建立一种嗜神经病毒跨突触标记大脑特定功能网络的方法

The certain functional circuit in the brain is utilized to process a certain brain function. This functional circuit is a part of the anatomy circuit that composed by synaptic connections. Current neural technology cannot resolve the structure of a functional circuit that is responsible to process a given behavior or a specific information. In this project, we try to build a tracing system to solve this problem: A) We plan to construct and prepare two kinds of glycoprotein gene deleted neurotropic viruses which express a fluorescent protein. We also plan to construct and prepare a series of adeno-associated helper virus which supply the necessary genes to help the neurotropic viruses to specific infection and assembly. B) We plan to establish a triple transgenic strain named c-fos-tTA/TetO-lacZ/Dat-cre. The integration of the project A and B form a tracing system to resolve the structure of a universal or specific cell type related functional circuits. C) To validate this tracing system, we choose tracing the functional circuit of the dopaminergic neurons, which play a key role in brain function and brain diseases in midbrain. We plan to integrate the drug-induced Tet system, neurotropic virus and AAV relayed specific infection and trans-synaptic tracing, reward or punish training. First of all, as neural activity marker, the expression of LacZ will reveals the excitement in the dopaminergic neurons and the related up-down stream of the functional network. Second, the expression of fluorescent proteins reveals the excited dopaminergic neurons related up-down stream of anatomy circuit. Third, the existing trans-mono-synaptic tracing reveals the structure of up-down stream of the anatomy circuit of universal dopaminergic neurons. The comprehensive comparative analysis will obtain the relationship between the three levels of the dopaminergic neurons. Finally, this new trans-synaptic tracing system will extract a functional circuit of a universal or given type of neurons that responsible for a specific brain function.

大脑行使特定功能所涉及的神经网络，即功能神经网络，是通过突触连接的结构网络的组成部分。现有技术尚不能解析结构网络中处理特定信息的功能网络。本项目针对这一问题：A）构建和制备并携带荧光蛋白的重组嗜神经病毒及腺相关辅助病毒；B）建立c-fos-tTA/TetO-lacZ/Dat-cre 小鼠品系；上述 (A+B) 形成一套可用于普适或细胞选择的特定功能网络标记体系。C）作为方法验证，以调节大脑功能和脑疾病密切相关的多巴胺能神经元为对象，在涉及奖赏或惩罚行为时，结合Tet药物诱导，由神经元活动控制嗜神经病毒感染及跨突触标记；以lacZ为神经活动的标示物，揭示兴奋的多巴胺神经元及其上下游均被兴奋的功能网络；以嗜神经病毒荧光蛋白为标示物，揭示兴奋多巴胺神经元上下游结构网络；结合现有跨单突触系统，揭示多巴胺神经元结构神经网络。综合比较分析，获得特定类型细胞执行特定任务的功能网络与相应的结构网络的关系

本项目围绕“特异性标记功能神经网络”这个技术需求为核心，发展系列新型嗜神经病毒，辅助腺相关病毒，转基因动物模型，动物行为训练模式，系统的建立一套研究模型，以转基因小鼠VTA 的多巴胺神经元为神经网络节点，比较其在条件惩罚和奖赏两种训练模式中分别涉及的上下游神经网络差异，从而揭示功能神经网络标记在神经科学研究中的重要意义。