MSH 4–10 improves motor unit reorganization during peripheral nerve regeneration in the rat

Peptides, Vol. 6, Suppl. i, pp. 7%83, 1985. ©Ankho International Inc. Printed in the U.S.A.

0196-9781/85 $3.00 + .00

ACTI4/MI4 4-10 ImprovesMotorUnit Reorganization During Peripheral Nerve Regeneration in the Rat CLAUDE

SAINT-COME

AND FLEUR

L. STRAND 1

D e p a r t m e n t o f Biology, N e w York University, Washington Square, N e w York, N Y 10003

SAINT-COME, C. AND F. L. STRAND. ACTH/MSH 4-10 improves motor unit reorganization daring peripheral nerve regeneration in the rat. PEPTIDES 6: Suppl. 1, 77-83, 1985.--ACTH/MSH 4-10 administration (10 ~g/48 hr IP for 7 days) enhances neuromuscular activity following crush denervation of rat extensor digitorum longus muscle. The number of regenerated functional motor units is greater in peptide treated rats than in saline treated denervated controls. Selective activation of responding motor units indicates that ACTH/MSH 4-10 preferentially accelerates the reformation and stabilization of small size motor units. These observed effects may be beneficial since they contribute to the early reestablishment of more organized motor units, thereby restoring f'me control of motor functions, in contrast to the disorderly reorganization of untreated regenerating systems. Possible mechanisms of peptide action (neurotransmitter synthesis and release, excitability changes, etc.) are discussed. ACTH/MSH 4-10

Neuromuscular activity

Regeneration

A C T H / M S H peptides have been shown to have beneficial effects on regenerating peripheral nerve and on the reinnervation of skeletal muscle. These effects include an acceleration of the growth of the regenerating axons and a more rapid recovery of motor and sensory function [37]. The ameliorative action of the peptides is neurogenic and independent of the adrenocortical steroids, which in fact have a deleterious influence on regenerating neurons. This extra-adrenal effect of A C T H peptides was convincingly demonstrated by Bijlsma et al. [3, 4, 5] using a series o f non-steroidogenic A C T H fragments (ACTH 4-10; A C T H 4--9: a synthetic A C T H 4-9 analogue, Org 2766; and A C T H 6-10) all of which enhanced functional recovery to a similar degree. These authors suggest that the stimulatory effect of ACTH 4-10 is on the number o f regenerating nerve fibers rather than on the rate of elongation, and that this effect is most pronounced during the early stages of regeneration. Another possibility is that A C T H may directly affect synaptogenesis [37]. Neuromuscular performance is highly dependent upon the architecture of the motor unit. Each motor unit is composed o f a spinal alpha motoneuron and the muscle fibers innervated by this neuron [14]. The number of muscle fibers within a motor unit, and hence its size, depends on the diameter of the innervating axon: large diameter axons innervate large units whereas small diameter axons innervate small motor units. The excitability of a neuron, and consequently its sequential participation in graded muscular activity, also depends on its size. Small motoneurons are more excitable than larger ones. Thus small motor units, inner-

Motor units

vated by small nerve fibers, have a lower threshold to stimulation than larger motor units and therefore are recruited first when trains of electrical stimuli are applied to central neuron pools [21, 22, 34]. These small motor units produce less tension but are less susceptible to fatigue than are the larger motor units [7,8]. While gradients in functional thresholds undoubtedly are closely related to the characteristics of the innervated muscle fibers, there is much evidence to show that these thresholds are not immutable and may, in fact be modulated by many extrinsic and intrinsic factors [10]. ACTH, M S H and related fragments have been shown to increase the excitability of central neurons [25, 26, 35, 41]. Thus changes in the size, threshold, tetanic tension, and fatiguability of regenerating motor units as affected by A C T H / M S H peptides were investigated in this study to more specifically determine the mode by which these neuropeptides accelerate the recovery of motor function following crush denervation. Another important facet of regeneration is the pattern of motor unit reorganization. In normal muscle, the muscle fibers of a motor unit are widely scattered throughout the muscle [ 15,27] so that there is considerable overlap of motor unit territory. This unexpected overlap is due to the process of elimination of superfluous nerve fibers during development. Until the second postnatal week, rat muscle fibers are polyinnervated, having approximately five nerve fibers for each muscle fiber. As rejection of four of these five fibers occurs randomly, overlapping and widespread motor unit fields result [32,39]. During regeneration, the axons of the

~Requests for reprints should be addressed to Professor Fleur L. Strand, Biology Department, Room 1009 Main Building, New York University, Washington Square, New York, NY 10003.

78

SAINT-COME AND STRAND

20 msec

5 0 msec

FIG. 1. Recruitment of motor units with finely graded increases in stimulus strength delivered to the peroneai nerve, a: intact; b: denervated plus 0.2 ml saline on the day of crush denervation and thereafter every 48 hr for 7 days; c: denervated plus 10 p.g ACTH/MSH 4--10 on the day of crush denervation and thereafter every 48 hr for 7 days. Motor unit recruitment in intact rats is characterized by small, discrete increments in the twitches. Crush denervation results in the appearance of large incremental steps in muscle twitches. ACTH/MSH 4--10 administration stimulates the reestablishment of small incremental steps in twitch amplitude.

crushed motoneurons send sprouts to reestablish connections with the denervated muscle fibers. Polyneuronal innervation is characteristic of the early stages of regeneration, as it is of development. As regeneration is completed, the extraneous nerve fibers are eliminated and the mature ratio of one nerve terminal to one muscle fiber is reestablished [1,28]. However, the size of the regenerated motor unit is usually larger than normal while the territory it occupies is more compact [27]. Functional reorganization of regenerating motor units of the extensor digitorum longus (EDL) muscle o f the adrenalectomized rat following crush denervation of the peroneal nerve is accelerated by chronic administration of A C T H 1-39 [33]. In these experiments, motor unit activity was evaluated in vitro and adrenalectomy was performed to control for possible corticosteroid effects. As motor unit responses, as normally elicited in the intact organism, are flexible and contigent upon complex neural circuitry [11] we decided to study the regenerating motor unit in situ as a more appropriate model. Motor unit distribution, number, mean size and response to changes in stimulus frequency were determined in regenerating E D L of rats that had been treated with the nonsteroidogenic A C T H / M S H 4-10 and compared to the saline treated regenerating E D L muscles of the controls. METHOD

Crush denervation was performed under sodium pentobarbital anaesthesia (45 mg/kg body wt injected IP). The left common peroneal nerve was exposed at its point of entrance into the peroneous longus muscle. The nerve was first marked by applying pressure with a silk thread (5-0) against a glass rod for 10 seconds. Then a 1 mm length from the marked point was crushed with number 5 watchmaker's forceps having a uniformly filed tip of 0.2 mm. Electromechanical Recording Seven days after crush denervation the animals were prepared for electromechanical recording under sodium pentobarbital anaesthesia (50 mg/kg body wt IP). Twenty percent o f the initial dosage were given when needed during the course of the experiment. The extensor digitorum longus (EDL) muscle was isolated from connective tissues. The distal tendons of the E D L muscle were attached to an isometric transducer (Grass FT10). The foot and femur were pinned to prevent movement. The skin surrounding the preparation was clipped to a ring to form a sling in which was placed mineral oil maintained at 37°C--1.0. The rat was rigidly attached in the prone position to a temperature-controlled surgical table. The sciatic nerve was exposed and all branches to muscles other than the E D L muscle were cut. The uncut nerve was suspended on a pair of hook electrodes (4 ram) with a 0.2 mm contact diameter.

Animals

Recruitment of Motor Units

Male Sprague Dawley rats, weighing 125--150 g were obtained from Blue Spruce Farms one week prior to the experiment. The animals were maintained on a balanced 12 hour light:12 hour dark cycle and were supplied with rat chow and water ad lib. The rats were divided into 3 groups. (1) intact; (2) denervated controls that received 0.2 ml saline IP on the day of the nerve crush and thereafter every 48 hours, for 7 days; (3) denervated and treated with A C T H / M S H 4-10 (Org OI63) (10/xg in 0.2 ml IP) on the day of the crush and thereafter every 48 hours for 7 days.

Contractions of single motor units were elicited by delivering square pulses generated by a constant current stimulator (Coulborn Instrument), to the intact sciatic nerve. Twitch responses of low-threshold motor units were obtained by stimulating the nerve with pulses of 200 p.sec duration repetitively at 1 Hz. A very small current was used until the first unit was activated. The stimulus that resulted in activation of the first putative motor unit was considered as threshold current. The strength of stimulation was then finely graded while twitch responses were monitored on a

A C T H / M S H 4-10 IMPROVES R E G E N E R A T I O N Tektronix (5103 N) storage oscilloscope. Series of putative motor units were recorded at the same compliance (sensitivity g/cm) until alternation (change in compliance) became necessary. Motor units were grouped according to current range: threshold, 2 times threshold, and up to 5 times threshold. Responses were photographed with a polaroid camera, measured under an enlarger, and the values entered into a TRS-80 model 4 microcomputer programmed to evaluate motor unit size, number, size distribution and frequency analysis of motor unit distribution. Motor unit number was evaluated according to the method introduced by McComas and associates [29]. Since the action potentials generated by motor units are polyphasic during regeneration, the mechanical responses were used to evaluate the number of motor units according to the method described by Harris and Wilson [20]. The number of functional motor units was evaluated by dividing the maximum twitch tension by the average increment in twitch responses. Motor Unit Activity Index

79

ir~oct */ . o f units

Denel'wotecl

100. *ACTH 4-10 80

1.4

Repetitive pulses of 200 /xsec duration and of maximal strength were delivered to the nerve at a frequency of 10 Hz for 1 min. The mean contraction amplitude after 1 min was taken as an index of motor unit activity at low frequency stimulation. Tetanic Tension at Optimum Frequency A train of pulses of supramaximal strength was delivered to the nerve for a period of 500 msec at different frequencies, starting with 10 Hz, at 5 minute intervals. The frequency was increased until complete fusion of the mechanical response was achieved. The amplitude o f the response at fusion frequency and at 200 msec from the beginning of the contraction was taken as the maximum tetanic tension at optimum frequency. Fatiguability of Motor Units Susceptibility to fatigue was determined by delivering train of pulses of supramaximal strength to the nerve for a period of 500 msec at 450 Hz. Statistical Analysis One way analysis of variance was used to detect significance of difference between groups. Significance between specific means was tested by means of the StudentNewman-Keuls test. RESULTS

Normal Distribution o f Motor Units in the Adult Rat EDL When stimuli of finely graded increases in strength are delivered to the peroneal nerve, the resulting twitches of the E D L muscle are also finely graded, indicating that the motor units are being recruited by small increments in an orderly manner (Fig. la). Frequency analysis of motor unit size, as determined by tension output, indicates that 50% of the motor unit population is less than 1.4 g; 20% is between 1.4 and 1.8 g; and the remaining 30% lies between 1.8 and 2.2 g (Fig. 2). The number of functional motor units in the E D L of the adult rat is 38---3.

I

4 0 - ,/////////. ~

1.8

22.

, ~

2.6

Twitch size

3.0

3.4

~.~

3B

((3)

FIG. 2. Distribution of motor unit twitch size in rat EDL muscle. Motor unit size of intact rat is skewed toward small size. Crush denervation results in a shift toward large size with very little variation within the population of motor units. ACTH/MSH 4-10 administration results in a shift from large motor unit size toward smaller motor unit size. The variation in unit size within the motor unit population is greater in ACTH/MSH 4-10 treated rats than in controls.

Effects o f Denervation and ACTH/MSH 4-10 Administration on Motor Unit Characteristics Motor unit size. Seven days after crush denervation, the fine gradations in twitch size that are characteristic of the normal intact muscle are replaced by large jumps (Fig. lb). The distribution of motor unit size has become asymmetrical, with 80% of the motor units between 2.2 and 3.8 g and the remainingmotor units lying _between 1.8 and 2.2 g (Fig. 2). The administration of A C T H / M S H 4-10 (10/~g/48 hr, IP dally from the day of the crush) results in the reappearance of stepwise gradations in twitch size similar to, but not as fine as those of the intact E D L muscle (Fig. lc). Administration of the peptide also results in a greater variation in the distribution of motor unit size than in the saline treated control animals. In the A C T H / M S H 4-10 treated rats, 60% of the newly innervated motor units are between 2.2 and 3.4 g; 40% lie between 1.4 and 2.2 g (Fig. 2). Number and mean size o f functional motor units. Crush denervation results in a significant drop (p<0.01) in the number of functional motor units when muscle function is investigated 7 days after the crush. The administration o f A C T H / M S H 4-10 stimulates the reappearance of additional functional motor units at this time (p<0.05) (Fig. 3). While there appears to be an accompanying increase in mean motor unit size in the saline treated controls, and a decrease in mean motor unit size in the peptide treated rats, these changes are not statistically significant. Activity index o f responding motor units at low frequency stimulation. When the activity index of responding MUs is tested by low frequency stimulation (10 Hz) over a period of 1 min, it is clearly seen that crush denervation markedly

80

SAINT-COME AND STRAND Number of units

TABLE 1 ACTIVITYINDEX OF INDIRECTLYSTIMULATEDEDL 7 DAYS AFTER NERVE CRUSH

50.

4o.

Intact Denervated + Saline Denervated + ACTH 4-10 (10/zg/48 hr for 7 days) n=6

T

30-

g puU/min

-+S.D.

22.1 11.5 15.1"

3.6 2.6 2.5

Values expressed as mean ___standard deviation. All denervated values significantly different from intact (p<0.01). *p<0.05 vs. denervated + saline. 20-

TABLE 2 MAXIMUMTETANICTENSIONOF INDIRECTLYSTIMULATEDEDL 7 DAYSAFTERNERVE CRUSH

lO.

a

g pull

-S.D.

160 40 65*

16 9 5

FIG. 3. Number of functional motor units, a: intact; b: denervated plus 10 /xg of ACTI-I/MSH 4-10 on the day of denervation and thereafter every 48 hr for 7 days; c: denervated plus 0.2 ml saline on the day of denervation and thereafter every 48 hr for 7 days. Motor unit number sharply declines 7 days after crush denervation (/7<0.01). Mean size (proportional to the width of the bar) increases but is not statistically significant. ACTH/MSH 4-10 administration stimulates recovery of more functional motor units (p<0.05) and results in a reduction in mean size (value not statistically significant) as compared to denervated plus saline.

Intact Denervated + Saline Denervated + ACTH 4--10 (10/zg/48 hr for 7 days)

reduces this parameter (0<0.01). ACTH/MSH 4-10 administration significantly increases the activity index of denervated muscle 7 days after crush (0<0.05) although recovery at this time is not complete (Table 1).

gressive increases in twitch amplitude are achieved through the orderly recruitment of motor units. Our estimate of 38 motor units compares well with that of Close who reported about 40 motor units [12]. As stimulation strength is increased from threshold to maximum, the estimated 38 motor units are progressively recruited in a logical manner according to their inherent characteristics. Since the number of muscle fibers in a motor unit determines its tension output, the latter may be used as an electromechanical measure of motor unit size. Crush denervation increases the size of the regenerating motor units of the EDL. Seven days after denervation, 80% of the regenerating motor units are considerably larger than any of those found in the intact EDL, and 20% of the newly formed motor units are approximately twice the size of the largest motor units of the intact EDL (Fig. 2). The expansion of motor unit size is at the expense of motor unit number, which is significantly reduced (Fig. 3). Since there are very few small motor units regenerated in the saline treated animals, gradual increases in stimulus strength can evoke only large increments (or jumps) in twitch tension. In this stage of reinnervation, the denervated muscle is able to achieve twitch tension in terms of ultimate strength but is unable to respond if the motor act demands fine control. In denervated animals treated with ACTH/MSH 4--10, reinnervation does not produce the extremely large motor units characteristic of the control denervated rats. Although the delicate range of small, intermediate and large motor units of the intact muscle is still absent, peptide treatment

Tetanic tension at optimum frequency stimulation. Seven days after crush denervation, the amplitude of tetanic tension as evoked by stimulation of the EDL muscle via the peroneal nerve at optimum frequency is considerably reduced (0<0.01) in saline treated controls. Table 2 shows that the administration of ACTH/MSH 4-10 to denervated animals elevates tetanic tension significantly over that of control denervated rats (0<0.05) although tension does not return to intact values at this time.

Tetanic tension at above optimum frequency stimulation. Regenerating motor units are unable to maintain tetanic tension amplitude when the frequency of stimulation is increased beyond optimum (450 Hz). Seven days after crush denervation the rate of decline in tetanic tension of EDL muscle was significantly greater than that observed in intact EDL muscle (0<0.05). This is illustrated in Fig. 4a and b. Figure 4c shows the improvement in the maintenance of tetanic tension when the regenerating system has been exposed to ACTH/MSH 4-10 treatment, an improvement that is most marked (0<0.05) during the last 300 msec of stimulation. DISCUSSION In the EDL muscle of the normal, intact adult rat, pro-

Values expressed as mean -+ standard deviation. All denervated values significantly different from intact (p<0.01). *p<0.01 vs. denervated + saline.

ACTH/MSH 4-10 IMPROVES REGENERATION

[.

81

f

1 2og

0.I sec FIG. 4. Fatiguability of motor units under high frequency stimulation (450 Hz) stimulation at supramaximal stimulus strength for 500 msec. a: intact; b: denervated plus 0.2 ml saline on the day of crush and thereafter every 48 hr for 7 days; c: denervated plus l0/zg ACTH/MSH4-10 on the day of denervation and thereafter every 48 hr for 7 days. The peptide partially restores the ability of denervated muscle to maintain tetanic tension.

generates more variation within the population of reformed motor units. The decrease in motor unit size is accompanied by an increase in the number of functional motor units. In these peptide treated animals the recruitment of motor units is considerably more controlled than in the denervated, saline treated rats, indicating a greater range of motor units capable of responding to strength and fine control demands. This permits the motor unit to function as an integrating device responsive to rapid changes in input [22]. While the fine control of recruitment characteristic of the intact animal is not achievedb__y_s_eve_ndays o f ~ p t i d e treatment of the denervated animal, the peptide clearly improves the pattern of motor unit reinnervation, bringing it closer to the norm of the intact muscle. Further studies are underway to follow reinnervation patterns as influenced by ACTH/MSH administration for longer periods of time. Differences in the size and number of motor units after denervation may also be correlated to the extent of the denervation. From studies on partial denervation, Gorio [19] has suggested that severe denervation increases sprouting more than does slight denervation, consequently increasing the number of nerve terminals available to innervate muscle fibers. Our studies involved crush denervation, a technique which, while permitting the retention of the nerve sheath for better alignment during reinnervation [23], nevertheless causes a complete separation of the axons in the region of the crush. One would therefore expect considerable axonal sprouting and large new motor units, few in number. The reduction in motor unit size and the concomittant increase in motor unit number under the influence of ACTH/MSH 4--10 are in agreement with the results of morphological studies by Bijlsma et al. [2,5] who report that this peptide increases the number of regenerating axons of small diameter. Since small axons tend to innervate fewer muscle fibers, this would explain the formation of small size motor units in ACTH/MSH 4--10 treated denervated rats. Crush denervation results in impaired neuromuscular performance when repetitive pulses are delivered to the regenerating nerve. The significant decline in the activity index may be due to a decrease in the excitability of the regenerating motor units (Gorio [18]). The inability of the reformed

motor unit to faithfully respond to repetitive stimulation may also be due to a delay in the recovery of the transmitter release mechanism. During the early stages of reinnervation, miniature endplate potentials (mepps) are scarce although transmitter is available in the terminals [17,18]. The involvement of ACTH/MSH neuropeptides in synaptic events is well documented [24, 40, 42]. ACTH/MSH 4--10 increases mepp frequency in rat phrenic nerve-diaphragm preparations in vitro [6] as well as in in situ sciatic nerve-skeletal muscle preparations [16]. Thus the improvement in the activity index observed in denervated, peptide treated animals reported here may be due to an enhancement of neurotransmitter mechanisms. Another possibility is that ACFH/MSH 4-10 increases the excitability of the motor units [26, 35, 38] thus indirectly affecting transmitter release or utilization. The significant decrease in tetanic tension that results from denervation can be attributed to a reduction in the number of functional motor units and/or the inability of the newly formed synapses to respond to high frequency stimulation. The administration of ACTH/MSH 4-10 during the first week of regeneration may result in the appearance of additional functional motor units even though they, like the motor units of the intact animal, are smaller than those of the saline treated controls. Another possible explanation for the increased tension generated by smaller motor units is that there may be better synchronous discharge of spinal motoneurons in the peptide treated rats: electrical stimulation of the peroneal nerve in situ results in both antidromic and orthodromic conduction. Burke [10] has shown that a major restructuring of motor unit threshold occurs when synaptic input is altered, or external or intrinsic factors change. The twitch tension produced by motor units varies almost linearly as a function of their threshold tension of recruitment [13,31]. In humans, it has been shown that the twitch tensions of motor units are smaller than normal during the early stages of regeneration and that voluntary movement in such patients, even in the later stages of regeneration when muscle strength is restored, recruits motor units in an apparently random manner that precludes fine motor control [30]. Thus both the reorganization of the motor unit in terms of the

82

SAINT-COME AND STRAND

muscle fibers it innervates, and the recruitment of the units by either voluntary or antidromic stimulation, m a y be disorderly in untreated regenerated systems. T r e a t m e n t o f patients with progressive spinal atrophy by infusions o f A C T H / M S H 4-10 p r e v e n t s the characteristic decline in e v o k e d muscle potentials [36], further e v i d e n c e for an ameliorative role for this peptide on damaged m o t o r units. Similarly, the i m p r o v e d ability to maintain tetanic tension, and the increase in the activity index of peptide treated animals, m a y depend on the increased availability of functional m o t o r units and the greater range o f m o t o r unit size, as c o m p a r e d to the saline treated d e n e r v a t e d rats. Fatigueresistant m o t o r units are m o r e readily excited than fatiguesensitive units [9]. The ability to maintain a high level of activity for prolonged periods o f time depends upon the small m o t o r unit population, the population o f units that is most fatigue-resistant and m o s t excitable. The effectiveness o f A C T H / M S H 4-10 in preventing fatigue may also be due to a direct role o f this peptide on spinal m o t o n e u r o n s , increas-

ing their excitability [26,38] and perhaps increasing transmitter release and resynthesis [24, 40, 42]. It would therefore appear that the beneficial effects of A C T H / M S H 4-10 during the early stages of regeneration, as e v i d e n c e d by the i m p r o v e m e n t o f many facets of m o t o r unit performance, orginate in the preferential formation of many small m o t o r units. In the absence of the peptide, reinnervation o f muscle occurs but the m o t o r units thus p r o d u c e d are large and disorganized. Since m a n y of the impairments in n e u r o m u s c u l a r function during reinnervation can be attributed to changes in the architecture of the m o t o r units, it is suggested that the i m p r o v e m e n t o b s e r v e d in response to A C T H / M S H 4-10 t r e a t m e n t may be due to an organizational effect o f the peptide on regenerating m o t o r units.

ACKNOWLEDGEMENT This study was supported by Organon, Oss, The Netherlands.

REFERENCES 1. Bennett, M. R. and A. G. Pettigrew. The formation of synapses in striated muscle during development. J Physiol 241: 515-545, 1974. 2. Bijlsma, W. A., E. van Asselt, H. Veldman, F. G. I. Jennekens, P. Schotman and W. H. Gispen. Ultrastructural study of the effect of ACTH 4-10 on nerve regeneration: outgrowing axons become larger in number and smaller in diameter. In press. 3. Bijisma, W. A., F. G. I. Jennekins, P. Schotman and W. H. Gispen. Corticotropin (ACTH) like peptides stimulate peripheral nerve regeneration. In: Functional Recovery from Brain Damage, edited by M. W. van Hof and G. Mohn. Amsterdam: Elsevier/North Holland, 1981, pp. 411-416. 4. Bijlsma, W. A., F. G. I. Jennekens, P. Schotman and W. H. Gispen. Effects of corticotrophin (ACTH) on recovery of sensorimotor function in the rat: structure-activity study. Eur J Pharmacol 76: 73-79, 1981. 5. Bijlsma, W. A., F. G. I. Jennekens, P. Schotman and W. H. Gispen. Stimulation by ACTH 4-10 of nerve fiber regeneration following sciatic nerve crush. Muscle Nerve 6: 102-110, 1983. 6. Birnberger, K. L., R. Rudel and A. Struppler. ACTH and neuromuscular transmission: Electrophysiological in vitro investigation of the effects of corticotropin and an ACTH fragment on neuromuscular transmission. Am Neurol 1: 270-275, 1979. 7. Burke, R. E., D. N. Levine, F. E. Zajac, P. Tsairis and W. K. Engel. Mammalian motor units, physiological histochemical correlation in three types in cat gastrocnemius. Science 174: 109-I 12, 1971. 8. Burke, R. E. and P. Tsairis. Anatomy and innervation ratios in motor units of cat gastrocnemius. J Physiol (Lond) 234: 749765, 1973. 9. Burke, R. E., R. P. Dum, M. J. O'Donovan, M. J. Toop and P. Tsairis. Properties of soleus muscle and of individual soleus motor units after cross-innervation by FDL motoneurons. Soc Neurosci Abstr 9: 765, 1979. 10. Burke, R. E. Motor unit recruitment: what are the critical factors. In: Motor Unit Types, Recruitment and Plasticity in Health and Disease, edited by J. E. Desmedt. Basel: Karger, 1981, pp. 61-84. 11. Clamann, P. H. The influence of different inputs on the recruitment order of muscles and their motor units. In: Motor Unit Types, Recruitment and Plasticity in Health and Disease. edited by J. E. Desmedt. Basel: Karger, 1981, pp. 176-183. 12. Close, R. Properties of motor units in fast and slow skeletal muscles of the rat. J Physiol 193: 45-55, 1967.

13. Desmedt, J. E. and E. Godaux. Fast motor units are not preferentially activated in rapid voluntary contractions in man. Nature 267: 717-719, 1977. 14. Eccles, J. C. and C. S. Sherrington. Numbers and contraction values of individual motor units examined in some muscles of the limb. Proc R Soc Biol 106: 326--357, 1930. 15. Edstrom, L. and E. Kugelberg. Histochemical composition, distribution of fibers and fatiguability of single motor units. Anterior tibial muscle of the rat. J Neurol Neurosurg Psychiatry 31: 424-433, 1968. 16. Gonzalez, E. R. and F. L. Strand. Neurotropic action of MSH/ACTH 4-10 on neuromuscular function in hypophysectomized rats. Peptides 2: Suppl l, 107-113, 1981. 17. Gorio, A. and A. Manro. Reversibility and mode of action of black widow spider venom on the vertebrate neuromuscular junction. J Gen Physiol 73: 245-263, 1979. 18. Gorio, A. Muscle innervation and reinnervation as a model for development and specificity of neuronal connections. In: Multidisciplinary Approach to Brain Development, edited by C. D. Benedetta, R. Balazs, G. Gombos and G. Porcellati. Amsterdam: Elsevier/North Holland Biomedical Press, 1980, pp. 439452. 19. Gorio, A., P. Marini and R. Zanoni. Muscle reinnervation III. Motoneuron sprouting capacity, enhancement by exogenous gangliosides. Neuroscience 8: 417-429, 1983. 20. Harris, J. B. and P. Wilson. Mechanical properties of dystrophic mouse muscle. J Neurol Neurosurg Psychiatry 34: 512-520, 1971. 21. Henneman, E., G. Somjen and D. O. Carpenter. Functional significance of cell size in spinal motor neurons. J Neurophysiol 28: 560-580, 1965. 22. Henneman, E. Recruitment of motor neurons: the size principle. In: Motor Unit Types, Recruitment and Plasticity in Health and Disease, edited by J. E. Desmedt. Basel: Karger, 1981, pp. 26-60. 23. Karlstrom, L. and A. Dahlstrom. The effect of different types of axonal trauma on the synthesis and transport of amino storage granules in rat sciatic nerve. J Neurobiol 4: 191-200, 1973. 24. Keim, K. L., F. L. Strand and E. B. Sigg. ACTH/MSH peptides increase myocardial contractility in vitro by a neurotropic action. In press. 25. Krivoy, W. and E. Zimmerman. A possible role of polypeptides in synaptic transmission. In: Chemical Modulation o f Brain Function, edited by H. C. Sabelli. New York: Raven Press, 1973, pp. 111-121.

ACTH/MSH 4-10 IMPROVES REGENERATION

26. Krivoy, W. A. and E. Zimmerman. An effect of fl-melanocyte stimulating hormone (/~-MSH) on motoneurons of cat spinal cord. Eur J Pharmacol 46: 315-322, 1977. 27. Kugelberg, E. The motor unit: morphology and function. In: Motor Unit Types, Recruitment and Plasticity in Health and Disease, edited by J. E. Desmedt. Basel: Karger, 1981, pp. 1-16. 28. McArdle, J. J. Complex end plate potentials at regenerating neuromuscular junction of the rat. Exp Neurol 49: 629-638, 1975. 29. McComas, A. J., P. R. W. Fawcett, M. J. Campbell and R. E. P. Sica. Electrophysiological estimation of the number of motor units within a human muscle. J Neurol Neurosurg Psychiatry 34: 121-131, 1971. 30. Milner-Brown, H. S., R. B. Stein, R. G. Lee and W. F. Brown. Motor unit recruitment in patients with neuromuscular disorders. In: Motor Unit Types, Recruitment and Plasticity in Health and Disease, edited by J. E. Desmedt. Basel: Karger, 1981, pp. 305-318. 31. Milner-Brown, H. S., R. B. Stein and R. Yemm. The contractile properties of human motor units during voluntary isometric contractions. J Physiol (Lond) 228: 288-306, 1973. 32. Redferu, P. A. Neuromuscular transmission in new-born rats. J Physiol 209: 701-709, 1970. 33. Saint-Come, C., G. R. Acker and F. L. Strand. Peptide influences on the development and regeneration of motor performance. Peptides 3: 439-449, 1982. 34. Somjen, G., D. O. Carpenter and E. Henneman. Responses of motor neurons of different sizes to graded stimulation of supraspinal centers of the brain. J Neurophysiol 2g: 958--965, 1965.

83

35. Strand, F. L., A. Cayer, E. Gonzalez and H. Stoboy. Peptide enhancement of neuromuscular function: Animal and clinical studies. Pharmacol Biochem Behav 5: Suppl 1,179-187, 1976. 36. Strand, F. L., H. Stoboy, G. Friedebold, W. Krivoy, H. Heyck and H. van Riezen. Changes in muscle action potentials of patients with diseases of motor units following infusion of a peptide fragment of ACTH. Arzneimittelforsch 27: 681-683, 1977. 37. Strand, F. L. and T. T. Kung. ACTH accelerates recovery of neuromuscular function following crushing of peripheral nerve. Peptides 1: 135-138, 1980. 38. Strand, F. L., T. T. Kung and C. Saint-Come. Regenerative ability of spinal motor systems as influenced by ACTH/MSH peptides. In: Functional Recovery for Brain Damage, edited by M. W. van Hof and G. Mohn. Amsterdam: Elsevier, 1981, pp. 369-409. 39. Thompson, W. and J. K. S. Jensen. The extent of sprouting of remaining motor units in partly denervated immature and adult rat soleus muscle. Soc Neurosci Abstr 2: 523-535, 1977. 40. Torda, C. and H. WoLff. Effect of pituitary hormones, cortisone and adrenalectomy on some aspects of neuromuscular systems and acetylcholine synthesis. Am J Physiol 169: 140-149, 1952. 41. Urban, I. and D. de Wied. Changes in excitability of the theta activity generating substrate by ACTH 4-10 in the rat. Exp Brain Res 24: 324-334, 1976. 42. Veals, J. and F. L. Strand. Adrenocorticotropic hormone effects on 14C-choline accumulation by rat brain synaptosomes. Physiologist 22: 75, 1979.