Scanning electron microscope study of neuromuscular junctions in different muscle fiber types in the zebra finch and rat

Examination by scanning electron microscopy revealed differences between neuromuscular junctions in the muscle fibers of the zebra finch (bird) and rat. The neuromuscular junctions between the anterior and posterior latissimus dorsi muscles of the zebra finch were compared. The junctions of the former, exclusively slow tonic fibers, were small and numerous along the long axis of a single muscle fiber. The synaptic depressions per junction were few. The junctions of the latter, exclusively fast twitch fibers, were large and consisted of more synaptic depressions than the former. Junctional folds were occasionally found in some depressions. The neuromuscular junctions between the extensor digitorum longus and soleus muscles of the rat were also compared. The former consisted almost entirely of fast twitch muscle fibers, whereas the latter consisted of both slow twitch fibers (75%) and fast twitch fibers (25%). The junctions in the extensor digitorum longus muscle were almost all labyrinthine gutters containing exclusively slit-like junctional folds. In the soleus muscle, two types of junctions were observed. One type was similar to that of the extensor digitorum longus muscle; the other was characterized by labyrinthine gutters containing sparse, narrow slit-like and pit-like junctional folds. We suggest from these structural differences of the subneural apparatuses that the junction of the fast twitch muscle is characterized by the subneural apparatus containing numerous slit-like junctional folds, and that of the slow twitch muscle fiber characterized by the apparatus containing sparse, narrow slit-like and pit-like junctional folds.     

Relative capabilities of sarcoplasmic reticulum in fast and slow mammalian skeletal muscles.

1. The calcium uptake capabilities of the sarcoplasmic reticulum (SR) of the fat-twitch muscles extensor digitorum longus (EDL) and tibialis anterior (TA) of the rat and the extensor digitorum longus of the cat have been compared with the same capabilities of the slow-twitch soleus muscles of the rat and cat. 2. For the ra the Vmax values of sarcoplasmic reticulum from tibialis anterior, extensor digitorum longus and from soleus muscles were 50, 51, and 10 micronmole Ca2+/g per minute, respectively. 3. For the extensor digitorum longus and soleus muscles of the cat the Vmax values were 34 and 5-6 micronmole Ca2+/g per minute, respectively. 4. These data were compared with mechanical data as reported in the literature for the same muscles. The relative calcium uptake capabilities of sarcoplasmic reticulum from slow and fast muscles corresponded closely to the relative rates of relaxation of these muscles.     

Contractile properties, fatiguability and glycolytic metabolism in fast- and slow-twitch rat skeletal muscles of various temperatures

The influence of muscle temperature (28 and 36 degrees C) on fatiguability and glycolytic metabolism was studied during 5 min of intermittent stimulation of motor nerves of the tibialis anterior, extensor digitorum longus (fast-twitch) and soleus (slow-twitch) muscles in the rat at 100 Hz (200 ms per s). The decline in isometric tension was not affected by muscle temperature either in fast- or in slow-twitch muscles. In fast-twitch muscles the utilization of glycogen during stimulation was the same at 28 and 36 degrees C, while in the soleus muscle it was lower at 28 degrees C. The concentration of glucose-6-phosphate immediately after stimulation was higher in the muscles at 28 degrees C than in those at 36 degrees C, whereas no difference in lactate concentration was found between the two temperature groups. These observations indicate that compared with the rate at 36 degrees C, the rate of glycogenolysis at 28 degrees C is unchanged in fast-twitch, but decreased in slow-twitch muscle. This might imply increased economy of ATP turnover during contraction in the soleus muscle at 28 degrees C.     

Demonstration of glutamate-like immunoreactivity at rat neuromuscular junctions by quantitative electron microscopic immunocytochemistry

Motor nerve terminals and adjacent structures in the extensor digitorum longus and soleus muscles of young adult rats were examined for their content of glutamate by means of quantitative, electron microscopic immunocytochemistry employing colloidal gold particles as markers. The level of glutamate immunoreactivity was stronger in the extensor digitorum longus terminals than in the soleus terminals. In both muscles the glutamate immunolabelling was stronger in the nerve terminals than in the synaptic clefts and the postsynaptic tissue separating the secondary clefts, but the differences were larger in the extensor digitorum longus than in the soleus muscle. The myofibrils of the soleus muscle were more densely labelled than those in the extensor digitorum longus muscle: The level of immunoreactivity was high in the Schwann cells of both muscles. By comparing the labelling intensity of motor nerve terminals with that of muscle fibres and hippocampal mossy fibres (compartments that have been analysed previously with respect to their glutamate content), the mean concentration of fixed glutamate in the extensor digitorum terminals was estimated to be in the range of 10–20 mmol/l. An association of glutamate immunoreactivity with synaptic vesicles was demonstrated in the most strongly labelled terminals. Whether these epitopes were localized in the interior of the vesicles or at their external surface could not be resolved with the present technique. These data indicate that motor nerve terminals contain glutamate, and that the enrichment of this amino acid is more pronounced in the terminals of the extensor digitorum longus muscle (a fast muscle) than in those of the soleus muscle (a slow muscle). A possible modulatory or trophic role of glutamate in the mammalian neuromuscular junction should be considered.     

Topographic comparison of neuromuscular junctions in mouse slow and fast twitch muscles

The neuromuscular junctions of mammalian slow and fast twitch muscles are activated differently in vivo and show corresponding physiological differences in vitro, but the structural basis or consequences of these differences are relatively unexplored. Therefore, neuromuscular junctions of mouse fast (extensor digitorum longus) and slow (soleus) twitch muscles were compared by use of new scanning and light microscopy techniques. In both muscles, the endplate appeared as an elliptical area raised to a variable extent above the surrounding sarcolemma and containing the primary clefts. In most soleus endplates, this raised surface area was considerably higher and wider and about three times larger than in extensor digitorum longus. In addition, the primary cleft area was about two-fold greater in soleus than in extensor digitorum longus, even though cleft length was the same. The primary clefts formed either an elliptical shape along the outer margin of the endplate with inward-directed branches or a group of relatively rectilinear dendritic branches orthogonally oriented to one another. The latter type was most frequent in soleus and the elliptical type in extensor digitorum longus. Corresponding patterns of nerve terminal arborizations were seen by light microscopy. Although nerve terminal areas were the same in fast and slow muscles, in the former, numerous diverticulae significantly increased the length of the nerve terminal outline. The possible physiological significance of the different synaptic structure of slow and fast muscle is discussed.     

Effects of perchlorate on myofibrillar calcium sensitivity in rat skinned skeletal muscles

The effects of perchlorate (1–20 mm ) on myofibrillar calcium responsiveness have been tested in Triton X-100-skinned fibre bundles from rat soleus (slow-twitch) and extensor digitorum longus (fast-twitch) skeletal muscles. In extensor digitorum longus and soleus, perchlorate dose-dependently shifted the pCa (-log[Ca²⁺])/tension relationship towards lower free calcium concentration (sensitizing effect) and maximal tension was unchanged. The degree of sensitization was greater in extensor digitorum longus than in soleus bundles. Reversibility after exposure to 12 mm perchlorate was complete in soleus but not in extensor digitorum longus muscles. In fact, the ‘return’ pCa/tension relationship in extensor digitorum longus was shifted to higher free calcium concentration (desensitizing effect) compared with control. Perchlorate (12 mm ) also enhanced myofibrillar calcium responsiveness of frog semitendinosus skinned skeletal fibres. Assuming a passive distribution of perchlorate across the sarcolemma, this sensitizing effect is probably not involved in perchlorate-induced potentiation of contractile responses of intact muscles and thereby supports the specificity of perchlorate as an agonist of the excitation/calcium release sequence in skeletal muscle fibres.     

Postnatal development of rat motor nerve terminals

Summary The nerve terminals of neuromuscular junctions in the rat diaphragm, extensor digitorum longus muscle and soleus muscle have been studied in animals between 3 weeks and 2.5 years of age using methylene blue stain and light microscopy. Dimensions, structure and organization of the nerve terminals were shown to change during life at various rates in different muscles and postnatal periods. The area and length of the terminals increase in all three muscles until young adult age. Later these dimensions continue to increase in the extensor digitorum longus and soleus muscles. In the diaphragm only the length increases, and this occurs late in adult life. The area also increases in relation to the diameter of the corresponding muscle fiber. Adult soleus terminals are more elongated than terminals in the diaphragm and extensor digitorum longus muscle. During adult life the extension of nerve terminals in relation to muscle fiber length increases in the extensor digitorum longus and soleus muscles, but is almost unchanged in the diaphragm. The nerve terminal branches are mainly coarse and irregular in young animals, but possess varying numbers of varicosities in adult animals. The number of varicosities is high in the extensor digitorum longus muscle and low in the diaphragm. In old animals the number of varicosities tends to be reduced. With increasing age the nerve terminal branches become organized in distinct groups with increasing distance between the groups. This is prominent in the soleus.     

Ultrastructural studies of young and old mouse neuromuscular junctions

Summary The ultrastructure of the neuromuscular junction of young and old male CBF-1 mice was analysed both qualitatively and quantitatively. The age-related findings were similar in both the phasic extensor digitorum longus muscle and the tonic soleus muscle but more pronounced in the latter. Presynaptic terminals of old mice compared to young showed decreases in nerve terminal area, mitochondria and synaptic vesicles, but increases in smooth endoplasmic reticulum, coated vesicles, cisternae, microtubules and probably neurofilaments. On the postsynaptic side there were increases in complexity of junctional folds and subsarcolemmal vesicles, and the appearance of lipofuscin deposits. Occasional denervated postsynaptic regions were encountered in old neuromuscular junctions, but the predominant characteristics of aging changes were not those of denervation. Rather, a unique and uniform process involving most of the population of nerve terminals, possibly of physiologically adaptive significance, appears to occur with age in both phasic and tonic limb muscles.     

Changes produced by chronic denervation in the temperature-dependent isometric contractile characteristics of rat fast and slow twitch skeletal muscles.

1. Influence of temperature (range 20-35 degrees C) on the isometric contractile properties of normal and chronically denervated fast-twitch extensor digitorum longus and slow-twitch soleus muscles of the rat have been studied in vitro. 2. The times to peak twitch tension of denervated muscles were longer than those of normal in both types of muscle. The denervated muscles were, however, identifiable as 'fast-twitch' or 'slow-twitch' throughout the entire temperature range. 3. The twitch tension/tetanic tension ratios of the denervated muscles at 35 degrees C were significantly higher than those of the normal muscles. 4. The twitch tension of the normal extensor digitorum longus muscles increased whereas that of the normal soleus muscles decreased with cooling from 35 to 20 degrees C. Such a qualitative difference did not exist between the denervated extensor digitorum longus and soleus muscles. The twitch tensions of both denervated muscles decreased with cooling. 5. Cooling to 20 degrees C resulted in a greater fall in tetanic tension in the denervated than in the normal muscles.     

Targeting functional subtypes of spinal motoneurons and skeletal muscle fibers in vivo by intramuscular injection of adenoviral and adeno-associated viral vectors

We report that functional subtypes of spinal motoneurons and skeletal muscle fibers can be selectively transduced using replication-defective adenoviral (ADV) or adeno-associated (AAV) viral vectors. After intramuscular injection in adult rodents, ADV vectors transduced both fast-twitch and slow-twitch skeletal muscle fibers. Intramuscular injection of ADV vectors also caused transduction of spinal motoneurons and dorsal root ganglion cells. However, only neurons innervating the injected muscle were transduced, as shown by co-injection of a retrograde axonal tracer. In adult male rats it is therefore possible to transduce fast or slow spinal motoneurons and muscle fibers selectively since in these animals, the extensor digitorum longus and soleus muscles contain almost exclusively fast or slow motor units, respectively. In rats, AAV vectors transduced muscle fibers in the predominantly fast extensor digitorum longus but not in the predominantly slow soleus muscle. We did not observe any transduction of spinal motoneurons following intramuscular injection of AAV vectors. These results show that physiologically and clinically important subpopulations of cells in the neuromuscular system can be selectively transduced by viral vectors.     

Effect of thyroid hormones on acetylcholinesterase mRNA levels in the slow soleus and fast extensor digitorum longus muscles of the rat

In the rat, the level of acetylcholinesterase messenger RNA in the typical slow soleus muscles is only about 20-30% of that in the fast extensor digitorum longus muscles. The expression of contractile proteins in muscles is influenced by thyroid hormones and hyperthyroidism makes the slow soleus muscle faster. The influence of thyroid hormones on the levels of acetylcholinesterase messenger RNA level in the slow soleus and fast extensor digitorum longus muscle of the rat was studied in order to examine the effect of thyroid hormones on muscle acetylcholinesterase expression. Hyperthyroidism was induced in rats by daily thyroid hormone injection or thyroid hormone releasing tablet implantation. Hind-limb suspension was applied to produce muscle unloading. Muscle denervation or reinnervation was achieved by sciatic nerve transection or crush. Acetylcholinesterase messenger RNA levels were analyzed by Northern blots and evaluated densitometrically. Hyperthyroidism increased the levels of acetylcholinesterase messenger RNA in the slow soleus muscles close to the levels in the fast extensor digitorum longus. The effect was the same in the unloaded soleus muscles. Acetylcholinesterase expression increased also in the absence of innervation (denervation), in the presence of changed nerve activation pattern (reinnervation), and under enhanced tonic neural activation of the soleus muscle (electrical stimulation). However, the changes were substantially smaller than those observed in the control soleus muscles. Enhancement of acetylcholinesterase expression in the soleus muscles by the thyroid hormones is, therefore, at last in part due to hormonal effect on the muscle itself. On the contrary, increased level of the thyroid hormones had no influence on acetylcholinesterase expression in the normal fast extensor digitorum longus muscles. However, some enhancing influence was apparent whenever the total number of nerve-induced muscle activations per day in the extensor digitorum longus muscle was increased. Thyroid hormones seem to be an independent extrinsic factor of acetylcholinesterase regulation in the slow soleus muscle.     

The influence of secretin on the secretion of pepsin in response to acid stimulants in the anaesthetized cat.

1. The problem of selectivity during reinnervation of skeletal muscle fibres was investigated in the rat using the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus muscles and their nerves. 2. After an operation on these nerves permitting them to compete for reinnervation of one or the other muscle (hereafter called Y-union), virtually the total isometric tetanic tension of EDL muscle could be elicited by stimulating the EDL nerve, while stimulating the soleus nerve yielded little or no tension. In the case of the soleus muscle, stimulation of either nerve elicited about half of the total isometric tetanic tension. 3. During the course of reinnervation of these muscles in non-competitive situations, the time course of increase in the ratio of tension elicited by nerve stimulation to that by direct stimulation was slower in the case of soleus nerve reinnervating EDL muscle, compared with cross-reinnervation in the reverse direction or reinnervation of each muscle by its own nerve. 4. Crushing the common peroneal nerve 12 days after a Y-union in an attempt to retard the EDL nerve did not favour reinnervation of the EDL by soleus nerve, but crushing the nerve again or just once at 1 month after the original operation produced substantial partial reinnervation of the EDL by the soleus nerve. 5. It is concluded that soleus nerve fibres form functioning neuromuscular synapses on EDL muscle fibres only with difficulty. The pattern of reinnervation reveals characteristic differences between fast-twitch and slow-twitch muscles on the one hand and between their respective nerves on the other.     

The distal hindlimb musculature of the cat. Patterns of normal use

Chronic recordings were made of electromyographic (EMG) activity, tension, and length of distal hindlimb muscles in six cats performing a variety of normal motor tasks. Muscles studied thoroughly or in part were medial gastrocnemius, lateral gastrocnemius, plantaris, soleus, flexor digitorum brevis, flexor digitorum longus, flexor hallucis longus, tibialis posterior, tibialis anterior, extensor digitorum longus, peroneus longus, and peroneus brevis. Postural and locomotor activities were examined, as well as jumping, landing, scratching, and paw shaking. In general, muscles could be assigned to traditional groupings (e.g. extensor, flexor) related to the demands of the motor task. Patterns of muscle activity were most often consistent with current understanding of muscle mechanics and neural coordination. However, purely functional distinctions between flexor digitorum longus and flexor hallucis longus ("anatomical synergists") were made on the basis of activity patterns. Likewise, the activity of plantaris and flexor digitorum brevis, which are attached in series, was differentiated in certain tasks. The rhythmical oscillatory patterns of scratching and paw shaking were found to differ temporally in a manner consistent with the limb mechanics. In several cases, mechanical explanations of specific muscle activity required length and force records, as well as EMG patterns. Future efforts to study motor patterns should incorporate information about the relationships between muscle activation, tension, length and velocity.     

The distal hindlimb musculature of the cat

Summary Chronic recordings were made of electromyographic (EMG) activity, tension, and length of distal hindlimb muscles in six cats performing a variety of normal motor tasks. Muscles studied thoroughly or in part were medial gastrocnemius, lateral gastrocnemius, plantaris, soleus, flexor digitorum brevis, flexor digitorum longus, flexor hallucis longus, tibialis posterior, tibialis anterior, extensor digitorum longus, peroneus longus, and peroneus brevis. Postural and locomotor activities were examined, as well as jumping, landing, scratching, and paw shaking. In general, muscles could be assigned to traditional groupings (e.g. extensor, flexor) related to the demands of the motor task. Patterns of muscle activity were most often consistent with current understanding of muscle mechanics and neural coordination. However, purely functional distinctions between flexor digitorum longus and flexor hallucis longus (anatomical synergists) were made on the basis of activity patterns. Likewise, the activity of plantaris and flexor digitorum brevis, which are attached in series, was differentiated in certain tasks. The rhythmical oscillatory patterns of scratching and paw shaking were found to differ temporally in a manner consistent with the limb mechanics. In several cases, mechanical explanations of specific muscle activity required length and force records, as well as EMG patterns. Future efforts to study motor patterns should incorporate information about the relationships between muscle activation, tension, length and velocity.Abbreviations EDL extensor digitorum longus - FDB flexor digitorum brevis - FDL flexor digitorum longus - FHL flexor hallucis longus - LG lateral gastrocnemius - MG medial gastrocnemius - PB peroneus brevis - PL peroneus longus - PLT plantaris - SOL soleus - TA tibialis anterior - TP tibialis posterior Limbs A ankle - K knee - LF left forelimb - LH left hindlimb - RF right forelimb - RH right hindlimb Step Cycle Phases E₁ first extension, late swing phase prior to footfall - E₂ second extension, early stance phase - E₃ third extension, late stance phase - F flexion, early swing phase     

Physiological properties and pattern of innervation of regenerated muscles in the rat

The regeneration of fast and slow muscles was compared following "mincing" and replacement into their own or alien muscle bed. At intervals varying from 2 to 9 weeks the tension developed by the regenerated muscles was assessed and compared to that developed by the muscles from the contralateral unoperated side. This parameter was then taken as an indication of recovery. The regenerated muscles never developed more than half of the tension of the control muscles. Muscles regenerated in the bed of extensor digitorum longus became fast-twitch muscles and muscles regenerated in the bed of soleus became slow-twitch muscles, no matter whether they originated from an extensor digitorum longus or soleus "mince". The regeneration of the muscle tissue in the place of extensor digitorum longus developed better than in the place of soleus. The pattern of innervation of the regenerated muscles was analysed using a combined cholinesterase silver stain. Many of the regenerated fibres had more than one end plate and some end plates more than one axon terminal. These results show that in adult animals muscle redevelopment can occur, but only to a limited extent. Moreover, on reinnervation of regenerated muscle fibres the axons do not assume their original pattern of innervation.     

Effects of nerve cross-union on rat intracellular potassium in fast-twitch and slow-twitch rat muscles

1. Electrolytes of normal, self-innervated and cross-innervated extensor digitorum longus and soleus muscles of rats have been determined.2. [K](i) was 173 m-equiv/l. for both normal and self-innervated extensor digitorum longus. In cross-innervated extensor digitorum longus it was reduced to 159 m-equiv/l.3. For normal and self-innervated soleus, [K](i) was 150 m-equiv/l. and 154 m-equiv/l. respectively. In cross-innervated soleus it was increased to 182 m-equiv/l.4. The content and distribution of most other electrolytes of cross-innervated soleus, as well as its weight, were not significantly different from those of controls. On the other hand, cross-innervated extensor digitorum longus weighed about half as much as controls and contained markedly elevated Na(+), Cl(-) and extrafibre water and reduced non-collagenous protein and intrafibre water.5. It is concluded that [K](i) of fast-twitch and slow-twitch muscle fibres are under neural regulation. Possible mechanisms for this regulation are discussed.     

Late reinnervation of the rat soleus muscle is differentially suppressed by chronic stimulation and by ectopic innervation

Soleus (SOL) and extensor digitorum longus (EDL) muscles in adult rats were kept denervated for 2 months by four repeated freezes at 2-week intervals of the sciatic nerve. Reinnervation was studied in the absence or presence of chronic muscle stimulation, starting 1 month before reinnervation began. In addition, reinnervation was studied in SOL muscles where a previously transplanted fibular (FIB) nerve had formed ectopic neuromuscular junctions outside the original endplate area. After repeated freezes only, reinnervation was complete judged by tension measurements and histochemical examinations in SOL (n = 7) and EDL (n = 8) muscles. In directly stimulated muscles reinnervation was incomplete, and the force tensions evoked from indirect stimulation was on average 87 (n = 5) and 82% (n = 5) of direct muscle stimulation in SOL and EDL muscles, respectively. Of ectopically innervated SOL muscle fibres, only 26% became reinnervated in 12 muscles. Denervation and reinnervation increased the number of muscle fibres in stimulated (n = 4) and unstimulated (n = 5) EDL muscles by 18 and 15%, respectively. In stimulated (n = 4) and unstimulated (n = 7) SOL muscles, on the other hand, the number of muscle fibres remained normal. The stronger suppression of reinnervation in ectopically reinnervated compared to chronically stimulated SOL fibres indicates that reinnervation can also be suppressed by activity independent influences from the foreign nerve.     

Effects of adrenaline on excitation-induced stimulation of the sodium-potassium pump in rat skeletal muscle

Experiments were performed on isolated rat soleus and extensor digitorum longus (EDL) muscles of 4-week-old rats. In the soleus, direct electrical stimulation for 10 min induced a frequency-dependent increase in the ouabain-suppressible 86Rb+ uptake, which was maximal (+110%) at a frequency of 2 Hz. In the EDL this frequency only induced a 31% increase. A supramaximal concentration of adrenaline (10 mumol l-1) stimulated ouabain-suppressible 86Rb+ uptake by 80% and 27% in soleus and EDL, respectively. The combined effect of stimulation at 2 Hz and adrenaline was not significantly larger than each of the interventions alone in either of the muscles. The fractional loss of 22Na+ from soleus muscle was increased by around 50% by the exposure to adrenaline, electrical stimulation at 2 Hz or a combination of both. The effect of electrical stimulation on 22Na+ efflux was not prevented by addition of propranolol (1 or 10 mumol l-1). The results indicate that the stimulation of active Na+-K+ transport induced by adrenaline or electrical stimulation is much more pronounced in soleus (slow-twitch) muscle than in EDL (fast-twitch) muscle. Since it has been suggested that an accumulation of K+ ions in the extracellular space may play a role in the development of fatigue (Bigland-Ritchie 1984), our findings might be related to the fact that slow-twitch muscles have a much higher resistance to fatigue than fast-twitch muscles (Burke et al. 1971).     

Segmental motor and sensory innervation of muscles in anterior leg compartment as revealed by retrograde transport of horseradish peroxidase

Horseradish peroxidase (HRP) tracing technique was used to label and localize motor and sensory neurons innervating tibialis anterior, extensor hallucis longus and extensor digitorum longus muscles of the anterior leg compartment of the rat. The tibialis anterior sensory neurons were located in the ipsilateral L4 and L5 spinal ganglia. Cells of origin of tibialis anterior motor endings were also found in the ipsilateral ventral horn of the same cord segments as the labeled sensory ganglia. Extensor hallucis longus sensory neurons were located in L4 to L6 spinal ganglia, while its labeled motor neurons were located in L4 and L5 spinal cord segments. The motor neurons innervating the extensor digitorum longus muscle were located in L4 to L6 spinal cord segments; its sensory neurons were previously localized. All labeled motor and sensory neurons were present on the ipsilateral side. Almost all motoneurons innervating the 3 muscles were present in the dorsolateral nucleus of the ventral horn.