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.     

Changes in number and distribution of orthogonal arrays during postnatal muscle development

Quantitative freeze-fracture electron microscopy was utilized to study the changes in number and distribution of orthogonal arrays (aggregates of 6-7 nm particles) of the sarcolemmas of the fast twitch extensor digitorum longus (EDL) and slow twitch soleus muscles during the first few weeks of postnatal development in the rat. In the adult rat, orthogonal arrays are present in high densities in the fast twitch type II fibers but only in low densities in slow twitch type I fibers. In this study, the changing histochemical profiles of fiber types in the EDL and soleus muscles were also determined for the first month of postnatal development and correlated with the changing number and distribution of orthogonal arrays during the same time frame. At day 3 postpartum, EDL and soleus fibers possessed few orthogonal arrays. The developing EDL fibers rapidly acquired additional orthogonal arrays until the approximate adult number and distribution were attained at postpartum day 25. In contrast, the slow twitch soleus fibers rapidly acquired orthogonal arrays and type IIA fibers until day 35 when both were in excess of adult values. Subsequently, the number of arrays and type IIA fibers declined to normal adult ranges. We suggest that the patterns of development of orthogonal arrays and fiber types are different in the EDL and soleus because the types of innervating motor units are different in the two muscles. The EDL is innervated almost entirely by fast motor units throughout early development and maturity. The soleus, however, is initially innervated by a more heterogeneous population of motor neurons. Thus, during the period of polyneuronal innervation which occurs normally during the first weeks of postnatal development, many individual soleus fibers may possess simultaneous innervation by axons from different motor neuron types. These dual influences may be responsible for the irregular pattern of development of orthogonal arrays and type IIA fibers in developing soleus fibers. Later, as the adult pattern of monosynaptic innervation is developed, expected adult values of orthogonal arrays and fiber types are attained.     

Choline acetyltransferase activity in muscles of old rats

The total activity of choline acetyltransferase (ChAc) in the rat extensor digitorum longus (EDL) and soleus muscles increased by 50 and 55%, respectively, between 3 and 9 months of age. In rats 28 to 29 months old, the activity of ChAc in EDL and soleus diminished to 41 and 40%, respectively, of the activity observed in 9-month-old animals. Age changes of ChAc activity in the diaphragm were not significant. The number of muscle fibers in EDL and soleus muscles of rats 28 to 29 months old decreased by 44 and 38% respectively, in comparison with younger animals. Mean muscle fiber diameters did not change between 3 and 9 months of age and decreased by 24, 35 and 9% in the EDL, soleus and diaphragm, respectively, in the 28- to 29-month-old rats. The activity of ChAc expressed in relation to one muscle fiber was about the same in the EDL and soleus muscles. It increased between 3 and 9 months and decreased between 9 and 28 to 29 months of age. The observation that ChAc activity per muscle fiber was identical in the fast EDL and slow soleus muscle suggests that the physiological differences between the two muscles are not caused by a difference in the capacity of their motor nerves to synthesize ACh. In the diaphragm the activity of ChAc per muscle fiber apparently did not diminish in old age. The decrease in the total ChAc activity in the limb muscles of old animals seems due both to a decrease in the number of nerve terminals in the muscles and to a decrease in the amount of enzyme present in individual terminals. We suggest that the maintenance of ChAc activity in the motor nerve terminals in the diaphragm of old rats is due to the continuous activity of this muscle and its motor nerves.     

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.     

Recovery of rat skeletal muscles after partial denervation is enhanced by treatment with nifedipine

Following partial denervation of adult rat skeletal muscle intact axons sprout to reinnervate denervated muscle fibres and increase their territory. The extent of this increase is limited and may depend on the ability of axon terminals to form and maintain synaptic contacts with the denervated muscle fibres. Here we tested the possibility whether reducing Ca²⁺ entry into presynaptic nerve terminals through dihydropyridine sensitive channels may allow more nerve–muscle contacts to be formed and maintained. Hindlimb muscles of adult Wistar rats were partially denervated by removing a small segment of the L4 or L5 spinal nerve on one side. A nifedipine-containing silastic rubber strip was subsequently implanted close to the partially denervated soleus or extensor digitorum longus (EDL) muscles in some animals. In control experiments silastic strips which did not contain nifedipine were used. Several weeks later isometric contractions were recorded, to determine the effect of (a) partial denervation and (b) nifedipine treatment on force output and motor unit numbers. The tension produced by nifedipine treated partially denervated muscles was 82% and 79% of the unoperated contralateral value for soleus and EDL, respectively. This was significantly greater than in untreated muscles, which only produced 61% and 48%, respectively. Mean motor unit force was also significantly larger with nifedipine treatment. Histological analysis revealed that a significantly larger proportion of the total number of muscle fibres remained in nifedipine-treated partially denervated muscles (soleus, 90% and EDL, 101%) compared with untreated muscles (soleus, 51% and EDL, 66%). Thus the number of neuromuscular contacts was increased with nifedipine treatment.     

Effect of precocious locomotor activity on the development of motoneurones and motor units of slow and fast muscles in rat

We have investigated the effect of precociously increasing locomotor activity during early postnatal development by daily treatment with the monoaminergic precursor L-DOPA on the survival of motoneurones supplying the slow soleus (SOL) muscle and the fast, tibialis anterior (TA) and extensor digitorum longus (EDL) muscles as well as the contractile and histochemical properties of these muscles. L-DOPA treatment resulted in a significant loss of motoneurones to the slow SOL muscle, but not to the fast TA and EDL muscles. Moreover, motoneurones to fast muscles also die as when exposed to increased activity in early life, if their axons are repeatedly injured. The loss of normal soleus motoneurones was accompanied by an increase in force of the remaining motor units and sprouting of the surviving axons suggesting a remodelling of motor unit organisation. The time to peak contraction of both SOL and EDL muscles from L-DOPA treated rats was prolonged at 8 weeks of age. At 4 weeks the soleus muscles of the L-DOPA treated animal developed more tension than the saline treated one. This difference between the two groups did not persist and by 8 weeks of age the muscle weight and tetanic tension from either group were not significantly different from control animals. The present study shows that early transient, precocious locomotor activity induced by L-DOPA is damaging to normal soleus but not to normal EDL/TA motoneurones.     

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.     

Elimination of Motor Nerve Terminals in Neonatal Mice Expressing a Gene for Slow Wallerian Degeneration (C57Bl/Wlds)

Degeneration of motor terminals after nerve section occurs much more slowly than normal in young adult mice of the C57BI/ Wld^S strain. This observation prompted us to re-examine the possible role of degeneration and intrinsic axon withdrawal during neonatal synapse elimination. Polyneuronal innervation was assayed by two methods: intracellular recording of end-plate potentials in cut-muscle fibre preparations of isolated hemidiaphragm and soleus muscles; and in silver-stained preparations of triangularis sterni and transversus abdominis muscle fibres. No differences in the rate of synapse elimination were detected in unoperated Wld^s compared with CBA, C3H/HE and BALB/c mice. At 3 days of age, >80% of fibres were polyneuronally innervated. By 7 days this declined to ∼20% of hemidiaphragm, 50% of triangularis sterni and 60% of soleus fibres. Nearly all fibres were mononeuronally innervated by 15 days. The mean number of terminals per triangularis sterni muscle fibre 7 days after birth was 1.55 ± 0.07 in Wlds and 1.56 ± 0.09 in wild-type mice. Three to 4 days after sciatic nerve section, near-normal numbers of motor units were evident in isometric tension recordings of the soleus muscle, and intracellular recordings revealed many polyneuronally innervated fibres. Mononeuronally and polyneuronally innervated fibres were also observed in silver-stained preparations of soleus and transversus abdominis muscles made 3–4 days after sciatic or intercostal nerve section. We conclude (i) that the Wld^s gene has no direct impact on the normal rate of postnatal synapse elimination, (ii) that Wallerian degeneration and synapse elimination must occur by distinct and different mechanisms, and (iii) that muscle fibres are able to sustain polyneuronal synaptic inputs even after motor axons have become disconnected from their cell bodies.     

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)     

Effects of training on skeletal muscle in protein-deprived rats

The effects of exercise on atrophy of muscle fibres and loss of mitochondria in the extensor digitorum longus (EDL) and soleus muscles were studied in protein deprived rats. They had smaller muscle fibres than aged-matched control rats, the difference being more evident in type 2 than type 1 fibres both in the fast EDL and slow soleus muscles. The loss of weight was more pronounced in the EDL muscle which is composed mainly of type 2 fibres than in the soleus muscle which is composed mainly of type 1 fibres. Protein deprived rats subjected to a programme of periodic running on a treadmill for 12 weeks showed less muscle atrophy than sedentary, protein deprived rats. This effect of exercise in diminishing the degree of atrophy was more pronounced in the type 2 than type 1 fibres. The protein deprived rats which had been sedentary showed a marked loss of subsarcolemmal mitochondria, which was not seen in protein deprived rats undergoing exercise.     

Calcitonin gene-related peptide-like immunoreactivity, in botulinum toxin-paralysed rat muscles

Changes in calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) at the motor endplates of botulinum toxin-paralysed rat muscles were investigated using immunohistochemistry. One day following toxin injection, a dramatic increase in CGRP-LI was detected at the motor endplates and within preterminal axons of the soleus and gastrocnemius muscles. The upregulation of CGRP-LI persisted throughout the period during which muscle fibres were paralysed and new neuromuscular junctions were being formed by the growing sprouts. Decline of CGRP-LI at the motor endplates coincided with clinical recovery. Both up- and down-regulation of CGRP-LI took place earlier in the soleus than in the gastrocnemius muscle. Up-regulation of CGRP-LI was also detected in a subpopulation of motor axons in the sciatic nerves and in the spinal motor neurons innervating the paralysed muscles. These results indicate that levels of CGRP are regulated, at least partly, by changes in the target innervation. They also suggest an important role for CGRP in the regenerative processes following muscle paralysis.     

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.     

IGF-I treatment improves the functional properties of fast- and slow-twitch skeletal muscles from dystrophic mice

Although insulin-like growth factor-I (IGF-I) has been proposed for use by patients suffering from muscle wasting conditions, few studies have investigated the functional properties of dystrophic skeletal muscle following IGF-I treatment. 129P1 ReJ-Lama2(dy) (129 ReJ dy/dy) dystrophic mice suffer from a deficiency in the structural protein, laminin, and exhibit severe muscle wasting and weakness. We tested the hypothesis that 4 weeks of IGF-I treatment ( approximately 2 mg/kg body mass, 50 g/h via mini-osmotic pump, subcutaneously) would increase the mass and force producing capacity of skeletal muscles from dystrophic mice. IGF-I treatment increased the mass of the extensor digitorum longus (EDL) and soleus muscles of dystrophic mice by 20 and 29%, respectively, compared with untreated dystrophic mice (administered saline-vehicle only). Absolute maximum force (P(o)) of the EDL and soleus muscle was increased by 40 and 32%, respectively, following IGF-I treatment. Specific P(o) (sP(o)) was increased by 23% in the EDL muscles of treated compared with untreated mice, but in the soleus muscle sP(o) was unchanged. IGF-I treatment increased the proportion of type IIB and type IIA fibres and decreased the proportion of type I fibres in the EDL muscles of dystrophic mice. In the soleus muscles of dystrophic mice, IGF-I treatment increased the proportion of type IIA fibres and decreased the proportion of type I fibres. Average fibre cross-sectional area was increased in the EDL and soleus muscles of treated compared with untreated mice. We conclude that IGF-I treatment ameliorates muscle wasting and improves the functional properties of skeletal muscles of dystrophic mice. The findings have important implications for the role of IGF-I in ameliorating muscle wasting associated with the muscular dystrophies.     

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.     

Choline acetyltransferase and acetylcholinesterase activities in muscles of aged mice

The activities of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were assayed in intact diaphragm, extensor digitorum longus (EDL), and soleus muscles or their homogenates of young (2-6 months) and aged (24-34 months) mice. CAT activity (per mg of protein) was significantly higher in diaphragm and soleus of old mice in comparison with the young but the age change in EDL was negligible. On the other hand, AChE activity (per mg of protein) was significantly higher in EDL of old mice but in diaphragm and soleus muscles the enzyme activity did not show any significant change statistically. The diaphragm muscle was divided into two fractions, one being neuromuscular (NM) fraction and the other the remainder of the muscle (M fraction). No appreciable change in the ratio of the enzyme activities of NM fraction to the one of M fraction was obtained between the young and aged preparations. Thus, it seems likely that there is an age-related change in CAT and AChE activities which might be affected by the degree to which muscle activity is maintained.     

Effects of tenotomy on muscles reinnervated by a foreign nerve

Tenotomy of the rat soleus (SOL) and gastrocnemius (MG) muscles produces a central degeneration in slow fatigue-resistant fibers, but not in similar fibers of muscles in the extensor and peroneal compartments. To investigate the part that innervation plays in rendering a particular fiber type in a particular muscle susceptible to this degeneration, the SOL, extensor digitorum longus (EDL), and MG muscles were experimentally reinnervated by foreign nerves and tenotomized. When the SOL was reinnervated by the common peroneal nerve, slow fatigue-resistant fibers showed lesions, but when the EDL was reinnervated by the nerve to the SOL, no lesions were found after tenotomy. When the MG was reinnervated by the nerve to the SOL, slow fatigue-resistant fibers that had differentiated in regions normally occupied almost entirely by fast fatigable fibers showed characteristic lesions. These results show that the failure of tenotomy to produce lesions in the EDL is not due to the nature of its innervation and that a fiber type not normally susceptible to the degenerative change will become susceptible when transformed to the slow fatigueresistant type.     

Stimulation of denervated rat soleus muscle with fast and slow activity patterns induces different expression of acetylcholinesterase molecular forms

The relative amount and distribution of acetylcholinesterase (AChE) molecular forms were studied in slow soleus and (less extensively) in fast extensor digitorum longus (EDL) muscles of the rat before and after denervation and direct stimulation. Normal EDL muscles showed higher total and specific AChE activity than normal soleus muscles and contained essentially three different molecular AChE forms (G1, G4, and A12) as opposed to six forms (G1, G2, G4, A4, A8, and A12) in the soleus. Denervation reduced AChE activity in both muscles. In the soleus direct stimulation starting 2 to 3 weeks after denervation increased the specific AChE activity markedly. The increase started 12 to 24 hr after the onset of stimulation, reached 3 to 5 times normal values after 2 to 7 days, and then declined gradually toward normal values over the next 2 weeks. Furthermore, the effect on the different molecular forms depended strongly on the stimulus pattern. Thus, intermittent 100 Hz stimulation (fast pattern) induced essentially the three forms typical of the normal EDL, whereas continuous 10 Hz stimulation induced the six forms characteristic of normal soleus muscles but with some differences in their relative proportions. In the EDL, 2 days of continuous 10 Hz stimulation (the only duration and pattern examined) failed to induce a similar increase in AChE activity.     

Ultrastructure and innervation of regenerated intrafusal muscle fibres in heterochronous isografts of the fast rat muscle

Extensor digitorum longus (EDL) muscles from 2- to 28-day-old rats were grafted into EDL muscles of adult inbred recipients (n = 8). At 1–6 months after the operation, experimental muscles were excised and the ultrastructure and innervation of regenerated muscle spindles was examined. Regenerated muscle spindles (n = 36) in isografted EDL muscles contained 4.3 ± 0.2 (mean ± SEM) encapsulated muscle fibres. These “intrafusal” muscle fibres lacked nuclear bag and nuclear chain accumulations, which are characteristic of normal muscle spindles; thus, they rather resembled thin encapsulated extrafusal muscle fibres. In the same sample, myelinated axons were found in 33 (92%) muscle spindles, but no sensory terminals were found. These findings demonstrate that regenerated spindles in isografted EDL muscles were not reinnervated by spindle-specific sensory axons, but exclusively by motor axons. Typical intracapsular motor endplates (MEPs) were found in one third of regenerated spindles examined. Their motor terminals contained accumulated mitochondria and synaptic vesicles. As is characteristic for MEPs, axolemma and sarcolemma were separated by a synaptic cleft about 60 nm wide that contained a basal lamina. The underlying sarcolemma formed either small infoldings or none at all, and the subsynaptic area contained only small subsarcolemmal accumulations of mitochondria. It is apparent that the structures described here as “regenerated muscle spindles” do not perform their normal physiological function as stretch receptors because they lack the sensory innervation. The present results show that regeneration and reinnervation in heterochronous isografts corresponds to that previously described in autotransplanted free muscle grafts. The results also show that, during muscle spindle regeneration, intrafusal satellite cells develop into extrafusal-like muscle fibres, apparently due to their motor innervation. Received: 15 June 1999 / Revised, accepted: 20 December 1999