NMDA受体NR1亚单位与GABA_A受体在精神分裂症小鼠海马齿状回颗粒细胞层的表达

目的:明确NMDA受体NR1亚单位、GABAA受体与精神分裂症(SP)小鼠海马齿状回颗粒细胞层新生颗粒细胞的共存模式;阐明NR1、GABAA受体在SP小鼠海马中的表达。方法:实验组小鼠腹腔注射MK-801(每日0.6 mg/kg),对照组注射等量的生理盐水,连续注射14 d后对两组动物分别进行BrdU标记,断头并进行如下处理:(1)免疫荧光染色,观察海马DG区中NR1和GABAA的表达以及与BrdU标记的新生颗粒细胞的共存模式;(2)利用RT-PCR技术,检测海马GABAA及NR1 mRNA表达水平的改变。结果:(1)停药后实验组与对照组比较,海马神经细胞的增殖率下降了23.1%(P0.05),GABAA、NR1两种神经细胞增殖数无差异性改变(P0.05);(2)实验组小鼠海马GABAAmRNA的表达量较对照组显著下降(P0.05),而NR1 mRNA的表达量较对照组明显增高(P0.05)。结论:(1)小鼠精神分裂症后可引起海马齿状回神经细胞增殖的降低;(2)小鼠精神分裂症后,在mRNA水平上,海马GABAA的表达降低,NR1的表达升高。     

侧脑室注射NMDA受体激动剂后精神分裂症小鼠海马齿状回内NR1和BrdU的改变

目的:探讨精神分裂症(schizophrenia,SP)小鼠侧脑室注射N-甲基-D-天门冬氨酸(NMDA)受体激动剂后,海马齿状回(dentate gyrus,DG)颗粒细胞层NR1和5-溴脱氧尿嘧啶核苷(BrdU)阳性细胞数量的改变。方法:选择健康6周龄雄性C57小鼠,地卓西平马来酸盐(MK-801)慢性给药制备精神分裂症动物模型,实验分为NMDA组、生理盐水组、SP组和空白对照组共四组,NMDA组侧脑室注射NMDA受体激动剂。BrdU标记后行免疫荧光染色,激光共聚焦显微镜下观察和计数DG颗粒细胞层NR1和BrdU阳性细胞的表达和数量变化。结果:(1)NR1阳性细胞数在NMDA组、空白对照组、SP组和生理盐水组分别为16.1±2.08,14.5±2.17,19.4±4.65,19.5±4.33,经比较,NMDA组和空白对照组NR1阳性细胞的数量明显少于SP组(P<0.05),但NMDA组与空白对照组相比无明显差异(P>0.05),SP组与生理盐水组相比也无明显差异(P>0.05)。(2)BrdU阳性细胞数在NMDA组、空白对照组、SP组和生理盐水组分别为5.2±2.53、5.3±0.82、3.4±1.58、3.5±1.35,经比较,NMDA组和空白对照组分别多于SP组(P<0.05),和生理盐水组(P<0.05);但NMDA组较空白对照组无明显差异(P>0.05),SP组较生理盐水组亦无明显差异(P>0.05)。结论:SP小鼠侧脑室注射NMDA受体激动剂后,可引起DG颗粒层NR1阳性细胞数减少和BrdU阳性神经细胞数的增多。     

癫痫大鼠海马NMDA受体亚基NR2A和BDNF mRNA水平的检测

目的 通过锂-匹罗卡品癫痫模型(ithium-pilocarpine seizures rats model of epilepsy,LPS),研究NMDA受体亚基NR2A、BDNF mRNA的表达,探讨NR2A、BDNF在LPS中的作用.方法 建立氯化锂-匹罗卡品大鼠模型,运用原位杂交技术检测致痫后各组不同时间点海马CA1、CA3及DG区NR2A与BDNF mRNA的表达.结果 LPS海马NR2A、BDNF mRNA在各观察时间点及部位模型组与正常对照组比较均有明显上调,且有显著统计学差异(P＜0.05).模型组NR2A mRNA的表达上调7 d达峰值(P＜0.05);而BDNF mRNA表达上调14 d达峰值.VPA干预组NR2A mRNA在大鼠海马不同时间及部位(除1 d的CA3区)的表达较模型组明显下调(P＜0.05);BDNF mRNA在大鼠海马不同时间及部位(除28 d的DG区)的表达较模型组明显下调(P＜0.05).结论 锂-匹罗卡品腹腔注射可诱导大鼠海马NR2A和BDNF mRNA的表达明显上调;NR2A mRNA表达的增强可能是诱导调控BDNF mRNA表达增强的重要机制之一,说明NMDA受体亚基NR2A可能成为抑制癫痫发作的新靶点.     

急性脑缺血通过激活EphB2/ephrin-B1/NMDA受体信号通路促进小鼠海马神经发生

目的:观察急性脑缺血对小鼠海马神经发生的影响,并探讨该过程是否涉及EphB2/ephrin-B1/NMDA受体信号通路的激活。方法:52只C57BL/6小鼠随机分为2组,即假手术组和急性脑缺血模型组,每组26只,其中每组用于Morris水迷宫实验6只,HE染色4只,BrdU免疫荧光染色4只,双皮质素(DCX)免疫荧光染色4只,RT-qPCR实验4只,Western blot实验4只。采用双侧颈总动脉夹闭法建立小鼠脑缺血模型。HE染色观察小鼠海马CA1区病理改变;Morris水迷宫实验检测小鼠学习与记忆等认知功能的改变;免疫荧光染色观察小鼠海马区BrdU阳性细胞和DCX蛋白表达以评估神经发生情况;RT-qPCR及Western blot检测小鼠海马EphB2、ephrin-B1、reelin、微管相关蛋白2(MAP-2)及NMDA受体亚基NR2A和NR2B的mRNA及蛋白表达。结果:脑缺血小鼠海马CA1区神经元损伤显著(P0.01),学习记忆功能显著下降(P0.01),提示脑缺血模型成功建立;海马区BrdU阳性细胞和DCX蛋白表达显著增加(P0.01),表明脑缺血后海马出现神经发生;同时海马区EphB2、ephrin-B1、reelin、MAP-2、NR2A和NR2B的表达水平也显著上调(P0.05)。结论:急性脑缺血能促进小鼠海马神经干细胞增殖及内源性神经发生,其促进神经发生的机制可能与激活EphB2/ephrin-B1/NMDA受体信号通路有关。     

CPZ介导急性脱髓鞘小鼠海马中nestin的表达

目的:探讨双环己酮草酰二腙(cuprizone,CPZ)介导急性脱髓鞘小鼠海马中nestin的表达。方法:用掺入0.2%CPZ的普通饲料饲养C57BL/6小鼠6周,制备脱髓鞘模型,然后使用免疫荧光染色、q RT-PCR、Western Blot方法,检测小鼠脑内髓鞘脱失后海马中nestin的表达变化。结果:(1)体重称量结果显示:CPZ组体重明显低于对照组(P0.01);(2)免疫荧光结果显示:CPZ组海马CA区和DG区颗粒细胞层中nestin阳性细胞明显低于对照组分别为:P0.05,P0.01;(3)Western Blot和q RT-PCR实验结果显示:CPZ组海马中nestin蛋白和m RNA含量均明显低于对照组(P0.05)。结论:CPZ介导小鼠急性脱髓鞘后海马内nestin表达降低,提示髓鞘脱失可能会抑制nestin表达。     

慢性复合应激对大鼠学习与记忆及海马NMDA受体亚基NR2A和NR2B表达的影响

为了观察慢性复合应激对大鼠学习与记忆的影响和海马内 NMDA受体亚基 NR2 A、NR2 B表达的变化。本研究将成年雄性 Wistar大鼠分为实验组和对照组 ,实验组动物每天交替暴露于复合应激原环境中达 6周 ,用 Morris水迷宫和 Y迷宫作业测试其空间学习与记忆成绩 ,再采用免疫组织化学和图像处理方法分析海马 CA1 、CA3、齿状回区的 NR2 A和 NR2 B的表达。结果显示 :( 1) Morris水迷宫测试 :慢性复合应激组大鼠寻找平台的潜伏期较对照组明显缩短 ,Y迷宫测试 :慢性复合应激组大鼠学会躲避电击的正确次数较对照组明显增多 ;( 2 )慢性复合应激组海马内 NMDA受体亚基 NR2 B表达水平较对照组明显上调 ,NR2 A表达水平无显著变化。结论 :慢性复合应激可增强学习与记忆能力 ,NMDA受体表达变化可能是影响学习与记忆的机制之一     

NMDA受体亚单位NR1、NR2A和NR2B在大鼠海马的免疫组织化学表达

目的 :观察N 甲基 D 门冬氨酸受体亚单位 1 (N methyl D aspartatereceptorsubunit 1 ,NR1 )、亚单位 2A(NR2A)和亚单位 2B(NR2B)在成年大鼠海马结构各区的表达特点 ,为研究三者在海马生理和病理过程中的作用提供形态学资料。方法 :大鼠脑 2 0 μm厚冰冻切片 ,免疫组织化学ABC法显色 ,图像分析。结果 :NR1、NR2A和NR2B在海马CA1～CA3区锥体细胞以及齿状回颗粒细胞普遍表达 ,三者中以NR1免疫组织化学反应最强 ,NR2A最弱 ,NR2B居中。NR1与NR2A在海马各区间的表达水平都无显著差异 ;NR2B在海马CA1区的表达明显强于其在CA3区及齿状回的表达 ,尤其是CA1区锥体细胞的顶树突在贯穿辐射层及腔隙分子层的全长中都呈高表达。结论 :NR1、NR2A和NR2B在正常海马结构各区的表达强度和形式存在差异 ,提示各区间天然N 甲基 D 门冬氨酸(N methyl D aspartate ,NMDA)受体的亚单位构成比例可能有所不同     

前脑缺血再灌流后大鼠海马 NMDA受体亚单位NR1蛋白的表达变化

目的　观察大鼠前脑缺血再灌流后海马各区域NMDA受体亚单位NR1表达的变化和差异 ,探讨NR1在缺血性脑损伤中的作用。　方法　免疫组织化学和图像处理技术。　结果　 1 在前脑缺血再灌流后早期 ,海马各区域NR1的表达水平显著下降 (P <0 0 5 )。CA1区 ,下降趋势持续存在且不可逆 ,直至再灌流后第 7d ,NR1在该区域的染色强度降至对照组的 17% (P <0 0 5 )。CA3区及齿状回 ,NR1的表达下降是可逆的 ,再灌流后 72h ,齿状回的表达恢复正常 ,再灌后第 7d ,CA3区的表达也恢复到对照组的 96 % ,两组间染色强度无显著差异。 2 在迟发性神经元坏死出现前的缺血再灌流的早期 ,NR1在CA1区、CA3区及齿状回表达下降的幅度不一致 ,再灌后 6h以前 ,CA1区的下降幅度明显小于CA3区及齿状回 (P <0 0 5 ) ,再灌后 12h ,CA1区的下降幅度仍低于CA3区 (P <0 0 5 )。结论　短暂性前脑缺血后 ,NR1在海马CA1区、CA3区及齿状回表达下降的幅度和可逆性存在显著差异 ,这种差异可能是造成CA1区缺血敏感性的重要原因。     

短暂性脑缺血对C57BL/6小鼠海马区神经发生的影响

目的:利用小鼠急性全脑缺血模型观察急性脑缺血对海马神经发生的影响。方法:将C57BL/6雄性小鼠(6~8周)随机分为脑缺血组和假手术组。采用双侧颈动脉结扎法建立短暂性全脑缺血模型;利用HE染色观察脑缺血后不同时间(1周,2周)海马区形态学变化;采用免疫组织荧光染色观察缺血后海马区caspase-3表达变化;利用Ed U染色观察缺血3 d、7 d、14 d和28 d后,海马区神经干细胞增殖变化;利用免疫组织荧光染色观察缺血3 d、7 d、14 d和28 d后,海马区nestin、胶质纤维酸性蛋白(GFAP)及少突胶质细胞特异蛋白(OSP)等蛋白表达变化,判断脑缺血对海马区神经干细胞分化的影响。结果:脑缺血7 d后,小鼠海马CA1区神经元数目减少,caspase-3阳性细胞增加(P0.05)。海马DG区Ed U阳性细胞数量在第3 d开始增加(P0.05),第1周达到峰值(P0.05);海马CA1区Ed U阳性细胞数量在缺血后1周开始增加(P0.05),缺血2周后逐渐降低。海马DG区nestin、GFAP及OSP阳性细胞数量均在缺血3 d后开始增加(P0.05),缺血1周后达到峰值(P0.05),2周后逐渐降低。海马CA1区GFAP及OSP阳性细胞数量在缺血3 d后开始增加(P0.05),缺血1周后达到峰值(P0.05),2周后逐渐降低。结论:脑缺血激活了海马DG区神经干细胞,使干细胞增殖,并向神经元、星形胶质细胞、少突胶质细胞分化,同时逐渐向CA1区迁移。     

5-Aza-cdR可能通过提高海马组织内 NR2B磷酸化水平改善小鼠学习和记忆能力

目的研究5-氮杂-2′-脱氧胞苷(5-Aza-CdR)对小鼠在跳台实验中的学习和记忆能力及其海马组织中NR2B 1472位点的酪氨酸磷酸化(p-Y1472 NR2B)的影响。方法将小鼠随机分为对照组(control)和5-Aza-CdR处理组(5-Aza-CdR)。侧脑室内注射10μmol/L 5-Aza-CdR,通过跳台实验检测小鼠的行为、学习和记忆能力。采用real-time PCR检测小鼠海马组织中NR2B mRNA的表达;Western blot和免疫荧光检测小鼠海马组织NR2B和磷酸化NR2B-Y1472(p-Y1472 NR2B)的表达水平。结果 5-Aza-CdR组小鼠在跳台实验中潜伏期增加,错误次数减少,学习和记忆能力明显提高(P0.05),同时,小鼠海马组织中NR2B mRNA和蛋白的表达水平均无明显变化,NR2B-Y1472的磷酸化水平升高(P0.05),CDK5活性降低(P0.05)。CA1区和CA3区NR2B荧光强度无差异,而CA1区p-Y1472 NR2B显著升高(P0.05)。结论 5-Aza-CdR对学习和记忆能力的影响可能与海马中p-Y1472 NR2B磷酸化相关。     

NMDA受体在癫痫发病机制中的作用

癫痫是慢性反复发作短暂脑功能失调综合征,是神经科常见疾病之一。目前有关癫痫的发病机制尚未阐明。研究表明,癫痫的发生与兴奋性神经递质和抑制性神经递质的失衡有关。谷氨酸作为一种主要的兴奋性神经递质,通过受体介导的兴奋性机制在癫痫的发生过程中具有重要作用。谷氨酸受体可以分为促离子型和促代谢型2类。     

Single channel properties of the N-methyl-D-aspartate receptor channel using NMDA and NMDA agonists: on-cell recordings

Summary The cell-attached patch-clamp mode has been applied in cultured rat hippocampal neurons to record single channel currents through the ion channel which is coupled to activation of the N-methyl-D-aspartate (NMDA) receptor. A channel, with a conductance of 37 pS, was studied with either NMDA or D-cis-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) in the patch pipette. The mean open time of the channels with NMDA in the pipette was near 5 ms at -80 mV and was diminished about 0.7 ms for each 20 mV of hyperpolarization. The mean open times with ACPD in the pipette were longer at all voltages studied and for both agonists the mean open times showed an exponential dependence on patch potential. Distributions for the channel open times were generally well-fit with single exponentials, whereas distributions for closed times required two-component fits. In some instances, the open distributions also showed a second, rapid time component. When Mg² was included in the pipette (40 or 100 M), the mean open times were significantly diminished with the effect increasing with the magnitude of the hyperpolarization. Both the amplitudes and mean open times of the NMDA channel were strongly modulated by temperature with Q₁₀ values in excess of 2.5.     

NMDA receptors in the basal ganglia

The basal ganglia consist of several interconnected nuclei located in the telecephalon, diencephalon and mesencephalon that are involved in a variety of motor and non-motor behavioural functions. Glutamate receptors play a major role in neurotransmission within the basal ganglia and are present in all nuclei of the basal ganglia. This review focuses on the contribution of the NMDA class of glutamatergic receptors to various movement disorders whose primary pathology lies within the basal ganglia and discusses how pharmacological manipulation of such receptors may be therapeutically useful.     

Dantrolene antagonizes the glycineB site of the NMDA receptor

In this study we tested the hypothesis that dantrolene, an established inhibitor of the skeletal muscle isoform of the ryanodine receptor, may interfere with activity of NMDA receptors in neurons. We assessed the effects of dantrolene on [(3)H]MK-801 and [(3)H]glycine binding to isolated rat cortical membranes. Dantrolene inhibited [(3)H]MK-801 binding in the presence of 100 microM NMDA with an IC(50) of 58.4 microM. The IC(50) value increased to 99.6, 343.0 and 364.6 microM in the presence of 10, 30 and 50 microM glycine, respectively, suggesting that dantrolene competes with glycine for binding site at the NMDA receptor complex. A binding assay using [(3)H]glycine confirmed this supposition: dantrolene inhibited strychnine-insensitive glycine binding in a dose-dependent way. Thus, our results show that dantrolene at concentrations of 50-100 microM and higher blocks the glycine binding site of the NMDA receptor complex and in this way inhibits activation of the NMDA ion channel. These data reveal a new mechanism of dantrolene action in neuronal tissue. Our results also suggest that the neuroprotective effect of dantrolene may be at least partly explained by its activity as a non-competitive antagonist of NMDA receptors.     

Channel gating of NMDA receptors

Glutamate receptors specifically activated by N-methyl-D-aspartate (NMDA receptors) are ion channels that play multiple fundamental roles in the physiology of vertebrate nervous systems. The mechanisms that control the opening and closing, or gating, of the channel of NMDA receptors are among the most basic determinants of receptor function, and yet are not well understood. Here we consider current understanding of the link between agonist binding and NMDA receptor channel gating, of the conformational changes that occur during gating, and of the location of the channel gate. Information is drawn from studies of NMDA receptors themselves, of other types of glutamate receptors, and of more distantly related potassium channels.