Pii: S0306-4522(99)00118-9

Prolonged flurazepam exposure regulates the expression of selected (a1, b2, b3) GABAA 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 GABAA receptor subunit (a1, a2, b1–3 and g2) 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 a1 and b3 subunit messenger RNA expression detected in a previous study, relative a1 and b3 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 b3 subunit protein levels in crude homogenates of the hippocampal dentate region from flurazepam-treated rats, an area which showed fairly uniform decreases in b3 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 a2, b1 or b2 subunit antibody immunostaining were found in any brain region. g2 subunit antibody staining was changed only in the dentate molecular layer. The selective changes in GABAA receptor subunit antibody immunostaining density in the hippocampus suggested that a change in the composition of GABAA receptors involving specific subunits (a1 and b3) may be one mechanism underlying benzodiazepine anticonvulsant tolerance. q 1999 IBRO. Published by Elsevier Science Ltd.