Plasticity of GABAA Receptors after Ethanol Pre-Exposure in Cultured Hippocampal Neurons

Alcohol use causes many physiological changes in brain with behavioral sequelae. We previously observed (J Neurosci 27: 12367-12377, 2007) plastic changes in hippocampal slice recordings paralleling behavioral changes in rats treated with a single intoxicating dose of ethanol (EtOH). Here, we were able to reproduce in primary cultured hippocampal neurons many of the effects of in vivo EtOH exposure on GABA(A) receptors (GABA(A)Rs). Cells grown 11 to 15 days in vitro demonstrated GABA(A)R delta subunit expression and sensitivity to enhancement by short-term exposure to EtOH (60 mM) of GABA(A)R-mediated tonic current (I-tonic) using whole-cell patch-clamp techniques. EtOH gave virtually no enhancement of mIPSCs. Cells pre-exposed to EtOH (60 mM) for 30 min showed, 1 h after EtOH withdrawal, a 50% decrease in basal I-tonic magnitude and tolerance to short-term EtOH enhancement of I-tonic, followed by reduced basal mIPSC area at 4 h. At 24 h, we saw considerable recovery in mIPSC area and significant potentiation by short-term EtOH; in addition, GABA(A)R currents exhibited reduced enhancement by benzodiazepines. These changes paralleled significant decreases in cell-surface expression of normally extrasynaptic delta and alpha 4 GABA(A)R subunits as early as 20 min after EtOH exposure and reduced alpha 5-containing GABA(A)Rs at 1 h, followed by a larger reduction of normally synaptic alpha 1 subunit at 4 h, and then by increases in alpha 4 gamma 2-containing cell-surface receptors by 24 h. Measuring internalization of biotinylated GABA(A)Rs, we showed for the first time that the EtOH-induced loss of I-tonic and cell-surface delta/alpha 4 20 min after withdrawal results from increased receptor endocytosis rather than decreased exocytosis.