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Multiple axon branches innervating single endplates of kitten soleus myofibers
Braht Reseurch, i]0(197o) t55 i,'~l
158
(~ Elsevier Scientitic Publishing Conapany, Amsterdam
Printed in The Nethefkmd~
Multiple axon branches innervating single endplates of kitten soleus myofibers
DAN A. RILEY
Department of Anatomy, University of Cali]brnia, San Franciso, Calif 94143 ( U. S.A. ) (Accepted March 22nd, 1976)
There is good physiological evidence that adult myofibers of mammalian skeletal muscles are unineuronally innervated4, 6m while developing myofibers are polyneuronally innervated 1,a& Bennett and Pettigrew a using a cholinesterasesilver stain identified one endplate per myofiber in the developing diaphragm muscles of rats and found that these endplates were innervated by more than one nerve terminal. In contrast, a preliminary gold chloride study of kitten muscles revealed multiple endplates per myofiber, each endplate apparently innervated by a different axon 9. Therefore, the present study further investigates developing soleus muscles of kittens to ascertain whether multiple fibers per endplate or multiple endplates per myofiber account for the polyneuronal innervation. Soleus muscles are studied because functional polyneuronal innervation is common in newborn muscles and essentially absent 6 weeks later 1. Striking changes in the pattern of innervation should occur during this period which can be correlated with the physiology. The present results indicate that most of the myofibers have a single endplate and these endplates are innervated by more than one axon during the period of functional polyneuronal innervation. Soleus muscles were removed from pairs of anesthetized kittens (newborn, 2, 4, and 6 weeks of age). Nerve fibers were stained en bloc using a modified de Castro silver technique 2. Teased bundles of whole myofibers were examined light microscopically to assess motor innervation patterns. Innervated endplates were examined to determine the number of endplates per myofiber and the number of axons from different neurons per endplate. Endplates were identified by collections of myonuclei occupying non-striated cytoplasm in the periphery of myofibers. To obtain a representative sample, 200 endplates per muscle were analyzed consecutively. Nerve fibers that arborized within the confines of the endplate as described by Gutmann and Young 7 were considered terminals of alpha motor axons. Examining the whole length of myofibers in the 2-, 4- and 6-week-old kittens revealed that one endplate per myofiber was the common pattern, in each group, not more than 1 °J~iof the myofibers possessed two endplates. There was less certainty
159 a b o u t the n u m b e r o f endplates per myofiber in newborn muscles because these myofibers were smaller and difficult to resolve as separate entities. However, the majority of myofibers appeared to possess a single endplate. The major problem in recognizing polyneuronal innervation o f endplates is to distinguish endplates innervated by multiple terminals o f different neurons from those innervated by multiple terminals o f a single neuron. Absolute p r o o f o f separate origin requires the impossible task o f tracing each fiber back to the soma of its parent neuron. In adult muscles, when an axon bifurcates and sends both branches to the same endplate, the majority of these axons branch between the endplates and the larger intramuscular nerve bundlesL Therefore, in the present study nerve fibers that remained distinct when traced proximally from an endplate to an intramuscular nerve bundle were considered to be f r o m different neurons. A n endplate supplied by multiple fibers that were branches o f a single axon was recognized as unineuronally innervated, Endplates of the 2-, 4- and 6-week-old animals were innervated by 1, 2, 3 or ~> 4 fibers (Table I). In each case, these fibers were readily followed from the endplates into the nerve bundles as separate entities. Branching was difficult to resolve in the newborn muscles because the distance between the endplates and the intramuscular nerve bundles was short (Fig. 1). Therefore, their endplates were classified as either singly innervated (1 terminal) or multiply innervated (2 or more terminals) realizing that in this group some of the multiple terminals represented preterminal branching o f single axons (Table I). Approximately 72 ~o of the endplates encountered in newborn muscles were innervated by 2 or more terminals. The percentage of endplates with multiple terminals progressively declined with age (Table I and Fig. 1). While endplates with 3 and >/ 4 terminals were present in the muscles of 2-week-old kittens, 2 terminals per endplate predominated (Table I). TABLE I P E R C E N T A G E S OF E N D P L A T E S I N N E R V A T E D BY T H E S P E C I F I E D N U M B E R O F N E R V E T E R M I N A L S
Age
Newborn* 2 weeks** 4 weeks 6 weeks
Kitten
1 2 I 2 I 2 1 2
Number of'nerve terminals per endplate 1
2
27.5 28.0 32.5 69.0 97.0 97.6 99.5 98.5
72.5 72.0 51.5 30.2 3.0 2.4 0.5 1.5
3
>~4
14.5 0.8
1.5
* For the newborn muscles alone, column 2 represents the percentage of endplates with >~ 1 fiber. Nerve branching was usually not resolvable in the group, but some clearly had 3 and 4 fibers per endplate. ** This pair of kittens were unequal in size, unlike the other pairs. The muscles of kitten 2 were 1.5 times longer than those of kitten 1. Kitten 2 was large for his chronological age which probably accounts for the smaller percentage of endplates with 2 or more terminals.
160
Fig. 1. Silver stained m o t o r nerve terminals o f developing kitten soleus muscles. A: newborn; miram u s c u l a r nerve bundle (IN[I). B: 2 weeks; the a r r o w h e a d points to an endplate in x~hich terminals of 3 a x o n s arborize. C: 4 weeks; 2 axons arborize at the endplate indicated by the arrowhead. These two a x o n s remained separate fibers when traced at high magnification into the larger nerve bundle. D: 6 weeks; endplate myonuclei (N), All magnifications are ,: 800.
161 The present study demonstrates that developing myofibers in kitten soleus muscles exhibit a single endplate rather than multiple endplates per myofiber. During the period of functional polyneuronal innervation 1, these endplates are innervated by more than one nerve fiber, Since these fibers fit the criteria for terminals of alpha motor axons 7 and were judged to come from different neurons, multiple terminals per endplate appears to be the anatomical substrate for polyneuronal innervation. Bagust, Lewis and Westerman I predicted from their physiological studies on kitten soleus muscles that 47 ~o of the myofibers were polyneuronally innervated at 2 weeks of age and very few (1.3°/,,) were so innervated at 6 weeks. The present histological findings correlate directly with these predicted percentages and support the assumption which was used in their calculations that the majority of polyneuronally innervated myofibers at 2 weeks are innervated by two axons rather than more than two. They further suggested that developing myofibers exhibited more than one endplate which could explain the polyneuronal innervation 9. This was not corroborated by the present findings because multiple endplates were uncommon at all of the stages examined. Thus, polyneuronal innervation appears explicable in large part by multiple nerve terminals innervating a single endplate per myofiber. Even if these terminals were unable to generate endplate potentials, multiple innervation allows motor neurons to interact on their end-organ. That is, the process of attaining unineuronal innervation is likely to involve competition between neurons resulting in the suppression and withdrawal of all but the appropriate nerve fiber a. This interaction may be ,~ssential in the establishment of motor units. I wish to thank Mrs. Edith Salazar for her excellent technical assistance. This research was supported by the Dubois, P.R.O.P.S. Endowment, and Academic Senate Research State funds. I BAGUST, J., LEWtS, D. M., AND WESTERMAN, Polyneuronal innervation of kitten skeletal muscle, J. Physiol. (Lend.), 229 (1973) 241-255. 2 BARKER, D., AND IP, M. C., A silver method for demonstrating the innervation of mammalian muscle in teased preparations, J. Physiol. (Lond.), 169 (1963) 73-74P. 3 BENNETT, M. R., AND PETT1GREW, A. G., The formation of synapses in striated muscle during development, J. Physiol. (Lond.), 241 (1974) 515 545. 4 BROWN, M. C., AND MATTHEWS, P. B. C., An investigation into the possible existence of polyneuronal innervation of individual skeletal muscle fibers in certain hind-limb muscles of the cat, J. Physiol. (Lond.), 151 (1960) 436-457. 5 FANGBONER, R. F., AND VANABLE, J. W., Jr., Formation and regression of inappropriate nerve sprouts during trochlear nerve regeneration in Xenopus laevis, J. comp. Neurol., 157 (1974) 391-406. 6 GUTH, L., Neuromuscular function after regeneration of interrupted nerve fibers into partially denervated muscle, Exp. Neurol., 6 (1962) 129 141. 7 GUTMANN, E., AND YOUNG, J. Z., The re-innervation of muscle after various periods of atrophy, J. Anat. (Lond.), 78 (1944) 15 43. 8 REDFERN, P. A., Neuromuscular transmission in newborn rats, J. Physiol. (Lond.), 209 (1970) 701-709. 9 WESTERMAN,R. A., LEWIS, D. M., BAGUST, J., EDJTEHADI, G. D., AND PALLOT, D., Communication between nerves and muscles: Postnatal development in kitten hind-limb fast and slow twitch muscle. In H. P. ZIPPEL (Ed.), Symposium olt Memory and TransJer ofln]brmation, Plenum Press, New York, 1972, pp. 255-291.
158
(~ Elsevier Scientitic Publishing Conapany, Amsterdam
Printed in The Nethefkmd~
Multiple axon branches innervating single endplates of kitten soleus myofibers
DAN A. RILEY
Department of Anatomy, University of Cali]brnia, San Franciso, Calif 94143 ( U. S.A. ) (Accepted March 22nd, 1976)
There is good physiological evidence that adult myofibers of mammalian skeletal muscles are unineuronally innervated4, 6m while developing myofibers are polyneuronally innervated 1,a& Bennett and Pettigrew a using a cholinesterasesilver stain identified one endplate per myofiber in the developing diaphragm muscles of rats and found that these endplates were innervated by more than one nerve terminal. In contrast, a preliminary gold chloride study of kitten muscles revealed multiple endplates per myofiber, each endplate apparently innervated by a different axon 9. Therefore, the present study further investigates developing soleus muscles of kittens to ascertain whether multiple fibers per endplate or multiple endplates per myofiber account for the polyneuronal innervation. Soleus muscles are studied because functional polyneuronal innervation is common in newborn muscles and essentially absent 6 weeks later 1. Striking changes in the pattern of innervation should occur during this period which can be correlated with the physiology. The present results indicate that most of the myofibers have a single endplate and these endplates are innervated by more than one axon during the period of functional polyneuronal innervation. Soleus muscles were removed from pairs of anesthetized kittens (newborn, 2, 4, and 6 weeks of age). Nerve fibers were stained en bloc using a modified de Castro silver technique 2. Teased bundles of whole myofibers were examined light microscopically to assess motor innervation patterns. Innervated endplates were examined to determine the number of endplates per myofiber and the number of axons from different neurons per endplate. Endplates were identified by collections of myonuclei occupying non-striated cytoplasm in the periphery of myofibers. To obtain a representative sample, 200 endplates per muscle were analyzed consecutively. Nerve fibers that arborized within the confines of the endplate as described by Gutmann and Young 7 were considered terminals of alpha motor axons. Examining the whole length of myofibers in the 2-, 4- and 6-week-old kittens revealed that one endplate per myofiber was the common pattern, in each group, not more than 1 °J~iof the myofibers possessed two endplates. There was less certainty
159 a b o u t the n u m b e r o f endplates per myofiber in newborn muscles because these myofibers were smaller and difficult to resolve as separate entities. However, the majority of myofibers appeared to possess a single endplate. The major problem in recognizing polyneuronal innervation o f endplates is to distinguish endplates innervated by multiple terminals o f different neurons from those innervated by multiple terminals o f a single neuron. Absolute p r o o f o f separate origin requires the impossible task o f tracing each fiber back to the soma of its parent neuron. In adult muscles, when an axon bifurcates and sends both branches to the same endplate, the majority of these axons branch between the endplates and the larger intramuscular nerve bundlesL Therefore, in the present study nerve fibers that remained distinct when traced proximally from an endplate to an intramuscular nerve bundle were considered to be f r o m different neurons. A n endplate supplied by multiple fibers that were branches o f a single axon was recognized as unineuronally innervated, Endplates of the 2-, 4- and 6-week-old animals were innervated by 1, 2, 3 or ~> 4 fibers (Table I). In each case, these fibers were readily followed from the endplates into the nerve bundles as separate entities. Branching was difficult to resolve in the newborn muscles because the distance between the endplates and the intramuscular nerve bundles was short (Fig. 1). Therefore, their endplates were classified as either singly innervated (1 terminal) or multiply innervated (2 or more terminals) realizing that in this group some of the multiple terminals represented preterminal branching o f single axons (Table I). Approximately 72 ~o of the endplates encountered in newborn muscles were innervated by 2 or more terminals. The percentage of endplates with multiple terminals progressively declined with age (Table I and Fig. 1). While endplates with 3 and >/ 4 terminals were present in the muscles of 2-week-old kittens, 2 terminals per endplate predominated (Table I). TABLE I P E R C E N T A G E S OF E N D P L A T E S I N N E R V A T E D BY T H E S P E C I F I E D N U M B E R O F N E R V E T E R M I N A L S
Age
Newborn* 2 weeks** 4 weeks 6 weeks
Kitten
1 2 I 2 I 2 1 2
Number of'nerve terminals per endplate 1
2
27.5 28.0 32.5 69.0 97.0 97.6 99.5 98.5
72.5 72.0 51.5 30.2 3.0 2.4 0.5 1.5
3
>~4
14.5 0.8
1.5
* For the newborn muscles alone, column 2 represents the percentage of endplates with >~ 1 fiber. Nerve branching was usually not resolvable in the group, but some clearly had 3 and 4 fibers per endplate. ** This pair of kittens were unequal in size, unlike the other pairs. The muscles of kitten 2 were 1.5 times longer than those of kitten 1. Kitten 2 was large for his chronological age which probably accounts for the smaller percentage of endplates with 2 or more terminals.
160
Fig. 1. Silver stained m o t o r nerve terminals o f developing kitten soleus muscles. A: newborn; miram u s c u l a r nerve bundle (IN[I). B: 2 weeks; the a r r o w h e a d points to an endplate in x~hich terminals of 3 a x o n s arborize. C: 4 weeks; 2 axons arborize at the endplate indicated by the arrowhead. These two a x o n s remained separate fibers when traced at high magnification into the larger nerve bundle. D: 6 weeks; endplate myonuclei (N), All magnifications are ,: 800.
161 The present study demonstrates that developing myofibers in kitten soleus muscles exhibit a single endplate rather than multiple endplates per myofiber. During the period of functional polyneuronal innervation 1, these endplates are innervated by more than one nerve fiber, Since these fibers fit the criteria for terminals of alpha motor axons 7 and were judged to come from different neurons, multiple terminals per endplate appears to be the anatomical substrate for polyneuronal innervation. Bagust, Lewis and Westerman I predicted from their physiological studies on kitten soleus muscles that 47 ~o of the myofibers were polyneuronally innervated at 2 weeks of age and very few (1.3°/,,) were so innervated at 6 weeks. The present histological findings correlate directly with these predicted percentages and support the assumption which was used in their calculations that the majority of polyneuronally innervated myofibers at 2 weeks are innervated by two axons rather than more than two. They further suggested that developing myofibers exhibited more than one endplate which could explain the polyneuronal innervation 9. This was not corroborated by the present findings because multiple endplates were uncommon at all of the stages examined. Thus, polyneuronal innervation appears explicable in large part by multiple nerve terminals innervating a single endplate per myofiber. Even if these terminals were unable to generate endplate potentials, multiple innervation allows motor neurons to interact on their end-organ. That is, the process of attaining unineuronal innervation is likely to involve competition between neurons resulting in the suppression and withdrawal of all but the appropriate nerve fiber a. This interaction may be ,~ssential in the establishment of motor units. I wish to thank Mrs. Edith Salazar for her excellent technical assistance. This research was supported by the Dubois, P.R.O.P.S. Endowment, and Academic Senate Research State funds. I BAGUST, J., LEWtS, D. M., AND WESTERMAN, Polyneuronal innervation of kitten skeletal muscle, J. Physiol. (Lend.), 229 (1973) 241-255. 2 BARKER, D., AND IP, M. C., A silver method for demonstrating the innervation of mammalian muscle in teased preparations, J. Physiol. (Lond.), 169 (1963) 73-74P. 3 BENNETT, M. R., AND PETT1GREW, A. G., The formation of synapses in striated muscle during development, J. Physiol. (Lond.), 241 (1974) 515 545. 4 BROWN, M. C., AND MATTHEWS, P. B. C., An investigation into the possible existence of polyneuronal innervation of individual skeletal muscle fibers in certain hind-limb muscles of the cat, J. Physiol. (Lond.), 151 (1960) 436-457. 5 FANGBONER, R. F., AND VANABLE, J. W., Jr., Formation and regression of inappropriate nerve sprouts during trochlear nerve regeneration in Xenopus laevis, J. comp. Neurol., 157 (1974) 391-406. 6 GUTH, L., Neuromuscular function after regeneration of interrupted nerve fibers into partially denervated muscle, Exp. Neurol., 6 (1962) 129 141. 7 GUTMANN, E., AND YOUNG, J. Z., The re-innervation of muscle after various periods of atrophy, J. Anat. (Lond.), 78 (1944) 15 43. 8 REDFERN, P. A., Neuromuscular transmission in newborn rats, J. Physiol. (Lond.), 209 (1970) 701-709. 9 WESTERMAN,R. A., LEWIS, D. M., BAGUST, J., EDJTEHADI, G. D., AND PALLOT, D., Communication between nerves and muscles: Postnatal development in kitten hind-limb fast and slow twitch muscle. In H. P. ZIPPEL (Ed.), Symposium olt Memory and TransJer ofln]brmation, Plenum Press, New York, 1972, pp. 255-291.