Nerve terminal damage by beta-bungarotoxin: its clinical significance.
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We report here original data on the biological basis of prolonged neuromuscular paralysis caused by the toxic phospholipase A2 beta-bungarotoxin. Electron microscopy and immunocytochemical labeling with anti-synaptophysin and anti-neurofilament have been used to show that the early onset of paralysis is associated with the depletion of synaptic vesicles from the motor nerve terminals of skeletal muscle and that this is followed by the destruction of the motor nerve terminal and the degeneration of the cytoskeleton of the intramuscular axons. The postjunctional architecture of the junctions were unaffected and the binding of fluorescein-isothiocyanate-conjugated alpha-bungarotoxin to acetylcholine receptor was not apparently affected by exposure to beta-bungarotoxin. The re-innervation of the muscle fiber was associated by extensive pre- and post-terminal sprouting at 3 to 5 days but was stable by 7 days. Extensive collateral innervation of adjacent muscle fibers was a significant feature of the re-innervated neuromuscular junctions. These findings suggest that the prolonged and severe paralysis seen in victims of envenoming bites by kraits (elapid snakes of the genus Bungarus) and other related snakes of the family Elapidae is caused by the depletion of synaptic vesicles from motor nerve terminals and the degeneration of the motor nerve terminal and intramuscular axons. (+info)
Functional repair of motor endplates after botulinum neurotoxin type A poisoning: biphasic switch of synaptic activity between nerve sprouts and their parent terminals.
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Blockade of acetylcholine release by botulinum neurotoxin type A at the neuromuscular junction induces the formation of an extensive network of nerve-terminal sprouts. By repeated in vivo imaging of N-(3-triethyl ammonium propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide uptake into identified nerve endings of the mouse sternomastoid muscle after a single intramuscular injection of the toxin, inhibition of stimulated uptake of the dye at the terminals was detected within a few days, together with an increase in staining of the newly formed sprouts. After 28 days, when nerve stimulation again elicited muscle contraction, regulated vesicle recycling occurred only in the sprouts [shown to contain certain soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAREs) and to abut acetylcholine receptors] and not at the parent terminals. Therefore, only these sprouts could be responsible for nerve-muscle transmission at this time. However, a second, distinct phase of the rehabilitation process followed with a return of vesicle turnover to the original terminals, accompanied by an elimination of the by then superfluous sprouts. This extension and later removal of "functional" sprouts indicate their fundamental importance in the repair of paralyzed endplates, a finding with ramifications for the vital process of nerve regeneration. (+info)
Effect of hypertonicity on augmentation and potentiation and on corresponding quantal parameters of transmitter release.
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Augmentation and (posttetanic) potentiation are two of the four components comprising the enhanced release of transmitter following repetitive nerve stimulation. To examine the quantal basis of these components under isotonic and hypertonic conditions, we recorded miniature endplate potentials (MEPPs) from isolated frog (Rana pipiens) cutaneous pectoris muscles, before and after repetitive nerve stimulation (40 s at 80 Hz). Continuous recordings were made in low Ca2+ high Mg2+ isotonic Ringer solution, in Ringer that was made hypertonic with 100 mM sucrose, and in wash solution. Estimates were obtained of m (no. of quanta released), n (no. of functional release sites), p (mean probability of release), and vars p (spatial variance in p), using a method that employed MEPP counts. Hypertonicity abolished augmentation without affecting potentiation. There were prolonged poststimulation increases in m, n, and p and a marked but transient increase in vars p in the hypertonic solution. All effects were completely reversed with wash. The time constants of decay for potentiation and for vars p were virtually identical. The results are consistent with the notion that augmentation is caused by Ca2+ influx through voltage-gated calcium channels and that potentiation is due to Na+-induced Ca2+ release from mitochondria. The results also demonstrate the utility of this approach for analyzing the dynamics of quantal transmitter release. (+info)
Waglerin-1 selectively blocks the epsilon form of the muscle nicotinic acetylcholine receptor.
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Neonatal mice resist the lethal effect of Waglerin-1. Because Waglerin-1 blocks the nicotinic acetylcholine receptor of mature end-plates, the appearance of lethality may result from the epsilon- for gamma-subunit substitution. In support of this hypothesis, adult knockout (KO) mice lacking the gene coding for the epsilon-subunit resist the lethal effect of Waglerin-1. In contrast, heterozygous litter mates respond to Waglerin-1 like adult wild-type mice. In vitro application of 1 microM Waglerin-1 inhibited spontaneous miniature end-plate potentials and evoked end-plate potentials of adult wild-type and heterozygous KO mice. Both miniature end-plate potentials and end-plate potentials of neonatal wild-type and adult homozygous KO mice resisted Waglerin-1. Waglerin-1 decreased the end-plate response of adult wild-type mice to iontophoretically applied acetylcholine (ACh) with an IC50 value of 50 nM; 1 microM Waglerin-1 decreased the ACh response to 4 +/- 1% of control for adult heterozygous KO mice. In contrast, 1 microM Waglerin-1 decreased the ACh response to 73 +/- 2% of control for wild-type mice less than 11 days old and had no effect on the ACh response of adult homozygous KO mice. Between 11 and 12 days after birth, the suppressant effect of Waglerin-1 on wild-type end-plate responses to ACh dramatically increased. Waglerin-1 reduced binding of alpha-bungarotoxin to end-plates of adult but not neonatal wild-type mice. These data demonstrate that Waglerin-1 selectively blocks the mouse muscle nicotinic acetylcholine receptor containing the epsilon-subunit. (+info)
Block of quantal end-plate currents of mouse muscle by physostigmine and procaine.
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of quantal end-plate currents of mouse muscle by physostigmine and procaine. Quantal endplate currents (qEPCs) were recorded from hemidiaphragms of mice by means of a macro-patch-clamp electrode. Excitation was blocked with tetrodotoxin, and quantal release was elicited by depolarizing pulses through the electrode. Physostigmine (Phys) or procaine (Proc) was applied to the recording site by perfusion of the electrode tip. Low concentrations of Phys increased the amplitude and prolonged the decay time constants of qEPCs from approximately 3 to approximately 10 ms, due to block of acetylcholine-esterase. With 20 microM to 2 mM Phys or Proc, the decay of qEPCs became biphasic, an initial short time constant taus decreasing to <1 ms with 1 mM Phys and to approximately 0.3 ms with 1 mM Proc. The long second time constant of the decay, taul, reached values of +info)
Characterization of a vertebrate neuromuscular junction that demonstrates selective resistance to botulinum toxin.
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Botulinum toxin blocks transmitter release by proceeding through a series of four steps: binding to cell surface receptors, penetration of the cell membrane by receptor-mediated endocytosis, penetration of the endosome membrane by pH-induced translocation, and intracellular proteolysis of substrates that govern exocytosis. Each of these steps is essential for toxin action on intact cells. Therefore, alterations in cell structure or cell function that impede any of these steps should confer resistance to toxin. In the present study, screening for susceptibility to four serotypes of botulinum toxin revealed that the cutaneous-pectoris nerve-muscle preparation of Rana pipiens is resistant to type B botulinum toxin. Resistance was demonstrated both by electrophysiologic techniques and by dye-staining techniques. In addition, resistance to serotype B was demonstrated at toxin concentrations that were 2 orders of magnitude higher than those associated with blockade produced by other serotypes. In experiments on broken cell preparations, type B toxin cleaved synaptobrevin from frog brain synaptosomes. However, the toxin did not bind to frog nerve membranes. These findings suggest that resistance is due to an absence of cell surface receptors for botulinum toxin type B. The fact that cutaneous-pectoris preparations were sensitive to other botulinum toxin serotypes (A, C, and D), as well as other neuromuscular blocking agents (alpha-latrotoxin, beta-bungarotoxin), indicates that botulinum toxin type B receptors are distinct. (+info)
Noradrenaline synchronizes evoked quantal release at frog neuromuscular junctions.
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1. Noradrenaline (NA) increases synaptic efficacy at the frog neuromuscular junction. To test the hypothesis that one of the actions of NA is to shorten the period over which evoked quanta are released, we measured the latencies of focally recorded uniquantal endplate currents (EPCs). 2. NA shortened the release period for evoked quantal release. The interval between the time when responses with minimal delay appeared and the point at which 90 % of all latencies had occurred was shortened in the presence of 1 x 10-5 M NA by about 35 % at 20 C and by about 45 % at 8 C. Inhibitor and agonist experiments showed that NA acts on a beta-adrenoreceptor. 3. The better synchronization of release significantly increased the size of reconstructed multi- quantal EPCs. This suggests that NA facilitates synaptic transmission by making the release of quanta more synchronous. 4. The synchronizing action of NA might potentiate neuromuscular transmission during nerve regeneration, transmitter exhaustion and other extreme physiological states where the quantal content is reduced, such as survival in cold and hibernation. (+info)
Empty synaptic vesicles recycle and undergo exocytosis at vesamicol-treated motor nerve terminals.
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We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were treated with 5 microM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s-1 in 35 KP but then declined to <1 s-1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1-43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals. (+info)