Hippocampal synaptogenesis in cell culture: developmental time course of synapse formation, calcium influx, and synaptic protein distribution

TLDR

The study examined hippocampal neuron cultures using electrophysiology, calcium imaging, and immunocytochemistry to track synapse formation. Inhibitory and excitatory synapses formed within 12 days, with calcium influx via N‑type channels appearing as synapses matured and synaptic proteins redistributed from diffuse to punctate patterns.

Abstract

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 proteins 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.