Structural changes after transmitter release at the frog neuromuscular junction.

TLDR

Membrane retrieval and recycling that reverse exocytosis occur more slowly, as noted in prior studies. The study used rapid freezing of frog muscle at various times after nerve stimulation, with 4‑aminopyridine to increase quanta, to capture the sequence of structural changes during synaptic vesicle exocytosis. Vesicle openings at active zones appear 3–4 ms after stimulation, peak at 5–6 ms, then vanish by 50–100 ms, with later openings larger; after disappearance, clusters of large intramembrane particles—matching undischarged vesicle membranes—remain, indicating that exocytotic vesicles collapse into the plasma membrane, a process that is variable and asynchronous, ultimately leading to particle coalescence and dispersion within the first second.

Abstract

The sequence of structural changes that occur during synaptic vesicle exocytosis was studied by quick-freezing muscles at different intervals after stimulating their nerves, in the presence of 4-aminopyridine to increase the number of transmitter quanta released by each stimulus. Vesicle openings began to appear at the active zones of the intramuscular nerves within 3-4 ms after a single stimulus. The concentration of these openings peaked at 5-6 ms, and then declined to zero 50-100 ms late. At the later times, vesicle openings tended to be larger. Left behind at the active zones, after the vesicle openings disappeared, were clusters of large intramembrane particles. The larger particles in these clusters were the same size as intramembrane particles in undischarged vesicles, and were slightly larger than the particles which form the rows delineating active zones. Because previous tracer work had shown that new vesicles do not pinch off from the plasma membrane at these early times, we concluded that the particle clusters originate from membranes of discharged vesicles which collapse into the plasmalemma after exocytosis. The rate of vesicle collapse appeared to be variable because different stages occurred simultaneously at most times after stimulation; this asynchrony was taken to indicate that the collapse of each exocytotic vesicle is slowed by previous nearby collapses. The ultimate fate of synaptic vesicle membrane after collapse appeared to be coalescence with the plasma membrane, as the clusters of particles gradually dispersed into surrounding areas during the first second after a stimulus. The membrane retrieval and recycling that reverse this exocytotic sequence have a slower onset, as has been described in previous reports.