Benzodiazepine-mediated regulation of α1, α2, β1-3 and γ2 GABAA receptor subunit proteins in the rat brain hippocampus and cortex

Prolonged flurazepam exposure regulates the expression of selected (alpha 1, beta 2, beta 3) GABA(A) receptor subunit messenger RNAs in specific regions of the hippocampus and cortex with a time-course consistent with benzodiazepine tolerance both in vivo and in vitro. In this report, the immunostaining density of six specific GABA(A) receptor subunit (alpha 1, alpha 2, beta 1-3 and gamma 2) antibodies was measured in the hippocampus and cortex, among other brain areas, in slide-mounted brain sections from flurazepam-treated and control rats using quantitative computer-assisted image analysis techniques. In parallel with the localized reduction in alpha 1 and beta 3 subunit messenger RNA expression detected in a previous study, relative alpha 1 and beta 3 subunit antibody immunostaining density was significantly decreased in flurazepam-treated rat hippocampal CA1, CA3 and dentate dendritic regions, and in specific cortical layers. Quantitative western blot analysis showed that beta 3 subunit protein levels in crude homogenates of the hippocampal dentate region from flurazepam-treated rats, an area which showed fairly uniform decreases in beta 3 subunit immunostaining (16-21%), were reduced to a similar degree (18%). The latter findings provide independent support that relative immunostaining density may provide an accurate estimate of protein levels. Consistent with the absence of the regulation of their respective messenger RNAs immediately after ending flurazepam administration, no changes in the density of alpha 2, p beta i or beta 2 subunit antibody immunostaining were found in any brain region. gamma 2 subunit antibody staining was changed only in the dentate molecular layer. The selective changes in GABA(A) receptor subunit antibody immunostaining density in the hippocampus suggested that a change in the composition of GABA(A) receptors involving specific subunits (alpha 1 and beta 3) may be one mechanism underlying benzodiazepine anticonvulsant tolerance. (C) 1999 IBRO. Published by Elsevier Science Ltd.