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.     

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.     

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.

     

Nonselective motor innervation of nuclear bag1 intrafusal muscle fibers in the cat

The motor nerve supply to cat nuclear bag1 intrafusal muscle fibers was reconstructed from light and electron microscopy of serial transverse sections of spindles in the tenuissimus muscle. Twenty-six of thirty poles of bag1 fibers that were examined received motor innervation. Every innervated bag1 pole received at least one (range 1-3) selective motor axon that supplied this fiber type only. Four of the innervated bag1 poles (15%) received additional motor supply from a nonselective motor axon that also innervated one nuclear chain fiber in the same spindle pole. The chain fibers co-innervated with bag1 fibers were among the longest chain fibers although they were shorter than two long chain fibers also present in the spindle poles. In cross-sections stained with toluidine blue they displayed 1-3 equatorial nuclei side by side, and there were fewer intermyofibrillar granules in their polar regions than in most of the other chain fibers. The endings of nonselective motor axons on the bag1 and chain fibers were morphologically and ultrastructurally dissimilar. It is suggested that instances of common innervation of the (dynamic) bag1 fiber and a (static?) chain fiber represent an integral and, presumably, functionally meaningful part of the motor pattern in some cat spindles.     

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

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

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.     

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.     

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.     

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.     

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

Muscle spindles were studied histochemically in serial transverse sections of specimens of the cat tenuissimus muscle. The nuclear chain intrafusal muscle fibers were separated into three subtypes, called long, intermediate and typical. The long chain and intermediate chain fibers tended to assume a particular position within the axial bundle of intrafusal fibers. The fibers were usually located in that layer of chain fibers that was positioned farthest away from the bag2 fiber. Furthermore, they were usually situated adjacent to the bag1 fiber throughout much of the extent of the spindle pole. Some long chain and intermediate chain fibers had several fiber nuclei abreast at the equator rather than a single row of central nuclei, as in most nuclear chain fibers. The relative position of intrafusal fibers within the cat spindle may reflect their order of formation during development, with the fibers retaining, to a variable degree, their association with the bag2 fiber which acted as template. Thus, the axial position of long chain and intermediate chain fibers suggests that they are among the first nuclear chain fibers to form. This may play a role in the known preferential innervation of these chain fibers by skeleto-fusimotor axons.     

Nonuniform expression of myosin heavy chain isoforms along the length of cat intrafusal muscle fibers

Summary The expression of four myosin heavy chain (MHC) isoforms, avian slow-tonic (ATO) or neonatal-twitch (ANT) and mammalian slow-twitch (MST) or fast-twitch (MFT) in intrafusal fibers was examined by immunocytochemistry of spindles in the tenuissimus muscle of adult eats. The predominant MHCs expressed by nuclear bag fibers were ATO and MST, whereas the MHCs prevalent in nuclear chain fibers were ANT and MFT. The expression of these isoforms of MHC was not uniform along the length of intrafusal fibers. In general, both bag and chain fibers expressed avian MHC in the intracapsular region and mammalian MHC in the extracapsular region. The nonuniform expression of MHCs observed along the length of bag and chain fibers implies that different genes are activated in myonuclei located in the intracapsular and extracapsular regions of the same muscle fiber. Regional differences in gene activation might result from a greater effect of afferents on myonuclei located near the equator of intrafusal fibers then on myonuclei outside the spindle capsule.     

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.     

The occurrence of "mixed" nuclear bag intrafusal fibers in the cat muscle spindle

A total of 147 muscle spindles was studied histochemically in serial transverse sections of 42 cat tenuissimus muscle specimens. Nuclear bag1, nuclear bag2 and nuclear chain intrafusal muscle fibers were distinguished by the differential staining resulting from the reactions for myosin adenosine 5'-triphosphatase and nicotinamide adenine dinucleotide tetrazolium reductase. The majority of intrafusal fibers were of the same histochemical type at both fiber poles. However, seven muscle spindles contained one nuclear bag fiber each that presented as a bag1 in one pole and as a bag2 in the other pole. These "mixed" nuclear bag fibers were found in spindles that also contained at least one bag1 and one bag2 fiber of equivalent histochemical presentation in both fiber poles. The "mixed" bag fibers displayed differences of apparent fiber diameter and relative polar length between the two fiber poles. The motor innervation pattern, as revealed by staining for cholinesterase, was also dissimilar between the two poles of "mixed" bag fibers. The study indicates that the spindle equatorial region may in some instances serve as a boundary between two morphologically and histochemically different poles of the same intrafusal fiber.