Highly multiplexed circuit mapping using barcoded rabies viruses and in situ sequencing.

摘要 了解神经回路的功能需要深入研究两个回路 元素：单元类型和连接性。轴突追踪与高通量相结合 对遗传条形码神经元的DNA测序使同步表征成为可能 神经元的解剖学和分子同一性及其投影场。然而，没有高- 目前存在的吞吐量工具允许我们映射突触前和 突触后神经元，同时识别映射神经元的细胞类型。 在这里，我们建议使用条形码狂犬病应用一系列新的基于测序的方法 病毒可实现两个主要目标：第一，允许表征投射神经元 大脑中所有投射到多个靶区的区域；第二，允许同时 将输入映射到转录上不同的神经元亚型。我们会把我们的井结合在一起- 建立单细胞逆行标记单细胞RNA-seq(scRNA-seq)管道 狂犬病条形码检测，以进行高度多元化的细胞类型表征 神经元投射到整个大脑的许多目标区域中的每一个。此外，我们将适应 我们的条形码解剖结构通过测序(BARseq)管道进行解析，以表征空间 这些投射神经元的分布和细胞类型(Retro-BARseq)。我们将进一步建立 研究神经回路的解剖和转录特征的方法学 利用scRNA-seq/BARseq追踪狂犬病的逆行跨突触回路 (TransR-seq和TransR-BARseq)。最后，我们将努力达到单神经元水平 应用优化的条形码狂犬病对野生型小鼠的连接体追踪和测序 病毒可以实现单个神经元的独特条形码。 该项目的成功将导致一套高通量、高分辨率的方法 因此，通过将条码狂犬病病毒与组学相结合来对连接体进行测序 揭示给定神经细胞内神经元的连通性和转录特性 巡回赛。这些技术，其中大部分应该适用于任何哺乳动物物种， 将在神经元电路的吞吐量和分辨率方面提供数量级的提高 分析，对神经科学的许多分支产生了革命性的影响。

ABSTRACT Understanding the function of neural circuits requires thorough investigation of two circuit elements: cell types and connectivity. The combination of axonal tracing with high-throughput DNA sequencing of genetically barcoded neurons has enabled the simultaneous characterization of anatomical and molecular identities of neurons and their projection fields. However, no high- throughput tools currently exist that allow us to map connections between presynaptic and postsynaptic neurons while identifying the cell types of mapped neurons. Here we propose to apply a family of novel sequencing-based approaches using barcoded rabies viruses to achieve two main goals: first, to allow characterization of projection neurons everywhere in the brain that project to multiple target regions; and second, to allow simultaneous mapping of the inputs to transcriptomically-distinct subtypes of neurons. We will combine our well- established single-cell RNA-seq (scRNA-seq) pipeline with retrograde labeling (Retro-seq) for rabies barcode detection in order to conduct highly multiplexed cell type characterization of neurons projecting to each of the many target areas across the brain. Furthermore, we will adapt our barcoded anatomy resolved by sequencing (BARseq) pipeline to characterize the spatial distribution and cell types of those projection neurons (Retro-BARseq). We will further establish the methodology to investigate the anatomical and transcriptomic features of neural circuitry revealed by rabies-based retrograde trans-synaptic circuit tracing using scRNA-seq/BARseq (TransR-seq and TransR-BARseq). Finally, we will strive to reach single-neuron-level connectome tracing and sequencing in wild-type mice by applying the optimized barcoded rabies viruses to achieve unique barcoding of individual neurons. Success of the project will result in a set of high-throughput, high-resolution methods for sequencing the connectome by combining barcoded rabies viruses with omics, therefore revealing both the connectivity and transcriptomic identities of neurons within a given neural circuit. These techniques, the majority of which should be applicable to any mammalian species, will provide orders of magnitude increases in both throughput and resolution of neuronal circuit analysis, with a transformative impact on many branches of neuroscience.