Maternal Separation Increased Male Off-springs' Depression-and Anxiety-like Behaviors When Exposed To Chronic Mild Stress And The Intervention Effect Of WNT Agonist

Objective: Depression is the most prevalent and debilitating psychiatric disorder,with a proportion of patients have treat-resistant to classic antidepressants.Recent researches have shown that the WNT signaling pathway in the nucleus plays a key role in the pathogenesis of depression.In this study,we focused on ?-catenin,a key molecule of the WNT signaling pathway,and its downstream transcription factorTCF4,to investigate the changes in WNT pathway activation when off-springs exposed to chronic stressors.In early life,the interaction of parents and children is critical to individual neurodevelopment and it can influence the adulthood responsiveness to environmental stress.Our study investigated the combined effects of two rodent models: Maternal separation model(MS)and Chronic mild stress model(CMS).To demonstrated the effects of maternal separation,we compared the differences between the behavioural results and biochemical indicators after exposed to single CMS model or combined stress model.ES and WNT agonist can further investigate the clinical significance of WNT signaling pathway and provide a new target for the treatment of depression.Methods: Ten males and twenty female mice were selected as breeders,then 63 male off-springs were included to establish the maternal separation model(MS)and the adult chronic mild stress(CMS).Sixty newly born male mice were randomly divided into MS group and control group.The methods of establishing maternal separation model were as follows: on postnatal day(PND)2-PND9,separation for 3 h per day;on PND 10-PND 16,separation for 4 h per day;on PND 17-PND 21,separation for 5 h per day.The MS started at 9 A.M,during this period,separated pups are isolated from each other,and mice of control group were raised normally.All off-springs received chronic mild stress in adulthood which totally lasted 3 weeks(PND60-PND80).The process of CMS includes food deprivation(12 h),water deprivation(12 h),empty cage(12 h),wet bedding(12 h),cage tilt(12 h),flashing light(12 h),reversed light/dark cycle(24h).Two stress models were randomly divided into three groups for drug intervention which last 21 days.ES: 10 mg/kg/d,WNT agonist: 10 mg/kg/d,and equal volume of normal saline: 10 mg/kg/d,all solutions were intraperitoneally injected.At this time,stressed off-springs were randomly divided into seven groups according to MS and drug administration.When the stressors were completed,all mice was tested by saccharine preference test,open field test,high plus maze and forced swimming test,to value the depression-and anxiety-like behaviour;and the Western Blot was used to detected the level of ?-catenin and its downstream transcription factor-TCF4 in mice hippocampus(HIP).Results: 1.Saccharine preference test and forced swimming test were used to evaluate the depression-like behavior.Statistical analysis showed that MS reduced the saccharine intake and increased the immobility swimming time when mice exposed to chronic stress and normal saline administration when compared to the blank group,indicating that mice which were exposed to childhood MS showed increased levels of depression when applied to adult chronic stress.Drug intervention(ES and WNT agonist)resulted in increased saccharine intake and deduced immobility time of stressed mice,and both drug interventions alleviated depressive state.In mice exposed to MS,the saccharine preference index with intervention of WNT agonist was lower than that of control group,and the immobility time with ES intervention was lower than that of control group;Indicating that MS reduced the intervention effects of ES and WNT agonist.2.Statistical analysis of the time and entries of mice appearing in the central zone of open field test showed that there was no post hoc test,so there was no significant difference in behavioral results among the six groups.3.The results of statistical analysis of the time and entries of mice entering open arms on the elevated plus maze showed that,when compared to blank group,mice exposed to MS showed a decrease in the entries of open arms and exploration time of open time,it was speculated that MS increased anxiety levels when adult mice exposed to chronic stress.After intervention of ES and WNT agonist,stressed mice exhibited an increase in time and entries of open arms,indicating that drug administration alleviated anxiety-like behaviors of mice and there was no significant difference between MS and anti-anxiety effect of drugs.4.Statistical analysis of WB found that compared to the control group,mice which exposed to MS showed reduced levels of ?-catenin and TCF4 after chronic stressors,indicating that MS in early life inhibited the expression of WNT signaling pathway.Drug intervention of ES and WNT agonist reversed the lower levels of ?-catenin and TCF4 in mice hippocampus,showed that drug intervention can increase the activity of the WNT pathway.In the meanwhile,MS deduced the increase of TCF4 in hippocampus,showed that MS could affect the therapeutic effect of WNT agonist.Conclusion: Maternal separation(MS)can affect the sensitivity of off-springs to adult stress,and increase the depression-and anxiety-like behavior when exposed to chronic mild stress;levels of ?-catenin and TCF4 in hippocampus also showed corresponding decrease.ES and WNT agonist can activate the expression of inhibited WNT pathway,and play an alleviating role in depression-and anxiety-like behavior.Regulating the WNT pathway may be the key to alleviate stress-induced behavior,and the WNT pathway can be used as a new target and new idea for antidepressant treatment.