Intrafusal fibre types in rat limb muscle spindles

Summary Morphological, histochemical and ultrastructural characteristics of intrafusal fibre types were studied in rat muscle spindles. The existence of three intrafusal fibre types, namely the typical bag, the intermediate bag and the chain fibres was confirmed. Intrafusal fibres differ in diameter, length and number of nuclei in the equatorial zone. Histochemically, typical bag fibres exhibit both alkali-and acid-stable ATPase activity and low SDH activity. Intermediate bag fibres possess low alkali-stable ATPase activity; after acid-preincubation, however, they have low activity only in the juxtaequatorial region, whereas in the polar zones they exhibit high acid-stable ATPase activity. The SDH activity varies from moderate to high. The chain fibres exhibit high alkali-stable and low acid-stable ATPase and high SDH activity in the extensor digitorum longus muscle, whereas in the soleus muscle the acid-stable ATPase activity varies from a low one to a high one, either among individual chain fibres in one spindle, and/or repeatedly along the fibre length.Since there are regional differences in morphological characteristics and in staining properties of intrafusal fibres, a reliable identification of intrafusal fibre types can only be achieved by an analysis of serial sections.     

Intrafusal fibre types in rat limb muscle spindles: morphological and histochemical characteristics.

Morphological, histochemical and ultrastructural characteristics of intrafusal fibre types were studied in rat muscle spindles. The existence of three intrafusal fibre types, namely the typical bag, the intermediate bag and the chain fibres was confirmed. Intrafusal fibres differ in diameter, length and number of nuclei in the equatorial zone. Histochemically, typical bag fibres exhibit both alkali- and acid-stable ATPase activity and low SDH activity. Intermediate bag fibres possess low alkali-stable ATPase activity; after acid-preincubation, however, they have low activity only in the juxtaequatorial region, whereas in the polar zones they exhibit high acid-stable ATPase activity. The SDH activity varies from moderate to high. The chain fibres exhibit high alkali-stable and low acid-stable ATPase and high SDH activity in the extensor digitorum longus muscle, whereas in the soleus muscle the acid-stable ATPase activity varies from a low one to a high one, either among individual chain fibres in one spindle, and/or repeatedly along the fibre length. Since there are regional differences in morphological characteristics and in staining properties of intrafusal fibres, a reliable identification of intrafusal fibre types can only be achieved by an analysis of serial sections.     

Effects of electrical stimulation upon post-hatching development of fibre types in normally innervated fast and slow latissimus dorsii muscles of the chicken

In chicken, the main characteristic properties of muscle fibre types in slow anterior (ALD) and fast posterior (PLD) latissimus dorsii are acquired during post-hatching development. At day 4 it becomes possible to distinguish between alpha' and beta' fibre types in ALD muscle. At the same time, mATPase staining and NADH-TR activity permit recognition of alpha w and alpha R fibres within PLD muscle. During further development, muscle fibre typology progressively changes towards the adult slow and fast type. Chronic stimulation at a slow rhythm (5 Hz) of PLD prevents the change in relative proportions of alpha R and alpha W fibres within the muscle that occurs in normal post-hatching development and increases the number of beta R fibres. Moreover, oxidative activity is increased in all muscle fibre types following stimulation. In ALD muscle, chronic stimulation at a fast rhythm (40 Hz) results in a decrease in oxidative activity and inhibits the differentiation of alpha' and beta' muscle fibre types. This study demonstrates that in young chicken, the pattern of activity influences the differenciation of fibre types in slow and fast muscles.     

Cytophotometry of post mortem glycogenolysis in different histochemical types of muscle fibres of the pig.

Synopsis Serial transverse sections of porcine longissimus dorsi muscle (18 pigs, 50 to 178 kg live weight) were reacted for NAD tetrazolium reductase and ATPase at pH9.4, and for glycogen with the periodic acid-Schiff (PAS) reaction. Three histochemical types of muscle fibre were identified; (1) strong ATPase and weak NADH oxidative activity; (2) strong ATPase and intermediate NADH oxidative activity; and (3) weak ATPase and strong NADH oxidative activity. Immediatepost mortem samples from one side of each animal were compared with a laterpost mortem sample from the other side by measuring the absorbance of PAS-stained glycogen at 570 nm with a microscope photometer. Laterpost mortem absorbance was expressed as a percentage of immediatepost mortem absorbance in each category of muscle fibre in order to compensate for distributional error and different starting levels of glycogen. Muscle fibres with weak ATPase and strong NADH oxidative activity showed a progressive decrease in absorbance of PAS-stained glycogenpost mortem. In some animals, fibres with strong ATPase and intermediate or weak NADH oxidative activity showed an initialpost mortem increase in absorbance of PAS-stained glycogen which was then followed by a progressive decrease. The maximum rates of decrease in absorbance in the three fibre types did not differ to any great extent.     

A histochemical analysis of mammalian oxidative skeletal muscle fibres using the enzymes of energetic metabolism

Summary Some histochemical parameters of the three main fibre types of rat vastus lateralis muscle were studied. Succinic dehydrogenase (SDH), creatine kinase (CK), sarcotubular ATPase (SR-ATPase) and mitochondrial ATPase activities were demonstrated in serial sections. The three fibre types, recognised by the distribution pattern of SDH activity, all show high CK activity. However, red Type I oxidative fibres when examined for ATPase and ATPase dependent CK activity, show distinct heterogeneity revealing sub-populations within the same homogeneous fibre type. Three distinct patterns were recognised in the red Type I fibres depending on the distribution of the final reaction product. The present histochemical evidence confirms the fact that subdivision of mammalian skeletal muscle into three fibre types is only approximate and probably more than three types exist.     

Transitional stages in the histochemical development of muscle fibres during post-natal growth

Synopsis Serial frozen sections of longissimus dorsi muscles from seven pigs at different live weights (13 to 127 kg) were reacted for ATPase by the calcium method at an alkaline pH and for NADH oxidative activity. One hundred muscle fibres from each animal were identified individually in serial sections and their staining intensity was measured with a microscope photometer at 600 nm. For each section, staining intensity of fibres (% tranmission) was measured and converted to the nearest one-tenth unit of the range from the darkest to the lightest staining fibres. Frequency of occurrence of fibre types was plotted on a 10×10 grid using the range co-ordinates for NADH oxidative activity (vertical) and ATPase activity (horizontal). The commonly recognized histochemical fibre types in this muscle appeared as crowded areas in the grid but, in many cases, these areas were part of a continuous L shaped distribution. In fibres having an ATPase staining intensity of 1.0 and 0.9 units of the range, a continuous but skewed distribution with regard to NADH oxidative activity was detected. In fibres with NADH oxidative activity of 0.6 to 1.0 units of the range, a continuous but irregular distribution with regard to ATPase activity was detected. Within this range, there was some evidence of a growth-related shift towards weaker ATPase activity.     

Histochemical profiles of guinea-pig intrafusal fibres in normal muscles and after denervation, cordotomy and tenotomy

Synopsis Histochemical profiles of intrafusal fibres were analysed in normal muscles and in denervated, cordotomized and tenotomized preparations. Based on ATPase activity at polar regions, normal intrafusal fibres were classified as (I) ATPase-light fibres showing low or low-moderate activity when pre-incubated in either an acid or alkaline medium; (2) ATPase-dark fibres demonstrating high activity when preincubated in either an acid or alkaline medium and (3) ATPase-reversing fibres displaying low to moderate activity when pre-incubated in an acid medium, but showing high activity when pre-incubated in an alkaline medium. Four weeks after nerve section contrasting responses were seen between intrafusal fibre types. The ATPase-reversing fibres showed large decreases in polar cross-sectional area and NADH-diaphorase (NADH-D) activity, whereas fibres of the ATPase-light and ATPase-dark types were less subject to atrophy and their NADH-D levels were frequently increased. This differential effect suggests that ATPase-reversing fibres are trophically more dependent on neural innervation than ATPase-light and ATPase-dark fibres. After cordotomy and tenotomy no such marked differential responses were noted between fibre types.     

Studies on the swimming musculature of the rainbow trout I. Fibre types

A histochemical study has been made on the myotomal musculature of the rainbow trout Salmo gairdneri Richardson. Four main types of fibre can be distinguished on the basis of differences in fibre size, lipid content and succinic dehydrogenase and myofibrillar adenosine triphosphatase activity (ATPase activity). Early histological studies have concluded that in addition to a superficial strip of muscle small diameter 'red' fibres occur throughout the trout myotome. These small diameter fibres occurring in the bulk of the myotome are shown to differ from the superficial fibres with respect to their oxidative metabolism and myofibrillar ATPase. The relevance of this finding to studies on the energetics and swimming efficiency of this species are discussed.     

Comparison of red and white muscles by cytophotometry of their muscle fibre populations

Synopsis Samples from two red muscles (vastus intermedius and vastus medialis) and two white muscles (biceps femoris and gluteus medius) were taken from four pigs. Serial transverse sections were reacted for ATPase and NADH oxidative activity. Sections were mapped with a projection microscope so that the staining intensity of individual fibres for the two reactions could be measured with a simple microscope photometer. Transmission values at 600 nm were converted to units of 0–10 for the range from darkest to lightest staining fibres on each section to cancel variation in staining intensity between sections. The aim of the study was to use simple cytophotometry instead of subjective judgement in the categorization of different histochemical types of muscle fibres. Cytophotometry enabled clear resolution of the major fibre types (types I and II using the ATPase reaction), partial resolution of more variable characteristics (NADH oxidative activity in type I and II fibres) and no resolution of subtle subtypes (IA and IB with the NADH oxidative reaction). However, between the major fibre types, cytophotometry revealed variable numbers of fibres with transitional characteristics. There were more of these fibres in red muscles. With sections reacted for ATPase, transmission values for low magnification fields containing 100 to 200 fibres were correlated (r=–0.91) with the ratio of type I:II fibres.     

A comparative microphotometric study of succinate dehydrogenase activity levels in type I,IIA and IIB fibres of mammalian and human muscles

Summary Activity levels of succinate dehydrogenase (SDH) were determined kinetically by means of comparative microphotometric measurements in situ. Activities were correlated with fibre types classified histochemically according to Brooke and Kaiser (1970). Analyses of tibialis anterior muscles in the mouse, rat, guinea pig, rabbit, cat and the human showed pronounced variations in the activity profiles of type I, type IIA and IIB fibres of these muscles. Large scattering of enzyme activity existed in the three fibre populations. Overlaps of varying extent were found for the SDH profiles between the different muscles. Type I fibres reveal species diffeences in aerobic oxidative capacity. Whereas the majority of the IIB fibres in rabbit muscle tended to be low in SDH activity, the main fraction of this fibre population was characterized by high activities in mouse muscle. Similarly, the IIA fibre populations revealed opposite properties in mouse and rabbit muscles. These extremes as well as intermediate activity patterns indicate that no general scheme exists according to which the histochemically assessable myosin ATPase is correlated with the aerobic oxidative capacity of muscle fibres in various mammalian muscles.     

The radial distribution of succinate dehydrogenase activity in porcine muscle fibres

Summary A microscope photometer with a computer-controlled scanning stage was used to map the distribution of succinate dehydrogenase (SDH) activity in transverse sections of skeletal muscle fibres of pigs from 9 to 29 weeks of age. Absorbance due to SDH activity was measured in successive concentric zones that converged on the central axis of the muscle fibre. In all fibres, there was less SDH activity in the axis of the fibre than in the periphery of the fibre. In fibres with strong adenosine triphosphatase (ATPase) activity and weak overall SDH activity, the radial gradient of SDH activity remained constant as fibres grew in cross-sectional area. However, in fibres with weak ATPase and strong SDH and in fibres with strong ATPase and strong SDH, the radial gradient of SDH activity increased as fibres grew larger. Changes in radial gradients were due to both decreased SDH activity in the axis and to increased SDH activity in the periphery. In all three fibre types, the overall SDH activity per fibre increased with age.     

Relationship among fibre type, myosin ATPase activity and contractile properties

Summary At least two types of skeletal muscle myosin have been described which differ in ATPase activity and stability in alkaline or acidic media. Differences in ATPase characteristics distinguish Type I and Type II fibres histochemically. In this study, ATPase activity of myosin from muscles of several species with known histochemical and contractile properties has been determined to test the hypothesis that (1) myosin ATPase activity, (2) histochemical determination of fibre types and (3) maximum shortening velocity, all provide equivalent estimates of contractile properties in muscles of mixed fibre types. Maximum shortening velocity appears to be proportional to ATPase activity as expected from previous reports by Barany. However, both myosin ATPase and the maximum shortening velocity exhibit curvilinear relationships to the fraction of cross-sectional area occupied by Type II fibres. Therefore, we reject the hypothesis and conclude that histochemically determined myofibrillar ATPase does not accurately reflect the intrinsic ATPase activity or shortening velocity in muscles of mixed fibre types. Our data are consistent with the presence of more than two myosin isozymes or with a mixture of isozymes within single muscle fibres.     

Slow and fast fatigable frog muscle fibres: electrophysiological and histochemical characteristics

Continuous activity of isolated frog gastrocnemius muscle fibres provoked by repetitive stimulation of 5 Hz was used as an experimental model for fatigue development in different fibre types. Parameter changes of the elicited intracellular action potentials and mechanical twitches during the period of uninterrupted activity were used as criteria for fatigue evaluation. Slow fatigable muscle fibre (SMF) and fast fatigable muscle fibre (FMF) types were distinguished depending on the duration of their uninterrupted activity, which was significantly longer in SMFs than in FMFs. The normalized changes of action potential amplitude and duration were significantly smaller in FMFs than in SMFs. The average twitch force and velocity of contraction and relaxation were significantly higher in FMFs than in SMFs. Myosin ATPase (mATPase) and succinate dehydrogenase activity were studied by histochemical assessment in order to validate the fibre type classification based on their electrophysiological characteristics. Based on the relative mATPase reactivity, the fibres of the studied muscle were classified as one of five different types (1-2, 2, 2-3, 3 and tonic). Smaller sized fibres (tonic and type 3) expressed higher succinate dehydrogenase activity than larger sized fibres (type 1-2, 2), which is related to the fatigue resistance. The differences between fatigue development in SMFs and FMFs during continuous activity were associated with fibre-type specific mATPase and succinate dehydrogenase activity.     

去神经对快,慢肌纤维肌球蛋白ATPase影响的组织化学观察

本文用组织化学方法观察了豚鼠比目鱼肌(SOL)和腓骨第三肌(PT)在去神经后其快、慢纤维肌球蛋白ATPase特性的变化。在正常肌肉中Ⅰ型(慢)纤维和Ⅱ型(快)纤维分别具有酸和碱稳定ATPase活性。慢纤维在去神经后出现了碱稳定ATPase活性,而快纤维则无明显变化。结果表明,只有慢纤维的肌球蛋白ATPase特性才与神经支配有关。     

A comparative histochemical study of intrinsic laryngeal muscles of ungulates and carnivores

The intrinsic laryngeal muscles of the horse, donkey, sheep, ox, pig, dog and cat were examined for myosin ATPase, following acid and alkali pre-incubation, SDH and M-alphaGPDH activities. In all laryngeal muscles two fibre types, betaR and alphaR, belonging to slow and fast-contracting, fatigue-resistant motor units (types S and FR) were present in different proportions. The alphaW fibre type, belonging to fast-contracting and fatigue-resistant motor units was absent (type FF). The alphaR fibres of the dog and the cat were subdivided into groups by the various degrees of acid stable myosin ATPase, oxidative and glycolytic activities. In the ox and pig laryngeal muscles, the same fibres showed an atypical myosin ATPase activity, as high as the fast-contracting fibres but acid-resistant like the slow-twitch fibres. The most uniform muscle was the CAD, which was formed of a higher percentage of slow-twitch fibres than the other laryngeal muscles of the same species. Also the VE muscle was very uniform in the dog, horse and donkey but the fast-twitch fibres were by far the most numerous, the highest in fact among all the laryngeal muscles. In the TA muscle of the cat, sheep and ox, the percentage of fast-twitch fibres was very high in the rostral portion decreasing gradually towards the caudal portion. Thus it was possible to separate histochemically the TA muscle in the rostral (pars ventricularis) and caudal (pars vocalis) portions which are related to the VE and the VO muscles of the dog, horse and donkey. In the VO muscle the slow-twitch fibres are more numerous than in the VE. The two portions of the TA were not detected by histochemical methods in the pig. However, this muscle has the highest percentage of fast-twitch fibres. The qualitative and quantitative data presented in this paper together with the data reported in the literature, enable us to correlate morphological and functional aspects of fibre composition among the species.     

Histochemical definition of muscle fibre types in the trunk musculature of a teleost fish (cod,Gadus morhua,L.)

Summary Cryostat sections incubated for myofibrillar ATPase, SDH, LDH, and -GPDH as well as p-phenylene-diamine stained semithin sections were used to define muscle fibre types in the trunk musculature of the cod (Gadus morhua, L.).Three zones (superficial, intermediate, deep) containing different muscle fibre types are present within both epaxial and hypaxial parts of each myomere subjacent to the lateral line.Atypical relations concerning myofibrillar ATPase activity probably reflects instability of myosin during storage of frozen tissue. The histochemical reaction does not distinguish between myofibrillar and mitochondrial ATPase in cod muscle.Based on ATPase and SDH activities, seven different histochemical profiles of muscle fibres can be identified in trunk musculature of this teleost fish. Attempts to homologize these fibre types with those in cyclostomes or those in higher animals proved futile. The higher number of histochemically defined muscle fibre types in cod might be explained by developmental processes and an admixture of immature fibres throughout life.     

Factors affecting the histochemical composition of reinnervated soleus muscle of rat.

The myofibrillar ATPase reaction was utilized to determine the relative proportion of type II fibres in reinnervated soleus muscle 6 months after transection and reunion of the nerve at various distances from the muscle. In self-reinnervated soleus muscle, a highly significant decrease in the percentage of type II fibres from 10.5 +/- 1.6% in normal muscle to 0.7 +/- 0.4% was noted. In randomly reinnervated soleus muscle the percentage of type II fibres gradually increased with the distance of the site of nerve transection from the muscle. After transection and reunion of the muscular branch of the tibial nerve, the type II fibres constituted 34.0 +/- 1.5% and that of the reinnervated soleus muscle after transection and reunion of the sciatic nerve stump was 73.7 +/- 1.7% of the total fibre population. Different factors which might be responsible for the observed differences in the degree of cytochemical transformation of muscle fibre types in the process of reinnervation are discussed.     

Nitric oxide synthase in human skeletal muscles related to defined fibre types

Skeletal muscle functions regulated by NO are now firmly established. However, the knowledge about the NO synthase (NOS) expression related to a defined fibre type in human skeletal muscles necessitates further clarification. To address this issue, we examined localization of NOS isoforms I, II and III, in human skeletal muscles employing immunocytochemical labeling with tyramide signal amplification complemented with enzyme histochemistry and Western blotting. The NOS immunoreactivity was related to fibre types of different classification systems: physiological classification into slow and fast, ATPase classification into I, IIA, IIAX, IIX, and physiological-metabolic classification into slow-oxidative (SO), fast-oxidative glycolytic (FOG) and fast-glycolytic (FG). We found a correlation of NOS I–III immunoreactivity to metabolic defined fibre types with strong expression in FOG fibres. This implies that NO as modulator of muscle function is involved in oxidative metabolism in connection with fast force development, which only occurs in FOG fibres. The NOS expression showed no correlation to ATPase fibre subtypes due to the metabolic heterogeneity of ATPase fibre types. Healthy and affected vastus medialis muscles after anterior cruciate ligament rupture revealed similar NOS expression level as shown by Western blotting with, however, different expression patterns related to the fibre types in affected muscles. This suggests an altered modulation of force development in the fibres of diseased muscles.     

pH lability of myosin ATPase activity permits discrimination of different muscle fibre types in crustaceans

Myofibrillar actomyosin ATPase activity has been studied histochemically in the closer muscle of the crab Eriphia spinifrons. Preincubation at pH 4.6 and 5.0 reveals differences in the lability of the ATPase. This permits the discrimination of four fibre types. Of these, three represent subgroups of rapidly contracting fibres. The histochemically defined fibre types correspond well with four groups defined according to electrophysiological criteria.     

Effects of body suspension on soleus muscle fibres and spinal motoneurones in the rat.

1. After 14 days of body suspension, fibre type composition and fibre cross-sectional area in the soleus muscle of 17-week-old male Sprague-Dawley rats were investigated. Oxidative enzyme activity of soleus motoneurones in the spinal cord was also examined. 2. After suspension, soleus muscle weight decreased by 44.2%, the cross-sectional area of SO and FOG fibres decreased by 60.4% and 58.6%, respectively. 3. The percentage of fibre types was not changed by suspension. However, ATPase activity after alkaline preincubation was markedly inhibited in FOG fibres. 4. Oxidative enzyme activity of soleus motoneurones was not changed by suspension. 5. This study demonstrates that using mature animals body suspension induces atrophy of fast- and slow-twitch fibres accompanied with the selective inhibition in ATPase activity of fast-twitch fibres, and without changes in histochemical profiles of the corresponding motoneurones.