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

Abstract

Prolonged flurazepam exposure regulates the expression of selected (α1, β2, β3) 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 (α1, α2, β1–3 and γ2) 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 α1 and β3 subunit messenger RNA expression detected in a previous study, relative α1 and β3 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 β3 subunit protein levels in crude homogenates of the hippocampal dentate region from flurazepam-treated rats, an area which showed fairly uniform decreases in β3 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 α2, β1 or β2 subunit antibody immunostaining were found in any brain region. γ2 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 (α1 and β3) may be one mechanism underlying benzodiazepine anticonvulsant tolerance.