- Email: [email protected]
The Elimination of Polyneuronal Innervation of End-Plates in Developing Rat Muscles with Altered Function
The Elimination of Polyneuronal Innervation of End-Plates in Developing Rat Muscles with Altered Function JIRINA ZELENA, F. VYSKOEIL and ISA JIRMANOVA Institute of Physiology, Czechoslovak Academy of Sciences, 142 20 Prague (Czechoslovakia)
INTRODUCTION It has been firmly established that focally innervated muscle fibres receive transitory polyneuronal innervation during development which is gradually reduced to a monoaxonal supply per end-plate (Redfern, 1970; Bagust et al., 1973; Bennett and Pettigrew, 1974; Brown et al. 1975, 1976; Jansen et al., 1975; Korneliussen and Jansen, 1976; Riley, 1976, 1977; Rosenthal and Taraskevich, 1977). However, the factors and mechanisms which bring about the reduction of the transient polyneuronal innervation are still not known. It has been postulated that the state of activity of the postsynaptic cell may control, in a retrograde manner, the selective stabilization of synapses during the process of maturation (Changeux et al., 1973). In developing rat skeletal muscles, tenotomy appeared to delay the regression of multiaxonal innervation, which had been interpreted as an indication that the state of activity of the nerve-muscle preparation regulates the evolution of its innervation (Benoit and Changeux, 1975). In order to test the above hypothesis (Benoit and Changeux, 1975), we have examined the development of motor end-plates in the rat extensor digitorum longus (EDL) muscles after having changed the state of their activity (1) by removal of the synergic anterior tibialis muscles, which induced compensatory hypertrophy in the EDL; (2) by de-afferentation combined with a high transection of the spinal cord, which restricted active movements of the hind limbs. The reduction of multiaxonal innervation was studied by electrophysiological and morphological methods 9 and 14 days after birth, after either operation performed in neonatal and 1-day-old rats. METHODS Operations
(1) In the first group of animals, the tibialis anterior muscle was totally removed from the right side in new-born male rats under cold anaesthesia. The EDL was left intact in situ, and the skin was sutured. The contralateral EDL
366 muscles, and EDLs of 1-2 unoperated rats of each litter served as control. (2) In the second group, 1-day-old male rats were operated under cold anaesthesia. The spinal cord was exposed by laminectomy from the level of Th8 caudally, and all lumbosacral dorsal roots were sectioned on both sides. In addition, the spinal cord was transected at the level of Th8-9. In this manner, the motoneurones of the lumbosacral region were deprived of most of their afferent input. A hyperextension of both hind limbs dcveloped and was maintained from the first day after the operation onwards, and active movements of the hind limbs were thus restricted. Unoperated littermates served as control.
Electrophysiology Nine and 14 days after the operation, EDL muscles were removed and immersed in oxygenated standard Liley solution, with a potassium content lowered from 5 t o 2.5 mM, in order t o ensure better nerve conductivity. to (+)-tubocurarine was added into the bath in a concentration of 0.5 X 2 X 10-6 M. The standard microelectrode technique was used for recording endplate potentials. The nerve was stimulated at its entry into the EDL with a double wire platinum electrode at a frequency of 0.5 Hz. As the intensity of stimulation was gradually increased, the amplitude of the end-plate potentials varied in a stepwise manner which made it possible t o reveal the number of axons with different thresholds innervating each end-plate examined (cf. VyskoCil and Magazanik, 1977). Thirty to 6 0 end-plates were examined in each muscle.
Morphology
For light microscopy, EDL muscles of 9- and 14-day-old rats were fixed in a 2.5% paraformaldehyde solution overnight. Longitudinal sections 30-40 pm thick were cut with a cryostat microtome, stained by Karnovsky’s method for end-plate Bcetylcholinesterase and impregnated with AgN03 according to a modified method of Mclsaac and Kiernan (1974). The number of axons per end-plate was determined under the light microscope in 50- 150 end-plates in each muscle. For electron microscopy, muscles were fixed in a solution of 1% paraformaldehyde and 1% glutaraldehyde in 0.4 M phosphate buffer for 2-3 h, post-fixed with 2% Os04 for 2 h, dehydrated and embedded in Durcupan. Regions containing end-plates were selected for ultrathin sectioning on transverse semithin sections stained with toluidine blue. Ultrathin sections were stained with 1% uranylacetate and 0.1% lead citrate, and examined with a JEM lOOB electron microscope. RESULTS (1) Differences in the degree o f end-plate maturation in experimental
and control muscles
Motor end-plates were examined in hypertrophic, de-afferented and control muscles 9 and 14 days after birth. The number of monoaxonally and poly-
367 END- PLATES WITH 1 AND 2.3 AXONS
9 DAYS ELECTROPHYSIOLOGY
€NO-PLATES WITH 1 AND 2.3 AXONS
14 DAYS ELECTROPHYSIOLOGY
100 . I .
--AS-
--
50
1
2.3
21476
CH
2.3 25118
1
C
1
2,3
QOP D
1
2
92P CH
HISTOLOGY
100-
1 2 128/5 C
1 2 8013 D
HISTOLOGY
7.
50 -
31014
35015
l0Ob
3-14
56015
lOOl1
Fig. 1 (left). Percentage of motor end-plates with mono- and multiaxonal innervation in EDL muscles 9 days after birth, determined by electrophysiological (upper row) and histological (lower row) methods. Ordinate: percentage of end-plates. Abscissa: paired columns indicating mean % k S.E.M. of monoaxonal(1) and multiaxonal(2, 3) end-plates in each muscle group; the 2nd column, if divided by a transverse line, comprises the percentage of endplates with 2 (above the line) and 3 axons (below the line). CH (hatched columns), muscles undergoing compensatory hypertrophy; C (white columns), control muscles; D (dotted columns), de-afferented muscles. The number of end-plates and muscles investigated in each group is given below each pair of columns. Significant differences between muscle groups, calculated according t o the t-test, are indicated at interrupted lines connecting the related columns; n.s., not significant. Fig. 2 (right). Percentage of motor end-plates with mono- and multiaxonal innervation in EDL muscles 14 days after birth. For explanation see text to Fig. 1.
axonally supplied end-plates was determined by electrophysiological and morphological methods and the results were expressed as percentage of monoaxonal and polyaxonal end-plates in each muscle group. The summarized results are shown in Figs. 1 and 2. The results obtained in the two control groups for hypertrophic and de-afferented EDLs were not significantly different, and both control groups are pooled in the figures. In EDL muscles undergoing compensatory hypertrophy , the muscle weight was increased by 70% on day 9 and by 25% on day 14 in comparison with the contralateral control muscles. The proportion of monoaxonal end-plates on day 9 was 70%0 higher in hypertrophic muscles than in control EDLs (Fig. 1). By day 14 the electrophysiologically revealed difference diminished to 15% (Fig. 2, Electrophysiology) and the small difference found by morphological method was no longer statistically significant (Fig. 2, Histology). In de-afferented muscles in which active movements were greatly restricted, the proportion of monoaxonal end-plates was 50% lower than in the control muscles on day 9 (Fig. 1). The difference was lost by day 14, although
Fig. 3. Transverse section of an end-plate with 1 large and several small terminals in a shallow groove o n the muscle fibre surface (m), from a de-afferented EDL muscle 1 4 days after birth. The postsynaptic membrane is thickened along t h e synaptic contact (between arrows) but not in the developing infoldings. Note that 2 tiny terminal profiles are n o t in contact with the basal lamina. A degenerated axonal profile (arrowhead) is seen engulfed within the cytoplasm of the Schwann cell (S) covering the end-plate. Fig. 4. A Schwann cell (S) with 1 large and 2 small axonal profiles near an end-plate on the muscle fibre (m) seen below; from a hypertrophic EDL 1 4 days after removal of the anterior tibia1 muscle. The small axon (arrowhead) enwrapped by Schwann cell processes is assumed t o be retracting from the end-plate.
Fig. 5. A larger magnification of the presumably retracting axon seen in Fig. 4, taken from another section. Note that the Schwann cell processes (S) are still in contact with basal lamina of the muscle fibre (m). Scale 1 p m .
3 69 de-afferented muscles had restricted movements during the whole investigated period. If we take the percentage of monoaxonal end-plates as a measure of endplate maturation, it can be concluded that the rate of maturation is accelerated in hypertrophic muscles and retarded in de-afferented muscles by day 9. In two week’s time, however, the majority of end-plates becomes monoaxonal both in experimental and control muscles. (2) Retraction o f redundant axons - a probable mode of reduction
of multiaxonal innervation
A sample of about 50 end-plates from control and experimental muscles was
examined in the electron microscope with the aim of elucidating the question of whether or not the reduction of multiaxonal innervation occurred by degeneration of redundant axons and terminals. None of the investigated endplates contained degenerating or degenerated terminals. However, occasional degenerated axonal profiles were observed in the end-plate region, encircled by Schwann cell processes or engulfed by the cytoplasm of Schwann cells covering the end-plate (Fig. 3). In muscles of 14-day-old animals, a small axonal profile was sometimes seen enwrapped by numerous Schwann cell processes, near a large-sized axon approaching the same end-plate (Figs. 4 and 5). Intramuscular nerve branches which mainly consisted of axons with incipient myelination by day 14, frequently contained one or two tiny axonal profiles on the outer circumference of the Schwann cells encircling the central axon. These ultrastructural findings suggest that terminals become detached and axons are retracted during the process of end-plate maturation. DISCUSSION A transient functional hyperinnervation of muscle fibres appears to be regular developmental feature preceding the establishment of monoaxonal contacts characteristic for mature neuromuscular junctions of focally innervated muscles. In the rat, polyneuronal innervation is being reduced during the first two weeks after birth, and the monoaxonal pattern is established between the second and third week postnatally (Redfern, 1970; Bennett and Pettigrew, 1974; Benoit and Changeux, 1975; Riley 1977; Rosenthal and Taraskevich, 1977). In our experiments the process of reduction of multiaxonal innervation was found to be accelerated in rat EDL muscles undergoing compensatory hypertrophy. A similar effect has recently been observed in developing rat soleus muscles after chronic nerve stimulation (O’Brien et al., 1977). However, no difference was found between stimulated and unstimulated muscles by histological method (Ostberg and Vrbovd, 1977), which was explained by the assumption that non-functional axons still maintained their contact with the muscle fibre. It is possible that redundant axons are retracted from the endplate with a delay, after their terminals have lost functional contact with the muscle fibre. In our study, the percentage of multiaxonal end-plates deter-
3 70 mined histologically was usually somewhat higher than that revealed electrophysiologically but the difference was small. In de-afferented muscles of animals with restricted movement of hind limbs, elimination of multiaxonal innervation was found to be retarded, as was previously described after tenotomy (Benoit and Changeux, 1975). Although chronic effects upon the neuromuscular activity of the model situation used in our experiments are not clearly defined (cf. Hnik, 1956; Gutmann, 1972; MackovA and Hnik, 1973), it is apparent that activity influences the process of elimination of redundant axons. It should be noted, however, that the retardation effect observed after de-afferentation was only transient. The developmental programme of end-plate maturation - which is presumably genetically encoded - was eventually carried out without significant delay in de-afferented muscles with reduced active movements, i.e. by motoneurones chronically deprived of their afferent input. As regards the mechanism of elimination of polyneuronal innervation during development, our findings contradict the conclusions of Rosenthal and Taraskevich ( 1 977) that redundant terminals degenerate as in denervated adult muscles. Our results rather indicate that redundant terminals are eliminated by retraction, as has been suggested previously (Korneliussen and Jansen, 1976; Riley, 1977). Although degenerated axons are occasionally observed in the endplate region, it is assumed that such degeneration occurs during the process of axonal retraction, when terminal parts of receding axons may be severed or pinched off and phagocytized by Schwann cells. The presence of tiny axonal profiles, found sometimes in intramuscular nerves outside the Schwann cells myelinating the growing axons, suggests that non-functional axons cease to grow, become atrophic and are retracted into persisting parent axons within the muscle. However, the problem concerning the factors affecting the detachment and retraction of supernumeraxy terminals requires further study. SUMMARY The elimination of polyneuronal innervation from developing end-plates was studied in rat EDL muscles with altered function (1) during compensatory hypertrophy and (2) after de-afferentation. (1) The EDL muscles became overloaded and underwent compensatory hypertrophy after removal of the tibialis anterior muscles in new-born rats. The maturation of end-plates was speeded up: electrophysiological examination revealed that 74% end-plates already had a monoaxonal supply by day 9, and 94% were monoaxonally supplied by day 14, as compared with 43% and 82% monoaxonal end-plates in corresponding control muscles. Similar results were obtained by histological method, but the percentage of monoaxonal end-plates was as a rule somewhat lower: 72% monoaxonal end-plates were found in hypertrophic muscles on day 9 and 85% on day 14, as compared with 49% and 77% monoaxonal end-plates in control muscles. (2) After bilateral section of lumbosacral dorsal roots and high transection of the spinal cord in lday-old rats, active movements were restricted and the maturation of end-plates in EDL muscles was retarded: only 24% and 37% end-
37 1 plates became monoaxonal by day 9 as determined by electrophysiological and histological method respectively. No difference was found between deafferented and control muscles by day 14, when about 80% of all end-plates investigated already had a monoaxonal supply. The maturation of motor end-plates thus appears t o be speeded up by compensatory hypertrophy and slowed down by de-afferentation. The retardation observed after de-afferen tation is only temporary: the majority of end-plates become monoaxonally supplied in two weeks time in both experimental and control muscles. Since no degenerating or degenerated motor terminals were found in our sample of 50 end-plates investigated in the electron microscope, our findings support the assumption that redundant terminals become detached and axons retract during postnatal development. Occasional axons may, however, degenerate when they are trapped and severed during the process of retraction. ACKNOWLEDGEMENTS The authors wish t o thank Mrs. Marie Sobotkovh, Mrs. MarkCta KrupkovA, Mr. H. Kunz and Ing. V. Pokomq for their skillful technical assistance, and Dr. P. H n i l for critical reading of the manuscript. REFERENCES Bagust, J., Lewis, D.M., Westerman, R.A. (1 973) Polyneuronal innervation of kitten skeletal muscle. J. Physiol. (Lond.), 229, 241-255. Bennett, M.R., Pettigrew, A.G. (1 974) The formation of synapses in striated muscle during development. J. Physiol. (Lond.), 241, 51 5-545. Benoit, P., Changeux, J . P . (1975) Consequences of tenotomy o n the evolution of multiinnervation in developing rat soleus muscle. Brain Res., 99, 354-358. Brown, M.C., Jansen, J.K.S. and Van Essen, D. (1975) A large-scale reduction in motoneurone peripheral fields during postnatal development in the rat. A c t a physiol. scand., 95, 3-4A. Brown, M.C., Jansen, J.K.S.and Van Essen, D. (1976) Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation. J. Physiol. (Lond.), 261,387-422. Changeux, J.-P., Courrege, P.H. and Danchin, A. (1973) A theory of the epigenesis of neuronal networks by selective stabilization of synapses. Proc. nat. Acad. Sci. (Wash.), 70, 2974-2978. Gutmann, E. (1 972) Comparative aspects of compensatory hypertrophy of skeletal and heart muscle. Folia Fac. med. Univ. Comenianae Bratisl., 10, Suppl., 229-239. Hnfk, P. ( 1956) Motor function disturbances and excitability changes following de-afferentation. Physiol. bohemoslova, 5, 305-3 15. Jansen, J.K.S., Van Essen, D. and Brown, M.C. (1975) Formation and elimination of synapses in skeletal muscles of rat. Cold Spring Harb. Symp. quant. Biol., 40, 425434. Korneliussen, H., Jansen, J.K.S. ( 1 976) Morphological aspects of the elimination of polyneuronal innervation of skeletal muscle fibres in newborn rats. J. Neurocyrol., 5, 591604. Mackovi, E., Hnik, P. (1973) Compensatory muscle hypertrophy induced by tenotomy of synergists is not true working hypertrophy. Physiol. bohemoslov., 22, 43-49. McIsaac, E. and Kiernan, J.A. ( 1 974) Complete staining of neuromuscular innervation with bromoindigo and silver. Stain Technol., 49, 2 1 1-214.
312 O’Brien, R.A.D., Purves, R.D., Vrbovl, G. ( 1 977) Effect of activity o n the elimination of multiple innervation in soleus muscles of rats. J. Physiol. (Lond.), 27 1, 54-55P. Ostberg, A.J.C., Vrbovh, G. (1977) Illustration of the disappearance of polyneuronal innervation of developing skeletal muscle. J. Physiol. (Lond.), 271, 6-7P. Redfern, P.A. (1 970) Neuromuscular transmission in newborn rats. J . Physiol. (Lond.), 209, 701-709. Riley, D.A. (1976) Multiple axon branches innervating single end-plates of kitten soleus myofibers. Brain Res., 1 10, 158- 16 1. Riley, D.A. (1 977) Spontaneous elimination of nerve terminals from the end-plates of developing skeletal myofibers. Brain Res., 134, 279-285. Rosenthal, J.L., Taraskevich, P.S. ( 1977) Reduction of multiaxonal innervation at the neuromuscular junction of the rat during development. J. Physiol. (Lond.), 270, 229-3 10. VyskoEil, F., Magazanik, L.G.(1977) Dual end-plate potentials at the single neuromuscular junction of adult frog. Pflugers Arch., 368, 271-273,
INTRODUCTION It has been firmly established that focally innervated muscle fibres receive transitory polyneuronal innervation during development which is gradually reduced to a monoaxonal supply per end-plate (Redfern, 1970; Bagust et al., 1973; Bennett and Pettigrew, 1974; Brown et al. 1975, 1976; Jansen et al., 1975; Korneliussen and Jansen, 1976; Riley, 1976, 1977; Rosenthal and Taraskevich, 1977). However, the factors and mechanisms which bring about the reduction of the transient polyneuronal innervation are still not known. It has been postulated that the state of activity of the postsynaptic cell may control, in a retrograde manner, the selective stabilization of synapses during the process of maturation (Changeux et al., 1973). In developing rat skeletal muscles, tenotomy appeared to delay the regression of multiaxonal innervation, which had been interpreted as an indication that the state of activity of the nerve-muscle preparation regulates the evolution of its innervation (Benoit and Changeux, 1975). In order to test the above hypothesis (Benoit and Changeux, 1975), we have examined the development of motor end-plates in the rat extensor digitorum longus (EDL) muscles after having changed the state of their activity (1) by removal of the synergic anterior tibialis muscles, which induced compensatory hypertrophy in the EDL; (2) by de-afferentation combined with a high transection of the spinal cord, which restricted active movements of the hind limbs. The reduction of multiaxonal innervation was studied by electrophysiological and morphological methods 9 and 14 days after birth, after either operation performed in neonatal and 1-day-old rats. METHODS Operations
(1) In the first group of animals, the tibialis anterior muscle was totally removed from the right side in new-born male rats under cold anaesthesia. The EDL was left intact in situ, and the skin was sutured. The contralateral EDL
366 muscles, and EDLs of 1-2 unoperated rats of each litter served as control. (2) In the second group, 1-day-old male rats were operated under cold anaesthesia. The spinal cord was exposed by laminectomy from the level of Th8 caudally, and all lumbosacral dorsal roots were sectioned on both sides. In addition, the spinal cord was transected at the level of Th8-9. In this manner, the motoneurones of the lumbosacral region were deprived of most of their afferent input. A hyperextension of both hind limbs dcveloped and was maintained from the first day after the operation onwards, and active movements of the hind limbs were thus restricted. Unoperated littermates served as control.
Electrophysiology Nine and 14 days after the operation, EDL muscles were removed and immersed in oxygenated standard Liley solution, with a potassium content lowered from 5 t o 2.5 mM, in order t o ensure better nerve conductivity. to (+)-tubocurarine was added into the bath in a concentration of 0.5 X 2 X 10-6 M. The standard microelectrode technique was used for recording endplate potentials. The nerve was stimulated at its entry into the EDL with a double wire platinum electrode at a frequency of 0.5 Hz. As the intensity of stimulation was gradually increased, the amplitude of the end-plate potentials varied in a stepwise manner which made it possible t o reveal the number of axons with different thresholds innervating each end-plate examined (cf. VyskoCil and Magazanik, 1977). Thirty to 6 0 end-plates were examined in each muscle.
Morphology
For light microscopy, EDL muscles of 9- and 14-day-old rats were fixed in a 2.5% paraformaldehyde solution overnight. Longitudinal sections 30-40 pm thick were cut with a cryostat microtome, stained by Karnovsky’s method for end-plate Bcetylcholinesterase and impregnated with AgN03 according to a modified method of Mclsaac and Kiernan (1974). The number of axons per end-plate was determined under the light microscope in 50- 150 end-plates in each muscle. For electron microscopy, muscles were fixed in a solution of 1% paraformaldehyde and 1% glutaraldehyde in 0.4 M phosphate buffer for 2-3 h, post-fixed with 2% Os04 for 2 h, dehydrated and embedded in Durcupan. Regions containing end-plates were selected for ultrathin sectioning on transverse semithin sections stained with toluidine blue. Ultrathin sections were stained with 1% uranylacetate and 0.1% lead citrate, and examined with a JEM lOOB electron microscope. RESULTS (1) Differences in the degree o f end-plate maturation in experimental
and control muscles
Motor end-plates were examined in hypertrophic, de-afferented and control muscles 9 and 14 days after birth. The number of monoaxonally and poly-
367 END- PLATES WITH 1 AND 2.3 AXONS
9 DAYS ELECTROPHYSIOLOGY
€NO-PLATES WITH 1 AND 2.3 AXONS
14 DAYS ELECTROPHYSIOLOGY
100 . I .
--AS-
--
50
1
2.3
21476
CH
2.3 25118
1
C
1
2,3
QOP D
1
2
92P CH
HISTOLOGY
100-
1 2 128/5 C
1 2 8013 D
HISTOLOGY
7.
50 -
31014
35015
l0Ob
3-14
56015
lOOl1
Fig. 1 (left). Percentage of motor end-plates with mono- and multiaxonal innervation in EDL muscles 9 days after birth, determined by electrophysiological (upper row) and histological (lower row) methods. Ordinate: percentage of end-plates. Abscissa: paired columns indicating mean % k S.E.M. of monoaxonal(1) and multiaxonal(2, 3) end-plates in each muscle group; the 2nd column, if divided by a transverse line, comprises the percentage of endplates with 2 (above the line) and 3 axons (below the line). CH (hatched columns), muscles undergoing compensatory hypertrophy; C (white columns), control muscles; D (dotted columns), de-afferented muscles. The number of end-plates and muscles investigated in each group is given below each pair of columns. Significant differences between muscle groups, calculated according t o the t-test, are indicated at interrupted lines connecting the related columns; n.s., not significant. Fig. 2 (right). Percentage of motor end-plates with mono- and multiaxonal innervation in EDL muscles 14 days after birth. For explanation see text to Fig. 1.
axonally supplied end-plates was determined by electrophysiological and morphological methods and the results were expressed as percentage of monoaxonal and polyaxonal end-plates in each muscle group. The summarized results are shown in Figs. 1 and 2. The results obtained in the two control groups for hypertrophic and de-afferented EDLs were not significantly different, and both control groups are pooled in the figures. In EDL muscles undergoing compensatory hypertrophy , the muscle weight was increased by 70% on day 9 and by 25% on day 14 in comparison with the contralateral control muscles. The proportion of monoaxonal end-plates on day 9 was 70%0 higher in hypertrophic muscles than in control EDLs (Fig. 1). By day 14 the electrophysiologically revealed difference diminished to 15% (Fig. 2, Electrophysiology) and the small difference found by morphological method was no longer statistically significant (Fig. 2, Histology). In de-afferented muscles in which active movements were greatly restricted, the proportion of monoaxonal end-plates was 50% lower than in the control muscles on day 9 (Fig. 1). The difference was lost by day 14, although
Fig. 3. Transverse section of an end-plate with 1 large and several small terminals in a shallow groove o n the muscle fibre surface (m), from a de-afferented EDL muscle 1 4 days after birth. The postsynaptic membrane is thickened along t h e synaptic contact (between arrows) but not in the developing infoldings. Note that 2 tiny terminal profiles are n o t in contact with the basal lamina. A degenerated axonal profile (arrowhead) is seen engulfed within the cytoplasm of the Schwann cell (S) covering the end-plate. Fig. 4. A Schwann cell (S) with 1 large and 2 small axonal profiles near an end-plate on the muscle fibre (m) seen below; from a hypertrophic EDL 1 4 days after removal of the anterior tibia1 muscle. The small axon (arrowhead) enwrapped by Schwann cell processes is assumed t o be retracting from the end-plate.
Fig. 5. A larger magnification of the presumably retracting axon seen in Fig. 4, taken from another section. Note that the Schwann cell processes (S) are still in contact with basal lamina of the muscle fibre (m). Scale 1 p m .
3 69 de-afferented muscles had restricted movements during the whole investigated period. If we take the percentage of monoaxonal end-plates as a measure of endplate maturation, it can be concluded that the rate of maturation is accelerated in hypertrophic muscles and retarded in de-afferented muscles by day 9. In two week’s time, however, the majority of end-plates becomes monoaxonal both in experimental and control muscles. (2) Retraction o f redundant axons - a probable mode of reduction
of multiaxonal innervation
A sample of about 50 end-plates from control and experimental muscles was
examined in the electron microscope with the aim of elucidating the question of whether or not the reduction of multiaxonal innervation occurred by degeneration of redundant axons and terminals. None of the investigated endplates contained degenerating or degenerated terminals. However, occasional degenerated axonal profiles were observed in the end-plate region, encircled by Schwann cell processes or engulfed by the cytoplasm of Schwann cells covering the end-plate (Fig. 3). In muscles of 14-day-old animals, a small axonal profile was sometimes seen enwrapped by numerous Schwann cell processes, near a large-sized axon approaching the same end-plate (Figs. 4 and 5). Intramuscular nerve branches which mainly consisted of axons with incipient myelination by day 14, frequently contained one or two tiny axonal profiles on the outer circumference of the Schwann cells encircling the central axon. These ultrastructural findings suggest that terminals become detached and axons are retracted during the process of end-plate maturation. DISCUSSION A transient functional hyperinnervation of muscle fibres appears to be regular developmental feature preceding the establishment of monoaxonal contacts characteristic for mature neuromuscular junctions of focally innervated muscles. In the rat, polyneuronal innervation is being reduced during the first two weeks after birth, and the monoaxonal pattern is established between the second and third week postnatally (Redfern, 1970; Bennett and Pettigrew, 1974; Benoit and Changeux, 1975; Riley 1977; Rosenthal and Taraskevich, 1977). In our experiments the process of reduction of multiaxonal innervation was found to be accelerated in rat EDL muscles undergoing compensatory hypertrophy. A similar effect has recently been observed in developing rat soleus muscles after chronic nerve stimulation (O’Brien et al., 1977). However, no difference was found between stimulated and unstimulated muscles by histological method (Ostberg and Vrbovd, 1977), which was explained by the assumption that non-functional axons still maintained their contact with the muscle fibre. It is possible that redundant axons are retracted from the endplate with a delay, after their terminals have lost functional contact with the muscle fibre. In our study, the percentage of multiaxonal end-plates deter-
3 70 mined histologically was usually somewhat higher than that revealed electrophysiologically but the difference was small. In de-afferented muscles of animals with restricted movement of hind limbs, elimination of multiaxonal innervation was found to be retarded, as was previously described after tenotomy (Benoit and Changeux, 1975). Although chronic effects upon the neuromuscular activity of the model situation used in our experiments are not clearly defined (cf. Hnik, 1956; Gutmann, 1972; MackovA and Hnik, 1973), it is apparent that activity influences the process of elimination of redundant axons. It should be noted, however, that the retardation effect observed after de-afferentation was only transient. The developmental programme of end-plate maturation - which is presumably genetically encoded - was eventually carried out without significant delay in de-afferented muscles with reduced active movements, i.e. by motoneurones chronically deprived of their afferent input. As regards the mechanism of elimination of polyneuronal innervation during development, our findings contradict the conclusions of Rosenthal and Taraskevich ( 1 977) that redundant terminals degenerate as in denervated adult muscles. Our results rather indicate that redundant terminals are eliminated by retraction, as has been suggested previously (Korneliussen and Jansen, 1976; Riley, 1977). Although degenerated axons are occasionally observed in the endplate region, it is assumed that such degeneration occurs during the process of axonal retraction, when terminal parts of receding axons may be severed or pinched off and phagocytized by Schwann cells. The presence of tiny axonal profiles, found sometimes in intramuscular nerves outside the Schwann cells myelinating the growing axons, suggests that non-functional axons cease to grow, become atrophic and are retracted into persisting parent axons within the muscle. However, the problem concerning the factors affecting the detachment and retraction of supernumeraxy terminals requires further study. SUMMARY The elimination of polyneuronal innervation from developing end-plates was studied in rat EDL muscles with altered function (1) during compensatory hypertrophy and (2) after de-afferentation. (1) The EDL muscles became overloaded and underwent compensatory hypertrophy after removal of the tibialis anterior muscles in new-born rats. The maturation of end-plates was speeded up: electrophysiological examination revealed that 74% end-plates already had a monoaxonal supply by day 9, and 94% were monoaxonally supplied by day 14, as compared with 43% and 82% monoaxonal end-plates in corresponding control muscles. Similar results were obtained by histological method, but the percentage of monoaxonal end-plates was as a rule somewhat lower: 72% monoaxonal end-plates were found in hypertrophic muscles on day 9 and 85% on day 14, as compared with 49% and 77% monoaxonal end-plates in control muscles. (2) After bilateral section of lumbosacral dorsal roots and high transection of the spinal cord in lday-old rats, active movements were restricted and the maturation of end-plates in EDL muscles was retarded: only 24% and 37% end-
37 1 plates became monoaxonal by day 9 as determined by electrophysiological and histological method respectively. No difference was found between deafferented and control muscles by day 14, when about 80% of all end-plates investigated already had a monoaxonal supply. The maturation of motor end-plates thus appears t o be speeded up by compensatory hypertrophy and slowed down by de-afferentation. The retardation observed after de-afferen tation is only temporary: the majority of end-plates become monoaxonally supplied in two weeks time in both experimental and control muscles. Since no degenerating or degenerated motor terminals were found in our sample of 50 end-plates investigated in the electron microscope, our findings support the assumption that redundant terminals become detached and axons retract during postnatal development. Occasional axons may, however, degenerate when they are trapped and severed during the process of retraction. ACKNOWLEDGEMENTS The authors wish t o thank Mrs. Marie Sobotkovh, Mrs. MarkCta KrupkovA, Mr. H. Kunz and Ing. V. Pokomq for their skillful technical assistance, and Dr. P. H n i l for critical reading of the manuscript. REFERENCES Bagust, J., Lewis, D.M., Westerman, R.A. (1 973) Polyneuronal innervation of kitten skeletal muscle. J. Physiol. (Lond.), 229, 241-255. Bennett, M.R., Pettigrew, A.G. (1 974) The formation of synapses in striated muscle during development. J. Physiol. (Lond.), 241, 51 5-545. Benoit, P., Changeux, J . P . (1975) Consequences of tenotomy o n the evolution of multiinnervation in developing rat soleus muscle. Brain Res., 99, 354-358. Brown, M.C., Jansen, J.K.S. and Van Essen, D. (1975) A large-scale reduction in motoneurone peripheral fields during postnatal development in the rat. A c t a physiol. scand., 95, 3-4A. Brown, M.C., Jansen, J.K.S.and Van Essen, D. (1976) Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation. J. Physiol. (Lond.), 261,387-422. Changeux, J.-P., Courrege, P.H. and Danchin, A. (1973) A theory of the epigenesis of neuronal networks by selective stabilization of synapses. Proc. nat. Acad. Sci. (Wash.), 70, 2974-2978. Gutmann, E. (1 972) Comparative aspects of compensatory hypertrophy of skeletal and heart muscle. Folia Fac. med. Univ. Comenianae Bratisl., 10, Suppl., 229-239. Hnfk, P. ( 1956) Motor function disturbances and excitability changes following de-afferentation. Physiol. bohemoslova, 5, 305-3 15. Jansen, J.K.S., Van Essen, D. and Brown, M.C. (1975) Formation and elimination of synapses in skeletal muscles of rat. Cold Spring Harb. Symp. quant. Biol., 40, 425434. Korneliussen, H., Jansen, J.K.S. ( 1 976) Morphological aspects of the elimination of polyneuronal innervation of skeletal muscle fibres in newborn rats. J. Neurocyrol., 5, 591604. Mackovi, E., Hnik, P. (1973) Compensatory muscle hypertrophy induced by tenotomy of synergists is not true working hypertrophy. Physiol. bohemoslov., 22, 43-49. McIsaac, E. and Kiernan, J.A. ( 1 974) Complete staining of neuromuscular innervation with bromoindigo and silver. Stain Technol., 49, 2 1 1-214.
312 O’Brien, R.A.D., Purves, R.D., Vrbovl, G. ( 1 977) Effect of activity o n the elimination of multiple innervation in soleus muscles of rats. J. Physiol. (Lond.), 27 1, 54-55P. Ostberg, A.J.C., Vrbovh, G. (1977) Illustration of the disappearance of polyneuronal innervation of developing skeletal muscle. J. Physiol. (Lond.), 271, 6-7P. Redfern, P.A. (1 970) Neuromuscular transmission in newborn rats. J . Physiol. (Lond.), 209, 701-709. Riley, D.A. (1976) Multiple axon branches innervating single end-plates of kitten soleus myofibers. Brain Res., 1 10, 158- 16 1. Riley, D.A. (1 977) Spontaneous elimination of nerve terminals from the end-plates of developing skeletal myofibers. Brain Res., 134, 279-285. Rosenthal, J.L., Taraskevich, P.S. ( 1977) Reduction of multiaxonal innervation at the neuromuscular junction of the rat during development. J. Physiol. (Lond.), 270, 229-3 10. VyskoEil, F., Magazanik, L.G.(1977) Dual end-plate potentials at the single neuromuscular junction of adult frog. Pflugers Arch., 368, 271-273,