Fedotova BMC Medical Genetics 2019, 20(Suppl 1):49
https://doi.org/10.1186/s12881-019-0774-2

RESEARCH

Open Access

Vitamin D3 treatment differentiallyaffects anxiety-like behavior in the oldovariectomized female rats and oldovariectomized female rats treated withlow dose of 17β-estradiol
Julia O. Fedotova
From 11th International Multiconference “Bioinformatics of Genome Regulation and Structure\Systems Biology” - BGRS\SB-2018
Novosibirsk, Russia. 20-25 August 2018

Abstract
Background: Estrogen deficiency effects on affective-related behavior are restricted to certain periods of age after
ovary removal. Among other nutraceuticals, one of such «natural» substances for treatment of affective-relateddiseases could be vitamin D3. It is a great interest to evaluate the effects of repeated cholecalciferol administration
on anxiety-related behavior in the old female rats with long-term estrogen deficiency. The present study was
performed to determine the behavioral effects of cholecalciferol treatment at different doses as an adjunctive
therapy alone or in a combination with low dose of 17β-estradiol on anxiety-like behavior of the old (16–18
months) female rats at 12 weeks after ovariectomy.
Methods: Vitamin D3 supplementation individually (as cholecalciferol at doses of 1.0, 2.5 or 5.0 mg/kg/day,s.c.) or in co-administration with of 17β-estradiol (17β-E2, 0.5 μg/rat, s.c.) were given to the old ovariectomized(OVX) rats at 12 weeks after ovariectomy. Anxiety-related state was tested in the elevated plus maze (EPM)
and light-dark test (LDT), as well behavioral reactivity was registered in the open field test (OFT). Moreover,
25-hydroxyvitamin D3 levels in the blood serum of these OVX rats treated with Vitamin D3 or Vitamin D3 plus
17β-E2 were measured.
Results: The results of the present study indicated that Vitamin D3 supplementation at dose of 1.0 mg/kg/day
decreased manifestations of anxiety-like profile in the old OVX rats. Treatment with Vitamin D3 (1.0 mg/kg/day)plus 17β-E2 in resulted in more profound anxiolytic-like effects the old OVX rats than effects of both drugs
administered alone. Moreover, treatment with cholecalciferol (1.0 mg/kg/day, s.c.) in the old ovariectomized
rats after ovariectomy at 12 weeks produced elevated estradiol and 25-OH-VD3 levels for these rats as compared to the
old OVX females treated with oil solvent.
Conclusions: Using the preclinical study, chronic cholecalciferol, 17β-E2 and their combination treatment were shown
to be effective for anxiety-like treatment in the old subjects with long-term estrogen deficiency.
Keywords: Vitamin D3, Anxiety, Menopause, Estradiol, Age, Old female rats

Correspondence: julia.fedotova@mail.ru
ITMO University, Saint-Petersburg, Russia© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Fedotova BMC Medical Genetics 2019, 20(Suppl 1):49

BackgroundAnxiety disorders are twice common in women than in
men and the risk is increased during the menopausal
transition more [1, 2]. Menopause is a phase of awoman’s life which indicates termination of her reproductive function. It is induced by the loss of ovarian follicular activity, which leads to the permanent cessationof menstruation [2, 3]. Menopause is often associatedwith an onset of mood disturbances, especially anxiety
and depression [3, 4]. For women, data suggest that estrogens are strongly implicated in the regulation ofmood and behavior, as well as in the pathophysiology ofmood disorders [5–7]. Nowadays, there is a strong tendency to rising of duration of living in the whole world,
however, the median age of programmed termination ofa woman’s reproductive life failed to altered [5–7]. Thus,
majority of women in the ageing population stays for asignificant period of their lifetime in a postmenopausal
state which is clearly associated with very low estrogenlevels that could one of the marked trigger factors fordevelopment of affective-related disorders [5, 7].
A strategy to alleviate the mood disorders associatedwith menopause is hormonal replacement therapy(HRT) [8]. However, controversial results related to theeffectiveness of such treatment have been frequently reported [9]. These discrepancies could be associated tovarious factors, one of them being the time when estrogen restitution is initiated after the beginning of menopause [10, 11].
There is growing interest about the potential of dietand nutrients to improve the mental health of thewomen population and for the treatment of psychiatricdisorders [10, 11]. In the case of mood disorders, thelimitations of psychotropic drugs to achieve adequate
rates of clinical remission and functional recovery havepromoted the search for complementary approaches[12–15]. Menopausal women are now choosing to takealternative and complementary therapies marketed as«natural» treatments that offer the positive health effects
of estrogens without the unwanted side effects [12, 13].
Effectiveness of α-tocopherol, ascorbic acid, β-carotene
were previously shown for wide group of disorders andforms of administration [16, 17]. Among other nutraceuticals, one of such «natural» substances for treatment of affective-related diseases could be vitamin D(VD) [14, 15].
VD is a neuroactive secosteroid with well-known
physiological role in skeleton formation and maintenance, as well as diverse «non-skeletal» functions [18, 19].
«Non-skeletal» functions of VD are connected with its
different range of outcomes in the central nervous system,
such as neuroplasticity, apoptosis, cell proliferation anddifferentiation [20, 21]. All functions of VD in the body(classical functions, i.e., effect upon calcium-phosphate

Page 46 of 56

management and the non-classical ones) are imposed by
the VD nuclear receptor (VDR), regulating directly thegene expression [22, 23]. Nuclear VDR are member of receptors family for transcription factors which are activatedby numerous ligands [22, 23]. VDR are present in mosttissues and cells in the body, and within the brain show
some specificity to the prefrontal cortex, hippocampus,
cingulate gyrus, thalamus, hypothalamus and substantia
nigra [24]. This is of relevance as many of those brain
regions have been implicated in the physiology ofaffective-related disorders.
Estrogen deficiency effects on affective-related behavior are restricted to certain periods of age after ovary removal [25–27]. Preclinical data suggest that onset age ofmenopause can be important to obtain behavioral positive or negative results. Thus, it is a great interest toevaluate the effects of repeated cholecalciferol administration on anxiety-related behavior in the middle-aged
and old female rats with long-term estrogen deficiency.
The aim of the present study was to determine if repeated systemic treatment with cholecalciferol affected
on anxiety-like behavior in the old female rats after
long-term ovariectomy.

Methods
Animals

Female albino Wistar rats (16–18 months, old rats,
weighing 260–270 g, respectively) from the special biocollection of Koltushi vivarium (St. Petersburg, Russia)
were used in the present study. All rats were allocated ingroups and were allowed to accommodate for 1 week inthe animal house at I.P. Pavlov Institute of Physiology, ofthe Russian Academy of Sciences, before subjecting
them to behavioral testing and pharmacological treatments. They were provided with a standard pellet dietand were given water ad libitum. The animals were kept
at a temperature of 23 °C ± 2 °C and a 12 h light/darkcycle as well as a constant relative humidity (50% ± 10%)during all experimental sessions. Female rats of different
age rats were randomly separated into experimentalgroups 4, including the control groups.
Vitamin D3 and 17β-E2 treatments, as well as anxietyrelated tests were carried out a double-blind method byusing rules of the Health guide for the care and use ofLaboratory animals (1978) formulated by the National
Institute of Health. Females of different age were placed
to the special room for behavioral trials at least 1 h prior
to the beginning of the experimental sessions whichwere performed from 09:00 am to 12:00 am. The experimental protocols of this study were approved by theInstitutional Animal Ethics Committee of I.P. Pavlov
Institute of Physiology, Russia (protocol 1095/1 from
June 25, 2012).

Fedotova BMC Medical Genetics 2019, 20(Suppl 1):49

SurgeryLong-term ovariectomy surgery was performed as previously described [28]. Briefly, middle-aged and old female
rats were anesthetized with ketamine (70 mg/kg b.w.)
mixed with xylazine (10 mg/kg b.w.). To avoid inflammation, the rats were administered with meloxicam (1 mg/
kg b.w.). The fallopian tube was crushed and the ovary
was removed by cutting. The effectiveness of long-term
ovariectomy or 17β-estradiol (17β-E2) application wasassessed by vaginal smears. The old ovariectomized(OVX) females were housed in groups of five in cages
separated by groups. To assure the long-term absence of
estrogens, all rats after surgery were remained to the
housing facilities for 12 weeks.
Drug treatments

17β-Estradiol, 17β-E2 (Sigma, USA) at low dose of
5.0 μg/rat [27, 28] and Vitamin D3 as cholecalcirefol(Sigma, USA) at several doses (1.0, 2.5 or 5.0 mg/kg)[29] were subcutaneously (s.c.) administered once daily
starting 14 days prior to the cognitive experiments.
17β-E2 was dissolved in sterile sesame oil, VD3 – in 95%
ethanol solvent, aliquoted and stored at -80 °C. The
stock of VD3 was dissolved in a sterile water, resulting ina solution of cholecalciferol with 2% ethanol. All drug
solutions were freshly prepared before each behavioral
testing. 17β-E2 and cholecalcirefol were injected in a volume of 0.1 ml. The estrogen, 17β-E2 (E-8875, Sigma
Chemical Co., St. Louis, MO, USA) was dissolved insterile sesame oil. Cholecalcirefol (C-9756, Sigma Chemical Co., St. Louis, MO, USA) was dissolved in 95% ethanol, aliquoted and stored at -80 °C. The stock ofcholecalciferol was diluted in a sterile water, resulting ina solution of cholecalciferol with 2% ethanol. 17β-E2 wasinjected subcutaneously (s.c. at a dose of 0.5 μg/rat). The
low dose of 17β-E2 (5.0 μg/rat subcutaneously, s.c.) waschosen from the studies performed by Estrada-Camarenaand co-workers [29, 30]. Three doses of cholecalciferol(1.0, 2.5 or 5.0 mg/kg, s.c.) were chosen from the behavioral study performed by Idrus and co-workers [31]. All
solutions were freshly prepared before each experimental
series. All preparations were administered in a volume of
0.1 ml. Following 12 weeks after ovariectomy, cholecalciferol, 17β-E2 and oil solvent were injected once daily for
14 days.

Page 47 of 562) old intact female rats + cholecalciferol 1.0 mg/kg(old intact-Vit D3 1.0),
3) old intact female rats + cholecalciferol 2.5 mg/kg(old intact-Vit D32.5),4) old intact female rats + cholecalciferol 5.0 mg/kg(old intact-Vit D3 5.0),
5) old OVX + solvent (old OVX-Sol),6) old OVX rats +17β-E2 (old OVX-17β-E2),7) old OVX rats + cholecalciferol 1.0 mg/kg (oldOVX-Vit D3 1.0),8) old OVX rats + cholecalciferol 2.5 mg/kg (oldOVX-Vit D3 2.5),9) old OVX rats + Vit D3 5.0 mg/kg (old OVX-Vit D3 5.0),
10) old OVX rats + cholecalciferol 1.0 mg/kg + 17β-E2(old OVX-Vit D3 1.0-17β-E2),11) old OVX rats + cholecalciferol 2.5 mg/kg + 17β-E2(old OVX-Vit D3 2.5-17β-E2),12) old OVX rats + cholecalciferol 5.0 mg/kg + 17β-E2(old OVX-Vit D3 5.0-17β-E2).
The treatment period for animals was 14 days, and at
the end of the treatment period (1 h after the last doseof solvent, Vitamin D3 or 17β-E2), all animals were subjected to the EPM, LDT and the open field test. During
testing sessions in all behavioral tests the control and experimental groups of rats were also given with solvent,Vitamin D3 or 17β-E2.
Behavioral tests
Elevated plus maze test

EPM is a commonly accepted as standard test ofanxiety-like behavior and was used to assess ananxiety-like behavioral responses [32, 33]. This test issensitive to putative anxiogenic-like and anxiolytic-like
drugs [34]. The EPM is consisted of two open arms
(50 × 10 cm2) and two closed arms (40 × 10 cm2) with acentral platform (10 × 10 cm2), and elevated 50 cm above
the floor level. All female rats from control and experimental groups were randomly placed at the center of the
EPM and allowed them to freely move in the apparatus
for 5 min. The number of entries and total time spent inopen arms were accepted as parameters of anxiolyticlike effects of treatments. The apparatus was cleaned
with damp cloth after each trial to avoid place preference and the influence of olfactory stimuli.

Animal groups

Light/dark test

Female rats (old intact and OVX) were randomly divided
into 24 groups, accordingly to their age, with 8 rats ineach group.
The following experimental groups for the old female
rats were created in the present study:

The apparatus was consisted of two identical boxes(30 × 40 × 40 cm), one of which with white walls andfloor and illuminated by a 60 W light from above, while
the other of the box was painted black and had a lid so
it was not illuminated [35, 36]. The number of entrances
and the total time in the light box were registered for 5
min [7]. The increase of the number of entrances and1) old intact female rats + solvent,Fedotova BMC Medical Genetics 2019, 20(Suppl 1):49

the total time in the light box were postulated as manifestation of anxiolytic-like effects of treatments. The apparatus was cleaned with damp cloth after each trial to
avoid place preference and the influence of olfactorystimuli.
Open field test

The effect of cholecalciferol on locomotor, rearing andgrooming activities was evaluated automatically using an
open-field computer-aided controlling system as described previously [37]. The apparatus consists a square
platform (80.0 cm × 80.0 cm; wall height 36.0 cm). The
floor of the platform was divided into 16 equal squaresof 19.5 cm × 19.5 cm. A video camera fixed at the top,
and the apparatus was illuminated by a light source of
120 Lux on the ceiling. Each rat was placed at the centerof the apparatus and allowed to explore freely for 5 min.
Total number of central and peripheral square crossings
were recorded for each animal. The apparatus wascleaned with damp cloth after each trial to avoid place
preference and the influence of olfactory stimuli.
Determination of estradiol, 25-OH-VD3 and calcium levelsin the blood serum

Blood samples were collected in tubes and centrifuged.
After centrifugation, serum was separated, frozen andstored at − 20 °C until biochemical assessment. Estradiol
levels was assessed using commercial available ELISA kit(DRG Diagnostics, Marburg, Germany). The sensitivity
for estradiol detection using ELISA kit was 3.0 pg/ml.
Measurement of 25-hydroxyvitamin D3 (25-OH-VD3)levels was performed by ELISA kit (CSB-E08098r, Cusabio Biotech Co., Ltd., Wuhan, P.R. China). Technicalvariability for 25-OH-VD3 ELISA kit was low with coefficients of variation of < 10% intra-assay and < 15%inter-assay. Detection range of 25-OH-VD3 levels was
20 μg/L-100 μg/L. The sensitivity of the 25-OH-VD3
ELISA kit was 5.0 μg/L. Calcium concentrations weredetected by spectrophotometric method using Calcium
assay colorimetric kit (ab102505, Abcam, France). The
sensitivity of the calcium kit was 0.1 m/M. All the procedures of estradiol, 25-OH-VD3 and calcium kits were conducted following the manufacturer’s instruction manual.
Statistical analysis

Data were expressed as means ± standard error (S.E.M).
Differences among means were postulated as significant
at р ≤ 0.05. Behavioral and biochemical data were analyzed using a two-way ANOVA and subsequent post hoc
analysis with Dunnett’s multiple comparison test. Statistical calculation was carried out using SPSS software 19
version (SPSS Inc., Chicago, IL., USA).

Page 48 of 56

Results
Vitamin D3 in different doses decreases anxiety-likeprofile of the old OVX and OVX rats given with 17βestradiol after long-term absence of estrogen as measured
in the EPM test

Two-way ANOVA test demonstrated a significant hormone condition × treatment interaction ([F(5,44) =11.44, P < 0.05] and [F(5,44) = 11.12, p < 0.01], respectively), significant effect of hormone conditions ([F(5,44)= 9.22, P < 0.01] and [F(5,44) = 16.88, p < 0.01], respectively) and significant effect for treatment ([F(5,44) =11.56, P < 0.05], and [F(5,44) = 12.56, p < 0.05], respectively) for the time spent into the open arms or the number of entries into the open arms of the old OVX rats.
Post-hoc analyses revealed differences among the groups
for the anxiety-like state during experimental sessions(p < 0.05).
The old intact rats treated with Vitamin D3 at doses of
1.0 mg/kg, 2.5 mg/kg or 5.0 mg/kg failed to alter the timespent into the open arms and the number of entries into
the open arms as compared to the old control rats
(Fig. 1ab, p > 0.05). The old OVX rats given with solvent displayed a significant decrease of the time spent
into the open arms and the number of entries into
the open arms as compared to the old control rats
(Fig. 1ab, p > 0.05).
Vitamin D3 supplementation administered in all doses
to the old OVX rats resulted in increase of the timespent into the open arms and the number of entries into
the open arms as compared to the old OVX rats treatedwith solvent (Fig. 1ab, p > 0.05). Combined administration of cholecalciferol at dose of 1.0 mg/kg with 17β-E2
to the old OVX rats more significantly increased thetime spent into the open arms and the number of entries
into the open arms as compared to the old OVX rats
treated with solvent or 17β-E2 (Fig. 1ab, p > 0.05). The
old OVX rats administered with cholecalciferol at dosesof 2.5 mg/kg and 5.0 mg/kg plus 17β-E2 showed similarvalues of the time spent into the open arms and thenumber of entries into the open arms like as old OVX
rats treated with 17β-E2 (Fig. 1ab, p > 0.05).
Vitamin D3 at different doses reverses anxiety-like profileof the old OVX and OVX rats given with 17β-estradiol after
long-term absence of estrogen as measured in light-dark box

Two-way analysis of variance showed a significant
hormone condition × treatment interaction ([F(5,44) =11.52, p < 0.01]), significant effect of hormone conditions([F(5,44) = 11.22, p < 0.05]) and significant effect fortreatment ([F(5,44) = 10.08, p < 0.01]) for time spent andnumber of entries in the light compartment of the old
OVX rats. Post-hoc analyses revealed differences among
the groups for the anxiety-like state during experimental
sessions (p < 0.05).

Fedotova BMC Medical Genetics 2019, 20(Suppl 1):49

Page 49 of 56

Fig. 1 Effects of Vitamin D3 administration on anxiety-like behavior of the old ovariectomized (OVX) rats following long-term estrogen deficiency
in the elevated plus maze. a – time spent into the open arms, sec; b – the number of entries into the open arms. The obtained results show themean ± standard error of the mean (SEM). *– p < 0.05 as compared to the control group of the old sham-operated rats; # – p < 0.05 as comparedto the old OVX rats treated with solvent; $ – p < 0.05 as compared to the old OVX rats treated with 17β-estradiol (17β-E2). Each group compriseda minimum of eight rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day subcutaneously (s.c.), once daily, for 14 days. The administered doseof 17β-estradiol was 0.5 μg/rat s.c., once daily, for 14 days

The old intact rats treated with Vitamin D3 at doses of
1.0 mg/kg, 2.5 mg/kg or 5.0 mg/kg failed to modify timespent and number of entries in the light compartment ascompared to the old control rats (Fig. 2ab, p > 0.05). The
old OVX rats given with solvent displayed a significant
decrease of time spent and number of entries in the lightcompartment as compared to the old control rats
(Fig. 2ab, p > 0.05).
Vitamin D3 supplementation administered in all doses
to the old OVX rats induced increase of the time spentand number of entries in the light compartment as compared to the old OVX rats treated with solvent (Fig. 2ab,p > 0.05). Combined administration of cholecalciferol atdose of 1.0 mg/kg with 17β-E2 to the old OVX rats moresignificantly increased time spent and number of entries
in the light compartment as compared to the old OVX
rats treated with solvent or 17β-E2 (Fig. 2ab, p > 0.05).

The old OVX rats administered with Vitamin D3 atdoses of 2.5 mg/kg and 5.0 mg/kg plus 17β-E2 showed
identical parameters of time spent and number of entries
in the light compartment like as old OVX rats treatedwith 17β-E2 (Fig. 2ab, p > 0.05).

Vitamin D3 administration changes behavioral reactivityof the old OVX and OVX rats treated with 17β-estradiol

Accordingly to the two-way ANOVA test, there weresignificant differences for the grooming behavior between hormone conditions ([F(5,44) = 8.12, p < 0.05]) between drug treatment ([F(5,44) = 12.51, p < 0.01]) and aninteraction between hormone condition and treatments([F(5,44) = 7.16, p < 0.01]) in the old OVX rats. Further
post-hoc test revealed differences for grooming between
experimental groups of the old OVX rats (p < 0.05).

Fedotova BMC Medical Genetics 2019, 20(Suppl 1):49

Page 50 of 56

Fig. 2 Effects of Vitamin D3 administration on anxiety-like behavior of the old OVX rats following long-term estrogen deficiency in the light/darktest. a – time spent in the light box, sec; b – the number of entrances in the light box. The obtained results show the mean ± standard error of
the mean (SEM). * – p < 0.05 as compared to the control group of sham-operated rats, # – p < 0.05 as compared to the old OVX rats treated withsolvent, $ – p < 0.05 as compared to the old OVX rats treated with 17β-estradiol. Each group comprised a minimum of eight rats. Cholecalciferol was
given at 1.0, 2.5 or 5.0 mg/kg/day s.c., once daily, for 14 days. The administered dose of 17β-estradiol (17β-E2) was 0.5 μg/rat s.c., once daily, for 14 days

Vitamin D3 injected at several doses failed to demonstrate any changes of behavioral reactivity of the old intact females in the OFT as compared to the control rats
(Table 1, p > 0.05).
A significant decrease of grooming behavior was registered in the old OVX rats given with solvent as compared to the control (Table 1, p < 0.05). 17β-E2significantly reduced grooming reactions in the old OVX
rats as compared to the middle-aged OVX rats (Table 1,
p < 0.05). The old OVX rats treated with Vitamin D3 in
all tested doses alone or in a combination with 17β-E2
did not demonstrate any modifications of motor andrearing activities as compared to the OVX rats given
with solvent (Table 1, p < 0.05).
However, the old OVX rats treated with Vitamin D3 atdoses of 1.0, 2.5 and 5.0 mg/kg demonstrated an increaseof grooming behavior as compared to the OVX rats. A

co-administration of Vitamin D3 at these doses with
17β-E2 decreased grooming behavior as compared to old
intact and OVX rats received with solvent or 17β-E2(Table 1, p < 0.05).
Modifications of 25-hydroxyvitamin D3, estradiol andcalcium levels in the blood serum following Vitamin D3
administration in the old OVX and OVX females treatedwith 17β-estradiol

The old intact rats treated with cholecalciferol at dosesof 1.0, 2.5 and 5.0 mg/kg failed to alter estradiol levels inthe serum blood as compared to the control rats (Fig. 3,
p > 0.05) and increased 25-OH-VD3 levels (Fig. 4, p <0.05). Long-term ovariectomy in the old female rats
resulted in a significant decrease of estradiol and25-OH-VD3 levels in the blood as compared to the oldcontrol females (Figs. 3 and 4, p < 0.05). The 17β-E2Fedotova BMC Medical Genetics 2019, 20(Suppl 1):49

Page 51 of 56

Table 1 Effects of Vitamin D3 administration on behavior of the old ovariectomized (OVX) rats following long-term estrogen deficiency
in the open field test for 5 minGroups

Crossing

Rearing

Grooming

Old control rats + solvent

69.7 ± 5.2

11.5 ± 0.3

2.9 ± 0.2

Old intact rats + cholecalciferol 1.0 mg/kg

71.8 ± 2.9

10.7 ± 0.3

3.2 ± 0.2

Old intact rats + cholecalciferol 2.5 mg/kg

66.9 ± 3.6

10.4 ± 0.2

3.1 ± 0.2

Old intact rats + cholecalciferol 5.0 mg/kg

69.0 ± 4.2

12.2 ± 0.8

3.0 ± 0.2

Old OVX rats + solvent (OVX/solvent rats)

72.1 ± 2.3

12.1 ± 0.6

1.0 ± 0.2*

Old OVX rats +17β-E2 (OVX/17β-E2 rats)

64.3 ± 4.6

11.7 ± 0.8

3.1 ± 0.3#

Old OVX rats + cholecalciferol 1.0 mg/kg

63.2 ± 3.5

12.6 ± 0.9

4.2 ± 0.2#

Old OVX rats + cholecalciferol 2.5 mg/kg

67.2 ± 5.2

10.2 ± 0.8

3.9 ± 0.2#

Old OVX rats + cholecalciferol 5.0 mg/kg

70.3 ± 4.4

11.5 ± 0.5

4.3 ± 0.2#

Old OVX rats + cholecalciferol 1.0 mg/kg + 17β-E2

72.1 ± 6.8

12.2 ± 0.6

0.7 ± 0.2* # $Old OVX rats + cholecalciferol 2.5 mg/kg + 17β-E278.5 ± 8.4

11.8 ± 0.4

0.6 ± 0.2* # $Old OVX rats + cholecalciferol 5.0 mg/kg + 17β-E2

69.4 ± 6.6

10.9 ± 0.8

0.9 ± 0.2* # $The obtained results show the mean ± standard error of the mean (SEM). *p < 0.05 as compared to the control group of the old sham-operated rats; #p < 0.05 ascompared to the old OVX rats treated with solvent; $p < 0.05 as compared to the old OVX rats treated with 17β-estradiol (17β-E2). Each group comprised a minimumof eight rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day subcutaneously (s.c.), once daily, for 14 days. The administered dose of 17β-estradiol was 0.5 μg/rats.c., once daily, for 14 days

supplementation (0.5 μg/kg, s.c.) failed to modify 25OH-VD3 levels in the blood of the old OVX rats as compared to the old OVX rats administered with solvent(Fig. 4, p > 0.05), and the value of this parameter in the
old OVX/17β-E2 females were lower than that of thevalue of old control rats. However, 17β-E2 supplementation significantly increased estradiol levels in the bloodof the old OVX rats as compared to the old OVX rats
given with solvent (Fig. 3, p < 0.05).
The old OVX rats treated with cholecalciferol at all
doses significantly increased 25-OH-VD3 and estradiol
levels in the serum blood as compared to the old OVX

rats treated with solvent (Fig. 4, p < 0.05). However, thevalues of 25-OH-VD3 and estradiol levels in the old
OVX rats treated with cholecalciferol at all doses werelower than that of the values of old control rats.
Co-administration of Vitamin D3 (1.0 mg/kg) and
17β-E2 markedly enhanced estradiol levels in the old
OVX rats as compared to the groups of old OVX rats
received with solvent or 17β-E2 (Fig. 3, p < 0.05). Vitamin D3 supplementation (2.5 mg/kg and 5.0 mg/kg) plus
17β-E2 did not modify estradiol concentrations in theserum blood of the old OVX rats as compared to the
OVX rats given with 17β-E2 (Fig. 3, p > 0.05). Cholecalciferol

Fig. 3 Effects of Vitamin D3 administration on estradiol level of the old ovariectomized (OVX) rats following long-term estrogen deficiency in theserum blood. The obtained results show the mean ± standard error of the mean (SEM). *– p < 0.05 as compared to the control group of the old
sham-operated rats; # – p < 0.05 as compared to the old OVX rats treated with solvent; $ – p < 0.05 as compared to the old OVX rats treated with
17β-estradiol (17β-E2). Each group comprised a minimum of eight rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day subcutaneously (s.c.),once daily, for 14 days. The administered dose of 17β-estradiol was 0.5 μg/rat s.c., once daily, for 14 days

Fedotova BMC Medical Genetics 2019, 20(Suppl 1):49

Page 52 of 56

Fig. 4 Effects of Vitamin D3 administration on 25-OH-VD3 level of the old ovariectomized (OVX) rats following long-term estrogen deficiency inthe serum blood. The obtained results show the mean ± standard error of the mean (SEM). *– p < 0.05 as compared to the control group of theold sham-operated rats; # – p < 0.05 as compared to the old OVX rats treated with solvent; $ – p < 0.05 as compared to the old OVX rats treatedwith 17β-estradiol (17β-E2). Each group comprised a minimum of eight rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day subcutaneously(s.c.), once daily, for 14 days. The administered dose of 17β-estradiol was 0.5 μg/rat s.c., once daily, for 14 days

at all doses in combination with 17β-E2 significantly increased 25-OH-VD3 levels when these old OVX rats werecompared with the old OVX/solvent and OVX/17β-E2groups (Fig. 4, p > 0.05).
The two-way ANOVA failed to show any significant differences in the calcium levels in the blood serum between
hormone conditions, drug treatments, and an interaction
between hormone condition and treatments in the old
OVX rats with long-term estrogen deficiency (Figs. 5, p >0.05). The post-hoc test did not find any differences among
the experimental groups for the calcium levels (p > 0.05).

Discussion
In the present study, the effects of chronic cholecalciferol treatment at different doses (1.0, 2.5 and 5.0 mg/kg,s.c.) for 14 days on anxiety-like behavior in the old female rats with long-term estrogen deficiency and 17β-E2
supplementation in a low dose were examined. Cholecalciferol at all investigated doses did not produce anychanges of anxiety-like behavior of the old intact female
rats in the EPM and LDT. Analyzing the results frombiochemical assay, an increase of 25-OH-VD3 concentrations and absence of any modifications of estradiol levels

Fig. 5 Effects of Vitamin D3 administration on calcium level of the old ovariectomized (OVX) rats following long-term estrogen deficiency in theserum blood. The obtained results show the mean ± standard error of the mean (SEM). *– p < 0.05 as compared to the control group of the old
sham-operated rats; # – p < 0.05 as compared to the old OVX rats treated with solvent; $ – p < 0.05 as compared to the old OVX rats treated with
17β-estradiol (17β-E2). Each group comprised a minimum of eight rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day subcutaneously (s.c.),once daily, for 14 days. The administered dose of 17β-estradiol was 0.5 μg/rat s.c., once daily, for 14 days

Fedotova BMC Medical Genetics 2019, 20(Suppl 1):49

in the serum blood of the middle-aged and old intact
rats given with different doses of cholecalciferol were
found.
These results suggest that cholecalciferol-induced theincreasing of 25-OH-VD3 levels in the blood serum of
the old intact-ovary rats are not associated with absenceof anxiety-like profile alterations in the behavioral tests.
Furthermore, the old ovary-intact female rats are alsoneeded to evaluate the behavioral effects of cholecalciferol administered at several doses in the EPM and LDT
paradigms.
The results showed that in the old OVX rats following
12 weeks of postovariectomy period, there were marked
anxiety-like behavior as assessed by EPM and LDT. Although 17β-E2 supplementation resulted in significant
anxiolytic-like effect of the old OVX rats with long-term
absence of estrogen, the 17β-E2 administration was notable to completely diminish anxiety-like behavior to thelevel of the old control intact animals. According tothese results, we conclude that old OVX rats following
12 weeks of postovariectomy period display significant
anxiety-related behavior, while 17β-E2 administration tothe old OVX rats attenuates the estrogen deficiencyinduced anxiety-like behavior to some extent. In fact,
these experiments showed that the effects of 17β-E2 supplementation on anxiety-like behavior did not associatedwith absence of its effects on 25-OH-VD3 levels in the
old OVX rats. The long-term effect of ovariectomy onanxiety-like behavior in old female rats were submitted
in a standard behavioral tests [26, 27, 38].
Cholecalciferol at all doses per se had a significant
anxiolytic-like effect in the old OVX rats following
long-term ovariectomy. Unexpectedly, that the old OVX
rats with 12 weeks postovariectomy period administeredwith cholecalciferol at doses 2.5 and 5.0 mg/kg/day in
combination with 17β-E2 showed similar anxiety-likeprofile like the OVX rats given with 17β-E2. Thus, we
did not observe any synergic anxiolytic-like effects ofcholecalciferol at doses of 2.5 mg/kg or 5.0 mg/kg in the
old OVX rats given with 17β-E2. It might supposed that
there are some concurrent relation between 17β-E2 andcholecalciferol at doses of 2.5 and 5.0 mg/kg/day. In fact,
application of 17β-E2 interfere with anxiolytic-like actionof cholecalciferol at doses of 2.5 mg/kg or 5.0 mg/kg
in the old OVX rats after long-term ovariectomy.
Simultaneously, cholecalciferol treatment in all tested
doses similarly increased grooming, did not change
locomotor activity and rearing of the old OVX rats
after long-term ovariectomy. Thus, the present results
suggest that anxiolytic-like effects of Vitamin D3 are
specific in the old OVX rats given with solvent or
17β-E2, since data of the OFT were not able to demonstrate any of alterations in motor or rearing activities in these rats.

Page 53 of 56

Biochemical analysis showed that administration ofcholecalciferol at all doses alone or in a combination
with 17β-E2 resulted in elevated 25-OH-VD3 levels inthe blood serum of the old OVX rats with long-term absence of estrogen. Cholecalciferol administered alone atall doses similarly increased estradiol levels in the blood
serum of the old OVX rats after long-term ovariectomy.
On the other hand, only application of cholecalciferol ata dose of 1.0 mg/kg with low dose of 17β-E2 induced
more profound increase of estradiol levels in the blood
serum of the old OVX rats. Moreover, cholecalciferol inseveral doses failed to induce any changes in calcium
concentrations in the blood serum of the old OVX rats
given with solvent or 17β-E2.
These data suggested that the different effects of cholecalciferol application per se in the old OVX rats withlong-term absence of estrogen on anxiety-like behavior
in the EPM and LDT did not associated with its effects
on estradiol, 25-OH-VD3 and calcium levels.
However, we can speculate that behavioral effects ofcholecalciferol treatment with low dose of 17β-E2 in the
EPM and LDT tests might connected with fluctuationsof estradiol levels in the blood serum of the old OVX
rats. It is possible that specific sites of action involved in
the anxiolytic-like effects of cholecalciferol that also
modulated by estrogens are affected by the endocrine
milieu that prevails at different period for the old female
rats. Moreover, after a long-time absence of ovarian fluctuations an adaptive process may contribute to a better
response for cholecalciferol administration at a dose of
1.0 mg/kg in the old female rats.
The role of ovarian hormones in anxiety and stresssensitivity is of great interest for women transitioning
through menopause [39, 40]. Mood disorders during
menopause could be partly due to loss of estrogen withmenopause because estrogen is known to have neuroprotective effects on brain [41, 42]. Menopausal hormonal therapy (MHT) may improve the symptoms ofaffective-related state in people or decrease the risk ofdeveloping mood disturbances in older women, but thisis unclear because in some studies MHT does not stop
the development of anxiety-like symptoms in elderly
postmenopausal women [43]. The exact role of estrogenstill needs to be defined.
Menopause are also at higher risk of developing VD
deficiency due to decreased dietary intake, less sun exposure, restricted outdoor activity and a decreased capacity to produce enough calcitriol as a result of an age
related decline in hydroxylation by kidneys [44–46].
Vitamin D3 is a hormone precursor which is transformed into 1,25-dihydroxyvitamin D (1,25-(OH)2D3) inthe liver and kidney [47]. Through decades This active
form of VD has been involved in the brain development
and functions of the central nervous system (CNS)Fedotova BMC Medical Genetics 2019, 20(Suppl 1):49[48, 49]. Hormonal form 1,25(OH)2D3 enters the brain
via the blood brain barrier to act directly on cells containing its nuclear receptor, the VDR [50, 51].
The presence of VD receptors (VDR) outside the skeletal system, enterocytes and renal tubular cells was
confirmed in many cell types including immune cells,
neurons, pancreatic cells, myocytes, cardiomyocytes,
endothelium cells, which stress pleiotropic activity of
VD [52, 53]. The active form of vitamin D is transferred
to astrocytes where it can bind to VDR and initiate genetranscription or be inactivated when in excess [50, 51].
Alternatively, 1,25(OH)2D3 can induce autocrine orparacrine rapid non-genomic actions since all brain cell
types express the other membrane receptor of VD [50].
It is possible that the behavioral effects of cholecalciferol
are mediated by multiple target regions, including brain
centers that are involved in the mechanisms of anxietylike behavior. Regardless, it cannot presently exclude
possible indirect effects of vitamin D3 on different
neurotransmitter circuits. Further research is required toinvestigate the underlying mechanisms in its anxiolyticlike effects.
It is well-established both systemic effects of VD on
calcium metabolism and neuroprotective effects of VD
on the brain [54]. Low VD levels has been implicated in
the pathophysiological mechanisms of cardiovasculardiseases, depression, anxiety, cognitive disorders, obesity,
metabolic syndrome, type 2 diabetes, various types ofcancer, immune disorders [10, 54]. According to Gaugris
and co-workers (2005) [55], the prevalence of low VDlevels appears to be high in postmenopausal women.
Additionally, the decline of estrogens after menopause
decreases the activity of 1α-OHase, what results in lower
synthesis of the active VD form [55, 56]. Application ofVitamin D3 in specific periods of women’s life, seems to
be of great importance, because it may reduce the risk ofaffective-related diseases during menopausal period[57–59]. However, the current data for Vitamin D3 application studies are very incomplete and need of more intensive investigations. The main point of question is to
examine how the interaction between Vitamin D3 and estradiol might alter at specific periods of women’s life, andthe impacts of such alterations elsewhere in the postmenopausal woman. VDR have been identified throughout thefemale reproductive tract [60, 61]. Some studies havedemonstrated a direct modulation by VD of estradiol, estrone, and progesterone production in human ovariancells [62–64]. It could be supposed that estrogens and VD
share similar targets of the brain to induce theiranxiolytic-like effects. However, the behavioral manifestations of VD at various doses are completely different in
the middle-aged and old OVX females. It is likely that VD
acts through a various mechanisms that are sensitive in female rats of old age with long-term absence of ovarian

Page 54 of 56

hormones. Moreover, it is completely needed to understand the precise mechanisms of how VD treatment alone
or in a combination with 17β-estradiol supplementation
may affect women’s anxiety-related state.
These points illustrate how the current state of VD
treatment research is incomplete and in need of more
intensive research. Working toward uncovering how theinteraction between VD and estradiol changes after
menopause, and the implications of these changes elsewhere in the postmenopausal woman, is necessary forproviding the most complete understanding of how VD
treatment alone or in a combination with 17β-estradiol
supplementation may affect women’s affective-related
state.
VD as changes in VDR impact on various brain neurotransmitters, and thus suggest a potential role of vitamin
D in causing and redressing mood disorders [65]. It
could be supposed, even though estrogens and cholecalciferol share similar targets on monoaminergic or another neurotransmitter systems to induce their anxiolyticlike effects, the behavioral manifestations of cholecalciferol are completely different in the old OVX females. It is
likely that cholecalciferol acts through a different mechanisms of action that is sensitive to the age of female rats
with long-term absence of ovarian hormones.
In conclusion, the results of this study can be summarized as follows: all tested doses of cholecalciferol given
alone are produced anxiolytic-like effects in the EPM
and LDT in the old OVX female rats; the one specificdose of cholecalciferol (1.0 mg/kg/day) in the old OVX
rats is able to induce synergic anxiolytic-like effect in the
EPM and LDT; effects of cholecalciferol on anxiety-likebehavior in the old OVX rats after long-term absence of
estrogen are dependent from absence or presence ofadditional hormonal treatment as 17β-E2. Further investigations is to be addressed in relation to such issues:
whether different effects of cholecalciferol on anxietylike behavior are dependent from different age of rats, orwhether different doses of cholecalciferol on anxiety-likebehavior in OVX rats with different age rats might leadto negative versus positive effects. Further research, withproperly designed experimental studies, is needed to test
this hypothesis. In addition, further research is needed
to elucidate the biochemical mechanism/(s) of cholecalciferol effects on the anxiety-like behavior and their
physiological relevance for development of mood impairment at estrogen deficiency at aging. Furthermore, themechanism by which cholecalciferol produces anxiolyticlike effect in the old OVX rats and the implications of thisin brain function need to be investigated in future research.
Moreover, further studies are needed to evaluate the association of VD with estrogen-related pathways and to conductdetail experiments together with biochemical studies ofthese subjects to verify the significance of this study.

Fedotova BMC Medical Genetics 2019, 20(Suppl 1):49

Conclusions
The present data of the preclinical study indicates thatchronic cholecalciferol at a dose of 1.0 mg/kg, s.c. treatment decreased anxiety-related behavior after impairment induced by long-term ovariectomy in the old
female rats with long-term absence of estrogen. Moreover, a combination of cholecalciferol at a dose of 1.0
mg/kg s.c. and 17β-E2 is more effective than 17β-E2alone in the old OVX rats inducing a more synergic
anxiolytic-like effects in the EPM and LDT. Furthermore, this is the first study to show a beneficial effect ofchronic cholecalciferol at dose of 1.0 mg/kg s.c. administration on anxiety-related states induced by long-term
ovariectomy in the old female rats. Importantly, theseresults suggest that 17β-E2 administration interferedwith anxiolytic-like action of cholecalciferol administered alone at doses of 2.5 mg/kg or 5.0 mg/kg to the old
OVX rats with long-term absence of estrogens. This
work promotes more effective creating of the novel
therapeutic targets and strategies for anxiety-like treatmentin the old subjects with long-term estrogen deficiency.
Funding
This work was financially supported by Government of Russian Federation,
Grant 08–08. The funding body played no role in the design of the study
and collection, analysis, and interpretation of data and in writing the manuscript.
Publication costs are funded by the author.
Availability of data and materials
Not applicable.
About this supplement
This article has been published as part of BMC Medical Genetics Volume 20
Supplement 1, 2019: Selected articles from BGRS\SB-2018: medical genetics. The
full contents of the supplement are available online at https://bmcmedgenet.
biomedcentral.com/articles/supplements/volume-20-supplement-1.
Author’s contributions
Author JOF designed the study, performed the experiments, analyzed thedata and wrote the protocol and manuscript. The author read and approved
the final manuscript.
Ethics approval
All the experiments performed this research were approved by the EthicalCommittee for Animal Research, I.P. Pavlov Institute of Physiology of theRussian Academy of Sciences (protocol 1095/1 from June 25, 2012). The field
experiments were conducted under local legislation and permissions.
Consent for publication
Not applicable.
Competing interests
The author declares that she has no competing interests.

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