| Objective To investigate the effects of chronic stress on emotion,cognitive function and hippocampal synaptic plasticity changes in childhood rat and the mechanism of RhoA-ROCK2 pathway involved in this process.Methods(1)3-week-old SPF healthy SD rats,26 in each group,were randomly divided into male group rearing(GR-M),female group rearing(GR-F),male separating rearing(SR-M)and female separating rearing(SR-F)groups;the juvenile chronic stress model was established by single-cage feeding;(2)The changes of cognitive function was detected by Intelli Cage behavior detection system(TSE),spatial learning and memory was detected by Morris water maze(MWM),the desire to explore new objects was detected by novel object recognition(NOR),,and elevated plus maze(EPM)was used to detect the emotional changes of rats.(3)The expression of FOSB/ΔFOSB were detected by immunohistochemistry(IHC)and Western Blot(WB)to observe the activation of brain regions in rats chronic stress;(4)Golgi staining was used to detect the changes of dendritic complexity,dendritic spine density,and transmission electron microscopy(TEM)was used to observe synaptic ultrastructure of hippocampal neurons;(5)Full-length transcriptome sequencing(Iso-Seq)technique was used to screen the differentially expressed genes which involved in the plasticity change in dendritic spines of hippocampal neurons,and the activation of the differentially expressed gene RhoA was verified by quantitative PCR(q-PCR),immunofluorescence staining(IF)and WB technology;(6)The molecules of RhoA-ROCK2 pathway p-ROCK2,p-LIMK and p-cofilin were detected by IF and WB techniques,the changes of F-actin,G-actin and ratio of F-actin/G-actin were analyzed.Results(1)Results of cognitive function TSE results: There was no difference in simple learning between GR and SR groups,but the spatial learning,punitive learning and memory decreased,and recognition memory impaired.MWM results: Compared with GR-F group,SR-F rats’ target quadrant movement distance decreased(P < 0.01).Compared with GR group,the target quadrant residence time of male and female rats in SR group decreased(P < 0.05).Compared with GR group,the frequency of male and female rats crossing the platform in SR group decreased(P < 0.05,P < 0.01).NOR results: the contact time to the new object was significantly longer than that of the old object in GR group(P < 0.01,P < 0.05),the contact time to the new object was significantly less than that of the old object of male and female rats in SR group(both P < 0.05).EPM results: Compared with GR group,the number and the residence time of rats entering the open arms in SR group were significantly less(both P <0.01),suggesting that the spatial learning and memory of rats in GR group decreased,the interest in exploring new objects decreased,and the anxiety-like emotion increased.(2)FOSB/ΔFOSB results: The results of IHC showed that FOSB immunopositive staining was observed in the cytoplasm and membrane of hippocampal neurons in all groups,and the immunopositive staining in the CA1,CA3 and DG regions in SR group was significantly deeper than that in GR group.The results of WB showed that the expression of ΔFOSB in the hippocampus in SR group was higher than that in GR group(both P < 0.01),suggesting that hippocampal neurons were activated.(3)Nissl staining results: Compared with GR group,Nissl staining in CA1,CA3 and DG regions in SR group was deepened,and the arrangement of pyramidal neurons in CA3 region were dispersed.(4)Golgi staining results: Compared with GR group,the number of dendrites in CA1 and DG regions of in SR group decreased(SR-F: P < 0.05,SR-M: P < 0.01,P < 0.05),dendrite length decreased in CA1 region(both P < 0.05),total dendrite spine density decreased in CA3 and DG region(SR-F: P < 0.01,SR-M: P < 0.05),compared with GR-F group,the total spine density in CA1 region of SR-F group was decreased(P < 0.05).In CA1,CA3 and DG region,mature dendrite spine density decreased(SR-F: P < 0.05,P < 0.01,P < 0.01,SR-M: P < 0.05,P < 0.01),the density of immature dendritic spines increased(SR-F: all P < 0.05,SR-M: all P< 0.01).(5)TEM results: The synaptic cleft of excitatory synapses of hippocampal neurons in SR-M and SR-F groups were blurred or disappeared,and the postsynaptic compact areas were narrowed and thickened.(6)Iso-Seq results: Compared with GR group,there were 1112 and 1017 up-regulated genes,998 and 2043 down-regulated genes respectively in the hippocampus of male and female rats in SR group,and the RhoA gene which related to the regulation of dendritic spine plasticity is up-regulated in both male and female rats in SR group.q-PCR results: Compared with GR group,RhoA m RNA level in hippocampus of male and female rats in SR group increased(both P < 0.01).(7)IF results: RhoA,p-LIMK and p-cofilin immunopositive staining could be observed in hippocampal neurons of all groups.RhoA was mainly expressed in the membrane and cytoplasm,p-LIMK was mainly in the membrane,and p-cofilin in the cytoplasm.Compared with GR group,RhoA expressions in CA1 and DG regions were increased(all P < 0.01),the expressions of p-LIMK and p-cofilin in CA1,CA3 and DG regions decreased of male and female rats in SR group(all P < 0.01),and in CA3 region RhoA expression was increased in SR-F group(P < 0.01).(8)WB results: Compared with GR group,the relative expressions of RhoA,p-RhoA and p-Rock2 were increased(all P < 0.01).the relative expressions of p-LIMK and p-cofilin were decreased(P < 0.01,P < 0.05,P < 0.05,P < 0.01),the F-actin/G-actin ratio in hippocampus of male and female rats in SR group were decreased(P < 0.01).Conclusion(1)The cognitive function of SR rats decreased,and the anxiety-like behavior increased;(2)In SR rats,the dendritic complexity of hippocampal neurons decreased,the total dendritic spines density and the mature dendritic spines density decreased,but the immature dendritic spines density increased;(3)The activation of RhoA-ROCK2 pathway in hippocampus maybe participate in the changes of hippocampal synaptic plasticity changes,emotional and cognitive disfunctions by regulating the imbalance of F-actin/G-actin ratio and plasticity of DS of juvenile chronic stress rat. |
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