Circadian programming of the ellipsoid body sleep homeostat in Drosophila.
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ABSTRACT:
SUBMITTER: Andreani T
PROVIDER: S-EPMC9270026 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:One output arm of the sleep homeostat in Drosophila appears to be a group of neurons with projections to the dorsal fan-shaped body (dFB neurons) of the central complex in the brain. However, neurons that regulate the sleep homeostat remain poorly understood. Using neurogenetic approaches combined with Ca2+ imaging, we characterized synaptic connections between dFB neurons and distinct sets of upstream sleep-regulatory neurons. One group of the sleep-promoting upstream neurons is a set of circadian pacemaker neurons that activates dFB neurons via direct glutaminergic excitatory synaptic connections. Opposing this population, a group of arousal-promoting neurons downregulates dFB axonal output with dopamine. Co-activating these two inputs leads to frequent shifts between sleep and wake states. We also show that dFB neurons release the neurotransmitter GABA and inhibit octopaminergic arousal neurons. We propose that dFB neurons integrate synaptic inputs from distinct sets of upstream sleep-promoting circadian clock neurons, and arousal neurons.
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Project description:Increasing ambient temperature reorganizes the Drosophila sleep pattern in a way similar to the human response to heat, increasing daytime sleep while decreasing nighttime sleep. Mutation of core circadian genes blocks the immediate increase in daytime sleep, but not the heat-stimulated decrease in nighttime sleep, when animals are in a light:dark cycle. The ability of per(01) flies to increase daytime sleep in light:dark can be rescued by expression of PER in either LNv or DN1p clock cells and does not require rescue of locomotor rhythms. Prolonged heat exposure engages the homeostat to maintain daytime sleep in the face of nighttime sleep loss. In constant darkness, all genotypes show an immediate decrease in sleep in response to temperature shift during the subjective day, implying that the absence of light input uncovers a clock-independent pro-arousal effect of increased temperature. Interestingly, the effects of temperature on nighttime sleep are blunted in constant darkness and in cry(OUT) mutants in light:dark, suggesting that they are dependent on the presence of light the previous day. In contrast, flies of all genotypes kept in constant light sleep more at all times of day in response to high temperature, indicating that the presence of light can invert the normal nighttime response to increased temperature. The effect of temperature on sleep thus reflects coordinated regulation by light, the homeostat, and components of the clock, allowing animals to reorganize sleep patterns in response to high temperature with rough preservation of the total amount of sleep.
| S-EPMC5080662 | biostudies-literature
Project description:Timing and duration of sleep are controlled by the circadian system, which keeps an ~24-h internal rhythm that entrains to environmental stimuli, and the sleep homeostat, which rises as a function of time awake. There is a normal distribution across the population in how the circadian system aligns with typical day and night resulting in varying circadian preferences called chronotypes. A portion of the variation in the population is controlled by genetics as shown by the single-gene mutations that confer extreme early or late chronotypes. Similarly, there is a normal distribution across the population in sleep duration. Genetic variations have been identified that lead to a short sleep phenotype in which individuals sleep only 4-6.5?h nightly. Negative health consequences have been identified when individuals do not sleep at their ideal circadian timing or are sleep deprived relative to intrinsic sleep need. Whether familial natural short sleepers are at risk of the health consequences associated with a short sleep duration based on population data is not known. More work needs to be done to better assess for an individual's chronotype and degree of sleep deprivation to answer these questions.
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Project description:The ellipsoid body (EB) is a major structure of the central complex of the Drosophila melanogaster brain. Twenty-two subtypes of EB ring neurons have been identified based on anatomic and morphologic characteristics by 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. Using an unbiased thermogenetic screen of EB drivers using female flies, we found the following: (1) multiple ring neurons are involved in the modulation of amount of sleep and structure in a synergistic manner; (2) analysis of data for ΔP(doze)/ΔP(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 GLM analysis correlating ring cell subtype and activity-dependent changes in sleep parameters across all lines identified several cell types significantly associated with specific sleep effects: R3p was daytime sleep-promoting, and R4m was nighttime wake-promoting; and (4) R3d cells present in 5HT7-GAL4 and in GAL4 lines, 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. The unique highly interconnected structure of the EB suggests a local-network model worth future investigation; understanding EB subtype interactions may provide insight how sleep circuits in general are structured.SIGNIFICANCE STATEMENT How 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 behaviors 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.
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Project description:Orientation, the spatial organization of animal behavior, is an essential faculty of animals. Bacteria and lower animals such as insects exhibit taxis, innate orientation behavior, directly toward or away from a directional cue. Organisms can also orient themselves at a specific angle relative to the cues. In this study, using Drosophila as a model system, we established a visual orientation conditioning paradigm based on a flight simulator in which a stationary flying fly could control the rotation of a visual object. By coupling aversive heat shocks to a fly's orientation toward one side of the visual object, we found that the fly could be conditioned to orientate toward the left or right side of the frontal visual object and retain this conditioned visual orientation. The lower and upper visual fields have different roles in conditioned visual orientation. Transfer experiments showed that conditioned visual orientation could generalize between visual targets of different sizes, compactness, or vertical positions, but not of contour orientation. Rut-Type I adenylyl cyclase and Dnc-phosphodiesterase were dispensable for visual orientation conditioning. Normal activity and scb signaling in R3/R4d neurons of the ellipsoid body were required for visual orientation conditioning. Our studies established a visual orientation conditioning paradigm and examined the behavioral properties and neural circuitry of visual orientation, an important component of the insect's spatial navigation.
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Project description:Sleep is controlled by homeostatic mechanisms, which drive sleep after wakefulness, and a circadian clock, which confers the 24-h rhythm of sleep. These processes interact with each other to control the timing of sleep in a daily cycle as well as following sleep deprivation. However, the mechanisms by which they interact are poorly understood. We show here that hugin+ neurons, previously identified as neurons that function downstream of the clock to regulate rhythms of locomotor activity, are also targets of the sleep homeostat. Sleep deprivation decreases activity of hugin+ neurons, likely to suppress circadian-driven activity during recovery sleep, and ablation of hugin+ neurons promotes sleep increases generated by activation of the homeostatic sleep locus, the dorsal fan-shaped body (dFB). Also, mutations in peptides produced by the hugin+ locus increase recovery sleep following deprivation. Transsynaptic mapping reveals that hugin+ neurons feed back onto central clock neurons, which also show decreased activity upon sleep loss, in a Hugin peptide-dependent fashion. We propose that hugin+ neurons integrate circadian and sleep signals to modulate circadian circuitry and regulate the timing of sleep.
| S-EPMC8617458 | biostudies-literature
Project description: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.
2021-10-20 | GSE186076 | GEO
Project description:The central complex (CX) is a midline-situated collection of neuropil compartments in the arthropod central brain, implicated in higher-order processes such as goal-directed navigation. Here, we provide a systematic genetic-neuroanatomical analysis of the ellipsoid body (EB), a compartment which represents a major afferent portal of the Drosophila CX. The neuropil volume of the EB, along with its prominent input compartment, called the bulb, is subdivided into precisely tessellated domains, distinguishable based on intensity of the global marker DN-cadherin. EB tangential elements (so-called ring neurons), most of which are derived from the DALv2 neuroblast lineage, predominantly interconnect the bulb and EB domains in a topographically organized fashion. Using the DN-cadherin domains as a framework, we first characterized this connectivity by Gal4 driver lines expressed in different DALv2 ring neuron (R-neuron) subclasses. We identified 11 subclasses, 6 of which correspond to previously described projection patterns, and 5 novel patterns. These subclasses both spatially (based on EB innervation pattern) and numerically (cell counts) summate to the total EB volume and R-neuron cell number, suggesting that our compilation of R-neuron subclasses approaches completion. EB columnar elements, as well as non-DALv2 derived extrinsic ring neurons (ExR-neurons), were also incorporated into this anatomical framework. Finally, we addressed the connectivity between R-neurons and their targets, using the anterograde trans-synaptic labeling method, trans-Tango. This study demonstrates putative interactions of R-neuron subclasses and reveals general principles of information flow within the EB network. Our work will facilitate the generation and testing of hypotheses regarding circuit interactions within the EB and the rest of the CX.
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Project description:Complex behaviors arise from neural circuits that are assembled from diverse cell types. Sleep is a conserved and essential behavior, yet little is known regarding how the nervous system generates neuron types of the sleep-wake circuit. Here, we focus on the specification of Drosophila sleep-promoting neurons-long-field tangential input neurons that project to the dorsal layers of the fan-shaped body neuropil in the central complex (CX). We use lineage analysis and genetic birth dating to identify two bilateral Type II neural stem cells that generate these dorsal fan-shaped body (dFB) neurons. We show that adult dFB neurons express Ecdysone-induced protein E93, and loss of Ecdysone signaling or E93 in Type II NSCs results in the misspecification of the adult dFB neurons. Finally, we show that E93 knockdown in Type II NSCs affects adult sleep behavior. Our results provide insight into how extrinsic hormonal signaling acts on NSCs to generate neuronal diversity required for adult sleep behavior. These findings suggest that some adult sleep disorders might derive from defects in stem cell-specific temporal neurodevelopmental programs.
| S-EPMC10592846 | biostudies-literature
Project description:In humans and many other animals, memory consolidation occurs through multiple temporal phases and usually involves more than one neuroanatomical brain system. Genetic dissection of Pavlovian olfactory learning in Drosophila melanogaster has revealed multiple memory phases, but the predominant view holds that all memory phases occur in mushroom body neurons. Here, we demonstrate an acute requirement for NMDA receptors (NMDARs) outside of the mushroom body during long-term memory (LTM) consolidation. Targeted dsRNA-mediated silencing of Nmdar1 and Nmdar2 (also known as dNR1 or dNR2, respectively) in cholinergic R4m-subtype large-field neurons of the ellipsoid body specifically disrupted LTM consolidation, but not retrieval. Similar silencing of functional NMDARs in the mushroom body disrupted an earlier memory phase, leaving LTM intact. Our results clearly establish an anatomical site outside of the mushroom body involved with LTM consolidation, thus revealing both a distributed brain system subserving olfactory memory formation and the existence of a system-level memory consolidation in Drosophila.
| S-EPMC3055246 | biostudies-literature