构建细胞选择、复制可控、顺行跨突触、结构与功能研究兼顾的单纯疱疹工具病毒体系

Elucidation of complex neural circuits is fundamental for understanding brain functions. Viral transneuronal tracing has become an increasingly powerful technique for defining the organization of neural networks. Currently, vast majority of neurotropic viruses spread retrogradely through neural circuits and few, anterogradely. The properties of specific angerograde transsynaptic spreading, large genome suitable for genetic modification and broad host range make herpes simplex virus type 1 H129 strain a unique, extremely valuable tool for mapping output neural pathways, but only a very limited transsynaptic H129 recombinants are available up to now and there still exists the shortcomings, such as high pathogenicity, low sensitivity, etc. In this project, we aim to modify the virus genome to construct attenuated, cell type-specific, replication-controllable H129 recombinants expressing fluorescent reporter and light-sensitive proteins for anterograde monosynaptic/polysynaptic circuit tracing, based on comprehensively applying the technology of molecular biology, optogenetics, genetic recombination and inducible expression elements such as Cre/LoxP and Tet-on. Such H129 recombinants will be applied to transgenic mice expressing Cre in targeted populations of neurons to analyze the structures and functions of the olfactory associated neural circuits. Through this project, a new set of cell-specific, flexible and high-efficient H129 recombinants system for anterograde transneuronal circuit tracing and for functional studies will be established.

解析神经环路的结构和功能是理解大脑工作机制的基础。嗜神经病毒跨突触追踪已成为解析神经网络结构的重要方法。目前大部分嗜神经工具病毒都是逆向跨突触，顺行跨突触的只有单纯疱疹病毒H129株和水疱性口炎病毒。H129高特异顺行跨突触、基因组荷载大、感染宿主范围广等特性使其具有独特的顺行环路示踪应用价值，但目前可用的H129重组病毒相当有限且存在毒力强、灵敏度低等问题。本项目综合运用分子生物学、光遗传学、同源重组等技术，拟将病毒主要毒力因子敲除、引入荧光蛋白和光感基因等，同时利用Cre/LoxP、Tet-on等诱导表达系统调控基因表达以构建低毒性、细胞选择、复制可控的顺行跨单/多突触H129重组病毒体系；作为方法验证，以Cre转基因小鼠为对象，揭示嗅觉神经系统主要环路在细胞水平上的结构与功能特性。通过项目实施旨在建立一套细胞特异、灵活、高效的H129病毒工具系统用于神经环路顺行追踪或相关功能研究。

神经环路结构和功能解析是理解大脑工作机制的基础。嗜神经病毒跨突触示踪已发展成为相关研究的重要方法。单纯疱疹病毒1型（HSV1） H129株具有特异顺行跨突触传播、外源基因荷载容量大、感染宿主范围广等优势，但目前H129工具病毒普遍存在毒性大、灵敏度低、感染细胞不特异等缺陷。针对这些问题，本项目综合运用分子生物学、病毒学、光遗传学等技术手段，主要通过1）HSV增亮；2）高效减毒；3）TK控制的跨突触可控；4）细胞选择性示踪等策略对H129病毒进行系列重组改造，构建减毒、细胞选择、复制可控的顺行跨单或多突触H129工具病毒。目前我们已成功构建获得顺行跨多突触示踪的高亮HSV，神经元结构细节标记清晰，其亮度基本可以与RNA示踪工具病毒媲美；构建的系列新型低毒HSV工具病毒毒性小、表达丰度强，中枢感染动物不会致病，达到了良好标记效果；建立了高效的HSV/AAV顺行跨单突触系统用于示踪特异类型神经元的一级输出网络；利用Cre/Loxp调控系统控制基因条件性表达实现了HSV细胞选择性跨突触示踪。总之，通过项目实施我们已建立低毒、高灵敏、细胞选择、跨突触可控的HSV工具病毒系统，为神经环路顺行跨突触示踪提供了一批新型高效的研究工具。