Subtype-specific roles of ellipsoid body ring neurons in sleep regulation in Drosophila

The ellipsoid body (EB) is a major structure of the central complex of the Drosophila melanogaster brain. 22 subtypes of EB ring neurons have been identified based on anatomical and morphological characteristics with light-level microscopy and EM connectomics. A few studies have associated ring neurons with the regulation of sleep homeostasis and structure. However, cell type-specific and population interactions in the regulation of sleep remain unclear. Employing a unbiased thermogenetic screen of collected EB drivers, we found: 1) multiple ring neurons are involved in the modulation of amount of sleep and structure in a synergistic manner; 2) analysis of data for {Delta}P(doze)/ {Delta}P(wake) using a mixed Gaussian model detected 5 clusters of GAL4 drivers which had similar effects on sleep pressure and/or depth: lines driving arousal contained R4m neurons, whereas lines that increased sleep pressure had R3m cells; 3) a general linear model analysis correlating ring cell subtype and activity-dependent changes in sleep parameters across all the lines identified several cell types significantly associated with specific sleep effects: R3p for daytime sleep promotion, and R4m for nighttime wake-promoting; and 4) another subclass, R3d cells present in 5HT7-GAL4+ neurons and in GAL4 lines from our screen which exclusively affect sleep structure, were found to contribute to fragmentation of sleep during both day and night. Thus, multiple subtypes of ring neurons distinctively control sleep amount and/or structure, and the unique highly interconnected structure of the EB and its connections with other regions of brain suggest a local-network model worth future investigation. SIGNIFICANCE STATEMENTHow multiple brain regions, with many cell types, can coherently regulate sleep remains unclear, but identification of cell type-specific roles can generate opportunities for understanding the principles of integration and cooperation. The ellipsoid body (EB) of the fly brain exhibits a high level of connectivity and functional heterogeneity yet is able to tune multiple behaviours in real-time, including sleep. Leveraging the powerful genetic tools available in Drosophila and recent progress in the characterization of the morphology and connectivity of EB ring neurons, we identify several EB subtypes specifically associated with distinct aspects of sleep. Our findings will aid in revealing the rules of coding and integration in the brain.