Am J Physiol Regul Integr Comp Physiol 294: R956–R965, 2008.
First published January 9, 2007; doi:10.1152/ajpregu.00637.2007.
Pituitary adenylate cyclase-activating polypeptide maintains neonatal
breathing but not metabolism during mild reductions in ambient temperature
Kevin J. Cummings, Chris Willie, and Richard J. A. Wilson
Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada
Submitted 4 September 2007; accepted in final form 2 January 2008
neonatal apnea; temperature; metabolism; hypoxia; neonatal apnea;
sudden infant death syndrome
THE PRIMARY ROLE OF THE RESPIRATORY control system is to
couple ventilation (V̇E) to changes in metabolic rate for the
maintenance of blood gases. In small neonatal rodents, a drop
in ambient temperature just outside the thermoneutral zone
initiates nonshivering thermogenesis and an increase in oxygen
consumption (V̇O2). Despite this metabolic response, the body
temperature of small neonatal rodents continues to fall with
ambient temperature, owing to their large surface area-tovolume ratio (28). This causes a potential problem for the
neonatal respiratory control system; a fall in body temperature
has a suppressive effect on neuronal function, including those
involved in the control of breathing [Q10 effect: (27, 36, 41)].
Neonates must therefore have compensatory mechanisms to
Address for reprint requests and other correspondence: R. J. A. Wilson,
Hotchkiss Brain Institute & Institute of Maternal and Child Health, Dept. of
Physiology and Biophysics, Univ. of Calgary, 3330 Hospital Dr. N.W.,
Calgary, Alberta T2N 4N1, Canada (e-mail: [email protected]).
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mitigate the Q10 effect and maintain the tight coupling between
V̇E and metabolic rate.
Pituitary adenylate cyclase-activating polypeptide (PACAP)
has potent modulatory effects on both the central and peripheral nervous systems. The primary structure of PACAP is
remarkably constrained throughout evolution, suggesting one
or more critical developmental and/or physiological roles (38).
PACAP stimulates three classes of G protein-coupled receptors
that utilize cAMP and phosphoinosotide species to produce its
cellular effects (42). At the system level, PACAP has been
implicated in the proliferation, differentiation, and survival of
neural progenitor cells within the central nervous system (30,
31, 43). However, PACAP is also involved in a range of acute
physiological processes with mounting evidence suggesting
PACAP may be vital for maintaining homeostasis in the face of
thermal and metabolic stress (1, 39); a large proportion of
PACAP⫺/⫺ mice do not survive the neonatal period when
raised in slightly cooler environments (12), and PACAP-deficient adult mice have impaired catecholaminergic counterregulatory responses to hypoglycemia (13) and hypotension (22).
Previously, we showed that postnatal day 4 (P4) PACAPdeficient neonates have blunted respiratory responses to hypoxia and hypercapnia. Furthermore, in slightly older (P7–P14)
PACAP⫺/⫺ neonates, anesthetic-induced hypothermia causes
long-duration apneas that culminate in atrioventricular block
(8). Based on these observations, PACAP might also be important for maintaining V̇E in unanesthetized animals during
small drops in ambient temperature that are similar to those
normally experienced in the litter. However, given the abundance of PACAP within the hypothalamus (2, 34), the inability
of older PACAP⫺/⫺ neonatal mice to maintain body temperature (12), and the suppressive effects of anesthetic on metabolism, it is equally possible that the breathing phenotypes that
we previously demonstrated in neonatal PACAP⫺/⫺ mice are
secondary to a suppression of metabolic rate.
Here, we test the hypothesis that PACAP is important for the
preservation of V̇E, but not metabolic rate, during small reductions in ambient temperature. Our results indicate that mild
ambient cooling is an environmental stressor that selectively
inhibits the breathing of PACAP-deficient P4 neonates, an
effect that is not related to abnormal responses of metabolic
rate or body temperature.
MATERIALS AND METHODS
Experimental procedures were approved by the University of
Calgary Animal Care Committee and were in accordance with national guidelines. A total of 225 neonates were used in this study.
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0363-6119/08 $8.00 Copyright © 2008 the American Physiological Society
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Cummings KJ, Willie C, Wilson RJ. Pituitary adenylate cyclaseactivating polypeptide maintains neonatal breathing but not metabolism during mild reductions in ambient temperature. Am J Physiol
Regul Integr Comp Physiol 294: R956–R965, 2008. First published
January 9, 2007; doi:10.1152/ajpregu.00637.2007.—Mild reductions
in ambient temperature dramatically increase the mortality of neonatal
mice deficient in pituitary adenylate cyclase-activating polypeptide
(PACAP), with the majority of animals succumbing in the second
postnatal week. During anesthesia-induced hypothermia, PACAP⫺/⫺
mice at this age are also vulnerable to prolonged apneas and sudden
death. From these observations, we hypothesized that before the onset
of genotype-specific mortality and in the absence of anesthetic, the
breathing of PACAP-deficient mice is more susceptible to mild
reductions in ambient temperature than wild-type littermates. To test
this hypothesis, we recorded breathing in one group of postnatal day
4 PACAP⫹/⫹, ⫹/⫺, and ⫺/⫺ neonates (using unrestrained, flowthrough plethysmography) and metabolic rate in a separate group
(using indirect calorimetry), both of which were exposed acutely to
ambient temperatures slightly below (29°C), slightly above (36°C), or
at thermoneutrality (32°C). At 32°C, the breathing frequency of
PACAP⫺/⫺ neonates was significantly less than PACAP⫹/⫹ littermates. Reducing the ambient temperature to 29°C caused a significant
suppression of tidal volume and ventilation in both PACAP⫹/⫺
and ⫺/⫺ animals, while the tidal volume and ventilation of
PACAP⫹/⫹ animals remained unchanged. Genotype had no effect on
the ventilatory responses to ambient warming. At all three ambient
temperatures, genotype had no influence on oxygen consumption or
body temperature. These results suggest that during mild reductions in
ambient temperature, PACAP is vital for the preservation of neonatal
tidal volume and ventilation, but not for metabolic rate or body
temperature.
PACAP MAINTAINS NEONATAL BREATHING DURING COOLING
Animals
Mice were housed at 22–24°C with a 12:12-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 is the same mixed line used in previous studies (8,
11, 12). Although there is some between-strain variability in terms of
phenotype severity, previous studies have suggested that high mortality in PACAP knockout neonatal mice is strain independent (11, 13,
14, 39). Littermates were used in all experiments. Genotyping of mice
was performed after data were analyzed; thus, experiments and
analysis were performed in a blinded fashion.
Plethysmography
generated from breathing in neonatal mice weighing ⬍3g [tidal
volume (VT) 0.05 ml (27)]. Ambient temperature was monitored
continuously using a thermocouple inserted through the top of the
chamber, which was checked periodically against a standard mercury
thermometer (Omega Engineering, Stamford, CT). The experimental
protocol is shown in Fig. 1A. Animals were placed into the animal
chamber, held at 32 ⫾ 0.5°C, and allowed a 5-min settle period.
Breathing was then recorded for 5 min after which the temperature
was either maintained at 32°C (thermoneutral control, n ⫽ 36), cooled
to 29 ⫾ 0.5°C (cool protocol, n ⫽ 54), or warmed to 36 ⫾ 0.5°C
(warm protocol, n ⫽ 45) by adjusting the position of the chamber
relative to a heat lamp. The rate of cooling and heating were ⬃0.6°C/
min and 0.4°C/min, respectively. Recordings were continued throughout the period of temperature change (⬃5 min for cooling, 10 min for
warming), and at the new steady-state temperature. The total time of
each protocol was 20 min. However, the rate of warming was slower
than the rate of cooling. Consequently, it took more of the protocol to
reach the steady-state warm temperature (36°C) than the steady-state
cool temperature (29°C), and thus, when comparing the two protocols,
please note that the duration at 36°C was slightly shorter than the
Fig. 1. Methodology for assessing breathing and metabolic
rate in pituitary adenylate cyclase-activating polypeptide
(PACAP) genotypes. A: three plethysmograph protocols were
used: 1) warm, 2) thermoneutral control, 3) cool. Ta, ambient
temperature. B: representative recording of changes in fractional inspired O2 (FIO2). C: mass of PACAP genotypes used
in plethysmography and metabolism experiments. Note that
the mass of PACAP⫹/⫺ postnatal day 4 (P4) neonates is not
statistically different than that of wild-type littermates, while
that of PACAP⫺/⫺ neonates is significantly lower than that of
both PACAP⫹/⫹ and ⫹/⫺ littermates (1-factor ANOVA: P ⬍
0.001; *Tukey post hoc PACAP⫹/⫹ vs. PACAP⫺/⫺: P ⬍ 0.001;
Tukey post hoc PACAP⫹/⫹ vs. PACAP ⫹/⫺: P ⫽ 0.700).
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Whole body, continuous flow, unrestrained plethysmography was
performed as described previously (8). Prior to experimentation,
animals were kept at an ambient temperature of 32 ⫾ 0.5°C, within
the thermoneutral range of animals of this age (4, 29), and close to the
ambient temperature within the litter (29). Two 20-ml chambers, one
acting as a reference chamber, were used to detect the pressure signal
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PACAP MAINTAINS NEONATAL BREATHING DURING COOLING
duration at 29°C. The chamber was flushed with a continuous flow of
room air, regulated by a pair of precision gas flow controllers, at a rate
sufficient to exchange the air within the chamber in ⬍1 min.
Data Analysis
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Of the 20 min of breathing recorded, three 2-min segments were
used for analysis (Fig. 1A): baseline (minutes 4 and 5), during
temperature change (minutes 7 and 8), new steady-state temperature
(minutes 19 and 20). Analysis was performed offline on a total of
123,973 breaths using semiautomated analysis software written in
VEE (by R. J. A. Wilson; Agilent Technologies, Palo Alto, CA), as
described previously (8). Parameters derived from the pressure waveform associated with an animal breathing within a barometric chamber were rate (breaths/min), index of VT, and index of V̇E. In addition,
the standard deviation of the breathing period (SDP) was analyzed to
determine whether temperature had any selective effects on the
stability of breathing across PACAP genotypes.
Note that in adults, the pressure changes associated with the
expansion of inspired air through warming and humidification dominate the pressure waveform associated with breathing. However, in
small rodents, the signal is dominated by pressure changes resulting
from the rarefaction and compression of gases within the airways (this
point is illustrated in Fig. 3B, showing that a sizeable respiratory
signal remained even when the ambient-to-body temperature differential was ⬍1°C). While pressure changes associated with rarefaction
and compression of inspired air are proportional to VT, they are also
influenced by airway resistance and changes in inspiratory time. Thus,
we refer to our measurements as “indices” (for detailed discussion
please see Ref. 8). A further caveat of employing whole body
plethysmography for this study is that the component of our signal
produced by humidification and warming of inspired gas is dependent
on the ambient-to-body temperature differential; reducing this differential by ambient warming is likely to have artificially decreased our
VT and V̇E indices. Cooling the ambient environment has the opposite
effect, increasing the differential (although to a much lesser extent) and
having the potential to increase the magnitude of the indices. For the
implications of this caveat to our main finding please see DISCUSSION.
Baseline room-air respiratory variables graphed in Fig. 2 were
obtained by combining data from the 32°C baseline segment (minutes
4 and 5) of all three treatment groups (29°C, 32°C, 36°C). VT data
over the entire experiment for each litter were normalized to the
average baseline (minutes 4 and 5) VT index of PACAP⫹/⫺ littermates (Figs. 2 and 3). In addition, to better illustrate the changes in V̇E
after cooling, ventilatory data from minutes 19 and 20 are also
expressed relative to each animal’s baseline (%change, Fig. 5).
At the end of the recording, animals were killed by decapitation,
and rectal temperature (body temperature) was measured immediately
thereafter using a thermocouple. Animals were then weighed and ear
clipped for postanalysis genotyping, as described previously (8).
Gaseous Metabolism (V̇O2)
To obtain the sensitivity required to record metabolic rate in freely
behaving neonatal mice weighing ⬍3 g, closed-loop, small volume,
indirect calorimetery was used. The circuit consisted of a 15-ml
chamber connected to an O2 analyzer (PA-1B O2 analyzer, Version 2;
Sable Systems, Henderson, NV) calibrated to known gas mixtures. A
pump (gas-analyzer, Sub-Sampler; Sable Systems), with constant
flow, was used to pull air through the meter and return it to the
chamber. The total volume of the circuit (⬃100 ml) was calculated by
observing the change in % O2 from room air, after injecting 1 ml of
100% O2 and allowing time for the gas to equilibrate.
Experiments were performed on three sets of mice. For measurements of gaseous metabolism at 29°C (n ⫽ 39) and 32°C (n ⫽ 51),
mice were separated from their mothers and preincubated in a small
holding container maintained at either temperature with a heat lamp.
The holding container was monitored continually with a thermocouple
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Fig. 2. Effects of PACAP genotype on baseline breathing while at thermoneutral ambient temperature (32°C). Data from minutes 4 and 5 of the baseline
period, pooled from the three experimental protocols (warm, thermoneutral
control, cool). Animals were breathing air and held at thermoneutral ambient
temperature (32°C). A: index of tidal volume (VT). B: breathing rate (breaths/
min). C: index of minute ventilation (V̇E). Littermate, P4 wild-type
(PACAP⫹/⫹; n ⫽ 30; black bars), heterozygous (PACAP⫹/⫺; n ⫽ 70; grey
bars), and knockout (PACAP⫺/⫺; n ⫽ 35; white bars) were used. In each
animal, mean values were obtained from minutes 4 and 5. Error bars in this and
subsequent figures are means ⫾ SE. Note that PACAP genotype affects
breathing rate (B) and overall V̇E index (C) but not VT index (A).
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Data Analysis
Data were analyzed in Clampfit Version 8.1. V̇O2 was calculated by
the following formula (derived from Fick’s principle): V̇O2 ⫽ (absolute value of the difference in fractional O2 concentration from start to
end of experiment) ⫻ (V ⫺ m) ⫻ 0.01, where V is the volume (ml)
of the closed loop and m is the volume (ml) of air displaced by the
animal (the volume of air displaced by an animal was assumed to be
approximately equal to the animal’s mass, in grams). Values for V̇O2
are normalized to the weight of the animal and expressed as ml
O2 䡠 min⫺1 䡠 kg⫺1, under conditions of standard temperature and pressure.
Statistical Analysis
Interactions between genotype and ambient temperature that affect
breathing, V̇O2, and body temperature were assessed using a twofactor, repeated-measures ANOVA, with Tukey post hoc comparisons
where appropriate. Between-genotype differences in baseline breathing and animal mass were analyzed with a between-subject, one-way
ANOVA. Where noted in the text, data that failed a normality test
were square-root transformed before being subjected to ANOVA.
Fig. 1C). However, there was no difference in body mass
between PACAP⫹/⫹ and PACAP⫹/⫺ animals (P ⫽ 0.61).
Effects of PACAP Deficiency on Breathing During
Thermoneutral Conditions
Minutes 4 and 5 of each of the three plethysmography
protocols (when all animals experienced identical conditions)
were combined to provide the maximum power to examine
initial baseline breathing at thermoneutral temperature (32°C,
Fig. 2, A–C). There were no significant differences in VT index
between genotypes (Fig. 2A; P ⫽ 0.153). However, both
PACAP⫹/⫺ and PACAP⫺/⫺ mice breathed at a slightly lower
rate (Fig. 2B; P ⫽ 0.018), reducing overall baseline V̇E significantly compared with wild-type littermates (Fig. 2C; P ⫽
0.044, data square root transformed). PACAP deficiency had
no effect on SDP during baseline (P ⫽ 0.172, not shown).
To assess the changes in breathing resulting from the time in
the chamber (time control), 36 animals were exposed for 20
min to a constant ambient temperature of 32°C. Similar to the
genotypic differences seen in minutes 4 and 5, both PACAP⫹/⫺
and PACAP⫺/⫺ neonates continued to have a reduced V̇E index
throughout the time control (P ⫽ 0.023, Fig. 3A, right).
Although a reduced rate may have contributed to this effect,
the effect of genotype on rate alone was not quite significant
(P ⫽ 0.08, Fig. 3A, middle). Also mirroring minutes 4 and 5,
over the entire 20-min control period genotype alone had no
significant effects on VT index (Fig. 3A, left) or breathing
stability (not shown).
While there were no significant effects of time alone on rate
or overall V̇E index (rate: P ⫽ 0.077, Fig. 3A, middle; index of
V̇E:: P ⫽ 0.09, Fig. 3A, right), there was a significant interaction between genotype and time on VT index (P ⫽ 0.021): the
wild-type VT index was significantly reduced at minutes 19 and
20, relative to baseline (minutes 4 and 5) (Fig. 3A, left, filled
bars; ⫺10.1% ⫾ 5.7%; Tukey post hoc: P ⫽ 0.012); however,
there was no effect of time on the VT index of PACAPdeficient animals (Fig. 3A, left; PACAP⫹/⫺: ⫹5.7% ⫾ 3.2%;
Tukey post hoc: P ⫽ 0.324; PACAP⫺/⫺: ⫹1.6% ⫾ 7.5%;
Tukey post hoc: P ⫽ 0.566).
In summary, PACAP-deficient animals have reduced V̇E at
thermoneutral ambient temperatures, but any changes with
time in rate, VT index, and V̇E index were slight at best and
below significance. The only significant effect of time was in
wild-type animals; despite being kept at their thermoneutral
ambient temperature, VT index dropped by 10%.
Effects of PACAP Deficiency on Breathing After a Mild
Increase in Ambient Temperature
RESULTS
Three 20-min plethysmography protocols were used in
which ambient temperature was: 1) increased from 32°C to
36°C (warm), 2) held constant at 32°C (thermoneutral control),
or 3) decreased from 32°C to 29°C (cool) (Fig. 1A). Neonates
used for the warm plethysmography protocol were subsequently subjected to the metabolism protocol at an ambient
temperature of 36°C. Two separate sets of animals were used
for measuring V̇O2 at 32°C and 29°C. The total ratio of
PACAP⫹/⫹, ⫹/⫺, and ⫺/⫺ genotypes for all experiments was
48:122:55, respectively, confirming our previous observation
that there was no increase in the mortality for PACAP-deficient
mice at P4 (8). The mass of PACAP⫺/⫺ neonates was ⬃88%
that of PACAP⫹/⫹ and PACAP⫹/⫺ littermates (P ⬍ 0.001;
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Increasing ambient temperature from 32°C to 36°C significantly affected all breathing parameters (VT index, rate, and V̇E
index decreased: P ⬍ 0.001 for each variable; Fig. 3B; SDP
increased: P ⬍ 0.001, not shown). However, PACAP genotype
had no significant effect on the magnitude of these responses
(genotype ⫻ temperature interaction; P ⬎ 0.05 for all variables).
Effects of PACAP Deficiency on Breathing After a Mild
Reduction in Ambient Temperature
When considering all animals together, lowering ambient
temperature had a significant overall effect on VT index (P ⫽
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(Omega Engineering), and, while in the holding container, mice were
prevented from huddling. Measurements of gaseous metabolism at
36°C were made from the mice used for the 36°C plethysmography
protocol (n ⫽ 50), immediately after the plethysmography protocol
was completed. Therefore, animals from all three groups experienced
a similar period of time at their respective temperatures prior to the
determination of metabolic rate.
For all experiments, single animals were placed in the preheated
metabolic chamber with the circuit open and allowed to settle for 5
minutes, after which the circuit was closed. Oxygen decay in the
chamber was recorded for 5 min; however, we analyzed V̇O2 only in
the first minute to minimize the influence of altered inspired gases on
metabolism (27, 37). Over this period, V̇O2 was linear, yielding R2
values ⬎0.95. Ambient temperature within the chamber was monitored continuously with a thermocouple and maintained at 36°C,
32°C, or 29°C (⫾ 0.5°C) by making minor adjustments to the position
of the heat lamp relative to the chamber. Although the metabolic rate
in neonatal rats is constant over long periods of maternal separation
(28), we minimized the total time of separation to a maximum of 4 h
for each litter tested. Most animals appeared to become accustomed to
the chamber quickly, resting without significant movement for the
entire recording.
Oxygen measurements were recorded on a computer equipped with
an A/D board (model TL2; Axon Instruments, Union City, CA) and
data acquisition software (AxoTape, Version 2.02; Axon Instruments). At the end of the recording, animals were killed, weighed, and
ear clipped for postanalysis genotyping (8).
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Fig. 3. Breathing of PACAP genotypes over time and after warming and cooling of the ambient temperature. Breathing during 20 min at thermoneutral (32°C)
ambient temperature (thermoneutral time control, n ⫽ 36; A), during and after warming the ambient temperature to 36°C (warming, n ⫽ 45; B), and during and
after cooling ambient temperature to 29°C (cooling, n ⫽ 54; C). Data shown are for index of VT, (left), breathing rate (Breaths/min, middle), and index of minute
V̇E (right) during minutes 4 and 5 (baseline), minutes 7 and 8 (temperature cooling or warming), and minutes 19 and 20 (new steady-state temperature) from
wild-type (PACAP⫹/⫹; black bars), PACAP heterozygous (PACAP⫹/⫺; grey bars), and PACAP knockout (PACAP⫺/⫺; white bars) P4 littermates. Significant
effects (P ⬍ 0.05) of time, genotype (G), and genotype-time interaction (G ⫻ time) were determined using two-factor, repeated-measures ANOVAs, and are
indicated in each graph. Note that 1) in PACAP-deficient animals, neither the index of VT nor the index of V̇E changes with time (A); 2) the absence of any
genotype-specific effects of warming on breathing (B), and that cooling decreases the indices of VT and V̇E in PACAP-deficient animals, but not in wild-type
littermates (C).
0.003, Fig. 3C, left), rate (P ⬍ 0.001, Fig. 3C, middle), and V̇E
index (P ⬍ 0.001, Fig. 3C, right). Cooling also significantly
reduced breathing stability (i.e., SDP increased, P ⫽ 0.016, not
shown). The effects of temperature on rate and variability were
independent of genotype (genotype ⫻ temperature interaction
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on rate and SDP: P ⫽ 0.730 and P ⫽ 0.08, respectively).
Importantly, however, the responses of VT and V̇E indices were
dependent on genotype. In wild-type animals, cooling had no
significant effect on either VT or V̇E indices, whereas in their
PACAP-deficient littermates, cooling caused a reduction in
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Effects of Initial V̇E on the Response of PACAP-Deficient
Animals to Cooling
At the start of the experiment (minutes 4 and 5), when all
three groups experience the same conditions, PACAP-deficient
animals in the thermoneutral control group had a slightly
reduced breathing rate compared with that of the PACAPdeficient animals in the cooling group (difference: 10% and
16% for PACAP⫹/⫺ and ⫺/⫺ animals, respectively; compare
Fig. 3A, middle with Fig. 3C, middle). We explored the effect
of this slight difference in initial phenotype to determine
whether the reduced V̇E experienced by PACAP-deficient animals with cooling was the result of an initial high level of V̇E.
Specifically, we analyzed the relationship between initial ventilatory frequency and change in V̇E over time alone (control
group, Fig. 7A) and during cooling (cooling group, Fig. 7B). In
the control group, the fall in V̇E over time was only weakly
related to the initial level of V̇E, and this effect was confined to
PACAP⫹/⫺ animals (Fig. 7A, PACAP⫹/⫺: R2 ⫽ 0.48, P ⬍
0.01). Moreover, in the cooling group, the reduction in V̇E
observed in PACAP-deficient animals was unrelated to the
initial rate (Fig. 7B; PACAP⫹/⫺: R2 ⫽ 0.04, P ⫽ 0.28;
PACAP⫺/⫺: R2 ⬍ 0.001; P ⫽ 0.98).
DISCUSSION
Effects of PACAP Deficiency on Gaseous Metabolism
and Body Temperature
Changing ambient temperature in either direction significantly increased V̇O2 in all genotypes (P ⬍ 0.001), suggesting
that 32°C was within the thermoneutral range for these animals
(Fig. 6A). However, all genotypes had the same metabolic
response to temperature changes (genotype ⫻ temperature
interaction: P ⫽ 0.501, Fig. 6A).
At ambient temperatures of 29°C, 32°C, and 36°C, the
body temperatures of neonates were ⬃32–33°C, 35–36°C,
and 37°C, respectively. There was neither an effect of
genotype alone (P ⫽ 0.580) nor an interaction between
genotype and ambient temperature on body temperature
(P ⫽ 0.321, Fig. 6B).
In mice, PACAP deficiency increases the chance of sudden
neonatal mortality in the second postnatal week, especially
when litters are exposed to mild thermal stress (12). Our
previous study demonstrated that during anesthetic-induced
hypothermia, PD7–14 PACAP-null mice, but not their wildtype littermates, experience long-duration apneas, ultimately
leading to atrioventricular block (8). However, breathing, metabolic rate, and body temperature are tightly coupled, and
PACAP has been implicated in the regulation of all three
physiological systems (1, 8, 12). In the present study, we
investigated the relative effect of PACAP-deficiency on
the adaptation of these systems to thermal stress free from the
confounding effects of anesthetic and at P4, a developmental
time point before the increased mortality for PACAP⫺/⫺ mice.
Fig. 4. Representative plethysmographic tracings, showing
the breathing pattern of wild-type and PACAP⫹/⫺ littermates before, during, and after ambient cooling. Recordings
of entire experiments are shown (top), including the 5 min
of baseline breathing (TA ⫽ 32°C), the period of cooling
(between dotted lines), and breathing at the reduced temperature (TA ⫽ 29°C). Expanded traces of individual
breaths from minutes 4 and 5 (TA ⫽ 32°C) and minutes 19
and 20 (TA ⫽ 29°C) are indicated below, illustrating the
reduction in VT experienced only by PACAP-deficient
animals with cooling, while the VT of the wild-type littermate is maintained. The VT response of PACAP⫺/⫺ littermates is similar to that of PACAP⫹/⫺ animals (not shown).
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both VT and V̇E indices (overall genotype ⫻ temperature
interaction: P ⫽ 0.019, Fig. 3C, left and P ⫽ 0.042, Fig. 3C,
right, respectively).
Example data demonstrating the differential effects of cooling on V̇E in wild-type and PACAP-deficient animals are
shown in Fig. 4 and the average VT and V̇E indices for each
genotype at 29°C (minutes 19 and 20), relative to 32°C (minutes 4 and 5), are shown in Fig. 5. A statistical analysis of the
data in Fig. 5 emphasizes the results above in that 1) VT index
of PACAP⫹/⫹ mice did not change significantly after cooling
(Tukey post hoc: P ⫽ 0.339), whereas the VT index of
PACAP⫹/⫺ and PACAP⫺/⫺ neonates fell 10.9% ⫾ 5.8%
(Tukey post hoc: P ⫽ 0.002) and 17.4% ⫾ 4.2% (Tukey post
hoc: P ⫽ 0.003), respectively; and 2) V̇E index of PACAP⫹/⫹
mice did not change significantly after cooling (Tukey post
hoc: P ⫽ 0.88), whereas V̇E index of PACAP⫹/⫺ and
PACAP⫺/⫺ neonates fell 18.9 ⫾ 6.2% (Tukey post hoc: P ⬍
0.001) and 28.8 ⫾ 3.8% (Tukey post hoc: P ⬍ 0.001),
respectively.
In summary, overall, cooling had effects on all three respiratory variables. However, the effect was genotype dependent;
changes in VT and V̇E during cooling were limited to PACAPdeficient animals.
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PACAP MAINTAINS NEONATAL BREATHING DURING COOLING
This study presents two novel findings. First, unlike their
wild-type littermates, the VT and V̇E of P4 mice with both
complete and partial PACAP deficiency are significantly reduced during cooling. Second, PACAP deficiency has no effect
on the metabolic and body temperature responses of P4 neonates to changing ambient temperature. Thus, while the inability to measure blood gases means we cannot conclude unequivocally that PACAP deficiency results in hypoventilation during
cooling, PACAP-deficient mice show a relative hypoventilation, suggesting that they are more prone to hypoxemia and
hypercapnia during cooling compared with wild-type littermates.
Methodological Considerations
There are several caveats that should be considered when
interpreting our data.
Maternal separation. For both plethysmography and indirect
calorimetry experiments, some animals were separated from
maternal care for several hours. The possibility exists, therefore, that the metabolism and/or breathing of some pups was
compromised prior to testing. To ensure there was no experimenter-introduced bias with regard to the average duration of
maternal separation for each genotype, we tested pups randomly while blinded to genotype. To determine whether the
PACAP genotype affected how animals respond to maternal
separation, we performed time-control experiments in which
animals were maintained at thermoneutral conditions. The
results indicate that V̇E was relatively constant when animals
were maintained at thermoneutral ambient temperature, regardless of genotype (Fig. 5A). Thus, PACAP genotypic differences in anxiety levels (33) or other physiological manifestations resulting from maternal separation cannot alone explain
the precipitous fall in V̇E observed in the cool-challenged,
PACAP-deficient animals.
AJP-Regul Integr Comp Physiol • VOL
Comparison with previous study. Although PACAP-deficient animals in both this study and a previous study (8) had
lower V̇E than their wild-type littermates during baseline conditions, in the present study, this was due to a reduction in
breathing rate, not VT as was found previously. We currently
do not know enough about the specific role of PACAP in the
control of breathing to explain this difference with any degree
of satisfaction. However, the effect is probably not due to a
difference in settling time since, in the present study, the VT of
each genotype converged over the course of the 20-min thermoneutral protocol (Fig. 3A). We note that strain can have a
considerable effect on breathing in neonatal rodents (5, 10) and
speculate that the proportion of 129SvJ alleles to C57/BL6
alleles at loci important for ventilatory control may have
changed between studies.
Effect of differences in body weight. The P4 PACAP⫺/⫺
neonates used in the present study were significantly smaller
than their wild-type and heterozygous littermates, a phenotypic
difference not observed in other studies using PACAP signaling-deficient mice (8, 11, 32). Therefore, the possibility exists
that the reduction in VT and V̇E in PACAP⫺/⫺ neonates with
cooling is related to a smaller body size or is secondary to a
developmental defect. However, we feel this is unlikely because PACAP⫹/⫺ neonates are the same size as wild-type
littermates but share the ventilatory phenotype of their
PACAP⫺/⫺ littermates.
Technical considerations in assessing respiratory measurements. As stated in METHODS, the pressure signal recorded from
small animals in a chamber is likely dominated by rarefaction
and compression of gases in the lungs. However, if the signal
includes a component derived from the heating and humidification of inspired air, then changing the ambient temperature
could have directly influenced the size of the recorded signal.
Indeed, we note that with warming, which reduces the ambient294 • MARCH 2008 •
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Fig. 5. Summary of changes in breathing and
metabolic rate from baseline conditions after
cooling. A: thermoneutral time control: change
in VT index (left) and V̇E index (right) of
PACAP genotypes over time, held at an ambient temperature of 32°C. B: change in VT
index (left) and V̇E index (right) of PACAP
genotypes after cooling to an ambient temperature of 29°C. Data are the VT index and V̇E
index at minutes 19 and 20 (either 32°C for
time control or 29°C for cool protocol) relative
to minutes 4 and 5 (baseline thermoneutral,
32°C). Within each genotype, significant effects (P ⬍ 0.05) of cooling on VT index or V̇E
index relative to baseline are indicated (^).
*Significant differences from wild-type after
cooling (minutes 19 and 20) . Note the lack of
effect of time alone on either the VT or V̇E
indices of PACAP-deficient P4 neonates and
the selective effect of cooling on the VT and
V̇E indices of PACAP⫹/⫺ and PACAP⫺/⫺
neonates.
PACAP MAINTAINS NEONATAL BREATHING DURING COOLING
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choconstriction than wild-type littermates and have a higher
airway resistance. Yet, PACAP-deficient mice had a smaller
VT index than wild-type littermates. This argument suggests
that our results might underestimate the reduction in V̇E caused
by cooling in PACAP-deficient animals.
Response of V̇O2 and Body Temperature to Changes
in Ambient Temperature and the Role of PACAP
The body temperature measurements made in the present
study demonstrate that, despite the augmentation of metabo-
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Fig. 6. Effect of mild changes in ambient temperature on the metabolic rate
and body temperature (TB) of PACAP genotypes. Oxygen consumption (V̇O2)
(A), and body temperature (B), at ambient temperatures of 29°C, 32°C, and
36°C in P4 PACAP genotypes. PACAP⫹/⫹: n ⫽ 8 (29°C); n ⫽ 10 (32°C); n ⫽
14 (36°C); black bars. PACAP⫹/⫺: n ⫽ 19 (29°C); n ⫽ 33 (32°C); n ⫽ 22
(36°C); grey bars. PACAP⫺/⫺: n ⫽ 12 (29°C); n ⫽ 8 (32°C); n ⫽ 14 (36°C);
white bars. Significant effects (P ⬍ 0.05) of ambient temperature (T), genotype, and genotype-ambient temperature interactions were determined with a
2-factor, repeated-measures ANOVA, and are indicated in each graph. Note
the lack of significant differences between PACAP genotypes with respect to
oxygen consumption and body temperature.
to-body temperature differential, both VT and V̇E indices fell,
despite the fact that metabolism, and therefore V̇E, should
increase with temperature. However, regarding our main finding that PACAP plays a role in maintaining V̇E during cooling,
we note 1) that the effect is genotype-specific [Given that
PACAP-genotype has no effect on body temperature (see Fig.
6), the effects of cooling on the proportion of the signal
sensitive to the ambient-to-body temperature differential would
be expected to affect all genotypes equally.]; and 2) that falls
in ambient temperature exacerbate the disparity between ambient and body temperature, which would be expected to
increase the component of the signal originating from the
heating of inspired air; all else being equal, the signal should
increase. However, the signal recorded for PACAP-deficient
animals decreased as temperature fell.
A related consideration is that the amplitude of the rarefaction- and compression-derived pressure signal is, in part, subject to airway resistance; the higher the resistance, the greater
the amplitude of the signal. PACAP is a bronchodilator (23)
and, thus, PACAP-deficient mice may be more prone to bronAJP-Regul Integr Comp Physiol • VOL
Fig. 7. Influence of initial breathing frequency on the ventilatory response of
PACAP genotypes. Relationship between initial ventilatory rate during the
baseline period (minutes 4 and 5 of protocol, TA ⫽ 32°C) and the change in V̇E
(% change from baseline to minutes 19 and 20 of the protocol) during the
thermoneutral time control (A) and after cooling (B) in PACAP⫹/⫺ (solid
circles), PACAP⫺/⫺ (open circles) animals, and wild-type littermates (triangles). During cooling, linear regression indicates no significant relationship for
either PACAP⫹/⫺ animals (R2 ⫽ 0.04, P ⫽ 0.28), PACAP⫺/⫺ animals
(R2⬍0.01, P ⫽ 0.99), or wild-type littermates (R2 ⫽ 0.18, P ⫽ 0.20). Thus, the
reduction in V̇E experienced by PACAP-deficient genotypes with cooling is
unrelated to an initially high level of V̇E.
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PACAP MAINTAINS NEONATAL BREATHING DURING COOLING
lism at reduced ambient temperature, neonatal mice depend
heavily on behavioral mechanisms for maintaining body temperature. Thus, they confirm studies suggesting neonatal rodents are poikilothermic (28).
The values we obtained for thermoneutral V̇O2 are within the
range previously reported for neonatal rodents of equivalent
age (3, 29), as are the changes observed in gaseous metabolism
after warming and cooling (28). The increase in V̇O2 at an
ambient temperature of 29°C is likely a result of adaptive
thermogenesis, while the increase at 36°C is likely a Q10 effect.
The lack of differences between genotypes for both V̇O2 and
body temperature imply that PACAP has no effect on adaptive
thermogenesis at this developmental stage.
Several recent studies have noted that absence of PACAP
and/or PAC1 receptors leads to a higher mortality prior to
weaning (11, 13, 14, 17), a condition exacerbated by raising the
animals in rooms kept at slightly lower temperatures (12). In
this paper, we show that nonanesthetized PACAP⫺/⫺ and
PACAP⫹/⫺ animals express a temperature-dependent respiratory phenotype by P4. However, while both PACAP⫺/⫺ and
PACAP⫹/⫺ animals share the abnormal temperature-dependent respiratory phenotype, only PACAP⫺/⫺ neonates are
reported to have significant mortality with mild reductions in
ambient temperature. Thus, it is likely that additional defects
associated with complete PACAP deficiency contribute to
death. These include a more pronounced blunting of the hypoxic and hypercapnic ventilatory responses at P4 (8) and
abnormalities in temperature regulation, fat metabolism, and
cardiac function that occur later on in postnatal development
(11–13, 32, 39). Interestingly, some of the characteristics of
PACAP⫺/⫺ and PAC1 receptor ⫺/⫺ neonates that appear later
on in development (11, 32) resemble those of neonatal rats
exposed to chronic hypoxia from birth. These similarities
include growth inhibition, pulmonary hypertension, and neonatal death starting at P5 (see Figs. 5.25 and 5.26 in Ref. 27).
Therefore, the phenotypes appearing after P4 in PACAP⫺/⫺
neonates may be secondary to chronic hypoxemia from birth,
related to insufficient ventilatory responses to environmental
stress.
Mechanism of Action of PACAP in Breathing
During Cooling
When P4 neonatal mice are exposed to a cool ambient
environment, body temperature falls, despite increased thermogenesis. Thus, a complement of factors that counteract the
inhibitory effects of Q10 on V̇E may be important. Although
PACAP appears to be one of these critical factors, the cellular
loci where PACAP is acting remains speculative. One possibility is that the impaired chemoreflex in PACAP-deficient
mice that we previously reported (8) is exacerbated by cooling.
Several reports suggest that intravenous injections of PACAP
can have acute effects on V̇E in adult animals, possibly by
acting directly on the carotid body (15, 35). Interestingly, the
carotid bodies have also been implicated in thermoregulation
(20). It is equally possible that PACAP maintains V̇E by
stimulating neurons that are activated by reductions in ambient
and/or body temperature but do not participate directly in the
AJP-Regul Integr Comp Physiol • VOL
Perspectives and Significance
In this study, we have demonstrated an important role for
PACAP signaling in the preservation of neonatal V̇E during
mild temperature stress. These observations may partially explain the temperature-dependent mortality of PACAP mice,
and are in keeping with a growing consensus that PACAP is
important for maintaining homeostasis in the face of acute
environmental stress: a ubiquitous requirement of all animals
that might contribute to its highly constrained structure
throughout evolution.
GRANTS
Salary support for this work was provided (to R. J. A. Wilson) by the Focus
On Stroke Program (a partnership between the Canadian Heart and Stroke
Foundation, the Canadian Stroke Network, the Canadian Institutes of Health
Research, and AstraZeneca), and the Alberta Heritage Foundation for Medical
Research. Salary support (for K. J. Cummings) was provided, in part, by a
Canadian Institutes of Health Research Fellowship and a Heart and Stroke
Foundation of Canada Research Fellowship Award. Operating funds for this
project were obtained from the Canadian Institutes of Health Research.
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