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1 Department of Physiology and Biophysics, University of Calgary, Calgary AB, Canada2 Department of Biology, University of Victoria, Victoria BC, Canada
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(Received 5 August 2003;
accepted after revision 3 November 2003;
first published online 7 November 2003)
Corresponding author R. J. A. Wilson: Respiratory Research Group, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr NW, Calgary, Alberta T2N 4N1, Canada. Email: wilsonr{at}ucalgary.ca
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Introduction |
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Several lines of evidence suggest that PACAP may be involved in respiratory control. Firstly, intravenous injection of PACAP in dogs is reported to cause an increase in ventilation that is abolished by transecting the carotid sinus nerves innervating the peripheral respiratory chemoreceptors (Runcie et al. 1995). Secondly, PACAP is located widely within the rat brain, including several areas containing neurones implicated in respiratory rhythm generation and/or respiratory chemosensitivity: the ventrolateral medulla, the ventral medullary surface, the raphe nucleus pallidus and the nucleus tractus solitarius (Hannibal, 2002). Thirdly, PACAP binds with high affinity to three distinct G-protein coupled receptors (PAC1, VPAC1 and VPAC2) that can activate adenylate cyclase leading to the production of cAMP and PKA activation (Vaudry et al. 2000). PKA and cAMP are potent modulators of both phrenic motor neurones and putative rhythm generating neurones in the ventral lateral medulla (Lalley et al. 1997; Arata et al. 1993; Bocchiaro et al. 2003; Shao et al. 2003).
In light of these data, we hypothesized that the higher neonatal mortality in PACAP-null mice was principally the result of defective respiratory control. To test this hypothesis, we evaluated ventilation in PACAP+/+, +/- and -/- littermates at an early stage of postnatal development (postnatal day 4 (P4)) using continuous-flow, unrestrained, whole-body plethysmography. We also determined whether genotype influenced ventilatory responses to hypoxia and hypercapnia. We found that PACAP deficiency leads to a significant reduction in ventilation and blunted responses to both hypoxia and hypercapnia. To determine whether this respiratory abnormality could lead to death, we studied the effects of PACAP deletion on respiration and cardiovascular function during the sensitive period of postnatal development (P7-P14) using electrocardiography (ECG). Under conditions that exacerbate the chance of sudden death of PACAP-null mice (isoflurane-induced hypothermia), we found that prolonged apnoea preceded atrio-ventricular block. These data are consistent with an important role for PACAP in respiratory control.
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Methods |
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Animals
Mice were housed at 24–26°C with a 12 h light–dark cycle and were given food and water ad libitum. PACAP+/+, +/- and -/- mice were bred from PACAP +/- mice, kept on a 129SvJ
C57BL/6 hybrid background. This PACAP deficient mouse line was generated by Gray et al. (2001). Mixed breeding strains were used as (a) they produce larger offspring with larger tidal volumes than pure strains, which facilitates measurement of respiratory parameters, and (b) previous reports indicate that this line has a similar phenotype to a C57BL/6 back-crossed line, suggesting that strain has little influence on the increased neonatal mortality in PACAP-deficient mice (Gray et al. 2001). To limit genetic variability, comparisons between littermates were used in all experiments.
Whole-body plethysmography
Respiration was assessed in non-anaesthetized PACAP+/+, +/- and -/- P4 pups using continuous-flow, unrestrained, whole-body plethysmography. In order to have the exquisite sensitivity required to record breaths of <0.05 ml (based on an adult 20 g mouse with tidal volume (VT) of 0.15 ml; Guyton, 1947; Mortola, 1984), we modified a commercial system designed for studies of larger rodents (Buxco Electronics Inc., Sharon, CT, USA). The apparatus consisted of two 20 ml chambers: one to house the animal and the other to act as a reference. A low dead space differential pressure transducer was used to monitor the pressures in the chambers, removing the contribution of environmental pressure fluctuations and thereby isolating pressure changes in the box related to breathing. This differential pressure signal was amplified, digitized and stored on a computer. The animal chamber incorporated gas inlet and outlet ports. Fresh gas was fed to the chamber by a pump. Exhaust gas was passed through CO2 and O2 gas analysers. Continuous flow of gas through the chamber, which was sufficient to exchange the gas in the chamber within a minute, was held constant by a pair of precision gas flow controllers. The animal chamber also contained humidity and temperature probes that were monitored continuously (Omega Engineering, Stamford, CA, USA). A heat lamp was used to hold the ambient temperature in the chamber within the thermoneutral range (33 ± 0.5°C), thus protecting against hypothermia in mice pups that have limited thermogenic capacity and normally rely on behavioural approaches for thermoregulation (Mortola, 2001).
Pups were subjected to either hypoxia (10% O2–90% N2) or hypercapnia (8% CO2–92% air) using the following protocol: 15 min calm-down period, 5 min baseline (room air) period, 5 min hypoxia or hypercapnia treatment, and 15 min washout (room air) period. Data from minutes 3–5 of baseline, minutes 2–3 and 4–5 of treatment and minutes 2–3 and 14–15 of washout were analysed. Analysis was performed offline using semiautomated analysis software written in VEE (by R.J.A.W.) (Agilent Technologies, Palo Alto, CA, USA). The software imported the raw pressure signals exported from the data acquisition system and identified and quantified the properties of breaths in the predefined sections of data. Breathing parameters studied were rate (breaths min-1), inspiratory time (TI), expiratory time (TE) and standard deviation of the period (period variability, V). Given that we used flow-through plethysmography, essentially a leaky box, the pressure signal we record is proportional to volume changes, i.e. flow (e.g. Mortola & Lanthier, 1996). Therefore, we integrated the area under the inspiratory pressure curve to obtain an index (see below) of VT.
We did not attempt to measure absolute tidal volume. Previous quantitative analysis of tidal volume has been based on the principle that air entering the lungs is heated and humidified, thereby increasing the pressure within the chamber as a whole (Drorbaugh & Fenn, 1955). In pilot experiments, we found that the breathing-related pressure changes recorded in our chamber were unaffected by increased humidity (from 20 to 95%) and temperature (from 33 to 36°C). Therefore, the breathing-related pressure changes we observed are more likely to result from the compression and rarefaction of external gases, occurring as a consequence of airway resistance, during inspiration and expiration, respectively (Enhorning et al. 1998; Mortola & Frappell, 1998). Consequently, our measurements provide an index of tidal volume that will be influenced by changes in airway resistance.
Experiments and analysis of breathing were completed before genotyping animals, ensuring both were performed blind. Statistical analysis of data was performed using two-factor, repeated-measures ANOVA to assess the effects of genotype, treatment (hypoxia or hypercapnia), and the interaction between genotype and treatment on breathing parameters. To determine the effect of genotype on baseline breathing (i.e. when animals were breathing air), baseline data from the two treatment groups were pooled. A one-way, between-subject ANOVA was used for statistical analysis. A one-way, between-subject ANOVA was also used to compare body weights of the three genotypes.
ECG
Surface electrocardiography (ECG) tracings were recorded from postnatal days 7–14 PACAP+/+ and PACAP-/- littermates. P7–14 neonates were used for this part of the study because: (a) the size of these animals is more convenient for instrumentation, and (b) this is the period of increased mortality of PACAP -/- mice and we were interested specifically in the cause of death. Animals were genotyped before experiments and PACAP-/- and +/+ littermates were paired, with pairs being exposed to identical experimental conditions.
Pups were briefly anaesthetized with 2 or 3% isoflurane under a heat lamp to prevent anaesthesia-induced hypothermia. A rectal temperature probe was inserted. The fractional concentration of isoflurane was then reduced to 1.5 or 2%. During the remainder of the experiment (see below), the heat lamp was regulated using a feedback circuit to clamp rectal temperature (Tb) at either a normothermic or a hypothermic level (30°C). We chose this level of hypothermia because: (a) the body temperature of neonatal mice depends largely on environmental temperatures (Alexander, 1975; Mortola, 2001), and (b) a previous study demonstrated that a reduction in ambient temperature of 3°C was sufficient to increase the mortality of PACAP-null mice, without increasing the mortality of wild-type animals (Gray et al. 2002).
ECG was performed by placing paws into three small plastic cylinders (one paw per cylinder) backfilled with conducting gel and containing silver/silver-chloride needle electrodes (EW-Wright, Guilford, CT, USA). ECGs were recorded using a custom-designed amplifier (bandwidth: 0.1 Hz–1 kHz). Data were digitized, analysed and stored using a data acquisition board (National Instruments, Austin TX, USA serial PCI-M10-16XE-10) and custom software written in LabView 5.1 (National Instruments) by Dr Anders Nygren. Breathing produced distinct bursts of ‘noise’ on ECG recordings caused by the movement of the animal's chest cavity.
Given that PACAP-/- mortality increases with reduced ambient temperature, a protocol was designed to assess the consequence of PACAP deficiency on heart function and breathing during anaesthesia-induced hypothermia. Initially, ECG recordings were made during 10 min of normothermia (1.5 or 2% isoflurane). The isoflurane concentration was then increased to 3% and the body temperature allowed to drop to 30°C. Once the rectal temperature was at 30°C, ECG recordings were continued for an additional 10 min. For analysis of breathing, breaths were counted over minute 6 of normothermia and hypothermia. Data were normalized (reciprocal transform) before a two-way ANOVA. For analysis of heart rate (beats·min-1), R-waves were counted over the same time intervals used to analyse breathing.
Ten PACAP+/+ and 10 PACAP-/- littermates were subjected to experimentation. However, 3/10 PACAP-/- animals died upon application of anaesthetic and no recording was obtained. In addition, another PACAP-/- animal had no recordable breathing during minute 6 of hypothermia. Therefore, data from these pups and their PACAP+/+ littermates were excluded from analysis, leaving data from six pairs of animals. Data was analysed for significant differences using two-way repeated-measures ANOVA.
Genotyping
PACAP genotype was determined using PCR with primers directed against signal peptide DNA (5'atgtgtagcggagcaaggctgg 3'), the PACAP-encoding region (5'cactcggacggcatcttcacagatag3') and the 3' untranslated region (5'gaacacgagtgatgactggtcagtc-3'). Using these three primers, it was possible to detect PACAP+/+, +/- and -/- animals with one reaction, as the PACAP-/- mice were lacking the PACAP-encoding region, but not the other two regions. The parameters were: 94°C (30 s), 64°C (30 s), 72°C (45 s) for 38 cycles with 1 µl of genomic DNA isolated from proteinase K-digested ear notches. PCR products were analysed in ethidium-bromide stained 1.5% agarose gels.
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Results |
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PACAP+/+, +/- or -/- P4 pups were identical in appearance and there was no significant difference in body weight between genotypes (one-way ANOVA: P = 0.20). Although there was a slightly smaller proportion of PACAP-/- littermates than would be predicted from Mendelian genetics (Table 1), this difference was not significant (
2; P = 0.423). This suggests that P4 is before the period in which a PACAP-/- genotype leads to increased neonatal mortality. To study breathing at this age, we used whole-body, continuous-flow plethysmography in two groups of unanaesthetized, unrestrained animals. One group was subject to a protocol that involved a 5 min exposure to hypercapnia (n= 47); the other group was subject to an identical protocol but exposed to hypoxia (n= 34).
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Baseline data from both treatment groups were pooled to determine whether genotype influenced respiration when animals breathed room air (Fig. 1). Unrestrained, non-anaesthetized wild-type P4 mice had an average breathing rate of 183 breaths min-1 while breathing air, within the range reported previously (Bissonnette & Knopp, 2001; Kazemian et al. 2001; Ptak et al. 2002). Genotype did not affect timing variables (TI, P = 0.21; TE, P = 0.24; rate, P = 0.19; and variability of the period, P = 0.41). However, the PACAP-/- mice had a smaller index of tidal volume (P < 0.01) than wild-type littermates resulting in a 25% reduction in minute ventilation (P < 0.01).
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Overall hypoxia (10% O2, balanced air) produced a marked reduction in TE(P < 0.001) (Fig. 2B), and a small but significant reduction in TI(P < 0.05) (Fig. 2A). Respiratory rate was increased and period variability was markedly reduced (P < 0.001 for each) (Fig. 2C and D). Hypoxia increased both VT(P < 0.01) and VE(P < 0.001) (Fig. 2E and F). Our results confirm the lack of ventilatory hypoxic depression in neonatal mice within the first days of birth and the presence of posthypoxic decline, which has been reported in P7 mice (Robinson et al. 2000; Bissonnette & Knopp, 2001).
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However, genotype did not affect either the hypoxia-mediated reduction in TE(P = 0.33) or the hypoxia-mediated increase in VT(P = 0.54). Despite the significant influence of genotype on the hypoxia-mediated changes in rate, genotype had no effect on the overall ventilatory response to hypoxia (i.e. VE, P = 0.57). The most likely explanation for this is that the influence of genotype on hypoxia-mediated changes in rate is not of sufficient magnitude to overcome the variability in the influence of genotype on hypoxia-mediated changes in VT. In summary, there is a genotype effect on overall ventilation but genotype does not change the response of VE to hypoxia.
Respiratory response to hypercapnia and the effect of genotype
Hypercapnia (8% CO2, balanced air) had no significant effect on TI(P = 0.45) but caused significant reductions in TE (P < 0.001; Fig. 3A and B). Rate was significantly increased by hypercapnia (P < 0.001) and there was a significant reduction in period variability (P < 0.001) (Fig. 3C and D). VT was also increased (P < 0.001) (Fig. 3E). Concomitantly, hypercapnia caused a significant increase in minute ventilation (VE, P < 0.001) (Fig. 3F).
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Effects of conditions that selectively exacerbate PACAP -/- neonatal mortality
To determine whether a respiratory phenotype may be one of the primary causes of neonatal death of PACAP-/- mice, we examined the physiology of the PACAP-/- genotype under life-threatening conditions. Specifically, we used ECGs to study respiratory and cardiac function in anaesthetized PACAP+/+ and -/- littermates (aged P7–14) during hypothermia, which has been shown to increase the mortality of PACAP-/- neonates (Gray et al. 2002). Respiratory events were observed clearly on ECG recordings as bursts of ‘noise’ corresponding to respiratory movements of the chest wall.
Under normothermic conditions, respiration in both PACAP+/+(n= 6) and -/-(n= 6) genotypes was regular, with no significant difference in respiratory rate (Tukey's post hoc test: P = 0.75) (Fig. 4A). A slight but significantly greater heart rate was observed in the PACAP-null neonates when compared to wild-type littermates (Tukey's post hoc test: P = 0.019) (Fig. 4C).
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Figure 5 shows examples of ECGs during the hypothermic part of the protocol from one of the 6 +/+/-/- pairs of animals used to make Fig. 4. Figure 5A2 and B2 (wild-type mice and PACAP-deficient mice, respectively) shows the effect of hypothermia in the 15 s immediately after the time at which rectal temperatures reached 30°C. Although heart rate is comparable between the genotypes, the respiratory rate of the PACAP-null mouse is much less than its wild-type littermate (compare Fig. 5A2 and 5B2).
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In total, 3 of 6 PACAP-/- neonates succumbed by the end of the hypothermic treatment with AV-block. These three mice had long duration apnoeas preceding AV-block, suggesting that chronic hypoxaemia led to cardiac arrest. A 4th mouse had no detectable breathing with an absence of AV-block. In contrast, none of the PACAP +/+ neonates died, and ECG tracings remained relatively normal, even after 10 min of hypothermia (e.g. Fig. 5A1).
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Discussion |
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Breathing in neonatal mice assessed by plethysmography
Advances in mouse genomics offer new approaches to assess the role of specific proteins in the ontogeny and control of breathing (Funk et al. 1997). To take full advantage of these new approaches the technical difficulties of recording breathing non-invasively in an animal weighing only a few grams, and having tidal volumes of <0.05 ml (Mortola, 1984), need to be overcome. The only techniques currently available are whole-body (e.g. Mortola & Lanthier, 1996; Ptak et al. 2002; Autret et al. 2002; Viemari et al. 2003) and head-out (Burton et al. 1997; Robinson et al. 2000; Kazemian et al. 2001) plethysmography. Head-out plethysmography provides a measure of tidal volume independent of airway resistance but this advantage must be weighed against the stress to the animal imposed by this technique, in which an animal is secured by an airtight band around its neck (Gaultier et al. 2003).
Given the accumulating evidence that PACAP may be involved in stress responses, we used unrestrained whole-body plethysmography for the current study. Consequently, a caveat that should be considered when interpreting our results is that the index of tidal volume we used (i.e. the area under the inspiratory pressure curve recorded in the chamber) depends, in part, on airway resistance: as airway resistance increases so would our index. Hypercapnia and hypoxia both cause bronchodilatation, reducing airway resistance (Wetzel et al. 1992; D'Angelo et al. 2001). Therefore, the actual increase in tidal volume when animals were challenged with either 8% CO2 or 10% O2 is likely to be greater than suggested by our index. Similarly, PACAP is a potent bronchodilator (Linden, 1999) so PACAP deficiency could result in a more broncho-constricted lung, with higher airway resistance. Our index of tidal volume might therefore overestimate the tidal volume of the PACAP-null mice, which, in turn, would mean our results underestimate the modulatory affects of PACAP on breathing.
Direct role for PACAP in respiratory control?
Our data suggest that PACAP influences respiration, both during baseline conditions and in response to hypoxic and hypercapnic challenges. The influence of PACAP may be direct, through neuromodulation of the respiratory circuit, or it may be indirect through augmentation of metabolism. Indeed, a proportion of PACAP-null mice that survive into the second week of life develop a wasting disorder characterized by abnormalities in both glucose and fat metabolism, fasting hyperinsulinaemia and a redistribution of fat stores from the peripheral adipose tissue to the liver and skeletal muscle (Gray et al. 2001). If PACAP is involved in maintaining the metabolic rate of neonates, PACAP deficiency would result in less CO2 production during baseline conditions, which in turn would be expected to reduce minute ventilation. A lower metabolism of PACAP-deficient animals may also explain why these mice have a blunted response (timing variables, not overall ventilation) to hypoxia in comparison to wild-type littermates, if the lower metabolism allows them to reside on a shallower portion of the O2-ventilatory response curve.
However, there are several reasons to suggest that the wasting disorder alone is not responsible for the respiratory phenotype we observed: First, blunted chemoresponse of PACAP-null neonates occurs at postnatal day 4, before the previously reported onset of the wasting disorder. Second, although the effect of PACAP on the metabolism of P4 littermates was not measured directly, we found no significant difference in body weight between the three genotypes. Nor did PACAP-null mice show any evidence of peripheral fat wasting or lipid accumulation in the liver upon gross morphological examination. Third, we probably minimize the effects of metabolism on respiration by keeping mice at thermoneutral temperatures during experiments (Mortola & Dotta, 1992). Fourth, while PACAP+/- neonates are normal with regards to lipid and glucose metabolism (4), values for VE during both hypoxia and hypercapnia are between those of PACAP+/+ and -/- littermates (Figs 2F and 3F). This suggests that there is a dose-dependent effect of PACAP on respiration, independent of lipid and/or glucose metabolism. Interestingly, the wasting disorder resembles the effect of chronic neonatal hypoxia and could, in fact, be secondary to a respiratory defect (Mortola, 2001). Finally, differences in metabolic rate are unlikely to explain the differential effects of genotype on the overall ventilatory response to CO2, as the ventilatory response to CO2 is linear throughout most of the physiological range (Loeschke & Gertz, 1958).
An alternate hypothesis is that PACAP has a role in respiratory chemosensitivity, acting directly on elements of the respiratory circuits to modulate their response to respiratory challenges. In a previous study in anaesthetized adult dogs, bilateral sectioning of the carotid sinus nerve innervating the carotid bodies abolished the stimulatory effect of intravenous PACAP27 injections on ventilation, leading the authors to suggest that PACAP was stimulating the peripheral carotid chemoreceptor (Runcie et al. 1995). Interestingly, cAMP has been shown to increase the excitability of Type 1 glomus cells within the carotid body (Stea et al. 1995). While the dog data provide compelling indirect evidence that PACAP may be involved in peripherally mediated chemosensitivity, at least in adults, the effects of PACAP on O2 and CO2 chemosensitivity per se were not directly tested. Nor do the data exclude the possibility that PACAP modulates other components in the neuronal pathway between the peripheral chemoreceptors and respiratory motor neurones.
We note that PACAP is widely distributed within the CNS and is located in brainstem regions implicated in mediating the response to peripheral chemoreceptor activation, the generation of respiratory rhythm and central chemosensitivity (e.g. nucleus of the solitary tract, ventro-lateral medulla, nucleus ambiguous and dorsal vagal nucleus; Hannibal, 2002). In addition, the hypothalamus, which contains the highest concentrations of PACAP and PACAP-specific receptors, has populations of neurones that (a) receive inputs from peripheral chemoreceptors (Thomas & Calaresu, 1972) (b) are sensitive to both CO2 and O2 (Waldrop & Porter, 1995) and (c) project directly to the phrenic motor nucleus (Kc et al. 2002). Therefore, we consider it likely that PACAP acts centrally to modulate respiration.
PACAP, being a potent stimulator of cAMP production and subsequent protein kinase-A (PKA) activation, is a good candidate for a central respiratory neuromodulator. Several groups have established that cAMP, cAMP analogues and cAMP-stimulating agents, such as forskolin, increase respiratory rate when applied to a number of brainstem regions (Arata et al. 1993; Mironov et al. 1999). PKA has also been implicated in the control of respiration by modulating the excitability of respiratory units. For example, PKA increases excitability of both inspiratory neurones within the pre-Botzinger complex (Shao et al. 2003), as well as hypoglossal (XII) motoneurones (Bocchiaro et al. 2003), by modulating postsynaptic AMPA (amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) currents. In addition, the cAMP–PKA pathway has been implicated in the modulation of expiratory neurone excitability in the medulla (Lalley et al. 1997).
PACAP and sudden neonatal death
In addition to a blunted chemoresponse in PACAP-deficient animals, we found that under hypothermic conditions, the rate of breathing in PACAP-null animals is significantly reduced when compared to wild-type littermates, leading to AV-block and death in 50% of the animals tested. These data suggest that the respiratory phenotype may account for the higher mortality of PACAP-null mice when housed at slightly lower ambient temperatures. An important caveat to these experiments is that we used hypothermia and isoflurane to increase the likelihood of neonatal death while making recordings of cardiac and ventilatory activity. While these conditions had no significant effect on either the heart rate or ventilation of wild-type littermates, we cannot exclude the possibility that the cause of death of PACAP-null mice exposed to acute isoflurane–hypothermia was different from that of PACAP-null mice housed at slightly reduced ambient temperatures. It should be noted, however, that, in all genotypes, isoflurane had only a mild effect on breathing and cardiac function before the body temperature was reduced.
Previously studies have demonstrated that the body temperature of small neonatal mammals is highly dependent on ambient temperatures, being regulated mainly by behavioural mechanisms rather than by non-shivering thermogenesis (Mortola, 2001). In neonatal rats, for example, non-shivering thermogenesis is absent during the first 3–4 days (Fowler & Kellogg, 1975). The body temperature of neonatal mice is therefore likely to fluctuate significantly. Our results suggest that PACAP may play a critical physiological role in coping with these fluctuations.
Interestingly, environmental stressors (including hyperthermia), a critical developmental period, abnormal respiratory chemosensitivity and a genetic predisposition have been implicated in human SIDS (Ackerman et al. 2001; Guntheroth & Spiers, 2002). PACAP-deficient mice (a) respond poorly to environmental stress, i.e. hypothermia (the effect of hyperthermia has not been determined), (b) have a critical period in which they are more prone to death than wild-type littermates, and (c) have abnormal responses to hypoxia and hypercapnia. Therefore, it may be constructive to determine whether congenital abnormalities in PACAP-dependent signalling pathways contribute to some cases of SIDS. However, given that (a) breathing abnormalities and a high rate of neonatal death is a phenotype common to several strains of knockout mice (Guillemot et al. 1993; Hummler et al. 1996; Tokieda et al. 1997) (b) brainstem abnormalities have been identified in some, but not all SIDS victims (Kinney & Filiano, 1988), and (c) a mutation in a gene encoding a cardiac sodium channel, which almost never occurs in the adult population, is present in 3% of SIDS victims (Ackerman et al. 2001), it seems likely that the genetic predisposition of SIDS might include congenital abnormalities in a large number of genes.
We have shown that PACAP is an important hormone for the ventilatory response to both hypoxia and hypercapnia in neonatal mice. This is the earliest phenotype for PACAP-deficient mice described to date and may contribute to their sudden-death phenotype. Our findings are in keeping with the recently described importance of PACAP in the mammalian response to stress. We hypothesize that congenital defects within PACAP-dependent signalling pathways lead to an increased susceptibility to infant death resulting from an abnormal regulation of breathing.
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