Influence of neonatal motor denervation on expression of myosin heavy chain isoforms in rat muscle spindles

In order to evaluate the effects of fusimotor elimination on the expression of myosin heavy chain (MHC) proteins in intrafusal fibres, we compared the muscle spindles in hind limb muscles of 3- to 6-week-old rats de-efferented at birth with those of their litter-mate controls. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal MHC isoforms, against synaptophysin, the neurofilament 68 kD subunit and laminin. We found that de-efferented intrafusal fibres differentiated, as in normal spindles, into nuclear bag and bag fibres both containing predominantly slow MHC, and nuclear chain fibres that contained fast and neonatal MHC. In both de-efferented and control intrafusal fibres the same MHCs were stained; the degree and extent of staining, however, varied. Both types of de-efferented bag fibres displayed a high content of slow tonic and slow twitch MHC along most of the fibre length, in contrast to the prominent regional variation in control bag fibres. In their encapsulated regions, the de-efferented bag fibres were more similar to each other in their reactivity to anti-fast twitch and anti-neonatal MHC antibodies than the control bag fibres. In these aspects they resembled more closely the bag fibres of newborn rats. The differences might be due to an arrest of "specialization" in the regional expression of the different MHC isoforms. Chain fibres developed MHC patterns identical to those of control spindles with all the antibodies used, even though they differentiated from the beginning in the absence of motor innervation. The structural differentiation of the capsule and sensory innervation in de-efferented muscle spindles, as shown by anti-laminin, anti-synaptophysin and anti-neurofilament staining, did not differ from the controls. We conclude, in agreement with previous studies, that the sensory innervation plays a key role in inducing and supporting the differentiation of intrafusal fibres and the specific expression of their MHC. However, we also show that motor innervation and/or muscle function seem to be necessary for the diversity in the expression and distribution of different slow and fast MHC isoforms in the bag and bag fibres.     

Rat muscle spindle immunocytochemistry revisited

Summary 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, wheras 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.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

Summary The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations.Nuclear bag₁ fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag₂ fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with antineonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag₁ fibers lacked staining for M-protein, whereas bag₂ fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag₁ fibers were less reactive and clear striations were not observed, in contrast to bag₂ and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition, (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested.Taken together, the data show that in adult rat solcus, slow tonic and neonatal myosin heavy, chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Development of immunohistochemical characteristics of intrafusal fibres in normal and de-efferented rat muscle spindles

Summary Intrafusal muscle fibres in adult muscle spindles differ in their myosin composition. After selective motor denervation intrafusal muscle fibres develop mature ultrastructural characteristics. In order to evaluate the role of fusimotor innervation on the maturation of the myosin composition of intrafusal muscle fibres we have examined with immunohistochemical techniques i) the postnatal development of muscle spindles in new-born rats and in 7–21 day old rats; ii) muscle spindles in the EDL of 21-day-old rats de-efferented at birth. For the characterization of myosins in intrafusal fibres we used three myosin antisera: antipectoral myosin, antiheart myosin and antiheart myosin adsorbed with muscle powder from the soleus muscle of guinea pig. We show in this study that during development intrafusal fibres change immunoreactivity and that in the absence of motor innervation bag fibres do not fully develop the myosin characteristics of control spindles. We conclude that the maturation of bag₁ and bag₂ fibres apparently requires next to the inductive influence of sensory axon terminals the presence and activity of fusimotor axons.     

Unusual intrafusal fibres in human muscle spindles

We have studied the morphology and pattern of expression of myosin heavy chain (MHC) isoforms of intrafusal fibres in a human first lumbrical muscle. Each intrafusal fibre type, namely nuclear bag1, nuclear bag2 and nuclear chain fibres, had a distinct MHC composition and distribution of different MHC isoforms along the whole length of intrafusal fibres. However, most muscle spindles analyzed also contained one or several intrafusal fibres exhibiting an extrafusal or mixed pattern of immunoreactivity which did not correspond to any of the described intrafusal fibre types. We conclude that the latter fibres do not represent new intrafusal fibre types, but their morphology and expression of MHC merely reflects the differences in their innervation owing to their unusual localization at the edge or outside the axial bundle of intrafusal fibres.     

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.     

Differentiation of supernumerary fibres in neonatally deefferented rat muscle spindles

Abstract. The myofibrillar ATPase (mATPase) activity and the pattern of expression of several myosin heavy chain (MHC) isoforms and of M-protein (Mr 165000) were studied in serial cross sections of neonatally deefferented 5- to 8-week-old rat hindlimb muscle spindles with supernumerary intrafusal fibres. In a sample of 5- to 6-week-old neonatally deefferented muscle spindles cut through the A region, the average number of intrafusal fibres per spindle was 8.4 in comparison to 4.2 in control spindles. Parent fibres extended throughout the whole encapsulated portion of the spindle, whereas supernumerary fibres were found only in the A region. The diameters of the supernumerary intrafusal fibres varied from less than 1 μ up to 10 μ approximately. On the basis of the mATPase activity and the pattern of expression of MHC isoforms and of M-protein, the vast majority of the supernumerary fibres could be classified as nuclear bag₂, bag₁ or chain fibres. However, some supernumerary fibres with small diameters exhibited features that did not fit any of the three known intrafusal fibre types. Two major processes, namely fibre splitting versus activation and fusion of satellite cells, might account for the formation of supernumerary fibres. The data presented suggest the existence of at least two types of intrafusal satellite cells. One type of satellite cell is related to the nuclear bag fibres and gives rise to myotubes which, if they have sensory innervation, can express slow tonic MHC and, therefore, differentiate into a phenotype similar to that seen in nuclear bag fibres. The other type of satellite cells form myotubes which attain a fast phenotype similar to that seen in nuclear chain fibres irrespective of the presence or absence of sensory innervation.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations. Nuclear bag1 fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag2 fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with anti-neonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag1 fibers lacked staining for M-protein, whereas bag2 fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag1 fibers were less reactive and clear striations were not observed, in contrast to bag2 and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested. Taken together, the data show that in adult rat soleus, slow tonic and neonatal myosin heavy chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Intrafusal myosin heavy chain expression of human masseter and biceps muscles at young age shows fundamental similarities but also marked differences

Muscle spindles are skeletal muscle mechanoreceptors that provide proprioceptive information to the central nervous system. The human adult masseter muscle has greater number, larger and more complex muscle spindles than the adult biceps. For a better knowledge of muscle diversity and physiological properties, this study examined the myosin heavy chain (MyHC) expression of muscle spindle intrafusal fibres in the human young masseter and young biceps muscles by using a panel of monoclonal antibodies (mAbs) against different MyHC isoforms. Eight MyHC isoforms were detected in both muscles-slow-tonic, I, IIa, IIx, foetal, embryonic, α-cardiac and an isoform not previously reported in intrafusal fibres, termed IIx′. Individual fibres co-expressed 2–6 isoforms. MyHC-slow tonic separated bag₁, AS-bag₁ and bag₂ fibres from chain fibres. Typically, bag fibres also expressed MyHC-I and α-cardiac, whereas chain fibres expressed IIa and foetal. In the young masseter 98 % of bag₁ showed MyHC-α cardiac versus 30 % in the young biceps, 35 % of bag₂ showed MyHC-IIx′ versus none in biceps, 17 % of the chain fibres showed MyHC-I versus 61 % in the biceps. In conclusion, the result showed fundamental similarities in intrafusal MyHC expression between young masseter and biceps, but also marked differences implying muscle-specific proprioceptive control, probably related to diverse evolutionary and developmental origins. Finding of similarities in MyHC expression between young and adult masseter and biceps muscle spindles, respectively, in accordance with previously reported similarities in mATPase fibre type composition suggest early maturation of muscle spindles, preceding extrafusal fibres in growth and maturation.     

Slow-tonic MHC expression in paralyzed hindlimbs of fetal rats

Summary 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.     

Expression of myosin heavy chain isoforms in developing rat muscle spindles

Summary The development of muscle spindles, with respect to the expression of myosin heavy chain isoforms was studied in rat hind limbs from 17 days of gestation up to seven days after birth. Serial cross-sections were labelled with antibodies against slow tonic, slow twitch and neonatal isomyosins, myomesin, laminin and neurofilament protein.At 17–18 days of gestation, a small population of primary myotubes expressing slow tonic myosin were identified as the earliest spindle primordia. These myotubes also expressed slow twitch and, to a lesser extent, neonatal myosin. At 19–20 days of gestation a second myotube became apparent; this staining strongly with anti-neonatal myosin. A day later this secondary myotube acquired reactivity to anti-slow tonic and anti-slow twitch myosins. By birth, a third myotube was present; this staining strongly with anti-neonatal myosin but otherwise unreactive with the other antibodies against myosin heavy chains. Three days after birth a fourth myotube, with identical reactivity to the third one, became apparent. Regional variation in the expression of isomyosins, which was present since birth in the two nuclear bag fibers was further enhanced: the nuclear bag₂ staining strongly with anti-slow tonic and antineonatal in the equatorial region and with decreasing intensity towards the poles, whilst with anti-slow twitch the stainability was low in the equatorial and high in the polar region. The nuclear bag₁ fiber showed a homogeneous staining: high with anti-slow tonic, moderate with anti-neonatal, and displayed stainability to antislow twitch myosin in the polar regions only. No regional variation was found along the chain fiber/myotube. At seven days after birth, the pattern of reactivity was similar to that found in the adult spindles, except for the bag₁ fiber which still expressed neonatal myosin.We show that slow tonic myosin is expressed from early development and it is a reliable marker of developing bag fibers. We suggest that muscle spindles are formed from special cell lineages of which the primary generation myotubes expressing slow tonic myosin represent the primordium of muscle spindles.     

Development of immunohistochemical characteristics of intrafusal fibres in normal and de-efferented rat muscle spindles

Intrafusal muscle fibres in adult muscle spindles differ in their myosin composition. After selective motor denervation intrafusal muscle fibres develop mature ultrastructural characteristics. In order to evaluate the role of fusimotor innervation on the maturation of the myosin composition of intrafusal muscle fibres we have examined with immunohistochemical techniques i) the postnatal development of muscle spindles in new-born rats and in 7-21 day old rats; ii) muscle spindles in the EDL of 21-day-old rats de-efferented at birth. For the characterization of myosins in intrafusal fibres we used three myosin antisera: antipectoral myosin, antiheart myosin and antiheart myosin adsorbed with muscle powder from the soleus muscle of guinea pig. We show in this study that during development intrafusal fibres change immunoreactivity and that in the absence of motor innervation bag fibres do not fully develop the myosin characteristics of control spindles. We conclude that the maturation of bag1 and bag2 fibres apparently requires next to the inductive influence of sensory axon terminals the presence and activity of fusimotor axons.     

One-bag-fiber muscle spindles in tenuissimus muscles of the cat

Summary Over 150 complete and 139 incomplete single muscle spindles were examined in serial transverse sections of cat tenuissimus muscles in search for spindles lacking one of the two types of nuclear bag intrafusal fiber. Several histochemical reactions were used to type the intrafusal muscle fibers and assess the spindle motor and sensory innervation. One complete spindle lacked a bag₁ fiber, and another spindle lacked a bag₂ fiber. Several incomplete spindles also lacked bag₁ fibers. In addition, ten double tandem spindles contained one capsular unit each that lacked the bag₁ fiber, and one triple tandem spindle had two such capsules. All one-bag-fiber spindles had primary sensory innervation, but none had secondary sensory innervation. Their motor innervation was similar to that of the usual two-bag-fiber spindles in the number and disposition of intrafusal motor endings. It is unclear whether the one-bag fiber spindles, either single or tandem-linked, are products of an aberrant spindle development or represent a true anatomical and functional subcategory of the cat muscle spindle.     

A histoenzymatic study of rat intrafusal muscle fibres

Summary The histochemical activities of myofibrillar adenosine triphosphatase (ATPase), succinic dehydrogenase (SDH) and alpha glycerophosphate dehydrogenase (-GPD) were studied in intrafusal muscle fibres of rat fast and slow muscles. The ATPase reaction was carried out after the three standard acid preincubations. The cold K₂-EDTA preincubated ATPase reaction product was similar to that seen following the regular or alkalipreincubated ATPase reaction, except that the intermediate bag fibres exhibited much higher activity after cold K₂-EDTA preincubation. Following either acetic acid solution or cold and room temperature K₂-EDTA-preincubation, followed by the ATPase reaction, chain fibres of the fast muscles vastus lateralis and extensor digitorum longus exhibited a very low amount of reaction product as compared with those of the slow soleus. Veronal acetate and K₂-EDTA preincubations (and equally preincubation in acetic acid solution) resulted in acid stable ATPase activity along the entire length of the typical bag fibres but only in the polar regions of the intermediate bag fibres. On the basis of differing -GPD reaction, two sub populations of nuclear chain fibres were discovered in one spindle. It is a matter of conjecture, to what extent the histochemical differences of intrafusal fibres from fast and slow muscles reflects functional distinctions in the response to stretch of muscle spindles from fast and slow muscles.     

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

Summary 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.     

Origin of intrafusal muscle fibers in the rat

Summary 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.     

Expression of an alpha cardiac-like myosin heavy chain in muscle spindle fibres

Summary In the present study we have investigated the reactivity of rat muscle to a specific monoclonal antibody directed against alpha cardiac myosin heavy chain. Serial cross sections of rat hindlimb muscles from the 17th day in utero to adulthood, and after neonatal denervation and de-efferentation, were studied by light microscope immunohistochemistry. Staining with anti- myosin heavy chain was restricted to intrafusal bag fibres in all specimens studied. Nuclear bag₂ fibres were moderately to strongly stained in the intracapsular portion and gradually lost their reactivity towards the ends, whereas nuclear bag₁ fibres were stained for a short distance in each pole. Nuclear bag₂ fibres displayed reactivity to anti- myosin heavy chain from the 21st day of gestation, whereas nuclear bag₁ fibres only acquired reactivity to anti- myosin heavy chain three days after birth. After neonatal de-efferentation, the reactivity of nuclear bag₂ fibres to anti- myosin heavy chain was decreased and limited to a shorter portion of the fibre, whereas nuclear bag₁ fibres were unreactive. We showed that a myosin heavy chain isoform hitherto unknown for skeletal muscle is specifically expressed in rat nuclear bag fibres. These findings add further complexity to the intricate pattern of isomyosin expression in intrafusal fibres. Furthermore, we show that motor innervation influences the expression of this isomyosin along the length of the fibres.     

Immunocytochemical characterisation of two generations of fibers during the development of the human quadriceps muscle

We have carried out a comprehensive study of the formation of muscle fibers in the human quadriceps in a large series of well dated human foetuses and children. Our results demonstrate that a first generation of muscle fibers forms between 8-10 weeks. These fibers all express slow twitch myosin heavy chain (MHC) in addition to embryonic and foetal MHCs, vimentin and desmin. Between 10-11 weeks, a subpopulation of these fibers express slow tonic MHC, being the first primordia of muscle spindles. Extrafusal fibers of a second generation form progressively and asynchronously around the primary fibers between 10-18 weeks, giving the muscle a very heterogeneous aspect due to different degrees of organization of their proteins. By 20 weeks, these second generation fibers become homogeneous and thereafter undergo a process of maturation and differentiation when they eliminate vimentin, embryonic and foetal MHCs to express either slow twitch or fast MHC. The differentiation of these second generation fibers into slow and fast depends upon different factors, such as motor innervation or level of thyroid hormone. Around the intrafusal first generation fibers, additional subsequent generations of fibers are also progressively formed. Some differ from the extrafusal second generation fibers by expressing slow tonic MHC, others by continuous expression of foetal MHC. The differentiation of intrafusal fibers is probably under the influence of both sensory and motor innervation.     

The occurrence of “mixed” nuclear bag intrafusal fibers in the cat muscle spindle

Summary A total of 147 muscle spindles was studied histochemically in serial transverse sections of 42 cat tenuissimus muscle specimens. Nuclear bag₁, nuclear bag₂ and nuclear chain intrafusal muscle fibers were distinguished by the differential staining resulting from the reactions for myosin adenosine 5-triphosphatase and nicotinamide adenine dinucleotide tetrazolium reductase. The majority of intrafusal fibers were of the same histochemical type at both fiber poles. However, seven muscle spindles contained one nuclear bag fiber each that presented as a bag₁ in one pole and as a bag₂ in the other pole. These mixed nuclear bag fibers were found in spindles that also contained at least one bag₁ and one bag₂ fiber of equivalent histochemical presentation in both fiber poles. The mixed bag fibers displayed differences of apparent fiber diameter and relative polar length between the two fiber poles. The motor innervation pattern, as revealed by staining for cholinesterase, was also dissimilar between the two poles of mixed bag fibers. The study indicates that the spindle equatorial region may in some instances serve as a boundary between two morphologically and histochemically different poles of the same intrafusal fiber.     

Expression of myosin heavy chain (MyHC) isoforms in rat intrafusal muscle fibres after neonatal deefferentation and subsequent denervation

The analysis of developing intrafusal fibres is not feasible in the absence of primary sensory axons, as neonatal denervation leads to the disintegration of muscle spindles. On the other hand, neonatal deefferentation does not arrest their differentiation and, moreover, it leads to the neomyogenesis of supernumerary intrafusal profiles. If the sciatic nerve was sectioned in 4-week-old rats deefferented at the birth, muscle spindles survived, the neomyogenesis proceeded and the denervated intrafusal fibres expressed the spindle specific slow tonic (STO) MyHC. The expression of MyHC pattern in individual fibres and the differentiation of the fibre type characteristics were, however, less obvious compared to the control or deefferented spindles. The newly formed intrafusal profiles (which differentiated from satellite cells in the absence of innervation) expressed the STO MyHC particularly when they developed in a spatial relation to nuclear bag fibres.