MINIATURE EXCITATORY JUNCTION POTENTIALS IN THE SOMATIC MUSCLE OF THE EARTHWORM, PHERETIMA COMMUNISSIMA, IN SODIUM FREE SOLUTION

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1 J. Exp. Biol. (1969), 50, With 11 textfigures Printed in Great Britain MINIATURE EXCITATORY JUNCTION POTENTIALS IN THE SOMATIC MUSCLE OF THE EARTHWORM, PHERETIMA COMMUNISSIMA, IN SODIUM FREE SOLUTION Y. ITO, H. KURIYAMA AND N. TASHIRO Department of Physiology, Faculty of Medicine and Dentistry, Kyushu University, Fukuoka, Japan {Received 8 October 1968) INTRODUCTION Neuromuscular transmission of excitation in invertebrates has been studied extensively by many investigators (cf. Bullock & Horridge, 1963). Miniature inhibitory junction potentials (m.i.j.p.s) and miniature excitatory junction potentials (m.e.j.p.s) could be recorded from the longitudinal layer of the somatic muscle in the earthworm. Field stimulation to the peripheral nerves elicited inhibitory junction potentials (i.j.p.s) and excitatory junction potentials (e.j.p.s). Generation of the m.i.j.p. and i.j.p. was blocked by treatment with picrotoxin (icr 6 g./ml.) and the equilibrium potentials for them ranged between 52 and 58 mv. The m.e.j.p.s and e.j.p.s were blocked by dtubocurarine (io~ 6 g./ml.) and the equilibrium potential for them was o mv. The former was thought to be due to release of yaminobutyric acid (GABA) from the peripheral inhibitory nerves and to selective increase of chloride permeability. The latter was thought to be due to release of acetylcholine which increases the sodium and potassium permeabilities of the postsynaptic muscle membrane. Generation of the m.e.j.p. was less common than that of the m.i.j.p., in spite of the double innervation to the muscle fibres. However, when the external sodium (Na) 0 was replaced with (Tris) 0, the membrane was hyperpolarized from 35 mv. to 55 mv. and frequently generated the m.e.j.p. (Hidaka et al. 1969b, c). The present experiments were intended to investigate the ionic mechanism involved in generation of the m.e.j.p.s in sodiumfree solution, and the results indicated that, in sodiumfree solution, generation of the m.e.j.p in a depolarizing direction is due mainly to increase in calcium permeability and, to a lesser extent, to increase in potassium permeability, brought about by the release of acetylcholine from the nerve terminals. METHOD The longitudinal layer of the somatic muscle of the earthworm, Pheretima cotnmunissima, 58 cm. in length, was used. The earthworm was pinned on the plate and dissected from the dorsal side along its length. The alimentary canal was carefully dissected from the body wall and removed. A portion of the remaining tissue, io 1 5 cm. in length, was fixed on a rubber plate with pins. The tissue was immersed in an organ bath, through which solutions at room temperature flowed continuously.

2 io8 Y. ITO, H. KURIYAMA AND N. TASHIRO The normal solution (Ringer's solution) used for this tissue was of the following composition: Na, 140 mm; K, 27 mm; Ca, i8 mm; Mg, io mm; Cl, 1483 HIM; and ph was adjusted from 73 to 75. The microelectrode was used for making the electrical recording as well as for stimulating by means of the Wheatstone bridge method. The range of the applied current was between io~ 10 and 5 x io~ 9 A. The microelectrodes were filled with 3 MKC1 for the measurement of the various properties of the membrane, and were filled with 2 M potassium citrate for the measurement of the reversal potential for the m.e.j.p. Sodiumfree solution was prepared using Tris(hydroxymethyl)aminomethane (C 4 H 11 NO 3 ) to maintain isosmoticity. The solution was titrated with a high concentration of HC1, and the ph was adjusted to 74. The general procedures of the experiment were the same as those described by Hidaka, Ito & Kuriyama (1969 a) and Hidaka et al. (1969J, c). The experiments were carried out through the year. However, during the winter season (NovemberMarch) the effective resistance of the muscle fibres in normal solution was nearly twice that measured during the summer season (AprilOctober). RESULT Generation of m.e.j.p. The membrane potential of muscle membrane was very low. However, the sodiumfree solution (sodium was substituted by Tris) hyperpolarized the membrane from 36 mv. (s.e. = ±05, n = 50) to ~54mV. (S.E. = ±o6, n = 50), and increased Control Nafree (Tris) Ca 2+ 2 mm 2x10"'A. r0 L 50 mv. 500 msec. Fig. 1. Effects of pulses of current delivered to the single musclefibreby the Wheatstone bridge method in normal and sodiumfree (Tris) solution, (a) Control; (6) sodiumfree solution. the input resistance of the membrane from 42 MQ (s.e. = + II, n = 20) to 63 MQ (s.e. = + O'9, n = 30) during the summer season. Figure 1 shows the effects of pulses of current delivered to the muscle membrane in normal and in sodiumfree solution. The inward and outward currents were applied to the fibres through a Wheatstone bridge. In both the solutions the spike with overshoot potential could be elicited, but

3 Junction potentials in the earthworm 109 (a) m.e.j.p. in sodiumfree solution (b) dtubocurarine 10"* g./ml. (c) After rinsed by sodiumfree solution r 50 mv. 500 msec. Fig. a. Effects of dtubocurarine (io~ 6 g./ml.) on the miniature excitatory junction potentials (m.e.j.p.s). (a) Control: sodiumfree solution; (6) dtubocurarine in sodiumfree solution (after 5 min.); (c) after washing out with sodiumfree solution Amplitude of m.e.j.p. 40 Frequency of 1 m.e.j.p i so 40 i j \ \ Tin t i f» I? 1 *» mv. 10 \ \ \ vucn, i Frequency/5 sec. Fig. 3. Distributions of amplitude and frequency of m.e.j.p.s measured from the single muscle fibre in sodiumfree solution. Continuous line shows the theoretical Poisson curve calculated from the measured values.

4 no Y. ITO, H. KURIYAMA AND N. TASHIRO afterhyperpolarization of the membrane was not observed in sodiumfree solution due to the hyperpolarization of the membrane. In sodiumfree solution the spontaneously generated miniature depolarizing potentials could be recorded more frequently than those in the normal solution. These small potential changes in the resting membrane were not influenced by treatment with picrotoxin (IO~ 6 IO~ 5 g./ml.) and atropine (IO~ 7 IO~ 6 g./ml.). However, treatment with dtubocurarine (IO~ 6 IO~ 5 g./ml.) completely abolished them. Figure 2 Nafree Nafree +10mV. hyper. Nafree +20 mv. hyper. 20 mv. 05 sec. Fig. 4. Generation of m.e.j.p.s in sodiumfree solution, (a) Sodiumfree solution; (6) 10 mv. hyperpolarization by inward current in sodiumfree solution; (c) 20 mv. hyperpolarization by inward current in sodiumfree solution. shows the effect of dtubocurarine (io~ 6 g./ml.) on the the generation of the miniature depolarizing potentials. Therefore, these potential changes are presumably m.e.j.p.s. The spontaneous spike discharges and the spikes elicited by the intracellular depolarizing currents could be observed in sodiumfree solution because the spike is due to inward movement of calcium ions during the active state of the membrane (Hidaka et al ). The amplitude and distribution of m.e.j.p.s measured from the single cell are shown in Fig. 3. The amplitude and frequency of the m.e.j.p.s varied in individual fibres. In this particular fibre, the mean amplitude, measured from larger than 05 mv,. was i8 mv.; the mean frequency was o34/sec. The distribution curve for the frequency of m.e.j.p.s agreed with the theoretical Poisson distribution as shown in Fig. 3, thus indicating the random generation of the m.e.j.p.s in sodiumfree solution as observed in the sodiumcontaining solution. The skew curve observed in the distribution of the amplitude might indicate a diffuse innervation of the excitatory nerve terminals to the muscle fibres. The effects of hyperpolarization of the

5 Junction potentials in the earthworm 111 muscle membrane on the amplitude and frequency of the m.e.j.p.s in sodiumfree solution were observed. Since the intensityvoltage relation of the membrane was linear in the ranges of the membrane potential from 90 mv. to 20 mv., this indicated that no change of the input resistance of the membrane occurred in the above ranges of the potential changes. Amplitude (50 sec.) Nafree solution Mean 19 mv. Nafree solution +10 mv. hyperpolarization Mean 23 mv. Nafree solution +20 mv. h/perpolarization _n Mean 28 mv. 80 z i I I I I I I i e I/I t « mv. mv. mv. Fig. 5. Histograms of the amplitude of m.e.j.p.s generated within 50 msec, in sodiumfree solution, (a) Sodiumfree solution; (b) 10 mv. hyperpolarization by inward current in sodiumfree solution; (c) 20 mv. hyperpolarization by inward current in sodiumfree solution. 159 Figure 4 shows the appearance of the m.e.j.p.s in sodiumfree solution with 10 mv. and 20 mv. hyperpolarization of the membrane. Figure 5 shows the histograms of the amplitude of the m.e.j.p.s which appeared within 50 sec. in the sodiumfree solution and with both 10 mv. and 20 mv. hyperpolarization of the membrane, and Fig. 6 shows the histograms of appearance of the m.e.j.p. within 50 sec. at the different membrane potential levels. The hyperpolarizing currents were applied as intracellular polarizing currents in the sodiumfree solution. To prevent the generation of m.i.j.p.s the tissue was previously treated with picrotoxin (io~ 6 g./ml.). Because the reversal potential level for the m.i.j.p.s was about 55 mv. and conditioning hyperpolarization of the membrane was up to 60 mv., the polarity of the m.i.j.p.s was reversed to the same direction as that of the polarity of the m.e.j.p.s. In this particular cell the mean m.e.j.p. amplitude was 1*9 mv. and the mean frequency was 4/8/sec. in the sodiumfree solution. When the membrane was hyperpolarized from 55 to 65 mv., the mean amplitude and frequency were increased to 23 and 6o mv./sec. respectively, and when the membrane was hyperpolarized to 75 mv. these values increased to 28 and 76 mv./sec. respectively. Increased frequency of the m.e.j.p.s in the hyperpolarized membrane might be due

6 ii2 Y. ITO, H. KURIYAMA AND N. TASHIRO to the increased driving force of the membrane, thus inducing the increased appearance of the m.e.j.p.s which were previously lost in the noise level. When the mean amplitude of the m.e.j.p. was plotted against the membrane potentials, the reversal potential level could be estimated from the extrapolated line, and this value was calculated to be 16 mv. from Fig. 5. On the other hand, when the reversal potential level for the m.e.j.p. was measured by the application of the inward current, this value was 20 mv. (s.e. = + 12, n = 11). The difference in the results obtained from these two methods was not significant. However, the main explanation _ ' ' Nafree solution r i Mean 48/sec. 1 4 / A, No./sec. (b) _ Appearance2 (50 sec.) ' Nafree solution + 10 mv. hyperpolarization Mean 6/sec. J n i No./sec. ' c ' Nafree solution +20 mv. hyperpolarization _ Mean 76/sec. r r 1 I fl J "..rf... In No./sec. Fig. 6. Histograms of the appearances of m.e.j.p.s within 50 msec, in sodiumfree solution, (a) Sodiumfree solution; (6) 10 mv. hyperpolarization by inward current in sodiumfree solution; (c) 20 mv. hyperpolarization by inward current in sodiumfree solution. for the low value of the reversal potential level measured by the conditioning hyperpolarization of the membrane might be the appearance of the small m.e.j.p.s which were previously lost in the noise level. Effect of acetylcholine on the input resistance and membrane potential In the previous paper Hidaka et al. (1969c) thought that the ionic mechanism involved in the generation of the m.e.j.p. and e.j.p. was centred upon increase of G Na and G K, because they could not measure the excitatory junction current by the voltageclamp method, but in the sodiumfree solution reversal potential level for the m.e.j.p. shifted from 0 mv. (s.e. = ± 14, n = 5) in the normal solution to 20 mv. (s.e. = ±i*2, n = 11). In the present experiment we observed that, even in the sodiumfree solution, acetylcholine (io~ 6 g./ml.) reduced the membrane resistance, and pretreatment with dtubocurarine (io~ 6 g./ml.) prevented this reduction of the membrane resistance caused by treatment with acetylcholine. Figure 7 shows effects of acetylcholine alone and acetylcholine together with dtubocurarine on the intensityvoltage relation of the membrane under application of the weak hyperpolarizing currents (io~ 10 2 x io~ 9 A). The graph shows the results obtained from six different

7 Junction potentials in the earthworm 113 experiments. Treatment with acetylcholine (io~ 6 g./ml.) consistently reduced the input resistance of the membrane and pretreatment with dtubocurarine (io~ 6 g./ml.) prevented this. This increased membrane conductance could not be explained by increased potassium conductance alone, because the membrane was slightly depolarized by treatment with acetylcholine (io~ 6 g./ml.) from 512 mv. (S.E. = ±o6, n = 25) to 482 mv. (S.E. = + 07, n = 25), thus indicating that increased permeability to another ion, namely calcium ion, must be involved. However, chloride ion is probably not involved, because (i) picrotoxin (IO~ 6 IO~ 5 g./ml.) did not influence the reversal potential level for the m.e.j.p.s, and (ii) in 56 mm chloride solution ((Cl) 0 was substituted by dglutamate) the reversal potential level for the m.e.j.p.s remained the same as in normal solution. 10 l T ^^~ (a) (b) 10 o'a 10 ACh 10"' g./ml. ^ o o^0 o Control / oo _ _o t # * 0 0o o O o0 0 * $ A. O ** w O DTC 10 6 g./ml. 0 Control b Fig. 7. Currentvoltage relation of the muscle fibres measured in the presence of acetylcholine (io~ fl g./ml.) alone and in the presence of acetylcholine (io~ 6 g./ml.) together with Dtubocurarine (io" 0 g./ml.). Six different fibres were used, (a) Control (O), acetylcholine (O); (6) control (O), effects of acetylcholine in the pretreatment with dtubocurarine ( ). Effects of calcium and potassium ions on the reversal potential for m.e.j.p. Effects of various concentrations of calcium ion on the reversal potential level for the m.e.j.p. were observed in the presence of picrotoxin (icr 5 g./ml.). In the calciumdeficient solution (i, 05, 02 mm) the muscle membranes were depolarized from 352 mv. (S.E. = ±o8, n = 25) to 324 mv. (S.E. = ±09, n = 25), to 29 mv. (S.E. = +o8, n = 25) and to 21 mv. (S.E. = ±o6, n = 25) respectively, and the membrane resistances were reduced from 68 MQ (S.E. = + 24, n = 6) to 54 MQ (S.E. = ± 25, n = 8), to 42 MQ (S.E. = + 23, n = 10) and to 37 MQ, (S.E. = ±21, n = 8) respectively. However, in the sodiumfree solution calcium concentrations ranging from five times to one fifth the normal concentration change neither the input resistance of the membrane nor the membrane potential. Furthermore, the intensityvoltage relation observed by the intracellular polarizing method under various calcium concentrations 8 Exp. Biol. 51, 1

8 114 Y. ITO, H. KURIYAMA AND N. TASHIRO in the sodiumfree solution showed no rectification by the membrane within ranges of membrane potential from 90 to 20 mv. Excess calcium concentrations lowered the reversal potential level from 20 mv. (s.e. = +12, n = 11) with 2 mm calcium ion to 13 mv. (s.e. = + 14, n = 10) > mm Kconcentration ocaconcentration Fig. 8. Reversal potential level for the m.e.j.p.s under various calcium (O) and potassium ( ) concentrations in sodiumfree solution. +20 T msec Fig. 9. Effects of intracellular polarization on the amplitude and polarity of the m.e.j.p.s in sodiumfree solution. Continuous line indicates extracellular potential level. KC1, 271HM; CaCl 2, 2 mm. Note: m.e.j.p. elicited the spike with overshoot. with 5 mm calcium ion. When the external calcium was reduced to one fifth of the normal concentration the reversal potential it was increased up to 36 mv. (s.e. = ± 05, n = 5). The change of the reversal potential produced by a tenfold change of the external calcium concentration was estimated to be 17 mv. in a solution containing 27 mm potassium. Figure 8 shows the reversal potentials for the m.e.j.p. under the,

9 Junction potentials in the earthworm 115 various external concentrations of calcium and potassium ions. Horizontal bars show twice the s.d. When the external potassium was reduced in the presence of picrotoxin (io~ 6 g./ml.), the reversal potential level shifted in a more negative direction, i.e. in the solution containing one tenth the normal potassium concentration ( , Fig. 10. Legend as for Fig. 9. KC1 and CaCl s are 054 and 2 mm respectively msec Fig. ii. Legend as for Fig. 9. KC1 and CaCl 2 are 2 # 7 and 5 mm respectively. the reversal potential level was measured to be 445 mv. (s.e. = ±24, n = 5). The change of the reversal potential level produced by a tenfold change of the external potassium concentration was 245 mv. in solutions containing 2 mm calcium. Figures 911 show the actual reversal potential levels for the m.e.j.p. measured in sodiumfree solution, i.e. Fig. 9 shows the reversal potential level for the m.e.j.p. measured in 2 mm calcium and 27 mm potassium by application of the intracellular polarizing currents, in 2 mm calcium and 0*54 mm potassium (Fig. 10) and in 5 mm Lcalcium and 27 mm potassium solutions (Fig. 11). During these experiments, to 82

10 u6 Y. ITO, H. KURIYAMA AND N. TASHIRO prevent the generation of repetitive spikes by the depolarization of the membrane during the application of the currents, the current was applied to the fibre with the minimal gradient. The shift of the reversal potential level in one tenth normal potassium concentration was at 24 mv., and in ten times normal calcium concentration was at 17 mv. DISCUSSION According to the results obtained by electron microscopic examination the longitudinal somatic muscle (diameter; 510/i, length; 1 mm.) of the earthworm is comparable with the striated muscle of vertebrates in that both contain interdigitating arrays of thick and thin filaments. The muscle of the earthworm might be called obliquely striated muscle to distinguish it from striated muscle and unstriated muscle. The cell membrane showed halfdesmosome or desmosome structure in several places but no tight junction was observed with the neighbouring cells. (Kawaguti & Ikemoto, 1958; Ikemoto, 1963; Nishihara, 1967). The muscle fibres were innervated by excitatory nerves which contain many small vesicles (diam A) and by inhibitory nerves which contain many cored vesicles (diameter A). The muscle membrane facing the nerve terminal had no special structure (Nishihara, 1967). The membrane potential of the longitudinal muscle was 35 mv. However, in sodiumfree solution the membrane was hyperpolarized to 54 mv. and showed increased input resistance. On the other hand, reduction of the calcium concentration to one tenth of normal reduced the membrane potential to 18 mv. and also reduced the input resistance. However, in sodiumfree solution changing the calcium concentration from onefifth normal to five times normal changed neither the membrane potential nor the input resistance of the membrane. In physiological solution the specific resistance of the membrane, calculated as a limited cable, was 12 kq cm. 2, and this value was much higher than that observed in the skeletal muscle of the frog (Katz, 1948; Hidaka et al. 1969a, b). Therefore, the low membrane potential of the muscle fibre might be due to relatively low potassium permeability and relatively high sodium permeability which was controlled by calcium ion. The m.e.j.p. from the muscle fibre was less commonly recorded than the m.i.j.p. However, in sodiumfree solution generation of the m.e.j.p. could be recorded as frequently as the m.i.j.p.s in normal solution; this might be due to the properties of the postsynaptic muscle membrane caused by the increased input resistance and hyperpolarization of the membrane. In the present experiments, especially in sodiumfree solution, measurements of the reversal potential levels for the m.e.j.p.s under the various ionic concentrations might give the ratio (a) of calcium permeability coefficient (P C a) against potassium permeability coefficient (P K )> ^ca^k = a > from the equation introduced by the constantfield theory (Hodgkin & Katz, 1949), since chloride permeability could be neglected by the pretreatment with picrotoxin (Hidaka et al c). Grundfest (1966) has also reviewed the action of picrotoxin in blocking synaptic and nonsynaptic chloride activation in the arthropod muscle membrane. Furthermore, it was assumed that the internal concentrations of free sodium ion and calcium ion were negligibly small, and the internal potassium concentration was estimated to be 32 mm from the potassium equilibrium potential of 60 mv. Under the above assumption, the value of a, was calculated under various calcium and potassium concentrations

11 Junction potentials in the earthworm 117 i.e. in 27 mm (KC1) O, a = 8 in 2 mm (Ca) 0 ; a = 7 in 5 HIM (Ca) 0, and a = 7 in 10 mm (Ca) 0. These values are mutually in agreement, thus indicating that the permeability coefficient of calcium is nearly eight times higher than that of potassium in the presence of acetylcholine. On the other hand, when the external calcium concentration was kept at 2 mm a = 8 in 27 mm (K) o as described previously, and a = 5 in 054 mm (K) o, and a = 4 in 027 mm (K) o. The calculated values varied from 8 to 4. However, the values observed in the various concentrations of potassium indicated that the calcium permeability is much higher than the potassium permeability in the presence of acetylcholine, thus generating the m.e.j.p.s in the depolarizing direction. More information about the mechanism involved on the generations of the m.e.j.p.s will be necessary for further theoretical considerations. It might be possible to conclude from the present experiment that acetylcholine increased sodium, potassium and calcium permeabilities of the longitudinal muscle membrane of the earthworm in physiological solution as observed in the frog skeletal muscle (Takeuchi & Takeuchi, 1959, 1960a; Takeuchi, 1963). However, in sodiumfree solution generation of the m.e.j.p. is due to increase in calcium permeability and, to a lesser extent, to increase in potassium permeability, by spontaneous release of acetylcholine from the nerve terminals. SUMMARY 1. Miniature excitatory junction potentials (m.e.j.p.s) could be recorded from the longitudinal muscle layer of earthworm in sodiumfree solution. 2. The amplitude and frequency of the m.e.j.p.s indicated the diffuse innervation and random release of the chemical transmitter from the nerve terminals. 3. Generation of the m.e.j.p.s was prevented by treatment with Dtubocurarine, but not by atropine and picrotoxin. 4. Hyperpolarizations of the membrane by applications of inward current increased the frequency and amplitude of the m.e.j.p.s in sodiumfree solution. 5. The reversal potential level for the m.e.j.p.s in sodiumfree solution was 20 mv., and this value was 20raV.negative to that measured in physiological solution. Lowpotassium solution shifted the reversal potential levels in a more negative and highcalcium in a less negative direction. 6. The change of the reversal potential produced by a tenfold change of the external potassium concentration was 245 mv., and that by change of the external calcium concentration was 17 mv. REFERENCES BULLOCK, T. H. & HORRIDGE, G. A. (1965). Structure and Function in the Nervous System of Invertebrates, vols. 1 and 11. San Francisco and London: W. H. Freeman and Co. GRUNDFEST, H. (1966). Comparative electrobiology of excitable membrane. In Advances in Comparative Physiology and Biochemistry, vol 2, pp Ed. O. Lowenstein. New York: Academic Press. HIDAKA, T., ITO, Y. & KURIYAMA, H. (1969a). Membrane properties of the somatic muscle (obliquely striated muscle) of the earthworm. J. exp. Biol. (in the Press). HIDAKA, T., ITO, Y., KURIYAMA, H. & TASHIRO, N. (19696). Effects of various ions on the resting and active membrane of the somatic muscle of earthworm. J. exp. Biol. (in the Press.) HIDAKA, T., ITO, Y., KURIYAMA, H. & TASHIRO, N. (1969 c). Neuromuscular transmission in the longitudinal layer of somatic muscle in the earthworm. J. Exp. Biol. (in the Press). HODGKIN, A. L. & KATZ, B. (1949). The effect of sodium ions on the electrical activity of the giant axon of the squid. J. Physiol. 108, 3777.

12 n8 Y. ITO, H. KURIYAMA AND N. TASHIRO IKEMOTO, N. (1963). Further studies in electron microscopic structures of the obliquestriated muscle of the earthworm, Eisenia Faetida. Biol.J. Okayama Univ. 9, KATZ, B. (1948). The electrical properties of the muscle fibre membrane. Proc. R. Soc. B 135, KAWAGUTI, S. & IKEMOTO, N. (1958). Electron microscopy on the smooth muscle of the leech Hirudo Nipponia. Biol. J. Okayama Univ. 4, NISHIHARA, H. (1967). The fine structure of the earthworm body wall muscle. Ada anat. nippon. 42, 389. TAKEUCHI, A. & TAKEUCHI, N. (1959). Active phase of frog's endplate potential. J. Neurophysiol. 22, 39541i TAKEUCHI, A. & TAKEUCHI, N. (1960a). On the permeability of the endplate membrane during the action of transmitter. J. Physiol. 154, TAKEUCHI, A. & TAKEUCHI, N. (19606). Further analysis of relationship between endplate potential and endplate current. J. Neurophysiol. 23, TAKEUCHI, N. (1963). Some properties of conductance changes at the endplate membrane during the action of acetylcholine. J. Physiol. 167,