Formation of mixed glycine and GABAergic synapses in cultured spinal cord neurons

In the spinal cord, GABA and glycine mediate inhibition at separate or mixed synapses containing glycine and/or GABA(A) receptors (GlyR and GABA(A)R, respectively). We have analysed here the sequence of events leading to inhibitory synapse formation during synaptogenesis of embryonic spinal cord neurons between 1 and 11 days in vitro (DIV). We used immunocytochemical methods to detect simultaneously an antigen specific to inhibitory terminals, the vesicular inhibitory amino acid transporter (VIAAT), and one of the following postsynaptic elements: GlyR, GABA(A)R or gephyrin, the anchoring protein of GlyR, which is also associated with GABA(A)R. Quantitative analysis revealed that until 5 DIV most gephyrin clusters were not adjacent to VIAAT-positive profiles, but became associated with them at later stages. In contrast, GlyR and GABAAR clustered predominantly in front of VIAAT-containing terminals at all stages. However, about 10% of receptor aggregates were detected at nonsynaptic loci. The two receptors colocalized in 66.2+/-2.5% of the inhibitory postsynaptic domains after 11 DIV, while 30.3+/-2.6% and 3.4+/-0.8% of them contained only GlyR and GABA(A)R, respectively. Interestingly, at 3 DIV GABA(A)R clustered at a postsynaptic location prior to gephyrin and GlyR; GABA(A)R could thus be the initiating element in the construction of mixed glycine and GABAergic synapses. The late colocalization of gephyrin with GABA(A)R, and the demonstration by other groups that, in the absence of gephyrin, postsynaptic GABA(A)R is not detected, suggest that gephyrin is involved in the stabilization of GABA(A)R rather than in its initial accumulation at synaptic sites.