Research
Molecular Mechanisms of Diazepam Tolerance

Investigator: Erminio Costa, M.D.

Tolerance to benzodiazepines (BZ) limits their clinical use as 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 PARTLkL allosteric modulators of GABA action at GABAAreceptors (FAM and PAM, respectively). Both drug classes act on GABAAreceptors that include one al-5 and one y2-3 subunit in their structure, but their intrinsic activity in ampliffing GABA action differs. Diazepam, (a FAM), but not imidazenil, (a PAM) induces tolerance in rodents. In preliminary studies, we have shown that in tolerant rats there is a change in the expression ofmRNAs encoding for a I, y2 and a5 GABAA receptor subunits which reverses when tolernace is reversed. Such changes are not present in rats receiving imidazenil in doses equipotent to those of diazepam that induce tolerance. These two drugs win be used as research tools to explore the mechanism of diazepam tolerance. We hypothesize that tolerance to diazepam is triggered by an alteration in GABAAreceptor 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 diazepant tolerance to its anticonvulsant, anticonflict and cognition disrupting action using as a contrast imidazenil. 2) PCR analysis changes in 14 subunit mRNA content in 28 brain areas at the onset and termination of diazeparn tolerance and test whether imidazenil produces similar changes or antagonizes diazepam changes. 3) Measure with gold immuncilabelling in brain slices the content of 8 different GABAAreceptor subunits in pertinent brain areas of diazepam tolerant rats using imidazenil as a contrast and/or as antagonist. 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-immunoiabelling with gold particles of different size as 3a. 3b.