Localization of Components of Glycinergic Synapses During Rat Spinal Cord Development I. COLIN, P. ROSTAING, A. AUGUSTIN, AND A. TRILLER* Laboratoire de Biologie Cellulaire de la Synapse Normale et Pathologique, INSERM U 497, Ecole Normale Supe ´ rieure, 75005 Paris, France ABSTRACT The sequence of events leading to the chemical matching of presynaptic neurotransmit- ters and postsynaptic transmitter receptors is investigated here in vivo for the spinal glycine receptor (GlyR) by using immunocytochemical methods. In the ventral horn of adult rat spinal cord, GlyRs are only present at glycinergic postsynaptic differentiations where they are stabilized by the associated protein gephyrin. With quantitative 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 number of cell surface gephyrin-immunoreactive (-IR) spots exceeds that of GlyR. They first match 10 days after birth. The densities of postsynaptic gephyrin- and GlyR-IR were quantified between birth and the adult stage with post-embedding immunogold staining. Immunostaining for gephyrin and GlyR was not detected in the extrasynaptic membrane. The density of staining in postsynaptic membrane increased progressively with development. The inhibitory amino- acid content of the presynaptic terminal boutons opposed to gephyrin-IR sites was also analyzed. In the newborn, postnatal day 10, and adult, more than 90% of these boutons were immunostained for glycine. As seen with serial sections, 38% and 51.2% of the terminals also contained -aminobutyric acid (GABA) in neonate and adult, respectively. These data indicate that around the time of birth, most glycine-containing boutons, some also containing GABA, are opposed to gephyrin-IR postsynaptic densities, whereas GlyRs are not present. Our results suggest that gephyrin determines subsynaptic loci on the plasma membrane where GlyR will subsequently accumulate. J. Comp. Neurol. 398:359–372, 1998.  1998 Wiley-Liss, Inc. Indexing terms: synaptic transmission; inhibition; glycine receptor; glycine; GABA; immunogold In the ventral spinal cord, the glycine receptor (GlyR) and the associated gephyrin colocalize and accumulate in front of the presynaptic active zones (Triller et al., 1985, 1987; Todd et al., 1996), thus defining functional microdo- mains on the cell surface of motoneurons. Our current knowledge about the sequence of events that leads to the dense packing of the receptors at postsynaptic sites de- rives from developmental studies on the muscular nico- tinic acetylcholine receptor and its cytoplasmic-associated protein, rapsyn (Cartaud and Changeux, 1993; Froehner, 1993). In contrast, the formation of central synapses is less well documented, and the GlyR-gephyrin complex pro- vides one of the best systems to address this problem. The GlyR, a ligand-gated channel mediating a chloride- dependent inhibition, is composed of three copies of a ‘‘ligand-binding’’  subunit and two copies of a ‘‘structural’’  subunit (Langosch et al., 1988). A membrane-associated protein, gephyrin, has been copurified with GlyR  and  subunits (Pfeiffer et al., 1982). Antisense experiments on cultured spinal neurons showed that gephyrin is directly involved in the accumulation of GlyR clusters at postsynap- tic sites (Kirsch et al., 1993b). Gephyrin binds strongly to polymerized tubulin (Kirsch et al., 1991) and interacts with the large cytoplasmic loop of GlyR  subunit (Meyer et al., 1995). Therefore, it is likely that gephyrin serves as a bridge between the  subunit and the subsynaptic cytoskeleton. This notion was reinforced by experiments with alkaloids affecting cytoskeletal protein polymeriza- Grant sponsors: Association Franc ¸aise contre les Myopathies, Institut de Recherche sur la Moelle Epinie `re, Ministe `re de la Recherche et de la Technologie, France. Dr. Colin’s current address: Institut de Biologie Cellulaire et de Morpholo- gie, Universite ´ de Lausanne, 9, rue du Bugnon, 1005 Lausanne, Suisse. *Correspondence to:Antoine Triller, Laboratoire de Biologie Cellulaire de la Synapse N&P, INSERM U497, Ecole Normale Supe ´ rieure, 46 Rue d’Ulm, 75005 Paris, France. E-mail: triller@wotan.ens.fr Received 1 December 1997; Revised 17 April 1998; Accepted 19 April 1998 THE JOURNAL OF COMPARATIVE NEUROLOGY 398:359–372 (1998)  1998 WILEY-LISS, INC.