High-throughput approaches to local and long-range synaptic connectivity

项目摘要/摘要 这项提议的首要目标是开发一种强大的方法来绘制大脑的连接图 快速、准确、经济实惠。过去为解决拆解建筑群的挑战所做的努力 哺乳动物大脑的连接体在吞吐量/效率和 决议。两种前沿神经元映射技术--基于条形码的连接映射(BARseq) 和扩展显微镜(EXM)-已经证明它们可以实现高效和高分辨率的连接 在哺乳动物神经组织内的映射。我们将优化并整合这两种技术来绘制地图 具有单一突触分辨率的本地和远程电路。在exm中，神经组织是物理上的 扩展，使其更容易消除附近的神经纤维的歧义和检测的准确位置 突触相关蛋白。这种方法非常适合于梳理出 密密麻麻的本地电路。使用BARseq，神经元表达独特的条形码标签，这甚至允许 远距离的过程要准确地追溯到它们的躯体起源。将BARseq与In Site相结合 通过免疫标记技术，我们还可以精确地识别每根纤维上突触的位置。在这里我们 建议优化这两种方法的组合，这将使一个平台能够生成 具有单一突触水平分辨率的全脑微连接组。这一努力的成功具有明显的影响 对于神经科学研究的未来，包括改变我们对正常 在神经精神障碍的背景下发生的大脑回路和特定的干扰。

Project Summary/Abstract The overarching objective of this proposal is to develop a robust approach to map the brain's connections quickly, accurately, and cost-effectively. Past efforts to address the challenge of teasing apart the complex connectome of the mammalian brain were subject to a steep trade-off between throughput/efficiency and resolution. Two cutting-edge neuronal mapping techniques—barcoding based connection mapping (BARseq) and expansion microscopy (ExM)—have proven they can achieve efficient and high-resolution connection mapping within mammalian neural tissue. We will optimize and then integrate these two techniques to map both local and long-range circuitry with a single-synapse resolution. In ExM, neural tissue is physically expanded, making it easier to disambiguate neural fibers in close proximity and to detect the precise location of synapse-associated proteins. This approach is ideally suited to teasing apart the paths and connections among densely packed local circuits. Using BARseq, neurons express unique barcoded tags, which allows even distant processes to be accurately traced to their somatic origins. Combining BARseq with in situ immunolabeling techniques, we can also precisely identify the location of synapses on each fiber. Here we propose to optimize the combination of these two approaches, which will enable a platform for generating a brainwide microconnectome with single-synapse level resolution. Success in this effort has clear implications for the future of neuroscience research, including the potential to transform our understanding of both normal brain circuitry and the specific disruptions that occur within the context of neuropsychiatric disorders.