Soluble and membrane-bound factors together account for target dependence of cultured parasympathetic neurons

The survival of avian ciliary ganglion (CG) neurons in culture depends upon an exogenous supply of trophic factor(s). Skeletal muscle, a normal ganglionic target tissue, is a well documented provider of survival-promoting activity, although the molecular basis for this ability to foster neuronal survival has not been thoroughly investigated. To identify the source of skeletal muscle support, dissociated neurons were plated into microwells containing either: a basal, trophically deficient medium; live pectoral muscle myotubes; medium conditioned by myotubes; membrane remnants of osmotically lysed myotubes; or, membrane remnants and conditioned medium. Neurons remaining in culture were counted after 1, 2, 5, and 7 days. The results reveal that neuronal survival is supported by both muscle conditioned medium and the membrane remnants of cultured myotubes. Each of these alone provides for only partial survival, while both combine to equal the activity of live myotubes. Treatment of the lysed membranes with either 1.5 M NaCI and/or 15 U heparin removed only 50-60% of the activity, suggesting that multiple factors are involved in the neuronal support obtained from lysed myotubes. This is in contrast to fibroblast remnants, which support some neuronal survival, but whose activity is wholly removed by NaCI. Conditioned medium also contains a heparin binding component which accounts for approximately 60% of its activity. These results indicate that full trophic support from the cultured target tissue requires at least two distinct active agents. The experiments further suggest that the target-derived factors responsible for neuronal survival in culture, and perhaps in vivo, are both soluble and membrane-associated molecules.