Prenatal nicotine alters maturation of breathing and neural circuits regulating respiratory control

Respiratory Physiology & Neurobiology 162 (2008) 32–40

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Prenatal nicotine alters maturation of breathing and neural circuits regulating respiratory control ` a,b,c , David Perrin a,b,c , Julie Peyronnet a,b,c , Aurelien ´ Sophie Mahliere Boussouar a,b,c , d e a,b,c Guy Annat , Jean-Paul Viale , Jacqueline Pequignot , Jean-Marc Pequignot a,b,c , Yvette Dalmaz a,b,c,∗ a

Universit´e de Lyon, Lyon F-69003, France Universit´e Lyon 1, Lyon F-69003, France c CNRS, UMR 5123, Physiologie Int´egrative, Cellulaire et Mol´eculaire, Villeurbanne F69622, France d Universit´e Lyon 1, Facult´e de M´edecine Grange Blanche, Lyon F-69003, France e Hospices Civils de Lyon, D´epartement Anesth´esie R´eanimation, Hˆ opital Croix-Rousse, Lyon, France b

a r t i c l e

i n f o

Article history: Accepted 24 March 2008 Keywords: Prenatal nicotine Development Catecholamines Chemoreflex Ventilation Hypoxic ventilatory response

a b s t r a c t While perinatal nicotine effects on ventilation have been widely investigated, the prenatal impact of nicotine treatment during gestation on both breathing and neural circuits involved in respiratory control remains unknown. We examined the effects of nicotine, from embryonic day 5 (E5) to E20, on baseline ventilation, the two hypoxic ventilatory response components and in vivo tyrosine hydroxylase (TH) activity in carotid bodies and brainstem areas, assessed at postnatal day 7 (P7), P11 and P21. In pups prenatally exposed to nicotine, baseline ventilation and hypoxic ventilatory response were increased at P7 (+48%) and P11 (+46%), with increased tidal volume (p < 0.05). Hypoxia blunted frequency response at P7 and revealed unstable ventilation at P11. In carotid bodies, TH activity increased by 20% at P7 and decreased by 48% at P11 (p < 0.05). In most brainstem areas it was reduced by 20–33% until P11. Changes were resolved by P21. Prenatal nicotine led to postnatal ventilatory sequelae, partly resulting from impaired maturation of peripheral chemoreceptors and brainstem integrative sites. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Maternal smoking during pregnancy induces a high incidence of ventilatory abnormalities in infants (apnea, delayed arousal responses, altered hypoxic ventilatory drive, or bronchopulmonary disease) with increased risk of Sudden Infant Death Syndrome (Mitchell and Milerad, 2006). Although the effects of prenatal nicotine on ventilation have been extensively studied in animals (Huang et al., 2004; Bamford et al., 1996; Bamford and Carroll, 1999; Simakajornboon et al., 2004), results remain conflicting, due to divergent experimental designs (species, nicotine administration route, time of pregnancy, duration and dose of nicotine administration, postnatal age) and types of ventilatory analysis (baseline data, apnea incidence, ventilatory response to hypoxia or hyperoxia). Most studies assessed changes in overall minute ventilation rather than in the two components: respiratory frequency and tidal

∗ Corresponding author at: UMR CNRS 5123, Physiologie Integrative, ´ Cellulaire et ´ Moleculaire, Universite´ Lyon 1, 43 Boulevard 11 Novembre 1918, 69622 Villeurbanne Cedex, France. Tel.: +33 4 72 43 11 71; fax: +33 4 72 43 11 72. E-mail address: [email protected] (Y. Dalmaz). 1569-9048/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2008.03.009

volume. In addition, the cellular mechanisms underlying the control of ventilation after prenatal nicotine exposure remain partially elucidated. The neural circuits regulating breathing belong to the chemoafferent pathway (Bianchi et al., 1995). In rats, the afferent chemosensory fibers arising from the carotid bodies project onto discrete areas of the medulla oblongata: mainly the caudal part of the nucleus tractus solitarius and, to a lesser extent, the ventrolateral medulla (Finley and Katz, 1992). The nucleus tractus solitarius and the ventrolateral medulla contain clusters of noradrenergic and adrenergic neurons: A2C2 and A1C1, respectively. The A2C2 cell group displays a functional subdivision: the caudal part (A2C2c) is influenced by peripheral chemosensory input and the rostral part (A2C2r) by barosensory input (Soulier et al., 1992). The A2 and A1 cell groups are involved in respiration through their connections to the adjacent dorsal and ventral respiratory groups. In addition, the A5 cell group, located in the ventrolateral pons and projecting onto the medulla oblongata and spinal cord, controls sympathetic output and respiratory events (Guyenet et al., 1993). The locus coeruleus (A6), the major noradrenergic cell group in the brain, belonging to the pontine tegmentum, with extensive descending projections onto the medulla oblongata and spinal cord, is involved in arousal

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and cardiorespiratory regulation and is part of the chemoreflex pathway (Guyenet et al., 1993; Hilaire et al., 2004). Catecholamines are the main neurotransmitters involved in chemoafferent pathway control. Firstly, dopamine is the most abundant neurotransmitter at the chemosensory synapse of the carotid body (Finley et al., 1992). Secondly, the central integration sites located in the nucleus tractus solitarius and the ventrolateral medulla are respectively associated with the A2C2 and the A1C1 catecholaminergic clusters (Finley and Katz, 1992). Thirdly, there is growing evidence that brainstem catecholaminergic neurons participate in the control of breathing. Mice invalidated for genes such as BDNF, mash, ret or rnx exhibited catecholaminergic impairment of carotid body, petrosal ganglion or brainstem noradrenergic system, systematically accompanied by ventilatory deficiency (Hilaire, 2006). Maturation of these neural circuits develops within the first postnatal weeks and is dependent on the perinatal environment (White et al., 1994; Gauda et al., 2004; Donnelly, 2005; Wong-Riley and Liu, 2005). Prenatal nicotine reduces the brainstem noradrenaline level and/or utilization rate until weaning (Navarro et al., 1988), and induces cell damage, cell loss and synaptic dysfunction in the developing brain (Slotkin, 1998, 2004; Slotkin et al., 2007). It also enhances the expression of protein kinase C which plays an important role in both excitatory and inhibitory respiratory neurons (Bandla et al., 1999), thus possibly contributing to altered ventilation (Simakajornboon et al., 2004). In rat carotid bodies, perinatal exposure to nicotine upregulates tyrosine hydroxylase (TH) mRNA (Holgert et al., 1995; Gauda et al., 2001) and depresses breathing by attenuating the carotid body drive (Holgert et al., 1995). Catecholamines are major components regulating ventilation at the peripheral and central levels (Finley and Katz, 1992; Bianchi et al., 1995), yet no studies have concomitantly investigated the effects of prenatal nicotine exposure on the development of breathing and catecholamine metabolism within the chemoafferent pathway. We therefore tested the hypothesis that prenatal nicotine exposure might cause abnormal breathing pattern development and that the impaired respiratory function might result in part from impaired development of the neural network regulating breathing. In the present study, nicotine was delivered in pellet form throughout gestation from embryonic day 5 (E5) until birth; functional sequelae on resting ventilation and ventilatory response to acute hypoxic challenge were analyzed at postnatal days 7 (P7), 11 (P11) and 21 (P21). To determine whether nicotine-induced developmental abnormalities in ventilation were associated with changes in the postnatal maturation of ventilatory control, in vivo TH activity was analyzed at P7, P11 and P21 in the peripheral and central catecholaminergic structures of the chemoafferent pathway: i.e., carotid bodies, A2C2 caudal (A2C2c) and rostral (A2C2r) part, A1C1, A5 and A6 brainstem cell groups. 2. Methods 2.1. Animals Male and female Sprague–Dawley rats (IFFA Credo, France) were mated at night, and the morning on which sperm-positive smears were obtained was defined as embryonic day 0 (E0). Pregnant rats (300–320 g) were then housed in an air-conditioned room at 26 ± 1 ◦ C with a 12-h light–dark cycle and free access to food and water. They were randomly assigned to two groups: nicotine and control. The nicotine group received the following treatment: pregnant dams were operated on at E5, so as not to disturb implantation of the embryo in the uterine wall. Dams were anesthetized with a single intraperitoneal injection of avertin (1 g tribromethanol dis-

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solved in a mixture of 0.5 ml pentanol, 6 ml ethanol and 67.5 ml saline serum) (1 ml 100 g−1 body weight). Nicotine pellets (Interchim, Montluc¸on, France) were implanted subcutaneously between the scapulae. The nicotine group (n = 45) received a pellet delivering 50 mg free-base nicotine over 21 days, corresponding to a dose rate of 6 mg kg−1 day−1 . These dams gave birth to the group of “prenatal nicotine” pups. The control group (n = 30), operated on at E5, received a placebo pellet (Interchim, Montluc¸on, France) delivering 50 mg vehicle for 21 days. These dams gave birth to the group of control pups. At birth, prenatal nicotine pups were redistributed to nursing females never exposed to nicotine, to avoid any possible effects of nicotine and cotinine in the milk. Pups were randomized among all litters within each treatment group and litter sizes were randomly culled to 10 pups (8 males, 2 females) to ensure standard nutrition. Pups were housed with their mother and siblings until study. All animals were housed at 26 ± 1 ◦ C with a 12-h light–dark cycle. Only male rats were analyzed, to avoid gender differences in ventilation (Joseph et al., 2000). Experiments were carried out according to the ethical principles ` de l’Agriculture) and EU Council laid down by the French (Ministere Directives for the care and use of laboratory animals (No. 02889). 2.2. Maternal plasma nicotine and cotinine measurement To determine plasma nicotine and cotinine levels, 18 pregnant rats from the nicotine group were anesthetized (pentobarbital 0.15 ml 100 g−1 body weight intraperitoneally) at embryonic days E5 (n = 3), E8 (n = 3), E13 (n = 3), E16 (n = 3) and E21 (n = 3) and on postnatal day P5 (n = 3). Samples were taken by cardiac puncture. Blood was taken into a heparinized tube, light-protected and centrifuged. Plasma was frozen and stored at −20 ◦ C until assayed. Nicotine and cotinine levels were measured by HPLC-UV as previously described (Hariharan et al., 1988). The detection threshold was set at 0.012 nmol/ml for plasma nicotine and cotinine. 2.3. Ventilation measurement Ventilation was measured in awake unrestrained male pups at P7, P11 and P21 using a barometric plethysmograph (Bartlett and Tenney, 1970). According to the protocol described previously (Peyronnet et al., 2000), the volume of the Plexiglas chambers was 0.22 l for P7 pups and 0.75 l for P11 and P21 pups. The Plexiglas plethysmographic chamber was connected to a reference chamber of the same size which was flushed with heated humidified air and both chambers were saturated with water vapor. Temperature and O2 and CO2 levels were continuously monitored. The system was calibrated by injecting a constant known volume of air with a syringe before placing an animal in the box, to avoid signal interference between calibration and respiration. Alternately, one control and one prenatal nicotine pups were weighed and placed in the chamber. Measurement began when the animal was calm. The gas flow was interrupted and the inlet and outlet tubes were closed. Breathing pressure fluctuations were recorded for 30–40 cycles with a differential pressure transducer (Celesco, CA, USA). The inspired CO2 fraction was typically <0.5% at the end of the gas exposure period. Values for respiratory frequency (fR , min−1 ), tidal volume (VT , ml 100 g−1 ) and minute ventilation (VE , ml min−1 100 g−1 ) were calculated breath-to-breath by computer analysis of the spirogram using standard methods. To assess the hypoxic ventilatory response (HVR), measurements were performed under baseline conditions (normoxia) and during hypoxic challenge (10% O2 for 10 min). Baseline measurements were made in quadruplets separated by 10 min intervals. The mean value was defined as the baseline

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(normoxic) ventilation. The HVR was determined using hypoxic challenge carried out by flushing the chamber with a mixture of 10% O2 + 90% N2 . Washout of the chamber required about 40 s to reach a new stable inspired O2 fraction. This was considered to be the beginning of the hypoxic challenge and rats were continuously exposed to hypoxia for 10 min. Once the 10% O2 level was reached inside the chamber, the system was clamped and the first measurement was made after 1 min of exposure. Values over 40–60 breath cycles were averaged. Analysis at 1 min of hypoxia assessed the involvement of the carotid body in the hypoxic ventilatory response. Subsequent recordings were made in the same way every 3 min at 4, 7 and 10 min after the beginning of hypoxic exposure. These measurements assessed the kinetics of the hypoxic ventilatory response and the involvement of both peripheral and central components (Powell et al., 1998). To eliminate circadian oscillations, measurements were performed between 9 a.m. and 2 p.m. To evaluate tidal volume under BPTS conditions (body pressure and temperature when saturated with water vapor) as reported by Bartlett and Tenney (1970), the colonic temperature of each pup was measured during hypoxic challenge. To collect reliable VT data, all recordings were carried out at the thermoneutrality temperature of 32 ± 1 ◦ C (7-day-olds) or 30 ± 1 ◦ C (11- and 21-day-olds). To eliminate small variations in ambient temperature leading to fluctuations in VT values, especially critical in small rodents (Mortola and Frappel, 1998), the gas (air or hypoxia mix) was heated or cooled as necessary before entry in the plethysmograph. Other groups of animals, different from those used for ventilation measurement, were used to record body temperature under baseline conditions (normoxia) and during the 10 min of hypoxic challenge. Each age group was composed of five prenatal nicotine and five control pups. Rectal temperature was monitored by a fine tungsten-constantan thermocouple (Chessel 4001). Rectal temperature was recorded at 1, 4, 7 and 10 min of hypoxia. These measurements of body temperature during hypoxic challenge were important for assessing the contribution of metabolic components to the hypoxic ventilatory response. 2.4. Tissue dissection Neurochemical assays and ventilatory measurements were performed on separate animals in order to eliminate the putative neurochemical changes resulting from the brief hypoxic challenge

used for HVR measurement. Control and prenatal nicotine pups were sacrificed by cervical dislocation after deep anesthesia with i.p. pentobarbital. The carotid bodies were dissected out under a dissection microscope, placed in 100 ␮l 0.1 M perchloric acid containing 2.7 mM EDTA–Na2 and stored at −80 ◦ C until assay. The brainstem was stored at −80 ◦ C at P7 and was cut into serial coronal slices at P11 and P21. Slice thickness was proportional to brain size and was adapted to age (288 ␮m at P11, 384 ␮m at P21). Visualization of the VII cranial nerve, the pyramidal decussation, the cerebral aqueduct and the III and IV ventricles was used as anatomical reference to select the relative positions of the various areas. The catecholaminergic cell groups A2C2, A1C1, A5 and A6 were then punched out bilaterally with a needle (0.9 mm inner diameter) according to the dissection procedure described by Palkovits and Brownstein (1988) and adapted to young animals (Roux et al., 2003). As previously described (Soulier et al., 1992; Peyronnet et al., 2000), the microtome blade was adjusted to the plane containing the point where pyramidal decussation appears. This slice is referred to as the zero plane. Serial slices were cut as shown in Fig. 1. The A2C2 cell group was punched out from 6 slices in the dorsal part and was divided into two subsets, caudal (A2C2c) and rostral (A2C2r) to the calamus scriptorius, in order to distinguish the area receiving respectively chemosensory input and barosensory fibers (Finley and Katz, 1992). The A1C1 cell group was punched out from five slices in the ventral part (Fig. 1). After adjustment of the microtome blade to the plane containing the point where the VII cranial nerve appears, the A6 cell group was punched out from four slices in the dorsal part and the A5 cell group from three slices in the ventral part (Fig. 1). The punches were placed in 100 ␮l 0.1 M perchloric acid containing 2.7 mM EDTA–Na2 and were stored at −80 ◦ C until assay. 2.5. Neurochemistry 2.5.1. Measurement of in vivo tyrosine hydroxylase (TH) activity In vivo TH activity was measured by accumulation of l-dihydroxyphenylalanine (l-DOPA) following inhibition of DOPA-decarboxylase with NSD 1015 (3-hydroxybenzylhydrazine dihydrochloride, Sigma) injected intraperitoneally (100 mg kg−1 ) 20 min before sacrifice. l-DOPA accumulation is considered to be a reliable index of in vivo TH activity and is used as a marker of the rate of catecholamine synthesis (Carlsson et al., 1972; Lachuer

Fig. 1. Schematic diagram (A) represents the consecutive coronal slices used for punches. Each number represents consecutive coronal slices. Each slice is numbered according to its position relative to that containing the pyramidal decussation (referred to as 0). The caudal and the rostral parts of the A2 cell group were punched out from 3 slices (respectively −1 to 1 and 2 to 4). The A1 cell group was punched out from five slices (0 to 4), the A5 cell group from three slices (8–10) and the A6 cell group from four slices (8–11) (Peyronnet et al., 2000). Coronal (B and C) and sagittal planes show the locations of catecholamine-containing neurons in the brainstem (Haxhiu et al., 2001). NTS, Nucleus Tractus Solitarius; A1C1, A2C2, A5 and A6, catecholaminergic cell groups.

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et al., 1992). TH activity was expressed in picomoles of l-DOPA formed per 20 min per structure. 2.5.2. DOPA assay The structures were assayed by high-performance liquid chromatography coupled with electrochemical detection (Peyronnet et al., 2000). Carotid bodies and brain punches were disrupted by ultrasound. The homogenates were centrifuged (8800 × g, 5 min) and 10 ␮l aliquot was injected into a reverse-phase column (Spherisorb ODS 2.5 ␮m, 125 mm × 4 mm, Macherey-Nagel, Hoerdt, France) with a mobile phase of 50 mM citric acid, 50 mM sodium acetate, 1 mM EDTA–Na2 , 437 mM acetic acid, 4.3 mM octane sulfonate and 5% methanol. The flow rate was 0.9 ml min−1 . l-DOPA was measured at +0.67 V versus the Ag+ /AgCl reference electrode (Eldec 102 Chromatofield, Chˆateauneuf-Les-Martigues, France). The detection limits, calculated by doubling the background noise level and expressed in terms of picomoles of injected amounts, were less than 0.03 pmol for all compounds and the intraassay coefficient was 0.02%. 2.6. Expression of results and statistical analysis Brain punches were dissected in excess, which ensured that all catecholaminergic cells were harvested from the selected brain region. Significant daily variations in brain protein levels have already been reported. It is therefore more appropriate to express in vivo TH activity per structure than per mg of protein (Poncet et al., 1994). Data are expressed as mean ± S.E.M. Because the studies were multivariate (prenatal treatment and age for neurochemistry, and prenatal treatment, age and time of hypoxic challenge for ventilation), data were first subjected to global ANOVA. Where a significant effect of treatment or interaction of treatment with age or hypoxic challenge was detected, lower order ANOVA was then carried out to determine the effect or interaction of time of hypoxic challenge and age. When interactions were significant, a post hoc test (Fisher protected least square difference) was used to compare individual groups. A p value <0.05 was considered statistically significant. 3. Results 3.1. Maternal plasma nicotine and cotinine concentrations The release of nicotine from the pellet was monitored by measuring plasma nicotine and cotinine levels over the 3-week time course. Following an initial peak on the day of pellet implantation (E5), plasma concentrations of nicotine and cotinine stabilized (Fig. 2). 3.2. Viability and growth Pregnant rats were exposed to nicotine 5 days after impregnation to avoid disturbing embryo implantation. Nevertheless, 7% of the nicotine-treated dams resorbed their fetuses. Administration of prenatal nicotine significantly reduced maternal weight

Fig. 2. Maternal plasma nicotine () and cotinine (䊉) concentrations from embryonic day 5 (E5) to the postnatal day 5 (P5) after nicotine pellet implantation in pregnant rats. Pellet implantation was performed at E5. Values are expressed as mean ± S.E.M. All concentrations were above the detection threshold (0.012 nmol/ml).

gain (+25 ± 11.7% in the nicotine group vs. +63 ± 8% in the control group, at end of gestation) and litter size (11.88 ± 1.29 pups/litter in the nicotine group versus 14.4 ± 0.5 in the controls group). 5% early postnatal deaths were observed among prenatal nicotine pups whereas all the control pups survived. Prenatal exposure to nicotine significantly reduced pup body weight within the first week and enhanced it at P21 (Table 1). 3.3. Ventilatory measurement The body temperature under baseline conditions and following the 10 min of hypoxic challenge was similar between controls and prenatal nicotine pups at all ages and in all conditions (Table 2). 3.3.1. Baseline ventilatory data Baseline ventilatory components (minute ventilation normalized to body weight, respiratory frequency, and tidal volume) are shown in Fig. 3. Compared with the age-matched control group, the nicotine group exhibited a significant increase in baseline minute ventilation at P7 (+48%) and P11 (+42%) (p < 0.05). This increase was due to a higher tidal volume, with no change in frequency. By P21, minute ventilation did not differ between the control and prenatal nicotine groups. 3.3.2. Hypoxic ventilatory response 3.3.2.1. Hypoxic ventilatory response of control and prenatal nicotine rats. After evaluation of ventilation in resting conditions (baseline data), control and prenatal nicotine pups were exposed to moderate hypoxia (10% O2 , 1, 4, 7, and 10 min) (Fig. 4). In control animals at each postnatal age, hypoxic exposure induced increased minute ventilation by the first minute, sustained throughout the 10 min of hypoxic exposure. While this increase in ventilation was secondary to an increase in respiratory frequency rather than in tidal volume at P7 and P21, increases in both respiratory frequency and tidal volume were both observed at P11. At P7, P11 and P21, prenatal nicotine pups responded to hypoxic challenge by increasing their overall minute ventilation; at P7 and P21, this involved an increase in respiratory frequency, whereas at P11 it was secondary to an increase in both respiratory frequency and tidal volume. In addition, at P11 minute ventilation

Table 1 Effect of prenatal nicotine exposure (50 mg free-base nicotine over 21 days) on the body weight (g) of rat pups Age (days)

Control Prenatal nicotine

P3

P7

P11

P21

9.82 ± 0.21 (n = 60) 9.11 ± 0.19* (n = 63)

14.92 ± 0.26 (n = 50) 16.51 ± 0.15 (n = 50)

23.02 ± 0.34 (n = 40) 23.63 ± 0.42 (n = 40)

44.51 ± 0.75 (n = 25) 50.30 ± 0.54* (n = 28)

Data are expressed as mean ± S.E.M. n = number of animals. Postnatal days 3 (P3), 7 (P7), 11 (P11) and 21 (P21). * p < 0.05 represents significant differences between the control group and the prenatal nicotine group.

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Table 2 Effects of hypoxia on the body temperature of control and prenatal nicotine pups on postnatal day 7 (P7), 11 (P11) and 21 (P21) P7

Basal Hypoxia (10 min, 10% O2 )

P11

P21

Control

Prenatal nicotine

Control

Prenatal nicotine

Control

Prenatal nicotine

36.63 ± 0.31 (n = 5) 36.56 ± 0.27 (n = 5)

36.32 ± 0.44 (n = 5) 36.31 ± 0.56 (n = 5)

35.81 ± 0.37 (n = 5) 35.68 ± 0.42 (n = 5)

36.86 ± 0.43 (n = 5) 36.63 ± 0.38 (n = 5)

36.78 ± 0.18 (n = 5) 36.86 ± 0.22 (n = 5)

36.76 ± 0.21 (n = 5) 36.04 ± 0.92 (n = 5)

Data are expressed as mean ± S.E.M. in control (n = 5 per age-group) and prenatal nicotine-exposed rats (n = 5 per age-group).

Fig. 3. Effect of prenatal nicotine exposure on ventilatory components in baseline conditions on postnatal day 7 (P7), 11 (P11) and 21 (P21). Data are expressed as mean ± S.E.M. VE : minute ventilation, VT : tidal volume, fR : respiratory frequency. *Significant difference between control and prenatal nicotine groups (p < 0.05).

was reduced after 4 min of hypoxic challenge compared to the first minute (p < 0.05), indicating an unstable hypoxic ventilatory response (Fig. 4). 3.3.2.2. Comparison of the hypoxic ventilatory response of the prenatal nicotine and control pups. Compared with age-matched controls, prenatal nicotine pups showed impaired hypoxic ventilatory response; this impairment was age- and component-dependent

(Fig. 4). At P7, minute ventilation was increased, secondary to increased tidal volume whereas respiratory frequency was significantly lowered (p < 0.05). At P11, the hypoxic ventilatory response was increased, associated with increased tidal volume but with no difference in respiratory frequency (Fig. 4). At P21, the hypoxic ventilatory response showed similar minute ventilation in both groups, but tidal volume was increased in the prenatal nicotine pups. Prenatal nicotine induced increased tidal volume in response to hypoxia

Fig. 4. Effect of prenatal nicotine on the ventilatory response to hypoxia of 7-, 11- and 21-day-old rat pups. Tests were performed with whole-body plethysmography. 0 min represents values obtained under normoxia. A mixture containing 10% O2 was used for the hypoxic challenge. Data are expressed as mean ± S.E.M. in control () and prenatal nicotine-exposed rats (䊉). VE : minute ventilation, VT : tidal volume, fR : respiratory frequency. *A significant difference between control and prenatal nicotine-exposed rats (p < 0.05). § A significant difference between 0 and 1 minute of hypoxia (p < 0.05). ‡ A significant difference between 1 and 4 min of hypoxia (p < 0.05).

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Fig. 5. Effect of prenatal nicotine exposure on in vivo tyrosine hydroxylase activity in carotid bodies at postnatal days 3, 7, 11 and 21 (P3, P7, P11, P21). Values are expressed as mean ± S.E.M. *Significant differences between the control and the prenatal nicotine-exposed rat pups (p < 0.05). § A significant difference from values obtained at P3 (p < 0.05).

at P7, P11 and P21 but blunted the respiratory frequency at P7. In consequence, the relative contribution of respiratory frequency to the hypoxic ventilatory response was reduced at P7.

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in central respiratory control mechanisms have been proposed (Slotkin et al., 1995, 1997), no study previously examined developmental change in the catecholaminergic neural circuits controlling respiration. The originality and relevance of the present study lie in the in vivo assessment of the chemoafferent pathway and the longitudinal evaluation of both respiratory behavioral and neurochemical events at postnatal days P7, P11 and P21, specifically in male pups, allowing a causal relationship to be established between the ventilatory and biochemical findings. The main findings were that prenatal nicotine exposure impaired postnatal development of breathing and of the neural catecholaminergic networks belonging to the chemoafferent pathway. Prenatal nicotine: (i) increased baseline minute ventilation and hypoxic ventilatory response until P11, by an increase in tidal volume; (ii) blunted hypoxic respiratory frequency response at P7; and (iii) altered the catecholaminergic pattern of the developing carotid bodies and brainstem areas. The changes were marked during the first postnatal week, attenuated during the second and resolved by the third. 4.1. Methodological consideration

3.4. Neurochemical data In vivo TH activity was measured in the carotid bodies and in the central structures of the chemoafferent pathway. 3.4.1. In vivo TH activity in carotid bodies In the carotid bodies of control pups, in vivo TH activity decreased during the first week of postnatal life (0.73 ± 0.07 pmol/20 min at P3, 0.49 ± 0.06 pmol/20 min at P7) and remained within the same range thereafter (Fig. 5). In the carotid bodies of prenatal nicotine pups, such a fall was not observed at P7 (0.66 ± 0.06 pmol/20 min, compared to 0.79 ± 0.09 pmol/20 min at P3), but was detected later, at P11 (0.28 ± 0.05 pmol/20 min). This maturation pattern gave an in vivo carotid body TH activity that was 20% higher in prenatal nicotine pups than in age-matched controls at P7 and 48% lower at P11. 3.4.2. In vivo TH activity in central brainstem catecholaminergic areas In vivo TH activity in central areas is reported in Table 3. In the whole brainstem of prenatal nicotine pups, in vivo TH activity was lower (−20%) than in age-matched controls at P7. This decrease was further observed at P11 in the A1C1 (−24%), A5 (−33%) and A6 (−31%) cell groups (Table 3). No difference in catecholaminergic cell group TH activity was detected between the prenatal and control groups at P21. 4. Discussion Although the ventilatory effects of prenatal nicotine exposure have been widely investigated and nicotine-induced changes

It is the first study to use nicotine administered from a pellet, delivering 50 mg free-base nicotine for 21 days (i.e., 6 mg kg−1 day−1 ). This dose is that delivered by the osmotic minipump used by most authors. The plasma levels of nicotine and cotinine delivered from the pellets were high on the first day following implantation and then stabilized, as previously reported with the osmotic minipump (Sanderson et al., 1993). Pellet-delivered nicotine led to plasma nicotine and cotinine levels similar to those detected in heavy smokers (Mercelina-Roumans et al., 1996). The kinetics of nicotine administration (21 days from E5) does not rule out nicotine exposure through breast milk until postnatal day 4. In order to eliminate this interference and focus on prenatal nicotine effects, nicotine-free lactating dams were used as nurses from birth. Nicotine treatment during pregnancy induces vasoconstriction by reducing blood flow to the fetus, decreasing its oxygen supply. Although an influence of nicotine-induced fetal hypoxia cannot be totally excluded, it seemed to be of minor importance in our protocol: a major feature of prenatal hypoxia is reduced pup body weight from birth to P7 (Mamet et al., 2002), not found in the present data. The respiratory control system undergoes significant postnatal maturational change, especially within the first 2 postnatal weeks (Carroll, 2003; Donnelly, 2005; Wong-Riley and Liu, 2005; Liu et al., 2006); our various postnatal time-points were therefore chosen in order to focus on the immature stage of the chemoafferent pathway, the critical maturational period of brainstem nuclei, and the period of metabolic change around weaning. Our ventilatory study evaluated minute ventilation at rest and during acute hypoxic challenge and measured the two components of minute ventilation: respiratory frequency and tidal volume. The accuracy of tidal volume calculation based on whole-body

Table 3 In vivo tyrosine hydroxylase activity in central areas of the chemoreflex pathway in control and prenatal nicotine pups at 7, 11 and 21 days of postnatal life P7

Brainstem A2C2c A2C2r A1C1 A5 A6

P11

Control

Prenatal nicotine

398 ± 28 (n = 10)

305 ± 30* (n = 10)

P21

Control 4.62 3.23 5.90 3.72 17.52

± ± ± ± ±

Prenatal nicotine 0.51 (n = 15) 0.33 (n = 15) 0.51 (n = 15) 0.39 (n = 15) 1.31 (n = 15)

3.83 3.31 4.51 2.48 12.04

± ± ± ± ±

0.42 (n = 12) 0.40 (n = 12) 0.39* (n = 12) 0.19* (n = 12) 1.43* (n = 12)

Control 8.14 10.04 10.29 7.21 12.51

± ± ± ± ±

Prenatal nicotine 0.63 (n = 12) 0.81 (n = 12) 0.78 (n = 12) 0.81 (n = 12) 1.04 (n = 12)

Data are expressed as picomoles of DOPA per 20 min per pair of cell groups or per brainstem and represent mean ± S.E.M. n = 10 for each group. * p < 0.05 vs. control.

6.91 9.05 9.48 7.41 12.48

± ± ± ± ±

0.62 (n = 11) 0.91 (n = 11) 0.79 (n = 11) 0.70 (n = 11) 1.29 (n = 11)

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plethysmography data has been questioned, especially in newborns (Mortola and Frappel, 1998). The absolute VT and VE values should be viewed with caution, but could be used appropriately to compare control and prenatal nicotine data. The 10 min duration of the hypoxic test induced carotid-body-mediated hypoxic ventilation, which develops promptly, avoiding the side-effects of longer hypoxic exposure, such as anapyrexia. The ventilatory response to hypoxia is a balance of excitatory and inhibitory input and a complex interplay between several distinct mechanisms, involving generation of sensory input in the peripheral chemoreceptors and their central integration. Commonly, the initial phase (during the first minute) of the response is attributed to chemoreceptor stimulation, reflecting peripheral arterial chemosensitivity, and the second phase to central inhibitory and peripheral excitatory interplay (Powell et al., 1998). Neurochemical and ventilatory measurements were performed on distinct animals, so as to eliminate the putative metabolic changes induced by the brief hypoxic challenge. Catecholaminergic area development was assessed by TH, the rate-limiting enzyme in catecholamine synthesis, rather than endogenous monoamines. Endogenous catecholamine levels result from various processes (synthesis, release, uptake, metabolism and turnover); absence of change in catecholamine levels thus does not preclude change in metabolic events (Muneoka et al., 1997). In contrast, change in release and/or metabolism results in parallel change in TH activity through feed-back regulation. Therefore, TH activity is the major index to assess the dynamics of catecholaminergic neurons (Lachuer et al., 1992).

4.2. Prenatal nicotine-enhanced baseline ventilation and impaired postnatal hypoxic ventilatory response maturation The effect of prenatal nicotine on respiratory physiology remains controversial, due to divergent experimental protocols (time and dose of nicotine administration, postnatal age studied, and duration of the nicotine challenge), and it is difficult to highlight a clear effect or draw a general conclusion. Prenatal nicotine exposure induced a postnatal decrease in baseline minute ventilation and no attenuation of the hypoxic ventilatory response (Huang et al., 2004; Bamford et al., 1996; Bamford and Carroll, 1999; Simakajornboon et al., 2004). In the present study, prenatal nicotine enhanced baseline ventilation and impaired postnatal hypoxic ventilatory response development and the involvement of both components of ventilatory output (respiratory frequency vs. tidal volume). The relative contribution of respiratory frequency to the hypoxic ventilatory response was reduced at P7, suggesting differential maturation of the mechanisms controlling fR and VT . At P11, prenatal exposure to nicotine induced instability in the hypoxic ventilatory response. Although we did not perform the Dejours test to evaluate the contribution of chemoreceptors to baseline ventilation, previous data suggest that increased baseline peripheral arterial chemoreceptor levels may be operative in destabilizing breathing in acute challenge (Al Matary et al., 2004; Gauda et al., 2007). WongRiley and Liu (2005) reported that the transient dominance of inhibitory over excitatory neurotransmission by this postnatal age may render the respiratory system sensitive to failure under stress. Although the ventilatory disturbances were resolved at weaning, subtle impairments might persist which could be revealed under other stress conditions such as immobilization (Peyronnet et al., 2002). In addition to ventilatory response, an important component of the response to hypoxia in small animals is a reduction in metabolic rate to maintain oxygen availability (Mortola, 2003). Changes in metabolism and ventilation during hypoxia in newborns mini-

mize the fall in arterial PO2 by decreasing energy utilization and temperature homeostasis. Previous data using prenatal nicotine (6 mg kg−1 day−1 ) from E2 to P7 reported no effect of nicotine on oxygen consumption under normoxia or hypoxia (Bamford et al., 1996). The present study found no change in the body temperature of control or nicotine pups under hypoxia at any age. Therefore, the enhanced hypoxic ventilatory response of the prenatal nicotine pups observed at P7 and P11 did not result from changes in metabolism.

4.3. Prenatal nicotine impaired the postnatal maturation of carotid bodies and brainstem areas: interrelationship with altered breathing pattern Peripheral chemoreceptors are essential to the development of respiratory control mechanisms (Donnelly and Haddad, 1991; Gonzalez et al., 1994). The present results showed that, during the first 2 postnatal weeks, prenatal nicotine altered the development of peripheral chemoreceptors and of brainstem integrative sites, thus contributing to ventilation impairment. In carotid bodies, prenatal nicotine countered the drop in TH activity within the first week, delaying it to P11. Thus, prenatal nicotine altered the well-known postnatal catecholaminergic pattern of the developing carotid body (Hertzberg et al., 1990). The rise in oxygen supply at birth leads the carotid body to change its sensitivity setting, partly by adjusting its dopamine metabolism (Donnelly and Doyle, 1994; Bairam and Carroll, 2005). Decreased postnatal dopamine turnover and THmRNA in carotid bodies have been suggested as factors contributing to increased carotid body sensitivity to hypoxia and carotid sinus nerve activity resulting in selective stimulation of respiratory frequency (Hertzberg et al., 1990; Holgert et al., 1995; Bairam et al., 2006; Gauda et al., 1996). In contrast, high levels of dopamine and THmRNA in the immature carotid body of newborns blunts the ventilatory chemoreflex response (Hertzberg et al., 1992; Holgert et al., 1995; Bairam and Carroll, 2005). Thus, the high level of in vivo TH activity observed at P7 in the carotid body of pups prenatally exposed to nicotine may contribute to the blunted hypoxic respiratory frequency response. At P11, the low level of TH activity may contribute to the normalization of hypoxic respiratory frequency response. In addition to catecholamines, acetylcholine (ACh) modulates breathing (Fitzgerald, 2000), by activating highly selective nicotinic acetylcholine receptors (nAChRs) which are present in the carotid bodies (Hirasawa et al., 2003) and brainstem nuclei (nucleus tractus solitarius and A6). In carotid bodies of the rat (Jackson and Nurse, 1998) and cat (Fitzgerald et al., 2003) in vitro, specific nicotinic receptors antagonists reduces the hypoxia-induced release of dopamine which indicates a cholinergic-catecholaminergic interrelationship via the nicotinic receptors and a key involvement of ACh and its receptors in the regulation of dopamine metabolism in carotid bodies. There have, however, been no observations of the functional properties of cholinergic receptors in the carotid body after chronic exposure to nicotine in vivo. The expression of nicotinic ␣3 , ␣4 , ␣7 and ␤2 receptors in carotid bodies is age-dependent (Bairam et al., 2007) and although the role of cholinergic system has been suggested in the maturation of carotid body function during the first weeks following birth (Bairam et al., 2001), the relationship between the age-related expression pattern of AChRs and in vivo function of nAChRs in developing rats remain unknown. Moreover, the effects of prenatal nicotine on the development of cholinergic receptors in carotid bodies remain unknown. The cholinergic component of the carotid body is dependent on development and environment (hypoxia, hyperoxia) (Shirahata et al., 2007). Therefore, we might suggest

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that prenatal nicotine acts on both the catecholaminergic and cholinergic components of developing rat carotid bodies, and by modifying the developmental balance of the two systems in the carotid bodies, may contribute to the altered postnatal ventilation. In the brainstem, prenatal nicotine exposure was found to alter the maturation of central areas of the chemoreflex pathway. The reduced noradrenergic activity in most of the brainstem areas until P11 may participate in the central control of ventilatory disturbance induced by prenatal nicotine. Among numerous factors regulating breathing (Bianchi et al., 1995; Simakajornboon and Kuptanon, 2005), noradrenaline in brainstem cell groups, by depressing the ventilatory output, is considered to be an essential modulator of ventilation and of the ventilatory response elicited by peripheral chemoreception (Hilaire et al., 2004; Hilaire, 2006). In absence of peripheral input, hypoxic ventilatory response recovery is characterized by a selective increase in tidal volume reflecting reorganization of respiratory centers (Roux et al., 2000). Therefore, the higher VT hypoxic response, especially at P7, in pups prenatally exposed to nicotine reflects the compensatory involvement of noradrenergic brainstem areas subsequent to the described immaturity of carotid body. In the brainstem, cholinergic receptors regulate inspiratory and pre-inspiratory neuronal function at the level of the rostral ventrolateral medulla and C4 neurons (Hatori et al., 2006) and prenatal nicotine exposure reduced the burst frequency of the fourth cervical ventral root (Luo et al., 2004). However, little is known about the development of the cholinergic system in brainstem respiratory nuclei. Chronic nicotine upregulated its own receptors in the rodent brain and increased the catecholamine release, evidencing in central areas, as in the carotid bodies, interactions between catecholaminergic and cholinergic systems. Concerning prenatal nicotine, it induces adverse effects on cholinergic development (Navarro et al., 1989) with central cholinergic and noradrenergic hypoactivity during the first 2 postnatal weeks (Slotkin, 2004) and it reduced expression of nAChR subunit mRNAs (Chen et al., 2005). Many nicotine side-effects result from decreased ␤-adrenergic responsiveness to agonist (Slotkin, 2004) and loss of function of ␤2 -containing nicotinic acetylcholine receptors (Cohen et al., 2005). Mutant mice lacking ␤2 nicotinic cholinergic receptor subunit exhibit increased hypoxic ventilatory response and unstable breathing (Cohen et al., 2002), similar to the present observations in pups prenatally exposed to nicotine. It can thus be hypothesized that prenatal nicotine exposure induced an impairment of cholinergic function, contributing to the described respiratory plasticity.

4.4. Pathophysiological significance and conclusion In conclusion, prenatal exposure to nicotine altered the development of ventilation and of chemosensory processes. The postnatal ventilatory sequelae may partly result from impaired maturation of peripheral chemoreceptors and of brainstem integration of chemoreceptor input. This reflects the involvement of adaptive processes to deal with the prenatal environmental nicotine insult and highlights developmental plasticity in respiratory control. The pathophysiological implications of these results are important. In humans, nicotine substitution using patches is a safe alternative, but needs careful re-assessment in pregnant women. The altered ventilatory pattern until P11 with no change later may be the footprint of prenatal nicotine sequelae, which could be revealed later by environmental disturbances (temperature, repeated apnea causing intermittent hypoxia, nicotine consumption at adolescence) (Slotkin et al., 2007).

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Acknowledgements This work was supported by the Centre National de la Recherche ´ eration ´ Scientifique, the Universite´ Claude Bernard, and the Fed “Naitre et Vivre” (France). David Perrin held a fellowship from ` ´ the Ministere de l’Enseignement Superieur et de la Recherche, Julie Peyronnet held grants from the Karolinska Institute, Swe´ den, and Aurelien Boussouar held grants from the Agence de l’Environnement et de la Maˆıtrise de l’Energie.

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