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Acetylcholine metabolism and release at the nerve-electroplaque junction
Brain Research, 62 (1973) 543-549
543
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
A C E T Y L C H O L I N E METABOLISM A N D RELEASE AT THE NERVEELECTROPLAQUE J U N C T I O N
YVES DUNANT D~partement de Pharmacologie, 20, rue de l'Ecole de Mddecine, 1211 Geneva 4 (Switzerland)
For generations of scientists, electric fishes have been an object of curiosity, sometimes of experimentation, often of controversies 4,6,9,12,14,25. Among them Torpedo marmorata, a common species of marine elasmobranch, is provided with two electric organs which can deliver rather strong discharges (Fig. 1). They are made of stacks of richly innervated electroplaques which are homologous both morphologically and with regard to the chemical transmitter to the motor endplate of voluntary muscle. Indeed the electric organs have high concentrations of acetylcholine (ACh) and acetylcholinesterase; infusion of ACh produces an electric response and endogenous ACh can be collected during nerve stimulation 1°,11,24. In Torpedo as in other marine species 5, the electroplaques are not able to produce any action potential. Curare completely abolishes their response to direct field stimulation. Then the discharge appears as a pure summation of postjunctional potentials1,2,4,12,13. Thanks to the large number of nerve terminals, purely cholinergic synaptic vesicles were isolated and purified from the electric organ of Torpedo. They have been shown to contain an appreciable amount of the neurotransmitter substance 17,~8. It then became possible to study the turnover and release of ACh during nervous activity at a subcellular level. This was done in a series of experiments in Paris with J. Gautron, M. Israel, B. Lesbats and R. Manaranche, and in Geneva with E. BabelGu6rin and P. Jirounek. Electrogenic tissue was stimulated in vivo after careful dissection of 1 of the 4 electric nerves (see Fig. 1); pieces of organ were also excised from the Torpedo and stimulated in vitro either through their nerve or by field stimulation8,1~. The response discharges were always recorded during the experiments to control the efficiency of stimulation. Glass microelectrodes were also used to record the local response of single electroplaques or the miniature postjunctional potentials. The latter showed several properties in common with their homologues of the neuromuscular junction 21. They were abolished by curare and their time course was prolonged by anticholinesterases (Fig. 2) confirming their cholinergic nature. Only a part of the total ACh of the electric organ was found to be associated with synaptic vesicles. In fact the transmitter is stored in two main compartments containing approximately equal amounts. Bound ACh is that which remains after the tissue has been homogenized; most of it is present in synaptic vesicles. Free ACh is
544
v. D U N A N [
7
// Fig. 1. Torpedo marmorata. Preparation of one of the two electric organs showing the 4 electric nerves coming out of the central nervous system. In inset, the natural discharge observed as a reflex after pinching the tail of the animal, and recorded in open circuit. Amplitude: 60 V.
the fraction hydrolysed by the strong esterases of the tissue when the nerve terminals are disrupted. Free ACh can therefore be determined as the difference between bound ACh and the total ACh found when the tissue is directly extracted with trichloroacetic acidS,16,1s,22,~3. When fragments of electric organ were incubated in the presence of a labelled precursor, the tissue was able to synthesize radioactive ACh in vitro. In the electric organ as in other preparations the localization of choline acetyltransferase, the synthesizing enzyme, seemed to be cytoplasmic 15,16. The new ACh reached both compartments but the specific radioactivity of free ACh remained always higher than that of bound ACh indicating that only a part of vesicular ACh is exchangeable with the free c o m p a r t m e n P 9,2°. This heterogeneity of bound ACh appeared due to a heterogeneity of each vesicle rather than to the presence of different populations of vesicles e3. When the electrogenic tissue was submitted to repetitive stimulation at frequencies ranging from 1/sec to 20/sec, the discharge fell off after 1500-2000 stimuli. Characteristically, the fall of the response always showed an intermediary plateau or a ' b u m p ' (Fig. 3). Measurements of ACh were done at the exhaustion of the discharge.
A C h RELEASE AT TI-IE NERVE-ELECTROPLAQUE JUNCTION
545
=
J
2mVL_ 2Orris
Fig. 2. Miniature postjunctional potentials in the electric organ of Torpedo. Upper picture: continuous record of miniatures with an extracellular microelectrode filled with 2 M NaC1. Other picture: superimposed oscilloscope traces showing miniatures recorded on the same piece of tissue with the same electrode in the presence (first line) or the absence (second line) of 10 -4 M neostigmine.
7.
546
DUNAN
1
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-
0
-g t,_ ¢0 e"
:6 -~
50-
u
o_
0
. . . . 0
I ,5
. . . .
I . . . . 10
I 15rain
400 Bound
0
. . . . 0
i 5
. . . .
I 10
. . . .
ACh
i 15rain
Fig. 3. Effect of repetitive field stimulation at the frequency of 1/sec on acetylcholine compartments in the electric organ of Torpedo. Upper graph, amplitude of the response discharge, falling down with the characteristic intermediary plateau or 'bump'. Other graph, corresponding variations of free acetylcholine and stability of bound acetytcholine during that stimulation. The values obtained in two different experiments are shown by the solid and interrupted lines respectively.
They showed a drop in the level of free ACh but neither that of bound ACh nor the number of synaptic vesicles counted on histological preparations was significantly modified. However, when stimulation was continued after exhaustion of the discharge or when measurements were made in the period of recovery, rather large changes (increases as well as decreases) of bound ACh and synaptic vesicles were observed. Stimulation was also given after preincubation with a labelled precursor. At the exhaustion of the response the specific radioactivity of ACh increased in the free compartment whose total amount decreased, indicating an acceleration of synthesis during the period of stimulation. The radioactivity of bound ACh was not modified even after several series of stimulation. The released transmitter was also analysed
ACh
RELEASE AT THE NERVE-ELECTROPLAQUE JUNCTION
547
in the superfusing solution and as choline in the tissue. Its radioactivity could only be explained as originating from the free compartment of ACh which was then considered as the most immediately available for release on stimulation7,s. The fact was corroborated in experiments where fragments of electric organs were incubated in the absence of calcium ions, known to be required for the release of neurotransmitter substances. Both the electrical response and the level of free ACh decreased and vanished, whereas bound ACh remained unchanged. When stimulation was given in vivo or in a normal solution, calcium ions entered the nerve terminals during their activity. Calcium is believed to play some role in transmission not only by triggering the release of the transmitter but also by regulating its intracellular repartitionL ACh compartments were also analysed at different stages during the fall of the discharge (Fig. 3 and ref. 19). The first exponential drop of the response was accompanied by a similar exponential drop of free ACh which reached a rather low level after 60--200 stimuli. Then occurred a rapid but transient synthesis of new ACh which was even able to increase the level of free ACh. This phase corresponds to the plateau or the 'bump' in the fall of the physiological response. Later on, free ACh decreased again and the discharge went to exhaustion. No significant changes of bound ACh were observed in these and in other similar experiments. Our conclusion that free ACh represents the most immediately available compartment of transmitter does not implicate an explanation of the mechanisms by which ACh is released from the nerve terminals. However, massive 'exocytosis' of the whole content of the total population of vesicles compensated by their rapid refilling was clearly excluded on the basis of our experiments using radioactivity. An exact description of the nature and properties of free ACh in the living terminals is not yet achieved. This compartment could be freely diffusible in cytoplasm, but also slightly bound to some subcellular component. Consequently, partial exocytosis by a small number of vesicles cannot be excluded. Other mechanisms can also be envisaged provided they take 3 facts into account: (1) The newly synthesized transmitter is the first available for release. (2) The recently liberated transmitter can be rapidly resynthesized and re-used at once. (3) ACh is probably released in the electric organ in a quantal fashion as it is at the neuromuscular junctionzl. In conclusion.
(1) The electric organ of Torpedo is very rich in acetylcholine (ACh), which is stored in two main compartments: bound ACh is associated with synaptic vesicles, free ACh is a labile fraction which is hydrolysed when the tissue is homogenized. Free ACh represents about half of the total ACh; its subcellular localization has not yet been elucidated. (2) Stimulation of the tissue in vivo and in vitro until exhaustion of the electrical response was accompanied by a drop in the level of free ACh, whereas neither the amount of bound ACh nor the number of synaptic vesicles were significantly altered.
548
¥. DUNAN I
(3) Both the free ACh and the physiological response diasppeared when the tissue was incubated in the absence of calcium. (4) During the activity there was a rapid but transient acceleration of ACh synthesis which partly compensated the loss of free ACh and modified the fall of the physiological discharge. (5) On pre-incubation of the tissue with labelled precursors, the acetylcholine stores became radioactive. The specific radioactivity of free ACh fluctuated on serial stimulations, whereas that of bound ACh remained stable under these conditions. (6) It is concluded that the free compartment of ACh is the most immediately available for release on stimulation. 1 ALBE-FESSARD,D., Propridtds 61ectriques passives du tissu 61ectrog~ne des poissons 61ectriques,
Arch. Sci. Physiol., 4 (1950) 413-434. 2 AUGER,D., ET FESSARD,A., Action du curare, de l'atropine et de l'6sdrine sur le prisme dlectrog~ne de la Torpille, C.R. Soc. Biol. (Paris), 135 (1941) 76-78. 3 BABEL-GUI~RIN,E., ET DUNANT, Y., Entr6e de calcium et libdration d'ac6tylcholine dans l'organe dlectrique de la Torpille, C.R. Acad. Sci. (Paris), 275 (1972) 2961-2964. 4 BENNETT, M. L. V., Comparative physiology: electric organs, Ann. Rev. Physiol., 32 0970) 471-528. 5 BENNETT, M. L. V., WURZEL, M., AND GRUNDFEST, H., Properties of electroplaques of Torpedo nobiliana, J. gen. Physiol., 44 (1961) 757-804. 6 CHAGAS, C., AND PALS DE CARVALHO, A. (Eds.), Bioelectrogenesis, a Comparative Survey of its' Mechanisms with Particular Emphasis on Electric Fishes, Elsevier, Amsterdam, 1959, 413 pp. 7 DUNANT, Y., GAUTRON,J., ISRAEL,M., LESBATS,B., ET MANARANCHE,R., Effet de la stimulation de l'organe 61ectrique de la Torpille sur les 'compartiments libre et lid' d'acdtylcholine, C.R. Acad. Sci. (Paris), 273 (1971) 233-236. 8 DUNANT, Y., GAUTRON, J., ISRAi~L, M., LESBATS,B., ET MANARANCHE,R., Les compartiments d'ac6tylcholine de l'organe dlectrique et leurs modifications par la stimulation, J. Neurochem., 19 (1972) 1987-2002. 9 FARADAY,M., Notice on the caracter and the direction of the electric force of Gymnotus, Phil. Trans. B, 129 (1839) 1-12. 10 FELDBERG, W., FESSARD, A., AND NACHMANSOHN,D., The cholinergic nature of the nervous supply to the electric organ of the Torpedo (Torpedo marmorata), J. Physiol. (Lond.), 97 (1940) 3P. 11 FELDBERG,W., AND FESSARD,A., The cholinergic nature of the nerves to the electric organ of the Torpedo (Torpedo marmorata), J. Physiol. (Lond.), 101 (1942) 200-216. 12 FESSARD,A., Some basic aspects of the activity of electric plate3, Ann. N. Y. Acad. Sci., 47 (1946) 501-514. 13 FESSARD,A., Recherches sur le fonctionnement des organes dlectriques. I. Analyse des formes de ddcharge obtenues par divers proc6d6s d'excitation, Arch. int. Physiol., 55 (1947) 1-26. 14 FESSARD, A., Les organes 61ectriques. In P. P. GRASSl~(Ed.), Trait~ de Zoologic, Tome Xlll, Masson, Paris, 1958, pp. 1143-1238. 15 FONNUM, V., AND MALTHE-SORENSSEN,O., Molecular properties of choline acetyltransferase and their importance for the compartmentation of acetylcholine synthesis. In P. B. BRADLEYAND R. W. BRIMBLECOMBE(Eds.), Biological and Pharmacological Mechanisms Underlying Behaviour, Progress in Brain Research, Vol. 36, Elsevier, Amsterdam, 1972, pp. 13-27. 16 ISRAEL, M., Localisation de l'acdtylcholine des synapses myoneurales et nerf-61ectroplaque, Arch. Anat. micr. Morph. exp., 59 (1970) 5-98. 17 ISRAEL,M., GAUTRON,J., ET LESBATS,B., Isolement des vdsicules synaptiques de l'organe dlectrique de la Torpille et localisation de l'ac,6tylcholine ~ leur niveau, C.R. Acad. Sci. (Paris), 266 (1968) 273-275. 18 ISRAEL, M., GAUTRON, J., ET LESBATS,B., Fractionnement de l'organe dlectrique de la Torpille: Localisation subcellulaire de l'ac6tylcholine, J. Neurochem., 17 (1970) 1441-1450. 19 ISRAi~L,M., LESBATS,B., ET MANARANCHE,R., Variations d'acdtylcholine en relation avec l'dvolution de la d6charge, pendant la stimulation de l'organe 61ectrique de la Torpille, C.R. Acad. Sci., (Paris), 275 (1972) 2957-2960.
ACh
RELEASE AT THE NERVE-ELECTROPLAQUE JUNCTION
549
20 ISRAF.L,M., HIRT, L., ET MASTOUR-FRACHON,P., M6tabolismes et 6changes d'ac6tylcholine dans les terminaisons nerveuses de l'organe 61ectrique de la Torpille, C. R. Acad. Sci. (Paris), 276 (1973) 2725-2728. 21 KATZ,B., The Release of Neural Transmitter Substances, Liverpool Univ. Press, Liverpool, 1969, 60 pp. 22 MARCHBANKS,R. M., ANDISRAgL,M., Aspects of acetylcholine metabolism in the electric organ of Torpedo marmorata, J. Neurochem., 18 (1971) 439-448. 23 MARCHBANKS,R. M., AND ISRAEL, M., The heterogeneity of bound acetylcholine and synaptic vesicles, Biochem. J., 129 (1972) 1049-1061. 24 MARNAY,A., Cholinest6rase dans l'organe 61ectrique de la Torpille, C.R. Soc. Biol. (Paris), 126 (1937) 573-574. 25 MATTEUCCI,C., Poisson 61ectriques. In Lemons sur les Ph~nom~nes Physiques des Corps Vivants, Masson, Paris, 1847, pp. 190-213.
543
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
A C E T Y L C H O L I N E METABOLISM A N D RELEASE AT THE NERVEELECTROPLAQUE J U N C T I O N
YVES DUNANT D~partement de Pharmacologie, 20, rue de l'Ecole de Mddecine, 1211 Geneva 4 (Switzerland)
For generations of scientists, electric fishes have been an object of curiosity, sometimes of experimentation, often of controversies 4,6,9,12,14,25. Among them Torpedo marmorata, a common species of marine elasmobranch, is provided with two electric organs which can deliver rather strong discharges (Fig. 1). They are made of stacks of richly innervated electroplaques which are homologous both morphologically and with regard to the chemical transmitter to the motor endplate of voluntary muscle. Indeed the electric organs have high concentrations of acetylcholine (ACh) and acetylcholinesterase; infusion of ACh produces an electric response and endogenous ACh can be collected during nerve stimulation 1°,11,24. In Torpedo as in other marine species 5, the electroplaques are not able to produce any action potential. Curare completely abolishes their response to direct field stimulation. Then the discharge appears as a pure summation of postjunctional potentials1,2,4,12,13. Thanks to the large number of nerve terminals, purely cholinergic synaptic vesicles were isolated and purified from the electric organ of Torpedo. They have been shown to contain an appreciable amount of the neurotransmitter substance 17,~8. It then became possible to study the turnover and release of ACh during nervous activity at a subcellular level. This was done in a series of experiments in Paris with J. Gautron, M. Israel, B. Lesbats and R. Manaranche, and in Geneva with E. BabelGu6rin and P. Jirounek. Electrogenic tissue was stimulated in vivo after careful dissection of 1 of the 4 electric nerves (see Fig. 1); pieces of organ were also excised from the Torpedo and stimulated in vitro either through their nerve or by field stimulation8,1~. The response discharges were always recorded during the experiments to control the efficiency of stimulation. Glass microelectrodes were also used to record the local response of single electroplaques or the miniature postjunctional potentials. The latter showed several properties in common with their homologues of the neuromuscular junction 21. They were abolished by curare and their time course was prolonged by anticholinesterases (Fig. 2) confirming their cholinergic nature. Only a part of the total ACh of the electric organ was found to be associated with synaptic vesicles. In fact the transmitter is stored in two main compartments containing approximately equal amounts. Bound ACh is that which remains after the tissue has been homogenized; most of it is present in synaptic vesicles. Free ACh is
544
v. D U N A N [
7
// Fig. 1. Torpedo marmorata. Preparation of one of the two electric organs showing the 4 electric nerves coming out of the central nervous system. In inset, the natural discharge observed as a reflex after pinching the tail of the animal, and recorded in open circuit. Amplitude: 60 V.
the fraction hydrolysed by the strong esterases of the tissue when the nerve terminals are disrupted. Free ACh can therefore be determined as the difference between bound ACh and the total ACh found when the tissue is directly extracted with trichloroacetic acidS,16,1s,22,~3. When fragments of electric organ were incubated in the presence of a labelled precursor, the tissue was able to synthesize radioactive ACh in vitro. In the electric organ as in other preparations the localization of choline acetyltransferase, the synthesizing enzyme, seemed to be cytoplasmic 15,16. The new ACh reached both compartments but the specific radioactivity of free ACh remained always higher than that of bound ACh indicating that only a part of vesicular ACh is exchangeable with the free c o m p a r t m e n P 9,2°. This heterogeneity of bound ACh appeared due to a heterogeneity of each vesicle rather than to the presence of different populations of vesicles e3. When the electrogenic tissue was submitted to repetitive stimulation at frequencies ranging from 1/sec to 20/sec, the discharge fell off after 1500-2000 stimuli. Characteristically, the fall of the response always showed an intermediary plateau or a ' b u m p ' (Fig. 3). Measurements of ACh were done at the exhaustion of the discharge.
A C h RELEASE AT TI-IE NERVE-ELECTROPLAQUE JUNCTION
545
=
J
2mVL_ 2Orris
Fig. 2. Miniature postjunctional potentials in the electric organ of Torpedo. Upper picture: continuous record of miniatures with an extracellular microelectrode filled with 2 M NaC1. Other picture: superimposed oscilloscope traces showing miniatures recorded on the same piece of tissue with the same electrode in the presence (first line) or the absence (second line) of 10 -4 M neostigmine.
7.
546
DUNAN
1
% 1oo
-
0
-g t,_ ¢0 e"
:6 -~
50-
u
o_
0
. . . . 0
I ,5
. . . .
I . . . . 10
I 15rain
400 Bound
0
. . . . 0
i 5
. . . .
I 10
. . . .
ACh
i 15rain
Fig. 3. Effect of repetitive field stimulation at the frequency of 1/sec on acetylcholine compartments in the electric organ of Torpedo. Upper graph, amplitude of the response discharge, falling down with the characteristic intermediary plateau or 'bump'. Other graph, corresponding variations of free acetylcholine and stability of bound acetytcholine during that stimulation. The values obtained in two different experiments are shown by the solid and interrupted lines respectively.
They showed a drop in the level of free ACh but neither that of bound ACh nor the number of synaptic vesicles counted on histological preparations was significantly modified. However, when stimulation was continued after exhaustion of the discharge or when measurements were made in the period of recovery, rather large changes (increases as well as decreases) of bound ACh and synaptic vesicles were observed. Stimulation was also given after preincubation with a labelled precursor. At the exhaustion of the response the specific radioactivity of ACh increased in the free compartment whose total amount decreased, indicating an acceleration of synthesis during the period of stimulation. The radioactivity of bound ACh was not modified even after several series of stimulation. The released transmitter was also analysed
ACh
RELEASE AT THE NERVE-ELECTROPLAQUE JUNCTION
547
in the superfusing solution and as choline in the tissue. Its radioactivity could only be explained as originating from the free compartment of ACh which was then considered as the most immediately available for release on stimulation7,s. The fact was corroborated in experiments where fragments of electric organs were incubated in the absence of calcium ions, known to be required for the release of neurotransmitter substances. Both the electrical response and the level of free ACh decreased and vanished, whereas bound ACh remained unchanged. When stimulation was given in vivo or in a normal solution, calcium ions entered the nerve terminals during their activity. Calcium is believed to play some role in transmission not only by triggering the release of the transmitter but also by regulating its intracellular repartitionL ACh compartments were also analysed at different stages during the fall of the discharge (Fig. 3 and ref. 19). The first exponential drop of the response was accompanied by a similar exponential drop of free ACh which reached a rather low level after 60--200 stimuli. Then occurred a rapid but transient synthesis of new ACh which was even able to increase the level of free ACh. This phase corresponds to the plateau or the 'bump' in the fall of the physiological response. Later on, free ACh decreased again and the discharge went to exhaustion. No significant changes of bound ACh were observed in these and in other similar experiments. Our conclusion that free ACh represents the most immediately available compartment of transmitter does not implicate an explanation of the mechanisms by which ACh is released from the nerve terminals. However, massive 'exocytosis' of the whole content of the total population of vesicles compensated by their rapid refilling was clearly excluded on the basis of our experiments using radioactivity. An exact description of the nature and properties of free ACh in the living terminals is not yet achieved. This compartment could be freely diffusible in cytoplasm, but also slightly bound to some subcellular component. Consequently, partial exocytosis by a small number of vesicles cannot be excluded. Other mechanisms can also be envisaged provided they take 3 facts into account: (1) The newly synthesized transmitter is the first available for release. (2) The recently liberated transmitter can be rapidly resynthesized and re-used at once. (3) ACh is probably released in the electric organ in a quantal fashion as it is at the neuromuscular junctionzl. In conclusion.
(1) The electric organ of Torpedo is very rich in acetylcholine (ACh), which is stored in two main compartments: bound ACh is associated with synaptic vesicles, free ACh is a labile fraction which is hydrolysed when the tissue is homogenized. Free ACh represents about half of the total ACh; its subcellular localization has not yet been elucidated. (2) Stimulation of the tissue in vivo and in vitro until exhaustion of the electrical response was accompanied by a drop in the level of free ACh, whereas neither the amount of bound ACh nor the number of synaptic vesicles were significantly altered.
548
¥. DUNAN I
(3) Both the free ACh and the physiological response diasppeared when the tissue was incubated in the absence of calcium. (4) During the activity there was a rapid but transient acceleration of ACh synthesis which partly compensated the loss of free ACh and modified the fall of the physiological discharge. (5) On pre-incubation of the tissue with labelled precursors, the acetylcholine stores became radioactive. The specific radioactivity of free ACh fluctuated on serial stimulations, whereas that of bound ACh remained stable under these conditions. (6) It is concluded that the free compartment of ACh is the most immediately available for release on stimulation. 1 ALBE-FESSARD,D., Propridtds 61ectriques passives du tissu 61ectrog~ne des poissons 61ectriques,
Arch. Sci. Physiol., 4 (1950) 413-434. 2 AUGER,D., ET FESSARD,A., Action du curare, de l'atropine et de l'6sdrine sur le prisme dlectrog~ne de la Torpille, C.R. Soc. Biol. (Paris), 135 (1941) 76-78. 3 BABEL-GUI~RIN,E., ET DUNANT, Y., Entr6e de calcium et libdration d'ac6tylcholine dans l'organe dlectrique de la Torpille, C.R. Acad. Sci. (Paris), 275 (1972) 2961-2964. 4 BENNETT, M. L. V., Comparative physiology: electric organs, Ann. Rev. Physiol., 32 0970) 471-528. 5 BENNETT, M. L. V., WURZEL, M., AND GRUNDFEST, H., Properties of electroplaques of Torpedo nobiliana, J. gen. Physiol., 44 (1961) 757-804. 6 CHAGAS, C., AND PALS DE CARVALHO, A. (Eds.), Bioelectrogenesis, a Comparative Survey of its' Mechanisms with Particular Emphasis on Electric Fishes, Elsevier, Amsterdam, 1959, 413 pp. 7 DUNANT, Y., GAUTRON,J., ISRAEL,M., LESBATS,B., ET MANARANCHE,R., Effet de la stimulation de l'organe 61ectrique de la Torpille sur les 'compartiments libre et lid' d'acdtylcholine, C.R. Acad. Sci. (Paris), 273 (1971) 233-236. 8 DUNANT, Y., GAUTRON, J., ISRAi~L, M., LESBATS,B., ET MANARANCHE,R., Les compartiments d'ac6tylcholine de l'organe dlectrique et leurs modifications par la stimulation, J. Neurochem., 19 (1972) 1987-2002. 9 FARADAY,M., Notice on the caracter and the direction of the electric force of Gymnotus, Phil. Trans. B, 129 (1839) 1-12. 10 FELDBERG, W., FESSARD, A., AND NACHMANSOHN,D., The cholinergic nature of the nervous supply to the electric organ of the Torpedo (Torpedo marmorata), J. Physiol. (Lond.), 97 (1940) 3P. 11 FELDBERG,W., AND FESSARD,A., The cholinergic nature of the nerves to the electric organ of the Torpedo (Torpedo marmorata), J. Physiol. (Lond.), 101 (1942) 200-216. 12 FESSARD,A., Some basic aspects of the activity of electric plate3, Ann. N. Y. Acad. Sci., 47 (1946) 501-514. 13 FESSARD,A., Recherches sur le fonctionnement des organes dlectriques. I. Analyse des formes de ddcharge obtenues par divers proc6d6s d'excitation, Arch. int. Physiol., 55 (1947) 1-26. 14 FESSARD, A., Les organes 61ectriques. In P. P. GRASSl~(Ed.), Trait~ de Zoologic, Tome Xlll, Masson, Paris, 1958, pp. 1143-1238. 15 FONNUM, V., AND MALTHE-SORENSSEN,O., Molecular properties of choline acetyltransferase and their importance for the compartmentation of acetylcholine synthesis. In P. B. BRADLEYAND R. W. BRIMBLECOMBE(Eds.), Biological and Pharmacological Mechanisms Underlying Behaviour, Progress in Brain Research, Vol. 36, Elsevier, Amsterdam, 1972, pp. 13-27. 16 ISRAEL, M., Localisation de l'acdtylcholine des synapses myoneurales et nerf-61ectroplaque, Arch. Anat. micr. Morph. exp., 59 (1970) 5-98. 17 ISRAEL,M., GAUTRON,J., ET LESBATS,B., Isolement des vdsicules synaptiques de l'organe dlectrique de la Torpille et localisation de l'ac,6tylcholine ~ leur niveau, C.R. Acad. Sci. (Paris), 266 (1968) 273-275. 18 ISRAEL, M., GAUTRON, J., ET LESBATS,B., Fractionnement de l'organe dlectrique de la Torpille: Localisation subcellulaire de l'ac6tylcholine, J. Neurochem., 17 (1970) 1441-1450. 19 ISRAi~L,M., LESBATS,B., ET MANARANCHE,R., Variations d'acdtylcholine en relation avec l'dvolution de la d6charge, pendant la stimulation de l'organe 61ectrique de la Torpille, C.R. Acad. Sci., (Paris), 275 (1972) 2957-2960.
ACh
RELEASE AT THE NERVE-ELECTROPLAQUE JUNCTION
549
20 ISRAF.L,M., HIRT, L., ET MASTOUR-FRACHON,P., M6tabolismes et 6changes d'ac6tylcholine dans les terminaisons nerveuses de l'organe 61ectrique de la Torpille, C. R. Acad. Sci. (Paris), 276 (1973) 2725-2728. 21 KATZ,B., The Release of Neural Transmitter Substances, Liverpool Univ. Press, Liverpool, 1969, 60 pp. 22 MARCHBANKS,R. M., ANDISRAgL,M., Aspects of acetylcholine metabolism in the electric organ of Torpedo marmorata, J. Neurochem., 18 (1971) 439-448. 23 MARCHBANKS,R. M., AND ISRAEL, M., The heterogeneity of bound acetylcholine and synaptic vesicles, Biochem. J., 129 (1972) 1049-1061. 24 MARNAY,A., Cholinest6rase dans l'organe 61ectrique de la Torpille, C.R. Soc. Biol. (Paris), 126 (1937) 573-574. 25 MATTEUCCI,C., Poisson 61ectriques. In Lemons sur les Ph~nom~nes Physiques des Corps Vivants, Masson, Paris, 1847, pp. 190-213.