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

Abstract

Abstract-Prolonged flurazepam exposure regulates the expression of selected (a1, b2, b3) 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 (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 GABA A receptor subunit antibody immunostaining density in the hippocampus suggested that a change in the composition of GABA A receptors involving specific subunits (a1 and b3) may be one mechanism underlying benzodiazepine anticonvulsant tolerance. ᭧ 1999 IBRO. Published by Elsevier Science Ltd.Key words: tolerance, flurazepam, quantitative immunohistochemistry, dentate gyrus, limbic system, CA1 pyramidal cells.Although effective anxiolytics and hypnotics, the clinical use of benzodiazepines as anticonvulsants is limited by tolerance development during prolonged administration. A large body of evidence has indicated that benzodiazepine tolerance is related to a reduction in GABA A receptor-mediated fast inhibitory synaptic transmission (for reviews see Refs 2 and 27). The native GABA A receptor is a pentamer composed of combinations of homologous proteins derived from five subunit families with multiple variants (a1-6, b1-4, g1-3, d1 and e1). 8,11,34 The GABA A receptor macromolecule contains binding domains for GABA and several allosteric modulators, including a well-characterized benzodiazepine binding site. 59Benzodiazepines potentiate GABA A receptor-mediated inhibitory function by increasing the opening probability of the integral Cl Ϫ channel, 52,62 and may increase Cl Ϫ channel conductance. 13Although the synaptic mechanisms mediating benzodiazepine tolerance have not been...

Short-term steroid treatment increases δ GABAA receptor subunit expression in rat CA1 hippocampus: Pharmacological and behavioral effects

“…In the present study, the steroid-induced decrease in α1 expression was reflected pharmacologically by a markedly reduced responsiveness of the GABAergic tonic current to zolpidem at a concentration selective for α1-containing GABAR (Horne et al, 1992;Vicini et al, 2001). Similar α4/α1 subunit substitutions have been reported following withdrawal from other GABA-modulatory compounds, such as ethanol (Kumar et al, 2003;Montpied et al, 1991;Liang et al, 2004) and BDZs (Chen et al, 2004), suggesting that this may represent a homeostatic mechanism common to GABA modulators. In fact, reduced responsiveness of the tonic current to these modulators has also been reported in the chronic intermittent model of ethanol intoxication (Liang et al, 2004).…”

Regulation of GABAAReceptor Subunit Expression by Pharmacological Agents

“…Treatment of cultured rat hippocampal cells with 10 M lorazepam or etizolam had no effect on the expression of ␣2 mRNA, but withdrawal (6 h) from them slightly increased the expression . The ␣2 polypeptide was not affected by long-term treatment with diazepam or flurazepam, but imidazenil down-regulated it in the frontoparietal cortex (Pesold et al, 1997;Chen et al, 1999). In contrast to ␣1, studies with most BZs suggest no effect of long-term BZ treatment or withdrawal from the treatment on ␣2 subunit expression.…”

“…Withdrawal (2 days) from long-term flurazepam treatment slightly reduced ␣1 mRNA in the CA1 region, the expression returning to control level in 7 days (Tietz et al, 1993(Tietz et al, , 1999a. Longterm treatment with flurazepam down-regulated the ␣1 polypeptide in hippocampal CA1 and CA3 regions and dentate gyrus (Chen et al, 1999). On day 2 of withdrawal from long-term treatment with flurazepam, down-regulation of ␣1 polypeptide persisted in the stratum oriens region of the CA1 (Tietz et al, 1999b).…”

“…Hippocampal ␤1 112 mRNA was not affected by long-term treatment with diazepam (Heninger et al, 1990) or flurazepam (Tietz et al, 1999a). Long-term treatment with flurazepam did not affect ␤1 polypeptide expression in the hippocampus (Chen et al, 1999). Triazolam up-regulated ␤1 mRNA in the cingulate cortex and hippocampal CA1 region and downregulated it in the interpeduncular nucleus (RamseyWilliams and Carter, 1996).…”

“…Withdrawal (2 days) from long-term flurazepam treatment reduced ␣1 mRNA in the cerebral cortical layers II to III and IV (Tietz et al, 1993), whereas on withdrawal day 7, ␣1 mRNA had returned to the control level (Tietz et al, 1999a) (Table 4). Long-term treatment with diazepam and flurazepam (but not imidazenil) reduced ␣1 polypeptide in the cerebral cortex Pesold et al, 1997;Chen et al, 1999) (Table 6). This reduction was not detected on day 2 of withdrawal from long-term flurazepam treatment (Tietz et al, 1999b) (Table 7).…”