- Email: [email protected]
Elimination of polyneuronal innervation in proximal and distal leg muscles of chick embryos
DevelopmentalBrain Research, 1 (1981) 299-302
299
© Elsevier/North-Holland Biomedical Press
Elimination of polyneuronal innervation in proximal and distal leg muscles of chick embryos
SUSAN POCKETT*
Departments of Physiology and Neurophysiology, University of Oslo, Oslo 1 (Norway) (Accepted October 15th, 1980)
Key words: polyneuronal synapse elimination - - chick embryo
In chick embryo leg muscles, elimination of polyneuronal innervation takes place one to 1.5 days earlier in the thigh muscle m. ambiens than in the foot muscle m. flexor hallucis brevis. Initial formation of synapses takes place 2-3 days earlier in the proximal than the distal muscle, so the length of time synapses have been active may be a factor in the control of elimination of polyneuronal innervation during development.
In mammals, skeletal muscle fibres are polyneuronally innervated at birth. Over the next two weeks, synapse elimination occurs until each muscle fibre is innervated by only one axon3,4,9. Polyaxonal innervation also occurs in embryo chick muscles 1. The mechanism whereby synapses are eliminated is not well understood. In the rabbit, elimination of polyneuronal innervation occurs at different times in different muscles, showing that a general hormonal signal is not responsible for initiating the process of elimination3. Neuromuscular activity is involved in determining when synapses will be eliminated during development2,s, 10. In the present study, the possibility that an important factor is the length of time a synapse has been active was investigated, by examining the evolution of multi-innervation in two different embryonic chick muscles which are first innervated on different days of development. According to Landmesser and Morris 7, the undifferentiated muscle masses in the thigh of chick embryos can first be activated by nerve stimulation at Stage 27-29 of the Hamburger and Hamilton6 scheme (5-6 days of incubation). Calf muscles are first innervated at about the same time, but muscles of the foot are not yet present and can not be activated by nerve stimulation until Stage 34 (8 days of incubation). Thus, if the time synapses have been active is an important factor in causing elimination of polyneuronal innervation, thigh muscles may lose their multiple innervation 2-3 days before foot muscles. The present investigation studied the disappearance of polyneuronal innervation * Present address • Department of Physiology, University of Auckland, Private Bag, Auckland, New Zealand.
300 in the ambiens muscle of the thigh and the flexor hallucis brevis of the foot of embryo chicks. The muscles, together with their nerves, were excised from 14- to 21-day-old chick embryos and pinned out in a recording chamber perfused with well-oxygenated Ringers of composition (in raM): NaC1, 137; KC1, 5; CaC12, 5; MgCIz, 1; NaH2PO4, 1; Tris.C1, 10; glucose, 10; buffered to pH 7.4; or alternatively Na ÷, 135; K ÷, 5; CI-, 124; Ca 2+, 5; Mg z÷, 1 ; HCO-3, 24; glucose, 11 ; glutamic acid, 0.3; glutamine, 0.4; choline, 5.0 mg/ml; cocarboxylase, 20 btg/1; oxygenated with 95 % O~/5 ~ CO2. The nerve was stimulated with a well-fitting suction electrode. Nerve-evoked contractions were blocked with 5 mM Mg 2÷and 1 × 10-6 g/ml D-tubocurarine chloride. Intracellular recordings were made with conventional glass microelectrodes. End-plate potentials (epps) were evoked by stimuli of graded strength applied to the nerve. Large fluctuations in the amplitude of individual epp components (see Fig. 1) made it impossible to distinguish reliably between, e.g. triple and quadruple innervation of a given fibre, so fibres were scored simply as polyneuronally innervated or singly innervated, and not according to how many axons innervated them. A fibre was taken to be polyneuronally innervated if superposition of 10-20 responses at threshold stimulation strength (50% epp failure) and 10-20 responses at 2-3 times threshold stimulation strength showed a clear increase of epp amplitude at suprathreshold strength.
A
I
2my
10 msec
iml Fig. 1. End-plate potentials from a polyneuronally innervated cell in a 16-day-old embryo ambiens muscle. A: superimposed sweeps at threshold nerve stimulation strength. B: superimposed sweeps at twice threshold stimulation strength. Records A and B are from the same cell.
301 ¢m
L~
(31J 100 O
E ~:~ 80
(4:31 (14:6) O O (19:5) O (19:5)
6o
(14:3) (8:5) O
o~°~ 40 LU Z
>-
g
20
(27: 6)
fL
0 14
• (1503)12~2) (18:4) i
I
i
i
I
i
15
16
17
18
19
AGE
OF
20
21
CHICK EMBRYO (DAYS)
Fig. 2. Percentage of polyneuronally innervated muscle fibres with age in O, m. flexor hallucis brevis (distal), and I , m. ambiens (proximal) muscles of chick embryos. Each point represents the average of a number of muscle fibres pooled from several muscles. The first of each pair of bracketed figures is the number of muscle fibres assessed and the second is the number of muscles.
In some experiments, muscles were stained for acetylcholinesteraseS; after formalin fixation small bundles of muscle fibres were teased out and examined by light microscopy. Both the muscles chosen for study had focal, as opposed to distributed innervation (which is more common in chick limbs). In both m. ambiens and m. flexor hallucis brevis, each muscle fibre had only a single AChE site and all epps recorded in a given fibre had a single rise time. Nerve conduction, particularly in the younger embryos, was quite labile and intermittent conduction (or release) failure at higher stimulus intensities often made it difficult to classify fibres as polyneuronally or singly innervated. This occurred in about 50 % of fibres and in such cases the fibre was not scored. Another difficulty was that the threshold voltages for stimulation of a given axon sometimes fluctuated. Particularly in earlier experiments, difficulty was also experienced in achieving stable penetrations of the muscle fibres with microelectrodes. Good penetrations showed resting membrane potentials of 80-90 mV. Although a systematic study of this was not undertaken, the resting membrane potential was not markedly affected by D-tubocurarine, as has been reported for rat embryo muscles 11. Because of the difficulties mentioned, it was not possible routinely to record the innervation state of 10-20 muscle fibres per muscle, as would have been desirable. Therefore, results from different muscles were pooled to give the time-courses of elimination shown in Fig. 2. In the toe muscle, m. flexor hallucis brevis, all the muscle fibres scored were polyneuronally innervated at 16 days of incubation and all were singly innervated by day 20. Elimination of polyneuronal innervation took place earlier in the thigh muscle, m. ambiens. For example, in 16-day-old embryos, 14 of 14 cells scored were polyneuronally innervated in m. flexor hallucis brevis while 4 of 19 cells were already singly innervated in m. ambiens. At 18 days of incubation 19 of 29 cells were polyneuronally innervated in m. flexor hallucis brevis and only 5 of 27 cells scored were polyneuronally innervated in m.
302 ambiens. In general, elimination a p p e a r e d to take place 1-1.5 days earlier in the p r o x i mal m. a m b i e n s t h a n the distal m. flexor hallucis brevis. The p o o l e d results were confirmed in experiments where m. flexor hallucis brevis a n d m. a m b i e n s muscles from the same leg o f the same e m b r y o were studied in the same r e c o r d i n g chamber. In one 18-day-old chick e m b r y o , 4 o f 5 muscle fibres assessed in the m. flexor hallucis brevis were p o l y n e u r o n a l l y innervated, while only one o f 5 cells s a m p l e d in the m. ambiens o f the same leg was p o l y n e u r o n a l l y innervated. In a second 18-day-old e m b r y o 3 o f 5 cells in m. flexor hallucis brevis were p o l y n e u r o n a l l y innervated a n d none o f 5 cells in m. ambiens was p o l y n e u r o n a l l y innervated. In one 16-day e m b r y o , 5 o f 5 cells o f m. flexor hallucis brevis were p o l y n e u r o n a l l y innervated and 4 o f 5 cells in m. a m b i e n s were p o l y n e u r o n a l l y innervated. These results suggest that at least the final phase o f elimination o f p o l y n e u r o n a l i n n e r v a t i o n takes place 1-1.5 days earlier in the p r o x i m a l leg muscle a m b i e n s t h a n in the distal leg muscle flexor hallucis brevis. Evidence is available 7 to indicate t h a t the p r o x i m a l muscle in this case is first innervated 2-3 days earlier t h a n the distal. Thus, the d a t a presented here s u p p o r t the idea that the length of time synapses have been active m a y be a f a c t o r in the c o n t r o l o f elimination o f p o l y n e u r o n a l innervation in development. T h a n k s are due to Drs. J. K. S. Jansen and T. L o m o for facilities and advice. The a u t h o r was s u p p o r t e d by an Overseas Research F e l l o w s h i p f r o m the M R C o f N e w Zealand.
1 Bennett, M. R. and Pettigrew, A. G., The formation of synapses in striated muscle during development, J. Physiol. (Lond.), 241 (1974) 515-545. 2 Benoit, P. and Changeux, J.-P., Consequences of tenotomy on the evolution of multiinnervation in developing rat soleus muscle. Brain Research, 99 (1975) 354-358. 3 Bixby, J. L. and Van Essen, D. C., Regional differences in the timing of synapse elimination in skeletal muscles of the neonatal rabbit, Brain Research, 169 (1979) 275-286. 4 Brown, M. C., Jansen, J. K. S. and Van Essen, D., Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation, J. Physiol. (Lond.), 261 (1976) 387-422. 5 Buckley, G. A. and Heaton, J. A., A quantitative study of cholinesterase in myoneural junctions from rat and guinea-pig extraocular muscles, J. Physiol. (Lond.), 199 (1968) 743-749. 6 Hamburger, V. and Hamilton, H. L., A series of normal stages in the development of the chick embryo, J. Morph., 88 (1951) 49-92. 7 Landmesser, L. and Morris, D. G., The development of functional innervation in the hind limb of the chick embryo, J. Physiol. (Lond.), 249 (1975) 301-326. 80'Brien, R. A. D., Ostberg, A. J. C. and Vrbova, G., Observations on the elimination of polyneuronal innervation in developing mammalian skeletal muscle, J. Physiol. (Lond.), 282 (1978) 571-582. 9 Redfern, P. A., Neuromuscular transmission in new-born rats, J. Physiol. (Lond.), 209 (1970) 701-709. 10 Thompson, W., Kuffler, D. P. and Jansen, J. K. S., The effects of prolonged reversible block of nerve impulses on the elimination of polyneuronal innervation of new-born rat skeletal muscle fibres, Neuroscience, 4 (1979) 271-281. 11 Ziskind, L. and Dennis, M. J., Depolarising effect of curare on embryonic rat muscles, Nature (Lond.), 276 (1978) 622-623.
299
© Elsevier/North-Holland Biomedical Press
Elimination of polyneuronal innervation in proximal and distal leg muscles of chick embryos
SUSAN POCKETT*
Departments of Physiology and Neurophysiology, University of Oslo, Oslo 1 (Norway) (Accepted October 15th, 1980)
Key words: polyneuronal synapse elimination - - chick embryo
In chick embryo leg muscles, elimination of polyneuronal innervation takes place one to 1.5 days earlier in the thigh muscle m. ambiens than in the foot muscle m. flexor hallucis brevis. Initial formation of synapses takes place 2-3 days earlier in the proximal than the distal muscle, so the length of time synapses have been active may be a factor in the control of elimination of polyneuronal innervation during development.
In mammals, skeletal muscle fibres are polyneuronally innervated at birth. Over the next two weeks, synapse elimination occurs until each muscle fibre is innervated by only one axon3,4,9. Polyaxonal innervation also occurs in embryo chick muscles 1. The mechanism whereby synapses are eliminated is not well understood. In the rabbit, elimination of polyneuronal innervation occurs at different times in different muscles, showing that a general hormonal signal is not responsible for initiating the process of elimination3. Neuromuscular activity is involved in determining when synapses will be eliminated during development2,s, 10. In the present study, the possibility that an important factor is the length of time a synapse has been active was investigated, by examining the evolution of multi-innervation in two different embryonic chick muscles which are first innervated on different days of development. According to Landmesser and Morris 7, the undifferentiated muscle masses in the thigh of chick embryos can first be activated by nerve stimulation at Stage 27-29 of the Hamburger and Hamilton6 scheme (5-6 days of incubation). Calf muscles are first innervated at about the same time, but muscles of the foot are not yet present and can not be activated by nerve stimulation until Stage 34 (8 days of incubation). Thus, if the time synapses have been active is an important factor in causing elimination of polyneuronal innervation, thigh muscles may lose their multiple innervation 2-3 days before foot muscles. The present investigation studied the disappearance of polyneuronal innervation * Present address • Department of Physiology, University of Auckland, Private Bag, Auckland, New Zealand.
300 in the ambiens muscle of the thigh and the flexor hallucis brevis of the foot of embryo chicks. The muscles, together with their nerves, were excised from 14- to 21-day-old chick embryos and pinned out in a recording chamber perfused with well-oxygenated Ringers of composition (in raM): NaC1, 137; KC1, 5; CaC12, 5; MgCIz, 1; NaH2PO4, 1; Tris.C1, 10; glucose, 10; buffered to pH 7.4; or alternatively Na ÷, 135; K ÷, 5; CI-, 124; Ca 2+, 5; Mg z÷, 1 ; HCO-3, 24; glucose, 11 ; glutamic acid, 0.3; glutamine, 0.4; choline, 5.0 mg/ml; cocarboxylase, 20 btg/1; oxygenated with 95 % O~/5 ~ CO2. The nerve was stimulated with a well-fitting suction electrode. Nerve-evoked contractions were blocked with 5 mM Mg 2÷and 1 × 10-6 g/ml D-tubocurarine chloride. Intracellular recordings were made with conventional glass microelectrodes. End-plate potentials (epps) were evoked by stimuli of graded strength applied to the nerve. Large fluctuations in the amplitude of individual epp components (see Fig. 1) made it impossible to distinguish reliably between, e.g. triple and quadruple innervation of a given fibre, so fibres were scored simply as polyneuronally innervated or singly innervated, and not according to how many axons innervated them. A fibre was taken to be polyneuronally innervated if superposition of 10-20 responses at threshold stimulation strength (50% epp failure) and 10-20 responses at 2-3 times threshold stimulation strength showed a clear increase of epp amplitude at suprathreshold strength.
A
I
2my
10 msec
iml Fig. 1. End-plate potentials from a polyneuronally innervated cell in a 16-day-old embryo ambiens muscle. A: superimposed sweeps at threshold nerve stimulation strength. B: superimposed sweeps at twice threshold stimulation strength. Records A and B are from the same cell.
301 ¢m
L~
(31J 100 O
E ~:~ 80
(4:31 (14:6) O O (19:5) O (19:5)
6o
(14:3) (8:5) O
o~°~ 40 LU Z
>-
g
20
(27: 6)
fL
0 14
• (1503)12~2) (18:4) i
I
i
i
I
i
15
16
17
18
19
AGE
OF
20
21
CHICK EMBRYO (DAYS)
Fig. 2. Percentage of polyneuronally innervated muscle fibres with age in O, m. flexor hallucis brevis (distal), and I , m. ambiens (proximal) muscles of chick embryos. Each point represents the average of a number of muscle fibres pooled from several muscles. The first of each pair of bracketed figures is the number of muscle fibres assessed and the second is the number of muscles.
In some experiments, muscles were stained for acetylcholinesteraseS; after formalin fixation small bundles of muscle fibres were teased out and examined by light microscopy. Both the muscles chosen for study had focal, as opposed to distributed innervation (which is more common in chick limbs). In both m. ambiens and m. flexor hallucis brevis, each muscle fibre had only a single AChE site and all epps recorded in a given fibre had a single rise time. Nerve conduction, particularly in the younger embryos, was quite labile and intermittent conduction (or release) failure at higher stimulus intensities often made it difficult to classify fibres as polyneuronally or singly innervated. This occurred in about 50 % of fibres and in such cases the fibre was not scored. Another difficulty was that the threshold voltages for stimulation of a given axon sometimes fluctuated. Particularly in earlier experiments, difficulty was also experienced in achieving stable penetrations of the muscle fibres with microelectrodes. Good penetrations showed resting membrane potentials of 80-90 mV. Although a systematic study of this was not undertaken, the resting membrane potential was not markedly affected by D-tubocurarine, as has been reported for rat embryo muscles 11. Because of the difficulties mentioned, it was not possible routinely to record the innervation state of 10-20 muscle fibres per muscle, as would have been desirable. Therefore, results from different muscles were pooled to give the time-courses of elimination shown in Fig. 2. In the toe muscle, m. flexor hallucis brevis, all the muscle fibres scored were polyneuronally innervated at 16 days of incubation and all were singly innervated by day 20. Elimination of polyneuronal innervation took place earlier in the thigh muscle, m. ambiens. For example, in 16-day-old embryos, 14 of 14 cells scored were polyneuronally innervated in m. flexor hallucis brevis while 4 of 19 cells were already singly innervated in m. ambiens. At 18 days of incubation 19 of 29 cells were polyneuronally innervated in m. flexor hallucis brevis and only 5 of 27 cells scored were polyneuronally innervated in m.
302 ambiens. In general, elimination a p p e a r e d to take place 1-1.5 days earlier in the p r o x i mal m. a m b i e n s t h a n the distal m. flexor hallucis brevis. The p o o l e d results were confirmed in experiments where m. flexor hallucis brevis a n d m. a m b i e n s muscles from the same leg o f the same e m b r y o were studied in the same r e c o r d i n g chamber. In one 18-day-old chick e m b r y o , 4 o f 5 muscle fibres assessed in the m. flexor hallucis brevis were p o l y n e u r o n a l l y innervated, while only one o f 5 cells s a m p l e d in the m. ambiens o f the same leg was p o l y n e u r o n a l l y innervated. In a second 18-day-old e m b r y o 3 o f 5 cells in m. flexor hallucis brevis were p o l y n e u r o n a l l y innervated a n d none o f 5 cells in m. ambiens was p o l y n e u r o n a l l y innervated. In one 16-day e m b r y o , 5 o f 5 cells o f m. flexor hallucis brevis were p o l y n e u r o n a l l y innervated and 4 o f 5 cells in m. a m b i e n s were p o l y n e u r o n a l l y innervated. These results suggest that at least the final phase o f elimination o f p o l y n e u r o n a l i n n e r v a t i o n takes place 1-1.5 days earlier in the p r o x i m a l leg muscle a m b i e n s t h a n in the distal leg muscle flexor hallucis brevis. Evidence is available 7 to indicate t h a t the p r o x i m a l muscle in this case is first innervated 2-3 days earlier t h a n the distal. Thus, the d a t a presented here s u p p o r t the idea that the length of time synapses have been active m a y be a f a c t o r in the c o n t r o l o f elimination o f p o l y n e u r o n a l innervation in development. T h a n k s are due to Drs. J. K. S. Jansen and T. L o m o for facilities and advice. The a u t h o r was s u p p o r t e d by an Overseas Research F e l l o w s h i p f r o m the M R C o f N e w Zealand.
1 Bennett, M. R. and Pettigrew, A. G., The formation of synapses in striated muscle during development, J. Physiol. (Lond.), 241 (1974) 515-545. 2 Benoit, P. and Changeux, J.-P., Consequences of tenotomy on the evolution of multiinnervation in developing rat soleus muscle. Brain Research, 99 (1975) 354-358. 3 Bixby, J. L. and Van Essen, D. C., Regional differences in the timing of synapse elimination in skeletal muscles of the neonatal rabbit, Brain Research, 169 (1979) 275-286. 4 Brown, M. C., Jansen, J. K. S. and Van Essen, D., Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation, J. Physiol. (Lond.), 261 (1976) 387-422. 5 Buckley, G. A. and Heaton, J. A., A quantitative study of cholinesterase in myoneural junctions from rat and guinea-pig extraocular muscles, J. Physiol. (Lond.), 199 (1968) 743-749. 6 Hamburger, V. and Hamilton, H. L., A series of normal stages in the development of the chick embryo, J. Morph., 88 (1951) 49-92. 7 Landmesser, L. and Morris, D. G., The development of functional innervation in the hind limb of the chick embryo, J. Physiol. (Lond.), 249 (1975) 301-326. 80'Brien, R. A. D., Ostberg, A. J. C. and Vrbova, G., Observations on the elimination of polyneuronal innervation in developing mammalian skeletal muscle, J. Physiol. (Lond.), 282 (1978) 571-582. 9 Redfern, P. A., Neuromuscular transmission in new-born rats, J. Physiol. (Lond.), 209 (1970) 701-709. 10 Thompson, W., Kuffler, D. P. and Jansen, J. K. S., The effects of prolonged reversible block of nerve impulses on the elimination of polyneuronal innervation of new-born rat skeletal muscle fibres, Neuroscience, 4 (1979) 271-281. 11 Ziskind, L. and Dennis, M. J., Depolarising effect of curare on embryonic rat muscles, Nature (Lond.), 276 (1978) 622-623.