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

项目总结/摘要 这项提案的首要目标是开发一种强大的方法来绘制大脑的连接 快速、准确、经济高效。过去的努力，以解决挑战戏弄分开复杂的 哺乳动物大脑的连接体受到吞吐量/效率之间的急剧权衡， 分辨率两种最前沿的神经元映射技术--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中的映射。我们将优化并整合这两种技术， 具有单突触分辨率的本地和远程电路。在ExM中，神经组织在物理上 扩大，使其更容易消除模糊的神经纤维在密切的接近和检测的精确位置， 突触相关蛋白这种方法非常适合于梳理 密集的本地电路使用BARseq，神经元表达独特的条形码标签，这甚至允许 遥远的过程，以准确地追踪到他们的躯体起源。BARseq与原位结合 通过免疫标记技术，我们还可以精确地识别每个纤维上突触的位置。这里我们 我建议优化这两种方法的结合，这将使一个平台， 具有单突触水平分辨率的全脑微连接体。这一努力的成功具有明显的意义 对于神经科学研究的未来，包括改变我们对正常和神经系统的理解的潜力， 大脑回路和神经精神疾病背景下发生的特定中断。

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.