单耳气传导阻滞模型大鼠下丘脑N-甲基-D-门冬氨酸受体各亚基mRNA的表达

实验利用单耳外耳道皮下缝合的方法建立单侧气传导持续阻滞大鼠模型，观察环境变化对生后9，23，37 d的SD大鼠听觉中枢神经系统下丘脑NMDA受体NR1，NR2A，NR2B和NR2C mRNA基因表达的影响。PT-PCR结果显示，单耳缝合后缝耳对侧下丘脑NR1，NR2A，NR2B亚单位与缝耳同侧下丘脑NR1，NR2B依赖的听觉神经元发育临界期均在23 d附近，但同侧下丘脑NR2A亚单位依赖的听觉神经元发育的临界期的结束可能接近出生后37 d。结果证实了下丘脑NMDA受体亚基可受听觉环境调控的假设。     

大鼠胎脑皮质神经干细胞体外培养及诱导分化的实验研究

目的 从孕龄15d SD胚胎鼠脑皮质中分离并培养神经干细胞（neural stem cells。NSCs），观察其生长、增殖及分化。方法 采用包含碱性成纤维细胞生长因子（bFGF）和表皮细胞生长因子（EGF）的无血清培养及单细胞克隆技术，对胚胎鼠脑皮质神经干细胞进行原代、传代培养及诱导其分化。用Nestin染色鉴定神经干细胞特性，用免疫组化方法（β-Ⅲ-tubulin、GFAP染色）检测神经干细胞分化为神经元及神经胶质细胞状况。结果 从孕龄15dSD胚胎鼠脑皮质中分离的组织，经原代及传代培养均可形成细胞克隆．切具有增殖能力。原代及传代培养细胞呈Nestin（神经上皮干细胞蛋白）表达阳性．诱导分化后的细胞表达神经元细胞、星形胶质细胞的特异性抗原。结论 本实验分离、培养的孕龄15dSD胚胎鼠脑皮质细胞Nestin表达阳性．分化后表达神经元和星形胶质细胞的标记物，是大鼠的神经干细胞，并具有多向分化潜能。     

大鼠大脑皮层损伤后N-甲基D-天门冬氨酸受体亚单位NR1、NR2A和NR2B的表达研究

目的 分析研究急性损伤后大鼠大脑皮层N-甲基D-天门冬氨酸受体(NMDA)亚单位NR1，NR2A和NR2B的表达。方法 建立开放式挫伤型机械脑损伤大鼠模型，采用NMDA受体的NR1，NR2A和NR2B3种亚单位的特异性抗体，对不同时间点的皮层组织3种亚单位蛋白作定量免疫印迹分析。结果 以健侧大脑皮层作为对照组，NR1亚单位在各时间点的含量保持基本稳定。NR2A的含量在3h内升高明显，至3h达到高峰，随后降至正常；NR2B在1h时略有上升后随即下降，至3h时最低，尔后又明显升高，于6h时达高峰，随后又恢复正常。结论 大鼠脑皮层损伤NMDA受体NR2A和NR2B亚单位的表达发生了改变。     

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.     

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.     

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     

低氧联合GDNF诱导大鼠神经干细胞分化为TH阳性神经元的实验研究

目的本试验在低氧环境下利用胶质源性神经营养因子(GDNF)对大鼠胚胎神经干细胞(NSCs)进行定向诱导分化,从而研究低氧对NSCs向多巴胺能神经元分化的影响,并获得相当数量的多巴胺能细胞,为下一步的细胞移植奠定基础。方法分离培养孕14 d～16 d Wistar大鼠胚胎皮质NSCs,通过传代培养获得单克隆生长的NSCs,在不同氧浓度下利用GDNF对大鼠NSCs进行定向诱导分化,通过免疫细胞化学方法检测TH阳性细胞表达。结果低氧可明显促进NSCs向TH阳性神经元分化,与常氧对照组比较有显著差异(P 0.01),且在3%低氧浓度时,NSCs向TH阳性神经元分化率最高。结论低氧可促进NSCs向TH阳性神经元分化,3%低氧浓度是促进NSCs向TH阳性神经元分化的最适氧浓度,低氧促进NSCs向TH阳性神经元分化的分化率与氧浓度梯度无依赖关系,低氧联合GDNF诱导NSCs分化为TH阳性神经元,诱导效能更为显著。     

胚胎大鼠神经干细胞培养及分化鉴定的实验研究

目的建立胚胎大鼠神经干细胞体外培养及分化鉴定的方法。方法分离不问孕龄(13- 18d)SD胎鼠脑室下区、中脑及海马等部位组织,在含有表皮生长因子和碱性成纤维细胞生长因子及N-2 添加剂的DMEM培养基中悬浮培养,形成神经球体后撤除生长因子,经消化传代于含胎牛血清培养基中贴壁生长。应用免疫荧光方法检测原代细胞特异性标记巢蛋白、神经元标记微管蛋白-β、胶质细胞标记胶质纤维酸性蛋白,以及少突胶质细胞标记半乳糖脑苷的表达。结果胎鼠脑组织不同部位均可分离出神经干细胞,体外培养可以向神经元和神经胶质细胞分化,孕龄13d与18d胎鼠产生神经干细胞球体的能力不同,前者优于后者。结论 (1)胚胎大鼠脑内不同部位来源的组织生成神经干细胞球体数量和质量基本相同,但是不同胎龄的鼠脑组织产生神经干细胞球体的能力有所差异。(2)神经干细胞具有向神经元和神经胶质细胞分化的潜能。     

大鼠胚胎脑皮层神经干细胞的分离和培养

目的探讨分离、培养、纯化大鼠胚胎神经干细胞(neuralstem cell,NSCs)的最佳条件,以获得充足的神经干细胞来源,用于中枢神经系统疾病的治疗.方法分离孕13～15 d胎鼠大脑皮层,在无血清含神经生长因子N2培养液中培养,利用有限稀释法单克隆培养和改良法连续传代纯化并扩增NSCs,免疫组织化学法对NSCs及分化细胞进行鉴定.结果可以通过体外培养获得大量神经源性干细胞,在体外经多次传代后仍具有很强的增殖能力和多向分化潜能.结论NSCs的存活和分裂依赖于神经生长因子和N2添加剂的浓度,胚胎脑组织和神经球分离方法影响NSCs的形成速度和数量.掌握NSCs的体外纯化培养和鉴定手段可为进一步研究NSCs生物学特性及神经系统损伤的治疗提供新方法.     

Developmental expression of NMDA and AMPA receptor subunits in vestibular nuclear neurons that encode gravity-related horizontal orientations

We examined the expression profile of subunits of ionotropic glutamate receptors [N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA)] during postnatal development of connectivity in the rat vestibular nucleus. Vestibular nuclear neurons were functionally activated by constant velocity off-vertical axis rotation, a strategy to stimulate otolith organs in the inner ear. These neurons indicated Fos expression as a result. By immunodetection for Fos, otolith-related neurons that expressed NMDA/AMPA receptor subunits were identified as early as P7, and these neurons were found to increase progressively up to adulthood. Although there was developmental invariance in the percentage of Fos-immunoreactive neurons expressing the NR1, NR2A, GluR1, or GluR2/3 subunits, those expressing the NR2B subunit decreased from P14 onward, and those expressing the GluR4 subunit decreased in adults. These double-immunohistochemical data were corroborated by combined immuno-/hybridization histochemical data obtained from Fos-immunoreactive neurons expressing NR2B mRNA or GluR4 mRNA. The staining of both NR2B and GluR4 in the cytoplasm of these neurons decreased upon maturation. The percentage of Fos-immunoreactive neurons expressing the other ionotropic glutamate receptor subunits (viz. NR1, NR2A, GluR1, and GluR2/3) remained relatively constant throughout postnatal maturation. Triple immunofluorescence further demonstrated coexpression of NR1 and NR2 subunits in Fos-immunoreactive neurons. Coexpression of NR1 subunit with each of the GluR subunits was also observed among the Fos-immunoreactive neurons. Taken together, the different expression profiles of ionotropic glutamate receptor subunits constitute the histological basis for glutamatergic neurotransmission in the maturation of central vestibular connectivity for the coding of gravity-related horizontal head movements.     

Functional excitatory GABAA receptors precede ionotropic glutamate receptors in radial glia-like neural stem cells

The involvement of neurotransmission in neuronal development is a generally accepted concept. Nevertheless, the precise regulation of neurotransmitter receptor expression is still unclear. To investigate the expression profiles of the most important ionotropic neurotransmitter receptors, namely GABA_A receptors (GABA_ARs), NMDA receptors (NMDARs), and AMPA receptors (AMPARs), quantitative RT-PCR, immunoblot analysis and patch clamp studies were performed in in vitro-generated neural stem cells (NSCs). This clearly defined cell line is closely related to radial glia cells, the stem cells in the neonate brain.We found functional GABA_ARs of the subunit composition α2, β3, and γ1 to be expressed. Unexpectedly, functional ionotropic glutamate receptors were absent. However, NSCs expressed the NMDAR subunits NR2A and NR3A, and the AMPAR subunit GluR4 at the protein level, and GluR3 at the mRNA level.The overexpression of functional NMDARs in NSCs led to an increased mRNA level of AMPAR subunits, indicating a role in synaptogenesis. Early neuronal markers remained unchanged. These data extend our knowledge about ionotropic neurotransmitter receptor expression during neuronal development and will aid further investigations on activity-dependent neurogenesis.     

Biochemical and molecular studies of NMDA receptor subunits NR1/2A/2B in hippocampal subregions throughout progression of Alzheimer's disease pathology

Alzheimer's disease (AD) is characterized by loss of specific cell populations within selective subregions of the hippocampus. Excitotoxicity, mediated via ionotropic glutamate receptors, may play a crucial role in this selective neuronal vulnerability. We investigated whether alterations in NMDA receptor subunits occurred during AD progression. Employing biochemical and in situ hybridization techniques in subjects with a broad range of AD pathology, protein levels, and mRNA expression of NR1/2A/2B subunits were assayed. With increasing AD neuropathology, protein levels and mRNA expression for NR1/2B subunits were significantly reduced, while the NR2A subunit mRNA expression and protein levels were unchanged. Cellular analysis of neuronal mRNA expression revealed a significant increase in the NR2A subunit in subjects with moderate neurofibrillary tangle neuropathology. This investigation supports the hypothesis that alterations occur in the expression of specific NMDA receptor subunits with increasing AD pathologic severity, which is hypothesized to contribute to the vulnerability of these neurons.     

Quantitative measurement of glutamate receptor subunit protein expression in the postnatal rat spinal cord

Glutamate is the major excitatory neurotransmitter in the CNS and its effects on neurons are dependent on the type and composition of glutamate receptors with which it interacts. In this study, the protein expression levels of several ionotropic glutamate receptor subunits (N-methyl-D-aspartate (NMDA) subunits NR1, NR2A, NR2B, and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor subunits GluR1, GluR2, GluR4) were quantified in particulate preparations from rat spinal cord at various ages after birth. We found that all six subunits showed high expression in the early postnatal period, followed by a subsequent decline as the rats matured to adults. The levels of two subunits (NR2A and GluR4) were found to initially increase during the first postnatal week prior to the decline to adult levels. The high levels of expression observed of these subunits in the early postnatal period may have implications for mechanisms of neural injury and cell death in the immature nervous system that involve cation influx through ionotropic glutamate receptors.     

Expression of functional NR1/NR2B-type NMDA receptors in neuronally differentiated SK-N-SH human cell line

The present study demonstrates that human SK-N-SH neuroblastoma cells, differentiated by retinoic acid (RA), express functional NMDA receptors and become vulnerable to glutamate toxicity. During exposure to RA, SK-N-SH cells switched from non-neuronal to neuronal phenotype by showing antigenic changes typical of postmitotic neurons together with markers specific for cholinergic cells. Neuronally differentiated cells displayed positive immunoreactivity to the vesicular acetylcholine transporter and active acetylcholine release in response to depolarizing stimuli. The differentiation correlated with the expression of NMDA receptors. RT-PCR and immunoblotting analysis identified NMDA receptor subunits NR1 and NR2B, in RA-differentiated cultures. The NR1 protein immunolocalized to the neuronal cell population and assembled with the NR2B subunit to form functional N-methyl-D-aspartate (NMDA) receptors. Glutamate or NMDA application, concentration-dependently increased the intracellular Ca2+ levels and acetylcholine release in differentiated cultures, but not in undifferentiated SK-N-SH cells. Moreover, differentiated cultures became vulnerable to NMDA receptor-mediated excitotoxicity. The glutamate effects were enhanced by glycine application and were prevented by the NMDA receptor blocker MK 801, as well as by the NR2B selective antagonist ifenprodil. These data suggest that SK-N-SH cells differentiated by brief treatment with RA may represent an unlimited source of neuron-like cells suitable for studying molecular events associated with activation of human NR1/NR2B receptors.     

The NMDA receptor subunit NR2B of neonatal rat brain: complex formation and enrichment in axonal growth cones

The N-methyl-d -aspartate (NMDA) subtype of ionotropic glutamate receptors comprises a family of highly homologous subunits which assemble into oligomeric protein complexes. Alterations in subunit composition are developmentally regulated, leading to functionally distinct receptor populations. Here, the contribution of the subunit NR2B to NMDA receptor complex formation was analysed in neonatal rat brain, employing polyclonal antibodies raised against NR2B-specific synthetic peptides. By hydrodynamic size fractionation of the solubilized receptor protein and chemical cross-linking, NR2B antigen was found to be associated with several protein species of up to 690 kDa molecular weight. These observations show NR2B to be part of a multimeric receptor complex. Fractionation of cortex homogenates from E18 rat embryos on sucrose density gradients revealed NR2B polypeptide to be highly enriched in axonal growth cones. A similar distribution was found by fluorescence microscopy of immature hippocampal neurons, showing a preferential accumulation of NR2B antigen in axonal growth cones and varicosities. In mature cells, NR2B antigen displayed a punctated distribution pattern with redistribution to somato-dendritic spheres. The association of NR2B with axonal growth cones and processes of immature neurons suggests a role of NMDA receptors in the regulation of neurite outgrowth and migration.     

N-Methyl-D-aspartate receptor subunit proteins and their phosphorylation status are altered selectively in Alzheimer's disease

The N-methyl-D-aspartate (NMDA) receptor is a subtype of the ionotropic glutamate receptor that plays a pivotal role in synaptic mechanisms of learning and memory. We tested the hypothesis that NMDA receptor protein levels are abnormal in Alzheimer's disease (AD). By immunoblotting, we assessed levels of both non-phosphorylated and phosphorylated receptor subunit proteins from four separate regions of 16 post-mortem brains. Three patient groups with thorough pre-mortem neuropsychological testing were evaluated, including AD, early AD (p-AD), and control patients. Protein levels and phosphorylation status of NMDA receptor subunits NR1, NR2A and NR2B were correlated with measurements of cognitive performance. Selective regional reductions in NMDA receptor subunit protein levels were found in AD compared to controls, but protein levels in the p-AD group were similar to controls. Reductions of NR1 (53%, P<0.05) and NR2B (40%, P<0.05) were identified in hippocampus. Reductions of NR2A (39%, P<0.05) and NR2B (31%, P<0.01) were found in entorhinal cortex. No reductions were noted in occipital cortex and caudate. Phosphorylated NR2A (30%, P<0.05) and NR2B (56%, P<0.01) were selectively reduced in entorhinal cortex in AD when compared to controls. Both phosphorylated and non-phosphorylated NMDA receptor protein levels in entorhinal cortex correlated with Mini-Mental Status Examination (MMSE) and Blessed (BIMC) scores. The losses of phosphorylated and non-phosphorylated NMDA receptor subunit proteins correlated with changes in synaptobrevin levels (a presynaptic protein), but not with age or post-mortem interval. Our results demonstrate that NMDA receptor subunits are selectively and differentially reduced in areas of AD brain, and these abnormalities correlate with presynaptic alterations and cognitive deficits in AD.