Expression of nestin, desmin and vimentin in intact and regenerating muscle spindles of rat hind limb skeletal muscles.

We describe the expression and distribution patterns of nestin, desmin and vimentin in intact and regenerating muscle spindles of the rat hind limb skeletal muscles. Regeneration was induced by intramuscular isotransplantation of extensor digitorum longus (EDL) or soleus muscles from 15-day-old rats into the EDL muscle of adult female inbred Lewis rats. The host muscles with grafts were excised after 7-, 16-, 21- and 29-day survival and immunohistochemically stained. Nestin expression in intact spindles in host muscles was restricted to Schwann cells of sensory and motor nerves. In transplanted muscles, however, nestin expression was also found in regenerating "spindle fibers", 7 and 16 days after grafting. From the 21st day onwards, the regenerated spindle fibers were devoid of nestin immunoreactivity. Desmin was detected in spindle fibers at all developmental stages in regenerating as well as in intact spindles. Vimentin was expressed in cells of the outer and inner capsules of all muscle spindles and in newly formed myoblasts and myotubes of regenerating spindles 7 days after grafting. Our results show that the expression pattern of these intermediate filaments in regenerating spindle fibers corresponds to that found in regenerating extrafusal fibers, which supports our earlier suggestion that they resemble small-diameter extrafusal fibers.

Neurotrophin-3 ameliorates sensory-motor deficits in Er81-deficient mice.

Two factors, the ETS transcription factor ER81 and skeletal muscle-derived neurotrophin-3 (NT3), are essential for the formation of muscle spindles and the function of spindle afferent-motoneuron synapses in the spinal cord. Spindles either degenerate completely or are abnormal, and spindle afferents fail to project to spinal motoneurons in Er81 null mice; however, the interactions between ER81 and NT3 during the processes of afferent neuron and muscle spindle development are poorly understood. To examine if overexpression of NT3 in muscle rescues spindles and afferent-motoneuron connectivity in the absence of ER81, we generated myoNT3;Er81(-/-) double-mutant mice that selectively overexpress NT3 in muscle in the absence of ER81. Spindle reflex arcs in myoNT3;Er81(-/-) mutants differed greatly from Er81 null mice. Muscle spindle densities were greater and more afferents projected into the ventral spinal cord in myoNT3;Er81(-/-) mice. Spindles of myoNT3;Er81(-/-) muscles responded normally to repetitive muscle taps, and the monosynaptic inputs from Ia afferents to motoneurons, grossly reduced in Er81(-/-) mutants, were restored to wild-type levels in myoNT3;Er81(-/-) mice. Thus, an excess of muscle-derived NT3 reverses deficits in spindle numbers and afferent function induced by the absence of ER81. We conclude that muscle-derived NT3 can modulate spindle density and afferent-motoneuron connectivity independently of ER81.

Fiber content and myosin heavy chain composition of muscle spindles in aged human biceps brachii.

The present study investigated potential age-related changes in human muscle spindles with respect to the intrafusal fiber-type content and myosin heavy chain (MyHC) composition in biceps brachii muscle. The total number of intrafusal fibers per spindle decreased significantly with aging, due to a significant reduction in the number of nuclear chain fibers. Nuclear chain fibers in old spindles were short and some showed novel expression of MyHC alpha-cardiac. The expression of MyHC alpha-cardiac in bag1 and bag2 fibers was greatly decreased in the A region. The expression of slow MyHC was increased in nuclear bag1 fibers and that of fetal MyHC decreased in bag2 fibers whereas the patterns of distribution of the remaining MyHC isoforms were generally not affected by aging. We conclude that aging appears to have an important impact on muscle spindle composition. These changes in muscle spindle phenotype may reflect an age-related deterioration in sensory and motor innervation and are likely to have an impact in motor control in the elderly.

Motor, sensory and autonomic nerve terminals containing NAP-22 immunoreactivity in the rat muscle.

Neuron-enriched acidic protein having a molecular mass of 22 kDa, NAP-22, is a Ca(2+)-dependent calmodulin-binding protein and is phosphorylated with protein kinase C (PKC). This protein is localized to the biological membrane via myristoylation and found in the membrane fraction of the brain and in the synaptic vesicle fraction. Recent studies showed that NAP-22 is localized in the membrane raft domain in a cholesterol-dependent manner and suggest a role for NAP-22 in maturation and/or maintenance of nerve terminals by controlling cholesterol-dependent membrane dynamics. The present study revealed the immunohistochemical distribution of NAP-22 in the peripheral nerves in rat muscles. In all examined muscles, nerve terminals in the motor endplates showed NAP-22 immunoreactivity associated with the membranes of synaptic vesicles and nerve terminals. In the muscle spindles, annulospiral endings, which made spirals around the intrafusal muscles, showed intense NAP-22 immunoreactivity. Autonomic nerve fibers around the intramuscular blood vessels also showed the immunoreactivity for NAP-22. NAP-22 immunoreactivity in these peripheral nerves was observed from birth to adulthood (100 days after birth). Though growth-associated protein-43 (GAP-43) immunoreactivity in these nerves was observed from birth, this immunoreactivity decreased from 20 days after birth. These findings suggest that NAP-22 is distributed and regulates functions in the motor, sensory and autonomic nerve terminals in the peripheral nervous system.

Muscle spindle reinnervation following phenol block.

Infusion of phenol into peripheral nerves is used clinically to manage spasticity. It produces relief of symptoms by chemical denervation. We simulated the clinical procedure by bathing the lateral plantar nerve of rats in 7% phenol solution for 20 min. We studied the innervation of muscle spindles in the plantar lumbrical muscles of untreated rats and in rats 4 and 6 weeks after a single phenol block. Spindles were identified by the immunoreactivity of nuclear bag(1) fibers to slow tonic myosin (antibody ALD 19). The integrity of the sensory and motor reinnervation of spindles was evaluated using a monoclonal antibody specific for a high molecular weight neurofilament protein. Four weeks after phenol block, muscle spindles were difficult to find, as their immunoreactivity to antibody ALD 19 was reduced. In those spindles studied, most (>80%) were completely denervated. The remainder of which were innervated by afferents only. None received efferent (gamma) innervation. After 6 weeks, spindles were readily identified and nearly all (>90%) received recognizable afferent innervation. A much smaller number (38%) received gamma innervation. Phenol block thus results in a complete denervation of muscle spindles, followed by a fairly rapid sensory reinnervation. Reinnervation by gamma motor neurons is either incomplete or significantly delayed.

Proportions of slow myosin heavy chain-positive fibers in muscle spindles and adjoining extrafusal fascicles, and the positioning of spindles relative to these fascicles.

Chicken leg muscles were examined to calculate the percentages of slow myosin heavy chain (MHC)-positive fibers in spindles and in adjacent extrafusal fascicles, and to clarify how the encapsulated portions of muscle spindles are positioned relative to these fascicles. Unlike mammals, in chicken leg muscles slow-twitch MHC and slow-tonic MHC are expressed in intrafusal fibers and in extrafusal fibers, suggesting a close developmental connection between the two fiber populations. In 8-week-old muscles the proportions of slow MHC-positive extrafusal fibers that ringed muscle spindles ranged from 0-100%. In contrast, proportions of slow MHC-positive intrafusal fibers in spindles ranged from 0-57%. Similar proportions in fiber type composition between intrafusal fibers and surrounding extrafusal fibers were apparent at embryonic days 15 and 16, demonstrating early divergence of extrafusal and intrafusal fibers. Muscle spindles were rarely located within single fascicles. Instead, they were commonly placed where several fascicles converged. The frequent extrafascicular location of spindles suggests migration of intrafusal myoblasts from developing clusters of extrafusal fibers toward the interstitium, perhaps along a neurotrophic gradient established by sensory axons that are advancing in the connective tissue matrix that separates adjoining fascicles.

Neurocalcin-immunopositive nerve terminals in the muscle spindle, Golgi tendon organ and motor endplate.

The present study revealed the immunohistochemical distribution of neurocalcin, a three EF-hand calcium-binding protein, in the rat muscles and tendons. In the muscle spindles, annulospiral endings, which made spirals around the intrafusal muscles, showed intense neurocalcin-immunoreactivity. In the Golgi tendon organs, immunopositive thick nerve fibers entered the collagenous fibers resulting in the projection of many swelling terminals. In all examined muscles, nerve terminals in the motor endplates showed neurocalcin-immunoreactivity associated with the membranes of synaptic vesicles and mitochondria. These findings suggest that neurocalcin is distributed and regulates calcium signaling in both afferent and efferent nerve terminals in the muscles and tendons.

Structural and functional maturation of the buccal stretch receptors in rats.

Postnatal functional and structural development of the buccal stretch receptor (BSR) of rats was investigated, using electrophysiological and morphological techniques. For functional analysis, sustained discharges in response to ramp-and-hold stretches were recorded from the BSRs isolated from animals aged 10 days to 10 weeks. The threshold amplitude of stretch for a sustained discharge fell significantly between 10 days and 3 weeks, reaching adult values at 5 weeks of age, while the static sensitivity increased conspicuously between 2 and 4 weeks after birth. On the other hand, between 1 and 4 weeks of age, apparent structural changes in the BSR were observed on the number of preterminal branches in a sensory unit, the size of the varicose-like swellings along the terminal axon, the density of collagen and elastic fibers around the core structure, and the content of the sub-capsular space. From these results, we suggest that the increase in the density of the connective tissue around the core structure is associated with an enhancement in the elasticity of the BSR in the early postnatal stages, decreasing the threshold amplitude of stretch for a sustained discharge. One possible explanation for the maturation of the static sensitivity of this receptor is growth of the sensory axon terminals filled with dense mitochondria.

Origin of intrafusal fibers from a subset of primary myotubes in the rat.

S46, a monoclonal antibody (mAb) specific for the SM-1 and SM-2 isoforms of avian slow myosin heavy chains (MHC), was used to study the earliest stages of development of intrafusal fibers in muscle spindles of the rat hindlimb. Spindles formed only in the regions of fetal muscles that contained primary myotubes reactive to mAb S46, such as the axial region of the tibialis anterior muscle. The first intrafusal fiber to form, the nuclear bag2 fiber, originated from within the population of S46-reactive primary myotubes. Binding of mAb S46 by myotubes giving rise to the bag2 fibers preceded the appearance of encapsulated spindles in the muscles by electron microscopy. However, reactivity to S46 intensified in the myotubes transforming into bag2 fibers after the innervation of the fibers by afferents, and dissipated in myotubes differentiating into slow-twitch (type I) extrafusal fibers. Thus, afferents may enhance intrafusal expression of the MHC isoform reactive to mAb S46. The pattern of S46 binding to nuclear bag and chain intrafusal fibers in both developing and adult spindles was the same as that reported for the mAb ALD19, suggesting that both antibodies bind to the same MHC isoform. This isoform is probably a developmental form of slow myosin, because it was transiently expressed during the development of type I extrafusal fibers. The origin of bag2 intrafusal and type I extrafusal fibers from a bipotential subpopulation of primary myotubes reactive to mAb S46 correlates with the location of muscle spindles in the slow regions of muscles in adult rat hindlimbs.

Sequences of intrafusal fiber formation are muscle-dependent in rat hindlimbs.

A rat muscle spindle typically contains four intrafusal fibers-one nuclear bag2, one nuclear bag1 and two nuclear chain fibers. We compared the sequence of formation of the three intrafusal fiber types among the tibialis anterior (TA), soleus (SOL) and medial gastrocnemius (MG) muscles using immunocytochemistry of spindle-specific myosin heavy chain isoforms. Spindles of the TA began to differentiate earlier and acquired the full complement of intrafusal fibers sooner than spindles of the SOL or MG muscles. At the onset of spindle assembly, the intrafusal myotubes expressed myosin heavy chains similar to those expressed by extrafusal myotubes. The first intrafusal myotube then differentiated into the bag2 fiber regardless of the muscle. However, the fate of the second-forming intrafusal myotube varied among the muscles studied. It usually differentiated into a chain fiber in the TA, into a bag1 fiber in the SOL, and into either a bag1 or a chain in the MG. The fate of the third-forming intrafusal myotube was reciprocal to that of the second; i.e. in those spindles in which the bag1 fiber was second to form, a chain was third, and vice versa. The fourth and last intrafusal myotube gave rise to a chain fiber. The inter- and intramuscular variability in the fate of intrafusal myotubes of the second and third generation argues against the existence of a program intrinsic to the myotubes that would mandate their differentiation along specific paths. Rather, an extrinsic regulatory factor, probably associated with the primary afferent neuron, may govern differentiation of pluripotential myotubes into particular types of intrafusal fiber. The fate of the intrafusal myotubes might then depend on the timing of the regulatory effect of afferents relative to the stage of development of the intrafusal bundle.

Immunohistochemical characterization of human masseter muscle spindles.

An enzyme- and immunohistochemical study has been performed on human masseter muscle spindles. Antibodies selective for different myosin heavy chain (MHC) isoforms and M-band proteins (M-protein, myomesin, and MM-CK) were used. The expression of these proteins was determined in the different intrafusal fiber types. Nuclear bag1 and nuclear bag2 fibers expressed predominantly slow-twitch and slow-tonic MHCs. The bag2 fibers in addition contained fetal MHC. Nuclear chain fibers coexpressed embryonic, fetal, and fast-twitch MHCs. The bag2 and chain fibers contained all three M-band proteins, whereas the bag1 fibers contained only myomesin. In general the MHC expression in the human masseter intrafusal fiber types was similar to that previously reported for limb muscles in man as well as for limb and masseter muscles in other species. However, the number of intrafusal fibers per spindle was unusually high (up to 36). This reinforces the idea that masseter muscle spindles have a strong proprioceptive impact during the control of jaw movements.

Electrophoretically defined myosin heavy chain patterns of single human muscle spindles.

At least four myosin heavy chain (MHC) isoforms were separated by SDS-PAGE in extracts of intrafusal fibers isolated by microdissection from human lumbrical muscles. The fastest migrating MHC represents a slow isoform. The slowest migrating MHC was identified as the embryonic MHCemb. A faint band, moving slightly faster than MHCemb, most likely represents a neonatal/fetal MHC isoform. A prominent band, moving between the latter and the slow isoform is suggested to represent a hitherto unidentified, spindle-specific MHC isoform, MHCif.

Distribution of dystrophin and neurofilament protein in muscle spindles of normal and Mdx-dystrophic mice: an immunocytochemical study.

Dystrophin is a high molecular weight protein localized under the sarcolemma of normal extrafusal muscle fibers but absent in skeletal muscle of Duchenne muscular dystrophy patients and mdx mice. Muscle spindles in the soleus of 32-week-old normal and age-matched mdx mice were examined by immunocytochemical methods to determine the localization of dystrophin in polar and equatorial regions of the intrafusal fibers. Spindles were serially sectioned in transverse and longitudinal planes, and were double-labelled with an antibody to dystrophin and with an antibody to a 200 kD neurofilament protein, which revealed their sensory innervation. By fluorescence microscopy, intrafusal fibers in the soleus of mdx mice were deficient in dystrophin throughout their lengths, whereas their sensory nerve terminals stained intensely with the nerve-specific antibody and appeared unaltered in dystrophy. In the normal soleus, intrafusal fibers displayed a regional variability in the distribution of dystrophin. Polar regions of bag and chain fibers exhibited a peripheral rim of sarcolemmal staining equivalent to that seen in the neighboring extrafusal fibers. Dystrophin labelling in equatorial regions of normal intrafusal fibers, however, showed dystrophin-deficient segments alternating in a spiral fashion with positive-staining domains along the sarcolemma. Double-labelling for dystrophin and neurofilament protein showed that these dystrophin-deficient sites were subjacent to the annulospiral sensory nerve wrappings terminating on the intrafusal fibers. These findings suggest that dystrophin is not an integral part of the subsynaptic sensory membrane in equatorial regions of normal intrafusal fibers and thus is not directly related to sensory signal transduction. The complete absence of this protein in mdx intrafusal fibers indicates that these fibers exhibit the same primary defect in muscular dystrophy as seen in the extrafusal fibers. However, because of their small diameters, capsular investment, and relatively low tension outputs, dystrophic intrafusal fibers may be less prone to the sarcolemmal membrane disruption that is characteristic of extrafusal fibers in this disorder.

Differential effects of neonatal denervation on intrafusal muscle fibers in the rat.

The response of developing muscle spindles to denervation was studied by sectioning the nerve to the medial gastrocnemius muscle of rats at birth. The denervated spindles were examined daily throughout the first postnatal week for changes in ultrastructure and expression of several isoforms of myosin heavy chain (MHC). Each of the three different types of intrafusal muscle fiber exhibited a different response to denervation. Within 5 days after the nerve section nuclear bag2 fibers degenerated completely; nuclear bag1 fibers persisted, but ceased to express the 'spindle-specific' slow-tonic MHC isoform and thereby could not be differentiated from extrafusal fibers; nuclear chain fibers did not form. The capsules of spindles disassembled, hence spindles or their remnants could no longer be identified 1 week after denervation. Neonatal deefferentation has little effect on these features of developing spindles, so removal of afferent innervation is presumably the factor that induces the loss of spindles in denervated muscles. Degeneration of the bag2 fiber, but not bag1 or extrafusal fibers, reflects a greater dependence of the bag2 fiber than the bag1 fiber on afferent innervation for maintenance of its structural integrity. This difference in response of the two types of immature bag fiber to denervation might reflect an origin of the bag2 fibers from a lineage of myogenic cells distinct from that giving rise to bag1 or extrafusal fibers, or a difference in the length of contact with afferents between the two types of bag fiber prior to nerve section.

Calbindin D-28k-immunoreactivity in rat muscle spindles during postnatal maturation and after denervation.

Calbindin D-28k-immunoreactivity has been demonstrated in some of the intrafusal muscle fibres and in the capsule of adult rat muscle spindles. In this study, the immunocytochemical localization of calbindin D-28k in the muscle spindles of triceps surae muscle was studied during postnatal maturation and after denervation. In young rats calbindin D-28k-immunoreactivity was seen in a few intrafusal fibres, first at the age of 4 days. At the 7th day, three calbindin D-28k-immunoreactive fibres and one unlabelled fibre were seen in most muscle spindles, as in adult rats. The spindle capsule and perineurial sheath of nerves were first seen to exhibit calbindin D-28k immunoreactivity at the age of 14 days, and thereafter the localization of calbinding D-28k-like immunoreactivity was similar to that in adult rats. After denervation, calbindin D-28k-immunoreactivity remained in intrafusal muscle fibres and the spindle capsule for a long period. After two months of denervation, calbindin D-28k immunoreactivity could still be seen in the spindle capsule, but the intrafusal fibres were not labelled. The innervation is known to have trophic effects on the intrafusal fibres. The present findings suggest that the expression of calbindin D-28k-immunoreactivity in maturating muscle spindles may be induced by the developing innervation. The decrease of calbindin D-28k-immunoreactivity in intrafusal fibres after denervation may be due to the loss of trophic factors released by the nerves.

Arrest of developmental conversion of type II to type I fibres after suspension hypokinesia.

The effects of hypokinesia and of the lack of gravity on muscle fibres, fibre type composition and myosin light chain pattern, as well as on muscle mechanoreceptors were investigated in the slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles of young growing and adult rats after suspension hypokinesia (SH) of their hind limbs. The animals were suspended by their tail so that their hind limbs were relieved of their normal weight-bearing function for 3-6 weeks. In normal 3- to 4-week-old rats the SOL contained about 50% type I fibres and their percentage increased up to about 80% until the 10th week, with simultaneous reduction of type IIA fibres. After 3 to 6 weeks of suspension treatment maintained from 3- to 4-week-old rats up to 6 to 10 weeks of age, the SOL still only contained about 50% of type I fibres. The content of fast LC1 and LC2 in the SOL of 6-week-old rats after 3 weeks of suspension was higher than that of control litter-mates reflecting the higher occurrence of IIA fibres in the suspended solei. No changes in fibre type composition were observed after SH performed in adult rats. SH thus leads, in young animals, to the arrest of conversion of type IIA to type I fibres resulting in the persistence of the fibre type composition and of the myosin light chain pattern corresponding to those present in the SOL at the time of the onset of suspension. In both young and adult rats, SH markedly decreased the mass and the mean cross-sectional area of the SOL, mainly due to the severe atrophy of type I fibres. We observed no signs indicating conversion of type I back to type IIA muscle fibres due to the SH either in young or adult animals. In contrast to profound changes in the SOL, no significant differences were found in the EDL in any of the parameters studied. No changes in the investigated parameters of muscle spindles and tendon organs were observed after SH, performed either in young or in adult rats. We thus conclude that SH leads to muscle atrophy and that it influences mainly or exclusively type I fibres in muscles with a postural function such as the SOL.(ABSTRACT TRUNCATED AT 400 WORDS)

Dystrophin: localization and presumed function.

To determine the localization and functional significance of dystrophin, we studied various tissues from almost the entire body of control and mdx mice, and control rats, using polyclonal antibodies against dystrophin. We observed a dystrophin reaction in synaptic regions such as neuromuscular junctions, the equatorial region of intrafusal muscle fibers, the outer plexiform layer of the retina, the myoepithelial cell layer of salivary and sweat glands, tactile nerve endings, and neurons in the brain. These dystrophin-positive regions reportedly contain actin filaments as a common characteristic, which is compatible with the dystrophin cDNA sequence. Dystrophin was absent in these regions in mdx mice. These results suggest that dystrophin plays an important physiological and/or structural role in cell motility as a trigger for propagating contractile force in, for example, the conduction system, with some relationship between actin filaments.

Rat muscle spindle immunocytochemistry revisited.

The expression of myosin heavy chain isoforms in muscle spindle fibres has been the subject of a number of immunocytochemical studies, some of them with discordant results. In order to assess whether these discrepancies are due to differences in the specificity and sensitivity of the antibodies used, we have compared the reactivity of rat muscle spindle fibres to two pairs of antibodies presumed to be directed against slow tonic (ALD 19 and ALD 58) and neonatal (NN5) and neonatal/fast (MF30) myosin heavy chains. Adult, developing and neonatally de-efferented muscle spindles from the rat hind limb muscles were studied in serial cross-sections processed for the peroxidase-antiperoxidase method. Important differences in the staining profiles of intrafusal fibres were noted when ALD 19 and ALD 58 were compared. ALD 19 stained the muscle spindle precursors from the seventeenth day in utero, whereas ALD 58 only did so by the twentieth day of gestation. In adult spindles ALD 19 stained the nuclear bag1 fibres along their entire length, whereas ALD 58 did not stain these fibres towards their ends. ALD 19 stained the nuclear bag2 fibres along the A, B and inner C region, but ALD 58 stained these fibres only in the A and the inner B regions. ALD 19 stained some nuclear chain fibres along a short equatorial segment, whereas ALD 58 did not stain the nuclear chain fibres at all. NN5 stained the nascent nuclear bag1 and chain fibre precursors at earlier stages of development than MF30. Clear differential staining between primary and secondary generation of both extra- and intrafusal myotubes was seen with NN5, whereas MF30 stained all myotubes alike. However, in postnatal spindles, MF30 was a very good negative marker of nuclear bag1 fibres. The staining profile of the adult fibres with NN5 and MF30 was rather similar. The staining pattern of neonatally de-efferented bag fibres obtained with ALD 19 and ALD 58 was practically identical and it differed from that of control spindles, confirming that motor innervation participates in the regulation of the expression of slow tonic MHC along the length of the nuclear bag2 fibres, as we have previously shown with ALD 19. The distinct staining patterns obtained with ALD 19 versus ALD 58 and with NN5 versus MF30 reflect differences in antibody sensitivity and specificity. These differences account, in part, for the discrepancies in the results of previous studies on muscle spindles, published by Kucera and Walro using ALD 58 and MF30, and by us using ALD 19 and NN5.

Slow-tonic MHC expression in paralyzed hindlimbs of fetal rats.

Whether nerve activity and active contraction of myotubes are essential for the assembly and initial differentiation of muscle spindles was investigated by paralyzing fetal rats with tetrodotoxin (TTX) from embryonic day 16 (E16) to E21, prior to and during the period when spindles typically form. TTX-treated soleus muscles were examined by light and electron microscopy for the presence of spindles and expression of myosin heavy chain (MHC) isoforms by the intrafusal fibers. Treatment with TTX did not inhibit the formation of a spindle capsule or the expression of a slow-tonic MHC isoform characteristic of intrafusal fibers, but did retard development of spindles. Spindles of TTX-treated E21 muscles usually consisted of one intrafusal fiber (bag2) only rather than two fibers (bag1 and bag2) typically present in untreated (control) E21 spindles. Intrafusal fibers of TTX-treated spindles also had only one sensory region supplied by multiple afferents, and were devoid of motor innervation. These features are characteristic of spindles in normal E18-E19 muscles. Thus, nerve and/or muscle activity is not essential for the assembly of muscle spindles, formation of a spindle capsule, and transformation of undifferentiated myotubes into the intrafusal fibers containing spindle-specific myosin isoforms. However, activity may promote the maturation of intrafusal bundles, as well as the maturation of afferent and efferent nerve supplies to intrafusal fibers.