Brain Research 917 (2001) 139–146www.elsevier.com/ locate /bres

Research report

Activity-dependent expression of GAD in the granule cells of the rat67

hippocampus*´ ´ ´Monica Ramırez, Rafael Gutierrez

´ ´ ´ ´Departamento de Fisiologıa, Biof ısica y Neurociencias, Centro de Investigacion y de Estudios Avanzados, del Instituto Politecnico Nacional,´Apartado Postal 14-740, Mexico D.F. 07000, Mexico

Accepted 11 July 2001

Abstract

In the normal granule cells of the dentate gyrus glutamate, GABA and glutamic acid decarboxylase (GAD ) coexist. After kindled67

seizures, this enzyme is transiently overexpressed and simultaneous glutamatergic and GABAergic transmission in the mossy fiberprojection occurs. Since this dual transmission is also seen after acutely-induced seizures, we decided to study the relationship betweenthe expression of GAD and the induction of simultaneous glutamatergic and GABAergic transmission by kindled or acutely induced67

seizures. We also explored whether kindling of the dentate gyrus in vitro, that does not induce epileptiform activity, could induce theexpression of GAD . We confirm that kindling epilepsy induces the expression of GAD in the dentate gyrus. Despite the emergence of67 67

GABAergic transmission in the mossy fiber projection after a single seizure, GAD expression in the dentate gyrus appeared similar to67

controls, however, in the mossy fibers an enhanced immunostaining was evident. Interestingly, kindling the dentate gyrus in vitro inducesa marked GAD staining in the granule cells. Our results show that after the activity-dependent emergence of simultaneous glutamatergic67

and GABAergic transmission from the mossy fibers, GAD is expressed in the mossy fibers and, upon long-lasting enduring stimulation67

periods, also in the dentate gyrus. Thus, this phenomenon does not depend on the presence of epileptic activity, but rather, on increasedexcitatory input onto the dentate gyrus. This can represent a protective mechanism that can sustain GABA synthesis in anactivity-dependent manner. 2001 Elsevier Science B.V. All rights reserved.

Keywords: GABA; Glutamate; Mossy fiber; Kindling; Epilepsy; Seizure; GAD

1. Introduction kainic acid-induced limbic seizures increase granule cellexpression of GAD mRNA, and GABA can be immuno-67

The confirmation that fast GABAergic transmission in cytochemically detected [9,10,17,22]. These data suggestedthe mossy fiber projection appears after kindled or acutely that GABAergic transmission could be present in theinduced seizures [5,6] supports the idea that although the mossy fiber synapse due to up-regulation of GAD and67

granule cells of the dentate gyrus (DG) are glutamatergic the consequent synthesis of GABA.[1], they can use GABA for fast neurotransmission. Immunohistological studies of GAD expression have67

Indeed, granule cells normally contain GABA [16] and its revealed a time lag between the experimental manipulationsynthesizing enzyme glutamic acid decarboxylase causing the seizures and the appearance of the enzyme in(GAD ) [20]. Recent electrophysiological evidence the granule cells. However, acutely-induced seizures in-67

strongly supports this hypothesis by showing that mono- duce simultaneous glutamatergic and GABAergic trans-synaptic GABAergic potentials can be evoked in CA3 mission in the mossy fiber projection, that can be recordedpyramidal cells by mossy fiber activation, with characteris- one hour after the seizure [5]. Moreover, direct or synaptictics typical of mossy fiber transmission [25]. Provoking kindling stimulation of the DG in vitro for 3 h induces thisseizures by long lasting electrical stimulation of the phenomenon in the absence of epileptiform activity. Thisperforant path (PP) up-regulates GAD expression [20] and induced GABAergic transmission from the mossy fibers,

evidenced during glutamate receptors’ blockade, is mono-synaptic, is sensitive to mGluR agonists and does not*Corresponding author. Tel.: 152-5-7477-000 ext. 5700; fax: 152-5-depend on the presence of a permanent epileptic state nor7477-105.

´ ´E-mail address: grafael@fisio.cinvestav.mx (R. Gutierrez). on the sole occurrence seizures (R. Gutierrez, unpublished

0006-8993/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 01 )02794-9

´ ´140 M. Ramırez, R. Gutierrez / Brain Research 917 (2001) 139 –146

observations). We have also ruled out the possibility that glass-insulated platinum wire (50 mm) electrode wasthe DG-evoked inhibitory responses in the kindled animals placed over the PP and test pulses of 0.1 ms duration wereare due to stimulation of sprouted interneurons or to their applied to fix the intensity for kindling stimulation at arecruitment through electrical synapses [5,21]. value that evoked 50% of the maximal field potential

It is intriguing that unlike control animals, GABAergic response in the DG. The kindling stimulation protocoltransmission in the mossy fiber synapse can be detected consisted of three trains of 0.1 ms pulses at 100 Hz duringwith intracellular recordings either after several seizures in 1 s, applied with an intertrain interval of 1 min, every 15epileptic animals, in which structural alterations remain for min, for 3 h (12 kindling trials). Throughout the experi-several months [2,19], or an hour after the presence of a ment, extracellular spontaneous activity of the granular cellsingle seizure in a normal rat [6]. This suggests that GABA layer of the DG and CA3 was recorded with glasssynthesis and its release mechanisms can be up-regulated microelectrodes (8–15 MV) filled with NaCl (155 mM)after enhanced excitability. Indeed, we have found an and an AxoClamp 2B amplifier.activity-dependent expression of GABA in mossy fiber

´terminals (G. Gomez-Lira, E. Trillo, M. Asai, M. Sitges,´R. Gutierrez, unpublished data) and of vesicular GABA 2.3. GAD immunocytochemistry67

transporter (VGAT) mRNA in mossy fiber terminals anddentate gyrus. For detection of GAD immunoreactivity in kindled67

In view of the aforementioned, in this work we analyze animals, we processed five rats a day after the fifth seizurethe temporal expression of GAD in response to the and four rats a month after the last kindled seizure. For67

experimental manipulations that are known to induce experiments of PTZ-treated rats, we processed, at 2, 24 andsimultaneous glutamatergic and GABAergic neurotrans- 48 h after the seizure, three rats for each time period.mission in the mossy fiber synapse, namely: after an Finally, we processed three slices intermittently stimulatedacutely-induced seizure, after several kindled seizures and for 3 h in vitro (see previous section). Each group wasafter PP kindling in vitro, a procedure that does not processed in parallel with slices taken from sham-operatedproduce epileptiform activity. control rats (n56). Under ether anesthesia, we carried out

intracardiac perfusion of 0.1 M phosphate buffer at pH 7.4(solution A) during 1–2 min (at 12 ml /min) followed by

2. Methods perfusion of paraformaldehyde (500 ml) at 4% in the samebuffer (solution B). The brains were dissected and further

2.1. Kindled and acutely-induced seizures fixed for 2 h in solution B and transferred to a buffer withsucrose (30%) for 36 h. In vitro-stimulated slices were

Male Wistar rats (230–250 g; Harlan Sprague Dawley, collected from the recording chamber and fixed in parafor-USA) were anesthetized with ketamine (60 mg/kg i.p.) and maldehyde at 4% for 24 h, after which they were trans-implanted with bipolar stainless steel electrodes (80 KV) ferred to the buffer with sucrose for 36 h. The brains andinto the left basolateral amygdala (AP 2.5; L 5; H 8.5) stimulated slices were frozen, and slices of 30 mm were cut[12]. Starting 7 to 10 days after the operation, the animals in a microtome and collected in phosphate-buffered salinewere daily stimulated with a 1 s train of square pulses (0.1 (PBS) (0.9% NaCl). The slices were free-floating incu-ms pulse duration; 60 Hz; 500 mA) until five generalized bated in PBS with 0.3% H O to block endogenous2 2

convulsive seizures (stage V) [14] were evoked. Acute peroxidase activity. After rinsing for three periods of 10seizures were induced with a single i.p. administration of min in PBS, they were incubated in 10% horse serumpentylenetetrazol (PTZ; 70 mg/kg; Sigma). All experimen- (Gibco), 1% gelatine (Sigma) and 0.2% Tween-20tal procedures were approved by the Committee on Ethical (Sigma), to avoid unspecific GAD staining. Slices wereAnimal Research of our Institution. then incubated in the presence of GAD antibody67

(Chemicon) at 1:1000 in PBS and horse serum (5%; 48C)2.2. Kindling in vitro during 36 h with agitation. After four 10-min washing

periods, they were incubated in the presence of an anti-Control rats were decapitated under deep ether anes- rabbit biotinylated IgG (1:800; Amersham Pharmacia

thesia, the brains were dissected, and combined entorhinal Biotech) The slices were then rinsed four times and.

cortex–hippocampus slices (400 mm) were cut with a processed with the ABC Kit (Vectastain; Vector Labs.) forvibroslicer (Campden Instruments, UK) submerged in 1 h at room temperature. After four 10-min washingoxygenated artificial cerebrospinal fluid (ACSF) at 48C. periods, the slices were developed with diaminobenzidineThe slices were then transferred to an interface recording (Sigma; 10 mg/25 ml 10 mM PBS) for 10 min andchamber and were constantly perfused with oxygenated contrasted with nickel sulphate (30%). MicroscopicalACSF at 358C containing (in mM): NaCl, 124; KCl, 3; observations and micrographs were taken on an AxiovertNaH PO , 1.25; MgSO , 1.8; CaCl , 1.6; NaHCO , 26; Zeiss microscope with the appropriate photographic equip-2 4 4 2 3

glucose 10; pH 7.35. For kindling stimulation, a bipolar ment.

´ ´M. Ramırez, R. Gutierrez / Brain Research 917 (2001) 139 –146 141

3. Results 3.1.3. PTZ-treated ratsSurprisingly, although we observed inhibitory transmis-

The electrophysiological results corresponding to kin- sion from DG to CA3 in the presence of glutamatergicdled and PTZ-induced seizures have been previously antagonists in the PTZ-treated rats [5], we did not findreported [5]. In these experiments, as well as in those using immunostaining in the granule cell layer of the DG after 2,

´the in vitro kindling protocol (R. Gutierrez, unpublished 24 or 48 h of the acutely-induced seizure (Fig. 1B).data), single-pulse electrical stimulation of the DG in the However, a higher density of the GAD signal was67

presence of NMDA and non-NMDA glutamate receptor observed in putative basket cells aligning in the borderblockers (APV, 30 mM; NBQX, 10 mM) evoked a mono- between the hilus and the granule cell layer (Figs. 2B andsynaptic IPSP. By contrast, recordings from more than 100 3B). Also, clearly stained interneurons were observed incontrol pyramidal cells show that perfusion of gluta- the CA3 region (Fig. 4C).matergic antagonists blocks the monosynaptic EPSP andthe fast and slow IPSPs. This is indicative that the IPSPs 3.1.4. Acutely stimulated slicesare polysynaptically driven. Pyramidal cells from kindled During the 3 h that lasted the kindling stimulationanimals that were recorded a month after the last seizure, protocol plus an hour after its completion, on-line continu-had the same responses than the control non-stimulated ous monitoring of extracellular activity showed no epi-animals [5,6]. leptiform activity in the stimulated slices (not shown). The

immunohistological studies on these slices showed clearimmunostaining of the stratum lucidum along the mossy

3.1. Immunocytochemical experiments fibers. Puncta of the mossy terminals were seen surround-ing the pyramidal cells in the CA3 region. Interestingly,

As control experiments for the immunocytochemical we found that the granule cell layer of the DG and hilarstudies, slices from control and kindled rats were pro- region were densely immunostained in all the acutelycessed at the same time, but lacking the first or second stimulated slices (Fig. 5B), whereas sister unstimulatedantibody. This manipulation prevented staining (not slices, perfused in the same recording chamber, were likeshown). any other control preparation (Fig. 5A).

In summary, our immunohistological results show thatafter a single PTZ-induced seizure, GAD is not ex-673.1.1. Control ratspressed in the DG beyond its control presence, butWe found clearly stained putative inhibitory inter-interneurons show higher staining than controls. However,neurons and basket cells in the border of the dentate gyrusif several seizures occur, GAD is expressed in the mossy67and in the hilar region, whereas the granule cells of thefibers and in the granule cell layer. This continuousdentate gyrus do not present immunostaining (Figs. 1A andpresence of hyperexcitatory input to the DG due to3A). In some of these preparations, few granule cells couldrepetitive seizures is also mimicked by the 3-h in vitrobe identified with a clear GAD staining. Some clearly67 kindling protocol applied over the PP, whereby GAD is67identifiable mossy fibers in stratum lucidum were alsoalso expressed in the granule cell layer and mossy fibersfound to be stained. In no case, pyramidal cells of the CAand after which GABAergic transmission occurs. Con-region were stained.comitantly, the interneuronal population overexpresses theenzyme. After a period of 1 month without seizures,

3.1.2. Kindled rats GAD expression in the DG seems to return to control67

Hippocampi processed a day after the last of several levels and GABAergic transmission is generally not ob-kindled seizures presented dense immunostaining of the served.dentate gyrus and hilar region (Figs. 2A and 3C). Themossy fibers were stained along the stratum lucidum andwithin CA3, where dark puncta, reminiscent of mossy 4. Discussionvaricosities, surrounded pyramidal cells (Fig. 4C). Also,besides the identification of stained granule cells, as judged From our immunocytochemical data we conclude thatby their density and shape, and of putative interneurons, a the granule cells of the DG are able to synthesize GAD67

higher background immunoreactivity was also apparent in in response to seizures, as previously reportedthe hilar region, the granule cell layer and the molecular [8,9,16,17,20,22] and we can relate this to conditions inlayer. Hippocampi processed a month after the last seizure which we have proven the existence of simultaneousshowed a similar pattern of GAD staining in the DG to glutamatergic and GABAergic transmission from the DG67

that observed in the control animals (Figs. 2B and 3D). to CA3 [5,6]. We also extend these observations byHowever, in this group, we found a higher immunostaining showing that a single PTZ-induced seizure and in vitroin the hilus and mossy fibers in the stratum lucidum than in kindling stimulation for 3 h, in the absence of epilepticcontrol rats (Figs. 3D and 4D). activity, also up-regulate the expression of GAD in the67

´ ´142 M. Ramırez, R. Gutierrez / Brain Research 917 (2001) 139 –146

Fig. 1. GAD stained sections of the hippocampus of a control rat (A) and of a PTZ-treated rat, 2 h after the seizure (B). In A, GAD-reactive interneurons,67

but no granule cells, are stained in the border between the DG and the hilar region. Two hours after an acutely induced seizure (B), an identifiable row ofGAD-reactive interneurons in the inner border of the granule cell layer is densely stained. Mossy fibers are also more evident. The calibration bar in Brepresents 400 mm and is valid for both panels.

mossy fibers and DG, respectively. Together, this evidence dependent effects of rapid kindling or electrically inducedsuggests that the temporal pattern of expression of GAD status epilepticus as well [18].67

in the granule cell layer and the mossy fibers differs Based on our previous electrophysiological findingsdepending on the time the system is subject to an increased [5,6], we propose that GABAergic transmission in theexcitatory input, whereby an apparent local synaptic mossy fiber projection is rapidly enhanced upon demandexpression of this enzyme upon rapid demands (a single and we now show that GAD expression follows this67

seizure) is followed by a later somatic expression upon demand. This assumption is supported by the fact thatlong-lasting enduring stimulation periods (repeated sei- seizures provoked by a single PTZ administration inducezures or 3 h kindling stimulation). A similar difference in simultaneous glutamatergic and GABAergic transmissionthe pattern of expression of GAD was also found in in the mossy fiber synapse, without the apparent expres-67

kainate treated rats [9]. Interestingly, an enhanced im- sion of GAD in the DG. Mossy fibers, on the other hand,67

munoreactivity to several peptides (somatostatin, neuro- appear clearly immunoreactive. This suggests that as thepeptide Y, neurokinin B and cholecystokinin) in the DG, enzyme is synthesized it is transported to the mossy fibers,hilar region and mossy fibers also reflect the activity- thus needing more time for it to be detected in the DG.

´ ´M. Ramırez, R. Gutierrez / Brain Research 917 (2001) 139 –146 143

Fig. 2. GAD stained sections of the hippocampus of kindled rats, 24 h (A) and 1 month (B) after the last seizure. The granule cell layer of the DG is67

densely immunostained 24 h after the last of several seizures (see magnification in A) and returns to control condition a month later (B). However, the hilarregion and the mossy fibers remain stained during this non-seizing period. The magnification in B shows the granule cell layer without immunoreactivity,whereas the output region of the granule cells is densely stained. For comparison see the control preparation, depicted in Fig. 1A. The amplification is thesame as in Fig. 1.

This notion is supported by the experiments showing that whole-cell voltage clamp recordings reveal monosynaptickindling of the PP in vitro, that does not induce epi- GABAergic responses in CA3 to mossy fiber stimulationleptiform activity, induces both GABAergic transmission [25]. However, intracellular recordings show no evidence

´to CA3 (R. Gutierrez, unpublished observations) and the of GABAergic transmission in the mossy fiber pathway,expression of GAD in the DG. When the system is not after blocking glutamatergic transmission. Thus, up-regula-67

longer stimulated (by repeated seizures), GAD product- tion of GABA synthesis seems likely to occur after67

ion and GABAergic transmission ceases [5,6,17] and seizures [22] or after enhanced input to the system.rekindling the system induces again the presence of Interestingly, an origin for GABA, other than the GAD-GABAergic transmission [5] and GAD staining in the mediated synthetic pathway, has been proposed to exist67

mossy fibers is observed. particularly in mossy fiber terminals [23,24]. With the usePrevious reports show a mild immunostaining of GAD of the mossy fiber synaptosomal preparation and in67

and GABA in the DG of control preparations [16,20] and agreement with other authors [23,24], we found that mossy

´ ´144 M. Ramırez, R. Gutierrez / Brain Research 917 (2001) 139 –146

Fig. 3. Immunoreactivity for GAD in the DG of a control rat (A), of a rat after a single PTZ-induced seizure (B), of a kindled rat 24 h after the last of67

several seizures (C) and of a kindled rat a month after the last of several seizures (D). The DG of control slices (A) lacks immunoreactivity in the granulecell layer, however some putative interneurons and granule cells are stained. Two hours after a single seizure (B), overexpression of GAD in the putative67

interneurons in the border between the granule cell layer and the hilar region is observed. Contrary to the single seizure condition, repeated kindled seizuresinduce overexpression of GAD in the granule cell layer (C), that returns to control conditions a month later (D). Abbreviations: ML, molecular layer;67

GCL, granule cell layer; H, hilar region. The bar represents 50 mm.

fiber synaptosomes contain GABA and we extend this One can state that the level of GAD that might be67

observation by establishing that its content is significantly normally present in granule cells, and which seems to be´enhanced in kindled animals (G. Gomez-Lira, E. Trillo, M. immunocytochemically undetectable, is sufficient to pro-

´Asai, M. Sitges, R. Gutierrez, unpublished observations). vide GABA for a rapid response. However, after a periodThese observations parallel the appearance of inhibitory of enhanced excitatory input that gives rise to simultaneoustransmission upon pyramidal cells, under glutamate re- glutamatergic and GABAergic transmission, GAD is67

ceptors blockade. overexpressed. The presence of seizures and the lack ofIt is interesting that after PTZ-induced seizures, the detection of GAD in the DG shortly after the seizures67

immunodetection of GAD in the DG is similar to that in has previously been reported [3]. It seems then that the67

controls. However, by contrast, a clearly stained row of synthesis of GAD that is required to synthesize more67

interneurons was evident in the inner part of the DG and a GABA to replenish the available pool takes some time.more stained neuropil is also seen in the DG and hilar This is further supported by an enhancement of GABAregion. In agreement with a previous report that shows that released in CA3 some time after seizures [3].interneurons have higher expression of GAD mRNA A question that immediately prompts after observing the67

after kainic acid-induced seizures [17], we also found more GABAergic transmission from the mossy fibers to CA3GAD immunoreactivity in these cells, which also corre- refers to the location of the receptors for the released67

spond to those reported to contain more GAD mRNA in GABA. It has been recently shown in cultured pyramidalcontrol and post-seizure conditions [4]. However, within hippocampal cells, that GABA receptors cluster onA

the hilar region, Obenaus et al. [11] reported a loss of pyramidal cells in the absence of GABA input, and evenGAD mRNA containing neurons. apposed to mismatched glutamatergic presynaptic ele-

´ ´M. Ramırez, R. Gutierrez / Brain Research 917 (2001) 139 –146 145

Fig. 4. Immunoreactivity for GAD in the CA3 region of a control rat (A), of a rat after a single PTZ-induced seizure (B), of a kindled rat 24 h after the67

last of several seizures (C) and of a kindled rat a month after the last of several seizures (D). Notice that contrary to controls (A), GAD immunoreactivity67

is evident in more interneurons and fibers after a single seizure (B). Kindling epilepsy induces overexpression of GAD in fibers and puncta surrounding67

pyramidal cells (C) that remains high even a month after the last seizure (D). Abbreviations: SP, stratum pyramidale; SR, stratum radiatum; SL, stratumlucidum. The bar represents 50 mm.

ments. The existence of such mismatched appositionssuggests the hypothesis that there is an element common toGABA and glutamate synapses [15]. It is therefore likelythat pyramidal cells do have GABA receptors in appositionto mossy fiber inputs. Indeed, DG stimulation is able toinduce fast IPSPs in the pyramidal cells, whose onsetlatency parallels that of the EPSP and whose reversalpotential can be easily obtained [5,6].

We conclude that enhanced excitatory input onto the DGresults in the expression of fast inhibition in the DG–CA3synapse, followed by an up-regulation of the synthesis ofGAD . The transient overexpression of GAD [8,17] and67 67

GABA synthesis in granule cells, and the consequentinhibitory transmission upon pyramidal cells of CA3 [5,6]are likely to serve as a protective mechanism after theestablishment of epilepsy, whereby further spread of

Fig. 5. Repetitive high-frequency synaptic activation of the DG by PP epileptic activity can be limited. Whether the expression ofstimulation induces overexpression of GAD in the DG. Sister slices67 the GABAergic transmission observed under glutamatergicperfused in the same chamber were processed in parallel. (A) Depicts a

blockade after seizures or kindling is a consequence of thecontrol slice, in which some putative interneurons are stained, but not theoverexpression of GAD cannot be disclosed with thegranule cell layer. (B) Depicts a slice stimulated for 3 h, in which a dense 67

staining of the DG and hilar region is evident. present experiments. However, noteworthy is the temporal

´ ´146 M. Ramırez, R. Gutierrez / Brain Research 917 (2001) 139 –146

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