Comparison of transmitter release properties of embryonic sympathetic neurons growing in vivo and in vitro

Abstract

The functional behavior of embryonic chick sympathetic neurons was determined by inducing release of [ 3H]norepinephrine by electrical stimulation of sympathetic neurons growing in the chick heart and in culture, with and without heart cells. A very close correspondence between the functional behavior of neurons developing with the heart cells, either in vivo or in vitro, was demonstrated. For example, the outflow of tritium from [ 3H]norepinephrine loaded sympathetic neurons of 15-day-old chick heart was about three times more at 10 Hz than at 1 Hz. In contrast, the outflow of tritium from 12-day-old [ 3H]norepinephrine loaded cultured sympathetic neurons was inversely related to the frequency of stimulation (outflow at 1 Hz was about three time more than at 10 Hz). When neurons were co-cultured with the heart cells, the frequency-outflow relationship reverted to that seen in the intact heart. Electrically-evoked outflow of tritium from the heart was reduced in a concentration-dependent manner by 3–30 nM tetrodotoxin, abolished in 0.25 mM Ca medium, and potentiated by 3mM tetraethylammonium. In sharp contrast, the outflow evoked by stimulation of cultured neurons was neither blocked by 30–300 nM tetrodotoxin, low Ca, nor potentiated by tetraethylammonium. However, when neurons were co-cultured with heart cells, the evoked outflow was blocked by 30 nM tetrodotoxin and low Ca, and potentiated by tetraethylammonium. Veratrine (10μM) had very little effect on the outflow from cultured neurons but induced a massive outflow from co-cultures as well as hearts. Neurons grown in a medium conditioned by the heart cells were not sensitive to tetrodotoxin and veratrine. It is implied that cultured sympathetic neurons are endowed mostly with Ca channels, and that the Na channels become functional only when neurons are grown with the target cells. This dramatic alteration in the functional behavior of neurons co-cultured with heart cells indicates that the effector organ has an important role in the development of ionic conductances of sympathetic neurons growing in the body and in culture.