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.     

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.     

Asymmetric molecular forms of acetylcholinesterase in mammalian skeletal muscles

Velocity sedimentation analysis of acetylcholinesterase (AChE) molecular forms was performed separately in endplate-rich and endplate-free regions of the diaphargm muscle of the rat, guinea pig, rabbit, dog, and pig, and in mm. erectores trunci and m. vastus lateralis in man. Several high-ionic-strength media were first tested to achieve better solubilization of AChE from rat muscles than by the usual 1 M NaCl- Triton X-100 medium. Ninety-five percent of the rat diaphragm was solubilized in a single extraction step by medium containing 1 M lithium chloride instead of NaCl. Homologous molecular forms of AChE were found in all species. The asymmetric forms were invariably present in the endplate regions of muscles but their activity in endplate-free regions was much lower than in endplate regions in all investigated mammals except in man. Essentially the same pattern of AChE molecular forms was present in both regions in human muscles. High extrajunctional activity of the asymmetric forms makes human muscles similar to immature rodent muscles in vivo and in culture. The pattern of AChE molecular forms in the endplate region of the diaphragm in senile 24-month-old rats was not significantly different from that in 3-month-old animals. The persistence of the asymmetric AChE forms in the diaphragm of senile rats suggests that neuromuscular interactions do not become deficient with age in this muscle.     

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.     

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.     

The cAMP-dependent protein kinase mediates the expression of AChE in chick myotubes

Calcitonin gene-related peptide (CGRP), a neuropeptide synthesized by motor neurons, stimulates the expression of AChR and AChE at the vertebrate neuromuscular junctions. The signaling mechanism of CGRP-induced AChE expression in muscle was determined both in vitro and in vivo. In cultured chick myotubes, the intracellular cAMP-dependent protein kinase (PKA) activity increased to approximately 2-fold after the application of CGRP or PKA activators; the induction was blocked by PKA inhibitors. in vivo transfection analysis on chick gastrocnemius muscles showed that the transfection of cDNA encoding constitutively active mutant Galphas increased the expression of AChE mRNA and protein to approximately 2-fold, while the constitutively active mutant Galphai cDNA transfection showed an opposite effect. The induced catalytic subunit of AChE at approximately 105 kDa was determined by specific antibody. These findings indicate that the CGRP-induced AChE expression in chick muscle is mediated by a PKA-dependent pathway.     

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.     

Two types of focal accumulations of acetylcholinesterase appear in noninnervated regenerating skeletal muscles of the rat

Muscle fibers in the soleus muscle of the rat, injured by bupivacaine and free autografting, were allowed to regenerate within their old basal laminae. Histochemical and cytochemical analysis of newly synthesized acetylcholinesterase (AChE) revealed that two kinds of focal accumulations of AChE appeared in regenerating myotubes. First, AChE gets concentrated at the sites of the former motor endplates. Accumulation of AChE starts in places where a tight contact between the remnants of the old junctional basal lamina and the budding surface of the myotube engulf the extracellular material. Appearance of these AChE accumulations can be prevented by papain treatment of the soleus muscle before autografting but not by predenervating it for 1 month. Focalization of AChE is probably induced by a component of the junctional basal lamina, possibly a protein, the existence of which is not dependent upon continuous presence of the motor nerve and may be produced by the muscle. This view is corroborated by the fact that an additional kind of AChE accumulation appeared in regenerating muscles in regions remote from the sites where motor endplates were located in the muscles of origin. Although differing in localization, size, and appearance, both kinds of AChE accumulations ultrastructurally resemble the postsynaptic specialization of the motor endplate: they consist of tubelike sarcolemmal invaginations containing AChE. The extrajunctional AChE accumulations seem to arise spontaneously and are usually located more than 750 micron away from the junctional ones as if some local inhibitory mechanism prevents their formation in the immediate vicinity.     

Acetylcholine receptors in extrajunctional regions of innervated muscle have a slow degradation rate.

Scanning EM autoradiography was used to determine the degradation rate of extrajunctional ACh receptors (AChRs) in innervated sternomastoid muscles of the mouse. We report that in innervated muscles, extrajunctional AChRs have a slow degradation rate (t1/2, approximately 8 d), similar to that seen at the neuromuscular junction. We conclude that slowly degrading AChRs (Rs) need not be localized at the specialized structure of the nerve-muscle junction. Degradation of extrajunctional as well as junctional AChRs may depend primarily on the state of innervation of the muscle.     

Different degradation rates of junctional and extrajunctional acetylcholine receptors of human muscle cultured in monolayer and innervated by fetal rat spinal cord neurons.

It is well demonstrated that in intact animals the degradation rate of the junctional acetylcholine receptor (AChR) is significantly slower than that of the extrajunctional receptor. Such data, however, are not available for human AChRs because the required experimentation cannot be performed in humans. We have now studied the degradation rate of the junctional and extrajunctional AChRs, utilizing our tissue culture model, in which well-differentiated neuromuscular junctions (NMJs) form on human muscle cultured in monolayer and innervated long-term by fetal rat spinal cord neurons. Half-life of AChRs was studied by a method utilizing the autoradiography of 125I-alpha bungarotoxin and computerized video image analysis. Extrajunctional AChRs degraded with a half-life of 1.3 days whereas junctional AChRs degraded with a half-life of 3.5 days. Our studies demonstrate for the first time that in innervated cultured human muscle: (a) the life span of human junctional AChR, is approximately 3 times longer than that of the extrajunctional AChR and (b) the stability of human AChR is neuronally regulated. This system can now be applied to evaluate the influence of pharmacologic agents on the stability of human junctional AChR, which is of potential importance in the treatment of myasthenia gravis and other diseases of the NMJ.     

Influence of innervation on molecular forms of acetylcholinesterase in regenerating fast and slow skeletal muscles

Nerve-intact muscle regenerates were prepared by ischemic-toxic injury of slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles of the rat. Rapid innervation of regenerating myotubes modified intrinsic patterns of AChE molecular forms, revealed by velocity sedimentation in linear sucrose gradients. Regarding their onset, the effects of innervation can be classified as early and late. The earliest changes in the SOL regenerates appeared a few days after innervation by their motoneurons: the activity of the 13 S AChE form (A 8) increased significantly in comparison to non-innervated regenerates. The pattern of AChE molecular forms became similar to that in the normal SOL muscle during the 2nd week after injury. In contrast, no major differences were observed between 8 day-old innervated and non-innervated EDL regenerates. Their patterns of AChE molecular forms resembled that in the normal EDL. However, the predominance of the 10 S AChE form (G 4) characteristic for the 2-week old non-innervated regenerates was prevented by innervation. Early effect of innervation observed in the SOL regenerates but not in the EDL may be due to intrinsically different response of the regenerating SOL myotubes to innervation. Rather high extrajunctional activity of the asymmetric 16 S (A 12) molecular form of AChE in early regenerates was reduced to adult level in about 3 weeks in the SOL, and nearly completely suppressed in 5 weeks after innervation in the EDL regenerates. This reduction is assumed to be a late effect of innervation, as well as a decrease of the activity of the 4 S AChE form (G 1) in the SOL regenerates. A suppressive mechanism is activated in the extra-junctional regions of the innervated muscle regenerates during their maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of spinal cord stimulation on the innervation pattern of muscle fibers in vivo.

In chick embryo, chronic stimulation of the brachial spinal cord at a fast rhythm from days 7 to 18 of development induced an increase in AChE activity sites and ACh receptor (AChR) clusters in slow anterior latissimus dorsi (ALD) muscle. Most AChR clusters and AChE spots were contacted by nerve endings. A previous study showed that such spinal cord stimulation causes changes in ALD muscle properties, especially the appearance of a high proportion of fast type II fibers (Fournier Le Ray et al., 1989). Analysis of the synaptic pattern in different fiber types of experimental ALD muscle indicated a decrease in the distance between successive AChE spots in slow type III fibers compared to controls, whereas the intersynaptic distance in fast type II fibers was very similar to that in the rare fast fibers developing in control ALD. Fast fibers of experimental muscles exhibited less AChR than did slow fibers. The increased number of neuromuscular junctions in ALD muscle after spinal cord stimulation appeared to be preferentially located in slow fibers. Electron microscopy showed no change in the number of axons in ALD nerve after spinal cord stimulation. The activity imposed on brachial motoneurons apparently caused terminal sprouting of ALD nerve in target muscle, thus accounting for the increase in neuromuscular contacts in ALD muscle fibers. Differences in the distribution of nerve contacts indicate that the type of muscle fiber innervated may play a critical role in the synaptic pattern during chick embryogenesis.     

Induction of synaptic extracellular matrix molecules at ectopic neuromuscular junctions

The induction of synapse-specific molecules recognized by peanut agglutinin (PNA) was examined at ectopic neuromuscular junctions in adult frog muscles using light and electron microscopy. In normal frog muscles, PNA specifically recognizes the extracellular matrix at neuromuscular junctions but not at extrajunctional regions. This report shows binding of PNA at ectopic neuromuscular junctions which were initially extrasynaptic and hence unrecognized by PNA. Results suggest that synapse-specific extracellular matrix molecules can be induced de novo at new junctional sites.     

Acetylcholinesterase activity of skeletal muscle in a non-immunogenic model for myasthenia gravis in rats

Summary. Myasthenia gravis is caused by an autoimmune attack to acetylcholine receptors of skeletal muscle. Acetylcholine release from motor nerve terminals is upregulated in patients with myasthenia gravis and also in rat "myasthenic" models, dependent on the reduction of the number of acetylcholine receptors. This study addresses the question as to whether at "myasthenic" endplates there are changes in the activity of acetylcholinesterase. To this end we studied acetylcholinesterase activity in junctional and extrajunctional regions of dilator naris, extensor digitorum longus, and hemidiaphragm muscles from rats with α-bungarotoxin-induced myasth-enia gravis. In all studied muscles from "myasthenic" rats there was no significant change of junctional acetylcholinesterase activity. In contrast, in dilator naris and extensor digitorum longus muscles, there was a 60% and 30% increase of extrajunctional acetylcholinesterase activity. There was no significant change in the extrajunctional activity in hemidiaphragm muscles. Velocity sedimentation analysis revealed that the increase in extrajunctional activity in extensor digitorum longus muscles could be attributed to an increase of the activity of the G₄ form of acetylcholinesterase. Treatment of rats with 6.4 μg h⁻¹ neostigmine bromide for 29 days had no influence on junctional and extrajunctional acetylcholinesterase activity of extensor digitorum longus muscles from rats with α-bungarotoxin-induced myasthenia gravis. Received August 26, 1998; accepted November 30, 1998     

The roles of disuse and loss of neurotrophic function in denervation atrophy of skeletal muscle

Denervated muscle fibers are characterized by a lowered resting membrane potential (RMP), increased extrajunctional acetylcholine (ACh) sensitivity, and decreased junctional acetylcholinesterase (AChE) activity. Whether these changes in denervated muscle result from cessation of contractile activity, from interruption of axonal transport, or from both is not known. Experiments were therefore designed to analyze whether or not the denervation changes could be ascribed solely to the loss of contractile activity. In one experiment, the hemidiaphragm of the rat was rendered quiescent for 1 to 3 weeks either by spinal hemisection at C2 (disuse) or by unilateral phrenicotomy (denervation). After denervation there was a spread of ACh sensitivity to extrajunctional regions, a decline in RMP, and a reduction in 16 S AChE (a measure of junctional AChE activity). Comparable changes did not occur after spinal hemisection, and we therefore conclude that inactivity alone does not induce these changes in denervated muscle. In another experiment, rats were chronically paralyzed by repeated administration of d-tubocurarine. During this time the extensor digitorum longus muscle of one hind limb was denervated. After 6 h of immobilization by d-tubocurarine, the RMP of denervated muscle fibers was significantly reduced whereas that of the contralateral innervated muscle fibers was unchanged. This result supports the previous interpretation, viz., that the change in RMP of denervated muscle fibers cannot be attributed solely to muscle inactivity. Experiments by others have shown that chronic disuse causes changes that are qualitatively but not quantitatively equivalent to those of denervation. Those observations, together with the present results, enable us to conclude that inactivity does not initiate the changes in extrajunctional ACh sensitivity, RMP, and junctional AChE activity seen in denervated muscle and that these properties of muscle are normally regulated by axonally transported neurotrophic influences.     

Is fast fiber innervation responsible for increased acetylcholinesterase activity in reinnervating soleus muscles?

During reinnervation of the completely denervated rat hind limb we observed previously a temporary overproduction of acetylcholinesterases in the soleus but not in the extensor digitorum longus muscle. In the present study, we investigated whether the predominantly slow soleus, which is low in AChE activity, is initially reinnervated by axons that originally innervated fast muscle fibers with high AChE activity, such as those of the extensor digitorum longus. Local denervation of the rat soleus was carried out to eliminate reinnervation by axons destined for other muscles. This produced an overshoot in AChE activity that was qualitatively similar to that observed with high sciatic crush. Local denervation of the soleus in the guinea pig was done because this muscle is composed solely of slow (type I) fibers, thereby virtually eliminating the possibility of homologous muscle fast fiber innervation. The overshoot in this preparation was qualitatively similar to that seen with distal denervation in the guinea pig and local and distal denervation in the rat. Thus, initial fast fiber innervation is not responsible for the patterns of change in AChE activity seen with reinnervation in the soleus. We concluded that the neural control of AChE is different in these two muscles and may reflect specific differences in the characteristics of AChE regulation in fast and slow muscle. How these neural influences are translated into muscle synthesis and degradation remains unknown.     

Utrophin mRNA Expression in Muscle Is Not Restricted to the Neuromuscular Junction

Utrophin is normally present exclusively in synaptic regions of skeletal muscle fibers, although it is expressed extrasynaptically in certain pathological situations, where it has been proposed to compensate for the absence of dystrophin in Duchenne muscular dystrophy patients andmdxmice. Recently there have been conflicting reports regarding the preferential expression of utrophin mRNA at the neuromuscular junction. Usingin situhybridization with RNA probes, we show a clear accumulation of autoradiographic labeling at more than 90% of neuromuscular junctions (identified by histochemical demonstration of cholinesterase activity). The intensity of this labeling is proportional to the number of junctional myonuclei in the section. Some clusters of labeling were found associated with nonmuscle nuclei (e.g., blood vessels, nerves), where utrophin is present. In addition, labeling for utrophin mRNA was associated with about 25% of extrajunctional myonuclei, where the protein is not present. The mean labeling per nucleus at junctional myonuclei was at least 10 times greater than at extrajunctional myonuclei. We discuss the possible regulatory mechanisms involved in the heterogeneous expression of utrophin mRNA in skeletal muscle.     

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.