戊四氮点燃大鼠中海马谷氨酸转运体的作用研究

目的 研究点燃形成过程中和点燃后谷氨酸转运体的变化,进一步探讨慢性癫痫的点燃机制。方法 将78只雄性成年Wistar 大鼠随机分为对照组（I组）和戊四氮（PTZ）组（Ⅱ组）。Ⅱ组腹腔注射阈下剂量的PTZ（35mg/kg),每日1次,直至达到点燃标准;Ⅰ组腹腔注射等量生理盐水。采用逆转录聚合酶链式反应（RT－PCR）方法检测海马区谷氨酸转运体－1（GLT－1）mRNA和兴奋性氨基酸载体－1（EAAC1）mRNA的表达。结果 GLT－1mRNA的表达在0h、48h时显著升高,随后下降;EAAC1mRNA的表达呈上升趋势。点燃后第60d时,基本恢复至对照组水平。结论 海马区GLT－1的下降和EAAC1的升高可能与癫痫敏感性的形成与维持有关。     

胶质细胞谷氨酸转运体在大鼠点燃效应中的作用研究

目的 研究点燃形成过程中和点燃后大鼠海马中氨酸天门氨酸转运体（GLAST）和谷氨酸转运体1（GLT－1）的变化,进一步探讨癫痫的形成机制。方法 将78只雄性成年Wistar大鼠随机分为对照组（I组）和戊四氮（PTZ）组（Ⅱ组）。Ⅱ组腹腔注射阈下剂量的PTZ（335mg/kg）,每日1次,直到达到点燃标准;I组腹腔注射等量生理盐水。采有RT－PCR方法检测海马区GLAST和GLT－1mRNA的表达。结果 PTZ组点燃后,GLASTmRNA的表达下降,60天时恢复至对照组;与对照组比较,PTZ组GLT－1mRNA的表达,在给药后15天时开始上升,点燃后0小时和48小时时显著升高,此后呈下降趋势。60天时,两组比较无明显差异。结论 海马区胶质细胞谷氨酸转运体的下降可能与癫痫敏感性的形成有关。     

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。     

海马颗粒细胞和苔藓纤维表达GABA的研究

颗粒细胞和苔藓纤维除了释放谷氨酸外,还能合成和释放γ-氨基丁酸(GABA).研究发现大鼠出生三周内可在海马颗粒细胞和苔藓纤维内发现GABA能标志物:GABA、谷氨酸脱羧酶、囊泡GABA转运体、囊泡GABA转运体mRNA和GABA膜转运体mRNA水平升高,但成年大鼠只能在高兴奋状态后检测到这些标志物的短暂升高.电生理学研究使海马CA3区锥体细胞内苔藓纤维释放GABA产生的IPSP区别于其他的干扰性IPSP,也揭示出此种释放的一些特点,如GABA与谷氨酸释放于不同囊泡,并能被Ⅲ型谷氨酸受体阻断.苔藓纤维GABA的释放可以产生神经保护、兴奋抑制和抗癫痫的作用.     

戊四氮点燃过程中大鼠海马胶质细胞增生和突触重建的研究

目的探讨戊四氮点燃过程中海马胶质细胞增生及突触重建与慢性癫痫发病机制的关系。方法大鼠随机分为对照组、非药物干预组(戊四氮35mg/kg,腹腔注射,每日一次)和药物干预组(苯巴比妥30mg/kg,戊四氮35mg/kg,均为腹腔注射,每日一次)。采用免疫组织化学方法观察胶质原纤维酸性蛋白(GFAP)和神经细胞粘附分子(NCAM)表达水平。结果非药物干预组大鼠注射戊四氮后在行为学未出现惊厥,脑电图未出现痫性放电的点燃前潜伏期内,出现突触重建和胶质细胞增生,以海马CA3区、门区明显,与对照组比较,有显著性差异(P<0.05);与药物干预组对应时间点比较,亦有显著性差异(P<0.05)。结论大鼠注射戊四氮后引起反应性胶质细胞增生和神经元可塑性改变,可能与形成异常神经元放电环路,最终诱发癫痫发作有关,苯巴比妥可抑制异常神经网络的建立,预防癫痫发生。     

戊四氮点燃过程中大鼠海马 BMP4表达与神经增殖关系

目的探讨戊四氮(PTZ)点燃过程中大鼠海马骨形成蛋白4(bone morphogenetic protein-4,BMP4)的表达变化与神经增殖的关系。方法将成年大鼠分为对照组与模型组,模型组大鼠又根据在点燃中的不同时相点分为9组。用免疫组织化学与原位杂交的方法检测海马齿状回BMP4mRNA与BrdU阳性细胞数的变化。结果正常成年大鼠BMP4阳性细胞主要分布于齿状回的门区、颗粒下层、CA3、CA1区。BrdU阳性细胞主要分布在齿状回颗粒下层。BMP4阳性细胞与BrdU阳性细胞在点燃过程中均明显增加,呈明显正相关,点燃后2月降至基线水平。结论BMP4可能通过影响成年大鼠海马神经发生在PTZ点燃过程中起重要作用。     

海马神经细胞活化在戊四氮点燃大鼠癫痫形成过程中的作用

目的 以核因子κB/P65(nuclear factor κB,NF-κB/P65)的核转位作为神经细胞活化的标志,观察在戊四氮(pentylenetetrazol,PTZ)点燃大鼠出现惊厥之前,即点燃过程中海马神经细胞NF-κB活化在癫痫形成过程中的作用.方法 将大鼠随机分为对照组、非药物干预组、药物干预组(苯巴比妥30 mg/kg, 腹腔注射,每日一次).除对照组外均以低于急性致惊剂量的PTZ(40mg/kg,腹腔注射,每日一次)点燃大鼠,用行为学观察和脑电图确定癫痫存在,免疫组织化学方法 检测大鼠癫痫形成过程中不同时间点海马CA各区和齿状回神经细胞NF-κB活化,用图像分析系统分析对照组、非药物干预组和药物干预组三大组间海马神经细胞NF-κB活化的差异.结果 非药物干预组大鼠均于17d～22d点燃,而药物干预组PTZ点燃大鼠所需时间明显延长(于30d～35d点燃),且行为学惊厥程度和脑电痫样放电明显轻于非药物干预组.非药物干预组大鼠在行为学未出现惊厥,脑电图未出现痫样放电的点燃前潜伏期内,其海马CA各区、齿状回NF-κB活化的阳性神经细胞数明显增加,与对照组相比两者有显著性差异(P<0.05);药物干预组在与非药物干预组相应时间点的海马CA各区、齿状回NF-κB活化的阳性神经细胞明显减少,两者有显著性差异(P<0.05).结论 海马神经细胞活化是PTZ点燃大鼠癫痫形成的重要机制之一,苯巴比妥可通过抑制神经细胞活化,预防癫痫发生.     

γ-氨基丁酸转运体与癫痫

GABA转运体可以快速摄取突触间隙的GABA ,同时又可以通过逆转运释放GABA。因此 ,转运体可以调节细胞外的GABA浓度及作用时程。本文就GABA转运体的分类及在癫痫发病中的作用进行综述。     

戊四氮点燃大鼠海马CA3区钙超载与亚低温对点燃大鼠脑保护作用的研究

目的 海马CA1区钙超载是否在癫痫的发病机制中起主要作用及亚低温对癫痫是否有治疗意义。方法用荧光倒置显微镜来测定癫痫状态下海马CA1、CA3区钙超载的情况，并用钙离子拮抗剂尼莫地平作用于海马脑片看钙超载的变化。然后将大鼠分为头皮温度为41℃、37℃、32℃、26℃的4组，通过对4组点燃大鼠行为学和海马组织病理学的观察来看亚低温对癫痫的脑保护作用。结果癫痫大鼠海马CA1，CA3区钙超载高于对照组．CA1区钙超载高于CA1区。在尼莫地平的作用下CA1区钙超载明显降低。组织病理学改变显示CA1区是海马区神经元变性、坏死最明显的部位。而亚低温下癫痫的发作程度和神经元变性、坏死最轻。结论钙超载参与了癫痫的发病机制，其中海马CA3区的钙超载在癫痫发病机制中起主要作用。而亚低温对脑有保护作用。     

海藻氨酸致痫大鼠海马谷氨酸转运体功能的变化

目的研究腹腔注射海藻氨酸致癫痫发作后海马谷氨酸转运体功能的动态变化,以探讨谷氨酸转运体在癫痫发生中的作用机制。方法60只健康成年雄性Wistar大鼠,随机分为海藻氨酸组和对照组。海藻氨酸组30只大鼠均腹腔注射海藻氨酸10mg/kg,分别于注射后4h、24h、48h、5d和7d依据Racine制定的行为学标准观察大鼠的行为学改变。同时还分别测定不同时间点海马突触膜颗粒和海马组织切片对3氢-左旋-谷氨酸(3H-L-Glu)的摄取量,以反映谷氨酸转运体于点燃后不同时间点的活性。结果与对照组相比,海藻氨酸组大鼠海马突触膜颗粒谷氨酸转运体功能于点燃后4h减弱,对3氢-左旋-谷氨酸的摄取量减少(P<0.05),并持续至注射后第5～7天(P<0.01);海马组织切片检查显示谷氨酸转运体功能在点燃后4～48h增强,于注射后第5～7天减弱(P<0.05)。结论谷氨酸转运体功能的变化与海藻氨酸致痫大鼠模型癫痫的发生及易感性有关。     

GABA受体基因转染对癫痫大鼠海马区病理变化的影响

目的探讨在下丘脑乳头体上核内转染γ-氨基丁酸(GABA)受体基因后对海人藻酸(KA)致痫大鼠海马病理变化的影响,从而为难治性癫痫的治疗开辟新的途径。方法在右侧杏仁核内注射KA制备癫痫动物模型作对照,GABA基因转染组则利用仙台病毒(HVJ)-脂质体转染法预先在下丘脑的乳头体上核内转染被脂质体包被的GABA受体基因,48h后在杏仁核内注射KA,两组大鼠分别进行HE染色观察。结果 GABA基因转染组大鼠海马区病理改变较KA对照组明显减轻。结论下丘脑内转染GABA受体基因后可以抑制癫痫发作的程度。     

血脑屏障上GABA转运体亚型的分布

目的 分离不含神经组织的高纯度脑微血管片段并研究其GABA转运体的亚型分布。方法 采用磁珠在体动脉灌注标记大鼠脑血管、机械和胶原酶消化相结合解离脑组织技术，经过筛去除大血管后，在磁场下分选出脑微血管片段。应用RT-PCR技术分析神经元特异表达基因microtubule—associated protein(MAP-2a)、胶质细胞特异表达基因glutamine synthetase、血管内皮细胞特异表达基因CD31及4种已发现的GABA转运体亚型基因的表达，克隆微血管片段表达的GABA转运体亚型基因并测序确证。结果 分离的脑微血管片段没有发现明显的神经组织附着；也没有检测到神经元和胶质细胞特异表达的mRNA。在该脑微血管片段检测到1种低亲和力的GABA转运体BGT-1和1种高亲和力的转运体GAT-2。结论 磁选获得的脑微血管片段适用于血脑屏障上转运体基因的检测与克隆研究。大鼠血脑屏障上存在GAT-2和BGT-1两种GABA转运体亚型。本研究没有在血脑屏障发现其他已报道的GABA转运体，是否存在新的GABA转运体，尚需进一步研究探讨。     

Spatial and temporal alterations in the GABA shunt in the gerbil hippocampus following transient ischemia

In the present study, we have identified the alteration in the expressions of GABA shunt-associated enzymes and the GABA transporter in order to determine the relationship between the neuronal damage and GABA metabolism following ischemia. At 30 min post-ischemia, the immunoreactivities of the glutamic acid decarboxylase (GAD) isoforms were markedly elevated in the CA1 region, as compared with the sham operated group. At 3-12 h post-ischemia, their immunoreactivities recovered at the sham level. These patterns were similarly observed up to 12 h following ischemia insult. However, the intensity of GAD67 was markedly increased at 24 h post-ischemic insult. The temporal changes in GABA transporter 1 (GAT-1) expressions were similar to that of GAD67, but not GAD65, expression, at least prior to 12 h after ischemic insults. GAT-1 immunoreactivity was significantly elevated in the CA1 region posterior to 12 h post-ischemia. Both succinic semialdehyde dehydrogenase (SSADH) and succinic semialdehyde reductase (SSAR) immunoreactivities were not altered in GABAergic neurons following ischemia. In contrast, in pyramidal cells, both SSADH and SSAR immunoreactivities showed chronological alterations in the CA1 region. Thus, our findings suggest that the differential alterations of GABA metabolism may be one of the important factors in neuronal damages induced by ischemia.     

海人酸致癫痫大鼠海马GABAB受体亚单位GBR1a mRNA表达的研究

目的　通过研究海人酸致癫痫大鼠海马 GABAB受体亚单位 GBR1a m RNA表达的变化 ,为进一步探明癫痫发病的受体分子生物学机制奠定基础。方法　在立体定位仪下 ,将海人酸注射至大鼠杏仁核制备癫痫模型。将大鼠随机分为正常组和海马致癫痫组 ,分别于不同时间取材 ,进行脑组织 GABAB受体亚单位 GBR1a m RNA原位杂交检测。结果　正常组海马各区均有极少量的 GABAB受体亚单位 GBR1a m RNA的分布 ;海人酸致癫痫组( 6 h,12 h,2 4h) GABAB受体亚单位 GBR1a m RNA水平显著高于对照组 ( P<0 .0 1)。结论　正常大鼠海马各区存在着少量的 GBR1a m RNA表达 ,海人酸致痫后 6 h,12 h,2 4h,GBR1a m RNA的表达水平上调。     

Reduced aggression in mice lacking GABA transporter subtype 1

Dysregulation of the brain GABAergic system has been implicated in the pathophysiology of violence and aggression. As a key regulator of central GABAergic activity, dysfunction of the GABA transporter subtype 1 (GAT1) represents a potential mechanism mediating pathologic aggression. We provide evidence that GAT1-/- mice and GAT1+/- mice exhibit lower aggressive behavior both in home cage resident-intruder test and neutral arena resident-intruder test, compared to wild-type mice (GAT1+/+). The pharmacologic effects of the GAT1 inhibitor, tiagabine and the GABA(A) receptor antagonist, bicuculline have been assessed in GAT1+/+ mice: tiagabine inhibits attacks but bicuculline induces attacks. Compared to GAT1+/- and +/+ mice, the GAT1-/- mice displayed a normal circadian pattern of home cage activity, but more activity overall. Meanwhile, reduced testosterone concentration was found in GAT1-/- mice compared to GAT1+/+ mice but not in GAT1+/+ mice treated with tiagabine, suggesting that testosterone is not directly involved in GAT1 mediated aggressive behavior regulation. These results showed that GAT1 is an important target involved in the regulation of aggressive behavior in mice, and long-term dysfunction of GAT1 may also result in the alteration of testosterone secretion.     

Anxiolytic-like effects of acute and chronic GABA transporter inhibition in rats

Summary. Acute GABA transporter inhibition can induce anxiolytic-like behaviors. The present analysis addressed whether chronic treatment (23 days via drinking water) with a GABA transporter inhibitor affects rat behavior similar to acute treatment and interferes with additional benzodiazepine-receptor agonistic treatment. Seventy-one rats divided into seven groups were acutely treated with either vehicle, diazepam (2 mg/kg), zolpidem (0.05 mg/kg), tiagabine (19 mg/kg) or chronically with tiagabine with or without acute diazepam or zolpidem. Animals were behaviorally characterized in an elevated plus-maze. None of the treatments induced changes in the activity of the animals. Acute and chronic treatment with tiagabine induced anxiolytic-like effects, similar to acute doses of diazepam. Acute diazepam did not enhance chronic tiagabine effects, whereas acute zolpidem attenuated the anxiolytic-like effects of chronic tiagabine. It is concluded that anxiolytic effects of acute GABA-uptake inhibition by tiagabine persist under chronic treatment and are sensitive to concomitant use of benzodiazepine receptor ligands. Received October 26, 2001; accepted January 30, 2002     

Protection of malonate-induced GABA but not dopamine loss by GABA transporter blockade in rat striatum

Previous work has shown that overstimulation of GABA(A) receptors can potentiate neuronal cell damage during excitotoxic or metabolic stress in vitro and that GABA(A) antagonists or GABA transport blockers are neuroprotective under these situations. Malonate, a reversible succinate dehydrogenase/mitochondrial complex II inhibitor, is frequently used in animals to model cell loss in neurodegenerative diseases such as Parkinson's and Huntington's diseases. To determine if GABA transporter blockade during mitochondrial impairment can protect neurons in vivo as compared with in vitro studies, rats received a stereotaxic infusion of malonate (2 micromol) into the left striatum to induce a metabolic stress. The nonsubstrate GABA transport blocker, NO711 (20 nmol) was infused in some rats 30 min before and 3 h following malonate infusion. After 1 week, dopamine and GABA levels in the striata were measured. Malonate caused a significant loss of striatal dopamine and GABA. Blockade of the GABA transporter significantly attenuated GABA, but not dopamine loss. In contrast with several in vitro reports, GABA(A) receptors were not a downstream mediator of protection by NO711. Intrastriatal infusion of malonate (2 micromol) plus or minus the GABA(A) receptor agonist muscimol (1 micromol), the GABA(A) Cl- binding site antagonist picrotoxin (50 nmol) or the GABA(B) receptor antagonist saclofen (33 nmol) did not modify loss of striatal dopamine or GABA when examined 1 week following infusion. These data show that GABA transporter blockade during mitochondrial impairment in the striatum provides protection to GABAergic neurons. GABA transporter blockade, which is currently a pharmacological strategy for the treatment of epilepsy, may thus also be beneficial in the treatment of acute and chronic conditions involving energy inhibition such as stroke/ischemia or Huntington's disease. These findings also point to fundamental differences between immature and adult neurons in the downstream involvement of GABA receptors during metabolic insult.     

自发性癫痫大鼠脑海马两种锌转运体表达的研究

目的 探讨反复癫痫发作后的自发性癫痫大鼠(SER)与正常Wistar大鼠脑海马锌转运体1和3的表达与癫痫的相关性,以及癫痫发作后脑海马内含锌神经元内锌稳态的改变情况.方法 应用Western blot和RT-PCR方法分析反复癫痫发作后的SER与正常Wistar大鼠脑海马锌转运体1和3表达情况.结果 SER反复癫痫发作后脑海马锌转运体1蛋白及mRNA表达均明显高于正常Wistar大鼠,差异有统计学意义(P<0.05);锌转运体3蛋白表达与正常Wistar大鼠相比无明显改变,差异无统计学意义(P>0.05).结论 反复癫痫发作后SER脑海马可能在神经元尤其突触后神经元内出现锌浓度增高现象,锌转运体1表达上调可通过降低神经元内Zn2+水平对抗癫痫发作引起的神经元损伤,从而起到保护神经元的作用.