示踪病毒工具在小鼠视觉环路各级神经元功能和形态研究中的应用  

Application of virus tracing tools in the study of function and morphology of neurons in the visual circuit

作　　者：沈科炯 沈吟[1] Shen Kejiong;Shen Yin(Eye Center,Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院眼科中心,430060

出　　处：《中华实验眼科杂志》2020年第9期746-753,共8页Chinese Journal Of Experimental Ophthalmology

基　　金：国家重点研发计划项目(2017YFE0103400)。

摘　　要：目的利用病毒工具研究视网膜到脑的神经环路中神经细胞的形态结构及细胞之间的联系方式,并根据其示踪方向性和跨细胞突触性质,探索其用于研究视觉环路中细胞形态和连接分布情况。方法应用随机数字表法将6~8周龄实验小鼠随机分组,每组4只。野生型C57BL/J小鼠用于荧光金、腺相关病毒(AAV)、伪狂犬病毒(PRV)和1型单纯疱疹病毒129株(H129)示踪实验;转基因鼠GAD2-Cre小鼠用于AAV的示踪实验;转基因鼠Thy1-Cre小鼠用于狂犬病毒(RV)跨单突触示踪实验。病毒方向选择性示踪:(1)逆向示踪在小鼠双侧上丘注射逆向神经示踪剂荧光金、AAV、PRV或RV,在特定时间点处死小鼠并取视网膜铺片,观察投射到脑的视网膜细胞形态分布和数量;(2)顺向示踪在小鼠玻璃体腔注射H129等顺向示踪剂,在相应时间点取小鼠的大脑切片,观察视网膜投射到脑的细胞形态分布和数量。结果逆向标记中,荧光金标记到的视网膜神经节细胞比AAV标记到的数量更多,但AAV标记的神经节细胞有更多细胞突起的精细结构,荧光金仅能标记细胞的胞体。玻璃体腔注射H129和视觉投射脑区定位注射PRV能跨多突触后显示环路中串联起来的细胞分级投射。跨单突触示踪病毒,RVG缺失的重组RV在结合辅助病毒后,可有效逆向跨1个突触,标记下一级的细胞。结论 AAV病毒通过其携带的荧光蛋白可研究细胞精细的形态学特征。H129、PRV和RV可用于研究视觉环路中神经连接。结合Cre/loxP、Caspase-3、DTA、Gcamp等基因元件技术,病毒工具能精确调控表达特定类型细胞的功能,同时进行非常细致的视觉功能和形态研究。Objective To utilize four viral tracer tools with different properties,to visualize cell morphology and connectome in visual pathways and explore and summarize the applications of different viral tracer tools in visual circuit,basing on the technology of neural circuitry tracing with viral vectors.Methods For cell tracing,C57BL/J mice were injected by fluorogold,adeno-associated virus(AAV),pseudorabies virus(PRV)or herpes simplex virus 1 strain 129(H129).GAD2-Cre mice were injected by AAV.Thy1-Cre mice were injected by rabies virus(RV).Four mice were contained in every group.Retrograde tracing:mice were injected with fluorogold,AAV,PRV or RV in SC regions.After a certain period,their retinas were isolated,expanded into flat-mounted on slides to observe the morphology and distribution of labelled retinal cells.Anterograde tracing:mice were injected with H129 in vitreous cavities.After a certain period,their brains were isolated to observe the morphology and distribution of labelled brain cells.Results The number of cells labelled by fluorogold was larger than that labelled by AAV.The morphology of cells labelled by AAV possessed more details than that by fluorogold.H129 and PRV injection could display numerous cells with synaptic connections in the visual circuit.Recombined RV could mono-transsynaptically spread and label cells with the support of helper virus.Conclusions AAV is more helpful in studying cell morphology.HSV-1,PRV and RV can be used to study cell connectomes.Cooperated with Cre/loxP system,Caspase-3,DTA,Gcamp or other neuroscience tools,viral tracer tools can be used to regulate the function of cells that belong to a specific cell type.

关 键 词：示踪 荧光金 病毒工具 视觉环路 

分 类 号：R774[医药卫生—眼科]