The respiratory variables of unrestrained one-day-old mice maintained in the recording chamber under normoxia remained stable throughout the recording period (n = 4). Baseline values were defined after 20 min of adaptation in the chamber and the following values of the respiratory variables expressed as the percentage baseline. At the end of the recording period, the mean f _R and mean V_T were 106.9 ± 14.3% and 120.9 ± 7.6% of control values, respectively, leading to a mean V˙_E of 131.2 ± 12.4% of control values. This observation shows the stability of the respiratory variables during the period of retention in the recording chamber.

The mean f _R was 166.1 ± 12.5 breaths·min⁻¹, the mean V_T 35.6 ± 9.6 μl·g⁻¹, and the mean V˙_E 5.4 ± 1.2 ml·g⁻¹·min⁻¹ during the normoxic control period in the unrestrained one-day-old mice that were subsequently submitted to hypoxia (n = 8). The mean V˙_E tended to peak at 158.1 ± 29.4% of control values 2 min after the onset of hypoxic exposure (Figure 1). It then decreased significantly dropping below control (p < 0.04) and below the high values observed at the onset of hypoxia (p < 0.03) to 68.9 ± 10.0% of control values 25 min after the onset of hypoxic exposure (Figure 1). Changes in V˙_E were related to the respective effects of hypoxia on both f _R and V_T. The mean f _R was at 115.4 ± 19.0% of control values 2 min after the onset of hypoxic exposure and at 86.5 ± 7.5% of control values 25 min after the onset of the hypoxic test. The mean V_T was at 138.8 ± 18.6% of control values 2 min after the onset of hypoxic exposure and at 73.7 ± 15.8% of control values 25 min after the onset of hypoxic exposure.

One-day-old mice maintained under normoxic conditions (control animals) had a relatively small number of c-FOS-positive cells both in respiratory related areas of the medulla oblongata: NTS, VLM, RTN/pFRG, PP, 10N, 12N, and medullary raphe nuclei (Table 1; Figures 2A,C,G,I,K), and related respiratory areas of the pons:. PB, KF, A5, LC, and SubC (Table 1; Figures 2E,M).

We measured respiratory variables using whole-body flow barometric plethysmography in freely moving newborn mice to limit confinement-related stress. This is a commonly used technique to measure pulmonary ventilation in unrestrained and non-anesthetized animals that consists of recording the changes of pressure in a chamber caused by breathing (Tree et al., 2014). As the inspired gas is warmed and humidified from the ambient to the pulmonary values, the total pressure in the recording chamber increases; the opposite occurs during expiration. The accuracy of V_T calculations based on whole-body plethysmography data of small animals has been questioned (Enhorning et al., 1998; Mortola and Frappell, 1998). The body temperature, humidity, and temperature of the recording chamber are difficult to control due to the small size of the devices used for such small animals, leading to highly variable absolute V_T and V˙_E values. These values should thus be viewed with caution. This has led researchers to base their analysis on the changing values of V_T and V˙_E as a percentage of control values. Furthermore, small mammals, particularly newborns, develop hypoxic hypothermia due to hypometabolism and increased heat loss. In our experiments, the temperature of the animal was evaluated before and after each experiment via an oral probe because of the difficulty to measure body temperature continuously with our home-made plethysmograph. Thus, our plethysmographic data should be taken with caution. Indeed, the plethysmography was used primarily to simply assure the presence of a biphasic respiratory response in one day old mice, as classically described in other newborns (Simakajornboon et al., 2004; Bairam et al., 2013).

In addition to these considerations, the pups were isolated from their mother for a long time during the plethysmographic recordings. We observed no significant modification of the respiratory variables in animals maintained under normoxia despite this long separation that may have led to physiological changes. This indicates that changes in the respiratory variables developed by newborn mice submitted to hypoxic conditions are effectively due to the reduction in O₂, and not their long retention.

Analysis of c-FOS protein revealed increased activity in neurons of the mPB and dSubC. Such an increase has not been reported in other newborn rodents (see the Figure 3 in Berquin et al., 2000b). In the fetus of sheep, the participation of cells located in the dorsolateral part of the pons to HVD, more precisely those in the mPB, KF, and SubC, has already been discussed in the literature (Gluckman and Johnston, 1987; Walker, 1995; Breen et al., 1997; Nitsos and Walker, 1999; Walker et al., 2000; Teppema and Dahan, 2010). Hypoxia induces an increase in c-FOS expression in neurons of the mPB and SubC in fetal, but not newborn, sheep (Breen et al., 1997; Nitsos and Walker, 1999). The hypoxia-activated cells of the SubC have been proposed to be O₂ sensors and to strongly inhibit breathing in fetal sheep, but to lose this effect after birth because of their inhibition by peripheral chemoreceptors (Breen et al., 1997). The present observed increase in c-FOS-positive cells in the dSubC, of which a significant portion are catecholaminergic, may be indicative of activated cells with similar properties to those identified in the fetus by Walker and collaborators (Walker, 1995; Breen et al., 1997; Nitsos and Walker, 1999). In such a model, they may be involved in HVD in one-day-old mice and constitute a non-inhibited fetal mechanism at birth different from that of other studied newborn mammals, such as newborn rats, which are more mature at birth than mice (Gauda, 2006). This feature of one-day-old mice may make this model particularly suitable for mechanistic studies on the occurrence of excessive HVD in premature newborns or even on the occurrence of SIDS. Indeed, SIDS has been hypothesized to result from the awakening of a defense mechanism of the fetus consisting of a strong depressive response to hypoxemia that limits O₂ consumption (Lavezzi, 2015). A recent publication by Lavezzi and collaborators emphasized hypoplasia and neurochemical alterations of KF neurons underlying the pathogenetic mechanisms of SIDS (Lavezzi, 2015). Our present data and previously published work in fetal and newborn sheep (Breen et al., 1997) underscore the interest in searching for possible alterations in the integrity of dSubC neurons in the brainstems of infants who died of SIDS.

In rats, SubC and mPB have been shown to contain pre-motoneurons that innervate hypoglossal motoneurons involved in maintaining upper airway patency during breathing (Dobbins and Feldman, 1995; Fay and Norgren, 1997). The presently observed decrease in c-FOS protein levels in the hypoglossal nucleus cells may be linked to an inhibitory drive coming from the dSubC and mPB during hypoxia. This hypothetic inhibitory pathway may contribute to HVD by reducing the airway opening when activated under hypoxia. Noradrenergic cells of the dSubC innervate the hypoglossal nucleus, of which the motoneurons are activated by noradrenalin (Aldes et al., 1992; Fenik et al., 2008; Funk et al., 2011). If dSubC cells are involved in the decrease in the expression of c-FOS in the hypoglossal nucleus, they would not be those immunoreactive for TH. Further experiments are necessary to determine the pharmacological phenotype of the c-FOS-positive, but TH-negative, cells of the dSubC.

We observed a large increase in the number of c-FOS-positive cells under hypoxia in the RMg. Such an increase has not been reported in newborns of other species (Breen et al., 1997; Teppema et al., 1997; Horn et al., 2000; Berquin et al., 2000b) or adults subjected to moderate hypoxia (Berquin et al., 2000a), but has been reported in adults under severe hypoxia (Erickson and Millhorn, 1994). Based on data from the literature, we suggest that hypoxia-activated RMg neurons contribute to HVD. Indeed, activation of RMg neurons attenuate the activation of NTS neurons by peripheral chemoreceptors (Perez and Ruiz, 1995) and RMg neurons exert a moderating influence on the V_T under hypoxic conditions (Gargaglioni et al., 2003). Thus, activation of RMg neurons in one-day-old mice may participate in the HVD. With the exception of a few cells, the hypoxic c-FOS-positive cells that we observed were not serotoninergic. In addition to serotoninergic neurons, the RMg contains GABAergic neurons that could be involved in HVD. This is supported by the fact that iv administration of bicuculline, a GABA_A antagonist, reduces the respiratory inhibition that occurs after electrical stimulation of the RMg (Cao et al., 2006).

Hypoxia induced an increase in the number of c-FOS-positive cells in areas recognized to be involved in early hypoxic hyperventilation triggered by stimulation of peripheral chemoreceptors (see for revue Teppema and Dahan, 2010) i.e., the cNTS, mNTS, and lPB. The cNTS and mNTS constitute the major central site for the integration of inputs from peripheral chemoreceptors whereas only few afferents from peripheral chemoreceptors project toward the VLM (Finley and Katz, 1992). The two subnuclei of the NTS are the first relay between peripheral afferences and the VLM and lPB (Nunez-Abades et al., 1993; Hayward and Felder, 1995). All of these connections conceivably provide a common basis for the hypoxic increase in c-FOS expression that we observed in these structures in one-day-old mice and that has been observed in other species at several stages of development (Teppema et al., 1997; Berquin et al., 2000b).

The dual detection of c-FOS and TH revealed that no, or only very few, neurons activated by hypoxia in the cNTS, mNTS, and VLM were catecholaminergic. Previous data about the catecholaminergic character of c-FOS-positive hypoxia-activated cells in these structures made in adult mammals have showed that no, or very few, c-FOS hypoxia-activated neurons in the NTS are also catecholaminergic, whereas a large portion of c-FOS hypoxia-activated neurons in the VLM are also immunoreactive for TH (Erickson and Millhorn, 1994; Soulier et al., 1997; Teppema et al., 1997). The only data from the literature concerning this issue in newborn animals is the study of c-FOS expression in newborn rats under high-altitude conditions showing that an altitude of 8000 m induces an increase in the number of c-FOS positive cells in the NTS and VLM with a large portion of them in the VLM also immunoreactive for TH (Kaur et al., 2001). Taking into account these data, our results suggest that the effect of hypoxia on the VLM differed in one-day-old mice from other studied newborn or adult mammals. This difference may depend on the maturation state of either VLM neurons or brainstem connections. The absence of VLM catecholaminergic neuron activation in one-day-old mice could conceivably participate in HVD, and even strengthen it in this model, as they have been associated with hypoxic hyperventilation (Erickson and Millhorn, 1994; Soulier et al., 1997; Teppema et al., 1997). If this observation is likely due to the degree of immaturity of the central nervous system of the one-day-old mice as we assume (Teppema et al., 1997; Gauda, 2006; Mallard and Vexler, 2015), a similar mechanism may be involved in exacerbating the respiratory problems encountered at birth in premature infants.

We observed an increase in the number of c-FOS-positive cells in the RTN/pFRG. The RTN/pFRG is involved in the respiratory adaptation to both hypercapnia (Teppema et al., 1997; Nattie, 2001; Okada et al., 2002; Guyenet et al., 2005; Stornetta et al., 2006; Teppema and Dahan, 2010; Guyenet and Bayliss, 2015) and hypoxia (Bodineau et al., 2000a, 2001; Bodineau and Larnicol, 2001; Takakura et al., 2006; Voituron et al., 2006, 2011; Teppema and Dahan, 2010). The RTN/pFRG contains both CO₂ and O₂ sensor cells (Guyenet et al., 2005; Voituron et al., 2006, 2011; Onimaru et al., 2008; Lazarenko et al., 2009; Guyenet and Mulkey, 2010) and also integrates chemosensory inputs from peripheral chemoreceptors (Berquin et al., 2000a; Bodineau et al., 2000b; Takakura et al., 2006). The well-identified CO₂-sensor cells of the RTN/pFRG (Lazarenko et al., 2009; Guyenet and Mulkey, 2010) are derived from neurons that express Phox2b, Atoh-1, and Egr-2, and are characterized by the presence of PHOX2B, NK1 receptors, VGLUT2, TASK-2, GPR4, and GALANIN (Weston et al., 2004; Stornetta et al., 2006; Onimaru et al., 2008; Dubreuil et al., 2009; Rose et al., 2009; Guyenet and Mulkey, 2010; Guyenet and Bayliss, 2015; Ruffault et al., 2015). In the present work, none of the c-FOS positive cells of the RTN/pFRG were PHOX2B-positive. This suggests that although the one-day-old mice displayed severe hypoventilation under hypoxia, hypoventilation may not entail an increase in CO₂ that should have been detected by the PHOX2B cells of the RTN/pFRG. Regardless of the mechanisms responsible for HVD, the concomitant hypometabolism that maintains isocapnia, which constitutes a feature of the newborn response to hypoxia (Mortola, 2004), appears to be extremely effective in one-day-old mice. The activation of PHOX2B-negative RTN/pFRG cells may depend on intrinsic O₂-sensing properties that we previously demonstrated by measuring c-FOS expression in brainstem spinal cord preparations from newborn rodents (Voituron et al., 2006, 2011). It is unlikely that the increase in the number of c-FOS-positive/PHOX2B-negative cells in RTN/pFRG depends on peripheral chemoreceptor inputs. Takakura and collaborators have shown that such indirect activation implicates cNTS glutamatergic neurons projecting to PHOX2B RTN/pFRG cells (Takakura et al., 2006). This suggests possible immaturity of the connection between cNTS cells activated by peripheral chemoreceptors and RTN/pFRG PHOX2B-positive cells.

We observed a greater increase in c-FOS expression in the more medial group, called PP, with 30% of the c-FOS-positive cells containing 5-HT, than for the RTN/pFRG. This group of cells has been suggested to be the positional homolog of the human medullary arcuate nucleus (Filiano and Kinney, 1992), a structure in which abnormalities have been reported in infants who died of SIDS (Filiano and Kinney, 1992; Paterson et al., 2006; Kinney et al., 2011). The increase in c-FOS protein levels induced by hypoxia in the PP is of particular interest given the possible involvement of both the arcuate nucleus and abnormalities in the respiratory response to hypoxia in SIDS (Paterson et al., 2006; Kinney et al., 2011). The PP contains GABAergic and serotoninergic neurons (Stornetta and Guyenet, 1999; Weston et al., 2004; Stornetta et al., 2005) and displays multiple sites of projections including the pre-Bötzinger complex and intermediolateral column (Holtman et al., 1990; Jansen et al., 1995). Functionally, the PP has been reported to be involved in autonomic regulation under hypoxia. Darnall and collaborators reported that the destruction of 5-HT medullary neurons, including those of the ROb and PP increase the arousal latency from sleep induced by hypoxia in newborn rats (Darnall et al., 2016). In the relatively immature one-day-old mouse (Gauda, 2006), we only observed an increase in c-FOS- and 5-HT-positive cells in the PP, but not in the ROb, where the number of c-FOS-positive cells tended to decrease. Thus, our results suggest that 5-HT PP neurons must have a critical role in arousal from sleep under hypoxia in immature newborn mammals. Our results combined with the fact that hypoplasia of this region has been shown in newborn death due to SIDS, suggests that the dysfunction of 5-HT neurons of the PP in infants, and particularly in premature infants, could result in a high risk situation due to the decrease of arousal from sleep during hypoxia.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.