Baroreflex involuntary FRANK Department

control diving M. SMITH

of arterial blood pressure during in ducks (Anas platyrhynchos var.) AND DAVID

of Zoology,

University

R. JONES of British

Columbia,

Smith, Frank M., and David R. Jones. Baroreflex control of arterial blood pressure during involuntary diving in ducks (Anas platyrhynchos var.). Am. J. Physiol. 263 (Regulatory Integrative Comp. Physiol. 32): R693-R702, 1992.-The dynamic role of arterial baroreceptors in control of mean arterial blood pressure(MAP), heart rate (HR), cardiac output (CO), hindlimb vascular (HLVR) and total peripheral (TPR) resistance responsesto forced dives wasinvestigated in acutely and chronically barodenervated ducks. To activate the baroreflex, the proximal end of one aortic nerve was stimulated electrically with bipolar electrodesthat had been implanted under pentobarbital sodium anesthesia.Predive nerve stimulation caused CO to fall (by reducing HR; stroke volume remained constant), producing a decreasein MAP to half the prestimulation level. During diving (for 2.5min periods) nerve stimulation did not affect HR and MAP after the first minute of submersion.Neither HLVR nor TPR contributed to the fall in MAP during aortic nerve stimulation before or during diving. The effects of nerve stimulation on HR and MAP were maintained to the end of dives in animals given 100% 0, to breathe before diving. In separateexperiments, increasing arterial chemoreceptorinput by perfusing one vascularly isolated carotid body with venous blood causeda reduction in the effects of aortic nerve stimulation on MAP. Arterial baroreceptors may thus act on HR to alter MAP early in the dive, but as the dive progressesthe baroreflex is attenuated by an increasein peripheral chemoreceptor drive. arterial baroreceptors; aortic nerve; bradycardia; total peripheral resistance; hindlimb vascular resistance; cardiac output; carotid body chemoreceptors BLOOD PRESSURE in dabbling ducks during forced submersion is maintained at ~80% of the level before submergence (4, 24) when the arterial baroreceptors are intact. When these receptors are deafferented, the mean arterial blood pressure (MAP) response to diving is variable, depending on the time elapsing between deafferentation and observation (8, 13, 16, 24). Although barodenervation experiments show that cardiovascular adjustments during diving can occur independently of baroreceptor input, such experiments do not provide a clear picture of the active role baroreceptor input may play in the development and maintenance of the diving responses. Millard (21) has suggested, on the basis of pharmacological evidence in birds, that the baroreflex operates during diving but proposed that its ability to respond to changes in MAP is reduced. Jones and West (15) and more recently Jones et al. (13) demonstrated, by electrically stimulating the cut central end of one aortic nerve, that the baroreflex is functional during diving in acutely (l-2 days) barodenervated ducks. This study also showed that the effects of aortic nerve stimulation on cardiovascular variables were attenuated as the dive progressed. Cardiovascular mechanoreceptors, peripheral arterial and central chemoreceptors, and exteroreceptors of the head are active during diving in birds (11), and interacARTERIAL

0363-6119/92

$2.00

Copyright

Vancouver,

British

Columbia

V6T 2A9, Canada

tions among several of these inputs can modify the expression of reflex circulatory adjustments (12). The strong chemoreceptor drive that develops during diving in ducks and that is largely responsible for the fall in heart rate (HR) (11) may interact with the baroreflex by overriding it centrally. The present experiments were therefore undertaken to investigate the role of input from arterial baroreceptors in cardiovascular control during diving. Electrical stimulation of one aortic nerve was used to simulate the input to the central nervous system that would result from changes in blood pressure at the baroreceptors. In addition, we examined the modulatory effects of altered arterial chemoreceptor input on baroreflex function in ducks during submergence and while spontaneously breathing air. Some studies of the cardiovascular responses to forced submersion have taken changes in HR as indexes of changes in cardiac output (CO), and resistance changes in one vascular bed (typically the hindlimb) to reflect changes in total peripheral resistance (TPR). Given that the effector limbs of the baroreflex are the same as those involved in the diving responses, there may be an uncoupling of the coordinated control of the cardiovascular system after baroreceptor loss that could render these assumptions invalid. To determine the veracity of these assumptions under our experimental conditions, we have simultaneously recorded HR and central blood flow to estimate CO and TPR, in addition to monitoring hindlimb blood flow to estimate hindlimb vascular resistance during diving in a subset of the animals in this study. MATERIALS

AND METHODS

A total of 18 female white Pekin ducks (Anas platyrhynchos var.) wasusedin theseexperiments. Animals werehousedsingly in cagesin a room with a 12-h light:12-h dark photoperiod at 20°C for the duration of the experiments. Food and water were available to the animals ad libitum. Experiment 1: Aortic Nerve Stimulation During Diving and Hypoxic Hypercapnia Preparation. In this set of experiments 12 animals [body

mass3.2 t 0.2 (SE) kg] were used.Unilateral barodenervation, electrodeimplantation, and the placementof a snarearound one aortic nerve for completing bilateral barodenervation were carried out under pentobarbital sodium (40 mg/kg, MTE Pharmaceuticals, Mississauga,Ontario) anesthesia.Animals were restrained on their backson an operating table, and the skin and underlying air sacmembranewere punctured in the midline to give accessto the cardiovascularstructures in the anterior thorax asdescribedby Smith and Jones(24). Surgical instruments and implanted deviceswere previously sterilized. The bipolar electrodes used for nerve stimulation in this study were similar to the “patch” electrodesdiscussedby Loeb and Gans (18) for recording electrical activity from the surface of skeletal musclein vivo. The free ends of the electrode leads

0 1992 the American

Physiological

Society

R693

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R694

BAROREFLEX

FUNCTION

were fitted with a connector that could be sutured to the skin. The left and right aortic nerves in ducks, carrying the only systemic arterial baroreceptor fibers in these animals (8, 23), course from the lower poles of the nodose ganglia of both vagus nerves to the root of the aorta. The stimulating electrode patch was secured with 6-O Vicryl sutures (Ethicon Sutures, Peterborough, Ontario) over the left aortic nerve where it ran within a sheet of fascia extending from the carotid artery to the dorsal body wall. A strain relief was formed by coiling the leads inside the chest and the remaining lead length and connector were brought out through the skin incision. After the electrodes were secured, ~3 mm of the same nerve was dissected free of surrounding tissue at a point 6-10 mm distal to the electrode site. Two ligatures of 5-O silk, separated by 2 mm, were tied around the exposed nerve segment, and this segment was then macerated thoroughly with a pair of forceps. This procedure was used to denervate the baroreceptors on the left side, while ensuring minimum disruption of the blood supply to the nerve at the electrode site proximal to the denervation ligatures. A snare of 4-O silk for later denervation of baroreceptors on the right side was then placed around the right aortic nerve (24), and the free end of the snarewas brought out through the skin incision. Three of the 12 animals in this study were fitted with ultrasonic Doppler blood flow probes (Parks Medical Electronics, Beaverton, OR) on the descendingaorta and the left brachiocephalic trunk while the chest was open, to estimate CO for calculating TPR. A plastic cuff of the sameinternal diameter as the probe on the left brachiocephalic trunk was placed in a correspondingposition on the right trunk. The probe leadswere formed into strain-relief loopsinside the chest and exteriorized through the skin incision. The air sacmembraneand skin were then repaired, and all electrical connectors and the free end of the snare were securedto the skin with sutures. The incision was then treated with a topical antibiotic (bacitracin, Glaxo Laboratories, Toronto, Ontario), and a gauze bandagewas applied. Intramuscular injections of a broad-spectrum antibiotic (250 mg Penbritin, Ayerst Laboratories, Montreal, Quebec) were given routinely after surgery. Forty-eight hours later an ultrasonic flow probe wasimplanted on the right or left ischiatic artery, and the right ulnar artery and vein were cannulated as describedby Smith and Jones (24). These operationswere carried out after local infiltration of lidocaine hydrochloride (2% Xylocaine, Astra Pharmaceuticals,Mississauga,Ontario). Animals were allowedto recover for a total of 7 days after the first operation before experiments were begun. Aortic nerve stimulation wasperformed with pairs of rectangular pulsesof opposite polarity (17) generatedby two stimulators (type S48, Grass Instrument, Quincy, MA), connected in tandem to a pair of GrassPSIU constant-current stimulus isolation units. The pulses[0.5-ms duration (25)] within each biphasic pair were separatedby 0.5 ms and were delivered in 20-s trains. The stimulus frequencieswere IO-20 Hz within trains, and intensity wasset in the rangeof 0.5-2 mA, dependingon the blood pressureresponsesof the animal. In our preparation, in which the relationship between the stimulating electrodesand the nerve could have changedfrom day to day over a period of severalweeks,it was not practical to usea fixed value of stimulus current for the duration of each experiment; we therefore used the principle of “biocalibration” describedby Swett and Bourassa(25) for setting stimulus parameters.At the beginning of the trials on each day, with the animals breathing air, stimulus parameterswere set to give a fall in pressureof 45-50% of the prestimulation value for the duration of a 20-s stimulus train. Theseparameterswere then usedfor all stimulation trials on that day. Experimental procedure. NQRMOXIC DIVES. After recovery from surgery, animals were loosely restrained ventral surface downward on a table. Phasic hindlimb blood flow was recorded

IN DIVING

on one channel of a Parks 806A Directional Doppler recording system,operating at a continuous-waveexcitation frequency of IO MHz. Mean blood flow wasobtained by electrically filtering the phasic flow signalwith a low-passfilter. The zero-flow referencebaselinewas set by observation of the flow trace during the prolongeddiastolic periodsobtained in the latter part of the first dive (10, 24). Hindlimb-vascular resistance(HLVR) was calculatedasthe quotient of MAP and meanischiatic flow. The samerecording system was used for monitoring blood flow in the descendingaorta and brachiocephalic trunk, the zero-flow baselinesagain being establishedin the long diastolic periods during the first dive (10,24). This zero-flow level wasconfirmed by comparisonwith that observedafter the death of the animal at the end of the experiment. CO wasestimatedby doublingthe flow obtained in one brachiocephalic trunk and adding this to the flow in the descendingaorta. TPR was then calculated as the quotient of MAP and CO. Phasic blood pressurewas recorded from the arterial cannula with a pressuretransducer (P23 Db, Statham Laboratories, Hato Rey, PR) fixed at the level of the heart. The transducer was calibrated against a mercury manometer. The arterial cannula-transducercombination had a dampednatural resonancefrequency ~40 Hz [determined by an impulsetest (20)], and the pressurerecording systemwas linear over the range of O-350 mmHg. MAP was obtained by electrically filtering the phasic pressuresignal with a low-pass filter. Phasic and mean blood pressureand flows as well as an electrocardiogram obtained from bipolar subcutaneous electrodes and an event marker signal were recorded on an eight-channel pen-recorder writing on rectilinear coordinates (Techni-Rite Electronics, Warwick, RI). The integrity of the right aortic nerve was checkedby monitoring the cardiac responseto transient hypertension produced by the intravenous injection of 25- to 50-pgbolusdosesof phenylephrine (Neo-Synephrine hydrochloride, Winthrop Laboratories, Aurora, Ontario) in avian saline. The responsesto forced submersion(“diving”) in unilaterally barodenervated animals were then tested by immersingthe headbeak down in a waterfilled funnel as described by Smith and Jones (24). A fully developedbradycardia during diving and a substantial fall in HR during pressor tests were taken as indications that the animals had recovered from the surgical procedures.Data recorded from the animals during these pressortests and dives were usedas control values for comparisonwith data from the sameanimalsafter completing bilateral barodenervation. Withdrawal of the snarearound the right aortic nerve destroyedthe nerve, thus removing all systemic arterial baroreceptor input. Pressortests were repeated24 h after nerve section. Successful barodenervation wasindicated by a lack of changein HR during pressortests. Before the first dive after bilateral barodenervation, aortic nerve stimulus current and frequency were set as previously described, and cardiovascular variables were recorded before and during a 20-s period of stimulation. These prestimulation recordingsconstituted the control valuesfor the effects of nerve stimulation predive and for comparisonwith values taken before stimulation in the dive. One minute after the end of the predive stimulation period the animals were dived. Then 20-s periods of aortic nerve stimulation were repeatedat 1 min and 2 min IO s after the start of the dive. At the end of the second stimulus period (2 min 30 s of submergence)the water was removed and the animalsresumedbreathing. This protocol was repeated at l- to 3-day intervals for up to 23 days after total barodenervation. Arterial blood samples(0.6 ml) were taken for blood gasanalysis before and at 2 min in the dive from animals before and after total barodenervation. Each samplewasreplacedwith an equivalent volume of avian saline.No hematocrit measurements were made in these experiments, but the total blood volume

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BAROREFLEX

FUNCTION

IN DIVING

R695

withdrawn on any day (1.2 ml) was ~0.3% of the whole blood volume of the animal. Arterial oxygen tension (Pao,), carbon dioxide tension (Pa co,), and pH (pH,) were measured in a blood gas analyzer (IL 13, Instrument Laboratories, Lexington, MA) with electrodesmaintained at the animal’s body temperature (41°C) by a thermostatted water jacket. Electrodes were calibrated with known gas mixtures and standard buffer solutions before each blood sample. At the end of the period of experiments each animal was deeply anesthetized with pentobarbital sodium and received 1,000 IU of heparin intravenously. The left vagus nerve was exposed and sectioned cranial to the electrode site, and the aortic nerve was stimulated using the sameparametersas during the last dive. No cardiovascular responsesto stimulation occurred in any of the animals, indicating that the electrodes activated afferent and not efferent vagal elements.The ischiatic artery flow probe was calibrated in situ by cannulating the artery distal to the probe site and withdrawing blood through the vesselin the probe at known flow rates with the aid of a syringe pump. The animal was then killed with an additional dose of pentobarbital sodium, and the zero-flow Doppler frequency shift, obtained by recording the signal from the in situ probe after death, provided the samebaselinereferenceasat end diastole during a dive. The flow probeson the descendingaorta and the brachiocephalic trunk were calibrated in vitro by removing each probe with its enclosedvesselsegment,cannulating both ends of the segmentand running heparinized blood through vesseland probe at known rates with a syringe pump. Calibration curves of volume flow against pen displacement were linear. The zero-flow baselinesfor theseprobes were also the sameafter death as during acetylcholine-induced and dive bradycardia. Barodenervation wasconfirmed, and the relationship of the electrodes to the aortic nerve was determined by microdissection,during postmortem examination. HYPEROXIC DIVES. One to two weeksafter completebarodenervation, four ducks were given 100% oxygen gas to breathe before diving to reducechemoreceptorinput (6,19). After 4 min of oxygen exposurethe aortic nerve was stimulated as outlined above to obtain predive responses.Animals were then dived for 2.5 min, and aortic nerve stimulations were repeated.Two hyperoxic dives on different days were performed on eachanimal. Arterial blood samplesdrawn just before and at 2 min after the start of the dive were analyzed for blood gaslevels. EXPOSURE TO HYPOXIC HYPERCAPNIA. The aortic nerves of three of the bilaterally barodenervated animalswere stimulated to give a fall in MAP of -50% while the animalsbreathed room air, and blood sampleswere taken for blood gasanalysis.These animalswerethen given a hypoxic (PO, 79 mmHg)-hypercapnic (Pco, 48 mmHg) gas mixture to breathe for 2 min, and aortic nerve stimulation was repeated. Arterial blood sampleswere withdrawn just before the nerve wasstimulated. This procedure was repeated in two of the three animals 24 h later, making a total of five sets of paired normoxic and hypoxic-hypercapnic responses.

facing toward the chemoreceptorsand the heart. Stimulating electrodes were placed on the left aortic nerve as described above, and the nerve was then sectioneddistal to the electrode site. The right vagus was located near the thoracic inlet, infiltrated with lidocaine, ligated, and cut to denervate baroreceptors and carotid chemoreceptorson the right side. The air sac membraneand the skin incision were then repaired after passing the electrodeleads,carotid cannula, and occludertube out of the chest. The right ulnar artery and vein were exposed and cannulated, and the incision wasclosed.The animal wasturned to an upright position on the operating table and lightly restrained. Venous and carotid artery cannulaswere led to a 50-ml syringe in an infusion-withdrawal pump (Harvard Apparatus, South Natick, MA) via an arrangementof stopcocksto which a pressuretransducer (Narco Biosystems, Houston, TX) and a saline-filled syringe for flushing were also attached. The pump syringe was kept at body temperature by a heating coil connected to a recirculating constant-temperature water bath. The ulnar arterial cannula wasconnectedto a secondpressuretransducer for recording arterial blood pressure. The animal was allowed to recover for 3-4 h before the experiment beganand was consciousthroughout the experiment. All incision sites were periodically infiltrated with local anesthetic throughout the recovery period and during the experiment. The parameters for aortic nerve stimulation were adjusted as describedabove, while the innervated left carotid body wasperfusedwith arterial blood from the heart (autoperfused). The blood pressureand HR responsesto aortic nerve stimulation during autoperfusion were taken ascontrol responsesfor theseexperiments. About IO ml of venous blood was withdrawn into the infusion pump syringe (no change in arterial pressureresulted from this procedure), and the carotid artery occluder wasthen inflated to vascularly isolate the carotid body from the arterial circulation. This isolation was verified by the immediate loss of pulsatile pressurefrom the carotid cannula and by the subsequentgradual decreasein mean pressureas the small volume of blood trapped in the carotid region drained through the venous outflow. Perfusion of the carotid body with venousblood from the syringe pump wasthen begun,the flow rate being set to produce the samemean pressurein the isolated carotid region as during the previous autoperfusion. The aortic nerve was stimulated again after l-2 min of perfusion with venous blood, and the circulatory responseswere recorded. Autoperfusion was then reestablished.Two or three trials with venous perfusion were done in each animal at 15 to 20-min intervals, then the responsesto aortic nerve stimulation were obtained while the carotid body was perfusedfrom the syringe with arterial blood obtained from the carotid artery. This served to evaluate the effects of the carotid body perfusion processitself on the responsesto aortic nerve stimulation. Samplesfor blood gasanalysis were withdrawn from the pump syringe during perfusion of the carotid body with both arterial and venous blood.

Experiment 2: Aortic Nerve Stimulation During Carotid Body Perfusion

Data Analysis

Six femaleducks (meanbody mass3.0 t 0.3 kg) were usedin separateacute experiments to test the effects of peripheral arterial chemoreceptorstimulation on the operation of the baroreflex. Surgical preparation for these experiments was performed under pentobarbital anesthesia,using a procedure similar to that describedby Jones et al. (12) for vascular isolation and perfusion of the carotid body. All side branches of the left carotid artery in the area of the carotid body were ligated, and a pneumatic occluder was placed on the carotid artery near its junction with the left brachiocephalictrunk. The carotid artery was cannulated distal to the carotid body, with the cannula tip

The changesin cardiovascular variables as a result of total barodenervation, the effects of diving, and the effects of stimulation of the aortic nerve on these variables before and during diving were analyzed using a blocked analysis of variance (ANOVA) model (26,27). For the purposesof this analysis,each animal was treated as one block (27) with one observation per animal per day group. On any day before or after barodenervation the predive and dive valuesof each cardiovascularvariable were analyzed as successivetreatments with time, and an F value wasobtained. If this value wassignificant, pair-wise comparisonsof meansweredone with Tukey’s multiple meanscomparison test. In an analysis of the effects of stimulation of the

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R696

BAROREFLEX

FUNCTION

IN DIVING

aortic nerve, the absolute value of each variable during stimulation was compared only with the prestimulation value for that dive time. Longitudinal analyses of variations over the days after denervation were performed using a similar ANOVA model to compare values obtained at the same dive times on different days. Values recorded before and during dives before denervation were treated as control values for comparison with values taken at those dive times on all days after nerve section. Significant differences between blood gas values before and after barodenervation, and before and during diving were estimated using paired t tests. All statistical tests were evaluated at a P level of 0.05, and all values are expressed as means t SE. For convenience in describing the results of these experiments in the text, some data are presented in the form of relative changes (expressed as per cent changes or proportional changes) for clarity. These relative changes represent statistically significant differences between absolute values. For the purposes of this study, the term predenervation refers to animals in which the left aortic nerve had been sectioned and the right aortic nerve left intact. The term barodenervation refers to the sameanimals after destruction of the right aortic nerve to completebilateral aortic nerve section.

min and by the end of a 2-min 30-s dive had dropped by 83% (Fig. 2). Over the first minute of these dives HLVR increased by 5.5 times and by end dive was 6.5 times the predive value (Fig. 3). Blood pressure was, however, not maintained (Fig. I), falling by 18% at 1 min and by 24% after 2 min 30 s of diving. For the first 4 days after complete barodenervation, the cardiac chronotropic response was decreased during submersion, HR at both 1 min and 2 min 30 s in the dive being more than twice the rate at corresponding times in control dives. After 4-6 days without baroreceptors the dive bradycardia deepened until HR fell to the same level as in dives before barodenervation. The mean HLVR response to diving, up to 14 days after nerve section, was the same as before denervation (Fig. 3). After 14 days the HLVR response was reduced significantly, resistance rising only 4.2 times the predive value, in contrast to increases of 5.5 times or more before and in the first 2 wk after barodenervation. End-dive MAP in barodenervates fell by 25-58%, the degree of hypotension increasing with time after barodenervation (Fig. 1). This progressive trend toward greater dive hypotension reflected the inRESULTS crease in intensity of bradycardia. That is, while the Aortic Nerve Stimulation During Diving bradycardia was reduced in the first few days after baroand Hypoxic Hypercapnia denervation, dive MAP in this period was significantly The average slope of the relationship between HR and higher than when the animals were dived >l wk after a phenylephrine-induced pressure rise was -1.0 t 0.1 nerve section; in the latter case HR fell to the same level beats mine1 . mmHg-l in unilaterally barodenervated as in dives before barodenervation, and dive hypotension ducks. After destruction of the remaining aortic nerve was maximal. there was no significant change in HR in response to Responses to stimulation of the aortic nerve before and transient hypertension. Figures l-3 represent data from during diving are represented by the solid bars accompa12 ducks before and up to 23 days after total barodenernying the open bars in Figs. l-3. Predive stimulation vation, which was produced by withdrawal of the snare induced mean HR reductions of 35-42% on all days postaround the right aortic nerve. MAP in animals breathing denervation (Fig. 2). HLVR, however, responded signifiair before submergence did not change significantly, with cantly to predive nerve stimulation only at 2 and 9-10 the exception of a transient increase at 9-10 days after days postdenervation (Fig. 3). These changes in HR and denervation, throughout the period of observation (Fig. peripheral resistance produced a uniform drop in blood 1). HR (Fig. ~2) was also not significantly affected by the pressure of 46% during predive stimulations performed denervation procedure with the exception of a rise in HR up to 14 days after nerve section; stimulation was slightly on days 3 and 4 postdenervation. HLVR (Fig. 3) was less effective in reducing resting MAP in the last day unaffected by barodenervation. group (days 15-23, Fig. 1). Normoxic dives. During dives made in animals before When the aortic nerve was stimulated early in the dive barodenervation, HR fell by 78% of the predive value at 1 in barodenervated animals, a greater proportional HR l

300Predive

O-

*

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Fig. 1. Mean arterial blood pressure (MAP) responses to aortic nerve stimulation before diving (predive) and at 1 min and 2 min 10 s during dives. Each bar represents mean of 12 observations in 12 animals. Error bars represent SE. Data recorded from unilaterally barodenervated animals are represented by hatched bars (preden). For each day group after bilateral barodenervation, values are presented just before stimulation (open bars) and during stimulation (solid bars). Number of days after denervation for which data are included in each day group is indicated under bars. * Prestimulation values significantly different from predive for that day group. 0 Values l in denervates significantly different from predenervation value at that dive time. + Significant effects of stimulation in each day group and at each dive time. Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (128.205.114.091) on December 1, 2018. Copyright © 1992 the American Physiological Society. All rights reserved.

BAROREFLEX

FUNCTION

Predive

100

R697

IN DIVING

Dive

2~10

Dive

OPre den

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2

3

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Fig. 2. Heart rate (HR) responses to aortic nerve stimulation before and during diving. Bar labels and symbols are same as for Fig. 1.

response (42-60% reduction) was obtained than could be produced by the same stimulus given predive (35-40% reduction, Fig. 2). The extent of stimulation-induced decrease in HR at this dive time was not dependent on the HR just before stimulation; the proportional effect of stimulation was the same when dive HR was high (l-4 days postdenervation) as when the dive bradycardia was more fully developed (5-23 days postdenervation). HLVR was not significantly affected by stimulation of the aortic nerve in the dive (Fig. 3) and thus did not contribute to stimulus-induced circulatory changes. Despite the enhanced cardiac response to stimulation, the resulting fall in MAP was less than that generated by the same stimulus given predive. Aortic nerve stimulation late in the dive was effective on HR only at 15-23 days after barodenervation. Even at this time the proportional reduction in HR was less than that occurring earlier in the dive or at predive. Stimulation had no significant effect on HLVR at any time after barodenervation. As a consequence of the reduction in cardiac response and the lack of a peripheral vascular response, stimulation of the aortic nerve had no significant effect on end-dive blood pressure in ducks at any time after barodenervation. Predive Pa o (85.3 t 1.6 mmHg), Pace (26.9 t 2.2 mmHg), and pfia (7.488 t 0.040) in unilaterally barodenervated ducks were not significantly affected by complete bilateral barodenervation (Pa0 86.5 t 0.8 mmHg, Pace 22.7 t 1.0 mmHg, pH, 7.469 $ 0.010). The fall in Pao2 during diving was the same in ducks after total barodeni ervation (to 59.0 t 1.5 mmHg) as it was in unilaterally barodenervated ducks (to 53.4 t 3.1 mmHg). The changes in Pa co, and pH, during diving were also not 7

7oc

affected by total barodenervation. Paco2 rose to 38.3 t 2.7 mmHg in unilateral denervates and to 36.2 t 1.4 mmHg in bilateral denervates after 2 min of submergence. pH, was 7.383 t 0.041 in unilateral denervates and 7.333 t 0.014 after total barodenervation at 2 min in the dive. Estimates of predive and dive cardiac stroke volume (SV) and CO as well as calculated HLVR and TPR are presented in Figs. 4 and 5 for three animals before and after total barodenervation. Although these animals were part of the previous experimental group, the data reported here were recorded during separate dives, and sometimes on different days, than the previous results. In addition, successful recording from the central flow probes could not be extended beyon .d 16 days a.fter denervation. For these reasons, the day groups are different for the data in Figs. 4 and 5 than in the Figs. l-3, although data analysis was carried out in the same manner. Before complete barodenervation, mean CO decreased by 84% in a 215-min dive, while SV was unchanged from the predive level (Fig. 4). TPR increased by 4.9 times at endldive, while HLVR rose by 5.8 times (Fig. 5). Complete barodenervation caused no changes in CO, SV, TPR, or HLVR in these animals, while stimulation of the aortic nerve before diving affected only CO significantly, reducing this by 25-35% (Fig. 4). During diving, CO fell by 70-87%, with greater decreases in CO occurring as more time elapsed after denervation. The effects of stimulation on CO during diving followed the same pattern as the HR responses to stimulation in the whole group of animals. Stimuli delivered at 1 min in the dive had the same proportional effect on CO as at predive on most days after barodenervation, but, at end dive, stimulation produced no significant change in CO at any time after r I

Predive

2110

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609 ,' 50 cl

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Fig. 3. Hindlimb vascular resistance (HLVR) responses to aortic nerve stimulation before and during diving. Bar labels and symbols are same as for Fig. I. Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (128.205.114.091) on December 1, 2018. Copyright © 1992 the American Physiological Society. All rights reserved.

R698

BAROREFLEX

FUNCTION

Predive

" Pre den

1

2

2.5-

I:00

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4

5-8

9-16

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1

IN DIVING 2:lO Dive

Dive

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I.00 Dive

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&

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Pre den

1

2

3 Day

Fig. 4. Cardiac output (CO) and stroke volume (SV) responses to aortic nerve stimulation before and during diving for 2 dives in each of 3 animals. Notation, bar shading, and symbols are same as for Fig. 1.

barodenervation (Fig. 4). Dive SV was not altered significantly from the predive level, nor was SV significantly influenced by aortic nerve stimulation in the dive at any time after denervation. Figure 5 shows that, while the change in TPR in the dive was reduced for the first 4 days after denervation, this response returned to the predenervation magnitude after 5 days. Baroreceptor removal did not, however, alter the magnitude of the HLVR response to diving over the entire 16-day period of the experiments in this group of animals, nor did HLVR or TPR respond to aortic nerve stimulation during diving. Hyperoxic &es. One to two weeks after total baroreceptor denervation, 4 of the 12 animals were given 100% oxygen to breathe before diving. This produced Pao, of 359.6 t 7.1 mmHg but did not significantly affect Pace (24.7 t 2.4 mmHg) or pH, (7.447 t 0.038). Blood samples taken during dives after exposure to oxygen (hyperoxic dives) showed that Pao decreased significantly to 264.0 2

Predive

+ 29.1 mmHg, but remained significantly above the Pao level measured in animals breathing air. Pace rose sig! nificantly to 48.2 t 5.3 mmHg in hyperoxic dives, while pH, fell significantly to 7.218 t 0.044. Stimulation of the aortic nerve in hyperoxic animals before diving provoked a 42% fall in HR but no change in HLVR, and a 54% decrease in MAP was observed (Fig. 6). By 1 min in the dive, HR in hyperoxic animals had decreased by 43%) while HLVR rose by 3.3 times and blood pressure was maintained. Nerve stimulation at this time produced no change in HLVR but evoked a 61% drop in HR, leading to the same fall in MAP as observed during predive stimulation. By the end of the dive HR had fallen by 66% with no further increase in HLVR above that seen at 1 min in the dive, and end-dive arterial pressure was not significantly different from that at 1 min of diving. However, end-dive aortic nerve stimulation reduced HR by half with no effect on HLVR, and produced a 42% fall in blood 2~10 Dive

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Fig. 5. Total peripheral resistance (TPR) and HLVR responses to aortic nerve stimulation before and during diving for 2 dives in each of 3 animals. Notation, bar shading, and symbols are same as for Fig. 1. Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (128.205.114.091) on December 1, 2018. Copyright © 1992 the American Physiological Society. All rights reserved.

BAROREFLEX

FUNCTION

R699

IN DIVING

duced a significant decrease of 28% in HR, to 239 t 16 beats/min. This value was also significantly above the HR obtained during aortic nerve stimulation in normoxia. Upon return of the animals to air breathing, aortic nerve stimulation again produced the same changes in HR and MAP as those obtained in the normoxic state before hypoxic hypercapnia. Aortic Nerve Stimulation Carotid Body Perfusion

‘T .-= 2500 E. -$ 200 z 150 -

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Pre dive

1.oo dive

2 :I0 dive

Fig. 6. Cardiovascular responses to aortic nerve stimulation before and during hyperoxic dives, as indicated by bar labels at bottom. MAP, HR, and HLVR values are given for 8 observations in 4 animals. Error bars represent SE. * Significant changes from predive. + At given dive time, values during stimulation (solid bars) were significantly different from prestimulation values (open bars).

pressure, to the same level as during stimulation predive and at 1 min in the dive. Exposure to hypoxic hypercapnia. Pao was 87.9 t 1.6 mmHg, Pace, was 25.5 t 1.8 mmHg, and pH, was 7.532 t 0.023 in ducks breathing air just before exposure to an hypoxic-hypercapnic gas mixture. Whole body hypoxic hypercapnia was produced in three animals by giving them a high PCO~-~OWPO, mixture to breathe for 2 min. The resulting Pa co was 62.3 t 1.9 mmHg, Pace was 43.8 t 4.3 mmHg, and pH, was 7.349 t 0.040; all changes in blood gas variables were significant. MAP before hypoxic hypercapnic exposure was 189 t 7 mmHg; during aortic nerve stimulation this fell 49% to 93 t 9 mmHg. HR in normoxia was 275 t 11 beats/min, falling to 150 t 15 beats/min during nerve stimulation, a significant decrease of 45%. MAP after 2 min of hypoxic hypercapnia was 203 t 11 mmHg, a value not significantly different from that obtained when the animals were breathing air. Repeat stimulation of the aortic nerve during hypoxic hypercapnia caused a 25% fall in MAP, to 152 t 9 mmHg, a value significantly greater than that during stimulation in normoxic animals. HR in hyperoxic hypercapnia was 321 t 14 be a t s/ min, a value not significantly different from the normoxic HR. Aortic nerve stimulation pro-

During

Mean Pao in the experimental animals 3-4 h after surgery, when the experiments were begun, was 92.1 t 2.1 mmHg; Pace was 19.2 t 2.9 mmHg, and pH, was 7.536 t 0.009. The’spontaneous ventilation rate during autoperfusion of the carotid body was 18.7 t 1.5 breaths/min. Stimulation parameters for aortic nerve activation were set to bring MAP down to approximately half the prestimulation value during autoperfusion, from 175 t 6 to 92 + 5 mmHg (mean decrease 47%); HR fell from 301 t 12 tO 180 t 15 beats/min during stimulation, a drop of 40%. When the carotid body was perfused with venous blood [venous oxygen tension (Pvoz) 45.5 2 3.2 mmHg, venous carbon dioxide tension (PvCo,) 48.5 t 2.9 mmHg, and venous pH (pH,) 7.347 t 0.0151, no significant change in MAP (178 t 7 mmHg) or HR (322 t 13 beats/ min) occurred, but when the aortic nerve was stimulated with the same parameters used during autoperfusion only a 33% fall in MAP, to 120 & 5 mmHg, resulted; this value was significantly higher than the corresponding value during autoperfusion. HR fell by 22% during stimulation, to 249 t 11 beats/min; this value was also significantly greater than the corresponding value during autoperfusion. That the carotid body chemoreceptor output increased during perfusion with venous blood was indicated by an elevation in the ventilation frequency to 25.5 t 1.1 breaths/min. When the carotid body was perfused with arterial blood from the syringe, ventilation rate, MAP, and HR were the same as during autoperfusion, and aortic nerve stimulation produced the same decrease in MAP and HR as did stimulation during autoperfusion. Peripheral resistance was not monitored in these animals, because this limb of the baroreflex did not contribute to blood pressure changes induced by aortic nerve stimulation during diving. DISCUSSION

In ducks, electrically stimulating one aortic nerve to simulate an increase in baroreceptor input causes a reduction in HR and CO, without affecting peripheral resistance, in the first minute of diving. The net result is a fall in MAP during stimulation. These circulatory effects of baroreceptor nerve activation are not constant throughout the dive; MAP is unaffected by subsequent nerve stimulation as the dive progresses beyond the first minute. However, when the input from arterial chemoreceptors in a dive is reduced by giving the animals oxygen to breathe before submersion, baroreflex influences on the heart and MAP remain effective to the end of 2-min 30-s dives. On the other hand, in ducks breathing air, baroreflex effects on MAP are sharply reduced by increases in peripheral arterial chemoreceptor drive. The

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R700

BAROREFLEX

FUNCTION

attenuation of baroreflex function during diving therefore appears to stem, at least in part, from increased chemoreceptor drive resulting from the progressive hypoxic hypercapnia that develops as the dive progresses. Stimulation of the aortic nerve early in the dive in acutely and chronically barodenervated ducks in the present study produced a depressor response expressed entirely through baroreflex action on CO; this must have been mediated by a fall in HR, because SV did not change during stimulation. The lack of baroreflex effects on CO, HR, or MAP after the first minute of the dive confirms the findings of Jones and West (15) and Jones et al. (13) in acutely barodenervated ducks. It is possible that the reduction in effectiveness of the baroreflex was apparent and not real, because the efferent limbs of the baroreflex (CO and peripheral resistance) are both reset independently of the baroreflex to levels close to their operating limits during the dive so that even a fully operative baroreflex may then be unable to influence MAP. In other words, during diving, HR may be forced to its lower limit and peripheral resistance to its upper limit; under these circumstances the baroreflex might not be able to further alter these effecters. However, this was not the case in the present experiments, as end-dive HR could be forced lower than the levels reported here by increasing the aortic nerve stimulus intensity, so the system had not reached its limits. The important point is that the relative effectiveness of the baroreflex on MAP, for the same level of simulated baroreceptor input as before diving, was drastically reduced by the end of a 2.5min dive. Additionally, the proportional effect of stimulation early in the dive was the same when HR was high (in the first few days after barodenervation) as when HR was low (2-3 wk after denervation), so this effect was not dependent on the starting HR. Our contention that the depression of baroreflex function during diving was due to strong chemoreceptor activation is supported by the results of experiments in which the baroreflex was activated under conditions of altered chemoreceptor drive. The efficacy of aortic nerve stimulation in altering both HR and MAP was maintained throughout dives in which peripheral chemoreceptor drive was reduced by arterial hyperoxia (Fig. 6), in contrast to the end-dive attenuation of the baroreflex seen in dives in which chemoreceptor drive reached high levels (Figs. I and 2). The residual end-dive bradycardia in oxygen-loaded ducks (Fig. 6) likely resulted from a moderate elevation of peripheral chemoreceptor drive caused by increased Pace, and pH,. Additional evidence for partial baroreflex occlusion by chemoreceptor input is provided by the finding that when blood gases in spontaneously breathing animals were set artificially at levels similar to those found during diving, the baroreflex was also attenuated by about the same degree as in ducks during diving. However, this could have resulted from the effects of hypoxic hypercapnia on the various central and peripheral neural elements involved in MAP control. To address this problem we limited hypoxic hypercapnic conditions solely to one set of peripheral chemoreceptors in an otherwise normoxic animal, in the carotid body perfusion experiments. Under these conditions, there was an ele-

IN DIVING

vated chemoreceptor drive during perfusion of the carotid body with venous blood, indicated by an increase in ventilatory rate. We attribute the decreased effects of aortic nerve stimulation on HR and MAP to partial inhibition of the baroreflex in the brainstem by increased peripheral chemoreflex activity. This was not an artifact of the perfusion procedure, because carotid body perfusion with arterial blood in a normoxic animal had no such effect on the baroreflex. The mechanism for this chemoreceptorbaroreceptor interaction is not known but may involve the algebraic summation of an inhibitory and an excitatory input on the final common pathway for cardiac control, as discussed by Abboud and Thames (1). When carotid chemoreceptor drive was elevated during spontaneous breathing, ventilation frequency was also increased, and this may have been an additional factor in reducing the effectiveness of baroreceptor nerve stimulation on blood pressure. Animals were apneic during diving, however, and reduced chemoreceptor drive in hyperoxic dives was accompanied by enhanced depressor and HR responses to aortic nerve stimulation; these responses were attenuated in dives after breathing air. These results suggest that the attenuation of the baroreflex in ducks in the present study may be independent of ventilation. Taken together, the results of these experiments provide strong evidence that chemoreceptor-baroreceptor interaction can occur during diving, reducing the effectiveness of baroreceptor control of MAP. This situation appears to be analogous to that in rabbits during arterial hypoxia, in which the gains of the baroreceptor-heart period and baroreceptor-blood pressure reflexes were reduced (7). These authors reached this conclusion only after they had done a thorough analysis of the baroreflexindependent resetting of cardiovascular variables during hypoxia. Such resetting also occurs during diving in barodenervated ducks because the cardiovascular responses during submersion are still expressed in the absence of arterial baroreceptors, even though dive MAP is not as well maintained as in baroreceptor-intact animals (Ref. 24 and the present study). Iriki and Korner (7) also reported that the gain of the baroreflex-sympathetic nerve activity reflex for the control of some vascular beds was enhanced during hypoxia. In our experiments, this was not apparent for either HLVR or TPR; stimulation of the baroreceptor input pathway with an intensity that produced substantial changes in blood pressure in normoxic animals before diving (Fig. 1) produced no significant changes in vascular resistance either before or during diving. In contrast to the results of the present study, Angel1 James et al. (2) reported that, in anesthetized seals in which diving responses were elicited by filling a face mask with water, the gain of both baroreceptor-MAP and baroreceptor-heart period reflexes was greater than in the predive state. The differences between these findings and those of the present study may be due to fundamental differences between mammalian and avian blood pressure control systems, but could also be due to differences in the method used to invoke the baroreflex. Angel1 James et al. (2) manipulated pressure at the receptors by injecting phenylephrine intravenously during diving, a method

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BAROREFLEX

FUNCTION

IN DIVING

R701

that may elicit an exaggerated cardiac response. was absent there was little change in the way that short Responses of CO to stimulation of the aortic nerve volleys of baroreceptor information was processed by the central neural circuitry of the baroreflex. No attempt was paralleled those of HR before and during diving. Given made to explore the effects of stimulus patterns that rethe lack of significant TPR or HLVR changes during sembled the phasic baroreceptor nerve discharge patterns stimulation either before or during diving, we conclude seen in vivo, because we were attempting to simulate a that all changes in MAP as a result of baroreflex activaconstant baroreceptor input over a wide range of HR. tion in our experiments were due to changes in HR. That The Pao, and pH, values in unilaterally and bilaterally is, if pressure in baroreceptor-intact ducks were to be barodenervated ducks are lower than those reported by altered over the same range as that simulated by aortic nerve stimulation in this study, the baroreflex would act other workers for chronically barodenervated ducks (16) via HR to control MAP. Changes in CO in dives before but the Pace levels in the present study are similar to those reported by Bouverot et al. (3) for restrained intact and after barodenervation in the subgroup of animals ducks. The values reported here are not likely to be assofitted with central flow probes followed a similar pattern ciated with respiratory alkalosis, because ventilatory freto HR changes during diving in the whole group. There was also no change in end-dive SV from the predive level. quency was unaffected by baroreceptor nerve interrupWe conclude that the cardiac chronotropic response dur- tion and Pa o, was not elevated in resting normoxic ing diving is therefore a reliable index of the behavior of barodenervates. Altered Pa co, and pH, may, however, have resulted from a disturbance in the ionic balance of CO. The lack of a significant change in SV during diving blood plasma as a consequence of barodenervation. Artein the present experiments extends the findings of earlier rial baroreceptors are an integral part of the ion regulastudies in intact ducks (9,14) to barodenervated animals. tion and acid-base control systems in the body, and The responses of TPR during diving were different disruptions in baroreflex function will affect the homeothan the HLVR responses in the same animals over the static abilities of these regulatory mechanisms (5). first 4 days after denervation. Dive HLVR after denervaIn conclusion, our experiments indicate that if the tion rose by the same factor as before denervation, but the baroreceptors see a sufficient change in blood pressure postdenervation response of TPR to diving was signifinear the start of a forced dive the baroreflex modulates cantly reduced compared with the response before denerthe chemoreceptor-generated fall in CO to help maintain vation. Obviously not all vascular beds responded with MAP in the face of increasing peripheral vasoconstricthe same degree of vasoconstriction as the hindlimb in tion. However, baroreceptor control of the circulation will dives made within a few days of barodenervation. This not be effective after the first minute of diving because of finding parallels the results of Butler and Jones (4), who an attenuation of baroreflex effects on HR and MAP by reported that in intact animals resistance to blood flow in progressively increasing peripheral chemoreceptor drive. the hindlimb during diving increased more, proportionally, than did resistance in vascular beds supplied by the This research was supported by grants to D. R. Jones from the and Natural Sciences and Engicarotid arteries. Additional evidence of this is provided by British Columbia Heart Foundation neering Research Council of Canada. Jones et al. (9), who reported nonuniform flow redistriAddress for reprint requests: F. M. Smith, Dept. of Anatomy, Faculty bution throughout the body in force-dived ducks, pointof Medicine, Dalhousie University, Halifax, Nova Scotia B3H 4H7, ing up the differential nature of regional vasoconstriction Canada. during diving in these animals. From 5 to 16 days post- Received 3 April 1991; accepted in final form 19 March 1992. denervation the TPR response to diving in the present REFERENCES experiments followed the same pattern as the predenervation response, whereas in the same animals the propor1. Abboud, F. M., and M. D. Thames. Interaction of cardiovastional HLVR response was unchanged from the predencular reflexes in circulatory control. In: Handbook of Physiology. The Cardiovascular System. Peripheral Circulation and Organ ervation response over the entire 16-day observation BZood FZow. Bethesda, MD: Am. Physiol. Sot., 1983, sect. 2, vol. 3, period. Thus, in barodenervated animals during the first pt. 2, chapt. 19, p. 675-753. few days after denervation, changes in HLVR during div2. Angel1 James, J. E., M. De B. Daly, and R. Elsner. Arterial ing are not reliable indexes of changes in TPR. baroreflexes in the seal and their modification during experimental dives. Am. J. Physiol. 234 (Heart Circ. Physiol. 3): H730-H739, Section of one aortic nerve did not appear to affect the 1978. sensitivity of the baroreceptor-HR reflex, because the av3. Bouverot, P., N. Hill, and Y. Jammes. Ventilatory responses erage slope of the pressure-HR relationship (-1.0 to COZ in chronically chemodenervated Peking ducks. Respir. beats min-l lmmHg-I) was the same as that in ducks Physiol. 22: 137-156, 1974. with both aortic nerves intact (-1.2 beats. min-l mm4. Butler, P. J., and D. R. Jones. The effect of variations in heart rate and regional distribution of blood flow on the normal pressor Hg-l; Ref. 24). These values are also similar to that reresponse to diving in ducks. J. Physiol. Lond. 214: 457-479, 1971. ported by Millard (- 1.17 beats min-l . mmHg-l, Ref. 5. Ferrario, C. M., A. Tramposch, Y. Kawano, and K. B. 21). It is not clear why the sensitivity of this reflex was Brosnihan. Sodium balance and the reflex regulation of barorenot reduced in unilaterally barodenervated animals; in ceptor function. CircuZation 75, Suppl. I: 1141-1148, 1987. mammals sequential sectioning of the nerves carrying 6. Gabbott, G. R. J., and D. R. Jones. Habituation of the cardiac response to involuntary diving in diving and dabbling ducks. J. baroreceptor afferents causes step reductions in the overExp. Biol. 131: 403-415, 1987. all gain of the reflex (22). Proportional cardiovascular 7. Iriki, M., and P. I. Korner. Central nervous interactions beresponses to aortic nerve stimulation in ducks were contween chemoreceptor and baroreceptor control mechanisms. In: sistent with time after barodenervation, implying that Integrative Functions of the Autonomic Nervous System, edited by even though tonic baroreceptor input to the brain stem C. M. Brooks, K. Koizumi, and A. Sato. Tokyo: Univ. of Tokyo l

l

l

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R702

BAROREFLEX

FUNCTION

Press, 1979, p. 415-426. D. R. Systemic arterial baroreceptors in ducks and the consequences of their denervation on some cardiovascular responses to diving. J. Physiol. Lond. 234: 499-518, 1973.

8. Jones,

Jones, Clark.

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Regional distribution

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and

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of blood flow during diving in the

duck (Anas platyrhynchos). Jones,

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D. R., and W. K. Milsom. Peripheral receptors affecting breathing and cardiovascular function in non-mammalian vertebrates. J. Exp. Biol. 100: 59-91, 1982. Jones, D. R., W. K. Milsom, and G. R. J. Gabbott. The role of central and peripheral chemoreceptors in diving responses of ducks. Am. J. Physiol. 243 (Regulatory Integrative Comp. Physiol. 12): R537-R545, 1982. Jones, D. R., W. K. Milsom, F. M. Smith, N. H. West, and 0. S. Bamford. Diving responses in ducks after acute barodenervation. Am. J. Physiol. 245 (Regulatory Integrative Comp. PhysioZ. 14): R222-R229, 1983. 14. Jones, D. R., and M. J. Purves. The carotid body in the duck

and the consequences of its denervation upon the cardiac responses to immersion. J. Physiol. Lond. 211: 279-294, 1970. 15. Jones, D. R., and N. H. West. The contribution of arterial chemoreceptors and baroreceptors to diving reflexes in birds. In: Respiratory Function in Birds, Adult and Embryonic, edited by J. Piiper. Berlin: Springer-Verlag, 1978, p. 95-104. 16. Lillo, R. S., and D. R. Jones. Control of diving responses by carotid bodies and baroreceptors in ducks. Am. J. Physiol. 242 (Regulatory Lilly, J. Stimulation

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C. The balanced pulsed-pair waveform. In: ELectrical of the Brain, edited by D. E. Sheer. Austin: Univ. of

IN DIVING

Texas Press, 1961, p. 60-64. 18. Loeb, G. E., and C. Gans. Electromyogruphy for Experimentalists, Chicago, IL: University of Chicago Press, 1986. 19. Mangalam, H. J., D. R. Jones, and A. M. A. Lacombe. Adrenal gland denervation and diving in ducks. Am. J. Physiol. 252 (Regulatory Integrative Comp. Physiol. 21): Rll43-R1151, 1987. 20. McDonald, D. A. Blood Flow in Arteries. London: Arnold, 1974. 21. Millard, R. W. Depressed baroreceptor-cardiac reflex sensitivity during simulated diving in ducks. Comp. Biochem. Physiol. A Comp. Physiol. 65: 247-249, 1980. 22. Sagawa, K. Baroreflex control of systemic arterial pressure and vascular bed. In: Handbook of Physiology. The Cardiovascular System. Peripheral Circulation and Organ Blood Flow. Bethesda, MD: Am. Physiol. Sot., 1983, sect. 2, vol. 3, pt. 2, p. 453-496. 23. Smith, During

F. M. Forced

dissertation). 1987. 24. Smith,

F. M.,

baroreceptor

Arterial Baroreceptor Control of the Circulation Dives in Ducks (Anas platyrhynchos var.)(PhD

Vancouver, and

Canada: Univ. of British

Columbia,

The effects of acute and chronic on diving responses in ducks. Am. J.

D. R. Jones.

denervation

Physiol. 258 (Regulatory Integrative Comp. Physiol. 27): R895R902, 1990. 25. Swett, J. E., and C. M. Bourassa. Electrical stimulation of peripheral nerve. In: Electrical Stimulation Research Techniques,

edited by M. M. Patterson and R. P. Kesner. New York: Academic, 1981, p. 244-298. 26. Wallenstein, S., C. L. Zucker, and J. L. Fleiss. Some statistical methods useful in circulation research. Circ. Res. 47: 1-9, 1980. 27. Zar, J. H. Biostatistical Prentice-Hall, 1984.

Analysis

(2nd ed.). Englewood Cliffs, NJ:

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