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.     

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.     

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.     

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.     

Histochemistry of rat intrafusal muscle fibers and their motor innervation.

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

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

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

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

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

Histochemistry of intrafusal muscle fibers outside the spindle capsule.

Regional differences in histochemical properties along the length of rat intrafusal muscle fibers were examined. Outside the muscle spindle capsule the nuclear bagfibers lose their hitherto characteristic ATPase activity and stain in a manner similar to that of extrafusal type I muscle fibers, perhaps in relationship to spindle skeletofusimotor innervation.     

Sensory and motor innervation of bird intrafusal muscle fibers

• 1.1. Most bird muscle spindles are supplied by only one primary afferent.
• 2.2. Secondary afferents occur irregularly.
• 3.3. Sensory terminals are covered by a basal lamina and a collagenous sheath.
• 4.4. Two types of motor terminal are recognized which can be referred to specific types of intrafusal fiber.
• 5.5. The sensory and motor innervation of bird intrafusal fibers is less understood than that of mammalian intrafusal fibers.

     

模拟失重条件下大鼠比目鱼肌肌梭神经营养因子3表达降低

本研究旨在探讨模拟失重对大鼠比目鱼肌肌梭神经营养因子3(neurotrophin-3,NT-3)表达的影响。采用大鼠尾部悬吊法建立模拟失重动物模型,按体重配对原则随机将大鼠分为5组,即尾悬吊3d组、7d组、14d组、21d组和正常同步对照组。采用免疫组织化学ABC染色法及酶联免疫吸附实验法(ELISA)检测大鼠比目鱼肌肌梭NT-3的表达。结果显示,大鼠比目鱼肌梭外肌中未见到NT-3表达;正常对照组大鼠比目鱼肌肌梭中,核袋1和核袋2纤维NT-3呈现强阳性表达;模拟失重后,梭内肌纤维的NT-3免疫染色反应进行性降低;NT-3的ELISA定量检测结果显示,正常组、尾悬吊3d组、7d组、14d组和21d组大鼠比目鱼肌NT-3的含量分别为(14.23±1.65)、(14.11±1.53)、(13.09±1.47)、(12.45±1.51)和(9.85±1.52)pg/mg。统计比较显示,尾悬吊14d后,大鼠比目鱼肌NT-3的含量较正常对照组明显减少(P<0.05);而尾悬吊21d后,大鼠比目鱼肌NT-3的表达进一步减少(P<0.01)。以上结果表明,模拟失重可致大鼠比目鱼肌肌梭NT-3的表达明显减少,并且随着模拟失重时...     

Arrangement of cytoskeletal filaments at the equator of chicken intrafusal muscle fibers

The organization of the cytoskeleton at the equator of chicken intrafusal fibers was examined with immunofluorescence light microscopy, using monoclonal antibodies against myosin heavy chains, desmin, actin and tropomyosin. Actin was localized in the cytosol and in equatorial nuclei, while myosin heavy chains, desmin and tropomyosin were only observed in the cytosol. Although all four proteins were present at the equator and at the pole, the fluorescence produced after incubation with the different antibodies varied considerably between the two regions. Staining at the pole was in the form of striations, but at the equator it was non-striated and more uniform. The observed fluorescent patterns suggest that at the equator filaments are assembled into looser arrays than in the sarcomeres of the pole. A flexible cytoskeleton at the equator would be an appropriate substrate for distorting the affixed sensory endings during an applied stress.     

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.     

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.     

A histoenzymatic study of rat intrafusal muscle fibres

Summary The histochemical activities of myofibrillar adenosine triphosphatase (ATPase), succinic dehydrogenase (SDH) and alpha glycerophosphate dehydrogenase (-GPD) were studied in intrafusal muscle fibres of rat fast and slow muscles. The ATPase reaction was carried out after the three standard acid preincubations. The cold K₂-EDTA preincubated ATPase reaction product was similar to that seen following the regular or alkalipreincubated ATPase reaction, except that the intermediate bag fibres exhibited much higher activity after cold K₂-EDTA preincubation. Following either acetic acid solution or cold and room temperature K₂-EDTA-preincubation, followed by the ATPase reaction, chain fibres of the fast muscles vastus lateralis and extensor digitorum longus exhibited a very low amount of reaction product as compared with those of the slow soleus. Veronal acetate and K₂-EDTA preincubations (and equally preincubation in acetic acid solution) resulted in acid stable ATPase activity along the entire length of the typical bag fibres but only in the polar regions of the intermediate bag fibres. On the basis of differing -GPD reaction, two sub populations of nuclear chain fibres were discovered in one spindle. It is a matter of conjecture, to what extent the histochemical differences of intrafusal fibres from fast and slow muscles reflects functional distinctions in the response to stretch of muscle spindles from fast and slow muscles.     

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.