The Effects of Vitamin D-3 During Pregnancy and Lactation on Offspring Physiology and Behavior in Sprague-Dawley Rats

Abstract

Recent findings show that developmental vitamin D deficiency leads to altered brain morphology and behavioral development in the rat offspring. We examined the effects of different dietary vitamin D levels in rat dams on behavior and biochemistry of the offspring. Females were divided into five conditions and received diets containing 0, 1,5, 3.3, 6.0, or 10.0 IU/g of vitamin D(3) from mating to weaning. Offspring were tested as juveniles and as adults for anxiety, social learning and behavior, and locomotion. Results show that both deficient and excessive levels of vitamin D(3) in juveniles lead to altered physiology and behavior. In juveniles but not adults, variations in vitamin D were related to variations in measures of anxiety and marginally, activity levels. For social behaviors, both juveniles and adults were affected by mothers' diets. In general, offspring of animals receiving abnormal concentrations of vitamin D showed the most deficits. © 2012 Wiley Periodicals, Inc. Dev Psychobiol.

Full text

The Effects of Vitamin D
3
During Pregnancy and
Lactation on Offspring
Physiology and Behavior in
Sprague–Dawley Rats
ABSTRACT: Recent findings show that developmental vitamin D deficiency leads
to altered brain morphology and behavioral development in the rat offspring. We
examined the effects of different dietary vitamin D levels in rat dams on behavior
and biochemistry of the offspring. Females were divided into five conditions and
received diets containing 0, 1,5, 3.3, 6.0, or 10.0 IU/g of vitamin D
3
from mating
to weaning. Offspring were tested as juveniles and as adults for anxiety, social
learning and behavior, and locomotion. Results show that both deficient and
excessive levels of vitamin D
3
in juveniles lead to altered physiology and behav-
ior. In juveniles but not adults, variations in vitamin D were related to variations
in measures of anxiety and marginally, activity levels. For social behaviors,
both juveniles and adults were affected by mothers’ diets. In general, offspring
of animals receiving abnormal concentrations of vitamin D showed the most def-
icits. ß2012 Wiley Periodicals, Inc. Dev Psychobiol 56: 12–22, 2014.
Keywords: vitamin D; lactation; development; behavior, biochemistry; rats
INTRODUCTION
There has been growing interest regarding the potential
effects of vitamin D
3
deficiency on mental health; in-
cluding depressive disorders (Humble, 2010), Seasonal
Affective Disorder (Gloth, Alam, & Hollis, 1999),
multiple sclerosis and other immune conditions
(Raghuwanshi, Joshi, & Christakos, 2008; Smolders,
Menheere, Kessels, Damoiseaux, & Hupperts, 2008).
Furthermore, developmental vitamin D (DVD) deficien-
cy has recently been linked to mental disorders such as
schizophrenia (McGrath, 1999; McGrath et al., 2010),
Parkinson’s Disease (Newmark & Newmark, 2007),
and autism (Grant & Soles, 2009).
Diseases with season of birth effects, such as schizo-
phrenia are found to be more prevalent in people born
in the winter and early spring months (McGrath, 1999).
This is a pattern that is also found in serum vitamin D
levels in relation to length of the photoperiod; with
lower levels in the winter months (Holick, 1995). The
developmental rat model of vitamin D deficiency has
recently been employed to examine its potential role in
the neurophysiology and behavior in the rat.
Vitamin D is integral to calcium and phosphate me-
tabolism and absorption. Further, vitamin D receptors
(VDR) are found in the rat brain throughout embryonic
life into adulthood (Prufer, Veenstra, Jirinkowski, &
Kumar, 1999; Veenstra et al., 1998), and have been
implicated in the development of the brain, mediating
various processes; such as neurogenesis, cell prolifera-
tion, neurotransmitter synthesis, and cell differentiation
Developmental Psychobiology
Pauline Pan
1,2
Daniel H.S. Jin
2
Munmun Chatterjee-Chakraborty
3
Katherine Halievski
2
Daeria Lawson
2
David Remedios
2
Cassandra Smetka
2
Vania Pinto
1
Esteban Parra
4
Alison S. Fleming
1
1
Department of Cell and Systems Biology
University of Toronto, Toronto
Ontario, Canada M1C1A4
E-mail: alison.fleming@utoronto.ca
2
Department of Psychology
University of Toronto at Mississauga
3359 Mississauga Rd N., Davis Building
Room 2037B, Mississauga, Ontario,
Canada L5L1C6
E-mail: alison.fleming@utoronto.ca
3
Centre for Addiction and Mental Health,
Toronto, Ontario, Canada M5S2S1
4
Department of Anthropology, University
of Toronto, Mississauga, Ontario, Canada
L5L1C6
Manuscript Received: 19 March 2012
Manuscript Accepted: 26 September 2012
Pauline Pan, Daniel H.S. Jin, and Munmun Chatterjee-
Chakraborty contributed equally to organization, analyses, and/or
writing.
Correspondence to: A. S. Fleming
Contract grant sponsor: NSERC to A.S. Fleming; Contract grant
sponsor: Research Opportunity Program in Psychology at the Univer-
sity of Toronto at Mississauga.
Article first published online in Wiley Online Library
(wileyonlinelibrary.com): 5 November 2012
DOI 10.1002/dev.21086 ß2012 Wiley Periodicals, Inc.

(Baksi & Hughes, 1982; Cui, McGrath, Burne,
Mackay-Sim, & Eyles, 2007; Garcion, Wion-Barbot,
Montero-Menei, Berger, & Wion, 2002; Kesby et al.,
2010; Wion, MacGrogan, Nevue, Houlgatte, & Brachet,
1991). Additionally, pups exposed to DVD deficiency
during the pre-weaning period develop longer brains
with larger lateral ventricles and a thinner cortex, as
well as reduced glial cell line-derived neurotropic fac-
tor and nerve growth factor expression (Eyles, Brown,
Mackay-Sim, McGrath, & Fe
´ron, 2003). Some of these
neural changes persist into adulthood (Fe
´ron et al.,
2005). Vitamin D deficiency in rats and mice also result
in various adverse effects such as reduced learning
and memory function (Becker, Eyles, McGrath, &
Grecksch, 2005), hyperlocomotion (Burne, Becker,
et al., 2004), increased anxiety (Kalueff, Lou, Laaksi,
& Tuohimaa, 2004), impaired spatial learning
(Altemus, Finger, Wolf, & Birge, 1987), and impaired
startle and sensori-motor gating or pre-pulse inhibition
(Brune, Fe
´ron, et al., 2004). Given the relation between
vitamin D
3
and dopamine function it is not surprising
that some of the behaviors affected by a deficiency in
vitamin D
3
are dopaminergically mediated (Kesby
et al., 2010).
In the rat, there is a direct relationship between die-
tary vitamin D
3
intake and serum vitamin D
3
level
(Fleet et al., 2008). Moreover, vitamin D
3
deficiency
throughout pregnancy directly results in depletion of
fetal vitamin D
3
, although serum calcium levels remain
unaltered, compensated for by maternal mechanisms
(O’Loan et al., 2007). Previous studies examining
maternal vitamin D
3
deficiency for different durations
during fetal development (Becker et al., 2005; Burne,
Becker, et al., 2004; Eyles et al., 2006; O’Loan et al.,
2007) show that vitamin D
3
deficiency in the later
stages of gestation alone is sufficient to provoke behav-
ioral changes in the offspring (O’Loan et al., 2007).
Given that pups vitamin D intake is dependent primari-
ly on the mother via storage obtained in utero as well
as the milk until at least 3 weeks of age (Clements &
Fraser, 1988), here we explore vitamin D
3
deficiency
during both gestation and lactation on basic physiology
and behavior of offspring.
In addition to the timing of maternal vitamin D defi-
ciency, recent evidence shows that high levels of DVD
may also have adverse effects in humans (McGrath
et al., 2010); we therefore undertook a dose–response
analysis and investigated the effects of excessive levels
of vitamin D
3
in addition to deficient levels. Thus, the
purpose of this study was to examine the effects of
administering various levels of vitamin D
3
during mat-
ing, pregnancy and lactation on behavior and physiolo-
gy in juvenile and adult offspring. We included
behavioral assessments during the juvenile period
because previous research found mostly subtle effects
of the vitamin D
3
deficiency in adulthood. Hence, we
wished to determine how behavior would be affected
closer to the time that the deficiency was in place as
well as when changes in behavior might first occur. We
predicted that prolonged vitamin D
3
deficiency would
have similar effects on behavior as it has on physiolo-
gy. Specifically, we expected to find that vitamin D
3
deficient diets would result in an increase in anxiety
and activity level, and impair social learning in juvenile
and adult offspring. We had no directional prediction
regarding the effect of the highest dosage of vitamin D.
METHODS
Subjects
Female Sprague–Dawley rats (n¼56) derived from a stock
originally obtained from Charles River Farms (St. Constant,
Quebec, Canada) were born and raised at the University of
Toronto at Mississauga animal facility. Animals were housed
in Plexiglas cages (20 cm 43 cm 22 cm) under 12:12 hr
light:dark cycle with lights on at 08:00 hr. Temperature and
humidity were kept constant between 22–248C and 60–65%,
respectively. Food and water were given ad libitum and
females remained on their respective dietary conditions begin-
ning at the time of mating until pups were weaned at post-
natal day (PND) 18. All procedures conformed to the Canadi-
an Council on Animal Care guidelines, and were approved by
the University of Toronto at Mississauga’s Local Animal Care
Committee.
Experimental Conditions
Females were randomly assigned to one of five dietary condi-
tions, each with a different concentration of vitamin D
3
: 0 IU/
g(n¼10), 1.5 IU/g (n¼10), 3.3 IU/g (n¼10), control
group; 3.3 IU/g is the standard amount of vitamin D
3
added
to compose the regular Rat Chow used for all rats in
our vivarium (see http://labdiet.com/pdf/5012.pdf), 6.0 IU/g
(n¼12) and 10.0 IU/g (n¼14). The diet was composed by
the TestDiet
1
division of LabDiet
1
(Purina Mills, LLC) un-
der the requirements of International Standards Organization.
Females were housed under non-UV emitting light and
remained on their respective diets (Rat Chow; LabDiet
1
)
from the start of mating until PND 18. Animals were housed
under identical UV conditions and given identical diets until
the start of our experimental manipulations.
For a subset of the mothers, food intake was monitored
daily between PND 2-14 by weighing in and out the pow-
dered diet every day. The 24 hr difference score constituted
the measure of daily food intake.
Mating, Parturition, and Offspring
Females were approximately 18 weeks old at the time of mat-
ing. Each male was placed with a female in her home cage
for 18 hr/day for 8 days. Males were returned to their home
Developmental Psychobiology Effects of Maternal Vitamin D on Offspring 13

cages for 6 hr each day to receive regular Rat Chow
(LabDiet
1
).
Animals were monitored for parturition 21 days after the
first day of mating. If pups were born before 16:00 hr, that
day was designated as day of parturition, that is, PND 0. Pups
were counted, sexed, and culled to as close to six male and
six females as possible on PND 1. Most animals gave birth
to litters of >12 pups and there was no significant effect of
vitamin D3 dosage on litter size (p¼.507; data not shown).
Pups remained with their mothers until PND 18, at which
time they were weaned and placed on a regular diet consisting
of 3.3 IU/g vitamin D
3
Rat Chow.
Blood Chemistry and Body Measurements
On PND 9, body weights of males and females from each
litter were measured in groups and their average weight was
calculated. On PND 18, two males and two females from
each litter were sacrificed via live decapitation for brain and
blood collection. Their body weight, brain weight, body
length, and head length were measured. Body lengths were
measured from nose-to-rump, head widths from ear-to-ear
and head lengths from nose to the end of skull.
Twenty-five (OH) vitamin D
3
in serum was assessed by a
competitive enzyme-linked immunoassay (ELISA), VitaKit
D
TM
(SciMed Technologies Inc., Alta, Edmonton, Alberta,
Canada). This kit was based on fluid milk samples and there-
fore an alternative extraction was needed from serum. Ten
microliter of 50% DMSO in phosphate buffered saline was
added to 10 ml of serum, mixed, and left at room temperature
for 15 min. Next, 50 ml of hexane was added and mixed,
followed by 20 min in a 48C centrifuge at 4,500 rpm. The
hexane layer (50 ml) was carefully extracted from the surface
and 10 ml of this hexane extract was used per well in
the ELISA assay. Absorbance was measured at 450 nm on a
spectrophotometer and the amount of vitamin D
3
in each
sample was determined from a standard curve. Measurements
were expressed by the ELISA kit in units of IU/ml.
Serum calcium was measured by a commercial calcium
kit (Pointe Scientific, Inc., Canton, MI). Calcium in the serum
reacts with Arsenazo III to form a purple complex that can be
detected by a spectrophotometer at 650 nm. Concentrations
were calculated from a standard sample and presented in units
of mg/dl.
Serum phosphate was measured using a commercial
inorganic phosphorus kit (Pointe Scientific, Inc.). Inorganic
phosphorus in serum reacts in acid solution with ammonium
molybdate to produce a complex that absorbs light at
340 nm. Samples were measured in a spectrophotometer and
concentrations were calculated from a standard sample and
presented in units of mg/dl.
Serum urea was measured using a commercial urea nitro-
gen kit (Pointe Scientific, Inc.). Serum urea reacts with the
enzyme urease to produce ammonia. This then reacts with
hypochlorite and phenol in an alkaline medium to produce a
complex that can absorb light at 630 nm. A spectrophotome-
ter was used to measure the reacted samples. Concentrations
were calculated from a standard sample and presented in units
of mg/dl.
Behavioral Tests
Subjects were tested as juveniles between PND 35–40, and as
adults between PND 100–105. Testing was done in both age
groups to assess the permanence of any effects of vitamin D
3
.
One male and one female from each litter were assigned to
the anxiety, social behavior and learning, and locomotor
activity test conditions. The assignment of animals’ behavior-
al tests remained consistent between the two phases of test-
ing. However, separate statistical analyses were undertaken
on the effects of dosage and sex at the two timepoints.
Elevated Plus Maze
Anxiety was tested using an elevated plus maze (EPM) modi-
fied from a previous design (Pellow, Chopin, File, & Briley,
1985). It consisted of two open arms (10 cm 50 cm 
45 cm) and two closed arms (10 cm 50 cm 45 cm)
that extended from a central platform (10 cm 10 cm).
The maze was constructed from wood, painted over in black
and was elevated 50 cm from the base. Subjects were trans-
ported from their colony room and tested in a separate
room between 10:00 and 13:00 hr. Each rat was placed in
the centre facing the experimenter at the beginning of the
test, and then allowed to explore the maze for 10 min. The
apparatus was cleaned with 70% ethanol and warm water
after each test. The amount of time spent in each arm and
the centre, along with grooming and rearing behaviors were
recorded using Behavioral Evaluation Strategy and Taxo-
nomy (BEST) software (Educational Consulting, Inc., Hobe
Sound, FL).
Social Behavior and Social Learning
Subjects were tested in their home cage in their home colony
room. Cage mates were removed from home cages and each
subject was allowed to interact with a same sex conspecific of
similar age and weight for two consecutive days: 30 min on
the first day and 10 min on the second day. On the second
day, subjects were randomly selected to receive either the
same conspecific as Day 1 (familiar) or a new conspecific
(unfamiliar) fitting the same criteria. The interactions were
video recorded and then coded using BEST. The following
measures were analyzed for both frequency and duration:
anogenital sniffing, body sniffing, head sniffing, play fighting,
and grooming (Macbeth, Edds, & Young, 2009).
Locomotor Activity
Subjects were tested for 1 hr each for 3 consecutive days in a
locomotor activity box (47 cm 26 cm 20 cm) between
10:00 and 13:00 hr. Each test box was connected to a com-
puter, which recorded the animal’s activity using infrared
lights nodes. On the bottom and top of each test box, there
are rows of corresponding infrared light nodes, each pair of
corresponding nodes connected to make a beam. Each time a
beam was interrupted, the program recorded a ‘‘Cut,’’ and
each time the animal traversed the entire length of one row of
nodes, the program recorded a ‘‘Length.’’ Data were collected
14 Pan et al. Developmental Psychobiology

into 5-min bins. Each test box was cleaned with 70% ethanol
between trials.
Statistical Analysis
For most outcome measures two-way ANOVAs were
performed, comparing the five vitamin D
3
groups as well as
between males and females, followed by Tukey’s post hoc
tests comparing pairs of groups. For behavioral measures,
figures are included for juvenile performance where group
differences were significant or marginally significant, and, for
comparison purposes, on adults on the same measures where
group differences were no longer found.
RESULTS
Biochemistry
There was a significant effect of maternal vitamin D
3
diet on serum vitamin D
3
concentrations in the off-
spring at PND 18 (see Fig. 1A). Two-way ANOVA
revealed a significant effect of vitamin D3 (F
(4,45)
¼
171.67, p<.001) but no effect of sex. As expected,
decreased vitamin D
3
dose in the maternal diet resulted
in partial depletion of serum vitamin D
3
in the off-
spring. Tukey’s post hoc analysis showed that differen-
ces among all groups were significant except between
0 and 1.5 IU/g groups (p<.05). Two-way, ANOVA
also showed a significant effect of dietary maternal
vitamin D
3
level on serum calcium levels in the off-
spring (F
(4,55)
¼18.812, p<.001; see Fig. 1B); again,
there was no main effect of sex or interaction. Post hoc
analysis revealed that the 10.0 IU/g group had signifi-
cantly higher serum calcium levels than the other
groups, while the 6.0 IU/g group had significantly
higher serum calcium levels compared to those in the
1.5 IU/g group (p<.05).
No effects were found in serum phosphate levels for
vitamin D
3
dosage or sex (F
(4,55)
¼2.429, p>.05;
F
(1,55)
¼.115, p>.05). Two-way ANOVA showed
both a main effect of vitamin D dosage (F
(4,55)
¼
4.025, p<.05) and of sex (F
(1,55)
¼32.059, p<.001)
on serum urea levels. Further investigation reveals that
vitamin D
3
dosage has a significant effect in males
(F
(4,27)
¼4.04, p<.05; data not shown) but not in
females.
Body Length, Body Weight, and Brain Weight
Two-way ANOVA showed no significant effect of
vitamin D
3
dosage or sex on pups’ body weight on
PND 9. However, by PND 18, there were significant
effects of maternal vitamin D
3
levels on pups’ body
lengths (F
(4,109)
¼4.617, p<.05; Fig. 2A), body
weight (F
(4,109)
¼6.19, p<.001; Fig. 2B), and brain
weight (F
(4,108)
¼5.87, p<.001; Fig. 2C). When
pups’ brain weights were corrected for pups’ body
weights, there is still a significant effect of vitamin D3
dosage (F
(9,107)
¼4.906, p¼.001; data not shown).
There was no effect of sex on pup’s body weight, body
length, or brain weight, and no interactions on PND
18. Tukey’s post hoc analysis revealed that rats in
10.0 IU/g groups weighed significantly less than those
in 3.3 and 6.0 IU/g groups (p<.05), while those in
10.0 IU/g group were shorter in body length compared
to 0, 3.3, and 6.0 IU/g groups (p<.05). Post hoc anal-
ysis also showed that the brains of animals in 10.0 IU/g
group weighed less than animals the other groups
(p<.05).
FIGURE 1 (A) Serum vitamin D concentration in the off-
spring at the time of weaning on PND 18 (mean SE).
Decreased vitamin D dose in the maternal diet resulted in par-
tial depletion of serum vitamin D in the offspring,
F
(4,44)
¼178. 36, p¼.000. Post hoc analysis shows that
animals in the 0 and 1.5 IU/g group had significantly lower
serum levels of vitamin D3 than those in the 3.3, 6.0, and
10.0 IU/g groups (

p<.05,

p<.01). (B) Serum calcium
levels in the offspring on PND 18 (mean SE). Higher levels
of dietary maternal vitamin D resulted in increased serum
calcium levels, F
(4,54)
¼19.864, p¼.000. Tukey’s post hoc
analysis showed that animals in the 6.0 IU/g and 10.0 IU/g
groups had significantly higher serum calcium levels com-
pared to those in the 0 IU/g and 1.5 IU/g groups. As well,
animals in the control (3.3 IU/g) group had significantly lower
serum levels of calcium than animals in the 10.0 IU/g group
(

p<.05,

p<.01).
Developmental Psychobiology Effects of Maternal Vitamin D on Offspring 15

Mothers’ Food Intake
One-way ANOVA showed no significant effect of
vitamin D
3
levels in mothers’ diets on mothers’ food
intake over the first 10 days after parturition. Whether
it does over the next week prior to weaning, we do not
know.
Behavioral Tests
Elevated Plus Maze. Two-way ANOVA revealed a sig-
nificant effect of vitamin D
3
levels in mothers’ diets on
grooming frequency in the EPM test in offspring
between PND 35-40 (F
(4,54)
¼2.916, p<.05; see
Fig. 3A), but no main effect of sex and no interaction.
Tukey’s post hoc analysis showed a significant differ-
ence between the 0 and 6.0 IU/g groups (p<.05);
animals in 0 IU/g group showed significantly more
grooming behaviors than those in the 6.0 IU/g group.
While there were no significant group effects at
p<.05 (2T) of vitamin D
3
on time or proportional
time spent in open arms, or number of arm changes,
for both the ratio of open arm entries (F
(4,54)
¼2.135
p¼.09 (.045, 1T, Fig. 3B) and total number of arm
changes in the maze (F
(4,54)
¼2.319, p¼.07 (.035,
1T, not shown), there were marginal overall group dif-
ferences. For the ratio score the 3.3 IU/g group showed
higher levels than both the lowest concentration group
(0 IU/g) and the highest concentration group (10.0 IU/
g). There was no effect of sex on any of the above
measures in the EPM. For comparison purposes, as
can be seen in Figure 3C,D, there were no significant
or marginal effects of vitamin D on any measure of
anxiety in adult offspring between PND 100-105.
Social Behavior and Learning. In the juveniles, there
was a group effect on social sniffing only in males on
the first day of testing (F
(4,28)
¼3.116, p<.05,
Fig. 4A), but no effect on social learning (reflected
in the differential responses to the familiar vs. the un-
familiar conspecific on Day 2 and the proportional
change in responses from Day 1 to 2). The 10.0 IU/g
male animals showed the highest levels of sniffing in
comparison to all other groups. In addition, there was a
significant group effect on frequency and duration of
self-grooming, with the 3.3 IU/g animals showing the
lowest levels and the extreme groups showing higher
levels (F
(4,49)
¼3.15, 3.45; p<.015–.022, Fig. 4B).
There were no other significant effects in juveniles.
However, among adults a somewhat different pattern
was shown. There was no group effect on sniffing
or grooming (Fig. 4C), but there was a significant
group effect on ‘‘playfighting’’ duration (F
(4,55)
¼2.65,
p<.05; see Fig. 4D). Post hoc analysis showed that
FIGURE 2 (A) Pup body length (nose to rump) measured
on PND 18 (mean SE). Pups that received the highest level
of dietary maternal vitamin D had reduced body length,
F
(4,108)
¼4.571, p¼.002. Tukey’s post hoc analysis showed
that offspring in the 10.0 IU/g group had significantly shorter
body lengths compared to those in the 0, 3.3, and 6.0 IU/g
groups (

p<.05). (B) Maternal dietary vitamin D levels
influenced pup body weight at the time of weaning on PND
18 (mean SE). Pups showed increased body weight with
higher levels of maternal vitamin D but the effect was
reversed in 10.0 IU/g group. F
(4,108)
¼6.361, p¼.000.
Tukey’s post hoc analysis showed that offspring in 3.3 and
6.0 IU/g groups weighed significantly more compared to
those in the 10.0 IU/g group (

p<.05). (C) Brain weight of
the pups on PND 18 was affected by the mother’s dietary
vitamin D level (mean SE), F
(4,107)
¼5.714, p¼.000.
Mothers that received the highest level of Vitamin D3 in diet
produced pups with the lowest brain weight. Post hoc analysis
indicated that the offspring in the 10.0 IU/g group had signifi-
cantly lower brain weight compared to all other groups
(

p<.05).
16 Pan et al. Developmental Psychobiology

rats in the sufficient groups (3.3, 6.0 IU/g) spent more
time playfighting than those in the 1.5 IU/g group.
Locomotor Activity. There was a significant effect
of vitamin D
3
levels in mothers’ diets on locomotor
activity levels in the offspring between PND 35–40
(F
(4,56)
¼3.217, p¼.021; see Fig. 5A); but no effect
of sex. Tukey’s post hoc reveals a marginally signifi-
cant difference between the 0 and 3.3 IU/g groups,
p¼.091, but no significant differences between any
other pairs of groups. There was a significant effect of
testing days on the mean distance travelled (p<.001)
in both males and females with a gradual decrease of
activity level over the three testing days. There was
also a significant days effect on the number of drop-
pings animals deposited in the test box (p<.05). Last-
ly, there were also no significant effects of maternal
vitamin D levels on locomotion in the adult offspring
(Fig. 5B).
DISCUSSION
The current study found that the administration of
different concentrations of vitamin D
3
at mating and
during pregnancy and lactation are transmitted to the
offspring through the mothers’ milk during nursing,
producing levels of vitamin D
3
and of calcium in the
offspring circulation that are related to the dose present
in the mothers’ diet. Furthermore, this shows that varia-
tions in vitamin D
3
are related to juvenile behavior,
such that very low levels administered to mothers pro-
duce in offspring (a) enhanced anxiety, as indicated by
increased grooming frequency and lowered probability
to enter the open arms; (b) enhanced anxiety in social
contexts as shown by increased self-grooming during
the social interaction task; and (c) reductions in
FIGURE 3 (A) Grooming frequency of pups in the EPM in
juveniles as a function of vitamin D dosage administered to
mothers. Deficient maternal vitamin D diet resulted in
increased anxiety level as measured by grooming frequency
in the EPM in the juvenile offspring, F
(4,54)
¼3.256,
p¼.019. The effect was reversed when the mother received
the highest level of dietary vitamin D (10.0 IU/g). Post hoc
analysis indicated that juvenile rats in 6.0 IU/g groomed
significantly less compared to those in 0 IU/g (

p<.05). (B)
Ratio of entries into open arms/total entries in the EPM in
juveniles as a function of vitamin D dosage administered to
mothers. Deficient vitamin D diet resulted in a marginal in-
crease in anxiety as measured by and ratio of open entries to
total in juvenile offspring (F
(4,54)
¼2.135, p¼.090). Rats
with 3.3 IU/g maternal vitamin D diet were the most likely to
enter the open arms (F
(4,54)
¼2.135 p¼.045, 1T)

p<.05.
(C) Grooming frequency of pups in the EPM in adulthood
in adults as a function of vitamin D dosage administered to
mothers. There were no significant group effects, F
(4,53)
¼
.023, p¼.999. (D) Ratio of entries into open arms/total
entries in the EPM in adults as a function of vitamin D dos-
age administered to mothers. There were no significant group
effects, F
(4,53)
¼1.17, p¼.336.
Developmental Psychobiology Effects of Maternal Vitamin D on Offspring 17

locomotion in the lowest vitamin D groups (0 and
1.5 IU/g) which showed marginal differences from the
control group (3.3 IU/g). In general, the animals show-
ing the most clearly adapted responses on these meas-
ures were those in the middle dosage 3.3 IU/g group
which represents the dosage that is present in usual rat
chow. Further, with the exception of social behaviors,
effects seen in most of the behaviors tested do not per-
sist into adulthood. For social behaviors, however,
DVD deficiency results in alterations in some social
behaviors that occurred during the juvenile period and
alterations in others (i.e., playfighting) in adulthood.
Finally, the current study shows that vitamin D
3
excess
also exerts negative effects both on growth and on
some behaviors, suggesting that high concentrations
may in fact be toxic.
While there was a strong dosage effect of DVD
levels on all body measurements, only the group that
received the highest level of maternal vitamin D
showed shorter body lengths, lower body, and brain
weights compared to those on the regular diet (3.3 IU/g
vitamin D
3
added). Groups receiving deficient doses of
vitamin D
3
did not show decreased measurements in
body weight, unlike what previous studies have shown
(Brune, Fe
´ron, et al., 2004; O’Loan et al., 2007). While
the biological basis for such effects is not immediately
clear, we suspect that the anti-proliferative effects of
vitamin D
3
contributed to the decreased body and brain
weights in the heavily supplemented group, as VDRs
are found throughout the brain as well as in various
types of cells throughout the body including muscle,
adipose tissue, and bone marrow (Norman, 2006; Prufer
et al., 1999; Veenstra et al., 1998). We know based on
the maternal food intake results that the effects on the
offspring did not result from differential eating by the
different groups of mothers during the first 10 days
postpartum. However, mothers’ eating could have af-
fected pup growths after the first 10 days although we
think this is unlikely. Since by the first 10 postpartum
days, animals would have been on the diets for a
month, we assume that any changes in mothers food
intake that were vitamin D dependent would be
revealed in the 10 days during which it was measured.
Based on this rationale, we assume that any differences
FIGURE 4 (A) Grooming frequency of pups during the
social interactions in juveniles as a function of dosage of
vitamin D administered to the mothers. F
(4,53)
¼3.452,
p¼1.015. (B) Social behavior as measured by the total dura-
tion of ‘‘playfighting’’ indicates no significant difference be-
tween vitamin D groups. F
(4,53)
¼.066, p¼.992. (C) Social
behavior in adults as measured by grooming frequency on
Day 1 of test days indicates no difference between Vitamin D
groups. F
(4,55)
¼1.223, p¼.313. (D) Deficient vitamin D
levels in the mother’s diet significantly reduced ‘‘playfight-
ing’’ duration in the adult offspring. Similar effects were seen
in groups that received the highest level of dietary maternal
vitamin D. Post hoc analysis showed that the 1.5 IU/g group
and the 10.0 IU/g group were significantly different from the
3.3 IU/g group (F
(4,55)
¼4.546, p¼.003).

p<.05.
18 Pan et al. Developmental Psychobiology

in serum vitamin D
3
levels in the offspring are a func-
tion of the concentration of vitamin D
3
in their moth-
ers’ diets and not a function of alterations in food
consumption by their mothers. We can also infer from
this that any adverse effects present in juvenile rats are
a function of the level of vitamin D
3
in their systems,
and not due to malnutrition induced by changed eating
habits in the lactating dams.
The observation that dams that received a diet
completely depleted of vitamin D
3
still produced pups
with detectable levels of serum vitamin D
3
may not be
surprising given that newborn rats depend primarily on
the stored vitamin D
3
obtained in utero and other stud-
ies suggest that the dietary administration beginning
6 weeks prior to conception would be necessary to ful-
ly eliminate maternal vitamin D
3
(O’Loan et al., 2007).
It is also worth noting that the litters in our study litters
were culled to 12 pups from litter sizes of up to 16
pups, a procedure that may have amplified the amount
of vitamin D
3
each pup received over what they would
have received had the litter sizes been larger. Finally,
the dams that received higher-than-normal dietary
vitamin D
3
produced pups with serum vitamin D
3
lev-
els that were less than expected by a linear relationship,
which may suggest that there is a ceiling effect in
which excessive levels of maternal vitamin D
3
are ei-
ther not being transferred to the pups or are being me-
tabolized by the pups. It is plausible that the inhibition
results from a preventative mechanism given the physi-
ological implications of excessive vitamin D
3
intake
such as progressive weight loss, difficulty in movement
and respiration, epistaxis, and subnormal body tempera-
ture in rats (Chavhan et al., 2011; Hathcock, Shao,
Vieth, & Heaney, 2007 for review).
Increased grooming activity both in the EPM and in
the social tests likely indicates increased levels of anxi-
ety in maternal vitamin D
3
deficient juvenile offspring.
Although grooming was not the central aspect of either
test, it nevertheless has been interpreted as a sign of
anxiety (van Erp, Kruk, Meelis, & Willekesens-Bramer,
1994). Similar to our findings, VDR null mutant mice
show an increase in grooming behavior in multiple anx-
iety tests (Kalueff et al., 2004). There was a marked
increase in grooming frequency at the 10.0 IU/g group
that was very similar to the trends seen in physiological
measures. While the reason for such an effect is
unclear, this trend suggests possible adverse effects of
excess DVD. However, this relationship was no longer
seen in adulthood, suggesting that effects of transient
DVD anomaly can be normalized by post-weaning rear-
ing on a diet containing normal levels of vitamin D
3
(3.3 IU/g). Unfortunately, in the present design, we did
not monitor estrous cyclicity of adult animals, where
vitamin D effects were reduced or absent, and it is pos-
sible that our tests did not occur randomly with respect
to the estrous cycle. This could have had an impact on
our results given that there is evidence that emotional
responsiveness and activity do change as a function of
stage of estrous cycle (Mora, Dussaubat, & Dı
´az-Ve
´liz,
1996). This issue would not be relevant in the case of
testing of pre-pubertal animals who are not yet cycling.
There was an effect of vitamin D
3
dosage on juve-
nile activity level, that is, a reduction in locomotor ac-
tivity level in the deficient groups as compared to those
in the group that received regular diet. Our result is in
contrast to previous findings in which the deficient
offspring showed hyperlocomotion in the adulthood
(Burne, Becker, et al., 2004; Eyles et al., 2006). The
present results are, however, consistent with results
based on VDR knockout mice in which the mutant
mice showed decreased motor activity levels (Burne,
FIGURE 5 (A) Locomotor activity levels in juveniles as
measured by total bottom beam breaks on the first day of test-
ing in a locomotor activity box (mean SE). Mother’s die-
tary vitamin D levels has a significant effect F
(4,56)
¼3.217,
p¼.021. Post hoc analysis shows no significant difference
between any particular pairs of groups. However, the juvenile
offspring that received the control maternal vitamin D diet
(3.3 IU/g) showed the highest level of activity. (B) Locomotor
activity levels in adults as measured by average beam breaks
across 3 days indicate no difference between vitamin D
groups. F
(4,54)
¼.845, p¼.504).
Developmental Psychobiology Effects of Maternal Vitamin D on Offspring 19

McGrath, Eyles, & Mackay-Sim, 2005; Kalueff et al.,
2004).
The close association of vitamin D and calcium
absorption may be informative in the interpretation of
our data, as there was a significant group effect on se-
rum calcium levels in the pups. Hence, the reduction in
serum calcium levels may have disrupted musculoskel-
etal development and led to reduced activity levels.
Notwithstanding, the largest reduction in calcium level
was only at 11% and it is still possible that this effect
can be attributed to dysregulation of the dopamine sys-
tem as suggested for increased anxiety. Interestingly,
there was a decline in activity level in groups that re-
ceived higher level of DVD than the control. We specu-
late that overexposure to vitamin D
3
may have resulted
in hypercalcemia, leading to a disruption in motor
activity. At present, it is difficult to determine whether
the effect was a result of brain anomaly resulting from
DVD toxicity. In keeping with other behaviors, all
locomotor anomalies disappeared in adulthood, sug-
gesting that postnatal vitamin D
3
supplementation
rescued any adverse behavioral effects.
Only social behavior showed a persistent, albeit sub-
tle, effect in adulthood. The duration of social ‘‘play-
fighting’’ was reduced in the deficient groups and in
the heavily supplemented group. It is important to note
that behaviors were changed in peri-pubertal rats and it
is possible that DVD deficiency may selectively affect
peri-pubertal behaviors only. While we cannot specu-
late on the neural correlates, alteration in social behav-
ior in both the pre- and peri-pubertal periods are
considered negative symptoms in the animal models of
schizophrenia (Becker et al., 2003).
There is evidence that many of the outcomes
reported here, including anxiety and locomotor activity,
may result from alterations in dopamine function
(Beninger, 1983; Koch, 1999; Serafim & Felicio,
2001). In fact, vitamin D has been implicated in the
synthesis of dopamine (Baksi & Hughes, 1982) and the
regulation of dopamine receptors and transporters
(Kesby et al., 2010; Peeyush et al., 2010) throughout
the limbic system and striatum (Walbert, Jirikowski, &
Prufer, 2001), structures known to be important for
emotion regulation, attention, and activity. Hence,
dopamine dysfunction may well be the primary mecha-
nism through which DVD affects behavior. Future
research should be conducted to analyze whether the
DVD deficiency of the mother affects dopamine recep-
tors or function in the offspring’s brain.
We found that high levels of vitamin D
3
may have
adverse effects. To our knowledge, we are the first to
examine the effects of high levels of vitamin D
3
on
behavior in rats. It is difficult to determine whether
the highest level given in the current study can be
considered a toxic level, although levels given in this
study are much lower than levels used in Chavhan
et al. (2011). However, our experimental conditions
ranged up to three times the amount given in the regu-
lar laboratory rat diet. While the exact mechanism by
which the excess amount of vitamin D
3
can lead to an
adverse behavioral outcome has yet to be studied, a
recent study has found that there is a U-shaped
relationship of prenatal vitamin D
3
level and risk of
schizophrenia in humans (McGrath et al., 2010).
Although the current study did not yield any direct
evidence for a relationship between DVD deficiency
and endophenotypes of schizophrenia, our behavioral
results point to the possible dysregulation of the dopa-
mine system. Future research investigating potential
toxicity effects seems warranted.
The design of our study was unique primarily in that
we investigated the effects of both deficient and exces-
sive levels of vitamin D
3
in comparison to a normal
control. This paradigm could have implications for
other mammalian species (including humans), as (a)
many mammalian offspring are naturally reared away
from sunlight and dependent mainly on the milk for
vitamin D supply in early postnatal development; and
(b) DVD is still a large health problem for pregnant
women and consequently their infants in many parts of
the world (Holick & Chen, 2008; Raiten & Picciano,
2004). In this rat model, we have demonstrated that
prolonged DVD deficiency from pregnancy and
throughout lactation has real, but usually transient,
effects on offspring behavior.
NOTES
This work was supported by an NSERC grant to A.S. Flem-
ing, and as part of the Research Opportunity Program in Psy-
chology at the University of Toronto at Mississauga. The
authors would like to thank all the members of the Fleming/
Kraemer lab for their assistance, and the University of
Toronto Mississauga animal facility staff for excellent animal
care.
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