Synaptogenesis by Single Identified Neurons in Vitro: Contribution of Rapidly Transported and Newly Synthesized Proteins

The object of these studies is to define the molecular events that occur during synaptogenesis. Our approach is to use single identified Aplysia neurons grown in culture under conditions where chemical synapses are formed. In this report we studied synapses established by R2, a giant cholinergic neuron, onto neurons R15 and L11, and a group of left upper quadrant (LUQ) cells. The detailed electrophysiology of these contacts was described in the preceding paper (Schacher, S., S. G. Rayport, and R. T. Ambron (1985) (J. Neurosci. 5: 2851-2856). Within the animal, R2 synapses on thousands of unicellular mucus glands in the skin. R2 growing in vitro will establish contacts with isolated mucus glands. Although we do not know whether a functional synapse is formed, electron microscopy shows that the membrane in the area of contact is differentiated and that the ending is filled with various types of vesicles. A single R2 regenerating neurites in vitro synthesizes more than 300 polypeptides containing [35S]methionine. Many of these are subsequently transported into the growing neurites. We compared the newly synthesized proteins made by R2 before and after synapse formation and found that the expression of a 68-kilodalton (kd) and 72-kd protein was markedly enhanced after synaptogenesis. The finding that only two proteins were affected implies that many of the proteins required for synapse formation are present in R2 prior to contacting a target cell. Support for this idea was obtained when we compared the proteins present in R2's neurites in vitro with those that are rapidly transported to R2's mature synapses in vivo (Ambron, R. T., S. Schacher, and S. G. Rayport (1985) J. Neurosci. 5: 2866-2873).(ABSTRACT TRUNCATED AT 250 WORDS)