Basal expression and induction of glutamate decarboxylase GABA in excitatory granule cells of the rat and monkey hippocampal dentate gyrus

THE JOURNAL OF COMPARATIVE NEUROLOGY 373~593-618 (1996)

Basal Expression and Induction of Glutamate Decarboxylase and GABA in Excitatory Granule Cells of the Rat and

Monkey Hippocampal Dentate Gyrus

ROBERT S. SLOVITER, MARC A. DICHTER, TARA L. RACHINSKY, EVELYN DEAN, JEFFREY H. GOODMAN, ANNE L. SOLLAS, AND DAVID L. MARTIN

Neurology Research Center, Helen Hayes Hospital, New York State Department of Health, West Haverstraw, New York 10993 (R.S.S., E.D., J.H.G., A.L.S.), Departments of

Pharmacology and Neurology, Columbia University, New York, New York 10032 (R.S.S.), Wadsworth Center for Laboratories and Research, New York State Department of Health,

Albany, Department of Environmental Health and Toxicology, State University of New York at Albany, Albany, New York 12201 (D.L.M.); Departments of Neurology and

Pharmacology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104 (M.A.D., T.L.R. )

ABSTRACT The excitatory, glutamatergic granule cells of the hippocampal dentate gyrus are presumed

to play central roles in normal learning and memory, and in the genesis of spontaneous seizure discharges that originate within the temporal lobe. In localizing the two GABA-producing forms of glutamate decarboxylase (GAD65 and GAD67) in the normal hippocampus as a prelude to experimental epilepsy studies, we unexpectedly discovered that, in addition to its presence in hippocampal nonprincipal cells, GAD67-like immunoreactivity (LI) was present in the excitatory axons (the mossy fibers) of normal dentate granule cells of rats, mice, and the monkey Macaca nemestrina. Using improved immunocytochemical methods, we were also able to detect GABA-LI in normal granule cell somata and processes. Conversely, GAD65-LI was undetectable in normal granule cells.

Perforant pathway stimulation for 24 hours, which evoked population spikes and epileptiform discharges in both dentate granule cells and hippocampal pyramidal neurons, induced GAD65-, GAD67-, and GABA-LI only in granule cells. Despite prolonged excitation, normally GAD- and GABA-negative dentate hilar neurons and hippocampal pyramidal cells remained immunonegative. Induced granule cell GAD65-, GAD67-, and GABA-LI remained elevated above control immunoreactivity for at least 4 days after the end of stimulation. Pre-embedding immunocytochemical electron microscopy confirmed that GAD67- and GABA-LI were induced selectively within granule cells; granule cell layer glia and endothelial cells were GAD- and GABA-immunonegative. In situ hybridization after stimulation revealed a similarly selective induction of GAD65 and GAD67 mRNA in dentate granule cells.

Neurochemical analysis of the microdissected dentate gyrus and area CA1 determined whether changes in GAD- and GABA-LI reflect changes in the concentrations of chemically identified GAD and GABA. Stimulation for 24 hours increased GAD67 and GABA concentrations sixfold in the dentate gyrus, and decreased the concentrations of the GABA precursors glutamate and glutamine. No significant change in GAD65 concentration was detected in the microdissected dentate gyrus despite the induction of GAD65-LI. The concentrations of GAD65, GAD67, GABA, glutamate and glutamine in area CA1 were not significantly different from control concentrations.

These results indicate that dentate granule cells normally contain two “fast-acting’’ amino acid neurotransmitters, one excitatory and one inhibitory, and may therefore produce both excitatory and inhibitory effects. Although the physiological role of granule cell GABA is

Accepted July 12, 1996. Address reprint requests to Dr. R.S. Sloviter, Neurology Research Center,

Helen Hayes Hospital, West Haverstraw, NY 10993.

:i, 1996 WILEY-LISS, INC.

594 R.S. SLOVITER ET AL

unknown, the discovery of both basal and activity-dependent GAD and GABA expression in glutamatergic dentate granule cells may have fundamental implications for physiological plasticity presumed to underlie normal learning and memory. Furthermore, the induction of granule cell GAD and GABA by afferent excitation may constitute a mechanism by which epileptic seizures trigger compensatory interictal network inhibition or GABA-mediated neurotrophic effects. 1996 WiIey-Liss, Inc.

Indexing terms: hippocampus, inhibition, epilepsy, seizures, in situ hybridization

Traditional views of neuronal network organization suggest that excitatory principal cells produce short-latency excitation of postsynaptic targets cells and, in turn, receive inhibitory inputs that govern their excitability (Eccles, 1973; Shepherd, 1979). Current knowledge indicates that synaptic excitation and inhibition are produced by separate neuron populations that contain either an excitatory or an inhibitory “fast-acting’’ neurotransmitter, but not both (Eccles, 1973; Shepherd, 1979; Schwartz, 1991). Within the brain, the principal fast-acting excitatory transmitter is glutamate, and the principal inhibitory transmitter is y-aminobutyric acid (GABA) (Schwartz, 1991). GABA is produced by the 65- and 67-kDa forms of glutamate decarboxylase (GAD65 and GAD67; Erlander et al., 19911, which are regarded as definitive markers of inhibitory neurons that utilize GABA as a transmitter (Wood et al., 1976; Ribak et al., 1978; Mugnaini and Oertel, 1985; Babb et al., 1988; Erlander et al., 1991; Feldblum et al., 1993; Esclapez et al., 1994).

Within the hippocampal formation, the dentate gyrus is thought to play a pivotal role in the normal hippocampal functions of learning and memory (Scoville and Milner, 1957; Alvarez et al., 1995), and to underlie the propensity of the hippocampus to generate seizure discharges (Dichter and Spencer, 1969; Spencer et al., 1990; Lothman et al., 1991; Williamson et al., 1995). The principal cells of the dentate gyrus are the excitatory (Andersen et al., 1966; Yamamoto, 1972, 1982; Brown and Johnston, 1983) and glutamatergic (Crawford and Connor, 1973; Storm-Mathi- sen et al., 1983; Soriano and Frotscher, 1994) granule cells, which receive direct synaptic input from the entorhinal cortex (Ramon y Cajal, 1893, 1995; Lorente de No, 1934; Andersen et al., 1966).

Dichter and colleagues recently reported the paradoxical finding that GAD mRNA is expressed in both inhibitory and excitatory hippocampal neurons grown in culture (Dichter et al., 1994; Cao et al., 1996). This finding led us to determine whether any of the excitatory hippocampal neuron populations express GAD or GABA in vivo in response to focal afferent stimulation that evokes hippocampal principal cell population spikes and epileptiform discharges. However, in preparation for these experimental epilepsy studies, we unexpectedly discovered that intense GAD67-like immunoreactivity (LI) was present in the excitatory axons of normal dentate granule cells. We have therefore re-examined the issue of GAD and GABA localization in the normal hippocampus of the rat, mouse, and monkey Macaca nemestrina using improved immunocytochemical methodology, and then determined whether seizure activity alters GAD and GABA expression in the rat. In this paper, we report the first immunocytochemical localization of GAD67-LI in the normal hippocampus, demonstrate that dentate granule cells express both GAD67 and GABA normally, and show that afferent excitation of all hippocam-

pal principal cell populations selectively induces the long lasting expression of GAD65-, GAD67-, and GABA-LI ir dentate granule cells. A preliminary report of this study ha; been presented in abstract form (Sloviter et al., 1995).

MATERIALS AND METHODS Animal treatment

Animals were treated in accordance with the guideline: set by the New York State Department of Health and the National Institutes of Health. Male Sprague-Dawley descen dent rats weighing 250-35Og were anesthetized with ure. thane (1.25 gikg S.C. initially and additional injections a: needed; 250 mg urethaneiml 0.9% saline) and placed in i Kopf stereotaxic device. Rectal temperature was main tained automatically at 37.0 ? 0.2”C with a heating pac (Harvard Apparatus). A bipolar stainless steel stimulatinb electrode (NE-200; Rhodes Medical) was placed in thr angular bundle of the perforant path (approximately 4.E mm lateral from the midline suture and immediate11 rostra1 to the lambdoid suture). Glass microelectrodes (1.5 mm outer diameter) were pulled on a Kopf electrode puller filled with 0.9% saline, lowered into the brain (approxi. mately 2 mm lateral from the midline suture, 3 mm rostra: to the lambda, and 3.5 mm below the brain surface), and placed in the dorsal blade of the granule cell layer b) optimizing the characteristic shape of the evoked potentia: (Andersen et al., 1966). Biphasic current pulses (0.1 msec duration) were generated by a Grass S88 stimulator with E Grass stimulus isolation unit. Stimulus voltage greater than that needed to evoke maximum amplitude populatior spikes was used, and was usually 20 or 30 V. Potential: were amplified by a Grass preamplifier, displayed on ii Nicolet Series 400 digital oscilloscope, and stored on dis. kette.

The intermittent stimulation paradigm used to evokt hippocampal population spikes and epileptiform discharge> has been described in detail previously (Sloviter, 1983 1991). Paired pulses were delivered 40 msec apart at 2 H r continuously throughout the 1-24-hour stimulation pe riod, and a 20-Hz/lO-second stimulus train of single pulses was delivered through the same stimulating electrode oncc per minute.

Subsets of control and stimulated rats were injected 1 hour before the start of 6 hours of stimulation with cycloheximide (Sigma) at a dose ( 2 mgikg s.c.) shown t c : inhibit protein synthesis (Ch’ih et al., 1977; Schreiber et al., 1993).

Perfusion-fixation Perforant path-stimulated rats, naive male rats, naive

male ICR mice (25-34 g; Harlan), and four male Macaw nemestrina monkeys (5-7 kg) that had been subjected to

GAD AND GABA IN THE HIPPOCAMPUS 595

behavioral conditioning as previously described (Wolpaw and Herchenroder, 1990) were sacrificed by perfusion- fixation through the aorta by gravity feed. Rats and mice were anesthetized with urethane, and perfused with a fixative optimal for GAD67 and GABA immunocytochemistry (0.9% saline for 1-2 minutes followed by 3.0% glutaral- dehydei 1.0% paraformaldehyde in 0.1M phosphate buffer, pH 7.4, for 10 minutes), or a fixative optimal for GAD65 and GAD67 immunostaining, as well as for in situ hybridization histochemistry (2% paraformaldehyde in 0.1M sodium acetate buffer, pH 6.5, for 3 minutes, followed by 15 minutes perfusion with 2% paraformaldehydeio. 1% glutaraldehyde in 0.1M sodium borate buffer, pH 8.5). This fixative exploits the low pHihigh pH principle of formaldehyde fixation that improves aldehyde penetration and yields superior immunocytochemical results (Berod et al., 1981). After storage in situ overnight a t 4"C, rat and mouse brains were removed and cut on a Vibratome in 0.1M Tris buffer, pH 7.6.

The monkeys were anesthetized with ketaminelatropine, then pentobarbital, and perfusion-fixed with 3.0%) glutaral- dehydei 1.0% paraformaldehyde in 0.1M phosphate buffer, pH 7.4, for 10 minutes. After storage in situ at 4°C for 2-9 days, the brains were removed, and the hippocampal forma- tions were dissected and stored in perfusate at 4°C until they were sectioned.

Brain sections from all animals were cut either 30 or 50 pm-thick on a Vibratome. For Nissl staining, 30-pm-thick sections were mounted directly onto chrom alum- and gelatin-coated glass slides. Slides were dehydrated in graded alcohols and xylene, rehydrated, stained with 1.0% cresyl violet, dehydrated, and coverslipped with Permount.

Immunocytochemical methods Sections cut 50 pm thick were washed in 0.1M Tris

buffer, pH 7.6 (Tris; 3 x 5 minutes), and then for 30 minutes in 1.0% hydrogen peroxide in Tris to inactivate endogenous peroxidase. Sections were then washed in Tris for 5 minutes, and in Tris containing 0.005%; bovine serum albumin (Tris-BSA) for 15 minutes. Triton X-100 detergent, which is universally added to the buffer-BSA solution to increase antibody penetration of the section, was found in pilot studies to interfere significantly with GAD and GABA stain sensitivity and quality, and was therefore omitted for GAD and GABA immunocytochemistry. Triton X-100 (0.1%) was included in the Tris-BSA solution only for calbindin and calretinin staining.

Sections intended for mouse monoclonal antibody solutions were first incubated for 1 hour in 10% normal horse serum (to block nonspecific staining as a result of later incubation in horse anti-mouse antiserum). For sections to be incubated in polyclonal rabbit antisera, this lo%> serum step was omitted because biotinylated protein A, which introduces negligible nonspecific staining, was used in place of the secondary antibody.

Sections were washed in Tris for 15 minutes, Tris-BSA for 15 minutes, and then incubated with agitation overnight a t 4°C in mouse monoclonal anti-GAD65 (1:5,000 dilution; clone GAD-6; Chang and Gottlieb, 1988; obtained from Boehringer Mannheim), in polyclonal rabbit anti- GAD67 (1:3-10,000 dilution; K2 antiserum; Kaufman eta]., 1986; obtained from Chemicon), or in one of three different antibodies raised against GABA coupled to BSA with glutaraldehyde (Chemicon rabbit GABA antiserum 131, dilutions: 1:5,000- 1:20,000; Sigma rabbit GABA antiserum A-2052, 1:5,000 dilution; ICN mouse monoclonal

GABA antibody clone 5A9, 1: 15,000 dilution). Monoclonal anti-calbindin antibody (1:500,000 dilution; clone CL-300) was obtained from Sigma. Rabbit anti-calretinin antisera were obtained from SWant (No. 7696; 1:1,000 dilution), Dr. John Rogers (Ab-6C; 1:2,000 dilution), and Dr. David Jacobowitz (1:2,000 dilution).

The next day, rabbit antisera-incubated sections were washed in Tris-BSA for 15 minutes and then incubated with agitation for 1 hour in biotinylated Protein A (diluted 1:400 in Tris-BSA). Monoclonal antibody-incubated sections were incubated with agitation for 1 hour in biotinylated horse anti-mouse IgG ( ~ 4 0 0 dilution in Tris-BSA; Vector Labs Elite Kit). All sections were then washed in 0.5M Tris-BSA for 15 minutes, and incubated with agitation for 1 hour in avidin-biotin-horseradish peroxidase complex (Vector Labs Elite; diluted 1:1,000 in 0.5M Tris- BSA). Sections were then washed in 0.1M Tris buffer (3 x 5 minutes) and placed in diaminobenzidine (DAB) solution (50 mg DAB from Polysciences, 0.3 mg glucose oxidase from Sigma, 40 mg ammonium chloride, and 200 mg B-D- glucose; q.s. to 100 ml with 0.1M Tris buffer, pH 7.6). Sections were washed in 0.1M Tris buffer, mounted, and coverslipped as described above for Nissl-stained sections.

Antibody specificity In addition to the available information on the specificity

and cross-reactivity of the two previously characterized GAD antibodies used in this study (Esclapez et al., 1994), we noted that 0.5 pg purified recombinant GAD67iml diluted GAD67 antiserum completely blocked GAD67 staining. However, 1.0 pg purified GAD67/ml had no effect on GAD65 staining. Conversely, whereas 0.5 pg purified recombinant GAD65iml diluted GAD65 antibody completely blocked GAD65 staining, 1.0 pg GAD65iml had no effect on GAD67 staining. Omission of all primary GAD65, GAD67, GABA, calbindin, and calretinin antibodies prevented specific staining.

Pre-embedding immunocytochemical electron microscopy

Immunocytochemically stained brain sections were rinsed in 0.1M cacodylate buffer, pH 7.4, postfixed in 1.5% potas- sium ferricyanidei l.O%, osmium tetroxide in 0.1 M cacodylate buffer for 90 minutes, rinsed in 0.1 M cacodylate buffer for 10 minutes, rinsed in distilled water for 10 minutes, and stained en bloc in 1%, uranyl acetate for 90 minutes. Sections were dehydrated in ethanol, cleared with propylene oxide, infiltrated in 1:1, then 1:3, propylene oxide: LX112 EPON (Ladd) for 3 hours, before going into 100% LX112 for 3 changes over 2 days. Sections were oriented on Teflon Spray (EMS)-coated glass slides. Previously polymer- ized LX112-filled and inverted Beem capsules were placed over the sections. Polymerization took place in a 60°C oven for 60 hours. Blocks were sectioned on a Reichert Ultracut microtome a t 60-90 nm. Sections from the immunocytochemically stained surface of the block were picked up on parlodion-coated, carbon-shadowed 1 mm x 2 mm slot grids, and examined and photographed in a JEOL lOOSX electron microscope a t an accelerating voltage of 60 kV.

GAD65 and GAD67 mRNA in situ hybridization histochemistry

For in situ hybridization histochemistry, rats were perfused with 2% paraformaldehyde in 0.1M sodium acetate

396 R.S. SLOVITER ET A:

buffer, pH 6.5, for 3 minutes, followed by 15 minutes perfusion with 2% paraformaldehydei0. 1% glutaraldehyde in 0.1M sodium borate buffer, pH 8.5. After brains were stored in situ overnight at 4"C, brains were infiltrated overnight at 4°C with a cryoprotective solution of 0.5M sucrose in phosphate buffered saline (PBS), frozen on dry ice, and sectioned 16-pm-thick on a cryostat. Sections were mounted on Fisher SuperfrostiPlus slides. In later experiments designed to improve histological section integrity, brains were simply removed from the skull and cut on a Vibratome into 30-pm-thick sections in PBS. Sections were mounted directly onto Fisher SuperfrostiPlus slides and air dried. Photographs of GAD mRNA localization are of cryostat sections.

RNA probes for GAD65 and GAD67 were synthesized by in vitro transcription by using cDNA templates containing the entire codingregion of each gene (Erlander et al., 1991). Transcripts were labeled with digoxigenin-11-UTP by using the Genius kit and protocols obtained from Boehringer Mannheim. Tissue sections were pretreated prior to hybridization according to the method of Simmons et al. (1989). Sections were incubated in 0.001% proteinase K for 30 minutes at 37°C in a buffer consisting of 0.1M Tris HC1 (pH 8.0), and 0.05M EDTA. Sections were then acetylated with 0.25% acetic anhydride in 0.1M triethanolamine, rinsed in distilled water, dehydrated in ascending concentrations of ethanol, and incubated overnight a t 55°C in a hybridization solution consisting of 50%. formamide, 5 x saline sodium citrate solution ( Ix SSC: 150 mM NaCl, 60 mM sodium citrate, pH 7.01, 8% dextran sulfate, l x Denhardt's solution, 5 mM EDTA, 0.1% CHAPS, 500 pgiml tRNA, and 2.5 ngi pl RNA probe. Following hybridization, sections were washed in 4~ SSC and incubated in 20 pgiml ribonuclease A at 37°C for 30 minutes. Stringency washes were per- formed with decreasing concentrations of SSC, ending with an incubation at 60°C in 0.1 x SSC for 30 minutes. Sections were then incubated for 1 hour at room temperature in a blocking buffer (0.1M Tris-HC1, pH 7.5, 0.1 M NaCl, 2 mM MgC12, and 3% bovine serum albumin) and then overnight at 4°C in antidigoxigenin antibody conjugated with alkaline phosphatase (Boehringer Mannheim) diluted 1:500 in the blocking buffer described above. Bound antibody was localized by using the chromagens Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (X-phosphate; both from Boehringer Mannheim) diluted according to manufac- turer's instructions in a buffer consisting of 0.1M Tris-HC1 (pH 9.51, 0.1 M NaCl, 50 mM MgC12, 0.1% Tween-20, and 1 mM levamisole. After the colored precipitate had formed ( 1-4 hours), sections were fixed in 4% paraformaldehyde in PBS to stop the reaction. Sections were coverslipped with either 90% glyceroli0.lM Tris HCl or glycerol jelly (Sigma).

Measurement of GAD65, GAD67, GABA, glutamate, aspartate, and glutamine

Paired control and stimulated rats were sacrificed immediately after the end of 24 hours of stimulation. Brains were rapidly removed and immersed in ice-cold Tris buffer (0.1M; pH 7.6). Six consecutive 600-pm-thick coronal sections corresponding approximately to the region between Plates 28 and 42 of the atlas of Paxinos and Watson (1986) were cut and stored in ice-cold buffer. After both brains were cut, hippocampi from each section were dissected under stereo microscopic control into two samples, frozen on dry ice, and weighed.

The dentate gyrus sample was obtained by cutting alor the stratum radiatumistratum lacunosum-moleculare bo der, as shown in Figure 1C. This was done to ensure th, the CA1 sample contained no dentate gyrus tissue. cut was then made along a line defined by the two later tips of the granule cell layer, and then cuts were ma( along the base of the granule cell layer to remove arc CA3c and most of the hilus. Therefore, the dentate gyri sample contained a small amount of tissue from stratu lacunosum-moleculare of CA1, and a small amount of hili tissue. The CA1 sample was obtained by a subsequei cut made perpendicular to the pyramidal cell layer medi to area CA2 (Fig. lC), thereby eliminating the possibilii that the CA1 sample contained granule cell mossy fib1 terminals.

GAD65 and GAD67 were measured by quantitative immi noblotting (Rimvall and Martin, 1994). Bands were stain€ with antiserum W887, which was raised against a peptic corresponding to a sequence shared by GAD65 and GADE (amino acids 570-585 of GAD65 and 578-593 of GAD6' Sheikh and Martin, 19961, and were visualized by enhance chemiluminescence (Amersham). Standard curves pri pared with recombinant GAD67 were run on each ge Films were scanned with a Pharmacia Imagemaster DT densitometer, and the relative amount of each form of GA was determined with Imagemaster software. Amino acic were measured in perchloric acid extracts of tissue samplt following precolumn derivitization by the o-phthalaldehyc method (Spink et al., 1986; Battaglioli and Martin, 1991 Protein was measured by the Coomassie blue metha (Bradford, 1976). Data were analyzed by ANOVA and tl- groups compared by a post-hoc Neuman-Keuls test.

RESULTS Immunocytochemical localization of GAD67

and GAD65 in the hippocampus Although GAD67 has been localized in many brai

regions (Esclapez et al., 19941, its localization in t h hippocampus has not been described. In addition to it expected presence in nonprincipal cells of all hippocamp: subregions, we unexpectedly found intense GAD67-LI i the two axonal projection fields of the dentate granule cell (Fig. 21, which are the hilus of the dentate gyrus, and t h stratum lucidum of area CA3 (Lorente de NO, 1934; Black stad, 1956; Amaral, 1978). Figure 1B shows normal calbir din-LI, which is expressed in dentate granule cells (Slovitei 1989), and illustrates the location of granule cell somat: dendrites, and axons. Note in Figure 2 that GAD67-I exhibits the same pattern of hilar and mossy fiber stainin as shown in Figure 1B for calbindin-11. Similar stainin patterns of GAD67-LI were observed in normal mice (dat not shown), and in the monkey Macaca nemestrina (Fig. virtually identical results were obtained in all 4 monkey evaluated).

In contrast to the presence of GAD67-LI in dentat granule cells and nonprincipal cells of all hippocampz subregions, GAD65,-LI was normally detectable only i nonprincipal cells (Fig. 41, and the pattern of staining wa identical to that described in previous studies that hay localized GAD65 (Wood et al., 1976; Ribak et al., 197t Mugnaini and Oertel, 1985; Babb et al., 1988). GAD65-I was undetectable in normal dentate granule cell somat.;,


Fig. 1. Normal rat hippocampus. ACresyl violet-stained hippocampus showing the principal cell layers, which include the granule cells (G) of the dentate gyrus and the pyramidal cells (PI of the hippocampus proper. B:Calhindin-like immunoreactivity (LIj in the normal rat hippocampus, which is contained throughout the cytoplasm of dentate granule cells. Note the location of calhindin-immunoreactive granule cell dendrites of the dentate molecular layer (ML), the immunoreactive somata of the granule cell layer (G) , the immunoreactive axon collaterals in the hilus (HI, and the calhindin-immunoreactive mossy fiber axons in stratum lucidum of area CA3 (arrows). C:Diagram of the two microdissected regions (shaded areas) used for neurochemical analysis. The dentate gyrus sample included the granule cell layer (GCI, the dentate molecular layer (ML), and the area CA1 stratum lacunosum- moleculare (SLM), which was included to ensure that the CA1 sample contained no dentate gyrus tissue (see Materials and Methods section). Magnification: x26. Scale bar = 0.5 mm.

and was notably minimal in the stratum lucidum of area CA3 compared to other strata (Fig. 4).

Adsorption of GAD67 antiserum with purified recombinant GAD67, but not GAD65, prevented specific staining for GAD67-LI (see Materials and Methods). Adsorption of GAD65 antiserum with purified recombinant GAD65, but not GAD67, prevented specific staining for GAD65-LI. GAD65 staining was not obtained in monkey hippocampal sections because the 3% glutaraldehyde used for perfusion- fixation eliminated GAD65 immunoreactivity.

Immunocytochemical localization of GABA in the hippocampus

The paradoxical finding that excitatory, glutamatergic dentate granule cells are normally GAD67-immunoreactive led us to reexamine the localization of GABA in the normal rat, mouse, and monkey hippocampus. By utilizing optimal fixation methods, and by eliminating the detergent com- monly used to facilitate antibody penetration into the section, we were able to detect specific staining for GABA-LI at greater intensity than previously obtained (Sloviter and Nilaver, 1987). Without detergent, GABA-LI was detectable in the dendrites, somata, and axons of granule cells in brain sections from normal rats (n = 36; Fig. 51, mice (n = 3; data not shown), and the monkey Macaca nernestrina (n = 4; Fig. 6). Identical immunocytochemical results were obtained with three different anti-GABA antibodies (two polyclonal and one monoclonal; see Methods). Granule cell GABA-LI was consistently more intense in hippocampal sections from all 4 monkeys examined (Fig. 6) than in sections from rats (Fig. 5) fixed and processed by identical methods.

In contrast to its presence in granule cells and scattered nonprincipal cells of all subregions, GABA-LI was undetectable in the majority of hilar neurons (Fig. 6B), or in pyramidal cells (Figs. 5 and 6). Figure 7 identifies the main population of monkey dentate hilar neurons that appear devoid of GABA immunoreactivity. In the course of staining monkey sections with a variety of antisera, we observed that all four monkeys exhibited calretinin-11 in the population of hilar neurons (presumed mossy cells) that apparently form the ipsilateral associational/commissural input to the inner molecular layer (Amaral, 1978; Ribak et al., 1985; Soriano and Frotscher, 1994). These large neurons have been shown to be glutamate-immunoreactive in the rat (Soriano and Frotscher, 1994). Figure 7C shows the location of the calretinin-positive axon plexus in the inner molecular layer, which presumably originates in the calretinin-positive hilar neurons. All three calretinin antisera produced similar results. Figure 7F shows in an adjacent section of the same hippocampus that these cells, as well as the adjoining CA3 pyramidal cells, are GABA-negative. Thus, as in the rat (Sloviter and Nilaver, 1987), the vast majority of monkey hilar neuron somata appear devoid of GABA-LI despite the increased sensitivity of the modified method. The presence of calretinin-LI in presumed hilar mossy cells of the monkey Macaca nernestrina, which is a pattern of staining that apparently does not occur in all monkeys (Seress et al., 1993), apparently reveals the extent of the hilus, and may therefore serve as a valuable demarca- tion line between the dentate hilus and the hippocampal CA3 pyramidal cell layer.

598 R.S. SLOVITER ET AL.

Fig. 2. Glutamate decarboqdase 67-like immunoreactivity (GAD67- LI ) in the normal rat hippocampus. AWhole hippocampusidentate gyrus showing GAD67-immunoreactive nonprincipal cells in all strata, and intense GAD67-LI in the region of the granule cell hilar (H) axon collaterals, and in the mossy fiber pathway (arrows). B:Higher magnifi- ration o f the dorsal eranule cell laver (G). hilus (H). and the Dvramidal

cells, as well as GAD67-immunoreactive nonprincipal cell somata in all subregions. C:GAD67-LI in area CA3a of the pyramidal cell layer. Note the GAD67-immunoreactive plexus surroundingimmunonegative pyra. midal cell somata (asterisks), and the GAD67-positive mossy fibei pathway, which innervates the proximal apical pyramidal cell dendrite: in stratum lucidum (SL). Marmification: x50 in A: x260 in B and (!


Fig. 3. GAD67-LI in the hippocampus of the monkey Macaca nemestrina. A.Cresy1 violet-stained hippocampus showing the granule cell layer (G) , the hilus of the dentate gyms (H), and the pyramidal cell layer ( P ) of the hippocampus proper. B:Calbindin-LI, showing the immunoreactive granule cells, their axon collaterals in the hilus, and

the mossy fiber pathway (arrows) that innervates the CA3 pyramidal cells. C:GAD67-LI. Note its presence in the granule cell axon plexus of the hilus (H), and in the mossy fiber pathway (arrows). Magnifications: x 14 in A and B; x28 in C. Scale bar = 1.0 mm in A and B, 0.5 mm in C.

GAD%-, GAD67-, and GABA-LI after prolonged perforant path stimulation

Because normal granule cells exhibited intense GAD67- and GABA-LI, we determined whether synaptic excitation

alters GAD and GABA expression in granule cells or induces it in any hippocampal neuron populations that do not normally express GAD and GABA. Perforant path stimulation reliably evoked population spikes and epileptiform discharges in the granule cell, CA3, and CA1 pyrami-


Fig 4. GAD65-LI in the normal rat hippocampus. AWhole hippo- presumed fibers that surround immunonegative principal cells. C: campus/dentate gyrus showing GAD65-immunoreactive nonprincipal GAD65-LI in the CA3a pyramidal cell layer. Note the GAD65- cells in all strata, and clearly absent immunoreactivity in the region of immunoreactive plexus surrounding immunonegative pyramidal cell the mossy fiber pathway (axons of the granule cells; arrows). B:Higher somata (asterisks), and the absence of GAD65-LI in stratum lucidum magnification of the dorsal granule cell layer (GI, hilus (HI, and CA3c (SL). Magnification: x50 in A; ~ 2 6 0 in Band C Scale bar = 0.5 mm in pyramidal cell layer (PI. Note that GAD65-LI in the cell layers is in A, 50 km in Band C.

Fig. 5. y-Aminobutyric acid-like immunoreactivity (GABA-LI) in the normal rat hippocampus. A:Whole hippocampusidentate gyrus showing GABA-immunoreactive nonprincipal cells in all strata, as well as the GABA-immunoreactive dentate granule cells (GI. Note intense GABA-LI in the granule cell dendritic region in the molecular layer (MLI, in the granule cell soma1 layer (GI, in the granule cell axon collateral projection to the hilus (HI, and in the mossy fiber pathway (arrowsi. B:Higher magnification of the dorsal granule cell layer (G) , hilus (HI, and the CA3c pyramidal cell layer (PI. Note GABA- immunoreactive granule cells and nonprincipal cells of the granule cell

layer, as well as GABA-positive (arrows) and GABA-negative (arrowheads) hilar neurons (HI. C:GABA-LI in the CA3a region. Note the GABA-immunoreactive plexus surrounding immunonegative pyramidal cell somata (asterisks), and the GABA-positive mossy fiber pathway in s t ra tum lucidum (SL). Note also that the apparent GABA-LI visible in some CA3 pyramidal cells in C is immunoreactivity on the other surface of the section and not in the focal plane. Conversely, granule cell GABA-LI in R is on the upper surface, within the focal plane. Magnification: x50 in A; x260 in Band C. Scale bar = 0.5 mm in A, 50 W r n in B and C.


Fig. 6. GABA-LI in the hippocampus of the monkey Macaca adjacent GABA-negative hilar neurons (arrowheads). Relatively few hilar neurons are GABA-immunoreactive despite improved methods that have increased immunocytochemical sensitivity. C:GABA-LI in area CA3a. Note the GABA-immunoreactive mossy fiber pathway of stratum lucidum (SL), which innervates the proximal dendrites of CA3 pyramidal cells (arrowheads). Magnification: x28 in A; x 130 in B and C. Scale bar = 0.5 mm in A, 100 Fm in Band C.

nemestrina. AWhole hippocampusidentate gyrus showing GABA- immunoreactive nonprincipal cells in all strata, as well as the GABA- immunoreactive dentate granule cells. Note intense GABA-LI in the dentate molecular layer (ML), the granule cell soma1 layer (G), in the granule cell axon collateral projection to the hilus (H), and in the mossy fiber pathway (mf; arrows). B:Higher magnification of the granule cell layer (GI and hilus (H). Note GABA-immunoreactive granule cells and


Fig. 7. Calretinin- and GAEiA-LI in the monkey hippocampus. A: Cresyl violet-stained hippocampus showing the dentate granule cells t G), dentate hilar neurons (HI, and the hippocampal pyramidal cells (P). B:The granule cell layer and hilus at higher magnification. C,D: Calretinin-LI. Note that calretinin-11 is present in a population of hilar neurons and in a hand of presumed axon terminals in the inner molecular layer (ML; arrows in C). If the calretinin-immunoreactive terminal staining in the molecular layer originates in the calretinin- positive hilar neurons, then calretinin immunostaining may be a marker in this species of the dentate hilar “mossy” cells (Amaral, 1978 1, which form the ipsilateral associationalicommissural projection to the inner dentate molecular layer (Rihak et al., 1985). E,F:GABA-LI

in an adjacent section showing that the calretinin-immunoreactive hilar neurons (shown in D) are devoid of detectable GABA-LI (arrowheads in F), whereas the adjacent granule cells ( G ) are intensely GABA-immunoreactive. That is, neither the calretinin-positive hilar somata, nor the calretinin-positive terminal field in the inner molecular layer, are GABA-immunoreactive in a section in which intense GABA immunoreactivity is apparent. Thus, GABA-LI is present only in dentate granule cells and some nonprincipal cells of all subregions; the vast majority of hilar neurons and all pyramidal cells appear devoid of GABA-LI. Magnification: ~ 2 6 , left column; x 130, right column. Scale bar=0.5mminA,C,andE, 1001~-minB,D,andF.

dal cell layers with latencies of approximately 6.0, 10.5, and 17.0 msec, respectively (Fig. 8). As shown previously, unilateral perforant path stimulation evokes population spikes and epileptiform discharges bilaterally (Sloviter,

1983), and primarily damages the ipsilateral hippocampus (Sloviter, 1983, 1991). Therefore, this study focuses on the seizure-associated induction of GAD and GABA on the contralateral side, and avoids the separate issue of seizure-

604

induced cell death, which is beyond the scope of this paper. Note that all photographs of brain sections from stimulated animals are of the side contralateral to stimulation. How- ever, all changes in GAD and GABA staining produced by unilateral perforant path stimulation occurred bilaterally.

Stimulation for 4,6, or 24 hours produced a highly reproducible induction of GAD67-LI throughout the somata and dendrites of dentate granule cells, and increased GABA-LI in granule cell somata, dendrites, and axons in all rats examined immediately after the end of stimulation (n = 6 at 4 hours, n = 10 at 6 hours, and n = 18 at 24 hours). The selective induction of GAD67- and GABA-LI in granule cells by afferent excitation is most clearly demonstrated by incubating sections in dilute GAD and GABA antisera, which produces faint staining in control sections and intense staining in identically co- processed sections from stimulated animals (Fig. 9). Whereas granule cells of stimulated rats exhibited intense GABA-LI, the adjacent hilar neurons remained GABA-immunonegative (Fig. 9H), indicating that the induction of GAD67- and GABA-LI is highly cell-specific. Stimulation for 1 or 2 hours induced granule cell somal GAD67 in rats examined 6 hours after the start of stimulation, but not in rats examined immediately after the end of 1 or 2 hours of stimulation ( n = 3 each; data not shown).

The induction of GAD67- and GABA-LI was produced throughout the dorsal (septal) dentate gyrus (Fig. 101, which matches the segment of the hippocampus that is excited and ultimately damaged by prolonged stimulation of the angular bundle of the perforant pathway (compare Fig. 10 with Fig. 4 of Sloviter, 1983). Figure 10 shows both the rostra1 (GAD67- and GABA-induced) and temporal (uninduced) granule cells in coronal sections that contain both segments of the dentate gyrus. This result circumvents the inherent interpretative problem of comparing stain intensity in experimental and control animals, which always exhibit some differences in fixation and stain quality. That is, different areas within the same brain section showed clear differences in 0 6 7 - and GABA-LI (Fig. 10) that cannot be due to inter-animal variability.

The induction of granule cell GAD67- and GABA-LI occurred bilaterally in all cases. Immediately after 4 or 6 hours of stimulation, GAD67- and GABA-LI were more intense on the stimulated side. After 24 hours of stimulation, GAD67- and GABA-LI were further increased in intensity and similar bilaterally. Granule cell mossy fiber GAD67- and GABA-LI remained obviously increased above control stain intensity 1, 2, and 4 days after the end of 24 hours of stimulation, but appeared not obviously distin- guishable from controls 7 days after stimulation (n = 2 each). However, because GAD67- and GABA-LI are normally present in the mossy fiber pathway, it is difficult to identify the exact poststimulation period when the intensity of GAD67-LI first appears to have returned to normal. Therefore, these qualitative descriptions are conservative approximations. The reasonable conclusion to be drawn is that granule cell GAD67- and GABA-LI remain elevated for several days after stimulation, and possibly longer.

GAIl65-LI Although granule cells exhibited no obviously detectable GAD65-LI normally or immediately after 1-6 hours of perforant path stimulation, rats stimulated for 24 hours exhibited clear evidence of granule cell somal GAD65-LI immediately after stimulation (Fig. 11). Unlike GAD67-LI, however, not all granule cell somata exhibited

GAD67- and GABA-LI.

A

1

2

3

B

1

2

3

C

1

2

3

R.S. SLOVITER ET AL

Granule cell layer

CA1 pyramidal cell layer

b* -------.-,.--~+-

CA3 pyramidal cell layer J L ~ ~ - - - - . - ~ , - - . * , - .

Fig. 8. Evoked responses of dentate granule cells and hippocampal pyramidal cells to focal stimulation of the perforant pathway in rats. A: Responses recorded in the granule cell layer. 1: Responses to paired pulses delivered 40 msec apart at 2 Hz ( 4 x expanded time base a t right before starting a 20-Hz train. 2: Granule cell population spikes evoked during a 10-seci20-Hz stimulus train. 3: Epileptiform response to 2 Hi. stimulus pair 3 seconds after the end of the stimulus train. B,C:Similai responses in the CAI and CA3 pyramidal cell layers to perforant path stimulus trains. Note that perforant path stimulation evokes popula tion spikes and epileptiform discharges in all three principal cell populations. Calibration bars = 16 mV in A, 4.4 mV in Band C, 50 msec in all panels.

unequivocal GAD65-LI. In addition, GAD65-LI was alway>. less intense than GAD67-LI in adjacent sections. Althougt GAD65-LI was absent from the somata 1, 2, 4 and 7 days after the end of stimulation (n = 2 at each point), GAD65-L,

Fig. 9. Dilute antiserum immunocytochemistry for GAD67-and GABA-LI after perforant path stimulation. Urethane-anesthetized rats were perfusion-fixed immediately after 24 hours of no stimulation (control; left column) or intermittent stimulation (experimental; right column) that evoked population spikes and epileptiform discharges in dentate granule cells, and in CA1 and CA3 pyramidal cells (evoked potentials shown in Fig. 8). Brain sections were incubated in dilute antisera to illustrate the differences between control and experimental GAD67- and GABA-LI. A,C:GAD67-LI in the control hippocampus and dentate gyrus, respectively. Note that at this high antiserum dilution (1:10,000), only faint immunoreactivity is evident in the control mossy fiber pathway (arrows in A); intense GAD67-LI is evident in control tissue only in dentate nongranule cells (arrows in C). B,D:After stimulation, granule cell somata and dendrites are intensely GAD67-

immunoreactive. E,G:GABA-LI in the control hippocampus and dentate gyrus, respectively. Note that at this high antiserum dilution (1:20,000), intense immunoreactivity is apparent only in presumed dentate nonprincipal cells (arrows in G); only faint GABA-LI is evident in the mossy fiber pathway (arrows in El. At this dilution, granule cell GABA-LI is undetectable. F,H:After stimulation, the same diluted antiserum yields intense GABA-LI in granule cell dendrites, somata, and axon terminals. Note that despite sustained excitation and induction of intense granule cell GABA-LI, hippocampal pyramidal neurons and hilar neurons (arrowheads in H) remain GABA-immunonegative. Note also that control and experimental animals were produced in parallel, and that sections from both brains were co-processed identically. Magnification: x26 in A,B,E, and F; ~ 2 6 0 in C,D,G, and H. Scale bar = 0.5 mm in A,B,E, and F, 50 pm in C,D,G, and H.

606 R.S. SLOVITER ET AL

Fig. 10. Septotemporal differences in induction of GAD67- and GABA-LI by 24 hours of perforant path stimulation. AGAD67-LI in a sham control hippocampal section incubated in dilute antiserum that was used to highlight the differences between control and stimulated animals. B:GAD67-LI immediately after 24 hours of perforant path stimulation. Note that induced GAD67-LI is greater in the rostra1 (septa11 portion of the hippocampus that is activated by stimulation of the angular bundle of the perforant path, whereas the ventral (temporal) hippocampus (arrows in A-C point to the ventral granule cell layer)

exhibits relatively normal stain intensity. C:GABA-LI in the same stimulated rat showing a similar location of induced GABA-LI. The selective induction of GAD67- and GABA-LI in the septal, but not temporal, portions of the same hippocampal section circumvents interpretative problems inherent in comparisons between control and experimental animals, which always exhibit some inter-animal variability in fixation and stain character. Magnification:x24. Scale bar = 1.0 mm.

was detectable in the normally GAD65-immunonegative probably the result of induction of GAD mRNA and GAD in granule cell axon terminal fields in the hilus and stratum normally GAD-positive nonprincipal cells (Feldblum et al., lucidum of area CA3 (Fig. 11E) for at least 4 days after the 19901, changes in immunocytochemical stain intensity end of 24 hours of stimulation. Thus, 24 hours of stimula- could also be the result of artifactual, stimulation-induced tion briefly evoked the appearance of somal GAD65-LI, changes that facilitate antibody penetration or binding. which presumablv became axon terminal GAD65-LI that persisted for days:

Although epileptiform discharges were also evoked in CA3 and CA1 pyramidal cells (Fig. 8) , pyramidal cells were devoid of detectable GAD65-, GAD67-, or GABA-LI at all time points evaluated (0, 3 hours, 1, 2, 4, and 7 days after the end of 24 hours of stimulation; n 2 2 a t each time point). Hippocampal nonprincipal cells, which are normally GAD- and GABA-immunoreactive, frequently appeared more darkly stained in sections from experimental rats than in sections from co-processed control animals. Al- though the increased stain intensity after stimulation is

Effect of cycloheximide pretreatment on GAD67 and GABA induction

Pretreatment of rats with the protein synthesis inhibitor cycloheximide prevented the induction of granule cell somal GAD67-LI in both hippocampi of rats stimulated for 4 hours. The similarity of GABA-LI in sections of each cycloheximide- or vehicle treated animal ( n = 4 each) indicated that despite preventing somal GAD67 induction, cycloheximide did not prevent induction of granule cell somal GABA-LI (data not shown). Thus, stimulation in the


Fig. 11. GAD65-LI in the control and 24-hour perforant path- stimulated rat hippocampus. A,B:GAD65-LI in the whole hippocampus and the dorsal granule cell layer (G) and hilus (HI, respectively, of a sham control rat. C,D:Induction of dentate granule cell GAD65-LI immediately after 24 hours of perforant path stimulation. Note that whereas control granule cells (in B) are devoid of GAD65-LI, granule cells of the stimulated rat (shown in D) exhibit perinuclear cytoplasmic GAD65-LI (arrows). Note also that incubation in chromagen was minimized to illustrate the difference between the control and experimental tissues. More intense control GAD65-LI is shown in Figure 4.

absence of cycloheximide presumably increased granule cell GABA via both pre-existing and newly synthesized GAD67. An attempt was made to determine the effect of cycloheximide on GAD- and GABA-LI after 24 hours of stimulation, but rats did not survive for 24 hours after cvcloheximide

E,F:Two days after the end of 24 hours of perforant path stimulation. Note in F that although dentate granule cell somata appear devoid of GAD65-LI a t this time point, GAD65-LI is now apparent in the hilar granule cell axon collaterals (H) and mossy fiber pathway (arrows in El. Thus, stimulation induces soma1 granule cell GAD65-LI, which quickly disappears from the soma, and apparently reappears and persists for days, in granule cell axon terminals. Magnifications: x26 in A,C, and E; x260 in B,D, and F. Scale bar = 0.5 mm in A,C, and E, 50 km in B,D, and F.

Immunocytochemical electron microscopy of GAD67- and GABA-LI

Pre-embedding immunocytochemical electron microscopy addressed the cellular specificity and intracellular

administration. distribution of GAD- and GABA-LI after stimulation.


GAD67-LI induced by 24 hours of perforant path stimulation was highly neuron-specific in that GAD67-LI was present within granule cell layer neurons, but not in surrounding glial or capillary endothelial cells (Fig. 12A). Within immunoreactive granule cell layer somata, the electron-dense reaction product was distributed throughout the cytoplasm. However, mitochondrial cristae and the nucleus were relatively immunonegative (Figs. 12, 13). Like GAD67-LI, GABA-LI was present in granule cells, but not in adjacent glial or endothelial cells (Fig. 12B). However, in contrast to the absence of nuclear GAD67-LI, GABA-LI was present throughout the granule cell cytoplasm and nucleoplasm (Figs. 12B, 14). The same non-nuclear distribution of GAD-LI, and the cytoplasmic and nuclear distribution of GABA-LI have been noted in studies of normal nonprincipal cells (Ribak et al., 1978; Oertel et al., 1981; Storm- Mathisen and Ottersen, 1983). GAD65-LI induced in granule cells by 24 hours of perforant path stimulation was not sufficiently intense to detect unambiguously at the electron microscopic level.

Mossy fiber terminals in synaptic contact with apical dendrites of CA3 pyramidal cells exhibited GABA-LI on synaptic vesicles and the outer mitochondrial membrane; mitochondrial cristae were relatively immunonegative (Fig. 14). However, the presence of antibody-bound reaction product on synaptic vesicles does not indicate that mossy fiber GABA is vesicle-bound in vivo. That is, vesicle membrane-bound GABA-LI may have originated from free cytoplasmic GABA that was covalently attached to all subcellular organelles by glutaraldehyde during the fixation process.

These ultrastructural data indicate only that the GAD- and GABA-immunoreactivities that appear at the light microscopic level to be within granule cells are, in fact, within granule cells and not within glial and endothelial cells. Whether the subcellular location of immunocytochemically deposited chromagen is an accurate indicator of the normal location of the molecules being localized is doubtful. More accurate localization of granule cell GAD and GABA will require postembedding immunocytochemical analysis.

G A D 6 and GAD67 in situ hybridization after afferent stimulation

Figure 15A and C show the normal locations of GAD65 and GAD67 mRNA-containing neurons of the dentate gyrus. Both mRNAs are present in dentate nonprincipal cells of all strata, and their distributions were identical to that described in detail in previously published reports (Feldblum et al., 1993; Houser and Esclapez, 1994). The results of localizing GAD mRNA in stimulated rat hippocampi were consistent with the immunocytochemical findings of induced GAD65- and GAD67-LI. Perforant path stimulation for 1 hour, with sacrifice 6 hours after the start of stimulation, evoked no obviously detectable expression of GAD65 or GAD67 mRNA (data not shown). Stimulation for 2 hours, with sacrifice 6 hours after the start of stimulation evoked GAD67 mRNA, but no detectable GAD65 mRNA, in dentate granule cells. Stimulation for 6 hours with immediate sacrifice evoked intense GAD67 mRNA expression in granule cells, but little or no detectable GAD65 mRNA expression. Stimulation for 24 hours evoked a further increase in GAD67 mRNA expression (Fig. 15D), and also evoked GAD65 mRNA a t this time point (Fig. 15B).

Hybridization staining for granule cell GAD67 mRNA 1 and 2 days after the end of 24 hours of stimulation was

decreased from the maximum observed immediately after stimulation (Fig. E D ) , but was still clearly elevated above controls, which exhibited no detectable GAD67 mRNA (Fig. 15C). Conversely, granule cell GAD65 mRNA, which was induced immediately after 24 hours of stimulation, was undetectable 1 or 2 days after the end of 24 hours of stimulation. GAD65 and GAD67 mRNA were not induced in any other normally GAD mRNA-negative cell populations (most dentate hilar neurons and hippocampal pyramidal cells). Nonprincipal cells in sections from experimental animals frequently exhibited darker staining for both GAD mRNAs compared to co-processed control sections. Al- though this light microscopic method is inherently nonquan- titative, it is likely that in addition to inducing GAD mRNA expression in dentate granule cells, prolonged perforant path stimulation increased nonprincipal cell GAD mRNAs, as has been reported in kainate-treated rats (Feldblum et al., 1990).

Measurement of GAD65, GAD67, GABA, glutamate, and glutamine concentration

To determine whether changes in GAD- and GABA-LI reflect changes in actual GABA and GAD concentration, we microdissected and froze samples of the dentate gyrus and hippocampal area CA1 for subsequent neurochemical analysis. For each sample, we measured GAD65 and GAD67 by quantitative immunoblotting, and also determined the concentrations of GABA and aspartate, as well as glutamate and glutamine, which are the main metabolic precursors of GABA (Reubi et al., 1978; Paulsen et al., 1988; Battaglioli and Martin, 1991). Stimulation for 24 hours increased dentate gyrus GAD67 concentration sixfold, but had no significant effect on GAD65 concentration (Fig. 16) despite an apparent induction of granule cell soma1 GAD65-LI (Fig. 11). The newly expressed GAD67 comigrated with full- length GAD67 (Fig. 16), and thus does not correspond to either of the two truncated embryonic forms of the enzyme (Bond et al., 1990; Szabo et al., 1994). GABA was similarly increased in the dentate gyrus by afferent stimulation, and we observed a significantly higher concentration of GABA in the left dentate gyrus (stimulated side) than in the right (contralateral side; Fig. 17; P < 0.0002). In contrast to the increase in GABA levels, 24 hours of stimulation reduced the concentrations of glutamate (by 24%; P < 0.01) and glutamine (by 34% ; P < 0.05) in the combined left and right dentate gyri samples. The concentration of glutamate was sinificantly lower in the left (stimulated side) than the right (contralateral) dentate gyrus. The concentration of aspartate was also significantly lower in the dentate gyrus of stimulated animals.

Despite the induction of epileptiform discharges in CA1 pyramidal cells, 24 hours of stimulation did not significantly affect the concentrations of GAD65, GAD67, GABA, glutamate, aspartate, or glutamine in the samples of area CA1 (Fig. 17). The failure to observe significant changes in area CA1 may be attributable to the small number of samples analyzed (n = 31, as the apparent pattern of changes in the amino acids in area CA1 was the same as in the dentate gyrus. However, there was no hint in the data that the content of GAD65 or GAD67 in CA1 was affected by stimulation (Fig. 161, and the change in GABA (Fig. 17, upper panels), if it had been significant, would have been much smaller than the increase in GABA in the dentate gyrus (Fig. 17, upper panels).

Fig. 12. Ultrastructural localization of GAD67- and GABA-LI imme- throughout the cytoplasm and nucleoplasm of granule cell layer neurons, which surround an immunonegative granule cell layer neuron (asterisk). Whether this immunonegative cell is an uninduced granule cell or a nongranule cell is undetermined. This figure shows that: 1) GAD67- and GABA reaction products are within granule cell layer neurons, and 21 GAD67- and GABA induction is highly cell specific for granule cell layer neurons. Magnification: ~4 ,000 . Scale bar = 5 km.

diately after the end of 24 hours of perforant path stimulation. A GAD67-LI is present in the cytoplasm, but not nuclei, of granule cell layer neurons (GI. Note that an adjacent glial cell (asterisk) and a capillary endothelial cell (arrow) are GAD-immunonegative, and that an adjacent presumed interneuron (Int) appears immunoreactive (shown at higher magnification in Fig. 131, but exhibits less cytoplasmic reaction product than granule cell layer cells (GI. B:GABA-LI is present

Fig. 13. GAD67-LI in the dentate gyrus after 24 hours of perforant path stimulation. A,B:Higher magnification photographs of the scene shown in Figure 12A. A: GAD67-LI in presumed granule cells (G) is restricted to cytoplasmic organelles. Note that polysomes and the external mitochondria1 membrane contain reaction product, whereas glial cell (asterisk) processes and myelinated axons are immunonegative. B: A GAD67-immunoreactive, presumed interneuron (Int) lying a t the base of the granule cell layer ( the same cell is shown a t lower

magnification in a serial section in Fig. 12A). Note that this neuron, which exhibits an infolded nucleus (Fig. 12A) characteristic of dentate nongranule cells (Ribak and Anderson, 1980), appears less GAD67- immunoreactive than the adjacent granule cells shown above in A. Note that. detectable GAD67-LI in this presumed interneuron is restricted mainly to the polysomes. Magnifications: x 7,500 in A; x 19,000 in B. Scale bar = 2.0 pm in A, 1.0 pm in B.

Fig. 14. GABA-LI in granule cell dendrites and axon terminals after 24 hours of' perforant path stimulation. A:The dentate inner molecular layer. Note that GABA-LI is mainly restricted to dendrites, and that synaptic terminals, myelinated axons, and a capillary endothelial cell iasteriski are GABA-negative. B:Stratum lucidum of area CA3a showing (:ABA-immunoreactive mossy fiber terminals (asterisks) surrounding a (;AHA-negative dendrite ID). Note tha t GABA immunoreaction

product appears to stain the vesicular membrane and the outer mitochondria1 membrane (arrow). An unstained mossy fiber terminal (mf t ) is shown at right bottom (mft ) for comparison. I t is unclear whether unstained terminals are devoid of GABA-LI, o r are simply beneath the stained surface of'the tissue block. Mapifications: x6.375 in A; x 2,750 in B. Scalc bars = 2.5 pn in A and B.


Fig. 15. GAD65 and GAD67 mRNA localization in the dentate gyrus after 24 hours of perforant path stimulation. A,C:GAD65 and GAD67 mRNA-containing neurons in a sham control animal. Note the presence of GAD mRNA in dentate nonprincipal cells, and their absence in the granule cell layer (G). B,D.Immediately after 24 hours of stimulation.

Note the appearance of GAD mRNA in the granule cell layer. GAD67 mRNA staining was always darker than GAD65 mRNA staining in sections co-processed by identical methods. Magnification: x 26. Scale bar = 0.5 mm.

DISCUSSION This study has yielded two findings of potential signifi-

cance for our understanding of hippocampal network function and dysfunction. First, the results indicate that the excitatory, glutamatergc dentate granule cells of rats, mice, and the monkey Macaca nemestrina are intensely immunoreactive for GAD67 and GABA in the normal state. Although evidence for functional GABA release from granule cells is lacking, this finding indicates that dentate granule cells normally contain two “fast-acting’’ amino acid neurotransmitters, one excitatory and one inhibitory, and may therefore produce both excitatory and inhibitory postsynaptic effects.

Second, our results show that afferent stimulation that induces population spikes and epileptiform discharges in the three hippocampal principal cell populations induces detectable GAD65 and GAD67 mRNA and protein, as well as GABA, only in the granule cells. These results imply that the spontaneous seizures of human temporal lobe epilepsy may induce granule cell GAD and GABA synthesis above basal levels, possibly influencing the excitability of the hippocampal network in epilepsy. Whether this hypoth- esized seizure-induced increase in granule cell GABA influences subsequent spontaneous clinical seizure occurrence, duration, and frequency, is a target of anticonwlsant drug

action, or plays a neurotrophic role (Hansen et al., 1984 Cherubini et al., 1991; LoTurco et al., 19951, remains to bt determined. However, the fact that the dentate granules cells of human hippocampi removed in the treatment 0’. temporal lobe epilepsy are 0 6 7 - and GABA-immunoreac . tive (R.S. Sloviter, R.R. Goodman, and T.A. Pedley, unpub- lished observation) suggests that extrapolation to the hu. man is plausible.

Relation of the results to previous findings The unexpected finding that excitatory hippocampal gran-

ule cells are intensely GAD67- and GABA-immunoreactive clarifies the existing literature on this subject. In terms rf GAD67-LI, no previous studies have reported GAD67 localization in the normal hippocampus. However, Schwarzer and Sperk (1995) recently reported the appearance cff GAD67 mRNA and protein immunoreactivity in granule cells after kainate-induced seizures, although they did not detect GAD67-LI in normal animals. Our ability to detect intense GAD67-LI in normal granule cells is apparently due simply to greater methodological sensitivity.

With regard to GABA in granule cells, most of the marg studies that have localized hippocampal GABA-LI ha\re found none in the granule cells or the mossy fiber pathway (Storm-Mathisen et al., 1983; Seguela et al., 1984; Somogyr


DG GAD67 -- A GAD65 /

L R L R C ST

0 L Q 2l

0

GAD67

C ST C ST DG CAI

Fig. 16. Measurements of GAD concentrations in dentate gyms (DGI and CA1 subregions of the hippocampus by quantitative immunoblotting. A: Immunoblots of GAD from the left (L; stimulated side) and right (R; unstimulated side) from one control (C) and one stimulated (STI rat killed immediately after 24 hours of stimulation or no

et al., 1985; Anderson et al., 1986; Gamrani et al., 1986; Sloviter and Nilaver, 1987; Soriano and Frotscher, 1993, 1994). However, a few studies have noted detectable GABA-LI in the stratum lucidum at the light microscopic level (Ottersen and Storm-Mathisen, 1984; Woodson et al., 1989), or in individual mossy fiber terminals a t the ultrastructural level (Sandler and Smith, 1991).

Ottersen and Storm-Mathisen (1984) were the first to describe GABA-LI in stratum lucidum. They concluded that the GABA-LI they detected was within the mossy fiber pathway, and noted that the intensity of GABA-LI in presumed mossy fiber boutons was less than in other GABA-immunoreactive puncta. Ben-Ari and colleagues sub- sequently provided clearer evidence of GABA-LI in the granule cell axon projection zones in the hilus and stratum lucidum (Woodson et al., 1989), but rejected the suggestion of Ottersen and Storm-Mathisen (1984) that this GABA-LI was within granule cell axons. Ben-Ari and colleagues concluded that the GABA-LI that appeared to be in granule cell axons was, in fact, within a dense axonal network originating in interneurons or extrinsic GABA neurons, not granule cells (Woodson et al., 1989). Finally, using postembedding GABA immunocytochemical electron microscopy, Sandler and Smith (1991) reported binding of gold-labelled GABA antibody to individual monkey and human mossy fiber terminals. These few reports of GABA-LI in the axon terminals of excitatory cells “known” to lack GAD, when

I

CAI

L R L R C ST

4

0 2:

T GAD65 -

C ST DG

C ST CAI

stimulation. B: GAD67 was elevated 6.7-fold in the dentate mrus samples of rats stimulated for 24 hours ( P < 0.001), hut was unchanged in area CA1. GAD65 was unaffected in both regions. Each bar repre- sents the mean values 2 SEM obtained by densitometric analysis of blots from 3 control and 3 experimental animals.

viewed in the context of the far more numerous reports of hippocampal GABA localization that did not detect GABA-LI in the mossy fibers, have caused the few reports of mossy fiber GABA-LI to be either ignored or reasonably assumed to reflect uptake of extracellular GABA by mossy fiber terminals (Houser and Esclapez, 1994). The present results with GAD67- and GABA-LI confirm that the observations of Ottersen and Storm-Mathisen (1984), and of Sandler and Smith (1991), were correct; GABA-LI is present within normal dentate granule cells.

GAD protein versus GAD mRNA as markers of inhibitory neurons

The observation that dentate granule cells normally contain both GAD67- and GABA-LI, but little or no GAD67 mRNA (Feldblum et al., 1993; Houser and Esclapez, 1994), raises questions about GAD mRNA visualization as a defining criterion for the identification of GABAergic neurons. Recent studies employing in situ hybridization methods have indicated that only hippocampal nonprincipal cells exhibit specific staining for GAD67 and GAD65 mRNA (Feldblum et al., 1993; Houser and Esclapez, 1994). How- ever, Houser and Esclapez (1994) and Schwarzer and Sperk (1995) noted recently that although they judged dentate granule cells to be devoid of specific staining for either GAD65 or GAD67 mRNA, faint labeling of granule cells for


200

150 C al 100

Q 50

.- c

2

1

Dentate Gyrus

I Right Len

* Sham vsStim t Left vs Riqht

Sham Stimulated Sham Stimulated

Area CAI ::E 10

5

0

6o F GLN

Sham Stimulated Sham Stimulated

Fig. 17. Amino acid concentrations in microdissected dentate gyms and CA1 subregions of the hippocampus. In the dentate gyrus (DG), stimulation produced a 6-fold elevation of GABA in the left ( P < 0.0002) and 3.7-fold elevation on the right side ( P < 0.0005) when cornpared to DG from sham animals, and there was a significant left-right difference in the stimulated animals ( P < 0.0002). Glutamate was significantly lower than sham control in the left ( P < 0.0005) and right DG tP < 0.051, and there was a significant left-right difference iP < 0.01). Glutamine (GLN) was significantly lower in the left ( P < 0.001) and right DG ( P < 0.01), but there was no significant left-right difference. Aspartate was significantly lower in the left ( P < 0.0005) and right DG ( P < 0.0005), and the left-right difference was significant tP < 0.05). There were no significant differences in area CA1 except for aspartate where the left side was different from sham ( P < 0.05). Results are means 2 SEM for six animals for the dentate gyrus and three animals for area CA1. Data were analyzed by ANOVA and the groups compared by a post hoc Neuman-Kuels test.

GAD67 mRNA raised the possibility that granule cells might contain low concentrations of GAD67 message. Clearly, the present finding of intense GAD and GABA immunoreactivity in granule cells suggests that the faint granule cell staining for GAD67 mRNA (Figs. 1B and 3 of Houser and Esclapez, 1994) is biologically significant, even if it is only barely detectable with the presently available methodology. The fact that easily detectable protein immunoreactivity can be present in cells that appear relatively devoid of their corresponding mRNA reinforces the main caveat concerning the interpretation of results of immunocytochemical and in situ hybridization studies. That is, these methods only identify the location where tissue

ligands reside in highest concentration. Lack of staining may indicate only that the ligand is below the limit of detection, not that it is absent.

Expression and functions of GAD65 and GAD67

Most GABAergic neurons appear to express both forms of GAD, and different cell types express different amounts of the two forms (Erlander et al., 1991; Mercugliano et al., 1992; Feldblum et al., 1993; Esclapez et al., 1994; Hendrick- sen et al., 1994; Houser and Esclapez, 1994; Vardi et al., 1994; Vardi and Auerbach, 1995). The dentate granule cell is, therefore, one of the few cell types that may normally express only one form of GAD. Of course, our inability to detect GAD65-LI in normal granule cells does not eliminate the possibility that it is normally expressed, but at concentrations too low to detect immunocytochemically. Although it is significant that granule cells respond to prolonged excitation by expressing GAD65, it is important to empha- size that the increase in GAD65-LI is much smaller than the increase in GAD67-LI. The validity of this conclusion drawn from the immunocytochemical data is evident from the in situ hybridization data (Fig. 151, and from the immunoblotting experiments in which we observed no detectable increase in dentate gyrus GAD65. Examination of the data shows that we would have easily observed an increase in GAD65 had it approached the magnitude of the increase in GAD67. We attribute our failure to observe a measureable increase in GAD65 by immunoblotting to the difficulty of measuring the small amount of induced GAD65 in granule cells in the presence of the large amount of GAD65 normally present in the terminals of dentate GAB- Aergic nonprincipal cells.

The greater induction of GAD67 than GAD65 in granule cells is consistent with studies showing that the expressions of the two forms are regulated independently (Rimvall and Martin, 1992; Soghomonian et al., 1992; Ma et al., 1994; Laprade and Soghomonian, 1995; Romijn et al., 1994; Feldblum et al., 19951, and is also consistent with those few studies in which experimentally induced changes in the expressions of GAD67 and GAD65 have been compared (Rimvall and Martin, 1992; Rimvall et al., 1993; Soghomo- nian et al., 1992; Romijn et al., 1994; Laprade and Soghomo- nian, 1995; Martin et al., 1996). In all of these studies, which involved a variety of brain regions and manipula- tions, the expression of GAD67 was much more responsive than that of GAD65. Although the mechanisms that regulate GAD67 expression are not well characterized, they operate at both the transcriptional and post-transcriptional levels (Soghomonian et al., 1992; Rimvall et al., 1993).

The normally intense staining for GAD67, and the lack of staining for GAD65 in the granule cell terminal fields of normal animals, has implications for our understanding of the functions of the two GAD isoforms. GAD65 has been generally associated with synaptic terminals and synaptic transmission because it is found in high concentrations in presynaptic endings and synaptosomes (Kaufman et al., 1991; Esclapez et al., 1994; Erlander et al., 1991; Rimvall and Martin, 1994), is targeted to the Golgi apparatus in model systems by a small sequence in its amino terminal domain (Solimena et al., 1993, 19941, and has been localized to vesicles (McLaughlin et al., 1974; Wood et al., 1976; Reetz et al., 1991). Although GAD67 has been generally associated with cell bodies and dendrites rather than terminals (Kaufman et al., 1991; Esclapez et al., 1994), it is


present in synaptosomes and nerve endings (Erlander et al., 1991; Esclapez et al., 1994; Rimvall and Martin, 19941, and our immunocytochemical results clearly indicate that both forms of GAD are present in somata, dendrites, and axons.

Because GAD67 forms heteromultimers with GAD65 (Sheikh and Martin, 1996) and GAD67 only appears in the Golgi apparatus of model systems if GAD65 is also present (Dirkx et al., 1995), it has been suggested that GAD67 is transported to nerve endings together with GAD65 ( D i r k et al., 1995). Our discovery of intense GAD67-LI in normal granule cell terminals in the absence of any detectable staining for GAD65 suggests that this view is probably simplistic, and that GAD67 may be transported to nerve endings in the absence of GAD65.

Stimulation-induced changes in amino acid concentrations

Because we observed an increase in GABA despite inhibition of soma1 GAD67 induction by cycloheximide, the stimulation-induced increase in granule cell GABA content probably results from an activation of pre-existing GAD, in addition to the activity of newly synthesized GAD67. The basis of the activation of pre-existing GAD is unknown, but could be due to the activation of apoGAD (Martin and Rimvall, 1993), changes in GAD phosphorylation (Bao et al., 1995), or a loss of mossy fiber Zn2+ (Assaf and Chung, 1984; Howell et al., 1984; Charton et al., 1985; Sloviter, 1985; Frederickson et al., 1988), which is a potent GAD inhibitor (Wu and Roberts, 1974).

Although the stimulation-induced reductions in the concentrations of glutamine, glutamate and aspartate may be causally related to the increase in GABA, our data do not establish such a relationship. The reductions in glutamine and glutamate may result from an inability to provide enough precursor to support the increased production of GABA, but glutamine concentrations may also fall because the high metabolic demands imposed by the unremitting excitation reduced the availability of the energy needed for glutamine synthesis. An additional possibility is suggested by the view that glutamate and glutamine are organic osmolytes, and that changes in their tissue concentration may be a homeostatic response to tissue swelling (Nagelhus et al., 1996).

Functional implications of basally expressed and seizure-induced GABA in dentate

granule cells The conclusion that dentate granule cells normally con-

tain GAD and GABA, and the finding that afferent excitation of granule cells induces additional GAD- and GABA-LI, imply that granule cells may normally possess both excitatory and inhibitory properties. However, our immunocytochemical data do not establish that granule cell GAD and GABA immunoreactivities reflect releasable GABA. Gran- ule cell GAD might serve to reduce granule cell glutamate by converting it to nonfunctional GABA, or mossy fiber GABA might have a nonsynaptic, noninhibitory role in cellular function or protection.

Because seizures lead to the production of lactic acid and the acidification of brain tissue (Prichard, 1994; Meric et al., 1994), we have considered the possibility that GABA production might serve to control pH changes that occur in dentate granule cells during prolonged excitation. The idea that GABA production can counteract acid production is derived from early studies indicating that bacteria produce

GABA and amines to limit the acidification that occurs as cultures approach stationary phase (Hanke and Koessler, 1924; Gale, 1940). GABA itself has no buffering capacity at physiological pH, but the production of each molecule of GABA by GAD consumes one proton and, thus, could neutralize the production of a corresponding amount of acid produced by other reactions. However, continued GABA production would be essential for pH control. Diver- sion of energy metabolites through the GABA shunt would not provide better pH control than the normal tricarboxyiic acid (TCA) cycle because the overall reactions of the GABA shunt and the by-passed segment of the TCA cycle are the same except for the production of one GTP. Thus, GABA production could not provide pH control within granule cells after it had reached a new metabolic steady state unless the GABA was exported for degradation by other cells such as astrocytes. After 24 hours of stimulation, the concentration of GABA increased by about 15 pmolig tissue in the left dentate gyrus. Assuming that granule cells occupy approximately 50% of the microdissected tissue volume, their intracellular GABA would increase by 30 mM. Although this is a substantial increase, it is by no means clear that this amount of GABA production could counterbalance an appreciable fraction of the acid produced over such a prolonged period of excitation.

Assuming for the purpose of discussion that granule cell GABA has been conserved phylogenetically for its inhibitory properties, GABA may be released from axons, somata, or dendrites, and produce inhibitory effects as a result. Dendritic GABA release might affect afferent input strength, and somatic release could conceivably contribute to tonic somatic inhibition and the regulation of granule cell excitability. If mossy fiber GABA is released at synaptic sites, it may contribute to the IPSP produced in CA3 pyramidal cells (Kehl and McLennan, 1985; Janigro and Schwartz- kroin, 1988). In addition, mossy fiber GABA release may regulate mossy fiber glutamate release via a presynaptic GABAB receptor-mediated effect. Clearly, these functional scenarios are purely conjectural, and the present results serve mainly as the rational basis for studies designed to localize mossy fiber synapse GABAA and GABAB receptors, and to determine whether mossy fiber terminals segregate transmitter pools of glutamate and GABA, whether mossy fiber GABA is vesicular, whether granule cells release GABA in response to depolarization, and whether the conditions for glutamate and GABA release differ in terms of afferent stimulus frequency.

GABA is not the only substance in the mossy fiber pathway that may play an inhibitory role. Recent studies indicate that dynorphin, which is normally present in granule cell axon terminals, may act presynaptically to inhibit afferent input into the hippocampus (Wagner et a]., 1993) or to inhibit neighboring mossy fiber terminal discharges (Weisskopf et al., 1993). In addition, neuropeptide Y, although not detectable in normal mossy fibers, is rapidly induced in mossy fibers by perforant path stimulation (Sloviter and Lowenstein, 1992) or kainate (Sperk et al., 19921, and has been shown to affect granule cell calcium channels (McQuiston et al., 1996). Thus, at least three inhibitory molecules, GABA, dynorphin, and neuropeptide Y, are basally or inducibly expressed in mossy fiber terminals and may play different inhibitory roles. Clearly, future physiological studies will have to overcome the challenge of discriminating between functional effects caused by mossy fiber GABA release, and those caused by other inhibitory


substances or GABA release from GABAergic nonprincipal cells.

In terms of their possible relevance to epilepsy, the present results suggest that seizure-induced mossy fiber GAD and GABA may alter hippocampal excitability and be possible targets of anticonvulsant drugs. In addition, a seizure-induced increase in GAD and GABA might reacti- vate neurotrophic processes that are influenced by GABA in development (Hansen et al., 1984; Cherubini et al., 1991; LoTurco et al., 19951, and which could conceivably play a role in granule cell proliferation and axonal reorganization. However, answers to questions about the functional conse- quences of these findings must await future studies.

ACKNOWLEDGMENTS This research was supported by NIH grants NS18201 to

R.S.S., NS24927 to M.A.D., and MH35664 to D.L.M. We thank Dr. J. Wolpaw of the New York State Department of Health for generously providing the monkey brains; Dr. J. Rogers of the Physiological Laboratory, Cambridge, and Dr. D. Jacobowitz of NIH for generously providing anti- calretinin antisera; S.B. Martin, G. Battaglioli, s. Neubort, and L. Kulkarni for expert technical assistance; and Drs. B. Sukhareva and K. Wilcox for useful discussions. We also thank Drs. D. Johnston, D.H. Lowenstein, O.P. Ottersen, T.A. Pedley, and H.E. Scharfman for useful discussions and constructive criticism of the manuscript.

LITERATURE CITED Alvarez, P., S. Zola-Morgan, and L.R. Squire (1995) Damage limited to the

hippocampal region produces long-lasting memory impairment in monkeys. J . Neurosci. 15:3796-3807.

Amaral, D.G. (1978) A Golgi study of cell types in the hilar region of the hippocampus in the rat. J. Comp. Neurol. 182851-914.

Andersen, P., B. Holmqvist, and P.E. Voorhoeve (1966) Entorhinal activation of dentate granule cells. Acta. Physiol. Scand. 66:448-460.

Anderson. K.J., B.E. Maley, and S.W. Scheff (1986) Immunocytochemical localization of y-aminohutyric acid in the rat hippocampal formation. Neurosci. Lett. 69:7-12.

Assaf, S.Y., and S.-H. Chung (1984) Release of endogenous zinc from brain tissue during activity. Nature 308:734-736.

Bahh. T.L.. J .K. Pretorius, W.R. Kupfer, and W.J. Brown (1988) Distribution of glutamate decarhoxylase-immunoreactive neurons and synapses in the rat and monkey hippocampus: light and electron microscopy. J. Comp. Neurol. 278:121-138.

Bao. J.. W.Y. Cheung, and J:Y. Wu 11995) Brain L-glutamate decarboxylase inhibition by phosphorylation and activation by dephosphorylation. J. Biol Chem. 270,6464-6467.

Battaglioli, G., and D.L. Martin (1991) GABA synthesis in brain slices is dependent on glutamine produced in astrocytes. Neurochem. Res. 16.151- 156.

Berod, A,, B.K. Hartman, and J.F. Pujol (1981) Importance of fixation in immunohistochemistry: Use of formaldehyde solutions a t variable pH for the localization of tyrosine hydroxylase. J . Histochem. Cytochem. 29844-850.

Blackstad, T.W. 11956) Commissural connections of the hippocampal region in the rat. J . Comp. Neurol. 105.417-538.

Bond, K.W., R.J. Wyhorski, and D.I. Gottlieh (1990) Developmentally regulated expression of an exon containing a stop codon in the gene for glutamic acid decarboxylase. Proc. Natl. Acad. Sci. U.S.A. 87:8771-8775.

Bradford, M.M. ( 1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72248-254.

Brown, T.H., and D. Johnston (1983) Voltage clamp analysis of mossy fiber synaptic input to hippocampal neurons. J. Neurophysiol. 50:487-507.

Cao, Y., K. Wilcox, C. Martin, T. Rachinsky, J. Eherwine, and M. Dichter 119961 Presence of mRNA for glutamic acid decarboxylax in hoth excitatory and inhibitory neurons. Proc. Natl. Acad. Sci. U.S.A., 93r9844-9849.

Chang, Y.-C., and D.I. Gottlieh (1988) Characterization of the proteins purified with monoclonal antibodies to glutamic acid decarhoxylase. J. Neurosci. 8:2123-2130.

Charton, G., C. Rovira, Y. Ben-Ari, and V. Leviel (1985) Spontaneous and evoked release of zinc in the hippocampal mossy fiber zone of the rat in situ. Exp. Brain. Res. 58:202-205.

Cheruhini, E., J.L. Gaiarsa, and Y. Ben-Ari (1991) GABA: an excitatory transmitter in early poshatal life. Trends Neurosci. I2:515-519.

Ch’ih, J.J., R. Procyk, and T.M. Devlin 11977) Regulation of mammalian protein synthesis in vivo. Biochem. J. 162:501-507.

Crawford, I.L., and J.D. Connor (1973) Localization and release of glutamic acid in relation to the hippocampal mossy fiber pathway. Nature 244:442--143.

Dichter, M.A., Y. Cao, K.S. Wilcox, and J. Eherwine (1994) Glutamic acid decarhoxylase mRNA is expressed in hoth excitatory and inhibitory neurons in cultured hippocampal neurons. SOC. Neurosci. Ahstr. 20; 1165.

Dichter, M., and W.A. Spencer (1969) Penicillin-induced interictal discharges from the cat hippocampus. 11. Mechanisms underlying origm and restriction. J. Neurophysiol. 32:663-687

D i r k , Jr., R., A. Thomas, L. Li, A. Lernmark, R.S. Sherwin, P. DeCamilli, and M. Solimena (1995) Targeting of the 67-kDa isoform of glutamic acid decarboxylase to intracellular organelles is mediated hy its interaction with the NH,-terminal region of the 65-kDa isoform of glutamic acid decarhoxylase. J. Biol. Chem. 270,2241-2246.

Eccles, J.C. (1973) The Understanding of the Brain. New York: McGraw- Hill.

Erlander, M.G., N.J.K. Tillakaratne, S. Feldhlum, N. Patel, and A.J. Tohin (1991) Two genes encode distinct glutamate decarboxylases. Neuron 7:491-100.

Esclapez, M., N.J.K. Tillakaratne, D.L. Kaufman, A.J. Tobin, and C.R. Houser (1994) Comparative localization of two forms of glutamic acid decarhoxylase and their mRNAs in rat brain supports the concept of functional differences between the forms. J. Neurosci. 14:1834-1855.

Feldhlum, S., R.F. Ackermann, and A.J. Tohin 11990) Long-term increase of glutamate decarhoxylase mRNA in a rat model of temporal lobe epilepsy. Neuron 5:361-371.

Feldhlum, S., A. Dumoulin, M. Anoal, F. Sandillon, and A. Privat (1995) Comparative distribution of GAD,, and GAD,, mRNAs and proteins in the rat spinal cord supports a differential regulation of these two glutamate decarhoxylases in vivo. J. Neurosci. Res. 42,742-757.

Feldhlum, S., M.G. Erlander, and A.J. Tohin 11993) Different distributions of GAD65 and GAD67 mRNAs suggest that the two glutamate decarhoxylases play distinctive functional roles. J . Neurosci. Res. 343589-706.

Frederickson, C.J., M.D. Hernandez, S.A. Goik, J.D. Morton, and J.F. McGinty (1988) Loss of zinc staining from hippocampal mossy fibers during kainic acid induced seizures: a histofluorescent study. Brain Res. 446:383-386.

Gale, E.F. 11940) The production of amines by bacteria. I. The decarhoxyl- ation of amino-acids by strains of bacterium coli. J . Biochem. 34:392- 413.

Gamrani, H., B. Onteniente, P. Sequela, M. Geffard, and A. Callas (1986) Gamma-aminohutyric acid immunoreactivity in the rat hippocampus. A light and electron microscopic study with anti-GABA antibodies. Brain Res. 264:30-38.

Hanke, M.M., and K.K. Koessler 11924) Studies of proteinogenous amines. XVIII. On the production of histamine, tyramine, and phenol in common laboratory media hy certain intestinal microorganisms. J . Biol. Chem. 59:855-866.

Hansen, G.H., E. Meier, and A. Schoushoe (1984) GABA influences the ultrastructure composition of cerebellar granule cells during development in culture. Int. J. Dev. Neurosci. 2247-257.

Hendrickson, A.E., N.J.K. Tillakaratne, R.D. Mehra, M. Esclapez, A. Erick- son, L. Vician. and A.J. Tobin (1994) Differential localization of two glutamic acid decarhoxylases (GAD65 and GAD67i in adult monkey visual cortex. J. Comp. Neurol. 343:566-581.

Houser, C.R., and M. Esclapez (1994) Localization of mRNAs encoding two forms of glutamic acid decarhoxylase in the rat hippocampal formation. Hippocampus 4:530-545.

Howell, G.A., M.G. Welch, and C.J. Frederickson 11984) Stimulation- induced uptake and release of zinc in hippocampal slices. Nature 308:736-738.

Janigro, D., and P.A. Schwartzkroin (1988) Effects of GABA on CA3 pyramidal cell dendrites in rabbit hippocampal slices. Brain Res. 453,265- 274.

GAD 4 N D GABA IN THE HIPPOCAMPUS

Kaufman, D.L., C.R. IIouser, and A.J. Tobin (1991) Two forms of the gamma-aminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions. J. Neurochem. 56:720-723.

Kaufman, D.L., J.F. McGinnis, N.R. Krieger, and A.J. Tobin (1986) Brain glutamate decarboxylase cloned in A@- 1l:fusion protein produces y-aminohutyric acid. Science 232:1138-1140.

Kehl, S J , and H. McLennan (1985) An electrophysiological characterization of inhibitions and postsynaptic potentials in rat hippocampal CA3 neurons in vitro. Exp. Brain Res. 60299-308.

Laprade, N.. and J .J . Soghomonian (1995) Differential regulation of mRNA levels encoding for the two isoforms of glutamate decarboxylase (GAD65 and GAD671 by dopamine receptors in the rat striatum. Mot. Brain Res. 34:65-74.

Lorente de NO, R. (1934) Studies on the structure of the cerebral cortex. 11. Continuation of the study of the ammonic system. J. Psychol. Neurol. 46.113-177.

Lothman, E.W., E.H. Bertram, and J.L. Stringer (1991) Functional anatomy of hippocampal seizures. Prog. Neurobiol. 37: 1-82.

LoTurco, J.J., D.F. Owens,M.J.S. Heath, M.B.E. Davis, andA.R. Kriegstein (1995 1 GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis. Neuron 15: 1287-1298.

Ma, W., T. Behar, L. Chang, and J.L. Barker (1994) Transient increase in expression of GADb, and GAD,? mRNAs during postnatal development of rat spinal cord. J . Comp. Neurol. 346:151-160.

Martin, D.L., and K. Rimvall (1993) Regulation of y-aminobutyric acid synthesis in the brain. J . Neurochem. 60:395-407.

Martin, D.L., S.N. Sheikh, S.B. Martin, and K. Dakshinamurti (1996) Manipulation of brain GABA levels modifies the expression of both GAD65 and GAD67 in rat cerebellum. J. Neurochem. 66:Suppl.l:S83.

McLaughlin, B.J., J.G.Wood, K. Saito, R. Barber, J .E. Vaughn, E. Roberts, and J.-Y. Wu (1974) The fine structural localization of glutamate decarboxylase in synaptic terminals of rodent cerebellum. Brain Res. 76:377-91

McQuiston, A.R., J .J . Petrozzino, J.A. Connor, and W.F. Colmers (1996) Neuropeptide Y I receptors inhibit N-type calcium currents and reduce transient calcium increases in rat dentate granule cells. J . Neurosci. 16:1422-1429.

Mercugliano, M., J.-J. Soghomonian, Y. &in, H.Q. Nguyen, S. Feldblum, M.G. Erlander, A.J. Tobin, and M.-F Chesselet (1992) Comparative distribution of messenger RNAs encoding glutamic acid decarboxylases IM, 65,000 and M, 67,000) in the basal ganglia of the rat. J. Comp. Neurol. 318245-254.

Meric, P., B. Barrere, M. Peres, B. Gillet, G. Berenger, J.C. Beloeil, and J. Seylaz ( 19941 Effects of kainate-induced seizures on cerebral metabo- lism: a combined 1H and 31P NMR study in rat. Brain Res. 638153-60.

Mugnaini, E., and W.H. Oertel (1985) An atlas of the distribution of GABAerDc neurons and terminals in the rat CNS as revealed by GAD immunohistochemistry. In A. Bjorklund and T. Hokfelt (eds): Handbook of Chemical Neuroanatomy. Amsterdam: Elsevier pp. 436-608.

Nagelhus, E.A., A. Lehmann, and O.P. Ottersen (1996) Neuronal and glial handling of glutamate and glutamine during hypoosmotic stress: a biochemical and quantitative immunocytochemical analysis using the rat cerebellum as a model. Neuroscience 721743-755.

Oertel, W.H., D.E. Schmechel, E. Mugnaini, M.L. Tappaz, and I.J. Kopin (1981) Immunocytochemical localization of glutamate decarboxylase in rat cerebellum with a new antiserum. Neuroscience 6:2715-2735.

Ottersen, O.P., and J. Storm-Mathisen (1984) Neurons containing or accumulating transmitter amino acids. In A. Bjorklund, T. Hokfelt, and M.J. Kuhar (eds): Handbook of Chemical Neuroanatomy. Amsterdam: Elsevi~r, pp. 141-246.

Paulsen. R.E., E. Odden, and F. Fonnum (1988) Importance of glutamine for y-aminobutyric acid synthesis in rat neostriatum in vivo. J. Neurochem. 511 1‘294-1299.

Paxinos. G., and C. Watson i 1986) The Rat Brain in Stereotaxic Coordinates. Sydney: Academic Press.

Prichard, J.W. (1994) Nuclear magnetic resonance spectroscopy of seizure states. Epilepsia 35:SuppL6:S14-S20.

RamOn y Cajal, S. (18931 Estructura del asta de Ammon Anat Soc Esp Histol Nat Madrid 2253-114.

RamOn y Cajal, S. (19951 Histology of the nervous system. Volume 11. New York: Oxford.

Reetz, A.. M. Solimena, M. Matteoli, F. Folli, K. Takei, and P. De Camilli (1991 I GABA and pancreatic B-cells: Colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion. EMBO J 10:1275-1284.

617

Reubi, J.C., C. Van Den Berg, and M. Cuenod (1978) Glutamine as precursor for the GABA and glutamate transmitter pools. Neurosci. Lett. IOrl7I- 174.

Rihak, C.E., and L. Anderson (1980) Ultrastructure of the pyramidal basket cells in the dentate g y m s of the rat. J Comp Neurol 192:903-916.

Ribak, C.E., L. Seress, and D.G. Amarali1985) The development, ultrastructure and synaptic connections of the mossy cells of the dentate gyrus. J. Neurocytol. 14335-857.

Ribak, C.E., J .E. Vaughn, and K. Saito (1978) Immunocytochemical localization of glutamic acid decarboxylase in neuronal somata following colchi- cine inhibition of axonal transport. Brain Res. 140:315-332.

Rimvall, K., and D.L. Martin i 1992) Increased intraeellular y-aminobutyric acid selectively lowers the level of the larger of two glutamate decarboxylase proteins in cultured GABAergic neurons from rat cerebral cortex. J . Neurochem. 58: 158-166.

Rimvall, K., and D.L. Martin (1994) The level of GAD,, protein is highly sensitive to small increases in intraneuronal y-aminobutyric acid levels. J . Neurochem. 62:1375-1381.

Rimvall, K., S.N. Sheikh, and D.L. Martin (1993) Effects of increased y-aminobutyric acid levels on GAD,, protein and mRNA levels in rat cerebral cortex. J . Neurochem. 60:714-720.

Romijn, H.J., A.W.J.W. Janszen, and J. Van Marle (1994) Quantitative immunofluorescence data suggest a permanently enhanced GAD,,/ GADs5 ratio in nerve endings in rat cerebral cortex damaged by early postnatal hypoxia-ischemia: A comparison between two computer- assisted procedures for quantification of confocal laser scanning microscopic immunofluorescence images. Brain Res. 657:245-257.

Sandler, R., and A.D. Smith (1991) Coexistence of GABA and glutamate in mossy fiber terminals of the primate hippocampus: an ultrastructural study. J. Comp. Neurol. 303:177-192.

Schreiber, S.S., G. Tocco, I. Najm, R.F. Thompson, and M. Baudry (1993) Cycloheximide prevents kainate-induced neuronal death and c-fos expression in adult rat brain. J . Mol. Neurosci. 4:149-159.

Schwartz, J.H. (19911 Chemical messengers: small molecules and peptides. In E.R. Kandel, J .H. Schwartz, and T.M. Jessell (eds): Principles of Neural Science. Norwalk Appleton and Lange, pp. 213-224.

Schwarzer, C., and G. Sperk (1995) Hippocampal granule cells express glutamic acid decarhoxylase-67 after limbic seizures in the rat. Neurosci- ence 69705-709.

Scoville, W.B., and B. Milner (1957) Loss of recent memory after bilateral hippocampal lesions. J. Neurol. Neurosurg. Psychiatry 20: 11-2 1

Seguela, P., M. Geffard, R.M. Buijs, and M. Le Moal (1984) Antibodies against y-aminobutyric acid: specificity studies and immunocytochemical results. Proc. Natl. Acad. Sci. U.S.A. 81:3888-3892.

Seress, L., K. Nitsch, and C. Leranth (1993) Calretinin immunoreactivity in the monkey hippocampal formation. I. Light and electron microscopic characteristics and co-localization with other calcium-binding proteins. Neuroscience 55: 775-796.

Sheikh, S.N., and D.L. Martin (1996) Heteromers of glutamate decarboxylase isoforma occur in rat cerebellum. J. Neurochem. 66:2082-2090.

Shepherd, G.M. (1979) The Synaptic Organization of the Brain. New York: Oxford.

Simmons, D.M., J .L. Arriza, and L.W. Swanson (1989) A complete protocol for in situ hybridization of messenger RNAs in brain and other tissues with radiolabeled single-stranded RNA probes. J . Histotechnol. 12:169- 181.

Sloviter, R.S. (1983) “Epileptic” brain damage in rats induced by sustained electrical stimulation of the perforant path. I . Acute electrophysiological and light microscopic studies. Brain Res. Bull. 10:675-697.

Sloviter, R.S. i 1985) A selective loss of hippocampal mossy fiber Timm stain accompanies granule cell seizure activity induced by perforant path stimulation. Brain Res. 330:150-153.

Sloviter, R.S. (1989) Calcium binding protein (Calbindin-D28K) and parval- bumin immunocytochemistry; localization in the rat hippocampus with specific reference to the selective vulnerability of hippocampal neurons to seizure activity. J. Comp. Neurol. 280: 183-196.

Sloviter, R.S. i 1991 1 Permanently altered hippocampal structure, excitability and inhibition after experimental status epilepticus in the rat; the “dormant basket cell” hypothesis and its possible relevance to temporal lobe epilepsy. Hippocampus 1:41-66.

Sloviter, R.S., and D.H. Lowenstein (1992) Heat shock protein expression in vulnerable cells of the rat hippocampus as an indicator of excitation- induced neuronal stress. J. Neurosci. 12:3004-3009.

Sloviter, R.S., and G. Nilaver (1987) Immunocytochemical localization of GABA-, cholecystokinin-, vasoactive intestinal polypeptide-. and somato-


statin-like immunoreactivity in the area dentata and hippocampus of the rat. J. Comp. Neurol. 256.42-60.

Sloviter, R.S., A.L. Sollas, E. Dean, and M.A. Dichter (19951 Immunocyto- chemical localization of 65- and 67kDa glutamic acid decarboxylase (GADI- and GABA-like immunoreactivities (LIl in the rat hippocampal formation; GAD67- and GABA-LI are present in normal dentate granule cells and are rapidly induced by afferent stimulation, kainic acid, or pilocarpine. SOC. Neurosci. Abstr. 21:1968.

Soghomonian, J .J . , C. Gonzales, and M.-F. Chesselet (1992) Messenger RNAs encoding glutamate decarboxylases are differentially affected by nigrostriatal lesions in subpopulations of striatal neurons. Brain Res. 576:68-79.

Solimena, M.. D. Aggujaro, C. Muntzel, R. D i r k , M. Butler, P. DeCamilli, and A. Hayday (1993) Association of GAD-65, but not of GAD-67, with the Golgi complex of transfected Chinese hamster ovary cells mediated by the N-terminal region. Proc. Natl. Acad. Sci. U S A . 90.3073-3077.

Solimena, M., R. Dirkx, Jr., M. Radzynski, 0. Mundigl, and P. DeCamilli (1994) A signal located within amino acids 1-27 of GAD65 is required for its targeting to the Golgi complex region. J. Cell. Biol. 126.331-341.

Somogyi, P., A.J. Hodgson, 1.W. Chubb, B. Penke, and A. Erdei (1985) Antisera to y-aminobutyric acid. 11. Immunocytochemical application to the central nervous system. J. Histochem. Cytochem. 33.240-248.

Soriano, E., and M. Frotscher (1993) GABAergic innervation of the rat fascia dentata: a novel type of interneuron in the granule cell layer with extensive axonal arborization in the molecular layer. J. Comp. Neurol. 334.385-396.

Soriano, E., and M. Frotscher (19941 Mossy cells of the rat fascia dentata are glutamate-immunoreactive. Hippocampus 4.65-70.

Spencer, S.S., P.D. Williamson, D.D. Spencer, and R.H. Mattson (1990) Combined depth and subdural electrode investigation in uncontrolled epilepsy. Neurology 46:74-79.

Sperk, G., J. Marksteiner, B. Gruber, R. Bellmann, M. Mahata, and M. Ortler (19921 Functional changes in neuropeptide Y and somatostatin containing neurons induced by limbic seizures in the rat. Neuroscience 50:831-846.

Spink, D.C., J.W. Swann, O.C. Snead, R.A. Waniewski, and D.L. Martin ( 1986) Analysis of aspartate and glutamate in human cerebrospinal fluid by high-performance liquid chromatography with automated precolumn derivatization. Anal. Biochem. 158:79-86.

Storm-Mathisen, J., A.K. Leknes, A.T. Bore, J.L. Vaaland, P. Edminson, F.-M.S. Haug, and O.P. Ottersen (1983) First visualization of glutamate and GABA in neurons by immunocytochemistry. Nature 301:517-520.

Storm-Mathisen, J., and O.P. Ottersen (1983) Immunohistochemistry of glutamate and GABA. In L. Hertz, E. Kvamme, E.G. McGeer, and A. Schousboe (eds): Glutamine, glutamate and GABA in the Central Nervous System. New York: Alan R. Liss Inc., pp. 185-201.

Szabo, G., Z. Katarova, and R. Greenspan (1994) Distinct protein forms are produced from alternatively spliced bicistronic glutamic acid decarboxylase mRNAs during development. Mol. Cell Biol. 14:7535-7545.

Vardi, N., and P. Auerbach (1995) Specific cell types in cat retina express different forms of glutamic acid decarboxylase. J. Comp. Neurol. 351:374- 384.

Vardi, N., D.L. Kaufman, and P. Sterling (1994) Horizontal cells in cat and monkey retina express different isoforms of glutamic acid decarboxylase. Visual Neurosci. 11:135-142.

Wagner, J.J., G.W. Terman, and C. Chavkin (1993) Endogenous dynorphins inhibit excitatory neurotransmission and block LTP induction in the hippocampus. Nature 36345 1-454.

Weisskopf, M.G., K.A. Zalutsky, and R.A. Nicoll (1993) The opioid peptide dynorphin mediates heterosynaptic depression of hippocampal mossy fibre synapses and modulates long-term potentiation. Nature 362423- 427.

Williamson, A,, S.S. Spencer, and D.D. Spencer (1995) Depth electrode studies and intracellular dentate granule cell recordings in temporal lobe epilepsy. Ann. Neurol. 38.778-787.

Wolpaw, J.R., and P.A. Herchenroder ( 19901 Operant conditioning of H-reflex in freely moving monkeys. J Neurosci Methods 31:145-152.

Wood, J.G., B.J. McLaughlin, and J.E. Vaughn (1976) Immunocytochemical localization of GAD in electron microscopic preparations of rodent CNS. In E. Roberts, T.N. Chase, and D.B. Tower (eds): GABA in Nervous System Function. New York: Raven, pp. 133-148.

Woodson, W., L. Nitecka, and Y. Ben-Ari (1989) Organization of the GABAergic system in the rat hippocampal formation: a quantitative immunocytochemical study. J. Comp. Neurol. 280:254-271.

Wu, J.-Y., and E. Roberts (1974) Properties of brain L-glutamate decarboxylase: Inhibition studies. J Neurochem 23:759-767.

Yamamoto, C. (1972) Activation of hippocampal neurons by mossy fiber stimulation in thin brain sections in vitro. Exp. Brain Res. 14:423-435.

Yamamoto, C. (1982) Quanta1 analysis of excitatory postsynaptic potentials induced in hippocampal neurons by activation of granule cells. Exp. Brain Res. 46.170-176.

Documents

Basal expression and induction of glutamate decarboxylase GABA in excitatory granule cells of the rat and monkey hippocampal dentate gyrus