Whole-Brain Functional Imaging and Analysis of Zebrafish Sleep

摘要 睡眠占据了我们生活的三分之一，与睡眠相关的疾病每年估计要花费1000亿美元，但 管理其监管的机制仍然知之甚少。尽管已经取得了实质性的进展， 在发现和理解特定的促进睡眠和觉醒的神经元和 分子途径，缺少的是对这些机制如何在 大脑调节睡眠和觉醒为全脑行为状态。为了实现这一目标，我们提出了 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，并响应引起这些状态的扰动，然后应用数学分析 以及通过建模来揭示睡眠背后的基本原理。这一探索性项目的主要目标是 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，以及 建立模型来解释睡眠背后的神经回路原理。我们将使用小的和 透明幼体斑马鱼，一种具有良好睡眠行为特征的脊椎动物模型，其调节是 与哺乳动物一样保守。使用该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，我们将进行全脑神经元活动的记录 自然和诱导睡眠和清醒状态下的细胞分辨率。然后我们将应用数学工具 从这些全脑记录中提取洞察力，以确定睡眠背后的神经底物。我们的 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 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 比例和分辨率。从这些研究中获得的独特见解将为更完整的 对睡眠背后的神经机制的理解，其功能障碍会造成重大负担 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.