NMDA受体亚单位NR2A在凝血酶诱导的脑出血后脑损伤中的研究

目的 探讨NMDA受体亚单位NR2A在脑出血后脑损伤中的作用机制.方法 成年雄性SD大鼠随机分为生理盐水组,脑出血组,凝血酶组,凝血酶＋阿加曲班组.各组在手术后48 h采用不同方法观察其分布及动态变化规律.结果 NMDA受体亚单位NR2A在72h达高峰,阿加曲班可以减少其在72 h时的表达.阿加曲班可以改善血脑屏障的通透性、减轻脑水肿.结论 NMDA受体亚单位NR2A参与了脑出血后脑损伤的病理生理过程;阿加曲班通过抑制NMDA受体亚单位NR2A的激活而发挥神经保护作用.     

脑缺血再灌注大鼠皮质NMDA受体亚单位NR2A/B蛋白的表达变化

目的 采用免疫细胞化学技术，探讨急性脑缺血再灌注不同时间段大鼠脑顶皮质NMDA受体亚单位NR2A及NR2B蛋白的表达变化。方法 雄性Wistar大鼠70只,随机分成正常对照组、假手术对照组、脑缺血再灌注对照组；以颈动脉引流法行全脑缺血7min再灌注造模，术后分6h、24h、72h3个时间段取脑．脑组织恒冷箱连续冠状切片,免疫细胞化学ABC反应，图像分析系统行顶皮质Ⅰ区V层免疫阳性面积检测。结果 （1）麻醉及假手术可导致顶皮质NR2A、NR2B蛋白表达短暂增多，24h内恢复正常；（2）缺血再灌注后6h前后形成表达高峰,24h恢复正常，随后表达急剧减少，持续至72h以后。结论（1）脑缺血再灌注可导致顶皮质神经元NR2A、NR2B蛋白表达变化，且表达存在明显的时间依赖性；（2）缺血再灌注早期皮质NR2A、NR2B蛋白高度表达可能是导致迟发性神经元丢失的重要原因。     

NMDA受体NR2A亚单位在凝血酶诱导的大鼠脑出血后脑损伤作用机制的研究

目的探讨N-甲基-D天冬氨酸受体(NMDA)受体NR2A亚单位在凝血酶诱导的大鼠脑出血后脑损伤中的作用机制。方法将健康雄性SD大鼠随机分为生理盐水对照组、凝血酶组、凝血酶+阿加曲班组,建立大鼠脑出血模型。造模后48h,采用伊文思蓝法检测血脑屏障(BBB)的通透性,干-湿重法测脑组织的含水量。采用Western-blot方法观察出血后各时间点(0h、0.5h、6h、24h、72h、120h)血肿周围脑组织NR2A分布及动态变化规律。结果 (1)阿加曲班可以明显改善大鼠脑出血BBB通透性(P<0.05),血肿周围脑水肿明显减轻(P<0.05)。(2)Western-blot结果显示,NR2A在6h时开始增加,72h达到高峰,在120h时基本恢复。阿加曲班可以明显减少NR2A在72h时的表达(P<0.05)。结论 (1)NR2A参与了凝血酶诱导的脑出血后脑损伤的病理生理过程。(2)凝血酶可能通过上调NR2A表达引起脑出血后脑组织的损伤。     

大鼠听皮质NMDA受体来单位NR2BmRNA年龄—依赖性表达

应用原位杂交技术，研究了大鼠生后发育过程中，听皮质神经元NMDA受体亚单位NR2B mRNA年龄－依赖性的表达变化。特异性DIG标记寡核苷酸探针检测显示，NR2B亚单位mRNA阳性神经元数量从出生后即有高水平表达，之后，随着天龄增长逐渐递减，在出生后14d出现一过性表达高峰，14－21d时表达水平急剧降低（＞50．0％），21d后保持低水平表达至成年。研究结果为进一步在皮质水平上探讨出生后听觉功能发育可塑性的分子机制提供了重要资料。     

NMDA受体亚单元NR2A和NR2B mRNA在缺血大鼠海马的表达及其与细胞凋亡的关系

目的 探讨短暂性前脑缺血后大鼠海马NR2A和NR2B mRNA的表达变化及其与海马缺血性细胞凋亡的关系。方法 四动脉阻断法建立脑缺血再灌注动物模型、原位杂交、TUNEL染色和图像分析与统计处理。结果 ①缺血后，NR2A和NR2B mRNA在海马各区呈现出一种相对一致的表达规律。在CA1区，NR2A和NR2B mRNA的表达分别在缺血再灌6h和12h降至低谷，然后回升，在缺血再灌48h都升至高峰，之后表达再次下降，直至缺血后7d；在CA3区，该变化规律依然存在，不同的是表达变化的幅度明显减小；而在齿状回，缺血再灌0．5～72h，二者的表达未见显著性变化；72h后，表达下降，直至缺血后7d。②缺血后24h，凋亡细胞出现，主要位于海马CA1区，进行性增多，48h增加更为明显，缺血后72h达高峰；然后凋亡细胞有所减少，但至缺血后7d，依然存在。结论 短暂性前脑缺血后，大鼠海马各区NR2A和NR2B mRNA的表达变化及细胞凋亡均存在着显著性差异；这种差异提示，缺血后，NR2A和NR2B mRNA的表达变化与海马的选择性易损现象和缺血性细胞凋亡之间可能存在着某种密切的关系。     

戊四氮点燃癫癎大鼠空间学习记忆改变及海马NR2B表达

目的观察戊四氮点燃癫癎大鼠空间学习记忆功能变化及海马NMDA2型受体(NR2)B亚单位(NR2B)表达,探讨二者的关系及PTZ致癎大鼠认知障碍发生的分子机制。方法采用戊四氮(PTZ)慢性癫癎(CE)模型,Y-迷宫对两组大鼠进行行为学检测,免疫组织化学方法观察两组大鼠海马CA3区NR2B表达的变化,反转录多聚酶链反应(RT-PCR)方法检测大鼠海马NR2B mRNA的表达。结果癫癎组大鼠空间学习记忆能力受损;其海马CA3区NR2B阳性细胞较对照组明显减少(P<0.01),同时伴有海马NR2B mRNA表达下降(P<0.01)。结论戊四氮点燃癫癎大鼠空间学习记忆受损可能与海马神经元NR2B的表达减少有关。     

盐酸多奈哌齐对拟VaD大鼠海马CA1区NR1及NR2B免疫组织化学表达的影响

目的探讨盐酸多奈哌齐对拟血管性痴呆大鼠海马神经元N-甲基-D-天门冬氨酸(NMDA)受体亚单位R1(NMDAR1,NR1)和R2B(NMDAR2B,NR2B)的免疫组织化学表达的影响。方法采用双侧颈总动脉反复夹闭、再通,并腹腔注射硝普钠法制备模型,用盐酸多奈哌齐溶液灌胃,Y-型迷宫试验观察其行为学改变,用免疫组织化学技术观测大鼠海马神经元NR1、NR2B的表达变化。结果盐酸多奈哌齐组大鼠海马CA1区NR1表达明显降低,与模型组比较有显著性差异(均P<0.01),与假手术组相比差异无显著性;NR2B表达较模型组明显增高(P<0.01),与假手术组比较无显著性差异。结论盐酸多奈哌齐有可能通过降低NR1的表达,提高海马NR2B的表达,从而改善拟血管性痴呆大鼠的学习、记忆成绩。     

大鼠听皮质NMDA受体亚单位NR2BmRNA年龄-依赖性表达

应用原位杂交技术 ,研究了大鼠生后发育过程中 ,听皮质神经元NMDA受体亚单位NR2BmRNA年龄 依赖性的表达变化。特异性DIG标记寡核苷酸探针检测显示 ,NR2B亚单位mRNA阳性神经元数量从出生后即有高水平表达 ,之后 ,随着天龄增长逐渐递减 ,在出生后 14d出现一过性表达高峰 ,14～ 2 1d时表达水平急剧降低(>5 0 .0 % ) ,2 1d后保持低水平表达至成年。研究结果为进一步在皮质水平上探讨出生后听觉功能发育可塑性的分子机制提供了重要资料     

大鼠听皮质NMDA受体亚单位NR2BmRNA年龄-依赖性表达

应用原位杂交技术,研究了大鼠生后发育过程中,听皮质神经元NMDA受体亚单位NR2B mRNA年龄-依赖性的表达变化.特异性DIG标记寡核苷酸探针检测显示,NR2B亚单位mRNA阳性神经元数量从出生后即有高水平表达,之后,随着天龄增长逐渐递减,在出生后14 d出现一过性表达高峰,14～21 d时表达水平急剧降低(＞50.0%),21 d后保持低水平表达至成年.研究结果为进一步在皮质水平上探讨出生后听觉功能发育可塑性的分子机制提供了重要资料.     

神经干细胞分化的神经元离子型谷氨酸受体NMDA亚单位的mRNA和蛋白表达

目的在体外研究由大鼠神经干细胞(NSCs)分化而来神经元细胞中离子型谷氨酸NMDA受体表达。方法分离培养孕14～16d胎鼠皮质和海马神经干细胞，对NSCs进行nestin和分化鉴定。通过RT—PCR、Western blot免疫印迹和免疫组化检测NSCs分化的神经元细胞中离子型谷氨酸NMDA受体亚单位NR1、NR2A和NR2B的mRNA和蛋白表达。结果从孕14～16d胎鼠大脑中分离培养出NSCs，NSCs分化后的神经元可以表达离子型谷氨酸NMDA受体亚单位NR1、NR2A和NR2B。结论由NSCs分化而来的神经元能表达离子型谷氨酸NMDA受体。     

NMDA receptor subunit gene expression in the rat brain: a quantitative analysis of endogenous mRNA levels of NR1Com, NR2A, NR2B, NR2C, NR2D and NR3A.

Electrophysiological recordings have shown NMDA receptors to be heterogenous structures capable of responding to selected antagonists and agonists in multiple ways. This diversity in functional response has led investigators to conclude that these channels are comprised of unique combinations of receptor subunits which determine a cell's functional NMDA-signature [H. Meguro, H. Mori, K. Araki, E. Kushiya, T. Kutsuwada, M. Yamazaki, T. Kumanishi, M. Arakawa, K. Sakimura, M. Mishina, Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs, Nature (London) 357 (1992) 70-74; T. Ishii, K. Moriyoshi, H. Sugihara, K. Sakurada, H. Kadotani, M. Yokoi, C. Akazawa, R. Shigemoto, N. Mizuno, S. Nakanishi, Molecular characterization of the family of the N-methyl-d-aspartate receptor subunits, J. Biol. Chem. 268 (1993) 2836-2843; K.A. Wafford, C.J. Bain, B. Le Bourdelles, P.J. Whiting, J.A. Kemp, Preferential co-assembly of recombinant NMDA receptors composed of three different subunits, NeuroReport 4 (1993) 1347-1349; T. Priestley, P. Laughton, J. Myers, B. Le Bourdelles, J. Kerby, P.J. Whiting, Pharmacological properties of recombinant human N-methyl-d-aspartate receptors comprising NR1a/NR2A and NR1a/NR2B subunit assemblies expressed in permanently transfected mouse fiberblast cells, Mol. Pharmacol. 48 (1995) 841-848; P.H. Seeburg, N. Burnashev, G. Kohr, T. Kuner, R. Sprengel, H. Monyer, The NMDA receptor channel: molecular design of a coincidence detector, Recent Prog. Horm. Res. 50 (1995) 19-34; A.L. Buller, D.T. Monagahan, Pharmacological heterogeneity of NMDA receptors: characterization of NR1a/NR2D heteromers expressed in Xenopus oocytes, Eur. J. Pharmacol. 320 (1997) 87-94]. In situ hybridization and immunocytochemical studies have shown that there is a spatio-temporal level of expression throughout the brain for each of the receptor subunits with some regions showing a strong preference for a particular subunit. Although these studies collectively show that there are regional differences with respect to NMDA receptor subunit expression in the brain, it has not been determined at what level(s) these genes are expressed or whether each region displays a unique NMDA-subunit signature. The present study was undertaken to examine the level of gene expression for the NR1, NR2A, NR2B, NR2C, NR2D and NR3A receptor subunits in isolated regions of rat brain using the nuclease protection assay. Results show that each of the brain regions examined expresses all six NMDA receptor subunits. The level of message expression for NR1 greatly exceeded that of the other subunits combined, with values ranging from 67-88% of the total subunit gene expression. The relative proportions of the other subunits (NR2A-D and NR3A) varied widely, suggesting that NMDA receptor composition is unique to each region of the brain.     

NR2 to NR3B subunit switchover of NMDA receptors in early postnatal motoneurons

The NR3B NMDA receptor subunit is selective to somatic motoneurons in the adult nervous system. Here we report its developmental expression in the mouse brain and spinal cord by in situ hybridization. NR3B mRNA was detected in few neural regions during embryonic and neonatal periods. It first appeared in motoneurons at postnatal day (P)10-P14, and attained the maximal level at P21 and adult stage. This developmental profile was reciprocal with that of NR2 subunits, of which NR2A mRNA was most predominant in embryonic and neonatal motoneurons and downregulated by P14. Interestingly, mRNA of the NR1 subunit, which is required for functional NMDA receptors, displayed a 'V'-shaped change, decreasing with the early postnatal decline of NR2 mRNAs and increasing with the subsequent appearance of NR3B mRNA. Therefore, the major regulatory subunit of NMDA receptors is likely to switch from NR2 to NR3B in somatic motoneurons during the early postnatal period.     

Distribution and expression of the subunits of N-methyl-d-aspartate (NMDA) receptors; NR1, NR2A and NR2B in hypoxic newborn piglet brains

The purpose of this study was to identify the distribution and the expression of the NR1, NR2A and NR2B subunits of the NMDA receptor after cerebral hypoxia. Ten piglets were divided into control and hypoxic groups (n=5, each). The control piglets were ventilated with normoxia for 1 h, and the hypoxic piglets were ventilated with hypoxia until p_aO₂ was below 20 mmHg. Tissue samples from the nine different regions of newborn piglet brain were obtained, and the protein amount of the NR1, NR2A, and NR2B subunits measured by immunoblot using the antibody to the NR1, NR2A, and NR2B subunits. The NR1, N2A, and NR2B subunits were distributed very differently; hippocampus and cortical area are more prominent than white matter and cerebellum. But the expression of the NR1, NR2A and NR2B subunits were not significantly different between the control and the hypoxic group, 1 h after hypoxic exposure, indicating no changes in the protein amount of NMDA receptor subunits. These results show a significantly higher amount of the NR1, NR2A and NR2B subunits in the hippocampus and the cerebral cortex of newborn brains, indicating that these structures could be highly vulnerable to excitotoxicity in the newborn brain.     

Cellular and synaptic distribution of NR2A and NR2B in macaque monkey and rat hippocampus as visualized with subunit-specific monoclonal antibodies

The functional and pharmacological attributes of the N-methyl-D-aspartate (NMDA) receptor are related to its subunit composition, thus resolving the subunit composition of NMDA receptors in specific classes of synapses is an important step in characterizing excitatory circuits. Toward this end, mouse monoclonal antibodies were raised against fusion protein antigens corresponding to the putative amino acid sequences of human NMDA receptor subunits NR2A and NR2B. The subunit specificity of these monoclonal antibodies was demonstrated with transfected human and rat NMDA receptor cDNAs, and their immunoreactivity was established in rat, macaque monkey, and human brain tissue. At the light microscopic level, both NR2A and NR2B exhibit a distribution in monkey and rat hippocampus very similar to NMDA receptor subunit NR1, and both are highly colocalized with NR1. Electron microscopic immunogold studies demonstrated that both NR2A and NR2B are often present in asymmetric synapses in CA1, commonly colocalized with NR1, and often colocalized with each other in the same asymmetric synapses. Both assembly and synthetic pools are present within spines and spine necks, respectively, particularly for NR2A. The confocal and ultrastructural data suggest that whereas NR1, NR2A, and NR2B are essentially uniformly colocalized in hippocampal projection neurons, there is extensive heterogeneity at the synaptic level that would lead to multiple functional classes of NMDA receptor-mediated synapses, and extensive capacity for plasticity at the synapse. Thus, the subunit profile of a given synapse may be dynamic, with regulation of local synthesis and insertion of different subunits into the synapse leading to a complex, heterogeneous, and shifting set of functional attributes of the NMDA receptor.     

Developmental changes in the expression of NMDA receptor subunits (NR1, NR2A, NR2B) in the cat visual cortex and the effects of dark rearing

The present study used Western blots to determine changes in the level of expression of the three major NMDA receptor subunits, NR1, NR2A, and NR2B, in relation to the 'critical period' in cat visual cortex. NR2A rose dramatically (10-fold) from very low levels at 1 week to a peak at 5 weeks and gradually declined into adulthood (twofold). NR2B showed a similar time course to NR2A, but the rise from 1 to 5 weeks was of lesser magnitude (twofold). NR1 was expressed at comparable levels at 1, 5, 10 weeks and declined markedly (fivefold) in older animals. No significant effects of dark rearing on the levels of NR2B and NR1 were found. However, NR2A expression was significantly elevated in normal compared to dark reared visual cortex (twofold) at 5 weeks and significantly elevated in dark reared compared to normal visual cortex at 20 weeks (twofold). The close agreement between NR2A expression and both the time course of the critical period and the effects of dark rearing on that time course further indicates a role of this subunit in visual cortical critical period plasticity.