The goal of the present study is to determine the functional significance of GABAA receptor (GABA-R) subunit changes produced by fluctuations in endogenous hormonal levels. Towards this end, we will assess mRNA and protein levels of alpha4 and delta subunits concomitant with evaluation of synaptic current as well as characterization of GABA-gated current in acutely dissociated CA1 hippocampal neurons upon withdrawal from the GABA-modulatory 3alpha,5alpha-THP (3alpha-OH-5alpha-pregnan-20-one). This hormone paradigm mimics hormonal conditions underling periods of endogenous hormone withdrawal, such as pre-menstrual syndrome. Findings supported by the previous funding period established that 3alpha,5alpha-THP withdrawal results in anxiogenic, pro-convulsant effects similar to those seen after withdrawal from other GABA-modulatory compounds, such as the benzodiazepines (BDZs). These behavioral effects were well-correlated with a marked decrease in the decay time for GABA-gated current, which resulted in a decrease in total integrated GABA current, an effect due specifically to upregulation of the alpha4 subunit of the GABA-R. This increase in the alpha4 subunit also resulted in a near total BDZ insensitivity. The goal of the present studies is to determine the effect of alpha4 subunit upregulation on synaptic currents recorded from the hippocampal slice preparation; mIPSCs as well as evoked IPSCs will be determined across hormone state and correlated with alpha4 subunit levels using Western blot procedures. We will also evaluate the kinetics of GABA-gated current using excised outside-out patches under non-equilibrium conditions of receptor saturation and ultra-fast GABA exposure (<1 msec). These conditions more closely mimic those of actual synaptic events. Decay time constants for GABA-gated current, as well as rate of desensitization, will be determined in cells with alpha4-containing GABA-R (hormone-induced or transfected HEK-293 cells) and compared with control alpha2-containing receptors. In addition, we will test the hypothesis that the 3alpha,5alpha-THP insensitivity we observe is due to upregulation of the delta subunit of the GABA-R, assessed using whole cell patch clamp techniques. In both cases, antisense-induced suppression of subunit expression will be employed to further verify the role of these subunits in modifying changes in synaptic current and 3alpha,5alpha-THP tolerance. These studies will test the hypothesis that alterations in intrinsic properties of GABA-R occur as a result of altered subunit composition after hormone exposure and withdrawal.
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