ELECTROPHYSIOLOGICAL INVESTIGATIONS OF THE HEART OF SQUILL A MANTIS

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1 J. Exp. Biol. (1964), 41, With 7 text-figures Printed in Great Britain ELECTROPHYSIOLOGICAL INVESTIGATIONS OF THE HEART OF SQUILL A MANTIS I. THE GANGLIONIC NERVE TRUNK BY HILARY F. BROWN Stazione Zoologica, Naples, and University Laboratory of Physiology, Oxford {Received 6 February 1964) GENERAL INTRODUCTION In most, possibly all, Crustacea the heart beat is neurogenic. Each beat is initiated by a burst of impulses from a small group of neurones whose cell bodies lie grouped in the ganglionic nerve trunk (g.n.t.) in the heart wall and whose axons run to the heart muscle. Many attempts have been made by electrophysiologists to discover how these neurones are integrated to fire rhythmic bursts (for example Maynard, 1953, 1955; Hagiwara & Bullock, 1957; Bullock & Terzuolo, 1957). However, apart from the study by Irisawa et al. (1962) of some aspects of heart muscle physiology in Squilla oratoria, analysis of the complete neuromuscular system of a crustacean heart does not appear to have been undertaken. In the present study such an analysis has been attempted in the case of the heart of the stomatopod, Squilla mantis. The relatively simple anatomical plan of the stomatopod heart makes it a more favourable preparation for electrophysiological study than is the heart of decapod Crustacea. The present paper describes the investigations made of the ganglionic nerve trunk of Squilla mantis. A second paper (Brown, 1964 a) will be concerned with the electrophysiological properties of the heart muscle of Squilla. In a third paper (Brown, 19646) the mode of action of extracts of the pericardial organs on the heart of Squilla will be considered in relation to the electrophysiology of untreated hearts. THE ANATOMY OF THE HEART OF SQU1J.LA MANTIS The stomatopod heart is a long tubular structure which extends about half the total length of the body; in Squilla mantis this amounts to about 8 cm. It shows obvious signs of segmentation in having 13 pairs of ostial orifices and 15 pairs of lateral arteries. Fig. 1 is a diagram of the heart of Squilla mantis, redrawn from Alexandrowicz (1934). In his paper Alexandrowicz gave a clear and detailed account of the innervation of the Squilla heart, of which the main points will be summarized here. The ganglionic nerve trunk (g.n.t.) is a chain of nerve cells which runs the length of the heart tube outside the muscle in the mid-dorsal line. For the most part there is one nerve cell body per heart segment, situated behind the ostial orifices. The largest cell bodies are in the sixth to thirteenth segments and reach 180 //, in diameter, while the cell bodies near either end of the chain are much smaller. Alexandrowicz described two large processes arising from the cell body of each

2 690 HILARY F. BROWN neurone, one running forwards and one backwards in the trunk. He did not find it possible to determine how far these processes ran in the trunk before giving off branches to the muscle, though in some cases they seemed to branch in the third segment from the cell body, and he found side branches of the processes of more than one neurone in the same heart segment. He reported seeingfiveor six axons in cross-sections of the g.n.t., except in the anterior and posterior regions of the trunk, where he found fewer. Dorsal, nerves Paired ostia Lateral arteries Ganglionic nerve trunk Posterior aorta Fig. 1. Diagram of the heart of Squilla mantis (redrawn from Alexandrowicz, 1934). Approx. twice natural size. Alexandrowicz also described two other types of cell processes: short dendrites which arise from the cell bodies and the proximal regions of the longitudinal processes and end on the muscle fibres close to the cell bodies, and thin collaterals which arise from the other types of cell processes and ramify within the g.n.t. Three pairs of dorsal nerves enter the g.n.t. from the central nervous system (Fig. 1).

3 Electrophysiological investigations of the heart of Squilla mantis 691,,.., METHODS Material Specimens of Squilla mantis were obtained locally at Naples and kept in running sea water, where they would live for up to 3 weeks. Isolation and perfusion of the heart The animals were decapitated and their claws were removed. The heart was exposed by cutting away the carapace and removing the dorsal muscle blocks. The posterior aorta was grasped with forceps and the heart was cut out from behind forwards. Some of the underlying gonad tissue was removed as well and was trimmed away later. The heart was quickly transferred to the perfusion dish and the posterior aorta was tied on to the inflow cannula. The valve at the base of the posterior aorta was opened with the cannula tip so that the fluid would flow into the heart by this route, counter to the natural direction of flow. Table 1. Composition of the perfusion fluid used for isolated Squilla hearts NaCl KC1 CaCl, MgCl a NaHCO, Urea 520 m-equiv./l. 13 m-equiv./l. 28 m-equiv./l. 49 m-equiv./l. toph g./l. The perfusion fluid was made up according to the formula given by Welsh (1939) for crustacean hearts (Table 1). A little glucose was added. During the summer the perfusion fluid was cooled to o C. before it was placed in the reservoir bottle, insulated with glass wool (a in Fig. 2). It then passed through an oxygenating chamber (d) and by the time it reached the perfusion dish (/) its temperature was about 17 0 C, which was still considerably cooler than the summer room-temperature in Naples. Perfusion dishes were of glass or Perspex, with two holes in the lateral walls into which were set the glass inflow cannula and an outflow tube leading to a filter pump, by means of which the fluid was kept at a constant level, about half a centimetre above the heart. The perfusion dish was mounted on the stage of a dissecting microscope, with illumination from below. Micromanipulators were used for placing the electrodes. After a few minutes of perfusion the isolated heart would start to beat regularly. It was always left for some time to adjust to perfusion conditions before recording was started. Some hearts would continue to beat for up to 6 hr. when isolated and perfused, while others, under seemingly identical conditions, died far sooner. These differences in vigour could not be correlated with the apparent condition of the animal. Often animals which seemed very active had weak hearts while vigorous hearts might come from weak animals. Recording from the ganglionic nerve trunk Only extracellular records were taken from the g.n.t. Conventional d.c. amplifiers were used and the resulting signal was displayed on a Cossor oscilloscope. The elec-

4 692 HILARY F. BROWN trodes used were thin hooks of chlorided silver wire. The g.n.t. of the Squilla heart is too fine and too closely attached to the muscle to be separated from it, so with fine scissors two longitudinal cuts were made in the muscle wall on either side of the g.n.t. for the distance of a few heart segments. As Alexandrowicz (1931) first observed in the heart of Ligia oceanica, cutting the heart muscle has no effect on the co-ordination of the crustacean heart beat as long as the g.n.t. is intact. The length of g.n.t. and muscle thus freed was hooked over the silver wire electrode. The electrode was mounted on the micro-manipulator arm (j) in Fig. 2. By moving the electrode up and down, the g.n.t. was lifted out of the fluid for recording and replaced at other times. A second chlorided silver electrode was placed in the perfusion dish and earthed. Fig. 2. Diagram of the apparatus used for electrical recording from the heart of Squilla mantis, a, Thermally insulated reservoir bottle; b, cooled perfusion fluid; c, fluid level indicator; d, aeration chamber; e, screw clip on rubber inflow lead; /, perfusion chamber; g, heart; h, movable stage of microscope; i, outflow to filter pump;/, microelectrode or chlorided silver hook electrode; k, micromanipulator arm; I, n, wires leading to cathode follower and amplifier; m, microscope; o, mirror. RESULTS Extracellular records from the ganglionic nerve trunk The average rate of beating of 10 isolated perfused hearts, soon after perfusion had been started, was 17 beats/min. External electrodes, placed as described above at any point on the g.n.t. recorded at each of these beats a certain number (N) of nerve impulses (a burst). N might be any number between 1 and 12 but was more or less constant for any one heart under given conditions (Fig. 3). A diagram of a sequence of three externally recorded g.n.t. bursts is given in Fig. 4 to show the precise limits of the durations and intervals which are referred to in discussing these records. The nerve impulses recorded externally from one point on the g.n.t. were simple

5 Electrophysiological investigations of the heart of Squilla mantis 693 triphasic spikes and were almost always exactly the same height throughout a sequence of several beats. (Any such sequence is termed a 'run'.) This suggests that repeated activity of the same structure was being recorded. On one occasion, a glass microelectrode was used to explore a region of a g.n.t. containing a cell body. An external record of the impulse burst, exactly similar to that recorded with hook electrodes, was obtained, but only when the electrode tip was close up against the cell body itself. This suggests that activity of the cell body rather than of cell processes was responsible for the recorded signals. Indirect support for this conclusion, too detailed to be given here, comes also from recordings from the ganglionic nerve trunks of decapod hearts. w. rmn rmt-1 1 sec. MmV. Fig. 3. Typical extracellular records from the ganglionic nerve trunks of spontaneously beating Squilla hearts, (a), (b), (c) recorded from the same heart at different times, (d) From the g.n.t. of a second heart. Spikes re-touched. Burst duration lm P U ' Se intervals Burst interval - 4+f- Fig. 4. Diagram of an extracellular record from the g.n.t. during a sequence of three heart beats, to illustrate the terms used. This is a' run' of three' bursts'. When two hook electrodes were placed at different points of the g.n.t., the second one recorded at a given beat the same number of impulses as did the first. Typically, the impulses in this second record were spaced at the same intervals as those recorded at the first electrode, but the whole record at the second site was slightly displaced in time (Fig. 5 (a)). It appears likely that all the nerve cells in the g.n.t. fire in succession N times per beat. In most of the nine hearts from which records were taken simultaneously from two regions of the g.n.t., each nerve impulse was invariably recorded at the same one of the two electrodes before being recorded at the other (as in Fig. 5(a)). In four of the nine hearts this was at the anterior recording site; in two of the nine hearts the impulses all appeared first at the posterior recording site. In the 45 Exp. Biol. 41, 4

6 694 HILARY F. BROWN remaining three hearts, though most of the impulses were first recorded at one and the same site, the last impulse or last few impulses of some or all bursts were first recorded at the other site (Fig. 5 (b)). In this last group the delay between the electrodes was the same for all the impulses (though reversible in sign) suggesting that the impulses are initiated in the anterior or posterior regions of the chain, beyond the two recording sites. In a typical case the recording sites were at the levels of cell 7 and cell 11. This conclusion is supported by the results of the cutting and ligaturing experiments described below. 1mV.[ 100 msec. Fig. 5. Simultaneous extracellular records from two points on the ganglionic nerve trunk of Squilla mantis hearts. Records from two hearts, (a) and (6). The dotted lines are drawn in to emphasize the time differences at the two recording points. In (6) the last 5 spikes reversed their direction of travel. In the top record of (b) the large, slow component is from the muscle. Tracings of original records. The site of burst initiation The perfused heart was cut transversely or ligatured at different points in an attempt to locate the region responsible for initiating the rhythm. Some portions of the g.n.t. so isolated would continue to initiate beats in the muscle isolated with them. When the heart was divided in two at about the level of cells 7 and 8, one half would continue to beat at the previous rate while the other half either beat more slowly or was completely quiescent. In portions which were initially quiescent sporadic beats would usually start up after a few minutes and beating would later become more regular and faster. The anterior and posterior portions of a few hearts were further subdivided by several transverse cuts or ligatures. The cells of the g.n.t. were afterwards stained with methylene blue to verify the positions of the cuts. Only some of the portions continued beating immediately after isolation and these (denoted by the cells they contained) are listed in Table 2. They beat considerably faster than the whole heart had done. The middle region of the heart (that containing cells 6-10) usually remained quiescent when isolated by transverse cuts but the amount of spontaneity varied in

7 Electrophysiological investigations of the heart of Squilla mantis 695 different hearts. Sometimes even single neurones of this region which had been separated from their neighbours would, some time after isolation, set up slow beating of the muscle isolated with them. In two cases (f in Table 2) several portions of the heart were left beating immediately after cutting. Despite this variability it seems that the cells with the greatest intrinsic rhythmicity and the highest spontaneous firing rate are those near the ends of the g.n.t. This is in keeping with the finding that within an intact g.n.t. the impulses appear to be initiated in the anterior or posterior portions of the chain. Table 2. Portions of Squilla hearts which continued beating when isolated The numbers refer to the contained cells. No. of Posterior halves hearts Cell 12 1 Cell 13 1 Cells 14 and 15 1 Cells 13, 14 and 15 2 Cells 11 \ 12 if 13, 14 and 15/ Anterior halves Cell 1 I Cells 1, 2 and 3 1 Cells 5 and 6 1 Cells Portion not further subdivided. + More than one portion of the heart left beating immediately after cutting. The number of impulses in the g.n.t. burst The number of impulses in the burst fired by the g.n.t. was related to the interval between the bursts. In any one heart the number was fairly constant over a short time (several minutes) but over a longer period the heart slowed and the number of impulses per burst increased. Fig. 6 illustrates this relationship for a single heart. The recordings were taken over a period of 1^ hr. The correlation coefficient for the points in Fig. 6 is 0-98, which is highly significant. Fig. 7 shows the relationship between the number of impulses per g.n.t. burst and the burst interval for many hearts plotted on the same graph. Each reading gives the average number of impulses per burst and the average burst interval for a run of 4-5 beats of a spontaneously beating heart. From some hearts more than one set of observations is included, taken at intervals of \ hr. or more. Fourteen readings from ten hearts are included in the graph. The records analysed included not only extracellular records from the g.n.t., where impulse number and burst interval can be seen directly, but also intracellular records from the heart muscle. In these latter, as is shown by recording simultaneously from the g.n.t. and from the muscle (see Brown, 1964a), the number and spacing of the peaks of the junction potential recorded in the muscle at a heart beat correspond exactly to those of the nerve impulses in the g.n.t. burst initiating them. One point in Fig. 7 differs widely from the others. It was taken from a heart 45-2

8 696 HILARY F. BROWN that was beating very slowly at the time. If this point is included, then the correlation coefficient for the readings is 0-39 and P > o-i. If it is omitted, the correlation coefficient for the remaining points is 0-90, which is highly significant (P < o*oi). Its exclusion seems justified, for the rate of beating had become very slow, and the heart was probably in poor condition. Two previous readings from the same heart both fall near the other points. 10 r 6 I o o Average number of impulses per burst Fig. 6. The average number of impulses per burst plotted against burst interval in a spontaneously beating Squilla heart as the rate of beating dropped over a period of ij hr. Each point on the graph represents the average values for a run of three to five bursts. There is a little evidence that the composition of the g.n.t. burst may be affected by proprioceptive impulses from the dendritic endings of the neurones on the muscle fibres. On one occasion when the muscle was being stimulated directly with long square pulses of current (1-5 sec. duration and up to 50/tA. intensity) it was observed that the g.n.t. fired every time the stimulus was given, though with a delay of 50 msec. This long delay indicates that the g.n.t. was not directly excited by the electrical stimulus (as when the stimulating electrode was placed directly on the g.n.t.) but that burst firing was being regulated by impulses from the proprioceptive endings, presumably stimulated mechanically by the muscle movement.

9 Electrophysiological investigations of the heart of Squilla mantis 697 The pattern of impulses within the g.n.t. burst The intervals between the impulses in a g.n.t. burst averaged 45 msec, but they were not all the same length. Within one run recorded from a heart the spacing of impulses within the bursts remained remarkably constant. When, as was usually the case, two bursts of a run were both composed of the same number of impulses, the comparable intervals (first, second, third, etc.) within the two bursts were of almost exactly equal s 6 o o o I o o o o o o Average number of Impulses per burst Fig. 7. The relation between the number of impulses per burst (abscissa) and the burst interval in seconds (ordinate) for ten spontaneously beating hearts of Squilla mantis. Each point represents average values for a run of three to five bursts. length. In all the runs recorded from any one heart the pattern of impulses within the bursts remained similar although the number of impulses per burst varied from run to run. Thus in the bursts shown in Fig. 3 (a) thefirstinterval between impulses was longer than the second, while the third and subsequent intervals lengthened again, and the sixth and last was noticeably the longest. Fig. 3 (b) and (c) show two other runs recorded from the same heart. The relative lengths of the intervals within the bursts followed the same pattern (second interval shorter than the first, then interval length progressively increasing) although in each run the number of impulses per burst differed. The impulse pattern of the bursts from another heart (Fig. 3 (d)) was not the same, for the first interval within the bursts was not consistently longer than the second, but

10 698 HILARY F. BROWN the bursts from this heart did show the same progressive lengthening of the later intervals. Measurements of the impulse intervals in bursts recorded from the g.n.t.'s of thirteen hearts showed that the only completely consistent feature of their pattern was that the last interval was always as long as, or longer than, the penultimate interval and was usually markedly longer. DISCUSSION The impulses recorded externally from any one point of the g.n.t. appear to represent repeated firings of the same structure, probably a cell body. This is borne out by the records taken simultaneously from two points of a g.n.t., which showed a constant delay between the two recording electrodes for all impulses, indicating that all the impulses travel along the same ' firing channel' within a g.n.t. and not in several parallel pathways. The simplest hypothesis is that the cells fire in order, one after another from the initiating cell forward or backward, each cell directly stimulating the one next to it to fire. This is hard to reconcile with Alexandrowicz's histological observations that the bipolar processes of the g.n.t. neurones run forwards and backwards within the g.n.t. for some segments before branching and that cross-sections of the g.n.t. showed five or six axons. However, there must be some branching nearer the cell body than this, for, in those heart segments which beat when isolated from the rest of the heart by two transverse cuts, some of the axons to the muscle must have left the g.n.t. within the segment containing the cell body. On the basis of his histological findings, Alexandrowicz suggested that excitation might pass from one g.n.t. neurone to the next by way of the muscle. Thus the firing of one neuron would cause contraction of the muscle fibres it innervated, which in turn would excite the dendrites of another neurone and so on. However, it can be assumed that the speed at which the nerve impulses travel along the processes in the g.n.t. is not faster than the conduction velocities found in general for crustacean peripheral nerves (between 1 and 5 m./sec). Since the average value for conduction velocity along the g.n.t. found in the present study was 1-5 m./sec., it seems unlikelythat there are more breaks in the 'firing channel' than simple synapses between neurones. Furthermore, careful removal of nearly all the muscle on either side of the g.n.t. for a distance of several segments did not disturb the firing of the nerve cells as long as the g.n.t. was unharmed, suggesting that the muscle is not a normal link in the excitation pathway. Irisawa & Irisawa (1957) cut the g.n.t. of the heart of Squilla oratoria transversely in front of the 13th cell and then further forward segment by segment. In the seven cases they tested, they found that the segment containing cell 13 always beat after isolation at the same rate as had the whole heart and that the rate of beating of segments further forward decreased progressively. Cell 11 was in three cases out of seven quiescent after isolation, cell 10 was quiescent in six cases out of seven. They suggested that the cell of the 13th segment is the pacemaker and governs the rate of the heart beat. But in the present investigation of Squilla mantis, although spontaneity was similarly found to be greatest in the cells towards either end of the g.n.t. chain, simultaneous recording from two points on the g.n.t. showed that in intact hearts, beating spontaneously, the bursts were initiated as frequently in the anterior as in the posterior region of the g.n.t. and that initiation could shift from one of these regions to the other in the same g.n.t.

11 Electrophysiological investigations of the heart of Squilla mantis 699 Maynard (1955) considered that every neurone in the lobster g.n.t. might be capable of spontaneous activity but that in a normal burst only the first to fire did so spontaneously and this drove the others. Similarly, in Squilla mantis, while most cells of the g.n.t. show some spontaneity in that even the quiescent cells in the middle of the chain would often start up slow firing some time after isolation, those which fire most readily when isolated are those towards the ends of the chain (i.e. in those regions where burst firing is initiated in the intact heart). The cell with greatest spontaneity following isolation may be one of a number of cells towards the front or back of the chain, which again suggests that no one cell in the chain of sixteen is invariably 'the pacemaker'. The nature of the spontaneity of the neurones and of their integration for coordinated burst firing needs more investigation. Although the muscle does not seem to be a link in the conduction path of impulses along the g.n.t., it remains possible that proprioceptive impulses from the dendritic endings of the g.n.t. cells on the muscle fibres can affect the pattern of impulses in the burst, as was apparently happening in the case reported above (see Results). Hagiwara & Bullock (1957) took intracellular recordings from the g.n.t. neurones of the lobster. They found that repetitive presynaptic stimulation results in synaptic potentials whose amplitude bears an inverse relation to frequency. From the larger synaptic potentials (at lower rates) more spikes arise than from the smaller ones. The spikes are initiated at the base of the axon and do not invade the soma of the neurone and 'wipe out' the depolarization there. Thus the synaptic potential can persist and determine the number of spikes in the burst. Two findings suggest that in Squilla the process which determines the composition of the burst is of a similar nature. First, there is the correlation between increase in the number of impulses per burst and increasing burst interval and, secondly, the lengthening of the impulse intervals towards the end of the bursts, which implies an exponential decline of the process initiating the impulses. However, the nerve impulses recorded from the g.n.t. of Squilla appear to be firings of the nerve cell bodies which cannot therefore be carrying graded synaptic potentials determining burst composition. It is possible that the region of graded potentials and impulse initiation is in some other part of the g.n.t. neurone in Squilla, say at the base of the dendrites, so that the soma as well as the axon here carries all-or-none impulses. Alternatively, the small cells of the front and back regions of the g.n.t. where the bursts are normally initiated may not carry all-or-none impulses in the same way as do the other cells. Recordings were not obtained from these cells, which are smaller and less accessible than the central neurones of the g.n.t. chain. SUMMARY 1. With an external hook electrode placed upon the ganglionic nerve trunk of the isolated heart of Squilla mantis a burst of a small number (3-12) of nerve impulses was recorded at each heart beat. 2. The number of impulses per burst showed a direct correlation with interval between bursts. 3. The only consistent feature of impulse pattern within the bursts was a lengthening of the intervals between impulses towards the ends of the bursts.

12 700 HILARY F. BROWN 4. Electrodes at two points on the ganglionic nerve trunk each recorded the same number of impulses at a burst. The delay between the two recording points was the same for all impulses, and usually all the impulses were, in a given heart, recorded travelling in the same direction, though this could be either forwards or backwards along the chain. 5. It is suggested that each cell in the chain of 16 fires in succession the same number of times during a burst and that the impulses travel along the same ' firing channel' within the ganglionic nerve trunk. 6. Cells near the two ends of the chain showed the greatest spontaneity when isolated by transverse cuts or ligatures. Coupled with the records obtained from two points, this suggests that the bursts are initiated in the front or back regions of the chain, but not invariably by the same one of the 16 cells. This work formed part of a D.Phil thesis for Oxford University. I should like to thank my supervisor Miss R. J. Banister for her help, the Director and staff of the Stazione Zoologica, Naples, for laboratory facilities, and the D.S.I.R. for the postgraduate studentship which supported me. REFERENCES ALEXANDROWICZ, J. S. (1931). Quelques experiences sur le fonctionnement du systeme nerveux du cceur des Crustaces. C.R. Soc. Biol., Paris, 108, ALEXANDROWICZ, J. S. (1934). The innervation of the heart of Crustacea. II. Stomatopoda. Quart. J. Micr. Sci. 76, BROWN, H. F. (1964a). Electrophysiological investigations of the heart of Squilla mantis. II. The heart muscle. J. Exp. Biol. 41, BROWN, H. F. (19644). Electrophysiological investigations of the heart of Squilla mantis. III. The mode of action of pericardial organ extract on the heart. J. Exp. Biol. 41, BULLOCK, T. D. & TERZUOLO, C. A. (1957). Diverse forms of activity in the somata of spontaneous and integrating ganglion cells. J. Physiol. 138, HAGIWARA, S. & BULLOCK, T. H. (1957). Intracellular potentials in pacemaker and integrative neurons of the lobster cardiac ganglion. J. Cell. Comp. Physiol. 50, IRISAWA, H. & IRISAWA, A. F. (1957). The electrocardiogram of a Stomatopod. Biol. Bull, Wood's Hole, 112, IRISAWA, H., IRISAWA, A., MATSUBAYASHI, T. & KOBAYASHI, M. (1962). The nervous control of the intracellular action potential of the Squilla heart. J. Cell. Comp. Physiol. 59, MAYNARD, D. M. (1953). Integration in the cardiac ganglion of Homarus. Biol. Bull., Wood's Hole, l<> MAYNARD, D. M. (1955). Activity in a crustacean ganglion. II. Pattern and interaction in burst formation. Biol. Bull., Wood's Hole, 109, WELSH, J. H. (1939). Chemical mediation in crustaceans. I. The occurrence of acetylcholine in nervous tissue and its action on the decapod heart. J. Exp. Biol. 16, Note added in proof Watanabe and Takeda (1963) have recorded intracellularly from the neurones of the ganglionic nerve trunk of the heart of Squilla oratoria. Their results suggest that firing of the cell body of each neurone is not essential for conduction along the chain, for parallel axons, functionally linked to one another and to the soma axon by sideconnections, carry propagated impulses past the somata before these have discharged. They conclude that transmission across the side-connections takes place with a high safety factor, and probably electrically so that the whole g.n.t. acts as a single unit. WATANABE, A. & TAKEDA, K. (1963). The spread of excitation among neurons in the heart ganglion of the Stomatopod, Squilla oratoria. J. Gen. Physiol. 46,