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Reduction of glutamate responses by caroverine at the crayfish neuromuscular junction
174
Brain Research, 266 (1983) 174 -~177 Elsevier Biomedical Press
~Uon
of ~
~
~ neuromu~
by car~wertr,e at the crayfish J,jnction
M. ISHIDA and H. SHINOZAKI
The Tokyo Metropolitan Institute of Medical Science, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113 (Japan) (Accepted January 4th, 1983)
Key words: glutamate - caroverine - open channel blocker - crayfish neuromuscular junction
Caroverine depressed the glutamate current induced by ionophoreticatly applied glutamate, but affected the neurally evoked excitatory junctional responses less. Caroverine caused more marked reduction of a conditioned glutamate current.'The results obtained suggest that caroverine is an open channel blocker for glutamate.
At the crayfish neuromuscular junction where glutamate is a leading candidate for an excitatory transmitter 6,7.1],]8,some compounds affect neither the average unit size nor decay of excitatory junctional currents in spite of the fact that they are powerful glutamate inhibitors 5,z5,~7. The mechanism of action of these compounds is unclear. It is desirable to know the exact mechanisms of action of these compounds to clarify further the possible role of glutamate at this site. Diltiazem is one of these compounds and has been shown to be a non-competitive glutamate antagonistS,tL Recently, we found another compound, caroverine, which depressed the glutamate response at the crayfish neuromuscular j unction. The chemical structure of caroverine is very similar to that of diltiazem. Qualitative effects of caroverine and its effective concentration range are similar to those of diltiazem at the crayfish neuromuscular junction. The nerve-evoked response was less affected by these drugs than the glutamate response. More interesting would be the reverse finding. It has been reported that some open channel blockers affect the nerve-evoked synaptic responses less than the responses to ionophoretically applied cholinomimetics at the vertebrate endplate 1.2. The methods used were similar to those re-
ported previously5. Potential changes of the crayfish opener muscle were recorded either intracellularly from the muscle fiber with a 3 M KCl-filled microelectrode or extraceUulady from the neuromuscular junction with a 2 M NaCl-filled microelectrode. In a number of experiments the membrane potential of the muscle fiber was clamped with an intracellular microCAROVERINE
~
OCH 3
I C2H5 CH2CH2N ~C2H5
DILTIAZEM
~ N
OCH 3
%O
/,CH 3 CH2CH2N <--.CH3 Fig. 1. Chemical structures ofcaroverine and diltiazem.
0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
175 electrode to record the glutamate current ~0. Caroverine was added to the perfusing fluid. Glutamate was ionophoreticaUy applied to the most sensitive site or was added to the bathing solution. Caroverine (0.2 mM) affected neither the resting membrane potential nor the input resistance of the crayfish opener muscle. However, at concentrations greater than 0.2 m M the resting membrane potential often became unstable, and local contraction of the muscle fiber was usually observed. Caroverine (0.2 mM) reduced the depolarization of the muscle fiber produced by bath-applied glutamate (0.1 mM) to about half. In this case the time to peak (rise time) of the glutamate response was slightly shortened and the depolarization was not maintained but declined within about 1 min to a steady level in spite of the fact that it was maintained in the absence of the drug. The action of the drug on glutamate responses was completely reversible after washing (5 min). The threshold concentration of caroverine needed to depress glutamate responses was less than 0.1 mM, being similar to that of diltiazem. Glutamate currents induced by ionophoretically applied glutamate at intervals of 10 s were reduced by caroverine in a dose-dependent manner and caroverine did not seem to be a competitive antagonist for glutamate. In some preparations the glutamate current was hardly affected by 0.1 m M caroverine. When the interpulse interval of a double pulse was very short ( < 500 ms), caroverine depressed the second glutamate current more markedly than the first. Even when the first response was hardly affected at the standard membrane potential ( - 8 5 mV) in the presence of caroverine, reduction of the second response was observed. As shown in Fig. 2, during a train of glutamate pulses successive glutamate currents gradually decrease in size. In the presence ofcaroverine (0.15 mM) the degree of the decrease in size became more marked. Similar data were obtained in the concanavalin A-treated muscle. As concanavalin A is a completely irreversible blocker of desensitization of the glutamate receptor°,14, the above results suggest that caroverine is an open channel blocker.
-q"
a
"i-'T 'l" 'l " i "r--
"l 5 0 n A
.i J i
m
l
500 msec Fig. 2. C a r o v e r i n e accelerates the d e p r e s s i o n s e e n in trains o f
glutamate currents. Glutamate currents were evoked by a train of 10stimuli (10 ms duration, 50 hA) at intervalsof 300 ms in the absenceand presenceof caroverine.Upper trace, monitoredinjectioncurrent, a, control; b, 0.15 mM caroverihe.
The number of the free reactive receptors is expected to be decreased in the presence of both caroverine and glutamate. Therefore the recovery from the refractory form of the glutamate receptor to the free reactive one was examined. The rate constant o f the recovery was calculated from the slope of semilogarithmic plots of (I¢It)/I¢ against the pulse interval t, where I¢ is the amplitude of the test response in the absence of the conditioning response and It the amplitude with a pulse interval t 1,16. The slope was slightly decreased in the presence of the drug. The reduction was statistically significant (P < 0.01, n = 5). In one preparation the values of the rate constant in the absence and presence of caroverine (0.2 mM) were 9.0 s-I and 7.3 s-I, respectively. Effects of some channel blockers have been shown to be dependent on membrane potential 1.2,4.Reduction of the glutamate current caused by caroverine became more prominent as the membrane was hyperpolarized, suggesting that effect of caroverine was voltage-dependent. At the standard membrane potential the decay of the glutamate current tail was close to a single
176 exponential, and caroverine slightly accelerated its decay (statistically significant at P ~ 0.01), the mean values of the decay time constant in the absence and presence of caroverine (0.15 mM) being 30.9 ___2.3 ms (n = 8) and 26.0 __. 0.6 ms (n = 5), respectively, when regressions were calculated between 80 and 20% of the peak amplitude of the glutamate current. Biphasic decay of the glutamate current was observed on rare occasions when the membrane was hyperpolarized in the presence ofcaroverine. Glutamate potentials and successive intracellular e.j.ps induced by trains of pulses at 10 Hz for 80 ms were alternately evoked at intervals of 4 s. A few minutes after the normal saline had been replaced by one containing caroverine (0.2 mM), the amplitude of glutamate potentials was reduced to less than half, while that of e.j.ps was slightly decreased. The number of quanta released per impulse was estimated from the number of failures of extracellularly recorded e.j.ps. The quantal content was decreased in the presence of caroverine (0.2 mM), the ratio to the control being 0.51 _+ 0.31 (n = 5). The average unit size and the decay time constant were slightly decreased by addition of caroverine (0.2 mM) to the perfusing fluid, the ratios to the control being 0.84 __ 0.12 (n = 5)and 0.84 ___ 0.14 (n = 7), respectively. However, there was no statistically significant difference between the control and the caroverine-treated preparation. The channel block theory predicts that transformation from the active open channel to the blocked open channel is a function of the blocker concentration ~. Therefore, it is desirable to examine the effect of caroverine at higher concentrations. However, it was not possible to perform the experiments because local contracture can be overcome by detubulation. The above results suggest that caroverine is an open channel blocker for glutamate, although the drug did not satisfy all necessary criteria for the open channel blocker. The decay time constant of the glutamate current tail was slightly decreased in the presence of caroverine and biphasic decay was sometimes observed. When a
conditioned glutamate current was examined, the amplitude of the test response was more affected than the first in the presence of caroverine. The response to bath applied glutamate was not maintained but declined during its constant application in the presence of caroverine. The model that has most commonly been proposed to account for the properties of channel blocking drugs in the following, A + R.
"~AR .---'-~AR* . ~
CAR*
In this scheme, A is an agonist molecule, R the receptor in its resting state, R* the receptor in its active open state, and C is the blocking agent, a /3 are the rate constants. Chlorisondamine is known as an open channel blocker in crustacea8:7 and depressed both the amplitudes of e.j.ps and glutamate responses to the same degree. In this case it seems that both values of the association rate constant (a) and the dissociation rate constant (/3) are very large. On the other hand, caroverine affected e.j.ps less than the glutamate current. Since exposure of the neurally released transmitter to receptors is much shorter than that of ionophoretically applied agonist, if both the values of a and/3 are small, it may be possible to explain the smaller effect ofcaroverine on e.j.ps. The experimental result that recovery from the refractory form of the glutamate receptor was only slightly delayed by caroverine suggests a relatively small value of/3. We have to consider another explanation for the caroverine action as well. It is that glutamate is not an excitatory transmitter at the crayfish neuromuscular junction. Many pharmacological problems at this site remain unsolved 13,17. Further studies of glutamate inhibitors will be required to understand the possible role of glutamate at this site. The authors wish to thank Dr. Y. Kudo, Mitsubishi-Kasei Institute of Life Sciences, for a gift of caroverine. This work was supported in part by Grant-in-Aid for Scientific Research, The Ministry of Education, Science and Culture.
177 1 Adams, P. R., Drug blockade of open end-plate channels, J. Physiol. (Lond.), 260 (1976) 531-552. 2 Albuquerque, E. X. and Gage, P. W., Differential effects of perhydrohistrionicotoxin on neurally and ionophoretically evoked endplate currents, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 1596-1599. 3 Dudel, J. and Kuffler, S. W., The quantal nature of transmission and spontaneous miniature potentials at the crayfish neuromuscular junction, J. Physiol. (Lond.), 155 (1961) 514-529. 4 Feltz, A., Large, W. A. and Trautmann, A., Analysis of atropine action at the frog neuromuscular junction, J. Physiol. (Lond.), 269 (1977) 109-130. 5 Ishida, M. and Shinozaki, H., Differential effects of diltiazem on glutamate potentials and excitatory junctional potentials at the crayfish neuromuscular junction, J. Physiol. (Lond.), 298 (1980) 301-319. 6 Kawagoe, R., Onodera, K. and Takeuchi, A., Release of glutamate from the crayfish neuromuscular junction, J. Physiol. (Lond.), 312 (198 l) 225-236. 7 Kawagoe, R., Onodera, K. and Takeuchi, A., On the quantal release of endogenous glutamate from the crayfish neuromuscular junction, J. Physiol. (Lond.), 322 (1982) 529-539, 8 Lingle, C., Eisen, J. S. and Marder, E., Block of glutamatergic excitatory synaptic channels by chlorisondamine, Molec. PharmacoL, 19 (1981) 349-353. 9 Mathers, D. A. and Usherwood, P. N. R., Concanavalin A blocks desensitization of glutamate receptors on insect muscle fibres, Nature (Lond.), 259 (1976) 409-411. 10 Onodera, K. and Takeuchi, A., Permeability changes produced by L-glutamate at the excitatory post-synaptic
membrane of the crayfish muscle, J. Physiol. (Lond.), 255 (1976) 669-685. I 10nodera, K. and Takeuchi, A., Distribution and pharmacological properties ofsynaptic and extrasynaptic glutamate receptors on crayfish muscle, Jr. Physiol. (Lond), 306 (! 980) 233- 250. 12 Rang, H. P., The action of ganglionic blocking drugs on the synaptic responses of rat submandibular ganglion cells, Brit. J. Pharmacol., 75 (1982) 15 l- 168. 13 Shinozaki, H., The pharmacology of the excitatory neuromuscular junction in the crayfish, Progr. Neurobiol., 14 (1980) 121-155. 14 Shinozaki, H. and Ishida, M., Pharmacological distinction between the excitatory junctional potential and the glutamate potential revealed by concanavalin A at the crayfish neuromuscular junction, Brain Research, 161 (1979) 493- 501. 15 Shinozaki, H. and Ishida, M., Glutamate potential: differences from the excitatory junctional potential revealed by diltiazem and concanavalin A in crayfish neuromuscular junction, J. Physiol. (Paris), 75 (1979) 623- 627. 16 Shinozaki, H. and Ishida, M., The recovery from desensitization of the glutamate receptor in the crayfish neuromuscular junction, Neurosci. Lett., 21 (1981) 293-296. 17 Shinozaki, H., Ishida, M. and Mizuta, T., Glutamate inhibitors in the crayfish neuromuscular junction, Comp. Biochem. Physiol.,72C (1982) 249-255. 18 Takeuchi, A., Excitatory and inhibitory transmitter actions at the crayfish neuromuscular junction. In S. Thesleft (Ed.), Motor Innervation of Muscle, Academic Press, London, 1976, pp. 231-261.
Brain Research, 266 (1983) 174 -~177 Elsevier Biomedical Press
~Uon
of ~
~
~ neuromu~
by car~wertr,e at the crayfish J,jnction
M. ISHIDA and H. SHINOZAKI
The Tokyo Metropolitan Institute of Medical Science, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113 (Japan) (Accepted January 4th, 1983)
Key words: glutamate - caroverine - open channel blocker - crayfish neuromuscular junction
Caroverine depressed the glutamate current induced by ionophoreticatly applied glutamate, but affected the neurally evoked excitatory junctional responses less. Caroverine caused more marked reduction of a conditioned glutamate current.'The results obtained suggest that caroverine is an open channel blocker for glutamate.
At the crayfish neuromuscular junction where glutamate is a leading candidate for an excitatory transmitter 6,7.1],]8,some compounds affect neither the average unit size nor decay of excitatory junctional currents in spite of the fact that they are powerful glutamate inhibitors 5,z5,~7. The mechanism of action of these compounds is unclear. It is desirable to know the exact mechanisms of action of these compounds to clarify further the possible role of glutamate at this site. Diltiazem is one of these compounds and has been shown to be a non-competitive glutamate antagonistS,tL Recently, we found another compound, caroverine, which depressed the glutamate response at the crayfish neuromuscular j unction. The chemical structure of caroverine is very similar to that of diltiazem. Qualitative effects of caroverine and its effective concentration range are similar to those of diltiazem at the crayfish neuromuscular junction. The nerve-evoked response was less affected by these drugs than the glutamate response. More interesting would be the reverse finding. It has been reported that some open channel blockers affect the nerve-evoked synaptic responses less than the responses to ionophoretically applied cholinomimetics at the vertebrate endplate 1.2. The methods used were similar to those re-
ported previously5. Potential changes of the crayfish opener muscle were recorded either intracellularly from the muscle fiber with a 3 M KCl-filled microelectrode or extraceUulady from the neuromuscular junction with a 2 M NaCl-filled microelectrode. In a number of experiments the membrane potential of the muscle fiber was clamped with an intracellular microCAROVERINE
~
OCH 3
I C2H5 CH2CH2N ~C2H5
DILTIAZEM
~ N
OCH 3
%O
/,CH 3 CH2CH2N <--.CH3 Fig. 1. Chemical structures ofcaroverine and diltiazem.
0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
175 electrode to record the glutamate current ~0. Caroverine was added to the perfusing fluid. Glutamate was ionophoreticaUy applied to the most sensitive site or was added to the bathing solution. Caroverine (0.2 mM) affected neither the resting membrane potential nor the input resistance of the crayfish opener muscle. However, at concentrations greater than 0.2 m M the resting membrane potential often became unstable, and local contraction of the muscle fiber was usually observed. Caroverine (0.2 mM) reduced the depolarization of the muscle fiber produced by bath-applied glutamate (0.1 mM) to about half. In this case the time to peak (rise time) of the glutamate response was slightly shortened and the depolarization was not maintained but declined within about 1 min to a steady level in spite of the fact that it was maintained in the absence of the drug. The action of the drug on glutamate responses was completely reversible after washing (5 min). The threshold concentration of caroverine needed to depress glutamate responses was less than 0.1 mM, being similar to that of diltiazem. Glutamate currents induced by ionophoretically applied glutamate at intervals of 10 s were reduced by caroverine in a dose-dependent manner and caroverine did not seem to be a competitive antagonist for glutamate. In some preparations the glutamate current was hardly affected by 0.1 m M caroverine. When the interpulse interval of a double pulse was very short ( < 500 ms), caroverine depressed the second glutamate current more markedly than the first. Even when the first response was hardly affected at the standard membrane potential ( - 8 5 mV) in the presence of caroverine, reduction of the second response was observed. As shown in Fig. 2, during a train of glutamate pulses successive glutamate currents gradually decrease in size. In the presence ofcaroverine (0.15 mM) the degree of the decrease in size became more marked. Similar data were obtained in the concanavalin A-treated muscle. As concanavalin A is a completely irreversible blocker of desensitization of the glutamate receptor°,14, the above results suggest that caroverine is an open channel blocker.
-q"
a
"i-'T 'l" 'l " i "r--
"l 5 0 n A
.i J i
m
l
500 msec Fig. 2. C a r o v e r i n e accelerates the d e p r e s s i o n s e e n in trains o f
glutamate currents. Glutamate currents were evoked by a train of 10stimuli (10 ms duration, 50 hA) at intervalsof 300 ms in the absenceand presenceof caroverine.Upper trace, monitoredinjectioncurrent, a, control; b, 0.15 mM caroverihe.
The number of the free reactive receptors is expected to be decreased in the presence of both caroverine and glutamate. Therefore the recovery from the refractory form of the glutamate receptor to the free reactive one was examined. The rate constant o f the recovery was calculated from the slope of semilogarithmic plots of (I¢It)/I¢ against the pulse interval t, where I¢ is the amplitude of the test response in the absence of the conditioning response and It the amplitude with a pulse interval t 1,16. The slope was slightly decreased in the presence of the drug. The reduction was statistically significant (P < 0.01, n = 5). In one preparation the values of the rate constant in the absence and presence of caroverine (0.2 mM) were 9.0 s-I and 7.3 s-I, respectively. Effects of some channel blockers have been shown to be dependent on membrane potential 1.2,4.Reduction of the glutamate current caused by caroverine became more prominent as the membrane was hyperpolarized, suggesting that effect of caroverine was voltage-dependent. At the standard membrane potential the decay of the glutamate current tail was close to a single
176 exponential, and caroverine slightly accelerated its decay (statistically significant at P ~ 0.01), the mean values of the decay time constant in the absence and presence of caroverine (0.15 mM) being 30.9 ___2.3 ms (n = 8) and 26.0 __. 0.6 ms (n = 5), respectively, when regressions were calculated between 80 and 20% of the peak amplitude of the glutamate current. Biphasic decay of the glutamate current was observed on rare occasions when the membrane was hyperpolarized in the presence ofcaroverine. Glutamate potentials and successive intracellular e.j.ps induced by trains of pulses at 10 Hz for 80 ms were alternately evoked at intervals of 4 s. A few minutes after the normal saline had been replaced by one containing caroverine (0.2 mM), the amplitude of glutamate potentials was reduced to less than half, while that of e.j.ps was slightly decreased. The number of quanta released per impulse was estimated from the number of failures of extracellularly recorded e.j.ps. The quantal content was decreased in the presence of caroverine (0.2 mM), the ratio to the control being 0.51 _+ 0.31 (n = 5). The average unit size and the decay time constant were slightly decreased by addition of caroverine (0.2 mM) to the perfusing fluid, the ratios to the control being 0.84 __ 0.12 (n = 5)and 0.84 ___ 0.14 (n = 7), respectively. However, there was no statistically significant difference between the control and the caroverine-treated preparation. The channel block theory predicts that transformation from the active open channel to the blocked open channel is a function of the blocker concentration ~. Therefore, it is desirable to examine the effect of caroverine at higher concentrations. However, it was not possible to perform the experiments because local contracture can be overcome by detubulation. The above results suggest that caroverine is an open channel blocker for glutamate, although the drug did not satisfy all necessary criteria for the open channel blocker. The decay time constant of the glutamate current tail was slightly decreased in the presence of caroverine and biphasic decay was sometimes observed. When a
conditioned glutamate current was examined, the amplitude of the test response was more affected than the first in the presence of caroverine. The response to bath applied glutamate was not maintained but declined during its constant application in the presence of caroverine. The model that has most commonly been proposed to account for the properties of channel blocking drugs in the following, A + R.
"~AR .---'-~AR* . ~
CAR*
In this scheme, A is an agonist molecule, R the receptor in its resting state, R* the receptor in its active open state, and C is the blocking agent, a /3 are the rate constants. Chlorisondamine is known as an open channel blocker in crustacea8:7 and depressed both the amplitudes of e.j.ps and glutamate responses to the same degree. In this case it seems that both values of the association rate constant (a) and the dissociation rate constant (/3) are very large. On the other hand, caroverine affected e.j.ps less than the glutamate current. Since exposure of the neurally released transmitter to receptors is much shorter than that of ionophoretically applied agonist, if both the values of a and/3 are small, it may be possible to explain the smaller effect ofcaroverine on e.j.ps. The experimental result that recovery from the refractory form of the glutamate receptor was only slightly delayed by caroverine suggests a relatively small value of/3. We have to consider another explanation for the caroverine action as well. It is that glutamate is not an excitatory transmitter at the crayfish neuromuscular junction. Many pharmacological problems at this site remain unsolved 13,17. Further studies of glutamate inhibitors will be required to understand the possible role of glutamate at this site. The authors wish to thank Dr. Y. Kudo, Mitsubishi-Kasei Institute of Life Sciences, for a gift of caroverine. This work was supported in part by Grant-in-Aid for Scientific Research, The Ministry of Education, Science and Culture.
177 1 Adams, P. R., Drug blockade of open end-plate channels, J. Physiol. (Lond.), 260 (1976) 531-552. 2 Albuquerque, E. X. and Gage, P. W., Differential effects of perhydrohistrionicotoxin on neurally and ionophoretically evoked endplate currents, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 1596-1599. 3 Dudel, J. and Kuffler, S. W., The quantal nature of transmission and spontaneous miniature potentials at the crayfish neuromuscular junction, J. Physiol. (Lond.), 155 (1961) 514-529. 4 Feltz, A., Large, W. A. and Trautmann, A., Analysis of atropine action at the frog neuromuscular junction, J. Physiol. (Lond.), 269 (1977) 109-130. 5 Ishida, M. and Shinozaki, H., Differential effects of diltiazem on glutamate potentials and excitatory junctional potentials at the crayfish neuromuscular junction, J. Physiol. (Lond.), 298 (1980) 301-319. 6 Kawagoe, R., Onodera, K. and Takeuchi, A., Release of glutamate from the crayfish neuromuscular junction, J. Physiol. (Lond.), 312 (198 l) 225-236. 7 Kawagoe, R., Onodera, K. and Takeuchi, A., On the quantal release of endogenous glutamate from the crayfish neuromuscular junction, J. Physiol. (Lond.), 322 (1982) 529-539, 8 Lingle, C., Eisen, J. S. and Marder, E., Block of glutamatergic excitatory synaptic channels by chlorisondamine, Molec. PharmacoL, 19 (1981) 349-353. 9 Mathers, D. A. and Usherwood, P. N. R., Concanavalin A blocks desensitization of glutamate receptors on insect muscle fibres, Nature (Lond.), 259 (1976) 409-411. 10 Onodera, K. and Takeuchi, A., Permeability changes produced by L-glutamate at the excitatory post-synaptic
membrane of the crayfish muscle, J. Physiol. (Lond.), 255 (1976) 669-685. I 10nodera, K. and Takeuchi, A., Distribution and pharmacological properties ofsynaptic and extrasynaptic glutamate receptors on crayfish muscle, Jr. Physiol. (Lond), 306 (! 980) 233- 250. 12 Rang, H. P., The action of ganglionic blocking drugs on the synaptic responses of rat submandibular ganglion cells, Brit. J. Pharmacol., 75 (1982) 15 l- 168. 13 Shinozaki, H., The pharmacology of the excitatory neuromuscular junction in the crayfish, Progr. Neurobiol., 14 (1980) 121-155. 14 Shinozaki, H. and Ishida, M., Pharmacological distinction between the excitatory junctional potential and the glutamate potential revealed by concanavalin A at the crayfish neuromuscular junction, Brain Research, 161 (1979) 493- 501. 15 Shinozaki, H. and Ishida, M., Glutamate potential: differences from the excitatory junctional potential revealed by diltiazem and concanavalin A in crayfish neuromuscular junction, J. Physiol. (Paris), 75 (1979) 623- 627. 16 Shinozaki, H. and Ishida, M., The recovery from desensitization of the glutamate receptor in the crayfish neuromuscular junction, Neurosci. Lett., 21 (1981) 293-296. 17 Shinozaki, H., Ishida, M. and Mizuta, T., Glutamate inhibitors in the crayfish neuromuscular junction, Comp. Biochem. Physiol.,72C (1982) 249-255. 18 Takeuchi, A., Excitatory and inhibitory transmitter actions at the crayfish neuromuscular junction. In S. Thesleft (Ed.), Motor Innervation of Muscle, Academic Press, London, 1976, pp. 231-261.