CONICET | Buscador de Institutos y Recursos Humanos

Benzodiazepine tolerance induced by chronic administration isthe result of adaptive changes in GABAA receptors which are examplesof neuronal plasticity. We have previously demonstrated that prolonged diazepamadministration in rats results in tolerance to the sedative and anxiolyticeffects that is accompanied with alterations in GABAA receptors inthe cerebral cortex. The aim of this work was to investigate the molecularmechanism of benzodiazepine tolerance in an invitro model of cultured neurons exposed to diazepam. To this end, primaryneuronal cultures, prepared from rat cerebral cortices, were exposed to 50 μMdiazepam for 48 h. At the end of this incubation cells were collected forbinding, western blot or quantitative real-time PCR experiments. The exposureto diazepam induced a 40 % decrease (p<0.05, one sample Student´s t test) in the potentiation of [3H]flunitrazepambinding by 1 mM GABA, named uncoupling. Uncoupling was prevented in thepresence of flumazenil or picrotoxin (p<0.05, one-way ANOVA and Tukey´s posthoc tests), suggesting that depends on the binding of the benzodiazepine to itsspecific recognition site and on the GABAA receptor activation.Uncoupling was also blocked by nifedipine, an inhibitor of L-type voltage-gatedcalcium channels (L-VGCC, p<0.05, one-way ANOVA and Tukey´s post hoc tests).In addition, diazepam treatment induced a 30 % decrease (p<0.05, one sampleStudent´s t test) in GABAAreceptor α1 subunit mRNA and protein levels that is prevented by nifedipine (p<0.05,one-way ANOVA and Tukey´s post hoc tests). These results suggest that themechanism of tolerance is mediated by repression of α1 subunit gene expressioninduced by Ca2+ influx through L-VGCC that would finally result inthe uncoupling of GABAA receptor allosteric interactions. Theunderstanding of the tolerance mechanism will contribute to the design of newdrugs that will maintain their efficacies after long treatments.