**3.4 Modifications of 25-hydroxyvitamin D3, estradiol and calcium levels in the blood serum following vitamin D3 administration in the middle-aged and old OVX and OVX females treated with 17β-estradiol**

The middle-aged intact rats treated with cholecalciferol at doses of 1.0, 2.5 and 5.0 mg/kg increased 25-OH-VD3 levels (**Figure 5**, *p* < 0.05) and failed to alter estradiol levels in the serum blood as compared to the control rats (**Figure 6**, *p* > 0.05).

Long-term ovariectomy in the middle-aged female rats resulted in a significant decrease of estradiol and 25-OH-VD3 levels in the blood as compared to the middleaged control females (**Figures 5** and **6**, *p* < 0.05). The 17β-E2 supplementation (0.5 μg/kg, SC) failed to modify 25-OH-VD3 levels in the blood of the middleaged OVX rats as compared to the middle-aged OVX rats administered with solvent (**Figure 5**, *p* > 0.05), and the value of this parameter in the middle-aged OVX/17β-E2 females were lower than that of the value of middle-aged control rats. However, 17β-E2 supplementation significantly increased estradiol levels in the blood of the middle-aged OVX rats as compared to the middle-aged OVX rats given with solvent (**Figure 6**, *p* < 0.05).

The middle-aged OVX rats treated with cholecalciferol at all tested doses significantly increased estradiol levels in the serum blood as compared to the middle-aged OVX rats treated with solvent (**Figure 6**, *p* < 0.05). However, the value of estradiol levels in the middle-aged OVX rats treated with cholecalciferol at these doses were lower than that of the value of middle-aged control rats. The middle-aged OVX rats treated with cholecalciferol at doses of 2.5 and 5.0 mg/kg significantly increased 25-OH-VD3 levels in the serum blood as compared to the middle-aged OVX rats


*\* 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.*

*The obtained results show the mean ± S.E.M. 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. 17β-Estradiol (17β-E2) was given at 0.5 μg/rat, s.c., once daily, during 14 days.*

#### **Table 1.**

*Fads and Facts about Vitamin D*

with different age (*p* < 0.05).

**Figure 4.**

(**Tables 1** and **2**, *p* < 0.05).

the middle-aged control rats (**Tables 1** and **2**, *p* > 0.05).

*administered dose of 17β-estradiol (17β-E2) was 0.5 μg/rat s.c., once daily, for 14 days.*

*p* < 0.01] and [F(5,44) = 12.56, *p* < 0.01], respectively) and an interaction between hormone condition and treatments ([F(5,44) = 7.16, *p* < 0.01] and [F(5,44) = 9.26, *p* < 0.01], respectively) in the middle-aged and old OVX rats. Further *post-hoc* test revealed differences for grooming between experimental groups of the OVX rats

*Effects of cholecalciferol administration on anxiety-like behavior of the old OVX rats following long-term estrogen deficiency in the light/dark test. (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 with solvent, ##—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* 

Vitamin D3 injected at several doses failed to demonstrate any changes of behavioral reactivity of the middle-aged and old intact females in the OFT as compared to

A significant decrease of grooming behavior was registered in the middle-aged and old OVX rats given with solvent as compared to the control (**Tables 1** and **2**, *p* < 0.05). 17β-E2 significantly reduced grooming reactions in the middle-aged and old OVX rats as compared to the middle-aged OVX rats (**Tables 1** and **2**, *p* < 0.05). The middle-aged and 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 and rearing activities as compared to the middle-aged OVX rats given with solvent

However, the middle-aged and old OVX rats treated with vitamin D3 at doses of 1.0, 2.5 and 5.0 mg/kg demonstrated an increase of grooming behavior as compared to the middle-aged OVX rats. A co-administration of vitamin D3 at these doses with 17β-E2 decreased grooming behavior as compared to both middle-aged, as well as old intact and OVX rats received with solvent or 17β-E2 (**Tables 1** and **2**, *p* < 0.05).

**44**

*Cholecalciferol influences on behavioral parameters of the middle-aged OVX rats following long-term estrogen deficiency in the open field test for 5 min.*


*\* 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.*

*The obtained results show the mean ± S.E.M. Each group comprised a minimum of 8 rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day, s.c., once daily, for 14 days. 17β-Estradiol (17β-E2) was given at 0.5 μg/rat, s.c., once daily, during 14 days.*

#### **Table 2.**

*Effects of cholecalciferol administration on behavioral impairments of the old OVX rats following long-term estrogen deficiency in the open field test for 5 min.*

#### **Figure 5.**

*Effects of cholecalciferol administration on 25-OH-VD3 level of the middle-aged ovariectomized (OVX) rats following long-term estrogen deficiency in the serum 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.*

treated with solvent (**Figure 5**, *p* < 0.05). The value of 25-OH-VD3 content in the middle-aged OVX rats treated with cholecalciferol at doses of 2.5 and 5.0 mg/kg were lower than that of the value of middle-aged control rats. Moreover, cholecalciferol administered at a dose of 1.0 mg/kg into the middle-aged OVX rats failed to change 25-OH-VD3 levels in the serum blood as compared to the middle-aged OVX rats treated with solvent (**Figure 5**, *p* > 0.05). Cholecalciferol treatment at doses of

**47**

**Figure 7.**

*once daily, for 14 days.*

*Behavioral Effects of Vitamin D3 at Estrogen Deficiency in Females of Different Age*

2.5 and 5.0 mg/kg in combination with 17β-E2 more significantly elevated 25-OH-VD3 levels for the middle-aged OVX rats as compared to the OVX females treated with oil solvent or 17β-E2 (**Figure 3**, *p* < 0.05). Combined administration of cholecalciferol at a dose of 1.0 mg/kg and 17β-E2 in the middle-aged OVX rats failed to change 25-OH-VD3 levels as compared to the OVX rats administered with 17β-E2 or solvent (**Figure 5**, *p* > 0.05). Cholecalciferol at all doses in combination with 17β-E2 significantly increased estradiol levels when middle-aged OVX rats/cholecalciferol in tested doses plus 17β-E2 rats were compared with the middle-aged OVX/solvent

*Effects of cholecalciferol administration on estradiol level of the middle-aged ovariectomized (OVX) rats following long-term estrogen deficiency in the serum 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* 

The old intact rats treated with cholecalciferol at doses of 1.0, 2.5 and 5.0 mg/kg increased 25-OH-VD3 levels (**Figure 7**, *p* < 0.05) and failed to alter estradiol levels in the serum blood as compared to the control rats (**Figure 8**, *p* > 0.05). Long-term ovariectomy in the old female rats resulted in a significant decrease of estradiol and 25-OH-VD3 levels in the blood as compared to the old control females (**Figures 7** and **8**, *p* < 0.05). The 17β-E2 supplementation (0.5 μg/kg, s.c.) failed to modify

*Effects of cholecalciferol administration on 25-OH-VD3 level of the old ovariectomized (OVX) rats following long-term estrogen deficiency in the serum 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.,* 

*DOI: http://dx.doi.org/10.5772/intechopen.82596*

**Figure 6.**

and OVX/17β-E2 rat groups (**Figure 6**, *p* > 0.05).

*17β-estradiol was 0.5 μg/rat s.c., once daily, for 14 days.*

*Behavioral Effects of Vitamin D3 at Estrogen Deficiency in Females of Different Age DOI: http://dx.doi.org/10.5772/intechopen.82596*

#### **Figure 6.**

*Fads and Facts about Vitamin D*

**46**

**Figure 5.**

*\**

*#*

**Table 2.**

*daily, during 14 days.*

*17β-estradiol was 0.5 μg/rat s.c., once daily, for 14 days.*

*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.*

*estrogen deficiency in the open field test for 5 min.*

treated with solvent (**Figure 5**, *p* < 0.05). The value of 25-OH-VD3 content in the middle-aged OVX rats treated with cholecalciferol at doses of 2.5 and 5.0 mg/kg were lower than that of the value of middle-aged control rats. Moreover, cholecalciferol administered at a dose of 1.0 mg/kg into the middle-aged OVX rats failed to change 25-OH-VD3 levels in the serum blood as compared to the middle-aged OVX rats treated with solvent (**Figure 5**, *p* > 0.05). Cholecalciferol treatment at doses of

*Effects of cholecalciferol administration on 25-OH-VD3 level of the middle-aged ovariectomized (OVX) rats following long-term estrogen deficiency in the serum 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* 

**Groups 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-E2 78.5 ± 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 ± S.E.M. Each group comprised a minimum of 8 rats. Cholecalciferol was given at 1.0, 2.5 or 5.0 mg/kg/day, s.c., once daily, for 14 days. 17β-Estradiol (17β-E2) was given at 0.5 μg/rat, s.c., once* 

*Effects of cholecalciferol administration on behavioral impairments of the old OVX rats following long-term* 

*Effects of cholecalciferol administration on estradiol level of the middle-aged ovariectomized (OVX) rats following long-term estrogen deficiency in the serum 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.*

2.5 and 5.0 mg/kg in combination with 17β-E2 more significantly elevated 25-OH-VD3 levels for the middle-aged OVX rats as compared to the OVX females treated with oil solvent or 17β-E2 (**Figure 3**, *p* < 0.05). Combined administration of cholecalciferol at a dose of 1.0 mg/kg and 17β-E2 in the middle-aged OVX rats failed to change 25-OH-VD3 levels as compared to the OVX rats administered with 17β-E2 or solvent (**Figure 5**, *p* > 0.05). Cholecalciferol at all doses in combination with 17β-E2 significantly increased estradiol levels when middle-aged OVX rats/cholecalciferol in tested doses plus 17β-E2 rats were compared with the middle-aged OVX/solvent and OVX/17β-E2 rat groups (**Figure 6**, *p* > 0.05).

The old intact rats treated with cholecalciferol at doses of 1.0, 2.5 and 5.0 mg/kg increased 25-OH-VD3 levels (**Figure 7**, *p* < 0.05) and failed to alter estradiol levels in the serum blood as compared to the control rats (**Figure 8**, *p* > 0.05). Long-term ovariectomy in the old female rats resulted in a significant decrease of estradiol and 25-OH-VD3 levels in the blood as compared to the old control females (**Figures 7** and **8**, *p* < 0.05). The 17β-E2 supplementation (0.5 μg/kg, s.c.) failed to modify

#### **Figure 7.**

*Effects of cholecalciferol administration on 25-OH-VD3 level of the old ovariectomized (OVX) rats following long-term estrogen deficiency in the serum 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.*

#### **Figure 8.**

*Effects of cholecalciferol administration on estradiol level of the old ovariectomized (OVX) rats following long-term estrogen deficiency in the serum 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.*

25-OH-VD3 levels in the blood of the old OVX rats as compared to the old OVX rats administered with solvent (**Figure 7**, *p* > 0.05), and the value of this parameter in the old OVX/17β-E2 females were lower than that of the value of old control rats. However, 17β-E2 supplementation significantly increased estradiol levels in the blood of the old OVX rats as compared to the old OVX rats given with solvent (**Figure 8**, *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 (**Figure 8**, *p* < 0.05). However, the values of 25-OH-VD3 and estradiol levels in the old OVX rats treated with cholecalciferol at all doses were lower 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 (**Figure 7**, *p* < 0.05). Vitamin D3 supplementation (2.5 and 5.0 mg/kg) plus 17β-E2 did not modify estradiol concentrations in the serum blood of the old OVX rats as compared to the OVX rats given with 17β-E2

#### **Figure 9.**

*Effects of cholecalciferol administration on calcium level of the middle-ged ovariectomized (OVX) rats following long-term estrogen deficiency in the serum 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.*

**49**

levels (*p* > 0.05).

*rat s.c., once daily, for 14 days.*

**Figure 10.**

**4. Discussion**

long-term absence of estrogen.

*Behavioral Effects of Vitamin D3 at Estrogen Deficiency in Females of Different Age*

(**Figure 7**, *p* > 0.05). Cholecalciferol at all doses in combination with 17β-E2 significantly increased 25-OH-VD3 levels when these old OVX rats were compared with

*Effects of cholecalciferol administration on calcium level of the old ovariectomized (OVX) rats following long-term estrogen deficiency in the serum 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/*

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 middle-aged and old OVX rats with long-term estrogen deficiency (**Figures 9** and **10**, *p* > 0.05). The *posthoc* test did not find any differences among the experimental groups for the calcium

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 middle-aged and old female rats with long-term estrogen deficiency and 17β-E2 supplementation in a low dose were examined. Endogenous estrogens were removed by ovariectomy and only after 12 weeks post-ovariectomy period, these rats were used in all experiments. The results of behavioral testing for the anxiety-related effects of cholecalciferol were compared in both old OVX rats and OVX female rats treated with 17β-E2. Simultaneously, the effects of cholecalciferol at similar doses on anxiety-like behavior were tested in middle-aged and old intact female rats. For this purpose, the elevated plus maze (EPM) and light-dark test (LDT) were made use in the present study. It was also investigated whether the effects of cholecalciferol at different doses were specific in the EPM and LDT, measuring its effects on the behavioral activity in the OFT of the middle-aged and old intact and OVX rats after

Cholecalciferol at all investigated doses did not produce any changes of anxiety-like behavior of the middle-aged and old intact female rats in the EPM and LDT. Analyzing the results from biochemical assay, an increase of 25-OH-VD3 concentrations and absence of any modifications of estradiol levels in the serum blood of the middle-aged

These results suggest that cholecalciferol induced the increasing of 25-OH-VD3 levels in the blood serum of the middle-aged and old intact-ovary rats are not associated with absence of anxiety-like profile alterations in the behavioral tests.

and old intact rats given with different doses of cholecalciferol were found.

the old OVX/solvent and OVX/17β-E2 groups (**Figure 8**, *p* > 0.05).

*DOI: http://dx.doi.org/10.5772/intechopen.82596*

*Behavioral Effects of Vitamin D3 at Estrogen Deficiency in Females of Different Age DOI: http://dx.doi.org/10.5772/intechopen.82596*

#### **Figure 10.**

*Fads and Facts about Vitamin D*

25-OH-VD3 levels in the blood of the old OVX rats as compared to the old OVX rats administered with solvent (**Figure 7**, *p* > 0.05), and the value of this parameter in the old OVX/17β-E2 females were lower than that of the value of old control rats. However, 17β-E2 supplementation significantly increased estradiol levels in the blood of the old OVX rats as compared to the old OVX rats given with solvent (**Figure 8**,

*Effects of cholecalciferol administration on estradiol level of the old ovariectomized (OVX) rats following long-term estrogen deficiency in the serum 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.,* 

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 (**Figure 8**, *p* < 0.05). However, the values of 25-OH-VD3 and estradiol levels in the old OVX rats treated with cholecalciferol at all doses were

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 (**Figure 7**, *p* < 0.05). Vitamin D3 supplementation (2.5 and 5.0 mg/kg) plus 17β-E2 did not modify estradiol concentrations in the serum blood of the old OVX rats as compared to the OVX rats given with 17β-E2

*Effects of cholecalciferol administration on calcium level of the middle-ged ovariectomized (OVX) rats following long-term estrogen deficiency in the serum 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* 

**48**

**Figure 9.**

*p* < 0.05).

*once daily, for 14 days.*

**Figure 8.**

lower than that of the values of old control rats.

*17β-estradiol was 0.5 μg/rat s.c., once daily, for 14 days.*

*Effects of cholecalciferol administration on calcium level of the old ovariectomized (OVX) rats following long-term estrogen deficiency in the serum 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.*

(**Figure 7**, *p* > 0.05). Cholecalciferol at all doses in combination with 17β-E2 significantly increased 25-OH-VD3 levels when these old OVX rats were compared with the old OVX/solvent and OVX/17β-E2 groups (**Figure 8**, *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 middle-aged and old OVX rats with long-term estrogen deficiency (**Figures 9** and **10**, *p* > 0.05). The *posthoc* test did not find any differences among the experimental groups for the calcium levels (*p* > 0.05).

### **4. 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 middle-aged and old female rats with long-term estrogen deficiency and 17β-E2 supplementation in a low dose were examined. Endogenous estrogens were removed by ovariectomy and only after 12 weeks post-ovariectomy period, these rats were used in all experiments. The results of behavioral testing for the anxiety-related effects of cholecalciferol were compared in both old OVX rats and OVX female rats treated with 17β-E2. Simultaneously, the effects of cholecalciferol at similar doses on anxiety-like behavior were tested in middle-aged and old intact female rats. For this purpose, the elevated plus maze (EPM) and light-dark test (LDT) were made use in the present study. It was also investigated whether the effects of cholecalciferol at different doses were specific in the EPM and LDT, measuring its effects on the behavioral activity in the OFT of the middle-aged and old intact and OVX rats after long-term absence of estrogen.

Cholecalciferol at all investigated doses did not produce any changes of anxiety-like behavior of the middle-aged and old intact female rats in the EPM and LDT. Analyzing the results from biochemical assay, an increase of 25-OH-VD3 concentrations and absence of any modifications of estradiol levels 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 the increasing of 25-OH-VD3 levels in the blood serum of the middle-aged and old intact-ovary rats are not associated with absence of anxiety-like profile alterations in the behavioral tests.

Furthermore, the ovary-intact female rats of different age are also needed to evaluate the behavioral effects of cholecalciferol administered at several doses in the EPM and LDT paradigms.

The results showed that in the middle-aged and old OVX rats following 12 weeks of post-ovariectomy period, there were marked anxiety-like behavior as assessed by EPM and LDT. Although 17β-E2 supplementation resulted in significant anxiolyticlike effect of the middle-aged and old OVX rats with long-term absence of estrogen, the 17β-E2 administration was not able to completely diminish anxiety-like behavior to the level of the middle-aged and old control intact animals. According to these results, we conclude that middle-aged and old OVX rats following 12 weeks of postovariectomy period display significant anxiety-related behavior, while 17β-E2 administration to the middle-aged and old OVX rats attenuates the estrogen deficiency-induced anxiety-like behavior to some extent. In fact, these experiments showed that the effects of 17β-E2 supplementation on anxiety-like behavior did not associated with absence of its effects on 25-OH-VD3 levels in the old OVX rats. The long-term effect of ovariectomy on anxiety-like behavior in female rats of different age that were submitted in a standard behavioral tests [24, 35, 36].

Cholecalciferol at dose of 5.0 mg/kg/day per se had a significant anxiolyticlike effect in the middle-aged OVX rats following long-term ovariectomy. On the contrary, cholecalciferol at doses of 1.0 and 2.5 mg/kg/day failed to induce any modifications of anxiety-like behavior in the middle-aged OVX rats with long-term absence of estrogen. Combined application of vitamin D3 (1.0 mg/kg/day) with 17β-E2-induced synergic anxiolytic-like action of both preparations in the anxiety-related tasks of the old OVX rats. 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 post-ovariectomy period administered with cholecalciferol at doses 2.5 and 5.0 mg/kg/day in combination with 17β-E2 showed similar anxiety-like profile like the OVX rats given with 17β-E2. Thus, we did not observe any synergic anxiolytic-like effects of cholecalciferol at doses of 2.5 or 5.0 mg/kg in the old OVX rats given with 17β-E2. It might suppose that there are some concurrent relation between 17β-E2 and cholecalciferol at doses of 2.5 and 5.0 mg/kg/day. In fact, application of 17β-E2 interfere with anxiolytic-like action of cholecalciferol at doses of 2.5 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 middle-aged and old OVX rats after long-term ovariectomy. Thus, the present results suggest that anxiolyticlike effects of vitamin D3 are specific in the OVX rats of different age 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. Combined application of vitamin D3 (5.0 mg/kg/day) with low dose of 17β-E2 synergistically decreased anxiety-like profile of the middle-aged OVX rats. However, vitamin D3 at all doses significantly decreased grooming behavior in the middle-aged OVX rats treated with 17β-E2.

Unexpectedly, the middle-aged OVX rats treated with vitamin D3 at doses 1.0 and 2.5 mg/kg/day in combination with 17β-E2 have practically identical parameters of anxiety-like state as the OVX rats received only 17β-E2. Thus, this study failed to show any anxiolytic-like effects of vitamin D3 at doses of 1.0 or 2.5 mg/kg in the middle-aged OVX treated with 17β-E2. The present results might speculate that there exists some concurrent relation between 17β-E2 and vitamin D3 at doses of 2.5 and 5.0 mg/kg/day for the brain structures implicated in the mechanisms of anxiety. In fact, additional application of 17β-E2 to the middle-aged OVX rats interfered with anxiolytic-like action of vitamin D3 when it administered at doses 1.0 or

**51**

*Behavioral Effects of Vitamin D3 at Estrogen Deficiency in Females of Different Age*

2.5 mg/kg. Thus, the effects of vitamin D3 administration in a combination with low dose of 17β-E2 on anxiety-like state of the middle-aged OVX rats are determined by

ELISA assays demonstrated that administration of cholecalciferol only at doses

of 2.5 and 5.0 mg/kg resulted in elevated 25-OH-VD3 levels in the blood serum of the middle-aged OVX rats with long-term absence of estrogen. Moreover, application of cholecalciferol at these doses with low dose of 17β-E2 induced more profound increase of 25-OH-VD3 levels in the blood serum of the middle-aged OVX rats. These data suggest that the different effects of cholecalciferol application in the middle-aged OVX rats with long-term absence of estrogen on anxiety-like behavior

in the EPM and LDT did not associated with its effects on 25-OH-VD3 levels.

Biochemical analysis showed that administration of cholecalciferol at all doses alone or in a combination with 17β-E2 resulted in elevated 25-OH-VD3 levels in the blood serum of the old OVX rats with long-term absence of estrogen. Cholecalciferol administered alone at all 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 at a 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 in several 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 suggest that the different effects of cholecalciferol application per se in the middle-aged and old OVX rats with long-term absence of estrogen on anxiety-like behavior in the EPM and LDT did not associated with its effects on

However, we can speculate that behavioral effects of cholecalciferol treatment with low dose of 17β-E2 in the EPM and LDT tests might connected with fluctuations of estradiol levels in the blood serum of the middle-aged and 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 middle-aged and 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 and for cholecalciferol at a dose of 5.0 mg/kg in the

The role of ovarian hormones in anxiety and stress sensitivity is of great interest for women transitioning through menopause [37, 38]. Mood disorders during menopause could be partly due to loss of estrogen with menopause because estrogen is known to have neuroprotective effects on brain [39, 40]. Menopausal hormonal therapy (MHT) may improve the symptoms of affective-related state in people or decrease the risk of developing mood disturbances in older women, but this is unclear because in some studies MHT does not stop the development of anxiety-like symptoms in elderly postmenopausal women [41]. The exact role of estrogen still

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

Vitamin D3 is a hormone precursor which is transformed into 1,25-dihydroxyvitamin D (1,25-(OH)2D3) in the liver and kidney [45]. Through decades, this active form of VD has been involved in the brain development and functions of the central nervous system (CNS) [46, 47]. Hormonal form 1,25(OH)2D3 enters the brain via the blood-brain barrier to act directly on cells containing its nuclear receptor, the

*DOI: http://dx.doi.org/10.5772/intechopen.82596*

estradiol, 25-OH-VD3 and calcium levels.

middle-aged female rats.

needs to be defined.

by kidneys [42–44].

VDR [48, 49].

dose of treatment.

#### *Behavioral Effects of Vitamin D3 at Estrogen Deficiency in Females of Different Age DOI: http://dx.doi.org/10.5772/intechopen.82596*

2.5 mg/kg. Thus, the effects of vitamin D3 administration in a combination with low dose of 17β-E2 on anxiety-like state of the middle-aged OVX rats are determined by dose of treatment.

ELISA assays demonstrated that administration of cholecalciferol only at doses of 2.5 and 5.0 mg/kg resulted in elevated 25-OH-VD3 levels in the blood serum of the middle-aged OVX rats with long-term absence of estrogen. Moreover, application of cholecalciferol at these doses with low dose of 17β-E2 induced more profound increase of 25-OH-VD3 levels in the blood serum of the middle-aged OVX rats. These data suggest that the different effects of cholecalciferol application in the middle-aged OVX rats with long-term absence of estrogen on anxiety-like behavior in the EPM and LDT did not associated with its effects on 25-OH-VD3 levels.

Biochemical analysis showed that administration of cholecalciferol at all doses alone or in a combination with 17β-E2 resulted in elevated 25-OH-VD3 levels in the blood serum of the old OVX rats with long-term absence of estrogen. Cholecalciferol administered alone at all 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 at a 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 in several 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 suggest that the different effects of cholecalciferol application per se in the middle-aged and old OVX rats with long-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 of cholecalciferol treatment with low dose of 17β-E2 in the EPM and LDT tests might connected with fluctuations of estradiol levels in the blood serum of the middle-aged and 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 middle-aged and 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 and for cholecalciferol at a dose of 5.0 mg/kg in the middle-aged female rats.

The role of ovarian hormones in anxiety and stress sensitivity is of great interest for women transitioning through menopause [37, 38]. Mood disorders during menopause could be partly due to loss of estrogen with menopause because estrogen is known to have neuroprotective effects on brain [39, 40]. Menopausal hormonal therapy (MHT) may improve the symptoms of affective-related state in people or decrease the risk of developing mood disturbances in older women, but this is unclear because in some studies MHT does not stop the development of anxiety-like symptoms in elderly postmenopausal women [41]. The exact role of estrogen still 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 [42–44].

Vitamin D3 is a hormone precursor which is transformed into 1,25-dihydroxyvitamin D (1,25-(OH)2D3) in the liver and kidney [45]. Through decades, this active form of VD has been involved in the brain development and functions of the central nervous system (CNS) [46, 47]. 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 [48, 49].

*Fads and Facts about Vitamin D*

EPM and LDT paradigms.

Furthermore, the ovary-intact female rats of different age are also needed to evaluate the behavioral effects of cholecalciferol administered at several doses in the

age that were submitted in a standard behavioral tests [24, 35, 36].

The results showed that in the middle-aged and old OVX rats following 12 weeks of post-ovariectomy period, there were marked anxiety-like behavior as assessed by EPM and LDT. Although 17β-E2 supplementation resulted in significant anxiolyticlike effect of the middle-aged and old OVX rats with long-term absence of estrogen, the 17β-E2 administration was not able to completely diminish anxiety-like behavior to the level of the middle-aged and old control intact animals. According to these results, we conclude that middle-aged and old OVX rats following 12 weeks of postovariectomy period display significant anxiety-related behavior, while 17β-E2 administration to the middle-aged and old OVX rats attenuates the estrogen deficiency-induced anxiety-like behavior to some extent. In fact, these experiments showed that the effects of 17β-E2 supplementation on anxiety-like behavior did not associated with absence of its effects on 25-OH-VD3 levels in the old OVX rats. The long-term effect of ovariectomy on anxiety-like behavior in female rats of different

Cholecalciferol at dose of 5.0 mg/kg/day per se had a significant anxiolyticlike effect in the middle-aged OVX rats following long-term ovariectomy. On the contrary, cholecalciferol at doses of 1.0 and 2.5 mg/kg/day failed to induce any modifications of anxiety-like behavior in the middle-aged OVX rats with

long-term absence of estrogen. Combined application of vitamin D3 (1.0 mg/kg/day) with 17β-E2-induced synergic anxiolytic-like action of both preparations in the anxiety-related tasks of the old OVX rats. 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 post-ovariectomy period administered with cholecalciferol at doses 2.5 and 5.0 mg/kg/day in combination with 17β-E2 showed similar anxiety-like profile like the OVX rats given with 17β-E2. Thus, we did not observe any synergic anxiolytic-like effects of cholecalciferol at doses of 2.5 or 5.0 mg/kg in the old OVX rats given with 17β-E2. It might suppose that there are some concurrent relation between 17β-E2 and cholecalciferol at doses of 2.5 and 5.0 mg/kg/day. In fact, application of 17β-E2 interfere with anxiolytic-like action of cholecalciferol at doses of 2.5 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 middle-aged and old OVX rats after long-term ovariectomy. Thus, the present results suggest that anxiolyticlike effects of vitamin D3 are specific in the OVX rats of different age 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. Combined application of vitamin D3 (5.0 mg/kg/day) with low dose of 17β-E2 synergistically decreased anxiety-like profile of the middle-aged OVX rats. However, vitamin D3 at all doses significantly decreased grooming behavior in the middle-aged OVX rats

Unexpectedly, the middle-aged OVX rats treated with vitamin D3 at doses 1.0 and 2.5 mg/kg/day in combination with 17β-E2 have practically identical parameters of anxiety-like state as the OVX rats received only 17β-E2. Thus, this study failed to show any anxiolytic-like effects of vitamin D3 at doses of 1.0 or 2.5 mg/kg in the middle-aged OVX treated with 17β-E2. The present results might speculate that there exists some concurrent relation between 17β-E2 and vitamin D3 at doses of 2.5 and 5.0 mg/kg/day for the brain structures implicated in the mechanisms of anxiety. In fact, additional application of 17β-E2 to the middle-aged OVX rats interfered with anxiolytic-like action of vitamin D3 when it administered at doses 1.0 or

**50**

treated with 17β-E2.

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 [50, 51]. The active form of vitamin D is transferred to astrocytes where it can bind to VDR and initiate gene transcription or be inactivated when in excess [48, 49]. Alternatively, 1,25(OH)2D3 can induce autocrine or paracrine rapid non-genomic actions since all brain cell types express the other membrane receptor of VD [48]. 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 anxiety-like behavior. Regardless, it cannot presently exclude possible indirect effects of vitamin D3 on different neurotransmitter circuits. Further research is required to investigate the underlying mechanisms in its anxiolytic-like effects.

It is well-established both systemic effects of VD on calcium metabolism and neuroprotective effects of VD on the brain [52]. Low VD levels has been implicated in the pathophysiological mechanisms of cardiovascular diseases, depression, anxiety, cognitive disorders, obesity, metabolic syndrome, type 2 diabetes, various types of cancer and immune disorders [10, 52]. According to Gaugris and co-workers [53], the prevalence of low VD levels appears to be high in postmenopausal women. Additionally, the decline of estrogens after menopause decreases the activity of 1α-OHase, which results in lower synthesis of the active VD form [53, 54]. Application of vitamin D3 in specific periods of women's life, seems to be of great importance, because it may reduce the risk of affective-related diseases during menopausal period [55–57]. 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, and the impacts of such alterations elsewhere in the postmenopausal woman. VDR have been identified throughout the female reproductive tract [58, 59]. Some studies have demonstrated a direct modulation by VD of estradiol, estrone and progesterone production in human ovarian cells [60–62]. It could be supposed that estrogens and VD share similar targets of the brain to induce their anxiolytic-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 different age with long-term absence of ovarian 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 the interaction between VD and estradiol changes after menopause, and the implications of these changes elsewhere in the postmenopausal woman, is necessary for providing 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 [63]. It could be supposed, even though estrogens and cholecalciferol share similar targets on monoaminergic or another neurotransmitter system 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

**53**

*Behavioral Effects of Vitamin D3 at Estrogen Deficiency in Females of Different Age*

EPM and LDT in the old OVX female rats; the one specific dose of cholecalciferol (1.0 mg/kg/day) in the old OVX rats and another one dose of cholecalciferol (5.0 mg/kg/day) in the middle-aged OVX rats are able to induce synergic anxiolytic-like effect in the EPM and LDT; effects of cholecalciferol on anxiety-like behavior in the middle-aged and old OVX rats after long-term absence of estrogen are dependent from absence or presence of additional hormonal treatment as 17β-E2. Further investigations is to be addressed in relation to such issues: whether different effects of cholecalciferol on anxiety-like behavior are dependent from different age of rats, or whether different doses of cholecalciferol on anxiety-like behavior in OVX rats with different age rats might lead to negative versus positive effects. Further research, with properly 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, the mechanism by which cholecalciferol produces anxiolytic-like effect in the middle-aged and old OVX rats and the implications of this in 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 conduct detail experiments together with biochemical studies of these

The present data of the preclinical study indicates that chronic cholecalciferol at a dose of 5.0 or 1.0 mg/kg, s.c. treatment decreased anxiety-related behavior after impairment induced by long-term ovariectomy in the middle-aged and old female rats with long-term absence of estrogen, respectively. The data also indicate that the combination of cholecalciferol at a dose of 5.0 mg/kg and 17β-E2 is more effective than 17β-E2 alone in the middle-aged OVX rats inducing more synergic anxiolytic-like effects in the EPM and LDT. Moreover, a combination of cholecalciferol at a dose of 1.0 mg/kg s.c. and 17β-E2 is more effective than 17β-E2 alone 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 of chronic 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, these results suggest that 17β-E2 administration interfered with anxiolytic-like action of cholecalciferol administered alone at doses of 2.5 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 treatment in the

middle-aged and old subjects with long-term estrogen deficiency.

The reported study was funded by Russian Science Foundation (RSF) accord-

*DOI: http://dx.doi.org/10.5772/intechopen.82596*

subjects to verify the significance of this study.

**5. Conclusions**

**Acknowledgements**

**Conflict of interest**

ingly to the research project No. 16-15-10053.

The author declares no conflict of interest.

*Behavioral Effects of Vitamin D3 at Estrogen Deficiency in Females of Different Age DOI: http://dx.doi.org/10.5772/intechopen.82596*

EPM and LDT in the old OVX female rats; the one specific dose of cholecalciferol (1.0 mg/kg/day) in the old OVX rats and another one dose of cholecalciferol (5.0 mg/kg/day) in the middle-aged OVX rats are able to induce synergic anxiolytic-like effect in the EPM and LDT; effects of cholecalciferol on anxiety-like behavior in the middle-aged and old OVX rats after long-term absence of estrogen are dependent from absence or presence of additional hormonal treatment as 17β-E2. Further investigations is to be addressed in relation to such issues: whether different effects of cholecalciferol on anxiety-like behavior are dependent from different age of rats, or whether different doses of cholecalciferol on anxiety-like behavior in OVX rats with different age rats might lead to negative versus positive effects. Further research, with properly 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, the mechanism by which cholecalciferol produces anxiolytic-like effect in the middle-aged and old OVX rats and the implications of this in 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 conduct detail experiments together with biochemical studies of these subjects to verify the significance of this study.

## **5. Conclusions**

*Fads and Facts about Vitamin D*

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 [50, 51]. The active form of vitamin D is transferred to astrocytes where it can bind to VDR and initiate gene transcription or be inactivated when in excess [48, 49]. Alternatively, 1,25(OH)2D3 can induce autocrine or paracrine rapid non-genomic actions since all brain cell types express the other membrane receptor of VD [48]. 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 anxiety-like behavior. Regardless, it cannot presently exclude possible indirect effects of vitamin D3 on different neurotransmitter circuits. Further research is required to investigate the underlying mechanisms in its anxiolytic-like effects. It is well-established both systemic effects of VD on calcium metabolism and neuroprotective effects of VD on the brain [52]. Low VD levels has been implicated in the pathophysiological mechanisms of cardiovascular diseases, depression, anxiety, cognitive disorders, obesity, metabolic syndrome, type 2 diabetes, various types of cancer and immune disorders [10, 52]. According to Gaugris and co-workers [53], the prevalence of low VD levels appears to be high in postmenopausal women. Additionally, the decline of estrogens after menopause decreases the activity of 1α-OHase, which results in lower synthesis of the active VD form [53, 54]. Application of vitamin D3 in specific periods of women's life, seems to be of great importance, because it may reduce the risk of affective-related diseases during menopausal period [55–57]. 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, and the impacts of such alterations elsewhere in the postmenopausal woman. VDR have been identified throughout the female reproductive tract [58, 59]. Some studies have demonstrated a direct modulation by VD of estradiol, estrone and progesterone production in human ovarian cells [60–62]. It could be supposed that estrogens and VD share similar targets of the brain to induce their anxiolytic-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 different age with long-term absence of ovarian hormones. Moreover, it is completely needed to understand the precise mechanisms of how VD treatment alone or in a combination with 17β-estradiol

These points illustrate how the current state of VD treatment research is incomplete and in need of more intensive research. Working toward uncovering how the interaction between VD and estradiol changes after menopause, and the implications of these changes elsewhere in the postmenopausal woman, is necessary for providing 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 [63]. It could be supposed, even though estrogens and cholecalciferol share similar targets on monoaminergic or another neurotransmitter system 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

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

supplementation may affect women's anxiety-related state.

**52**

absence of ovarian hormones.

The present data of the preclinical study indicates that chronic cholecalciferol at a dose of 5.0 or 1.0 mg/kg, s.c. treatment decreased anxiety-related behavior after impairment induced by long-term ovariectomy in the middle-aged and old female rats with long-term absence of estrogen, respectively. The data also indicate that the combination of cholecalciferol at a dose of 5.0 mg/kg and 17β-E2 is more effective than 17β-E2 alone in the middle-aged OVX rats inducing more synergic anxiolytic-like effects in the EPM and LDT. Moreover, a combination of cholecalciferol at a dose of 1.0 mg/kg s.c. and 17β-E2 is more effective than 17β-E2 alone 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 of chronic 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, these results suggest that 17β-E2 administration interfered with anxiolytic-like action of cholecalciferol administered alone at doses of 2.5 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 treatment in the middle-aged and old subjects with long-term estrogen deficiency.

### **Acknowledgements**

The reported study was funded by Russian Science Foundation (RSF) accordingly to the research project No. 16-15-10053.
