A quantitative electron microscopic study on synapse formation in dissociated fetal rat cerebral cortex in vitro

Occipital cortex of 19-day-old fetal rats was dissociated and cultured in vitro for 2-5 weeks in horse serum supplemented Eagle's MEM. The outgrowing neurons rapidly formed a dense network which started to degenerate after 3 weeks in vitro. By means of electron microscopy the numerical development of 6 different categories of synapses was followed during the time in culture. This approach revealed a sigmoid growth curve emerging over the first 3 weeks for the category of axo-dendritic synapses (which comprised the bulk of all synapses counted). Thereafter a decline in number set in. Chronic exposure of these dissociated cerebral cortex cultures to 50 microgram/ml xylocaine (a concentration adequate to block all measurable bioelectrical activity) did not prevent the formation of functional synaptic networks having normal synaptic ultrastructure. These results are in agreement with previous studies on fetal cerebral explants in culture. However, in some groups of our cultures, xylocaine led to a retardation in neurite outgrowth and in numerical synapse formation. Since these xylocaine effects were dose-related at a concentration double that required to silence the cultures, the growth retardation was probably not due to selective suppression of bioelectric activity, but rather to some general cytotoxic effect of the drug. In one of the xylocaine-treated groups (50 microgram/ml) which showed an exceptionally rapid neuronal outgrowth by the use of horse serum obtained from a different source than in the other groups, no deficit was noted in the number of synapses formed. Extracellular recording in this latter drug-treated group of cultures during the third week revealed (after return to control medium) spontaneous isolated action potentials, burst patterns and slow waves which were indistinguishable from the bioelectric activity seen in the control cultures. It is concluded that bioelectric activity in dissociated cortex cultures is not a prerequisite for the formation of apparently normal, functional synaptic networks.