PROJECT SUMMARY Sleep disorders are pervasive and cost approximately $100 billion a year, yet the mechanisms that regulate sleep are still poorly understood. Previous sleep research has focused on neurons and some neuronal factors that regulate the homeostatic process of sleep. However, an understanding of how astrocytes, a type of glial cell, contribute to the sleep homeostat is less well understood. We propose a series of experiments to investigate the role of astrocytes in regulating homeostatic sleep need which exploit key features of larval zebrafish. The advantages of using larval zebrafish for this purpose is their amenability to whole-brain calcium imaging, with single cell resolution, which is possible due to their transparency and relatively small but conserved vertebrate brain, as well as their amenability to large-scale behavioral assays. Using larval zebrafish and our custom-built two-photon selective plane illumination microscope (2P-SPIM), we will perform several imaging and perturbation experiments that record whole-brain astrocyte activity in both natural and induced sleep and wake states. We then will apply computational tools to segment individual astrocytes and identify astrocytes that may encode the sleep homeostat. In addition, we will use optogenetic, chemogenetic, and loss-of-function perturbation approaches to test for functional roles of astrocytes in sleep. This diversity supplement application describes an experimental and conceptual career development plan for a graduate student whose experimental goals are to (1) test the hypothesis that astrocytes encode homeostatic sleep need in zebrafish, (2) test the hypothesis that stimulation of astrocytes results in increased sleep, and (3) test the hypothesis that inhibition or loss of astrocytes results in decreased sleep. We also describe a detailed career development plan that addresses key gaps in the graduate student’s training. This diversity supplement application directly relates to the parent grant that proposes to explore neuronal mechanisms that underlie zebrafish sleep by extending these studies to astrocytes. Thus, the experiments described in the diversity supplement application are separate from, yet synergize with, those described for neuronal mechanisms in the parent grant. Together, the parent grant and diversity supplement have the potential to transform our understanding of mechanisms that regulate sleep homeostasis, which is important as sleep disorders impose physical and economic burdens on society.