the central and reflex control of respiration in the frog

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J. Physiol. (939) 95, 305-327 612.28:597.8

THE CENTRAL AND REFLEX CONTROLOF RESPIRATION IN THE FROG

BY D. H. SMYTH

From the Department of Physiology, Pharmacology and Biochemistry,University College, London

(Received 14 December 1938)

THE experiments of Heymans & Heymans [1927], Heymans, Bouckaert& Dautrebande [1930], Schmidt [1932], Selladurai & Wright [1932] andmany subsequent workers have made it clear that a centre situated inthe medulla can no longer be held solely responsible for the chemicalcontrol of respiration, and the parts played by the carotid sinus andaortic regions are now well recognized. Chemical stimulation of receptorsin these areas, quite distinct from pressure changes [Bogue & Stella,1935], produce reflex alterations in breathing, and indeed the responseto oxygen lack seems to depend largely on these reflexes.

Most of the work done has been concerned with the relative partsplayed by the centre and the extramedullary chemoreceptors undervarious conditions, or with the mode of stimulation in each case.Furthermore, the experiments deal almost exclusively with mammalianrespiration. A new line of approach to the problem is offered by aninvestigation of the respiratory reflexes of other less highly organizedvertebrates, and a study of the amphibian respiration seems desirableas presenting an early stage in the development of air-breathing re-spiratory organs. The frog is chosen as representative of this class ofanimals, and further interest is added by the fact that whereas olderwork suggested that the respiratory centre of the frog was fundamentallydifferent from that of the mammals, certain morphological and experi-mental evidence points to the probable existence of carotid sinus andaortic reflexes at least in connexion with the circulation.

The scope of these investigations therefore includes (1) the nature ofrespiration in the frog and the possibilities of recording it, (2) thealterations in respiration in response to changes in the composition of theair breathed, (3) the possibility of such changes being controlled by a

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centre in the medulla sensitive to carbon dioxide and oxygen lack, (4) theevidence for the existence of respiratory reflexes comparable with thoseoriginating in the mammalian carotid sinus and aortic area.

The mechanism of respiration in the frog. The large amount of workon the respiratoryprocesses in the frog has been reviewedby Babak [1921].The mechanism is briefly as follows. Respiration is effected partlythrough the skin and partly through the lungs. In addition a smallamount of gaseous exchange may occur through the mucous membraneof the mouth and pharynx, but this is relatively unimportant. Thecutaneous respiration consists of inward diffusion of oxygen and outwarddiffusion of carbon dioxide through the general surface of the skin, theblood being carried to the skin by a common pulmo-cutaneous arterywhich also carries blood to the lungs. The skin respiration does notrequire any specific respiratory movements. The pulmonary respirationis a much less simple process. The driving force is provided by themuscles of the floor of the mouth-chiefly the petrohyoids, geniohyoids,hyoglossi, genioglossi, sternohyoids and omohyoids. These by rhythmiccontraction and relaxation alter the capacity of the mouth cavity. Airleaving or entering the mouth cavity may do so either through theglottis connecting with the lungs, or through the nostrils connectingwith the outside. The normal sequence of events is the following. Withthe lungs filled with air and the glottis closed, small rhythmic contractionsof the muscles of the floor of the mouth occur and air enters and isexpelled through the open nostrils. These movements take place with afrequency of about 80-120 per min. At intervals a much more extensivemovement occurs. With the nostrils closed and the glottis open, airenters the mouth cavity from the lungs and is forced back again by astrong contraction of the mouth muscles. This may occur several timesor only once, the glottis is then closed, and the finer movements arecontinued. These two types of movement are referred to as oscillationsand lung movements respectively, and while the former renew the airin the mouth cavity with fresh air from outside the latter ventilate thelungs with the air in the mouth cavity. It is clear that both movementsare essential for ventilation of the lungs with fresh air. The relation ofthe two types of movement to each other is by no means constant. Theremay be only single lung movements interrupting the rhythm of theoscillations, and this appears to be the commonest condition. There maybe complete absence of lung movements as, for example, at low tem-peratures.. The lung movements may be very frequent, occurring ingroups without oscillations during the groups. The nostrils may open

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with each lung movement, allowing the air to pass from the lung to theoutside. In addition, abnormal types of respiration occur which will bereferred to later.

These respiratory movements depend on centres in the brain, andaccording to Babak a centre in the medulla controls the lung movementswhile a higher centre, probably in the mid-brain, is responsible for theoscillations.

METHODSThe general method used was to record the respiratory movements of

the frog in response to the administration of gas mixtures containingvarying amounts of oxygen and carbon dioxide. The frogs used were Ranaesculenta and the experiments were mostly done with spring and summerfrogs. The records obtained with normal animals were compared withthose obtained on animals in which various procedures had been carriedout with a view to eliminating possible reflex sources of stimulation.

As regards the actual recording of the respiration there are the twomore or less independent sets of movement-the oscillations and thelung movements. It is evident that the lung movements have not thesame significance as the mammalian lung movements for several reasons.They are in themselves incomplete, i.e. without the oscillations theycannot lead to a continued ventilation of the lungs. Furthermore, sincethe same air may pass to and fro several times between lungs and mouthcavity, the actual pulmonary ventilation would be of little significance.There is also the skin respiration going on continuously independent ofany respiratory movements. Nevertheless, it was decided to record thelung movements and use them as a criterion of activity of the respiratorycentre, since they are most likely to be analogous with the respiratorymovements of the mammal. The oscillation movements were not recordedsince it was found that attempts to do so interfered with the movementsthemselves, and also the type of movement is rather different from anyprocess in mammalian respiration. The results will show how far thisview has been justified.

Many complicated methods have been evolved for the recording ofthe frog's respiration. Most of these, as Babak [1921] has pointed outin his review, have the great disadvantage that they involve someoperative interference, such as cannulation of the lungs or nostrils, whichdisturbs the normal respiratory processes. It was especially desired toavoid all such procedures and also the use of any anaesthetic or narcoticwhich might have an influence on the respiratory centre. Accordinglythe following technique was employed. A small rubber bag was fitted

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round the body of the frog, which was unanaesthetized and in every wayquite normal. The bag was held in position by a rubber jacket whichfitted the animal loosely, and was connected with a 1 c.c. float volumerecorder. As the movements were very delicate they were recordedphotoelectrically, a selenium cell and valve amplifier being used, andthe movements recorded with a Weston relay. Since it was of importanceto keep the magnification of the movements constant throughout anexperiment, and since movements of the frog other than respiratory oneswould alter the zero position of the float recorder, an arrangement wasprovided so that the connexion between the frog and the recorder wasautomatically opened at regular intervals so that the pressure came toatmospheric and the recorder back to a constant zero position. The frogwith the rubber bag was enclosed in a glass chamber, where it was underdirect observation. Through the chamber a constant stream of air wascirculated, and for the air other gas mixtures could be substituted asdesired. A sampling tube led from the chamber directly to a Haldane'sgas analysis apparatus, so that analysis could be made of the compositionof the air to which the frog was exposed. For experiments involvingchanges in temperature, the required alteration was made by heatingthe air passing into the chamber.

It was found that this arrangement gave satisfactory results andthe frog usually remained quite still and appeared normal. Any othermovement other than the respiratory ones were of course also recorded,but these were readily distinguishable by their greater amplitude, andtheir irregularity; they were also directly observed and noted.

An examination of the records reproduced shows a considerablevariation of the normal respiration both as regards amplitude andfrequency. The differences in amplitude from experiment to experimentare not of significance, since only flank movements are recorded, andthe size of the movement will depend in each case upon the preciseposition of the rubber bag relative to the animal's body. The alterationsin frequency are in accordance with what has already been pointed out,namely, the great irregularity of the lung movements. In each case theeffects produced by any particular gas mixture is seen as a relativeincrease or decrease in amplitude or frequency of the respirations.

The various operative procedures to be described for deafferentingthe respiratory centre were carried out under ether anaesthesia. In allcases where respiration was tested after operation several days wereallowed to elapse for recovery from the operation and anaesthetic beforethe respiration was investigated.

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RESULTSThe two important cases investigated were the response to increased

amounts of carbon dioxide and to oxygen lack. The important questionin each case is whether the response observed is due to a central or aperipheral action. There is here a possibility of confusion of terminology,as the term "peripheral action" in this sense in mammals refers only toreceptors in the carotid sinus or aortic areas, while in the case of thefrog it has been used to mean stixjulation from the skin or mucousmembranes without any reference to the carotid sinus. In the followingdiscussion of the effects of carbon dioxide excess and oxygen lack theterm peripheral action will be used in the same sense as in the literatureon the frog, i.e. to denote effects originating at the surface of the body,while the term central action shall include possible effects through thecarotid or aortic areas. At a later stage an attempt will be made todistinguish between the effects originating in these chemoreceptors andthose produced through medullary centres directly. The effects of carbondioxide excess and oxygen lack will be discussed separately.

Excess of carbon dioxideIt can be readily shown that if a frog is put into an atmosphere

containing a relatively high concentration of carbon dioxide it showsincreased respiratory movements, but there is doubt as to the causeof the production of hyperpnoea and also few figures are available inregard to the sensitivity. Von Budenbrock [1928] gives 10% as theconcentration of carbon dioxide necessary to produce hyperpnoea, andin most of the work done apparently high concentrations have been used.In these experiments it has been found that the sensitivity is muchgreater. A considerable increase in respiratory activity is constantlyproduced by 5% carbon dioxide, and definite effects by as low as 2 %.Records illustrating the effects of carbon dioxide in several differentfrogs are shown in Fig. 1. It is seen that the first effect is not a hyperpnoea,or at least the hyperpnoea does not begin immediately. There is usuallya short delay up to about 2 min., or there may be a definite initialinhibition of respiration. It would appear, therefore, that carbondioxide has two effects, an initial inhibitory one and later a stimulatoryaction. In the two records in Fig. 1, in which the frog was exposed tothe higher concentrations of carbon dioxide, the large movements duringadministration of the gas were caused by general body movements otherthan respiratory ones. These are most marked in the case of 8*9% carbon

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dioxide. With still higher concentrations these movements were somarked as to render recording of respiration impossible. After the initialinhibition and irregular movements, a regular hyperpnoea follows andonly slowly returns to normal again.

Fig. 1. Effect of carbon dioxide, A, 2-9%; B, 4-7%; and C, 8-9% respectively, on therespiratory movements of normal frogs. In these and subsequent records the signalsshow the time of administration of the carbon dioxide or other gas, the time when thiswas discontinued and between these the period during which a sample of gas was takenfor analysis. The results of the analysis are also indicated. In B and C the largemovements during and just subsequent to the administration of carbon dioxide aredue to movements other than respiratory ones. Time in 10 sec. intervals.

There has been some doubt about accepting the stimulating actionof carbon dioxide on the respiration as being due to a central action;and it has been ascribed to action on the skin or mucous membrane ofthe mouth. Winterstein [1900] claimed to have settled the question byexposing the spinal cord, painting it with 2% carbolic which was saidto paralyse the sensory elements without affecting the motor, and thenshowing that carbon dioxide produced no hyperpnoea. Such an experi-ment is associated with a great deal of shock and trauma and there is

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also the possibility of absorption of phenol. Other methods weretherefore sought in order to cut off peripherally originating impulses.

The possible sources of such impulses are:(1) the general surface of the skin, supplied by cutaneous nerves,

which reach the medulla by the spinal cord;(2) the skin of the head in particular, the conjunctiva or other parts

supplied by the Vth cranial nerve;(3) the mucous membrane of the mouth supplied chiefly by the

palatal branches of the VJIth nerve and by the glossopharyngeal nerve.These possibilities were examined in turn as follows. A frog was

anaesthetized with ether and decerebrated by removing the roof of thecranium and cutting off the cerebral hemispheres. The skin was nowcompletely removed from the whole body after ligature of its mainvessels, and also as far as possible from the head, being cut close to theeyes, nostrils and edges of the jaws. A number of the animals recoveredfrom the anaesthetic and assumed a normal posture and in a few casesthey survived long enough for respiration to be tested. The result of onesuch experiment is seen in Fig. 2 A, and it is evident that the adminis-tration of carbon dioxide is followed by a hyperpnoea, the source ofwhich could not be receptor organs in the skin. A similar result wasobtained in three separate animals, and in a number of cases wherecarbon dioxide produced no effect the animal was found to be moribund.

Other less drastic methods of removing the cutaneous influence onrespiration were also tried. The frog was painted over with a solutionof 5 % percaine, and after a minute or so washed with water. If this wasnot done quickly, enough percaine was absorbed to produce generaleffects. Fig. 2 B shows the response to carbon dioxide of a frog treatedwith percaine, and again it is evident that a hyperpnoea follows. Thisexperiment is rather unsatisfactory in that one has no means of testingwhether the skin is completely anaesthetized or not.

A more certain way of removing the skin impulses is to divide all thenerve pathways involved. The frog was anaesthetized with ether. Thearticulation between the 1st and 2nd vertebrae was located and thespinal cord divided between them with the points of a pair of scissors.Since it was uncertain whether the second spinal nerve was thusseparated from the medulla the brachial nerve and its roots as faras possible were divided from the ventral side. The ophthalmic andmaxillary divisions of the Vth cranial nerve were then divided. This isreadily done through an incision posterior and medial to the eyeball.The maxillary division is first isolated as a common trunk containing

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also the mandibular division. It is followed laterally till it divides andthen the anterior division-the maxillary branch-is cut. The mandibulardivision must be left intact, since it supplies the masseter muscles and,if divided, the lower jaw drops and normal respiratioxi is impossible. As

Fig. 2. A, effect of 4.5% carbon dioxide on respiration of decerebrated frog after removalof the skin. B, effect of 4-2% carbon dioxide after painting the skin of a frog with5% percaine. C, effect of 6-3% carbon dioxide on respiration after section of spinalcord, ophthalmic and maxillary divisions of Vth nerves, rami palatini of VIIth nerves,and IXth nerves. Time in 10 sec. intervals.

it is said to contain few sensory fibres [Gaup, 1896] it is assumed thatit cannot play a great part in carrying cutaneous impulses. The skinincisions were sutured and after recovery from the operation therespiration was tested.

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Preparations of this kind were made and tested for the response tocarbon dioxide, and in all cases hyperpnoea resulted.

It seems, therefore, unlikely that the skin is the source of reflexesresponsible for the carbon dioxide hyperpnoea. The mucous membraneof the mouth was next investigated. This was denervated by cutting therami palatini of the VIIth nerves, the glossopharyngeal nerves, and thefirst two divisions of the Vth nerves. The ramus palatinus can be cutfrom the roof of the mouth by making a small central incision in themucous membrane. The glossopharyngeal nerves are cut from theventral side. A transverse crescentic incision is made in the skin andbody wall at the level of the sternum. If now the upper edge of theincision is lifted up, the branches of the aorta can be followed. Theglossopharyngeal and hypoglossal nerves are seen in close relationshipto the glomus caroticus and the glossopharyngeal nerves are divideddistal to the glomus caroticus. The first two divisions of the Vth aredivided as they send branches which communicate with the ramuspalatinus of the VIIth. The denervation of the palate leaves unalteredthe hyperpnoea in response to carbon dioxide. The palate denervationwas then combined with the skin denervation, and Fig. 20 shows theresponse to carbon dioxide of a frog in which the spinal cord had beendivided, together with the brachial nerve, the first and second divisionsof the trigeminal, the rami palatini of the facial and the glossopharyngealnerves. It seems likely that here one has eliminated most of the possiblesuperficial reflex sources of the carbon dioxide hyperpnoea.

The initial inhibitory effect of carbon dioxide on the respiration hasalready been noted, and was now more fully investigated. It would seemlikely that this effect of carbon dioxide has a reflex origin, as it resemblesthe reflex inhibition of respiration produced in mammals by stimulationof the Vth nerve endings. A convenient method of studying this reflexwas to produce first a slight degree of hyperpnoea and then administerthe carbon dioxide, when the inhibitory effect was more marked. Forthis purpose the frog was exposed to an atmosphere of about 10% oxygenin nitrogen, which, as will be shown in the next part, causes an increasein respiration, and during this hyperpnoea carbon dioxide was ad-ministered. Attempts were now made to eliminate reflex sources by themethods already described and Fig. 3 shows the results obtained in anumber of cases. It is clear that in no case has the reflex inhibitionbeen eliminated.

These findings at once raise a question, which is difficult to answersatisfactorily. If the inhibitory effect of carbon dioxide, which is

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probably a reflex one, still persists in spite of the denervations carriedout, there is presumably some reflex source sensitive to carbon dioxidestill left, which might also be responsible for the hyperpnoea. To provethat the one effect was central and the other reflex would require somedenervation to be carried out which would abolish the inhibitory effect

Fig. 3. Records illustrating the inhibitory effects of carbon dioxide on respiration. A, 4-9%carbon dioxide administered to normal frog during the mild hyperpnoea producedby 9-1% oxygen in nitrogen. B, 5% carbon dioxide administered to decerebratedfrog with skin removed. C, 4-8% carbon dioxide and 5% carbon dioxide given aftersection of Vth (first two divisions) rami palatini of VIIth and IXth nerves. D, frogafter section of spinal cord, Vth nerve (first two divisions), rami palatini of VIIth andIXth nerves. 5-5% carbon dioxide administered during the mild hyperpnoea causedby 12% oxygen in nitrogen. In B and C the large movements during administrationof carbon dioxide were due to movements other than respiratory ones. Time in10 sec. intervals.

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and leave the hyperpnoea. This it has not been found possible to do.Some evidence, however, for the fundamental difference between the twoeffects was obtained in the following way. A probable source of theinhibitory reflex is in the mucous membrane of the glottis and larynxsupplied by vagal afferents. It is not possible to denervate these sincethe vagus is also motor to the larynx and the cutting of the vagalbranches to the larynx interferes with normal respiration. If, however,the concentration of carbon dioxide at the nerve endings is increasedwithout increasing the concentration of carbon dioxide in the general

1~~~~~~~~~~~~

a!~~~~ _

I stS

Fig. 4. Inhibition of respiration by carbon dioxide without subsequent stimulationproduced by a local increase in carbon dioxide content of the air in the region of thenostrils. The signal shows the period during which carbon dioxide was administered.Time in 10 sec. intervals.

atmosphere, the elimination of carbon dioxide through the skin wouldproceed normally and therefore one might expect to get the inhibitoryeffect without any general effect, i.e. without any hyperpnoea. This wasdone by keeping the frog in an atmosphere of air, and directing with asyringe a few c.c. of carbon dioxide to the nostril region. Fig. 4 showsthe results obtained by such a procedure and here one sees the inhibitoryeffect without any general hyperpnoea.

Considerable evidence has thus been given for the belief that carbondioxide produces hyperpnoea not by a reflex source originating at thebody surface, but by some sort of central action; while it also producesa reflex inhibition probably by vagal afferents in the larynx.

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Effects of oxygen lackUp to the work of Babak [1911] it seemed very doubtful whether

oxygen lack could stimulate respiration in the frog. Rosenthal [1864]had described a hyperpnoea in response to diminished oxygen pressure,but other workers [Aubert, 1882; Sokolows & Luchsinger, 1880;Pfluiger, 1875] described the effects of oxygen lack on frogs withoutmentioning an acceleration of the respiratory rhythm. Bethe [1903]concluded that the respiratory centre of the amphibian is essentiallydifferent from that of the higher animals in regard to chemical controlof respiration. Babak reinvestigated the question and found definitelythat the frog increased its respiration in response to oxygen lack. Heobtained these results by careful observation of the animal without anyattempt to record the actual movements, and the failure of others toobtain similar findings he attributed either to the employment forrecording the respirations of methods which interfered with the normalmovements, or to the failure to distinguish between the two types ofmovements, the oscillations and the lung movements, since in the anoxichyperpnoea only the latter are increased, the former being actuallyreduced. Babak produced the anoxaemia by putting the animal in anatmosphere of hydrogen, and according to him the following course ofevents occurred. There is first"a decrease of the oscillation movements.This is accompanied by gradual increase in the lung movements bothin amplitude and frequency. The lung movements may then occur inisolated groups. Later a new type of lung movement occurs in whichair is forced into the lungs several times without being permitted toescape between, so that the animal blows up its lungs. The oscillationsmay now disappear altogether, and the lung movements themselvesbecome less frequent. At this stage Babak readmitted air, and now anuninterrupted rhythm of lung movements began which only graduallybecame reduced in frequency and amplitude. The oscillations graduallyreturned and ultimately normal respiration was again established. Babakclaimed that two stages of hyperpnoea are represented, the first is theincrease in lung movements at the beginning of the experiment, thesecond is the steady rhythm of lung movements after air has beenreadmitted. Between these two is a stage of depression during whichthe centre is depressed by oxygen lack.

These results of Babak's have been confirmed in these experiments,and are illustrated in the records shown (Fig. 5), which are, it will beremembered, only the lung movements, and do not take into con-

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sideration the oscillations. The first change is a gradual increase in thelung movements. Later these become grouped into periods. It was foundthat if one continued until the lung movements became less frequentthere was usually some general excitation and body movements, whichinterfered with the recording, so that air was usually readmitted whenthe definite periodic breathing had set in. On readmitting the air a greatincrease in respiration occurred which only gradually diminished-theuninterrupted rhythm described by Babak.

Fig. 5. Effects of anoxia on the respiration of normal frogs, produced by administrationof nitrogen. Time in 10 sec. intervals.

It is reasonable to conclude, as Babak concluded, that this picturerepresents a hyperpnoea comparable to that seen in higher animals.What appears especially interesting is that one can see the transitionfrom stimulation to depression of the centre, as represented by theperiodic breathing, and again, the transition from depression to stimu-lation when the air is readmitted. The assumption is that the oxygencan diffuse through the skin on readmission of air and thus the centrecan recover sufficiently to be able to respond to the diminished oxygensupply. Such an effect is only possible in an animal which has a secondmeans of maintaining its gaseous exchanges apart from the lungs.

The hyperpnoea in these cases was produced by passing nitrogeninto the chamber. A further series of experiments was now carried outto determine the effects of lesser degrees of anoxia, with a view toestimating the sensitivity of the centre to this form of stimulation.Fig. 6 shows the response to various oxygen tensions in different animals.It will be seen that about 10% oxygen is sufficient to produce a definite

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increase of respiration. As a rule with these lesser degrees of oxygen lackthere is only stimulation without a stage of depression, as presumablythe threshold of damage to the centre is not exceeded. In a few casesthe response to even 10% oxygen was in the nature of definite periodicbreathing (Fig. 6), but this was the exception rather than the rule. It is

Fig. 6. Effects of partial anoxia (10.4% oxygen, 6% oxygen, 6% oxygen and 11% oxygenin nitrogen respectively) on respiration of normal frogs. At M the mixture of oxygenand nitrogen was administered. The lowest record shows definite periodic breathingproduced by 11% oxygen in nitrogen. Time in 10 sec. intervals.

evident then that not only can the respiration of the frog be stimulatedby oxygen lack, but also that the sensitivity is not greatly different fromthat of the mammal.

Since Babak accepted Winterstein's evidence that carbon dioxideproduced no central effect, he concluded that oxygen lack must befundamentally different from carbon dioxide excess in its effects on themedulla, and furthermore he decided that the oxygen tension in thefrog's blood is the factor controlling respiration. He sought to confirm

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this by experiments on the effects of temperature, and found that a risein temperature produced a hyperpnoea similar to that of oxygen lack.Such a hyperpnoea had two possible sources. The increased temperaturewould increase metabolism, increase the usage of oxygen and productionof carbon dioxide, and the diminished oxygen content of the blood wouldstimulate respiration. (He assumed that carbon dioxide had no centralaction.) The second possibility was a direct effect of temperature on therespiratory centre. To differentiate between these possibilities he warmed

Fig. 7. A, respiratory movements of normal frog in air at 360 C. B, the same frog inatmosphere of oxygen at 360 C. C, effect of replacing air by oxygen during thehyperpnoea produced by rise in temperature, and of subsequent change to air again.Time in 10 sec. intervals.

the head alone and found no increase in respiration. He considered thisas evidence supporting the control by oxygen lack, but did not think itcomplete enough to be decisive.

We repeated Babak's temperature experiments and found that anincrease in respiration followed a rise in temperature. Usually a definiteincrease occurred when the temperature in the chamber rose from roomtemperature, about 18-20° C., to about 27° C., but whether the frog hadattained this temperature or not was not known. On cooling the chamberthe respirations returned to normal.

The experiment was now repeated in an atmosphere of oxygen insteadof air, the stream of oxygen being warmed as it entered the chamber.There was now no increase in respiration. Fig. 7 shows the contrast ofthe effects in oxygen and in air. The same thing was more strikingly

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shown by warming the frog in air and during the hyperpnoea sendingin oxygen without altering the temperature. The hyperpnoea appears tobe controlled by the presence or absence of oxygen. That the effect oftemperature on the respiration is caused by oxygen lack would indicatethat the effect of oxygen lack is not on peripheral structures but dependsalso on a central mechanism. This was further shown by the fact thathyperpnoea could be produced by oxygen lack in the preparation whichhas been described in the carbon dioxide experiments, i.e. after cuttingspinal cord, Vth and VIIth nerves and glossopharyngeal nerves.

There is thus evidence for believing (1) that the frog shows a truehyperpnoea in response to oxygen lack, (2) that the effect is not dueto a reflex originating at the surface of the body, but is of central origin,(3) that the increased respiration produced by a rise in temperaturedepends on the anoxaemia produced and is not due to the direct effectof temperature on the medulla.

Discussion of the parts played by oxygen and carbon dioxide in thechemical regulation of the respiration of the frog

It has been shown that an increase in respiration in the frog can beproduced by either an increase in the carbon dioxide pressure or adecrease in the oxygen pressure in the air breathed. The effect in eachcase appears to depend not on reflexes originating at the surface of thebody, but on central mechanisms. The findings would fit in with theassumption of centres controlling the respiration of a similar nature tothose in the mammal. The case of the temperature experiments seemsat first sight an exception to this hypothesis, as here the increase inrespiration was seen to be dependent on oxygen lack; whereas if thecentre were similar to the mammalian one it might be supposed thatthe carbon dioxide produced as the result of the increased metabolismwould have more effect on respiration than a corresponding degree ofanoxaemia. A plausible answer is given by a consideration of the effectof the skin respiration. The respective roles of the skin and lungs in thegaseous exchanges have been studied by Krogh [1904] who estimatedthe amounts of oxygen and carbon dioxide taken up and excretedthrough the skin and lungs at different seasons of the year. He foundthat, apart from the breeding season, the lungs play a relatively smallpart in respiration. During this period the oxygen taken up by the lungsrises to a high peak. On the other hand, the oxygen taken up by theskin remains constant throughout the year. In contrast to this thecarbon dioxide excretion through the skin rises at the breeding season,

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as does that also through the lungs, the latter remaining, however, atother seasons of the year extremely low.

The following explanation seems to fit the facts better than thosequoted by Babak, viz. retention of carbon dioxide in the lungs [Krogh],vasomotor changes in the skin [Maar] or action of glands in the skin[Harley]. The oxygen intake in the skin represents the maximumpossible at the normal oxygen tension of the atmosphere. Consequentlyincrease in oxygen intake can only be provided by the lungs which arestimulated by the oxygen lack acting on the centre. The carbon dioxideexcretion through the skin at quiescent periods of the year does notrepresent the maximum possible rate of diffusion, as is proved by thefact that it rises during the breeding season. An increase in carbondioxide production leads, therefore, to an increased excretion throughthe skin alone without the assistance of the lungs. The fact that thecarbon dioxide excretion by the lungs does rise in the breeding seasonfollows automatically from the increased pulmonary ventilation, which,however, is brought about by the oxygen lack. Thus the lung movementsare regulated by oxygen lack and not by excess of carbon dioxide. If thefrog is exposed to an atmosphere containing an increased amount ofcarbon dioxide the excretion of carbon dioxide by the skin is reduced,the carbon dioxide in the blood rises and respiration is stimulated.

A consideration of the anatomy of the circulation shows more clearlythe sequence of events. The venae cavae bring to the sinus venosusvenous blood from the body generally and also blood from the skinthrough the venae cutanae magnae. The blood from the skin has alreadygiven off much of its carbon dioxide, so that the mixed venous bloodentering the heart has already lost a considerable amount of its carbondioxide. This blood collects in the right auricle while the oxygenatedblood from the lungs collects in the left auricle. During ventricularsystole the blood from the two auricles is not mixed, but is so directedby the time relations of the events of the cardiac cycle and a complicatedsystem of septa in the truncus arteriosus, that the first blood to be ejectedfrom the heart is the most venous and goes to the pulmocutaneousartery. The last phase of ventricular systole ejects the oxygenated bloodfrom the left auricle, and this goes to the carotid artery. Between theseextremes the blood, partly venous and partly arterial, goes to the aorta.It thus happens that blood containing a high percentage of carbondioxide never enters the carotid, unless the skin elimination is interferedwith. On the other hand, oxygen lack will at once affect the blood fromthe lungs, and therefore the blood passing to the carotid artery. The fact

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that the production of carbon dioxide does not control the respirationis not due to the insensitivity of the centre to carbon dioxide, but to thevascular arrangement and the excretion of carbon dioxide by the skin,so that the blood containing excess carbon dioxide never arrives at theparts supplied by the carotid artery.

The carotid sinus mechanismIt has been made clear that in demonstrating a "central" control

of respiration in the frog, no distinction has been drawn between thecentre situated in the medulla and possible extra-medullary chemo-receptors such as would correspond with those in the carotid and aorticregions in the mammal. It is now necessary to investigate whether suchperipheral receptors are present in the frog and if so, what part theycontribute to the chemical control of respiration. This possibility issuggested by both morphological and experimental evidence. Koch[1931] pointed out that the sensory areas in the carotid sinuses and aortawere derived from parts of the branchial arches of the embryo. Thiswould suggest a similar function in all vertebrates for structures of thesame derivation and, since the source of derivation is the branchialarches, would suggest a respiratory function. Experimental evidenceon the similarity of the functions of the branchial arches and of homo-logous structures in higher vertebrates exists, but concerns mostly thecirculatory functions. Lutz & Wyman [1932] showed that increase inpressure in the branchial vessels could produce cardiac inhibition in theelasmobranch. Irvine, Solandt & Solandt [1935] recorded the actionpotentials in all five branchial nerves in the dogfish in response to increasedpressure in the branchial vessels. As regards the amphibian, Kuno &v. Brucke [1914] showed that, in the frog, changes in intra-aortic pressurecould affect the blood pressure and heart rate, and they took this to beanalogous with the depressor reflex in mammals. The main interest inthe frog, however, is in connexion with the carotid sinus region. Thereis situated at the bifurcation of the common carotid artery of the froga small swelling known as the glandula carotica. To this structure havebeen ascribed various functions, chief of which was the regulation of theblood flow so that the carotid artery receives the blood from the lastphase of ventricular contraction. Another suggestion has been that ofan accessory heart. It is especially interesting now to note that Huschke[1831] traced its development from one of the gill arches of the tadpoleand suggested that it probably had a respiratory function. Its develop-ment has been studied in more detail by Maurer [1888] and Marshall

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[1893], and these authors have described it as coming from the thirdbranchial arch, i.e. the same one as the carotid sinus in the mammal.Meyer [1927] showed that it receives fibres from the glossopharyngealnerve and also that a stimulation of this nerve proximal to the glandcaused a fall in blood pressure while stimulation distal to the gland hadno effect. Boyd [1933] demonstrated the close relationship of thedeveloping carotid gland in the tadpole to the nerve of the third branchialarch and suggested the possibility of its functional similarity to thecarotid sinus of the mammal. Ask-Upmark [1935] in a comprehensivestudy of the carotid vascular arrangements in a wide range of vertebratesconcluded that morphologically the carotid gland of the frog was homo-logous with the carotid sinus of the mammal. It remains thereforeto investigate whether respiratory reflexes can be shown to originatein this structure.

Experiments on the carotid glandThe method of investigation was to record the respiration of the frog

in response to carbon dioxide and anoxia as has already been described,before and after denervation of the carotid gland with or withoutdenervation of the aortic areas. The denervation was carried out underether anaesthesia. The carotid regions were exposed by a crescenticexcision of the skin and body wall at the level of the sternum, and thenby pulling up the sternum and body wall the carotid gland was freed fromsurrounding structures so that its only attachments were the common,external and internal carotid arteries. If desired the aortic arches andthe origins of the pulmocutaneous arteries were cleaned, the vagus beingseparated from the vessels but not divided. In some cases the parts tobe denervated were also painted with either absolute alcohol or phenol.They were then washed with saline and the incision sutured. At least24 hr. was allowed for recovery and the respiration was tested. Theoperation was accompanied by a considerable mortality and of thesurviving animals only those were tested which appeared normal andresponded briskly to cutaneous stimulation.

Results obtainedAs no accurately comparable measurements of the respiration were

possible, the differences in the behaviour of the animals before and afterdenervation can only be judged from the general appearance of therecords obtained. As regards carbon dioxide, the response of thedenervated animals did not appear to differ definitely from the same

323

D. H. SMYTH

animals before denervation. The impression was gained in a number ofcases that the sensitivity was reduced, but that the animals could stillrespond there was no doubt. Fig. 8 shows records obtained in suchexperiments.

As regards the effects of oxygen lack there was a definite reductionin the hyperpnoea following denervation. This varied from almostcomplete absence of response to a greatly diminished response in com-parison with that previously obtained. The eighteen frogs which weretested after denervation included seven in which the vessels had beencleared but not otherwise treated, five in which the vessels had been

Fig. 8. A, effect of 8-7% carbon dioxide on respiratory movements after denervation ofcarotid gland by painting with phenol. B, effect of 5-35% carbon dioxide after cleaningaortic arches and carotid gland. The large movements in A during administration ofcarbon dioxide are due to movements other than respiratory ones. Time in 10 sec.intervals.

cleared and painted with absolute alcohol and six in which the vesselshad been cleared and painted with phenol. In sixteen of these there wasdefinite reduction of the response; in the other two there was littledifference before and after denervation. In nine of the experimentswhich showed a diminution in the anoxic response the glands alone hadbeen denervated with as little disturbance as possible of the aortae.In one of the two cases where there was no diminution in the responsethis had also been done. It appeared therefore that the carotid glandwas of more importance than the aortic region in the response toanoxaemia. Fig. 9 illustrates records obtained in one experiment. Theseshow a comparison of the effects of carbon dioxide and oxygen lack ina frog before and after denervation of the carotid gland.

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The records indicate that the glandula carotica plays a very definitepart in the response to anoxia; while the hyperpnoea in response tocarbon dioxide seems to be almost if not quite independent of it. It isalso seen that the centre itself can respond to some extent to oxygenlack, even after denervation of the possible reflex sources.

DIscusSIONThese results show a much closer relationship between the amphibian

and mammalian respiratory control than has hitherto been advanced.The respiratory centre of the frog, like that of the mammal, is sensitiveto changes in both the carbon dioxide and oxygen content of its bloodsupply. The fundamental difference lies rather in the elimination ofcarbon dioxide by the skin in the frog, and not in the centre itself.

It has also been shown that the derivatives of the branchial archestake a part in the control of respiration in the frog; so that the glandulacarotica is in this respect homologous with the carotid body of themammal. The results obtained may be compared with the very similarchronic experiments in mammals by Schmidt [1932], Gemmill & Reeves[1933], Wright [1936] and Smyth [1937]. In the frog, just as in themammal, the response to carbon dioxide remains after denervation ofthe carotid area; while that to oxygen lack is greatly reduced. Thedifference in the case of oxygen lack is a much more rapid depression ofrespiration in the mammal, and frotn this it would seem that the centreof the frog is more robust and less easily damaged by oxygen lack. It isalso of interest to note that the carotid area is more important than theaortic area in the frog, as has been found in the mammal.

SUMMARY1. A method has been described for investigating the respiration of

the frog and the effects of carbon dioxide and oxygen lack.2. Carbon dioxide produces two effects, an initial inhibitory one

probably depending on vagal afferents in the larynx, and a generalstimulating effect of central origin.

3. Oxygen lack also produces a hyperpnoea and this depends at leastpartly on reflexes originating in the glandula carotica. There is thusevidence that the glandula carotica of the frog is analogous in thisrespect with the carotid body of the mammal.

4. There is no reason to assume that the respiratory centre of thefrog is fundamentally different from that of the mammal. On accountof the excretion of carbon dioxide through the skin, however, and because

326


of the special vascular arrangements, the depth of pulmonary respirationis regulated by the degree of oxygen required, and not by the amountof carbon dioxide produced.

My thanks are due to Mr J. R. Squires for assistance with the photoelectrical recording.The expenses of this investigation were defrayed by a grant from the Thomas Smythe

Hughes Medical Research Fund.

REFERENCES

Ask-Upmark, E. [1935]. Acta p8ychiat., Kbh., 6 supp.Aubert, H. [1882]. Pftisg. Arch. ges. Phy8iol. 27, 570.Babak, E. [1911]. Folia neurobiol., Lpz., p. 539.Babak, E. [1921]. Winterstein's Handbuch der vergleichenden Phy8iologie, Jena, 1, 706.Bethe, A. [1903]. Allgemeine Anatomie und Physiologie des Nerven8yetem, p. 393. Leipzig.Bogue, J. Y. & Stella, G. [1935]. J. Physiol. 83, 459.Boyd, J. D. [1933]. J. Anat., Lond., 68, 154.v. Budenbrock, W. [1928]. Grundries der vergleichenden Physiologie, p. 362. Berlin.Gaup, E. [1896]. Anatomie dee Froechee. Braunschweig.Gemmill, C. L. & Reeves, D. L. [1933]. Amer. J. Physiol. 105, 487.Heymans, C., Bouckaert, J. J. & Dautrebande, L. [1930]. Arch. int. Pharmacodyn. 39, 400.Heymans, J. F. & Heymans, C. [1927]. Arch. int. Pharmacodyn. 33, 273.Huschke [1831]. Treviranus Z. Phy8iol. 4.Irvine, L., Solandt, D. Y. & Solandt, 0. M. [1935]. J. Phy8iol. 84, 187.Koch, E. [1931]. Die Reflektorieche Selbst8teuerung de8 Kreielaufes. Dresden.Krogh, A. [1904]. Skand. Arch. Phy8iol. 15, 328.Kuno, Y. & von Brucke, E. T. [1914]. Pflug. Arch. gem. Phy8iol. 157, 117.Lutz, B. R. & Wyman, L. C. [1932]. Biol. Bull. Wood'8 Hole, 62, 10.Marshall, A. M. [1893]. Vertebrate Embryology. London.Maurer, F. [1888]. Gegenbaure Jb. 14, 175.Meyer, F. [1927]. Pflug. Arch. gms. Phy8iol. 215, 545.PflUger, E. [1875]. Pflug. Arch. ges. Phyeiol. 10, 316.Rosenthal, J. [1864]. Arch. Anat. Physiol. Wiee. Med. p. 462.Schmidt, C. F. [1932]. Amer. J. Phyeiol. 102, 94.Selladurai, S. & Wright, S. [1932]. Quart. J. exp. Phyeiol. 22, 233.Smyth, D. H. [1937]. J. Phy8iol. 88, 425.Sokolows, 0. & Luchsinger, B. [1880]. Pfltig. Arch. gas. Phy8iol. 23, 283.Winterstein, H. [1900]. Arch. Phy8iol. Supp. p. 177.Wright, S. [1936]. Quart. J. exp. Phy8iol. 26, 63.

the central and reflex control of respiration in the frog

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