Modulation Of Extrasynaptic ?-GABA_A Receptor By Single-dose Alcohol Withdrawal In Hippocampal Neurons

The effects of alcohol on the central nervous system are thought to play important roles in alcohol tolerance,dependence and withdrawal symptoms.Considerable evidence suggests that ?-aminobutyric acid type A receptors(GABAARs)are one of the major targets for alcohol in the brain.Previous reports have shown that GABAAR subunit composition,function and expression are altered after chronic intermittent administration of alcohol in vivo,accompanied by the corresponding behavior changes;while temporary plastic changes in GABAARs after withdrawal from single-dose alcohol are occurred both in vivo and in vitro.The mechanisms by which the relative subunit composition,localization and function of specific GABAARs are altered by alcohol withdrawal are not clear.Studies have shown that extrasynaptic ?-GABAAR endocytosis is the possible trigger for alcohol-induce GABAAR plasticity,and protein phosphorylation regulation of extrasynaptic ?-GABAAR mediated by protein kinase C(PKC)is considered to be an important underlying mechanism.However,the the nature of their roles remains to be elucidated and the possible PKC subtype involved in is still to be clarified.In the study,we used electrophysiological,protein biochemical,molecular biology and fluorescence imaging techniques to investigate the regulation mechanism of the rapid internalization of extrasynaptic GABAA receptor induced by single dose ethanol withdrawal in cultured hippocampal neurons from rat.We observed a significant decrease in Itonic amplitudes and Itonic responsiveness to acute ethanol in neurons at 30-min withdrawal from short-term ethanol exposure(60 mM for 30 min).Okadaic acid pre-exposure(100 nM for 30 min)and withdrawal(30 min)also induced a smaller but significant decrease in Itonic amplitudes and Itonic responsiveness to acute ethanol.These results suggest that protein phosphorylation is involved in short-term ethanol withdrawal-induced decrease in extrasynaptic ?-GABAAR function.PKC inhibition prevents ethanol withdrawal-induced alterations in Itonic amplitude and Itonic responsiveness to acute ethanol,as well as reduction in extrasynaptic GABAAR ?expression,suggesting PKC activation is involved in short-term ethanol exposure/withdrawal regulation of extrasynaptic ?-GABAARs.Ethanol withdrawal notably increased PKC8 but not PKC? and PKC? expression in cultured hippocampal neurons,indicating that PKC? may be involved in short-term ethanol exposure/withdrawal regulation of ?-GABAARs.We subsequently found that PKC8 inhibition prevents ethanol withdrawal-induced decreases in GABAAR ? cell-surface expression,recombinant GABAAR ? fluorescence intensity,and extrasynaptic Itonic responsiveness to acute ethanol.In summary,these data suggest that PKC? activation is involved in regulation of extrasynaptic ?-GABAAR function and expression by a single-dose ethanol withdrawal.PKC8 activation is likely to be an important basis for alcohol withdrawal-induced GABAA receptor plasticity.Clarifying the mechanisms in rapid regulation of extrasynaptic ?-GABAAR by a single dose alcohol withdrawal will help us to find out new targets and ideas for the clinical therapies for alcohol tolerance,dependence and AWS.