1. The Mechanisms Involved In Depression-like Behavior Induced By Estrogen Deficiency2. The Effects Of Prenatal Glucocorticoids Exposure On Depression-like Behavior In Offspring

Depression is a global mental health problem, which is characteristiced with high incidence, high mortality and high morbidity. Epidemiological surveys show that, many factors such as low estrogen level and adverse intrauterine environment can increase the incidence of depression significantly. Animal experiments also show that, estrogen deficiency can increase depression-like behavior, while giving estrogen replacement therapy can alleviate this phenomenon. Meanwhile, adult offspring emerge increased depression-like behavior if stimulated by stress or exposed to excess glucocorticoids (GCs) during pregnancy. However, the mechanisms of increased depressive-like behavior induce by estrogen deficiency and adverse intrauterine environment is still unclear.Therefore, in the first part of this subject, we builded ovariectomized rat model (Ovx rats model) to simulate the low estrogen level of body, then we used sucrose consumption test and open-field test to observe the depression-like behavior changes in rats. On this basis, we used Real-time PCR, Western blot and Elisa method to study the expression of5-HT, inflammatory factors in brain of Ovx rats. Then we analysised the correlation between these indexes and the content of estrogen in serum, clarified the internal mechanism of depressive behavior due to estrogen deficiency in rats.In the second part of this project, firstly, we constructed a prenatal GCs exposure rat model in late pregnancy and used sucrose consumption test, open-field test to observe depression-like behavior on adult offspring rats. On this basis, we used sucrose consumption test, open-field test and tail suspension test to observate wether depressive-like behavior induced by prenatal GCs exposure in late pregnancy can inherit to the second generation or not. Then we used real-time PCR and Western blot method to study CRH and its receptors expression in offspring rats which exposure to GCs in prenatal stage and clarified the internal mechanisms of this kind of depressive behavior.Main results:Part one:The mechanisms involved in depression-like behavior induced by estrogen deficiency.We builded three group rats:Sham group, Ovx group and Ovx+E2group, then weighed body weight, uterine weight and detected serum estradiol levels of these groups. The experimental results showed that, compared with Sham group, the weight of Ovx group rats was significantly increased, uterine weight and serum E2levels were significantly decreased. Given E2therapy for12weeks can significantly reversed all of the above phenomena. These results suggested that the ovx rat model we builded was success.1. Depression-like behavior increased induced by estrogen deficiency in rats.We used sucrose consumption test and open-field test to observe the animal behavior of these three groups. Experimental results showed that, compared with the Sham group, Ovx rats showed significantly reduced24hours sucrose consumption percentage, decreased the number of poking into holes, rearing times and the distance traveled in center in OFT. Given E2therapy for12weeks can significantly reverse all above phenomenon. The results suggested that depression-like behavior of ovariectomized rats is increased, given E2can reduce the depression-like behavior alternatively.2. The mechanisms involved in depression-like behavior induced by estrogen deficiency.(1) First we used Elisa method to detect the changes of5-HT, IDO in hippocampus and prefrontal cortex of three groups. The results showed that, compared with Sham group5-HT content in hippocampus of Ovx rats was significantly decreased, while IDO was significantly increased, estrogen was able to reverse these effects. The level of5-HT and IDO of the three groups did not change significantly in prefrontal cortex. Statistical correlation analysis show that the content of5-HT in hippocampus and level of E2in serum were positively correlated, the content of IDO in hippocampus and level of E2in serum or5-HT level in hippocampus are negatively correlated. These results indicated that the estrogen deficency can lead to increasing of IDO content in hippocampus, and then cause tryptophan metabolism abnormal which results in decreasing of5-HT content in hippocampus.(2) Furthermore, we used Elisa method to detect proinflammatory cytokines IFN-y, IL-6and TNF-α expression in hippocampus and prefrontal cortex. The results showed that, IFN-y, IL-6significantly increased in hippocampus of Ovx rats, TNF-a did not change significantly. Administration of exogenous E2can also reverse the upregulation of IFN-y and IL-6. The expression of these proinflammatory cytokine did not change significantly in cortex of the three groups. Correlation analysis showed that IFN-y, IL-6levels in hippocampus and serum E2concentration is negatively correlated.(3) We used Real-time-PCR, Western blot to detect TLR-4mRNA and protein, pNF-κB, NF-κB protein expression in hippocampus and prefrontal cortex. The results indicated that, the expression of TLR-4mRNA, protein and phosphorylated NF-κB (p65 subunit) is significantly up-regulated in hippocampus of Ovx group when compared with Sham group, exogenous E2can reverse this effect. TLR-4mRNA and protein, phosphorylated NF-κB (p65subunit) protein did not changed significantly in prefrontal cortex.(4) We used Elisa method to detected corticosterone and ACTH changes in serum of three group rats. The results show that ACTH and corticosterone in Ovx rats did not change significantly when compared with the other two groups. These results suggest that the mechanism of increased depression-like behavior caused by estrogen deficiency does not involve excessive activation of the HPA axis.All of the above results suggest that estrogen deficiency increases depression-like behavior may be related to the activation of TLR-4and pNF-KB in hippocampus which leading to increase the expression of IFN-y, IL-6, IDO and decrease of5-HT.Part two:The effects of prenatal glucocorticoid exposure on depression-like behavior in offspring.1. Depression-like behavior increased induced by prenatal GCs exposure.(1) We weighed body weight after born at1day,1week,3weeks,6weeks,9weeks respective. We found that rat body weight of prenatal exposure to GCs is not significantly lower than control groupat1day. Body weight of prenatal exposure GCs offspring male rat appear lighter than the control group from1week, female rats appear lighter in weight than the control group from3weeks. Body weight of prenatal exposure to GCs offspring rats was still significantly lower than the control group until9weeks.(2) We used sucrose consumption test and open field test to observe depression-like behavior in rats which expose to GCs in prenatal stage. Results showed that, compared with control group, offspring exposed to GCs showed significantly reduced sucrose consumption percentage in24hours, the number of poking into holes, rearing times and the distance traveled in center in OFT. All of the above behavior experimental results show that depression-like behavior in rats which expose to GCs in prenatal stage is increased.2. The impact of prenatal exposure to GCs on depression-like behavior on second generation.We used sucrose consumption test, open field test and tail suspension test to observe the depression-like behavior in second generation and determine wether the impact of prenatal exposure to glucocorticoid on the depression-like behavior can be inherited to next generation or not. Our results show that: (1) Tail suspension test showed that, male offspring of second generation who come from Con/Dex increased immobility time. This result indicated that Con/Dex second generation rats reduced its activity.(2) Open field test showed that, offspring of second generation who come from Con/Dex decreased of central distance and increased of immobility time. These results indicated that rats of second generation reduced its exploratory behavior.(3) Sucrose consumption test showed that, female offspring of second generation who come from Con/Dex and Dex/Dex group showed decreased of sucrose consumption percentage in24hours. Female decreased percentage is more obvious than male. This results indicated that second generation rats emerge anhedonia.These behavior results suggest that the increase depressive-like behavior induced by exposure to GCs in late pregnancy can be inherit to next generation, depressive-like behavior may be through "Motherhood" genetic to the next generation.3. Imprinting effect of prenatal GCs exposure on the expression of CRH and its receptor in hippocampus(1) We used Real-time PCR and Western blot method to detect CRH, CRHR), CRHR2mRNA and protein level changes in hippocampus and cortex of offspring rats. We found that prenatal exposure to GCs can significantly increased CRH and CRHR1mRNA expression in hippocampus, while CRHR2mRNA expression dose not change significant. Meanwhile, prenatal exposure to GCs significantly increased rat hippocampal CRHR1protein expression when compared with the control group. CRHR2protein expression dose not change significantly.(2) Next, We used BSP method to examine the methylation status of CpG islands in CRH and CRHR1promoter region of female hippocampus. The results showed that CpG methylation level of CRHR1promoter region in DEX group is significantly lower than that in the control group, while CpG methylation level of CRH promoter region in DEX group has no significantly with that in the control group. These results suggest that the increase of CRHR1expression is regulated by the methylation of CpG islands in CRHR1promoter region.Conclusions:1. Estrogen deficiency can lead to the activation of inflammatory reaction in hippocampus and effect tryptophan metabolism to decrease the content of5-HT in hippocampus which resulting in animal depression-like behavior. 2. Prenatal GCs exposure can increase depression-like behavior of adult offspring, and this increase of depressive-like behavior can be inherited to the next generation. Depression-like behavior of offspring is related to the increase of CRHR1expression which may be regulated by the methylation of CpG in CRHR1promoter region.