Development of Excitability in Embryonic Chick Skeletal Muscle Cells MASAAKIRA KANO Department of Physiology, School of Medicine, Xitasato University, Sagamihara, Kanagawa, J a p a n
ABSTRACT During embryonic and early postnatal development, the chick leg muscle cells undergo a series of changes in their electrical responses in the following sequence : passive response, plateau response, plateau plus spike response and spike response. This suggests that the electrogenetic mechanism of muscles matures during development; a mechanism producing the plateau may first be induced, and then that producing the spike. The plateau is sensitive to manganese or cobalt ions, while the spike to tetrodotoxin. This suggests that the plateau is related to the increase in permeability to calcium ions, while the spike to sodium ions.
The initiation of action potentials is one horn) were incubated at 38°C. The emof the unique features of nerve and muscle bryonic limbs were removed at various cell membranes. The electrical activity of stages of development from 13 days to 21 mature chick skeletal muscle cells is char- days (hatching) ; some experiments were acterized by short-duration spike potential; done on young chicks after hatching. The this is true even in multiple-innervated muscles of the crus, digital flexor group, slow muscle cells (Ginsborg, '60). In cul- were used. They were held in a chamber tured skeletal muscle cells of both the by gripping with a pair of forceps at atchick and rat, another type of electrical tached bones and tendons, bathed in oxypotential, the plateau potential, has also been elicited (Li et al., '59; Kano et al., genated saline (154.0 mM NaC1, 5.6 mM '72; Kano and Shimada, '73; Kidokoro, KC1, 2.2 mM CaC1, and 5 mM Tris-HC1, '73). Thus a wide spectrum of responses pH 7.4), and maintained at 2 5 r l " C . Muscle cells were penetrated with a sinhas been observed in different cultured muscle cells. These include passive, pla- gle glass microelectrode, filled with 3 M teau, plateau plus spike, and spike re- KC1 and having a resistance of 30 to 100 sponses. It has been supposed that this MR. The microelectrode was mounted in variety of electrogenesis may result from the input stage of the modified bridge cirdifferences i n maturity and/or special en- cuit so that it could be used simultanevironment produced by culture (Kano et ously to pass current and to record voltage (Takahashi, '64). Current and potential al., '72). The present study is a n extension of the across the cell membrane were monitored previous studies on the cultured chick with a penwriter and displayed on a n osskeletal muscle cells (Kano et al., '72; cilloscope. The current pulses were about Kano and Shimada, '73); this was under- 100 msec in duration and were usually taken mainly in order to follow up the de- delivered every six to ten seconds. velopment of electrogenesis in the intact RESULTS skeletal muscle cells during ontogeny. The When the microelectrode penetrated the results indicated that the same variety of electrogenesis was encountered during de- muscle cells, a n abrupt change in potenvelopment of skeletal muscle cells in vivo tial was obtained. The recorded potential often declined gradually to a lower steady as that in those in vitro. value but usually remained steady at the MATERIALS AND METHODS initial value for at least first several tens Fertilized chicken eggs (White LegReceived Dec. 19, '74. Accepted Mar. 24, '75. J. CELL. PHYSIOL., 86: 503-510.
503
504
MASAAKIRA KANO
of seconds following penetration. In some cases the potential increased over a short period following penetration and reached to a steady value (fig. 3C). Resting membrane potentials were therefore measured as the amplitude of the initial voltage deflection (Boethius and Knutsson, '70) or the highest value reached. The mean resting membrane potential increased during embryonic and early postnatal development, as shown in figure 1. A marked increase took place between the fifteenth and twentieth embryonic days, from about --40 to about -60 mV, and thereafter the rate of increase was more gradual. Exactly the same data has been reported by Boethius and Knutsson ('70) for developing thigh muscle cells of the chick. The impaled cells were stimulated with pulses of outward current delivered through the recording microelectrode. There were occasions in which no apparent irreversible damage to the muscle cell was observed after penetration by the microelectrode and stimulation through it. In these cases, as stated above, the membrane
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potential remained steady at the initial value or increased to a higher steady value. The excitable properties of the membrane were tested in these adequately penetrated cells. Recording periods ranged from the usual of several minutes to more than 30 minutes. Of 1,177 cells that were impaled, 765 could be tested. Various types of responses were recorded from the muscle cells at different stages of development (fig. 2 ) . They could be classified into four types according to the presence or absence of plateau and/or spike potentials : passive (the response without electrical activity, fig. 2 A ) , plateau (the response consistng of a plateau potenial alone, fig. 2B), plateau plus spike (the response consisting of plateau and spike potentials, fig. 2C, D ) , and spike (the response consisting of spike potential alone, fig. 2E). This variety of responses is similar to that which has been observed in the cultured chick skeletal muscle cells (Kano et al., '72). Each response recorded from a given muscle cell was fairly constant in size and shape, although variations were found from one cell to another.
*
-
12 20 -
40
"
A
0
13
hatching
4
15
17
19
21
2
4
6
8
10
12
14
16
18
days Fig. 1 The resting potential as a function of developmental age. The values are mean SD. Numbers in parentheses represent number of muscle cells examined. *The differences from the value in 13-day embryos are significant with P 0.001; **P 0.01; ***P 0.05. -t
ABSTRACT During embryonic and early postnatal development, the chick leg muscle cells undergo a series of changes in their electrical responses in the following sequence : passive response, plateau response, plateau plus spike response and spike response. This suggests that the electrogenetic mechanism of muscles matures during development; a mechanism producing the plateau may first be induced, and then that producing the spike. The plateau is sensitive to manganese or cobalt ions, while the spike to tetrodotoxin. This suggests that the plateau is related to the increase in permeability to calcium ions, while the spike to sodium ions.
The initiation of action potentials is one horn) were incubated at 38°C. The emof the unique features of nerve and muscle bryonic limbs were removed at various cell membranes. The electrical activity of stages of development from 13 days to 21 mature chick skeletal muscle cells is char- days (hatching) ; some experiments were acterized by short-duration spike potential; done on young chicks after hatching. The this is true even in multiple-innervated muscles of the crus, digital flexor group, slow muscle cells (Ginsborg, '60). In cul- were used. They were held in a chamber tured skeletal muscle cells of both the by gripping with a pair of forceps at atchick and rat, another type of electrical tached bones and tendons, bathed in oxypotential, the plateau potential, has also been elicited (Li et al., '59; Kano et al., genated saline (154.0 mM NaC1, 5.6 mM '72; Kano and Shimada, '73; Kidokoro, KC1, 2.2 mM CaC1, and 5 mM Tris-HC1, '73). Thus a wide spectrum of responses pH 7.4), and maintained at 2 5 r l " C . Muscle cells were penetrated with a sinhas been observed in different cultured muscle cells. These include passive, pla- gle glass microelectrode, filled with 3 M teau, plateau plus spike, and spike re- KC1 and having a resistance of 30 to 100 sponses. It has been supposed that this MR. The microelectrode was mounted in variety of electrogenesis may result from the input stage of the modified bridge cirdifferences i n maturity and/or special en- cuit so that it could be used simultanevironment produced by culture (Kano et ously to pass current and to record voltage (Takahashi, '64). Current and potential al., '72). The present study is a n extension of the across the cell membrane were monitored previous studies on the cultured chick with a penwriter and displayed on a n osskeletal muscle cells (Kano et al., '72; cilloscope. The current pulses were about Kano and Shimada, '73); this was under- 100 msec in duration and were usually taken mainly in order to follow up the de- delivered every six to ten seconds. velopment of electrogenesis in the intact RESULTS skeletal muscle cells during ontogeny. The When the microelectrode penetrated the results indicated that the same variety of electrogenesis was encountered during de- muscle cells, a n abrupt change in potenvelopment of skeletal muscle cells in vivo tial was obtained. The recorded potential often declined gradually to a lower steady as that in those in vitro. value but usually remained steady at the MATERIALS AND METHODS initial value for at least first several tens Fertilized chicken eggs (White LegReceived Dec. 19, '74. Accepted Mar. 24, '75. J. CELL. PHYSIOL., 86: 503-510.
503
504
MASAAKIRA KANO
of seconds following penetration. In some cases the potential increased over a short period following penetration and reached to a steady value (fig. 3C). Resting membrane potentials were therefore measured as the amplitude of the initial voltage deflection (Boethius and Knutsson, '70) or the highest value reached. The mean resting membrane potential increased during embryonic and early postnatal development, as shown in figure 1. A marked increase took place between the fifteenth and twentieth embryonic days, from about --40 to about -60 mV, and thereafter the rate of increase was more gradual. Exactly the same data has been reported by Boethius and Knutsson ('70) for developing thigh muscle cells of the chick. The impaled cells were stimulated with pulses of outward current delivered through the recording microelectrode. There were occasions in which no apparent irreversible damage to the muscle cell was observed after penetration by the microelectrode and stimulation through it. In these cases, as stated above, the membrane
60 80
potential remained steady at the initial value or increased to a higher steady value. The excitable properties of the membrane were tested in these adequately penetrated cells. Recording periods ranged from the usual of several minutes to more than 30 minutes. Of 1,177 cells that were impaled, 765 could be tested. Various types of responses were recorded from the muscle cells at different stages of development (fig. 2 ) . They could be classified into four types according to the presence or absence of plateau and/or spike potentials : passive (the response without electrical activity, fig. 2 A ) , plateau (the response consistng of a plateau potenial alone, fig. 2B), plateau plus spike (the response consisting of plateau and spike potentials, fig. 2C, D ) , and spike (the response consisting of spike potential alone, fig. 2E). This variety of responses is similar to that which has been observed in the cultured chick skeletal muscle cells (Kano et al., '72). Each response recorded from a given muscle cell was fairly constant in size and shape, although variations were found from one cell to another.
*
-
12 20 -
40
"
A
0
13
hatching
4
15
17
19
21
2
4
6
8
10
12
14
16
18
days Fig. 1 The resting potential as a function of developmental age. The values are mean SD. Numbers in parentheses represent number of muscle cells examined. *The differences from the value in 13-day embryos are significant with P 0.001; **P 0.01; ***P 0.05. -t
