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J M Walro

Publications and source records attributed to J M Walro.

At least 37 records · Page 2Linked to original sources
PubMed1993

Influence of muscle cell substrates on differentiation of intrafusal fiber types in neonatal rats.

Rat muscle spindles contain one nuclear bag2, one nuclear bag1, and two nuclear chain fibers. The three different types of intrafusal fiber in spindles may be a reflection of concomitant changes in proportions of slow primary, slow/fast secondary, and fast secondary myotubes during the period of spindle development. We examined whether experimentally altering the available muscle substrates would impact the intrafusal fiber type composition of spindles. De novo formation of spindles in muscles devoid of primary myotubes was induced by crushing the nerve to the medial gastrocnemius muscle in newborn rats and administering nerve growth factor for ten days afterwards. Encapsulated fibers of the reinnervated muscles examined one month after nerve crush had myofibrillar adenosine triphosphatase and myosin heavy chain profiles similar to normal bag2, bag1, or chain intrafusal fibers. However, spindles in reinnervated muscles contained fewer fibers than controls. Most experimental spindles contained chain and/or bag1 fibers, the two fiber types which ordinarily arise during secondary myogenesis. In contrast, bag2 fibers, fibers that normally form concomitant with primary myogenesis, were absent from nearly 90% of spindles in reinnervated muscles. The paucity of bag2 fibers may reflect the absence of primary myotubes, whereas the prevalence of chain and/or bag1 fibers may reflect that secondary myotubes or myofibers that descended from the secondary myotubes were the principal muscle substrates available for spindle formation in the nerve-crushed muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1992

Formation of muscle spindles in the absence of motor innervation.

Whether muscle spindles can form in muscles innervated only by afferents was investigated by removing the lumbosacral segment of the spinal cord immediately after crushing the nerve to the medial gastrocnemius (MG) muscle in newborn rats, and administering nerve growth factor for 10 days afterwards. The nerve-crushed MG muscles reinnervated by afferents in the absence of motor innervation were examined at postnatal (P) days 7, 9 and 30 for the presence of spindles by light and electron microscope. Reinnervated MG muscles contained spindle-like encapsulations of 1-4 fibers at 7, 9 and 30 days after the nerve crush. The number of spindles exceeded that of normal MG muscles, suggestive of de novo formation of spindles. All nerve-muscle contacts in the spindles had features of sensory endings, and intrafusal fibers expressed the spindle-specific slow-tonic myosin heavy chain (MHC) isoform at P30. No motor endplates were visible on any muscle fibers and extrafusal fibers were atrophied, as would be predicted in the absence of motor innervation. Thus, efferents are not essential for the formation and differentiation of muscle spindles in reinnervated muscles of neonatal rats.

Animals↗
PubMed1992

Superfluousness of motor innervation for the formation of muscle spindles in neonatal rats.

Muscle spindles form de novo in reinnervated muscles of neonatal rats treated with nerve growth factor. Whether the spindles can also form in muscle reinnervated only by afferents was investigated by removing the lumbosacral segment of the spinal cord immediately after crushing the nerve to the medial gastrocnemius muscle at birth, and administering nerve growth factor for 10 days afterwards. As predicted, the medial gastrocnemius muscles were reinnervated by afferents, but not efferents. No motor endplates were visible on any muscle fibers, and extrafusal fibers were atrophied. The reinnervated muscles contained spindle-like encapsulations of one to four fibers at 5, 7, 9 and 30 days after the nerve crush. The number of spindles as well as encapsulated fibers exceeded that of normal medial gastrocnemius muscles. The encapsulated fibers resembled typical intrafusal fibers. They had normal sensory-muscle contacts, but no motor endings. The fibers displayed equatorial clusters of myonuclei and expressed the spindle-specific slow-tonic myosin heavy chain isoform at postnatal day 30. Thus, efferents are not essential for the formation and differentiation of muscle spindles in reinnervated muscles of neonatal rats.

Animals↗
PubMed1992

Expression of type-specific MHC isoforms in rat intrafusal muscle fibers.

Myosin heavy chain (MHC) expression by intrafusal fibers was studied by immunocytochemistry to determine how closely it parallels MHC expression by extrafusal fibers in the soleus and tibialis anterior muscles of the rat. Among the MHC isoforms expressed in extrafusal fibers, only the slow-twitch MHC of Type 1 extrafusal fibers was expressed along much of the fibers. Monoclonal antibodies (MAb) specific for this MHC bound to the entire length of bag2 fibers and the extracapsular region of bag1 fibers. The fast-twitch MHC isoform strongly expressed by bag2 and chain fibers had an epitope not recognized by MAb to the MHC isoforms characteristic of developing muscle fibers or the three subtypes (2A, 2B, 2X) of Type 2 extrafusal fibers. Therefore, intrafusal fibers may express a fast-twitch MHC that is not expressed by extrafusal fibers. Unlike extrafusal fibers, all three intrafusal fiber types bound MAb generated against mammalian heart and chicken limb muscles. The similarity of the fast-twitch MHC of bag2 and chain fibers and the slow-tonic MHC of bag1 and bag2 fibers to the MHC isoforms expressed in avian extrafusal fibers suggests that phylogenetically primitive MHCs might persist in intrafusal fibers. Data are discussed relative to the origin and regional regulation of MHC isoforms in intrafusal and extrafusal fibers of rat hindlimb muscles.

Aging↗
PubMed1992

Morphometric analysis of coated vesicles in developing rat muscle spindles.

The incidence of coated vesicles under sarcolemmal surfaces of equatorial, juxta-equatorial and polar regions in developing and adult spindles of the rat soleus muscle was examined by quantitative morphometry of transverse ultrathin sections. Coated vesicles were more numerous: 1) under primary sensory endings than under other types of neuromuscular contacts; 2) under the appositional sarcolemma between neighbouring intrafusal fibres than under free surfaces of the sarcolemma; and 3) in developing than in mature spindles. Factors such as location and age of the animal often interacted to produce an additive effect on the incidence of coated vesicles. Although there was a high incidence of coated vesicles at the postsynaptic surface under sensory terminals of bag2 fibres in 18 and 19 day gestational embryonic rats, it peaked in 4 day postnatal animals. The high incidence of coated vesicles at sensory endings supports the view that coated vesicles mediate neurotrophic interactions between afferents and intrafusal fibres during the critical late gestation and early postnatal time period, as sensory axons first contact their target fibres and exert a maximal directing influence on the differentiation of intrafusal fibre types. In addition, the preferential localization of coated vesicles under appositional rather than free surfaces of developing intrafusal fibres in 0-4 day rats suggests that they play a role in the transport of active substances among intrafusal fibres exhibiting different stages of maturity.

Aging↗
PubMed1991

Non-neural and neural expression of myosin heavy chains by regenerated intrafusal fibers of rats.

Expression of myosin heavy chain (MHC) isoforms was studied in rat soleus (SOL) and extensor digitorum longus (EDL) muscles which regenerated in the presence or absence of innervation. Frozen sections of two 5 day denervated SOL and EDL grafts, two 40 day denervated SOL and EDL grafts, and two reinnervated 40 day SOL and EDL grafts were processed for demonstration of motor endplates, sensory endings, myosin adenosine triphosphatase (mATPase) and for expression of 4 MHCs. No qualitative differences in MHC expression were noted between 5 day or 40 day denervated grafts of the SOL and EDL muscles. All regenerated intrafusal and extrafusal myotubes or myofibers reacted to antibodies against neonatal and fast-twitch MHCs, but not to antibodies against slow-twitch and slow-tonic MHCs in these grafts. These data indicate that MHCs expressed by regenerated intrafusal myotubes do not parallel those expressed by myotubes which give rise to the three types of intrafusal fibers during development and that MHC expression by regenerated intrafusal myotubes parallels that of regenerated extrafusal myotubes prior to innervation. However, some regenerated intrafusal fibers in 40 day nerve-intact grafts bound antibodies to slow-twitch and slow-tonic MHCs, thus expressions of these two MHCs are nerve-dependent in regenerated muscle spindles.

Animals↗
PubMed1991

Neural organization of spindles in three hindlimb muscles of the rat.

The neuroanatomical organization of the dynamic (bag1) and static (bag2 and chain) intrafusal systems was compared by light and electron microscopy of serial sections among 71 poles of muscle spindle in soleus (SOL), extensor digitorum longus (EDL), and lumbrical (LUM) muscles in the rat. Eighty-four percent of 195 fusimotor (gamma) axons to the spindles innervated either the dynamic bag1 fiber or the static bag2 and/or chain fibers. Sixteen percent of the gamma axons coinnervated the dynamic and static intrafusal fibers. Some of these nonselective axons were branches of effernts that also gave rise to axons selective to either the dynamic or static types of intrafusal fibers in one or more spindles. Thus activation of individual stem gamma efferents might not have a purely dynamic or purely static effect on the integrated afferent outflow from spindles of a hindlimb muscles in the rat. In addition, primary afferents in all muscles had terminations that cross-innervated the dynamic bag1 and static bag1 and/or chain intrafusal fibers in individual spindles, an arrangement that may enhance the mixed dynamic/static behavior of afferents when different intrafusal fibers are activated concurrent. Spindles of the slow SOL and fast EDL muscles had similar features, whereas differences were observed in the organization of the proximal (SOL and EDL) and distal (LUM) muscles. Spindles in LUM muscles had fewer static intrafusal fibers, a higher ratio of dynamic to static gamma axons, and a higher incidence of skeletofusimotor (beta) innervation to intrafusal fibers than spindles in the SOL or EDL muscles. Thus, the relative contribution of dynamic and static systems to muscle afferent outflow may differ among spindles located in different segments of the rat hindlimb. However, the dynamic and static intrafusal systems of spindle were less sharply demarcated in each of the three hindlimb rat muscles than in the cat tenuissimus muscle.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Aggregation of myonuclei and the spread of slow-tonic myosin immunoreactivity in developing muscle spindles.

The pattern of regional expression of a slow-tonic myosin heavy chain (MHC) isoform was studied in developing rat soleus intrafusal muscle fibers. Binding of the slow-tonic antibody (ATO) began at the equator of prenatal intrafusal fibers where sensory nerve endings are located, and spread into the polar regions of nuclear bag2 and bag1 fibers but not nuclear chain fibers during ontogeny. The onset of the ATO reactivity coincided with the appearance of equatorial clusters of myonuclei (nuclear bag formations) in bag1 and bag2 fibers. Moreover, the intensity of the ATO reaction was strongest in the region of equatorial myonuclei and decreased with increasing distance from the equator of bag1 and bag2 fibers at all stages of prenatal and postnatal development. The polar expansion of ATO reactivity continued throughout the postnatal development of bag1 fibers, but ceased shortly after birth in bag2 fiber coincident with innervation by motor axons. Thus, afferents that innervate the equator might induce the slow-tonic MHC isoform in bag2 and bag1 fibers by regulating the myosin gene expression by equatorial myonuclei, and efferents or twitch contractile activity might inhibit the spread of the slow-tonic MHC isoform into the poles of bag2 but not bag1 fibers. Absence of ATO binding in chain fibers suggests that chain myotubes may not be as susceptible to the effect of afferents as are myotubes that develop into bag2 and bag1 fibers. The different patterns of slow-tonic MHC expression in the three types of intrafusal fiber may therefore result from the interaction of three elements: sensory neurons, motor neurons, and intrafusal myotubes.

Aging↗
PubMed1990

Origin of intrafusal muscle fibers in the rat.

The expression of several isoforms of myosin heavy chain (MHC) by intrafusal and extrafusal fibers of the rat soleus muscle at different stages of development was compared by immunocytochemistry. The first intrafusal myotube to form, the bag2 fiber, expressed a slow-twitch MHC isoform identical to that expressed by the primary extrafusal myotubes. The second intrafusal myotube to form, the bag1 fiber, expressed a fast-twitch MHC similar to that initially expressed by the secondary extrafusal myotubes. At subsequent stages of development, the equatorial and juxtaequatorial regions of bag2 and bag1 intrafusal myofibers began to express a slow-tonic myosin isoform not expressed by extrafusal fibers, and ceased to express some of the MHC isoforms present initially. Myotubes which eventually matured into chain fibers expressed initially both the slow-twitch and fast-twitch MHC isoforms similar to some secondary extrafusal myotubes. In contrast, adult chain fibers expressed the fast-twitch MHC isoform only. Hence intrafusal myotubes initially expressed no unique MHCs, but rather expressed MHCs similar to those expressed by extrafusal myotubes at the same chronological stage of muscle development. These observations suggest that both intrafusal and extrafusal fibers develop from common pools of bipotential myotubes. Differences in MHC expression observed between intrafusal and extrafusal fibers of rat muscle might then result from a morphogenetic effect of afferent innervation on intrafusal myotubes.

Animals↗
PubMed1989

The re-expression of two myosin heavy chains in regenerated rat muscle spindles.

The vascular supply and tendons of the extensor digitorum longus (EDL) muscles of two adult rats were unilaterally severed and the muscles were allowed to regenerate for 40 days. Serial frozen sections of muscle grafts were cut and stained for enzymes that delineated fiber type, sensory endings and motor endings. MF30 and ALD58, two antibodies which react only to intrafusal fibers in normal rat muscle, were reacted against sections of nerve-intact muscle grafts. Data were compared to that from muscles of normal rats. Encapsulated fibers devoid of sensory innervation and some extrafusal fibers in muscle grafts had a weak to moderate reaction to MF30, but no reaction to ALD58. Regenerated, encapsulated fibers with sensory innervation bound both MF30 and ALD58. These data indicate that afferents which reinnervate regenerated spindles retain the capacity to induce expression of spindle-specific myosin isoforms in rats.

Animals↗
PubMed1989

Role of nerve and muscle factors in the development of rat muscle spindles.

The soleus muscles of fetal rats were examined by electron microscopy to determine whether the early differentiation of muscle spindles is dependent upon sensory innervation, motor innervation, or both. Simple unencapsulated afferent-muscle contacts were observed on the primary myotubes at 17 and 18 days of gestation. Spindles, encapsulations of muscle fibers innervated by afferents, could be recognized early on day 18 of gestation. The full complement of spindles in the soleus muscle was present at day 19, in the region of the neuromuscular hilum. More afferents innervated spindles at days 18 and 19 of gestation than at subsequent developmental stages, or in adult rats; hence, competition for available myotubes may exist among afferents early in development. Some of the myotubes that gave rise to the first intrafusal (bag2) fiber had been innervated by skeletomotor (alpha) axons prior to their incorporation into spindles. However, encapsulated intrafusal fibers received no motor innervation until fusimotor (gamma) axons innervated spindles 3 days after the arrival of afferents and formation of spindles, at day 20. The second (bag1) intrafusal fiber was already formed when gamma axons arrived. Thus, the assembly of bag1 and bag2 intrafusal fibers occurs in the presence of sensory but not gamma motor innervation. However, transient innervation of future bag2 fibers by alpha axons suggests that both sensory and alpha motor neurons may influence the initial stages of bag2 fiber assembly. The confinement of nascent spindles to a localized region of the developing muscle and the limited number of spindles in developing muscles in spite of an abundance of afferents raise the possibility that afferents interact with a special population of undifferentiated myotubes to form intrafusal fibers.

Animals↗
PubMed1989

Postnatal expression of myosin heavy chains in muscle spindles of the rat.

The immunocytochemical expression of two myosin isoforms in intrafusal muscle fibers was examined in soleus muscles of neonatal (zero to six days postpartum) and adult rats. Monoclonal antibodies specific for myosin heavy chains of the slow-tonic anterior latissimus dorsi (ALD58) and fast-twitch pectoralis (MF30) muscles of the chicken were used. In adults ALD58 bound to the intracapsular regions of bag1 and bag2 fibers and MF30 bound to the intracapsular regions of bag2 and chain fibers. The extracapsular regions of intrafusal fibers and all extrafusal fibers did not react to ALD58 or MF30. Bag1 and bag2 fibers of neonatal rats expressed immature myosin patterns but chain fibers did not. The adult pattern of immunoreactivity of intrafusal fibers developed by the fourth postnatal day, when the patterns of motor but not sensory innervation in the spindle are still immature. Data suggest that the expression and maintenance of the specific anti-myosin immunoreactivity of intrafusal fibers during postnatal development of rat spindles is dependent upon sensory but not motor innervation. Moreover, afferents might regulate the gene expression responsible for synthesis of myosins isoforms specific to intrafusal fibers only in those myonuclei located within the capsule, but not in the myonuclei in extracapsular regions of intrafusal fibers.

Animals↗
PubMed1989

Innervation of regenerated spindles in muscle grafts of the rat.

Features of the nerve supply and the encapsulated fibers of muscle spindles were assessed in grafted and normal extensor digitorum longus (EDL) muscles of rats by analysis of serial 10-microns frozen transverse sections stained for enzymes which delineated motor and sensory endings, oxidative capacity and muscle fiber type. The number of fibers was significantly more variable, and branched fibers were more frequently observed in regenerated spindles than in control spindles. Forty-eight percent of regenerated spindles received sensory innervation. Spindles reinnervated by afferents had a larger periaxial space than did spindles which were not reinnervated by afferents. Regenerated fibers innervated by afferents had small cross-sectional areas, equatorial regions with myofibrils restricted to the periphery of fibers, unpredictable patterns of nonuniform and nonreversible staining along the length of the fiber for 'myofibrillar' adenosine triphosphatase (mATPase) after acid and alkaline preincubation. In contrast, regenerated fibers devoid of sensory innervation resembled extrafusal fibers in that they usually exhibited myofibrils throughout the length of the fiber, no central aggregations of myonuclei, uniform staining for mATPase and a reversal of staining for mATPase after preincubation in an acid or alkaline medium. Approximately thirty percent of encapsulated fibers devoid of sensory innervation stained analogous to a type I extrafusal fiber, a pattern of staining never observed in intrafusal fibers of normal spindles. Groups of encapsulated fibers all exhibiting this pattern of staining reflect that either these fibers may have been innervated by collaterals of skeletomotor axons that originally innervated type I extrafusal fibers or that fibers innervated by only fusimotor neurons express patterns of staining for mATPase similar to extrafusal fibers in the absence of sensory innervation. Sensory innervation may also influence the reestablishment of multiple sites of motor endings on regenerated intrafusal fibers. Those regenerated fibers innervated by afferents had more motor endings than did regenerated fibers devoid of sensory innervation. Differences in size, morphology, and patterns of staining for mATPase and numbers of motor endings between fibers innervated by afferents and fibers devoid of sensory innervation reflect that afferents can influence the differentiation of muscle cells and the reestablishment of motor innervation other than during the late prenatal/early postnatal period when muscle spindles form and differentiate in rats.

Adenosine Triphosphatases↗
PubMed1989

Nonuniform expression of myosin heavy chain isoforms along the length of cat intrafusal muscle fibers.

The expression of four myosin heavy chain (MHC) isoforms, avian slow-tonic (ATO) or neonatal-twitch (ANT) and mammalian slow-twitch (MST) or fast-twitch (MFT) in intrafusal fibers was examined by immunocytochemistry of spindles in the tenuissimus muscle of adult cats. The predominant MHCs expressed by nuclear bag fibers were ATO and MST, whereas the MHCs prevalent in nuclear chain fibers were ANT and MFT. The expression of these isoforms of MHC was not uniform along the length of intrafusal fibers. In general, both bag and chain fibers expressed avian MHC in the intracapsular region and mammalian MHC in the extracapsular region. The nonuniform expression of MHCs observed along the length of bag and chain fibers implies that different genes are activated in myonuclei located in the intracapsular and extracapsular regions of the same muscle fiber. Regional differences in gene activation might result from a greater effect of afferents on myonuclei located near the equator of intrafusal fibers then on myonuclei outside the spindle capsule.

Animals↗
PubMed1988

The effect of neonatal deafferentation or deefferentation on the immunocytochemistry of muscle spindles in the rat.

Immature muscle spindles were either deafferented or deefferented by selectively severing the sensory or motor nerve supply to the soleus muscle in neonatal rats. Experimental spindles were examined two months after the surgery using monoclonal antibodies specific for myosin heavy chains of slow-tonic and fast-twitch chicken muscles. The deefferented spindles exhibited a pattern of antibody binding that closely resembled that of normal adult intrafusal fibers, whereas deafferented intrafusal fibers were unreactive with the two antibodies. These observations suggest that sensory innervation is responsible for the expression of myosins in developing intrafusal muscle fibers of rat.

Animals↗
PubMed1988

Innervation of developing intrafusal muscle fibers in the rat.

The chronology of development of spindle neural elements was examined by electron microscopy in fetal and neonatal rats. The three types of intrafusal muscle fiber of spindles from the soleus muscle acquired sensory and motor innervation in the same sequence as they formed--bag2, bag1, and chain. Both the primary and secondary afferents contacted developing spindles before day 20 of gestation. Sensory endings were present on myoblasts, myotubes, and myofibers in all intrafusal bundles regardless of age. The basic features of the sensory innervation--first-order branching of the parent axon, separation of the primary and secondary sensory regions, and location of both primary and secondary endings beneath the basal lamina of the intrafusal fibers--were all established by the fourth postnatal day. Cross-terminals, sensory terminals shared by more than one intrafusal fiber, were more numerous at all developmental stages than in mature spindles. No afferents to immature spindles were supernumerary, and no sensory axons appeared to retract from terminations on intrafusal fibers. The earliest motor axons contacted spindles on the 20th day of gestation or shortly afterward. More motor axons supplied the immature spindles, and a greater number of axon terminals were visible at immature intrafusal motor endings than in adult spindles; hence, retraction of supernumerary motor axons accompanies maturation of the fusimotor system analogous to that observed during the maturation of the skeletomotor system. Motor endings were observed only on the relatively mature myofibers; intrafusal myoblasts and myotubes lacked motor innervation in all age groups. This independence of the early stages of intrafusal fiber assembly from motor innervation may reflect a special inherent myogenic potential of intrafusal myotubes or may stem from the innervation of spindles by sensory axons.

Animals↗