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  1. Evaluation Summary:

    This manuscript addresses important questions: what is the relationship between circadian and homeostatic regulation of sleep, and how do the circuits underlying these levels of control interact? The authors have designed a very elegant method to answer these questions in Drosophila: a new sleep-deprivation protocol that allows them to test sleep rebound over the course of the day. Interesting observations are made, such as time-of-day dependence of sleep homeostasis, identification of important neural pathways modulating sleep rebound in a time-dependent manner, and molecular and physiological variations that might drive time-dependent sleep homeostasis. Experiments establishing a link between the circadian clock/neurons and molecular and physiological changes observed in sleep homeostat neurons would help to provide support for the claims made in the title and abstract.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    Andreani et al. perform an array of experiments to identify putative interactions between neural circuits comprising the circadian clock and the sleep homeostat in the Drosophila nervous system.

    The authors' core hypothesis is that the circadian clock modulates the magnitude of sleep rebound (an output of the sleep homeostat) following sleep deprivation (SD) in a time-of-day-dependent manner. This is certainly a fascinating hypothesis that would be of great interest to address.

    By performing short episodes of SD across differing timepoints during the day/night cycle, they suggest that clock-dependent control of sleep homeostat output does occur. They identify a variety of circadian circuits (and downstream output pathways) that they propose bi-directionally regulate the degree of sleep rebound in the morning (high rebound) versus the evening (low rebound). They then utilise cell-specific RNAseq data and quantitative immuno-staining of presynaptic protein expression to propose a clock-dependent enhancement of excitability or synaptic plasticity in Ellipsoid Body (EB) R5-neurons - an important cellular component of the sleep homeostat (Liu et al., (2016) Cell).

    The strengths of this manuscript include:

    - The design of a novel paradigm to examine temporal changes in sleep rebound.
    - The generation of new tools to examine sleep-relevant neural circuits.
    - The production of a large gene expression dataset from EB R5-neurons (the subject of intense study in the Drosophila sleep field) across different timepoints and sleep levels.

    Two concerns are noted about the current manuscript. Firstly, one concern is that the differences in morning vs evening sleep rebound simply reflect the fact that during the evening period, female flies are initially highly active (during evening anticipation), and hence do not lose sleep during SD; and subsequently sleep at near maximal levels after lights-off, with little room for additional rebound. During the morning rebound phase, in contrast, the lower levels of sleep provide more room for rebound to occur. Secondly, the idea that subsets of the clock neuron network drive temporal changes in excitability and gene expression within sleep homeostat EB R5 neurons is not experimentally supported.

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  3. Reviewer #2 (Public Review):

    Sleep is regulated by two major processes: homeostasis and control of sleep timing by the circadian clock. The present manuscript addresses important questions: what is the relationship between circadian and homeostatic regulations of sleep, and how do the circuits underlying these levels of control interact? The authors used the fruit fly Drosophila to address these questions. Indeed, Drosophila is a remarkably potent model organism to dissect the basic mechanisms underlying sleep.

    A major strength of the manuscript is the well-designed protocol to test sleep homeostasis over the course of the day. The results are very convincing that sleep rebound is much stronger in the morning than in the evening, and that the circadian clock controls this rhythm in sleep homeostasis. Also convincing is the data uncovering a pathway linking "morning" circadian neurons (DN1s) to sleep control centers, and how this circuit promotes morning sleep rebound. The gene expression studies in homeostat neurons (EB-R5) interestingly identify genes linked to neural activity that show differential expression in the morning and evening. Moreover, molecular and physiological markers support the idea of differential activity of these neurons as a function of time-of-day, which could explain how sleep homeostasis is controlled in a time-dependent manner.

    The main weaknesses of this manuscript are the lack of direct evidence that molecular and physiological changes observed in R5 neurons are under clock and circadian neuron control, and the uncertain nature of how the "evening' neurons communicate with the sleep homeostat circuit.

    In summary, this manuscript presents very interesting observations related to sleep homeostasis and its circadian control, but it is not yet clear how these observations fit together to explain how the circadian clock controls sleep.

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