Hypoxia exposure can have distinct physiological effects between early developmental and adult life stages, but it is unclear how the effects of hypoxia may progress during continuous exposure throughout life. We examined this issue in deer mice (Peromyscus maniculatus) from a population native to high altitude. Mice were bred in captivity in one of three treatment groups: normoxia (controls), life-long hypoxia (∼12 kPa O2 from conception to adulthood) and parental hypoxia (normoxia from conception to adulthood, but parents previously exposed to hypoxia). Metabolic, thermoregulatory and ventilatory responses to progressive stepwise hypoxia and haematology were then measured at post-natal day (P) 14 and 30 and/or in adulthood. Life-long hypoxia had consistent effects across ages on metabolism, attenuating the declines in O2 consumption rate (V̇O2) and body temperature during progressive hypoxia compared with control mice. However, life-long hypoxia had age-specific effects on breathing, blunting the hypoxia-induced increases in air convection requirement (quotient of total ventilation and V̇O2) at P14 and P30 only, but then shifting breathing pattern towards deeper and/or less frequent breaths at P30 and adulthood. Hypoxia exposure also increased blood–O2 affinity at P14 and P30, in association with an increase in arterial O2 saturation in hypoxia at P30. In contrast, parental hypoxia had no effects on metabolism or breathing, but it increased blood–O2 affinity and decreased red cell haemoglobin content at P14 (but not P30). Therefore, hypoxia exposure has some consistent effects across early life and adulthood, and some other effects that are unique to specific life stages.
Environmental conditions experienced during early development can play an important role in shaping adult phenotypes. Developmental plasticity is defined as the process where phenotypes are altered by early life experiences, and these effects can often be persistent into adult life, and are irreversible (Burggren, 2020; Burggren and Reyna, 2011; Moczek et al., 2011). Evidence suggests that the phenotypic responses to environmental stressors during development are not always the same as those during adult life, and responses can even vary depending on the developmental stage over which stressors are experienced (e.g. prenatal versus postnatal development) (Burggren and Reyna, 2011; Carroll, 2003). Additionally, conditions experienced by parents can affect their offspring (e.g. multi-generational or transgenerational effects), even if those offspring never experienced the conditions directly at any stage of development (Burton and Metcalfe, 2014; Ho and Burggren, 2012). Developmental plasticity has been shown to alter many aspects of animal physiology, including systems-level determinants of metabolism and O2 transport (Bavis, 2005; Burggren and Reyna, 2011; Carroll, 2003; Ho and Burggren, 2012; Vulesevic and Perry, 2006). However, many questions about the persistence and life stage-specificity of developmental plasticity remain unresolved.
Low O2 (hypoxia) exposure during early life can lead to plastic changes in respiratory physiology, but the responses to hypoxia differ when exposure occurs in prenatal versus postnatal development. For example, prenatal hypoxia in utero increases total ventilation and the magnitude of the hypoxic ventilatory response (HVR) when measured early after birth in rats (16 h to 7 days old) (Gleed and Mortola, 1991; Peyronnet et al., 2000, 2007), and these effects have been shown to persist for several weeks after offspring are born and raised in normoxia. In contrast, early postnatal hypoxia exposure blunts or even abolishes the HVR in some species (e.g. rats and llamas) (Brooks and Tenney, 1968; Lumbroso and Joseph, 2009; Okubo and Mortola, 1990), which could be a result of disruptions in the normal development of the carotid bodies or other parts of the hypoxic chemoreflex (Bavis, 2005; Brooks and Tenney, 1968; Lumbroso and Joseph, 2009; Peyronnet et al., 2000; Sterni et al., 1999; Yilmaz et al., 2005). Indeed, this early post-natal stage also coincides with a critical period of development in the neural networks that control breathing (Wong-Riley et al., 2019). However, little is known about the interaction between life stage-specific effects and hypoxia across prenatal and early postnatal development, or if hypoxia then persists further into adult life. Furthermore, few studies have examined how parental exposure to hypoxia affects respiratory physiology in offspring.
High altitude is characterized by both hypoxia and cold temperatures, challenging the ability of organisms to maintain a high enough O2 supply to support exercise and thermoregulation. Control of breathing by hypoxia is altered in many high-altitude taxa compared with low-altitude taxa, and in some cases, these changes have an evolved (i.e. genetic) basis. Several highland taxa breathe more (sometimes as a result of a deeper breathing pattern) and have an enhanced HVR compared with their low-altitude counterparts, including Tibetan humans, deer mice (Peromyscus maniculatus), plateau pika (Ochotona curzoniae) and bar-headed geese (Anser indicus) (Beall et al., 1997; Ivy and Scott, 2017a, 2018; Lague et al., 2016; Moore, 2000; Pichon et al., 2009; Scott and Milsom, 2006). Many high-altitude taxa also exhibit increased haemoglobin (Hb)–O2 affinity (Galen et al., 2015; Natarajan et al., 2015a,b, 2016, 2018; Projecto-Garcia et al., 2013; Signore et al., 2019; Tufts et al., 2015; Zhu et al., 2018). These changes have not occurred in all highland taxa (Beall, 2000; Brutsaert et al., 2005; Ivy et al., 2018; Lague et al., 2017; Schwenke et al., 2007; Storz, 2016; Tashi et al., 2014), but in the taxa in which they have arisen, they are likely valuable for augmenting O2 uptake and transport in hypoxia. Few studies have examined the ontogenetic development of the unique respiratory physiology of high-altitude natives, but recent studies in deer mice suggest that the physiology of highlanders starts diverging from that of lowlanders during the development of endothermy over the first few weeks of life (Ivy et al., 2020; Robertson et al., 2019). However, the effects of hypoxia exposure during early development on the respiratory physiology of high-altitude natives is not well understood.
The objective of this study was to investigate the plasticity of metabolism, thermoregulation and respiratory physiology in response to hypoxia exposure throughout development and adulthood in deer mice (Peromyscus maniculatus), and to determine whether parental hypoxia exposure had any persistent effects on its own. Deer mice are broadly distributed across North America and can be found from sea level (∼21 kPa O2) to over 4300 m (∼12 kPa O2) elevation in the Rocky Mountains (Hock, 1964; Natarajan et al., 2015b; Snyder et al., 1982). Adults at high altitude sustain high metabolic rates in the wild (Hayes, 1989), and have evolved a higher aerobic capacity (V̇O2,max) in hypoxia than low-altitude deer mice and white-footed mice (P. leucopus, a congeneric species that is restricted to low altitudes) (Cheviron et al., 2012, 2013, 2014; Lui et al., 2015; Tate et al., 2017, 2020). This is associated with evolved changes across the O2 transport pathway, including increases in Hb–O2 affinity and changes in various other traits that enhance O2 supply and utilization by thermogenic tissues, such as skeletal muscle (Dawson et al., 2018; Lau et al., 2017; Lui et al., 2015; Mahalingam et al., 2017; Scott et al., 2015; Snyder et al., 1982; Storz et al., 2009, 2010; Tate et al., 2017, 2020). Evolved changes in the control of breathing have also arisen in high-altitude deer mice. Highlanders exhibit higher total ventilation and a more effective breathing pattern (deeper but less frequent breaths) than their lowland counterparts in comparisons made between mice that are born and raised in normoxia (Ivy and Scott, 2017a, 2018; Ivy et al., 2020). However, the ventilatory phenotype of high-altitude deer mice does not change in response to chronic hypoxia exposure during adulthood, in contrast to the robust increases in total ventilation that are exhibited by low-altitude mice (Ivy and Scott, 2017a,b, 2018). Here, we sought to examine whether high-altitude deer mice were also unresponsive to hypoxia exposure in early life, or whether developmental plasticity might lead to unique adaptive changes in the control of breathing and in other metabolic and respiratory traits that do not occur as a result of plasticity during adulthood.
MATERIALS AND METHODS
Mouse populations and breeding design
Captive breeding populations were established from wild populations of deer mice [Peromyscus maniculatus rufinus (Wagner 1845)] native to high altitude near the summit of Mount Evans, CO, USA (39°35′18″N, 105°38′38″W; 4350 m above sea level). Wild adults were transported to McMaster University (∼50 m above sea level) and housed in common laboratory conditions, and were used as parental stock to produce first generation (G1) laboratory progeny. Breeding pairs were held in individual cages, the male was removed when the female was visibly pregnant, and pups were weaned and moved to separate cages at post-natal day (P) 21. G1 mice were similarly used as parental stock to produce second generation (G2) progeny. Experiments were conducted on several distinct families of G2 mice. Mice were generally held at 24–25°C under a photoperiod of 12 h:12 h light:dark, and were provided with unlimited access to standard rodent chow and water. All animal protocols followed guidelines established by the Canadian Council on Animal Care and were approved by the McMaster University Animal Research Ethics Board (AUP 16-01-02).
We used a standardized breeding design to expose G2 mice to normoxia (21 kPa O2) or hypoxia (12 kPa O2) (Fig. 1). Each breeding pair was first allowed to raise four litters, in order to avoid potential effects of variation in litter size and resource allocation that may arise across the first few litters (Kirkland and Layne, 1989). Different breeding pairs were used for studies of young deer mice (P14–P30) than for studies of adult deer mice. In studies of young mice, each pair conceived and raised litter 5 in standard cage conditions of normobaric normoxia (which we call the ‘normoxia control group’). After weaning, the pups of litter 5 remained in normoxia but the mother and father were moved to hypobaric hypoxia (barometric pressure of 60 kPa, ∼12 kPa O2; simulating the hypoxia at an elevation of 4300 m). Parents were then allowed to conceive litter 6, which was born and raised in hypobaric hypoxia (the ‘life-long hypoxia group’). After weaning litter 6, the pups of litter 6 remained in hypoxia but the breeding pairs were returned to normoxia and allowed to conceive and birth litter 7 in normoxia. Litter 7 was then raised in normoxia (the ‘parental hypoxia group’). Studies of adult mice were carried out in the same manner, except that litters 8 and 9 were used for the life-long hypoxia and parental hypoxia groups, respectively. Each litter and treatment group contained a mix of both female and male pups. Exposures to hypobaric hypoxia were conducted using specially designed hypobaric chambers (Lui et al., 2015; McClelland et al., 1998). Cages were cleaned twice a week, which for hypoxia exposures required that mice be returned to normobaria for a brief period (<20 min). Measurements on young mice were generally conducted at ages P14 and P30, but owing to temporary suspension of research activities during the COVID-19 pandemic, two of five individuals from the parental hypoxia group had to be measured at P27 instead of P30. Measurements on adult mice were made between 6 and 8 months of age.
Acute hypoxia responses
Hypoxia responses were measured in unrestrained mice using barometric plethysmography, respirometry and pulse oximetry techniques that we have used in previous studies (Ivy and Scott, 2017b, 2018; Ivy et al., 2020). Mice were placed in a whole-body plethysmograph chamber (530 ml) that was supplied with normoxic air (21 kPa O2, balance N2) at flow rates appropriate for the size and metabolic rate of the mouse (300 ml min−1 for P14, 450 ml min−1 for P27/30, and 600 ml min−1 for adults), such that metabolism in normoxia led to changes in O2 and CO2 pressures of only ∼0.05–0.2 kPa. Experiments were performed at room temperature (24–25°C), which is below the lower limit of the thermoneutral zone, as lower critical temperatures for deer mice are ∼28–30°C depending on the population (Brower and Cade, 1966; Hayward, 1965). Mice were given 20–40 min to adjust to the chamber and experimental conditions until stable breathing and O2 consumption were observed. Mice remained at 21 kPa O2 for an additional 20 min, followed by exposure to acute stepwise reductions in inspired O2 pressure (PO2: 16, 12, 10, 9 and 8 kPa O2) for 20 min at each step. Incurrent gas composition was set by mixing dry compressed gases using precision flow meters (Sierra Instruments, Monterey, CA, USA) and a mass flow controller (MFC-4, Sable Systems, Las Vegas, NV, USA). Body temperature was measured at the beginning and end of the experiments (immediately when mice were removed from the chamber) using a mouse rectal probe (RET-3-ISO, Physitemp).
Breathing and O2 consumption rates (V̇O2) were determined using the last 10 min at each inspired PO2 when responses had reached a steady state. Gas composition was measured continuously in incurrent and excurrent air flows that were subsampled at 200 ml min−1. For incurrent air, the subsampled air was dried with prebaked Drierite and then the O2 fraction was measured using a galvanic fuel cell O2 analyzer (FC-10, Sable Systems). For excurrent air, water vapour pressure was measured using a thin-film capacitive water vapour analyzer (RH-300, Sable Systems), the gas stream was dried with prebaked Drierite, the O2 fraction was measured as above, and the CO2 fraction was measured using an infrared CO2 analyzer (CA-10, Sable Systems). These data were used to calculate V̇O2, expressed in volumes at standard temperature and pressure (STP) using appropriate equations for dry air as described by Lighton (2008). Chamber temperature was continuously recorded with a thermocouple (TC-2000, Sable Systems) through the lid of the chamber. Breathing frequency, tidal volume and total ventilation were measured using whole-body plethysmography as previously described (Ivy and Scott, 2017b, 2018; Ivy et al., 2020). Total ventilation and tidal volume data are expressed in volumes at body temperature and pressure of saturated air (BTPS). Air convection requirement is the quotient of total ventilation and V̇O2. All of the above data were acquired using a PowerLab 16/32 and Labchart 8 Pro software (ADInstruments, Colorado Springs, CO, USA). Arterial O2 saturation (SaO2) was measured in young mice (but not in adult mice) using MouseOx Plus pulse oximeter collar sensors and data acquisition system (Starr Life Sciences, Oakmont, PA, USA). This was enabled by removing fur from around the neck ∼2 days before experiments.
Blood was collected for haematology immediately following acute hypoxia experiments (as described above). All mice were euthanized with an overdose of isoflurane followed by decapitation. Blood Hb content was measured using Drabkin's reagent (Sigma-Aldrich, Mississauga, ON, Canada) according to the manufacturer's instructions, and haematocrit was measured by centrifuging blood in a heparinized capillary tube at 12,700 g for 5 min. Mean cell Hb content was the quotient of Hb concentration and haematocrit. In young mice (but not in adult mice), we also generated O2 dissociation curves at 37°C using a Hemox Analyzer (TCS Scientific, New Hope, PA, USA) using 10 µl of whole blood in 5 ml of buffer containing 100 mmol l−1 Hepes, 50 mmol l−1 EDTA, 100 mmol l−1 KCl, 0.1% bovine serum albumin and 0.2% antifoaming agent at pH 7.4 (TCS Scientific). The O2 affinity of the erythrocytes (red cell P50, the PO2 at 50% saturation) was calculated using Hemox Analytic Software (TCS Scientific).
For acute hypoxia responses, we used two-factor ANOVA to examine the main effects of treatment (e.g. normoxia control group versus life-long hypoxia group) and inspired PO2 (repeated measure) as well as their interaction within each age group. For body mass and haematology, we used two-factor ANOVAs to evaluate the effects of treatment, age and their interaction. Holm–Šidák post hoc tests were used throughout as appropriate. All data met the normality and equal variance assumptions of two-factor ANOVAs. Values are reported as means±s.e.m. and all statistical analyses were conducted with SigmaStat software (v3.5) with a significance level of P<0.05.
Effects of life-long hypoxia on metabolic, thermoregulatory and ventilatory responses to acute hypoxia
Life-long hypoxia consistently blunted the depression of O2 consumption rate and body temperature during acute hypoxia exposure across ages in high-altitude deer mice (Fig. 2, Table 1). Hypoxia-exposed mice maintained significantly higher O2 consumption rates in severe hypoxia (below 9–12 kPa) compared with controls at P14 (PO2×treatment, P<0.001), P30 (PO2×treatment, P=0.008) and in adulthood (PO2×treatment, P=0.049) (Fig. 2A,D,G). O2 consumption rate declined by ∼26-70% from normoxic levels at or below ∼12 kPa O2 in control mice, and the magnitude of the decline was greater in younger mice than in older mice. However, O2 consumption rates only declined by ∼8–44% from normoxic levels at or below 12 kPa O2 in mice raised in hypoxia. Similarly, mice exposed to life-long hypoxia exhibited little change in body temperature in severe hypoxia, in contrast to the significant decline in body temperature observed in normoxic controls (Fig. 2B,E,H), and there were significant PO2×treatment interactions at every age (Table 1).
Effects of life-long hypoxia on total ventilation and air convection requirement differed across ages (Fig. 3, Table 1). Total ventilation increased in response to acute hypoxia, as reflected by significant main effects of inspired PO2, with significant PO2×treatment interactions at all ages (P<0.005) that appeared to be driven by higher total ventilation in severe hypoxia (8–12 kPa) in the life-long hypoxia group (Fig. 3A,C,E). However, when total ventilation was expressed relative to O2 consumption (the air convection requirement), this metric was reduced in young mice from life-long hypoxia groups (PO2×treatment: P=0.013 at P14; P=0.015 at P30), but then returned to being equal or slightly greater than control levels in adult mice exposed to life-long hypoxia (Fig. 3B,D,F).
Life-long hypoxia also appeared to have age-specific effects on breathing pattern (Figs 4 and 5, Table 1). At P14, the life-long hypoxia group had an altered breathing frequency response to acute hypoxia (PO2×treatment, P<0.001) but exhibited no differences in tidal volume (Fig. 4A,B). However, this variation appeared to result from the effects of developmental hypoxia on total ventilation, because there was a similar relationship between total ventilation and tidal volume between treatment groups (Fig. 5A). By P30, the life-long hypoxia group began to exhibit differences in breathing pattern, because hypoxia exposure tended to reduce breathing frequency (treatment effect, P=0.054; PO2×treatment interaction: P=0.004) and deepen tidal volume (treatment effect, P=0.003; PO2×treatment interaction: P=0.022) (Fig. 4C,D), and there was a divergence in the relationship between total ventilation and tidal volume between treatment groups (Fig. 5B). Differences in breathing pattern were also apparent in adulthood, as reflected by a further divergence in the total ventilation–tidal volume relationship between control and life-long hypoxia groups (Fig. 5C), and life-long hypoxia tended to be associated with higher tidal volumes (PO2×treatment, P=0.015) (Fig. 4E,F).
The differences in metabolic and ventilatory responses to hypoxia between treatments did not result from differences in body mass, which increased as expected with age (age effect, P<0.001) but was similar between normoxic controls (P14, 7.07±0.68 g; P30, 14.40±1.75 g; adults, 22.26±0.98 g) and life-long hypoxia groups (P14, 7.13±0.57 g; P30, 14.02±0.31 g; adults, 21.94±1.74 g) (Table 2).
Effects of life-long hypoxia on arterial O2 saturation and heart rate during acute hypoxia
There were age-specific differences in arterial O2 saturation and heart rate in life-long hypoxia groups, which were measured during acute stepwise hypoxia in P14 and P30 mice (Fig. 6, Table 1). In early development (P14), the life-long hypoxia group exhibited no significant differences in arterial O2 saturation in hypoxia (Fig. 6A) but decreased heart rate less in response to severe hypoxia than the control group (PO2×treatment, P<0.001) (Fig. 6B). This was in contrast to the effects of hypoxia exposure later in development (P30), when the life-long hypoxia group exhibited a smaller reduction in arterial O2 saturation in hypoxia compared with control mice (PO2×treatment, P=0.019), but only in severe hypoxia (8 kPa) (Fig. 6C). However, the groups did not differ significantly in their heart rate responses to hypoxia (Fig. 6D).
Effects of life-long hypoxia on haematology
Life-long hypoxia also affected haematology (Fig. 7, Table 2). Life-long hypoxia significantly increased haematocrit across all ages of mice (treatment effect, P<0.001; treatment×age: P=0.034) (Fig. 7A). However, although blood Hb content increased with age, it was not significantly different between treatment groups (Fig. 7B), as a result of variation in mean cell Hb content (Fig. 7C). Mice in the life-long hypoxia group also had increased O2 affinity of red cells (main effect of treatment: P<0.001): P50 remained unchanged at ∼5.5 kPa O2 from P14 to P30 in normoxia controls, but in the life-long hypoxia groups it was only ∼4.6 kPa at P14 and ∼5.0 kPa at P30 (Fig. 7D).
Effects of parental hypoxia exposure on metabolism and respiratory physiology
In contrast to the strong effects of life-long hypoxia on several metabolic and respiratory phenotypes, parental hypoxia had almost no persistent effects at any age. The parental hypoxia group was statistically indistinguishable from normoxic controls for measurements of O2 consumption rate, body temperature, total ventilation, air convection requirement, breathing frequency, tidal volume, arterial O2 saturation, heart rate and haematocrit (Tables S1–S4). However, P14 mice in the parental hypoxia group had lower Hb contents and P50 than normoxic controls, which drove the nearly significant or significant effects of parental hypoxia exposure on blood and mean cell Hb contents (treatment×age interactions: P=0.061 and 0.034, respectively; Fig. 8B,C) and on red cell O2 affinity (age effect, P=0.034; Fig. 8D, Table S4). However, this parental treatment effect was abolished by P27/30, by which time Hb contents and P50 of the parental hypoxia group had increased to control levels.
We have shown that exposure to hypoxia throughout life has some effects on metabolism and breathing that are persistent throughout development and some other effects that are specific to a subset of life stages. Life-long hypoxia exposure reduced the magnitude of hypoxic metabolic depression in mice from early life through to adulthood, but effects of hypoxia exposure throughout life on breathing pattern only became apparent in juveniles (P30) and adults. The latter effects of hypoxia are in stark contrast to our previous observations in adult deer mice from high altitude, which exhibit no changes in breathing pattern in response to chronic hypoxia (Ivy and Scott, 2017a, 2018). In contrast, parental hypoxia alone had no persistent effects on the control of metabolism or breathing. Therefore, plasticity in response to developmental hypoxia probably contributes to the ability of deer mice to cope with hypoxia at high altitudes.
The attenuation of metabolic and body temperature depression in response to acute hypoxia that was observed here after life-long hypoxia exposure (Fig. 2) is similar to our previous findings on the effects of hypoxia acclimation in adult deer mice from high altitude (Ivy and Scott, 2017a). Acute hypoxia exposure leads to declines in metabolism and/or body temperature in many species of mammals and birds (Dzal and Milsom, 2019; Scott et al., 2008; Tattersall and Milsom, 2003; Tattersall et al., 2002), likely as a result of a controlled reduction in the body temperature set-point (Tattersall and Milsom, 2009). Part of the decline in metabolism observed here was probably a result of decreases in active thermogenesis, because mice were below their lower critical temperature (Brower and Cade, 1966; Hayward, 1965), and resting metabolic rates in normoxia were above the basal metabolic rate of deer mice (Russell and Chappell, 2007). Glutamatergic neurons in the median preoptic nucleus of the hypothalamus appear to play a key role in regulating metabolism and body temperature in rodents (Hrvatin et al., 2020; Takahashi et al., 2020), but the mechanism underlying how these neurons integrate hypoxia signals and elicit metabolic and body temperature depression in acute hypoxia remains unresolved. It is possible that chronic hypoxia augments O2 supply to the hypothalamus and/or to the tissues that send afferent information to the hypothalamus, thus blunting the signal for metabolic and body temperature depression. It is also possible that chronic hypoxia leads to neuroplasticity in metabolic control networks or to plasticity in thermogenic tissues, thus blunting the efferent response to hypoxic signals. Whatever the mechanisms at play, our findings in deer mice are consistent with findings in some other rodent species exposed to chronic hypoxia during prenatal development (Peyronnet et al., 2000). These findings suggest that the effects of chronic hypoxia on the control of metabolism and body temperature are similar throughout life, rather than being a unique response of a particular life stage.
Chronic hypoxia blunted the hypoxic ventilatory response during early postnatal life, as reflected by the attenuated response of air convection requirement to acute hypoxia, but this effect did not persist into adulthood (Fig. 3). Developmental hypoxia exposure is known to induce plastic changes in the control of breathing, but whereas prenatal hypoxia increases total ventilation and the hypoxic ventilatory response (Gleed and Mortola, 1991; Peyronnet et al., 2000, 2007), postnatal hypoxia leads to the opposite effects (Brooks and Tenney, 1968; Lumbroso and Joseph, 2009; Okubo and Mortola, 1990). Therefore, our results on the effects of combined exposure to prenatal and postnatal hypoxia in early life mirror the effects of postnatal hypoxia. Exposure to postnatal hypoxia elicits its effects by disrupting the normal development of the carotid bodies or other components of the hypoxic chemoreflex (Bavis, 2005; Brooks and Tenney, 1968; Lumbroso and Joseph, 2009; Peyronnet et al., 2000; Sterni et al., 1999; Yilmaz et al., 2005). If developmental hypoxia has similar effects in high-altitude deer mice, it could exacerbate the slower carotid body growth that these mice exhibit during early development in normoxia compared with low-altitude deer mice (Ivy et al., 2020). The effects of developmental hypoxia on ventilation that were observed here could have also been influenced by mild cold exposure, because measurements were made at temperatures slightly below the thermoneutral zone (Brower and Cade, 1966; Hayward, 1965). Nevertheless, the effects of developmental hypoxia appear to be reversed or otherwise compensated for after continued exposure to hypoxia, such that air convection requirements in adult mice after life-long hypoxia are much like those in normoxic mice.
Developmental hypoxia had no influence on the relationship between breathing frequency and tidal volume in early postnatal life, but differences in breathing pattern arose with continued exposure to hypoxia into juvenile and adult life. In particular, mice exposed to hypoxia until juvenile or adult life stages tended to breathe deeper but less frequently at multiple given levels of total ventilation (Figs 4 and 5), a breathing pattern that should augment alveolar ventilation. These findings contrast our previous observations of adult deer mice from high altitude, in which hypoxia acclimation resulted in no changes to total ventilation, breathing frequency or tidal volume, unlike low-altitude mice that do increase total ventilation and deepen breathing pattern during hypoxia acclimation (Ivy and Scott, 2017a,b, 2018). These results suggest that plasticity in response to developmental hypoxia has unique effects on breathing pattern that are not elicited by hypoxia acclimation in adulthood.
Hypoxia exposure during development increased blood–O2 affinity (Fig. 7). The effects of developmental hypoxia (as well as parental hypoxia) on blood–O2 affinity at P14 could be partially explained by the apparent reduction in mean cell Hb content, which could reflect red cell swelling that might have reduced the intracellular concentration of negative allosteric modifiers such as 2,3-diphosphoglycerate. Another possibility is that developmental hypoxia induced the post-natal expression of embryonic Hb isoforms, which tend to have a higher O2 affinity than the adult-expressed isoforms and are not expressed after birth in either high- or low-altitude deer mice raised in normoxia (Ivy et al., 2020). However, the hypoxia-induced increases in blood–O2 affinity did not improve arterial O2 saturation until juvenile life stages. This could reflect the fact that arterial O2 saturation is a result of the combined effects of blood–O2 affinity and pulmonary O2 transport. Developmental hypoxia can constrain lung growth during the first weeks of life in domestic mice and rats (Jochmans-Lemoine et al., 2018), and our results here and the work of others (Brooks and Tenney, 1968; Lumbroso and Joseph, 2009; Okubo and Mortola, 1990) suggests that post-natal hypoxia can reduce total ventilation (absolute and relative to metabolism). Therefore, increases in Hb–O2 affinity may help offset effects of reductions in air convection requirement and possibly pulmonary O2 diffusing capacity early after birth, and then later help augment arterial O2 saturation once breathing increases and lung growth has advanced.
Studies of high-altitude natives hold great promise for uncovering naturally evolved strategies for overcoming hypoxia. Here, we have shown that developmental plasticity resulting from hypoxia exposure during early life likely contributes to the ability of deer mice to cope with hypoxia at high altitudes. The effects of plasticity on hypoxia responses and tolerance are overlaid upon the effects of adaptive evolution in high-altitude populations (Ivy and Scott, 2017a, 2018; Ivy et al., 2020). Whether high-altitude adaptation has led to evolved changes in developmental plasticity remains unclear, as the response to developmental hypoxia has yet to be compared between high-altitude and low-altitude mice. Future research targeting the genetic basis of the evolution and plasticity of respiratory physiology in high-altitude deer mice could provide appreciable insight into the mechanisms underlying nature's solutions to O2 deprivation.
Conceptualization: C.M.I., G.R.S.; Methodology: C.M.I., G.R.S.; Formal analysis: C.M.I.; Investigation: C.M.I.; Resources: G.R.S.; Data curation: C.M.I.; Writing - original draft: C.M.I.; Writing - review & editing: C.M.I., G.R.S.; Visualization: G.R.S.; Supervision: G.R.S.; Project administration: G.R.S.; Funding acquisition: G.R.S.
C.M.I. was supported by a Natural Science and Engineering Research Council of Canada (NSERC) PGS-D and an Ontario Graduate Scholarship; G.R.S. was supported by an NSERC Discovery Grant and the Canada Research Chairs Program.
The authors declare no competing or financial interests.