Altered GABA(A) receptor subunit and splice variant expression in rats treated with chronic intermittent ethanol

Background: Intermittent chronic administration of ethanol to rats has been shown previously to produce a hyperexcitable, kindling-like state, accompa nied by reduced inhibitory synaptic transmission in the hippocampus and cha nges in gamma -aminobutyric acid type A (GABA(A)) receptors. Further inform ation is needed on the detailed changes in GABA(A) receptors and their time course and persistence, as is comparison to changes after chronic, continu ous ethanol. Methods: GABA(A) receptors were analyzed in the rat brain after chronic int ermittent ethanol (CIE) by using radioligand binding, photoaffinity labelin g of polypeptides, and estimates of messenger RNA (mRNA) levels of receptor subunits by reverse transcriptase-polymerase chain reaction (RT-PCR) and i n situ hybridization. Results: CIE rats were confirmed to have increased GABA(A) receptor binding of the benzodiazepine partial inverse agonist and ethanol antidote ligand Ro15-4513, due to increased expression of the alpha6 subunit polypeptide in the cerebellum, shown by photoaffinity labeling. Estimates of mRNA levels by use of RT-PCR did not reveal any significant increase in alpha6 or in se veral other receptor subunits in several brain regions, but a decrease in t he ratio of the long and short splice variants (L/S) of the gamma2 subunit was detected in the hippocampus, especially the CA1 region. Conclusions: Changes in GABA(A) receptors were found in rats given CIE. Inc reased alpha6 subunit in the cerebellum was demonstrated by using both the binding to diazepam-insensitive sites for [H-3]Ro15-4513 and increased leve ls of the 57-kDa alpha6 polypeptide after photoaffinity labeling with this ligand. This increase appeared after 30 doses of ethanol and decayed to nor mal 1 week after ethanol was discontinued. The transient change in cerebell ar alpha6 subunit-containing receptors, also reportedly seen after chronic continuous ethanol, is thus unlikely to account for the persistently hypere xcitable, kindled, seizure-susceptible state seen in CIE. However, the sign ificant decrease in gamma2 subunit L/S splice variant ratio in the hippocam pus implies changes in GABA(A) receptor function, possibly involving protei n phosphorylation by protein kinase C. Altered receptor trafficking and tur nover associated with synaptic plasticity may contribute to the observed re duced inhibition in the hippocampus and other signs of alcohol dependence p roduced by CIE.