NMDA受体亚单位在离体大鼠海马神经干细胞中的表达

为了研究早期离体培养的胎鼠海马神经干细胞(NSCs)中NMDA受体亚单位NR1、NR2A和NR2B的表达,分离、培养、传代孕18～19d胎鼠海马NSCs,对NSCs进行nestin和分化鉴定。通过免疫荧光反应和RT-PCR法检测原代培养、传代1次、传代2次的NSCs中NMDA受体亚单位NR1、NR2A和NR2B的蛋白和mRNA表达。结果显示,从孕18～19d的胎鼠大脑海马分离培养出的NSCs,NMDA受体亚单位NR1、NR2A和NR2B的免疫荧光反应均呈阳性,这三种受体亚单位的mRNA在海马NSCs上均被检测到。上述结果提示,离体培养的胎鼠早期海马NSCs能稳定表达NMDA受体亚单位NR1、NR2A和NR2B。     

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     

生后早期大鼠海马NMDA受体亚单位NR1、NR2A和NR2B的表达变化

运用免疫组织化学反应和图象分析处理技术研究生后 1d、4d、1周、2周、3周、4周、5周、6周的 SD大鼠海马结构中NMDA受体亚单位 NR1、NR2 A、NR2 B三种蛋白质的表达变化规律。结果表明 ,生后各时间点海马结构各区锥体细胞及颗粒细胞胞体中均有 NR1、NR2 A、NR2 B的表达 ,NR2 B还在锥体细胞的顶树突中有较强表达。 NR1与 NR2 B的生后表达变化模式相似 ,在生后 1d和 4d,两者在 CA3区的表达均高于 CA1 区 ;生后 1周后两者在 CA1 区的表达则高于 CA3区 ;到生后 2～ 3周其表达达到峰值。而 NR2 A却与此不同 ,生后 1d、4d时其在海马结构各区的表达较高 ,随发育时间延长表达逐渐下降 ,大约在生后 4周降至谷底。整个发育过程中 ,NR1在海马结构各区的表达始终高于 NR2 A和 NR2 B的表达。这些结果提示 ,生后早期大鼠海马结构 NR1、NR2 A、NR2 B的表达具有发育性时空差异和亚单位差异 ,这种差异可能与发育早期海马学习功能的特异性以及海马各区对缺血敏感性不同有关。     

大鼠听皮质NMDA受体亚单位NR2B蛋白质的年龄-依赖性表达

目的：研究sD大鼠生后发育过程中,听皮质神经元NMDA受体亚单位NR2B蛋白质的表达规律。方法：采用免疫组织化学反应和蛋白质印迹技术,分别检测生后1、2、3周和成年动物听皮质神经元NMDA受体亚单位NR2B蛋白质的表达。结果：NR2B阳性神经元在生后第1周密度最高,随着生后周龄增长,NR2B阳性神经元密度递减．3周龄后降至成年动物的低表达水平。结论：大鼠生后发育过程巾．听皮质NR2B亚单位蛋白质呈现年龄-依赖性表达,此结果与mRNA水平上的表达趋势一致。     

NR2B反义寡核苷酸对大鼠海马CA1区NR2B mRNA表达的影响

目的：观察NMDA受体2B亚单位（NR2B）反义寡核苷酸（ANR2B）对海马CA1区NR2B mRNA表达的影响,筛选有效的ANR2B,探讨改变NR2B mRNA表达的新方法.方法：正常SD大鼠,海马CA1区立体定位注射ANR2B,灌注取脑,连续冰冻切片,原位杂交组织化学方法染色,光镜下观察NR2B mRNA在ANB2B作用下的表达变化.结果：正常大鼠海马CA1区立体定位注射ANR2B后,注射点及其周围NR2B mRNA原位杂交染色强度明显下降,仅有少量锥体细胞和颗粒细胞散在分布;而在注射NR2B正义寡核苷酸（SNR2B）、生理盐水（NS）及NS插针不注射（NSNO）的海马切片上,海马CA1区的染色特征与注射ANR2B有明显差别,其作用区锥体细胞、颗粒细胞及顶树突的NR2B原位杂交染色强度无明显变化.结论：ANR2B能够降低NR2B mRNA在正常大鼠海马CA1区的基础性高表达.     

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     

NR2B反义寡核苷酸对脑缺血海马CA1区NR2B mRNA表达的影响

为研究NMDA受体2B亚单位(NR2B)反义寡核苷酸(antisense oligonucleotide to NR2B,ANR2B)对短暂性脑缺血后海马CA1区NR2B mRNA表达的影响,分别向成年SD大鼠海马CA1区内立体定位注射ANR2B、NR2B正义寡核苷酸(SNR2B)、无菌生理盐水(NS),或者插针不注射(NSNO),24h后行四动脉阻断前脑缺血手术(缺血15min、再灌注24h),经心冲灌固定取脑,连续冰冻切片,原位杂交组织化学方法染色,光镜下观察各组每侧鼠脑NR2B mRNA的表达变化,并用LEICAQWin进行图像分析。结果显示,单纯缺血组海马各区的NR2B mRNA显色强度明显增加;缺血再灌组、假手术组和正常组海马CA1区内ANR2B注射点及其周围的NR2B mRNA显色明显下降;而在注射SNR2B、NS或NSNO的各组海马切片上,NR2B mRNA显色均无明显变化。结果表明ANR2B可以特异性地在体局部防止缺血后NR2B mRNA的高水平表达。     

快速老化小鼠海马突触体内NMDA受体的表达变化

目的:观察NMDA受体在SAMP8小鼠海马突触体内的表达变化。方法:首先应用生物化学的方法分离海马突触蛋白,并对其进行鉴定。其次,Western Blot检测NMDA受体的主要亚基NR1、NR2A和NR2B在SAMP8小鼠海马突触体内的表达变化。结果:PSD-95和synaptophysin特异性抗体检测显示突触蛋白的分离是成功的。SAMP8小鼠海马内NR1、NR2A和NR2B在突触的表达均显著低于SAMR1小鼠。进一步分析NR1、NR2A和NR2B蛋白在突触的表达量占总表达量的比值,SAMP8小鼠同样显著低于SAMR1小鼠,而SAMR1和CD-1小鼠间没有显著性差异。结论:SAMP8小鼠海马突触体内NR1、NR2A和NR2B的蛋白表达水平均显著性降低,推测NMDA受体在突触表达水平的降低可能是导致受体功能失调,激发突触功能损伤信号途径的原因之一,进而导致SAMP8小鼠学习记忆功能的下降。     

Bcl-2、Bax蛋白在NRs抗体预处理后缺氧缺糖海马脑片CA_1区的表达变化

运用海马脑片培养技术、海马脑片缺氧缺糖方法、免疫组织化学染色和图像分析处理技术观察用 NMDA受体亚单位抗体预处理后再缺氧缺糖的海马脑片 CA1 区 Bcl-2和 Bax蛋白的表达变化 ,以探究其亚单位与海马脑片缺氧缺糖性损伤的关系。结果显示 ,各实验组海马脑片 CA1 区均有不同程度 Bcl-2、Bax蛋白表达和细胞缺失形成的空洞。 Bcl-2蛋白在 NR1+ OGD组、NR2 A+ OGD组和 NR2 A + NR2 B+ OGD组 CA1 区的表达均明显弱于 OGD组 (3组均 P<0 .0 5 ) ;其在 NR2 B+ OGD组和HOTC组的表达则强于 OGD组 (两者 P<0 .0 5 )。 Bax蛋白在 NR1+ OGD组、NR2 A+ OGD组和 NR2 A+ NR2 B+ OGD组的表达均强于 OGD组 (NR2 A+ OGD组 P<0 .0 5 ) ;在 NR2 B+ OGD组和 HOTC组其表达则明显弱于 OGD组 (后者 P<0 .0 5 )。结论 :单纯缺氧缺糖可引起海马脑片 CA1 区锥体细胞的迟发性损伤 ,同时引起 Bcl-2蛋白和 Bax蛋白的表达变化 ;预加 NMDA受体亚单位 NR1、NR2 A抗体和 NR2 A+ NR2 B抗体可以加重缺氧缺糖性海马脑片 CA1 区细胞损伤程度 ;预加 NR2 B抗体则可减轻其损伤程度。提示 NMDA受体亚单位成分的变化可以调节 Bcl-2和 Bax蛋白在缺氧缺糖性海马脑片 CA1 区的表达 ,从而调节CA1 区神经元的损伤程度     

NR2A亚单位C末端的部分缺失对 NMDA受体功能和表达的影响

目的研究NMDA受体NR2A亚单位羧基末端（以下简称为C末端）不同部位缺失对该受体表面表达和功能的影响。方法以GFP-NR2A为起始质粒构建了4个依次缺失部分c末端的GFP标记的NR2A亚单位表达载体：NR2A-ΔC1（缺失897L至1017S）、NR2A-ΔC2（缺失1024D至1142P）、NR2A-ΔC3（缺失1149D至1347G）及NR2A-ΔC4（缺失1354S至1464V）;通过活细胞免疫化学染色分析这些载体与NR1-1a亚单位共转染后在HEK293细胞和海马神经元的表面表达,并通过电生理检测转染HEK293细胞NMDA受体的功能。结果当NR2A-ΔC1、NR2A-ΔC2、NR2AΔC3、NR2A-ΔC4分别与NR1-1a共转染HEK293细胞时,均可获得细胞膜表面表达,其表面染色阳性的细胞在绿色荧光蛋白细胞中的百分比与共转染NR1-1a／GFP-NR2A时的百分比相比均无显著性降低;并且NR1-1a／NR2A-ΔC2或NR1-1a／NR2A-ΔC4转染的HEK293细胞均能记录到谷氨酸诱发的NMDA受体通道电流。在海马神经元中,当转染NR2A-ΔC1、NR2A-ΔC2或NR2A-ΔC3时,单位长度树突表面表达的受体簇数量均显著低于转染GFP-NR2A的神经元,而转染NR2A-ΔC4的树突表面表达的受体簇数量却无显著性降低。结论在HEK293细胞中,C末端部分缺失的NR2A亚单位不影响含NR2A亚单位的NMDA受体在膜表面表达;而在海马神经元中,含NR2A的NMDA受体的表面表达数量受NR2AC末端的调控。     

In vivo blockade of N-methyl-D-aspartate receptors induces rapid trafficking of NR2B subunits away from synapses and out of spines and terminals in adult cortex

We investigated the role of in vivo synaptic activity upon trafficking of the N-methyl-D-aspartate (NMDA) receptor subunit, NR2B, at mature synapses by electron microscopic immunocytochemistry. In vivo blockade of NMDA receptors was achieved by applying the NMDA receptor antagonist, D-2-amino-5-phosphonovalerate (D-APV), onto the cortical surface of one hemisphere of anesthetized adult rats. Inactive L-2-amino-5-phosphonovalerate (L-APV) was applied to the contralateral hemisphere for within-animal control and to assess basal level of NR2B subunits at synapses. Within 30 min of D-APV treatment, we observed a decrease in the number of layer I axo-spinous asymmetric synapses that are positively immuno-labeled for the NR2B subunits. This decrease was paralleled by reductions in the absolute number of immuno-gold particles found at these synapses. The decrease of NR2B labeling was detectable in all five animals examined. Significant reductions were seen not only at post-synaptic densities, but also within the cytoplasm of spines and axon terminals. The data demonstrate that blockade of NMDA receptors induces trafficking of NR2B subunits out of synaptic membranes, spines, and terminals. This is in sharp contrast to a previous observation that NR2A subunits move into spines and axon terminals following in vivo blockade with D-APV. These findings point to yet unknown, NMDA receptor activity-dependent mechanisms that separately regulate the localization of NR2A and NR2B subunits at synapses.     

Delineation of additional PSD-95 binding domains within NMDA receptor NR2 subunits reveals differences between NR2A/PSD-95 and NR2B/PSD-95 association

N-methyl-D-aspartate (NMDA) receptors are clustered at synapses via their association with the PSD-95 (post-synaptic density-95) membrane associated guanylate kinase (MAGUK) family of scaffolding proteins. PSD-95 is the best characterized of this family. It is known to associate with NMDA receptor NR2 subunits via a conserved ES(E/D)V amino acid sequence located at their C-termini and thus to promote the clustering, regulation and the trafficking of assembled NR1/NR2 NMDA receptors at synapses. Here we have investigated in more detail NMDA receptor NR2/PSD-95 protein-protein association. Wild-type NR1 and PSD-95alpha were co-expressed with a series of rodent C-terminal truncated constructs of either NR2A or NR2B subunits in human embryonic kidney (HEK) 293 cells and the association of PSD-95alpha with assembled receptors determined by immunoprecipitation. Additional PSD-95 binding domains that differed between NR2A and NR2B subunits were identified. These domains mapped to the amino acid sequences NR2A (1382-1420) and NR2B (1086-1157). These results suggest that NR2A and NR2B may associate with PSD-95 but with different affinities. This may be important in the determination of the lateral mobility of NMDA receptor subtypes in post-synaptic membranes.     

Expression of the N-methyl-D-aspartate receptor NR1 splice variants and NR2 subunit subtypes in the rat colon

N-methyl-D-aspartate (NMDA) receptors and the expression of their different splice variants and subunits were previously characterized in the brain and spinal cord. However, knowledge on the NMDA receptor expression and function in the enteric nervous system is limited. Previous work suggested that NMDA receptors were involved in a rat model of visceral hypersensitivity. The aim of this study was to characterize the expression of the NMDA receptor NR1 splice variants and the NR2 subunit subtypes in the rat colon. We visualized the expression of NR1 protein in the rat submucosal and myenteric plexuses. The NR1 splice variants found in the colon of rats lacked the N1 and C1 cassettes and contained the C2 and C2' cassettes (NR1(000) and NR1(001)). The NR2B and NR2D subunits were also found in the rat colon. Moreover, NMDA receptors in the rat colon were heteromeric, since NR1 was co-localized with NR2B and NR2D subunits using fluorescent immunohistochemistry. The identification of the NMDA receptors in the enteric nervous system could lead to the development of drugs that selectively modulate bowel function.     

Functional NR2B- and NR2D-containing NMDA receptor channels in rat substantia nigra dopaminergic neurones

NMDA receptors regulate burst firing of dopaminergic neurones in the substantia nigra pars compacta (SNc) and may contribute to excitotoxic cell death in Parkinson's disease (PD). In order to investigate the subunit composition of functional NMDA receptors in identified rat SNc dopaminergic neurones, we have analysed the properties of individual NMDA receptor channels in outside-out patches. NMDA (100 n m ) activated channels corresponding to four chord conductances of 18, 30, 41 and 54 pS. Direct transitions were observed between all conductance levels. Between 18 pS and 41 pS conductance levels, direct transitions were asymmetric, consistent with the presence of NR2D-containing NMDA receptors. Channel activity in response to 100 n m or 200 μ m NMDA was not affected by zinc or TPEN ( N,N,N',N '-tetrakis-[2-pyridylmethyl]-ethylenediamine), indicating that SNc dopaminergic neurones do not contain functional NR2A subunits. The effect of the NR2B antagonist ifenprodil was complex: 1 μ m ifenprodil reduced open probability, while 10 μ m reduced channel open time but had no effect on open probability of channels activated by 100 n m NMDA. When the concentration of NMDA was increased to 200 μ m , ifenprodil (10 μ m ) produced the expected reduction in open probability. These results indicate that NR2B subunits are present in SNc dopaminergic neurones. Taken together, these findings indicate that NR2D and NR2B subunits form functional NMDA receptor channels in SNc dopaminergic neurones, and suggest that they may form a triheteromeric NMDA receptor composed of NR1/NR2B/NR2D subunits.     

Differential expression of NMDA and AMPA receptor subunits in rat dorsal and ventral hippocampus

Several studies have demonstrated anatomical and functional segregation along the dorsoventral axis of the hippocampus. This study examined the possible differences in the AMPA and NMDA receptor subunit composition and receptor binding parameters between dorsal and ventral hippocampus, since several evidence suggest diversification of NMDA receptor-dependent processes between the two hippocampal poles. Three sets of rat dorsal and ventral hippocampus slices were prepared: 1) transverse slices for examining a) the expression of the AMPA (GluRA, GluRB, GluRC) and NMDA (NR1, NR2A, NR2B) subunits mRNA using in situ hybridization, b) the protein expression of NR2A and NR2B subunits using Western blotting, and c) by using quantitative autoradiography, c(1)) the specific binding of the AMPA receptor agonist [(3)H]AMPA and c(2)) the specific binding of the NMDA receptor antagonist [(3)H]MK-801, 2) longitudinal slices containing only the cornus ammonis 1 (CA1) region for performing [(3)H]MK-801 saturation experiments and 3) transverse slices for electrophysiological measures of NMDA receptor-mediated excitatory postsynaptic potentials. Ventral compared with dorsal hippocampus showed for NMDA receptors: 1) lower levels of mRNA and protein expression for NR2A and NR2B subunits in CA1 with the ratio of NR2A /NR2B differing between the two poles and 2) lower levels of [(3)H]MK-801 binding in the ventral hippocampus, with the lowest value observed in CA1, apparently resulting from a decreased receptor density since the B(max) value was lower in ventral hippocampus. For the AMPA receptors CA1 our results showed in ventral hippocampus compared with dorsal hippocampus: 1) lower levels of mRNA expression for GluRA, GluRB and GluRC subunits, which were more pronounced in CA1 and in dentate gyrus region and 2) lower levels of [(3)H]AMPA binding. Intracellular recordings obtained from pyramidal neurons in CA1 showed longer NMDA receptor-mediated excitatory postsynaptic potentials in ventral hippocampus compared with dorsal hippocampus. In conclusion, the differences in the subunit mRNA and protein expression of NMDA and AMPA receptors as well as the lower density of their binding sites observed in ventral hippocampus compared with dorsal hippocampus suggest that the glutamatergic function differs between the two hippocampal poles. Consistently, the lower value of the ratio NR2A/NR2B seen in the ventral part would imply that the ventral hippocampus NMDA receptor subtype is functionally different than the dorsal hippocampus subtype, as supported by our intracellular recordings. This could be related to the lower ability of ventral hippocampus for long-term synaptic plasticity and to the higher involvement of the NMDA receptors in the epileptiform discharges, observed in ventral hippocampus compared with dorsal hippocampus.     

Nitration is a mechanism of regulation of the NMDA receptor function during hypoxia

The present study tested the hypothesis that nitration is a mechanism of hypoxia-induced modification of the N-methyl-D-aspartate (NMDA) receptor. To test this hypothesis the effect of hypoxia on the nitration of the NR1, NR2A and NR2B subunits of the NMDA receptor was determined. Furthermore, the effect of administration of a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine (NNLA) on the hypoxia-induced nitration of the NMDA receptor subunits as well as the NMDA receptor-mediated Ca2+ influx, an index of NMDA receptor-ion channel function, were determined in cortical synaptosomes. Studies were performed in newborn piglets divided into normoxic, hypoxic and hypoxic-NNLA groups. Hypoxia was induced by decreasing the FiO(2) to 0.07-0.09 for 60 min. Cerebral tissue hypoxia was confirmed by determining the levels of high energy phosphates ATP and phosphocreatine. Nitration of the NMDA receptor subunits was determined by immunoprecipitation using specific antibodies and western blot analysis. NMDA receptor-ion channel-mediated Ca2+ influx was determined using 45Ca2+. There was a significant increase in the nitrated NR1, NR2A and NR2B subunits following hypoxia: 104+/-11 vs. 275+/-18 optical density (OD)xmm(2) for NR1 (P<0.05), 212+/-36 vs. 421+/-16 ODxmm(2) for NR2A (P<0.05) and 246+/-44 vs. 360+/-26 ODxmm(2) for NR2B (P<0.05). This increase in nitrated NR1, NR2A and NR2B subunits of the NMDA receptor was prevented by the administration of NNLA prior to hypoxia (NR1 160+/-19, P=NS, NNLA vs. normoxic; NR2A 304+/-49, P=NS, NNLA vs. normoxic, and NR2B 274+/-19, P=NS, NNLA vs. normoxic). The increase in nitration of the NR1, NR2A and NR2B subunits of the NMDA receptor increased as a function of decreased cerebral high-energy phosphates, ATP and phosphocreatine, during hypoxia. Furthermore, NOS blockade prior to hypoxia resulted in prevention of the hypoxia-induced increase in NMDA receptor-mediated Ca2+ influx. Our results demonstrate that hypoxia results in increased nitration of the NMDA receptor subunits and that administration of an NOS inhibitor prior to hypoxia prevents the hypoxia-induced nitration of the NMDA receptor subunits as well as the hypoxia-induced increase in NMDA receptor-mediated Ca2+ influx. We conclude that nitration is a mechanism of modification of the NMDA receptor function during hypoxia in the newborn piglet brain.     

Reduced NR2A expression and prolonged decay of NMDA receptor-mediated synaptic current in rat vagal motoneurons following axotomy

To elucidate characteristic changes in the N -methyl- d -aspartate (NMDA) receptor on neurons following axotomy, subunit expressions and functional features of the NMDA receptor were examined in the dorsal motor nucleus of vagus (DMV) of rats receiving vagal axotomy at the neck. Western blotting analysis demonstrated that the expression of NR2A decreased 2–3 days after in vivo axotomy, while expression of NR1 and NR2B, NR2C and NR2D subunits did not change significantly. To examine the functional changes, patch clamp recordings in whole-cell mode were employed on the axotomized DMV neurons identified by retrograde labelling with fluorescent dye. The amplitude ratios of ifenprodil-sensitive components of NMDA response and d , l -2-amino-5-phosphovaleric acid (APV)-sensitive evoked postsynaptic current increased after axotomy. In addition, APV-sensitive postsynaptic currents exhibited a longer decay time in identified axotomized vagal motoneurons than in control neurons. No significant differences in the current density of the NMDA response and the peak amplitude of APV-sensitive synaptic currents were observed between axotomized and intact DMV neurons. In conclusion, a decrease in NR2A expression results in the appearance of functional characteristics of the NMDA receptor predominantly containing the NR2B subunit. This might lead to a long-term increase of the susceptibility of neurons to excitotoxicity.