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Effect of age and muscle type on regeneration of neuromuscular synapses in mice
Brain Research, 372 (1986) 163-166 Elsevier
163
BRE 21522
Effect of age and muscle type on regeneration of neuromuscular synapses in mice WILLIAM G. HOPKINS, JANET LIANG and ELIZABETH J. BARRETT Department of Physiology, School of Medicine, Universityof Auckland, Auckland, (New Zealand) (Accepted January 7th, 1986) Key words: neuromuscular synapse - - aging- - muscle- - nerve regeneration - - sprouting - - multiple innervation
A silver stain was used to investigate the regeneration of nerve terminals in mouse diaphragm, superior gluteus and tensor fasciae latae (TFL) muscles following nerve crush injury at different ages (9-530 days). The development of myelinated terminal branches and the development and elimination of terminal sprouts were little affected by age or muscle type followingreinnervation. However, multiple axonal innervation developed on up to 50% of the gluteus and TFL muscle fibres, and this was subsequently eliminated only in the youngest animals.
When motor axons regenerate into muscle there is a period during which synapse morphology is abnormal. Gutmann and Young 3 reported the presence of 'escaped' nerve terminals or terminal sprouts in reinnervated rat muscles, and two or more axons contacting individual muscle fibres at the site of the original synapse have also been observed 3,5. It was shown subsequently with intracellular recording that focal multiple innervation was present transiently in reinnervated muscle fibres 6. However, there has been no systematic investigation of the effects of animal age and muscle type on the remodelling of regenerating neuromuscular synapses. We report here on the changes in regenerating synapses following nerve crush injury in mice of four different ages: neonatal (age 9-12 days), juvenile (25-35 days), mature (100-150 days) and elderly (400-530 days). We have analyzed three muscles which differ markedly in their morphology and in the morphology of their synapses4: the diaphragm, an oxidative muscle with small, structurally simple nerve terminals; the tensor fasciae latae (TFL), a glycolytic muscle with large nerve terminals and frequent myelinated nerve terminal branches; and the superior gluteus, a muscle with mixed fibres and mixed nerve terminals. Mice were anaesthetized with halothane and the
muscle nerves were crushed with fine forceps at the point of insertion into the muscle. The animals were killed by cervical dislocation 7-85 days later, the muscles excised, stained with silver to reveal intramuscular axons and examined as whole mounts in the light microscope; 100 terminals in each muscle were scored either as simple or as possessing a myelinated terminal branch, a terminal sprout, or two or more distinct terminal axons entering the synapse from an intramuscular nerve branch (Fig. 1) as previously described 4. Fig. 2 shows that age had no significant effect on t h e establishment of myelinated nerve terminal branches in the three muscles: by 60 days post-operation the frequency of a myelinated terminal branch had risen to 5 - 1 5 % in the diaphragm, 30-55% in the gluteus, and 35-80% in the TFL, which are only slightly higher than the values in normal adult muscles4. The period of approximately 60 days for the development of the myelinated branches is also very similar to the period during which myelinated branches develop in the normal animal (between 30 and 100 days of age4). Terminal sprouts developed at the nerve terminals as soon as axons regenerated into the muscles, and as Fig. 3 shows, reached a significantly (P < 0.05) higher level initially in the diaphragm than in the gluteus
Correspondence: W.G. Hopkins, Department of Physiology, School of Medicine, University of Auckland, Auckland, New Zealand. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
164 SIMPLE
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:
MYELINATED
MULTIPLE
BRANCH
AXONS
:
:
ir T T TERMINAL
SPROUT
:
peared to rise to slightly lower maxima approximately 5 days later than in the neonates. In the diaphragm the maximum was less than 20% and this fell subsequently to less than 10%; however, in the gluteus and TFL there was no subsequent elimination of multiple axonal innervation. The frequencies of multiple axonal innervation in the mature and elderly muscles showed similar time courses to those of the juvenile muscles except that the initial rise occurred up to 10 days later than in the neonates, and the frequencies in gluteus and TFL remained elevated at a significantly higher level that in juveniles. (Mean frequency + S.E.M. for all gluteus and TFL muscles more than 20 days post-operation: elderly and mature, 29.5 + 1.2%; juvenile, 16.8 + 2.3%; neonate 3.4 + 1.0%. These are all significantly different, P <
0.01). The delay in the appearance of sprouts and multiple axonal innervation in the older muscles can be explained at least partly by the fact that the older animals are larger and therefore a greater time is re-
+o]
DIAPHRAGM
40
20
== Fig. 1. Representation of silver-stained appearance of motor nerve terminals with either a simple morphology or with a myelinated branch, multiple axons, or a terminal sprout. Thickened axon represents myelin.
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or TFL (mean frequency + S.E.M. for all age groups 0-30 days post-operation: diaphragm, 39.2 + 7.2%; gluteus + TFL, 22.8 + 3.2%). The sprouts were eliminated subsequently with frequencies dropping to less than 10% by 60 days post-operation in all muscles. The development and elimination of sprouts appeared to be slightly delayed (by 1-2 weeks) in the elderly animals. Age and muscle type had marked effects on the development and elimination of multiple axonal innervation at synapses (Fig. 4). In all neonatal muscles the frequency of multiple axonal innervation rose to a maximum of 30-40% as soon as axons returned to the muscles (7 days post-operation), then fell to less 'than 5% by 35 days post-operation. In juvenile mice the frequency of multiple axonal innervation ap-
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Fig. 2. Frequency of nerve terminals with a myetinated branch in diaphragm (upper), gluteus (middle) and TFL (lower) muscles reinnervated for various times following denervation at neonatal (O'"O), juvenile (C)..... C)), mature (A--A) or elderly (+ +) ages.
165
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before it reaches the muscle fibre 4. Following denervation these additional branches would remain as additional denervated endoneurial pathways for guiding regenerating axons 2. Two axons regenerating to the muscle could occupy these pathways separately and might therefore resist the mechanism, whatever it might be, that normally leads to the elimination of multiple innervation. In summary these results show that the capacity for neuromuscular synapses to develop myelinated branches and to eliminate terminal sprouts following nerve regeneration is not affected by age, but that the re-establishment of normal single axonal innervation is reduced in gluteus and TFL muscles as animals get older. Different endoneurial pathways in the intramuscular nerves may account partly for the age and muscle-specific differences.
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This work was supported by the N Z Medical Research Council.
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Fig. 3. Frequency of nerve terminal sprouts in diaphragm (upper), gluteus (middle) and TFL (lower) muscles reinnervated for various times following denervation at neonatal (O.O), juvenile (O..... O), mature (A---A) or elderly (+ +) ages. O x
quired for the axons to regenerate the extra distance. The average rate of regeneration may also be reduced in old animals 7. The different patterns of terminal sprouting and multiple axonal innervation in the oxidative and glycolytic muscles have some similarity to the pattern of terminal sprouting and nodal sprouting in slow and fast muscles following partial denervation or paralysis with botulinum toxin 1. Terminal sprouts are more frequent in the slow soleus muscle, as they are here in the oxidative diaphragm, whereas nodal sprouts are more frequent in the fast peroneus tertius as is multiple axonal innervation in the gluteus and TFL. The reason for these differences between muscles is not known. However, there may be a straightforward explanation for the lack of elimination of multiple axonal innervation in the older TFL and gluteus muscles. In these muscles, prior to denervation about half of the nerve terminals have myelinated terminal branches, many of which arise on the terminal axon
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Fig. 4. Frequency of multiple axonal innervation in diaphragm (upper), gluteus (middle) and TFL (lower) muscles reinnervated for various times following denervation at neonatal (O"'"Q), juvenile (O..... O), mature (A---A) or elderly (+ +) ages.
166 1 Brown, M.C., Holland, R.L. and Ironton, R., Nodal and terminal sprouting from motor nerves in fast and slow muscles of the mouse, J. Physiol. (London), 306 (1980) 493-510. 2 Brown, M.C. and Hopkins, W.G., Role of degenerating axon pathways in regeneration of mouse soleus motor axons, J. Physiol. (London), 318 (1981) 365-373. 3 Gutmann, E. and Young, J.Z., The reinnervation of muscle after various periods of atrophy, J. Anat., 78 (1944) 15-43. 4 Hopkins, W.G., Brown, M.C. and Keynes, R.J., Post-natal growth of motor nerve terminals in muscles of the mouse, J. Neurocytol., 14 (1985) 525-540.
5 Kiernan, J.A. and Mclsaac, E., Complete staining of neuromuscular innervation with bromo-indigo and silver, Stain Technol., 49 (1974) 211-214. 6 McArdle, J.J., Complex end-plate potentials at the regenerating neuromuscular junction of the rat, Exp. Neurol., 49 (1975) 629-638. 7 Pestronk, A., Drachman, D.B. and Griffin, J.W., Effects of aging on nerve sprouting and regeneration, Exp. Neurol., 70 (1980) 65-82.
163
BRE 21522
Effect of age and muscle type on regeneration of neuromuscular synapses in mice WILLIAM G. HOPKINS, JANET LIANG and ELIZABETH J. BARRETT Department of Physiology, School of Medicine, Universityof Auckland, Auckland, (New Zealand) (Accepted January 7th, 1986) Key words: neuromuscular synapse - - aging- - muscle- - nerve regeneration - - sprouting - - multiple innervation
A silver stain was used to investigate the regeneration of nerve terminals in mouse diaphragm, superior gluteus and tensor fasciae latae (TFL) muscles following nerve crush injury at different ages (9-530 days). The development of myelinated terminal branches and the development and elimination of terminal sprouts were little affected by age or muscle type followingreinnervation. However, multiple axonal innervation developed on up to 50% of the gluteus and TFL muscle fibres, and this was subsequently eliminated only in the youngest animals.
When motor axons regenerate into muscle there is a period during which synapse morphology is abnormal. Gutmann and Young 3 reported the presence of 'escaped' nerve terminals or terminal sprouts in reinnervated rat muscles, and two or more axons contacting individual muscle fibres at the site of the original synapse have also been observed 3,5. It was shown subsequently with intracellular recording that focal multiple innervation was present transiently in reinnervated muscle fibres 6. However, there has been no systematic investigation of the effects of animal age and muscle type on the remodelling of regenerating neuromuscular synapses. We report here on the changes in regenerating synapses following nerve crush injury in mice of four different ages: neonatal (age 9-12 days), juvenile (25-35 days), mature (100-150 days) and elderly (400-530 days). We have analyzed three muscles which differ markedly in their morphology and in the morphology of their synapses4: the diaphragm, an oxidative muscle with small, structurally simple nerve terminals; the tensor fasciae latae (TFL), a glycolytic muscle with large nerve terminals and frequent myelinated nerve terminal branches; and the superior gluteus, a muscle with mixed fibres and mixed nerve terminals. Mice were anaesthetized with halothane and the
muscle nerves were crushed with fine forceps at the point of insertion into the muscle. The animals were killed by cervical dislocation 7-85 days later, the muscles excised, stained with silver to reveal intramuscular axons and examined as whole mounts in the light microscope; 100 terminals in each muscle were scored either as simple or as possessing a myelinated terminal branch, a terminal sprout, or two or more distinct terminal axons entering the synapse from an intramuscular nerve branch (Fig. 1) as previously described 4. Fig. 2 shows that age had no significant effect on t h e establishment of myelinated nerve terminal branches in the three muscles: by 60 days post-operation the frequency of a myelinated terminal branch had risen to 5 - 1 5 % in the diaphragm, 30-55% in the gluteus, and 35-80% in the TFL, which are only slightly higher than the values in normal adult muscles4. The period of approximately 60 days for the development of the myelinated branches is also very similar to the period during which myelinated branches develop in the normal animal (between 30 and 100 days of age4). Terminal sprouts developed at the nerve terminals as soon as axons regenerated into the muscles, and as Fig. 3 shows, reached a significantly (P < 0.05) higher level initially in the diaphragm than in the gluteus
Correspondence: W.G. Hopkins, Department of Physiology, School of Medicine, University of Auckland, Auckland, New Zealand. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
164 SIMPLE
/
:
MYELINATED
MULTIPLE
BRANCH
AXONS
:
:
ir T T TERMINAL
SPROUT
:
peared to rise to slightly lower maxima approximately 5 days later than in the neonates. In the diaphragm the maximum was less than 20% and this fell subsequently to less than 10%; however, in the gluteus and TFL there was no subsequent elimination of multiple axonal innervation. The frequencies of multiple axonal innervation in the mature and elderly muscles showed similar time courses to those of the juvenile muscles except that the initial rise occurred up to 10 days later than in the neonates, and the frequencies in gluteus and TFL remained elevated at a significantly higher level that in juveniles. (Mean frequency + S.E.M. for all gluteus and TFL muscles more than 20 days post-operation: elderly and mature, 29.5 + 1.2%; juvenile, 16.8 + 2.3%; neonate 3.4 + 1.0%. These are all significantly different, P <
0.01). The delay in the appearance of sprouts and multiple axonal innervation in the older muscles can be explained at least partly by the fact that the older animals are larger and therefore a greater time is re-
+o]
DIAPHRAGM
40
20
== Fig. 1. Representation of silver-stained appearance of motor nerve terminals with either a simple morphology or with a myelinated branch, multiple axons, or a terminal sprout. Thickened axon represents myelin.
GI LU Z
0
+otQLOT 0s 4O
~,~
20
or TFL (mean frequency + S.E.M. for all age groups 0-30 days post-operation: diaphragm, 39.2 + 7.2%; gluteus + TFL, 22.8 + 3.2%). The sprouts were eliminated subsequently with frequencies dropping to less than 10% by 60 days post-operation in all muscles. The development and elimination of sprouts appeared to be slightly delayed (by 1-2 weeks) in the elderly animals. Age and muscle type had marked effects on the development and elimination of multiple axonal innervation at synapses (Fig. 4). In all neonatal muscles the frequency of multiple axonal innervation rose to a maximum of 30-40% as soon as axons returned to the muscles (7 days post-operation), then fell to less 'than 5% by 35 days post-operation. In juvenile mice the frequency of multiple axonal innervation ap-
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Fig. 2. Frequency of nerve terminals with a myetinated branch in diaphragm (upper), gluteus (middle) and TFL (lower) muscles reinnervated for various times following denervation at neonatal (O'"O), juvenile (C)..... C)), mature (A--A) or elderly (+ +) ages.
165
: 1 ~~~/
before it reaches the muscle fibre 4. Following denervation these additional branches would remain as additional denervated endoneurial pathways for guiding regenerating axons 2. Two axons regenerating to the muscle could occupy these pathways separately and might therefore resist the mechanism, whatever it might be, that normally leads to the elimination of multiple innervation. In summary these results show that the capacity for neuromuscular synapses to develop myelinated branches and to eliminate terminal sprouts following nerve regeneration is not affected by age, but that the re-establishment of normal single axonal innervation is reduced in gluteus and TFL muscles as animals get older. Different endoneurial pathways in the intramuscular nerves may account partly for the age and muscle-specific differences.
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This work was supported by the N Z Medical Research Council.
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10
20 30 40 50 60 DAYS POST-DENERVATION
70
80
90
o° t0oAHA•.
Fig. 3. Frequency of nerve terminal sprouts in diaphragm (upper), gluteus (middle) and TFL (lower) muscles reinnervated for various times following denervation at neonatal (O.O), juvenile (O..... O), mature (A---A) or elderly (+ +) ages. O x
quired for the axons to regenerate the extra distance. The average rate of regeneration may also be reduced in old animals 7. The different patterns of terminal sprouting and multiple axonal innervation in the oxidative and glycolytic muscles have some similarity to the pattern of terminal sprouting and nodal sprouting in slow and fast muscles following partial denervation or paralysis with botulinum toxin 1. Terminal sprouts are more frequent in the slow soleus muscle, as they are here in the oxidative diaphragm, whereas nodal sprouts are more frequent in the fast peroneus tertius as is multiple axonal innervation in the gluteus and TFL. The reason for these differences between muscles is not known. However, there may be a straightforward explanation for the lack of elimination of multiple axonal innervation in the older TFL and gluteus muscles. In these muscles, prior to denervation about half of the nerve terminals have myelinated terminal branches, many of which arise on the terminal axon
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Fig. 4. Frequency of multiple axonal innervation in diaphragm (upper), gluteus (middle) and TFL (lower) muscles reinnervated for various times following denervation at neonatal (O"'"Q), juvenile (O..... O), mature (A---A) or elderly (+ +) ages.
166 1 Brown, M.C., Holland, R.L. and Ironton, R., Nodal and terminal sprouting from motor nerves in fast and slow muscles of the mouse, J. Physiol. (London), 306 (1980) 493-510. 2 Brown, M.C. and Hopkins, W.G., Role of degenerating axon pathways in regeneration of mouse soleus motor axons, J. Physiol. (London), 318 (1981) 365-373. 3 Gutmann, E. and Young, J.Z., The reinnervation of muscle after various periods of atrophy, J. Anat., 78 (1944) 15-43. 4 Hopkins, W.G., Brown, M.C. and Keynes, R.J., Post-natal growth of motor nerve terminals in muscles of the mouse, J. Neurocytol., 14 (1985) 525-540.
5 Kiernan, J.A. and Mclsaac, E., Complete staining of neuromuscular innervation with bromo-indigo and silver, Stain Technol., 49 (1974) 211-214. 6 McArdle, J.J., Complex end-plate potentials at the regenerating neuromuscular junction of the rat, Exp. Neurol., 49 (1975) 629-638. 7 Pestronk, A., Drachman, D.B. and Griffin, J.W., Effects of aging on nerve sprouting and regeneration, Exp. Neurol., 70 (1980) 65-82.