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.
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