Biology subjects

Shafer, O. T.

Publications and source records attributed to Shafer, O. T..

9 recordsLinked to original sources

bioRxiv2025

The recognition of behaviorally distinct sleep stages in Drosophila melanogaster uncovers previously obscured homeostatic and circadian control of sleep.

Understanding the mechanisms underlying homeostatic sleep regulation is a central unmet goal of sleep science. Our comprehension of such regulation in mammals has required recognizing distinct sleep stages. Drosophila melanogaster is an important genetic model system for studying sleep. Since the discovery of sleep-like states in the fly 25 years ago, the field has treated sleep as a unitary state consisting of any inactivity lasting 5 minutes or longer, despite convergent work suggesting the existence of multiple sleep states. Here, we establish that three distinct sleep states in flies can be classified based on simple inactivity duration criteria. We show that the daily initiation of these sleep states is temporally distinct, with long sleep occurring immediately following the largest daily period of wakefulness. We also report that rebound in response to mechanical sleep deprivation is present only in long sleep and comes at the expense of shorter sleep states. Deprivation-induced decreases in shorter sleep states obscure homeostatic sleep rebound, but only when sleep is measured using traditional methods. We observe distinctly timed ultradian oscillations of fly sleep states, reminiscent of mammalian sleep cycles. Our results indicate that the recognition of such sleep states will be necessary to fully realize the promise of the Drosophila model system for identifying conserved genetic mechanisms underlying such regulation.

animal behavior and cognition↗

bioRxiv2023

Stem cell-specific ecdysone signaling regulates the development and function of a Drosophila sleep homeostat

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.

neuroscience↗

bioRxiv2022

Homeostatic control of deep sleep in Drosophila: Implications for Discovering Correlates of Sleep Pressure

Homeostatic control of sleep is typically addressed through mechanical stimulation-induced forced wakefulness and the measurement of subsequent increases in sleep. A major confound attends this approach: biological responses to deprivation may reflect a direct response to the mechanical insult rather than to the loss of sleep. Similar confounds accompany all forms of sleep deprivation and represent a major challenge to the field. Here we describe a new paradigm for sleep deprivation in Drosophila that fully accounts for sleep-independent effects. Our results reveal that deep sleep states are the primary target of homeostatic control and establish the presence of multi-cycle sleep rebound following deprivation. Furthermore, we establish that specific deprivation of deep sleep state results in state-specific homeostatic rebound. Finally, by accounting for the molecular effects of mechanical stimulation during deprivation experiments, we show that serotonin levels track sleep pressure in the flys central brain. Our results illustrate the critical need to control for sleep-independent effects of deprivation when examining the molecular correlates of sleep pressure and call for a critical reassessment of work that has not accounted for such non-specific effects.

animal behavior and cognition↗

bioRxiv2022

Circadian Regulation of Sleep in Drosophila: Insights from a Two-Process Model

Sleep is controlled by two processes - a circadian clock that regulates its timing and a homeostat that regulates the drive to sleep. Drosophila has been an insightful model for understanding both processes. For four decades, Borbely and Daans two-process model has provided a powerful framework for understanding how circadian and homeostatic processes regulate sleep. However, the field of fly sleep has not employed such a model as a framework for the investigation of sleep. To this end, we have adapted the two-process model to the fly and establish its utility by showing that it can provide empirically testable predictions regarding the circadian and homeostatic control of fly sleep. We show that the ultradian rhythms previously reported for loss-of-function clock mutants are a predictable consequence of a functional sleep homeostat in the absence of a functioning circadian system. We find that a model in which the circadian clock speed and homeostatic rates act without influencing each other provides imprecise predictions regarding how clock speed influences the strength of sleep rhythms and the amount of daily sleep. We also find that quantitatively good fits between empirical values and model predictions were achieved only when clock speeds were positively correlated with rates of decay of sleep pressure. Our results indicate that longer sleep bouts better reflect the homeostatic process than the current definition of sleep as any inactivity lasting five minutes or more. This two-process model represents a powerful framework for future work on the molecular and physiological regulation of fly sleep.

animal behavior and cognition↗

bioRxiv2022

Parametric effects of light acting via multiple photoreceptor types contribute significantly to circadian entrainment in Drosophila melanogaster.

Circadian rhythms in physiology and behavior have near 24-hour periodicities that must adjust to the exact 24-hour geophysical cycles on earth to ensure adaptive daily timing. Such adjustment is called entrainment. One major mode of entrainment is via the continuous modulation of circadian period by the prolonged presence of light. Although Drosophila melanogaster is a prominent insect model of chronobiology, there is little evidence for such continuous effects of light in the species. In this study, we demonstrate that prolonged light exposure at specific times of the day shapes the daily timing of activity in flies. We also establish that continuous blue- and UV-blocked light lengthens the circadian period of Drosophila and provide evidence that this is produced by the combined action of multiple photoreceptors which, includes the cell autonomous photoreceptor cryptochrome. Finally, we introduce ramped light cycles as an entrainment paradigm that produces light entrainment that lacks the large light-driven startle responses typically displayed by flies and requires multiple days for entrainment to shifted cycles. These features are reminiscent of entrainment in mammalian models systems and make possible new experimental approaches to understanding the mechanisms underlying entrainment in the fly.

genetics↗

bioRxiv2022

Connectomic Analysis of the Drosophila Lateral Neuron Clock Cells Reveals the Synaptic Basis of Functional Pacemaker Classes.

The circadian clock orchestrates daily changes in physiology and behavior to ensure internal temporal order and optimal timing across the day. In animals, a central brain clock coordinates circadian rhythms throughout the body and is characterized by a remarkable robustness that depends on synaptic connections between constituent neurons. The clock neuron network of Drosophila, which shares network motifs with clock networks in the mammalian brain yet is built of many fewer neurons, offers a powerful model for understanding the network properties of circadian timekeeping. Here we report an assessment of synaptic connectivity within a clock network, focusing on the critical lateral neuron (LN) clock neuron classes. Our results reveal that previously identified anatomical and functional subclasses of LNs represent distinct connectomic types. Moreover, we identify a small number of clock cell subtypes representing highly synaptically coupled nodes within the clock neuron network. This suggests that neurons lacking molecular timekeeping likely play integral roles within the circadian timekeeping network. To our knowledge, this represents the first comprehensive connectomic analysis of a circadian neuronal network.

neuroscience↗

bioRxiv2021

PHASE: A MATLAB Based Program for the Analysis of Drosophila Phase, Activity, and Sleep under Entrainment

The problem of entrainment is central to circadian biology. In this regard, Drosophila has been an important model system. Owing to the simplicity of its nervous system and the availability of powerful genetic tools, the system has shed significant light on the molecular and neural underpinnings of entrainment. However, much remains to be learned regarding the molecular and physiological mechanisms underlying this important phenomenon. Under cyclic light/dark conditions, Drosophila melanogaster displays crepuscular patterns of locomotor activity with one peak anticipating dawn and the other anticipating dusk. These peaks are characterized through an estimation of their phase relative to the environmental light cycle and the extent of their anticipation of light transitions. In Drosophila chronobiology, estimations of phases are often subjective, and anticipation indices vary significantly between studies. Though there is increasing interest in building flexible analysis software tools in the field, none incorporates objective measures of Drosophila activity peaks in combination with the analysis of fly activity/sleep in the same program. To this end, we have developed PHASE, a MATLAB-based program that is simple and easy to use and (i) supports the visualization and analysis of activity and sleep under entrainment, (ii) allows analysis of both activity and sleep parameters within user-defined windows within a diurnal cycle, (iii) uses a smoothing filter for the objective identification of peaks of activity (and therefore can be used to quantitatively characterize them), and (iv) offers a series of analyses for the assessment of behavioral anticipation of environmental transitions.

animal behavior and cognition↗

bioRxiv2020

Dopamine signaling in wake promoting clock neurons is not required for the normal regulation of sleep in Drosophila

Dopamine is a wakefulness promoting neuromodulator in mammals and fruit flies. In D. melanogaster, the network of clock neurons that drives sleep/activity cycles comprises both wake and sleep promoting cell types, indicating that the sleep-wake circuitry is intimately linked to the circadian clock. The large and small ventrolateral neurons (l-LNvs and s-LNvs) have been identified as wake-promoting neurons within the clock neuron network. The l-LNvs are innervated by dopaminergic neurons, and earlier work proposed that dopamine signaling raises cAMP levels in the l-LNvs and thus induces excitatory electrical activity (action potential firing), which results in wakefulness and inhibits sleep. Here, we test this hypothesis by combining cAMP imaging and patch-clamp recordings in isolated brains. We find that dopamine application indeed increases cAMP levels and depolarizes the l-LNvs, but surprisingly, it does not result in increased firing rates. Down-regulation of the excitatory dopamine receptor, Dop1R1 in the l-and s-LNvs, but not of Dop1R2, abolished the depolarization of l-LNvs in response to dopamine. This indicates that dopamine signals via Dop1R1 to the l-LNvs. Down-regulation of Dop1R1 or Dop1R2 receptors in the l- and s-LNvs does not affect sleep. Unexpectedly, we find a moderate decrease of daytime sleep with down-regulation of Dop1R1 and of nighttime sleep with down-regulation of Dop1R2. Since the l-LNvs do not utilize Dop1R2 receptors and the s-LNvs respond also to dopamine, we conclude that the s-LNvs are responsible for the observed decrease in nighttime sleep. In summary, dopamine signaling in the wake-promoting LNvs is not required for daytime arousal, but likely promotes nighttime sleep via the s-LNvs. Significance statementIn insect and mammalian brains, sleep promoting networks are intimately linked to the circadian clock, and the mechanisms underlying sleep and circadian timekeeping are evolutionarily ancient and highly conserved. Here we show that dopamine, one important sleep modulator in flies and mammals, plays surprisingly complex roles in the regulation of sleep by clock containing neurons. Dopamine inhibits neurons in a central brain sleep center to promote sleep and excites wake-promoting circadian clock neurons. It is therefore predicted to promote wakefulness through both of these networks. Nevertheless, our results reveal that dopamine acting on wake promoting clock neurons promotes sleep, revealing a previously unappreciated complexity in the dopaminergic control of sleep.

neuroscience↗

bioRxiv2019

Sites of Circadian Clock Neuron Plasticity Mediate Sensory Integration and Entrainment.

Networks of circadian timekeeping in the brain display marked daily changes in neuronal morphology. In Drosophila melanogaster, the striking daily structural remodeling of the dorsal medial termini of the small ventral lateral neurons has long been hypothesized to mediate endogenous circadian timekeeping. To test this model, we have specifically abrogated these sites of daily neuronal remodeling through the reprogramming of neural development and assessed the effects on circadian timekeeping and clock outputs. Remarkably, the loss of these sites has no measurable effects on endogenous circadian timekeeping or on any of the major output functions of the small ventral lateral neurons. Rather, their loss reduces sites of glutamatergic sensory neurotransmission that normally encodes naturalistic time-cues from the environment. These results support an alternative model: structural plasticity in critical clock neurons is the basis for proper integration of light and temperature and gates sensory inputs into circadian clock neuron networks.

neuroscience↗