Histochemical and immunohistochemical investigation of muscle fibres in the sturgeon (Chondrostei; Acipenser)

The arrangement and innervation of lateral muscle and adductor mandibulae (AM) complex were examined histochemically and immunohistochemically in the sturgeon Acipenser transmontanus. Lateral muscle was arranged in myotomes in which the superficial slow-red muscle layer and the deep fast-white muscle layer were separated by an intermediate layer of pink muscle. The AM complex was composed of a small slow-red muscle layer, but the bulk of the muscle was composed of fast-white fibres. Pink fibres appeared both in an intermediate layer and scattered throughout the fast-white muscle. The innervation appeared to resemble that of elasmobranchs and some teleosts. Slow-red fibres of lateral muscle were multiply innervated, whereas fast-white muscle fibres were focally innervated at the myoseptal end. Pink fibres of lateral muscle and fast-white fibres of AM were focally innervated in the mid-region of the fibres. There were no significant differences between 5 month old and adult sturgeons except for a much better developed intermediate (pink) layer surrounding the lateral line in the adult.     

The presynaptic active zones in three different types of fibres in frog muscle

Presynaptic active zones were studied in slow, fast and intermediate types of frog muscle fibres in freeze-fracture replicas. In fast fibres, the double rows of paired particles are present on active zone ridges perperdicular to the longitudinal axis of the nerve whereas in slow fibres active zone ridges are rudimentary or absent and double rows of particles occur in all directions, mostly paired, sometimes single. In the intermediate type of muscle fibres both types of active zone deployment coexist on a single muscle fibre.     

Effects of training on axial muscle of two cyprinid species: Chondrostoma nasus (L.) and Leuciscus cephalus (L.)

The effect of an endurance training program lasting 17 weeks was studied in two cyprinid species, Chondrostoma nasus (L.) and Leuciscus cephalus (L.). Red, intermediate and white axial muscle were investigated. Morphometrical analysis revealed that training induced, in both species, increased red and intermediate muscle mass, fibre diameter and capillarization. Differences between species in the response to training were observed for volume densities of mitochondria and lipid. In contrast to C. nasus, L. cephalus show higher values for these compartments in red and intermediate fibres. The results are considered adaptational changes which increase the aerobic capacity of red and intermediate muscle fibres to meet higher sustained swimming activities.     

Morphology and organization of muscle fibres in the thoracic coxal muscle receptor organ and the associated promotor muscle, in a crayfish, Cherax destructor, and mud crab, Scylla serrata

Using confocal laser scanning and conventional light microscopy, the morphology and organization of the muscle fibres in a proprioceptor, the thoracic coxal muscle receptor organ (TCMRO), and the associated 'extrafusal' promotor muscle were investigated in two species of decapod crustacea, the crayfish Cherax destructor and the mud crab Scylla serrata . The diameter of the TCMROs was shown to increase distally, with an increase up to 350% recorded for the crayfish. The tapered shape of the crayfish TCMRO was demonstrated to amplify movements mechanically at the transducer region where the afferent nerves attach. Serial sectioning of the TCMROs, showed that the fibre number increased in the proximal to distal direction from 14 to 30 fibres in the crayfish and from 7 to 20 in the crab. Optical sectioning with the laser scanning confocal microscope revealed that the increase in fibre numbers was the result of muscle fibres branching in the distal third section of the TCMRO. The percentage of muscle tissue in the cross-sectional area in the TCMRO was found to be only 35.2% and 64.6% in the crayfish and crab, respectively. Longitudinal sectioning using laser scanning confocal microscopy revealed the average sarcomere length of the TCMRO muscle fibres of both species to be in the intermediate range for crustacean muscle fibres (4.1 ± 0.1 µm and 4.55 ± 0.34 µm for the crayfish and crab) compared with the long sarcomere muscle fibres in the associated promotor muscles (7.87 ± 0.2 and 10.6 ± 0.6 µm). The distinct morphology of the TCMRO muscle fibres – smaller diameter, intermediate sarcomere length and branching of fibres compared to the larger, long sarcomere promotor fibre muscle fibres – suggest that the TCMRO muscle fibres are specialized in their role of proprioception.     

The histochemistry of muscle in juvenile Atlantic salmon, Salmo solar L.

In fry, 1- and 2-year old juvenile Atlantic salmon, relatively small superficial red muscle fibres staining well for glycogen and succinic dehydrogenase (SDH) but with little myofibrillar ATPase, were evident on either side of the lateral line, in June. Well differentiated relatively large white fibres contained lower concentrations of glycogen than the red fibres, higher ATPase and no SDH, except traces in the larger 1- and 2-year-olds. Intermediate size pink fibres, which were also intermediate between red and white fibres in their staining properties, occurred in a thin diffuse layer along the red-white boundary, thickest at the apex near the vertebral column, and most evident in the younger fish.     

Studies on the nature of the Z-discs in skeletal muscle fibres of sharks, Etmopterus spinax L. and Galeus melastomus Rafinesque-Schmaltz

The ultrastructural details of Z-discs from red, intermediate, and white axial muscle fibres from the sharks Etmopterus spinax and Galeus melastomus are described. Red fibre Z-discs contain the most amorphous matrix material, and are thicker (100–115 nm) than intermediate (85–88 nm) and white Z-discs (75–80 nm). Four sets of oblique bars extend tangentially from each thin filament. In each set two bars are present, those of white fibres are close together (approximately 5 nm), while those of red fibres are separated by a distance of 15 nm. A model of shark Z-disc structure is proposed. The denaturation (heat transition) temperatures of the muscle proteins were studied by differential scanning calorimetry (DSC). The heat transitions of collagen, actin, and myosin were identified; the actin heat transition temperature increased in the sequence red, intermediate, and white. The total protein pattern of red and white muscle were studied by SDS electrophoresis. A protein with a molecular weight of about 55000 may represent a Z-disc protein.     

Organization of skeletal muscle in the urodele Triturus cristatus: muscle fibre types and motor units

Four muscle fibre types are described in the biceps and extensor digitorum communis muscles of the newt forelimb. The histological criteria forming the basis for the distinctions include differential staining with p-phenylenediamine and succinate dehydrogenase histochemistry and electron microscopy. In addition, three distinctive motor unit types are described for the biceps muscle. These are fast units, slow units and intermediate units. The structure of muscle fibre and the physiological characteristics of muscle fibres belonging to each motor unit, have been correlated by using iontophoretic passage of Lucifer yellow into muscle fibres belonging to physiologically characterized motor units and their subsequent histological identification by the succinate dehydrogenase reaction. The three motor unit types correspond to slow muscle fibres, intermediate muscle fibres and two classes of fast muscle fibres.     

Characterization of swimming muscle in the Atlantic mackerel, Scomber scombrus L.

Both red and white muscle were removed from juvenile and adult Atlantic mackerel, Scomber scombrus L., for histochemical characterization of the muscle fibre types. Staining of white muscle for myosin ATPase, SDH, NADH diaphorase, GPDH and LDH revealed that these fibres are homogeneous. Red muscle was shown to be heterogeneous, of at least two fibre types recognizable on the basis of myosin ATPase staining with preincubation at a pH of 9·8. These two red types are dispersed throughout the red muscle and are present in both juveniles and adults. Red muscle is located both deep within the myotomes and as a superficial layer of muscle fibres. A third group of muscle fibres, intermediate in nature, was distinguished at the apex of the red muscle 'triangle,' between the epaxial and hypaxial muscle, using NADH diaphorase and myosin ATPase stains. This paper discusses the possibility that functionally different muscle fibres occur in the red swimming muscle of the Atlantic mackerel.     

A study of the peripheral innervation and muscle fibre types of Ictalurus nebulosus (Lesueur) and Ictalurus punctatus (Rafinesque)

The innervation pattern and fibre types of the axial musculature of two closely related catfish species with differing lifestyles, Ictalurus nebulosus (Lesueur) and I. punctatus (Rafinesque) were investigated. Both fish displayed the multiple innervation pattern in the red muscle. However, the white muscle of I. nebulosus demonstrated terminal innervation while I. puncrurus displayed multiply innervated white muscle fibres. Fibre typing utilizing histochemical techniques for glyco-gen, lipid, succinic dehydrogenase and glucose-6-phosphate dehydrogenase revealed the typical teleostean distribution of red, intermediate and white muscle fibres in both fish. Staining was greatest in the red muscle fibres and least in the white muscle fibres. The white muscle fibres of I. punctatus stained slightly more for lipid than the white fibres of I. nebulosus which may be correlated with a greater aerobic capacity related to lifestyle and possibly innervation.     

A histochemical and biometric comparison of the long peroneal muscle and its innervation in Gallus gallus and Coturnix c. japonica]

Following a previous comparison of the peroneus longus muscle of the quail and the starling, the present paper deals with a comparative study of this muscle in two birds of the order Gallinaceae, the quail and the bantam, bearing in mind certain data found in the starling. The study deals with the types of muscle fibres, their frequency in various parts of the muscle, their lipid content and their innervation. In the quail, two types of fibres are found, with a low and high lipid content respectively, while the bantam and starling have also a third, intermediate type. These intermediate fibres have a characteristically intermediate lipid content, peripherally situated nuclei and areas with a myofibrillar structure. The proportion of the two of three types of fibres varies with the species and in the distal and proximal parts of the muscle. The innervation of the peroneus longus muscle is different in the three species. In the quail, the two types of fibres have only one arboriform motor end-plate per fibre. In the bantam, the two types of homologous fibres also have only one motor end-plate, but with fewer arborifications. The intermediate fibre type, on the other hand, is innervated by several small nerve endings for each muscle fibre. This type of multiple innervation is also found in the starling. The peroneus longus muscle is thus functionally different in the two birds of the order Gallinaceae, whereas the rapid and slow system of innervation is found in the bantam and the starling. In the quail and the bantam, there is a strong positive correlation between the diameter of the muscle fibre and the longitudinal extent of the motor end-plate. This correlation is not marked in the starling. The characteristics of the innervation revealed by the cholinesterase activity concentrated in the synaptic grooves were confirmed by a direct study of the nerve fibres, using the Bielschowsky-Gros method. In the quail only 'en plaque' endings are found, while in the bantam and the starling both 'en plaque' and 'en grappe' endings are present. A parallel is drawn between the differences in function of the peroneus longus muscle and the characteristic features of its histology and innervation in the three species.     

Loss of muscle fibres in a landlocked dwarf Atlantic salmon population

Growth of fast myotomal muscle in teleosts involves the continuous production of muscle fibres until some genetically pre-determined length. The dwarf landlocked (Bleke) population of Atlantic salmon (Salmo salar L.) from Byglands-fjord, Southern Norway mature at about 25 cm fork length and reach a maximum size of only 30 cm in the wild. The maximum diameter (D(max)) of fast muscle fibres in 4-year-old Bleke salmon (25-28 cm fork length) was 118 microm and not significantly different from that found in immature migratory salmon of a similar size. In contrast no evidence for active fibre recruitment was found in the Bleke salmon, such that the maximum fibre number, FN(max), was only 21-30% of that reported in typical farmed and wild migratory populations, respectively. We hypothesise that, once established, the physiological consequences of the dwarf condition led to rapid selection for reduced fibre number, possibly to reduce the maintenance costs associated with ionic homeostasis.     

Fibre types in locomotory muscles of the cartilaginous fish Chimaera monstrosa

The cartilaginous fish Chimaera monstrosa swims slowly by means of pectoral fin movements, and fast by undulations of the tail. In order to compare the fibres in the corresponding muscles, they were studied by histochemistry and electron microscopy. Three fibre types were identified by microphotometry and morphometry. Most of the axial muscles are white fibres, containing little mitochondria and glycogen. Red fibres, with glycogen and about 5 % mitochondria constitute a thin sheet in the axial muscles, composed of one fibre layer only. Pink fibres, with intermediate amounts of glycogen and mitochondria are situated between these two types, but are often not covered by red fibres. Pectoral muscles contain numerous red and intermediate fibres, partially mixed, superficially, and white fibres deeper. Pectoral muscle red fibres contain about 25 % mitochondria, half of which are situated in subsarcolemmal accummulations. The sarcotubular system has T-tubules at the Z discs, and the terminal cisternae are partially divided by regularly spaced clefts.     

Changes in ATPase and SDH reactions of the rat extrafusal and intrafusal muscle fibres after preincubations at different pH

Summary The dependence of adenosine-triphosphatase (ATPase) and succinic dehydrogenase (SDH) histochemical reactions on the pH of the preincubation medium was studied in serial cross sections of 1- to 6-month-old rat extensor digitorum longus (EDL) and soleus (SOL) muscles.The use of a wide spectrum of pH values confirmed the previous results showing that: (1) according to their ATPase and SDH reactions 3 types of extrafusal muscle fibres, i.e., fast-twitch glycolytic (FG), fast-twitch oxidative-glycolytic (FOG) and slow-twitch oxidative (SO) and 3 types of intrafusal muscle fibres, i.e. typical and intermediate nuclear bag fibres and nuclear chain fibres were observed; (2) only acid preincubation (pH 4.35) is necessary to demonstrate the reversal of the ATPase reaction; while (3) alkali preincubation (pH 10.4) does not provide any new important information as compared with ATPase without preincubation. Furthermore, it was shown that: (4) fast-twitch muscle fibres exhibited high ATPase activity on preincubations at pH 4.9 to 10.4, slow-twitch fibres had very high ATPase activity on preincubation at pH 4.3 and 4.5; (5) after preincubation at pH 4.5 two types of FOG fibres were observed, differing in their ATPase activity; (6) in both muscles there were fibres with intermediate ATPase activity both after acid and/or alkali preincubations; (7) the intrafusal muscle fibres exhibited some specific characteristics when compared with extrafusal fibres.In contrast to the ATPase reactions, SDH activity was decreased equally, in both extra- and intrafusal fibres, with increasing acidity and alkality of the preincubation medium.     

Changes in ATPase and SDH reactions of the rat extrafusal and intrafusal muscle fibres after preincubations at different pH.

The dependence of adenosine-triphosphatase (ATPase) and succinic dehydrogenase (SDH) histochemical reactions on the pH of the preincubation medium was studied in serial cross sections of 1- to 6-month-old rat extensor digitorum longus (EDL) and soleus (SOL) muscles. The use of a wide spectrum of pH values confirmed the previous results showing that: (1) according to their ATPase and SDH reactions 3 types of extrafusal muscle fibres, i.e., fast-twitch glycolytic (FG), fast-twitch oxidative-glycolytic (FOG) and slow-twitch oxidative (SO) and 3 types of intrafusal muscle fibres, i.e. typical and intermediate nuclear bag fibres and nuclear chain fibres were observed; (2) only acid preincubation (pH 4.35) is necessary to demonstrate the reversal of the ATPase reaction; while (3) alkali preincubation (pH 10.4) does not provide any new important information as compared with ATPase without preincubation. Furthermore, it was shown that: (4) fast-twitch muscle fibres exhibited high ATPase activity on preincubations at pH 4.9 to 10.4, slow-twitch fibres had very high ATPase activity on preincubation at pH 4.3 and 4.5; (5) after preincubation at pH 4.5 two types of FOG fibres were observed, differing in their ATPase activity; (6) in both muscles there were fibres with intermediate ATPase activity both after acid and/or alkali preincubations; (7) the intrafusal muscle fibres exhibited some specific characteristics when compared with extrafusal fibres. In contrast to the ATPase reactions, SDH activity was decreased equally, in both extra- and intrafusal fibres, with increasing acidity and alkality of the preincubation medium.     

Evolution of the arthropod neuromuscular system. 1. Arrangement of muscles and innervation in the walking legs of a scorpion: Vaejovis spinigerus (Wood, 1863) Vaejovidae, Scorpiones, Arachnida

(1) The musculature of the walking legs is analysed with regard to both morphology and function in the scorpion, Vaejovis spinigerus (Wood, 1863) (Vaejovidae, Scorpiones, Arachnida), and selected other species. Conspicuous features are multipartite muscles, muscles spanning two joints, and partial lack of antagonistic muscles. The muscle arrangement is compared to that in the walking limbs of other Arthropoda and possible phylogenetic implications are discussed. (2). Histochemical characterisation of selected leg muscles indicates that these are composed of layers of slow, intermediate and fast muscle fibres. Anti-GABA immunohistochemistry shows that mainly the intermediate fibres receive innervation from putative inhibitory motoneurons. (3). Intracellular recording from muscle fibres reveals both excitatory and inhibitory muscle innervation. Individual muscle fibres may receive input from more than one inhibitory motoneuron, as indicated by different IPSP amplitudes. (4). The motoneuron supply of the leg muscles is analysed by retrograde fills of motor nerves. The general arrangement of leg motoneurons in the central nervous system and motoneuron anatomy conforms to the situation in pterygote insects and decapod crustaceans. For example, there are an anterior and a posterior group of leg motoneurons in each hemineuromere, and two contralateral somata near the ganglion midline. Between 12 and 20 motoneurons are found to supply each muscle. Most motoneuron cell bodies supplying a given muscle are arranged in a single cluster with a specific location.     

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.     

Histochemical localization of octopamine- and proctolin-sensitive adenylate cyclase activity in a locust skeletal muscle

A histochemical technique for the localization of adenylate cyclase activity has been applied to the extensor-tibiae muscle of the hindleg of the locust, Schistocerca gregaria to localise the sites of action of the modulatory compounds octopamine and proctolin. Octopamine-sensitive adenylate cyclase activity can be demonstrated in fast and intermediate type muscle fibres but not in the limited number of purely slow muscle fibres (3-6) in the fan region at the proximal end of the muscle. In contrast the latter fibres are the only ones in the muscle to exhibit proctolin-sensitive adenylate cyclase activity. In both cases the bulk of the reaction product is localised in the sarcoplasmic reticulum component of the dyads, with lesser amounts occurring beneath the sarcolemmal membrane, in the non-dyad sarcoplasmic reticulum and in the T-tubule system. The results are consistent with physiological data suggesting that proctolin, but not octopamine, mediates its effects on the myogenic rhythm of contraction and relaxation in this muscle by changing the levels of cyclic AMP in the small group of slow muscle fibres which act as the pacemaker for this rhythm.     

Ultrastructure of frog muscle fiber thick filaments at rest and during potassium contracture]

Isolated slow and intermediate frog muscle fibres were fixed in the rest state and under potassium contracture (50-100 mM KC1). The longitudinal and cross sections of two types of fibres were investigated. It was shown that at the rest the thick filaments of different fibres had similar length (1.6-1.65 mum), diameter (160-165 A) and the amount of subunits (12-13). Under potassium contracture the length of the thick filaments of both fibre types was shortened by 25-30% of the rest-length, the diameter of the slow fibres increased to 180-185 A, the diameter of the intermediate fibres to 200-220 A. The amount of subunits increased to 14-15 in slow fibres and to 17-18 in intermediate fibres. We believe that the ultrastructural changes observed in the thick filaments are a result of molecular transformation in these filaments, which seems to be important for maintaining the contracture.     

Muscle development in cultured blackspot seabream Pagellus bogaraveo: preliminary histochemical and immunohistochemical data on the fibre types

We studied muscle ontogeny and fibre type characteristics in the blackspot seabream, a new species for commercial aquaculture. Myosin ATPase and SDH histochemistry and immunohistochemistry were tested at different ontogenetic stages, using a panel of antibodies to myosin isoforms and parvalbumin. In general, deep white muscle was parvalbumin‐positive, and superficial 'red' muscle was parvalbumin‐negative at all ages examined. At 6 days of age (transition from endogenous to exogenous feeding) three layers of muscle fibres were observed with different antimyosin reactivities: superficial monolayer, presumptive slow red (present only as a small group of fibres adjacent to the lateral line nerve), and presumptive fast‐white (forming the bulk of the muscle). The superficial monolayer and presumptive slow fibres were positive for SDH. At 60 days of age (transition from live to artificial feeding) an additional fibre type was identified: a typical 'pink' or intermediate layer. In juveniles, the axial muscle consisted mainly of fast white fibres covered by a slow‐red layer and between them a pink layer. Surprisingly, the red layer could be resolved into two distinct types by myosin immunostaining. Red fibres were also present along the horizontal septum, near the notochord. Both red and white muscle layers showed a mosaic appearance, which was confirmed by ATPase reaction. The work was financed by British Council, CRUP, and FCT (PhD Grant SFRH‐BD‐14068–2003).     

Histochemical localization of octopamine- and proctolin-sensitive adenylate cyclase activity in a locust skeletal muscle

Summary A histochemical technique for the localization of adenylate cyclase activity has been applied to the extensortibiae muscle of the hindleg of the locust, Schistocerca gregaria to localise the sites of action of the modulatory compounds octopamine and proctolin. Octopamine-sensitive adenylate cyclase activity can be demonstrated in fast and intermediate type muscle fibres but not in the limited number of purely slow muscle fibres (3–6) in the fan region at the proximal end of the muscle. In contrast the latter fibres are the only ones in the muscle to exhibit proctolinsensitive adenylate cyclase activity. In both cases the bulk of the reaction product is localised in the sarcoplasmic reticulum component of the dyads, with lesser amounts occurring beneath the sarcolemmal membrane, in the non-dyad sarcoplasmic reticulum and in the T-tubule system. The results are consistent with physiological data suggesting that proctolin, but not octopamine, mediates its effects on the myogenic rhythm of contraction and relaxation in this muscle by changing the levels of cyclic AMP in the small group of slow muscle fibres which act as the pacemaker for this rhythm.