Formation of synaptic specializations in the inner plexiform layer of the developing chick retina

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Abstract
The development of synapse‐like specializations was investigated in the inner plexiform layer of the developing chick retina by using light and electron microscopy. Six monoclonal antibodies, directed against glycine and γ‐aminobutyric acid (GABA)A receptor subunits, the intracellular receptor‐associated protein gephyrin, synaptotagmin, and synaptophysin were used to determine the initial appearance and distribution of their antigens. Synaptophysin and synaptotagmin immunoreactivity was detected in the retina concurrent with the formation of the inner plexiform layer at embryonic day 7. This early appearance before synaptic differentiation, together with the transient expression of synaptotagmin immunoreactivity in the synapse‐free optic fiber layer, suggests that in the developing central nervous system (CNS) these proteins are not confined to synapses. The first immunofluorescence signal detected with specific antibodies against the β2 and β3‐subunits of the GABAA receptor, the glycine receptor, and gephyrin appeared at embryonic day 12. In contrast, the α1‐subunit of the adult‐type glycine receptor heteromeric complex was detectable only at later stages of development, after embryonic day 16, suggesting a change in the subunit composition of some glycine receptor complexes. The staining was clearly punctate, indicating the clustering of the α1‐subunit at synapses. Electron microscopic investigation revealed the first postsynaptic densities and active zones in the inner plexiform layer of the retina at embryonic day 12. These results reveal different patterns of development for the investigated pre‐ and postsynaptic proteins and indicate a parallel appearance of gephyrin, glycine receptor, and the β2 and β3‐subunits of the GABAA receptor with the first synaptic specializations in the inner plexiform layer of the developing chick retina.