Studies on rat sympathetic neurons developing in cell culture: I. Growth characteristics and electrophysiological properties

Abstract

In this series of three papers, we describe electrophysiological and pharmacological studies on sympathetic principal neurons developing in cell culture. This paper is concerned with the methods for growing and recording from the neurons and with observations on some of their electrical properties. The succeeding papers are concerned with functional synapses which the neurons form with one another. Superior cervical ganglia of newborn rats were dissociated into single cells and small cell clusters, and the resulting cell suspension of principal neurons and a much smaller number of non-neuronal cells was cultured at low density in medium containing nerve growth factor ( D. Bray, 1970, Proc. Nat. Acad. Sci. USA. 65, 905–910; R. E. Mains and P. H. Patterson, 1973a, J. Cell Biol. 59, 329–345 ). As in the previous studies the multiplication of the non-neuronal cells could be controlled so that the neurons grew in the presence of an increasing number of non-neuronal cells or in the virtual absence of other cell types. Another method for obtaining mixed cultures was to plate the initial cell suspension onto a preexisting layer of cells dissociated from some other tissue (e.g., heart). Neurons grown for 3 weeks or longer in the presence of non-neuronal cells had resting potentials, passive electrical properties, and action potentials generally similar to those reported for principal neurons of the superior cervical ganglia of adult rats. Through the use of tetrodotoxin, tetraethylammonium, and cobalt, evidence was obtained for the presence of potential-sensitive sodium, potassium, and calcium channels. Frequently the action potential was followed by a prolonged after-hyperpolarization whose properties suggested the presence of potassium channels controlled by calcium ions. When the neurons were grown in the absence of non-neuronal cells, the action potentials were similar, but the prolonged after-hyperpolarization was rarely seen, and the neurons usually discharged repetitively in response to a steady depolarization.