S. Thesleff

Effects of botulinum toxin on neuromuscular transmission in the rat.

1. Botulinum toxin (BoTx) type A partially blocks spontaneous transmitter release from nerve terminals in the rat. Minature end‐plate potentials (m.e.p.p.s) are present at all end‐plates, initially with a low frequency but increasing with time after posoning. Their amplitude distribution is at first skew with a predominace of very small m.e.p.p.s but, after a few days, larger than normal m.e.p.p.s appear. 2. Tetanic nerve stimulation, Black Widow Spider Venom, the Caionophore A 23187 or mechanical damage to nerve terminals increases the frequency of m.e.p.p.s and alters the amplitude distribution of m.e.p.p.s towards a normal Gaussian one; the m.e.p.p. size approaches that seen at normal end‐plates. This was seen at any time after poisoning. 3. Nerve stimulation gives rise to end‐plate potentials (e.p.p.s) of low amplitude and high failure rate. Statistical analysis indicates that evoked release is quantal in nature and follows Poisson statistics, quantum size being initially very small, but after a few days approaching normal size. Short‐term tetanic nerve stimulation reversibly increases the quantum content of e.p.p.s and during early stages of paralysis long‐term (2 hr) stimulation causes an apparently permanent increase in quantum size. 4. Raising the extracellular Ca concentration from 2 to 16 mM increases the frequency of m.e.p.p.s in normal muscle but not in BoTx poisoned ones. K‐free medium or ouabain, which are believed to raise the intracellular Ca concentration in nerve terminals, similarly increases m.e.p.p. frequency in normal but not in poisoned muscles. When the Ca‐ionophore A 23187 is used together with high extracellular Ca (greater than 4 mM) massive release of transmitter occurs from poisoned terminals. 5. The extracellular Ca concentration which causes a certain level of transmitter release in reponse to nerve impulses is considerably higher at BoTx poisoned end‐plates than at normal ones. The slope value for Ca dependence of transmitter release is about 1‐5 compared with about 3 at normal end‐plates. 6. Tetraethylammonium (TEA) greatly increases the amount of transmitter released by nerve impulses and restores neuromuscular transmission during all stages of poisoning, although it has not effect on spontaneous transmitter release. In the presence of TEA the power relation between Ca concentration and quantum content at the BoTx poisoned end‐plate is similar to that seen at normal end‐plates. 7. It is suggested that in BoTx poisoning the mechanism for transmitter release has a reduced sensitivity to Ca, and the level for activation by intracellular Ca is elevated. Once the intracellular concentration of Ca is raised to this level, by tetanic nerve stimulation, mechanical injury to nerve terminals, the Ca‐ionophore or the prolongation of the nerve action potential with TEA, augmented transmitter release occurs, similar to that which occurs in normal nerve terminals at a lower level of Ca.

Antagonism of the paralysis produced by botulinum toxin in the rat ☆: The effects of tetraethylammonium, guanidine and 4-aminopyridine

The injection of botulinum toxin type A into the hind-leg of adult rats causes complete paralysis of the leg lasting for several weeks. In the extensor digitorum longus (EDL) muscle transmitter release is reduced to a level of less than 1% of normal. Tetraethylammonium (TEA) and guanidine in concentrations of about 3 mM restore, in EDL muslces in vitro, neuromuscular transmission to about the normal level, provided that the external calcium concentration is 4 mM or higher. 4-Aminopyridine (4-AP) has similar restorative effect but is about 20-30 times more potent. Unlike TEA and guanidine, 4-AP is effective when the ambient calcium concentration is 2 mM; this drug is therefore also active in vivo. The intravenous injection of 4-AP (5 mg/kg body weight) restores neuromuscular transmission from complete paralysis by botulinum toxin to a normal level as shown by the recording of almost normal twitch and tetanic tensions in the EDL muscle. In rats paralysed by a lethal dose of botulinum toxin, the intraperitoneal administration of 4-AP restores general motor activity, the effect lasting 1-2 hours. A study of the effects of these drugs on spontaneous and evoked transmitter release suggests that all three compounds increase the level of free calcium inside the nerve terminals. In botulinum poisoning the transmitter release mechanism appears to be intact, but a reduced sensitivity to calcium has been shown (Cull-Candy et al. 1976), and this could explain why the drugs restore evoked transmitter release in botulinum poisoning.

Potency of 3,4-Diaminopyridine and 4-aminopyridine on mammalian neuromuscular transmission and the effect of pH changes

3,4-Diaminopyridine (3,4-DAP) like 4-aminopyridine (4-AP) greatly increased stimulus-evoked transmitter release, recorded as end-plate potentials, in single fibers of the isolated mouse nerve-hemidiaphragm preparation. 3,4-DAP was 6–7 times more potent in this respect than 4-AP. 3,4-DAP was able to restore, from complete paralysis, neuromuscular transmission in isolated botulinum toxin (type A)-poisoned rat muscles and in this respect also was 6–7 times more potent than 4-AP. Changing the pH of the external solution from 7.4 to 9.0 had no significant effect on nerve-evoked transmitter release. However, both 4-AP and 3,4-DAP, which have pKa values of about 9, were about 3 times more potent in increasing transmitter release at pH 9.0 than at pH 7.4. This was interpreted to mean that the two molecules are more effective in their unionized form and therefore possibly exert their effect on transmitter release at an intracellular site in the nerve terminal. The slope values for the calcium dependence of nerve-evoked transmitter release were similar at pH 7.4 and 9.0 and were not altered by 3,4-DAP or 4-AP. Like 4-AP, 3,4-DAP converted the end-plate potential produced by electronic depolarization of tetrodotoxin-blocked frog nerve terminals into an all-or-none response, implying that the drugs enhance transmitter release by greatly increasing the voltage-dependent calcium conductance of the nerve terminal membrane.

The effects of taipoxin and notexin on the function and fine structure of the murine neuromuscular junction

The isolated neurotoxins taipoxin and notexin from the venoms of the Elapidae, Oxyuranus scutellatus and Notechis scutatus scutatus respectively cause a neuromuscular block when administered to the mouse in vivo or to the phrenic nerve-hemidiaphragm preparation in vitro. The block is preceded by a latency period during which the toxins bind irreversibly to the nerve. The period is shortened by nerve activity. The frequency of the miniature end-plate potentials is gradually reduced, almost to zero, and their amplitude distribution is altered; small and very large miniature endplate potentials appearing. Ultrastructurally the endplates are altered in the presynaptic portion but not in the postsynaptic part. In an early stage of poisoning the axolemma has an increased number of omega-shaped indentations similar in size to synaptic vesicles. At a later stage, when the animals die of respiratory paralysis, the axolemmal indentations are more numerous and the synaptic vesicles greatly reduced in number, the remaining vesicles having a variable and frequently larger than normal size. When impulse activity in the phrenic nerve is stopped by cutting the nerve before the administration of toxin there is no reduction in the number of synaptic vesicles, only the appearance of an increased number of axolemmal indentations. It is suggested that taipoxin and notexin irreversibly interfere with the formation of synaptic vesicles by arresting vesicle membrane recycling at the level of the axolemma. When the pre-existing store of vesicles is depleted, by nerve activity, a neuromuscular block results.

Studies on the trophic influence of nerve on skeletal muscle

Colchicine is a drug known to block axoplasmic transport in myelinated nerves. When injected under the perineurium of the sciatic nerve of the rat, the drug induced denervation-like changes in the extensor digitorum longus muscle. These changes, consisting of the appearance of extra-junctional cholinergic sensitivity and of action potentials resistant to the blocking effect of tetrodotoxin, developed despite the fact that nerve impulse conduction, transmitter release and neuromuscular transmission were intact. The results indicate that trophic substances, necessary for the prevention of denervation changes, are transported by axoplasmic flow. β-Bungarotoxin, a purified neurotoxin from Bungarus multicinctus acts selectively on motor nerve terminals blocking the release of acetylcholine from the nerve. S.c. injection of the toxin into the hind leg of rats produced paralysis and complete blockade of spontaneous transmitter release in the extensor digitorum longus muscle. The toxin also induced the aforementioned denervation-like changes in the muscle membrane. It is suggested that β-bungarotoxin in addition to blocking transmitter release blocks the release of trophic substances from the nerve or alternatively that neuromuscular transmission and resulting muscle activity constitute trophic influences necessary for the prevention of the appearance of denervation changes.

PHYSIOLOGICAL EFFECTS OF DENERVATION OF MUSCLE

Following denervation the physiological properties of mammalian skeletal muscle undergo a number of characteristic changes. Since the effects of denervation on the dynamic properties are treated elsewhere42 in this monograph, the present paper will be limited to the electrophysiological changes. As a further limitation, the effects presented will mainly be those that are observed during the first few clays, that is, within one week after sectioning of the nerve. During that early period, atrophy is not yet prominent and, in general, it is not necessary to consider its influence on electrophysiological membrane properties. The most striking effects of denervation are as follows: (1) a fall in the resting membrane potential; (2) the appearance of extrajunctional cholinergic receptors: and (3) quantitative and qualitative changes in the action potential.

4‐AMINOPYRIDINE AND EVOKED TRANSMITTER RELEASE FROM MOTOR NERVE ENDINGS

1 In the presence of tetrodotoxin, electrotonic depolarization of frog motor nerve terminals causes the appearance of stimulus‐graded endplate potentials. When 4‐aminopyridine is added, the graded endplate potential is converted into a triggered all‐or‐none response resulting in giant endplate potentials of about 70 mV amplitude and 50 ms duration. The triggered endplate potentials are abolished in Ca2+ ‐free saline and are blocked by Mn2+ ions. Sr2+ but not Ba2+ can replace Ca2+ in supporting transmitter release. Mg2+ fails, even in concentrations as high as 32 mm, to affect the amplitude and the shape of the endplate potential but abolishes it when the Ca2+ concentration is reduced to 0.2 mm. 2 Despite the large amplitude of the triggered endplate potential in the presence of 4‐aminopyridine and tetrodotoxin, repetitive stimulation up to 10 Hz causes only a small decline in amplitude of successive endplate potentials. However, in the presence of (+)‐tubocurarine or gallamine, repetitive nerve stimulation produces a marked decline in successive endplate potential amplitude. The fall is counteracted when evoked transmitter release is reduced in the presence of 0.2 mm Ca2+. The results suggest that in the presence of 4‐aminopyridine such large amounts of transmitter are released that even during repetitive stimulation (5 to 10 Hz) endplate potentials are of maximal amplitude. 3 4‐Aminopyridine causes a parallel shift to the right of the dose‐response curve to Mg2+ for blockade of nerve impw/se‐evoked transmitter release (in the absence of tetrodotoxin). A similar parallel shift occurs in the presence of tetraethylammonium and guanidine. 4 It is concluded that 4‐aminopyridine increases transmitter release by enhancing the transport efficacy for Ca2+ across the nerve terminal membrane during nerve terminal depolarization.

An electrophysiologic study of the neuromuscular junction in myasthenia gravis

After a study of some of the electrophysiological properties of the neuromuscular junction in isolated intercostal muscles obtained from patients with no known neuromuscular disorder (Elmqvist, Johns & Thesleff, 1960), a similar study was undertaken in patients with myasthenia gravis. The intercostal muscles were obtained by biopsy under regional anaesthesia from six patients with chronic and non-localized myasthenia gravis. Immediately upon removal the muscles were studied by the use of intracellular electrodes. Evidence will be presented for a pre-junctional deficiency of transmitter formation or transmitter release.

A new type of transmitter release at the neuromuscular junction

Examination of spontaneous miniature endplate potentials (MEPPs) in murine skeletal muscle has revealed that in conditions such as botulinum poisoning, during nerve terminal regeneration or in the presence of the drug 4-aminoquinoline, two types of acetylcholine release are responsible for the MEPPs. In addition to the MEPPs which correspond to the quantal component of a nerve impulse-evoked endplate potential a second type of acetylcholine release occurs. The latter type of transmitter release gives rise to MEPPs with a more prolonged time-to-peak and frequently a larger than normal amplitude. It is unaffected by nerve terminal depolarization and transmembrane Ca2+ fluxes. The relationship between MEPP frequency and temperature has a Q10 of about 12 compared to 2-3 for normal MEPPs. In botulinum-poisoned muscles this secretory type of transmitter release dominates, being exclusively present in muscles where nerve stimulation fails to release transmitter. In normal muscle such a release is induced by 4-aminoquinoline which may cause up to 45% of all the spontaneous MEPPs to be of that kind. It is suggested that the described spontaneous secretion of acetylcholine serves in inductory and neurotrophic function.

Studies on the nature of the cholinergic receptor

Abstract The effects of a number of proteolytic enzymes on the acetylcholine sensitivity of the chronically deniervated musue membrane and on the cholinesterase activity of the innervated end-plate were examined. None of the enzymes affected the acetylcholine sensitivity, but all of them inactivated cholinesterase. A sulfhydryl blocker ( p -chloro-mercuribenzoate) and a disulfide bond reducing agent (dithioerythritol) both reduced the acetylcholine sensitivity of the muscle membrane, the effect being only slowly reversed by washing. It was concluded that the ability of p -chloro-mercuribenzoate and of dithioerythritol to inhibit the cholinergic receptor favors the view that a protein is a part of the receptor structure. The failure of proteolytic enzymes to affect the receptor suggests that either the receptor protein is not denaturated by these enzymes or that its hydrolysis does not interfere with the receptor function.