Distribution of GABAergic cells and fibers in the hippocampal formation of the Macaque monkey: An immunohistochemical and in situ hybridization study

Distribution of GABAergic Cellsand Fibers in the Hippocampal Formation

of the Macaque Monkey:An Immunohistochemical and In Situ

Hybridization Study

ANA L. JONGEN-RELO,1 ASLA PITKANEN,2 AND DAVID G. AMARAL1*1Department of Psychiatry and Center for Neuroscience, University of California-Davis,

Davis, California 956162A.I. Virtanen Institute, University of Kuopio, Kuopio FIN-70211, Finland

ABSTRACTThe g-aminobutyric acid (GABAergic) system of the hippocampal formation of Macaca

fascicularis monkeys was studied immunohistochemically with a monoclonal antibody toGABA and with nonisotopic in situ hybridization with cRNA probes for glutamic aciddecarboxylase 65 (GAD65) and GAD67. The highest densities of labeled cells were observed inthe presubiculum, parasubiculum, entorhinal cortex, and subiculum, whereas the CA3 fieldand the dentate gyrus had the lowest densities of positive neurons. Within the dentate gyrus,most of the GABAergic neurons were located in the polymorphic layer and in the deep portionof the granule cell layer. GABAergic terminals were densest in the outer two-thirds of themolecular layer. GABAergic neurons were seen throughout all layers of the hippocampus.Terminal labeling was highest in the stratum lacunosum-moleculare. A higher terminallabeling was observed in the subiculum than in CA1 and was particularly prominent in layerII of the presubiculum. A bundle of GABAergic fibers was visible deep to the cell layers of thepresubiculum and subiculum. This bundle could be followed into the angular bundleipsilaterally and was continuous with stained fibers in the dorsal hippocampal commissure.This pattern of labeling is reminiscent of the presubicular projections to the contralateralentorhinal cortex. GABAergic cells were observed in all layers of the entorhinal cortexalthough the density was higher in layers II and III than in layers V and VI. The in situhybridization preparations largely confirmed the distribution of GABAergic neurons in allfields of the hippocampal formation. J. Comp. Neurol. 408:237–271, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: dentate gyrus; entorhinal cortex; glutamic acid decarboxylase; parasubiculum;

presubiculum; subiculum

g-Aminobutyric acid (GABA) is the major inhibitoryneurotransmitter in the mammalian brain (Storm-Mathi-sen and Fonnum, 1971; Storm-Mathisen, 1972). GABA isproduced mainly through an enzymatic decarboxylation ofglutamic acid by glutamic acid decarboxylase (GAD). PutativeGABAergic neurons were first demonstrated in the hippo-campal formation by using antibodies specific to GAD (Ribaket al., 1978) and later by using antibodies to GABA (Storm-Mathisen et al., 1983). These studies have been confirmedand extended in numerous immunohistochemical studiescarried out mainly in the rodent brain (Kohler and Chan-Palay, 1983; Seress and Ribak, 1983; Somogyi et al., 1983b,1984, 1985; Kosaka et al., 1984; Sloviter and Nilaver, 1987;Gamrani et al., 1986; Woodson et al., 1989).

In rats, GABA immunoreactivity is found in a morpho-logically diverse population of nonprincipal neurons that

Grant sponsor: California Regional Primate Research Center; Grantnumber: RR 00169; Grant sponsor: National Institutes of Health; Grantnumber: NS16980; Grant sponsor: Human Frontier Science Program;Grant number: LT-315.

A.L.J.R is currently at the Laboratory of Behavioural Biology, SwissFederal Institute of Technology, Zurich, Schwerzenbach, CH-8603, Switzer-land. E-mail: [email protected]

*Correspondence to: Dr. David G. Amaral, Center for Neuroscience,University of California-Davis, 1544 Newton Court, Davis, CA 95616.E-mail: [email protected]

Received 12 August 1997; Revised 4 August 1998; Accepted 1 December1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 408:237–271 (1999)

r 1999 WILEY-LISS, INC.

are distributed throughout all portions of the hippocampalformation (Seress and Pokorny, 1981; Ribak and Seress,1983, 1988; Seress and Ribak, 1983; Somogyi et al.,1983a,b, 1985; Schwartzkroin and Kunkel, 1985; Schlanderet al., 1987; Lacaille and Schwartzkroin, 1988; Seress andFrotscher, 1991; Soriano et al., 1990, 1993; Halasy andSomogyi, 1993; Soriano and Frotscher, 1993; Ribak et al.,1993). These neurons are distinguishable not only on thebasis of somal size and shape but also by their input/output characteristics (Freund and Buzsaki, 1996). Someneurons, such as the basket cells, give rise to axons thatterminate preferentially on neuronal cell bodies, whereasothers, such as axoaxonic or chandelier cells, primarilyinnervate axonal initial segments. There are even someGABAergic cells that exclusively innervate other GABAer-gic interneurons (Freund and Buzaski, 1996; Gulys et al.,1996). GABAergic neurons in the rat also constitute aheterogeneous population with regard to their colocaliza-tion with other neuroactive substances. The neuropeptidescoexpressed with GABA include cholecystokinin, somato-statin, neuropeptide Y, and vasoactive-intestinal peptide(Somogyi et al., 1984; Kosaka et al., 1985, 1988; Leranthand Frotscher, 1986, 1987; Esclapez and Houser, 1995;Sloviter and Nilaver, 1987). In addition, GABAergic neu-rons contain several calcium-binding proteins, such ascalbindin D28-Kda, parvalbumin, and calretinin (Kosaka etal., 1987; Nitsch et al., 1989; Sloviter, 1989; Ribak et al.,1990; Gulyas et al., 1991, 1992; Miettinen et al., 1992;Acsady et al., 1993; Seress et al., 1993a,b).

Two different GAD enzymes have been identified inmammalian brain, each derived from a separate gene(Erlander et al., 1991; Erlander and Tobin, 1991; Bu et al.,1992). In the rat brain, one gene produces a protein with amolecular weight of 66,600 (GAD67), and the other geneproduces a protein with a molecular weight of 65,200(GAD65) (Erlander et al., 1991). The localization of GADmRNAs is a reliable and sensitive marker for GABAergicneurons and has been used as a marker in the hippocam-

pal formation of rats (Houser and Esclapez, 1994), in themonkey amygdala (Pitkanen and Amaral, 1994) and visualcortex (Hendrickson et al., 1994), and in several otherregions of the rat brain (Mercugliano et al., 1992; Esclapezet al., 1993; 1994; Retaux et al., 1993). Although both forms ofGAD are present in most brain regions, the amount of mRNAsfor each form may vary both across and within specific brainregions (Feldblum et al., 1993; Esclapez et al., 1994;Hendrickson et al., 1994; Houser and Esclapez, 1994).

Although there is an extensive literature on the distribu-tion and description of GABAergic neurons in the ratbrain, only a few studies have described these cells in theprimate hippocampal formation (Bakst et al., 1985; Babbet al., 1988; Leranth and Ribak, 1991; Nitsch and Leranth,1991; Seress et al., 1991, 1993a,b, 1994; Sloviter et al.,1991, 1996; Pitkanen and Amaral, 1993; Ribak et al.,1993). To our knowledge, there are no descriptions of thedistribution of GABAergic cells and fibers in severalregions of the hippocampal formation, such as the entorhi-nal cortex. Therefore, in the present study, we present asurvey of the distribution of GABAergic cells in themonkey hippocampal formation based on immunohisto-chemical preparations using a monoclonal antibody raisedagainst GABA-bovine serum albumin (BSA) complex (Sza-bat et al., l992) as well as in situ hybridization histochemi-cal preparations using probes of mRNAs encoding GAD65and GAD67.

MATERIALS AND METHODS

Immunohistochemistry

Tissue preparation. The distribution of GABAergicprofiles was evaluated in the macaque monkey hippocam-pal formation. The brains of three Macaca fascicularismonkeys (M17–91, M18–91, and M19–91) were preparedfor the immunohistochemical demonstration of GABA asdescribed previously (Pitkanen and Amaral, 1994). Briefly,two monkeys (M17–91 and M18–91) were perfused intra-cardially with 0.9% sodium chloride solution (250 ml/minute, for 2 minutes) followed by a fixative containing alow concentration of glutaraldehyde (0.1% glutaraldehydeand 4% paraformaldehyde in 100 mM sodium phosphatebuffer, pH 7.4), and one monkey (M19–91) was perfusedwith a fixative containing a higher concentration of glutar-aldehyde (2.5% glutaraldehyde and 1% paraformaldehydein 100 mM sodium phosphate buffer, pH 7.4) for 10minutes at a flow rate of 250 ml/minute and then at a flowrate of 100 ml/minute for 50 minutes. In all cases, animalswere deeply anesthetized with ketamine HCl (10 mg/kg,i.m.) followed by sodium pentobarbital (50 mg/kg, i.p.). Thebrains were immediately blocked stereotaxically and post-fixed in the same fixative for 6 hours. The brains werecryoprotected first with 10% and then 20% glycerol with2% dimethylsulfoxide, frozen, cut into 30-µm-thick sec-tions, and stored in tissue-collecting solution (TCS; 30%ethylene glycol, 25% glycerol in 50 mM sodium phosphatebuffer, pH 7.4) at 270°C until used. All experimentalprocedures were carried out according to protocols ap-proved by the University of California-Davis and theCalifornia Regional Primate Research Center Institu-tional Animal Care and Use Committee.

Immunohistochemical procedures. Sections from twoof the brains (M17–91 and M18–91) were stained by usingthe peroxidase-antiperoxidase (PAP) method, and sections

Abbreviations

35 area 35 of the perirhinal cortexa alveusCA1–CA3 fields CA1–CA3 of the hippocampusDG dentate gyrusE entorhinal cortexEC entorhinal cortex, caudal divisionECL entorhinal cortex, caudal limiting divisionEI entorhinal cortex, intermediate divisionELc entorhinal cortex, caudal portion of the lateral divisionELr entorhinal cortex, rostral portion of the lateral divisionEO entorhinal cortex, olfactory divisionER entorhinal cortex, rostral divisionf fimbriaGL, GCL dentate gyrus, granule cell layerIML dentate gyrus, inner molecular layerML dentate gyrus, molecular layerOML dentate gyrus, outer molecular layerPaS parasubiculumPCL pyramidal cell layerPL dentate gyrus, polymorphic layerPrS presubiculumS subiculumSL stratum lucidumSLM stratum lacunosum-moleculareSO stratum oriensSR stratum radiatum

238 A.L. JONGEN-RELO ET AL.

from the third brain (Ml9–91) were stained by using theavidin-biotin technique as described by Pitkanen andAmaral (1994). Briefly, for the PAP method, free-floatingcoronal sections were washed three times in 20 mMpotassium phosphate buffer, pH 7.4 (KPBS), and incu-bated in a blocking solution containing 10% normal rabbitserum and 0.5% Triton X-100 in KPBS for 4 hours at roomtemperature. The sections were then incubated in a solu-tion that contained a monoclonal mouse anti-GABA anti-body (115AD5; provided by Dr. Ismo Virtanen, Departmentof Anatomy, University of Helsinki, Helsinki, Finland;Szabat et al., 1992) diluted 1:30, 0.5% Triton X-100 and 1%normal rabbit serum in KPBS for 48 hours at 4°C. Thecharacterization of the monoclonal GABA antibody used inthese studies has been described elsewhere in detail(Szabat et al., 1992; see also Pitkanen and Amaral,1994).The sections were then washed in KPBS and incu-bated in a solution containing rabbit anti-mouse immuno-globulin G (IgG; 1:30; ICN, Costa Mesa, CA), 1% normalrabbit serum, and 0.2% Triton X-100 in KPBS for 4 hoursat room temperature. The sections were again washed andthen incubated overnight at 4°C in a solution that con-tained mouse PAP (1:100; Sternberger Monoclonals, Balti-more, MD), 1% normal rabbit serum, and 0.2% TritonX-100 in KPBS. The sections were then reacted withdiaminobenzidine (DAB; Pierce, Rockford, IL; 0.05% DABand 0.04% H2O2 in KPBS, pH 7.4), rinsed, mounted ongelatin-coated slides, and intensified with OsO4 and thio-carbohydrazide by using the method of Lewis et al. (1986)or with the silver intensification method described byQuinn and Graybiel (1990). For the avidin-biotin method,the sections were incubated for 2 days in mouse monoclo-nal anti-GABA antiserum (diluted 1:400). The incubationprotocol followed the same procedure described previouslyfor the identification of parvalbumin-immunoreactive cells(Pitkanen and Amaral, 1993).

In situ hybridization histochemistry

Tissue preparation. Tissue for this study was ob-tained from three Macaca fascicularis monkeys (M6–94,M7–94, and M8–94). The animals were deeply anesthe-tized and perfused intracardially with 0.9% sodium chlo-ride solution (250 ml/minute for 2 minutes) followed by afixative containing 4% paraformaldehyde in 100 mM so-dium phosphate buffer, pH 7.4, for 10 minutes at a flowrate of 250 ml/minute and then at a flow rate of 100ml/minute for 50 minutes. The brains were blocked stereo-taxically and postfixed in the same fixative for 6 hours. Thebrains were cryoprotected and cut as described above andwere stored in TCS at 270°C until processing for in situhybridization histochemistry. Before use, the TCS wastreated with 0.05% diethylpyrocarbonate (DEPC) andautoclaved to inactivate RNase activity.

Probe synthesis and in situ hybridization. The hu-man GAD65 and GAD67 RNA probes were obtained by invitro transcription of two previously described GAD cDNAs(kindly provided by Dr. A. Tobin, UCLA). RNA probes wereproduced by transcription of GAD67 DNA and GAD65DNA by using a nonradioactive RNA labeling kit (Boeh-ringer Mannheim, Indianapolis, IN) according to a proto-col described by Houser and Esclapez (1994).

Free-floating sections were processed for GAD65 andGAD67 in situ hybridization as described by Houser andEsclapez (1994). Briefly, sections were subjected to a series

of pretreatment steps to enhance penetration of the probes.After the pretreatment, the sections were incubated for 1hour at room temperature in a prehybridization solutioncontaining 50% formamide, 750 mM NaCl, 25 mM EDTA,25 mM piperazine-N,N8-bis (2-ethanesulfonic acid), 0.2%sodium dodecyl sulfate, 0.02% Ficoll, 0.02% polyvinylpyr-rolidone, 0.02% BSA, 250 µg/ml poly A, 250 µg/ml salmonsperm DNA, and 250 µg/ml t-RNA. Sections were thenhybridized for 16–18 hours in a humid chamber at 50°C inthe hybridization solution that consisted of the prehybrid-ization solution with the addition of approximately 0.2–0.4ng/µl digoxigenin-labeled RNA probe, 100 mM dithiothrei-tol, and 4% dextran sulfate. Following hybridization,sections were submitted to a series of stringency washesconsisting of decreasing concentrations of a saline sodiumcitrate solution. Sections were then processed for immuno-detection of the digoxigenin label with the basic reagentsof the nonradioactive nucleic acid detection kit (Boeh-ringer Mannheim).

Previous work has indicated that human GAD65 andGAD67 cDNAs hybridize selectively to two different cellu-lar mRNAs of 5.7 kb and 3.7 kb, respectively, and do notcross hybridize with the alternate mRNA at high strin-gency (Erlander et al., 1991; Bu et al., 1992). The specific-ity of the hybridization reaction was checked by processingsome sections as described above but with labeled senseprobes instead of antisense probes. No staining was ob-served in these conditions.

Analysis of immunohistochemically prepared tissue.

For each experimental case, a one-in-eight series of immu-nohistochemically prepared sections throughout the fullrostrocaudal extent of the entorhinal cortex and otherareas of the hippocampal formation was analyzed withbrightfield optics by using a Leitz Dialux 20 microscope(Wetzlar, Germany). The distribution of GABA-immunore-active cell bodies in a representative series of coronalsections (four levels through the entorhinal cortex and twolevels through the hippocampus) from one of the cases(M17–91) was plotted by using a computer-aided digitizingsystem (MD-2; Minnesota Datametrics, Shoreview, MN).The distributions of GAD67 and GAD65 mRNA-express-ing neurons were analyzed in sections selected to matchthe rostrocaudal levels analyzed in the GABA immunohis-tochemical preparations. These sections were taken fromcases M7–94 and M8–94, respectively. Labeled neuronswere plotted by using the Neurolucida computer-aidedplotting system (MicroBrightfield, Colchester, VT). Thelaminar and regional boundaries of the areas of thehippocampal formation were drawn from adjacent Nissl-stained sections. The camera lucida drawings and thedistribution plots were superimposed by using the Canvas5.0 (Daneba Software, Miami, FL) software package on aMacintosh computer (Apple Computers, Cupertino, CA).To illustrate the variety of subtypes of the GABA-immunoreactive cells, camera lucida drawings were madeof representative cells from several hippocampal fields.

Low-power, brightfield photomicrographs were takenwith a Nikon Multiphot 4 3 5 inch camera system (Tokyo,Japan). High-power photomicrographs were taken by us-ing a Leitz DMRB photomicroscope (Wetzlar, Germany).The illustrations were made by scanning the 4 3 5 inchnegatives and the 35 mm negatives using a Polaroid 45and a Polaroid SprintScan 35 Plus scanner, respectively.The digital images were subsequently edited by using

GABA IN THE MONKEY HIPPOCAMPAL FORMATION 239

Adobe Photoshop 5.0 software (Adobe Systems, MountainView, CA) on a Macintosh computer and were printed witha Fuji Pictrography 3000 color printer (Tokyo, Japan).

Quantitative analysis of the GABA immunostaining.

To analyze the sizes of the various subtypes of GABA-immunoreactive cells, camera lucida drawings were madefrom representative cells (n 5 957). The feret diameter(defined as the diameter of a fictitious circular object thathas the same area as the current object) for the multipolar,pyramidal-shaped, and spheroidal cells; the major/minoraxes for the fusiform cells; and the cross-sectional areas forall cells were measured with the Sigma Scan software(Jandel Scientific Software, San Raphael, CA) on anMS-DOS computer by using a digitizing tablet. Differencesin the densities of GABAergic neurons in the various fieldsof the hippocampal formation were assessed by plottingand counting the labeled cells in sections at five represen-tative levels through the rostrocaudal extent of the hippo-campus and entorhinal cortex. Adjacent Nissl-stainedsections were used to delineate the boundaries of thedifferent regions and layers of the hippocampal formationin the distribution plots. The cross-sectional areas of thedifferent fields of the hippocampal formation were mea-sured with the Sigma Scan software on an MS-DOScomputer. The entorhinal cortex at each level was subdi-vided further into 500 µm radial bins, and the numbers oflabeled cells per mm2 were measured in each bin sepa-rately for layers I, II/III, V, and VI.

RESULTS

Nomenclature of the monkeyhippocampal formation

The nomenclature and cytoarchitectonic subdivisions ofthe hippocampal formation have been described previ-ously (Pitkanen and Amaral, 1993). In the term hippocam-pal formation, we include the dentate gyrus, hippocampus,subiculum, presubiculum, parasubiculum, and entorhinalcortex. The regions of the monkey hippocampal formationare indicated in the outline of a representative Nissl-stained section in Figure 1A,B. The dentate gyrus iscomprised of three layers: a cell-dense granule cell layer, arelatively cell-free molecular layer that lies superficial tothe granule cell layer and extends to the hippocampalfissure or ventricle, and a rather narrow polymorphic layerlocated subjacent to the granule cell layer. The hippocam-pus is divided into three distinct fields: CA3, CA2, and CA1(Fig. 1A,B). CA3 and CA2 are characterized by largepyramidal cells that are located in a relatively compactprincipal cell layer; CA2 is differentiated from CA3 by thelack of a mossy fiber input. CA1 has smaller pyramidalcells in its principal cell layer, which is substantiallythicker than in CA2 and CA3. The hippocampus is subdi-vided further into several laminae that run parallel to thepyramidal cell layer. In all hippocampal fields, the termsuperficial means towards the pia (or hippocampal fis-sure), and the term deep is used to indicate the oppositedirection. Deep to the pyramidal layer is the cell-poorstratum oriens, and deep to this is the fiber-rich alveus.Superficial to the pyramidal cell layer in CA3 is thestratum lucidum, in which some of the mossy fibers travel.In CA2 and CA1, the region just superficial to the pyrami-dal cell layer (and in CA3 superficial to the stratum

lucidum), is the stratum radiatum: the stratum lacunosum-moleculare is superficial to the stratum radiatum.

The border of CA1 with the subiculum is quite obliqueand is sometimes marked by a narrow, obliquely oriented,cell-free zone near which slightly increased numbers ofsmall neurons are located. The stratum radiatum ends atthe superficial CA1/subiculum border, and the relativelycell-free zone superficial to the pyramidal cell layer in thesubiculum is called the molecular layer. The presubiculumand parasubiculum have a cell-free layer I and a denselycellular layer II. Layer II of the presubiculum can bedifferentiated into a thinner superficial and a thicker deepsublamina on the basis of a number of histochemical andimmunohistochemical staining procedures. There are scat-tered, large cells deep to layer II of the presubiculum andparasubiculum that are often included as deep layers ofthese regions. It is unclear in the monkey, however,whether these cells are associated with these regions or,instead, are an extension of the deep layers of the entorhi-nal cortex (Amaral et al., 1987).

The monkey entorhinal cortex is divided into sevencytoarchitectonically distinct divisions (Amaral et al., 1987):the olfactory division of the entorhinal cortex (EO); therostral division (ER); the rostral portion of the lateraldivision (ELr); the caudal portion of the lateral division(ELc); the intermediate division (EI); the caudal division(EC); and the caudal limiting division (ECL). The entorhi-nal cortex is divided further into six layers. These includelayer I, a cell-poor layer beneath the pia; layer II, a thinlayer of darkly stained multipolar cells that are sometimesgrouped into islands; layer III, a broad, densely cellularlayer in which the cells tend to be organized in patchesrostrally but are more columnar caudally; layer IV, anarrow cell-free layer (the lamina dissecans) that is clearlyvisible only in EI; layer V, a band of large pyramidal cellsthat can be subdivided into two superficial laminae (Vaand Vb) with cells of different sizes and a deeper, largelyacellular layer (layer Vc); and layer VI, which is a rela-tively broad cellular layer that, at caudal levels, has theappearance of coiled rows of cells.

General features

Distribution of GABA immunoreactivity. GABA im-munoreactivity was found to be associated with cell bodies,fibers, and terminals throughout the whole hippocampalformation (Figs. 2, 3, 7). In the brains that were perfusedwith the fixative containing the low concentration ofglutaraldehyde, neuronal cell bodies were labeled preferen-tially, and initial portions of dendrites could be visualized.The brain that was fixed with the high glutaraldehydeconcentration showed a greater neuropil staining (varicosefibers and varicosities), the reaction product tended tohave a granular appearance, and dendrites were notlabeled.

The density of GABA-positive neurons varied substan-tially across the different fields of the hippocampal forma-tion (Fig. 10). Layer II of the presubiculum showed thehighest density of GABAergic neurons in the hippocampalformation, followed by layer II of the parasubiculum, alllayers of the entorhinal cortex, and the subiculum. Thelowest densities of labeled neurons were found in thehippocampus and dentate gyrus.

The GABAergic cells throughout the hippocampal forma-tion constituted three major classes. The largest class


Fig. 1. A: Photomicrograph of a Nissl-stained coronal sectionillustrating the different cytoarchitectonic fields of the monkey hippo-campal formation. B: Outline of the Nissl-stained section shown in Ademarcating the various laminae of the dentate gyrus, the hippocam-pus, the subiculum, the presubiculum, the parasubiculum, and the

entorhinal cortex. The hatched area here and in succeeding illustra-tions indicates the temporal horn of the lateral ventricle. Romannumerals indicate the layers in various cortical areas. For abbrevia-tions, see list. Scale bar 5 1 mm.

Fig. 2. Photomicrograph and plot of the distribution of GABAergiccells in a representative coronal section through the rostral hippocam-pal formation A: Brightfield photomicrograph of a section processedfor GABA immunohistochemistry. B: Outline of an adjacent sectionprocessed for Nissl staining superimposed on a computer-generatedplot of the distribution of GABA-immunoreactive neurons observed inthe section shown in A. Each dot represents one labeled neuron. Note

the heavy fiber labeling of the end bulb of the mossy fiber projection ofCA3 (arrowhead in A). Note also the higher neuropil labeling in theouter two-thirds (oml) than in the inner one-third (iml) of themolecular layer of the dentate gyrus. Arrows in A indicate the highimmunoreactivity in the stratum oriens of the subiculum. For abbre-viations, see list. Scale bar 5 1 mm.

Fig. 3. A,B: Photomicrograph and plot, respectively, of the distribu-tion of GABAergic cells in a representative coronal section through thecaudal hippocampal formation. Arrowhead in A points to the increased

staining in stratum lucidum associated with the mossy fibers. Arrowin A points to an area of higher density of GABA-immunoreactive cellsin the CA2 field. For abbreviations, see list. Scale bar 5 1 mm.

consists of multipolar cells. The multipolar cells variedmarkedly in their somal shape and size. The most distin-guishable multipolar cells had spheroidal, oval, triangular,or pyramidal cell bodies. The second major class of GABAergiccells consisted of fusiform cells. The fusiform cells hadoval-shaped cell bodies with two thick dendrites originat-ing from opposite poles of the somata. The fusiform cellbodies also varied from very small to very large. The thirdmajor class of GABAergic cells consisted of stellate cells.These cells are distinguished from other multipolar cellsbecause of the regular, stellate distribution of their mul-

tiple, thin dendrites, which originate from the soma. Thesestellate cells were most prominent in the CA3 field of thehippocampus (hilar portion).

Distribution of GAD65 and GAD67 mRNA-expressing

neurons. Neurons positive for GAD65 and GAD67 mRNAwere detected in all layers of the hippocampal formation(Figs. 4, 5, 6A). By using the same probe concentration,GAD65 mRNA-positive neurons showed a lower labelingintensity than GAD67 mRNA-labeled neurons. There alsoappeared to be fewer neurons that were positive forGAD65 mRNA than for GAD67 mRNA throughout the

Fig. 4. Plots of the distribution of cells expressing mRNA forglutamic acid decarboxylase 65 (GAD65) in coronal sections at tworostrocaudal levels through the hippocampal formation arranged fromrostral (A) to caudal (B). The outlines of an adjacent section processed

for Nissl staining are superimposed on the computer-generated plotsof the distribution of GAD65-positive neurons. Each dot representsone labeled neuron. For abbreviations, see list. Scale bar 5 1 mm.


hippocampal formation. The distribution of GAD mRNA-positive cells corresponded well to the distribution ofGABA immunoreactive neurons. Thus, the presubiculum,parasubiculum, entorhinal cortex, and subiculum showedthe highest densities of GAD65 mRNA- and GAD67 mRNA-labeled neurons, whereas the lowest densities were ob-served in field CA3 and in the dentate gyrus.

Although no attempt was made to quantify the intensityof the color reaction product, there were qualitative differ-ences between the intensity of labeling for the two GAD

mRNAs. To monitor the development of the color reactionproduct, a few sections were examined periodically duringthe incubation with the color substrate. The first cells thatappeared to be labeled for GAD65 and GAD67 mRNA werefound in the dentate gyrus (after 1–2 hours in the colorsubstrate), followed by the hippocampus. The last cells todevelop color reaction were the neurons in the entorhinalcortex after incubation in the color substrate for 5–8 hours.Because there were very few differences in the distributionof GAD65 and GAD67 mRNA-positive neurons, their

Fig. 5. Plots of the distribution of cells expressing mRNA forGAD67 in coronal sections at two rostrocaudal levels through thehippocampal formation arranged from rostral (A) to caudal (B). Theoutlines of an adjacent section processed for Nissl staining are

superimposed on the computer-generated plot of the distribution ofGAD67-positive neurons. Each dot represents one labeled neuron. Forabbreviations, see list. Scale bar 5 1 mm.


Fig. 6. A: Brightfield photomicrograph of a section processed withnonisotopic in situ hybridization histochemistry for mRNA for GAD67.Higher magnifications of the areas indicated in the boxes are shown inB and C. B: GAD67 mRNA-positive neurons in the dentate gyrus. Note

the lack of signal in the granule cell layer. C: GAD67 mRNA-positiveneurons in the pyramidal cell layer at the border region between theCA2 and CA1 fields. For abbreviations, see list. Scale bars 5 1 mm inA, 50 µm in C (also applies to B).


distributions are described together below. The few pro-nounced differences between the patterns of GAD65 andGAD67 mRNA-labeled neurons also are discussed.

Dentate gyrus

Distribution of GABA-immunoreactive fibers and ter-

minals. The highest fiber densities in the dentate gyruswere found within the granule cell layer (Figs. 2, 3, 19A). Adense plexus of varicose axons surrounded the lightlystained granule cells. The molecular layer generally had alow density of varicose fibers that was markedly higher inthe outer two-thirds of the layer (Fig. 2A). The polymor-phic layer had slightly denser fiber and terminal labelingthan the adjacent portion of CA3.

Distribution and description of GABA-immunoreac-

tive and GAD mRNA-positive neurons in the dentate

gyrus: Immunohistochemical findings. GABAergicneurons were found in all layers of the dentate gyrus, withthe highest densities in the polymorphic layer (Figs. 2, 3,10, 11) . The density of GABAergic neurons in the polymor-phic layer was similar at all rostrocaudal levels, except forthe caudal pole, where it increased (Fig. 11A). The densityof labeled neurons in the molecular layer was highest atthe most rostral level examined (Fig. 11A). The density oflabeled cells associated with the granule cell layer wasroughly similar at all rostrocaudal levels.

The GABAergic neurons in the molecular layer hadspheroidal or multipolar cell bodies with dendrites typi-cally oriented in a stellate fashion (Fig. 14, cells 1–3). Theirdendrites varied from thin and barely visible to thick andwell stained. Some multipolar cells located in the innermolecular layer had all of their dendrites oriented towardthe hippocampal fissure (Fig. 14, cell 3). A second, lesscommon class of GABAergic molecular layer neurons hadfusiform cell bodies and dendrites oriented parallel to thegranule cell layer. These cells typically were located closeto the hippocampal fissure and had thick and occasionallybeaded dendrites.

Two major types of GABAergic neurons were observed inthe polymorphic layer. The most prominent type consistedof multipolar cells, with several radiating dendrites (Fig.14, cells 11, 12, 14, and 15). Some of the dendrites enteredthe granule cell and molecular layers and occasionallyextended into the hilar portion of CA3. The second mosttypical polymorphic layer GABAergic cell type had large,fusiform-shaped cell bodies. At least two subtypes offusiform cells were observed: one subtype had a horizon-tally oriented cell body with dendrites running parallel tothe granule cell layer (Fig. 14, cell 13), and the secondsubtype had a vertically oriented soma with dendritesextending through the granule cell layer into the molecu-lar layer (Fig. 15, cell 5). The horizontal fusiform cellsoften were faintly labeled.

Although these were the most commonly observed celltypes, there were other types of GABAergic neurons foundin the polymorphic layer. One was a large, pyramid-shapedneuron located just subjacent to the granule cell layer (Fig.14, cell 11). Another, rare GABAergic cell type had a small,spheroidal soma with short, thin dendrites that could notbe followed far from the cell body. Caudally, at the medialand lateral ‘‘Vs’’ of the dentate gyrus, two unique types ofGABAergic cells were identified. One was observed ini-tially in parvalbumin preparations, the so called ‘‘fork-like’’ cells (Fig. 15, cells 3, 4, and 6; Fig. 20D; see also Fig.

21B in Pitkanen and Amaral, 1993). These cells had two ormore thick, parallel dendrites emerging from one pole ofthe cell body that extended into the molecular layer; thedendrites originating from the other pole remained withinthe polymorphic layer. Another interesting cell type had anoval-shaped cell body with dendrites originating from onepole of the soma and extending up into the molecular layer(Fig. 15, cells 1 and 2; Fig. 20E,F). These cells had a thinaxon leaving the soma that extended into the polymorphiclayer. This type of unipolar GABAergic cell has not beendescribed previously in the monkey, but it was seen inGolgi preparations of the rat dentate gyrus (Amaral andWoodward, 1977; Amaral, 1978). We will call this cell typethe polymorphic layer unipolar GABAergic (PLUG) cell.Other multipolar cell types were observed mainly atcaudal levels of the polymorphic layer. This cell hastriangular- or diamond-shaped somata (Fig. 14, cell 12)with long, unbeaded dendrites that occasionally enter themolecular layer.

Most of the labeled neurons in the granule cell layerwere located at the interface between the granule cell layerand the polymorphic layer (Fig. 14, cells 4–10; Fig. 20A,B).Many of these cells resembled the basket cells described byRibak and Seress (1983). Among the most commonlyobserved cells were the pyramidal basket cell, the fusiformbasket cell, and the multipolar basket cell. The pyramidalbasket cells varied substantially in size (Table 1) and had atriangular-shaped soma with the apex inserted into thedeep half of the granule cell layer. The apical (main)dendrite ascended through the granule cell layer into themolecular layer, and basal dendrites ramified in the poly-morphic layer (Fig. 14, cell 6; Fig. 20A,B). The fusiformbasket cells had fusiform-shaped cell bodies of variablesizes (Table 1) that typically were located in the lower halfof the granule cell layer or just subjacent to it. Two types offusiform basket cells were observed. One type was orientedvertically, with apical and basal dendrites oriented perpen-dicular to the granule cell layer (Fig. 14, cell 9). The apicaldendrite extended through the granule cell layer andbranched in the molecular layer. Other, vertically orientedfusiform cells with smaller cell bodies were located in theupper half of the granule cell layer. The other major type offusiform basket cell had cell bodies and dendrites thatwere oriented horizontally along the interface of thepolymorphic layer and the granule cell layer (Fig. 14,cell 13).

Multipolar basket cell bodies with soma sizes varyingfrom small to large (Table 1) were distributed throughoutthe granule cell layer (Fig. 14, cells 7, 8, and 10). Somedendrites of these multipolar cells entered the molecularlayer, and some descended into the hilus. A variant of themultipolar cells with cell bodies located in the lower half ofthe granule cell layer had all of their dendrites directedinto the molecular layer. Another multipolar cell type wastypically seen at the ‘‘Vs’’ of the granule cell layer; thesecells had oval-shaped cell bodies with dendrites that oftenextended through the granule cell layer into the molecularcell layer (Fig. 14, cells 4 and 5).

Small spheroidal, multipolar cells were found in thelower half of the granule cell layer, and these had thin,radiating dendrites that remained within the granule celllayer. Some spheroidal cells had beaded dendrites extend-ing into the molecular layer and others extending into thehilus. Occasionally, an axon could be identified originatingfrom the cell body and branching into the molecular layer.


Fig. 7. A–H: Photomicrographs and plots of the distribution ofGABAergic cells in coronal sections through the entorhinal cortexarranged from rostral (A) to caudal (G). A, C, E, and G are brightfieldphotomicrographs of sections processed for GABA immunohistochem-istry. In B, D, F, and H, the outlines of adjacent Nissl-stained sectionswere superimposed on the computer-generated plots of the distribu-

tion of GABA-immunoreactive neurons. Each dot represents onelabeled neuron. Arrowheads indicate the borders of the subfields of theentorhinal cortex. Roman numerals indicate the layers of the entorhi-nal cortex. For abbreviations, see list. Scale bars 5 1 mm in D (alsoapplies to A–C) and H (also applies to E–G).


Other small spheroidal cells had axons leaving the granulecell layer and descending into the hilus.

Visualization of fibers and terminal plexuses, as discussedabove, were better in the material that was prepared with thehigh glutaraldehyde perfusion. The number of labeled

neurons, however, was substantially lower. This was veryclear in the polymorphic layer, in which many GABAergiccells were found with the low glutaraldehyde fixation, butvery few were seen in the high glutaraldehyde material. Itis interesting to note that, whereas the brains that were

Figure 7 (Continued)


perfused with the low concentration of glutaraldehydeshowed little or no immunostaining of the granule cells,some granule cells were clearly positive for GABA in thehigh glutaraldehyde-fixed material.

Sloviter and colleagues (1996) also reported intenseGABA immunoreactivity in some granule cells of themonkey (Macaca nemestrina) dentate gyrus. There werethree main differences in their protocol compared with theprocedures used in the present study: 1) the animals wereperfused with a higher concentration of glutaraldehyde(3% glutaraldehyde/1% paraformaldehyde); 2) the immuno-histochemical incubations were performed without addingthe detergent Triton X-100 to the buffers; and 3) a differentprimary GABA antibody was used. To investigate whetherthe differences in the intensity of the GABAergic stainingof the granule cells were inherent to the methods used, weprepared sections from the animals perfused with the lowconcentration of glutaraldehyde either with the monoclo-nal anti-GABA antibody used in our studies or with theantibody used by Sloviter and colleagues (Chemicon rabbitanti-GABA antiserum 131 diluted 1:10,000 in 0.1 M Tris-buffer, pH 7.6, containing 0.005% BSA). In addition,

adjacent sections destined for staining with each antibodywere incubated in the presence or absence of Triton X-100(0.3%) in the buffers.

The sections that were incubated with Triton X-100showed better dendritic and terminal labeling regardlessof which antibody was used. The GABAergic basket termi-nals around the granule cells, for example, clearly werelabeled better with the Triton X-100 treatment (Fig. 19A).However, a more remarkable difference was observed inthe GABAergic immunoreactivity of the granule cell layer.In our standard protocol (low glutaraldehyde fixation with0.3% Triton X-100 in the buffers), as discussed above, thegranule cells did not demonstrate any significant immuno-staining (Fig. 19A). In the sections that were incubatedwithout Triton X-100, however, the granule cells weremoderately GABA immunoreactive (Fig. 19B). Similarresults also were observed by using the Chemicon poly-clonal antibody (not shown).

The granule cell layer was the only area of the hippocam-pal formation in which additional cellular staining wasobserved when Triton X-100 was eliminated. We did notfeel obliged, therefore, to provide additional comments on

Fig. 8. A–D: Plots of the distribution of cells expressing the GAD65mRNA in the entorhinal cortex arranged from rostral (A) to caudal (D).The outlines of an adjacent section processed for Nissl staining aresuperimposed on the computer-generated plots of the distribution of

GAD65-positive neurons. Arrows indicate the borders of the subfieldsof the entorhinal cortex. Each dot represents one labeled neuron. Forabbreviations, see list. Scale bar 5 1 mm.


Fig. 9. A–D: Plots of the distribution of cells expressing the GAD67mRNA in the entorhinal cortex arranged from rostral (A) to caudal (D).The outlines of an adjacent section processed for Nissl staining aresuperimposed on the computer-generated plots of the distribution of

GAD67-positive neurons. Each dot represents one labeled neuron.Arrows indicate the borders of the subfields of the entorhinal cortex.For abbreviations, see list. Scale bar 5 1 mm.

Fig. 10. Summary histogram illustrating the densities of GABAer-gic neurons in the cytoarchitectonic fields of the hippocampal forma-tion. Bars represent the number of labeled cells per mm2 for eachregion averaged over five rostrocaudal levels. Note that the highest

densities of GABAergic neurons were found in the presubiculum (PrS).The CA3 region of the hippocampus and the dentate gyrus (DG)showed the lowest densities of labeled neurons. For abbreviations, seelist.

this type of staining in other regions. Similarly, becausethe distribution of cellular and fiber/terminal labeling wassimilar with the Virtanen and Chemicon antibodies when

Triton X-100 was added to incubation buffers, all of thedescriptions below are based on material that was pre-pared with the Virtanen antibody.

Fig. 11. A–D: Densities of GABAergic neurons in different laminaeand at different rostrocaudal levels of the dentate gyrus (A) andhippocampus (B–D). In A, note the increased density of GABAergicneurons at the most caudal level of the polymorphic layer of thedentate gyrus. In the CA3 (B) and CA2 (C) fields of the hippocampus,the highest densities of GABAergic cells generally were seen rostrally.In CA1 (D), there were no rostrocaudal differences in the density of

labeled neurons in the stratum radiatum or in the pyramidal celllayer. Whereas the density of GABAergic cells in the stratum lacuno-sum-moleculare was higher rostrally than caudally, in the stratumoriens, the density of labeled neurons increased gradually along itsrostrocaudal extent. White and black bars represent the most rostraland caudal levels, respectively, that were examined, and gray barsrepresent intermediate levels. For abbreviations, see list.

TABLE 1. Mean Area and Diameters of the Major Classes of GABAergic Cells in the Dentate Gyrus and Hippocampus1

Region/cell typeMean area

(min-max) µm2Mean feret diameter

(min-max) µmMean major axis

(min-max) µmMean minor axis

(min-max) µm

Dentate gyrusMultipolar 75.8 (35.4–186.3) 9.7 (6.7–15.4)Fusiform 82.7 (44.3–166.3) 17.3 (11.1–30.4) 7.0 (4.2–11.7)Pyramidal basket 83.9 (49.6–137.8) 10.2 (7.9–13.2)Fusiform basket 81.1 (54.9–110.5) 15.9 (11.6–22.0) 8.0 (7.3–11.7)Multipolar basket 70.3 (35.9–146.4) 9.3 (6.8–13.6)

CA3Multipolar 83.6 (35.8–180.1) 10.2 (6.7–15.1)Fusiform 100.3 (223.2–37.9) 18.6 (9.1–31.8) 8.0 (5.0–12.0)Stellate 87.6 (52.1–143.0) 10.5 (8.1–13.5)

CA2Multipolar 103.7 (48.9–235.0) 11.3 (7.9–17.3)Fusiform 128.2 (61.6–238.6) 22.2 (12.1–39.6) 8.6 (5.4–14.0)

CA1Multipolar 109.4 (33.9–233.8) 11.6 (6.6–17.3)Fusiform 123.0 (45.5–259.1) 21.8 (8.8–39.7) 8.8 (4.7–14.9)

1GABA, g-aminobutyric acid; min-max, minimum-maximum.


In situ hybridization findings. GAD mRNA-positiveneurons were found in all layers of the dentate gyrus (Figs.4, 5, 6A,B) with slightly fewer labeled neurons in themolecular and granule cell layers than in the polymorphiclayer. We did not detect above background levels of GADmRNA within the granule cells, despite the reports ofGAD67 immunoreactivity in the granule cells of the ratand Macaca nemestrina monkey (Sloviter et al., 1996).Many labeled neurons were found at the border betweenthe granule cell layer and the polymorphic layer in theregion occupied by the basket cells. Because the colorreaction product was present mainly in the somal cyto-plasm and rarely extended into the dendrites (Fig. 6B), itwas difficult to correlate the types of neurons that wereseen by using this methodology with those that werevisualized by using GABA immunohistochemistry. Never-theless, labeled, pyramidal-shaped cells, presumed pyrami-dal basket cells, were identified at the interface betweenthe granule cell layer and the polymorphic layer (Fig. 6B).Because immunohistochemistry and in situ hybridizationwere performed on sections from different animals, noquantitative comparisons were made between the twomethodologies. Qualitatively, however, it appeared thatthe numbers of basket cells were similar in both prepara-tions. Fusiform GAD mRNA-containing neurons could beidentified also in the polymorphic layer. Labeled, angularneurons were observed also in the dentate gyrus, andthese resembled the multipolar cells (Fig. 6A,B)

Hippocampus


minals in the hippocampus.

CA3. In the hilar portion of CA3, the mossy fibersappeared heavily GABA immunoreactive (Figs. 2A, 3A). Itis interesting to note that the mossy fibers were onlylightly labeled within the polymorphic layer but becamemore distinctly labeled as they assembled into fascicles

within CA3. In the hilar portion of CA3, the labeled mossyfibers ran partially through the pyramidal cell layer,forming the intrapyramidal bundle. More distally, themossy fibers ran just superficial to the pyramidal celllayer, forming the suprapyramidal bundle, i.e., the stra-tum lucidum (Figs. 2A, 3A). High neuropil labeling wasobserved in the stratum lacunosum-moleculare and con-sisted of a dense network of fibers and varicosities. In thestratum oriens, labeled fibers were oriented mostly paral-lel to the pyramidal cell layer. The alveus also showed ahigh density of GABAergic fibers running parallel to thepia (Figs. 2A, 3A).

CA2. The density of neuropil labeling in CA2 wasslightly higher than in CA1 and CA3 (Figs. 2A, 3A). In thepyramidal cell layer, a high density of GABAergic termi-nals was seen around unlabeled pyramidal cells (Fig. 22).This staining was less pronounced in CA3 and CA1.

CA1. In CA1, most labeled fibers were confined to theborder region between the stratum lacunosum-moleculareand the stratum radiatum (Figs. 2A, 3A). The stratumradiatum showed a lower density of labeled fibers andterminals than the stratum lacunosum-moleculare andthe pyramidal cell layer (Figs. 2A, 3A). The neuropil of thestratum oriens generally was stained more darkly thanthe pyramidal cell layer and consisted of more numerousvaricosities (Figs. 2A, 3A). Most of the labeled fibers in thestratum oriens traveled parallel to the alveus. In contrastto CA3 and CA2, very few labeled fibers were found in thealveus of CA1 (Figs. 2A, 3A). At the border of CA1 with thesubiculum, there was a conspicuous increase of neuropillabeling (Figs. 2A, 3A, 23A).


tive, GAD mRNA-positive neurons in the hippocampus:

Immunohistochemical findings.

CA3. Within CA3, the stratum lacunosum-moleculareand the pyramidal cell layer showed a slightly higherdensity of GABAergic neurons than the stratum radiatum

Fig. 12. Histogram illustrating the densities of GABAergic neurons along the rostrocaudal extent ofthe subiculum, presubiculum, and parasubiculum. White and black bars represent the most rostral andcaudal levels, respectively, and gray bars represent intermediate levels.


and stratum oriens (Fig. 10). The density of labeledneurons in all lamina of CA3 was highest rostrally (Fig.11B).

In the hilar portion of CA3, two peculiar types ofGABAergic cells were common. One was a stellate, multi-polar cell that was found intermingled with the labeledmossy fibers. This cell type had medium-sized, spheroidalcell bodies (Table 1) and numerous, fine, slightly varicosedendrites (Fig. 16, cells 3, 5, and 6; Fig. 21A,B). Thesestellate cells were present almost exclusively in the proxi-mal part of CA3. The second cell type was a multipolar cellwith a large, diamond-shaped cell body (Fig. 16, cell 8).These multipolar cells had smooth and beaded dendritesradiating in all directions. Some of these cells had long,beaded dendrites that occasionally entered the polymor-phic layer of the dentate gyrus.

In the stratum lacunosum-moleculare, most of the GABA-ergic neurons had a spheroidal, multipolar appearancewith few visible dendrites. Typically, they were located inthe deepest portions of the stratum lacunosum-molecu-lare. Occasionally, fusiform cells were found in the stratumlacunosum-moleculare (Fig. 16, cell 14).

In the stratum radiatum, two types of GABAergic cellswere prominent. One consisted of large, vertically orientedfusiform cells with basal dendrites extending into thepyramidal cell layer and apical dendrites extending intothe stratum lacunosum-moleculare (for soma sizes, seeTable 1; Fig. 16, cell 13). The other type was a class ofmultipolar cells that varied substantially in size (Table 1;Fig. 16, cells 10–12). The large, multipolar cells typicallywere located in the deeper portions of the stratum radia-tum, and their dendrites often extended into the pyrami-

Fig. 13. A,B: The density of GABAergic neurons in the entorhinalcortex at two rostrocaudal levels. The entorhinal cortex was subdi-vided into 500-µm bins along its transverse axis, and the density ofGABAergic neurons in layers I, II/III, V, and VI were counted for eachbin. Note the higher density of GABAergic neurons in layer I laterally

than medially. Note also that layers II/III showed higher densities oflabeled neurons than layers V and VI. The two rostrocaudal levelsillustrated in A and B are shown in Figure 7E,F and in Figure 7G,H,respectively.


dal cell layer. Other than the fusiform and multipolarneurons, other types of cells were seen occasionally in thestratum radiatum consisting of small, spheroidal cellswith fine dendritic arborization and modified pyramidalcells. Typically, there was a marked accumulation of largefusiform and multipolar GABAergic cells superficially inthe stratum radiatum, just at the border with the stratumlacunosum-moleculare.

Several types of labeled neurons were found in thepyramidal cell layer (Fig. 16, cells 3–9). One prominenttype had a pyramidal-shaped cell body with apical den-drites extending into the stratum radiatum and basaldendrites extending into the stratum oriens. Another

major type was the multipolar cell (for cell sizes, see Table1; Fig. 16, cell 8) with some dendrites extending into thestratum radiatum. The small multipolar neurons typicallyhad more spherically shaped cell bodies than the largemultipolar neurons and had their dendrites confined to thepyramidal cell layer or to the stratum oriens (Fig. 16, cell7). There also were horizontally oriented fusiform cells inthe pyramidal cell layer, with dendrites running intostratum oriens. The fusiform neurons varied substantiallyin size (Table 1). GABAergic neurons located in the mostsuperficial part of the pyramidal cell layer had dendritesextending into and through the stratum lucidum. Modi-fied, inverted, pyramidal-shaped cells also were seen in the

Fig. 14. Camera lucida drawings of GABAergic cell types in thedentate gyrus. Cells 1, 2, and 3 are examples of spheroidal, multipolarcells. Note that cell 3 is located at the border of the granule cell layer(GCL) and the molecular layer but has all of its dendrites orientedtoward the molecular layer. Cells 4 and 5 are oval-shaped, multipolarcells located in the granule cell layer, with multiple dendrites ascend-ing into the molecular layer and descending to the polymorphic layer(PL). Cell 6 is a typical pyramidal basket cell with one prominentdendrite in the granule cell layer that enters the molecular layer andtwo basal dendrites in the polymorphic layer. Cells 7, 8, and 10 areexamples of multipolar basket cells in the granule cell layer. Note thatthe dendrites of these cells are oriented toward both the molecular

layer and the polymorphic layer. Cells 9 and 13 are horizontal andvertical fusiform basket cells, respectively. Cell 9 is located in thegranule cell layer and has one prominent apical dendrite orientedtoward the molecular layer and a basal dendrite entering the polymor-phic layer. Cell 13 is located subjacent to the granule cell layer and hasdendrites oriented parallel to the granule cell layer. The axons(asterisks) of cells 4 and 9 descend for a short distance into thepolymorphic layer. Cells 11, 12, 14, and 15 are large multipolar cellswith multiple dendrites radiating in all directions. OML, outermolecular layer of the dentate gyrus; IML, inner molecular layer of thedentate gyrus. Scale bar 5 20 µm.


pyramidal cell layer (Fig. 16, cells 4 and 9). The dendritesof these cells sometimes extended into the stratum oriens(Fig. 16, cell 4).

In the stratum oriens, there were mainly multipolarneurons (Fig. 16, cells 1 and 2). The dendrites of these cellsoften remained in the stratum oriens or extended into thedeep portions of the pyramidal cell layer. Occasionally,vertically and horizontally oriented fusiform cells wereobserved in the stratum oriens.

Very few, if any, labeled neurons were seen within thestratum lucidum. Occasionally, a neuron was seen at themost superficial portion of the stratum lucidum withdendrites extending through it towards the pyramidal celllayer. These cells were mostly small and multipolar andhad spheroidal cell bodies. Occasionally, large, oval-shaped GABAergic neurons were found in the stratumlucidum. Labeled neurons were seen very rarely in thealveus.

CA2. CA2 had the highest density of labeled neuronsin the hippocampus (Figs. 2, 3, 10). Within CA2, thehighest densities of GABAergic neurons were observed inthe pyramidal cell layer, and the lowest densities wereobserved in the stratum lacunosum-moleculare (Fig. 10).The highest densities of GABAergic neurons were found atthe most rostral levels (Fig. 11C).

In the stratum lacunosum-moleculare, the most promi-nent GABAergic cell type was a small, multipolar cell witha spherical cell body. Fusiform cells were seen less fre-quently in this layer. The immunopositive cell types foundin the stratum radiatum included modified pyramidal cellsand small-to-large multipolar cells. Large, vertically ori-ented, fusiform cells also were observed in the stratumradiatum. These neurons varied substantially in size(Table 1). Similar to what was seen in CA3, there was anaccumulation of large, fusiform, GABAergic neurons at theborder between the stratum radiatum and the stratumlacunosum-moleculare. Dendrites belonging to these cellsoften extended into the stratum lacunosum-moleculare.

In the pyramidal cell layer, the majority of the GABAer-gic neurons had either pyramidal, modified pyramidal,fusiform, or multipolar cell bodies. The pyramidal-shapedcells were rather uniform in size. Small spheroidal cellswere present also in the pyramidal cell layer. The stratumoriens of CA2 had cell types similar to those found in CA3.

CA1. Within CA1, all lamina showed similar densitiesof GABAergic neurons. The stratum lacunosum-molecu-lare demonstrated slightly higher densities of labeled cellsthan the other hippocampal fields (Figs. 2, 3, 10), and thehighest densities of labeled neurons were found rostrally(Fig. 11D). A peculiarity of CA1 was that there was ahigher density of labeled neurons in its distal one-third(closer to the subiculum) than in its proximal one-third.

In the stratum lacunosum-moleculare, most GABAergiccells were multipolar with spheroidal cell bodies (Fig. 17,cell 13). There was a heterogeneous population of GABAer-gic neurons in the stratum radiatum consisting of fusiformneurons, irregularly shaped multipolar neurons, pyrami-dal-shaped cells, and spheroidal multipolar neurons (Fig.17, cells 11, 12, and 14–16). The fusiform neurons wereoriented perpendicular to the pyramidal cell layer (Fig. 17,cells 8 and 11, Fig. 21E). Their long, beaded dendritesextended into the pyramidal cell layer and often enteredthe stratum lacunosum-moleculare. There were manymedium-to-large multipolar cells in the stratum radiatum(see Table 1; Fig. 17, cells 12 and 14–16; Fig. 21C,E).Pyramidal-shaped and modified pyramidal cells and small,spheroidal multipolar cells (Fig. 21D–F) also were com-mon. The multipolar cells had both smooth and beadeddendrites, with apical dendrites that sometimes extendedinto the stratum lacunosum-moleculare and basal den-drites that extended into the pyramidal cell layer. Largefusiform cells and large multipolar cells were observed atthe border of the stratum radiatum and the stratumlacunosum-moleculare. Some large multipolar cells hadtriangular- to diamond-shaped cell bodies, and their den-drites extended into the pyramidal cell layer (Fig. 17, cells14 and 15; Fig. 21E).

The population of GABAergic neurons in the pyramidalcell layer was comprised of a diverse group of types andsizes, including multipolar cells (Table 1; Fig. 17, cells 2–7and 9), pyramidal-shaped cells, and fusiform cells (Fig. 17,cell 10). The stellate, multipolar cells had many, thin,slightly varicose dendrites (Fig. 17, cells 2 and 3). Thepyramidal-shaped cells had medium-to-large cell bodieswith prominent apical and basal dendrites. Neurons lo-cated superficially in the pyramidal cell layer had beadeddendrites that extended through the stratum radiatumand into the stratum lacunosum-moleculare and that alsodescended into the stratum oriens (Fig. 17, cells 9 and 10).Cell bodies located more deeply in the pyramidal cell layer

Fig. 15. Camera lucida drawing of GABAergic cell types in thepolymorphic layer (PL) of the dentate gyrus. These cells are located inthe medial ‘‘V’’ of the dentate gyrus. Cells 1 and 2 are the so-calledpolymorphic layer unipolar GABAergic cells (PLUG), which have all oftheir dendrites oriented toward the granule cell layer (GCL) andextending into the molecular layer (ML). A descending axon (asterisk)travels for a short distance in the polymorphic layer. Cells 3, 4, and 6are fork-like cells. Typically, those cells have two or more thick,parallel dendrites emerging from one pole of the cell body andextending from the polymorphic layer into the molecular layer; thedendrites originating from the other pole remain in the hilus. Cell 5 isa vertical fusiform cell. Scale bar 5 20 µm.


Fig. 16. Camera lucida drawings of various GABAergic cell typesin CA3. Cells 1 and 2 are small and large multipolar cells, respectively,located in the stratum oriens (SO). Cells 3, 5, and 6 are stellate cellsfound in the hilar portion of CA3. These cells have multiple, thindendrites radiating in all directions. Cells 4, 9, 10, and 12 are modified,pyramidal-shaped cells located in the pyramidal cell layer (PCL) andthe stratum radiatum (SR), respectively. Note that the dendrites of cell

4 extend into the stratum oriens. Cells 7 and 8 are examples of a smalland a large multipolar cell, respectively. Cell 11 is an example of alarge multipolar cell in the stratum radiatum. Cells 13 and 14 aresmall, vertical fusiform cells located in the stratum radiatum and thestratum lacunosum-moleculare (SLM), respectively. a, alveus. Scalebar 5 20 µm.


Fig. 17. Camera lucida drawings of various GABAergic cell typesin CA1. Cell 1 is a large, horizontal, fusiform cell typically found in thestratum oriens. Cells 2 and 3 are stellate cells located in the deepportions of the pyramidal cell layer. Cells 4–7 and 9 are multipolarcells in the pyramidal cell layer. Note the diversity of cell sizes andshapes among these multipolar cells. Cell 9 is located superficially inthe pyramidal cell layer and has long, beaded dendrites extending intostratum lacunosum-moleculare. Cell 10 is a vertical fusiform cell

located in the pyramidal cell layer with bifurcating dendrites thatextend into the stratum oriens. Cells 8 and 11 are small and largevertical fusiform cells, respectively, located in the stratum radiatum.Cells 12 and 14–16 are large multipolar cells found in the stratumradiatum. Cell 14 has a pyramidal-shaped cell body, and cell 15 has adiamond-shaped cell body. Cell 13 is a small, spheroidal cell located inthe stratum lacunosum-moleculare. Scale bar 5 20 µm.


had dendrites that ascended into the stratum radiatumand descended at least into the stratum oriens and thealveus (Fig. 17, cell 5).

There was a slightly higher density of GABAergic neu-rons in the stratum oriens of CA1 than in CA3 and CA2.Most of these neurons were large, horizontally oriented,fusiform cells that had long dendrites oriented parallel tothe alveus (Fig. 17, cell 1). These fusiform neurons weresomewhat larger than those found in CA3 and CA2.

At the transition between CA1 and the subiculum, thedensity of large, fusiform neurons increased markedly.There also were large multipolar cells and small spheroi-dal cells in the stratum oriens.

In situ hybridization findings. Although GAD65 andGAD67 mRNA-positive cells were seen in all layers of thehippocampus, there were some differences in the distribu-tion of the two populations (Figs. 4, 5, 6A,C). The density ofGAD67 mRNA-positive neurons was higher in the pyrami-dal cell layer than in the stratum radiatum and stratumlacunosum-moleculare, whereas GAD65 mRNA-labeledneurons were more numerous in the stratum radiatumand stratum lacunosum-moleculare.

Most of those cells had angular-shaped cell bodies thatvaried in size from large to small (Fig. 6C). Large, fusiformneurons were seen in the stratum radiatum of CA1 (Fig.6C). It is interesting to note that the intensity of labeling ofthe positive neurons varied substantially within the differ-ent laminae of CA2. The GAD67 mRNA-positive neuronslocated in the deep portions of the pyramidal layer andstratum oriens showed a stronger labeling intensity thanthe labeled neurons in the stratum radiatum and stratumlacunosum-moleculare. This was also true for GAD65mRNA. In CA1, many horizontal, fusiform neurons wereseen in the stratum oriens. As in CA2, the stainingintensity of labeled neurons in the stratum oriens and inthe pyramidal cell layer was stronger than that for neu-rons in the stratum radiatum.

Subiculum, presubiculum, and parasubiculum


minals.

Subiculum. The density of labeled fibers and terminalsincreased markedly in the pyramidal cell layer of thesubiculum relative to CA1 (Figs. 2A, 3A, 23A,B). Thischange in immunoreactivity clearly demarcated the ob-lique border of CA1 with the subiculum (Fig. 23A,B). In themolecular layer, there was a slightly lower density oflabeled fibers, but the fibers were more varicose than inother layers. The ‘‘stratum oriens’’ of the subiculum showeda markedly higher density of labeled fibers and terminals(Figs. 2A, 3A, 23A).

Presubiculum. The density of labeled fibers and termi-nals in the presubiculum was slightly higher than in thesubiculum (Figs. 2A, 3A). Layer II had a higher density oflabeled fibers than layer I, but layer I showed a dense,punctate neuropil labeling.

Parasubiculum. The density of labeled fibers and termi-nals was lower in the parasubiculum than in the presubicu-lum (Figs. 2A, 3A). Nonetheless, numerous labeled fiberswere distributed throughout layers I and II, with a higherdensity in layer II. Similar to what was seen in thepresubiculum, layer I of the parasubiculum showed adenser accumulation of varicosities.


tive and GAD mRNA-positive neurons: Immunohisto-

chemical findings.

Subiculum. The density of GABA-immunoreactive neu-rons in the subiculum was among the highest in thehippocampal formation (Figs. 2, 3, 10). Comparable densi-ties of labeled neurons were found in the molecular andpyramidal cell layers (Figs. 10, 12). These densities did notvary substantially along the rostrocaudal extent of thesubiculum (Fig. 12). Most of the numerous GABAergicneurons in the molecular layer of the subiculum had smallto medium-sized, spherical cell bodies (Table 2) and weredarkly immunoreactive. Their dendrites rarely were vis-ible.

In the pyramidal cell layer, GABAergic neurons weredistributed mainly in two broad, obliquely oriented bandsthat were located in the superficial one-third and in thedeep one-third (Figs. 2A, 3A, 23A). In the deep one-third, avariety of immunoreactive cell types was observed. Theseincluded multipolar cells and fusiform cells (Table 2). Inthe superficial one-third of the pyramidal cell layer, mostlabeled neurons had small, spherical cell bodies. The cellbodies of these spheroidal, multipolar cells were staineddarker than any other cells in the hippocampal formation,although their dendrites were not labeled clearly. Occasion-ally, large multipolar and fusiform GABAergic neuronswere seen in the superficial portion of the pyramidal celllayer. Immediately at the border of CA1 and the subicu-lum, the distribution of GABAergic neurons was some-what different. There, large multipolar and stellate cellswere found to populate the transition zone (Fig. 23A,B).The dendrites of these large multipolar cells remainedmostly in the pyramidal cell layer of the subiculum.

Presubiculum. The presubiculum, particularly layerII, demonstrated the highest densities of GABAergic neu-rons in the hippocampal formation. (Figs. 2, 3, 10). Small,spheroidal GABAergic cells were very abundant in layer I(Table 2). The majority of these cells resembled the darklystained, small, spheroidal cells found in the molecular

TABLE 2. Mean Area and Diameters of the Major Classes of GABAergic Cells in the Subiculum, Presubiculum, Parasubiculum, and Entorhinal Cortex

Region/cell typeMean area

(min-max) µm2Mean feret diameter

(min-max) µmMean major axis

(min-max) µmMean minor axis

(min-max) µm

SubiculumMultipolar 87.0 (39.6–177.0) 10.4 (7.1–15.0)Fusiform 106.6 (45.9–161.2) 18.2 (11.5–26.9) 8.9 (5.1–13.9)

PresubiculumMultipolar 71.9 (28.5–177.2) 9.4 (6.0–15.0)Fusiform 83.3 (50.1–141.8) 15.4 (10.2–21.9) 8.2 (6.4–13.0)

ParasubiculumMultipolar 71.6 (39.0–112.5) 9.5 (7.0–12.0)

Entorhinal cortexMultipolar 68.7 (35.6–130.3) 9.3 (6.7–12.9)Fusiform 82.2 (39.9–154.7) 15.8 (9.2–25.3) 7.7 (5.6–11.6)


layer of the subiculum. Occasionally, large multipolar cellswere seen in layer I. The labeled cells in layer II wereorganized in a laminar fashion that differed in the medialand lateral portions of the presubiculum. In the narrower,lateral portion, GABAergic neurons were distributedthroughout layer II. In the thicker, more medial portion,where layer II is clearly bilaminate even in Nissl-stainedmaterial, most of the labeled neurons were found in thedeep half of the layer. The most commonly found GABAer-gic cell type in layer II was a small to medium-sized,spheroidal, multipolar neuron with very few stained den-drites. Occasionally, small to medium-sized, fusiform andmultipolar cells were observed.

In the area deep to layer II, labeled cells were generallylarger than those in layer I and II. Most of these GABAer-gic cells were multipolar, and their dendrites often ex-tended into layer II. Occasionally, vertically oriented fusi-form cells were observed in the deep layers of thepresubiculum.

Parasubiculum. The density of GABAergic neurons inlayer II of the parasubiculum was the second highest inthe hippocampal formation (Figs. 2, 3, 10). Within theparasubiculum, the density of labeled neurons in layer IIwas markedly higher than in layer I (Figs. 10, 12). Thedensity of labeled neurons in layer I was lower than in thepresubiculum (Fig. 10). The types of GABAergic neuronsin the parasubiculum were similar to those found in thepresubiculum. Most of the GABAergic neurons in layer Ihad small, spherical cell bodies.

The most common GABAergic cell type in layer II was asmall, spheroidal cell. These cells were stained very darklyand had very thin dendrites. Occasionally, fusiform andmultipolar cells were seen in the deeper portion of layer II,with dendrites extending into more superficial parts oflayer II. The multipolar cell bodies varied from small tomedium-sized, and the fusiform cells generally were small(Table 2).

In situ hybridization findings.

Subiculum. The density of GAD65 mRNA and GAD67mRNA neurons in the subiculum was substantially higherthan in the adjacent CA1 (Figs. 4, 5, 6A). Fusiform-shapedneurons as well as small, oval-shaped cell bodies were seenin the pyramidal layer. In the molecular layer, the labeledneurons generally were stained more lightly than those inthe pyramidal cell layer, and they had smaller cell bodies.

Presubiculum. Similar to what was seen in the GABAimmunohistochemical preparations, the presubiculumshowed the highest densities of GAD mRNA-positive neu-rons in the hippocampal formation (Figs. 4, 5). GAD65mRNA and GAD67 mRNA neurons were found in all layersof the presubiculum, although the highest densities werefound in layer II. The labeled neurons in layer I showed aslightly lower intensity of staining than those in layer II.Most GAD mRNA-positive neurons in the presubiculumhad small, oval-shaped cell bodies. Occasionally, large,fusiform GAD mRNA-positive neurons were observed inlayer II.

Parasubiculum. The parasubiculum also demonstrateda very high density of GAD mRNA-positive neurons (Figs.4, 5). Like the presubiculum, labeled neurons were foundin layer I and layer II (Figs. 4, 5). The most commonlylabeled neuronal type had a small, spherical cell body.

Entorhinal cortex


minals. GABA immunoreactivity in the entorhinal cor-tex revealed both regional and laminar differences. Atrostral levels, there was a higher density of labeled fibersand terminals laterally in the entorhinal cortex (Figs.7A,C), whereas, at progressively more caudal levels, thetransverse gradient was not as evident (Fig. 7E,G).

Throughout the whole rostrocaudal extent of the entorhi-nal cortex, layer I showed the highest densities of varicosi-ties, whereas the highest densities of labeled fibers werefound in layers II and III (Fig. 7A,C). In layer II, a denseterminal plexus was observed to surround unlabeled cells.Dense clusters of labeled fibers were observed in layers IIand III, particularly in the lateral parts of the entorhinalcortex. Often, but not always, these clusters were associ-ated with clusters of GABA-immunoreactive cells, giving apatchy appearance to the distribution of GABA immuno-staining in this location. Layer V had lower levels oflabeled fibers and terminals, but dense terminal labelingwas still evident around the unlabeled pyramidal cellbodies. Layer VI showed the lowest density of stainedneuropil.


tive and GAD mRNA-positive neurons: Immunohisto-

chemical findings. The entorhinal cortex demonstrateda fairly high density of GABA-immunoreactive neurons(Fig. 10). The density of labeled neurons did not differsubstantially at different rostrocaudal levels (Fig. 7A–H).In layer I, there was a higher density of GABAergicneurons in the lateral subdivisions of the entorhinal cortex(ELr, ELc, and ECL) than in the medial parts (EO, ER, EI,and EC; Figs. 7A–H, 13A,B). No lateromedial gradientswere observed in any other layers (Fig. 13A,B).

Throughout the entire rostrocaudal extent of the entorhi-nal cortex, the density of labeled cells was higher in thesuperficial layers (layers II and III) than in the deep layers(layers V and VI; Fig. 13A,B). At rostral levels, the labeledneurons in layer III were organized in patches, whereas, atmore caudal levels, they showed a more columnar appear-ance (Fig. 7A–H).

Layer I was populated mainly by small, spheroidal,multipolar cells. These cells had short dendrites distrib-uted in a stellate fashion (Fig. 18, cells 1 and 2). Therewere also a few small, fusiform cells and multipolar cells inlayer I. In layer II, the predominant types of GABAergiccells were small, multipolar cells and fusiform neurons.These neurons were found commonly within the cellislands that are typical of ER and EI in layer II, and theirdendrites often extended into layer III (Fig. 18, cells 3 and5). However, some islands did not have any GABAergicneurons. Fusiform and multipolar GABAergic cells wereobserved occasionally in the cell-poor regions between thecell islands. Smaller, multipolar (Fig. 18, cell 4) andfusiform neurons also were seen in layer II. The dendritesof the fusiform cells were oriented horizontally, parallel tothe pial surface.

Layer III showed a substantial diversity of labeledneuronal types. The most common type was the multipolarcell. These cells varied substantially in size (Table 2) andhad dendrites radiating in all directions (Fig. 18, cells6–8). Some GABAergic cells located in the deeper portions


Fig. 18. Camera lucida drawings of various GABAergic cell typesin the entorhinal cortex. Cells 1 and 2 are small, spheroidal, multipo-lar cells found in layer I. Cells 3 and 5 are large, multipolar cellsobserved in the layer II of the rostral portion of the lateral division ofthe entorhinal cortex. Cell 4 is a small spheroidal cell located in layer

II. Cells 6–8 are large, multipolar cells in layer III. Cells 9 and 10 aresmall and large multipolar cells, respectively, found in layer V. Cell 11is a large, inverted pyramidal-shaped cell in layer VI. Cell 12 is a large,oval, multipolar cell. Cell 13 a small, stellate cell. Scale bar 5 20 µm.


of layer III had long dendrites extending into layer II.Pyramidal-shaped cells with apical dendrites extendinginto the superficial layers also were found in layer III. Avariety of other cell types was observed in layer III, such assmall spheroidal cells and fusiform cells.

There were very few labeled neurons in layer IV. In layerV, the most prominent cell type had a fusiform cell body,varying from small to medium-sized (Table 2), with apicaldendrites extending into layer III and with basal dendritesextending into the deeper layers. There also were small,spheroidal neurons and multipolar cells in layer V (Fig. 18,cells 9 and 10). Occasionally, a pyramidal-shaped cell couldbe observed with apical dendrites extending into layer III.There also were many labeled cells in the cell-poor sub-layer Vc, including fusiform, pyramidal-shaped, and multi-polar cells.

The GABAergic neurons in layer VI generally werelarger than those found in the other layers (Fig. 18, cells11–13). The most common type was a fusiform cell. Theapical dendrites of the fusiform cells often extended intothe superficial layers, and the basal dendrites descendedto the subcortical white matter. Multipolar and pyramidalcells also were seen frequently in layer VI (Fig. 18, cells 12and 13). The deeper portions of layer VI also containedsmall spheroidal cells and small fusiform cells.

In situ hybridization findings. In agreement with thedistribution of the GABA-immunopositive neurons, theentorhinal cortex demonstrated a high density of GADmRNA-positive cells (Figs. 8, 9). At the most rostral levels,more GAD65 and GAD67 mRNA-positive neurons wereobserved in the ELr subdivision than in the ER or EOsubdivisions (Figs. 8A,B, 9A,B). This difference graduallydisappeared at more caudal levels (Figs. 8C,D, 9C,D).

Although GAD mRNA-positive neurons were abundantin all layers, layers II and III showed the highest densitiesof labeled neurons (Figs. 8, 9). The density of labeled cellsin layer III was higher in the ELr subdivision than in theER and EO subdivisions. This difference was more pro-nounced at the most rostral levels (Figs. 8A,B, 9A,B).

The GAD mRNA-positive neurons in layer III werestained more darkly than those in layers I, V, and VI.Labeled neurons throughout the entorhinal cortex tendedto have angular cell bodies. The population of labeled cellswas comprised of large fusiform and multipolar neuronsand an occasional pyramidal-shaped neuron in the deepportions of layer III.

Fimbria and other fiber bundles

In the fornix, distinct clusters of small, spherical, GABA-ergic neurons could be observed among the immunoreac-

Fig. 19. Photomicrographs using Nomarski optics of the GABAimmunostaining in the dentate gyrus to illustrate the effect of TritonX-100 on the staining pattern of the granule cells. A: This section wasincubated with 0.3% Triton X-100 in the incubation buffers. Note thatthe granule cells are not stained for GABA but are surrounded by

labeled terminals, presumably from the basket cells. B: This sectionwas incubated in the absence of Triton X-100. Note that the majority ofthe granule cells are now stained for GABA. Note also that theterminal labeling around the granule cells is less visible in thiscondition. For abbreviations, see list. Scale bar 5 100 µm.


Fig. 20. A–F: Brightfield photomicrographs of GABA-immunoreac-tive cells in the dentate gyrus. The cells in A and B (arrowhead) aretypical pyramidal basket cells in the deep part of the granule cell layer.Arrow in B points to a fusiform basket cell located within the granulecell layer. The cell in C is a small fusiform cell located at the borderbetween the polymorphic layer and the hilar region of the CA3. The

cell in D is an example of the fork-like cell in the medial ‘‘V’’ of thedentate gyrus. Note that the dendrites (arrows) enter the granule celllayer. The cells in E and F are examples of the polymorphic layerunipolar GABAergic (PLUG) cell. For abbreviations, see list. Scalebar 5 50 µm.

Fig. 21. A–F: Brightfield photomicrographs illustrating GABAer-gic cell types in the hippocampus. A and B are stellate cells located inthe proximal CA3. Note the thick bundles of stained mossy fibersrunning through the pyramidal cell layer (arrows in A). C: Example ofa multipolar cell in the stratum radiatum of CA1. D: A pyramidal-shaped cell in the stratum radiatum of the CA1. E: A field in the

stratum radiatum of CA1 illustrating the heterogeneity of the GABAer-gic cells: multipolar cells; a small spheroidal, multipolar cell with long,beaded dendrites (arrowhead); a fusiform cell (small arrow); and alarge, diamond-shaped cell (large arrow). F: A large, pyramidal-shapedGABAergic cell in the pyramidal cell layer of CA1. Scale bar 5 50 µm.

tive fibers (Fig. 24A,B). Many of the labeled cells hadprominent dendrites (Fig. 24B). GABAergic cells andlabeled fibers also were seen in the angular bundle and inthe subcortical white matter. These cells were multipolarand spheroidal and had cell bodies of various sizes.

Deep to the subiculum and to the presubiculum, abundle of GABAergic fibers was visible and was continu-ous with labeled fibers running in the angular bundle(Figs. 2A, 23A). It is interesting to note that labeled fibersalso were found in the dorsal hippocampal commissure(Fig. 25A). These GABAergic fibers were located in thesame region that contained commissural fibers from thepresubiculum to the contralateral entorhinal cortex (Fig.25A,B).

DISCUSSION

This report presents a comprehensive analysis of thedistribution and morphology of GABAergic neurons in themacaque monkey hippocampal formation. Both immuno-histochemistry for the detection of GABA and in situhybridization for the detection of GAD65 and GAD67mRNA were used for these studies. Although similarstudies carried out in the monkey amygdaloid complex(Pitkanen and Amaral, 1994) have indicated that the insitu procedures were more sensitive for detecting GABAer-gic neurons than the immunohistochemical procedures,

the current studies demonstrated a remarkable correspon-dence between the distributions represented in prepara-tions for GAD65 mRNA, GAD67 mRNA, and GABA immu-nohistochemistry. Previous studies in the primate brainhave used antibodies against GAD, parvalbumin, calbin-din, calretinin, or somatostatin (Bakst et al., 1985; Babb etal., 1988; Leranth and Ribak, 1991; Seress et al., 1991,1993a,b, 1994; Sloviter et al., 1991, 1996; Pitkanen andAmaral, 1993; Ribak et al., 1993). Although each of thecalcium-binding proteins or peptides appears to label asubset of the GABAergic cell population, this study pro-vides the first report of the full complement of GABAergicneurons distributed throughout the entire primate hippo-campal formation.

Quantitative aspects of GABAergiccell distribution

Both qualitative impressions and our quantitative analy-ses of cell density indicated that there were substantialregional differences in the density of GABAergic neuronsin the different fields of the hippocampal formation (Fig.10). GABAergic neuron density was particularly low in thedentate gyrus and hippocampus. The fields of the subicu-lar complex and entorhinal cortex, by contrast, have muchhigher densities of GABAergic neurons. This is interest-ing, in that the fields of the hippocampus and the dentategyrus are much more vulnerable to the excitotoxic conse-quences of seizure activity and ischemia than the otherhippocampal fields. It is interesting to note that the CA2field of the hippocampus, the so-called resistant sector(Meldrum and Corsellis, 1984), had the highest density ofGABAergic neurons of any hippocampal field. Moreover,the subiculum has nearly twice the density of GABAergicneurons as the CA1 field. Perhaps the increased inhibitionavailable to the subiculum may explain in part the strikingborder between CA1, which can be lost entirely in humanischemic trauma (Zola-Morgan et al., 1986), and thenear-complete preservation of the subiculum. By using amonkey model of global transient ischemia, we previouslyfound that the proximal portion of the CA1 field was mostvulnerable to this trauma (Zola-Morgan et al., 1992).Unfortunately, neither the density of GABAergic neuronsnor that of GABAergic fibers and terminals can explainthese differences in neuronal vulnerability.

Presubiculum contains the highest densityof GABAergic neurons in the

hippocampal formation

This report presents the first description of the GABAer-gic system in primate retrohippocampal structures, suchas the presubiculum and entorhinal cortex. We havedemonstrated that layer II of the presubiculum has thehighest density of GABAergic neurons in the hippocampalformation. The density is more than five times higher thanin the pyramidal cell layer of the CA1 field of the hippocam-pus. We also observed a dense band of GABA-immunoreac-tive fibers that appeared to leave layer II of the presubicu-lum and enter both the angular bundle and the alveus.These fibers outlined a trajectory that was reminiscent ofthe projections from the presubiculum to the ipsilateraland contralateral entorhinal cortex (Amaral et al., 1984).In particular, there were GABA-immunoreactive fibers inthe location within the dorsal hippocampal commissure, inwhich the contralaterally directed presubicular fibers arefound. This raises the possibility, therefore, that some of

Fig. 22. Higher magnification photomicrograph illustrating thedense terminal labeling (arrows) around unstained pyramidal cells(asterisks) in the pyramidal cell layer of CA2. Scale bar 5 20 µm.


Fig. 23. Brightfield photomicrographs of the border region be-tween CA1 and the subiculum. A: GABA-stained section showing themarked increase in immunoreactivity at the border between CA1 andthe subiculum. The border is indicated approximately by straightarrows. Curved arrows indicate stained fibers in the stratum oriens ofthe subiculum that continue into the presubiculum. These fibers may

contribute to the commissural connection of the presubiculum to theentorhinal cortex. B: Nissl-stained section adjacent to the one pro-cessed for GABA. Note the increased density and oblique orientation ofthe cells at the border between CA1 and the subiculum (arrows). Forabbreviations, see list. Scale bars 5 200 µm.

Fig. 24. A: Photomicrograph illustrating GABA immunoreactivityin the fornix. Note the presence of many labeled neurons (arrows).Note also the bundles of heavily labeled fibers, particularly in the mostlateral aspects of the fornix. B: High-magnification photomicrograph

using Nomarski optics to illustrate GABAergic cells in the fornix. Anarrow points to the dendrite of one of these cells. Scale bars 5 500 µmin A, 20 µm in B.

Fig. 25. A: Camera lucida drawing illustrating the distribution ofGABAergic immunostained fibers in the dorsal hippocampal commis-sure (arrow). These fibers are in a position occupied by fibers thatoriginate in the presubiculum and terminate in the contralateralentorhinal cortex (compare with B). B: Darkfield photomicrographshowing the organization of the commissural projections of thehippocampal formation after 3H-amino acid injections into the dentategyrus and underlying presubiculum (adapted from Amaral et al.,1984). The injection site was in the right hemisphere (right side of

image). A large arrow points to the major bundle of labeled fibers thatarises primarily in the presubiculum. The small horizontal arrowindicates labeled fibers crossing the midline, and the shorter smallarrow points to fibers that have crossed the midline and are destinedfor the contralateral entorhinal cortex. Arrowheads indicate a secondbundle of labeled fibers in the fimbria on both sides that presumablyoriginated from the uncal and rostral portions of the hippocampus andthe dentate gyrus (for detailed explanation, see Amaral et al., 1984).cc, Corpus callosum. Scale bars 5 1 mm.


the numerous GABAergic neurons in the presubiculumactually may be projection neurons and may bilaterallyinnervate the entorhinal cortex. This possibility, again,has interesting implications for the analysis of medialtemporal lobe epilepsy, because the primary termination ofthe presubicular fibers is in layer III of the entorhinalcortex, and this layer is most vulnerable to pathologyconsequent to seizure activity (Du et al., 1993, 1995).

Support for at least part of this conjecture has come fromrecent work in the rat. Van Haeften et al. (1997) havedemonstrated that GABAergic neurons in the rat presu-biculum project to the ipsilateral entorhinal cortex. Theirattempts at demonstrating a contralateral projection, how-ever, were not successful. Thus, in the rat, it appears thatGABAergic presubicular cells project only to layer III ofthe ipsilateral entorhinal cortex. Studies are currentlyunder way to determine the organization of these presu-bicular pathways in the monkey brain.

GABA staining of fiber systems

We have confirmed previous findings that the mossyfibers are immunoreactive for GABA (Ottersen and Storm-Mathisen, 1984; Woodson et al., 1989; Sandler and Smith,1991; Sloviter et al., 1996). This has been somewhatsurprising, because the mossy fibers are also immunoreac-tive for glutamate (Storm-Mathisen et al., 1983) and havebeen presumed to constitute one of the major excitatoryconnections of the hippocampal formation. Stimulation ofthe granule cells, in fact, produces an excitatory postsynap-tic potential followed by an inhibitory postsynaptic poten-tial in CA3 pyramidal cells (Yamamoto, 1972). The excita-tory postsynaptic potential is mediated through glutamateby using an a-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptor (Collingridge et al., 1992; Williamsonand Wheal, 1992), and the inhibitory postsynaptic poten-tial can be blocked with bicuculline, suggesting that it ismediated by GABA (Frotscher et al., 1988). The inhibitorypostsynaptic potential has been thought generally to bedue to a feed-back inhibitory synapse from interneuronslocated in the vicinity of the mossy fibers and not fromrelease of GABA from the mossy fibers (Buzsaki, 1984;Frotscher, 1989). Sloviter et al. (1996) demonstrated GABAand GAD67 immunoreactivity in the mossy fibers of ratsand Macaca nemestrina monkeys and further demon-strated that the levels of both GAD65 and GAD67 as wellas GABA could be elevated following 24 hours of electricalstimulation of the perforant path projection to the dentategyrus in rats. They provided a detailed discussion of thepotential role of GABA in the synaptic economy of themossy fibers, and we can provide no further evidence toeither support or refute the possibility of GABAergicneurotransmission in the mossy fiber system. We shouldpoint out, however, that Gutierrez and Heinemann (1997)have provided preliminary data indicating that mossyfibers may release glutamate and GABA, at least afteramygdala kindling.

Regarding the staining of GABA markers in the granulecells, we do have a few methodological comments. Granulecells display detectable levels of GABA-immunostainingonly if the animal is perfused with a high concentration ofglutaraldehyde (2.5% in our study; 3.0% in the study bySloviter et al., 1996) or when the detergent Triton X-100 isleft out of the incubation buffers. High concentrations ofglutaraldehyde in the fixative also produce high back-ground in immunohistochemistry (Kosaka et al., 1986),

which could account for some of the granule cell stainingseen in both studies. However, increased backgroundcannot explain the increased levels of staining seen in theepilepsy models. Schwarzer and Sperk (1995), for example,provided evidence for synthesis of GABA in the granulecells 6–24 hours after kainic acid injections. There was atransient increase in the GAD67 mRNA levels in thegranule cells, followed by a long-lasting, enhanced GADimmunoreactivity in the mossy fibers. These data, as notedabove, have been replicated and extended by Sloviter andcolleagues (1996). Thus, at least after stimulation orseizures, it would appear that granule cells in the rat arecapable of producing GAD67 and GAD65 and, subse-quently, GABA. We should reiterate, however, that, in ourhands, the granule cells in the dentate gyrus of theprimate hippocampal formation did not show any specificsignal for either GAD65 or GAD67 mRNA. During pro-longed incubation in the color substrate, a light GAD67mRNA labeling of the granule cells was observed. Houserand Esclapez (1994) also noticed low levels of GAD67mRNA in the granule cells of the rat hippocampal forma-tion, but those authors pointed out that nonspecific stain-ing is not uncommon with in situ hybridization studies inregions of high cell density, such as the granule cell layer.

With respect to the differences in the staining patternsobserved with and without Triton X-100 in the buffers, it isnot clear why the omission of Triton X-100 would causestrong staining of one particular cell type, i.e., the granulecell (staining in other cell types that were negative withlow concentrations of glutaraldehyde did not occur). Lightmicroscopic immunohistochemical procedures usually re-quire the addition of a detergent to improve the penetra-tion of antisera into the tissue. In the present study, whenwe compared sections incubated with or without TritonX-100, it was clear that the Triton X-100 treatmentimproved penetration of the tissue by the antisera, whichresulted in more complete staining of dendrites and termi-nal varicosities. Because the goal of this study was toprovide a description of the morphology and distribution ofGABAergic neurons, we have based our description primar-ily on the preparations that included Triton X-100 treat-ment.

In situ hybridization for GAD65 mRNA andGAD67 mRNA in the hippocampal formation

Although the relevance of the light staining for GADmRNA in the dentate granule cells remains to be deter-mined, nonprincipal cells that clearly were positive forGAD65 mRNA or GAD67 mRNA were detected in allregions of the primate hippocampal formation. Moreover,with few exceptions, the distributions of the neuronslabeled for GAD65 and GAD67 mRNA were very similar.These results are in agreement with recent studies inrodents (Feldblum et al., 1993; Houser and Esclapez,1994). However, we did observe some subtle differencesbetween the distributions of neurons labeled for each ofthe forms of GAD. There was a higher density of GAD67mRNA-expressing neurons than GAD65 mRNA-positiveneurons throughout the hippocampal formation.Adifferen-tial expression of the two forms of GAD mRNA also hasbeen demonstrated in the monkey visual cortex (Hendrick-son et al., 1994). By using double-labeling in situ hybridiza-tion with the same probes used in the present study,Hendrickson and colleagues showed that 18% of the corti-cal neurons express GAD67 mRNA, whereas only 13%


express GAD65 mRNA, suggesting that a population ofGABAergic neurons might lack GAD65 mRNA.

The level of neuronal labeling in the monkey hippocam-pal formation that was achieved with probes for GAD67mRNA was generally higher than that achieved withprobes for GAD65 mRNA. Hendrickson and colleagues(1994) also found differences in the levels of labeling forGAD65 and GAD67 mRNA among the GABAergic neuronsin the monkey visual cortex. The majority of the GABAer-gic cells in layer VI contained both GAD mRNAs, but theintensity of labeling of the GAD67 mRNA was substan-tially higher than that of GAD65 mRNA. The secondpopulation had equivalent and high levels of both messen-gers, and a third, small population contained only GAD67mRNA (Hendrickson et al., 1994).

Even within the staining pattern for one or the otherforms of GAD mRNA, there were differences in the inten-sity of labeling among GAD mRNA-positive neurons indifferent regions of the hippocampus. Labeled neurons inthe stratum oriens, for example, were labeled more in-tensely than those in the superficial portion of the pyrami-dal cell layer, the stratum radiatum, or the stratumlacunosum-moleculare. In rats, no such difference in label-ing intensity of GAD mRNAs has been reported. Houserand colleagues (Esclapez et al., 1993; Houser and Escla-pez, 1994) found that the levels of labeling for GAD65 andGAD67 mRNA in the hippocampus were quite similar,with the exception of some positive cells in the hilus thatshowed a slightly higher staining intensity for the GAD65mRNA. Generally, differences in labeling intensity areconsidered to reflect differences in relative amounts ofGAD mRNAs within the neurons (Esclapez et al., 1993).

Comparison with other species

There is rapidly increasing evidence that the populationof GABAergic interneurons in the rodent hippocampalformation is highly heterogeneous. Interneurons havebeen defined on the basis of their axonal and dendriticorganization, their inputs, or the precise regional andpostsynaptic location of their terminations (Han et al.,1993; Sik et al., 1995; Acsady et al., 1996a,b; Freund andBuzaski, 1996; Hajos et al., 1996). In the dentate gyrus, forexample, at least five types of inhibitory neurons havebeen identified based on the laminar distribution of theirdendrites and axons and their postsynaptic elements: thehilar-commissural-associational pathway-related (HI-CAP) cells, the (hilar perforant path-associated (HIPP)cells, the molecular layer perforant path-associated (MOPP)cells, the basket cells, and the axoaxonic or chandelier cells(Halasy and Somogyi, 1993; Han et al., 1993). Although weobserved GABAergic cells that share features, such aslocation of cell bodies and dendritic characteristics, withseveral of these cell types, it is difficult to establish tightlinks between the cells we observed and those defined inthe rat without the added information afforded by intracel-lular filling, double-labeling studies, and analyses of theprecise inputs to the interneurons. The present studyprovides baseline information for carrying out these moredetailed analyses in future studies.

ACKNOWLEDGMENTS

The authors thank Ms. Haydee Vercesi for assistancewith the in situ hybridization procedures. This work wasconducted in part at the California Regional Primate

Research Center (grant RR 00169) and was support by agrant to D.G.A. from the National Institutes of Health(NS16980) and by a fellowship to A.L.J.-R. from theHuman Frontier Science Program (LT-315).

LITERATURE CITED

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Documents

Distribution of GABAergic cells and fibers in the hippocampal formation of the Macaque monkey: An immunohistochemical and in situ hybridization study