Determination of synaptic phenotype: insulin and cAMP independently initiate development of electrotonic coupling between cultured sympathetic neurons.

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Abstract
Electrotonic coupling between pairs of sympathetic neurons dissociated from superior cervical ganglia of neonatal rats is rare when cells are cultured for 2 wk in a nutrient medium plus serum and is common when cells are cultured for the same period in serum-free defined medium. This defined medium is the same nutrient medium with 5 added factors (progesterone, transferrin, putrescine, insulin, and Se). When added singly to serum-containing medium, insulin and, to a lesser extent, Se promote the development of electrotonic and dye coupling. The insulin effect is obtained with doses as low as 0.01 .mu.g/ml and is maximal after exposures of 3-5 days. The incidence of electrotonic coupling is also enhanced by exposure cells to dibutyryl cAMP. This effect is obtained with doses as low as 0.1 mM, is faster (being maximal at .apprxeq. 12 h exposure), and is prolonged in the presece of the phosphodiesterase inhibitor caffeine. Butyrate itself promotes coupling to a small extent, but cAMP involvement is confirmed by similar effects of other membrane permeant analogs. Endogenous levels of cAMP are significantly elevated in cultures grown in the defined medium but not in those in serum-containing medium to which insulin or Se are added. The promotion of coupling by cAMP and by insulin or Se are independent. The develoment of coupling in the defined medium thus seems to be a consequence of the addition of promoting substances (insulin, Se) and the removal of an inhibitory effect of serum on cAMP levels.