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.

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 (IKIR) and early slowly inactivating (ID) 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 IKIR and ID 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.

An increasing number of studies in newborn mice are being performed to determine the mechanisms of sleep apnea, which is the hallmark of early breathing disorders. Whole body plethysmography is the method of choice, as it does not require immobilization, which affects behavioral states and breathing. However, activity inside the plethysmograph may disturb the respiratory signal. Visual classification of the respiratory signal into ventilatory activity, activity-related disturbances, or apneas is so time-consuming as to considerably hamper the phenotyping of large pup samples. We propose an automatic classification of activity based on respiratory disturbances and of apneas based on spectral analysis. This method was validated in newborn mice on the day of birth and on postnatal days 2, 5, and 10, under normoxic and hypoxic (5% O(2)) conditions. For both activity and apneas, visual and automatic scores showed high Pearson's correlation coefficients (0.92 and 0.98, respectively) and high intraclass correlation coefficients (0.96-0.99), supporting strong agreement between the two methods. The present results suggest that breathing disturbances may provide a valid indirect index of activity in freely moving newborn mice and that automatic apnea classification based on spectral analysis may be efficient in terms of precision and of time saved.

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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 Phox2bϩ/Ϫ 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: Ϫ37% (SD 13) and Ϫ25% (SD 18), respectively, P Ͻ 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 ϭ 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.control of breathing; chemosensitivity; apnea CENTRAL CONGENITAL HYPOVENTILATION syndrome (CCHS or Ondine's curse) is a rare disease, generally present from birth and characterized by hypoventilation during sleep in the absence of primary neuromuscular or lung disease or of brain stem lesions (3). Throughout life, patients with CCHS have absent or markedly reduced ventilatory responses to sustained hypercapnia (29) and, to a lesser extent, to sustained hypoxia (29). These respiratory impairments are generally ascribed to impaired central integration of chemosensory inputs at the brain stem level, rather than to failure of chemoreceptor activity, which is at least partially present (15,22,37). The genetic basis for CCHS was discovered recently: most patients with CCHS have a heterozygous mutation of the PHOX2B gene (2,23,41,43). PHOX2B is a master regulator of the noradrenergic phenotype and of all neuronal relays of autonomic medullary reflex pathways (30), including peripheral chemosensitive pathways (9).Mice with a single functional Phox2b allele (i.e., Phox2bϩ/Ϫ mice) provide a unique opportunity to investigate the genotype-phenotype relationship in CCHS. Our laboratory's previous studies showed that Phox2bϩ/Ϫ mice had longer sleep apnea times than their wild-type littermates on postnatal day (P) 5 (P5) (11) and a weaker response to CO 2 on P2, followed by normalization before P10 (9). These ventilatory impairments were reminiscent of CCHS. However, the hyperpneic response t...

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.

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.

Fifteen million babies are born preterm every year and a significant number suffer from permanent neurological injuries linked to white matter injury (WMI). A chief cause of preterm birth itself and predictor of the severity of WMI is exposure to maternal-fetal infection-inflammation such as chorioamnionitis. There are no neurotherapeutics for this WMI. To affect this healthcare need, the repurposing of drugs with efficacy in other white matter injury models is an attractive strategy. As such, we tested the efficacy of GSK247246, an H3R antagonist/inverse agonist, in a model of inflammation-mediated WMI of the preterm born infant recapitulating the main clinical hallmarks of human brain injury, which are oligodendrocyte maturation arrest, microglial reactivity, and hypomyelination. WMI is induced by mimicking the effects of maternal-fetal infection-inflammation and setting up neuroinflammation. We induce this process at the time in the mouse when brain development is equivalent to the human third trimester; postnatal day (P)1 through to P5 with i.p. interleukin-1β (IL-1β) injections. We initiated GSK247246 treatment (i.p at 7 mg/kg or 20 mg/kg) after neuroinflammation was well established (on P6) and it was administered twice daily through to P10. Outcomes were assessed at P10 and P30 with gene and protein analysis. A low dose of GSK247246 (7 mg/kg) lead to a recovery in protein expression of markers of myelin (density of Myelin Basic Protein, MBP & Proteolipid Proteins, PLP) and a reduction in macro- and microgliosis (density of ionising adaptor protein, IBA1 & glial fibrillary acid protein, GFAP). Our results confirm the neurotherapeutic efficacy of targeting the H3R for WMI seen in a cuprizone model of multiple sclerosis and a recently reported clinical trial in relapsing-remitting multiple sclerosis patients. Further work is needed to develop a slow release strategy for this agent and test its efficacy in large animal models of preterm infant WMI.