大鼠全脑缺血再灌注后海马CA1区神经元凋亡机制的研究

目的 研究大鼠全脑短暂缺血再灌注后海马CA1区椎体神经元的死亡机制.方法 采用经典的四血管夹闭法制作大鼠全脑缺血再灌注模型,取缺血再灌注后不同时间(6 h、24 h、48 h)的海马CA1区脑组织,用ELISA检测Caspase-3的活性变化.同时运用免疫印记方法检测PKC、NMDA受体NR2A及NR2B亚基磷酸化水平的变化.结果 与对照组相比,Caspase-3检测显示缺血再灌注6 h、24 h、48 h海马CA1区椎体神经元呈现逐渐凋亡增多的迟发性死亡.磷酸化蛋白激酶C在缺血6 h即有明显增高,并且于24 h、48 h达到表达高峰(P<0.05);NMDA受体亚基NR2A和R2B的磷酸化水平缺血再灌注后均呈现明显升高趋势.讨论 PKC、NR2B、CaMⅡ级联反应的发生可能是大鼠全脑缺血再灌注后海马CA1区锥体神经元发生迟发性死亡的重要机制.     

二氮嗪预处理对Aβ_(1~42)作用神经元NR2B亚基蛋白表达的影响

目的研究ATP敏感的钾离子通道开放剂二氮嗪(diazoxide)预处理对Aβ1~42作用原代培养神经元N-甲基-D-天冬氨酸(NMDA)受体2B(NR2B)亚基蛋白表达的影响。方法原代培养大鼠皮层海马神经元并进行鉴定,将细胞随机分为对照组、单纯Aβ1~42干预组、二氮嗪预处理1h后Aβ1~42干预组(Aβ1~42+diazoxide)、单纯二氮嗪预处理组,并采用免疫印迹检测不同时间点(24 h、72 h)细胞NR2B亚基蛋白表达水平的变化。结果 Aβ1~42(2μmol/L)作用神经元24 h后,与对照组相比,单纯Aβ1~42干预组和Aβ1~42+diazoxide组的NR2B亚基蛋白表达均无明显改变。Aβ1~42作用神经元72 h后,与对照组比较,单纯Aβ1~42组NR2B亚基蛋白表达量显著升高(P0.05);而与单纯Aβ1~42干预组相比,Aβ1~42+diazoxide组NR2B亚基蛋白表达明显降低(P0.05)。结论 Aβ1~42作用原代培养神经元72 h,能够显著增加神经细胞NR2B亚基蛋白的表达量;同时,二氮嗪能拮抗Aβ1~42所引起的NR2B亚基蛋白表达量升高,提示二氮嗪可能通过NMDA受体通路影响Aβ1~42的细胞毒性作用。     

炎症后内脏高敏感大鼠前扣带回皮质NR2A、NR2B表达变化

背景:研究显示N-甲基-D-天冬氨酸(NMDA)受体参与了伤害性信号的传递,中枢前扣带回皮质(ACC)在内脏高敏感大鼠内脏疼痛反应的调节中起重要作用,该作用是由NMDA受体活性增强介导的。目的:检测炎症后内脏痛觉过敏大鼠ACC区域NMDA受体亚基NR2A、NR2B的表达变化,探讨两者在炎症后内脏高敏感形成中的作用。方法:以去氧胆酸结肠灌注建立炎症后内脏痛觉过敏大鼠模型。造模3周后观察实验组和对照组结肠组织病理学改变,以对结直肠扩张(CRD)的内脏运动反射(VMR)幅值为指标评价内脏痛敏感性的变化,以免疫荧光法和蛋白质印迹法检测ACC区域NR2A、NR2B表达情况。结果:实验组和对照组大鼠结肠组织均未见明显病理学改变。CRD压力为60 mm Hg(伤害性刺激)时,实验组VMR幅值显著高于对照组(P0.05)。与对照组相比,实验组ACC区域NR2A、NR2B荧光强度和蛋白表达量均显著上调(P0.05)。结论:ACC区域NMDA受体亚基NR2A、NR2B表达上调在炎症后内脏高敏感的形成中发挥重要作用。     

NMDA受体NR1、NR2A/B在丘脑前核-海马CA1、CA3脑区和齿状回的分布与表达

目的 探讨NMDA受体NR1、NR2A/B在丘脑前核-海马CA1、CA3脑区和齿状回的分布与表达,以及丘脑前核-海马神经元的学习记忆功能及作用机制.方法 运用原位杂交检测技术观测丘脑前核及海马CA1、CA3和齿状回内NMDA受体NR1、NR2A 及NR2B mRNA的分布特点.结果 ①原位杂交阳性产物呈棕黄色,主要分布在神经元的胞浆中,胞核基本不着色.②在丘脑前核,阳性神经元分布较密集,细胞形态较一致.③在海马锥体层阳性神经元分布较多,呈带状.在分子层、多形层分布少.④NR1、NR2A/B在丘脑前核和海马CA1、CA3脑区及齿状回均有表达,其中NR1在齿状回表达水平最强,NR2B在丘脑前核、海马CA1、CA3和齿状回表达水平基本相同.结论 在丘脑前核-海马的局部神经元环路中NMDA受体NR1、NR2A及NR2B mRNA分布广泛.其中NR2B mRNA在丘脑前核和海马CA1、CA3脑区及齿状回表达水平基本相同,可能与此环路学习记忆有关.     

人参皂苷Rd预处理对局灶性脑缺血再灌注大鼠NR2B受体和核酸内切酶G表达的影响

目的:观察人参皂苷Rd预处理对局灶性脑缺血再灌注大鼠基底节区N-甲基-D-天冬氨酸(NMDA)受体亚单位NR2B蛋白和核酸内切酶G(EndoG)表达变化的影响,探讨人参皂苷Rd治疗缺血性卒中的可能机制.方法:栓线法建立大鼠大脑中动脉闭塞模型,免疫组织化学染色和图像分析法检测局灶性脑缺血1 h再灌注1、6、24、72 h后基底节区NR2B和EndoG表达,评价人参皂苷Rd对NR2B和EndoG表达和脑梗死体积的影响.结果:缺血再灌注组缺血侧基底节区NR2B阳性表达显著增加,EndoG在细胞核内表达显著;再灌注不同时间点人参皂苷Rd处理组NR2B和EndoG阳性表达均显著降低(P<0.05或P<0.01),脑梗死体积显著缩小(P<0.01).结论:脑缺血再灌注后NMDA受体亚单位NR2B和凋亡诱导因子EndoG表达显著增加;人参皂苷Rd预处理能显著降低NR2B和EndoG表达,通过抑制兴奋性神经毒性和阻断神经细胞凋亡,缩小脑梗死体积,从而起神经保护作用.     

电针对脑缺血再灌注模型大鼠纹状体NMDA受体NR2亚基蛋白表达的影响

目的 观察脑缺血再灌注模型大鼠受损侧纹状体组织中NMDA受体调节亚单位NR2A和NR2B蛋白表达及其电针干预后的变化,探讨电针对缺血再灌注兴奋性氨基酸毒性的拮抗作用,进一步阐明电针治疗缺血性脑血管疾病的机制.方法 用数字随机表将15只SD大鼠随机分成3组:假手术组、模型组、电针组,每组各5只.采用改良Longa线栓法制作大脑中动脉短暂性缺血再灌注模型,电针组大鼠在再灌同时电针“百会”、“大椎”两穴(连续波,频率3 Hz,电流强度1～3 mA),30 min,深度均为10 mm.免疫组织化学法及图像处理系统检测分析大鼠受损侧纹状体NR2A、NR2B蛋白表达情况.结果 与假手术组相比,模型组大鼠纹状体组织中NR2A的表达量明显下降,而NR2B的表达量明显升高,给予电针“百会”、“大椎”穴后的大鼠纹状体组织NR2A表达细胞数及含量均明显高于模型组,表达NR2B的细胞数及含量均明显降低.结论 电针可能通过激活NR2A受体蛋白表达,抑制NR2B受体蛋白的过量表达,从而调节NMDA受体复合物的整体功能活性,减少NMDA受体介导的兴奋性氨基酸毒性反应,减轻脑缺血再灌注引起的局灶性脑损伤,发挥一定的脑神经保护作用.     

NMDA受体介导的缺血性脑损伤的治疗进展

NMDA受体介导的神经兴奋毒性在缺血性脑损伤中起着关键性作用。目前 ,从NMDA受体出发 ,通过抑制受体激动剂的释放、拮抗受体的活性、下调受体蛋白的基因表达等途径对缺血性脑损伤的治疗进行了大量的实验研究。     

NMDA受体NR2B亚型基因对海马成年新生神经元形态发生的影响

目的研究NMDA受体NR2B亚型基因对海马成年新生颗粒细胞形态发生的影响。方法通过Cre-loxp重组酶系统构建NMDA受体NR2B亚型基因单细胞敲除模型。运用Neurolucida软件系统对野生型、NR2B基因敲除成年新生神经元进行形态重建,观察树突长度、树突复杂度以及棘突的变化。结果 NR2B基因敲除成年新生神经元外形与WT神经元相似,树突长度接近,棘突减少。Sholl分析显示在以胞体为中心,距胞体不同距离的树突亚区域内,树突复杂性(树突交叉)减低,与此同时棘突分布密度减低(P<0.05)。结论海马成年新生颗粒神经元NR2B基因敲除对新生细胞树突长度影响甚微,但降低了树突复杂性,减少棘突形成,从而导致了功能性整合入海马特定神经信息网络的障碍。     

脑通胶囊对VD大鼠海马组织NMDA受体1亚基和2B亚基mRNA表达的影响

目的观察脑通胶囊对血管性痴呆(VD)模型大鼠学习记忆、海马组织N-甲基-D-天冬氨酸(NMDA)受体1亚基和2B亚基mRNA表达的影响。方法采用改良的四血管法(14-VO)制备VD模型,Morris水迷宫测定大鼠学习、记忆能力,实时荧光定量PCR检测NMDA受体1亚基和2B亚基mRNA的表达情况。结果与模型组比较,脑通胶囊大、中剂量组逃避潜伏期缩短,穿越原平台次数增加,NMDA受体1亚基mRNA表达降低,NMDA受体2B亚基mRNA表达升高(P<0.05,P<0.01)。结论脑通胶囊可降低NMDA受体1亚基mRNA的表达,提高NMDA受体2B亚基mRNA的表达,从而改善VD大鼠学习、记忆能力。     

NR2B受体拮抗剂及其在缺血性脑血管病中的应用

脑缺血时的神经损伤与谷氨酸过度兴奋N-甲基-D-天门冬氨酸（NMDA）受体有关。由于非选择性NMDA受体拮抗剂能影响所有NMDA受体而产生不良反应,其临床应用受到很大限制,因此选择性NMDA受体在近年来受到越来越多的重视。NR2B亚单位拈抗剂主要分为哌啶衍生物、酰胺衍生物、脒衍生物、氨基喹啉衍生物等,主要代表药物有艾芬地尔、依利罗地。它们能选择性作用于NMDA受体NR2B亚单位,有望成为临床上安全有效的神经保护剂。     

Dual regulation of NR2B and NR2C expression by NMDA receptor activation in mouse cerebellar granule cell cultures

In the developing cerebellum, switching of the subunit composition of NMDA receptors occurs in granule cells from NR2B-containing receptors to NR2C-containing ones. We investigated the mechanisms underlying switching of NR2B and NR2C subunit composition in primary cultures of mouse granule cells at the physiological KCl concentration (5 mM). Granule cells extensively extended their neuritic processes 48 h after having been cultured in serum-free medium containing 5 mM KCl. Consistent with this morphological change, NR2B mRNA and NR2C mRNA were down- and up-regulated, respectively, in the granule cells. This dual regulation of the two mRNAs was abrogated by blocking excitation of granule cells with TTX. This neuronal activity–dependent regulation of NR2B and NR2C mRNAs was abolished by the addition of selective antagonists of AMPA receptors and NMDA receptors. Furthermore, the dual regulation of NR2B and NR2C mRNAs in TTX-treated cells was restored by the addition of NMDA in the presence of the AMPA receptor antagonist, but not by that of AMPA in the presence of the NMDA receptor antagonist. Importantly, the NMDA receptor activation drove the NR2B/NR2C switching of NMDA receptors in the cell-surface membrane of granule cells. This investigation demonstrates that stimulation of NMDA receptors in conjunction with the AMPA receptor–mediated excitation of granule cells plays a key role in functional subunit switching of NMDA receptors in maturing granule cells at the physiological KCl concentration.     

Enhanced ethanol inhibition of recombinant N-methyl-D-aspartate receptors by magnesium: role of NR3A subunits

Background:  The effects of ethanol on brain function are thought to be partly because of altered activity of ion channels that regulate synaptic activity. Results from previous studies from this lab and others have shown that ethanol inhibits the function of the N -methyl- d -aspartate (NMDA) receptors, a calcium-permeable ion channel activated by the neurotransmitter glutamate. Factors that influence the acute sensitivity of NMDA receptors to ethanol may be critical in determining how neurons and neuronal networks respond to the presence of ethanol. In this study, we have examined the effect of physiologically relevant concentrations of magnesium on the ethanol sensitivity of recombinant NMDA receptors and how ethanol inhibition under these conditions is influenced by the NR3A subunit.
Methods:  Recombinant cDNAs encoding NMDA receptor subunits were expressed in human embryonic kidney 293 cells. Whole-cell patch-clamp electrophysiology was used to measure currents induced by rapid application of glutamate in the absence and presence of ethanol.
Results:  In magnesium-free recording solution, ethanol inhibited glutamate-mediated currents in cells transfected with NMDA receptor subunits. The magnitude of ethanol inhibition was significantly enhanced when recordings were carried out in media containing 1 mM magnesium. This effect was reversible and required magnesium-sensitive receptors. Magnesium did not enhance ethanol inhibition of glycine-activated NR1/NR3A/NR3B receptors. However, NR3A co-expression prevented the enhancement of ethanol's inhibitory effect on receptors composed of NR2A but not NR2B subunits.
Conclusions:  These results suggest that under physiological conditions, NR3A may be an important regulator of the acute ethanol sensitivity of brain NMDA receptors.     

Chronic ethanol upregulates NMDA and AMPA, but not kainate receptor subunit proteins in rat primary cortical cultures

The present study examined the effects of chronic ethanol exposure on the expression of N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxalone (AMPA) and kainate receptor subunit proteins in rat cortical neuronal cultures grown in media containing 2 mM (high) or 0.1 mM (low) glutamine. Immunoblot analysis of NMDA (NR1, NR2A, NR2B, and NR2D), AMPA (GluR1 and GluR2/3), and kainate (GluR6/7) subunit polypeptides in 3-, 5-, 8-, 10-, and 12 day-old-cultures showed that NMDA receptor subunits NR1, NR2A, and NR2B and AMPA receptor subunits GluR2/3 progressively increased as a function of time, whereas levels of NMDA subunit NR2D were high at day 3 and progressively declined to barely detectable levels by day 12. Levels of AMPA subunit GluR1 and the kainate subunit GluR6/7 remained stable throughout the time course. Replacing the culture media with low glutamine media at culture day 5 did not alter the levels of subunit proteins measured at culture days 9 and 13. However, exposure of low glutamine cultures to 100 mM ethanol for 4 days (starting at culture day 9) significantly increased the levels of NMDA receptor subunits (NR1, NR2A, and NR2B) and AMPA receptor subunits (GluR1 and GluR2/3), but had no effect upon kainate receptor subunits (GluR6/7) or the synapse-associated proteins synapsin I and PSD-95. In contrast, chronic ethanol did not alter the levels of any of these subunit proteins in cells grown in high glutamine. These data demonstrate that under certain experimental conditions, prolonged exposure to ethanol upregulates NMDA and AMPA receptor subunit proteins, but has no effect upon kainate receptor subunit proteins. Because we have previously shown that acute ethanol can inhibit NMDA and AMPA, but not kainate, receptor function in these cultures, the increase in subunit expression likely reflects an adaptive response to the inhibitory effects of ethanol and suggests that both NMDA and AMPA receptors may play an important role in adaptation of the CNS to chronic ethanol.     

NMDA receptor function is regulated by the inhibitory scaffolding protein, RACK1

Phosphorylation regulates the function of ligand-gated ion channels such as the N-methyl d-aspartate (NMDA) receptor. Here we report a mechanism for modulation of the phosphorylation state and function of the NMDA receptor via an inhibitory scaffolding protein, RACK1. We found that RACK1 binds both the NR2B subunit of the NMDA receptor and the nonreceptor protein tyrosine kinase, Fyn. RACK1 inhibits Fyn phosphorylation of NR2B and decreases NMDA receptor-mediated currents in CA1 hippocampal slices. Peptides that disrupt the interactions between RACK1, NR2B, and Fyn induce phosphorylation and potentiate NMDA receptor-mediated currents. Therefore, RACK1 is a regulator of NMDA receptor function and may play a role in synaptic plasticity, addiction, learning, and memory.     

The molecular events occur during MK-801-induced cytochrome oxidase subunit II down-regulation in GT1-7 cells

Previously, we showed that predominant expression of the N-methyl-D-aspartate (NMDA) receptor in the neurons of the sexually dimorphic nucleus of the preoptic area of male rats plays an important role in preventing neurons from apoptosis during sexual development. Blocking of the NMDA receptor by dizocilpine ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-iminemaleate (MK-801) causes down-regulation of some survival-related genes including cytochrome oxidase subunit II (COII), a mitochondria-encoded complex IV subunit, which in turn induces ATP depletion and the occurrence of apoptosis. The aim of this study is to investigate the molecular events during down-regulation of the COII gene expression induced by blocking of the NMDA receptor. Treatment of the GnRH cell line (GT1-7) with MK-801 caused 1) a decrease of intracellular calcium concentration ([Ca2+]i) after 20 h; 2) significant decreases of the levels of peroxisome proliferator-activated receptor coactivator-1 (PGC-1) mRNA and protein after 24 h; 3) down-regulation of COII mRNA after 36 h; and 4) the occurrence of neuronal apoptosis after 48 h. Accordingly, we hypothesize that blocking of the NMDA receptor may cause a decrease of the [Ca2+]i, which in turn inhibits the expressions of PGC-1 and COII and then leads to subsequent neuronal apoptosis.     

Effects of c-Src tyrosine kinase on ethanol sensitivity of recombinant NMDA receptors expressed in HEK 293 cells

N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that are important mediators of the actions of the excitatory amino acid neurotransmitter glutamate. Previous studies have shown that ethanol inhibits the function of both wild-type receptors found in neurons and recombinant NMDA receptors expressed in heterologous cells, such as oocytes and transfected mammalian cells. Although some studies have reported that certain subunit combinations display an enhanced sensitivity to ethanol, this effect is not observed in all experimental systems. This discrepancy may be due to varying levels of endogenous modulators, such as kinases, between different cell preparations. In this study, we investigated the effects of tyrosine phosphorylation on the ethanol sensitivity of NMDA receptor function using a recombinant cell system where levels of both NMDA subunits and protein kinases can be more carefully controlled. Human embryonic kidney (HEK 293) cells were transfected with different NMDA receptor subunits and a c-Src-green fluorescent protein (GFP) fusion protein that could be directly visualized in living cells. Agonist-stimulated calcium flux was measured in single cells using fura-2 video imaging. As expected, cells transfected with the NR1/NR2B subunits were more sensitive to inhibition by the NR2 selective antagonist ifenprodil than those transfected with NR1/ NR2A or NR1/NR2A/NR2B subunits. All receptor combinations were inhibited by ethanol (25 and 100 mM), with the NR1/NR2B combination being slightly more sensitive than NR1/NR2A or NR1/NR2A/NR2B. Control and NMDA-receptor transfected HEK 293 cells displayed a low degree of tyrosine phosphorylation as measured by immunofluorescence and Western immunoblotting using an antiphosphotyrosine antibody. Phosphorylation was markedly enhanced in cells transfected with the c-Src-GFP fusion protein. The sensitivity of NMDA receptors to either 25 or 100 mM ethanol, or 10 microM ifenprodil, was not significantly altered by co-transfection with c-Src-GFP. These results indicate that, although NMDA receptors can be a target of c-Src tyrosine kinase, tyrosine phosphorylation by this enzyme does not modulate the inhibitory effects of ethanol on NMDA-activated currents.     

氯离子通道与缺血性卒中

缺血性卒中是发病率和致残率最高的疾病之一.高血压是公认的缺血性卒中最重要的独立危险因素,在高血压发展过程中出现的血管重构是引发缺血性卒中的病理学基础.研究表明,血管平滑肌细胞异常增殖和凋亡都将导致血管重构.此外,脑缺血再灌注可导致神经元损伤和凋亡.近来的研究表明,血管重构和神经元凋亡都与氯离子通道有关.至少3种氯离子通道参与这些过程:容积调控的氯离子通道、钙激活的氯离子通道以及囊性纤维跨膜电导调节体.文章就这3种氯离子通道在血管重构、神经元凋亡以及缺血性卒中中的作用进行了综述.     

Effects of 8 different NR1 splice variants on the ethanol inhibition of recombinant NMDA receptors

BACKGROUND: N-Methyl-D-aspartate (NMDA) receptors are glutamate-activated ion channels that are assembled from NR1 and NR2 subunits. These receptors are highly enriched in brain neurons and are considered to be an important target for the acute and chronic effects of ethanol. NR2 subunits (A-D) arise from separate genes and are expressed in a developmental and brain region-specific manner. The NR1 subunit has 8 isoforms that are generated by alternative splicing of a single gene. The heteromeric subunit makeup of the NMDA receptor determines the pharmacological and biophysical properties of the receptor and provides for functional receptor heterogeneity. Although results from previous studies suggest that NR2 subunits affect the ethanol sensitivity of NMDA receptors, the role of the NR1 subunit and its multiple splice variants is less well known. METHODS: In this study, all 8 NR1 splice variants were individually coexpressed with each NR2 subunit in human embryonic kidney 293 (HEK293) cells and tested for inhibition by ethanol using patch-clamp electrophysiology. RESULTS: All 32 subunit combinations tested gave reproducible glutamate-activated currents and all receptors were inhibited to some degree by 100 mM ethanol. The sensitivity of individual receptors to ethanol was affected by the specific NR1 splice variant expressed with receptors containing the NR1-3 and NR1-4 subunits among the least inhibited by ethanol. CONCLUSIONS: These results suggest that regional, developmental, or compensatory changes in the expression of NR1 splice variants may significantly affect ethanol inhibition of NMDA receptors.