Cytochemical study of developing neurotransmitter properties of dissociated sympathetic neurons grown in co-culture with dissociated pineal cells

Abstract

We studied the development of neurotransmitter phenotype in sympathetic neurons grown in the presence of pinealocytes, a target tissue having adrenergic but not cholinergic receptors. Neurons, dissociated from neonatal rat superior cervical ganglia, were grown in co-culture with dissociated pineal cells. Both ganglionic and pineal non-neuronal background cells were allowed to grow nearly to confluency. Electron microscopic cytochemical techniques were used to examine sets of co-cultures at weekly intervals over 5 weeks. Adrenergic vesicles were identified by their dense granular precipitate following potassium permanganate fixation. We found that the percentage of small granular vesicles, both in synaptic boutons onto other neurons and in axonal varicosities, declined very little over 5 weeks. After an initial drop from 75 to 65%, the percentage of small granular vesicles remained remarkably constant. Throughout the 5 weeks, more than 70% of the boutons and varicosities contained a predominance of small granular vesicles; fewer than 20% contained a predominance of clear vesicles. Although both somal synaptic boutons and axonal varicosities retained a predominantly adrenergic ultrastructure, at certain weeks there was a statistically significant shift in the percent distribution of adrenergic vesicles in somal boutons compared with the distribution in axonal varicosities. Because these cultures were grown under conditions known to favor an induction of acetylcholine metabolism and a suppression of catecholamine metabolism, we conclude that the maintainence of adrenergic ultrastructure over 5 weeks may be due to the presence of the pineal cells.