilczynski
et al., 2005; Leary, 2009; Zornik and Kelley, 2011). Male calling
is not always expressed in lock-step with varying androgen
levels, however, especially when naturally behaving animals
are studied. Solís and Penna (1997) found that androgen
concentrations and vocalization rates were correlated in a field
population of Batrachyla taeniata. Studies in other species have
produced conflicting results. In some cases, androgens are
lower in calling anurans relative to noncallers (Mendonça
et al., 1985), whereas in other species, androgens are higher
in calling males (Townsend and Moger, 1987; Marler and
Ryan, 1996). One of the reasons for the variety of findings is
that natural calling behavior is influenced by many factors.
Burmeister and Wilczynski (2001) provided some insight
into this variability in a study that distinguished spontaneous
calling from evoked calling, that is, calling when males were
isolated from other callers versus when males heard the calls
of other males. There, as expected, castration eliminated all
calling. In intact and castrated frogs implanted with testos-
terone capsules, testosterone levels correlated with sponta-
neous calling, but not evoked calling in the same animals.
When hearing other males’ calls, males with sufficient testos-
terone vigorously responded by calling at high rates. This
pattern of antiphonal calling is a standard feature of frog social
behavior (Wells, 1977; Gerhardt and Huber, 2002). Similarly,
although spontaneous calling declined along with testosterone
in laboratory-held green treefrogs, evoked calling rates (calling
when chorus sounds were present) did not change over the
breeding season (O’Bryant and Wilczynski, 2010), suggesting
again that social cues can override whatever motivational effect
androgens may have. In a separate study (Burmeister et al.,
2001), a male’s latency to resume calling after capture was
also correlated with circulating androgen level. In total, this
work suggests that testosterone has a permissive effect and
increases the motivation to call, but once social cues are added,
male calling is at least, if not more, driven by the calls of
competitor males.
Several aspects of female reproductive behavior also vary
with fluctuating gonadal steroid levels particularly as females
with prolonged breeding seasons pass through different phases
of their ovulatory cycle (Lea et al., 2000; Bosch and Boyero,
2004; Lynch et al., 2006). Female sexual behavior most often
includes phonotaxis, or the orientation to and movement
toward conspecific calling males (Ryan, 1985; Gerhardt and
Huber, 2002), but, depending on the species, can also include
producing vocalizations (Tobias et al., 1988; Roy et al., 1995;
Emerson and Boyd, 1999), or exhibiting behaviors that reject
males, such as release calls and leg extensions (Diakow
and Nemiroff, 1981; Kelley, 1982; Boyd, 1992). Because
phonotaxis is such a common and robust indicator of female
receptivity, it is most often used in studies of female social
behavior. As for male calling, female phonotaxis toward
conspecific calls is expressed during the breeding season
when estrogens and progesterone are high. Lynch and
Wilczynski (2005) tracked the gonadal steroid levels over
the natural ovulatory cycle of field-caught túngara frogs
(Physalaemus (¼Engystomops) pustulosus). Females collected at
104 Neuroendocrine Control of Social Behavior in Frogs

a breeding site prior to mating had low levels of estrogens and
progesterone. Females that had entered into amplexus (the
condition in which a male tightly grasps a female to align their
bodies for sperm and egg release), indicating they were ready to
release eggs, had peak estrogens and progesterone levels.
Immediately after egg release, estrogens and progesterone
rapidly fell to their lowest levels during the breeding season.
Female phonotaxis behavior can assess the behavioral variation
that occurs with these natural cycles or after experimental
manipulation of hormone levels and has been used in natu-
rally cycling and in hormonally treated túngara frog females
(Figure 2; Lynch and Wilczynski, 2005; Lynch et al., 2006;
Chakraborty and Burmeister, 2009). Receptivity, as defined
by an approach to a recording of a male advertisement call,
increases and decreases with the rise and fall of gonadal
hormones, as happens in other cycling female vertebrates. At
the same time, phonotaxis toward relatively unattractive male
calls (‘permissiveness’) also increases when estrogens are
high. However, females never lose the ability to discriminate
between attractive multisyllable calls and unattractive single
syllable or artificially manipulated calls made to sound like
hybrid signals regardless of estrogens levels and reproductive
state. That is, when estrogens and progesterone levels are
high, which coincides with the point at which eggs are ready
to be released, females are highly motivated to select a calling
male and are more accepting of calls that, under lower
hormone levels, they would reject. However, regardless of
how receptive or permissive females become, they always elect
to go to an attractive call over an unattractive call if they have
the choice. Chakraborty and Burmeister (2009) found that
estrogens alone could account for these behavioral changes.
As female frogs reach the point when their eggs are mature
enough to be released, they are under strong pressure to find
a suitable male. At some point, the egg mass becomes so large
that it will be released regardless of whether mating is
underway to fertilize it. If this happens, females have wasted
the considerable resources that went into growing the egg
mass, and wasted what may be one of their few, and perhaps
only, opportunities to procreate. As Lynch et al. (2006) argued,
estrogen-driven receptivity drives the female to search for and
accept males. Estrogen-driven decreases in choosiness change
the search and decision process so that she will accept a wider
variety of males as the point of egg release becomes eminent. At
no time, however, does the female lose her ability to pick the
more ‘attractive’ male among the choices she has.
Other studies have implicated progesterone (Gordon and
Gerhardt, 2009), luteinizing hormone itself (Kelley, 1982),
and prostaglandin (Diakow and Nemiroff, 1981; Schmidt,
1985; Weintraub et al., 1985) in some aspects of female frog
reproductive social behavior. It is also possible that in those
frog species where females produce advertisement vocaliza-
tions, testosterone might regulate this behavior as it does in
males (Emerson and Boyd, 1999). There is, however, very little
direct evidence that this is the case.

Receptivity
As estrogens increase:

Noise Normal
call
Response to a
normal male call
Permissiveness

Hybrid call Noise

Response to a
normally ignored call
Discrimination

One Two
syllable syllable
call call
Go to the more
attractive call

Figure 2 Diagram of phonotaxis experiments used in Lynch et al. (2005, 2006) to show changes in female túngara frog (Physalaemus pustulosus)
behavior as plasma levels of estrogens change. Females are placed in the center of a large experimental chamber and stimuli are broadcast from
speakers on either side. Receptivity (top panel) can be measured by the proportion of females moving toward a male call. Permissiveness (middle
panel) is measured by the proportion of females moving toward a normally unattractive call, such as an artificial call constructed as an acoustic
hybrid of a P. pustulosus and a Physalaemus enesfae call. Discrimination (bottom panel) is measured by the proportion of females moving toward
a two-syllable versus one-syllable call; receptive túngara females will display phonotaxis behavior to either when presented alone, but normally find
multisyllable calls more attractive. As estrogens increase receptivity increases (females are more likely to respond to any call), and permissiveness
increases (females are more likely to respond to a hybrid call), but females maintain the ability to discriminate between attractive two-syllable calls
and less attractive one-syllable calls by preferentially going toward the two-syllable call.
 Neuroendocrine Control of Social Behavior in Frogs 105

2.06.2.3 Seasonal Variation in Other Hormones: Adrenal It may be, of course, that corticosterone levels can reach so
Steroids high a level that reproductive behavior ceases. However, an
alternative view of corticosteroid action may be that it too
Adrenal steroids, specifically corticosteroids like corticosterone,
has a seasonal pattern that like gonadal steroids is, on average,
are most often identified as ‘stress’ hormones rather than
higher in the breeding season than out of it. Rather than func-
seasonally varying endocrine products influencing social
tioning as an antagonist to calling or other social behaviors, it
behavior. But in fact, corticosteroids are linked to social
facilitates reproductive social behavior by supporting the
behavior and are found to be elevated during breeding periods
demand for increased energy mobilization during this impor-
in many amphibians (Licht et al., 1983; Mendonça et al., 1985;
tant period of an animal’s life (Eikenaar et al., 2012). Leary
Orchinik et al., 1988; Harvey et al., 1997; Romero, 2002;
et al. (2008) and de Assis et al. (2012) have in fact demon-
Moore and Jessop, 2003; Eikenaar et al., 2012). Frog calling
strated in different frog species that corticosterone levels corre-
is an extremely energetically demanding behavior (Taigen
late with vocal effort as expected if adrenal steroids are
and Wells, 1985; Marler and Ryan, 1996; Wells, 2001). As
mobilized to support this energetically intensive behavior. Of
such, the energetic stress resulting from intense vocal displays
course, the seasonal difference in adrenal steroid levels may
would be expected to trigger elevated corticosterone levels. In
not be environmentally controlled as for gonadal steroids,
fact, calling males do have high levels of corticosterone (Leary
but may be secondary to the energetic effort attendant to calling
et al., 2006, 2008; de Assis et al., 2012). What is the implication
and other reproductive efforts.
of high corticosteroids in frogs?
The Energetics-Hormone-Vocalization model remains an
Experimental manipulations of corticosterone in many
important insight, as something must trigger switches between
different vertebrates have resulted in a decrease in reproduction
behavioral states. If not adrenal steroids, are there other signals
and the social behavior supporting it. These studies have also
of energetic or related physiological condition that could
found that experimental elevation of corticosteroids yields
mediate this switch? As yet, these are unknown. Understanding
a suppression of testosterone levels in males. This observation
the interaction of metabolic regulation and social behavior is
gave rise to the ‘Energetics-Hormone-Vocalization’ hypothesis
important, and given the strong energetic demands of at least
(Emerson, 2001; Emerson and Hess, 2001) that corticosterone
male social behavior in frogs, these vertebrates could be useful
levels mediate a switch between calling and noncalling in male
models for investigating that process.
frogs. The idea is that calling effort results in energetic stress,
The work on corticosteroids and behavior in frogs has
which is signaled by raising corticosterone levels. As corticoste-
largely focused on males and with them, on the interaction
rone levels rise, androgen levels fall. At some point, androgen
of these adrenal steroids with androgens. The energetic stress
levels fall below the threshold for calling, and males switch
profile of females is unknown, and interactions between corti-
from reproductive behavior to foraging behavior. Once energy
costeroids and gonadal steroids in females is unclear except for
stores are rebuilt, corticosterone levels fall, testosterone levels
one study in green treefrog females (Davis and Leary, 2015)
rise, and males switch back from foraging to reproduction.
that found no effect of experimentally elevated corticosterone
This reasonable model is based on numerous laboratory
on female estrogens, progesterone, testosterone, or dihydrotes-
studies in many vertebrates showing that treatment with exog-
tosterone. That study did find, however, that high levels of
enous corticosteroids or infliction of stress of some kind
corticosterone reduced the strength of their preference among
reduces testosterone levels. In some seasonally breeding
male calls (Figure 3), but did not seem to change their motiva-
amphibians, chronic or acute corticosterone administration
tion to mate in phonotaxis tests. Davis and Leary speculated
inhibits courtship (Moore and Miller, 1984; Marler and Ryan,
that the corticosterone-mediated decrease in choosiness may
1996; Burmeister et al., 2001). Furthermore, smaller males in
be a mechanism for reducing risky extended searches for males
poorer condition that adopt noncalling satellite behavior in
under stressful conditions. In fact, factors such as high preda-
frog choruses have elevated corticosterone levels, as well as
tion risk have been argued to increase the cost of mate sampling
lower testosterone levels, compared to robustly calling males
and alter search strategies (Crowley et al., 1991; Rand et al.,
(Leary and Harris, 2013). Beyond this, however, the
1991).
Energetics-Hormone-Vocalization hypothesis has received little
support from studies that measure natural corticosterone–
2.06.2.4 Seasonal Variation in Other Hormones: Melatonin
androgen relationships. During the time of reproductive social
behavior, corticosterone and testosterone are most often seen Melatonin is a pineal hormone released in circadian and
to be elevated together and either positively correlated (Licht seasonal patterns in many vertebrates. In amphibians (and
et al., 1983; de Assis et al., 2012) or unrelated (Leary et al., some birds), the retina produces melatonin as well in particu-
2006, 2008, 2015) in naturally behaving male frogs. Moreover, larly high levels and may contribute significantly to circulating
exogenous arginine vasotocin treatment in male green treefrogs levels of this hormone (Acuña-Castroviejo et al., 2014).
stimulates calling (Burmeister et al., 2001), likely through its Amphibian melatonin cycles are the same as in other vertebrates,
effect on central nervous system areas (see also Coddington, with high levels at night and higher levels in winter, when the
Chapter 2.07, The Vertebrate Brain Stem as the Primary Site dark phase is longer, than in summer (Delgado and
of Behavior Command: Insights from an Amphibian), but it Vivien-Roels, 1989; d’Istria et al., 1994). In ectotherms tempera-
also increases levels of circulating corticosterone, likely through ture is another potent regulator of seasonal melatonin patterns
AVT/AVP’s well-known peripheral effect as a stimulator of (Rawding and Hutchison, 1992; Lutterschmidt and Mason,
adrenal steroid release. The increasing corticosterone does not 2009). Melatonin has been found to regulate reproduction in
negatively affect the calling induced by AVT. many animals (Revel et al., 2009; Barrett and Bolborea, 2012),
106 Neuroendocrine Control of Social Behavior in Frogs
Probability of choosing call broadcast at high
1
call rate (2.5 call/s) 0.8
0.6
0.4
0.2
0
Control Saline
(n=9) (n=7)
Figure 3 The probability of female green treefrogs (Hyla cinerea) in
phonotaxis tests moving toward a call at a high call rate (2.5 calls/s)
versus a low call rate (1.2 calls/s) following no treatment, treatment
with saline vehicle, or different doses of corticosterone (CORT). Dotted
line indicates equal choice for each stimulus. At the highest corticoste-
rone levels, female choice approaches random and is significantly
different from the other treatments. Asterisks indicate significant differ-
ence from control (no treatment) condition. From Davis, A.G., Leary,
C.J., 2015. Elevated stress hormone diminishes the strength of female
preferences for acoustic signals in the green treefrog. Horm. Behav. 69
(0), 119–122.
including courtship behavior, gonadal physiology, and sex
steroid hormone concentrations in some ectothermic vertebrates
(de Vlaming et al., 1974; Krohmer and Lutterschmidt, 2011).
Experimental manipulation of melatonin levels in frogs, so far
only in males, has failed to find any effect of melatonin on circu-
lating testosterone (Pierantoni et al., 1986; Lutterschmidt and
Wilczynski, 2012). This suggests that the seasonal decline in
testosterone that occurs as frogs move out of the breeding season
is not due to the increasing level or nightly duration of mela-
tonin that occurs in the winter, nonbreeding season.
No work has related melatonin with social behavior in
frogs. However, melatonin does influence the amount of the
neuromodulatory peptide arginine vasotocin (AVT) in brain
areas related to social behavior. AVT and its mammalian
homolog arginine vasopressin have been implicated in many
types of male social behavior, particularly social signaling.
The important role of AVT in amphibian courtship behavior
will be discussed at length in the chapter by Coddington in
this volume (Coddington, Chapter 2.07, The Vertebrate Brain
Stem as the Primary Site of Behavior Command: Insights from
an Amphibian) and is reviewed in Emerson and Boyd (1999)
and in Wilczynski et al. (2005). Briefly, as in other vertebrates,
AVT cell number is sexually dimorphic and higher in males.
Treatments that elevate AVT stimulate advertisement calling
in males and result in advertisement calls that females find
more attractive. Implantation of melatonin capsules in
breeding-condition green treefrogs in the summer results in
a decrease in the number of AVT immunoreactivity in two
brain areas, the nucleus accumbens and the suprachiasmatic
nucleus (Lutterschmidt and Wilczynski, 2012). The effect
was restricted to males; brain AVT seemed relatively insensitive
to melatonin changes in females. Furthermore, the same treat-
ment in winter males in nonbreeding condition also results in
no significant changes in AVT immunoreactive cells in any
* *
4 µg CORT 8 µg CORT 16 µg CORT
(n=8) (n=11) (n=10)
Figure 4 Number of melatonin type 1 receptor immunoreactive cells
(MT1-IR) in the nucleus accumbens of male and female green treefrogs
(Hyla cinerea) in summer and winter. Overall numbers are significantly
higher in the summer, when a significant sex difference is also
apparent. From Howard, C.M., Lutterschmidt, D.I., 2015. The effects of
melatonin on brain arginine vasotocin: relationship with sex and
seasonal differences in melatonin receptor type 1 in green treefrogs
(Hyla cinerea). J. Neuroendocrinol. 27 (8), 670–679.
brain area (Howard and Lutterschmidt, 2015). The different
sex and season effects of melatonin appear to be due to differ-
ences in melatonin receptor profiles. Amphibians, like
mammals, have three types of brain melatonin receptors
(Wiechmann and Wirsig-Wiechmann, 1993; Li et al., 2013),
one of which, MT1(or Mel1a), has a role in regulating seasonal
reproduction in other vertebrates (Dubocovich, 1988; Pre-
ndergast, 2010; Luridiana et al., 2015). MT1 immunoreactive
cells are more numerous in the nucleus accumbens in males
than in females and are less numerous there and in all brain
regions in winter males than in summer males (Figure 4).
The sex and season differences in the effects of melatonin on
the brain AVT system thus appears to be due to sex and season
differences in the levels of MT1 receptors (Howard and Lut-
terschmidt, 2015).
It is important to remember that females also have seasonal
changes in reproductive behavior. AVT in general does not
seem to have as important a role in regulating female frog
social behavior, although AVT treatment does inhibit female
release calls (Diakow and Nemiroff, 1981; Boyd, 1992) and
may increase female phonotaxis in some species (Schmidt,
1984; Boyd, 1994). No other neural peptide has been found
to influence female frog social behavior.
2.06.3 Steroid Hormone Receptors in the Brain
Across vertebrates, receptors for gonadal steroids are wide-
spread across the brain. Androgen and estrogen receptors are
particularly noticeable within the forebrain areas connected
into brain networks for social decision making (Goodson,
2005; Goodson and Thompson, 2010) and reward (Berridge
and Kringelbach, 2013). The pattern of these and other neuro-
chemical markers are conserved across vertebrates (O’Connell
and Hofmann, 2012). Androgen and estrogen receptors are
seen here in frogs, as well as in motor and sensory areas,
and in the thalamus and midbrain, suggesting that as in other
vertebrates, gonadal steroids have widespread modulatory
 Neuroendocrine Control of Social Behavior in Frogs 107

effects on multiple aspects of social behavior. The distribution
of sex steroid receptors indicates that gonadal hormones influ-
ence both the vocal and the auditory components of the frog
social system. The torus semicircularis, a large midbrain area
homologous to the inferior colliculus (Wilczynski and Ende-
pols, 2007) where auditory sensory and motor systems
come together, is rich in androgen, estrogen, and progesterone
receptors (Figure 5).
The anatomical organization of the frog auditory system is
reviewed in detail in Wilczynski and Capranica (1984) and
in Wilczynski and Endepols (2007). Like the mammalian
inferior colliculus, the amphibian torus is the site where
ascending auditory input from lower brainstem auditory areas
converges and where auditory information is then relayed to
a variety of thalamic nuclei as well as sent via downstream
connections to the brainstem reticular formation and auditory
areas. Neuronal responses with complex spectral and temporal
properties emerge in the torus (reviewed in Rose and Gooler,
2007). These feature-detecting properties of toral neurons are
linked to the detection of the species’ advertisement calls,
accentuating a bias toward responding to call-related acoustic
signals seen throughout the frog auditory system. Significant
sex differences in auditory responses also appear in the
midbrain (Hoke et al., 2008, 2010; Wilczynski and Ryan,
2010). The response of the torus to acoustic stimuli as a whole
closely mirrors behavioral responses to conspecific signals
(Hoke et al., 2008; Mangiamele and Burmeister, 2011).
With its strategic anatomical position and neurophysiological
bias toward responding to call features, the torus acts as a key
sensory motor interface, and more generally a control point
regulating connections between auditory input, and motor,
endocrine, and social responses (Wilczynski and Endepols,
2007; Wilczynski and Ryan, 2010).
The torus is composed of several subdivisions, the laminar,
principal, and magnocellular nuclei being the most prominent.
Toral nuclei are rich in receptors for a variety of hormones
and neuromodulators (Endepols et al., 2000; Wilczynski and
Endepols, 2007) including receptors for gonadal steroids
(Figure 5). The laminar nucleus, which provides a significant
portion of the torus’ ascending and descending outputs, has
the highest concentration of estrogen and androgen receptors
within the torus (Kelley et al., 1975; Morrell et al., 1975;
Kelley, 1980; Chakraborty and Burmeister, 2010). Androgen,
ERalpha, and ERbeta receptors are found there, as well as in
the principal nucleus (Chakraborty and Burmeister, 2010).
Progesterone receptors are also expressed in the laminar
nucleus, as well as in the magnocellular nucleus (O’Connell
et al., 2011). Although there is no sex difference in the locations
of toral steroid receptors, the expression levels do differ
(Chakraborty and Burmeister, 2010). Females express more
ERbeta in the laminar nucleus and more ERalpha in the prin-
cipal nucleus; males express more androgen receptor in the
principal nucleus.
Brain areas caudal to the midbrain show little evidence
of sex steroid binding in frogs. Androgen receptors are
present in the pretrigeminal nucleus (also called the DTAM),
a key part of the frog vocal control system, as well as on the
motor neurons in the XI-X complex innervating the larynx
(Emerson and Boyd, 1999; see also Kelley, Chapter 2.08,
Hormones and Vocal Systems: Insights from Xenopus).
Neither androgen nor estrogen receptors have been
described in the amphibian brainstem auditory areas,
although progesterone receptor protein, as identified by
immunocytochemistry, seems to be widespread throughout
brainstem reticular areas. Forebrain areas, on the other hand,
are rich in all gonadal steroid receptors (Kelley et al., 1975;

Figure 5 Locations of androgen (AR), estrogen (ER) (reported in Chakraborty and Burmeister, 2010 based on in situ hybridization), and proges-
terone receptors (PR) (reported in O’Connell et al., 2011 based on immunocytochemistry) in the torus semicircularis (the auditory midbrain) and
surrounding areas in túngara frogs (Physalaemus pustulosus). Abbreviations: TE, optic tectum; Teg, tegmental areas; Tl, laminar nucleus of the torus
semicircularis; Tm, magnocellular nucleus of the torus semicircularis; Tp, principal nucleus of the torus semicircularis.
108 Neuroendocrine Control of Social Behavior in Frogs

(a) MP
(AR,ER)

(ER) Auditory
St TS brainstem
A
C (AR,ER)
(AR, P
ER) VM
Sep SI

(AR,
POA/Hy
ER)

Endocrine
control

(b)
MP

St Auditory
(AR, A (ER) TS brainstem
ER) C (AR,ER)
P
(AR)
Sep VM SI

POA/Hy

Motor
systems

Figure 6 Diagram of the audioendocrine (a) and audiomotor (b) systems of the frog brain defined Wilczynski and Endepols (2007) based on
ascending connections from the torus semicircularis. Presence of moderate to high levels of androgen (AR) and estrogen (ER) receptor expression
within the systems are indicated. Abbreviations: A, anterior thalamic nucleus; C, central thalamic nucleus; Hy, hypothalamus; MP, medial pallium;
P, posterior thalamic nucleus; POA, preoptic area; Sep, septal complex; Si, secondary isthmal nucleus; St, striatopallidal complex; TS, torus semi-
circularis; VM, ventromedial thalamic nucleus.

Morrell et al., 1975; Chakraborty and Burmeister, 2010;
O’Connell et al., 2011). This includes the targets of ascending
auditory connections, and the motor and basal forebrain
areas implicated in the control of vocalization (Emerson
and Boyd, 1999). Wilczynski and Endepols (2007)
conceptualized ascending circuits emerging from the torus as
comprising audiomotor, audioendocrine, and audiolimbic
components based on their forebrain targets (Figure 6). Each
includes several nuclei with estrogen, androgen, and
progesterone receptors, including thalamic nuclei, areas of
the basal forebrain implicated in male and female social
behavior such as the preoptic area and ventral hypothalamus,
and limbic centers such as the medial septum and the medial
pallium (the amphibian homolog of the mammalian
hippocampal complex) (Chakraborty and Burmeister, 2010;
O’Connell et al., 2011).
Glucocorticoids also influence social behavior in frogs,
and presumably work at least in part through neural systems
to do so. They interact with brain arginine vasotocin systems
to modulate courtship behavior in newts, which is described
in detail by Coddington (Coddington, Chapter 2.07, The
Vertebrate Brain Stem as the Primary Site of Behavior
Command: Insights from an Amphibian), although whether
they do the same in frogs is unknown. Yao et al. (2008)
found glucocorticoid receptor protein widely distributed
throughout the brain in Xenopus with particularly high levels
in forebrain limbic and hypothalamic areas. Receptors were
also found throughout the midbrain and hindbrain. No
particular association with vocal or auditory centers is
apparent, including no strong presence in the toral nuclei
with high gonadal steroid receptor expression. The nature
and distribution of glucocorticoid receptors and other
components of the brain’s stress signaling system in
amphibians deserves further attention, especially given
new work showing significant effects of adrenal steroids
on acoustic communication in frogs (Leary et al., 2006;
Davis and Leary, 2015) and the substantial previous work
on their strong influence on olfactory-driven courtship
behavior in salamanders (reviewed in Coddington, Chapter
2.07, The Vertebrate Brain Stem as the Primary Site of
Behavior Command: Insights from an Amphibian).
2.06.4 Hormone Effects on Hearing
The auditory system detects and analyzes the acoustic social
cues guiding frog social interactions and transmits this infor-
mation to motor, endocrine, and limbic areas responsible for
orchestrating the behavioral and physiological components
of frog social behavior (Figure 6). The sensory systems respon-
sible for inputting information about social signals are often
thought of as the stable component of social neural networks.
In fact, growing evidence shows that sensory processing is
remarkably plastic. Several lines of evidence support the idea
that hormone-dependent changes in hearing are present in
amphibians and that this results in changes in the detection

and response to the male advertisement call. This may be
a major component of the hormonal regulation of anuran
reproductive social behavior.
2.06.4.1 Seasonal Changes in Hearing Track Seasonal
Changes in Sex Steroids and Behavior
Social behavior waxes and wanes seasonally in frogs along
with their levels of circulating sex steroid hormones. In
nonbreeding seasons, frogs are less responsive to their species’
vocal communication signals. Changes in the auditory system
may contribute to this. By several measures, midbrain
responses to auditory stimulation are more robust in the
summer breeding season than when frogs are in a nonbreeding
condition. Goense and Feng (2005) found that more toral
auditory cells could be isolated in Lithobates (¼Rana) pipiens
when recording in the summer, latencies were shorter, and
phase locking to amplitude modulated signals stronger. Earlier
work found greater suprathreshold multiunit responses in the
torus in fire-bellied toads (Bombina bombina; Walkowiak,
1980) and gray treefrogs (Hyla chrysoscelis; Hillary, 1984)
during the breeding season. Seasonal threshold changes at
the single neuron level may (Hillary, 1984) or may not
(Penna et al., 1992; Goense and Feng, 2005) be present.
However, the overall picture of the anuran midbrain is that
stronger auditory responses are present in the summer,
when hormone levels are high, than in the winter, when levels
are low.
In females, estrogen levels drop precipitously immedi-
ately after eggs are laid, and along with this, females become
unreceptive to male advertisement calls (Lynch et al., 2006).
Toral auditory responses change cyclically in a way that
matches the change in receptivity and which is also consis-
tent with the seasonal changes. In postmated female green
treefrogs, toral response strength to band-limited noise
stimuli is significantly less than in premated females
(Miranda and Wilczynski, 2009a). The effect is restricted to
lower frequencies in the frog’s hearing range, which are ulti-
mately derived from the ear’s amphibian papilla, one of two
main auditory end organs in the amphibian ear (for reviews
of the amphibian ear, see Zakon and Wilczynski, 1988;
Simmons et al., 2007). Responses to the higher frequencies
derived from stimulation of the frog’s second auditory end
organ, the basilar papilla, were stable throughout the repro-
ductive cycle. Similarly, Goense and Feng (2005) found that
seasonal changes were apparent in lower frequencies,
whereas responses to the higher basilar papilla frequencies
were unchanged across seasons.
The midbrain may not be the first auditory processing stage
at which seasonal and reproductive-cycle changes in hearing
occur. Zhang et al. (2012) found that peripheral auditory
thresholds were lower during the breeding season than the
nonbreeding season in the Emei Music frog (Babina daunchina).
This is the only frog in which a seasonal change in the periph-
eral auditory system has been documented. However, seasonal
or hormone-dependent changes to the ear’s sensitivity have
been described in several vertebrates (Sisneros and Bass,
2003; Sisneros et al., 2004; Caras, 2013; Gall et al., 2013).
Further studies on the effects of steroid hormones on the ear
itself are clearly warranted.
Neuroendocrine Control of Social Behavior in Frogs 109
2.06.4.2 Experimental Manipulation of Circulating Gonadal
Steroids Modifies the Auditory System
Several studies have directly manipulated androgens and estro-
gens to show the effect these hormones have on auditory pro-
cessing. Nearly all have focused on the midbrain, where
estradiol injections were reported to increase the amplitude
of toral-evoked potentials to tones (Yovanof and Feng,
1983). Testosterone’s effects have been more inconsistent.
Penna et al. (1992) found no testosterone effects on midbrain
thresholds, which may not be surprising given that most
studies of seasonal variation in midbrain auditory responses
find little or no change in thresholds. Testosterone appears to
induce more complicated sex- and stimulus-specific changes
than simple threshold changes in toral auditory responses.
Testosterone treatment yielded no statistically significant
changes in midbrain thresholds in male green treefrogs
(Miranda and Wilczynski, 2009b). In females, however, testos-
terone increased thresholds to tones with frequencies within
the spectral bands of the male advertisement call, but not to
those outside those bands, and also increased thresholds to
the advertisement call itself. These results bear an interesting
relationship to a natural sex difference in green treefrog sensi-
tivity to the male call. In the hormone state appropriate for
breeding condition animals, female toral multiunit responses
to the male call have lower thresholds than do the responses
of males to the same stimulus (Miranda and Wilczynski,
2009b). After testosterone treatment, female thresholds
increase to match those of males, eliminating the sex difference
in sensitivity.
Quantification of immediate early gene (IEG) mRNA
expression also shows the stimulatory effect of gonadal
hormones on toral auditory activity. Treating túngara frog
females with gonadotropin (Lynch and Wilczynski, 2008),
which elevates estrogen levels, or with estrogen itself (Chakra-
borty and Burmeister, 2015) results in an increased midbrain
response to conspecific advertisement calls (Figure 7). Both
endocrine treatments cause an increase in IEG expression
even without accompanying auditory stimulation. There is
thus an additive effect of hormones and stimulus presentation,
resulting in a higher level of midbrain activity to calls when
estrogens are high (in females; a matching study of males
and testosterone has not been done). It is not clear what might
be the electrophysiological correlate of the estrogen-induced
elevation of IEG expression in the absence of an auditory stim-
ulus. Hormone-dependent changes in spontaneous activity, as
measured by single- or multiunit spike counts, have not been
consistently reported. It may be, however, that gonadal
hormones induce subthreshold depolarizations or represent
other, nonelectrophysiological, changes in midbrain cells that
adds to the response strength of midbrain auditory neurons
to calls under the endocrine conditions found in reproductively
active frogs.
Chakraborty and Burmeister (2015) is the only study that
extended the analysis of hormone effects on hearing beyond
the midbrain. They found that the facilitating, additive effects
of estradiol on the responses to conspecific calls extended to
many areas of the limbic forebrain that are sensitive to auditory
stimulation. In most cases, as in the torus, both conspecific calls
and estradiol alone resulted in an elevation of IEG expression,
110 Neuroendocrine Control of Social Behavior in Frogs

(a) (b)

(c) (d)

Figure 7 Photomicrographs of immediate early gene egr-1 mRNA expression in the laminar nucleus of the torus semicircularis in female túngara
frogs (Physalaemus pustulosus). (a) Egr-1 expression in saline-treated frogs with no acoustic stimuli; (b) Egr-1 expression in saline-treated frogs
hearing chorus sounds; (c) Egr-1 expression in saline-treated frogs treated with human chorionic gonadotropin (HCG) with no acoustic stimuli; (d)
Egr-1 expression in HCG-treated frogs hearing chorus sounds. From Lynch, K.S., Wilczynski, W., 2008. Reproductive hormones modify reception of
species-typical communication signals in a female anuran. Brain Behav. Evol. 71 (2), 143–150.

and together the effects were additive. The one exception was
the preoptic area. There, a statistical call by hormone interac-
tion was apparent, suggesting that preoptic auditory responses
are dependent on estradiol level, not just additive to it.
Gonadal steroids (or at least estrogens in females, which is
the only condition studied to date) appear to influence the
strength of auditory responses beyond the midbrain, to the
forebrain areas to which it feeds information.
2.06.4.3 Implications for Social Behavior
Social behavior in frogs, or in any animal, consists of detecting
social signals and responding with appropriate behaviors.
Between signal detection and response integrative processes
link sensory, motor, and physiological systems and modify
a variety of motivational, affective, and cognitive responses.
Hormones can and do influence the process at any stage. The
input, or sensory, side of the system is modified on a seasonal
and cyclic basis by gonadal hormones. The auditory system
changes make the advertisement call more detectable, more
stimulatory, and thus more salient when gonadal steroids are
elevated. These changes should enhance the probability of
both males and females to respond to the call in their own
sex-specific way.
2.06.5 Hearing Effects on Hormones
A traditional approach in behavioral endocrinology is to inves-
tigate how hormones modulate behavior. But behavior also
modulates hormonal state. Engaging in any social behavior
triggers an endocrine response of some kind. In anurans,
acoustic signals in the form of male advertisement calls are
the most common and most salient sensory cues associated
with social behavior. Hormonal condition influences how
strongly the nervous system responds to those cues. Hearing
those cues, in turn, influences the endocrine state of both males
and females.
2.06.5.1 Anatomical Links between the Auditory Midbrain
and Hypothalamic Areas Define an Audioendocrine Circuit
Neuroanatomical pathways emerging from the torus semicircu-
laris provide the substrate by which auditory stimulation by
calls can modulate the endocrine system. Ascending toral effer-
ents, mainly from the laminar nucleus, reach multiple thalamic
nuclei (Wilczynski et al., 1993; Wilczynski and Endepols,
2007) that together relay auditory information to the basal
ganglia, limbic pallial and subpallial areas, and directly to
hypothalamic and preoptic areas (Figure 6). Wilczynski and
Endepols (2007) termed the toral-thalamic-hypothalamic
pathway the audioendocrine system. Three distinct circuits
comprise this system. Auditory inputs to the ventral hypothal-
amus arise from the central and anterior thalamic nuclei (Neary
and Wilczynski, 1986; Hall and Feng, 1987; Neary, 1988;
Allison and Wilczynski, 1991). Connections to the anterior
preoptic area arise mainly from the anterior thalamic nucleus,
supplemented by a small projection from the central thalamic
nucleus. Auditory input to both the ventral hypothalamus and
anterior preoptic area also comes from the secondary isthmal
nucleus (Neary and Wilczynski, 1986; Neary, 1988; Allison
and Wilczynski, 1991), an area of the isthmal tegmentum

that receives a heavy toral input (Neary, 1988). The secondary
isthmal area is similar in location to the mammalian parabra-
chial region, which is recognized as relaying sensory informa-
tion to the hypothalamus. It is studied in mammals mainly
in regards to the transmission of behaviorally relevant chemo-
sensory or pain signals. Connections from thalamic and isth-
mal sensory areas are not unique to frogs, but the
connections relaying auditory information are exceptionally
robust in these vertebrates.
The acoustic sensitivity of preoptic and hypothalamic
areas have been confirmed by electrophysiology (Urano and
Gorbman, 1981; Wilczynski and Allison, 1989; Allison, 1992)
and by studies using immediate early gene expression as a mar-
ker of neural activity (Hoke et al., 2005, 2007; Chakraborty and
Burmeister, 2015). Response properties of preoptic and hypo-
thalamic neurons are characterized by long latencies and firing
rates that declines gradually after the termination of the stimulus
(Figure 8). In both areas, most acoustically sensitive neurons are
insensitive to tones or noise bursts, but fire strongly when stim-
ulated with a conspecific call. IEG studies show additional
complexity in the responses in these and other hypothalamic
and limbic centers. Responses in hypothalamic areas are often
a combination of auditory- and movement-related activation
Figure 8 Poststimulus–time histograms showing single unit
responses of a preoptic area (a) and a ventral hypothalamic (b) neuron
in the green treefrog (Hyla cinerea). Repetitive white noise (wn) and
mating call (mc) presentations alternate with no stimulus period (ns).
Responses to the conspecific call are marked by long latency, persis-
tent responses; spontaneous activity can remain elevated after the call
stimulus ends. From Allison, J., 1992. Acoustic modulation of neural
activity in the preoptic area and ventral hypothalamus of the green tree-
frog (Hyla cinerea). J. Comp. Physiol. A 171 (3), 387–395.
Neuroendocrine Control of Social Behavior in Frogs 111
(Hoke et al., 2007), and in both preoptic and hypothalamic
areas gonadal hormones add to or interact with the acoustically
driven IEG expression (Chakraborty and Burmeister, 2015).
The preoptic area and ventral hypothalamus region respon-
sive to acoustic call stimuli are part of the neuroendocrine
system controlling gonadotropin release. The extent of the
anatomical connections and the widespread acoustic sensitivity
across hypothalamic nuclei suggest that acoustic social signals,
and perhaps other auditory stimuli, may have a broader impact
on endocrine and physiological regulation. This, however, has
not been investigated.
2.06.5.2 Hearing Calls Elevates Levels of Circulating Steroid
Hormones
Observations across multiple vertebrate species have docu-
mented that engaging in social interactions stimulates the
reproductive system. In frogs, male Rana esculenta maintained
mature testes longer if they were exposed to conspecific calls
compared to control males exposed to nonsocial sound stimuli
or no sound (Brzoska and Obert, 1980), and in female midwife
toads, Alytes muletensis, hearing conspecific calls facilitated
oocyte retention (Lea et al., 2000). There is substantial direct
evidence that hearing conspecific advertisement calls results
in an elevation of gonadal steroids in both males and females.
Hearing conspecific calls assembled into a simulated chorus
over 10 nights increases circulating androgen levels in male
green treefrogs (Burmeister and Wilczynski, 2000) (Figure 9).
Hearing random tones matched in time and amplitude have
no such effect, and in fact result is no difference from remaining
in silence for the duration of the experiment. The same occurs
in southern leopard frogs, Rana sphenocephala (Chu and
Wilczynski, 2001). Interestingly, the effect appears to be a direct
result of hearing, rather than calling or engaging in any other
behavior. Both testosterone and dihydrotestosterone increase
in treefrogs after hearing calls regardless of whether or not the
listener calls back, and males hearing tones have no elevation
in androgens whether or not they themselves call. Gonadal
steroids are also elevated in female túngara frogs exposed to
conspecific male advertisement calls with the same 10-day
exposure (Lynch and Wilczynski, 2006) (Figure 9). In those
females, estrogens, but not testosterone, increased significantly.
In male treefrogs, the elevation in testosterone coincides with
an increase in the number of GnRH-immunoreactive cells in
the preoptic area (Burmeister and Wilczynski, 2005). The
strong connections from auditory nuclei to the preoptic area
and other hypothalamic areas would provide the substrate by
which the constant stimulation by conspecific social signals
can stimulate production of GnRH and ultimately the
increased release of androgens and estrogens downstream of
GnRH release. A similar coordinated elevation of both GnRH
and estrogens, mediated by social interactions, occurs in
newts (Propper and Moore, 1991).
2.06.5.3 Short-Term Exposure to Aggressive or
Advertisement Calls Has Different Effects
Social stimulation of reproductive physiology and endocrine
state occurs under conditions that mimic the state of frogs in
a breeding chorus, where males and females hear a changing
112 Neuroendocrine Control of Social Behavior in Frogs

Figure 9 Increases in plasma testosterone (a) in green treefrog (Hyla cinerea) males and estradiol (b) in túngara frog (Physalaemus pustulosus)
females after 10 consecutive nights of hearing chorus sounds. Similar exposure to tones (and silence in males) has no effect. Male data from
Burmeister, S., Wilczynski, W., 2000. Social signals influence hormones independently of calling behavior in the treefrog (Hyla cinerea). Horm. Behav.
38 (4), 201–209; female data from Lynch, K.S., Wilczynski, W., 2006. Social regulation of plasma estradiol concentration in a female anuran. Horm.
Behav. 50 (1), 101–106.

pattern of conspecific advertisement calls for many hours each testosterone levels than winners. It is difficult to know whether
night for many nights. But within a chorus, behaving individuals losing triggered the fall in testosterone or whether the low
can engage in rapid, short-range interactions as well. Male frogs testosterone was a preexisting condition causing the losing
in many species, for example, occupy and defend call sites from
which they respond with matching advertisement calls to males (a) 30
calling close-by and will escalate their vocalization and occasion-
a
ally engage in physical fights when calling intruders encroach on 25
Corticosterone (ng ml–1)

their site. Fewer researchers have explored the hormonal conse-

quences of these rapid, short-range interactions, but those that 20
have find a decrease, rather than an increase, in androgens.
Experimental simulation of a territorial intrusion in the neotrop- 15
ical treefrog, Hypsiboas faber, by presenting the male with taped
advertisement calls for 10 min caused a rapid decrease in testos- 10 a
terone in the threatened male (de Assis et al., 2012). The same
effect occurred in male green treefrogs after exposure to conspe- 5
cific advertisement calls in a similar simulated vocal intruder
paradigm (Leary, 2014). There are clearly complicated and 0
Pre-broadcast Post-broadcast
context-dependent patterns of behaviorally induced hormone (b)
changes. 22.5
a b
Behavioral triggers of adrenal steroids were unknown until 20
recently, until Leary (2014) explored the effects of aggressive
17.5 b
Testosterone (ng ml–1)

calls in male green treefrogs. Green treefrogs produce a separate

15 a
aggressive call when engaged in escalating, short-range interac-
tions with other calling males. Hearing aggressive calls triggered 12.5
an increase in corticosteroid levels within 45 min of the stim- 10
ulus (Figure 10). The corticosterone increase was accompanied
7.5
by a rapid reduction in androgen levels. Hearing advertisement
calls, on the other hand, did not stimulate adrenal steroid 5
changes in this species or in male H. faber (de Assis et al., 2.5
2012). It is not known if changes of this type occur in females.
0
The consequence of natural aggressive interactions between Pre-broadcast Post-broadcast
males may also lead to a rapid shift in steroid hormone levels.
In many vertebrates, there is a rapid elevation of testosterone Figure 10 Plasma corticosterone (a) and testosterone (b) levels
when a male wins an aggressive interaction; losers of these chal- before and after a 45 min presentation of nearby aggressive calls
(black), advertisement calls (gray), or silent control (white) in male
lenges often see a slower decrease in testosterone. Calling male
green treefrogs (Hyla cinerea). Testosterone decreases after hearing
frogs will act aggressively toward nearby males intruding on
either the advertisement or aggressive call, whereas corticosterone
their calling site; losers of these fights stop calling and adopt levels increase after hearing the aggressive calls; a and b above bars
a silent satellite strategy (reviewed in Gerhardt and Huber, indicates significant differences in pre- and post-treatment hormone
2002; Wells, 2007). Leary and Harris (2013) reported that levels within treatment groups. From Leary, C.J., 2014. Close-range
males that lost short-range aggressive vocal interactions with vocal signals elicit a stress response in male green treefrogs: resolution
other males (that is, fell silent or moved away) had lower of an androgen-based conflict. Anim. Behav. 96, 39–48.

condition; but in either case, the relationship between
hormone level and social status is very interesting.
2.06.5.4 Implications for Social Behavior
Frog social behavior is dependent on adequate levels of
gonadal steroid hormones. Frog social behavior also requires
males and females to listen to a variety of conspecific advertise-
ment calls in varying patterns over a long period of time. This
experience elevates sex steroid hormones, resulting in a positive
feedback system that can help maintain high hormone levels,
behavior, and reproductive capacity throughout a breeding
season and slow the inevitable decline in endocrine state that
leads to the termination of the breeding season (O’Bryant
and Wilczynski, 2010).
For males, the interaction between androgens and calling
helps them coordinate their social breeding behavior. Frog
choruses are structured as lek-like communal displays in which
males advertise and compete for females (for a review of leks,
see Höglund and Alatalo, 1995). As argued by Wilczynski and
Burmeister (2016), reproductive success in a lek depends on
a combination of cooperation and competition. A successful
lek depends on multiple males displaying at the same time
and place, as females are attracted to sites with large numbers
of displaying males. Within the lek, however, males are only
successful if they outcompete other males, that is, display
more vigorously or more often, than neighboring males. A posi-
tive feedback system between hormones and behavior maintains
calling behavior in the presence of other calling males. It also
stimulates, or at least helps maintain, reproductive physiology
when competition, in the form of other calling males, is intense.
For females, the implications are more complicated.
Behavioral-endocrine interactions help coordinate the level
of social behavior with reproductive physiology and egg
maturation and help increase the drive to receptivity when
vigorously calling males are present. On the other hand,
female reproductive stimulation by male calls can be viewed
as a way for males to manipulate female physiology, and
with it their behavior, to make them more receptive and
driven to accept a male. As reviewed in Section 2.06.2.1, as
female estrogens rise, receptivity increases, but choosiness
decreases, meaning that the strength of female preference
declines (Lynch et al., 2006; Chakraborty and Burmeister,
2009). A male under intense male–male competition may
benefit from driving a female toward amplexus both behav-
iorally and physiologically.
The short-term endocrine effects of hearing advertisement
and aggressive calls are quite different from the effects of
chronic exposure to a chorus. The elevation of corticosterone
and the decrease in androgens may be part of a behavioral
system in which male agonistic interactions lead to behav-
ioral state differences within the chorus, with males adopting
different strategies. Frogs with extended breeding seasons
and large choruses often have males with different behav-
ioral strategies (Wells, 2007). Some call persistently from
defended sites, some adopt satellite behavior, in which
they stay near calling males, remain silent, and attempt to
intercept females moving toward the calling male, and
some move away from callers to find another call site from
which to broadcast their vocal signals. Behaviorally adjusted
Neuroendocrine Control of Social Behavior in Frogs 113
steroid levels may play some part in these shifts (Leary et al.,
2006; Leary and Harris, 2013).
2.06.6 Summary and Conclusions
The effect of hormones on frog social behavior aligns with
effects seen in seasonal vertebrates generally. Amphibian social
behavior offers a way to examine this in the context of verte-
brates heavily dependent on acoustic communication, and
with a strong sex difference in the way males and females
produce and respond to those signals. With that in mind,
work on the behavioral neuroendocrinology of anuran social
behavior can be considered in light of the three aspects of their
social behavior noted in Section 2.06.1.
Reproduction is seasonal, and under that pattern, gonadal
steroids vary seasonally and with them so does reproductive
social behavior in both males and females. Both correlative
data from naturally behaving frogs and experimental work
manipulating hormones provide compelling evidence that
androgens in males and estrogens in females drive the expres-
sion of male calling and female responses to the call, respec-
tively. More recent work being done on adrenal steroids lays
the foundation for interesting research to untangle the role
these hormones play in naturally occurring social behavior.
This includes the question of whether baseline corticosterone
levels change seasonally in response to external seasonal cues
in the same way gonadal steroids are regulated or whether the
elevations seen when animals are in their social reproductive
state are a reaction to the specific social, energetic, or other
conditions encountered by the individual at that time. The
way in which melatonin influences social behavior is also ripe
for further studies, as are the potential effects of other mono-
amine and peptide hormones. Of particular interest is the ques-
tion of what endocrine or neuromodulatory agents regulate
shifts in behavioral state from reproduction to foraging or other
behavioral modes, an important topic in the field of environ-
mental physiology (Lutterschmidt and Maine, 2014), but
which is unexplored in amphibians except for work testing
the Energetics-Hormone-Vocalization hypothesis.
Acoustic communication is the foundation for anuran social
behavior, and we can see from the work on frogs that endocrine
modulation influences both the sensory and the motor compo-
nents of this communication system to change the way frogs
hear and respond to conspecific vocal signals when they are
engaged in seasonal reproductive social behavior. The social
behavior and communication system of frogs offers an opportu-
nity to explore this phenomenon further, and in particular the
mechanisms behind the changes in both components of a social
communication, and how the input and output changes interact
through the integrative networks of the brain. The work on audi-
tory changes highlights a growing appreciation that social behav-
ior’s input side is as plastic as its output side and that steroid
hormones have a particularly strong influence on auditory func-
tions from the ear to the forebrain (Bass et al., 2016).
Acoustic communication is a sexually differentiated behavior in
frogs, from the large sex difference in behavior to more subtle
differences in the response properties of the ear and higher
areas of the auditory system. With this comes the opportunity
to examine the morphological, physiological, neural, and
114 Neuroendocrine Control of Social Behavior in Frogs
behavioral sex differences in specific components of a social
system in a truly integrative way. Work on the vocal production
component of frog social behavior is an exceptional example of
this (Kelley, Chapter 2.08, Hormones and Vocal Systems:
Insights from Xenopus) and a model for pursuing research on
the sensory side of this social system.
Work on the behavioral endocrinology and neuroethology of
amphibian behavior has made significant contributions to
understanding general principles of vertebrate social behavior
and acoustic communication. Amphibians continue to be valu-
able model organisms for understanding how the sensory,
integrative, and motor components of social behavior interact
and are coordinated by a variety of hormones.
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 2.07 The Vertebrate Brain Stem as the Primary Site of Behavior Command:
Insights from an Amphibian
Emma Coddington, Willamette University, Salem, OR, USA
Ó 2017 Elsevier Inc. All rights reserved.

2.07.1 Scope of This Chapter 117

2.07.2 Broadening the Theoretical Framework Underlying Our Understanding of Sex Behaviors 117
2.07.2.1 A Broader Framework for the Evolutionary Significance of Sex Behaviors 117
2.07.2.2 A Modular Approach to Understanding Sex Behaviors 118
2.07.3 Current Understanding of Motor Control of Vertebrate Behaviors and Taricha Clasping 119
2.07.3.1 Taricha Clasp Behavior 119
2.07.3.2 Motor Control of Vertebrate Behaviors Including Clasp Behavior 119
2.07.3.3 Salamanders Provide an Ideal Model to Examine Brainstem Command of Behavior 122
2.07.4 Neuromodulation of Clasping 123
2.07.4.1 VT Enhances Social Behaviors, Including Clasping 123
2.07.4.2 Acute Stress Rapidly Suppresses Sex Behavior Through CORT Actions 124
2.07.4.3 How Does CORT Rapidly Modify Behaviors? 124
2.07.4.3.1 Context-Dependent Interactions of CORT and VT 125
2.07.4.3.2 CORT Modifies VT V1a Receptor Availability in mRF Neurons 125
2.07.4.4 Identity of Membrane CORT Receptors 126
2.07.5 Outlook 126
Acknowledgments 126
References 127

2.07.1 Scope of This Chapter 2.07.2 Broadening the Theoretical Framework

This chapter begins by asking us to broaden the scope of how
we define and discuss sex behaviors in order to identify previ-
ously unconsidered questions, allowing us to grow our under-
standing of the evolutionary and proximal mechanisms that
mediate sex behaviors. The current understanding of motor
control of sex behaviors in vertebrates is summarized and dis-
cussed in the context of neural control of clasping in the newt,
Taricha granulosa, a urodele amphibian. This chapter is centered
on the key role of the rostromedial medullary reticular forma-
tion (mRF) in the vertebrate brainstem and its principal output
cells, the reticulospinal neurons. The concept of the brainstem
as an essential region in motor control of behavior is extended
by current data that show how neuromodulation and
metamodulation provide for situationally relevant expression
of clasping. There are at least two key hormones, corticosterone
(CORT) and vasotocin (VT), that act on mRF neurons to
modulate context-dependent expression of sex behavior in
newts (clasping). Studies in Taricha examining the nature of
the interaction between these two hormones expose two
main findings: (1) endocannabinoid signaling is critical for
CORT effects on sensorimotor processing leading to
clasping and (2) CORT suppresses V1a receptor availability
in a novel mechanism of metamodulation. Given the conser-
vation of motor circuits and neuromodulatory controls of
behavior, we can derive and apply fundamental principles
learned from the study of Taricha clasping to a broader under-
standing of how basic behaviors of vertebrates are controlled
by brainstem neural circuits and modulated to allow for
context-appropriate expression.
Underlying Our Understanding of Sex Behaviors
2.07.2.1 A Broader Framework for the Evolutionary
Significance of Sex Behaviors
Sex behaviors are often referred to synonymously with repro-
ductive behaviors, motivated toward gamete exchange. If we
define behavior as the musculoskeletal movements that animals
make, then sex/reproductive behaviors are the suite of coordi-
nated musculoskeletal movements made toward locating,
attracting, competing, choosing, and persuading a conspecific(s)
to join them in sexual interactions. Classically, sex behaviors
have been defined and limited to the act of gamete exchange,
with a large amount of effort expended toward understanding
penile erection and ejaculation, resource competition, and
mate selection, in concert with understanding female receptivity
to male attentions. The significance of these studies requires us
to agree that the only and primary drive motivating sex behav-
iors is to exchange gametes. We could, however, consider that
the suite of behaviors involved in sexual interactions may also
serve an arguably even more fundamental animal drive, the
drive for affiliation and social connection through touch.
From this perspective, our working definition of sex behavior
can encompass same-sex, heterosexual, and affiliative behaviors
that can be, but not necessarily always, used toward sexual inter-
actions (Zuk, 2006). This directly applies to how we are begin-
ning to view Taricha clasping behavior, and how we could view
sex behaviors across all vertebrates.
I am compelled to adopt this broadened definition of sex
behaviors because it allows us to ask previously unconsidered
questions and therefore grow our understanding of the

Hormones, Brain, and Behavior, 3rd edition, Volume 2 http://dx.doi.org/10.1016/B978-0-12-803592-4.00025-0 117

118 The Vertebrate Brain Stem as the Primary Site of Behavior Command: Insights from an Amphibian
evolutionary and proximal mechanisms mediating behaviors
in general. If we view sex behaviors on a continuum from
completely solitary to paired sexual interactions, then several
inferences can be made. The suite of sexual behaviors
possible, and used in one species, will vary according to
evolutionary constraints – defining the neural, hormonal,
and musculoskeletal architecture and therefore specific behav-
iors that any one individual has to work with. The notion of
social selection has been offered to better explain the evolu-
tion of diverse sexual strategies and behaviors observed in
the animal kingdom (Roughgarden et al., 2006; Milam et
al., 2011; Roughgarden, 2012). The manifestation of specific
behaviors at a given time point in any one individual will
vary according to ontogenetic, social, environmental, and
immediate physiological influences that are coded by a flex-
ible combination of neural and endocrine systems.
How we define the suite of sexual behaviors involved, and
the selective forces acting on them, will shift how we view the
behaviors and underlying neural control and will therefore
shift how we ask questions and reveal any emergent under-
standing of fundamental principles. The species-specific and
sex-specific manifestation of sex behaviors is typically viewed
as different behaviors, yet there are modules of behaviors and
neural circuits that are common across species and sexes
(Dominici et al., 2011). For example, female and male newts
both employ clasping behavior, where the hind- and fore-
limbs are bilaterally and rhythmically engaged to grasp
something between their limbs. Females use this behavior
to lay eggs on the underside of sticks and reeds in ponds;
males use this same behavior to grasp and persuade the
female to ultimately exchange gametes, but males and
females also use clasping behavior with conspecifics of any
sex when exuberantly responding to food placement in their
tanks.
The modular nature of the motor control circuits in combi-
nation with the ability of neuromodulators to shift the
command allows for the exhibition of apparently ‘different’
behaviors. If we begin to realize the common elements
composing these behaviors we can begin to examine the under-
lying fundamental principles of neural control and hormone
action. The behavior itself becomes goal-directed, sex- and
species-specific, when engaged within a specific window of
time – during specific life stages, seasons, and circadian time
frames.
2.07.2.2 A Modular Approach to Understanding Sex
Behaviors
In order to derive fundamental principles of neuronal and
hormonal regulation of behaviors, it is also useful to redefine
how we (de)construct, and therefore measure, behaviors in
general and sex behaviors specifically. Interestingly, a recent
survey revealed that behavioral biologists do not yet agree
on the definition of behavior (Levitis et al., 2009). We
suggest that this is largely because behaviors are often
defined based on their functional endpoint: sexual interac-
tions that result in gamete exchange, territorial defense,
foraging, parental or affiliative behaviors. Yet these behaviors
listed above are more accurately termed ‘behavioral suites’
and consist of a concert of many discrete modules of
behaviors, the smallest functional unit of behaviors akin to
genes, being organized in a predictable order toward an
easily identifiable goal. Behavioral biologists construct etho-
grams to capture the predictable and precise sequence of
behavioral modules composing a behavioral suite, such as
sex or feeding behavior. Some modules are common across
animals within entire taxa, some are species-specific, some
can be skipped or repeated an individual’s ethogram, and
some can occur across behavioral suites of the same species
across sexes. Applying the comparative approach to this
view of behaviors would allow us to examine a module
common across species, across sexes within a species, or
within a sex across different ontogenetic stages or situational
contexts, revealing fundamental principles of neural function
and hormone action. This modular approach to under-
standing behaviors has been employed in robotics as
a bottom-up basis of designing behaviorally adaptive robots
(Lenser et al., 2001) and has been suggested as a useful
framework to understand the neurobiological basis of sex
differences in behavior (Yang and Shah, 2014). If behavioral
biologists view discrete behaviors as modules, then we can
begin to realize that courtship behavior is distinct from affili-
ative or feeding behaviors, not due to the specific modules
per se, but due to the order of modules employed with the
addition or elimination of specific modules. Examining the
neural and endocrine regulation of Taricha clasping behavior
as a behavioral module offers the opportunity to apply this
modified comparative method to reveal previously unappre-
ciated proximal mechanisms.
Understanding the fundamental principles of how the
neural and endocrine systems work in concert to facilitate
reflexive or goal-directed behaviors is not trivial. The nervous
and endocrine systems are both dynamic; they influence
each other in highly flexible ways and function across very
different spatial and temporal scales. Historically, researchers
have approached questions in this field by focusing on either
the neural or the endocrine control of behaviors. Few models
bring the two fields together. The most comprehensively
examined models exploit the relative simplicity of inverte-
brates such as the crayfish stomatogastric system controlling
food digestion behaviors, nudibranch escape response, and
leech locomotion. Favored vertebrate models include bird-
song with an emphasis on cortical circuits, rodent lordosis
behavior with an emphasis on the hypothalamus, and
teleost and Xenopus vocalization circuits that include the
brainstem.
We are examining questions about the role of the brain-
stem in a urodele amphibian, rough-skinned newts (T. gran-
ulosa). Our overarching working hypothesis asserts that (1)
clasping behavior can be considered a modified version of
locomotion; (2) a given hormone will modulate behavior
in a species-, sex-, and context-specific manner, enabling
a different order and frequency of modules employed to
manifest a specific goal-directed behavioral suite; (3) a given
hormone will activate different behavior suites in the same
organism because it uses a cascade of different cellular
mechanisms acting on different timescales from milliseconds
to seasons; and (4) metamodulation of hormone actions
is essential for the expression of context-appropriate
behaviors.
 The Vertebrate Brain Stem as the Primary Site of Behavior Command: Insights from an Amphibian
2.07.3 Current Understanding of Motor Control
of Vertebrate Behaviors and Taricha Clasping
2.07.3.1 Taricha Clasp Behavior
Taricha is a urodele amphibian that exhibits a robust sex
behavior, amplectic clasping, which is modulated by experi-
ence and exposure to hormones (Moore and Rose, 2002;
Moore et al., 2005). The clasp itself is one element of a complex
sequence of behavioral modules underlying a number of
behaviors in Taricha including reproductive behaviors. Court-
ship clasping by males entails a sequence of events that are
guided by various sensory modalities (vision, olfaction,
somatic sensation) (Propper, 1991). The suite of courtship
behaviors include a coordinated sequence of behavioral
modules in which the male pursues and clasps the female
and then rubs the female’s snout with his chin. The clasp is
stereotyped: an unstressed mature male grasps the back of
a sexually attractive ovulating female and synchronously
tightens his hind legs around the female’s dorsally presented
torso (Propper, 1991; Moore and Rose, 2002). This response
is robustly triggered and maintained by tactile somatosensory
pressure applied to the cloaca, located between the hind limbs.
The clasp as a modified version of locomotion
The clasp behavior can be considered a fundamental behavioral
module that engages the same limbs as other forms of locomotion,
recruiting the same muscles, motoneurons, spinal central pattern
generators (CPGs), and rostromedial medullary reticular formation
(mRF), but in a behavior-specific order and rhythmicity. Viewing
the clasp from this perspective informs how we consider all vertebrate
behaviors that involve limb movements and torso postures. The basic
neural circuitry driving the behavior involves the same brainstem and
spinal cord regions and the same neuromuscular units. The mRF is
one of the primary sites where vertebrates manifest their urgent
behavior decisions to fuck/fight/flight/or freeze, and the decision is
made based on modulation and metamodulation of the local
circuitry. The reticulospinal neurons provide the primary output
from the mRF, dictating the order of CPG recruitment and rate of
rhythmicity. The most rostral reticular region, the mesencephalic
locomotor region (MLR), provides drive to the mRF, because the
MLR receives and integrates most of the inputs coming from cortical
and subcortical brain regions. These supra-brainstem regions provide
important goal-directed salience and greater flexibility to the basic
suite of behaviors encoded by the brainstem and spinal cord.
2.07.3.2 Motor Control of Vertebrate Behaviors Including
Clasp Behavior
All behaviors, including clasping, are enacted by the coordinated
engagement of muscles on each limb, torso, head, neck, and tail
required to enact the behavior. Drawing on the collective knowl-
edge of locomotor circuitry from studies of lamprey (Grillner,
2006; Grillner et al., 2008; Kozlov et al., 2014), amphibians
(Rose, 1990a,b; Rose et al., 1995; Chevallier et al., 2008; Ryczko
et al., 2010; Ryczko and Dubuc, 2013; Sánchez-Camacho et al.,
2001; Ten Donkelaar, 1990), fish (Goodson and Bass, 2002;
Bass et al., 2008; Korn and Faber, 1996, 2005; Fetcho, 1992;
Finger, 2009; Eaton et al., 2001), and mammal locomotion
(Grillner, 2011; Humphries et al., 2007; Jordan et al., 2008;
Kimura et al., 2013; McCrea and Rybak, 2008; Tresch, 2010;
Whelan, 1996; Siegel, 1979) in combination with our current
understanding of behavioral neuroendocrine control of sex
119
behaviors (Beach, 1967; Kelley and Pfaff, 1976; Moralí et al.,
2003; Crews and Moore, 2005; Adkins-Regan, 1990, 2009;
Carter, 1998; Pfaus et al., 2001; Ball and Balthazart, 2004; Moore
and Rose, 2002; Coddington and Moore, 2003; Hull and Dom-
inguez, 2007; Lutterschmidt and Maine, 2014), we can under-
stand that sexual interactions are composed of a suite of
behaviors that are initiated in a seasonally appropriate manner,
modulated to be situationally appropriate, and maintained by
interconnected microcircuits of neurons in the brainstem and
spinal cord. Seasonally, circadian, and physiologically appro-
priate motivation (goal-directed information) is provided by
inputs from basal ganglia, striatum, cerebellum, hypothalamus,
thalamus, and cortex to the mRF of the brainstem (Grillner,
2006, 2011). The reticulospinal neurons in the mRF provide
the primary output commands to initiate and maintain specific
behaviors in all vertebrates (Grillner, 2011; Humphries et al.,
2007). In salamanders specifically, descending reticulospinal
neurons influence the onset, offset, and duration of the clasp
motor reflex (Naujoks Manteuffel and Manteuffel, 1988; Rose
et al., 1995). The mRF region is subject to multisensory and
hormonal neuromodulation and metamodulation (Bowsher
et al., 1968; Rose et al., 1993, 1995; Martin et al., 2010, 2011).
Neuromodulation effectively broadens the operational range
of neuronal circuits allowing for flexible modification of basic
behaviors (Katz, 1999; Mesce, 2002; Marder and Prinz, 2003;
Marder et al., 2014), while metamodulation is the second-
order modulation of neuromodulator actions (Katz, 1999;
Mesce, 2002). Collectively, these provide modulatory input
that allows for context-appropriate behaviors.
The brainstem and spinal cord circuitry in communication
with the musculoskeletal system are necessary and sufficient
to produce situationally specific behaviors in all vertebrates
(Figure 1). Original studies in cats and dogs by Sherrington
(1910) and nonhuman primates by Lawrence and Kuypers
(1968a,b) revealed the essential and sufficient role of the
medulla oblongata in the motor control of the trunk and prox-
imal and distal limb segments. Decerebrate cats and dogs
maintain the ability to walk, run, jump, and climb. However,
the speed of movements was far from normal. These coordi-
nated behaviors are entirely achievable by an intact brainstem
and spinal cord, with no input from rostral parts of the central
nervous system (CNS). When decerebration leaves the hypo-
thalamus intact with brainstem, these decerebrate animals
can also homeostatically regulate functions such as body
temperature and hunger (Lawrence and Kuypers, 1968b).
Since these early studies, however, most investigations inter-
ested in understanding motor control of behavior shifted their
focus to supra-brainstem structures underpinned by the arguably
false assumption that ‘higher vertebrates’ are dominated by
‘higher brain function.’ This has led to tunnel vision and the
brainstem being hidden in a collective blind spot. Only recently
has the focus broadened again – heavily influenced and inspired
by the discoveries in lamprey of a brainstem command circuit
for locomotor behaviors (Grillner et al., 2008; Grillner, 2011)
and in teleosts, a brainstem Mauthner neuron command for
the C-start escape response (Korn and Faber, 1996; 2005;
Eaton et al., 2001). For example, an intriguing hypothesis has
been proposed that reticulospinal neurons within the nucleus
reticularis gigantocellularis (NGC, also referred to as the mRF)
might be evolutionary analogs of Mauthner neurons, and the
120 The Vertebrate Brain Stem as the Primary Site of Behavior Command: Insights from an Amphibian

Figure 1 Representation of the vertebrate nervous system involved in sensorimotor selection, generation, and neuromodulation of behaviors.
Green boxes indicate regions that are essential for the generation of basic motor programs and a broad selection of postures and limb move-
ments, such as walking, clasping/hugging, or swimming. These brain regions also provide information regarding the onset and offset of the
behavior, maintenance, and intensity. mRF, rostromedial medullary reticular formation located in the rostral aspect of the medulla oblongata;
MLR, mesencephalic locomotor region located at the boundary between the brainstem and midbrain; and CPG, central pattern generator, a distrib-
uted network of neurons that are flexibly connected and together coordinate a set of muscles toward an endpoint, such as one limb movement.
Gray symbols indicate sensory input for initiation and feedback for ongoing modulation of behavior; from top down: olfaction, vision, hearing and
lateral line, taste, touch, and proprioception. Tan boxes indicate rostral brain regions that provide modulatory neuronal input to the brainstem
motor circuits. The removal of inhibitory input from the basal ganglia allows motor programs to proceed. There is anatomical evidence that the
cortex and vestibular systems also have some direct connections to the spinal cord as well. Orange ovals indicate brain regions known to provide
neuroendocrine modulatory input to the mRF, providing broader context to the motor programs including intrinsic and extrinsic information such
as physiological state and seasonal information. The final motor output to muscles is provided by the spinal motoneurons located within spinal
CPGs innervating muscle fibers.

NGC/mRF contributes to decision-making by functioning as
a primary site of general arousal (Machne et al., 1955; Pfaff
et al., 2012). This hypothesis is supported by the findings that
the brainstem and neurons within the mRF respond to and
provide emotional context (Coimbra et al., 2006; Vertes and
Miller, 1976).
The mRF is highly conserved across taxa (Grillner, 2011;
Jordan et al., 2008; Matsuyama et al., 2004a) and can be
viewed as the major sensorimotor integration region in all
vertebrates mediating socially and situationally appropriate
behaviors. Situationally relevant information to the mRF is
received from three key neuromodulatory inputs: (1) multi-
modal sensory information and feedback (Pfaff et al.,
2012; Rose et al., 1995; Martin et al., 2010; Azim et al.,
2014); (2) general physiology and seasonal information
from the medial hypothalamus, periaqueductal gray (PAG),
and lateral hypothalamus (Jordan, 1998; Jordan et al.,
2008); and (3) acute hormone changes in response to the
immediate situation (Coddington et al., 2007; Rose et al.,
1995, 1998; Rose and Moore, 2002). Sensory inputs arriving
 The Vertebrate Brain Stem as the Primary Site of Behavior Command: Insights from an Amphibian
at the mRF originate from a broad range of sources
(Figure 1) and provide necessary and sufficient sensory
input to affect immediate context-appropriate behavioral
commands. These include tactile/somatosensory (Rose
et al., 1995; Rose and Sutin, 1973; Saadé et al., 1983), olfac-
tory (Bowsher et al., 1968; Thompson et al., 2008), visual
(Martin et al., 2010), auditory (Bowsher et al., 1968; Martin
et al., 2010), lateral line (Bodznick and Northcutt, 1980),
vestibular (Ladpli and Brodal, 1968; Peterson and Abzug,
1975), thermal (Farham and Douglas, 1985), nociceptive
(Fort et al., 1994; Willis, 1985), visceral (Brown et al.,
2002; Curtis et al., 2002; Kaddumi and Hubscher, 2006),
baroreceptive (Guilbaud et al., 1973; Haxhiu and Loewy,
1996), and chemosensory stimuli (Andreatta-Van Leyen
et al., 1990; Guyenet et al., 2005; Silver and Finger, 2009).
This sensory information is integrated at the mRF with
socially relevant information (Shelley et al., 2006), energy
balance (Grill and Hayes, 2012), and the need for general
vigilance and anxiety as conveyed by corticotropin-releasing
hormone (Lowry et al., 1996; Hubbard et al., 2010). Collec-
tively, the neuromodulatory input provides the situational
information required to the mRF.
The integrated context-appropriate motor commands are
sent to the spinal cord by the projection neurons of the
mRF, the reticulospinal neurons. Also highly conserved across
taxa (Murakami et al., 2004; Fetcho, 1992), reticulospinal
neurons are sometimes referred to as second-order premotor
neurons. However, this terminology belies the enormously
integrative functions they provide. These neurons are related
to Mauthner neurons, the enormous neurons located in the
mRF that are responsible for the startle response in fish,
tadpoles, and salamanders (Eaton et al., 2001; Korn and
Faber, 2005; Oda, 2015; Sillar, 2009; Zottoli and Faber,
2000). Smaller and more numerous than Mauthner neurons,
reticulospinal neurons typically range from 25 to 50 mm in
length and project in two directions: rostrally to supra-
brainstem sites and caudally to the spinal cord (Newman
et al., 1983; Roberts and Alford, 1986; Sakai et al., 2009).
Their descending output to the spinal cord provides the key
information to select behaviors by modulating the order and
frequency of motor rhythm produced by the spinal central
pattern generators (CPGs) (Grillner, 2006; Grillner and Wal-
len, 1985; Grillner et al., 2008; Matsuyama et al., 2004b;
McCrea and Rybak, 2008).
CPGs are evolutionarily conserved across the animal
kingdom; a fundamental functional unit for the motor control
of invertebrates and vertebrates (Marder and Bucher, 2001).
They are a functionally connected and spatially distributed
group of neurons that coordinate a subset of movements. In
vertebrates, CPGs in the spinal cord coordinate limb move-
ments, while those in the hindbrain coordinate movements
by the torso, head, and neck (Grillner, 2006). The coordinated
rhythmic activity of neurons in CPGs underlies the rhythmic
patterns of behaviors. A key feature of CPG function is that
they are essentially modular and can function in isolation
and be flexibly connected with each other to varying degrees
of strength. This flexibility allows for variability in the order
of activation (left, right), regulates the intensity of the move-
ment, and therefore the behavior is enacted (Buchanan,
2011; Grillner, 2006).
121
Reticulospinal neurons are responsible for modifying the
strength of connections between CPGs and the timing of ipsi-
lateral- and contralateral-CPG communication (Grillner,
2006). Therefore, the mRF provides key information to the
CPGs that results in specific behavior of limbs and posture.
For example, a walk gait would involve asymmetric limb
flexion–extension patterns in a distinctly different order
compared with a gallop (Grillner, 2006). In Taricha, reticulo-
spinal neurons respond to multimodal sensory and hormone
information and are modulated by hormone input to produce
the key output that commands walking, swimming, or clasping
(Moore and Rose, 2002; Rose et al., 1993, 1995). Clasping
behavior involves strong, rhythmic, bilateral flexion of fore-
and hind limbs and therefore requires recruitment of the
same limb CPGs that control each limb in a pattern and order
distinctly different from that used for walking or swimming. To
understand the relationship between reticulospinal neurons,
CPGs, and the subsequent behavior, we suggest that clasping
is a modified form of locomotion that employs reticulospinal
neurons and limb and postural CPGs in a species-specific
manner to produce symmetrical bilateral flexion of paired
limbs.
There are inputs to the mRF from more rostral brainstem
regions that provide additional nuance and salience to any
motor program. The mRF output is directly influenced by
the mesencephalic locomotor region (MLR) (Figure 1).
This highly conserved structure located at the brainstem–
midbrain boundary contributes to locomotion in all verte-
brates investigated to date (Bachmann et al., 2013; Cabel-
guen et al., 2003; Garcia-Rill et al., 1987; Garcia-Rill and
Skinner, 1987; Shik et al., 1967; Sirota et al., 2000). Early
studies performed in cats revealed that unilateral or bilateral
stimulation of neurons in MLR elicit switching between
walking, trotting, and galloping in high-decerebrate cats
(Shik et al., 1967). The MLR mediates its effects through
monosynaptic projections to the mRF where a majority of
the mRF neurons are activated in synchrony with MLR output
(67%) and behavioral pattern (Orlovskiĭ, 1970a,b). This
highly reliable circuit is conserved from teleosts to primates
(Grillner, 2006; Ryczko et al., 2016). Voluntary selection
(goal-directed selection) of motor output is provided by
output from the basal ganglia to the MLR and is also
conserved across all vertebrates (Cabelguen et al., 2010; Grill-
ner, 2006, 2011; Ryczko et al., 2016).
Studies of decerebrate mammals have demonstrated that
supra-brainstem regions are not necessary for the full
suite of limb and torso motor commands; nevertheless,
supra-brainstem input is important for the greatly nuanced
versions of the basic motor programs provided by the
brainstem þ spinal cord. More recent studies in rodents
confirm that spinal interneurons receive very weak input
from corticospinal neurons, but are powerfully innervated
on both the cell bodies and dendrites by reticulospinal
neurons (Mitchell et al., 2016). This is not to say that supra-
brainstem inputs are unimportant, but that their roles are
key for appropriate expression of skilled goal-directed behav-
iors. Brainstem motor networks receive direct descending
dopaminergic inputs that potentiate the locomotor output
(Ryczko et al., 2016), and recurrent information from spinal
cord interneurons to mRF and cortex, in parallel with spinal
122 The Vertebrate Brain Stem as the Primary Site of Behavior Command: Insights from an Amphibian

cord to cerebellum, play a crucial role in skilled hand þ arm
reaching (Azim et al., 2014).
2.07.3.3 Salamanders Provide an Ideal Model to Examine
Brainstem Command of Behavior
Taricha granulosa are ideal model organisms for neuroethology
studies because (1) the males have robust, ethologically rele-
vant behaviors that are measurably modulated by hormones
(Moore et al., 2005; Moore and Zoeller, 1985; Rose and Moore,
2002); (2) the neural system regulating those behaviors is iden-
tified and amenable to interrogation because the neurons
controlling the clasp behavior are large (20–100 mm) and
highly accessible for functional imaging and electrophysiology
(Rose et al., 1993); (3) the molecular and pharmacological
identity of many receptors and signaling molecules has been
identified, including the cannabinoid type 1 receptor (CB1)
(Hollis et al., 2006; Soderstrom, 2009; Soderstrom et al.,
2000) and V1a receptors (Searcy et al., 2011); and (4) these
animals are abundant in the Pacific Northwest of the
USA and offer a rare opportunity to observe organisms’
behavior in nature.
Salamanders, in general, provide an ideal model to examine
brainstem command of behavior and therefore the role of mRF
and MLR in selecting and mediating locomotion and clasping
behaviors for several reasons. Primarily, the control of motor
output is highly conserved; therefore general principles about
neural control of tetrapod behaviors can be derived from inves-
tigations in salamanders (Grillner, 2006; Ryczko et al., 2016;
Sánchez-Camacho et al., 2001). Secondly, there are some
taxon-specific anatomical arrangements that render the sala-
mander an ideal model to examine mRF: the cells of the mRF
are presented on the floor of the fourth ventricle unobscured
by the cerebellum and available for in vivo single-unit and
whole-cell electrophysiological examination, and there is
a wealth of knowledge in this species regarding hormonal
modulation of clasping and locomotion to rely on. To date,
what is known about the mRF and its functional role in sala-
mander clasp and locomotor behavior modules comes primarily
from in vivo single-unit electrophysiology studies (Coddington
et al., 2007; Hubbard et al., 2010; Lewis et al., 2005; Lewis
and Rose, 2003; Lowry et al., 1996; Rose, 2000; Rose et al.,
1995, 1998, 1993; Ryczko et al., 2016; Smetana et al., 2010).
The mRF in the hindbrain initiates and maintains clasping
(Moore and Rose, 2002; Rose et al., 1993, 1995: Figure 2).
In vivo single-unit recordings from freely behaving animals
have revealed that the neurons in the mRF respond to clasp-
inducing somatosensory stimulation to the cloaca, and the

Figure 2 Neuroethology model of Taricha clasping behavior. (a) Schematic of neural control of clasping. (b) Transverse section of a male Taricha
brainstem at the level of the mRF region, located in the rostromedial aspect of the medulla oblongata. Abbreviations indicate specific regions – mRF,
rostromedial medullary reticular formation; ra, raphe nucleus; rp, parvocellular reticular formation; VIIIvn, eighth cranial nerve. Box indicates location
of image in c. Scale bar ¼ 100 mm. (c) Location of Taricha CB1 receptors in the hindbrain mRF (in situ hybridization), scale bar ¼ 20 mm. (d) Repre-
sentative traces of single-unit recordings from mRF neurons of awake and behaving Taricha illustrating how CORT diminishes spontaneous and
sensory stimulated responses. Gray bars below spike traces indicate the timing (onset to offset) of pressure applied to the cloaca designed to simu-
late cloacal stimulation, a necessary and sufficient somatosensory stimulus to elicit clasp behavior. The lower trace indicates time in 1 s divisions.
Reprinted with permission from Coddington, E., Lewis, C., Rose, J.D., Moore, F.L., 2007. Endocannabinoids mediate the effects of acute stress and
corticosterone on sex behavior. Endocrinology 148, 493–500.
 The Vertebrate Brain Stem as the Primary Site of Behavior Command: Insights from an Amphibian
reticulospinal neurons provide the output that is coincident
with motor control of the clasp. By focusing on the
somatosensory-evoked clasp, the neurobehavioral model
system is refined to a single, readily controlled sensory stimulus
and simple, readily elicited, and quantifiable neural and behav-
ioral responses.
2.07.4 Neuromodulation of Clasping
The manifestation of courtship behaviors by males and females
is seasonally and ontogenetically constrained and modulated
by hormones. The specific roles of a variety of hormones and
transmitters in activating courtship behaviors have been well
characterized in Taricha: sex-specific gonadal steroid actions,
rapid actions of CORT and its membrane receptor, sex-
specific and seasonal variation in VT, and dopamine have
been reviewed in earlier publications (Moore et al., 2005;
Moore and Rose, 2002).
The incidence and maintenance of clasping is enhanced by
VT (Moore and Miller, 1984; Rose et al., 1995) and suppressed
by exposure to acute stress, an effect mediated by CORT
(Coddington et al., 2007; Coddington and Moore, 2003;
Moore and Zoeller, 1985; Rose and Moore, 1999). Our more
recent work has established that suppression of clasping by
acute stress and CORT will not occur if CB1 receptors are
blocked (Coddington et al., 2007). These receptors are located
in the mRF (Hollis et al., 2006), and their pharmacology has
been well characterized in Taricha (Soderstrom et al., 2000).
Importantly, VT and CORT both provide information about
situational context to the brain, and their effects are themselves
context-dependent (Coddington and Moore, 2003): if an
animal is exposed to CORT or acute stress first, VT does not
enhance clasping.
Several lines of evidence have revealed that VT and CORT
interact in a time-dependent manner to provide different
context-appropriate behavioral outcomes, and at the core of
this switch is a third signaling molecule, eCB (Coddington
et al., 2007; Coddington and Moore, 2003). Endocannabinoids
are best known for their unique role in suppressing presynaptic
neurotransmitter release to postsynaptic neurons (Heifets and
Castillo, 2009; Kreitzer and Regehr, 2001; Wilson and Nicoll,
2001). They are also well known to play a critical role in medi-
ating vertebrate responses to stress and anxiety (Coddington
et al., 2007; Hill, 2012; McEwen and Gianaros, 2011; Riebe
and Wotjak, 2011; Taber and Hurley, 2009). By focusing on
the peptide VT and the steroid hormone, this section examines
the interactions between these two hormones, which promote
switching between very different behavioral outcomes: VT
promotes prosocial behaviors, including promoting courtship
behaviors, while CORT promotes stress-avoidance and stress-
coping behaviors in all vertebrates, including blocking court-
ship behaviors.
2.07.4.1 VT Enhances Social Behaviors, Including Clasping
VT is a nonmammalian homologue of vasopressin (VP); VT/VP
are nonapeptide hormones that have conserved functions
influencing peripheral physiology via release from the
neurohypophysis and behaviors via release from neurons
123
broadly distributed within the CNS. Both VT and VP affect
a variety of social behaviors in vertebrates (Goodson and
Bass, 2001): mammals (Young et al., 2001), avians (Panzica
et al., 2001), teleosts (Bass and Grober, 2001), and amphibians
(Deviche and Moore, 1987; Emerson and Boyd, 1999; Iwata
et al., 2000; Moore et al., 1992).
In Taricha, VT acts as a neuromodulator to regulate male
clasping behavior. The action of VT at the mRF is proposed
to occur by influencing sensorimotor integration of socially
relevant sensory information by mRF neurons. This principle
is based on multiple lines of evidence. Administration of VT
increases the incidence of male clasping behaviors and VT
antagonists suppress these behaviors (Moore and Miller,
1984; Moore and Zoeller, 1979). Reticulospinal and interneu-
rons within the mRF respond robustly to intracranial adminis-
tration of VT with an increase in spontaneous activity and
sensory responsiveness to clasp-triggering cloacal stimulation
(Moore and Rose, 2002; Rose et al., 1995). Interestingly, VT’s
influence is not limited to sexually specific stimuli. The behav-
ioral responses of males to visual and olfactory stimuli origi-
nating from food or sexual partners are markedly enhanced
by VT administration (Thompson and Moore, 2000). Further-
more, the current notion is that social experience results in
elevated VT at the mRF. Female Taricha sex pheromones
promote courtship clasping in males (Thompson and Moore,
2000) and do so by amplifying the sensory responsiveness of
mRF neurons to clasp-triggering somatosensory stimulation
of the cloaca (Thompson et al., 2008). This neuromodulation
of olfactory signals is dependent on endogenously released
VT, because V1a antagonist blocks pheromone-induced behav-
ioral amplification (Thompson et al., 2008).
While V1a receptors for VT are broadly distributed
throughout the CNS (Evans et al., 2000), single-unit
electrophysiology predicted that a primary site of action is the
mRF circuitry (Rose et al., 1995). Neuroanatomical location of
these receptors was, however, unknown until recently,
because there were no acceptable antibodies available for
immunohistochemical identification of V1a receptor proteins.
In lieu of this, a functional imaging method was developed
using VT conjugated to a fluorophore, VT–Oregon Green
(VT-OG) (Lewis et al., 2004). This method is predicated on
the understanding that when VT binds its V1a receptor, they
behave like all G-protein-coupled receptors analyzed thus far,
by clustering with other V1a–VT complexes, and undergoing
receptor-mediated endocytosis (Fukunaga et al., 2006). Proof
of concept experiments verified that the VT-OG molecule
retains physiological function identical to unlabeled VT, and
its action is blocked by V1a antagonists (Lewis et al., 2004).
The advantage of this method is that we can use functional
imaging to assess the location and availability of functional
V1a receptors by tracking the VT-OG molecule as it is
packaged into vesicles visible by confocal microscopy (Lewis
et al., 2005). VT-OG is endocytosed into 70% of mRF
reticulospinal neurons and 80% mRF interneurons,
supporting the notion that a large population of mRF
neurons can respond to this peptide (Davis et al., 2015; Lewis
et al., 2005). Collectively, evidence from behavioral,
electrophysiological, and functional imaging support the
notion that VT is elevated in response to social
experience and modulates sensorimotor integration in mRF
124 The Vertebrate Brain Stem as the Primary Site of Behavior Command: Insights from an Amphibian
neurons to modify the expression of clasp behavior in a context-
appropriate manner.
2.07.4.2 Acute Stress Rapidly Suppresses Sex Behavior
Through CORT Actions
Activation of the hypothalamo–pituitary–adrenal (HPA) axis
by acute stress leads to a rapid release of CORT into circulation.
A majority of research has focused on the negative effects of
CORT in the induction of chronic disease states that develop
after prolonged stress (Hunter et al., 2015; McEwen and
Gianaros, 2011). These effects are mediated by the intracellular
glucocorticoid receptor (GR) and mineralocorticoid receptor
(MR), which act as ligand-activated transcription factors to
influence genomic responses. However, high concentrations of
CORT, which are released into general circulation after an acute
threat, affect behavior on a much faster timescale than transcrip-
tional control: a few seconds to 5 min to initiate behavior
changes and 20–60 min to peak, before declining equally
precipitously (Rankin et al., 2012). These acute effects of
CORT are crucial to an animal’s ability to respond appropriately
to a broad range of perceived threats. Rapid adaptive behavioral
responses are therefore thought to be mediated by nongenomic
mechanisms mediated through membrane receptors.
A fundamental premise of my framework is that a primary
response to a perceived threat is behavioral and that neural
correlates of CORT actions on behavior must be present.
Many different behavior suites are modified by acutely
elevated CORT, including reproductive behaviors in
mammals (Etgen and Barfield, 1986), teleosts (Remage-
Healey and Bass, 2006), and amphibians (Moore and Zoeller,
1985); locomotor activity of mammals (Meijer et al., 1998)
and birds (Breuner et al., 1998; Breuner and Wingfield,
2000); and aggression in mammals (Bertsch et al., 2011). At
a neural systems level, these changes in behavior are
hypothesized to be mediated by nongenomic actions of
CORT on the processing of external sensory information
(environment) and/or motor output, leading to context-
appropriate behaviors (Coddington and Moore, 2003). Simi-
larly, CORT rapidly induces changes in the animal’s attention
from ongoing activity to the perceived threat (Putman et al.,
2007). These behavioral and perceptive changes predict that
CORT directly impacts neuron function, although this predic-
tion has rarely been directly tested.
2.07.4.3 How Does CORT Rapidly Modify Behaviors?
The challenge to understanding rapid actions of CORT is due to
behavioral and cellular responses being context- or state-
dependent, and we have yet to consider the possibility of
response and recovery involving a cascade of mechanisms
that can occur within the ‘rapid’ millisecond-to-minute time
frame. Early work in Taricha revealed that CORT rapidly
suppresses male clasping behavior in a dose-dependent
manner (Orchinik et al., 1991, 1994). The cellular mecha-
nism(s) by which CORT might rapidly influence neuronal pro-
cessing and immediate behavioral responses are too rapid to be
genomic and occur in the absence of gene transcription
(Evanson et al., 2010; Haller et al., 1998; Mikics et al., 2004;
Orchinik et al., 1991).
Parsimony would predict that there is a single mechanism
that mediates the rapid response to CORT. For example,
some researchers have argued that intracellular GRs and MRss
alone explain the rapid response to CORT (Joëls and Van
Riel, 2004), while others argue that a novel membrane receptor
is responsible (Moore, 2003; Orchinik et al., 1991; Tasker et al.,
2006). I propose that parsimony, in this case, is limiting our
full understanding of rapid responses to stress. Instead, CORT
must engage a cascade of effects across different timescales,
from milliseconds to days and seasons, and multiple receptors
and mechanisms must be employed for a context-appropriate
response to stress and for recovery from stress to occur. Our
research suggests that CORT mediates behavioral responses to
acute stress through one established mechanism and one novel
mechanism acting on two different ‘rapid’ timescales: (1)
CORT modifies synaptic communication by upregulating
endogenous cannabinoid (eCB) signaling and (2) CORT
modifies subsequent modulatory influence by interfering
with other receptors’ availability.
eCBs act as major neuromodulatory signaling molecules
that regulate many different physiological and behavioral
functions and have been identified as a key mediator of rapid
CORT effects in a variety of brain functions necessary for appro-
priate decision-making during stress. They are critical for extinc-
tion of fear memories (Marsicano and Lafenetre, 2009;
Marsicano et al., 2002), relief from anxiety (Riebe and Wotjak,
2011), providing negative feedback to the HPA axis (Di et al.,
2003, 2005), habituation of the HPA axis to repeated stress
(Hill, 2012; Hill et al., 2010a), and modifying behavioral
responses to stress (Coddington et al., 2007). Studies on the
mechanism of CORT–eCB interaction have focused on the rat
hypothalamus, where CORT is the trigger for typical eCB
signaling (Evanson et al., 2010; Hill et al., 2010b; Hill and
Tasker, 2012); where CORT initiates the synthesis and release
of eCBs from postsynaptic neurons, and eCBs then travel retro-
gradely to suppress neurotransmitter release by presynaptic
neurons (Crosby and Bains, 2012; Crosby et al., 2011; Di
et al., 2003, 2005; Hill et al., 2010b). This retrograde eCB
signaling mechanism is a remarkably elegant way in which
postsynaptic neurons can switch off one presynaptic pathway
of information and therefore tune into the remaining salient
inputs.
The CORT-initiated eCB signaling observed in rodents also
occurs in Taricha and is one of the primary mechanisms
employed by CORT to exert rapid effects in sensorimotor
processing necessary for context-appropriate clasping.
Administration of a CB1 receptor antagonist blocks the
typically suppressive actions of CORT on male clasping
behavior. CB1 receptor antagonist also blocks CORT’s
inhibitory effect on mRF neurons; in fact CORT post-CB1
blockade resulted in 25% of mRF neurons actually increasing
in spontaneous activity and sensory responsiveness to clasp-
generating tactile stimuli (Coddington et al., 2007). A
reasonable conclusion from these studies in Taricha is that
CORT acts in the mRF in a manner similar to that observed
in rodent hypothalamus – binding a receptor on presynaptic
membranes, initiating synthesis and secretion of an
endocannabinoid that travels retrogradely to inhibit
neurotransmitter release from presynaptic neurons. The
added information that we gain from the Taricha model is
 The Vertebrate Brain Stem as the Primary Site of Behavior Command: Insights from an Amphibian
that we have learned that eCB signaling provides a key
molecular switch allowing CORT to have variable effects on
behavioral outcomes on the same sensorimotor circuitry.
Additional support for this mechanism comes from char-
acterization of Taricha-specific cannabinoid receptors. Taricha
CB1 receptors were cloned, sequenced, and pharmacologically
identified (Soderstrom et al., 2000). They are densely
expressed in Taricha brain tissue and have a high sequence
homology with mammalian CB1 receptors. Importantly, Tar-
icha CB1 receptors functionally bind agonists and antagonists
in a pharmacological signature identical to mammalian CB1
receptors (Soderstrom et al., 2000). A plausible hypothesis
was that CORT, an equally small lipophilic molecule, might
co-opt CB1 receptors to effect rapid neuronal changes.
However, this hypothesis was ruled out by pharmacological
studies (Soderstrom et al., 2000). In situ hybridization reveals
that the density and distribution of CB1 receptors is consistent
with other vertebrates, and fundamental to our working
model they are densely expressed in the mRF (Hollis et al.,
2006). Therefore, in support of our working model, func-
tional CB1 receptors are found in the hindbrain region, which
is known to mediate clasping, and respond to CORT
administration.
2.07.4.3.1 Context-Dependent Interactions of CORT and VT
The suppressive effect of CORT on clasping behavior is only
apparent when administered to solitary males; if males
have already recently engaged in courtship behavior, then
neither acute stress nor exogenous CORT suppresses clasping
(Coddington and Moore, 2003). A parallel study where court-
ship experience was replaced with administration of VT prior to
acute stress or CORT led to a similar outcome: VT completely
blocked the typically suppressive effects of CORT on clasping
(Coddington et al., 2007).
The context-dependent effect of acute stress on clasping
behavior is time sensitive and involves two primary hormones
(VT and CORT) that seem to convey opposing information; VT
says go, and CORT says no. In vivo single-unit electrophysiolog-
ical recordings of mRF neurons in male Taricha show that the
typical suppressive effects of CORT are not seen if VT is admin-
istered between 10 and 60 min prior to CORT administration
(Rose et al., 1995). The converse is also true: the typically
enhancing effects of VT are not apparent if CORT is adminis-
tered between 2 and 20 min prior to administration of VT
(Rose et al., 1995). Based on these early electrophysiological
findings, we supposed that effects observed at the single-cell
level would be reproduced at the behavioral levels. In other
words, if the animals experienced prosocial behaviors prior to
exposure to a threat, then the driving force on the animal
would be to continue employing prosocial behaviors.
Conversely, if the animals were solitary and then exposed to
an acute threat, then they would fall back onto the three ‘f’s’:
fight, flight, or freeze. Behavioral studies confirmed this
hypothesis. Male Taricha allowed to engage in courtship with
a female for 1 h (courtship typically extends for 3–20 h in
lab), readily reengaged in courtship after separation and expo-
sure to 2 min restraint stress or administration of CORT (Cod-
dington and Moore, 2003). This is remarkable given that 95–
100% of solitary males exposed to 2 min restraint stress or
CORT administration will not engage in courtship. In addition,
125
administration of VT in lieu of courtship experience with
a female mirrored the effect of prosocial behavior, preventing
the typically suppressive effects of CORT on clasping (Codding-
ton and Moore, 2003).
A parsimonious explanation for this time-dependent inter-
action relates to the time constraints involved in eCB synthesis,
release, and subsequent modification of presynaptic release of
neurotransmitters. Evidence definitely supports the notion that
this mechanism is in play in sensorimotor processing involved
in the decision process leading to clasping or escape. However,
it is helpful to adopt a broader view and consider whether there
are other mechanisms involved in the escape – clasp, CORT–VT
interplay that might also occur in the ‘rapid’ nongenomic time
frame.
2.07.4.3.2 CORT Modifies VT V1a Receptor Availability in mRF
Neurons
Another plausible mechanism that would explain the time
span of the interactions observed between CORT and VT
includes CORT decreasing the availability of V1a receptors
and therefore inhibiting subsequent VT-mediated effects. We
tested this hypothesis with the use of VT conjugated to
a fluorescent label that is endocytosed (internalized in
neurons) through a receptor-mediated process when the
ligand binds to V1a receptors (Fukunaga et al., 2006; Wieffer
et al., 2009). VT endocytosis was blocked by mid- and high
doses (akin to levels during an acute stress) of CORT in the
clasp-controlling region of the hindbrain, mRF (Figure 3;
Davis et al., 2015). The percent of VT-containing vesicles
significantly declined with increasing doses of CORT, which
indicates that there were fewer receptor-mediated endocytosis
events, and the intensity per vesicle also significantly
decreased, indicating that there were fewer receptor–VT pairs
per vesicle (Figure 3). These data collectively suggest that
CORT is affecting the availability of V1a receptors. The
specific subcellular mechanism remains unclear but could be
explained by (1) a decrease in the number of V1a receptors
expressed at the membrane, (2) a decrease in the affinity of
extant V1a receptors, and/or (3) a suppression of the
machinery by which receptor-mediated endocytosis occurs.
The behavioral significance of these data was strengthened
by two further findings: (1) the influence of CORT on VT endo-
cytosis in the mRF is only apparent during the breeding season,
i.e., when VT is modulating the expression of clasping behavior,
and (2) the CORT-induced suppression of VT endocytosis
observed in the mRF was not observed in the inferior
reticular formation (iRF) (Davis et al., 2015) – a region
thought to be homologous to one of the premotor regions
that regulate vocalization in all vertebrates (Bass et al., 2008).
In contrast to the mRF breeding-season data where CORT
significantly reduced key measures of endocytosis (number of
vesicles containing VT, the total intensity of VT/mm3, and
percent area covered by vesicles), there was no significant
effect of CORT on these three measures in the iRF during the
breeding season. But there was a significant increase in
amount of VT found per endocytosed vesicle in the iRF after
pretreatment with high-stress CORT concentrations – polar
opposite effects in the two functionally different reticular
regions. Collectively these functional imaging data suggest
that CORT metamodulates VT effects by modifying V1a
126 The Vertebrate Brain Stem as the Primary Site of Behavior Command: Insights from an Amphibian

Figure 3 Corticosterone (CORT) reduces the availability of V1a receptors as indicated by a decrease in receptor-mediated endocytosis of VT conju-
gated to Oregon green (VT-OG) into hindbrain mRF cells. (a and b) are representative confocal images captured at 63x. Orange arrows indicate DAPI-
labeled nuclei. Fuchsia arrows indicate putative vesicles that have endocytosed VT-OG with V1a receptors. (c) CORT significantly decreased the
average number of endocytosed vesicles containing VT-OG compared with brains pretreated with vehicle control solution. (d) CORT significantly
decreased the intensity of VT-OG per vesicle. Collectively, these data reveal that CORT reduced the availability of functional V1a receptors and affected
the endocytic machinery so that fewer pairs of V1a receptor þ VT-OG were internalized per vesicle. Quantification of images was achieved using
Axiovision software to analyze maximum projection images of confocal slices (N ¼ 4 newts per group). Scale bars ¼ 20 mm. Graphs show data as
mean  SE, and asterisks above bars indicate significant differences between groups (p < 0.01). Reproduced based on Davis, A., Abraham, E.,
Mcevoy, E., Sonnenfeld, S., Lewis, C., Hubbard, C.S., Dolence, E.K., Rose, J.D., Coddington, E., 2015. Corticosterone suppresses vasotocin-
enhanced clasping behavior in male rough-skinned newts by novel mechanisms interfering with V1a receptor availability and receptor-mediated
endocytosis. Horm. Behav. 69, 39–49.

receptor availability. This effect is consistent with the natural
and context-dependent expression of clasping behavior, as
CORT only modified VT endocytosis in the mRF and not in
the iRF, and in a season-dependent manner. We suggest that
metamodulation of other hormone receptors’ availability is
an unappreciated mechanism of hormone action common
among vertebrates (Von Zastrow and Williams, 2012) and is
a major player in the neural processing leading to context-
appropriate behaviors.
was able to block the kappa-mediated suppression of
clasping (Lombana et al., 2015). Therefore, while the in vitro
data provide evidence that the mCR might be a kappa-opiate-
like receptor, our in vivo behavioral data suggest that kappa-
mediated suppression of clasping occurs via a different subset
of membrane receptors, and endocannabinoids do not
mediate the kappa-induced suppression of clasping. The
molecular identity of mCR remains elusive to this date.

2.07.5 Outlook
2.07.4.4 Identity of Membrane CORT Receptors
The role of the brainstem is pivotal for the manifestation of
CORT influences clasping by binding a G-protein-coupled
context-appropriate behaviors, yet we know very little about
receptor located at the cell surface (Haller et al., 2008; Orchinik
this highly conserved brain region in any vertebrate and
et al., 1994). There is conflicting evidence concerning the
even less in tetrapod vertebrates. Harnessing what is known
nature of this receptor: it could be a new membrane receptor
about the brainstem, sensorimotor control of vertebrate loco-
specific to CORT (Orchinik et al., 1991) or a kappa-opioid-
motion, and hormone effects on sex behaviors, we can better
like receptor (Bradford et al., 2005; Evans et al., 2000; Moore,
understand the evolutionary and proximal mechanisms that
2003). Early behavioral studies revealed that kappa agonists
drive sex behaviors specifically and behaviors involving
suppress clasping behavior in a dose-dependent manner
limb and torso in general. Using whole-cell electrophysiology
(Deviche and Moore, 1987), in a manner identical to CORT.
in concert with functional imaging, we can reveal previously
Additionally, in vitro assays showed that membrane CORT
unappreciated mechanisms that neural circuits and hormones
receptors (mCRs) recognize kappa-opioid receptor agonists
are using to modify behaviors in the rapid nongenomic time
and antagonists (Bradford et al., 2005; Evans et al., 2000).
frame. There is much to be learned through the lens of Taricha
Combined, these studies suggested that CORT and kappa
clasping behavior.
agonists might suppress clasping behavior via the same
pathway: an mCR-initiated upregulation of eCB signaling.
However, further behavioral pharmacological examination Acknowledgments
revealed that kappa agonists suppress clasping via a pathway
independent of eCB signaling, which is independent of the Gratitude to Dan Yaeger and Jim Rose for conversations about the
pathway activated by CORT. Pretreatment with a CB1 vertebrate brainstem. And thanks to Barbara Stebbins-Boaz and Jennifer
antagonist failed to block the kappa-mediated suppression of Butler for their editorial support. This work is funded by NSF (IOS-
clasping, whereas a typical kappa-opiate antagonist, nor-BNI, 1351129).
 The Vertebrate Brain Stem as the Primary Site of Behavior Command: Insights from an Amphibian
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 2.08 Hormones and Vocal Systems: Insights from Xenopus
Erik Zornik, Reed College, Portland, OR, USA
Darcy B Kelley, Columbia University, New York, NY, USA
Ó 2017 Elsevier Inc. All rights reserved.
This chapter is a revision of the previous edition chapter by E.J. Yang, D.B. Kelley, volume 1, pp. 693–706, Ó 2009, Elsevier Inc.

2.08.1 Introduction 131

2.08.2 Mating and Vocal Behaviors 132
2.08.2.1 Vocalizations Depend on Social Context 132
2.08.2.1.1 Male Advertisement Calls 132
2.08.2.1.2 Female Unreceptive Calls 132
2.08.2.1.3 Female Rapping and Male Answer Call 132
2.08.2.1.4 Amplectant Call 132
2.08.2.1.5 Male–Male Interaction Calls 132
2.08.2.2 Clasping, Reproductive Status, and Social Hierarchies 134
2.08.2.2.1 Female Receptivity 134
2.08.2.2.2 Male–Female Clasping 134
2.08.2.2.3 Male–Male Clasping 134
2.08.2.2.4 Vocal Dominance 135
2.08.2.3 Hormone-Dependence of Vocal Behaviors 135
2.08.2.3.1 Advertisement Calls 135
2.08.2.3.2 Female Calling 135
2.08.2.3.3 Male Interaction Calls 137
2.08.2.3.4 Insights from Endocrine Disrupting Compounds 137
2.08.3 Sex Differences and Hormone Targets in the Vocal System 137
2.08.3.1 Sex Differences and Hormone Targets in the Larynx 137
2.08.3.2 Sex Differences and Hormone Targets in the Vocal Circuit 137
2.08.3.2.1 Motor Circuit 137
2.08.3.2.2 Auditory Pathways 138
2.08.4 Generating Sexually Differentiated Vocalizations 138
2.08.4.1 Generating Fast and Slow Calls 138
2.08.4.1.1 Larynx 138
2.08.4.1.2 Central Pattern Generator 139
2.08.4.2 Generating Complex and Simple Vocal Patterns 140
2.08.4.2.1 Biphasic versus Monophasic Calling 140
2.08.4.2.2 Intensity Modulation of Vocal Trills 140
2.08.5 Processing Vocal Signals 141
2.08.5.1 Sexually Differentiated Auditory Processing 141
2.08.5.2 Generating Socially Appropriate Vocal Responses 142
2.08.6 Evolution of Vocal Effectors in Xenopus 142
2.08.7 Conclusion 142
References 143

2.08.1 Introduction mechanisms at each level must be complemented with studies

The stunning diversity of patterned movements that make up
behavioral repertoires are governed by neural circuits at
multiple levels and supported by specialized features of muscle
effectors. In vertebrates, behaviors that are specific to each sex
are also regulated, during development and in adulthood, by
the endocrine signals that control reproduction. The complex
nature of hormone action, with multiple targets and tempo-
rally distinct but overlapping sensitive periods, creates a major
challenge in deciphering the causal relevance of hormone
signaling at each level of control. To overcome this hurdle,
investigations of the physiological, cellular, and molecular
that integrate across mechanism and level of organization.
In this chapter, we review research on the sexually differen-
tiated vocal behaviors of South African clawed frogs, Xenopus
laevis. The Xenopus vocal system is unusual because Xenopus is
entirely aquatic throughout its life cycle; vocalization is
produced under water and is not powered by air movement
(breathing). The essential involvement of breathing circuits in
terrestrial vertebrates greatly complicates the study of hind-
brain control of vocalization (McLean et al., 2013; Tupal
et al., 2014). Vocalization without breathing in Xenopus
provides many experimental advantages that include two
powerful reduced experimental preparations: the isolated

Hormones, Brain, and Behavior, 3rd edition, Volume 2 http://dx.doi.org/10.1016/B978-0-12-803592-4.00023-7 131

132 Hormones and Vocal Systems: Insights from Xenopus
(ex vivo) larynx and the isolated brain. ‘Fictive’ vocal behaviors
can be evoked in either preparation independently of the other
(Tobias and Kelley, 1987; Rhodes et al., 2007). The ex vivo
larynx preparation has shed light on how the periphery
constrains sound production, as well as on how hormones
support the production of sex-specific behaviors (Zornik and
Kelley, 2011). The ex vivo brain preparation has provided
important insights into the essential components of the vocal
central pattern generator (CPG) and the neural circuits that
generate different vocal rhythms (Rhodes et al., 2007; Yu and
Yamaguchi, 2009, 2010; Yamaguchi et al., 2010; Zornik and
Yamaguchi, 2012). In vivo studies of intact animals comple-
ment findings ex vivo. Lesion studies, combined with the
ex vivo brain preparation, have provided insight into forebrain
mechanisms that govern context-dependent social signaling
(Hall et al., 2013). Finally, recent advances in understanding
auditory processing will facilitate future integrative syntheses
of sensory, sensorimotor, and motor levels of control (Elliott
et al., 2011; Hall et al., 2016).
2.08.2 Mating and Vocal Behaviors
In X. laevis, the most extensively studied Xenopus species,
mating occurs over a prolonged period (6 months), and
only a small percentage of females are sexually receptive at
any given time (Tobias et al., 1998b). Because these fully
aquatic frogs mate under water at night and typically live in
turbid bodies of water with many conspecifics, vocal signals
drive courtship as they are well suited to locating a suitable
mate. Each call is made up of brief (5–10 ms; Vignal and Kel-
ley, 2007) sound pulses produced with different temporal
patterns. Eight unique call types have been described (Figure 1),
and each can be distinguished through a combination of the
pulse rate, duration, and period (repetition rate) of each trill,
as well as the pulse intensity (loudness) and frequency (pitch).
2.08.2.1 Vocalizations Depend on Social Context
2.08.2.1.1 Male Advertisement Calls
The male advertisement call (Figure 1(a)) is the most spectrally
and temporally complex in the X. laevis repertoire. It can be
divided broadly into two phases: the fast trill and the slow trill.
Fast and slow trills alternate during calling bouts. Across the
fast phase (60 Hz; 200–300 ms duration), sound pulses
increase in amplitude. For the slow phase, sound pulses are
produced at 30 Hz; several ‘loud slow’ pulses often precede
the longer-duration (700–800 ms) slow trills. Fast trill sound
pulses contain two dominant frequencies (DF1: 1.9 kHz;
DF2: 2.2 kHz), while slow trill pulses contain a third, lower
DF 1.2 kHz (Figure 2).
During the peak reproductive season (August in Cape Town,
South Africa), the male-specific advertisement call can be
recorded using an underwater microphone for prolonged
periods (>45 min; Tobias et al., 2004). In the lab, males also
call for prolonged periods when alone. When paired with
a conspecific, males switch from advertisement to answer
calling (Figure 1(b)). In the laboratory, broadcasts of advertise-
ment calls suppress calling by other males (Tobias et al., 2004)
accounting for vocal dominance and the prevalence of calling
by a single male in field recordings (Tobias et al., 2010). The
male advertisement call also attracts gravid females (Picker,
1983) and evokes a specific call, rapping (Figure 1(b); Tobias
et al., 1998b).
2.08.2.1.2 Female Unreceptive Calls
During the breeding season, most female X. laevis are sexually
unreceptive at any given time. When clasped by a male, nongra-
vid females produce a slow series of sound pulses at a rate of 4–
8 Hz called ticking (Figure 1(c)). Unlike male advertisement
calling, the sound pulses in ticking are spectrally broad, with
a single DF around 1.2 kHz (Figure 2). The temporal pattern
is simple, consisting of an ongoing, monotonous trill, and
pulses do not vary systematically in intensity as the call prog-
resses (Tobias et al., 1998b). Nongravid females also extend
their hindlimbs when clasped (Kelley, 1982). When males
are exposed to playbacks of female unreceptive calls during
advertisement calling, they exhibit temporary vocal suppres-
sion (Tobias et al., 2004; Elliott and Kelley, 2007).
2.08.2.1.3 Female Rapping and Male Answer Call
Female X. laevis also advertise for mates using the rapping call.
Rapping can be evoked in gravid females by advertisement
calls, and pairs of sexually receptive males and females produce
rapping/advertisement calling duets (Tobias et al., 1998b).
Rapping trill rates average 12 Hz. Playbacks of rapping evoke
advertisement and answer calling. The answer call, a modified
advertisement call, contains elongated fast trills, shortened
slow trills, and enhanced intensity modulation (Figure 1(b)).
2.08.2.1.4 Amplectant Call
Upon clasping a female or another male (amplexus), males
generate a low-intensity amplectant call consisting of two to
three sound pulses at 150 ms intervals (Figure 1(d); Picker,
1980; Zornik and Kelley, 2008). These sounds coincide with
male forelimb contraction and ventrally directed head move-
ments, potential signals for synchronizing egg and sperm release.
2.08.2.1.5 Male–Male Interaction Calls
All of the six call types given by males can be recorded from
male/male pairs; three (chirping, growling, and male ticking)
are directed exclusively toward males (Tobias et al., 2004).
Chirping, a brief (5 pulses) 60–80 Hz burst repeated
approximately four times per second (Figure 1(e)), can be
recorded as one male approaches another and is reliably
produced by the clasping male (Tobias et al., 2004) as well
as in response to playbacks of synthetic male-like calls (Vignal
and Kelley, 2007). Chirping could be a male–male aggressive
signal, though as noted above, advertisement calls suffice for
vocal suppression.
In male/male pairs, the clasped male produces growling,
a rapid, variable-length trill (Figure 1(e); 40–80 Hz Tobias
et al., 2004). Unlike the sound pulses of other male calls,
growling sound pulses have a lower DF around 1 kHz (Tobias
et al., 2004).
Male ticking is a rare call, and it is not clear whether it is eli-
cited by amplexus, and if so, whether the clasper or claspee
produces the call (Tobias et al., 2004). The sound pulse rate
in male ticking is 4 Hz, as in female ticking (Figure 1(c)),
 Hormones and Vocal Systems: Insights from Xenopus 133

Male calls Female calls

Advertisement calling
(a)

Fast trill Slow trill

500 ms

(b)
Answer calling Rapping

Fast trill Slow trill 500 ms

(c) Ticking

500 ms

Amplectant calling
(d)

500 ms

Chirping

(e)

250 ms
Growling

250 ms

Figure 1 The social context-dependent Xenopus laevis vocal repertoire. (a) Male X. laevis produce advertisement calls in isolation, as well as in the
presence of conspecific females and males. Advertisement calls are temporally complex. Sound pulses occur in alternating trills: fast trill pulses are
produced at a rate of 60 Hz, while slow trill pulse rates average 30 Hz. Sound pulse intensity changes within each call. Calls produced by a single
animal can continue uninterrupted for up to 45 min. (b) When sexually receptive females are about to oviposit, they often generate a call, rapping,
consisting of an 12 Hz pulse rate that attracts males. When males hear rapping, they approach the female (or loudspeaker playing an artificial
call) and generate a modified version of the advertisement call, answer call. Answer calling consists of elongated fast trills and truncated slow trills.
(c) When unreceptive females are clasped by a male, they extend their hindlimbs and generate a slower, 4 Hz, call: ticking. Ticking suppresses male
calling. Ticking has also been recorded from male/male pairs (Tobias et al., 2004). While the sound pulse rate (4 Hz) does not differ from female
ticking, sound pulses frequencies (1.7–2.3 kHz) in male ticking are higher than in female ticking (1.2 kHz). (d) When females are reproductively
active, they respond to male clasps with flexion of the hindlimbs, which promotes fertilization during oviposition. During this prolonged amplexus,
males generate a low-intensity, low pulse rate call: amplectant call. (e) Males produce all call types in the presence of other males. Two call types
most commonly associated with male–male interactions are chirping and growling, which are believed to represent agonistic signals. Chirping
appears to be largely associated with dominant males, while growling is produced by clasped males. Pulse intervals for both calls are fast
(40–80 Hz), with distinctive temporal patterns; chirps are brief and repeated multiple times per second, each growling trill tend to last several
hundred milliseconds. Reproduced from Zornik, E., Kelley, D.B., 2011. A neuroendocrine basis for the hierarchical control of frog courtship vocaliza-
tions. Front. Neuroendocrinol. 32, 353–366.

but spectral frequencies of male pulses are higher (1.8 kHz)
than in females (1.2 kHz).
The distinctive features of different calls given by the sexes
allow the social context in which a recording was obtained to
be determined from the calls alone. For example, a laboratory
recording that includes advertisement calling and growling
indicates a male/male pair in which one is clasping the other
(and thus sexually active). A recording that includes rapping
and answer calling indicates interactions between a sexually
active female/male pair. Support for the reliability of these
134 Hormones and Vocal Systems: Insights from Xenopus
4000 Hz
3000 Hz
2000 Hz
1000 Hz
Fast trill Slow trill
Figure 2 Spectrograms of sexually differentiated Xenopus laevis calls. Left, male advertisement calls are spectrally complex. Fast trill sound pulses
contain two dominant frequencies (DFs) at 1.9 and 2.2 kHz. Slow trill sound pulses contain a lower frequency component 1.2 kHz. Right,
female ticking sound pulses have less finely tuned frequency ranges, with a single DF at 1.2 kHz. Scale bars: 250 ms.
inferences comes from the ability to reproduce the outcomes of
social interactions in isolated frogs using sound broadcasts.
Rapping broadcasts, for example, stimulate male answer calling
(Vignal and Kelley, 2007) while intense advertisement calling
produces prolonged vocal suppression in males (Tobias et al.,
2010). The broadcast paradigm has been useful in functional
dissection of forebrain influences on vocal responses, described
below (Hall et al., 2013).
2.08.2.2 Clasping, Reproductive Status, and Social
Hierarchies
2.08.2.2.1 Female Receptivity
Nearly a century of experimental work has probed the role of
steroids in female X. laevis reproduction. Early studies found
that anterior pituitary extracts could induce ovulation in
X. laevis females, while hypophysectomy lead to dramatic
ovarian retrogression (Hogben et al., 1931). These results
set the stage for the widespread use of Xenopus as a test for
pregnancy; injection of urine containing human chorionic
gonadotropin (hCG, a hormone produced by the placenta)
leads to egg ripening and oviposition in Xenopus (Elkan,
1938). Although little is known about the relation between
hormonal fluctuations in adult female X. laevis to their repro-
ductive cycles in nature, one study found a strong positive
correlation between hypothalamic GnRH (also known as
LHRH) and breeding season (King and Millar, 1979). Thus,
it is likely that seasonal elevation of GnRH leads to gonado-
tropin (LH)-induced oocyte maturation and oviposition,
two events that can be recapitulated by exposure to hCG.
Although it was initially assumed that hCG injections
promote oocyte maturation via progesterone signaling, recent
experiments indicate that plasma progesterone is an order of
magnitude lower than androgens and that androgens instead
appear responsible for oocyte maturation (Lutz et al., 2001).
Both hCG and GnRH induce a receptive posture – flexion
of the hindlimbs – in intact females clasped by males
(Figure 1(d); Kelley, 1982). Ovariectomy eliminates this
behavior, which can be reinstated by injections of progesterone
and estradiol together, although each hormone by itself does
not induce the receptive response (Kelley, 1982). These results
do not rule out a role for androgens in female responses to
clasping, as progesterone has been shown to be rapidly
metabolized to the androgen androstenedione (Lutz et al.,
2001). Injection of GnRH into ovariectomized females treated
with progesterone and estradiol increased the likelihood of
clasping by males and led to prolonged amplexus, an effect
not present after hCG administration. Thus GnRH might be
acting directly on brain targets rather the pituitary to elicit
receptive behaviors.
Prostaglandin E2 (PGE2) injection has also been shown to
increase female receptivity (Weintraub et al., 1985). PGE2
injections led to dramatic and rapid (30 s–3 min) reduction
of unreceptive leg extensions. Because these effects occur in
both intact and ovariectomized females, it is likely that
a PGE2 acts directly on neuronal targets to mediate these
behavioral changes. Similar rapid actions of prostaglandins
have been described in fish (Stacey and Goetz, 1982; Juntti
et al., 2016).
2.08.2.2.2 Male–Female Clasping
Clasping is a critical component of Xenopus mating behavior; in
the laboratory, amplexus can be maintained for several days
(Kelley, 1982). Male clasping is androgen-dependent. Castra-
tion abolishes clasping, while T or DHT implants reinstate
the behavior (Kelley and Pfaff, 1976). Although intact females
do not clasp males, gonadectomized females treated with
either T or DHT do clasp males, and clasp durations do not
differ from hormone-replaced males. The finding that DHT
can induce clasping in both sexes suggests androgens are suffi-
cient to induce clasping; E on the other hand does not induce
clasping.
2.08.2.2.3 Male–Male Clasping
Group-housed male X. laevis are often observed clasping each
other, sometimes for prolonged periods. A recent study
suggests that these male–male interactions do not necessarily
reflect aggression or failure to discriminate sex, but may instead
represent an alternative mating strategy (Rhodes et al., 2014).
In triad experiments, in which two males were tested with
one female, most males preferentially clasped females;
however, a subset of males preferentially directed clasping
toward other males. Males that are less likely to clasp a male
in male–male pairs are more likely to ‘win’ clasps of females
in subsequent male–male–female triads. Conversely, males
that preferentially clasped the other male in the male–male

pair rarely gained access to the female, but continued to clasp
the other male. Thus clasping in this study does not appear
to represent a means of demonstrating or establishing repro-
ductive dominance. Together, these results support the hypoth-
esis that some males use an alternative mating strategy in which
they gain physical proximity to gravid females via clasping an
amplexing male; such physical proximity could allow fertiliza-
tion of some eggs as they are laid by the female.
2.08.2.2.4 Vocal Dominance
The finding that, in any given pond, only one or a few males
produce advertisement calls each night prompted laboratory
investigations into the mechanisms regulating calling. Tobias
et al. (2004) showed that pairing two males led to vocal
silencing of one. Transitive, linear vocal dominance hierarchies
can be inferred from tests of multiple male pairs and remain
stable for up to 2 weeks (Tobias et al., 2010). Physical contact
is not required to establish vocal dominance, and the amount
of male clasping did not match vocal dominance patterns. Play-
back of male advertisement calls alone was sufficient to vocally
suppress all exposed males (Tobias et al., 2010). Vocal suppres-
sion was achieved whether the playback recordings were
derived from a vocally dominant or subordinate animal; low-
intensity playbacks of either call were less effective in vocal
suppression than high-intensity playbacks. Quantity or inten-
sity of calling thus appears to be a determining factor in estab-
lishing dominance.
Whether the establishment or maintenance of vocal domi-
nance is hormone-dependent has yet to be investigated.
Because advertisement calling depends on circulating andro-
gens (see below), differential levels of circulating androgens
could be correlated with vocal dominance, as observed in social
dominance in other species such as cichlids (Maruska, 2014),
electric fish (Cuddy et al., 2012), and rodents (Shen et al.,
2015). Another possible contributor is corticosterone, as the
stress-related glucocorticoids are known to be correlated
with social dominance in many species (Abbott et al., 2003;
Goymann and Wingfield, 2004; Jeffrey et al., 2012; Ode
et al., 2015). Because of its prolonged nature, advertisement
calling could be energetically costly, perhaps contributing to
the scarcity of calling in any given population during the
breeding season. In a corticosterone-regulated pattern of vocal
dominance, males in an optimal physiological state (i.e., well-
fed, metabolically stable, low stress) would be most likely
to gain vocal dominance, leading to greater reproductive
success.
2.08.2.3 Hormone-Dependence of Vocal Behaviors
2.08.2.3.1 Advertisement Calls
Male advertisement calling depends on endocrine state. Most
castrated males show a complete loss of advertisement calling
after about 1 month (Wetzel and Kelley, 1983), although
some animals do produce small amounts of abnormal calling
as long as 1 year after castration (Zornik and Yamaguchi,
2011). Normal calling in castrated males resumes following
treatment with either T or DHT (Wetzel and Kelley, 1983).
In the lab, male calling is greatly enhanced by injection of
hCG (Wetzel and Kelley, 1983; Tobias et al., 2004; Yang
et al., 2007). Very little calling is observed in uninjected male
Hormones and Vocal Systems: Insights from Xenopus 135
pairs. However, when one or both males in a pair is injected
(100 IU, 24 h and 6–8 h before each trial), the amount of
calling is greatly increased, with all six call types represented.
Although some of this effect is likely due to an increase in circu-
lating androgens, hCG also appears to affect calling by directly
activating vocal nuclei in the brain. The original evidence for
a central mechanism of hCG function came from experiments
in castrated, hormone-replaced frogs (Wetzel and Kelley,
1983). As described above, calling was rapidly eliminated
following castration, but could be reinstated by replacing T or
DHT. Calling rates in hormone-replaced animals were signifi-
cantly lower than intact frogs. However, when these animals
were injected with hCG, time spent calling increased as it did
during presurgery control recordings. Since the testes were no
longer present, an extragonadal target was implicated.
Later experiments provided strong support for the hypoth-
esis that gonadotropins enhance advertisement calling via
direct action in the brain (Yang et al., 2007). Intracerebroven-
tricular (ICV) injection of hCG was effective at enhancing
calling at far lower doses than systemic injections. This effect
appears to be transduced via luteinizing hormone receptors,
which are expressed in the central amygdala (CeA), a known
vocal control region (Figure 3; Brahic and Kelley, 2003; Hall
et al., 2013). Exposure of the isolated brain to hCG induced
upregulation of the immediate early gene egr-1 in the CeA,
even when synaptic transmission was blocked. Endogenous
gonadotropin signaling must not be sufficient to induce
calling, however, because hCG injections in castrated males
do not enhance male calling (Zornik and Yamaguchi,
2011). Thus, androgens and gonadotropins are both required
in the brain to allow normal production of male advertise-
ment calls.
As described above, linear vocal hierarchies between males
can be stable for up to 2 weeks, but subordinate males only
suppress their calling when in the presence of a dominant
male. The mechanisms that underlie rapid suppression of
calling is unknown, but must involve processing of auditory
information, given that playbacks alone are sufficient to
suppress calling. A likely candidate nucleus for regulating vocal
suppression is the CeA. Because this region receives auditory
information (Figure 3(a); Hall et al., 2013) and is a target of
gonadotropin (Yang et al., 2007), one intriguing possibility is
that vocal suppression may be a result of changes in endoge-
nous gonadotropin signaling in the CeA.
2.08.2.3.2 Female Calling
While females do not use vocalizations when paired with other
females (Tobias et al., 2004), reproductive state is reflected in
their vocalizations when paired with a male. Unreceptive
females produce the slow call, ticking, while receptive females
produce the faster call, rapping (Figure 1; Tobias et al., 1998b).
Although hormones that promote vitellogenin synthesis (e.g.,
estradiol), oocyte maturation (e.g., androgens) and female
receptivity (GnRH, progesterone, and estradiol) have been
described, we do not have a complete picture of how endoge-
nous hormones modulate female vocal behaviors. The time
course of changes in female vocal behaviors induced by hCG
in the presence of an advertising, clasping male has been fol-
lowed in intact females (Wu et al., 2001). All uninjected
females ticked when clasped; 6 h after injection, all females
136 Hormones and Vocal Systems: Insights from Xenopus

(a)
TS
CT
DTAM
CeA n.IX-X

VN

LHR ER AR
(b)

CeA
A TS
S

n.IX-X
DTAM
VN

Figure 3 Auditory and vocal pathways in Xenopus laevis. (a) Diagram (top) and photograph (bottom) of a sagittal view of the X. laevis brain and
anterior spinal cord (anterior is left, dorsal is up). Key auditory, sensorimotor and vocal central pattern generator (CPG) nuclei are shown with their
known connections. The midbrain torus semicircularis (TS) processes auditory information and projects to the central thalamus (CT), which in turn
projects to the central amygdala (CeA). CeA projections to the hindbrain are thought to regulate social context-dependent activation of the vocal CPG.
CPG nuclei DTAM and n.IX-X are reciprocally connected. (b) Diagram (top) and photograph (bottom) of a dorsal view of the X. laevis brain and
anterior spinal cord (anterior is left). Color coded nuclei indicate adult patterns of hormone receptor expression (LHR, luteinizing hormone receptor;
ER, estrogen receptor; AR, androgen receptor). Laryngeal motor neuron axons exit the brain via the caudal root of cranial nerve IX-X, the vocal
nerve (VN).

were silent when clasped. Rapping in response to male calling
appeared in a smaller subset of females by 9 h postinjection
and was recorded out to 18 h postinjection.
What are the mechanisms underlying suppression of ticking
and promotion of rapping? A potential endocrine suppressor
of ticking is prostaglandin; PGE2 rapidly suppresses ticking
behaviors when given exogenously but does not elicit rapping
(Weintraub et al., 1985). In cichlid fish, PGF2a activates female
reproductive behaviors and appears to act directly on CNS
targets via a gonadal hormone-induced PGF2 receptor (Juntti
et al., 2016). A similar scenario might operate via PGE2 in
Xenopus.
What is the trigger for rapping? hCG-induced serum steroid
hormones in females are dominated by androgens, and their
local action triggers the final reduction divisions of meiosis
responsible for oocyte formation (Lutz et al., 2001). While T
and DHT treatment masculinize female vocal behavior, the
primary circulating androgen in females, androstenedione,
might preferentially act on a subset of neural target cells that
promote rapping. An acute increase in circulating androgen,
associated with imminent oviposition, might activate neural
targets responsible for producing rapping. Another possibility
is that rapping arises in response to gonadotropin signaling
in the CeA. Recent experiments in the ex vivo brain
preparation (described below) showed that CeA electrical
stimulation preferentially induce rappinglike vocal patterns,
suggesting that increased activity in the CeA may promote
receptive vocalizations in females (Ballagh, 2014).

2.08.2.3.3 Male Interaction Calls
As described above, Rhodes et al. (2014) observed that males
clasping other males (male preference) were less likely to clasp
females in a male–male–female triad, indicating that male–
male clasping may represent an alternative (i.e., ‘sneaker
male’) mating strategy rather than an agonistic interaction.
Because they did not report vocal patterns in their study,
however, it is not clear whether vocal dominance is associated
with female-preference or male-preference clasping.
In an earlier study, Tobias et al. (2004) showed that vocally
dominant males produce many chirps while clasping, while
vocally subordinate males clasping another male produced
few chirps. These results support the idea that chirping is an
aggressive vocal behavior that functions in regulating reproduc-
tive dominance. Clasping by a dominant male may represent
aggression, while clasping by a subordinate male may represent
a ‘sneaker’ strategy. Males in the Rhodes et al. (2014) study
were not injected with hCG, while both males in the Tobias
et al. (2004, 2010) paired recordings were. The hCG treatments
may have led to aggressive clasping absent from the Rhodes
et al.’s study.
These laboratory results leave open the question of how,
and under what circumstances, endogenous gonadotropins
act to promote aggressive clasping and chirping. Field record-
ings in South Africa reveal that while advertisement calling is
produced at high levels throughout the breeding season, chirp-
ing peaks at the end of the season (Tobias et al., 2004).
Growling, produced by males clasped by other males, drops
immediately prior to the surge in chirping and answer calling.
This rapid change could reflect a population-wide surge in
endogenous gonadotropin in the late South African winter, as
has been observed in females (King and Millar, 1979). Testing
this hypothesis will require replication of the vocal results
together with hormone measurements.
2.08.2.3.4 Insights from Endocrine Disrupting Compounds
Most hormone manipulation studies in Xenopus have addressed
long-term (i.e., weeks to months) effects. Recently, a series of
studies investigating the effects of endocrine disrupting
compounds have begun to address short-term effects of endo-
crine signaling on vocal behaviors. In one study, exposure of
hCG-injected males to the antiandrogen flutamide (10 6 M)
significantly reduced the amount of advertisement calling
compared to hCG-injected controls 3 and 4 days after
exposure, resulting in calling levels as low as non-hCG-
injected animals (Behrends et al., 2010). Similar reductions in
calling were observed during exposure to 10 6 M of the
antiandrogen vinclozolin; advertisement calling and chirping
were reduced while ticking and growling were increased
(Hoffmann and Kloas, 2010). In contrast, 4-day exposure to
low doses (30.5 ng l 1, 3.05 mg l 1, 30.5 mg l 1) of the xeno-
androgen methyldihydrotestosterone (MDHT) induced
significant increases in the percentage of advertisement calling
(Hoffmann and Kloas, 2012a). The increase in percentage of
advertisement calls produced by males was paralleled by
a significant decrease in a call the authors referred to as
rasping. The spectrogram of rasping reveals a call that appears
similar to an elongated (over 5 s) slow trill uninterrupted by
fast trills (Hoffmann and Kloas, 2012b). Interestingly,
a similar pattern of calling was observed in a study of the
Hormones and Vocal Systems: Insights from Xenopus 137
effects of long-term (5–18 months) castration (Zornik and
Yamaguchi, 2011). These calls (referred to as ‘isolated slow
trill’ in Zornik and Yamaguchi) therefore may be generated by
males with relatively low androgen signaling, but can be
rapidly eliminated by exogenous androgens. The behavioral
relevance of ‘isolated slow trills’ is not understood.
2.08.3 Sex Differences and Hormone Targets in the
Vocal System
The vocal behaviors of X. laevis are highly differentiated between
the sexes. Nearly all neural and muscular components of the
vocal circuits are regulated by one or more steroid hormones.
The vocal organ, which contains the single muscle group needed
to generate vocal sounds, differs markedly in males and females
(Sassoon and Kelley, 1986). In the brain, many nuclei related to
auditory processing and vocal motor production were first iden-
tified by their ability to concentrate steroid hormones (Kelley
et al., 1975; Morrell et al., 1975; Kelley, 1980) and have been
shown to express mRNA for the androgen receptor (Pérez
et al., 1996). More recently, functional contributions of these
vocal system components and the mechanisms whereby steroid
hormones influence the vocal system have been investigated. In
the following section we describe known components of the
vocal system and briefly describe the hormones that regulate
their development and function.
2.08.3.1 Sex Differences and Hormone Targets in the Larynx
The adult X. laevis vocal organ is markedly sexually dimorphic
both in size and in tissue components and is functionally
specialized for producing sex-specific vocal behaviors
(described in detail below). Male larynges contain a greater
number of muscle fibers than female larynges and express
a number of structural and functional adaptations that support
the production of fast rhythmic vocalizations. Female laryngeal
features support the production of slower calls (Tobias and
Kelley, 1987; Sassoon and Kelley, 1986; Sassoon et al., 1987;
Marin et al., 1990; Tobias et al., 1995). Larynges of both sexes
express androgen and estrogen receptors (Segil et al., 1987;
Fischer et al., 1995; Wu et al., 2003). At metamorphosis,
male and female larynges are structurally and functionally
similar; sex-specific traits arise during juvenile development.
All identified sexually distinct laryngeal traits are established
and maintained by either androgens or estrogens. Although
some traits gradually lose hormone sensitivity, most sex-
specific traits remain at least partially steroid sensitive in adult-
hood (Zornik and Kelley, 2011).
2.08.3.2 Sex Differences and Hormone Targets in the Vocal
Circuit
2.08.3.2.1 Motor Circuit
Laryngeal muscle fibers are innervated by motor neurons located
in cranial nerve nucleus (n.) IX-X in the caudal hindbrain
(Kelley, 1980; Wetzel et al., 1985). Molecular markers
(transcription factors and neurotransmitter enzymes) support
the homology of the Xenopus n.IX-X to nucleus ambiguus of
mammals (Albersheim-Carter et al., 2016). As in the sexually
138 Hormones and Vocal Systems: Insights from Xenopus
dimorphic larynx with its greater number of muscle fibers in
males, the number of male vocal motor neurons in n.IX-X is
greater than in females (Kay et al., 1999). Unlike the larynx,
however, differences in laryngeal motor neuron number
develop before metamorphosis (Kelley and Dennison, 1990),
arising through an androgen-dependent reduction in cell death
in males compared to females, a process that can be partially
rescued in females by androgen exposure (Kay et al., 1999). In
adulthood, motor neurons remain sensitive to androgens, and
many, but not all, of the laryngeal motor neurons express
androgen receptors (Perez et al., 1996). Following axotomy in
adult males, the total number of n.IX-X motor neurons is
greatly reduced, and cell loss can be partly prevented by DHT
treatment in both males and females (Pérez and Kelley, 1996).
Androgens upregulate expression of the calcium-binding
protein calbindin in axotomized female vocal motor neurons,
suggesting a possible mechanism for its neuroprotective effects
(Perez and Kelley, 1996).
Vocal rhythms are generated by a hindbrain CPG (Rhodes
et al., 2007). The two primary vocal CPG nuclei are n.IX-X in
the caudal medulla, which contains both motor neurons and
interneurons, and a nucleus, DTAM (used as a proper noun),
located in the anterior brain stem (Figure 3(a)). These two
CPG nuclei are reciprocally connected via n.IX-X projection
neurons (Wetzel et al., 1985; Zornik and Kelley, 2007), and
communication between them is critical for generating vocal
rhythms (Rhodes et al., 2007; Zornik et al., 2010). In
addition to motor neurons, some interneurons in n.IX-X also
express androgen receptors, as do neurons in DTAM (Pérez
et al., 1996). Thus the two primary CPG nuclei are both
under direct control of androgens (Figure 3(b)).
2.08.3.2.2 Auditory Pathways
Androgen receptors are expressed in the auditory ganglion,
indicating that peripheral coding of auditory information is
regulated by gonadal steroids (Pérez et al., 1996). An important
auditory processing nucleus, the laminar nucleus of the torus
semicircularis (the homologue of the mammalian inferior
colliculus) located in the midbrain, concentrates three classes
of steroid hormones: androgens, estrogens, and progesterone
(Figure 3(b); Morrell et al., 1975; Kelley, 1980). The basic
sensory–motor integration pathways have been identified
anatomically. Auditory information appears to primarily travel
from the torus to central thalamus (CT) and then on the central
amygdala (CeA), which completes the vocal pathway circuit by
projecting back to the CPG via DTAM (Figure 3(b); Hall et al.,
2013). Neurons in the CeA express estrogen receptors and
luteinizing hormone receptors (Figure 3(b); Morrell et al.,
1975; Yang et al., 2007). Thus, nearly every component of
the anatomically identified auditory circuit is sensitive to
reproductive hormones, indicating that sensory processing of
vocal signals and sensorimotor integration are likely to be as
sexually distinct as the behaviors themselves.
2.08.4 Generating Sexually Differentiated
Vocalizations
Vocal behaviors of males and females differ markedly in at least
three ways.
1. Rate: Except for ticking and amplectant calls, male vocali-
zations consist of rapid sound pulses, between 30 and
80 Hz, whereas the fastest female call does not exceed
12 Hz.
2. Complexity: Male advertisement and answer calls are
temporally complex: trills with different durations and
pulse repetition rate alternate. Both female calls are simpler
trains of sound pulses.
3. Spectral features: Male advertisement and answer calls are
spectrally complex: each sound pulse includes two well-
defined frequency peaks. Sound pulses in both female
calls contain a single, less sharply defined peak.
In this section, we discuss each class of sexually differenti-
ated vocal features, focusing on the operation of multiple levels
of mechanistic control and the role of hormonal regulation.
2.08.4.1 Generating Fast and Slow Calls
The most dramatic vocal difference between the sexes is pulse
rate. The biphasic male advertisement call consists of two
rate-specific phases, fast trill and slow trill. The typical fast trill
pulse rate is 60 Hz (interpulse interval: 16 ms), while the
slow trill pulse rate is 30 Hz (interval, 30 ms). In contrast,
rates of sound pulses in the female receptive call, rapping, range
from 8 to 12 Hz (interval, 80–125 ms), much slower than
male advertisement calls. The female unreceptive call, ticking,
is even slower, with pulse rates at 4 Hz. How are these sex
differences in sound pulse rhythms (biphasic vs monophasic)
and rates (fast vs slow) produced?
Two reduced preparations – the isolated larynx and brain –
have greatly advanced our understanding of the mechanisms
underlying sex-specific vocal behaviors. We review our current
understanding of how sex-specific vocalizations are generated
and perceived and discuss experiments aimed at determining
the precise role of hormones in shaping vocal output.
2.08.4.1.1 Larynx
Xenopus vocalizations are produced under water by the contrac-
tion of a single muscle group, the laryngeal dilators. Each
sound pulse in a call is generated by a synchronous bilateral
contraction of these muscles (Tobias and Kelley, 1987; Yager,
1992). Investigations of laryngeal mechanisms of sound
production have been greatly facilitated by the fact that the iso-
lated larynx can generate sounds in vitro when both laryngeal
nerves are stimulated synchronously (Tobias and Kelley,
1987). This experimental preparation – the ‘vox in vitro’ – has
allowed the identification of numerous sex-specific traits of
male and female vocal muscle.
Sound pulses are generated when laryngeal cartilages, the
arytenoid discs, are rapidly pulled apart by a tendon into which
the laryngeal muscle inserts; before the next pulse can be gener-
ated, muscles must relax completely to allow laryngeal carti-
lages to regain contact (Yager, 1992). Speed of contraction
and relaxation are major functional differences between male
and female larynges. Male laryngeal muscle fibers are fast
twitch, and the muscles can fully contract and relax when stim-
ulated at fast trill rates, 60 Hz (Tobias and Kelley, 1987). In
contrast, female laryngeal muscle cannot generate sound pulses
faster than 30 Hz (associated with the upper limit for pulse

intervals in rapping; Tobias and Kelley, 1987; Tobias et al.,
1998b). Above these sex-typical rate ceilings, the fused tension
that can be recorded from the tendon prevents the arytenoid
discs from regaining contact; subsequent sound pulses cannot
be generated in response to nerve activity.
The sexually differentiated expression pattern of a larynx-
specific myosin heavy chain is a major factor underlying sex-
specific contraction rates. Adult male larynges contain a single
fiber type – medium-sized, fast-twitch muscle fibers – whereas
female larynges contain a mixture of fast-twitch and slow-
twitch fibers (Sassoon and Kelley, 1986). The sex difference
in fiber types reflects expression of a laryngeal-specific myosin
heavy chain (LM) in all male muscle fibers, but only in a subset
of female muscle fibers (Catz et al., 1992). The development of
ubiquitous LM expression in males requires androgen, and
DHT-exposure in juvenile females upregulates LM expression.
In addition, androgen treatment in females leads to
a dramatic increase in the number of histologically identified
fast-twitch fiber types (Sassoon et al., 1987) and also leads to
rapid increase in contraction and relaxation rate of the
muscle (Potter et al., 2005). Although long-term castration of
adult males does not affect the histological profiles of male
muscle fibers (Sassoon et al., 1987), there is a dramatic
slowing of muscle contraction and an accompanying increase
in fused tension during nerve stimulation trains at fast trill
rates (Zornik and Yamaguchi, 2011). Whether this castration-
induced slowing of the male larynx is due to effects on LM
(as opposed to other components of the muscle’s contractile
machinery) is not known. In summary, larynges of males and
females support the sex-specific calls: male larynges are
specialized for generating fast calls, while female vocal organs
can generate only relatively slow calls. These laryngeal
characteristics are androgen-dependent and many remain
hormone-dependent in adulthood.
2.08.4.1.2 Central Pattern Generator
Laryngeal muscles provide constraints on the rates of sound
pulse generation but do not shape most features of vocal
patterning; these are generated within the brain. Yamaguchi
and Kelley (2000) recorded from the laryngeal nerve of male
and female frogs during vocalization and found that each
sound pulse is preceded by a compound action potential
(CAP). For both sexes and all call types, synchronous firing
of laryngeal motor neurons in the brain generates a CAP in
the nerve that, in turn, induces rapid contraction of the laryn-
geal dilators and a resulting sound pulse. Thus, the temporal
features of each Xenopus call primarily reflect patterns of brain
activity.
Because the temporal patterns of nerve activity and sound
are matched, vocal ‘intention’ can be identified unambiguously
by measuring nerve activity. This feature of the Xenopus vocal
system has been put to effective use through the development
of a ‘singing brain in a dish’ preparation (Rhodes et al., 2007).
The CNS from forebrain to hindbrain is removed from the
frog, pinned to a coated dish, and maintained in an oxygenated
artificial cerebrospinal fluid while recording from the laryngeal
nerve, the most caudal nerve rootlet of N.IX-X (Simpson et al.,
1986). Bath-application of serotonin (5-HT) reliably induces
‘fictive vocalizations’ – nerve activity that matches the sound
pulse patterns of singing frogs (Rhodes et al., 2007); some
Hormones and Vocal Systems: Insights from Xenopus 139
spontaneous vocal-like nerve activity patterns can also be
recorded from the isolated brain (e.g., fictive amplectant
calling; Zornik and Kelley, 2008). Though 5-HT initiates fictive
calling in both sexes, the patterns are sex-specific as they are
in vivo; 5-HT induces fictive advertisement calls in male brains
and fictive ticking in female brains.
The isolated brain preparation facilitates localization of the
vocal pattern generator to major brain regions. Transections of
the isolated brain at the border between the midbrain and
hindbrain do not eliminate 5-HT-induced fictive calls, indi-
cating that the vocal CPG is located in the hindbrain (Yu and
Yamaguchi, 2010). The vocal CPG includes two reciprocally
connected nuclei: n.IX-X in the caudal hindbrain and
a premotor nucleus, DTAM, in the rostral hindbrain (Brahic
and Kelley, 2003; Rhodes et al., 2007). In Xenopus, both vocal
CPG nuclei were first identified by their ability to concentrate
androgen (Kelley, 1980), and both are sexually dimorphic
(Wetzel et al., 1985; Kay et al., 1999; Brahic and Kelley,
2003). Homologues of these two nuclei are also believed to
participate in generating vocal rhythms in a distantly related
frog species, Rana pipiens (Schmidt, 1992), and expiration in
lamprey (‘paratrigeminal respiratory group’ nucleus; Cinelli
et al., 2013; Leininger and Kelley, 2015) and mammals
(parabrachial complex; Dick et al., 1994; Song and Poon,
2009). It is likely that vocal CPGs in many, if not all, frog
species comprise these two nuclei and that both contribute to
coordinating vocalization with breathing in mammals.
2.08.4.1.2.1 Motor Neurons
As described above, many, but not all, laryngeal motor neurons
express androgen receptors (Kelley, 1980; Pérez et al., 1996),
which suggests an ongoing role for androgens in maintaining
appropriate motor neuron function throughout adulthood.
Motor neuron firing rates are significantly higher during fictive
vocalizations in males than in females, and membrane proper-
ties and firing patterns of motor neurons express sexually
distinct features for generating their sex-specific motor patterns.
The majority of male motor neurons spike with short latency
following current injection and are thus well suited to
the demands of rapid firing with high temporal precision
(Yamaguchi et al., 2003). In contrast, female motor neurons
have longer spike onset times (Yamaguchi et al., 2003), which
may account for the less precise firing patterns, and resulting
longer nerve CAPs, than males during both in vivo and ex vivo
vocalizations (Yamaguchi and Kelley, 2000; Rhodes et al.,
2007). Patch-clamp recordings in slice preparations also
revealed that male motor neurons have higher capacitance
than female motor neurons (Yamaguchi et al., 2003). Larger
motor neuron somata volumes in male motor neurons could
contribute to these sex differences (Potter et al., 2005).
The size difference is highly plastic, and female motor neurons
increase to male size after only 1 week of testosterone
treatment.
2.08.4.1.2.2 Premotor Neurons
The major source of input to laryngeal motor nucleus is the
premotor nucleus, DTAM, located in the anterior hindbrain
(Wetzel et al., 1985; Zornik and Kelley, 2007). Whole-cell
patch-clamp recordings in isolated male brains during
fictive advertisement calling have revealed a population of
140 Hormones and Vocal Systems: Insights from Xenopus
premotor neurons that are active only during fictive fast trills
(Zornik and Yamaguchi, 2012). These fast trill neurons
produce long-lasting depolarizations that coincide with each
fast trill. During these depolarizations, fast trill neurons produce
action potentials that precede each fast trill CAP recorded from
the nerve. Most fast trill neurons project directly to n.IX-X,
suggesting that these neurons provide premotor signals that
drive motor output. Earlier physiological studies showed that
DTAM neurons provide monosynaptic glutamatergic excitatory
input to laryngeal motor neurons (Zornik and Kelley, 2008),
supporting the notion that synchronous firing of fast trill
neurons is responsible for generating each fast trill CAP.
When depolarized by current steps, fast trill neurons
preferentially generate spikes around 60 Hz, the approximate
rate of fast trills (Zornik and Yamaguchi, 2012). Intrinsic fast
trill neuron membrane properties may be tuned to fire at
60 Hz, and thus be critical for setting the fast trill rhythm.
DTAM neurons express androgen receptors (Kelley, 1981; Pérez
et al., 1996), and it is likely that fast trill neurons in particular
are directly regulated by androgens. When androgens were
ablated by castration, the rate of fictive fast trills was reduced
over a period of months, indicating a role for androgens in
maintaining normal fast trill rhythms (Zornik and Yamaguchi,
2011). We hypothesize that androgens act directly on fast
trill neurons to ensure high levels of expression of androgen-
regulated proteins that permit firing at 60 Hz for long
periods and that the loss of these properties in the absence of
androgen shifts vocal rhythms. Exactly how androgens act to
regulate fast trill neurons and possible roles for an activity-
dependent mechanism remain to be explored.
The sensitivity of sexually differentiated behavioral systems
to reproductive hormones depends on developmental stage:
early actions can produce permanent changes in the behavioral
repertoire, while actions during adulthood can be transient
(Phoenix et al., 1959). Sensitivity to androgens is often studied
by manipulating hormone levels across development. For
X. laevis, androgen promotion of clasping serves as an example
of a transient action of a hormone: abolished in adult males by
gonadectomy, reinstated by androgen replacement, and
induced, in a form not distinguishable from that of males, in
adult females by androgen treatment (Kelley, 1982). Similarly,
male calling is abolished by castration and reinstated by
androgen replacement (Wetzel and Kelley, 1983). However,
masculinization of calling in females appears less malleable.
Watson and Kelley (1992) provided testis implants or
androgen pellets to juveniles at a number of postmetamorphic
stages. They noted a decline in the ability to masculinize call
temporal patterns with age in females but not in males.
Long-term testis implants and androgen treatment (10–
15 months) in adult females, however, did induce some
biphasic advertisement calls. Potter et al. (2005) found that
within 8 weeks, androgen-treated adult females produced
biphasic, advertisement call-like vocalizations. Their fast trills
approached, but did not quite equal, the 60 Hz rates typical
of males. These results confirm that the adult female CPG can
be masculinized to generate male-like vocal rhythms, albeit
incompletely. While the processes underlying this androgen-
induced masculinization of adult females is unclear, cells fated
to become fast trill neurons are likely participants. Identifying
androgen-induced functional, structural, and/or synaptic
changes in these neurons that drive the ontogeny of fast trill
rhythms is a challenge for the future. A goal is to identify
both CPG components that remain androgen-sensitive, as
well as those that lose androgen-sensitivity and thus constrain
masculinization in adulthood.
2.08.4.2 Generating Complex and Simple Vocal Patterns
2.08.4.2.1 Biphasic versus Monophasic Calling
Fast trill neurons in DTAM produce long-lasting depolariza-
tions that coincide with each fast trill (Zornik and Yamaguchi,
2012). These depolarizations require NMDA receptor
signaling and are blocked by NMDA receptor antagonists. In
the presence of TTX (to block spike-mediated synaptic trans-
mission) and NMDA (to activate NMDA receptors), fast trill
neurons generate membrane potential oscillations in which
the duration and period of each depolarization is similar to
the duration and period of fast trills. The constellation of ion
currents in DTAM must support membrane potential oscilla-
tions with advertisement call patterns: that is, about one call
per second, with each fast trill lasting about 300 ms. Slow trills
only occur while fast trill neurons are hyperpolarized. Thus fast
trill neurons may be largely responsible for controlling the
temporal patterns of male advertisement calls.
Because fast trill neurons produce these membrane poten-
tial oscillations synchronously, population–level activity in
DTAM can be readily recorded as local field potential (LFP)
waves that correspond to fast trills. DTAM activity in females
does not reveal the LFP waves seen in males (Ballagh, 2014).
During androgen treatment, however, females gradually
develop biphasic calls. Masculinization of existing neurons in
female DTAM probably involves the rearrangement of ion
currents to promote long-lasting depolarizations that lead to
membrane oscillations with an 1 Hz period.
During 5-HT evoked fictive ticking in the ex vivo female
brain, CAP rates recorded from the laryngeal nerve occur
at 4 Hz (Rhodes et al., 2007). In the initial stages of
testosterone-induced masculinization, single sound pulses
transition to doublet, triplet, and quadruplet pulse bursts
(Hannigan and Kelley, 1986; Potter et al., 2005). The appear-
ance of pulse bursts could be due to prolonged depolarization
in female premotor neurons.
Which neuronal properties are most strongly regulated by
androgens and which neuronal properties are most closely
associated with overall circuit activity? Elongating bursts may
be correlated with increasing NMDA receptor current density.
Alternatively, NMDA currents may change little during mascu-
linization; instead, an upregulation of slower outward currents
could underlie changes in temporal patterns of vocal output.
Combining androgen-induced masculinization treatments in
adult females with physiological recordings in the isolated
brain will permit the identification of steroid-induced neuronal
traits that contribute to sex-specific vocal characteristics.
2.08.4.2.2 Intensity Modulation of Vocal Trills
2.08.4.2.2.1 Synaptic Strength in the Larynx
In addition to rhythm and rate, the intensity modulation of
male and female calls also differs. In male advertisement and
answer calls, sound pulses early in fast trills are of low
intensity and become gradually louder throughout each trill;

female ticking and rapping sound pulses, in contrast, do not
vary systematically in intensity (Figure 1; Tobias et al.,
1998b). The laryngeal neuromuscular synapse contributes to
intensity modulation. When the male laryngeal nerve is stimu-
lated at advertisement call rates ex vivo, amplitudes of the elec-
tromyogram, tension, and sound amplitudes all increase
progressively throughout the stimulus train (Tobias and Kelley,
1987). This increase in amplitudes reflects the use-dependent
facilitation of the weak male laryngeal synapse (Tobias et al.,
1995; Ruel et al., 1998). In contrast, females have strong laryn-
geal synapses, well suited to ensuring that slower rates of nerve
activity are faithfully converted to sound. The sex difference in
synaptic strength arises presynaptically and is due to elevated
quantal content at the synaptic terminals of female motor
neurons (Tobias et al., 1995). Strong female laryngeal synapses
arise during juvenile development and require prolonged
elevated estrogen levels; ovariectomy leads to a decrease in
synaptic strength (Tobias et al., 1998a). In contrast, androgen
treatment weakens female synapses (Tobias and Kelley,
1995). In adult males, long-term castration appears to reduce
male-typical EMG potentiation during nerve stimulation
trains, reflecting castration-dependent synaptic strengthening
(Zornik and Yamaguchi, 2011).
Laryngeal neuromuscular junctions are well suited to gener-
ating intensity-modulated male calls via weak facilitating
synapses and slower female calls via strong synapses, both of
which depend on appropriate androgen and estrogen levels
during development and in adulthood. Thus, as is the case
for clasping behavior, synaptic strength displays endocrine-
regulated flexibility, even in adulthood.
2.08.4.2.2.2 Motor Neuron Recruitment
Laryngeal nerve recordings in singing frogs revealed that CAP
amplitudes during fast trills also progressively increase
throughout each trill (Yamaguchi and Kelley, 2000), and this
pattern is preserved in fictive vocalizations (Rhodes et al.,
2007). The most likely mechanism is sequential recruitment of
motor neurons. The neural basis for this recruitment appears
to have at least two components. First, the premotor fast trill
neurons are gradually recruited across each trill. Early in a trill
only a few fast trill neurons spike; the number of spiking
neurons then increases, and nearly all fast trill neurons spike
at the end of a trill (Zornik and Yamaguchi, 2012). Second,
the DTAM-to-motor neuron synapses facilitate; when DTAM is
electrically stimulated in trains from 20 to 60 Hz, nerve CAPs
progressively increase in amplitude, as is also seen during
in vivo calling (Zornik and Kelley, 2008). Thus, intensity
modulation of fast trills is coded in three ways: premotor
neuron activation, premotor-to-motor synaptic facilitation,
and neuromuscular junction facilitation. We know that the
weak, facilitating male neuromuscular synapse is regulated by
hormones. Because both DTAM and motor neurons express
androgen receptors, it is likely that some aspects of this
trait are also androgen-dependent. Finally, because of the
redundant mechanisms underlying intensity modulation, this
aspect of fast trills could be a behaviorally critical component
of the communication signal, a hypothesis supported by the
even more dramatic intensity modulation of the answer call
during male/female duets (Tobias et al., 1998b).
Hormones and Vocal Systems: Insights from Xenopus 141
2.08.5 Processing Vocal Signals
2.08.5.1 Sexually Differentiated Auditory Processing
Auditory sensitivity also appears to differ in females and males.
While no sex differences in spectral preference were identified
in auditory nerve fibers using single-cell electrophysiological
recordings (N.VIII; Elliott et al., 2007), auditory brain stem
response (ABR) measurements have revealed sex-specific
frequency preferences (Hall et al., 2016). Xenopus laevis male
sound pulses contain two major DFs: 1.9 and 2.2 kHz
(Figure 2). As would be predicted from the matched-filter
hypothesis (Frishkopf et al., 1968; Capranica and Moffat,
1983; Moreno-Gomez et al., 2013; Simmons, 2013), in
females, ABR thresholds for combinations of these two
frequencies are lower than either frequency alone; small shifts
in either abolish this enhanced sensitivity. This enhanced sensi-
tivity is absent in males. Instead, ABR sensitivity peaks at lower
frequencies, just under 1.5 kHz.
These results indicate that, at the level of the hindbrain, the
female auditory system is more highly tuned to male call
frequencies than is the male auditory system; this acoustic
advantage may facilitate locating a mate. Males also communi-
cate vocally, but most of their vocal interactions are close
range, often while clasping, and enhanced sensitivity may be
less important. However, males do establish vocal dominance
hierarchies, which are sensitive to advertisement call intensity
(Tobias et al., 2010). Relative insensitivity to advertisement
calls could release males from a vocally subordinate condition
at shorter distances from a dominant male. Field recordings
do indicate that very few frogs advertise on any given night
(Tobias et al., 2004); the number may depend on how well
sound travels in particular bodies of water.
An alternative possibility is that slow trills, which contain
a lower frequency band below 1.5 kHz not present in fast trill
sound pulses (Figure 2; Yamaguchi et al., 2010), may be the
more salient feature for establishing dominance. This hypoth-
esis has some support from experimental evidence: a subset
of males was preferentially vocally suppressed by artificial
slow-trill-only stimuli, which were more effective than either
fast-trill-only stimuli or natural advertisement call playbacks
(Tobias et al., 2010). If true, these findings may indicate
a sex-specific tuning to distinct features of the male advertise-
ment call: females attending to fast trills, males attending to
slow trills. This arrangement is analogous to sex-specific prefer-
ences in the tree frog, Eleutherodactylus coqui, in which the male
auditory system preferentially responds to the first ‘co’ note of
a male calls, while female auditory systems preferentially
respond to the second ‘qui’ component (Narins and Capranica,
1976).
Male auditory sensitivity may also be tuned to female
frequencies, which have a DF 1.2 kHz (Vignal and Kelley,
2007). Although the unreceptive call, ticking, is used at close
range, the female ‘advertisement call,’ rapping, is a high-inten-
sity signal that should carry longer distances under water,
allowing males to find distant receptive mates (Tobias et al.,
1998b).
Sex-specific auditory sensitivities are steroid-dependent.
Ovariectomy masculinized female sensitivity to DF1–DF2
dyads and to frequencies between 1 and 1.5 kHz, while
androgen treatment with DHT prevented this masculinization
142 Hormones and Vocal Systems: Insights from Xenopus
(Hall et al., 2016). These results are consistent with the finding
that androgens are the primary circulating steroids in female
X. laevis (Lutz et al., 2001). These results also suggest a possible
functional role for the observation that androgen receptors are
expressed in the auditory ganglion (Pérez et al., 1996), but one
would then expect a similar change in males. It is possible that-
male and female auditory ganglia respond differently to
androgen exposure, or that there is a dose–response difference
between the sexes. Alternatively, sex-specific production of
the DHT metabolite 5a-androstane-3b,17b-diol (3b-diol),
a potent agonist of ERb, could be a DHT-dependent source of
estrogen (Oliveira et al., 2007). Although ERb expression in
the Xenopus auditory periphery has not been explored, if
estrogen receptors are expressed in hair cells of the inner ear,
as they are in vocal fish (Forlano et al., 2015), then selective
production of 3b-diol only in female hair cells could account
for DHT-dependent feminization of the auditory periphery.
2.08.5.2 Generating Socially Appropriate Vocal Responses
To facilitate effective social interactions, vocal responses of
a receiver must be appropriately based on auditory information
from the sender. Examples of socially appropriate vocal
responses in X. laevis include the stimulation of male answer
calling by female rapping, the transient suppression of male
calling by female ticking, and the prolonged suppression of
male calling by intense advertisement calling of another male
(Figure 1; Tobias et al., 1998b, 2004; Elliott and Kelley,
2007). Activation of CNS auditory regions must be sufficient
to promote appropriate vocal responses since broadcast calls
are effective in eliciting these responses. The CeA plays a critical
role in matching auditory signals to call-type responses (Hall
et al., 2013). Neurons in the CeA receive synaptic input from
the CT, which in turn receives inputs from the auditory
midbrain (Figure 3). When the CeA is lesioned, males continue
to generate spontaneous advertisement calls, and thus are not
mute. Lesioned males also can still respond to intense adver-
tisement call broadcasts with prolonged vocal suppression,
and thus are not deaf. However, both ticking and rapping
also elicit prolonged vocal suppression, a response that intact
males do not normally make to either female call. In addition
to socially inappropriate vocal suppression to female calling,
CeA-lesioned males also produce a lower proportion of
answer calls when paired with another male. These results indi-
cate that the CeA may be a positive regulator of vocal responses,
as such responses are diminished after lesions. Because the
CeA is reciprocally connected with the CPG nucleus DTAM
(Figure 3), there is a direct anatomical pathway by which
CeA may activate socially appropriate vocal responses.
2.08.6 Evolution of Vocal Effectors in Xenopus
The ex vivo brain and larynx preparations have been used to
probe the mechanisms underlying the evolutionary convergence
of a rare vocal pattern in Xenopus (Leininger and Kelley, 2013;
Leininger et al., 2015). Each Xenopus species has a unique adver-
tisement call that can be distinguished by call temporal patterns
and spectral properties of sound pulses (Tobias et al., 2011).
Three classes of call pattern can be identified: fast and
long-lasting (trill-type (as in X. laevis)), fast and brief (burst-
type), and slow and ongoing (click-type). Each type occurs in
multiple branches of the phylogeny, and parsimony suggests
that the ancestral call type was burst-type. The click-type pattern
occurs in two species – Xenopus boumbaensis and Xenopus borealis
– which are found on distantly related branches of the Xenopus
phylogeny, indicating that their click-type calls evolved indepen-
dently. As in X. laevis, the X. boumbaensis larynx is strongly
sexually dimorphic; male fibers are large and fast twitch, while
female fibers are small and mixed twitch type. Because male
X. boumbaensis laryngeal fibers are mostly fast twitch, they could
support the production of fast trills as found in X. laevis.
However, the CPG generates very short vocal bursts consisting
of two CAPs in the nerve (Leininger and Kelley, 2013). These
doublets are translated into single sound pulses due to the laryn-
geal synapse’s requirement for facilitation (see Section
2.08.4.2.2.1). In X. borealis the larynges are less dramatically
sexually dimorphic. Male and female muscle fibers are both
a mixture of fast and slow twitch, though male fibers are larger
and greater in number (Leininger et al., 2015). The X. borealis
vocal CPG produces only single CAPS. Because the male
X. borealis laryngeal synapse does not require facilitation, the
single CAPs in the nerve faithfully produce single sound pulses.
Thus, the vocal CPG rhythm and the laryngeal synapse
in X. borealis are similar to features associated with ticking in
female X. laevis. Since male laryngeal muscle fibers in the very
distantly related species Xenopus tropicalis are entirely fast twitch
(Baur et al., 2008), this feature is presumed to represent the
ancestral state. The convergent evolution of click-type calls in
X. borealis and X. boumbaensis are thus due to distinct mecha-
nisms: in X. boumbaensis, the number of CAPs produced by the
CPG is greatly reduced, while in X. borealis, the developmental
program for the CPG, the laryngeal synapse, and muscle fiber
types are all demasculinized (Leininger and Kelley, 2015).
2.08.7 Conclusion
Despite the conceptual value of the organization-activation
dichotomy (Phoenix et al., 1959), this stark distinction is often
an oversimplification with many exceptions (Arnold and
Breedlove, 1985; Tobet and Fox, 1992; Arnold, 2009; McCarthy
and Nugent, 2013). Hormonal regulation occurs throughout
development and into adulthood. While some traits are irre-
versibly set during a narrow critical period, others retain full
hormone sensitivity throughout life, while still others retain
intermediate sensitivity. Xenopus vocalizations are complex,
context-dependent and sexually differentiated, making them
excellent subjects for identifying the role of reproductive
hormones in regulating behaviors. The two ex vivo vocal prepa-
rations, the isolated larynx and brain, have permitted
numerous advances in understanding the neural and muscular
mechanisms of vocal production. Each can produce fictive
vocalizations independent of the other, a feature that has
enabled identification of separate hormonal effects on discrete
vocal effectors that work together in the animal. For example,
although the larynges of many androgen-treated females are
not capable of generating sound pulses at fast trill rates, the
isolated brain preparation permits an accurate assessment of
the hormone sensitivity of the vocal CPG in adults. Similarly,

castration leads to nearly complete elimination of in vivo male
calling, yet fictive vocalizations can still be generated by the
isolated brain, allowing the identification of castration-
induced functional changes in the CPG. Because the full
acoustic communication pathway from ear to brain to vocal
organ has been outlined, with nearly every component known
to be hormone-sensitive, the Xenopus vocal system provides an
experimentally powerful preparation for discovering novel
roles for hormone-directed behaviors and should offer new
insights about fundamental mechanisms for sexually differen-
tiated vertebrate behaviors.
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 2.09 Endocrinology of Complex Life Cycles: Amphibians
Robert J Denver, The University of Michigan, Ann Arbor, MI, USA
Ó 2017 Elsevier Inc. All rights reserved.

2.09.1 Most Amphibian Species Have Complex Life Cycles 146

2.09.2 Endocrinology of Metamorphosis 146
2.09.2.1 Overview 146
2.09.2.2 Thyroid Hormone 148
2.09.2.2.1 Role of Thyroid Hormone in Amphibian Development 148
2.09.2.2.2 Thyroid Gland Development and Hormone Production 148
2.09.2.2.3 Thyroid Hormone Metabolism 148
2.09.2.2.4 Thyroid Hormone Transport in Blood 151
2.09.2.2.5 Cellular Uptake of Thyroid Hormone 152
2.09.2.2.6 Mechanisms of Thyroid Hormone Action 152
2.09.2.3 Corticosteroids 154
2.09.2.3.1 Development of and Hormones Produced by Amphibian Interrenal Glands 154
2.09.2.3.2 Roles of Corticosteroids in Amphibian Growth and Development 154
2.09.2.4 Neuroendocrine Control of Metamorphosis 155
2.09.2.4.1 Developmental Expression and Regulation of TSH 156
2.09.2.4.2 Neurohormones Regulating TSH 156
2.09.2.4.3 Other Neurohormones That Regulate TSH Secretion 157
2.09.2.4.4 Negative Feedback Regulation of TSH 158
2.09.2.4.5 Developmental Expression and Regulation of Adrenocorticotropic Hormone 159
2.09.2.4.6 Prolactin and Growth Hormone 159
2.09.3 Role of the Neuroendocrine System in Mediating Environmental Influences on the Timing
of Metamorphosis 160
2.09.4 Future Directions 161
Acknowledgment 162
References 162

Glossary
Adrenocorticotropic hormone (ACTH) Small
polypeptide hormone derived from a larger precursor
(proopiomelanocortin) produced by the anterior
pituitary gland that stimulates the adrenal cortex
(interrenal glands in nonmammalian species) to produce
corticosteroids (primarily glucocorticoids).
Corticotropin-releasing hormone (CRH) Forty-one-
amino-acid polypeptide produced in the hypothalamus
and extrahypothalamic sites that stimulates the release of
ACTH (all vertebrates studied) and TSH (nonmammalian
vertebrates) by the anterior pituitary gland. CRH is
a member of a family of peptides of similar size that
include the urocortins, urotensin I, and sauvagine. CRH-
like peptides play central roles in developmental,
behavioral, and physiological responses to stressors.
Growth hormone (GH) Simple polypeptide hormone
produced by the anterior pituitary gland that stimulates
growth in all vertebrate species studied.
Metamorphic climax The final and most rapid phase of
morphological change when thyroid activity is at its peak.
Paedomorphosis Reproductive maturity is attained while
in a larval or branchiate form.
Premetamorphosis Stage of amphibian larval
development when the animal grows but little or no
morphological change occurs; plasma thyroid hormone
concentrations are low.
Prolactin (PRL) Simple polypeptide hormone produced
by the anterior pituitary gland that stimulates lactation
in mammals and has antimetamorphic effects in
amphibians.
Prometamorphosis Stage of amphibian larval
development when metamorphosis begins. Hindlimb
growth and development is evident externally. The thyroid
gland becomes active and secretes thyroid hormone in
response to increasing plasma concentrations of pituitary
thyroid stimulating hormone (TSH).
Thyroid stimulating hormone (TSH) Glycoprotein
hormone comprised of two subunits produced by the
anterior pituitary gland that stimulates the production of
thyroid hormone by the thyroid gland.
Thyrotropin-releasing hormone (TRH) Tripeptide
produced in the hypothalamus and extrahypothalamic
sites that stimulates the release of TSH by the anterior
pituitary gland.

Hormones, Brain, and Behavior, 3rd edition, Volume 2 http://dx.doi.org/10.1016/B978-0-12-803592-4.00024-9 145

146 Endocrinology of Complex Life Cycles: Amphibians
2.09.1 Most Amphibian Species Have Complex Life
Cycles
Amphibians exhibit considerable diversity in behavioral, phys-
iological, and life history strategies. They are geographically
widespread, occupying a diverse range of habitats. The life
history strategies of amphibian species include complex life
cycles (e.g., metamorphosis) and direct development (Callery
et al., 2001). The majority of amphibian species exhibit
complex life cycles, and thus have two very different life stages
that are affected differently by environmental factors. Most
anuran (frog) larvae are aquatic, and tadpoles are found in
a wide variety of habitats, ranging from water-filled crevices
in rocks, logs, or leaves to larger ponds or streams. Most then
undergo morphological, biochemical, and physiological trans-
formation into adults, which are sensitive to different environ-
mental variables than larvae, due to this shift in habitat
(Duellman and Trueb, 1994). Some amphibians have lost
the larval form and develop directly into the adult morphology
(direct development); others do not metamorphose but repro-
duce in the aquatic habitat while retaining the larval
morphology (paedomorphosis).
Metamorphosis is a stage of many amphibian life cycles
characterized by dramatic morphological transformation that
is accompanied by a transition in ecological niche and behav-
ioral mode. Amphibians that undergo a metamorphosis
exhibit strong variation, both between and within species, in
the duration of the larval period (Wilbur and Collins, 1973;
Werner, 1986). Larvae encounter diverse ecological conditions
during development. Variation in abiotic factors (e.g., water
availability, temperature, photoperiod) as well as biotic factors
(e.g., intra- and interspecific competition, predation) can
interact in complex ways to influence larval growth and devel-
opment (Semlitsch, 1987; Sredl and Collins, 1992; Rowe and
Dunson, 1995; Taylor and Scott, 1997; Alvarez and Nicieza,
2002; Relyea, 2002; Downie et al., 2004; Relyea, 2007). The
timing of metamorphosis is a central amphibian life history
trait that likely reflects the quality and relative permanence of
the larval habitat. Species that breed in predictable habitats
(i.e., permanent or semipermanent lakes and ponds) tend to
have longer larval periods. Species that breed in unpredictable
habitats (i.e., ephemeral pools) generally have much shorter
larval periods (Denver, 1997b).
Wilbur and Collins (1973) suggested that there is
a threshold of minimum body size that must be reached before
metamorphosis is possible and that larval growth rates deter-
mine the timing of metamorphosis after this minimum size
has been attained. Werner (1986) added mortality risk in the
larval and adult habitat to the list of factors that ultimately
influence metamorphosis. The effects of environmental factors
may differ depending on the animal’s stage of development.
These observations led Day and Rowe (2002) to incorporate
developmental thresholds into the Wilbur–Collins model.
Environmental factors that influence growth rate or mortality
risk therefore should alter the timing of metamorphosis, and
the effects of the environment may be influenced by the stage
of development that has been achieved. For example, the
same factor may be inhibitory to growth if present early in
the larval phase or stimulatory to development if present
during metamorphosis (e.g., population density, food
availability, pond drying or predation – reviewed by Denver,
1997b). Thus, body size and stage of development may interact
in complex ways to determine the phenotypic response to
specific environmental variables.
Amphibian larvae exhibit plasticity in the timing of
metamorphosis and can capitalize on favorable conditions
for growth as long as such conditions last (up until a genetically
determined upper limit to the length of the larval period)
(Newman, 1992; Rudolf and Rodel, 2007). Such plasticity
may permit amphibian larvae to match their phenotype
(morphology, physiology, metamorphic timing) to prevailing
environmental conditions. Animals capable of phenotypic
plasticity may have a higher probability of surviving in unpre-
dictable habitats compared with those with a genetically fixed,
or canalized phenotype (Stearns, 1989; Newman, 1992;
Van Buskirk, 2002; Gomez-Mestre and Buchholz, 2006).
The upper and lower limits to the length of the larval period
are determined by genetic factors that are subject to natural
selection. The plasticity of larval period length within these
limits is also subject to natural selection and is influenced at
both proximate and ultimate levels by the environment. While
metamorphic timing is determined by both genetic and envi-
ronmental factors, its expression depends on the development
and activity of endocrine glands and the actions of the
hormones that these glands produce (see below).
Among the most extreme evolutionary modifications of the
ancestral, complex life history is paedomorphosis. Most
amphibian larvae undergo a metamorphosis to an adult form
before becoming sexually mature. Some species of urodele
amphibians (e.g., salamanders, newts) exhibit paedomor-
phosis, where reproductive maturity is attained while in a larval
or branchiate form. Paedomorphosis refers to the retention of
juvenile characteristics in sexually mature adults (Gould,
1977). Paedomorphosis can either be obligate or facultative
depending on the species. Obligate paedomorphs never
undergo metamorphosis and remain in an aquatic habitat their
entire lives (e.g., Necturus, Proteus, Amphiuma, Ambystoma mexi-
canum – axolotl). Facultatively paedomorphic species can
either become paedomorphic and remain in the aquatic
habitat, or metamorphose and move into the terrestrial
environment where they become sexually mature (e.g., Ambys-
toma tigrinum, Ambystoma talpoideum, Ambystoma gracile,
Notophthalmus viridescens) (Duellman and Trueb, 1994). The
developmental ‘decision’ to become paedomorphic or to meta-
morphose in facultative species depends on the prevailing envi-
ronmental conditions rather than the animal’s genotype
(Harris, 1987; Semlitsch, 1987; Licht, 1992; Jackson and
Semlitsch, 1993; Denoel and Poncin, 2001) and may be
controlled by the interplay of antagonistic hormonal pathways
(Wakahara, 1994, 1996; Rosenkilde and Ussing, 1996). The
reader is referred to a recent review by Johnson and Voss
(2013) for a detailed discussion of the hormonal basis for
paedomorphosis in salamanders.
2.09.2 Endocrinology of Metamorphosis
2.09.2.1 Overview
Hormones orchestrate the diverse morphological and physio-
logical changes that occur during metamorphosis and also
 Endocrinology of Complex Life Cycles: Amphibians 147

function as mediators of environmental effects on develop-
ment. A striking characteristic of amphibian metamorphosis
is that a single signaling molecule produced by the thyroid
gland (thyroid hormone – TH) can orchestrate the entire suite
of molecular, biochemical, and morphological changes.
Gudernatsch (1912) first showed that the vertebrate thyroid
gland contained a factor that could induce precocious meta-
morphosis if fed to tadpoles. This compound, later identified
as 3,5,30 ,50 -tetraiodothyronine (thyroxine) (Kendall, 1915;
Harrington, 1926; Harrington and George, 1927) and referred
to as thyroid hormone (TH), is now known to be the primary
hormone controlling amphibian metamorphosis. Thyroid
hormone is required for amphibian metamorphosis (Brown
and Cai, 2007); the hormone initiates gene expression
programs in diverse tissues that lead to cell proliferation, death,
differentiation, or migration (Brown and Cai, 2007). While
hormones produced by the anterior pituitary gland and the
interrenal glands (amphibian homologs of the mammalian
adrenal cortex; corticosteroids – CS) influence the rate of meta-
morphosis by controlling TH production and action on target
tissues, exogenous TH alone can induce the entire suite of
tissue transformations. Furthermore, chemical or surgical
thyroidectomy results in metamorphic stasis (Dodd and
Dodd, 1976; Kikuyama et al., 1993). Neurohormones
produced in the hypothalamus control hormone biosynthesis
and secretion by the pituitary gland, and the hypothalamus
mediates the interaction between the external and internal
environments, and the production of hormones that control
metamorphosis.
The work of William Etkin laid much of the foundation for
our current understanding of the endocrine control of meta-
morphosis. Etkin, (1968) proposed a model for the hormonal
changes that occur during amphibian metamorphosis. He also
coined the terms in common use today among amphibian
endocrinologists for describing the stages of anuran develop-
ment: ‘premetamorphosis,’ when the larvae grow but little or
no morphological change occurs and plasma TH concentra-
tions are low; ‘prometamorphosis,’ when hindlimb growth
accelerates and plasma TH concentration rises; ‘metamorphic
climax,’ the final and most rapid phase of morphological
change when thyroid activity is at its peak (Dodd and Dodd,
1976; White and Nicoll, 1981; Table 1).
The objective of the following section is to describe the cast
of endocrine characters that interact to control metamorphosis.

Table 1 A comparison of three of the most widely cited staging tables for postembryonic, feeding stages of anuran larvae

NF a staging for Major, common diagnostic

Xenopus laevis features/morphological changes TK staging b Gosner staging c Terminology of Etkin d

1–45 Nonfeeding stages (comparable to 1–25

Shumway stagese 1–24)
46 I 26 Premetamorphosis
47–48 Feeding begins II 27
49–50 III 28
51 IV 29
V 30
52 Foot paddle stages
VI 31
53 VII 32
VIII 33
54 IX 34
X 35
55 Hindlimb stages XI 36 Prometamorphosis
XII 37
56 XIII 38
57–58 XIV–XVI 39–40
59 Tadpole reaches maximum length XVII 40
60 XVIII 41
XIX
61 XX
62 Rapid tail resorption begins, front XXI 42 Climax
limbs erupt*
63 XXII 43
64 XXIII 44
65 Stump of tail remains XXIV 45
66 Tail completely resorbed, juvenile frog XXV 46

Note: Table is derived from similar tables published by Nieuwkoop and Faber (1956), Dodd and Dodd (1976), and Kikuyama et al. (1993) with the
addition of Gosner staging. Note that the table is modified somewhat with respect to the table published by Kikuyama et al. (1993) with deference to
the comparison between Xenopus laevis and the staging of Rana pipiens (Taylor and Kollros, 1946) made by Nieuwkoop and Faber (1956).
Comparison of Taylor and Kollros (1946) with Gosner (1960) staging tables is based on that of Gosner (1960).
*In Xenopus species the front limbs do not erupt, but instead develop outside of the body cavity.
a
Stages of Nieuwkoop and Faber (1956).
b
Stages of Taylor and Kollros (1946).
c
Stages of Gosner (1960).
d
Terminology of Etkin (1968).
e
Stages of Shumway (1940).
148 Endocrinology of Complex Life Cycles: Amphibians
For each endocrine axis involved in metamorphosis I will first
examine its developmental schedule. This should allow predic-
tions of when the endocrine system is sufficiently developed to
allow the animal to become competent to respond to the
external environmental. I will also examine the multiple levels
at which the activity and functioning of each endocrine axis can
be regulated. The goal, which is addressed in Section 2.09.3 on
integration, is to understand how the endocrine system deter-
mines the timing of metamorphosis and mediates environ-
mental effects on amphibian development.
2.09.2.2 Thyroid Hormone
2.09.2.2.1 Role of Thyroid Hormone in Amphibian
Development
Perhaps the most striking characteristic of amphibian meta-
morphosis, from the perspective of hormonal control, is that
a single signaling molecule, produced by a highly restricted
group of cells (the thyroid epithelial cells), can orchestrate
the entire suite of molecular, biochemical, and morphological
changes. Depending on the tissue, TH can induce cell prolifer-
ation, cell death, differentiation, or migration. Target cells for
TH are now known to activate both similar and different sets
of genes according to the concentration of this single signaling
molecule. Specific tissues exhibit different dose sensitivities to
TH, and the challenge for investigators studying the molecular
basis of TH action during metamorphosis is to determine how
and why individual tissues respond differently to the hormone
and exhibit differential dose responses (see Brown and Cai,
2007). Thyroid hormone regulation of metamorphosis has
been best studied in anuran amphibians, which is the primary
focus of this chapter, but it is important to note that TH is also
the primary morphogen in metamorphosing urodele (sala-
mander) species (reviewed by Johnson and Voss, 2013).
2.09.2.2.2 Thyroid Gland Development and Hormone
Production
The thyroid gland develops early in the amphibian embryo
when the anlage consists of a thickening of the pharyngeal
epithelium; these cells are capable of synthesizing small iodo-
proteins (reviewed by Dodd and Dodd, 1976; Regard et al.,
1978). The gland matures functionally at the time of hatching
when it separates into two distinct lobes and is essentially
completely developed by late premetamorphosis/early prome-
tamorphosis (Nieuwkoop and Faber, 1956; Saxen et al.,
1957a,b; Kaye, 1959, 1961; Dodd and Dodd, 1976; Regard
et al., 1978). Multiple measures of thyroid activity including
radioiodine uptake, gland ultrastructure, and plasma concen-
tration or tissue content of THs show that thyroid activity
increases markedly during prometamorphosis (Table 1;
Figure 1), peaks at metamorphic climax, and declines there-
after to reach an ‘adult’ level of activity (Kaye, 1959, 1960;
Dodd and Dodd, 1976; Regard et al., 1978; Kikuyama et al.,
1993). Ultrastructural analyses show a dramatic increase in
thyroid follicular cell height during prometamorphosis with
a peak at metamorphic climax that corresponds to the peak
in plasma concentrations (and tissue content) of THs (Dodd
and Dodd, 1976; Regard et al., 1978).
When Etkin proposed his endocrine-based model for
metamorphosis, investigators at the time did not have
sensitive and quantitative methods for determining plasma
TH concentrations. Early methods relied on the determination
of protein-bound iodide to estimate plasma TH titers (Just,
1972). Subsequently, sensitive and specific radioimmunoas-
says (RIAs) were developed that allowed determinations of
plasma thyroxine (T4; the primary product of the thyroid
gland) and 3,5,30 -triiodothyronine (T3; derived from T4 by
monodeiodination in target tissues) concentrations during
metamorphosis. These studies confirmed earlier studies and
the predictions of Etkin by demonstrating low-to-nondetect-
able plasma TH concentrations during premetamorphosis,
increasing concentrations during prometamorphosis, and
a dramatic peak at metamorphic climax (Figure 1(b); Leloup
and Buscaglia, 1977; Miyauchi et al., 1977; Regard et al., 1978;
Mondou and Kaltenbach, 1979; Suzuki and Suzuki, 1981;
Weil, 1986; Niinuma et al., 1991a; Weber et al., 1994; Denver,
1998a; Krain and Denver, 2004).
Because of the difficulty of obtaining blood from small
tadpoles for analysis by RIA, until recently only those species
with tadpoles large enough to obtain a serum sample were
analyzed. Thus, most blood measurements have been done
on ranid species (e.g., Rana catesbeiana; Rana clamitans;
however, Leloup and Buscaglia (1977) and Tata et al. (1993)
measured THs in plasma pools of Xenopus laevis; see also
Buscaglia et al. (1985) for measures of plasma T3 and T4 in
other Xenopus spp.). In species with small tadpoles, develop-
mental changes in TH content of whole bodies and individual
tissues have been determined. These analyses have shown that
changes in whole body TH content in the smaller species essen-
tially parallel changes observed in the plasma of tadpoles of the
larger species (Bufo japonicus (Niinuma et al., 1991a); Spea
hammondii (Denver, 1993, 1998a); X. laevis (Krain and Denver,
2004); Bufo marinus (Weber et al., 1994)). The peak in whole-
body T3 and T4 coincides with peak uptake of 131I in B. japonicus
(Niinuma et al., 1991a). Thus, it is likely that determination of
whole-body hormone content provides a reasonable estimate
of physiological changes in TH production in species for which
blood samples are unobtainable.
2.09.2.2.3 Thyroid Hormone Metabolism
The major product of the amphibian thyroid gland is T4 with
minor amounts of T3 produced (Rosenkilde, 1978; Buscaglia
et al., 1985). The result is that plasma T4 concentration tends
to be an order of magnitude greater than T3 (Regard et al.,
1978; Larras-Regard et al., 1981). The only case where this rela-
tionship may not hold is for X. laevis where the reported plasma
concentrations of T3 and T4 are very similar, and the T3/T4 ratio
may even exceed one at metamorphic climax (Leloup and
Buscaglia, 1977; Buscaglia, 1985). Measures of tissue content
of T4 and T3 in various species show that the two hormones
are present in roughly similar amounts (Niinuma et al.,
1991a; Weber et al., 1994; Denver, 1997a, 1998a). Although
a comprehensive analysis of both blood concentrations and
tissue contents of THs has not been done for any species, it is
likely that the higher T3:T4 ratio in tissues compared with
T3:T4 ratio in blood reflects high tissue 50 -monodeiodinase
activity (see below).
An important point of control of TH bioactivity is at the
target tissues, where monodeiodinase enzymes convert T4 to
T3, or inactivate T4 and T3 (Figure 2). The monodeiodinases
 Endocrinology of Complex Life Cycles: Amphibians 149

Figure 1 Changes in thyroid hormones and pituitary thyroid stimulating hormone (TSH) throughout metamorphosis in Xenopus laevis. (a) Tadpoles
at Nieuwkoop–Faber (NF) stages 52, 58, and 64 (photos by David Bay). (b) Changes in whole-body thyroxine (T4) and 3,5,30 -triiodothyronine (T3)
throughout metamorphosis. Asterisks denote stage at which there was a statistically significant increase in whole-body hormone content. (Modified
from Krain, L.P., Denver, R.J., 2004. Developmental expression and hormonal regulation of glucocorticoid and thyroid hormone receptors during
metamorphosis in Xenopus laevis. J. Endocrinol. 181 (1), 91–104.) (c) and (d) Changes in TSHb and the common glycoprotein hormone subunit
(a-GSU) mRNAs in the pituitary gland of X. laevis throughout metamorphosis as determined by semiquantitative RT-PCR. Different letters indicate
significant differences among means. (Modified from Manzon, R.G., Denver, R.J., 2004. Regulation of pituitary thyrotropin gene expression during
Xenopus metamorphosis: negative feedback is functional throughout metamorphosis. J. Endocrinol. 182 (2), 273–285.)

catalyze two basic reactions: a 50 monodeiodination (outer
ring) that results in bioactivation and a 5 monodeiodination
(inner ring) that results in bioinactivation of the substrate, T4
or T3. There are three types of vertebrate deiodinases (types I,
II, and III) that differ in their substrate specificity, kinetics,
and sensitivity to inhibitors. Type I catalyzes both 5 and 50 ,
type II 50 , and type III 5 deiodination (St Germain et al.,
2009). Types II and III, but not type I, enzyme activities have
been detected in tadpole tissues, and although frogs have
a type I (Dio1) gene, little is known about its expression or
function (Becker et al., 1997; Dubois et al., 2006; Kuiper
et al., 2006). An iodotyrosine deiodinase that is involved
with recycling of iodine from mono- and diiodotyrosine is
expressed in the thyroid gland and is strongly induced by TH
in the intestine during metamorphosis via a TH response
element (TRE) located in the promoter of the gene (Fujimoto
et al., 2012).
Three deiodinase genes have been isolated in amphibian
species (Brown, 2005). The Dio2 (type II) and Dio3 (type III)
genes exhibit tissue-specific and developmental stage-specific
expression patterns (Becker et al., 1997; Cai and Brown,
2004; Brown, 2005; Duarte-Guterman et al., 2012). The expres-
sion patterns correlate with the asynchronous tissue
morphogenesis and the roles that the deiodinases play in
modulating intracellular T3 concentration during metamor-
phosis (Brown, 2005; St Germain et al., 2009). In many cells
150 Endocrinology of Complex Life Cycles: Amphibians

Figure 2 Central and peripheral organization of the thyroid and stress endocrine axes controlling amphibian metamorphosis. Shown is a schematic
representation of the hypothalamo–pituitary–thyroid (HPT) and hypothalamo–pituitary–interrenal (HPI; stress) axes in amphibian tadpoles, their regu-
lation by input from the external environment, transduction of this input by neural and neuroendocrine pathways, and synergistic interactions among
thyroid hormones and corticosteroids in target cells leading to the promotion of metamorphosis. The two endocrine axes are controlled centrally by
CRH which acts on the anterior pituitary gland (AP) to stimulate the release of thyrotropin (TSH) and corticotropin (ACTH). TSH acts on the thyroid
gland to stimulate release of thyroxine (T4) and 3,5,30 -triiodothyronine (T3). Thyroid hormones are transported in the blood bound by serum binding
proteins (transthyretin, thyroxine binding globulin, and albumin). ACTH acts on interrenal cortical cells in the interrenal glands to stimulate biosyn-
thesis and release of glucocorticoids which are transported in the blood bound to corticosteroid binding globulin. Cellular uptake of T3 and T4 is ach-
ieved by organic anion, monocarboxylate, and amino acid transporters; there is also evidence that thyroid hormones may enter cells bound to
transthyretin via a receptor-mediated process. Glucocorticoids enter cells by passive diffusion across the plasma membrane. Upon entering the cell,
thyroid hormone is bound by cytosolic binding proteins, some of which (the monodeiodinases) convert the hormone to either active (T3; deiodinases
types I and II) or inactive forms (reverse T3 (rT3), diiodothyronine (T2); deiodinase type III). Thyroid hormone receptors (TR) form heterodimers with
retinoid X receptors (RXR) and are bound to DNA in the unliganded form where they actively repress gene transcription. Upon thyroid hormone

both enzymes may be expressed, and the relative expression
levels may establish a type of push–pull mechanism that regu-
lates intracellular T3 concentration (St Germain et al., 2009).
Alternatively, in some tissues the two genes show different
temporal dynamics, leading to hormone inactivation or activa-
tion at different developmental stages.
For example, Dio3 mRNA is expressed in several cell types in
tadpole tail, but not in tail muscle cells (Berry et al., 1998); both
Dio3 mRNA and 5 deiodinase activity increase during late
prometamorphosis (Nieuwkoop-Faber (NF) stages 59–61;
refer Table 1 for a definition of developmental stages) but
then decline sharply at metamorphic climax (St Germain
et al., 1994; Brown et al., 1996). This pattern of Dio3 expression
may protect the tadpole tail, an essential locomotory organ,
from premature resorption (Brown, 2005). By contrast, Dio2
expression, which occurs mainly in tail fibroblasts, is undetect-
able before late prometamorphosis at which time expression
increases markedly and is maintained through the end of meta-
morphosis (Cai and Brown, 2004). This late expression of Dio2
is hypothesized to generate bioactive T3 at an appropriate devel-
opmental stage to accelerate tail resorption. Neither Dio2 nor
Dio3 mRNAs are expressed in tail muscle cells (Berry et al.,
1998; Bonett et al., 2010); nonmuscle tail cells may inactivate
T4 during pre- and prometamorphosis to protect tail muscle
cells from apoptosis and subsequently generate high local
concentrations of T3 to promote tail muscle cell apoptosis at
metamorphic climax.
Tissue transformations during metamorphosis are asynchro-
nous: some tissues respond early to low plasma concentrations
of TH (e.g., hindlimb, brain), while other tissues respond later
in development and require high TH concentration (e.g., intes-
tine, tail – discussed above). The expression patterns of the
monodiodinase genes may play a key role in establishing tissue
competence to respond to the TH signal. For example, for tissues
that respond early in metamorphosis to TH such as the retina
and hindlimb, Dio2 expression was high during early prometa-
morphosis but declined at metamorphic climax. The importance
of 50 deiodinase activity for hindlimb development is supported
by findings that T4 has no effect on the hindlimb in the presence
of the deiodinase inhibitor iopanoic acid (Brown, 2005). Dio2
mRNA expression showed a progressive decline in the brain
throughout metamorphosis, while brain Dio3 mRNA increased
during late prometamorphosis and metamorphic climax
(Hogan et al., 2007). Thyroid hormone induces cell proliferation
in the early prometamorphic tadpole brain, but cells of the
neurogenic zone become refractory to TH action on cell prolifer-
ation as metamorphic climax approaches, which may be
explained by the temporal patterns of Dio2 and Dio3 expression
and actions (Cai and Brown, 2004; Denver et al., 2009).
Thyroid hormone regulates the expression of the Dio2 and
Dio3 genes. The Dio3 gene appears to be a direct T3 response
=
binding to TR gene transcription is derepressed and activated. Upon entering the cell glucocorticoids bind to corticosteroid receptors (glucocorticoid
receptor (GR) or mineralocorticoid receptor (MR)) that are located in the cytosol bound to heat shock proteins (the ‘foldosome’). Hormone binding
causes a conformational change in the receptor, the release of heat shock proteins, and dimerization and translocation of receptors to the nucleus
where they bind DNA to activate or repress target genes. When cells are exposed to low concentrations of thyroid hormone plus glucocorticoids,
genes such as the TRs, deiodinase type 2, and the thyroid hormone-inducible transcription factor Klf9 are activated in a synergistic manner. This
leads to enhanced sensitivity of cells to the actions of thyroid hormone, which accelerates metamorphosis. þ, indicates an increase; , indicates
a decrease in the regulated variable.
Endocrinology of Complex Life Cycles: Amphibians 151
gene based on its rapid induction in response to T3, and accu-
mulation of the mRNA in response to T3 treatment is resistant
to protein synthesis inhibition; i.e., it does not require a new
protein to be made to activate the gene, only preexisting TRs
(Wang and Brown, 1993; St Germain et al., 1994; Becker
et al., 1995; Denver et al., 1997; Kawahara et al., 1999; Hogan
et al., 2007; Das et al., 2009; Duarte-Guterman et al., 2012).
Thyroid hormone positively regulates 50 deiodinase
activity and Dio2 mRNA in tadpoles (Buscaglia, 1985; Brown,
2005; Hogan et al., 2007; Duarte-Guterman et al., 2012).
However, unlike Dio3, which is an early T3 response gene,
Dio2 exhibits delayed response kinetics; the gene was not iso-
lated in screens for early response, direct TR target genes in Xen-
opus tadpole tissues (Brown, 2005; Das et al., 2006; Buchholz
et al., 2007; Bonett et al., 2010). The dependence of Dio2 expres-
sion on TH may vary among tissues. Tissues in which cell
proliferation occurs as an early response to TH constitutively
express relatively high levels of Dio2 (e.g., neurogenic zones of
the brain and spinal cord, limb buds); whereas, in tissues that
transform later, Dio2 is upregulated by TH (Cai and Brown,
2004). Treatment of early prometamorphic tadpoles with T4
can induce cell proliferation in these tissues, which can be
blocked by the deiodinase inhibitor iopanoic acid (Cai and
Brown, 2004). Dio2 mRNA in brain and spinal cord declines
at metamorphic climax, as does TH-dependent cell
proliferation (Cai and Brown, 2004; Denver et al., 2009).
While the decline in Dio2 mRNA may be permissive for the
reduction in TH-dependent cell proliferation in the brain, it
does not alone explain why the brain becomes refractory to
TH action since treatment with T3 could not increase cell
proliferation (which normally declines) as the animals
approach metamorphic climax (Denver et al., 2009). The
decline in cell proliferation is likely due to processes, probably
under TH control, that lead to a reduction in the stem cell/
progenitor pool in the ventricular/subventricular zones of the
tadpole brain. Dio2 expression then appears in late-responding
tissues such as the intestine, tail, and anterior pituitary and
may be induced at this time by rising plasma titers of TH
(Huang et al., 2001; Cai and Brown, 2004; Manzon and
Denver, 2004; discussed more below). Physiological roles for
tissue monodeiodinases in the timing of metamorphosis are
supported by experiments with iopanoic acid and transgenesis
overexpression of Dio3 (Buscaglia, 1985; Galton, 1989; Becker
et al., 1997; Huang et al., 1999, 2001; Marsh-Armstrong et al.,
1999; Cai and Brown, 2004).
2.09.2.2.4 Thyroid Hormone Transport in Blood
Thyroxine synthesized by thyroid follicular cells diffuses into
the bloodstream where it is reversibly bound by plasma
proteins that transport the hormone from the site of produc-
tion to its target tissues (Figure 2). Two plasma binding
152 Endocrinology of Complex Life Cycles: Amphibians
proteins that bind T4 and T3 with moderate to high affinities
have been identified in vertebrates. Thyroxine-binding globulin
(TBG) binds T4 with high affinity and low capacity but is found
only in large, eutherian mammals (Power et al., 2000). Trans-
thyretin (TTR; also known as prealbumin) is found in all verte-
brates and it binds T4 with moderate affinity and intermediate
capacity. Both TBG and TTRs can also bind T3, although in
most cases with 10 times lower affinity than T4 (Power et al.,
2000). However, the situation in amphibia is the reverse, where
TTR binds T3 with greater affinity than T4 (Yamauchi et al.,
1993, 1998, 1999, 2000). The two primary sites for TTR expres-
sion in vertebrates are the liver and the choroid plexus
(although it is expressed at other sites (Power et al., 2000)).
In amphibians TTR is expressed primarily in the liver (Power
et al., 2000). An essential function of TTR is its interaction
with retinol-binding protein, which acts as a carrier for all-
trans-retinol in the blood. The functional significance of
this interaction is not known, but it is intriguing that T3 and
9-cis-retinoic acid (which is a metabolite of all-trans-retinol)
serve as ligands for the TR-retinoid X receptor (RXR)
heterocomplex that regulates TH target genes. Serum albumin
also binds T3 and T4 in many species with low affinity and
high capacity, and Power et al. (2000) suggested that
albumin might be the principal T4-binding protein in
amphibia.
Circulating TTR protein is present in tadpoles during preme-
tamorphosis and prometamorphosis when thyroid activity is
increasing, but declines at metamorphic climax (Yamauchi
et al., 1998, 2000; Prapunpoj et al., 2000). The free hormone
hypothesis (Mendel, 1989; Ekins, 1990) leads to the prediction
that TTR during pre- and early prometamorphosis serves to
reduce the free fraction of TH in blood thus limiting bioavail-
ability. Conversely, hormone-binding proteins can serve as
a reservoir for hormone in the blood; TTR could therefore
help to sustain increasing plasma TH concentrations prior to
the acceleration of thyroid gland activity induced by rising
plasma TSH titers. The TTR concentration in the blood declines
at metamorphic climax when plasma TH concentration is
maximal. The continued rise in TH synthesis by the thyroid
gland, paired with a decline in TTR, could result in an increase
in the free hormone fraction in the blood. At the same time, the
rate of clearance of T3 from the circulation would likely
increase. However, because the thyroid synthetic rate is high
at metamorphic climax, total plasma T3 concentration
continues to rise. Thus, one would predict that, not only does
the hormone production rate increase at metamorphic climax,
but so does the proportional availability of T3 to the target
tissues. To my knowledge, T3 or T4 clearance rates have not
been calculated in tadpoles at different stages of development.
Based on TTR expression profiles one would predict that clear-
ance rates would be lower during prometamorphosis
compared with premetamorphosis or metamorphic climax.
Furthermore, given the lower affinity of TTR for T4 compared
with T3, one would predict that the clearance rate for T4 would
be higher than that of T3.
2.09.2.2.5 Cellular Uptake of Thyroid Hormone
Tadpole cells have the capacity to actively take up TH (see Krain
and Denver, 2004), and this activity may be regulated during
metamorphosis. Saturable, carrier-mediated uptake of THs
has been demonstrated in tadpole red blood cells (Galton
et al., 1986; Yamauchi et al., 1989; Murata and Yamauchi,
2005). The genes that encode TH transport proteins could be
important loci for the modulation of the timing of
metamorphosis.
There are three general classes of proteins that allow for
active uptake of TH by cells: the organic anion transporters
(OATC), monocarboxylate transporters (MCT), and the L
amino acid permeases (LAT) (Ritchie et al., 1999, 2003;
Friesema et al., 2005; Jansen et al., 2005; Visser et al., 2008).
Orthologs of OATC, MCT, and LAT genes have been isolated
from frogs, and patterns of expression throughout metamor-
phosis have been described (Shi and Brown, 1993; Liang
et al., 1997; Connors et al., 2010; Choi et al., 2015a). Only
the amino acid permeases have so far received attention with
regard to their function during tadpole metamorphosis. The
T3-inducible gene IU12 from X. laevis intestine (Shi and Brown,
1993; Liang et al., 1997) encodes a subunit of a heterodimeric
amino acid permease complex (System L (Torrents et al., 1998;
Ritchie et al., 2003)). This permease complex efficiently trans-
ports T3 and T4 when expressed in the Xenopus oocyte expres-
sion system, but is inhibited by reverse T3 (Ritchie et al.,
1999). Overexpression of System L in Xenopus oocytes
increased cytoplasmic and nuclear delivery of THs from the
external medium and enhanced transcriptional activation by
TH receptors (TRs) (Ritchie et al., 2002). By contrast, blocking
endogenous System L activity in mammalian cells reduced
both TH uptake and TR function (Ritchie et al., 2003). The
fact that IU12 is a T3-inducible gene suggests that it could
play a role in mediating T3 uptake by cells during tadpole meta-
morphosis (Liang et al., 1997). Choi et al. (2015a) recently
showed that forced expression of LAT1 in tadpole tail muscle
cells accelerated T3-induced cell loss.
Upon entering cells THs may bind to a series of intracel-
lular binding proteins, termed cytoplasmic TH binding
proteins (CTHBPs) that represent several classes of multifunc-
tional proteins. These proteins may have a variety of enzy-
matic activities; for example, three genes isolated from
X. laevis code for: (1) a cytosolic aldehyde dehydrogenase
(ALDH1) which catalyzes the formation of retinoic acid
(Yamauchi and Tata, 1994), (2) an M2 pyruvate kinase
(PKM2) (Shi et al., 1994), and (3) protein disulfide isomerase
(PDI; induced by T3 in tadpole brain (Denver et al., 1997)),
which catalyzes disulfide bond formation, and human PDI
has been shown to bind TH with high affinity (Cheng et al.,
1987; Yamauchi et al., 1987). These CTHBPs may transport
THs within the cytoplasm to the nucleus to bind to TRs, or
alternatively, they may serve as chelators to limit the cellular
free TH concentration (Shi, 2000a). It is also possible that
TH may regulate the enzymatic activity of these proteins
(Ashizawa and Cheng, 1992). Choi et al. (2015a) recently
showed that forced expression in tadpole tail muscle cells of
PKM2 and another CTHBP that has not been previously char-
acterized in amphibians, the mu-crystallin homolog CRYM
accelerated T3-induced cell loss, supporting that these
proteins facilitate TH action.
2.09.2.2.6 Mechanisms of Thyroid Hormone Action
Tadpoles become competent to respond to exogenous TH at
the time of hatching (Tata, 1968). This establishment of

competence to respond to the hormone likely depends on the
expression of TRs (Shi et al., 1996). TRs are ligand-activated
transcription factors that belong to the steroid hormone
receptor superfamily (Mangelsdorf et al., 1995). There are
two TR genes, termed a and b, in all vertebrates studied to
date; owing to its pseudotetraploidy, X. laevis possesses four
TR genes, two as and two bs (Buchholz et al., 2006). The two
X. laevis TRa genes each appear to give rise to single, unique
proteins; whereas, alternative mRNA splicing of TRb transcripts
can give rise to two different receptor isoforms for each TRb
gene (Buchholz et al., 2006).
The TRa genes are first expressed shortly after hatching in
X. laevis, and their expression rises during premetamorphosis
and remains high throughout metamorphosis (Baker and
Tata, 1990; Yaoita and Brown, 1990; Banker et al., 1991;
Kawahara et al., 1991). It has been hypothesized that the early
expression of TRa establishes hormone responsiveness of
tadpole tissues (Baker and Tata, 1990; Shi et al., 1996). TRb
mRNA is not detected until early prometamorphosis, but its
expression increases during prometamorphosis in parallel
with TH synthesis (Yaoita and Brown, 1990; Kawahara
et al., 1991; Baker and Tata, 1992; Kanamori and Brown,
1992). Several studies have shown that the TR genes are upre-
gulated by T3 in X. laevis and R. catesbeiana (Yaoita and Brown,
1990; Kawahara et al., 1991; Schneider and Galton, 1991;
Helbing et al., 1992) (a phenomenon termed ‘autoinduction’;
see Tata et al., 1993). A thyroid hormone response element to
which TRs can bind and regulate transcription has been
identified in the X. laevis TRbA gene (Ranjan et al., 1994;
Machuca et al., 1995). Autoinduction may require the upregu-
lation of accessory transcription factors such as the immediate
early, TH-inducible gene Krüppel-like factor 9 (Klf9;
aka basic transcription element binding protein 1; BTEB1)
(Bagamasbad et al., 2015; Hu et al., 2016).
A central role for TRs in metamorphosis is supported by
transgenic studies in X. laevis. For example, transgenic expres-
sion of a dominant negative TR blocks metamorphosis
(Schreiber et al., 2001; Buchholz et al., 2003), while expres-
sion of a dominant positive TR promotes metamorphic
changes in the absence of TH (Buchholz et al., 2004). Aran
et al. (2014) recently showed that autoinduction of TR
a and b and the actions of exogenous T3 on epibranchial
remodeling were strongly reduced or eliminated in paedomor-
phic versus metamorphic populations of the Oklahoma sala-
mander Eurycea tynerensis. These findings provide further
support for the essential role of TH and TRs in metamorphosis,
and their roles in the expression of alternative developmental
modes in amphibians.
Specific functions for the different receptor subtypes in
amphibia are poorly understood. Expression studies suggest
differential roles, as do pharmacological studies with TR
subtype-selective agonists. For example, studies with the TRa-
selective agonist CO23 support that this receptor subtype is
involved in cell proliferation (Ocasio and Scanlan, 2006;
Denver et al., 2009), while studies with the TRb selective
agonists GC1 and GC24 support that this subtype is primarily
involved with tissue resorption (apoptosis) and cell differenti-
ation (Furlow et al., 2004; Ocasio and Scanlan, 2006; Denver
et al., 2009). Experimental studies addressing specific
functions for the different TRs in amphibians were limited
Endocrinology of Complex Life Cycles: Amphibians 153
by the inability to delete, or knockdown, the TRs. Recent tech-
nological developments using transcription activator-like
effector nucleases (TALENS) for genome editing have shed light
on the role of TRa in tadpole development and metamor-
phosis. Two groups recently used TALENS to introduce muta-
tions into the TRa locus in Xenopus tropicalis (Choi et al.,
2015b; Wen and Shi, 2015). Mutations in TRa led to enhanced
growth of premetamorphic tadpoles and the acceleration of the
onset of metamorphosis (Wen and Shi, 2015). Although the
timing of the initiation of metamorphosis was accelerated,
these mutant tadpoles were resistant to exogenous T3 and
exhibited a delay in spontaneous metamorphosis. Gene expres-
sion analyses showed increased baseline mRNA levels, but the
loss of T3 response in TR target genes (Choi et al., 2015b; Wen
and Shi, 2015). Together, these findings support that the apor-
eceptor normally represses metamorphic genes in larvae, and
when removed shortens the larval period. However, liganded
TRa is required for establishing tissue responsiveness to the
hormone during metamorphosis, and so these studies provide
critical support for the dual function model for TR actions
during metamorphosis (Buchholz et al., 2006; Shi et al.,
2012; Grimaldi et al., 2013a). Future studies using TALENS
and CRISPR/Cas9 genome editing should shed further light
on the different roles of the TR subtypes in tadpole
development.
Thyroid hormone receptors function as dimers; that is, the
DNA consensus sequences that TRs bind to are six nucleotides
in length and are referred to as ‘half sites.’ Two of these half
sites comprise a TRE (Williams and Brent, 1995). These TREs
can be located within the promoter, within the structural part
of the gene, or upstream of the transcription start site. Homo-
dimers of TRa or TRb can form on most TREs, but the preferred
configuration appears to be as a heterodimer with retinoid X
receptor (RXR) (Wong and Shi, 1995; PuzianowskaKuznicka
et al., 1997). TR-RXR heterodimers bind DNA and
transactivate TRE-containing genes much more effectively
than TR homodimers. In the unliganded form, the TR-RXR
complex functions as a transcriptional repressor (Wong and
Shi, 1995). The TR-RXR heterocomplex recruits cofactor
proteins that mediate the repressive or activational actions of
the complex (Shi, 2000b; McKenna and O’Malley, 2002). The
unliganded receptor may have important developmental
functions in the premetamorphic tadpole (discussed below).
The TR and RXR genes exhibit more or less coordinate
regulation during metamorphosis, and this coordination may
be essential to the timing of tissue-specific changes (Wong
and Shi, 1995). Current studies are focused on identifying
TREs throughout the genome in different tadpole tissues
using chromatin immunoprecipitation (ChIP) combined
with high-throughput sequencing (Grimaldi et al., 2013b).
Hormone binding to the TR-RXR receptor complex induces
gene expression in target tissues. The TRs cause modifications
of chromatin through recruitment of coactivator and
corepressor proteins (Shi et al., 2012; Grimaldi et al., 2013a).
In the unliganded state, TRs function primarily as
transcriptional repressors, recruiting corepressors such as
NCoR and SMRT which then recruit histone deacetylases
(Tomita et al., 2004). This leads to a compact, repressive
chromatin structure (Sachs et al., 2001; Jones and Shi, 2003).
When TH binds to TRs, corepressors are exchanged for
154 Endocrinology of Complex Life Cycles: Amphibians
coactivators, leading to transcriptional derepression and
transactivation. Many of the coactivators have intrinsic
histone acetyl transferase activity, leading to the addition of
acetyl groups to lysine residues on histone tails. Coactivator
expression and recruitment to target genes is correlated with
tissue transformation and gene activation (Paul and Shi,
2003; Paul et al., 2005b), and transgenic analysis suggests
that the recruitment of coactivators by TRs is essential for
metamorphosis to proceed (Paul et al., 2005a, 2007). A role
for unliganded TR in tadpole development, whereby it
represses the expression of adult genes (Sachs et al., 2002;
Sato et al., 2007), has led to the development of the dual
function model for TR action during metamorphosis. This
model proposes that unliganded TRs (the aporeceptors) play
an important role in development by repressing adult genes
in the tadpole prior to the onset of thyroid activity, but
activate expression of metamorphosis-associated genes when
ligand is present (reviewed by Buchholz et al., 2006;
Grimaldi et al., 2013a; Shi, 2013). The aporeceptors have
also been implicated in heart development and the
maturation of the brain and other organs prior to the onset
of thyroid gland function in mammals (Bernal and Morte,
2013). Discussion of the characteristics of the gene regulation
cascades and the functions of the gene products induced by
TH in different tissues during metamorphosis is beyond the
scope of this chapter. For detailed discussion of these topics
the reader is referred to the following reviews: Furlow and
Neff (2006), Buchholz et al. (2006), Brown and Cai (2007),
and Grimaldi et al. (2013a).
2.09.2.3 Corticosteroids
In addition to TH, corticosteroids (CS), the primary verte-
brate stress hormones, play important roles in amphibian
metamorphosis. Like TH, the production of CS changes
with development and li
2.09.2.3.1 Development of and Hormones Produced
by Amphibian Interrenal Glands
The interrenal gland is generally less active in early, premeta-
morphic developmental stages and more active during prome-
tamorphosis and metamorphic climax (Dodd and Dodd,
1976). The ultrastructural appearance of X. laevis interrenal
cells indicates relative inactivity in mid-prometamorphs,
increasing to peak activity at metamorphic climax (reviewed
by Dodd and Dodd, 1976; however, see below for contrary
evidence). Activity of the interrenal enzyme, D5-3b-hydroxyste-
roid dehydrogenase (HSD) is present throughout development
in R. catesbeiana and X. laevis but increases at metamorphic
climax in R. catesbeiana (Hsu et al., 1980; Kang et al., 1995).
Carr and Norris (1988) found a similar pattern for plasma
corticosterone and interrenal HSD activity in the tiger sala-
mander, A. tigrinum.
Radioimmunoassays for corticosteroids have been done on
plasma samples collected throughout the metamorphic period
for several amphibian species: R. catesbeiana (Jaffe, 1981; Krug
et al., 1983; Kikuyama et al., 1986); B. japonicas (Niinuma et al.,
1989); X. laevis (Jolivet-Jaudet and Leloup-Hatey, 1984);
A. tigrinum (Carr and Norris, 1988). Whole-body measures of
corticosteroid content have also been determined throughout
development: S. hammondii (Denver, 1998a); X. laevis (Kloas
et al., 1997; Krain and Denver, 2004) (Glennemeier and
Denver, 2002a); Rana pipiens (Glennemeier and Denver,
2002a). The majority of these studies showed low-to-nonde-
tectable corticosteroids during premetamorphosis, and
a marked increase at metamorphic climax, more or less in
parallel with the rise in THs. The only exception to this rule
is whole-body corticosteroid content in X. laevis. Kloas et al.
(1997) reported that whole-body corticosterone content in
X. laevis increases during premetamorphosis to reach a peak
at NK stage 48 and then declines during prometamorphosis
and is low at metamorphic climax. Kloas et al. (1997) also