cellular composition and cytoarchitecture of the adult ......cellular composition and...

Cellular Composition and Cytoarchitectureof the Adult Human Subventricular Zone:

A Niche of Neural Stem Cells

ALFREDO QUINONES-HINOJOSA,1* NADER SANAI,1 MARIO SORIANO-NAVARRO,2

OSCAR GONZALEZ-PEREZ,1 ZAMAN MIRZADEH,1 SARA GIL-PEROTIN,2

RICHARD ROMERO-RODRIGUEZ,1 MITCHELL S. BERGER,1

JOSE MANUEL GARCIA-VERDUGO,2AND ARTUR0 ALVAREZ-BUYLLA1*

1Department of Neurological Surgery, Brain Tumor Research Center, Developmental StemCell Biology Program, University of California, San Francisco, San Francisco, California 941432Departamento de Biologıa Celular, Facultad de Biologıa, Universidad de Valencia, Burjassot-

46100, Valencia, Spain

ABSTRACTThe lateral wall of the lateral ventricle in the human brain contains neural stem cells throughout

adult life. We conducted a cytoarchitectural and ultrastructural study in complete postmortem brains(n � 7) and in postmortem (n � 42) and intraoperative tissue (n � 43) samples of the lateral walls ofthe human lateral ventricles. With varying thickness and cell densities, four layers were observedthroughout the lateral ventricular wall: a monolayer of ependymal cells (Layer I), a hypocellular gap(Layer II), a ribbon of cells (Layer III) composed of astrocytes, and a transitional zone (Layer IV) intothe brain parenchyma. Unlike rodents and nonhuman primates, adult human glial fibrillary acidicprotein (GFAP)� subventricular zone (SVZ) astrocytes are separated from the ependyma by thehypocellular gap. Some astrocytes as well as a few GFAP-cells in Layer II in the SVZ of the anteriorhorn and the body of the lateral ventricle appear to proliferate based on proliferating cell nuclearantigen (PCNA) and Ki67 staining. However, compared to rodents, the adult human SVZ appears tobe devoid of chain migration or large numbers of newly formed young neurons. It was only in theanterior SVZ that we found examples of elongated Tuj1� cells with migratory morphology. Weprovide ultrastructural criteria to identify the different cells types in the human SVZ including threedistinct types of astrocytes and a group of displaced ependymal cells between Layers II and III.Ultrastructural analysis of this layer revealed a remarkable network of astrocytic and ependymalprocesses. This work provides a basic description of the organization of the adult human SVZ. J.Comp. Neurol. 494:415–434, 2006. © 2005 Wiley-Liss, Inc.

Indexing terms: human; SVZ; ependymal cells; neurogenesis; cytoarchitecture; stem cells

Neurogenesis persists in two germinal regions of theadult mammalian brain: the subventricular zone (SVZ)(Alvarez-Buylla and Garcia-Verdugo, 2002) on the walls ofthe lateral ventricle, and the subgranular layer of thedentate gyrus in the hippocampus (Kempermann, 2002).Cell proliferation in the SVZ has been demonstrated in

many vertebrate species including mice, rats, rabbits,voles, dogs, cows, monkeys, and humans (Blakemore,1969; Lewis, 1968; Blakemore and Jolly, 1972; McDermottand Lantos, 1990; Eriksson et al., 1998; Huang et al.,1998; Gould et al., 1999; Kornack and Rakic, 2001;Rodriguez-Perez et al., 2003; Sanai et al., 2004). In rodents

Grant sponsor: National Institutes of Health; Grant number:1F32NS047011-01 (to A.Q.H.); Grant number: RO1 HD032116 (to A.A.B.);Grant sponsor: Howard Hughes Medical Institute Medical Student Fellow-ship (to N.S.); Grant sponsor: David B. Anderson American Brain TumorAssociation Fellowship (to N.S.); Grant sponsor: Ministry of Science andTechnology of Spain; Grant number: SAF 03229 (to J.M.G.V.); Grantsponsor: Fondo de Investigaciones Sanitarias Instituto de Salud Carlos III,Madrid; Grant number: Exp. 01/9513 (to S.G.P.); Grant sponsor: Univer-sity of California Institute for Mexico and the United States (to O.G.P.).

Alfredo Quinones-Hinojosa’s current address is Department of Neuro-surgery, Johns Hopkins University, 4940 Eastern Avenue, B 121, Balti-more, MD 21224.

*Correspondence to: Alfredo Quinones-Hinojosa, Department of Neuro-surgery, Johns Hopkins University, 4940 Eastern Avenue, B 121, Balti-more, MD 21224; Email: [email protected] or Arturo Alvarez-Buylla,University of California, San Francisco, Department of Neurological Sur-gery, Brain Tumor Research Center, 505 Parnassus Ave., Moffitt HospitalRoom M779, Box 0112, San Francisco, CA 94143-0112.E-mail: [email protected]

Received 11 March 2005; Revised 11 May 2005; Accepted 11 July 2005DOI 10.1002/cne.20798Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 494:415–434 (2006)

© 2005 WILEY-LISS, INC.

the SVZ is the source of a large number of new neuronsdestined for the olfactory bulb. Cells born in this germinalregion migrate through an extensive network of intercon-nected pathways (Doetsch and Alvarez-Buylla, 1996) toreach the rostral migratory stream (RMS). The RMS ends inthe olfactory bulb, where young neurons differentiate intolocal interneurons in the granule. This process has beendemonstrated in the brain of adult rodents (Lois andAlvarez-Buylla, 1994) and primates ( nack and Rakic, 2001;Pencea et al., 2001), but apparently does not occur in adulthuman brain (Sanai et al., 2004). The SVZ is also a source ofoligodendrocytes during development (Levison and Gold-man, 1993) and in the adult brain after demyelinating le-sions (Picard-Riera et al., 2002). For all these reasons thereis considerable interest in the SVZ as a site of neural pro-genitors for brain repair (Alvarez-Buylla et al., 2000; Bernieret al., 2000; Sanai et al., 2004).

SVZ astrocytes (Type B cells) have been identified as theprimary progenitors of new neurons in the adult rodentbrain (Doetsch et al., 1999a; Imura et al., 2003). Thesecells generate intermediate precursors (Type C cells) thatfunction as transit amplifying cells for the generation oflarge number of new neurons. Cells that behave in vitro asneural stem cells can be isolated from adult brain tissue(Weiss et al., 1996; Gage, 2000). Interestingly, SVZ astro-cytes isolated from the adult human brain can also func-tion as neural stem cells and can generate new neurons invitro (Sanai et al., 2004). Some SVZ astrocytes divide inthe adult human SVZ, but the function of these dividingastrocytes is not known.

The organization of the adult human SVZ is signifi-cantly different than that of rodents. In adult rodents,SVZ astrocytes (Type B cells) are located next to theependymal layer and ensheath chains of migrating youngneurons (Doetsch et al., 1997b; Peretto et al., 1997). Incontrast, in the adult human brain SVZ astrocytes are notfound adjacent to the ependyma and no chains of migrat-ing neuroblasts are found in this region. Instead, the cellbodies of human SVZ astrocytes accumulate in a band orribbon separated from the ependymal layer by a gap thatis largely devoid of cells (Sanai et al., 2004).

This information is derived from analysis of small tissuesamples of the lateral wall of the lateral ventricle. How-ever, the lateral wall of the lateral ventricles in adulthuman brain is very extensive. To date, there is no com-prehensive map of the organization of this germinal wallin humans. The extent of this astrocyte ribbon and itsrelationship to other cells in the adult human SVZ re-mains unknown. The ultrastructural organization of thehuman SVZ, and in particular of the hypocellular gap thatseparates astrocytes from the ependymal layer, is also notknown. The possibility of migratory paths from the SVZ toregions of the adult human brain, other than the olfactorybulb, has also not been explored. Given the interest in theSVZ as an important source of new neurons and oligoden-drocytes in the adult mammalian brain, we undertook ananatomical study of the organization of the adult humanSVZ in the entire lateral wall of the lateral ventricle.

MATERIALS AND METHODS

Anatomical partitioning of the walls of thelateral ventricle

The walls of the lateral ventricles were divided into fourregions along the rostrocaudal axis: frontal horn, body,

atrium/occipital horn, and temporal horn (Table 1; Figs.1–4). The walls of the lateral ventricles were further sub-divided into zones along the ventral-dorsal axis: ventral(V), intermediate, and dorsal (D). The ventral zone com-prises the base of the caudate and the floor of the ventricle;the intermediate zone overlies the caudate and encom-passes the caudate vein; the dorsal zone encompasses theregion facing the corpus callosum and the beginning of thecaudate. The temporal horn was subdivided into a dorsal(D), ventral (V), and a hippocampal zone. The dorsal zonecontains the roof of the ventricle (including the tail of thecaudate), the ventral zone mainly the lateral wall.

Intraoperative specimens

Human SVZ specimens (n � 43) were excised as part ofthe planned margin of resection surrounding an intrapa-renchymal or periventricular lesion during neurosurgicalprocedures from July 2001 to July 2004. These lesionsincluded epileptic foci, gliomas, and arteriovenous malfor-mations. Parenchymal or ventricular wall abnormalitiesare not associated with these vascular phenomena(Zabramski et al., 1999). All tissue (typically �0.5 cm3)was resected by a neurosurgeon under an operating mi-croscope. The specimens from each of the five regionsdescribed above were lifted from the lateral ventricle walland placed in 4% paraformaldehyde (PFA) or 2% glutar-aldehyde � 2% PFA. The location of each intraoperativespecimen was recorded with stereotactic 3D surgical nav-igation (Stealth System, Sofamor Danek, Memphis, TN).Collection of intraoperative specimens was in accordancewith the guidelines of the University of California, SanFrancisco (UCSF) Committee on Human Research (Ap-proval #H11170-19113-03B).

Postmortem specimens

Seven brains (average age 40 years, range 10–68 years;average postmortem delay 8.4 hours, range 6–12 hours)were collected at autopsy. Another 42 different specimens(typically �1 cm3) were collected from different regions

TABLE 1. Regions and Descriptions of the Lateral Ventricle (Based onRhoton, 2002)

Region Definitions

Anterior horn Part of the lateral ventricle located anterior tothe foramen of Monro. Has a medial wallformed by the septum pellucidum and alateral wall composed of the head of thecaudate nucleus.

Body Extends from the posterior edge of theforamen of Monro to the point where theseptum pellucidum disappears and thecorpus callosum and fornix meet. Thelateral wall is formed by the body of thecaudate nucleus

Atrium and occipital horn Together these two form a triangular cavity,with the apex posteriorly in the occipitallobe and the base anteriorly in the pulvinar.The atrium opens anteriorly above thethalamus into the body and anteriorly belowthe thalamus into the temporal horn. Thelateral wall has an anterior part, formed bythe caudate nucleus as it wraps around thelateral margin of the pulvinar, and aposterior part, formed by the fibers of thetapetum as they sweep anterio-inferiorlyalong the lateral margin of the ventricle.

Temporal horn Extends forward from the atrium below thepulvinar in the medial part of the temporallobe and ends blindly in an anterior wallthat is situated immediately behind theamygdaloid nucleus.

416 A. QUINONES-HINOJOSA ET AL.

throughout the lateral ventricular walls. All postmortemspecimens were obtained within 12 hours of death frompatients without clinical or postmortem evidence of brainpathology. Three of seven complete brains were processedfor light microscopy. The cerebral hemispheres were fixedby bilateral perfusion with 4% PFA in 0.1 M phosphate-buffered saline (PBS), pH 7.4, through the internal carotidarteries and cut coronally into 1.5–2.0-cm slabs. Theseslabs were immersed in 4% PFA for 2 weeks and theneither cryoprotected in 30% sucrose for cryostat sectioningor rinsed overnight in 0.1 M PBS for vibratome sectioning.Blocks of brain surrounding the SVZ, �2.5 cm3 in size,were excised along with deep brain nuclei and/or corticallandmarks in order to identify spatial relationships. Fourbrains were used for electron microscopy and were im-mersed in 2% glutaraldehyde � 2% PFA for 24 hours andsubsequently rinsed with PB 0.1 M. The wall of the lateralventricle was dissected in 0.5-cm3 blocks along the rostro-caudal and dorsal-ventral axis. Collection of pathologyspecimens was in accordance with the guidelines of theUCSF Committee on Human Research (Approval#H11170-19113-03B).

Immunohistochemistry

All fixed specimens were cut on a vibratome (50-�msections) or cryostat (10–50-�m sections). For cryostatsectioning, tissue was frozen in OCT compound (Tissue-Tek, Sakura Fine Tek, Torrance, CA). For immunohisto-chemistry (IHC), tissue sections were thoroughly rinsed inPBS and incubated for 1 hour at room temperature inblocking solution (PBS, 0.1% Triton, 10% normal goatserum). Sections were then incubated for 24 hours at 4°Cin primary antibody diluted in blocking solution (Table 2).Following primary antibody incubation, sections wererinsed in PBS and then incubated in the appropriate flu-

orescent secondary antibody (Molecular Probes, Eugene,OR; Jackson Laboratories, West Grove, PA) at a dilutionof 1:500 for 24 hours at 4°C. After extensive rinsing, sec-tions were counterstained with DAPI (40 ng/ml) for 5minutes at room temperature and mounted in Aqua Poly-mount (Polysciences, Warrington, PA). For every primaryantibody and brain sample we stained the tissue with andwithout primary antibody; omission of the primary anti-body resulted in no labeling. In addition, we also dilutedthe primary antibody up to 10-fold and the staining inidentical tissue and fixation conditions went from dense tolight and then to imperceptible staining. Positive controlsfor all the antibodies used revealed only the stained pat-tern that has been reported before (Doetsch et al., 1997a;Eriksson et al., 1998; Mizuguchi et al., 1999; Ligon et al.,2004; Jin et al., 2004) (Table 3). Western blot character-ization of the antibodies used for this study has beenpreviously reported: for glial fibrillary acidic protein(GFAP), a single 51 kDa band (Debus et al., 1983); forvimentin, a single 55 kDa band (Alvarez-Buylla et al.,1987); for �III-tubulin (TuJ1 antibody), a single 50 kDaband (Gass et al., 1990); for PSA-NCAM, a broad bandmigrating above 180 kDa (Bouzioukh et al., 2001; Jin etal., 2004); for doublecortin, a doublet around 45 kDa (Mi-zuguchi et al., 1999; Jin et al., 2004); for NeuN, 2–3 bandsin the 46–48 kDa range, and possibly another band at�66 kDa (Jin et al., 2004); for Ki67, a double band at 345and 395 kDa (Key et al., 1993); for proliferating cell nu-clear antigen (PCNA), a 36 kDa band (Waseem and Lane,1990); for Olig 2, two bands of 38 and 40 kDa (Ligon et al.,2004).

Brightfield images were taken with a digital camera(SPOT camera, Diagnostic Instruments, Sterling Heights,MI) on a light microscope (Olympus, AX70) and then ex-

TABLE 2. Antibodies Used

Antibody Host Dilution Source/catalog # Imuunogenpeptide

sequence Description

GFAP Mousemonoclonal

1:500 Chemicon/MAB 3402 Purified glial filament Unknown Astrocyte marker

Vimentin Mousemonoclonal

1:1 Developmental StudiesHybridoma Bank/40E-C

Vimentin protein 55 kDa Unknown Intermediate filamentprotein frequently usedas a markerneuroepithelial cells

TuJ1 Mousemonoclonal

1:500 Covance/MMS-435P Microtubules derivedfrom rat brain

CEAQGPK Early neuronal marker

Doublecortin Guinea pigpolyclonal

1:1000 Chemicon/AB 5910 Synthetic peptide ofmouse and humandoublecortin

LYLPLSLDDSDSLGDSM

Marker of migratingneurons

NeuN Mousemonoclonal

1:500 Chemicon/MAB 377 Cell nuclei from mousebrain

Unknown Neuron-specific nuclearprotein

Ki67 Rabbitpolyclonal

1:1000 Novocastra/NCL-Ki67p Prokaryotic recombinantfusion proteincontaining 1086bp ofthe Ki67 gene

Proprietary Marker of proliferatingcells

PCNA Mousemonoclonal

1:50 Dako/M0879 Proliferating cell nuclearantigen 36 kDa

VSDYEMKLMDLDVEQ

Marker of proliferatingcells

PSA NCAM Mousemonoclonal

1:400 Abcys/AbC0019 Meningococcus group B(strain 355)

carbohydrate Marker of migratingneuroblasts

Olig-2 Rabbitpolyclonal

1:10 000 Donation1 GST-OLIG2 fusionprotein

MDSDASLVSSRPSSPEPDDLFLPARSKGGSSSGFTGGTVSSSTPSDCPPELSSELRGAMGASGAHPGDKLGGGGFKSSSSSTSSSTSSAATSSTKKDKKQMTEPELQ

bHLH transcriptionfactor expressed inoligodendrocytes andgliomas

1Ligon et al., 2004.

417ORGANIZATION OF THE HUMAN SVZ

ported to PhotoShop (Adobe Systems, San Jose, CA). Allfigures were compiled using PageMaker (Adobe Systems).

Electron microscopy

Whole (n � 4) and sectioned (n � 5) postmortem humanbrains, as well as intraoperative SVZ specimens (n � 9),were fixed in 2% glutaraldehyde � 2% paraformaldehyde.These specimens were cut on a vibratome in transverse orsagittal 200-�m sections. The sections were postfixed in2% osmium for 2 hours, rinsed, dehydrated, and embed-ded in Araldite (Durcupan, Fluka, Buchs, Switzerland).Serial 1-�m semithin sections were stained with 1% To-luidine blue and examined under the light microscope(Zeiss) to study the overall organization of the SVZ. Ul-trathin (0.05 �m) sections were cut with a diamond knife,stained with lead citrate, and examined under a Jeol100CX electron microscope to identify individual celltypes.

Postembedding immunogold staining forGFAP

Blocks of tissue were fixed by immersion in 2% parafor-maldehyde, 1% glutaraldehyde as previously reported(Blasco-Ibanez et al., 1998). For GFAP immunolabeling,tissue was immersed in 1% periodic acid (H5IO6) for 10minutes, 2% sodium metaperiodate (NaIO4) for 10 min-utes, 1% borohydride for 15 minutes, rinsed in Tris-buffered saline (TBS; pH 7.4), 1% ovalbumin dissolved inTBS for 30 minutes, TBS containing NGS for 10 minutes,rabbit anti-GFAP antiserum (1:200, Dako, Carpinteria,CA) in NGS/TBS for 15 minutes, TBS for 20 minutes, Trisbuffer (pH 7.4) containing 1% bovine serum albumin for 10minutes, goat antirabbit IgG-coated colloidal gold (Sigma,St. Louis, MO; 1:20 in NGS/TBS) for 15 minutes, satu-rated uranyl acetate for 30 minutes, then stained withlead citrate for 1 minute. Between each step specimenswere rinsed with sterile water except after blocking. Allsteps were performed on droplets of solutions in humidPetri dishes. When the primary antibody was replacedwith normal rabbit serum (1:200) we did not observe anyaccumulation of colloidal gold particles.

Serial analysis with Cresyl violet and withLuxol fast blue staining

For a morphological overview of the human SVZ at thelight microscope, serial 30–50 �m sections were cut on thecryostat and stained with Cresyl violet and with Luxolfast blue � Cresyl violet. These sections were prepared

from postmortem tissue blocks (2.5 cm3) in the coronalplane prepared as indicated above.

RESULTS

Light microscopy: histology andimmunohistochemistry

We focused this study on the lateral wall of the lateralventricle, where proliferation has been described in adulthumans (Sanai et al., 2004) and where neurogenesis per-sists in other adult mammalian species. The only excep-tion was the temporal horn, where in addition to thelateral wall we also studied the wall that directly overlaysthe hippocampus. We subdivided the lateral wall of thelateral ventricle into four rostrocaudal domains (Rhoton,2002): anterior horn (Fig. 1), body of the ventricle (Fig. 2),atrium/occipital horn (Fig. 3), and temporal horn (Fig. 4).The walls of the ventricles in each of these regions weresubdivided into three zones: ventral (V), intermediate, anddorsal (D).

Anterior horn region. At the anterior horn (Fig. 1A–I), ependymal cells (Layer I) were arranged as a one-cellthick epithelium. Adjacent to the ependymal layer therewas a predominantly hypocellular region heavily popu-lated by GFAP� processes. We will refer to this secondlayer (Layer II) as the gap or hypocellular layer. Next tothe hypocellular gap there was a dense ribbon of cellbodies (Layer III). Many of these cell bodies in Layer IIIwere immunopositive for GFAP and had a stellate mor-phology, although considerable variation in morphologywas observed. Some astrocytes had many primary pro-cesses with few ramifications and others had fewer pri-mary processes with many ramifications. There was noconsistent orientation of the astrocytic processes. Furtheraway from the ventricular surface the cellularity dimin-ished and the appearance resembled that of the underly-ing striatal brain parenchyma. It is this transitional re-gion that we refer to as Layer IV and it is here that we findthe first evidence of neurons.

The region of GFAP� processes in Layer II thickened asit progressed towards both the dorsal and ventral zones ofthe ventricle. The ribbon of astrocytes in the intermediatezone was thicker (Fig. 1H). Towards the dorsal and ven-tral zones this ribbon of astrocytes increased in distancefrom the ventricular surface, forming a wedge-like struc-ture (Fig. 1G,I). In these dorsal and ventral regions thelayer of cells bodies (Layer III) becomes intermixed withthe layer of GFAP� processes, forming a thick network of

TABLE 3. Positive Controls and Results for the Antibodies

Antibody Positive control tissue Result

GFAP Human cortex, human cerebellum, mice brain tissue Stellate-shaped cells were identified in all tissues and only stained thepattern that has been reported before (Doetsch et al., 1997)

Vimentin Mice brain tissue Found to stain the ependymal cells as well as cells in the RMS in thesame pattern that has been reported before (Doetsch et al., 1997)

TuJ1 and PSA-NCAM Mice brain tissue in the RMS Found on the RMS in the same pattern that has been reported before(Doetsch et al., 1997)

Doublecortin Human fetal brain tissue Found to stain immature neurons in the same pattern that has beendescribed before (Mizuguchi et al., 1999)

NeuN Human cortex and hippocampus as well mice brain Found to stain immature neurons in the same pattern that has beendescribed before (Eriksson et al., 1998)

Ki67 and PCNA Human fetal brain tissue, meningioma tumor tissue andglioblastoma multiforme brain tumor tissue

Found to stain newborn cells that express cell cycle proteins in thesame pattern that has been described before

Olig-2 Anaplastic oligodendrogliomas Found to stain cells in the same pattern that has been describedbefore (Ligon et al., 2004)


processes and cell bodies. The interface of Layer II and III,which is very thin in the middle region, becomes veryprominent towards the dorsal and ventral borders of theventricle.

Body of the ventricle region. The organization of thewalls in the body of the ventricle was similar to that of theanterior horn (Fig. 2). Layer III was also thicker in thedorsal and ventral aspects of this wall (Fig. 2G,I). How-ever, in contrast to the anterior horn the ependymal layer(I) formed prominent infoldings at the ventral zone (Fig.2F). These infoldings were observed in every specimenstudied, although their location in rostrocaudal sectionsvaried from one individual to another. Layer II (the hypo-cellular gap) was also prominent in this region, and wasnot significantly different from its appearance in the an-terior horn (Fig. 2G–I). The astrocytic ribbon was dis-cretely separated from Layer II by a region of lower GFAPreactivity and astrocytes did not extend far into the un-derlying body of the caudate. This made the astrocyticribbon in Layer III best defined in this intermediate zone

of the body of the ventricle (Fig. 2H) compared to all otherregions or zones studied.

Atrium and occipital horn region. In the atrium andthe occipital horn (Fig. 3) the ependymal layer remainedmonocellular; no ependymal infoldings were observed. Inthis region the hypocellular gap (Layer II) was thinner inthe dorsal (Fig. 3F) and intermediate zones (Fig. 3G) of theventricle and thicker in the ventral zone of the ventricle(Fig. 3G–I). In the dorsal aspects of the posterior regionsthe hypocellular layer becomes thin and cells bodies in thesubventricular zone come close to the ependymal layer(Fig. 3A). However, we always found a layer of stronglyimmunoreactive GFAP fibers (Fig. 3G). Unlike the ante-rior horns and body of the ventricle, where the SVZ islargely surrounded by gray matter (caudate), in theseoccipital regions the SVZ is lined by optic radiation fibers(Fig. 3E). The astrocytic layer also differed significantlyfrom those in more anterior regions of the ventricle. Manyastrocytes had a radial disposition (Fig. 3H), unlike thestellar organization of the astrocytes in the anterior horn

Fig. 1. Anterior horn. A–C:Semithin sections of the humanbrain stained with Toluidine. Inthis region, Layer I, ependymallayer (E), was separated fromLayer III by Layer II, a hypocellu-lar gap (H), a region heavily popu-lated by GFAP� processes, mostlyhypocellular. D: The region of theventricle studied. E: Coronal crosssection of a human brain specimenstudied demonstrates the dorsal,intermediate, and ventral zonesstudied. F: Cresyl violet slide ofthe ventral zone at this region ofthe ventricle. G–I: Immunocyto-chemistry slides showing GFAP inred and DAPI in blue at this regionof the ventricle. These GFAP�processes band thickened towardthe dorsal and ventral zones of theventricle. Layer III, facing the cau-date nucleus, intermediate zone(H), had a large concentration ofGFAP� astrocytes. Towards thedorsal (G) and ventral (I) zonesthis ribbon of astrocytes ran fur-ther away from the ventricularsurface, forming a wedge-likestructure. The organization of thedifferent layers was most evidentin the intermediate zone of theventricle, where there was a layerof GFAP� astrocytes separatedfrom the ventricle by a band ofGFAP positivity (H). R, ribbon ofcells; H, hypocellular gap; E,ependymal layer. Scale bars � 25�m in A–C; 50 �m in F; 40 �m inG–I.


and body of the ventricle. The astrocytic layer was veryprominent in the intermediate zone and ventrally, but wasless prominent dorsally (cf. Fig. 3F,G with H). GFAP�

astrocytes were positioned deeper in the brain paren-chyma, and their radial processes (Fig. 3G,H) intermixwith the fiber tracks of the underlying white matter. In-

Fig. 2. Body of the ventricle. A–C: Semithin sections of thehuman brain stained with Toluidine. In this region, Layer III, theribbon of cells (R) was also thicker in the dorsal and ventral aspectsof this wall. Cell bodies were sparsely present in Layer II asobserved in Toluidine-stained semithin sections (arrow in A).D: The region of the ventricle studied. E: Coronal cross section of ahuman brain specimen studied demonstrates the dorsal, interme-diate, and ventral zones studied. F: The ependymal Layer I, ven-

trally, formed prominent infoldings. G–I: Immunocytochemistryslides showing GFAP in red and DAPI in blue at this region of theventricle. These GFAP� processes band thickened towards thedorsal (G) and ventral (I) zones of the ventricle, forming a wedge-like structure. The astrocytic ribbon, in Layer III, was also bestdefined in this intermediate zone compared to all other regions orzones (H). R, ribbon of cells; H, hypocellular gap; E, ependymallayer. Scale bars � 25 �m in A–C; 75 �m in F; 40 �m in G–I.


Fig. 3. Atrium and occipital horn. A–C: Semithin sections of thehuman brain stained with Toluidine. The ependymal layer re-mained monocellular; no ependymal infoldings were observed. Inthe dorsal aspects of the posterior regions the hypocellular layerbecomes thin and cells bodies in the subventricular zone come closeto the ependymal layer (A). D: The region of the ventricle studied.E: Coronal cross section of a human brain specimen studied dem-onstrates the dorsal, intermediate, and ventral zones studied. Un-like the anterior horns and body of the ventricle, where the SVZ islargely surrounded by gray matter (caudate) in these occipitalregions, the SVZ is lined by the fibers of the optical radiations.F–H: Immunocytochemistry slides showing GFAP in red and DAPIin blue at this region of the ventricle. In this region, the hypocel-

lular gap (Layer II) was thinner in the dorsal (F), and intermediate(G) zones of the ventricle and thicker in the ventral zone of theventricle (H). The astrocytic layer also differed significantly fromthose in more anterior regions of the ventricle. Many astrocyteshad a radial disposition (G,H) unlike the stellar organization of theastrocytes in the anterior horn and body of the ventricle. GFAP�astrocytes were positioned deeper in to the brain parenchyma andtheir radial processes (G,H) intermix with the fiber tracks of theunderlying white matter. In the dorsal region, astrocytes are lessdense and are organized tangentially (F), whereas in the interme-diate zone (G) we observed that astrocytes were organized intoradially oriented clusters. R, ribbon of cells; H, hypocellular gap; E,ependymal layer. Scale bars � 25 �m in A–C; 40 �m in F–H.


terestingly, in the dorsal region astrocytes are less denseand are organized tangentially (Fig. 3F), whereas in theintermediate zone (Fig. 3G) we observed that astrocyteswere organized into radially oriented clusters.

Temporal horn region. The temporal horn surroundsthe hippocampus dorsally. The lateral wall facing mostlywhite matter (radiations from the geniculate and tempo-ral cortex) is topographically equivalent to the lateral wall

Fig. 4. Temporal horn. A–C: Semithin sections of the human brainstained with Toluidine. Layer I, the ependymal layer, was composedof a single layer of ependymal cells without ependymal infoldings.Layer II, the hypocellular gap, was most prominent dorsally (A) andthinner ventrally (B). D: The region of the ventricle studied. E: Coro-nal cross section of a human brain specimen studied demonstrates thedorsal, ventral, and hippocampus zones studied. F–I: Immunocyto-chemistry slides showing GFAP in red and DAPI in blue at this region

of the ventricle. Astrocytes do not form a discrete ribbon, but weremore dispersed dorsally (G) and ventrally (H). The face of the ventri-cle facing the hippocampus (C,F,I) had a monolayer of ependymal cellswithout infolding and had a well-defined GFAP-rich acellular LayerII. Astrocytes bordering Layer II on the wall facing the hippocampuswere smaller and dispersed into the underlying parenchyma (I). R,ribbon of cells; H, hypocellular gap; E, ependymal layer. Scale bars �25 �m in A–C; 80 �m in F; 40 �m in G–I.


of the rest of the ventricular system described above. As inother regions of the lateral ventricle, Layer I, the ependy-mal layer, was composed of a single layer of ependymalcells (Fig. 4A–C); no ependymal infoldings were noted inthese regions. Layer II, the hypocellular gap, was mostprominent dorsally and thinner ventrally. A wide layer ofastrocytes (Layer III) bordered Layer II. In this part of theventricle astrocytes do not form a discrete ribbon but weremore dispersed. Yet they are clearly associated with theperiventricular parenchyma, as deeper into the tissue thislayer of astrocytes that are well stained by GFAP antibod-ies is lost. We also studied the wall of the ventricle facingthe hippocampus directly (Fig. 4C,F,I). This wall is topo-graphically continuous with the medial wall more anteri-orly. It has been suggested that this wall of the ventricularcan regenerate neurons destined to the CA1 region of theadult hippocampus in rat (Nakatomi et al., 2002). In hu-mans, the wall facing the hippocampus had a monolayer ofependymal cells without infoldings and had a well-definedGFAP�-rich acellular Layer II (Fig. 4C,F,I). Astrocytesbordering Layer II on the wall facing the hippocampuswere smaller and dispersed into the underlying paren-chyma.

Characterization of the SVZ with other markers.

As illustrated above, the different layers of the periven-tricular parenchyma can be visualized by GFAP staining.Other markers also provide useful information on thecellular composition of the human SVZ. Polysialic acidneural cell adhesion molecule (PSA-NCAM), a markerassociated with neuronal migration and plasticity in theadult brain (Rutishauser et al., 1988; Rousselot et al.,1995; Bernier et al., 2000) was expressed by a subset ofcells in all regions of the lateral wall of the lateral ventri-cle studied (Fig. 5A–D; Table 4). These cells were alwayssituated in Layers II or III in the anterior horn. Thesecells were infrequent, but a few were consistently ob-served in each section in the dorsal, intermediate, or ven-tral zones of the SVZ. Many of these PSA-NCAM� cellshad an elongated or bipolar morphology. However, thesecells were not observed forming chains (closely apposed,elongated neuroblasts connected by membrane specializa-tions) as seen in rodents (Lois et al., 1996; Doetsch et al.,1997a). Interestingly, one of the two processes was fre-quently bifurcated and tipped with growth cones. In addi-tion to the solitary cells described above, we observed adense network of PSA-NCAM� fibers in Layer III of thedorsal wall of the temporal ventricle (Fig. 5D). Thesefibers had many varicosities and, in some regions, resem-bled long dendrites covered by spines. These processeswere consistently oriented perpendicular to the ventricle.The origin of these fibers could not be determined, as theywere not associated with PSA-NCAM-immunopositive cellbodies.

In order to investigate which cells in the different layersof the human SVZ divide, we used Ki67 and PCNA stain-ing (Gerdes et al., 1991; Bedard and Parent, 2004). Cellslabeled with Ki67 or PCNA were rare compared to therodent SVZ. However, we reliably observed Ki67- andPCNA-labeled cells in the lateral ventricular wall in theanterior horn as well as in the body, atrium, and temporalhorns. These cells were localized to Layers II and III (Fig.5E–H). Some of the Ki67� cells in Layer III also immu-nostained with GFAP, indicating that a small subpopula-tion of astrocytes in the SVZ divide (Fig. 5F, red arrow).We subsequently performed double-immunostaining for

Ki67� and PCNA (Fig. 5H, inset). Ki67 labels cellsthroughout the cell cycle, whereas PCNA is downregu-lated in late G2 and during mitosis, and is again upregu-lated in G1. Consistently, PCNA� cells were also Ki67�and most of these cells were in Layer III. In contrast, notall PCNA� cells were Ki67�; cells labeled only by PCNAwere more common in Layer II. These findings suggestthat dividing cells may shift their position from Layer IIIto II during the cell cycle.

Cells and processes labeled by Tuj1 antibodies, whichstains �-III tubulin expressed by immature neurons(Moody et al., 1989; Katsetos et al., 1991; Easter et al.,1993), were only observed in the anterior horn and body ofthe lateral ventricles (Fig. 5I,J). We did not observe Ki67or PCNA� cells double-labeled for Tuj1, suggesting that ifthese cells are produced in the adult, they are postmitotic.Whereas Tuj1� fibers with varicosities were abundant(more common in the ventral regions of the lateral wall),cell bodies were extremely rare. These fibers and cellswere localized mainly in Layer II close to III and werealways oriented tangentially. The Tuj� fibers are similarto those previously described by Bernier et al. (2000). Themorphology of the few cell bodies observed was very elon-gated. Interestingly, in one of these cells we observed aleading process with a growth cone (Fig. 5J).

Electron microscopy

Ependymal layer (Layer I). The ependymal layerwas very similar in all regions and zones of the lateralventricle studied. Ependymal cells were found to be cuboi-dal with round-to-oval nuclei, sparse chromatin, and 1–2nucleoli. Their cytoplasm contains numerous organelles,unlike rodent ependymal cells, which possess far fewer. Inthe apical portion these cells had abundant microvilli andcilia that displayed a 9�2 microtubule organization. Lat-erally, the cells present numerous, deep interdigitationswith other ependymal cells with desmosomes and tightjunctions. Many of the ependymal cells had basal expan-sions. These expansions were oriented perpendicularly,away from the ventricle, but it is not clear whether theseexpansions traverse the hypocellular layer in its entirety(Fig. 6A,B). Other expansions turn in the hypocellular gapand run parallel to the ventricle wall (Fig. 6A). Ependymalcell processes in the hypocellular gap region could be dis-tinguished from those of astrocytes; they had fewer inter-mediate filaments compared to those of astrocytes, hadabundant mitochondria and vesicles (endoplasmic reticu-lum), numerous small Golgi, and occasional lipofuscingranules (Fig. 6C).

Hypocellular layer (Layer II). The hypocellular gapabuts the ependymal cell layer (Fig. 6). This layer ismainly formed by expansions from the ependymal cellsand by many processes of astrocytes, but some astrocytecell bodies are occasionally present in this layer. As men-tioned above, the thickness of the hypocellular gap andnumber of cell bodies in this layer varies between regions.The immunocytochemical staining at the light microscopesuggests that this layer contains numerous GFAP� ex-pansions from astrocytes. We were interested in under-standing in more detail the organization of this layer andthe types of contacts between processes. For this we usedintraoperative specimens of the ventricular wall of theanterior horn, body of the ventricle, occipital, and tempo-ral horn, which allowed optimal ultrastructural preserva-tion (Fig. 6). In this material, astrocytic and ependymal


Figure 5


expansions, and numerous ramifications, of these pro-cesses were observed. Processes from astrocytes containeddense bundles of GFAP� intermediate filaments, as con-firmed by immunogold EM (Fig. 6I). GFAP� processescontain few organelles and some mitochondria, as well assmall vesicles distributed along each expansion. Occasion-ally, glycogen particles were present in these processes(Fig. 6E). Astrocytic processes in Layer II form a remark-able network of interconnected processes joined to eachother by gap junctions and large desmosomes (Fig. 6D,F).Gap junctions and desmosomes were also present alongthe soma of the few displaced astrocytes occasionallypresent in Layer II (Fig. 7G,H). Dendrites, axons, andsynaptic contacts were rare in Layer II, but some wereobserved in the anterior horn and the body of the ventri-cle. Neuronal somata were extremely rare in the hypocel-lular gap and were only observed in the anterior horn andbody of the ventricle. These neurons had large nuclearinvaginations and numerous cytoplasmic organelles andmitochondria (Fig. 8A,B), similar to inhibitory interneu-rons of the underlying striatum. Synaptic contacts wereobserved on the soma and dendrites of these cells (Fig.8C).

Ribbon of cells (Layer III). Layer III was mainlycomposed of the cell bodies of large astrocytes. Three as-trocytic types were distinguished in this layer at the EM(see below). The ribbon of cells varied in thickness withlocation; it was most prominent in the anterior horn aswell as in the body of the ventricle (facing the caudatenucleus). Myelinated axons defined the transition be-tween Layer III and the underlying brain parenchyma(Layer IV). Synaptic contacts and neuronal somata, whichwere rare in Layer III, began to be present at this transi-

tion and became progressively more common deeper intoLayer IV. Some oligodendrocytes were also observed inLayer III (Fig. 7F, asterisk). Importantly, occasional mi-toses were observed in the ribbon of cells on the ventric-ular wall facing the caudate (anterior horn and body of theventricle) (Fig. 7C).

Interestingly, in the ventral portion and floor of theventricle of the anterior horn and body of the ventricle, inaddition to astrocytes and the occasional oligodendrocyte,we also found cell bodies of ependymal cells within LayerIII (Fig. 8D–F). These displaced ependymal cells, firstidentified in semithin sections at the light microscope andconfirmed to be ependymal cells at the EM level, did notface the ventricle, but formed small clusters in Layer III(Fig. 8E). They were observed in all the subjects studied,young and old, and in both pre- and postmortem samples.Displaced ependymal cells formed small round-oval ro-settes containing 4–14 cells (based on reconstructions ofserial sections) surrounding a central complex containingabundant microvilli, cilia, and junctional complexes (Fig.8E,F). Displaced ependymal cells in Layer III hadelectron-dense junction complexes but lacked well-developed tight junctions. Their cilia contained a 9�2microtubule organization (Fig. 8D,d). Furthermore, somedisplaced ependymal cells had short processes, but theseprocesses did not exhibit gap junctions and/or desmo-somes; these junctions appear to be confined to the cellbodies and to the clusters of displaced ependymal cells.Displaced ependymal cells were not found deeper in LayerIII or at the interface with Layer IV, where myelin isfound.

Astrocyte types

Since astrocytes are such an important component ofthe adult human SVZ, we studied this population of cellsin more detail (Fig. 7). Three types of astrocytes wereobserved between Layers II and III of the human SVZwith the EM. The first type is a small astrocyte with long,tangentially oriented horizontal projections (Fig. 7B). Thiswas the predominant type of astrocyte in Layer II. Thesecells contain scarce cytoplasm and few organelles, but hadvery dense bundles of intermediate filaments in their cy-toplasm and processes. The second type of astrocyte islarge and mainly found at the interface of the hypocellularlayer and the ribbon of cells (Layer II/III) and withinLayer III (Fig. 7A,a). These astrocytes had large expan-sions, large cell bodies, and abundant organelles (mito-chondria, intermediate filament, endoplasmic reticulumvesicles). These astrocytes were most common in the mid-dle portion of the lateral wall at the level of the body of thelateral ventricle. The third and last type of astrocyte isalso large (Fig. 7D,E), but has few cytoplasmic organellesand a much lighter cytoplasm, and is mainly found over-

TABLE 4. Summary of Immunohistochemistry Results for the Different Regions of the Lateral Ventricle1

GFAP Vim Tuj1 DCX NeuN Olig2 Ki67 PCNAPSA-

NCAM

Anterior horn �� – � � � � ��Body �� Atrium/occipital horn �� Temporal horn ��

1The intensity of immunocytochemical staining is represented by �, light; ��, intermediate, ��, dark staining; –, no staining; n.d., not determined.

Fig. 5. Characterization of the human SVZ with multiple markers.A–D: PSA-NCAM was expressed by a subset of cells in all regions ofthe lateral wall of the lateral ventricle studied from the anterior hornto the temporal horn. In the anterior horn (A), PSA-NCAM� cellswere found in the dorsal, intermediate, and ventral zones of the SVZ,some of the PSA-NCAM� cells had morphology similar to that ofmigrating cells. However, these cells were not observed formingchains, as has been observed in other vertebrates. In the dorsal wallof temporal horn (D), there were also PSA-NCAM� fibers that ap-peared perpendicular to the ventricle. Some PSA-NCAM� cell bodieswere also identified in the dorsal and lateral walls of the temporalhorn. E–H: Some Ki67� cells colabeled with GFAP (F) and PCNA (H).Most of the Ki67� cells were observed in Layer III. However, some ofthe Ki67� and PCNA� cells were also present in Layer II. I–J: Tuj1�staining was only found in regions of the anterior horn and the bodyof the ventricle, in the SVZ, at the transition between Layers II andIII. Most of the Tuj1� cells were found in Layer II and had anelongated morphology (J). A Horn, anterior horn; body, body of theventricle; A/O Horn, atrium and occipital horn; T Horn, temporalhorn. Scale bars � 15 �m in A,F; 20 �m in B,D,J; 5 �m in C,G, and Hinset; 8 �m in E; 30 �m in H; 25 �m I.


Figure 6


lying the hippocampus, in the ventral zone of the temporalhorn in Layer III.

It is unlikely that these different ultrastructures resultfrom fixation or preservation artifacts, as the three sub-types of astrocytes were observed in both intraoperative(immediately fixed) and postmortem tissue. However,slight deterioration of the gap junctions, desmosomes, andintermediate filaments, as well as some changes in mito-chondria and chromatin preservation, were observed inthe postmortem tissue (cf. Fig. 7F,G). These alterationswere more common in specimens that had the largestpostmortem intervals before fixation.

DISCUSSION

Here we present a detailed analysis of the cytoarchitec-ture and cellular organization of the adult human SVZ.This is the largest germinal region of the adult brain in allmammalian species studied, but remains poorly under-stood in humans. The principal aim of the present analysiswas to provide a basic description of the architecture andultrastructural of the SVZ of the lateral ventricle. Thiswork extends previous studies using samples of restrictedareas of the lateral ventricular wall (Bernier et al., 2000;Weickert et al., 2000; Sanai et al., 2004) by providing adetailed anatomical description of the entire lateral wallof the lateral ventricle using intraoperative as well aspostmortem samples. Based on these data we propose amodel for the cellular organization of the human SVZ (Fig.9). In order to further elucidate possible mechanisms tostimulate cell division and migration of SVZ progenitors todirect these cells to areas of injury or degeneration (LeBelle and Svendsen, 2002; Parent, 2003; Lindvall et al.,2004; Lindvall and Kokaia, 2004), we need a sound under-standing of this germinal region.

Unique anatomical features of the humanlateral ventricular walls

One of the most intriguing and interesting aspects of thehuman SVZ is that it differs significantly from that of anyother studied mammalian species. In the SVZ of mice(Doetsch et al., 1997b), dog (Blakemore and Jolly, 1972),

and primates (Kornack and Rakic, 2001; Pencea et al.,2001), astrocytes lie directly next to the ependymal layer.In contrast, human SVZ astrocytes are separated from theependyma by a region largely devoid of cell bodies andvery rich in processes from astrocytes and ependymalcells. Although this organization appears unique to theadult human brain, some features may be comparable tothat reported in other vertebrates. For example, in a re-cent study of the bovine SVZ (Rodriguez et al., 2003), alayer separating the ependyma from the SVZ astrocytes isalso observed in regions of the lateral ventricle wall facingthe caudate nucleus. Interestingly, many small Tuj1�cells populate the bovine layer separating ependymal cellsfrom the underlying astrocytes. The hypocellular gap inhumans was present throughout the lateral wall of thelateral ventricle, but it varied in thickness from region toregion and it did not contain a population of Tuj1� cells.

Compared to other mammals, the adult human SVZappears to contain no chains of migrating neuroblasts. Inthe rodent brain, a widespread network of pathways forchain migration extends throughout most of the lateralwall of the lateral ventricle (Doetsch and Alvarez-Buylla,1996). These chains are visualized with antibodies to PSA-NCAM and Tuj1. Many of these chains join the RMSleading to the olfactory bulb (Doetsch and Alvarez-Buylla,1996; Alonso et al., 1999). Similar chains have also beendescribed in rhesus (Pencea et al., 2001) and macaquemonkeys (Kornack and Rakic, 2001). In humans, however,we did not find chains of migrating neuroblasts in any ofthe ventricular walls studied. Some individual TuJ1�cells were observed in the human SVZ. TuJ1� cells in theSVZ have also been observed by others (Bernier et al.,2000; Weickert et al., 2000; Sanai et al., 2004) and themajority of these cells have an elongated morphology.Interestingly, in some of these cells a leading processtipped by a growth cone was observed (e.g., Fig. 5F). Thissuggests that some migrating young neurons exist withinthe human SVZ. However, these cells were rare, were onlyobserved in the anterior horn and the body of the ventriclefacing the striatum, and compared to other species theydid not form chains. A prior study by Bernier et al. (2000)revealed small oval thickenings along TuJ1� fibers in theSVZ and similar swellings were observed in our material(e.g., Fig. 5C). Bernier et al. suggested that these swell-ings corresponded to the cell bodies of neuroblasts migrat-ing in chains. However, when we counterstained our sec-tions with DAPI we found no evidence of nuclei in thesethickenings. At the EM we also found no evidence ofchains of migrating cells. We believe that these thicken-ings represent varicosities along TuJ1� neuronal pro-cesses.

Chains of migrating cells in the adult rodent and mon-key brain can be visualized with PSA-NCAM antibodies(Bonfanti and Theodosis, 1994; Rousselot et al., 1995;Kornack and Rakic, 2001). In the adult human SVZ somePSA-NCAM� cells were observed, but the majority ofthese cells did not have a migratory morphology and werenot arranged in chains. These cells were present in LayersIII and IV between the astrocytic cell bodies. In some ofthese cells processes with dendritic spines were observed(e.g., Fig. 5I), suggesting a more mature neuronal mor-phology. This population of PSA-NCAM� cells was notlabeled by any of the other markers we used, includingTuJ1 antibodies. PSA-NCAM expression has been associ-ated with cellular plasticity (Rutishauser and Land-

Fig. 6. Characterization of the ependymal and hypocellular layers.A,B: Ependymal cells were cuboidal with round-to-oval nuclei, sparsechromatin, and 1–2 nucleoli. Their cytoplasm contains numerousorganelles. Basal expansions from the ependymal cells adopt a par-allel and/or perpendicular direction to the ventricle, but it is not clearwhether these expansions traverse the hypocellular layer completely(white arrows in B). Inset in A shows the vimentin� staining ofependymal cells. C: The processes of ependymal cells (dots) differedfrom those of the astrocytes (dashed lines) in several respects sincethese ependymal cells display fewer intermediate filaments, abun-dant mitochondria, and vesicles. D: Astrocyte processes in Layer IIappear to form a remarkable network of interconnections via gapjunctions (arrow) and desmosomes (arrowhead). E: Occasionally, gly-cogen particles were present in these astrocytic processes. F–H: Gapjunctions (illustrated by solid arrows in F and in by broken arrow inG) and desmosomes (illustrated by solid arrows in G and H) were alsopresent on the soma of the fewer displaced astrocytes occasionallypresent in Layer II. I: The intense GFAP� staining observed in thisregion at the light microscope level suggested that these intermediatefilaments contain GFAP. This inference was confirmed with GFAPimmunogold staining in EM. E, ependymal; *, astrocyte. Scale bars �10 �m in A; 5 �m in B; 2.5 �m in C; 0.5 �m in D–I.


Figure 7


messer, 1996) and it is possible that this population ofPSNA-CAM� cells in the human brain is involved indynamic processes. Further work is required to identifythese cells and determine their identity and function.However, it is clear that they do not form chains of mi-grating neuroblasts, as observed in other mammals. Aprevious study reports clusters of PSA-NCAM� cell in thehuman SVZ of children less than 1 year old, but did not seesimilar clusters in adult specimens (Weickert et al., 2000).Chain migration in humans may be a transient phenom-enon that only occurs during a restricted periods of infantdevelopment.

Organization of the hypocellular layer

The adult human brain SVZ not only contains few mi-grating cells, but also had fewer proliferating cells com-pared to similar regions of the adult rodent or monkeySVZ. What could be the function of this region in humans?The hypocellular gap is a prominent component of theadult human brain SVZ. This region remains poorly un-derstood. For this reason we carried out a detailed high-resolution EM analysis of this region. Our results reveal aremarkable network of interconnected astrocytic pro-cesses in the hypocellular gap. This is consistent withobservations at the LM level revealing a dense band ofintense GFAP staining in the hypocellular gap. We con-firmed that the astrocytic processes seen at the EM areGFAP� (Fig. 6I). These processes are linked by a varietyof junctional complexes. Prominent gap junctions and des-mosomes were observed between astrocytic processes.Clearly, this is a region where astrocytic processes cometogether and possibly exchange signals.

We do not know the origin of these astrocytic processes,as they are highly contorted and in our material we couldonly follow them for a short distance. In some sections weobserved astrocytes in Layer III sending processes thatcurved, or ramify, along the hypocellular gap. It is likelythat many of the processes within Layer II originate inLayer III, but we do not know if astrocytes deeper withinthe brain parenchyma also send processes that reach thehypocellular gap. What we did observe was many ependy-mal cells that project short processes into the hypocellular

gap. Interestingly, these ependymal processes also inter-act closely with the processes of astrocytes (Fig. 6C) andestablish specialized contacts (desmosomes and smallunions similar to tight junctions). Many of the ependymalprocesses appear to branch or ramify within the hypocel-lular gap, increasing their contacts with astrocytic pro-cesses, but we did not find evidence of gap junctions be-tween astrocytes and ependymal cells in the hypocellulargap.

Layer II (the hypocellular gap) appears to be a region ofinteraction between astrocytes and between astrocytesand ependymal cells. The functional reason why thesecells are linked together within Layer II remains un-known. It has been suggested that astrocytes are inter-connected, forming networks that may regulate neuronalfunctions (Haydon, 2001; Fields and Stevens-Graham,2002; Pascual and Haydon, 2003) and this may be a func-tion of the interconnections we observe in the hypocellulargap. Alternatively, the interconnections between astro-cytes and between astrocytes and ependymal cells maycontribute to ionic, neurotransmitter, and metabolic ho-meostasis of brain parenchyma. For example, extracellu-lar K� in regions surrounding the SVZ may be shuntedinto the cerebrospinal fluid (CSF) via a Layer II-rich net-work of interconnections between astrocytes and ependy-mal cells. Occasionally, some synaptic contacts were ob-served in the hypocellular gap. It is possible that somefunctions of the hypocellular gap are under neural control,as previously suggested for the periventricular region ofthe hypothalamus (Kobayashi et al., 1970; Guldner andWolff, 1973; Paull et al., 1979).

Finally, the hypocellular gap may be a remnant of in-tense germinal activity during an earlier period of life. Asmentioned above, in some regions of the young bovine SVZa Layer II also exists and in some areas this layer ispopulated by numerous TuJ1-positive cells that may cor-respond to young neurons (Rodriguez-Perez et al., 2003).It is possible that in the SVZ of human infants Layer II isalso populated by many young neurons and, as this pop-ulation is consumed, a layer of astrocytic processes isretained. Some of the astrocytes in the human SVZ LayerIII continue to divide in the adult. While previous workindicates that GFAP� cells have neural stem cell proper-ties in vitro (Sanai et al., 2004), we do not know the preciselocation of these neural stem cells in the adult humanSVZ, or whether proliferating GFAP� cells function asstem cells in vivo. A small subpopulation of cells alsoproliferates in Layer II, but these cells appear to beGFAP–. Some of these cells may also have stem cell po-tential or be derived from neural stem cells in vivo. It hasbeen hypothesized that SVZ astrocytes in rodents interactclosely with ependymal cells and touch the ventricle whenthey are activated (Doetsch et al., 1999b; Lim et al., 2000).In adult human brain we observed some astrocytes thatextend processes through Layer II and contact the ependy-mal layer and ventricle (Sanai et al., 2004). In the presentanalysis we also observed astrocyte processes within theependymal layer and contacting the ventricle at the EM(not shown). The hypocellular gap may separate neuralstem cells from the ventricle and this may regulate theirproliferation and possible neurogenesis. It will be inter-esting to determine if proliferating cells in Layer III haveany projections that extend into the ependymal layer andpossibly reach the ventricle.

Fig. 7. Astrocyte characteristics. Three types of astrocytes in Lay-ers II and III of the human SVZ were identified. A: Large astrocytes,mainly found in the interface of the hypocellular, and the ribbon ofcells, layer (Layer II/III). These astrocytes had large expansions, largecell bodies, and abundant organelles (mitochondria, intermediate fil-ament, endoplasmic reticulum vesicles). These astrocytes are alsopresent within Layer III and were very common in the middle portionof the lateral wall at the level of the body of the lateral ventricle. a: Aninternal cilium (white arrow). B: Small astrocyte with long tangen-tially oriented horizontal projections. This type of astrocyte was thepredominant type in Layer II. These cells contain scarce cytoplasmand few organelles, but were rich in intermediate filaments. C: Occa-sional mitosis were found in the ribbon of cells layer. Light (D) andEM (E) pictures show a type of astrocyte that is also large, but has fewcytoplasmic organelles and a much lighter cytoplasm, and is mainlyfound overlying the hippocampus, in the ventral zone of the temporalhorn in Layer III. F: Immediately fixed postmortem tissue, (6 hours)shows a good preservation of the astrocytes (*, oligodendrocyte).G: Fixed tissue 10 hours postmortem shows slight deterioration somechanges in mitochondria and chromatin preservation were observedin the postmortem tissue. Scale bars � 2.5 �m in A,C,E; 1.0 �m in a;3.5 �m in B; 10 �m in D; 2.0 �m in F,G.


Figure 8


Displaced ependymal cells

In the adult human brain, mature ependyma regulatethe transport of ions, small molecules, and water betweenthe CSF and neuropil, thereby serving as an important

barrier between CSF and brain parenchyma (Bruni,1998). Human ependymal cells have been studied by im-munohistological methods (Gould and Howard, 1987;Gould et al., 1990), and by EM (Hirano and Matsui, 1975;Gould et al., 1990). Previous studies of the ependymallayer in adult humans have focused on cells at this inter-face and are always described as the lining of the ventric-ular cavity (Roessmann et al., 1980; Gould et al., 1990). Itwas for this reason that we were surprised to find smallclusters of ependymal cells within the SVZ. These cellswere clearly ependymal cells, as they retained their tuftsof cilia with 9�2 microtubule organization. We foundthese displaced ependymal cells in all studied brain spec-imens irrespective of age or sex and, interestingly, theywere always in a similar ventral location.

The origin of these displaced ependymal cells is notknown. It is possible that some ependymal cells becomedisplaced during their differentiation from radial glia(Spassky et al., 2005). In a study of ependymal cells inthe developing human brain, however, no evidence of

Fig. 9. Proposed model of thehuman SVZ organization in thecoronal plane. Model showingthe organization of the humanSVZ with Layers I–IV. Layer I,ependyma; Layer II, hypocellulargap; Layer III, ribbon of cells;Layer IV, transitional zone to thebrain parenchyma. Astrocytes(blue cytoplasm and light-bluenucleus) with processes at thebase of the SVZ mainly found inLayer III but some also in LayerII. Displaced ependymal cells(gray cytoplasm and light-graynucleus) form clusters and aremainly found between Layers IIand III. Neurons (red cytoplasmand light-red nucleus) are foundmainly along the interface be-tween Layers III and IV withprocesses sent to Layers II andIII. In green are the synapsesfound in Layers II and III.

Fig. 8. Characteristics of neurons and displaced ependymal cells.A: Large neurons, with large invaginations and numerous cytoplas-mic organelles and mitochondria, were observed in the hypocellularLayer II. B: A large dendritic expansion and three synaptic contacts(arrows) are observed. C: Synaptic contacts were found over the somaand dendrites. D,d: The cilia of displaced ependymal cells displayed a9�2 configuration (arrows in e) with union complexes. E: Displacedependymal cells were first identified in semithin sections with opticalmicroscopy. F: Electron microscopy revealed that these displacedependymal cells formed small spherical or oval groups of 4–14 cellsand surrounded central complexes that were formed by abundantmicrovilli, cilium, and union complexes. Circles demonstrate a zonewhere cilia exist which indicates that these are displaced ependymalcells. Scale bars � 2.5 �m in A,D; 2 �m in B; 0.5 �m in C; 1 �m in d;5 �m in E; 4 �m in F.


displaced ependymal cells was observed (Gould et al.,1990). A more likely explanation is that these displacedependymal cells become trapped with the SVZ by theblending of ependymal foldings, as previously suggestedin regions of the cerebral aqueduct and fourth ventriclein humans (Alvarez et al., 1987). In fact, we noted thatventral regions of the anterior horn and body of theventricle the ependymal layer are folded (Fig. 2F).Union of the superficial ependymal cell layer within thepeaks of infoldings may leave some ependymal cellstrapped within the troughs. Studies of the ratependyma appear to indicate that adhesion and fusionof the ependyma occur between the hippocampus andcorpus callosum (Kawamata et al., 1995). In this case, itis the opposing walls of the ventricular wall that blend,resulting in the formation of small cysts or groups ofependymal cells that are no longer associated with aventricular cavity.

It is not known whether displaced human ependymalcells have any function. Clearly, the role of ependymalcells in the propulsion of cerebrospinal fluid cannottake place within the SVZ, when these cell’s cilia are notexposed to the ventricular cavity. However, they mayretain interaction with SVZ astrocytes and may contrib-ute to exchange of signals that take place in this niche.

Categorization of human SVZ astrocytes

We report three distinct types of astrocytes along thelateral wall of the human lateral ventricles. These as-trocytes differ not only in their location within the SVZ,but also in their size, ultrastructure, and relationship tothe ependyma. We know that a subpopulation of SVZastrocytes proliferate in vivo, and a small subpopulationbehaves as multipotent stem cells in vitro (Sanai et al.,2004). In adult rodents, different subpopulations of SVZastrocytes (Type B cells) have been described. We do notknow if only one of the populations has stem cell poten-tial or whether all do. It is also possible that differentsubpopulations correspond to astrocytes in differentcompetent states or different stages of the cell cycle.Recent work in rodents suggest that neural stem cells inthe SVZ correspond to a population of astrocytes thatcontain only one or two primary processes, in contrast tothe many primary processes present in stellate astro-cytes (Garcia et al., 2004). The cells that were labeledwith Ki67 in our material were relatively complex andhad highly ramified primary processes. However, it isdifficult to categorize astrocytes based purely on theirmorphology, as these cells attain very different struc-tures, depending on the region of the brain where theyreside. It is also not possible to compare the astrocytesin different species, as the brain size and structurechanges the morphology of astrocytes also changes. Infact, the astrocytes we observe in the SVZ of adulthumans differ significantly with those we and otherspreviously described in the adult rodent brain SVZ (Pri-vat and Leblond, 1972; Doetsch et al., 1997a; Peretto etal., 1997). The data presented here is a first step toclassify the different types of astrocytes within theadult human SVZ. This will help to identify those as-trocytes that correspond to the neural stem cells infuture studies.

CONCLUSIONS

The architecture and function of the adult human SVZdiffers significantly from that described in other mam-mals. While in rodents a large component of the SVZcorresponds to young neurons en route to the olfactorybulb, this migration does not appear to occur in adulthumans. The SVZ in adult humans may generate neuronsthat migrate to locations other than the olfactory bulb.However, in the present analysis of serial sections of thelateral wall of the lateral ventricle we did not find evi-dence for chain migration of young neurons to other re-gions of the adult brain. Recent work in adult rabbitsuggest that some SVZ cells migrate ventrally in the cau-dal telencephalon (Luzzati et al., 2003). We did not findevidence of a similar migration in adult humans. Someproliferating cells, as revealed by Ki67 and PCNA stain-ing, are present in the SVZ of adult humans, including thesamples from older patients. This indicates that the SVZin humans retains germinal potential. However, the levelof proliferation in the adult human SVZ seems small com-pared to what is observed in other mammals. While wefound no evidence of massive chain migration in the adulthuman SVZ, it is possible that a small number of youngneurons may be produced here and that these cells mi-grate as individual cells. Indeed, a few Tuj1� cells withmigratory morphologies were found in the SVZ of theanterior horn and the body of the ventricle, but not inother regions of the SVZ studied. Interestingly, it was onlyin this anterior region that we found proliferating GFAP�astrocytes in Layer III. It will be interesting to determinethe ultimate fate of the putative migrating young neuronsand determine if they originate from these proliferatingastrocytes. Hence, this work provides a comprehensiveanalysis of adult human SVZ anatomy, a niche whereadult neural stem cells reside.

ACKNOWLEDGMENTS

We thank the faculty of the UCSF Department of Neu-rological Surgery for providing intraoperative specimensand Drs. Andrew Bollen, Scott R. VandenBerg, and TarikTihan for assistance with postmortem specimens. Wethank Florian Merkle for insightful comments. The au-thors declare that they have no competing financial inter-ests.

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