Akers et al. Molecular Brain 2011, 4:29
http://www.molecularbrain.com/content/4/1/29

RESEARCH

Open Access

Fetal alcohol exposure leads to abnormal
olfactory bulb development and impaired
odor discrimination in adult mice
Katherine G Akers1, Steven A Kushner1,2, Ana T Leslie1, Laura Clarke3, Derek van der Kooy3, Jason P Lerch1,4,5 andPaul W Frankland1,6,7*

Abstract
Background: Children whose mothers consumed alcohol during pregnancy exhibit widespread brain abnormalities
and a complex array of behavioral disturbances. Here, we used a mouse model of fetal alcohol exposure toinvestigate relationships between brain abnormalities and specific behavioral alterations during adulthood.
Results: Mice drank a 10% ethanol solution throughout pregnancy. When fetal alcohol-exposed offspring reachedadulthood, we used high resolution MRI to conduct a brain-wide screen for structural changes and found that thelargest reduction in volume occurred in the olfactory bulbs. Next, we tested adult mice in an associative olfactorytask and found that fetal alcohol exposure impaired discrimination between similar odors but left odor memory
intact. Finally, we investigated olfactory bulb neurogenesis as a potential mechanism by performing an in vitro
neurosphere assay, in vivo labeling of new cells using BrdU, and in vivo labeling of new cells using a transgenic
reporter system. We found that fetal alcohol exposure decreased the number of neural precursor cells in the
subependymal zone and the number of new cells in the olfactory bulbs during the first few postnatal weeks.
Conclusions: Using a combination of techniques, including structural brain imaging, in vitro and in vivo cell
detection methods, and behavioral testing, we found that fetal alcohol exposure results in smaller olfactory bulbs
and impairments in odor discrimination that persist into adulthood. Furthermore, we found that theseabnormalities in olfactory bulb structure and function may arise from deficits in the generation of new olfactory
bulb neurons during early postnatal development.
Keywords: fetal alcohol exposure, MRI, olfactory bulb, subependymal zone, odor discrimination, odor memory,
neurospheres, neurogenesis

Background
Approximately 1% of children in the United States display symptoms of Fetal Alcohol Spectrum Disorder(FASD) [1], a classification encompassing the full rangeof subtle to severe birth defects resulting from the consumption of alcohol during pregnancy [2]. Althoughsome symptoms are outwardly visible, such as stunted
growth and craniofacial abnormalities, one of the mostpronounced effects is damage to the developing brain.
Neuroimaging studies reveal that children with FASD
* Correspondence: paul.frankland@sickkids.ca
1
Neurosciences and Mental Health, Hospital for Sick Children, Toronto,
Ontario, Canada
Full list of author information is available at the end of the article

possess widespread brain abnormalities including reductions in the volume of the corpus callosum, cerebellum,basal ganglia, and hippocampus [3-5]. Presumably, thesebrain anomalies contribute to the complex array of
behavioral disturbances observed in children with FASD
including hyperactivity, attention deficits, impaired
learning and memory, social problems, anxiety, anddepression [5-9].
The high incidence of FASD warrants further study
into its biological basis and behavioral manifestations,but it is often difficult to disentangle the links between
gestational alcohol exposure and developmental
abnormalities. For instance, pregnant women may notaccurately report the extent of their drinking [10], and© 2011 Akers et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

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maternal alcohol use is often concurrent with other factors that pose risk to the developing fetus such as smoking, illicit drug use, poor nutrition, and psychiatricillness [11-13]. Many difficulties inherent in human studies, however, can be overcome by animal models of
fetal alcohol exposure [14], which allow for control over
the amount and timing of alcohol intake as well as theisolation of drinking from other confounding factors.
Furthermore, animal models provide opportunities toinvestigate the neurobiological mechanisms underlying
fetal alcohol effects and to determine if those effects
persist across the lifespan.
Here, we used a mouse model to investigate theimpact of fetal alcohol exposure on brain development
and behavioral outcome. Throughout pregnancy, mousemothers drank a 10% ethanol (EtOH) solution. Whenoffspring reached adulthood, we used high-resolution
magnetic resonance imaging (MRI) to perform a brainwide screen for structural abnormalities and found thatthe olfactory bulbs (OB) exhibited the largest reduction
in volume following fetal alcohol exposure. Next, weused an associative olfactory task to assess odor discrimination and odor memory and found that adult micewith fetal alcohol exposure displayed impairment inability to discriminate between odors with a high degreeof similarity. Finally, using an in vitro neurosphere assay
and in vivo cell labeling techniques, we found that the
reduced OB volume and olfactory deficits in mice withfetal alcohol exposure were associated with disruptionsin postnatal OB neurogenesis.

Results
Fetal alcohol exposure results in structural brain
abnormalities in adult miceChildren whose mothers consumed alcohol during pregnancy possess structural abnormalities in several brain
regions [3-5], many of which are also observed in mouseembryos exposed to alcohol during gestation [15]. To
determine whether these structural abnormalities persist
into adulthood, we used MRI due to its whole brain
coverage and excellent soft tissue contrast to screen for
anatomical differences between control and fetal alcohol-exposed (FAE) mice on postnatal day (P) 60. We
found that fetal alcohol exposure produced a negligible
decrease in total brain volume (d = -0.2). After normalizing for total brain size, however, FAE mice exhibited
both decreases and increases in the volume of individual
brain regions (Figure 1). The regions exhibiting the largest decreases in volume were the olfactory bulbs (OB),the granule cell layer of the dentate gyrus region of the
hippocampus, and the fourth ventricle. The regions
exhibiting the largest increases in volume were the basal
forebrain, the posterior part of the anterior commissure,the stria medullaris of the thalamus, and the amygdala.

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These findings suggest that structural brain abnormalities resulting from fetal alcohol exposure persist into
adulthood and may contribute to long-lasting behavioral
alterations.
Assessment of olfactory behavior using associative
olfactory task

Because the OB exhibited the largest reduction involume, we investigated whether fetal alcohol exposure
produces deficits in olfactory behavior during adulthood.
Starting at P60, adult mice were trained in an associative
olfactory task in which one odor (+ odor) was always
paired with a sugar reward and the other odor (-odor)
was never paired with sugar (Figure 2a). If mice could
discriminate between the + and -odors and selectively
associate the + odor with sugar, then they were expected
to dig preferentially in the bedding on top of the + odor
during a post-training probe test. To vary the difficultyof the discrimination, we used increasingly similar binary mixtures of R-carvone (spearmint) and S-carvone(caraway) as the + and -odors (Figure 2b). In the easiest
version of the task (i.e., 100:0), mice were trained to discriminate between an odor composed of 100% R-carvone/0% S-carvone and an odor composed of 0% Rcarvone/100% S-carvone. In a more difficult version (i.e.,
80:20), mice were trained to discriminate between an
odor composed of 80% R-carvone/20% S-carvone andan odor composed of 20% R-carvone/80% S-carvone. Tovary memory demands, the delay between training andthe probe test was 10 min, 24 hours, or 7 days. Thus, byusing a single task and varying two of its parameters, we
could assess ability to discriminate between similarodors as well as ability to remember an association
between an odor and a reward.
Before testing fetal alcohol-exposed mice, we firstassessed how well normal mice could discriminate
between odors of increasing similarity. Separate groupsof untreated mice were trained in all six versions of
the task. During training, mice spent more time
digging at the + odor compared to the -odor regardlessof task version (Figure 2c) (version × odor × day analysis of variance (ANOVA); odor main effect: F(1,59) =740.14, p < .001). Furthermore, the amount of timespent digging at the + odor increased across days,
whereas the amount of time spent digging at the -odor
remained low (version × odor × day ANOVA; day ×odor interaction: F(3,177) = 53.30, p < .001). Notably,
mice spent more time digging at the + odor than the-odor even when the two odor mixtures were identical(50:50), suggesting that it was the presence of sugar
and not the ability to discriminate between odors that
determined whether mice persisted in digging during
training. During the probe test 24 hours later, when nosugar was present, mice dug preferentially at theAkers et al. Molecular Brain 2011, 4:29
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Figure 1 Effect of fetal alcohol exposure on brain volume. High-resolution MRI was used to screen for volumetric differences between adultcontrol (n = 10) and FAE (n = 10) mice in 62 brain regions. Negative and positive effect sizes indicate fetal alcohol exposure-induced decreases
and increases in volume, respectively, with an effect size of ≥ 0.8 or ≤ -0.8 considered large (black bars).

+ odor only in certain versions of the task (Figure 2d)(version × odor ANOVA; version × odor interaction:
F(5,46) = 2.81, p = .027). Specifically, mice in the 100:0,
90:10, and 80:20 versions of the task spent more time
digging at the + odor compared to the - odor (100:0:t(9) = 2.27, p = .049; 90:10: t(8) = 4.05, p = .004; 80:20:t(7) = 2.91, p = .023), indicating that they discriminatedbetween odors that were relatively distinct. Mice in the
70:30, 60:40, and 50:50 versions of the task, however,spent equivalent amounts of time digging at the + and-odors, indicating that they did not discriminate
between odors that were relatively similar or identical.
To further characterize task performance, we computed
a discrimination index with ‘1’ denoting digging exclusively at the + odor and ‘0’ denoting equivalent amountsof digging at the + and -odors (i.e., chance level). We
found that the discrimination index differed dependingon task version (version ANOVA; version main effect:
F(5,46) = 3.70, p = .007). Only mice in the 100:0, 90:10,
and 80:20 versions of the task had discrimination
indices significantly above chance level (Figure 2e)(100:0: t(9) = 5.03, p = .001; 90:10: t(8) = 4.82, p = .001;
80:20: t(7) = 4.54, p = .003).

Fetal alcohol exposure impairs odor discrimination in
adult mice

To examine whether fetal alcohol exposure impairs ability to discriminate between odors, we trained control
and FAE mice in either the 100:0 or 80:20 version of
the task. These versions were chosen because they werethe easiest and most difficult versions, respectively, thatwere successfully performed by untreated mice. Also, toexamine whether fetal alcohol exposure impairs ability
to remember an association between an odor and areward, we varied the delay between training and theprobe test (Figure 3a). During training, mice spent moretime digging at the + odor compared to the -odor
regardless of task version (Figure 3b) (version × group ×
odor × day ANOVA; odor main effect: F(1,65) = 520.04,
p < .001), and the amount of time spent digging at the+ odor but not the -odor increased across days (version
× group × odor × day ANOVA; day × odor interaction:
F(3,195) = 32.70, p < .001). Importantly, we found nodifferences between groups during training, indicatingthat both control and FAE mice detected the sugar in
the bedding during + trials and had similar levels of
motivation to perform the task.

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Figure 2 Associative olfactory task. (a) Mice were trained to discriminate between two odors using an associative olfactory task in which the+ odor but not the -odor was paired with sugar. (b) The + and -odors were composed of increasingly similar binary mixtures of R-carvone andS-carvone. (c) During training, mice spent more time digging at the + odor compared to the -odor regardless of odor similarity (100:0: n = 10;
90:10: n = 9; 80:20: n = 8; 70:30: n = 8; 60:40: n = 9; 50:50: n = 8). (d) During a probe test given 24 hours after training, mice spent more time
digging at the + odor compared to the -odor when the odors were relatively distinct (100:0, 90:10, 80:20) but not when the odors wererelatively similar (70:30, 60:40) or identical (50:50). (e) Mice had discrimination indices above chance level only when the odors were relatively
distinct (100:0, 90:10, 80:20).

During the probe test for the 100:0 task, we foundthat both control and FAE mice spent more time digging at the + odor compared to the -odor regardless ofthe delay between training and testing (Figure 3c)(group × odor × delay ANOVA; odor main effect: F(1,42) = 69.04, p < .001). Both control and FAE mice
had discrimination indices significantly above chancelevel at all delays (Figure 3d) (control 10 min: t(6) =3.70, p = .010; control 24 hours: t(7) = 10.91, p < .001,
control 7 days: t(8) = 7.07, p < .001; FAE 10 min: t(7) =6.89, p < .001, FAE 24 hours: t(7) = 3.91, p = .006, FAE
7 days: t(7) = 5.57, p = .001). Therefore, when the + and-odors were relatively distinct, both control and FAE
mice discriminated between the odors, selectively associated the + odor with sugar, and remembered theodor-sugar association for at least 7 days.
During the probe test for the 80:20 task, however, weobserved differences between control and FAE mice inthe amount of time spent digging at the + and -odors(Figure 3e) (group × odor × delay ANOVA; group mainAkers et al. Molecular Brain 2011, 4:29
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Figure 3 Effect of fetal alcohol exposure on associative olfactory task performance. (a) Adult control and FAE mice were trained todiscriminate between two relatively distinct odors (100:0) or two relatively similar odors (80:20), with a 10 min (100:0: control n = 7, FAE n = 8;
80:20: control n = 9, FAE n = 9), 24 hour (100:0: control n = 8, FAE n = 8; 80:20: control n = 9, FAE n = 9), or 7 day (100:0: control n = 9, FAE
n = 9; 80:20: control n = 10, FAE n = 9) delay between training and testing. (b) Both control and FAE mice spent more time digging at the +odor compared to the -odor during training. (c,d) Both control and FAE mice discriminated between + and -odors when the odors wererelatively distinct (100:0 vs. 0:100), regardless of the delay between training and testing. (e,f) FAE mice failed to discriminate between + and-odors when the odors were relatively similar (80:20 vs. 20:80).

effect: F(1,49) = 7.40, p = .009; group × delay interaction: F(2,49) = 8.58, p = .001; odor main effect: F(1,29)= 17.90, p < .001). Control mice exhibited higher discrimination indices than FAE mice regardless of the delay
between training and testing (Figure 3f) (group × delay
ANOVA; group main effect: F(1,49) = 14.00, p < .001).
Whereas control mice had discrimination indices significantly above chance level at all delays, despite evidenceof slight forgetting at the longest delay (10 min: t(8) =5.03, p = .001; 24 hours: t(8) = 9.69, p < .001; 7 days: t(9) = 3.70, p = .005), FAE mice had discrimination
indices not significantly different from chance level atall delays. Therefore, when the + and -odors were relatively similar, FAE mice failed to discriminate between
odors. Together, these results suggest that the reduction
in OB volume by fetal alcohol exposure is associatedwith a specific impairment in perceptual discrimination
between similar odors, leaving ability to learn andremember associations between an odor and a reward
intact.
Associative olfactory task performance is hippocampalindependent

Because the associative olfactory task involves both perceptual discrimination between odors and the association of an odor with a reward, it might require not only
the OB but also other brain regions involved in learningand memory such as the hippocampus. As we observed

volumetric reductions in both the OB and the granulecell layer of the dentate gyrus, it is possible that the
impairment in performance observed among mice withfetal alcohol exposure could be attributed to abnormal
development of the hippocampus rather than the OB.
To determine whether performance in the associative
olfactory task requires the hippocampus, untreated micereceived extensive N-methyl-D-aspartic acid (NMDA)
lesions of the hippocampus (Figure 4a) and were trainedin the 80:20 version of the task. During the probe test
given 24 hours after training, both sham and hippocampal-lesion (HPC) mice spent more time digging at the +odor compared to the -odor (Figure 4b) (group × odor
ANOVA; odor main effect: F(1,15) = 26.00, p < .001),
and both sham and HPC mice had discrimination
indices significantly above chance level (Figure 4c)(sham: t(7) = 4.04, p = .005; HPC: t(8) = 3.64, p = .007),
indicating that HPC mice learned to discriminate
between two similar odors. Also, we observed that HPC
mice spent more time digging at the + odor compared
to sham-lesion mice (Figure 4b) (group × odor
ANOVA; group × odor interaction: F(1,15) = 5.29, p =.036), consistent with previous reports of hyperactivity
following hippocampal lesions [16,17].
To verify the effectiveness of the lesions, we next
tested HPC mice in the open field and the water maze.
As expected based on previous studies [18,19], HPC
mice showed no habituation of activity levels in theAkers et al. Molecular Brain 2011, 4:29
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Figure 4 Associative olfactory task performance does not require the hippocampus. (a) Representative images of sham and hippocampal(HPC) lesions. (b) Both sham (n = 8) and HPC (n = 9) mice spent more time digging at the + odor compared to the -odor. (c) Both sham and
HPC mice had discrimination indices above chance level. (d) HPC mice did not show habituation of activity levels in the open field. (e,f) HPC
mice did not learn the location of a hidden platform in the water maze. (g) Density plots for grouped data showing where mice concentrated
their searches for the hidden platform. The color scale represents number of visits.

open field (Figure 4d) (group × minute ANOVA; group× minute interaction: F(14,210) = 5.49, p < .001), no
improvement in latency to find a hidden platform acrosswater maze training (Figure 4e) (group × day ANOVA;
group × day interaction: F(4,60) = 6.89, p < .001), andno evidence of a spatial bias for the platform location
after water maze training (Figure 4fg) (group ANOVA;
group main effect: F(1,15) = 21.68, p < .001), thereby
minimizing the possibility that successful performancein the associative olfactory task was supported by residual hippocampal tissue. The finding that the hippocampus is not necessary for associative olfactory task
performance, together with the observation that otherolfactory areas of the brain (e.g., olfactory tubercule, lateral olfactory tract) show relatively small changes involume following fetal alcohol exposure, strengthens thelikelihood that the impairment in odor discrimination
observed among FAE mice is due to abnormal development of the OB as opposed to other brain regions.
Fetal alcohol exposure reduces OB neurogenesis during
early postnatal development

As the OB is one of the brain regions to which newneurons continue to be added after birth and into adulthood [20,21], we next investigated whether deficits inpostnatal neurogenesis may contribute to the reduced

OB volume and impaired odor discrimination among
FAE mice.
Postnatally-generated OB neurons originate in the
subependymal zone (SEZ) of the lateral ventricles before
migrating to the OB via the rostral migratory stream[22]. To examine whether fetal alcohol exposure reduces
the pool of neural precursor cells in the SEZ, we performed an in vitro neurosphere assay. SEZ tissue was
extracted from mice between P4 and P14, dissociatedinto single cells, and cultured under proliferative conditions for 7 days (Figure 5a). Through bulk culturing, wefound that FAE mice had fewer neural precursor cells
compared to age-matched control mice (Figure 5b) (t(8)= 3.89, p = .005). Furthermore, culturing at clonal density demonstrated that FAE mice had fewer small (50100 μm) neurospheres (t(8) = 3.54, p = .008) but similar
numbers of large (> 100 μm) neurospheres compared tocontrol mice (Figure 5c). Given that small neurospheresare believed to arise from neural progenitor cells withfinite capacity for mitotic cell divisions, whereas large
neurospheres are believed to arise from neural stem
cells with unlimited capacity for self-renewal [23], thesefindings suggest that fetal alcohol exposure specificallyreduces the number of SEZ neural progenitor cells.
A reduction in the number of SEZ neural progenitor
cells could lead to a decrease in the number of new

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Figure 5 Effect of fetal alcohol exposure on olfactory bulb neurogenesis. (a) Postnatal OB neurogenesis was assessed using 3 methods: (i)in vitro neurosphere assay, (ii) in vivo labeling with BrdU, and (iii) in vivo labeling with LacZ using a transgenic reporter system. (b,c) FAE mice (n= 9) had fewer in vitro neural precursor cells and fewer small neurospheres in the SEZ compared to control mice (n = 9) between P4 and P14.
(d) FAE mice had fewer BrdU+ cells in the granule cell layer of the OB compared to control mice P7 (control n = 9, FAE n = 8) and P21 (control
n = 8, FAE n = 9). (e) There was no difference in BrdU+ cells between control (n = 8) and FAE mice (n = 8) at P60. (f) FAE reporter mice had
fewer LacZ+ cells in the granule cell layer of the OB at P30 (control n = 8, FAE (10%) n = 6, FAE (15%) n = 9).

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neurons arriving at the OB. To examine whether fetal
alcohol exposure reduces the number of new OB neurons, we performed in vivo labeling of new cells with
the nucleoside analog 5-bromo-2’-deoxyuridine (BrdU)(Figure 5a). Mice received a single injection of BrdU at
P7 or P21 and were perfused 4 weeks later. Using
stereological techniques, we quantified the number of
BrdU+ cells in the granule cell layer of the OB, the destination of nearly all postnatally-generated OB neurons[24]. We found that the number of BrdU+ cells declinedwith age for both control and FAE mice (Figure 5d)(group × age ANOVA; age main effect: F(1,30) = 172.03,
p < .001). However, FAE mice had fewer BrdU+ cells
than control mice across both ages (group × age
ANOVA; group main effect: F(1,30) = 10.45, p = .003;
P7: (t(15) = 2.37, p = .031; P21: t(15) = 2.63, p = .019),
indicating that fetal alcohol exposure reduces the number of new cells in the OB during the early postnatal
period. To determine whether the decrease in OB neurogenesis persists into adulthood, mice received multiple
BrdU injections on P60-62 and were perfused 4 weeks
later. At this age, we found no difference between control and FAE mice in the number of BrdU+ cells (Figure5e), suggesting that the effect of fetal alcohol exposure
on OB neurogenesis is transient. Together, these findings indicate that deficits in early postnatal OB neurogenesis may underlie the reduction in OB volume andimpairment in odor discrimination observed among
adult mice with fetal alcohol exposure.
As an additional test of whether fetal alcohol exposure
reduces the number of new OB neurons, we performedin vivo labeling of new cells using a genetic reporter system (Figure 5a). NestinCreERT2 × RosaLacZ transgenic
mice were injected with tamoxifen (TAM) on P30-34,
leading to expression of the LacZ transgene in nestin+
cells [25,26], and were perfused 4 weeks later. We foundthat FAE mice with mothers that drank 10% EtOH during pregnancy exhibited a small but non-significant
decrease in the number of LacZ+ cells in the granulecell layer of the OB compared to control mice (Figure5f). However, mice with mothers that drank 15% EtOH
exhibited a larger, significant decrease in the number of
LacZ+ cells (group ANOVA; group main effect: F(2,20)= 6.18, p = .008; 15% vs. control: t(15) = 3.42, p = .004).
These findings suggest that the magnitude of the deficit
in postnatal OB neurogenesis following fetal alcohol
exposure depends on the amount of alcohol consumed
during pregnancy.

Discussion
Using a mouse model, we aimed to establish a connection between fetal alcohol-induced changes in brain
development and alterations in behavioral outcome by
first identifying the structural brain abnormalities that

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persist into adulthood and then linking those brain
abnormalities with specific modifications in behavior.
Using high-resolution MRI, we observed both increases
and decreases in the size of particular brain regions,
including the basal forebrain, anterior commissure,amygdala, and the granule cell layer of the dentate
gyrus. Amid these widespread changes in brain volume,
however, the largest reduction in volume occurred in
the OB. We found that this decrease in OB volume during adulthood may arise from deficits in OB neurogenesis occurring specifically during early development, as
mice with fetal alcohol exposure possessed fewer neuralprogenitor cells in the SEZ and fewer new cells in the
granule cell layer of the OB during the first few postnatal weeks, but normal numbers of new cells in the OB
during adulthood. To determine the long-term consequences of abnormal OB development for olfactory
behavior, we tested adult mice in an associative olfactorytask designed to assess both odor discrimination andodor memory. We found that although fetal alcoholexposed mice learned and remembered an association
between an odor and a reward, they failed to discriminate between odors with a high degree of similarity.
Therefore, using a combination of techniques, including
structural brain imaging, in vitro and in vivo cell detection methods, and behavioral testing, we found that fetal
alcohol exposure affects the initial formation of the OB
and produces deficits in olfactory behavior that are evident during adulthood. These findings demonstrate that
exposure to alcohol during gestation can have a significant impact on brain development, thereby leading to
disturbances in behavior that persist into adulthood.
Fetal alcohol exposure produces abnormalities in OB
development in rodents

Our finding of reduced OB volume in fetal alcoholexposed mice is consistent with previous studies examining the effect of alcohol on brain development. Grossobservations [27] and MRI studies [28,29] of mouseembryos show that the OB is one of the brain regionsthat is reduced in volume following fetal alcohol exposure. Also, studies estimating volume from serial brain
sections show that exposure to alcohol during gestation(equivalent to the 1st and 2nd trimesters in humans) or
the first ten postnatal days (equivalent to the 3rd trimester in humans) reduces OB volume in pups [30-33] andadult rodents [34]. In the present study, we performed abrain-wide MRI screen for structural changes in adultmice with fetal alcohol exposure and found that out of
62 brain regions, the OB was the region exhibiting thelargest reduction in volume. Thus, whereas most previous studies observed reductions in OB volume shortly
after alcohol exposure (i.e., in prenatal or early postnatal
rodents), we found that this fetal alcohol-induced

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reduction in OB volume is not only long-lasting, but isalso one of the most prominent structural changes evident during adulthood. Taken together, therefore, thesestudies indicate that the OB is a brain region that is particularly vulnerable to fetal alcohol exposure, with alcohol-induced structural abnormalities emerging early indevelopment and persisting into adulthood.
This reduction in OB volume could result from multiple contributing mechanisms, including an increase incell death or a decrease in the generation of new cells.
Here, we explored whether fetal alcohol exposure
decreases the addition of new cells to the granule cell
layer of the OB. Granule cells are the most populous
cell type in the OB [35] and are predominantly generated during the first few weeks of postnatal life [36]. We
found that young mice with fetal alcohol exposure possessed fewer newly-generated granule cells in the OB,
which agrees with previous studies showing that pups
with fetal alcohol exposure have fewer granule cells in
the OB [30] and a disproportionate decrease in thevolume of the granule cell layer compared to other cell
layers in the OB [37]. Also, in accord with a previous
report that fetal alcohol exposure reduces the numberof neural stem cells in the SEZ [38], we found thatyoung mice with fetal alcohol exposure possessed fewer
SEZ neural progenitor cells, suggesting that the decreasein developmentally-generated OB granule cells resultsfrom a deficiency in SEZ cell proliferation. Consistent
with a change in the number of granule cells, which are
inhibitory neurons that synthesize brain-derived neurotrophic factor (BDNF) [39,40], young rodents with fetal
alcohol exposure also exhibited alterations in GABA
levels [41], GABA-related gene expression [42], andBDNF levels in the OB [33]. Therefore, a decrease in
the number of granule cells may lead not only to a
reduction in overall OB volume but also to alterations
in neurotransmission and neurotrophin expression
within the OB.
Fetal alcohol exposure produces disturbances in olfactory
behavior in rodents

The observation of fetal alcohol-induced abnormalities
in OB development raises the question of whether
rodents with fetal alcohol exposure display disturbancesin olfactory-related behavior. Previous studies indicatethat rodents exposed to alcohol during gestation display
a preference for the odor of ethanol later in life, particularly during adolescence [43,44]. Furthermore, fetal alcohol exposure has been observed to result in
impairments in nonassociative and associative odor
learning in very young rodents. Specifically, compared
to control pups, alcohol-exposed pups displayed lesshabituation of a cardiac response to an odor acrossrepeated presentations [45,46]. Furthermore, fetal

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alcohol-exposed pups failed to associate an odor witheither an appetitive or an aversive stimulus [47]. Adult
rodents with fetal alcohol exposure, however, showednormal associative odor learning [47], suggesting that
fetal alcohol-induced impairments in odor learning
might dissipate with age. In the present study, we useda more sensitive olfactory task that allowed us to assess
memory for an odor-reward association as well as perceptual discrimination between increasingly similarodors. We found that, consistent with a previous study[47], adult mice with fetal alcohol exposure displayed
normal odor learning, as they were able to associate anodor with a reward and remember this association for atleast 7 days. However, they failed to discriminate
between odors with a high degree of similarity. Therefore, we found that exposure to alcohol during gestation
impairs ability to perceive subtle differences between
odors and, furthermore, that this impairment persists
into adulthood. Because discrimination between similarodors is facilitated through granule cell-mediated lateral
inhibition in the OB [48,49], we speculate that the poor
odor discrimination among fetal alcohol-exposed mice
could arise from non-optimal levels of lateral inhibition
resulting from fewer numbers of developmentally-generated granule cells in the OB.
Contribution of developmental vs. adult neurogenesis toolfactory behavior

The role of OB neurogenesis in olfactory behavior iscurrently under debate, with some studies reporting that
reduction of neurogenesis impairs odor memory [50-53]
and others reporting that reduction of neurogenesis
impairs odor discrimination [54-56]. As suggested by
Lazarini and colleagues [53], this apparent discrepancy
could be explained by the time in development at which
OB neurogenesis is reduced. When OB neurogenesis
was reduced throughout the lifespan using unconditional genetic mutations, impairments in odor discrimination were observed [54-56]. However, when OB
neurogenesis was reduced only during adulthood using
irradiation or antimitotic drugs, odor discrimination waspreserved, but odor memory was disrupted [50-53]. Thissuggests that developmentally-generated, but not adultgenerated, OB neurons are important for odor discrimination. The present study offers additional support forthis hypothesis. Specifically, FAE mice showed deficits in
OB neurogenesis during the first few postnatal weeks
but normal OB neurogenesis during adulthood, and thisselective reduction in developmentally-generated OB
neurons was associated with impairment in odor discrimination but not odor memory. Together, therefore,
these findings suggest that the initial formation of theOB via the production of new neurons during early
development may be critical for the ability toAkers et al. Molecular Brain 2011, 4:29
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discriminate between odors, whereas the continued
addition of new neurons to the OB during adulthood
may support odor memory.

Page 10 of 15

provides a model system that can be used to further
understand relationships between brain and behavior[79] and points toward new clinical directions in thedetection and treatment of FASD.

Brain-wide effects of fetal alcohol exposure in rodents

Similar to the widespread changes in brain structure
observed among children with FASD [3-5], we foundbrain-wide changes in the volume of several regions inadult mice with fetal alcohol exposure, including the
granule cell layer of the dentate gyrus, basal forebrain,anterior commissure, and amygdala. In particular, ourobservation of a decrease in the volume of the granulecell layer of the dentate gyrus is consistent with previous, well-documented findings of altered hippocampal structure and function among adult rodents withfetal alcohol exposure [57,58], including lower cellcounts [59,60], reduced neurogenesis [61,62], deficits
in synaptic plasticity [63,64], and impaired performance in water maze and contextual fear memorytasks [65-69]. Our observation of an increase in thevolume of the basal forebrain during adulthood also
extends previous findings of abnormal basal forebrain
structure [32] and cholinergic neurotransmission [70]in rodent pups following fetal alcohol exposure. Interestingly, cholinergic input to the OB has been found tomodulate perceptual discrimination between similarodors [71,72]. Therefore, future studies could investigate whether fetal alcohol exposure-induced alterations
in cholinergic transmission from the basal forebrain to
the OB might contribute to impairments in odor
discrimination.

Conclusions
Using a mouse model, we found that exposure to alcohol during gestation results in smaller OBs and impairments in odor discrimination that persist into
adulthood. Furthermore, we found that these abnormalities in OB structure and function may arise from deficits in the generation of new OB neurons from the SEZ
during the first few postnatal weeks. Although controversial [73], there is evidence that, like mice, humans
also exhibit postnatal OB neurogenesis, with new cells
originating in the SEZ and migrating to the OB [74,75].
Therefore, humans exposed to alcohol during gestation
may exhibit changes in OB development similar to
those observed in mice. Indeed, autopsies of children
with severe FASD revealed anomalies in OB structure[76]. Although it is unknown whether children with
FASD exhibit deficits in olfaction, observations that
individuals with alcoholism or Korsakoff’s syndrome areunable to discriminate between odors [77,78] is suggestive of this possibility. Therefore, our finding of a connection between fetal alcohol-induced abnormalities in
OB development and alterations in olfactory behavior

Methods
Mice

All procedures were approved by the Animal Care Committee at The Hospital for Sick Children. Mice werebred in the colony at The Hospital for Sick Children
and maintained on a 12 hr light/dark cycle (lights on at
0700 hrs). Control and FAE mice were obtained from across between female 129Svev and male C57Bl/6 mice
(Taconic). In one experiment, control and FAE mice
were obtained from a cross between female wild-type
129Svev and male CreER T2 heterozygous × RosaLacZ
homozygous mice in a C57Bl/6 background. Genotypes
were determined using PCR analysis of tail DNA samples. After weaning on P21, mice were group-housed (25 per cage) in transparent plastic cages (31 × 17 × 14cm) and allowed free access to food and water unlessotherwise specified. To reduce the possibility of litter
effects on dependent measures [80], mice in eachexperimental group were sampled from between 3 and 6
different litters.
Fetal alcohol exposure

The fetal alcohol exposure procedure was adapted from
Allan and colleagues [81-83]. Individually-housed female
mice (n = 29) were given water containing 10% EtOH +0.1% saccharin in place of regular drinking water. Before
pregnancy, the concentration of EtOH in the water was
gradually increased to 10% (2 days at 2%, 2 days at 5%,
7 days at 10%). Mice continued to receive 10% EtOH
during a 4-day period of cohabitation with a male breeder and throughout pregnancy. Starting on the day ofbirth (P0), the concentration of EtOH was gradually
decreased (2 days at 5%, 2 days at 2%) and then replacedwith regular water. Control mice (n = 29) received watercontaining 0.1% saccharin throughout pregnancy. In the
CreERT2 × RosaLacZ experiment, a subset of mice (n =3) received 15% EtOH + 0.1% saccharin, with the concentration of EtOH gradually increased before pregnancy (2 days at 4%, 2 days at 8%, 7 days at 15%) anddecreased starting on P0 (2 days at 8%, 2 days at 4%).
Control, 10%, and 15% mice consumed an average of
4.14 ± 0.09 g, 3.80 ± 0.07 g, and 3.55 ± 0.05 g (mean ±SEM) fluid each day during pregnancy, resulting in an
average daily EtOH dose of 0 ± 0 g/kg, 13.60 ± 0.23 g/
kg, and 20.87 ± 1.65 g/kg, respectively. Although micecontinued to drink a reduced amount of EtOH during
the first few days after giving birth, the amount of EtOH
passed on to pups through lactation is low (~2% ofmaternal dose) [84]. Therefore, it is expected that theAkers et al. Molecular Brain 2011, 4:29
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amount of EtOH received by pups during this period
was minimal.
Consistent with a previous study showing that drinking 10% EtOH does not reduce food consumption [81],we found no differences among groups in body weightat the onset of pregnancy (control: 24.46 ± 0.85 g, 10%:24.59 ± 0.77 g, 15%: 23.70 ± 2.70 g; group ANOVA,
group main effect: p = .49) or weight gained acrosspregnancy (control: 11.68 ± 0.53 g, 10%: 12.61 ± 0.53 g,
15%: 16.13 ± 1.59 g; group ANOVA, group main effect:p = .08), although there was a non-significant trend
toward greater weight gain in 15% EtOH mice compared
to control (Tukey’s post-hoc test: p = .06) or 10% EtOH
mice (Tukey’s post-hoc test: p = .09). Also, we observed
no differences among groups in litter size (control: 6.26
± 0.38 pups, 10%: 6.32 ± 0.39 pups, 15%: 7.67 ± 0.88
pups; group ANOVA, group main effect: p = .54).
Furthermore, consistent with a previous study showingthat drinking 10% EtOH during pregnancy does not disrupt maternal care behaviors, including licking andgrooming of pups and time spent on the nest [81], we
observed no differences among groups in pup weightacross the pre-weaning period (P3: control: 2.64 ± 0.09
g, 10%: 2.79 ± 0.08 g, 15%: 2.49 ± 0.09 g; P10: control:
7.28 ± 0.23 g, 10%: 7.46 ± 0.24 g, 15%: 7.28 ± 0.29 g;
P21: control: 14.14 ± 0.33 g, 10%: 14.38 ± 0.38 g, 15%:
13.90 ± 0.35 g; age × group ANOVA, age × group interaction: p = .96, group main effect: p = .61, age main
effect: F(2, 116) = 1060.08, p < .001).
This drinking procedure in mice [81,83] (and similar
procedures in rats [63,85]) offers a number of advantages over other EtOH administration methods. First,
EtOH is delivered through drinking water instead of via
gastric intubation or injection, which reduces theamount of stress to mothers or pups. Second, mice havefree access to regular food at all times instead of receiving a liquid diet as their sole nutrient source. Third, thesteady decrease in EtOH dose during the first few days
after birth allows for gradual withdrawal from EtOH
without disrupting maternal care-giving behaviors.
Therefore, this drinking procedure lessens or eliminates
the need for a pair-fed condition or cross-fostering ofpups to non-EtOH-consuming dams, while producingchanges in offspring brain and behavior that replicate
those observed following EtOH-containing liquid diets[64,66,67].
MRI

MRI methods were similar to those previously
described by Lerch et al. [86,87]. On P60, male mice
were anaesthetized with choral hydrate and perfusedtranscardially with phosphate buffered saline (PBS) + 1
μl/ml heparin (Pharmaceutical Partners of Canada) + 2
mM ProHance (Bracco Diagnostics Inc.) followed byPage 11 of 15

4% paraformaldehyde (PFA) + 2 mM ProHance. Skulls
were separated from the bodies, and the skin, lower
jaw, ears, and cartilaginous nose tip were removed.
The remaining skull structures containing the brain
were postfixed overnight in PFA + 2 mM ProHance
and stored at 4°C in PBS + 0.02% sodium azide + 2
mM ProHance.
A multi-channel 7.0-T MRI scanner (Varian Inc.)
with a 6-cm inner bore diameter insert gradient wasused to acquire anatomical images of brains withinskulls. Before imaging, skulls were removed from solution, blotted, and placed into plastic tubes (13 mm diameter) filled with a proton-free susceptibility-matching
fluid (Fluorinert FC-77, 3 MCorp). Three custom-built
solenoid coils (14 mm diameter, 18.3 cm length) withover-wound ends were used to image three brains inparallel. Parameters used in the scans were optimized
for grey/white matter contrast: a T2-weighted, 3D fast
spin-echo sequence with 6 echoes, with TR/TE = 325/
32 ms, four averages, field-of-view 14 × 14 × 25 mm3,
and matrix size = 432 × 432 × 780 giving an image
with 32 μm isotropic voxels. Total imaging time was
11.3 hours. Geometric distortion due to position of thecoils inside the magnet was calibrated using a precision
machined MR phantom. To assess anatomical differences between brains from control and FAE mice, animage registration-based approach was used to compute volumes for 62 structures from each brain using
an anatomical atlas [88].
Associative olfactory task

Starting at P60, male and female mice were trained andtested in an associative olfactory task adapted from
Schellinck et al. [89]. Forty-eight hours before training,mice were placed on food restriction to maintain a 10%
reduction in free-feeding body weight. To familiarize
mice with the reward used during training, ~20 sugarpellets (sucrose, 10 mg each, TestDiet) were given toeach mouse 24 hours before training. Training and testing occurred in transparent plastic cages (31 × 17 × 14cm). Odors were diluted in mineral oil (1:100), applied
to filter paper (50 μl), and placed inside of petri dishes(15 mm diameter) with 10 small holes drilled in the lid.
Dishes were placed on the bottom of the cage and covered with bedding. Odors consisted of binary mixturesof the enantiomers R-(-)-carvone (98% purity, Sigma)
and S-(+)-carvone (96% purity, Sigma). During reinforced trials, one of the odors (+ odor) was presented
with 6 sugar pellets placed under the bedding on top of
the dish. During non-reinforced trials, the other odor (odor) was presented with no sugar pellets Mice received
one 5-min reinforced trial and one 5-min non-reinforced trial per day for 4 days, with trial order counterbalanced across days. Within each group of mice,Akers et al. Molecular Brain 2011, 4:29
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particular odor mixtures served as + and -odors aroughly equal number of times.
Ten min, 24 hours, or 7 days after training, micereceived a 10-min probe test during which the + and-odor were presented simultaneously. No sugar pellets
were present during the probe test. Mice were maintained on food restriction if the delay between training
and testing was 10 min or 24 hours. If the delay
between training and testing was 7 days, food restriction
was terminated after training, and mice were deprivedof food 24 hours before the probe test. Amount of timespent digging at the + and -odors (i.e., moving bedding
directly on top of the dishes with snout or forepaws)
was recorded with a video camera and coded manually.
Discrimination between odors was measured using a
discrimination index defined as (digging+ odor -diggingodor)/(digging+ odor + digging- odor).
Hippocampal lesions

Female mice were treated with atropine (0.1 mg/kg) andanesthetized with chloral hydrate (400 mg/kg). To prevent seizure activity associated with excitotoxic lesions,mice were also pretreated with diazepam (5 mg/kg).
Using stereotaxic procedures, NMDA (10 mg/ml) was
infused into the following 8 sites (4 per hemisphere)with respect to bregma (posterior, lateral, ventral): -2.0mm, ± 1.5 mm, 1.8 mm; -2.5 mm, ± 1.8 mm, 2.0 mm;-3.0 mm, ± 2.7 mm, 3.0 mm; and -3.0 mm, ± 2.7 mm,
3.5 mm. A volume of 0.1 μl was infused at the 3 mostdorsal sites, and a volume of 0.15 μl was infused at themost ventral site. NMDA was delivered via a 32-gauge
microsyringe (Hamilton). A pump maintained the infusion rate at 0.1 μl/min and the syringe was left in placefor 5 minutes after each infusion. Sham mice were treated identically except no NMDA was infused. Mice were
postoperatively treated with ketoprofen (5 mg/kg).
After 7 days of recovery, mice underwent training andtesting in the 80:20 version of the associative olfactorytask as previously described. Mice also underwent testing in the open field and the water maze. For the open
field, mice were released into the center of a square
plastic chamber (45 × 45 × 20 cm) and allowed to
explore for 15 min under dim light. Total distance traveled was recorded by an overhead video camera andanalyzed using automated software (Actimetrics). For
the water maze, a circular plastic pool (120 cm diameter, 50 cm height) was filled to a depth of 40 cm
with water maintained at ~26°C. Water was made opaque by the addition of nontoxic paint. A circular escapeplatform (10 cm diameter) was submerged 0.5 cm below
the water surface in a fixed location within the center of
one of the pool quadrants. The pool was surrounded bycurtains, located at least 1 m from the pool wall, thatwere painted with distinct geometric cues. Mice received

Page 12 of 15

3 trials per day for 5 days. Trials started when mice
were released into the pool, facing the wall, from one of
4 possible points. A different release point was used foreach trial on each day, and the order of release points
varied pseudorandomly across days such that each point
was used a roughly equal number of times. Trials ended
when mice reached the platform or 60 s elapsed. If amouse failed to find the platform, it was guided by theexperimenter. The inter-trial interval was ~10 min.
After the final training trial, mice received a 60-s probe
test with the platform removed from the pool. Swim
paths were recorded by an overhead video camera.
Latency to reach the platform during training and number of platform crosses during the probe trial was analyzed using Actimetrics software. Density plots weregenerated using custom software created in ourlaboratory.
After behavioral testing, mice were anesthetized withchloral hydrate and perfused transcardially with PBS followed by 4% PFA. Brains were postfixed in 4% PFA
overnight, transferred to 30% sucrose, and stored at 4°C.
Coronal sections (50 μm) across the entire anterior-posterior extent of the hippocampus were cut using a cryostat. Sections were mounted on gelatin-coated slides,
stained with neutral red, and cover-slipped with Cytoseal. Using Stereo Investigator software, the entire hippocampus and the area of the hippocampus sustaining
damage (i.e., cell loss or absence of tissue) were outlined
separately for every fourth section. The proportion of
total hippocampal tissue damaged by NMDA infusion
was 67.90 ± 4.90%. Spared tissue was mainly restricted
to the most posterior and ventral regions of the hippocampus, with the overlying neocortex exhibiting little orno damage.
Neurosphere assay

An in vitro neurosphere assay [90] was performed on
SEZ tissue extracted from male and female mice
between P4 and P14. Tissue underwent enzymatic digestion (1.33 mg/ml trypsin + 0.67 mg/ml hyaluronidase +0.13 mg/ml kynurenic acid) at 37°C for 50 min and was
isolated in serum-free media (SFM) with trypsin inhibitor. The tissue was mechanically dissociated into a single cell suspension, and cell density was determinedusing trypan blue exclusion. SEZ cells were cultured inbulk and at clonal density using SFM containing 20 ng/
ml epidermal growth factor (mouse submaxillary,
Sigma) + 10 ng/ml fibroblast growth factor-2 (humanrecombinant, Sigma) + 2 μg/ml heparin (Sigma). For thebulk culture, 2.2 × 105 cells per mouse were cultured in
25 ml of SFM. After 7 days, the total number of cells
was calculated from the cell density, which was determined using trypan blue exclusion following mechanicaldissociation into a single-cell suspension. For the clonal

Akers et al. Molecular Brain 2011, 4:29
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density culture, 2 cells/μl were plated for each mouse in
24-well uncoated plates (0.5 ml SFM per well). After 7
days, the total number of small (50-100 μm diameter)
and large (> 100 μm diameter) spheres formed in eachwell was counted.
BrdU and LacZ labeling and quantification

At P7 or P21, male and female mice received a single
injection of BrdU (50 mg/kg) dissolved in PBS. At P60,
mice received 2 injections of BrdU per day (100 mg/kg,
5 hours apart) for 3 days. Four weeks later, mice wereanesthetized with chloral hydrate and perfused transcardially with PBS followed by 4% PFA. Brains were postfixed overnight in PFA, transferred to PBS + 0.01%
sodium azide, and stored at 4°C. Coronal sections (40μm) were cut from the anterior tip of the main OB until
the intrusion of the accessory OB. Sections were treatedwith 1 N HCl at 45°C for 30 min, 1% H 2 0 2 at room
temperature for 15 min, and 0.2 M glycine in PBS atroom temperature for 10 min. Sections were incubatedwith the primary antibody (rat anti-BrdU monoclonal,
1:1000, Accurate Chemicals) at room temperature overnight and the secondary antibody (Alexa-488 goat antirat, 1:1000, Molecular Probes) at room temperature for2 hrs. Antibodies were diluted in blocking solution containing 2.5% bovine serum albumin + 5% goat serum +0.3% Triton X-100 dissolved in PBS. Sections werecounterstained with 4’,6-diamidino-2-phenylindole(DAPI, 1:10000, Sigma) and mounted on slides with Permafluor anti-fade medium.
At P30, double transgenic Nestin-CreERT2 × RosaLacZ
mice received 1 injection of TAM (180 mg/kg, Sigma)
dissolved in 10% EtOH/90% sunflower seed oil per day
for 5 consecutive days. Mice were perfused 4 weeks
later. OB tissue was prepared as previously described.
Sections were treated with 1% SDS for 10 min, 1% H202
for 15 min, and 0.2 M glycine in PBS for 10 min. Sections were incubated with the primary antibody (rabbit
polyclonal anti-LacZ, 1:250, Molecular Probes) at room
temperature overnight and the secondary antibody(Alexa-488 goat anti-rabbit, 1:500, Molecular Probes) atroom temperature for 2 hrs. Sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI,
1:10000, Sigma) and mounted on slides with Permafluor
anti-fade medium.
BrdU and LacZ quantification was performed using an
Olympus BX61 epifluorescent microscope and Stereo
Investigator software. The total number of BrdU+ or
LacZ+ cells in the granule cell layer of the OB was estimated using optical fractionator. With a 60 × objective,
BrdU+ or LacZ+ cells were counted from every sixth
section within 60 μm × 60 μm counting frames equally
spaced across a 300 μm × 300 μm grid. Cells that fell

Page 13 of 15

along the leftmost and bottommost edges of the counting frame were excluded.
Statistical analysis

For MRI data, we compared differences between control
and FAE mice across all 62 brain regions using a measure of effect size, Cohen’s d, for which values ≥ 0.8 or≤ -0.8 indicate a large effect size [91]. For neurosphereassay data, values for FAE mice were expressed as thepercentage of values for control mice, and differences
between groups were tested using one-sample t-tests.
For all other data, ANOVAs were performed and followed by unpaired or one-sample t-tests. In the experiments in which both males and females were used(associative olfactory task testing, BrdU labeling, and
LacZ labeling), we initially included sex as a factor in
the ANOVAs. As we found no significant effects involving sex, however, this factor was dropped from analysis.
Acknowledgements
This work was supported by grants from the Canadian Institutes of HealthResearch (CIHR, MOP86762) and NARSAD to P.W.F. K.G.A. was supported bya postdoctoral fellowship from CIHR. We thank Christine Laliberte forrunning the MRI scans, Joel Ross for assistance with the neurosphere assay,and Henry Ma for assistance with behavioral testing. We also thank Don
Wilson for helpful discussions.
Author details
1
Neurosciences and Mental Health, Hospital for Sick Children, Toronto,
Ontario, Canada. 2Department of Psychiatry, Erasmus Medical Center,
Rotterdam, The Netherlands. 3Department of Molecular Genetics, Universityof Toronto, Toronto, Ontario, Canada. 4Mouse Imaging Centre, Hospital for
Sick Children, Toronto, Ontario, Canada. 5Department of Medical Biophysics,
University of Toronto, Toronto, Ontario, Canada. 6Department of Physiology,
University of Toronto, Toronto, Ontario, Canada. 7Institute of Medical Science,
University of Toronto, Toronto, Ontario, Canada.
Authors’ contributions
KGA, SAK, DV, and PWF conceived of and designed the experiments. KGA
carried out the EtOH treatment, ran the behavioral tests, performed
surgeries, conducted immunohistochemistry, and analyzed the data. SAK
and LC performed the neurosphere assay. ATL participated in EtOH
treatment and immunohistochemistry. JPL coordinated the MRI scans andanalyzed the MRI data. KGA and PWF wrote the manuscript. All authors readand approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 3 June 2011 Accepted: 7 July 2011 Published: 7 July 2011
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doi:10.1186/1756-6606-4-29
Cite this article as: Akers et al.: Fetal alcohol exposure leads toabnormal olfactory bulb development and impaired odor
discrimination in adult mice. Molecular Brain 2011 4:29.