Gephyrin is critical for glycine receptor clustering but not for the formation of functional GABAergic synapses in hippocampal neurons.

The role of the scaffolding protein gephyrin at hippocampal inhibitory synapses is not well understood. A previous study (Kneussel et al., 1999) reported a complete loss of synaptic clusters of the major GABA(A)R subunits alpha2 and gamma2 in hippocampal neurons lacking gephyrin. In contrast, we show here that GABA(A)R alpha2 and gamma2 subunits do cluster at pyramidal synapses in hippocampal cultures from gephyrin-/- mice, albeit at reduced levels compared with control neurons. Synaptic aggregation of GABA(A)R alpha1 on interneurons was identical between the culture types. Furthermore, we recorded miniature IPSCs (mIPSCs) from gephyrin-/- neurons. Although the mean mIPSC amplitude was reduced (by 23%) compared with control, the frequency of these events was unchanged. Cell surface labeling experiments indicated that gephyrin contributes, in part, to aggregation but not to insertion or stabilization of GABA(A)R alpha2 and gamma2 in the plasma membrane. Thus, a major gephyrin-independent component of hippocampal inhibitory synapse development must exist. We also report that glycine receptors cluster at GABAergic synapses in a subset of hippocampal interneurons and pyramidal neurons. Unlike GABA(A)Rs, synaptic clustering of glycine receptors was completely abolished in gephyrin-/- neurons. Finally, artificial extrasynaptic aggregation of GABA(A)R was able to redistribute and cocluster gephyrin by a mechanism requiring a neuron-specific modification or intermediary protein. We propose a model of hippocampal inhibitory synapse development in which some GABA(A)Rs cluster at synapses by a gephyrin-independent mechanism and recruit gephyrin. This clustered gephyrin may then recruit glycine receptors, additional GABA(A)Rs, and other signal-transducing components.