Mechanism Of Hippocampus Impairment And Depressive-like Behavior In Male Offspring Induced By Chronic Stress During Pregnancy

Objective To explore the mechanism of hippocampus impairment and depressive-Like behavior in male offspring induced by chronic stress during pregnancy。Experiment 1Methods:A total of 20 adult male and 40 female C57/BL6J mice were used as parent mice in the experiments for studying,each cage was housed with female and male rats at a ratio of 2:1 at 7:00pm.Vaginal plugs were investigated on the following morning 7:00am.When the vaginal suppository was found,the mice were designated as day 1 of pregnancy and raised separately.After a week,20 pregnant mice were selected for chronic stress during pregnancy（CSDP）.Stressors consisted of tail pinch（2 cm apart from the end of the tail）for 5 min,restraint for 2 h,cold swimming at 4-8℃ for 5 min and then towel-drying,warm swimming at 28-35℃ for 15 min and then towel-drying,reverse day and night cycles,water deprivation for 24 h,food deprivation for 24 h.Since gestational day 7,one of these stressors was applied daily to each rat in a random order until delivery.At postnatal day 21,male offspring mice were randomly assigned to NC+NC group,NC+COS group,CSDP+NC and CSDP+COS group,each group n=8.Male offspring mice in NC+COS group and CSDP+COS group were subjected to chronic stress in the same way as the pregnant mice during the postnatal day 21-35.We examined the depression related indices in male offspring mice through the FST（forced swimming test）,EPM（elevated plus maze）and OFT（open field test）.Detection of CRH concentrations in serum by ELISA.The Golgi Cox staining was used to survey the dendrite morphological changes of hippocampal neurons.Neuronal apoptosis in the hippocampal CA3 regions was detected by using TUNEL staining.P-mTOR（Ser2448）,mTOR were detected by western blotting to calculate mTOR kinase activity.The mRNA levels of mTOR were measured by RT-PCR.Results:Behavioral results showed that:compared with NC+NC group,the swimming immobility time of CSDP+NC group was appreciably increased（P<0.01）,the moving speed of CSDP+NC group were appreciable reduction（P<0.05）and the open arm entry rate of CSDP+NC group were significantly decreased（P<0.05）,the open arm entry rate of CSDP+COS group were significantly increased（P<0.001）;Compared with the CSDP+NC group,the swimming immobility time of CSDP+COS group was appreciably decreased（P<0.01）,the open arm entry rate of CSDP+COS group and NC+COS group were significantly increased（P<0.001）.Furthermore,compared with NC+COS group,the open arm entry rate of CSDP+COS group was appreciably increased（P<0.01）.None of the extra observed differences between the groups was statistically significant.ELISA results showed that:compared with NC+NC group、NC+COS group and CSDP+COS group,the concentrations of CRH in CSDP+NC group was elevated but not achieving significance.Golgi Cox staining showed that:compared with NC+NC group,the CSDP+NC group showed a significantly decreased number of dendritic intersections in the 30-90μm concentric circle（30μm:P<0.01;40μm:P<0.001;50μm:P<0.001;60μm:P<0.001;70μm:P<0.01;80μm:P<0.05;90μm:P<0.05）,the CSDP+COS group showed a significantly decreased number of dendritic intersections only at the 40μm and 90μm concentric circle（40μm:P<0.05;90μm:P<0.05）.Compared with CSDP+NC group,the number of dendritic intersections in CSDP+COS group was significant increased（30μm:P<0.05;40μm:P<0.01;50μm:P<0.05;60μm:P<0.05;70μm:P<0.05）.Between NC+COS group and CSDP+NC group,NC+COS group showed a significantly grow number of dendritic intersections in the 40-90μm concentric circle（40μm:P<0.01;50μm:P<0.01;60μm:P<0.01;70μm:P<0.01;80μm:P<0.05;90μm:P<0.05）.TUNEL staining showed that:compared with NC+NC group,the apoptotic index in CSDP+NC group were significant increased（P<0.001）;compared with CSDP+NC group,the apoptotic index in CSDP+COS group were significant decreased（P<0.001）.Additionally,the index of NC+COS group was much lower than the CSDP+NC group,and there was extremely significant difference（P<0.001）.No statistically significant difference of the apoptosis index was found between NC+NC,NC+COS and CSDP+COS group.WB and RT-PCR results showed that:compared with NC+NC group,the mTOR activity of CSDP+NC group was significant decreased（P<0.01）,the mTOR activity of CSDP+COS group was significant increased（P<0.01）,the mTOR activity of NC+COS group was significant decreased（P<0.05）;compared with CSDP+NC group,the mTOR activity of CSDP+COS group was extremely significant increased（P<0.001）.Furthermore,the mTOR activity of NC+COS group compared with the CSDP+NC group do not have any significant difference;the mTOR activity of NC+COS group compared with the CSDP+COS group showed a significantly decrease（P<0.001）.The results of RT-PCR were in agreement with WB,showing the same trends,but with no statistically significant differences.Experiment 2:Methods:Establishing conditional CRHR1 knockout mice by CRISPR/Cas9 and LoxP/Cre system.According to the results of gene identification,the male offspring were divided into the normal wild type（N+WT）group,the normal CRHR1 gene knockout（N+KN）group,the chronic stress during pregnancy+wild type（CSDP+WT）group and the chronic stress during pregnancy+CRHR1 gene knockout（CSDP+KN）group.Chronic unpredictable stress was initiated on day 7 of gestation until delivery day（Seven different types of stressors were used,and the random number table method was used to select the type of stress in each day.The same as in Experiment 1）.Behavioral testing of offspring depression,began on day 40.We used the SPT,OFT,FST and TST to assess depressive-like behavior in the male offspring mice.Detection of CRH concentrations of offspring was conducted by ELISA.The Golgi Cox staining was used to survey the dendrite morphological changes of hippocampal neurons.Neuronal apoptosis in the hippocampal CA3 regions was detected by using TUNEL staining.P-mTOR（Ser2448）,mTOR were detected by western blotting to calculate mTOR kinase activity.P-AKT andβ-actin were detected by western blotting to calculate the expression of p-AKT.Results:Behavioral results showed that:compared with N+WT group,the sucrose preference rate of CSDP+WT group was appreciably decreased（P<0.001）,the number of entries in the central zone of CSDP+WT group were significantly decreased（P<0.001）,the swimming immobility time of CSDP+WT group was appreciably increased（P<0.01）,the immobility time in the tail suspension test of CSDP+WT group were appreciable elevated（P<0.001）;compared with CSDP+WT group,the sucrose preference rate of CSDP+KN group was appreciably increased（P<0.001）,the number of entries in the central zone of CSDP+KN group were significantly increased（P<0.01）,the swimming immobility time of CSDP+KN group was appreciably decreased（P<0.01）,the immobility time in the tail suspension test of CSDP+KN group were appreciable decreased（P<0.05）;Behavioral indicators like:the sucrose preference rate,the number of entries in the central zone,the swimming immobility time,the immobility time in the tail suspension test,those indicators do not have statistical differences between N+WT group,N+KN group and CSDP+KN group.ELISA results showed that:compared with N+WT group,the CRH level of CSDP+WT group was appreciably increased（P<0.01）,the CRH level of CSDP+KN group were significantly increased（P<0.05）;compared with N+KN group,the CRH level of CSDP+WT group was appreciably increased（P<0.001）,the CRH level of CSDP+KN group were significantly increased（P<0.01）;the CRH level do not have statistical differences between N+WT group and N+KN group;the CRH level do not have statistical differences between CSDP+WT group and CSDP+KN group.Golgi Cox staining showed that:compared with N+WT group,the CSDP+WT group showed a significantly decreased number of dendritic intersections in the 20-90μm concentric circle（20μm:P<0.01;30μm:P<0.001;40μm:P<0.001;50μm:P<0.001;60μm:P<0.001;70μm:P<0.001;80μm:P<0.05;90μm:P<0.05）;Compared with CSDP+WT group,the number of dendritic intersections in CSDP+KN group was significant increased（20μm:P<0.05;30μm:P<0.01;40μm:P<0.001;50μm:P<0.001;60μm:P<0.05;70μm:P<0.001;80μm:P<0.01）;There were no significant differences among the three groups（N+WT,N+KN,CSDP+KN）.TUNEL staining showed that:compared with N+WT group,the apoptotic index in CSDP+WT group were significant increased（P<0.001）;compared with CSDP+WT group,the apoptotic index in CSDP+KN group were significant decreased（P<0.001）;No statistically significant difference of the apoptosis index was found between N+WT,N+KN and CSDP+KN group.WB results showed that:compared with N+WT group,the expression of p-AKT of CSDP+NC group was significant decreased（P<0.01）,the mTOR activity of CSDP+NC group was significant decreased（P<0.01）;compared with CSDP+WT group,the expression of p-AKT of CSDP+NC group was significant increased（P<0.01）,the mTOR activity of CSDP+COS group was extremely significant increased（P<0.001）.There was no difference in mTOR activity or p-AKT expression between N+WT,N+KN and CSDP+KN group.Furthermore,compared with N+KN group,the expression of p-AKT of CSDP+WT group was significant decreased（P<0.001）,the mTOR activity of CSDP+WT group was significant decreased（P<0.05）.Conclusion:CSDP induced depressive symptoms in male offspring accompanied by neuronal damage in the hippocampus and reduced mTOR activity.The COS ameliorated the depression-like behavior and pathological damage of hippocampus in male offspring male.The CSDP and COS has an antagonistic role in inducing male offspring depression.The COS ameliorated the depression-like behavior and pathological damage in male offspring male caused by CSDP.CSDP induced depressive symptoms in male offspring accompanied by neuronal damage in the hippocampus.CSDP might through up-regulating the p-AKT/mTOR signaling pathway by the elevated CRH which by acting on hippocampal CRHR1 damage the hippocampal structure and function,then induce the depression-like behavior and pathological damage in hippocampus in offspring.