Effect of ethanol on young nerve endings

The ethanol changes the quantal spontaneous release of acetylcholine and it affects the reinnervation time course. The effects of ethanol on regenerated nerve endings have been tested. 20 days after crushing sciatic nerve, the m.e.p.p. frequency at the end plate of rat extensor digitorum longus muscle keep in Ringer solution without and with ethanol has been estimated by intracellular recordings. The increase of the m.e.p.p. frequency produced by ethanol is greater in immature, than in normal nerve endings.     

Changes in the synaptic activity in sciatic nerve-extensor digitorum longus (preparations induced by ethanol)

The aim of this research is the study of the modification of synaptic activity caused by ethanol in the rat sciatic nerve-extensor digitorum longus (EDL) muscle preparation. For such a purpose, intracellular recordings have been carried out, keeping the muscle immersed in normal Ringer solution and in Ringer solutions containing ethanol at different concentrations up to 0,8 M. Therefore, the resting potential of muscle cells and the frequency of m.e.p.p.s were measured. Qualitative observations of m.e.p.p.s shape were also carried out. Ethanol increases the frequency of m.e.p.p.s in the rat sciatic nerve - EDL muscle preparation. The logarithm of relative frequency (frequency in Ringer solution with ethanol/frequency in normal Ringer solution) is linear with respect to the concentration of ethanol, with a slope of 1.44. Furthermore, ethanol increases the amplitude and lengthens the time course of m.e.p.p.s. The muscle cells undergo a hyperpolarization of about 2-3% at the lowest concentrations of ethanol tested.     

Recovery of sensation and reflex activity during treatment with ethanol

Ethanol greatly modifies synaptic function and it affects the reinnervation time course. In order to better clarify the effects of ethanol on nerve regeneration, we have observed the recovery of algesthesia and reflex activity in the rat during the treatment with ethanol. For this purpose the posterior leg of Sprague Dawley rats was denervated by crushing the sciatic nerve and the recovery of algesthesia and reflex activity was tested at the level of the metatarsus-falanx articulation of the homolateral paw. During the reinnervation period, the animals were treated daily with ethanol 3 g/Kg of body weight. The recovery of algesthesia and reflex activity comes about in a medio-lateral direction and proceeds linearly. It is completed on the 25th day after the denervation. Ethanol does not cause changes in the time course of algesthesia recovery, however it does cause a slight delay in the recovery of the reflex activity.     

Skeletal muscle reinnervation by reduced axonal numbers results in whole muscle force deficits

Patients sustaining a peripheral nerve injury will frequently experience residual muscle weakness after muscle reinnervation, even if the nerve repair is performed under optimal circumstances to allow rapid muscle reinnervation. The mechanisms responsible for this contractile dysfunction remain unclear. It is hypothesized that after peripheral nerve injury and repair, a reduced number of axons are available for skeletal muscle reinnervation that results in whole muscle force and specific force deficits. A rat model of peroneal nerve injury and repair was designed so that the number of axons available for reinnervation could be systematically reduced. In adult rats, the peroneal nerve to the extensor digitorum longus muscle was either left intact (sham group, n = 8) or divided and repaired with either 50 percent (R50 group, n = 7) or 100 percent (R100 group, n = 8) of the axons in the proximal stump included in the repair. Four months after surgery, maximal tetanic isometric force was measured and specific force was calculated for each animal. Mean tetanic isometric force for extensor digitorum longus muscles from R50 rats (2765.7 +/- 767.6 mN) was significantly lower than sham (4082.8 +/- 196.5 mN) and R100 (3729.0 +/-370.2 mN) rats (p < 0.003). Mean specific force calculations revealed significant deficits in both the R100 (242.1 +/- 30 kN/m2) and R50 (190.6 +/- 51.8 kN/m2) rats compared with the sham animals (295.9 +/- 14 kN/m2) (p < 0.0005). These data support our hypothesis that after peripheral nerve injury and repair, reinnervation of skeletal muscle by a reduced number of axons results in a reduction in tetanic isometric force and specific force. The greater relative reduction in specific force compared with absolute force production after partial nerve repair may indicate that a population of residual denervated muscle fibers is responsible for this deficit.     

Age-Related Responses of Skeletal Muscle After Ectopic Innervation, with Particular Reference to 16S Acetylcholinesterase, in Adult Rats

Abstract: The formation of ectopic junctions between the foreign fibular nerve and the soleus muscle of young (35-day-old) and mature (200-day-old) adult rats was induced by severing the normal nerve 4 weeks after implanting the foreign nerve. The various molecular forms of ace-tylcholinesterase (AChE) were studied both at the implanted region and at the original denervated endplates. The velocity of contraction was also studied. In young rats the 16S form was first detected in the ectopic junctions around day 5 after reinnervation; this form rapidly increased during the following weeks, reaching a plateau by day 20. By contrast, in mature rats the appearance of the 16S AChE was dramatically delayed; in fact, it could not be observed before day 80 after reinnervation. (The 16S AChE form appeared at day 20 after reinnervation in the original denervated endplates of young rats; however, at the same time, no effect was observed in mature animals.) The original, slow muscle fibers of the soleus became faster upon reinnervation; this change occurred also much earlier in younger than in mature rats. Our results indicate a loss of plasticity in the skeletal muscle of mature rats. We suggest caution in the use of the ectopic innervation model to study development in mature adult rats.     

Long-term changes in neurotrophic factor expression in distal nerve stump following denervation and reinnervation with motor or sensory nerve

Several factors have been proposed to account for poor motor recovery after prolonged denervation, including motor neuron cell death and incomplete or poor regeneration of motor fibers into the muscle. Both may result from failure of the muscle and the distal motor nerve stump to continue expression of neurotrophic factors following delayed muscle reinnervation. This study investigated whether regenerating motor or sensory axons modulate distal nerve neurotrophic factor expression. We found that transected distal tibial nerve up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) mRNA, down-regulated neurotrophin-3 and ciliary neurotrophic factor mRNA, and that although these levels returned to normal with regeneration, the chronically denervated distal nerve stump continued to express these neurotrophic factors for at least 6 months following injury. A sensory nerve (the cutaneous saphenous nerve) sutured to distal tibial nerve lowered injury-induced BDNF and GDNF mRNA levels in distal stump, but repair with a mixed nerve (peroneal, containing muscle and cutaneous axons) was more effective. Repair with sensory or mixed nerves did not affect nerve growth factor or neurotrophin-3 expression. Thus, distal nerve contributed to a neurotrophic environment for nerve regeneration for at least 6 months, and sensory nerve repair helped normalize distal nerve neurotrophic factor mRNA expression following denervation. Furthermore, as BDNF and GDNF levels in distal stump increased following denervation and returned to control levels following reinnervation, their levels serve as markers for the status of regeneration by either motor or sensory nerve.     

Cytotactin is involved in synaptogenesis during regeneration of the frog neuromuscular system.

The expression of cytotactin, an extracellular matrix glycoprotein involved in morphogenesis and regeneration, was determined in the normal and regenerating neuromuscular system of the frog Rana temporaria. Cytotactin was expressed in adult brain and gut as two major components of Mr 190,000 and 200,000 and a minor form of higher molecular weight, but was almost undetectable in skeletal muscle extract. However, cytotactin was concentrated at the neuromuscular junctions as well as at the nodes of Ranvier. After nerve transection, cytotactin staining increased in the distal stump along the endoneurial tubes. In preparations of basal lamina sheaths of frog cutaneous pectoris muscle obtained by inducing the degeneration of both nerve and muscle fibers, cytotactin was found in dense accumulations at original synaptic sites. In order to define the role of cytotactin in axonal regeneration and muscle reinnervation, the effect of anti-cytotactin antibodies on the reinnervation of the basal lamina sheaths preparations was examined in vivo. In control preparations, regenerating nerve terminals preferentially reinnervate the original synaptic sites. In the presence of anti-cytotactin antibodies, axon regeneration occurred with normal fasciculation and branching but with altered preterminal nerve fibers pathways. Ultrastructural observations showed that synaptic basal laminae reinnervation was greatly delayed or inhibited. These results suggest that cytotactin plays a primordial role in synaptogenesis, at least during nerve regeneration and reinnervation in the adult neuromuscular system.     

Inducible Expression of Neurotrophic Factors by Mesenchymal Progenitor Cells Derived from Traumatically Injured Human Muscle

Peripheral nerve damage frequently accompanies musculoskeletal trauma and repair of these nerves could be enhanced by the targeted application of neurotrophic factors (NTFs), which are typically expressed by endogenous cells that support nerve regeneration. Injured muscle tissues express NTFs to promote reinnervation as the tissue regenerates, but the source of these factors from within the muscles is not fully understood. We have previously identified a population of mesenchymal progenitor cells (MPCs) in traumatized muscle tissue with properties that support tissue regeneration, and our hypothesis was that MPCs also secrete the NTFs that are associated with muscle tissue reinnervation. We determined that MPCs express genes associated with neurogenic function and measured the protein-level expression of specific NTFs with known functions to support nerve regeneration. We also demonstrated the effectiveness of a neurotrophic induction protocol to enhance the expression of the NTFs, which suggests that the expression of these factors may be modulated by the cellular environment. Finally, neurotrophic induction affected the expression of cell surface markers and proliferation rate of the MPCs. Our findings indicate that traumatized muscle-derived MPCs may be useful as a therapeutic cell type to enhance peripheral nerve regeneration following musculoskeletal injury.     

The Impact of Motor Axon Misdirection and Attrition on Behavioral Deficit Following Experimental Nerve Injuries

Peripheral nerve transection and neuroma-in-continuity injuries are associated with permanent functional deficits, often despite successful end-organ reinnervation. Axonal misdirection with non-specific reinnervation, frustrated regeneration and axonal attrition are believed to be among the anatomical substrates that underlie the poor functional recovery associated with these devastating injuries. Yet, functional deficits associated with axonal misdirection in experimental neuroma-in-continuity injuries have not yet been studied. We hypothesized that experimental neuroma-in-continuity injuries would result in motor axon misdirection and attrition with proportional persistent functional deficits. The femoral nerve misdirection model was exploited to assess major motor pathway misdirection and axonal attrition over a spectrum of experimental nerve injuries, with neuroma-in-continuity injuries simulated by the combination of compression and traction forces in 42 male rats. Sciatic nerve injuries were employed in an additional 42 rats, to evaluate the contribution of axonal misdirection to locomotor deficits by a ladder rung task up to 12 weeks. Retrograde motor neuron labeling techniques were utilized to determine the degree of axonal misdirection and attrition. Characteristic histological neuroma-in-continuity features were demonstrated in the neuroma-in-continuity groups and poor functional recovery was seen despite successful nerve regeneration and muscle reinnervation. Good positive and negative correlations were observed respectively between axonal misdirection (p<.0001, r²=.67), motor neuron counts (attrition) (p<.0001, r²=.69) and final functional deficits. We demonstrate prominent motor axon misdirection and attrition in neuroma-in-continuity and transection injuries of mixed motor nerves that contribute to the long-term functional deficits. Although widely accepted in theory, to our knowledge, this is the first experimental evidence to convincingly demonstrate these correlations with data inclusive of the neuroma-in-continuity spectrum. This work emphasizes the need to focus on strategies that promote both robust and accurate nerve regeneration to optimize functional recovery. It also demonstrates that clinically relevant neuroma-in-continuity injuries can now also be subjected to experimental investigation.     

Variations in the hyperpolarization potential of ethanol on muscle during reinnervation

Ethanol causes the hyperpolarization of the excitable membranes. In the Extensor Digitorum Longus (EDL) muscle of the rat the increase of resting membrane potential is 2-5% and is independent of the concentration of alcohol between 0.2 and 0.4 M, while at higher concentrations the membrane potential falls to levels equal or inferior to the normal potential. We have studied the hyperpolarization action of ethanol on the denervated muscle by crushing the sciatic nerve. Also under these conditions in which, as is known, there is a drop in the resting potential, ethanol causes hyperpolarization, however it is in general greater and it is dependent upon the concentration between 0.2 and 0.8 M.     

Morphological changes in rat skeletal muscle following reinnervation

Morphological changes appearing in the course of muscle regeneration after reinnervation of denervated M. soleus (slow) and M. tibialis anterior (fast) rat skeletal muscle were investigated. It was found that pathological changes typical for denervation atrophy (seen on the 10th day after crushing the sciatic nerve) and symptoms of regeneration (beginning about the 15th day) were much more pronounced in the soleus than in the tibialis muscle. Some stages of regeneration in the soleus muscle could be distinguished. The contractile material destructions were the first pathological changes that disappeared after the beginning of regeneration. In the second stage other denervation changes disappeared and intensive regeneration of muscle fibres was observed. In the next stage regeneration slowed down, and the reduction of the excess of muscle nuclei was visible. Four months after crushing the nerve, regeneration proceeded to completion with only some traces of the passed processes: in the soleus muscle, chains of sarcolemmal nuclei, satellite cells and newly formed muscle fibres were more often seen than in contralateral muscle; in the tibialis, collagen depots were present around the vessels and between muscle fascicles.     

The effect of cortisol on the excitability of the rat muscle fibre membrane and neuromuscular transmission.

The present experiments show that cortisol when applied in vitro, exerted two different effects on the electrical excitability of the diaphragm muscle fibre membrane and on the neuromuscular transmission depending on the concentration used. At low concentrations (2.5X10(-6) mol.l-1) it potentiated action potentials, increased resting membrane polarization by 3--4 mV and did not affect neuromuscular transmission. Higher concentrations (10(-2) mol.l-1) suppressed the action potential to a certain extent, depolarized the muscle fibre membrane by 6 mV and reduced the amplitudes of m.e.p.p.s and e.p.p.s as well as those of iontophoretically evoked acetylcholine potentials. It was concluded that the effect of low concentrations of cortisol is primary and is probably due to the enhancement of resting membrane permeability for K+ ions and to the changes in ion channels. Cortisol in high doses increased muscle oxygen consumption, so that its suppressing effect might be due to inhibition of energy metabolism.     

Post-tetanic potentiation of miniature end-plate potential frequency at neuromuscular junction of the rat soleus muscle

1. Changes in miniature end-plate potential (m.e.p.p.) frequency by repetitive nerve stimulation were examined in the rat soleus muscle. 2. The increase of m.e.p.p. frequency was induced by repetitive stimulation and persisted for several minutes after the tetanus. That is, post-tetanic potentiation (PTP) of neuromuscular transmission was first demonstrated here in the rat soleus muscle. 3. The time course of the decay of m.e.p.p. frequency after the tetanus showed a double exponential curve which consisted of a fast decaying component (augmentation) and a slow decaying component (potentiation). 4. The magnitude of PTP depended on the stimulation frequency and its duration. It increased with the increase of duration and was at its maximum at a frequency of 100 Hz. 5. No PTP was elicited by repetitive stimulation under conditions in which end-plate potential (e.p.p.) was completely suppressed, and, moreover, m.e.p.p. frequency tended to decrease after the tetanus.     

Microglial changes accompanying the promotion of retinal ganglion cell axonal regeneration into peripheral nerve grafts

Intravitreal injection of the microglia inhibitor tuftsin 1-3 leads to an increase in retinal ganglion cell axonal regeneration into peripheral nerve grafts and a decrease in phagocytic cells in the retina. However, the relation of phagocytic cells and particularly microglia towards axonal regeneration remains unclear. Initially, to assess this, tuftsin 1-3's effect on axonal regeneration was reexamined by doing a dose-response study. Optimal doses were found to be 2.5 μg/ml and 250 μg/ml in rats and hamsters respectively. We then studied retinal phagocytic cells in rats. Microglial cells were classified as resting or activated based on their morphology following OX42 immunolabelling. In controls, most microglial cells were in the resting state. Optic nerve cut led to an increase in the total number of microglia and a ten-fold elevation in the proportion of activated cells; changes were more pronounced at the optic nerve stump. Anastomosis of an autologous segment of sciatic nerve to the stump of the freshly cut optic nerve minimized the overall increase in microglia, and combined with 2.5 μg/ml tuftsin 1-3, lead to a marked blunting of activation. Preservation within the retina of a higher proportion of resting over active form of microglia, and not the prevention of microglial proliferation per se, may be a crucial factor in allowing additional retinal ganglion cell axons to regenerate into peripheral nerve grafts.     

Muscle spindle formation and differentiation in regenerating rat muscle grafts

Muscle spindle development and function are dependent upon sensory innervation. During muscle regeneration, both neural and muscular components of spindles degenerate and it is not known whether reinnervation of a regenerating muscle results in reestablishment of proper neuromuscular relationships within spindles or whether sensory neurons may exert an influence upon differentiation of these spindles. Muscle spindle regeneration was studied in bupivacaine-treated grafts of rat extensor digitorum longus (EDL) muscles. Three types of EDL graft were performed in order to manipulate the extent to which regenerating spindles might be reinnervated: (1) grafts reinnervated following severance of their nerve supply (standard grafts); (2) grafts in which intact nerve sheaths appear to facilitate reinnervation (nerveintact grafts); and (3) grafts in which reinnervation was prevented (nonreinnervated grafts). Complete degeneration of muscle fibers occurred in all grafts prior to regeneration. Initial formation of spindles in regenerating EDL grafts is independent of innervation; intrafusal muscle fibers degenerate and regenerate within spindle capsules that remain intact and viable. The extent of spindle differentiation was evaluated in each type of graft using criteria that included nucleation and ATPase activity, both of which have been shown to be regulated by sensory innervation, as well as the number of muscle fibers/spindle and morphology of spindle capsules.While most spindles contained normal numbers of muscle fibers, most of these fibers were morphologically and histochemically abnormal. Alterations of ATPase activity occurred in all spindles, but were least severe in nerve-intact grafts. While fully differentiated nuclear bag and chain fibers were not observed in regenerated spindles, large, vesicular nuclei, similar to those of normal intrafusal fibers, were present in a small number of spindles in nerve-intact grafts. Sensory nerve terminations were observed only in those spindles that also contained the distinctive nuclei. This study suggests that a specific neurotrophic influence is necessary for regeneration of normal intrafusal muscle fibers and that this influence corresponds to the properly timed sensory neuron-muscle interaction which directs muscle spindle embryogenesis. However, the infrequent occurrence of characteristics unique to intrafusal muscle fibers indicates that reinnervation of regenerating muscle grafts by sensory neurons is inadequate and/or faulty.     

4-aminoquinoline-induced 'giant' miniature endplate potentials at mammalian neuromuscular junctions

4-Aminoquinoline (4-AQ) in concentrations around 200 micrometers induces, within minutes of its application to isolated mouse or rat neuromuscular junctions, the appearance of a population of miniature endplate potentials (m.e.p.ps) with a larger than normal amplitude, so-called giant m.e.p.ps (g.m.e.p.ps). With amplitudes 2-12 times the modal value of m.e.p.p. amplitude, the population of g.m.e.p.ps varied between 15 and 45% of the total population of m.e.p.ps. There was no increase in the frequency of m.e.p.ps but a positive correlation between the frequency of g.m.e.p.ps and the total frequency of m.e.p.ps. In many instances the rise time and decay time of g.m.e.p.ps were prolonged compared to normal. Elevated extracellular calcium concentrations increased the frequency of m.e.p.ps but had no effect on g.m.e.p.p. frequency. High extracellular potassium concentrations markedly increased m.e.p.p. frequency but failed to influence g.m.e.p.p. frequency. Similar observations were made with ethanol 0.1 M, ouabain 200 micrometers or black widow spider venom. Botulinum toxin type A markedly reduced total m.e.p.p. frequency but 4-AQ still induced g.m.e.p.ps. Nerve stimulation failed to release quanta corresponding to the g.m.e.p.ps. G.m.e.p.ps seemed to originate from quantal acetylcholine release from the nerve terminal since they were abolished by surgical denervation and by the addition of d-tubocurarine to the medium. Blockade of voltage-sensitive calcium or sodium channels by, respectively, manganese ions or tetrodotoxin failed to affect the appearance and the frequency of g.m.e.p.ps. The electrophysiological findings and a statistical analysis of the characteristics of the m.e.p.ps indicate that they belong to two populations. One population is accelerated by the depolarization-release coupling mechanism responsible for evoked transmitter release and is characterized by an amplitude distribution and a process in time that indicate that they correspond to releases occurring at 'active zones' in the nerve terminal. The second population of m.e.p.ps is uninfluenced by nerve terminal depolarization and transmembrane calcium fluxes. This population apparently originates from sites dispersed in the nerve terminal membrane and outside the 'active zones'. 4-AQ increases the frequency of this second m.e.p.p. population without affecting the first population.     

Regeneration of specific innervation in Xenopus pectoralis muscle

We investigated the motor unit organization and precision of reinnervation in the Xenopus pectoralis muscle following different manipulations, including crush or section of the posterior pectoralis nerve, foreign nerve innervation, and crush coupled with activity modulation or block. Most fibers have two neuromuscular junctions, and multielectrode recordings were used to identify the axonal origin of all inputs to both junctions on most or all fibers covering about 25% of the muscle surface. Following simple nerve crush, a highly organized innervation pattern was restored, indistinguishable from the normal pattern, including selective innervation of fibers of similar input resistance (R_in), compact motor unit organization, and high incidence of exclusive innervation of both end plates on each fiber by the same axon (distributed mononeuronal innervation, or a/a pattern). Initial reinnervation was equally precise when nerve conduction in the regenerating nerve was blocked by tetrodotoxin. More distant or repeated nerve crush or nerve section delayed and reduced the precision of reinnervation, but the majority of fibers still received input to both end plates by the same axon, often in combination with others. A foreign nerve, the pectoralis sternalis, which in its own muscle forms only single end plates, showed less precise reinnervation, but still had an incidence of a/a innervation far above chance. These data imply the expression and recognition of remarkably precise chemospecific cues even in mature animals, superimposed on which is a further refinement by synapse elimination, probably based on an activity-dependent process. © 1996 John Wiley & Sons, Inc.     

Denervated muscle fibers explain the deficit in specific force following reinnervation of the rat extensor digitorum longus muscle

The authors tested the hypothesis that, after denervation and reinnervation of skeletal muscle, observed deficits in specific force can be completely attributed to the presence of denervated muscle fibers. The peroneal nerve innervating the extensor digitorum longus muscle in rats was sectioned and the distal stump was coapted to the proximal stump, allowing either a large number of motor axons (nonreduced, n = 12) or a drastically reduced number of axons access to the distal nerve stump (drastically reduced, n = 18). A control group of rats underwent exposure of the peroneal nerve, without transection, followed by wound closure (control, n = 9). Four months after the operation, the maximum tetanic isometric force (Fo) of the extensor digitorum longus muscle was measured in situ and the specific force (sFo) was calculated. Cross-sections of the muscles were labeled for neural cell adhesion molecule (NCAM) protein to distinguish between innervated and denervated muscle fibers. Compared with extensor digitorum longus muscles from rats in the control (295 +/- 11 kN/m2) and nonreduced (276 +/- 12 kN/m2) groups, sFo of the extensor digitorum longus muscles from animals in the drastically reduced group was decreased (227 +/- 15 kN/m2, p < 0.05). The percentage of denervated muscle fibers in the extensor digitorum longus muscles from animals in the drastically reduced group (18 +/- 3 percent) was significantly higher than in the control (3 +/- 1 percent) group, but not compared with the nonreduced (9 +/- 2 percent) group. After exclusion of the denervated fibers, sFo did not differ between extensor digitorum longus muscles from animals in the drastically reduced (270 +/- 20 kN/m2), nonreduced (301 +/- 13 kN/m2), or control (303 +/- 10 kN/m2) groups. The authors conclude that, under circumstances of denervation and rapid reinnervation, the decrease in sFo of muscle can be attributed to the presence of denervated muscle fibers.     

Habitual exercise enhances neuromuscular transmission efficacy of rat soleus muscle in situ.

Rat motor nerve terminals and the endplates they interact with exhibit changes to varying patterns of use, as when exposed to increased activation in the form of endurance exercise training. The extent to which these changes affect neuromuscular transmission efficacy is uncertain. In this study, the effects of habitual exercise on the electrophysiological properties of neuromuscular transmission in rat soleus muscle were investigated using a novel in situ approach. Consistent with previous reports, miniature endplate potential frequency was enhanced by habitual exercise. Other passive properties, such as resting membrane potential, miniature endplate potential amplitude, and "giant" miniature endplate potential characteristics were unaltered by the training program. Full-size endplate potentials were obtained by blocking soleus muscle action potentials with mu-conotoxin GIIIb. Quantal content values were 91.5 and 119.9 for control and active groups, respectively (P < 0.01). We also measured the rate and extent of endplate potential amplitude rundown during 3-s trains of continuous stimulation at 25, 50, and 75 Hz; at 50 and 75 Hz, we found both the rate and extent of rundown to be significantly attenuated (10--20%) in a specific population of cells from active rats (P < 0.05). The results establish the degree of activity-dependent plasticity as it pertains to neuromuscular transmission in a mammalian slow-twitch muscle.