Distinct mechanisms regulate GABAA receptor and gephyrin clustering at perisomatic and axo‐axonic synapses on CA1 pyramidal cells

Non‐Technical Summary To be effective, synaptic transmission requires precise alignment of the presynaptic terminal, releasing the neurotransmitter, with the postsynaptic density, where receptors are present at high density. Complex molecular mechanisms ensure this interplay between neurons and, in addition, stabilize receptors in the postsynaptic membrane. To explore these mechanisms at GABAergic synapses, which mediate inhibitory neurotransmission in the brain, we investigated here the consequences of ‘removing’ the receptors, using targeted gene deletion. Our results show that the receptors are dispensable for synapse formation, but are required for the postsynaptic aggregation of several proteins involved in receptor trafficking, anchoring and regulation. Defects in the molecular regulation of GABAergic synapses have been associated with neurodevelopmental disorders, mental retardation, anxiety and mood disorders, underscoring the relevance of fine tuning of GABAergic inhibition for proper brain function. Pyramidal cells express various GABAA receptor (GABAAR) subtypes, possibly to match inputs from functionally distinct interneurons targeting specific subcellular domains. Postsynaptic anchoring of GABAARs is ensured by a complex interplay between the scaffolding protein gephyrin, neuroligin‐2 and collybistin. Direct interactions between these proteins and GABAAR subunits might contribute to synapse‐specific distribution of GABAAR subtypes. In addition, the dystrophin–glycoprotein complex, mainly localized at perisomatic synapses, regulates GABAAR postsynaptic clustering at these sites. Here, we investigated how the functional and molecular organization of GABAergic synapses in CA1 pyramidal neurons is altered in mice lacking the GABAAR α2 subunit (α2‐KO). We report a marked, layer‐specific loss of postsynaptic gephyrin and neuroligin‐2 clusters, without changes in GABAergic presynaptic terminals. Whole‐cell voltage‐clamp recordings in slices from α2‐KO mice show a 40% decrease in GABAergic mIPSC frequency, with unchanged amplitude and kinetics. Applying low/high concentrations of zolpidem to discriminate between α1‐ and α2/α3‐GABAARs demonstrates that residual mIPSCs in α2‐KO mice are mediated by α1‐GABAARs. Immunofluorescence analysis reveals maintenance of α1‐GABAAR and neuroligin‐2 clusters, but not gephyrin clusters, in perisomatic synapses of mutant mice, along with a complete loss of these three markers on the axon initial segment. This strik