The formation of chemical synapses between hippocampal neurons in primary cell culture was studied using electrophysiology, calcium imaging, and immunocytochemical approaches. Inhibitory and excitatory synapses formed within 12 d in cell culture (DIC) that were sensitive to the N-type calcium channel blocker omega-conotoxin GVIA (omega-CgTx). At 4 DIC, immature connections were present in which spontaneous, but rarely evoked, synaptic currents were detected. At both 4 and 12 DIC, the synaptic

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... oteins rab3a, synapsin I, and synaptotagmin were present in hippocampal neurons, but the subcellular distribution changed from one in which immunoreactivity was initially distributed within soma and neurites to a punctate varicose appearance. Correlated with the transformation from immature to mature synaptic states was the onset of omega-CgTx-sensitive calcium influx. Taken together, these data suggest that the expression of functional omega-CgTx-sensitive calcium influx is temporally coincident with synapse formation, and that during the maturation of the synapse there is a redistribution of synaptic proteins.