Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

Abstract

We applied the quick-freezing technique to investigate the precise temporal coincidence between the onset of quantal secretion and the appearance of fusions of synaptic vesicles with the prejunctional membrane. Frog cutaneous pectoris nerve-muscle preparations were soaked in modified Ringer's solution with 1 mM 4-aminopyridine, 10 mM Ca 2+, and 10 -4 M d-Tubocurarine and quick-frozen 1-10 ms after a single supramaximal shock. The frozen muscles were then either freeze-fractured or cryosubstituted in acetone with 13% OsO4 and processed for thin section electron microscopy. Temporal resolution of <1 ms can be achieved using a quick-freeze device that increases the rate of freezing of the muscle after it strikes the chilled copper block (15°K) and that minimizes the precooling of the muscle during its descent toward the block. We minimized variations in transmission time by examining thin sections taken only from the medial edge of the muscle, which was at a fixed distance from the point of stimulation of the nerve. The ultrastructure of the cryosubstituted preparations was well preserved to a depth of 5-10/~m, and within this narrow band vesicles were found fused with the axolemma after a minimum delay of 2.5 ms after stimulation of the nerve. Since the total transmission time to this edge of the muscle was ~3 ms, these results indicate that the vesicles fuse with the axolemma precisely at the same time the quanta are released.Freeze-fracture does not seem to be an adequate experimental technique for this work because in the well-preserved band of the muscle the fracture plane crosses, but does not cleave, the inner hydrophobic domain of the plasmalemma. Fracture faces may form in deeper regions of the muscle where tissue preservation is unsatisfactory and freezing is delayed.Neurotransmitters are released from nerve terminals in two ways: by the continuous leakage of individual molecules across the nerve terminal membrane (23,33,41,50) and by synchronous secretion of few thousands molecules in packages, or quanta (14,15,20). Quantal secretion causes the changes in membrane potential that excite the postsynaptic cell, and transmission at a synapse is critically dependent upon this mechanism of release. The vesicle hypothesis holds that each quantum is confined within one of the synaptic vesicles that populate the nerve terminal and is released by exocytosis when the membrane of that vesicle fuses with the axolemma at one of the many specialized regions called "active zones" (8,13,51).A number of workers have shown that at frog neuromuscular junctions vesicles fuse with the axolemma of stimulated 1386 terminals and some of the remaining vesicles become labeled with extracellular tracers (9)(10)(11)25). Although these observations indicate that vesicles fuse with and are recovered from the axolemma when quanta are actively secreted, the slowness of chemical fixation precludes demonstrating that vesicle fusion and transmitter release are coincident. The images of fusion seen after chemical fixation represent...

Bilayers merge even when exocytosis is transient

“…It has been proposed that synaptic vesicles also can reseal after they release transmitter (20,39). In this model of exocytosis, the vesicle does not exchange membrane components with the plasma membrane (21).…”

High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells.

“…After 1 h, they were washed carefully and then postfixed for an additional hour with cold 2% OS04 in 0.12 M phosphate buffer, pH 7.3. Dehydration, infiltration, and embedding in Araldite were done as described previously (Watanabe et al, 1983 (Torri Tarelli et al, 1985). Frozen specimens were then transferred into an ultra-highvacuum chamber (Cryofract 190) and freeze-dried under controlled conditions as described previously (Grohovaz et al, 1996).…”

Effect of alpha‐latrotoxin on the frog neuromuscular junction at low temperature.

“…Although recent quick-freeze studies of frog terminals treated with 4-aminopyridine have demonstrated that vesicles fuse with the presynaptic membrane a few milliseconds after the arrival of a single nerve impulse (Heuser, Reese, Dennis, Jan, Jan & Evans, 1979;Torri-Tarelli, Grohovaz, Fesce & Ceccarelli, 1985), controversy still exists over whether the secreted quanta represent ACh that is released from the interiors of the fused vesicles (Tauc, 1982). An alternative hypothesis is that the quanta represent axoplasmic ACh which diffuses in pulses through channels in the presynaptic membrane that become transiently permeable to it (Tauc, 1982;Israel & Manaranche, 1985).…”