Whole-Brain Functional Imaging and Analysis of Zebrafish Sleep

摘要 睡眠占据了我们生命的三分之一，与睡眠有关的疾病每年花费约1000亿美元， 对其管理机制的了解仍然很少。尽管取得了实质性进展， 在发现和理解特定的睡眠促进和唤醒促进神经元， 分子途径，缺少的是对这些机制如何在 大脑将睡眠和清醒作为全脑行为状态来调节。为了实现这一目标，我们提出了一个 概念简单但功能强大的方法：记录正常睡眠期间大脑中每个神经元的活动 和尾流状态，并响应于引起这些状态的扰动，然后应用数学分析 和建模来揭示睡眠的基本原理。这个探索性项目的主要目标是 开发和验证成像、分析和建模工具，这些工具将作为后续 更大规模的应用，将全面识别和表征睡眠调节电路， 生成模型来解释睡眠背后的神经元回路原理。我们将使用小型和 透明的斑马鱼幼体，一种具有良好特征的睡眠行为的脊椎动物模型，其调节是 与哺乳动物的相似。使用这个模型和我们定制开发的双光子选择平面 照明显微镜（2P-SPIM）平台，我们将进行神经元活动的全脑记录， 在自然和诱导睡眠和唤醒状态期间的细胞分辨率。然后我们将应用数学工具 从这些全脑记录中提取见解，以识别睡眠的神经基质。我们 分析将使我们能够测试现有的睡眠调节模型，并根据我们的研究提出新的模型。 数据该项目将首次实现对脊椎动物睡眠的全面观察和分析， 规模和分辨率。从这些研究中获得的独特见解将为更完整的 了解睡眠的神经机制，其功能障碍造成了重大负担 对社会的影响

ABSTRACT Sleep occupies a third of our lives and sleep-related ailments cost an estimated $100 billion per year, yet the mechanisms governing its regulation remain poorly understood. Despite the substantial progress that has been made in the discovery and understanding of specific sleep-promoting and wake-promoting neuronal and molecular pathways, what is missing is an integrated understanding of how these mechanisms work together in the brain to regulate sleep and wake as whole-brain behavioral states. Toward this goal, we propose a conceptually simple yet powerful approach: record the activity of every neuron in the brain during normal sleep and wake states, and in response to perturbations that induce these states, then apply mathematical analysis and modeling to uncover fundamental principles that underlie sleep. The main goals of this exploratory project are to develop and validate imaging, analysis, and modeling tools that will serve as a foundation for a subsequent larger-scale application that will comprehensively identify and characterize sleep-regulating circuits, and generate models to explain the neuronal circuit principles that underlie sleep. We will use the small and transparent larval zebrafish, a vertebrate model with well-characterized sleep behavior whose regulation is conserved with that of mammals. Using this model and our custom-developed two-photon selective plane illumination microscopy (2P-SPIM) platform, we will perform whole-brain recordings of neuronal activity with cellular-resolution during both natural and induced sleep and wake states. We will then apply mathematical tools to extract insights from these whole-brain recordings to identify the neural substrates that underlie sleep. Our analysis will allow us to both test existing models of sleep regulation and to propose new models based on our data. This project will be the first to achieve comprehensive observation and analysis of vertebrate sleep at such scale and resolution. The unique insights gained from these studies will pave the way toward a more complete understanding of the neuronal mechanisms that underlie sleep, whose dysfunction imposes a significant burden on society.