Neurotrophic regulation of dynamic properties of skeletal muscle: effects of botulinum toxin and denervation.

In order to determine the role of acetylcholine (ACh) transmission in neurotrophic regulation of dynamic properties of muscle, the effects of botulinum toxin treatment were compared with those of denervation. The extensor digitorum longus (EDL) and soleus muscles of rats were either denervated or injected with botulinum toxin. At times up to 25 days the isometric properties of these muscles were determined. 2. Both botulinum treatment and denervation produced progressive slowing of the time to peak of the twitch (TPT) and half-relaxation time of the twitch (1/2 RT), which was more pronounced in the EDL than in the soleus. 3. Both treatments produced slowing of the relaxation curve following tetanic contraction, more marked in the EDL than in the soleus muscle. This indicates a slowing of relaxation, and suggests a prolongation of the active state of the muscle. 4. The maximum rate of rise of the tetanus did not change significantly in the EDL and soleus muscles after botulinum treatment or denervation. This suggests that there is no major change in the speed of contraction under conditions of botulinum treatment or denervation. 5. The changes produced by botulinum treatment and denervation were virtually identical in all parameters tested. This is interpreted meaning that cholinergic transmission (including muscle usage), or some other factor closely related to cholinergic transmission, accounts for the motor nerve's trophic influence in maintaining these dynamic properties of skeletal muscles.     

Effects of botulinum toxin induced muscle paralysis on endocytosis and lysosomal enzyme activities in mouse skeletal muscle

The effects of botulinum toxin (type A) induced muscle paralysis on endocytosis and lysosomal enzyme activities in skeletal muscle were compared with the effects of surgical denervation. Muscle atrophy, measured as decrease in total muscle protein content, was as large or larger after botulinum toxin treatment as after denervation. Endocytic activity, measured as the in vitro uptake of horseradish peroxidase, and the specific activities of the lysosomal enzymes N-acetyl--d-glucosaminidase and cathepsin D were all increased six days after denervation. Only the specific activity of cathepsin D was increased six days after botulinum toxin poisoning. The uptake of horseradish peroxidase and the specific activity of N-acetyl--d-glucosamidase were also increased eleven days after poisoning. Transverse sections of eleven days botulinum poisoned muscles from animals injected with horseradish peroxidase showed fibres with dense peroxidase staining similar to those seen in denervated muscle although they seemed to occur less frequently.The results show that increases in endocytic activity and lysosomal enzyme activities may occur in skeletal muscle without the presence of degenerating axons. The differences in effects of surgical denervation and botulinum toxin induced paralysis are discussed in terms of what is known about the mechanism of action of botulinum toxin and the possible functional roles of the two lysosomal enzymes studied.     

The distribution of acetylcholine receptors in the normal and denervated neuromuscular junction of the frog

Summary A combined light and electron microscopic investigation was performed to study the distribution and fate of clusters of horseradish peroxidase (HRP)-labelled or rhodamine-labelled alpha-bungarotoxin binding sites in normal and denervated cutaneus pectoris muscles of the frog.After staining for axon and cholinesterase (ChE) it appears that, in normal muscles, binding sites for rhodamine alpha-bungarotoxin are strictly confined to those parts of the synaptic gutter occupied by the nerve terminal. Binding sites are missing in axon-abandoned gutters or gutters occupied only by the Schwann cell. Similarly, in partially occupied gutters, HRP-toxin binding sites are confined to the parts of the muscle fibre membrane apposed to the nerve; they are missing at axon-free lateral parts at which secondary clefts and ChE are present. These observations suggest that junctional acetylcholine (ACh) receptor clusters are strictly controlled by the nerve.Thirty-five days after denervation, receptor density was apparently reduced in some parts of the gutters while other parts of the same gutters showed high receptor density. In addition, the length of gutter totally devoid of receptor clusters increased from an average fraction of 9% in control muscles to 14% in denervated muscles. Loss of receptors occurred both at Schwann cell-free and at Schwann cell-occupied gutters. In muscles denervated for 500–750 days, no toxin binding sites could be detected in the junctional membrane, whereas ChE was still present. Schwann cells had apparently abandoned some gutters but were present at others. Upon arrival of spontaneously reinnervating axons, muscle fibres accumulate ACh receptor clusters at the junctional membrane. Patches intensely labelled with alpha-bungarotoxin and associated with ChE reaction product were found near former junctions in long-term denervated muscles.It is concluded that after long-term denervation the muscle fibre cannot maintain junctional ACh receptor clusters by itself. In normally innervated muscles, receptor clusters are actively maintained by nerve-borne factors near the transmitter release sites.     

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.     

Muscle enzymatic changes induced by blockage of axoplasmic transport.

The activity of malic dehydrogenase, pyruvic kinase, and phosphorylase b was measured in the geniohyoid muscle of the cat after injection of 10 10 mM colchicine into the hypoglossal nerve. Experiments performed 1-60 days after the injection showed that the activity of the three enzymes gradually decreased (day 4-5), reached a maximum fall (day 10-25), and subsequently returned to control values (day 30-60). Concomitantly to these enzymatic alterations, the muscles showed fibrillation and ACh hypersensitivity; however, in contrast to denervation, the drug had no effect on nerve conduction, effective neuromuscular transmission, and ultrastructure of motor end plates. Experiments with [3H]colchicine indicated that the observed changes were brought about by the drug acting directly on the motor axons rather than on the muscle cells. The transsynaptic effects induced by colchicine treatment to the nerve can be ascribed to a temporary interruption of axoplasmic transport. It is suggested that neurotrophic regulation of some muscle-soluble enzymes partly depend on the normal operation of the axoplasmic transport system.     

Effect of implantation of an extra nerve on the recovery of neuromuscular transmission from botulinum toxin.

1. The common peroneal nerve was implanted into soleus in the mouse and 2 weeks later a sublethal dose of botulinum toxin injected causing a block of neuromuscular transmission at the terminals of the soleus nerve. Most muscle fibres became innervated by the common peroneal nerve. 2. Recovery of neuromuscular transmission at the soleus nerve terminals was delayed in the common peroneal nerve implanted muscles. 3. Stimulation of the soleus nerve after botulinum-evoked subthreshold end-plate potentials (e.p.p.s) in virtually every fibre tested in unoperated muscles. In common peroneal nerve-implanted muscles stimulation of the soleus nerve failed to evoke e.p.p.s in about 40% of fibres tested and where e.p.p.s were recorded their amplitudes were generally smaller. 4. When the common peroneal nerve was cut 2 months after botulinum, neuromuscular transmission at soleus nerve terminals occurred after 4 weeks. When the common peroneal nerve was cut 6 months after botulinum, transmission was found at soleus nerve terminals within 1 week. 5. Recovery of transmission at soleus nerve terminals from the effects of botulinum toxin is delayed if the muscle fibres become innervated by the common peroneal nerve and a proportion of soleus nerve terminals cease to release acetylcholine (ACh) until after the peroneal nerve has been cut.     

The denervated muscle: facts and hypotheses. A historical review

Denervation changes in skeletal muscle (atrophy; alterations of myofibrillar expression, muscle membrane electrical properties, ACh sensitivity and excitation–contraction coupling process; fibrillation), and their possible causes are reviewed. All changes can be counteracted by muscle electrostimulation, while denervation-like effects can be caused by the complete conduction block in muscle nerve. These results do not support the hypothesis that the lack of neurotrophic, non-motor factors plays a role in denervation phenomena. Instead they support the view that the lack of neuromotor discharge is the only cause of the phenomena and that neuromotor activity is an essential factor in regulating muscle properties. However, some experimental results cannot apparently be explained by the lack of neuromotor impulses, and may still suggest that neurotrophic influences exist. A hypothesis is that neurotrophic factors, too feeble to maintain a role in completely differentiated, adult muscles, can concur with neuromotor activity in the differentiation of immature, developing muscles.     

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.

     

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.     

Two factors responsible for the development of denervation hypersensitivity

1. Innervated adult skeletal muscle is sensitive to acetylcholine at the end-plate region only. After denervation the entire muscle membrane becomes chemosensitive. The period of greatest increase in sensitivity in rat soleus muscles following section of the sciatic nerve in the thigh is between 48 and 72 hr post-operatively.2. Direct electrical stimulation was found to prevent the onset of the development of denervation hypersensitivity during the first 2-3 days after nerve section. Thereafter, electrical stimulation only reduced the sensitivity of denervated muscles to acetylcholine (ACh).3. The period of greatest increase in sensitivity follows loss of transmission and degeneration of the nerve terminals. Once this degeneration is under way, electrical stimulation is no longer as effective in preventing the development of denervation hypersensitivity.4. Hypersensitivity is also seen in muscles on which a small piece of thread or degenerating nerve has been placed. Hypersensitivity following these procedures declines within a few days, unlike denervation hypersensitivity which persists until innervation is restored.5. The present results suggest that activity alone cannot prevent the development of hypersensitivity in the presence of degenerating nerve fibres, or muscle damage. Activity does however counteract increased sensitivity. It is suggested that two factors interact to produce denervation hypersensitivity; the presence of degenerating nerve tissue and concomitant cellular changes bring about changes in the muscle fibre membrane causing it to become hypersensitive; and the loss of muscle activity, resulting in the persistence of hypersensitivity until innervation is restored.     

Repeated stimuli for axonal growth causes motoneuron death in adult rats: the effect of botulinum toxin followed by partial denervation

Axons of motoneurons to tibialis anterior and extensor digitorum longus muscles of adult rats were induced to sprout by injecting botulinum toxin into them, by partial denervation or by a combination of the two procedures. Ten weeks later, the number of motoneurons innervating the control and operated tibialis anterior and extensor digitorum longus muscles was established by retrograde labelling with horseradish peroxidase. In the same preparations, the motoneurons were also stained with a Nissl stain (gallocyanin) to reveal motoneurons in the sciatic pool. Examination of the spinal cords from animals treated with botulinum toxin showed that the number of retrogradely labelled cells and those stained with gallocyanin in the ventral horn on the treated compared to the control side was unchanged. In rats that had their L4 spinal nerve sectioned on one side, the number of retrogradely labelled cells on the operated side was 48+/-3% (n = 5) of that present in the control unoperated ventral horn. Thus, just over half the innervation was removed by cutting the L4 spinal nerve. Counts made from gallocyanin-stained sections showed that 94+/-4% (n = 5) of motoneurons were present in the ventral horn on the operated side. Thus, section of the L4 spinal nerve did not lead to any death of motoneurons. In rats that had their muscles injected with botulinum toxin three weeks prior to partial denervation, the number of retrogradely labelled cells was reduced from 48+/-3% (n = 5) to 35+/-4% (n = 5). Moreover, only 67+/-5% (n = 5) of motoneurons stained with gallocyanin, suggesting that a proportion of motoneurons died after this combined procedure. This result was supported by experiments in which motor unit numbers in extensor digitorum longus muscles were determined by measurements of stepwise increments of force in response to stimulation of the motor nerve with increasing stimulus intensity. In partially denervated extensor digitorum longus muscles, 16.6+/-0.7 (n = 5) motor units could be identified, and in animals treated with botulinum toxin prior to partial denervation only 13.3+/-0.9 (n = 3) motor units were present. Taken together, these results show that treatment with botulinum toxin followed by partial denervation causes motoneuron death in adult rats.     

Catabolic effect of anabolic steroid in skeletal muscle under conditions of impaired neurotrophic regulation

Effect of anabolic steroid nandrolone (Phenobolin) on fast (plantar) and slow (soleus) skeletal muscles was studied in guinea pigs with impaired neurotrophic regulation (denervation, axonal transport blockade) or after tenotomy. Immunohistochemical analysis with monoclonal antibodies to fast myosin heavy chains showed that injection of anabolic steroid did not modify the relative content of fast and slow muscle fibers in the studied muscles under all experimental conditions. Injection of anabolic steroid did not modify the weight of the studied muscles and did not prevent its drop after denervation or tenotomy. Axonal transport blockade by colchicine application on the nerve induced the appearance of fast muscle fibers in the slow soleus muscle and an increase in its weight; in the slow muscle, nandrolone did not prevent the induction of fast myosin synthesis. Under conditions of axonal transport blockade, the agent exerted a catabolic effect and considerably reduced the muscle weight. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol.127, No. 5, pp. 569–572, May, 1999     

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.     

Repeated stimuli for axonal growth causes motoneuron death in adult rats: the effect of botulinum toxin followed by partial denervation

Axons of motoneurons to tibialis anterior and extensor digitorum longus muscles of adult rats were induced to sprout by injecting botulinum toxin into them, by partial denervation or by a combination of the two procedures. Ten weeks later, the number of motoneurons innervating the control and operated tibialis anterior and extensor digitorum longus muscles was established by retrograde labelling with horseradish peroxidase. In the same preparations, the motoneurons were also stained with a Nissl stain (gallocyanin) to reveal motoneurons in the sciatic pool. Examination of the spinal cords from animals treated with botulinum toxin showed that the number of retrogradely labelled cells and those stained with gallocyanin in the ventral horn on the treated compared to the control side was unchanged. In rats that had their L4 spinal nerve sectioned on one side, the number of retrogradely labelled cells on the operated side was 48±3% (n=5) of that present in the control unoperated ventral horn. Thus, just over half the innervation was removed by cutting the L4 spinal nerve. Counts made from gallocyanin-stained sections showed that 94±4% (n=5) of motoneurons were present in the ventral horn on the operated side. Thus, section of the L4 spinal nerve did not lead to any death of motoneurons. In rats that had their muscles injected with botulinum toxin three weeks prior to partial denervation, the number of retrogradely labelled cells was reduced from 48±3% (n=5) to 35±4% (n=5). Moreover, only 67±5% (n=5) of motoneurons stained with gallocyanin, suggesting that a proportion of motoneurons died after this combined procedure. This result was supported by experiments in which motor unit numbers in extensor digitorum longus muscles were determined by measurements of stepwise increments of force in response to stimulation of the motor nerve with increasing stimulus intensity. In partially denervated extensor digitorum longus muscles, 16.6±0.7 (n=5) motor units could be identified, and in animals treated with botulinum toxin prior to partial denervation only 13.3±0.9 (n=3) motor units were present.Taken together, these results show that treatment with botulinum toxin followed by partial denervation causes motoneuron death in adult rats.     

Adaptive changes in locomotor activity following botulinum toxin injection in ankle extensor muscles of cats.

The present study investigated the adaptations made in motor behavior following a temporary reduction in ankle extensor activity in the walking cat. Temporary muscle weakness was induced by injecting botulinum toxin into the lateral gastrocnemius (LG), plantaris (PL), and soleus (SOL) muscles, or SOL alone. The medial gastrocnemius (MG) muscle was not injected. Adaptations in the level of muscle activity were recorded using chronically implanted electromyographic (EMG) electrodes. Serial recordings were made prior to botulinum toxin injections and for several days following the injections. Kinematic analysis of ankle joint movements was made from video records to assess the impact of the botulinum toxin injections on the function of the ankle joint during walking. Following injection of the LG, PL, and SOL muscles with botulinum toxin, the amplitude of the MG burst increased over a period of a few days to a week. This increase was similar to the previously reported changes produced in MG following transection of the nerves serving LG, PL, and SOL. Following the weakening of the ankle extensor muscles, there was a temporary deficit in ankle function during walking as evidenced by a marked increase in the amount of ankle flexion that occurred at stance onset. This functional deficit recovered relatively quickly and was not associated with a return of the EMG pattern to the preinjection pattern. After recovery from the initial injections, a second injection of botulinum toxin into SOL alone was performed. No functional deficits were observed in the ankle movements during walking following this second injection. However, weakening SOL produced increases in the burst amplitudes of the MG, LG, and PL muscles over a period of a few days. This suggests that normal movements at the ankle during walking can be generated with more than one pattern of ankle extensor activity and that there is flexibility in how the necessary torque is produced. A final procedure, transection of the nerves serving LG, PL, and SOL, failed to produce any functional deficits in ankle movements. The implication is that adaptations to the neural control of ankle extensor activity that were induced by the initial procedure persisted after the recovery of the injected muscles and were sufficient to compensate for the subsequent challenges.     

A study on early post-denervation changes of non-quantal and quantal acetylcholine release in the rat diaphragm

The d-tubocurarine (dTC) induced hyperpolarization of antiesterase-treated muscles at the endplate zone, miniature endplate potentials (mepps), resting membrane potentials (RMPs) and the input resistances of single muscle fibres (R_in) were measured in rat diaphragm at various times after denervation. The dTC-induced hyperpolarization decreased in two phases: 2 h after denervation it decreased transiently to 25%, after 4 h it had partially recovered to 60% and from 6 h it progressively decreased up to 12 h after which time it changed to depolarization. The initial fall and recovery were also present in muscles from sham-operated animals. The frequency of mepps decreased by 25% and the amplitude diminished by 10% within the first 2–4 h. After 10 h the frequency had decreased by 35% and the amplitude by 65%. After 12 h no mepps were present. The RMP was not significantly changed during the first 16 h after denervation. From 16 to 24 h the membrane became depolarized at a rate of about 1 mV/h. The input resistance of a single muscle fibre was constant for 12 h after denervation and from 12 to 24 h it increased by 25%. It is concluded that the early decrease in the dTC-induced hyperpolarization is probably due to the desensitization of acetylcholine (ACh) receptors caused by stress-activated non-quantal ACh release. The later decrease of dTC-hyperpolarization reflects a fall in the non-quantal ACh release. The depolarization of the resting membrane after denervation is related to the decrease in passive membrane permeability which is a secondary consequence of transmission failure. The present results do not distinguish between non-quantally released and quantally released ACh as possible trophic agents, since both types of release disappear at the same time.     

Increased extrajunctional acetylcholine sensitivity produced by chronic acetylcholine sensitivity produced by chronic post-synaptic neuromuscular blockade.

1. Anaesthetized rats were paralysed for periods of up to 3 days by chronic administration of D-tubocurarine (DTC), succinylcholine or alpha-bungarotoxin. 2. After 3 days of treatment with DTC, the phrenic nerve remained active. Neuromuscular transmission and spontaneous miniature end-plate potentials (m.e.p.p.s) were restored after removal of the DTC. Resting potentials and input resistances of muscle fibres that had been paralysed for 3 days were similar to those in denervated fibers. 3. Chronic neuromuscular blockade increased the binding of [125-I]-alpha-bungarotoxin by extrajunctional regions of muscle. The time course of the increase was similar to that seen after denervation. Binding to muscles from animals that were anaesthetized and respirated, but not paralysed, was not increased. 4. Three days of paralysis increased the sensitivity of the extrajunctional muscle membrane to acetylcholine (ACh) applied by iontophoresis. 5. Approximately the same proportion of muscle fibres from muscles paralysed for 3 days gave overshooting action potentials in the presence of tetrodotoxin 10-minus 6 g/ml. as did fibres form muscles denervated for 3 days. 6. Chronic paralysis did not change the accumulation of acetylcholinesterase above a ligation in the sciatic nerve. 7. These results are consistent with the idea that extrajunctional ACh sensitivity is normally controlled by muscle activity.     

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.     

Effects of X-irradiation on axonal sprouting induced by botulinum toxin

The effect of X-irradiation on axonal sprouting of motor nerves induced by botulinum toxin was examined. Muscles of one leg in the mouse were X-irradiated (15 Gy) prior to the injection of a locally paralysing dose of botulinum toxin. It was found that axonal sprouting occurred as expected, but the sprouts remained unmyelinated and many degenerated. Fewer new end-plates were formed, muscles remained more severely atrophied and supersensitive to acetylcholine and recovery of neuromuscular transmission was greatly delayed when compared with the effects of botulinum toxin alone.The experiments show that X-irradiation did not prevent sprouting but, probably by impairing Schwann cell proliferation, altered axon-Schwann cell relationships and prevented the maturation of newly-formed axons and the differentiation of new end-plates.     

Cholinergic receptors at denervated mammalian motor end-plates.

The number of acetylcholine receptors at normal and denervated end-plated in rat soleus muscles was studied using the binding of [125A] alpha-bungarotoxin as a quantitative assay. Normal end-plates bound several thousand times as much toxin as equal areas of extra-synaptic muscle membrane. After short-term denervation (up to 2.4 weeks) the extrajunctional binding increased, but there was no change in specific binding to the motor end-plate. Denervation for longer periods (up to 7 weeks) reduced binding sites at the end-plate by up to 60-70%. Direct electrical stimulation of these muscles for the entire period of denervation did not prevent the loss of junctional binding sites even though it was adequate to abolish the increase in extrajunctional toxin binding. In contrast, denervated end-plates on muscle fibres cross-innervated by a foreign nerve at a distant location continued to bind normal amounts of toxin for over four months.