BRAIN CONNECTS: Mapping brain-wide connectivity of neuronal types using barcoded connectomics

大脑连接：使用条形码连接组学绘制全脑神经元类型的连接

• 批准号: 10663723
• 负责人: XIAOYIN CHEN
• 金额: $ 220.4万
• 依托单位: ALLEN INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-22 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10663723
• 关键词: Anatomy; Animals; Axon; BRAIN initiative; Bar Codes; Behavioral; Brain; Brain Mapping; Brain region; Cells; Censuses; Characteristics; Cognitive; Complement; Computer Models; Consumption; Data; Data Set; Electron Microscopy; Equipment; Foundations; Gene Expression; Goals; Individual; Injections; Label; Laboratories; Link; Macaca; Maps; Messenger RNA; Methods; Microscopy; Mus; Neuroanatomy; Neurons; Neuropil; Primates; Process; RNA; RNA Processing; RNA Sequences; Rabies; Rabies virus; Reagent; Resolution; Rodent; Sindbis Virus; Speed; Structure; Synapses; Techniques; Testing; Time; Tropism; Viral; Visual; Visual Cortex; Work; cell type; connectome; cost; design; extrastriate visual cortex; improved; in situ sequencing; light microscopy; neuronal cell body; nonhuman primate; novel strategies; postsynaptic neurons; presynaptic neurons; rabies viral tracing; scale up; transcriptomics

Project Summary Mapping the brain-wide connections of neurons provides a foundation for understanding the structure and functions of a brain. Neuroanatomical techniques based on light-microscopy or electron microscopy have advanced tremendously in throughput and cost in recent years, but it remains challenging to scale them up to systematically interrogate large non-human primate (NHP) brains. Here we propose to develop sequencing-based neuroanatomical approaches to achieve high throughput and highly multiplexed brain- wide mapping of neuronal projections and synaptic connectivity in NHPs at cellular resolution. Unlike microscopy-based techniques, which rely on visually tracing individual axons from the somas to axonal termini, sequencing-based approaches label neurons with unique virally encoded RNA sequences, or “barcodes.” Sequencing and matching barcodes thus reveals the projections and/or synaptic connectivity of neurons. Thus, by transforming projection and connectivity mapping into sequencing problems, sequencing-based neuroanatomical approaches are not constrained by the same tradeoffs that plague microscopy-based techniques. Specifically, we will develop and optimize three techniques for brain-wide mapping. First, we will optimize BRICseq (brain-wide individual animal connectome sequencing), which can currently map the projections of tens to hundreds of thousands of neurons in a single mouse brain at cellular resolution. We aim to adapt BRICseq for NHP brains, further reduce cost and increase throughput, to achieve the ability to map a million neurons per brain at cellular resolution at extremely low cost per neuron. Second, we will optimize BARseq (barcoded anatomy resolve by sequencing) for NHP brains. BARseq uses in situ sequencing of the same viral barcodes used in BRICseq to achieve higher resolution in projection mapping and to also read out gene expression in the same neurons. Thus, BARseq can associate neuronal projections with cell types defined by gene expression in individual neurons. We will automate in situ sequencing, reduce probe cost, and scale up BARseq to achieve the ability to map brain- wide projections in NHP brains. Finally, we will develop barcoded rabies virus-based monosynaptic tracing to achieve highly multiplexed mapping of synaptic connectivity of neuronal types at cellular resolution. Determining the synaptic connectivity of neuronal types will powerfully constrain and test computational models of circuit function beyond what knowing the axonal projections allows. We will apply all three techniques to generate a multi-resolution projection and synaptic connectivity map of the macaque visual cortex. With the ability to generate massive single-neuron datasets and the ability to link projections and synaptic connectivity to neuronal types, our proposed techniques complement mature techniques deployed at BRAIN CONNECTS centers to achieve an unprecedented view of NHP brains.

项目概要 绘制神经元的全脑连接图为理解神经元的结构和 基于光学显微镜或电子显微镜的神经解剖技术。 近年来在吞吐量和成本方面取得了巨大进步，但扩大规模仍然具有挑战性 系统地询问大型非人类灵长类动物（NHP）大脑在这里我们建议开发。 基于测序的神经解剖学方法，以实现高通量和高度复用的脑 在细胞分辨率下对 NHP 中的神经投射和突触连接进行广泛映射。 基于显微镜的技术，依赖于视觉追踪从体细胞到轴突的单个轴突 末端，基于测序的方法用独特的病毒编码的RNA序列标记神经元，或者 因此，对条形码进行测序和匹配可以揭示投影和/或突触连接。 因此，通过将投影和连接映射转化为排序问题， 基于测序的神经解剖学方法不受困扰的相同权衡的限制 具体来说，我们将开发和优化三种全脑技术。 首先，我们将优化 BRICseq（全脑个体动物连接组测序）。 目前可以绘制单个小鼠大脑中数万至数十万个神经元的投影 我们的目标是使 BRICseq 适用于 NHP 大脑，进一步降低成本并提高吞吐量， 实现以极低的成本以细胞分辨率绘制每个大脑一百万个神经元的能力 其次，我们将为 NHP 大脑优化 BARseq（条形码解剖解析）。 BARseq 使用 BRICseq 中使用的相同病毒条形码的原位测序来实现更高分辨率 投影映射并读出相同神经元中的基因表达，因此，BARseq 可以。 我们将把神经投射与由单个神经元的基因表达定义的细胞类型联系起来。 自动化原位测序、降低探针成本并扩大 BARseq 规模，以实现脑图谱绘制的能力 最后，我们将开发基于条形码的狂犬病病毒单突触追踪。 以细胞分辨率实现神经类型突触连接的高度多重映射。 确定神经类型的突触连接将有力地约束和测试计算能力 我们将应用所有三个模型来了解轴突投影之外的电路功能模型。 生成猕猴视觉的多分辨率投影和突触连接图的技术 具有生成大量单神经元数据集以及链接投影和数据的能力。 突触连接到神经类型，我们提出的技术补充了成熟的技术 部署在 BRAIN CONNECTS 中心，以实现 NHP 大脑的前所未有的视图。