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Down syndrome (DS) is a genetic disease characterized by a supernumerary chromosome 21. Intellectual deficiency (ID) is one of the most prominent features of DS. Central nervous system defects lead to learning disabilities, motor and language delays, and memory impairments. At present, a prenatal treatment for the ID in DS is lacking. Subcutaneous administration of synthetic preimplantation factor (sPIF, a peptide with a range of biological functions) in a model of severe brain damage has shown neuroprotective and anti-inflammatory properties by directly targeting neurons and microglia. Here, we evaluated the effect of PIF administration during gestation and until weaning on Dp(16)1Yey mice (a mouse model of DS). Possible effects at the juvenile stage were assessed using behavioral tests and molecular and histological analyses of the brain. To test the influence of perinatal sPIF treatment at the adult stage, hippocampus-dependent memory was evaluated on postnatal day 90. Dp(16)1Yey pups showed significant behavioral impairment, with impaired neurogenesis, microglial cell activation and a low microglial cell count, and the deregulated expression of genes linked to neuroinflammation and cell cycle regulation. Treatment with sPIF restored early postnatal hippocampal neurogenesis, with beneficial effects on astrocytes, microglia, inflammation, and cell cycle markers. Moreover, treatment with sPIF restored the level of DYRK1A, a protein that is involved in cognitive impairments in DS. In line with the beneficial effects on neurogenesis, perinatal treatment with sPIF was associated with an improvement in working memory in adult Dp(16)1Yey mice. Perinatal treatment with sPIF might be an option for mitigating cognitive impairments in people with DS., (© 2024. The Author(s).)

Whether peripheral chemoreceptor response is altered in congenital central hypoventilation syndrome (CCHS) remains debated. Our aim was to prospectively evaluate both peripheral and central CO 2 chemosensitivity and to evaluate their correlations with daytime Pco 2 and arterial desaturation during exercise in CCHS. To this end, tidal breathing was recorded in patients with CCHS allowing the calculation of loop gain and its components {steady-state controller (assumed to mainly be peripheral chemosensitivity) and plant gains using a bivariate [end-tidal Pco 2 ([Formula: see text]) and ventilation] constrained model}, a hyperoxic, hypercapnic ventilatory response test (central chemosensitivity), and a 6-min walk test (arterial desaturation). The results of loop gain were compared with those previously obtained in a healthy group of similar age. The study prospectively included 23 subjects with CCHS, without daytime ventilatory support; the subjects had a median age of 10 (5.6 to 27.4) yr (15 females) with moderate polyalanine repeat mutation (PARM: 20/25, 20/26, n = 11), severe PARM (20/27, 20/33, n = 8), or non-PARM ( n = 4). As compared with 23 healthy subjects (4.9-27.0 yr), the subjects with CCHS had a decreased controller gain and an increased plant gain. Mean daytime [Formula: see text] level of subjects with CCHS correlated negatively to both Log(controller gain) and the slope of CO 2 response. Genotype was not related to chemosensitivity. Arterial desaturation on exercise correlated negatively with Log(controller) gain but not with the slope of the CO 2 response. In conclusion, we demonstrate that peripheral CO 2 chemosensitivity is altered in some patients with CCHS and that the daytime [Formula: see text] depends on central and peripheral chemoreceptor responses. NEW & NOTEWORTHY Altered central CO 2 chemosensitivity is a hallmark of congenital central hypoventilation syndrome (CCHS). Peripheral CO 2 chemosensitivity can be partly assessed by controller gain measurement obtained from tidal breathing recording. In young subjects with CCHS, this study shows that both central and peripheral CO 2 sensitivities independently contribute to daytime Pco 2 . Hypocapnia during nighttime-assisted ventilation is associated with higher peripheral chemosensitivity that is further associated with lesser arterial desaturation at walk.

Background: Whether dysfunctional breathing (DB) subtype classification is useful remains undetermined. The hyperventilation provocation test (HVPT) is used to diagnose DB. This test begins with a 3-min phase of hyperventilation during which fractional end-tidal CO 2 (FETCO 2 ) decreases that could be an assessment of plant gain, which relies on CO 2 stores. Our aim was to assess 1) whether the children suffering from different subtypes of DB exhibit decreased plant gain and 2) the relationships between HVPT characteristics and plant gain. Methods: We retrospectively selected 48 children (median age 13.5 years, 36 females, 12 males) who exhibited during a cardiopulmonary exercise test either alveolar hyperventilation (transcutaneous PCO 2 < 30 mmHg, n = 6) or inappropriate hyperventilation (increased VE'/V'CO 2 slope) without hypocapnia ( n = 18) or dyspnea without hyperventilation ( n = 18) compared to children exhibiting physiological breathlessness (dyspnea for sports only, n = 6). These children underwent tidal-breathing recording (ventilation and FETCO 2 allowing the calculation of plant gain) and a HVPT. Results: The plant gain was significantly higher in the physiological group as compared to the dyspnea without hyperventilation group, p = 0.024 and hyperventilation without hypocapnia group, p = 0.008 (trend for the hyperventilation with hypocapnia group, p = 0.078). The slope of linear decrease in FETCO 2 during hyperventilation was significantly more negative in physiological breathlessness group as compared to hyperventilation without hypocapnia group ( p = 0.005) and dyspnea without hyperventilation group ( p = 0.049). Conclusion: The children with DB, regardless of their subtype, deplete their CO 2 stores (decreased plant gain), which may be due to intermittent alveolar hyperventilation, suggesting the futility of our subtype classification., Competing Interests: 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., (Copyright © 2023 Bokov, Peiffer, Gallego, Pautrat, Matrot and Delclaux.)

Background: Autonomic nervous system (ANS) dysregulation has been described in congenital central hypoventilation syndrome (CCHS). The objectives were to describe heart rate variability (HRV) analyses in children suffering from CCHS both while awake and asleep and their relationships with both ambulatory blood pressure (BP) and ECG monitoring results., Methods: This retrospective study enrolled children with CCHS (n = 33, median age 8.4 years, 18 girls) who had BP and ECG monitored during the same 24 h. From the latter, HRV analyses were obtained during daytime and nighttime., Results: The prevalences of hypertension and sinus pauses were 33% (95% confidence interval [CI]: 18-52) and 18% (95% CI: 7-35), respectively. The decrease in systolic BP at night negatively correlated with an increase in very low frequency (VLF) and LF powers at night, and the longest RR interval positively correlated with daytime VLF and LF powers. Among the three groups of children (polyalanine repeat expansion mutation [PARM], moderate [20/25 and 20/26], severe [20/27 and 20/33], and non-PARMs), the prevalence of elevated BP or hypertension was different: in PARM subjects: 6/18 moderate, 7/9 severe versus 0/6 in non-PARM (p = 0.002)., Conclusion: Modifications of cardiac ANS are associated with systemic hypertension and the occurrence of sinus pauses in CCHS., Impact: Children with congenital central hypoventilation syndrome (CCHS) exhibit an increased prevalence of hypertension and sinus pauses that are linked to cardiac autonomic nervous system dysfunction. Sinus pauses are the main manifestation of sinus nodal dysfunction in children with CCHS. The increased prevalence of hypertension, especially at nighttime, is a new finding in CCHS. Sinus nodal dysfunction can be due to the sole impairment of the cardiac autonomic nervous system. Ambulatory blood pressure and ECG monitoring are mandatory in patients with CCHS., (© 2022. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Introduction: Congenital Central Hypoventilation Syndrome, a rare disease caused by PHOX2B mutation, is associated with absent or blunted CO 2 /H + chemosensitivity due to the dysfunction of PHOX2B neurons of the retrotrapezoid nucleus. No pharmacological treatment is available. Clinical observations have reported non-systematic CO 2 /H + chemosensitivity recovery under desogestrel., Methods: Here, we used a preclinical model of Congenital Central Hypoventilation Syndrome, the retrotrapezoid nucleus conditional Phox2b mutant mouse, to investigate whether etonogestrel, the active metabolite of desogestrel, led to a restoration of chemosensitivity by acting on serotonin neurons known to be sensitive to etonogestrel, or retrotrapezoid nucleus PHOX2B residual cells that persist despite the mutation. The influence of etonogestrel on respiratory variables under hypercapnia was investigated using whole-body plethysmographic recording. The effect of etonogestrel, alone or combined with serotonin drugs, on the respiratory rhythm of medullary-spinal cord preparations from Phox2b mutants and wildtype mice was analyzed under metabolic acidosis. c-FOS, serotonin and PHOX2B were immunodetected. Serotonin metabolic pathways were characterized in the medulla oblongata by ultra-high-performance liquid chromatography., Results: We observed etonogestrel restored chemosensitivity in Phox2b mutants in a non-systematic way. Histological differences between Phox2b mutants with restored chemosensitivity and Phox2b mutant without restored chemosensitivity indicated greater activation of serotonin neurons of the raphe obscurus nucleus but no effect on retrotrapezoid nucleus PHOX2B residual cells. Finally, the increase in serotonergic signaling by the fluoxetine application modulated the respiratory effect of etonogestrel differently between Phox2b mutant mice and their WT littermates or WT OF1 mice, a result which parallels with differences in the functional state of serotonergic metabolic pathways between these different mice., Discussion: Our work thus highlights that serotonin systems were critically important for the occurrence of an etonogestrel-restoration, an element to consider in potential therapeutic intervention in Congenital Central Hypoventilation Syndrome patients., Competing Interests: 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., (Copyright © 2023 Casciato, Bianchi, Reverdy, Joubert, Delucenay-Clarke, Parrot, Ramanantsoa, Sizun, Matrot, Straus, Similowski, Cayetanot and Bodineau.)

Instable ventilatory control is an endotypic trait of obstructive sleep apnea syndrome (OSAS). This study aimed to evaluate the relationships between the anatomical compromise of the upper (oro- and naso-pharynx) and lower airways and ventilatory control (measured by chemical loop gain) in otherwise healthy children suffering from moderate to severe OSAS (apnea hypopnea index ≥ 5/hour). The children underwent ear, nose and throat examination, measurement of impedance of the respiratory system that allowed characterizing peripheral lung mechanics using the extended Resistance-Inertance-Compliance model. Physiologically constrained analytical model based on tidal breathing analysis allowed for the computation of steady-state plant gain, steady-state controller gain (CG0) and steady-state loop gain (LG0). Medium-frequency components of the feedback control system were then deduced. Fifty children (median age 11.2 years) were enrolled. Oropharyngeal obstruction was associated with decreased CG0 (0.6 [0.2; 1.0] vs 1.5 [0.5; 6.6] L.s - 1 .mmHg - 1 , p = 0.038) and LG0 (0.4 [0.2; 1.1] vs 1.2 [0.4; 9.3], p = 0.027), while nasal obstruction did not modify ventilatory control parameters. In a multivariate analysis Medium-Frequency PG was negatively related to minute ventilation and respiratory system compliance. Both upper (tonsil hypertrophy) and lower (compliance of respiratory system) airways are linked to ventilatory control in children with moderate to severe OSAS., (© 2022. The Author(s).)

Intrauterine growth restriction (IUGR) increases the risk of bronchopulmonary dysplasia (BPD), one of the major complications of prematurity. Antenatal low-protein diet (LPD) exposure in rats induces IUGR and mimics BPD-related alveolarization disorders. Peroxisome proliferator-activated receptor-γ (PPARγ) plays a key role in normal lung development and was found deregulated following LPD exposure. The objective of this article was to investigate the effects of nebulized curcumin, a natural PPARγ agonist, to prevent IUGR-related abnormal lung development. We studied rat pups antenatally exposed to an LPD or control diet (CTL) and treated with nebulized curcumin (50 mg/kg) or vehicle from postnatal ( P ) days 1 to 5 . The primary readouts were lung morphometric analyses at P21 . Immunohistochemistry ( P21 ) and microarrays ( P6 and P11 ) were compared within animals exposed to LPD versus controls, with and without curcumin treatment. Quantitative morphometric analyses revealed that LPD induced abnormal alveolarization as evidenced by a significant increase in mean linear intercept (MLI) observed in P21 LPD-exposed animals. Early curcumin treatment prevented this effect, and two-way ANOVA analysis demonstrated significant interaction between diet and curcumin both for MLI [ F (1,39) = 12.67, P = 0.001] and radial alveolar count at P21 [ F (1,40) = 6.065, P = 0.0182]. Immunohistochemistry for fatty acid binding protein 4 (FABP4), a major regulator of PPARγ pathway, showed a decreased FABP4 + alveolar cell density in LPD-exposed animals treated by curcumin. Transcriptomic analysis showed that early curcumin significantly prevented the activation of profibrotic pathways observed at P11 in LPD-exposed animals. Nebulized curcumin appears to be a promising strategy to prevent alveolarization disorders in IUGR rat pups, targeting pathways involved in lung development.

Background: Optimal management of anesthesia-induced respiratory depression requires identification of the neural pathways that are most effective in maintaining breathing during anesthesia. Lesion studies point to the brainstem retrotrapezoid nucleus. We therefore examined the respiratory effects of common anesthetic/analgesic agents in mice with selective genetic loss of retrotrapezoid nucleus neurons (Phox2b mice, hereafter designated "mutants")., Methods: All mice received intraperitoneal ketamine doses ranging from 100 mg/kg at postnatal day (P) 8 to 250 mg/kg at P60 to P62. Anesthesia effects in P8 and P14 to P16 mice were then analyzed by administering propofol (100 and 150 mg/kg at P8 and P14 to P16, respectively) and fentanyl at an anesthetic dose (1 mg/kg at P8 and P14 to P16)., Results: Most mutant mice died of respiratory arrest within 13 min of ketamine injection at P8 (12 of 13, 92% vs. 0 of 8, 0% wild type; Fisher exact test, P < 0.001) and P14 to P16 (32 of 42, 76% vs. 0 of 59, 0% wild type; P < 0.001). Cardiac activity continued after terminal apnea, and mortality was prevented by mechanical ventilation, supporting respiratory arrest as the cause of death in the mutants. Ketamine-induced mortality in mutants compared to wild types was confirmed at P29 to P31 (24 of 36, 67% vs. 9 of 45, 20%; P < 0.001) and P60 to P62 (8 of 19, 42% vs. 0 of 12, 0%; P = 0.011). Anesthesia-induced mortality in mutants compared to wild types was also observed with propofol at P8 (7 of 7, 100% vs. 0 of 17,7/7, 100% vs. 0/17, 0%; P < 0.001) and P14 to P16 (8 of 10, 80% vs. 0 of 10, 0%; P < 0.001) and with fentanyl at P8 (15 of 16, 94% vs. 0 of 13, 0%; P < 0.001) and P14 to P16 (5 of 7, 71% vs. 0 of 11, 0%; P = 0.002)., Conclusions: Ketamine, propofol, and fentanyl caused death by respiratory arrest in most mice with selective loss of retrotrapezoid nucleus neurons, in doses that were safe in their wild type littermates. The retrotrapezoid nucleus is critical to sustain breathing during deep anesthesia and may prove to be a pharmacologic target for this purpose.

The loop gain (LG) is defined as the ratio of a ventilatory response over the perturbation in ventilation, and it is used to analyze ventilatory control stability. The LG can be derived from minute ventilation (V̇e), end-tidal Pco 2 ([Formula: see text]), and end-tidal Po 2 ([Formula: see text]) values. Several methods of LG assessment have been developed, which have never been compared. We evaluated the computability, the short-term repeatability, and the agreement of six published (or slightly modified) models for LG determination. These models included three unconstrained autoregressive models, univariate (V̇e), bivariate (V̇e, [Formula: see text]), and trivariate (V̇e, [Formula: see text], and [Formula: see text]), and three analytical transfer function constrained models based on V̇e, V̇e and CO 2 -sensitivity, and V̇e and central and peripheral CO 2 sensitivities, respectively. The models were tested with tidal breathing data in 37 awake healthy subjects (median age 35 yr; 23 women, 14 men). Modeling failed in 11, 0, and 0 subjects for the three unconstrained models, respectively, and 4, 1, and 9 subjects for the three constrained models, respectively. Bland and Altman analyses of the LG values in the medium frequency range of two separate recordings demonstrated good repeatability for four models, excluding univariate and trivariate unconstrained models. The four repeatable models gave LG values that were in agreement (medium frequency LG, median 0.100-0.210), although the constrained model based on V̇e systematically overestimated LG values. The variances explained by these models were ∼20%. In conclusion, model-based analyses of tidal breathing were performed with different approaches that gave comparable results for chemical LG and explained variance. NEW & NOTEWORTHY Several methods of chemical loop gain measurement have been published but never compared. We show that a better repeatability is obtained with analytical constrained models compared with autoregressive unconstrained models and that the repeatable models gave comparable results of loop gain, even if the calculation based on ventilation-only recording gave higher values than those obtained with both ventilation and end-tidal Pco 2 recording. The explained variance of ventilation was similar whatever the model.

Pituitary growth hormone (GH) and insulinlike growth factor (IGF)-1 are anabolic hormones whose physiological roles are particularly important during development. The activity of the GH/IGF-1 axis is controlled by complex neuroendocrine systems including two hypothalamic neuropeptides, GH-releasing hormone (GHRH) and somatostatin (SRIF), and a gastrointestinal hormone, ghrelin. The neurotransmitter acetylcholine (ACh) is involved in tuning GH secretion, and its GH-stimulatory action has mainly been shown in adults but is not clearly documented during development. ACh, together with these hormones and their receptors, is expressed before birth, and somatotroph cells are already responsive to GHRH, SRIF, and ghrelin. We thus hypothesized that ACh could contribute to the modulation of the main components of the somatotropic axis during development. In this study, we generated a choline acetyltransferase knockout mouse line and showed that heterozygous mice display a transient deficit in ACh from embryonic day 18.5 to postnatal day 10, and they recover normal ACh levels from the second postnatal week. This developmental ACh deficiency had no major impact on weight gain and cardiorespiratory status of newborn mice. Using this mouse model, we found that endogenous ACh levels determined the concentrations of circulating GH and IGF-1 at embryonic and postnatal stages. In particular, serum GH level was correlated with brain ACh content. ACh also modulated the levels of GHRH and SRIF in the hypothalamus and ghrelin in the stomach, and it affected the levels of these hormones in the circulation. This study identifies ACh as a potential regulator of the somatotropic axis during the developmental period.

Apnea of prematurity (AOP) is considered a risk factor for neurodevelopmental disorders in children based on epidemiological studies. This idea is supported by studies in newborn rodents in which exposure to intermittent hypoxia (IH) as a model of AOP significantly impairs development. However, the severe IH used in these studies may not fully reflect the broad spectrum of AOP severity. Considering that hypoxia appears neuroprotective under various conditions, we hypothesized that moderate IH would protect the neonatal mouse brain against behavioral stressors and brain damage. On P6, each pup in each litter was randomly assigned to one of three groups: a group exposed to IH while separated from the mother (IH group), a control group exposed to normoxia while separated from the mother (AIR group), and a group of untreated unmanipulated pups left continuously with their mother until weaning (UNT group). Exposure to moderate IH (8% O2) consisted of 20 hypoxic events/hour, 6 h per day from postnatal day 6 (P6) to P10. The stress generated by maternal separation in newborn rodents is known to impair brain development, and we expected this effect to be smaller in the IH group compared to the AIR group. In a separate experiment, we combined maternal separation with excitotoxic brain lesions mimicking those seen in preterm infants. We analyzed memory, angiogenesis, neurogenesis and brain lesion size. In non-lesioned mice, IH stimulated hippocampal angiogenesis and neurogenesis and improved short-term memory indices. In brain-lesioned mice, IH decreased lesion size and prevented memory impairments. Contrary to common perception, IH mimicking moderate apnea may offer neuroprotection, at least in part, against brain lesions and cognitive dysfunctions related to prematurity. AOP may therefore have beneficial effects in some preterm infants. These results support the need for stratification based on AOP severity in clinical trials of treatments for AOP, to determine whether in patients with moderate AOP, these treatments are beneficial or deleterious.

The ALK (Anaplastic Lymphoma Kinase) gene encodes a tyrosine kinase receptor preferentially expressed in the central and peripheral nervous systems. A syndromic presentation associating congenital neuroblastoma with severe encephalopathy and an abnormal shape of the brainstem has been described in patients harbouring de novo germline F1174V and F1245V ALK mutations. Here, we investigated the phenotype of knock-in (KI) mice bearing the AlkF1178L mutation (F1174L in human). Although heterozygous KI mice did not reproduce the severe breathing and feeding difficulties observed in human patients, behavioral tests documented a reduced activity during dark phases and an increased anxiety of mutated mice. Matings of heterozygotes yielded the expected proportions of wild-type, heterozygotes and homozygotes at birth but a high neonatal lethality was noticed for homozygotes. We documented Alk expression in several motor nuclei of the brainstem involved in the control of sucking and swallowing. Evaluation of basic physiological functions 12 hours after birth revealed slightly more apneas but a dramatic reduced milk intake for homozygotes compared to control littermates. Overall, our data demonstrate that Alk activation above a critical threshold is not compatible with survival in mice, in agreement with the extremely severe phenotype of patients carrying aggressive de novo ALK germline mutations.

Neonates respond to hypoxia initially by increasing ventilation, and then by markedly decreasing both ventilation (hypoxic ventilatory decline) and oxygen consumption (hypoxic hypometabolism). This latter process, which vanishes with age, reflects a tight coupling between ventilatory and thermogenic responses to hypoxia. The neurological substrate of hypoxic hypometabolism is unclear, but it is known to be centrally mediated, with a strong involvement of the 5-hydroxytryptamine (5-HT, serotonin) system. To clarify this issue, we investigated the possible role of VGLUT3, the third subtype of vesicular glutamate transporter. VGLUT3 contributes to glutamate signalling by 5-HT neurons, facilitates 5-HT transmission and is expressed in strategic regions for respiratory and thermogenic control. We therefore assumed that VGLUT3 might significantly contribute to the response to hypoxia. To test this possibility, we analysed this response in newborn mice lacking VGLUT3 using anatomical, biochemical, electrophysiological and integrative physiology approaches. We found that the lack of VGLUT3 did not affect the histological organization of brainstem respiratory networks or respiratory activity under basal conditions. However, it impaired respiratory responses to 5-HT and anoxia, showing a marked alteration of central respiratory control. These impairments were associated with altered 5-HT turnover at the brainstem level. Furthermore, under cold conditions, the lack of VGLUT3 disrupted the metabolic rate, body temperature, baseline breathing and the ventilatory response to hypoxia. We conclude that VGLUT3 expression is dispensable under basal conditions but is required for optimal response to hypoxic stress in neonates.

Adult motor coordination requires strong coincident cortical excitatory input to hyperpolarized medium spiny neurons (MSNs), the dominant neuronal population of the striatum. However, cortical and subcortical neurons generate during development large ongoing patterns required for activity-dependent construction of networks. This raises the question of whether immature MSNs have adult features from early stages or whether they generate immature patterns that are timely silenced to enable locomotion. Using a wide range of techniques including dynamic two-photon imaging, whole cell or single-channel patch clamp recording in slices from Nkx2.1-GFP mice, we now report a silencing of MSNs that timely coincides with locomotion. At embryonic stage (as early as E16) and during early postnatal days, genetically identified MSNs have a depolarized resting membrane potential, a high input resistance and lack both inward rectifying (IK(IR)) and early slowly inactivating (I(D)) potassium currents. They generate intrinsic voltage-gated clustered calcium activity without synaptic components. From postnatal days 5-7, the striatal network transiently generates synapse-driven giant depolarizing potentials when activation of cortical inputs evokes long lasting EPSCs in MSNs. Both are mediated by NR2C/D-receptors. These immature features are abruptly replaced by adult ones before P10: MSNs express IK(IR) and I(D) and generate short lasting, time-locked cortico-striatal AMPA/NMDA EPSCs with no NR2C/D component. This shift parallels the onset of quadruped motion by the pup. Therefore, MSNs generate immature patterns that are timely shut off to enable the coordination of motor programs.

The Phox2b genesis necessary for the development of the autonomic nervous system, and especially, of respiratory neuronal circuits. In the present study, we examined the role of Phox2b in ventilatory and thermoregulatory responses to hypoxic stress, which are closely related in the postnatal period. Hypoxic stress was generated by strong thermal stimulus, combined or not with reduced inspired O(2). To this end, we exposed 6-day-old Phox2b(+/-) pups and their wild-type littermates (Phox2b(+/+)) to hypoxia (10% O(2)) or hypercapnia (8% CO(2)) under thermoneutral (33°C) or cold (26°C) conditions. We found that Phox2b(+/-) pups showed less normoxic ventilation (V(E)) in the cold than Phox2b(+/+) pups. Phox2b(+/-) pups also showed lower oxygen consumption (VO(2)) in the cold, reflecting reduced thermogenesis and a lower body temperature. Furthermore, while the cold depressed ventilatory responses to hypoxia and hypercapnia in both genotype groups, this effect was less pronounced in Phox2b(+/-) pups. Finally, because serotonin (5-HT) neurons are pivotal to respiratory and thermoregulatory circuits and depend on Phox2b for their differentiation, we studied 5-HT metabolism using high pressure liquid chromatography, and found that it was altered in Phox2b(+/-) pups. We conclude that Phox2b haploinsufficiency alters the ability of newborns to cope with metabolic challenges, possibly due to 5-HT signaling impairments.

Breathing is a spontaneous, rhythmic motor behavior critical for maintaining O(2), CO(2), and pH homeostasis. In mammals, it is generated by a neuronal network in the lower brainstem, the respiratory rhythm generator (Feldman et al., 2003). A century-old tenet in respiratory physiology posits that the respiratory chemoreflex, the stimulation of breathing by an increase in partial pressure of CO(2) in the blood, is indispensable for rhythmic breathing. Here we have revisited this postulate with the help of mouse genetics. We have engineered a conditional mouse mutant in which the toxic PHOX2B(27Ala) mutation that causes congenital central hypoventilation syndrome in man is targeted to the retrotrapezoid nucleus, a site essential for central chemosensitivity. The mutants lack a retrotrapezoid nucleus and their breathing is not stimulated by elevated CO(2) at least up to postnatal day 9 and they barely respond as juveniles, but nevertheless survive, breathe normally beyond the first days after birth, and maintain blood PCO(2) within the normal range. Input from peripheral chemoreceptors that sense PO(2) in the blood appears to compensate for the missing CO(2) response since silencing them by high O(2) abolishes rhythmic breathing. CO(2) chemosensitivity partially recovered in adulthood. Hence, during the early life of rodents, the excitatory input normally afforded by elevated CO(2) is dispensable for life-sustaining breathing and maintaining CO(2) homeostasis in the blood.

In newborns, hypoxia elicits increased ventilation, arousal followed by defensive movements, and cries. Cold is known to affect the ventilatory response to hypoxia, but whether it affects the arousal response remains unknown. The aim of the present study was to assess the effects of cold on the ventilatory and arousal responses to hypoxia in newborn mice. We designed an original platform measuring noninvasively and simultaneously the breathing pattern by whole body plethysmography, body temperature by infrared thermography, as well as motor and ultrasonic vocal (USV) responses. Six-day-old mice were exposed twice to 10% O(2) for 3 min at either cold temperature (26 degrees C) or thermoneutrality (33 degrees C). At 33 degrees C, hypoxia elicited a marked increase in ventilation followed by a small ventilatory decline, small motor response, and almost no USVs. Body temperature was not influenced by hypoxia, and oxygen consumption (Vo(2)) displayed minimal changes. At 26 degrees C, hypoxia elicited a slight increase in ventilation with a large ventilatory decline and a large drop of Vo(2). This response was accompanied by marked USV and motor responses. Hypoxia elicited a small decrease in temperature after the return to normoxia, thus precluding any causal influence on the motor and USV responses to hypoxia. In conclusion, cold stimulated arousal and stress responses to hypoxia, while depressing hypoxic hyperpnea. Arousal is an important defense mechanism against sleep-disordered breathing. The dissociation between ventilatory and behavioral responses to hypoxia suggests that deficits in the arousal response associated with sleep breathing disorders cannot be attributed to a depressed hypoxic response.

Congenital central hypoventilation syndrome (CCHS) is a rare disease with variable severity, generally present from birth and chiefly characterized by impaired chemosensitivity to hypercapnia. The main cause of CCHS is a mutation in the PHOX2B gene, which encodes a transcription factor involved in the development of autonomic medullary reflex pathways. Temperature regulation is abnormal in many patients with CCHS. Here, we examined whether ambient temperature influenced CO(2) sensitivity in a mouse model of CCHS. A weak response to CO(2) at thermoneutrality (32 degrees C) was noted previously in 2-day-old mice with an invalidated Phox2b allele (Phox2b+/-), compared with wild-type littermates. We exposed Phox2b+/- pups to 8% CO(2) at three ambient temperatures (TAs): 29 degrees C, 32 degrees C, and 35 degrees C. We measured breathing variables and heart rate (HR) noninvasively using a novel whole body flow plethysmograph equipped with contact electrodes. Body temperature and baseline breathing increased similarly with TA in mutant and wild-type pups. The hypercapnic ventilatory response increased linearly with TA in both groups, while remaining smaller in mutant than in wild-type pups at all TAs. The differences between the absolute increases in ventilation in mutant and wild-type pups become more pronounced as temperature increased above 29 degrees C. The ventilatory abnormalities in mutant pups were not associated with significant impairments of heart rate control. In both mutant and wild-type pups, baseline HR increased with TA. In conclusion, TA strongly influenced the hypercapnic ventilatory response in Phox2b+/- mutant mice. These findings suggest that abnormal temperature regulation may contribute to the severity of respiratory impairments in CCHS patients.

Brief oxygen therapy is commonly used for resuscitation at birth or prevention of hypoxaemia before procedures during the neonatal period. However, O(2) may severely depress breathing, especially when administered repeatedly. The aim of the present study was to test the effects of repeated hyperoxia on breathing control in newborn mice. A total of 97 Swiss mouse pups were assigned to O(2) or air on post-natal day 0, 1 or 2. Each pup in the O(2) group was subjected to four hyperoxic tests (100% O(2) for 3 min followed by 12 min normoxia), whereas pups in the air group were maintained in normoxia. Breathing variables were measured using flow-through barometric plethysmography. O(2) significantly decreased minute ventilation as seen in a decrease in respiratory rate. This decrease became significantly larger with repeated exposure and ranged -17- -26% for all ages combined. Furthermore, hyperoxia increased total apnoea duration, as compared with the baseline value. In newborn mice, repeated hyperoxia increasingly depressed breathing. This finding further supports a need for stringent control of oxygen therapy, most notably repeated oxygen administration in the neonatal period for premature newborn infants and those carried to term.

Heterozygous mutations of the transcription factor PHOX2B have been found in most patients with central congenital hypoventilation syndrome, a rare disease characterized by sleep-related hypoventilation and impaired chemosensitivity to sustained hypercapnia and sustained hypoxia. PHOX2B is a master regulator of autonomic reflex pathways, including peripheral chemosensitive pathways. In the present study, we used hyperoxic tests to assess the strength of the peripheral chemoreceptor tonic drive in Phox2b+/-newborn mice. We exposed 69 wild-type and 67 mutant mice to two hyperoxic tests (12-min air followed by 3-min 100% O2) 2 days after birth. Breathing variables were measured noninvasively using whole body flow plethysmography. The initial minute ventilation decrease was larger in mutant pups than in wild-type pups: -37% (SD 13) and -25% (SD 18), respectively, P<0.0001. Furthermore, minute ventilation remained depressed throughout O2 exposure in mutants, possibly because of their previously reported impaired CO2 chemosensitivity, whereas it returned rapidly to the normoxic level in wild-type pups. Hyperoxia considerably increased total apnea duration in mutant compared with wild-type pups (P=0.0001). A complementary experiment established that body temperature was not influenced by hyperoxia in either genotype group and, therefore, did not account for genotype-related differences in the hyperoxic ventilatory response. Thus partial loss of Phox2b function by heterozygosity did not diminish the tonic drive from peripheral chemoreceptors.