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Next generation sequencing of isolated R5 ellipsoid body neurons of Drosophila in the morning and evening with and without sleep deprivation

ABSTRACT: Purpose: After identifying a difference in the response to sleep deprivation between the morning and evening, we assayed gene expression in a key homeostatic circuit in the Drosophila brain: the R5 ellipsoid body neurons. We sought to identify changes in gene expression in this circuit that are critical for the behavioral output. Methods: Flies expressing GFP via a restrictive R5 driver were entrained to a 12:12 light:dark paradigm at 25C. Mated females were then loaded into individual tubes with sucrose food and placed into behavior boards from Trinkinetics. Control flies were not subjected to any sleep deprivation and were collected in either the morning or evening. Experimental flies were subjected to 2.5 hrs of mechanical sleep deprivation (SD) via vortexing just before either the morning or evening. Following SD (or at the appropriate control timepoint), 40-50 brains were dissected out in dissecting saline with TTX, AP5, and DNQX to inhibit sodium channels, NMDA receptors, and AMPA receptors respectively. The brains were transferred to SM Active medium with channel blockers after dissection, then washed with dissecting saline before being digested with papain. Digestion was stopped after 30 minutes by the addition of SM Active medium with channel blockers. The brains were then triturated by manual pipetting to produce a single cell suspension. GFP+ cells were immediately sorted by fluorescence activated cell sorting into a single tube. 350-500 cells were collected per experiment. In total, we collected 12 samples, three replicates for each timepoint and condition. Cells were lysed in extraction buffer from the Arcturus PicoPure kit and stored at -80C until ready to process. RNA was extracted from each set of cells using the PicoPure kit, and then reverse transcribed into cDNA using a T7/polydT primer. One round of in vitro transcription using the MEGAScript kit was performed to amplify the RNA. Following IVT, library prep was performed using the TruSeq Stranded mRNA Library Prep Kit. We generated 30 million reads per sample on a NovaSeq. Analysis was performed using kallisto and sleuth.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE186076 | GEO | 2021/10/20

REPOSITORIES: GEO

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Project description:Extended periods of waking result in physiological impairments in humans, rats, and flies. Sleep homeostasis, the increase in sleep observed following sleep loss, is believed to counter the negative effects of prolonged waking by restoring vital biological processes that are degraded during sleep deprivation. Sleep homeostasis, as with other behaviors, is influenced by both genes and environment. We report here that during periods of starvation, flies remain spontaneously awake but, in contrast to sleep deprivation, do not accrue any of the negative consequences of prolonged waking. Specifically, the homeostatic response and learning impairments that are a characteristic of sleep loss are not observed following prolonged waking induced by starvation. To identify the genes responsible for the protective effects of starvation we conducted transcription profiling of sleep deprived flies that accrue sleep debt compared to starved siblings that do not. Genes involved in lipid metabolism were highly enriched in our dataset of 84 differentially regulated transcripts. Follow up genetic studies established that 6 genes involved in lipid metabolism strongly influence sleep homeostasis. Two of these genes, brummer (bmm) and Lipid storage droplet 2 (Lsd2), are in the same lipolysis pathway but exert antagonistic effects on lipid storage. bmm mutant flies have excess fat stores and display a large homeostatic response following sleep deprivation. In contrast, Lsd2 mutant flies, which phenocopy aspects of starvation as measured by low triglyceride stores, do not exhibit a homeostatic response following sleep loss. Importantly, Lsd2 mutant flies are not learning impaired after sleep deprivation. These results provide the first genetic evidence, to our knowledge, that lipid metabolism plays an important role in regulating the homeostatic response and can protect against neuronal impairments induced by prolonged waking. Two-condition experiments: sleep deprived vs starved. RNA from 8 biological replicates for each condition was pooled in groups of 2 to create 4 samples. Each of the 4 samples is run in duplicate with untreated circadian matched controls.

Project description:Although the specific functions of sleep have not been completely elucidated, the literature has suggested that sleep is essential for proper homeostasis. Sleep loss is associated with changes in behavioral, neurochemical, cellular, and metabolic functions as well as impaired immune response. We evaluated the gene expression profiles of healthy volunteers submitted to 48 hours of prolonged wakefulness (PW) followed by 12 hours of sleep recovery (SR) using high-resolution microarrays. Peripheral whole blood was collected in the morning before the initiation of sleep deprivation (baseline), after the second night of PW, and one night after SR. the identified differentially expressed genes were related to immune response, DNA damage and repair as well as inflammation. Examples of these include: killer cell lectin-like receptors family granzymes and T-cell receptors, which play important roles in host defense. These results support the idea that sleep loss can lead to alteration of molecular processes that drive perturbation of cellular immunity, induction of inflammatory response and homeostatic imbalance. Moreover, down-regulation of multiple genes after prolonged wakefulness (in comparison with baseline condition) and up-regulated after sleep recovery (in comparison with prolonged wakefulness condition) were observed, suggesting an attempt of the body to re-establish internal homeostasis. In silico validation of alterations in the expression of CETN3, DNAJC, IGFR2B and CEACAM genes, confirmed the previous findings related to the molecular effects of sleep deprivation. It is clear that confirmatory studies will be necessary to fully validate the potential candidate genes and functional networks identified. Nevertheless, the present findings confirm that the effects of sleep deprivation are not restricted to the brain and can occur intensely in peripheral tissues. The peripheral blood from each volunteer (nine individuals) were collected in the baseline night and every morning after PW and after the night of SR.

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Next generation sequencing of isolated R5 ellipsoid body neurons of Drosophila in the morning and evening with and without sleep deprivation

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