培养大鼠海马神经元树突发育的活细胞成像和量化分析

目的:采用活细胞成像法测量培养海马神经元的树突生长、分支复杂程度和树突丝的运动性,以及定量分析树突树发育的形态学特征。方法:出生后第1天进行海马神经元培养,于第5天转染绿荧光蛋白(GFP)标记的纤维型肌动蛋白(GFP-F-Actin)和定位在膜上的GFP(F-GFP),观察培养第7~14天树突的发育情况并进行量化分析。结果:培养第7~9天可以观察到高密度的树突丝活跃的运动,树突丝的平均密度分别为(10.78±3.78)个/100μm、(10.68±2.96)个/100μm、(9.99±3.67)个/100μm(P>0.05),有(30.18±14.03)%到(87.36±20.88)%的树突丝运动活跃(P<0.001);第7~14天树突树的总长度从(410.74±185.98)μm增长为(1238.21±418.32)μm(P<0.001),树突树的总分支数从(18.93±7.23)支增加为(33.60±10.46)支(P<0.001);培养第14天树突棘的密度为(37.17±6.46)个/100μm。结论:结合荧光蛋白转染和活细胞成像的方法,可动态、连续地观察,并定量分析发育过程中树突丝、树突树和树突棘的形态学特征,是体外观察神经元发育的理想方法。

Live imaging and quantitative analysis of dendritic development of cultured rat hippocampal neurons

OBJECTIVE: To measure mobility of dendritic filopodia, complexity of dendritic arborization using method of live imaging in cultured rat hippocampal neurons and to analyze their morphological characters quantitatively. METHODS: Vectors expressing Green Fluorescent Protein- Fibrous Actin (GFP-F-Actin) and F-GFP were co-transfected into cultured rat hippocampal neurons at 5 d in vitro (DIV 5). Neurons expressing GFP were photographed and analyzed with Metamorph software. RESULT: Dendritic filopodia was observed to move actively from DIV 7 to DIV 9. The mean density of filopodia was (10.78 +/-3.78)/100 microm, (10.68 +/-2.96)/100 microm and (9.99 +/-3.67)/100 microm (P >0.05), and there were (30.18 +/-14.03)% to (87.36 +/-20.88)% filopodia were mobile (P <0.001). During DIV 7-DIV 14, the total length of dendritic branches grew from (410.74 +/-185.98) microm to (1238.21 +/-418.32)microm (P <0.001) and the number of dendritic branches increased from 18.93 +/-7.23 to 33.60 +/-10.46 (P<0.001). The density of spine was (37.17 +/-6.46)/100 microm at DIV 14. CONCLUSION: The combination of live imaging with quantitative analysis is a useful method to study dendritic morphological development in vitro, including indicators of dendritic filopodia, dendritic arborization and spines.