开发DNA条形码单细胞分辨率环路示踪技术解析内侧前额叶皮层精细环路构筑

The functions of the nervous system rely on the precise organization of neural circuits. We have obtained certain understanding on the connectivity and function of neural circuits mainly at the mesoscopic level. However, the precise connectivity patterns of neural circuits is poorly known, which limits the accurate interpretation for the neural network information processing patterns and deep understanding on the pathology of psychiatric disorders, such as autism, schizophrenia et al. Therefore, it is necessary to develop a single-neuron resolution tracing system to decipher the fine architecture of neural circuits. In the current project, based on our expertise on neurotropic virus engineering, we are planning to develop a novel single-cell resolution neural circuits tracing method by combining retrograde tracing of DNA-barcode viruses with multi-round in situ hybridization, which will realize precise delineation on the output connection patterns of a certain nucleus at single-neuron resolution. With the help of this method, we will map the fine architecture of medial prefrontal cortex output network， including single-neuron projection pattern and neurotransmitter property. These studies will help us to better understand the fine structure, specific functions and pathological changes of the medial prefrontal cortex.

神经系统的功能依赖于神经元的精密环路构筑。我们对神经环路在介观水平的连接和功能有了一定的认识，然而对神经环路精细连接模式的解析还十分有限，限制了对神经网络信息处理模式的精确解读和对自闭症、精神分裂症等精神疾病发病机制的深度理解。因此，亟待开发一套单神经元分辨率的精细神经环路解析工具。本项目基于申请人在嗜神经病毒改造的长期积累，利用DNA序列条形码逆向示踪病毒，结合多轮原位杂交技术开发一套单细胞分辨率的神经环路示踪方法，以实现对特定核团输出环路单神经元精细连接模式的解析。我们将用该方法解析内侧前额叶皮层输出环路的精细构筑,包括单神经元水平的投射模式和神经递质类型，为研究内侧前额叶皮层精细结构、特定功能和病理变化打下基础。

神经系统的功能依赖于神经元精密环路，然而，我们对神经环路的精细连接模式（单神经元精度）解析还十分有限，限制了对神经网络信息处理模式的精确解读。因而，有必要从单神经元分辨率解析神经环路的精细结构。我们将基于AAV病毒构建带有DNA条形码示踪病毒库，每个条形码（Barcode）序列由特定DNA序列组成，将条形码示踪病毒库标记特定环路的神经元群，实现单神经元的条形码病毒标记，结合单细胞测序技术和原位测序技术解读单个神经元中的条形码序列及该细胞的转录组表达谱。我们利用以上方法对内侧前额叶皮层输出环路的精细构筑进行解析，从分子水平揭示神经环路构筑基础。