Molecular Determinants of Extrasyn. GABA Receptor Fcn on Cerebellar Granule Cells

该提案的广泛目标是检验两个假设，（i）小脑颗粒细胞的突触外抑制是由GABAA受体（GABAR）亚基α 6、β 3和δ组成的受体介导的，以及（ii）该亚基组合物的受体是某些全身麻醉剂、神经活性类固醇和乙醇的主要靶点。该提案 依赖于分子方法和电生理学分析的结合。本研究对β 3和δ亚基基因缺陷小鼠和携带颗粒细胞特异性GABAR基因α 6点突变（R100Q）的新产生的“敲入”大鼠的颗粒细胞GABAR功能进行了仔细的研究。将进行三个具体目标：1）突触和突触外GABAR信号的电生理学分析将测试在缺乏β 3和δ亚基基因的小鼠中突触外抑制是否被特异性破坏。2)测量野生型和α 6R100Q“敲入”大鼠中的突触和突触外信号，以评价突触外GABAR是否是小脑中乙醇、全身麻醉剂和神经活性类固醇作用的行为相关浓度的关键决定因素。3)将设计用于增强或削弱对乙醇或全身麻醉剂的敏感性的突变的荧光标记的GABAR亚基引入野生型和敲除颗粒细胞中，并测量突触外GABAR信号。这些多学科实验严格评估突触外抑制的分子基础，并测试确定乙醇，麻醉剂和神经活性类固醇作用的分子决定因素。对麻醉和酒精中毒机制的理解的进步将产生深远的临床和社会效益，包括改善精神疾病的治疗和更好的治疗严重酒精滥用。

The broad goals of this proposal test two hypotheses, (i) that extrasynaptic inhibition of cerebellar granule cells is mediated by receptors made up of the GABAA receptor (GABAR) subunits alpha6, beta3, and delta, and (ii) that receptors of this subunit composition are primary targets for certain general anesthetics, neuroactive steroids, and ethanol. The proposal relies on a combination of molecular approaches and electrophysiological analysis. This project carefully examines granule cell GABAR function in mice deficient in the beta3, and delta subunit genes and in a newly generated "knock-in" rat carrying a point mutation (R100Q) in the granule-cell specific GABAR gene alpha6.The R100Q mutation is hypothesized to enhance the sensitivity to various GABAR modulators including ethanol and benzodiazepines. Three specific aims will be undertaken: 1) Electrophysiological analysis of synaptic and extrasynaptic GABAR signals will test whether extrasynaptic inhibition is specifically disrupted in mice lacking the genes for the beta3 and delta subunits. 2) Measurement of synaptic and extrasynaptic signals in wild type and in the alpha6R100Q "knock-in" rat will be used to evaluate whether extrasynaptic GABARs are key determinants of behaviorally relevant concentrations of ethanol, general anesthetic, and neuroactive steroid action in cerebellum. 3) Mutant, fluorescently-tagged GABAR subunits designed to enhance or impair sensitivity to ethanol or general anesthetics will be introduced into wild type and knockout granule cells and extrasynaptic GABAR signals will be measured. These multidisciplinary experiments rigorously evaluate the molecular basis for extrasynaptic inhibition and test identify molecular determinants of ethanol, anesthetic, and neuroactive steroid action. Advancement in understanding the mechanisms responsible for anesthesia and ethanol intoxication will yield far-reaching clinical and societal benefits including improved treatments for psychiatric disorders and better therapies for severe alcohol abuse.