Prenatal nicotine exposure increases apnoea and reduces nicotinic potentiation of hypoglossal inspiratory output in mice

Abstract

We examined the effects of in utero nicotine exposure on postnatal development of breathing pattern and ventilatory responses to hypoxia (7.4 % O₂) using whole-body plethysmography in mice at postnatal day 0 (P0), P3, P9, P19 and P42. Nicotine delayed early postnatal changes in breathing pattern. During normoxia, control and nicotine-exposed P0 mice exhibited a high frequency of apnoea (f_A) which declined by P3 in control animals (from 6.7 ± 0.7 to 2.2 ± 0.7 min⁻¹) but persisted in P3 nicotine-exposed animals (5.4 ± 1.3 min⁻¹). Hypoxia induced a rapid and sustained reduction in f_A except in P0 nicotine-exposed animals where it fell initially and then increased throughout the hypoxic period. During recovery, f_A increased above control levels in both groups at P0. By P3 this increase was reduced in control but persisted in nicotine-exposed animals. To examine the origin of differences in respiratory behaviour, we compared the activity of hypoglossal (XII) nerves and motoneurons in medullary slice preparations. The frequency and variability of the respiratory rhythm and the envelope of inspiratory activity in XII nerves and motoneurons were indistinguishable between control and nicotine-exposed animals. Activation of postsynaptic nicotine receptors caused an inward current in XII motoneurons that potentiated XII nerve burst amplitude by 25 ± 5 % in control but only 14 ± 3 % in nicotine-exposed animals. Increased apnoea following nicotine exposure does not appear to reflect changes in basal activity of rhythm or pattern-generating networks, but may result, in part, from reduced nicotinic modulation of XII motoneurons.

It is generally accepted that cigarette smoking during pregnancy is harmful to fetal development and is associated with an increased risk of miscarriage, perinatal death and sudden infant death syndrome (SIDS) (Mitchell et al. 1992; DiFranza & Lew, 1995). While tobacco contains a plethora of toxic chemicals, nicotine is strongly implicated as a causative link due to its adverse effects upon the development of the central nervous system (CNS) (see recent reviews by Slotkin, 1998; Dempsey & Benowitz, 2001). Nicotine exposure is associated with a premature switch from neuronal proliferation to cell differentiation (Navarro et al. 1989; Nordberg et al. 1991) and a disruption in the developmental expression patterns of numerous transmitter systems (Slotkin, 1998; Slotkin et al. 1999). The best documented of these is the upregulation and desensitization of high-affinity nicotinic receptors in the CNS (Bhat et al. 1991; Nordberg et al. 1991; Pauly et al. 1991; Peng et al. 1994).

The consequences of these changes for respiratory control are unknown, but of considerable interest due to the relationship between smoking and SIDS and the long-standing hypothesis that an abnormality in the control of breathing, particularly in the mechanisms that defend against severe hypoxaemia, contributes to SIDS (Hunt, 1992; Poets et al. 1993). Previous in vivo studies in rat have yielded variable results, but a consensus is growing that altered respiratory control (Fewell & Smith, 1998; Bamford & Carroll, 1999; St John & Leiter, 1999) contributes to the reduced ability of nicotine-exposed animals to tolerate hypoxia (Slotkin et al. 1995). Most studies have focused on overall changes in ventilation, but paid minimal attention to breathing pattern. In addition, with few exceptions (St John & Leiter, 1999), responses have not been examined in the earliest neonatal periods when immature control mechanisms are more likely to contribute to unstable breathing patterns, frequent apnoeas and hypoxic episodes (Mortola, 1984).

The first goal of this study was therefore to investigate the effects of prenatal nicotine exposure on the development of breathing pattern and the ventilatory response to hypoxia in mice in vivo that range in age from newborn (P0) to adult. Most previous work in this area has been performed in rats. Our use of mice is based on the fact that they are an increasingly important model system for study of respiratory control. Not only do mice facilitate transgenic approaches (Funk et al. 1997a; Katz & Balkowiec, 1997; Bond et al. 2000; Bou-Flores et al. 2000), the ability to produce rhythmically active medullary slice preparations from animals up to 3 weeks of age has increased their use in developmental analyses of rhythm generation, pattern formation and synaptic modulation (Funk & Feldman, 1995; Funk et al. 1997c). In addition, our recent studies in normal mice indicate a high prevalence of apnoea in newborns under control conditions that increases markedly during recovery from hypoxia (Robinson et al. 2000). The instability of the murine breathing pattern at birth and its rapid stabilization during development make mice well suited for exploring mechanisms of apnoea and how teratogens such as nicotine affect the development and stability of central respiratory circuits. Understanding this relationship is especially important due to the correlation of SIDS with both smoking (Mitchell et al. 1992; DiFranza & Lew, 1995) and apnoea (Gaultier, 1995).

The second goal of this study was to determine whether nicotine-induced changes in breathing pattern in vivo are associated with changes in central networks that generate respiratory rhythm and pattern, or motoneurons that control the upper airway. Nicotine exposure alters development of peripheral chemoreceptors (Holgert et al. 1995) and the hypoxic/hyperoxic responses they mediate (Milerad et al. 1995; Bamford et al. 1996; Fewell & Smith, 1998; St John & Leiter, 1999). Effects on rhythm- and pattern-generating systems have not been examined but are likely. Even in the absence of premature neuronal differentiation, nicotine-mediated disruption of cholinergic receptor expression in the brainstem (Pauly et al. 1991; Slotkin et al. 1999) is likely to affect respiratory activity because cholinergic systems modulate medullary respiratory neurons and networks (Bradley & Lucy, 1983; Bohmer et al. 1987; Monteau et al. 1990; Shao & Feldman, 2000; Shao & Feldman, 2001), including inspiratory motoneurons (Bellingham & Berger, 1996; Zaninetti et al. 1999; Bellingham & Funk, 2000). Cholinergic systems are also important in the state-dependent control of breathing, as first proposed by Hobson et al. (Hobson et al. 1975). To assess the actions of nicotine exposure on the behaviour of central rhythm-generating networks and respiratory motoneurons, we used medullary slice preparations from neonatal mice that generate rhythmic respiratory-related activity in vitro. These preparations allow the effects of nicotine exposure on rhythm- and pattern-forming components of the respiratory circuit to be examined separately, and in isolation from, its influences on peripheral, homeostatic control components.

METHODS

Animals

All experiments were carried out on Swiss CD-1 mice using methods approved by the Animal Ethics Committee of the University of Auckland. Animals were fed ad libitum water and dry pellets, weaned at 19 days and kept in a quiet room at 21–22 °C and 50–65 % relative humidity under a 12 h light/12 h dark cycle.

Selection of Swiss CD mice was important for these experiments because the dams tolerate disturbance during pregnancy and in the first few hours after delivery, facilitating osmotic micropump implantation and repeated examination of neonates in vivo.

Prenatal nicotine exposure

Timed mating of females was performed at ∼6 weeks of age and fetal nicotine exposure was achieved using an Alzet 1007D osmotic micropump (Alza Corp., CA, USA) which was implanted under aseptic conditions in the dam at gestational day 10. The pumps were charged with either distilled water (control animals) or 240 mg ml⁻¹ nicotine and had a nominal infusion rate of 0.5 μl h⁻¹ for 8 days. The dam was anaesthetised with halothane (3 % for induction, 1–2 % for maintenance) and the pump inserted subcutaneously on the dorsal midline between the shoulder blades. The incision was sutured and the mouse was removed from the anaesthetic machine and allowed to recover. All incisions healed without infection within a few days and no antibiotic treatment was required. Pups were weighed immediately after birth and prior to in vivo experimentation throughout development. At the end of the study surviving dams and offspring were humanely killed by CO₂ anaesthesia and cervical dislocation.

HPLC analysis of plasma nicotine

To determine the effect of nicotine infusion on maternal plasma nicotine concentration, four pregnant mice were anaesthetised with diethylether, decapitated and exsanguinated 7 days after pump insertion. Blood was collected in chromic acid-washed glassware containing 100 units of heparin, transferred by plastic pipettes to centrifuge tubes and centrifuged at 13 000 r.p.m. in a Micro Centaur bench centrifuge (Sanyo MSE, UK) for 10 min. The plasma was carefully transferred from the centrifuge tube to another tube and stored at −70 °C until analysis.

Alumina columns (15 ml, Extrelut 3, Merck, Germany) were used to extract nicotine and its primary metabolite cotinine (Benowitz et al. 1983) from stored plasma samples prior to analysis by HPLC. The alumina columns were washed with 10 ml dichloromethane (HPLC grade, Mallinkrodt) 24 h prior to use. They were allowed to dry by standing. Internal standard, 100 μl 5-methyl-cotinine, was added to 1 ml plasma along with1 ml 0.5 m NaOH. The mixture was transferred to the pre-washed alumina columns and allowed to bind for 15 min. The cotinine and nicotine were eluted with 10 ml of a 10 : 1 mixture of dichloromethane and isopropanol. The eluates were acidified with 100 μl of 25 mm methanolic HCl. Solvents were then evaporated under nitrogen and the residue reconstituted in 100 μl of the HPLC mobile phase.

The nicotine and cotinine in the sample extracts were analysed by reversed-phase ion-paired HPLC. The chromatograph consisted of a 996 photodiode array detector, a 717 autosampler and a 510 pump and the detection wavelength was set at 259 nm. The system was computer controlled with a Millenium³² chromatography manager version 3.05.01 via an IEEE 488 Buslace card and a pump control module. All the components were supplied by Waters Associates (Milford, MA, USA). The separation was performed on a Prodigy OD5 (3) column, 150 × 3.2 mm, 5 μm particle size, 100 Å pore size, with a security guard cartridge (Phenomonex, Torrance, CA, USA). The mobile phase comprised 85 % v/v 0.05 m phosphate buffer at pH 3.0 with 15 % v/v acetonitrite (HPLC grade, Hallinkrodt) modified with 3 g l⁻¹ octylsulphonate sodium salt (HPLC grade, BDH Laboratory Supplies, Poole, UK). At a flow rate of 0.5 ml min⁻¹, cotinine eluted in 7.2 min and nicotine in 10.9 min. Peak identity and purity were achieved by spectral comparison with pure compounds. Nicotine was obtained from Research Biochemicals International (Natick, MA, USA), cotinine from Sigma (St Louis, MO, USA) and 5-methyl-cotinine was kindly donated by Professor Neal Benowitz (Division of Clinical Pharmacology and Experimental Therapeutics, Department of Medicine, UCSF). Recoveries of nicotine and cotinine were 83 % and 91 % respectively. This is similar to those measured previously using similar methods (Zuccaro et al. 1993).

In vivo studies

Measurement of ventilation

Baseline ventilatory parameters and responses to hypoxia were measured in P0 (n = 6), P3 (n = 7), P9 (n = 8), P19 (n = 13) and adult (> P42, n = 6) unanaesthetized, nicotine-exposed mice using continuous head-out, whole body plethysmography as described previously (Robinson et al. 2000). Briefly, the plethysmograph consisted of separate head (10 ml) and body chambers (45 ml) separated by a flexible latex seal (Dentsply, Australia). An animal was placed in the body chamber with its head poking through an adjustable hole in the latex seal into the head chamber. Integrity of the neck seal was verified before and after each experiment by ensuring that the body/plethysmograph chamber maintained constant positive pressure when 50 μl air was injected into it. Data were excluded if the seal was not maintained throughout the trial. Zero flow was defined at the beginning of each experiment by opening a low-resistance port to atmosphere.

Chamber temperature was maintained in thermoneutral ranges, 35–36 °C for P0 to P9 animals and 32–33 °C for P19 and adult animals (Spiers & Adair, 1986) – by a Bat-12 Digital Thermometer, TCAT-1 temperature controller (Physitemp, NJ, USA) and infrared lamp. Normoxic and hypoxic gases (see below) were drawn through the head chamber at 250 ml min⁻¹ to a Datex (Helsinki, Finland) O₂/CO₂ gas analyser. The body chamber was connected to the atmosphere through a high-resistance pneumotachograph head (Fleisch, Switzerland) connected to an MP 45–1 pressure transducer (Validyne Engineering Corp, CA, USA). The output of the pressure transducer was then passed through a CD15 Carrier Demodulator (Validyne Engineering Corp, CA, USA).

The output voltage of both the CD15 Carrier Demodulator and the gas analyser were digitized by a MacLab A–D converter. Data were recorded at 40 Hz using Chart 3.3.8 (AD Instruments Pty Ltd, Australia) running on a Power PC Macintosh 7300/180 under Mac OS 8.0 (Apple Computer Inc., CA, USA). Flow/volume calibration was performed at the beginning and end of each experiment by the injection of air into the chamber from 10, 50 and 100 μl glass syringes (Hamilton, NV, USA).

Inspired gas mixtures

Normoxic gas (medical air, 21 % O₂) was obtained commercially. Hypoxic gas (7.4 % O₂) was produced by dilution of medical air with oxygen-free nitrogen using a mixing pump (Wösthoff, Germany). This level of hypoxia produced a consistent, robust hypoxic ventilatory response in animals of all ages, including neonates (Robinson et al. 2000). Gases were stored in 100 l Douglas bags and humidified prior to delivery. The composition of the gas leaving the head chamber was continuously monitored by the Datex O₂/CO₂ gas analyser, recorded on computer, and remained stable throughout experiments.

Experimental protocol

Once the mice were resting quietly (minimum 5 min, sleep state was not assessed), control data were acquired for 5 min. Inspired gas was switched from normoxia to hypoxia via a three-way valve. Changeover of inspired gases within the head chamber was complete within 5 s. The period of hypoxic exposure was 12 min to ensure that sufficient time was allowed for the hypoxic depression of ventilation to develop (Robinson et al. 2000). After 12 min of hypoxia, normoxia was restored and recovery data collected for 10 min.

Plethysmography data analysis

The Chart 3.3.8 records were saved as text files, converted to binary files and analysed by a purpose-written Qbasic program running under DOS on a PC 386 (Robinson et al. 2000).

Values of minute ventilation (V̇_E), tidal volume (V_T), respiratory frequency (f_R), inspiratory (T_I) and expiratory time (T_E), frequency of apnoea (f_A) and percentage of total time spent apnoeic (T_A) were averaged for each 30 s period of the 27 min protocol. To measure T_E, the analysis programme defined the flow reversal associated with the change from inspiration to expiration and T_E was defined as the interval extending from this point (end inspiration) to the beginning of the next inspiration. Expiratory pauses of zero flow greater than 3 s in duration were defined as apnoea (Jacquin et al. 1996). The hypoxic ventilatory response was analysed in two phases: Phase I between minutes one and three of hypoxia, and Phase II between minutes 9 and 11 of hypoxia. Posthypoxic responses were assessed by averaging values over the first 3 min following the return to normoxia. Data are reported in absolute terms and relative to normoxic control values.

Experimental runs in which there was movement artifact occupying > 5 % of the total time were rejected (Robinson et al. 2000). Given the difficulty of obtaining stable breathing responses from these animals for the lengthy protocol (> 27 min), the ratio of rejected to successful experimental runs was approximately 4 : 1 leaving the final numbers reported in Table 1. We did not attempt to ensure that measurements were made on the same animal at all developmental stages and therefore they were treated statistically as independent measures.

Table 1.

Means ± s.e.m. of respiratory variables measured in control and nicotine-exposed neonatal, juvenile and adult mice during the normoxic control period

Age	n	Mass (g)	VT (ml kg−1)	TI (s)	TE (s)	V̇E(ml min−1 kg−1)	fR (min−1)	fA (min−1)	TA (%)
Control
P0	9	1.6 ± 0.1	12.7 ± 1.3	0.28 ± 0.04	0.48 ± 0.06	690 ± 90	55 ± 7	6.7 ± 0.7	29.0 ± 6.0
P3	7	2.9 ± 0.1*	11.2 ± 0.9	0.12 ± 0.01*	0.33 ± 0.03*	1530 ± 250*	130 ± 13*	2.2 ± 0.7*	5.1 ± 2.4 *
P9	6	6.0 ± 0.4*	9.9 ± 1.3	0.13 ± 0.01	0.25 ± 0.04*	1600 ± 160	169 ± 21*	0.3 ± 0.1*	0.1 ± 0.1*
P19	6	9.4 ± 0.5*	12.6 ± 1.3	0.14 ± 0.01	0.19 ± 0.02*	2460 ± 210*	182 ± 12*	0.1 ± 0.1	0.0 ± 0.0
Adult	6	25.8 ± 1.3*	11.0 ± 1.9	0.13 ± 0.02	0.16 ± 0.02	2170 ± 430	207 ± 20 *	0.0 ± 0.0	0.0 ± 0.0
Nicotine exposed
P0	6	1.7 ± 0.1	15.4 ± 1.0	0.18 ± 0.02	0.44 ± 0.07	910 ± 187	70 ± 11	8.1 ± 1.7	25.1 ± 4.8
P3	7	2.4 ± 0.1*	11.7 ± 1.4	0.14 ± 0.01*	0.40 ± 0.04*	1071 ± 140	98 ± 16*	5.4 ± 1.3*†	14.4 ± 5.5*†
P9	8	5.9 ± 0.2*	12.0 ± 1.6	0.10 ± 0.01*	0.24 ± 0.03*	2175 ± 315*	177 ± 15*	0.6 ± 0.4*	0.0 ± 0.0*
P19	13	8.3 ± 0.5*	15.3 ± 1.1	0.15 ± 0.01*	0.19 ± 0.02*	2652 ± 265*	186 ± 14	0 ± 0	0.0 ± 0.0
Adult	6	22.5 ± 0.3*	10.4 ± 0.7	0.16 ± 0.01	0.19 ± 0.01	1697 ± 112*	165 ± 5*	0 ± 0	0.0 ± 0.0

^*Significantly (P < 0.05) different from the younger age group

^†significantly (P < 0.05) different from control group. Control data reproduced with permission from Robinson et al. (2000).

In vitro studies

Experiments were performed on rhythmically active transverse medullary slice preparations isolated from control or nicotine-exposed P0–P3 Swiss CD-1 mice. Techniques for preparation of rhythmically active brain slices are described in detail elsewhere (Funk et al. 1994). Briefly, mice were anaesthetized with diethylether, decerebrated, and the brainstem-spinal cord isolated in a 15 ml dissection chamber containing artificial cerebrospinal fluid (aCSF) (mm: 120 NaCl, 3 KCl, 1 CaCl₂, 2 MgSO₄, 26 NaHCO₃, 1.25 NaH₂PO₄, 20 d-glucose; pH 7.45) bubbled with 95 % O₂–5 % CO₂ at room temperature. The brainstem-spinal cord was pinned to a wax chuck and sectioned using a vibratome (Pelco-101, Ted Pella, CA, USA). Serial sections (100–200 μm) were cut in the rostro-caudal direction and examined for neuroanatomical landmarks until the compact division of nucleus ambiguus and the rostral border of the inferior olive were observed, whereupon a single 600 μm section was cut. This slice extended from the rostral margin of the pre-Bötzinger complex (pre-BötC) to obex and contained the pre-BötC, rostral-ventral respiratory group, XII motor nuclei and rostral XII nerve rootlets (Fig. 4A).

Figure 4. No effect of nicotine exposure on baseline behaviour of respiratory networks in vitro.

A, schematic of transverse medullary slice preparation. Arrangement of suction electrodes for recording inspiratory activity from XII nerves (∫XII; nerve activity is typically recorded bilaterally), pressure ejection pipette for local application of drugs and whole-cell recording pipettes for recording membrane voltage (V_m) or current (I_m) from individual inspiratory motoneurons under current- or voltage-clamp respectively. (NA, nucleus ambiguus; IO, inferior olive; 5 SP, spinal trigeminal nucleus; XII, hypoglossal nucleus; pre-BötC, pre-Bötzinger complex). B, long time-scale recordings of integrated inspiratory burst activity (∫XII) recorded from the hypoglossal nerve in control and nicotine-exposed P3 mice. C, expanded time-scale recording showing inspiratory burst profile recorded from the hypoglossal nerve in slices from control and nicotine-exposed P3 mice. Burst envelopes represent averages of 10 consecutive inspiratory cycles. D, return maps where the period of cycle n is plotted against the period of cycle n + 1 for nine control and nine nicotine-exposed mice (20 consecutive cycles per slice). Dashed outlines in return maps border the same parameter space and indicate similar cycle-to-cycle variation in respiratory period for slices taken from control and nicotine-exposed animals. E, rhythmic inspiratory synaptic currents recorded under voltage-clamp conditions (holding potential of −60 mV) from XII motoneurons in control and nicotine-exposed slices. Synaptic currents shown occurred consecutively and in conjunction with bursts of activity in the XII nerve (not shown). Dashed lines between bursts indicate quiescent period deleted between cycles. F, expanded time-scale traces of single inspiratory synaptic current envelopes recorded from a control and a nicotine-exposed slice.

For extracellular studies, slices were fixed caudal surface up in a recording chamber (10 ml volume) and superfused with 26–27 °C aCSF solution at a flow rate of 20 ml min⁻¹. In whole-cell recording experiments, slices were placed caudal surface up under a nylon mesh and perfused at 1–2 ml min⁻¹ in a 1 ml chamber. Thirty minutes prior to the start of data collection extracellular K⁺ concentration was raised from 3 to 9 mm to maintain long-term respiratory network activity (Funk et al. 1993).

Recording of XII nerve activity

Bilateral extracellular recording of XII nerve activity was performed using suction electrodes with internal tip diameters ranging from 40 to 80 μm. Recordings were amplified (50 000 times), band-pass filtered (0.1–3 kHz), full-wave rectified and integrated (τ = 50 ms). The output of the integrator was displayed on a storage oscilloscope (Tetronix 5111, Beverton, OR, USA), recorded on a chart recorder (Gould Instruments 2400S, Valley View, OH, USA) and, along with raw data, stored via pulse code modulation on VCR cassette (Vetter 402, PA, USA).

Analysis of XII nerve output

Rectified, integrated nerve recordings were analysed for inspiratory burst frequency as well as ipsi- and contralateral XII nerve inspiratory burst amplitudes using a custom-written LabVIEW (National Instruments Corporation, Austin, TX, USA) acquisition and analysis programme. Burst amplitude was quantified, in arbitrary voltage units, as the distance from baseline to the peak of the integrated burst envelope. In cases where drug application was associated with an increase in tonic nerve discharge and a shift in the baseline, burst amplitude was calculated by subtracting the baseline shift from the peak amplitude. While some of the baseline shift may represent tonic activation of respiratory motoneurons, baseline correction was chosen to reduce the possibility of overestimating the potentiating actions of nicotine on inspiratory burst amplitude.

Contralateral nerve activity was recorded to monitor the degree of drug diffusion to the contralateral motor nucleus. Responses to 10 μm nicotine were exclusively unilateral. Nicotine-mediated increases in frequency, indicative of nicotine diffusion to rhythm-generating regions of the slice in the ventrolateral medulla (Shao & Feldman, 2001), were never observed.

The magnitude of responses was determined by comparing control burst amplitude (averaged over 2 min prior to drug application) with the highest value of a 30 s moving average recorded during the first minute following onset of drug application.

To optimize conditions for comparison, two slices (one from a control and one from a nicotine-exposed animal) were placed side by side in the recording chamber and examined on the same day under virtually identical conditions. Responses to local nicotine application were then assessed in random order.

Whole-cell recording

Whole-cell recordings from XII motoneurons (n = 10) were established under direct visualization using infra-red differential interference contrast microscopy (Stuart et al. 1993). Patch electrodes (resistance 4.0–5.0 MΩ; tip size 1.5–2 μm) were pulled on a horizontal puller (Sutter Model P-97, Novato, CA, USA) from 1.2 mm o.d. filamented borosilicate glass (Clark Electromedical, Reading, UK) and filled with potassium gluconate solution containing (mm): K⁺-gluconate, 125; NaCl, 5; CaCl₂, 1; Hepes, 10; BAPTA, 10; Mg²⁺-ATP, 2. Intracellular solution pH was adjusted to 7.2–7.3 with 5 m KOH. Intracellular signals were amplified with a patch-clamp amplifier (5 kHz Bessel filter, Axopatch 200B, Axon Instruments, Foster City, CA, USA). Seal resistances prior to membrane rupture ranged from 1.5 to 6 GΩ.

Series resistance and whole-cell capacitance were estimated under voltage-clamp conditions by using short voltage pulses (100 Hz, −10 mV, 3.0 ms). Series resistance averaged 17.4 ± 1.0 MΩ. Series resistance was monitored throughout the experiment and compensated up to 80 %. Data were discarded if series resistance increased by more than 10 % between control and test conditions. Current–voltage (I–V) relationships were examined by applying a series of command voltage or current pulses (600 ms) controlled by a 166 MHz Pentium PC running pCLAMP 6.0 software. In rhythmically active slices (prior to application of TTX), pulses were triggered from integrated XII nerve activity following a 2 s delay to ensure that injected and inspiratory synaptic currents did not overlap.

Neuron input resistance (R_N) was calculated from the inverse of the slope of a least squares regression line fitted to the I–V curves. Membrane potentials have not been adjusted for liquid junction potentials. All signals were recorded continuously via pulse code modulation (Vetter 402, PA, USA) for off-line analysis.

Whole-cell data analysis

Selected portions of data were digitized off-line at 1–20 kHz using pCLAMP 6.0/Axoscope 1.1 (Axon Instruments, Foster City, CA, USA) software in conjunction with a Digidata 1200A A–D board, and stored on computer for subsequent analysis. Currents generated in response to voltage steps were measured in Axograph 3.0 (Axon Instruments, Foster City, CA, USA).

Motoneuron identification

Criteria for XII motoneuron identification have been described elsewhere (Funk et al. 1993, 1997b) and include: presence of rhythmic synaptic drive currents/depolarizations in synchrony with rhythmic bursts of activity in XII nerve roots; multipolar soma located in the ventromedial portion of the XII nucleus; stable resting potentials with leak currents < −100 pA at holding potentials of −60 mV; R_N values between 100 and 150 MΩ.

Drugs and drug application

Drugs used included nicotine (nicotine di-d-tartrate, Research Biochemicals International, Natick, MA, USA, 10–100 μm), and tetrodotoxin (TTX; Alomone Labs, Jerusalem, Israel, 0.5–1.0 μm). All drugs were made up in aCSF and stored as frozen aliquots.

Local application of nicotine was performed via pressure injection using triple-barrelled ejection pipettes positioned within 20 μm of the ventromedial aspect of the XII nucleus. Drugs were delivered at 70 kPa (10 p.s.i.) and injection protocols were controlled via a programmable stimulator (Digitimer, Devices Type 3290, UK). For extracellular studies, the concentration range and duration of nicotine applications were established in a preliminary set of experiments. At 1 μm, effects of nicotine on inspiratory burst activity were not apparent while at 100 μm tonic effects predominated and partially obscured inspiratory activity. Since the main objective was to examine the effects of nicotine on inspiratory burst amplitude, 10 μm nicotine was used to examine effects on burst amplitude. Thirty-second applications were selected to allow measurement of several inspiratory cycles during the peak of the nicotine effect. Applications were not extended for longer than 30 s to minimize reductions in inspiratory burst amplitude (due to depolarization block) that often occurred when nicotine was applied for long periods. For antagonist experiments with hexamethonium, 100 μm nicotine was used. Consecutive applications were associated with a small degree of run-down, even when they were separated by an interval of 15 min, probably reflecting receptor desensitization. Thus, only the response to the first application was analysed. For the intracellular studies both 10 and 100 μm nicotine was applied.

Note that the concentrations and duration of drug applications used in the present study should not be directly compared with those in experiments where similar agents are bath-applied for two reasons. Firstly, diffusion limitations in these relatively thick brain slices slow response kinetics relative to isolated cells. Secondly, drug concentration decreases exponentially with distance from the pipette tip (Nicholson, 1985). Previous experiments with this preparation indicate that drug concentration in the pipette must be approximately 10-fold greater than the bath-applied concentration to produce similar effects (Liu et al. 1990).

Statistical analysis

Statistical analysis was conducted using SAS 6.1 (SAS Institute, Cary, NC, USA). Raw data were tested for normality using the Shapiro–Wilk statistic and, where appropriate, subjected to a two-way ANOVA. Differences among interventions were sought (at the 95 % level of confidence, P < 0.05) using mutually orthogonal contrast coefficients to partition the treatment sum of squares.

Non-normal data were subjected to the Kruskal–Wallis test and, where appropriate, differences between groups were sought using Wilcoxon signed-rank tests with Bonferroni adjusted P values. Data are presented as means ± s.e.m. In vivo respiratory data for some age groups are omitted from some of the figures for clarity.

RESULTS

Plasma nicotine

Previous studies in mice have required nicotine infusion rates 8–10-fold higher than those used in rats to produce similar levels of nicotinic receptor up-regulation (van de Kamp & Collins, 1994). However, the effect of nicotine infusion on plasma nicotine concentration has not previously been reported in mice. We therefore directly measured the concentration of nicotine and its primary metabolite, cotinine, in the plasma of mice using HPLC. Plasma concentrations of nicotine and cotinine produced by infusion rates of 0.12 mg h⁻¹ (0.5 μl h⁻¹ of nicotine solution at 240 mg ml⁻¹) for 7 days were 233 ± 45 and 327 ± 84 ng ml⁻¹ respectively (n = 4).

Chronic nicotine exposure does not influence fetal or postnatal growth

Prenatal nicotine exposure did not alter gross measures of prenatal development. The litter size (control 15 ± 1.5; nicotine 12 ± 1.0, n = 3) and birth weight (control 1.31 ± 0.02 g, n = 45, nicotine 1.32 ± 0.03 g, n = 39) were similar in control and nicotine-exposed animals. Postnatal increases in body weight, assessed at postnatal day 0 ± 0 (P0; within 12 h of birth), 3 ± 0 (P3), 9 ± 0 (P9), 19 ± 1 (P19) and 45 ± 1 (adult), were also unaffected by nicotine exposure (Table 1).

Prenatal nicotine exposure increases incidence of apnoea in neonatal mice

Baseline measurements of weight-corrected respiratory parameters averaged for 5 min prior to hypoxic exposure are presented in Table 1 for nicotine-exposed and control animals. The developmental changes in breathing pattern were generally similar in control and nicotine-exposed animals. The most profound changes in breathing pattern occurred in the period between P0 and P9 (Table 1). There was a doubling in V̇_E over this time frame, due largely to an increase in average f_R since weight-corrected V_T did not change significantly between P0 and adult. The increase in f_R between P0 and P9 in both groups was primarily due to a developmental reduction in the frequency of apnoea, f_A, and therefore the percentage of time spent apnoeic, T_A, as well as reductions in T_I and T_E. Beyond P9 apnoeas were virtually absent and there was a continued increase in f_R due to a further reduction in T_E. V̇_E also continued to increase such that at P19 it was significantly greater than at P3 and P9. This trend reversed by adulthood as f_R and V̇_E declined (Table 1, Figs 1 and 2).

Figure 1. Development of the ventilatory response to hypoxia.

Time course of the relative changes in minute ventilation (V̇_E), respiratory frequency (f_R) and tidal volume (V_T) during an hypoxic test protocol (5 min air breathing, 12 min of hypoxia and 10 min of recovery). Responses were recorded from nicotine-exposed mice of ages P0, P3, P9, P19 and adult and are reported relative to mean normoxic values.

Figure 2. Ventilatory and apnoeic responses to hypoxia in nicotine-exposed neonatal mice.

Time course of changes in: minute ventilation (V̇_E) (A), respiratory frequency (f_R) (B), frequency of apnoea (f_A) (C), and amount of time spent apnoeic (T_A) (D) in P0 and P3 nicotine-exposed animals during an hypoxic test protocol (5 min air breathing, 12 min of hypoxia and 10 min of recovery). Broken lines are the mean responses recorded in P0 and P3 control animals, reproduced with permission from Robinson et al. (2000).

The main difference between the two groups was that reductions in apnoea and increases in V̇_E that occurred between P0 and P3 in control animals did not occur until after P3 in nicotine-exposed animals (Table 1). The two groups demonstrated similar levels of apnoea at P0. The f_A and T_A were 6.7 ± 0.7 and 29 ± 6 % for control animals and 8.1 ± 1.7 and 25 ± 5 % in nicotine-treated animals respectively. This period of instability, which characterizes the breathing pattern of newborns of many species (Mortola, 1984), was prolonged in nicotine-exposed animals. The f_A and T_A decreased only a minor amount by P3 in nicotine-exposed animals to 5.4 ± 1.3 cycles min⁻¹ and 14 ± 6 %. In contrast, over a similar time frame in control animals the f_A fell to 2.2 ± 0.7 cycles min⁻¹ to occupy only 5 ± 2 % of total time. A delayed development of respiratory networks is also supported by the observation that V̇_E doubles between P0 and P3 and does not change between P3 and P9 in control animals, whereas in nicotine-exposed animals, V̇_E does not increase between P0 and P3 but doubles between P3 and P9.

Hypoxic ventilatory response in nicotine-exposed animals

The time course of the changes in V̇_E, f_R and V_T relative to normoxic control is plotted for nicotine-exposed animals of all age groups in Fig. 1. With the onset of hypoxia, elevations in f_R and V_T (Figs 1 and 2B), combined with reductions in f_A and T_A (Figs 2C and D), were responsible for the rapid increase in V̇_E observed in all age groups. The absolute magnitude of this initial increase during Phase I (between minutes one and three of hypoxia) was similar in all age groups. In relative terms, however, it was largest in P0 and P3 animals (Fig. 1) due to a higher incidence of apnoea that decreased with onset of hypoxia (Figs 2C and D). After the initial increase, f_R, V_T and V̇_E declined continuously by varying amounts such that levels during Phase II (between 9 and 11 min of hypoxia) were at or slightly above levels observed prior to hypoxia (Fig. 1). The degree of hypoxic roll-off between Phases I and II varied developmentally and was greatest for P0 mice, reflecting a progressive increase in f_A from 1.8 ± 0.5 to 7.1 ± 2.9 min⁻¹ and T_A from 4 ± 2 % to 17 ± 6 %. An increase in f_A during hypoxia was not observed in mice from other age groups. P3 and P0 mice showed a similar relative increase in V̇_E during Phase I but the magnitude of the roll-off was significantly reduced in P3 mice since f_A and T_A remained constant throughout the hypoxic period (Figs 2C and D). The Phase I increase in relative V̇_E and roll-off during Phase II were similar in P9, P19 and adult mice, but smaller compared with responses in P0 and P3 mice (Fig. 1).

Immediately upon the return to normoxia, V̇_E decreased significantly relative to normoxic control levels in all age groups except adults (Fig. 1). The magnitude of this posthypoxic depression, assessed by averaging values over the first 3 min after the return to normoxia, decreased with age. Relative V̇_E declined to 0.39 ± 0.07, 0.55 ± 0.15, 0.63 ± 0.14, 0.80 ± 0.10 and 1.03 ± 0.18 of control in P0, P3, P9, P19 and adult animals respectively. The duration of the posthypoxic depression also varied developmentally. In P0 animals V̇_E remained below normoxic control levels for the entire 10 min recovery period, largely due to increases in f_A. In P3 and P9 animals V̇_E returned towards control levels while in P19 and adult animals V̇_E rebounded beyond, and remained greater than, normoxic control levels for the remainder of the 10 min recovery period.

While the ventilatory responses of control (Robinson et al. 2000) and nicotine-exposed mice to hypoxia were qualitatively similar, two main features distinguished the hypoxic ventilatory responses of these two groups. These differences were apparent only in the two youngest age groups (P0 and P3) and were related to differences in the f_A and T_A. Data are depicted in Fig. 2, which compares the time course of V̇_E, f_R, f_A and T_A for P0 and P3 control and nicotine-exposed animals. In addition, Fig. 3 summarizes the developmental changes in f_A during normoxic breathing (Fig. 3A), during Phase II of hypoxia (Fig. 3B) and during the early stages of recovery (Fig. 3C) for control and nicotine-exposed mice. First, in P0 animals the f_A and T_A increased significantly during the 12 min hypoxic period in nicotine-exposed animals only (Fig. 2). In control animals, the f_A and T_A remained stable throughout the hypoxic exposure (Phase I, 2.2 ± 0.4 min⁻¹ and 6 ± 2 %; Phase II, 2.8 ± 0.3 min⁻¹ and 6 ± 2 %). In contrast, in nicotine-exposed animals f_A increased from 1.8 ± 0.5 to 7.1 ± 2.9 min⁻¹ and T_A increased from 4 ± 2 % to 17 ± 6 % between Phases I and II of hypoxia. The result was a significantly greater roll-off in V̇_E and f_R in the P0 nicotine-exposed group. Second, posthypoxic responses at P0 and P3 differed between control and nicotine-exposed animals. When compared with the prehypoxia control period, the significant increase in f_A and T_A during the posthypoxic period was actually proportionately greater for control (f_A 6.7 ± 0.7 prehypoxia increased to 10.8 ± 0.8 min⁻¹ posthypoxia representing a 162 % increase; T_A 29 ± 6 prehypoxia increased to 50 ± 6 % posthypoxia representing a 172 % increase) than nicotine-exposed (f_A 8.1 ± 1.7 prehypoxia increased to 11.3 ± 1.3 min⁻¹ posthypoxia representing a 140 % increase; T_A 25 ± 4.8 prehypoxia to 49 ± 5 % posthypoxia representing a 196 % increase) animals (Figs 2 and 3). It was also proportionately greater in P3 control (f_A 2.2 ± 0.7 prehypoxia increased to 5.3 ± 0.1 min⁻¹ representing an increase of 240 %; T_A 5.1 ± 2.4 prehypoxia increased to 30 ± 7 % posthypoxia representing a ∼600 % increase) compared with nicotine-exposed animals (f_A 5.4 ± 1.3 prehypoxia increased to 8.5 ± 1.0 min⁻¹ posthypoxia representing an increase of 157 %; T_A 14.4 ± 5.5 % prehypoxia increased to 44 ± 8 % posthypoxia representing an increase of ∼300 %). In absolute terms, however, the posthypoxic increase in f_A and T_A was similar in P0 control (f_A 10.8 ± 0.8 min⁻¹, T_A 50 ± 6 %) and nicotine-exposed (f_A 11.3 ± 1.3 min⁻¹, T_A 49 ± 5 %) animals, while P3 nicotine-exposed animals (f_A 8.5 ± 1.0 min⁻¹, T_A 44 ± 8 %) experienced a significantly greater incidence of apnoea during the posthypoxic recovery period than control animals (f_A 5.3 ± 0.1 min⁻¹, T_A 30 ± 7 %) (Figs 2 and 3). By P9, apnoea was a rare event appearing with similarly low frequencies in control and nicotine-exposed mice.

Figure 3. Developmental changes in the frequency of apnoeas during different phases of the hypoxic response.

Average values of frequency of apnoea (f_A) recorded during the fourth minute of the normoxic control period (A), minute 10 of hypoxia (B), and the first minute of recovery (C) for control and nicotine-exposed mice of various ages. *Significant difference (P < 0.05).

Minimal effects of prenatal nicotine exposure on behaviour of central respiratory networks in vitro

Prenatal nicotine exposure does not affect rhythmic activity of respiratory circuits in vitro

To examine whether the differences in respiratory behaviour observed in vivo between control and nicotine-exposed animals reflect changes in the behaviour of central networks underlying rhythm generation or transmission of this activity to respiratory motoneurons, we compared the rhythmic activity of medullary slices isolated from P3 control and nicotine-exposed mice.

Effects of nicotine exposure on behaviour of rhythm-generating systems were assessed by looking at the nature and regularity of the respiratory-related rhythm (Fig. 4B and D). Baseline respiratory-related activity of slices isolated from control (n = 9) and nicotine-exposed mice (n = 9) were indistinguishable, as shown for 50 s traces (Fig. 4B). Average burst frequency for control (0.21 ± 0.03 Hz) and nicotine-exposed (0.21 ± 0.02 Hz) animals were similar. Since the increased incidence of apnoea in nicotine-exposed animals may result from greater instability of rhythm-generating networks, we examined regularity of rhythmic output in slices from control and nicotine-exposed mice. First, return maps were constructed by plotting the period of one cycle (n) against that of the subsequent cycle (n + 1) for 20 consecutive respiratory cycles from nine control and nine nicotine-exposed slices (Fig. 4D). This method provides a convenient graphical method of assessing variability in rhythmic behaviour since dispersion of data points increases with increasing variability. Data from control and nicotine-exposed groups, however, showed similar distributions. Coefficients of variation for interburst intervals (18 % in control and 22 % in nicotine-exposed slices) further suggest that cycle-to-cycle variability in respiratory period was similar between groups.

Effects of nicotine on pattern-forming systems (including drive transmission pathways and XII motoneurons) were assessed by comparing in control and nicotine-exposed slices the inspiratory-related synaptic drive currents received by XII motoneurons (Fig. 4E and F) and the pattern of XII nerve output (i.e. the burst envelope, Fig. 4C). Inspiratory currents recorded from XII motoneurons in control and nicotine-exposed slices were similar in pattern, duration and magnitude (Fig. 4E and F). These rhythmic synaptic inputs produced similar rapidly incrementing, slowly decrementing bursts of activity in XII nerves of both groups. This can be seen in Fig. 4C where the nerve burst envelopes averaged from 10 consecutive cycles of a control and nicotine-exposed slice are superimposed. The mean nerve burst durations were 482 ± 35 ms and 479 ± 38 ms while average synaptic current was 263 ± 30 vs. 257 ± 13 pA and charge transfer was 23.6 ± 1.9 vs. 22.0 ± 0.9 pA s in control and nicotine-exposed slices respectively.

Prenatal nicotine exposure alters XII motoneuron responses to modulatory inputs in vitro

The failure of nicotine exposure to alter baseline behaviour of inspiratory networks does not exclude the possibility that it may alter responses to modulatory inputs. We therefore tested the hypothesis that nicotine exposure would alter the efficacy of nicotine in modulating XII inspiratory activity. We first established that XII motoneurons express postsynaptic nicotine receptors by locally applying nicotine (100 μm, 30 s) to inspiratory XII motoneurons during whole-cell recording (Fig. 5). Nicotine induced an inward current (Fig. 5A) in ten of ten inspiratory XII motoneurons examined. The current averaged −35 ± 6 pA and peaked within 30 s of application. Current-voltage relationships were obtained by applying a series of 5 mV voltage pulses (600 ms) between −90 and −25 mV. Current–voltage relationships for the nicotine current were obtained by subtracting the control I–V curve from that obtained in the presence of nicotine. In three of 10 cells the current was associated with a small decrease in R_N of 7 ± 2 MΩ from a control value of 111 ± 8 MΩ, and reversed near 0 mV (Fig. 5Ba and C). In the remaining seven of 10 cells nicotine had no significant effect on R_N (ΔR_N = 2 ± 2 MΩ) and currents did not reverse, reflecting either small currents, the small range over which voltage dependence of the current was examined in these cells or space-clamp problems. In the one cell examined, nicotine (100 μm) had no effect on peak synaptic current (control 172 ± 7; nicotine 171 ± 9 pA) or charge transfer (control 22.9 ± 1.3; nicotine 22.9 ± 1.2 pA).

Figure 5. Nicotine induces a hexamethonium-sensitive, TTX-insensitive inward current in XII motoneurons.

A, voltage-clamp recording of a XII motoneuron showing changes in membrane current produced by local application of 100 μm nicotine over the hypoglossal nucleus before, during and after a 2 min pre-application of 500 μm hexamethonium bromide. B, nicotine-induced inward current (Ba) and depolarization (Bb) persist in the presence of TTX (1 μm). C, current–voltage (I–V) relationships for a XII motoneuron before (control) and during (nicotine) application. The I–V relationship for the nicotine current was obtained by subtracting the control from the nicotine I–V relationship.

To confirm that the inward currents were caused by nicotine, the nicotinic receptor antagonist hexamethonium bromide (Gerzanich et al. 1995; 500 μm) was applied for 2 min prior to the administration of nicotine. Hexamethonium reduced the nicotine-induced current by 90 % (Fig. 5A) from −67 to only −7 pA (n = 1). After a 5 min wash-out period, the nicotine-induced current returned to 50 pA, 75 % of control.

To confirm postsynaptic receptor location, two inspiratory XII motoneurons were examined in the presence of 1 μm bath-applied TTX (Fig. 5B). TTX abolished both the inspiratory synaptic currents to these motoneurons and XII nerve output, but nicotine-induced inward currents (or depolarizations) persisted, averaging −23 ± 9 pA at −60 mV (Fig. 5B; n = 2). Under TTX, extrapolation of the I–V relationship for the nicotine current, which was calculated via linear regression based on responses to voltage steps between −90 mV and −25 mV, indicated that the nicotine-induced current reversed near +7 mV (Fig. 5C).

Having established that XII motoneurons possess the substrate upon which nicotine can act, we examined the effects of prenatal nicotine exposure on the ability of nicotine to modulate XII nerve inspiratory activity. Nicotine was locally applied ipsilaterally over the XII motor nucleus and changes in XII nerve output monitored. As shown for a single control slice in Fig. 6A, nicotine (10 μm) increased tonic discharge (evident in the thickening and upward shift in the baseline of the ∫XII nerve recording) and potentiated inspiratory burst amplitude by 25 ± 5 % (Fig. 6A, n = 9). In nicotine-exposed animals, however, nicotine (10 μm) had minimal effect on tonic discharge and the 14 ± 3 % (n = 9) potentiation of burst amplitude was significantly less than that observed in control slices. That these increases in tonic discharge and burst amplitude potentiation were due to activation of nicotinic receptors was confirmed in four control slices where the tonic discharge and a 32 ± 5 % potentiation of burst amplitude induced by 100 μm nicotine were blocked by hexamethonium (Fig. 6B). The fact that responses to 100 μm nicotine were completely blocked by hexamethonium established that the responses elicited by 10 μm were due to activation of nicotinic receptors.

Figure 6. Chronic nicotine exposure reduces response to nicotine receptor activation.

A, long time-scale recordings of integrated, hypoglossal nerve activity in control and nicotine-exposed medullary slices from P3 mice showing activity before, during and after 30 s application of 10 μm nicotine. Histogram plots mean (± s.e.m.) nicotine-mediated potentiation of XII nerve inspiratory burst amplitude for control (n = 11) and nicotine-exposed slices (n = 9). B, integrated hypoglossal nerve recording (∫XII) from a different slice to that shown in A illustrates response to a 30 s application of 100 μm nicotine before, during and after addition of 500 μm hexamethonium bromide to the bathing solution. Histogram represents the mean (± s.e.m.) nicotine-mediated potentiation of burst amplitude observed in the presence and absence of hexamethonium (n = 4). *Significant difference between nicotine-mediated potentiation in control compared with nicotine-exposed slices (P < 0.05).

DISCUSSION

Nicotine exposure increases incidence of apnoea in newborn mice

Our experiments provide the first detailed developmental analysis of how prenatal nicotine exposure affects breathing pattern and the hypoxic ventilatory response in mice. The key observation is that while overall ventilatory responses were qualitatively similar between control and nicotine-exposed animals in all age groups, nicotine exposure reduced breathing pattern stability in the youngest animals. This instability was first apparent in P0 nicotine-exposed animals in the progressive increase in the incidence of apnoea that occurred during hypoxia. It was also apparent at P3 where the high incidence of apnoea present in both groups under normoxic conditions at P0 persisted until P3 in nicotine-exposed mice. Finally, the posthypoxic increase in apnoea was significantly greater in P3 nicotine-exposed animals, resembling the response of P0 control animals. Thus, our data suggest that normal developmental changes in breathing pattern are delayed following prenatal nicotine exposure. These increases in apnoea following nicotine exposure have obvious implications for survival during and immediately following an hypoxic insult.

Previous studies in rat indicate a range of changes in respiratory behaviour and hypoxic responsiveness following prenatal nicotine exposure that may all contribute to the increased mortality of nicotine-exposed rats exposed to long periods (> 1 h) of severe hypoxia (5 % O₂) (Slotkin et al. 1995). For example, the contribution of peripheral chemoreceptor drive to basal ventilation, as determined by the 100 % O₂ test, is reduced following both acute postnatal (Holgert et al. 1995; Milerad et al. 1995) and chronic prenatal nicotine exposure (Bamford & Carroll, 1999). Some, but not all studies (Bamford et al. 1996; Bamford & Carroll, 1999), including ours, indicate that the ventilatory response to hypoxia is blunted (St John & Leiter, 1999). The ability to autoresuscitate after repeated, but not single (St John & Leiter, 1999), anoxic episodes is also reduced in nicotine-exposed animals (Fewell & Smith, 1998).

The different effects reported most likely reflect the type and severity of hypoxic stimulus (e.g. single or repeated episodes; moderate vs. severe hypoxia) and, since the response to hypoxia changes developmentally (Jansen & Chernick, 1983; Mortola, 1996), the developmental stage at which responses were measured. For example, with one exception (St John & Leiter, 1999), respiratory behaviour of rats exposed prenatally to nicotine has rarely been examined in animals younger than P3. Our separate analysis of P0 and P3 mice was motivated by evidence that respiratory networks, neurons and modulatory systems change substantially even over this relatively short developmental window (Funk et al. 1994; Selvaratnam et al. 1998; Hilaire & Duron, 1999), and the likelihood that the effects of a teratogen will be most apparent in the newborn when breathing pattern is most unstable (Mortola, 1984). Clearly differences observed in the present study between control and nicotine-exposed animals would have gone undetected if the P0 and P3 age groups had been excluded from analysis.

Differences in nicotine dosage must also be considered. Dosages reported in rat studies were selected to produce a level of cholinergic receptor upregulation similar to that observed in human smokers (Benwell et al. 1988). They are relatively consistent between studies, ranging between 2 and 7 mg kg⁻¹ day⁻¹, but 6 mg kg⁻¹ day⁻¹ is typical (Slotkin et al. 1995; Bamford et al. 1996; Fewell & Smith, 1998; Bamford & Carroll, 1999; St John & Leiter, 1999). Thus, dosage is not likely to be a significant source of variation between these studies.

In the present study, we selected an infusion rate of 60 mg kg⁻¹ day⁻¹ because infusion rates almost 10-fold greater than used in rats are required in mice to achieve similar up-regulation of [³H]nicotine binding in the fetus (van de Kamp & Collins, 1994). In addition, this level of nicotine exposure in mice upregulates nicotinic receptor expression in respiratory-related regions of the brainstem, particularly the medullary reticular formation and XII nuclei (Pauly et al. 1991). Our measurements of plasma nicotine, indicating only 2-fold greater levels in spite of 10-fold higher infusion rates (Richardson & Tizabi, 1994), confirm that much higher infusion rates are required to elevate nicotine in the plasma of mice. This requirement is proposed to reflect pharmacokinetic differences in the metabolism of nicotine in mice vs. rats. Indeed the half-life of plasma nicotine in mouse is 5–7 min (Petersen et al. 1984) whereas that for rat is about 54 min (Plowchalk et al. 1992; for humans it is 2–2.5 h; Benowitz & Jacob, 1993). We cannot exclude the possibility that the higher plasma nicotine levels reached in our mice contributed to the unique effects of nicotine exposure on the stability of breathing pattern. However, as discussed above, these infusion rates are similar to those required in mice to reproduce the nicotinic receptor upregulation observed in rats (van de Kamp & Collins, 1994). Doses were also non-toxic. The number of neonates per litter, neonatal weight at birth and rate of postnatal weight gain were not affected. For most strains of mouse, it is not until doses reach 96 mg kg⁻¹ day⁻¹ that significant effects on heart rate, startle responses, performance in a Y-maze, and body temperature are consistently observed (Marks et al. 1986a, b). We therefore propose that the destabilizing action of nicotine exposure on respiratory rhythm described here does not reflect a difference in nicotine dosage, but rather the fact that we analysed breathing pattern in the period immediately after birth (P0–P3).

Mechanism of nicotine's action on respiratory behaviour

The adverse effects of prenatal nicotine exposure on nervous system development are well established. They are mediated in part through activation of native receptors by exogenous nicotine which results in premature neuronal differentiation, reduction in neuron numbers (Navarro et al. 1989; McFarland et al. 1991) and disruption of cholinergic (and catecholaminergic) receptor expression patterns (Slotkin, 1998; Slotkin et al. 1999). The consequences of these actions for respiratory pattern in mouse and other rodents in vivo have been summarized above yet the underlying changes in the central respiratory control system responsible for the altered behaviour have received little attention. The increased incidence of apnoea observed in vivo could be central or obstructive in nature with multiple factors contributing including nicotine-mediated disruption of: central rhythm-generating networks, transmission pathways that transmit rhythmic drive to respiratory motoneurons, respiratory motoneurons and their modulatory systems, afferent control systems (chemosensitive or mechanosensitive) and descending inputs from higher centres normally involved in maintaining respiratory rhythm in vivo.

To assess whether disruption of rhythm-generating circuits, transmission pathways or motoneurons involved in controlling airway patency (XII motoneurons) are likely to contribute to the increased incidence of apnoea in vivo, we compared behaviour of rhythmic medullary slice preparations taken from control and nicotine-exposed animals. Rhythmic medullary slices were chosen for these experiments because they contain the ‘kernel’ of the circuit required for rhythm generation, drive transmission pathways (pattern-forming systems) and XII motoneurons. Moreover, they generate rhythm in the complete absence of afferent input, allowing the effects of nicotine on medullary circuit elements to be examined in isolation from the potentially confounding influences of nicotine on afferent control systems and higher, non-medullary centres. We hypothesized that increased variability in rhythmic activity of nicotine-exposed vs. control slices would support disruption of rhythm-generating circuits, differences in the pattern of inspiratory synaptic currents would suggest disruption of either pattern-forming systems or motoneuron glutamate receptors that mediate inspiratory drive (Funk et al. 1993), while variations in the inspiratory burst envelope recorded from XII nerves in the absence of differences in rhythm-generating networks or inspiratory synaptic currents would suggest altered XII motoneuron excitability.

In spite of the fact that respiratory rhythm, and respiratory neurons in the ventrolateral medulla, are sensitive to cholinergic (Bradley & Lucy, 1983; Bohmer et al. 1987; Monteau et al. 1990; Shao & Feldman, 2001) and catecholaminergic modulation (Johnson et al. 1996; Hilaire & Duron, 1999), and that nicotine exposure alters expression of these two receptor systems within the brainstem (Pauly et al. 1991; Slotkin et al. 1999), chronic prenatal exposure did not appear to affect baseline behaviour of the central rhythm-generating networks or drive transmission pathways. The baseline respiratory related activity generated by nicotine-exposed slices was indistinguishable from control. The average frequency, coefficient of variation and cycle-to-cycle variability were all unaffected by nicotine exposure. Inspiratory synaptic inputs to XII motoneurons in control and nicotine-exposed slices were also similar. The greater instability of rhythm in nicotine-exposed animals (reflected in increased apnoea) in vivo is therefore unlikely to reflect a change in the basal activity of rhythm- or pattern-generating circuits.

We next examined the possibility that XII motoneuron behaviour was adversely affected by nicotine. Appropriate activity in this motoneuron pool is important in preventing airway obstruction and apnoea during sleep (Sauerland & Harper, 1976; Remmers et al. 1978). Thus, these experiments were designed to assess the likelihood that improper control of the upper airway contributed to the nicotine-mediated increase in apnoea observed in vivo. Abundant evidence indicates that XII motoneurons possess the substrate upon which exogenous nicotine could act to alter motoneuron excitability. They express functional nicotinic receptors (Fig. 5; Zaninetti et al. 1999) and are modulated by cholinergic (Swanson et al. 1987; Zaninetti et al. 1999; Bellingham & Funk, 2000; Shao & Feldman, 2001) and catecholaminergic systems (Aldes et al. 1992; Funk et al. 1994; Parkis et al. 1995; Selvaratnam et al. 1998). In spite of this, XII motoneuron behaviour was unaffected by nicotine exposure. The inspiratory inputs to these motoneurons in control and nicotine-exposed animals were similar in pattern and magnitude, suggesting no change in glutamatergic transmission or receptor expression at the level of the motoneuron. Repetitive firing behaviour was also minimally affected. This was not examined at the single-cell level. However, the inspiratory burst envelopes recorded from XII nerves, which represent the average output of the XII motoneuron pool, were similar in control and nicotine-exposed slices. Thus, alterations in baseline properties of XII motoneurons are unlikely to contribute to the increased apnoea observed in vivo.

Nicotinic modulation of XII motoneuron excitability

Although nicotine exposure did not appear to alter baseline properties of rhythm-generating networks, pattern-forming circuits or XII motoneurons, the possibility remained that the increased apnoea in vivo results in part from nicotine-mediated disruption of neuromodulatory systems. As a first step in exploring this possibility we compared the effects of nicotine on XII motoneuron and inspiratory activity in slices from control and nicotine-exposed animals. We focused on nicotinic transmission within the XII nucleus for three reasons. First, disruption of cholinergic transmission by nicotine is well established (Marks et al. 1986b; Bhat et al. 1991; Pauly et al. 1991; Peng et al. 1994; Slotkin et al. 1999). Second, the homogeneous nature of the XII motoneuron pool (Viana et al. 1990), which contrasts with heterogeneous respiratory cell groups in the ventrolateral medulla (Bianchi et al. 1995), would maximize response consistency and facilitate detection of any nicotine-mediated change in cholinergic modulation. Third, it has been hypothesized that increased excitatory cholinergic inputs to upper airway motoneurons during sleep play an important role in maintaining airway patency and preventing apnoea (Bellingham & Funk, 2000). Disruption of such an excitatory input through nicotine exposure could compromise airway control and contribute to apnoea.

Consistent with this hypothesis, production of a TTX-resistant, hexamethonium-sensitive inward current in XII motoneurons of mouse established that, as in rat (Zaninetti et al. 1999), nicotine is excitatory. A reversal potential in three of 10 motoneurons near 0 mV is similar to that observed for nicotine-mediated currents in dorsal motor vagal neurons (Neff et al. 1995), and facial and XII motoneurons of rat (Zaninetti et al. 1999), and compatible with a non-selective cationic conductance.

The excitatory nature of nicotine and the potential importance of nicotinic modulation in controlling XII motoneuron excitability and maintaining airway patency was further demonstrated by nicotinic potentiation of XII nerve inspiratory activity. This potentiation is likely to reflect, at least in part, nicotine-mediated depolarization, since nicotine was without effect on neuronal input resistance. Whether facilitation of glutamatergic neurotransmission (Bordey et al. 1996; Neff et al. 1998) contributes to this potentiation requires further examination. Limited observations reported here suggest that inspiratory synaptic currents are not potentiated by nicotine.

Most important in the context of this study was that the potentiating actions of nicotine on inspiratory-related XII output were reduced by prenatal nicotine exposure. Under control conditions, XII motoneuron excitability and airway tone are established through an interaction between cholinergic, aminergic and possibly glycinergic mechanisms. During sleep, there occurs a shift in the balance between these different modulatory systems due to state-dependent increases in activity of cholinergic pontine neurons (el Mansari et al. 1989; Steriade et al. 1990), glycinergic neurons (Yamuy et al. 1999; Fung et al. 2000) and decreases in activity of serotonergic raphe neurons (Jacobs & Fornal, 1991; Leung & Mason, 1999) and noradrenergic locus coeruleus neurons (Aston-Jones & Bloom, 1981). The net consequence of these changes for excitability of XII motoneurons is apparent in the well established reduction in airway tone that occurs during sleep (Sauerland & Harper, 1976; Remmers et al. 1978). At the level of XII motoneurons, this decrease in tone probably reflects disfacilitation due to withdrawal of excitatory 5-HT (Woch et al. 1996) and noradrenergic inputs, and M2 receptor-mediated presynaptic inhibition (Bellingham & Berger, 1996) of inspiratory drive. Increased glycinergic inhibition may also contribute to the reduction in airway tone (Yamuy et al. 1999; Fung et al. 2000; but see Kubin et al. 1993). The in vitro analyses performed here emphasize the potential importance of postsynaptic nicotinic modulation to airway control. Increased nicotinic receptor activation during sleep may provide the only excitatory mechanism capable of counteracting or attenuating the numerous factors that combine to reduce XII motoneuron excitability and airway tone during sleep. Attenuation of the excitatory nicotinic effect by prenatal nicotine exposure, through upregulation (Pauly et al. 1991) of desensitized nicotine receptors (Peng et al. 1994), reduced ACh synthesis (Zahalka et al. 1992) and increased acetylcholinesterase activity (Chang et al. 1973; Mizobe & Livett, 1983), may increase vulnerability of the airway to occlusion and contribute to the increased apnoea observed in nicotine-exposed animals in vivo.

Given that airway control is determined through the interaction of numerous factors, the actual contribution of altered nicotinic modulation of XII motoneurons to the increased apnoea observed in vivo is not known. The increased apnoea may reflect the actions of nicotine on afferent processing systems, higher centres including cholinergic cell groups excluded from the slice, or non-cholinergic modulatory systems. For example, alteration in chemoreceptor function is implicated by the observations that nicotine exposure reduces dopamine content and increases tyrosine hydroxylase expression in the carotid bodies (Holgert et al. 1995), reduces hypoxic sensitivity in neonatal rats (St John & Leiter, 1999) and reduces the fall in ventilation associated with breathing 100 % O₂ (Holgert et al. 1995; Milerad et al. 1995; Bamford & Carroll, 1999). Recent evidence in rats that nicotine modulates rhythm-generating circuits (Shao & Feldman, 2001) also raises the possibility that the increased apnoea in vivo reflects disruption of nicotinic inputs to the pre-Bötzinger complex.

Implications for sudden infant death syndrome

SIDS has long been hypothesized to result from inadequate cardiorespiratory responses to acute or repeated episodes of hypoxia that occur due to airway obstruction or sleep apnoea (Hunt, 1992; Poets et al. 1993). The most significant risk factor for SIDS is maternal smoking where nicotine is a likely causative agent (Mitchell et al. 1992; DiFranza & Lew, 1995). A model that accounts for the strong statistical association between SIDS and maternal smoking (Slotkin et al. 1995) proposes that following nicotine exposure, the hypoxia-induced autonomous catecholamine release from adrenal chromaffin cells that modulates cardiovascular, respiratory and metabolic responses to hypoxia is lost before reflex control of this response is established (Slotkin et al. 1995). A developmental window of vulnerability is proposed to open when this protective mechanism is compromised. Our data indicate that during early development nicotine exposure increases incidence of apnoea. Thus, the risk of experiencing an hypoxic episode, the event that may actually trigger SIDS, is increased in nicotine-exposed animals.

Impaired ability to recover from prolonged apnoea could also increase vulnerability to SIDS (Hunt, 1987, 1992). In humans, this recovery is thought to occur early during the apnoea through normal arousal mechanisms or later in the apnoea through hypoxic gasping (autoresuscitation; Fewell & Smith, 1998). Infants of smoking mothers have an impaired arousal response to hypoxia (Lewis & Bosque, 1995). In addition nicotine impairs the ability of rat pups to autoresuscitate from repeated (Fewell & Smith, 1998), but not single (St John & Leiter, 1999), episodes of hypoxia. In this light our observation that the incidence of apnoea is greatest during recovery from hypoxia is important as it will increase the potential for repeated hypoxic exposure. Thus, we propose that maternal smoking increases the risk of SIDS by removing the autonomous adrenomedullary catecholamine release that co-ordinates protective responses to hypoxia, reducing the ability to arouse/autoresuscitate from hypoxia, while at the same time increasing the incidence of apnoea.

In summary, results indicate that prenatal nicotine exposure in mice delays development of breathing pattern, increasing the incidence of apnoea in the youngest animals before, during and after hypoxia. This increased apnoea does not reflect alterations in the baseline behaviour of rhythm-generating or pattern-forming circuits but may result from the reduced efficacy with which nicotine potentiates XII motoneuron activity.