GABAA receptors play a crucial role in mediating fast inhibitory neurotransmission in the central nervous system. These receptors are targets of numerous pharmacological agents used clinically to control neuronal excitability in different neurological disorders. Sustained stimulation of GABAA receptors by endogenous or exogenous modulators leads to adaptive homeostatic alterations in the receptor function. In particular, chronic benzodiazepine administration results in tolerance to most of the behavioral effects, limiting the clinical use of these drugs for long-term treatments. In previous studies, we found that prolonged exposure of rat cerebrocortical neurons to diazepam produces uncoupling of GABA/benzodiazepine site interactions and decreased expression of the GABAA receptor α1 subunit gene, mediated by a mechanism involving the activation of L-type voltage-gated calcium channels (L-VGCCs). This work aimed to further explore the molecular basis of GABAA receptor regulation induced by prolonged benzodiazepine stimulation. Our findings indicate that diazepam increases intracellular calcium levels, confirming the dependence of benzodiazepine-induced GABAA receptor regulation on calcium entry through L-VGCCs. Immunocytochemical analyses revealed that sustained diazepam treatment reduces the expression of α1-containing GABAA receptors on the cell surface, which likely impacts functional receptors. By knocking-down α1 subunit expression, we demonstrated that α1 downregulation alone results in minor, non-significant uncoupling, suggesting that additional GABAA receptor modifications contribute to the observed uncoupling. Altogether, our results suggest that persistent exposure of GABAA receptors to benzodiazepines produces uncoupling and downregulation of functional α1-containing GABAA receptors through two distinct mechanisms, both initiated by calcium influx through L-VGCCs.
Keywords: Benzodiazepines; Cerebrocortical neurons; Tolerance.
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