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

In the spinal cord, GABA and glycine mediate inhibition at separate or mixe d synapses containing glycine and/or GABA(A) receptors (GlyR and GABA(A)R, respectively). We have analysed here the sequence of events leading to inhi bitory synapse formation during synaptogenesis of embryonic spinal cord neu rons between 1 and 11 days in vitro (DIV). We used immunocytochemical metho ds to detect simultaneously an antigen specific to inhibitory terminals, th e vesicular inhibitory amino acid transporter (VIAAT), and one of the follo wing postsynaptic elements: GlyR, GABA(A)R or gephyrin, the anchoring prote in of GlyR, which is also associated with GABA(A)R. Quantitative analysis r evealed that until 5 DIV most gephyrin clusters were not adjacent to VIAAT- positive profiles, but became associated with them at later stages. In cont rast, GlyR and GABA(A)R clustered predominantly in front of VIAAT-containin g terminals at all stages. However, about 10% of receptor aggregates were d etected 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% a nd 3.4 +/- 0.8% of them contained only GlyR and GABA(A)R, respectively. Int erestingly, 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 co nstruction 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 tha t gephyrin is involved in the stabilization of GABA(A)R rather than in its initial accumulation at synaptic sites.