Morphology and histochemistry of the ambiens muscle of the red-eared turtle (Pseudemys scripta)

Six fiber types have been described in the ambiens muscle of red-eared turtles. These include one slow oxidative type, two fast oxidative types, two fast oxidative and glycolytic types, and one fast glycolytic type. Fiber types are non-randomly distributed throughout cross sections of the muscle. There is a decreasing gradient of oxidative staining and an increasing gradient of glycolytic staining along an axis from the superficial to deep regions of the muscle. The slow oxidative fibers are predominantly located within one or two fascicles of the superficial surface of the muscle. The fast glycolytic fibers are predominant in deep fascicles. In contrast to previous reports of histochemically monotypic intrafusal fibers in turtle muscle, ambiens muscle spindles have been observed containing one to eleven intrafusal fibers, including two fiber types. Fiber diameter and area are consistently smaller than observed in most extrafusal fibers. Spindles are predominantly located in superficial and cranial fascicles of the ambiens muscle and are located in regions characterized by extrafusal fibers with high oxidative activity.     

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.     

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.     

Distribution of polymorphic forms of troponin components in extra- and intrafusal fibers of an avian slow muscle

Summary By indirect immunofluorescence microscopy, the reactivities of extra- and intrafusal muscle fibers with antibodies against troponin (TN) components were studied in an avian slow muscle, the anterior latissimus dorsi (ALD) of the chicken. Serial cross sections of the muscle were exposed to antibodies specific to TN components (TN-T, -I, and -C) from adult chicken breast and ventricular muscles. In extrafusal fibers, four distinct categories were identified on the basis of differential reactivity with these antibodies. The predominant population of fibers (> 95%) reacted weakly only with antiventricular TN-C. The second type of fibers (< 5%) was stained with antibodies raised against breast TN components. The third group of fibers (< 1%) was labeled not only with antibreast TN components, but also with antiventricular TN-T and -C. The last class of fibers (< 1%) reacted with antibodies directed against ventricular TN-T and -C. These results were correlated with myofibrillar ATPase staining patterns of fibers. In intrafusal muscle fibers of this muscle, the same four types of fibers were observed; in these fibers, however, there appeared to be a longitudinal variation in the reactivity. In conclusion, the slow ALD muscle of the adult chicken contains populations of both extrafusal and intrafusal fibers which are heterogeneous in reactivity with TN component antibodies.     

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.     

Immunohistochemical localization of alpha(1a)-adrenoreceptors in muscle spindles of rabbit masseter muscle

The expression of alpha(1a)-adrenoreceptors (alpha(1a)-ARs) within the muscle spindles of rabbit masseter muscle was investigated. The alpha(1a)-ARs were detected by immunohistochemical fluorescent method and examined along the entire length of 109 cross serially sectioned spindles. The sympathetic fibers were visualized by the immunofluorescent labeling of the noradrenaline synthesizing enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). In order to recognize the intrafusal muscle fiber types, antibodies for different myosin heavy chain isoforms (MyHCI) were used. TH and DBH immunolabeled nerve fibers have been observed within the capsule lamellar layers, in the periaxial fluid space and close to intrafusal muscle fibers. The alpha(1a)-ARs were detected on the smooth muscle cells of the blood vessels coursing in the muscle and in the capsule lamellar layers or within the periaxial fluid space of the spindles. Moreover, at the polar regions of a high percentage (88.1%) of muscle spindles a strong alpha(1a)-ARs immunoreactivity was present on the intrafusal muscle fibers. In double immunostained sections for alpha(1a)-ARs and MyHCI it was evidenced that both bag, and nuclear chain fibers express alpha(1a)-ARs. The receptors that we have detected by immunofluorescence may support a direct control by adrenergic fibers on muscle spindle.     

Expression of type-specific MHC isoforms in rat intrafusal muscle fibers.

Myosin heavy chain (MHC) expression by intrafusal fibers was studied by immunocytochemistry to determine how closely it parallels MHC expression by extrafusal fibers in the soleus and tibialis anterior muscles of the rat. Among the MHC isoforms expressed in extrafusal fibers, only the slow-twitch MHC of Type 1 extrafusal fibers was expressed along much of the fibers. Monoclonal antibodies (MAb) specific for this MHC bound to the entire length of bag2 fibers and the extracapsular region of bag1 fibers. The fast-twitch MHC isoform strongly expressed by bag2 and chain fibers had an epitope not recognized by MAb to the MHC isoforms characteristic of developing muscle fibers or the three subtypes (2A, 2B, 2X) of Type 2 extrafusal fibers. Therefore, intrafusal fibers may express a fast-twitch MHC that is not expressed by extrafusal fibers. Unlike extrafusal fibers, all three intrafusal fiber types bound MAb generated against mammalian heart and chicken limb muscles. The similarity of the fast-twitch MHC of bag2 and chain fibers and the slow-tonic MHC of bag1 and bag2 fibers to the MHC isoforms expressed in avian extrafusal fibers suggests that phylogenetically primitive MHCs might persist in intrafusal fibers. Data are discussed relative to the origin and regional regulation of MHC isoforms in intrafusal and extrafusal fibers of rat hindlimb muscles.     

Pax7 shows higher satellite cell frequencies and concentrations within intrafusal fibers of muscle spindles

Intrafusal fibers within muscle spindles make up a small subpopulation of muscle fibers. These proprioceptive fibers differ from most extrafusal fibers because, even in maturity, their diameters remain small, and they retain expression of developmental myosins. Although both extrafusal and intrafusal fibers contain satellite cells (SCs), comparatively little is known about intrafusal SCs. Analyzing chicken fast-phasic posterior (PLD) and slow-tonic anterior (ALD) latissimus dorsi muscles, we show that SCs of both intrafusal and extrafusal fibers express Pax7. We further test the hypotheses that intrafusal fibers display parameters reflective of extrafusal immaturity. These hypotheses are that intrafusal fibers contain (a) higher SC frequencies (number of SC nuclei/all nuclei within basal lamina) and concentrations (closer together) and (b) smaller myonuclear domains than do adjacent extrafusal fibers. IHC techniques were applied to PLD and ALD muscles excised at 30 and 138 days posthatch. The hypotheses were validated, suggesting that intrafusal fibers have greater capacities for growth, regeneration, and repair than do adjacent extrafusal fibers. During maturation, extrafusal and intrafusal fibers show similar trends of decreasing SC frequencies and concentrations and increases in myonuclear domains. Thus, extrafusal and intrafusal fibers alike should exhibit reduced capacities for growth, regeneration, and repair during maturation.     

Morphology and morphometry of motor endings on macaque intrafusal fibers

The ultrastructural studies have shown three types of motor endings in the macaque intrafusal fibers: 1) unindented axon terminals with smooth or shallowly folded postsynaptic membrane; 2) indented terminals with few postsynaptic folds; and 3) indented terminals with heavily folded postsynaptic membrane. The terminals on bag 1 and chain fibers were generally more indented than those on the bag 2 fibers. Deeply indented terminals with highly folded postsynaptic membranes were noticed on the bag 1 and chain endings in spindles from lumbrical but not the biceps muscle. In the individual intrafusal fibers from the biceps and lumbrical spindles, the degree of indentation did not correlate with the extent of postsynaptic folding (P greater than .01). Endings on bag 1 and chain fibers in the lumbrical spindles showed a positive correlation between indentation of terminals and their distance from the primary sensory endings (P less than .01), whereas the lumbrical bag 2 endings and the biceps intrafusal endings did not (P greater than .01). The shape of the intrafusal motor endings thus is independent of their location but dependent on the type of intrafusal fibers.     

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.     

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.     

Origin of intrafusal muscle fibers in the rat

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.     

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.     

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.     

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.     

Expression of dystrophin on the cell surface membrane of intrafusal fibers of human skeletal muscle

Summary We examined the morphological expression of dystrophin in the intrafusal muscle fibers in skeletal muscle from normal human and Duchenne muscular dystrophy (DMD) patients, using antisera against the N-terminal and C-terminal regions of dystrophin. The intrafusal fibers of normal muscle express dystrophin on their cell surface membrane, but those of DMD muscle do not.Abbreviation DMD Duchenne muscular dystrophy     

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.     

Ultrastructure of Muscle Spindles (Mechanoreceptors) of Rat m. soleus after Support Unloading and Its Combination with Hypergravity

We used electron microscopy to evaluate the effect of support unloading of m. soleus in adult Wistar rats (restrained in antiorthostatic position for 23–24 h/day within 24 days) on the ultrastructure of the intrafusal fibers and motor neuromuscular junctions of the muscle spindles, as well as the efficiency of intermittent hypergravity (+2G_Z; 1 h/day for 19 days in a centrifuge in hypokinetic cages) as a countermeasure used in conditions of support unloading of this muscle. In the absence of support on the hind limbs, most of intrafusal fibers of m. soleus preserved the typical ultrastructure, while the axon terminals of the neuromuscular junctions accumulated a lot of synaptic vesicles (including large vesicles); the coated vesicles were absent due to unloading of the muscle and its muscle spindles (no contractions of the intrafusal fibers). A short-term effects of hypergravity at the background of support unloading of m. soleus mostly induced static loading of the muscle inducing different responses of the intrafusal fibers in different regions of the muscle spindles: local lysis of myofilaments was observed in single intrafusal fibers of the equatorial and intracapsular motor regions, while myofibrils remained intact in most fibers in the intra- and extracapsular regions of the spindles. The revealed adaptive response of the intrafusal fibers is, on the one hand, due to their specific innervation and ultrastructure and, on the other hand, to positive effect of hypergravity on the motor and extracapsular regions of the muscle spindles. Hypergravity decreased the number of synaptic vesicles and induced appearance of the coated vesicles in the axon terminals of the neuromuscular junctions of the intrafusal fibers in the animals restrained in antiorthostatic position (support unloading of m. soleus), which is due to increased functional load of the muscle. The ultrastructure of the muscle spindles adequately reflected the functional status of the postural m. soleus both during support unloading and support unloading combined with hypergravity load.     

Presence in chicken tibialis anterior and extensor digitorum longus muscle spindles of reactive and unreactive intrafusal fibers after incubation with monoclonal antibodies against myosin heavy chains

Cross and longitudinal sections from the encapsulated portions of chicken tibialis anterior and extensor digitorum longus muscle spindles were examined to determine whether their intrafusal fibers were a structurally homogeneous or heterogeneous population. The techniques used were the histochemical actomyosin (mATPase) reaction, and fluorescence immunohistochemistry employing two monoclonal antibodies, CA-83 and CCM-52, that are specific for myosin heavy chains. After incubation with antibody CCM-52, intrafusal fibers fluoresced either strongly or weakly to moderately. Antibody CA-83 was even more selective. In addition to identifying the strongly reactive category, it clearly separated the remaining fibers into unreactive and moderately reactive groups. As a whole, after incubation for mATPase, pH 9.6 preincubation, unreactive fibers stained darker than strongly reactive fibers. Moreover, the cross-sectional area of the unreactive fibers was significantly larger than that of the strongly reactive fibers. In the average-size muscle spindle with six intrafusal fibers, there were four unreactive fibers and two strongly reactive fibers. In about one-third of the receptors examined, one moderately reactive fiber was present. Taken together, the data indicate that intrafusal fibers of chicken tibialis anterior and extensor digitorum longus muscles are not structurally homogeneous. The observed variations can be better explained in terms of different fiber types than of continuous gradients within one type of fiber.