LOCALIZATION OF COMPONENTS OF GLYCINERGIC SYNAPSES DURING RAT SPINAL-CORD DEVELOPMENT

The sequence of events leading to the chemical matching of presynaptic neurotransmitters and postsynaptic transmitter receptors is investiga ted here in vivo for the spinal glycine receptor (GlyR) by using immun ocytochemical methods. In the ventral horn of adult rat spinal cord, G lyRs are only present at glycinergic postsynaptic differentiations whe re they are stabilized by the associated protein gephyrin. With quanti tative confocal microscopy, we found that gephyrin is detected before GlyRs at embryonic day (E)13-E14 and at E15, respectively, inside the cytoplasm and at plasmalemmal loci. Around the time of birth, the numb er of cell surface gephyrin-immunoreactive (-IR) spots exceeds that of GlyR. They first match 10 days after birth. The densities of postsyna ptic gephyrin- and GlyR-IR were quantified between birth and the adult stage with post-embedding immunogold staining. Immunostaining for gep hyrin and GlyR was not detected in the extrasynaptic membrane. The den sity of staining in postsynaptic membrane increased progressively with development. The inhibitory aminoacid content of the presynaptic term inal boutons opposed to gephyrin-IR sites was also analyzed. In the ne wborn, postnatal day 10, and adult, more than 90% of these boutons wer e immunostained for glycine. As seen with serial sections, 38% and 51. 2% of the terminals also contained gamma-aminobutyric acid (GABA) in n eonate and adult, respectively. These data indicate that around the ti me of birth, most glycine-containing boutons, some also containing GAB A, are opposed to gephyrin-IR postsynaptic densities, whereas GlyRs ar e not present. Our results suggest that gephyrin determines subsynapti c loci an the plasma membrane where GlyR will subsequently accumulate. J. Comp. Neurol. 398:359-372, 1998. (C) 1998 Wiley-Liss, Inc.