Anoxic block and recovery of axoplasmic transport and electrical excitability of nerve.

Axoplasmic transport of cat sciatic nerves was studied in vitro in a chamber in which maximal alpha action potentials could also be elicited. After initiation of N2 anoxia, electrical responses fell to zero at an average time of 22 min. A shorter time to zero of 11 min was seen during a second period of anoxia. A good recovery of both action potential responses and axoplasmic transport occurs after a period of anoxia lasting 1--1.5 hr. An apparent failure of recovery of axoplasmic transport was seen after 2 hr of anoxia with a good recovery of electrical responses. Axoplasmic transport tended to return toward normal when more time was allowed for recovery after anoxia. An adequate supply of approximately P was shown to be present by measurement of ATP and creatine phosphate levels. The delay in recovery of transport thus signifies a failure of utilization of approximately P by the transport mechanism. Longer periods of anoxia and recovery were limited in vitro and for this reason, ischemic anoxia was produced in vivo. Blood pressure cuffs were placed on the upper thigh of cats and maintained for times of 1--8 hr at pressures of 300-310 mm Hg. Then, recovery times up to 7 days were allowed. It was shown that axoplasmic transport could gradually recovery after an anoxia lasting up to 6-7 hr if sufficient recovery times were allowed. A possible explanation for the delay in the recovery of axoplasmic transport and the disassociation in the earlier recovery of electrical responses as against the recovery of transport was discussed.     

Low temperature slowing and cold-block of fast axoplasmic transport in mammalian nerves in vitro

1) Fast axoplasmic transport in mammalian nerve in vitro was studied using an isotope labeling technique. The rate of outflow in cat sciatic nerve fibers of 410 mm/day in vitro was reduced at temperatures below 38°C with a Q₁₀ of 2.0 in the range 38–18°C and a Q₁₀ of 2.3 at 38–13°C. 2) At a temperature of 11°C a partial failure of transport occurred. At temperatures below 11°C a complete block of fast axoplasmic transport occurred, a phenomenon termed “cold-block.” No transport at all was seen over the temperature range of 10–0°C for times lasting up to 48 hr. 3) Transport was resumed after a period of cold-block lasting up to 22 hr when the nerves were brought back to a temperature of 38°C. Some deleterious effects due to cold-block were seen in the recovery phase as indicated by a reduction in crest amplitude, change in its form, and slowed rate. 4) The ∼P level (combined ATP and creatine phosphate) remained near control level in nerves kept at low or cold-block temperatures for times as long as 64 hr. The reduction in fast axoplasmic transport rate seen at low temperatures for times up to 22 hr was therefore considered due to a decrease in the utilization of ATP, a concept in accord with the “transport filament” model proposed to account for fast axoplasmic transport. 5) The sloping of the front of the crest over the temperature range of 18–13°C suggests an additonal factor at the lower temperatures. A disassembly of microtubules is discussed as a possible explanation of the cold-block phenomenon.     

Relation of ATP and creatine phosphate to fast axoplasmic transport in mammalian nerve

—ATP and creatine phosphate (CP) levels in cat sciatic nerve maintained in vitro were measured. Anoxia produced by N₂ or NaCN or the uncoupling of phosphorylation with DNP reduced the combined levels of ATP + CP to approximately one-half of control levels within 15 min. These agents also blocked fast axoplasmic transport in vitro within 15 min. A block of glycolysis with iodoacetic acid (IAA) reduced the combined levels of ATP + CP to approximately one half of control levels within 1.5–2 h and exposure of nerve in vitro to IAA caused a block of fast axoplasmic transport within the same interval. The correlation of the time at which block of transport occurred with the fall in the level of high-energy phosphates is consistent with the hypothesis that ATP supplies the energy required by the mechanism underlying fast exoplasmic transport.     

Block of axoplasmic transport in vitro by vinca alkaloids

The three potent antimitotic vinca alkaloids: vincristine (VCR), vinblastine (VLB), and vindesine (VDS) were compared for their effect in blocking axoplasmic transport in vitro using a desheathed preparation of the peroneal branch of cat sciatic nerve. A range of vinca alkaloid concentrations from 1–100μM was examined. The relative order of potency in blocking axoplasmic transport was VCR > VLB > VDS at a concentration of 25μM. At the higher concentrations block occurred so rapidly that a statistically significant difference between these agents could not be obtained. The relation of vinca block ot the transport mechanism is discussed.     

A COMPARISON OF AXONALLY TRANSPORTED PROTEINS IN THE RAT SCIATIC NERVE BY IN VITRO AND IN VIVO TECHNIQUES

An in vitro system for studying fast axonal transport in mammalian nerves has been developed. The viability of in vitro nerve preparations was established on the basis of three criteria: electron microscopy, electrical properties, and the activities of two marker enzymes, 5'-nucleotidase and total ATPase. The specific activity of transported proteins was greater using the in vitro procedure, and the level of locally incorporated radioactivity lower, when compared to in vivo transport experiments. Separation of solubilized transported proteins on polyacrylamide gels in the presence of sodium dodecyl sulfate showed that a large number of polypeptides are transported. Using a double label procedure which employed L-[³H]methionine and L-[³⁵S]methionine, proteins transported in vitro and in vivo were compared. No differences in the electrophoretic distribution of transported proteins from the two systems was seen. The major component of transported proteins electrophoresed with an apparent molecular weight of 105,000 ± 24,000. Using the in vitro system, transported proteins were compared to those labelled locally in either Schwann cells or cells of the dorsal root ganglion. Large differences in the labelling patterns were observed in both comparisons. We conclude that in vitro procedures provide a valid means of studying rapid axoplasmic transport. The proteins carried by rapid axoplasmic transport differ from those synthesized in either the Schwann cells of the sciatic nerve or the cells of the dorsal root ganglion.     

Inhibition of Axoplasmic Transport in the Optic System by Kainic Acid

Axoplasmic transport along the optic axons was studied after intraocular injections of kainic acid (KA). Transport of labeled material did not initiate from the eye when KA was injected simultaneously with the protein precursor [3H]proline. When KA was injected after axoplasmic transport of labeled proteins had begun, no additional radioactive material moved out of the retinal ganglion cells. However, the labeled material already present in the optic nerve at the time of KA injection continued to move, and accumulated at the nerve endings. Although KA reduces the incorporation of precursor, this effect of KA on axoplasmic transport appears to be more than a consequence of inhibition on precursor uptake or protein synthesis. Recovery from this KA action began 6 h after exposure to KA and was about 50% recovered by 36 h. The extent of the recovery remained at this level for as long as a week, which suggested a partial recovery of the ganglion cells. A second exposure to KA after the inner plexiform layer had virtually disappeared was as effective as the first exposure in preventing the appearance of transported protein in the optic nerve, suggesting a direct action of KA on the ganglion cells. We interpreted the results to indicate that KA interferes with the initiation phase of axoplasmic transport in ganglion cells and this effect is partially reversible.     

Metabolite concentrations in electrically stimulated lugworms Arenicola marina

• 1.1. The concentrations of lactate, succinate, alanine, aspartate, acetate and propionate in the lugworm Arenicola marina were measured after 1, 2.5 and 12 hr of continuous electrical stimulation under aerobic and anaerobic conditions.
• 2.2. A continuous increase of the concentrations of alanine and acetate, and a decrease of aspartate occurred during the first 2.5 hr of electrical stimulation. A marked rise of succinate and propionate was observed only in experiments lasting longer than 2.5 hr.
• 3.3. No changes were detected in the concentrations of lactate. Under anoxia the metabolites accumulated at significantly higher rates than under aerobic conditions.

     

DEPENDENCE OF FAST AXOPLASMIC TRANSPORT IN NERVE ON OXIDATIVE METABOLISM

—A crest of labelled activity moving down the sciatic nerve at 401 ± 35 mm/day after injection of the L7 dorsal root ganglion of the cat with L-[³H]leucine characterizes fast axoplasmic transport of materials and has been studied with regard to its dependence on oxidative metabolism. Transport of labelled materials in vitro occurred if the nerve was supplied with O₂ or 95 % O₂+ 5 % CO₂. Transport was not dependent upon continuity of the fibres with the ganglionic soma. Asphyxiation (N₂) rapidly blocked fast transport in vitro. Likewise NaCN or dinitrophenol in an O₂ atmosphere both effectively block fast transport within 15 min. Tetrodotoxin and procaine, agents which block excitation of the membrane, had no effect on fast transport. The inference is that oxidative metabolism supplies the energy required by the molecular mechanism underlying fast axoplasmic transport.     

AXOPLASMIC TRANSPORT OF CHOLINE ACETYLTRANSFERASE ACTIVITY IN MICE: EFFECT OF AGE AND NEUROTOMY

—We studied the axoplasmic transport of choline acetyltransferase (CAT) activity in sciatic nerves of normal mice of various ages. For at least 3 days after unilateral ligation of sciatic nerves of 6 and 30-week-old mice, the CAT activity in the ligated nerve increased as a linear function of time and the increase was confined to the 3 mm length of nerve immediately proximal to the ligature. The rate of increase of CAT activity in the ligated nerves of the 30-week-old mice was only 45 ± 6% that of the 6-week-old mice, whereas the CAT activity of non-ligated sciatic nerves of the older mice was 87 ± 6% more than that of the younger mice (n = 18, P < 0·001). The average velocity of axoplasmic transport of CAT activity was five times greater in the younger mice (1·5 ± 0·2 mm/day vs 0·3 ± 0·1 mm/day, n = 6, P < 0·01). Even greater differences were observed between still younger and older animals: the av velocity of axoplasmic transport of 2-week-old mice (3·5 ± 0·2 mm/day) was 17·5 times greater than that of 36-week-old mice (0·2 ± 0·1 mm/day). We also studied the axoplasmic transport of CAT activity in 6-week-old mice after unilateral section of the sciatic nerve. For at least 3 months after the operation, there were no differences between the sectioned nerves and the intact contralateral nerves with respect to the increase in CAT activity immediately proximal to a ligature placed at various times after neurotomy and one day before sacrifice. On the other hand, there was a reduction in the CAT activity of more proximal segments of the sectioned nerves. The reduction of CAT activity was maximal (52 ± 3%) 3 weeks after the operation when the maximum increase (2·5-fold) in the av velocity of axoplasmic transport of CAT activity was recorded (n = 6, P < 0·001). The inclusion of purified (100-fold) mouse brain CAT activity in the assays for the CAT activity of nerve segments demonstrated that the differences in content and rate of transport were not due to the presence of activators or inhibitors of CAT activity. These differences probably reflect physiologic changes in the axoplasmic transport of cholinergic neurons during development and regeneration.     

Mechanisms of antibody formation: I. Early in vivo proliferation of mouse spleen T cells as an initial step in antibody formation

Spleen cultures prepared from mice injected 24 hr earlier with 2 × 10⁶?2 × 10⁸ sRBC and challenged in vitro with sRBC produced 10 times more anti-sRBC IgM PFC than cultures prepared from uninjected mice. The effect was specific for the particular species of foreign RBC injected in vivo. In vitro responses to TNP were also increased in spleen cultures prepared from animals injected 24 or 12 hr earlier with carrier RBC alone, directly implicating carrier-specific T cells in this process. Similar enhancements of PFC formation occurred in cultures prepared from mice which had been injected with sRBC 24 and 48 hr earlier, but which were exposed to lethal irradiation at 1 hr after injection of antigen, if their spleens were shielded extracorporeally during irradiation. This finding indicated that in vivo recruitment of antigen-reactive extrasplenic X-ray-sensitive cells from the circulating lymphocyte pool by the spleen could not account for the observed enhancement.Proliferation in the spleen of antigen-reactive T cells, commencing 12–20 hr after the administration of antigen, was demonstrated by the tritiated thymidine pulse technique. An 8-hr hot-pulse given to spleen cell cultures from normal animals at 20 hr after in vitro challenge with antigen did not affect the rate of generation of IgM-producing cells; however, administration of a similar pulse to cultures which were initiated at 12 or at 20 hr after the in vivo injection of sRBC eliminated the enhanced generation of PFC and delayed the in vitro response to sRBC by 24 hr.Spleen cell cultures were prepared from mice which had been injected in vivo with sRBC at 12, 20, and 70 hr earlier, and 8- to 10-hr hot pulses were given immediately after initiation of the cultures. The cultures were then challenged with sRBC-TNP; antibody responses to TNP were greatly reduced in hot-pulsed cultures prepared from mice injected in vivo with carrier RBC at 12 or 20 hr prior to initiation of the cultures. In contrast, antibody responses to TNP observed in hot-pulsed cultures prepared from mice which had been injected with carrier RBC at 70 hr prior to initiation of the cultures were generally similar to those of nonpulsed 70 hr control cultures. This result suggests that the onset of T helper cell proliferation begins within 12–20 hr after injection of antigen, but subsides in vivo within 70 hr. By that time, the antigen-reactive T cells have already differentiated to perform their helper function.In spite of the triggering of T-cell proliferation during the first 24 hr after injection of antigen, spleen cell cultures prepared from mice which had been injected 24 hr earlier in vivo with 2 × 10⁸ sRBC produced only minimal numbers of anti-sRBC PFC if no antigen was added to the cultures. The presence of unprocessed antigen thus appears to be a requirement for B-cell proliferation in vitro, even after T-cell division has been triggered. This finding is consistent with earlier suggestions that the function of “helper” T cells may not be limited to passive transport of antigenic determinants to B cells. Evidence is also presented to support the contention that the antigen-reactive T cell involved in this process may have to undergo cell division in order to develop “helper” capacity.     

Regeneration of peripheral nerves and intracerebral transplantation]

Axoplasmic flow is essential to the regeneration of peripheral nerves. We observed a mean of 12 mm/day for the slow axoplasmic flow and a mean of 410mm/day for the fast axoplasmic flow. In the process of regeneration of peripheral nerves, however, slow transport increased to 14.7mm/day and fast transport to 572mm/day on day 7. We reviewed the relevant literature on the axoplasmic flow and described the topics in this report. Some central nerves may show poor regeneration but it has been confirmed that nerve cells grow and survive by intracerebral nerve transplantation, and this technique has been applied to the treatment of Parkinson's disease. Further development can be expected for the regeneration of central nerves through transplantation.     

The effects of blue and red light on Acetabularia mediterranea after long exposure to darkness

After Acetabularia mediterranea cells were kept in darkness for 2–8 weeks, all the cellular processes were arrested and the algae did not grow. In particular, the transcellular electrical potential (V_AB) decreased to almost zero and cytoplasmic streaming was arrested. Upon illumination with continous blue light (BL), the first events were (as with white light (WL)), immediate increase in V_AB and movements of water, followed, after a lag period of 1–3 min, by transient recovery of cytoplasmic streaming which lasted about 16 min. After 10 min (earlier than in WL), the frequency of the spontaneous action potentials increased much more than in WL. Then, after 1.5–4 hr during which V_AB often decreased to zero while the cytoplasmic movements stopped, both activities resumed with diurnal oscillations. BL stimulated (as WL) rRNA synthesis, migration of rRNA from nucleus towards apex and cell growth. Upon illumination with red light (RL), V_AB also increased, but water movements were much less pronounced than in BL. The transient streaming phase was shorter. The spontaneous action potentials increased in frequency much later (several hr) and much less than in BL or WL. V_AB did not decreased at any time and was maintained at particularly high values. Cytoplasmic streaming resumed, but showed very attenuated or no rhythm. rRNA synthesis and migration remained low. Cell growth did not resume during the experiments. By comparing our results with those of other studies relating to growth, morphogenesis and photosynthesis, we suggest that BL and RL could affect all these processes by differentially modifying the cytoplasmic concentrations of ions which may influence the functions of the cytoskeleton.     

The presence of transfer RNA in the axoplasm of the squid giant axon

Previous work has revealed that 4S RNA is the primary species of RNA in the axoplasm from the giant axons of the squid and Myxicola. This study shows that axoplasmic 4S RNA from the squid giant axon has the functional properties of tRNA. Axoplasmic RNA was charged with amino acids by aminoacyl-tRNA synthetases prepared from squid brain. The aminoacylation was prevented by incubating the RNA with RNase prior to running the reaction. The amino acid-RNA complex was labile at pH 9, which is characteristic of the acyl linkage between an amino acid and its tRNA. Aminoacyl-tRNA synthetase activity was also present in the axoplasm, primarily in the soluble fraction.     

Physiological and biochemical changes in frog sciatic nerve during anoxia and recovery

Frog (Rana pipiens) sciatic nerve was incubated, with and without stimulation, in an oil bath. The correlation between changes in the magnitude of the compound action potential (α and β) and changes in metabolites, particularly energy reserves, during anoxia and recovery from anoxia was studied. The time to extinction of the action potential in anoxia was frequency-dependent. The action potential could not be restored, nor its extinction delayed, by washing the nerve in O₂-free Ringer's solution. Therefore, in this system extracellular K⁺ accumulation was not a significant factor in blocking impulse conduction. At the time of complete nerve block resulting from anoxia (90 min at rest), ATP, P-creatine and glucose were 30, 10 and 10 per cent, respectively, of initial levels. Glycogen did not fall below 42 per cent of control levels even after 5 h of anoxia. Changes in the levels of energy reserves during anoxia were used to calculate the metabolic rate of nerves at rest and during stimulation. In one series of experiments, the resting metabolic rate was 0·12 mequiv. of ‘high-energy phosphate’ (～P)/kg/min. Stimulation increased the metabolic rate to 0·22 mequiv. of ～P/kg/min at 30 Hz and to 0·29 mequiv. of ～P/kg/min at 100 Hz. The change in metabolic rate when the nerve passed from the resting to the stimulated state was quite abrupt, an observation suggesting that the slow transition observed with methods monitoring O₂, consumption was largely instrumental. In nerve stimulated to exhaustion in the absence of O₂, neither ATP nor P-creatine had fully recovered within 60 min after O₂, was readmitted, although the action potential reached supranormal levels 15 min after return to O₂. The ratio of lactate: pyruvate, which increased as expected during anoxia, paradoxically increased even further after O₂, was readmitted. The rate of energy utilization during recovery was 0·30 mequiv. of ～P/kg/min. Nerves stimulated at 100–200 Hz in O₂, exhibited no changes in levels of P-creatine, ATP or lactate, an observation implying that the nerve could not be made to use ～P faster than oxidation of glucose could provide it. This meant that the maximal metabolic rate was not limited by the rate of supply of chemical energy. Instead, the limitation may have arisen as a result of a limited rate at which ionic imbalance can result from stimulation or a limited pump capacity of the axonal membrane. Nerves stimulated at 200 Hz in O₂ for 20 min and then transferred to an O₂-free environment without further stimulation exhibited an increase in the rate of energy utilization (nearly two-fold) over the resting rate, a finding that suggested a metabolic (ionic?) debt as a result of activity which could not be met even though the energy supply was adequate. Therefore, restriction of energy expenditure by a limiting pumping rate seemed to be the most likely explanation. The resting metabolic rate of frog sciatic nerve was only one-quarter to one-third of the rate for rat sciatic nerve, when compared at the same temperature (25°C).     

THE ULTRASTRUCTURE OF THE NEUROMUSCULAR JUNCTIONS OF MAMMALIAN RED, WHITE, AND INTERMEDIATE SKELETAL MUSCLE FIBERS

Distinct ultrastructural differences exist at the neuromuscular junctions of red, white, and intermediate fibers of a mammalian twitch skeletal muscle (albino rat diaphragm). The primary criteria for recognizing the three fiber types are differences in fiber diameter, mitochondrial content, and width of the Z line. In the red fiber the neuromuscular relationship presents the least sarcoplasmic and axoplasmic surface at each contact. Points of contact are relatively discrete and separate, and axonal terminals are small and elliptical. The junctional folds are relatively shallow, sparse, and irregular in arrangement. Axoplasmic vesicles are moderate in number, and sarcoplasmic vesicles are sparse. In the white fiber long, flat axonal terminals present considerable axoplasmic surface. Vast sarcoplasmic surface area is created by long, branching, closely spaced junctional folds that may merge with folds at adjacent contacts to occupy a more continuous and widespread area. Axoplasmic and sarcoplasmic vesicles are numerous. Both axoplasmic and sarcoplasmic mitochondria of the white fiber usually contain intramitochondrial granules. The intermediate fiber has large axonal terminals that are associated with the most widely spaced and deepest junctional folds. In all three fiber types, the junctional sarcoplasm is rich in free ribosomes, cisternae of granular endoplasmic reticulum, and randomly distributed microtubules.     

A novel action of collapsin: Collapsin-1 increases antero- and retrograde axoplasmic transport independently of growth cone collapse

Chick collapsin-1, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Collapsin-1 induces growth cone collapse via a pathway which may include CRMP-62 and heterotrimeric G proteins. CRMP-62 protein is related to UNC-33, a nematode neuronal protein required for appropriately directed axonal extension. Mutations in unc-33 affect neural microtubules, the basic cytoskeletal elements for axoplasmic transport. Using computer-assisted video-enhanced differential interference contrast microscopy, we now demonstrate that collapsin-1 potently promotes axoplasmic transport. Collapsin-1 doubles the number of antero- and retrograde-transported organelles but not their velocity. Collapsin-1 decreases the number of stationary organelles, suggesting that the fraction of time during which a particle is moving is increased. Collapsin-1-stimulated transport occurs by a mechanism distinct from that causing growth cone collapse. Pertussis toxin (PTX) but not its B oligomer blocks collapsin-induced growth cone collapse. The holotoxin does not affect collapsin-stimulated axoplasmic transport. Mastoparan and a myelin protein NI-35 induce PTX-sensitive growth cone collapse but do not stimulate axoplasmic transport. These results provide evidence that collapsin has a unique property to activate axonal vesicular transport systems. There are at least two distinct pathways through which collapsin exerts its actions in developing neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 316–328, 1997     

Effect of anoxia and reoxygenation on antioxidant enzyme activities in immortalized brain endothelial cells

Summary The effects of anoxia and reoxygenation on major antioxidant enzyme activities were investigatedin vitro in immortalized rat brain endothelial cells (RBE₄ cells). A sublethal anoxic period of 12 h was assessed for RBE₄ cells using the neutral red uptake test. Anoxia markedly influenced the specific activity of catalase and superoxide dismutase, with no major effect on glutathione peroxidase or glutathione reductase. After 24 h postanoxia, the superoxide dismutase activity modulated by the presence or absence of oxygen returned to control value. Damage and recovery of RBE₄ immortalized rat brain endothelial cells in culture after exposure to free radicals and other oxygen-derived species provides a usefulin vitro model to study anoxia-reoxygenation trauma at the cellular level.     

Neural control of the expression of a Ca2+-activated K+ channel involved in the induction of myotonic-like characteristics

Summary 1. Expression of the apamin-sensitive K⁺ channel (SK⁺) in rat skeletal muscle is neurally regulated. The regulatory effect of the nerve over the expression of some muscle ion channels has been attributed to the electrical activity triggered by the nerve and/or to a trophic effect of some molecules transported from the soma to the axonal endings. 2. SK⁺ channels apparently are involved in myotonic dystrophy (MD), therefore understanding the factors that regulate their expression may ultimately have important clinical relevance. 3. To establish if axoplasmic transport is involved in this process, we used two experimental approaches in adult rats: (a) Both sciatic nerves were severed, leaving a short or a long nerve stump attached to the anterior tibialis (AT). (b) Colchicine or vinblastine (VBL), two axonal transport blockers of different potencies, was applied on one leg to the sciatic nerve. To determine whether electrical activity affects the expression of SK⁺ channels, denervated AT were directly stimulated. The corresponding contralateral muscles were used as controls. 4. With these experimental conditions we measured (a) apamin binding to muscle membranes, (b) muscle contractile characteristics, and (c) electromyographic activity. 5. In the short- and long-nerve stump experiments, 5 days after denervation¹²⁵I-apamin binding to AT membranes was 2.0 times higher in the short-stump side. This difference disappeared at longer times. The delayed expression of SK⁺ channels in the muscle left with a longer nerve stump can be attributed to the extra axoplasm contained in the longer stump, which maintains a normally repressive signal for a longer period of time. Ten to 15 days after application of axonal transport blockers we found that the muscle half-relaxation time increased in the drug-treated side and apamin partially reverted the prolonged relaxation. Myotonic-like discharges specifically blockable by apamin were always present in the drug-treated leg.¹²⁵I-Apamin binding, which is undetectable in a microsomal preparation from hind leg control muscles, was increased in the drug-treated preparations. Apamin binding to denervated and stimulated AT muscles was lower than in the contralateral unstimulated muscles [3.3±1.0 vs 6.8±0.8 (n=4) fmol/mg protein]. 6. Our results demonstrate that electrical activity and axoplasmic transport are involved in the control of expression of SK⁺ in rat skeletal muscle. However, the increased expression of this channel induces myotonic-like characteristics that are reversed by apamin. This myotonic activity could be a model for MD.