The development of tolerance to benzodiazepine (BZ) anticonvulsant actions limits their clinical value and may relate to patterns of chronic abuse. BZs potentiate GABA inhibition at the GABAA receptor (GABAR) increasing Cl conductance. Regulation of the GABAR following chronic BZ treatment is well established as one mechanism underlying BZ tolerance, yet the sequence of events at brain GABA synapses which result in BZ tolerance are not well understood. Findings of electrophysiological studies in in vitro hippocampal slices, ongoing GABAR autoradiographic studies, and our initial in situ hybridization and immunohistochemical studies of GABAR subunit mRNA and protein have established that the BZ tolerant rat hippocampus provides a useful model for studying the synaptic mechanisms of BZ tolerance and have provided a basis for the proposed studies. Studies designed to evaluate the temporal relation between the functional changes associated with chronic BZ treatment and the regulation of GABARs will be carried out in the hippocampus at several time-points after discontinuing 1 week oral flurazepam (FZP) treatment. Molecular biological, immunohistochemical and electrophysiological methods will be used to address three hypotheses: 1) GABAR subunit composition is modified by chronic BZ treatment; 2) BZ and GABA actions are attenuated by chronic BZ treatment as a function of changes in presynaptic, as well as postsynaptic, GABA transmission; and 3) that changes in GABAR composition resulting from changes in mRNA expression, thus subunit protein expression, are localized to hippocampal layers associated with GABA-mediated inhibition. The magnitude and time-course of the development and reversal of chronic BZ-induced changes in the GABAergic inhibitory system and BZ and GABA agonist sensitivity are related to the degree and time-course of changes in GABAR subunits. A change in the expression of the genes encoding GABAR subunits, thus a change in subunit composition is proposed as one mechanism for GABAR regulation, therefore the expression of mRNAs for GABAR subunits (alpha (1-5), beta (1-3) and gamma (1-2)) will be systematically studied using quantitative in situ hybridization methods in hippocampal layers and temporally correlated with changes in subunit proteins using quantitative immunohistochemical methods developed in our lab. Presynaptic GABA release will be indirectly measured by a change in the frequency of mini IPSCs. The functional consequences of chronic FZP treatment on GABA and BZ pharmacology will be measured using whole-cell patch-clamp methods to measure GABA-induced currents in CAl pyramidal cells in hippocampal slices and in acutely dissociated CAl neurons. Changes in diazepam and zolpidem's effects to potentiate GABA currents will also be assessed in these two models. A functional and molecular reorganization of GABAR synapses may provide a basis for BZ tolerance.
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