Morphological and biochemical studies on the development of cholinergic properties in cultured sympathetic neurons. I. Correlative changes in choline acetyltransferase and synaptic vesicle cytochemistry.

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Abstract
Under certain culture conditions, neonatal rat superior cervical ganglion neurons display not only numerous expected adrenergic characteristics but certain cholinergic functions, e.g., the development of hexamethonium-sensitive synaptic contacts and accumulation of choline acetyltransferase (ChAc). Whether the entire population of cultured neurons was acquiring cholinergic capabilities or whether this phenomenon was restricted to a subpopulation was investigated. After 1-6 and 8 wk in culture, neurons were fixed in KMnO4 after incubation in norepinephrine and prepared for EM analysis of synaptic vesicle content to determine whether vesicles were dense cored or clear. ChAc, acetylcholinesterase (AChE) and dopa-decarboxylase (DDC) activities were assayed in sister cultures. In the period from 1-8 wk in culture, the average ChAc activity per neuron increased 1100-fold and the DDC and AChE activities increased 20- and 30-fold, respectively. After 1 wk in culture, 48 of 50 synaptic boutons contained predominantly dense-cored vesicles, but by 8 wk the synaptic vesicle population was predominantly of the clear type. At intermediate times, the vesicle population in many boutons was mixed. The morphology of the synaptic contacts on neuronal surfaces was characteristic of autonomic systems, with no definite clustering of the vesicles adjacent to the area of contact. Increased vesicle size correlated with increasing age in culture and the presence of a dense core. These cultures apparently contain 1 initially adrenergic population of neurons. This population develops cholinergic mechanisms. That a neuron may at a given time express both cholinergic and adrenergic mechanisms is suggested by the approximately equal numbers of clear and dense-cored vesicles in the boutons found at the intermediate times.