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.     

Histochemical profiles of cat intrafusal muscle fibers and their motor innervation

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.     

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.     

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.     

Flow cytometric analysis of chromosomes and cells using a modified BrdU-hoechst method

Summary The sensory innervation of 46 poles of long chain intrafusal muscle fibers was studied histochemically by staining for NADH-TR in periodic, 8 m thick transverse sections of cat muscle spindles. Each long chain fiber carried terminals of the primary sensory axon, and 23 of the fiber poles also displayed secondary sensory endings. With the NADH-TR reaction there was no apparent difference in the cross-sectional appearance of sensory endings on the long chain and on other nuclear chain fibers. However, the contact area between the secondary endings and the muscle fiber tended to be shorter on the long chain than on the neighboring chain fibers of shorter polar length. This was also the case for one long chain fiber in which the sensory innervation was examined in serial, 1 m thick sections stained with toluidine blue. Discharges of the secondary sensory axons in cat spindles may be affected more by contraction of the shorter nuclear chain fibers than by activation of the long chain fibers.     

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.     

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.     

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.     

The fine structural localization of acetylcholinesterase in the muscle spindle of the rat

Summary Muscle spindles from lumbricalis muscles of the rat were incubated for acetylcholinesterase with a modified thiocholine-method of Lewis and Shute and examined by light and electron microscopy.All types of motor nerve ending showed heavy deposits of reaction product in the synaptic cleft. The underlying sarcoplasmic reticulum, transverse tubular system, and, when present, the envelope of sole plate nuclei were also stained.In the sensory region, the reaction was negative in the interface between the plasma membranes of the primary sensory terminal and muscle. One of two secondary sensory endings identified showed distinct reaction product in the cleft; the other secondary sensory ending showed no such reaction.Precipitates were present on the sarcolemma of the intrafusal muscle fibers in the polar and adjacent myotube regions, but not at the spindle equator. Extrafusal and intrafusal myelinated -nerve fibers and preterminal motor axons showed staining of the axolemma. Fibers with thick myelin sheaths and preterminal sensory axons were free of acetylcholinesterase activity, as were the unmyelinated nerve fibers.We wish to thank Mrs. D. Schilling and Mrs. Ch. Beyer for technical assistanc     

Histochemical study of an unusual cat intrafusal muscle fiber

A cat tenuissimus muscle spindle that contained two long chain intrafusal fibers in its distal pole is described. One of the fibers (lc1) had a histochemical profile (ATPase, NADH-TR, ChE reactions) of the kind which is characteristic for long chain fibers. The other fiber (lc2) consisted of two separate segments. The inner lc2 segment included the sensory equatorial region and was histochemically normal. The outer lc2 segment carried a motor plate, and did not stain for NADH-TR in the same way as the inner lc2 segment and the lc1 fiber. It is suggested that the unusual enzyme staining properties of the outer lc2 segment stemmed from its lack of sensory innervation, a situation which may have permitted the full expression of influences mediated by its motor nerve supply.     

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.     

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.     

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.     

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

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

The Fine Structure of Muscle Spindles in the Lumbrical Muscles of the Rat

Lumbrical muscles of young rats were fixed with OsO₄ and embedded in methacrylate for electron microscopy. The spindle capsule was found to be continuous with and similar in structure to the sheath of Henle surrounding the nerves supplying the spindle. The capsule consists of several closely applied concentric cytoplasmic sheets. Each sheet is about 1,000 A thick and has no fenestrations. Many caveolae and vesicles in the cytoplasm suggest active transport through the sheets. The periaxial space fluid contains much solid material. It is suggested that the capsule and periaxial space regulate internal chemical environment. The interfibrillar structures are less evident in the polar regions of intrafusal fibres than in extrafusal fibres. Simple motor end-plates occur on the polar regions of intrafusal fibres. In the myotube region of the intrafusal fibre a peripheral zone of myofibrils surrounds a cytoplasmic core containing nuclei, mitochondria, Golgi bodies, reticulum, and a few lipid-like granules. Naked sensory endings lie on the myotube "in parallel" with the underlying myofilaments. Naked processes of the primary sensory ending deeply indent the muscle plasma membrane and the underlying wisps of myofilament in the nuclear bag region. The plasma membranes of sensory nerve ending and intrafusal muscle fibre are about 200 A apart.     

Histochemical study of an unusual cat intrafusal muscle fiber

Summary A cat tenuissimus muscle spindle that contained two long chain intrafusal fibers in its distal pole is described. One of the fibers (1 c₁) had a histochemical profile (ATPase, NADH-TR, ChE reactions) of the kind which is characteristic for long chain fibers. The other fiber (1 c₂) consisted of two separate segments. The inner 1 c₂ segment included the sensory equatorial region and was histochemically normal. The outer 1 c₂ segment carried a motor plate, and did not stain for NADH-TR in the same way as the inner 1 c₂ segment and the 1 c₁ fiber. It is suggested that the unusual enzyme staining properties of the outer 1 c₂ segment stemmed from its lack of sensory innervation, a situation which may have permitted the full expression of influences mediated by its motor nerve supply.     

Development of chicken intrafusal muscle fibers

The first sign of developing intrafusal fibers in chicken leg muscles appeared on embryonic day (E) 13 when sensory axons contacted undifferentiated myotubes. In sections incubated with monoclonal antibodies against myosin heavy chains (MHC) diverse immunostaining was observed within the developing intrafusal fiber bundle. Large primary intrafusal myotubes immunostained moderately to strongly for embryonic and neonatal MHC, but they were unreactive or reacted only weakly with antibodies against slow MHC. Smaller, secondary intrafusal myotubes reacted only weakly to moderately for embryonic and neonatal MHC, but 1–2 days after their formation they reacted strongly for slow and slow-tonic MHC. In contrast to mammals, slow-tonic MHC was also observed in extrafusal fibers. Intrafusal fibers derived from primary myotubes acquired fast MHC and retained at least a moderate level of embryonic MHC. On the other hand, intrafusal fibers developing from secondary myotubes lost the embryonic and neonatal isoforms prior to hatching and became slow. Based on relative amounts of embryonic, neonatal and slow MHC future fast and slow intrafusal fibers could be first identified at E14. At the polar regions of intrafusal fibers positions of nerve endings and acetylcholinesterase activity were seen to match as early as E16. Approximately equal numbers of slow and fast intrafusal fibers formed prenatally; however, in postnatal muscle spindles fast fibers were usually in the majority, suggesting that some fibers transformed from slow to fast.     

Effects of Latrodectus spider venoms on sensory and motor nerve terminals of muscle spindles

The effects of the venoms of the spiders Latrodectus mactans tredecimguttatus (black widow) and Latrodectus mactans hasselti (red back) on sensory nerve terminals in muscle spindles were studied in the mouse. A sublethal dose of venom was injected into tibialis anterior and extensor digitorum longus muscles of one leg. After survival from 30 minutes to 6 weeks muscles were examined in serial paraffin sections impregnated with silver or by electron microscopy. Sensory endings became swollen, some within 30 minutes, while over the next few hours there was progressive degeneration of annulospiral endings. By 24 hours every spindle identified by light or electron microscopy was devoid of sensory terminals. Degenerated nerve endings were taken up into the sarcoplasm of intrafusal muscle fibres. Regeneration of sensory axons began within 24 hours, new incomplete spirals were formed by 5 days and by 1 week annulospiral endings were almost all normal in appearance. Intrafusal motor terminals underwent similar acute degenerative and regenerative changes. These experiments show that intrafusal sensory and motor terminals are equally affected by Latrodectus venoms. Sensory nerve fibres possess a capacity for regeneration equal to that of motor fibres and reinnervate intrafusal muscle fibres close to their original sites of 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.     

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.