Constraints on the interpretation of nonquantal acetylcholine release from frog neuromuscular junctions

In the frog nerve-muscle preparation, there is evidence for nonquantal release of acetylcholine (ACh) at a level 100 times that attributable to spontaneous quantal events (miniature endplate potentials). It is widely assumed that nonquantal release occurs near the sites of quantal release (active zones) in the nerve terminal, close to the postsynaptic muscle membrane. This high level of nonquantal ACh release has led to the suggestion that it may serve a trophic function at the nerve-muscle junction. However, the precise origin and mechanism of nonquantal release have not been determined. We have used outside-out patches of ACh receptor-rich membrane as a sensitive technique for the direct measurement of ACh release from highly localized regions of enzymatically treated nerve terminals and have found little detectable ACh "leakage" from active-zone regions. If all of the nonquantal ACh release were localized to the under surface of the nerve terminal, we estimate that we would have detected more than 10 times the low level detected with our patch probe. Furthermore, although quantal release was easily measurable, vesicular exocytosis (hypothesized to insert ACh transport proteins into the plasma membrane, thereby producing the leak) did not increase single-channel activity in the patch probe above that attributable to quantal release. We conclude that, at rest, the active-zone region of nerve terminals is not a major source of nonquantal ACh release and that vesicular exocytosis does not noticeably increase the level of nonquantal release from the nerve terminal. Thus, with biochemical measurements that indicate that spontaneous ACh release is relatively unchanged by prior denervation, these results question the assumed source and mechanism of nonquantal release and the suggestion that leakage of ACh from active-zone regions plays a trophic role in nerve-muscle interaction.