Morphological and cytochemical studies of synapses formed in culture between isolated rat superior cervical ganglion neurons

Abstract Neurons from the superior cervical ganglia (SCG) of perinatal rats were mechanically dissociated and established in culture by a method modified from that of Bray ('70). The neurons attached to the collagen substrate with some degree of reaggregation, and their rapidly growing processes soon formed an interconnecting network. Light and electron microscope examination showed that the morphology of the cultured cells resembled that of the SCG principal cell; neither satellite cells nor the small intensely fluorescent interneurons were observed. After six to seven days in culture, synapses were seen on both the principal cell somata and on their processes. After aldehyde fixation, the synapses contained clear, pleiomorphic vesicles; after KMnO 4 fixation a variable number of the vesicles contained dense cores. After reserpine treatment and KMnO 4 fixation the synaptic vesicles of the cultured networks contained few dense cores. Similarly treated cultured networks showed an increase in the number of synaptic vesicles with dense cores if incubated with dihydroxyphenylalanine prior to fixation, but a suppression of this increase in the presence of a specific inhibitor of dopamine B‐hydroxylase, FLA 63. The morphology of synapses in the cultured networks was compared to that of endings in the intact rat SCG and iris in vivo, both before and after treatment with norepinephrine and 5‐hydroxydopamine. The vesicles in the synaptic endings in cultures were found to resemble those of the adrenergic endings of the rat iris, and to be similar to those seen within neuronal processes in the rat SCG. Our findings indicate that adrenergic synapses are formed between isolated SCG principal neurons in culture, and thus support recent evidence that such synapses occur in the intact rat SCG. The absence of other tissues which could receive innervation, and of ensheathing satellite cells, may provide a possible explanation for their frequency in the culture situation.