High-throughput sequencing of synaptic partnerships and gene expression at single-cell resolution in vivo

项目摘要 大脑功能依赖于特定类型神经元之间突触连接的形成和维持， 然而，对细胞类型连接性和指导这些关系的分子的系统描述仍然存在， 因为我们缺乏必要的工具。测量突触的传统方法 连接和网络-如全细胞电生理学和解剖重建-样本 只有少数细胞或小的组织体积，不容易扩展到许多动物或基因型， 确定每个细胞的分子类型和状态。为了弥补这一差距，我开发了一个条形码狂犬病 基于病毒的方法称为SBARRO，它将突触伙伴关系数据存储在每个细胞的RNA中， 在数十万个细胞中同时测量突触网络和基因表达 使用高通量单细胞RNA测序。SBARRO体外实验表明， 细胞类型特异性连接性的定量模型可以系统地生成并用于发现 与连接特性相关的基因表达特征。然而，与细胞相关的技术限制 采样，重组腺相关“辅助”病毒（rAAV）和狂犬病病毒生物学已经排除 完整脑组织的分析 在这里，我建议解决这些限制，并推进SBARRO生成突触的定量模型， 基于以下目标的体内网络：1）创建基于SBARRO的解剖学信息版本 空间分辨单细胞RNA测序（称为“Slide-SBARRO”）; 2）开发一类新的CRE 重组酶敏感的rAAV，其递送转基因，同时报告表达的重组状态， 评估狂犬病毒从突触后到突触前传递的突触选择性 细胞我将重点放在小鼠纹状体上，因为：1）纹状体细胞群现在已经被我很好地描述， 其他; 2）对内在纹状体细胞类型的突触组织知之甚少; 3）广泛的小鼠 遗传工具使Slide-SBARRO连接模型能够通过细胞类型特异性 解剖学和电生理学。

PROJECT SUMMARY Brain function depends on forming and maintaining synaptic connections between neurons of specific types, yet systematic descriptions of cell-type connectivity and the molecules that instruct these relationships remain challenging because we lack some necessary tools. Traditional approaches for measuring synaptic connections and networks – such as whole-cell electrophysiology and anatomical reconstructions – sample only a few cells or small tissue volumes, do not readily scale to many animals or genotypes, and do not ascertain the molecular type and state of each cell. To bridge this gap, I have developed a barcoded rabies virus-based method called SBARRO which stores synaptic partnership data in each cells' RNA, allowing synaptic networks and gene expression to be measured simultaneously in hundreds of thousands of cells using high-throughput single-cell RNA sequencing. SBARRO experiments in vitro have demonstrated that quantitative models of cell type-specific connectivity can be systematically generated and used to discover gene expression signatures associated with connectivity properties. Yet technical limitations related to cell sampling, recombinant adeno-associated “helper” viruses (rAAVs) and rabies virus biology have precluded analyses of intact brain tissue. Here, I propose to address these limitations and advance SBARRO to generate quantitative models of synaptic networks in vivo based on the following aims: 1) create an anatomically-informed version of SBARRO based on spatially-resolved single-cell RNA sequencing (called “Slide-SBARRO”); 2) develop a new class of CRE recombinase-sensitive rAAVs which deliver transgenes while reporting the recombination state of expressed RNA; and 3) evaluate the synapse-selectivity of rabies virus transmission from postsynaptic to presynaptic cells. I will focus on mouse striatum because: 1) striatal cell populations are now well-characterized by me and others; 2) little is known about the synaptic organization of intrinsic striatal cell types and 3) extensive mouse genetic tools enable Slide-SBARRO connectivity models to be carefully tested through cell-type-specific anatomy and electrophysiology.