Serum-free culture of dissociated, purified adult and aged sympathetic neurons and quantitative assays of growth and survival

In vitro studies of dissociated neurons have provided crucial data regarding the regulation of plasticity in embryonic and perinatal neurons from both central and peripheral nervous systems. There have been few attempts to apply these methods to adult or aged neurons and the methods that have been reported have not been able to dissect the possible confounding contributions of non-neuronal cells and serum. Furthermore, quantitative assays of cultured neurons, particularly of their growth: have rarely been described. We report here the development of a novel method for the dissociation, purification and culture of sympathetic neurons from the adult and aged rat SCG under serum free conditions and in defined media. The technique results in a relatively high yield of viable, growing neurons. We describe methods for assaying the total yield of neurons, the proportion of surviving neurons and the proportion of neurons initiating neurite outgrowth after plating. A novel semiautomated assay of neurite outgrowth is outlined using image analysis of composite images of immunofluorescence-stained single neurons. (C) 2001 Elsevier Science B.V. All rights reserved.