J Physiology (Paris) (1998) 92, 53-57 © Elsevier, Paris

Arcachon and cholinergic transmission

Victor P. Whittaker*

Max-Planck-lnstitut fiir biophysikalische Chemic, D-37070 GOttingen, Germany

Abstract -- The cholinergic nature of transmission at the electromotor synapse of Torpedo marmorata was established at Arcachon in 1939 by Feldberg, Fessard and Nachmansohn (J. Physiol. (Lond.) 97 (1939/1940) 3P-4P) soon after transmission at the neuromuscular junction had been shown to be cholinergic. In 1964, after a quarter of a century of neglect, workers in Cambridge, then in Paris, Gtittingen and elsewhere, began to use this system, 500-1000 times richer in cholinergic synapses than muscle, for intensive studies of cholinergic transmission at the cellular and molecular level. (@Elsevier, Paris)

Resum6 -- Arcachon et la transmission cholinergique. La nature cholinergique de la transmission ~ la synapse 61ectromotrice de la torpille a 6t6 6tablie ~ Arcachon en 1939 par Feldberg, Fessard et Nachmansohn (J. Physiol. (Lond.) 97 (1939/1940) 3P-4P), peu aprrs la drcouverte du caractrre cholinergique de la transmission h la jonction neuromusculaire. Aprrs une prriode assez longue de d6su6tude, les chercheurs de Cambridge, Paris, Grttingen et d'ailleurs ont commencr, vers 1964, h utiliser ce systrme, 500-1000 fois plus riche en terminaisons cholinergiques que le muscle, pour 6tudier intensivement la transmission cholinergique au niveau cellulaire et molr- culaire. (@Elsevier, Paris) Torpedo marmorata I electric organ / electromotor synapse / cholinergic transmission

1. Introduction However, in terms of availability, amount of tissue per specimen and density of synaptic material, the The electric ray, Torpedo marmorata, is fairly Torpedinidae, especially T. marmorata (Eastern At- common in the Bay of Biscay (Baie de Gascoigne): lantic), T. ocellata (Mediterranian) and T. californica although a slow breeder (6-12 young every 2 years), (Pacific), are the family of choice. it is not fished commercially. Its chief interest for Electric organs have excited the interest of bio- the neurobiologist is that it possesses paired electric logists since earliest times: the puzzling nature of organs on each side of the midline which are capable their discharge was only resolved with the discovery of giving shocks (up to 40 V in air), strong enough of electricity and indeed led to the invention of the to stun prey and deter predators. These organs are battery or Voltaic pile which A. Volta (for attribu- under the control of the electromotor nerves which originate in the electric lobes, paired nuclei on the tions in this form, see Whittaker [20] for original stem placed caudally to the cerebellum. The references) himself described, in a memoire to the electromotor are in turn under the control Royal Society of London published in 1800, as an of the oval nucleus in the brain-stem. This integrates artifical electric organ. J. Bernstein correctly postu- sensations of hunger, detection of prey and nocicep- lated in 1912 that the discharge was caused by a tive stimuli and decides whether to release a train transient increase in the permeability of the electro- of electric shocks (short trains at a frequency of plaque membrane to positively charged ions. 100-300 Hz). The electric organ consists of stacks It has been known since the 1870s, mainly from of flattened cells known as electrocytes, electropla- the work of A. Babuchin in 1876, that the electro- ques or electroplax; these are densely innervated on cytes are derived embryonically from myoblasts; it their ventral surfaces by fine branches of the elec- is therefore not surprising that the electromotor sy- tromotor . napse is cholinergic. Grundfest [5] in a definitive Torpedo spp. are not the only fish to have electric review, reinterpreted Bernstein's theory of the organs; members of at least seven other diverse fa- discharge in the light of contemporary knowledge milies have acquired them by a process of conver- of the excitation of muscle; he described it as the gent evolution (for a recent review see [21]). summation in series and in parallel of normal exci- tatory postsynaptic potentials (EPSPs) evoked by the release of transmitter from the presynaptic nerve ter- minals. Unlike some other electric organs, the elec- *Address for corre~spondence: 197 Huntingdon Road, Cam- trocytes of Torpedinidae are electrically inexcitable; bridge CB3 0DL, UK thus the discharge in this type of electric organ is 54 V. R Whittaker

pure summed EPSP unmodified by a conducted res- substance from the electromotor nerve terminals, ponse corresponding to a muscle action potential. probably , as in muscle. Apart from a few autonomic vascular endings, the Nachmansohn, a medical graduate of Berlin Uni- innervation of the Torpedo electric organ is purely versity, had received his early research training with cholinergic with a synaptic content 500-1000 times O. Meyerhof, who had done classical work on the that of muscle; indeed the organ resembles a huge energy-yielding metabolic reactions sustaining mus- mass of hypertrophied motor endplates. With cular contractions. Nachmansohn thought that bio- 400-500 g of tissue per fish to work with, the elec- chemical methods should be applied to nerve activity tric organ is an ideal resource for studies of the cel- and argued that if acetylcholine were indeed a trans- lular and molecular biology of all aspects of mitter at the neuromuscular junction, the released cholinergic transmission: the synthesis and storage transmitter must be destroyed by cholinesterase of transmitter, its release and postsynaptic action, its within the refractory period of muscle. With his stu- hydrolysis to inactive products (choline and acetate) dent-assistant Annette Mamay he showed that the and their eventual reutilization. The electric lobes, cholinesterase activity of the innervated portion of though available in smaller quantities (ca. 400 the frog sartorius muscle was several thousand times mg/specimen), contain large (-40 lam diameter) cho- greater than that of the non-innervated portion and linergic cell bodies studded with putative glutamer- great enough to destroy released acetylcholine within gic nerve terminals; they contain the complete the muscle's refractory period. Being aware that the genome for cholinergic function and the mechanisms electrocytes of electric tissue resembled motor end- for the formation and transport of synaptic vesicles. plates, he asked Mamay to test for the presence of In only one important respect does the electromotor cholinesterase in electric tissue. She found very high terminal differ from terminals in muscle (including levels of activity in this tissue, exceeding that of Torpedo muscle): its synaptic vesicles are larger (-90 muscle several hundred-fold. nm in diameter versus -50 nm in muscle). This fa- Fessard heard about these results and invited cilitates a study of their function which appears to Nachmansohn to work with him at Arcachon on che- be similar in all respects to that of their smaller con- mical transmission in the electric organ. However, geners. if the techniques the Dale group had used so suc- cessfully with muscle were to be applied to the electric organ, a third collaborator who could assay acetylcholine in tissue and perfusates and apply it 2. Discovery of the cholinergic nature of to the organ by close arterial injection would be transmission at the electromotor synapse needed. Accounts differ [20] on how Feldberg came to be The discovery that transmission at the electromo- invited to join Fessard and Nachmansohn but no-one tor synapse is cholinergic was made during 3 weeks better qualified than he could have been chosen, in of research in June 1939 as the coming World War view of his participation in the work on muscle. cast its shadow over Europe. Three neurobiologists The results showed unequivocally that transmis- of very different backgrounds came together at the sion at the electromotor synapse is cholinergic. Pub- Station Biologique d'Arcachon where Torpedo from lication was delayed by the outbreak of the Second the Bassin was readily available. They were A. Fes- World War in September 1939 and Nachmansohn's sard, an electrophysiologist from the College de hasty departure for America, but the results were France, Paris, D. Nachmansohn, a biochemist and eventually written up by Feldberg and were publish- German Jewish emigrant working at the Sorbonne, ed, together with some additional work Feldberg did Paris, and W. Feldberg, a pharmacologist and also in London on extracts of electric organ he brought a German Jewish emigrant working with H.H. Dale back with him, in a now classical paper in the Jour- in London. nal of Physiology [4]. Fessard had worked extensively with electric tis- Three lines of evidence justify the conclusion that sue (for literature citations see Feldberg and Fessard transmission at the electromotor synapse is choliner- [4]). He showed that excitation could only be gic. Firstly, extracts of electric organ under condtions brought about via the nerve - denervated tissue was which prevent the breakdown of acetylcholine con- unexcitable mechanically and electrically - and that tain a substance which precisely matches the action nerve action could be blocked by drugs (curare, of acetylcholine in a variety of assay systems: the eserine) that block the cholinergic transmission in frog rectus abdominis muscle; the dorsal muscle of muscle discovered by H.H. Dale, W. Feldberg and the leech; slowing of the frog's heart; a fall in the M. Vogt in 1936. He surmised that the discharge cat's blood pressure; adrenaline release from the must be triggered by the release of a depolarizing adrenal medulla. No other known substance would Xth International Symposium on Cholinergic Mechanisms 55

match acetylcholine in all of them. The substance preparations of synaptic vesicles were obtained. Pu- behaved like acetylcholine in its lability in alkaline rer preparations of the latter were subsequently ob- solutions and its sensitivity to cholinesterase. The tained by M. IsraEl on his return to France. acetylcholine equivalence of the substance (400 Meanwhile, the large-scale isolation of pure synaptic nmol g of tissue -1) is within the range found by later vesicles was made possible [25] by three technical authors. In the second line of evidence it is shown innovations: comminution of the tissue by crushing that what we can now assume to be acetylcholine after rendering it brittle by freezing it at low tem- is released into perfusates of the organ on stimula- peratures; extraction of the crushed tissue with so- tion and in the third, close arterial injection of au- lutions iso-osmotic to Torpedo fluids; and thentic acetylcholine excited discharges similar to high-resolution separation of the resultant cytoplas- those evoked by nerve stimulation. mic extracts on continuous density gradients in a Some years later Woodin (cited by Whittaker large-capacity zonal rotor. This enabled the compo- [19]) confirmed the identity of the acetylcholine-like sition, biophysics and recycling of synaptic vesicles substance in electric organ as acetylcholine by paper to be intensively studied [20]. Briefly, it was estab- chromatography and more recently, it has been un- lished by the G6ttingen group that acetylcholine equivocally identified by gas chromatography-mass exists in two pools: the cytoplasmic, where it is syn- spectrometry [18]. thesized, and the vesicular, in which it is stored and Nachmansohn continued to work on electric or- from which it is released. Small differences in den- gans in the US, but switched to the electric eel, Gym- sity and composition enabled three pools of vesicles notus electricus, a denizen of the Amazon. His views to be separated: largely empty vesicles newly arrived on the role of acetylcholine in the nervous system from the cell body by fast axonal transport [6], a began to deviate sharply from those of Dale and reserve pool and a stimulus-induced recycling pool most other neurobiologists (for a critique and refer- [29] which preferentially takes up acetylcholine ences, see Whittaker [20]); nevertheless, his group newly synthesized in the cytoplasm. By using cho- made many important contributions to cholinergic line analogues it was possible to label the cytoplas- neurobiology. The comparative electrophysiology of mic and the two transmitter-containing vesicular electric organs attracted sporadic attention during the pools differentially and to show that transmitter re- 1950s and with the coming of electron microscopy, leased by stimulation arises exclusively from the re- the fine structure of the electromotor synapse was cycling pool [7]. studied in several Torpedinidae: Narcine [17]; The vesicular uptake of acetylcholine is driven by T. marmorata [9, 13]. Nevertheless, the potentialities a proton gradient generated by a vacuolar-type of the electromotor system for the investigation of ATPase [28] and is facilitated by a vesicular acetyl- the cellular and molecular biology of cholinergic choline transporter (vAChT), the sequence of which transmission remained largely neglected for 25 is encoded within the first intron of the gene locus years. for choline acetyltransferase [3].

3.2. Synaptosomes and presynaptic plasma 3. The electromotor innervation of Torpedo as a membranes model cholinergic system Improved methods of preparing synaptosomes 3.1. The electromotor synapse." cytoplasmic and were developed both by our group and that of IsraEl; vesicular pools of acetylcholine when well sealed these were able to develop mem- brane potentials comparable to those in intact neu- My own involvement with the Torpedo electric rons [10, 11]. The specificity and properties of the organ started in 1963 soon after my discovery of choline uptake system were extensively studied [2, the synaptosome and the successful isolation from 23]. The plasma membranes are rich in a family of synaptosomes of pure preparations of mammalian minor gangliosides containing a sialylated N-acetyl- cortical synaptic vesicles, a proportion of which sto- galactosamine residue; these are specific for mam- red acetylcholine in amounts consistent with their malian cholinergic neurons and have permitted the being the morphological basis of quantal transmitter immunoisolation of cholinergic synaptosomes from release [24, 26]. In 1963 a colleague, R.D. Keynes, a mixed population (for review see [22]). suggested that Torpedo electric organ might be a bet- ter source of cholinergic vesicles than mammalian 3.3. Electromotor cell bodies and axons brain. The tissue proved difficult to homogenize, but with M.N. Sheridan and M. IsraEl [14, 27] synapto- The cell bodies of electromotor neurons are somes in low yield and reasonably homogeneous -40 ~tm in diameter and thus among the largest ver- 56 V.P. Whittaker

tebrate motor neurons known. They are readily iso- and mode of catalysis are known. The choline moie- lated and though shorn of their dendrites are well ty of acetylcholine is firmly held in a trough lined sealed. Their choline acetyltransferase activity is with 14 aromatic residues whose rt electrons interact twice that of cholinergic cell bodies in Aplysia and with the positive charge on the quaternary nitrogen about 40 times greater than mammalian ganglion of acetylcholine. Hydrolysis is effected by a charge cells, but only one quarter that of electromotor nerve relay system; the catalytic triad has been identified terminals. as Glu-327, His-440 and Ser-200. These widely se- The perikarya of these cells are rich in Golgi parated residues are brought together by folding membranes and synaptic vesicles and contain readily [151. translatable mRNA which code inter alia for several Massouli6 and coworkers (for review see Massou- proteins [12]. Choline acetyltrans- lid and Toutant [8]) have shown that - ferase is transported down the at the slow rate terase can exist in several forms in electric organ [1]; synaptic vesicles are transported at the fast rate and other tissues. The catalytic subunit (Gj) is a and take up acetylcholine only when they reach the highly glycosylated soluble globular protein of mo- terminal [6]. lecular mass 70-85 kDa. In tissues a tetramer (G4) The nerve terminals in the lobe are those of af- of this unit is normally conjugated through S-S ferents from the command or oval nucleus. They are bonds to a collagen tail giving an asymmetric form probably glutamergic and have been isolated in low (A4). Further aggregation via interaction of the tails yield by conventional methods. gives larger forms culminating in Al2, an aggregate of three A4 units. Such forms are extractable by so- 3.4. Cotransmission in the electromotor synapse lutions of high ionic strength. In another conjugate, dimeric catalytic units are linked through ethanola- Many cholinergic neurons are now known to uti- mine, glycan and glucosamine to phosphatidylinosi- lize various neuropeptides as co-transmitters. The tol (PI); this form is inserted via the lipid end of its electromotor neurons are no exception; as in mam- tail into lipoprotein membranes. Such tails are found malian brain and autonomic ganglia, VIP is the co- in several unrelated membrane-bound molecules transmitter. It is transported to the terminal in with the common property of being solubilized by dense-cored vesicles (for references see Whittaker PI-specific phospholipase C (PIPLC). [20]). Another vesicular constituent which may act as a cotransmitter or regulator is ATE present in ve- sicles in a 1:5 ratio to acetylcholine. 4. The development of the electromotor system

3.5. The nicotinic acetylcholine (nAChR) This has been extensively studied by J. Melllinger in Rheims and by the G6ttingen group (for refer- In one of the major advances of modern neuro- ences see Whittaker [20]). Nineteenth-century obser- biology the acetylcholine receptor in the muscle and vations have been confirmed and extended by electrocyte postsynaptic membrane is now known to electronmicroscopic, electrophysiological and bio- be a ligand-gated pentameric ion channel composed chemical techniques. Briefly, four stages in deve- of four polypeptide subunits, ~, [~, y, 5 of known lopment have been recognized: I, myogenic; II, sequences in which the ~ unit is represented twice. electrocytogenic; III, synaptogenic divided into Ilia, Early work was done on Electrophorus (Gymnotus) penetration of stacks of electrocytes by ingrowing electric organ but when it was realised (from about neurites, and IIIb, functional synaptogenesis. During 1975) that Torpedo membranes are much richer in phases II and Ilia, extensive apoptosis of electromo- receptor than Electrophorus, there was a shift to Tor- tor neurons takes place as neurites are matched with pedo spp. (7:. californica and T. marmorata) by the their targets. Two trophic factors have been identi- many groups working in this field. The complete re- fied: a heat-stable neuronotrophic factor essential for ceptor is a rivet-like structure which undergoes a til- neuronal survival and a heat-sensitive cholinotrophic ting conformational change as it opens and closes factor that stimulates the expression of the choliner- [16]. gic phenotype. The molecular structure of these fac- tors has not been identified. 3.6. Acetylcholinesterase

The type of cholinesterase present in electrocytes 5. Conclusion hydrolyses acetylcholine faster than other choline es- ters and is thus known as an acetylcholinesterase. It Although a vast amount of important work has has been fully sequenced and its tertiary structure been done with the electromotor system of Torpedo Xth International Symposium on Cholinergic Mechanisms 57

since its cholinergic character was first established [13] Sheridan M.N., The fine structure of the electric organ of Torpedo marmorata, J. Cell Biol. 24 (1965) 129-141. by Feldberg, Fessard and Nachmansohn, its poten- tiality is far from exhausted. While fish may not be [14] Sheridan M.N., Whittaker V.E, Isolated synaptic vesicles: morphology and acetylcholine content, J. Physiol. 175 so convenient to work with as small rodents, any (1964) 25P-26E difficulties in transporting and keeping them can be [15] Sussman J.L., Harel M., Frolow F., Oefner C., Goldman A., overcome by careful organization. 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