MOLECULAR MECHANISMS OF DIAZEPAM TOLERANCE

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DESCRIPTION (Adapted from applicant's abstract): Tolerance to benzodiazepines (BZ) limits their clinical use of anticonvulsants, anxiolytic and antipanic agents. In the clinical practice to maintain the level of drug efficacy the BZ doses are increased to overcome tolerance and this often leads to physical dependence. We must understand the mechanisms of BZ tolerance in order to overcome the problems in their clinical use and we believe that this will also bring about an initial understanding of how GABAergic transmission is regulated. BZ can be classified as FULL or PARTIAL allosteric modulators of GABA action at GABA-a receptors (FAM and PAM, respectively). Both drug classes act on GABA-a receptors that include one alpha1-5 and one gamma2-3 subunit in their structure, but their intrinsic activity in amplifying GABA action differs. Diazepam, (a FAM), but not imidazenil, (a PAM) induces tolerance in rodents. In preliminary studies, we have shown that in tolerance rats there is a change in the expression of mRNAs encoding for alpha1, gamma2 and alpha5 GABA-a receptor subunits which reverses when tolerance is reversed. Such changes are not present in rats receiving imidazenil in doses equipotent to those of diazepam that induce tolerance. These two drugs will be used as research tools to explore the mechanism of diazepam tolerance. We hypothesize that tolerance to diazepam is triggered by an alteration of GABA-a receptor subunit expression restricted to functionally dedicated brain areas. We propose a systematic investigation of the mechanism of diazepam tolerance in rats with the following aims: (1) Determine onset and duration of diazepam tolerance to its anticonvulsant, anticonflict and cognition disrupting action using as a contrast imidazemil, (2) PCR analysis changes in 14 subunit mRNA content in 28 brain areas at the onset and termination of diazepam tolerance and test whether imidazenil produces similar changes or antagonizes diazepam changes, (3) Measure with gold immunolabeling in brain slices the content of 8 different GABA-a receptor subunits in pertinent brain areas of diazepam tolerant rats using imidazenil as a contrast and/or as antagonist and (4) Study changes of subunit expression in neuronal membranes of dissociated cultures prepared from pertinent brain areas of tolerant rats; determine receptor coexpression of pertinent subunits with double-immunolabelling with gold particles of different size.