GAD在颞叶癫痫大鼠海马内源性促痫机制中的作用

目的:探讨GAD65、GAD67在颞叶癫痫发生后海马内源性促痫机制中的作用。方法:112只雄性SD大鼠随机分为实验组(n=70)与对照组(n=42),实验组大鼠选用海人酸腹腔注射法建立颞叶癫痫模型,对照组大鼠腹腔注射无菌生理盐水。选取腹腔注射后3小时、6小时、12小时、24小时、48小时、7天、30天为研究的时间点,颞叶海马的CA1区、CA3区、齿状回为研究部位。腹腔给药后每天观察大鼠的行为学变化,大鼠处死前进行EEG描记。用原位杂交方法检测不同时间点海马不同区域GAD65、GAD67mRNA的表达,免疫组织化学法检测GAD65、GAD67蛋白的表达。结果:实验组大鼠海马GAD65 mRNA及其蛋白的表达随时间呈逐渐增高趋势,致痫后48小时～30天,GAD65 mRNA及其蛋白表达较对照组增高(48小时P<0.05;7～30天P<0.01);海人酸致痫后6小时、24小时实验组大鼠海马的GAD67mRNA及其蛋白表达较对照组增高(分别为P<0.01、P<0.05)。结论:颞叶癫痫急性期海马GAD67表达的增高及慢性期海马GAD65表达的增高是癫痫发生后机体的内源性抗痫机制。     

海人酸致痫大鼠海马及颞叶皮质区域NMDA受体亚单位R1蛋白的表达

背景：研究表明天门冬氨酸受体亚单位R1与癫痫的发生有关,但其具体的表达情况与癫痫脑损伤的关系尚不明确。 目的：观察海人酸致痫大鼠海马及颞叶皮质区域天门冬氨酸受体亚单位1蛋白表达的变化。 设计、时间及地点：随机对照动物实验,于2002-03/2003-03在吉林大学基础医学院生理学和病理生物学教研室完成。 材料：兔抗天门冬氨酸受体受体亚单位1多克隆抗体购自武汉博士德生物科技公司。 方法：将80只雄性健康鼠龄22周的Wistar大鼠随机分为模型组70只和假手术组10只。模型组注射海人酸1 μL至右侧杏仁核,制备癫痫动物模型,分别于造模后2,6,24,72 h及7,15,30 d各取10只动物处死。假手术组在大鼠右侧杏仁核注入PBS 1 μL。 主要观察指标：致痫后2,6,24,72 h及7,15,30 d应用流式细胞仪技术及免疫组织化学的方法观察大鼠脑组织海马及颞叶皮质天门冬氨酸受体受体亚单位1蛋白表达的变化。 结果：假手术组海马各区及颞叶皮质有极少量的天门冬氨酸受体受体亚单位1蛋白阳性细胞分布,海人酸致痫后2 h大鼠海马各区及颞叶皮质天门冬氨酸受体受体亚单位1蛋白表达迅速增加,6 h明显升高,7 d略有下降（CA3区及颞叶皮质）但仍高于假手术组,并持续至30 d（P<0.01）。致痫后海马CA3区、齿状回与颞叶皮质相比,NMDAR1阳性细胞数增多 (P < 0.01)。 结论：海人酸致痫后大鼠海马及颞叶皮质天门冬氨酸受体受体亚单位1蛋白的表达水平均上调,在海马CA3及齿状回较为明显,并持续至致痫后的30 d。天门冬氨酸受体受体亚单位1可能参与了癫痫发生和癫痫脑的长时程的兴奋过程。     

海人酸癫痫大鼠模型的TNF-α及其mRNA表达特征

目的立体定向手术建立海人酸颞叶癫痫模型,检测癫痫大鼠海马内TNF-α及其mRNA的表达, 评价其意义。方法大鼠一侧海马CA3区注射海人酸,观察其行为学特征及HE染色的病理学改变,免疫组化和原位杂交法检测大鼠海马内TNF—α蛋白和mRNA的动态表达。结果大鼠注射海人酸后出现湿狗样抖动、头面部肌阵挛、肢体阵挛及全面强直阵挛发作等,病理可见海马神经元变性、缺失及胶质细胞增生,海马内TNF-α蛋白与 mRNA表达时程基本一致,3h出现,12h达高峰,而后逐渐下降,7d后回归至对照组表达水平,15d,30d又高于对照组。结论在一侧海马注射海人酸的大鼠癫痫模型中,内源性TNF—α参与了癫痫的发病机制。     

构建海人酸急性致痫小鼠模型

目的 建立海人酸诱导的小鼠急性癫痫模型,并探讨其特点.方法 实验取健康雄性昆明小鼠99只,随机分为生理盐水组(n=33)和致痫组(n=66),痫组腹腔注射海人酸10mg/kg,生理盐水组腹腔注射生理盐水35μl/g.注射后连续5h观察小鼠是否有痫性发作并分级.当小鼠持续痫性发作达1h时给予地西泮4mg/kg腹腔注射,对照组3只和致痫组9只同时描记脑电图,并取脑切片后苏木精-伊红染色法观察海马各区病理学改变.结果 ①行为学表现,模型组小鼠注射海人酸后可出现湿狗样抖动、头面部肌肉阵挛、肢体阵挛及全面强直阵挛发作.生理盐水对照组未见癫痫发作.②脑电图表现,癫痫持续状态小鼠表现为持续性节律性棘波、棘慢波或高波幅慢波.③病理学研究,双侧海马均可出现神经元变性,以CA1和门区为主.结论 腹腔注射海人酸致痫小鼠急性模型同相应的大鼠模型一样,具有制作简便、痫性发作潜伏期短、致痫率高等特点,其所产生的急性癫痫模型具有与人类颞叶癫痫相似的行为、脑电图与神经病理改变.     

癫痫动物模型的建立及多药耐药基因产物P-糖蛋白的表达

目的 建立局限性癫痫动物模型,探讨多药耐药基因产物P-糖蛋白的表达规律以及癫痫耐药机制。方法 42只Wistar大鼠随机分为对照组,海人酸致痫组,苯妥英钠(phenytoin sodium,PHT)+苯巴比妥(phenobarbital,PB)和加巴喷丁(gabapentin)抗癫痫药物干预组。致痫组大鼠于右侧海马区注射海人酸,于给药后第6h、24h、3d、5d和7d处死;干预组大鼠在注射海人酸前30min,分别行加巴喷丁(20mg/kg)或PHT(3～90mg/kg)+PB(30mg/kg)腹腔注射,然后注射海人酸,给药后第6h、3d、5d和7d处死。所有动物均采用免疫组织化学法检测多药耐药基因-1的表达产物P-糖蛋白。结果 对照组大鼠无癫痫发作。致痫组大鼠均出现癫痫发作,经抗癫痫药物干预的大鼠,癫痫发作的开始时间延迟、持续时间缩短(P<0.01)。不同剂量PHT组大鼠癫痫发作开始和持续时间有所不同,组间比较差异有显著性意义(P<0.01);随PHT剂量的增加,P-糖蛋白表达增高。干预组阳性细胞数高于致病组(P<0.01)。注射海人酸后6h,出现P-精蛋白强烈表达,3～5d后减弱,7d后完全消失;但干预组大鼠,在给药后第3d仍强烈表达,并逐渐增强,直至第7d仍无减弱趋势。结论 (1)一侧海马区注射海人酸,可成功地建立癫痫模型;(2)抗癫痫药物可使多药耐药基因-1的表达产物P-糖蛋白表达增强,且随药物剂量增加而     

海人酸致痫大鼠海马IL-1β、TNF-α的表达

目的 立体定向手术建立海人酸(KA)颞叶癫痫大鼠模型,检测海马内IL-1β、TNF-α蛋白及其mRNA的表达.方法 大鼠随机分为空白对照组、生理盐水对照组和模型组.模型组大鼠一侧海马CA3区注射KA (生理盐水组注射生理盐水),观察其行为学特征,HE染色和Nissl染色以及电镜观察其病理学改变,免疫组化法检测大鼠海马内IL-1β、TNF-α蛋白的表达,原位杂交法检测TNF-α mRNA的动态表达.结果 大鼠注射KA后出现湿狗样抖动、头面部肌阵挛、肢体阵挛及全面强直阵挛发作等,病理结果显示海马神经元变性、缺失及胶质细胞增生,模型组IL-1β在致痫后3 、6 h表达水平明显增加并于12 h达高峰,之后逐渐下降,7 d后与对照组相比差异无统计学意义(P＞0.05);TNF-α蛋白与mRNA表达时程基本一致,3 h出现,12 h达高峰,而后逐渐下降,7 d后回归至对照组表达水平,15 、30 d又高于对照组(P＜0.05).结论 (1)大鼠一侧海马注射KA是人类颞叶癫痫理想的动物模型;(2)内源性IL-1β、TNF-α参与了癫痫发病机制.     

中枢组胺对戊四氮点燃大鼠海马GABA能神经元的影响

目的:探讨中枢组胺抗癫痫的作用机制。方法:应用免疫组织化学方法研究中枢组胺对戊四氮(PTZ)致癫痫大鼠海马神经元GABA、GAD67表达的影响。结果:戊四氮致痫组大鼠海马神经元GABA、GAD67的表达量明显低于正常对照组,L-组胺酸干预组明显高于戊四氮致痫组,L-组胺酸干预组与正常对照组之间差异无显著性意义。结论:中枢组胺通过激活海马GABA能神经元来抑制癫痫的发生和发展。     

海人酸致痫大鼠海马细胞外氨基酸类神经递质的变化

目的 分析海马细胞外氨基酸递质在癫痫发生中的作用,探讨海人酸致痫模型大鼠癫痫发生的机制.方法 应用立体定向方法建立海人酸大鼠颞叶癫痫模型,观察大鼠行为学和电生理变化,应用电镜观察大鼠海马超微结构,应用微透析获取大鼠海马细胞外液,高压液相色谱法测定透析液中的兴奋性氨基酸谷氨酸、抑制性氨基酸牛磺酸及γ-氨基丁酸的含量.结果 海人酸注射后大鼠出现典型的颞叶癫痫发作,皮层脑电显示痫性发作,电镜显示兴奋性神经递质增加,高效液相色谱分析显示海马细胞外谷氨酸、牛磺酸和γ-氨基丁酸含量明显高于对照组(P＜0.05),虽然谷氨酸、γ-氨基丁酸都升高,而谷氨酸升高的更明显.结论 兴奋性氨基酸与抑制性氨基酸的失衡在海人酸致痫大鼠模型的癫痫发生过程中发挥重要作用,是癫痫发生的原因之一.     

A型钾通道Kv4.1在戊四唑致痫大鼠海马区的表达

目的通过检测A型钾通道Kv4.1在戊四唑(PTZ)致痫大鼠海马CA1、CA3及齿状回区的表达变化,探讨A型钾通道在癫痫发病机制中的作用。方法 SD大鼠40只,随机分为正常组、致痫后1 h、24 h、72 h组。腹腔注射PTZ制备大鼠癫痫模型,应用免疫组化及Western Blot技术检测Kv4.1在各时间段海马CA1、CA3及齿状回区的蛋白表达情况。结果致痫组大鼠海马区Kv4.1蛋白表达水平在致痫后1 h、24 h、72 h三个时间段均明显高于正常组(P<0.05);各致痫组之间Kv4.1蛋白表达水平无明显差异(P>0.05)。结论大鼠癫痫模型海马区A型钾通道Kv4.1蛋白表达增多,Kv4.1的表达上调可能在癫痫的发生中起作用。     

钙结合蛋白与颞叶癫痫大鼠海马神经元易损性的关系研究

目的 探讨微白蛋白(Parvalbumin,PV)、钙视网膜蛋白(Calretinin,CR)、钙结合蛋白-D28K(Calbindin-D28k,CB)与颞叶癫痫大鼠海马神经元易损性的关系及在颞叶癫痫的发生发展中的作用.方法 采用氯化锂-匹罗卡品颞叶癫痫大鼠模型,应用免疫组化动态观察海马神经元内PV、CR、CB的表达.结果 实验组大鼠PV阳性中间神经元的数量在CA3区无明显变化(P>0.05);在CA1区,呈进行性下降,到7d时达最低值(P<0.01);在齿状回及门区,先进行性下降,15d时达最低值(P<0.01),30d后开始上升,到60d几乎达正常水平(P>0.05).大鼠各区CR阳性中间神经元的数量较对照组均明显下降;在CA3区,急性期和慢性期下降最明显(P<0.01);在CA1区24h组的数量最少(P<0.01);在齿状回的门区15d组的数量最低(P<0.01).CB阳性中间神经元的数量在CA3区与对照组无明显变化(P>0.05);在CA1区,6h后开始减少(P<0.05),7d后达最低值(P<0.01),然后稍有回升,但仍明显低于对照组(P<0.05);门区CB阳性中间神经元的数量较对照组增加(P<0.05).结论 氯化锂-匹罗卡品颞叶癫痫动物模型中海马内含PV、CR、CB的GABA能中间神经元存在选择性易损性,CA1区含PV、CR、CB的GABA能中间神经元的急剧减少可能诱发了颞叶癫痫的急性发作;含PV、CB的GABA能中间神经元在齿状回的改变可能在颞叶癫痫自发性复发性发作的发生和发展中扮演了非常重要的角色.     

Up-regulation of GAD65 and GAD67 in remaining hippocampal GABA neurons in a model of temporal lobe epilepsy.

In the pilocarpine model of chronic limbic seizures, subpopulations of glutamic acid decarboxylase (GAD)-containing neurons within the hilus of the dentate gyrus and stratum oriens of the CA1 hippocampal region are vulnerable to seizure-induced damage. However, many gamma-aminobutyric acid (GABA) neurons remain in these and other regions of the hippocampal formation. To determine whether long-term changes occur in the main metabolic pathway responsible for GABA synthesis in remaining GABA neurons, the levels of mRNA and protein labeling for the two forms of GAD (GAD65 and GAD67) were studied in pilocarpine-treated animals that had developed spontaneous seizures. Qualitative and semiquantitative analyses of nonradioactive in situ hybridization experiments demonstrated marked increases in the relative amounts of GAD65 and GAD67 mRNAs in remaining hippocampal GABA neurons. In addition, immunohistochemical studies demonstrated parallel increases in the intensity of terminal labeling for both GAD65 and GAD67 isoforms throughout the hippocampal formation. These increases were most striking for GAD65, the isoform of GAD that is particularly abundant in axon terminals. These findings demonstrate that, in a neuronal network that is capable of generating seizures, both GAD65 and GAD67 are up-regulated at the gene and protein levels in the remaining GABA neurons of the hippocampal formation. This study provides further evidence for the complexity of changes in the GABA system in this model of temporal lobe epilepsy.     

Glutamate decarboxylase 67 is expressed in hippocampal mossy fibers of temporal lobe epilepsy patients

Recently, expression of glutamate decarboxylase-67 (GAD67), a key enzyme of GABA synthesis, was detected in the otherwise glutamatergic mossy fibers of the rat hippocampus. Synthesis of the enzyme was markedly enhanced after experimentally induced status epilepticus. Here, we investigated the expression of GAD67 protein and mRNA in 44 hippocampal specimens from patients with mesial temporal lobe epilepsy (TLE) using double immunofluorescence histochemistry, immunoblotting, and in situ hybridization. Both in specimens with (n = 37) and without (n = 7) hippocampal sclerosis, GAD67 was highly coexpressed with dynorphin in terminal areas of mossy fibers, including the dentate hilus and the stratum lucidum of sector CA3. In the cases with Ammon's horn sclerosis, also the inner molecular layer of the dentate gyrus contained strong staining for GAD67 immunoreactivity, indicating labeling of mossy fiber terminals that specifically sprout into this area. Double immunofluorescence revealed the colocalization of GAD67 immunoreactivity with the mossy fiber marker dynorphin. The extent of colabeling correlated with the number of seizures experienced by the patients. Furthermore, GAD67 mRNA was found in granule cells of the dentate gyrus. Levels, both of GAD67 mRNA and of GAD67 immunoreactivity were similar in sclerotic and nonsclerotic specimens and appeared to be increased compared to post mortem controls. Provided that the strong expression of GAD67 results in synthesis of GABA in hippocampal mossy fibers this may represent a self-protecting mechanism in TLE. In addition GAD67 expression also may result in conversion of excessive intracellular glutamate to nontoxic GABA within mossy fiber terminals.     

电压门控性钠通道mRNA在海人酸致痫大鼠表达的研究

目的:探讨电压门控性钠通道在癫痫发病机制中的作用。方法:采用海人酸颞叶癫痫模型,运用原位杂交技术检测不同时点海马DG区、CA1区、CA2区和CA3区SCN2A、SCN3A mRNA的表达。结果:SCN2A和SCN3AmRNA均表达于海马的DG区、CA1、CA2、CA3区。海人酸致痫后3小时在海马各区表达开始增强,6小时明显增强(P<0.05),12小时达到高峰(P<0.01),24小时开始下降,48小时恢复至正常水平。结论:电压门控性钠通道SCN2AmRNA和SCN3A mRNA的表达增加可能参与了颞叶癫痫急性期的发病。     

颞叶癫痫大鼠海马区NR2B／PSD-95的动态表达变化

目的:观察N-甲基-D-天冬氨酸受体2亚基B(NR2B)和突触后致密物95(PSD-95)蛋白在锂-匹罗卡品致大鼠海马表达的动态变化,探讨其在颞叶癫癎发生、发展中的作用。方法:采用锂-匹罗卡品颞叶癫癎大鼠模型,用免疫组织化学法观察不同时间点大鼠海马CA1、CA3、DG区NR2B和PSD-95蛋白表达。结果:NR2B和PSD-95蛋白在大鼠海马分布广泛,表达丰富;大鼠腹腔注射锂-匹罗卡品后,NR2B和PSD-95蛋白在海马各区表达逐渐减少,24h降至低谷,与对照组比较差异有显著意义(P<0.01);此后逐渐回升,但仍低于对照组;30d在海马CA1、CA3区表达再次降低,与对照组比较有显著意义(P<0.05)。结论:NR2B和PSD-95蛋白表达下调可能分别参与颞叶癫癎急性期和慢性自发发作期的保护性机制。     

杏仁核点燃癫痫鼠GAD67mRNA的表达

目的 探讨大鼠杏仁核点燃癫痫后脑组织谷氨酸脱羧酶（GAD）mRNA的表达及其在癫痫发作后表达变化的意义。方法 通过建立与人类癫痫发生,形成具有高度相似性的杏仁核点燃大鼠癫痫模型。采用原位杂交技术检测癫痫鼠颞叶及海马组织不同点燃时程GAD67mRNA表达。结果 点燃后1d,GAD67mRNA表达增多并且表达信号增强,至7d时达高峰,以后表达逐渐下降,但在点燃后49d,表达仍高于正常,与对照组及手术对照组相比有统计学差异。结论 在慢性癫痫发作中,GABA能神经元的活性增强,考虑是由于癫痫过程中兴奋性增强,引起GABA能神经元抑制功能代偿性增加的结果,即癫痫发作后GABA能神经元介入的抑制功能代偿性增加的,这可能是机体内源性抗癫痫机制增强的一种反应。     

Various modifications of the intrahippocampal kainate model of mesial temporal lobe epilepsy in rats fail to resolve the marked rat-to-mouse differences in type and frequency of spontaneous seizures in this model

Temporal lobe epilepsy (TLE) is the most common type of acquired epilepsy in adults. TLE can develop after diverse brain insults, including traumatic brain injury, infections, stroke, or prolonged status epilepticus (SE). Post-SE rodent models of TLE are widely used to understand mechanisms of epileptogenesis and develop treatments for epilepsy prevention. In this respect, the intrahippocampal kainate model of TLE in mice is of interest, because highly frequent spontaneous electrographic seizures develop in the kainate focus, allowing evaluation of both anti-seizure and anti-epileptogenic effects of novel drugs with only short EEG recording periods, which is not possible in any other model of TLE, including the intrahippocampal kainate model in rats. In the present study, we investigated whether the marked mouse-to-rat difference in occurrence and frequency of spontaneous seizures is due to a species difference or to technical variables, such as anesthesia during kainate injection, kainate dose, or location of kainate injection and EEG electrode in the hippocampus. When, as in the mouse model, anesthesia was used during kainate injection, only few rats developed epilepsy, although severity or duration of SE was not affected by isoflurane. In contrast, most rats developed epilepsy when kainate was injected without anesthesia. However, frequent electrographic seizures as observed in mice did not occur in rats, irrespective of location of kainate injection (CA1, CA3) or EEG recording electrode (CA1, CA3, dentate gyrus) or dose of kainate injected. These data indicate marked phenotypic differences between mice and rats in this model. Further studies should explore the mechanisms underlying this species difference.     

GAD65‐Ab encephalitis and subtle focal status epilepticus

Aims. To delineate common epilepsy features associated with the presence of glutamic acid decarboxylase autoantibodies (GAD65‐Ab). Methods. Three consecutive cases of GAD65‐Ab encephalitis patients, followed in our neurological department, were investigated with regards to clinical semiology and EEG. Results. These patients presented new‐onset subtle ictal clinical features. Patients 1 and 2 described prolonged and transitory feelings of “déjà vu ‐ déjà vécu” and a “dreamy state”. Patient 3 was admitted for subsequent transient aphasia events followed by paroxysmal behavioural disturbances. Epileptic origin of the symptoms was confirmed using either a standard EEG (observation of temporal status epilepticus in one case) or a prolonged EEG (focal epileptiform activity during an asymptomatic period for two patients). All patients suffered from clinical focal status epilepticus. Patients 1 and 2 presented with temporo‐mesial seizures in agreement with the definition for limbic encephalitis, whereas Patient 3 presented with neocortical (lateral temporal and frontal lobe) seizures arguing for a non‐limbic encephalitis. A high level of GAD65‐Ab was found in cerebral spinal fluid, confirming a diagnosis of epilepsy associated with GAD65‐Ab encephalitis. Conclusion. Encephalitis seems to be a frequent neurological syndrome associated with GAD65‐Ab disorders. Epilepsy may be more frequent and severe than currently suggested, as ictal semiology may be subtle for these outpatients in whom standard EEG is commonly falsely reassuring. Subtle focal status epilepticus is a particular semiology of the GAD65‐Ab encephalitis spectrum.     

Transient increase of P-glycoprotein expression in endothelium and parenchyma of limbic brain regions in the kainate model of temporal lobe epilepsy

Several recent studies have shown that the multidrug transporter P-glycoprotein (PGP) is over-expressed in endothelial cells from brain blood vessels of patients with refractory temporal lobe epilepsy (TLE), suggesting that altered drug permeability across the blood-brain barrier (BBB) may be involved in pharmacoresistance to antiepileptic drugs (AEDs). Furthermore, over-expression of PGP has been found in astrocytes of epileptogenic tissue. However, it is not known in which regions of the temporal lobe PGP over-expression occurs and whether the over-expression is a result of uncontrolled seizures, of the mechanisms underlying epilepsy, or of chronic administration of AEDs. In the present study, we used the rat kainate model of TLE to study the time-course of PGP expression in capillary endothelium and parenchyma of the hippocampus and several other limbic brain regions thought to be involved in TLE. Kainate was administered at a dose which produced a generalized convulsive status epilepticus (SE), which was limited to a duration of 90 min by diazepam. PGP was detected by immunohistochemistry either 24 h or 10 days after SE, using a monoclonal PGP antibody. In both kainate-treated rats and controls, PGP staining was observed mainly in microvessel endothelial cells and, to a much lesser extent, in parenchymal cells. Twenty-four hours after SE, significant increases in PGP expression were determined in endothelial cells of the dentate gyrus and in parenchymal cells of the CA1 and CA3 sectors of the hippocampus. Furthermore, increased PGP expression was observed in the amygdala, piriform, and parietal cortex, but not in the substantia nigra. Ten days after the kainate-induced SE, except for an increase in parenchymal PGP expression in the dentate hilus and CA1 sector, no significant differences to controls were determined, indicating that most PGP increases seen 24 h after SE were only transient. The data indicate that PGP over-expression is a transient result of seizures and occurs in several regions of the temporal lobe. Seizure-induced over-expression of PGP in capillary endothelial cells of the BBB is likely to reduce the penetration of AEDs into brain parenchyma, which could explain the drug-refractoriness of seizures in TLE.     

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

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 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 Wiley-Liss, Inc.