Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine

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 Ca2+, 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 less than 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 degrees 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 micron, 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 approximately 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.