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Inhibition by α-amanitin of development of tetrodotoxin-sensitive spike induced by brain extract in cultured chick skeletal muscle cells
674
Developmental Brain Research, 3 (1982) 674-678 Elsevier Biomedical Press
Inhibition by a-amanitin of development of tetrodotoxin-seneitive spike induced by brain extract in cultured chick skeletal muscle cells
MASAAKIRA KANO and NOBUYUKI SUZUKI Department of Physiology, School of Medicine, Kitasato University, Sagamihara, Kanagawa 228 (Japan) (Accepted December 17th, 1981) Key words: neurotrophism - - trophic substance - - a-amanitin - - RNA synthesis - - tetrodotoxinsensitive spike - - chick skeletal muscle culture
The enhancing effect of brain extract on the development of tetrodotoxin-sensitive sodium channels in cultured chick skeletal muscle cells is blocked by treatment of culture with a-amanitin, an RNA polymerase inhibitor. The result is interpreted as indicating that the trophic substance in the brain extract exerts its effect on the development of sodium channels through regulation of gene transcription. The motor nerves exert trophic influences on muscle development and maintenance a. Recent evidence indicates that neurohumoral substances released from nerves subserve this function~,6,13,14,ts,19, 22-24, although their effects are modified by muscle activity or nerve transmission a. Recently, some such trophic substances were isolated from nervous tissues and characterized as a protein ta,16,22 or a peptide s,~l. Furthermore, it is suggested that such trophic substances may act at the level of gene expression (refs. 1, 5, 17). The rate of gene transcription by R N A polymerases is, then, one regulatory site which may be influenced by the trophic substances. The action potential of adult innervated vertebrate skeletal muscle fibers is operated by tetrodotoxin (TTX)-sensitive sodium channels. The development and maintenance of the channels in the muscle cell membrane are under the trophic control of innervation 7,20. Using skeletal muscle cultures, it was shown that cell-free nerve extracts can duplicate at least a part of the effect of innervation on the development and maintenance of the TTX-sensitive sodium channels in uninnervated skeletal muscle cellsg,~z,t3. Thus, the results indicate that the trophic control of the channels of muscle cells might well be mediated by a t r o p h i c substance produced by nervous tissues. It was of interest to see whether or not the trophic substance in the nerve extracts could exert its effect on the development and maintenance of TTX-sensitive sodium channels in muscle cells through regulation of gene transcription. We now report that the enhancing effect of the nerve extract on the development of TTX-sensitive sodium channels in cultured chick skeletal muscle cells is completely eliminated by treatment of the culture with low concentrations of a-amanitin. This mushroom toxin 0165-3806/82/0000-0000/$02.75 © Elsevier Biomedical Press
675 is a potent inhibitor of RNA synthesis; at low concentrations it specifically inhibits DNA-dependent RNA polymerase II, which is responsible for the transcription of messenger (m) RNA precursor 2,15,9"1. It can be therefore suggested that the nerve extract enhances the development of TTX-sensitive sodium channels in cultured muscle cells by inducing the specific mRNA transcription. Muscle cell cultures were prepared from thigh muscles of 11-day-old chick embryos as previously describeda, 10. The basic culture medium was Eagle's minimal essential medium supplemented with horse serum (10 ~ by volume), nerve-free chick embryo extract ( 5 ~ by volume), glutamine (1 mM), penicillin (50 U/ml), and streptomycin (50 #g/ml). The nerve-free chick embryo extract was made from 10-dayold chick embryos deprived of brain and spinal cord according to the method described previouslya, i.e. all the possible neurotrophic materials were eliminated from the basic culture medium. This basic culture medium was utilized for control cultures. To enhance the development of TTX-sensitive sodium channels in muscle cells developing in cell culture, brain extract was added to the muscle cultures throughout the entire cultivation period. The brain extract was prepared from whole brains of 19-day-old chick embryos by centrifuging a 1:2 homogenate in Tyrode's solution at 2000 g for 10 min. The supernatant was used as the brain extract; it contained approximately 10 mg of protein/ml and was added to the basic culture medium to a final concentration of 0.3 mg of extract protein/ml of culture medium. This concentration of brain extract has been shown to be effective to enhance the development of TTX-sensitive sodium channels in cultured muscle cells9. Muscle cells were maintained in the absence or presence of brain extract for 13 or 14 days in culture. At the end of the culture period, the maximum rate of rise of TTX-sensitive spike of muscle cells was measured. The rate of rise of spike was obtained by differentiating the potential with a circuit having a time constant of 200/,s. Since the spike of the cultured chick skeletal muscle cells has been found to be highly sensitive to low concentrations of TTX, the maximum rate of rise of the spike can be used as an index of the density of TTXsensitive sodium channels in the muscle cell membrane0,10. Procedure for the measurement of the maximum rate of rise of the TTX-sensitive spike was previously described9,10. Briefly, the cultures were studied on the stage of an inverted phasecontrast microscope, and during experiments the cells were bathed in oxygenated saline buffered with Tris-HC1 (pH 7.4) and maintained at 35 °C. A fully differentiated muscle cell with cross-striations was selected and penetrated with closely adjacent conventional recording and current-passing microelectrodes. To minimize the effect of variation in the resting membrane potential and to ensure maximal spike generation, the membrane potential was preset in each case at --80 mV by passing a steady hyperpolarizing current, and a spike was evoked by a superimposed depolarizing current pulse, adjusted in each case to generate a spike with 5-10 ms latency. The mean maximum rate of rise of TTX-sensitive spike was about 40 V/s in control cultures grown in the basic culture medium in the absence of brain extract. The addition of brain extract to the culture medium of muscle cells increased the mean value to about 75 V/s, confirming our earlier observationa. This increase in the
676 TABLE I Prevention by a-amanitin o f the brain extract-induced increase in the maximum rate o f rise o f tetrodotoxin-sensitive spike o f muscle cells in 13- or 14-day-old cultures
Cultures were grown in the absence and presence of brain extract (0.3 mg of extract protein/ml) for 13 or 14 days, and some of these two groups of cultures were treated with a-amanitin (1.0/~g/ml) from seventh day of culture onward. Each value is the mean ± S.E.M. for indicated number of cells from two different batches of cultures. Treatment
Number o f cells
Maximum rate o f rise (V/s)
Control Control ÷ a-amanitin Brain extract Brain extract + a-amanitin
28 25 22 43
40.8 ± 2.5 37.0 ± 2.3 74.2 4- 6.5* 46.9 ± 2.7
* Indicates a statistically significant increase (P < 0.001) from the control. maximum rate of rise of TTX-sensitive spike by the brain extract was almost completely prevented by exposure of the cultures to a-amanitin (Sigma) at a concentration of 1 #g/mi of culture medium from the seventh day of culture onward; the mean value was almost similar to that obtained in control cultures. The same treatment of control cultures with ct-amanitin, however, had no significant effect on the mean value. Table I summarizes these results. In pilot experiments we examined the time and dose required for a-amanitin to block the effect of brain extract on the development of the maximum rate of rise of TTX-sensitive spike. It was previously shown that in medium containing brain extract, morphologically matured muscle fibers with characteristic cross-striations were seen within 7 days of culture; in medium without brain extract, muscle cells matured more slowly, but long and wide myotubes were already present by the seventh day and some of them had faint cross-striations 9,1°. For the study of the effect of a-amanitin on the development of TTX-sensitive sodium channels, but not on the morphological development, the muscle cells were given time to develop morphologically during the first 7 days in culture without a-amanitin. Increase in the maximum rate of rise of TTX-sensitive spikes by brain extract was almost completely prevented when aamanitin was added to the cultures at a concentration of 1 #g/ml of culture medium from the seventh day of culture onward. When the toxin (1 #g/ml) was added between 7 and 10 days in culture, a less, though still substantial effect was obtained; but exposure to the toxin (1/zg/ml) for 1 day on the seventh day in culture had no effect on the maximum rate of rise of TTX-sensitive spike. We also looked at the effects of various dosages of a-amanitin (0.008, 0.04, 0.2, 1.0, 5.0, 15.0 #g/ml of culture medium) in the exposure from the seventh day of culture onward. It was found that dosage of aamanitin over 0.2/zg/ml of culture medium was sufficient to prevent the enhancing effect of brain extract on the development of the channels and that at concentrations of 5.0 or 15.0 ~g/ml of culture medium, the destruction of some muscle fibers was seen. In the present experiments, therefore, a concentration of a-amanitin of 1 /~g/ml was chosen and added to the cultures from the seventh day of culture onward; the data
677 shown in Table I was obtained by this toxin-treatment. This toxin-treatment had no effects on the morphology of muscle cells in culture. The results reported here demonstrate that the development of TTX-sensitive sodium channels in control cultures was not inhibited by the treatment with a-amanitin, whereas the enhancement of the development of TTX-sensitive sodium channels by brain extract was completely inhibited by the toxin-treatment. Furthermore, the cultures treated with the toxin were morphologically indistinguishable from the untreated cultures. It is likely, therefore, that this inhibition by the toxin is specific to the enhancement by brain extract and is not due to a general deterioration of the muscle cells. The cell-free synthesis of transfer or ribosomal R N A is not inhibited by aamanitin at the concentration of 1.0 #g/m121, the dose chosen for our studies. Therefore, our findings suggest that the brain extract enhances the development of TTX-sensitive sodium channels in cultured muscle cells by inducing the synthesis of m R N A responsible for the development of TTX-sensitive sodium channels. Since as noted before a-amanitin did not inhibit the development of TTX-sensitive sodium channels in control cultures without brain extract, it would appear that the m R N A responsible for the development of the channels in control cultures was intrinsically synthesized and already present before the toxin-treatment. We wish to thank Prof. M. Kawakami for encouragement and advice, Dr. S. Hasegawa for critical reading of the manuscript, Dr. S. Yamazaki for help in the preparation of the manuscript, and Ms. M. Y a m a m o t o for technical assistance in preparing the cultures. This work was supported in part by grants from the Japanese Ministry of Education, Science and Culture, and from the National Center for Nervous, Mental and Muscular Disorders ( N C N M M D ) of the Japanese Ministry of Health and Welfare.
1 Fambrough, D. M., Hartzell, H. C., Powell, J. A., Rash, J. E. and Joseph, N., On the differentiation and organization of the surface membrane of a postsynaptic cell - - the skeletal muscle fiber. In M. V. L. Bennett (Ed.), Synaptic Transmission and Neuronal Interaction, Raven Press, New York, 1974, pp. 285-313. 2 Fiume, L. and Wieland, Th., Amanitins. Chemistry and action, FEBS Lett., 8 (1970) 1-5. 3 Guth, L., 'Trophic' influences of nerve on muscle, PhysioL Rev., 48 (1968) 645-687. 4 Guth, L. and Albuquerque, E. X., The neurotrophic regulation of resting membrane potential and extrajunctional acetylcholine sensitivity in mammalian skeletal muscle. In A. Mauro (Ed.), Muscle Regeneration, Raven Press, New York, 1979, pp. 405-415. 5 Gutmann, E., Neurotrophic relations, Ann. Rev. Physiol., 38 (1976) 177-216. 6 Harris, A. J., Inductwe functions of the nervous system, Ann. Rev. Physiol., 36 (1974) 251-305. 7 Harris, J. B. and Marshall, M. W., Tetrodotoxin-resistant action potentials in newborn rat muscle, Nature New Biol., 243 (1973) 191-192. 8 Jessell, T. M., Siegel, R. E. and Fischbach, G. D., Induction of acetylcholine receptors on cultured skeletal muscle by a factor extracted from brain and spinal cord, Proc. nat. Acad. Sci. (U.S.A.), 76 (1979) 5397-5401. 9 Kano, M., Suzuki, N. and Ojima, H., Neurotrophic effect of nerve extract on development of tetrodotoxin-sensitive spike potential in skeletal muscle cells in culture, J. cell. Physiol., 99 (1979) 327-332.
678 10 Kano, M. and Yamamoto, M., Development of spike potentials in skeletal muscle cells differentiated in vitro from chick embryo, J. cell. Physiol., 90 (1977) 439-444. 11 Kuromi, H., Gonoi, T. and Hasegawa, S., Partial purification and characterization of neurotrophic substance affecting tetrodotoxin sensitivity of organ-cultured mouse muscle, Brain Res., 175 (1979) 109-118. 12 Kuromi, H., Gonoi, T. and Hasegawa, S., Neurotrophic substance develops tetrodotoxin-sensitive action potential and increases curare-sensitivity of acetylcholine response in cultured rat myotubes, Develop. Brain Res., 1 (1981) 369-379. 13 Kuromi, H. and Hasegawa, S., Neurotrophic effect of spinal cord extract on membrane potentials of organ-cultured mouse skeletal muscle, Brain Res., 100 (1975) 178-181. 14 Lentz, T. L., Effect of brain extracts on cholinesterase activity of cultured skeletal muscle, Exp. Neurol., 45 (1974) 520-526. 15 Lindell, T. J., Weinberg, F., Morris, P. W., Roeder, R. G. and Rutter, W. J., Specific inhibition of nuclear R N A polymerase II by a-amanitin, Science, 170 (1970) 447-449. 16 Markelonis, G. and Oh, T. H., A sciatic nerve protein has a tropbic effect on development and maintenance of skeletal muscle cells in culture, Proc. nat. Acad. Sci. (U.S.A.), 76 (1979) 24702474. 17 McComas, A. J., Neuromuscular Function and Disorders, Butterworths, London, 1977, pp. 85-88. 18 Oh, T. H., Neurotrophic effects of sciatic nerve extracts on muscle development in culture, Exp. Neurol., 50 (1976) 376-386. 19 Podleski, T. R., Axelrod, D., Ravdin, P., Greenberg, I., Johnson, M. M. and Salpeter, M. M., Nerve extract induces increase and redistribution of acetylcholine receptors in cloned muscle cells, Proc. nat. Acad. Sci. (U.S.A.), 75 (1978) 2035-2039. 20 Redfern, P. and Thesleff, S., Action potential generation in denervated rat skeletal muscle. II. The action of tetrodotoxin, Actaphysiol. scand., 82 (1971) 70-78. 21 Roeder, R. G., Eukaryotic nuclear R N A polymerases. In R. Losik and M. Chamberlin (Eds.), RNA Polymerase, Cold Spring Harbor Laboratory, New York, 1976, pp. 285-329. 22 Singer, M., Neurotrophic control of limb regeneration in the newt, Ann. N.Y. Acad. Sci., 228 (1974) 308-322. 23 Younkin, S. G., Brett, R. S., Davey, B. and Younkin, L. H., Substances moved by axonal transport and released by nerve stimulation have an innervation-like effect on muscle, Science, 200 (1978) 1292-1295. 24 Warnick, J. E., Albuquerque, E. X. and Guth, L., The demonstration of neurotrophic function by application of colchicine or vinblastine to the peripheral nerve, Exp. Neurol., 57 (1977) 622-636.
Developmental Brain Research, 3 (1982) 674-678 Elsevier Biomedical Press
Inhibition by a-amanitin of development of tetrodotoxin-seneitive spike induced by brain extract in cultured chick skeletal muscle cells
MASAAKIRA KANO and NOBUYUKI SUZUKI Department of Physiology, School of Medicine, Kitasato University, Sagamihara, Kanagawa 228 (Japan) (Accepted December 17th, 1981) Key words: neurotrophism - - trophic substance - - a-amanitin - - RNA synthesis - - tetrodotoxinsensitive spike - - chick skeletal muscle culture
The enhancing effect of brain extract on the development of tetrodotoxin-sensitive sodium channels in cultured chick skeletal muscle cells is blocked by treatment of culture with a-amanitin, an RNA polymerase inhibitor. The result is interpreted as indicating that the trophic substance in the brain extract exerts its effect on the development of sodium channels through regulation of gene transcription. The motor nerves exert trophic influences on muscle development and maintenance a. Recent evidence indicates that neurohumoral substances released from nerves subserve this function~,6,13,14,ts,19, 22-24, although their effects are modified by muscle activity or nerve transmission a. Recently, some such trophic substances were isolated from nervous tissues and characterized as a protein ta,16,22 or a peptide s,~l. Furthermore, it is suggested that such trophic substances may act at the level of gene expression (refs. 1, 5, 17). The rate of gene transcription by R N A polymerases is, then, one regulatory site which may be influenced by the trophic substances. The action potential of adult innervated vertebrate skeletal muscle fibers is operated by tetrodotoxin (TTX)-sensitive sodium channels. The development and maintenance of the channels in the muscle cell membrane are under the trophic control of innervation 7,20. Using skeletal muscle cultures, it was shown that cell-free nerve extracts can duplicate at least a part of the effect of innervation on the development and maintenance of the TTX-sensitive sodium channels in uninnervated skeletal muscle cellsg,~z,t3. Thus, the results indicate that the trophic control of the channels of muscle cells might well be mediated by a t r o p h i c substance produced by nervous tissues. It was of interest to see whether or not the trophic substance in the nerve extracts could exert its effect on the development and maintenance of TTX-sensitive sodium channels in muscle cells through regulation of gene transcription. We now report that the enhancing effect of the nerve extract on the development of TTX-sensitive sodium channels in cultured chick skeletal muscle cells is completely eliminated by treatment of the culture with low concentrations of a-amanitin. This mushroom toxin 0165-3806/82/0000-0000/$02.75 © Elsevier Biomedical Press
675 is a potent inhibitor of RNA synthesis; at low concentrations it specifically inhibits DNA-dependent RNA polymerase II, which is responsible for the transcription of messenger (m) RNA precursor 2,15,9"1. It can be therefore suggested that the nerve extract enhances the development of TTX-sensitive sodium channels in cultured muscle cells by inducing the specific mRNA transcription. Muscle cell cultures were prepared from thigh muscles of 11-day-old chick embryos as previously describeda, 10. The basic culture medium was Eagle's minimal essential medium supplemented with horse serum (10 ~ by volume), nerve-free chick embryo extract ( 5 ~ by volume), glutamine (1 mM), penicillin (50 U/ml), and streptomycin (50 #g/ml). The nerve-free chick embryo extract was made from 10-dayold chick embryos deprived of brain and spinal cord according to the method described previouslya, i.e. all the possible neurotrophic materials were eliminated from the basic culture medium. This basic culture medium was utilized for control cultures. To enhance the development of TTX-sensitive sodium channels in muscle cells developing in cell culture, brain extract was added to the muscle cultures throughout the entire cultivation period. The brain extract was prepared from whole brains of 19-day-old chick embryos by centrifuging a 1:2 homogenate in Tyrode's solution at 2000 g for 10 min. The supernatant was used as the brain extract; it contained approximately 10 mg of protein/ml and was added to the basic culture medium to a final concentration of 0.3 mg of extract protein/ml of culture medium. This concentration of brain extract has been shown to be effective to enhance the development of TTX-sensitive sodium channels in cultured muscle cells9. Muscle cells were maintained in the absence or presence of brain extract for 13 or 14 days in culture. At the end of the culture period, the maximum rate of rise of TTX-sensitive spike of muscle cells was measured. The rate of rise of spike was obtained by differentiating the potential with a circuit having a time constant of 200/,s. Since the spike of the cultured chick skeletal muscle cells has been found to be highly sensitive to low concentrations of TTX, the maximum rate of rise of the spike can be used as an index of the density of TTXsensitive sodium channels in the muscle cell membrane0,10. Procedure for the measurement of the maximum rate of rise of the TTX-sensitive spike was previously described9,10. Briefly, the cultures were studied on the stage of an inverted phasecontrast microscope, and during experiments the cells were bathed in oxygenated saline buffered with Tris-HC1 (pH 7.4) and maintained at 35 °C. A fully differentiated muscle cell with cross-striations was selected and penetrated with closely adjacent conventional recording and current-passing microelectrodes. To minimize the effect of variation in the resting membrane potential and to ensure maximal spike generation, the membrane potential was preset in each case at --80 mV by passing a steady hyperpolarizing current, and a spike was evoked by a superimposed depolarizing current pulse, adjusted in each case to generate a spike with 5-10 ms latency. The mean maximum rate of rise of TTX-sensitive spike was about 40 V/s in control cultures grown in the basic culture medium in the absence of brain extract. The addition of brain extract to the culture medium of muscle cells increased the mean value to about 75 V/s, confirming our earlier observationa. This increase in the
676 TABLE I Prevention by a-amanitin o f the brain extract-induced increase in the maximum rate o f rise o f tetrodotoxin-sensitive spike o f muscle cells in 13- or 14-day-old cultures
Cultures were grown in the absence and presence of brain extract (0.3 mg of extract protein/ml) for 13 or 14 days, and some of these two groups of cultures were treated with a-amanitin (1.0/~g/ml) from seventh day of culture onward. Each value is the mean ± S.E.M. for indicated number of cells from two different batches of cultures. Treatment
Number o f cells
Maximum rate o f rise (V/s)
Control Control ÷ a-amanitin Brain extract Brain extract + a-amanitin
28 25 22 43
40.8 ± 2.5 37.0 ± 2.3 74.2 4- 6.5* 46.9 ± 2.7
* Indicates a statistically significant increase (P < 0.001) from the control. maximum rate of rise of TTX-sensitive spike by the brain extract was almost completely prevented by exposure of the cultures to a-amanitin (Sigma) at a concentration of 1 #g/mi of culture medium from the seventh day of culture onward; the mean value was almost similar to that obtained in control cultures. The same treatment of control cultures with ct-amanitin, however, had no significant effect on the mean value. Table I summarizes these results. In pilot experiments we examined the time and dose required for a-amanitin to block the effect of brain extract on the development of the maximum rate of rise of TTX-sensitive spike. It was previously shown that in medium containing brain extract, morphologically matured muscle fibers with characteristic cross-striations were seen within 7 days of culture; in medium without brain extract, muscle cells matured more slowly, but long and wide myotubes were already present by the seventh day and some of them had faint cross-striations 9,1°. For the study of the effect of a-amanitin on the development of TTX-sensitive sodium channels, but not on the morphological development, the muscle cells were given time to develop morphologically during the first 7 days in culture without a-amanitin. Increase in the maximum rate of rise of TTX-sensitive spikes by brain extract was almost completely prevented when aamanitin was added to the cultures at a concentration of 1 #g/ml of culture medium from the seventh day of culture onward. When the toxin (1 #g/ml) was added between 7 and 10 days in culture, a less, though still substantial effect was obtained; but exposure to the toxin (1/zg/ml) for 1 day on the seventh day in culture had no effect on the maximum rate of rise of TTX-sensitive spike. We also looked at the effects of various dosages of a-amanitin (0.008, 0.04, 0.2, 1.0, 5.0, 15.0 #g/ml of culture medium) in the exposure from the seventh day of culture onward. It was found that dosage of aamanitin over 0.2/zg/ml of culture medium was sufficient to prevent the enhancing effect of brain extract on the development of the channels and that at concentrations of 5.0 or 15.0 ~g/ml of culture medium, the destruction of some muscle fibers was seen. In the present experiments, therefore, a concentration of a-amanitin of 1 /~g/ml was chosen and added to the cultures from the seventh day of culture onward; the data
677 shown in Table I was obtained by this toxin-treatment. This toxin-treatment had no effects on the morphology of muscle cells in culture. The results reported here demonstrate that the development of TTX-sensitive sodium channels in control cultures was not inhibited by the treatment with a-amanitin, whereas the enhancement of the development of TTX-sensitive sodium channels by brain extract was completely inhibited by the toxin-treatment. Furthermore, the cultures treated with the toxin were morphologically indistinguishable from the untreated cultures. It is likely, therefore, that this inhibition by the toxin is specific to the enhancement by brain extract and is not due to a general deterioration of the muscle cells. The cell-free synthesis of transfer or ribosomal R N A is not inhibited by aamanitin at the concentration of 1.0 #g/m121, the dose chosen for our studies. Therefore, our findings suggest that the brain extract enhances the development of TTX-sensitive sodium channels in cultured muscle cells by inducing the synthesis of m R N A responsible for the development of TTX-sensitive sodium channels. Since as noted before a-amanitin did not inhibit the development of TTX-sensitive sodium channels in control cultures without brain extract, it would appear that the m R N A responsible for the development of the channels in control cultures was intrinsically synthesized and already present before the toxin-treatment. We wish to thank Prof. M. Kawakami for encouragement and advice, Dr. S. Hasegawa for critical reading of the manuscript, Dr. S. Yamazaki for help in the preparation of the manuscript, and Ms. M. Y a m a m o t o for technical assistance in preparing the cultures. This work was supported in part by grants from the Japanese Ministry of Education, Science and Culture, and from the National Center for Nervous, Mental and Muscular Disorders ( N C N M M D ) of the Japanese Ministry of Health and Welfare.
1 Fambrough, D. M., Hartzell, H. C., Powell, J. A., Rash, J. E. and Joseph, N., On the differentiation and organization of the surface membrane of a postsynaptic cell - - the skeletal muscle fiber. In M. V. L. Bennett (Ed.), Synaptic Transmission and Neuronal Interaction, Raven Press, New York, 1974, pp. 285-313. 2 Fiume, L. and Wieland, Th., Amanitins. Chemistry and action, FEBS Lett., 8 (1970) 1-5. 3 Guth, L., 'Trophic' influences of nerve on muscle, PhysioL Rev., 48 (1968) 645-687. 4 Guth, L. and Albuquerque, E. X., The neurotrophic regulation of resting membrane potential and extrajunctional acetylcholine sensitivity in mammalian skeletal muscle. In A. Mauro (Ed.), Muscle Regeneration, Raven Press, New York, 1979, pp. 405-415. 5 Gutmann, E., Neurotrophic relations, Ann. Rev. Physiol., 38 (1976) 177-216. 6 Harris, A. J., Inductwe functions of the nervous system, Ann. Rev. Physiol., 36 (1974) 251-305. 7 Harris, J. B. and Marshall, M. W., Tetrodotoxin-resistant action potentials in newborn rat muscle, Nature New Biol., 243 (1973) 191-192. 8 Jessell, T. M., Siegel, R. E. and Fischbach, G. D., Induction of acetylcholine receptors on cultured skeletal muscle by a factor extracted from brain and spinal cord, Proc. nat. Acad. Sci. (U.S.A.), 76 (1979) 5397-5401. 9 Kano, M., Suzuki, N. and Ojima, H., Neurotrophic effect of nerve extract on development of tetrodotoxin-sensitive spike potential in skeletal muscle cells in culture, J. cell. Physiol., 99 (1979) 327-332.
678 10 Kano, M. and Yamamoto, M., Development of spike potentials in skeletal muscle cells differentiated in vitro from chick embryo, J. cell. Physiol., 90 (1977) 439-444. 11 Kuromi, H., Gonoi, T. and Hasegawa, S., Partial purification and characterization of neurotrophic substance affecting tetrodotoxin sensitivity of organ-cultured mouse muscle, Brain Res., 175 (1979) 109-118. 12 Kuromi, H., Gonoi, T. and Hasegawa, S., Neurotrophic substance develops tetrodotoxin-sensitive action potential and increases curare-sensitivity of acetylcholine response in cultured rat myotubes, Develop. Brain Res., 1 (1981) 369-379. 13 Kuromi, H. and Hasegawa, S., Neurotrophic effect of spinal cord extract on membrane potentials of organ-cultured mouse skeletal muscle, Brain Res., 100 (1975) 178-181. 14 Lentz, T. L., Effect of brain extracts on cholinesterase activity of cultured skeletal muscle, Exp. Neurol., 45 (1974) 520-526. 15 Lindell, T. J., Weinberg, F., Morris, P. W., Roeder, R. G. and Rutter, W. J., Specific inhibition of nuclear R N A polymerase II by a-amanitin, Science, 170 (1970) 447-449. 16 Markelonis, G. and Oh, T. H., A sciatic nerve protein has a tropbic effect on development and maintenance of skeletal muscle cells in culture, Proc. nat. Acad. Sci. (U.S.A.), 76 (1979) 24702474. 17 McComas, A. J., Neuromuscular Function and Disorders, Butterworths, London, 1977, pp. 85-88. 18 Oh, T. H., Neurotrophic effects of sciatic nerve extracts on muscle development in culture, Exp. Neurol., 50 (1976) 376-386. 19 Podleski, T. R., Axelrod, D., Ravdin, P., Greenberg, I., Johnson, M. M. and Salpeter, M. M., Nerve extract induces increase and redistribution of acetylcholine receptors in cloned muscle cells, Proc. nat. Acad. Sci. (U.S.A.), 75 (1978) 2035-2039. 20 Redfern, P. and Thesleff, S., Action potential generation in denervated rat skeletal muscle. II. The action of tetrodotoxin, Actaphysiol. scand., 82 (1971) 70-78. 21 Roeder, R. G., Eukaryotic nuclear R N A polymerases. In R. Losik and M. Chamberlin (Eds.), RNA Polymerase, Cold Spring Harbor Laboratory, New York, 1976, pp. 285-329. 22 Singer, M., Neurotrophic control of limb regeneration in the newt, Ann. N.Y. Acad. Sci., 228 (1974) 308-322. 23 Younkin, S. G., Brett, R. S., Davey, B. and Younkin, L. H., Substances moved by axonal transport and released by nerve stimulation have an innervation-like effect on muscle, Science, 200 (1978) 1292-1295. 24 Warnick, J. E., Albuquerque, E. X. and Guth, L., The demonstration of neurotrophic function by application of colchicine or vinblastine to the peripheral nerve, Exp. Neurol., 57 (1977) 622-636.