Whole-Brain Functional Imaging and Analysis of Zebrafish Sleep

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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.