Influence of developmental nicotine exposure ... - Wiley Online Library

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J Physiol 593.23 (2015) pp 5201–5213

Influence of developmental nicotine exposure on the ventilatory and metabolic response to hyperthermia Jonathan Ferng1 and Ralph F. Fregosi1,2 1 2

Department of Physiology Department of Neuroscience, The University of Arizona, Tucson, AZ, USA

Key points

r Both developmental nicotine exposure and hyperthermia are associated with breathing abnormalities in neonates.

The Journal of Physiology

r Whether or not gestational nicotine exposure interacts with heat stress to alter the normal ventilatory response to hyperthermia is unclear.

r With moderate thermal stress, nicotine-exposed rat pups breathed less and had longer apnoeas r r

than control pups, although this was true at both normal and high temperatures, suggesting an effect of gestational nicotine exposure that is independent of body temperature. With severe thermal stress, nicotine-exposed pups failed to increase pulmonary ventilation, and had a delayed recovery from hyperthermia-induced gasping The results show that gestational nicotine exposure has subtle but physiologically significant effects on the ventilatory response to severe hyperthermia, which could impact upon both gas exchange and temperature regulation in infants with fever, excessive swaddling or exposure to hot environmental temperatures.

Abstract To determine whether developmental nicotine exposure (DNE) alters the ventilatory and metabolic response to hyperthermia in neonatal rats (postnatal age 2–4 days), pregnant dams were exposed to nicotine (6 mg kg−1 of nicotine tartrate daily) or saline with an osmotic mini-pump implanted subdermally on day 5 of gestation. Rat pups (a total of 72 controls and 72 DNE pups) were studied under thermoneutral conditions (chamber temperature 33°C) and during moderate thermal stress (37.5°C). In all pups, core temperature was similar to chamber temperature, with no treatment effects. The rates of pulmonary ventilation (V˙ I ), O2 consumption (V˙ O2 ) and CO2 production (V˙ CO2 ) did not change with hyperthermia in either control or DNE pups. However, V˙ I was lower in DNE pups at both chamber temperatures, whereas the duration of spontaneous apnoeas was longer in DNE pups than in controls at 33°C. The V˙ I /V˙ O2 ratio increased at 37.5°C in control pups, although it did not change in DNE pups. To simulate severe thermal stress, additional pups were studied at 33°C and 43°C. V˙ I increased with heating in control pups but not in DNE pups. As heat stress continued, gasping was evoked in both groups, with no effect of DNE on the gasping pattern. Over a 20 min recovery period at 33°C, V˙ I returned to baseline in control pups but remained depressed in DNE pups. In addition to altering baseline V˙ I and apnoea duration, DNE is associated with subtle but significant alterations in the ventilatory response to hyperthermia in neonatal rats. (Received 27 July 2015; accepted after revision 22 September 2015; first published online 2 October 2015) Corresponding author R. F. Fregosi: Department of Physiology, College of Medicine, The University of Arizona, Tucson, AZ 84724, USA. Email: [email protected] Abbreviations DNE, developmental nicotine exposure; P, postnatal day; RER, respiratory exchange ratio; Tchamber , chamber temperature; Tcore , core temperature; V˙ I , pulmonary ventilation rate; V˙ O2 , oxygen consumption rate; V˙ CO2 , carbon dioxide production rate; VT , tidal volume.

C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

DOI: 10.1113/JP271374

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J. Ferng and R. F. Fregosi

Introduction Approximately 500,000 infants are born to tobacco smoking mothers in the USA each year (Adams et al. 2012). A large epidemiological evaluation published in 2010 shows that 13–19% of low birth weight deliveries, 23–34% of sudden infant death syndrome deaths and 5–7% of deaths associated with preterm birth were attributable to prenatal smoking (Dietz et al. 2010). Infants and children born to mothers that smoked during and after pregnancy have delayed growth and a higher incidence of behavioural, intellectual and cardiorespiratory abnormalities (Lassen & Oei, 1998; Hafstrom et al. 2005; Huang et al. 2006; Winzer-Serhan, 2008; Braun et al. 2009; Mitchell et al. 2012). Tobacco smoke contains over 4000 chemicals, many of which are confirmed neuroteratogens and/or carcinogens. Among these, nicotine is a critical neuroteratogen, which, by itself, profoundly influences brain development in neonatal animals (Roy et al. 1998; Roy et al. 2002; Smith et al. 2010) and is associated with an increased incidence of cardiorespiratory abnormalities, including more frequent and longer spontaneous apnoeas, delayed arousal responses, and diminished responses to hypoxia and hypercapnia (Huang et al. 2004; Hafstrom et al. 2005; Huang et al. 2010; Xia et al. 2010). This is not surprising because neuronal nicotinic ACh receptors are expressed very early in gestation (Hellstrom-Lindahl et al. 1998; Slotkin et al. 2005), and some studies suggest that activation of nicotinic ACh receptors in utero alters normal mitotic and apoptotic processes (Roy et al. 1998), as well as synaptogenesis (Navarro et al. 1989; Zahalka et al. 1992; Hellstrom-Lindahl et al. 2001; Slotkin et al. 2004; Pauly & Slotkin, 2008; Dwyer et al. 2009; Smith et al. 2010). Remarkably, many physicians prescribe nicotine patches to pregnant smokers (Stead et al. 2008), with the goal of avoiding the other toxins in tobacco smoke. Unfortunately, our understanding of the harmful effects of this replacement therapy is uncertain. Heat stress is also associated with breathing instability in infants and animal models (Daily et al. 1969; Perlstein et al. 1970; Fleming et al. 1992), including a prolongation of apnoea duration in infants (Berterottiere et al. 1990). These observations raise the possibility that developmental nicotine exposure (DNE) exacerbates the adverse influence of thermal stress on cardiorespiratory control. In support of this, prenatal exposure to cigarette smoke prolongs the depression of respiratory frequency observed following hypoxic hyperthermia in 1-week-old rat pups (Pendlebury et al. 2008) and prolongs the reflex apnoea evoked by the combination of hyperthermia and laryngeal stimulation (Xia et al. 2010). These studies suggest that prenatal exposure to cigarette smoke or nicotine amplifies cardiopulmonary reflex responses when stressors such as hypoxia or laryngeal stimulation are combined with hyperthermia. Despite these important

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observations, comprehensive information on the impact of DNE on the thermoregulatory, ventilatory and metabolic response to hyperthermia alone is lacking. Accordingly, we tested the hypothesis that DNE alters the thermoregulatory, ventilatory and metabolic response to moderate and severe thermal stress, as occurs in fever, exposure to environmental heat stress and excessive swaddling of infants (Sunderland & Emery, 1981; Stanton, 1984; Nelson & Taylor, 1989).

Methods Animals

All data were obtained from experiments approved by the Institutional Animal Care and Use Committee at The University of Arizona and in accordance with guidelines specified by the Journal of Physiology. As summarized in Table 1, a total of 164 neonatal rats (Sprague–Dawley, Charles River Laboratories, Wilmington, MA, USA), aged postnatal day (P) 2, 3 and 4 (P2–P4) were studied. The neonates were derived from 30 saline-exposed and 27 nicotine-exposed dams. One to eight pups, approximately half male and half female, were taken from each litter. The pulmonary ventilation rate (V˙ I ) was measured in 36 saline-exposed and 36 nicotine-exposed neonates (Experiment 1) (Table 1), and metabolic rate was measured in a separate set of 36 saline-exposed and 36 nicotine-exposed pups (Experiment 2) (Table 1). In each cohort of 36 pups, there were 12 neonates from each of the three age groups (Table 1). The ventilatory response to severe thermal stress was examined in a separate group of pups (Experiment 3) (Table 1), aged P3 and P4 (seven saline exposed; seven DNE). Finally, we subjected an additional six pups (three saline, three DNE) to severe thermal stress, followed by rapid removal from the chamber for the measurement of core temperature (Tcore ) (Experiment 4) (Table 1). Each neonate was studied only once, on P2, P3 or P4, because we were concerned that heat exposure may alter the development of ventilatory control and could confound the results obtained on subsequent exposures to thermal stress. We also carried out t tests on V˙ I , frequency and O2 consumption (V˙ O2 ) for males vs. females, and found no significant differences in either treatment group. Accordingly, the data from animals of both sexes were combined for all analyses. Pups were rendered unconscious by prolonged hypothermia and death was confirmed by pneumothorax to ensure that the animals had ceased breathing and the heart had stopped beating. Developmental nicotine exposure

As described previously (Luo et al. 2007; Huang et al. 2010; Pilarski et al. 2011, 2012; Jaiswal et al. 2013), dams were C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

Hyperthermia and breathing in neonates

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Table 1. Number of litters and neonatal animals that were used for each of the experimental protocols, including the number of neonates used at each of the three postnatal ages (P2, P3 and P4) Number of P2 neonates used

Number of Litters Experiment (1) Lung ventilation, moderate thermal stress (2) Metabolic rate, moderate thermal stress (3) Lung ventilation, severe thermal stress (4) Measurement of peak Tcore , severe thermal stress Total

Number of P3 neonates used

Number of P4 neonates used

Saline

DNE

Saline

DNE

Saline

DNE

Saline

DNE

13

13

12

12

12

12

12

12

10

8

12

12

12

12

12

12

6

5

2

2

5

5

1

1

3

3

30

27

32

32

implanted with 28-day osmotic mini-pumps on day 5 of gestation to ensure that DNE began early in the embryonic period. Our pumps release fluid at a rate of 2.5 µl h−1 , and are set to deliver 1.62 mg of nicotine tartrate salt per day. In our hands, the pregnant dams typically weigh 200 g on the day of pump implantation and 340 g at parturition. Thus, the dose of nicotine is 8.1 mg kg−1 day−1 just after implantation, and 4.8 mg kg−1 day−1 at parturition. Assuming linear growth throughout pregnancy, the dams weigh 270 g at the mid-point of pregnancy, and so the calculated average dose over the course of pregnancy is 6 mg kg−1 day−1 , which is equivalent to an average value of 1.95 mg kg−1 day−1 of free base nicotine (Matta et al. 2007). Control dams received a pump filled with physiological saline delivered at the same rate (2.5 µl h−1 ). Because gestation in the dams is 21 days, neonates were exposed to nicotine or saline through the placenta on gestational days 5–21, and via breast milk during the postnatal period (Matta et al. 2007).

Measurements

Experiments were done between 08.00 h and 17.00 h. Head-out plethysmography (Fig. 1A) was used to measure V˙ I , as described previously (Huang et al. 2004; Huang et al. 2010). The chamber had ports for a thermocouple probe, for volume calibration and for connection to the pneumotachometer (model 8431; Hans-Rudolph Inc., Kansas City, MO, USA). The two ports of the pneumotachometer were attached to the positive and negative inlets of a pressure transducer with a range of ± 2 cm H2 O (DP45-16; Validyne Engineering, Northridge, CA, USA). The pressure signal, which is proportional to the airflow rate, was sent in parallel to an analogue integrator (model 7; Grass Instruments, Quincy, MA, USA) and an A/D board. The inspiratory phase of the airflow signal was integrated to derive the C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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24

26

26

inspired tidal volume (VT ). The digitized airflow signal and inspired VT were displayed on the computer screen (Fig. 4) and stored using Spike II software (Cambridge Electronic Design, Cambridge, UK). The thermocouple probe was attached to a control unit (TCAT-1A Temperature Controller; Physitemp, Clifton, NJ, USA) that maintained chamber temperature (Tchamber ) with a heat lamp (Fig. 1). For thermoneutral conditions, Tchamber was maintained at 33 ± 0.15°C (mean ± SD), corresponding to normal nesting conditions for neonatal rats (Mortola, 1984; Mortola & Dotta, 1992). Moderate and severe heat stress were defined as Tchamber values of 37.5 ± 0.53 and 43°C, respectively. Metabolic rate was measured with whole-body plethysmography as described previously (Fig. 1B). The pups were weighed and placed in a homemade, 83 ml Plexiglass cylinder. A port on each end of the cylinder allowed air to be pulled through the chamber at a constant rate of 40 ml min−1 . Temperature was monitored and maintained as described above. The gas exiting the chamber passed through a canister (Dri-Rite, Chicago, IL, USA) and then to an airflow, O2 and CO2 analyser (Raytek, Santa Cruz, CA, USA). Flow, CO2 and O2 levels were digitized using the Spike II A/D board and software.

Experimental protocols

For measurement of inspired VT and breathing frequency, neonates were placed in the head-out plethysmograph and, once they were reasonably still, we recorded airflow for 700 s under thermoneutral conditions. The rat was then removed from the chamber and Tcore was measured within 20–30 s. During the subsequent 15 min rest period, Tchamber was recalibrated to 37.5°C, the animal was placed back in the chamber and recording continued for another 700 s, at which time the animal was removed from the chamber and Tcore was recorded. We are confident

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that the animal’s Tcore did not change appreciably over the 20–30 s period between removal from the chamber and insertion of the probe, given that the time constant for passive heat transfer in P2 neonatal rats is >2 min (Mortola & Dotta, 1992) and that, in pilot studies, Tcore remained approximately constant for 2–3 min following removal from the chamber. Thus, our measurements of Tcore provide a realistic estimate of the actual Tcore of the animal in the chamber. For measurement of metabolic rate, neonates were placed in the whole-body plethysmograph, with recordings taken when the O2 and CO2 levels in the gas exiting the plethysmograph were stable for at least 60 s, which typically required 4–8 min. After recordings were obtained, the animal was removed from the chamber and Tcore was measured. Tchamber was then increased to 37.5°C, and the experiments were repeated. To examine the ventilatory response to severe hyperthermia, a third group of pups were used. After measuring baseline Tcore , the animal was placed in the head-out plethysmograph and airflow was recorded for 5 min at a Tchamber of 33°C. The plethysmograph was then warmed to 43°C, with breathing being recorded

A A/D Board

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continuously. When the animal began gasping (defined as a breathing frequency at least three times lower than the breathing frequency at baseline, with very large VT ) (Fig. 8C), we waited another 30 s before dropping Tchamber back to 33°C to monitor the recovery from gasping. Recording continued for an additional 20 min, the animal was removed from the plethysmograph and Tcore was measured. Finally, this protocol was repeated in an additional three saline-exposed and three nicotine-exposed rats that were removed from the plethysmograph immediately after the onset of gasping so that peak Tcore could be measured. Data analysis

The first author (JF) conducted the experiments and all the analyses, and was not blinded to the treatment groups. Inspired VT and breathing frequency were obtained by identifying periods of stable breathing (i.e. with an absence of movement artefact, compare Fig. 8A and B) and computing the average value of 10–20 breaths within these periods. This was carried out for each of five epochs throughout the 660 s recording, and a grand average was

Demodulator Analog Integrator Pneumotachometer

Pressure Transducer

Thermocouple Probe Control Unit Calibration Syringe

B

Body Chamber

Demodulator

Neck Collar

Heat Lamp

A/D Board

Pressure Transducer Pneumotachometer

Thermocouple Probe

Control Unit

Flow, O2, CO2 Analyzer Body Chamber

Heat Lamp

Figure 1. Head-out and whole body plethysmography systems A, head-out plethysmography. Temperature is servo-controlled with a heat lamp and control unit. Flow entering and exiting the chamber with breathing passed through a pneumotachometer that was connected to a differential pressure transducer. The voltage output from the pressure transducer was sent in parallel to an A/D board and an analogue integrator that was set to record inspired flow. The output from the analogue integrator was also sent to the A/D board, and inspired breath volume was calibrated by injecting known volumes of air into the chamber. B, whole-body plethysmography was used to record metabolic rate. An analyser pulled gas through the chamber and across Dri-rite and the O2 and CO2 analysers. Chamber gas flow was monitored by a pneumotach placed in series with the analyser flow output. The pneumotachometer was calibrated with a rotameter. Voltage outputs from the pressure transducer (flow) and the O2 and CO2 analysers were sent to the A/D board for visualization and analysis.



computed for breathing frequency, inspired VT and V˙ I . As described previously (Huang et al. 2004), apnoeas were defined as the absence of breathing for two or more typical baseline cycle periods (Fig. 4), and were counted over a 10 min period, which began 60 s into the recording and extended for 10 min. For each animal, we computed the number of apnoeas in 10 min, and the average duration of each apnoea. Metabolic rate recordings were analysed by calculating the difference between the percentages of O2 and CO2 in inspired gas and the steady-state values measured at the chamber exit port. These differences were multiplied by the constant flow rate of 40 ml min−1 to calculate the oxygen consumption rate (V˙ O2 ) and carbon dioxide production rate (V˙ CO2 ) in each epoch. During severe thermal stress, we measured breathing frequency, VT , and V˙ I by averaging 10–20 breathing cycles in each 2 min epoch throughout the recording, which

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included baseline, heat stress and gasping periods. As a result of the presence of movement artefacts during heat stress (Fig. 8B), we sometimes had to average fewer than 10 breath cycles. We analysed each breath cycle in the recovery period, and computed a composite average value for the entire 20 min period in each animal.

A Temperature (°C)

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Saline 42 40

Tcore

38 36 34 32

Tchamber

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100 125 150

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Litter DNE 2.5

Figure 3. Relation between core and chamber temperatures Changes in Tcore and Tchamber in saline-exposed (A) and DNE pups (B) under thermoneutral conditions (Tchamber = 33°C), and during moderate thermal stress (Tchamber = 37.5°C). Each animal is represented twice: once at a Tchamber of 33°C and once at 37.5°C. There are 72 pups in each treatment group, resulting in 144 observations in each group. For a detailed explanation, see text.

•

V I (ml/min/g)

2.0 1.5 1.0 0.5 0.0

0

2

4

6 Litter

Figure 2. Analysis of litter effects The weight corrected pulmonary ventilation rate under baseline (thermoneutral) conditions, plotted as a function of the litter from which animals were derived. The data represent the experiments from protocol 1 (Table 1), where 13 saline-exposed control litters and 13 DNE litters were used. The regression line (continuous line) and 95% confidence limits are included. Neither regression slope differed from zero, which is consistent with an absence of litter effects. Moreover, the variance within litters is similar to the variance between litters. For more details, see text. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

Figure 4. Airflow recording from the head-out plethysmograph in a saline-exposed P4 neonatal rat, under thermoneutral conditions (Tchamber = 33°C) The airflow trace (bottom trace) is uncalibrated, with inspiratory flow upwards. The upper trace shows the integrated inspiratory airflow signal, with a 100 µL calibration bar in the inset. One apnoea is shown, which is defined as the absence of breathing for greater than or equal to two cycle periods recorded under thermoneutral conditions.

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Statistical analysis

Breathing frequency, V˙ I , VT , V˙ O2 , V˙ CO2 , apnoea number, apnoea duration and the respiratory exchange ratio (RER, V˙ CO2 /V˙ O2 ) were analysed using a two-factor, mixed-model ANOVA, with treatment (saline vs. DNE) and temperature (thermoneutral vs. hyperthermic conditions) as the two factors. However, when the test for normality of distributions failed, indicating ineffective matching, we used ordinary two-factor ANOVA. In either case, if the ANOVA was significant, paired contrasts were

A

Analysis of litter effects

# V I (ml/min/g)

2.0

Because we used from one to eight pups per litter (median of three pups per litter for both treatment groups), it is important to demonstrate that the variance in key dependent variables, such as V˙ I , is approximately the same both within and across litters. This is because

#

1.5

.

analysed with the Bonferroni post hoc procedure. An unpaired t test was used to examine treatment effects for body weight, time to gasping onset and gasping duration. For all tests, P < 0.05 was considered statistically significant. To mitigate effects of differences in body weight, and for ease of comparison with published values, V˙ I and VT are expressed per gramme of body weight in all pups, whereas V˙ O2 and V˙ CO2 are expressed per kilogramme body weight. For all summary data (Figs 5 to 7 and 9), we show individual data for every animal, with the mean represented by horizontal lines. Results

Saline DNE

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1.0 0.5 37.5 °C

A

Tchamber (°C)

B

VT (μl /g)

20

#

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Apneas in 10 min

33 °C

10

75 60

Saline DNE

40 20 0

33 °C 5 33 °C

37.5 °C

B 4

frequency (min–1)

Apnea duration (sec)

Tchamber (°C)

C

37.5 °C

Tchamber (°C)

250 200 150

#

3 2.5 2.0 1.5 1.0 0.5 0.0

100

33 °C

37.5 °C

Tchamber (°C)

50 33 °C

37.5 °C

Tchamber (°C) Figure 5. Ventilatory response to moderate thermal stress Individual values for V˙ I (A), VT (B) and frequency (C), at Tchamber of 33 and 37.5°C, in saline (filled circles) and DNE (open squares) pups. V˙ I did not change with temperature, although it was lower in DNE pups at each Tchamber . This was the result of a lower VT (B) at a Tchamber of 37.5°C. There were no treatment or temperature effects on breathing frequency. # , DNE different from saline (P < 0.05). Horizontal lines represent the group mean value.

Figure 6. Number and duration of apnoeas as a function of chamber temperate Individual values showing the number of apnoeas in 10 min (A) and the duration of each apnoea (B) at a Tchamber of 33 and 39°C in saline (filled circles) and DNE (open squares) pups. Two-way ANOVA revealed a significant temperature effect for apnoea number, although no treatment effects or temperature-treatment interaction, indicating that both treatment groups had more apnoeas with heating (see text). There was a significant treatment effect for apnoea duration at 33°C but not at 37.5°C. # , DNE different from saline (P < 0.05). Horizontal lines represent the group mean value. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society


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the data from each animal used are assumed to represent an independent observation, whether they originate from the same litter or not. However, this is not always the case simply because pups from a single litter are genetically similar, and they develop in a unique intrauterine environment (Holson & Pearce, 1992). We examined the weight corrected pulmonary ventilation rate (i.e. ml min–1 g–1 ), measured under baseline (thermoneutral) conditions, as a function of the litter in which the animal was derived. The data shown in Fig. 2 represent the experiments from protocol 1 (Table 1), where 13 saline-exposed control litters and 13 DNE litters were used. The regression line (continuous line) and 95% confidence limits are included. Neither regression slope differed from

A

.

VO2 (ml/min/kg)

100 80

Saline DNE *

60 40 20 0 33 °C 37.5 °C Tchamber (°C)

B

.

VCO2 (ml/min/kg)

100 80 60 40

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zero, which is consistent with an absence of litter effects. Moreover, variance within litters is similar to variance between litters. Thus, we assume that any treatment effects reported in the present study are the result of DNE, and not litter effects. Body weight and Tcore

Values for body weight and Tcore for pups in the ventilation (n = 36 saline and 36 DNE) and metabolic rate measurement groups (n = 36 saline and 36 DNE) were the same within each of the treatment groups. Therefore, to examine treatment effects on body mass and Tcore , the data were combined, yielding 72 pups in each treatment group. Saline-exposed control pups weighed significantly more than DNE pups (Saline 9.4 ± 0.2 g; DNE 8.6 ± 0.2 g; P = 0.0046). After analysing the data by age and treatment with two-factor ANOVA, we found that the difference was significant on P2 (P = 0.0035), although not on P3 or P4; thus, the difference in body mass between saline and DNE pups when data from all age groups are combined is largely the result of growth retardation on P2 in the DNE pups. Figure 3 shows the relation between Tchamber and Tcore for all saline-exposed (Fig. 3A) and DNE pups (Fig. 3B). Each animal is represented twice: once at an average Tchamber of 33 ± 0.01°C and once at 37.50 ± 0.04°C. Note that Tchamber was very consistent across experiments, and that, in both groups, Tcore was either just above (at low Tchamber ), or approximately equal to Tchamber . The group average data show that, although both control and DNE pups increased Tcore significantly with heating (Temperature effect, F = 317, P < 0.0001), there were no treatment effects. Indeed, average Tcore was identical

20 0 33 °C 37.5 °C Tchamber (°C)

C

1.2 1.0

RER

0.8 0.6 0.4 0.2 0.0 33 °C 37.5 °C Tchamber (°C) Figure 7. Metabolic response to moderate thermal stress Individual values for V˙ O2 (A), V˙ CO2 (B) and RER (C) at a Tchamber of 33 and 37.5°C in saline (filled circles) and DNE (open squares) pups. There were no significant treatment effects, although the temperature effect was significant for V˙ O2 in control animals. ∗ , different from 33°C within a treatment group (P < 0.05). C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

Figure 8. Representative airflow and tidal volume recording from the head-out plethysmograph in a P4 saline-exposed neonatal rat The airflow trace (bottom trace) is uncalibrated, with inspiratory flow upwards. As in Fig. 4, the upper trace shows the integrated inspiratory airflow signal, with a 10 µL calibration bar shown in the inset. A, thermoneutral conditions (Tchamber = 33°C). B, Tchamber of 43°C. C, during sustained gasping. D, recovery from gasping (Tchamber = 33°C).

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in both treatment groups at a Tchamber of 33°C (Saline 34.54 ± 0.16°C; DNE 34.52 ± 0.16°C) and 37.5°C (Saline 36.98 ± 0.14°C; DNE 36.91 ± 0.19°C). Ventilatory response to hyperthermia

A representative recording from a P4 saline-exposed neonate under thermoneutral conditions (Tchamber = 33°C) is shown in Fig. 4. We were able to obtain high fidelity recordings of airflow and VT in all pups, and apnoeic episodes, as defined in the Methods, were easily identified. ANOVA revealed a significant treatment effect for V˙ I (F = 13.45, P < 0.0001), although there were no temperature effects or treatment–temperature interactions. Post hoc analyses showed that V˙ I was slightly but significantly lower in DNE pups at a Tchamber of both 33 and 37.5°C (Fig. 5A). Accordingly, DNE alters V˙ I , although this effect is independent of Tcore . The lower V˙ I in DNE pups is the result of a slightly lower VT , although this was significant only at a Tchamber of 37.5°C (Fig. 5B). There were no temperature or treatment effects on breathing frequency (Fig. 5C). B *

45 40

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e

0

0

There were no treatment effects for V˙ O2 , V˙ CO2 or RER, and no temperature effects for V˙ CO2 and RER in either treatment group (Fig. 7). By contrast, V˙ O2 fell with heating in control pups, and this temperature effect was significant (ANOVA, temperature effect, F = 6.86, P = 0.006). Ventilatory response to severe hyperthermia

25

* *

Apnoea number and duration were also computed (Fig. 4) under thermoneutral and hyperthermic conditions, with data for each animal shown in Fig. 6. The number of apnoeas observed over the 10 min recording period in control and DNE pups was the same at each Tchamber (Fig. 6A). Although ANOVA revealed a significant temperature effect (F = 6.09, P = 0.016), Bonferroni post hoc tests did not reveal a significant difference between 33 and 37.5°C in either treatment group, suggesting that the temperature effect was the same in both groups; this is supported by the absence of a treatment–time interaction. By contrast, there was a significant treatment effect for apnoea duration (F = 11.65, P = 0.0011), with post hoc tests showing that the difference was at a Tchamber of 33°C, and that the duration was prolonged in DNE animals (Fig. 6B). Given the possibility that this difference was biased by the single outlier seen in Fig. 6B, we removed that data point and repeated the statistical analysis. Even with the outlier removed, the difference was significant (F = 10.72, P = 0.0013), indicating that the finding is robust. There were no temperature effects and no interaction between treatment group and temperature, suggesting that the longer apnoea duration is an effect of DNE independent of body temperature. Metabolic response to hyperthermia

0

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frequency (min–1)

*

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G

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A

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Figure 9. Temperature and ventilatory responses to severe thermal stress A–D, Tcore and ventilatory response to severe thermal stress in saline-exposed (filled circles) and DNE (open squares) pups. Tchamber was 33°C at baseline, and then increased to 43°C, and was maintained at that level until gasping ensued. Thirty seconds after gasping onset, Tchamber was adjusted back to 33°C, and recovery data were recorded for 20 min. Tcore was measured after the 20 min recovery period ended. To obtain values for peak Tcore at a Tchamber of 43°C, this protocol was repeated in six rats (three saline and three DNE), although the pups were removed directly after the onset of gasping so that Tcore could be measured. # , DNE different from saline (P < 0.05); ∗ , different from 33°C within a treatment group (P < 0.05). Horizontal lines represent the group mean value.

Figure 8 shows representative airflow and inspiratory VT recordings from a P4, saline-exposed neonate under baseline conditions (Fig. 8A), during severe hyperthermia (Tchamber = 43°C) but before the onset of gasping (Fig. 8B), during hyperthermia-induced gasping (Fig. 8C) and during recovery from gasping (Fig. 8D), as Tchamber was falling back towards 33°C. During severe hyperthermia, the breathing pattern became irregular, as characterized by a high frequency and a low VT , as well as obvious movement artefacts appearing in the airflow signal (Fig. 8B). Gasping was characterized by a very slow frequency, with large VT (Fig. 8C) and an absence of movement artefact. Recovery from gasping, which occurred in all but three pups, was characterized by very regular, shallow breathing (Fig. 8D). Individual and average values for Tcore , V˙ I , frequency and VT measured before, during and after severe hyperthermia are shown in Fig. 9. Tcore was significantly higher at 43°C and in recovery than at 33°C in both control C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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and DNE pups (Fig. 9A, asterisks). Interestingly, Tcore was significantly higher in DNE pups than in controls at a Tchamber of 33°C (Fig. 9A, hash symbol), although there were no treatment effects at a Tchamber of 43°C or during recovery. Raising Tchamber from 33 to 43°C increased V˙ I in control pups but not in the DNE pups (Fig. 9B). Frequency increased significantly with heating in both groups (Fig. 9C), although VT did not change significantly at a Tchamber of 43°C in either treatment group (Fig. 9D). During the period of hyperthermia-induced gasping, pups in both groups had significantly higher VT (Fig. 9D) but lower V˙ I (Fig. 9B) and frequency (Fig. 9C) compared to baseline levels, and ANOVA did not reveal significant treatment effects for any of these variables. Over a 20 min recovery period at 33°C, V˙ I was not different from baseline in control pups but remained significantly lower than baseline in DNE pups (Fig. 9B). Frequency and VT in the recovery period were statistically the same as baseline in both treatment groups. Finally, there were no treatment effects for the time to gasping onset (Saline 838 ± 265 s; DNE 607 ± 198 s; P = 0.407) or the duration of gasping (Saline 1078 ± 318 s; DNE 842 ± 15 s; P = 0.49) in response to severe thermal stress. Discussion We studied the ventilatory and metabolic responses to moderate and severe thermal stress in neonatal rat pups that were exposed to nicotine in utero and after birth (DNE). Under thermoneutral conditions (Tchamber = 33°C), V˙ I was lower and apnoea duration longer in DNE than in control pups. Moderate thermal stress did not change V˙ I significantly in either treatment group, although the average V˙ I was significantly lower in DNE pups than in controls. Estimates of V˙ I /V˙ O2 showed an increase with moderate thermal stress in control pups but no change in DNE pups. With severe heat stress, V˙ I initially increased in control pups, fell significantly during the period of hyperthermic gasping and returned to baseline levels in the recovery period. By contrast, DNE pups failed to increase V˙ I significantly with heating and, although their V˙ I fell to the same level as controls during gasping, it remained significantly below baseline levels in the recovery period. Consistent with recent studies in animal models and clinical observations made in infants, our results suggest that DNE is associated with subtle but significant alterations in the ventilatory response to thermal stress in neonates. Critique of methods Nicotine dosing. Our model exposes rat pups to nicotine in utero via the placental transfer of nicotine, and after birth via breast milk. Similar to humans, rats metabolize C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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nicotine within a few hours, with some being eliminated via the kidneys and the remainder converted to cotinine, nicotine-N -oxide and 3 -hydroxycotinine (Matta et al. 2007). By contrast, cotinine has a half-life of 20–24 h (Sastry et al. 1995), and there is a positive correlation between plasma nicotine and cotinine levels (Florescu et al. 2009). We have previously shown that the same dosing regimen used in the present study results in plasma cotinine levels ranging from 60–92 ng ml–1 in P2–P4 neonates (Powell et al. 2015), which is similar to the average cotinine levels of 88 ng ml–1 found in the umbilical cord blood of newborns from mothers who smoked an average of 95 cigarettes week–1 (Berlin et al. 2010). These literature values confirm that our dosing regimen mimics the nicotine levels in the newborns of pregnant human smokers, and is thus physiologically meaningful. Measuring V˙ I and metabolic rate in separate groups of pups. We did this to provide the most natural

environment possible during recording. Combining the two techniques, such as by using head-out plethysmography and placing a mask on the rat to simultaneously measure metabolic rate, would provide direct measurement of the ratio of minute ventilation to oxygen consumption or carbon dioxide production (V˙ I /V˙ O2 or V˙ I /V˙ CO2 ), which, in turn, would provide some insight into the influence of DNE on the complex interactions between body temperature, pulmonary ventilation and metabolic rate. However, we were concerned that the facemask, especially under thermal challenge would severely stress the animals and confound the results. ˙ 2 by combining V˙ I Accordingly, we estimated V˙ I / VO ˙ and VO 2 data obtained in different animals. As discussed ˙ 2 are very close to those below, our values for V˙ I and VO reported by others, increasing our confidence in these estimates. Measurement of Tcore . Tcore was measured before and after (but not during) heat stress. Monitoring Tcore throughout the recordings would have given temporal information about how changes in Tcore related to ventilation and metabolic rate. However, a surface temperature sensor would not accurately represent Tcore , and keeping a rectal probe in the rats during the recording would introduce undue stress. In future studies, implantable temperature sensors and a telemetry system could perhaps be used to obtain continuous measurements of Tcore . We note that the latter method, although powerful, does require surgical intervention in very small animals, which creates additional problems. Developmental influences. We combined data from 2-, 3and 4-day-old pups, raising the possibility that we missed developmental changes on the influence of DNE on the

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response to thermal stress. The narrow age range studied was chosen to gather information about the influence of DNE in the early neonatal period, and, as a result we do not know whether the influences of DNE persist or resolve as the pups develop. We conducted a three-factor ANOVA with treatment, temperature and age the main factors and, although some age effects were significant, the magnitude and direction of change were the same in both treatment groups, indicating that the effects were the natural result of growth and development, and not DNE. We did find a very small but significant difference in body weight between saline-exposed controls and DNE pups on P2, although not on P3 or P4. The influence of DNE on body weight in neonatal rats studied over the first week of life is equivocal. We found no influence of DNE body weight in our previous studies (Huang et al. 2004; Luo et al. 2004; Luo et al. 2007; Pilarski & Fregosi, 2009; Huang et al. 2010; Pilarski et al. 2011, 2012; Jaiswal et al. 2013; Jaiswal et al. 2015; Powell et al. 2015), which is consistent with some previous studies (Holloway et al. 2005; Mitchell et al. 2012), although not others (Bamford & Carroll, 1999; Xiao et al. 2007). We note that, in our earlier studies, we used fewer animals than employed in the present study, suggesting that the effects of DNE on body weight are small and difficult to detect. This is consistent with a study in 65 control rat pups and 31 nicotine-exposed rat pups studied on P3, showing a small but significantly lower weight in nicotine-exposed pups (8.4 vs. 7.6 g) (Bamford & Carroll, 1999). The influence of DNE on body weight probably also depends on the details of the study, including species used, nicotine dose, duration of exposure, route of nicotine delivery, etc. Nevertheless, to ensure that even these small differences in body weight did not influence the interpretation of the data, we expressed pulmonary ventilation rate and metabolic rate as a function of body weight. We also found a significant interaction between age and treatment for breathing frequency as a result of a slightly lower frequency in DNE pups at both 33 and 37.5°C on P2. Given that the responses in pups studied on P2, P3 or P4 were so similar, and do not impact ventilatory or metabolic responses to thermal stress, we decided to combine ages to increase statistical power and to increase the odds of detecting significant differences between control and DNE animals. DNE, heat stress and Tcore

Tcore increased significantly and similarly in both treatment groups when Tchamber was raised from 33 – 37.5°C, and the values that we recorded are consistent with published data (Mortola & Dotta, 1992; Merazzi & Mortola, 1999; Xia et al. 2008; Xia et al. 2009; Xia et al. 2010). Importantly, we confirm earlier work showing that Tcore closely tracks ambient temperature (Mortola & Dotta, 1992), suggesting that neonatal rodents are not able

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to thermoregulate efficiently. Tcore of the DNE pups used in the severe thermal stress experiments was slightly but significantly higher than in control pups at a Tchamber of 33°C, although this difference remains unexplained. DNE and the ventilatory and metabolic response to heat stress

The V˙ I , breathing frequency and VT values are in agreement with previous data reported both by ourselves and others (Saetta & Mortola, 1985; Bamford et al. 1996; Malcolm & Henderson-Smart, 1996; St-John & Leiter, 1999; Huang et al. 2004; Cummings et al. 2009; Huang et al. 2010; Niane et al. 2012), and so we are confident that any treatment or temperature effects are real. Nonetheless, DNE caused only subtle alterations in the ventilatory or metabolic response to moderate thermal stress. Although V˙ I was lower at a Tchamber of 33 and 37.5°C, there was no temperature effect or treatment–temperature interaction. A lower baseline V˙ I in nicotine-exposed rat pups has been observed previously (St-John & Leiter, 1999; Huang et al. 2004). Also consistent with previous work is the longer apnoea duration in DNE pups (Huang et al. 2004) but, as with the lower V˙ I , statistical analysis showed that this is a consequence of DNE that is independent of body temperature; the mechanisms underlying these DNE-mediated alterations in ventilation and apnoea duration remain unexplained. Metabolic rate did not differ significantly in control and DNE pups at a Tchamber of 33 and 37.5°C. However, V˙ O2 fell with heating in control pups but not in the DNE animals. Given that V˙ I did not change significantly with heating, the drop in V˙ O2 would be associated with mild hyperventilation, which may be driven by the need for heat dissipation rather than regulation of blood gases and pH (Mortola & Frappell, 2000). In addition, the drop in V˙ O2 would reduce heat production, which would also mitigate increases in Tcore during heat stress. The DNE pups did not make these homeostatic adjustments, suggesting that DNE causes subtle but potentially significant alterations in the metabolic and ventilatory response to thermal stress. Given the subtlety of the effects of DNE on the response to moderate heat stress, we examined the influence of DNE on the ventilatory response to severe hyperthermia (Tchamber = 43°C). Severe thermal stress was associated with a significant, frequency-mediated increase in V˙ I in control pups but not in the DNE pups. This is consistent with data obtained in other mammalian species, including piglets, kittens and mice, showing that hyperthermia results in an increase in breathing frequency, a decrease in VT and an increase in V˙ I (Galland et al. 1993; Ni et al. 1996; Parmeggiani et al. 1998). These data imply that DNE somehow alters the ventilatory response to severe, but not more moderate, heat stress. Moreover, although V˙ I remained depressed in both groups following C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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recovery from severe hyperthermia, this was significant only in the DNE pups, consistent with delayed restoration of the normal breathing rhythm following severe heat exposure. This observation is also consistent with data showing delayed recovery from combined hypoxia and hyperthermia in cigarette smoke-exposed, P7 neonatal rats (Pendlebury et al. 2008). In addition to studying the onset of hyperthermia and recovery, we also studied breathing during hyperthermic gasping. Under these conditions, the frequency and VT responses were the same in both treatment groups. The hyperthermia-induced gasping pattern that we observed is consistent with the gasping pattern evoked by hyperthermic hypoxia in neonatal rats (Pendlebury et al. 2008). Moreover, the lack of influence of DNE on gasping initiation, duration and pattern with severe hyperthermia is consistent with previous work showing that gasping evoked by hyperthermic hypoxia was not altered in smoke-exposed neonatal rats (Pendlebury et al. 2008). Taken together, these data support the idea that the gasping rhythm is fundamental, is largely invariant regardless of how it is evoked (St John, 1996) and is not influenced by DNE or prenatal exposure to cigarette smoke. Significance and conclusions

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Additional information Competing interests The authors declare that they have no competing interests. Author contributions JF collected and analysed all of the data and also wrote the initial draft of the manuscript. RF designed the experiments, prepared the graphs and figures, conducted the statistical analyses and wrote the final version of the manuscript. Both authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Funding This work was funded by the US Public Health Service, NIH R01 HD 071302 (to RF) and the University of Arizona Graduate Program in Physiological Sciences (to JF). Acknowledgements We wish to thank Seres Cross and Marina Cholanian, PhD, for surgically implanting the osmotic mini pumps into the pregnant dams. We also thank Dr M. Cholanian and Lila B. Wollman for reading an initial version of the manuscript.