Hyperosmolar D-mannitol reverses the increased membrane excitability and the nodal swelling caused by Caribbean ciguatoxin-1 in single frog myelinated axons

The effects of hyperosmolar D-mannitol were studied on single frog myelinated nerve fibres previously poisoned with Caribbean ciguatoxin-1 (C-CTX-1), a new toxin isolated from the pelagic fish Caranx latus inhabiting the Caribbean region. In current-clamped myelinated axons, C-CTX-1 (50-120 nM) caused spontaneous and repetitive action potential discharges after a short delay. In addition, the toxin produced a marked swelling of nodes of Ranvier of myelinated axons that reached a steady state within about 90 min, as revealed by using confocal laser scanning microscopy. The increased excitability and the nodal swelling caused by C-CTX-1 were prevented or reversed by an external hyperosmotic solution containing 100 mM D-mannitol. Moreover, the C-CTX-1-induced nodal swelling was completely prevented by the blockade of voltage-sensitive sodium channels by tetrodotoxin (TTX). It is suggested that C-CTX-1, by increasing nerve membrane excitability, enhances Na(+) entry into nodes of Ranvier through TTX-sensitive sodium channels, which directly or indirectly disturb the osmotic equilibrium between intra- and extra-axonal media resulting in an influx of water that was responsible for the long-lasting nodal swelling. The fact, that hyperosmolar D-mannitol either reversed or prevented the neurocellular actions of C-CTX-1, is of particular interest since it provides the rational basis for its use to treat the neurological symptoms of ciguatera fish poisoning in the Caribbean area.     

Hyperosmolar -mannitol reverses the increased membrane excitability and the nodal swelling caused by Caribbean ciguatoxin-1 in single frog myelinated axons

The effects of hyperosmolar -mannitol were studied on single frog myelinated nerve fibres previously poisoned with Caribbean ciguatoxin-1 (C-CTX-1), a new toxin isolated from the pelagic fish Caranx latus inhabiting the Caribbean region. In current-clamped myelinated axons, C-CTX-1 (50–120 nM) caused spontaneous and repetitive action potential discharges after a short delay. In addition, the toxin produced a marked swelling of nodes of Ranvier of myelinated axons that reached a steady state within about 90 min, as revealed by using confocal laser scanning microscopy. The increased excitability and the nodal swelling caused by C-CTX-1 were prevented or reversed by an external hyperosmotic solution containing 100 mM -mannitol. Moreover, the C-CTX-1-induced nodal swelling was completely prevented by the blockade of voltage-sensitive sodium channels by tetrodotoxin (TTX). It is suggested that C-CTX-1, by increasing nerve membrane excitability, enhances Na⁺ entry into nodes of Ranvier through TTX-sensitive sodium channels, which directly or indirectly disturb the osmotic equilibrium between intra- and extra-axonal media resulting in an influx of water that was responsible for the long-lasting nodal swelling. The fact, that hyperosmolar -mannitol either reversed or prevented the neurocellular actions of C-CTX-1, is of particular interest since it provides the rational basis for its use to treat the neurological symptoms of ciguatera fish poisoning in the Caribbean area.     

Pandinus imperator scorpion venom blocks voltage-gated potassium channels in nerve fibers

We have examined the effects of venom from the scorpion Pandinus imperator on the membrane currents of voltage-clamped frog myelinated nerve fibers using the Vaseline-gap method. Crude venom, applied externally in concentrations from 50 to 500 micrograms/ml, selectively blocked the voltage-gated potassium currents without affecting nodal sodium currents or resting conductances. Block of potassium channels by Pandinus venom was highly dependent on the membrane voltage, being greater at negative potentials than at positive potentials. The blocking effects of Pandinus venom were irreversible on the time scale of our experiments; however, even high concentrations of venom failed to block potassium currents completely at positive potentials. These results suggest that Pandinus venom contains a component(s) that interacts specifically and strongly with a subpopulation of axonal potassium channels.     

Presynaptic origin of penicillin after discharges at mammalian nerve terminals.

The site of origin and mechanism underlying the generation of repetitive after-discharges produced by penicillin was studied in the isolated rat phrenic nerve-hemidiaphragm preparation. Application of low concentrations of sodium penicillin to the bathing solution initiated bursts of antidromic action potentials originating at or near the motor nerve terminals following single orthodromic stimuli to the nerve. Afterdischarges could not be elicited by direct stimulation of the muscle fibers alone, or when the nerve trunk was isolated from the neuromuscular junction and exposed to penicillin. D-Tubocurarine applied in doses sufficient to abolish postsynaptic responses did not diminish penicillin-induced after discharges. At concentrations which most reliably produced repetitive firing (5000 IU/ml; 8.5 mM), penicillin did not accelerate the frequency of spontaneous transmitter release (MEPPs), yet significantly increased the relative excitability of nerve endings to extracellular stimulation. It is concluded that penicillin acts directly and preferentially on presynaptic nerve terminals to induce repetitive afterdischarges which arise independently of postsynaptic depolarization, transmitter-mediated potassium efflux, or muscle fiber contraction. The results suggest that the convulsant effects of penicillin at a mammalian neuromuscular junction are due to non-depolarizing alterations in the intrinsic excitability of the terminal membrane which increase the probability of suprathreshold depolarizations during the recovery period of spike electrogenesis. Several models of the mechanisms which might produce hyperexcitability at presynaptic nerve terminals are discussed.     

Effect of calcium channel blockaders (verapamil, D-600 and manganese ions) on mediator release from frog muscle motor nerve endings]

The reduction in the EPPs quantum content produced by manganese ions (0.4-5.0 mM) was observed in the frog sartorius muscle. In contrast to the inhibitory action on the evoked release, manganese ions increased a spontaneous transmitter release. Verapamil (1-10(-6)-5-10(-5) g/ml) and D-600 (2.5-10(-5) g/ml) did not inhibit the evoked release but increased the spontaneous one. All the calcium antagonists studied were able to prevent the facilitatory effect of imidazole (3 mM) on neuromuscular transmission. Verapamil (5-10(-6)-5-10(-5) g/ml) disturbed the action potential generation during the repetitive stimulation of the motor nerve. Manganese ions were ineffective in this respect. A conclusion is made that the calcium ionic channels in the nerve terminals differ from the calcium channels in some other tissues (heart, soma of neurons etc.).     

Suppression by phenytoin of convulsant-induced afterdischarges at presynaptic nerve terminals

The mechanisms underlying the induction of afterdischarges at presynaptic nerve terminal by convulsant aminopyridines and their suppression by the anticonvulsant drug phenytoin were studied at the frog neuromusclar preparation. Addition of aminopyridine to the perfusing solution induced the appearance of afterdischarges in motor nerve fibres following their primary response to a single nerve stimulus. The afterdischarges seemed to originate at or near the nerve terminals and to propagate both antidromically and orthodromically. The latter resulted in repetitive activation of the neuromuscular synapse. Focal recordings of nerve terminal potentials suggested that aminopyridines may induce afterdischarges by slowing spike repolarization and thereby producing a prolonged depolarization of nerve terminals. Phenytoin suppressed the aminopyridine-induced afterdischarges and the resultant repetitive excitation of the postsynaptic muscle fibres. This effect of phenytoin was associated with a depression of the action potential at the motor nerve terminals but not at their parent axons. These results single the presynaptic nerve terminals as preferential sites for convulsant and anti-convulsant actions.     

Distribution of Ca2+ channels on frog motor nerve terminals revealed by fluorescent omega-conotoxin.

Tetramethylrhodamine-conjugated omega-conotoxin was used as a fluorescent stain (Jones et al., 1989) to determine the spatial distribution of voltage-gated Ca2+ channels along frog motor nerve terminals. Like native omega-conotoxin, the fluorescent toxin blocked neuromuscular transmission irreversibly. The fluorescent staining was confined to the neuromuscular junction and consisted of a series of narrow bands (in face views) or dots (in side views) approximately 1 micron apart. This characteristic staining pattern was prevented by pretreatment with omega-conotoxin and by prior denervation for 5-7 d. Combined fluorescence and phase-contrast optics indicated that the stain was on the synaptic rather than the nonsynaptic side of the nerve terminal. The bands and dots of stain proved to be in spatial register with the postsynaptic junctional folds, as revealed by combined staining of ACh receptors. It is concluded that the voltage-gated Ca2+ channels on frog motor nerve terminals are concentrated at active zones. The findings are consistent with the suggestion (Heuser et al., 1974; Pumplin et al., 1981) that the large intramembraneous particles seen at freeze-fractured active zones are voltage-gated Ca2+ channels.     

Neurophysiologic assessment of disorders affecting the neuromuscular junction

The neuromuscular junction (NMJ) is a specialized synapse with a complex structural organization. Muscle contraction involves several steps: (1) nerve conduction to depolarize the motor nerve terminals, (2) opening of voltage-gated calcium channels (VGCCs) in the presynaptic membrane, (3) generation of endplate potential in the postsynaptic membrane via acetylcholine receptors, (4) depolarization of muscle sodium channels, and (5) excitation-contraction (E-C) coupling. Each step can be affected by various diseases. Guillain-Barre syndrome involves distal axons and possibly the presynaptic NMJ. The abnormalities can be detected by nerve conduction studies and single-fiber electromyography (SFEMG). Myasthenia gravis (MG) with anti-acetylcholine receptor (AChR) antibodies, is the most common NMJ disorder, and ~5% of myasthenia patients are positive for anti-muscle specific kinase (MuSK) antibodies. Patterns and severity of neuromuscular transmission failure detected by repetitive nerve stimulation test and SFEMG are somewhat different in AChR-MG and MuSK-MG. Excitation-contraction (E-C) coupling can be affected by MG, possibly via antibodies against ryanodine receptors. The E-C coupling time can be assessed with an accelerometer. Lambert-Eaton myasthenic syndrome is caused by antibodies against presynaptic VGCCs. This review will focus on neurophysiological testing, including SFEMG, and measurements of E-C coupling time with an accelerometer. In addition to confirming or excluding the diagnosis, these techniques can provide new insights into the pathophysiology of a variety of neuromuscular disorders.     

Protein and enzyme distribution in microsomal and myelin fractions from rat and jimpy mouse brain

4-Aminopyridine (4-AP) powerfully increases transmitter release from motor nerve terminals of rat and frog skeletal muscle in response to single nerve impulses. The drug also enhances transmitter release during repetitive nerve activity but, at D-tubocurarine-blocked endplates, only the first impulses cause increased transmitter release at stimulation frequencies at or above 50 Hz. At magnesium- and botulinum-poisoned endplates, 4-AP potentiates transmitter release at every stimulus during tetanic nerve stimulation and restores neuromuscular transmission. Spontaneous transmitter release in the rat is not affected by the drug, but at some frog endplates miniature endplate potential (mepp) frequency increases. The drug has no post-synaptic action, as evidenced by its lack of effect on amplitude or time course of mepps. Decreasing the temperature from 37 to 15 degrees C does not abolish the effect of 4-AP on neuromuscular transmission. In the presence of 4-AP, single nerve impulses produce repetitive spontaneous activity in the nerve terminal of the frog nerve-muscle preparation. Experiments on the mode of action of 4-AP suggest that the drug increases transmitter release by enhancing the influx of calcium ions during depolarization of the nerve terminal.     

Non-uniform release at the frog neuromuscular junction: evidence of morphological and physiological plasticity

The frog neuromuscular junction (NMJ) is a fusiform structure parallel to the muscle fiber with a few secondary and tertiary branches. Both sprouting and regression can occur on the same nerve terminal, suggesting a continuous on-going remodelling of the mature neuromuscular junction. Thus, the frog NMJ is a dynamic structure. Ultrastructural observations of the nerve terminal suggest that the active zones are distributed equally along the mature nerve terminal. Disorganized active zones have however been observed in distal regions. The density of synaptic vesicles is also uniform throughout the whole structure. However, mitochondria appear to be more abundant in the very distal regions of the nerve terminal. The postjunctional folds and the cholinergic receptors are also uniformly distributed along the NMJ. However, during remodelling periods, the distributions of postjunctional folds and of cholinergic receptors are not uniform in the degenerating and regenerating regions. Fig. 1 summarizes these morphological data. The frequency of spontaneous release (MEPPs) at the NMJ is higher in the proximal region than in the distal regions and recent evidence suggests that the mean MEPP amplitude is higher in the proximal than in the distal portions. Evoked transmitter release is also non-uniform along the frog NMJ. As for spontaneous release, it is higher in the proximal regions than in the distal regions. Failures of the active propagation of the PNAP at low safety points, such as the end of the myelinated axon and the branching points, may be one of the mechanisms responsible for unequal evoked release. It is also possible that the PNAP does not actively invade the whole extend of the nerve terminal since Na+ channels are absent from the distal regions. Fig. 2 summarizes these physiological data.     

Neurotonia: impulse-induced repetitive discharges in motor nerves in peripheral neuropathy

Delayed relaxation of muscle following voluntary contraction was an unusual feature of a mild chronic sesorimotor peripheral neuropathy in an adult. This abnormality resulted from rapid repetitive discharges in motor nerves occurring only in response to passing impulses; there was no spontaneous nerve discharge. Voluntary contraction of affected muscles generated involuntary high-frequency discharges of motor unit potentials, which persisted briefly after attempted relaxation. Nerve blocks localized independent zones of hyperexcitability in distal and proximal sections of nerve from which such repetitive discharges could be triggered. Intravenous administration of phenytoin diminished the discharge. Examination of intramuscular nerve bundles revealed loss of myelinated nerve fibers with numberous sprouting and remyelinating axons. These findings, along with muscle biopsy changes of neurogenic atrophy and type grouping, strongly favor an axonal neuropathy. An explanation for the repetitive nerve discharge is slow waning of heightened excitability of a motor nerve after passage of an impulse.     

Endotoxin alters spontaneous transmitter release at the frog neuromuscular junction.

The direct neurotoxic effects of E. coli endotoxin (ETX) on spontaneous transmitter release were tested at the frog sartorius muscle neuromuscular junction. Spontaneous transmitter release was monitored by intracellularly recording miniature end-plate potentials (MEPPs). Junctions were continuously exposed to standard concentrations of 10 microgram/ml of 3 ETX samples, 2 of which produced a significant elevation of MEPP frequency followed by a decline of frequency to very low rates. The third ETX sample, known to have a decreased canine lethality, was without effect on MEPP frequency. No significant changes in MEPP amplitude were evident. The rate of change in MEPP frequency, but not the peak frequency, was reduced by lowering ETX concentrations. Alterations in MEPP frequency induced by ETX were prevented by removing Ca++ and antagonized by high [K+]out. The results suggest that ETX alters ion conductance channels, particularly those for Ca++, in the presynaptic terminal membrane.     

Selective Depletion of Clear Synaptic Vesicles and Enhanced Quantal Transmitter Release at Frog Motor Nerve Endings Produced by Trachynilysin, a Protein Toxin Isolated from Stonefish (Synanceia trachynis) Venom

Our previous observation that low concentrations of stonefish (Synanceia trachynis) venom elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals prompted our present study to purify the quantal transmitter-releasing toxin present in the venom and to characterize the toxin's ability to alter the ultrastructure and immunoreactivity of frog motor nerve terminals. Fractionation of S. trachynis venom by sequential anion exchange fast protein-liquid chromatography (FPLC) and size-exclusion FPLC yielded a highly purified preparation of a membrane-perturbing (haemolytic) protein toxin, named trachynilysin. Trachynilysin (2–20 μg/ml) significantly increased spontaneous quantal acetylcholine release from motor endings, as detected by recording miniature endplate potentials from isolated frog cutaneous pectoris neuromuscular preparations. Ultrastructural analysis of nerve terminals in which quantal acetylcholine release was stimulated to exhaustion by 3 h exposure to trachynilysin revealed swelling of nerve terminals and marked depletion of small clear synaptic vesicles. However, trachynilysin did not induce a parallel depletion of large dense-core vesicles. Large dense-core vesicles contained calcitonin gene-related peptide (CGRP), as revealed by colloidal gold immunostaining, and trachynilysin-treated nerve endings exhibited CGRP-like immunofluorescence similar to that of untreated terminals. Our results indicate that the ability of stonefish venom to elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals is a function of trachynilysin, which selectively stimulates the release of small clear synaptic vesicles and impairs the recycling of small clear synaptic vesicles but does not affect the release of large dense-core vesicles. Trachynilysin may be a valuable tool for use in other secretory terminals to discriminate between neurotransmitter and neuropeptide release.     

The Kv7 potassium channel activator flupirtine affects clinical excitability parameters of myelinated axons in isolated rat sural nerve

Flupirtine is an activator of Kv7 (KCNQ/M) potassium channels that has found clinical use as an analgesic with muscle relaxant properties. Kv7 potassium channels are expressed in axonal membranes and pharmacological activation of these channels may restore abnormal nerve excitability. We have examined the effect of flupirtine on the electrical excitability of myelinated axons in isolated segments of rat sural nerve. Axonal excitability was studied in vitro with the same parameters used by clinical neurophysiologists to assess peripheral nerve excitability in situ . Application of flupirtine in low micromolar concentrations resulted in an increase in threshold current, a reduction of refractoriness and an increase in post-spike superexcitability. These effects are consistent with an increase in Kv7 conductance and membrane hyperpolarization. Flupirtine also enhanced and prolonged the late, long-lasting period of axonal subexcitability that follows a short burst of action potentials. This effect was blocked by XE 991 (10 µM), an antagonist of Kv7 channels. In summary, flupirtine affects measures of excitability that are altered in the myelinated axons of patients with peripheral nerve disorders. This indicates that neuropathies with abnormal nerve excitability parameters corresponding to those affected by flupirtine may benefit from activation of axonal Kv7 potassium channels.     

Concurrent chronic motor axonal polyneuropathy and synaptic impairment of neuromuscular junction

Polyneuropathies may exhibits clinical, electrophysiologic signs of neuromuscular junction impairment. Distal motor nerve terminals and neuromuscular junction contain pre or postsynaptically specific targets for circulating autoantibodies, if present in neuropathies. Motor nerve terminal blockade either reversible or permanent is a putative factor of muscle weakness. A 59-year-old patient exhibited oropharyngeal, facial, extremity weakness, fluctuating fatigability, and areflexia. Elecectrophysiologic studies showed purely motor axonal polyneuropathy. Thenar, facial slow rate repetitive stimulation revealed up to 47% decrement of compound muscle action potential size. Single fiber electromyography on voluntary activation confirmed increased jitter and impulse blocking in all muscles examined in one third of the fibers. Repeated testings for antibodies to gangliosides, acetylcholine, muscle tyrosine kinase receptors, voltage-gated calcium channels were negative. Oral pyridostigmine bromide improved bulbar symptoms. Pulse intravenous immunoglobulin, oral steroids, and azathioprine had steady benefit. Impairment of neuromuscular transmission if occurring in chronic axonal neuropathies highlights mechanisms and significance of neuromuscular chronic "synaptopathies."     

Neuromuscular transmission in the murine mutants "motor end-plate disease" and "jolting"

Mice with the inherited disorder "motor end-plate disease" suffered from a progressive neuromuscular weakness and muscular wasting. The weakness resulted from a failure of evoked transmitter release from the motor nerve terminals. The failure in transmission was all-or-nothing in nature. The numbers of muscle fibres in skeletal muscle and myelinated axons in several major nerve trunks were no different from normal. The loss in muscle bulk was caused by the neuromuscular defect and not from a loss of motor units or muscle fibres. The inherited murine disorder "jolting" was allelic with "motor end-plate disease". Affected "jolting" mice suffered no detectable morphological abnormality in skeletal muscle or peripheral nerve. The physiological properties of skeletal muscle and the characteristics of neuromuscular transmission were indistinguishable from normal.     

Sodium channels accumulate at the tips of injured axons

The axolemmal distribution of voltage-gated sodium channels largely determines the regions of axonal electrical excitability. Using a wellcharacterized anti–sodium channel antibody, we examined peripheral nerve fibers focally injured by exposure to the neurotoxic agent, potassium tellurite (K₂TeO₃). Immunocytochemical and radioimmunoassay data showed a focal accumulation of sodium channels within the tips of injured axons. The major increase in sodium channel concentration occurred between 7 and 11 days after toxin exposure; however, immunocytochemically, excess sodium channels persisted in several axonal endings for a much longer time. The accumulation of sodium channels at injured axonal tips may be responsible, in part, for ectopic axonal excitability and the resulting abnormal sensory phenomena (especially pain and paresthesias) which frequently complicate peripheral nerve injury in humans. © 1994 John Wiley & Sons, Inc.     

Ion channels in axon and Schwann cell membranes at paranodes of mammalian myelinated fibers studied with patch clamp.

While recent studies have established the presence of voltage-gated ion channels on Schwann cells in culture and on freshly isolated fibers from mature mammals, an important issue not yet explored is whether Schwann cell channels are regionally specialized. In the nodal region, the intimate association between the Schwann cell and its axon suggests that this is a likely site for functional specialization. Here, we examine whether there is a localized expression of channels in the Schwann cell paranodal regions, in a manner similar to that already shown for the nodal axon. Cell-attached and outside-out patch-clamp recordings were made from paranodal regions of rat myelinated sciatic nerve fibers where the myelin on both sides of the node was retracted by enzymatic treatment. Even though no myelin was visible on the surface of the retracted paranode, significant portions of this surface were found to stain positively with a marker (anti-galactocerebroside) for Schwann cell membranes, suggesting that part of the axon still was covered by glial membranes. Using Lucifer yellow in the recording pipettes, we observed that the dye diffused into either axons or Schwann cells when the membrane under the tip was ruptured. Using this as a criterion to identify membranes obtained from retracted paranodes, we found delayed and inwardly rectifying potassium channels on both axon- and Schwann-derived patches. However, sodium channels were detected only in axon patches. This is the first report that voltage-gated glial channels are present in immediate vicinity to axons of the PNS. This finding, coupled with earlier reports that functional channels are absent in soma of mature myelinating Schwann cells, suggests that ion channels in these cells are regionally specialized for functional interaction with axons.     

Nitric oxide synthase in rat neuromuscular junctions and in nerve terminals of torpedo electric organ: Its role as regulator of acetylcholine release

The distribution of nitric oxide synthase on peripheral motor system was studied using a specific antibody against the neuronal isoform of nitric oxide synthase (nNOS). The immunoreactivity for nNOS was detected on the sarcolemmal surface of muscle cells, in intramuscular axons and in neuromuscular synapses. At the neuromuscular junctions, ultrastructural immunolabeling demonstrated that nNOS immunoreactivity was localized mainly into the presynaptic nerve terminals as well as adjacent postsynaptic muscle membrane. Similar immunostaining pattern was present in frog muscles and Torpedo electric organs. After chronic muscle denervation, nNOS immunoreactity at endplate level decreased during the first week but it was upregulated after 30 days of denervation. In denervated endplates, nNOS immunoreactivity was localized in the terminal Schwann cells covering the degenerated neuromuscular junctions whereas nNOS was not detected in Schwann cells under normal conditions. In Torpedo synaptosomes, acetylcholine (ACh) release elicited by potassium depolarization was inhibited by NO donors such as sodium nitroprusside. In contrast, application of inhibitors of NOS activity, aminoguanidine (AMG) and Nω-Nitro-L-arginine methyl esther (L-NAME) increased acetylcholine release. These results indicate that nNOS is present at the motor nerve terminals in a variety of vertebrates and that it may be involved in the physiological modulation of ACh release and in the regulation of muscle response to nerve injury.     

Distribution and possible abnormality in antigenic composition of sodium channels in peripheral axons of dystrophic mice

In some dystrophic mice (Bar-Harbour 129 dy/dy), axons of the sciatic nerve are a-myelinated but are capable of carrying action potentials. In this study, we showed by immunofluorescence that such excitability is supported by the presence of voltage-gated sodium channels along the a-myelinated axon. In addition, the number of sodium channels measured by radioimmunoassay in sciatic nerves of these dystrophic mice is significantly higher. Furthermore, the composition of sodium channel epitopes is abnormal. This suggested a link between the disease and the biogenesis of the sodium channels.