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.

Electrogenesis of embryonic chick skeletal muscle cells differentiated in monolayer cultures was investigated. Muscle fibers in uitro generate spike potentials similar to those of fibers in wino. However, other responses, plateaux resembling those i n heart muscle, are also elicited. These results suggest that a functional differentiation exists i n cultured muscle fibers.

The embryonic chick skeletal muscle cells differentiated in cell culture from trypsin-dissociated myoblasts produce a spike response which is tetrodotoxin-sensitive. It has been found that many cells also produce a plateau response which is resistant to tetrodotoxin. The plateau response frequently occurs even in the muscle cells which do not normally exhibit the spike response. During the plateau response membrane resistance is greatly reduced below its resting value. The current-voltage relation in muscle cells with the plateau response is always S-shaped. It is suggested that the plateau arises from a voltage-dependent increase in permeability to external cations whose influx produce the maintained depolarization, and from low level of repolarizing potassium outflux. The plateau response is sensitive to manganese ions. This finding, together with resistibility to tetrodotoxin, suggests that calcium ions are the dominant carriers for the depolarizing current.The electrical activity of mature chick skeletal muscle fibers in vivo is characterized by short-duration spike potential; this is true even in multiple-innervated slow muscle fibers (Ginsborg, '60). Similar spike potentials could be elicited in chick muscle fibers both in organ (Li et al., '59) and cell culture (Fischbach et al., '71; Kano et al., '72). In cultured chick muscle fibers, another type of electrical potential, the plateau potential, has also been elicited (Li et al., '59; Kano et al., '72). It has been observed that the plateau potentials of muscle fibers in cell culture are resistant to tetrodotoxin (Kano et al., '72). The present paper reports the characteristics of the plateau elicited in chick skeletal muscle fibers differentiated in vitro from trypsin-dissociated myoblasts. MATERIALS AND METHODSTissue culture. Suspensions of myoblasts were obtained from thigh muscle of 12-day chick embryos by standard trypsinization procedures (Moscona, '61). The cells were dispersed in the following culture medium: Eagle's minimum essential medium with glutamine, 10 % horse serum, 10% embryo extract, and penicillinstreptomycin in concentration of 50 units and 50 pglml, respectively. For monolayer cultures, the cells in suspension were inoculated onto collagen-coated (Bornstein, '58) plastic culture dishes. Inoculation densities ranged from 105 to 106 cells per 35 or 60 mm dish. Cultures were maintained at 37" C in a 5 % COz-air atmosphere saturated with water. The culture medium was changed every two days.Electrophysiological arrangements. Prior to electrophysiological experiments, the medium was decanted and the cultures were washed twice in Tyrode's solution. Each culture dish received 2 (35 mm dish) or 6 ml (60 mm dish) of Tyrode's solution and was kept at about 36°C during the experiments. A muscle fiber was penetrated under visual control with two glass microelectrodes filled with 3M-KCl; one was used for recording membrane potentials and the other for passing pulses of current through the fiber membrane. The microelectrode resistances ...

Trypsin-dissociated myoblasts from leg muscle of 12-day chick embryos have been cultured in monolayers. After four days the muscle cultures have been confronted with fragments of the spinal cord of six-day chick embryos.Electrophysiological and morphological analysis demonstrate that characteristic neuromuscular transmission can develop in these cultures. Electrical stimulation of the cord fragment evokes contractions of innervated muscle fibers, from which end plate potentials and miniature end plate potentials with average frequency around one per second or more can be recorded. D-tubocurarine (1 Irgl ml) suppresses reversibly these synaptic potentials.Non-innervated muscle fibers are sensitive to acetylcholine over all their surface, while innervated muscle fibers are sensitive at the regions where structures suggestive of motor end plate ("bulb-type") are found.We can conclude that neuromuscular connections developed in vitro in our experiments are functional in respect of transmission of impulses but also in respect of neurotrophic influences for restriction of chemosensitivity.

In the presence of tetraethylammonium or barium ions, the larval muscle fibers of Drosophila melanogaster were found to produce an all-or-none action potential operated by the calcium channels. The development of this distinctive membrane property during the maturation of muscle cells was studied by measuring the maximum rate of rise of the action potential in the larval muscle fibers at different stages of development from the sixteenth to ninety-sixth hours after hatching. The value increased significantly with age until a peak was reached at the sixty-fourth hour, although it became lower again as puparium formation neared at about the ninety-sixth hour. This suggests that during larval development the muscle fibers develop the ability to generate an action potential due to an inward current through the calcium channels, although the ability became lower at the later stage of larval development.