The Effect Of Melatonin On HPA Axis And Neurogenesis In Young Mice Experiencing Maternal Separation

Part I The effect of meternal separation on HPA axis and neurogenesis in young mice Objective:To investigate the effect of meternal separation(MS)on the HPA axis and neurogenesis in young mice.Methods:12 pregnant mice were randomly divided into maternal separation groups(MS groups)and control group.The MS groups were divided into three subgroups including 7 d,14 d and 21 d.The neonatal mice and mothers were separated for 3 hours everyday in 7 d、14 d and 21 d subgroups(neonatal mice in 7 d group were separated from postanal day 1 to postanal day 7 for 3 hours eyery day,and so on).The control group always cage with mother without any processing after birth.All the mice were weaned in postanal day 23.Separating female and male mice,and male mice fed according to the principle of 5~7 in each cage were killed and taken the brain in postanal day 42.Brain tissue is routinely embedded in paraffin.The expression and distribution of glucocorticoid receptor(GR)and doublecortin(DCX)in hippocampus and prefrontal cortex of each group were detected by immunohistochemical staining and ImageJ image analysis technique.Results:GR immunoreactive products were brown granules and were evenly distributed in the cytoplasm and nucleus of neurons.GR immunoreactive cells were arranged in a strip inhippocampus and were widely distributed in the prefrontal cortex.Compared with the control group,the expression and the density of GR immunoreactive cells in the hippocampus and prefrontal cortex were decreased and there was a significant decreasing trend with the extension of the repetition time.The mean optical density in MS groups were significantly lower than that in control group(P <0.05).The difference between MS groups were statistically significant(P <0.05).DCX positive products were brown and expressed in the cytoplasm and protrusions of cells.DCX positive cells were spindle-shaped,with single leading protrusions and most of the neurites directed toward and extended into the molecular layer.The expression of DCX positive cells in each group was different in the hippocampal dentate gyrus subgranular zone of mice,but not in the prefrontal cortex.Compared with the control group,the DCX positive cells began to decrease significantly in 7 d group of MS and there was a significant decreasing trend with the extension of the repetition time.The mean optical density of DCX immunohistochemistry in MS groups were lower than that in control group(P <0.05).The difference was significant between MS groups(P <0.05).Conclusion:MS decreased the expression of GR in hippocampus and prefrontal cortex and reduced neurogenesis in the hippocampal dentate gyrus subgranular zone in young mice with the prolongation of separation time,the decreasing trend.So,adverse early life experience of MS decreased the expression of GR in hippocampus and prefrontal cortex,increased HPA axis responsiveness to stress and inhibited neurogenesis in young mice.It shows that the longer the separation time,the greater the effect on HPA axis and neurogenesis.Part Ⅱ The effect of melatonin on HPA axis and neurogenesis in young mice experiencing maternal separation Object:To investigate the effect of melatonin(MT)on HPA axis and neurogenesis in young mice experiencing maternal separation(MS).Method:Nine pregnant mice were randomly divided into maternal separation+melatonin intervention group(MS+MT group),maternal separation+saline group(MS+NS group)and control group.The newborn mice were separated from the female rat for 3 hours every day in the MS+MT group and the MS+NS group during the period of P1 ~ P21.The newborn mice in MS+MT group and MS+NS group were injected intraperitoneally with melatonin(10ml.kg-1.d-1)and normal saline(10ml.kg-1.d-1)at 17 o’clock from P 7 for 14 days respectively,and the newborn mice in the control group were fed with the mothers without any treatment.All the newborn mice were weaned in P23.Separating female mice from male mice,and the male mice fed according to the principle of 5~7 in each cage were killed and taken the brain in P42.The brains were routinely embedded in paraffin.The expression and distribution of GR and DCX in hippocampus and prefrontal cortex were detected by immunohistochemical staining and ImageJ image analysis.Result:GR immunoreactive products were brown granules,and were evenly distributed in the cytoplasm and nucleus.GR immunoreactive cells were arranged in a strip in hippocampus,and were widely distributed in the prefrontal cortex.GR positive cells in the hippocampus and prefrontal cortex had different degree expression in different groups.GR immunoreactive products were mainly located in the cytoplasm of hippocampus andprefrontal cortex in MS+NS group,and the mean optical density were lower than that in the control group(P<0.05).The expression of GR in the hippocampus and prefrontal cortex were significantly increased after MT intervention,and there was significant difference compared with that in the MS+NS group and the control group(P<0.05).DCX positive products were brown,and expressed in the cytoplasm and protrusions of the cells.DCX positive cells were spindle-shaped.They had a single leading protrusions,and most of the protrusions were toward and deeped into the molecular layer.DCX positive cells of dentate gyrus in hippocampus were expressed in different levels in each groups,and no expression was found in the prefrontal cortex.Compared with the control group,the number of DCX immunoreactive cells in the hippocampal dentate gyrus in MS+NS group were significantly lower than that in the control group,and the average optical density were significantly lower than that in the control group(P<0.05).The positive expression of DCX in hippocampal dentate gyru were significantly increased after MT intervention,and there was significant difference compared with the MS+NS and the control group(P<0.05).Conclusion:MT can increase the expression of GR in the hippocampus and prefrontal cortex,and enhance neurogenesis.MT participates in the body stress response,inhibite the activity of HPA axis,and improve the level of hippocampal neurogenesis in young mice experiencing maternal separation.MT can reverse permanent damage of brain in mice which experience adverse early life event.