Response of dystrophic muscles to reduced load

We have previously reported that, in dystrophic mice, functional overload has a damaging effect on the tibialis anterior (TA) muscle. In the present study, we have examined the effect of a load reduction on the TA and extensor digitorum longus (EDL) muscles. Our results show that reducing the passive load to which these muscles are subjected in dystrophic mice by resecting the Achilles tendon has a beneficial effect. The force output of the "released" EDL muscle improved, while the time course of contraction and relaxation of the "released" TA muscle became faster. Also in this muscle, resistance to fatigue became significantly greater. Low frequency electrical stimulation of the "released" muscles via implanted electrodes had little effect on their force output. It led, however, to a relative speeding of their time course of contraction and relaxation and to a further increase in their resistance to fatigue. Taken together, our results suggest that the beneficial effect of low frequency electrical stimulation on the force output of weak dystrophic muscles, described in the preceding paper, might be conditioned by the load to which these muscles are subjected.     

A study of the increased sensitivity of denervated and re-innervated muscle to depolarizing drugs

1. The response of re-innervated muscles to depolarizing blocking drugs was studied.

2. During the early stages of re-innervation the `new' neuromuscular junctions are more sensitive to decamethonium and suxamethonium. The recovery from the block and the repolarization of the end-plate are markedly slowed.

3. This increased sensitivity does not appear to be characteristic of all newly formed neuromuscular junctions. It was found that in new-born kittens the muscles are very insensitive to neuromuscular blocking drugs.

4. It is concluded that the increased sensitivity of re-innervated muscles to depolarizing blocking drugs and the slow repolarization are due to a persistence of changes which developed during denervation.

     

Changes in chemosensitivity of developing chick muscle fibres in relation to endplate formation

1. The sensitivity to acetylcholine (ACh) of the fast posterior latissimus dorsi (PLD) and slow anterior latissimus dorsi (ALD) during embryonic development was studied and compared. The sensitivities were expressed as a ratio of the maximal tetanic tension and tension developed in response to ACh. 2. Up to the 17th day of incubation both muscles are sensitive to ACh to a similar extent; at the 18th day the sensitivity of the PLD muscle decreases and continues to do so until hatching and thereafter. 3. Since the decrease in sensitivity of PLD muscles takes place a few days after innervation, it is suggested that this is caused by activity of the motor nerve. To test this curare (dTc) and hemicholinium (HC-3), drugs that interfere with neuromuscular transmission, were injected into the yolk sac of the embryos when nervemuscle connections are usually established. In the curare and HC-3 treated embryos the desensitization of the PLD muscles did not take place. 4. The distribution of endplates on PLD muscles from drug treated 20-21 day old embryos was compared to that of untreated controls. Whereas control PLD muscles have only one band of endplates, muscles from curarized embryos and HC-3 treated embryos have several bands of endplates, and many muscle fibres with multiple innervation were found. 5. It is suggested that nerve fibres which make connections with PLD muscle fibres bring about a decline in chemosensitivity by releasing more transmitter, and thereby prevent further nerve muscle connections from being made along the same muscle fibre.     

The use of local anaesthetics to produce prolonged motor nerve block in the study of denervation hypersensitivity

1. Silastoseal cuffs containing 25% and 45% lignocain, 25% marcain and 25% NaCl were placed onto the sciatic nerve. The effects of this on the sciatic nerve as well as the soleus and extensor digitorum longus (EDL) muscles was studied. 2. Cuffs containing local anaesthetics caused both early and late phases of paralysis, cuffs containing NaCl caused only the late phase of paralysis, showing that the late phase of paralysis was not due to the anaesthetic properties of the drugs. The paralysis produced by these procedures was irreversible. 3. Responses to indirect and direct stimulation of soleus and EDL muscles were compared to assess the degree of denervation. In case of cuffs containing 25% marcain and 45% lignocain more than half of the muscle fibres were denervated. 4. Acetylcholine sensitivity was assesses in all experiments. Soleus and EDL muscles from all animals that had cuffed nerves containing either local anaesthetics or 25% NaCl were hypersensitive to ACh. The degree of hypersensitivity could be correlated to the degree of "denervation" as assessed by comparing the directly and indirectly elicited twitch tensions. 5. It was found that the sciatic nerve undergoes degenerative changes when exposed to cuffs containing either local anaesthetics or 25% NaCl.     

The effect of long-term stimulation of fast muscles on their blood flow,metabolism and ability to withstand fatigue

Chronic stimulation of fast rabbit muscles (tibialis anterior, extensor digitorum longus and the peroneal muscle group) at a frequency naturally occurring in nerves to slow muscles increased their ability to withstand fatigue. Isometric tension decreased during a 10-min period of contractions at 4 Hz by 75% in control muscles, but only 55% in muscles chronically stimulated for 4 days, and 23% in muscles stimulated for 28 days. Chronic stimulation had little effect on resting blood flow, oxygen or glucose consumption. The output or consumption of lactate and free fatty acids (FFA) at rest were also unaffected. The glycogen content was regularly increased, and was apparent after only 2 days of stimulation. The activity of fatty acid activating enzyme was increased after 28 days. During a 10-min period of isometric contractions at 4 Hz, there was a markedly greater increase in blood flow and oxygen consumption in muscles stimulated for 14-28 days than in control muscles; lactic acid output was lower in muscles stimulated for 28 days, and the uptake of FFA was significantly higher. It is therefore suggested that muscles chronically stimulated for 14-28 days use fats as the main source of energy during isometric contractions. The predominantly oxidative metabolism is probably facilitated by the higher density of capillaries. The latter also enables more efficient delivery of oxygen, and therefore smaller fatiguability, already after 4 days of chronic stimulation.     

Two factors responsible for the development of denervation hypersensitivity

1. Innervated adult skeletal muscle is sensitive to acetylcholine at the end-plate region only. After denervation the entire muscle membrane becomes chemosensitive. The period of greatest increase in sensitivity in rat soleus muscles following section of the sciatic nerve in the thigh is between 48 and 72 hr post-operatively.2. Direct electrical stimulation was found to prevent the onset of the development of denervation hypersensitivity during the first 2-3 days after nerve section. Thereafter, electrical stimulation only reduced the sensitivity of denervated muscles to acetylcholine (ACh).3. The period of greatest increase in sensitivity follows loss of transmission and degeneration of the nerve terminals. Once this degeneration is under way, electrical stimulation is no longer as effective in preventing the development of denervation hypersensitivity.4. Hypersensitivity is also seen in muscles on which a small piece of thread or degenerating nerve has been placed. Hypersensitivity following these procedures declines within a few days, unlike denervation hypersensitivity which persists until innervation is restored.5. The present results suggest that activity alone cannot prevent the development of hypersensitivity in the presence of degenerating nerve fibres, or muscle damage. Activity does however counteract increased sensitivity. It is suggested that two factors interact to produce denervation hypersensitivity; the presence of degenerating nerve tissue and concomitant cellular changes bring about changes in the muscle fibre membrane causing it to become hypersensitive; and the loss of muscle activity, resulting in the persistence of hypersensitivity until innervation is restored.     

The importance of the motor nerve for the development of chemosensitivity at the neuromuscular junction

Experiments were performed on kittens to study the role of innervation on the development of the pharmacological responses of the neuromuscular junction. The importance of the nerve for the development of the differing properties of fast and slow muscles was also studied.It was confirmed that the different responses to depolarising drugs of the slow soleus and fast flexor hallucis longus muscles are not apparent during the first week of the animal's life.Even when the motor nerves to these muscles were crossed at this time the alien innervation did not affect the subsequent development of the different responses of fast and slow muscles to suxa- and decamethonium. This suggests that the different properties of the postsynaptic membrane are established very early in the animal's life and that they cannot be changed by altering the innervation.The neuromuscular junctions of these young animals are very insensitive to suxa- and decamethonium. When innervation is delayed, during the early postnatal period, the development of the high sensitivity of the neuromuscular junctions to the blocking and depolarising actions of these drugs was retarded. It is therefore concluded that the motor nerve induces the high chemosensitivity of the adult endplate region.     

Time course of changes in EMG activity of fast muscles after partial denervation

After partial denervation, the remaining motor units (MUs) of adult fast extensor digitorum longus muscle (EDL) expand their peripheral field. The time course of this event was studied using tension measurement and recordings of electromyographic (EMG) activity. The results show that after section of the L4 spinal nerve, when only 5.3 ± 0.63 of the 40 MUs normally supplying EDL muscle remain, the force of individual motor units starts to increase between the 1st and 2nd week after the operation and continues to do so for a further week. The drastic reduction of the number of motoneurones supplying the fast EDL leads to an increase in activity of the remaining MUs. In the 1st week after partial denervation, there was a sharp increase in the EMG activity of remaining motor units. During the next 12 days, this increase became less marked, but EMG activity remained nevertheless significantly higher than that of the unoperated EDL muscle. Many MUs became tonically active during posture. The EMG activity pattern during locomotion was also altered, so that the burst duration was positively correlated with the step cycle duration. Moreover, shortly after partial denervation, the interlimb coordination was disturbed but returned to its original symmetrical use 1–2 weeks later. Received: 17 September 1996 / Accepted: 3 November 1997     

Expression of cholinergic phenotype by embryonic ventral horn neurons transplanted into the spinal cord in the rat

Solid grafts of E12 embryonic spinal ventral horn were transplanted into motoneuron-depleted adult lumbar spinal cord in the rat. A muscle was implanted parallel to the vertebral column with its nerve inserted into the lumbar cord at the site of transplantation so as to provide a target for innervation by the grafted neurons. Previous retrograde labelling studies have shown that modest numbers of grafted motoneuron-like cells participate in the muscle's reinnervation and these are often found outside the graft within the host spinal cord. However, Nissl stained sections show that larger numbers of neurons survive within tissue recognisable as being of graft origin. In this study we have examined the expression of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) by neurons within the graft. These enzymes are involved in cholinergic neurotransmission and are characteristic of motoneurons. Thirty-four to seventy days following transplantation the grafts contained numerous neurons with acetylcholinesterase (AChE) activity. Different patterns of AChE staining were observed which probably reflected the degree of differentiation and maturation within the graft. AChE positive neurons were found in isolation or in groups resembling developing motor pools. Most of the AChE-positive neurons appeared immature with scant cytoplasm. However, neurons could be found which appeared relatively mature with a regularly shaped nucleus, prominent nucleolus and Nissl bodies. The grafts contained few AChE-positive axons and no dense plexuses of varicose fibres around the neurons such as are found around motoneurons in the mature ventral horn. Comparisons between the size of AChE-positive neurons in the graft and the size of AChE-positive neurons in the developing ventral horn found that the size of grafted neurons to be intermediate between the sizes of spinal motoneurons at E19 and P0. Far fewer grafted neurons were found to be immunoreactive for choline acetyltransferase (ChAT) than histochemically reactive for AChE. This was consistent with our findings in the spinal cord during normal development where we found that fixation and staining procedures which labelled adult motoneurons failed to reliably demonstrate ChAT immunoreactivety in normal motoneurons prenatally, although AChE histochemical reactivity could be demonstrated as early as E16. We conclude that the grafts contain numbers of immature motoneurons which fail to proceed beyond a certain stage of development, perhaps because of a failure to form appropriate efferent and afferent connections.     

The role of Ca2+ in the elimination of polyneuronal innervation of rat soleus muscle fibres

The mechanism by which nerve - muscle contacts are reduced during postnatal development of the rat soleus muscle was investigated using electrophysiological methods. Between days 7 and 9 after birth, soleus muscle fibres lose 0.19–0.24 terminals per muscle fibre within 24 h. A much more rapid loss of contacts is seen when muscles are exposed in vitro to acetylcholine (10⁻³ g/ml). In this case 0.67–0.87 terminals per muscle fibre lose contact within 2 h. The loss of neuromuscular contacts induced by acetylcholine can be reduced by preincubating the muscles in solutions containing acetoxymethyl ester of 1,2-bis(2-amino-phenoxylethane-N,N₁;N₁-tetraacetic acid (BAPTA-AM), a Ca²⁺ chelating agent that enters cells and reduces the Ca²⁺ transients inside the cell. Treatment of muscles with nifedipine, which blocks dihydropyridine-sensitive (L-type) Ca²⁺ channels, also reduced the acetylcholinesterase-induced loss of neuromuscular contacts. The results indicate that transient increases in Ca²⁺ inside nerve terminals contribute to loss of neuromuscular contacts, and that these increases occur by Ca²⁺ entry through L-type channels.