Sustained treatment with an α5 GABA A receptor negative allosteric modulator delays excitatory circuit development while maintaining GABAergic neurotransmission

α5 subunit GABA type A receptor (GABA_AR) preferring negative allosteric modulators (NAMs) are cognitive enhancers with antidepressant-like effects. α5-NAM success in treating mouse models of neurodevelopmental disorders with excessive inhibition have led to Phase 2 clinical trials for Down syndrome. Despite in vivo efficacy, no study has examined the effects of continued α5-NAM treatment on inhibitory and excitatory synapse plasticity to identify mechanisms of action. Here we used L-655,708, an imidazobenzodiazepine that acts as a highly selective but weak α5-NAM, to investigate the impact of sustained treatment on hippocampal neuron synapse and dendrite development. We show that 2-day pharmacological reduction of α5-GABA_AR signaling from DIV12-14, when GABA_ARs contribute to depolarization, delays dendritic spine maturation and the NMDA receptor (NMDAR) GluN2B/GluN2A developmental shift. In contrast, α5-NAM treatment from DIV19-21, when hyperpolarizing GABA_AR signaling predominates, enhances surface synaptic GluN2A while decreasing GluN2B. Despite changes in NMDAR subtype surface levels and localization, total levels of key excitatory synapse proteins were largely unchanged, and mEPSCs were unaltered. Importantly, 2-day α5-NAM treatment does not alter the total surface levels or distribution of α5-GABA_ARs, reduce the gephyrin inhibitory synaptic scaffold, or impair phasic or tonic inhibition. Furthermore, α5-NAM inhibition of the GABA_AR tonic current in mature neurons is maintained after 2-day α5-NAM treatment, suggesting reduced tolerance liability, in contrast to other clinically relevant GABA_AR-targeting drugs such as benzodiazepines. Together, these results show that α5-GABA_ARs contribute to dendritic spine maturation and excitatory synapse development via a NMDAR dependent mechanism without perturbing overall neuronal excitability.