Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release.

TLDR

The authors built a rapid‑freezing device that contacts tissues with a cold metal block and uses a timing circuit to stimulate frog neuromuscular junctions milliseconds before freezing, and they employed 4‑aminopyridine to boost quantal release, performed spatial statistics on vesicle fusion sites, and validated the method’s temporal resolution (≤2 ms) with a new capacitance measurement. Freeze‑fracture replicas captured synaptic vesicles undergoing exocytosis, with the number of vesicles frozen increasing proportionally to 4‑AP‑induced quanta, and spatial analysis showed independent fusion events, confirming quick‑freezing as a reliable tool for visualizing transient synaptic transmission.

Abstract

We describe the design and operation of a machine that freezes biological tissues by contact with a cold metal block, which incorporates a timing circuit that stimulates frog neuromuscular junctions in the last few milliseconds before thay are frozen. We show freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synpatic vesicles caught in the act of exocytosis. We use 4-aminopyridine (4-AP) to increase the number of transmitter quanta discharged with each nerve impulse, and show that the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that one vesicle undergoes exocytosis for each quantum that is discharged. We perform statistical analyses on the spatial distribution of synaptic vesicle discharge sites along the "active zones" that mark the secretory regions of these nerves, and show that individual vesicles fuse with the plasma membrane independent of one another, as expected from physiological demonstrations that quanta are discharged independently. Thus, the utility of quick-freezing as a technique to capture biological processes as evanescent as synaptic transmission has been established. An appendix describes a new capacitance method to measure freezing rates, which shows that the "temporal resolution" of our quick-freezing technique is 2 ms or better.