THE RESPIRATORY CENTRE IN THE TENCH(TINCA TINCA L.)

I. THE EFFECTS OF BRAIN TRANSECTION ON RESPIRATION

BY G. SHELTON*

Department of Zoology and Comparative Physiology,Birmingham University

{Received 13 September 1958)

I. INTRODUCTION

Though the concept of a respiratory centre is a very old one, investigations of sucha centre, in the vertebrate animals at least, have been confined almost entirely tothe mammals. The centre is usually thought of as a group of neurones, situatedlargely in the bulb, which is responsible for all the respiratory integration. Sensorymessages, from stretch and gas-tension receptors, impinge on these neurones andaffect the rhythmic activity they produce. These changes in turn, when trans-mitted to the respiratory effectors via connexions in the cord and the spinal motorneurones, cause modifications of the respiratory movements. The exact anatomicallocation of such a centre has not been universally agreed on, however, and even itsexistence has been doubted by some authors. Liljestrand (1953), for example,points out that the possibility of respiratory integration occurring at the spinal levelhas been almost completely disregarded. In the mammal one of the factors com-plicating any investigation of the site of respiratory integration is that the majorsensory input of the respiratory complex goes into the medulla whilst the motorsupply to the respiratory muscles comes from the spinal cord. Consequently, aconsiderable portion of the central nervous system has to be intact for respirationto continue. In the teleost fish a more compact arrangement appears to exist, withthe important respiratory pathways, both afferent and efferent, being carriedlargely in the Vth and Vllth cranial nerves. It would be interesting, therefore, tosee whether integrating neurones exist outside the direct connexions between thesensory and motor nuclei of these cranial nerves.

The technique of making transactions is one of the simplest means of delimitingthe respiratory areas of the brain and it has been used a great deal in investigationsof the mammalian respiratory centre (Lumsden, 1923; Stella, 1938; Hoff &Breckenridge, 1949). One of the principal limitations of the method is that it ispossible for respiratory breakdown to occur when parts of the brain, other thanthose directly concerned in respiratory integration, are removed. It has beendemonstrated that systems existing in the brain stem can affect the performanceof quite separately co-ordinated activities, such as the simple reflexes (Magoun,

• Present address: Department of Zoology, Southampton University.

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1944). A system of this sort, though its removal or damage might cause modifica-tion of respiration, would not be considered to be part of a respiratory centre. Inspite of this difficulty the transection technique is still a valuable method fordelimiting those parts of the brain which can co-ordinate the normal breathingmovements.

A few transection experiments have been done on elasmobranchs. Hyde (1904)showed that the respiratory centre in the skate is located in the medulla. Sheclaimed that the centre in these forms is segmentally arranged, the units associatedwith the sensory and motor areas of the Vllth, IXth and Xth cranial nerves beingcapable of independent rhythmic activity when separated by transverse cuts.Springer (1928), working on dogfish, was unable to confirm the segmental inde-pendence and found that the respiratory region occupied a much greater area ofthe medulla. No investigations of this sort have been done on teleosts, though it isclear from some of the results of the early workers (see review by Healey, 1957) thatdamage to the medulla stops the respiratory movements.

Fig. 1. The clamp used for holding the fish during experiments on therespiratory centre. For further details see text.

II. METHODS

The experiments were done on fifty-six tench, the majority of which were 15-20 cm.in length and 45-60 g. in weight. In a few later experiments, in which the labyrinthreflexes were tested, slightly larger fish were used because mirrors had to beattached to their eyes. The fish were fixed in a holder (Fig. 1) which has proved tobe satisfactory in several different types of experiment on fish respiration. Theholder consists basically of three elements, the trunk clamp A and the head clamps

Respiratory centre in the tench. I 193

B and C. The trunk clamp is made of wood, grooves being cut out in the two halvesto match the body of the fish. One half of the clamp is fixed to the base plate andthe other half is adjustable on the two bolts. The head clamps B and C are retainedon the fixed half of the trunk clamp. The clamp C consists of two \ in. diameterrods in the ends of which are cut V-shaped notches of a size suitable to fit on to thebony supra-orbital ridges of the fish. The rod fixing on to the left supra-orbitalridge is looped over the cranium and slides on the rod of the right side as thediagram shows. This arrangement leaves the left side of the head free from anyobstruction which might interfere with recording apparatus. The operative parts ofclamp B are the two pieces of metal which can slide apart on their supporting rodsin much the same way as a surgeon's retractor. In this way the ends of these metalclaws can be made to grip the bone of the skull at the edges of the hole made toexpose the brain. Both clamps B and C are independently adjustable so thatdifferent sizes of fish can be accommodated in the holder. When all three clampsare used the head of the fish can be rigidly fixed, although the respiratory move-ments are not interfered with in any way. In many experiments, including most ofthose described in this paper, it was not necessary to hold the head of the fish quiteso firmly and in these cases the clamp B was not used.

The holder was fastened to the bottom of a Perspex tank which had a watercapacity of 1000 ml., the water being constantly aerated throughout an experiment.All the experiments were done at room temperature (18-20° C). The movementsof the lower jaw and of an opercular flap were recorded on a smoked drum usingvery light levers so that the movements were not visibly impeded. As far as possiblethe records were taken before and after the operation to expose the brain, and thenafter recovery from each transection so that a clear picture of the normal patternand the subsequent modifications was obtained.

During the operation to expose the brain, and when subsequently the braintransections were being made, the fish were deeply anaesthetized in 0-5-1-0%urethane solution. They were allowed to recover to a lighter level of anaesthesia(0-2 % urethane approx.) when the recordings of the movements were taken. Thetransections themselves were made with a cataract knife or with mounted razorblade fragments. A Marconi MME 3 cautery was also used to produce the lesionsin some cases, though the results were not noticeably different from those obtainedby cutting. After transection there was usually a period of shock when the fish didnot breathe and during this period water was passed over the gills from a cannulainserted into the buccal cavity. When respiration had ceased permanently as theresult of a brain transection the gills were perfused continuously until the end ofthe experiment.

III. RESULTS

A. The effects of brain transections on breathing movements

The experiments involved transections of the nervous system in both the midbrainand the spinal cord—posterior medulla regions. In the figures which show theresults, the brain is drawn as seen from the dorsal surface and the transection levels

13 Exp. BioL 36, 1

194 G. SHELTON

are represented by the transverse lines. The order of the lettering in the diagramsrepresents the sequence in which the sections were made at the various levels. Theparts of the brain involved in these vertical transections can be seen in Fig. 2 whichshows the brain from a dorso-lateral aspect.

Op.L

IV V.Olf. L

Sp.C.

IXII C.N.

V and VIIC.N.

Fig. 2. Tench brain seen from a dorso-lateral aspect. Sp.C, spinal cord; Vag.L., vagal lobes;Fac.L., facial lobe; Cer., cerebellum; Op.L., optic lobes; Olf.L., olfactory lobes; C.N., cranialnerves; Inf.L., inferior lobe; IVV., IVth ventricle.

Fig. 3. Respiratory movements before (a) and after (A, c, d, e) brain transections. Upper trace—movements of operculum (up on trace = operculum closing). Lower trace—movements ofmouth (up to trace = mouth opening). Time marker on all figures—10 sec. intervals.

Respiratory centre in the tench. I 195

Sections through the optic region lobe, though causing a considerable loss ofblood in some cases, had very little effect on the respiratory rhythm after the initialshock period. Certainly the variations were not outside those that normally occurredin the intact animal which had been deeply anaesthetized and then allowed torecover. Transections were made down to the level of the anterior border of thecerebellum, with surprisingly little change in the respiratory pattern (Fig. 3 a, b).Section at a level lower than this became impossible without damaging the Vth andVllth cranial nerves, but it was possible to remove the cerebellum completely(Fig. 3 c) without affecting respiration. Attempts were made to remove, by suction,the more dorsal parts of the medulla beneath the cerebellum and these were alwaysfollowed by a considerable change in the respiratory pattern. Usually the breathingmovements stopped, but on two occasions rhythmic movements were producedeven though lesions were made in this way approximately to the level of the Vthmotor nuclei.

Fig. 4. Respiratory movements before (a) and after (b, c, d) spinal cord and brain transectionsUpper trace—movements of operculum (up on trace = operculum closing). Lower trace—movements of mouth (up on trace = mouth opening).

Transections in the region of the spinal cord and posterior parts of the medullawere more easily performed without causing undue haemorrhage. High sections ofthe spinal cord in tench never caused respiratory breakdown. In the mammal suchsections disrupt important connexions between the medulla and the respiratorymotor neurones and the breathing movements cease. In the teleost fish the onlyconnexions which are disrupted by section of the cord or medulla are those to the

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196 G. SHELTON

spino-occipital efferents and this means that the hypoglossal musculature (m.sternohyoideus in the tench) can no longer function in respiration. This failure hadvery little effect on the breathing movements as a whole, other muscles being ableto participate in opening the mouth (Fig. 46). Transections of the medulla at thelevel of the obex (Fig. 4 c) and, further forward, in the middle of the exposed part ofthe IVth ventricle (Fig. ^d) were also possible without affecting the ability of therespiratory complex to produce rhythmic activity. The sections in the region of theIVth ventricle did result in modifications of the breathing rhythm in most cases and

Fig. 5. Respiratory movements before (a) and after (b, c, d) brain transections. Upper trace—movements of operculum (up on trace = operculum opening). Lower trace—movements ofmouth (up on trace=mouth closing).

in some animals the breathing movements ceased altogether. However, the criticallevel of transection, after which normal breathing movements ceased in all cases,was at the level of the posterior border of the facial lobe (Fig. 56). After such asection a considerable change occurred in the movement pattern; the usual rhythmicactivity disappeared completely and the mouth (and to a lesser extent the oper-culum) usually made quivering movements. This type of movement continued tosome extent with higher sections (Fig. 5 c), though recovery was often slow andfrequently no movements were produced at all.

A very striking feature of the movement pattern, produced after transections hadbeen made in these regions of the posterior medulla, was the appearance of pro-longed (5-10 sec.) opercular abductions recurring rhythmically every \ to 2 min.(Fig. 4c, d; Fig. $b-d). The movements of the mouth during these periods of

Respiratory centre in the tench. I 197

opercular abduction varied somewhat in different individuals. If rhythmic re-spiratory movements had not been stopped by the transection then usually themouth stopped moving in the closed position (Fig. 4 c, d). If, however, the normalbreathing had ceased as a result of a posterior facial lobe transection and themouth was making quivering movements then there was no change in the mouthmovements in some individuals, whilst in others the opercular abduction wasaccompanied by an increase in the intensity of the mouth quivering. With stillhigher transection the mouth again closed during the opercular transection (Fig.5 c). The slow rhythm persisted even after transection at the posterior border of thecerebellum (Fig. 5 d).

Fig. 6. Respiratory movements after spinal cord (a) IXth and Xth nerves (6), IVth ventricle (c),and facial lobe (d) transections. Upper trace—movements of operculum (up on trace = oper-culum opening). Lower trace—movements of mouth (up on trace = mouth closing).

Because of their position on the brain stem the IXth and Xth cranial nerves were,of necessity, progressively removed during the hind-brain transections. It waspossible therefore that the breakdown of respiration or the appearance of the slowabductions (or possibly both) was due to damage to the nerves rather than to thebrain itself. Indeed, Powers & Clark (1942) had concluded that in teleost fish theIXth and Xth cranial nerves, particularly the former, were of fundamental import-ance in the initiation of the respiratory rhythm. These authors suggested thatafferent volleys in the nerves converted tonic activity in the respiratory centre to thenormal rhythmic pattern. To decide which of the possible explanations was correct,experiments were done in which the IXth and Xth cranial nerves were sectionedbefore any brain transections were made. The IXth and Xth nerves were approachedat their origin from the brain stem via holes made in the skull in the region of the

198 G. SHELTON

labyrinth. The nerve roots were lifted up on glass hooks and then cut with scissorsor knife. This ensured that no fragments were left intact. The only effect thatsections of these nerves had on the breathing movements was to increase theiramplitude; the normal respiratory rhythm continued and the prolonged opercularabductions did not appear (Fig. 6 b). The amplitude of the movements returnedto the normal level over a period of 1-2 hr. Transections at a higher level than thespinal cord did result in the appearance of the abductions (Fig. 6 c), and even highertransections stopped breathing in the same animal (Fig. bd). It is very likely there-fore that the activity seen after transection of the posterior medulla is due to thedamage caused to the brain itself and not to the removal of any sensory or motorcomponents carried in the IXth and Xth cranial nerves.

It was of interest to see whether the slow rhythmic abductions of the opercula,like the respiratory movements themselves, were produced by activity in themedulla, or whether they were the result of activity in the higher centres such as theoptic tectum. In experiments in which the whole of the fore- and midbrains and thecerebellum were removed it was found that a subsequent transection at the faciallobe level would result in the appearance of the slow rhythm (Fig. 3 e). The neuronesinstrumental in the production of this activity must lie in the anterior part of themedulla.

B. The effects of brain transections on a vestibular-eye reflex

In the mammal it has been shown that certain areas of the brain reticular for-mation contain neurones having a facilitator or suppressor effect on reflex activityin general (Magoun, 1950). Hoff & Breckenridge (1949, 1954) have proposed thatthe inspiratory cramps, ensuing after section of the brain at the pontine level, arecaused by removal of a generalized suppressor area of the brain stem and are notdue to removal of a pneumotaxic centre as proposed by Lumsden (1923) and Pitts(1946). Liljestrand (1953) has extended this concept and suggests that the regionswithin the medial reticular formation of the bulb, long accepted as the site of therespiratory centre, are themselves generalized facilitator areas. Similarly, thenature of the neural mechanism, which is situated in the facial lobe region of thefish brain and whose removal causes such serious breakdown of the normal breathingrhythm, is not obvious. It may be a vital part of the respiratory centre itself or itmay be part of a more generalized system in the reticular formation.

Since any interference with such generalized suppressor or facilitator areas shouldresult in the modification of all reflexes, it should be possible to decide between thepossibilities outlined above. The static vestibular-eye reflexes were chosen as beingthe most suitable for experiments on fish. It must be noted that investigations ofthe brain-stem reticular formation of the mammal have been largely restricted toexamination of its influences on cortical activity or on motor activity from thespinal cord. However, there seems to be no reason to suppose that the eye musclemotor neurones, or the interneurones having synaptic contact with them, areimmune from these influences. One reflex at least, the mammalian blink reflex,which is mediated by neurones within the brain, can be suppressed by bulbarstimulation (Magoun, 1944).

Respiratory centre in the tench. I 199

In the present experiments the fish was rotated on its long axis from the normalupright position through about 60° (in io° steps) to a position of right eye down, lefteye up. Mirrors were fixed to the left eye and to the body by means of rubbersolution and light levers were used to measure both the angle through which thebody was rotated and the angle of the eye deflexion. The deflexions were measuredbefore and after the spinal cord and medulla had been transected at various levels,several measurements being made at each level. There was considerable variationbetween individuals even with intact central nervous systems. One of the sixanimals tested showed an angular displacement of 220 ± 7° of the eye relative to thetrunk, when the trunk was rotated through 6o°. This was the smallest deflexionmeasured, and, at the other extreme, one fish showed an eye displacement of340 ± 30 for the same trunk rotation. After stopping respiratory movements witha transection through the IVth ventricle region, the eye deflexions in each individualwere found to be both qualitatively and quantitatively the same as before, providedan adequate supply of water was maintained to the gills. A complicating factorwhich made the measurement of eye deflexion more difficult after transection at anylevel was the occurrence of a lot of eye movement particularly in the horizontalplane. The resting position of the eye from which these excursions were made wasstill obvious and only when it was in this position were the measurements made.Transection caused no enhancement or inhibition of this one reflex which wastested.

IV. DISCUSSION

The fact that the slow rhythmic abductions can occur concurrently with a normalrespiratory rhythm and can then persist when normal breathing has failed demon-strates that the nervous elements producing the two are largely independent. Thisslow rhythm cannot therefore be considered as a development of the primaryrespiratory rhythm as is suggested for gasping respiration in the medullary prepara-tion in the mammal (Brodie & Borison, 1957). It must be an expression either ofanother pre-existing activity, probably much modified by the effects of transection,or of an entirely new pattern of nervous activity. The evidence favours the formerof these two possibilities, since the rhythmically occurring cough is an example ofsuch a slow rhythm in the intact animal and examination does reveal some simi-larities between the cough and the slow abduction. The frequency range over whichthe two occur is roughly the same, though the cough in the intact animal is usuallymore frequent than the slow abduction. Furthermore, after low transections whennormal breathing is continued, it is sometimes possible to see transitional statesbetween the normal cough and the prolonged opercular abduction (Fig. ja-c).Finally, it is perhaps significant that, during the initial part of the cough, the oper-culum opens whilst the mouth closes (Hughes & Shelton, 1958) and the sameattitude is usually adopted by these two structures in the prolonged activity aftertransection. It is suggested, therefore, that the slow abductions represent theactivity of neurones situated beneath the cerebellum in the anterior part of themedulla, and concerned in the intact animal with co-ordination of rhythmic

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coughing movements. The normal, brief cough is not produced unless the lowerlevels of the medulla are intact, however, and some part of the integrative mechanismnecessary for normal activity must be situated here. Moreover, this posterior partof the mechanism is independent of input from the IXth and Xth cranial nervesand so is entirely central in location (Fig. 6b). It is interesting to note in passingthat the cough does persist after section of the IXth and Xth cranial nerves, althoughit is usually thought of as a reflex action initiated by foreign matter on the gills.

Though the transection experiments do not allow an exact anatomical locus to begiven to the respiratory neurones, it is clear that these neurones must be containedwithin the medulla between the transection levels having little or no effect on therespiratory movements. They occur, therefore, below the region where the Vth andVllth cranial nerves emerge from the brain. A more exact rostral limit to therespiratory neurones cannot be given by this method because these nerves mustremain intact for respiration to continue. The caudal limits, on the other hand, canbe set more exactly. The experiments on the vestibular-eye reflex show that allreflex activity is not affected by brain transection at the facial lobe level. It isunlikely, therefore, that respiratory failure is due to damage to a generalized sup-pressor or facilitator area of the medulla. It is also unlikely that this failure is theresult of direct injury to the tissue of the brain, causing for example massed injurydischarges in neurones unrelated to respiration. Similar injury effects must havebeen caused by the more posterior brain sections, some of which were very near thecritical level, and yet these had no fundamental effect on the breathing rhythm.Application of procaine to the cut surface of a brain, transected at the facial lobelevel, had no effect on the random quivering movements of the mouth and operculauntil it was present in sufficient concentration to act as a general anaesthetic, whenall movements stopped. Furthermore, no indication of the normal rhythm was everseen, though a fish was kept alive up to 3 hr. after facial lobe transections. Duringthis time an effect due to an injury discharge should have disappeared.

In this case, therefore, respiratory failure after brain transection at the posteriorborder of the facial lobe is apparently due to removal of part of the system directlyinvolved in respiratory integration. There is very little interference with the sensoryor motor pathways of the Vth and Vllth cranial nerves as the nuclei of these nervesare situated in the anterior part of the medulla. The descending ramus of the Vthcranial nerve, and a sensory ramus of the Vllth cranial nerve ending in the largefacial lobe, are the only components extending back into the region of the medullainvolved in these transections. The descending ramus of the Vth runs back to asecondary gustatory nucleus and is sectioned at lower levels than those stoppingthe breathing movements. It can also be shown that transections causing respiratoryfailure need not involve the facial lobe. Furthermore, the respiratory neurones orconnexions situated at this critical level are not part of an essential reflex involvingthe IXth and Xth cranial nerves, as the experiments have shown. Therefore, asdirect sensory and motor pathways are not involved, transection in the facial loberegion removes neurones which are part of an intermediate integrating systembetween the sensory and motor nuclei of the cranial nerves involved in respiration.

Respiratory centre in the tench. I 201

These neurones are essential in the production of the normal respiratory rhythmand so are part of what could properly be called a respiratory centre. It is unlikelythat the whole of such a centre is removed by transection at the facial lobe level.The centre probably consists of a large number of interacting neurones and removalof a relatively small number of these would be sufficient to cause respiratory failure.The work on mammals would suggest that the neurones are situated in the reticularformation of the medulla. However, the site of the neurones and their extensionwithin the region of the medulla delimited by the transections are problems whichcan be solved only by the use of other techniques.

SUMMARY

1. The effects of brain transections on the breathing movements of the tench aredescribed.

2. The whole of the mid- and forebrain, and the cerebellum, can be removedwithout producing any change in the breathing movements.

3. Normal movements continue after section of the IXth and Xth cranial nerves.4. Transections of the spinal cord and posterior medulla are without effect on the

breathing rhythm until they reach a level just behind the facial lobe. The breakdownof respiration produced by transection at this level is interpreted as being due toremoval of part of the respiratory centre.

5. Rhythmically repeated movements in which the opercula abduct and themouth closes are seen after transection in the posterior parts of the medulla. Thesemovements are thought to be due to activity in neurones which are responsible forco-ordination of the coughs in the intact animal. These neurones are situated in theanterior part of the medulla, beneath the cerebellum.

I wish to record my thanks to Prof. O. E. Lowenstein, F.R.S., and to Dr G. M.Hughes for valuable discussion and encouragement. Some of the work describedwas carried out in the Department of Zoology, Cambridge, while the author helda Junior Research Grant from the Department of Scientific and Industrial Research.

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New York: Academic Press Inc.HOFF, H. E. & BRECKENRIDCE, C. G. (1949). The medullary origin of respiratory periodicity in the

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Neurol. Psychiat. Chicago, 73, 11—42.HUGHES, G. M. & SHELTON, G. (1958). The mechanism of gill ventilation in three freshwater

teleosts. J. Exp. Biol. 35, 807-23.HYDE, I. H. (1004). Localization of the respiratory centre in the skate. Amer.J. Physiol. 10, 236—58.LiLjBSTRAND, A. (1953). Respiratory reactions elicited from the medulla oblongata of the cat. Acta

physiol. scand. 29 (Supplement 106), 321-93.LUMSDEN, T. (1923). Observations on the respiratory centres in the cat, J. Physiol. 57, 153-60.MAGOUN, H. W. (1944). Bulbar activity and facilitation of motor activity. Science, 100, 549-50.

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MAOOUN, H. W. (1950). Caudal and cephalic influences of the brain stem reticular formation.Pkytiol. Rev. 30, 459-74.

PITTS, R. F. (1946). Organization of the respiratory centre. Pkytiol. Rev. 26, 600-30.POWERS, E. B. & CLARK, E. T. (1943). Control of normal breathing in fishes by receptors located in

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STELLA, G. (1938). On the mechanism of production and the physiological significance of apneusia.J. Phytiol. 93, 10-23.