Arousal response to hypoxia in newborns: Insights from animal models

Biological Psychology 84 (2010) 39–45

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Biological Psychology journal homepage: www.elsevier.com/locate/biopsycho

Review

Arousal response to hypoxia in newborns: Insights from animal models Jorge Gallego a,b,*, Boris Matrot a,b a b

Inserm, UMR676, Hoˆpital Robert Debre´, 75019 Paris, France Universite´ Paris 7 Denis Diderot, Faculte´ de Me´decine, 75019 Paris, France

A R T I C L E I N F O

A B S T R A C T

Article history: Received 24 April 2009 Received in revised form 30 November 2009 Accepted 2 December 2009 Available online 11 December 2009

In newborns, the inability to initiate an arousal response to hypoxia is associated with apnea of prematurity, sudden infant death syndrome, and rare genetic disorders of respiratory control. Despite intensive research, the mechanisms of this response are poorly understood. This paper provides an overview of studies investigating the arousal response to hypoxia, with special emphasis on newborn mouse models. Mutant mouse models can provide valuable information regarding the pathogenesis of genetically determined disorders affecting arousal response to hypoxia, although data remain sparse. In mice, the arousal response to hypoxia emerges immediately after birth, when the ventilatory response to hypoxia is still immature. Habituation of the arousal response occurs after repeated hypoxic episodes. Newborn mice can learn to associate novel odors to hypoxia and respond to those odors by producing alerting responses, suggesting that the arousal response to hypoxia may be shaped by learning processes. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Breathing Conditioning Habituation Ultrasonic vocalizations

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Arousal response to hypoxia in human newborns . Clinical significance . . . . . . . . . . . . . . . . . . . . . . . . . Arousal response to hypoxia in newborn rodents . Mechanisms of the arousal response . . . . . . . . . . . Effects of prenatal nicotine exposure . . . . . . . . . . . Role of temperature . . . . . . . . . . . . . . . . . . . . . . . . . Habituation effects. . . . . . . . . . . . . . . . . . . . . . . . . . Conditioning effects . . . . . . . . . . . . . . . . . . . . . . . . . Genetic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Arousal from sleep in response to potentially harmful stimuli is a key feature of breathing control in newborns. Ventilatory efforts against obstructed upper airways (Masa et al., 2003), hypercapnia (excess carbon dioxide in the blood) (Ayas et al., 2000) or hypoxia (deficient oxygen supply to the tissues) (Parslow et al., 2003) may elicit arousal and subsequent defensive behaviors to prevent hypoxic damage. As detailed below, failure to arouse in response to hypoxia is regarded as a possible mechanism for several respiratory control disorders in newborns, including sudden

* Corresponding author at: Inserm, UMR676, Hoˆpital Robert Debre´, 48 Boulevard Se´rurier, 75019 Paris, France. Tel.: +33 1 40 03 19 75. E-mail address: [email protected] (J. Gallego). 0301-0511/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.biopsycho.2009.12.001

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infant death syndrome (SIDS). The highly complex arousal response involves all components of breathing control: the brainstem respiratory rhythm generator, central and peripheral chemoreceptors, mechanoreceptors located in the respiratory tract and muscles, brainstem and midbrain arousal networks, and cortically mediated motor responses. In newborns, rapid maturation of these components further increases the complexity of the mechanisms underlying the arousal response to hypoxia. For these reasons, experimental studies aimed at understanding the mechanisms of the arousal response to hypoxia are difficult to conduct in human infants; animal models must be used instead. In the present short review, we summarize the main features of the arousal response to hypoxia in human newborns and their clinical significance. We then discuss the validity of newborn animal

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models and review mechanistic information from animal studies. Similar to several other vital functions, the arousal response to hypoxia originates in both genetic and acquired processes. These processes are illustrated by physiological experiments in genetically engineered mice, which are given special attention in the present review, and by a relatively small number of conditioning studies. 1. Arousal response to hypoxia in human newborns In human newborns, hypoxia elicits increased ventilation and arousal from sleep, followed by defensive movements and alerting cries (Fewell, 2005; Horne et al., 2005; Thach, 2002). Hypoxic episodes in newborns are frequently caused by apneas, which are defined as cessation of respiration for more than 20 s or cessation of respiration of any duration accompanied by bradycardia (heart rate below 100 beats/min). Apneas may be central or obstructive. In central apneas, the central nervous system stops activating the respiratory muscles, causing breathing movements to cease. In obstructive apneas, the respiratory muscles are activated, but airflow is blocked by collapsed upper airways. Apneas are very common in newborn mammals, especially during sleep, because their respiratory control systems, including airway patency control, are immature (Martin and Abu-Shaweesh, 2005). The arousal response to hypoxia stimulates breathing and ensures avoidance of life-threatening events, such as positional asphyxia (Phillipson and Sullivan, 1978). The ability to initiate appropriate responses to hypoxia is considered a vital reflex in newborns. Most apneas are terminated by complete or partial arousal from sleep (Leiter and Bohm, 2007). Thus, apnea duration and the consequent degree of blood oxygen desaturation are closely related to arousal ability. The arousal response to hypoxia is highly dependent on sleep state. Three sleep states are defined in human newborns: active sleep (AS), quiet sleep (QS), and indeterminate sleep (Lehtonen and Martin, 2004). AS is characterized by low EEG voltage, irregular breathing, eye movements, body movements, and low muscle tone. QS is characterized by closed eyes, high EEG voltage, regular breathing, absent eye movements, absent body movements, and high muscle tone. Indeterminate sleep is defined as a state that does not meet the criteria for either AS or QS. In human newborns, during the first 6 postnatal months, arousal in response to a mild hypoxic stimulus (15% O2) occurs consistently during AS, which is the dominant sleep stage throughout this period, but it occurs inconsistently during QS (Richardson et al., 2006, 2007). Another common phenomenon in newborn mammals is periodic breathing, which is characterized by the alternation of hyperventilation and hypoventilation episodes. In human term or preterm infants, periodic breathing is associated with low PaO2 values (Al-Matary et al., 2004; Rigatto and Brady, 1972a,b). The rate of the O2 saturation drop and the saturation reached during hypoxic episodes are critical for arousal to occur (Richardson et al., 2007). Since blood gas levels depend on the ventilatory response to hypoxia, the ventilatory and arousal responses are related to each other. However, the occurrence of the arousal response to hypoxia depends on numerous physiological and behavioral factors, including previous experience of hypoxic states, as discussed below. This contribution of psychological factors adds to the complexity of the response. 2. Clinical significance As previously mentioned, an inability to initiate the arousal response to hypoxia is considered a possible cause of SIDS, the main cause of infant mortality in industrialized countries (Dunne et al., 1992; Horne et al., 2005; Hunt, 2005; Kahn et al., 2002; McCulloch et al., 1982; Sawaguchi et al., 2005; Thach, 2005; Ward

et al., 1992). The arousal response to hypoxia is the last defensive stage before self-resuscitation, which consists in recovering from apnea by producing gasps (i.e., deep irregular inspiratory movements with a low frequency involving the whole body and a wide open mouth). An inability to initiate the arousal response to hypoxia is also involved in apparently life-threatening events (ALTEs), which are characterized by a combination of apnea, color change (usually cyanosis or pallor), marked change in muscle tone (usually prominent limpness), and choking or gagging. The incidence of ALTEs is estimated at 0.05–6% of infants in the general population (Harrington et al., 2002). Absence of the arousal response to hypoxia is a feature of several rare genetic respiratory control disorders. In congenital central hypoventilation syndrome (CCHS or Ondine’s syndrome), for instance, arousal fails to occur in response to endogenous challenges of isolated hypercapnia or hypoxia or to stimulation by combined hypercapnia and hypoxia (Chen and Keens, 2004). CCHS is a rare disorder that typically presents during the neonatal period. Affected patients have little or no ventilatory sensitivity to hypercapnia and hypoxia during sleep or wakefulness and also exhibit autonomic disorders, although these disorders present considerable interindividual variability in pattern and severity (Huffman et al., 1999; Marazita et al., 2001; Vanderlaan et al., 2004). The identification of PHOX2B as the disease-causing gene in CCHS (Amiel et al., 2003; Cummings et al., 2009; Matera et al., 2004; Sasaki et al., 2003) generated considerable interest in the role for this gene in CCHS as well as in prevalent conditions such as sleep apnea. PHOX2B mutations were recently found in adults with sleep-related hypoventilation and, in some instances, central apneas and severe hypoxemia, i.e., deficient blood oxygenation (Antic et al., 2006; Barratt et al., 2007; Diedrich et al., 2007; Doherty et al., 2007; Dubreuil et al., 2008; Gaultier et al., 2004; Hoppenbrouwers et al., 2005; Matera et al., 2004; Trochet et al., 2005). Thus, respiratory control disorders associated with PHOX2B mutations may be more prevalent than previously inferred from the very low incidence of CCHS in newborns. The role of PHOX2B in respiratory control and particularly in the arousal response to hypoxia has been examined using mouse genetic models, which are presented in detail below. Prader-Willi syndrome is a rare genetic disorder in which arousal thresholds during hypercapnic and hypoxic episodes are abnormal. Features include hypotonia and poor weight gain in infancy, rapid and excessive weight gain between 1 and 6 years of age (eventually leading to morbid obesity), dysmorphic facial features, hypogonadism, and developmental delay. Many patients display sleepdisordered breathing with apneas, which may occur in the neonatal period, and episodes of hypoventilation (Nixon and Brouillette, 2002). The underlying genetic abnormality is the lack of expression of several genes, including the gene encoding neurally differentiated embryonal carcinoma-derived protein (NECDIN) (Nakada et al., 1998). The NECDIN gene is a ‘‘growth-suppressor’’ gene that may facilitate cell-cycle exit and maintenance of the neuronal postmitotic state. Daytime breathing is impaired in Prader-Willi syndrome and, importantly, ventilatory responses to hypoxia and hypercapnia are blunted. Blunting of the ventilatory responses is present in normal-weight individuals with Prader-Willi syndrome but is exacerbated by obesity, restricted lung volume consequent to scoliosis, and thoracic muscle weakness. Sleep apneas of mixed central and obstructive origin are common in Prader-Willi syndrome (Camfferman et al., 2006; Festen et al., 2008). In addition to the above rare diseases, failure to initiate an appropriate arousal response to hypoxia is associated with sleep apnea syndrome, which is particularly common in preterm infants (Harrington et al., 2002; Thach, 2002; Waters and Tinworth, 2005). In high-income countries, the incidence of preterm birth is estimated at 5–12% (Wen et al., 2004). Apnea of prematurity occurs in more than 85% of preterm infants who are less than 34

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weeks of gestational age, especially during sleep, and often persists beyond the postconceptional age corresponding to term gestation (Eichenwald et al., 1997). The apneas are due to immaturity of both respiratory rhythm genesis and chemosensitivity. In preterm humans, apneas that are associated with a decrease in heart rate cause cerebral hypoxia via both O2 desaturation of arterial blood and decreased cerebral blood flow (Lehtonen and Martin, 2004). Re-oxygenation after restoration of breathing may induce oxidative stress, thereby aggravating the pathophysiological process (Prabhakar et al., 2001). Apnea of prematurity is considered a risk factor for brain injuries and developmental disorders in humans (Bass et al., 2004; Janvier et al., 2004). Caffeine is the treatment of choice for apnea of prematurity. Recent studies suggest that the temporary adverse effects of caffeine are outweighed by improved survival without neurodevelopmental disability at 18–21 months. Nevertheless, longer follow-up is warranted, as the neurodevelopmental outcome at 18–21 months is not predictive of later neurodevelopmental outcome and school performance (Stevenson, 2007). Both full-term and preterm infants show a progressive decrease in apnea episodes over time. Apnea in preterm infants generally returns to normal levels (i.e., those found in term infants) at 43–44 weeks of postconceptional age (Horne et al., 2004). 3. Arousal response to hypoxia in newborn rodents Despite intensive research over the last three decades (Horne et al., 2005), the mechanisms of the arousal response to hypoxia remain elusive (Richardson et al., 2006; Thoppil et al., 1991). For ethical reasons, these mechanisms are difficult to investigate in human infants. Therefore, newborn animal models that reflect both the autonomic and behavioral components of the ventilatory response to hypoxia, hereafter termed the hypoxic response, must be used. When investigating breathing and arousal patterns in freely behaving newborn mammals, non-invasive approaches are recommended. Furthermore, the determination of sleep–wake states (wakefulness, AS, QS, and undetermined sleep) using electroencephalography or electro-oculography is feasible in newborn rodents only after 8 days of postnatal age (Karlsson and Blumberg, 2002). However, QS and AS can be distinguished in newborn rats and mice based on neck muscle electromyograms, motor twitches, and coordinated movements (Durand et al., 2005; Karlsson and Blumberg, 2002). A recent study in 7-day-old mice confirms that sleep–wake states can be assessed using behavioral markers without electromyography (Balbir et al., 2008). Sleep in newborn rats or mice nearly satisfies the criteria used to characterize sleep across species (Blumberg et al., 2005). The proportions of AS and QS in 5-day-old mouse pups were within the ranges found in human preterm infants (Durand et al., 2005). However, the precise correspondence between sleep-stage development in human and rodent newborns is still unknown. The arousal response to hypoxia in newborn mice is characterized by a stereotyped motor response consisting of sudden neck and forepaw extensions (Dauger et al., 2001). This response is present as soon as 3 h after birth (Dauger et al., 2001). After 2 days of postnatal age, this pattern of motor activity is followed by headraising (Dauger et al., 2001). This behavioral pattern is reminiscent of the thrashing movements that occur during the arousal sequence in human infants (Lijowska et al., 1997; McNamara et al., 1998). The strength of the arousal response is reflected by the short latency relative to hypoxia onset and the long movement duration. The only non-invasive method available for ventilatory measurements is whole-body plethysmography. The animal is placed in a chamber, and the pressure changes in the chamber produced by the animal’s breathing are measured. The pressure

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increases during inspiration, both because the inspired gas is warmed from the temperature in the chamber to that in the alveoli, and because water vapor is added to the inspired gas. However, pressure changes may be influenced by compression and rarefaction of external gases in the measurement chamber, caused by airway resistance during inspiration and expiration, respectively (Enhorning et al., 1998; Mortola and Frappell, 1998). Whole-body plethysmography provides semi-quantitative measurements of tidal volume and ventilation while allowing valid measurements of breathing rate and apneas. A recently developed integrated setup for phenotyping newborn mice combines plethysmographic measurement of breathing variables (as described above) with non-invasive electrocardiogram recordings (using electrodes embedded in the floor of the plethysmograph chamber (Ramanantsoa et al., 2007)), body temperature (by infrared thermography (Bollen et al., 2009)), movements (based on respiratory signal analysis (Matrot et al., 2005)), and ultrasonic vocalizations (Bollen et al., 2009), thus allowing accurate characterization of the arousal response to hypoxia. 4. Mechanisms of the arousal response In newborn mammals, the ventilatory response to hypoxia is triggered by oxygen-sensitive chemoreceptors located in the carotid bodies near the carotid artery bifurcation. Chemoreceptors detect changes in arterial blood oxygen pressure (PaO2) and send sensory information to the nucleus tractus solitarius (NTS) in the brainstem. Absence of excitatory input from the peripheral chemoreceptors in newborn mammals is associated with increased apneas, decreased arousals, and longer arousal latency (Gauda et al., 2007). Within seconds after a PaO2 decrease is sensed, ventilation (V0 E) increases. If hypoxia persists, V0 E decreases and the metabolic rate falls (Bissonnette, 2000; Bollen et al., 2009; Martin et al., 1998; Richardson et al., 2006). This biphasic pattern of the ventilatory response (initial hyperpnea followed by a decrease in ventilation that may fall below the pre-hypoxic level) is characteristic of newborn mammals. In humans, failure to rapidly restore PaO2 via these autonomic adjustments (either increased O2 supply or decreased O2 consumption) leads to the initiation of an arousal response, including cortical arousal from sleep, movements, and crying (Fewell, 2005; Galland et al., 2003; Horne et al., 2005). The arousal response to hypoxia, which is accompanied by increases in heart rate, arterial pressure, and ventilation, is reminiscent of the ‘‘fight or flight’’ reaction (Horne et al., 2004). In humans, the ventilation increase produced by hypoxia is accompanied by a concomitant increase in the sensory input from mechanoreceptors in the lung (the pulmonary stretch receptors), upper airways or respiratory muscles. This increased input is thought to play a primary role in the arousal response to hypoxia (Berry and Gleeson, 1997). However, studies in newborn mice suggest that the arousal response to hypoxia may not be chiefly determined by afferent signals from mechanoreceptors under any circumstances. First, the arousal response to hypoxia is present 3 h after birth (before postnatal resetting of the peripheral chemoreceptors (Carroll and Kim, 2005)), at a time when the ventilatory response to hypoxia is almost nonexistent (Dauger et al., 2001). The arousal response to hypoxia is substantially delayed relative to the peak of ventilatory activity (Dauger et al., 2001). Furthermore, cold stimulates the arousal response to hypoxia while at the same time depressing the hyperpneic response to hypoxia (Bollen et al., 2009). In human infants aged 5–6 months, the arousal and ventilatory responses to hypoxia may be differently affected by prenatal nicotine exposure. In fact, maternal smoking does not affect ventilatory responses preceding hypoxia-induced arousal but does depress arousability (Parslow et al., 2004). Finally, newborn mice with one inactivated Phox2b allele (Phox2b+/) show

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a normal biphasic response to hypoxia but have blunted arousal responses to hypoxia compared to their wild-type littermates (Ramanantsoa et al., 2009; unpublished data). 5. Effects of prenatal nicotine exposure Maternal smoking during pregnancy is one cause of blunting of the arousal response to hypoxia in human newborns. During QS, infants who were exposed to nicotine in utero have abnormally high arousal thresholds after obstructive apneas, with decreased arousability in response to hypoxia (Hafstrom et al., 2005). In human infants aged 2–5 weeks, 2–3 months, and 5–6 months, even moderate levels of maternal cigarette smoking depress the arousal responses to hypoxia, although there is no effect on the ventilatory responses occurring immediately before arousal, as noted above (Parslow et al., 2004). The mechanism by which prenatal nicotine exposure affects postnatal arousal to hypoxia has been investigated in developing animals. In 5-day-old lambs exposed to nicotine prenatally, the arousal response to hypoxia during QS is delayed (Hafstrom et al., 2002). Prenatal nicotine exposure impairs arousal responses not only to hypoxia, but also to other stimuli as well (e.g., touch). In adolescent mice, impairment of the arousal response to hypoxia is mediated by the effects of nicotine on acetylcholine receptors (nAChRs), most notably those containing the b2 subunit, a major component of high-affinity nicotine binding sites in the nervous system (Cohen et al., 2005). Newborn mice exposed to nicotine in utero exhibit unstable breathing and depressed arousal responses to hypoxia; this latter effect is reversed in newborn mice lacking b2-containing nAChRs, confirming the role for the b2 subunit in the effects of prenatal nicotine exposure (Cohen et al., 2005). 6. Role of temperature In human infants, high ambient temperatures and bundling in blankets or clothing increase the risk of SIDS (Kinney, 2009). The Back to Sleep campaign, advocating the supine sleep position and light clothing, was associated with an approximately 50% decrease in the incidence of SIDS over 10 years (Task Force on Sudden Infant Death Syndrome, 2005; Vennemann et al., 2006). Among other insights into the pathophysiology of SIDS, this major achievement supports a link between ambient temperature and the arousal response to hypoxia in newborns. This link was recently examined in newborn mice exposed to hypoxia (10% O2) under either cold or warm conditions (Bollen et al., 2009). Under warm conditions, hypoxia elicited a strong hyperpneic ventilatory response, contrasting with a moderate motor response and absence of vocalizations (Bollen et al., 2009). Cold had opposite effects on ventilatory responses and the arousal responses to hypoxia. When exposed to hypoxia under cold conditions, 6-day-old mice produced ultrasonic vocalizations and vigorous motor activity, contrasting with a markedly depressed hyperpneic response. The ultrasonic response to hypoxia was reminiscent of the cries produced by human infants at the end of the arousal sequence. This dissociation between ventilatory and behavioral responses to hypoxia confirmed previous findings (Dauger et al., 2001) and suggested that arousal response impairments associated with sleep-breathing disorders might not always be ascribable to a depressed hypoxic response. Taken together, these results suggest that mechanisms other than increased ventilatory effort may contribute to triggering the arousal response to hypoxia. 7. Habituation effects An intriguing aspect of the arousal response to hypoxia is that it is shaped by past experience. Habituation is a non-associative

learning process by which repeated exposure to a stimulus lessens the reflex response to this stimulus. By definition, habituation is not the result of sensory adaptation but rather represents a learned inhibition of the response to the stimulus. Infants may habituate to repeated hypoxic events and, therefore, may fail to respond after a number of events (Horne et al., 2005). This effect was investigated in a more systematic fashion in animal models. Exposure to repeated acute hypoxia in newborn animals led to habituation of the arousal response, whose frequency decreased in response to a fixed stimulus (reviewed in Waters and Tinworth, 2005). For example, newborn lambs became less arousable after repeated hypoxic tests during sleep (Fewell and Konduri, 1989; Johnston et al., 1999). The increase in arousal latency that occurs with repeated hypoxic stimulation is generally regarded as a habituation process. Sensory adaptation at the chemoreceptor level is unlikely, as there is usually no decrease in the magnitude of V0 E changes. For example, Waters and Tinworth reported that arousal strength had largely recovered at the beginning of each sequence of intermittent hypoxic tests but decreased markedly with test repetition (Waters and Tinworth, 2005). Thus, the decrease in hypoxic response (the main characteristic of habituation) is not ascribable to desensitization or response blunting. Interestingly, repeated exposure to hypercapnia in newborn lambs does not induce progressive depression of the arousal response (Johnston et al., 2007). Conceivably, delayed arousal to intermittent hypoxia may be a protective mechanism to avoid sleep disruption. Habituation may also be part of a general postnatal strategy against hypoxia by which oxygen consumption is adapted to oxygen availability by reducing breathing and heart frequencies, body temperature, metabolic rate, and defensive responses. Habituation effects in response to hypoxia are difficult to investigate in human newborns. Previous attempts using a small number of mild hypoxic exposures (15% O2, five tests) failed to demonstrate habituation of the arousal response to hypoxia (Richardson et al., 2006). However, for ethical reasons, experiments in newborns are extremely difficult to conduct. Experiments involving replication of the hypoxic episodes associated with repetitive apneas would be unacceptable. 8. Conditioning effects In contrast to habituation studies, conditioning studies showed that the defensive response to hypoxia was potentiated by previous experience. In a recent conditioning experiment (Bollen et al., 2007), newborn mice were exposed to two artificial odors (conditioned stimuli, CS). For acquisition (two trials), pups were exposed to one odor (CS+) in a hypoxic gas mixture (10% O2, which was the unconditioned stimulus, US) and to another odor (CS-) in air. The pups were then exposed to each odor while breathing air. Newborn mice produced significantly more ultrasonic vocalizations when exposed to the odor previously paired with hypoxia (CS+) than to the control odor (CS-). This result indicates that the behavioral response to hypoxia may be shaped by conditioning processes soon after birth and that newborn mice can anticipate hypoxic stress (Bollen et al., 2007). It also suggests that blunting of the arousal response to hypoxia in some preterm infants may be due not only to respiratory control immaturity, but also to deficits in the cognitive abilities that underlie anticipatory defensealerting behaviors. Under natural conditions, conditioning may be produced by the natural forerunners of hypoxia-related perceptions, such as the facial stimuli that accompany the face-down position that is potentially responsible for positional asphyxia or ventilatory efforts against airway obstruction. Conditioning of the alerting response to hypoxia and conditioning of the ventilatory response

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to hypoxia (Nsegbe et al., 1998; Nsegbe et al., 1999) may complement each other to shape defense responses to hypoxia. 9. Genetic factors At present, the prevailing hypothesis for explaining SIDS involves defective serotonin-mediated neurotransmission in the medulla. Serotonin receptor binding is consistently diminished in babies who died of SIDS (Leiter, 2009). Impairments in medullary 5-HT neurons may disrupt several important homeostatic functions, including control of breathing, blood pressure, temperature, and arousal (Kinney, 2009). Based on these data, several mouse models have been developed in an attempt to replicate at least part of the sequence of events leading to SIDS in human infants. Unexpectedly, mice with serotonin deficiencies induced by pharmacological or genetic interventions did not consistently exhibit impairments in respiratory or cardiovascular control that would have supported hypotheses about the pathogenesis of SIDS (Leiter, 2009). One possible explanation is that mutant mice used as SIDS models were not studied at an age corresponding to the peak incidence of SIDS in humans (2–4 months). For example, mice with a targeted disruption of the serotonin transporter (Li and Nattie, 2008), overexpression of the serotonin 1A autoreceptors (Audero et al., 2008) or absence of central 5-HT neurons (Hodges et al., 2008) were investigated in adulthood; in addition, sex differences, which are present in the incidence of SIDS and may differently affect newborn and adult animals, were not considered in these studies. Furthermore, arousal responses to hypoxia were not assessed. The arousal response to hypoxia depends on many factors that undergo profound developmental changes in the early postnatal period (chemosensitivity, organization of sleep–wake states, thermoregulation abilities, etc.). These quantitative and qualitative changes in arousal processes complicate comparisons of the arousal response to hypoxia between adult and newborn animals. However, a recent study showed that newborn mice that selectively lacked serotonin neurons (Lmx1bf/f/p) displayed frequent and severe apnea, a marked decrease in ventilation, and high perinatal mortality (Hodges et al., 2009). These results extended previous results in Pet-1 knock-out newborn mice, which have 30% of the normal number of 5-HT neurons (Erickson et al., 2007). Further studies may investigate whether the arousal response to hypoxia was impaired in these mutant mouse models. Prader-Willi syndrome is also characterized by impaired arousal to hypoxia (Arens et al., 1996). The role for NECDIN deficiency in the respiratory control abnormalities seen in PraderWilli syndrome was investigated in Necdin-null mutant newborn mice (Greer and Wevrick, 2008). These mice exhibited respiratory instability at birth with apneas and died soon after birth from severe hypoventilation. However, their arousal response to hypoxia was not studied. The finding that PHOX2B was the main disease-causing gene for CCHS was consistent with previous knowledge of the role for this gene in respiratory control development (Gallego and Dauger, 2008). The transcription factor Phox2b is required for the development of most neuronal types in the central and peripheral nervous systems (Brunet and Pattyn, 2002; Pattyn et al., 1997; Tiveron et al., 1996). Phox2b is expressed by a chain of neurons involved in the integration of peripheral and central chemoreception, which includes the carotid bodies, chemoreceptor afferents, chemoresponsive projections of the NTS to the ventrolateral medulla, and central chemoreceptors located in the retrotrapezoid nucleus (Stornetta et al., 2006). Newborn mice bearing the mutation in Phox2b that causes CCHS in humans display a ventilatory phenotype that is very similar to the human disease: breathing is irregular, there is no

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response to CO2, and the animals die soon after birth from central apnea (Dubreuil et al., 2008). However, the early mortality of these mutant mice precludes analysis of the arousal response. By contrast, mice with one inactivated Phox2b allele (Phox2b+/ mice) do not replicate the CCHS phenotype, instead exhibiting normal development and fertility, but they provide interesting information regarding the role for Phox2b in the arousal response to hypoxia. In 2-day-old Phox2b+/ mice, the latency of the arousal response to hypoxia (5% O2) is not significantly different from that observed in wild-type littermates (Dauger et al., 2003). However, recent studies in 6-day-old Phox2b+/+ and Phox2b+/ pups exposed to 10% O2 showed significantly shorter durations of defensive movements and ultrasonic vocalizations (Ramanantsoa et al., 2009; unpublished data). These differences were observed under both cold (26 8C) and warm (33 8C) conditions, although the ventilatory responses to hypoxia were not different between mutant and wild-type pups under warm conditions, as noted above. Therefore, Phox2b may play a specific role in the arousal response to hypoxia in newborns, which is separate from its effect on ventilatory control. 10. Conclusion The arousal response to hypoxia is a vital reflex that is impaired in several neonatal disorders, some of which can be fatal. The sequence of ventilatory and behavioral responses to hypoxia in newborn mice closely resembles that in human infants. Although the responses are related, impaired arousal is not necessarily ascribable to a depressed response to hypoxia. The arousal response to hypoxia may be shaped by both non-associative and associative learning processes. Newborn mice constitute a powerful tool for investigating the mechanisms of this response and, more specifically, for unraveling the genetic determinants of arousal impairments. Acknowledgments This study was supported by the Institut National de la Sante´ et de la Recherche Me´dicale, the Chancellerie des Universite´s de Paris (Legs Poix), the Fondation Garches, and the Fondation Marguerite Marie Delacroix. We are greatly indebted to Yann Rotrou for his original technological contribution to newborn mouse phenotyping. References Al-Matary, A., Kutbi, I., Qurashi, M., Khalil, M., Alvaro, R., Kwiatkowski, K., et al., 2004. Increased peripheral chemoreceptor activity may be critical in destabilizing breathing in neonates. Seminars in Perinatology 28, 264–272. Amiel, J., Laudier, B., Attie-Bitach, T., Trang, H., de Pontual, L., Gener, B., et al., 2003. Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome. Nature Genetics 33, 459–461. Antic, N.A., Malow, B.A., Lange, N., McEvoy, R.D., Olson, A.L., Turkington, P., et al., 2006. PHOX2B mutation-confirmed congenital central hypoventilation syndrome: presentation in adulthood. American Journal of Respiratory and Critical Care Medicine 174, 923–927. Arens, R., Gozal, D., Burrell, B.C., Bailey, S.L., Bautista, D.B., Keens, T.G., et al., 1996. Arousal and cardiorespiratory responses to hypoxia in Prader-Willi syndrome. American Journal of Respiratory and Critical Care Medicine 153, 283–287. Audero, E., Coppi, E., Mlinar, B., Rossetti, T., Caprioli, A., Banchaabouchi, M.A., et al., 2008. Sporadic autonomic dysregulation and death associated with excessive serotonin autoinhibition. Science 321, 130–133. Ayas, N.T., Brown, R., Shea, S.A., 2000. Hypercapnia can induce arousal from sleep in the absence of altered respiratory mechanoreception. American Journal of Respiratory and Critical Care Medicine 162, 1004–1008. Balbir, A., Lande, B., Fitzgerald, R.S., Polotsky, V., Mitzner, W., Shirahata, M., 2008. Behavioral and respiratory characteristics during sleep in neonatal DBA/2J and A/J mice. Brain Research 1241, 84–91. Barratt, S., Kendrick, A.H., Buchanan, F., Whittle, A.T., 2007. Central hypoventilation with PHOX2B expansion mutation presenting in adulthood. Thorax 62, 919– 920.

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