gabaergic axon terminals at perisomatic and dendritic inhibitory sites show different...

GABAergic Axon Terminalsat Perisomatic and Dendritic Inhibitory

Sites Show Different ImmunoreactivitiesAgainst Two GAD Isoforms, GAD67

and GAD65, in the Mouse Hippocampus:A Digitized Quantitative Analysis

TAKAICHI FUKUDA,1* YUSUKE AIKA,1 CLAUS W. HEIZMANN,2

AND TOSHIO KOSAKA1

1Department of Anatomy and Neurobiology, Faculty of Medicine, Kyushu University,Fukuoka 812-8582, Japan

2Department of Pediatrics, Division of Clinical Chemistry and Biochemistry,University of Zurich, CH-8032 Zurich, Switzerland

ABSTRACTGlutamic acid decarboxylase (GAD), the g-aminobutyric acid (GABA)-synthetic enzyme,

consists of two isoforms, GAD67 and GAD65.Although distributions of the two GAD isoforms at thesomatic level are known to be heterogeneous among different subpopulations of GABAergicneurons, those at the synaptic level have not been investigated. In order to analyze quantitativelythe two GAD-isoform immunoreactivities in axon terminals, we combined confocal laser scanningmicroscopy with digitized image analysis to measure the gray levels of immunofluorescent signalsfor the two GAD isoforms in a large number of individual boutons in each hippocampal and dentatelayer of the mouse. Synaptic boutons exhibited lamina-specific immunoreactivities against the GADisoforms. Boutons in the principal cell layers (stratum pyramidale of the hippocampus proper andthe granule cell layer of the dentate gyrus) showed more intense immunoreactivity against GAD67than those in the dendritic layers (strata lacunosum-moleculare, radiatum, and oriens of thehippocampus proper and the molecular layer of the dentate gyrus). By contrast, boutons in thedendritic layers showed more intense immunoreactivity against GAD65 than those in the principalcell layers. Such differential distributions could be correlated to the GAD-isoform immunoreactivi-ties in the axon terminals originating from parvalbumin-containing neurons, a particular subpopu-lation of hippocampal GABAergic neurons mainly innervating the perisomatic domain of principalneurons. In addition to previously reported physiological and pharmacological differences betweenthe GABAergic synapses on perisomatic domain and those on distal dendrites, the present resultssuggest a functional differentiation of GABAergic synapses between these two inhibitory sites.J. Comp. Neurol. 395:177–194, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: GABA; parvalbumin; interneuron; synapse; confocal microscopy

Gamma-aminobutyric acid (GABA)-containing neuronsare the major inhibitory neurons in the brain and consti-tute the vast majority of nonprincipal neurons in thehippocampal formation. While principal neurons are rela-tively uniform within each hippocampal subregion, non-principal GABAergic neurons are characterized by theirdiversity in morphological, chemical, and physiologicalfeatures. GABAergic neurons contain glutamic acid decar-boxylase (GAD), the GABA-synthetic enzyme (Ribak et al.,1978; Seress and Ribak, 1983; Mugnaini and Oertel, 1985).Recent studies have shown that GAD consists of twoisoforms, GAD67 and GAD65 (for reviews: Erlander and

Tobin, 1991; Martin and Rimvall, 1993). The intensities ofimmunocytochemical and mRNA signals against the twoGAD isoforms appear to be heterogeneous among different

Grant sponsor: Japanese Ministry of Education, Science and Culture;Grant number: 09480213; Grant sponsor: Uehara Memorial Foundation;Grant sponsor: Mitsubishi Foundation.

*Correspondence to: T. Fukuda, Department of Anatomy and Neurobiol-ogy, Faculty of Medicine, Kyushi University, Maidashi, Higashi-ku, Fukuoka812–8582, Japan. E-mail: [email protected]

Received 3 October 1997; Revised 13 January 1998; Accepted 20 January1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 395:177–194 (1998)

r 1998 WILEY-LISS, INC.

subpopulations of GABAergic neurons within many brainareas (Esclapez et al., 1993, 1994; Houser and Esclapez,1994; Hendrickson et al., 1995; Vardi and Auerbach, 1995;Fukuda et al., 1997; Yuan et al., 1997).

In our previous study, we demonstrated that hippocam-pal GABAergic neurons containing a calcium-binding pro-tein parvalbumin (PV) show a unique GAD-isoform immu-noreactivity in their somata (Fukuda et al., 1997). PV-positive neurons frequently showed very weakimmunoreactivity against GAD65 despite moderate tointense immunoreactivity against GAD67, whereas PV-negative GABAergic neurons in the hippocampus propergenerally exhibited moderate to intense immunoreactivi-ties against both GAD isoforms. PV neurons are character-ized by a distinct set of morphology (Kosaka et al., 1996),and they overlap largely with the population of basket cellsand axoaxonic cells (Kosaka et al., 1987; Katsumaru et al.,1988a), which make synaptic contacts on perisomaticregions of principal neurons (Ramon y Cajal, 1911; Lorentede No, 1934; Somogyi et al., 1983). Therefore PV neuronscan be included in the category of so-called somatic inhibi-tory neurons that have been proposed to control thegeneration of sodium-dependent action potentials on theirtarget cells (Miles et al., 1996). By contrast, the axons ofdendritic inhibitory neurons such as bistratified cells(Buhl et al., 1994) terminate on the dendritic domain oftarget cells in conjunction with the terminal field ofspecific excitatory input pathways (Halasy and Somogyi,1993; Han et al., 1993; Buckmaster and Schwartzkroin,1995; Gulyas and Freund, 1996; Halasy et al., 1996) andare thought to modify dendritic electrogenesis (Miles et al.,1996). Since recent studies revealed prominent physiologi-cal and pharmacological differences between perisomaticand dendritic inhibition (Lambert and Wilson, 1993; Pearce,1993; Buhl et al., 1994; Soltesz et al., 1995; Miles et al.,1996), it is of interest whether synaptic boutons in thesetwo inhibitory sites contain different GAD isoforms. Theweak GAD65 immunoreactivity observed in PV-containingsomata suggests that there might be small amounts ofGAD65 in the terminals that abut on perisomatic domainof the principal cell membranes. However, it has beenrather difficult to test this hypothesis and estimate theamount of GAD isoforms in ‘‘individual’’ axon terminals bythe conventional morphological and biochemical methods.

In the present study, we attempted to reveal the distribu-tions of the two GAD isoforms in axon terminals bycombining confocal laser scanning microscopy (CLSM) anddigitized image analysis. To survey, as an initial step,the entire population of GABAergic terminals in thehippocampus proper and the dentate gyrus, we measuredthe GAD-isoform immunoreactivities in thousands ofbouton profiles whose CLSM images were obtained fromall hippocampal and dentate layers under the constantCLSM conditions; then collected data were comparedbetween different layers, separatery for each GAD isoform.Next we focused on the GAD-isoform immunoreactivitiesin PV-containing terminals. We found that both GAD65and GAD67 immunoreactivities are different betweenperisomatic and dendritic axon terminals and that thedifference could be related to PV-containing terminals.These findings raise the possibility that GABA synthesis iscontrolled differently in perisomatic vs. dendritic axonterminals.

MATERIALS AND METHODS

Fixation and tissue preparation

Seven male C57BL/6J mice (20–30 g, 8–15-weeks-old)were used in the present study in accordance with theinstitutional guidance for animal welfare. Under deepanesthesia with an intraperitoneal injection of sodiumpentobarbital (10 mg/100 g body weight), the animals werequickly perfused through the ascending aorta with 10 mMphosphate-buffered saline (PBS, pH 7.2), followed by 50 mlof a fixative containing 4% paraformaldehyde, 0.1% glutar-aldehyde, and 0.2% picric acid in 0.1 M phosphate buffer(PB), pH 7.2 at room temperature. Serial sections trans-versing the dorsal hippocampus, 40 µm in thickness, werecut with a Vibratome (Technical Products, St. Louis, MO)and processed for immunocytochemistry using the free-floating method.

Confocal laser scanning light microscopy

After cryoprotection with 30% sucrose in PB and freeze-thaw procedure using liquid N2, sections were incubatedovernight in 1% bovine serum albumin (BSA) in PBS, for 7days with a mixture of rabbit polyclonal antibody K2(dilution 1:2,000) against GAD67 (Kaufman et al., 1991;Chemicon, Temecula, CA) and mouse monoclonal antibodyGAD6 (1:1,000) against GAD65 (Chang and Gottlieb,1988), then with biotinylated horse anti-mouse IgG (1:200;Vector, Burlingame, CA) overnight, and finally with amixture of fluorescein isothiocyanate (FITC)-conjugateddonkey anti-rabbit IgG (1:100; Jackson, West Grove, PA)and streptavidin-conjugated Texas Red (1:200; Amersham,Buckinghamshire, UK) overnight, all steps at 20°C. Allprimary and secondary antibodies were diluted in 1%BSA-PBS. Sections were mounted in the anti-fading me-dium Vectashield (Vector; Longin et al., 1993). Sectionswere examined with a confocal laser scanning microscopeMRC-1000 (BioRad, Herts, UK) equipped with a Krypton-Argon ion laser and mounted on a light microscope (Opti-photo, Nikon, Tokyo, Japan). All CLSM images wereobtained using a 603 oil immersion objective lens (N.A. 51.40, Nikon) and 23 zoom factor. Under these conditions 1pixel corresponds to 0.133 µm when the frame size is 640 3480 pixels. Control sections were prepared by omission ofprimary antibodies and by mismatching secondary antibod-ies; both provided only weak nonspecific staining. Singlelaser beams, 488 nm and 568 nm in wavelength, werealternately used to collect images for different fluorescentsignals, instead of simultaneous dual beams, in order toavoid bleed-through of one fluorescent signal to another;the bleed-through was confirmed to be negligible by usingthe mixer program of the instrument for observations ofsingle stained sections that were prepared simultaneouslywith double-stained sections. Single stained sections werealso used to rule out the possibility that double labelingcould have compromised the labeling for one GAD isoformin the structures where intense staining for another GADisoform occurred. There was no appreciable difference ineach GAD-isoform immunoreactivity between double- andsingle-stained sections.

To test the effect of detergent in the incubating solutionson the quality of immunostaining, two types of sampleswere prepared, one processed with 0.1% Triton and theother without Triton, simultaneously from the same ani-mal, and were observed under the same confocal laserscanning microscopy (CLSM) conditions. We ascertained

178 T. FUKUDA ET AL.

that the quality of immunocytochemical staining washigher in the sections not treated with Triton, particularlyfor GAD65 staining, although Triton did improve thepermeation of antibodies into the section. Therefore wecollected images for quantitative study from the mostsuperficial part of the detergent-free sections.

As a next step we compared the GAD65 or the GAD67immunoreactivity between PV-positive and -negative ter-minals. Primary antibodies were combined as follows: 1)mouse GAD6 (1:1,000) and rabbit anti-rat PV antibody(1:5,000; Kagi et al., 1987); 2) rabbit K2 (1:2,000) and goatanti-rat PV antibody (1:10,000); the specificity of the latterwas tested by Western blot analysis and there was no crossreactivity with other calcium-binding proteins (data notshown). In case 1, sections were further processed withhorse biotinylated anti-mouse IgG (1:200) overnight andthen with a mixture of donkey FITC-conjugated anti-rabbit IgG (1:100; Jackson) and streptavidin-conjugatedTexas Red (1:200) overnight. In case 2, sections wereincubated with donkey biotinylated anti-goat IgG (1:500,Jackson) overnight, and then with a mixture of donkeyFITC-conjugated anti-rabbit IgG (1:100) and streptavidin-conjugated Texas Red (1:200) overnight.

Quantitative analysis of GAD67 and GAD65immunoreactivities in boutons

Intensities of GAD67- and GAD65-immunofluorescentsignals in individual boutons were measured in the CLSMimages. Conditions for acquiring images were standard-ized for each section by adjusting several parameters insuch a way that the quality of bouton profiles in thedentate granule cell layer was optimal in each GAD-isoform imaging, with a maximum number of specificallystained boutons and a low background of nonspecificstaining. The aperture size of the pinhole was set as smallas possible. The granule cell layer was chosen because onlythis layer contained both intensely GAD67-immunoreac-tive (-IR) boutons and intensely GAD65-IR boutons. CLSMconditions were readjusted, if necessary, by observing theCA1 stratum pyramidale for GAD67 imaging and the CA1stratum lacunosum-moleculare for GAD65 imaging; thegain was lowered if saturation in signal intensities oc-curred in these layers. Following these procedures, CLSMconditions were kept constant in each section until allimages necessary for quantitative analysis were collectedfrom different layers. Single sections were selected fromfour mice each, and two images at different locations weretaken from each layer in individual sections. Images weretransferred to a personal computer (Power Macintosh8100, Apple, Cupertino, CA) by using NIH Image software.

Single optical slices taken by Kalman averaging (fourtimes) were used to measure signal intensities in indi-vidual boutons. We defined ‘‘GAD-IR’’ boutons as theboutons immunoreactive for at least one of the two GADisoforms, and selected the profiles of GAD-IR boutons bydigitized image processing (Fig. 1). We first obtainedbinary images of GAD67-positive boutons (Fig. 1C) andthose of GAD65-positive boutons (Fig. 1C8), each sepa-rately, from the same double-fluorescently stained sectionsby using NIH Image. Briefly, gray levels of images wereinverted (no signal 5 0, maximum level 5 255) andbackground was subtracted by the ‘‘Rolling Ball’’ commandin NIH Image. These inverted and subtracted images (Fig.1B,B8) were stored in magneto-optical discs and thenthresholded at a constant gray level, 80 in the present

study, to obtain binary images. After noise was reducedfrom the binary images by 3 3 3 ‘‘Opening’’ filtering,profiles whose areas were smaller than 8 pixels wereremoved. This threshold size corresponded to the area of acircle whose diameter is approximately 0.5 µm, the size ofthe smallest labeled axon terminals in the correlatedCLSM-electron microscopy (linear shrinkage factor inCLSM was assigned to be 0.94 based on the direct measure-ment). Then binary particles corresponding to GAD67-positive boutons (Fig. 1C) and those corresponding toGAD65-positive boutons (Fig. 1C8) were put together byusing ‘‘image mathematics’’ (GAD67-positive binary par-ticles) OR (GAD65-positive binary particles) to obtainbinary images of GAD-IR (GAD67- and/or GAD65-IR)boutons (Fig. 1D). Finally we extracted profiles of indi-vidual boutons from either GAD67-immunostained (Fig.1B) or GAD65-immunostained (Fig. 1B8) images by usingimage mathematics (binary GAD-IR boutons [Fig. 1D])AND (inverted and subtracted image for GAD67 [Fig.1B];Fig.1E), or (binary GAD-IR boutons [Fig.1D]) AND (in-verted and subtracted image for GAD65 [Fig. 1B8]; Fig.1E8). By the last procedure the gray levels in Figure 1B orB8 were kept unchanged in the pixels that were selected inFigure 1D while they were assigned to be zero in the pixelsthat were not selected. The frame of 250 3 250 pixels insize was positioned in each extracted image so as not toinclude somatic profile nor large blood vessels. Mean graylevels in the individual bouton profiles were measured bythe ‘‘Analyze Particle’’ command. Data were collectedusing macro-programs written by T.F. and Y.A. and ana-lyzed by Excel (Microsoft).

Data for analyzing GAD-isoform immunoreactivities inPV-positive and -negative boutons were collected in thesame way as above, with slight modifications; binaryprofiles whose areas were larger than 150 pixels (3 µm2)were excluded from analysis in order to remove profiles ofPV-IR dendrites. Four sections, two for GAD65 immunore-activity and the other two for GAD67 immunoreactivity,were selected from an animal, and five images at differentlocations were taken from each layer in individual sec-tions.

For preparing illustrations for presentation, selectedimages were processed by using image-editing software(Adobe Photoshop 3.0; Adobe Systems, Inc., MountainView, CA) and combined into plates. Only the contrast andbrightness were adjusted. Figures were directly printed ona Pictrography (Fuji-Xerox).

Correlated immunoelectron microscopy

Some double-immunofluorescent sections were rinsed inPBS after CLSM observations and incubated with VectorABC complex (1:200) for 2 hours for detection of GAD65 orwith rabbit peroxidase-antiperoxidase complex (1:200;Dako, Denmark) overnight for detection of GAD67. Aftercolor development with 3,38diaminobenzidine tetrahydro-chloride (DAB), sections were processed for usual electronmicroscopy, including osmification lasting 1.5 hours on iceand staining with uranyl acetate for 1.5 hours beforedehydration. Sections were flat-embedded in Epon-Araldite, ultrathin sections were cut from the most super-ficial part of the specimens, lightly stained with uranylacetate and lead citrate, and examined in a transmissionelectron microscope (Hitachi H-7100).

GAD65 AND GAD67 IN AXON TERMINALS 179

Fig. 1. Procedures for extracting the profiles of gutamic aciddecarboxylase-immunoreactivity (GAD-IR; GAD67-IR and/or GAD65-IR) boutons (see Materials and Methods). A,A8: Original confocal laserscanning microscopy (CLSM) images showing GAD67 (A) and GAD65(A8) immunoreactivities in the outer molecular layer of the dentategyrus. While a small number of boutons show intense immunoreactivi-ties against both GAD isoforms (arrow), the majority exhibit weakGAD67 immunoreactivity and intense GAD65 immunoreactivity(crossed arrows). B,B8: Images in which original gray levels areinverted and background signals are subtracted by using the ‘‘RollingBall’’ command in NIH Image. C,C8: Binary images obtained by

thresholding images B and B8 at the gray level of 80, followed by‘‘Opening Filtering’’ and by removal of particles whose areas aresmaller than eight pixels. D: Combined binary image from C and C8 byimage mathematics ‘‘(C) OR (C8)’’, corresponding to the boutons thatare immunoreactive for GAD67 and/or GAD65. Two identical panelscommon to both GAD67 and GAD65 immunostainings are shown.E,E8: Extracted profiles of individual boutons by image mathematics‘‘(B) AND (D)’’, and ‘‘(B8) AND (D)’’. The mean gray levels for GAD67and GAD65 immunoreactivities were measured in individual profilesof bouton extracted in E and E8. Scale bar 5 5 µm.


RESULTS

General remarks

Both GAD67- and GAD65-immunoreactive (-IR) struc-tures were distributed throughout the hippocampus properand the dentate gyrus (Fig. 2). They were composed ofnumerous bouton-like structures, many scattered somata,and occasional neuronal processes (presumptive axonsand dendrites). Morphological features and distributionsof labeled somata were similar to those described earlierfor hippocampal GAD-IR neurons (Ribak et al., 1978;Seress and Ribak, 1983; Somogyi et al., 1984; Kosaka etal., 1985; Esclapez et al., 1993, 1994; Fukuda et al., 1997)and were not repeated here. However, when CLSM condi-tions were so adjusted as to prevent saturation (halation)in the brightest signals in boutons (see Materials andMethods), GAD65 signals in somata generally appearedweak, because those conditions were suboptimal for so-matic imaging. This may probably reflect the fact thatGAD65 is contained in axon terminals at significantlyhigher level than in somata, which we could not haverevealed in our previous observations of DAB-coloredspecimens. On the other hand, GAD67 signals in somatawere not so weak under present CLSM conditions, prob-ably due to relatively high contents of GAD67 in somata.

Distribution patterns of bouton-like structures analyzedby CLSM were also different from those described previ-ously on a basis of observations in DAB-colored specimens.Whereas GAD-IR boutons in DAB specimens were distrib-uted ubiquitously in all hippocampal and dentate layerswithout apparent laminar difference in their immunoreac-tivities (Fukuda et al., 1997), CLSM clearly revealed thatintensities of signals in boutons were considerably differ-ent among layers in each GAD-isoform immunostaining(Figs. 2, 4, 5). To analyze these laminar patterns moreprecisely and objectively, we measured gray levels ofimmunostaining in individual boutons for each GAD iso-form and compared them across layers. It is important toemphasize here that the present method did not allow usto compare directly the content of one GAD isoform withthe other within each bouton, because we used differentprimary and secondary antibodies, different fluorescentdyes, and different CLSM channels for detecting the twoGAD isoforms. Therefore we could only compare theintensity of immunoreactivity, separately for each GADisoform, between different layers and between differentsubpopulations of GABAergic neurons.

As shown in the previous study (Sloviter et al., 1996),the mossy fiber terminals in the stratum lucidum of theCA3 region and those in the hilus of the dentate gyrusshowed intense immunoreactivity against GAD67, but notagainst GAD65 (Fig. 2). Thus the stratum lucidum and thehilus were excluded from the quantitative analysis be-cause of difficulty in discriminating GABAergic boutonsfrom GAD67-IR mossy fiber terminals located in theselayers.

Correlated CLSM-EM study

As an initial step we used electron microscopy (EM) toidentify bouton-like profiles seen in CLSM. In GAD67 andGAD65 immunostainings individual boutons detected inCLSM were identified again by EM and were found in mostcases to be presynaptic axon terminals containing synapticvesicles (Fig. 3). Moreover, symmetrical synapses werefrequently observed between the labeled terminals and

their postsynaptic targets (Fig. 3C,E–G). On the otherhand, axon terminals forming asymmetrical synapseswere devoid of immunoreactivity against each GAD iso-form (Fig. 3D) with the solitary exception in the GAD67-IRmossy fiber terminals (see above; Sloviter et al., 1996).Based on these correlated CLSM-EM examinations weconcluded that most of the bouton-like profiles detected inCLSM could be safely regarded as GAD-IR axon terminals.

Both GAD67-labeled boutons and GAD65-labeled onesvaried in size. In EM the smallest ones were about 0.5 µmin diameter along the major axis (Fig. 3F), the lengthcorresponding to the diameter of a circle whose area waseight pixels in CLSM. This size was used in the followingbinary image processing as a threshold for removingthinner axonal profiles as well as noise signals (see Materi-als and Methods).

Qualitative observations of GAD67 andGAD65 immunoreactivities in boutons

Hippocampus proper. Distributions of the two GADisoforms in boutons showed considerable laminar differ-ences (Figs. 2A,B, 4). The GAD67 immunoreactivity wasmost prominent in the stratum pyramidale of both CA1and CA3 regions, whereas it was relatively weak in thestrata oriens, radiatum, and lacunosum-moleculare (Fig.4A–D). Conversely, the GAD65 immunoreactivity in bou-tons was rather weak in the stratum pyramidale but moreintense in the other layers (Fig. 4E–H). When doublefluorescent CLSM images were merged into pseudo-colorimages (Fig. 2), GAD67 immunoreactivity (green) wasoutstanding in the stratum pyramidale of both CA1 andCA3 regions whereas GAD65 immunoreactivity (red) wasdominant in the remaining hippocampal layers. Whenobserved in more detail, individual boutons within eachlayer exhibited blended colors of various intensities for thetwo GAD isoforms, from greenish through yellowish toreddish (Fig. 2A,B). However, a particular color tone wasdominant in each layer as above. Thus the majority ofboutons in the stratum pyramidale (the principal celllayer) exhibited intense GAD67 immunoreactivity andweak GAD65 immunoreactivity whereas those in thedendritic layers (strata oriens, radiatum, and lacunosum-moleculare) exhibited intense GAD65 immunoreactivityand weak GAD67 immunoreactivity. The CA3 stratumlucidum was colored green because it contained mossyfiber terminals that were intensely immunostained forGAD67 but not stained for GAD65 at all.

Dentate gyrus. Patterns of GAD67 and GAD65 immu-noreactivities in boutons in the dentate gyrus were gener-ally similar to those in the hippocampus proper, but somedifferences were also noted. GAD67 immunoreactivity wasprominent in the majority of boutons located in the gran-ule cell layer (principal cell layer) whereas it was weak inmost of the boutons in the molecular layer (dendritic layer;Figs. 2C, 5A–D). This contrast in GAD67 immunoreactiv-ity between the principal cell layer and the apical dendriticlayer was the same as that observed between the corre-sponding layers in the hippocampus proper. On the otherhand, GAD65 immunoreactivity was intense in most of theboutons in the molecular layer whereas it was relativelyweak in many intensely GAD67-IR boutons in the granulecell layer (Figs. 2C, 5E-H), again following the patternobserved in the hippocampus proper. However, the granulecell layer also contained intensely GAD65-IR/very weaklyGAD67-IR boutons (Fig. 5D,H), which could be visualized


Figure 2 and Figure 10


as reddish particles in the merged two-color images (Fig.2C). This type of boutons was much fewer in the stratumpyramidale of the hippocampus proper. The hilus of thedentate gyrus was largely filled with intensely GAD67-IRstructures that lacked GAD65 immunoreactivity and werecolored green. This staining characteristic was consistentwith that in the stratum lucidum of the CA3 region; bothareas contained abundant mossy fiber terminals.

Quantitative analysis of GAD65 and GAD67immunoreactivities in boutons

The intensities of immunofluorescent signals for the twoGAD isoforms were measured in individual boutons by theprocedures described in Materials and Methods (Fig. 1)and compared between layers (Figs. 6–9). Scattergrams inFigure 6 demonstrate the relationship of the GAD65 andGAD67 immunoreactivities in individual boutons in eachlayer. Gray levels in each GAD-isoform immunostainingwere arbitrarily classified into two categories; those lessthan 100 were defined as ‘‘low’’ while those equal to or morethan 100 as ‘‘high,’’ and data in the scattergrams weredivided into four groups by combining this classificationfor GAD65 immunoreactivity with that for GAD67 immu-noreactivity (Table 1).

Consistent with the above qualitative observations, graylevels of both GAD65 and GAD67 immunoreactivities inboutons in the principal cell layers were in sharp contrastto those in the dendritic layers. In the hippocampusproper, the majority of boutons in the stratum pyramidale(53 and 51% in the CA1 and CA3 regions, respectively)showed relatively low gray levels (, 100) of immunoreac-tivity against GAD65 while high ($ 100) gray levels ofimmunoreactivity against GAD67 (Fig. 6 and Table 1). Bycontrast, 50 to 80% of boutons in the hippocampal den-dritic layers (strata lacunosum-moleculare, radiatum, andoriens) clustered in the quadrant of GAD65-high and

GAD67-low gray levels of immunoreactivities. Particu-larly in the stratum lacunosum-moleculare, 77% (CA1)and 72% (CA3) of boutons showed such immunoreactivi-ties. When compared separately for each GAD isoformacross the hippocampal layers of the CA1 (Fig. 7) and CA3(Fig. 8) regions, boutons in the stratum pyramidale showedsignificantly lower gray levels for GAD65 and higher graylevels for GAD67 than those in other hippocampal layers(P , 0.001 in two sample z-test).

The dendritic layer of the dentate gyrus was divided intothree sublayers, outer, middle, and inner third of themolecular layer for the quantitative analysis since thesethree sublayers are known to receive different excitatoryas well as inhibitory inputs. In all these dendritic sublay-ers, the majority of boutons showed relatively high graylevels of GAD65 immunoreactivity and relatively low graylevels of GAD67 immunoreactivity (Fig. 6): 60 to 80% ofboutons showed such immunoreactivities, with the gradi-ent of the highest percentage in the outer molecular layer(Table 1). The scattergram in the granule cell layer differedconsiderably from those in the three dendritic sublayers(Fig. 6) but was similar to those in the CA1 and CA3stratum pyramidale. Some differences were also noted,however, between the dentate and the hippocampal princi-pal cell layers. First, although dots in the scattergram forthe granule cell layer accumulated within the range ofGAD65-low/GAD67-high gray levels as in the stratumpyramidale, other populations of boutons showing higherGAD65 and low GAD67 gray levels were also recognized inthe granule cell layer, corresponding well to the qualitativeobservation described above. Second, a population of bou-tons showing high gray levels for GAD67 appeared to havehigher gray levels for GAD65 in the granule cell layer thanin the CA1 and CA3 stratum pyramidale (Fig. 6). Based onthese two features, three populations of boutons (GAD65-low/GAD67-high; GAD65-high/GAD67-high; GAD65-high/GAD67-low) were nearly equal in percentages in thegranule cell layer (Table 1). When compared across layers(Fig. 9), however, boutons in the granule cell layer showedsignificantly lower gray levels for GAD65 and higher graylevels for GAD67 than those in the three dendritic sublay-ers (P , 0.001 in two sample z-test).

GAD67 and GAD65 immunoreactivitiesin PV-positive boutons

Previously we demonstrated that hippocampal PV-containing GABAergic neurons show weak immunoreactiv-ity against GAD65 and moderate to intense immunoreac-tivity against GAD67 in their somata (Fukuda et al.,1997). Since PV-containing axon terminals are preferen-tially located in the principal cell layers, the presentresults showing abundance of weakly GAD65-IR/intenselyGAD67-IR boutons in these layers suggest the possibilitythat unique GAD-isoform immunoreactivities in the hippo-campal and dentate principal layers are closely related tothose in PV-containing GABAergic terminals. Therefore,we directly investigated the GAD65 and GAD67 immuno-reactivities in PV-positive boutons by combining PV andGAD65, or PV and GAD67, in double immunofluorescentstainings.

PV-containing boutons in both the stratum pyramidaleand the granule cell layer displayed relatively weak immu-nostaining for GAD65 and intense immunostaining forGAD67 (Fig. 10: next to Fig. 2). Moreover, although theselayers contained some boutons showing intense immunore-

Fig. 2. Montages of pseudo-color confocal laser scanning micros-copy (CLSM) images showing GAD67 (green) and GAD65 (red)immunoreactivities in the CA1 (A) and CA3 (B) regions of the dorsalhippocampus and the dentate gyrus (C). Montages were made fromimages taken through a 603 oil immersion objective under constantCLSM conditions and visualized in print by identical image-processing and photographical procedures. Note that numerousGAD-IR boutons are distributed throughout the areas but they showlamina-specific patterns in their immunoreactivities. GAD67-intense/GAD65-weak immunoreactivity (greenish) is prominent in the princi-pal cell layers, namely, the stratum pyramidale (sp) of the hippocam-pus proper (A,B) and the granule cell layer (gr) of the dentate gyrus(C), whereas GAD67-weak/GAD65-intense immunoreactivity (red-dish) is prominent in the dendritic layers, i.e., the strata lacunosum-moleculare (slm), radiatum (sr), and oriens (so) of the hippocampusproper (A,B), and the outer (om), middle (mm) and inner (im)molecular layer of the dentate gyrus (C). Intense GAD67 immunoreac-tivity along the mossy fiber pathway is also seen in the hilus (h) andthe CA3 stratum lucidum (lc). The stratum lacunosum-moleculare andthe outer part of the dentate molecular layer are also characterized bythe larger number of boutons per unit area. Scale bar 5 10 µm.

Fig. 10. Pseudo-color confocal laser scanning microscopy (CLSM)images showing parvalbumin (PV; green) and GAD65 (red) immunore-activities (A), and PV (green) and GAD67 (red) immunoreactivities(B), in the CA1 stratum pyramidale. A: Most of the PV-positiveboutons are greenish, indicating weak immunoreactivity againstGAD65, whereas intensely GAD65-IR boutons (red) are PV negative.B: In contrast with the result in A, most of the PV-positive boutons areyellowish, indicating intense immunoreactivity against GAD67. Scalebar 5 5 µm.


activity against GAD65 (Fig. 10A), almost all of them werePV-negative. These observations were supported by thequantitative analysis (Fig. 11). As was apparent in thehistograms, PV-containing boutons exhibited relativelylow gray levels (, 100) of GAD65 immunoreactivity buthigh gray levels ($ 100) of GAD67 immunoreactivity ineach of the three principal cell layers.

Numerical densities of GAD-IR boutons

The present method of analyzing GAD-isoform immuno-reactivities in individual boutons enabled us to estimatethe relative abundance of GAD-IR boutons in each hippo-campal and dentate layer. Although this approach is not asaccurate as in the direct counting of axon terminals in EM,

Fig. 3. Correlated confocal laser scanning microscopy (CLSM)-electron microscopic (EM) observations of GAD65-IR boutons in thestratum lacunosum-moleculare of the CA1 region. A,B: CLSM imagesin the two single optical slices, 0.3 µm apart along z-axis of the section,showing profiles of labeled boutons and a soma. Several boutons(arrows) were examined directly by EM. C–G: Ultrastructures of theboutons (asterisk) indicated by arrows in A and B. Note that all these

boutons can be identified as presynaptic terminals (asterisk) contain-ing synaptic vesicles and that some of them form symmetricalsynapses (arrowheads). On the other hand, axon terminals makingasymmetrical synapses (a) are devoid of immunoreactivity. d labelsdendrite arising from the GAD positive soma in A. Scale bars 5 5 µmin A and B, 0.5 µm in C–G.


Fig. 4. Four pairs of confocal laser scanning microscopy (CLSM)images showing GAD67 (A-D) and GAD65 (E–H) immunoreactivitiesin the strata lacunosum-moleculare (A,E), radiatum (B,F), pyrami-dale (C,G), and oriens (D,H) in the CA1 region. Images are taken fromthe same material under fixed CLSM conditions across layers andvisualized in print by identical image-processing and photographical

procedures. Although a small number of boutons show intense immu-noreactivities against both GAD isoforms (arrows), the majorityexhibit intense immunoreactivity against only one of the two GADisoforms, GAD67-intense/GAD65-weak immunoreactivity (arrow-heads) in the stratum pyramidale and GAD67-weak/GAD65-intenseimmunoreactivity (crossed arrows) in other layers. Scale bar 5 5 µm.


Fig. 5. Four pairs of confocal laser scanning microscopy (CLSM)images showing GAD67 (A–D) and GAD65 (E–H) immunoreactivitiesin the outer (A,E), middle (B,F), and inner (C,G) third of the molecularlayer and in the granule cell layer (D,H) of the dentate gyrus. Imagesare taken from the same material under fixed CLSM conditions andvisualized by identical procedures. Just as in the correspondingdendritic layers of the hippocampus proper (Fig. 4), the majority ofboutons throughout the three sublayers of the molecular layer

(A–C,E–G) exhibit GAD67-weak/GAD65-intense immunoreactivity(crossed arrows). Arrows indicate the minority of boutons exhibitingintense immunoreactivities against both GAD isoforms. In the granulecell layer (D,H), boutons showing GAD67-intense/GAD65-weak immu-noreactivity (arrowheads) are more numerous than in other layers,but those showing GAD67-weak/GAD65-intense immunoreactivity(crossed arrows) as well as those showing intense immunoreactivitiesagainst both GAD isoforms (arrows) are also frequent. Scale bar 5 5 µm.


we could examine many more boutons and sample broaderareas than would be possible with usual electron micro-scopic observations. Both CLSM images (Figs. 2, 4, 5) andthe scattergrams (Fig. 6) suggested the higher incidence ofGAD-IR boutons in the most distal part of the apicaldendritic layers, namely, the stratum lacunosum-molecu-lare of both the CA1 and CA3 regions and the outer third ofthe dentate molecular layer. The numerical densities ofGAD-IR boutons in these layers were significantly higherthan those in other layers within the same regions (Fig.12).

DISCUSSION

In the present study we revealed the heterogeneousdistributions of the two GAD isoforms in axon terminals bythe quantitative method using CLSM and digitized imageanalysis. Our major findings can be summarized as fol-lows: 1) the majority of axon terminals in each hippocam-pal and dentate layer showed lamina-specific immunoreac-tivities against both GAD isoforms although someexceptional cases also occurred within each layer; 2) theGAD67 immunoreactivity in axon terminals was generallymore intense in the principal cell layers than in thedendritic layers; 3) the GAD65 immunoreactivity in axonterminals was generally more intense in the dendriticlayers than in the principal cell layers; 4) PV-IR axonterminals in the principal cell layers showed intenseimmunoreactivity against GAD67 but relatively weakimmunoreactivity against GAD65.

Methodological consideration

As already mentioned at the beginning of Results, wemade a comparative study for each GAD isoform betweenlayers and could not compare directly the immunoreactiv-ity for the two GAD isoforms in individual boutons.Another important point is that the gray levels we used forquantifying immunofluorescent signals reflected just therelative amount of GAD molecules, not the absolute con-tents, because of the lack of calibration curves for estimat-ing the latter. In spite of this limitation the presentquantitative data clearly pointed out the laminar differ-ences in the GAD-isoform immunoreactivities in axonterminals between somatic and dendritic layers.

Quantitative analysis of immunoreactivities by CLSMrequires special consideration, because intensities of sig-nals detected in CLSM are subject to many factors. Forexample, adjustment of parameters for acquiring imagesin CLSM, such as aperture size of the pinhole and gain ofthe detector, greatly influenced the intensity of signals.Moreover, imaging conditions that are optimal in one layermight be suboptimal in other layers, leading to the satura-tion in gray levels or the failure in obtaining properimages. Another problem was that optimal CLSM condi-tions differed somewhat between sections, since intensityof immunostaining varied from section to section. There-fore it was necessary to standardize the CLSM conditionsby some reasonable criteria (see Materials and Methods).By doing so the labeling intensities could be comparedunder the same condition across different layers withineach section. Furthermore, images taken from differentsections could be compared under relatively constantconditions by adjusting the parameters using the equiva-lent anatomical structures.

Profiles of GAD65-immunoreactive perisomatic boutonsshown here might appear considerably different fromthose obtained from DAB-colored specimens, where bou-tons in both somatic and dendritic layers exhibited moder-ate to intense reaction (Fukuda et al., 1997). Such appar-ent discrepancy between DAB-colored images and CLSMprofiles might be derived from the possibility that DABreaction may saturate somehow during developing pro-cess, thereby making potentially diverse immunoreactiv-ity in various structures homogeneous. Comparison ofGAD65 immunostaining between somatic and terminallevels could explain this. GAD65 immunoreactivity insomata was very weak in the present CLSM conditionswhile terminal labeling in dendritic layers was generallyintense in the same sections (Fig. 2). On the other hand,GAD65 immunostaining in somata was moderate to in-tense in the DAB specimens, close to the level of terminallabeling (Fukuda et al., 1997). Therefore it became ratherdifficult to detect the difference between somatic andterminal DAB reactions by conventional methods, usuallyby eyes. Under such conditions the difference in GAD65immunoreactivity between perisomatic and dendritic ter-minals clearly seen in CLSM might also become obscure.This hypothesis is further supported by our present obser-vations in the correlated CLSM-EM study, where differen-tial GAD65 immunoreactivity in the perisomatic anddendritic terminals in CLSM became indistinct after DABprocessing. We think that CLSM is more suitable forquantifying intensity of immunoreactivity whereas DABspecimens have the advantage in detecting weak signals;weak DAB reaction can be enhanced by several methodswhile more intense DAB reaction do not ‘‘overshoot’’ (freefrom halation) after enhancement. Present study usingCLSM revealed lamina-specific distribution of the twoGAD isoforms at the bouton level that could not bedemonstrated by previous methods. Yet we should empha-size again that our quantitative data reflect just therelative amounts of enzyme and that the present resultsshould be interpreted comparatively in conjunction withthe observations based on other methods.

GAD65 immunoreactivity

We previously demonstrated that GABAergic neuronsshowing weak to almost no GAD65 immunoreactivity intheir somata, in spite of signal enhancement in DABspecimens, constitute a relatively large population ofGABAergic neurons in the stratum pyramidale and aremostly PV-containing neurons (Fukuda et al., 1997). Wealso confirmed the relatively weak GAD65 immunoreactiv-ity in PV-containing somata in the dentate gyrus, particu-larly at the border between the granule cell layer and thehilus (unpublished observations). PV neurons in theseprincipal cell layers have been thought to coincide largelywith basket cells and axoaxonic cells (Kosaka et al., 1987;Katsumaru et al., 1988a) with axonal endings terminatingprimarily on perisomatic regions of principal neurons.Since the majority of axon terminals in principal celllayers showed relatively weak GAD65 immunoreactivity,there appeared to be some consistency in GAD65 immuno-reactivity between the somata and the axon terminals ofPV neurons. In fact, we could confirm the weak GAD65immunoreactivity in PV-containing terminals directly indouble-fluorescent CLSM imaging. On the other hand,GAD65 immunoreactivity in boutons located outside theprincipal cell layers was generally more intense than that


Figure 6


in the principal cell layers. This was also in accordancewith the observations at the somatic level, because themajority of PV-negative somata, either inside or outsidethe principal cell layers, showed moderate to intenseimmunoreactivity for GAD65 (Fukuda et al., 1997), andthe greater part of these neurons is thought to innervatepredominantly the dendritic layers of the hippocampus,i.e., the layers outside the principal cell layers. Consider-ing that the vast majority of postsynaptic targets in theselayers are dendrites of principal neurons, it can be con-cluded from the present results that the level of GAD65 isgenerally higher in axon terminals located on principal cell

dendrites than in those located on perisomatic regions ofthe principal cells.

Intraneuronal distribution of GAD could be related to atleast two factors: synthesis rate within soma and transportrate from soma to axon terminals. Although we have nodata concerning synthesis rate of GAD65 mRNA and ofGAD65 protein, relatively weak GAD65 immunostainingin both somata and terminals of PV neurons suggest thatpossibly low level of synthesis may be more responsible fortheir unique GAD65 immunoreactivity than involvementof transport rate.

GAD67 immunoreactivity

Just as in the distribution of GAD65, the GAD67-immunoreactivity in axon terminals showed a markedlaminar difference. Axon terminals located outside theprincipal cell layers, the great majority of which werePV-negative, displayed relatively weak immunoreactivityagainst GAD67, while boutons abutting on principal cellsomata showed an intense GAD67 immunoreactivity. Wecould conclude that the level of GAD67 is higher in axonterminals abutting on perisomatic regions of the principalcells than in terminals located on principal cell dendrites.

In contrast with GAD65 distribution, moderate to in-tense GAD67 immunoreactivity in somata was observed inmost of hippocampal and dentate GABAergic neurons bothin CLSM and in DAB sections (Fukuda et al., 1997).Quantitative analysis of the contents of GAD67 and itsmRNA at the somatic level will be helpful for understand-

Fig. 7. Histograms comparing the gray levels of GAD-isoform immunoreactivities in boutons acrossdifferent layers in the CA1 region of the hippocampus proper. Abscissa, gray level for each GAD-isoformimmunoreactivity; ordinate, number of boutons.

Fig. 6. Scattergrams showing gray levels for GAD67 and GAD65immunostainings in individual boutons in different layers of CA1 (A)and CA3 (B) regions and of the dentate gyrus (C). Each dot representsa single bouton. Abscissa, gray level for GAD65 immunoreactivity;ordinate, gray level for GAD67 immunoreactivity. Note the frequentoccurrence of boutons characterized by high ($ 100) gray levels ofGAD65 immunoreactivity and low (, 100) gray levels of GAD67immunoreactivity in the strata lacunosum-moleculare (slm), radia-tum (sr), and oriens (so) in CA1 and CA3 regions and throughout thethree sublayers of dentate molecular layer, i.e., outer (om), middle(mm), and inner (im) molecular layer. By contrast, the majority ofboutons in the stratum pyramidale (sp) of both the CA1 and CA3regions are accumulated in the quadrant of low GAD65 gray levels andhigh GAD67 gray levels. In the granule cell layer (gr) the majority ofboutons show high gray levels of GAD67 immunoreactivity as in thestratum pyramidale, but GAD65 immunoreactivity of these boutons issomewhat higher than that in the stratum pyramidale.


ing the mechanisms of differential intracellular distribu-tion of GAD67 between perisomatic and dendritic inhibi-tory neurons.

Heterogeneity within each layer

In spite of the general laminar patterns of GAD-isoformimmunoreactivities as above, we must also consider excep-tions. First, intensely GAD65-IR boutons were also ob-served in the principal cell layers. The origin of theseboutons might be non-PV basket neurons, e.g., thosecontaining other chemical substances such as CCK and/orVIP (Harris et al., 1985; Nunzi et al., 1985; Acsady et al.,1996). Second, a small number of PV-IR boutons were alsolocated outside the principal cell layers, namely, in thestrata radiatum and oriens of both CA1 and CA3 regions,but not in the stratum lacunosum-moleculare or in thedentate molecular layer. Preliminary double immunofluo-rescent CLSM revealed that these boutons exhibited vari-ous GAD-isoform immunoreactivities. Although the originof these PV-IR boutons, whether from intrinsic neurons orfrom extrahippocampal sources, remains to be identified,we could at least say that some heterogeneity exists inPV-positive boutons. Likewise GAD-isoform immunoreac-tivities were more or less heterogeneous within each layer.Further studies are needed to reveal in more detail thediversity in GAD immunoreactivities in the hippocampalformation and we should refrain from oversimplification inany aspects of potentially diverse hippocampal nonprinci-pal neurons.

Functional implications

Mammalian GAD requires a cofactor pyridoxal 58-phosphate (PLP) for enzymatic activity (Roberts andFrankel, 1951). The two GAD isoforms have distinctcofactor interactions; in mouse brain extracts GAD67 isnearly saturated with PLP while GAD65 is about half-saturated with PLP (Kaufman et al., 1991). Since theassociation of apo-GAD (inactive GAD) and PLP to formactive holo-GAD (PLP bound) is an important regulator ofGAD activity (Martin, 1987), lamina-specific distributionsof the two GAD-isoform immunoreactivities in boutonsmight be related to the functional differences between theperisomatic and dendritic inhibitory sites as discussedbelow.

Dominance of the active enzyme form in the GAD67molecules suggests that GAD67 may be rich in terminalswhere synaptic activities are continuously high. The pre-sent study revealed preferential occurrence of GAD67 inaxon terminals that abut on the perisomatic domain ofprincipal cells. Physiological data suggest that perisomaticinhibitory cells may discharge at considerably higherfrequencies than do dendritically projecting cells (Miles etal., 1996). In fact, basket cells in vivo were shown tocontinue firing with rhythmic fast bursts of 30 to 60 Hzduring theta activity (Sik et al., 1995; Ylinen et al., 1995b).Moreover, a tonic barrage of randomly occurring spontane-ous inhibitory events (miniature IPSCs) at relatively highfrequencies originate mainly from GABAergic synapses

Fig. 8. Histograms comparing the gray levels of GAD-isoform immunoreactivities in boutons acrossdifferent layers in the CA3 region of the hippocampus proper. Abscissa, gray level for each GAD-isoformimmunoreactivity; ordinate, number of boutons.


close to the soma (Soltesz et al., 1995). In light of thesephysiological data, our results on GAD67 distributionagree with the above presumptive correlation betweentonic synaptic activity and GAD67 immunoreactivity. An-other basis for this correlation is the relatively large size ofperisomatic terminals (Miles et al., 1996), particularlythose containing PV with many mitochondria (our unpub-lished observations), since larger terminals are thought to

correlate with an increased probability of transmitterrelease (Pierce and Lewin, 1994).

On the other hand, axon terminals located on thedendritic domain of principal neurons showed higherimmunoreactivity against GAD65 than those on the periso-matic domain. About half of GAD65 is present in brain asapoenzyme, thereby providing a reservoir of inactive GADthat can be drawn on when additional GABA synthesis isrequired (Martin et al., 1991; Kaufman et al., 1991). Thecyclic interconversion of apo- and holo-GAD is stronglyregulated by physiological concentrations of polyanionssuch as ATP and by inorganic phosphate (Pi); ATP pro-motes the conversion of holo- to apo-GAD while Pi pro-motes conversion of apo- to holo-GAD. Through this andother enzymatic mechanisms it has been assumed thatGAD65 is specialized to respond to short-term changes indemand for GABA (Martin and Rimval, 1993). Based onthese molecular properties, our present data support theproposal by Houser and Esclapez (1994) that GAD65within terminals in the dendritic regions could be con-verted from the apo- to the holo-enzyme form in responseto local demands for greater GABAergic control. Domi-nance of the inducible form of GAD in axon terminals onthe dendritic domain of principal neurons might be relatedto the segregation of the specific excitatory inputs terminat-ing on this domain (Amaral and Witter, 1995), whereinhibitory actions might be local and transient rather thangeneral and continuous.

Fig. 9. Histograms comparing the gray levels of GAD-isoform immunoreactivities in boutons acrossdifferent layers in the dentate gyrus. Abscissa, gray level for each GAD-isoform immunoreactivity;ordinate, number of boutons.

TABLE 1. Percentages of Glutamic Acid Decarboxylase(GAD)-Immunoreactive Boutons Categorized by GAD67

and GAD65 Immunoreactivities1

GAD67 high/GAD65 low

GAD67 high/GAD65 high

GAD67 low/GAD65 high

GAD67 low/GAD65 low Total

CA1slm 1.8 21.3 76.6 0.2 100sr 10.7 29.4 59.6 0.3 100sp 52.8 28.5 16.6 2 100so 7.4 29.6 63 0 100

CA3slm 1.4 26.2 72.1 0.2 100sr 5.5 39 55.4 0 100sp 51 32.1 16.2 0.7 100so 7.4 43.1 49.2 0.2 100

DGom 0.8 22.8 76.4 0.1 100mm 1.8 28.4 69.6 0.2 100im 4.5 33.7 61.5 0.2 100gr 30.9 31.5 36.9 0.6 100

1CA1, CA1 region; CA3, CA3 region; DG, dentate gyrus; gr, granule cell layer; im, innermolecular layer; mm, middle molecular layer; om, outer molecular layer; slm, stratumlacunosum-moleculare; so, stratum oriens; sp, stratum pyramidale; sr, stratum radia-tum.


Fig. 11. Histograms showing gray levels of GAD65 (A) and GAD67(B) immunoreactivities of parvalbumin- (PV)-positive boutons in theprincipal cell layers; CA1 and CA3 stratum pyramidale (sp), and

dentate granule cell layer (gr). The majority of PV-positive boutonsshow low gray levels of GAD65 immunoreactivity (A) and high graylevels of GAD67 immunoreactivity (B) in each principal cell layer.

Fig. 12. Comparison of the numerical densities of GAD-IR boutonsbetween different layers of CA1 (A) and CA3 (B) regions and of thedentate gyrus (C). Data were obtained from the same materials as inFigure 6. Numerical density is expressed in an arbitrary unit (number

of boutons per unit area of 250 3 250 pixels which corresponds to areaof 33.3 µm 3 33.3 µm). In each of three regions the most distal part ofthe dendritic layers shows significantly higher density of boutons thanother layers (P , 0.01 in two sample t-test).


In addition to their specified manner of axonal termina-tion as basket and axoaxonic cells (Kosaka et al., 1987;Katsumaru et al., 1988a), PV-containing neurons haveother characteristic properties; they form gap junctionswith each other (Katsumaru et al., 1988b) and they alsoform dense mutual connections through perisomatic synap-tic contacts (Sik et al., 1995; Fukuda et al., 1996; Cobb etal., 1997). Supported partly by these morphological fea-tures as well as by computer simulation analyses, recentphysiological findings have raised the possibility thatPV-neurons are crucially involved in the induction andmaintenance of synchronized membrane potential oscilla-tions such as theta and gamma in principal neuronsthrough GABAA-receptor mediated summed IPSPs (Solteszand Deschenes, 1993; Bragin et al., 1995; Buzsaki andChrobak, 1995; Cobb et al., 1995; Traub, 1995; Whitting-ton et al., 1995; Ylinen et al., 1995a,b; Traub et al., 1996;Wang and Buzsaki, 1996; Cobb et al., 1997). Basket cellshave been proposed to control membrane potential oscilla-tions not only through their actions on principal neuronsbut also through dense interconnections among them-selves. Because virtually all GAD67 molecules are thoughtto be in an active form, the preferential localization ofGAD67 in the perisomatic PV-containing terminals ap-pears suitable for continuous GABA synthesis that mightbe required during oscillatory activities.

ACKNOWLEDGMENTS

The monoclonal antibody GAD6 was obtained from theDevelopmental Studies Hybridoma Bank maintained bythe Department of Pharmacology and Molecular Science,Johns Hopkins University School of Medicine, Baltimore,MD, and the Department of Biological Sciences, Univer-sity of Iowa, Iowa City, IA, under contract N01-HD-6-2915from the NICHD. We are grateful to Dr. P.S. Buckmaster(University of California, Davis) for his helpful commentsand critical reading of the manuscript. We thank Ms. Odafor her secretarial assistance.

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gabaergic axon terminals at perisomatic and dendritic inhibitory sites show different...

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