Inhibition of calpains, by treatment with leupeptin, improves motoneuron survival and muscle function in models of motoneuron degeneration

The effect of treatment with leupeptin, a calpain inhibitor, on motoneuron survival and muscle function was examined in in vitro and in vivo models of motoneuron degeneration. Exposure of primary rat motoneurons to alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) is an established in vitro model of excitotoxic motoneuron death. Here we show that leupeptin treatment improved motoneuron survival following exposure to AMPA (50 microM). Application of leupeptin (100 microM) to AMPA treated cultures rescued many motoneurons so that 74% (+/-3.4 S.E.M., n=5) survived compared with only 49% (+/-2.4 S.E.M., n=5) in untreated cultures. The effect of treatment with leupeptin on motoneuron survival and muscle function was also examined in vivo. In 3 day-old rats, the sciatic nerve was crushed and at the time of injury, a silicon implant containing leupeptin was inserted onto the lumbar spinal cord. The effect on long-term motoneuron survival and muscle function was assessed 12 weeks after injury. The results showed that there was long-term improvement in motoneuron survival in the leupeptin treated group. Thus, in untreated animals 12 weeks after nerve crush only 30% (+/-2.8. S.E.M., n=3) of sciatic motoneurons survived compared with 43% (+/-1.5 S.E.M., n=3) in the leupeptin-treated group. This improvement in motoneuron survival was reflected in a significant improvement in muscle function in the leupeptin-treated group. For example in the soleus muscle of treated rats 20.8 (+/-1.40 S.E.M., n=5) motor units survived compared with only 14.6 (+/-1.21 S.E.M., n=5) in untreated animals. Thus, treatment with leupeptin, a calpain inhibitor, rescues motoneurons from cell death and improves muscle function following nerve injury.     

Reducing transmitter release from nerve terminals influences motoneuron survival in developing rats

Motoneurons in neonatal rats die following injury to the peripheral nerve. However, this vulnerability to nerve injury declines rapidly so that nerve injury at five days of age results in little if any motoneuron death. We have proposed that the role of the target during this critical period of development is to up-regulate the release of transmitter from developing motor nerve terminals. Here we show that reducing the release of acetylcholine from nerve terminals in neonatal rats can affect motoneuron maturation and survival. The soleus muscle in neonatal rats was treated with either magnesium or hemicholinium, and the number of motoneurons that survived was established 10 weeks later by retrograde labelling. Following treatment with magnesium, only 58.1% (+/-10.4 S.E.M., n=5) of the motoneurons in the soleus motor pool survived, although hemicholinium had no effect on motoneuron survival. However, those motoneurons that survived following treatment with either magnesium or hemicholinium did not develop normally since they remained susceptible to axotomy-induced cell death for longer than normal. In adult animals in which the sciatic nerve was crushed at five days of age following prior treatment with either magnesium or hemicholinium, only 27.6% (+/-6.2 S.E.M., n=5) and 44% (+/-6.1 S.E.M., n=4) of motoneurons in the sciatic motor pool survived, respectively, although no motoneurons died following injury alone or when injury was preceded by treatment with control implants containing NaCl. These results indicate that the release of acetylcholine from motor nerve terminals plays an important role in the development and survival of motoneurons.     

The effect of muscle activity on motor unit size in partially denervated rat soleus muscles

It has been shown previously that after section of L5 ventral ramus at 5 days the intact axons of L4 ventral ramus retain their large neonatal peripheral field in the rat soleus muscles. Soleus muscles of 5-day-old rats were partially denervated by section of their major neural input, L5 ventral ramus, and in addition paralysed with alpha-bungarotoxin for 3-5 days. The motor unit size was examined 2 months later. The tension developed by individual motor units from muscles that were partially denervated and in addition temporarily paralysed was much less than that after partial denervation alone. This reduced tension output was not due to muscle atrophy but to a smaller number of muscle fibres supplied by individual axons. Thus, unlike after partial denervation only, motoneurons were unable to maintain their large neonatal territory when the muscle was temporarily paralysed and they were unable to reoccupy this territory after the muscles recovered from the paralysis. The possibility that arrested muscle maturation due to paralysis has a permanent effect on motor unit size is discussed.     

Motor-unit properties following cross-reinnervation of cat lateral gastrocnemius and soleus muscles with medial gastrocnemius nerve. I. Influence of motoneurons on muscle

This study addresses two questions: is reinnervation of mammalian skeletal muscle selective with respect to motor-unit type? And to what degree may muscle-unit contractile properties be determined by the motoneuron? Properties of individual motor units were examined following cross-reinnervation (X-reinnervation) of lateral gastrocnemius (LG) and soleus muscles by the medial gastrocnemius (MG) nerve in the cat. We examined animals at two postoperative times: 9-10 wk (medX) and 9-11 mo (longX). For comparison, properties of normal LG and soleus motor units were studied. Motor units were classified on the basis of their contractile response as fast contracting fatigable, fast intermediate, fast contracting fatigue resistant, or slow (types FF, FI, FR, or S, respectively) (13,29). Muscle fibers were classified on the basis of histochemical properties as fast glycolytic, fast oxidative glycolytic, or slow oxidative (types FG, FOG, or SO, respectively) (61). Reinnervation of LG and soleus was not selective with respect to motor-unit type. Both muscles were innervated by a full complement of MG motoneuron types, apparently in normal MG proportions. MG motoneurons determined LG muscle fibers' properties to a similar degree as reinnervated MG muscle fibers. In contrast, soleus muscle fibers "resisted" the influence of MG motoneurons. Thus, although longX-reinnervated LG muscle (longX LG) had a motor-unit type distribution similar to normal or self-reinnervated MG, longX soleus contained predominantly type S motor units. Overall mean values for muscle-unit contractile properties reflected this motor-unit type distribution. Muscle units in longX LG and longX soleus had contractile properties typical of the same motor-unit type in normal LG or soleus, respectively. Motor-unit types were recognizable at 10 wk X-reinnervation, although muscle-unit tensions were lower than after 10 mo. The proportions of fast and slow motor units in medX LG were similar to longX LG, although a greater proportion of fast units were resistant to fatigue at 10 wk. There were fewer fast units in medX soleus than longX soleus, which suggested that motor-unit type conversion or innervation of muscle fibers by fast motoneurons is not complete at 10 wk. We conclude that reinnervation of the LG and soleus muscles by MG motoneurons was not selective with respect to motor-unit type. MG motoneurons determined LG muscle properties to a similar degree as self-reinnervated MG muscle fibers. Soleus muscle fibers resisted the influence of MG motoneurons, representing a limit to neural determination of muscle properties.     

Cross-reinnervated motor units in cat muscle. I. Flexor digitorum longus muscle units reinnervated by soleus motoneurons

The properties of flexor digitorum longus (FDL) muscles and of individual motor units were studied in cats 30-50 wk after self-reinnervation by FDL motoneurons (FDL----FDL) or cross-reinnervation by soleus (SOL) motoneurons (SOL----FDL). Individual motor units were functionally isolated by intracellular recording and stimulation of identified SOL alpha-motoneurons. Glycogen-depletion methods permitted histochemical study of muscle fibers belonging to physiologically characterized muscle units. The observations were compared with data from normal cat FDL muscles and motor units (27). Intentionally self-reinnervated FDL muscles (FDL----FDL; n = 5) were normal in size and wet weight. FDL----FDL motor units could be classified into the same physiological categories found in normal FDL [types: fast contracting, fatigable (FF), fast contracting, fatigue resistant (FR), and slow (S); n = 24], with approximately the same proportions as normal. The histochemical muscle fiber types associated with these categories were also qualitatively normal although there was evidence of marked distortion of the normal histochemical mosaic. These data confirm other studies of self-reinnervation and suggest that self-reinnervation can produce complete interconversion of muscle fiber types. Cross-reinnervation of FDL muscle by SOL motoneurons (SOL----FDL; n = 12) produced muscles that were smaller (about half the normal wet weight) and more red than normal. SOL----FDL muscle contracted more slowly than normal or FDL----FDL muscles and had much higher proportions of histochemical type I muscle fibers. In those SOL----FDL muscles, in which little or no unwanted self-reinnervation could be demonstrated, greater than 95% of the muscle fibers were type I. Forty-one individual motor units in SOL----FDL muscles were isolated by intracellular penetration in functionally identified SOL alpha-motoneurons. Their muscle units were all type S by physiological criteria (absence of "sag" in unfused tetani and marked resistance to fatigue). SOL----FDL muscle units had contraction times and fatigue properties that were essentially identical to those of type S units in the normal FDL. All of the seven units, successfully studied by glycogen depletion, exhibited histochemical type I fibers. SOL motoneurons that innervated FDL muscle units had slightly shorter afterhyperpolarization durations than normal SOL cells, but axonal conduction velocities were normal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cross-reinnervated motor units in cat muscle. II. Soleus muscle reinnervated by flexor digitorum longus motoneurons

The properties of whole soleus (SOL) muscles and of individual motor units were studied in cats 30-50 wk after self-reinnervation by soleus (SOL) motoneurons (SOL----SOL) or cross-reinnervation by flexor digitorum longus (FDL) motoneurons (FDL----SOL). As in the preceding paper (22), intracellular and glycogen-depletion methods were used to examine the physiological and histochemical properties of individual motor units. The results were compared with data from normal SOL motor units (8, 12). Intentionally self-reinnervated SOL muscles (SOL----SOL; n = 6) were normal in size and wet weight, and all of the five SOL----SOL motor units studied had physiological and histochemical characteristics that matched those of normal SOL units. Cross-reinnervation of SOL by FDL alpha-motoneurons (FDL----SOL; n = 7) produced muscles with wet weights and appearance essentially identical to normal SOL. However, whole-muscle twitch contraction times were much shorter (mean 60.4 ms) than those of normal (mean 136.9 ms, n = 18) or SOL----SOL muscles (mean 115.3 ms; n = 6). Despite this difference, none of the FDL----SOL muscles contained more than 7% histochemical type II muscle fibers, all of which were type IIA. Normal cat SOL muscles can contain up to 5% type IIA fibers, but none of our SOL----SOL muscles showed any type II fibers. Two FDL----SOL muscles had significant amounts of unintended self-reinnervation, permitting side-by-side comparison of FDL----SOL and SOL----SOL muscle fibers. The twitch contraction times of the two populations differed markedly, but they were histochemically indistinguishable except for the fact that SOL----SOL fibers had high neutral fat content (as do normal SOL fibers), whereas FDL----SOL showed much lower fat content. The 23 FDL----SOL muscle units studied were classified as physiological type S by criteria ("sag" test and fatigue resistance) used to identify motor-unit types in normal cat muscles. All five of the FDL----SOL units studied histochemically after glycogen depletion showed the type I histochemical profile, which is characteristic of the normal cat SOL. In marked contrast to the preceding study, cross-reinnervation of cat SOL by FDL motoneurons produced no conversion of muscle-unit properties into those associated with fast-twitch unit types, despite significant decreases in isometric twitch contraction time. The altered twitch speed was not associated with evident changes in conventional myofibrillar adenosine triphosphatase (ATPase) histochemistry.(ABSTRACT TRUNCATED AT 400 WORDS)     

Motor-unit properties following cross-reinnervation of cat lateral gastrocnemius and soleus muscles with medial gastrocnemius nerve. II. Influence of muscle on motoneurons

We tested whether the muscle innervated may influence the expression of motoneuron electrical properties. Properties of individual motor units were examined following cross-reinnervation (X-reinnervation) of cat lateral gastrocnemius (LG) and soleus muscles by the medial gastrocnemius (MG) nerve. We examined animals at two postoperative times: 9-10 wk (medX) and 9-11 mo (longX). For comparison, normal LG and soleus motoneuron properties were also studied. Motor units were classified on the basis of their contractile responses as fast contracting fatigable, fast intermediate fast contracting fatigue resistant, and slow types FF, FI, FR, or S, respectively) (9, 21). Motoneuron electrical properties (rheobase, input resistance, axonal conduction velocity, afterhyperpolarization) were measured. After 9-11 mo, MG motoneurons that innervated LG muscle showed recovery of electrical properties similar to self-regenerated MG motoneurons. The relationships between motoneuron electrical properties were largely similar to self-regenerated MG. For MG motoneurons that innervated LG, motoneuron type (65) predicted motor-unit type in 74% of cases. LongX-soleus motoneurons differed from longX-LG motoneurons or self-regenerated MG motoneurons in mean values for motoneuron electrical properties. The differences in overall means reflected the predominance of type S motor units. The relationships between motoneuron electrical properties were also different than in self-regenerated MG motoneurons. In all cases, the alterations were in the direction of properties of type S units, and the relationship between normal soleus motoneurons and their muscle units. Within motor-unit types, the mean values were typical for that type in self-regenerated MG. Motoneuron type (65) was a fairly strong predictor of motor-unit type in longX soleus. MG motoneurons that innervated soleus displayed altered values for axonal conduction velocity, rheobase, and input resistance, which could indicate incomplete recovery from the axotomized state. However, although mean afterhyperpolarization (AHP) half-decay time was unaltered by axotomy (25), this parameter was significantly lengthened in MG motoneurons that innervated soleus muscle. There were, however, individual motoneuron-muscle-unit mismatches, which suggested that longer mean AHP half-decay time may also be due to incomplete recovery of a subpopulation of motoneurons. Those MG motoneurons able to specify soleus muscle-fiber type exhibited motoneuron electrical properties typical of that same motoneuron type in self-regenerated MG.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nerve growth in botulinum toxin poisoned muscles

The rate of growth of motor axon sprouts in muscles fully paralysed with botulinum toxin was studied using the zinc iodide—osmium tetroxide stain. In the mouse soleus and peroneus muscles, the proportions of endplates with nerve terminal sprouts rose to almost 100% in two to three weeks. In both muscles, the initial rate of terminal sprout growth was rather slow—about 3 μm per day. Two to three weeks after the injection the rate had risen to about 15 μm per day. Substantial numbers of polyneuronally innervated muscle fibres could be demonstrated in the soleus, but not the peroneus, by twelve days after the injection. The soleus remained polyneuronally innervated over a year after the injection.The time course of the tension recovery by the blocked soleus nerve was compared with that of the tension development by a fibular nerve implanted into a fully paralysed or completely denervated soleus muscle. The rate of tension recovery by the fibular nerve was the same whether the soleus was denervated or simply blocked. After an initial delay, the recovery of tension by the paralysed soleus nerve followed a course very similar to that of the implanted nerves.The implanted nerve did not affect the initial terminal sprouting of the paralysed soleus endplates. However, after three weeks, sprouts from soleus nerve endplates on many fibular innervated muscle fibres had disappeared. The implanted nerve did not affect the sprouting of soleus endplates on the muscle fibres which it did not innervate. The recovery of tension by the soleus nerves to muscles innervated by implanted nerves was reduced.It is concluded (1) that recovery from paralysis after botulinum toxin poisoning is slow because formation of new extrajunctional synapses is slow; (2) that motor nerve terminals are more readily induced to sprout by changes occurring on their own muscle fibres than by inactivity induced changes in muscle fibres elsewhere in the same muscle; (3) that paralysis of the mouse soleus renders the muscle as receptive to innervation extrajunctionally as does denervation.     

Synchronization of motor units in human soleus muscle during standing postural tasks

During standing posture, the soleus muscles acts to control sway in the anteroposterior (AP) direction. The soleus muscles bilaterally share a common function during standing tasks. We sought to determine whether common descending inputs, as evidenced by the synchronization of bilateral motor unit pairs, were employed as a strategy to control this common function. Single motor units were recorded from the soleus muscles in subjects who stood on adjacent force platforms for 5 min with their eyes open or closed. While standing with the eyes open, only 4/39 bilateral motor unit pairs showed significant synchronization. Similarly, only 3/36 motor unit pairs were significantly synchronized during the eyes closed task. The low incidence of synchronization was observed despite a high correlation in the amount of sway in the AP direction between legs in both the eyes open and eyes closed tasks (rho = 0.80 and rho = 0.83, respectively). When the extent of synchronization was assessed between pairs of motor units within the same leg with the eyes open, 10/12 pairs were synchronized. Furthermore, when pairs of soleus motor units were recorded both bilaterally and unilaterally during voluntary isometric ankle plantarflexion, only 4/30 bilateral pairs showed significant synchronization, whereas 19/24 unilateral pairs had significant synchronization. In this study, there was little evidence of the existence of synchronization between bilateral soleus motor unit pairs in either postural tasks or voluntary isometric contractions. In cases in which bilateral synchronization was observed, it was considerably weaker than the synchronization of motor units within a single soleus muscle. The results of this study reveal that it is rather uncommon for bilateral soleus motoneurons to receive common descending synaptic inputs, whereas two motoneurons within a single soleus muscle do.     

Repeated stimuli for axonal growth causes motoneuron death in adult rats: the effect of botulinum toxin followed by partial denervation

Axons of motoneurons to tibialis anterior and extensor digitorum longus muscles of adult rats were induced to sprout by injecting botulinum toxin into them, by partial denervation or by a combination of the two procedures. Ten weeks later, the number of motoneurons innervating the control and operated tibialis anterior and extensor digitorum longus muscles was established by retrograde labelling with horseradish peroxidase. In the same preparations, the motoneurons were also stained with a Nissl stain (gallocyanin) to reveal motoneurons in the sciatic pool. Examination of the spinal cords from animals treated with botulinum toxin showed that the number of retrogradely labelled cells and those stained with gallocyanin in the ventral horn on the treated compared to the control side was unchanged. In rats that had their L4 spinal nerve sectioned on one side, the number of retrogradely labelled cells on the operated side was 48+/-3% (n = 5) of that present in the control unoperated ventral horn. Thus, just over half the innervation was removed by cutting the L4 spinal nerve. Counts made from gallocyanin-stained sections showed that 94+/-4% (n = 5) of motoneurons were present in the ventral horn on the operated side. Thus, section of the L4 spinal nerve did not lead to any death of motoneurons. In rats that had their muscles injected with botulinum toxin three weeks prior to partial denervation, the number of retrogradely labelled cells was reduced from 48+/-3% (n = 5) to 35+/-4% (n = 5). Moreover, only 67+/-5% (n = 5) of motoneurons stained with gallocyanin, suggesting that a proportion of motoneurons died after this combined procedure. This result was supported by experiments in which motor unit numbers in extensor digitorum longus muscles were determined by measurements of stepwise increments of force in response to stimulation of the motor nerve with increasing stimulus intensity. In partially denervated extensor digitorum longus muscles, 16.6+/-0.7 (n = 5) motor units could be identified, and in animals treated with botulinum toxin prior to partial denervation only 13.3+/-0.9 (n = 3) motor units were present. Taken together, these results show that treatment with botulinum toxin followed by partial denervation causes motoneuron death in adult rats.     

Motoneuron and muscle-unit properties after long-term direct innervation of soleus muscle by medial gastrocnemius nerve in cat

1. This study addresses the following questions. 1) In a previous experiment, when the combined lateral gastrocnemius-soleus nerve was cross-innervated by the medial gastrocnemius (MG) nerve, was the predominance of slow muscle units in soleus muscle a result of selective routing of slow motor axons into soleus? 2) Is MG-nerve-induced conversion of soleus muscle fibers from slow to fast more complete at very long (18 mo vs. 9-11 mo) postoperative times? 3) Do MG motoneurons that cross-innervate soleus muscle recover their normal membrane electrical properties at very long postoperative times? 2. The proximal portion of approximately one-third of the MG nerve was coapted directly with the distally isolated soleus nerve. The MG muscle remained innervated by the unoperated portion of the MG nerve. At 6, 10, or 18 mos postoperative, motoneuron and/or muscle-unit properties were determined for MG motoneurons innervating MG, soleus, or neither muscle, and for axotomized soleus motoneurons. 3. In the partially denervated MG muscle, the proportions of motor units of each type were normal. This suggests that the population of MG motor axons that had been directed to the soleus nerve also contained a representative distribution of MG motoneuron types. 4. Most motor units (74%) in cross-innervated soleus (Xsoleus) were type S (based on muscle-unit contractile properties), in spite of the soleus nerve's having been cross-connected by approximately 75% fast MG motoneurons. Thus, even at very long postoperative times, slow soleus muscle units resisted conversion by fast MG motoneurons. 5. Thirty-two percent of MG motoneurons that had been cross-connected to soleus nerve elicited no measurable muscle contraction, compared with approximately 10% in previous reinnervation experiments in which the MG nerve was coapted with the MG or lateral gastrocnemius-soleus nerve. Thus MG motoneurons may be disadvantaged in their ability to innervate soleus muscle fibers. 6. It appears that at long postoperative times, those fast MG motoneurons tha had innervated large soleus muscle units had failed to convert those muscle fibers to fast types and had failed also to recover their normal motoneuron electrical properties. Conversion and recovery did occur for fast MG motoneurons that innervated small soleus muscle units and for slow MG motoneurons.     

Over-expression of parvalbumin in transgenic mice rescues motoneurons from injury-induced cell death

Following nerve injury in neonatal rats, a large proportion of motoneurons die, possibly as a consequence of an increase in vulnerability to the excitotoxic effects of glutamate. Calcium-dependent glutamate excitotoxicity is thought to play a significant role not only in injury-induced motoneuron death, but also in motoneuron degeneration in diseases such as amyotrophic lateral sclerosis (ALS). Motoneurons are particularly vulnerable to calcium influx following glutamate receptor activation, as they lack a number of calcium binding proteins, such as calbindin-D(28k) and parvalbumin. Therefore, it is possible that increasing the ability of motoneurons to buffer intracellular calcium may protect them from cell death and prevent the decline in motor function that usually occurs as a consequence of motoneuron loss. In this study we have tested this possibility by examining the effect of neonatal axotomy on motoneuron survival and muscle force production in normal and transgenic mice that over-express parvalbumin in their motoneurons.The sciatic nerve was crushed in one hindlimb of new-born transgenic and wildtype mice. The effect on motoneuron survival was assessed 8 weeks later by retrograde labelling of motoneurons innervating the tibialis anterior muscle. Following nerve injury in wildtype mice, only 20.2% (+/-2.2, S.E.M.; n=4) of injured motoneurons survive long term compared with 47.2% (+/-4.4, S.E.M.; n=4) in parvalbumin over-expressing mice. Surprisingly, this dramatic increase in motoneuron survival was not reflected in a significant improvement in muscle function, since 8 weeks after injury there was no improvement in either maximal twitch and tetanic force, or muscle weights.Thus, inducing spinal motoneurons to express parvalbumin protects a large proportion of motoneurons from injury-induced cell death, but this is not sufficient to restore muscle function.     

Reinnervation and recovery of mouse soleus muscle after long-term denervation

Reinnervation and recovery of the mouse soleus muscle were studied 2-10 months after denervation periods of about 7 months. To maintain denervation the right sciatic nerve was frozen 14 times at 2-week intervals. Though initially intermittent muscle reinnervation occurred, contractile force of denervated muscles was reduced to less than 10% of the contralateral muscles by the fifth nerve freezing and further declined thereafter. Following reinnervation, recovery of soleus muscle force proceeded slowly to reach plateau values after 5-6 months. Tetanic muscle force reached on average 72% (range 58-86%, n = 12) of contralateral muscles after 5-10 months, (P less than 0.01, t-test for absolute values) and 87% of unoperated animals after 10 months (P less than 0.05, n = 5). Muscle fibre diameters were significantly reduced in reinnervated muscles, but frequency distributions were normal and similarly shaped in reinnervated and control muscles, suggesting complete muscle reinnervation and the absence of denervated fibres even at 2 months of reinnervation. Total numbers of muscle fibres were similar in reinnervated (842 +/- 73 S.D., n = 15), contralateral (854 +/- 104 S.D., n = 15) and control soleus muscles (853 +/- 77 S.D., n = 5). The number of myelinated axons in regenerating soleus nerves reached control values by 3 months after the last freezing, continued to increase till 6 months (150% of control), and declined thereafter (125% at 9-10 months). In the contralateral soleus nerves the number of myelinated axons remained constant during this period. Nerve fibre diameters remained abnormally small; even after 10 months of reinnervation fibre diameters were unimodally distributed with a mean diameter of 3.3 microns in contrast to the bimodal distribution in intact nerves (mean values 3.9 and 9.0 microns, respectively). Total fibre cross-section area per nerve increased with time but reached only 54% +/- 6 S.D., (n = 3) of contralateral nerves by 10 months. The relative thickness of the myelin sheath (g-ratio) returned to normal after 9-10 months. Anatomically, muscle reinnervation appeared to be complete by 7-8 weeks since unusually small muscle fibre profiles were absent.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of physiological and histochemical properties of motor units after cross-reinnervation of antagonistic muscles in the cat hindlimb

1. Physiological and histochemical properties of the cat ankle extensor muscles, the lateral and medial gastrocnemius, and the soleus were studied after cross-reinnervation by flexor motoneurons. 2. Tibial and common peroneal nerves were cut and cross-united in the popliteal fossa of 2- to 6-mo-old cats. Eighteen to 24 mo later, single motor units were isolated by dissection and stimulation of ventral root filaments and classified into four types: fast-twitch, fatigable (FF), fast-twitch with intermediate fatigue resistance (FI), fast-twitch, fatigue-resistant (FR), and slow, fatigue-resistant (S). Muscle fibers were classified as fast glycolytic (FG), fast, oxidative glycolytic (FOG), and slow oxidative (SO) on the basis of histochemical staining. 3. Although motor-unit force was normally well correlated with the size of the innervating motor axon in the cross-reinnervated muscles, the force of different unit types overlapped considerably. The reinnervated motor units also showed a higher than normal degree of fatigability. 4. The range of muscle unit forces in cross-reinnervated triceps surae muscles was the same as in the normally innervated triceps surae muscles. This range is 2-3 times greater than the flexor muscles, which the common peroneal nerve normally supplies. The range of contraction speed of units in the cross-reinnervated extensor muscles was comparable to that in the flexor muscles, consistent with a motoneuron-specific determination of muscle speed (28). 5. SO and FOG muscle fibers were found in all reinnervated triceps surae muscles, but FG fibers were only found in reinnervated medial gastrocnemius (MG) and lateral gastrocnemius (LG) muscles, consistent with previous findings of the resistance of soleus muscles to complete conversion (10, 16, 20, 21). Type grouping of muscle fibers was characteristic of the reinnervated muscles. 6. Reinnervated SO muscle fibers were larger than the corresponding fibers in normally innervated muscles as were the estimated number of muscle fibers innervated by slow motor axons. Nonetheless, the force generated by the S motor units remained relatively smaller than FR and FF units. The relative contributions of the number, cross-sectional area and specific tension to the force generation of reinnervated motor units are discussed.     

Fatigue properties of human thenar motor units paralysed by chronic spinal cord injury

Human muscles paralysed chronically by spinal cord injury (SCI) fatigue excessively. Whether these reductions in force reflect a decrease in the fatigue resistance of the motor units is unknown. Our aim was to determine the fatigability of thenar motor units paralysed chronically (10 ± 2 years) by cervical SCI. Surface electromyographic activity (EMG) and force were recorded from 17 paralysed motor units ( n = 7 subjects) in response to intraneural motor axon stimulation (13 pulses at 40 Hz, 1 s⁻¹ for 2 min). Unit force decreased progressively, reaching 8–60% of initial after 2 min, whereas both the amplitude and area of the first EMG potentials in the trains increased significantly (both P < 0.05). Thus, transmission of neural signals to the sarcolemma was effective and the reduction in force must reflect impaired processes in the muscle fibres. The median fatigue index for paralysed units (0.31), the ratio of the force at 2 min compared to the initial force, was significantly lower than that for units from control subjects (0.85, P < 0.05), but the distribution of fatigue indices for each population had a similar shape (ranges: 0.08–0.60 and 0.41–0.95, respectively). Hence, chronic paralysis did not limit the range of fatigability typically found for thenar units, only its magnitude. These findings suggest that all paralysed units underwent similar reductions in fatigue resistance. After fatigue, paralysed unit forces were reduced at all frequencies (1–100 Hz, P < 0.05). Twitch contraction and half-relaxation times were increased, as was the frequency needed to produce half maximal force ( P < 0.05). Thus, stimulation protocols used to produce functional movements in paralysed muscles need to accommodate the significant and rapid fatigue of the motor units.     

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.     

Delayed riluzole treatment is able to rescue injured rat spinal motoneurons

The effect of delayed 2-amino-6-trifluoromethoxy-benzothiazole (riluzole) treatment on injured motoneurons was studied. The L4 ventral root of adult rats was avulsed and reimplanted into the spinal cord. Immediately after the operation or with a delay of 5, 10, 14 or 16 days animals were treated with riluzole (n=5 in each group) while another four animals remained untreated. Three months after the operation the fluorescent dye Fast Blue was applied to the proximal end of the cut ventral ramus of the L4 spinal nerve to retrogradely label reinnervating neurons. Three days later the spinal cords were processed for counting the retrogradely labeled cells and choline acetyltransferase immunohistochemistry was performed to reveal the cholinergic cells in the spinal cords. In untreated animals there were 20.4+/-1.6 (+/-S.E.M.) retrogradely labeled neurons while in animals treated with riluzole immediately or 5 and 10 days after ventral root avulsion the number of labeled motoneurons ranged between 763+/-36 and 815+/-50 (S.E.M.). Riluzole treatment starting at 14 and 16 days after injury resulted in significantly lower number of reinnervating motoneurons (67+/-4 and 52+/-3 S.E.M., respectively). Thus, riluzole dramatically enhanced the survival and reinnervating capacity of injured motoneurons not only when treatment started immediately after injury but also in cases when riluzole treatment was delayed for up to 10 days. These results suggest that motoneurons destined to die after ventral root avulsion are programmed to survive for some time after injury and riluzole is able to rescue them during this period of time.     

Responses of ankle extensor and flexor motoneurons to transcranial magnetic stimulation

Transcranial magnetic stimulation (TMS) of the motor cortex excites limb muscles of the contralateral side of the body. Reports of poorly defined, or a complete lack of systematic excitatory responses of soleus motoneurons compared with those of tibialis anterior (TA) motoneurons has led to the proposal that while all ankle flexor motoneurons receive strong corticomotoneuronal connections, very few soleus motoneurons do. In addition, the connections to these few motoneurons are weak. The nature of corticomotoneuronal connections onto these two motoneuron pools was re-evaluated in the following experiments. The leg area of the left motor cortex was stimulated with a large double-cone coil using Magstim 200, while surface electromyographic (EMG) and single motor unit (SMU) responses were recorded from soleus and TA muscles of healthy adult subjects. Under resting conditions, the onset (25-30 ms) and duration of concomitantly recorded short latency motor evoked potentials (MEPs) in surface EMG from both muscles were similar. The input-output relationships of the simultaneously recorded soleus and TA EMG responses showed much greater increases in TA MEPs compared with soleus MEPs with identical increases in stimulus intensity. Under resting and nonisometric conditions, a later peak with onset latency of approximately 100 ms was observed in soleus. During isometric conditions or with vibration of the TA tendon, the second soleus peak was abolished indicating reflex origin of this peak. Recordings from 42 soleus and 39 TA motor units showed clear response peaks in the peristimulus time histograms (PSTHs) of every unit. Two statistical tests were done to determine the onset and duration of response peaks in the PSTHs. With chi(2) test, the duration was 6.9 +/- 4.2 ms (mean +/- SD) for soleus and 5.1 +/- 2.1 ms for TA. Using the criterion of discerning a peak by bin counts being three SDs above background, the duration was 10.0 +/- 4.4 ms for soleus and 7.8 +/- 2.6 ms for TA. Results of these experiments do not suggest a lack of systematic corticomotoneuronal connections on soleus motoneurons when compared with those on TA, though some differences in the strengths of corticomotoneuronal connections onto the two pools do exist.     

Functional and structural changes of rat plantaris motoneurons following compensatory hypertrophy of the muscle

The conduction velocity and histological structure of motoneurons innervating normal and hypertrophied rat plantaris muscles were investigated. Hypertrophy was produced by ablation of synergist muscles. Single motor units were obtained by ventral root dissection and conduction velocities measured. The structure of neurons was investigated following retrograde labeling with horseradish peroxidase. A combined silver, gold and cholinesterase staining method was developed to study the motor endplate. In addition, the peripheral nerve was fixed, embedded in Araldite, and sectioned for determination of axonal size and myelin thickness. Conduction velocity of motor axons decreased following hypertrophy of the skeletal muscle (control CV = 75.8 +/- 8.9 m s-1, n = 94, hypertrophy CV = 69.0 +/- 12.3 m s-1, n = 84). However, no alteration in the size of motor axons or myelin thickness could account for this alteration in conduction velocity. Mean motoneuronal soma size decreased following muscle hypertrophy (soma diameter: control 36.1 +/- 4.6 microns, n = 283, hypertrophy 32.9 +/- 4.5 microns, n = 294). The complexity of the motor endplate increased following hypertrophy with an increased occurrence of nodal sprouts. In addition, the area of cholinesterase staining increased following hypertrophy (control 588.1 +/- 297.2 microns 2, n = 269, hypertrophy 857.7 +/- 357.0 microns 2, n = 269). This study found that both the morphological and physiological parameters of motoneurons innervating a hypertrophied muscle were shifted toward those of normal rat slow motor units.     

The effects of tenotomy and compensatory hypertrophy on the postnatal development of soleus motor units in the cat

Kittens, 5-7 days old, were subjected to tenotomy of either the soleus muscle or all its Achilles tendon synergists. When the cats had reached the adult stage, the physiological properties of the soleus motor units were investigated in both the atrophic and hypertrophic situations. Tenotomy resulted in a marked muscle weight loss and overload due to tenotomy of synergists in a marked gain in muscle weight compared to the contralateral side. The motor units of the tenotomized soleus muscles exhibited a moderate shortening of the twitch contraction time, and also a change in twitch shape, related to the degree of atrophy. In the motor units of the hypertrophic soleus muscles, an increased fatiguability could be demonstrated. Further, in this group, there was a tendency towards differentiation of the normally uniform motor units into two groups with different features. The functional implications of this process are discussed. The development of motor units in both the tenotomized and the hypertrophic muscles were only marginally influenced by the operations, and it is concluded that the basic features of their development are largely unaffected by the functional manipulations imposed in the present study.