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Neurotrophic effect of spinal cord extract on membrane potentials of organ-cultured mouse skeletal muscle
178
Bra#l Research, 100 (1975) 178-181 ~:) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Neurotrophic effect of spinal cord extract on membrane potentials of organcultured mouse skeletal muscle
HIROSHI KUROMI AND SHUJI HASEGAWA Brain Research Institute, School of Medicine, Chiba University, Chiba (Japan)
(Accepted August 26th, 1975)
Some membrane properties of skeletal muscle appear to be controlled by motoneurones 4. When mammalian skeletal muscle is denervated, the resting membrane potential and the maximal rates of rise and fall of action potential are reduced1,1°, le. In innervated muscle, generation of action potentials is completely blocked by tetrodotoxin (TTX), while in denervated muscle the action potential is resistant to TTX 2,9,11. The change in membrane properties of denervated muscle may be due to a loss of 'trophic' substance(s) which are normally transmitted from the motoneurone to the muscle 3. We examined the effects of addition of spinal cord extract on membrane properties of organ-cultured skeletal muscle. The extensor digitorum longus muscle (EDL) of male mice (20-25 g) was denervated by cutting the deep peroneal nerve close to the knee joint. Three days later the mice were anaesthetized with sodium pentobarbital (40 mg/kg, i.p.). The EDL was carefully removed and mounted on a U-shaped glass rod by binding its tendons to the ends of the rod (which were separated by 13 mm). It was then cultured for 3 days at 37 °C in a glass vial containing 4 ml of medium under an atmosphere of 95 % 02-5 ~,~ CO2. The medium consisted of 85 % Eagle's minimal essential medium and 15 % horse serum containing penicillin (50 U/ml) and streptomycin (50 #g/ml). The medium was changed after the first hour and thereafter every 24 h. Mouse spinal cord and liver were removed and homogenized with an equal volume of Tyrode's solution and centrifuged at 2000 × g for 10 rain. The supernatants were used as tissue extracts. An extract equivalent to 100 mg of the tissue wet weight was added to 4 ml of the culture medium. At the end of the culture period, E D L was mounted in a bath containing Eagle's minimal essential medium bubbled with 95 % 02-5 % CO2 at 37 °C, and the membrane properties of the muscle were investigated. Two glass microelectrodes filIed with 3 M KCI were inserted into the same surface muscle fibre about 50 #m apart; one was used for passing current and the other for recording the potential changes. Constant anodal current was passed through the electrode to locally polarize the muscle fibre from --85 to - - 9 0 inV. This current was interrupted for a 5 msec duration by applying a cathodal shock, adjusted in each case to produce an action potential with 1-3 msec latency. The rates of rise and fall of the
Ox
TABLE I in vivo
EFFECT OF TISSUE EXTRACT ON THE MAXIMAL RATES OF RISE AND FALL OF ACTION POTENTIAL GENERATED IN ORGAN-CULTURES OF MUSCLE THAT HAD BEEN PREVIOUSLY DENERVATED FOR 3 DAYS
Days in culture
0 3 3 3
N
40 27 25 29
± 1.00 -+- 1.20 ± 0.79 q- 0.69
Resting potential ( m Ix)
56.7 57.8 57.7 57.2
± 44±
15.6 18.2 18.1"* 16.1
Maximal rate o f rise (V/sec)
512 458 593 420
± 8.3 4- 9.5 4- 6.9** ± 4.9*
Maximal rate of fall (V/sec)
167 143 190 114
58.9 62.8 59.0 56.9
± ± ± ±
2.45 0.84 1.26 1.04
Resting potential (miX)
10 aM T T X N
32 30 26 25
67.7 60.0 37.2 64.5
± ± 44-
4.2 3.9 3.3** 3.0
Maximal rate o f rise (%)
79.5 76.9 38.0 68.9
-+- 6.9 ± 6.8 4- 4.4** ± 4.6
Maximal rate of fall (%)
The rates in the presence of T T X are expressed as a percentage of the values obtained in the same muscle in the absence of TTX. Each value is the mean ± S.E.M. N represents number of fibres examined.
Addition o f extract
None None Spinal cord Liver
* Differs from 3-day control, P < 0.01. ** P < 0.005.
180 action potential were obtained by using an RC derivating circuit (100 pF; 100 k~)). TTX was added to the bathing fluid at a concentration of 10-6 M. The resting membrane potential on the third day following denervation was reduced from 79.8 ± 1.36 mV (N ~ 20) to 56.7 ± 1.00 mV (N = 40), and this value was little changed for another 5 days of the period which we examined. The mean values of the maximal rate of rise and fall of action potential generated in innervated EDL were 622 ± 20.3 and 328 ± 19.3 V/sec (N = 20), respectively. After a 3-day denervation in vivo, the maximal rates of rise and fall of the action potential were reduced (Table I). These levels were little changed for another 5-day period. EDL denervated for 3 days in vivo was transferred into the culture condition and maintained for 3 days in vitro. In order to eliminate the muscle fibres which had been damaged during cultivation, fibres with resting potentials larger than --50 mV were selected for measurement of action potential generation. The maximal rates of rise and fall of action potential slightly decreased in the EDL cultured in the control medium (Table I). Whereas in the EDL cultured in the medium containing the spinal cord extract, both the maximal rates of rise and fall of action potential increased to 130 ~ of the 3-day control cultures (Table I). Furthermore, the maximal rate of rise of action potential was significantly higher after cultivation in the medium supplemented with the spinal cord extract for 3 days than it was on the third day following denervation (593 vs. 512, P < 0.005). On the other hand, the addition of liver extract to cultures only decreased the maximal rate of fall of action potential. TTX resistivity was expressed as the ratios of the values of the maximal rates of rise and fall of action potential in the presence of TTX (10 -6 M) to that in the absence of TTX. In freshly dissected normal EDL, the ratios of the maximal rates of rise and fall of the action potential were 5.4 ± 0.89 and 3.3 ~: 1.2 ~ (N = 15), respectively. The values increased on the third day following denervation bt vivo, and thereafter scarcely changed during cultivation in control medium (Table I). However, the addition of spinal cord extract significantly decreased TTX resistivity during cultivation (Table I). The addition of liver extract had no effect on TTX resistivity of cultured muscle (Table I). Our results show that spinal cord extract reversed the decrease in the maximal rates of rise and fall of action potential and its sensitivity to TTX which had been caused by the nerve section and by cultivation in control medium, and that liver extract was ineffective. These findings suggest the presence ofneurotrophic substance(s) in the spinal cord which regulate those properties of the muscle membrane which are responsible for generating the action potential. This interpretation supports a similar conclusion from in vitro experiments which showed that nerve extracts maintain biochemical (cholinesterase) and morphological properties of the muscle 5-s. We thank Profs. Y. Hagihara, M. Kano and Y. Shimada for valuable discussion and for preparing this manuscript.
181 1 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, ,4cta physiol, scand., 73 (1968) 471-480. 2 ALBUQUERQUE,E. X., AND WARNICK, J. E., The pharmacology of batrachotoxin. IV. Interaction with tetrodotoxin on innervated and chronically denervated rat skeletal muscle, J. Pharmacok exp. Ther., 180 (1972) 683-697. 3 ALBUQUERQUE,E. X., WARNICK, J. E., TASSE, J. R., AND SANSONE,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. 4 GUTH, L., 'Trophic' influences of nerve on muscle, Physiol. Rev., 48 (1968) 645-687. 5 LENTZ, T. L., Nerve trophic function: in vitro assay of effects of nerve tissue on muscle cholinesterase activity, Science, 171 (1971) 187-189. 6 LENTZ, T. L., Effect of brain extracts on cholinesterase activity of cultured skeletal muscle, Exp. Neurol., 45 (1974) 520-526. 7 OH, T. H., Neurotrophic effects: characterization of the nerve extract that stimulates muscle development in culture, Exp. NeuroL, 46 (1975) 432-438. 8 OH, T. H., JOHNSON,D. D., AND KIM, S. U., Neurotrophic effect on isolated chick embryo muscle in culture, Science, 178 (1972) 1298-1300. 9 REDFERN,P., LUNDH, H., AND THESLEFF,S., Tetrodotoxin resistant action potentials in denervated rat skeletal muscle, Europ. J. Pharmacol., l l (1970) 263-265. 10 REDFERN, P., AND TH~SLEFF, S., Action potential generation in denervated rat skeletal muscle. I. Quantitative aspects, Acta physiol, scand., 81 (1971) 557-564. 11 REDrERN, P., AND THESLEFF,S., Action potential generation in denervated rat skeletal muscle. I1. The action of tetrodotoxin, Acta physiol, scand., 82 (1971) 70-78. 12 WARE, F., JR., BENNETT,A. L., AND MCINTYRE,A. R., Membrane resting potential of denervated mammalian skeletal muscle measured in vivo, ,4mer. J. Physiol., 177 (1954) 115-118.
Bra#l Research, 100 (1975) 178-181 ~:) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Neurotrophic effect of spinal cord extract on membrane potentials of organcultured mouse skeletal muscle
HIROSHI KUROMI AND SHUJI HASEGAWA Brain Research Institute, School of Medicine, Chiba University, Chiba (Japan)
(Accepted August 26th, 1975)
Some membrane properties of skeletal muscle appear to be controlled by motoneurones 4. When mammalian skeletal muscle is denervated, the resting membrane potential and the maximal rates of rise and fall of action potential are reduced1,1°, le. In innervated muscle, generation of action potentials is completely blocked by tetrodotoxin (TTX), while in denervated muscle the action potential is resistant to TTX 2,9,11. The change in membrane properties of denervated muscle may be due to a loss of 'trophic' substance(s) which are normally transmitted from the motoneurone to the muscle 3. We examined the effects of addition of spinal cord extract on membrane properties of organ-cultured skeletal muscle. The extensor digitorum longus muscle (EDL) of male mice (20-25 g) was denervated by cutting the deep peroneal nerve close to the knee joint. Three days later the mice were anaesthetized with sodium pentobarbital (40 mg/kg, i.p.). The EDL was carefully removed and mounted on a U-shaped glass rod by binding its tendons to the ends of the rod (which were separated by 13 mm). It was then cultured for 3 days at 37 °C in a glass vial containing 4 ml of medium under an atmosphere of 95 % 02-5 ~,~ CO2. The medium consisted of 85 % Eagle's minimal essential medium and 15 % horse serum containing penicillin (50 U/ml) and streptomycin (50 #g/ml). The medium was changed after the first hour and thereafter every 24 h. Mouse spinal cord and liver were removed and homogenized with an equal volume of Tyrode's solution and centrifuged at 2000 × g for 10 rain. The supernatants were used as tissue extracts. An extract equivalent to 100 mg of the tissue wet weight was added to 4 ml of the culture medium. At the end of the culture period, E D L was mounted in a bath containing Eagle's minimal essential medium bubbled with 95 % 02-5 % CO2 at 37 °C, and the membrane properties of the muscle were investigated. Two glass microelectrodes filIed with 3 M KCI were inserted into the same surface muscle fibre about 50 #m apart; one was used for passing current and the other for recording the potential changes. Constant anodal current was passed through the electrode to locally polarize the muscle fibre from --85 to - - 9 0 inV. This current was interrupted for a 5 msec duration by applying a cathodal shock, adjusted in each case to produce an action potential with 1-3 msec latency. The rates of rise and fall of the
Ox
TABLE I in vivo
EFFECT OF TISSUE EXTRACT ON THE MAXIMAL RATES OF RISE AND FALL OF ACTION POTENTIAL GENERATED IN ORGAN-CULTURES OF MUSCLE THAT HAD BEEN PREVIOUSLY DENERVATED FOR 3 DAYS
Days in culture
0 3 3 3
N
40 27 25 29
± 1.00 -+- 1.20 ± 0.79 q- 0.69
Resting potential ( m Ix)
56.7 57.8 57.7 57.2
± 44±
15.6 18.2 18.1"* 16.1
Maximal rate o f rise (V/sec)
512 458 593 420
± 8.3 4- 9.5 4- 6.9** ± 4.9*
Maximal rate of fall (V/sec)
167 143 190 114
58.9 62.8 59.0 56.9
± ± ± ±
2.45 0.84 1.26 1.04
Resting potential (miX)
10 aM T T X N
32 30 26 25
67.7 60.0 37.2 64.5
± ± 44-
4.2 3.9 3.3** 3.0
Maximal rate o f rise (%)
79.5 76.9 38.0 68.9
-+- 6.9 ± 6.8 4- 4.4** ± 4.6
Maximal rate of fall (%)
The rates in the presence of T T X are expressed as a percentage of the values obtained in the same muscle in the absence of TTX. Each value is the mean ± S.E.M. N represents number of fibres examined.
Addition o f extract
None None Spinal cord Liver
* Differs from 3-day control, P < 0.01. ** P < 0.005.
180 action potential were obtained by using an RC derivating circuit (100 pF; 100 k~)). TTX was added to the bathing fluid at a concentration of 10-6 M. The resting membrane potential on the third day following denervation was reduced from 79.8 ± 1.36 mV (N ~ 20) to 56.7 ± 1.00 mV (N = 40), and this value was little changed for another 5 days of the period which we examined. The mean values of the maximal rate of rise and fall of action potential generated in innervated EDL were 622 ± 20.3 and 328 ± 19.3 V/sec (N = 20), respectively. After a 3-day denervation in vivo, the maximal rates of rise and fall of the action potential were reduced (Table I). These levels were little changed for another 5-day period. EDL denervated for 3 days in vivo was transferred into the culture condition and maintained for 3 days in vitro. In order to eliminate the muscle fibres which had been damaged during cultivation, fibres with resting potentials larger than --50 mV were selected for measurement of action potential generation. The maximal rates of rise and fall of action potential slightly decreased in the EDL cultured in the control medium (Table I). Whereas in the EDL cultured in the medium containing the spinal cord extract, both the maximal rates of rise and fall of action potential increased to 130 ~ of the 3-day control cultures (Table I). Furthermore, the maximal rate of rise of action potential was significantly higher after cultivation in the medium supplemented with the spinal cord extract for 3 days than it was on the third day following denervation (593 vs. 512, P < 0.005). On the other hand, the addition of liver extract to cultures only decreased the maximal rate of fall of action potential. TTX resistivity was expressed as the ratios of the values of the maximal rates of rise and fall of action potential in the presence of TTX (10 -6 M) to that in the absence of TTX. In freshly dissected normal EDL, the ratios of the maximal rates of rise and fall of the action potential were 5.4 ± 0.89 and 3.3 ~: 1.2 ~ (N = 15), respectively. The values increased on the third day following denervation bt vivo, and thereafter scarcely changed during cultivation in control medium (Table I). However, the addition of spinal cord extract significantly decreased TTX resistivity during cultivation (Table I). The addition of liver extract had no effect on TTX resistivity of cultured muscle (Table I). Our results show that spinal cord extract reversed the decrease in the maximal rates of rise and fall of action potential and its sensitivity to TTX which had been caused by the nerve section and by cultivation in control medium, and that liver extract was ineffective. These findings suggest the presence ofneurotrophic substance(s) in the spinal cord which regulate those properties of the muscle membrane which are responsible for generating the action potential. This interpretation supports a similar conclusion from in vitro experiments which showed that nerve extracts maintain biochemical (cholinesterase) and morphological properties of the muscle 5-s. We thank Profs. Y. Hagihara, M. Kano and Y. Shimada for valuable discussion and for preparing this manuscript.
181 1 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, ,4cta physiol, scand., 73 (1968) 471-480. 2 ALBUQUERQUE,E. X., AND WARNICK, J. E., The pharmacology of batrachotoxin. IV. Interaction with tetrodotoxin on innervated and chronically denervated rat skeletal muscle, J. Pharmacok exp. Ther., 180 (1972) 683-697. 3 ALBUQUERQUE,E. X., WARNICK, J. E., TASSE, J. R., AND SANSONE,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. 4 GUTH, L., 'Trophic' influences of nerve on muscle, Physiol. Rev., 48 (1968) 645-687. 5 LENTZ, T. L., Nerve trophic function: in vitro assay of effects of nerve tissue on muscle cholinesterase activity, Science, 171 (1971) 187-189. 6 LENTZ, T. L., Effect of brain extracts on cholinesterase activity of cultured skeletal muscle, Exp. Neurol., 45 (1974) 520-526. 7 OH, T. H., Neurotrophic effects: characterization of the nerve extract that stimulates muscle development in culture, Exp. NeuroL, 46 (1975) 432-438. 8 OH, T. H., JOHNSON,D. D., AND KIM, S. U., Neurotrophic effect on isolated chick embryo muscle in culture, Science, 178 (1972) 1298-1300. 9 REDFERN,P., LUNDH, H., AND THESLEFF,S., Tetrodotoxin resistant action potentials in denervated rat skeletal muscle, Europ. J. Pharmacol., l l (1970) 263-265. 10 REDFERN, P., AND TH~SLEFF, S., Action potential generation in denervated rat skeletal muscle. I. Quantitative aspects, Acta physiol, scand., 81 (1971) 557-564. 11 REDrERN, P., AND THESLEFF,S., Action potential generation in denervated rat skeletal muscle. I1. The action of tetrodotoxin, Acta physiol, scand., 82 (1971) 70-78. 12 WARE, F., JR., BENNETT,A. L., AND MCINTYRE,A. R., Membrane resting potential of denervated mammalian skeletal muscle measured in vivo, ,4mer. J. Physiol., 177 (1954) 115-118.