Effects And Mechanism Of Early Life Stress On The Learning And Remembering Functions Of Hippocampus

BackgroundEarly life stress is associated with changes in behavior, physiological responses tostress and susceptibility to psychopathology in adults. Studies on people and animals haveshown that brain is particularly sensitive to stress during early childhood. Stressexperienced during this period will lead to long-lasting alterations on neural development,which has relationship with mental disorders in adulthood. A widely-used animal model forearly life stress is maternal separation (MS), which is thought to affect development andcause alterations in neuroendocrine stress reactivity and emotionality lasting into adulthood.It has been widely used to investigate neurobiological changes associated with the etiologyand vulnerability to psychiatric diseases. The hippocampus has been considered to beimplicated in many of the functions that are altered by early adverse experience. First, thehippocampus mediates negative feedback of the HPA axis. Lesions of the hippocampus orthe fornix, its efferent hypothalamic projection, result in a slower restoration of basalmeasures of the HPA axis after stress. Second, the hippocampus is an important structure inregulating behavioral measures of anxiety. Rats with hippocampal lesions appear to be lessanxious when behaviors such as exploration of a new environment, social interaction andneophagia are measured. Third, the hathippocampus has a well-described role in spatialnavigation learning. Based on the fact that all these functions in the adult are influenced bymaternal separation during early life, abnormal hippocampal development may underliesome, if not all, of these aspects.Recently,studies on effect and mechanism of MD on hippocampus structure andfunction are not well detailed and completed. Thus many respects remain unclear. On thebasis of previous studies, we intend to investigate the effects and mechanism of MD on thelearning and remembering functions of hippocampus, especially when considering MDparadigms and animal genders. Objective1. To investigate the effects of MD on neurogenesis of hippocampus in both male andfemale offspring.2. To examine the effects of different MD paradigms on the expression ofhippocampal glucocorticoids receptors,calretinin and calbindin-D28k in male and femaleadolescent rats.3. To investigate the effects of MD on the stress response of hippocampus in bothmale and female adults.Methods(1) To build three different MD models. MD: All pups were separated from their damat9:00from PND2to PND14for3hours. MD4: All pups were separated from their dam atPND4for24hours (from9:00a day to9:00the next day). MD9: All pups were separatedfrom their dam at PND9for24hours (from9:00a day to9:00the next day).(2)To comparethe body weight of rats from MD group and CON group on PND21.(3) To investigte theneurogenesis of hippocampus in rats from MD group and CON group on PND21.Immunohistochemistry for ki-67was used to assess proliferation, and doublecortin to assessthe number of immature neurons.(4) To compare the exploratory behavior and generalactivity of rats from MD group and CON group by using the open field test on PND21.(5)To investigate the expression of hippocampal GR and GR mRNA in rats from MD, MD4,MD9and CON groups by using western blot and QT-PCR on PND21.(6) To investigatethe expression of hippocampal CALR and CALR mRNA in rats from MD, MD4, MD9andCON groups by using western blot and QT-PCR on PND21.(7) To investigate theexpression of hippocampal CALB and CALB mRNA in rats from MD, MD4, MD9andCON groups by using western blot and QT-PCR on PND21.(8) To investigate anxiety-likebehaviors in rats from MD, MD4, MD9and CON groups by using the elevated plus mazetest on PND21.(9) To investigate spatial learning and memory in rats from MD group andCON group by using the Morris water maze test on PND25.(10) To investigate theexpression of hippocampal CALR in rats from MD group and CON group on PND35,including those who suffered from MWM test and did not suffered from MWM test.(11) Toinvestigate the expression of hippocampal CALB in rats from MD group and CON groupon PND35, including those who suffered from MWM test and those who did not. Results1. No significant difference was found on body weight between males and females onPND21. Both males and females from MD group had lower weight than those from CONgroup (p<0.001).2. On PND21, males have more DCX+cells than females (p<0.001). For males, ratsfrom MD group had much more DCX+cells than those from CON group (p<0.001). Forfemales, rats from MD group had less DCX+cells than those from CON group (p<0.001).3. No significant difference was found on ki67+cells between males and females onPND21. For males, rats from MD group had less ki67+cells than those from CON group(p<0.001). While there was no difference on ki67+cells between females from MD groupand CON group.4. In open field test, males moved morel distance and spent more time moving thanfemales (p<0.05). For males, rats from MD group moved less distance and spent less timemoving than those from CON group (p<0.05). No difference was found in females.5. A significantly higher basic expression of GR was observed in female than malerats [F(1,47)=63.29, p<0.001]. Maternal deprivation from males with the following twoprotocols, i.e. repeated3h separations from PND2to14in MD group, and a single24hseparation on PND9in MD9group caused a significant upregulation of GR (p<0.05andp<0.001, respectively). However, no significant change in GR expression was foundbetween the four groups of female rats upon maternal deprivation under all protocols.6. Post hoc comparison revealed that the CALR level of male rats from both MDgroup and from MD9group was significantly higher than that of control rats, whereas therewas no significant difference between CON group and MD4group. The CALR of male ratsfrom MD group was higher than that of males from MD9group. For the female rats, thedensity of CALR from MD, MD4and MD9group all increased significantly compared withthe control rats. While the CALR level of female rats from the MD group was higher thanthat of females from the MD4group, no significant difference was observed betweenfemale MD4rats and female MD9rats.7. A dramatically higher CALB expression was also observed in female than maleadolescent rats [F(1,47)=75.67, p<0.001] on PND21. For male rats, the density of CALBfrom both MD group and MD9group increased prominently compared with the control rats, whereas the CALB density from MD4group decreased significantly. We also detected ahigher CALB expression in MD than MD9males. Among female groups, the CALB densityfrom MD group was significantly higher than that of the control rats. In contrast, MD4andMD9rats exhibited markedly decreased density of CALB, among which the CALB level inMD9rats was even lower.8. Two-way ANOVA revealed that compared with female rats, male rats spent moretime on the open arms and stretched more times in300seconds. For males,(a) rats bothfrom MD group and MD9group had higher percent of time spent on the open arms, reared,head-dipped and stretched more times at the EPM (elevated plus maze) than rats from CONgroup;(b) rats from MD4group had no significant difference with rats from CON group.For females,(a) rats both from MD group and MD9group reared more times at the EPMthan rats from CON group;(b) no significant difference was found in percent of time spenton the open arms and frequencies of head dips and stretched-attend in300seconds amongthe four groups (CON, MD, MD4and MD9).9. In Morris water maze test: For rats from CON group, females had more latency onthe first day than males (p<0.01), and no difference was found on the rest four days. Forrats from MD group, there was no difference between males and females. For males, ratsfrom MD group spent more time in finding the escape table than those from CON groupboth on the first day (p<0.05) and the last day (p<0.01). For females, rats from MD grouphad longer latency on the first day than those from CON group (p<0.01), and no differencewas found on the rest four days.10. A significant difference on the expression of CALR was observed in female thanmale rats (p<0.05). For males: rats who suffered from MD and MWM test had lower CALRlevel than those only suffered from MD (p<0.05); rats suffered from MD and MWM testhad lower CALR level than those from CON group (p<0.05); rats who suffered from MDand MWM test had lower CALR level than those just suffered from MWM test (p<0.01).For females: rats just suffered from MD had higher CALR level than those from CONgroup (p<0.05); rats which suffered from MD and MWM test had higher CALR level thanthose just suffered from MD (p<0.05); rats which suffered from MD and MWM test hadhigher CALR level than those just suffered from MWM test (p<0.05); rats which sufferedfrom MD and MWM test had higher CALR level than those from CON group (p<0.001). 11. A significant difference on the expression of CALR was observed in female thanmale rats (p<0.001). For males: rats just suffered from the MWM test had higher CALBlevel than those from CON group (p<0.001); rats just suffered from the MWM test hadhigher CALB level than those just suffered from MD (p<0.001); rats just suffered from theMWM test had higher CALB level than those suffered from MD and MWM test (p<0.001);rats suffered from MD and MWM test had lower CALB level than those from CON group(p<0.001); rats suffered from MD and MWM test had lower CALB level than those justsuffered from MD (p<0.001). For females: rats just suffered from the MWM test had higherCALB level than those from CON group (p<0.01); rats suffered from MD and MWM testhad higher CALB level than those from CON group (p<0.01); rats just suffered from MDhad higher CALB level than those from CON group (p<0.001).Conclusion1. Maternal deprivation (3hours, from PND2to PND14) alters hippocampusneurogenesis in a sex-dependent manner.2. Different MD paradigms have different sex-related effects on the expression ofhippocampal GRs, CALR and CALB.3. Maternal deprivation hampers adult spatial learning and memory in some extent. Itwill lead to the increase of anxious-like behaviors and decrease of exploratory behaviors.4. Female rats who suffered from maternal deprivation are much more sensitive tostress during adulthood, while male rats seem adaptive.