The Electromotor System of Torpedo A Model Cholinergic System V.P. Whittaker* Abteilung Neurochemie, Max-Planck-Institnt fiir Biophysikalische Chemie, D-3400 GSttingen

The electric organ of Torpedo, besides providing abundant amounts of cholinoceptive post-synaptic membrane for the isolation of the acetylcholine receptor protein, is a rich source of cholinergic nerve terminals. Using perfused, innervated tissue blocks from which synaptic vesicles in different functional states can be isolated, much information can be obtained about synaptic-vesicle dynamics. So far this is consistent with the view that the synaptic vesicles are the source of transmitter released on stimulation and that uptake of newly synthesized transmitter by the vesicles is dependent on their having discharged their previous charge of transmitter in at least one cycle of exo, and endocytosis. Studies of the protein composition of the vesicle membrane, especially when combined with similar information about the external presynaptic membrane, purified samples of which are now available from synaptosome (T-sac) preparations, promise to throw new light on the molecular mechanism underlying vesicle exo-/endocytosis.

The human nervous system is the most ,complex biological system we know, and thus, to understand many aspects of neural function - axonal conduction, synaptic transmission, habituation, even learning and m e m o r y - w e must often turn to simpler organisms where the processes we are interested in are presented in forms more accessible to experimentation: the giant axons and synapses of squid, the large ganglion cells of sea slugs, snails and leeches, the electromotor systems of electric fish are just a few examples of preparations which have provided the neurobiologist with the model systems he needs to investigate nervous function at the cellular and molecular level. * Summary o f a lecture given at an international Symposium on Cholinergic Mechanisms held at La Jolla, Calif., USA, March 1977 606

For the study of cholinergic transmission, the electromotor synapse of Torpedo-closely related developmentally to the neuromuscular junction-provides just the simplified and greatly hypertrophied system needed. As was realized by Fritsch [1] and other early workers, the electrocytes of the Torpedodevelop from myoblasts, and the electric lobes, which contain the perikarya of the electromotor neurons, are homologous with the hypoglossal and other cranial motor nuclei. The adult organ, in fact, contains up to 1000 times more nervous tissue than muscle making possible the isolation of cholinergic synaptic components that would be difficult to isolate from the latter; although mammalian brain is 10 times and squid brain 1000 times richer in cholinergic components than muscle, many other transmitters are present in addition to acetylcholine whereas electric tissue is purely cholinergic [2]. Electrophysiological researches [3, 4] have confirmed that single electrocytes generate postsynaptic potentials (psps) quite comparable to those of the motor end plate; the discharge of the organ is neither more or less than the summation, in series and in parallel, of the individual psps. The essential similarity of the electromotor synapses to other chemical synapses has been confirmed electron microscopically [5, 6]. But apart from some early work on the cholinesterase of the organ [7] few biochemical studies were made until 1964 when Sheridan and Whittaker [8] later joined by Israel [9] succeeded in isolating synaptic vesicles from homogenates of the electric organ by density-gradient centrifuging. Calculations based on vesicle counts and the acetylcholine content of the vesicle fraction revealed remarkably high acetylcholine concentrations in these vesicles; about 40000 molecules per vesicle corresponding to a concentration within the vesicle core of 0.4 M acetylcholine and comparable to the concentration of catecholamines in chromaffin granules, Using zonal rotors, and a procedure based on some improvements of the original techniques introduced by Israel et al. Naturwissenschaften 64, 606-611 (1977)

9 by Springer-Verlag 1977

[10], milligram quantities of pure synaptic vesicles were isolated from electric tissue in single runs [11] and this method, with later variants, has formed the basis of our continuing studies of vesicle composition. The original estimate for the acetylcholine content of the vesicles [8] has been revised upward; 100 000 molecules of acetylcholine per vesicle is now believed to be about the limiting concentration, considerably more than is found in mammalian vesicles [12-14]. A second low-molecular-weight constituent of Torpedo synaptic vesicles, in addition to acetylcholine, is ATP [15]. The packaging of these two substances together in the same storage vesicle has an interesting parallel in the chromaffin granule, which contains ATP and adrenaline (or noradrenaline). Variations in the molar ratio of acetylcholine to ATP on stimulation and across the vesicle peak obtained in zonal density gradients after centrifuging [16] show that the vesicle population is, in general, not homogeneous, and this has been confirmed in morphological studies of progressively stimulated terminals [17, 18]. Electric organs have been used not only for the study of cholinergic function, but also for cholinoceptive function. The electrocytes are cholinoceptive cells; in the Torpedo the entire ventral surface (excluding invaginations) of these cells is covered with nerve terminals. It is thus not surprising that the electrocytes are a rich source (again, 1000 times richer than muscle) of the acetylcholine receptor [19, 20]. This article will be concerned with recent work on the electromotor system, much of it unpublished or in process of publication, by members of the Abteilung ftir Neurochemie. For further background information the reader is referred to recent reviews [21, 221.

Chemical Embryology of the Electromotor System Figure 1 shows~ in diagrammatic form, the arrangement of the electromotor system. The perikarya of the electromotor neurons are found in the electric lobes, paired, yellowish eminences on the brain stem immediately behind the cerebellum. About 50% of the lobe tissue is made up of these large cell bodies which can be isolated in bulk [23-25] by cell-fractionation techniques. It is thought that transmission at the axodendritic synapses is non-cholinergic, but the transmitter has not yet been positively identified. During the summer of 1976 we were fortunate in obtaining a complete series of Torpedo embryos and Figure 2 summarizes the main results [25]. The lobe is detectable at 10 mm embryo length as a protuberance of the neural tube; by 20 ram, the neuronal population is complete and cell division stops. Biochemical differentiation does not occur till

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Fig. 1. The main components of the electromotor system much later: at about 60 mm, after which there is a steady increase in choline acetyltransferase to just before birth. Synaptogenesis is a rather late event, taking place at 60-70 mm. The period between the end of morphological differentiation and the onset of chemical differentiation should be an interesting one for further study. In the organ, one of the main questions to be solved is: at what stage does the differentiation of the future electrocyte begin to deviate from that of muscle ? Do electrocytes differentiate from true myotubes, or is there a putative electroblast, resembling the myoblast, but different from it? Recent electron-microscopic studies [25] have confirmed earlier light microscopy [1] by showing that the development of electrocytes is indeed indistinguishable from that of muscle up to the formation of the myotubes : columns of typical myoblasts form from mesoderm; these contain myofibrils and undergo fusion to form.myotubes; and only then do the flattening and horizontal elongation with loss of myofibrils, characteristic of the final stage of differentiation of the electrocyte, take place. Biochemical evidence confirms this description; at 40ram, the myoblasts in the region of the future electric organ contain a typical muscle myosin; at 50ram, at which differentiation of the electrocyte is almost complete, the myosin has fallen to a third of its original value and appears also to have changed its character. The control mechanisms involved in this differentiation are at present obscure. Synaptogenesis in the electric organ begins at about 60 mm embryo length and is preceded by the differentiation of the postsynaptic membrane. Choline acetyltransferase rises slowly and is at only 50% of the adult value at birth. However, nerve fibres are present in the organ from the beginning. As the head of the embryo extends sideways, axons grow out from the lobe cells between the gill arches and lie between

Naturwissenschaften64, 606--611 (1977) 9 by Springer-Verlag1977

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Fig. 2. Morphological and chemical differentiation of the electric lobes and electric organ in Torpedo marmorata. This species is ovoviviparous, the embryos with attached yoke-sac (not shown) being retained within a ~uterus' until birth. Choline acetyltransferase (CAT) is a marker for cholinergic neurones; acetylcholinesterase (ACHE), in the electric organ, is mainly a marker for the developing post-synaptic membrane. There is some evidence for a two-step rise in CAT corresponding to the ingrowth of fine axon terminations between the electrocytes (at 60-70 mm embryo length) and a further rise as synaptogenesis is completed [25]

the columns of myoblasts which develop between the extending upper and lower sheets of mesoderm. These columns, each one representing a future stack of electrocytes, form like stalagtites, by the fusion of outgrowths of the ventral and dorsal layers of mesoderm. The process continues until there are as many columns of myoblasts as there will be stacks of electrocytes in the fully developed organ. Additional axons grow down the tracts; growth cones may be observed within the nerve trunks and later, between the electrocytes. If the technical problems of supply and in-vitro culture of embryos, tissues and cells can be solved, this preparation should prove a useful one for studying the factors involved in the differentiation of a cholinergic motoneuron and its accompanying cholinoceptive effector cell. The Perfused Innervated Electric Organ

The electric organ has many advantages, due to the richness of its cholinergic innervation, over heart, 608

sympathetic ganglia or muscle, as a model system in which to study the mechanisms of transmitter storage and release during rest and activity. Our first experiments [17, 18] were performed in vivo, using fish anaesthetized with Tricaine methane sulphonate and stimulating the electric organ through electrodes placed on the lobes. These showed that the terminals could be extensively depleted of transmitter, ATP, and vesicles by repetitive stimulation at 5 Hz and the recovery process followed_ It was noted that stimulation caused a diminution in the mean profile diameter of the surviving vesicle population which was reversed on recovery. By excising, freezing and crushing the electric tissue at various stages after stimulation or recovery, and extracting and separating the vesicles according to our standard procedure, changes in acetylcholine, ATP, and protein content of the isolated vesicle fraction could be measured and correlated with the observed whole tissue changes. H o w ever, the preparation lacked the advantages possessed by perfused preparations, in that the medium in contact with the presynaptic nerve terminals could not

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9 by Springer-Verlag 1977

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Fig. 4. Evidence for exocytosis/endocytosis of Torpedo synaptic vesicles using dextran particles as a marker. The effect of stimulation at 0.1 Hz is to cause the progressive appearance of a population of vesicles the mean profile diameter of which is about 20% less than that of those in unstimulated blocks and at the same time to cause labelingwith an endocytosismarker. Black blocks diameter distribution of profiles containing electron-dense particles. No. of profiles measured is given in parentheses [30]

be readily varied in composition and the application of radioactive precursors, in particular, was difficult to control; accordingly, a perfused electric-organ preparation was devised [26-30], consisting of a block of tissue comprising the territory of a single electrom o t o r nerve and its accompanying blood vessels. Experiments in vivo [17, 18] had shown that whereas full recovery of the response of the electric organ to repetitive stimuli after prolonged stimulation to exhaustion via the lobe might take several days and might therefore be dependent on axonal flow of materials into the electromotor synapses, partial recovery t o o k place rapidly~ It would thus appear that at least a proportion of synaptic vesicles are reutilized (Scheme II, Fig. 3), and that release of transmitter does not involve total exocytosis as would normal exocrine secretion (Scheme I, Fig. 3). In order to detect whether one or more cycles of exo- and endocytosis had occurred, recourse was had to the perfused preparation and dextran of mol. wt. 10 000, readily detectable as electron-dense granules in ultrathin sections, was included in the perfusion medium. There was little or no uptake of dextran into vesicles in unstimulated blocks, but by stimulating at 0.1 Hz which causes little depletion of vesicle numbers, up to 80% of the vesicles could be labeled with dextran particles. At the same time, the characteristic reduction of mean profile diameter took place as had previously been seen in vivo (Fig. 4). No dextran-filled cisternae were seen as would have been expected if

the Heuser and Reese [31] scheme (III, Fig. 3) was operating. When vesicles were isolated from stimulated blocks using zonal centrifugation, the normally single vesicle peak separated into two (Fig. 5), VP 1 and VP2. Analysis of profile diameters showed that the vesicle profiles containing dextran particles were confined to VP2 and the mean diameter of these profiles was about 20% less than that of VP 1 (Fig..5, insert). Moreover, dextran-containing profiles accounted for about 80% of the profiles with diameters in this range. The capacity of the two vesicle populations to take up newly formed transmitter was tested by adding either radioactive acetate or choline to the perfusion medium., It will be seen from Figure 5 that only VP 2 is significantly labeled. This shows that the vesicular uptake of newly synthesized transmitter is coupled to the release of previously stored transmitter through at least one cycle of exo/endocytosis. The presence of dextran in the perfusion fluid was not found to be essential for the separation of the two types of vesicle: the 0.2-M sucrose and the p H of the perfusion medium [32] appear to be the important factors. In recent years it has been suggested (for critical review and references see [33]) that the quantal release of acetylcholine might well be brought about by a ' g a t e ' in the external presynaptic membrane and that the role of vesicles might be simply that of an internal ' b u f f e r ' system whose function is to maintain the

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line is linked to loss of preexisting stores of acetylcholine by exo- and endocytosis, are fully consistent with the vesicular hypothesis. Additional evidence consistent with the vesicular hypothesis has been forthcoming from a study of a false transmitter, N-2-hydroxyethyl-N-dimethylpyrrolidinium [22] which is taken up into vesicles and released on stimulation. There thus seems to be no need to postulate a 'gate' or carrier molecule of unique design in the external membrane to account for the known facts of vesicle properties and transmitter release at the electromotor synapse.

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concentration of cytoplasmic acetylcholine. However, the results with Torpedo, namely, that as in brain [34] the vesicle population is metabolically heterogeneous and further, that vesicular uptake of acetylcho610

The proteins of Torpedo electric organ synaptic vesicles have been reexamined using improved methods [35]. Using fiat-bed polyacrylamide-gel electrophoresis (Fig. 6) the main protein components of a number of preparations of vesicles made by different methods have been compared. It has been found that vesicles prepared in a single zonal run from a cytoplasmic extract of frozen and crushed electric organ and essentially free from contamination by morphological criteria contain several minor components and one major component (identified as tubulin) which are absent if the vesicles are first submitted to preliminary purification on a step gradient (compare gels 1 and 2, Fig. 6). It has previously been recognized [11, 14] that trace contamination of vesicles by soluble proteins and membrane fractions is not eliminated by a single zonal run. These highly purified vesicles contain no myosin (cf. [36]), but they do contain actin. When vesicles were isolated from terminals that had been stimulated at 0.1 Hz for 5 h, a condition which causes little change in vesicle numbers but under which at least 80% of vesicles have undergone at least one cycle of exo-/endocytosis, some small but significant changes were seen in the gel pattern; component S (tool. wt. 160000) was reduced relative to the other components and a protein apparently identical with the heavy chains of myosin now made its appearance (Mh in gel). One may perhaps speculate that actin, which it has so far not been possible to remove from the vesicle preparation, is a genuine constituent perhaps in the form of attached fibrils, and that reformation of vesicles during stimulation requires that activation of these fibrils by the attachment of myosin. Clathrin (tool. wt. 180000), the coat protein of coated vesicles [37] was not present in either type of vesicle. These results, especially when combined with similar information about the presynaptic plasma membrane, now available from synaptosomal (T-sac) preparation [38] promise to throw new light on the molecular mechanisms underlying vesicle exo-/endocytosis.

Naturwissenschaften 64, 606-611 (1977)

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The presence of a Mg z+/Ca 2+-activated ATPase in Torpedo vesicles has been demonstrated [39, 40]; in the presence of an optimum (5 mM) Mg z + concentration, acetylcholine (20 raM) produced a further 20% activation of the enzyme, but choline had no effect. This suggests that the ATPase may be part of a transport mechanism for acetylcholine.

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1. Fritsch, G.: Die elektrischen Fische. Leipzig: yon Veit 1890 2. Feldberg, W., Fessard, A.: J. Physiol. 101, 200 (1942) 3. Fessard, A., in: Trait6 de Zoologie, Vol. 13, p. 1143 (ed. P.P. Grass6). Paris: Masson 1958 4. Grundfest, H.: Prog. Biophys. Molec. Biol. 7, 1 (1959) 5. Luft, J.H.: Exp. Cell Res. Suppl, 5, 158 (1958) 6. Mathewson, R., eta1., in: Bioelectrogenesis, p. 25 (eds. C, Chagas, A. Paes de Carvalho). Amsterdam: Elsevier 1959 7. Nachmansohn, D., Lederer, E. : Bull. Soc. Chim. Biol. (Paris) 21, 797 (1939) 8. Sheridan, M.N., Whittaker, V.P.: J. Physiol. 175, 25P (1964) 9. Sheridan, M.N., et al.: Z. Zellforsch. 74, 291 (1966) 10. IsraN, M., et al.: J. Neurochem. 17, 144t (1970) 11. Whittaker, V.P., et al.: Biochem. J. 128, 833 (1972) 12. Whittaker, V.P., Sheridan, M.N. : J. Neurochem, 12, 363 (1965) 13. Wilson, W.S., et al. : ibid. 20, 659 (1973) 14. Nagy, A., et al. : Brain Res. 109, 285 (1976) 15. Dowdall, M.J., et al.: Biochem. J. 140, 1 (1974) 16. Dowdall, M.J., Zimmermann, H.: Brain Res. 71, 160 (1974) 17. Zimmermann, H., Whittaker, V.P.: J. Neurochem. 22, 435 (1974) 18. Zimmermann, H., Whittaker, V,P.: ibid. 22, 1109 (1974) 19. Raftery, M.A., et al., in: Biochemistry of Sensory Functions, p. 541 (ed. L. Jaenicke). New York" Springer 1975 20. Heilbronn, E., in: Cholinergic Mechanisms, p. 343 (ed. P.G. Waser). New York: Raven Press 1975 21. Whittaker, V.P., Zimmermann, H., in: Biochemical and Biophysical Perspectives in Marine Biology, Vol. 3, p. 67 (eds. D.C. Malins, J.R. Sargent). London: Academic Press 1976 22. Dowdall, M.J., et al. : Cold Spring Harbor Syrup. 40, 65 (1975) 23. Whittaker, V.P., et al. : Exp. Brain Res. 24, 22 (1975) 24. Fiore, L., Whittaker, V.P. : Abstr. 5th int. Meet. int. Soc. Neurochem. Barcelona, p. 130 (1975) 25. FOx, G.Q., etal.: Hoppe Seyler's Z. physiol. Chem. 358, 234 (1977) 26. Zimmermann, H., Dowdall, M.J. : Exp. Brain Res. 24, 23 (1975) 27. Zimmermann, H., Dowdall, M.J.: Abstract. 5th int. Meet. int. Soc. Neurochem. Barcelona, p. 128 (1975) 28. Zimmdi'mann, H., Dowdall, M.J. : Exp: Brain Res. 23, (Suppl.), 225 (1975) 29. Zimmermann, H., Baker, R.R.: Pfliigers Arch. 359 (Suppl.), R82 (1975) 30. Zimmermann, H. : Habilitationsschrift, G6ttingen 1976 31. Heuser, J.E., Reese, T.S.: J. Cell Biol. 57, 315 (1973) 32. Babel-Gu&in, E.:J. Neurochem. 23, 525 (1974) 33. Macintosh, F.C., Collier, B.: Hdb. exp. PharmakoL 42, 99 (1976) 34. Barker, L.A., et al.: Biochem. J. 130, 1063 (1972) 35. Stadler, H., Tashiro, T.: Hoppe-Seyler's Z. physiol. Chem. 358, 311 (1977) 36. Berl, S., et al. : Science 179, 441 (1973) 37. Pearse, B.M.F.: Proc. Nat. Acad. Sci. USA "73, 1255 (1976) 38. Dowall, M.J., Zimmermann, H.: Exp. Brain Res. 24, 7 (1975) 39. Breer, H. : Hoppe-Seyler's Z. physiol. Chem. 358, 219 (1977) 40. Hosie, R.J.A. : Biochem. J. 96, 404 (1965)

9 by Springer-Verlag 1977

Received March 18, 1977 611