Molecular Determinants of Extrasynaptic GABA Receptor Function on Cerebellar Gran

这项提议的广泛目标检验了两个假设，(I)突触外抑制小脑颗粒细胞是由由GABAA受体(GABAR)亚单位α6、β3和Delta组成的受体介导的，以及(Ii)这个亚单位组成的受体是某些全身麻醉药、神经活性类固醇和乙醇的主要靶标。这项建议 依赖于分子方法和电生理分析的结合。该项目仔细检查了缺乏Beta3和Delta亚单位基因的小鼠的颗粒细胞GABAR功能，以及在颗粒细胞特有的GABAR基因α6中携带点突变(R100Q)的新产生的“敲入”大鼠。R100Q突变被假设为增强对包括乙醇和苯二氮卓类在内的各种GABAR调节剂的敏感性。将进行三个具体目标：1)对突触和突触外GABAR信号进行电生理学分析，以测试在缺乏Beta3和Delta亚基基因的小鼠中，突触外抑制是否受到特异性破坏。2)在野生型和字母6R100Q“敲入”大鼠中，突触和突触外信号的测量将被用来评估突触外GABAR是否是小脑中行为相关浓度的乙醇、全身麻醉剂和神经活性类固醇作用的关键决定因素。3)旨在增强或削弱对乙醇或全身麻醉药敏感性的突变型、荧光标记的GABAR亚基将被引入野生型和敲除颗粒细胞，并将测量突触外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。在了解麻醉和酒精中毒的机制方面取得进展将产生深远的临床和社会益处，包括改善精神疾病的治疗和更好地治疗严重酒精滥用。

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.