The Ionic Basis of Axonal Conduction in the Central Nervous System of Viviparus Contectus (Millet) (Gastropoda: Prosobranchi A)

J. Exp. Biol. (1972), 57. 41-53 41 With 10 text-figures printed in Great Britain

THE IONIC BASIS OF AXONAL CONDUCTION IN THE CENTRAL NERVOUS SYSTEM OF VIVIPARUS CONTECTUS (MILLET) (GASTROPODA: PROSOBRANCHI A)

BY D. B. SATTELLE A.R.C. Unit of Invertebrate Chemistry and Physiology, Department of Zoology, Downing Street, Cambridge

{Received 10 December 1971)

INTRODUCTION The giant axon of the squid nervous system (Young, 1963 a, b) has become the classical preparation for studies on the ionic basis of nerve excitation (cf. Hodgkin, 1951, 1964). The squid nervous system functions in a bathing medium which approxi- mates in composition to sea water in having a high concentration of sodium relative to that of potassium and other inorganic cations. The situation in this cephalopod mollusc is not, however, universally encountered in molluscan species. For example, the nervous tissues of freshwater lamellibranchs and gastropods are bathed in an extremely dilute blood of low sodium and relatively high calcium concentration (cf. Potts & Parry, 1964). This raises the question of the ionic basis of excitation in these animals. The freshwater lamellibranch Anodonta cygnea possesses the most dilute body fluids in the Animal Kingdom, with a sodium concentration which averages i5-6mM/kg (Potts, 1954). Axonal function is, nevertheless, maintained by a sodium-dependent mechanism (Treherne, Mellon & Carlson, 1969). It has been suggested that there exists, within the central nervous system, a reservoir of sodium ions available to maintain function in the larger diameter (2-6 /im) fibres when the tissue is exposed to sodium-deficient solutions for extended periods (Carlson & Treherne, 1969; Treherne, Carlson & Gupta, 1969). Blood of similarly low osmotic pressure (40-60 mM/1 sodium chloride) and a sodium concentration of about 30 mM/1. has been reported in the freshwater prosobranchs of the genus Viviparus (Obuchowicz, 1958; Little, 1965). Results of a previous investigation (Sattelle & Lane, 1972) have indicated that there is a rapid access to the axonal surface of sodium ions in the fluid bathing the outer surface of the central nervous system of V. contectus. No detailed information is available, however, on the ionic basis of excitation in this gastropod mollusc. The present investigation employs electrophysiological techniques in order to establish the possible role of various monovalent and divalent cations in the maintenance of axonal conduction in the pleural-supraintestinal connective of V. contectus. The results are discussed in the light of the findings obtained for other molluscs. D. B. SATTELLE C.G. A.A.C.

Fig. i. A simplified diagram of the central nervous system of Viviparus contectus showing the distribution of the main ganglia and their connectives in relation to the anterior portion of the alimentary canal. Ganglia are indicated as follows: C.G., cerebral; P.G., pedal; Pl.G., pleural; Sb.G., subintestinal; Sp.G., supraintestinal; V.G., visceral. Other abbreviations: A.A.C, anterior alimentary canal; C, pleural-supraintestinal connective. (X3'5 approx.)

MATERIAL AND METHODS Specimens of Viviparus contectus, obtained from L. Haig and Co. Ltd., were maintained in large aquaria containing tap water under continuous flow conditions. Snails survived for long periods with a low mortality rate. The central nervous system of Viviparus {Paludina) has been described by Bouvier (1887) and is distinctive for the presence of long connectives (Fig. 1; cf. also Fretter and Graham, 1962). The absence of large-diameter cell bodies and axons has been demonstrated (Gorf, 1961) and attempts at intracellular impalement of cell bodies and axons in this study have been unsuccessful. Investigation of the ionic basis of excitation in V. contectus is therefore confined to extracellular recording techniques. Experiments were performed on isolated, ligatured lengths (8-10 mm) of the pleural- supraintestinal connective (Fig. 1), dissected under Ringer from the unanaesthetized animal. The nerve sheath was not dissected away, since a previous investigation demonstrated a rapid access to the axonal surface of ions in the bathing medium (Sattelle & Lane, 1972). These studies on the ionic basis of axonal conduction have, therefore, been performed on the sheathed connective. This preparation was placed across the four bridges of a Perspex bath (Fig. 2), and petroleum jelly seals were applied to isolate the five compartments. Each chamber was filled with either Viviparus blood or Ringer, and stimulating and recording electrodes were inserted into the end compartments. Stimuli (i-o-5-omsec pulses) were delivered to the connective via an isolation transformer, and electrical responses were led to an Isleworth A 101 pre- amplifier, coupled to a Tektronix 502A or 561 oscilloscope. Records were filmed using a Cossor or Nihon-Kohden oscilloscope camera. Axonal conduction in C.N.S. of Viviparus 43

Vaseline seal / Connective

Fig. 2. The nerve chamber devised to obtain extracellular recordings from isolated pleural- supraintestinal connectives. Abbreviations: C, outlet channel; S.E., stimulating electrodes; R.E., recording electrodes.

Test solutions were introduced to the central chamber of the preparation bath (Fig. 2) by means of a syringe and removed via the outlet channel by a second syringe. In this process three complete rinses of the central chamber preceded a final change of the test fluid. The process could be completed within 10-15 sec- Solutions in all chambers were replaced every 15 min. Tests with coloured dyes showed that there was no leakage between compartments. The ionic composition of the blood of V. contectus is not known in detail. A Ringer solution has been devised, based on that used for V. viviparus (Little, 1965). This solution, which successfully maintains the activity of isolated connectives of V. contectus, has the following composition: 24-0 Mm/1 NaCl; 9-0 nrn/l NaHCO3; 1-2 mni/1 KC1; 5-5 mM/1 CaCl2; pH 7-4. Axonal diameters in the pleural-supraintestinal connective have been obtained using representative fields of electron micrographs (N. J. Lane & D. B. Sattelle, unpublished observations).

RESULTS The compound action potential The compound action potential, recorded from the isolated pleural-supraintestinal connectives (Fig. 3) has two distinct components, a large, slow, somewhat variable component with an average conduction velocity (6) of about 0-02 m/sec, and a faster component with a conduction velocity of o-io m/sec for the fastest fibres (figures for 0 at 23 °C). Fig. 3 relates the compound action potential to the axon population of the connective. The square of the conduction velocity (6Z) has been plotted against the potential in microvolts recorded for different components of the compound action potential. Rushton (1951) has shown that the conduction velocity (8) in non-myeli- nated fibres is proportional to the square root of the axon diameter; 62 is therefore a function of axonal diameter. These results have been compared with a plot of the 44 D. B. SATTELLE 80 -i

60 -

a 20 c/i

0005 0010 0015 61

20 30 Axon diameter (jvm) Fig. 3. An attempt to relate the compound action potential recorded from the pleural-supraintestinal connective of V. contectus to the axon population of the connective. The upper graph is a plot of spike amplitude (in microvolts) against the square of the conduction velocity (0a), for different components of the compound action potential. The scale bars referring to the displayed record of a compound action potential represent 100 msec (horizontal) and 100 fiV (vertical). The lower graph shows the distribution of axonal diameters in the connective. Data on axonal diameters has been obtained using representative fieldso f electron micrographs (N. J. Lane & D. B. Sattelle, unpublished observations). distribution of axon diameters within the pleural-supraintestinal connective (data obtained from representative fields of electron micrographs) following the method employed by Treherne, Mellon & Carlson (1969) for the cerebro-visceral connectives of Anodonta. In this way the fast component of the compound action potential was related to the small population of larger-diameter fibres and the larger, slow component to the bulk Axonal conduction in C.N.S. of Viviparus 45 Blood Ringer 1-2 F*>

: o-8 i 60 [0-6 3 40 •0-4 20 0-2

a. .4567 10 t/3 Time (h) Fig. 4. Graph of relative conduction velocity (tftest/^normni) and spike amplitude (measured in microvolts) for the fast component of the compound action potential plotted against the time in hours. Viviparus blood in the test chamber is replaced by normal Ringer after 30 min. of smaller axons in the connective. Isolated connectives produced compound action potentials in Viviparus blood or Ringer for many hours, with only a slight fall in relative conduction velocity* and spike amplitude. Fig. 4 shows the change with time of these two parameters for the fast component of the compound action potential.

Effects of sodium-free solutions When equivalent concentrations of tris chloride and choline chloride were used to replace the sodium salts in Viviparus Ringer, conduction block was rapidly achieved. With dextran replacing sodium salts, however, the activty of both fast and slow fibres persisted for extended periods (Fig. 5). This persistence of activity has been investigated with particular reference to the fast fibres in which activity, blocked by perfu- sion with tris Ringer, was restored by exposure to dextran Ringer without further access to sodium ions in the bathing medium (Fig. 6). In such experiments slow fibres were also active in dextran Ringer but not in tris Ringer.

Effects of tetrodotoxin Tetrodotoxin (TTX) blocks action potentials in neurones by arresting the influx of sodium ions (Narahashi, Moore & Scott, 1964; Nakamura, Nakajima & Grundfest, 1965). TTX applied at concentrations of io"6 M, in normal Ringer and calcium-free Ringer did not produce conduction block, although there was some reduction of the compound action potential particularly in calcium-free Ringer. Applied in dextran Ringer at this concentration, TTX was able to produce conduction block (Fig. 7). At io~6 M, and higher concentrations, complete cessation of action potentials was achieved for all groups of fibres (Fig. 8). Under these conditions TTX acted more

• Relative conduction velocity (^test/^aormai) of a given component of the compound action potential is the ratio of the conduction velocity obtained in the test solution to that obtained in normal Ringer. D. B. SATTELLE

Substitute for Normal Sodium-free Ringer Normal sodium salts Ringer Ringer

Tris chloride

Choline chloride T

Dextran t T V Omin 30 sec Smin Fig. 5. Effects on the compound action potential of replacing the sodium salts of normal Ringer by isotonic concentrations of tris chloride, choline chloride and dextran. Scale bars represent 500 msec (horizontally) and 100 fiV (vertically).

Sodium Ringer Tris Ringer Dextran Ringer 1 Sodium Ringer

1-2 -1

•—•-

0-4 -

0-2 -

0 -1

1 1 1 15 30 45 60 Time (min) Fig. 6. Recovery of fast action potentials in dextran Ringer following exposure to tris Ringer. A plot of the relative conduction velocity of the fast fibres against time with normal Ringer, tris Ringer and dextran Ringer as the bathing solutions. Axonal conduction in C.N.S. of Viviparus 47

Ringer Ringer+TTX (10"6M) Ringer Ringer I r composition

Normal

Sodium-free

Omin 30 sec 5min 1 min

Fig. 7. Effects of tetrodotoxin (io~6 M) on the compound action potential. Scale bars represent 500 msec, (horizontally) and 200 /tV (vertically).

Ringer Ringer+TTX (1Q-5M) Ringer Ringer composition

Normal

Calcium-free

Sodium-free

Omin 30 sec 5 min 1 min Fig. 8. Effects of tetrodotoxin (io~5 M) on the compound action potential. Scale bars represent 500 msec (horizontally) and 300 /tV (vertically). D. B. SATTELLE

Table i Fast action potentials in different Ringer solutions Action potentials at various time intervals following sodium or calcium substitution

5 mm 30 min Sodium Na+ substitutes LJ+ Tris+ Choline+ TEA+ Dextran Calcium Ca2+ substitutes Mga+ Baa+ Sucrose The presence (+) and absence (—) of fast action potentials is recorded at various times following the substitution of either sodium or calcium salts. Apart from the substitutions indicated, other components of the Ringer are as described for normal Viviparus Ringer. rapidly in sodium-free (dextran) Ringer than in calcium-free Ringer. Applied in normal Ringer, TTX (at io~6 M) acted more slowly than in calcium-free Ringer. The effects of tetrodotoxin at both concentrations were reversible.

Effects of various cations The following sections deal with the effects on conduction processes of replacing the sodium and calcium of normal Ringer by various cationic and non-electrolyte substitutes. Experiments are confined to studies on the less variable fast component of the compound action potential which has been related to the larger-diameter axons of the connective (Fig. 3), and the results are summarized in Table 1.

(a) Effects of monovalent cations Replacing the sodium salts in Viviparus Ringer by isotonic concentrations of tris chloride and choline chloride produced, in both cases, a rapid conduction block in the fast axons (Fig. 5 and Table 1). Similarly, tetraethylammonium chloride (TEA-Cl), which appears to act as a sodium substitute in certain excitable cells (cf. Tasaki, 1968), did not support fast action potentials (Fig. 9). TEA-Cl produced cessation of activity in the fast fibres of Viviparus much more rapidly than in the fast fibres of Anodonta (Carlson & Treherne, 1969). Lithium has been observed to substitute for sodium in a number of conventional excitable systems (cf. Hodgkin, 1951; Keynes & Swan, 1959). When lithium chloride replaced the sodium salts in Viviparus Ringer, the fast action potentials were evoked for a 30 min. period with very little decline in their conduction velocity (Fig. 9).

(b) Effects of divalent cations The calcium chloride component of normal Ringer was replaced by sucrose, barium chloride and magnesium chloride, at concentrations which maintained iso- tonicity. Complete conduction block was in no case observed (cf. Table 1) although Axonal conduction in C.N.S. of Viviparus 49

Ringer Sodium-free Ringer Ringer

1-2

r -O o — S—-8 0-8

Li • : 0-6 TEA-

0-2

15 30 45 60 Time (min) Fig. 9. Effects on the fast action potentials of replacing the sodium salts of normal Ringer by isotonic concentrations of tetraethyl-ammonium (TEA) chloride and lithium chloride expressed as plots of the relative conduction velocity (tftest/^normai) against time (min).

Mn2+ (5 mM/1)

10 20 30 40 Time (min) Fig. 10. Effects on the size of the fast action potential of the addition of manganese chloride (5 mM/1) to normal Ringer. the effect of all such calcium substitutions was to produce a slight reduction in amplitude of the fast action potential. Manganous ions, which have been shown to block calcium-dependent conduction processes (Hagiwara & Nakajima, 1965), slightly reduced the spike amplitude of the fast fibres when applied at a concentration of 5*0 mM/1 in normal Ringer (Fig. 10).

DISCUSSION The compound action potential obtained from the pleural-supraintestinal connective of Viviparus contectus consists of fast and slow components associated with axon populations of large and small diameter respectively. The form of the action potential EXB 57 50 D. B. SATTELLE is similar to that obtained for the cerebro-visceral connectives of the lamellibrancri molluscs Cristaria plicata (Nakajima, 1961) and Anodonta cygnea (Treherne, Mellon & Carlson, 1969). The slow fibres of Viviparus have an average conduction velocity {6) of about c-02 m/sec (at 23 °C), similar to the figures (between 0-03 and 0-04 m/sec) obtained for Anodonta slow fibres (Treherne, Mellon & Carlson, 1969). The fast component, with a maximum value for 6 of o-io m/sec (at 23 °C) is rather slower than Anodonta fast fibres, and much slower than the maximum values of 6 reported for other gastropods investigated (cf. Bullock & Horridge, 1965). Figures for gastropod molluscs similar to those obtained for Viviparus fast fibres are the maximum values for 6 of 0-4 m/sec from the parapodial nerve (Frohlich, 1910) and c-50 m/sec (Krijgs- man, 1940) from the pedal nerve of Aplysia limacina. The rapid abolition of the compound action potential in tris and choline Ringer solutions indicates that the presence of sodium is important for normal action potential production in both fast and slow fibres of the connective of Viviparus. In addition, the observed blocking action of tetrodotoxin, a substance known to block the sodium conductance mechanism of conventional excitable tissues (Narahashi et al. 1964; Nakamura et al. 1965; and others), suggests that a sodium-dependent mechanism of spike generation operates. Sensitivity to tetrodotoxin is rather low (io~6 M), but comparable to that found in other gastropod molluscs in which sodium ions are involved in the inward current component of the action potential (Junge, 1967; More- ton, 1968 a). When tris and choline are employed to substitute for sodium in normal Ringer, conduction of the fast action potential is rapidly blocked. Likewise, tetraethyl ammonium (TEA) ions are unable to sustain the function of fast axons in the absence of sodium ions. This latter observation contrasts with the findings for frog nerve B and C fibres (Lorente de No, 1949), crustacean muscle (Fatt & Katz, 1951) and other conventional excitable systems (cf. Tasaki, 1968), where TEA, replacing sodium, maintains conduction processes for extended periods. TEA ions are able to maintain the function of the fast axons of Anodonta for very limited periods (10-15 min) (Carlson & Treherne, 1969). Lithium is a cation which can often replace sodium in conventional excitable systems (Hodgkin, 1951; Keynes & Swan, 1959) and it substitutes effectively for sodium in maintaining fast action potentials of Viviparus with little attenuation of conduction velocity during 30 min exposures. Comparable experiments on the giant neurones of the pulmonate gastropod Helix aspersa (Gardner & Kerkut, 1968) reveal that lithium supports action potentials fully for a period of about 15 min, after which cells quickly become inexcitable. Gardner & Kerkut proposed that lithium can carry the inward action current but that it does not stimulate the active sodium-pump and is not, therefore, a viable long-term substitute for sodium. It remains a possibility, however, that the sodium-pumps of the fast axons of Viviparus are incapable of distinguishing between sodium and lithium. Removal of calcium from the bathing medium slightly increases the sensitivity of axons of Viviparus to tetrodotoxin. This could be interpreted either as an effect of calcium on the binding of the toxin to its site of action in the nerve membrane, or, alternatively, as evidence for a small calcium contribution to the axonal action potential. When the calcium content of normal Ringer is replaced by sucrose, mag- Axonal conduction in C.N.S. of Viviparus 51 nesium or barium, conduction block is not produced in the larger axons even after '30 min exposures to calcium-free Ringer. Also manganous ions, which block calcium- dependent conduction processes (Hagiwara & Nakajima, 1965), produced a small reduction in the spike amplitude of fast axons when applied in normal Ringer at 5 mM/1. concentrations. These findings suggest that any role for calcium in carrying the action current of the fast action potential is very small compared to that of sodium. Such a strong dependence of action potential production upon the presence of sodium ions in the bathing medium is of interest in the light of the low sodium concentration observed in the blood of freshwater prosobranchs of the genus Viviparus (Obuchowicz, 1958; Little, 1965); this suggests that a conventional sodium gradient is maintained across the axolenuna by the presence of a very low intracellular level of sodium activity. The situation in V. contectus is not unparalleled in the molluscs, however, for a similarly conventional ionic basis of excitation has been described for axons of the cerebro-visceral connective of Anodonta cygnea. This freshwater lamellibranch possesses the most dilute blood in the Animal Kingdom (55 m-osmoles) with a sodium concentration of 15-6 mM/kg (Potts, 1954). The results of experiments on Viviparus connectives in which the sodium of normal Ringer is replaced by either electrolyte or non-electrolyte sodium-substitutes do not, however, appear entirely consistent with the picture presented so far of an axonal membrane with a conventional ionic basis of excitation functioning in an unregulated ionic micro- environment (cf. also Sattelle & Lane, 1972). Using tris or choline as the sodium substitute conduction block is rapidly achieved in all fibres. Dextran Ringer, however, maintains action potentials in all fibres for extended periods and will restore activity lost on exposure to tris Ringer without further access to sodium ions. That this does not reflect a change in the ion carrying the inward current of the action potential in dextran Ringer is indicated by the increased sensitivity of all axons to tetrodotoxin under these conditions. Similar results obtained for the larger axons of the connective of Anodonta have led to the proposal that part of the sodium available to maintain their function is associated with indiffusible anions situated close to the axon surface. Such a source of sodium would clearly be replaced by tris but not dextran (Treherne, Carlson & Gupta, 1969). In addition, these authors proposed a sequestered source of sodium close to the larger axons capable of restoring action potentials in dextran following their abolition in tris solutions. The site of such a sequestered store has been tentatively located in glial cells which, in the central nervous system of this lamellibranch, show a much more frequent association with the larger axons than with the smaller axons (Gupta, Mellon & Treherne, 1969). Radio-isotopic experiments on Anodonta revealed a small sodium fraction which does not normally exchange with labelled sodium in the bathing medium but which could be reduced during repetitive stimula- tion of the connective (Treherne, Carlson & Gupta, 1969). It is attractive to postulate a similar regulatory mechanism to account for the effects of electrolyte and non- electrolyte sodium-free solutions on Viviparus axons. There remain, however, two serious objections. First, in all axons of the pleural-supraintestinal connectives of Viviparus, activity is sustained by dextran Ringer, whereas this is only the case for the relatively small population of larger axons in Anodonta connectives. Secondly, a previous investigation revealed a sparse distribution of glial elements in the connective

4-2 52 D. B. SATTELLE of V. contectus and no evidence of a high incidence of glial association with the larger, axons (Sattelle & Lane, 1972). Further experimentation is required, therefore, on thJ effects of electrolyte and non-electrolyte sodium substitutes on axonal conduction processes in V. contectus.

SUMMARY 1. The compound action potential recorded from the pleural-supraintestinal connective of Viviparus contectus consists of a large, slow component with an average conduction velocity of about 0-02 m/sec (at 23 °C) and a faster component with a conduction velocity of o-io m/sec (at 23 °C) for the fastest fibres. 2. Both fast and slow action potentials are rapidly abolished by the substitution of tris chloride and choline chloride for the sodium salts of normal Ringer. Tetrodo- toxin, applied at io~5 M rapidly abolishes action potentials in all fibres.I t is, therefore, concluded that a largely sodium-dependent mechanism of spike generation operates in all axons of the connective. 3. Lithium ions effectively substitute for sodium ions in maintaining the fast action potentials for extended periods, whereas tetraethylammonium ions do not. 4. When the calcium chloride of normal Ringer is replaced by sucrose, magnesium chloride or barium chloride, conduction of fast action potentials is maintained. A small increase in the sensitivity of all axons to tetrodotoxin is observed in calcium-free Ringer; a slight reduction in the spike amplitude of fast action potentials follows the application of manganous ions at 5 mni/l in normal Ringer. It is concluded that any possible contribution of calcium to the generation of the action current of the fast action potential is very small compared to that of sodium. 5. All axons of the connective function for extended periods in sodium-free (dextran) Ringer. Under these conditions, tetrodotoxin blocks conduction in all fibres at concentrations of io~6 M, suggesting that function in dextran Ringer is maintained by a sodium-dependent mechanism.

I am indebted to Dr J. E. Treherne for his advice and constructive criticism throughout the course of this work and Dr R. W. Meech for his comments on the manuscript. I thank the Science Research Council for financial support.

REFERENCES BOUVIER, E. L. (1887). Systeme nerveux der prosobranches. Ann. Sci. Nat. Zool. 3, 1-510. BULLOCK, T. H. & HORRIDGE, G. A. (1965). Structure and Function in the Nervous Systems of Inverte- brates. San Francisco and London: Freeman. CARLSON, A. D. & TREHERNE, J. E. (1969). The ionic basis of the fast action potentials in the isolated cerebro-visceral connective of Anodonta cygnea. J. exp. Biol. 51, 297-318. FATT, P. & KATZ, B. (1951). Conduction of impulses in crustacean muscle fibres. J. Physiol. 115, 45.P. FRETTER, V. & GRAHAM, A. (1962). British Prosobranch Molluscs. London: Ray Society. FR&HLICH, F. W. (1910). Experimented Studien am Nervensystem der Mollusken. 13. l)ber die durch das Pedal Ganglion von Aplysia limacina, vermittiete ' Reflexverkettung'. Z. allg. Physiol. II, 351-70. GARDNER, D. R. & KERKUT, G. A. (1968). A comparison of the effects of sodium and lithium ions on action potentials from Helix aspersa neurones. Comp. Biochem. Physiol. 25, 33-48. GORF, A. (1961). Untersuchungen iiber Neurosekretion bei der Sumpfdeckelschnecke Vivipara vivipara L. Zool. Jb. 69, 379-404. GUPTA, B. L., MELLON, D., JR. & TREHERNE, J. E. (1969). The organization of the central nervous connectives in Anodonta cygnea (Linnaeus) (Mollusca: Eulamellibranchia). Tissue and Cell 1, 1-30. Axonal conduction in C.N.S. of Viviparus 53 HAGIWARA, S. & NAKAJIMA, S. (1965). Differences in Na and Ca spikes as examined by application of tetrodotoxin, procaine and manganese ions. J. gen. Physiol. 49, 793-806. HODGKIN, A. L. (1951). The ionic basis of electrical activity in nerve and muscle. Biol. Rev. 26, 339-409. HODGKIN, A. L. (1964). The Conduction of the Nervous Impulse. Liverpool University Press. JUNGE, D. (1967). Multi-ionic action potentials in molluscan giant neurones. Nature, Lond. 215, 546-8. KEYNES, R. D. & SWAN, R. C. (1959). The effect of external sodium concentration on the sodium fluxes in frog skeletal muscle. J. Physiol. 147, 591-625. KRJJGSMAN, B. J. (1940). Untersuchungen iiber die Erregungsleitung in Gastropodnerven. Proc. Acad. Sci. Antst. 43, 1065-77. LITTLE, C. (1965). Osmotic and ionic regulation in the prosobranch gastropod mollusc, Viviparus viviparus Linn. J. exp. Biol. 43, 23-37. LORENTE DE No, R. (1949). On the effect of certain quaternary ammonium ions upon frog nerve. J. cell comp. Physiol. 33, 1-231. MORETON, R. B. (1968a). Ionic mechanism of the action potentials of giant neurones of Helix aspersa. Nature, Lond. 219, 70-1. NAKAJIMA, Y. (1961). Electron microscope observations on the nerve fibres of Cristaria plicata. Z. Zell- forsch. mikrosk. Anat. 54, 262—74. NAKAMURA, Y., NAKAJIMA, S. & GRUNDFEST, H. (1965). The action of tetrodotoxin on electrogenic components of squid giant axons. J. gen. Physiol. 48, 985-96. NARAHASHI, T., MOORE, J. W. & SCOTT, W. R. (1964). Tetrodotoxin blockage of sodium conductance increase in lobster giant axons. J. gen. Physiol. 47, 965—74. OBUCHOWICZ, L. (1958). The influence of osmotic pressure of medium on oxygen consumption in Viviparus fasciatus. Bull. Soc. Amis Sci. Lett. Poznan 14, 367-70. POTTS, W. T. W. (1954). The inorganic composition of the blood oiMytilus edulis and Anodonta cygnea. J. exp. Biol. 31, 376-85. POTTS, W. T. W. & PARRY, G. (1964). Osmotic and Ionic Regulation in Animals. London: Pergamon. RUSHTON, W. A. H. (1951). A theory of the effects of fibre size in medullated nerve. J. Physiol. 115, 101-22. SATTELLE, D. B. & LANE, N. J. (1972). Architecture of gastropod central nervous tissues in relation to ionic movements. Tissve and Cell 4 (in the press). TASAKI, I. (1968). Nerve Excitation. A Macromolecular Approach. Springfield, Illinois: Charles C. Thomas. TREHERNE, J. E., CARLSON, A. D. & GUPTA, B. L. (1969). Extra-neuronal sodium store in central nervous system of Anodonta cygnea. Nature, Lond. 223, 377-80. TREHERNE, J. E., MELLON, D. & CARLSON, A. D. (1969). The ionic basis of axonal conduction in the central nervous system of Anodonta cygnea (Mollusca: Eulamellibranchia). J. exp. Biol. 50, 711-22. YOUNG, J. Z. (1936 a). The giant nerve-fibres and epistellar body of cephalopods. Quart. J. Micr. Sci. 78, 367-86. YOUNG, J. Z. (19366). Structure of nerve-fibres and synapses in some invertebrates. Cold Spr. Harb. Symp. Quant. Biol. 4, 1-6.