Prenatal Exposure to Nicotine Impairs Performance of
the 5-Choice Serial Reaction Time Task in Adult Rats
Tomasz Schneider*,1, Nicholas Ilott2, Giovana Brolese3, Lisiane Bizarro3, Philip JE Asherson2and
Ian P Stolerman1
1Section of Behavioural Pharmacology, Institute of Psychiatry P049, King’s College London, London, UK;2MRC Social Genetic and Developmental
Psychiatry, Institute of Psychiatry P080, King’s College London, London, UK;3Departamento de Psicologia do Desenvolvimento e da
Personalidade, Instituto de Psicologia, Universidade Federal do Rio Grande do Sul, Porto Alegre-RS, Brazil
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.
Neuropsychopharmacology (2011) 36, 1114–1125; doi:10.1038/npp.2010.249; published online 2 February 2011
Keywords: nicotine; gestation; attention; impulsivity; dopamine system; ADHD
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 compo-
nent 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 psychia-
tric 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 B5% of
children (Polanczyk et al, 2007) and persisting into adult
life in B65% of cases (Faraone et al, 2006). Heritability for
ADHD is B76% (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 intracel-
lular transcription factor (NR4A2; Smith et al, 2005). More
recently, rare copy number variants 4500kb were found
to be over-represented in ADHD cases compared with
controls, implicating neurodevelopmental processes in the
etiology of ADHD (Williams et al, 2010).
Received 3 June 2010; revised 8 December 2010; accepted 8
University of Oxford, South Parks Road, Oxford OX1 3UD, UK,
Tel: + 44 1865 271367, Fax: + 44 1865 310447
DrT Schneider,Experimental Psychology,
Neuropsychopharmacology (2011) 36, 1114–1125
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& 2011 American College of Neuropsychopharmacology.
A range of neuropsychological performance deficits is
associated with ADHD, although none have been unequi-
vocally 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
(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,
Comparable aspects of cognitive performance can be
measured in animals. Reaction time mean and variability,
accuracy errors, omission errors, and anticipatory re-
sponses, 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).
SUBJECTS AND METHODS
Both male (N¼25) and female (N¼67) 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, n¼19, foster mothers,
n¼37) 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 (n¼11) 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±11C) 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-contain-
ing water was adjusted to the pH of drinking water (pH 7)
with 0.001N NaOH. Doses are presented as those of nicotine
Nicotine Consumption and Nicotine Blood Level
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 o10ml 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 (n¼37)
continued to receive tap water. The females (n¼11) 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
Gestational exposure to nicotine
T Schneider et al
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 o10ml 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 48 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 develop-
ment and neuromuscular maturation. Tests were selected
from standard neurobehavioral developmental test batteries
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
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 1801 turn
within 30s when placed in a head-down position on a 251
inclined wooden surface. Rats were given two consecutive
trials per day and the mean was calculated. Negative
geotaxis reflects vestibular function, motor development,
Forelimb grip strength was assessed on PND17. A steel
wire (20cm long and B0.3cm 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 erup-
tion (PND 7–10), fur appearance (PND9), and eye opening
(PND12). Eyes were recorded as open only when both eyes
Tests in Adulthood
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 venti-
lated 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 (NIC¼10, Con¼12) 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
Gestational exposure to nicotine
T Schneider et al
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 425%.
Several performance measures were recorded: percentage
of correct responses (accuracy)¼100?(correct responses/
(correct+incorrect responses) as a measure of spatial
attention; percentage of omission errors (omissions)¼
100?(omission errors/stimuli presented), reflecting atten-
tion but also influenced by the general rate of responding;
latency of correct responses¼the mean time between
stimulus onset and a nose-poke in the correct hole; latency
of incorrect responses¼the mean time between stimulus
onset and a nose-poke in an incorrect hole; anticipatory
responses as percentage of trials¼100?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
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
A separate group of 22 adult rats (NIC¼10, Con¼12)
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
Gene Expression Studies
Sample preparation. A total of 10 rats aged 5 months from
control (n¼10) and NIC (n¼10) 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 ?801C 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 contamina-
tion. 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).
Quantitative measurement of gene expression using
qRT-PCR. Housekeeping gene (HK) selection was performed
using geNorm kits (PrimerDesign, Southampton, UK).
Table 1 Consecutive Steps During 5-CSRTT Training
Gestational exposure to nicotine
T Schneider et al
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 20ml
reactions using oligoT priming and Moloney murine
leukemia virus (MMLV) reverse transcription (PrimerDe-
sign). SYBR green chemistry was used to quantify HK mRNA
following the manufacturer’s guidelines for cycling condi-
tions, 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 20ml 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 (DDCt) applied
(Livak and Schmittgen, 2001). Animals were excluded from
the analysis of all genes if they showed expression values
that were 42 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
a¼0.05 using Unistat 5.6 (Unistat, London, UK). All data
are presented as mean±SEM if not otherwise stated.
Nicotine Exposure Before and During Pregnancy
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)¼15.1, po0.001). During
the last week of habituation, when the final concentration
of nicotine solution was used, both solution (F(1,25)¼
109.2, po0.001) and food consumption (F(1,25)¼5.29,
po0.05) per kg body weight were decreased in the nicotine-
exposed group.Lower body
297.7±4.0; F(1,25)¼46.4, po0.001) and decreased solu-
tion consumption (69.6±1.4 vs 146.8±2.5ml/kg; F(1,25)¼
71.7, po0.001), but not decreased food consumption (65.1
+0.8 vs 67.1+0.9g/kg; F(1,25)¼1.59, NS), were also
observed in pregnant animals exposed to nicotine.
Nicotine Blood Levels
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
Postnatal Growth and Maturation
Prenatal nicotine exposure had no effect on the body weight
gain of the offspring (F(4,80)¼1.51, NS) but birth weights
were lower in exposed animals (Figure 1a; F(1,20)¼24.8,
po0.001). The other maturational measures used in this
Table 2 Primers Used for Amplification of Five Target Genes
Gene Sense primer (50-30) Antisense primer (50-30) Product length (bp)
ThCCCTACCAAGATCAAACCTACC CTGGATACGAGAGGCATAGTTC 96
NR4A2CTTCACAACTTCCACCACCAGAACTA GGGGCGACTGCTTAAAGGA 103
DAT1TCCAGTTACAATAAGTTCACCAATAA CGACGAAGCCAGAGGAGAA 94
Primers were designed and supplied by PrimerDesign.
Gestational exposure to nicotine
T Schneider et al
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)¼40.3,
po0.001). 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)¼27.2, po0.001).
There was no group?PND interaction.
Similarly, the ontogeny of negative geotaxis was sig-
nificantly delayed in rats prenatally exposed to nicotine
(Figure 1c; F(1,20)¼5.92, po0.03). Both groups decreased
the latencies to turn 1801 over the consecutive sessions
(F(3,60)¼15.8, po0.001). There was no group and PND
Rats prenatally exposed to nicotine also showed de-
creased grip strength on PND17 (Figure 1d; F(1,20)¼9.24,
Locomotor Activity in Adulthood
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)¼6.25, po0.03; Figure 2a); smaller
numbers of reinforcers earned (F(1,20)¼6.11, po0.03;
Figure 2c); and an increased percentage of anticipatory
responses (F(1,20)¼22, po0.0001; Figure 2d). There
was also a trend toward increased omission errors
(F(1,20)¼3.02, po0.1) and a significant group?day
interaction (F(11,220)¼1.90, po0.05; 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)¼1.81, p¼0.06), accu-
racy (F(11,220)¼1.59, p¼0.1), and the number of
reinforcers earned (F(11,220)¼1.54, p¼0.1), and there
was no between-group difference in the speed of responding
for either correct or incorrect responses (F(1,11)¼3.31,
p¼0.1 and F(1,11)¼0.04, p¼0.8, respectively). There was
a significant effect of day for all variables shown in Figure 2
(smallest F(11,220)¼5.38, po0.001) that was attributable
to a progressive improvement of performance over the 12
days for accuracy, numbers of reinforcers, and anticipa-
tions; 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)¼3.49, po0.07; group?day
interaction: F(11,220)¼2.0, po0.03; Figure 3) with sig-
nificantly increased variability on days 3 and 10. The
variability of response times for correct responses was
are shown as means±SEM (white bars, controls, n¼14; black bars, nicotine exposed, n¼8). Litter was used as a unit for analysis. *po0.05 from post hoc
tests of between-group effects by least significance difference.
Decreased birth weight (a), impairment of motor coordination (b, c) and muscle strength (d) in male rats prenatally exposed to nicotine. Data
Gestational exposure to nicotine
T Schneider et al
negatively correlated with accuracy (r¼?0.52, po0.001)
and positively correlated with anticipation rate (r¼0.42,
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
percentage correct responses (a), number of anticipations, percentage omission errors (b), number of reinforcers earned (c), and percentage numbers of
anticipatory responses (d) for 12 days when a 1-s stimulus duration was used (means±SEM).
Impairments in attentional performance in the 5-CSRTT in adult control rats (n¼12) and in nicotine-exposed rats (n¼10). Data are shown for
correct responses in the 5-CSRTT in adult control and nicotine-exposed
rats. The SD of latency for correct responses is shown as means±SEM.
Other details as for Figure 2.
Increased intraindividual variability (SD) of response times for
discounting (controls, n¼12; nicotine exposed, n¼0). Data are shown as
Lack of effect of gestational exposure to nicotine on delay-
Gestational exposure to nicotine
T Schneider et al
(delay: F(7,18)¼43.1, po0.001); however, there was no
significant effect of nicotine exposure on choice behavior at
different delays (group: F(1,18)¼1.29, NS; delay?group:
There was a significant increase in the expression of DRD5
mRNA in the striatum of animals prenatally exposed to
nicotine (U¼8, po0.006). There were no further differ-
ences 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-discount-
Nicotine Exposure and Litter Characteristics
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 consump-
tion 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
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
dopamine-related genes of adult rats; striatum (a), prefrontal cortex (b).
Data obtained by RT-PCR are shown as means±SEM for control (n¼9;
*Mann–Whitney U-test results significant at least at Po0.05.
Effects of prenatal exposure to nicotine on the expression of
(n¼8, blackbars) animals.
Gestational exposure to nicotine
T Schneider et al
of the motor and vestibular systems of the brain, but this
needs further study.
Deficits in Tests of Attention and Impulsivity
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 atten-
tion, impulsive responding, variability of reaction times,
and delay discounting using the 5-CSRTT and delay-
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 methylpheni-
date (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 (po0.1) 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 (po0.05). 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
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
did notshow significant
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, suggest-
ing 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 unex-
pected, 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.
Gene Expression Analysis
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
Gestational exposure to nicotine
T Schneider et al
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 path-
ways 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. Never-
theless, 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 (OR¼1.34, 95% CI 1.21–1.50,
p¼8?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 home-
ostasis 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 condi-
tions 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
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 compensa-
tion 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.
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