| Objective: Thyroid hormones play an important role in the development and function of the central nervous system.Thyroid hormone deficiency during development or adulthood can lead to impairment of hippocampal structure and learning and memory function.The most important mechanism of thyroid hormones is to regulate the expression of target genes by binding to their nuclear receptors,and further exert the biological effects.In this study,we successfully constructed a mouse model of thyroxine nuclear receptor alpha 1(TR?1-E403X)gene mutation.Previous studies have observed impaired learning and memory function in hippocampus of the mutant mice.This study will further explore the effects of TR?1-E403 X mutation on the morphological development and function of hippocampus in mice,and preliminarily explore its mechanism of action.In this study,the expression of thyroid hormone nuclear receptor alpha 1(TR?1)in hippocampus of mice at different developmental stages will be detected by RNA scope? in situ hybridization(ISH)with highly sensitive and specific.Methods: The mutant model mice of TR?1-E403 X prepared by our research group were used for mating and breeding of male mice of various genotypes.The experimental time points were divided into 0 days,7 days,3 weeks of age,6 weeks of age and 16 weeks of age after birth.Items: 1.The expression of TR?1 in hippocampus of mice at different developmental stages was observed by RNA scope? in situ hybridization(ISH).2.The development of hippocampal morphology and structure in P0,P7,3,6 and 16-week-old mice was observed by Nissl staining of brain tissue;3.The learning and memory function,fear,anxiety and depression levels were assessed by new object recognition zero maze,open field,and tail suspension experiments in 16-week-old mice;4.The expression profiles of hippocampal tissue were sequenced in 3 and 16-week-old mice,and the hippocampal structure development and functionally related differentially expressed genes were screened.Then were further validated by real-time quantitative polymerase chain reaction(RT-PCR)in the hippocampus of mice at 3 and 16 weeks.Result: 1.RNA scope analysis of in situ hybridization(ISH)results: The expression of TR?1 was observed in the primitive neuroepithelial cells and hippocampus during the whole process from the development of the primitive neural tube in the early embryonicstage to the morphological maturation of the hippocampus.The expression of TR?1 was also at the peak of the morphological development of the hippocampus.2.Morphological experiment results: At P0: Compared with wild-type mice,homozygous mutant TR?1-E403 X mice had smaller hippocampal volume and thicker neuroepithelial layer in hippocampal area.At this time,pyramidal cells migrating to hippocampal CA1 area occupied the whole hippocampal CA1 area,the stratum oriens layer of hippocampal CA1 area had not yet appeared,and pyramidal cells had not migrated and settled in the stratum pyramidal layer,lingered in the middle layer and dispersed in the stratum pyramidal layer of hippocampal CA1 region.Heterozygous mutant mice had similar hippocampal morphology to wild type mice,but most of their pyramidal cells remained in the middle layer.Only a small number of pyramidal cells migrated to the pyramidal cell layer of CA1 region,but the number of pyramidal cells that had completed migration and settled was small,and the cells were scattered and not concentrated.P7: Nissl staining of hippocampal tissue in homozygous mutant mice,compared with the hippocampal morphology of wild-type mice,some neuroepithelial cells in hippocampal alveolar region have not yet completed migration to hippocampal CA1 region,stratum pyramidal layer in hippocampal CA1 region have partially completed migration and settlement,and many cells are still migrating and staying in the middle layer.At this time,the three-layer structure of hippocampal CA1 region were gradually clear.The pyramidal cells in hippocampal CA1 region which migrated and settled were smaller and less numerous than those in wild type mice.The results of Nissl staining in the hippocampus of heterozygous mutant mice were between wild type mice and homozygous mutant mice.At the age of 3 weeks: Compared with the hippocampal morphology of wild type mice,the stratum pyramidal layer in CA1 region of hippocampus of heterozygous mutant mice and homozygous mutant mice completed migration and settlement,and the three-layer structure of CA1 region of hippocampus was clear,but the pyramidal cells of hippocampal CA1 stratum pyramidal layer were scattered and not concentrated.6-week-old: Compared with the hippocampal morphology of wild-type mice,the three-layer structure of hippocampal CA1 was clear,but the pyramidal cells of hippocampal CA1 stratum pyramidal layer were scattered and not concentrated.16-week-old: Compared with wild-type mice,heterozygous mutantmice had clear three-layer structure in hippocampal CA1 region,and there was no significant difference in pyramidal cell morphology and arrangement in hippocampal CA1 region between heterozygous mutant mice and wild-type mice.3.Behavioral experiment results: 16-week-old heterozygous mutant mice in the zero maze test,the total movement distance of mice was shortened,the proportion of stiffness time increased,the number of times entering the arm and probe decreased,the number of excreted stools increased,and the difference was significant.In tail suspension test,the stiffness time of 16-week-old heterozygous mutant mice was prolonged,but there was no significant difference.Heterozygous mutant mice were not found to exhibit significant abnormalities in other experimental items.4.Sequencing and validation of hippocampal tissue expression profiles: Calml4 and RC3 expression in the hippocampus of 3-week-old homozygous mutant mice decreased,while NGF and c-Fos expression showed no difference.The expression of Calml4 and c-Fos in hippocampus of 3-week-old heterozygous mutant mice decreased,while that of c-Fos in hippocampus of 16-week-old heterozygous mutant mice decreased.Conclusion: 1.TR?1 was expressed in the primary neuroepithelial cells of the neural tube and the subareas of the hippocampal tissue from the early stage of embryonic neural tube formation to the development and maturation of the hippocampus,and the expression of TR?1 was increased at the peak of hippocampal CA1 development.2.TR?1E403X gene mutation delayed the migration of neurons in CA1 region of hippocampus of mutant mice,reduced the number of neurons,scattered and irregular arrangement.3.TR alpha 1-E403 X mutant mice had impairment of learning and memory ability,and were more prone to fear and anxiety,and also had a certain degree of depression tendency when there was stress environment outside.4.TR alpha 1-E403 X mutation down-regulates the expression of synaptic plasticityrelated molecules in the hippocampus of mutant mice,suggesting that the mutation of TR alpha 1-E403 X may be involved in the regulation of the expression of these molecules in the hippocampus,leading to hippocampal-related functional damage in mutant mice. |
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