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

The form of terminations of fusimotor (γ) and skeletofusimotor (β) 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 γ or β 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.     

Variations in intrafusal fiber size within histochemically identified types of intrafusal fibers in the pigeon

The myofibrillar adenosine triphosphatase reaction with acid preincubation allowed the identification of three types of intrafusal fibers in pigeon flexor carpi ulnaris muscle spindles. Measurements of cross-sectional areas at the polar region showed much overlap in fiber size among populations of each type.     

Morphology and morphometry of motor endings in primate intrafusal fibers

Summary The pre- and postsynaptic structure of 243 axon terminals of bag 1, bag 2, and chain fibers were studied in cynomolgus monkey skeletal muscle spindles. The motor endings of the biceps and gastrocnemius spindles (long limb muscle) were compared to the motor endings of lumbricals and opponens pollicis (intrinsic hand muscle) spindles. In both muscle groups the only significant difference observed in the presynaptic features was in the presynaptic membrane length. The postsynaptic features of bag 1, bag 2 and chain endings were similar in the long limb muscle spindles. In the intrinsic hand muscle, however, the bag 1 and chain endings showed complex postsynaptic structure which resembled the extra fusal endings while the postsynaptic structure of bag 2 endings was much simpler. From these studies we conclude that the postsynaptic structure of various intrafusal fiber types is dissimilar in different muscles.     

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.     

Glycogen-depletion method of intrafusal distribution of gamma-axons that increase sensitivity of spindle secondary endings

1. The glycogen-depletion method was used in cat tenuissimus muscles to investigate whether the action of static gamma-axons that increase the sensitivity of spindle secondary endings to muscle length is associated with a specific pattern of distribution of these axons to intrafusal muscle fibers. 2. In each experiment, a single gamma-axon acting on a secondary ending of a precisely located spindle was repetitively stimulated, and subsequently the intrafusal muscle fibers of that spindle were examined for glycogen depletion. 3. The gamma-axons whose repetitive stimulation increased the length sensitivity of secondary endings depleted all the chain fibers in one or both poles of the spindle, with the bag fibers being inconstantly involved. The gamma-axons whose stimulation did not exert this effect produced much more restricted patterns of glycogen depletion. 4. The length sensitivities of two secondary endings belonging to the same spindle were similarly affected by a single gamma-axon, whereas a gamma-axon could increase the sensitivity of a secondary ending without altering that of the primary ending of the same spindle. 5. The action exerted by single gamma-axons on secondary endings appears related to their intrafusal distribution since enhancement of the secondary endings sensitivity was observed only when all the chain fibers of at least one spindle pole were activated. 6. If several static gamma-axons supplying a given spindle are firing together, an increase in the length sensitivity of the secondary endings of that spindle can be expected since probably all chain fibers are activated.     

An ultrastructure investigation of intrafusal muscle fibers in myotonic dystrophy

Summary Ultrastructure features of muscle spindles from two patients with myotonic dystrophy are described. Intrafusal muscle fibers exhibit extensive splitting with nuclear bag fibers affected more so than nuclear chain fibers. No sensory endings are present on nuclear chain fibers nor on one nuclear bag fiber throughout the equatorial and myotube regions. Small motor end plates are evident on various segments of split intrafusal fibers in the polar region and some of these extend into the myotube region. Satellite cells are numerous on both nuclear bag and nuclear chain fibers. These frequently occupy the cleft space between segments of split intrafusal fibers. The myotonic dystrophy muscle spindle ultrastructure features seem to closely resemble the appearance of developing mammalian muscle spindles as illustrated with opossum fetal tissue.Supported in part by federal grants 5TO1 AMO5401-12 and 5RO1 HDO2788-06     

Myosin heavy chain expression in developing rat intrafusal muscle fibers

The immunocytochemical expression of several isoforms of myosin heavy chains (MHC) was determined in developing intrafusal and extrafusal fibers of the soleus muscle of prenatal and postnatal rats. At the onset of spindle assembly, both bag2 intrafusal myotubes and primary extrafusal myotubes bound a slow-twitch MHC antibody, whereas the bag1 and chain myotubes expressed a fast-twitch MHC isoform identical to that expressed by secondary extrafusal myotubes. Subsequently, developing intrafusal fibers began to express unique myosin isoforms, and ceased to express some of the myosin isoforms present initially. The initial similarity in MHC composition of intrafusal and extrafusal fibers suggests that these two kinds of mammalian muscle cell originate from a common pool of bipotential myotubes. Differences in MHC expression by intrafusal and extrafusal fibers in adult muscles might result from the effect of sensory neurons on the developing intrafusal myotubes.     

Regional differences in the contractile apparatus of intrafusal muscle fibers

In spindles of rat muscles, the particular class of intrafusal fiber that is unreactive over its entire striated length when incubated for alkali-stable “myofibrillar” ATPase, exhibits pronounced regional differences in staining following incubation for acid-stable ATPase. This variation along the contractile polar regions of the fiber can easily lead to misinterpretations of intrafusal fiber type, as well as the erroneous suggestion of two distinct populations of muscle spindles. Furthermore, such regional staining differences are strongly suggestive of variations in the contractile apparatus along certain intrafusal muscle fibers.     

Histochemical dichotomy of extrafusal and intrafusal fibers in an avian slow muscle

The avian anterior latissimus dorsi (ALD) is unique amongst vertebrates since it has been considered to be a true slow (or tonic) skeletal muscle. While the structure and function of the extrafusal fibers in the ALD have been intensively investigated, the intrafusal fibers of its muscle spindles and their relationship to the surrounding extrafusal fibers in this muscle have been virtually neglected. Serial frozen sections of this muscle from normal adult domestic chickens were tested for two separate enzymes: myosin ATPase after preincubations at differing pH (Brooke and Kaiser, '69) and NADH-tetrazolium reductase, a mitochondrial-bound oxidative enzyme. Both enzyme reactions were able to detect two distinct categories of extrafusal fibers in this muscle, as well as two classes of intrafusal fibers in its muscle spindles. Neither of the extrafusal fiber-types reacted like typical fast (or twitch) fibers for myosin ATPase; they did not show a characteristic reversal in their relative staining patterns throughout the alkaline (pH 9.4) to acid (pH 4.6 and 4.3) preincubation range. The majority of fibers (type 1) were significantly larger in their cross-sectional size, consistently stained lightly for ATPase, and showed high NADH-TR activity. They represented about 84% of the total fiber population (n = 3540 ± 75). The other set of extrafusal fibers (type 2) constituted the remaining 16% of the total fiber population. They were smaller in diameter, exhibited high myosin ATPase activity, and reacted less intensely for the oxidative enzyme. The histochemical characteristics of the two kinds of intrafusal fibers were profoundly different from those observed in the extrafusal fibers. Their crosssectional fiber diameters were not significantly different from each other, and they exhibited a reversal in their staining reactions for myosin ATPase following acid preincubation for this enzyme. The results of this study concur with recently published reports of extrafusal fiber heterogeneity in the slow ALD muscle of the chicken. In addition, this work clearly demonstrates a histochemical dichotomy amongst the intrafusal fibers of muscle spindles in this muscle.     

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 oberserved variations can be better explained in terms of different fiber types than of continuous gradients within one type of fiber.     

Alloantibodies that react with subsets of human T cells

Using the two color fluorescence (TCF) method, alloantibodies against subsets of T cells could be detected in sera from pregnant women with strong HLA antibodies. To preclude interference of these HLA antibodies with the recognition of the T cell antibodies, serum donors were selected which were HLA-Al, -B8, -DRw3. Their sera were tested on a panel of individuals homozygous for HLA-Al, -B8, -DRw3. By enriching peripheral mononuclear blood lymphocytes for Tgamma cells it could be shown that some of the sera reacted mainly with Tgamma and others with Tmu lymphocytes, while some sera reacted with both.     

Reactivity of rat and rabbit intrafusal fibers with monoclonal antibodies directed against myosin heavy chains

Serial cross and longitudinal sections from the intracapsular portions of intrafusal fibers of rat and rabbit tibialis anterior muscles were examined by fluorescence microscopy with a library of monoclonal antibodies directed against different epitopes on myosin heavy chains. Intrafusal fiber types were identified with the histochemical reactions for acid-stable and alkali-stable actomyosin ATPase. Three antibodies, known to react with avian heart and slow-tonic myosins, produced fluorescent staining in intrafusal fibers. Nuclear bag2 fibers reacted with all three antibodies, chain fibers with two, and nuclear bag1 fibers with only one. These results indicate that in rat and rabbit tibialis anterior muscle spindles nuclear bag2 fibers and chain fibers contain more than one myosin isoform. They also demonstrate that, in addition to the histochemical actomysin ATPase reaction, nuclear chain fibers and the two types of nuclear bag fibers can be identified by the selective reactivities of their myosin heavy chains.     

Differential effects of neonatal denervation on intrafusal muscle fibers in the rat

The response of developing muscle spindles to denervation was studied by sectioning the nerve to the medial gastrocnemius muscle of rats at birth. The denervated spindles were examined daily throughout the first postnatal week for changes in ultrastructure and expression of several isoforms of myosin heavy chain (MHC). Each of the three different types of intrafusal muscle fiber exhibited a different response to denervation. Within 5 days after the nerve section nuclear bag₂ fibers degenerated completely; nuclear bag₁ fibers persisted, but ceased to express the spindle-specific slow-tonic MHC isoform and thereby could not be differentiated from extrafusal fibers; nuclear chain fibers did not form. The capsules of spindles disassembled, hence spindles or their remnants could no longer be identified 1 week after denervation. Neonatal deefferentation has little effect on these features of developing spindles, so removal of afferent innervation is presumably the factor that induces the loss of spindles in denervated muscles. Degeneration of the bag₂ fiber, but not bag₁ or extrafusal fibers, reflects a greater dependence of the bag₂ fiber than the bag₁ fiber on afferent innervation for maintenance of its structural integrity. This difference in response of the two types of immature bag fiber to denervation might reflect an origin of the bag₂ fibers from a lineage of myogenic cells distinct from that giving rise to bag₁ or extrafusal fibers, or a difference in the length of contact with afferents between the two types of bag fiber prior to nerve section.     

Variation in histochemical enzyme profile and diameter along human masseter intrafusal muscle fibers

The histochemical enzyme profile of human masseter intrafusal muscle fibers was analyzed in consecutive serial cross sections along the individual fibers. Two hundred intrafusal fibers in 21 muscle spindles were classified. On the basis of equatorial nucleation, myosin ATPase-staining reactions after alkaline and acid preincubations and diameter, four different populations or types of intrafusal fiber were identified: large-diameter alkaline-stable and acid-stable fibers, bag2; two types of fiber with intermediate-diameter, alkaline-labile and acid-labile fibers corresponding to bag1 and alkaline-labile and acid-stable fibers designated as AS-bag1; and small-diameter alkaline-stable and acid-stable (pH 4.6)-acid-labile (pH 4.3) fibers called chain fibers. Regional variability in staining and diameter along the individual fibers was noted. In general, intrafusal fibers showed stronger oxidative reactions than did extrafusal fibers. The enzyme profile of the human masseter intrafusal fibers differed from that of extrafusal fibers in jaw, limb, and trunk muscles and also from that reported for spindles in limb and trunk muscles in man. The result suggests unique properties of human jaw muscle spindles and the jaw motor system.     

Shared motor innervation between dynamic and static intrafusal muscle fibers in the monkey

Distributions of 25 motor axons to 60 intrafusal muscle fibers of 10 poles of monkey spindle were reconstructed from serial 1 micron thick transverse sections of lumbrical muscles. About 44% of motor axons co-innervated two or more types of intrafusal fiber. The (dynamic) bag1 fiber shared motor innervation with the (static) bag2 or chain fibers in about 50% of spindle poles. Activation of single intrafusal fibers independent of the other fibers of the same intrafusal bundle occurs to a lesser degree in spindles of monkeys than in spindles of cats. Functional implications of this pattern of motor innervation are discussed.     

Nonselective motor innervation of intrafusal fibers in muscle spindles of the rat

Summary Distributions of motor axons to different types of intrafusal fiber were reconstructed from serial 1-m thick transverse sections of six poles of muscle spindle in the rat soleus. Motor axons innervated (dynamic) bag₁ fibers, or (static) bag₂ fibers in conjunction with chain fibers. However, approximately forty percent of axons that supplied the spindles synapsed on both bag₁ and bag₂, or bag₁ and chain fibers. The significance of this co-innervation of dynamic and static intrafusal fibers is discussed relative to the general organization and function of mammalian spindles.     

Histochemical properties of intrafusal fibers in the soleus muscle of the aged rat

ATPase reaction profiles of intrafusal fibers in the muscle spindle of the soleus muscle of 135-week-old rats were examined. Nuclear bag1 fibers contained an acid- and alkaline-labile form of the enzyme or an acid-labile and alkaline-stabile form, nuclear bag2 fibers contained an acid- and alkaline-stabile form, and nuclear chain fibers contained an acid-labile and alkaline-stabile form. These results indicate that the enzyme histochemical heterogeneity of intrafusal fibers is well-preserved during ageing.     

Resting membrane potentials and miniature postsynaptic potentials of frog intrafusal muscle fibers

By recording intracellular potentials the electrophysiological characteristics of the intrafusal muscle fibers of the frog muscle spindle were investigated. Analysis of the distribution of amplitudes of the miniature postsynaptic potentials indicates heterogeneity of the intrafusal muscle fibers. It is concluded that the frog muscle spindle contains three types of muscle fibers: those with single innervation, and those combining the two types of innervation.Laboratory of Neuromuscular Physiology, Physiological Institute, Leningrad University. (Presented by Academician V. N. Chernigovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 2, pp. 136–139, February, 1977.