Increased NMDAR activity and GluN2A-NMDAR silent synapse expansion induced by chronic benzodiazepine treatment.

Abstract

Benzodiazepines (BZDs) are critical sedative, anticonvulsant, and anxiolytic drugs that potentiate inhibitory GABAergic neurotransmission. However, clinical utility is hampered by drug tolerance and a hyperexcitable withdrawal syndrome characterized by neuronal excitation/inhibition (E/I) imbalance. Although enhanced excitation is implicated in BZD tolerance, the homeostatic changes to glutamatergic receptors remain undefined. Here, we report the impact of chronic (7-day) BZD treatment on excitatory synapse and NMDA receptor (NMDAR) function, expression, and subcellular localization in cortical neurons. Chronic treatment with the BZD diazepam (DZP) resulted in an increase in NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs). Confocal imaging studies revealed a DZP-induced enrichment of GluN2B-containing NMDARs at functional synapses (expressing AMPA receptors, AMPARs) while GluN2B subunit expression was otherwise unaltered. Conversely, localization of GluN2A-containing NMDARs (GluN2A-NMDARs) to functional synapses was unchanged, while GluN2A-NMDAR total protein levels and surface accumulation were enhanced. Intriguingly, we demonstrate for the first time the BZD-induced enrichment and expansion of GluN2A-NMDAR coverage at silent (AMPAR-lacking) synapses. Finally, biochemical fractionation analysis of the translation elongation protein eEF2, known to control E/I balance, detected lower levels of deactivated, phosphorylated eEF2 in the synaptic fraction of DZP-treated neurons, indicative of enhanced local translation. Collectively, our findings suggest that chronic BZD treatment triggers compensatory mechanisms which 1) enhance NMDAR function via increased GluN2B-NMDARs at functional synapses, and 2) promote the expression, surface localization, and accumulation of GluN2A-NMDARs at silent synapses, augmenting the potential for further synaptic plasticity.