基于Patch-seq从单细胞水平分析中脑-边缘多巴胺亚群神经元的分子特性及其与MA成瘾的关系

Mesencephalic dopaminergic neurons are projected to different brain regions with embryonic development. They differentated into meso-limbic, meso-cortical and mesostriatal dopaminergic subtype neurons and controled motor, mental and reward activities, respectively. Among these dopaminergic projections, the mesolimbic pathway is the most recognized neuroanatomical basis for drug addiction. The analysis of the heterogeneity of the DA subtype neurons helps to understand its physiological functions and to accurately treat drug addiction. However, the molecular characteristics of these mesencephalic DA subtype neurons are not clear. In our preliminary experiment, the distribution and localization of mesolimbic DA neurons are analyzed with the fluorescence retrograde tracing. And their electrophysiological properties are recored through patch clamp. Moreover, using Patch-seq technique, we profile the mesolimbic DA subtype neurons and perform the RNA-seq from a single cell, get a batch of circRNA molecules. Next, we will focus functional studies on the mesolimbic DA neural circuit, and analyze the specific circRNA molecules in DA neuron differentiation, DA projection and their relationship with methamphetamine addiction. Our study will refine the classification of DA neurons in the mesencephalon and will be possible to find target molecules of methamphetamine drug addiction.

中脑多巴胺能神经元随胚胎发育投射至不同的脑区，分化为不同的亚群神经元，执行运动、精神和奖赏等功能。其中，中脑边缘（mesolimbic）多巴胺神经环路是药物成瘾最公认的神经解剖学基础。分析该群DA神经元有助于理解其生理功能并对药物成瘾进行针对性治疗。然而，中脑边缘DA亚群神经元的分子特性一直不清。本项目前期实验通过神经逆行示踪分析了mesolimbic DA亚群神经元在中脑内的分布及定位，经Patch-seq分析了其电生理学特性，同时汲取了标记DA亚群细胞，从单细胞角度进行了RNA-seq，获取了一批circRNA分子。接下来，将针对mesolimbic DA神经环路进行功能研究，经脑内活细胞转染分析特异性circRNA及其关联的miRNA和mRNA对DA神经元分化、投射中的作用及与MA成瘾的关系。本项目将细化中脑DA神经元的分类并有望找出药物成瘾的靶分子。

中脑多巴胺能神经元随胚胎发育投射至不同的脑区，分化为不同的亚群神经元，执行运动、精神和奖赏等功能。其中，中脑边缘（mesolimbic）多巴胺神经环路是药物成瘾最公认的神经解剖学基础。本项目分析了该亚群神经元的电生理学和分子学特性。采用神经元示踪技术，定位了DA亚类神经元的定位分布，通过脑片钳技术分析了标记DA神经元的电生理学特征；通过Patch-seq技术汲取了标记亚类神经元，并进行了转录组分析，获得了一批差异基因分子，并进行了表达验证实验。对筛选了的circRNA和基因进行了功能分析。通过生信学分析了circRNA的关联靶基因，研究了circRNA通过靶基因的作用机制。本项目进一步细化了中脑DA神经元的分类，为寻找药物成瘾相关的靶分子提供了实验依据。