Ultrastructural study of reaggregated culture of brainstem: synaptogenesis

The synaptogenesis and the morphological differentiation of neural cells were studied in aggregating cultures. Brainstems of 14-15 days old rat embryos were removed and the area located between the mesencephalic flexure and the caudal portion of metencephalon was dissected and mechanically dissociated to single cells. These cells reassociated forming highly organized aggregates in which differentiation took place. Samples were harvested after different time periods, fixed and processed for electron-microscopic study. After one day in culture the aggregates were composed by rounded undifferentiated cells. These cells had a high nuclear/cytoplasmic relation, were devoid of processes and were separated by great intercellular spaces. At the end of the first week of culture cell differentiation and extension of processes were evident. A loose neuropil appeared: it was composed by abundant growing neurites and growth cones. Later, the neuropil became more compact and glial processes and synaptic terminals filled with vesicles appeared. The early appearance of vesicles in the synaptic endings was the first evidence of synaptogenesis. Post and presynaptic membrane densities appeared later, and fully mature synaptic contacts were seen by the end of the 3rd week in culture. Scarce myelin sheaths were observed after 35 days in vitro.