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 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.     

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.     

The transverse tubular system of cat intrafusal muscle fibres

Summary A modified staining technique for transverse tubular and sarcoplasmic reticular systems was used to investigate their occurrence in different types of intrafusal muscle fibres in cat tail spindles. Intrafusal muscle fibres can be divided into three basic regions, namely, the periaxial space (A-region), intracapsular area (B-region) and the extracapsular area (C-region); the components of these systems were seen to vary in structure and distribution. The occurrence of these systems also varied among the different types of intrafusal muscle fibres, namely, the bag₁, bag₂ and chain fibres. In bag₁ fibres components were sparse in the A-region, increased slightly in the B-region, but were most developed in the C-region; triads were consistently located at the border between A- and I-bands. In bag₂ fibres membrane components were noted in the A-region but were more abundant in the B-region where some tubular components showed transverse and longitudinal branches linked together in the form of a network; membrane systems diminished towards the C-region. The majority of triads were located within the A-bands. In chain fibres the membrane systems occurred more commonly in the A-region, while in the B- and C-regions, the transverse tubular system possessed numerous transverse and longitudinal branches forming irregularly distributed tubular networks. Some tubular branches were dilated, while other branches terminated as sacs among arrays of the sarcoplasmic reticular system in I-bands. Some transverse tubules bifurcated into two branches with numbers of dilated sarcoplasmic reticular cisternae lying on either side, or between, the branches. Triads sometimes occurred between A- and I-bands, but, generally, were situated well within A-bands.     

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.     

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.     

Histochemical heterogeneity of intrafusal muscle fibres in slow and fast skeletal muscles of the rat

Summary Intrafusal muscle fibres of the slow soleus (Sol) and fast vastus lateralis (VL) muscles of the rat were studied histochemically. Serial transverse sections were incubated for the localization of succinate dehydrogenase (SDH), alpha glycerophosphate dehydrogenase (GPD) and adenosine triphosphatase (ATPase). The latter was examined further after preincubation in acidic solution held at either low or room temperature (R_T). The bag₂ intrafusal fibres in both muscles displayed high regular and acid stable ATPase, but low SDH and GPD activities. Bag₁ intrafusal fibres showed low to moderate regular ATPase, a regional heterogeneity after R_T acid preincubation (low activity in juxtaequatorial and high in polar zones), moderate SDH, but low GPD reactions. In both muscles the chain fibres usually exhibited high ATPase for both regular and cold acid preincubated reactions, but usually low activity after R_T acid preincubation; they had high SDH but variable GPD activities. In Sol muscle, however, approximately 25% of spindles contained chain fibres that showed high acid-stable ATPase reaction after both cold and R_T acid preincubation. In contrast, chain fibres in some VL spindles had a characteristically low ATPase reaction even after cold acid preincubation. This study, therefore, has delineated the existence of an inherent heterogeneity among chain fibres (with respect to their histochemical reactions) in muscle spindles located within slow and fast muscles and also between those found within populations of either Sol or VL muscle spindles.     

A histoenzymatic study of rat intrafusal muscle fibres.

The histochemical activities of myofibrillar adenosine triphosphatase (ATPase), succinic dehydrogenase (SDH) and alpha glycerophosphate dehydrogenase (alpha-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 alkali-preincubated 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 alpha-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.     

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.     

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.     

A new grouping of intrafusal muscle fibers based on developmental studies of muscle spindles in the cat.

The development of muscle spindles was studied using the tenuissimus muscle of the cat. Observations show that the intrafusal muscle fibers develop as two separate groups: one group represented by a single nuclear bag fiber while the second group comprises the second nuclear bag fiber in association with all the nuclear chain fibers. This grouping is most pronounced in the fetus and is clearly seen in neonatal kittens (i.e., up to 2 weeks of age). As the intrafusal fibers begin to separate from each other, the groupings become less noticeable, although this basic pattern is often retained in the adult. The pattern of intrafusal fiber grouping is most noticeable in the equatorial regions of the spindle and least noticeable in the polar regions. This is not the grouping of fibers which would have been expected from a consideration of existing reports on muscle spindles. The implications for spindle form and function are considered.     

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.     

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.