152 Exp Physiol 94.1 pp 152–161

Experimental Physiology – Research Paper

Effects of hypoxic and hyperoxic incubation on thereactivity of the chicken embryo (Gallus gallus) ductusarteriosi in response to catecholamines and oxygen

Jennifer Copeland and Edward M. Dzialowski

Department of Biological Sciences, 1155 Union Circle 305220, University of North Texas, Denton, TX 76203, USA

Development in chronic hypoxia has been shown to have a significant negative impact on thedeveloping cardiovascular system. The developing chicken embryo has two ductus arteriosi(DA) that shunt blood away from the lungs to the systemic circuit and constrict duringhatching in response to an increase in arterial partial pressure of O2. The goal of this studywas to determine the influence of O2 levels during incubation on the vascular reactivity of theO2-sensitive DA using the chicken as a novel model system. In addition, we measured blood gasand air cell O2 during these developmental stages. Chicken embryos were incubated in hypoxia(15% O2), normoxia (21% O2) or hyperoxia (30% O2) and examined on incubation days 16 and18 and as internally pipped and externally pipped embryos. The vasoreactivity of the DA wasmeasured in response to an increase in O2 and during stepwise increases in noradrenaline (NA)and phenylephrine (PE). The DA from embryos incubated in hypoxic conditions contractedin response to O2 at a later hatching stage than the DA from embryos incubated in normoxicor hyperoxic conditions. The DA from day 18 embryos incubated in hypoxic conditions hada significantly weaker contractile response to NA and PE when compared with the DA fromembryos incubated in normoxic or hyperoxic conditions. Blood gas and air cell O2 were lowestfor embryos incubated in hypoxic conditions and highest for embryos incubated in hyperoxicconditions. Incubation in hypoxia significantly delays the maturation of DA, while incubationin hyperoxia accelerates development.

(Received 11 July 2008; accepted after revision 19 September 2008; first published online 19 September 2008)Corresponding author J. Copeland: Department of Biological Sciences, University of North Texas, 1155 Union Circle305220, Denton, TX 76203, USA. Email: jmc0342@unt.edu

The ductus arteriosus (DA) is an embryonic blood vesselthat connects the pulmonary arteries to the aorta inmammals, reptiles and birds (Bergwerff et al. 1999).This ensures adequate gas exchange by allowing a right-to-left shunt of right ventricular output away fromthe unventilated lungs and towards the embryonic gasexchanger, which is the placenta in mammals and thechorioallantoic membrane in birds. During hatching inbirds, the paired DA constrict preferentially proximal tothe pulmonary artery (Belanger et al. 2008), resulting inthe disappearance of the right-to-left shunt and increasedblood flow through the pulmonary arteries to the lungs. Inmammals, DA closure is stimulated by an increase in bloodgas partial pressure of O2 (PO2 ) that occurs at birth whenthe animal begins to breathe with its lungs (Heymann &

Rudolph, 1975; Berger et al. 1990; Smith, 1998). Hatchingin chickens is initiated on incubation day 20 when theembryo internally pips (IP) the air cell of the egg withits beak and begins breathing hypoxic air cell gas (Tazawaet al. 1983). However, it is not until the embryo breaksthe eggshell and begins to breathe normoxic air duringexternal pipping (EP) that blood gas PO2 increases andthe DA is stimulated to close (Agren et al. 2007; Belangeret al. 2008). Studies suggest that the responsiveness ofthe mammalian and avian DA to O2 is developmentallyregulated. The preterm DA from rabbits, guinea pigs andlambs exhibited a weaker contractile response to O2 whencompared with the DA from full-term animals (Noel &Cassin, 1976; Clyman et al. 1978; Thebaud et al. 2004;Kajimoto et al. 2007). Similarly, studies performed in

DOI: 10.1113/expphysiol.2008.044214 C© 2008 The Authors. Journal compilation C© 2009 The Physiological Society

Exp Physiol 94.1 pp 152–161 Hypoxia, hyperoxia and the chicken ductus arteriosus 153

birds found that the DA from older animals exhibiteda stronger O2-induced contraction when comparedwith the response from younger animals (Agren et al.2007; Belanger et al. 2008; Dzialowski & Greyner,2008).

It is well known that development in chronic hypoxiaproduces significant abnormalities in the developingcardiovascular system, as well as increased mortality anddecreased body weight (Dzialowski et al. 2002; Rouwetet al. 2002; Villamor et al. 2004). Additionally, thevascular phenotype of the developing chicken embryoresponds to chronic hypoxic exposure. Villamor et al.(2004) found that chicken embryos developing in chronichypoxia exhibited reduced pulmonary arterial contractileresponses to noradrenaline (NA), endothelin-1 and thethromboxane A2 mimetic, U-46619. Chronic hypoxicincubation also produced sympathetic hyperinnervationof the arterial system and an increase in heart-to-body mass ratio in chicken embryos (Ruijtenbeek et al.2000; Rouwet et al. 2002). Since the DA is an O2-sensitive tissue, changes in environmental O2 levels duringdevelopment are predicted to have significant effectson the developmental trajectory of the response of thisvessel to O2.

In approximately 70% of preterm human infants(<27 weeks gestation; Clyman, 2006), the DA fails toclose, leading to a patent DA (PDA). Developmentunder in utero hypoxaemic stress, such as occurs withintrauterine growth restriction or development athigh altitude, increases the incidence of a PDA inpreterm neonates (29–32 weeks gestation; Radza et al.2007). This may be especially important for the140 million individuals who reside above 2500 m and arechronically exposed to hypoxic conditions during in uterodevelopment (Niermeyer et al. 2001). However, no studyto date has examined how the mammalian or avian DAdevelops under chronic hypoxia or hyperoxia.

The goal of this study was to use the chicken DA asa novel model to characterize the effects of developmentin chronic hypoxia or hyperoxia on DA reactivity to O2

and catecholamines. Since the DA response to O2 andcatecholamines is developmentally regulated in chickens(Agren et al. 2007), we tested the DA of day 16 andday 18 non-lung-ventilating embryos, as well as IP andEP embryos breathing with their lungs, to examinehow chronic incubation O2 levels affected contractileresponses. The chicken embryo provides an excellentmodel for developmental studies of chronic O2 effects,because the embryo develops in ovo, allowing foreasy manipulation of the developmental environment(Sutendra & Michelakis, 2007). We hypothesized thatincubation in hypoxia would delay the maturation ofthe DA, while incubation in hyperoxia would acceleratedevelopment.

Methods

Eggs and incubation

White Leghorn chicken (Gallus gallus) eggs whereobtained from Texas A & M University and incubatedat 37.5◦C and a relative humidity of 70%. Eggs wereincubated in hovabator incubators (Hova-Bator, G.Q.F.Manufacturing, Savannah, GA, USA) maintained at threelevels of O2: 15% O2 (hypoxia), 21% O2 (control) or30% O2 (hyperoxia). The O2 level in the incubators wasmaintained using Engineering Systems O2/N2 controllers(model 1600; Newark, DE, USA) and checked regularlywith a Sable System FOX O2 analyser (Las Vegas, NV,USA). Embryos were killed by isoflurane inhalation orby decapitation. Preliminary experiments found that themethod of killing did not influence the response ofthe DA to O2. All experiments were approved by theUniversity of North Texas Institutional Animal Care andUse Committee.

Blood gas

Blood gases were measured on day 16, day 18 and in IPembryos incubated at the three O2 levels. On the day ofmeasurement, eggs were moved to a hypoxia/hyperoxiachamber maintained at 37.5◦C and the appropriateincubation O2 level. Eggs were allowed to equilibrate for2 h in the hypoxia/hyperoxia chamber prior to sampling.The O2 level in the sampling chamber was regulatedby a Porter Instrument gas mixer and monitored witha Sable Systems FOX O2 analyser (Hatfield, PA, USA).The embryos were candled to locate major chorioallantoicveins and arteries. A small portion of the shell and outermember over the vessel was removed, and 150 μl of bloodwas withdrawn from a major chorioallantoic membranevein or artery using a heparanized 31 gauge needle andsyringe. Blood PO2 and partial pressure of CO2 (PCO2 )were measured with a Radiometer ABL5 blood gas meter(Westlake, OH, USA).

Air cell

Air cell PO2 was measured using the technique describedby Tazawa et al. (1980). Air cell measurements were madeon day 16, day 18 and IP eggs. A small hole was drilledin the shell over the air cell, and a port for a syringewas glued into the shell. A glass syringe was attachedto the port, and the embryo was placed back into theincubator for at least 12 h. This allowed the gas in thesyringe to equilibrate with the air cell gas. The syringewas removed, and the content of the syringe was injectedinto an O2 electrode (Microelectrodes Inc., Bedford,NH, USA) maintained at 37.5◦C and recorded using anADInstruments Powerlab 8SP and Chart 5.4.2 software.

C© 2008 The Authors. Journal compilation C© 2009 The Physiological Society

154 J. Copeland and E. M. Dzialowski Exp Physiol 94.1 pp 152–161

In vitro vessel physiology

The left DA was removed from day 16, day 18, IP and EPembryos and placed in a physiological saline solution (PSS,composed of 120.5 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4,1.6 mM CaCl2, 1.2 mM NaH2PO4, 20.4 mM NaHCO3 and10 mM glucose) equilibrated with 95% N2 and 5% CO2,and any remaining connective tissue was removed. Sincethe chicken DA has two distinct morphological regionsalong its length (Belanger et al. 2008), the proximal anddistal portions of the left DA were separated, based onvisual inspection of their vessel morphology and diameter.A clear transition between the proximal portion and distalportion can be seen in the middle of each vessel (Agrenet al. 2007; Belanger et al. 2008). In this study, only themuscular proximal portion of the DA that corresponds tothe mammalian DA was used. Prior to removal of the DA,yolk-free body mass was measured.

In vitro contraction was measured on the isolated vesselsusing a four-chamber 610M Danish Myo Technologiesmyograph (Aarhns, Denmark). The excised vessels,ranging in length from 0.3 to 2 mm, were mounted intomyograph chambers by threading two 40 μm diameterstainless-steel wires through the lumen of the vessel. Onewire was attached to a force transducer and the otherto a micromanipulator that allowed for the adjustmentof vessel tension. The force generated by each vessel wasrecorded as the active wall tension (in mN mm−1) using anADInstruments Powerlab 8SP and Chart 5.4.2 software.Vessel contractile data are presented as the net activetension generated in response to the contractile agent.

Each chamber was filled with 5 ml of PSS and bubbledwith a 95% N2–5% CO2 gas mixture, resulting in a PO2

of 30 mmHg (4%) and a PCO2 of 40 mmHg. Bath PO2

and PCO2 were monitored with a Radiometer ABL5 bloodgas meter. The vessels were allowed to equilibrate for30 min, and the baseline tension for all vessels was set to atension that produced the largest contraction in responseto 120 mM KCl as determined in preliminary experiments.During this time, the vessels were contracted twice with120 mM KCl–PSS followed by three PSS rinses. All in vitromeasurements were made under low light conditions.

Contractile response

In day 18 (prepipped) and IP embryos, DA contraction ofisolated vessels was measured in response to cumulativestepwise increases in phenylephrine (PE; 10−8–10−4 M)and noradrenaline (NA; 10−8–10−5 M). After the additionof each contractile agent, the vessel was allowed to stabilizebefore the next concentration was added to the chamber.The contractile response of the left DA from day 16embryos was measured only in response to 10−4 M PEand 10−5 M NA.

The vessel contraction of day 16, day 18, IP and EPembryos was measured in response to an increase inorgan bath O2 from 4 to 25%. We did not measure thecontractile response from the EP hyperoxic DA becauseit was functionally closed by this point in hatching.Following the initial 30 min equilibration to 4% O2, thegas mixture bubbling the organ bath was then increased toachieve an organ bath containing 25% O2, and the vesseltension was measured once it reached a plateau.

To determine whether NA augments the response ofthe DA to O2 in day 16, day 18, IP and EP embryos, NAwas added to the organ bath after an increase in O2. TheO2-induced contraction was measured in response to anincrease in organ bath O2 from 4 to 25%, followed by theaddition of 10−5 M NA to the organ bath.

Chemicals

Phenylephrine and NA were obtained from SigmaChemical Co. (St Louis, MO, USA). Initial stock solutionsof PE and NA were made daily with distilled water followedby further dilutions in PSS.

Statistics

The effect of NA and PE on vessel tension was tested usingtwo-way repeated measures ANOVAs with age/incubationO2 level and concentration as factors. The effect of KCl,O2, blood gas and air cell O2 level was tested using atwo-way ANOVA with age and incubation O2 levels asfactors. All ANOVAs were followed by a Sidak–Holmpost hoc test of pairwise comparisons. Data are reportedas the means ± S.E.M. The level of significance for alltests was P < 0.05. Statistics were run on SAS 9.1 (SAS,Cary, NC, USA) and Sigmastat 3.5 (SyStat, Chicago, IL,USA). Determinations of EC50 were carried out withSigmastat 3.5.

Results

Blood gas and air cell

Arterial and venous PO2 were dependent on theincubation O2 level in which the embryo developed.Hyperoxic embryos had significantly higher arterialand venous PO2 levels than normoxic embryos(Table 1). Hypoxic embryos had significantly lowerarterial and venous PO2 levels than normoxic embryos(Table 1). Within a given incubation O2 treatment, therewere no significant differences in arterial PO2 betweenthe different stages of development. In contrast, venousPO2 significantly decreased as the embryos aged, withthe exception of the hypoxic embryos, in which PO2

remained constant. Internally pipped normoxic embryos

C© 2008 The Authors. Journal compilation C© 2009 The Physiological Society

Exp Physiol 94.1 pp 152–161 Hypoxia, hyperoxia and the chicken ductus arteriosus 155

Table 1. Blood gas, air cell and embryo mass measurements taken from day 16, day 18 and internally pipped embryos incubated inhypoxia (15% O2), normoxia (21% O2) or hyperoxia (30% O2)

Arterial PO2 Venous PO2 Arterial PCO2 Venous PCO2 Air cell PO2 Mass (g)(mmHg) (mmHg) (mmHg) (mmHg) (mmHg)

HypoxiaDay 16 22.6 ± 1.2∗ 48.4 ± 3.0∗ 30.1 ± .9∗ 21.7 ± 1.5∗ 72.9 ± 1.9∗ 13.8 ± 0.5∗

n = 14 n = 11 n = 14 n = 11 n = 15 n = 11Day 18 20.5 ± 1.3 38.9 ± 3.1∗ 33.4 ± 1.3∗ 26.2 ± 2.2∗ 70.7 ± 2.0∗ 16.6 ± 0.5∗†

n = 12 n = 9 n = 12 n = 9 n = 13 n = 31IP 18.7 ± 1.4∗ 45.4 ± 3.9 32.4 ± 1.8∗ 19.9 ± 1.1∗ 57.0 ± 2.5∗† 28.0 ± 0.6∗†

n = 10 n = 9 n = 10 n = 9 n = 11 n = 17NormoxiaDay 16 28.0 ± 0.7 63.9 ± 1.8 42.8 ± 1.4 28.2 ± 1.2 119.3 ± 2.6 16.3 ± 0.5

n = 13 n = 17 n = 13 n = 17 n = 8 n = 12Day 18 23.6 ± 0.9 57.3 ± 2.8 47.1 ± 1.1 33.1 ± 1.9 118.1 ± 4.0 22.8 ± 0.5†

n = 22 n = 11 n = 22 n = 11 n = 8 n = 22IP 25.9 ± 1.5 46.6 ± 2.1† 48.8 ± 2.7 34.6 ± 1.5† 107.6 ± 2.4 32.4 ± 0.6†

n = 13 n = 10 n = 13 n = 10 n = 12 n = 26HyperoxiaDay 16 36.6 ± 2.5∗ 85.7 ± 3.1∗ 47.7 ± 2.3∗ 34.9 ± 1.8∗ 217.5 ± 2.7

∗16.6 ± 0.4

n = 15 n = 15 n = 15 n = 15 n = 16 n = 16Day 18 35.1 ± 2.1∗ 76.3 ± 3.1∗† 44.5 ± 1.4 32.8 ± 1.5 198.9 ± 5.6∗† 25.5 ± 0.9∗†

n = 15 n = 20 n = 15 n = 20 n = 12 n = 13IP 32.6 ± 2.1∗ 67.9 ± 3.0∗† 51.8 ± 3.3 41.1 ± 2.0∗† 180.3 ± 6.5∗† 34.7 ± 0.5∗†

n = 11 n = 13 n = 11 n = 13 n = 18 n = 18

Blood gas samples were obtained from major chorioallantoic veins or arteries. ∗ Indicates a significant difference compared with theage-matched normoxic values at P < 0.05. † Indicates significant differences from day 16 within a given incubation treatment at P < 0.05.

had significantly lower venous PO2 than day 16 and day 18embryos, whereas venous PO2 decreased significantly ateach stage in hyperoxic embryos (P < 0.05).

Arterial and venous PCO2 were dependent on theincubation O2 level in which the embryo developed.Hyperoxic and normoxic embryos had significantlygreater arterial PCO2 levels than hypoxic embryos(Table 1). Day 16 hyperoxic embryos had significantlygreater arterial PCO2 levels than normoxic embryos(Table 1). Within each incubation O2 treatment, the onlysignificant difference in arterial PCO2 was found in theIP hyperoxic embryos, which had a significantly greaterarterial PCO2 than day 18 hyperoxic embryos (P < 0.05).Hyperoxic embryos had significantly greater venous PCO2

levels than normoxic embryos except on day 18 (P < 0.05).Normoxic embryos had a significantly greater venousPCO2 level than hypoxic embryos (P < 0.05). In hypoxicembryos, there were no significant differences in venousPCO2 between the different stages of development. Innormoxic embryos, IP embryos had a significantly greatervenous PCO2 than day 16 embryos (P < 0.05). Internallypipped hyperoxic embryos had a significantly greatervenous PCO2 than day 16 or day 18 embryos (P < 0.05;Table 1).

Embryo mass was greatest in embryos from thehyperoxic treatment and lowest in eggs from the hypoxictreatment (Table 1). Day 18 and IP hyperoxic embryomasses were significantly greater than their normoxicand hypoxic counterparts (P < 0.05). Hypoxic embryo

mass was significantly lower than that of normoxicand hyperoxic embryos at each developmental stage(P < 0.05). In each treatment, embryo mass increasedsignificantly with development (P < 0.05).

Air cell O2 tension was greatest in embryos from thehyperoxic treatment and lowest in eggs in the hypoxictreatment (Table 1). Internally pipped hypoxic eggs hadsignificantly lower O2 air cell content than day 16 orday 18 hypoxic eggs (P < 0.05). Oxygen air cell contentof normoxic eggs remained constant. Day 18 hyperoxiceggs had a significantly lower O2 air cell content thanday 16 eggs (P < 0.05). Internally pipped hyperoxic eggshad significantly lower O2 air cell content than day 16 orday 18 hyperoxic eggs (P < 0.05).

Contractile response to KCl

The DA response to KCl was dependent on both age andincubation O2 level (Table 2). The contractile response ofthe DA from hypoxic and normoxic embryos did not differsignificantly during any developmental stage. However,the DA from IP hyperoxic embryos had a significantlygreater contractile response to KCl than the DA fromnormoxic and hypoxic IP embryos (P < 0.05).

The DA response to KCl increased significantly asthe embryo aged (Table 2). The contractile response ofthe DA from day 16 and day 18 embryos did not differsignificantly in any incubation O2 level. The DA from IPand EP hypoxic embryos exhibited a significantly greater

C© 2008 The Authors. Journal compilation C© 2009 The Physiological Society

156 J. Copeland and E. M. Dzialowski Exp Physiol 94.1 pp 152–161

Table 2. Contractile responses to 120 mmol l−1 KCl of the left proximal DA in day 16, day 18,internally pipped and externally pipped embryos incubated in hypoxia (15% O2), normoxia (21%O2) or hyperoxia (30% O2)

Hypoxia (mN mm−1) Normoxia (mN mm−1) Hyperoxia (mN mm−1)

Day 16 0.28 ± 0.03 0.23 ± 0.04 0.37 ± 0.02n = 8 n = 8 n = 8

Day 18 0.35 ± 0.06 0.55 ± 0.04 0.81 ± 0.09n = 8 n = 8 n = 8

IP 0.87 ± 0.10† 0.86 ± 0.14 1.83 ± 0.19∗†n = 8 n = 8 n = 8

EP 1.37 ± 0.56† 1.4 ± 0.16†n = 8 n = 7

∗Indicates a significant difference compared with the age-matched normoxic values at P < 0.05.† Indicates significant differences from day 18 within a given incubation treatment at P < 0.05.

d

d

c c

b

a

c

b

a

b

c

c

A

B Noradrenaline [log M]

-8 -7 -6 -5

Ac

tiv

e T

en

sio

n (

mN

mm

–1)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Day 18 Hypoxic

Day 18 Normoxic

Day 18 Hyperoxic

IP Hypoxic

IP Normoxic

IP Hyperoxic

Phenylephrine [log M]

-8 -7 -6 -5 -4

Acti

ve T

en

sio

n (

mM

mm

–1)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Figure 1. Contractile response of the DA to adrenergic agonistsA, contractile responses of the left proximal DA in day 18 andinternally pipped embryos incubated in hypoxia (15% O2; day 18,n = 11; IP, n = 9), normoxia (21% O2; day 18, n = 11; IP, n = 10) orhyperoxia (30% O2; day 18; n = 12; IP, n = 13) to a stepwise increasein NA. Letters denote significantly different groups, P < 0.05.B, contractile responses of the left proximal DA in day 18 andinternally pipped embryos incubated in hypoxia (15% O2; day 18,n = 14; IP, n = 10), normoxia (21% O2; day 18, n = 11; IP, n = 10) orhyperoxia (30% O2; day 18, n = 11; IP, n = 15) to a stepwise increasein PE. Letters denote significantly different groups, P < 0.05.

contractile response to KCl than day 18 hypoxic embryos(P < 0.05). The DA from EP normoxic embryos exhibiteda significantly greater contractile response to KCl thanday 18 embryos (P < 0.05). The DA from IP hyperoxicembryos exhibited a significantly greater contractileresponse to KCl than day 18 embryos (P < 0.05).

Contractile response to adrenergic agonists

The DA response to NA was dependent on both ageand incubation O2 levels (Fig. 1A). There were significanteffects of incubation O2 on the DA contractile response toNA in day 18 embryos. The DA from both normoxic andhyperoxic day 18 embryos exhibited a significant dose-dependent contractile response to NA. In contrast, NAdid not produce a significant contraction in the hypoxicday 18 DA at any concentration (Fig. 1A). In all treatments,the DA contractile response to NA increased with age(Fig. 1A). Internally pipped embryos from all incubationO2 treatments exhibited a significantly stronger contractileresponse to NA than day 18 embryos within the sameincubation treatment (Fig. 1A). There were no significanteffects of embryo age or O2 incubation level onEC50 for NA (hypoxic IP, −6.5 ± 0.3 logM; normoxicday 18, −6.5 ± 0.5 logM; normoxic IP, −6.3 ± 0.2 logM;hyperoxic day 18, −6.5 ± 0.2 logM; and hyperoxic IP,−6.7 ± 0.2 logM).

There were significant effects of incubation O2 leveland age on the DA contractile response to PE (Fig. 1B).In day 18 embryos, PE produced a significantly strongercontraction in the hyperoxic DA than in the normoxicand hypoxic DA (P < 0.05). The DA of hypoxic embryosexhibited a significantly weaker contractile responseto PE than that of normoxic embryos (P < 0.05).Internally pipped embryos from all incubation O2

treatments exhibited significantly stronger contractileresponses than day 18 embryos within the same incubationtreatment (Fig. 1B). The DA contractile response to

C© 2008 The Authors. Journal compilation C© 2009 The Physiological Society

Exp Physiol 94.1 pp 152–161 Hypoxia, hyperoxia and the chicken ductus arteriosus 157

PE of hypoxic IP embryos was significantly weakerthan that of normoxic and hyperoxic IP embryos(Fig. 1B). The maximal DA contractile responses ofnormoxic and hyperoxic IP embryos to PE were notsignificantly different (Fig. 1B). The DA from day 18normoxic embryos responded at a significantly higherconcentration than the DA from IP normoxic embryos.There were no other significant effects of embryo age orincubation O2 level on the EC50 for PE (hypoxic day 18,−5.1 ± 0.2 logM; hypoxic IP, −5.8 ± 0.2 logM; normoxicday 18, −5.0 ± 0.1 logM; normoxic IP, −5.7 ± 0.2 logM;hyperoxic day 18, −5.3 ± 0.2 logM; and hyperoxic IP,−5.8 ± 0.1 logM).

Contractile response to O2 and noradrenalinewith O2

The DA contractile response to an acute increase in O2

increased with age and incubation O2 level (Fig. 2). Theresponse of the DA from hyperoxic embryos to an acuteincrease in O2 increased significantly with developmentalage (P < 0.05). In the DA from normoxic embryos, thecontractile response to an acute increase in O2 significantlyincreased from day 16 to day 18 and from IP to EP(P < 0.05). The first significant O2-induced contractionin the hypoxic DA occurred at IP. During the IP stage,an acute increase in O2 produced a significantly greatercontraction in the DA of hyperoxic embryos than in theDA of hypoxic or normoxic embryos (P < 0.05). The DAcontractile response to an acute increase in O2 was similarfor hyperoxic IP and hypoxic EP embryos, and hyperoxicIP and normoxic EP embryos (Fig. 2). At EP, the DAfrom normoxic embryos produced significantly greatercontraction in response to an acute increase in O2 thanthe DA from hypoxic embryos (P < 0.05).

An acute increase in O2 augmented the adrenergicresponse of the DA in all incubation O2 treatments. Onday 16, the DA contractile response to NA alone and toNA with an acute increase in O2 were not different inany treatment (Fig. 3). On day 18 and IP, NA produceda significantly greater DA contractile response in thepresence of 25% O2 than 4% O2 in all treatments(P < 0.05). On day 18, the DA of hypoxic embryos hada significantly weaker response to NA in the presence of25% O2 than the DA of normoxic and hyperoxic embryos.This was reversed in IP hypoxic embryos, which had asignificantly greater response to NA in the presence of25% O2 than their normoxic and hypoxic counterparts(P < 0.05). As the embryo developed, the DA responseto NA in both 4 and 25% O2 increased significantly(Fig. 3).

Discussion

In certain conditions, such as during intrauterine growthrestriction or at high altitude, the mammalian fetusdevelops in hypoxic conditions. The influence of alteredoxygen levels on the development and physiology ofthe O2-sensitive DA is unknown. Here we have takenadvantage for the first time of the self-contained chickenegg as a model to examine the influence of chronic hypoxicand chronic hyperoxic exposure during developmenton the maturation of the DA. Incubation in chronichypoxia is known to cause significant cardiovascularabnormalities in the developing chicken embryo (Rouwetet al. 2002; Villamor et al. 2004). Here we have shownthat this includes an alteration in the developmentaltrajectory of the DA. Embryos incubated in hypoxiawere exposed to lower O2 concentrations, thus haddecreased blood gas PO2 and air cell PO2 , whereas thereverse was true for embryos incubated in hyperoxia.The responsiveness of the DA to O2 and adrenergicagonists was developmentally regulated. The DA fromday 16 embryos in all treatments did not respondto NA, PE or O2, whereas by day 18, the DA fromnormoxic and hyperoxic embryos exhibited significantcontractile responses. Incubation in hypoxia produced adevelopmental delay in the contractile response of thechicken DA to O2 and catecholamines. Oxygen augmentedthe adrenergic response of the DA in day 18 and IPembryos in all treatments.

Figure 2. Contractile response of the DA to oxygenOxygen induced contraction of the left proximal DA in day 18,internally pipped and externally pipped (hypoxic and normoxic)embryos incubated in hypoxia (15% O2; day 16, n = 8; day 18;n = 10; IP, n = 11; EP, n = 12), normoxia (21% O2; day 16, n = 10;day 18, n = 17; IP, n = 18; EP, n = 11) or hyperoxia (30% O2; day 16,n = 9; day 18, n = 12; IP, n = 11) to an increase in organ bath O2 from4 to 25%. Open symbols denote a significant increase over baseline,P < 0.05. Letters denote significantly different groups, P < 0.05.

C© 2008 The Authors. Journal compilation C© 2009 The Physiological Society

158 J. Copeland and E. M. Dzialowski Exp Physiol 94.1 pp 152–161

Blood gas, air cell and body mass

Even though many studies have examined thedevelopment of the chicken in response to altered oxygenlevels, this is the first study to present blood gas valuesduring hypoxic and hyperoxic incubation. Blood gasvalues were dependent upon both age and incubationO2 level. The blood gas values obtained from normoxicembryos were similar to those obtained by Tazawaet al. (1983). As expected, chorioallantoic arterial PO2

and PCO2 were lowest in hypoxic embryos and greatestin hyperoxic embryos. Chorioallantoic venous PO2 inboth normoxic and hyperoxic embryos decreased withdevelopment; however, in hypoxic embryos venous PO2

remained relatively constant. The difference betweenarterial and venous PO2 , the driving force for O2

delivery to the tissues, of hypoxic embryos ranged from26.3 mmHg on day 16 to 18.4 mmHg on day 18. Thesevalues are only 75 and 55% of the normoxic valuesat these same times. At IP, venous PO2 levels of bothO2 normoxic and hypoxic embryos were similar, eventhough the embryos were exposed to markedly differentlevels of O2 in the incubators. Just before chickenembryos internally pip, they experience an increasein circulating catecholamines (Wittman & Prechtl,1991). This increase in catecholamines corresponds witha period of increased hypoxia in the embryo andaids the embryos by decreasing 2,3-diphosphoglyceratelevels, which facilitates an increase in O2 saturation ofhaemoglobin (Wittman & Prechtl, 1991). Higher levels ofcirculating catecholamines may help hypoxic embryos tomaintain venous PO2 throughout development. However,the levels of circulating catecholamines during chronichypoxic or hyperoxic incubation have never beenrecorded.

Oxygen content of the air cell was dependent uponboth age and incubation O2 level. Within hypoxic andnormoxic O2 treatments, the O2 contents of day 16 andday 18 eggs were similar; however, hyperoxic day 18 eggshad a significantly lower O2 content than day 16 eggs. Onceembryos began to breathe the gas in the air cell during IP,the O2 content significantly decreased in all treatments.Eggs incubated in hypoxic conditions had the lowest aircell O2 content, whereas eggs incubated in hyperoxicconditions had the highest air cell O2 content. Ournormoxic values were consistent with both Wangensteen &Rahn (1970–1971) and Tazawa et al. (1980). Our hypoxicvalues were similar to those obtained by Wangensteen et al.(1974) from chicken embryos that were incubated at highaltitude. From our data, we see that animals with higher aircell O2 contents typically have higher venous PO2 . The onlyexception is IP hypoxic embryos, which have significantlylower air cell O2 content than IP normoxic embryos, butsimilar venous PO2 levels. This suggests that hypoxic IP

embryos are more efficient in taking up O2 from the aircell, which may be due to an increase in lung ventilationrate of the embryo exposed to hypoxia when comparedwith the embryo exposed to normoxia. In response toacute hypoxia during IP, normoxic embryos have beenshown to increase tidal volume (Sbong & Dzialowski,2007).

Incubation in hypoxia and hyperoxia had significanteffects on body mass throughout incubation. As othershave shown, incubation in hypoxia slows growth, resultingin a smaller embryo at each stage of incubation(Dzialowski et al. 2002; Rouwet et al. 2002; Villamoret al. 2004; Azzam & Mortola, 2007), whereas hyperoxicincubation produced significantly larger embryos ateach stage of incubation. The decrease in body massduring incubation in hypoxia is associated with a lowervenous–arterial PO2 difference than embryos incubated innormoxia and results in an increase in incubation lengthfor embryos incubated in hypoxic conditions (Azzam et al.2007). Embryos incubated in hypoxic conditions tendedto IP a day later than the normoxic and hyperoxic embryos(J. Copeland, unpublished observation).

Figure 3. The effect of oxygen on the DA response to NAThe effect of oxygen on the left proximal DA in response to10−4 M NA in day 16, day 18 and internally pipped embryos incubatedin hypoxia (15% O2; n values for 4% O2 are day 16, n = 9; day 18,n = 11; IP, n = 9; and for 25% O2 are day 16, n = 8; day 18, n = 10;IP, n = 11), normoxia (21% O2; n values for 4% O2 are day 16, n = 8;day 18, n = 11; IP, n = 10; and for 25% O2 are day 16, n = 10;day 18, n = 12; IP, n = 11) or hyperoxia (30% O2; n values for 4% O2

are day 16, n = 9; day 18, n = 12; IP, n = 13; and for 25% O2 areday 16, n = 9; day 18, n = 9; IP, n = 8). Vessel response was measuredat 4 and 25% O2. ∗ Indicates a significant difference in net activetension of the 4% O2 group compared with the 25% O2 group for agiven age, P < 0.05.

C© 2008 The Authors. Journal compilation C© 2009 The Physiological Society

Exp Physiol 94.1 pp 152–161 Hypoxia, hyperoxia and the chicken ductus arteriosus 159

Potassium chloride-mediated contractions

Both age and incubation O2 level affected the receptor-independent DA contractile response to KCl. The DA fromday 16 embryos exhibited a weak contractile response toKCl which increased throughout development. This issimilar to the findings of Agren et al. (2007) that theDA from day 21 embryos exhibited a greater contractileresponse to KCl than the DA from day 15 or day 19embryos. The DA from hypoxic and normoxic embryosresponded in a similar manner throughout development,whereas the DA from IP hyperoxic embryos exhibited asignificantly greater contractile response. This suggeststhat the DA from hypoxic embryos does not experiencea developmental delay in the receptor-independentcontraction, but a developmental delay in the pathwaysinvolved in the response to O2 and catecholamines.

Adrenergic agonists

Both age and incubation O2 level affected the DAcontractile response to adrenergic agonists. The DA fromday 16 embryos in all treatments did not respond to NA.This confirms the findings of Agren et al. (2007) thatthe DA from day 15 chicken embryos did not respondto NA or PE, suggesting that up to day 16 of incubationthe DA lacks an active adrenergic contractile pathway.The DA from day 18 normoxic and hyperoxic embryosexhibited similar significant contractile responses to NA.However, the DA from hypoxic day 18 embryos still did notrespond to NA, suggesting a developmental delay of theadrenergic contractile pathway in the DA when incubatedin hypoxia. The DA of day 18 embryos incubated inhyperoxia exhibited the strongest contractile response toPE, and the DA of day 18 embryos incubated in hypoxiaexhibited the weakest response to PE. Agren et al. (2007)found that the DA from day 19 embryos exhibited astronger contractile response to PE than that from day 15embryos. We showed that the DA from IP embryos in alltreatments had a stronger response to NA than on day 18,suggesting further maturation of the adrenergic pathway.The DA of IP embryos incubated in hypoxic conditionshad a similar contractile response to NA as the DAfrom both normoxic and hyperoxic embryos, suggestingthat any developmental delays due to incubation inhypoxia disappeared by IP. However, embryos incubatedin hypoxic conditions internally pip approximately 1 dayafter the embryos incubated in normoxic and hyperoxicconditions, allowing for the maturation of the adrenergicpathway to catch up with embryos incubated in normoxicand hyperoxic conditions (J. Copeland, unpublishedobservation).

Several studies have examined the effect of incubationin chronic hypoxia on reactivity to NA and PE in othervessels from developing chicken embryos and fetal and

newborn sheep. Rouwet et al. (2002) found that themesenteric resistance arteries of day 19 chicken embryosexhibited a contractile response to NA that was similar inembryos chronically incubated in normoxic and hypoxicconditions. In contrast, Villamor et al. (2004) found thatchronic hypoxia reduced pulmonary arterial contractilereactivity to NA in day 19 chicken embryos. After spendingthe last 2 days of incubation in normoxia, the arterialreactivity of the embryos incubated in hypoxic conditionswas similar to that of the control pulmonary arteries(Villamor et al. 2004). An increased contractile responseto NA was observed in the pulmonary artery of fetal sheepexposed to chronic hypoxia (Xue et al. 2008). An increasedcontractile response to both NA and PE was observedin the small femoral arteries of newborn sheep exposedto chronic hypoxia (Herrera et al. 2007). In contrast,Longo & Pearce (1998) found that chronic hypoxia infetal sheep caused a decrease in the contractile responseof cerebral arteries to NA. In addition, they found thatchronic hypoxia decreased α1-adrenergic receptor densityin common carotid and intracranial arteries. The DAfrom hypoxic IP embryos exhibited a significantly weakercontraction to PE than that from normoxic or hyperoxicembryos. This suggests that incubation O2 level may havean effect on the maturation of α-adrenergic receptors inthe DA.

Response to O2 and noradrenaline with O2

There is a clear maturation of the O2-induced contractileresponse of the chicken DA during hatching (Fig. 2). TheDA of day 16 embryos in all treatments did not respondto an acute increase in O2, whereas significant increasesin contractile response to an acute increase in O2 wereobserved as development progressed. In contrast, Agrenet al. (2007) found that the DA from day 19 and EPchicken embryos produced a similar contractile responseto O2. However, Agren et al. (2007) made measurementson tissue containing both proximal and distal DA tissuemorphologies together, whereas we used only proximalDA tissue. A similar increase in contractile response to O2

has been observed in emu embryos (Dzialowski & Greyner,2008) and a number of mammalian species, such as rabbitsand guinea pigs (Noel & Cassin, 1976; Thebaud et al. 2004).This maturation of the O2-induced contractile responseprepares the DA for closure once the animal begins tobreathe with its lungs and blood gas PO2 increases (Tazawaet al. 1983).

The O2 levels experienced by the embryo duringincubation had a significant effect on the maturation ofthe O2-induced contractile response of the DA in thechicken embryo (Fig. 2). The DA from both normoxic andhyperoxic day 18 embryos exhibited a significant responseto O2. There was a delay in the maturation of the O2-

C© 2008 The Authors. Journal compilation C© 2009 The Physiological Society

160 J. Copeland and E. M. Dzialowski Exp Physiol 94.1 pp 152–161

induced contractile response of the DA of hypoxic embryosuntil IP. In contrast, the O2-induced contractile responseof the DA matured earlier in the embryos incubatedin hyperoxic conditions. We were unable to study theresponse of the DA from EP hyperoxic embryos becausethe vessel was almost completely closed, presumably inresponse to the increased O2 levels experienced duringhyperoxic incubation. The normoxic DA response to O2

begins to increase just before IP, the point at whichthe embryo begins to breathe with its lungs. The DAfrom EP normoxic embryos had a significantly greaterO2-induced contraction than the DA from IP normoxicembryos, which corresponds with the timing of the PO2

increase in the embryos as they respire normoxic gas(Tazawa et al. 1983; Meena & Mortola, 2002; Belanger et al.2008).

Belanger et al. (2008) have shown that the proximalportion of the DA in chicken embryos begins to closeduring EP. A relationship can be seen between an increasedcontractile response to O2 and accelerated closure in theproximal portion of the DA in hyperoxic embryos. Whencompared with the DA of normoxic embryos, the DAof the hyperoxic embryos had an accelerated maturationof the O2-induced constriction, whereas the DA of thehypoxic embryos experienced a developmental delay inmaturation of the O2-induced constriction. Coughlin &Husson (1960) exposed hatching chicken embryos tohypoxia and found that hypoxia was associated with adelay in DA closure. Ductus arteriosus closure in mammalsis stimulated by an increase in blood PO2 that occurs asthe animal begins to breathe with its lungs (Heymann& Rudolph, 1975; Smith, 1998). As shown in Table 1, IPhyperoxic embryos have significantly lower venous PO2

than day 16 embryos; however, this value is greater thanthat of the normoxic and hypoxic embryos. This suggeststhat in hyperoxic embryos, the pathway governing the O2-induced contraction of the DA matured with O2 levelsthat would normally constrict the mature vessel fromnormoxic embryos, and closure during IP might not needa subsequent increase in O2 above the already high levelsneeded to stimulate contraction.

Oxygen augmented the response of the DA to NA(Fig. 3). The DA from day 18 and IP embryos in alltreatments produced a significantly greater contractileresponse to NA when exposed to 25% O2 compared with4% O2. The DA from IP hypoxic embryos exposed to NAin 25% O2 exhibited the greatest contractile response. Thisis in contrast to the study by Agren et al. (2007), where theyfound that pretreatment with NA did not have an effect onthe O2 response; however, the study by Agren et al. (2007)used both proximal and distal tissues, whereas the presentstudy used only proximal tissue. Smith & McGrath (1988)found that in rabbits, NA did not augment the DA responseto O2. Our study suggests that circulating catecholaminesmay augment the O2-induced constriction in the DA of the

chicken embryo. However, the plasma concentrations ofNA and adrenaline have begun to decrease by the time theDA becomes most sensitive to NA in the chicken embryo(Wittman & Prechtl, 1991; Mulder et al. 2002).

Conclusion

The present study examines the effect of O2 incubationlevel on the vasoreactivity of the chicken DA. Bloodgas and air cell data follows the expected pattern, withhypoxic embryos having the lowest O2 levels and hyperoxicembryos having the highest O2 levels. Incubation O2 levelhas an effect on the maturation of the DA vasoreactivity tocatecholamines. Embryos incubated in hypoxia experiencea delayed response to both NA and PE. There is a similar,but more pronounced trend observed in the DA responseto O2. Embryos incubated in hypoxia respond to O2

later in development and have a significantly weakercontractile response than their normoxic and hyperoxiccounterparts. The DA from hyperoxic embryos exhibitan accelerated response to O2 and begin to close early,during IP. Incubation O2 level plays an important role inthe development of the chick DA. Incubation in hypoxiadelays the DA response to catecholamines and O2.

References

Agren P, Cogolludo AL, Kessels CG, Perez-Vizcaino F, De MeyJG, Blanco CE & Villamor E (2007). Ontogeny of chickenductus arteriosus response to oxygen and vasoconstrictors.Am J Physiol Regul Integr Comp Physiol 292, R485–R496.

Azzam MA & Mortola JP (2007). Organ growth in chickenembryos during hypoxia: implications on organ “sparing”and “catch-up growth”. Respir Physiol Neurobiol 159,155–162.

Azzam MA, Szdzuy K & Mortola JP (2007). Hypoxicincubation blunts the development of thermogenesis inchicken embryos and hatchlings. Am J Physiol Regul IntegrComp Physiol 292, R2373–R2379.

Belanger C, Copeland J, Muirhead D, Heinz D & Dzialowski E(2008). Morphological changes in the chicken ductusarteriosi during closure at hatching. Anat Rec 291,1007–1015.

Berger PJ, Horne RSC, Stoust M, Walker AM & Maloney JE(1990). Breathing at birth and the associated blood gas andpH changes in the lamb. Respir Physiol 82, 251–266.

Bergwerff M, DeRuiter MC & Gittenberger-de Groot AC(1999). Comparative anatomy and ontogeny of the ductusarteriosus, a vascular outsider. Anat Embryol 200, 559–571.

Clyman RI (2006). Mechanisms regulating the ductusarteriosus. Biol Neonate 89, 330–335.

Clyman RI, Mauray F, Wong L, Heymann MA & Rudolph AM(1978). The developmental response of the ductus arteriosusto oxygen. Biol Neonate 34, 177–181.

Coughlin FE & Husson GS (1960). Effect of hypoxia on theclosure of the ductus arteriosus in the chick. Am J Diseases100, 531.

C© 2008 The Authors. Journal compilation C© 2009 The Physiological Society

Exp Physiol 94.1 pp 152–161 Hypoxia, hyperoxia and the chicken ductus arteriosus 161

Dzialowski E & Greyner H (2008). Maturation of thecontractile response of the Emu ductus arteriosus. J CompPhysiol [B] 178, 401–412.

Dzialowski E, von Plettenberg D, Elmonoufy NA & BurggrenWW (2002). Chronic hypoxia alters the physiological andmorphological trajectories of developing chicken embryos.Comp Biochem Physiol A Mol Integr Physiol 131, 713–724.

Herrera EA, Pulgar VM, Riquelma RA, Sanhueza EM, ReyesRV, Ebensperger G, Parer JT, Valdez EA, Giussani DA,Blanco CE, Hanson MA & Llanos AJ (2007). High-altitudechronic hypoxia during gestation and after birth modifiescardiovascular responses in newborn sheep. Am J PhysiolRegul Integr Comp Physiol 292, R2234–R2240.

Heymann MA & Rudolph AM (1975). Control of the ductusarteriosus. Physiol Rev 55, 62–78.

Kajimoto H, Hashimoto K, Bonnet SN, Haromy A, Harry G,Moudgil R, Nakanishi T, Rebeyea I, Thebaud B, MichelakisED & Archer SL (2007). Oxygen activated theRho/Rho-kinase pathway and induces RhoB and ROCK-1expression in human and rabbit ductus arteriosus byincreasing mitochondria-derived reactive oxygen species: anewly recognized mechanism for sustaining ductalconstriction. Circulation 115, 1777–1788.

Longo LD & Pearce WJ (1998). High altitude, hypoxic-inducedmodulation of noradrenergic-mediated responses in fetaland adult cerebral arteries. Comp Biochem Physiol A MolIntegr Comp Physiol 119, 683–694.

Meena TM & Mortola JP (2002). Metabolic control ofpulmonary ventilation in the developing chick embryo.Respir Physiol 130, 43–55.

Mulder ALM, Miedema A, De Mey JGR, Gussani DA & BlancoCE (2002). Sympathetic control of the cardiovascularresponse to acute hypoxemia in the chick embryo. Am JPhysiol Regul Integr Comp Physiol 282, R1156–R1163.

Niermeyer S, Zamudio S & Moore LG (2001). The people. InHigh Altitude, ed. Hornbein TF & Schoene RB, pp. 43–100.Marcel Dekker, Inc., New York.

Noel S & Cassin S (1976). Maturation of contractile response ofductus arteriosus to oxygen and drugs. Am J Physiol 231,240–243.

Radza T, Magnenant E, Klosowski S, Tourneux P, Bachiri A &Storme L (2007). Early hemodynamic consequences ofpatent ductus arteriosus in preterm infants with intrauterinegrowth restriction. J Pediatr 151, 624–628.

Rouwet EV, Tintu AN, Schellings MWM, van Bilsen M,Lutgens E, Hofstra L, Slaaf DW, Ramsey G & le Noble FAC(2002). Hypoxia induces aortic hypertrophic growth, leftventricular dysfunction, and sympathetic hyperinnervationof peripheral arteries in the chicken embryo. Circulation 105,2791–2796.

Ruijtenbeek K, le Noble FAC, Janseen GMJ, Kessels CGA, FazziGE, Blanco CE & de May JGR (2000). Chronic hypoxiastimulates periarterial sympathetic nerve development inchicken embryos. Circulation 102, 2892–2897.

Sbong S & Dzialowski EM (2007). Respiratory andcardiovascular responses to acute hypoxia and hyperoxia ininternally pipped chicken embryos. Comp Biochem Physiol AMol Integr Physiol 148, 761–768.

Smith GC & McGrath JC (1988). Indomethacin, but notoxygen tension, affects the sensitivity of isolated neonatalrabbit ductus arteriosus, but not aorta, to noradrenaline.Cardiovasc Res 22, 910–915.

Smith GC (1998). The pharmacology of the ductus arteriosus.Pharmacol Rev 50, 35–58.

Sutendra G & Michelakis ED (2007). The chicken embryo as amodel for ductus arteriosus developmental biology: crackinginto new territory. Am J Physiol Regul Integr Comp Physiol292, R481–R484.

Tazawa H, Ar A, Rahn H & Piper J (1980). Repetitive andsimultaneous sampling from the air cell and blood vessels inthe chick embryo. Respir Physiol 39, 25–32.

Tazawa H, Visschedijk AHJ, Whitman J & Piper J (1983). Gasexchange, blood gases and acid-base status in the chickbefore, during and after hatching. Respir Physiol 53, 173–185.

Thebaud B, Michelakis ED, Wu XC, Moudgil R, Kuzyk M, DyckJR, Harry G, Hashimoto K, Haromy A, Rebeyka I & ArcherSL (2004). Oxygen-sensitive Kv channel gene transfer confersoxygen responsiveness to preterm rabbit and remodeledhuman ductus arteriosus: implications for infants withpatent ductus arteriosus. Circulation 110, 1372–1379.

Villamor E, Kessels CGA, Ruijtenbeek K, van Suylen RJ, Belik J,de May JGR & Blanco CE (2004). Chronic in ovo hypoxiadecreases pulmonary arterial contractile reactivity andinduces biventricular cardiac enlargement in the chickenembryo. J Physiol Regul Integr Comp Physiol 287, R642–R651.

Wangensteen OD & Rahn H (1970–1971). Respiratory gasexchange by the avian embryo. Respir Physiol 11, 31–45.

Wangensteen OD, Rahn H, Burton RR & Smith AH (1974).Respiratory gas exchange of high altitude adapted chickembryos. Respir Physiol 21, 61–70.

Wittman J & Prechtl J (1991). Respiratory function ofcatecholamines during the late period of avian development.Respir Physiol 83, 375–386.

Xue Q, Ducsay CA, Longo LD & Zhang L (2008). Effect oflong-term high-altitude hypoxia on fetal pulmonary vascularcontractility. J Appl Physiol 104, 1786–1792.

Acknowledgements

This study was supported by National Science Foundation grantno. IOS0417205 awarded to E.M.D.

C© 2008 The Authors. Journal compilation C© 2009 The Physiological Society