ETHANOL INHIBITION OF NMDA RECETOR MEDIATED RESPONSES

这项研究的目的是为了加深我们对乙醇(Etoh)导致急性中毒的神经元效应的理解。乙醇可有效抑制N- 甲基-D-天冬氨酸型谷氨酸受体(NMDAR)。到目前为止，在大多数被检查的神经元制剂中，这种抑制相对于其他谷氨酸受体是选择性的。谷氨酸 是哺乳动物中枢神经系统中的主要兴奋性神经递质，NMDAR通过 这种神经递质与中枢神经系统的许多功能有关，包括运动控制。 和信息存储。大量证据表明，乙醇抑制NMDAR功能与急性中毒有关，包括认知障碍 还有镇静剂。乙醇对NMDARs的作用机制尚不完全清楚。此外，文献中几乎没有关于分子性质的明确信息。 提供乙醇敏感性的NMDAR。本申请中提出的研究将解决 通过检验两个假说来解决这些问题。第一个目标将检验Etoh的假设 从缺乏epsilon 1或epsilon 2亚基的小鼠特定脑区分离出的神经元，或者在这些亚基的t端区域，NMDAR功能的抑制将发生改变。 子单位已被删除。这将通过检测在细胞培养中急性分离或生长的中枢神经系统神经元的全细胞记录中的乙醇对受体的抑制来检验。神经元 从野生型和突变小鼠的新皮质和小脑皮质中提取的蛋白质进行检测。预计乙醇将更有效地抑制野生型新皮质神经元中的NMDAR 小鼠相对于epsilon 2基因敲除和c-末端截短的动物。Epsilon 1基因敲除和c末端截短的小鼠应该表现出新皮质神经元对NMDAR的乙醇敏感性的发育变化的丧失。与野生型小鼠相比，epsilon1突变小鼠小脑颗粒细胞对NMDA受体的乙醇敏感性可能增强。NMDAR介导的突触传递在海马脑片的CA1区也将在野生型和epsilon 1突变小鼠中进行检测。据预测，乙醇将产生更大的抑制作用 在野生型中的传播比在epsilon 1基因敲除和c末端截短的小鼠中的传播要好。第二个目标将检验在孔环和第三膜上的关键氨基酸残基的假设 NMDAR1亚单位的跨(TMIII)结构域使NMDAR对乙醇具有敏感性。这 将通过全细胞电生理实验在HEK 293细胞中表达 含有突变型或野生型NMDAR1亚基的重组受体。在孔环和TMIII区域发生单点突变的野生型和受体的乙醇敏感性将 下定决心。将对突变受体的生物物理和药理学特性进行彻底检查，以确定突变是否专门影响乙醇的敏感性。拟议的实验将增加乙醇对NMDAR影响的分子基础的知识。人们希望，这些实验的结果将为开发能够抵消乙醇的一些破坏性神经影响的治疗方法提供基础。

The goal of this research project is to further our understandingof the neuronal effects of ethanol (EtOH) that contribute toacute intoxication. Ethanol potently inhibits the function of N- methyl-D-aspartate type glutamate receptors (NMDARs). In most neuronal preparations examined to date, this inhibition is selective with respect to other glutamate receptors. Glutamate is the major excitatory neurotransmitter in the mammalian CNS, and activation of NMDARs by this neurotransmitter has been implicated in a number of CNS functions including motor control and information storage. There is a wealth of evidence indicating that EtOH inhibition of NMDAR function contributes to aspects of acute intoxication, including cognitive impairment and sedation. The mechanism of EtOH action on NMDARs is not fully understood. In addition, there is little definitive information in the literature about the molecular properties of NMDARs that confer EtOH sensitivity. The studies proposed in this application will address these issues by testing two hypotheses. The first aim will test the hypothesis that EtOH inhibition of NMDAR function will be altered in neurons isolated from selected brain regions of mice lacking the epsilon1 or epsilon2 subunits or in which the t-terminal region of these subunits has been deleted. This will be tested by examining EtOH inhibition of receptors in whole-cell recordings from CNS neurons acutely isolated or grown in cell culture. Neurons from the neocortex and cerebellar cortex of wild-type and mutant mice will be examined. It is expected that EtOH will more potently inhibit NMDARs in neocortical neurons from wild-type mice relative to epsilon2 knockout and c-terminal truncated animals. The epsilon1 knockout and c-terminal truncated mice should show a loss of developmental changes in EtOH sensitivity of NMDARs in neocortical neurons. Ethanol sensitivity of NMDA receptors is likely to be enhanced in cerebellar granule cells from epsilon1 mutant mice relative to wild-type mice. NMDAR-mediated synaptic transmission in the CA1 region of hippocampal brain slices will also be examined in the wild-type and epsilon1 mutant mice. It is predicted that EtOH will produce greater inhibition of transmission in wild-type than in epsilon1 knockout and c-terminal truncated mice. The second aim will test the hypothesis that key amino acid residues in the pore-loop and third membrane spanning (TMIII) domains of the NMDAR1 subunit confer EtOH sensitivity on the NMDAR. This will be examined by whole cell electrophysiological experiments in HEK 293 cells expressing recombinant receptors containing mutant or wild-type NMDAR1 subunits. Ethanol sensitivity of wild-type and receptors with single-point mutations in the pore-loop and TMIII regions will be determined. Thorough examination of biophysical and pharmacological properties of mutant receptors will be carried out to determine if mutations specifically affect EtOH sensitivity. The proposed experiments will add to ow knowledge of the molecular basis of EtOH effects on NMDARs. It is hoped that the outcome of these experiments will provide a basis for the development of treatments that can counteract some of the damaging neural effects of EtOH.