The expression and localization of synaptic vesicle antigens at neuromuscular junctions in vitro

To examine the biochemical differentiation of presynaptic nerve terminals in vitro, we studied the expression and localization at synapses of two synaptic vesicle-specific antigens, synapsin I and protein p65. We purified these proteins from brain and raised a rabbit antiserum against each of them. Chick synapsin I had a slightly smaller molecular weight than its mammalian homologue, although the two share several properties. With the anti-p65 serum, the protein p65 could be detected consistently in presynaptic terminals at the neuromuscular junction in vivo, as had been previously shown for synapsin I. These antisera proved sufficiently sensitive to allow a study of the developmental expression of the antigens in embryonic chick brain, using protein blots. The two antigens are not coordinately regulated; protein p65 was detected substantially sooner in development that was synapsin I. Both synapsin I and protein p65 are expressed by ciliary ganglion neurons in vitro, as assessed by immunofluorescence using the affinity-purified antisera. The two antigens co-localized at all times in culture. Neurons grown alone were reliably stained only after 4 to 5 days in culture, but comparable levels of staining were found after 1 day when neurons were co-cultured with embryonic myotubes. In the co-cultures, staining was initially high in growth cones and neurites, but the brightest staining became confined to sites of nerve-muscle contact over 4 to 5 days in culture. In mature cultures, patches of bright staining for the vesicle antigens coincided with patches of acetylcholine receptors, suggesting that the antigens had become localized at synapses. The time course of this localization process suggests that it corresponds to the morphological maturation of synapses. It should be possible to exploit this system to obtain information about the molecules and processes involved in the induction of presynaptic differentiation.