SYNAPTIC MECHANISMS OF BENZODIAZEPINE TOLERANCE

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Support is being requested through an ADAMHA Research Scientist Development Award (Level II) for studies designed to systematically investigate the synaptic mechanisms underlying benzodiazepine (BZ) tolerance. The application is based on ongoing, NIDA funded, extracellular electrophysiological and autoradiographic binding studies in hippocampus of chronic flurazepam (FZP) treated rats. To broaden these investigations, intracellular and in situ hybridization studies are proposed as a part of the candidate's research career development plan. The studies proposed are a logical extension of the preliminary studies summarized and important direction for future research. To achieve these goals, the use of resources and consultants within the candidate's institution and other research institutions is outlined. Regulation of the various binding sites on the GABA/BZ/C1 channel complex has been implicated in BZ tolerance. It is hypothesized that drug-induced changes in BZ and GABA agonist sensitivity with chronic BZ treatment are a function of modifications at the GABA complex. The temporal relation between the functional changes associated with chronic BZ treatment and the regulation of GABA complex will be evaluated in the hippocampus, a brain site uniquely suited to test this and associated hypotheses, at several time-points after discontinuing a 1 or 4 week flurazepam (FZP) treatment. Functional changes in BZ and GABA agonist sensitivity will be measured electrophysiologically in in vitro hippocampus using the GABA-ergic recurrent and feed forward inhibitory circuits in CA1 region as a substrate. Extracellular analyses will include studies of paired-pulse inhibition and concentration-effect studies of BZ and GABA agonists. Extracellular studies in hippocampal slices from rats sacrificed 48 hr after 1 week FZP treatment indicate reduced paired-pulse inhibition, subsensitivity to GABAA agonists and tolerance to diazepam. The nature of the changes in local synaptic potentials which may be related to these extracellular findings will be investigated intracellularly in CA1 pyramidal cells. Alterations in the sensitivity to GABA agonists will be studied intracellularly using iontophoretic techniques. Associated modification of binding sites (GABA, BZ and C1 channel) on the GABA/BZ/C1 channel complex and site-site interactions (coupling) will be investigated using autoradiographic methods. Regulation of the expression of mRNAs for GABAA receptor subunits (alpha, beta and gamma) will be studied using in situ hybridization methods. GABA complex regulation and gene expression will be systematically studied in 5 histologically identified laminae and temporally correlated with the functional consequences of chronic FZP treatment on BZ and GABA agonists effects on CA1 pyramidal cell evoked responses measured electrophysiologically.