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性。 的 乙醇产生行为效应的确切分子机制， 耐受性和身体依赖性尚未被定义。 然而，在这方面， 几条证据表明GABA和NMDA受体系统参与了 这些事件。 本提案将审查长期、 和间歇性的乙醇处理。 拟议 研究将在哺乳动物培养的皮层神经元中进行， 受控条件，不依赖于药代动力学变异性， 使用涉及结合研究的综合方法， 偶联相互作用、功能测定和特异性 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点是 它们反映了完整CNS神经元细胞类型的多样性， 为研究慢性药物作用提供了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， 以及这一区域在调节乙醇作用方面的重要性。 由于GABA/A和NMDA受体以多种亚型存在， 慢性的，间歇性的，酒精治疗可能会产生一种 受体亚单位的差异性改变，其中一些增加， 在一些亚基中减少，和/或在其他亚基中没有变化。 这可能导致 在配体有或没有明显变化的受体中， 约束力 以下具体目标将检验这一假设： 比较和描述慢性和间歇性乙醇的影响 对配体与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会改变 与GABA/A和NMDA相关的各个位点之间的偶联 受体; III）慢性和间歇性乙醇治疗是否改变 GABA/A和NMDA受体介导的传递的功效; IV） 确定慢性和间歇性乙醇治疗对 GABA/A和NMDA受体亚基mRNA;和V）确定 慢性和间歇性乙醇处理对GABA/A和NMDA受体的影响 使用Western印迹和免疫沉淀法测定亚基多肽水平 问题研究

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.