Development of glycine receptor alpha subunit in cultivated rat spinal neurons: An immunocytochemical study

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Ontogenesis of the inhibitory glycine receptor was studied up to 12 days in vitro in spinal neurons placed previously in culture at embryonic day 14. The alpha subunit of the receptor was detected using standard and confocal immunofluorescence and a specific monoclonal antibody. The immunostaining was compared to that of synaptophysin, a synaptic vesicle antigen, which was taken as an index of synaptic maturity. Glycine receptors could be detected intracellularly, and not at the cellular surface in some cells as early as 2-3 days in vitro (DIV) prior to any synaptic contact. At 4-5 DIV, the number of cells which expressed the immunoreactivity and the fluorescence intensity increased. At this stage, spherical fluorescent blobs started to migrate in the neurites. From 6 DIV, the glycine receptor alpha subunit was detected at the neuronal surface and was organized in clusters whose number increased progressively with time. From 7 DIV, the intrasomatic immunoreactivity decreased, and at day 12, the pattern of labelling was similar to that observed in the adult spinal cord. A diffuse presence of the receptor at the surface of neurons could never be visualised, and when detected, the glycine receptors were always clustered. Thus, the increasing expression of clusters of glycine receptors at the neuronal surface was paralleled by that of synaptophysin in neuritic varicosities. These data suggest that transport of glycine receptors to the plasmamembrane and the formation of aggregates occurs simultaneously to synaptogenesis.