LSS Knockout (+/-) Improves Fearing Memory Of Mice

Background and aim: Fear memory is a research hotspot in the field of neurobiology.Fear memory is a brain activity coordinated by multiple brain functional areas,the most clearly studied is the amygdala.Also known as the amygdala,it's almond-shaped and located in the limbic system of the brain.It is the brain organization that produces emotion,regulates emotion,controls fear and memory.Many studies have found that the amygdala seems to be related to early childhood autism [1].At present,the pathogenesis is not very clear,and there is a lack of effective treatment,but more and more evidence indicates that abnormal cholesterol metabolism is closely related to this disease [2].Therefore,one of the main objectives of this study is to further study the role of cholesterol metabolism in fear memory in animals and its related molecular mechanisms.In this study,we used non-conditional heterozygous gene knockout mice model of cholesterol metabolism disorder(LSS+/-)to test their behavioral memory ability and combined with multi-omics study,in order to reveal the relationship between cholesterol metabolism disorder and the occurrence and development of neurological diseases.This work provides a link between cholesterol metabolism and fear memory and identifies LSS as a potential target for drug therapy.Methods:(1)LSS knockout(+/-)mouse model was constructed and behavioral differences between LSS knockout(+/-)mice and WT C57BL/6 wild-type mice were observed.Animal behavior experiments were conducted,including conditioned fear experiment,platform jump experiment,open field experiment,suspended tail experiment,elevated cross maze experiment and Morris water maze experiment.To determine whether the difference was statistically significant.Mature C57BL/6J mouse strains aged 8 weeks were used in animal behavior experiments.All animal experiments were approved by the Animal Ethics Committee of Anhui Medical University.Sixty sp F-born female 8-week-old Wide type(WT)and LSS knockout mice(LSS + /-)were divided into 6 groups for behavioral tests.Prior to the behavioral test,all laboratory animals were brought into the behavioral laboratory for at least one week after being raised to 8 weeks of age in SPF standard environment to eliminate environmental interference.Behavioral experiments were conducted by randomized double-blind experiment to exclude the influence of subjective factors of experimenter on behavioral experiments.Behavioral tests of mice were conducted at 16:00 every day to exclude the influence of sleep on animal behavior.All tests were conducted in the light Cycle Behavior lab from 08:00 to 20:00.To rule out the effects of different behaviors on the animals,only one behavioral test was given to each group.All videos were analyzed using behavioral software.(2)Transcriptome sequencing RNA-SEq analysis of differentially expressed genes in LSS knockout(+/-)mice and WT C57BL/6 wild-type mice brain tissues.(3)Immunohistochemical staining and immunofluorescence were used to observe the differences in the expression of specific proteins.Transmission electron microscopy imaging analysis showed differences in synaptic cleft between LSS knockout(+/-)mice and WT C57BL/6 wild-type mice.Western blotting(WB)method was used to detect the expression of related proteins related to fear memory,CCKBR,ZIF268 proteins,ERK and AKT signaling pathway related proteins.Results:(1)Behavioral test of mice: Compared with the litter wild group,LSS knockout mice showed more obvious anti-anxiety like behavior(P <0.05)and higher activity(P <0.05),and also showed better memory ability in spatial localization memory test(P <0.05).(2)Rn A-SEQ analysis of differentially expressed genes in whole brain tissues of LSS knockout(+/-)mice and WT C57BL/6 wild-type mice suggested: LSS heterozygous knockout resulted in the differences of 320 genes in the mouse brain,including 145 up-regulated genes and 175 down-regulated genes.KEGG enrichment analysis showed that these differentially expressed genes involved in neural activation receptor and ligand pathways,hippopotamus signaling pathways and glutamate synaptic pathways.(3)BY RT-QPCR analysis,CCKBR and Ador A2 were up-regulated by about 8 times and 6 times,respectively,while SLC6A3,Htr5 b,Glra3and Chrna were down-regulated by more than 3 times,respectively,suggesting that most of these genes are related to emotion,suggesting that they may affect the mood of mice.(4)Western blot results showed that Ca MK2 D,CCKBR,ZIF268 and KCCNH4 genes were significantly overexpressed in total protein of all brain tissues of LSS knockout(+/-)mice.(5)Electron microscope results LSS knockout(+/-)mouse brain tissue synaptic cleft widening.Conclusion: LSS knockout(+/-)improved fear memory and more significant anti-anxiety-like behavior in the mouse brain,partly through molecular mechanisms related to the metabolism of phospholipids in cholesterol.