Respiration Physiology (1979) 37, 201-218 © Elsevier/North-Holland Biomedical Press

O2-CHEMOREFLEX

DRIVE OF VENTILATION

IN AWAKE BIRDS AT REST

P. B O U V E R O T

~ a n d Ph. S F . B E R T 2

Laboratoire de Physiologie Respiratoire du C.N.R.S., associ# gt l'UniversitO Louis Pasteur, 23 rue Becquerel, 67087 Strasbourg, France

Abstract. Three varieties of birds (a non-flyer, the chicken; a diver, the duck; a flyer, the pigeon) were studied in resting conditions at neutral ambient temperature, while awake, either intact or sham operated, or after bilateral chronic carotid body denervation. Tidal volume (VT), ventilatory period (T) and minute volume (~' = V1"/T) measured plethysmographically, were recorded breath-by-breath in steady state at two levels of oxygenation, and in the course of transient pure 02 inhalation (60-sec O2-test). 02 partial pressures in the inspired and expired gases (Pio," and PETo2), and in the arterial blood (Pao,) were also measured. In intact and sham operated birds, the abrupt switch from a normoxic gas mixture (Flo, = 0.21) to pure 02 resulted shortly in rapid increases of Plo2, PETo2 and Pao2, and in a fall of ~ which was completed within 20-30 sec and accounted for about 30% of the control minute volume. In the animals previously made hypoxic (FIo2 = 0.12) and hyperventilating, the O2-test provoked a 50-60% fall of ~/. in chickens, a decreased V'r and an increased T contributed to the transient ventilatory changes; in ducks, mainly VT changed, and in the pigeon only T changed. In the birds with denervated carotid bodies, there were no ventilatory responses to hypoxia and to the O2-test. A few of these birds developed a tachypneic response to hypoxia with no apparent change in the effective pulmonary ventilation, which was partly overcome during the O2-test. It is concluded that in the three varieties of birds, the O2-chemoreflex drive from the carotid bodies controlled about 30% of the resting minute volume near sea level, and 50-60% in hypoxic conditions duplicating an altitude exposure to 4000 m. Arterial chemoreceptors Chicken Control of breathing Duck

Hypoxia Normoxia Oxygen breath tests Pigeon

Accepted for publication 16 February 1979 ! Reprint requests to Dr. P. Bouverot: Laboratoire de Physiologie Respiratoire du C.N.R.S., 23 rue Becquerel, 67087 Strasbourg, France. 2 Supported by Grant from the Direction G/re&ale de la Recherche Scientifiquc et Technique. Present address: Laboratoire de Physiologie Animale, U.E.R. Sciences. Universit6 de Bretagne Occidentale, Avenue le Gorgeu, 29200 Brest, France. 201

2,02

P. BOUVEROT AND Ph. S~BERT

Ventilatory reflexes originating in the carotid bodies in response to intravascular injections of NaCN as well as to changes in arterial Po: and Pco: have been described in the chicken (Bouverot and Leitner, 1972; Magno, 1973) and the duck (Jones and Purves, 1970b; Bouverot et al., 1974b). However, the part played by the arterial chemoreflex mechanisms in normal avian respiratory control remains unknown. The present study was undertaken to determine the reflex O2-drive (also called hypoxic drive) to pulmonary ventilation in three varieties of awake birds at rest: a terrestrial, sedentary bird, the chicken; a diver, the duck; and a flyer, the pigeon. The animals were studied at two levels of oxygenation, normoxic and hypoxic, and they were either intact, or had undergone chronic bilateral denervation of the carotid bodies. Breath-by-breath recording of ventilation, using a plethysmographic method, allowed analysis of the latency, the rate of development and the magnitude of the ventilatory changes immediately consecutive to the transient switch from the normoxic or hypoxic gas mixture to pure oxygen (02 test method; Dejours, 1957).

Methods ANIMALS

The experiments were performed on 5 Pekin ducks, 6 chickens and 11 pigeons, domesticated varieties of Anas platyrhynchos, Gallus gallus and Columbia livia respectively; mean body mass, BM, is given in table 1. They were housed as a flock in an outdoor aviary, under natural climatic and photoperiod conditions, and received commercial dry feed (ducks and chickens) or mixed grain (pigeons) and water ad libitum for bathing (ducks) and drinking. These birds will be referred to as intact. Carotid body denervation was carried out on 7 ducks, 5 chickens and 5 pigeons of the same broods as the intact birds, and raised the same way. The birds were anesthetized by intramuscular injection of Equithesin (Gandal, 1969) and artificially ventilated via an endotracheal tube. In the duck, the surgical procedure, via a single median opening of the clavicular air sac, was similar to that described by Jones and Purves (1970a) except that the firculum, sternum and suspensory muscles of the syrinx were left intact. In the chicken and pigeon, the procedure was derived from that described by Tummons and Sturkie (1969) for vagotomy in the thorax: the nodose ganglion of the vagus nerve was reached via a lateral incision along the dorsal edge of the pectoral muscle, then via the natural lateral groove where the fixed ribs attach to the floating ribs. Carotid body denervation consisted of transection of the neural filaments from the nodose ganglion to the carotid body regions on both sides. In preliminary experiments on the chicken, bilateral denerration in a single day resulted in death during or shortly after operation. Thereafter, only one carotid body was denervated, and, after 4-7 days recovery, the other side

AVIAN O:-CHEMOREFLEX CONTROL OF BREATHING

203

was denervated. The air sacs, muscles and skin were closed in layers. The animals were placed in a cage until completely recovered, a minimum of one week, before being returned to the flock. They appeared normal, survived for several weeks or months; and at the completion of the study, were killed with an overdose of Nembutal for anatomical control. These animals will be referred to as chemodenervated or denervated birds. To confirm that surgical maneuvers had not impaired the mechanics of breathing, in 5 ducks, 2 chickens and 2 pigeons (same mean body mass as in table 1), the neural filaments from the nodose ganglion were exposed on both sides but not cut. These birds will be referred to as sham operated. About 16 hours before each experiment, the animal was isolated in a cage and starved, but given free access to drinking water. A given animal underwent only one experiment a day, but could be used for several daily experiments. The experiments were all carried out between 9:00 a.m. and noon, mostly in winter for the pigeons and ducks, and in summer for the chickens. Mean values of barometric pressure and ambient temperature are shown in table I for intact birds; corresponding values for denervated and sham-operated animals were identical.

EXPERIMENTS

As shown in fig. 1 for a cock, the awake bird was placed in a brass and lucite chamber, divided in two parts" a body compartment and a head compartment. A rubber collar and saline gel made an airtight seal around the neck. Plastic PIo2

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Fig. 1. Diagram showing the body plethysmograph, open-circuit system and recording instruments for study of steady or transient ventilatory characteristics in awake birds. For details see Methods.

204

P. BOUVEROT AND Ph. SI~BERT

cheeks prevented the rubber membrane from oscillating and the bird from pulling its neck up or down through the opening. The body compartment was used as a plethysmograph (see Measurements). The head compartment (inner volume 1.2 L for ducks and chickens, 0.4 L for pigeons) was darkened and connected to an open circuit. Upstream the head compartment was connected via a three-way stopcock to either one of two wide T-tubes (inner diameter 2 cm) through which gases circulated in excess at constant identical flow rates (20 L. rain -1 for ducks and chickens, 7 L-min -I for pigeons). Gases were prepared from compressed pure nitrogen and oxygen by means of a set of rotameters and humidifying gas-mixing chambers, MI and M2, at room temperature; M~ delivered a gas mixture more or less enriched with 02; M2 delivered pure 02 (as shown in fig. 1) or the same gas mixture as M,. The free arm of each T-tube Oength 1 m) opened to the outside. Downstream from the head compartment, a suction pump (P) via a regularising capacity (not shown in fig. 1) withdrew gas flowing through the arm of the T-tube openinginto the head compartment at a constant flow rate (16 L. min- ! or 5 L. rain ~ I, depending on the bird); then, the gas flowing in excess through the T-tube escaped via its free arm. Between the gas-mixing devices and the pump, rubber bags reduced possible changes of pressure around the bird's head to less than 1 mm of water. Before reaching the animal, the gas mixture M~ (occasionally also M2) was sampled at constant pressure and flow through a paramagnetic O2-analyzer. Each day's experiment consisted of successive studies at two different levels of oxygenation. The bird breathing room air was allowed to equilibrate for about 30 min in the plethysmograph with the three-way cock disconnected from the head compartment. Then, the animal inhaled a normoxic gas mixture (M~; Flo, = 0.21 for 60 min). About 10 min after the beginning of inhalation, resting ventilatory measurements started (see below). When at least 10 respiratory cycles had been recorded, the bird was switched in the course of an expiration to pure oxygen for 60 sec (.prolonged O2-tes0; recording continued for the duration of the test. Several O2-tests were performed; between tests the animal breathed the M1 gas mixture for 4-7 min, till the ventilatory values (read on a video-terminal; see Measurements) returned to control levels. Blank tests were also occasionally performed; then, pure oxygen ( M 2 circuit in fig. l) was replaced by the gas mixtu:e flowing through the Mt circuit. In the second part of the daily experiment, the birds were exposed to a hypoxic gas mixture (Flo2 = 0.12) from Mt for 10 min and the O2-tests were repeated as above. The animal was then returned to the flock. All tests were analyzed unless interrupted by the bird's moving or struggling, which rarely occurred.

MEASUREMENTS

(1) Tidal volume (VT) and ventilatory period (T), from which were derived the respiratory frequency (f) and minute volume of ventilation (-~r), were measured by

AVIAN O2-CHEMOREFLEX CONTROL OF BREATHING

205

plethysmography. In the chicken and duck experiments, a volumetric plethysmograph was used, as shown in fig. 1. The body compartment was open to the atmosphere; thoracic movements of the bird caused air displacements through a No. 0 or a No. 1 Fleisch pneumotachograph connected to a + 2 mbar differential inductance manometer. For pigeons, the ventilation was measured by barometric plethysmography; the body compartment (14 L capacity) was closed, and the pressure changes related to the thoracic movemer~ts were directly measured with the inductance manometer. In either case, the output signal of the manometer fed simultaneously a photographic recorder (via an integration circuit in the experiments on chickens and ducks) and a minicomputer analog-to-digital converter. For each breath, the computing routine calculated VT (mlBTPS) from the output signal of the manometer and T (sec) by measuring the elapsed time between trigger pulses; f (min-~), determined as 60/T; and V on a breath-by-breath basis by multiplying VT by 60/T. The ventilatory values were displayed on a videoterminal together with the time of occurrence of each expiration, pata was stored on magnetic tape for further statistical treatment. Before each experiment, the system was calibrated by replacing the bird by an inert body of equal volume, and its neck in the airtight seal by a rubber stopper. A hand-driven syringe introduced and withdrew 10 fixed volumes at the usual frequency of the bird's respiration. The standard error was less than +. 1~ in the range of frequencies up to 50 cycles, min -J. Pressure changes in the body box were negligible in the experiments on ducks and chickens, and never exceeded 1 cm H~O above atmospheric pressure in the experiments on pigeons: thus there was no important elastic ventilatory load. (2) The O: and CO2 partial pressures in the ventilated gas were recorded in the ducks and chickens with a fast polarographic O2-analyzer and an infrared CO2-analyzer. As schematically shown in fig. 1, a plastic catheter, passing through the wall of the head compartment and taped onto the beak at the nostril, was connected to the inlets of the analyzers via a Y tube. The outlet of each gas meter was connected to a pump which sampled the gas at a constant flow rate (50 ml. min-J). The output signals of the analyzers were displayed simultaneously on the polygraph and, for purposes of experimental supervision, on the X-Y coordinates of an oscilloscope. (3) Arterial blood samples were occasionally taken from the brachial artery via a chronically implanted plastic catheter passing through the wall of the body compartment. Before an Oz-test and at various intervals from the start of oxygen breathing, blood samples were withdrawn into heparinized capillary tubes via a 10-channel cock. Free bleeding prior to sampling eliminated the problem of dead space. The circulation time from the brachial artery to the outlet of the sampling device was corrected for, but the circulation time from the lung capillaries to the brachial artery was not. The capillary tubes were sealed and stored on ice. Po_, was measured within 10 min by an IL microelectrode thermostatted at 41 °C.

206

P. BOUVEROT AND Ph. SI~BERT

ANALYSIS OF DATA

In the analysis of each O2-test, the averaged values of VT, T, f and ~f during the 10 ventilatory cycles before the onset of 02 breathing (time zero) were taken as the control values. The mean values prior to all the O2-tests performed in a Oven experiment at a given level of oxygenation were then averaged to obtain the mean control values for that experiment. The observed breath-by-breath ventilatory changes in the course of O2-tests were averaged in the same way, and plotted against time on a linear scale as in fig. 2. The individual mean values were averaged to yield the mean values for a Oven bird species (as in table 1 and fig. 4). In an effort to characterize more accurately the time course of ventilatory changes during O2-tests, a procedure similar to that described by Dowries and Lambertsen (1966) was used. In all tests, the ventilatory changes observed within the first 20-25 sex after the switch to pure 02 were plotted on a logarithmic scale as a function of time. They decreased from the control value (~/ref) to approach the residual, or minimal, ventilation (Vres) observed in the course of an O2-test. For each test, a regression line was calculated and drawn through the plotted values ln0~r - Vres) as a function of time from the onset of 02 breathing (fig. 5). From this line, extrapolated back to control, the time at which ventilatory depression began could be estimated (latency in table 1). Its slope described the rate of development of the ventilatory depression. The reciprocal of the slope gave the time constant, i.e. the time required for 63% of the maximal depression to occur (0.63 A Xrmax in table 1, where A ~fmax = ~rref- ~rres). In a few experiments, the rise of Po, in the inspired and expired gas and in the arterial blood from their control values to the maximal values (Paoe max) observed in the course of 02test were similarly analyzed, and In (Pao~ m a x - Pao~) was plotted as a function of time (fig. 5).

Results (1) MEAN RESTING RESPIRATORY VALUES

Mean resting respiratory values for intact birds are given in table 1: lines 5 to 10 refer to normoxic animals and lines 15 to 20 to hypoxic animals breathing 129/o oxygen in nitrogen. Values in the sham operated animals were not significantly different.

(2) VENTILATORY RESPONSES TO O2-TESTS

(a) Changes in the minute volume (~1) are illustrated in fig. 2 for one typical intact chicken. Switching the bird from either normoxic or hypoxic gas mixture to pure

207

A V I A N O2-CHEMOREFLEX CONTROL O F B R E A T H I N G TABLE l

Resting respiratory values, latency in onset, rate of development and magnitude of venti!atory changes in the course of transient O2-breathing in three species of awake intact birds previously either normoxic or hypoxic (mean values _+2 SE) Pigeons l

N

2 3 4

BM Ps T box

Chickens

ll kg Ton" °C

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0.55 749 20.2

145 105 34 8.1 33.7 270 70 2.4 4.2 26

83 52 28.9

8.2 43.3 350 180 2.4 3.8 51

Ducks

6 0.023 3.1 0.28

1.81 750 23.8

5 0.092 1.8 0.39

2.83 752 20.8

0.280 2.2 0.28

1.2 1.2 0.65 3.40 19.7 10.9 0.23 0.72

145 90 33.9 41.5 12.1 500 156 2.8 9.8 31

0.96 0.63 2.37 1.18 28.0 8.2 1.15 1.13

145 101 28.7 76.4 14A 1100 300 4.3 7.3 27

2.0 0.79 11.39 2.17 105.9 13.6 1.26 1.26

1.2 1.56 0.86 5.74 22.4 14.8 0.54 0.59

83 48 25.2 37 21.5 795 370 6. I 6.8 47

0.58 0.67 3.3 3.07 47.0 18.2 O.79 0.45

83 54 20. l 93 15.1 1400 800 3,2 6,5 57

1.37 0.37 7.8 7 0.84 104.8 25.8 0.97 2.42

oxygen (fig. 2A and 2B respectively) resulted in an abrupt increase of Po2 in the inspired gas 0~Io2) and, after a short time lag, in the end-tidal gas (PETo:) and arterial blood (Pao~); V started falling within a few seconds to reach a minimal value about 30 sec after the onset of oxygen breathing. In the lower part of fig. 2, the dashed line visualizes the residual, or minimal ventilation (Vres), expressed as the mean of 2-4 consecutive breaths with the minimal minute volume. If fig. 2B (hypoxic experiment) and fig. 2A (normoxic experiment) are compared, it may be seen that when the chicken breathed 12% oxygen in nitrogen, the control minute volume (~' on the left of time zero) was much higher than in normoxia. The residual ventilation in the course of the O2-test was approximately the same in normoxia and hypoxia. Consequently, the ventilatory depression due to O2-breathing was greater in the chicken when it was hypoxic.

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Fig. 2. Transient ventilatory effects in one intact awake chicken of the sudden inhalation of pure oxygen for 60 seconds (O2-tests). Ordinate: From the top down, tractional concentration of 02 in the inhaled gas (Fl02), partial pressures of 02 in the inspired gas (~), end-tidal gas (ET) and the arterial blood (a); pulmonary ventilation (~'). Abscissa: time, starting from the onset of breathing pure 02. (A) The bird breathed a normoxic gas mixture (Ho, = 0.21) prior to the O2-tests. (13)The animal breathed 12% oxygen in nitrogen (Fl02 = 0.12) for at least 10 min prior to the O2-tests. Pl02, PETo2 and 'V were measured breath-by-breath (see Methe~s); arterial blood was sampled at various intervals as indicated by thick horizontal bars. Continuous curves in upper part fitted by eye. Lines perpendicular to horizontal bars represent :!:2 SE ( n - 5 ) . Dashed straight lines indicate the residual minute volume (Vres) determined as the mean of several consecutive breaths a t minimal ~.

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Fig. 3. Mean ventilatory eh,-mges in the course of O2-tests in three species of awake birds with intact innervation of the carotid bodies. Abscissa: time, starting from the onset of 02 breathing. Ordinate: mean change in the minute volume per unit body mass. On the left, before time zero, the birds breathed (A) a normoxic gas mixture (Fie2 = 0.21) or (B) 12% oxygen in nitrogen (Fie2 ==0.12). At time zero, the animals were switched to pure oxygen for 60 sec; only the early phase of the response is shown. Solid lines, chickens (n = 6); dashed lines, ducks (n = 5); dotted lines, pigeons (n ffi ll). Vertical bars represent + 2 SE.

AVIAN O2-CHEMOREFLEX CONTROL OF BREATHING VT mlaTpS

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Fig. 4. Mean changesin the breathing pattern of unanesthetizedpigeons, chickensand ducks in the course of transient O2-breathing. Ordinate: tidal volume, VT; abscissa: ventilatory period, T; oblique lines: isoventilation lines, '¢. Open symbols, previously normoxic birds; closed symbols, previously hypoxic birds. Arrows visualize the overall pathways of breath-by-breath changes from control values to the maximal ventilatory depression observed in O2-tests. Symbols are for intact animals, and the size indicates mean value +2 SE. Large ellipses, ducks (n = 5); rectangles, chickens (n = 6); small ellipses, pigeons (n = 11).

A ventilatory depression in the course of transient O2-breathing was observed in all the intact or sham-operated chickens, ducks and pigeons. For purposes of species comparison, the observations made in intact animals are summarized in fig. 3, where the changes in the minute volume (A'v') have been expressed per unit body mass. Figure 3A shows the transient changes observed within the first 30 sec after the ~witch from a normoxic mixture to pure oxygen, and fig. 3B after the switch from 12% oxygen in nitrogen to pure oxygen. In the three species, the ventilatory depression started within a few seconds of the onset of 02 breathing, and was maximal within 30 seconds; the lower the initial level of oxygenation, greater the fall of ventilation. (b) The changes in the breathing pattern from the control to the residual minute volume during O~-tests in intact birds are illustrated in fig. 4. The changing breathing pattern during the ventilatory depression appears to differ from one species to the other. In the ducks, the fall of V was essentially due to a reduction of the tidal volume (VT). In the pigeons, it resulted exclusively from an increase in the ventilatory period, T (its reciprocal, the respiratory frequency f, decreased). In normoxic chickens, VT decreased and T increased. However, in the previously hypoxic chickens (closed symbols), the changes in VT were not significant and the ventilatory depression resulted mainly from a longer respiratory period. (3) MAGNITUDE OF VENTILATORY DEPRESSION

The maximal ventilatory depression in the course of O2-test (A ~max) was evaluated by subtracting the residual ventilation observed after the onset of oxygen breathing

210

P. BOUVEROT AND Ph. SI~BERT

(Vres) from the control ventilation just before the 02-test ('Vref). The mean values ofA~rmax ffi ~'ref - ~'res for intact birds are given in table I (lines 11 and 21). When expressed as percent of the control ventilation (table 1, lines 14 and 24), AVmax accounted for approximately 30% of the control minute volume in normoxic animals, and for 50-60% in the hypoxic ones, whatever the species. The values were similar in sham-operated birds.

(4) TIME COURSE OF VENTILATORY DEPRESSION

Figure 3 (A and B) indicates the time courses of the ventilatory changes in response to abrupt 02 inhalation. At least in hypoxic conditions (fig. 3B), pulmonary ventilation was more quickly depressed in the pigeons than in the other birds. Since these graphs resemble an exponential function, the method of exponential peeling was used in an attempt to characterize the latency and response rate of the In ~IDomax Pao2(t)) inverted u02

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20

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Fig. 5. Regression analysis of the early phase of the transient respiratory responses to O2-tests shown in fig. 2 for an intact awake chicken. Abscissa: time on linear scale, starting from the onset of 0 2 breathing. Ordinate: Top, semi-log plot (inverted to visualize the rise of P02) of the difference between the maximal value observed at the end of O2-tests (Pao, max) and either the control value (on the left of time zero) or the values obtained by discontinuous measurements during the O2-test. Bottom, semi-log plot of the difference between values of the minute volume, either during the control period or measured breathby-breath ('~(t)) during the O2-test, and the minima I value observed about 30 sex after the onset of O2-breathing. Open symbols, mean values (n - 5) in the previously normoxic bird (FI02 - 0.21); closed symbols, hypoxic bird (Fl02 - 0.12). Oblique lines are regression lines determined by the least squares methud. Intersection of regression lines with horizontal lines drawn through control data points indicates latency; the reciprocal of the slope of regression line gives the time constant of response.

AVIAN Oe-CHEMOREFLEX CONTROL OF BREATHING

211

ventilatory changes in the first 30 sec following the onset of 02 breathing. Even though neither the changes of ~/(a product of two functions) nor the changes of arterial Po: (dependent, at least partly, upon the complex kinetics of air sacs wash-in; see Scheid et al., 1974) should result in a single exponential. The procedure (see Methods) is illustrated in fig. 5 for the responses of the intact ohicken shown in fig. 2. It may be seen that the principal determinant affecting the latency of ventilatory changes is closely related to the rise of Poe in the arterial blood. This analysis was applied to all intact birds, and an averaged response latency and a mean time constant calculated. The values appear in table 1, lines 12 and 13 for normoxic experiments, and lines 22 and 23 for hypoxic ones. The longest latency for a ventilatory response, about 6 sec after the onset of 02 breathing, was observed in the hypoxic chickens; in other birds, the delay for 63% of the maximal ventilatory depression was around 10 sec in the normoxic chickens, and ranged from 3.8 to 7.3 sec in the other birds.

(5) CHEMODENERVATED BIRDS

Figure 6 shows the mean changes in the minute volume (A V, expressed per unit body mass) observed in the course of transient 02 breathing in birds chronically

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Fig. 6. Mean ventilatory changes in the course of Oe-tests in three species of awake hypoxic birds with chronic bilateral carotid body denervation. Abscissa: time, starting from the onset of Oe-breathing. Ordinate: mean changes in the minute volume expressed per unit body mass; scale identical to that in fig. 3. On the left of the vertical line at time zero, the birds breathed 12% oxygen in nitrogen (Fio2 = 0.12). At time zero, the animals were switched to pure oxygen for 60 sec; only the early phase of Oe-test is shown. n = 5, 3 and 5 in pigeons, chickens and ducks respectively.

212

P. BOUVEROT AND Ph. SEBERT

deprived of afferents from the carotid bodies, and previously made hypoxic by inhalation of 12% oxygen in nitrogen. If fig. 6 and fig. 3B are compared, it may be seen that hypoxic denervated birds did not hypoventilate during O2-tests as hypoxic intact animals did.

Discussion (1) O2-CHEMOREFLEX MECHANISM OF VENTILATORY DEPRESSION IN BIRDS

Bouverot and Leitner (1972) showed that giving a few tidal volumes of pure oxygen to breathe to anesthetized hypoxic hens resulted in a transient increase of arterial Po:, a decrease in the afferent activity of vagal fibers, and a decrease of pulmonary ventilation. These findings demonstrated that there is an arterial chemoreceptor mechanism involved in the control of breathing in birds. The aim of the present study was to assess, in conscious birds at neutral temperature, the magnitude of the hypoxic chemoreflex drive (also called O:-drive), i.e. the degree to which the O2-chemoreflex mechanism contributes to the overall ventilatory activity in physiological conditions, and to do so by means of the O2-test method (Dejours,

1957 . If, at a given level of oxygenation, there is a hypoxic chemoreflex drive to the respiratory centers, a decrease in ventilation must occur in the few seconds following a transient switch to pure oxygen, because Po: increases in the parabronchial gas and in the arterial blood (see fig. 2) perfusing the carotid bodies, whose afferent activity then decreases (Bouverot and Leitner, 1972; Bamford and Jones, 1976). In our chickens, ducks and pigeons, either intact or sham-operated, we observed a significant hypoventilation in the course of transient 02 breathing, as did Jones and Purves (1970b) in Khaki Campbell and White Aylesbury ducks. It may be assumed, on the basis of studies on mammals (see Dejours, 1962), that pure 05 inhalation did not modify the mechanical properties of the ventilatory apparatus. If the observed fall of ventilation during the O2-test really resulted from the decrease or from the suppression of a drive originating in the arterial chemoreceptors, a decreased ventilation should not be observed in chemodenervated birds. Figure 6, to be compared with fig. 3B for intact birds, shows that transient 02 breathing elicited no significant ventilatory changes in our birds chronically deprived of carotid body afferents when they were hypoxic, and normoxic chemodenervated birds did not respond either. The findings on chemodenervated birds also agree with those of Jones and Purves (1970b) and of Bouverot et al. (1974b). Since all normoxic or hypoxic sham-operated birds developed an early fall of ventilation in the course of O2-tests~ it can be inferred that the mechanics of breathing were not impaired by the surgical maneuvers themselves. It must be noted that a few denervated birds (2 chickens out of 5 and 2 ducks out of 7) exhibited a slight ventilatory depression, approximately half that observed in intact

AVIAN O2-CHEMOREFLEX CONTROL OF BREATHING VT, ml BTPS

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i 4

T, sec

Fig. 7. Mean changes in the breathing pattern of awake chickens chronically deprived of afferents from the carotid bodies. Ordinate: tidal volume, VT; abscissa: ventilatory period, T; oblique solid lines: isoventilation lines, V. The birds, when normoxic, showed no ventilatory reaction to O2-tests (white triangle; no arrow); but, when hypoxic, exhibited a tachypnea (black triangle) which declined during O2-tests (dashed arrow from the black triangle to the cross representing the maximal ventilatory depression). A dotted line drawn through the triangles and extrapolated to the left intercepts the ordinate at 8 ml, a value quite close to the dead space vglume for chickens of 2 kg body mass. Mean values in 2 animals (see Discussion).

animals when they were switched from a hypoxic mixture to pure oxygen. These birds, exposed to hypoxia, developed a tachypneic response with concomitant decreases in both the tidal volume and ventilatory period (compare solid and open triangles in fig. 7). The ensuing transient O2-inhalation caused the breathing pattern to return partly towards normoxic values (cross in fig. 7). Miller and Tenney (1975) also observed hypoxic tachypnea in the carotid-deafferented awake cat, and concluded that hypoxia acted as a powerful stimulant to the central respiratory frequency generator and as a depressant of the tidal volume. The fundamental mechanism by which hypoxia could act centrally to produced tachypnea and the functional significance of the tachypnea remain, however, to be elucidated. On the other hand, the effective parabronchial ventilation, 9'p eff, was almost certainly not affected during the tachypneic response. A line drawn through the triangles and cross of fig. 7 and extrapolated to the left intercepts the Y-axis at a value (around 8 ml) close to the expected dead space volume, VD (Kuhlmann and Fedde, 1976). The general equation of the line can be written VT = VD + aT. Dividing each member by T, and rearranging, leads to a = V - V D , and thus to a ffi Vp eff. This means that the dotted line in fig. 7 represents all VT, T combinations leading to constant effective parabronchial ventilation. In other words, certain of the denervated birds could modify their minute volume in hypoxia and during the O2-test without changi~Jg their parabronchial ventilation. This is a very different ventilatory response from that of intact animals; fig. 7 clearly shows that the changes of VT during 02tests in the denervated chickens were opposite to those in the intact animals shown in fig. 4.

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In co~tclusion, the fall of ventilation observed in the course of O2-tests in intact awake birds can be attributed to the decrease or to the suppression of a drive originating in the arterial chemoreceptors. The integrity of the afferent fibers from the carotid bodies is a prerequisite for the normal ventilatory depression to occur in response to transient O~-breathing.

(2) MAGNITUDE OF THE AVIAN HYPOXIC-CHEMOREFLEX DRIVE, CHANGES IN THE BREATHING PATTERN

The 'continuous O2-test' (Dejours, 1962, 1975), in which the O, inhalation is pi-olonged for some time while ventilatory changes are measured breath-by-breath, is the best method to estimate the magnitude of the hypoxic drive. In preliminary experiments it was found that the birds had to breathe at least five tidal volumes of 02 (i.e. during 10-30 sec depending on the species) for the ventilation to be maximally depressed. To make sure this point was passed, the birds breathed 02 for 60 sec. The ventilatory changes, however, were completed within 20-30 sec in the three varieties of birds studied (see ~rres in fig. 2, and maximal A V in fig. 3). In this early phase of the response to 02 , at the moment of maximal disruption, the transient ventilatory changes can be related directly to the 02 stimulus (Dejours, 1962); consequently, the observed maximal fall of ventilation (A emax) can be used as an index of the magnitude of the hypoxic chemoreflex drive which existed just prior to the onset of O~-breathing. The index, however, is not very accurate: acidification ofthe blood related to changes in the Hb/HbOe ratio cannot be avoided; moreover, as soon as ventilation declines in the course of Oz-test, there are concomitant increases in both the Pco~ value and H+-ion concentration in the arterial blood, increases which tend to oppose the ventilatory effects of increasing arterial Poe (Dejours, 1962). Consequently, values of A Vmax or 100 A ~max/V (respectively lines 11 and 14 for normoxic experiments, 21 and 24 for hypoxic experiments in table 1) underestimate the hypoxic drive. With this reservation, our study does indicate that arterial chemoreceptors controlled about 30% of the minute volume in resting conscious chickens, ducks and pigeons breathing air near sea level at neutral ambient temperature (see line 14 in table I), and about 50-60% in the birds breathing an hypoxic gas mixture equivalent to air breathing at approximately 4000 m (see line 24 in table 1). If arterial chemosensitive afferents contribute similarly to the control of minute volume in three avian species, the same cannot be said for the control of the ventilatory pattern. Indeed, abrupt release of the hypoxic chemoreflex drive by O~-breathing depressed mainly the tidal volume in the duck, the ventilatory period in the pigeon, and both VT and T in the chicken (fig. 4). The mechanisms underlying these species differences are not clear. To summarize, despite species variations in the breathing pattern, the degree to which the excitatory O2-related chemoreflex drive contributes to the setting of

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215

the minute volume appears to be the same in the terrestrial, diving or flying birds studied, in normal conditions of life near sea level. There is a tonic drive from the arterial chemoreceptors to the respiratory centers, which is active in normoxia. The lower the control arterial Po2 value, the stronger the drive; and under hypoxic conditions simulating exposure to an altitude of 4000 m, the avian pulmonary ventilation can be more than halved by transient O2-breathing which reduces or suppresses the chemoreflex drive.

(3) RESIDUALVENTILATION =

The Pao2 threshold for stimulation of the avian arterial chemoreceptors remains to be precisely determined. Figure 8 indicates that an O2-test provoked no ventilatory change in a duck previously made hyperoxic by breathing more than about 35% oxygen in nitrogen; thus there was no drive from the arterial chemoreceptors for inspired Po: values greater than about 240 Torr. Since arterial Po_, does rise above these values in the course of O2-tests (see fig. 2), the residual ventilation then observed must be the result of excitatory influences acting on the respiratory centers which do not originate from arterial chemoreceptor O2-stimulation. In our intact birds, the non-O2-chemoreflex drive in normoxia controlled a residual ventilation (~/res) which accounted for about 70% the minute volume (subtract each value of line 14 in table 1 from 100). Vres in hypoxic birds can be easily determined by subtracting A~/max (line 21 of table 1) from the control hypoxic minute volume (line 20), and expressing the difference as a percentage of the normoxic minute volume (given in line 10). This calculation indicates that the non-O.,-chemoLsTPS.min 2

0(~

,

I

100

I

200

I

300

I

P[o2 Torr

Fig. 8. Threshold for ventilatory reaction to O2-tests in an awake intact Pekin duck. Abscissa: partial pressure of oxygen in the gas mixture inhaled steadily by the bird before O2-tests. Ordinate: minute volume measured breath-by-breath. Circles and solid line refer to steady measurements during control pe[iods. Squaresand dashed fine refer to the minimal, or residual ventilation observed about 30 see after the on~t of O2-tests (Pio2 around 690 Ton'). Size of the symbols indicates mean values + 2 SE (number of measurements= 12). The vertical distance between a given circle and the related square indicates the magnitude of the ventilatory fall in the course of transient O2-breathing. Note that pure O2-breathing had no significant ventilatory effect on the bird previously made slightly hyperoxic due to steady inhalation of 35% 02 in nitrogen or more (PIo2/>240 Ton').

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reflex drive of hypoxic birds controlled a part of the ventilatory motor output which corresponded to 63?/0, 85~ and 55?/0 of the normoxic minute volume in pigeons, chickens and ducks respectively. Note that in the pigeons and ducks, the non-Oe-chemoreflex drive controlled some 10~ less of the minute volume in hypoxia than in normoxia. Both hypocapnia and central hypoxic depression could explain this observation. Arterial Pco: did decrease in the hyperventilating hypoxic birds (compare lines 17 and 7 in table 1), and a lesser ventilatory COs-stimulus could have been responsible for a ventilatory depression through either central or peripheral mechanisms, or both. Peripheral mechanisms could involve arterial chemoreceptors and intrapulmonary CO2sensitive units (see Bouverot, 1978, for review). Hypoxia may have a depressive central effect in mammals (Davenport et al., 1947; Lahiri, 1976). It acts concomitantly with the excitatory drive from the arterial chemoreceptors, and dominates when arterial chemoreceptor activity is suppressed in the course of pure O2-breathing. Such a mechanism may well have been involved during the hypoxic experiments on intact birds. In contrast, in the chickens, the non-O2-chemoreflex drive controlled some 157/o more of the minute volume in hypoxia than in normoxia. This may possibly be due to a thermal drive in the hypoxic chickens. These birds were studied in summertime and the temperature inside the plethysmograph, which could not be maintained constant, progressively increased from an average value of 22.0°C during the first part of the daily experiment, in normoxia, to 24.6 °C in the second, hypoxic part of the experiment (the Tbox value, given in line 4 of table 1, is the overall mean). It is conceivable that physical heat transfer from the body core to ambient air through the wail of the plethysmograph could have been hampered during the hypoxic experiments, with some compensatory increase in the pulmonary ventilation and respiratory evaporative cooling as a consequence (Bouverot et al., 1974a).

(4) DYNAMIC CHARACTERISTICS OF THE IMMEDIATE VENTILATORY RESPONSE TO 02 BREATHING

The time course of the respiratory depression following abrupt oxygen inhalation is determined by a variety of factors reviewed by Dejours (1962; see also Downes and Lambertsen, 1966). In the present study, by turning a three-way stopcock (fig. 1), oxygen was delivered during an expiratory movement to the open-circuit device in which the bird's head was enclosed; from the stopcock maneuver, 6.9 _+0.86 sec were required for 95~o of the maximal change in inspired Poe to occur. It cannot truly be said that the birds breathed pure oxygen abruptly. Consequently the mean overall latencies, 3.2 and 3.9 sec in normoxic and hypoxic experiments respectively (determined by averaging the three values of lines 12 and 22 of table 1), are almost certainly too high. Such short lat¢ncies, and the fast average time course

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217

(7.1 in normoxia and 5.7 sec in hypoxia for 63% of the maximal A V to occur, obtained by averaging the three values in lines 13 and 23 respectively) indicate that the drive from the arterial chemoreceptors may provide a temporal fine adjustment in the chemical control of avian respiration. In conclusion, the changes in the minute volume, which immediately follow sudden inhalation of pure oxygen, indicate that terrestrial, diving or flying birds do possess an important ventilatory drive from the arterial chemoreceptors when resting at neutral ambient temperature. This chemoreflex drive, inversely related to arterial Po:, controls about 30% of the pulmonary ventilation in normoxia, and 50--60% in hypoxic conditions duplicating exposure to 4000 m altitude.

Acknowledgements We gratefullyacknowledge the technical help of Miss Lacaisse during the experiments. We also thank J.-P. Gendner for his assistance in the computer operations, and Mrs. M.-L. Harmelin for her help in preparing the manuscript.

References Bamford, O. S. and D. R. Jones (1976). The effects of asphyxia on afferent activity recorded from the cervical vagus in the duck. Pfliigers Arch. 366: 95-99. Bouverot, P. and L.M. Leitner (1972). Arterial chemoreceptors in the domestic fowl. Respir. Physiol. 15: 310-320. Bouverot, P., G. Hildwein and D. Le Golf (1974a). Evaporative water loss, respiratory pattern, gas exchange and acid-base balance during thermal panting in Pekin ducks exposed to moderate heat. Respir. Physiol. 21: 255-269. Bouverot, P., N. Hill and Y. Jammes (1974b). Ventilatory responses to CO2 in intact and chronically chemodenervated Pekin ducks. Respir. Physiol. 22: 137-156. Bouverot, P. (1978). Control of breathing in birds compared wit mammals. Physiol. Rev. 58: 604-655. Davenport, H.W., G. Brewer, A.H. Chambers and S. Goldschmidt (1947). The respiratory responses to anoxemia of unanesthetized dogs with chronically denervated aortic and carotid chemoreceptors and their causes. Am. J. Physiol. 148: 408-416. Dejours, P. (1957). Int6r~t m6thodologique de i'6tude d'un organisme vivant ~i la phase initiale de rupture d'un 6quilibre physiologique. C.R. Acad. Scl. (Paris) 245: 1946-1948. Dejours, P. (1962). Chemoreflexes in breathing. Physiol. Rev. 42: 335-358. Dejours, P. (1975). Principles of Comparative Respiratory Physiology. Amsterdam, North-Holland Publishing Company, 253 p. Downes, J.J. and C.J. Lambertsen (1966). Dynamic characteristics of ventilatory depression in man on abrupt administration of 02. J. Appl. Physiol. 21 : 447-453. Gandal, C. P. (1969). Avian anesthesia. Fed. Proc. 28: 1533-1534. Jones, D. R. and M. J. Purves (1970a). The carotid body in the duck and the consequences of its denervation upon the cardiac responses to immersion. J. Physiol. (London) 211 : 279-294. Jones, D.R. and M.J. Purves (1970b). The effect of carotid body denervation upon the respiratory response to hypoxia and hypercapnia in the duck. J. Physiol. (London) 211 : 295-309. Kuhlmann, W.D. and M.R. Fedde (1976). Upper respiratory dead space in the chicken: its fraction of the tidal volume. Comp. Biochem. Physiol. 54A: 409-411.

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Lahiri, S. (1976). Depressant effect of acute and chronic hypoxia on ventilation. In: Morphology and Mechanisms of Chemoreceptors, edited by A. S. Paintal. Delhi, Vallabhbhai Patel Chest Institute, pp. 138-146. Magno, M. (1973). Cardio-respiratory responses to carotid body stimulation with NaCN in the chicken. Respir. Physiol. 17: 220-226. Miller, M.J. and S.M. Tenney (1975). Hypoxiaoinduced tachypnea in carotid-deafferented cats. Respir. Physiol. 23:"31-39. Scheid, P., H. Slama and H. Willmer (1974). Volume and ventilation of air sacs in ducks studied by inert gas wash-out. Respir. Physiol. 21: 19-36. Tummons, J. L. and P.D. Sturkie (I 969). Nervous control of heart rate during excitement in the adult white L,:gborp cock. Am. J. Physiol. 216: 1437-1440.