| BackgroundSchizophrenia is a common severe mental illness,which often occurs in young and middle-aged people,the course of disease is more prolonged,and the disability rate is high.The etiology and pathological mechanism of schizophrenia are not yet clear.Epidemiological studies have shown that infection during maternal pregnancy is an important environmental risk factor for mental illness in the offspring.The viral analog polyinosinic cytidine(Poly I:C)is administered during pregnancy to activate maternal immunity,and the offspring can better simulate the clinical and pathological phenomena of schizophrenia.Clinical studies have shown that patients with schizophrenia have abnormal function of the 5-HT system.This phenomenon has also been reported in the rat model of infected offspring during pregnancy,but the specific mechanism of its occurrence is unknown.ObjectivesIn order to investigate the possible mechanism of 5-HT system abnormality in the model by preparing the maternal immune activated offspring rat model,analyzing its behavior and 5-HT related molecular expression changes at different developmental nodes of the offspring,and to provide clues for the mechanism study of 5-HT system disorder associated with schizophrenia.Methods1.SD pregnant rats were randomly divided into Poly I:C group and NaCl group,and were injected with 10 mg/kg Poly I: C and equal volume of normal saline on the 9th day of pregnancy.Three hours after injection,the levels of inflammatory factors in the plasma of pregnant rats were detected to assess the immune activation status.After weaning,we conduct behavioral experiments such as open field experiment,elevated cross maze experiment,Y maze experiment,PPI and other behavioral tests in adolescence and adulthood to analyze the behavioral changes of offspring rats.2.The differentially expressed genes were analyzed by transcriptome sequencing,and the content of 5-HT in cerebrospinal fluid was analyzed by ultra performance liquid chromatography-mass spectrometry(UHPLC-MS/MS),and the disorder state of the 5-HT system of progeny infected during pregnancy was preliminarily analyzed.3.The prefrontal cortex and hippocampus tissue were selected as the study brain area,and real-time fluorescent quantitative PCR,immunoblotting and immunofluorescence were used to detect two groups of rats:(1)5-HT synthesis(TPH2),transport(VMAT2)and reuptake(SERT)Changes in the expression of related proteins,clarify the possible causes of abnormal 5-HT content;(2)Changes in the expression of 5-HT-related receptors(5-HT2AR),and analyze the downstream effects that may result.4.Correlation analysis was used to investigate the relationship between abnormal expression of 5-HT pathway molecules and abnormal behavior.Results1.The expression levels of inflammatory factors TNF-α(P=0.017),IL-1β(P=0.003)and IL-6(P=0.005)in the plasma of pregnant rat in Poly I:C group were higher than those in NaCl group,indicating that the model of maternal immune activation was successfully constructed.2.Animal behavioral results showed that infection with poly I:C during pregnancy may lead to schizophrenia like behaviors in offspring,such as increased anxiety level,impaired memory function and impaired sensory gating function,and abnormal behaviors are prominent with the increase of age.The results were as follows: the open field and elevated cross maze tests showed that the anxiety level of Poly I:C group rats increased,in which the movement distance of Poly I:C group rats in the central area decreased significantly in the open field test(P=0.001);the number and percentage of time entering the open arm decreased in the elevated cross maze test(P=0.02,0.008).The Y maze and passive avoidance experiments showed that the memory function of Poly I:C group rats was damaged.In the Y maze test,the number and time of entering novel arm of Poly I:C group adolescent rats were significantly reduced(P=0.047,0.022),the number of entering novel arm of poly I:C group adult rats was significantly reduced(P=0.008).In the passive avoidance test,the latency period T1 of poly I:C group rats entering the dark box was significantly reduced(P=0.018).The results of the prepulse inhibition test showed that the inhibition rates of Poly I:C group at 75 dB(P=0.012),80 dB(P=0.001)and 85 dB(P=0.002)were significantly reduced,suggesting that the sensory gating function of rats was impaired.3.The transcriptome sequencing results showed that there were 461 differentially expressed genes in the adolescent rats of Poly I:C group,including 262 up-regulated genes and 199 down-regulated genes;the enrichment analysis of KEGG pathway of differentially expressed genes involved 104 signaling pathways,including 5-HT signaling pathway.In addition,the content of 5-HT in CSF of Poly I:C group was significantly lower than that of NaCl group(P=0.041).The results suggested that there was a disorder of 5-HT system in infected offspring rats during pregnancy.4.Real-time quantitative PCR results showed that compared with the NaCl group,the expression level of SERT gene in the hippocampus of weaned rats in Poly I:C group was significantly down-regulated(P=0.007),the gene expression level of 5-HT2 A receptor in the hippocampus of adolescent rats was significantly up-regulated(P=0.012),but the gene expression level of SERT and TPH2 in the hippocampus of adult rats was significantly lower(P=0.006,P=0.002).5.The results of Western blot showed that compared with NaCl group,the protein expression level of SERT and VMAT2 in hippocampus of neonatal Poly I:C group rats decreased(P=0.038,0.021),and that of VMAT2 and 5-HT2 A receptor in prefrontal cortex decreased(P=0.024,0.040);the expression levels of SERT,TPH2 and 5-HT2 A receptors in the hippocampus of weaned Poly I:C group rats decreased(P=0.029,0.01,0.03),and that of TPH2 and VMAT2 in prefrontal cortex decreased significantly(P=0.045,0.037);the expression level of SERT and TPH2 protein in hippocampus of adolescent Poly I:C group rats decreased(P=0.018,0.001),while the expression level of 5-HT2 A receptor protein increased(P=0.004),and that of SERT in the prefrontal cortex decreased statistically(P=0.018);the protein expression level of VMAT2 in hippocampus of adult Poly I:C group rats decreased(P=0.023),and that of VMAT2 in prefrontal cortex decreased(P=0.03).6.Immunohistochemistry results showed that compared with the NaCl group,the TPH2 fluorescence intensity in hippocampal CA3 area of the adolescent rats in the Poly I:C group was significantly reduced(P=0.0001),and the TPH2 fluorescence intensity in the prefrontal cortex of weaned and adult rats in the Poly I:C group decreased significantly(P=0.003,0.0002);the fluorescence intensity of 5-HT2 AR in hippocampal CA3 area of the adolescent rats in Poly I:C group increased significantly(P=0.008).7.Correlation analysis showed that the protein levels of 5-HT2 A receptor in the hippocampus and prefrontal lobe of adult rats were negatively correlated with the inhibition rates of 75 dB and 80 dB in the prepulse inhibition experiment(P=0.02,0.03;P=0.009,0.021,respectively).ConclusionsProgeny rats infected with Poly I:C during pregnancy have dysfunction of 5-HT synthesis,5-HT transport,reuptake,and compensatory expression of 5-HT downstream receptors,which may be one of the important molecular mechanisms of behavioral and cognitive dysfunction in schizophrenia. |
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