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Contents lists available at ScienceDirect
Behavioural Brain Research
journal homepage: www.elsevier.com/locate/bbr
Research report
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High maternal choline consumption during pregnancy and nursing
alleviates deficits in social interaction and improves anxiety-like
behaviors in the BTBR T + Itpr3tf/J mouse model of autism
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Erika A. Langley 1 , Marina Krykbaeva 1 , Jan Krzysztof Blusztajn, Tiffany J. Mellott ∗
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Q2 Department of Pathology and Laboratory Medicine, Boston University School of Medicine, 72 East Concord St L810A, Boston, MA 02118, USA
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h i g h l i g h t s
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Deficits in social interaction were rescued by perinatal choline supplementation.
Choline supplementation lowered anxiety levels in mice in the OF and EPM.
Marble burying behavior was reduced in B6 and BTBR mice by choline supplementation.
Several choline and 1-carbon metabolism genes are found in BTBR autism-related QTL.
These include: Pcyt1b, Chpt1, Ppap2c, Pld1, Mthfd1, Mthfs, and Slc19a1.
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a r t i c l e
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a b s t r a c t
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Article history:
Received 7 April 2014
Received in revised form
23 September 2014
Accepted 28 September 2014
Available online xxx
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Keywords:
Choline
Autism
Social behavior
Anxiety
Mouse model
BTBR
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1. Introduction
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Autism is a neurodevelopmental disorder with multiple genetic and environmental risk factors. Choline
is a fundamental nutrient for brain development and high choline intake during prenatal and/or early
postnatal periods is neuroprotective. We examined the effects of perinatal choline supplementation on
social behavior, anxiety, and repetitive behaviors in the BTBR T + Itpr3tf/J (BTBR) mouse model of autism.
The BTBR or the more “sociable” C57BL/6J (B6) strain females were fed a control or choline-supplemented
diet from mating, throughout pregnancy and lactation. After weaning to a control diet, all offspring were
evaluated at one or two ages [postnatal days 33–36 and 89–91] using open field (OF), elevated plus
maze (EPM), marble burying (MB), and three-chamber social interaction tests. As expected, control-diet
BTBR mice displayed higher OF locomotor activity, impaired social preference, and increased digging
behavior during the MB test compared to control-diet B6 mice. Choline supplementation significantly
decreased digging behavior, elevated the percentage of open arm entries and time spent in open arms
in the EPM by BTBR mice, but had no effect on locomotion. Choline supplementation did not alter social
interaction in B6 mice but remarkably improved impairments in social interaction in BTBR mice at both
ages, indicating that the benefits of supplementation persist long after dietary choline returns to control
levels. In conclusion, our results suggest that high choline intake during early development can prevent
or dramatically reduce deficits in social behavior and anxiety in an autistic mouse model, revealing a
novel strategy for the treatment/prevention of autism spectrum disorders.
© 2014 Published by Elsevier B.V.
Autism spectrum disorder (ASD) is a heterogeneous group of
neurodevelopmental conditions characterized by behavioral deficiencies in social interactions, impairments in verbal and nonverbal
communication, stereotyped, repetitive patterns of behaviors and
∗ Corresponding author. Tel.: +1 617 638 4850; fax: +1 617 638 5400.
E-mail addresses: tmellott@bu.edu, tmellott@gmail.com (T.J. Mellott).
1
Contributed equally to this manuscript.
interests, and cognitive rigidity seen in childhood and frequently
continuing into adolescence and adulthood [1–5]. Abnormal development of multiple brain structures (including cerebral cortex,
amygdala, and cerebellum) has been observed in ASD with the use
of both in vivo magnetic resonance imaging (MRI) techniques and
postmortem brain analyses [6–8]. ASD exhibits high heritability and
genetic studies identified multiple loci that may increase the risk of
ASD. Interestingly this large number of genes can be systematized
into transcriptional networks that modulate the development of
synapses and cortical laminae [9,10]. Despite the high heritability, it is clear that ASD is not a simple genetic disorder and that
http://dx.doi.org/10.1016/j.bbr.2014.09.043
0166-4328/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Langley EA, et al. High maternal choline consumption during pregnancy and nursing alleviates deficits
in social interaction and improves anxiety-like behaviors in the BTBR T + Itpr3tf/J mouse model of autism. Behav Brain Res (2014),
http://dx.doi.org/10.1016/j.bbr.2014.09.043
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environmental factors may be equally as significant in its etiology. One such environmental factor is maternal nutrition during
the periconceptual period, pregnancy and nursing. In particular
there is good evidence that low maternal intake of folic acid during
these times increases the risk of having an autistic child [11,12].
Moreover, children who carry a common gene variant of folic acid
metabolism, methylene tetrahyfrofolate reductase (MTHFR, 677TT)
or whose mother are carriers of this variant that is known to
reduce the activity of the enzyme and confers increased demand
for dietary folate had an even higher risk of ASD [12]. Folate is a central enzyme cofactor in one-carbon metabolic pathways. Another
essential nutrient in these pathways is choline. However, while
folate is available not only in its natural food sources, in vitamin
supplements, and, thanks to a major public health initiative, in
multiple foods that are supplemented with this vitamin by law,
significant amounts of choline can only be obtained by a pregnant
mother via a normal diet. As foods vary widely in choline content so
does its intake by the population in the United States. Most recent
estimates indicate that a vast majority of people consumes less
choline than recommended [13–16]. As is the case for folate, high
choline consumption during pregnancy reduces the risk of neural
tube defects in offspring [17,18].
While autism may be uniquely human, animal models have been
developed to mimic symptoms and components of the disorder.
The inbred BTBR T + Itpr3tf/J (formerly named BTBR T + tf/J) mouse
strain is characterized as displaying an autism-like behavioral phenotype, such as deficits in reciprocal social interactions [19,20],
impaired communication, and repetitive behaviors [e.g. repetitive
self-grooming] as compared with high sociability and low selfgrooming reference strain C57BL/6J (B6) [21–24]. The BTBR mice
also show decreased accuracy in learning tasks, indicating a lower
attention capacity [25].
In this study, we examined the effects of perinatal choline supplementation on the behavioral deficits displayed by BTBR mice.
Extensive literature on the effects of early-life choline availability in rats and mice indicates that high choline intake during
the perinatal period is neuroprotective in a variety of models of
neuronal dysfunction, including that evoked by aging [26–28],
seizures [29–32], alcohol consumption [33–38] and genetic variation [39–45] including one of the ASD-associated conditions, Rett
syndrome [39–43]. Here we show that maternal dietary choline
supplementation in BTBR mice during pregnancy and lactation – a
period that spans most of rodent brain development in offspring
– can alleviate anxiety-like behavior in the EPM, reduce digging
behavior in the MB test, and improve deficits in social behavior
in the three-chamber testing paradigm in adolescent (P33-36) and
adult (P89-91) progeny.
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2. Materials and methods
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2.1. Animal subjects
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C57BL/6J (B6) and BTBR T + Itpr3tf/J (BTBR) mice were obtained
from The Jackson Laboratory (Bar Harbor, ME) for breeding. Animals were housed in rooms with a 12 h light/dark cycle with lights
on at 6:00 h. All animal procedures were in compliance with the
Institutional Animal Care and Use Committee of Boston University.
Breeding pairs of each strain of mice were divided into two groups:
control and choline supplemented. At mating, pairs were given
either a control (AIN76A diet containing 8 mmol/kg of choline chloride) or supplemented diet (36 mmol/kg). Females were examined
for vaginal plugs and the weights of the females were monitored
to follow the pregnancy. However, males were removed 4 days
after the breeding pairs were set up if a pregnancy was not confirmed. In case a plug was missed, the female remained on the
diet and her weight was closely monitored. If she did not show
signs of pregnancy, she was given a control diet for at least 2
weeks before breeding was attempted again. Any animals that were
given a choline-supplemented diet were never used for control-diet
breeding to avoid any additive effects of the diet. All breeders from
both strains were exposed to similar number of days (±3 days) on
the diets regardless of time required to become pregnant. Females
and their litters remained on the specified diets until weaning on
postnatal day (P) 21. All offspring were placed on the control diet
following weaning. Juveniles were weaned and housed with samesex, age-mated cagemates in groups of four mice per cage. The
majority of B6 litters contained 7–9 pups per litter. We culled litters
of 10 or more and litters of less than 7 were used as “stranger” mice
in social behavioral testing. In contrast, the majority of BTBR litters
produced 5–8 pups per litter. We did not use litters of 4 or fewer
and culled litters over 8 pups. There were no significant effects of
diet on litter sizes of either strain. For the first experimental cohort,
1–2 males and 1–2 females per litter were used for behavioral testing. In the second cohort, 2–3 males and 2–3 females per litter (at
least 1 of each sex per age group) were used for behavioral testing.
In total, there were 18 B6 litters (9 of each diet) and 21 BTBR litters
(10 control-diet and 11 choline-supplemented).
2.2. Behavioral testing
For these studies, two experimental cohorts of animals were
used. We tested approximately equal numbers of male and female
mice per group. At P33-36, the first cohort of animals (B6: Control
N = 20, Supplemented N = 18; BTBR: Control N = 24, Supplemented
N = 27) was behaviorally tested using the open field test, marble
burying test, and elevated plus maze. Animals were transferred to
the behavioral testing room 1 h before the initiation of testing. The
open field test was performed first, animals were then returned
to their home cages for 1 h before marble burying testing. After
returning to their home cages again for 3 h, the same animals were
tested on the elevated plus maze.
Social interaction and marble burying tests were performed on
the second cohort of mice during two developmental periods: P3336 (adolescence) (B6: Control N = 18, Supplemented N = 19; BTBR:
Control N = 20, Supplemented N = 21) and P89-91 (adulthood) (B6:
Control N = 16, Supplemented N = 14; BTBR: Control N = 19, Supplemented N = 17). Animals were transferred to the behavioral testing
room 1 h before the initiation of testing. Animals were returned to
their home cages for 1 h between tests. All behavioral procedures
were conducted between 12:00 h and 18:00 h.
2.2.1. Open field (OF)
Mice were tested for general exploratory locomotion in a rectangular open field arena (40 cm × 30 cm × 30 cm) for a 30 min session,
under an illumination of 30 lx. Total distance traveled in the arena,
velocity, time spent in central and peripheral zones and time spent
grooming was scored using Ethovision 9.0 software (Noldus Information Technology). The apparatus was cleaned using 70% ethanol
after each animal and allowed to dry for at least 30 min before the
next mouse was tested.
2.2.2. Marble burying (MB)
This test was used to assess levels of repetitive behaviors. Subjects were introduced into a clean, sterilized large acrylic cage
(40 cm × 30 cm × 30 cm) filled with woodchip bedding to a depth
of 4 cm and topped with 20 blue glass marbles (1.5 cm in diameter)
evenly spaced apart in four rows of 5 marbles for 30 min [46]. The
test was performed under low illumination of 20 lx. The number of
marbles covered by greater than 50% by bedding were tallied at the
conclusion of the test by researchers blind to sex and treatment
Please cite this article in press as: Langley EA, et al. High maternal choline consumption during pregnancy and nursing alleviates deficits
in social interaction and improves anxiety-like behaviors in the BTBR T + Itpr3tf/J mouse model of autism. Behav Brain Res (2014),
http://dx.doi.org/10.1016/j.bbr.2014.09.043
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group. The apparatus was cleaned using 70% ethanol after each
animal and refilled with bedding.
2.2.3. Elevated plus maze (EPM)
Animals were tested for anxiety-like behaviors in the elevated
plus-maze test using previously described methods [47–50]. The
elevated plus-maze consisted of two open arms (30 cm × 8 cm) and
two closed arms (30 cm × 8 cm × 25 cm) extending from a central
area (8 cm × 8 cm) and was raised 40 cm from the floor. The closed
arms were surrounded by high (20 cm), black opaque walls. The
entire arena was consistently lit from above at an illumination level
of 75 lx. The test began when the subject mouse was placed in the
center. The mouse was allowed to freely explore the maze for 5 min.
Time spent in the open arms, center, and closed arms and the number of entries into the open arms and closed arms were scored when
all four paws of the mouse were within a given region. Test parameters were scored by viewers who were blind to sex and treatment
group. The apparatus was cleaned using 70% ethanol after each
animal.
2.2.4. Social interaction
The three-chamber paradigm described by Crawley (2007) was
used to study social affiliation. The test was performed under low
illumination (20 lx). Test subjects were first habituated to the center of the test chamber with the walls between chambers closed for
5 min. The walls were then opened and the mouse was allowed to
freely explore all three chambers for an additional 10 min. The walls
were reinserted with the mouse in the center chamber before the
start of the test. The test consisted of two 10 min sessions. For the
first part, an age-matched, same-sex B6 mouse or “stranger mouse”
was placed inside the wire cage located in one of the side chambers. The other wire cage in the opposite side chamber remained
empty. The walls between chambers were removed to start the test.
For 10 min, the following parameters were recorded: 1. “Chamber
time/entries” defined as the duration and number of entries into
each compartment, 2. “Social approach” or “social sniffing” defined
as the duration and number of active (direct interaction with the
control animal, sniffing behavior or stretching of the body of the
subject toward the stranger mouse in an area 3–5 cm around the
containment unit) contacts between the subject animal and the
wire containment unit (with or without the control animal), and
3. Duration and number of other behaviors such as self-grooming,
“freezing” (no movements for more than 5 s), jumping, and repetitive behaviors. For the second part of the test, the subject mouse
was returned to the center chamber with the walls reinserted and
a novel stranger mouse was placed into the previously empty wire
cage. The second test was initiated when the walls are removed and
the test parameters are recorded as before. Test parameters were
scored by at least two viewers blind to sex and treatment group.
The apparatus was cleaned using 70% ethanol after each animal and
allowed to dry for at least 30 min before the next mouse was tested.
2.3. Data analysis
Data, presented as means ± SEM, were analyzed by a two-way
or three-way ANOVA, as appropriate. Post hoc analyses were performed with a Tukey’s test. Although previous studies reported no
sex differences in sociability or self-grooming in B6 and BTBR mice
[21,22,24,51–53], we tested male and female mice of each strain
to determine if the effects of choline supplementation were sexually dimorphic. Statistical analysis was performed on data from
individual sexes, but sex did not significantly influence the results
of any test performed in this study nor did it have a significant
impact on the effectiveness of choline supplementation. Therefore,
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the results for all experiments are presented as the combined data
obtained from both male and female subjects.
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3. Results
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3.1. Experimental Cohort 1
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3.1.1. Open field test
The open field test was conducted in order to assess general
locomotor activity and exploratory behavior. More anxious mice
avoid spending time in the center of an open field, and velocity
usually decreases as the mouse becomes habituated to the novel
environment [54]. We measured total distance traveled, average
velocity, and time spent in the center and peripheral zones per
5 min interval (data for time spent in peripheral zones are not
shown). Total distance traveled and velocity of both B6 and BTBR
strains declined across the 30 min session as expected, representing habituation to the novel open field (Fig. 1A, B). During the
first two intervals, BTBR mice exhibited higher distances as compared to B6 mice, consistent with previous studies [21,22,51,55,56].
Perinatal choline availability did not affect total distance scores
or velocity in either strain, indicating the absence of confounding
hypo- or hyperactivity effect by choline status. An increase in the
traveled distances by BTBR mice can be appreciated in the track
visualization images of representative animals from each strain
and dietary group (Fig. 1C). Choline supplementation also significantly increased the time spent in the center of the open field
by both B6 and BTBR mice, possibly indicative of decreased anxiety or an increase in exploratory behavior (Fig. 1D). Self-grooming
behavior was also monitored during this test. BTBR mice had longer
grooming times than B6 mice as predicted from previous studies
[21,22,51,55,56]; however, choline supplementation did not significantly alter grooming behavior (data not shown).
3.1.2. Marble burying test
This task was typically used as a measure for anxiety in rodents;
however, it was recently reclassified as an evaluation of digging as
a repetitive behavior, such as self-grooming, due to the lack of correlation with performance in other standard approaches to assess
anxiety-like behavior including the OF and EPM. As previously
reported, BTBR mice buried significantly more marbles compared
to B6 mice (Fig. 2) [46]. Perinatally choline-supplemented B6 and
BTBR mice buried significantly fewer marbles compared to mice of
the same strain whose mothers consumed a control diet and there
was no difference in the number of marbles buried by mice of either
strain following perinatal choline supplementation (Fig. 2), indicating that this treatment may reduce the occurrence or frequency of
repetitive behaviors.
3.1.3. Elevated plus maze
Mice with higher levels of anxiety tend to avoid exposed,
brightly-lit spaces such as the open arms and prefer to spend time in
the shelter of the closed arms. This effect is increased by the danger
of falling from the open arms [57]. During this 5 min test, the duration of time spent in the open and closed arms as well as the center
of the plus maze was measured. In addition, the number of entries
into each arm was recorded. Consistent with the results from the
open field analysis, BTBR mice, regardless of perinatal diet, displayed increased locomotor activity as compared to B6 mice, which
can be observed in the higher number of total arm entries (Fig. 3A).
BTBR mice of the control group, however, had a significantly lower
percentage of entries into the open arms (Fig. 3B), and thus spent
less time in the open arms than B6 mice of the same dietary group
(Fig. 3C). However, BTBR mice spent significantly more time in the
center of the EPM than B6 mice (Fig. 3C). Although supplementation with choline did not alter the behavior of the B6 mice in the
Please cite this article in press as: Langley EA, et al. High maternal choline consumption during pregnancy and nursing alleviates deficits
in social interaction and improves anxiety-like behaviors in the BTBR T + Itpr3tf/J mouse model of autism. Behav Brain Res (2014),
http://dx.doi.org/10.1016/j.bbr.2014.09.043
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A 2500 P33-36
8 P33-36
C57BL/6J
Control
B6
Control
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Supplemented
B6
Supplemented
BTBR Control
BTBR
Control
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Total Distance, cm
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Control
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Control
C57BL/6J
Supplemented
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BTBR Control
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Control
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C57BL/6J
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Control
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Fig. 1. Perinatal choline supplementation did not affect locomotor function. Control and perinatally choline-supplemented B6 and BTBR mice were evaluated using the
open field test at P33-36. Total distance traveled (A), average velocity (B), movement and time in the center of the arena (C, D) were recorded over a 30 min session. (A) As
determined by three-way ANOVA using diet, strain, and time intervals as independent variables, there were significant overall effects of strain (p < 0.005) and time interval
(p < 0.0001) on total distance traveled. (B) Similarly, strain (p < 0.01) and time interval (p < 0.0001), but not diet, significantly influenced velocity as determined by a three-way
ANOVA. (C) Red trace lines show movement of the mouse in the peripheral zone and yellow trace lines show movement in the center zone in representative images of mice
from each dietary group and strain. (D) Using a three-way ANOVA, diet (p < 0.0005), strain (p < 0.005), and time interval (p < 0.000001) had a significant overall effect on the
time spent in the center of the arena by all mice. There was a significant effect of diet on the time spent in the center by B6 mice (p < 0.05) and BTBR mice (p = 0.005), as
determined by two-way ANOVAs using diet and time intervals as independent variables for each strain. (For interpretation of the references to color in this figure legend,
Q4 the reader is referred to the web version of the article.)
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EPM, the percentage of open arm entries and the time spent in
open arms by BTBR mice were increased in a significant fashion by
choline (Fig. 3). Overall, our results indicate that BTBR strain, while
displaying higher locomotor activity, have increased tendency to
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Number of Marbles Buried
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avoid open areas suggesting higher levels of general anxiety during this test which can be alleviated by increased availability of
choline during perinatal development.
3.2. Experimental Cohort 2
C57BL/6J Control
C57BL/6J Supplemented
BTBR Control
BTBR Supplemented
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P33-36
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0
Fig. 2. Perinatal choline supplementation reduced marble burying behavior. The
marble burying test was conducted on P33-36 using B6 and BTBR mice which were
perinatally exposed to either a control or choline-supplemented diet. Data were analyzed by two-way ANOVA using diet and strain as independent variables, followed
by a post-hoc Tukey test. There was a significant overall effect of diet (p < 0.0001).
There was a significant increase in the number of marbles buried by the control-diet
BTBR mice as compared to the B6 mice (*, p = 0.05). Choline supplementation significantly decreased the number of marbles buried in both B6 († , p < 0.0001) and BTBR
mice († , p < 0.0001).
3.2.1. Social interaction and marble burying test
The three-chambered task was designed to qualitatively and
quantitatively assess the social approach behaviors of mice and
evaluate impairments in sociability or appropriate social interaction in animal models of disorders such as autism. Using this
behavioral test, we examined the effects of perinatal choline
supplementation on B6 and BTBR mice at two ages: P33-36 (adolescent) and P89-91 (adult). At P33-36, the B6 mice of both dietary
groups displayed a significant preference for the chamber with
the novel mouse compared to the chamber with the empty object
(Fig. 4A). In contrast, control BTBR mice spent significantly more
time in the chamber with the novel object than with the novel
mouse and times spent in both chambers significantly differed from
those of control-diet B6 mice (Fig. 4A). The chamber times of perinatally choline-supplemented BTBR mice, however, resembled those
of the B6 mice of either diet and were significantly different from
BTBR mice of the control group (Fig. 4A). The social approach times
of B6 mice and BTBR mice of the control groups were consistent
with previous reports [19,21,55,58], such that B6 mice spent significantly more time engaged in social interaction with the stranger
Please cite this article in press as: Langley EA, et al. High maternal choline consumption during pregnancy and nursing alleviates deficits
in social interaction and improves anxiety-like behaviors in the BTBR T + Itpr3tf/J mouse model of autism. Behav Brain Res (2014),
http://dx.doi.org/10.1016/j.bbr.2014.09.043
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Fig. 3. Perinatal choline supplementation reduced anxiety in the elevated plus
maze in the BTBR strain. The EPM was used to test control and perinatally cholinesupplemented B6 and BTBR mice at P33-36. The total number of entries into open
and closed arms (A), the percentage of open arm entries (B), and the time spent in
open and closed arms (C) were determined for each animal for the 5 min session.
Data were analyzed by two-way ANOVA using diet and strain as independent variables, followed by a post-hoc Tukey test. (A) There was a significant overall effect
of diet (p < 0.005) and strain (p < 0.0001) on the total number of entries. BTBR mice
had a significantly higher number of entries than B6 mice in both the control (*,
p = 0.0001) and choline supplemented groups ($ , p < 0.01). (B) There was a significant
overall effect of strain on the percentage of open arm entries (p < 0.0005). The percentage of open arm entries was significantly reduced in control-diet BTBR mice as
compared to control B6 mice (*, p < 0.001) and was dramatically increased by choline
supplementation of BTBR mice to near B6 levels († , p < 0.005). (C) Overall, there was
a significant effect of diet and strain on the time spent in the closed arms (p < 0.0001
and p < 0.005, respectively) and on time in the center (p < 0.01 and p < 0.0001, respectively), but only an effect of diet on time in open arms (p < 0.01). In the control group,
BTBR mice spent significantly less time in the open arms (*, p = 0.001) than B6 mice,
but more time in the center (*, p < 0.005). Choline supplementation decreased time
spent in closed arms († , p < 0.0001) and increased the time in open arms († , p < 0.005)
and center († , p < 0.05) by BTBR mice compared to control-diet BTBR mice. Cholinesupplemented BTBR mice also spent less time in closed arms ($ , p < 0.0005) than B6
mice of the same diet, but more time in the center ($ , p < 0.0005).
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mouse than with the novel object and that BTBR mice displayed a
significant impairment (Fig. 4B). Choline supplementation had no
effect on the social behavior of B6 mice but significantly increased
the social approach times in BTBR mice to a level similar to B6 mice.
Although, the social novelty session did not yield any significant differences in the chamber times between animals of different
strain or perinatal diet (Fig. 4C), we did observe the expected
preference for social interaction with stranger 2 and a noticeable
5
impairment in this preference in BTBR mice of the control group
(Fig. 4D). There was a significant reduction in the amount of time
engaged in social interaction with stranger 2 by control-diet BTBR
mice, but this deficit was ameliorated by choline supplementation.
In order to determine if social interaction would alter the frequency of digging behaviors and the level of anxiety, we subjected
the mice to the MB test following the social interaction test. BTBR
mice whose mothers consumed a control diet showed signs of
higher anxiety and/or more repetitive behaviors than B6 mice of
the same diet as seen by a significant increase in the number of
marbles buried (Fig. 4E). As in animals of cohort 1, perinatal cholinesupplementation not only significantly decreased the number of
marbles buried by animals of either strain when compared to mice
of the same strain exposed to a control diet throughout development, but also ameliorated the differences observed between the
BTBR and B6 strains. In addition, B6 and BTBR mice from the control
group showed a 30–50% increase in the number of marbles buried
following the social interaction test (as compared to Fig. 2).
To determine if the social behavioral deficits continue to be
observed with age and if the effects of perinatal choline supplementation continues months after the animals were switched to
a control diet, we tested adult B6 and BTBR mice at P89-91. The
observed chamber times for the older animals were similar to those
at the younger age in the social preference test (Figs. 5A, 4A). Again,
the control BTBR mice displayed a significant preference for the
chamber containing the novel object rather than the chamber with
the novel mouse in contrast to the preference observed in B6 mice.
The social approach times of adult mice were consistent with those
observed in the younger mice during the social preference task
and the expected results from previous studies [19,21,59]. Controldiet BTBR mice spent significantly less time with the stranger
mouse compared to B6 mice of the same diet group, demonstrating their low sociability (Fig. 5B). When perinatally supplemented
with choline, BTBR mice interacted with the stranger mouse for a
significantly greater amount of time than control-diet BTBR mice,
indicating that the improvements in social behavior in BTBR mice
by choline supplementation during perinatal development persists
for months after dietary choline was returned to control levels.
In the social novelty task in the P89-91 age group, a tendency
for a preference for a novel stranger mouse was observed for all
groups of mice in terms of time spent in each chamber and social
approach times (Fig. 5C, D). BTBR mice continuously exposed to
a control diet were not impaired in regard to their preference to
interact with stranger 2 at this age.
Again, adult control-diet BTBR mice buried significantly more
marbles than B6 marbles of the control group, and the significant
effects of choline supplementation on marble burying behaviors
were also observed in these mice (Fig. 5E). P89-91 animals (Fig. 5E)
buried approximately 20% more marbles than P33-36 animals
(Fig. 4F) in control groups.
4. Discussion
These results demonstrate that choline supplementation of
maternal diets during pregnancy and lactation can improve the offspring’s social interaction, reduce anxiety, and lessen the display
of some repetitive behaviors in a mouse model of autism. Using
the OF test to assess exploratory behavior and general activity,
our results confirmed previously published reports describing an
initial hyperactivity by BTBR mice, as demonstrated by increased
velocity and total distance traveled during the first two intervals
and then habituation to the novel environment [21,22,51,55,56].
Choline supplementation did not alter general locomotor activity
or habituation to the OF in either strain, indicating a lack of any confounding effects of choline on locomotion. The number of crosses
Please cite this article in press as: Langley EA, et al. High maternal choline consumption during pregnancy and nursing alleviates deficits
in social interaction and improves anxiety-like behaviors in the BTBR T + Itpr3tf/J mouse model of autism. Behav Brain Res (2014),
http://dx.doi.org/10.1016/j.bbr.2014.09.043
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Fig. 4. Perinatal choline supplementation improved social interaction in BTBR mice at P33-36. The three-chamber social interaction and marble burying tests were conducted
on control and perinatally choline-supplemented B6 and BTBR mice on P33-36. Social preference (A, B) and social novelty (C, D) were tested. The time spent in each chamber
(A, C); and the time engaged in direct social interaction, or “social approach,” (D) were recorded. Data were analyzed by two-way ANOVA using diet and strain as independent
variables, followed by a post-hoc Tukey test. (A) There was a significant effect of diet on the chamber times with the stranger mouse (p < 0.05), and time spent in the chamber
with the novel object was significantly influenced by strain (p < 0.05). Control B6 mice spent significantly more time in the chamber with the stranger mouse than the chamber
with the novel object (# , p < 0.001), as did mice from the choline-supplemented B6 (# , p < 0.05) group. In contrast, control-diet BTBR mice spent significantly more time in
the chamber with the novel object instead of with the stranger mouse (# , p < 0.0001). They spent significantly less time in the chamber with stranger 1 (*, p < 0.05) and more
time in the chamber with the novel object (*, p < 0.005) as compared to control-diet B6 mice. Choline supplementation of BTBR mice significantly increased the time spent in
the chamber with the stranger mouse († , p < 0.05) and decreased the time spent in the chamber with the novel object († , p < 0.05). (B) The time engaged in social interaction
with the stranger mouse was significantly influence by diet (p < 0.05). Control B6, choline-supplemented B6, and choline-supplemented BTBR mice spent significantly more
time interacting with the stranger mouse than the novel object (# : p < 0.0005, p < 0.005, and p < 0.00005, respectively). In the control group, BTBR mice spent significantly
less time interacting with the stranger mouse compared to the B6 mice (*, p < 0.005) and more time interacting with the novel object (*, p < 0.05). Choline supplementation
of BTBR mice significantly increased the amount of interaction time with the stranger mouse († , p < 0.001) and reduced the time spent interacting with the novel object
(† , p < 0.01). (C) There were no significant differences in chamber times between the groups of mice. (D). There was a significant effect of diet on social interaction times
with stranger 2 only (p < 0.05). In mice on a control diet, the interaction with stranger 2 was significantly reduced in BTBR mice compared to B6 (*, p < 0.005), but choline
supplementation significantly increased the interaction time with stranger 2 by BTBR mice († , p < 0.01). (E) Marble burying behavior was significantly influenced by diet
(p < 0.0001). Control-diet BTBR mice buried a significantly larger number of marbles than B6 mice (*, p < 0.01). Choline supplementation significantly reduced marble burying
behavior in both B6 († , p < 0.0005) and BTBR mice († , p < 0.0001).
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into the center of the arena and/or the time spent in the center can
be indicative of the level of exploratory behavior or anxiety displayed by a mouse. In our study, choline supplementation notably
increased the time spent in the center during the OF test by both
strains suggesting that increased dietary choline can either enhance
exploratory behavior or reduce anxiety. In fact, perinatal choline
supplementation has already been shown to attenuate age-related
declines in exploratory behavior [60] and decrease anxiety-like
behavior in rats [61].
Although the MB test was originally developed as a measure
of anxiety such that the number of marbles buried was influenced by treatment with anxiolytic and anxiogenic compounds,
recent studies have shown that it may be more accurate test to
assess levels of repetitive behavior rather than anxiety [62]. Surprisingly, the MB test exposed the most substantial effects of
choline supplementation, particularly since no significant effects
of choline were observed on grooming behavior during the OF test.
As expected, control-diet BTBR mice displayed significantly more
Please cite this article in press as: Langley EA, et al. High maternal choline consumption during pregnancy and nursing alleviates deficits
in social interaction and improves anxiety-like behaviors in the BTBR T + Itpr3tf/J mouse model of autism. Behav Brain Res (2014),
http://dx.doi.org/10.1016/j.bbr.2014.09.043
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Fig. 5. Perinatal choline supplementation improves social interaction behaviors in BTBR mice at P88-91. Control and choline-supplemented B6 and BTBR mice were tested
using the three-chamber social interaction and marble burying test at P88-91. Social preference (A, B) and social novelty (C, D) were tested. The time spent in each chamber
(A, C); and the time engaged in direct social interaction, or “social approach,” (D) were recorded. Data were analyzed by two-way ANOVA using diet and strain as independent
variables, followed by a post-hoc Tukey test. (A) There was a significant effect of strain and diet on the chamber times with both the stranger mouse (p < 0.05 and p < 0.05,
respectively) and the novel object (p < 0.05 and p < 0.05, respectively). Control B6 mice spent significantly more time in the chamber with the stranger mouse than the chamber
with the novel object (# , p < 0.05), as did mice from the choline-supplemented B6 (# , p < 0.005) and BTBR groups (# , p < 0.005). Control-diet BTBR mice spent significantly less
time in the chamber with stranger 1 (*, p < 0.05) and more time in the chamber with the novel object (*, p < 0.01) as compared to control-diet B6 mice. Choline supplementation
of BTBR mice decreased the time spent in the chamber with the novel object († , p < 0.05). (B) The time engaged in social interaction with the stranger mouse was significantly
influence by diet (p < 0.05). Control B6, choline-supplemented B6, and choline-supplemented BTBR mice spent significantly more time interacting with the stranger mouse
than the novel object (# : p < 0.01, p < 0.001, and p < 0.05, respectively). In the control group, significantly less time was spent interacting with the stranger mouse by BTBR mice
compared to the B6 mice (*, p < 0.05), but choline supplementation of BTBR mice significantly increased the amount of interaction time with the stranger mouse († , p < 0.05).
(C) There were no significant differences in chamber times between the groups of mice. (D). There was a significant effect of diet on social interaction times with stranger 1
only (p < 0.05). (E) Overall, marble burying behavior was significantly effected by diet (p < 0.0001). A significantly larger number of marbles was buried by control-diet BTBR
mice than by B6 mice (*, p < 0.0001). Choline supplementation significantly reduced marble burying behavior in both B6 († , p < 0.0001) and BTBR mice († , p < 0.0001).
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digging behavior than B6 mice. Remarkably, choline supplementation reduced marble burying in both B6 and BTBR mice. Given
that locomotor activity of either strain during the OF test was not
affected by choline, the data would suggest that the presentation of
certain repetitive behaviors was reduced by the supplementation
of choline during early development. This effect was also observed
in the results of the MB test following social testing. In fact, choline
supplementation was even more effective in reducing marble burying behaviors in both strains of mice following social anxiety, which
increased digging behaviors by 30–50%.
Previous studies have reported a large variability in the performance of BTBR mice in the EPM, ranging from the absence of
anxiety-like traits to anxiety scores similar to B6 mice to exaggerated behavioral responses to stress [19,50,53,55,63,64]. In this
study, control BTBR mice had a higher number of total entries in the
EPM, but had a significantly smaller percentage of open arm entries
and spent less time in the open arms indicating an avoidance of
the open areas and higher anxiety-like behavior. Interestingly, the
total time in the center of the EPM were significantly higher in both
control and choline-supplemented BTBR compared to B6 mice of
either diet, suggesting that both groups of BTBR mice displayed an
increased interest in exploring the open arms but control BTBR mice
failed to completely cross into the open arms with all four paws as
frequently. The BTBR mice in our study often displayed a stretched
Please cite this article in press as: Langley EA, et al. High maternal choline consumption during pregnancy and nursing alleviates deficits
in social interaction and improves anxiety-like behaviors in the BTBR T + Itpr3tf/J mouse model of autism. Behav Brain Res (2014),
http://dx.doi.org/10.1016/j.bbr.2014.09.043
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posture between the center and open arms of the EPM, consistent
with the observations of Pobbe et al. [19] that BTBR displayed significantly more risk assessment behaviors such as stretch attend
and stretched head-out postures than B6 mice in the EPM. Since
the EPM was performed on mice following OF and MB testing, it is
possible that the level of stress was elevated in these mice due to
handling, albeit minimal, and previous behavioral testing. Recently,
Benno et al. [63] showed that administration of acute stress before
the EPM altered the behavior of BTBR mice such that time spent
in the open arms was significantly reduced compared to stressed
B6 mice, as well as non-stressed B6 and BTBR mice, suggesting that
BTBR mice may be unusually stress-reactive and exhibit heightened
behavior in response to stress. Another factor that may influence
EPM performance may be age; specifically, mice tested in this study
were significantly younger (approximately 4 weeks old compared
to a range of 12–17 weeks old from other studies). Finally, experimental conditions such as lighting may have a significant effect on
EPM behavior, especially in the BTBR strain. Yang et al. [53] reported
increased percentage of time in open arms, open arm entries, and
total number of entries in BTBR mice when compared to B6 mice,
while Pobbe et al. [19] reported decreased time in open arms and
increased level of risk assessments, such as stretched posturing)
for BTBR mice. Yang et al. [53] conducted their assays in dimmer
lighting (20 lx) while Pobbe et al. [19] used 55 lx. This correlation of
behavior to light level may also indicate an increased anxiety-like
response of the BTBR strain in a situation with a more aversive stimulus, i.e. the more brightly-lit open arms. During our study, the EPM
was positioned in a brightly-lit environment, whereas the OF and
MB test were performed under dimly-lit conditions. Regardless,
when BTBR mice are supplemented with choline, the anxiety-like
behavior of avoiding open areas in the EPM is completely reversed,
signifying an anxiolytic effect of choline supplementation. It is possible that higher levels of dietary choline during brain development
may not only reduce basal anxiety levels in mice, but may also
render a mouse more equipped to handle aversive stimuli.
Low sociability in the BTBR strain has been consistently
reported in the three-chamber social test in several studies
[19,21,22,24,51,52,55,64–69], and the well-characterized deficits
in social preference by BTBR mice have become a prevalent
tool used to investigate the efficacy of pharmacological agents,
nutritional therapies, and even environmental factors on the
modification of social behavior in these mice. At P33-36 and P8991, both control and choline-supplemented B6 mice displayed a
strong preference for the stranger mouse over the novel object,
as observed in both the chamber times and social approach or
“sniffing” times. Consistent with previous studies, BTBR mice
showed a severe impairment in social preference at both ages
[19,21,22,24,51,52,55,64–69]; however, this deficiency in social
interaction was ameliorated by perinatal choline supplementation.
In testing for the preference for social novelty, both control and
choline-supplemented B6 mice displayed a distinct preference for
social interaction with a novel stranger mouse over the more familiar mouse at P33-36. At this age, social novelty preference was also
impaired in control BTBR mice, but this deficit could be ameliorated
by the supplementation of choline in the mother’s diet. The results
of the social novelty test at P88-91 were less clear in that BTBR
mice were not impaired in social novelty at this age. Other studies
have reported that BTBR mice, while impaired in social preference
over a novel object, display preference for social novelty similar
to B6 mice, i.e. chamber and/or sniffing times [55,64,68]. Moreover, Pearson et al. [70] suggested that the social novelty test may
not necessarily exclusively measure an intention to initiate social
interaction in mice, but may instead reflect the exploration of a
novel environmental stimulus. Their studies showed that by placing stranger 2 in the location previously occupied by stranger 1
and placing stranger 1 in the previously empty cage removes any
preference for either mouse, suggesting that the social preference
test may be a better measure of the intention to interact socially
as the preference test precludes any possibility of the position of
the mouse as a factor in the test subject’s preference [70]. While
our data on the impairments in social behavior of BTBR mice are
consistent with previously published reports, our results reveal an
effective treatment to improve the deficits in social interactions in
these mice. Although the mechanism is still unclear, it is apparent from our data that additional dietary choline during perinatal
development can be neuroprotective and can prevent some of the
behavioral abnormalities associated with ASD.
As noted above, ASD is highly heritable and there is evidence
that genetic polymorphism in MTHFR (a gene within the one-carbon
metabolic pathway), as well as low intake of folic acid during early
development increases the risk of ASD. The inbred BTBR mouse
strain used as a model of autism thus offers an opportunity to
investigate genetic determinants of the autism-like phenotypes
using linkage analysis. By generating F2 cross of B6 and BTBR mice
and linkage mapping, Jones-Davis et al. identified quantitative trait
loci (QTL) related to abnormal social behaviors of the BTBR mouse
model of autism [66]. These QTL were found on chromosomes
1, 3, 9, 10, 12 and X [note that the loci on chromosomes 9 and
X were transgressive]. The regions are large containing multiple
genes. Jones-Davis et al. focused their investigations on possible
polymorphic genes in those QTL that are expressed in cerebral cortex and encode developmental proteins, protein kinases, receptors
and synaptic proteins as well as immune and heat shock proteins.
We performed a search on these QTL for genes encoding proteins
related to the metabolism of choline, and one-carbon metabolism
including folic acid and methyl group transfer. We hypothesize that
polymorphisms in such genes could alter the animals’ requirement
for choline and/or the signaling pathways that involve cholinecontaining compounds (e.g. acetylcholine). Interestingly, the QTL
contained several genes encoding enzymes of phosphatidylcholine
turnover and of folate transport and metabolism. In addition
the QTLs contained three genes encoding nicotinic cholinergic
receptors (Chrna3, Chrna5, Chrnb5) a DNA methylation modulator, Dnmt3l and a methylated DNA binding protein, Mecp2 (Fig. 6).
Not surprisingly, most of the genes contained multiple single
nucleotide polymorphisms (SNPs) that distinguished the B6 and
BTBR strains (http://www.informatics.jax.org/strains SNPs.shtml).
Remarkably the genes of phosphatidylcholine turnover encode
enzymes that constitute almost complete pathway of its synthesis and breakdown and include Pcyt1b, Chpt1, Pld1 and Ppap2c.
Pcyt1b encodes the rate-limiting enzyme of phosphatidylcholoine
synthesis, phosphocholine cytidylyltransferase that catalyzes the
synthesis of CDP-choline [71]. The enzyme is highly expressed in
brain and has a critical function in axonal growth [71]. CDP-choline
and diacylglycerol then combine to generate phosphatidylcholine
in a reaction catalyzed by cholinephosphotransferase encoded by
Chpt1 [71]. One of the enzymes that hydrolyzes phosphatidylcholine is Pld1 that generates free choline and phosphatidic acid.
The choline molecule produced in this reaction can be recycled
back into the phospholipid pathway or converted to acetylcholine [72,73]. We have previously shown that high choline
intake during fetal development in rats increases PLD activity in hippocampal slices [74]. The QTL also contained Ppap2c
that catalyzes the conversion of phosphatidic acid to diacylglycerol, thus allowing recycling of the latter substrate for
phosphatidylcholine synthesis catalyzed by Chpt1. In addition, the
QTL contained three genes of folate metabolism (Slc19a1, Mthfs,
and Mthfd1). Slc19a1 encodes the reduced folate carrier (RFC) that
transports several folate species from the circulation into cells
[75]. Mthfs encodes 5,10-methenyltetrahydrofolate synthetase that
converts 5-formyltetrahydrofolate (5-formylTHF, folinic acid), considered a storage folate pool, to 5,10-methenyltetrahydrofolate
Please cite this article in press as: Langley EA, et al. High maternal choline consumption during pregnancy and nursing alleviates deficits
in social interaction and improves anxiety-like behaviors in the BTBR T + Itpr3tf/J mouse model of autism. Behav Brain Res (2014),
http://dx.doi.org/10.1016/j.bbr.2014.09.043
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Chrna3
Chrna5
Chrnb4
Methionine
Acetylcholine
Phosphocholine
Choline
THF
5-formylTHF
Mthfs
Mthfd1
Betaine
5,10-methyleneTHF
5,10-methenylTHF
Mthfd1
Pcyt1b
CDP-choline
Homocysteine
Ppap2c
Diacylglycerol
Chpt1
5-methylTHF
Phosphatidate
Pld1
Phosphatidylcholine
Slc19a1
SAH
Methylation reactions,
e.g. DNA methylation
Dnmt3l
Mecp2
SAM
Fig. 6. Genes related to choline metabolism contained in QTL for autism-related behaviors in BTBR mice. The figure assembles the QTL genes into a metabolic and functional
network. The metabolic pathways highlight the salient features of the network and are not meant to be comprehensive. For clarity only the names of the QTLs genes are
shown (red labels) and mouse gene (rather than protein) conventions are used. The genes are found in QTL on the following chromosomes: chr 3 (Pld1); chr 9 (Chrna3, Chrna5,
Chrnab4, Mthfs); chr 10 (Chpt1, Dnmt3l, Ppap2c, Slc19a1); chr 12 (Mthfd1); chr X (Mecp2, Pcyt1b). Dotted arrow indicates that the pathway includes intermediates; dashed
arrow indicates transmembrane transport. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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(5,10-methenylTHF). Interestingly, in mice consuming a diet devoid
of choline and folate, the hepatic and renal expression of MTHFS
protein was less than half that seen in animals on the control diet [76]. The product of MTHFS can also be generated by
methylenetetrahydrofolate dehydrogenase encoded by Mthfd1 that
uses tetrahydrofolate (THF) as a substrate for this reaction. The
enzyme also catalyzes the conversion of 5,10-methenylTHF to
5,10-methylenetetrahydrofolate (5,10-methyleneTHF). MTHFD1 is
polymorphic in humans. Remarkably, in a clinical study Kohlmeier
et al. found that premenopausal women with the common
MTHFD1-1958A allele polymorphism had 15 times increased susceptibility to developing organ dysfunction on a low-choline diet
as compared subjects who were not carriers of this allele [77].
The QTL also contained two genes whose products modify chromatin (Dnmt3l and Mecp2). Dnmt3l encodes a catalytically-inactive
member of DNA methyltransferase enzyme family [78]. Its product DNA methyltransferase 3-like binds to active DNMT3a [78] and
is critical for establishing the sites of de novo DNA methylation
[79]. We have previously shown that in rat fetuses whose mothers consumed a choline-deficient diet, Dnmt3l mRNA levels were
two-fold higher than in controls [80]. The Mecp2 gene encodes the
methyl CpG binding protein 2 whose mutant alleles cause Rett
syndrome—a form of mental retardation classified as a member
of autism spectrum disorders. Several studies showed that early
postnatal supplementation with choline improves symptoms and
ameliorates several brain measures in genetic mouse models of Rett
syndrome [39–43]. Overall the observations that the behavioralphenotype-related QTL in BTBR mice contain genes associated with
choline and one-carbon metabolism whose expression responds to
dietary choline and folate supply or whose polymorphism is associated with altered requirements for choline and folate, support the
notion that dietary intake of these nutrients during gestational and
early postnatal development may affect the risk of autism.
Acknowledgements
This work was supported by a grant from the Simons Foundation
awarded to TJM. We would also like to thank Mithilia Mahesh for
her assistance with the behavior testing performed in this study.
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