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Acetylcholine supersensitivity: the role of neurotrophic factors
Brain Research, 58 (1973) 255-259
255
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Acetylcholine supersensitivity: the role of neurotrophic factors A. CANGIANO* MRC Group in Developmental Neurobiology, Department of Neurosciences, MeMaster University, Hamilton, Ontario (Canada)
(Accepted May 1st, 1973)
The trophic influence of neurones has been extensively studied by investigating the effects of denervation on skeletal muscle. Perhaps the most striking result of this procedure is the development of an intense sensitivity to the chemical transmitter acetylcholine (ACh) over the entire surface of the muscle fibres 5,15. It has been recently shown that the muscular inactivity that follows denervation plays an important role in the development of ACh supersensitivity 13. However, this does not exclude the possibility that the distribution of ACh-receptors and other properties of the muscle fibre membrane are also under the control of chemical influences from the motor nerve. The existence of these influences is suggested by the effects of partial denervation 15 and by the importance of the length of the nerve stump in determining the time of onset of denervation phenomenal,9,14. Such a mechanism of neurotrophic control could well involve the transfer of chemical information by axoplasmic flow down to the nerve terminals. Recent evidence indicates that colchicine, known to block axoplasmic transport6,11, mimics nerve section in causing peripheral sprouting of adjacent nerves in salamanders, leaving the endings of the treated nerve functionally unaffected 1. The present study investigates the effects of colchicine treatment of mammalian peripheral nerves on skeletal muscle, using doses which would not interfere with the normal functioning of that muscle. In anaesthetized Wistar rats (150--430 g in body weight), colchicine (B.D.H. Ltd., M.W. 399.4) was applied to one sciatic nerve either by injecting 1-5 #1 of a 0.2 M solution under the epineurium 11 or by slipping around the nerve a silicone rubber sleeve is impregnated with the drug (0.1-0.2 ~ w/w). At various times after colchicine application, rats with no obvious signs of paresis of the treated leg were selected for acute experiments. The extensor digitorum longus (EDL) muscle with the innervating sciatic nerve was mounted in a chamber and perfused 12 at room temperature (22-25 °C). Conventional microelectrode techniques were used to record intracellularly and to pass current in muscle fibres, and ACh-sensitivity was measured from the endplate to the myotendinous junction by microiontophoresis 7. * Present address: Istituto Di Fisiologia Umana, Universit~i Di Pisa, Pisa, Italy.
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Fig. 1. Records from a single EDL muscle fibre 4 days after injection of colchicine (152 #g) in the ipsilateral sciatic nerve (163 g rat). A-F: upper traces show 'acetylcholine potentials' recorded at different sites along the same fibre, from the tendon region (A) to the end-plate region (F). The figure shown in each record indicates the distance in millimetres from the end-plate. Iontophoretic pulses are displayed on the lower traces: pulse duration is 10 msec for all records except for B and C, where it is 20 msec. G: record taken at position A, soon after the 'acetylcholine potential' shown in A was evoked; without moving the recording electrode, the sciatic nerve was stimulated central to the site of colchicine application, and a conducted muscle action potential recorded. The disturbance seen after the spike is due to the twitch of the muscle. H: MEPPS recorded at position F. F o u r days after the injection o f colchicine (see below for doses) 8 0 - 1 0 0 ~ o f the superficial fibres tested (5-12 per muscle) on the treated side had become sensitive to A C h over their entire surface. The extrajunctional ACh-sensitivity ranged f r o m 0.01 to 39 units (1 unit = 1 mV peak depolarization per 10 -9 C o f charge passed through the A C h pipette15), whereas the junctional sensitivity ranged from 2 to 48 units (other authors report 2-24 units 8, or 20-230 units 8,13, for the junctional sensitivity o f rat soleus and E D L muscles). Furthermore, in the great majority o f muscle fibres, direct stimulation elicited action potentials in the presence o f 1 × 10 -6 M tetrodotoxin. These changes are similar to those seen after denervation 5,15,17. H o w ever, in contrast to denervated fibres, these fibres not only had normal miniature end plate potentials (MEPPS) but also showed transmitted action potentials following nerve stimulation. In 16 different experiments, a total of 68 fibres exhibiting spread o f ACh-sensitivity were found to be excitable by nerve stimulation. A n example is shown in Fig. 1. In every experiment the E D L muscle o f the contralateral (untreated) side was also isolated, with its nerve, to assess possible systemic effects o f colchicine. R a t h e r surprisingly, the fibres o f the contralateral muscle consistently showed extrajunctional
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A
B
C
D
E
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'11x1"~-7A 0.1s
0.5s
H
F
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20/s
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Fig. 2. Upper row: EDL muscle responses (165 g rat) 4 days after the injection in the ipsilateral sciatic nerve of 152 #g of colchicine. A, B and C are tensions recorded isometrically in vivo with single and repetitive stimulation of the sciatic nerve. D shows the acetylcholine sensitivity measured near the tendon region in a fibre of the same muscle (duration of the iontophoretic pulse was 5 msec). E displays the action potential following sciatic nerve stimulation, recorded in the same fibre at the same position• In this muscle all the tested fibres (8) gave similar results, except for one fibre which was also sensitive extrajunctionally to A C h but was not excitable by nerve stimulation. The contralateral muscle behaved in the same manner both mechanically and electrically (not shown). Lower row: tension records (G, F and H) from the EDL muscle of an untreated rat of similar weight (156 g), for comparison.
ACh-sensitivity. After the injection into one sciatic nerve of 300-320 #g of colchicine, in rats weighing 320-440 g (6 experiments), 24 out of 29 ipsilateral fibres and 42 out of 55 contralateral fibres showed extrajunctional ACh-sensitivity. When body weight was taken into account~ an approximate dose-dependency of the phenomenon was apparent. With one-half the above dose used in 6 similar rats, only 4 out of 35 ipsilateral fibres showed spread of ACh-sensitivity, and 2 out of 50 contralateral fibres. If the same 'low dose' of colchicine was injected into one sciatic nerve of smaller rats (153-167 g), the spread of ACh-sensitivity was again found on both sides (98 ~o of tested fibres ipsilaterally and 96 ~ contralaterally in 4 animals). The extrajunctional ACh-sensitivity, as measured near the tendon, was the same for the two sides, i.e., 8.6 ± 1.5 units (mean ~ S.E.M.; n = 45) ipsilaterally and 6.9 ~ 0.9 units (n ---- 48) contralaterally. Because of these findings, the effects of systemic injections of the same doses of the drug (intramuscular or para-arterial) were investigated. The results were comparable to those described above for the nerve-injection experiments. It is concluded that colchicine injected under the epineurium of the sciatic nerve of one side produces its effects, on both sides, essentially through the systemic circulation. In experiments in which colchicine was applied to one sciatic nerve by means of a silicone rubber sleeve (containing about 200 ~g of the drug), spread of AChsensitivity occurred in fibres which exhibited an action potential following nerve stimulation. However, with this technique the ACh-sensitivity of fibres in the contra-
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lateral E D L muscle was unaffected except in one instance (a fibre which gave a small but detectable response to A C h in the tendon region). These experiments show that colchicine can produce some o f the characteristic 'denervation' changes in muscle without impairment of neuromuscular transmission. Motility of the animals and the tone o f their hind-limb muscles including E D L (as judged by the degree o f active spread of the digits) appeared normal. The tetanic tension developed by the affected E D L muscles was also normal after colchicine injection (Fig. 2). These observations indicate that 'denervation-like' changes can occur in the presence o f normal muscular activity. The results obtained are consistent with the interpretation that colchicine blocks some c o m p o n e n t o f axoplasmic flow, so interfering with neurotrophic control of muscle. However, some caution should be exercised until the possibility that diffusing colchicine acts directly on muscle (or specifically on nerve terminals), even with the silicone sleeve technique, is ruled out. This possibility has to be considered, especially in view o f the results o f colchicine injection (in this case the drug, after dilution in the body, is having its effects in a concentration o f about 10 -6 M or less). Investigations are under way to test whether, in injection experiments, the intra-axonal m o v e m e n t of labelled proteins 16 along the sciatic nerves is impaired, and to see if the result o f the silicone sleeve experiments can be explained entirely by a drug action at the site o f application. Note added in p r o o f I thank Dr. Jack D i a m o n d for support and continuous interest in this work. During the preparation of this manuscript two related papers were published. Albuquerque et al. 4, utilizing the sleeve technique, obtained findings similar to those described above for that technique; H o f m a n n and Thesleff 10 also obtained similar results using the injection technique, but limited their report to the treated side.
1 AGUILAR,C. E., BISBY, M. A., AND DIAMOND,J., Impulses and the transfer of trophic factors in nerves, J. Physiol. (Lond.), 226 (1972) 60P. 2 ALBUQUERQUE,E. X., SCHUH,F. T., ANDKAUFFMAN,F. C., Early membrane depolarization of the fast mammalian muscle after denervation, Pfliigers Arch. ges. Physiol., 328 (1971) 36-50. 3 ALBUQUERQUE,E. X., AND THESLEFF,S., A comparative study of membrane properties of innervated and chronically denervated fast and slow skeletal muscles of the rat, ActaphysioL scand., 73 (1968) 471-480. 4 ALBUQUERQUE,E. X., WARNICK,J. E., TASSE,J. R., ANDSANSONE,F. M., Effects of vinblastine and colchicine on neural regulation of the fast and slow skeletal muscles of the rat, Exp. NeuroL, 37 (1972) 607-634. 5 AXELSSON,J., AND THESLEEF,S., A study of supersensitivity in denervated mammalian skeletal muscle, J. Physiol. (Lond.), 147 (1959) 178-193. 6 DAHLSTROM,A., Effect of colchicine on transport of amine storage granules in sympathetic nerves of rat, Europ. J. Pharmacol., 5 (1968) 111-113. 7 DEE CASTILLO,J., AND KATZ, B., On the localization of acetylcholine receptors, J. Physiol. (Lond.), 128 (1955) 157-181. 8 FISCHBACH,G., AND ROBBINS,N., Effect of chronic disuse of rat soleus neuromuscular junctions on postsynaptic membrane, J. Neurophysiol., 34 (1971) 562-569. 9 HARRIS, J.B., AND THESLEEE,S., Nerve stump length and membrane changes in denervated skeletal muscle, Nature New Biol., 236 (1972) 60-61.
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10 HOFMANN,W. W., AND THESLEFF,S., Studies on the trophic influence of nerve on skeletal muscle, Europ. J. PharmacoL, 20 (1972) 256-260. 11 KREUTZBERG,G. W., Neuronal dynamics and axonal flow, IV. Blockage of intra-axonal enzyme transport by colchicine, Proc. nat. Acad. Sci. (Wash.), 62 (1969) 722-728. 12 LILEY, A. W., An investigation of spontaneous activity of the neuromuscular junction of the rat, J. Physiol. (Lond.), 132 (1956) 650-666. 13 LOMO,T., AND ROSENTHAL,J., Control of ACh sensitivity by muscle activity in the rat, J. PhysioL (Lond.), 221 (1972) 493-513. 14 Loco, J. V., AND EYZAGUIRRE,C., Fibrillation and hypersensitivity to ACh in denervated muscle: effect of length of degenerating nerve fibres, J. NeurophysioL, 18 (1955) 65-73. 15 MILEDI, R., The acetylcholine sensitivity of frog muscle fibres after complete or partial denervation, J. PhysioL (Lond.), 151 (1960) 1-23. 16 OCHS, S., Rate of fast axoplasmic transport in mammalian nerve fibres, J. Physiol. (Lond.), 227 (1972) 627-645. 17 REDFERN, P., AND THESLEFF, S., Action potential generation in denervated rat skeletal muscle. II. The action of tetrodotoxin, Acta physioL scand., 82 (1971) 70-78. 18 ROBERT, E. D., AND OESTER, Y.T., Absense of supersensitivity to acetylcholine in innervated muscle subjected to a prolonged pharmacologic block, J. Pharmacol. exp. Ther., 174 (1970) 133-140.
255
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Acetylcholine supersensitivity: the role of neurotrophic factors A. CANGIANO* MRC Group in Developmental Neurobiology, Department of Neurosciences, MeMaster University, Hamilton, Ontario (Canada)
(Accepted May 1st, 1973)
The trophic influence of neurones has been extensively studied by investigating the effects of denervation on skeletal muscle. Perhaps the most striking result of this procedure is the development of an intense sensitivity to the chemical transmitter acetylcholine (ACh) over the entire surface of the muscle fibres 5,15. It has been recently shown that the muscular inactivity that follows denervation plays an important role in the development of ACh supersensitivity 13. However, this does not exclude the possibility that the distribution of ACh-receptors and other properties of the muscle fibre membrane are also under the control of chemical influences from the motor nerve. The existence of these influences is suggested by the effects of partial denervation 15 and by the importance of the length of the nerve stump in determining the time of onset of denervation phenomenal,9,14. Such a mechanism of neurotrophic control could well involve the transfer of chemical information by axoplasmic flow down to the nerve terminals. Recent evidence indicates that colchicine, known to block axoplasmic transport6,11, mimics nerve section in causing peripheral sprouting of adjacent nerves in salamanders, leaving the endings of the treated nerve functionally unaffected 1. The present study investigates the effects of colchicine treatment of mammalian peripheral nerves on skeletal muscle, using doses which would not interfere with the normal functioning of that muscle. In anaesthetized Wistar rats (150--430 g in body weight), colchicine (B.D.H. Ltd., M.W. 399.4) was applied to one sciatic nerve either by injecting 1-5 #1 of a 0.2 M solution under the epineurium 11 or by slipping around the nerve a silicone rubber sleeve is impregnated with the drug (0.1-0.2 ~ w/w). At various times after colchicine application, rats with no obvious signs of paresis of the treated leg were selected for acute experiments. The extensor digitorum longus (EDL) muscle with the innervating sciatic nerve was mounted in a chamber and perfused 12 at room temperature (22-25 °C). Conventional microelectrode techniques were used to record intracellularly and to pass current in muscle fibres, and ACh-sensitivity was measured from the endplate to the myotendinous junction by microiontophoresis 7. * Present address: Istituto Di Fisiologia Umana, Universit~i Di Pisa, Pisa, Italy.
256
SHORT COMMUNICATIONS
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9:9
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Fig. 1. Records from a single EDL muscle fibre 4 days after injection of colchicine (152 #g) in the ipsilateral sciatic nerve (163 g rat). A-F: upper traces show 'acetylcholine potentials' recorded at different sites along the same fibre, from the tendon region (A) to the end-plate region (F). The figure shown in each record indicates the distance in millimetres from the end-plate. Iontophoretic pulses are displayed on the lower traces: pulse duration is 10 msec for all records except for B and C, where it is 20 msec. G: record taken at position A, soon after the 'acetylcholine potential' shown in A was evoked; without moving the recording electrode, the sciatic nerve was stimulated central to the site of colchicine application, and a conducted muscle action potential recorded. The disturbance seen after the spike is due to the twitch of the muscle. H: MEPPS recorded at position F. F o u r days after the injection o f colchicine (see below for doses) 8 0 - 1 0 0 ~ o f the superficial fibres tested (5-12 per muscle) on the treated side had become sensitive to A C h over their entire surface. The extrajunctional ACh-sensitivity ranged f r o m 0.01 to 39 units (1 unit = 1 mV peak depolarization per 10 -9 C o f charge passed through the A C h pipette15), whereas the junctional sensitivity ranged from 2 to 48 units (other authors report 2-24 units 8, or 20-230 units 8,13, for the junctional sensitivity o f rat soleus and E D L muscles). Furthermore, in the great majority o f muscle fibres, direct stimulation elicited action potentials in the presence o f 1 × 10 -6 M tetrodotoxin. These changes are similar to those seen after denervation 5,15,17. H o w ever, in contrast to denervated fibres, these fibres not only had normal miniature end plate potentials (MEPPS) but also showed transmitted action potentials following nerve stimulation. In 16 different experiments, a total of 68 fibres exhibiting spread o f ACh-sensitivity were found to be excitable by nerve stimulation. A n example is shown in Fig. 1. In every experiment the E D L muscle o f the contralateral (untreated) side was also isolated, with its nerve, to assess possible systemic effects o f colchicine. R a t h e r surprisingly, the fibres o f the contralateral muscle consistently showed extrajunctional
257
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A
B
C
D
E
0.ls
'11x1"~-7A 0.1s
0.5s
H
F
TWITCH
20/s
5ms
\
100/s
Fig. 2. Upper row: EDL muscle responses (165 g rat) 4 days after the injection in the ipsilateral sciatic nerve of 152 #g of colchicine. A, B and C are tensions recorded isometrically in vivo with single and repetitive stimulation of the sciatic nerve. D shows the acetylcholine sensitivity measured near the tendon region in a fibre of the same muscle (duration of the iontophoretic pulse was 5 msec). E displays the action potential following sciatic nerve stimulation, recorded in the same fibre at the same position• In this muscle all the tested fibres (8) gave similar results, except for one fibre which was also sensitive extrajunctionally to A C h but was not excitable by nerve stimulation. The contralateral muscle behaved in the same manner both mechanically and electrically (not shown). Lower row: tension records (G, F and H) from the EDL muscle of an untreated rat of similar weight (156 g), for comparison.
ACh-sensitivity. After the injection into one sciatic nerve of 300-320 #g of colchicine, in rats weighing 320-440 g (6 experiments), 24 out of 29 ipsilateral fibres and 42 out of 55 contralateral fibres showed extrajunctional ACh-sensitivity. When body weight was taken into account~ an approximate dose-dependency of the phenomenon was apparent. With one-half the above dose used in 6 similar rats, only 4 out of 35 ipsilateral fibres showed spread of ACh-sensitivity, and 2 out of 50 contralateral fibres. If the same 'low dose' of colchicine was injected into one sciatic nerve of smaller rats (153-167 g), the spread of ACh-sensitivity was again found on both sides (98 ~o of tested fibres ipsilaterally and 96 ~ contralaterally in 4 animals). The extrajunctional ACh-sensitivity, as measured near the tendon, was the same for the two sides, i.e., 8.6 ± 1.5 units (mean ~ S.E.M.; n = 45) ipsilaterally and 6.9 ~ 0.9 units (n ---- 48) contralaterally. Because of these findings, the effects of systemic injections of the same doses of the drug (intramuscular or para-arterial) were investigated. The results were comparable to those described above for the nerve-injection experiments. It is concluded that colchicine injected under the epineurium of the sciatic nerve of one side produces its effects, on both sides, essentially through the systemic circulation. In experiments in which colchicine was applied to one sciatic nerve by means of a silicone rubber sleeve (containing about 200 ~g of the drug), spread of AChsensitivity occurred in fibres which exhibited an action potential following nerve stimulation. However, with this technique the ACh-sensitivity of fibres in the contra-
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lateral E D L muscle was unaffected except in one instance (a fibre which gave a small but detectable response to A C h in the tendon region). These experiments show that colchicine can produce some o f the characteristic 'denervation' changes in muscle without impairment of neuromuscular transmission. Motility of the animals and the tone o f their hind-limb muscles including E D L (as judged by the degree o f active spread of the digits) appeared normal. The tetanic tension developed by the affected E D L muscles was also normal after colchicine injection (Fig. 2). These observations indicate that 'denervation-like' changes can occur in the presence o f normal muscular activity. The results obtained are consistent with the interpretation that colchicine blocks some c o m p o n e n t o f axoplasmic flow, so interfering with neurotrophic control of muscle. However, some caution should be exercised until the possibility that diffusing colchicine acts directly on muscle (or specifically on nerve terminals), even with the silicone sleeve technique, is ruled out. This possibility has to be considered, especially in view o f the results o f colchicine injection (in this case the drug, after dilution in the body, is having its effects in a concentration o f about 10 -6 M or less). Investigations are under way to test whether, in injection experiments, the intra-axonal m o v e m e n t of labelled proteins 16 along the sciatic nerves is impaired, and to see if the result o f the silicone sleeve experiments can be explained entirely by a drug action at the site o f application. Note added in p r o o f I thank Dr. Jack D i a m o n d for support and continuous interest in this work. During the preparation of this manuscript two related papers were published. Albuquerque et al. 4, utilizing the sleeve technique, obtained findings similar to those described above for that technique; H o f m a n n and Thesleff 10 also obtained similar results using the injection technique, but limited their report to the treated side.
1 AGUILAR,C. E., BISBY, M. A., AND DIAMOND,J., Impulses and the transfer of trophic factors in nerves, J. Physiol. (Lond.), 226 (1972) 60P. 2 ALBUQUERQUE,E. X., SCHUH,F. T., ANDKAUFFMAN,F. C., Early membrane depolarization of the fast mammalian muscle after denervation, Pfliigers Arch. ges. Physiol., 328 (1971) 36-50. 3 ALBUQUERQUE,E. X., AND THESLEFF,S., A comparative study of membrane properties of innervated and chronically denervated fast and slow skeletal muscles of the rat, ActaphysioL scand., 73 (1968) 471-480. 4 ALBUQUERQUE,E. X., WARNICK,J. E., TASSE,J. R., ANDSANSONE,F. M., Effects of vinblastine and colchicine on neural regulation of the fast and slow skeletal muscles of the rat, Exp. NeuroL, 37 (1972) 607-634. 5 AXELSSON,J., AND THESLEEF,S., A study of supersensitivity in denervated mammalian skeletal muscle, J. Physiol. (Lond.), 147 (1959) 178-193. 6 DAHLSTROM,A., Effect of colchicine on transport of amine storage granules in sympathetic nerves of rat, Europ. J. Pharmacol., 5 (1968) 111-113. 7 DEE CASTILLO,J., AND KATZ, B., On the localization of acetylcholine receptors, J. Physiol. (Lond.), 128 (1955) 157-181. 8 FISCHBACH,G., AND ROBBINS,N., Effect of chronic disuse of rat soleus neuromuscular junctions on postsynaptic membrane, J. Neurophysiol., 34 (1971) 562-569. 9 HARRIS, J.B., AND THESLEEE,S., Nerve stump length and membrane changes in denervated skeletal muscle, Nature New Biol., 236 (1972) 60-61.
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259
10 HOFMANN,W. W., AND THESLEFF,S., Studies on the trophic influence of nerve on skeletal muscle, Europ. J. PharmacoL, 20 (1972) 256-260. 11 KREUTZBERG,G. W., Neuronal dynamics and axonal flow, IV. Blockage of intra-axonal enzyme transport by colchicine, Proc. nat. Acad. Sci. (Wash.), 62 (1969) 722-728. 12 LILEY, A. W., An investigation of spontaneous activity of the neuromuscular junction of the rat, J. Physiol. (Lond.), 132 (1956) 650-666. 13 LOMO,T., AND ROSENTHAL,J., Control of ACh sensitivity by muscle activity in the rat, J. PhysioL (Lond.), 221 (1972) 493-513. 14 Loco, J. V., AND EYZAGUIRRE,C., Fibrillation and hypersensitivity to ACh in denervated muscle: effect of length of degenerating nerve fibres, J. NeurophysioL, 18 (1955) 65-73. 15 MILEDI, R., The acetylcholine sensitivity of frog muscle fibres after complete or partial denervation, J. PhysioL (Lond.), 151 (1960) 1-23. 16 OCHS, S., Rate of fast axoplasmic transport in mammalian nerve fibres, J. Physiol. (Lond.), 227 (1972) 627-645. 17 REDFERN, P., AND THESLEFF, S., Action potential generation in denervated rat skeletal muscle. II. The action of tetrodotoxin, Acta physioL scand., 82 (1971) 70-78. 18 ROBERT, E. D., AND OESTER, Y.T., Absense of supersensitivity to acetylcholine in innervated muscle subjected to a prolonged pharmacologic block, J. Pharmacol. exp. Ther., 174 (1970) 133-140.