Synaptic vesicle recycling at the neuromuscular junction in the presence of a presynaptic membrane marker

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Staining of the presynaptic axonal membrane of the neuromuscular junction with horseradish peroxidase-labeled α-bungarotoxin was utilized as a marker for observing directly the fate of this membrane during the process of synaptic vesicle release and recycling. The neuromuscular junctions of frog sartorius-sciatic nerve preparations were stained with horseradish peroxidase-α-bungarotoxin and stimulated by electrical stimulation of the nerve, high concentration of external potassium ions, and black widow spider venom. Some preparations were stimulated in the presence of exogenous horseradish peroxidase tracer after incubation in the conjugate and were found to contain horseradish peroxidase within many synaptic vesicles, indicating that the conjugate did not affect the process of synaptic vesicle recycling. Stimulation was followed by depletion of synaptic vesicles and appearance of axolemmal infoldings and membranous cisternae. With rest after electrical and potassium stimulation, synaptic vesicles were reconstituted and terminals assumed a more normal appearance. Membrane staining after stimulation occurred in the axolemmal infoldings, some of the intra-axonal cisternae, and in a few coated vesicles. However, all synaptic vesicles were unreactive, in either rested or unrested terminals. Thus, axonal membrane labeled with horseradish peroxidase-α-bungarotoxin did not become incorporated into new synaptic vesicles. These observations support a mechanism of recycling of synaptic vesicles by specific retrieval of vesicle membrane or constituents from the axolemma.