ETHANOL INHIBITION OF NMDA RECEPTOR MEDIATED RESPONSES

本研究项目的目的是进一步了解乙醇（EtOH）对急性中毒神经元的影响。 乙醇能有效抑制N- 甲基-D-天冬氨酸型谷氨酸受体（NMDAR）。 在大多数神经元制剂检查到目前为止，这种抑制是选择性的其他谷氨酸受体。 谷氨酸 是哺乳动物CNS中的主要兴奋性神经递质， 这种神经递质与许多中枢神经系统功能有关，包括运动控制 和信息存储。 有大量证据表明，乙醇抑制NMDAR功能有助于急性中毒的各个方面，包括认知障碍 和镇静剂 EtOH对NMDAR的作用机制尚未完全了解。 此外，在文献中几乎没有关于以下的分子性质的确定性信息： 赋予EtOH敏感性的NMDAR。 本申请中提出的研究将解决 这些问题通过测试两个假设。 第一个目标是检验EtOH 在从缺乏ε 1或ε 2亚基的小鼠的选定脑区域分离的神经元中，或在这些亚基的T-末端区域中， subunits已被删除。 这将通过检查急性分离或在细胞培养物中生长的CNS神经元的全细胞记录中受体的EtOH抑制进行检测。 神经元 将检查来自野生型和突变小鼠的新皮层和小脑皮层。 预期EtOH将更有效地抑制来自野生型神经元的新皮层神经元中的NMDAR。 小鼠相对于ε 2敲除和C-末端截短的动物。 ε 1敲除和C末端截短的小鼠应显示新皮层神经元中NMDAR的EtOH敏感性的发育变化丧失。 与野生型小鼠相比，ε 1突变小鼠小脑颗粒细胞中NMDA受体的乙醇敏感性可能增强。 还将在野生型和ε 1突变小鼠中检查海马脑切片的CA 1区中NMDAR介导的突触传递。 据预测，EtOH将产生更大的抑制作用， 在野生型小鼠中比在ε 1敲除和C-末端截短小鼠中更易传播。 第二个目的是检验孔环和第三层膜中的关键氨基酸残基 NMDAR 1亚基的跨（TMIII）结构域赋予NMDAR EtOH敏感性。 这 将通过在HEK 293细胞中的全细胞电生理学实验进行检查， 含有突变型或野生型NMDAR 1亚基的重组受体。 野生型和在孔环和TMIII区域具有单点突变的受体的乙醇敏感性将被评估。 测定 将对突变受体的生物物理学和药理学特性进行全面检查，以确定突变是否特异性影响EtOH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。 希望这些实验的结果将为开发可以抵消EtOH的一些破坏性神经效应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.