Gephyrin expression and clustering affects the size of glutamatergic synaptic contacts.

We have recently shown that disrupting the expression and post-synaptic clustering of gephyrin in cultured hippocampal pyramidal cells, by either gephyrin RNAi (RNA interference) or over-expression of a dominant negative gephyrin-enhanced green fluorescent protein (EGFP) fusion protein, leads to decreased number of post-synaptic gephyrin and GABA(A) receptor clusters and to reduced GABAergic innervation of these cells. On the other hand, increasing gephyrin expression led to a small increase in the number of gephyrin and GABA(A) receptor clusters and to little or no effect on GABAergic innervation. We are now reporting that altering gephyrin expression and clustering affects the size but not the density of glutamatergic synaptic contacts. Knocking down gephyrin with gephyrin RNAi, or preventing gephyrin clustering by over-expression of the dominant negative gephyrin-enhanced green fluorescent protein fusion protein, leads to larger post-synaptic PSD-95 clusters and larger pre-synaptic glutamatergic terminals. On the other hand, over-expression of gephyrin leads to slightly smaller PSD-95 clusters and pre-synaptic glutamatergic terminals. The change in size of PSD-95 clusters were accompanied by a parallel change in the size of NR2-NMDA receptor clusters. It is concluded that the levels of expression and clustering of gephyrin, a protein that concentrates at the post-synaptic complex of the inhibitory synapses, not only has homotypic effects on GABAergic synaptic contacts, but also has heterotypic effects on glutamatergic synaptic contacts. We are proposing that gephyrin is a counterpart of the post-synaptic glutamatergic scaffold protein PSD-95 in regulating the number and/or size of the excitatory and inhibitory synaptic contacts.