Incorporation of synaptotagmin II to the axolemma of botulinum type-A poisoned mouse motor endings during enhanced quantal acetylcholine release.

The involvement of terminal sprouts in neurotransmitter release by in vivo botulinum type-A toxin poisoned motor endings was investigated 15 to 40 days after a single injection of the toxin onto the levator auris longus muscle of the mouse. Enhanced quantal acetylcholine release was induced by alpha-latrotoxin or La3+ in conditions that prevent endocytosis, and an antibody directed against the lumenal domain of synaptotagmin II (Syt II) was used in the presence or absence of Triton X-100. We showed that, under resting conditions, the intravesicular domain of Syt II requires Triton X-100 to be labelled, whereas it becomes exposed to the outside of the axolemma of both the original terminal arborization and the newly formed sprouts during enhanced exocytosis. These data were taken to indicate that, when sprouting is prominent, the whole modified terminal arborization, including the original branches and the sprouts, possesses the machinery for Ca2+-independent exocytosis.

Gambiertoxin (CTX-4B), purified from wild Gambierdiscus toxicus dinoflagellates, induces Na(+)-dependent swelling of single frog myelinated axons and motor nerve terminals in situ.

The effects of gambiertoxin (CTX-4B), purified from the dinoflagellate Gambierdiscus toxicus, were assessed on the morphology of both frog myelinated axons and motor nerve terminals, using confocal laser scanning microscopy. During the action of the toxin (24 and 30 nM), a marked swelling of nodes of Ranvier and motor nerve terminals was observed. The CTX-4B-induced swelling could be prevented by blocking voltage-dependent Na+ channels with tetrodotoxin, and could be partly reversed by an external hyperosmotic solution containing 100 mM D-mannitol. The results suggest that CTX-4B, by modifying voltage-dependent Na+ channels, increases internal Na+ concentration of axons and nerve terminals and consequently induces water influx to compensate such an increase. It is suggested that stimulated transmitter release by CTX-4B, as well as by hyperosmotic dmannitol, contribute also to the swelling of the terminals through an increase in their surface area.

Nerve terminal sprouting in botulinum type-A treated mouse levator auris longus muscle.

The marked outgrowth of the motor nerve terminal arborization triggered by an in vivo local injection of Clostridium botulinum type-A toxin in the mouse levator auris longus muscle was studied with morphological and immunochemical approaches. The increase in total nerve terminal length depended on the time elapsed after toxin administration and was due to both increased number of terminal branches and branch length as revealed by a quantitative morphological analysis of whole mounts using the combined cholinesterase-silver stain. Nerve terminal sprouts increased in number, length and complexity even after the functional recovery of neuromuscular transmission had occurred as revealed by electrophysiological examination. Although we cannot exclude that transmitter release sites from the original nerve terminal arborization may still be functional after botulinum type-A toxin (BoTx-A) treatment, it is likely that newly formed functional release sites on the sprouts play a major role in the functional recovery of neuromuscular transmission. The presence of an immunoreactivity to synaptophysin and synaptotagmin-II, integral proteins of synaptic vesicles, gives support to our previous findings suggesting that nerve terminal sprouts have the molecular machinery for acetylcholine release.

Nodal swelling produced by ciguatoxin-induced selective activation of sodium channels in myelinated nerve fibers.

Ciguatoxin-1b, the major toxin involved in ciguatera fish poisoning, and D-mannitol were examined on frog nodes of Ranvier using confocal laser scanning microscopy and conventional current- and voltage-clamp techniques. During the action of 10 nM ciguatoxin-1b, an increase in nodal volume was observed as determined by digital image processing and three-dimensional reconstruction of axons. The increase was prevented by blocking Na+ channels with tetrodotoxin. Ciguatoxin-1b (10 nM) induced high frequency action potential discharges up to 70-100 Hz. Analysis of Na+ current revealed that the toxin modified a current fraction which was activated at resting membrane potential and failed to inactivate. Increasing the osmolality of the external solution by about 50% with D-mannitol restored the nodal volume to its control value and suppressed spontaneous action potentials. In addition, D-mannitol affected unmodified and ciguatoxin-1b-treated Na+ currents in a similar manner causing a reduction of maximum conductance, negative shifts of current reversal potential and modification of the voltage-dependence of current activation and inactivation. In conclusion, ciguatoxin-1b induced a tetrodotoxin-sensitive swelling of nodes of Ranvier and selectively affected the Na+ current of myelinated axons. It is proposed that ciguatoxin-1b, by modifying Na+ current, increased intracellular Na+ concentration which caused water influx and nodal swelling. This may explain some of the reported symptoms of ciguatera fish poisoning. D-mannitol, an agent used for ciguatera treatment, was found to reverse the effects of ciguatoxin-1b by reducing Na+ entry and increasing the efflux of water through its osmotic action. It is the first time that osmotic changes produced by the selective activation of ionic channels, i.e. Na+ channels, are reported.

Synaptotagmin II immunoreactivity in normal and botulinum type-A treated mouse motor nerve terminals.

The distribution of synaptotagmin II, a synaptic vesicle protein, was examined by immunohistochemistry at normal mouse motor nerve terminals and after botulinum type-A treatment. An immunoreactivity to synaptotagmin II was detected in both control and botulinum type-A treated motor nerve terminals including newly formed sprouts. These data, together with other reports showing the absence of synaptotagmin I at the neuromuscular junction, suggest that synaptotagmin II is the isoform involved in transmitter release at motor nerve terminals.

Mouse motor nerve terminal immunoreactivity to synaptotagmin II during sustained quantal transmitter release.

An antibody directed against the lumenal NH2-terminus of synaptotagmin II was used to examine the distribution of this vesicular protein either after spontaneous acetylcholine release or after sustained release induced by La3+ or alpha-latrotoxin, in conditions that prevent endocytosis. The detection of the epitope was examined in the presence or absence of Triton X-100. We show that, in resting conditions of transmitter release, permeabilization of nerve terminal membranes is required for obvious detection of synaptotagmin Ii immunoreactivity whereas during sustained rates of quantal release, permeabilization is not necessary. These data indicate that, in the latter conditions, synaptotagmin II is incorporated into the terminal axolemma and its intravesicular domain exposed at the extracellular nerve terminal surface.