Differentiation of sympathicoblasts in cultures of chick ganglia: light and electron microscopic, fluorescence and enzyme histochemical observations.

Abstract

Immature sympathetic ganglia prepared from 5 1/2-or 6-day-old chick embryos were cultured up to one month. The in vitro development was followed by phase microscopy, electron microscopy and using histochemistry for catecholamines, monoamine oxidase and cholinesterases. During the first week of culture extensive plexuses of nerve fibres were formed between and around the clusters of nerve cells. Mature-looking neurons were observed in the cultures by phase microscopy after three weeks, at which age the mean diameter of the perikarya was more than doubled. Varying catecholamine fluorescence was observed in the perikarya during the entire culture period. The nerve fibres showed usually only weak fluorescence, but, in the older cultures, bright varicosities were regularly found in the fibres. Monoamine oxidase activity was demonstrated already at three days of culture and the reaction was maintained positive. Weak or moderate acetyl-cholinesterase activity was demonstrated in the sympathicoblasts and young sympathetic neurons and their processes. The axolemma showed acetylcholinesterase activity also around the nerve terminals containing small dense cored vesicles. Reactions for the non-specific cholinesterases were negative. Electron microscopy of the 30-day-old cultures revealed that the clusters of nerve cells consisted of mature sympathetic neurons, which contained large (60-200 nm) and small (35-60 nm) granular catecholamine-storing vesicles. Glial cells were almost totally lacking. Large numbers of nerve terminals containing both large and small granular vesicles were observed in the clusters, often in synaptic contact with the sympathetic neurons. It is concluded that the primitive sympathicoblasts are, in favourable conditions, capable of differentiation in culture up to mature sympathetic neurons.