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Transient disruption of nerve-muscle interaction shortly after birth permanently alters the development of the rat soleus muscle
DEVELOPMENTAL BRAIN RESEARCH
ELSEVIER
Developmental Brain Research 94 (1996) 152-158
Research report
Transient disruption of nerve-muscle interaction shortly after birth permanently alters the development of the rat soleus muscle Linda Greensmith, Department
Angela
H. Hind, Gerta Vrbovi
ofAnatomy and Developmental Biology, Centre for Neuroscience,
*
Unioersity College London, Gower Street, London WCIE 6BT. UK
Accepted 23 January 1996
Abstract Transient paralysis of the rat soleus muscle shortly after birth leads to a permanent loss of motoneurones as revealed by retrograde labelling. Here we show that this loss of motoneurones is reflected in a reduction in the number of motor units. Soleus muscles in normal adult rats were found to have 27 ( + 0.6 S.E.M., IZ= 9) motor units. However, in muscles which had been treated with o-bungarotoxin (BTX) at birth and 3 days of age, causing paralysis lasting for 6-8 days, only 15 (k 0.6 S.E.M., n = 5) motor units remain. The effects of paralysis on the ability of the adult soleus muscle to develop force was also tested. Following treatment with a single BTX implant at
birth, causing paralysis for 2-3 days, soleus muscles develop less tension (73.7% +4.5 S.E.M., n = 8) and weigh less (88.2% k3.8 S.E.M., II = 13) than their unoperated controls. This loss of muscle force is caused by a loss of muscle fibres, which in muscles that had been paralysed at birth was 81.4% ( k4.1 S.E.M., n = 5) of control. Prolonging the duration of paralysis led to a greater reduction in force production, weight and the number of muscle fibres. Those muscles which had been paralysed at birth also took longer to relax during single twitch contractions. In addition, whereas normal soleus muscles contain around 20% of muscle fibres that do not react with antibodies to slow myosin HC, in soleus muscles paralysed at birth, 100% of the fibres reacted with this antibody. This study shows that disruption of neuromuscular interaction for a brief period after birth leads to a loss of motoneurones and a permanent impairment of muscle function. Keywords:
Motoneuron; Nerve-muscle interaction; Activity; Paralysis; Motor unit; Development
1. Introduction Following nerve injury in neonatal rats, a large proportion of motoneurones die [ 12,191 and as a consequence there is a severe impairment of muscle development and function [4]. Although after nerve crush, the axons of the surviving motoneurones are able to grow back to their target and establish contact with a number of muscle fibres, they are unable to increase their peripheral field and innervate the many denervated muscle fibres available to them [l l]. It is possible that nerve injury during a critical period in postnatal development limits the ability of injured axons to grow sufficient branches to reinnervate more fully the denervated muscle fibres. However, this is
* Corresponding author. Fax: (44) (171) 380 7349. 0165-3806/96/$15.00
0 1996 Elsevier Science B.V. All rights reserved
PII SO165-3806(96100037-5
unlikely, since previous results have indicated that after a nerve crush at 5 days of age, which results in little or no motoneurone death, muscle recovery is poor [ll]. The axons of these motoneurones are able to form many branches but these make inappropriate contacts with already innervated muscle fibres [12]. Thus, factors other than either the loss of motoneurones or injury to the nerve determine the success of recovery. We have therefore proposed that it is not nerve injury but rather the loss of neuromuscular interaction during a critical period of postnatal development that is detrimental to the development of muscles and motoneurones. Neuromuscular interaction can be disrupted by blocking the postsynaptic acetylcholine receptor (AChR) with (Ybungarotoxin (BTX). This snake toxin binds irreversibly to the AChR, and thereby prevents neuromuscular transmission. In previous experiments we have paralysed the soleus muscle of newborn rats with BTX and examined the effects of this treatment on the neuromuscular junction [7].
L. Greensmith et al. /Developmental
Shortly after recovery from paralysis, at 4-5 days of age, there is a reduction in the number of neuromuscular contacts, followed by a transient increase of contacts 9-18 days later. In normal animals of 2-3 weeks of age polyneuronal innervation typical of younger animals has already disappeared and the adult pattern of 1 axon terminal per endplate has emerged [ 1,16,18]. Thus, transient muscle paralysis induces nerve axons to grow during a time when synapse elimination is normally complete. This growth was induced without any physical damage to the axon. The study of the effects of this extended period of axonal growth on the survival of motoneurones revealed that a large proportion of motoneurones failed to survive [8]. However, whether the motoneurones that survive are capable of compensating for those that die and colonise the denervated muscle fibres is not known. The present study was designed to investigate firstly, whether the loss of motoneurones following neonatal muscle paralysis observed with retrograde labelling occurs in alpha as well as gamma motoneurones, and secondly, whether the motoneurones which survive are able to expand their peripheral territory and reinnervate muscle fibres that have lost their input.
Brain Research 94 (19961 152-1.58
were implanted into control animals and contained and 240 p,g NaCl respectively. 2.3. Isometric
153
120 kg
tension recordings
Two to ten months after birth, the animals were anaesthetised with chloral hydrate (4% one ml/100 g body weight, i.p.) and the soleus muscles in both the operated and contralateral control legs were prepared for assessment of their contractile properties in situ. The soleus muscles in each hindleg were dissected free of their surrounding tissue, the legs rigidly secured to a table with stainless steel pins, and the distal tendons attached to isometric force transducers (Dynamometer UFI, Devices) via a silk thread. The soleus motor nerve was dissected free and prepared for stimulation. All nerves to other muscles of the leg as well as the L4 and L5 ventral rami were cut. The length of the muscle was adjusted so that it developed maximal twitch tension. Isometric contractions were elicited by stimulating the motor nerve using a pulse width of 0.02 ms. Tetanic contractions were elicited by stimulating the soleus muscle at 20, 40 and 80 Hz for 800 ms. 2.4. Motor unit numbers and sizes
2. Material and methods 2.1. Surgical procedures Using ether anaesthesia and sterile conditions, a small silicon rubber strip containing either o-bungarotoxin (Sigma) or NaCl, was implanted alongside the soleus muscle in the right hindlimb of neonatal Wistar rats, within 6-12 h of birth. The silicon strip was positioned between the soleus and the flexor hallucis longus muscles away from the point of nerve entry to the muscle. The contralatera1 leg was left unoperated and used as a control. When the animals had fully recovered from the anaesthesia they were returned to their mothers and reared as normal. In some of these operated animals, a second silicon strip was implanted 3 days after the initial operation. Under the same conditions as previously described, the first silicon strip was located and removed from the hindleg and replaced with a larger implant containing either BTX or NaCl, as appropriate. These animals were then returned to their mothers. 2.2. Preparation
of implants
The preparation of BTX containing implants has been previously described [7]. Silicon strips weighing 0.3 mg were implanted into the hindleg of newborn rats, and contained approximately 7 p_g of BTX and 120 p_g NaCl. Strips weighing 0.75 mg were implanted into the hindlegs of 3 day old rats, and these implants contained 16 kg BTX and 240 Fg NaCl. Control implants of similar weights
To estimate the number of motor units in each muscle, the motor nerves of the operated and, in some experimental animals contralateral soleus muscles were stimulated every 4 s. The stimulus strength was gradually increased to obtain stepwise increments of twitch tension, as individual motor axons are recruited. The number of stepwise increments was counted to give an estimate of the number of motor axons present in the nerve. 2.5. Histology and immunocytochemistry After all the tension experiments had been completed, the control and operated soleus muscles were dissected out from the rats and weighed. The dissected soleus muscles were stretched to a length similar to that in the leg and control and operated muscles mounted side by side for each animal and frozen in isopentane cooled with liquid nitrogen. To determine the number, size and types of muscle fibres present, transverse cryostat sections (10 km) were collected onto gelatinized slides (0.5%) and stained for succinate dehydrogenase (SDH) activity [ 141. Immunocytochemistry was performed using a specific antibody against slow myosin (provided as a gift from Dr. G. Dhoot). The total number of muscle fibres and the number of slow myosin positive fibres per muscle was counted in experimental and control muscles. The cross-sectional area of operated and contralateral control muscles in both the NaCl- and BTX-treated animals was measured using a camera lucida and graphic digitizing tablet linked to a computer.
154
L. Greensmith et al./Deuelopmental
3. Results 3.1. Effects of temporary paralysis at birth on the deuelopment of the rat soleus muscle 3. I. I. Immediate consequences of transient muscle paralysis Routine haematoxylin-van Gieson staining of transverse sections from soleus muscles revealed that the implant containing either BTX or NaCl caused no damage to muscle fibres - all muscle nuclei were located peripherally and the only visible effects of the implant was the presence of a small amount of connective tissue along the surface of the muscle in the area of the implant. Treatment of the soleus muscle with BTX in neonatal rats in the manner described here has previously been reported to abolish neuromuscular transmission. Placing a BTX implant onto the muscle at birth caused complete paralysis for the first 24 h. The muscles gradually recov-
Brain Research 94 (1996) 152-158
ered from the paralysis and by 8-9 days postnatally transmission was fully restored [7]. Treatment with a second BTX implant at 3 days of age prolonged the duration of muscle paralysis for a further 3-4 days, so that in 8 day old animals the soleus muscles were still partially paralysed [8]. Muscle paralysis is likely to affect muscle development and its ability to develop force. Oxidative enzymes are known to be linked to muscle activity [lo]. Therefore, in the present study, succinate dehydrogenase (SDH) was visualised in operated and contralateral control soleus muscles. Muscles treated at birth with BTX showed less intense staining for SDH when compared to controls 14 and 28 days after BTX treatment. This finding is illustrated in Fig. lA,B for muscles after 14 days, where the staining intensity of untreated soleus muscle (Fig. 1A) is compared to that observed in the BTX-treated soleus muscle taken from the same animal (Fig. IB), and in Fig. lC,D for soleus muscles 28 days after BTX treatment. Even at this
Fig. 1. This figure shows examples of cross-sections taken from control and BTX-treated soleus muscles and stained for SDH. (A) shows a section from a control and (B) from a BTX-treated muscle taken from the same 14 day old animal. (C) shows a section from a control and (D) from a BTX-treated animal at 28 days of age. The scale bar represents 20 pm in (A) and (B), and 50 p,rn in (C) and CD).
L. Greensmith et al./Deuelopmental
155
Bruin Research 94 (1996) 152-158
C
1w
NaCl
BTX
2BTX
NaCl
BTX
2BTX
I
Fig. 2. Twitch and tetanic contractions developed by adult soleus muscles in response to stimulation of their motor nerves are shown. (A) and (B) arc records from a 3 month old rat whose soleus muscle on one side (B) was treated with a single BTX implant at birth and (C) is from the control side of rat which had its soleus muscle on one side (D) treated with BTX at birth and 3 days of age.
time the soleus muscles had less intense staining than control muscles. 3.1.2. Long-term sis
consequences
for SDH I
of transient muscle paraly-
3.1.2.1. Muscle tensions and weights. When the animals were between 2 and 10 months old, long after the muscle paralysis had worn off, the animals were re-anaesthetised and the soleus muscles in the treated and contralateral control legs prepared for in situ assessment of muscle force. For each experiment the maximum tetanic tension (MTT) of the control and treated soleus muscle was recorded and the MTT of the treated muscle was expressed as a percentage of that produced by the contralateral control. Table 1 and Fig. 2 show that soleus muscles that had been transiently paralysed shortly after birth and were examined when adult, develop significantly less tension than control NaCl-treated muscles. In animals where muscle paralysis was prolonged by treatment with a second BTX implant at 3 days of age the decrease of tension output was significantly greater. Muscles treated with a single BTX implant at birth developed 74% (f4.5 S.E.M., n = 13) of the tension developed by the contralateral control muscles, and muscles subjected to a more prolonged
Fig. 3. The block diagrams show changes of (A) maximum tetanic tension and (B) muscle fibre numbers expressed as a percentage of the control unoperated soleus muscles, taken from adult animals that had their soleus muscles on one side treated with either NaCI, a single BTX implant at birth or 2 BTX implants at birth and 3 days of age.
paralysis (2 BTX) developed only 47% (k3.3 S.E.M., n = 6) of the force produced by their contralateral controls (Mann-Whitney U-test, P < 0.01 for both groups). The results are summarised in Fig. 3A, which clearly illustrates that the tension developed by the muscles treated with 2 BTX implants was significantly less than that developed by those treated with a single implant (Mann-Whitney U-test, P < 0.01). At the end of the tension recording experiments, the experimental and the contralateral control soleus muscles were removed from the animals and weighed. The reduction in force output observed in those muscles which had been paralysed at birth was reflected in a reduction in muscle weight of these muscles (see Table 1). Thus, following treatment with a single BTX implant at birth,
Table 1 Mean values (+ S.E.M.) of maximum tetanic tension and muscle weights from adult animals treated with either NaCl, a single BTX implant at birth or two BTX implants at birth and 3 days are shown Treatment
NaCl BTX 2BTX
n
4 8 4
The % operated/control
Maximum
n
tetanic tension
Control
Operated
%op/con
116.5 + 7.5 132.4 + 14.8 192.0 + 18.6
146.5 f 15.7 102.1 * 11.1 88.4 f 5.8
117.Ok7.3 73.7 f 4.5 49.5 f 4.3
was calculated
9 13 8
Muscle weight Control
Operated
%op/con
0.163 kO.011 0.169 + 0.014 0. I84 f 0.029
0.163 + 0.012 0.150 * 0.013 0.120 & 0.017
101.3 f 4.4 88.2 + 3.8 66.0 * 3.4
for each animal and the mean values (+ S.E.M.) are given; n shows the number of animals in each group,
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L. Greensmith et al./Deuelopmental
Brain Research 94 (1996) 152-158
Table 2 The mean total numbers (+S.E.M.) of muscle fibres from adult soleus muscles treated with either NaCI, a single BTX implant at birth or two BTX implants at birth and 3 days are shown Treatment
NaCl BTX 2BTX
n
Number of muscle fibres
3 5 3
Control (left)
Operated (right)
%op/con
2887+ 35 2821 f 234 2659+110
2793 + 103 2278+200 1576+ 150
96.7 * 2.4 81.4k4.1 56.8 f 4.4
The % operated/control was calculated for each animal and the mean values (*S.E.M.) are given; n shows the number of animals in each group.
soleus muscles in adult animals weighed 88% (k3.7 S.E.M., n = 13) of their contralateral control muscles, and those soleus muscles subjected to a prolonged muscle paralysis weighed significantly less, 66% ( * S.E.M., n = 8; Mann-Whitney U-test, P < 0.05 and < 0.001, respectively). 3.1.2.2. Muscle fibre numbers. The relative reduction of force output of muscles treated with BTX described above may be due to either a decrease in the size of muscle fibres or to a reduction in the number of muscle fibres. To distinguish between these two possibilities, the total number of muscle fibres in control and BTX-treated muscles was established and the results are summarised in Table 2 and Fig. 3B. The number of muscle fibres found in normal and unoperated contralateral soleus muscles ranged between 2500-2900, which is in good agreement to data previously reported [2,13]. In soleus muscles paralysed with BTX at birth and examined later, there is a 19% decrease in the number of muscle fibres. This decrease is significant when compared to the contralateral muscles (Mann-Whitney U-test, P < 0.04). Following a more prolonged muscle paralysis, by treatment with two BTX implants, there is a greater decrease in the number of muscle fibres, so that 47% of fibres in the treated muscle are lost (Mann-Whitney U-test, P < 0.03). The number of muscle fibres present in soleus muscles treated with NaCl implants was not significantly different from normal, untreated soleus muscles or contralateral control soleus muscles. It appears therefore, that the reduction in tension developed by the BTX-treated soleus muscles is caused by a reduction of the number of muscle fibres in these muscles. A comparison of Fig. 3A and 3B shows that the reduction in Table 3 The times to peak twitch tension and half relaxation of single twitch contractions in one leg treated with either NaCl, BTX at birth or BTX at birth and 3 days Treatment
NaCl BTX 2BTX
n
5 7 4
The % operated/control
3.1.2.3. Motor unit number. The reduction in force output and the loss of muscle fibres in soleus muscles treated with BTX may have been caused by a loss of motoneurones. Previous results with retrograde labelling show that nearly 40% of the total number of motoneurones to the soleus muscle die after treatment with BTX at birth, and nearly 65% die following prolonged muscle paralysis [8]. In order to assess the effect of transient muscle paralysis on the survival of a-motoneurones only, estimates of the number of motor units in the treated and contralateral control soleus muscles were made by counting the increments of twitch tension elicited from the muscles by stimulation of the soleus motor nerve with stimuli of increasing intensity. Since the effect on muscle force and fibre number were greater following treatment with 2 BTX implants, motor unit number was assessed only in those animals which had been paralysed for a prolonged period after birth. In control experiments, the normal soleus muscle was found to have a mean of 27 ( f 0.6 S.E.M., n = 9) motor units. In soleus muscles subjected to prolonged paralysis at birth (2BTX) the number of motor units present was much less, with a mean of 15 (+ 0.6 S.E.M., n = 5) which is significantly different from that found in the control soleus muscles (Mann-Whitney U-test, P = 0.005). Thus, application of (-w-BTX during early postnatal development leads to a permanent loss of o-motoneurones. 3.1.2.4. Contractile properties and muscle fibre characteristics. Table 3 summarises the results of measurements of time to peak twitch tension and half relaxation obtained from the various experimental groups studied. The results show that the time to half relaxation was prolonged in muscles treated with BTX at birth, and this effect was greater in animals that were subjected to a prolonged
are shown for soleus muscles in adult animals that had the soleus muscle n
Time to peak Control
Operated
%op/con
80 + 8.4 106.4 f 8.4 76.3 f 6.3
76 k8.1 98.9 f 6.3 86.3 k 8.5
95.3 k 2.9 97.4 * 11.3 118.3 i 22.5
was calculated
muscle fibre number clearly reflects the decrease in muscle force observed after either a single or double BTX implant. It is nevertheless possible that a change in muscle fibre area may influence the force output in the BTX-treated muscles. Therefore, in some animals the muscle fibre areas were measured from haematoxylin-van Gieson stained transverse sections of the control and experimental muscles. The mean muscle fibre area in BTX-treated muscles was 95% (i 19.6 S.E.M., n = 3) of the untreated contralateral control muscles, and 93% (_t 2.6 S.E.M., n = 4) from muscles treated with two BTX implants.
5 5 4
Time to half relaxation Control
Operated
%op/con
111 k11.2 96 k5.1 67.5 + 4.8
99 * 13.3 126 + 19.6 110 f 18.7
88.9 f 5.2 88.2 f 3.8 160.7 + 19.0
for each animal and the mean values ( f S.E.M.) are given; n shows the number of animals in each group.
L. Greensmith et al./Deuelopmental
paralysis. Thus, it appears that paralysing the soleus muscle with BTX at birth enhances the ‘slow’ characteristics of this muscle. Whether this change is reflected in the distribution of fibre types in the adult soleus muscle was also studied. Staining of control soleus muscles for SDH, a marker of oxidative capacity, revealed the presence of large fibres of intermediate staining intensity in addition to a few small, darker stained fibres. Following temporary paralysis of the soleus muscle at birth, there were fewer darkly stained fibres and the overall level of staining for SDH appeared to be less than in controls. In addition, prolonging the duration of paralysis results in a greater reduction in SDH staining. Treated and contralateral control muscles were also studied using an antibody to the slow myosin heavy chain (kindly donated by Dr. G. Dhoot). Contralateral control soleus muscles stained with the slow myosin antibody showed that approximately 80% of the total number of muscle fibres expressed slow myosin, so that around 20% of fibres did not stain for slow myosin, while muscles subjected to a period of transient muscle paralysis by treatment with either 1 or 2 BTX implants after birth, were composed entirely of muscle fibres that stained positively for slow myosin.
4. Discussion Transient disruption of neuromuscular transmission in neonatal rats has been previously shown to result in the death of a large proportion of motoneurones to the soleus muscle [8]. These results were obtained by retrograde labelling, so that both alpha and gamma motoneurones were included in the analysis of motoneurone survival. The present study, using physiological assessment of the number of motor units present in BTX-treated soleus muscles, specifically examines the extent of survival of alpha motoneurones only and shows (a) that alpha motoneurones are lost following this period of early muscle paralysis, and (b) that those motoneurones which do survive this disruption of nerve:muscle interaction are unable to expand their peripheral field to occupy the many denervated muscle fibres available to them. What may be the cause of motoneurone death following a period of muscle paralysis at birth? We have recently shown that treatment with BTX at birth renders motoneurones susceptible to the toxic effects of the glutamate agonist NMDA [9]. Furthermore, treatment with MK-801 prevents the death of a proportion of motoneurones that would otherwise die following BTX treatment at birth [9]. Thus, preventing neuromuscular interaction during a critical period of development, either by nerve injury or treatment with BTX, results in an increased sensitivity of the affected motoneurones to the toxic effects of glutamate. Not only are motoneurones to paralysed muscles more sensitive to NMDA, the surviving motoneurones are also
Brain Research 94 (1996) 152-158
157
likely to be more active than normal motoneurones. One indication that the motoneurones to the BTX-treated muscles are more active is the finding that these soleus muscles have a prolonged time to half relaxation and contain no muscle fibres which stain positively for fast myosin. In other experimental situations where a reduction in the number of motor units is observed, either following partial denervation or nerve crush injury, EMG recordings demonstrate that there is an increase in the activity of the surviving motoneurones [15,20,21]. Such an increase in motoneurone activity may further contribute to the death of motoneurones deprived of interaction with their target. It is possible that this increased activity of the surviving motoneurones is also a contributing factor to their inability to expand their peripheral field. There is much evidence which shows that activity can induce the loss of neuromuscular contacts during the elimination of polyneuronal innervation which normally takes place during the first two weeks of postnatal life [.5,6,16,17]. The period in which the elimination of superfluous nerve terminals from muscle fibres takes place is altered in muscles paralysed with BTX at birth. As the muscles recover from the paralysis and neuromuscular activity resumes, weak synaptic contacts are lost [7] and as a consequence many muscle fibres die, which is reflected in the small size of the remaining units, as seen in the present study. It was previously shown in partially denervated muscles treated with BTX, that a large number of newly formed sprouts initially made contact with the denervated muscle fibres, but these contacts were lost when the muscles recovered from the paralysis and neuromuscular activity was resumed [3]. A similar loss of such weak contacts upon resumption of activity may also occur in the present experiments and this may explain why the motoneurones which survive after treatment with BTX at birth do not have an enlarged peripheral field. The results presented here clearly demonstrate that interaction between the motoneurone and its target muscle during early postnatal development is essential not only for motoneurone survival, but also for the ability of those motoneurones which do survive target deprivation to expand and maintain a large peripheral field.
Acknowledgements We are grateful to The Wellcome Trust and Action Research for their support. A.H. was in receipt of a SERC studentship. We would also like to thank Mr J. Dick for his excellent technical assistance.
References [l] Brown, M.C., Jansen, J.K.S. and Van Essen, D.C., Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation, _I. Physiol., 261 (1976) 387-422. [2] Connold, A.L. and VrbovB, G., Temporary loss of activity prevents
158
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[lo]
[11]
L. Greensmith
et al. /Deuelopmental
the increase in motor unit size in partially denervated rat soleus muscles, J. Physiol., 434 (1991) 107-119. Connold, A.L. and Vrbov& G., Neuromuscular contacts of expanded motor units in rat soleus muscles are rescued by leupeptin, Neuroscience, 63 (1995) 327-338. Dick, J., Greensmith, L. and VrbovB, G., The effects of nerve injury in neonatal rats on motor unit numbers and properties are prevented by blocking NMDA receptors, Neuromusc. Disord., 5 (1995) 371382. Duxson, M.J., The effect of postsynaptic block on development of the neuromuscular junction in postnatal rats, J. Neurocytol., 11 (1982) 395-408. Duxson, M.J. and VrbovB, G., Inhibition of acetylcholinesterase accelerates axon terminal withdrawal at the developing rat neuromuscular junction, J. Neurocytol., 14 (1985) 337-363. Greensmith, L. and VrbovB, G., Neuromuscular contacts on the developing rat soleus depend upon muscle activity, Deu. Brain Res., 62 (1991) 121-129. Greensmith, L. and VrbovB, G., Alterations of nerve-muscle interactions during postnatal development influence motoneurone survival in rats, Deu. Brain Res., 69 (1992) 125-131. Greensmith, L., Sanusi, J., Mentis, G.Z. and VrbovB, G., Transient muscle paralysis in neonatal rats renders motoneurones susceptible to NMDA-induced neurotoxicity, Neuroscience, 64 (1995) 109-l 15. Kugelberg, E. and Lindegren, B., Transmission and contraction fatigue of rat motor units in relation to succinate dehydrogenase activity of motor unit fibres, J. Physiol. 288 (1979) 285-300. Lowrie, M.B., Krishnan, S. and VrbovB, G., Recovery of slow and fast muscles following nerve injury during early postnatal development in the rat, J. Physiol., 331 (1982) 56-66.
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94 (1996) 152-158
[12] Lowrie, M.B., Subramaniam Krishnan and VrbovB, G., Permanent changes in muscle and motoneurones induced by nerve injury during a critical period of development of the rat, Dec. Brain Res., 310 (1987) 91-101. [13] McArdle, J.J. and Sansone, F.M., Reinnervation of fast and slow twitch muscle following nerve crush at birth, J. Physiol., 271 (1977) 567-586. 1141 Nachlas, M.M., Tson, K.C., DeSouza, E., Chang, C.H. and Seligman, A.M., Cytochemical demonstration of succinate dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole, J. Hisrochem.
Cy’tochem., 5 (1957)
[15] Navarrete,
[16]
[17]
[18] [I91 [20]
[21]
420-436.
R. and VrbovB, G., Differential effect of nerve injury at birth on the activity pattern of reinnervated slow and fast muscles of the rat, J. Physiol., 351 (1984) 675-685. O’Brien, R.A.D., &tberg, A.J.C. and VrbovB, G., Observations on the elimination of polyneuronal innervation in developing mammalian skeletal muscle, J. Physiol., 282 (1978) 571-582. O’Brien, R.A.D., &tberg, A.J.C. and Vrbovl, G., The effect of acetylcholine on the function and structure of the developing mammalian neuromuscular junction, Neuroscience, 5 (1980) 1367- 1379. Redfern, P.A., Neuromuscular transmission in newborn rats, J. Physiol., 209 (1970) 70 l-709. Romanes, G.J., Motor localisation and the effects of nerve injury on the ventral horn cells of the spinal cord, J. Anat., 80 (1946) 1 l- 13 1, Slawibska, U., Navarrete, R., Kasicki, S. and VrbovL, G., Motor activity patterns in rat soleus muscle after neonatal partial denervation, Neuromusc. Disord., 5 (1995) 179-I 86. Tyc’, F. and VrbovB, G., The effect of partial denervation of developing rat fast muscles on their motor unit properties, J. Physiol., 482.3 (1995)
65 I-660.
ELSEVIER
Developmental Brain Research 94 (1996) 152-158
Research report
Transient disruption of nerve-muscle interaction shortly after birth permanently alters the development of the rat soleus muscle Linda Greensmith, Department
Angela
H. Hind, Gerta Vrbovi
ofAnatomy and Developmental Biology, Centre for Neuroscience,
*
Unioersity College London, Gower Street, London WCIE 6BT. UK
Accepted 23 January 1996
Abstract Transient paralysis of the rat soleus muscle shortly after birth leads to a permanent loss of motoneurones as revealed by retrograde labelling. Here we show that this loss of motoneurones is reflected in a reduction in the number of motor units. Soleus muscles in normal adult rats were found to have 27 ( + 0.6 S.E.M., IZ= 9) motor units. However, in muscles which had been treated with o-bungarotoxin (BTX) at birth and 3 days of age, causing paralysis lasting for 6-8 days, only 15 (k 0.6 S.E.M., n = 5) motor units remain. The effects of paralysis on the ability of the adult soleus muscle to develop force was also tested. Following treatment with a single BTX implant at
birth, causing paralysis for 2-3 days, soleus muscles develop less tension (73.7% +4.5 S.E.M., n = 8) and weigh less (88.2% k3.8 S.E.M., II = 13) than their unoperated controls. This loss of muscle force is caused by a loss of muscle fibres, which in muscles that had been paralysed at birth was 81.4% ( k4.1 S.E.M., n = 5) of control. Prolonging the duration of paralysis led to a greater reduction in force production, weight and the number of muscle fibres. Those muscles which had been paralysed at birth also took longer to relax during single twitch contractions. In addition, whereas normal soleus muscles contain around 20% of muscle fibres that do not react with antibodies to slow myosin HC, in soleus muscles paralysed at birth, 100% of the fibres reacted with this antibody. This study shows that disruption of neuromuscular interaction for a brief period after birth leads to a loss of motoneurones and a permanent impairment of muscle function. Keywords:
Motoneuron; Nerve-muscle interaction; Activity; Paralysis; Motor unit; Development
1. Introduction Following nerve injury in neonatal rats, a large proportion of motoneurones die [ 12,191 and as a consequence there is a severe impairment of muscle development and function [4]. Although after nerve crush, the axons of the surviving motoneurones are able to grow back to their target and establish contact with a number of muscle fibres, they are unable to increase their peripheral field and innervate the many denervated muscle fibres available to them [l l]. It is possible that nerve injury during a critical period in postnatal development limits the ability of injured axons to grow sufficient branches to reinnervate more fully the denervated muscle fibres. However, this is
* Corresponding author. Fax: (44) (171) 380 7349. 0165-3806/96/$15.00
0 1996 Elsevier Science B.V. All rights reserved
PII SO165-3806(96100037-5
unlikely, since previous results have indicated that after a nerve crush at 5 days of age, which results in little or no motoneurone death, muscle recovery is poor [ll]. The axons of these motoneurones are able to form many branches but these make inappropriate contacts with already innervated muscle fibres [12]. Thus, factors other than either the loss of motoneurones or injury to the nerve determine the success of recovery. We have therefore proposed that it is not nerve injury but rather the loss of neuromuscular interaction during a critical period of postnatal development that is detrimental to the development of muscles and motoneurones. Neuromuscular interaction can be disrupted by blocking the postsynaptic acetylcholine receptor (AChR) with (Ybungarotoxin (BTX). This snake toxin binds irreversibly to the AChR, and thereby prevents neuromuscular transmission. In previous experiments we have paralysed the soleus muscle of newborn rats with BTX and examined the effects of this treatment on the neuromuscular junction [7].
L. Greensmith et al. /Developmental
Shortly after recovery from paralysis, at 4-5 days of age, there is a reduction in the number of neuromuscular contacts, followed by a transient increase of contacts 9-18 days later. In normal animals of 2-3 weeks of age polyneuronal innervation typical of younger animals has already disappeared and the adult pattern of 1 axon terminal per endplate has emerged [ 1,16,18]. Thus, transient muscle paralysis induces nerve axons to grow during a time when synapse elimination is normally complete. This growth was induced without any physical damage to the axon. The study of the effects of this extended period of axonal growth on the survival of motoneurones revealed that a large proportion of motoneurones failed to survive [8]. However, whether the motoneurones that survive are capable of compensating for those that die and colonise the denervated muscle fibres is not known. The present study was designed to investigate firstly, whether the loss of motoneurones following neonatal muscle paralysis observed with retrograde labelling occurs in alpha as well as gamma motoneurones, and secondly, whether the motoneurones which survive are able to expand their peripheral territory and reinnervate muscle fibres that have lost their input.
Brain Research 94 (19961 152-1.58
were implanted into control animals and contained and 240 p,g NaCl respectively. 2.3. Isometric
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tension recordings
Two to ten months after birth, the animals were anaesthetised with chloral hydrate (4% one ml/100 g body weight, i.p.) and the soleus muscles in both the operated and contralateral control legs were prepared for assessment of their contractile properties in situ. The soleus muscles in each hindleg were dissected free of their surrounding tissue, the legs rigidly secured to a table with stainless steel pins, and the distal tendons attached to isometric force transducers (Dynamometer UFI, Devices) via a silk thread. The soleus motor nerve was dissected free and prepared for stimulation. All nerves to other muscles of the leg as well as the L4 and L5 ventral rami were cut. The length of the muscle was adjusted so that it developed maximal twitch tension. Isometric contractions were elicited by stimulating the motor nerve using a pulse width of 0.02 ms. Tetanic contractions were elicited by stimulating the soleus muscle at 20, 40 and 80 Hz for 800 ms. 2.4. Motor unit numbers and sizes
2. Material and methods 2.1. Surgical procedures Using ether anaesthesia and sterile conditions, a small silicon rubber strip containing either o-bungarotoxin (Sigma) or NaCl, was implanted alongside the soleus muscle in the right hindlimb of neonatal Wistar rats, within 6-12 h of birth. The silicon strip was positioned between the soleus and the flexor hallucis longus muscles away from the point of nerve entry to the muscle. The contralatera1 leg was left unoperated and used as a control. When the animals had fully recovered from the anaesthesia they were returned to their mothers and reared as normal. In some of these operated animals, a second silicon strip was implanted 3 days after the initial operation. Under the same conditions as previously described, the first silicon strip was located and removed from the hindleg and replaced with a larger implant containing either BTX or NaCl, as appropriate. These animals were then returned to their mothers. 2.2. Preparation
of implants
The preparation of BTX containing implants has been previously described [7]. Silicon strips weighing 0.3 mg were implanted into the hindleg of newborn rats, and contained approximately 7 p_g of BTX and 120 p_g NaCl. Strips weighing 0.75 mg were implanted into the hindlegs of 3 day old rats, and these implants contained 16 kg BTX and 240 Fg NaCl. Control implants of similar weights
To estimate the number of motor units in each muscle, the motor nerves of the operated and, in some experimental animals contralateral soleus muscles were stimulated every 4 s. The stimulus strength was gradually increased to obtain stepwise increments of twitch tension, as individual motor axons are recruited. The number of stepwise increments was counted to give an estimate of the number of motor axons present in the nerve. 2.5. Histology and immunocytochemistry After all the tension experiments had been completed, the control and operated soleus muscles were dissected out from the rats and weighed. The dissected soleus muscles were stretched to a length similar to that in the leg and control and operated muscles mounted side by side for each animal and frozen in isopentane cooled with liquid nitrogen. To determine the number, size and types of muscle fibres present, transverse cryostat sections (10 km) were collected onto gelatinized slides (0.5%) and stained for succinate dehydrogenase (SDH) activity [ 141. Immunocytochemistry was performed using a specific antibody against slow myosin (provided as a gift from Dr. G. Dhoot). The total number of muscle fibres and the number of slow myosin positive fibres per muscle was counted in experimental and control muscles. The cross-sectional area of operated and contralateral control muscles in both the NaCl- and BTX-treated animals was measured using a camera lucida and graphic digitizing tablet linked to a computer.
154
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3. Results 3.1. Effects of temporary paralysis at birth on the deuelopment of the rat soleus muscle 3. I. I. Immediate consequences of transient muscle paralysis Routine haematoxylin-van Gieson staining of transverse sections from soleus muscles revealed that the implant containing either BTX or NaCl caused no damage to muscle fibres - all muscle nuclei were located peripherally and the only visible effects of the implant was the presence of a small amount of connective tissue along the surface of the muscle in the area of the implant. Treatment of the soleus muscle with BTX in neonatal rats in the manner described here has previously been reported to abolish neuromuscular transmission. Placing a BTX implant onto the muscle at birth caused complete paralysis for the first 24 h. The muscles gradually recov-
Brain Research 94 (1996) 152-158
ered from the paralysis and by 8-9 days postnatally transmission was fully restored [7]. Treatment with a second BTX implant at 3 days of age prolonged the duration of muscle paralysis for a further 3-4 days, so that in 8 day old animals the soleus muscles were still partially paralysed [8]. Muscle paralysis is likely to affect muscle development and its ability to develop force. Oxidative enzymes are known to be linked to muscle activity [lo]. Therefore, in the present study, succinate dehydrogenase (SDH) was visualised in operated and contralateral control soleus muscles. Muscles treated at birth with BTX showed less intense staining for SDH when compared to controls 14 and 28 days after BTX treatment. This finding is illustrated in Fig. lA,B for muscles after 14 days, where the staining intensity of untreated soleus muscle (Fig. 1A) is compared to that observed in the BTX-treated soleus muscle taken from the same animal (Fig. IB), and in Fig. lC,D for soleus muscles 28 days after BTX treatment. Even at this
Fig. 1. This figure shows examples of cross-sections taken from control and BTX-treated soleus muscles and stained for SDH. (A) shows a section from a control and (B) from a BTX-treated muscle taken from the same 14 day old animal. (C) shows a section from a control and (D) from a BTX-treated animal at 28 days of age. The scale bar represents 20 pm in (A) and (B), and 50 p,rn in (C) and CD).
L. Greensmith et al./Deuelopmental
155
Bruin Research 94 (1996) 152-158
C
1w
NaCl
BTX
2BTX
NaCl
BTX
2BTX
I
Fig. 2. Twitch and tetanic contractions developed by adult soleus muscles in response to stimulation of their motor nerves are shown. (A) and (B) arc records from a 3 month old rat whose soleus muscle on one side (B) was treated with a single BTX implant at birth and (C) is from the control side of rat which had its soleus muscle on one side (D) treated with BTX at birth and 3 days of age.
time the soleus muscles had less intense staining than control muscles. 3.1.2. Long-term sis
consequences
for SDH I
of transient muscle paraly-
3.1.2.1. Muscle tensions and weights. When the animals were between 2 and 10 months old, long after the muscle paralysis had worn off, the animals were re-anaesthetised and the soleus muscles in the treated and contralateral control legs prepared for in situ assessment of muscle force. For each experiment the maximum tetanic tension (MTT) of the control and treated soleus muscle was recorded and the MTT of the treated muscle was expressed as a percentage of that produced by the contralateral control. Table 1 and Fig. 2 show that soleus muscles that had been transiently paralysed shortly after birth and were examined when adult, develop significantly less tension than control NaCl-treated muscles. In animals where muscle paralysis was prolonged by treatment with a second BTX implant at 3 days of age the decrease of tension output was significantly greater. Muscles treated with a single BTX implant at birth developed 74% (f4.5 S.E.M., n = 13) of the tension developed by the contralateral control muscles, and muscles subjected to a more prolonged
Fig. 3. The block diagrams show changes of (A) maximum tetanic tension and (B) muscle fibre numbers expressed as a percentage of the control unoperated soleus muscles, taken from adult animals that had their soleus muscles on one side treated with either NaCI, a single BTX implant at birth or 2 BTX implants at birth and 3 days of age.
paralysis (2 BTX) developed only 47% (k3.3 S.E.M., n = 6) of the force produced by their contralateral controls (Mann-Whitney U-test, P < 0.01 for both groups). The results are summarised in Fig. 3A, which clearly illustrates that the tension developed by the muscles treated with 2 BTX implants was significantly less than that developed by those treated with a single implant (Mann-Whitney U-test, P < 0.01). At the end of the tension recording experiments, the experimental and the contralateral control soleus muscles were removed from the animals and weighed. The reduction in force output observed in those muscles which had been paralysed at birth was reflected in a reduction in muscle weight of these muscles (see Table 1). Thus, following treatment with a single BTX implant at birth,
Table 1 Mean values (+ S.E.M.) of maximum tetanic tension and muscle weights from adult animals treated with either NaCl, a single BTX implant at birth or two BTX implants at birth and 3 days are shown Treatment
NaCl BTX 2BTX
n
4 8 4
The % operated/control
Maximum
n
tetanic tension
Control
Operated
%op/con
116.5 + 7.5 132.4 + 14.8 192.0 + 18.6
146.5 f 15.7 102.1 * 11.1 88.4 f 5.8
117.Ok7.3 73.7 f 4.5 49.5 f 4.3
was calculated
9 13 8
Muscle weight Control
Operated
%op/con
0.163 kO.011 0.169 + 0.014 0. I84 f 0.029
0.163 + 0.012 0.150 * 0.013 0.120 & 0.017
101.3 f 4.4 88.2 + 3.8 66.0 * 3.4
for each animal and the mean values (+ S.E.M.) are given; n shows the number of animals in each group,
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L. Greensmith et al./Deuelopmental
Brain Research 94 (1996) 152-158
Table 2 The mean total numbers (+S.E.M.) of muscle fibres from adult soleus muscles treated with either NaCI, a single BTX implant at birth or two BTX implants at birth and 3 days are shown Treatment
NaCl BTX 2BTX
n
Number of muscle fibres
3 5 3
Control (left)
Operated (right)
%op/con
2887+ 35 2821 f 234 2659+110
2793 + 103 2278+200 1576+ 150
96.7 * 2.4 81.4k4.1 56.8 f 4.4
The % operated/control was calculated for each animal and the mean values (*S.E.M.) are given; n shows the number of animals in each group.
soleus muscles in adult animals weighed 88% (k3.7 S.E.M., n = 13) of their contralateral control muscles, and those soleus muscles subjected to a prolonged muscle paralysis weighed significantly less, 66% ( * S.E.M., n = 8; Mann-Whitney U-test, P < 0.05 and < 0.001, respectively). 3.1.2.2. Muscle fibre numbers. The relative reduction of force output of muscles treated with BTX described above may be due to either a decrease in the size of muscle fibres or to a reduction in the number of muscle fibres. To distinguish between these two possibilities, the total number of muscle fibres in control and BTX-treated muscles was established and the results are summarised in Table 2 and Fig. 3B. The number of muscle fibres found in normal and unoperated contralateral soleus muscles ranged between 2500-2900, which is in good agreement to data previously reported [2,13]. In soleus muscles paralysed with BTX at birth and examined later, there is a 19% decrease in the number of muscle fibres. This decrease is significant when compared to the contralateral muscles (Mann-Whitney U-test, P < 0.04). Following a more prolonged muscle paralysis, by treatment with two BTX implants, there is a greater decrease in the number of muscle fibres, so that 47% of fibres in the treated muscle are lost (Mann-Whitney U-test, P < 0.03). The number of muscle fibres present in soleus muscles treated with NaCl implants was not significantly different from normal, untreated soleus muscles or contralateral control soleus muscles. It appears therefore, that the reduction in tension developed by the BTX-treated soleus muscles is caused by a reduction of the number of muscle fibres in these muscles. A comparison of Fig. 3A and 3B shows that the reduction in Table 3 The times to peak twitch tension and half relaxation of single twitch contractions in one leg treated with either NaCl, BTX at birth or BTX at birth and 3 days Treatment
NaCl BTX 2BTX
n
5 7 4
The % operated/control
3.1.2.3. Motor unit number. The reduction in force output and the loss of muscle fibres in soleus muscles treated with BTX may have been caused by a loss of motoneurones. Previous results with retrograde labelling show that nearly 40% of the total number of motoneurones to the soleus muscle die after treatment with BTX at birth, and nearly 65% die following prolonged muscle paralysis [8]. In order to assess the effect of transient muscle paralysis on the survival of a-motoneurones only, estimates of the number of motor units in the treated and contralateral control soleus muscles were made by counting the increments of twitch tension elicited from the muscles by stimulation of the soleus motor nerve with stimuli of increasing intensity. Since the effect on muscle force and fibre number were greater following treatment with 2 BTX implants, motor unit number was assessed only in those animals which had been paralysed for a prolonged period after birth. In control experiments, the normal soleus muscle was found to have a mean of 27 ( f 0.6 S.E.M., n = 9) motor units. In soleus muscles subjected to prolonged paralysis at birth (2BTX) the number of motor units present was much less, with a mean of 15 (+ 0.6 S.E.M., n = 5) which is significantly different from that found in the control soleus muscles (Mann-Whitney U-test, P = 0.005). Thus, application of (-w-BTX during early postnatal development leads to a permanent loss of o-motoneurones. 3.1.2.4. Contractile properties and muscle fibre characteristics. Table 3 summarises the results of measurements of time to peak twitch tension and half relaxation obtained from the various experimental groups studied. The results show that the time to half relaxation was prolonged in muscles treated with BTX at birth, and this effect was greater in animals that were subjected to a prolonged
are shown for soleus muscles in adult animals that had the soleus muscle n
Time to peak Control
Operated
%op/con
80 + 8.4 106.4 f 8.4 76.3 f 6.3
76 k8.1 98.9 f 6.3 86.3 k 8.5
95.3 k 2.9 97.4 * 11.3 118.3 i 22.5
was calculated
muscle fibre number clearly reflects the decrease in muscle force observed after either a single or double BTX implant. It is nevertheless possible that a change in muscle fibre area may influence the force output in the BTX-treated muscles. Therefore, in some animals the muscle fibre areas were measured from haematoxylin-van Gieson stained transverse sections of the control and experimental muscles. The mean muscle fibre area in BTX-treated muscles was 95% (i 19.6 S.E.M., n = 3) of the untreated contralateral control muscles, and 93% (_t 2.6 S.E.M., n = 4) from muscles treated with two BTX implants.
5 5 4
Time to half relaxation Control
Operated
%op/con
111 k11.2 96 k5.1 67.5 + 4.8
99 * 13.3 126 + 19.6 110 f 18.7
88.9 f 5.2 88.2 f 3.8 160.7 + 19.0
for each animal and the mean values ( f S.E.M.) are given; n shows the number of animals in each group.
L. Greensmith et al./Deuelopmental
paralysis. Thus, it appears that paralysing the soleus muscle with BTX at birth enhances the ‘slow’ characteristics of this muscle. Whether this change is reflected in the distribution of fibre types in the adult soleus muscle was also studied. Staining of control soleus muscles for SDH, a marker of oxidative capacity, revealed the presence of large fibres of intermediate staining intensity in addition to a few small, darker stained fibres. Following temporary paralysis of the soleus muscle at birth, there were fewer darkly stained fibres and the overall level of staining for SDH appeared to be less than in controls. In addition, prolonging the duration of paralysis results in a greater reduction in SDH staining. Treated and contralateral control muscles were also studied using an antibody to the slow myosin heavy chain (kindly donated by Dr. G. Dhoot). Contralateral control soleus muscles stained with the slow myosin antibody showed that approximately 80% of the total number of muscle fibres expressed slow myosin, so that around 20% of fibres did not stain for slow myosin, while muscles subjected to a period of transient muscle paralysis by treatment with either 1 or 2 BTX implants after birth, were composed entirely of muscle fibres that stained positively for slow myosin.
4. Discussion Transient disruption of neuromuscular transmission in neonatal rats has been previously shown to result in the death of a large proportion of motoneurones to the soleus muscle [8]. These results were obtained by retrograde labelling, so that both alpha and gamma motoneurones were included in the analysis of motoneurone survival. The present study, using physiological assessment of the number of motor units present in BTX-treated soleus muscles, specifically examines the extent of survival of alpha motoneurones only and shows (a) that alpha motoneurones are lost following this period of early muscle paralysis, and (b) that those motoneurones which do survive this disruption of nerve:muscle interaction are unable to expand their peripheral field to occupy the many denervated muscle fibres available to them. What may be the cause of motoneurone death following a period of muscle paralysis at birth? We have recently shown that treatment with BTX at birth renders motoneurones susceptible to the toxic effects of the glutamate agonist NMDA [9]. Furthermore, treatment with MK-801 prevents the death of a proportion of motoneurones that would otherwise die following BTX treatment at birth [9]. Thus, preventing neuromuscular interaction during a critical period of development, either by nerve injury or treatment with BTX, results in an increased sensitivity of the affected motoneurones to the toxic effects of glutamate. Not only are motoneurones to paralysed muscles more sensitive to NMDA, the surviving motoneurones are also
Brain Research 94 (1996) 152-158
157
likely to be more active than normal motoneurones. One indication that the motoneurones to the BTX-treated muscles are more active is the finding that these soleus muscles have a prolonged time to half relaxation and contain no muscle fibres which stain positively for fast myosin. In other experimental situations where a reduction in the number of motor units is observed, either following partial denervation or nerve crush injury, EMG recordings demonstrate that there is an increase in the activity of the surviving motoneurones [15,20,21]. Such an increase in motoneurone activity may further contribute to the death of motoneurones deprived of interaction with their target. It is possible that this increased activity of the surviving motoneurones is also a contributing factor to their inability to expand their peripheral field. There is much evidence which shows that activity can induce the loss of neuromuscular contacts during the elimination of polyneuronal innervation which normally takes place during the first two weeks of postnatal life [.5,6,16,17]. The period in which the elimination of superfluous nerve terminals from muscle fibres takes place is altered in muscles paralysed with BTX at birth. As the muscles recover from the paralysis and neuromuscular activity resumes, weak synaptic contacts are lost [7] and as a consequence many muscle fibres die, which is reflected in the small size of the remaining units, as seen in the present study. It was previously shown in partially denervated muscles treated with BTX, that a large number of newly formed sprouts initially made contact with the denervated muscle fibres, but these contacts were lost when the muscles recovered from the paralysis and neuromuscular activity was resumed [3]. A similar loss of such weak contacts upon resumption of activity may also occur in the present experiments and this may explain why the motoneurones which survive after treatment with BTX at birth do not have an enlarged peripheral field. The results presented here clearly demonstrate that interaction between the motoneurone and its target muscle during early postnatal development is essential not only for motoneurone survival, but also for the ability of those motoneurones which do survive target deprivation to expand and maintain a large peripheral field.
Acknowledgements We are grateful to The Wellcome Trust and Action Research for their support. A.H. was in receipt of a SERC studentship. We would also like to thank Mr J. Dick for his excellent technical assistance.
References [l] Brown, M.C., Jansen, J.K.S. and Van Essen, D.C., Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation, _I. Physiol., 261 (1976) 387-422. [2] Connold, A.L. and VrbovB, G., Temporary loss of activity prevents
158
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[lo]
[11]
L. Greensmith
et al. /Deuelopmental
the increase in motor unit size in partially denervated rat soleus muscles, J. Physiol., 434 (1991) 107-119. Connold, A.L. and Vrbov& G., Neuromuscular contacts of expanded motor units in rat soleus muscles are rescued by leupeptin, Neuroscience, 63 (1995) 327-338. Dick, J., Greensmith, L. and VrbovB, G., The effects of nerve injury in neonatal rats on motor unit numbers and properties are prevented by blocking NMDA receptors, Neuromusc. Disord., 5 (1995) 371382. Duxson, M.J., The effect of postsynaptic block on development of the neuromuscular junction in postnatal rats, J. Neurocytol., 11 (1982) 395-408. Duxson, M.J. and VrbovB, G., Inhibition of acetylcholinesterase accelerates axon terminal withdrawal at the developing rat neuromuscular junction, J. Neurocytol., 14 (1985) 337-363. Greensmith, L. and VrbovB, G., Neuromuscular contacts on the developing rat soleus depend upon muscle activity, Deu. Brain Res., 62 (1991) 121-129. Greensmith, L. and VrbovB, G., Alterations of nerve-muscle interactions during postnatal development influence motoneurone survival in rats, Deu. Brain Res., 69 (1992) 125-131. Greensmith, L., Sanusi, J., Mentis, G.Z. and VrbovB, G., Transient muscle paralysis in neonatal rats renders motoneurones susceptible to NMDA-induced neurotoxicity, Neuroscience, 64 (1995) 109-l 15. Kugelberg, E. and Lindegren, B., Transmission and contraction fatigue of rat motor units in relation to succinate dehydrogenase activity of motor unit fibres, J. Physiol. 288 (1979) 285-300. Lowrie, M.B., Krishnan, S. and VrbovB, G., Recovery of slow and fast muscles following nerve injury during early postnatal development in the rat, J. Physiol., 331 (1982) 56-66.
Brain Research
94 (1996) 152-158
[12] Lowrie, M.B., Subramaniam Krishnan and VrbovB, G., Permanent changes in muscle and motoneurones induced by nerve injury during a critical period of development of the rat, Dec. Brain Res., 310 (1987) 91-101. [13] McArdle, J.J. and Sansone, F.M., Reinnervation of fast and slow twitch muscle following nerve crush at birth, J. Physiol., 271 (1977) 567-586. 1141 Nachlas, M.M., Tson, K.C., DeSouza, E., Chang, C.H. and Seligman, A.M., Cytochemical demonstration of succinate dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole, J. Hisrochem.
Cy’tochem., 5 (1957)
[15] Navarrete,
[16]
[17]
[18] [I91 [20]
[21]
420-436.
R. and VrbovB, G., Differential effect of nerve injury at birth on the activity pattern of reinnervated slow and fast muscles of the rat, J. Physiol., 351 (1984) 675-685. O’Brien, R.A.D., &tberg, A.J.C. and VrbovB, G., Observations on the elimination of polyneuronal innervation in developing mammalian skeletal muscle, J. Physiol., 282 (1978) 571-582. O’Brien, R.A.D., &tberg, A.J.C. and Vrbovl, G., The effect of acetylcholine on the function and structure of the developing mammalian neuromuscular junction, Neuroscience, 5 (1980) 1367- 1379. Redfern, P.A., Neuromuscular transmission in newborn rats, J. Physiol., 209 (1970) 70 l-709. Romanes, G.J., Motor localisation and the effects of nerve injury on the ventral horn cells of the spinal cord, J. Anat., 80 (1946) 1 l- 13 1, Slawibska, U., Navarrete, R., Kasicki, S. and VrbovL, G., Motor activity patterns in rat soleus muscle after neonatal partial denervation, Neuromusc. Disord., 5 (1995) 179-I 86. Tyc’, F. and VrbovB, G., The effect of partial denervation of developing rat fast muscles on their motor unit properties, J. Physiol., 482.3 (1995)
65 I-660.