1. The effect of extracellular lanthanum on neuromuscular transmission was studied in cockroach leg muscle and larval mealworm ventral muscle by means of microelectrodes.

  2. Miniature excitatory postsynaptic potential (MEPSP) frequency was markedly increased after lanthanum was added, in the presence and absence of calcium. The potentiation by La3+ was suppressed in a high Ca2+ saline and enhanced in the absence of Ca2+.

  3. Lanthanum ions blocked neuromuscular transmission at a concentration as low as 0-1 mm. The quantal content estimated by the failure method was reduced by 80% in the presence of 0·1 mm-La3+. The reduction in the EPSP amplitude by La3+ may be due to a decrease in the amount of transmitter released by a nerve impulse.

  4. The response to L-glutamate applied iontophoretically was also reduced in the presence of La3+. It seems unlikely that La3+ and L-glutamate were competing for a common binding site on the postsynaptic membrane since the apparent maximum of the dose-response curve for glutamate-induced depolarization was reduced in the presence of La3+.

  5. External recording of MEPSPs showed that adding lanthanum to the bathing medium increased the time constant of decay of the potential.

  6. These results suggest that lanthanum does indeed have a postsynaptic action in addition to its prejunctional action in insect muscle fibres.

Lanthanum produces blockage of neuromuscular transmission, perhaps due to a prevention of the influx of calcium ions into presynaptic nerve terminals (Miledi, 1966; Heuser & Miledi, 1971; Miledi, 1971). However, lanthanum also gives rise to a substantial increase in discharges of acetylcholine quanta (Blioch, Glagoleva, Liberman & Nenashev, 1968; Heuser & Miledi, 1971; DeBassio, Schnitzler & Parsons, 1972).

Recently, cobalt ions were found to have a dual effect on the spontaneous release of transmitter at insect motor nerve terminals, an inhibitory action being followed by an acceleratory action (Washio, 1982a). There was an antagonism by calcium of the inhibitory effect of cobalt. Since both cobalt (Weakly, 1973) and lanthanum (Blioch et al. 1968; Heuser & Miledi, 1971; DeBassio et al. 1972) are more effective than magnesium in suppressing evoked transmitter release, we have studied the presynaptic effect of lanthanum at insect neuromuscular junctions. In this paper evidence is also presented to suggest that lanthanum has a postsynaptic action on the insect neuromuscular synapse.

Experiments were performed on coxal depressor muscles of the cockroach, Periplaneta americana, muscle 178 from the metathoracic leg and 136 from the mesothoracic leg (Carbonell, 1947), and on the longitudinal ventral muscles of the larval mealworm, Tenebrio molitor. The standard bathing solution for cockroach muscles had the following composition (mm): NaCl, 158; KC1, 10·8; CaCl2, 5; HEPES, 5; adjusted to pH 7·0 with NaOH. For mealworm muscles composition was NaCl, 70; KC1, 30; CaCl2, 0·7; MgCh, 14·3; glucose, 445; HEPES, 5; adjusted to pH 7·2. Test solutions were prepared by substituting the appropriate cation chloride, for NaCl in the cockroach saline, and for glucose in the mealworm saline.

MEPSPs of cockroach muscle fibres were recorded intracellularly and extracellularly with micropipettes filled with 3 M-KCI and 2M-NaCl, respectively. The EPSPs in Tenebrio muscle fibres were evoked by stimulating the segmental nerve innervating the muscle with supramaximal square pulses (0·01 ms) using a pair of silver hook electrodes. The iontophoretic electrodes were filled with 1 M-Na L-glutamate at pH 8·0 and had resistances of about 80 MΩ. The preparation was constantly perfused at a rate of 5·0 ml min−1. Experiments were all performed at 20—24 °C.

Spontaneous release of transmitter

The experiments on spontaneous release of transmitter were performed on the cockroach muscle in order to compare with the effect of cobalt at the cockroach neuromuscular junction (Washio, 1982a), whereas the experiments on transmitter release by nerve impulses and on the response to L-glutamate applied iontophoreti-cally were performed at the Tenebrio neuromuscular junction, since these mechanisms have been well documented in those muscles (Yamamoto & Washio, 1980).

In the presence of 5mm-Ca2+, increasing the concentration of La3+ caused an increase in frequency of MEPSPs recorded intracellularly. The higher the concentration, the faster the rate of rise of the frequency and the shorter the time lag between the application of the test solution and increase in the frequency (Fig. 1). Although the effect of external La3+ was almost the same in both normal and high K+ (20·8 HIM) saline, it seemed that the time lag and time course were shorter in high K+ saline (Fig. 1). The high frequency caused by 5 mm-lanthanum lasted for less than 1 h at a K+ concentration of either 10·8 or 20·8 mm (Fig. 1). Raising Ca2+ concentration while keeping La3+ concentration constant reduced the potentiation, but not below a control level (Fig. 2B). For example, when 0·1 mm-La3+ was introduced (at 0·1 mm-Ca2+ and 20·8 mm-K+), it produced a sharp rise (more than one order of magnitude) in the frequency of occurrence of MEPSPs after a lag of several minutes (Fig. 2A). After reaching a peak, MEPSP frequency declined to a very low level along a roughly exponential time course with a time constant of about 14 min in this fibre.

Effect of elevating La3+ concentration on MEPSP frequency in saline containing 5 mm-Ca2+ on cockroach muscle fibres. (A) In a normal K+ (10·8 HIM) saline. (B) In a high K+ (20·8 mm) saline. La3+ concentrations are 0·1 mm(▵), 0·5 DIM (●), l·0 mw (×) and 5·0 mm(▴). The frequency on the ordinate in (B) is expressed as the ratio of the frequency in the test solution (Ft) to the frequency (F) in La3+-free saline per 10s. An arrow indicates the changeover point.

(A) Time course of the effect of 0·1 mm-La3+ on the MEPSP frequency in cockroach muscle in a saline containing 0·1 rriM-Ca2+. (B) Effect of external calcium concentration on the potentiation of MEPSP frequency by 0·1 mm-La. The frequency was measured 10—15 min after application of the La3+ saline. Arrows in (A) indicate the changeover points. Symbols on the ordinate in (B) as in Fig. 1B. Vertical bars indicate s.D. of each mean (N = 5). Both experiments were performed in high K+ (20·8mm) saline.

The time constant of the decay of the potentials recorded extracellularly depended on La3+ concentration. For example, in Fig. 3B, the time constant of the decay of the largest MEPSP was increased by 5 mm-La3+ from 0·61 ±0·05 ms (mean ± s.d., N= 12) to 0·97 ±0·18 ms (N = 10), (P<0·05, Student’s t-test). The frequency of MEPSPs recorded extracellularly was much lower than that recorded intracellularly in insect muscle fibres (Washio & Inouye, 1980).

Effect of 5 mm-La3+ on the time course of MEPSPs recorded extracellularly from a cockroach muscle fibre in saline containing Snw-Ca2* and 20·8mm-K+. (A), (B) Examples of the MEPSP, control and 15 min after application of La3+ respectively. (C) Semilogarithmic plot of the decay of the potentials before (○) and 15 min after (●) application of 5 mm-La3+. Time constants of the decay are 0·58 ms (○) and 0·72ms (●).

Transmitter release by nerve impulses

As in frog muscle (Miledi, 1966; Heuser & Miledi, 1971), La3+ could not replace Ca2+ as a requirement for transmitter release in Tenebrio muscle. Release was not restored by 0·5 mm-La3+ after blockage in Ca-deficient medium.

Fig. 4A illustrates the reduction of the excitatory postsynaptic potential (EPSP) of the Tenebrio muscle fibre during the application of 0·5 mm-La3+. The amplitude of the EPSP elicited by repetitive stimulation at 0·5 Hz was suppressed by 60% with perfusion for 2 min. The suppression of EPSP amplitude was almost irreversible (Fig External MEPSP amplitude (%) 4A3). The relationship between the amplitude of EPSP and extracellular La3+ concentration, and the effect of La3+ on the time course of the EPSP are shown in Figs 5 and 6A, respectively. The reduction in the amplitude of EPSP produced by La3+ could be antagonized to some degree by elevation of the external Ca2+ concentration (Fig. 7).

Comparisons of the effect of 0·5 mm-La3+ on EPSP (A) and the response to the iontophoreti-cally applied L-glutamate (glutamate potential. B). The EPSP and glutamate potential were simultaneously recorded from the same Tenebrio muscle fibre. A1, B1, before; A2, B3, 2min after the application; A3, B3 7·5 mmafter washing out with La3+-free saline containing 0·7 mm-Ca2+ and 14·3 mm-Mg2+.

Relation between EPSP amplitude (●), or the response to the iontophoretically applied L-glutamatc (○), and extracellular La3+ concentration in a saline containing 0·7 mm-Ca2+ and 14·3 mm-Mg2+. Amplitudes (mean ± s.D.) were measured at 10 min after application of La3+ to five Tenebrio muscle fibres.

Time course of the effect of La3+ concentration on EPSP amplitude (A) and on the glutamate potential (GP) amplitude (B) in saline containing 0·7mm-Ca2+ and 14·3mm-Mg2+ in Tenebrio muscle fibres. Ordinate represents percentage of the amplitude after the application of La3+ to the control. Arrows indicate washing out with a La3+-free saline.

Effect of external calcium concentration on the amplitude of EPSPs in the absence (●) and presence (○) of 0·1 mm-La3+ with 15mm-Mg2+ in a Tenebrio muscle fibre.

The quanta! content was calculated from the number of failures of EPSP (del Castillo & Katz, 1954) in the absence and presence of La3+ at low Ca2+ concentration (0·2mm-Ca2+ and 14·8mm-Mg2+) during repetitive nerve stimulation at 0·5 Hz (Table 1). The quantal content was reduced by 80% in the presence of 0·1 mm-La3+ Thus, the reduction in the EPSP amplitude by La3+ may be due to a decrease in the amount of transmitter released by a nerve impulse.

Table 1.

Effect of lanthanum on quantal content (m)

Effect of lanthanum on quantal content (m)
Effect of lanthanum on quantal content (m)

The response to L-glutamate applied iontophoretically

Since glutamate is a putative transmitter in the Tenebrio muscle (Yamamoto & Washio, 1980), we studied the effect of La3+ on sensitivity to L-glutamate applied iontophoretically.

During the application of 0-5 mm-La3+, the amplitude of the glutamate potential decreased by 50% in 2 min after the application and recovered completely 10 min after washing with a La3+-free saline (Fig. 4B). The time course of the recovery after washing was quite different from that of the neurally evoked response (cf. Fig. 4A and B). Also the threshold concentration of La3+ on the glutamate potential was found to be lower than that for the EPSP (Fig. 5). The difference of the threshold concentration and time course of the recovery between the response to L-glutamate applied iontophoretically and the EPSP may be attributed to the fact that the former is produced at a restricted junctional site while the latter is the integrated potential change of the junctions distributed over the whole muscle fibre.

Lanthanum produced a reduction in the apparent maximum of the dose-response curve for glutamate-induced depolarization (Fig. 8). Double logarithmic plots of the amplitude of the depolarizing response to L-glutamate against coulomb strength of the ejection currents showed that the limiting slope values were 2·6 and 1 ·8 in the absence and presence of 0·1mm-La3+ respectively (Fig. 8B). Thus, it seems unlikely that glutamate and lanthanum are competing for a common binding site on the postsynaptic membrane.

Effect of 01mm-La3+ on the dose-response curve to L-glutamate from a single Tenebrio muscle fibre. Semi-(A) and double (B) logarithmic plot of glutamate potential against the coulomb strength before (○), 10min (●) and 15min (×) after application of La3+ in the Ca2+-free saline containing 15 mm-Mg2+. The limiting slopes values in B are 2·6 (○) and 1·8 (×).

The present study in insect muscle fibres confirms the finding previously reported at frog neuromuscular junctions (Heuser & Miledi, 1971; DeBassio et al. 1972) that La3+ produces a substantial increase in spontaneous release of transmitter and blockage of neuromuscular transmission. Furthermore, our results indicate that La3+ brings about a reduction in the response to L-glutamate applied iontophoretically. The depression of the EPSP amplitude by La3+ could be accounted for by a decrease in the quantal content released by nerve impulses.

Although transmitter release by nerve impulses was decreased by extracellular La3+, spontaneous release of transmitter was substantially increased. The potentiation of MEPSP frequency by La3+ was suppressed in a high Ca2+ saline and enhanced in the absence of Ca2+. Thus, there might be an antagonism by Ca2+ of the La3+ acceleration of spontaneous release. DeBassio et al. (1972) have reported an antagonism between them and suggested that the competition might occur at anionic binding sites on the external surface of the nerve terminal. An inhibitory action on the spontaneous release of transmitter which has been found in the presence of Ca2+ (Washio, 1982a) has not been clear in the presence of La3+. The increase in the frequency was recorded in the absence of external Ca2+ or the presence of a very low concentration. Therefore, it seems more likely that La3+ acts on an intracellular site (DeBassio et al. 1972; Washio, 1982b). For example, La3+ has been reported to be an extremely potent inhibitor of the energy-linked accumulation of Ca2+ by mitochondria (Mela, 1968, 1969) and thereby increases the intracellular calcium concentration (Rahamimoff, 1978). However, MEPSP frequency was found to be lowered slightly in the presence of 5 mm-Ca2+ just after the application of 0·5 mm-La3+ in some cockroach muscle fibres partially depolarized with high K+ (Fig. IB). It might be possible that an inhibitory effect of La3+ on the external surface of the nerve terminal was followed by the acceleratory effect which masked the former.

Our results also showed that lanthanum ions decreased the amplitude of the transient depolarization (glutamate potential) produced by iontophoretically applied L-glutamate. Double logarithmic plots of the amplitude of the depolarizing response to L-glutamate against coulomb strength of the ejection currents showed that La3+ reduced the value for the slope of the junctional receptors. Therefore, it seems unlikely that L-glutamate and La3+ are competing for a common binding site on the post-synaptic membrane. Alternatively, La3+ might affect the kinetics of the channels activated by L-glutamate, since the trivalent ion can modify the time course of the MEPSP recorded extracellularly. It has been shown that the kinetics of L-glutamate-induced channels may be altered by the species of permeable cations (Cull-Candy & Miledi, 1982) as well as the kinetics of acetylcholine-induced ones (Gage & Van Helden, 1979; Bregestovski, Miledi & Parker, 1979; Magleby & Weinstock, 1980). On the other hand, it has been proposed that the change in the decay time of the endplate current can be explained by screening fixed negative charges on the end-plate membrane (Mallart & Molgó, 1978; Cohen & Van der Kloot, 1978). The kinetics of the channels depend upon the voltage gradient through the postsynaptic membrane. In insect muscles, hyperpolarization shortens the decay time of MEPSCs and depolarization lengthens the decay time (Cull-Candy & Miledi, 1982) which is in the opposite direction to the kinetics in vertebrate muscles (Anderson, Cull-Candy & Miledi, 1978) and to the behaviour expected from screening fixed negative charges (Cohen & Van der Kloot, 1978). Thus, the results obtained in insect muscles are incompatible with the surface charge interpretation. Recently, (+)-tubocurarine-induced block of the glutamate synapse resulting from plugging the open channels was reported in Tenebrio muscle fibres (Yamamoto & Washio, 1983). Thus, it might be possible that La3+ can alter the kinetics of glutamate channels by acting on channel properties as a non-competitive antagonist, like a local anaesthetic, or by changing the rates with which L-glutamate interacts with receptors, like acetylcholine and strontium (Miledi & Parker, 1980).

The authors wish to thank Professor David M. J. Quastel, Department of Pharmacology, University of British Columbia, for his helpful comments on the manuscript. Thanks are also due to Miss Junko Kaneko for her technical assistance.

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