The topography of long nuclear chain intrafusal fibers in the cat muscle spindle

Muscle spindles were studied histochemically in serial transverse sections of specimens of the cat tenuissimus muscle. The nuclear chain intrafusal muscle fibers were separated into three subtypes, called long, intermediate and typical. The long chain and intermediate chain fibers tended to assume a particular position within the axial bundle of intrafusal fibers. The fibers were usually located in that layer of chain fibers that was positioned farthest away from the bag2 fiber. Furthermore, they were usually situated adjacent to the bag1 fiber throughout much of the extent of the spindle pole. Some long chain and intermediate chain fibers had several fiber nuclei abreast at the equator rather than a single row of central nuclei, as in most nuclear chain fibers. The relative position of intrafusal fibers within the cat spindle may reflect their order of formation during development, with the fibers retaining, to a variable degree, their association with the bag2 fiber which acted as template. Thus, the axial position of long chain and intermediate chain fibers suggests that they are among the first nuclear chain fibers to form. This may play a role in the known preferential innervation of these chain fibers by skeleto-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.     

Histochemical profiles of cat intrafusal muscle fibers and their motor innervation

Summary Muscle spindles were examined histochemically in serial transverse sections of cat tenuissimus muscles. The myofibrillar adenosine triphosphatase (ATPase) staining reaction was used to identify nuclear bag₁, bag₂ and nuclear chain intrafusal muscle fibers. Regional differences in ATPase staining occurred along the bag₁ and bag₂ fibers but not along the chain fibers. All intrafusal fiber types displayed regional variability in staining for nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR). Motor nerve terminals were demonstrated along the poles of bag₁, bag₂ and chain fibers by staining for cholinesterase (ChE). There was no consistent spatial correlation between the intensity of regional ATPase staining along the bag fibers and location, number or type of motor endings. However, most ChE deposits occurred in intrafusal fiber regions that displayed the greatest NADH-TR variability. Some fiber poles or whole intrafusal fibers were devoid of any ChE deposits but their ATPase and NADH-TR content was comparable to that of fibers bearing ChE deposits. The observations suggested that motor nerve fibers per se may not play a major role in determining the histoenzymatic content of intrafusal fibers.     

Distribution of sarcoplasmic reticulum Ca-ATPase and of calsequestrin at the polar regions of rat,rabbit and cat intrafusal fibers

Summary Sarcoplasmic reticulum (SR) Ca²⁺-pumping ATPase (Ca-ATPase) and calsequestrin (CaS) were visualized by indirect immunofluorescence at the polar regions of adult rat, rabbit and cat intrafusal fibers. The immunohistochemical reaction products were regarded as histochemical markers of the SR and as valid indicators of the distribution of the two Ca²⁺sequestering proteins. Static nuclear bag₂ fibers displayed lower levels of both Ca-ATPase and CaS than the other two intrafusal fiber types. Nuclear chain fibers presented the highest Ca-ATPase levels and, together with dynamic nuclear bag₁ fibers, they also exhibited relatively high amounts of CaS. The level of Ca-ATPase was lower in bag₁ fibers than in nuclear chain fibers, but not as low as in bag₂ fibers. The comparatively high levels of Ca-ATPase and CaS seen in nuclear chain fibers coincided with their reported faster contractile speeds compared to nuclear bag fibers.Dedicated to Professor Dr. T.H.Schiebler on the occasion of his 65th birthday     

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.     

Immunohistochemical demonstration of embryonic myosin heavy chains in adult mammalian intrafusal fibers

Summary Serial cross sections of rat, rabbit and cat intrafusal fibers from muscle spindles of normal adult hindlimb muscles were incubated with a monoclonal antibody against embryonic myosin heavy chains. Intrafusal fiber types were identified by noting their staining patterns in adjacent sections incubated for myofibrillar ATPase after acid or alkaline preincubation. In rat and rabbit muscle spindles dynamic nuclear bag₁ fibers reacted strongly at the polar and juxtaequatorial regions. Static nuclear bag₂ fibers reacted weakly or not at all at the polar region, but showed a moderate amount of activity at the juxtaequator. At the equatorial region both types of nuclear bag fibers displayed a rim of fluorescence surrounding the nuclear bags, while the areas occupied by the nuclear bags themselves were negative. Nuclear chain fibers in rat and rabbit muscle spindles were unreactive with the specific antibody over their entire length. In cat muscle spindles both types of nuclear bag fibers presented profiles which resembled those of the nuclear bag fibers in the other two species, but unlike in rat and rabbit spindles, cat nuclear chain fibers reacted as strongly as dynamic nuclear bag₁ fibers.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

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.     

Ultrastructure of dynamic and static skeletofusimotor endings in a cat muscle spindle

Summary Endings of four skeletofusimotor axons in a spindle of the cat tenuissimus muscle were examined in semithin (1-m thick) and ultrathin transverse serial sections. Two (dynamic) axons terminated on the nuclear bag₁ intrafusal muscle fiber and on extrafusal fibers of the dark type. Two (static) axons terminated on the nuclear chain intrafusal fibers and extrafusal fibers of the intermediate type. The degree of indentation of axon terminals into the muscle surface, thickness of the sole plate and extent of folding of subjunctional membranes differed among intrafusal and extrafusal terminations of the same axon. Endings of axons on the bag₁ and chain fibers were also morphologically dissimilar. Motor axons may not determine ending morphology. Rather the form and structure of a bag₁ or chain ending may be determined by the type of intrafusal fiber on which the ending lies and the ending's distance from the primary sensory axon.     

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.     

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.     

Comparison of motor endings of the cat's muscle spindle stained for NADH-tetrazolium reductase and cholinesterase

Summary Cat muscle spindles were examined histochemically in serial transverse sections of tenuissimus muscles stained for ATPase, NADH-TR and ChE alternating sequentially. Motor nerve terminals on nuclear bag₁, bag₂ and nuclear chain intrafusal muscle fibers were identified in periodic sections stained for ChE. Intrafusal fiber regions that carried ChE-active areas were then examined in staining for NADH-TR. The motor endings on the three types of intrafusal fiber differed in their apparent histochemical content of both ChE and NADH-TR. The observations suggest that functional differences may exist among motor nerve terminals on the various intrafusal fiber types.     

Ultrastructure of Type 2A Extrafusal Muscle Fibers and Muscle Spindle Intrafusal Fibers of Adult Rats after Prolonged Swimming

We compared the ultrastructure of type 2A extrafusal muscle fibers, the nuclear chain, and other intrafusal fibers of muscle spindle (muscle stretch mechanoreceptor) in adult rats after prolonged swimming (5–10 h/day, 10 days). The Golgi apparatus was expressed moderately in type 2A extrafusal fibers and hypertrophied in the motor B zone of nuclear chain intrafusal fibers. Intense development of the Golgi apparatus in the nuclear chain intrafusal fiber appears to be related to glycogenolysis in the autophagous vacuoles, involvement in the lysosome activity, and plasma membrane renewal. Recapitulation of the mechanism of glycogen autophagy, which is observed in newborn rats during mobilization of glycogen from the liver and muscle, was demonstrated in adult rats under the influence of physical load in the nuclear chain and nuclear bag₂ intrafusal fibers and in type 2A extrafusal fibers. This is accounted for by a weak differentiation of the intrafusal muscle fibers: structurally, they are similar to myotubes and have specific features of blood supply and innervation. Individual features of experimental adult rats may also play a certain role.     

Morphometric studies on tenuissimus muscle spindles in the cat

Muscle spindles were studied histochemically in serial transverse sections of 42 cat tenuissimus muscle specimens. Staining for myofibrillar adenosine triphosphatase was employed to identify nuclear bag 1, nuclear bag 2, and nuclear chain intrafusal muscle fibers. The nuclear chain fibers were further subdivided into three categories according to their polar length and the intensity of their staining for nicotinamide adenine dinucleotide tetrazolium reductase. A total of 430 spindle poles were surveyed. The mean spindle content of bag 1, bag 2, and chain fibers was established. The mean polar length of intrafusal fibers as well as that of the intracapsular and extracapsular spindle regions was determined. A cholinesterase (ChE) staining technique was used to demonstrate the termination sites of motor axons along intrafusal fibers. Two types of circumscribed ChE deposits. The "rim" and the "plate," occurred on the fibers. The nuclear chain fibers usually carried both the ChE rims and plates, while most nuclear fibers displayed only the plates. The ChE plates were assessed in term of their appearance, staining intensity, length, and location along the fibers. The mean number of ChE plates found along the fibers was established for each of the various intrafusal fiber types. These histochemical observations are discussed with regard to the current concepts of cat spindle morphology and motor innervation. The results suggest a degree of predictability in the spindle fiber content and in the distribution of motor nerve terminals along intrafusal muscle fibers, at least in the tenuissimus muscle.     

Motor innervation of the cat muscle spindle studied by the cholinesterase technique

Summary Muscle spindles were traced in serial transverse sections of cat tenuissimus muscles. Myofibrillar adenosine triphosphatase staining reaction was used to identify nuclear bag₁, nuclear bag₂ and nuclear chain intrafusal muscle fibers. Typical chain fibers and long chain fibers were distinguished, the latter extending for more than 1,000 m beyond the termination of the spindle capsule. Simple rim and more elaborate plate deposits were demonstrated histochemically along the poles of the typical chain fibers in staining for cholinesterases. They were considered to correspond, respectively, to the trail and plate motor nerve terminals. Most long chain fibers and the majority of nuclear bag fibers had their motor innervation limited to plate-type endings. In addition, faint diffuse cholinesterase staining occurred along the spindle capsule and the surface of some intrafusal fibers. These histochemical observations are discussed with regard to the current concepts concerning the morphological and functional organization of the motor innervation of the cat muscle spindle.     

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.     

Nonselective motor innervation of nuclear bag1 intrafusal muscle fibers in the cat

The motor nerve supply to cat nuclear bag1 intrafusal muscle fibers was reconstructed from light and electron microscopy of serial transverse sections of spindles in the tenuissimus muscle. Twenty-six of thirty poles of bag1 fibers that were examined received motor innervation. Every innervated bag1 pole received at least one (range 1-3) selective motor axon that supplied this fiber type only. Four of the innervated bag1 poles (15%) received additional motor supply from a nonselective motor axon that also innervated one nuclear chain fiber in the same spindle pole. The chain fibers co-innervated with bag1 fibers were among the longest chain fibers although they were shorter than two long chain fibers also present in the spindle poles. In cross-sections stained with toluidine blue they displayed 1-3 equatorial nuclei side by side, and there were fewer intermyofibrillar granules in their polar regions than in most of the other chain fibers. The endings of nonselective motor axons on the bag1 and chain fibers were morphologically and ultrastructurally dissimilar. It is suggested that instances of common innervation of the (dynamic) bag1 fiber and a (static?) chain fiber represent an integral and, presumably, functionally meaningful part of the motor pattern in some cat spindles.     

Observations on the primary sensory ending of tenuissimus muscle spindles in the cat

Summary The arrangement of preterminal and terminal axon branches in the primary sensory endings of cat tenuissimus muscle spindles was studied using whole-mount and serial-section techniques. Although in every case one firstorder preterminal branch was supplied exclusively to the bag₁ type of intrafusal muscle fibre, the preterminal branching patterns differed considerably in detail.Terminals varied widely in size and location. Their precise form varied according to their position on the intrafusal muscle fibres rather than their relationship to preterminal branches. Terminals derived from separate preterminal branches remained separate and did not fuse with themselves or each other. Individually bag₁ fibres had most terminals, chain fibres least. The surface of the muscle fibres were differentially indented by the terminals, least in bag₁ fibres and most in chain fibres.The results are discussed in relation to mechanosensory transduction and to the factors involved in determining the form of the primary ending.     

Immunohistochemical differences in myosin composition among intrafusal muscle fibres

Summary Mammalian intrafusal fibre types (nuclear chain, nuclear bag₁ and nuclear bag₂ fibres) are known to differ in their ultrastructure, intensity of the myofibrillar histochemical ATP-ase reaction, type of innervation and time course of contraction. The present study concerns the myosin composition of these intrafusal fibre types in the soleus muscle (mouse) and the extensor digitorum longus muscle (rat). We used an immunohistochemical method with three myosin antisera raised in rabbits: anti chicken pectoral myosin, anti chicken heart myosin (1) and anti chicken heart myosin (2) (=anti chicken heart myosin (1) adsorbed with muscle powder from soleus muscle of guinea pig). The results showed that three intrafusal fibre types differed in their myosin composition. A comparison of intrafusal fibre types with extrafusal fibre types for the histochemical myofibrillar ATP-ase reactivity and the reactivity with myosin antisera showed a resemblance of nuclear chain fibres with extrafusal type II fibres and a difference between nuclear bag₁ and nuclear bag₂ fibres and all other fibre types.     

Distribution of SERCA isoforms in human intrafusal fibers

The sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) is a membrane protein that plays a crucial role in muscle relaxation by transporting cytosolic Ca²⁺ into the lumen of the sarco/endoplasmic reticulum. In this study, the presence of SERCA1 and SERCA2 was investigated in human intrafusal fibers by immunocytochemistry. Nuclear bag₁ fibers contained both SERCA1 and SERCA2 isoforms, with predominant staining seen with SERCA2 in the A and B regions. Most nuclear bag₂ fibers also contained SERCA1 and SERCA2 isoforms and their coexistence frequently occurred in the A region. SERCA1 was present whereas SERCA2 was generally absent in the nuclear chain fibers. The staining intensity seen with the SERCA1 monoclonal antibody varied in the order of chain>bag₁>bag₂. The expression of SERCA1 isoform was found to correlate with the presence of fast myosin heavy chain (MyHC) isoform in nuclear chain fibers, whereas for nuclear bag fibers there was no such apparent correlation between patterns of expression of SERCA and MyHC isoforms. The phenotype revealed for the human bag fibers was very sophisticated and adapted to attain a very wide range of contraction and relaxation velocities.     

A new grouping of intrafusal muscle fibers based on developmental studies of muscle spindles in the cat.

The development of muscle spindles was studied using the tenuissimus muscle of the cat. Observations show that the intrafusal muscle fibers develop as two separate groups: one group represented by a single nuclear bag fiber while the second group comprises the second nuclear bag fiber in association with all the nuclear chain fibers. This grouping is most pronounced in the fetus and is clearly seen in neonatal kittens (i.e., up to 2 weeks of age). As the intrafusal fibers begin to separate from each other, the groupings become less noticeable, although this basic pattern is often retained in the adult. The pattern of intrafusal fiber grouping is most noticeable in the equatorial regions of the spindle and least noticeable in the polar regions. This is not the grouping of fibers which would have been expected from a consideration of existing reports on muscle spindles. The implications for spindle form and function are considered.