Journal of Behavioral and Brain Science, 2013, 3, 85-99
http://dx.doi.org/10.4236/jbbs.2013.31009 Published Online February 2013 (http://www.scirp.org/journal/jbbs)
Neurodevelopmental Timing of Ethanol Exposure May
Contribute to Observed Hetero geneity of Behavioral
Deficits in a Mouse Model of Fetal Alcohol Spectrum
Katarzyna Mantha, Morgan Kleiber, Shiva Singh*
Department of Biology, Molecular Genetics Unit, Western University, London, Canada
Email: firstname.lastname@example.org, email@example.com, *firstname.lastname@example.org
Received November 8, 2012; revised December 10, 2012; accepted December 17, 2012
Maternal drinking during pregnancy can result in a wide spectrum of cognitive and behavioral abnormalities termed
fetal alcohol spectrum disorders (FASD). The heterogeneity observed in FASD-related phenotypes can be attributed to
a number of environmental and genetic factors; however, ethanol dose and timing of exposure may have significant
influences. Here, we report the behavioral effects of acute, binge-like ethanol exposure at three neurodevelopmental
times corresponding to the first, second, and third trimester of human development in C57BL/6J mice. Results show
that developmental ethanol exposure consistently delays the development of basic motor skill reflexes and coordination
as well as impairs spatial learning and memory. Observed changes in activity and anxiety-related behaviors, however,
appear to be dependent on timing of alcohol exposure. The variability in behaviors between different treatment models
suggests that these may be useful in evaluating the mechanisms disrupted by ethanol at specific neurodevelopmental
times. The results provide further evidence that, regardless of developmental stage, the developing brain is acutely sen-
sitive to alcohol exposure.
Keywords: Fetal Alcohol Spectrum Disorder (FASD); Ethanol; Behavior; Neurodevelopment; Mouse Model;
Maternal alcohol consumption during pregnancy can
result in morphological, behavioral and neurological ab-
normalities, collectively termed Fetal Alcohol Spectrum
Disorders (FASD) [1,2]. The prevalence of FASD is es-
timated to be approximately 1 in 100 live births in North
America, and the occurrence and severity of FASD phe-
notypes, including variable behavioral effects, have been
attributed to the timing and dosage of alcohol [3-6]. The
nature of the heterogeneity associated with variability of
manifestation is poorly understood, as is the mechanism
that causes these phenotypes to persist throughout the
lifetime of an individual. These phenotypes often include
delayed early-life development of motor control and co-
ordination, hyperactivity, increased risk for anxiety-re-
lated psychopathologies, impulsivity, inattentiveness and
intellectual impairment [7-13]. Further, many children
with FASD show deficits in cognition and learning, ex-
ecutive functioning, memory and social adaptation [13-
A number of animal models have been used to explore
the relationship between ethanol exposure and specific
FASD-related phenotypes [11,18-21]. Specifically, the
generation of well-established behavioral battery proto-
cols has led to a better characterization of behaviors and
cognition in animals that have been prenatally exposure
to alcohols. In particular, the C57BL/6J mouse has been
shown to be acutely sensitive to both the physiological
and the behavioral effects of neurodevelopmental ethanol
exposure. This strain of mouse has been successfully used
to replicate a number of FASD-relevant phenotypes in-
cluding impairments in cognitive function, activity levels,
novel-environment anxiety, and depression-related beha-
viors [22-27]. Such results are considered representative
of humans for a variety of reasons, including the fact that
rodent and human neurodevelopmental timelines are com-
parable [28-30], which allows for experimentation on
timing-of-exposure [31-33]. Most previous work, how-
ever, differs substantially in experimental factors such as
ethanol dosage, timing of exposure, time of testing, be-
opyright © 2013 SciRes. JBBS
K. MANTHA ET AL.
havioral phenotype evaluated, and testing protocol, mak-
ing comparisons across studies difficult. Thisstudy is no-
vel as it assesses the effects of exposure at specific neu-
rodevelopmental times using a consistent dosage regi-
ment. The results will provide a realistic framework for
Specifically, we have used the C57BL/6J (B6) mouse
strain to model the behavioral effects of acute, binge-like
alcohol exposure at three neurodevelopmental time po-
ints corresponding to human trimesters one (T1), two (T2)
and three (T3). There is evidence that binge alcohol ex-
posures are prevalent in pregnant women of certain high-
risk groups, and can lead to FASD-related clinical diag-
noses in resulting children [34,35]. We evaluated the re-
sulting offspring across a battery of behavioral tests rele-
vant to FASD-associated phenotypes from early neonatal
development to adulthood. The results offer a perspective
on the range of neurological and cognitive functions af-
fected by alcohol that is dependent on timing of expo-
2. Materials and Methods
2.1. Animals and Breeding
Male and female C57BL/6J mice were originally ob-
tained from Jackson Laboratories (Bar Harbor, ME, USE)
and subsequently bred at the Health Sciences Animal
Care Facility at the University of Western Ontario. All
procedures involving the use of mice met the ethical
standards outlined by the Canadian Council on Animal
Care and were approved by the Animal Use Subcommit-
tee of the University. Mice were housed in standard same-
sex colony cages with a temperature range of 21˚C -
24˚C and at 14-h light/10-h dark schedule, with free ac-
cess to food and water. For breeding, females of appro-
ximately eight weeks of age were housed in individual
cages and time-mated overnight with 8 - 12 weeks old
males. At the end of the mating period, females were
examined for presence of a vaginal plug, indicating ges-
tational day 0 (G0), and males were removed from cages.
2.2. Ethanol Injections
Pregnant females were randomly assigned to two groups
in each of the three trimesters: control dams injected
subcutaneously (abdomen) with 0.15 M saline solution
and ethanol-treated dams injected with 20% ethanol in
the saline solution at the same site. An injection of 2.5
g/kg was given twice, spaced two hours apart to model a
heavy, binge-like exposure (blood alcohol concentration
or BAC remains over 200 mg/dl for at least 4 hours) .
Subcutaneous injections were given on G8 and G11 to
model the first trimester [36,37], and on G14 and G16 to
model the second trimester equivalent [31,38]. In order
to model for the third trimester equivalent, pups at post-
natal day 4 (P4) and P7 were subcutaneously injected us-
ing the same dosage. This stage parallels the human equi-
valent of third trimester alcohol exposure [28,29,39]. It is
not possible to perfectly match such stages across species.
Consequently, these timings should be viewed as the best
approximation for the human equivalent. Dams were ran-
domly assigned to control and ethanol-treated groups.
Third trimester pups from each litter were matched where
possible to control for litter effects. The number of litters
used for T1 include 4 ethanol and 3 control litters, and 6
ethanol and 4 control litters for T2. For T3 we were able
to assign pups from the same litter to two treatments
from a total of 6 litters. Dams and pups were monitored
following ethanol injections until full recovery, and pups
were weaned on P21 into colony cages of two to four
same-sex littermates. All pups were run through the same
battery of tests and treated as statistically independent
2.3. Early Postnatal Development
From P2 to P21, pups were assessed for the ability to
reach critical developmental milestones evaluating the
appearance of age-appropriate motor skills such as bal-
ance, motor coordination, strength and reflexes, follow-
ing Hill and colleagues . Tests were performed at the
same time each day until the pup was able to perform the
task in the prescribed amount of time for two consecutive
days . The tests used included surface righting, nega-
tive geotaxis, cliff aversion, forelimb grasp, auditory star-
tle, ear twitch, open field traversal, air righting and eye
opening. Pups from T3 (P4 and P7) underwent develop-
mental milestone testing beginning at 9:30 am, followed
by injections at 12:00 pm.
2.4. Open-Field Locomotor Activity to Test
Activity and Anxiety
At P25 pups were assessed for activity in a novel open-
field environment using the infrared Actimeter system
and measured using Acti-Track software (Panlab, Barce-
lona, Spain) . We chose mice at young adolescence
following previous reports that this testing during this
period results in measurable differences between etha-
nol-treated and control mice . Also, this timing cor-
responds to the prepubescent period of development in
mice . The prepubescent period is the time markedly
used in human studies assessing disorders involving hy-
peractive behaviors [43,44]. The open field arena con-
sisted of a 45 cm (W) × 45 cm (L) surface constructed of
black plexiglass enclosed by four 35 cm-high clear acry-
lic walls, as well as an infrared frame that produced a 16
× 16 grid of intersecting beams used to track the move-
ment of each mouse. Infrared beam-break data were used
Copyright © 2013 SciRes. JBBS
K. MANTHA ET AL. 87
to calculate locomotor activity. Movement data were also
analyzed by dividing the arena into an outer periphery
zone and a central zone to allow for the evaluation of
thigmotaxis. Testing was conducted during the light phase
between 1000 h and 1300 h, and the lighting of the arena
was 100 lx. Each mouse was placed in the same corner of
the arena when beginning the trial, and was allowed to
freely explore for 15 min. At the end of the testing, the
mouse was removed and returned to its home cage. Be-
tween trials, the arena was cleaned with 30% isopropa-
2.5. Home Cage Activity Testing
Activity in a familiar environment was measured using
the infrared Actimeter system (Panlab, Barcelona, Spain).
At P35, mice were placed individually into 38 cm (L) ×
24 cm (W) × 14 cm (H) transparent plastic cages (Inno-
vive, San Diego, CA, USA) with standard woodchip bed-
ding and free access to food and water. Following a 24 h
acclimation period, cages were placed in the Actimeter
frame and testing was conducted overnight from 1900 h
to 0600 h, spanning the dark phase of the light/dark cycle
and 1 h of light at the beginning. Recordings were taken
for two consecutive nights and averaged. Mice were re-
housed in their original cages at the end of activity test-
2.6. Light/Dark Box for Testing Anxiety
The light/dark box was used as a measure of exploratory
behavior and anxiety in a novel, illuminated environment
to further characterize the thigmotaxis behavior observed
during the open-field test. The apparatus was constructed
following Crawley and colleagues  and included two
compartments consisting of a 27 cm (L) × 27 cm (W) ×
27 cm (H) light arena and a 18 cm (L) × 27 cm (W) × 27
cm (H) dark arena, with a 7.5 cm × 7.5 cm opening be-
tween the light and dark regions. At age P40, each mouse
was placed in the light arena facing the opening, and al-
lowed to freely explore both light and dark areas for 5
min. The overhead light in the room was 200 lx and all
trials were recorded by a ceiling-mounted camera. Any-
Maze digital tracking software (Stoelting, Wood Dale, IL,
USA) was used to analyze movements and track the
amount of time spent in the light arena versus the dark
arena. Following testing, the mouse was returned to its
2.7. Barnes Maze to Test Spatial Learning and
A modified version of Barnes maze for mice was con-
structed following Sunyer and colleagues  and was
conducted as previously described . Briefly, the test
consisted of four 3-minute training trials per day, spaced
15 minutes apart, over four consecutive days beginning
at P50 (acquisition days). For each trial, the mouse was
placed in a start chamber in the centre of the platform,
and after 10 seconds, an overhead 220 lx light and 85 dB
computer-generated white noise were turned on, and the
mouse was released to explore the platform. The trial
ended once the mouse successfully located the target and
entered the escape box. The light and white noise were
terminated immediately after entry. If the 3 minutes had
elapsed without the mouse entering the escape box, the
mouse was guided into the box by the experimenter and
left for 1 minute undisturbed. Between trials, the plat-
form was cleaned with 30% isopropanol. Trials were also
qualitatively scored based on whether the mouse used a
direct strategy (mouse moves directly to the escape hole
or an adjacent hole), a serial strategy (the first visit to the
escape box was preceded by visiting at least two adjacent
holes in a serial manner, in a clockwise or counter-clock-
wise direction), or a mixed strategy (hole explorations
were separated by crossing through the centre of the
maze or unorganized search).
The day following the last acquisition day (day 5 of
testing), the escape box was covered and the mouse was
allowed to explore the maze for 1 minute before being
returned to its home cage (probe trials). The number of
errors and explorations to the target hole were recorded.
The same probe trial protocol was also performed seven
days following the first probe trial (day 12 of testing)
without further training between these days. Trials were
recorded using a ceiling-mounted camera and analyzed
using AnyMaze digital tracking software (Stoelting, Wood
Dale, IL, USA) for latency to reach the escape box. Ex-
plorations and search strategy was scored manually by an
independent observer unaware of the treatment group.
2.8. Statistical Analysis
Data were analyzed using appropriate analysis of vari-
ance methods depending of the number of independent
variables (sex, treatment, repeated day of testing). Where
data were analyzed across days, repeated-measures ana-
lysis of variance (ANOVA) with treatment as the be-
tween-subjects factor and day as the within-subjects fac-
tor was used. For overnight activity, we performed hypo-
thesis-based, step-down analyses and corrected for mul-
tiple testing. All data are reported as mean ± standard
error of the mean. For the Barnes maze acquisition days,
data were log3-transformed to account for the differences
in variance between latencies to reach the target across
days. Data analysis was stratified by sex if sexually di-
morphic effects were observed. Effects of litter were ana-
lyzed using “litter” as a covariate in the ANOVA, how-
ever, no litter effects were observed for any of the beha-
Copyright © 2013 SciRes. JBBS
K. MANTHA ET AL.
vioral measures. All statistical analyses were performed
using SPSS v.16 (SPSS Inc, Chicago, IL, USA).
3.1. Postnatal Developmental Milestones
All pups were evaluated from P2 to P21 for the abilities
to reach critical neurodevelopmental milestones. The ave-
rage day each specific milestone was achieved by each
treatment group is shown in Table 1. The ethanol expo-
sure during T1 significantly delayed the ability of pups to
surface right themselves when placed on their backs
(F1,46 = 29.90, p < 0.001), grasp a rod with their fore-
limbs (F1,46 = 15.50, p < 0.001), extinguish pivoting be-
havior and transverse out of a 15 cm-diameter circle
(F1,46 = 15.10, p < 0.001), and right themselves in the air
when dropped from upside down from 5 cm (F1,46 =
23.71, p < 0.001). Interestingly, a significant interaction
between sex and treatment was observed for the ability of
the pup to right itself in the air when dropped (F1,46 =
7.60, p = 0.008), with control males taking longer to right
themselves than control females but exposed females tak-
ing longer to right themselves than exposed males. T2
ethanol-treated pups were significantly delayed in time it
took to surface right (F1,54 = 4.93, p = 0.03), turn 180˚
upward when placed downward on a screen set at 45˚
(F1,54 = 34.88, p < 0.001), crawl away from the edge of a
cliff (F1,54 = 25.10, p < 0.001), grasp the rod (F1,54 = 6.65,
p = 0.01), transverse out of the circle (F1,54 = 17.02, p <
0.001), right themselves in the air (F1,54 = 10.06, p =
0.003) and open their eyes for the first time (F1,54 = 6.21,
p = 0.02). Finally, T3 ethanol-treated pups had signifi-
cant delays in surface righting (F1,39 = 97.15, p < 0.001),
grasping the rod (F1,39 = 10.96, p = 0.002), reacting to a
handclap at a distance of 10 cm (F1,39 = 9.52, p = 0.004),
flattening their ear in response to an applicator (F1,39 =
6.34, p = 0.02), traversing outside of the circle (F1,39 =
27.67, p < 0.001), opening their eyes (F1,39 = 12.72, p <
0.001), and righting in the air (F1,39 = 63.24, p < 0.001).
3.2. Locomotor Activity and Anxiety-Related
Spontaneous activity of juvenile (P25) offspring from
control and ethanol-treated mice for each trimester was
assessed over a 15 minute period in a novel open-field
environment. Two-way ANOVA did not result in a sig-
nificant effect of sex or an interaction between sex and
treatment, therefore the groups were collapsed and a one-
way ANOVA with treatment as the main factor was ap-
plied. It showed a significant effect of treatment in T1
(F1,46 = 6.48, p = 0.01) and T2 (F1,55 = 69.89, p < 0.001),
where the number of beam breaks was increased in etha-
nol-treated mice versus control mice (Figures 1(a) and
(b)). No significant effect of treatment was observed for
T3 mice (Figure 1(c)).
To evaluate the effects of novelty induced anxiety-re-
lated traits in the open field task, we also examined dif-
ferences in thigmotaxis during open-field testing. Al-
though no statistically significant effect of treatment was
observed for T1 mice (Figure 2(a)), a significant main
effect of treatment was observed between T2 exposed
and control mice (F1,55 = 59.65, p < 0.001), with ethanol-
treated offspring spending significantly more time in the
centre zone than control mice (Figure 2(b)). Interest-
ingly, mice treated with ethanol during the T3 equivalent
spent significantly less time than control mice in the centre
Figure 1. Locomotor activity in a novel open-field environ-
ment. Mean (±SEM) infrared beam breaks of ethanol-trea-
ted and control mice from trimester 1 (a), trimester 2 (b)
and trimester 3 (c) over a 15 min period (n = 21 - 31 mice
per group). *p < 0.05; ***p < 0.001.
Copyright © 2013 SciRes. JBBS
K. MANTHA ET AL. 89
Figure 2. Time spent in the centre of a novel open-field en-
vironment to assess anxiety-related behaviours. Mean (±SEM )
time in seconds spent in the centre of the apparatus of etha-
nol-treated and control mice from trimester 1 (a), trimester
2 (b) and trimester 3 (c) over a 15 min period (n = 21 - 31
mice per group). **p < 0.01; ***p < 0.001.
zone of the open-field arena (F1,164 = 10.40, p = 0.002)
Home-cage (familiar environment) locomotor activity
was also assessed in ethanol-exposed and control adole-
scent mice between P30-40. Repeated-measures two-way
ANOVA detected significant main effects of hour and
treatment, but no significant effects of hour and treatment
or sex and treatment. A significant main effect of treat-
ment in T1 was found (F1,46 = 12.09, p = 0.001) with etha-
nol-treated mice demonstrating increased activity through-
out the nocturnal phase versus control mice (Figures 3(a)
and (b)). A significant main effect of treatment was found
in T3 with ethanol-exposed mice demonstrating increa-
sed activity versus control mice.
Finally, we used a light/dark box apparatus to further
evaluate the anxiety-related phenotypes we had observed
in (novel) open field activity. Here, T2 ethanol-treated
mice spent significantly (F1,55 = 7.08, p = 0.01) more
time (average: 152.46 ± 4.65 s) in the light area of the
box than control mice (average: 134.50 ± 4.89 s). Mice
from T1 were not significantly different (F1,46 = 1.82, p =
0.18) between control mice (160.90 ± 7.92 s) and etha-
nol-treated offspring (146.12 ± 7.58 s). Analysis of T3
data also did not result in a significant difference (F1,24 =
3.67, p = 0.067) between ethanol-treated offspring (141.06
± 6.80 s) and controls (121.86 ± 7.35 s).
3.3. Barnes Maze Task for Spatial Learning and
Mixed-model ANOVA showed a significant interaction
of treatment by acquisition day on latency to reach the
escape box for all trimester treatments. There was no sig-
nificant effect of sex or an interaction between sex and
treatment observed for any treatment time. Mice treated
with ethanol in T1 displayed increased latency to reach
the escape box than control mice on acquisition days 3
(F1,46 = 12.54, p = 0.001) and 4 (F1,46 = 9.86, p = 0.003)
(Figure 4(b)). Mice treated with ethanol in T2 had sig-
nificantly increased latency to reach the escape box on
day 1 (F1,55 = 10.60, p = 0.002) versus control mice but
performed similarly to control mice on days 2 to 4 (Fig-
ure 4(c)). Ethanol-exposed mice from T3 had increased
latency to reach the escape box on days 2 (F1, 39 = 12.78,
p = 0.001), 3 (F1,39 = 42.77, p < 0.001) and 4 (F1,39 =
35.99, p < 0.001) in comparison to control mice (Figure
4(d)). The efficiency of learning can be measured by the
strategy a mouse uses to locate a target, with an organ-
ized (direct) search providing evidence of spatial mem-
ory retention [47,48] We qualitatively scored search stra-
tegies used by each mouse (data not shown). Mice from
T1 were not observably different in the search strategies
used over trial days. Control T2 mice showed a general
trend of increased direct search strategies and a decrea-
sed percentage of mixed strategies, while their ethanol
treated counterparts used mixed strategies in a large pro-
portion of trials across all learning days. T3 control mice
showed a pronounced trend of increased direct search
strategies and decreased serial strategies across every
learning day as compared to ethanol-treated mice that
more often used mixed strategies.
Short-term and long-term retention of memory in the
Barnes maze was assessed on days 5 and 12, respectively,
by calculating the number of explorations to the target
hole location. On probe day 5, T1 ethanol-treated mice
Copyright © 2013 SciRes. JBBS
K. MANTHA ET AL.
Copyright © 2013 SciRes. JBBS
Figure 3. Locomotor activity in a familiar home-cage environment stratified by sex. Mean (±SEM) infrared beam breaks by
ethanol-exposed and control mice from trimester 1 males (a) and females (b), trimester 2 males (c) and females (d), and tri-
mester 3 males (e) and females (f) over an 11-h period (n = 9 - 20 mice per group). *p < 0.05; **p < 0.01; ***p < 0.001.
were not significantly different from controls in the num-
ber of explorations to the escape box (Figure 4(a)). Ge-
nerally, ethanol-treated mice displayed increased explo-
rations to the opposite end of the target than control mice
(Figures 5(a) and (b)). On probe day 12, we found a
main effect of sex (F1,46 = 4.22, p = 0.046), with females
exhibiting more explorations to the target (average fe-
male: 2.64 ± 0.15 explorations) than males (average male:
2.21 ± 0.15 explorations).
No significant effects of sex or an interaction between
sex and treatment was observed for the explorations of
ethanol-treated and control T2 mice on probe day 5. Sig-
nificant effects of treatment were observed for the ex-
plorations to the escape hole location (F1,55 = 5.21, p =
0.03) and hole position −1 (F1,55 = 14.27, p < 0.001), with
control mice displaying increased exploration around the
target than exposed mice (Figure 5(c)). Ethanol-treated
ice exhibited more explorations on the opposite site of m
K. MANTHA ET AL. 91
Figure 4. Latency to reach the target in the Barnes maze (a) used for spatial learning and memory. Average latencies to the
target represent mean (±SEM) of four trials per day across four acquisition days for ethanol-treated and control mice from
trimester 1 (b), trimester 2 (c) and trimester 3 (d). Data show n are c oll apse d acr oss se x ( n = 21 - 31 mice per group). **p < 0.01;
***p < 0.001.
the target zone than control mice (Figure 5(c)). Signifi-
cant effects of treatment were observed for the explora-
tions to the escape hole (F1,55 = 12.29, p = 0.001), with
control mice spending more time within the target area
than ethanol-treated mice (Figure 5(d)).
For T3 probe days 5 and 12 explorations, we did not
observe any significant effects of sex or an interaction
between sex and treatment. A significant effect of treat-
ment was observed on probe day 5 for explorations to the
target (F1,39 = 14.22, p < 0.001) and positions near the
target, with control mice displaying significantly more
explorations around the target than ethanol-treated mice
(Figure 5(e)). We also found a significant effect of treat-
ment on probe day 12 for the target hole (F1,39 = 7.80, p =
0.008), with control mice spending more time in the tar-
get area than ethanol-treated mice Figure 5(f)).
This study evaluates the effects of heavy binge-like etha-
nol exposure at neurodevelopmental times approximating
human trimesters one, two and three across a battery of
behavioral assays. The phenotypes assessed fall into three
categories: motor skill development, locomotor activity
including behaviors relevant to anxiety, and spatial learn-
ing and memory. The specific assays are well-established
and have been used extensively in the literature to model
human behaviors that are most relevant to FASD [40,46,
49-53]. Our results show that ethanol exposure causes
changes in a number of behaviors examined and that the
extent of alterations may be dependent upon the gesta-
tional timing of ethanol treatment.
4.1. Motor Skill Development
The results show that ethanol exposure at any time dur-
ing gestation may cause delays in motor skill and reflex
development. Specifically, our results show delays in sur-
face righting, open-field traversal and air righting (Table
), which follows the literature [54,55]. Such phenotypes 1
Copyright © 2013 SciRes. JBBS
K. MANTHA ET AL.
Figure 5. Number of explorations for control and ethanol-treated mice during Barnes maze on days 5 and 12 to represent
short-term and long-term memory retention, respectively. Mean (±SEM) number of explorations to each hole of the Barnes
maze for ethanol-treated and control mic e on day 5 for trimester 1 (a), trimester 2 (c) and trimester 3 (e), and day 12 for tri-
mester 1 (b), trimester 2 (d) and trimester 3 (f). The position of numbered holes (x-axis) of Barnes maze are shown in Figure
3(a). Data shown here are collapsed across sex (n = 21 - 31 mice per group). *p < 0.05; **p < 0.01; ***p < 0.001.
are early indicators of the rostro-caudal gradient of limb
coordination maturation . Ethanol exposure during the
trimester three equivalent appears to produce delays in
most measures, followed closely by trimester two, while
ethanol exposure at the first trimester produced subtle
effects, with less than half of the milestones significantly
altered. This pattern may suggest that the brain regions
responsible for the development of these neuro-motor
skills may be less sensitive to ethanol at early stages of
neurodevelopment, such as neurulation and cell prolif-
eration . Also, fetal ethanol exposure can cause cell
death and neuronal reduction [57-62], that is known to
lead to motor skill deficits . Further, the impairments
caused by late gestation alcohol exposure may be attrib-
uted to specific brain region development including syn-
apse formation in the cerebellum and prefrontal cortex
[33,61,63-65]. Such results are relevant to FASD as
young children with FASD also show delayed motor de-
velopment and fine-motor dysfunction including weak
grasp, poor hand-eye coordination and poor balance [66-
70], which is thought to result from damage to the cere-
ellum given its involvement in motor function control b
Copyright © 2013 SciRes. JBBS
K. MANTHA ET AL. 93
Table 1. Achievement of developmental milestones of postnatal day ethanol-exposed and control offspring.
T1 (n = 11)
(n = 18)
(n = 10)
(n = 12)
(n = 10)
(n = 12)
T1 (n = 13)
(n = 11)
(n = 9)
T1 (n = 14)
(n = 20)
(n = 12)
SR 6.5 ± 0.4 8.7 ± 0.3 7.3 ± 0.26.4 ± 0.4 8.4 ± 0.37.5 ± 0.3***9.1 ± 0.4*8.9 ± 0.3***9.6 ± 0.3***8.7 ± 0.5 *9.7 ± 0.3 ***9.8 ± 0.2
NG 7.0 ± 0.4 6.2 ± 0.4 5.4 ± 0.27.5 ± 0.4 7.4 ± 0.35.8 ± 0.26.6 ± 0.5***9.5 ± 0.76.0 ± 0.26.6 ± 0.4 ***9.4 ± 0.35.9 ± 0.2
CA 9.0 ± 0.4 7.9 ± 0.5 5.1 ± 0.39.7 ± 0.4 6.4 ± 0.95.5 ± 0.29.1 ± 0.5***9.7 ± 0.45.4 ± 0.28.7 ± 0.3 ***10.4 ± 0.45.8 ± 0.2
FG 9.6 ± 0.6 10.0 ± 0.7 10.8 ± 0.39.3 ± 0.4 9.4 ± 0.811.1 ± 0.3 ** *11.5 ± 0.711.1 ± 0.5**12.0 ± 0.3***11.9 ± 0.5 11.6 ± 0.3 **11.8 ± 0.3
AS 14.7 ± 0.2 13.9 ± 0.4 10.3 ± 0.3 13.8 ± 0.5 14.0 ± 0.3 10.3 ± 0.313.1 ± 0.513.4 ± 0.8**11.1 ± 0.313.8 ± 0.6 14.2 ± 0.4 **11.2 ± 0.2
ET 9.8 ± 0.3 9.3 ± 0.3 9.6 ± 0.29.5 ± 0.3 10.4 ± 0.59.7 ± 0.29.3 ± 0.410.4 ± 0.3*10.3 ± 0.29.9 ± 0.5 10.4 ± 0.3 *10.2 ± 0.3
OFT 10.6 ± 0.6 10.9 ± 0.4 11.7 ± 0.1 11.4 ± 0.4 10.0 ± 0.4 11.8 ± 0.2 ***12.5 ± 0.3** *12.4 ± 0.6** *13.0 ± 0.3***12.8 ± 0.4 ***12.5 ± 0.4***13.3 ± 0.4
EO 14.7 ± 0.1 13.8 ± 0.2 14.6 ± 0.2 14.3 ± 0.1 14.2 ± 0.3 14.9 ± 0.214.5 ± 0.1*14.6 ± 0.2***15.4 ± 0.214.6 ± 0.2 *14.4 ± 0.2***15.5 ± 0.2
AR 12.9 ± 0.7 12.1 ± 0.6 10.4 ± 0.3 11.0 ± 0.6 12.4 ± 0.5 10.8 ± 0.9 ***14.1 ± 0.5**14.0 ± 0.7***14.0 ± 0.6** *15.2 ± 0.4 **14.2 ± 0.4***13.6 ± 0.5
Table 1. Milestones include surface righting (SR), negative geotaxis (NG), cliff aversion (CA), forelimb grasp (FG), auditory startle (AS), ear twitch (ET), open
field traversal (OFT), eye opening (EO) and air righting (AR). Mean (±SEM) number of days to complete each task is compared across males and females from
trimester one (T1), trimester two (T2) and trimester 3 (T3), ethanol-exposed and control pups. *p < 0.05; **p < 0.01; ***p < 0.001.
[63,71]. The cerebellum is also known to be quite sensi-
tive to the apoptotic effects of ethanol, particularly dur-
ing later neurodevelopmental stages . Our results sup-
port these findings, and suggest that ethanol exposure la-
ter in gestation may be more detrimental to the develop-
ment of motor skills, reflexes and coordination.
4.2. Locomotor Activity and Anxiety-Related
The literature is divided on the impact of prenatal alcohol
exposure on activity levels [49,73,74]. Results included
in this report suggest that this apparent discrepancy may
be due, at least in part, to the timing of exposure during
neurodevelopment (Figure 1). Our findings are consis-
tent with other research that has observed increased ac-
tivity in animal models following ethanol administration
during earlier stages of fetal development [32,49,74,75],
but the same pattern was not observed for mice treated
during the trimester three equivalent. However, given that
increased activity levels as well as anxiety-related traits
are commonly found both in children and animal models
of FASD [10,24,49,76], we sought to differentiate the ef-
fects of alcohol on activity versus novelty-induced stress
by measuring nocturnal activity levels of the mouse (Fig-
ure 3). In a familiar, home-cage setting, the ethanol-
treated mice, independent of timing of exposure, are sig-
nificantly more active than control mice at certain peaks
throughout the night, which agrees with the literature [7,
20,25,41,49]. Although the mechanism by which ethanol
exposure leads to increased activity levels is unknown,
however children with FASD often develop Attention De-
ficit Hyperactivity Disorder (ADHD) comorbidity [77-
79]. This phenotype may be associated with neuronal
reduction within the basal ganglia, cerebellum, and the
prefrontal cortex [80,81] or abnormal cortical thickness
. These abnormalities have been observed in both
FASD and ADHD, and are implicated in executive func-
tioning and motor activity [83,84].
Interestingly, our results indicate that anxiety-related
traits may confound analysis of locomotor behaviors in
FASD models, particularly in novel environments. In this
study, we utilized two independent measures of both
anxiety and activity, with the light/dark box test to con-
firm the thigmotaxis observations of the open-field assay
and the home cage activity test to validate our open-field
locomotor observations. The thigmotaxis we observed in
the open-field locomotor tests was dependent upon the
timing of ethanol treatment (Figure 2), although the ob-
servation of a decrease in anxiety-related behaviors in T2
does not follow previous reports in the literature. It is
possible that trimester two exposure may not reduce an-
xiety, per se, but increases the risk of other FASD-related
phenotypes, such as impulsivity , and/or an anxioly-
tic effect . Conversely, increased anxiety-related be-
haviors, such as those in our T3 data, have been com-
monly reported in FASD-related literature [11,13,14, 86].
This may be due to ethanol’s ability to affect the devel-
opment and function of the hypothalamic-pituitary-adre-
nal (HPA) axis [87,88], leading to increased vulnerability
to anxiety-like phenotypes during adolescence and adult-
4.3. Spatial Learning and Memory
Our results (Figure 4) provide further support for deficits
in learning and memory caused by prenatal alcohol ex-
posure [15,16]. Results from T1 and T3 (Figures 4(b)
Copyright © 2013 SciRes. JBBS
K. MANTHA ET AL.
and (d)) support other studies that have observed mice
treated with ethanol either early in prenatal development
or early neonatal development show significant impair-
ment on spatial learning tasks [41,90-92]. This may be
due to ethanol-induced neurodegeneration at these de-
velopmental times in specific brain regions, such as the
hippocampus and prefrontal cortex [93-95] that are asso-
ciated with learning and memory deficits in young adult
Interestingly, mice exposed to ethanol during mid-ges-
tation (T2) took longer than control mice to learn the lo-
cation of the target on the first acquisition day, but were
able to improve over subsequent training days (Figure
4(c)). This pattern is consistent with the suggestion that,
with repetition, ethanol-exposed mice are able to perform
as well as control mice by the end of the acquisition days
. These results are consistent with reinforcement
learning in some children with FASD . Search strate-
gies used by T2 and T3 ethanol-treated mice provide fur-
ther evidence that ethanol-treated mice have difficulty in
remembering the location of the target in this task, which
may be attributed to the direct or indirect effect of etha-
nol during neurodevelopment. This follows previous re-
ports in children with FASD who show altered develop-
mental transitioning from visual to verbal memory stra-
tegies , and who also improve on learning and mem-
ory tasks when given an implicit learning strategy .
4.4. Sources of Error
We recognize that our study may include some still un-
recognized effects that are not easily eliminated. These
include potential litter and circadian effects. Also, we
have relied on BAC data from previous studies from ro-
dents, including C57BL/6 mice [29,94,99,100], rather
than directly measuring BAC in our experimental ani-
mals. This has allowed us to predict blood alcohol levels
to be above a critical threshold of neurodegeneration
(200 mg/dl) . In addition, the results from direct al-
cohol treatment of pups in trimester three, compared to
the indirect treatment of fetuses in trimesters one and two,
must be interpreted with caution. The dose used in our
experiment accommodates the weight of the animals;
however, our experimental paradigm does not match the
direct exposure of pups in trimester three with the other
treatments. Therefore, the pups’ sensitivity to alcohol may
contribute to the severity of behavioral results.
We have examined the effects of alcohol exposure during
specific neurodevelopmental times approximating the
three human trimesters using a consistent dosage. We
show that acute ethanol exposure during neurodevelop-
ment consistently leads to delays in achievement of mo-
tor skill coordination and spatial learning, regardless of
timing of exposure. However, activity and anxiety phe-
notypes may be more sensitive to the timing of alcohol
exposure and potentially confounded with one another,
as well as other FASD-associated endophenotypes. This
study provides a framework for a comparison of pheno-
typic outcomes using a consistent treatment paradigm
across major neurodevelopmental stages, which may be
useful in examining the underlying biological mecha-
nisms. The results add to the growing body of evidence
that the brain is sensitive to alcohol throughout neurode-
velopment, and that the timing of exposure may offer an
explanation for the extensive heterogeneity associated
with developmental spectrum disorders including FASD.
Project was developed by MLK, KM and SMS. KM car-
ried out experiments (T1 and T2), collected data, com-
pleted and compiled data analysis, and wrote the manu-
script. MLK carried out experiments, collected data, com-
pleted data analysis for T3. The three authors contributed
to the manuscript preparation. They would like to thank
Benjamin Laufer and Eric Diehl for their thorough re-
views of the manuscript. This research was supported by
a Queen Elizabeth II Scholarship in Science and Tech-
nology (QEIISST) to MLK and grants from the Natural
Sciences and Engineering Research Council of Canada
(NSERC), Canadian Institute of Health Research (CIHR),
and Ontario Mental Health Foundation (OMHF) to SMS.
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