SODIUM-DEPENDENT INCREASE IN QUANTAL SECRETION INDUCED BY BREVETOXIN-3 IN CA2-FREE MEDIUM IS ASSOCIATED WITH DEPLETION OF SYNAPTIC VESICLESAND SWELLING OF MOTOR-NERVE TERMINALS IN-SITU()

Brevetoxin-3 at nanomolar concentrations markedly enhanced spontaneous quantal transmitter release from neuromuscular junctions equilibrated in a Ca2+-free EGTA medium. After about 3 h, the sustained increase i n miniature endplate potential frequency led to an exhaustion of trans mitter release. This increase still occurred after loading the nerve t erminals with the Ca2+ chelator bis(aminophenoxy)ethanetetra-acetate o r after pretreatment with various pharmacological agents known to prev ent Ca2+ release from intracellular pools, but was completely prevente d by the Na+ channel blocker tetrodotoxin. Brevetoxin-3 also increased miniature endplate potential frequency from junctions treated with bo tulinum type-A toxin, but to a smaller extent than at normal junctions . At normal junctions, brevetoxin-3 exposure for 2h increased the thre e-dimensional projected area of living motor nerve terminals in situ b y about 74% while at botulinum type-A poisoned junctions a similar tox in exposure caused only a 29% increase. Tetrodotoxin prevented such ef fects, indicating that they are related to both Na+ entry into the ter minals and increased quantal transmitter release. Ultrastructural exam ination of nerve terminals from junctions exposed for 3 h to brevetoxi n-3 revealed profound depletions of clear and large dense core synapti c vesicles and an increase in coated vesicles and axolemma infoldings. These results indicate that brevetoxin-3 impairs the recycling of cle ar synaptic vesicles and are consistent with our immunofluorescent obs ervations showing that synaptophysin epitopes can be revealed without nerve terminal permeabilization. In contrast, no such changes were det ected in nerve terminals poisoned with botulinum type-A toxin which, a fter 3 h exposure to brevetoxin-3, retained their synaptic vesicles an d had a normal appearance. We conclude that tetrodotoxin-sensitive Na entry into motor nerve terminals induced by brevetoxin-3 triggers ext ernal Ca2+-independent asynchronous quantal transmitter release, block s synaptic vesicle recycling and induces swelling of the terminals. We suggest that an excess of cytoplasmic Na+ pei se can activate the asy nchronous neurotransmitter release process. (C) 1997 IBRO.