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

Open Access
Publisher Website
Google Scholar
Abstract
The design and operation of a machine is described 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 they are frozen. Freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synaptic vesicles caught in the act of exocytosis are shown. 4-Aminopyridine (4-AP) is used to increase the number of transmitter quanta discharged with each nerve impulse; the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that 1 vesicle undergoes exocytosis for each quantum that is discharged. Statistical analyses are performed 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 1 another, as expected from physiological demonstrations that quanta are discharged independently. The utility of quick-freezing as a technique to capture biological processes as evanescent as synaptic transmission is established. A new capacitance method measures freezing rates, which shows that the temporal resolution of this quick-freezing technique is 2 ms or better.