High Quality
Open the downloaded document, and select print from the file menu (PDF reader required).
Cellular/Molecular
Uniquely Hominid Features of Adult Human Astrocytes
NancyAnnOberheim,
1,5
TakahiroTakano,
1
XiaoningHan,
1
WeiHe,
1
JaneH.C.Lin,
2
FushunWang,
1
QiwuXu,
1
JeffreyD.Wyatt,
3
WebsterPilcher,
1
JeffreyG.Ojemann,
4
BruceR.Ransom,
5
StevenA.Goldman,
1
andMaikenNedergaard
1
1
Center for Translational Neuromedicine, Departments of Neurology and Neurosurgery, University of Rochester Medical Center, Rochester, New York14642,
2
Department of Pathology, New York Medical College, Valhalla, New York 10595,
3
Department of Comparative Medicine, University of Rochester,Rochester, New York 14642, and Departments of
4
Neurosurgery and
5
Neurology, University of Washington, Seattle, Washington 98195
Defining the microanatomic differences between the human brain and that of other mammals is key to understanding its uniquecomputational power. Although much effort has been devoted to comparative studies of neurons, astrocytes have received far lessattention.Wereportherethatprotoplasmicastrocytesinhumanneocortexare2.6-foldlargerindiameterandextend10-foldmoreGFAP(glial fibrillary acidic protein)-positive primary processes than their rodent counterparts. In cortical slices prepared from acutely re-sectedsurgicaltissue,protoplasmicastrocytespropagateCa
2
waveswithaspeedof36
m/s,approximatelyfourfoldfasterthanrodent.Human astrocytes also transiently increase cystosolic Ca
2
in response to glutamatergic and purinergic receptor agonists. The humanneocortex also harbors several anatomically defined subclasses of astrocytes not represented in rodents. These include a population of astrocytes that reside in layers 5–6 and extend long fibers characterized by regularly spaced varicosities. Another specialized type of astrocyte,theinterlaminarastrocyte,abundantlypopulatesthesuperficialcorticallayersandextendslongprocesseswithoutvaricositiesto cortical layers 3 and 4. Human fibrous astrocytes resemble their rodent counterpart but are larger in diameter. Thus, human corticalastrocytes are both larger, and structurally both more complex and more diverse, than those of rodents. On this basis, we posit that thisastrocytic complexity has permitted the increased functional competence of the adult human brain.
Introduction
Recent studies of glial cell have produced a wealth of informa-tion regarding their functional roles and structural organiza-tion. However, these studies have been based primarily onobservations in the rodent brain. It is generally assumed thathuman brain shares a multitude of similarities with rodentand that studies in mice and rats can serve as a model forsimilar measures in humans. However, it is essential to evalu-ate the similarities and differences between human brain andour experimental models.Study of human glia dates back to 1858 when the concept of “Nervenkitt” or neuroglia was first introduced by Rudolf Vir-chow (Kettenmann and Verkhratsky, 2008). Although Virchowbelieved that neuroglia was primarily connective tissue, he de-scribed cellular elements within this nerve glue. Late in the 19thcentury,astrocytesweredefinedbyVonLenhossekandclassifiedas either protoplasmic or fibrous based on morphology by Kol-likerandAndrizen(KettenmannandVerkhratsky,2008).Ramon y Cajal studied the morphology of astrocytes within human andother mammalian brains (Ramon y Cajal, 1897). AlthoughRamonyCajal’sdrawingsdepictthecomplexityofhumanastro-cytes, it was more recently appreciated that human and rodentastrocytes differ. A comparative study of Colombo and cowork-ersrevealedthatinterlaminarastrocytesareprimate-specificcells(Colombo et al., 1995; Colombo and Reisin, 2004). Interlaminarastrocytes were originally described by Andriezen and Retzius assmall glial cells, which reside in the upper cortical layers, andextend long glial fibrillary acidic protein-positive (GFAP
) pro-cesses through cortical layers 2–4 (Andriezen, 1893; Retzius,1894). The existence of millimeter long glial processesin primatecortex is distinctly different from the evenly spaced uniform as-trocytes in rodent cortex. Nonetheless, a comprehensive analysisof human versus rodent astrocytes has to date been performed,despite numerous studies on the evolutionary changes of neu-rons (Nimchinsky et al., 1999; DeFelipe et al., 2002; Yanez et al.,2005; Prabhakar et al., 2006). The aim of this study was thus todirectly compare human and rodent cortical astrocytes.By immunostaining thick sections of freshly resected surgicalsamples of human temporal neocortex for GFAP and then usingconfocal imaging to follow and reconstruct single cells, we foundthat human astrocytes are significantly larger and structurally more complex than their rodent counterparts. Furthermore, hu-man brain contains multiple classes of GFAP-immunoreactivecells with distinct morphological features, in particular, humanbrain is characterized by a novel astrocytic phenotype that wedesignate varicose projection astrocytes. These cells extend 1- to5-mm-long projection fibers that harbor regularly spaced vari-cosities. In addition to their morphological specializations, wenoted that functionally, human astrocytes exhibited increasedintracellular Ca
2
responses to purinergic and metabotropic re-
Received Sept. 30, 2008; revised Jan. 13, 2009; accepted Jan. 24, 2009.This work was supported by the G. Harold and Leila Y. Mathers Charitable Foundation and grants from theNational Institute of Neurological Disorders and Stroke (M.N., S.A.G., B.R.R.).Correspondence should be addressed to Dr. Maiken Nedergaard, Division of Glial Disease and Therapeutics,Center for Translational Neuromedicine, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester,NY 14642. E-mail: nedergaard@urmc.rochester.edu.DOI:10.1523/JNEUROSCI.4707-08.2009Copyright © 2009 Society for Neuroscience 0270-6474/09/293276-12$15.00/0
3276
•
The Journal of Neuroscience, March 11, 2009
•
29(10):3276–3287
ceptoragonistsandwereabletosupportCa
2
wavesatvelocitiessignificantly greater than in the rodent.
MaterialsandMethods
Tissue.
Human brain tissue was acquired from a total of 30 patientsduringsurgicalresectionforavarietyofpathologies,includingmedically refractory epilepsy (
n
23), tumor (
n
6), and decompression attrib-utable to abscess (
n
1). The age range of patients was 4–69 years old,withameanof32.4years.Onlytissuesdissectedbeyondtheprimaryareaof pathology were used. Adult primate cortical brain tissue was takenfrom a perfusion-fixed adult squirrel monkey [4% paraformaldehyde(PFA)], two perfusion-fixed adult rhesus macaques, and a Formalin-fixed adult common chimpanzee (Pan troglodytes). The chimpanzeedied at 55 years of age from a cardiac condition. Adult FVB/NJ mice andSpragueDawleyrats(TheJacksonLaboratory)wereusedasrodentcom-parisons. All experiments using human tissues were approved by theUniversityofRochesterResearchSubjectsReviewBoardandtheUniver-sity of Washington Research Subjects Review Board; all animal studieswere approved by the Institutional Animal Care and Use Committees of both the University of Rochester and the University of Washington.
Immunofluorescence.
Humantissuefromsurgicalresectionwasimme-diatelyplacedinPBScontaining4%PFAandfixedovernight.Itwasthenwashed in PBS for 4–6 h. Rhesus macaque monkeys, the squirrel mon-key, mice, or rats were deeply anesthetized with a mixture of ketamine(60 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and fixed
in situ
throughperfusionof4%PFAinPBS.Brainswereremovedandwashedovernightin PBS at 4°C. For additional examination of primates and for examina-tion the vasculature in rodents, brains were acutely isolated and imme-diately placed in 4% PFA, or Formalin in the case of the chimpanzeebrain, and fixed a minimum of 12 h, similar to processing of the humansurgical resections. After washing, all tissue was sectioned at 200–400
m using a vibratome and immersed in PBS at 4°C. Sections were thenincubated in 5% normal donkey serum (NDS) (Jackson ImmunoRe-search)and0.5–1%TritonX-100(Sigma-Aldrich)for1h.Sectionswereincubated in primary antibody in 1% NDS and 0.1–0.5% Triton X-100overnight at 4°C. Primary antibodies used were monoclonal anti-GFAP(mAB360,1:500;Millipore)andpolyclonalchickenanti-MAP2(ab5392,1:5000; Abcam). Sections were then washed and incubated in secondary antibodies (Jackson ImmunoResearch) and counterstained with 4,6-diamindino-2-phenylindole (DAPI) (D-21490, 1:10,000; Invitrogen) orsytox (S7020, 1:5000; Invitrogen). Images were collected with a confocallaser-scanning microscope (FV500 or FV1000; Olympus) (Wang et al.,2004). For immunoelectron microscopy, GFAP and aquaporin-4 anti-bodies (AB3068; Millipore) were used, and sections were prepared fromhuman tissue directly from surgical resection as described previously (GoldmanandNedergaard,1992).ImageswerecollectedwithanHitachi7100 electron microscope and analyzed with NIH ImageJ.
Diolistic labeling.
The Helios Gene Gun System (165-2431; Bio-Rad)was used to deliver gold (1.0
m) and tungsten particles coated withlipophilic dyes, DiI (D282; Invitrogen) or DiD (D307; Invitrogen), intolightly fixed brain slices from surgical resection and perfusion-fixed ro-dent brain. Preparations of the DiOlistic bullets were based on themethodofGanetal.(2000)andBenediktssonetal.(2005).Twohundredto400-
m-thickfixedbrainsliceswereplacedon2%agaroseblocksinaculture dish and shot with both DiI and DiD bullets using 100 psi of helium pressure. Slices were counterstained with sytox and mounted forimaging. When immunofluorescence was combined with diolistic label-ing, immunofluorescence was performed first, without Triton X-100,and then, after secondary antibody incubation, the sections were shotwith DiI and DiD bullets and counterstained.
Image collection and analysis.
Images were collected with 20 and 40
oil objective lenses (numerical aperture 0.75 and 1.3, respectively) withFluoView (Olympus) software and analyzed with both Fluoview andNIH ImageJ software. Stacks of 1
m inclement depth were used foranalysis. Diameter of the cell was defined as the longest axis through thenucleus of the domain of the cell based on GFAP immunostaining. Thethickness of the GFAP-positive processes was defined as the diameter of GFAPmeasuredbetweentwobranchingpoints,andthelengthoflongestprocesswasdefinedasthedistancefromthecenterofthenucleitothetipof the extended GFAP-positive processes. The domain of the cell basedondiolisticlabelingwasdeterminedbythecompilationofeachextensionof the diolistically labeled processes. Overlapping area of two neighbor-ing astrocytic domains over 10 1-
m stacks was averaged (Ogata andKosaka, 2002). As a second measure of overlap, processes that trespassedinto the domain of the adjacent astrocyte were traced to obtain eachlength. The summation of the lengths of these processes over 10 1-
mstacks was also averaged to determine the degree of the domain organi-zation (Oberheim et al., 2008). The means, SDs, and SEMs were calcu-lated, and the differences between groups were tested with a Student’s
t
test or ANOVA with Bonferroni’s
post hoc
test, with significance deter-mined to occur when
p
0.05 using a two-tailed test.
Calcium imaging.
Human tissue was prepared similar to rodent tissueasdescribedpreviously(Kangetal.,1998;Tianetal.,2005).Briefly,tissuetaken directly from surgical resection was placed in ice-cold solutioncontaining the following (in m
M
): 240 sucrose, 2.5 KCl, 0.5 CaCl
2
, 10MgCl
2
,26NaHCO
3
,1.25NaH
2
PO
4
,and10glucose(saturatedwith95%O
2
/5% CO
2
). Transverse sections of 400
m were then cut using a vi-bratome. Slices were then placed in oxygenated artificial CSF (ACSF) (inm
M
: 125 NaCl, 2.5 KCl, 2 CaCl
2
, 1 MgCl
2
, 26 NaHCO
3
, 1.25 NaH
2
PO
4
,and10glucose)wereallowedtorecoverfor
1h.Sliceswereloadedwiththecalciumindicatorfluo-4AM(Invitrogen)at30–32°Cfor60–80mininACSFcontainingfluo-4AM(10
M
)and0.02–0.04%pluronicF-127,in the presence or absence of
o
-nitrophenyl (NP)-EGTA AM (200
M
)(Invitrogen). The slices were then mounted in a perfusion chamber andwere viewed using a custom-built laser-scanning microscope (BX61WI,FV300; Olympus) attached to Mai Tai laser (SpectraPhysics). Photolysiswas performed using a custom-built system that released an ultravioletpulse3
mindiameteras10trains[threepulseswithadurationof10msand an interval of 10–30 ms; 30–40 mW; 355 nm, (DPSS Lasers)]. Cal-cium analysis was performed by quantifying the number of cells thatincreasedflorescence
2SDsabovebaselineflorescenceafterapplicationof drug or photolysis. Drugs used included ATP (100
M
-5 m
M
), gluta-mate (1–15 m
M
), (
)-1-aminocyclopentane-
trans
-1,3-dicarboxylic acid(t-ACPD)(30
M
),andTTX(1
M
)andwereallpurchasedfromSigma-Aldrich. Wave velocity was calculated as the distance from the targetdivided by the time-to-peak increase in florescence after uncaging andwas averaged for all cells within one wave.
Results
At least four morphologic classes of GFAP-positive cells arepresent in the human cortex
At least four major morphologic subclasses of GFAP-immunoreactive cells were identified in the adult human tempo-ral lobe (Fig. 1). Layer 1 is characterized by densely packed inter-laminar astrocytes, which extend millimeter long processesterminatinginlayers2–4.Layers2–6arecharacterizedbyproto-plasmicastrocytesofalargersizethanfoundinrodents.Inlayers5–6, a small number of varicose projection astrocytes reside.Thesecellsextendlongprocessesinaseeminglyrandommanner.Finally, fibrous astrocytes populate the white matter. Similarly,examination of the chimpanzee cortex revealed four subtypes of GFAP
cells (supplemental Fig. 1, available at www.jneurosci.org as supplemental material), although the cellular complexity didnotcomparewiththoseofhumanbrain.Incontrast,cortexof lower-orderprimates,includingtherhesusmacaqueandsquirrelmonkeys, contained three subtypes of astrocytes, whereas only protoplasmic and fibrous astrocytes were identified in rodents(supplemental Fig. 1, available at www.jneurosci.org as supple-mental material).
Varicose projection astrocytes: a novel GFAP-positive cell inhigher-order primate cortex
One of the most striking features distinguishing humans andchimpanzee from other lower primate and rodent astrocyteswas the presence of a previously undescribed pool of morpho-
Oberheim et al.
•
Complexity of Human Astrocytes J. Neurosci., March 11, 2009
•
29(10):3276–3287
• 3277
logically distinct GFAP
cells residing inlayers 5–6, characterized by long fiberswith prominent varicosities (Fig. 2
A
).The main GFAP
processes of these var-icose projection astrocytes are straighterthan those of protoplasmic astrocytes,whose primary processes are typically tortuous and highly branched (comparewith Fig. 4
B
). Additionally, these cellsalso extend one to five long processes of up to 1 mm in length, which may termi-nate in the neuropil or on the vascula-ture (Fig. 2
A
). The processes of theseprojection astrocytes are characterizedby varicosities that are spaced approxi-mately every 10
m (Fig. 2
A
,
C
) (see Fig.3
D
). The processes frequently penetratethe process-delimited domains of neigh-boring astrocytes. These varicose projec-tion astrocytes were relatively sparse butwere present in all 30 specimens exam-ined. In our analysis of primate tissue,we were able to locate a small number of varicose projection astrocytes withinlayers 5 or 6 of the chimpanzee cortex(Fig. 2
A
, inset). These cells differed fromthose seen in human in that they weresmaller and less complex, with fewermain GFAP
processes. However, webelieve that they are the same cell type inthat they were located in the same corti-cal loci and laminae, and similarly ex-tended one to three long processes char-acterized by evenly spaced varicosities.Wewereunabletoperformmorphomet-ric analysis to compare these chimpan-zee cells directly with their counterparts,given the paucity of higher-order pri-mate tissue available. In other species of primates, including rhesus macaque andsquirrel monkeys, we were unable toidentify this cell type, suggesting thatvaricose projection astrocytes are spe-cific to higher-order primates and hu-mans (supplemental Fig. 1, available atwww.jneurosci.org as supplemental ma-terial). To our knowledge, astrocyteswith this morphological phenotype havenot been reported in any other species,and the function of their long processesis not known.GFAP immunolabeling reveals only the filamentous skeleton of an astrocyte.Tovisualizethefullstructureofvaricoseprojectionastrocytes,we therefore used DiI-mediated diolistic labeling (biolistic in-sertion of DiI into cells) of lightly fixed human tissue to bettervisualize the full extent of single astroglia (Fig. 2
B–F
). DiI-tagged varicose projection astrocytes resemble protoplasmicastrocytes, with one to five long thin processes extending froma relatively symmetrical cell (Fig. 2
B–F
). The fine processes of these cells are, however, spiny and sharp, unlike the bulbousprocesses described previously in rodent protoplasmic astro-cytes (Bushong et al., 2002; Ogata and Kosaka, 2002; Ober-heim et al., 2008) (see Fig. 4
F
). Many of the processes fromvaricose projection astrocytes contacted the vasculature, ei-ther through the direct extension of long processes to vascularwalls or by en passant contact of glial processes with the cap-illary microvasculature (Fig. 2
F
).
Primate-specific interlaminar fibers in the human cortex
The GFAP-labeled human cortex exhibited an abundance of in-terlaminar astrocytes (Fig. 3
A–C
,
E
). Interlaminar astrocytes areprimate-specific cells that are densely packed and reside solely in
Figure 1.
There are four classes of GFAP
cells in the human cortex. Human brains were immunolabeled with GFAP andanalyzedthroughoutalllayersofthecortextodeterminesubclassesofhumanastrocytes.Layer1iscomposedofthecellbodiesof interlaminar astrocytes, whose processes extend over millimeter lengths through layers 2–4 and are characterized by theirtortuous morphology. Protoplasmic astrocytes, the most common, reside in layers 2–6. Polarized astrocytes are found only inhumansandareseensparselyinlayers5–6.Theyextendmillimeterlongprocessesthatarecharacterizedbyvaricosities.Fibrousastrocytes are found in the white matter and contain numerous overlapping processes. Yellow lines indicate areas in which thedifferent classes of astrocytes reside. Scale bar, 150
m.
3278
•
J. Neurosci., March 11, 2009
•
29(10):3276–3287 Oberheim et al.
•
Complexity of Human Astrocytes
Add a Comment