Mechanisms of Ligand-Induced GABA Receptor Plasticity

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The objectives of this component are to analyze the molecular mechanisms of chronic GABAergic ligand-induced plasticity in the central nervous system (CNS). Various plasticity-inducing treatments produce changes in levels of the GABAA receptor (GABAR) subunit alpha4 and its associated partners, gamma2 or 8, accompanied by altered sensitivity to endogenous neurosteroids. In this program, we have determined that alpha4beta3delta subunit-containing GABAR are extrasynaptically localized, involved in tonic inhibition, and the major target of modulation of CNS excitability by neurosteroids and anesthetics including ethanol, while being insensitive to benzodiazepines. In particular we showed by several lines of evidence that the alpha6beta3delta subtype of GABAR is very likely the target of action of ethanol in the cerebellum. This important discovery needs substantiation and forms the rationale for Aim I. We suggest that the alpha4/6beta3delta GABAR are both susceptible to plasticity and positioned to regulate excitability via neurosteroid-modulated tonic inhibition. In this project Aim II we will compare and contrast changes in GABAR following chronic steroid (extrasynaptic delta subunit-containing target) and chronic BZ (synaptic gamma2 subunit-containing target). In drug-treated mice, we will measure biochemically the levels of GABAR subunits in hippocampus and cerebellum and partnering of subunits using antibodies, as well as binding assays for GABAR levels and allosteric modulation indicative of subunit switches. In drug-treated primary cultured hippocampal neurons, we will measure the content of cell surface GABAR subunits and subunit partners using biotinylation. Plastic changes following chronic GABAergic drugs are relevant not only to the development of tolerance to clinically useful agents like benzodiazepines (BZ) as well as withdrawal and dependence, but also to the control of excitability by the GABA system. This in turn is critically important to normal CNS information processing and changes that occur in response to usual or unusual experiences (plasticity), including epileptogenic phenomena.