Morphological studies of synapses and varicosities in dissociated cell cultures of sympathetic neurons.

Abstract

Neurons dissociated from the superior cervical ganglia of newborn rats can be grown under conditions which support either adrenergic or cholinergic differentiation. In both cases, the neurons form numerous morphologically specialized synaptic terminals or synapses as well as relatively unspecialized varicosities. The ultrastructure of both types of terminal was compared in mature neuronal cultures and the effects of growth conditions on terminal morphology examined. After aldehyde-osmium fixation, synapses in cultures grown under adrenergic or cholinergic conditions were characterized by asymmetrical membrane specializations comparable to type I or asymmetric synapses; bismuth iodide and ethanolic phosphotungstic acid impregnation of neuronal cultures revealed the presence of characteristic synaptic membrane specializations: a presynaptic grid of dense projections and a wide postsynaptic dense band of uniform thickness. No membrane specializations were apparent in varicosities after aldehyde-osmium fixations or with these stains. Intramembranous particle distributions were examined in freeze-fracture replicas of neurons. Aggregates of large, 10-12 nm particles were found on P-face membrane leaflets of cell bodies and large diameter processes; this distribution is the same as that of synapses in thin-sectioned preparations. These particle aggregates may represent postsynaptic membrane specializations or acetylcholine receptors. The cytoplasmic leaflet of boutons contained large, 12-14 nm particles, which appeared to be concentrated at the region of synaptic contact at putative synapses, but were diffusely distributed in varicosity membranes. Similar large particles were also seen at a much lower density in the membrane E-face. None of these ultrastructural characteristics appeared to vary with transmitter identity or growth conditions. Synaptic vesicle shape, however, did vary in glutaraldehyde-fixed cultures. At all ages examined, neurons grown on monolayers of heart cells contained predominantly round vesicles, whereas neurons grown in the virtual absence of non-neuronal cells possessed pleiomorphic synaptic vesicles. This difference in vesicle shape appeared to be correlated more closely with growth in the presence of non-neuronal cells than with the transmitter present at the time of fixation.