Synapse formation and morphological differentiation of neuron types in embryonic rat dentate gyrus explants in vitro

Cultured explants obtained from the dentate gyrus of rat embryos (embryonic day 19-20) were used to investigate synapse formation and morphological differentiation of neuron types in the absence of extrinsic afferents. Synaptogenesis was studied by whole-cell recordings of postsynaptic currents and by ultrastructural analysis. Neurons were visualized using Lucifer Yellow filling or staining with DiI. In short-term (3-5 days) cultured explants postsynaptic currents were rarely evoked by extracellular stimulation and synapses were almost completely absent at the ultrastructural level. After 6-10 days in vitro, the incidence of evoking postsynaptic currents mediated by glutamate and GABA(A) receptors was strongly increased. At the ultrastructural level, the density of synapses increased more than 20-fold. These results demonstrate de novo formation of synapses in cultured embryonic dentate gyrus explants. Neuron types could be discriminated by their dendritic arborizations and by their electrophysiological properties. After 6-10 days in vitro, mossy-like cells exhibited 3-4 primary dendrites branching in a characteristic pattern and showed moderate spike-frequency adaptation. Application of serotonin (5-HT) to cultured explants elicited GABA(A)-receptor-mediated postsynaptic currents in mossy-like cells, indicating synaptic GABA release from local interneurons. Comparison to 5-HT evoked GABA release in mossy cells in age-matched, acute slices revealed only slight quantitative differences. In contrast to mossy cells, granule cells showing several primary dendrites originating at one cell pole were almost completely absent in cultured explants, suggesting an involvement of extrinsic afferents in the differentiation of granule cells.