Nerve growth in botulinum toxin poisoned muscles

The rate of growth of motor axon sprouts in muscles fully paralysed with botulinum toxin was studied using the zinc iodide—osmium tetroxide stain. In the mouse soleus and peroneus muscles, the proportions of endplates with nerve terminal sprouts rose to almost 100% in two to three weeks. In both muscles, the initial rate of terminal sprout growth was rather slow—about 3 μm per day. Two to three weeks after the injection the rate had risen to about 15 μm per day. Substantial numbers of polyneuronally innervated muscle fibres could be demonstrated in the soleus, but not the peroneus, by twelve days after the injection. The soleus remained polyneuronally innervated over a year after the injection.The time course of the tension recovery by the blocked soleus nerve was compared with that of the tension development by a fibular nerve implanted into a fully paralysed or completely denervated soleus muscle. The rate of tension recovery by the fibular nerve was the same whether the soleus was denervated or simply blocked. After an initial delay, the recovery of tension by the paralysed soleus nerve followed a course very similar to that of the implanted nerves.The implanted nerve did not affect the initial terminal sprouting of the paralysed soleus endplates. However, after three weeks, sprouts from soleus nerve endplates on many fibular innervated muscle fibres had disappeared. The implanted nerve did not affect the sprouting of soleus endplates on the muscle fibres which it did not innervate. The recovery of tension by the soleus nerves to muscles innervated by implanted nerves was reduced.It is concluded (1) that recovery from paralysis after botulinum toxin poisoning is slow because formation of new extrajunctional synapses is slow; (2) that motor nerve terminals are more readily induced to sprout by changes occurring on their own muscle fibres than by inactivity induced changes in muscle fibres elsewhere in the same muscle; (3) that paralysis of the mouse soleus renders the muscle as receptive to innervation extrajunctionally as does denervation.     

Chronic effects of botulinum toxin on neuromuscular transmission and sensitivity to acetylcholine in slow and fast skeletal muscle of the mouse

1. A sublethal dose of botulinum toxin (type A) was injected into the muscles of one hind limb of the mouse causing local paralysis.

2. Neuromuscular transmission and muscle sensitivity to acetylcholine (ACh) were studied in vitro in soleus and extensor digitorium longus (EDL) from 6 hr to 4 months after the injection of toxin.

3. Both soleus and EDL failed to respond to nerve stimulation within 6 hr of the injection of toxin.

4. In muscle fibres in which neuromuscular transmission was blocked, subthreshold end-plate potentials (e.p.p.s) were recorded. The amplitude of the e.p.p.s increased during recovery from the effects of the toxin and both muscles contracted in response to nerve stimulation after 2-3 weeks.

5. For about 2 months muscles fatigued more rapidly than normal during repetitive nerve stimulation because of the low quantal content of e.p.p.s.

6. Supersensitivity to ACh developed in 3-5 days and persisted after the return of neuromuscular transmission. Muscle sensitivity to ACh became normal when the rate of fatigue during nerve stimulation was normal.

     

The effects of tetanus toxin on neuromuscular transmission and on the morphology of motor end-plates in slow and fast skeletal muscle of the mouse

1. A sublethal dose of tetanus toxin was injected into the muscles of one hind leg of the mouse and caused local tetanus which persisted for 4 weeks.2. Neuromuscular transmission was studied in vitro in nerve-muscle preparations of soleus, a slow muscle, and extensor digitorum longus (EDL), a fast muscle, from 1 day to 6 months after the injection of toxin.3. Soleus failed to respond to nerve stimulation, became supersensitive to acetylcholine and showed spontaneous fibrillations for several weeks before returning to normal. EDL did not show these changes. A higher dose of tetanus toxin, lethal within 24 hr, caused paralysis of EDL as well as soleus.4. In muscle fibres in which neuromuscular transmission was blocked spontaneous miniature end-plate potentials (m.e.p.p.s) were recorded. The frequency of m.e.p.p.s was increased by repetitive nerve stimulation but not by raising the external potassium concentration.5. The amplitude of spontaneous m.e.p.p.s showed a skew distribution because of a disproportionate number of potentials of less than 0.2 mV.6. Raising the external calcium concentration did not restore neuromuscular transmission.7. Histological examination of soleus showed atrophy of muscle fibres with normal preterminal axons. There was sprouting from motor nerve terminals and subsequently new motor end-plates were formed. These changes were not found in EDL.8. The results indicate that, in the mouse, tetanus toxin causes a presynaptic block of neuromuscular transmission and ;functional denervation' of muscle. Slow muscle is more sensitive to the effects of the toxin than fast.     

Dissociation between nerve-muscle transmission and nerve trophic effects on rat diaphragm using type D botulinum toxin.

Small doses of botulinum toxin can produce partial blockage of transmitter release at the nerve--muscle junction. 2. Subthreshold e.p.p.s, 3--10 days after poisoning, show a distribution of amplitudes that is fitted by Poisson statistics. Successive e.p.p.s. in a short train show a marked facilitation. 3. Two weeks or more after poisoning with a dose of toxin that paralyses the whole muscle, when nerve--muscle transmission is in course of recovery, subthreshold e.p.p.s have an amplitude distribution that is fitted by binomial statistics. This property of transmission is similar to those described in newly formed nerve--muscle junctions, during embryogenesis or regeneration. 4. Muscle fibres with subthreshold transmission in the 5--10 day group of muscles were all supersensitive to ACh, as were a number of fibres in which nerve stimulation still produced an action potential. 5. Two weeks or more after poisoning, muscle fibres with subthreshold transmission had lost their extrajunctional ACh-sensitivity, as had many fibres with m.e.p.p.s of roughly normal frequency but no response to nerve stimulation. 6. In diaphragm muscles poisoned with botulinum toxin between 1 and 4 days previously, the rate of fast axonal transport of radioactively labelled proteins down the phrenic nerve is not greatly affected, but the amount of materials carried is reduced to about one quarter of normal. These labelled proteins accumulate in the intramuscular portion of the phrenic nerve, in or near the nerve terminals, to a much greater extent than in controls, showing that the normal release of some of these materials has been prevented by the toxin. 7. It is concluded that the blockage of the trophic effects of nerves by botulinum toxin is due to a blockage of release of trophic factors other than ACh. 8. The muscle nerve cannot maintain a muscle in its normal state simply by activation of contraction, and a regenerating nerve terminal can restore a muscle towards its normal state before it can release enough ACh to produce muscle contraction.     

Physiological characteristics of re-innervation of skeletal muscle in the mouse

1. Re-innervation of soleus was studied in the mouse after either crushing the sciatic nerve or re-implanting the nerve to soleus outside the original end-plate region.2. During the early stages of re-innervation subthreshold end-plate potentials (e.p.p.s) were recorded in muscle fibres in response to nerve stimulation. Later the e.p.p.s became large enough to evoke action potentials in muscle fibres.3. The rate of recovery of the release of acetylcholine (ACh) as estimated from the quantal content of e.p.p.s was faster when nerves re-innervated the old end-plate region after nerve crush than after re-implantation so that new neuromuscular junctions were formed.4. During re-innervation soleus fatigued more rapidly than normal during repetitive nerve stimulation. The fatigue was due to failure of neuromuscular transmission associated with an impaired release of ACh.5. During re-innervation soleus was supersensitive to ACh until nerve stimulation was capable of evoking action potentials in muscle fibres.     

Motor unit numbers,motor unit sizes and innervation of single muscle fibres in hyperinnervated adult mouse soleus muscle

The sural and the lateral plantar nerves were implanted simultaneously into the denervated soleus muscle of adult mice. Each of these nerves contained approximately the normal number of soleus motor axons. This procedure therefore allowed a study of how an initial excessive number of motor axons provided by two different, foreign nerves and terminating into the soleus muscle affected the final pattern of muscle innervation. In muscles examined two months or more after the implantation of the foreign nerves all muscle fibres were innervated. The fraction of the muscle innervated by either nerve varied widely from one preparation to another. However, all the motor axons which were implanted into the muscle appeared to make permanent synapses. Moreover, the distribution of motor unit sizes of each foreign nerve relative to the total number of muscle fibres innervated by that nerve was similar to the distribution of motor unit sizes in muscles cross-innervated by that nerve alone, although the absolute motor unit sizes were reduced. Estimated by intracellular recording, 20–30% of the muscle fibres were polyneuronally innervated. A similar fraction of teased muscle fibres stained for acetylcholinesterase had more than one endplate.     

The influence of secretin on the secretion of pepsin in response to acid stimulants in the anaesthetized cat.

1. The problem of selectivity during reinnervation of skeletal muscle fibres was investigated in the rat using the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus muscles and their nerves. 2. After an operation on these nerves permitting them to compete for reinnervation of one or the other muscle (hereafter called Y-union), virtually the total isometric tetanic tension of EDL muscle could be elicited by stimulating the EDL nerve, while stimulating the soleus nerve yielded little or no tension. In the case of the soleus muscle, stimulation of either nerve elicited about half of the total isometric tetanic tension. 3. During the course of reinnervation of these muscles in non-competitive situations, the time course of increase in the ratio of tension elicited by nerve stimulation to that by direct stimulation was slower in the case of soleus nerve reinnervating EDL muscle, compared with cross-reinnervation in the reverse direction or reinnervation of each muscle by its own nerve. 4. Crushing the common peroneal nerve 12 days after a Y-union in an attempt to retard the EDL nerve did not favour reinnervation of the EDL by soleus nerve, but crushing the nerve again or just once at 1 month after the original operation produced substantial partial reinnervation of the EDL by the soleus nerve. 5. It is concluded that soleus nerve fibres form functioning neuromuscular synapses on EDL muscle fibres only with difficulty. The pattern of reinnervation reveals characteristic differences between fast-twitch and slow-twitch muscles on the one hand and between their respective nerves on the other.     

Formation of ectopic neuromuscular junctions in adult rats

The formation of ectopic neuromuscular junctions between a transplanted foreign nerve (the superficial fibular nerve) and the denervated soleus muscle was examined in adult rats. Formation of new junctions was induced by denervating the soleus muscle by cutting the tibial nerve. Junctional transmission began 3 days after denervation when denervation was done 3 weeks after transplantation of the foreign nerve, and progressively later when denervation was done 8, 16 and 24 weeks after transplantation. The rate at which the transmission, once started, acquired mature characteristics was approximately the same in each case. Initially, spontaneous m. e.p. p.s were infrequent, long lasting with a skewed amplitude distribution. The e. p.p. s evoked by stimulation of the foreign nerve were then commonly below threshold for eliciting action potentials and were occasionally no larger than the size of the spontaneous m. e.p. p.s. M.e. p.p. characteristics became normal in the 2nd week after transmission had started. Fully effective evoked transmission with every innervated fibre responding with overshooting action potential occurred 1–3 months after onset of transmission.     

Observations on the action of type A botulinum toxin on frog neuromuscular junctions

1. Progressive block of neuromuscular transmission in frog sartorius and gastrocnemius preparations by haemagglutinin-free crystalline Type A botulinum toxin (BTX) was investigated by in vitro application and by injection of the toxin into living animals.2. Neuromuscular block was characterized by (a) decline in amplitude of evoked twitch contractions, (b) decline in amplitudes of end-plate potentials (e.p.p.s) and (c) changes in statistical characteristics of spontaneous miniature end-plate potentials (m.e.p.p.s).3. Progress of the block was enhanced by nerve stimulation.4. A decrease in frequency to less than 0.1/sec and decreased average amplitudes of m.e.p.p.s preceded observable impairment of neuromuscular transmission. These changes occurred as early as 3 hr after injection of the toxin into dorsal lymph sacs.5. The amplitude distributions of m.e.p.p.s changed from a normal distribution to one that showed an increased skewness toward smaller amplitudes as the block progressed. These changes were first detectable as early as 75 min following addition of the toxin to the bath.6. At later stages of toxin action, e.p.p.s began to decrease in amplitude and eventually failed altogether. E.p.p.s showed a normal quantal variation at very early stages in the block in Mg(2+)-treated preparations. At later stages of the block, it was not possible to test the quantal make-up of the e.p.p.7. At all stages before complete failure it was possible to obtain normal or greater than normal degrees of synaptic facilitation with paired stimuli to the nerve. This aspect of the coupling of nerve terminal depolarization to transmitter release appears to be relatively unaffected by BTX.8. Electrical depolarization of nerve terminals in partially blocked preparations evoked a maintained discharge of m.e.p.p.s with an amplitude distribution similar to that of the spontaneous m.e.p.p.s; hyperpolarization of the terminals evokes a distinctly larger class of m.e.p.p.s. In fully blocked preparations, depolarization of the terminals does not evoke transmitter release whereas hyperpolarization continues to yield the larger class of m.e.p.p.s.9. It is proposed that the neuromuscular block caused by BTX is due to impairment of a process by which vesicles become charged with transmitter before release.     

The neuropathology of DFP at cat soleus neuromuscular junction

Summary The fine structure of the cat soleus neuromuscular junction was studied following a single intra-arterial injection of di-isopropylfluorophosphate (DFP) into the right femoral artery. DFP induced separate subacute and delayed morphologic changes in soleus non-myelinated motor nerve terminals. Three days after DFP administration motor nerve terminals were reduced in number. Subacute DFP damage was also noted in the subneural apparatus and in the immediate subjacent muscle. Both pre- and post-junctional subacute changes were resolved two weeks post-DFP. One week following this initial regeneration, soleus motor nerve terminals underwent a delayed transient degeneration, followed by reinnervation of damaged endplates 6–8 weeks following DFP.Quantitative analysis of methylene blue-stained intramuscular nerves indicated that both subacutely and chronically denervated soleus muscle fibres were reinnervated by regeneration of the original motor axon. Reinnervation by means of collateral sprouting was insignificant. This mechanism of reinnervation and the rapidity with which it occurred suggests that both subacute and delayed soleus motor nerve damage is initiated from local actions of DFP on the non-myelinated terminal. The subacute reaction probably results from a direct cytotoxic action of DFP at pre- and post-junctional sites. The delayed nerve terminal degeneration may also stem from an acute effect not immediately detrimental to nerve function.     

The effect of tetanus toxin at the neuromuscular junction in the goldfish

1. The effect of tetanus toxin on neuromuscular transmission in the abductor superficialis muscle of the goldfish fin has been investigated.2. The abductor superficialis muscle is multiply innervated and junction potentials (j.p.s) and miniature junction potentials (min.j.p.s) can be recorded with an intracellular micro-electrode at any point of impalement. Intracellular recordings have been made from muscles in which neuromuscular transmission has been blocked, either completely, or partially, by I.M. injection of tetanus toxin. In addition, the tension response of both acutely and chronically toxin-blocked muscles to carbachol (1 x 10(-4)M) has been investigated.3. As the neuromuscular block proceeds, the frequency of min.j.p.s falls, and some time after the muscle has stopped responding to nerve stimulation the min.j.p.s disappear.4. In muscles in which the block has not yet proceeded to completion, it has been found that the reduction in the frequency of the min.j.p.s is unaccompanied by any change in their range of amplitudes.5. The min.j.p. frequency can be greatly increased in such incompletely blocked preparations by repetitive stimulation of the nerve (at, for example, 100/sec for 10 sec). The min.j.p.s obtained are indistinguishable from those seen in the absence of stimulation. Additionally, min.j.p.s can be evoked by similar repetitive stimulation in muscles which are completely blocked, and in which no min.j.p.s are seen without stimulation, so long as the block has only been complete for a short time.6. The tension response to carbachol of muscles which have been paralysed by tetanus toxin for only a few days is identical with that of normal muscles. In contrast, chronically toxin-paralysed muscles contract more rapidly, usually more vigorously, and relax more rapidly than normal muscles.7. It is concluded that tetanus toxin prevents both the nerve-stimulated and spontaneous release of acetycholine from the presynaptic terminals in the abductor superficialis muscle of the goldfish fin.     

Cross-innervation of fast and slow-twitch muscles by motor axons of the sural nerve in the mouse

The extensor digitorum longus (EDL) or soleus muscles of adult mice were cross-innervated by the sural nerve (SN) and deprived of their original innervation. The number and sizes of motor units and the location of endplates in these muscles were studied 1.5 to 16 months later. In the EDL muscle, the SN cross-innervated the original endplates. Very few ectopic endplates were seen, even when the nerve was implanted well outside of the original endplate area. Only 3% of the fibres were polyneuronally innervated. In the soleus muscle, however, the SN formed large numbers of ectopic endplates whether the nerve was implanted in the original endplate zone or outside of it. In addition, 20% of the muscle fibres were polyneuronally innervated. The SN cross-innervated both EDL and soleus muscles completely. There was no preference for a particular group of the SN motoneurones since all the cross-innervated muscles were innervated by all SN motor axons and the motor unit sizes of the SN were similar in the cross-innervated EDL and soleus muscles. It is concluded that intrinsic properties of a muscle determine the ability to form ectopic synapses. The distribution of the motor unit sizes is determined by the particular pool of motoneurones which innervates the muscle.     

Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation.

1. The events taking place during the elimination of polyneuronal innervation in the soleus muscle of new-born rats have been studied using a combination of electrophysiological and anatomical techniques. 2. Each immature muscle fibre is supplied by two or more motor axons which converge on to a single end-plate. There was no sign of electrical coupling between muscle fibres receiving multiple synaptic inputs. By the end of the second week after birth virtually all muscle fibres are innervated by only a single motor axon. 3. The average tension produced by individual motor units, measured in terms of the percentage of the total muscle twitch tension, declined dramatically during the first 2 weeks after birth. During this period there was no significant change in the number of motor neurones innervating the soleus muscle. Thus, the disappearance of polyneuronal innervation reflects a decrease in the number of peripheral synapses made by each motor neurone. 4. The decline in motor unit size was delayed, but not ultimately prevented, by the early surgical removal of all but a few motor axons to the soleus muscle. This procedure also caused a delay in the removal of polyneuronal innervation involving the remaining motor units. 5. Following a crush of the soleus nerve in neonatal animals, regenerating axons usually returned to the original end-plates. Polyneuronal innervation was extensive at early stages of re-innervation and it disappeared during the second week after birth just as in normal muscles. 6. Cross-innervation of neonatal muscles by an implanted foreign nerve caused a rapid disappearance of cholinesterase at denervated original end-plates and in most fibres prevented re-innervation by the original nerve. In the small proportion of fibres that did become innervated through both the foreign and original nerves the end-plates were more than 1 mm apart, and both foreign and original nerve end-plates could persist indefinitely. 7. Many cross-innervated fibres received multiple inputs through the foreign nerve. Some foreign end-plates were separated by distances ranging up to 1 mm. Polyneuronal innervation through the foreign nerve was completely eliminated during maturation but over a slightly longer period than in normal muscles. Apparently the elimination process can act over a distance up to but not much more than 1 mm. 8. These observations suggest that there are several factors influencing the elimination of redundant inputs in immature muscles. Individual motor neurones appear to have an inherent tendency to withdraw the majority of their original complement of peripheral terminals. The determination of which particular synapses are to survive, however, seems to be made in the periphery by a selection among all the synapses that innervate a limited region of each muscle fibre. There may be a competitive interaction among synapses in which those belonging to smaller motor units are less likely to be eliminated, thereby leading to a relatively uniform size of the motor units in the soleus.     

Differential expression of tenascin after denervation, damage or paralysis of mouse soleus muscle

Summary The expression of the extracellular matrix molecule tenascin was studied by immunocytochemistry and Western blotting in soleus muscles of adult mice after nerve damage (denervation), muscle injury (induced by enforced running or freezing) and functional block of synaptic transmission (botulinum toxin). Enhanced expression of tenascin in the extracellular spaces around focally damaged muscle fibres was found already 10 h after onset of running on a motor-driven treadmill which causes muscle injury in soleus muscle. Tenascin expression reached a peak at 2–3 days post-exercise, after which it declined gradually and became undetectable by two weeks after injury. Similarly, cryo-damage of soleus musclesin situ led to upregulation of tenascin. Chronic muscle denervation after sciatic nerve transection caused a persistent (studied up to 31 days) expression of tenascin at denervated endplates and in intramuscular nerve branches but not in other tissue compartments. Local application of botulinum toxin Type A, which results in muscle inactivity but not in tissue degeneration, however, did not induce tenascin expression 12 h to 12 days post-injection. Expression of tenascin after denervation and muscle damage, but its absence after paralysis, were verified by SDS-PAGE and Western blot analysis. Independent of the type of injury (muscle, nerve or both) the known major isoforms of mouse tenascin, as judged by M_r comparison, were re-expressed, with no preponderance of individual M_r forms. These results show that tenascin expression in adult muscles is induced by both axon and muscle fibre damage but not by muscle inactivity. In contrast, NCAM, in accordance with previous observations, showed enhanced expression both as a result of inactivity and in association with tissue repair.     

Multiple types of calcium channels mediate transmitter release during functional recovery of botulinum toxin type A-poisoned mouse motor nerve terminals

The involvement of different types of voltage-dependent calcium channels in nerve-evoked release of neurotransmitter was studied during recovery from neuromuscular paralysis produced by botulinum toxin type A intoxication. For this purpose, a single subcutaneous injection of botulinum toxin (1 IU; DL50) on to the surface of the mouse levator auris longus muscle was performed. The muscles were removed at several time-points after injection (i.e. at one, two, three, four, five, six and 12 weeks). Using electrophysiological techniques, we studied the effect of different types of calcium channel blockers (nitrendipine, omega-conotoxin-GVIA and omega-agatoxin-IVA) on the quantal content of synaptic transmission elicited by nerve stimulation. Morphological analysis using the conventional silver impregnation technique was also made. During the first four weeks after intoxication, sprouts were found at 80% of motor nerve terminals, while at 12 weeks their number was decreased and the nerve terminals were enlarged. The L-type channel blocker nitrendipine (1 microM) inhibited neurotransmitter release by 80% and 30% at two and five weeks, respectively, while no effects were found at later times. The N-type channel blocker omega-conotoxin-GVIA (1 microM) inhibited neurotransmitter release by 50-70% in muscles studied at two to six weeks, respectively, and had no effect 12 weeks after intoxication. The P-type channel blocker omega-agatoxin-IVA (100 nM) strongly reduced nerve-evoked transmitter release (>90%) at all the time-points studied. Identified motor nerve terminals were also sensitive to both nitrendipine and omega-conotoxin-GVIA. This study shows that multiple voltage-dependent calcium channels were coupled to transmitter release during the period of sprouting and consolidation, suggesting that they may be involved in the nerve ending functional recovery process.     

Motor responses of the urinary bladder and skeletal muscle in Botulinum intoxicated rats

1. Type A or type D botulinum toxin administered to rats did not produce a generalized paralysis of skeletal muscles at the time of ventilatory arrest. However, if survival was extended by artificial ventilation complete blockade of neuromuscular transmission developed 6.5 hr after 100 MLD of type D and 5 hr after 1000 MLD of type A toxin. The onset of paralysis of a muscle was shortened by repetitive stimulation of the motor nerves.2. There was no consistent blockade of parasympathetically innervated viscera in animals dying after type A toxin. Animals given type D toxin displayed mydriasis and urinary retention before death.3. Motor responses to electrical stimulation, of bladder preparations in vitro were more vulnerable to type D than to type A toxin. When somatic paralysis was complete in animals treated with type A or type D toxin the excised bladders produced pressure elevations 45 and 25%, respectively, of control preparations.4. During electrical stimulation of bladder preparations nearly paralysed by either toxin, the ACh release was significantly diminished from controls. In the rat bladder botulinum toxin specifically disrupted the liberation of mediator from post-ganglionic nerve endings.     

The interaction between foreign and original motor nerves innervating the soleus muscle of rats.

1. The fibular nerve was transplanted on to the soleus muscle of the rats. Interruption of the original soleus nerve then permitted cross-innervation, and subsequently, over a period of weeks, re-innervation by the original nerve. 2. Individual muscle fibres were often innervated by both the original and the foreign nerve. The original and foreign end-plates were located in separate regions of the muscle. There were no indications that the original nerve could displace or repress the foreign innervation. 3. The extent of re-innervation by the original nerve depended upon the method of denervation. A single crush of the nerve was followed by virtually complete re-innervation, even of muscle fibres already innervated by the foreign nerve. When re-innervation was delayed by resection of a segment of the nerve only muscle fibres without foreign nerve innervation were re-innervated. Denervation by a simple nerve cut gave an intermediate result. 4. Re-innervation by the original nerve can take place without measurable extrajunctional sensitivity to ACh. 5. The original end-plate region could retain high and localized sensitivity to ACh for several months despite degeneration of its motor nerve terminal and activity of the muscle fibre. 6. Established foreign end-plates were re-innervated by the foreign nerve on muscle fibres with intact original innervation. 7. The factors controlling synapse formation in skeletal muscles are discussed.     

Demonstration of glutamate-like immunoreactivity at rat neuromuscular junctions by quantitative electron microscopic immunocytochemistry

Motor nerve terminals and adjacent structures in the extensor digitorum longus and soleus muscles of young adult rats were examined for their content of glutamate by means of quantitative, electron microscopic immunocytochemistry employing colloidal gold particles as markers. The level of glutamate immunoreactivity was stronger in the extensor digitorum longus terminals than in the soleus terminals. In both muscles the glutamate immunolabelling was stronger in the nerve terminals than in the synaptic clefts and the postsynaptic tissue separating the secondary clefts, but the differences were larger in the extensor digitorum longus than in the soleus muscle. The myofibrils of the soleus muscle were more densely labelled than those in the extensor digitorum longus muscle: The level of immunoreactivity was high in the Schwann cells of both muscles. By comparing the labelling intensity of motor nerve terminals with that of muscle fibres and hippocampal mossy fibres (compartments that have been analysed previously with respect to their glutamate content), the mean concentration of fixed glutamate in the extensor digitorum terminals was estimated to be in the range of 10–20 mmol/l. An association of glutamate immunoreactivity with synaptic vesicles was demonstrated in the most strongly labelled terminals. Whether these epitopes were localized in the interior of the vesicles or at their external surface could not be resolved with the present technique. These data indicate that motor nerve terminals contain glutamate, and that the enrichment of this amino acid is more pronounced in the terminals of the extensor digitorum longus muscle (a fast muscle) than in those of the soleus muscle (a slow muscle). A possible modulatory or trophic role of glutamate in the mammalian neuromuscular junction should be considered.     

Inhibition of acetylcholinesterase accelerates axon terminal withdrawal at the developing rat neuromuscular junction

Summary In developing skeletal muscles, the rate at which superfluous innervation is lost from the endplates depends on the general level of neuromuscular activity. Whether it is activity of the presynaptic or postsynaptic structures (or both) that is critical is not well established. In this work, we transitorily inhibited the AChE of soleus muscle in postnatal rats, in order to increase postsynaptic activity, without directly altering activity of the nerve terminals. We then followed the time course of disappearance of axon terminals from the endplates of treated and normal muscles, using electron-microscope techniques. Three hours after inhibition of AChE, the muscle fibres exhibited local supercontracture and ultrastructural damage in the region of the endplate, consistent with local elevation of Ca²⁺ levels. At the same time, small electron-opaque vesicles, apparently of muscular origin, appeared in the synaptic cleft. The nerve terminals, however, were entirely normal in number and appearance. One day after treatment, endplates of esteraseinhibited muscles showed accelerated loss of nerve terminals, compared to endplates of normally developing muscles. No further loss of nerve terminals occurred, once AChE activity returned at the endplate. These results suggest that the rate at which superfluous nerve terminals retract from the developing neuromuscular junction is regulated by the level of activation of the muscle. It seems likely that activity of postsynaptic sites may similarly regulate changes in innervation patterns, in other developing or adapting neuro-neuronal or neuro-effector systems.     

Electrophysiological studies of neuromuscular transmission in hereditary `motor end-plate disease' of the mouse

1. Studies have been made on isolated nerve-muscle preparations from mice with hereditary ;motor end-plate disease'.2. Spontaneous fibrillation was observed in the isolated preparation.3. Muscles gave only a weak twitch or failed to contract in response to nerve stimulation. Direct stimulation of muscles caused a twitch response which had a slower time course than normal. Peripheral nerve conduction was normal.4. Intracellular recording from single muscle fibres showed that with longer survival of the animal an increasing proportion of fibres failed to show end-plate potentials or action potentials in response to nerve stimulation.5. Miniature end-plate potentials (m.e.p.p.s) were recorded in almost all muscle fibres including those in which neuromuscular transmission had failed. The frequency of m.e.p.p.s was greater than normal, was not increased by tetanic stimulation of the nerve but was increased by a raised external potassium concentration.6. Muscle fibres were supersensitive to acetylcholine.7. The results suggest that the muscular weakness in this disease is due to the failure of nerve action potentials to invade motor nerve terminals so that muscle fibres become ;functionally denervated'.