Incorporation of vesicular antigens into the presynaptic membrane during exocytosis at the frog neuromuscular junction: a light and electron microscopy immunochemical study.

We have studied the incorporation of vesicular membrane antigens into the presynaptic membrane during exocytosis of neurotransmitter at the frog neuromuscular junction. In a preliminary series of experiments, we first confirmed by electron microscopy that the synaptic vesicles are labelled following incubation with rabbit antisynaptic vesicle antibody of neuromuscular junction cross sections (cytoplasm and organelles reached by the antibodies). In a second series of experiments, intact neuromuscular junctions were stimulated with black widow spider venom and fixed with paraformaldehyde. The presence or absence of vesicular antigens in the presynaptic membrane was monitored with rabbit antisynaptic vesicle antibody and revealed with a second antibody coupled to peroxidase. In light microscopy, the labelled neuromuscular junctions are almost completely restricted to muscles stimulated with black widow spider venom and incubated with rabbit antisynaptic vesicle antibody. Only a few control muscles (not stimulated with black widow spider venom, but incubated with rabbit antisynaptic vesicle antibody) had labelled neuromuscular junctions. All control neuromuscular junctions, not incubated with rabbit antisynaptic vesicle antibody were unlabelled. Electron microscopy indicated that it is the presynaptic membrane of intact stimulated neuromuscular junctions which is labelled. In these intact neuromuscular junctions, the synaptic vesicles are usually unlabelled. Electron microscopy also indicated that the presynaptic membrane of only one type of control junctions (not stimulated with black widow spider venom, but incubated with rabbit antisynaptic vesicle antibody) is rarely and weakly labelled. Other types of controls (not incubated with rabbit antisynaptic vesicle antibody) are never labelled. Therefore our results are consistent with the incorporation of vesicular antigens into the plasma membrane during exocytosis produced by the black widow spider venom. The low level of labelling of unstimulated neuromuscular junctions suggest a rather complete retrieval of the vesicular proteins during endocytosis.