CHRONIC/INTERMITTENT ETHANOL AND GABA AND NMDA RECEPTORS

长期使用乙醇会导致耐受性和身体依赖。这个 酒精产生行为影响的确切分子机制， 容忍度和身体依赖的定义尚未确定。然而， 几条证据表明GABA和NMDA受体系统在 这些事件。目前的提案将审查慢性、 对这些受体进行间歇性的乙醇处理。建议数 研究将在哺乳动物培养的皮质神经元中进行，在 受控条件，与药代动力学变异性无关，并由 使用涉及绑定调查的综合方法， 偶联相互作用、功能分析和特定基因的表达 GABA/A和NMDA受体亚单位mRNA和多肽水平，以及 免疫沉淀研究。培养神经元的一个主要优势是 它们反映了完整的中枢神经系统神经元细胞类型的多样性，以及 为研究慢性药物的作用提供理想的体外模型体系 治疗。我们使用大脑皮层培养物的原因是 该区域存在高密度的GABA/A和NMDA受体， 以及这个区域在调节乙醇的作用中的重要性。 由于GABA/A和NMDA受体以几种不同的形式存在，因此它是可行的 慢性和间歇性的乙醇治疗可能会产生 受体亚基的不同变化，随着某些亚基的增加， 在一些亚基中减少，和/或在其他亚基中没有变化。这可能会导致 在配体有或没有明显变化的改变的受体中 有约束力的。以下具体目标将检验这一假设：i) 比较和描述慢性和间歇性乙醇的影响 配体与GABA/A和NMDA受体结合的处理 复杂；ii)慢性和间歇性乙醇治疗是否改变了 GABA/A和NMDA相关的不同位点之间的偶联 受体；iii)慢性和间歇性乙醇处理是否会改变 GABA/A和NMDA受体介导的转导效应；IV) 确定慢性和间歇性乙醇治疗对 GABA/A和NMDA受体亚单位mRNAs；以及V)决定了 慢性和间歇性乙醇处理对GABA/A和NMDA受体的影响 免疫印迹和免疫沉淀法测定亚基多肽水平 学习。

Chronic use of ethanol leads to tolerance and physical dependence. The exact molecular mechanisms by which ethanol produces behavioral effects, tolerance, and physical dependence have yet to be defined. However, several lines of evidence implicate GABA and NMDA receptor systems in these events. The present proposal will examine the effect of chronic, and intermittent, ethanol treatments on these receptors. The proposed studies will be conducted in mammalian cultured cortical neurons, under controlled conditions, independent of pharmacokinetic variability, and by using a comprehensive approach that involves investigation of binding, coupling interactions, functional assays, and the expression of specific GABA/A and NMDA receptor subunit mRNA, and polypeptide levels, and immunoprecipitation studies. A major advantage of the cultured neurons is that they reflect the diversity of cell types of intact CNS neurons, and provide an ideal in vitro model system to study the effect of chronic drug treatment. Our reason for using cultures from cerebral cortex is based on the presence of high density of GABA/A and NMDA receptors in this region, and the importance of this region in mediating the actions of ethanol. Since GABA/A and NMDA receptors exist in several isoforms, it is feasible that chronic, and intermittent, ethanol treatments may produce a differential alteration of receptor subunits, with increases in some, decreases in some, and/or no changes in other subunits. This could result in altered receptors with or without an apparent change in the ligand binding. The following specific aims will test this hypothesis: I) compare and characterize the effects of chronic, and intermittent, ethanol treatment on the binding of ligands to the GABA/A and NMDA receptor complex; II) does chronic, and intermittent, ethanol treatments alter the coupling between various sites associated with the GABA/A and NMDA receptors; III) does chronic, and intermittent, ethanol treatments alter the efficacy of GABA/A and NMDA receptor-mediated transmission; IV) determine the effects of chronic, and intermittent, ethanol treatment on GABA/A and NMDA receptor subunit mRNAs; and V) determine the effects of chronic, and intermittent, ethanol treatment on GABA/A and NMDA receptor subunit polypeptide levels using Western blot and immunoprecipitation studies.