褪黑素对阿尔茨海默病模型大鼠学习记忆能力及海马CA3区神经元的保护作用

目的 观察褪黑素(MT)对阿尔茨海默病(AD)模型大鼠学习记忆及海马CA3区神经元的保护作用.方法 随机将60只雄性Wistar大鼠分为正常对照组、AD模型组与MT治疗组.采用右侧海马CA1区注射β-淀粉样蛋白1～40(Aβ1～40)构建AD动物模型,MT治疗组以MT(10mg/kg·d)腹腔注射,正常对照组与AD模型...     

免疫调节剂对致痫大鼠脑内神经元及小胶质细胞内谷氨酸和蛋白激酶C免疫反应的影响

为了探讨免疫调节作用对癫痫发病的影响与相关机制,应用免疫细胞化学技术研究免疫调节剂干预戊四氮(pentyle-netetrazol,PTZ)致痫时,大鼠脑内及培养的小胶质细胞内谷氨酸(Glu)和蛋白激酶C(PKC)的表达变化。结果显示:在体实验中大鼠大脑皮质及海马,Glu和PKC在生理盐水对照组(NS组)、戊四氮组(PTZ组)、左旋咪唑+戊四氮组(LMS+PTZ组)、地塞米松+戊四氮组(DEX+PTZ组)均有不同的表达,且二种物质变化趋势基本一致。其中PTZ组较NS组表达增强,LMS+PTZ组进一步增强,DEX+PTZ组较PTZ组和LMS+PTZ组明显减弱。离体实验中,PTZ作用小胶质细胞2h即可引起Glu和PKC表达增多,6h达峰值,12h表达有所减弱,且PTZ组Glu和PKC表达较NS组明显增多,免疫增强剂(levamisole,LMS)可进一步增强Glu和PKC的表达;免疫抑制剂(dexamethasone,DEX)则下调其表达。以上结果提示免疫调节剂可能通过作用于神经元和小胶质细胞内Glu和PKC的表达而共同影响癫痫的发病机制和发病状态。     

大鼠脑内代谢型谷氨酸受体2和3亚型的分布

用免疫组化技术观察了代谢型谷氨酸受体２和３亚型阳性神经元胞体和纤维在大鼠脑内的定位分布。证明此二亚型阳性胞体和纤维密集分布于小脑颗粒细胞层；中等密度淡染的阳性胞体和纤维见于大脑皮质浅层、屏状核吻侧部、尾壳核、苍白球、腹侧苍白球、斜角带核垂直部、隔区、终纹床核和外侧网状核等处；小脑Ｐｕｒｋｉｎｊｅ细胞层和分子层内仅有少量淡染的阳性纤维。     

梓醇对谷氨酸诱导SD大鼠海马神经元损伤的保护作用

目的梓醇对谷氨酸诱导SD大鼠海马神经元损伤的影响。方法将培养7～9d的SD大鼠海马细胞进行NSE鉴定,然后随机分为:正常对照组;谷氨酸损伤组(Glu);谷氨酸损伤前予梓醇组(CAT+Glu)。采用倒置显微镜观察细胞形态学变化,噻唑蓝(MTT)法测定细胞存活率,比色法测定培养液中乳酸脱氢酶(LDH)含量,流式细胞术测定细胞凋亡率。结果与正常对照组比较,0.1mmol·L-1谷氨酸作用于海马神经元24h,出现明显细胞损伤,细胞存活率下降,培养液中LDH含量明显增加,细胞凋亡率增高(<0.01);0.2、1.0、5.0mg·L-1的梓醇可不同程度的改善谷氨酸损伤引起的神经细胞形态的改变,提高细胞存活力,减少LDH的漏出,降低细胞凋亡率,并呈一定的剂量依赖性。结论梓醇对谷氨酸诱导的海马神经细胞损伤有保护作用。     

EF-Tumt和EF-Tsmt在实验性癫痫大鼠脑内的表达

目的：了解线粒体功能与癫痫发病之间的关系。方法：建立大鼠癫痫持续状态模型,应用免疫组织化学法观察在癫痫发作时,大脑皮质、海马和纹状体中线粒体蛋白质翻译延长因子Tu和Ts表达的变化。结果：(1)在大脑皮质和海马中,EF-Tumt表达随癫痫发作时间延长而增加。在癫痫发作后4h时EF-Tumt的表达数量达到最高值,在48h时其表达又下降。在大鼠纹状体中0．5、4和48h表达均升高。(2)大脑皮质和海马中的EF-Tsmt表达随癫痫发作时间的延长而增加,在癫痫发作后4h达到最高值,48h又有所下降。纹状体中的EF-Tsmt表达则一直呈上升趋势。结论：癫痫持续发作可导致大鼠有关脑区线粒体蛋白质的合成功能增强。     

脑内炎症反应对大鼠黑质多巴胺能神经元长时程毒性作用的研究

目的观察黑质部位炎症反应对多巴胺能神经元的长时程毒性作用,探讨脑内炎症反应在黑质多巴胺能神经元慢性变性过程中的作用。方法健康SD雄性大鼠28只,随机分为生理盐水组和10μg、25μg、50μg脂多糖组。定位注射生理盐水或脂多糖入右侧脑室,术后24周观察大鼠行为学改变,免疫组织化学染色观察黑质部位小胶质细胞的激活情况及酪氨酸羟化酶阳性神经元的变化。结果1.大鼠行为学观察显示,10μg、25μg和50μg脂多糖组大鼠的平均运动速度与对照组相比,差异无统计学意义。2.OX-42免疫组织化学染色显示,10μg和25μg脂多糖组大鼠黑质部位小胶质细胞激活明显。50μg脂多糖组大鼠黑质部位小胶质细胞激活不明显。3.酪氨酸羟化酶免疫组织化学染色显示,10μg和25μg脂多糖组大鼠黑质酪氨酸羟化酶阳性神经元与对照组相比,胞体变小,突起减少甚至消失,染色变浅;50μg脂多糖组大鼠酪氨酸羟化酶阳性神经元形态与对照组相比没有明显改变。4.酪氨酸羟化酶阳性细胞计数显示,10μg和25μg脂多糖组大鼠与对照组比较,分别减少15.5%(P<0.01)和20.1%(P<0.01);50μg脂多糖组大鼠与对照组比较没有统计学差异。结论脑室注射脂多糖引发的脑内炎症可导致黑质多巴胺能神经元变性,这一过程呈慢性迟发性;同时,低剂量脂多糖激活小胶质细胞对黑质多巴胺能神经元的慢性毒性作用更为明显;该病理过程较好地模拟了帕金森病的发病特点。     

外源性硫化氢保护实验性癫痫持续状态大鼠海马神经元作用的研究

目的:为研究外源性硫化氢(H_2S)对实验性癫痫持续状态(status of epilepticus,SE)大鼠海马神经元的保护作用,我们用供体Na HS对氯化锂-匹罗卡品所致幼鼠SE海马神经元的影响,并对SE引起的脑损伤进行靶点干预。方法:用氯化锂-匹罗卡品建立大鼠SE模型,并分为正常+Na HS组,癫痫组,癫痫+Na HS组和癫痫+羟胺组,另设正常组(对照组),每组6只。用比色法测定血浆和海马H_2S浓度,用Real-Time PCR检测海马胱硫醚-β合酶(CBS)基因mRNA表达,用HE染色和Nissl染色观察海马神经元形态。结果:癫痫组血浆和海马组织H_2S浓度和CBS mRNA表达明显低于正常组(P0.01),癫痫+Na HS组血浆和海马组织H_2S浓度和CBS mRNA表达明显高于癫痫组(P0.01),癫痫+羟胺组血浆和海马组织H_2S浓度和CBS mRNA表达显著低于癫痫组(P0.05)。HE染色显示,癫痫组海马CA3区锥体细胞明显减少,细胞层次紊乱,形态不规则;尼氏染色显示,癫痫组海马CA3区尼氏体明显减少,锥体细胞排列紊乱;与癫痫组比较,癫痫+Na HS组海马尼氏体缺失和锥体细胞排列紊乱明显改善。结论:外源性H_2S对实验性癫痫持续状态大鼠海马组织CBS mRNA表达具有上调作用,并提高血浆和海马组织H_2S含量。同时,外源性H_2S对实验性癫痫大鼠海马组织神经元具有保护作用。     

养血清脑颗粒对蒙古沙鼠全脑缺血再灌注后海马CA1区神经元的治疗作用

目的 探讨蒙古沙鼠全脑缺血再灌注后养血清脑颗粒(Yangxueqingnaokeli,YXQNKL)的治疗作用.方法 采用蒙古沙鼠两侧颈总动脉结扎法,缺血30min再灌注5d模型(分为假手术组、缺血再灌注组、养血清脑颗粒治疗组).用Nissl染色法观察海马CA1区神经元的形态和数量,用免疫组织化学方法观察海马CA1区神经元神经钙离子感应蛋白1(NCS-1)、天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)和谷氨酸盐合成酶(Glusyn)的表达情况.结果 与缺血再灌注组比较,缺血再灌注 0.4g/kg养血清脑颗粒治疗组和缺血再灌注 0.8g/kg养血清脑颗粒治疗组Nissl染色显示海马CA1区神经元数量明显增加(P＜0.05);免疫组织化学法显示海马CA1区神经元细胞NCS-1、caspase-3和Glusyn的阳性细胞数明显减少(P＜0.05).结论 全脑缺血再灌注后,给予养血清脑颗粒能显著增加海马CA1区神经元数量,这与其减少海马CA1区神经元NCS-1、caspase-3和Glusyn的表达有密切的联系.     

红景天苷类似物MADP对谷氨酸诱导的大鼠海马神经元凋亡的保护作用

目的:观察新型红景天苷(salidroside,Sal)类似物4-甲氧基苯甲基-2-乙酰氨基2-脱氧-β-D-吡喃糖苷(4-methoxy-2-acetamido-2-deoxy-β-D-pyranoside,MADP)对谷氨酸(glutamate)损伤海马神经元的保护作用。方法:原代培养大鼠胚胎海马神经元,与浓度分别为60、120、240μmol/L的MADP或240μmol/L的Sal共同孵育24 h,加入125μmol/L谷氨酸损伤海马神经元15 min。使用相差显微镜观察海马神经元的形态变化;四甲基偶氮唑盐(methyl thiazolyl tetrazolium salt colorimetry,MTT)比色法测定细胞活力;甲基百里香酚蓝(methyl thymol blue,MTB)法测定细胞内游离的钙离子浓度;采用实时荧光定量PCR技术检测细胞凋亡相关基因Bcl-2,Bax,Caspase-3的表达水平。结果:MADP或Sal预处理24 h可改善谷氨酸损伤后神经元胞体的肿胀和突起的淡化,抑制胞内游离钙离子浓度的上升,提升Bcl-2/Bax mRNA的表达水平,降低Caspase-3 mRNA的表达水平。MADP对大鼠海马神经元的保护作用优于Sal。结论:与Sal相比较,MADP更好的发挥了拮抗谷氨酸对海马神经元损伤的作用,其机制可能与抑制钙离子内流,上调Bcl-2/Bax mRNA及下调Caspase-3 mRNA的表达水平相关。     

脑内炎症对黑质多巴胺能神经元的选择性损伤作用

目的 探讨脑内炎症反应对中脑黑质多巴胺能神经元的选择性变性作用.方法 健康SD雄性大鼠10只,随机分为实验组和对照组,实验组行脂多糖(LPS)右侧脑室定位注射,对照组注射生理盐水.注射后48周用免疫组织化学或组织化学法观察黑质多巴胺能、中缝核5-羟色胺能及基底核胆碱能3种不同类型神经元的变性及上述不同脑区小胶质细胞的激活情况.结果 免疫组织化学染色显示,LPS组在黑质、海马、纹状体、中缝核部位均可见到OX6阳性小胶质细胞,说明不同脑区均出现炎症反应.不同类型神经元染色结果显示,LPS组黑质多巴胺能神经元胞体变小、染色变浅、突起减少甚至消失,神经元数量比对照组减少40.1%(P<0.01);5-羟色胺能神经元及胆碱能神经元形态及数量均无明显改变.结论脑室注射LPS导致的脑内炎症反应可选择性引起黑质多巴胺能神经元变性损伤.     

Increased expression of GAD mRNA during the chronic epileptic syndrome due to intrahippocampal tetanus toxin

Summary A few mouse minimum lethal doses (MLD) of tetanus toxin injected into rat hippocampus triggers prolonged changes in neuronal function. Spontaneously recurring epileptic discharges arise in both the injected and the contralateral, uninjected hippocampus. The seizures remit after about 6 weeks, to be succeeded by a permanent depression of hippocampal neuronal responses. There is no evidence of any loss of pyramidal cells at this low dose of toxin. Here we studied presumptive inhibitory, GABAergic neurons, using in situ hybridization (ISH) with a probe directed against the mRNA encoding glutamic acid decarboxylase (GAD), at each of 1,2,4 and 8 weeks after injection of tetanus toxin. Epileptic activity was recorded from hippocampal slices prepared from both injected and contralateral hippocampi of rats at each time point, unexpectedly persisting until 8 weeks. There were no significant differences in the numbers of neurons containing GAD mRNA between toxin- and vehicle-injected and control rats in any hippocampal subfield, at any survival time, except for an apparently transient loss of hilar signal in vehicle-injected rats at 1 and 2 weeks which we attribute to a significant, transient loss of neuronal GAD mRNA to below the threshold for detection by ISH using this probe. In contrast there was a marked increase in GAD mRNA in the toxin-injected group, which reached a peak at 4 weeks, and returned to control levels by 8 weeks. The changes were bilateral and were most marked in the hilus of the dentate area, but were also significant in CA3 and CA1. Upregulation of GAD mRNA was preceded by an increase in the levels of the mRNA for the subunit of the GTP binding protein, Gs (Gs), at 2 weeks which affected the GABAergic neurons selectively, and not the pyramidal or granule cells. These marked changes in GAD mRNA may contribute to putative adaptive responses within GABAergic neurons, which would help contain epileptic activity in these chronic foci. The changes in GAD expression may be due to mechanisms acting through an increase in mRNA encoding Gs.     

Inhibition of altered peptide ligand-mediated antagonism of human GAD65-responsive CD4+ T cells by non-antagonizable T cells

Altered peptide ligands derived from T cell-reactive self antigens have been shown to be protective therapeutic agents in animal models of autoimmunity. In this study we identified several altered peptide ligands derived from the type 1 diabetes-associated autoantigen human glutamic acid decarboxylase 65 (hGAD65) epitope that were capable of antagonizing a subset of a panel of human CD4(+) GAD65 (555-567)-responsive T cell clones derived from a diabetic individual. While no altered peptide ligand was able to antagonize all six clones in the T cell panel, a single-substituted peptide of isoleucine to methionine at position 561, which resides at the TCR contact p5 position, was able to antagonize five out of the six hGAD65-responsive clones. In a mixed T cell culture system we observed that altered peptide ligand-mediated antagonism is inhibited in a dose-dependent manner by the presence of non-antagonizable hGAD65 (555-567)-responsive T cells. From an analysis of the cytokines present in the mixed T cell cultures, interleukin-2 was sufficient to inhibit altered peptide ligand-induced antagonism. The inhibition of altered peptide ligand-mediated antagonism of self-antigen-responsive T cells by non-antagonizable T cells has implications in altered peptide ligand therapy where T cell antagonism is the goal.     

大鼠GAD65重组腺病毒载体的构建和鉴定

目的构建大鼠GAD65重组腺病毒载体。方法将目的基因大鼠GAD65 cDNA亚克隆到穿梭质粒pShutile启动子CMV的下游,再通过I-CeuⅠ和PI-SceⅠ两个稀有酶切位点,将目的基因GAD65与腺病毒质粒DNA(pAdeno-X)进行体外连接,获得含目的基因GAD65重组腺病毒质粒DNA,后者经限制性内切酶PaeⅠ切割后,两端露出反向末端重复序列(rTR),利用脂质体转染293细胞,获得含目的基因重组腺病毒上清。PCR双引物检测含有目的基因GAD65的片段。结果在挑选的5个空斑中,有4个含GAD65 cDNA阳性重组腺病毒。GAD65重组腺病毒可感染293细胞并在293细胞内进行有效的复制。PCR双引物检测证明含有目的基因GAD65片段,表明利用体外连接法成功构建了含目的基因GAD65重组腺病毒转移载体。结论成功构建了GAD65重组腺病毒,为进一步研究GAD65重组腺病毒的功能提供了条件。     

Comparison through immunoblotting of glutamic acid decarboxylase (GAD) from newborn and adult rat brain

Immunochemical characterization of glutamic acid decarboxylase (GAD) from brain extracts of newborn and adult rats was investigated using a GAD antiserum that was previously raised against brain GAD from adult rats. According to the immunoprecipitation and saturation curves, no significant differences could be found as to the recognition of newborn and adult GAD by the antiserum. On immunoblots, both extracts revealed the same two immunolabelled bands (mol.wt. 59,000 and 62,000 ± 2000 Da). In both cases, the lightest band showed the strongest staining. Quantitative analysis of the immunolabelling indicated that each immunolabelled band was enriched about 10-fold in the adult brain extract. These data did not reveal any difference between newborn and adult GAD that was reminiscent of the difference found in an earlier study between GAD from lower and higher vertebrates. Whatever the regulatory mechanism responsible for the presence of two forms of GAD in the adult brain, it is already fully operative in newborn animals.     

Anticardiolipin,glutamic acid decarboxylase,and antinuclear antibodies in epileptic patients

To explore the hypothesis that raised anticardiolipin antibodies, glutamic acid decarboxylase, and antinuclear antibodies may be associated with epilepsy and/or pharmacoresistance, we studied titers in 74 epileptic patients and 50 controls. Epileptic patients were divided into two groups according to their response to anticonvulsant therapy. Group I included 52 children (30 females and 22 males with a mean age±SD of 7.0±2.4 years) suffering from different types of epilepsy who were treated with various anticonvulsants. Group II included 22 children (10 females and 12 males with a mean age of 6.2±3.6 years) suffering from therapy resistant epilepsy. We found that the prevalence of anticardiolipin antibodies was significantly higher in epileptic patients than in controls, while there was no significant difference between patients who were seizure free and those with uncontrolled epilepsy. No significant difference was found in glutamic acid decarboxylase antibodies between epileptic children and controls, and between patients who were seizure free and those with uncontrolled epilepsy. A significant difference in the incidence of antinuclear antibodies was found between epileptic children and controls, while no difference was found between well-controlled and drug-resistant epilepsy. In conclusion, the prevalence of anticardiolipin and antinuclear antibodies was higher in patients with epilepsy than in controls. There was no significant difference in serum glutamic acid decarboxylase antibodies between epileptic children and controls, and between patients who were seizure free and those with uncontrolled epilepsy. Received: 10 October 2002 / Accepted: 18 February 2003 Correspondence to A. Verrotti     

Glutamic acid decarboxylase (GAD)-immunoreactive structures in the adult human lateral geniculate nucleus

Summary The distribution of glutamic acid decarboxylase (GAD)-like immunoreactivity in the lateral geniculate nucleus (LGN) of the human adult was studied in vibratome sections (50–60 urn thick) using the avidinbiotin-peroxidase method. The tissue was obtained at autopsy from five individuals without any known neurological disorders. Only few GAD-immunoreactive neurons were present in the layers of the LGN, even less in the interlaminar zones. The numerical density of GAD-immunoreactive neurons and puncta (probably synaptic boutons and/or cross sectioned cell processes) in the magnocellular layers was larger than in the parvocellular layers. Furthermore, no striking differences between the individual parvocellular layers were noted. The immunoreactive somata were polygonal or triangular, occasionally pear-shaped, and ranged in size from 15 to 25 urn. They gave off two to four short, thick, straight primary dendrites. A preferred orientation of dendrites was not recognized. After bleaching the chromogen 4-chloro-1-naphthol and staining for lipofuscin pigment granules and basophilic material, 254 unequivocally relocated GAD-immunoreactive nerve cells could be classified as belonging to the lipofuscin pigment granules-containing class of interneurons.     

Neural elements containing glutamic acid decarboxylase (GAD) in the dorsal lateral geniculate nucleus of the rat; immunohistochemical studies by light and electron microscopy

Immunoreactive constituents of the dorsal lateral geniculate nucleus of adult albino rats were examined by light- and electron-microscopy, using the unlabelled antibody enzyme method, following treatment of brain slices with a purified antibody to glutamic acid decarboxylase. The neuropil of the dorsal lateral geniculate nucleus displayed a conspicuous granular immunoreactivity. In addition, the antibody was bound to a class of small neurons of characteristic morphology. These cells possessed few (commonly 2-4) sparsely branched, long dendrites from some of which immunoreactive appendages were traced. Many cells were bipolar in form, and the dendrites of some appeared to be preferentially orientated. The immunoreactive cells closely resembled intrinsic interneurons characterized in previous Golgi studies of this nucleus. By electron-microscopy, immunoreactive presynaptic elements were present both in the extraglomerular neuropil and in the synaptic glomeruli. The former were axon terminals containing flattened synaptic vesicles and making Gray type II axo-dendritic synaptic contact; they appeared to correspond to axon terminals whose origin in the thalamic reticular nucleus has been established in previous studies, but it is possible that some were axon terminals of intrinsic interneurons. The immunoreactive glomerular components also contained flattened vesicles, were presynaptic to presumptive projection cell dendrites, postsynaptic to retinal axon terminals, and participated in triplet (triadic) and other complex synaptic arrangements. They corresponded in all respects to the synaptic portions of the complex dendritic appendages of intrinsic interneurons, identified and characterized in previous studies. The finding that there are high levels of glutamic acid decarboxylase in the cell bodies, dendritic shafts and dendritic appendages of intrinsic interneurons in the dorsal lateral geniculate nucleus of the rat, and in the axon terminals of fibres projecting to this site from the thalamic reticular nucleus, allows us to conclude that the inhibitory inputs to the geniculo-cortical projection cells from both of these sources are probably mediated by gamma-aminobutyric acid.     

Anatomical relationships of dopaminergic and GABAergic systems with the GnRH-systems in the septo-hypothalamic area

Summary Immunohistochemical double staining for gonadotropin releasing hormone (GnRH) and tyrosine hydroxylase (TH) or glutamic acid decarboxylase (GAD) reveals in the septo-preopticdiagonal band complex of the rat brain close spatial associations between GnRH-immunoreactive perikarya and TH and GAD immunoreactive fibers. In the organum vasculosum laminae terminalis, no close spatial relationships could be observed between TH-or GAD-positive fibers and the GnRH-containing system. In contrast, in the median eminence substantial overlap exists in the distribution of GnRH with TH and GAD containing nerve fibers. This overlap is most intense for TH throughout the lateral palisade zone, while for GAD it is more restricted to the outermost portion of the external palisade zone. The results suggest that dopamine and GABA influence GnRH secretion via axosomatic contacts in the septo-preoptic-diagonal band complex, as well as via axo-axonic interactions in the median eminence, while no such interactions seem to exist in the organum vasculosum laminae terminalis. Since dopaminergic cell bodies in the ventral hypothalamus are closely apposed by GnRH and GAD containing fibers, the existence of feedback circuits among GnRH, dopamine and GABA systems is proposed.     

GABAergic inhibition in the rat pontine nuclei is exclusively extrinsic: evidence from an in situ hybridization study for GAD67 mRNA

 As clearly indicated by our electrophysiological work, GABAergic inhibition plays a powerful role in the pontine nuclei (PN), the major link between cerebral cortex and the cerebellum. Using the technique of in situ hybridization for the mRNA encoding for the γ-aminobutyric acid (GABA)-synthesizing isoenzyme glutamic acid decarboxylase₆₇ (GAD₆₇), we demonstrate here the total absence of potentially GABAergic neurons from the rat PN. This negative finding supports the notion that GABAergic inhibition in the PN of rats, unlike that of higher mammals, is exclusively based on extrapontine GABAergic afferents. Received: 20 August 1998 / Accepted: 29 October 1998     

Comparison of glutamic acid decarboxylase 67 immunoreactive neurons in the hippocampal CA1 region at various age stages in dogs

The hippocampus is a main brain region concerning learning and memory processes. It is imperative to determine the extent of alterations in number and function of inhibitory GABAergic interneurons in the hippocampus as a function of age. We examined changes in GABAergic neurons in the hippocampal CA1 region at various ages of dogs using glutamic acid decarboxylase 67 (GAD67), which is a rate-limiting enzyme for GABA synthesis. We found only one band in the brain homogenates in dogs as well as mice and rats. GAD67 immunoreactive neurons in 1-year-old dogs were mainly detected in the stratum oriens. In the 6-year-old group, GAD67 immunoreactive neurons were evenly distributed in the CA1 region, and numbers of the neurons were highest among all experimental groups. Thereafter, GAD67 immunoreactive neurons were significantly decreased region with age: GAD67 immunoreactive neurons were scarcely found in the CA1 region in 10-year-old dogs. The reduction of GAD67 immunoreactive neurons in the hippocampal CA1 region may be closely related to highly susceptibility to memory loss in old aged dogs.