Gamma-aminobutyric/A (GABA/A) receptors form part of a major neurotransmitter system in the brain that contributes to the regulation of anxiety, sleep and the pathogenesis of many neurological disorders. Recent developments in molecular cloning of GABA/A receptors have revealed an impressive heterogeneity of molecular forms of constituent subunits. There is growing evidence that an aspect of this large heterogeneity could be a functional heterogeneity of inhibitory synapses. Furthermore, the selectivity of drug action for distinct molecular forms of GABA/A receptors raise hopes for better treatment of disorders related to inhibitory GABAergic pathways. We have evidence that in cerebellum two functionally and pharmacologically distinct populations of inhibitory synapses can be found in relation to specific cell types. We propose to test the hypotheses that the GABA/A receptor subunits composing postsynaptic receptor-channel complexes play a major role at these synapses as determinants of synaptic strength. GABA-mediated synapses in visually identified neurons in the rat cerebellum, will be analyzed using whole-cell voltage-clamp recordings of synaptic currents. We will attempt to correlate functional differences of cerebellar synapses with the presence of the mRNA for specific GABA/A receptor subunits in these neurons determined with single-cell polymerase chain reaction and with the study of the expression of these subunits revealed by immunocytochemical techniques. This work will be complementary to patch-clamp recordings of GABA-activated channel currents in outside- out patches excised from neurons in brain slices and from cells transiently transfected with specific subunits of GABA/A receptors. We will also test the hypothesis that the presence of structurally distinct GABA/A receptor subtypes in the cerebellum, are the basis of specific pharmacological properties of agonists, antagonists as well as partial agonists of the benzodiazepine receptor. Lastly, we propose to provide the correlation between long-term changes in rat cerebellar inhibitory synapses and pharmacological treatments with clinically relevant compounds such as benzodiazepines, with acute and chronic ethanol treatments and with these experimental conditions combined. Data obtained from these specific aims will permit us to distinguish the contribution of the various molecular forms of GABA/A receptors as determinants of synaptic functions that underlie behavior and that are relevant in various CNS disorders.
