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Ventilatory responses to hypercapnia and hypoxia in heterozygous c-ret newborn mice
Respiratory Physiology & Neurobiology 131 (2002) 213– 222 www.elsevier.com/locate/resphysiol
Ventilatory responses to hypercapnia and hypoxia in heterozygous c-ret newborn mice Sophie Aizenfisz a,c, Ste´phane Dauger a,c, Estelle Durand a, Guy Vardon d, Be´atrice Levacher a, Michel Simonneau a, Vassilis Pachnis e, Claude Gaultier a,b, Jorge Gallego a,* Laboratoire de Neurologie et Physiologie du De´6eloppement, Hoˆpital Robert-Debre´, 48 Boule6ard Se´rurier, 75019 Paris, France b Ser6ice de Physiologie, Hoˆpital Robert-Debre´, 48 Boule6ard Se´rurier, 75019 Paris, France c Ser6ice de Pe´diatrie-Re´animation, Hoˆpital Robert-Debre´, 48 Boule6ard Se´rurier, 75019 Paris, France d Unite´ de Recherches sur les Adaptations Physiologiques et Comportementales, Uni6ersite´ de Picardie, 80036 Amiens, France e Di6ision of Molecular Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK
a
Accepted 16 April 2002
Abstract The c-ret proto-oncogene encodes a tyrosine-kinase receptor involved in survival and differentiation of neural crest cell lineages. Previous studies have shown that homozygous c-ret− / − mice die soon after birth and have impaired ventilatory responses to hypercapnia. Heterozygous c-ret + / − mice develop normally, but their respiratory phenotype has not been described in detail. We used whole-body flow plethysmography to compare baseline breathing and ventilatory and arousal responses to chemical stimuli in unrestrained heterozygous c-ret + / − newborn mice and their wild-type c-ret +/+ littermates at 10–12 h of postnatal age. The hyperpnoeic and arousal responses to hypoxia and hypercapnia were not significantly different in these two groups. However, the number and total duration of apnoeas and periodic breathing episodes were significantly higher in c-ret+ / − than in c-ret+ / + pups during hypoxia and post-hypoxic normoxia. These results are further evidence that respiratory control at birth is heavily dependent on genes involved in the neural determination of neural crest cells. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Development; pattern of breathing; Gene; proto-oncogene; c-ret; Mammals; mouse; Pattern of breathing; c-ret +/ −
1. Introduction The c-ret proto-oncogene encodes a tyrosine-kinase receptor involved in survival and differentia* Corresponding author. Tel.: + 33-1-40-03-47-81; fax: + 33-1-40-03-47-70. E-mail address: [email protected] (J. Gallego).
tion of neural crest cell lineages. The respiratory phenotype of c-ret+ /− newborn mice deserves careful investigation because heterozygous mutations in c-ret and its ligand Glial-Derived-Neurotrophic-Factor have been found in patients with central congenital hypoventilation syndrome (CCHS) (Amiel et al., 1998; Sakai et al., 2001), a
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disorder characterized by impaired respiratory control. In an earlier study, homozygous c-ret − / − mice died soon after birth, possibly from respiratory failure, and showed impaired ventilatory responses to hypercapnia (Burton et al., 1997). In contrast, heterozygous c-ret+/ − mice developed normally. On average, their ventilatory responses to hypercapnia and hypoxia showed no significant differences with those of wild-type c-ret + /+ newborns (Burton et al., 1997). However, important aspects of the postnatal maturation of breathing in mice were not taken into account in this previous study (Burton et al., 1997). First, in mice, the ventilatory response to hypoxia is small before postnatal chemoreceptor resetting and, consequently, should be examined after this event, which occurs at 6– 12 h of postnatal age (PNA) (Renolleau et al., 2001a). Second, the initial hyperpnoeic response to hypoxia is followed by a decline (hypoxic ventilatory decline, HVD) that outlasts the end of hypoxia and is associated with more numerous and longer apnoeas (Dauger et al., 2001a). These two components of the hypoxic response involve different neuronal systems (Gozal et al., 2000) and should, therefore, be investigated separately. Third, the ventilatory responses to hypoxia and hypercapnia are modulated by arousal processes starting at birth, and these processes may also be affected by the mutation (Dauger et al., 2001a). In the present study, we tested the hypothesis that the heterozygous c-ret mutation may impair respiratory control in newborn mice. To do this, we compared baseline breathing and ventilatory and arousal responses to hypoxia and hypercapnia in heterozygous c-ret +/ − newborn mice and in their wild-type c-ret+/ + littermates. We conducted the ventilatory tests after peripheral chemoreceptor resetting (between H10 and H12 PNA), and we measured breathing variables non-invasively using whole-body plethysmography to avoid possible breathing changes related to restraining the animals (Dauger et al., 1998).
2. Methods
2.1. Animals The c-ret− deficient mice were kept heterozygous on a 129SvEv× C57BL/10 hybrid background from heterozygous breed pairs. The mice were housed at 24 °C with a normal 12-h light–dark cycle and were given food and water ad libitum. Newborn mice were obtained by mating heterozygous c-ret+ /− males (provided by Pachnis, National Institute for Medical Research, London) with wild-type females from the same 129Sv hybrid background. Vaginal plugs were examined on the morning of the next day, which was counted as embryonic day 0 (E0). Time of birth was determined by checking the mice from E19 onwards, at intervals of 1 h during the day and of 4–6 h during the night. Seventy-four newborn mice were obtained and were tested between H10 and H12 of PNA.
2.1.1. Genotyping After ventilatory measurements, each newborn was weighed and sacrificed by neck section. Leg and tail tissue fragments were taken for c-ret, as well as for sry genotyping to determine the gender. These samples were subjected to PCR under standard conditions. The PCR primer sequences for the wild allele were exons 6 and 7 from the c-ret gene (P6: 5%-TGG GAG AAG GCG AGT TTG GAA A-3%; and P7: 5%-TTC AGG AAC ACT GGC TAC CAT G-3%). The PCR primer sequences for the mutant allele were exon 12 from the c-ret gene (5%-TTC CTC AGC ATG TAT CCC AT-3%) and one exon from the neomycin gene (5%-CGT GAT CGA CAA GAC CGG CTT3%). The amplification program comprised a denaturation cycle (5 min at 94 °C), followed by 35 cycles (of which each consisted of 40 sec at 94 °C, 40 sec at 58 °C, and 40 sec at 72 °C) and final elongation (2 min at 72 °C). PCR products were analysed in 1% agarose gel. 2.1.2. Determination of gender of newborn mice Gender was determined by PCR detection of the chromosome Y sry gene. Primer sequences were sry1 (5%-GAG AGC ATG GAG GGC CAT-
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3%) and sry2 (5%-CCA CTC CTC TGT GAC ACT3%). The amplification program involved a denaturation cycle (3 min at 94 °C), followed by 35 cycles (of which each consisted of 30 sec at 94 °C, 30 sec at 58 °C, and 30 sec at 72 °C) and final elongation (2 min at 72 °C). PCR products were analysed in 1% agarose gel.
2.2. Whole-body flow plethysmography Breath duration (TTOT, sec), tidal volume (VT, ml/g), and ventilation (VE, calculated as VT/TTOT and expressed in ml/sec/g) were measured non-invasively using whole-body flow barometric plethysmography, based on Drorbaugh and Fenn’s principle as previously described (Dauger et al., 2001a). The plethysmography comprised two Plexiglas cylinders serving as animal (40 ml) and reference (100 ml) chambers. Both chambers were immersed in a thermoregulated water-bath that maintained their temperature at 30.5 °C. A 50-ml/min flow of dry air (Bronkhorst Hi-Tec airflow stabilizer, Uurlo, The Netherlands) was divided into two 25-ml/min flows through the chambers, thus avoiding CO2 and water accumulation. Body temperature was assumed to be stable at 32 °C. The differential pressure between the animal and the reference chambers (EFFA transducer, Asnie`res, France; range 90.1 mbar) was filtered (bandwidth, 0.05– 15 Hz at − 3 dB), converted to a digital signal (MacAdios A/D 12-bits converter, GW-Instruments, Somerville, MA) at a sample rate of 100 Hz, and processed by customwritten software (Software Superscope II, GW-Instruments). Calibration was done before each session by injecting 2 ml of air into the animal chamber from a Hamilton syringe. The pressure rise induced by this injection was of similar magnitude to that induced by the VT of a newborn mouse.
2.3. Procedure Each animal underwent one hypoxic or one hypercapnic test. After 30 sec of familiarization inside the measurement chamber, baseline ventilation was recorded for 3 min. Then, the airflow through the plethysmography was automatically
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switched to a hypoxic (3 min at 5% O2 + 95% N2) or hypercapnic flow (10 min at 8% CO2 + 21% O2 + 71% N2) at a flow level of 25 ml/min, for 3 min. The hypoxic test was followed by 6 min of normoxia. For the sake of brevity, we use ‘hypoxia’ to designate the period from the switch to hypoxia to the switch back to air, disregarding the initial transitory changes in gas concentrations, and ‘post-hypoxia’ for the period following the switch back to air. We measured mouth temperature and body weight after the tests.
2.4. Beha6ioural arousal Behaviour was scored continuously throughout the test, as previously described (Dauger et al., 2001a). Sleep was defined as immobility in the recumbent position (eye opening occurs at 14 days PNA in mice and, consequently, all mice had their eyes closed throughout the study period). Behavioural arousal was defined as a stereotyped motor response characterized by sudden neck and forepaw extension. This motor response lasted several seconds, during which the respiratory signal was not available, and sleep generally resumed soon after this transient motor activity. Myoclonic twitches were not considered indicative of arousal. Arousal latency was calculated from the switch between air and hypoxia.
2.5. Signal analysis Ventilatory data free from artefacts were selected visually by discarding portions of the tracings without individualized breaths or with large drifts exceeding twice the mean amplitude of the volume signal (Dauger et al., 2001a). TTOT, VT, and V: E were averaged over successive 10-sec periods. Sequences of valid breaths were used after exclusion of apnoeas, defined as ventilatory pauses longer than twice the duration of the preceding breath. Apnoea type (central, obstructive, or mixed) could not be determined. We calculated the number and total duration of apnoeas during a given period (normoxia, hypoxia, post-hypoxia, and hypercapnia). The entire air and hypoxic periods (3 min), and the last 3 min of the hypercapnia or post-hypoxic normoxia periods were used for these calculations.
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The peak value of the VE-response to hypoxia was determined over the entire hypoxic stimulus and expressed as the percent of baseline VE. To assess the post-hypoxic ventilatory decline (PHVD), the mean VE over the last 3 min of post-hypoxia was expressed as the percentage of baseline VE.
level weight category using the weight distribution quartiles as the breakpoint values. However, this four level category yielded the same statistical results as the two level-category and, consequently, will not be discussed further.
3. Results
2.5.1. Periodic breathing Periodic breathing was investigated visually over the entire respiratory volume signal of hypoxic tests. We adapted morphological criteria for periodic breathing in newborn infants (Weintraub et al., 2001), defining periodic breathing as alternation of breathing with apnoeas lasting longer than 2 sec and occurring at intervals of less than 10 sec. The following were measured: onset of the episode of periodic breathing, durations of the first and last apnoeas, and duration of the intermediate breathing phase. We also identified respiratory events characterized by an increase followed by a decrease in the volume signal amplitude (crescendo–decrescendo patterns, Weintraub et al., 2001). 2.6. Statistics Variables were subjected to analyses of variance (ANOVA) with genotype, sex, and weight as between-subject factors (Superanova Software, Abacus Concepts, Berkeley, CA). Weight was introduced in the ANOVA as a category using the median (two-level category: ‘small’ and ‘large’ for the sake of convenience). We also defined a four-
3.1. Effects of the heterozygous c-ret + /− mutation on de6elopment Sample sizes, mean weight, and temperature for each test are presented in Table 1. The c-ret+ /− and c-ret+ /+ pups had similar weights, showing that the heterozygous c-ret mutation had no major effects on gross body development. Inter-individual variability in weight was accounted for by differences across litters: the mean weight of animals ranged across litters from 1.4 to 1.9 g (PB 0.0001). Litter size, which ranged from 2 to 10 animals, was inversely correlated with weight (r 2 = 0.47, PB 0.0001). The genotype of the pups was not statistically related to litter size. Body temperature was very similar in c-ret+ /+ and c-ret+ /− pups. The c-ret+ /− pups were normal in appearance and behaviour.
3.2. Ventilatory response to hypoxia Baseline VE was very similar in c-ret+ /− and c-ret+ /+ pups (13.39 3.8 and 13.294.0 ml/sec/ g, respectively). VE displayed a biphasic pattern in response to hypoxia without significant differ-
Table 1 Characteristics of the newborn mice Hypercapnia
Hypoxia
Genotype
c-ret+/+
c-ret+/−
c-ret+/+
c-ret+/−
Number M/F Weight (g) Temperature (°C)
10 5/5 1.509 0.2 32.490.9
9 6/3 1.60 90.1 32.8 9 0.4
33 15/18 1.48 9 0.2 31.8 9 0.6
22 11/11 1.55 9 0.2 31.9 9 0.5
Body weights, age and mouth temperature were not significantly different between heterozygous c-ret+/− and c-ret+/+ or between males and females. Values are means 9S.D.
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Fig. 1. Ventilatory responses to 5% O2 (left panel) and 8% CO2 (right panel) in c-ret+/ + and c-ret +/ − newborn mice. The tests were conducted at 10 – 12 h of PNA. Successive 10-sec means were averaged over 1-min periods for clarity. The ventilatory response to hypoxia showed a biphasic pattern in both groups. Arrows indicate the arousal response. No differences were observed between the two genotype groups. Values are group means 9 SEM.
ences between c-ret +/ − and c-ret+/ + pups (Fig. 1, left panel). Genotype had no significant effects on peak VE increase (62971% and 429 52%, respectively) or PHVD (− 299 26% and − 34952%, respectively). Arousal occurred in all pups, and time from hypoxia onset to arousal was closely similar in c-ret +/ − and c-ret +/ + pups (769 22 and 76924 sec, respectively). Weight and gender had no effects on either variable.
3.3. Apnoeas All apnoeas, including those within periodic breathing episodes, were considered for this analysis. Illustrative examples of respiratory traces are shown in Fig. 2. In both genotype groups, the pups had more apnoeas and longer total apnoea duration during post-hypoxic normoxia than during normoxia or hypoxia (PB 0.0001 for both comparisons, Fig. 3). The ‘small’ pups had more
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Fig. 2. Illustrative examples of breathing pattern in normoxia, hypoxia, and post-hypoxic normoxia in one typical c-ret +/ − newborn (top) and one typical c-ret + /+ (bottom). The traces during hypoxia corresponded to the hyperpnoeic phase of the biphasic hypoxic response. The traces during post-hypoxic normoxia were selected 2 min after the switch back to air. In both pups, breathing was depressed and irregular during post-hypoxic normoxia, but only the c-ret +/ − pup exhibited apnoeas and periodic breathing.
apnoeas than the ‘large’ pups, irrespective of genotype, and their total apnoea duration was longer (Table 2). The number and total duration of apnoeas were twice as large in c-ret +/ − as in c-ret +/ + newborn mice (Fig. 3, P =0.032 and P = 0.025, respectively). These genotype-related differences were significant during hypoxia (P = 0.023 and P = 0.009, respectively) and during post-hypoxic normoxia (P=0.048 and P = 0.026). They did not reach significance during normoxia (P = 0.086 and P=0.09, phase-by-genotype interaction, NS). Genotype-related effects on apnoea number and duration were independent from body weights, which were very similar in c-ret +/ + and c-ret+ / − mice (Table 1 and Table 2). In addition, the genotype-by-weight interactions were not significant for apnoea number or total apnoea duration (Table 2). Gender had no significant effects on these variables.
occasionally (number during the entire test: 1.89 1.4 in c-ret+ /+ and 1.79 1.7 in c-ret+ /− ), with no between-group difference in mean duration (21.59 24.2 and 26.69 21.8 sec, respectively; NS). Duration of periodic breathing was significantly longer in the male pups than in the female
3.4. Periodic breathing Periodic breathing was analysed over the entire hypoxic test (air, hypoxia, post-hypoxia). The number and the total duration of periodic breathing episodes were 2- to 3-fold larger in ‘small’ than in ‘large’ pups and were twice as large in c-ret +/− as in c-ret +/ + pups (Table 2). The effects of weight and genotype were independent (similar weights in c-ret +/ + and c-ret +/ −, with a non-significant weight-by-genotype interaction). Crescendo–decrescendo patterns occurred
Fig. 3. Number and total duration of apnoeas during 3-min normoxia, 3-min hypoxia (5% O2) and 6-min post-hypoxic normoxia in c-ret +/ + and c-ret +/ − newborn mice at 10 – 12 h of PNA. The corresponding numbers of apnoeas per min were 0.21, 0.12, and 0.59 in c-ret+/ − mice and 0.08, 0.01, and 0.34 in c-ret+/ + mice. Apnoeas were more numerous (P= 0.032) and their total duration was longer (P= 0.025) in c-ret +/ − than in c-ret +/ + mice. See text for complementary analyses. Values are group means 9SEM.
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Table 2 Summary ANOVAs of the number and total duration of apnoeas, and of the number and total durations of periodic breathing episodes, as a function of genotype and weight c-ret+/+ (n=33) Small (n= 14) Large (n=19)
c-ret+/− (n =22) Small (n =9) Large (n = 13)
P value +/+ vs. +/−
Weight (g) Apnoeas number Group mean
1.33 90.10 3.89 2.8 2.39 2.5
1.63 9 0.11 1.3 91.7
1.33 9 0.04 6.7 9 1.9 4.69 5.6
1.70 9 0.14 3.1 9 5.1
NS 0.032 0.03
0.0001 0.006
Total duration (sec) Group mean
11.7 912.7 6.29 9.6
2.2 92.7
28.4 934.2 15.3 925.4
6.3 911.4
0.024 0.025
0.001
Periodic breathing number Group mean
0.57 91.02
0.21 9 0.53
1.33 91.41
0.46 90.52
0.04
0.01
Duration (sec) Group mean
6.69 12.0 4.2 9 9.2
0.36 90.78
0.82 9 1.05 2.5 96.3
20.0 922.5 10.8 916.4
Small vs. large
0.040 4.5 9 5.3
0.02 0.023
0.004
Values are means 9S.D. Weight-by-genotype interactions were not significant for either variable.
pups, in both genotype groups (3.69 6.8 and 10.5916.8, PB0.04, genotype-by-gender interaction, NS).
3.5. Ventilatory response to hypercapnia Baseline VE was similar in c-ret +/ − and cret +/+ pups (14.29 3.1 and 14.39 4.0 ml/sec/g, respectively). Neither ventilatory responses to hypercapnia nor arousal latencies were significantly different between c-ret+/ − and c-ret +/ + mice (Fig. 1, right). Both groups showed a sustained VE increase during hypercapnia (77953 and 78 930%, respectively; NS) related mainly to a VT increase. Arousal occurred during the VE increase about 2 min after stimulus onset in both groups (1229 66 and 1469 67 sec, respectively; NS). One c-ret+/− animal (out of 9) and one c-ret +/+ animal (out of 10) did not arouse in response to hypercapnia. Apnoeas and periodic breathing were exceedingly uncommon during hypercapnia. Weight and gender had no significant effects on either variable.
4. Discussion The main results of this study are that c-ret +/ − newborn mice had (1) normal baseline ventila-
tion and hyperpnoeic and arousal responses to hypoxia and hypercapnia and (2) increased respiratory instability characterized by more numerous apnoeas with or without periodic breathing during hypoxia and post-hypoxic normoxia. These results support the hypothesis that the heterozygous c-ret mutation may impair ventilatory control at birth.
4.1. Genotype-related differences in respiratory control Whereas homozygous c-ret− /− mutant mice exhibit severe respiratory impairments and die within the first hours of life, our heterozygous c-ret+ /− mice developed normally, were fertile, and produced c-ret+ /− pups that were normal in weight and general appearance at 10– 12 h PNA. Despite their normal development, these heterozygous pups showed respiratory instability characterized by an increased frequency of apnoeas and periodic breathing. This effect was significant during hypoxia and the PHVD, when apnoeas occurred more frequently than during normoxia. However, a trend was found during normoxia, indicating that the unstable pattern in c-ret+ /− pups was not specific of hypoxia-induced depression. Genotype did not influence the relationship between weight and apnoeas. There-
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fore, c-ret+ /− should prove valuable for studying postnatal impairments in respiratory control independently from confounding related to major developmental disorders. The present results are at variance with a report by Burton et al. that neither c-ret +/ − nor cret + /+ newborn pups showed consistent ventilation changes in response to hypoxia (Burton et al., 1997). This discrepancy may stem from several methodological differences in the age of the pups, measurement methods, and determination of the hypoxic response. First, Burton et al. tested the pups over the entire day of birth, whereas we performed our tests only at H10– H12, i.e. after peripheral chemoreceptor resetting (Renolleau et al., 2001a). Second, in the study by Burton et al., the VE-response to hypoxia was averaged over a 10-min period of hypoxia, which included the HVD (Dauger et al., 2001a,b). In contrast, we averaged breathing variables over successive 10sec periods, so that we could determine the timecourse of the ventilatory response and distinguish the hyperpnoeic response from the HVD. Third, the head-out plethysmograph used by Burton et al. requires restraining the pups, whereas the whole-body plethysmography used in our study allows the pups to move freely. Restraint is a potent stressor that can influence the breathing pattern, as previously shown in adult mice (Dauger et al., 1998). The high frequency of apnoeic episodes and periodic breathing in c-ret+/ − pups contrasted with the normal breathing rate of these animals under normoxic, hypoxic and hypercapnic conditions in the intervals separating the apnoeas. In earlier studies, apnoeas were associated with an abnormally low breathing rate in newborn Krox20 −/− mice (Jacquin et al., 1996) and with an abnormally high breathing rate in newborn Rnx −/− mice (Shirasawa et al., 2000). Furthermore, Mash-1 − /− newborn mice had an increased breathing rate, with normal apnoea duration and frequency (Dauger et al., 2001b). Taken together, these results suggest that apnoea characteristics and breathing rate may be under different neural control mechanisms that may be selectively impaired by gene mutations.
4.2. Potential mechanisms underlying the c-ret + /− respiratory phenotype The c-ret gene is expressed in several brainstem structures involved in respiratory control (Dauger et al., 2001b): the rostral ventrolateral medulla (rVLM), which plays a crucial role in respiratory rhythmogenesis (Rekling and Feldman, 1998); the nucleus tractus solitarius, which receives afferents from peripheral chemoreceptors and is, therefore, closely involved in the hyperpnoeic component of the hypoxic response; and the A5 and A6 (locus coeruleus) nuclei, which inhibit the medullary respiratory rhythm generator in the rVLM (Arata et al., 1998; Moore et al., 1996). In addition to exerting inhibitory modulation on the respiratory rhythm generator via a2-adrenoceptors located in the rVLM, noradrenaline directly facilitates the phrenic motoneurones by activating the cervical a1-adrenoceptors (Hilaire and Duron, 1999; Bianchi et al., 1995; Al-Zubaidy et al., 1996). The c-ret heterozygous mutation may delay the maturation of noradrenergic structures involved in respiratory control, thus shifting the balance between stimulation and inhibition of breathing. In keeping with this possibility, the heterozygous mutation in Mash-1, which is upstream of c-ret in the noradrenergic brainstem neurons, caused respiratory impairments in newborn pups but not in adult mice (Dauger et al., 1999). Respiratory impairments in newborns, but not in adult mice were also found in heterozygous mutants for the endothelin-converting-enzyme, another gene involved in neural crest development (Renolleau et al., 2001b). A longitudinal study on the timecourse of respiratory impairments in c-ret+ /− mice would perhaps clarify this issue. On the other hand, the normal sustained hyperpnoeic response to hypercapnia in the c-ret+ /− pups indicates that the higher rate of apnoeas during the hypoxic test was not ascribable to an inability to maintain high levels of ventilation. Our data did not allow to determine sleep states, which have an important influence on the breathing pattern, including apnoeas. Therefore, it is unclear whether apnoeas occurred during active sleep, as is the rule in newborn mammals, or whether sleep architecture was affected by the
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c-ret + /− mutation. We cannot preclude that the higher rate of apnoeas in c-ret+/ − newborn mice was related to a larger amount of active sleep.
4.3. Conclusion Newborn mice with the c-ret +/ − genotype survive and develop normally, but show impairments of respiratory control characterized by more frequent and longer apnoeas than their wildtype littermates. This result is further evidence that genes involved in the neural determination of neural crest cells may play a role in the development of respiratory control. On the other hand, the normal hyperpnoeic responses to hypoxia and hypercapnia in c-ret +/ − mice contrast with the lack of chemosensitivity that characterizes CCHS patients. As noted above, c-ret heterozygous mutations have been found in some CCHS patients. However, the respiratory phenotype differences between c-ret+ /− mice and CCHS patients regarding chemosensitivity further supports the multigenic origin of CCHS.
Acknowledgements This study was supported by the Fondation pour La Recherche Me´ dicale, the Institut de Recherches Internationales Servier (grant awarded to S. Aizenfisz), the Universite´ Paris VII (Legs Poix), and EEC Biomed grant BMH4-972107.
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Bianchi, A.L., Denavit-Saubie, M., Champagnat, J., 1995. Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters. Physiol. Rev. 75, 1 – 45. Burton, M.D., Kawashima, A., Brayer, J.A., Kazemi, H., Shannon, D.C., Schuchardt, A., Costantini, F., Pachnis, V., Kinane, T.B., 1997. RET proto-oncogene is important for the development of respiratory CO2 sensitivity. J. Auton. Nerv. Syst. 63, 137 – 143. Dauger, S., Nsegbe, E., Vardon, G., Gaultier, C., Gallego, J., 1998. The effects of restraint on ventilatory responses to hypercapnia and hypoxia in adult mice. Respir. Physiol. 112, 215 – 225. Dauger, S., Renolleau, S., Vardon, G., Nepote, V., Mas, C., Simonneau, M., Gaultier, C., Gallego, J., 1999. Ventilatory responses to hypercapnia and hypoxia in Mash-1 heterozygous newborn and adult mice. Pediatr. Res. 46, 535 – 542. Dauger, S., Aizenfisz, S., Durand, E., Renolleau, S., Vardon, G., Gaultier, C., Gallego, J., 2001a. Arousal response to hypoxia in newborn mice. Respir. Physiol. 128, 235 – 240. Dauger, S., Guimiot, F., Renolleau, S., Levacher, B., Boda, B., Mas, C., Ne´ pote, V., Simonneau, M., Gaultier, C., Gallego, J., 2001b. MASH-1/RET pathway involvement in development of brainstem control of respiratory frequency in newborn mice. Physiol. Genomics 7, 149 – 157. Gozal, D., Simakajornboon, N., Czapla, M.A., Xue, Y.D., Gozal, E., Vlasic, V., Lasky, J.A., Liu, J.Y., 2000. Brainstem activation of platelet-derived growth factor-beta receptor modulates the late phase of the hypoxic ventilatory response. J. Neurochem. 74, 310 – 319. Hilaire, G., Duron, B., 1999. Maturation of the mammalian respiratory system. Physiol. Rev. 79, 325 – 360. Jacquin, T.D., Borday, V., Schneider-Maunoury, S., Topilko, P., Ghilini, G., Kato, F., Charnay, P., Champagnat, J., 1996. Reorganization of pontine rhythmogenic neuronal networks in Krox-20 knockout mice. Neuron 17, 747 – 758. Moore, P.J., Ackland, G.L., Hanson, M.A., 1996. Unilateral cooling in the region of locus coeruleus blocks the fall in respiratory output during hypoxia in anaesthetized neonatal sheep. Exp. Physiol. 81, 983 – 994. Rekling, J.C., Feldman, J.L., 1998. PreBotzinger complex and pacemaker neurons: hypothesized site and kernel for respiratory rhythm generation. Annu. Rev. Physiol. 60, 385 – 405. Renolleau, S., Dauger, S., Autret, F., Vardon, G., Gaultier, C., Gallego, J., 2001a. Maturation of baseline breathing and of hypercapnic and hypoxic ventilatory responses in newborn mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R1746 –R1753. Renolleau, S., Dauger, S., Vardon, G., Levacher, B., Simonneau, M., Yanagisawa, M., Gaultier, C., Gallego, J., 2001b. Impaired ventilatory responses to hypoxia in mice deficient in endothelin-converting-enzyme-1. Pediatr. Res. 49, 705 – 712.
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Sakai, T., Wakizaka, A., Nirasawa, Y., 2001. Congenital central hypoventilation syndrome associated with Hirschsprung’s disease: mutation analysis of the RET and endothelin-signaling pathways. Eur. J. Pediatr. Surg. 11, 335 –337. Shirasawa, S., Arata, A., Onimaru, H., Roth, K.A., Brown, G.A., Horning, S., Arata, S., Okumura, K., Sasazuki, T.,
Korsmeyer, S.J., 2000. Rnx deficiency results in congenital central hypoventilation. Nat. Genet. 24, 287 – 290. Weintraub, Z., Cates, D., Kwiatkowski, K., Al-Hathlol, K., Hussain, A., Rigatto, H., 2001. The morphology of periodic breathing in infants and adults. Respir. Physiol. 127, 173 – 184.
Ventilatory responses to hypercapnia and hypoxia in heterozygous c-ret newborn mice Sophie Aizenfisz a,c, Ste´phane Dauger a,c, Estelle Durand a, Guy Vardon d, Be´atrice Levacher a, Michel Simonneau a, Vassilis Pachnis e, Claude Gaultier a,b, Jorge Gallego a,* Laboratoire de Neurologie et Physiologie du De´6eloppement, Hoˆpital Robert-Debre´, 48 Boule6ard Se´rurier, 75019 Paris, France b Ser6ice de Physiologie, Hoˆpital Robert-Debre´, 48 Boule6ard Se´rurier, 75019 Paris, France c Ser6ice de Pe´diatrie-Re´animation, Hoˆpital Robert-Debre´, 48 Boule6ard Se´rurier, 75019 Paris, France d Unite´ de Recherches sur les Adaptations Physiologiques et Comportementales, Uni6ersite´ de Picardie, 80036 Amiens, France e Di6ision of Molecular Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK
a
Accepted 16 April 2002
Abstract The c-ret proto-oncogene encodes a tyrosine-kinase receptor involved in survival and differentiation of neural crest cell lineages. Previous studies have shown that homozygous c-ret− / − mice die soon after birth and have impaired ventilatory responses to hypercapnia. Heterozygous c-ret + / − mice develop normally, but their respiratory phenotype has not been described in detail. We used whole-body flow plethysmography to compare baseline breathing and ventilatory and arousal responses to chemical stimuli in unrestrained heterozygous c-ret + / − newborn mice and their wild-type c-ret +/+ littermates at 10–12 h of postnatal age. The hyperpnoeic and arousal responses to hypoxia and hypercapnia were not significantly different in these two groups. However, the number and total duration of apnoeas and periodic breathing episodes were significantly higher in c-ret+ / − than in c-ret+ / + pups during hypoxia and post-hypoxic normoxia. These results are further evidence that respiratory control at birth is heavily dependent on genes involved in the neural determination of neural crest cells. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Development; pattern of breathing; Gene; proto-oncogene; c-ret; Mammals; mouse; Pattern of breathing; c-ret +/ −
1. Introduction The c-ret proto-oncogene encodes a tyrosine-kinase receptor involved in survival and differentia* Corresponding author. Tel.: + 33-1-40-03-47-81; fax: + 33-1-40-03-47-70. E-mail address: [email protected] (J. Gallego).
tion of neural crest cell lineages. The respiratory phenotype of c-ret+ /− newborn mice deserves careful investigation because heterozygous mutations in c-ret and its ligand Glial-Derived-Neurotrophic-Factor have been found in patients with central congenital hypoventilation syndrome (CCHS) (Amiel et al., 1998; Sakai et al., 2001), a
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disorder characterized by impaired respiratory control. In an earlier study, homozygous c-ret − / − mice died soon after birth, possibly from respiratory failure, and showed impaired ventilatory responses to hypercapnia (Burton et al., 1997). In contrast, heterozygous c-ret+/ − mice developed normally. On average, their ventilatory responses to hypercapnia and hypoxia showed no significant differences with those of wild-type c-ret + /+ newborns (Burton et al., 1997). However, important aspects of the postnatal maturation of breathing in mice were not taken into account in this previous study (Burton et al., 1997). First, in mice, the ventilatory response to hypoxia is small before postnatal chemoreceptor resetting and, consequently, should be examined after this event, which occurs at 6– 12 h of postnatal age (PNA) (Renolleau et al., 2001a). Second, the initial hyperpnoeic response to hypoxia is followed by a decline (hypoxic ventilatory decline, HVD) that outlasts the end of hypoxia and is associated with more numerous and longer apnoeas (Dauger et al., 2001a). These two components of the hypoxic response involve different neuronal systems (Gozal et al., 2000) and should, therefore, be investigated separately. Third, the ventilatory responses to hypoxia and hypercapnia are modulated by arousal processes starting at birth, and these processes may also be affected by the mutation (Dauger et al., 2001a). In the present study, we tested the hypothesis that the heterozygous c-ret mutation may impair respiratory control in newborn mice. To do this, we compared baseline breathing and ventilatory and arousal responses to hypoxia and hypercapnia in heterozygous c-ret +/ − newborn mice and in their wild-type c-ret+/ + littermates. We conducted the ventilatory tests after peripheral chemoreceptor resetting (between H10 and H12 PNA), and we measured breathing variables non-invasively using whole-body plethysmography to avoid possible breathing changes related to restraining the animals (Dauger et al., 1998).
2. Methods
2.1. Animals The c-ret− deficient mice were kept heterozygous on a 129SvEv× C57BL/10 hybrid background from heterozygous breed pairs. The mice were housed at 24 °C with a normal 12-h light–dark cycle and were given food and water ad libitum. Newborn mice were obtained by mating heterozygous c-ret+ /− males (provided by Pachnis, National Institute for Medical Research, London) with wild-type females from the same 129Sv hybrid background. Vaginal plugs were examined on the morning of the next day, which was counted as embryonic day 0 (E0). Time of birth was determined by checking the mice from E19 onwards, at intervals of 1 h during the day and of 4–6 h during the night. Seventy-four newborn mice were obtained and were tested between H10 and H12 of PNA.
2.1.1. Genotyping After ventilatory measurements, each newborn was weighed and sacrificed by neck section. Leg and tail tissue fragments were taken for c-ret, as well as for sry genotyping to determine the gender. These samples were subjected to PCR under standard conditions. The PCR primer sequences for the wild allele were exons 6 and 7 from the c-ret gene (P6: 5%-TGG GAG AAG GCG AGT TTG GAA A-3%; and P7: 5%-TTC AGG AAC ACT GGC TAC CAT G-3%). The PCR primer sequences for the mutant allele were exon 12 from the c-ret gene (5%-TTC CTC AGC ATG TAT CCC AT-3%) and one exon from the neomycin gene (5%-CGT GAT CGA CAA GAC CGG CTT3%). The amplification program comprised a denaturation cycle (5 min at 94 °C), followed by 35 cycles (of which each consisted of 40 sec at 94 °C, 40 sec at 58 °C, and 40 sec at 72 °C) and final elongation (2 min at 72 °C). PCR products were analysed in 1% agarose gel. 2.1.2. Determination of gender of newborn mice Gender was determined by PCR detection of the chromosome Y sry gene. Primer sequences were sry1 (5%-GAG AGC ATG GAG GGC CAT-
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3%) and sry2 (5%-CCA CTC CTC TGT GAC ACT3%). The amplification program involved a denaturation cycle (3 min at 94 °C), followed by 35 cycles (of which each consisted of 30 sec at 94 °C, 30 sec at 58 °C, and 30 sec at 72 °C) and final elongation (2 min at 72 °C). PCR products were analysed in 1% agarose gel.
2.2. Whole-body flow plethysmography Breath duration (TTOT, sec), tidal volume (VT, ml/g), and ventilation (VE, calculated as VT/TTOT and expressed in ml/sec/g) were measured non-invasively using whole-body flow barometric plethysmography, based on Drorbaugh and Fenn’s principle as previously described (Dauger et al., 2001a). The plethysmography comprised two Plexiglas cylinders serving as animal (40 ml) and reference (100 ml) chambers. Both chambers were immersed in a thermoregulated water-bath that maintained their temperature at 30.5 °C. A 50-ml/min flow of dry air (Bronkhorst Hi-Tec airflow stabilizer, Uurlo, The Netherlands) was divided into two 25-ml/min flows through the chambers, thus avoiding CO2 and water accumulation. Body temperature was assumed to be stable at 32 °C. The differential pressure between the animal and the reference chambers (EFFA transducer, Asnie`res, France; range 90.1 mbar) was filtered (bandwidth, 0.05– 15 Hz at − 3 dB), converted to a digital signal (MacAdios A/D 12-bits converter, GW-Instruments, Somerville, MA) at a sample rate of 100 Hz, and processed by customwritten software (Software Superscope II, GW-Instruments). Calibration was done before each session by injecting 2 ml of air into the animal chamber from a Hamilton syringe. The pressure rise induced by this injection was of similar magnitude to that induced by the VT of a newborn mouse.
2.3. Procedure Each animal underwent one hypoxic or one hypercapnic test. After 30 sec of familiarization inside the measurement chamber, baseline ventilation was recorded for 3 min. Then, the airflow through the plethysmography was automatically
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switched to a hypoxic (3 min at 5% O2 + 95% N2) or hypercapnic flow (10 min at 8% CO2 + 21% O2 + 71% N2) at a flow level of 25 ml/min, for 3 min. The hypoxic test was followed by 6 min of normoxia. For the sake of brevity, we use ‘hypoxia’ to designate the period from the switch to hypoxia to the switch back to air, disregarding the initial transitory changes in gas concentrations, and ‘post-hypoxia’ for the period following the switch back to air. We measured mouth temperature and body weight after the tests.
2.4. Beha6ioural arousal Behaviour was scored continuously throughout the test, as previously described (Dauger et al., 2001a). Sleep was defined as immobility in the recumbent position (eye opening occurs at 14 days PNA in mice and, consequently, all mice had their eyes closed throughout the study period). Behavioural arousal was defined as a stereotyped motor response characterized by sudden neck and forepaw extension. This motor response lasted several seconds, during which the respiratory signal was not available, and sleep generally resumed soon after this transient motor activity. Myoclonic twitches were not considered indicative of arousal. Arousal latency was calculated from the switch between air and hypoxia.
2.5. Signal analysis Ventilatory data free from artefacts were selected visually by discarding portions of the tracings without individualized breaths or with large drifts exceeding twice the mean amplitude of the volume signal (Dauger et al., 2001a). TTOT, VT, and V: E were averaged over successive 10-sec periods. Sequences of valid breaths were used after exclusion of apnoeas, defined as ventilatory pauses longer than twice the duration of the preceding breath. Apnoea type (central, obstructive, or mixed) could not be determined. We calculated the number and total duration of apnoeas during a given period (normoxia, hypoxia, post-hypoxia, and hypercapnia). The entire air and hypoxic periods (3 min), and the last 3 min of the hypercapnia or post-hypoxic normoxia periods were used for these calculations.
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The peak value of the VE-response to hypoxia was determined over the entire hypoxic stimulus and expressed as the percent of baseline VE. To assess the post-hypoxic ventilatory decline (PHVD), the mean VE over the last 3 min of post-hypoxia was expressed as the percentage of baseline VE.
level weight category using the weight distribution quartiles as the breakpoint values. However, this four level category yielded the same statistical results as the two level-category and, consequently, will not be discussed further.
3. Results
2.5.1. Periodic breathing Periodic breathing was investigated visually over the entire respiratory volume signal of hypoxic tests. We adapted morphological criteria for periodic breathing in newborn infants (Weintraub et al., 2001), defining periodic breathing as alternation of breathing with apnoeas lasting longer than 2 sec and occurring at intervals of less than 10 sec. The following were measured: onset of the episode of periodic breathing, durations of the first and last apnoeas, and duration of the intermediate breathing phase. We also identified respiratory events characterized by an increase followed by a decrease in the volume signal amplitude (crescendo–decrescendo patterns, Weintraub et al., 2001). 2.6. Statistics Variables were subjected to analyses of variance (ANOVA) with genotype, sex, and weight as between-subject factors (Superanova Software, Abacus Concepts, Berkeley, CA). Weight was introduced in the ANOVA as a category using the median (two-level category: ‘small’ and ‘large’ for the sake of convenience). We also defined a four-
3.1. Effects of the heterozygous c-ret + /− mutation on de6elopment Sample sizes, mean weight, and temperature for each test are presented in Table 1. The c-ret+ /− and c-ret+ /+ pups had similar weights, showing that the heterozygous c-ret mutation had no major effects on gross body development. Inter-individual variability in weight was accounted for by differences across litters: the mean weight of animals ranged across litters from 1.4 to 1.9 g (PB 0.0001). Litter size, which ranged from 2 to 10 animals, was inversely correlated with weight (r 2 = 0.47, PB 0.0001). The genotype of the pups was not statistically related to litter size. Body temperature was very similar in c-ret+ /+ and c-ret+ /− pups. The c-ret+ /− pups were normal in appearance and behaviour.
3.2. Ventilatory response to hypoxia Baseline VE was very similar in c-ret+ /− and c-ret+ /+ pups (13.39 3.8 and 13.294.0 ml/sec/ g, respectively). VE displayed a biphasic pattern in response to hypoxia without significant differ-
Table 1 Characteristics of the newborn mice Hypercapnia
Hypoxia
Genotype
c-ret+/+
c-ret+/−
c-ret+/+
c-ret+/−
Number M/F Weight (g) Temperature (°C)
10 5/5 1.509 0.2 32.490.9
9 6/3 1.60 90.1 32.8 9 0.4
33 15/18 1.48 9 0.2 31.8 9 0.6
22 11/11 1.55 9 0.2 31.9 9 0.5
Body weights, age and mouth temperature were not significantly different between heterozygous c-ret+/− and c-ret+/+ or between males and females. Values are means 9S.D.
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Fig. 1. Ventilatory responses to 5% O2 (left panel) and 8% CO2 (right panel) in c-ret+/ + and c-ret +/ − newborn mice. The tests were conducted at 10 – 12 h of PNA. Successive 10-sec means were averaged over 1-min periods for clarity. The ventilatory response to hypoxia showed a biphasic pattern in both groups. Arrows indicate the arousal response. No differences were observed between the two genotype groups. Values are group means 9 SEM.
ences between c-ret +/ − and c-ret+/ + pups (Fig. 1, left panel). Genotype had no significant effects on peak VE increase (62971% and 429 52%, respectively) or PHVD (− 299 26% and − 34952%, respectively). Arousal occurred in all pups, and time from hypoxia onset to arousal was closely similar in c-ret +/ − and c-ret +/ + pups (769 22 and 76924 sec, respectively). Weight and gender had no effects on either variable.
3.3. Apnoeas All apnoeas, including those within periodic breathing episodes, were considered for this analysis. Illustrative examples of respiratory traces are shown in Fig. 2. In both genotype groups, the pups had more apnoeas and longer total apnoea duration during post-hypoxic normoxia than during normoxia or hypoxia (PB 0.0001 for both comparisons, Fig. 3). The ‘small’ pups had more
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Fig. 2. Illustrative examples of breathing pattern in normoxia, hypoxia, and post-hypoxic normoxia in one typical c-ret +/ − newborn (top) and one typical c-ret + /+ (bottom). The traces during hypoxia corresponded to the hyperpnoeic phase of the biphasic hypoxic response. The traces during post-hypoxic normoxia were selected 2 min after the switch back to air. In both pups, breathing was depressed and irregular during post-hypoxic normoxia, but only the c-ret +/ − pup exhibited apnoeas and periodic breathing.
apnoeas than the ‘large’ pups, irrespective of genotype, and their total apnoea duration was longer (Table 2). The number and total duration of apnoeas were twice as large in c-ret +/ − as in c-ret +/ + newborn mice (Fig. 3, P =0.032 and P = 0.025, respectively). These genotype-related differences were significant during hypoxia (P = 0.023 and P = 0.009, respectively) and during post-hypoxic normoxia (P=0.048 and P = 0.026). They did not reach significance during normoxia (P = 0.086 and P=0.09, phase-by-genotype interaction, NS). Genotype-related effects on apnoea number and duration were independent from body weights, which were very similar in c-ret +/ + and c-ret+ / − mice (Table 1 and Table 2). In addition, the genotype-by-weight interactions were not significant for apnoea number or total apnoea duration (Table 2). Gender had no significant effects on these variables.
occasionally (number during the entire test: 1.89 1.4 in c-ret+ /+ and 1.79 1.7 in c-ret+ /− ), with no between-group difference in mean duration (21.59 24.2 and 26.69 21.8 sec, respectively; NS). Duration of periodic breathing was significantly longer in the male pups than in the female
3.4. Periodic breathing Periodic breathing was analysed over the entire hypoxic test (air, hypoxia, post-hypoxia). The number and the total duration of periodic breathing episodes were 2- to 3-fold larger in ‘small’ than in ‘large’ pups and were twice as large in c-ret +/− as in c-ret +/ + pups (Table 2). The effects of weight and genotype were independent (similar weights in c-ret +/ + and c-ret +/ −, with a non-significant weight-by-genotype interaction). Crescendo–decrescendo patterns occurred
Fig. 3. Number and total duration of apnoeas during 3-min normoxia, 3-min hypoxia (5% O2) and 6-min post-hypoxic normoxia in c-ret +/ + and c-ret +/ − newborn mice at 10 – 12 h of PNA. The corresponding numbers of apnoeas per min were 0.21, 0.12, and 0.59 in c-ret+/ − mice and 0.08, 0.01, and 0.34 in c-ret+/ + mice. Apnoeas were more numerous (P= 0.032) and their total duration was longer (P= 0.025) in c-ret +/ − than in c-ret +/ + mice. See text for complementary analyses. Values are group means 9SEM.
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Table 2 Summary ANOVAs of the number and total duration of apnoeas, and of the number and total durations of periodic breathing episodes, as a function of genotype and weight c-ret+/+ (n=33) Small (n= 14) Large (n=19)
c-ret+/− (n =22) Small (n =9) Large (n = 13)
P value +/+ vs. +/−
Weight (g) Apnoeas number Group mean
1.33 90.10 3.89 2.8 2.39 2.5
1.63 9 0.11 1.3 91.7
1.33 9 0.04 6.7 9 1.9 4.69 5.6
1.70 9 0.14 3.1 9 5.1
NS 0.032 0.03
0.0001 0.006
Total duration (sec) Group mean
11.7 912.7 6.29 9.6
2.2 92.7
28.4 934.2 15.3 925.4
6.3 911.4
0.024 0.025
0.001
Periodic breathing number Group mean
0.57 91.02
0.21 9 0.53
1.33 91.41
0.46 90.52
0.04
0.01
Duration (sec) Group mean
6.69 12.0 4.2 9 9.2
0.36 90.78
0.82 9 1.05 2.5 96.3
20.0 922.5 10.8 916.4
Small vs. large
0.040 4.5 9 5.3
0.02 0.023
0.004
Values are means 9S.D. Weight-by-genotype interactions were not significant for either variable.
pups, in both genotype groups (3.69 6.8 and 10.5916.8, PB0.04, genotype-by-gender interaction, NS).
3.5. Ventilatory response to hypercapnia Baseline VE was similar in c-ret +/ − and cret +/+ pups (14.29 3.1 and 14.39 4.0 ml/sec/g, respectively). Neither ventilatory responses to hypercapnia nor arousal latencies were significantly different between c-ret+/ − and c-ret +/ + mice (Fig. 1, right). Both groups showed a sustained VE increase during hypercapnia (77953 and 78 930%, respectively; NS) related mainly to a VT increase. Arousal occurred during the VE increase about 2 min after stimulus onset in both groups (1229 66 and 1469 67 sec, respectively; NS). One c-ret+/− animal (out of 9) and one c-ret +/+ animal (out of 10) did not arouse in response to hypercapnia. Apnoeas and periodic breathing were exceedingly uncommon during hypercapnia. Weight and gender had no significant effects on either variable.
4. Discussion The main results of this study are that c-ret +/ − newborn mice had (1) normal baseline ventila-
tion and hyperpnoeic and arousal responses to hypoxia and hypercapnia and (2) increased respiratory instability characterized by more numerous apnoeas with or without periodic breathing during hypoxia and post-hypoxic normoxia. These results support the hypothesis that the heterozygous c-ret mutation may impair ventilatory control at birth.
4.1. Genotype-related differences in respiratory control Whereas homozygous c-ret− /− mutant mice exhibit severe respiratory impairments and die within the first hours of life, our heterozygous c-ret+ /− mice developed normally, were fertile, and produced c-ret+ /− pups that were normal in weight and general appearance at 10– 12 h PNA. Despite their normal development, these heterozygous pups showed respiratory instability characterized by an increased frequency of apnoeas and periodic breathing. This effect was significant during hypoxia and the PHVD, when apnoeas occurred more frequently than during normoxia. However, a trend was found during normoxia, indicating that the unstable pattern in c-ret+ /− pups was not specific of hypoxia-induced depression. Genotype did not influence the relationship between weight and apnoeas. There-
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fore, c-ret+ /− should prove valuable for studying postnatal impairments in respiratory control independently from confounding related to major developmental disorders. The present results are at variance with a report by Burton et al. that neither c-ret +/ − nor cret + /+ newborn pups showed consistent ventilation changes in response to hypoxia (Burton et al., 1997). This discrepancy may stem from several methodological differences in the age of the pups, measurement methods, and determination of the hypoxic response. First, Burton et al. tested the pups over the entire day of birth, whereas we performed our tests only at H10– H12, i.e. after peripheral chemoreceptor resetting (Renolleau et al., 2001a). Second, in the study by Burton et al., the VE-response to hypoxia was averaged over a 10-min period of hypoxia, which included the HVD (Dauger et al., 2001a,b). In contrast, we averaged breathing variables over successive 10sec periods, so that we could determine the timecourse of the ventilatory response and distinguish the hyperpnoeic response from the HVD. Third, the head-out plethysmograph used by Burton et al. requires restraining the pups, whereas the whole-body plethysmography used in our study allows the pups to move freely. Restraint is a potent stressor that can influence the breathing pattern, as previously shown in adult mice (Dauger et al., 1998). The high frequency of apnoeic episodes and periodic breathing in c-ret+/ − pups contrasted with the normal breathing rate of these animals under normoxic, hypoxic and hypercapnic conditions in the intervals separating the apnoeas. In earlier studies, apnoeas were associated with an abnormally low breathing rate in newborn Krox20 −/− mice (Jacquin et al., 1996) and with an abnormally high breathing rate in newborn Rnx −/− mice (Shirasawa et al., 2000). Furthermore, Mash-1 − /− newborn mice had an increased breathing rate, with normal apnoea duration and frequency (Dauger et al., 2001b). Taken together, these results suggest that apnoea characteristics and breathing rate may be under different neural control mechanisms that may be selectively impaired by gene mutations.
4.2. Potential mechanisms underlying the c-ret + /− respiratory phenotype The c-ret gene is expressed in several brainstem structures involved in respiratory control (Dauger et al., 2001b): the rostral ventrolateral medulla (rVLM), which plays a crucial role in respiratory rhythmogenesis (Rekling and Feldman, 1998); the nucleus tractus solitarius, which receives afferents from peripheral chemoreceptors and is, therefore, closely involved in the hyperpnoeic component of the hypoxic response; and the A5 and A6 (locus coeruleus) nuclei, which inhibit the medullary respiratory rhythm generator in the rVLM (Arata et al., 1998; Moore et al., 1996). In addition to exerting inhibitory modulation on the respiratory rhythm generator via a2-adrenoceptors located in the rVLM, noradrenaline directly facilitates the phrenic motoneurones by activating the cervical a1-adrenoceptors (Hilaire and Duron, 1999; Bianchi et al., 1995; Al-Zubaidy et al., 1996). The c-ret heterozygous mutation may delay the maturation of noradrenergic structures involved in respiratory control, thus shifting the balance between stimulation and inhibition of breathing. In keeping with this possibility, the heterozygous mutation in Mash-1, which is upstream of c-ret in the noradrenergic brainstem neurons, caused respiratory impairments in newborn pups but not in adult mice (Dauger et al., 1999). Respiratory impairments in newborns, but not in adult mice were also found in heterozygous mutants for the endothelin-converting-enzyme, another gene involved in neural crest development (Renolleau et al., 2001b). A longitudinal study on the timecourse of respiratory impairments in c-ret+ /− mice would perhaps clarify this issue. On the other hand, the normal sustained hyperpnoeic response to hypercapnia in the c-ret+ /− pups indicates that the higher rate of apnoeas during the hypoxic test was not ascribable to an inability to maintain high levels of ventilation. Our data did not allow to determine sleep states, which have an important influence on the breathing pattern, including apnoeas. Therefore, it is unclear whether apnoeas occurred during active sleep, as is the rule in newborn mammals, or whether sleep architecture was affected by the
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c-ret + /− mutation. We cannot preclude that the higher rate of apnoeas in c-ret+/ − newborn mice was related to a larger amount of active sleep.
4.3. Conclusion Newborn mice with the c-ret +/ − genotype survive and develop normally, but show impairments of respiratory control characterized by more frequent and longer apnoeas than their wildtype littermates. This result is further evidence that genes involved in the neural determination of neural crest cells may play a role in the development of respiratory control. On the other hand, the normal hyperpnoeic responses to hypoxia and hypercapnia in c-ret +/ − mice contrast with the lack of chemosensitivity that characterizes CCHS patients. As noted above, c-ret heterozygous mutations have been found in some CCHS patients. However, the respiratory phenotype differences between c-ret+ /− mice and CCHS patients regarding chemosensitivity further supports the multigenic origin of CCHS.
Acknowledgements This study was supported by the Fondation pour La Recherche Me´ dicale, the Institut de Recherches Internationales Servier (grant awarded to S. Aizenfisz), the Universite´ Paris VII (Legs Poix), and EEC Biomed grant BMH4-972107.
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