The expression of myosin heavy chain isoforms in normal and hypertrophied chicken slow muscle.

Hypertrophy was produced in the anterior latissimus dorsi (ALD) muscle of 5-wk-old chickens by application of a load to the humerus. After 4 wk, hypertrophied ALD muscles were greater than 2.5 times heavier than contralateral control ALD muscles. Two isomyosins are distinguishable in normal ALD muscles by their different electrophoretic mobilities. It is shown here that the faster migrating SM-1 isomyosin decreases in abundance with age and that the application of an overload enhances both the rate and extent of this process. Monoclonal antibodies were selected by an immunotransfer technique that were specific for the heavy chains associated with either SM-1 or SM-2, or cross-reacted with both isoforms. The cellular distribution of the SM-1 and SM-2 isomyosins was analyzed by immunofluorescent technique using these antibodies. Anti-SM-1 and anti-SM-2 antibodies reacted with separate populations of cells, whereas the third antibody reacted with all myocytes in the normal ALD muscle. These data suggest that there is an exclusive cellular distribution of myosin heavy chains associated with SM-1 and SM-2 proteins. Immunofluorescent analysis of hypertrophied muscle showed the anti-SM-2-specific antibody reacting with all myocytes, whereas the anti-SM-1-specific antibody reacted with none. This is consistent with the elimination of the SM-1 isoform in hypertrophied muscles.

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.

Effect of precocious locomotor activity on the development of motoneurones and motor units of slow and fast muscles in rat.

We have investigated the effect of precociously increasing locomotor activity during early postnatal development by daily treatment with the monoaminergic precursor L-DOPA on the survival of motoneurones supplying the slow soleus (SOL) muscle and the fast, tibialis anterior (TA) and extensor digitorum longus (EDL) muscles as well as the contractile and histochemical properties of these muscles. L-DOPA treatment resulted in a significant loss of motoneurones to the slow SOL muscle, but not to the fast TA and EDL muscles. Moreover, motoneurones to fast muscles also die as when exposed to increased activity in early life, if their axons are repeatedly injured. The loss of normal soleus motoneurones was accompanied by an increase in force of the remaining motor units and sprouting of the surviving axons suggesting a remodelling of motor unit organisation. The time to peak contraction of both SOL and EDL muscles from L-DOPA treated rats was prolonged at 8 weeks of age. At 4 weeks the soleus muscles of the L-DOPA treated animal developed more tension than the saline treated one. This difference between the two groups did not persist and by 8 weeks of age the muscle weight and tetanic tension from either group were not significantly different from control animals. The present study shows that early transient, precocious locomotor activity induced by L-DOPA is damaging to normal soleus but not to normal EDL/TA motoneurones.

Modification of motoneuron size after partial denervation in neonatal rats.

Our previous studies have shown that partial denervation of extensor digitorum longus muscle (EDL) in the rat at 3 days of age causes an increase in the activity of the intact motoneurons. The originally phasic pattern of activity of EDL became tonic after partial denervation. These modifications of motoneuron activity were associated with the change in the phenotype of the muscle from fast to slow contracting and with a conversion of the muscle fibres from a fast to a slow type. The present study investigates whether the size of the cell body of the active EDL motoneurons change in parallel with the altered muscular activity. The study involved partial denervation of rat EDL muscle by section of the L4 spinal nerve at 3 days of age. Then the remaining motoneurons from L5 spinal nerve supplying the EDL muscle were retrogradly labelled with horseradish peroxidase two months later. The results show a reduction in motoneuron size in parallel with an increase in activity of the motoneurons after partial denervation of EDL muscle.

Motoneurone survival: a functional approach.

The role of the target in the survival of developing motoneurones is discussed. The contribution of neurotrophic factors is re-evaluated in view of the following: (1) motoneurone numbers are affected only slightly in transgenic mice following deletions of genes coding for neurotrophins or their receptors; and (2) continued treatment with neurotrophins fails to achieve long-term motoneurone survival. Evidence that motoneurone survival depends on the induction of transmitter release initiated by contact between nerve and muscle is presented. The release of transmitter and the ensuing retraction of many axon branches transforms the motoneurone from a growing cell into a transmitting cell. It is suggested that only when motoneurones have undergone this transition can they survive within the mature CNS.

Transplants of embryonic motoneurones to adult spinal cord: survival and innervation abilities.

One goal of transplantation experiments involving damaged spinal cords is to reconstruct a functional innervation to muscles in the periphery. Embryonic spinal cord grafts have been shown to survive transplantation into adult spinal cord lacking motoneurones. Motoneurones from the graft appear to be able to innervate muscle tissue by being encouraged to grow across a bridge of peripheral nerve. Integration of grafted motoneurones appears to involve their migration from the graft into the host ventral horn, thus replacing depleted host neurones. These results suggest possible strategies of research that might lead to treatments of spinal cord injuries and disorders in which motoneurone loss occurs, such as amyotrophic lateral sclerosis, spinal muscular atrophies and poliomyelitis.

Dependence of postnatal motoneurones on their targets: review and hypothesis.

Motoneurones are known to die (1) during embryonic development (naturally occurring cell death), (2) early in postnatal development after axonal injury, and (3) as a consequence of disease, such as spinal muscular atrophy or (in later life) amyotrophic lateral sclerosis. Naturally occurring motoneurone death has been extensively investigated, and interaction with the target muscle has emerged as an important factor for survival of embryonic motoneurones. Evidence that this target dependence of motoneurones continues postnatally is discussed in this review, as is the possible nature of the retrograde signal from the muscle. An explanation for the role of the muscle in motoneurone survival is also proposed, which may be applicable in situations where motoneurone death occurs postnatally. This proposal takes into account the changing functional demands imposed on motoneurones as a result of the gradual maturation of the CNS, and suggests that during development the muscle induces the motoneurones to become competent to carry out these requirements.

Activity-dependent interactions between motoneurones and muscles: their role in the development of the motor unit.

In this review article we have attempted to provide an overview of the various forms of activity-dependent interactions between motoneurones and muscles and its consequences for the development of the motor unit. During early development the components of the motor unit undergo profound changes. Initially the two cell types develop independently of each other. The mechanisms that regulate their characteristic properties and prepare them for their encounter are poorly understood. However, when motor axons reach their target muscles the interaction between these cells profoundly affects their survival and further development. The earliest interactions between motoneurones and muscle fibres generate a form of activity which is in many ways different from that seen at later stages. This difference may be due to the immature types of ion channels and neurotransmitter receptors present in the membranes of both motoneurones and muscle fibres. For example, spontaneous release of acetylcholine may influence the myotube even before any synaptic specialization appears. This initial form of activity-dependent interaction does not necessarily depend on the generation of action potentials in either the motoneurone or the muscle fibre. Nevertheless, the ionic fluxes and electric fields produced by such interactions are likely to activate second messenger systems and influence the cells. An important step for the development of the motor unit in its final form is the initial distribution of synaptic contacts to primary and secondary myotubes and their later reorganization. Mechanisms that determine these events are proposed. It is argued that the initial layout of the motor unit territory depends on the matching of immature muscle fibres (possibly secondary myotubes) to terminals with relatively weak synaptic strength. Such matching can be the consequence of the properties of the muscle fibre at a particular stage of maturation which will accept only nerve terminals that match their developmental stage. Refinements of the motor unit territory follows later. It is achieved by activity-dependent elimination of nerve terminals from endplates that are innervated by more than one motoneurone. In this way the territory of the motor unit is established, but not necessarily the homogeneity of the physiological and biochemical properties of its muscle fibres. These properties develop gradually, largely as a consequence of the activity pattern that is imposed upon the muscle fibres supplied by a given motoneurone. This occurs when the motor system in the CNS completes its development so that specialized activity patterns are transmitted by particular motoneurones to the muscle fibres they supply.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of cross-innervation on the tropomyosin composition of rabbit skeletal muscle.

Soleus, semitendinosus and crureus muscles of the rabbit were found to contain alpha- and beta-tropomyosin subunits and additional forms that have been provisionally designated gamma and delta. Extensor digitorum longus and psoas muscles contained only alpha and beta subunits, the relative proportions of which varied between single fibres of psoas muscle. On cross-innervation of rabbit soleus and extensor digitorum longus muscles, the fraction of the total tropomyosin present as the beta subunit remained constant. The relative proportions of alpha, gamma and delta subunits changed as would be expected from the change in speed that occurred.

The Effect of Inhibiting the Calcium Activated Neutral Protease, on Motor Unit Size after Partial Denervation of the Rat Soleus Muscle.

Rat soleus muscles were partially denervated by removal of the L5 ventral ramus at either 4 - 6 days or 17 - 19 days. Local application of leupeptin, a potent inhibitor of the calcium activated neutral protease to these operated muscles, resulted in a significantly greater maximal tetanic tension and motor unit size, when compared to untreated partially denervated muscles. This was achieved in the 4 - 6 day operated animals by an increased number of terminals and in the 17 - 19 day old animals by increased number of axonal sprouts that maintain contact with muscle fibres. In both groups of operated animals in the leupeptin treated muscles large numbers of motor units were able to maintain or achieve an expanded territory, whilst the size of the largest motor unit did not appear to be increased. It is proposed that leupeptin exerts its effect by inhibiting the degradative action of the neuronal calcium activated neutral protease on the axonal cytoskeleton. Such inhibition may act to prevent or decrease the degradation of cytoskeletal structures in the nerve terminal, and so provide protection for weak terminals at a synapse and growth cones of sprouting axons following partial denervation.

Plasticity of acid phosphatase (FRAP) afferent terminal fields and of dorsal horn cell growth in the neonatal rat.

Peripheral nerve section results in depletion of fluoride-resistant acid phosphatase (FRAP) from the nerve terminals in the dorsal horn of the spinal cord (Schoenen et al., '68) and this has been used in the past to map the termination field of individual nerves (Rustioni et al., '71; Devor and Claman, '80). In the present study we show that a similar central depletion occurs following sciatic nerve section or crush in neonatal rats. Unlike adults, however, the area of depletion is rapidly filled by sprouting of FRAP-containing afferent terminals from nearby intact peripheral nerves. The sprouting is extensive but never completely fills the depleted area. After nerve crush there is some recovery of FRAP from the sciatic nerve terminals themselves as well as from nearby nerve terminals. The source of recovered FRAP is demonstrated by resectioning or recrushing the nerves. The sprouting occurred when the sciatic was injured on day 1 but failed to take place when the injury was applied on or after day 10. Sciatic nerve section on day 1 also produces marked growth retardation of the ipsilateral dorsal horn gray matter that becomes more apparent as the rat matures. Nerve crush produces a less marked shrinkage that is slower in onset. If the nerve is crushed repeatedly, however, so that regeneration is prevented, the shrinkage is analogous to that following nerve section. No shrinkage occurs if the nerve is cut or crushed on day 10. The results show that separation of the spinal cord from its peripheral input at a critical stage in development results in disruption of the somatotopic organization of the C fibre afferent input to the dorsal horn and in slowing of growth of the dorsal horn gray matter.

Disturbances of neuromuscular interaction may contribute to muscle weakness in spinal muscular atrophy.

During a critical period of development, motoneurones and muscles are dependent on functional interaction with each other for their survival. In certain neuromuscular disorders such as spinal muscular atrophy (SMA), motoneurones die and muscle development is seriously affected. Recently advances have been made towards understanding the genetic basis of this disease, and a particular gene, i.e. the survival motoneurone gene (SMN), has been identified and found missing in SMA patients. Nevertheless the function of this gene is not clear, it may be involved in the control of the development of either the motoneurone or the muscle. Here we report that disrupting neuromuscular interaction during the early postnatal period has similar consequences on the development of muscles and motoneurones to those seen in patients with SMA, in that there is a loss of motoneurones and muscle function is severely impaired. In view of this, we discuss the possibility that these symptoms in SMA patients may be due to a disturbance of neuromuscular interaction during a critical stage of development.

Possible strategies for treatment of SMA patients: a neurobiologist's view.

This paper discusses possible strategies that might prevent or alleviate muscle weakness of SMA patients and hence improve their condition. The strategies discussed are as follows. (1) Prevention of motoneurone death. To achieve this two main approaches have been applied. Firstly, trophic factors have been used to prevent motoneurone death after nerve injury and clinically in diseases such as motoneurone disease. The results of these attempts will be described. Secondly, the possibility that injured motoneurones die as a result of the excitotoxic effects of the excitatory transmitter glutamate will be explored. Evidence will be presented which indicates that blocking glutamate receptors can rescue injured motoneurones from death. (2) Replacement of lost motoneurones by embryonic grafts. Motoneurones from grafts of embryonic spinal cord have been shown to survive in the adult spinal cord and are able to reinnervate skeletal muscles. The potential and practical problems of this approach will be discussed. (3) Expansion or motor unit territory of surviving motoneurones. Such an expansion of the territory occupied by individual motor units can be achieved by encouraging sprouting and ensuring that the newly formed connections between the motoneurone and muscle fibres are maintained, so that individual motor units are capable of developing more force. Strategies to achieve such an expansion of motor unit territory will be described. Finally, combinations of some of these approaches are considered.

Grafts of embryonic tissue into spinal cord: a possible strategy for treating neuromuscular disorders.

The article describes various approaches used to bring about repair of damaged spinal cord by using embryonic grafts of neuronal tissue. One approach is to stimulate the host's neuronal elements to grow and regenerate. Indeed embryonic grafts have been found to reduce the effects of spinal cord injury, and promote regrowth of axons across a lesion site at least to a limited extent. Attempts have also been made to restore the loss of supraspinal influences with grafts from embryonic brain, and transplants of aminergic neurones have been shown to compensate for the loss of aminergic supraspinal inputs. Finally, it is possible to replace loss of highly specialised cells such as motoneurones by grafts of embryonic spinal cord. Grafted embryonic motoneurones are able to survive within adult host cord although both their chances of survival and maturation seem improved by prior depletion of the host motoneurones. They are able to innervate a skeletal muscle via its peripheral nerve if this is co-implanted at the site of grafting but no axon growth has yet been detected into the host ventral root. However, grafted embryonic neurones are able to migrate away from the graft to sites once occupied by missing motoneurones in the host anterior horn. Within the context of the treatment of neuromuscular disease, the research described suggests possible stratagems for the treatment of disorders such as amyotrophic lateral sclerosis, spinal muscular atrophies or poliomyelitis either by employing grafts that could release neuroactive substances which might prevent existing cells from dying, or even by replacing missing motoneurones with transplanted embryonic motoneurones.

Blocking of NMDA receptors during a critical stage of development reduces the effects of nerve injury at birth on muscles and motoneurones.

Blocking of NMDA receptors during a critical stage of development reduces the effects of nerve injury at birth on muscles and motoneurones. Injury to the sciatic nerve at birth causes many motoneurones to soleus and extensor digitorum longus (EDL) muscles of rats to die. This is reflected in a reduction of motor units in these muscles. In the soleus only 4 (12.3%) motor units remain while 10 (24.3%) remain in the EDL, showing that soleus alpha motoneurones are more sensitive to nerve injury at birth. Treatment with MK-801, an NMDA receptor blocker, rescues a proportion of motor units in both muscles, so that in the soleus 11 (36%) and in the EDL 17 (42%) of motor units survive. This loss of motor units results in muscle weakness and a reduction in force of both muscles. Treatment with MK-801 reduces the effect of nerve injury, so that muscles of treated animals are stronger and weigh more. Cross-sectional area and muscle fibre number in EDL muscles were assessed and found to be dramatically reduced after nerve injury at birth, so that the area was 20% of control, with only 13% of fibres remaining. Moreover the majority of the remaining EDL muscle fibres which are normally fast are converted into slow type I fibres, with 68% of fibres expressing slow myosin compared with 3% in control EDL muscles. In animals treated with MK-801 only 47% of muscle fibres are lost after nerve injury at birth, hence the area of the muscle is greater (51% of control). The change of muscle phenotype induced by nerve injury is prevented and the muscle fibre composition resembles that of normal EDL muscles in that 4% of muscle fibres express slow myosin compared with 3.5% in control EDL muscles. Thus, blocking NMDA receptors with MK-801 shortly after nerve injury at birth reduces the loss of motor units and this is directly reflected in an improved performance of the affected muscles.

Motor activity patterns in rat soleus muscle after neonatal partial denervation.

In normal rats the development of organized patterns of hind limb movements takes place during the first three weeks of life. After removal of a part of the rat soleus muscle's innervation in 5-day-old animals, the remaining motoneurones occupy a large peripheral field. The possibility that the development of the normal activity patterns of these motor units may be altered was studied. The EMG activity of the soleus muscles partially denervated at five days was compared to that of the contralateral unoperated muscles during spontaneous locomotion and induced reflex activity in animals at various ages. Like a normal soleus the partially denervated soleus developed with age a tonic activity pattern but the aggregate activity recorded from the partially denervated soleus was less than that in the control muscle. However, the amount of activity per motor unit was higher in the operated than in the control muscles, since these had only one-third to half of their normal complement of motor units. During locomotion both soleus muscles were activated like typical ankle extensors during the stance phase of the step cycle, but the burst duration of the operated muscle was significantly shorter. We conclude that partial denervation shortly after birth leads to an overall increase in activity of the remaining soleus motor units but does not drastically alter their temporal pattern of use during locomotion.

The use of a neurotoxic lectin, volkensin, to induce loss of identified motoneuron pools.

In this study we investigated degeneration of defined motor pools in the adult rat spinal cord and the associated changes in spinal cord in dorsal root ganglia and peripheral nerve. Degeneration of motoneurons was induced by the neurotoxic lectin, volkensin. This substance is taken up by the axons and retrogradely transported to the cell body, where it inhibits proteosynthesis and kills the neuron. Accordingly, in adult Wistar rats the peroneal or the sciatic nerve was injected with 5.0 ng volkensin, and the effect of this single injection was investigated at different intervals after the operation. Retrograde labelling by horseradish peroxidase was used to reveal the extent of cell death and glial repair was studied by immunostaining with different glial cell markers. Degenerating cells were observed in the ventral horn of the lumbar spinal cord and L4 and L5 dorsal root ganglia as early as four days after volkensin treatment and by two weeks no retrogradely labelled motoneurons could be found in the treated peroneal pool. These changes were accompanied by severe muscle weight loss. Examination of the ventral horn of the spinal cord on the treated side revealed many hypertrophic astrocytes and reactive microglial cells expressing an increased level of complement receptor type 3 immunoreactivity. In the volkensin-injected peripheral nerve, distinct signs of Wallerian-like degeneration could be observed. Schwann cells identified by immunostaining to S-100 protein appeared to be preserved. Interestingly, at later stages after volkensin injection (four to eight weeks), some retrogradely labelled motoneurons were seen in the peroneal pool; their number occasionally reached 18.4% of the control pool. The dorsal root ganglia showed extensive loss of neurons and numerous abnormal neurons were found throughout the period of the study. These findings suggest that some motoneurons are able to recover from exposure to volkensin and temporary arrest of proteosynthesis. Despite this, volkensin-induced selective motoneuron death in the adult rat can be a useful experimental model for degenerative motoneuron disease.

Competitive reinnervation of the rat superior cervical ganglion by foreign nerves.

The efficacy of foreign nerves to form synapses was studied morphologically and physiologically in the superior cervical ganglion of the rat. The denervated ganglion was anastomosed to the central stump of either the vagus, the hypoglossal or both nerves together. The degree of reinnervation was assessed two to ten months later. We measured the strength of contraction of the nictitating membrane after each type of operation and compared this to the number, type and distribution of synapses in the same ganglion. Both the vagus and hypoglossal nerves preferentially reinnervated a population of neurones that are situated in the cranial pole of the superior cervical ganglion and supply the nictitating membrane. When both nerves were connected to the ganglion only the vagus nerve could be shown to reinnervate it, and no reinnervation by the hypoglossal nerve was detected. However, in this experiment neither foreign nerve did as well in competition as each did alone and the overall result was reduced functional efficiency. We conclude that not all sympathetic neurones are equivalent and that, just like sympathetic afferents, the foreign nerves are capable of selectively reinnervating preferred target cells.

Replacement of missing motoneurons by embryonic grafts in the rat spinal cord.

The ventral quadrant of embryonic spinal cord with its motoneurons prelabelled by 5-bromo-2'-deoxyuridine was grafted into the spinal cord of adult rats. The ventral horn of the host had been previously partially depleted of its own motoneurons by a neonatal nerve lesion. To enhance the chances of survival of the transplanted embryonic motoneurons a target muscle was provided for their axons. Two to three months after the grafts were inserted into the cord nuclei containing 5'-bromo-2'-deoxyuridine were found in the graft and in the host's spinal cord. Many of the stained nuclei were much larger than those of embryonic motoneurons, and their size distribution was similar to that of nuclei from control motoneurons. Retrograde labelling with horseradish peroxidase, injected into the target muscle provided for the embryonic motoneurons, showed that some motoneurons had reached the muscle and presumably made contact with it. Physiological and histological examination of the target muscle showed that it was innervated and that it contained at least three different types of muscle fibres. Thus embryonic motoneurons can survive and develop in the adult spinal cord. Moreover, they seem to be able to make functional connections with skeletal muscle fibres. The heterogeneity of the muscle indicates that the motoneurons that supply them are able to differentiate into various types of cells.

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.