Molecular Determinants of Extrasynaptic GABA Receptor Function on Cerebellar Gran

小脑粒细胞突触外 GABA 受体功能的分子决定因素

• 批准号: 7390252
• 负责人: Thomas S Otis
• 金额: $ 23.19万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7390252
• 关键词: Alcohol abuse; Alcoholic Intoxication; Anesthesia procedures; Anesthetics; Behavioral; Benzodiazepines; Binding; Biological Assay; Blood alcohol level measurement; Breeding; Cerebellum; Clinical; Complement; Data; Ethanol; Etomidate; GABA Receptor; General anesthetic drugs; Genes; Goals; Grant; Knock-in Mouse; Knock-out; Link; Measurement; Measures; Mediating; Mental disorders; Molecular; Molecular Probes; Mus; Mutation; Oocytes; Point Mutation; Publishing; Range; Rattus; Receptor Gene; Receptor Signaling; Research Personnel; Role; Signal Transduction; Sindbis Virus; Steroids; Synapses; Testing; Transfection; Transgenic Mice; Xenopus oocyte; alcohol effect; alcohol sensitivity; base; design; gamma-Aminobutyric Acid; granule cell; improved; interdisciplinary approach; multidisciplinary; mutant; programs; receptor; receptor function; research study; synaptic inhibition; tetrahydrodeoxycorticosterone

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.

该提案的主要目标是测试两个假设，(i) 小脑颗粒细胞的突触外抑制是由 GABAA 受体 (GABAR) 亚基 α6、β3 和 δ 组成的受体介导的，以及 (ii) 该亚基的受体成分是某些全身麻醉剂、神经活性类固醇和乙醇的主要目标。提案 依赖于分子方法和电生理学分析的结合。该项目仔细检查了缺乏 beta3 和 delta 亚基基因的小鼠以及在颗粒细胞特异性 GABAR 基因 α6 中携带点突变 (R100Q) 的新生成的“敲入”大鼠中的颗粒细胞 GABAR 功能。R100Q 突变假设可以增强对各种 GABAR 调节剂（包括乙醇和苯二氮卓类药物）的敏感性。将实现三个具体目标：1）突触和突触外 GABAR 信号的电生理学分析将测试突触外抑制是否在缺乏 beta3 和 delta 亚基基因的小鼠中被特异性破坏。 2) 野生型和 alpha6R100Q“敲入”大鼠中突触和突触外信号的测量将用于评估突触外 GABAR 是否是小脑中乙醇、全身麻醉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) 旨在增强或削弱对乙醇或全身麻醉剂敏感性的突变、荧光标记的 GABAR 亚基将被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。对麻醉和乙醇中毒机制的进一步了解将产生深远的临床和社会效益，包括改进精神疾病的治疗方法和更好地治疗严重酗酒。