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The relationship between heart beat and respiration in teleost fish
Comp. Biochem. Physiol.,
1962, Vol. 7, pp. 237 to 250. Pergamon Press Ltd., London. Printed in Great Britain
THE RELATIONSHIP BETWEEN HEART BEAT AND R E S P I R A T I O N IN T E L E O S T F I S H G. SHELTON and D. J. RANDALL Department of Zoology, University of Southampton (Received
27 A u g u s t 1962)
A tendency for the heart to beat in the mouth-closing phase of the breathing movements is often seen in the resting tench. Synchrony is seldom perfect, except in the anaesthetized animal, and disappears if the fish is stimulated in any way. 2. When the breathing movements stop in the tench and other teleosts because of a natural pause, deep anaesthesia or curare-induced paralysis, the heart rate decreases. This inhibition of the heart, probably of reflex origin, can also be produced by perfusion of the gills with de-oxygenated water and is caused by lack of oxygen at the gills. 3. The breathing movements also appear to have a stimulating effect on the heart rate separate from the oxygen reflex.
Abstract--1.
INTRODUCTION
IT I8 reasonable to predict that, in the interests of economy of effort, a close relationship will be maintained between the amount of oxygen presented to an exchanging surface by an animal's breathing m o v e m e n t s and the amount of blood p u m p e d through that surface to transfer the oxygen to the tissues. In aquatic animals the relationship ought perhaps to be clearer than in terrestrial forms because of the considerable amount of work which must be done in ventilating the respiratory surface (Hughes & Shelton, 1962). A discrepancy between the capacity of the water stream to bring oxygen to the exchanging surface and the blood stream to take it away would lead to a serious waste of effort on the part of an aquatic animal, particularly if the lack of balance was due to excess ventilation. Very little is known of the exact quantitative features of the heart o u t p u t - ventilation ~olume relationship in aquatic or indeed in any animals. One of the major difficulties which has been encountered is that of determining the heart output and blood flow through the respiratory organ. However, some work has been done on fish and the existence of a relationship has been confirmed. In elasmobranchs it has been known for a long time that there is a simple numerical relationship between heart and breathing rates, the heart beat occurring every 1, 2, 3 or 4 breathing cycles (Lyon, 1926; Lutz, 1930). Satchell (1960) has extended the earlier work and has suggested that, in the dogfish, the heart beat m a y occur in relation to the breathing m o v e m e n t s in such a way as to make coincident the periods of m a x i m u m water flow over the gills and of m a x i m u m blood flow i6
237
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G. SHELTONAND D. J. RANDALL
through them. This precise type of relationship appears not to hold in teleost fish. Serfaty & Raynaud (1957a) have recorded respiratory movements and electrocardiographs from the tench and found that synchronization between heart beat and breathing is very rare and persists for a short time only. To some extent there is a similar lack of correlation in the trout (Hughes, 1961), though in this animal quite good synchronization has been observed occasionally with the heart beating at a specific point in the respiratory cycle. However, synchronization of such exactitude is not the only relationship which may exist. Indeed it would appear perhaps the least likely because it has the disadvantage of permitting adjustment to take place in a stepped instead of a continuously variable fashion. Obviously there can be a close correlation between heart output and ventilation volume without synchrony. Both depend on two variable parameters, the amplitude and frequency of the movements, and so the details of this correlation may be complex and variable from time to time and in different individuals. In spite of this complexity more details of the extent of the correlation between heart beat and breathing movements ought to be forthcoming from experiments in which the environment is changed in ways having a differential effect on the two systems. This, and a succeeding article (Randall & Shelton 1962), describe the effects of various factors on the heart rate and breathing movements. METHODS The majority of the experiments were done on tench (Tinca tinca) varying between 50 and 200 g in weight, though some eels (Anguilla vulgaris), trout (Salmo trutta) and roach (Rutilus rutilus) were also used. The experiments were carried out in Perspex tanks between 1 and 21. in capacity through which continuous water flow could be maintained. The flow through the tanks was from the tail of the fish towards the head in those experiments in which the fish was breathing normally, to avoid any forced ventilation of the gills. In the experiments in which the breathing movements were stopped by various means, artificial respiration was given by forcibly ventilating the gills via a cannula in the mouth. In all cases the flow rate was kept appreciably higher than that necessary to satisfy the oxygen demands of the fish. The experiments on unanaesthetized animals were done at tapwater temperature (13-15°C), those in which anaesthetic was dissolved in the water and a closed circuit maintained were done at room temperature (17-20°C). The temperature was constant to within I°C during any one experiment. The movements of the lower jaw were taken as representative of the breathing movements as a whole. Originally they were recorded on a smoked drum but in some experiments a mechanoelectric transducer was used, the output being taken to an Ediswan pen recorder. The heart rate was determined by recording the electrocardiogram (E.C.G.). This was detected by means of a unipolar system, using a steel electrode insulated with varnish except at the tip. This electrode pierced the skin mid-ventrally in front of the pectoral girdle, the tip being pushed forward to lie beneath the heart. An indifferent electrode was placed in the water contained in the tank. The signal was amplified in an Ediswan EPA amplifier and
T H E R E L A T I O N S H I P B E T W E E N H E A R T BEAT A N D R E S P I R A T I O N I N T E L E O S T F I S H
239
the output connected to a second pen on the Ediswan recorder. Alternatively a Tektronix 502 oscilloscope was used for display, records being taken with a Cossor oscilloscope camera. The primary object in recording the E.C.G. in these experiments was to determine the heart rate and not to study the E.C.G. itself. Descriptions of the E.C.G. obtained from a variety of teleosts have already been given (Kisch, 1948 ; Oets, 1950 ; Serfaty & Raynaud, 1956, 1957a) and we have nothing to add to these. The voltage and configuration of the E.C.G. varied considerably with the position of the detecting electrode in the ventral musculature. The electrode was adjusted until an adequate signal to noise ratio was obtained. By filtering off low frequencies in the amplifier a simple E.C.G. was obtained, and by including the low frequencies it was possible to record E.C.G. superimposed on voltage changes produced by the breathing movements. By this means it was possible to study some aspects of the heart and breathing relationship on a single trace. RESULTS The relationship of the heart beat to the breathing movements The heart rate of the resting, unanaesthetized tench is in the range 15-30 beats/min, that of the eel from 40-50 beats/min. The breathing rate is slightly higher and, as in the case of the heart rate, variable from animal to animal and in the same animal at different times. The resting tench breathes at a frequency between 30 and 60 respirations/min, the eel between 30 and 50, though in the latter there are long pauses in the breathing rhythm. Although some of our results have confirmed the conclusion of previous workers that no direct relationship exists between heart beat and breathing in teleosts, this is by no means universally true. A closer analysis of the position of the ventricular phase of the E.C.G. in relation to the breathing cycle of the tench showed that, while respiratory and cardiac frequencies might differ, under some conditions the heart beat appeared more often during the "mouth closing" phase of the cycle. This relationship was true particularly of fish which were obviously at rest and not disturbed in any way. Even minor disturbances tend to make the relationship less obvious, and high levels of carbon dioxide, for example, obliterate the pattern completely (Fig. 1). The records from nine animals have been examined in some detail and in four cases the pattern was very clear (Fig. l(a)), in four cases fairly clear (Fig. l(b)), and only in the single carbon dioxide experiment analysed (Fig. l(c)) was there no trace of "mouth closing" domination. Where breathing and heart rate differed, a relationship was maintained by the lengthening or shortening of the interval between heart beats. The lengthening of interval between heart beats was also noticed in many of the fish in which coughs broke up the smooth rhythm of breathing. A similar pattern was found in the eel with the heart beat appearing more often during the "mouth closing" phase of the breathing cycle and again giving a clear indication that co-ordination between breathing and heart beat does exist in the intact, unanaesthetized teleost. Breathing in the eel can be spasmodic, as van Dam (1938) has pointed out, the fish breathing quite regularly for a while and then
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G. SrtELTON AND D. J. RANDALL
pausing for periods up to 5 min in duration in extreme cases. W e found that the pauses of more than 30 sec duration were rare under normal conditions of oxygenation, temperature, etc. During the periods when the animal was breathing normally,
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FIG. l. Histograms of the occurrence of heart beat in relation to the breathing cycle of the tench. Successive breathing cycles are divided into ten equal parts and the occurrence of heart beats in these parts is plotted on a percentage basis in the histograms. (a) Clear relationship with "mouth closing" phase. Number of heart beats in analysis--182. Breathing rate--66 per rain. Heart rate--22 per rain. (b) Some evidence of "mouth closing" dominance. Number of heart beats in analysis--136. Breathing rate--39'5 per rain. Heart rate--16-5 per rain. (c) Respiration stimulated with CO2. No clear relationship, though fewer heart beats occur in "mouth opening" phase. Number of heart beats in analysis--ll8. Breathing rate--54 per min. Heart rate--46 per rain. the heart rate was about 50/min but when breathing stopped the heart rate dropped immediately to half the original value. T h e rate returned to the higher value as soon as the eel began ventilating the gills once more. T h i s relationship is m u c h more obvious than the one described previously for the breathing fish, and, though it is possible that one is an extreme case of the other, it is equally likely that two separate mechanisms are involved.
The effects of anaesthesia T h e main interest in the effects of anaesthetics centred on their influence at high concentrations on breathing rate and heart rate. O f the three anaesthetics used, namely urethane, nembutal and tricaine methane sulphate ( M S 222), only the latter had very obvious effects at light levels of anaesthesia. T h e concentrations of this anaesthetic required for a given level of sedation were variable but, in general, bathing a tench in a solution of 20 m g M S 222 per 1. made the animal unreactive to external stimuli, whilst 100 m g per 1. caused rapid loss of equilibrium with no m o v e m e n t s except those of breathing. T h e effects of M S 222 on the heart of the carp (Serfaty et al., 1959) and the tench (Randall, 1962) have been described previously. I n agreement with this previous work it was found that all concentrations
241
THE R E L A T I O N S H I P B E T W E E N HEART BEAT AND RESPIRATION IN TELEOST FISH
of M S 222 caused an increase in heart rate and, usually to a lesser extent, in respiratory rate and amplitude (Fig. 2). T h e effects on heart and breathing were simultaneous and noticeable within a minute of running the solution into the tank. IO0 90 8O
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FIG. 3. T he effect of water flow and succinyl choline on the heart rate and E.C.G. in the tench. (a) E.C.G. synchronous with breathing in lightly anaesthetized animal (25 mg MS 222 per 1.). Down on t r a c e - - m o u t h closing. (b) 200 sec after injection of 0-1 ml 1 % solution succinyl choline chloride. T h e major breathing movements have stopped. E.C.G. is longer in duration and of smaller amplitude. (c) Immediate effect of flow stoppage. Record taken 35 rain after injection during which time the gills have been perfused at a rate of 100 ml per min. T h e water flow is stopped during the period indicated by the marker line. (d) Record taken 80 sec after (c), flow still stopped. T h e heart rate has fallen and become irregular. (e) T he effect of restarting water flow. Record taken 220 sec after (d). Flow started where marker line stops. T i m e marker--1 second intervals.
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THE RELATIONSHIPBETWEENHEARTBEATANDRESPIRATIONIN TELEOSTFISH
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intramuscularly per 100 g fish) were similarly without effect on the heart and respiration. Insufficient experiments were done to enable any conclusions to be drawn about the possible effects of these two anaesthetics on the heart-breathing synchrony. All three anaesthetics, when present in higher concentrations than those given above, were effective in causing the breathing movements to become irregular and ultimately to stop altogether. In Fig. 2 the effects of a high concentration of MS 222 on the heart rate and breathing of the tench are shown. After 12 min in the anaesthetic solution the animal stopped breathing and the heart rate fell quickly to a level close to that seen in the unanaesthetized animal. The fact that the heart rate did not fall below the normal value can probably be explained in terms of the accelerating influence of MS 222. Anaesthetics not having this effect do in fact cause a fall in heart rate below the unanaesthetized frequency when the animal's breathing fails. At least part of the fall in frequency occurs immediately the breathing movements stop, but only after a delay is the full extent of the decrease in rate seen. A feature of the low heart rate which results from respiratory failure in deep anaesthesia is its somewhat irregular nature. This was not seen in the unanaesthetized eel during a respiratory pause; in this animal the heart beat remained regular. In both the tench and the eel, recovery of the breathing results in a simultaneous recovery of the heart rate. In Fig. 2 a short period of breathing at point E is reflected in the higher value for heart rate when next measured.
The effects of paralysing agents A definite connexion has been established between heart rate and breathing rate by the foregoing experiments but what is not certain is the extent to which the various components of this connexion are inter-related. It is possible that the same or different co-ordinating mechanisms are responsible for the relationship in the unanesthetized animal, in the lightly anaesthetized animal showing synchrony of breathing and heart beat, and in the non-breathing animal. The co-ordination may be a purely central nervous phenomenon or a peripheral effect of either a simple mechanical or a reflex nature. If the relationship is reflexly mediated the important afferent impulses may be associated with the breathing movements, the water stream over the exchanging surface, or the levels of the respiratory gases in the body or the medium. Finally it must be recognized that a relationship between heart and breathing can be effected not only by the breathing in some way influencing heart beat, as the foregoing comments assume, but also by an influence working in the reverse direction. In order to resolve some of these difficulties in interpretation, experiments were done on fish which had been paralysed, the paralysis being produced in two different ways by injection either of tubocurarine chloride (0-5-1.0 mg. per 100 g fish) or of succinyl choline (0.1-0.3 mg per 100 g fish). The curare dose is high by mammalian standards, the succinyl choline dose roughly comparable though the duration of the paralysis with the latter drug is greater than in mammals. Both drugs work at the endplate region but act in different ways. They were used in an
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G. SHELTONAND D. J. RANDALL
attempt to make sure that the important feature in the experiments was the paralysis and not the paralysing agent. The effect of a paralysing dose of one of the above drugs injected intramuscularly into an anaesthetized tench was to stop all but very slight movement of the respiratory musculature in 1-2 min. There are some components of the respiratory complex which are quite resistant to both curare and succinyl choline. These have not as yet been identified but they can produce slight movements which are noticeable particularly on the opercular valve. Satchell (1961) has also commented on the resistance to curare of some of the dogfish respiratory muscles. In the tench these small movements may continue for some time but eventually stop, with the doses used, some 15-30 rain after injection. Long before this, however, soon after the major breathing movements stop, there is a marked fall in heart rate and the beat becomes irregular. With succinyl choline it was also found that about 5 min after injection the character of the E.C.G. changed, with the amplitude of the components decreasing and the potential changes being made more slowly (Fig. 3(b)). This effect persisted for about 30 rain during which no experiments were done but the gills were perfused with water. Ultimately the E.C.G. recovered its normal appearance. Curare had no effect on the E.C.G. but, though the drugs differed in this respect, the results of the experiments were otherwise the same whichever drug was used. In the paralysed animal the heart rate could be restored to regularity, and a frequency within the normal range, by perfusion of the gills via a cannula in the mouth. In this preparation sudden stoppage of the perfusion caused the same result as before but it was clear that the fall in heart rate was not immediate (Fig. 3(c), (d)). This, and the irregularity, appeared gradually after stopping the water flow. Reinstatement of the flow, on the other hand, caused an immediate return of regularity and increase in frequency, usually to a level slightly higher than before the stoppage (Fig. 3(e)). It was found that variations in the flow rate of water over the gills did not greatly affect the heart if the flow was above a certain level. For example, a fourfold increase in flow from 60 to 230 ml per rain produced almost no change in the heart rate of a 107 g tench. Below a critical level of water flow the heart rate went down and the beat became irregular; in the case cited above there was a slight change at a flow rate of 40 ml per min. This is about the ventilation volume which might be expected in a resting fish of this size, though it is unlikely that the obvious inference can be drawn from this observation because the apparatus permitted measurement of water flow through the cannula and not through the fish's gills. For a variety of reasons ventilation and gas exchange are very much more efficient when the animal is breathing normally than they are when produced artificially in an immobilized animal. These experiments suggest that flow rate by itself may be unimportant and that the respiratory gases may be more directly involved in the failure of the heart. In confirmation of this it was found that the heart could be maintained at a normal level by very short bursts of water flow through the buccal cavity and gills with periods of no flow in between. In addition, on four of the paralysed tench,
THE RELATIONSHIP BETWEEN HEART BEAT AND RESPIRATION IN TELEOST FISH
245
experiments were done in which the gills were perfused at various rates with completely de-oxygenated water through which nitrogen was kept bubbling. As
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FIG. 4b. Fro. 4. T h e effect of oxygen lack on the h e a r t rate of a t e n t h paralysed w i t h succinyl choline. (a) C o n t i n u o u s p e r f u s i o n of gills w i t h 200 ml w a t e r per rain. P e r i o d of oxygen lack p r o d u c e d b y p e r f u s i o n w i t h d e - o x y g e n a t e d water. Delay in r e s p o n s e is due to time taken for oxygenated w a t e r to be w a s h e d away. (b) Period of oxygen lack p r o d u c e d b y s t o p p i n g the flow of water. Delay in r e s p o n s e is due to oxygen e o n t a i n e d in the w a t e r s u r r o u n d i n g the gills. I n b o t h cases recovery occurs i m m e d i a t e l y oxygen enters the gill region.
246
G. SHELTONAND D. J. RANDALL
soon as the de-oxygenated water reached the gills no matter at what rate it was flowing, the heart beat became irregular and the rate went down (Fig. 5). The possibility that the lack of oxygen in these experiments was having a direct effect upon the heart, perhaps via the coronary blood vessels, was eliminated when it was found that flow of de-oxygenated water over the gills had no effect on animals in which the tenth cranial nerves had been sectioned (Fig. 5). Stoppage of water flow was similarly without effect on the vagotomized preparation.
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F[c. 5. The effect of oxygen lack on the heart rate of a paralysed tench in which the Xth cranial nerves have been sectioned. Oxygen lack produced by perfusion with de-oxygenated water (i) and stopping the flow of water (ii). In some of the paralysed tench, rhythmic discharges of nerve impulses were recorded from respiratory neurones in the medulla oblongata. T h o u g h this work will be described in a later article, one point of interest emerged which has some relevance here. It was found that on some occasions, whilst the gills of the paralysed and anaesthetized (MS 222) animal were being perfused, there was synchrony between the discharges from the respiratory centre and the heart beat (Fig. 6). DISCUSSION It has been pointed out in the introduction that the most interesting quantitative measurements on the heart and ventilating mechanism would be ones which gave the Volume of blood and volume of water pumped at any time. These are the most important features of any relationship which may exist between circulation and the ventilating mechanism but unfortunately they are not easily determined. In the present work the amplitude and frequency of the breathing movements have been recorded and the ventilation volume is undoubtedly related to these, though not in a simple manner. In the case of the heart, however, only the frequency of beat has been determined and, in so far as heart output can obviously change
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Ft(;. 6. Records of r e s p i r a t o r y activity in the m e d u l l a o b l o n g a t a (lower trace) a n d of the E . C . ( ; . ( u p p e r trace) in a t e n c h paralysed w i t h succinyl choline a n d w i t h the gills artificially perfused. S y n c h r o n y of the h e a r t beat a n d the respiratory discharge can be seen. T w o m a j o r u n i t s w h i c h are to some e x t e n t i n d e p e n d e n t can be seen in the r e s p i r a t o r y discharge. S y n c h r o n y of the h e a r t beat is w i t h the smaller of the two.
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THE R E L A T I O N S H I P B E T W E E N HEART BEAT AND R E S P I R A T I O N I N TELEOST F I S H
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without the frequency of beat being affected, a complete analysis of the relationship is not possible with the present information. The relationship is nevertheless worth examining in the light of the results obtained since frequency of beat is clearly a very significant parameter having considerable bearing on the link between breathing and circulation. In the resting teleost there is a tendency for the heart beat and breathing to become synchronized. The synchrony is by no means perfect and, though the heart beats more frequently in the "mouth closing" phase, there is usually a large number of occasions when the beat occurs in other phases. A more marked synchrony is seen in anaesthetized fish and, in some, a very strict relationship has been seen over long periods of time. The fact that the unanaesthetized animal must be at rest and undisturbed for the described relationship to appear, suggests that a similar mechanism is at work as in the anaesthetized animal. The results in both cases are in some ways reminiscent of the synchrony, which has been described by yon Hoist (1934a, b), between breathing, movements of the trunk and movements of the pectoral fins in the goldfish. In explanation of this synchrony yon Hoist suggested that automatic cells in the CNS were responsible for the generation of a basic rhythmic process which could affect several systems. This view has been criticized (see review by Healey, 1957) as a general interpretation of all rhythmic activity. In some cases, however, it seems reasonable to identify yon Hoist's rhythmic neurones with the respiratory neurones in the medulla. These interact to produce a rhythmic discharge under many conditions including levels of anaesthesia which are sufficient to prevent all other movements. The constitution of the respiratory network appears to be variable (Shelton, 1961) and it is possible that under some conditions recruitment of neurones could result in musculature outside that normally associated with breathing being affected by the rhythmicity. A tendency towards the spreading of activity in a nervous system otherwise at a fairly low level of excitation could account for synchrony of heart beat, breathing and body movement under the conditions which have been described. The synchrony between heart and respiratory discharge in the curarized animal shows that, in this case at least, the relationship is not a simple mechanical one, nor does it depend on feedback from the respiratory musculature. Whether there is any greater significance to the heart-beat-breathing synchrony than as a side effect of function within a moderately inactive nervous system is difficult to say. Nothing is known of the relation between heart beat and blood flow through the gills and so it is impossible to decide whether synchrony during the mouth-closing phase confers an advantage, as Satchell (1960) has suggested for the dogfish. The synchrony in the teleost is easily lost and so is clearly subordinate to other factors in the co-ordination of breathing and heart output. Very little indeed is known of these factors, though heart output ought to be related largely to the needs of the tissues, and ventilation volume largely to fulfilling the gas exchange requirements of the blood flowing through the gills. But no matter what the tissue requirements are, there would be little point in the heart pumping more blood than could be saturated by the ventilation stream. To avoid excess activity,
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particularly at times when the amount of oxygen in the ventilation stream is insufficient to saturate the blood, it would be necessary for information about the oxygen content of the ventilation stream or of the blood to be relayed in some way to the heart. A mechanism of this general type appears to exist in the diving mammal (Scholander, 1940), in the elasmobranch (Satchell, 1961), and it also seems that low levels of oxygen produce a marked inhibition of the heart in teleost fish. This occurs whether the lack of oxygen is produced by a natural pause in breathing, by over-anaesthesia, by paralysis of the breathing musculature or by ventilation of the gills with de-oxygenated water. Inhibition may also occur if the oxygen lack is produced by removing the animal from water; Leivestad et al. (1957), Serfaty & Raynaud (1958), and Garey (1962) have all found that exposure to air causes cardiac slowing in teleost fish, though Serfaty & Renaud (1957b) have claimed that this response is not due to oxygen lack. In addition to the inhibition of the heart which is produced by oxygen lack at the gill region, probably via a reflex mechanism, there appears to be another more direct effect of the failure of breathing movements. In the paralysed animal where the effect of oxygen can be seen isolated from any relationship with the breathing movements themselves, a stoppage of flow through the gills does not immediately influence heart rate. A short time elapses before the heart begins to slow and it is assumed that there is a delay before the oxygen contained in the water around the gills is depleted. However, in the unanaesthetized eel a pause in breathing is followed immediately by a slight fall in heart rate. This is also true of an anaesthetized tench showing periodic breathing. In fact there appears to be an immediate effect of a stoppage of breathing in addition to the longer-term influence of lack of oxygen. There is no evidence to indicate whether this direct effect is produced by the central nervous system, by a reflex or by the direct influence of the contraction of respiratory musculature in the region of the heart. The relationship between breathing and cardiac output is obviously a complicated one. Even within the limitations of the present experiments at least three factors have appeared as participants in the overall pattern. Some suggestions have been made as to how these constituent factors may operate but any detailed analysis of the nervous pathways involved is impossible with our present knowledge of cardiac innervation and control. It is often stated that cardiac augmentor nerves are absent in teleosts (Nicol, 1952) and that the only innervation is the inhibitory one of the vagus. Jullien & Ripplinger (1950) have suggested that the vagus has two inhibitory effects, one chronotropic affecting frequency, and the other tonotropic affecting the muscle tone. If it is accepted that the only innervation of the heart is via the vagus, then it is necessary on the present evidence to suppose a cyclical inhibition in rhythm with the breathing movements to control heartbreathing synchrony, and a maintained inhibition to control absolute rate. Variations in the level of this maintained inhibition could be held responsible for the frequency changes seen with excitement, MS 222, lack of oxygen, etc. A system working entirely on inhibitory control is rather unusual and there have been suggestions that cardio-accelerator systems exist outside endocrine sources of
T H E R E L A T I O N S H I P B E T W E E N H E A R T B E A T A N D R E S P I R A T I O N IN T E L E O S T F I S H
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accelerating drugs (Mott, 1957). Section of the vagus nerve of a tench anaesthetized in M S 222 can result in a decrease in heart rate (Randall, 1962). T h i s suggests that the increase in heart rate produced by M S 222 is not due entirely to a decrease in the maintained level of chronotropic inhibition but that there mav be some cardio-accelerator fibres in the vagus nerve if not elsewhere. SUMMARY 1. Simultaneous recordings of the breathing m o v e m e n t s and the electrocardiogram have been taken f r o m the tench and three other teleosts. 2. In the resting animal there is a tendency for the heart to beat in the " m o u t h closing" phase of the breathing cycle. Synchrony is not perfect in the unanaesthetized animal and the heart often beats in other parts of the cycle. In the lightly anaesthetized fish perfect synchrony of heart and breathing has frequently been seen. Stimulation results in the disappearance of the relationship in both unanaesthetized and anaesthetized animals. Spread of activity from the rhythmically firing respiratory centre may account for the synchrony. 3. Deep anaesthesia, using M S 222, urethane or nembutal, ultimately causes respiratory failure and this is immediately followed by a fall in heart rate. 4. Injection of either succinyl choline chloride or tubocurarine chloride in sufficient quantity to paralyse the breathing muscles also results in a fall in heart rate. Perfusion of the gills with oxygenated water is followed by return of the heart rate to a normal level. Perfusion with de-oxygenated water slows the heart. it is suggested that the heart is reflexly inhibited whenever the oxygen supply at the gills is insufficient to enable the animal to maintain oxygen equilibrium. 5. In addition to the effect of oxygen supply on the heart, it also appears that the breathing m o v e m e n t s themselves have a stimulating effect on heart rate. Achno~cledgements--We are indebted to the Department of Scientific and Industrial Research for financial support; one of us (G. S.) is in receipt of an apparatus grant, the other (D. J. R.) of a research studentship grant.
REFERENCES GAREYW. F. (t962) Cardiac responses of fish in asphyxic environments. Biol. Bull., Wood's Hole 122, 362-386. I:tEALE'ZE. G. (1957) The nervous system. In The Physiology of Fishes (Edited by BBow.~ M.E.). Academic Press, New York. HUGHESG. M. (1961) Gill ventilation in fish. Rep. Challenger Soc. 13, No. 3, 23 24. ttUGHES G. M. & SHELTONG. (1962) Respiratory mechanisms and their nervous control in fish. In Advances in Comparative Physiology and Biochemistry, Vol. 1 (Edited by LOWENSTEINO. E.). Academic Press, New York. JVLLIENA. & RIVPHNCERJ. (1950) Le rameau cardiaque du pneumogastrique des poissons est form~ d'au moins deux nerfs : un neff chronotrope, cholinergique et un nerf tonotrope, non cholinergique. C.R. Acad. Sci. Paris 230, 867-886. KtscH B. (1948) Electrographic investigations of the heart of fish. Exp. ~/[ed. Surg. 6, 31-62. Ll~IVESTADH., ANDERSONH. & SCHOLANOERP. F. (1957) Physiological response to air exposure in codfish. Science 126, 505.
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G. SHELTON AND D. J. RANDALl,
LUTZ B. R. (1930) Respiratory rhythm in the elasmobranch Scyllium canicula. Biol. Bull., Woods Hole 59, 179-186. LYON E. P. (1926) A study of the blood pressure, circulation and respiration of sharks. J. Gen. Physiol. 8, 279-290. MOTT J. C. (1957) The cardiovascular system. In The Physiology of Fishes (Edited by BROWN M. E.). Academic Press, New York. NICOL J. A. C. (1952) Autonomic nervous systems in lower chordates. Biol. Rev. 27, 1-49. OETS J. (1950) Electrocardiograms of fishes. Physiol. Comp. 2, 181-186. RANDALL D. J. (1962) The effect of an anaesthetic on the heart and respiration of teleost fish. Nature, London, 195, 506. RANDALLD. J. & SHELTONG. (1962) The effects of varying environmental gas tensions on the breathing and heart rate of a teleost fish. In preparation. SATCHELL G. H. (1960) The reflex co-ordination of the heart beat with respiration in the dogfish. J. Exp. Biol. 37, 719-731. SATCHELL G. H. (1961) The response of the dogfish to anoxia. J. Exp. Biol. 38, 531-543. SCHOLANDER P. F. (1940) Experimental investigations on the respiratory function in diving mammals and birds. Hvalrdd. Skr. No. 22. SERFATYA., LABATR. ~; QUILLIERR. (1959) Les r6actions cardiaques chez la carpe (Cyprinus carpio) au cours d'une anesthdsie prolongde. Hydrobiologia 13, 144-151. SERFATY A. ~; RAYNAUDP. (1956) Electrocardiographe du poisson dans son milieu naturel. Bull. Soc. Zool. Ft. 81, 121-126. SERFATY A. & RAYNAUD P. (1957a) Quelques donn6es sur l'61ectrocardiogramme de la tanche (Tinca tinca L.) en eau douce. Bull. Soc. Zool. Ft. 82, 49-56. SERFATY A. & RAYNAUDP. (1957b) R6flexe a6ro-cardiaque chez la truite de rivi~re (Salmo trutta L.). J. Physiol. Path. Gdn. 49, 378-381. SERFATY A. &; RAYNAUD P. (1958) Le rdflexe a6ro-cardiaque chez la carpe commune (Cyprinus carpio L.) et le ph6nom~ne d'6chappement. Hydrobiologia 12, 38-42. SHELTON G. (1961) The respiratory centre in the tench (Tinca tinca L . ) - - I I . Respiratory neuronal activity in the medulla oblongata. J. Exp. Biol. 38, 79-92. VAN DAM L. (1938) On the utilisation of oxygen and regulation of breathing in some aquatic animals. Thesis, Groningen. VON HOLST E. (1934a) Reflex und Rhythmus im Goldfischrtickenmark. Zool. Anz. (Suppl. 7), 93-95. VON HOLST E. (1934b) Studien fiber Reflexe und Rhythmen beim Goldfisch (Carassius auratus). Z. Vergl. Physiol. 20, 582-599.
1962, Vol. 7, pp. 237 to 250. Pergamon Press Ltd., London. Printed in Great Britain
THE RELATIONSHIP BETWEEN HEART BEAT AND R E S P I R A T I O N IN T E L E O S T F I S H G. SHELTON and D. J. RANDALL Department of Zoology, University of Southampton (Received
27 A u g u s t 1962)
A tendency for the heart to beat in the mouth-closing phase of the breathing movements is often seen in the resting tench. Synchrony is seldom perfect, except in the anaesthetized animal, and disappears if the fish is stimulated in any way. 2. When the breathing movements stop in the tench and other teleosts because of a natural pause, deep anaesthesia or curare-induced paralysis, the heart rate decreases. This inhibition of the heart, probably of reflex origin, can also be produced by perfusion of the gills with de-oxygenated water and is caused by lack of oxygen at the gills. 3. The breathing movements also appear to have a stimulating effect on the heart rate separate from the oxygen reflex.
Abstract--1.
INTRODUCTION
IT I8 reasonable to predict that, in the interests of economy of effort, a close relationship will be maintained between the amount of oxygen presented to an exchanging surface by an animal's breathing m o v e m e n t s and the amount of blood p u m p e d through that surface to transfer the oxygen to the tissues. In aquatic animals the relationship ought perhaps to be clearer than in terrestrial forms because of the considerable amount of work which must be done in ventilating the respiratory surface (Hughes & Shelton, 1962). A discrepancy between the capacity of the water stream to bring oxygen to the exchanging surface and the blood stream to take it away would lead to a serious waste of effort on the part of an aquatic animal, particularly if the lack of balance was due to excess ventilation. Very little is known of the exact quantitative features of the heart o u t p u t - ventilation ~olume relationship in aquatic or indeed in any animals. One of the major difficulties which has been encountered is that of determining the heart output and blood flow through the respiratory organ. However, some work has been done on fish and the existence of a relationship has been confirmed. In elasmobranchs it has been known for a long time that there is a simple numerical relationship between heart and breathing rates, the heart beat occurring every 1, 2, 3 or 4 breathing cycles (Lyon, 1926; Lutz, 1930). Satchell (1960) has extended the earlier work and has suggested that, in the dogfish, the heart beat m a y occur in relation to the breathing m o v e m e n t s in such a way as to make coincident the periods of m a x i m u m water flow over the gills and of m a x i m u m blood flow i6
237
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G. SHELTONAND D. J. RANDALL
through them. This precise type of relationship appears not to hold in teleost fish. Serfaty & Raynaud (1957a) have recorded respiratory movements and electrocardiographs from the tench and found that synchronization between heart beat and breathing is very rare and persists for a short time only. To some extent there is a similar lack of correlation in the trout (Hughes, 1961), though in this animal quite good synchronization has been observed occasionally with the heart beating at a specific point in the respiratory cycle. However, synchronization of such exactitude is not the only relationship which may exist. Indeed it would appear perhaps the least likely because it has the disadvantage of permitting adjustment to take place in a stepped instead of a continuously variable fashion. Obviously there can be a close correlation between heart output and ventilation volume without synchrony. Both depend on two variable parameters, the amplitude and frequency of the movements, and so the details of this correlation may be complex and variable from time to time and in different individuals. In spite of this complexity more details of the extent of the correlation between heart beat and breathing movements ought to be forthcoming from experiments in which the environment is changed in ways having a differential effect on the two systems. This, and a succeeding article (Randall & Shelton 1962), describe the effects of various factors on the heart rate and breathing movements. METHODS The majority of the experiments were done on tench (Tinca tinca) varying between 50 and 200 g in weight, though some eels (Anguilla vulgaris), trout (Salmo trutta) and roach (Rutilus rutilus) were also used. The experiments were carried out in Perspex tanks between 1 and 21. in capacity through which continuous water flow could be maintained. The flow through the tanks was from the tail of the fish towards the head in those experiments in which the fish was breathing normally, to avoid any forced ventilation of the gills. In the experiments in which the breathing movements were stopped by various means, artificial respiration was given by forcibly ventilating the gills via a cannula in the mouth. In all cases the flow rate was kept appreciably higher than that necessary to satisfy the oxygen demands of the fish. The experiments on unanaesthetized animals were done at tapwater temperature (13-15°C), those in which anaesthetic was dissolved in the water and a closed circuit maintained were done at room temperature (17-20°C). The temperature was constant to within I°C during any one experiment. The movements of the lower jaw were taken as representative of the breathing movements as a whole. Originally they were recorded on a smoked drum but in some experiments a mechanoelectric transducer was used, the output being taken to an Ediswan pen recorder. The heart rate was determined by recording the electrocardiogram (E.C.G.). This was detected by means of a unipolar system, using a steel electrode insulated with varnish except at the tip. This electrode pierced the skin mid-ventrally in front of the pectoral girdle, the tip being pushed forward to lie beneath the heart. An indifferent electrode was placed in the water contained in the tank. The signal was amplified in an Ediswan EPA amplifier and
T H E R E L A T I O N S H I P B E T W E E N H E A R T BEAT A N D R E S P I R A T I O N I N T E L E O S T F I S H
239
the output connected to a second pen on the Ediswan recorder. Alternatively a Tektronix 502 oscilloscope was used for display, records being taken with a Cossor oscilloscope camera. The primary object in recording the E.C.G. in these experiments was to determine the heart rate and not to study the E.C.G. itself. Descriptions of the E.C.G. obtained from a variety of teleosts have already been given (Kisch, 1948 ; Oets, 1950 ; Serfaty & Raynaud, 1956, 1957a) and we have nothing to add to these. The voltage and configuration of the E.C.G. varied considerably with the position of the detecting electrode in the ventral musculature. The electrode was adjusted until an adequate signal to noise ratio was obtained. By filtering off low frequencies in the amplifier a simple E.C.G. was obtained, and by including the low frequencies it was possible to record E.C.G. superimposed on voltage changes produced by the breathing movements. By this means it was possible to study some aspects of the heart and breathing relationship on a single trace. RESULTS The relationship of the heart beat to the breathing movements The heart rate of the resting, unanaesthetized tench is in the range 15-30 beats/min, that of the eel from 40-50 beats/min. The breathing rate is slightly higher and, as in the case of the heart rate, variable from animal to animal and in the same animal at different times. The resting tench breathes at a frequency between 30 and 60 respirations/min, the eel between 30 and 50, though in the latter there are long pauses in the breathing rhythm. Although some of our results have confirmed the conclusion of previous workers that no direct relationship exists between heart beat and breathing in teleosts, this is by no means universally true. A closer analysis of the position of the ventricular phase of the E.C.G. in relation to the breathing cycle of the tench showed that, while respiratory and cardiac frequencies might differ, under some conditions the heart beat appeared more often during the "mouth closing" phase of the cycle. This relationship was true particularly of fish which were obviously at rest and not disturbed in any way. Even minor disturbances tend to make the relationship less obvious, and high levels of carbon dioxide, for example, obliterate the pattern completely (Fig. 1). The records from nine animals have been examined in some detail and in four cases the pattern was very clear (Fig. l(a)), in four cases fairly clear (Fig. l(b)), and only in the single carbon dioxide experiment analysed (Fig. l(c)) was there no trace of "mouth closing" domination. Where breathing and heart rate differed, a relationship was maintained by the lengthening or shortening of the interval between heart beats. The lengthening of interval between heart beats was also noticed in many of the fish in which coughs broke up the smooth rhythm of breathing. A similar pattern was found in the eel with the heart beat appearing more often during the "mouth closing" phase of the breathing cycle and again giving a clear indication that co-ordination between breathing and heart beat does exist in the intact, unanaesthetized teleost. Breathing in the eel can be spasmodic, as van Dam (1938) has pointed out, the fish breathing quite regularly for a while and then
240
G. SrtELTON AND D. J. RANDALL
pausing for periods up to 5 min in duration in extreme cases. W e found that the pauses of more than 30 sec duration were rare under normal conditions of oxygenation, temperature, etc. During the periods when the animal was breathing normally,
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FIG. l. Histograms of the occurrence of heart beat in relation to the breathing cycle of the tench. Successive breathing cycles are divided into ten equal parts and the occurrence of heart beats in these parts is plotted on a percentage basis in the histograms. (a) Clear relationship with "mouth closing" phase. Number of heart beats in analysis--182. Breathing rate--66 per rain. Heart rate--22 per rain. (b) Some evidence of "mouth closing" dominance. Number of heart beats in analysis--136. Breathing rate--39'5 per rain. Heart rate--16-5 per rain. (c) Respiration stimulated with CO2. No clear relationship, though fewer heart beats occur in "mouth opening" phase. Number of heart beats in analysis--ll8. Breathing rate--54 per min. Heart rate--46 per rain. the heart rate was about 50/min but when breathing stopped the heart rate dropped immediately to half the original value. T h e rate returned to the higher value as soon as the eel began ventilating the gills once more. T h i s relationship is m u c h more obvious than the one described previously for the breathing fish, and, though it is possible that one is an extreme case of the other, it is equally likely that two separate mechanisms are involved.
The effects of anaesthesia T h e main interest in the effects of anaesthetics centred on their influence at high concentrations on breathing rate and heart rate. O f the three anaesthetics used, namely urethane, nembutal and tricaine methane sulphate ( M S 222), only the latter had very obvious effects at light levels of anaesthesia. T h e concentrations of this anaesthetic required for a given level of sedation were variable but, in general, bathing a tench in a solution of 20 m g M S 222 per 1. made the animal unreactive to external stimuli, whilst 100 m g per 1. caused rapid loss of equilibrium with no m o v e m e n t s except those of breathing. T h e effects of M S 222 on the heart of the carp (Serfaty et al., 1959) and the tench (Randall, 1962) have been described previously. I n agreement with this previous work it was found that all concentrations
241
THE R E L A T I O N S H I P B E T W E E N HEART BEAT AND RESPIRATION IN TELEOST FISH
of M S 222 caused an increase in heart rate and, usually to a lesser extent, in respiratory rate and amplitude (Fig. 2). T h e effects on heart and breathing were simultaneous and noticeable within a minute of running the solution into the tank. IO0 90 8O
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THE RELATIONSHIPBETWEENHEARTBEATANDRESPIRATIONIN TELEOSTFISH
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intramuscularly per 100 g fish) were similarly without effect on the heart and respiration. Insufficient experiments were done to enable any conclusions to be drawn about the possible effects of these two anaesthetics on the heart-breathing synchrony. All three anaesthetics, when present in higher concentrations than those given above, were effective in causing the breathing movements to become irregular and ultimately to stop altogether. In Fig. 2 the effects of a high concentration of MS 222 on the heart rate and breathing of the tench are shown. After 12 min in the anaesthetic solution the animal stopped breathing and the heart rate fell quickly to a level close to that seen in the unanaesthetized animal. The fact that the heart rate did not fall below the normal value can probably be explained in terms of the accelerating influence of MS 222. Anaesthetics not having this effect do in fact cause a fall in heart rate below the unanaesthetized frequency when the animal's breathing fails. At least part of the fall in frequency occurs immediately the breathing movements stop, but only after a delay is the full extent of the decrease in rate seen. A feature of the low heart rate which results from respiratory failure in deep anaesthesia is its somewhat irregular nature. This was not seen in the unanaesthetized eel during a respiratory pause; in this animal the heart beat remained regular. In both the tench and the eel, recovery of the breathing results in a simultaneous recovery of the heart rate. In Fig. 2 a short period of breathing at point E is reflected in the higher value for heart rate when next measured.
The effects of paralysing agents A definite connexion has been established between heart rate and breathing rate by the foregoing experiments but what is not certain is the extent to which the various components of this connexion are inter-related. It is possible that the same or different co-ordinating mechanisms are responsible for the relationship in the unanesthetized animal, in the lightly anaesthetized animal showing synchrony of breathing and heart beat, and in the non-breathing animal. The co-ordination may be a purely central nervous phenomenon or a peripheral effect of either a simple mechanical or a reflex nature. If the relationship is reflexly mediated the important afferent impulses may be associated with the breathing movements, the water stream over the exchanging surface, or the levels of the respiratory gases in the body or the medium. Finally it must be recognized that a relationship between heart and breathing can be effected not only by the breathing in some way influencing heart beat, as the foregoing comments assume, but also by an influence working in the reverse direction. In order to resolve some of these difficulties in interpretation, experiments were done on fish which had been paralysed, the paralysis being produced in two different ways by injection either of tubocurarine chloride (0-5-1.0 mg. per 100 g fish) or of succinyl choline (0.1-0.3 mg per 100 g fish). The curare dose is high by mammalian standards, the succinyl choline dose roughly comparable though the duration of the paralysis with the latter drug is greater than in mammals. Both drugs work at the endplate region but act in different ways. They were used in an
244
G. SHELTONAND D. J. RANDALL
attempt to make sure that the important feature in the experiments was the paralysis and not the paralysing agent. The effect of a paralysing dose of one of the above drugs injected intramuscularly into an anaesthetized tench was to stop all but very slight movement of the respiratory musculature in 1-2 min. There are some components of the respiratory complex which are quite resistant to both curare and succinyl choline. These have not as yet been identified but they can produce slight movements which are noticeable particularly on the opercular valve. Satchell (1961) has also commented on the resistance to curare of some of the dogfish respiratory muscles. In the tench these small movements may continue for some time but eventually stop, with the doses used, some 15-30 rain after injection. Long before this, however, soon after the major breathing movements stop, there is a marked fall in heart rate and the beat becomes irregular. With succinyl choline it was also found that about 5 min after injection the character of the E.C.G. changed, with the amplitude of the components decreasing and the potential changes being made more slowly (Fig. 3(b)). This effect persisted for about 30 rain during which no experiments were done but the gills were perfused with water. Ultimately the E.C.G. recovered its normal appearance. Curare had no effect on the E.C.G. but, though the drugs differed in this respect, the results of the experiments were otherwise the same whichever drug was used. In the paralysed animal the heart rate could be restored to regularity, and a frequency within the normal range, by perfusion of the gills via a cannula in the mouth. In this preparation sudden stoppage of the perfusion caused the same result as before but it was clear that the fall in heart rate was not immediate (Fig. 3(c), (d)). This, and the irregularity, appeared gradually after stopping the water flow. Reinstatement of the flow, on the other hand, caused an immediate return of regularity and increase in frequency, usually to a level slightly higher than before the stoppage (Fig. 3(e)). It was found that variations in the flow rate of water over the gills did not greatly affect the heart if the flow was above a certain level. For example, a fourfold increase in flow from 60 to 230 ml per rain produced almost no change in the heart rate of a 107 g tench. Below a critical level of water flow the heart rate went down and the beat became irregular; in the case cited above there was a slight change at a flow rate of 40 ml per min. This is about the ventilation volume which might be expected in a resting fish of this size, though it is unlikely that the obvious inference can be drawn from this observation because the apparatus permitted measurement of water flow through the cannula and not through the fish's gills. For a variety of reasons ventilation and gas exchange are very much more efficient when the animal is breathing normally than they are when produced artificially in an immobilized animal. These experiments suggest that flow rate by itself may be unimportant and that the respiratory gases may be more directly involved in the failure of the heart. In confirmation of this it was found that the heart could be maintained at a normal level by very short bursts of water flow through the buccal cavity and gills with periods of no flow in between. In addition, on four of the paralysed tench,
THE RELATIONSHIP BETWEEN HEART BEAT AND RESPIRATION IN TELEOST FISH
245
experiments were done in which the gills were perfused at various rates with completely de-oxygenated water through which nitrogen was kept bubbling. As
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246
G. SHELTONAND D. J. RANDALL
soon as the de-oxygenated water reached the gills no matter at what rate it was flowing, the heart beat became irregular and the rate went down (Fig. 5). The possibility that the lack of oxygen in these experiments was having a direct effect upon the heart, perhaps via the coronary blood vessels, was eliminated when it was found that flow of de-oxygenated water over the gills had no effect on animals in which the tenth cranial nerves had been sectioned (Fig. 5). Stoppage of water flow was similarly without effect on the vagotomized preparation.
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F[c. 5. The effect of oxygen lack on the heart rate of a paralysed tench in which the Xth cranial nerves have been sectioned. Oxygen lack produced by perfusion with de-oxygenated water (i) and stopping the flow of water (ii). In some of the paralysed tench, rhythmic discharges of nerve impulses were recorded from respiratory neurones in the medulla oblongata. T h o u g h this work will be described in a later article, one point of interest emerged which has some relevance here. It was found that on some occasions, whilst the gills of the paralysed and anaesthetized (MS 222) animal were being perfused, there was synchrony between the discharges from the respiratory centre and the heart beat (Fig. 6). DISCUSSION It has been pointed out in the introduction that the most interesting quantitative measurements on the heart and ventilating mechanism would be ones which gave the Volume of blood and volume of water pumped at any time. These are the most important features of any relationship which may exist between circulation and the ventilating mechanism but unfortunately they are not easily determined. In the present work the amplitude and frequency of the breathing movements have been recorded and the ventilation volume is undoubtedly related to these, though not in a simple manner. In the case of the heart, however, only the frequency of beat has been determined and, in so far as heart output can obviously change
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Ft(;. 6. Records of r e s p i r a t o r y activity in the m e d u l l a o b l o n g a t a (lower trace) a n d of the E . C . ( ; . ( u p p e r trace) in a t e n c h paralysed w i t h succinyl choline a n d w i t h the gills artificially perfused. S y n c h r o n y of the h e a r t beat a n d the respiratory discharge can be seen. T w o m a j o r u n i t s w h i c h are to some e x t e n t i n d e p e n d e n t can be seen in the r e s p i r a t o r y discharge. S y n c h r o n y of the h e a r t beat is w i t h the smaller of the two.
I
E , C . G .
THE R E L A T I O N S H I P B E T W E E N HEART BEAT AND R E S P I R A T I O N I N TELEOST F I S H
247
without the frequency of beat being affected, a complete analysis of the relationship is not possible with the present information. The relationship is nevertheless worth examining in the light of the results obtained since frequency of beat is clearly a very significant parameter having considerable bearing on the link between breathing and circulation. In the resting teleost there is a tendency for the heart beat and breathing to become synchronized. The synchrony is by no means perfect and, though the heart beats more frequently in the "mouth closing" phase, there is usually a large number of occasions when the beat occurs in other phases. A more marked synchrony is seen in anaesthetized fish and, in some, a very strict relationship has been seen over long periods of time. The fact that the unanaesthetized animal must be at rest and undisturbed for the described relationship to appear, suggests that a similar mechanism is at work as in the anaesthetized animal. The results in both cases are in some ways reminiscent of the synchrony, which has been described by yon Hoist (1934a, b), between breathing, movements of the trunk and movements of the pectoral fins in the goldfish. In explanation of this synchrony yon Hoist suggested that automatic cells in the CNS were responsible for the generation of a basic rhythmic process which could affect several systems. This view has been criticized (see review by Healey, 1957) as a general interpretation of all rhythmic activity. In some cases, however, it seems reasonable to identify yon Hoist's rhythmic neurones with the respiratory neurones in the medulla. These interact to produce a rhythmic discharge under many conditions including levels of anaesthesia which are sufficient to prevent all other movements. The constitution of the respiratory network appears to be variable (Shelton, 1961) and it is possible that under some conditions recruitment of neurones could result in musculature outside that normally associated with breathing being affected by the rhythmicity. A tendency towards the spreading of activity in a nervous system otherwise at a fairly low level of excitation could account for synchrony of heart beat, breathing and body movement under the conditions which have been described. The synchrony between heart and respiratory discharge in the curarized animal shows that, in this case at least, the relationship is not a simple mechanical one, nor does it depend on feedback from the respiratory musculature. Whether there is any greater significance to the heart-beat-breathing synchrony than as a side effect of function within a moderately inactive nervous system is difficult to say. Nothing is known of the relation between heart beat and blood flow through the gills and so it is impossible to decide whether synchrony during the mouth-closing phase confers an advantage, as Satchell (1960) has suggested for the dogfish. The synchrony in the teleost is easily lost and so is clearly subordinate to other factors in the co-ordination of breathing and heart output. Very little indeed is known of these factors, though heart output ought to be related largely to the needs of the tissues, and ventilation volume largely to fulfilling the gas exchange requirements of the blood flowing through the gills. But no matter what the tissue requirements are, there would be little point in the heart pumping more blood than could be saturated by the ventilation stream. To avoid excess activity,
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G. SHELTONAND D. J. RANDALL
particularly at times when the amount of oxygen in the ventilation stream is insufficient to saturate the blood, it would be necessary for information about the oxygen content of the ventilation stream or of the blood to be relayed in some way to the heart. A mechanism of this general type appears to exist in the diving mammal (Scholander, 1940), in the elasmobranch (Satchell, 1961), and it also seems that low levels of oxygen produce a marked inhibition of the heart in teleost fish. This occurs whether the lack of oxygen is produced by a natural pause in breathing, by over-anaesthesia, by paralysis of the breathing musculature or by ventilation of the gills with de-oxygenated water. Inhibition may also occur if the oxygen lack is produced by removing the animal from water; Leivestad et al. (1957), Serfaty & Raynaud (1958), and Garey (1962) have all found that exposure to air causes cardiac slowing in teleost fish, though Serfaty & Renaud (1957b) have claimed that this response is not due to oxygen lack. In addition to the inhibition of the heart which is produced by oxygen lack at the gill region, probably via a reflex mechanism, there appears to be another more direct effect of the failure of breathing movements. In the paralysed animal where the effect of oxygen can be seen isolated from any relationship with the breathing movements themselves, a stoppage of flow through the gills does not immediately influence heart rate. A short time elapses before the heart begins to slow and it is assumed that there is a delay before the oxygen contained in the water around the gills is depleted. However, in the unanaesthetized eel a pause in breathing is followed immediately by a slight fall in heart rate. This is also true of an anaesthetized tench showing periodic breathing. In fact there appears to be an immediate effect of a stoppage of breathing in addition to the longer-term influence of lack of oxygen. There is no evidence to indicate whether this direct effect is produced by the central nervous system, by a reflex or by the direct influence of the contraction of respiratory musculature in the region of the heart. The relationship between breathing and cardiac output is obviously a complicated one. Even within the limitations of the present experiments at least three factors have appeared as participants in the overall pattern. Some suggestions have been made as to how these constituent factors may operate but any detailed analysis of the nervous pathways involved is impossible with our present knowledge of cardiac innervation and control. It is often stated that cardiac augmentor nerves are absent in teleosts (Nicol, 1952) and that the only innervation is the inhibitory one of the vagus. Jullien & Ripplinger (1950) have suggested that the vagus has two inhibitory effects, one chronotropic affecting frequency, and the other tonotropic affecting the muscle tone. If it is accepted that the only innervation of the heart is via the vagus, then it is necessary on the present evidence to suppose a cyclical inhibition in rhythm with the breathing movements to control heartbreathing synchrony, and a maintained inhibition to control absolute rate. Variations in the level of this maintained inhibition could be held responsible for the frequency changes seen with excitement, MS 222, lack of oxygen, etc. A system working entirely on inhibitory control is rather unusual and there have been suggestions that cardio-accelerator systems exist outside endocrine sources of
T H E R E L A T I O N S H I P B E T W E E N H E A R T B E A T A N D R E S P I R A T I O N IN T E L E O S T F I S H
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accelerating drugs (Mott, 1957). Section of the vagus nerve of a tench anaesthetized in M S 222 can result in a decrease in heart rate (Randall, 1962). T h i s suggests that the increase in heart rate produced by M S 222 is not due entirely to a decrease in the maintained level of chronotropic inhibition but that there mav be some cardio-accelerator fibres in the vagus nerve if not elsewhere. SUMMARY 1. Simultaneous recordings of the breathing m o v e m e n t s and the electrocardiogram have been taken f r o m the tench and three other teleosts. 2. In the resting animal there is a tendency for the heart to beat in the " m o u t h closing" phase of the breathing cycle. Synchrony is not perfect in the unanaesthetized animal and the heart often beats in other parts of the cycle. In the lightly anaesthetized fish perfect synchrony of heart and breathing has frequently been seen. Stimulation results in the disappearance of the relationship in both unanaesthetized and anaesthetized animals. Spread of activity from the rhythmically firing respiratory centre may account for the synchrony. 3. Deep anaesthesia, using M S 222, urethane or nembutal, ultimately causes respiratory failure and this is immediately followed by a fall in heart rate. 4. Injection of either succinyl choline chloride or tubocurarine chloride in sufficient quantity to paralyse the breathing muscles also results in a fall in heart rate. Perfusion of the gills with oxygenated water is followed by return of the heart rate to a normal level. Perfusion with de-oxygenated water slows the heart. it is suggested that the heart is reflexly inhibited whenever the oxygen supply at the gills is insufficient to enable the animal to maintain oxygen equilibrium. 5. In addition to the effect of oxygen supply on the heart, it also appears that the breathing m o v e m e n t s themselves have a stimulating effect on heart rate. Achno~cledgements--We are indebted to the Department of Scientific and Industrial Research for financial support; one of us (G. S.) is in receipt of an apparatus grant, the other (D. J. R.) of a research studentship grant.
REFERENCES GAREYW. F. (t962) Cardiac responses of fish in asphyxic environments. Biol. Bull., Wood's Hole 122, 362-386. I:tEALE'ZE. G. (1957) The nervous system. In The Physiology of Fishes (Edited by BBow.~ M.E.). Academic Press, New York. HUGHESG. M. (1961) Gill ventilation in fish. Rep. Challenger Soc. 13, No. 3, 23 24. ttUGHES G. M. & SHELTONG. (1962) Respiratory mechanisms and their nervous control in fish. In Advances in Comparative Physiology and Biochemistry, Vol. 1 (Edited by LOWENSTEINO. E.). Academic Press, New York. JVLLIENA. & RIVPHNCERJ. (1950) Le rameau cardiaque du pneumogastrique des poissons est form~ d'au moins deux nerfs : un neff chronotrope, cholinergique et un nerf tonotrope, non cholinergique. C.R. Acad. Sci. Paris 230, 867-886. KtscH B. (1948) Electrographic investigations of the heart of fish. Exp. ~/[ed. Surg. 6, 31-62. Ll~IVESTADH., ANDERSONH. & SCHOLANOERP. F. (1957) Physiological response to air exposure in codfish. Science 126, 505.
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LUTZ B. R. (1930) Respiratory rhythm in the elasmobranch Scyllium canicula. Biol. Bull., Woods Hole 59, 179-186. LYON E. P. (1926) A study of the blood pressure, circulation and respiration of sharks. J. Gen. Physiol. 8, 279-290. MOTT J. C. (1957) The cardiovascular system. In The Physiology of Fishes (Edited by BROWN M. E.). Academic Press, New York. NICOL J. A. C. (1952) Autonomic nervous systems in lower chordates. Biol. Rev. 27, 1-49. OETS J. (1950) Electrocardiograms of fishes. Physiol. Comp. 2, 181-186. RANDALL D. J. (1962) The effect of an anaesthetic on the heart and respiration of teleost fish. Nature, London, 195, 506. RANDALLD. J. & SHELTONG. (1962) The effects of varying environmental gas tensions on the breathing and heart rate of a teleost fish. In preparation. SATCHELL G. H. (1960) The reflex co-ordination of the heart beat with respiration in the dogfish. J. Exp. Biol. 37, 719-731. SATCHELL G. H. (1961) The response of the dogfish to anoxia. J. Exp. Biol. 38, 531-543. SCHOLANDER P. F. (1940) Experimental investigations on the respiratory function in diving mammals and birds. Hvalrdd. Skr. No. 22. SERFATYA., LABATR. ~; QUILLIERR. (1959) Les r6actions cardiaques chez la carpe (Cyprinus carpio) au cours d'une anesthdsie prolongde. Hydrobiologia 13, 144-151. SERFATY A. ~; RAYNAUDP. (1956) Electrocardiographe du poisson dans son milieu naturel. Bull. Soc. Zool. Ft. 81, 121-126. SERFATY A. & RAYNAUD P. (1957a) Quelques donn6es sur l'61ectrocardiogramme de la tanche (Tinca tinca L.) en eau douce. Bull. Soc. Zool. Ft. 82, 49-56. SERFATY A. & RAYNAUDP. (1957b) R6flexe a6ro-cardiaque chez la truite de rivi~re (Salmo trutta L.). J. Physiol. Path. Gdn. 49, 378-381. SERFATY A. &; RAYNAUD P. (1958) Le rdflexe a6ro-cardiaque chez la carpe commune (Cyprinus carpio L.) et le ph6nom~ne d'6chappement. Hydrobiologia 12, 38-42. SHELTON G. (1961) The respiratory centre in the tench (Tinca tinca L . ) - - I I . Respiratory neuronal activity in the medulla oblongata. J. Exp. Biol. 38, 79-92. VAN DAM L. (1938) On the utilisation of oxygen and regulation of breathing in some aquatic animals. Thesis, Groningen. VON HOLST E. (1934a) Reflex und Rhythmus im Goldfischrtickenmark. Zool. Anz. (Suppl. 7), 93-95. VON HOLST E. (1934b) Studien fiber Reflexe und Rhythmen beim Goldfisch (Carassius auratus). Z. Vergl. Physiol. 20, 582-599.