Two spatially and temporally distinct sources of calcium contribute to vesicle release at the zebrafish neuromuscular junction

The neuromuscular junction is a classic model synapse that has proved to be a rich source of scientific discovery for many decades. Accessibility made this synapse a favorite for researchers in the early days of electrophysiology. Zebrafish neuromuscular junction retains this key feature, but is also an optimal system for modern day molecular biology, genetics, and optical techniques. Moreover, the possibility to patch clamp motor neurons and simultaneously record from voltage clamped target muscle cells makes this a truly unique preparation. In this dissertation I will describe our efforts to examine two modes of vesicle release whose existence has been recognized since those early days of physiology on frog neuromuscular synapses. With paired recordings the distinction between synchronous and asynchronous release becomes clear. However, particularly with asynchronous transmission, the cause is decidedly less clear. At the zebrafish neuromuscular junction we have managed to isolate asynchronous release pharmacologically which has greatly facilitated its study. In the process, optical measurements of calcium dynamics throughout the motor neuron has uncovered a mechanism which explains key hallmarks of asynchronous release. Namely, why this mode of release is delayed, persistent and sensitive to the slow calcium chelator, EGTA.