ABSTRACT
Miniature excitatory junction potentials (m.e.j.p.s) could be recorded from the longitudinal muscle layer of earthworm in sodium-free solution.
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.
Generation of the m.e.j.p.s was prevented by treatment with D-tubocurarine, but not by atropine and picrotoxin.
Hyperpolarizations of the membrane by applications of inward current increased the frequency and amplitude of the m.e.j.p.s in sodium-free solution.
The reversal potential level for the m.e.j.p.s in sodium-free solution was −20 mV., and this value was 20 mV. negative to that measured in physiological solution. Low-potassium solution shifted the reversal potential levels in a more negative and high-calcium in a less negative direction.
The change of the reversal potential produced by a tenfold change of the external potassium concentration was 24.5 mV., and that by change of the external calcium concentration was 17 mV.
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 (10−6 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 d-tubocurarine (10−6 g./ml.) and the equilibrium potential for them was o mV. The former was thought to be due to release of γ-aminobutyric 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 sodium-free solution, and the results indicated that, in sodium-free 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 communissima, 5−8 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, 1·0−1·5 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.
The normal solution (Ringer’s solution) used for this tissue was of the following composition: Na, 140 mM; K, 2·7 mM; Ca, 1·8 mM; Mg, 1·0 mM; Cl, 148·3 mM; and pH was adjusted from 7·3 to 7·5. 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 10−10 and 5 × 10−9 A. The microelectrodes were filled with 3 M-KCI 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. Sodium-free solution was prepared using Tris-(hydroxy-methyl)-aminomethane (C4H11NO3) to maintain isosmoticity. The solution was titrated with a high concentration of HC1, and the pH was adjusted to 7·4. The general procedures of the experiment were the same as those described by Hidaka, Ito & Kuriyama (1969 a) and Hidaka et al. (1969b, c). The experiments were carried out through the year. However, during the winter season (November-March) the effective resistance of the muscle fibres in normal solution was nearly twice that measured during the summer season (April-October).
RESULT
Generation of m.e.j.p
The membrane potential of muscle membrane was very low. However, the sodium-free solution (sodium was substituted by Tris) hyperpolarized the membrane from − 36 mV. (S.E. = ±0·5, n = 50) to-54 mV. (S.E. = ±0·6, n = 50), and increased the input resistance of the membrane from 42 MΩ (S.E. = + 1·1, n = 20) to 63 MΩ (S.E. = + 0·9, n = 30) during the summer season. Figure 1 shows the effects of pulses of current delivered to the muscle membrane in normal and in sodium-free 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 after-hyperpolarization of the membrane was not observed in sodium-free solution due to the hyperpolarization of the membrane.
In sodium-free 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 (10−6−10−6g./ml.) and atropine (10−7−10−5g./ml.). However, treatment with d-tubocurarine (10−6−10−5 g./ml.) completely abolished them. Figure 2 shows the effect of d-tubocurarine (10−6 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 sodium-free solution because the spike is due to inward movement of calcium ions during the active state of the membrane (Hidaka et al. 1969b). 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 0·5 mV,, was 1·8 mV.; the mean frequency was 0·34/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 sodium-free solution as observed in the sodium-containing 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 muscle membrane on the amplitude and frequency of the m.e.j.p.s in sodium-free solution were observed. Since the intensity-voltage 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.
Figure 4 shows the appearance of the m.e.j.p.s in sodium-free 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 sodium-free 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 sodium-free solution. To prevent the generation of m.i.j.p.s the tissue was previously treated with picrotoxin (10−6 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 sodium-free solution. When the membrane was hyperpolarized from −55 to −65 mV., the mean amplitude and frequency were increased to 2·3 and 6·0 mV./sec. respectively, and when the membrane was hyperpolarized to − 75 mV. these values increased to 2·8 and 7·6 mV./sec. respectively.
Increased frequency of the m.e.j.p.s in the hyperpolarized membrane might be due 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. = ± 1·2, n = 11). The difference in the results obtained from these two methods was not significant. However, the main explanation 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 GNa and GK, because they could not measure the excitatory junction current by the voltage-clamp method, but in the sodium-free solution reversal potential level for the m.e.j.p. shifted from 0 mV. (S.E. = ± 1·4, n = 5) in the normal solution to —20 mV. (S.E. = ± 1·2, n = 11). In the present experiment we observed that, even in the sodium-free solution, acetylcholine (io−6 g./ml.) reduced the membrane resistance, and pre-treatment with d-tubocurarine (10−6 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 d-tubocurarine on the intensityvoltage relation of the membrane under application of the weak hyperpolarizing currents (10−10 − 2 × 10−9 A). The graph shows the results obtained from six different experiments. Treatment with acetylcholine (10−6 g./ml.) consistently reduced the input resistance of the membrane and pre-treatment with d-tubocurarine (10−6 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 (10−6 g./ml.) from − 51·2 mV. (S.E. = ±0·6, n − 25) to −48·2 mV. (S.E. = + 0·7, 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 (10−6−10−5 g./ml.) did not influence the reversal potential level for the m.e.j.p.s, and (ii) in 5·6 HIM chloride solution ( (Cl)0 was substituted by d-glutamate) the reversal potential level for the m.e.j.p.s remained the same as in normal solution.
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 (10−5 g./ml.).
In the calcium-deficient solution (1, 0·5, 0·2 mM) the muscle membranes were depolarized from −35·2 mV. (S.E. = ±0·8, n = 25) to − 32·4 mv. (S.E. = ±0·9, n = 25), to −29 mV. (S.E. = ±0·8, n = 25) and to −21 mV. (S.E. = ±0·6, n = 25) respectively, and the membrane resistances were reduced from 68 MΩ (S.E. = + 2·4, n = b) to 54 MΩ (S.E. = ± 2·5, n = 8), to 42 MΩ (S.E. = ± 2·3, n = 10) and to 37 MΩ (S.E. = ± 2·1, n = 8) respectively.
However, in the sodium-free 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 intensity-voltage relation observed by the intracellular polarizing method under various calcium concentrations in the sodium-free 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. ± 1·2, n = 11) with 2 mM calcium ion to − 13 mV. (S.E. = +1·4, n = 10) 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. = ± 0·5, 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 2·7 mM potassium. Figure 8 shows the reversal potentials for the m.e.j.p. under the, 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 (10−6 g./ml.), the reversal potential level shifted in a more negative direction, i.e. in the solution containing one tenth the normal potassium concentration (0·27 mM) the reversal potential level was measured to be −44·5 mV. (S.E. = ±2·4, n = 5). The change of the reversal potential level produced by a tenfold change of the external potassium concentration was 24·5 mV. in solutions containing 2 mM calcium.
Figures 9−11 show the actual reversal potential levels for the m.e.j.p. measured in sodium-free solution, i.e. Fig. 9 shows the reversal potential level for the m.e.j.p. measured in 2 mM calcium and 2·7 mM potassium by application of the intracellular polarizing currents, in 2 mM calcium and 0·54 mM potassium (Fig. 10) and in 5 mM.calcium and 2·7 mM potassium solutions (Fig. 11). During these experiments, to 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; 5−10μ, 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 half-desmosome 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. 300−500 Å) and by inhibitory nerves which contain many cored vesicles (diameter 900−1800 Å). 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 sodium-free 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 sodium-free solution changing the calcium concentration from one-fifth 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 kil 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 sodium-free 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 post-synaptic muscle membrane caused by the increased input resistance and hyperpolarization of the membrane. In the present experiments, especially in sodium-free solution, measurements of the reversal potential levels for the m.e.j.p.s under the various ionic concentrations might give the ratio (α) of calcium permeability coefficient (PCa)against potassium permeability coefficient (PK), PCa / PK = α. from the equation introduced by the constant-field theory (Hodgkin & Katz, 1949), since chloride permeability could be neglected by the pre-treatment with picrotoxin (Hidaka et al. 1969 c). Grundfest (1966) has also reviewed the action of picrotoxin in blocking synaptic and non-synaptic 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 α was calculated under various calcium and potassium concentrations e. in 2 · 7 mM (KC1)0, α = 8 in 2 mM (Ca)0 ; α = 7 in 5 mM (Ca)0, and α = 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 2-7 mM (K)o as described previously, and a = 5 in 0 · 54 mM (K)0, and α =4 in 0 · 27 mM (K)0. 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, 1960 a; Takeuchi, 1963). However, in sodium-free 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.