Development of tolerance to benzodiazepine (BZ) effects, in particular their anticonvulsant actions, limits their clinical usefulness. Tolerance development is probably also a factor in determining patterns of chronic abuse. BZs act at receptors which are modulatory sites on the GABA receptor to increase GABA-mediated Cl- flux. Regulation of the various binding sites on the GABA/BZ/Cl- channel complex has been implicated in tolerance, yet results of studies are inconsistent. Using a standard chronic BZ treatment, behavioral subsensitivity to local injection of BZ and GABA agonists in substantia nigra pars reticulata (SNpr) showed different time-courses of tolerance development and reversal suggesting multiple mechanisms involving regulation of the GABA complex might mediate alterations in BZ neuronal sensitivity. Receptor autoradiography revealed BZ and GABA agonist sensitivity with chronic BZ treatment are a function of modifications at the GABA complex. The hippocampus is the brain site uniquely suited to test this and associated hypotheses. Local changes in BZ and GABA agonist sensitivity will be sought using extracellular electrophysiological methods using the GABAergic inhibitory circuits in hippocampal formation as a substrate. Associated modification of binding sites and site-site interactions on the GABA/BZ/Cl- channel complex will be systematically studied in parallel in 5 histologically identified laminae of hippocampus using autoradiographic binding methods. The effect of chronic BZ treatment will be evaluated at several time-points after discontinuing a 1 or 4 week flurazepam (FZP) treatment. Electrophysiological measurements in in vitro hippocampal slice will evaluate changes in BZ and GABA potency and efficacy by (A) evaluating the function of the endogenous GABAergic system using """"""""paired-pulse"""""""" inhibition, and (B) concentration effect studies of prototype BZs (diazepam, FZP, desalkylFZP and clonazepam) and GABA agonists (GABA, muscimol, THIP and isoguavacine). Autoradiographic measurements will be made of (A) GABA ([3H]muscimol and [3H]bicuculline), BZ ([3H]Ro15-1788), and Cl- channel ([35S]t-butylbicyclophosphothionate, TBPS) binding, and (B) GABA/BZ ([3H]flunitrazepam + GABA) and GABA/Cl- channel ([35S]TBPS + GABA) coupling. This work will provide further information about the nature of GABA/BZ/Cl- channel interactions in the long-term neuronal adaptations which occur to BZs with chronic use.
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