Satellite cells in slow and fast rat muscles differ in respect to acetylcholinesterase regulation mechanisms they convey to their descendant myofibers during regeneration

The hypothesis of satellite cell diversity in slow and fast mammalian muscles was tested by examining acetylcholinesterase (AChE) regulation in muscles regenerating (1) under conditions of muscle disuse (tenotomy, leg immobilization) in which the pattern of neural stimulation is changed, and (2) after cross-transplantation when the regenerating muscle develops under a foreign neural stimulation pattern. Soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat were allowed to regenerate after ischemic-toxic injury either in their own sites or had been cross-transplanted to the site of the other muscle. Molecular forms of AChE in regenerating muscles were analyzed by velocity sedimentation in linear sucrose gradients. Neither tenotomy nor limb immobilization significantly affected the characteristic pattern of AChE molecular forms in regenerating SOL muscles, suggesting that the neural stimulation pattern is probably not decisive for its induction. During an early phase of regeneration, the general pattern of AChE molecular forms in the cross-transplanted regenerating muscle was predominantly determined by the type of its muscle of origin, and much less by the innervating nerve which exerted only a modest modifying effect. However, alkali-resistant myofibrillar ATPase activity on which the separation of muscle fibers into type I and type II is based, was determined predominantly by the motor nerve innervating the regenerating muscle. Mature regenerated EDL muscles (13 weeks after injury) which had been innervated by the SOL nerve became virtually indistinguishable from the SOL muscles in regard to their pattern of AChE molecular forms. However, AChE patterns of mature regenerated SOL muscles that had been innervated by the EDL nerve still displayed some features of the SOL pattern. In regard to AChE regulation, muscle satellite cells from slow or fast rat muscles convey to their descendant myotubes the information shifting their initial development in the direction of either slow or fast muscle, respectively. The satellite cells in fast or slow muscles are, therefore, intrinsically different. Intrinsic information is expressed mostly during an early phase of regeneration whereas later on the regulatory influence of the motor nerve more or less predominates. © 1994 Wiley-Liss, Inc.     

Expression of superfast myosin in aneural regenerates of cat jaw muscle

We investigated whether innervation is necessary for the expression of superfast myosin in regenerating cat jaw-closing muscle. Strips of jaw muscle were permitted to regenerate bilaterally in the beds of a fast limb muscle with innervation on one side being prevented surgically. Immunocytochemical analyses using anti-myosin heavy chain antibodies were done at various times postoperatively, the latest being after 78 days. We found little difference between innervated and uninnervated regenerates up to 27 days postoperatively. All regenerating myotubes expressed fetal myosin. In addition, some myotubes stained for superfast or slow myosin, while others stained for both superfast and slow myosins. Subsequently, uninnervated myotubes became atrophic but continued to express fetal, slow, and superfast myosins while innervated myofibers suppressed fetal and slow myosin expression. These results are consistent with the notion that satellite cells of jaw-closing muscles are committed to express superfast myosin during myogenesis, and that the expression of this program is independent of innervation.     

Changes in the cholinergic system of rat sciatic nerve and skeletal muscle following suspension-induced disuse

Muscle disuse-induced changes in the cholinergic system of sciatic nerve, slow-twitch soleus (SOL), and fast-twitch extensor digitorum longus (EDL) muscles were studied in rats. Rats with hind limbs suspended for 2 to 3 weeks showed marked elevation in the activity of choline acetyltransferase in sciatic nerve (38%), in the SOL (108%), and in the EDL (67%). Acetylcholinesterase (AChE) activity in the SOL increased 163% without changing the molecular forms pattern of 4S, 10S, 12S, and 16S. No significant (P greater than 0.05) changes in the activity and molecular forms pattern of AChE were seen in the EDL or in AChE activity of sciatic nerve. Nicotinic receptor binding of [3H]acetylcholine was increased in both muscles. When measured after 3 weeks of hind limb suspension the normal distribution of type I fibers in the SOL (87%) was reduced (to 58%) and a corresponding increase in types IIa and IIb fibers occurred. In the EDL no significant change in fiber proportion was observed. Muscle activity, such as loadbearing, appeared to have a greater controlling influence on the characteristics of the slow-twitch SOL muscle than on the fast-twitch EDL muscle.     

Reinnervation of a denervated slow muscle triggers high extrajunctional expression of the asymmetric molecular forms of acetyicholinesterase

Expression of acetylcholine receptor and of the asymmetric molecular forms of acetylcholinesterase (AChE) in the extrajunctional regions of rat muscles is suppressed during early postnatal development. In mature muscles, the extrajunctional synthesis of acetylcholine receptor, but not of the asymmetric molecular forms of AChE, becomes reactivated after denervation. The hypothesis that a denervated muscle needs reinnervation in order to revert transiently to an immature state characterized by high extrajunctional production of the asymmetric AChE forms, was examined in rat muscles recovering after nerve crush. Molecular forms of AChE were analysed by velocity sedimentation. Activity of the asymmetric A12 AChE form in the extrajunctional regions of the slow soleus (SOL) muscle increased during the first week after reinnervation to about 9 times its control level, remained high for about one week, and declined towards normal thereafter. If the nerve was crushed close to the muscle and reinnervation occured very rapidly, the extrajunctional increase of the A₁₂ AChE form still occured but was less pronounced than after late reinnervation. In contrast, a transient paralysis of the SOL muscle due to acetylcholine receptor blockade by α-bungarotoxin, followed by spontaneous recovery of muscle activity after 3–5 days, did not revert AChE regulation into an immature state. Disuse of the SOL muscle caused by leg immobilization, which is known to change the tonic pattern of neural stimulation of the SOL muscle into a phasic one, did not prevent the reversion of AChE regulation during reinnervation. This indicates that neural stimulation pattern is not crucial for this reversion. In contrast to slow SOL, the fast extensor digitorum longus muscle did not revert to an immature state in respect to AChE regulation after reinnervation. This muscle type-specific response may be due to intrinsic differences between the myogenic cells of slow and fast muscle fibres. © 1995 Wiley-Liss, Inc.     

Continuous low amplitude direct current stimulation of the crushed peripheral nerve accelerates the early recovery of choline acetyltransferase but not of acetylcholinesterase activity in fast and slow muscles

We investigated if continuous 1 μA direct current stimulation of the injured nerve, with the cathode electrode at the distal end of the nerve crush injury (cathode stimulation), accelerated the recovery of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity in transiently denervated extensor digitorum longus (EDL) and soleus (SOL) rat muscles. ChAT is a specific marker of cholinergic nerve terminals and may reflect axon ingrowth, and AChE reflects the re-establishment of neuromuscular junctions and recovery of muscle activity. Compared to sham operated animals, the cathode (CA) stimulated rats had a statistically significant larger ChAT activity in the EDL and SOL muscles on days 12 and 14 after nerve crush (P < 0.01, n = 6). The difference in ChAT activity between the groups decreased thereafter. Regarding recovery of muscle AChE, CA stimulation of the crushed sciatic nerve did not detectably accelerate the normalization of activity and pattern of AChE molecular forms in the EDL and SOL muscles. This means that the early rise in ChAT muscle activity in CA stimulated rats was not followed by an accelerated normalization of the neuromuscular transmission in the same group. It is more likely that the higher ChAT activity observed after cathode stimulation indicates a higher ChAT content in regenerating motor nerve endings, rather than a greater number of motor axons entering the muscles. It seems possible that cathode stimulation increased ChAT axonal transport, causing the early increase of ChAT content in the nerve endings. This raises the possibility that the axon transport and subsequent secretion of a trophic factor(s) from the nerve to the reinnervated muscle are enhanced as well, thus shortening the overall time of muscle force recovery in the absence of an appreciable acceleration of recovery of the neuromuscular transmission.     

Muscle activity-resistant acetylcholine receptor accumulation is induced in places of former motor endplates in ectopically innervated regenerating rat muscles

Expression of acetylcholine receptors (AChRs) in the extrajunctional muscle regions, but not in the neuromuscular junctions, is repressed by propagated electric activity in muscle fibers. During regeneration, subsynaptic-like specializations accumulating AChRs are induced in new myotubes by agrin attached to the synaptic basal lamina at the places of former motor endplates even in the absence of innervation. We examined whether AChRs still accumulated at these places when the regenerating muscles were ectopically innervated and the former synaptic places became extrajunctional. Rat soleus muscles were injured by bupivacaine and ischemia to produce complete myofiber degeneration. The soleus muscle nerve was permanently severed and the muscle was ectopically innervated by the peroneal nerve a few millimeters away from the former junctional region. After 4 weeks of regeneration, the muscles contracted upon nerve stimulation, showed little atrophy and the cross-section areas of their fibers were completely above the range in non-innervated regenerating muscles, indicating successful innervation. Subsynaptic-like specializations in the former junctional region still accumulated AChRs (and acetylcholinesterase) although no motor nerve endings were observed in their vicinity and the cross-section area of their fibers clearly demonstrated that they were ectopically innervated. We conclude that the expression of AChRs at the places of the former neuromuscular junctions in the ectopically innervated regenerated soleus muscles is activity-independent.     

Regenerated EDL muscle of rats requires innervation to maintain AChE molecular forms

Extensores digitorum longi of rats, infarcted and denervated by different surgical procedures, were used to analyze by biochemical and cytochemical methods the acetylcholinesterase (AChE) changes during muscle degeneration, regeneration, and early or delayed reinnervation. Biochemical tests showed that the regenerating muscle produces globular AChE forms (36% of controls) and small amounts of A12 (16S) asymmetric form (5% of controls); at the end of the regeneration, innervation and electromechanical function are required for the complete recovery of globular forms, and are absolutely critical to prevent A12 (16S) disappearance. Cytochemical observations showed that, unlike nicotinic receptor, AChE deposited at the neuromuscular junction before ischemic necrosis is protected from breakdown, as is the basal lamina of muscle fibers. Taken together, these observations contribute to the understanding of the factors that play a critical role in muscle repair and are, therefore, of clinical relevance.     

Specific impulse patterns regulate acetylcholinesterase activity in skeletal muscles of rats and rabbits

In rats, acetylcholinesterase (AChE) activity in the fast muscles is several times higher than in the slow soleus muscle. The hypothesis that specific neural impulse patterns in fast or slow muscles are responsible for different AChE activities was tested by altering the neural activation pattern in the fast extensor digitorum longus (EDL) muscle by chronic low-frequency stimulation of its nerve. In addition, the soleus muscle was examined after hind limb immobilization, which changed its neural activation pattern from tonic to phasic. Myosin heavy-chain (MHC) isoforms were analyzed by gel electrophoresis. Activity of the molecular forms of AChE was determined by velocity sedimentation. Low-frequency stimulation of the rat EDL for 35 days shifted the profile of MHC II isoforms toward a slower MHCIIa isoform. Activity of the globular G₁ and G₄ molecular forms of AChE decreased by a factor of 4 and 10, respectively, and became comparable with those in the soleus muscle. After hind limb immobilization, the fast MHCIId isoform, which is not normally present, appeared in the soleus muscle. Activity of the globular G₁ form of AChE increased approximately three times and approached the levels in the fast EDL muscle. In the rabbit, on the contrary to the rat, activity of the globular forms of AChE in a fast muscle increased after low-frequency stimulation. The results demonstrate that specific neural activation patterns regulate AChE activity in muscles. Great differences, however, exist among different mammalian species in regard to muscle AChE regulation. J. Neurosci. Res. 47:49–57, 1997. © 1997 Wiley-Liss, Inc.     

Regulation by exercise of the pool of G4 acetylcholinesterase characterizing fast muscles: opposite effect of running training in antagonist muscles

Fast muscles of rodents characteristically differ from their slow-twitch counterparts by exhibiting high levels of G4, i.e., the tetrameric acetylcholinesterase (AChE) molecular form. Converging evidence suggests that this additional G4 pool is specifically regulated by the type of activity actually performed by the muscle. This hypothesis was tested by studying the effect of a chronic increase in neuromuscular activity on the AChE content and distribution of molecular forms of functionally antagonist rat hindlimb muscles. They included the fast ankle extensors gastrocnemius (GAST) and plantaris (PL), the fast ankle flexors tibialis anterior (TA) and extensor digitorum longus (EDL), as well as the slow-twitch soleus (SOL). Neuromuscular activity was enhanced by subjecting the rats to a 12-week training program consisting of repeated sessions of prolonged endurance running on a rodent treadmill. This exercise regimen preferentially affected the G4 pool characterizing fast muscles which exhibited marked and opposite changes according to the functional role of the muscles. The amount of G4 was increased by more than 50% in the ankle extensors GAST and PL, which play a dynamic role, and reduced by about 40% in the ankle flexors TA and EDL, which exhibit a predominant tonic activity during running. The asymmetric forms A12 and A8 were slightly elevated in the fast muscles. In the case of the slow-twitch SOL, running training resulted in a small, nonspecific decrease in AChE content which affected most of the molecular forms. These data indicate that the size of the G4 pool characteristic of fast muscles depends on the type, dynamic or tonic, of activity actually performed. The present results support the conclusion that this G4 pool fulfills a specific and essential function, distinct from that of A12.     

Comparison of contractile properties between developing and regenerating soleus muscle: Influence of external calcium concentration upon the contractility

In newborn rat skeletal extensor digitorum longus (EDL) muscle, it has been found that an influx of calcium from the extracellular medium is necessary for contraction, in contrast to the situation observed in adult EDL muscle. The aim of the present study was to determine the influence of the extracellular calcium concentration ([Ca]o) upon the contractile responses elicited in developing as well as in regenerating (notexin-injected) soleus (SOL) muscle. A morphological study was performed to follow the steps of postnatal development and regeneration in SOL muscle. In nominally calcium-free solution, the amplitudes of the twitch and tetanic tensions were greatly reduced in 1–14-day-old developing SOL muscles, as well as in notexin-injected SOL muscles. With longer times after birth, twitch and tetanic tensions of SOL muscle were less affected by the absence of calcium. This contrasts with notexin-injected SOL muscle in which the amplitudes of the contractions remained strongly dependent on [Ca]o. The present finding suggests that some functional characteristics are different in regenerating muscle fibers and may be of interest in the evaluation of the contractile properties of muscles in which injections of genetically engineered or not autologous myoblasts or viral vector have been performed. © John Wiley & Sons, Inc.     

Congruity of acetylcholine receptor,acetylcholinesterase, and Dolichos biflorus lectin binding glycoprotein in postsynaptic-like sarcolemmal specializations in noninnervated regenerating rat muscles

Noninnervated regenerating muscles are able to form focal postsynaptic-like sarcolemmal specializations either in places of the former motor endplates ( “junctional” specializations) or elsewhere along the muscle fibers (extrajunctional specializations). The triple labeling histochemical method was introduced to analyse the congruity of focalization in such specializations of 3 synaptic components: acetylcholinesterase (AChE), acetylcholine receptor (AChR), and a specific synaptic glycoprotein which binds Dolichos biflorus lectin (DBAR). Noninnervated regenerating soleus and extensor digitorum longus (EDL) muscles of the rat were examined and compared with denervated muscles of neonatal and adult rats. All junctional sarcolemmal specializations in noninnervated regenerating muscles accumulated AChE and AChR. Localization of the 2 components was identical within the limits of resolution of the method. DBAR could not be demonstrated in junctional specializations in 17-day-old regenerating muscles. It seems that an agrin-like inducing substance in the former junctional basal lamina invariably triggers the accumulation of both AChE and AChR in the underlying sarcolemma of the regenerating muscle fiber. However, accumulation of DBAR would probably require the presence of the motor nerve. In most of the extrajunctional sarcolemmal specializations in 8-day-old regenerating soleus and EDL muscles, both AChE and AChR accumulated. However, about 10 percent of AChE accumulations lacked AChR and about 35% of AChR accumulations lacked AChE. Even greater variability was observed in 17-day-old regenerating muscles. The presence of DBAR in the extrajunctional postsynaptic-like sarcolemmal specializations could not be demonstrated. Similar extrajunctional sarcolemmal specializations were observed in denervated postnatal rat muscles. About 70% contained both AChE and AChR, and 30% contained only AChR, but none contained DBAR. In denervated mature muscles, sparse extrajunctional AChR accumulations did not contain detectable amounts of AChE. The ability to form complex postsynaptic-like sarcolemmal specializations in the absence of nerve, which is probably inherent to noninnervated immature muscle fibers, may be reduced with muscle maturation. Variable accumulation of individual components in the postsynaptic-like specializations indicates that different triggering factors may be involved in their accumulation or, at least, the mechanisms of their accumulation can function relatively independently. © 1993 Wiley-Liss, Inc.     

Abnormal distribution of skeletal muscle acetylcholinesterase molecular forms in dystrophic mice

Acetylcholinesterase (AChE) activities and molecular forms, as separated by density gradient centrifugation, were studied in dystrophic and clinically normal mouse muscle. Dystrophic hemidiaphragms exhibited normal AChE activity, but there was little or no 10 S enzyme, a form that constitutes 27% of control tissue AChE. The 10 S-AChE abnormality was similarly present in dystrophic extensor digitorum longus (EDL) muscle, but this muscle exhibited significantly reduced AChE activity. The EDL muscles also had reduced 16 S-AChE but normal 4 S enzyme activity. Chronic denervation of EDL muscles resulted in proportionally similar reductions of weight, total AChE, and 16 S enzyme in dystrophic and control muscles. We conclude that murine dystrophy involves some alterations that resemble denervation, but that there are major qualitative and quantitative differences in AChE that cannot be explained by a denervation-like effect.     

Neural regulation of muscle acetylcholinsterase: effects of batrachotoxin and 6-aminonicotinamide.

Batrachotoxin (BTX), which causes increased Na+ permeability and blocks axoplasmic transport, or 6-aminonicotinamide (6-AN), which causes neuronal damage, was injected into the subarachnoid space of rat lumbar spinal cord. The activity of acetylcholinesterase (AChE) was measured in homogenates of the fast-twitch extensor digitorum longus (EDL) muscle and the slow-twitch soleus (SOL) muscle 10 days after injection. Both drug treatments significantly decreased AChE in EDL and SOL. Correlative electrophysiological measurements were made in intact EDL and SOL after injection of BTX or 6-AN. The results support the hypothesis that AChE in muscle is neurotrophically controlled.     

Muscle activity pattern regulates postnatal development of acetylcholinesterase molecular forms in normal mice and mice with motor endplate disease

We have studied the relative contributions of muscle activity and nerve-supplied materials to the regulation of AChE molecular forms during postnatal development of muscles in normal mice and in mice with motor endplate disease (med mice). Onset of this hereditary disease causes a progressive failure of evoked release of ACh from the motor neuron, which prevents contraction in muscles such as biceps and soleus. In these innervated but inactive muscles, one can examine the consequences of inactivity on the distribution of AChE forms. In normal mouse biceps the distribution of AChE forms, as shown by sucrose-gradient analysis, change substantially after birth; the most dramatic alteration is an increase in G4 AChE from 15 to 45% of total AChE during the third postnatal week. AChE profiles in normal or med biceps are indistinguishable until 10-12 d after birth, but the changes in distribution of AChE forms does not occur in med biceps nor in normal biceps denervated 2 weeks after birth. In contrast, the distributions of AChE forms in a predominantly slow muscle, the soleus, are similar in med and normal mice both early (10 d) and late (20 d) in the course of the disease, and the distributions are affected little by denervation. The profiles of AChE forms seen in normal soleus at all times studied resembled those seen in newborn biceps or biceps inactivated by denervation or the med disease. We conclude that neither innervation, age-dependent changes intrinsic to muscle, nor muscle activity is sufficient to induce the changes we seen in AChE forms in biceps. These results support the hypothesis that neonatal, inactive, or tonically active muscles produce an intrinsic pattern of AChE molecular forms, and that a phasic pattern of activity induces a postnatal redistribution of the AChE molecular forms expressed by the muscle.     

Acetylcholinesterase molecular forms in C57BL/6J dystrophic mice

Acetylcholinesterase (AChE) was extracted from normal and dystrophic C57BL/6J mouse hindlimb muscles and its molecular forms fractionated by sucrose density gradient ultracentrifugation. In the soleus muscles from 6- to 7-week-old mice an increase in the 3 Svedberg unit (S) and a decrease in the 16S AChE molecular forms was observed in dystrophic animals compared to controls. At 12-13 weeks of age, no major significant differences in the relative proportions of AChE molecular forms were noted. In the extensor digitorum longus (EDL) muscles of 6- to 7-week-old dystrophic mice a significant decrease in the proportion of the 10S AChE molecular form and an increase in the 3S and 5S forms was observed. At 12-13 weeks, the dystrophic EDL muscles again displayed a decrease in the 10S form; however, the increase in the 3S and 5S AChE forms, while still apparent, was not significant. These results provide evidence for a biochemical abnormality in the distribution of specific AChE molecular forms, and a differential expression of this abnormality in the soleus and EDL muscles.     

The effect of spontaneous electromechanical activity on the metabolism of acetylcholinesterase in cultured embryonic rat myotubes

We have investigated the effect of electromechanical activity on the molecular forms of acetylcholinesterase (AChE) in cultured embryonic rat myotubes. Both globular and asymmetric forms of AChE are present on the 5th day of culture when myotubes are just beginning to fibrillate. Between days 5 and 8, the 4 S (G1), 10 S (G4), and 16 S (A12) forms increase dramatically, and appreciable 12.5 S (A8) AChE appears. When fibrillation is prevented by adding tetrodotoxin on day 4, the increases in the A12 and A8 forms are prevented, and the increases in the G4 and G1 forms are significantly impaired. At 8 days, fibrillating myotubes have 19 times more A12 AChE and over 4 times more G1 and G4 enzyme than do nonfibrillating myotubes. The effect of tetrodotoxin is reversible. When tetrodotoxin is removed at 7 days, fibrillation resumes promptly, and globular and asymmetric forms recover. Light microscopic examination of fibrillating and nonfibrillating myotubes showed that tetrodotoxin does not affect the gross morphological development of the myotubes. Titration of AChE-active sites with O-ethyl-S2-diisopropyl methyl-phosphonothionate demonstrated that the increase in AChE activity associated with fibrillation is due to an increase in the number of AChE molecules present and not to an increase in the rate at which individual AChE molecules turn over acetylcholine. To evaluate AChE metabolism in fibrillating and nonfibrillating myotubes, we examined the enzyme after inactivating it with paraoxon. Paraoxon readily penetrates cells and diethylphosphorylates a serine in the active site of AChE, thereby inactivating it. The diethylphosphorylated enzyme is stable, but it can be reactivated rapidly and quantitatively with pyridine-2-aldoxime methiodide (2-PAM). After inactivating AChE with paraoxon, we simultaneously evaluated synthesis (by following the newly synthesized active AChE) and turnover (by following the 2-PAM-reactivatable AChE). Our results show that globular and asymmetric forms of AChE are both synthesized more rapidly in fibrillating than in nonfibrillating myotubes.     

Accumulation of acetylcholine receptors is a necessary condition for normal accumulation of acetylcholinesterase during in vitro neuromuscular synaptogenesis

To study a step of the very complex processes of the formation of the neuromuscular junction (NMJ), we have analysed the clustering of acetylcholine receptors (AChR) and acetylcholinesterase (AChE) in myotubes cultured in various conditions. On the surface of rat myotubes cultured in the presence of spinal cord cells from embryonic rat, numerous AChE clusters appeared. Such clusters are always co-localized with AChR clusters, but the reverse is not true: the number of AChR clusters largely exceeds that of AChE clusters. Very few AChE clusters formed when such co-cultures were treated with monoclonal antibodies (mAbs) against the main immunogenic region (MIR) of the AChR, which provoke internalization and degradation of the AChRs of the muscular membrane. The total levels of AChE and proportions of molecular forms were unaffected. We also used non-innervated myotubes in which addition of agrin, a protein normally synthesized by motoneurons, transported to nerve terminals and inserted into the synaptic basal lamina, induces the formation of small clusters of AChE. When added to rat myotubes devoid of membrane AChR, agrin-induced AChE clusters did not form. Finally, we analysed the capacity of the variant of the C2 mouse muscle cell line deficient in AChR (1R-) to form clusters of AChE in co-cultures with spinal cord cells from rat: no formation of AChE clusters could be observed. In all these different systems of cultures, the conditions which prevented clustering of AChR (anti-AChR antibodies, deficiency of the variant C2 cell line) also suppressed AChE clustering. We concluded that clustering of AChR is a prerequisite for clustering of AChE, so that NMJ formation implies the sequential accumulation of these two components.     

Expression and Localization of PRiMA-Linked Globular Form Acetylcholinesterase in Vertebrate Neuromuscular Junctions

Acetylcholinesterase (AChE) is well known to process different molecular forms via the distinct interacting partners. Proline-rich membrane anchor (PRiMA)-linked tetrameric globular AChE (G4 AChE) is mainly found in the vertebrate brain; however, recent studies from our laboratory have suggested its existence at neuromuscular junctions (nmjs). Both muscle and motor neuron express AChE at the nmjs. In muscle, the expression of PRiMA-linked AChE is down-regulated during myogenic differentiation and by motor neuron innervation. As compared with muscle, spinal cord possessed higher total AChE activity and contained PRiMA-linked AChE forms. The spinal cord expression of this form increased during development. More importantly, PRiMA-linked G4 AChE identified as aggregates localized at nmjs. These findings suggest that the restricted localization of PRiMA-linked G4 AChE at the nmjs could be contributed by the pre-synaptic motor neuron and/or the post-synaptic muscle fiber.     

A distinct difference between slow and fast muscle in acetylcholinesterase recovery after reinnervation in the rat

The changes in acetylcholinesterase (AChE) and choline acetyltransferase (CAT) activity in nerve proximal and distal to the crush site as well as in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle were studied during denervation and reinnervation in rat. Within 24 h after nerve crush, conduction in the distal nerve and neuromuscular transmission was lost. In the distal nerve segment, AChE and CAT activity showed no initial increase and was reduced to 25% 14 days after crush. During the reinnervation period, AChE and CAT activity recovered to 50% (AChE) and 80% (CAT) of control values and CAT activity in the EDL and SOL muscles followed closely the changes in distal nerve. In muscle, AChE activity was reduced to 15% by 2 weeks postoperatively. Enzyme activity in EDL recovered to 50% of control activity in 5 weeks after crush. In the SOL, end-plate and non-end-plate regions' AChE activity recovered at a faster rate, resulting in a temporary increase in AChE activity to more than control values during the third and fourth week. By the end of the fifth week, AChE activity had returned to control activity. Turnover values for AChE based on the reinnervation data showed a half-life value for AChE in proximal nerve of 32 days, in distal nerve 42 days, in EDL 23 days, and for SOL 5.1 days. The half-life for AChE in both muscles was significantly shorter than that of the nerve, indicating that the nerve did not supply AChE to the muscles. Half-lives for CAT calculated on the basis of the reinnervation data were 11.6 days for proximal nerve, 18.4 days for distal nerve, and 30 days for SOL and EDL muscles. It is concluded that the ability to synthesize AChE in end-plate and non-end-plate regions of muscle is an endogenously programmed event in the development of both fast and slow muscles. The nerve initiates and maintains the synthesis and can modify the rate of synthesis in individual muscle fibers. The mechanism by which the nerve stimulates and maintains AChE synthesis in muscle may be related to the release of trophic factors muscle activity or to a combination of these and other factors still to be investigated.     

Effects of denervation and immobilisation during development upon [3H]ouabain binding by slow- and fast-twitch muscle of the rat

The effect of innervation and of muscle inactivity upon the normal production of Na+-K+-ATPase sites, assayed by [3H]ouabain binding, in muscle surface membranes has been determined for the rat. In both slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles a large increase was found to occur in [3H]ouabain binding per unit weight of muscle over the first 3 weeks of life. Interruptions of development, brought about by fixation of muscles at different lengths at 5 days of age, had no significant effect upon [3H]ouabain binding by EDL. In contrast, fixation led to a decrease in binding in SOL. When fixed in a shortened position profound morphological changes occurred, although these were not apparent when SOL was fixed in a stretched position. Denervation of SOL at 5 days of age significantly reduced the age related increase in the density of [3H]ouabain binding, whilst denervation of EDL had little effect. It was concluded that normal development of SOL is dependent upon innervation and possibly the resulting muscle activity, whereas development of EDL was relatively independent of innervation.