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Cigarette smoking is associated with a wide variety of adverse reproductive outcomes, including increased infant mortality and decreased birth weight. Prenatal exposure to tobacco smoke, of which nicotine is a major teratogenic component, has also been linked to the acceleration of the risk for different psychiatric disorders, including conduct disorder and attention deficit hyperactivity disorder (ADHD). Whether this increased risk is influenced by the direct effects of gestational nicotine exposure on the developing fetus remains uncertain. In this study we provide experimental evidence for the effects of prenatal nicotine exposure on measures of attention and impulsivity in adult male rats. Offspring of females exposed during pregnancy to 0.06mg/ml nicotine solution as the only source of water (daily consumption: 69.6±1.4ml/kg; nicotine blood level: 96.0±31.9ng/ml) had lower birth weight and delayed sensorimotor development measured by negative geotaxis, righting reflex, and grip strength. In the 5-choice serial reaction time test, adult rats showed increased numbers of anticipatory responses and omissions errors, more variable response times, and lower accuracy with evidence of delayed learning of the task demands when the 1s stimulus duration was introduced. In contrast, prenatal nicotine exposure had no effect on exploratory locomotion or delay-discounting test. Prenatal nicotine exposure increased expression of the D5 dopamine receptor gene in the striatum, but did not change expression of other dopamine-related genes (DRD4, DAT1, NR4A2, and TH) in either the striatum or the prefrontal cortex. These data suggest a direct effect of prenatal nicotine exposure on important aspects of attention, inhibitory control, or learning later in life.
Cigarette smoking is associated with a wide variety of adverse reproductive outcomes (Jauniaux and Burton, 2007), including increased infant mortality and decreased birth weight (Ernst et al, 2001; Winzer-Serhan, 2008). Prenatal exposure to nicotine, a major teratogenic component of tobacco, modulates neurotransmitter release, gene expression, neuronal outgrowth, cell survival, and synapse formation and maturation (Dwyer et al, 2008); and has also been linked to increased risk for childhood onset psychiatric disorders including attention deficit hyperactivity disorder (ADHD) (for review, see Pauly and Slotkin, 2008; Cornelius and Day, 2009). Recent literature suggests that the association with ADHD might be mediated by genetic effects rather than the direct toxic effects of nicotine (Thapar et al, 2009; D'Onofrio et al, 2008), but this has yet to be evaluated in an animal model.
ADHD is characterized by developmentally inappropriate and impairing levels of inattentive, hyperactive, and impulsive behaviors (Kuntsi et al, 2006) affecting ~5% of children (Polanczyk et al, 2007) and persisting into adult life in ~65% of cases (Faraone et al, 2006). Heritability for ADHD is ~76% (Faraone et al, 2005). Candidate gene studies have identified associations with genetic variants within or close to dopamine (DA) system genes including the D4 and D5 receptor genes (Li et al, 2006). Other DA system genes potentially associated with ADHD include the D1 receptor (DRD1; Misener et al, 2004), the DA transporter (DAT1; Asherson et al, 2007), and the DA-related intracellular transcription factor (NR4A2; Smith et al, 2005). More recently, rare copy number variants >500kb were found to be over-represented in ADHD cases compared with controls, implicating neurodevelopmental processes in the etiology of ADHD (Williams et al, 2010).
A range of neuropsychological performance deficits is associated with ADHD, although none have been unequivocally implicated in the etiology of ADHD symptoms (Johnson et al, 2009). Furthermore, there is considerable heterogeneity in the pattern of associated cognitive deficits, leading to contemporary models of ADHD that emphasize the role of two or more independent processes (Johnson et al, 2009; Kuntsi et al, 2010). Twin studies find partially overlapping etiological influences on the two core symptom domains of inattention and hyperactivity–impulsivity (McLoughlin et al, 2007). Overall, these findings indicate that ADHD is a heterogeneous condition with distinct etiological influences conferring risk to different behavioral and neuropsychological components of the disorder.
Cognitive performance impairments are seen on tasks measuring response inhibition and sustained attention such as the continuous performance test (Johnson et al, 2009; Willcutt et al, 2005). Compared with healthy controls, individuals with ADHD make more errors of omission (index of sustained attention) and commission (index of response inhibition), and have slower and more variable response times thought to reflect impairments in arousal or cognitive-energetic processes (Epstein et al, 2001, 2003; Klein et al, 2006; Uebel et al, 2010; Andreou et al, 2007; Johnson et al, 2009). Cognitive performance deficits have also been observed in choice impulsivity measured as the tendency to choose small rewards sooner than larger rewards later (Marco et al, 2009; Paloyelis et al, 2009). The ADHD combined subtype has been linked to the tendency to discount rewards more steeply, although evidence to date is limited and somewhat inconsistent (Barkley et al, 2001; Scheres et al, 2006; Paloyelis et al, 2009).
Comparable aspects of cognitive performance can be measured in animals. Reaction time mean and variability, accuracy errors, omission errors, and anticipatory responses, thought to reflect processes related to attention and impulsivity, can be assessed with the 5-choice serial reaction time test (5-CSRTT); choice impulsivity can be assessed in delay-discounting paradigms (Winstanley et al, 2006). These aspects of cognitive function have yet to be studied in animals prenatally exposed to nicotine, although other experimental measures have been investigated. Identified effects include intolerance to hypoxia (Slotkin et al, 1995), hyperactivity (Tizabi et al, 1997; Pauly et al, 2004), cognitive impairments (choice accuracy in spatial spontaneous alteration: Levin et al, 1993; acquisition and retention of the avoidance behavior: Vaglenova et al, 2008; radial-arm maze choice accuracy: Sorenson et al, 1991), increased anxiety (Vaglenova et al, 2004), and delayed development and maturation (Peters and Ngan, 1982; Murrin et al, 1987; Schneider et al, 2010). However, these findings are not entirely consistent as some studies found no decrement in avoidance behavior and spatial learning (Bertolini et al, 1982; Paulson et al, 1993), as well as hypoactivity (LeSage et al, 2006). Prenatal nicotine exposure has also been found to produce alterations in the development of neurochemical markers for DA in offspring (Fung, 1989; Ribary and Lichtensteiger, 1989; Muneoka et al, 1999).
In this study we evaluate in an animal model whether prenatal nicotine exposure influences cognitive functions related to ADHD in adult life. In addition, maturational and developmental data were collected and the activity level in a novel environment was measured in adults. Because of the strong a priori hypothesis of altered DA regulation in ADHD, we also determined mRNA expression for markers of DA function in frontal cortex and striatum, the regions known to be involved in ADHD (Durston et al, 2010).
Both male (N=25) and female (N=67) Lister hooded rats (Harlan Olac, Bicester, UK) were used. They were housed individually (except during mating) and had ad libitum access to food and drinking fluids (tap water or nicotine solutions). Females (224–303g at the beginning of the study) were weighed three times during the week preceding the start of the experiment. The average weight was calculated for each rat. A total of 56 females were divided into two groups (NIC exposure, n=19, foster mothers, n=37) balanced according to their body weight. Nineteen of the foster mothers were randomly chosen for use as a control group for comparisons of pregnancy and litter characteristics. An additional group of females (n=11) was used to assess nicotine blood levels in pregnant animals. The national and institutional guidelines for housing and treatment were followed. Animals were maintained in a temperature-controlled environment (21±1°C) at 50% humidity and on a 12-h light/dark cycle.
Nicotine bitartrate (Sigma, St Louis. MO) was dissolved in drinking water at varying concentrations. Nicotine-containing water was adjusted to the pH of drinking water (pH 7) with 0.001N NaOH. Doses are presented as those of nicotine base.
The procedure was based on the methods of Schneider et al (2010) with some modifications. In brief, 19 females were habituated to increasing concentrations of nicotine solution (0.02, 0.04, and 0.06mg/ml) in tap water as the only source of fluid for 3 weeks before mating. The final concentration used was 0.06mg/ml. Females drinking <10ml of nicotine solution per day had supplementary access to water. Nicotine treatment was terminated on the day that pups were delivered. The female used as foster mothers (n=37) continued to receive tap water. The females (n=11) used to evaluate nicotine blood levels during the second week of pregnancy were exposed to nicotine in an identical manner and nicotine concentrations were determined using tail vein blood and gas chromatography.
Females were controlled according to their estrous cycle. Females in proestrus and estrous were mated during the dark phase of the day at the beginning of the fourth week of nicotine exposure. Nicotine solution was not withheld before mating. The day on which a vaginal plug or spermatozoa were found in the vaginal smear was defined as gestational day 0.
Pregnant females from the nicotine and control groups were weighed twice weekly. A 0.06mg/ml nicotine solution was used throughout pregnancy and its consumption was assessed daily. Rats drinking <10ml of nicotine solution on any particular day were given access to tap water for 3min. Food consumption was evaluated three times a week.
All dams were checked twice daily (before 0800 and after 1630hours) starting a few days before delivery. Deliveries completed by 0800hours were assigned to postnatal day 1 (PND1). Pups born later that day were assigned to PND1 on the following morning. Litters were examined on PND1 for obvious morphological anomalies (eg, missing digits, facial malformations, and so on), sexed by relative anogenital distance, and, in the case of litters with >8 offspring, culled randomly to eight pups with equal numbers of males and females per litter whenever possible. Both nicotine-exposed and control litters were cross-fostered to nonexposed foster mothers within 24h after birth and the pups were evaluated throughout the lactation period in terms of reflex development and neuromuscular maturation. Tests were selected from standard neurobehavioral developmental test batteries (Adams, 1986).
In all, 14 control litters (53 males) and 8 NIC-exposed litters (20 males) were used to assess development and maturation in offspring. The dam was first removed from the home cage and specific tests measuring reflex development, motor coordination, and muscle strength were applied to the offspring. All testing was conducted between 0900 and 1600hours.
To assess righting reflex, each pup was given two successive trials per day from PND 2 to 5, and the time from being placed in a supine position until it righted itself onto all four feet was recorded. The cutoff time was 30s. Surface righting reflects the development of labyrinthine and body righting mechanisms as well as vestibular function and motor development.
Negative geotaxis was observed daily from PND7 to PND10; pups were timed for completing a 180° turn within 30s when placed in a head-down position on a 25° inclined wooden surface. Rats were given two consecutive trials per day and the mean was calculated. Negative geotaxis reflects vestibular function, motor development, and activity.
Forelimb grip strength was assessed on PND17. A steel wire (20cm long and ~0.3cm thick) was supported between two poles of wood 25cm above the table covered with soft towels. The latency to fall off the wire grasped by both forepaws was measured with a maximum time of 20s and is a measure of muscle strength.
Pups from each litter were weighed on PND 1, 5, 10, 15, and 20. The emergence of physical maturation landmarks were noted, including pinnae detachment (PND3), incisor eruption (PND 7–10), fur appearance (PND9), and eye opening (PND12). Eyes were recorded as open only when both eyes were open.
Tests in adulthood were conducted on groups of 10 (NIC) to 12 (Con) animals coming from 8 (NIC) and 12 (Con) litters.
The number of cage crosses was assessed in 2-month old animals during a 60-min test session in photocell activity cages measuring 30 × 30 × 30cm (Schneider et al, 2010). The animals had no previous exposure to the cages.
Aluminum operant conditioning chambers (Cenes, Cambridge, UK) were illuminated by house lights and housed in ventilated enclosures. The curved rear wall of each chamber contained five square holes. At the entrance of each hole, a photocell monitored interruptions of an infrared-light beam and at the rear there was a green light-emitting diode. A tray for delivering food pellets was located in the opposite wall, equidistant from each aperture.
The training phases of the experiments were based on procedures described elsewhere (Hahn et al, 2002). A total of 22 adult rats (NIC=10, Con=12) aged 3 months were assessed in the 5-CSRTT. They were housed singly 1 week before starting the 5-CSRTT. The mean weight of each animal was calculated as the average of the three weights from that week. The start point for each individual rat on the growth curve was identified and the body weight of each rat was reduced to 85% of its free-feeding weight by restricting the amount of food given during the following week. The experiment started on the fourth day of food restriction. Training was initiated by habituation to the chamber and magazine training, followed by attentional training beginning with response holes illuminated for 10s (stimulus duration), followed by the introduction of progressively more demanding task parameters (Table 1). In the final stage of training, a stimulus light in a randomly chosen hole was illuminated for 1s. If a subject nose-poked into a hole while it was illuminated or within 5s after the light had terminated (limited hold), a 45mg food pellet (BioServ, Frenchtown, NJ) was delivered into the food tray and a correct response was registered.
A response into any other hole during that time was recorded as an incorrect response and resulted in a 5-s time-out during which the house light was extinguished. A failure to respond before the end of the limited hold was registered as an omission error and had no programmed consequences until animals reached step 3 of the procedure, when a time-out of 5s duration was introduced (Table 1).
The next trial was initiated immediately after a correct response was made or at the end of the time-out that followed an incorrect response. The mean duration of the intertrial interval (ITI) was 5s; individual ITIs varied randomly within the range of 0.625–9.375s. Responses during ITIs were recorded as anticipatory responses and resulted in a time-out of 3s duration starting from step 3 of the procedure (responses during the time-outs were not counted as anticipatory responses). All training and test sessions lasted for 30min. Rats were advanced into consecutive experimental stages when their accuracy (percentage of correct responses) reached 70% and number of omissions was not >25%.
Several performance measures were recorded: percentage of correct responses (accuracy)=100 × (correct responses/(correct+incorrect responses) as a measure of spatial attention; percentage of omission errors (omissions)=100 × (omission errors/stimuli presented), reflecting attention but also influenced by the general rate of responding; latency of correct responses=the mean time between stimulus onset and a nose-poke in the correct hole; latency of incorrect responses=the mean time between stimulus onset and a nose-poke in an incorrect hole; anticipatory responses as percentage of trials=100 × total number of responses in ITIs/number of trials, as a measure of impulsive responding; reinforcers earned, equal to absolute number of correct responses in a session, as a measure of overall success of task performance. A measure of the variability of correct response times was introduced. Sessions were divided into three periods of 10min for each of which the mean latency was recorded. The measure of variability was the SD of the mean latencies for the three 10-min periods.
Standard experimental chambers (Campden Instruments, London, UK) were contained in sound-insulated, ventilated enclosures. The chambers were fitted with two retractable levers separated by a recess in which 45mg pellets of food could be presented. White noise was present at all times to mask external sounds. The experiments were controlled by programs written with the Arachnid system (Paul Fray, Cambridge, UK) running under RISC OS on Acorn computers.
A separate group of 22 adult rats (NIC=10, Con=12) aged 3 months were assessed in the delay-discounting test. They were habituated to experimental chambers during two 30min sessions with reward pellets being delivered every 30s. Training was conducted over three phases and was based on previously described experimental procedures (Winstanley et al, 2004). In the first phase, rats were trained to press the left or right levers on alternate sessions to receive a 45mg food pellet (BioServ). Each 30min session consisted of 60 trials. Subjects were trained for four sessions until all earned at least 50 rewards per session. In the second phase, rats were trained in 45min sessions divided into three blocks. During the first two blocks, 50 trials each, only one lever, either right or left, was presented. During the third block, two forced trials (only one lever presented) were followed by 48 free choice trials (two levers presented). The second phase lasted for 6 days until all animals had reached 0% of omissions on two consecutive days. During the third phase each rat had one lever designated as the ‘immediate' delivery lever (one pellet) and one lever as the ‘delay' delivery lever (5 pellets), with a delay of 2s. Each session consisted of 24 trials, divided into 3 blocks of 8 trials, with trials spaced apart by 100s. Each 8-trial block began with 2 ‘forced' trials in which either the left or the right lever was presented in random order for every pair of trials, followed by 6 ‘choice' trials in which both levers were presented. Levers assignments were counterbalanced across groups. The third phase lasted for 4 days until all animals had reached 90% preference for delayed larger reward on two consecutive days.
The main delay-discounting procedure was identical to the one used in the third phase of training, except that the delay to the larger reward was increased daily according to the sequence of 2, 6, 18, 36, 48, 54, 60, and 66s. Choice ratios (delay-lever presses/total lever presses) were calculated for each rat at each delay using the choice trial responses (ie, excluding single lever trials) summed across the three consecutive blocks.
A total of 10 rats aged 5 months from control (n=10) and NIC (n=10) groups were killed by decapitation and the brains were immediately dissected. The striatum and a 2-mm slice from the frontal cortex were removed, snap frozen on dry ice, and stored at −80°C until RNA extraction. RNA was extracted using Qiagen AllPrep RNA/DNA minikits (Qiagen, Crawley, UK). During the extraction procedure, RNA columns were treated with RNase-free DNase1 to eliminate genomic DNA contamination. Purity of RNA samples was assessed via the 260/280-wavelength ratio using a NanoDrop spectrophotometer. All ratios were of acceptable quality (RNA range; 1.88–2.38).
Housekeeping gene (HK) selection was performed using geNorm kits (PrimerDesign, Southampton, UK). The stability of 11 commonly used HK genes was assessed using 500ng total RNA from 4 samples of each group. Samples were first strand reverse transcribed in 20μl reactions using oligoT priming and Moloney murine leukemia virus (MMLV) reverse transcription (PrimerDesign). SYBR green chemistry was used to quantify HK mRNA following the manufacturer's guidelines for cycling conditions, with all samples run in duplicate (www.primerdesign.co.uk). GeNorm, a Visual Basic application tool in Excel, was used to statistically model the stability of the HK genes for accurate normalization of target genes. The geNorm output provides the user with the two most stably expressed HK genes, along with stability values for all genes analyzed. We chose three HK genes for normalization of target genes in each tissue: Cyc1, Mdh1, and Ywhaz for striatum and Cyc1, Mdh1, and Gapdh for frontal cortex.
Primers and PerfectProbe technology (PrimerDesign) were used to quantify five target genes; Th, Nr4a2, Slc6a3, Drd4, and Drd5, along with the three HK genes for each tissue. Primer sequences for target genes are given in Table 2. Total RNA was first strand cDNA synthesized in 20μl reactions using oligoT priming and MMLV reverse transcription; qRT-PCR reactions were performed in triplicate.
Behavioral data were analyzed using one- or two-factor ANOVA followed by Bonferroni modified least significant difference test (LSD) for post hoc analysis. For maturational and developmental data, litter (only males) was used as the unit for statistical analysis. Thus, the data subjected to statistical analyses were means for entire litters rather than results for individual animals within litters. The 5-CSRTT percentage data for accuracy and omissions were arcsine transformed, and latency data were log transformed (Hahn et al, 2002). Spearman's rank correlation test was used to correlate measures obtained in the 5-CSRTT. For those variables assessed multiple times, age (PND) and day of training were used as repeated measures.
Gene expression results (qRT-PCR data) were compared using the Mann–Whitney test. The Grubbs method was applied to identify outliers from triplicate samples (Burns et al, 2005) after which arithmetic means were taken across replicates and the comparative Ct method (ΔΔCt) applied (Livak and Schmittgen, 2001). Animals were excluded from the analysis of all genes if they showed expression values that were >2 SD from the mean in a given group for at least two genes (one animal from control and two animals from NIC group). All tests of significance were performed at α=0.05 using Unistat 5.6 (Unistat, London, UK). All data are presented as mean±SEM if not otherwise stated.
Three weeks of pre-exposure to increasing doses of nicotine as the only source of water resulted in decreased body weight before mating (F(1,25)=15.1, p<0.001). During the last week of habituation, when the final concentration of nicotine solution was used, both solution (F(1,25)=109.2, p<0.001) and food consumption (F(1,25)=5.29, p<0.05) per kg body weight were decreased in the nicotine-exposed group. Lower body weight (255.8±4.7 vs 297.7±4.0; F(1,25)=46.4, p<0.001) and decreased solution consumption (69.6±1.4 vs 146.8±2.5ml/kg; F(1,25)=71.7, p<0.001), but not decreased food consumption (65.1+0.8 vs 67.1+0.9g/kg; F(1,25)=1.59, NS), were also observed in pregnant animals exposed to nicotine.
The mean plasma nicotine blood level during the second week of pregnancy was 96.0±31.9ng/ml (mean±SD). There was no difference in mean nicotine solution consumption per kg body weight per day between the groups of nicotine-exposed pregnant females used for nicotine blood tests or for offspring delivery (67.9±8.9 vs 69.6±1.4ml/kg, corresponding to 4.07±0.05 vs 4.17±0.08mg/kg of nicotine, respectively).
There was no difference between control litters and those prenatally exposed to nicotine in any of the measures used: the number of live litters (11 cf 14), the percentage of live litters (57.9 cf 73.7), number of animals per litter (5.9±0.6 cf 5.7±0.8), the numbers of females and males per litter (2.1±0.3 vs 3.0±0.5 and 2.9±0.6 vs 2.0±0.4, respectively), and numbers of dead or malformed animals (1.36±0.5 vs 0.73±0.6).
Prenatal nicotine exposure had no effect on the body weight gain of the offspring (F(4,80)=1.51, NS) but birth weights were lower in exposed animals (Figure 1a; F(1,20)=24.8, p<0.001). The other maturational measures used in this study (pinnae detachment, fur appearance, incisor eruption, and eye opening) did not differ between the groups.
The ontogeny of the righting reflex was delayed in animals prenatally exposed to nicotine (Figure 1b; F(1,20)=40.3, p<0.001). Rats in both groups showed decreased latencies to right themselves onto all four feet from a supine position over the consecutive sessions (F(3,60)=27.2, p<0.001). There was no group × PND interaction.
Similarly, the ontogeny of negative geotaxis was significantly delayed in rats prenatally exposed to nicotine (Figure 1c; F(1,20)=5.92, p<0.03). Both groups decreased the latencies to turn 180° over the consecutive sessions (F(3,60)=15.8, p<0.001). There was no group and PND interaction.
Rats prenatally exposed to nicotine also showed decreased grip strength on PND17 (Figure 1d; F(1,20)=9.24, p<0.01).
There was no difference between nicotine-exposed and control animals in the number of cage crosses during a 60-min session (57.4±8.8 vs 59.9±9.3, respectively).
There was no difference between control and nicotine-exposed animals during acquisition of the task when the duration of the visual stimuli was either 10 or 5s. However, at the final stage when a 1-s stimulus duration was used, the performance of rats prenatally exposed to nicotine was compromised (Figure 2). Under this condition, adult rats prenatally exposed to nicotine exhibited: decreased accuracy (F(1,20)=6.25, p<0.03; Figure 2a); smaller numbers of reinforcers earned (F(1,20)=6.11, p<0.03; Figure 2c); and an increased percentage of anticipatory responses (F(1,20)=22, p<0.0001; Figure 2d). There was also a trend toward increased omission errors (F(1,20)=3.02, p<0.1) and a significant group × day interaction (F(11,220)=1.90, p<0.05; Figure 2b); the numbers of omission errors were increased during the first 2 days after introduction of the 1-s stimulus duration and on day 5. There was no group × day interaction for anticipatory responses (F(11,220)=1.81, p=0.06), accuracy (F(11,220)=1.59, p=0.1), and the number of reinforcers earned (F(11,220)=1.54, p=0.1), and there was no between-group difference in the speed of responding for either correct or incorrect responses (F(1,11)=3.31, p=0.1 and F(1,11)=0.04, p=0.8, respectively). There was a significant effect of day for all variables shown in Figure 2 (smallest F(11,220)=5.38, p<0.001) that was attributable to a progressive improvement of performance over the 12 days for accuracy, numbers of reinforcers, and anticipations; only the pattern of omission errors did not show an orderly relationship over days.
Rats prenatally exposed to nicotine showed signs of an increased variability of response times for correct responses (group: F(1,20)=3.49, p<0.07; group × day interaction: F(11,220)=2.0, p<0.03; Figure 3) with significantly increased variability on days 3 and 10. The variability of response times for correct responses was negatively correlated with accuracy (r=−0.52, p<0.001) and positively correlated with anticipation rate (r=0.42, p<0.001).
Both nicotine-exposed and control animals chose the large reward on almost every trial when the delay to the large reward was 2s (Figure 4). As the delay to the large reward increased, the preference of both groups of rats shifted toward the smaller but more immediate reward (delay: F(7,18)=43.1, p<0.001); however, there was no significant effect of nicotine exposure on choice behavior at different delays (group: F(1,18)=1.29, NS; delay × group: F(7,129)=0.73, NS).
There was a significant increase in the expression of DRD5 mRNA in the striatum of animals prenatally exposed to nicotine (U=8, p<0.006). There were no further differences between the two groups for any genes in either tissue (Figure 5a and b).
In this study we present the first experimental evidence of a link between prenatal nicotine exposure and cognitive performance deficits on the 5-CSRTT in adult rats. Following gestational exposure to nicotine, the offspring were found not only to have lower birth weight and delayed sensorimotor development, but also to be impaired during adulthood with respect to several measures of performance of the 5-CSRTT. In contrast, nicotine exposure had no effect on the locomotor activity of adult rats in a novel environment or on impulsive choice in the delay-discounting test.
The daily nicotine consumption of the pregnant mothers of 4.61±0.54mg/kg resulted in nicotine blood levels of 96±31.9ng/ml, which is at the upper end of the dose range for heavy smokers (Benowitz et al, 2009). In line with previous animal studies (eg, Murrin et al, 1987; Schneider et al, 2010), females exposed to a nicotine solution as the only source of fluid during pregnancy showed decreased body weight gain and lower solution and food consumption, although the latter was not significant in the present study. The implications of the reduced weights of the nicotine-exposed mothers and decreased food and water consumption need further investigation. Prenatal exposure to nicotine had no effect on the number of live litters, litter size, numbers of males and females per litter, or the numbers of malformed or dead offspring, suggesting only mild teratogenicity of the nicotine dose regimen used in this study.
Birth weight was decreased by prenatal exposure to nicotine, although there was no difference in weight gain during development (Figure 1a). This was expected and is similar to the results of human studies (Eskenazi et al, 1995). The offspring of animals exposed to nicotine in utero consistently show lower birth weights (Paulson et al, 1993; Peters and Ngan, 1982; Schneider et al, 2010); and in humans, the direct impact of prenatal nicotine exposure on birth weight remains after controlling for maternal genetic influences (Thapar et al, 2009). The long-term significance of lower birth weight is still unclear, but studies in humans have found associations between low birth weight and long-term cognitive deficits (Hack, 2006; Gianni et al, 2007) and behavioral disorders including ADHD (Winzer-Serhan, 2008). Recent evidence from monozygotic twin pairs shows that low birth weight confers a direct risk of ADHD that is independent of genetic effects (Greven et al, 2010).
Other maturational measures used in this experiment (pinnae detachment, fur appearance, incisor eruption, and eye lid opening) were spared in offspring prenatally exposed to nicotine. In contrast, developmental measures were all compromised. Significant delay of the righting reflex and negative geotaxis, as well as a shorter latency to fall in the grip strength test, were observed in rats prenatally exposed to nicotine, suggesting impairment of motor coordination and muscle strength (Figure 1). Our results are in line with previous studies showing deficits in righting reflex and negative geotaxis in rats and mice exposed to similar doses of nicotine (Peters and Ngan, 1982; Ajarem and Ahmad, 1998; Schneider et al, 2010). The delay in attaining these skills is probably because of damage or poor development of the motor and vestibular systems of the brain, but this needs further study.
Previous studies have demonstrated deficits in learning and memory in adult rats prenatally exposed to nicotine (Vaglenova et al, 2008; Levin et al, 1993), whereas the present report investigates possible impairments in attention, impulsive responding, variability of reaction times, and delay discounting using the 5-CSRTT and delay-discounting tasks.
The development of the 5-CSRTT for rats was initially stimulated by the need to understand, at a preclinical level, the nature of the deficits shown by children with ADHD and the effects of psychostimulant drugs such as methylphenidate (Robbins, 2002). The task is modeled after Leonard's 5-CSRTT used to study human attentional processes and is considered to have similarities with the continuous performance test of attention (Robbins, 2002). When stimulus duration in the 5-CSRTT is as short as 1s, the procedure is regarded as a means for assessing sustained attention rather than simply discriminated responding.
In the prenatal exposed nicotine group, we observed a trend (p<0.1) in the rate of omission errors in the 1s stimulus condition, which improved during the course of the 12 days of testing, with a significant group by day interaction (p<0.05). The observed impairment therefore reflects a delayed ability to learn a task with a high attentional load, which could reflect a deficit of attentional processing or more general learning difficulties. However, the group × day interaction was significant only for omission errors and only in the 1s stimulus condition, suggesting that the learning difficulty was restricted to a task condition that demanded high levels of sustained attention. This interpretation should be balanced by the possibility that the study might be underpowered to detect significant day by group interactions for the other variables, which would then indicate a more general learning difficulty.
In considering whether the pattern of increased omission errors in the 5-CSRTT is comparable with findings in ADHD, the study design with repeated daily measures needs to be taken into account. To the authors' knowledge, no comparable studies have been performed in ADHD with repeated daily measures, and hence it is not known whether performance in children and adults with ADHD would improve and catch up with the performance of healthy controls.
The other significant impairments that emerged in the 1s stimulus condition, which did not show significant group × day interactions, included decreased accuracy, increased anticipatory responses, smaller number of earned rewards, and response time variability (RTV). Accuracy in the task is thought to represent processes related to sustained attention, whereas anticipatory responses during the intratrial periods are thought to reflect a form of impulsive responding. Neither of these measures has been widely adopted in ADHD research, and hence it is difficult to make direct comparisons. Accuracy is rarely included in ADHD studies because there are marked ceiling effects in equivalent human tasks such as the fast task (Andreou et al, 2007), with both cases and controls showing very low rates of accuracy errors. In contrast, anticipatory responses have been evaluated in a few studies and are found to be significantly increased in children with ADHD compared with healthy controls (Bedard et al, 2003; Wada et al, 2000).
The RTV in the rats correlated negatively with accuracy scores and positively with anticipatory responses, suggesting that a general deficit might underlie the pattern of findings that link RTV to changes in attention and anticipatory responses. However, the change in RTV in the rat model may not reflect the same processes that lead to increased RTV in human disorders such as ADHD. First, increased RTV in humans with ADHD occurs under slow unrewarded conditions and tend to normalize under rewarded conditions (Andreou et al, 2007; Uebel et al, 2010), whereas responses in the 5-CSRTT are rewarded. Second, the measure of RTV used in this study is the SD of data averaged across three 10-min periods, which is different from the trial-by-trial variability associated with ADHD (Klein et al, 2006).
In the delay-discounting task, which measures a specific aspect of choice impulsivity, there was no difference detected between nicotine-exposed and controls rats. Human research suggests an association between ADHD and performance on delay-discounting tasks in children, although this is not consistently found in all children with ADHD (Marco et al, 2009; Paloyelis et al, 2009) and has not been studied in adults with ADHD. The discrepancy in our findings between impulsive responding indexed by anticipatory responses in the 5-CSRTT and the delay-discounting test is not unexpected, because these measure entirely different aspects of impulsivity, consistent with the nonunitary nature of impulsive behavior in humans (Evenden, 1999; Moeller et al, 2001; McDonald et al, 2003; Patton et al, 1995) and animals (see Winstanley et al, 2006 for review).
In utero nicotine exposure has also been associated with ‘hyperactivity' in humans as measured by a combined parental rating of restlessness, being fidgety, unable to settle, and easily distracted (Kotimaa et al, 2003), but no studies have used actigraph data. Overactivity in ADHD has been shown to be more pronounced under constant (habituated) and unstimulating conditions and to normalize in novel or stimulating environments (Antrop et al, 2000; Sagvolden et al, 1998). In this study we evaluated activity during a single activity test session, reflecting exploratory activity in a novel environment. Furthermore, the lack of effect of prenatal exposure to nicotine on exploratory locomotor activity in adult rats contrasts with some previous reports (Tizabi et al, 1997; Pauly et al, 2004; Ajarem and Ahmad, 1998), but agrees with others (LeSage et al, 2006; Romero and Chen, 2004). We did however observe increased locomotor activity after repeated testing of adolescent rats exposed prenatally to nicotine (Schneider et al, 2009), which accords better with the human literature on ADHD.
The most probable direct effects of prenatal nicotine exposure would be on nicotinic acetylcholine (ACh) systems (Slotkin, 2004) but given the close anatomic association of the ACh and the DA systems, it is likely to have secondary effects on the DA system (Shea and Steiner, 2008). In this study we focused on the DA system because dysregulation of DA signaling has been clearly implicated in processes leading to deficits of attention and impulsive responding. Animal studies indicate that prenatal exposure to nicotine has lasting effects on behaviors regulated by DA, including locomotor activity, stereotypy, and drug self-administration (Tizabi et al, 1997; Ajarem and Ahmad, 1998; Levin et al, 2006; Paz et al, 2007; Franke et al, 2008). This study looked for long-lasting effects of prenatal nicotine exposure on quantitative expression of the DA-related genes NR4A2, TH, DAT1, DRD4, and DRD5 that index DA regulatory function or have been reported to be associated with ADHD in genetic association studies (Waldman and Gizer, 2006; Gizer et al, 2009). We investigated gene expression in the rat striatum and frontal cortex because cortico-striatal pathways have been strongly implicated in ADHD (Castellanos, 2001) as well as attention and impulsive decision-making processes (Muir et al, 1996; Rogers et al, 2001; Cardinal, 2006; Winstanley et al, 2006).
There was little evidence for expression differences between the two groups for any of the genes studied in either tissue, although there was a small increase in DRD5 mRNA expression in the striatum of nicotine-exposed animals. Whether such a small difference is capable of influencing behavior remains an open question. Nevertheless, human studies suggest that DRD5 might be an important gene for ADHD, with evidence for the association of a specific genetic marker close to the DRD5 gene providing some of the strongest evidence for association with ADHD in children (OR=1.34, 95% CI 1.21–1.50, p=8 × 10−8) in a meta-analysis of nine independent studies (Li et al, 2006). Furthermore, the allele-specific association was recently replicated in a sample of adult patients with ADHD (Johansson et al, 2008). Interestingly, the DRD5 repeat polymorphism was reported to be associated with lower performance scores on the TOVA continuous performance test in ADHD patients and their parents (Manor et al, 2004).
This study has two main limitations. First, the possible teratogenic effects of prenatal exposure to nicotine cannot be clearly distinguished from the potential effects of dehydration and stress on the rodents given nicotine. For example, restriction of water intake during pregnancy induces marked alterations in maternal–fetal fluid homeostasis and reduces birth weights in newborns (Ross and Desai, 2005). Direct tests on the behavioral effects of gestational dehydration on rats do not seem to have been published and an impact on the cognitive performance measures used in this study cannot be excluded. The nicotine-exposed offspring were also low in birth weight, and low birth weight has been associated with several neuropsychological disorders including ADHD (Casper, 2004). Further studies are therefore needed to control for these potential confounds.
Second, although it was clear that performance of the 5-CSRTT was impaired on several parameters, the long-term persistence of effects was not demonstrated and the nature of the impairments therefore remains uncertain. As task performance was not stable when impairments were seen, these effects may have involved learning processes that are not specific to attentional tasks.
The findings indicate a direct impact of the prenatal environment on important aspects of cognition and inhibitory control later in life. The precise mechanisms by which such long-term impacts on behavior arise remain unknown, but are likely to involve epigenetic changes induced by exposure to the environmental factors (Mill and Petronis, 2008). The preclinical data presented in this study challenge the conclusion that the observed association between ADHD and maternal smoking in pregnancy is mediated entirely by genetic effects (Thapar et al, 2009; D'Onofrio et al, 2008), by showing that direct experimental manipulation of the prenatal environment, under conditions where genetic variance is controlled by the use of the same rat strain in the experimental and control samples, leads to cognitive changes that could contribute to components of the ADHD phenotype; including impulsive responding and an increase in errors during tasks with a high attentional load. Further research is required to control for potential confounding factors; yet, these data indicate the importance of the prenatal environment for aspects of inattentive and impulsive behavior in adulthood.
The research was supported by a grant from the Wellcome Trust (079314). We are grateful to Dr Jonna Kuntsi for helpful discussions.
Philip JE Asherson has received funding for his work on advisory boards, consultancy, or industry-sponsored educational activities from Janssen-Cilag, Eli-Lilly, Shire, and Flynn Pharma. He does not possess any relevant financial holdings. Ian P Stolerman has received compensation in the past 3 years for professional services to Institut de Recherches Servier, European Monitoring Centre for Drugs and Drug Addiction, Elsevier Science Publishers, Springer-Verlag, and the US National Institute on Drug Abuse. He does not possess any relevant financial holdings. The other authors declare no conflict of interest.