We have shown that ethanol dependence is associated with a reduction in GABAA receptor-mediated chloride channel function that may contribute to the development of physical dependence upon ethanol. The mechanism of these functional effects does not appear to involve a reduction in the number of GABA receptor-gated chloride channels. Prolonged ethanol exposure selectively and differentially alters the expression of several GABAA receptor subunit mRNAs and polypeptides in cortex and cerebellum. We have proposed that alterations in GABAA receptor subunit composition may be an important mechanism that underlies the reduction in GABAA receptor function and the development of dependence upon ethanol.
Our first aim i s to determine if chronic ethanol administration alters the subunit assembly of GABAA receptors using a combination of quantitative RT-PCR analysis of mRNA levels, Western blot analysis of polypeptide levels and immunoprecipitation studies with selective antibodies. Nuclear run-off experiments will be conducted to determine if changes in gene expression can be attributed to alterations in transcription rates. The next aim is to study the effects of chronic ethanol administration on GABAA receptor subunit expression in various brain regions where GABAA receptors differ in their sensitivity to ethanol. Studies of GABAA receptor expression at the cellular level are proposed using immunobinding assays on rat brain tissue sections. Our third goal is to investigate whether genetic differences in ethanol withdrawal seizure sensitivity are correlated with an alteration in a) the expression of GABAA receptor subunits in ethanol-naive mice or b) the effect of chronic ethanol administration on GABAA receptor subunit expression. Differences in GABAA receptor expression between the selected lines will then be subjected to genetic analysis to determine if the changes segregate with withdrawal seizure sensitivity. Our final goal is to investigate whether ethanol-induced alterations in GABAA receptor subunit expression are responsible for alterations in GABAA receptor function and CNS excitability. We plan to use selective antisense oligonucleotides to determine whether specific alterations in GABAA receptor expression in vivo are associated with the same alterations in GABAA receptor function and behavioral sensitivity that are observed with ethanol dependence. Future studies will address the same question in recombinant expression systems where the receptor subunit composition can be more precisely controlled. The results of these studies should have important implications for our understanding of the neurobiology of ethanol dependence and withdrawal.
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