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

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.     

Myofibrillar ATPase activity of intrafusal fibers in chronically de-afferented rat muscle spindles

Dorsal root ganglia L4, L5 were removed to accomplish long-term (1 year) de-afferentation of the rat soleus muscle. Muscle spindles in the muscles deprived of sensory innervation were morphologically and histochemically abnormal. The spindle periaxial fluid space was greatly diminished with a thicker capsular investment. De-afferented intrafusal muscle fibers lacked either "nuclear bags" "nuclear chains" at their midlengths. The intracapsular myofibrillar ATPase staining pattern of de-afferented nuclear bag fibers resembled that which the bag fibers normally display in their extracapsular regions. These abnormalities are discussed with respect to the regulatory functions of spindle sensory and motor nerves.     

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.     

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

Over 300 complete and incomplete cat muscle spindles were examined in serial transverse sections of tenuissimus muscles in search of spindles with more than two nuclear bag intrafusal muscle fibers. Several histochemical and histological stains were used to identify the intrafusal fibers and assess their motor and sensory innervation. About 13% of the spindles contained either three or four bag fibers rather than the usual two. Every multiple-bag-fiber spindle possessed at least one nuclear bag1 and one nuclear bag2 fiber. The supernumerary bag fibers were either another bag1 and/or bag2 fiber, or a mixed bag fiber. The extra bag fibers had the usual morphologic and histochemical properties of cat nuclear bag fibers. All multiple-bag spindles received primary sensory innervation, and most had secondary sensory endings in addition. Their motor pattern was similar in the number, appearance and disposition of intrafusal motor endings to that of the usual two-bag-fiber spindles. Bag fibers of the same kind shared motor nerve supply in three multiple-bag spindles in which tracings of individual motor axons were obtained histologically. It is unclear whether any functional advantage is conveyed to a muscle spindle by its having more than one bag1 and one bag2 fiber.     

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

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.     

Muscle spindle formation and differentiation in regenerating rat muscle grafts

Muscle spindle development and function are dependent upon sensory innervation. During muscle regeneration, both neural and muscular components of spindles degenerate and it is not known whether reinnervation of a regenerating muscle results in reestablishment of proper neuromuscular relationships within spindles or whether sensory neurons may exert an influence upon differentiation of these spindles. Muscle spindle regeneration was studied in bupivacaine-treated grafts of rat extensor digitorum longus (EDL) muscles. Three types of EDL graft were performed in order to manipulate the extent to which regenerating spindles might be reinnervated: (1) grafts reinnervated following severance of their nerve supply (standard grafts); (2) grafts in which intact nerve sheaths appear to facilitate reinnervation (nerveintact grafts); and (3) grafts in which reinnervation was prevented (nonreinnervated grafts). Complete degeneration of muscle fibers occurred in all grafts prior to regeneration. Initial formation of spindles in regenerating EDL grafts is independent of innervation; intrafusal muscle fibers degenerate and regenerate within spindle capsules that remain intact and viable. The extent of spindle differentiation was evaluated in each type of graft using criteria that included nucleation and ATPase activity, both of which have been shown to be regulated by sensory innervation, as well as the number of muscle fibers/spindle and morphology of spindle capsules.While most spindles contained normal numbers of muscle fibers, most of these fibers were morphologically and histochemically abnormal. Alterations of ATPase activity occurred in all spindles, but were least severe in nerve-intact grafts. While fully differentiated nuclear bag and chain fibers were not observed in regenerated spindles, large, vesicular nuclei, similar to those of normal intrafusal fibers, were present in a small number of spindles in nerve-intact grafts. Sensory nerve terminations were observed only in those spindles that also contained the distinctive nuclei. This study suggests that a specific neurotrophic influence is necessary for regeneration of normal intrafusal muscle fibers and that this influence corresponds to the properly timed sensory neuron-muscle interaction which directs muscle spindle embryogenesis. However, the infrequent occurrence of characteristics unique to intrafusal muscle fibers indicates that reinnervation of regenerating muscle grafts by sensory neurons is inadequate and/or faulty.     

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.     

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.     

Histochemistry of rat intrafusal muscle fibers and their motor innervation.

Muscle spindles were followed in serial transverse sections of freshly frozen rat soleus muscles. Adenosine triphosphatase (ATPase) histochemical staining reaction was used to identify nuclear bag1, nuclear bag2 and nuclear chain intrafusal muscle fibers. Regional differences in ATPase staining occurred along bag1 and bag2 fibers but not along chain fibers. Bag1 fibers displayed ultrastructural heterogenity when their intra- and extracapsular regions were compared. Simple "diffuse" and more elaborate "plate" motor nerve terminals were demonstrated histochemically along the poles of bag1 and bag2 fibers by staining for cholinesterase. One motor terminal of the "plate" appearance was present on a chain fiber pole. There was no consistent spatial correlation between the intensity of regional ATPase staining along the nuclear bag fibers and the location, number and type of motor endings. Other factors, such as intrafusal fiber sensory innervation and regional differences in active and passive functional recruitment of nuclear bag fibers during muscle activity, may contribute to the ATPase staining variability along the intrafusal fibers.     

Treatment with beta bungarotoxin blocks muscle spindle formation in fetal rats

Sensory and motor fibers of peripheral nerves were irreversibly destroyed in fetal rats by administering beta bungarotoxin (BTX) on embryonic day 16 or 17, after assembly of primary myotubes, but before the formation of muscle spindles. Soleus muscles of toxin-treated fetuses and their untreated littermates were removed just prior to birth and were examined by light microscopy of serial transverse sections for the presence of spindles and immunocytochemical expression of several isoforms of myosin heavy chains (MHC). Untreated muscles exhibited numerous spindles that were innervated by branches of intramuscular nerves and contained muscle fibers expressing a slow-tonic MHC isoform characteristic of the intrafusal but not extrafusal fibers. Toxin-treated muscles were devoid of intramuscular nerve bundles and perineurial structures. Encapsulations of muscle fibers resembling spindles were absent and no myotubes expressed the slow-tonic MHC isoform associated with intrafusal fibers in beta BTX-treated muscles. Thus, the assembly of muscle spindles, formation of the spindle capsule, and transformation of undifferentiated myotubes into the intrafusal fibers that contain spindle-specific myosin isoforms all depend on the presence of innervation in prenatal rat muscles.     

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.     

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.     

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

Summary 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 bag₁ and bag₂ 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 bag₁ and bag₂ fibres.     

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.     

The effect of neonatal deafferentation or defferentation on myosin heavy chain expression in intrafusal muscle fibers of the rat

Muscle spindles were either deafferented or deefferented by selectively severing the sensory or motor nerve supply to neonatal soleus muscles of rats at a time when spindles are formed but when intrafusal muscle fibers are structurally and immunocytochemically immature. Experimental muscles were excised two months after nerve section. Control and experimental spindles were examined using monoclonal antibodies specific for myosin heavy chains of slow-tonic (ALD58) and fast-twitch (MF30) chicken muscles. Only intrafusal fibers bound these antibodies in intact soleus muscles. The deefferented spindles exhibited a pattern of ALD58 and MF30 binding similar to that of normal adult intrafusal fibers, whereas deafferented intrafusal fibers were unreactive with the two antibodies. Thus intact sensory innervation is essential for myosin heavy chain expression in intrafusal muscle fibers during postnatal development of rat spindles.     

Motor innervation of intrafusal fibers in rat muscle spindles: incomplete separation of dynamic and static systems

Distributions of 53 motor axons to different types of intrafusal fibers were reconstructed from serial 1-micron-thick transverse sections of 13 poles of spindles in the rat soleus muscle. The mean number of motor axons that innervated a spindle pole was 4.1. Approximately 60% of motor axons lost their myelination prior to or shortly after entry into the periaxial fluid space of spindles. Motor innervation to the juxtaequatorial portion of nuclear bag fibers (particularly the bag1) consisted of groups of short, synaptic contacts that were terminations of thin, unmyelinated axons. In contrast, motor endings on both the bag1 and bag2 fibers were platelike in the polar intracapsular region. Chain fibers had a single midpolar platelike ending. The ratio of motor axons that innervated the bag1 fiber exclusively to axons that innervated bag2 and/or chain fibers was 1:1. However, one-fourth of motor axons coinnervated the dynamic bag1 fiber in conjunction with static bag2 and/or chain fibers. Thus the complete separation of motor control of the dynamic bag1 and static bag2 intrafusal systems observed in cat tenuissimus spindles is neither representative of the pattern of motor innervation in all other species of mammals nor essential to normal spindle function.     

The effect of neonatal deafferentation or deefferentation on myosin heavy chain expression in intrafusal muscle fibers of the rat

Summary Muscle spindles were either deafferented or deefferented by selectively severing the sensory or motor nerve supply to neonatal soleus muscles of rats at a time when spindles are formed but when intrafusal muscle fibers are structurally and immunocytochemically immature. Experimental muscles wereexcised two months after nerve section. Control and experimental spindles were examined using monoclonal antibodies specific for myosin heavy chains of slow-tonic (ALD58) and fast-twitch (MF30) chicken muscles. Only intrafusal fibers bound these antibodies in intact soleus muscles. The deefferented spindles exhibited a pattern of ALD58 and MF30 binding similar to that of normal adult intrafusal fibers, whereas deafferented intrafusal fibers were unreactive with the two antibodies. Thus intact sensory innervation is essential for myosin heavy chain expression in intrafusal muscle fibers during postnatal development of rat spindles.     

Appearance of sensory nerve terminals in cat muscle spindles stained for NADH-tetrazolium reductase

Cat muscle spindles were studied histochemically in serial transverse sections of the tenuissimus muscle stained for myofibrillar ATPase, cholinesterase or NADH-tetrazolium reductase. The terminal sites of the primary and secondary axons on intrafusal muscle fibers could be demonstrated due to their high NADH-TR activity. This sensory NADH-TR reactivity at the equator and in the juxtaequatorial regions disappeared following spindle chronic de-afferentation, but not after de-efferentation. Spindle poles that carried both primary and secondary sensory endings had a longer periaxial fluid space than poles with primary endings only, and their motor innervation, as determined by staining for ChE, was positioned at the greater distance from the equator. Some of the secondary endings occurred in intrafusal regions that displayed surface fiber ChE activity. The histochemical reaction for NADH-TR represents a simple, rapid and reliable method for studies of the distribution of sensory nerve terminals in the spindle.     

Appearance of sensory nerve terminals in cat muscle spindles stained for NADH-tetrazolium reductase

Summary Cat muscle spindles were studied histochemically in serial transverse sections of the tenuissimus muscle stained for myofibrillar ATPase, cholinesterase or NADH-tetrazolium reductase. The terminal sites of the primary and secondary sensory axons on intrafusal muscle fibers could be demonstrated due to their high NADH-TR activity. This sensory NADH-TR reactivity at the equator and in the juxtaequatorial regions disappeared following spindle chronic de-afferentation, but not after de-efferentation. Spindle poles that carried both primary and secondary sensory endings had a longer periaxial fluid space than poles with primary endings only, and their motor innervation, as determined by staining for ChE, was positioned at a greater distance from the equator. Some of the secondary endings occurred in intrafusal regions that displayed surface fiber ChE activity. The histochemical reaction for NADH-TR represents a simple, rapid and reliable method for studies of the distribution of sensory nerve terminals in the spindle.     

Histological and histochemical comparisons of muscle spindles in three hind limb muscles of the guinea pig.

Guinea pig soleus, medial gastrocnemius and vastus lateralis muscles were compared for spindle density and distribution, number of intrafusal fibers per spindle and histochemical appearance of the axial bundle. A total of 326 spindles was used in the comparisons. Spindle density was over four times greater in the soleus than in either the medial gastrocnemius or vastus lateralis. In the soleus the spindles were distributed at random, but in the other two muscles no spindles were found in those fascicles in which fast-twitch glycolytic extrafusal fibers predominated. The average number of intrafusal fibers per spindle varied by less than 5% between the three kinds of muscles. About 80% of all spindles located had four intrafusal fibers, two of the nuclear bag type and two of the nuclear chain type. The histochemical appearance of the axial bundle was the same in each kind of muscle. Based on intensities of the myofibrillar adenosine triphosphatase reaction product at polar regions nuclear bag fibers were separable into two histochemical groups; nuclear chain fibers were of only one histochemical type.