Ventilatory response to hypoxia in chicken hatchlings: A developmental window of sensitivity to embryonic hypoxia

Respiratory Physiology & Neurobiology 165 (2009) 49–53

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Ventilatory response to hypoxia in chicken hatchlings: A developmental window of sensitivity to embryonic hypoxia Kirsten Ferner 1 , Jacopo P. Mortola ∗ Department of Physiology, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, H3G 1Y6 Canada

a r t i c l e

i n f o

Article history: Accepted 6 October 2008 Keywords: Developmental plasticity Epigenetic adaptation Hypercapnia Hypometabolism Hypoxic ventilatory response

a b s t r a c t We had reported previously [Szdzuy, K., Mortola, J.P., 2007b. Ventilatory chemosensitivity of the 1day-old chicken hatchling after embryonic hypoxia. Am. J. Physiol. (Regul. Integr. Comp. Physiol.) 293, R1640–R1649] that hypoxia during incubation blunted ventilatory chemosensitivity in the hatchling. Because the carotid bodies become functional in the last portion of incubation, we asked whether these last days were the critical period for the effects of hypoxia on the development of ventilatory chemosensitivity. White Leghorn chicken eggs were incubated at 38 ◦ C either in 21% O2 (Controls) or in 15% O2 for the whole 3-week incubation (HxTot) or for only the 1st (Hx1), 2nd (Hx2) or 3rd week of incubation (Hx3). Hatching time had a delay of half a day in HxTot, and was normal in the other groups. Body weight was similar in ˙ were measured at about 20 h all hatchlings. Oxygen consumption (V˙ O2 ) and pulmonary ventilation (Ve) ˙ post-hatching. Ventilatory chemosensitivity was evaluated from the degree of hyperpnea (increase in Ve) ˙ V˙ O2 ) during acute hypoxia (15 and 10% O2 , 20 min each) and acute and hyperventilation (increase in Ve/ hypercapnia (2 and 4% CO2 , 20 min each). The responses to hypoxia were similarly decreased in HxTot and in Hx3 compared to controls, and were normal in the other experimental groups; those to hypercapnia ˙ were blunted only in HxTot. The results are in agreement with the idea that prenatal hypoxia blunts Ve chemosensitivity by interfering with the normal development of the carotid bodies. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Several studies have indicated that the development of respiratory control can be modulated by external influences. Of particular importance for both biological and clinical reasons is the influence played by changes in oxygenation during the perinatal period (Ling et al., 1997a; Carroll, 2003). For example, in kittens and rats, sustained hyperoxia (Ling et al., 1996, 1997b) or hypoxia (Okubo and Mortola, 1988, 1990; Hanson et al., 1989; Bavis et al., 2004; Reeves et al., 2006) in the neonatal period have resulted in a reduction in the hypoxic ventilatory response. These effects are reminiscent of the blunted ventilatory response to hypoxia in humans born and living at high altitude compared to sea level natives (Sørensen and Severinghaus, 1968; Lahiri et al., 1976). Differently, very few studies have addressed the effects of hypoxia prenatally on the development of ventilatory chemosensitivity, and obtained discordant results (Scotto et al., 1988; Gleed and Mortola, 1991; Peyronnet et

∗ Corresponding author. Tel.: +1 514 3984335 fax: +1 514 3987452. E-mail addresses: [email protected] (K. Ferner), [email protected] (J.P. Mortola). 1 Current address: Institute for Systematic Zoology, Museum of Natural History, Humboldt-University of Berlin, Invalidenstraße 43, 10115 Berlin, Germany. 1569-9048/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2008.10.004

al., 2000). Some difficulties with embryonic exposures in mammals arise from the hormonal and ventilatory responses of the mother and the placental adaptation to hypoxia, which can mask or modify the fetal responses. The avian model circumvents some of the interpretative issues of mammalian preparations, because the eggshell offers a constant diffusion barrier and the chorioallantoic membrane provides for the embryo’s gas exchange. Despite the structural differences, ventilatory control and the mechanisms of the responses to hypoxia and hypercapnia share numerous similarities between birds and mammals (Bouverot, 1978; Scheid and Piiper, 1986). Using the avian embryo model, we found that prenatal hypoxia blunted the ventilatory chemosensitivity of the hatchling (Szdzuy and Mortola, 2007b). Out of various possibilities, the most likely mechanism for this effect was considered a derangement of the normal function of the O2 -sensitive chemoreceptors, structures homologous to the carotid bodies of mammals (Milsom and Burleson, 2007). More recently, we found that also sustained embryonic hypercapnia, that is, a non-hypoxic sustained stimulation of the chemoreceptors, produced similar effects (Szdzuy and Mortola, 2008). In the avian embryo the carotid bodies develop morphologically during the middle third of incubation (Murillo-Ferrol, 1967; Kameda, 1994) and become functional probably only close to term (Mortola, 2004). Therefore, we reasoned that if a derangement of the functional

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development of the carotid body was the main mechanism for the effects of embryonic hypoxia on ventilatory chemosensitivity, then, the most obvious effects should occur with hypoxia during the last portion of incubation. Accordingly, we measured the ventilatory responses to acute hypoxia and hypercapnia in hatchlings exposed to embryonic hypoxia either for the whole three-week duration of incubation or for only the first, second and third week. 2. Methods Freshly laid fertilized eggs of White Leghorn chickens (Gallus gallus) were obtained from a local supplier. After noting the weight, at midday (embryonic day 0, E0) the eggs were placed in incubators set at the temperature (T) of 38 ◦ C and 60% relative humidity. Control eggs were in normoxia for the whole incubation (21% O2 ; Nx). Experimental eggs were incubated in hypoxia (15% O2 ) either for the whole incubation (20–21 days, HxTot), or for only the first (Hx1), second (Hx2) or third (Hx3) week of incubation. Embryos of HxTot and Hx3 hatched in hypoxia. The desired level of hypoxia (15% O2 ) was obtained by leaking a small stream of warmed and humidified N2 into the incubator from a pressurised tank, under the control of a flowmeter. The O2 concentration of the incubator was analysed continuously (Foxbox fuel cell gas analyser, Sable Systems Int., Las Vegas, NV) and displayed on a computer monitor. Incubation T and relative humidity were monitored by a data logger and a hygrometer placed inside the incubator; the former collected the T-data every 10 min, while humidity was read daily. Measurements were conducted on the day of hatching, on average about 20 h post-hatching (Table 1). 2.1. Pulmonary ventilation The breathing pattern was measured with the barometric technique originally proposed by Drorbaugh and Fenn (1955), adapted to the chick embryo and hatchling (Menna and Mortola, 2002, 2003). The approach stands on the fact that, when an animal is breathing inside a sealed chamber, the air inspired is warmed and humidified from the ambient to the pulmonary values, raising the chamber pressure; the opposite occurs in expiration. In the case of the hatchling, the animal chamber was separated into two sections, a smaller animal compartment, acting as “nest”, of about 100 ml, where the hatchling was positioned, and a larger outer compartment, of about 200 ml. This separation permitted the hatchling to stay at its customary temperature, about 37.5 ◦ C, kept constant by circulating water from a servo-controlled water bath. A transmitter powered by an external energiser-receiving unit monitored the nest T. The outer compartment was kept at a lower T, around

29 ◦ C, in order to maintain a sufficiently large T difference from the hatchling, desirable to improve the accuracy of the measurement (Mortola and Frappell, 1998). Body temperature was measured rectally, with a fine tungsten-constantan thermocouple, before and at the end of each section of the experiment. Further details of the methodology, possible problems and validation have been presented and discussed elsewhere (Szdzuy and Mortola, 2007a). Three polyethylene tubes passed through the lead of the chamber; two were for continuous flushing with the desired gas mixtures, the third line was connected to a sensitive pressure transducer for the recording of the pressure oscillation (P) related to breathing. The inflow line was connected to air or to a gas impermeable 10-l bag for the delivery of hypoxic (10 or 15% O2 ) or hypercapnic (2 or 4% CO2 ) gases. These gas mixtures were prepared by blending the pure gases from pressurised tanks, and checking their final concentration with calibrated gas analysers. The outflow line was connected to a suction pump, which maintained a steady flow of 150 ml/min, under the control of a precision needlevalve flowmeter. All signals (chamber and nest T, relative humidity, P and flow, O2 and CO2 concentrations) were converted digitally, acquired on line by a minicomputer at the sampling rate of 100 Hz and displayed breath-by-breath on a computer monitor. The volume calibration K of the chamber was obtained by injecting a known volume (Vcal) and recording the corresponding change in pressure (Pcal), K = Vcal/Pcal. For the recording of the breathing pattern, the flow through the chamber was momentarily interrupted by solenoid valves for the duration of about 2 min. This period of occlusion had negligible effects on the composition of the gases. Breathing frequency (f, breaths/min) was computed from the breath-by-breath total cycle duration of the P recording. Tidal volume (Vt, ␮l, at BTPS, body temperature, pressure and saturation conditions) was calculated from the P amplitude, K, body temperature and the T and water vapour pressure of the respirometer ˙ ml/min, (Drorbaugh and Fenn, 1955). Pulmonary ventilation (Ve, BTPS) equals f × Vt. 2.2. Gaseous metabolism Immediately before sealing the chamber for the measurements ˙ metabolic rate was measured by indirect calorimetry (oxyof Ve, gen consumption V˙ O2 , and carbon dioxide production, V˙ CO2 ) with an open-flow methodology (Frappell et al., 1992) adapted to the avian embryo and hatchling (Menna and Mortola, 2002). A steady gas flow of 150 ml/min was continuously delivered through the respirometer, and the inflow and outflow O2 and CO2 concentrations were monitored by calibrated gas analysers (Sable Systems International Fox, Henderson, NV), after the gas had passed through

Table 1 Average values during air breathing.

Number of animals Egg weight, g Hatching day Age at experiment, h Body weight, g Body temperature, ◦ C Tidal volume, ␮l Frequency, breaths/min ˙ Ve, ml/min V˙ O2 , ml/ min V˙ CO2 , ml/ min ˙ Ve/V O2 , BTPS/STPD

Nx

Hx1

Hx2

Hx3

HxTot

30 59.2 ± 0.6 20.5 ± 0.1 20.2 ± 0.5 41.0 ± 0.5 40.2 ± 0.1 267 ± 13 62.9 ± 0.2 16.1 ± 0.6 0.76 ± 0.02 0.54 ± 0.01 21.5 ± 0.8

15 58.1 ± 0.7 20.4 ± 0.2 20.0 ± 1.1 40.0 ± 0.7 40.1 ± 0.1 294 ± 20 66.2 ± 2.8 18.9 ± 1.0 0.81 ± 0.03 0.56 ± 0.02 23.8 ± 1.5

15 60.0 ± 0.8 20.5 ± 0.1 19.6 ± 0.8 42.0 ± 0.7 40.3 ± 0.2 262 ± 18 70.9 ± 3.6 17.7 ± 0.9 0.82 ± 0.04 0.58 ± 0.04 21.8 ± 1.1

15 60.6 ± 0.8 20.6 ± 0.1 20.2 ± 1.0 41.8 ± 0.7 40.5 ± 0.2 309 ± 18 64.5 ± 2.6 19.5 ± 0.8 0.91 ± 0.03a 0.61 ± 0.02 22.0 ± 1.3

15 58.1 ± 0.9 21.0 ± 0.1a,b 20.8 ± 1.2 39.6 ± 0.8 40.1 ± 0.2 294 ± 21 80.8 ± 4.1a,b,d 23.0 ± 1.4a,b,c 0.83 ± 0.03 0.60 ± 0.02 27.5 ± 1.3a,c,d

˙ pulmonary ventilation. V˙ O2 , oxygen consumption. V˙ CO2 , carbon dioxide production. Nx, normoxic incubation. Hx1, Hx2, Hx3, HxTot refer to Values are means ± 1 SEM. Ve, incubation in hypoxia (15% O2 ) during, respectively, the first, second or third week of incubation, or for the whole incubation. a,b,c,d Statistical difference from, respectively, Nx, Hx1, Hx2, Hx3 (ANOVA with post hoc Bonferroni for all possible comparisons, P < 0.05).

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a drying column. The output of the analysers was displayed during on-line acquisition. V˙ O2 and V˙ CO2 were derived from the flow rate and the inflow–outflow concentration difference. The values (at STPD, standard temperature, pressure, and dry conditions) are presented in ml/min. 2.3. Protocols All measurements were conducted at the ambient T of 37.5 ◦ C. After 30 min for acclimation, the inspired gas was switched to hypoxia (15% O2 and 10% O2 in this order, 20 min each) or to hypercapnia (2% CO2 and 4% CO2 in this order, 20 min each), with a period of air (30 min) in between the two exposures. The exposures to hypoxia and hypercapnia were in alternate order among animals, and data were collected during the last 2–3 min of each exposure. 2.4. Number of animals, analysis and statistics Experiments were conducted on 30 control hatchlings and 15 for each of the four hypoxic groups (Table 1). The hyperpneic and hyperventilatory responses to hypoxia and hypercapnia were analysed as the percent change from the air value of, respectively, ˙ and the ventilatory equivalent (Ve/ ˙ V˙ O ). This latter parameVe 2 ter takes into account the effects of changes in metabolic rate ˙ (Mortola and Gautier, 1995); indeed, especially in developon Ve ing organism, hypometabolism is a key response to hypoxia and ˙ (Mortola, can be even more important than the increase in Ve 1999). Data are presented as means ±1 SEM. Statistical comparisons among the five groups were done by one-way ANOVA, with post hoc Bonferroni’s limitations for the four comparisons between the experimental groups and controls. A difference was considered statistically significant at P < 0.05.

˙ Fig. 1. Changes in ventilatory equivalent (Ve/V O2 ) from the air value (21% inspired O2 and 0% inspired CO2 ) during exposure to hypoxia (low inspired oxygen concentration, Fio2 ) or hypercapnia (high inspired carbon dioxide concentration, Fico2 ), in hatchlings. Values (means ± 1 SE) are expressed in percent of the air value and refer to controls (air-incubation, open symbols, N = 30) and to the four experimental groups (filled symbols, N = 15 per group).

3. Results The eggs of the five groups did not differ in initial weight (Table 1). The embryos of Hx1, Hx2 and Hx3 hatched at the same time, and with similar body weight, as controls (Table 1). Differently, the HxTot group hatched about half a day later, as previously noted (Azzam et al., 2007; Szdzuy and Mortola, 2007b). This indicates that one week of hypoxia did not blunt the embryonic metabolic and growth trajectories to the extent of delaying hatching. During air breathing, body temperature, metabolic rate (V˙ O2 ˙ the breathing pattern and Ve/ ˙ V˙ O of Hx1, Hx2 and and V˙ CO2 ), Ve, 2 Hx3 did not differ from controls, the sole exception being a slightly higher V˙ O2 in Hx3 (Table 1). On the other hand, the hatchlings of the ˙ conHxTot group presented normal metabolic rate but elevated Ve, firming the previous data (Szdzuy and Mortola, 2007b). This modest ˙ V˙ O = 27.5 instead of 21.5; Table 1) is resting hyperventilation (Ve/ 2 reminiscent of what observed in rat pups after hypoxic gestation (Gleed and Mortola, 1991) and could be due to the persistence of right–left vascular shunts. In the control hatchlings, the hyperventilation during hyper˙ while capnia was approximately the same as the increase in Ve, in hypoxia the hyperventilation much exceeded the increase ˙ in Ve, because in hypoxia, differently from hypercapnia, the hypometabolism plays a major role in the hyperventilation (Menna and Mortola, 2003; Szdzuy and Mortola, 2007b). After hypoxia sustained for the whole incubation (HxTot) a marked reduction in the hyperventilatory responses to hypoxia and hypercapnia became evident. In hypoxia, this was due to the smaller increase in Vt (110% instead of 131%, averaging both hypoxic levels) and to a lesser hypometabolism (−9% instead of −15%). In hypercapnia, the lower hyperventilation was solely due to the smaller increase in Vt (148%

instead of 169%, averaging both hypercapnic levels). On the other ˙ and Ve/ ˙ V˙ O of Hx1, Hx2 and Hx3 either hand the data points of Ve 2 overlapped those of controls or fell between those of controls and ˙ and Ve/ ˙ V˙ O responses HxTot (Fig. 1). To compare statistically the Ve 2 ˙ chemosensitivity by sinwe found it convenient to express the Ve gle numbers, that is, by the slopes of the linear regressions through the data points of the hypoxic and hypercapnic tests (Szdzuy and ˙ response to hypoxia Mortola, 2008). The hyperpneic (increase in Ve) ˙ per each % drop of the HxTot group averaged 2.7% (±0.5) rise in Ve in O2 , which was about half the control value (5.5% ±0.7; P < 0.05; Fig. 2). The corresponding values for the hyperventilatory response ˙ to hypoxia (increase in Ve) were 4.4% (±0.7) in HxTot and 9.3% ±1.2 in controls (P < 0.01). In hypercapnia, the hyperpnea of the HxTot averaged 21.4% (±2.6), or about two thirds of the control value (34.8 ± 2.6%, P < 0.01). The same difference occurred for the hyperventilatory response (HxTot: 23% ±1.9, controls: 37.3 ± 2.2%; P < 0.001). The average hypoxic and hypercapnic responses of Hx1, Hx2 were not statistically different from controls. On the contrary, in Hx3 the hypoxic hyperpnea (2.8 ± 0.6%) and hyperventilation (4.9 ± 0.7%) had values similar to those of HxTot and in both cases significantly lower than in controls (P < 0.05; Fig. 2). As for the HxTot group, also in the case of the Hx3 group the lower hypoxic hyperventilation was due to a combination of a smaller increase in Vt and a lesser hypometabolism. 4. Discussion These results, while confirming that prenatal hypoxia affects the development of ventilatory chemosensitivity (Szdzuy and Mortola,

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˙ filled bars) and hyperFig. 2. Slope of the hyperpneic (increase in ventilation, Ve, ˙ ventilatory responses (increase in ventilatory equivalent, Ve/V O2 , open bars) during hypoxia (top) or hypercapnia (bottom) in control hatchlings and in those of the four hypoxic experimental conditions. The slopes are the percent change per unitary change in inspired O2 or CO2 concentration. Hx1, Hx2, Hx3, HxTot refer to incubation in hypoxia (15% O2 ) during, respectively, the first, second or third week of incubation, or for the whole incubation. Columns are mean values, bars represent 1 SEM. * Statistically significant difference from controls (P < 0.05, ANOVA, with post hoc Bonferroni limitations for the four comparisons).

2007b), indicate that the last third of incubation is the sensitive period for the blunting of the hypoxic hyperventilation. Several arguments and considerations had suggested previously that prenatal hypoxia may blunt the hatchling’s chemosensitivity via an alteration in the normal development of the carotid bodies. First, postnatally in mammals substantial evidence points toward an impairment of the peripheral chemoreceptors as the most likely mechanism for the plasticity of the hypoxic ventilatory response after a sustained alteration of the O2 level during the neonatal period (Ling et al., 1997a; Carroll, 2003; Powell, 2007). Second, hypercapnia during incubation diminished the ventilatory response to CO2 of adult finches and quails (Williams and Kilgore, 1992; Bavis and Kilgore, 2001) and the hypoxic chemosensitivity of chicken hatchlings (Szdzuy and Mortola, 2008). Hypercapnia has a variety of effects on acid-base not shared by, or even opposite to those of hypoxia, but, like hypoxia, acts on the chemorecep˙ chemosensitivity tors. Hence, the finding that the blunting of Ve occurred similarly after prenatal hypoxia or hypercapnia suggested an involvement of the peripheral chemoreceptors. Also, it is known that prolonged hypoxia in the chicken causes numerous alterations in the ultrastructural and immunoreactive characteristics of the glomus cells (Kameda et al., 1998), although similar analyses in hypoxic embryos have never been done. The fact that with hyper˙ levels almost twice those capnia the HxTot hatchlings reached Ve attained in hypoxia (Fig. 1) indicated that their blunted hypoxic hyperventilation could not be attributed to neuro-mechanical impediments. The current results give further support to the idea that the carotid bodies are the major culprit for the blunting effects of ˙ chemosensitivity. In fact, prenatal hypoxia on the newborn’s Ve in the chicken embryo, the primordial buds of the carotid bodies reach their final location at the bifurcation of the brachiocephalic artery only by day 8, and the glomus cells can be recognized only a few days later (Murillo-Ferrol, 1967; Kameda, 1994). Functional, ˙ chemo-responses can be recognized during although minimal, Ve the earlier phases (internal and external “pipping phases”) of the

hatching process (Pettit and Whittow, 1982; Menna and Mortola, 2003; Szdzuy and Mortola, 2007a). Hence, the fact that embryonic ˙ chemosensitivity only when occurring during hypoxia affected Ve the last third of incubation (Hx3 or HxTot) and had no effects when occurring at earlier embryonic times (Hx1 or Hx2) strongly suggests an involvement of the carotid body. Furthermore, the fact that in Hx3, differently from HxTot, hatching occurred at the normal time indicates that the blunting of the embryonic growth trajectory in ˙ HxTot (Azzam et al., 2007) is not responsible for the diminished Ve chemosensitivity of the hatchling. There is too little information to explain why the sensitivity to hypercapnia was compromised in HxTot but was not in Hx3, other than simply considering the fact that the hypercapnic response involves a larger and more disperse group of chemosensors than the hypoxic response does. As in mammals, also in birds ˙ response to hypercapnia the carotid bodies contribute to the Ve (Bouverot, 1978; Scheid and Piiper, 1986). The intrapulmonary CO2 sensitive receptors, equivalent to the mammalian intrapulmonary stretch receptors, are known to be active at hatching (Pilarski and Hempleman, 2007) but their role on the regulation of the breathing pattern and chemoresponses is not known. Equally unknown is the possible contribution of the centrally located CO2 -sensory regions. Continuous hypoxia has broad-ranging effects on the development of the embryo, affecting its growth and metabolic trajectory and the development of the internal organs (Azzam and Mortola, 2007; Azzam et al., 2007; Mortola and Cooney, 2008). Hence, it is possible that, in addition to the carotid bodies, continuous hypoxia ˙ may impact on receptors or sites of integration involved in the Ve response to CO2 that are left untouched when hypoxia is of shorter duration. In conclusion, hypoxic exposure for the whole incubation or for just the last week of incubation blunted the ventilatory response to hypoxia of the hatchling, while hypoxic exposures earlier in development had no effects. Hence, these results give further support to the idea that prenatal hypoxia interferes with the normal development of the carotid bodies. References Azzam, M.A., Mortola, J.P., 2007. Organ growth in chicken embryos during hypoxia: implications on organ “sparing” and “catch-up growth”. Respir. Physiol. Neurobiol. 159, 155–162. Azzam, M.A., Szdzuy, K., Mortola, J.P., 2007. Hypoxic incubation blunts the development of thermogenesis in chicken embryos and hatchlings. Am. J. Physiol. (Regul. Integr. Comp. Physiol.) 292, R2373–R2379. Bavis, R.W., Kilgore, D.L., 2001. Effects of embryonic CO2 exposure on the adult ventilatory response in quail: does gender matter? Respir. Physiol. 126, 183–199. Bavis, R.W., Olson, E.B., Vidruk, E.H., Fuller, D.D., Mitchell, G.S., 2004. Developmental plasticity of the hypoxic ventilatory response in rats induced by neonatal hypoxia. J. Physiol. 557, 645–660. Bouverot, P., 1978. Control of breathing in birds compared with mammals. Physiol. Rev. 58, 604–655. Carroll, J.L., 2003. Plasticity in respiratory motor control. invited review: developmental plasticity in respiratory control. J. Appl. Physiol. 94, 375–389. Drorbaugh, J.E., Fenn, W.O., 1955. A barometric method for measuring ventilation in newborn infants. Pediatrics 16, 81–87. Frappell, P.B., Lanthier, C., Baudinette, R.V., Mortola, J.P., 1992. Metabolism and ventilation in acute hypoxia: a comparative analysis in small mammalian species. Am. J. Physiol. (Regul. Integr. Comp. Physiol.) 262, R1040–1046. Gleed, R.D., Mortola, J.P., 1991. Ventilation in newborn rats after gestation at simulated high altitude. J. Appl. Physiol. 70, 1146–1151. Hanson, M.A., Kumar, P., Williams, B.A., 1989. The effect of chronic hypoxia upon the development of respiratory chemoreflexes in the newborn kitten. J. Physiol. 411, 563–574. Kameda, Y., 1994. Electron microscopic study on the development of the carotid body and glomus cell groups distributed in the wall of the common carotid artery and its branches in the chicken. J. Comp. Neurol. 348, 544–555. Kameda, Y., Miura, M., Hayashida, Y., 1998. Different effects of prolonged isocapnic hypoxia on the carotid body and the glomus cells in the wall of the common carotid artery of the chicken. Brain Res. 805, 191–206. Lahiri, S., DeLaney, R.G., Brody, J.S., Simpser, M., Velasquez, T., Motoyama, E.K., Polgar, C., 1976. Relative role of environmental and genetic factors in respiratory adaptation to high altitude. Nature 261, 133–135.

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