Changes in the cholinergic system of rat sciatic nerve and skeletal muscle following suspension-induced disuse

Muscle disuse-induced changes in the cholinergic system of sciatic nerve, slow-twitch soleus (SOL), and fast-twitch extensor digitorum longus (EDL) muscles were studied in rats. Rats with hind limbs suspended for 2 to 3 weeks showed marked elevation in the activity of choline acetyltransferase in sciatic nerve (38%), in the SOL (108%), and in the EDL (67%). Acetylcholinesterase (AChE) activity in the SOL increased 163% without changing the molecular forms pattern of 4S, 10S, 12S, and 16S. No significant (P greater than 0.05) changes in the activity and molecular forms pattern of AChE were seen in the EDL or in AChE activity of sciatic nerve. Nicotinic receptor binding of [3H]acetylcholine was increased in both muscles. When measured after 3 weeks of hind limb suspension the normal distribution of type I fibers in the SOL (87%) was reduced (to 58%) and a corresponding increase in types IIa and IIb fibers occurred. In the EDL no significant change in fiber proportion was observed. Muscle activity, such as loadbearing, appeared to have a greater controlling influence on the characteristics of the slow-twitch SOL muscle than on the fast-twitch EDL muscle.     

Contractile properties, fiber types, and myosin isoforms in fast and slow muscles of hyperactive Japanese waltzing mice

This study focuses on the effects of neuromuscular hyperactivity on the contractile properties, fiber type composition, and myosin heavy chain (MHC) isoform expression of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles in Japanese waltzing mice (JWM) of the C57BL/6J-v2J strain. The same properties were studied in the homologous muscle of control CBA/J mice (CM). In comparison to CM, the JWM exhibited (i) longer activity periods, prolonged bouts of running and a higher food intake, (ii) slower twitch and tetanic contractions of both EDL and SOL muscles, decreased cold and post-tetanic potentiation of the EDL, as well as increased cold and post-tetanic depressions of the SOL. Electrophoretic analyses of MHC isoform revealed a shift toward slower isoforms in both EDL and SOL muscles of JWM as compared to the homologous muscles of CM, namely, a shift from the fastest MHCIIb to the MHCIId/x isoform in the EDL muscle and a shift from MHCIIa to MHCI in the SOL muscle. The latter also contained a higher percentage of type I fibers and displayed a higher capillary density than the SOL muscle of CM. These findings show that the inherently enhanced motor activity of the JWM leads to fiber type transitions in the direction of slower phenotypes. JWM thus represent a suitable model for studying fast-to-slow fiber transitions under the influence of spontaneous motor hyperactivity.     

Rat alpha- and gamma-motoneuron soma size and succinate dehydrogenase activity are independent of neuromuscular activity level

The chronic level of neuromuscular activity, that is, activation and loading, strongly influences the morphological, metabolic, phenotypic, and physiological properties of skeletal muscles. The effects on the innervating motoneurons, however, are less established. We determined and compared the effects of 30 days of decreased activity (induced by a complete mid-thoracic spinal cord transection, ST) or near inactivity (induced by spinal cord isolation, SI) on the soma size and succinate dehydrogenase (SDH) activity of motoneurons innervating a predominantly slow ankle extensor (soleus) and a predominantly fast ankle flexor (tibialis anterior) muscle of adult rats. Soleus and tibialis anterior motoneuron pools were labeled retrogradely using nuclear yellow. The alpha- and gamma-motoneurons were classified based on soma size. Mean number of labeled motoneurons, and mean soma size and SDH activity for both alpha- and gamma-motoneurons were similar in control, ST, and SI rats. Compared to previous reports showing significant decreases in muscle fiber size and adaptations toward a "faster" metabolic profile following ST and SI, the results indicate that, unlike the muscles they innervate, the motoneurons are relatively unresponsive to chronic reductions in neuromuscular activity. The implication of these results is that mean size and SDH activity are independent of the number of action potentials generated by both alpha- and gamma-motoneurons and that even the absence of afferent input to the spinal cord has no influence on size and oxidative metabolic potential of the motoneuron soma.     

Electrophysiologic differences between mouse extensor digitorum longus and soleus

Miniature end-plate potentials (MEPPs) and indirectly elicited action potentials were recorded in vivo at 37°C from surface fibers of the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus (SOL) muscles of 3- to 4-month-old Bar Harbor 129 mice. The EDL MEPPs exhibited a significantly higher frequency, smaller amplitude, and shorter duration than the MEPPs of the SOL. Action potentials of EDL fibers exhibited a significantly greater amplitude and shorter duration than SOL fibers. A single stimulus elicited several action potentials from an EDL fiber but only one action potential from a SOL fiber. Fast- and slow-twitch muscle fibers can thus be identified and distinguished on the basis of these electrophysiologic parameters. There was no significant difference in resting membrane potentials between EDL and SOL fibers.     

Effects of denervation and immobilisation during development upon [3H]ouabain binding by slow- and fast-twitch muscle of the rat

The effect of innervation and of muscle inactivity upon the normal production of Na+-K+-ATPase sites, assayed by [3H]ouabain binding, in muscle surface membranes has been determined for the rat. In both slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles a large increase was found to occur in [3H]ouabain binding per unit weight of muscle over the first 3 weeks of life. Interruptions of development, brought about by fixation of muscles at different lengths at 5 days of age, had no significant effect upon [3H]ouabain binding by EDL. In contrast, fixation led to a decrease in binding in SOL. When fixed in a shortened position profound morphological changes occurred, although these were not apparent when SOL was fixed in a stretched position. Denervation of SOL at 5 days of age significantly reduced the age related increase in the density of [3H]ouabain binding, whilst denervation of EDL had little effect. It was concluded that normal development of SOL is dependent upon innervation and possibly the resulting muscle activity, whereas development of EDL was relatively independent of innervation.     

Muscle-specific beta-enolase concentrations after cross- and random innervation of soleus and extensor digitorum longus in rats

The concentration of beta-enolase, a highly specific marker of the skeletal muscle of rats, was determined in a slow-twitch muscle, the soleus (SOL) and a fast-twitch muscle, the extensor digitorum longus (EDL) after cross-innervation, random reinnervation, or denervation. The beta-enolase concentration is normally high in EDL and low in SOL. When the nerves entering into these muscles were cross-sutured, the beta-enolase concentration in EDL decreased and that in SOL increased to reach an almost equal value in 20 weeks and by the 35th week the SOL ultimately had a higher beta-enolase concentration than the EDL. When the sciatic nerve trunk was completely transected and sutured immediately, the beta-enolase concentration in EDL decreased and that of SOL increased; in 20 weeks SOL had a beta-enolase concentration similar to that of the EDL. When these muscles were denervated by cutting the sciatic nerve trunk, their beta-enolase concentrations were markedly lowered, but EDL still retained on the 12th week a beta-enolase value comparable to the normal SOL. Possible mechanisms behind the observed changes in beta-enolase concentration are discussed.     

Neural regulation of muscle acetylcholinsterase: effects of batrachotoxin and 6-aminonicotinamide.

Batrachotoxin (BTX), which causes increased Na+ permeability and blocks axoplasmic transport, or 6-aminonicotinamide (6-AN), which causes neuronal damage, was injected into the subarachnoid space of rat lumbar spinal cord. The activity of acetylcholinesterase (AChE) was measured in homogenates of the fast-twitch extensor digitorum longus (EDL) muscle and the slow-twitch soleus (SOL) muscle 10 days after injection. Both drug treatments significantly decreased AChE in EDL and SOL. Correlative electrophysiological measurements were made in intact EDL and SOL after injection of BTX or 6-AN. The results support the hypothesis that AChE in muscle is neurotrophically controlled.     

Regulation by exercise of the pool of G4 acetylcholinesterase characterizing fast muscles: opposite effect of running training in antagonist muscles

Fast muscles of rodents characteristically differ from their slow-twitch counterparts by exhibiting high levels of G4, i.e., the tetrameric acetylcholinesterase (AChE) molecular form. Converging evidence suggests that this additional G4 pool is specifically regulated by the type of activity actually performed by the muscle. This hypothesis was tested by studying the effect of a chronic increase in neuromuscular activity on the AChE content and distribution of molecular forms of functionally antagonist rat hindlimb muscles. They included the fast ankle extensors gastrocnemius (GAST) and plantaris (PL), the fast ankle flexors tibialis anterior (TA) and extensor digitorum longus (EDL), as well as the slow-twitch soleus (SOL). Neuromuscular activity was enhanced by subjecting the rats to a 12-week training program consisting of repeated sessions of prolonged endurance running on a rodent treadmill. This exercise regimen preferentially affected the G4 pool characterizing fast muscles which exhibited marked and opposite changes according to the functional role of the muscles. The amount of G4 was increased by more than 50% in the ankle extensors GAST and PL, which play a dynamic role, and reduced by about 40% in the ankle flexors TA and EDL, which exhibit a predominant tonic activity during running. The asymmetric forms A12 and A8 were slightly elevated in the fast muscles. In the case of the slow-twitch SOL, running training resulted in a small, nonspecific decrease in AChE content which affected most of the molecular forms. These data indicate that the size of the G4 pool characteristic of fast muscles depends on the type, dynamic or tonic, of activity actually performed. The present results support the conclusion that this G4 pool fulfills a specific and essential function, distinct from that of A12.     

Influence of temperature on isometric tension development in mouse fast- and slow-twitch skeletal muscles

The influence of temperature (range 35 to 20°C) on the isometric contractile properties of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles of the male C57 mice was studied in vitro, with direct stimulation. Cooling from 35 to 25°C resulted in an average twitch tension potentiation of 42% in EDL and 13% in SOL muscles. Further cooling to 20°C did not produce further potentiation in EDL muscles. The time-to-peak tension and the time to half-relaxation increased 2.4 to 3.2 times for a 10°C cooling in both muscles. The maximum tetanic tension was little changed in cooling from 35 to 30°C, but was depressed 16 to 19% in cooling to 20°C in both muscles. These results from mouse fast and slow muscles were compared with previously published data from muscles of the rat and the cat. The behaviors of the slow SOL muscles in the three species were found to be significantly different, but they could be explained on the basis of their muscle fiber-type composition.     

Slowing of fast-twitch muscle in the dystrophic mouse

Isometric twitch tension was measured in fast-twitch and slow-twitch muscles of normal and dystrophic ( ) mice in vivo. In dystrophic mice more than 6 months old the fast-twitch extensor digitorum longus (EDL) showed a prolongation of the time to peak tension as well as the time to relax to one-half peak tension ( ) compared with age-matched controls. In younger dystrophic mice (4 to 6 weeks) the time to peak tension was prolonged but not significantly so. This apparent “slowing” of dystrophic fast-twitch muscle was accompanied by a reduction in both cooling potentiation and post-tetanic potentiation toward values typical of slow-twitch muscle. Slow-twitch soleus muscle (SOL) of old mice was almost unaffected by the dystrophic process with regared to its contractile characteristics. However, there appeared to be a slight, but significant “speeding” of young dystrophic SOL compared with age-matched control muscles. This was apparent in reduced times to peak tension and half-relaxation as well as an enhanced cooling potentiation. We suggest that the altered contractile characteristics result from a change in some intrinsic property of the muscle fibers rather than from extrinsic factors such as the additional perimysial connective tissue seen in these muscles.     

Changes in isometric contractile properties of fast-twitch and slow-twitch skeletal muscle of C57BL/6J dy2J/dy2J dystrophic mice during postnatal development

Our primary aim was to determine if there exists a preferential involvement of the fast-twitch or slow-twitch skeletal muscle fibers in the dy2J/dy2J strain of murine dystrophy. The changes in the contractile properties of the slow-twitch soleus (SOL) and the fast-twitch extensor digitorum longus (EDL) muscles of normal and dystrophic mice were studied at 4, 8, 12, and 32 weeks of age. Isometric twitch and tetanus tension were decreased in the 4- and 8-week-old dystrophic EDL compared with controls, this situation being reversed in the older animals. At 12 weeks, the dystrophic EDL generated 15% more tetanic tension than normal EDL and by 32 weeks no significant difference was seen between normal and dystrophic EDL twitch or tetanus tension. By 8 weeks, dystrophic EDL exhibited a prolonged time-to-peak twitch tension (TTP) and half-relaxation time (1/2RT) of the isometric twitch which continued to 32 weeks. For the dystrophic SOL, decreased twitch and tetanus tension was observed from 4 to 32 weeks. At 8 and 12 weeks, TTP and 1/2RT of dystrophic SOL were prolonged. However, by 32 weeks there was no longer a significant difference seen in TTP or 1/2RT between normal and dystrophic SOL. Our results appear to indicate that a loss of the primary control which is determining the fiber composition of the individual muscles is occurring as the dystrophic process advances.     

Core myofibers and related alterations induced in rats' soleus muscle by immobilization in shortened position

Experimental induction of core myofibers by tenotomy or local tetanus suggests that mechanical factors such as muscle tension loss, shortening or immobilization may play a role in core fiber formation. To test this hypothesis, we investigated the morphologic alterations induced in soleus (SOL) and extensor digitorum longus (EDL) muscles following immobilization of rats' hindlimb in various positions. The SOL and EDL muscles were immobilized in either shortened or lengthened state by applying wire-meshed plaster cast for 1, 2 and 3 weeks. The muscles were dissected out, measured, weighed and examined by histochemistry and electron microscopy. Gross atrophy was noted in all muscles but was greatest in shortened SOL. The SOL atrophy was diffuse and associated with relative increase in type 2 fibers. In EDL, the atrophy selectively involved fibers with low oxidative enzyme activity. Core myofibers were seen mainly in shortened SOL and consisted of myofibrillar derangement, loss of myofilaments and streaming of Z bands. The preferential involvement of shortened SOL (tonic, fatigue-resistant, slow-twitch muscle) suggests that the functional length, loss of tension subsequent to shortening and intrinsic biochemical properties of the muscle are important in core fiber formation.     

Analysis of dystrophin in fast- and slow-twitch skeletal muscles from mdx and dy2J mice at different ages

Muscles from mdx, control, and dy2J/dy2J mice at different ages were analyzed for dystrophin in an attempt to relate the chronology of the protein expression with the final phenotypes in regenerated, normal, and dystrophic muscle, respectively. Immunostaining and gold staining of electrophoresis gels were carried out in the investigation. At 5, 25, and 219 days of age, control muscles exhibited dystrophin bands in both the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus (SOL) muscles. Muscles from the mdx mice at comparable ages (8, 28, and 217 days) never exhibited bands for dystrophin, although titin, nebulin, myosin, and other protein bands were present at intensities comparable to those in control muscles. The dystrophin band was present in both the EDL and SOL from dy2J/dy2J dystrophic mice. As indicated by the present study, the dystrophin deficiency from mdx tissue is not transient. This suggests that dystrophin is not necessary for the success of mdx muscle regeneration.     

Isometric contractile properties of fast- and slow-twitch muscles in normal and spastic mice

Isometric contractile properties of the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles of 24 male C57 mice were studied in vitro at 35°C. Ten animals exhibited the hereditary movement disorder known as spasticity. EDL muscles of spastic and normal mice developed similar specific tensions and they had similar twitch tension:tetanic tension ratios. The time-to-peak tension and the time to half relaxation in the twitch of spastic EDL muscles were, on average, slightly longer. SOL muscles of spastic and normal mice also developed similar specific tensions and their twitch contractions had similar times to half-relaxation. The time-to-peak tension in the twitch and the twitch tension: tetanic tension ratio of spastic SOL muscles were, on average, higher than in normal SOL. The significance of our findings remains somewhat uncertain.     

3H]Ouabain binding in normal and dystrophic mouse skeletal muscles and the effect of age

The numbers of Na+-K+ ATPase sites in skeletal muscles of normal and dystrophic mice between 3 and 17 months of age have been estimated using [3H]ouabain binding assays. In normal mice, at all ages, slow twitch muscle, soleus (SOL), bound significantly more [3H]ouabain than fast-twitch muscle, extensor digitorum longus (EDL). [3H]Ouabain binding did not alter in either SOL or EDL from normal mice over the age range studied. The numbers of Na+-K+ ATPase sites did alter in muscles taken from dystrophic mice (C57BL/6J dy2J/dy2J). In EDL there was an increase and in SOL a decrease in [3H]ouabain binding. This may be related to a change in muscle fibre metabolism from glycolytic to oxidative or to an altered activity pattern. Increasing age resulted in a progressive reduction in [3H]ouabain binding of both SOL and EDL from dystrophic mice. Part of this reduction may be only apparent and due to an increase in connective tissue composition of dystrophic muscles. A limited study of muscles from neonate dystrophic mice indicated that abnormal [3H]ouabain binding was not present in EDL before two weeks of age.     

EFFECTS OF DEXAMETHASONE ON FIBRE SUBTYPES IN RAT MUSCLE

Livingstone I., Johnson M.A. & Mastaglia F.L. (1981) Neuropathology and Applied Neurobiology 7, 381–398 Effects of dexamethasone on fibre subtypes in rat muscle The extent to which dexamethasone treatment produced atrophy of fast-twitch (EDL) and slow-twitch (SOL) muscles in the rat was investigated. The mean weight of steroid-treated EDL muscles was decreased as compared to normal, whereas SOL muscles from normal and dexamethasone-treated animals showed no significant difference. Muscle fibre diameters also showed comparatively minor changes in SOL, which consists of Type 1 (slow oxidative) and Type 2A (fast oxidative/glycolytic) fibres. Rat EDL contains, in addition to Type 1 and Type 2A fibres, two sub-populations of fast glycolytic fibres (Types 2B and 2B'). These fibre types showed the most severe degree of atrophy both after dexamethasone treatment and after denervation. The mean ratio of the weights of denervated to innervated EDL muscles was lower in steroid-treated rats than in normal animals suggesting that the atrophy produced by steroid treatment in conjunction with denervation was more than simply additive. Analysis of the proportions of histochemical fibre types in SOL and EDL showed that dexamethasone treatment produced no major alterations in the fibre type constitution of these muscles. However, further histochemical studies showed that there was relatively severe impairment of myophosphorylase activity in Type 2B' (fast glycolytic) fibres as compared to other fibre types; conversely Type 1 fibres frequently contained increased myophosphorylase. Levels of β-hydroxybutyrate dehydrogenase were low in both normal and steroid-treated EDL but high in SOL which also showed higher general oxidative activity. It is suggested that the particular susceptibility of fast glycolytic fibres to atrophy as a result of steroid treatment may be linked to: 1 the relatively severe reduction of myophosphorylase activity in these fibres and 2 their comparative inability to utilize alternative energy sources, especially substrates derived from free fatty acids.     

Relationship between soma diameter and oxidative enzyme activity of alpha-motoneurons.

In order to determine whether there is a relationship between soma diameter and oxidative enzyme activity of alpha-motoneurons, we investigated the alpha-motoneurons innervating the different portions within a rat gluteus medius muscle. Two fluorescent neuronal tracers, Nuclear yellow and Fast blue, were used for labeling motoneurons innervating the deep (predominance of oxidative fibers) and superficial (predominance of non-oxidative fibers) portions of the muscle. An inverse relationship between soma diameter and oxidative enzyme activity was not seen in the motoneuron pool innervating either the deep or superficial portions. When the two portions were taken together, however, the inverse relationship was seen. The inverse relationship seemed to be demonstrated in a motoneuron pool which intermingled motoneurons innervating oxidative fibers and motoneurons innervating non-oxidative fibers. These results suggest that the oxidative enzyme activity of alpha-motoneurons is not correlated strictly with their soma size. We consider that the oxidative enzyme activity of alpha-motoneurons is correlated with oxidative enzyme activity of the muscle unit.     

Abnormal distribution of proteins in the soleus and extensor digitorum longus of dystrophic mice

Proteins of the whole muscle homogenates of the slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) of normal and dystrophic C57BL/6J mice at 4, 8, 12, and 32 weeks of age were resolved on polyacrylamide isoelectric focusing gels. Gels of the normal SOL proteins at all ages contained two bands specific to SOL and not represented in EDL. Gels of normal EDL contained three bands highly amplified in EDL but barely detectable in SOL. The distribution of proteins in dystrophic SOL was abnormal at all age groups studied due, in part, to a decrease in the proportion of SOL-specific proteins relative to other proteins in the muscle. The distribution of proteins in dystrophic EDL appeared abnormal first at 12 weeks due to a decrease in the relative proportion of EDL-amplified proteins. Due to these and other changes, at 32 weeks the dystrophic SOL and EDL were almost indistinguishable on the basis of their proteins' distributions.     

Satellite cells in slow and fast rat muscles differ in respect to acetylcholinesterase regulation mechanisms they convey to their descendant myofibers during regeneration

The hypothesis of satellite cell diversity in slow and fast mammalian muscles was tested by examining acetylcholinesterase (AChE) regulation in muscles regenerating (1) under conditions of muscle disuse (tenotomy, leg immobilization) in which the pattern of neural stimulation is changed, and (2) after cross-transplantation when the regenerating muscle develops under a foreign neural stimulation pattern. Soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat were allowed to regenerate after ischemic-toxic injury either in their own sites or had been cross-transplanted to the site of the other muscle. Molecular forms of AChE in regenerating muscles were analyzed by velocity sedimentation in linear sucrose gradients. Neither tenotomy nor limb immobilization significantly affected the characteristic pattern of AChE molecular forms in regenerating SOL muscles, suggesting that the neural stimulation pattern is probably not decisive for its induction. During an early phase of regeneration, the general pattern of AChE molecular forms in the cross-transplanted regenerating muscle was predominantly determined by the type of its muscle of origin, and much less by the innervating nerve which exerted only a modest modifying effect. However, alkali-resistant myofibrillar ATPase activity on which the separation of muscle fibers into type I and type II is based, was determined predominantly by the motor nerve innervating the regenerating muscle. Mature regenerated EDL muscles (13 weeks after injury) which had been innervated by the SOL nerve became virtually indistinguishable from the SOL muscles in regard to their pattern of AChE molecular forms. However, AChE patterns of mature regenerated SOL muscles that had been innervated by the EDL nerve still displayed some features of the SOL pattern. In regard to AChE regulation, muscle satellite cells from slow or fast rat muscles convey to their descendant myotubes the information shifting their initial development in the direction of either slow or fast muscle, respectively. The satellite cells in fast or slow muscles are, therefore, intrinsically different. Intrinsic information is expressed mostly during an early phase of regeneration whereas later on the regulatory influence of the motor nerve more or less predominates. © 1994 Wiley-Liss, Inc.     

Dendritic distributions of motor neurons innervating fast and slow muscles of the hind limb of rats

The purpose of the study is to determine the difference, if any, between the dendritic distributions (field) of slow and fast muscle motoneurons. A cholera toxin subunit conjugated horseradish peroxidase (CTHRPs can label retrogradely the dendrites of motoneurons and was injected into a slow muscle (soleus, SOL) and a fast muscle (extensor digitorum longus, EDL) of the hind limbs of rats. The results show that the dendrites of EDL motoneurons project into Rexed lamina VI, VII, and IX while that of SOL into V, VI, VII, VIII, and IX of the gray matter of lumbosacral spinal cord. SOL motoneurons also have many long dendrites extending into ventrolateral and ventral white matter while such extensions from EDL motoneurons are short and scanty. Thus, the dendritic field of SOL motoneurons is wider than that of EDL motoneurons. It is suggested that this difference may be related to the fact that SOL is a slow, fatigue resistant muscle and is more often used than EDL which is fast, fatigable muscle and less often used in routine muscle activity.