Long-term survival and development of dissociated parasympathetic neurons in culture

Ciliary ganglion neurons from chick embryo were grown in cell culture following enzymatic and mechanical dissociation. The culture conditions necessary for long-term (greater than 21 days) survival of the isolated neurons were investigated. Neurons could be cultured with and without non-neural cells by adjusting the culture substrate. Chick embryo extract was found to be essential in the growth medium, and the inclusion of horse serum had an additional beneficial effect. Also, non-neural cells increased the survival of these neurons in culture. Thus, there appear to be several factors involved in the survival of these neurons in culture. Assay of choline acetyltransferase activity revealed a 100-fold increase in activity over the first two weeks in vitro, and the developmental pattern of the enzyme activity was initially similar to that seen in vivo. The cultured neurons also retained many of the electrophysiological properties of ganglionic neurons in vivo. These included normal resting transmembrane potential, action potential amplitude and an afterpotential. The passive membrane properties of the cultured neurons (membrane resistance, capacitance and time constant) differed somewhat from those of neurons in ganglia. These results suggest that if proper culture conditions are provided, these parasympathetic neurons adapt well to cell culture and develop many of the properties of normal ciliary ganglion neurons in vivo.