The Effect of Electrical Stimulation on Neuronal Outgrowth and the Development of a New Method for Chronic Long-Term Stimulation and Recording from Groups of Neurons in Culture

In this dissertation, I shall examine the response of neurite outgrowth from cultured rat superior cervical ganglion (SCG) neurons to electrical stimulation and to changes in cytoplasmic calcium. Previous studies have shown that suprathreshold electrical stimulation arrests axonal growth from mouse dorsal root ganglion (DRG) and Helisoma neurons (Fields et al., 1990; Cohan and Kater, 1986). Cohan and collaborators (1987) have attributed the arrest of neurite outgrowth from Helisoma neurons to a rise in the growth-cone calcium concentration, [Ca]gc. In the experiments presented in this dissertation, neurite outgrowth from neonatal rat SCG neurons continued unabated during continuous suprathreshold electrical stimulation at 10 Hz for up to one hour. As in previous studies, the internal calcium concentration rose during stimulation. Fura-2 measurements showed that growth cone calcium levels rose from about 100 nM to greater than 500 nM, before settling at about 350 nM during stimulation. Despite this increase, neurite outgrowth continued. My results suggest that electrical activity is not a universal signal for neurons to stop growing and that a rise in internal calcium does not always arrest the migration of growth cones. I was also able to record from and stimulate rat SCG neurons using a new device that allows maintained two-way communication between neurons and electronic circuitry. The new device or "neuron well array" holds individual neurons in surface micromachined holes. A self-supporting overhanging grillwork restrains the neurons in the holes. Each hole has an electrical contact which allows recording from and stimulation of the cell trapped therein. Neurons that grow in the holes appeared to suffer no observable ill effects of entrapment. Future neuronal development studies are planned with the wells.