上调海马区GluN3A表达对小鼠行为学及髓鞘表达的影响

Effects of GluN3A overexpression in the bilateral hippocampus on mouse behaviors and myelination

目的:探究海马区N-甲基-D-天冬氨酸受体(N-methyl-d-aspartate receptor,NMDAR)3A亚基(N-methyl-D-aspartate receptor subtype 3A,GluN3A)在焦虑、学习记忆和抑郁样行为中的作用及其可能的机制。方法:选取7~8周龄雄性C57BL/6N小鼠30只,随机分成GluN3A基因过表达组(GluN3A overexpression,GluN3A OE组)和对照组,分别注射过表达GluN3A病毒和对照空病毒至双侧海马,注射3周后,进行旷场实验、Y迷宫、强迫游泳和悬尾实验行为学测试,随后以Western blot和qRT-PCR检测GluN3A、髓鞘碱性蛋白(myelin basic protein,MBP)、髓鞘相关糖蛋白(myelin associated glycoprotein,MAG)和少突胶质细胞转录因子2(oligodendrocyte Lineage Transcription factor 2,Olig2)的表达情况;以免疫荧光技术检测少突胶质细胞发育状况。结果:旷场实验中,2组小鼠间总运动距离(P=0.256)、中心区域停留时间(P=0.487)差异均无统计学意义;Y迷宫实验中,GluN3A OE组小鼠进臂总次数低于对照组小鼠(P=0.015),但进臂交替率在2组小鼠间差异无统计学意义(P=0.381);强迫游泳实验(P=0.347)和悬尾实验(P=0.471)显示,不动时间在2组小鼠间差异均无统计学意义。双侧海马区域注射rAAV-GluN3A后,海马区GluN3A蛋白表达较对照组明显增加(P=0.005),海马区MBP蛋白(P=0.019)、MAG蛋白(P=0.022)和Olig2蛋白(P=0.022)表达降低,海马区MBP mRNA(P=0.043)、MAG mRNA(P=0.032)和Olig2 mRNA(P<0.001)的表达与蛋白表达下降趋势相一致,均降低;免疫荧光染色显示:GluN3A OE组小鼠海马区结肠腺瘤样息肉标记成熟少突胶质细胞密度降低(P<0.001),但血小板衍生生长因子受体标记少突胶质前体细胞密度无明显变化(P=0.542)。结论:在双侧海马区域过表达GluN3A亚基基因并未引发小鼠出现学习记忆能力改变、焦虑行为及抑郁样行为,仅引发海马区域髓鞘相关蛋白低表达及成熟少突胶质细胞减少。

Objective:To explore the role and possible regulatory mechanism of hippocampal N-methyl-D-aspartate receptor subtype 3A(GluN3A)subunit in anxiety-related behaviors,ability of learning and memory,and depression-like behaviors.Methods:Thirty male C57BL/6N mice aged 7-8 weeks were randomly divided into GluN3A overexpression group(GluN3A OE)and control group.Re⁃combinant adeno-associated viruses with(rAAV-GluN3A)and without(rAAV-control)the gene encoding GluN3A were injected into the bilateral hippocampus of mice in the GluN3A OE and control groups,respectively.At 3 weeks after virus injection,behavioral as⁃sessments were conducted using the open field test,Y maze test,forced swimming test,and tail suspension test.Subsequently,Western blot and qRT-PCR were performed to determine the expression levels of GluN3A,myelin basic protein(MBP),myelin-associated gly⁃coprotein(MAG),and oligodendrocyte transcription factor 2(Olig2).Moreover,immunofluorescence staining was carried out to evalu⁃ate the development of oligodendrocytes.Results:In the open field test,there were no significant differences in the total moving dis⁃tance(P=0.256)and the time spending in central area of open field(P=0.487)between the two groups.In the Y maze test,the total number of arm entries of mice was significantly lower in the GluN3A OE group than in the control group(P=0.015),while no significant difference was observed in alternating rate between the two groups(P=0.381).Both forced swimming test(P=0.347)and tail suspension test(P=0.471)showed no significant differences in immobility time between the two groups.After injection of rAAVGluN3A in the bilateral hippocampus,the expression of GluN3A protein in the hippocampus was significantly increased compared to that of control group(P=0.005).The expression levels of MBP pro⁃tein(P=0.019),MAG protein(P=0.022),and Olig2 protein(P=0.022)were significantly decreased in mice of the GluN3A OE group compared to mice of the control group.The expression levels of MBP mRNA(P=0.043),MAG mRNA(P=0.032),and Olig2 mRNA(P<0.001)were significantly decreased in mice of the GluN3A OE group,which was consistent with the decreased expression of MBP,MAG,and Olig2 proteins.Immunofluorescence showed that the density of CC1+mature oligodendrocytes was significantly decreased in the hippocampus of mice in the GluN3A OE group(P<0.001),while the density of PDGFRɑ+oligodendrocyte precursor cells displayed negligible variation(P=0.542).Conclusion:Overexpression of GluN3A subunit gene in the bilateral hippocampus did not lead to changes in ability of learning and memory,anxious behaviors,and depression-like behaviors in mice,but led to the low expression of myelin-related proteins and decreased density of mature oligodendrocytes in the hippocampus.