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.     

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.     

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.     

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.     

Factors that determine the form of neuromuscular junctions of intrafusal fibers in the cat

The form of terminations of fusimotor (gamma) and skeletofusimotor (beta) axons on intrafusal fibers was analyzed in serial sections of 20 spindles of the cat tenuissimus muscle. Seven synaptic features were assessed either qualitatively or quantitatively from electron micrographs of transverse sections of 184 intrafusal and 30 extrafusal endings. Features were compared among endings that were terminations of gamma or beta axons on different types of intrafusal fiber at different distances from the spindle equator. These comparisons indicated that interactions of several factors, and not the motor axon alone, determine the form of motor endings. Intrafusal muscle fiber type is dominant to the motor axon in regulation of the number and depth of postsynaptic folds. Separation of the influence of the motor axon from the muscle fiber was less clear with respect to the size of ending. Complete expression of the muscle fiber-motor axon interaction reflected by the form of motor endings is dependent upon location of the ending relative to the sensory region. Both depth of the primary synaptic cleft and size of the soleplate of motor endings increased with increasing distance of the ending from the spindle equator. A system of classification of cat intrafusal motor endings that reflects the multiplicity of factors that determine the form of endings, and one that simplifies the current terminology, is proposed.     

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.     

Examination of chronically de-efferented cat muscle spindles for cholinesterase activity

Summary Cat tenuissmus muscles were deprived of motor nerve supply for three months by sectioning of the appropriate ventral spinal roots. Muscle spindles were located in the chronically de-efferented muscles and examined histochemically in serial transverse sections. Staining for nicotinamide adenine dinucleotide tetrazolium reductase showed that the spindle sensory innervation was preserved. The de-efferented intrafusal muscle fibers retained their differential staining with the reaction for myosin adenosine triphosphatase. However, all cholinesterase-active areas that are normally found along nuclear bag and nuclear chain intrafusal fibers demonstrated loss of the enzyme activity in the chronically de-efferented spindles. It is concluded that all histochemically demonstrable cholinesterase activity within the cat muscle spindle is dependent upon the continuous presence of motor innervation.     

Heterogeneity of spindle units in the cat tenuissimus muscle

Three tandem spindles and their nerve supplies, reconstructed by light microscopy of serial transverse sections of the cat tenuissimus muscle, were compared to single spindle units. Each tandem spindle consisted of one large unit containing a dynamic bag1, a static bag2, and several static chain fibers (b1b2c unit) linked by the bag2 fiber to a small unit containing only a bag2 and chain fibers (b2c unit). Most features of primary afferents, secondary afferents, and motor neurons were qualitatively and quantitatively similar in both single and tandem b1b2c units. However, b1b2c units of tandem spindles had a lower density of skeletofusimotor innervation than did single b1b2c spindles. The b2c spindle units differed greatly from single or tandem b1b2c units. The b2c spindle units had fewer intrafusal fibers and incoming axons than either the tandem or single b1b2c units. The motor innervation of b2c units was typified by nonselective gamma axons that coinnervated both bag2 and chain fibers, in contrast to the regular occurrence of both selective and nonselective motor axons in b1b2c spindle units. The afferent located at the equator of b2c units differed in size, branching pattern, and intrafusal distribution of its ending from both the primary and secondary sensory axons of b1b2c units and, therefore, might represent a third category of spindle afferent. Thus, cat tenuissimus muscles contain three types of spindle units that differ in the number and organization of muscular and neural elements. These differences in structure and neural organization among tenuissimus spindle units may be a source for generation of different sensory signals in response to common mechanical or fusimotor stimuli.     

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.     

Histological study of motor innervation to long nuclear chain intrafusal fibers in the muscle spindle of the cat

Several muscle spindles of the cat tenuissimus muscle were cut in serial, 1-micron thick transverse sections and stained with toluidine blue in search for long nuclear chain intrafusal muscle fibers. Five complete poles of the long chain fibers were located. Each fiber pole displayed one plate-type motor ending situated in the extracapsular fiber region. The endings were supplied by myelinated motor axons that originated from intramuscular nerve fascicles containing motor axons to extrafusal muscle fibers. One of the endings was innervated by a collateral from a motor axon that supplied an extrafusal end-plate. Ultrastructurally, the long chain endings resembled extrafusal end-plates. They were more complex, in terms of prominence of sole-plate and degree of post-junctional folding, than any other intrafusal ending present in the spindles. The motor endings of the long chain fibers were assumed to be the terminals of static (fast) skeletofusimotor axons, which preferentially innervate the longest nuclear chain fibers of cat muscle spindles.     

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.     

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.     

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.     

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.     

Autonomic innervation of receptors and muscle fibres in cat skeletal muscle

Cat hindlimb muscles, deprived of their somatic innervation, have been examined with fluorescence and electron microscopy and in teased, silver preparations; normal diaphragm muscles have been examined with electron microscopy only. An autonomic innervation was found to be supplied to both intra- and extrafusal muscle fibres. It is not present in all muscle spindles and is not supplied at all to tendon organs. Fluorescence microscopy revealed a noradrenergic innervation distributed to extrafusal muscle fibres and some spindles. On the basis of the vesicle content of varicosities the extrafusal innervation was identified as noradrenergic (32 axons traced), and the spindle innervation as involving noradrenergic, cholinergic and non-adrenergic axons (14 traced). Some of the noradrenergic axons that innervate spindles and extrafusal muscle fibres are branches of axons that also innervate blood vessels. We cannot say whether there are any noradrenergic axons that are exclusively distributed to intra- or extrafusal muscle fibres. The varicosities themselves may be in neuroeffective association with striated muscle fibres only, or with both striated fibres and the smooth muscle cells in the walls of blood vessels. The functional implications of this direct autonomic innervation of muscle spindles and skeletal muscle fibres are discussed and past work on the subject is evaluated.     

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.     

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.     

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.     

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

Summary Muscle spindles were studied histochemically in serial transverse sections of specimens of the cat tenuissimus muscle. The nuclear chain intrafusal muscles 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.