On the possible origin of giant or slow-rising miniature end-plate potentials at the neuromuscular junction.

Giant or slow-rising miniature end-plate potentials (GMEPPs) caused by vesicular release of acetylcholine (ACh) occur at any time in about 50% of mouse diaphragm neuro muscular junctions, but generally at frequencies less than 0.03 s-1. Their frequency is, unlike that of miniature end-plate potentials (MEPPs), not affected by nerve terminal depolarization. Unlike MEPPs and stimulus-evoked end-plate potentials, GMEPPs have a prolonged time-to-peak and show an increase in time-to-peak with amplitude. By using these differences in amplitude and time course, GMEPPs can be separated from MEPPs. In contrast to MEPPs, GMEPPs are not blocked by botulinum neurotoxin type A. GMEPPs have a greater temperature sensitivity than MEPPs, disappearing at temperatures below 15 degrees C. Long-term paralysis by botulinum toxin and certain drugs which inhibit protein kinase C or affect actin filament polymerization (cytochalasins) enhance the frequency of GMEPPs. End-plate current recordings show that similar postsynaptic ACh receptors are activated by MEPPs and GMEPPs. It is suggested that GMEPPs are not caused by mechanisms involved in regulated neurotransmitter release but are generated by constitutive secretion.

Effects of stonefish (Synanceia trachynis) venom on murine and frog neuromuscular junctions.

The neuromuscular toxicity of stonefish (Synanceia trachynis) venom was characterized by electrophysiological and electron microscopic examination of isolated murine and frog nerve-skeletal muscle preparations exposed to various concentrations of venom. Low concentrations of venom (2.5-10 micrograms/ml) acted presynaptically by causing release and depletion of neurotransmitter from the nerve terminal. The response was Na+ channel-independent (resistant to tetrodotoxin), required the presence of either Ca2+ or Mg2+, and was observed with botulinum neurotoxin-paralyzed nerve-muscle preparations. Higher concentrations of venom (100-300 micrograms/ml) acted postsynaptically and presynaptically. They caused irreversible depolarization of muscle cells and microscopically observable muscle and nerve damage. We conclude that the previously observed neuromuscular toxicity of stonefish venom is a consequence of the venom's dose-dependent, presynaptic and postsynaptic actions at the myoneural junction.

Ultrastructure of botulinum type-A poisoned frog motor nerve terminals after enhanced quantal transmitter release caused by carbonyl cyanide m-chlorophenylhydrazone.

The effects of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on spontaneous quantal transmitter release and nerve terminal ultrastructure were studied on isolated cutaneous pectoris nerve-muscle preparations from frogs that were completely paralysed by a single sublethal dose of Clostridium botulinum type A toxin (BoTx). CCCP enhanced miniature endplate potential frequency at poisoned junctions and caused a reduction in the density of clear synaptic vesicles and of large dense core vesicles in motor nerve terminals. However, the intensity of these effects was much less important than that previously reported at unpoisoned junctions. The moderate depletion of synaptic vesicles can be related to the low levels of transmitter release detected with CCCP at BoTx-poisoned terminals.

Tetrahydroaminoacridine (tacrine) stimulates neurosecretion at mammalian motor endplates.

1. Tacrine (20 microM) induced, like 4-aminoquinoline (4-AQ, 200 microM), the appearance of a population of miniature endplate potentials (m.e.p.ps) with more than twice the normal amplitude or time-to-peak. The times-to-peak of nerve impulse-evoked endplate potentials were not similarly affected. 2. Cholinesterase inhibition by edrophonium (25 microM) did not prevent tacrine or 4-AQ from inducing this population of m.e.p.ps. 3. Nerve-muscle preparations in which the normal calcium-sensitive quantal release of acetylcholine had been blocked by botulinum neurotoxin type A also responded to tacrine by an increase in the frequency of giant or slow m.e.p.ps. 4. Reduction of the temperature from 30 degrees to 14 degrees C reduced the frequency of giant or slow m.e.p.ps induced either by tacrine or by 4-AQ. A similar effect was obtained by colchicine (5 mM). This supports the idea that proximo-distal axonal transport is required for the secretory activity. 5. The neurosecretion evoked by tacrine could explain the therapeutic effects of the drug claimed in the treatment of Alzheimer's type of dementia.

Presynaptic actions of botulinal neurotoxins at vertebrate neuromuscular junctions.

1. In the present paper we review some presynaptic aspects of the mode of action of botulinal toxins (BoTxs) at vertebrate neuromuscular junctions with emphasis on studies carried out in our laboratories using electrophysiological and morphological techniques. 2. Spontaneous quantal transmitter release recorded as miniature end-plate potentials is drastically affected by BoTxs. The low probability of release at poisoned terminals can be enhanced by carbonyl cyanide m-chlorophenylhydrazone (CCCP), Cd2+ and La3+. However, CCCP and La3+ which drastically deplete clear synaptic vesicles from unpoisoned terminals failed to markedly affect the density of synaptic vesicles at poisoned terminals. It is concluded that poisoned terminals have a reduced sensitivity to the release-promoting action of Ca2+, Cd2+ and La3+. 3. When comparing the effect of the various BoTxs on nerve-impulse evoked transmitter release it appears that increasing phasic Ca2+ entry into the terminals enhances evoked synchronized quantal release only from terminals poisoned with serotypes A and E. In contrast, enhanced Ca2+ entry into terminals poisoned with serotypes B, D and F induced a period of high frequency asynchronous release suggesting that these BoTxs may affect a presynaptic step beyond the influx of Ca2+, that may be involved in the synchronization of transmitter quanta. These data suggest that the actions of BoTxs involve several steps of the acetylcholine release process. 4. The analysis of presynaptic currents which depend on both Ca2+ entry and intraterminal background Ca2+ levels strongly suggests that neither Ca2+ entry nor intraterminal Ca2+ levels are altered by BoTxs. Furthermore, poisoned terminals are no more efficient than unpoisoned ones in dealing with Ca2+ overloads. 5. Finally, the morphological examination of junctions paralysed by BoTx-A indicates that the toxin triggers a particularly important overgrowth of the nerve terminals and suggests that the in vivo functional recovery may occur from an extension of the original nerve terminal arborization and the concomitant remodelling of postsynaptic structures.

Trophic interrelations at the neuromuscular junction as revealed by the use of botulinal neurotoxins.

1. From denervation studies the trophic influence of the motor nerve on the muscle cell is well documented while little is known about the influence of the muscle on the nerve. Sectioning the axon invariably destroys the nerve terminals and produces nerve degeneration products which themselves may affect nerve and muscle properties. With regard to those difficulties we believe that the botulinal neurotoxins (BoTx) are valuable complements to denervation since they selectively interrupt impulse transmission across the synapse without damaging its morphology. 2. Paralysis of mouse or rat skeletal muscle in vivo with BoTx type A causes marked growth of motor nerve terminals. The sprouting terminals are rich in large dense-core synaptic vesicles containing various neuropeptides and they spontaneously release large quanta of ACh. Thus, it appears that paralysis by BoTx is a strong stimulus for motor nerve growth and the delivery of "trophic" substances to the nerve terminals. 3. Postsynaptically, in extrajunctional areas, paralysis by BoTx induces all the changes observed following denervation, i.e. atrophy, appearance of extra-junctional ACh receptors, TTX-resistant action potentials, a fall of resting membrane potential, fibrillation potentials and the disappearance of extrajunctional acetylcholinesterase activity. Endplate properties are, however, largely maintained. 4. BoTx blockade delays and prevents the retraction of polyneuronal innervation and motoneurone death during development. This supports the suggestion that the paralysed muscle secretes factors essential for growth and for the survival of motoneurones. 5. Like denervated muscle, BoTx paralysed ones, express a high endocytotic activity restricted to a segment in the endplate region.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the actions of botulinum neurotoxin type E at the rat neuromuscular junction.

Botulinum neurotoxin (BoTx) serotype E blocks spontaneous and evoked quantal release of acetylcholine at the rat neuromuscular junction. Increasing extracellular Ca2+ to 8 mmol l-1 or substituting Ca2+ with La3+ (0.1 and 1.0 mmol l-1) or depolarizing the nerve terminals by 20 mmol l-1 K+ markedly increases miniature end-plate potential frequency in normal muscle, but in BoTx-E poisoned preparations none of these ions, with the exception of 1 mmol l-1 La3+, was able to restore spontaneous quantal transmitter release to levels recorded at unpoisoned junctions. In absolute values the enhancement with La3+ was much less than that reported at normal junctions. Nerve stimulation in the presence of 3,4-diaminopyridine (10-20 mumol l-1) and high calcium (8 mmol l-1) evoked multiquantal end-plate potentials and muscle twitches. We conclude that the neuromuscular block produced by BoTx serotype E is similar to that previously described for BoTx serotype A but differs from that produced by BoTx serotypes B, D and F in not causing desynchronization of nerve impulse-evoked transmitter release. 3,4-Diaminopyridine might be useful in the treatment of poisoning by BoTx serotype E since it markedly enhanced synchronous transmitter release from poisoned motor nerve terminals.

A study of synchronization of quantal transmitter release from mammalian motor endings by the use of botulinal toxins type A and D.

1. The effects of botulinum toxin (BoTx) types A and D on spontaneous and evoked phasic transmitter release were studied in the isolated extensor digitorum longus muscle of the rat or the levator auris longus muscle of mice. 2. The toxins were injected subcutaneously into the hindleg of adult rats or the dorsal aspect of the neck of mice. At various times after the injection the muscles were removed from the anaesthetized animal and neuromuscular transmission examined in vitro by conventional intracellular techniques. 3. Both toxins reduced spontaneous transmitter release recorded as the frequency of miniature end-plate potentials but BoTx type D was less effective in that respect than the type A toxin. 4. With both toxins the block of evoked phasic transmitter release, recorded as end-plate potentials, was almost complete. As previously reviewed by Simpson (1986) the block produced by BoTx type A was partially reversed by procedures which elevate the intraterminal level of calcium ions. However, in BoTx type D-paralysed muscles such procedures failed to restore phasic transmitter release but caused a period of high-frequency asynchronous transmitter release following each nerve impulse. 5. To investigate if the lack of synchronization of evoked transmitter release observed in BoTx type D-paralysed muscles was due to alterations in presynaptic currents we examined, by perineural recordings, the Na+, fast K+, slow K+, K+-Ca2+-dependent and the Ca2+ currents in BoTx type D-paralysed muscles. These presynaptic currents were not altered as compared to unpoisoned controls. 6. We suggest that there exists a presynaptic process, which in addition to Ca2+ influx participates in transmitter synchronization and which is a main target for BoTx type D action.

Is the internal calcium regulation altered in type A botulinum toxin-poisoned motor endings?

The hypothesis according to which botulinum A toxin blocks acetylcholine release from motor endings by stimulating intracellular Ca2+ disposal systems was tested by recording presynaptic membrane currents from poisoned muscles. Calcium and calcium-activated potassium currents displayed amplitudes, time courses and stimulation frequency-dependent inactivation similar to those observed in unpoisoned preparations. This indicates that poisoned endings are no more efficient than normal ones in dealing with Ca2+ overloads.

Effects of cadmium on quantal transmitter release and ultrastructure of frog motor nerve endings.

Exposure of frog cutaneous pectoris nerve-muscle preparations to cadmium (0.1-1 mM) results in an increase in miniature end-plate potential (m.e.p.p.) frequency. The increase is dependent on the concentration, the time of exposure and the co-presence of other divalent cations in the extracellular fluid. The stimulatory effect of cadmium is most marked in a calcium-free medium. Increased levels of calcium (4-10 mM) or of magnesium (10 mM) reduce the stimulatory effect suggesting that those cations interfere with the entry of cadmium into nerve endings. Once the effect of cadmium on m.e.p.p. frequency is attained, washing with a cadmium-free solution fails to abolish its effect. The action of cadmium on m.e.p.p. frequency slowly declines towards zero after about 3 hrs. An ultrastructural study of nerve terminals exposed for one hr to 1 mM cadmium reveals that neither in calcium-containing nor in a nominally calcium-free medium are there any significant changes in the number of synaptic vesicles as compared to controls. However, after 3 hrs of cadmium action in a calcium-free medium there is about 65% depletion of synaptic vesicles, while in calcium-containing media there is only about 25% depletion. The results suggest that cadmium by itself can support transmitter release but not synaptic vesicle recycling which instead might depend upon calcium.

Spontaneous transmitter release at the neuromuscular junction.

The classical studies of Katz and co-workers have shown that nerve impulses release quanta of acetylcholine at the neuromuscular junction. This release is regulated by presynaptic calcium and accounts for the trans-synaptic transmission of nerve impulses. In resting conditions it gives rise to small spontaneous potentials, i.e. miniature endplate potentials. In addition these investigators described a spontaneous molecular leakage of acetylcholine from the motor nerve. I have studied a third type of acetylcholine release. It is a spontaneous intermittent secretion of acetylcholine which postsynaptically causes large, generally slow rising potentials. This release is unaffected by presynaptic calcium and is therefore not influenced by nerve activity. The acetylcholine responsible for these potentials comes from the same pool of transmitter as that liberated by nerve impulses. The observation that the release is blocked by drugs that prevent the accumulation of acetylcholine into synaptic vesicles indicates that the secretion originates from clusters of vesicles or large vesicle-like structures in the nerve terminal. This type of release is present at a low frequency at normal neuromuscular junctions. It is markedly accelerated whenever the calcium-dependent quantal release of acetylcholine is blocked or impaired. The drug 4-aminoquinoline selectively stimulates this release. I speculate that this type of transmitter secretion is important for the development of synaptic connexions.

Aminoglycosides and 3,4-diaminopyridine on neuromuscular block caused by botulinum type A toxin.

Impulse-evoked transmitter release was greatly reduced at frog neuromuscular junctions 3-20 days after botulinum type A toxin (BoTx) poisoning. The reduction in transmitter release was accompanied by an increased variability in the latency between the presynaptic spike and the release of transmitter. The aminoglycoside antibiotics amikacin, gentamycin, and bekanamycin, when applied at concentrations within their therapeutic levels, markedly enhanced the blockade of transmitter release in BoTx-poisoned junctions. 3,4-diaminopyridine strongly antagonized the effects of BoTx at early stages of poisoning, and the combined presynaptic effects of BoTx and aminoglycoside antibiotics provided that transmitter release was not completely blocked by the toxin. The antagonism was apparent at all frequencies of stimulation. Since the aminoglycoside antibiotics enhanced the neuromuscular block caused by BoTx, these drugs should be avoided in patients suspected of poisoning by this toxin.

In botulinum type A-poisoned frog motor endings ouabain induces phasic transmitter release through Na+-Ca2+ exchange.

Ouabain (100 microM) applied for 60 min to botulinum A (BoTx) poisoned motor junctions increases, in a time-dependent manner, the mean number of acetylcholine quanta released by nerve stimulation and enhances the delayed transmitter release. The drug does not affect spontaneous quantal release. The observed effects on evoked transmitter release cannot be explained by changes in the configuration of presynaptic currents recorded from motor terminals. They suggest that in BoTx-poisoned motor endings the level of intraterminal Ca2+, lower than that required for the activation of quantal transmitter release, can be effectively increased through the reversed operation of an Na+-Ca2+ exchange system that normally uses the Na+ gradient to extrude Ca2+.

The nature and origin of calcium-insensitive miniature end-plate potentials at rodent neuromuscular junctions.

1. To study the nature and origin of slow-rising, Ca2+-insensitive miniature end-plate potentials (m.e.p.p.s) in mammalian muscle we used intracellular recording techniques and drugs which block acetylcholine (ACh) synthesis or the uptake of ACh into synaptic vesicles. Slow m.e.p.p.s were induced in vivo by paralysing the extensor digitorum longus muscle of the rat with botulinum toxin type A or in vitro by the application of 4-aminoquinoline to the mouse diaphragm nerve-muscle preparation. 2. Hemicholinium-3, which blocks ACh synthesis, reduced the amplitude of all synaptic potentials including slow m.e.p.p.s, but only if the nerve was stimulated. 3. 2(4-phenylpiperidino)cyclohexanol (AH-5183), which blocks the active uptake of ACh into synaptic vesicles, reduced both the frequency and the amplitude of slow m.e.p.p.s and did so without requiring nerve stimulation. 4. No correlation was observed between the molecular leakage of ACh from the motor nerve and the frequency and amplitude of slow m.e.p.p.s. 5. We conclude that slow m.e.p.p.s are caused by the release of ACh from the nerve terminal, possibly from a small pool of synaptic vesicle-like structures.