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.     

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)     

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.     

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.     

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.     

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.     

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.     

Reinnervation of normal and dystrophic skeletal muscle

A comparative study was conducted of resting membrane potential (RMP), extrajunctional acetylcholine (ACh) sensitivity, spontaneous and neurally evoked transmitter release, and directly and indirectly elicited action potentials in posterior latissimus dorsi (PLD) muscles of normal (line 412) and dystrophic (line 413) chickens during reinnervation after nerve crush. Control (nondenervated) dystrophic muscle fibers had a significantly greater RMP (-77.2 vs. -74.1 mV), extrajunctional ACh sensitivity (0.34 vs. 0.04 mV/nC), and incidence of repetitive firing of directly elicited action potentials than did normals. Miniature end-plate potential (MEPP) amplitude in dystrophic fibers was significantly reduced (0.26 vs. 0.38 mV). Six to 7 days after nerve crush, muscle fibers from both lines of chickens showed a significant reduction in RMP and an increase in extrajunctional ACh sensitivity. During this time spontaneous MEPPs were absent and the incidence of repetitive firing was decreased. No significant difference was noted between chicken lines in any of the properties studied. The return of normal properties associated with reinnervation occurred primarily between days 9 and 40. Repolarization of the RMP was clearly evident by day 9 in both lines, but dystrophic fibers showed a slightly earlier and greater degree of repolarization. Similarly, initial decreases in extrajunctional ACh sensitivity and the reappearance of MEPPs were observed on day 9 with dystrophic and day 12 with normal fibers. Neurally elicited action potentials were first recorded on day 11 for dystrophic and day 12 for normal fibers. Finally, multiple firing of directly elicited action potentials associated with reinnervation attained the same incidence (20 to 21% of fibers) on day 12 in dystrophic and day 14 in normal fibers. The results suggest that dystrophic chicken muscle has an enhanced capacity for reinnervation following nerve crush.     

Physiological properties of the innervated and denervated neuromuscular junction of hibernating and nonhibernating ground squirrels.

The physiological status of the neuromuscular junction of hibernating and nonhibernating 13-lined ground squirrels was studied to determine whether or not the metabolic changes during hibernation would alter the muscle's response to denervation. It was anticipated that the observations might clarify some aspects of the trophic interrelationship between nerve and muscle. The properties of innervated muscles were not significantly altered after the animals entered hibernation. The strength of contraction, speed of contraction, and resting membrane potential remained unchanged. In addition, extrajunctional sensitivity to acetylcholine did not develop. Because the muscles are inactive during hibernation, we conclude that muscle activity alone does not maintain the physiological properties of muscles. Denervation of muscles from nonhibernating animals resulted in loss of neuromuscular transmission, cessation of miniature end-plate potentials, partial muscle membrane depolarization, and appearance of extrajunctional sensitivity to acetylcholine. In contrast, muscles whose nerves were transected during the hibernating state showed unimpaired neuromuscular transmission and normal miniature end-plate potentials. However, the muscle became partially depolarized, indicating that the regulation of the resting membrane potential is under neurotrophic control and is not influenced solely by the release of acetylcholine (which had remained unchanged). The denervated muscles of hibernating animals did not develop extrajunctional sensitivity to acetylcholine; this probably reflects the low rate of protein turnover in tissues maintained at the low (7°C) body temperature of hibernating animals. Transection of the sciatic nerve of hibernating animals produced histologically demonstrable retrograde changes in the motor neurons of the lumbar spinal cord. It thus appears that hibernation does not adversely affect certain fundamental functions of the nervous system, such as transmission of nerve impulses, anterograde transmission of neurotrophic influences, and the retrograde transmission of signals which initiate the cell body's reaction to injury.     

The distribution of slow myosin in rat muscles after neonatal nerve crush

Following neonatal nerve injury fast skeletal muscles recover less well than slow ones. This is because many muscle fibers are lost during reinnervation. Since fast muscles normally contain a small population of slow muscle fibers, we have used a monoclonal antibody to slow myosin heavy chains (SMHC) to study their number and pattern of distribution in fast muscles following temporary denervation at 5-6 days of age and subsequent reinnervation. During this time the original distribution of slow fibers changed to one showing irregular grouping, indicating that reinnervation of muscles after neonatal nerve injury is as nonselective as it is after nerve injury in adults. Despite a large reduction in the total number of muscle fibers during reinnervation, the number of slow fibers did not decrease. Thus muscle fiber loss was at the expense of the fast motor units alone.     

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.     

Nerve regulation of class I and class II-asymmetric forms of acetylcholinesterase in rat skeletal muscles

Two classes of collagen-tailed, asymmetric forms (A-forms) of acetylcholinesterase (AChE) have been described in skeletal muscles of vertebrates. They are distinguished by their different solubilization requirements: class I A-forms are solubilized in the presence of high salt, whereas class II A-forms require in addition a chelating agent for solubilization. We report here that class II A-forms are less sensitive to nerve section than are class I A-forms. The latter form decreases faster and to a lower level of activity after denervation. The decay of both AChE classes is more rapidly in short-stump nerves than in long ones. The effect of nerve section on class II A-forms appears to be dependent on the particular muscle group being studied. Both classes of A-forms reappear after muscle reinnervation, but the class I A-forms recovered earlier. More interestingly, both classes of A-forms increase in normally innervated skeletal muscles after contralateral nerve injury. In this case, however, the class II A-forms change first. Muscular disuse induced by contralateral tenotomy is also followed by a rise in class II A-forms. Our results, showing differences in response and flexibility in the changes of the two classes of A-forms in several experimental conditions, represent a relevant contribution to the understanding of the regulation and functional role of the asymmetric forms of AChE in vertebrate skeletal muscles.     

Thyroid hormone enhances transected axonal regeneration and muscle reinnervation following rat sciatic nerve injury

Improvement of nerve regeneration and functional recovery following nerve injury is a challenging problem in clinical research. We have already shown that following rat sciatic nerve transection, the local administration of triiodothyronine (T3) significantly increased the number and the myelination of regenerated axons. Functional recovery is a sum of the number of regenerated axons and reinnervation of denervated peripheral targets. In the present study, we investigated whether the increased number of regenerated axons by T3‐treatment is linked to improved reinnervation of hind limb muscles. After transection of rat sciatic nerves, silicone or biodegradable nerve guides were implanted and filled with either T3 or phosphate buffer solution (PBS). Neuromuscular junctions (NMJs) were analyzed on gastrocnemius and plantar muscle sections stained with rhodamine α‐bungarotoxin and neurofilament antibody. Four weeks after surgery, most end‐plates (EPs) of operated limbs were still denervated and no effect of T3 on muscle reinnervation was detected at this stage of nerve repair. In contrast, after 14 weeks of nerve regeneration, T3 clearly enhanced the reinnervation of gastrocnemius and plantar EPs, demonstrated by significantly higher recovery of size and shape complexity of reinnervated EPs and also by increased acetylcholine receptor (AChRs) density on post synaptic membranes compared to PBS‐treated EPs. The stimulating effect of T3 on EP reinnervation is confirmed by a higher index of compound muscle action potentials recorded in gastrocnemius muscles. In conclusion, our results provide for the first time strong evidence that T3 enhances the restoration of NMJ structure and improves synaptic transmission. © 2010 Wiley‐Liss, Inc.     

Biochemical,morphological, and functional changes during peripheral nerve regeneration

The success of axon regeneration after nerve injury should be judged by the extent to which the target organs regain their function. Recovery of muscle contraction involves axon regeneration, reestablishment of nerve-muscle connections, recovery of transmission, and muscle force. All these processes were investigated under the same experimental conditions and correlated in order to better understand their time-course and interdependence. The sciatic nerve of a rat was crushed in the thigh. The ingrowth of regenerating motor axons into the soleus (SOL) and extensor digitorum longus (EDL) muscles was monitored by measuring the activity of choline acetyltransferase (ChAT), a marker enzyme for cholinergic nerve terminals, in the muscles. The electron microscopic cytochemistry of acetylcholine esterase (AChE) was used to estimate the reestablishment of neuromuscular junctions in these two muscles. The recovery of muscle contraction was followed by measuring the force of isometric contraction in the triceps surae muscle in vivo. The pattern of ChAT recovery during reinnervation was similar in the EDL and SOL. The statistically significant increase of ChAT activity in these muscles, 14 d after the nerve crush, signified the entry of regenerating axons into the calf muscles. Electron microscopic cytochemistry revealed the first small nerve endings in contact with the denervated end plates 12 d after denervation. Subsequently, the number of reinnervated motor end plates and the surface area of the neuromuscular junctions steadily increased. The recovery of muscle force started between d 14 and 21 after the nerve crush. Thirty-five days after denervation, the difference between the muscle force of the reinnervated muscle and the control became statistically insignificant. Morphological normalization of the motor end plates was practically complete 33 d after denervation, concomitant with the normalization of the muscle force. At that time, however, ChAT activity in both muscles was still clearly subnormal (33.5% in EDL and 45% of the control in SOL) and therefore does not reflect the true extent of muscle force recovery. Yet, it seems that in spite of this, the regenerated nerve terminals contained sufficient amounts of acetylcholine (ACh) to trigger normal muscle contractions.     

Effect of nerve extract on number of acetylcholine receptors in denervated muscles of rats

We have shown elsewhere that injection of an extract of peripheral nerves reduces the atrophy of denervated muscle fibers in vivo. Denervated muscle fibers exhibit supersensitivity to acetylcholine owing to the production of extrajunctional acetylcholine receptors. We sought to determine whether or not injection of nerve extract can influence the numbers of acetylcholine receptors in normal, immobilized, or denervated extensor digitorum longus muscles of rats. The receptors were assayed by measuring the binding of ¹²⁵I-α-bungarotoxin. Normally innervated muscles injected with nerve extract exhibited slightly increased binding of the toxin, but this was due to the injections per se. Immobilization caused a small, transient increase in binding of α-bungarotoxin, whereas denervated muscles bound considerably more toxin than innervated controls. The nerve extract did not reduce or prevent the increase in acetylcholine receptors caused by denervation but instead caused an even greater increase. We concluded that the neurotrophic factor extracted from peripheral nerve that is responsible for the maintenance of the sizes of the fibers probably does not down-regulate extrajunctional acetylcholine receptors. The limitation of acetylcholine receptors to the end-plate regions is probably effected by a different mechanism which has yet to be elucidated.     

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.     

Growth and denervation response of skeletal muscle fibers of newborn rats

The cross-sectional area of the fibers of hindlimb muscles of rats increased 10-40 times during the first 6 weeks after birth. Denervation at birth stopped the growth of the muscle fibers. The number of satellite cells decreased, and eventually all fibers vanished. Reinnervation, if any, was poor. Partial denervation did not induce collateral reinnervation. Some denervated gastrocnemius muscles were reinnervated and after 8-12 months contained hypertrophic fibers and signs of necrosis and regeneration. When soleus muscles were completely denervated and cross-reinnervated after 4 weeks by the peroneal nerve, only half as many fibers became reinnervated after neonatal denervation as compared to muscles denervated at the age of 4 weeks. The experiments suggest that immature muscle fibers are less apt to become reinnervated than mature fibers. The few reinnervated fibers may be overloaded and therefore hypertrophy and eventually necrotize. Regeneration is abortive because satellite cells are scarce. These results may be relevant for the understanding of neuromuscular disorders with early (fetal) onset.