Neonatal Isolation Modulates Synaptic Plasticity Of Hippocampal And Amygdala Neurons In PTSD Rats

Objective:Early life adversity(ELA)can induce stress related behavior and increase susceptibility to stress injury in adults,which may be related to the pathogenesis of post-traumatic stress disorder(PTSD).PTSD is a kind of delayed and persistent mental disorder,which is caused by individual experience,and encountered or witnessed stress events or situations that involve themselves or others to endanger their lives.The dysfunction of hypothalamus-pituitary-adrenal(HPA)axis which is involved in controlling stress response is an important feature of the pathogenesis of PTSD.Glucocorticoid(GC)in blood circulation participates in the negative feedback regulation of HPA axis by acting on glucocorticoid receptor(GR).It has also been reported that ELA can lead to programmed changes of HPA axis,so the increase of pressure sensitivity in adulthood may be due to the continuous changes of HPA axis activity and abnormal expression of GR.The GR-rich amygdala and hippocampus have pivotal roles in regulation of the HPA axis.The amygdala is the key nucleus to regulate the acquisition,expression and regression of conditioned fear,while the hippocampus as an integrated part of stress response is an important brain area for regulating cognitive,emotional and behavioral functions.They play fundamental roles in animal learning and memory,and have a high degree of synaptic plasticity.The changes of environmental factors in embryonic period and early stage of birth can affect the construction and function of synaptic structure in hippocampus and amygdala,and lead to the damage of postnatal brain function.Synapsin I in the presynaptic membrane regulates the release of neurotransmitters and participates in the formation and maintenance of synaptic connections between neurons in the central nervous system.Postsynaptic density proteins-95(PSD-95)plays an important role in synaptic structure,function and plasticity.In addition to synapsin I and PSD-95,cell adhesion molecules(CAMs)located in synapses can not only affect synapse formation,but also regulate synaptic function through protein interaction or signal cascade reaction and strengthen the transmission of signals between synapses.For example,neurexin I andneuroligin-1/-2 can alter the long-term memory storage and fear regulation of hippocampal and amygdala neurons by affecting the balance between excitatory and inhibitory neurotransmitters in the brain,thus leading to cognitive impairment.Therefore,we propose whether the short-term repeated isolation mode of neonatal isolation(NI)as an early source of psychological stress will induce synaptic homeostasis changes in hippocampal and amygdala neurons in adult PTSD model rats constructed by single continuous stress through GRs.The ratio of excitatory/inhibitory synapses and the balance of excitatory and inhibitory currents are affected,which leads to abnormal connections of neural networks and affects cognitive or emotional function and learning or memory abilities in adulthood,thus contributing to the susceptibility of PTSD.In this study,three behavioral testing of open field(OF)locomotor,elevated plus maze(EPM)and Morris water maze(MWM)tests were used to detect spontaneous exploratory behavior,anxiety level and spatial learning or memory ability in NI-SPS rats.The changes of GR,synapsin I,PSD-95,neurexin I and neuroligin-1/2 expressed in hippocampus and amygdala of NI and SPS rats were explored in terms of morphology,protein and gene level,and to provide a basis for further understanding the pathogenesis of PTSD.Combined with the behavior determination of NI-SPS rats,to study whether ELA can regulate the neural plasticity of PTSD rats through GR and participate in the pathophysiological process of stress response,memory and affective disorder,providing evidence for revealing the pathogenesis of PTSD.Methods: 1.Animal model,experimental grouping and physiological index measurement: the female Wistar pregnant mice were fed with free drinking water until birth.The day of delivery was designated as postnatal day 0(PND 0).In this study,100 young male rats were randomly divided into 4 groups(25 rats per group): 1)Control group: no interference;2)NI group: only treated with NI during PND 2~9,but not stimulated by SPS;3)SPS group: no NI treatment,only SPS stimulation at PND 56;4)NI+SPS group: first treated with NI during PND 2~9,and then stimulated by SPS at PND 56.The young rats were weaned at PND 22 and measured at a specific time point(PND 22,PND 40 and PND 60).The rats were tested and sampled from PND 63.2.Behavioral test: The behavioral testing of OF,EPM and MWM wereused to detect spontaneous exploratory behavior,anxiety and spatial learning or memory of rats in each group.3.Immunohistochemistry: the immunohistochemical method was used to observe the changes of GR,synapsin I and PSD-95 immunoreactivity in hippocampus and amygdala of rats.4.Enzyme linked immunosorbent assay(ELISA): plasma corticosterone(CORT)levels were measured by ELISA kit in rats during stress and anxiety.5.Western blotting: the western blotting was used to detect the expression of GR,synapsin I,PSD-95,neurexin I,neuroligin-1/-2 protein in hippocampus and amygdala.6.Real-Time PCR : The expression of synapsin I、PSD-95、neurexin I、neuroligin-1/-2 mRNA in hippocampus and amygdala was detected by the Real-Time PCR method.7.Immunofluorescence technique: the immunofluorescence was used to detect the expression of neuroligin-1/-2 in hippocampus.Results:1.Measurement of physiological indexes in rats: cmpared with the control group,the intake and drinking water of NI and SPS rats were not significantly changed,and NI or SPS had no significant effect on the weight gain of rats.2.Behavioral test results: in the OF test,neither NI nor SPS had significant effects on the overall horizontal movement of rats,but the entries in the central area and the percentage of central distance travelled in the NI+SPS group were significantly lower than NI or SPS group;In the EPM test,the percentage of entries into open arms and time spent in open arms in SPS group were significantly lower than control and NI group,and the NI+SPS group were significantly lower than SPS group;In the MWM test,NI and SPS had no significant effect on the total swimming distance,but had significant effects on the target crossing and the percentage of distance travelled in the target quadrant,and the NI+SPS group was significantly higher than the SPS group.3.Immunohistochemical results: the immunoreaction of GR in hippocampus and amygdala of rats was located in the nucleus and cytoplasm of neurons.The positive expression of GR in hippocampus of experimental group was higher than control group,but the positive expression in amygdala was lower than control group;The positive reaction of synapsin I in the hippocampus and amygdala of the control group was relatively light,mainly distributed in the membrane and cytoplasm of neurons.After NI,the immunoreactivity of synapsin I in hippocampus and amygdala increased.The positive expression of synapsin I in hippocampus of SPS group was increased,but the positive expression in amygdala was lower than control group;Theimmunohistochemical staining of PSD-95 antibody positive cells was brown,and the immunoreaction was mainly observed in the cytoplasm and processes of hippocampal and amygdala neurons.The positive expression of PSD-95 in hippocampus and amygdala in NI group was higher than control group.Meanwhile,the positive expression in hippocampus of SPS group was also increased,but the positive expression in amygdala was decreased;4.ELISA: there was no significant difference between the control and NI group in the level of plasma basal CORT,but SPS induced a significant increase of plasma CORT.In addition,the level of plasma CORT in NI+SPS group was significantly higher than NI group.5.Western blotting: the expression of GR protein in hippocampus of NI and SPS group was significantly higher than control group,while the expression in amygdala was significantly lower than control group.The changes of GR in NI+SPS group were significantly higher than SPS group;The expression of Synapsin I and neurexin I in hippocampus of NI and NI+SPS group was significantly lower than control group,but the expression of SPS group was significantly increased.The expression of synapsin I and neurexin I in the amygdala of NI and SPS group was significantly lower than control group,and the NI+SPS group was significantly lower than SPS group;Compared with the control group,the PSD-95 and the ratio of neuroligin-1/-2 expression in hippocampus of NI group and SPS group was significantly increased,and NI+SPS group was significantly higher than SPS group.The expression of PSD-95 in amygdala in NI group was significantly higher than control group,but significantly decreased in SPS group.However,the expression of neuroligin-1/-2 in amygdala of NI and SPS group was significantly lower than control group,and NI+SPS group was significantly lower than SPS group.6.Real Time PCR: the expression of synapsin I mRNA in hippocampus stimulated by SPS increased significantly,but decreased significantly in the NI and NI+SPS group.The expression of synapsin I mRNA in amygdala after NI and SPS stimulation was significantly lower than control group;The expression of PSD-95 mRNA in hippocampus of the experimental group was significantly higher than the control group.The expression of PSD-95 mRNA in amygdala of NI group was significantly higher than control group,while SPS group was significantly lower than control group;After stimulation with NI and SPS,neurexin I mRNA in hippocampus and amygdala decreased significantly,and the ratio of neuroligin-1/-2mRNA expression in hippocampus was significantly higher than control group,but in amygdala was significantly lower than control group.7.Immunofluorescence results:In the control group,neuroligin-1 and-2 were overexpressed in some cells of hippocampus.They were co-expressed in the cytoplasm and process,but the axon staining was less.In the NI group,most of the cells in hippocampus showed very low neuroligin-1 and-2 immunoreactivity and no expression in neurite.In the SPS group,most of the cells co-expressed neuroligin-1-and-2 in many longer neurites.The majority of neuroligin-1 and-2 colocalization in the cellular processes were at a lower level in the hippocampus of the NI+SPS group,and the number of nerve fibers was less than SPS group.Conclusion: 1.Compared with the control group,the NI rats showed no significant changes in anxiety,but NI+SPS rats showed higher anxiety levels in OF and EPM tests than SPS rats.It suggested that early-stressed rats were more likely to be affected by similar negative experiences in adulthood.2.The early NI had no effect on plasma CORT level in adult rats,but the plasma CORT levels increased in SPS and NI+SPS groups,so GR became a key regulator of HPA axis function in the animal model of early stress.3.The expression of GR in hippocampus of NI and SPS rats was significantly increased,but the changes in amygdala were opposite to hippocampus,and the NI increased these differences in hippocampus and amygdala.Meanwhile,the NI aggravated the changes of postsynaptic membrane in the hippocampus and presynaptic membrane in the amygdala of adult SPS rats.It also changed the ratio of excitatory-to-inhibitory synapses in hippocampus and amygdala of SPS rats.In conclusion,the changes of HPA axis and synaptic plasticity in hippocampus and amygdala induced by early stress may continue to adulthood,which may aggravate the changes of central nervous system in SPS rats and increase the susceptibility of PTSD.