Diazepam-induced loss of inhibitory synapses mediated by PLCδ/Ca2+/calcineurin signalling downstream of GABAA receptors

Benzodiazepines facilitate the inhibitory actions of GABA by binding to gamma-aminobutyric acid type A receptors (GABA(A)Rs), GABA-gated chloride/bicarbonate channels, which are the key mediators of transmission at inhibitory synapses in the brain. This activity underpins potent anxiolytic, anticonvulsant and hypnotic effects of benzodiazepines in patients. However, extended benzodiazepine treatments lead to development of tolerance, a process which, despite its important therapeutic implications, remains poorly characterised. Here we report that prolonged exposure to diazepam, the most widely used benzodiazepine in clinic, leads to a gradual disruption of neuronal inhibitory GABAergic synapses. The loss of synapses and the preceding, time-and dose-dependent decrease in surface levels of GABA(A)Rs, mediated by dynamin-dependent internalisation, were blocked by Ro 15-1788, a competitive benzodiazepine antagonist, and bicuculline, a competitive GABA antagonist, indicating that prolonged enhancement of GABA(A)R activity by diazepam is integral to the underlying molecular mechanism. Characterisation of this mechanism has revealed a metabotropic-type signalling downstream of GABA(A)Rs, involving mobilisation of Ca2+ from the intracellular stores and activation of the Ca2+/calmodulin-dependent phosphatase calcineurin, which, in turn, dephosphorylates GABA(A)Rs and promotes their endocytosis, leading to disassembly of inhibitory synapses. Furthermore, functional coupling between GABA(A)Rs and Ca2+ stores was sensitive to phospholipase C (PLC) inhibition by U73122, and regulated by PLCd, a PLC isoform found in direct association with GABA(A)Rs. Thus, a PLC delta/Ca2+/calcineurin signalling cascade converts the initial enhancement of GABA(A)Rs by benzodiazepines to a long-term downregulation of GABAergic synapses, this potentially underpinning the development of pharmacological and behavioural tolerance to these widely prescribed drugs.