Charing Cross Hospital, London, W.C. 2 (Received 28 September 1972)

Transcription

1 J. Physiol. (1973), 230, pp With 7 text-ftgurem Printed in Great Btitain STUDIES OF THE PULMONARY VAGAL CONTROL OF CENTRAL RESPIRATORY RHYTHM IN THE ABSENCE OF BREATHING MOVEMENTS By A. BARTOLI, EWA BYSTRZYCKA, A. GUZ, S. K. JAIN, M. I. M. NOBLE AND DIANA TRENCHARD From the Department of Medicine, Charing Cross Hospital Medical School, London, W. 6, and the Department of Thoracic Surgery, Charing Cross Hospital, London, W.C. 2 (Received 28 September 1972) SUMMARY 1. Breuer's hypothesis that the vagus nerves exert a tonic control of respiratory rhythm, in addition to the phasic control, was examined. 2. Closed-chest cardiopulmonary bypass was instituted in dogs weighing kg anaesthetized with chloralose. Respiratory rhythm was recorded from a phrenic electroneurogram. 3. Complete muscular paralysis induced with gallamine triethiodide produced an increase in the duration of inspiration and an increase in the amplitude of the integrated phrenic electroneurogram. There was no consistent effect on expiratory duration. Gallamine produced no effect when given after vagotomy. 4. In the paralysed state, an increase in lung volume of ml. for sec produced a sustained increase in the duration of expiration and a decrease of respiratory rate: there was little effect on inspiratory duration, or the amplitude of the integrated phrenic electroneurogram. 5. A decrease in lung volume of the same order of magnitude for the same period produced a sustained decrease in the duration of expiration and an increase of respiratory rate: there was little effect on inspiratory duration or the amplitude of the integrated phrenic electroneurogram. 6. The phenomena described in (5) and (6) constitute a high gain respiratory frequency controller. They did not occur after bilateral cervical vagotomy. 7. Bilateral cervical vagotomy during complete muscular paralysis produced a further increase in the duration of inspiration and in the amplitude of the integrated phrenic electroneurogram; there was no consistent effect on expiratory duration.

2 450 A. BARTOLI AND OTHERS 8. The results confirmed Breuer's hypothesis and showed that inspiratory duration and expiratory duration are controlled independently. INTRODUCTION Breuer's concept of 'the self-steering of breathing by the vagus nerves' (Breuer, 1868) has been the subject of renewed interest in recent years (Ciba Foundation Symposium, 1970). The aspect of this feedback control which has received the greatest attention is that due to the phasic increase in discharge from pulmonary stretch receptors during inspiration (Adrian, 1933), this being thought to terminate the inspiratory phase of the breathing cycle. This mechanism is one explanation of the fact that breathing becomes slower and deeper after bilateral vagal section. However, Breuer also postulated that the vagi have a control of respiratory frequency which is effective even when the lungs are not subject to breathing movements. Such an afferent vagal mechanism might provide a means by which changes in end-expiratory lung volume could influence the cycling frequency of the central respiratory controller. Breuer obtained experimental support for this second idea by studying open-chest rabbits which he kept alive with a constant flow of air passing through the trachea and out through multiple holes in the exposed lung surface. In this situation, with no lung movement apart from some minor 'bubbling' at the surface, cervical vagal section caused a slowing of central respiratory rate as judged by a reduction in the frequency of the respiratory movements of the nares. D. Trenchard & A. Guz (unpublished observations) repeated this experiment using 100 % oxygen instead of air. In spite of this, the arterial Po2 was always less than 40 mm Hg and the P0co2 above 70 mm Hg. The lungs showed gross haemorrhage. Under these circumstances, these authors were unable to interpret the results of cervical vagal section. In order to study this problem, we wished to (1) cut the vagi with the lungs static, i.e. repeat Breuer's experiment, (2) do this with normal arterial blood gas tensions and in the absence of lung pathology, and (3) change lung volume in the absence of breathing with the vagi intact, and without arterial blood gas change, to see if lung volume per se is a respiratory rate controller. In order to achieve these aims of a static lung with normal and constant blood gas tensions it was necessary to use cardiopulmonary bypass. METHODS Mongrel and Alsatian dogs, weighing kg, were used. Anaesthesia was induced with thiopentone and the animals were intubated with the largest possible endotracheal tube (size 14-16); the seal between the inflated rubber cuff and the trachea

3 VAGAL CONTROL OF RESPIRATORY FREQUENCY 451 was improved by coating the cuff with white paraffin. Anaesthesia was maintained with 1-2 % halothane in 50% N20/02, while the surgical procedures were carried out; this was then replaced with i.v. chloralose (Merck, 40 mg/kg). Polyvinyl catheters (internal diameter 1-5 mm) were inserted into the left carotid artery and external jugular vein; the latter was advanced until its tip was within the thorax. The following structures were exposed and dissected free from surrounding tissues: (1) both cervical vagi, (2) C 5 root of right phrenic nerve, (3) right external jugular vein, (4) right external iliac artery and vein. Haemostasis was carefully secured as heparin was to be administered. Cardiopulmonary bypass Closed-chest cardiopulmonary bypass was established by the method of Proctor (1966) using a bubble oxygenator based on the model described by Proctor & De Bono (1965). Large polyethylene (Portex) tubes of internal diameter 5-10 mm and outside diameter 6-11 mm were prepared by sealing one end with heat and smoothing the tip. Eight to ten holes of 3-4 mm diameter were made in the wall of the tube over a length of 8 cm, commencing 1 cm from the tip. The prepared catheter was then inserted into the external iliac vein above the inguinal ligament, and advanced until the resistance of the superior wall of the right atrium could be felt. The diameter of the tube chosen was the greatest that it was possible to insert into the vein. The catheter was connected with polyvinyl tubing to the entry part of the bubble oxygenator, situated at least 1 m below the mid thoracic level of the dog, resting in the supine position on a table. The exit part of the oxygenator was connected to a short metal cannula in the external iliac artery with polyvinyl tubing via a roller pump. Intravenous heparin (300 i.u./kg) was given before the insertion of the cannulae and whole body heparinization was maintained throughout the period ofcardiopulmonarybypass (150 i.u./kg. hr). The extracorporeal apparatus consisted of a bubble oxygenator with a heat exchanger within the bubble chamber in order to reduce the volume of priming fluid required to a minimum of 750 ml. The priming fluid consisted of a mixture of 50% Dextran (mol. wt. 40,000) and 50% of a solution containing the following millimolar concentrations: Na+ 131, K+ 5, Ca2+ 8, Cl- 111 and lactate 29. Venous blood was equilibrated in the bubble chamber with a mixture of 3-5% CO2 in 02, the precise concentration of CO2 being adjusted until the Pco, of the arterial blood was approximately 40 mm Hg. The concentration of CO2 required depended on the extracorporeal blood flow rate. The PX0, depended entirely on the extracorporeal circuit, i.e. it was completely independent of pulmonary ventilation. The gas flow was 6 I./min and this was adequate to lift blood from the bottom to the top of the bubble chamber, where it entered a defoaming chamber, containing siliconized wire mesh. At this point the oxygenation of the hemoglobin was complete; a hole was provided for the escape of CO2 and excess 02. The blood was then filtered through two steel-gauze filters into a reservoir, after which it was pumped into the external iliac artery by the roller pump. The pump flow, adjusted to keep a defined level in the reservoir constant, was dependent on the venous return to the oxygenator. This in turn depended critically on the diameter of the central venous cannula and on the correct placement of its tip. If this drainage was inadequate, then some blood was pumped forward by the right heart through the lungs and left heart. This could be detected by the presence of an ejection pulse on the arterial pressure trace. Such blood flow through the lungs was normally oxygenated during spontaneous breathing, but in paralysed animals (see below) the lungs acted as a right-to-left shunt, and this resulted in gradients of oxygen and carbon dioxide tensions between the blood sampled in the carotid artery and blood pumped into the iliac artery (Fig. 1). If this situation was found, a second venous cannula was inserted into the right atrium via the right external jugular vein

4 452 A. BARTOLJ AND OTHERS and connected in parallel with the iliac venous cannula. The improvement in venous return invariably resulted in a reduction of the forward flow through the lungs to an undetectable level. 3-5% CO2O/ Fig. 1. Schematic diagram of closed-chest cardiopulmonary bypass. RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle. Interrupted line shows right to left shunt that may develop with an inadequate bypass. The water moving through the heat exchanger was kept at approximately 410 C and circulated by a Churchill thermocirculator at a flow rate of 8 L./min. This ensured that the temperature of the blood entering the animal was C. Oesophageal temperature was above C. All temperature measurements were monitored with an electrothermometer (Ellab Instruments). Arterial blood ph, PCO2 and Po2 were monitored using a Radiometer electrode assembly. If the ph fell below 7-25, ml. of 82% sodium bicarbonate was administered. Management of the cardiopulmonary bypass was aided by measuring the arterial and central venous pressures; in addition, the electrocardiogram was also monitored. The degree of haemolysis was measured by taking serial samples of blood for haematocrit and plasma haemoglobin estimation. Recording techniques Central respiratory rhythm was monitored by means of the phrenic electroneurogram. The cut central end of the exposed C 5 phrenic root was placed on a bipolar electrode after removing the nerve sheath. The electrical activity of the nerve was then recorded using a Devices type 3 pre-amplifier/oscilloscope unit (high frequency 3 db, 2-5 khz; low frequency 3 db, 8 Hz) together with an analogue magnetic-tape recorder (Ampex SP 300, FM 0-38 m/sec or direct m/sec). The recorded signal

5 VAGAL CONTROL OF RESPIRATORY FREQUENCY 453 was played back on to a Cambridge multi-channel physiological recorder (oscilloscope photography) to obtain permanent visual records. At the same time the signal was rectified by a diode, and integrated using a Fenlow operational amplifier. The integral was not calibrated. It remained constant for periods of 5-10 min. Over larger periods oftime the amplitude decreased due to the slow accumulation ofblood at the electrodes. The control amplitude could therefore vary, especially if the electrodes had to be reapplied. Tracheal airflow passed through a pneumotachograph (Fleisch, no. 1) attached to the endotracheal tube; the differential pressure across the pneumotachograph was measured with a Statham PM 97 strain gauge, and the signal integrated using a Philbrick operational amplifier to give a continuous record of tidal volume. Airway pressure was measured through a needle in the endotracheal tube with a Statham P 23 Db strain gauge. The arterial and venous pressures were also measured with P 23 Db gauges. In some experiments the acute effect of the bypass procedure on the size of the functional residual capacity (F.R.C.) was assessed. Fifteen seconds before starting bypass, the endotracheal tube was switched into a wedge-type of spirometer (Oxford Medical Devices) containing 51. air. All variables were recorded on a multi-channel pen recorder (Cardiac Recorders) in addition to the analogue tape-recorder. Mfea&urements and stahistwal analysts Arterial pressure could be measured with an accuracy of 5 mm Hg and venous and airway pressures with an accuracy of 0 5 cm H20. Tidal volume and change in F.E.C. could both be measured with an accuracy of 10 ml. The amplitudes of the integrated phrenic electroneurogram ranged from 5 to 70 mm deflexion. Time could be measured with an accuracy of 0-1 sec. Inspiratory duration was measured from the points of inflexion of the integrated electrical activity of the phrenic nerve; an error of 0-1 see was possible at the beginning and the end of each integral. Expiratory duration was measured as the remainder of the respiratory cycle with the same error as for inspiratory duration. All values presented in the tables were obtained by taking the average over at least ten breaths before and after each intervention. A comparison of the statistical significance of differences between groups of data was made using a 1-tailed Wilcoxon matched-pairs signed-ranks test (Siegal, 1956). The null hypothesis was rejected at a probability value of 0-05 or less. Protocol After the establishment of cardiopulmonary bypass, no experiments were done for at least 30 min, during which time a steady state was reached with respect to cardiovascular variables and blood gas tensions. The integrity of the Hering-Breuer inflation reflex was checked at frequent intervals throughout each experiment by observing the apnoeic pause following constant pressure inflation of the lungs from a weighted Douglas bag (Widdicombe, 1961). The effect of the following manoeuvres on the central respiratory rhythm was studied. (a) Paralysis. Respiratory movements were abolished by the i.v. administration of gallamine triethiodide (Flaxedil) 14 mg/kg. In some experiments, the drug was given slowly so that central respiratory rhythm could be studied as paralysis developed over the course of a few breaths. The paralysis remained complete for at least 3 hr. To assess any possible direct effect on central respiratory rhythm, the same dose of gallamine was repeated on two occasions, about 2 hr after the initial dose at a time

6 454 A. BARTOLI AND OTHERS when paralysis was still complete. In addition, in one experiment, the drug was first given only after bilateral cervical vagotomy. (b) Change of lung volume. With the animal paralysed, lung volume was increased or decreased around its resting value with a syringe containing air in steps of ml. Each step began from the lung volume in the paralysed position: the gas volume was put in or taken out over periods of 1 sec, and an attempt was made to keep the new volume constant for periods of sec. Changes in Pc,2 during change in lung volume, such as would have occurred in the method of Woldring (1965) were eliminated by means of the cardiopulmonary bypass. (c) Vagotomy. With the animal paralysed, both cervical vagi were cut; lignocaine 2 g/100 ml. was then applied to the cut surfaces. In addition, vagotomy was performed in two dogs on cardiopulmonary bypass, who were breathing spontaneously; no gallamine had been given. Po8t-mortem examination At the end of each experiment, the lungs were excised and fixed by pouring a solution of 10 g formaldehyde, 0-9 g NaCI/100 ml. into the trachea, to replace the air within the lungs. Histological sections were prepared and submitted to a pathologist for assessment. The pathologist had no knowledge of the experimental procedure. RESULTS A successful bypass was established in eleven out of fourteen animals. Breathing continued, although tidal volume and respiratory frequency were often reduced by up to 25 %. A reduction in tidal volume and respiratory frequency occurred particularly if the institution of bypass lowered the P. 0co from a spontaneous level of mm Hg (anaesthetic depression). Respiratory frequency ranged from 8 to 33/min, and tidal volume from 120 to 310 ml. Pump flow was in the range I/min (mean 2-2 I/min) and the mean systemic arterial pressure was in the range mm Hg (mean 114 mm Hg). The Pa02 was greater than 200 mm Hg in all animals except one, where it was 95 mm Hg. The Pa, co2 entirely depended on the concentration of CO2 in the equilibrating gas mixture; the range achieved was usually mm Hg. With constant extracorporeal blood flow and constant oxygenator gas flow, the P., co2 changed by less than 1 mm Hg. The arterial ph was kept in the range The initial blood haematocrit ranged from 0-26 to 0 40 and fell to a final haematocrit ranging from 0-23 to Plasma haemoglobin was mg % at the beginning of bypass and rose to values of mg % at the end (3-6 hr later). Effect of cardiopulmonary bypass on functional residual capacity With the commencement of cardiopulmonary bypass, the functional residual capacity rose by a range of ml. The magnitude of the increase roughly paralleled the magnitude of the fall in central venous pressure which occurred at the same time. The increase in lung air volume

7 VAGAL CONTROL OF RESPIRATORY FREQUENCY 455 was presumably due to a reduction in central blood volume as a result of the bypass procedure. Hering-Breuer inflation reflex The Hering-Breuer inflation reflex was present before and after cardiopulmonary bypass and of similar magnitude to that shown for the dog by Widdicombe (1961). The magnitude of the apnoeic response to inflation remained approximately the same throughout each experiment until bilateral cervical vagotomy had been performed, after which there was no response. Effect of neuromuscular blockade There was no change in Pa, co2 and Pa, 02 during neuromuscular blockade producing apnoea for periods up to 3 hr. With the onset of paralysis, there were progressive decreases in tidal volume; the duration of inspiration and the amplitude of the phrenic electroneurogram increased in proportion to the reduction in tidal volume (Fig. 2a). When breathing movements ceased, a steady state was reached (Fig. 2 b). There was no consistent change in expiratory duration whereas inspiratory duration and the amplitude of the integrated phrenic electroneurogram always increased (Table 1). These changes in inspiratory and expiratory duration usually resulted in a fall of respiratory frequency. In one dog in which gallamine was not given until after the vagus nerves had been sectioned, there was no effect on central respiratory rhythm (Fig. 2c). In other dogs, additional similar doses of gallamine given while paralysis was still complete approximately 2 hr after the first dose had no effect on any of the measured variables. Effect of changes in lung volume There were no changes in Paco2 following a change in lung volume. A change in lung volume produced marked changes in expiratory duration but no consistent effect on inspiratory duration and the amplitude of the integrated phrenic electroneurogram (Figs. 3, 4). An increase in lung volume produced an increase in expiratory duration and a slowing of respiratory frequency, while a decrease in lung volume produced the reverse. The response was maintained over the period of change in lung volume lasting sec (Figs. 3, 4). During each maintained change in lung volume, the airway pressure drifted slightly back towards zero (Figs. 3, 4), suggesting leakage around the endotracheal tube. As airway pressure fell slightly, during a period of increased lung volume, there was a decrease in expiratory duration

8 456 a A. BARTOLI AND OTHERS Z +500 E500 F *> VT [ i500 ml. b A Phr int. Phr. 500 ml. B V ml./sec E A Phr. int. C 5ecI Phr. 0 E +1000r -100 O~b. 1 VT 1000 ml. B ml./sec [ 10 aeg Fig. 2a, b and c. For legend see opposite.

9 VAGAL CONTROL OF RESPIRATORY FREQUENCY 457 (Fig. 5); the reverse sequence occurred during a period of decreased lung volume. Analysis of the average values during each intervention showed that there was a close relationship between changes in expiratory duration and lung volume and respiratory rate (Fig. 6a, b). There was little or no effect on ispiratory duration (Fig. 6a). The results in all the dogs in which this manoeuvre was performed are shown in Table 2. In dogs 9 and 12 (Table 2), there were enough points available to perform a linear regression of change in breathing frequency (Af) on change in lung volume (AV). For dog 9 the linear regression equation was Af = AV (r = 0*942). For dog 12 the equation was Af = AV (r = 0.972). Thus, on average, a change of 1 breath/min was produced by a 14 ml. change in lung volume in dog 9 and by 15 ml. in dog 12. In every case, the effects of changing lung volume were abolished after bilateral cervical vagotomy. TABLE 1. Effect of paralysis on central respiratory activity during cardiopulmonary bypass with intact vagus nerves Amplitude of Resp. rate Inspiratory Expiratory integrated phrenic (mi*-1) duration (sec) duration (sec) ENG* (mm) Dog, I AA no. Control Paralysis Control Paralysis Control Paralysis Control Paralysis * P * Electroneurogram. Legend to Fig. 2. Fig. 2. Records of the effect of muscular paralysis. From the top, the traces are (1) integrated phrenic electroneurogram, periodically discharged to the top of the record, inspiration downwards, (2) phrenic electroneurogram, (3) air flow through the endotracheal tube (1k), (4) tidal volume (VT) obtained byintegration of V. (a) Gallamine given atthearrow.noteprogressiveincrease of inspiratory duration as tidal volume diminishes. (b) A, before paralysis, B, 10 min after paralysis. Note increase in inspiratory duration and amplitude of integrated phrenic electroneurogram in B. Expiratory duration is little affected. (c) A, before paralysis; B, 10 min after paralysis in a dog with bilateral cervical vagotomy. Note irregular breathing pattern which is unaffected by paralysis.

10 458 A. BARTOLJ AND OTHERS Effetd of cervical vagotomy With one or two exceptions, bilateral vagotomy produced an increase in inspiratory duration (P = 0.005) and an increase in the amplitude of the integrated phrenic electroneurogram (P > 0-025). There was no consistent +50 ml.- E 'ie' I 6a E E 10 sec Fig. 3. Records during paralysis. Tracings are phrenic electroneurogram and its integral as in Fig. 2 and airway pressure (AP). Lungs inflated with 50 ml. air from syringe at arrow. Time of release to F.R.C. indicated on AP trace. Continuous record mounted in three sections from above downwards. effect on expiratory duration (P > 0.05). The resultant change in respiratory rate was usually a decrease, but this effect just failed to reach the level of statistical significance (P 0.05) (Fig. 7, Table 3). In order to compare these effects with those of cervical vagotomy during spontaneous breathing, two dogs on complete cardiopulmonary by pass were subject to bilateral vagotomy without any gallamine having been administered. The effects in these dogs were similar to those in paralysed dogs (Table 3). It should be noted that the control measurements of inspiratory duration before vagotomy during paralysis were slightly different from the

11 VAGAL CONTROL OF RESPIRATORY FREQUENCY mi. -.1 I E a-'j Fig. 4. Records during paralysis. Tracings are phrenic electroneurogram and its integral as in Fig. 3 and airway pressure (AP). 100 nil. air withdrawn from lungs at arrow; return to F.R.C. indicated on AP trace. Continuous record mounted in two sections ml. I EU % I +50 ml. +25 ml. 5m. +50 ml. +50 ml. A LI I I I I I I I I I Expiratory time (sec) Fig. 5. Relationship between airway pressure and expiratory duration during periods of lung inflation of the magnitudes indicated. measurements made immediately after the onset of paralysis; this is not surprising in view of the lapse of 1-3 hr between the two interventions. Hi8tological 8tudie8 of the lung8 Macroscopically, the lungs appeared pale but otherwise normal. The basal pleural discoloration, typical of lungs from dogs who have been lying on their backs for several hours under anaesthesia, was also observed.

12 ~A. BARTOLI AND OTHERS Histological examination confirmed the paucity of pulmonary vascular contents. Perivascular or peribronchial oedema was usually absent. There was no alveolar oedema. TABLE 2. Effect of a change in lung volume on respiratory rhythm in the completely paralysed dog with intact vagus nerves Amplitude of Inspiratory dura- Expiratory dura- integrated phrenic tion (sec) tion (sec) ENG (mm) Change in A~ ~(~ Expt. lung volume During During During no. (AV) (ml.) Control AV Control AV Control AV *2 1* * * * * P *2 1* P * * * *0 2* * * * * * * * * * *6 1* DISCUSSION The slow deep breathing which follows cervical vagotomy is well known (Hering, 1868; Breuer, 1868). Our experiments have attempted to analyse the components of this phenomenon. During the course of this work an extremely sensitive vagal control of central respiratory frequency was demonstrated resulting from changes in static lung volume.

13 VAGAL CONTROL OF RESPIRATORY FREQUENCY 461 The characteristics of this rate controller are as follows. (1) The control is achieved purely through an effect on expiratory duration. (2) The controller has very high gain; clear-cut changes in respiratory frequency are caused by changes in lung volume of the order of 1-2 % of the resting value (this assumes that resting lung volume in this size of dog is approximately 1 1. (Crosfill & Widdicombe, 1961). (3) The control is effected (a) Dog 12 At (sec) A +6 - 'C x x x x x x I AV(ml.) +200 (b) Dog Af (min-') * I AV (ml.) S Fig. 6. The change in (a) duration (At) of inspiration (filled circles) and expiration (crosses), and (b) respiratory frequency (Af), produced by change in lung volume (A V) during paralysis, vagi intact, dog 12. Note that the effect of A V on respiratory frequency is almost entirely due to changes in the duration of expiration. Note marked influence of lung volume on respiratory frequency in the absence of lung movements. Regression equation Af = A V-0-55 (r = 0.972), i.e. AV of + 15ml. causes Afof ± 1 breath/min.

14 462 A. BARTOLI AND OTHERS through a deviation up and down around the resting lung volume, the sign of the effect being opposite to the sign of the lung volume deviation. (4) The effect on respiratory frequency is a linear function of the lung volume deviation on either side of the resting level, possibly suggesting Left vagotomy Right vagotomy I,_j 10 sec Fig. 7. Effect of vagotomy on respiratory rhythm during paralysis. Phrenic electroneurogram shows increase in inspiratory duration after vagotomy. Records continuous. Note increase in amplitude of integrated phrenic electroneurogram. T.ABx 3. Steady-state values for central respiratory rhythm taken before and at least 5 min after bilateral cervical vagotomy Amplitude of Respiratory Inspiratory dura- Expiratory dura- integrated phrenic rate min- tion (see) tion (see) ENG (mm) Expt. Vagono. Control tomy P * * * Dog not paralysed (no gallamine given). Control Vago- Control tomy Vago- Control tomy > Vagotomy < 0-025

15 VAGAL CONTROL OF RESPIRATORY FREQUENCY 463 a single mechanism. (5) The time constant of the control system is extremely long in relation to the duration of one respiratory cycle at physiological breathing frequencies (we were unable to achieve changes in lung volume which were absolutely maintained and cannot therefore make any firm statement about adaptation within the control loop). It is not unreasonable to suppose that the afferent information for this control arises from a modulation of the discharge still present at functional residual capacity from slowly adapting pulmonary stretch receptors (Adrian, 1933). Paintal (1966) has shown in the cat, that at least 60% of these receptors are still firing at the end-expiratory level. Bystrzycka & Huszczuk (1973) concluded from indirect evidence that stretch receptor discharge during expiration may control respiratory frequency. It is exceedingly unlikely that irritant receptors (Mills, Sellick & Widdicombe, 1970) are involved in the afferent pathway because (1) the slow manner in which the changes in lung volume were achieved is unlikely to have excited them, (2) their rapid adaptation does not fit with the long time constant of the response, (3) their increased rate of discharge produced by both inflation and deflation does not fit with the fact that the respiratory frequency response to inflation in our experiments was opposite to the response to deflation. Our experiments in which lung volume was changed clearly demonstrate that expiratory duration is controlled by different factors from inspiratory duration. This evidence in the dog is at variance with the suggestion of Clark & Euler (1972) that at least in the cat, expiratory duration is predominantly a function of inspiratory duration. The separate control of inspiratory and expiratory duration is further demonstrated by our experiments with muscular paralysis and cervical vagotomy. Pulmonary stretch receptors show an increase in discharge with each inspiration usually superimposed on a steady level of discharge (endexpiratory level of discharge, see above). Vagotomy removes the entire pattern whereas muscular paralysis removes only the phasic inspiratory component. The only consistent effect of muscular paralysis was to prolong inspiratory duration and increase the total output of the phrenic motoneurones. This is compatible with the hypothesis that the increase in stretch receptor discharge with each inspiration is inhibitory to inspiration and terminates it. The results during the onset of paralysis demonstrate that this mechanism is present on a breath to breath basis. Our experiments exclude any direct effect on central respiratory rhythm by the neuromuscular blocking drug used. The repetition of Breuer's experiment of cutting the vagi with the lungs static, under more physiological conditions showed that the effect of vagotomy on inspiratory duration and total phrenic motoneurone output

16 464 A. BARTOLI AND OTHERS was of similar magnitude to the effect of paralysis and was additive. The effect on inspiratory events of vagotomy after paralysis, and the lack of effect on these same events of change in lung volume, inply that there is tonic non-modulated vagal information which is inhibitory to inspiration. Paralysis and vagotomy had similar inconsistent effects on expiratory duration. If paralysis changes lung volume to a variable degree, variable changes in expiratory duration would be expected from the previously demonstrated, highly sensitive, expiratory duration/lung volume relationship. In the only experiment in which a spirogram was recorded at the time (Dog 9), the onset of paralysis was accompanied by a 72 ml. increase in lung volume above the end-expiratory level; this was associated with a prolongation of expiratory duration. The expiratory duration/lung volume relationship passes through a neutral point at which vagotomy would be expected to produce no effect on expiratory duration. The variable effect of vagotomy on expiratory duration which we found would be explicable in terms of variable deviations of lung volume from this neutral point at the moment of cutting the vagi. In those experiments in which we measured change in functional residual capacity at the institution of cardiopulmonary bypass, there was a variable increase in functional residual capacity above the pre-bypass value, presumably due to a variable reduction in the central blood volume. It could be argued that vagotomy during paralysis abolishes an influence arising in J-type receptors (Paintal, 1969). We think this is unlikely in these experiments because (1) there were no pathological abnormalities in the lungs; under these circumstances J-type receptors are probably quiescent (Guz & Trenchard, 1971; Trenchard, Gardner & Guz, 1972), (2) re-examination of records obtained in cats and rabbits with active J-type receptors (D. Trenchard & A. Guz, unpublished observations) reveals that the bradypnoea produced by blocking them is due to changes in both inspiratory and expiratory duration and not to a predominant increase in inspiratory duration as in the present study. M. I. M. N. was the recipient of a Wellcome Trust University Award. E. B. was the recipient of a personal grant from the Wellcome Trust. S. K.J. was the holder of a Commonwealth Fellowship. The work was supported by grants from the Chest and Heart Association and the Clinical Research Sub-Committee ofthe Board of Governors of Charing Cross Hospital. We are indebted to Dr E. Proctor of the Royal College of Surgeons for assistance in the establishment of an effective cardiopulmonary bypass procedure and to Professor Lynne Reid of the Institute of Diseases of the Chest for the histological assessments.

17 VAGAL CONTROL OF RESPIRATORY FREQUENCY 465 REFERENCES ADRIAN, E. D. (1933). Afferent impulses in the vagus and their effect on respiration. J. Physiol. 79, BREUER, J. (1868). Die Selbsteuering der Athmung durch den Nervus Vagus. Sber. Akad. Wise. Wien 58, BYsTRzYcKA E. & HuszczuK, A. (1973). Studies on the central effects of Hering- Breuer reflexes. Acta neurobiol. exp. 33, (CIBA FOUNDATION SYrPOSswM (1970). Breathing: Hering-Breuer Centenary Symposium, ed. RUmH PORTER. London: J. and A. Churchill. CLARK, F. J. & EULER, C. VON (1972). On the regulation of depth and rate of breathing. J. Physiol. 222, CROSPFILT, M. L. & WIDDICOMBE, J. E. (1961). Physical characteristics of the chest and lungs and the work of breathing in different mammalian species. J. Physiol. 158, GUZ, A. & TRENCHARD, D. (1971). The role of non-myelinated vagal afferent fibres from the lungs in the genesis of tachypnoea in the rabbit. J. Physiol. 213, HERING, E. (1868). Die Selbsteuering der Athmung durch den Nervus Vagus. Sber. Akad. Wiss. Wien 57, MILLs, J. E., SELLICK, H. & WIDDICOMBE, J. G. (1970). Ciba Foundation Symposium, pp London: Churchill. PAINTAL, A. S. (1966). Re-evaluation of Respiratory Reflexes. Q. Ji exp. Physiol. 51, PA=NTAL, A. S. (1969). Mechanism of stimulation of type J pulmonary receptors. J. Physiol. 203, PROCTOR, E. (1966). Closed-chest circulatory support by pump oxygenator in experimental ventricular fibrillation at normal temperature. Thorax 21, PROCTOR, E. & DE BONO, A. H. (1965). A low priming volume oxygenator for bloodless priming in cardiopulmonary bypass. Thorax 20, 540. SIEGAL, S. (1956). Nonparametric Statistics for the Behavioural Sciences. Tokyo: McGraw-Hill. TRENCHARD, D., GARDNER, D. & Guz, A. (1972). Role of pulmonary vagal afferent nerve fibres in the development of rapid shallow breathing in lung inflammation. Clin Sci. 42, WIDDICOMBE, J. G. (1961). Respiratory reflexes in man and other species. Clin. Sci. 21, WOLDRING, S. (1965). Inter-relation between lung volume, arterial CO2 tension and respiratory activity. J. apple. Physiol. 20,