Circadian clock neurons constantly monitor environmental temperature to set sleep timing.

Circadian clocks coordinate behaviour, physiology and metabolism with Earth's diurnal cycle. These clocks entrain to both light and temperature cycles, and daily environmental temperature oscillations probably contribute to human sleep patterns. However, the neural mechanisms through which circadian clocks monitor environmental temperature and modulate behaviour remain poorly understood. Here we elucidate how the circadian clock neuron network of Drosophila melanogaster processes changes in environmental temperature. In vivo calcium-imaging techniques demonstrate that the posterior dorsal neurons 1 (DN1ps), which are a discrete subset of sleep-promoting clock neurons, constantly monitor modest changes in environmental temperature. We find that these neurons are acutely inhibited by heating and excited by cooling; this is an unexpected result when considering the strong correlation between temperature and light, and the fact that light excites clock neurons. We demonstrate that the DN1ps rely on peripheral thermoreceptors located in the chordotonal organs and the aristae. We also show that the DN1ps and their thermosensory inputs are required for the normal timing of sleep in the presence of naturalistic temperature cycles. These results identify the DN1ps as a major gateway for temperature sensation into the circadian neural network, which continuously integrates temperature changes to coordinate the timing of sleep and activity.

Parametric effects of light acting via multiple photoreceptors contribute to circadian entrainment in Drosophila melanogaster.

Circadian rhythms in physiology and behaviour have near 24 h periodicities that must adjust to the exact 24 h 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 UV- and blue-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.

Dopamine Signaling in Wake-Promoting Clock Neurons Is Not Required for the Normal Regulation of Sleep in Drosophila.

Dopamine is a wake-promoting neuromodulator in mammals and fruit flies. In Drosophila melanogaster, the network of clock neurons that drives sleep/activity cycles comprises both wake-promoting and sleep-promoting cell types. The large ventrolateral neurons (l-LNvs) and small ventrolateral neurons (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. Downregulation of the excitatory D1-like dopamine receptor (Dop1R1) in the l-LNvs 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. Downregulation of Dop1R1 or Dop1R2 in the l-LNvs and s-LNvs does not affect sleep in males. Unexpectedly, we find a moderate decrease of daytime sleep with downregulation of Dop1R1 and of nighttime sleep with downregulation of Dop1R2. Since the l-LNvs do not use Dop1R2 receptors and the s-LNvs also respond 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 STATEMENT In 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.

RNA-seq analysis of Drosophila clock and non-clock neurons reveals neuron-specific cycling and novel candidate neuropeptides.

Locomotor activity rhythms are controlled by a network of ~150 circadian neurons within the adult Drosophila brain. They are subdivided based on their anatomical locations and properties. We profiled transcripts "around the clock" from three key groups of circadian neurons with different functions. We also profiled a non-circadian outgroup, dopaminergic (TH) neurons. They have cycling transcripts but fewer than clock neurons as well as low expression and poor cycling of clock gene transcripts. This suggests that TH neurons do not have a canonical circadian clock and that their gene expression cycling is driven by brain systemic cues. The three circadian groups are surprisingly diverse in their cycling transcripts and overall gene expression patterns, which include known and putative novel neuropeptides. Even the overall phase distributions of cycling transcripts are distinct, indicating that different regulatory principles govern transcript oscillations. This surprising cell-type diversity parallels the functional heterogeneity of the different neurons.

A Neural Network Underlying Circadian Entrainment and Photoperiodic Adjustment of Sleep and Activity in Drosophila.

UNLABELLED: A sensitivity of the circadian clock to light/dark cycles ensures that biological rhythms maintain optimal phase relationships with the external day. In animals, the circadian clock neuron network (CCNN) driving sleep/activity rhythms receives light input from multiple photoreceptors, but how these photoreceptors modulate CCNN components is not well understood. Here we show that the Hofbauer-Buchner eyelets differentially modulate two classes of ventral lateral neurons (LNvs) within the Drosophila CCNN. The eyelets antagonize Cryptochrome (CRY)- and compound-eye-based photoreception in the large LNvs while synergizing CRY-mediated photoreception in the small LNvs. Furthermore, we show that the large LNvs interact with subsets of "evening cells" to adjust the timing of the evening peak of activity in a day length-dependent manner. Our work identifies a peptidergic connection between the large LNvs and a group of evening cells that is critical for the seasonal adjustment of circadian rhythms. SIGNIFICANCE STATEMENT: In animals, circadian clocks have evolved to orchestrate the timing of behavior and metabolism. Consistent timing requires the entrainment these clocks to the solar day, a process that is critical for an organism's health. Light cycles are the most important external cue for the entrainment of circadian clocks, and the circadian system uses multiple photoreceptors to link timekeeping to the light/dark cycle. How light information from these photorecptors is integrated into the circadian clock neuron network to support entrainment is not understood. Our results establish that input from the HB eyelets differentially impacts the physiology of neuronal subgroups. This input pathway, together with input from the compound eyes, precisely times the activity of flies under long summer days. Our results provide a mechanistic model of light transduction and integration into the circadian system, identifying new and unexpected network motifs within the circadian clock neuron network.

A homeostatic sleep-stabilizing pathway in Drosophila composed of the sex peptide receptor and its ligand, the myoinhibitory peptide.

Sleep, a reversible quiescent state found in both invertebrate and vertebrate animals, disconnects animals from their environment and is highly regulated for coordination with wakeful activities, such as reproduction. The fruit fly, Drosophila melanogaster, has proven to be a valuable model for studying the regulation of sleep by circadian clock and homeostatic mechanisms. Here, we demonstrate that the sex peptide receptor (SPR) of Drosophila, known for its role in female reproduction, is also important in stabilizing sleep in both males and females. Mutants lacking either the SPR or its central ligand, myoinhibitory peptide (MIP), fall asleep normally, but have difficulty in maintaining a sleep-like state. Our analyses have mapped the SPR sleep function to pigment dispersing factor (pdf) neurons, an arousal center in the insect brain. MIP downregulates intracellular cAMP levels in pdf neurons through the SPR. MIP is released centrally before and during night-time sleep, when the sleep drive is elevated. Sleep deprivation during the night facilitates MIP secretion from specific brain neurons innervating pdf neurons. Moreover, flies lacking either SPR or MIP cannot recover sleep after the night-time sleep deprivation. These results delineate a central neuropeptide circuit that stabilizes the sleep state by feeding a slow-acting inhibitory input into the arousal system and plays an important role in sleep homeostasis.

Differentially timed extracellular signals synchronize pacemaker neuron clocks.

Synchronized neuronal activity is vital for complex processes like behavior. Circadian pacemaker neurons offer an unusual opportunity to study synchrony as their molecular clocks oscillate in phase over an extended timeframe (24 h). To identify where, when, and how synchronizing signals are perceived, we first studied the minimal clock neural circuit in Drosophila larvae, manipulating either the four master pacemaker neurons (LNvs) or two dorsal clock neurons (DN1s). Unexpectedly, we found that the PDF Receptor (PdfR) is required in both LNvs and DN1s to maintain synchronized LNv clocks. We also found that glutamate is a second synchronizing signal that is released from DN1s and perceived in LNvs via the metabotropic glutamate receptor (mGluRA). Because simultaneously reducing Pdfr and mGluRA expression in LNvs severely dampened Timeless clock protein oscillations, we conclude that the master pacemaker LNvs require extracellular signals to function normally. These two synchronizing signals are released at opposite times of day and drive cAMP oscillations in LNvs. Finally we found that PdfR and mGluRA also help synchronize Timeless oscillations in adult s-LNvs. We propose that differentially timed signals that drive cAMP oscillations and synchronize pacemaker neurons in circadian neural circuits will be conserved across species.

Remote control of renal physiology by the intestinal neuropeptide pigment-dispersing factor in Drosophila.

The role of the central neuropeptide pigment-dispersing factor (PDF) in circadian timekeeping in Drosophila is remarkably similar to that of vasoactive intestinal peptide (VIP) in mammals. Like VIP, PDF is expressed outside the circadian network by neurons innervating the gut, but the function and mode of action of this PDF have not been characterized. Here we investigate the visceral roles of PDF by adapting cellular and physiological methods to the study of visceral responses to PDF signaling in wild-type and mutant genetic backgrounds. We find that intestinal PDF acts at a distance on the renal system, where it regulates ureter contractions. We show that PdfR, PDF's established receptor, is expressed by the muscles of the excretory system, and present evidence that PdfR-induced cAMP increases underlie the myotropic effects of PDF. These findings extend the similarities between PDF and VIP beyond their shared central role as circadian regulators, and uncover an unexpected endocrine mode of myotropic action for an intestinal neuropeptide on the renal system.

A two-process model of Drosophila sleep reveals an inter-dependence between circadian clock speed and the rate of sleep pressure decay.

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, Borbély and Daan's two-process model has provided a powerful framework for understanding sleep regulation. 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 established 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 in the fly are robustly detectable and 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 5 minutes or more. This two-process model represents a powerful framework for work on the molecular and physiological regulation of fly sleep.

Sensitive Timing: A Reappraisal of Chronobiology's Foundational Texts.

The origin of experimental chronobiology can be traced to observations made in the 18th and 19th centuries on the sensitive plant Mimosa, which were described in two seminal reports: Jean-Jacques d'Ortous de Mairan's "Observation Botanique" (A Botanical Observation) and Augustin Pyramus de Candolle's "Du sommeil des feuilles" (On the sleep of leaves). Both report observations of the striking daily closing and opening of Mimosa leaves in controlled environments. This review presents translations of both texts with the aim of staying as faithful as possible to the original French texts. We also present the historical context in which these texts were written and link them to subsequent experiments that aimed at testing the veracity of their central conclusions. In particular, we definitely establish that Mairan himself presented his work to the French Royal Academy of Sciences, while the published report of his observation was authored by Fontenelle, the Secretary of the Academy. In addition, we offer a translation of Mairan's own presentation, based on the hand-written minutes of the academy. Finally, we discuss the decades of work on plant rhythms that laid the foundation for modern experimental chronobiology, including translations and discussion of the insightful and prescient reports by Charles François de Cisternay Dufay, Henri Louis Duhamel du Monceau, Johann Gottfried Zinn, and Wilhelm Pfeffer, which describe their efforts to reproduce and extend Mairan's pioneering observations.

Homeostatic control of deep sleep and molecular correlates of sleep pressure in Drosophila.

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 states 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 fly's 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.

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.

Reciprocal cholinergic and GABAergic modulation of the small ventrolateral pacemaker neurons of Drosophila's circadian clock neuron network.

The relatively simple clock neuron network of Drosophila is a valuable model system for the neuronal basis of circadian timekeeping. Unfortunately, many key neuronal classes of this network are inaccessible to electrophysiological analysis. We have therefore adopted the use of genetically encoded sensors to address the physiology of the fly's circadian clock network. Using genetically encoded Ca(2+) and cAMP sensors, we have investigated the physiological responses of two specific classes of clock neuron, the large and small ventrolateral neurons (l- and s-LN(v)s), to two neurotransmitters implicated in their modulation: acetylcholine (ACh) and γ-aminobutyric acid (GABA). Live imaging of l-LN(v) cAMP and Ca(2+) dynamics in response to cholinergic agonist and GABA application were well aligned with published electrophysiological data, indicating that our sensors were capable of faithfully reporting acute physiological responses to these transmitters within single adult clock neuron soma. We extended these live imaging methods to s-LN(v)s, critical neuronal pacemakers whose physiological properties in the adult brain are largely unknown. Our s-LN(v) experiments revealed the predicted excitatory responses to bath-applied cholinergic agonists and the predicted inhibitory effects of GABA and established that the antagonism of ACh and GABA extends to their effects on cAMP signaling. These data support recently published but physiologically untested models of s-LN(v) modulation and lead to the prediction that cholinergic and GABAergic inputs to s-LN(v)s will have opposing effects on the phase and/or period of the molecular clock within these critical pacemaker neurons.

Analysis of functional neuronal connectivity in the Drosophila brain.

Drosophila melanogaster is a valuable model system for the neural basis of complex behavior, but an inability to routinely interrogate physiologic connections within central neural networks of the fly brain remains a fundamental barrier to progress in the field. To address this problem, we have introduced a simple method of measuring functional connectivity based on the independent expression of the mammalian P2X2 purinoreceptor and genetically encoded Ca(2+) and cAMP sensors within separate genetically defined subsets of neurons in the adult brain. We show that such independent expression is capable of specifically rendering defined sets of neurons excitable by pulses of bath-applied ATP in a manner compatible with high-resolution Ca(2+) and cAMP imaging in putative follower neurons. Furthermore, we establish that this approach is sufficiently sensitive for the detection of excitatory and modulatory connections deep within larval and adult brains. This technically facile approach can now be used in wild-type and mutant genetic backgrounds to address functional connectivity within neuronal networks governing a wide range of complex behaviors in the fly. Furthermore, the effectiveness of this approach in the fly brain suggests that similar methods using appropriate heterologous receptors might be adopted for other widely used model systems.

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 within the Janelia hemibrain dataset. Our results reveal that previously identified anatomical and functional subclasses of LNs represent distinct connectomic types. Moreover, we identify a small number of non-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.

Most organisms on Earth possess an internal timekeeping system which ensures that bodily processes such as sleep, wakefulness or digestion take place at the right time. These precise daily rhythms are kept in check by a master clock in the brain. There, thousands of neurons ­ some of which carrying an internal 'molecular clock' ­ connect to each other through structures known as synapses. Exactly how the resulting network is organised to support circadian timekeeping remains unclear. To explore this question, Shafer, Gutierrez et al. focused on fruit flies, as recent efforts have systematically mapped every neuron and synaptic connection in the brain of this model organism. Analysing available data from the hemibrain connectome project at Janelia revealed that that the neurons with the most important timekeeping roles were in fact forming the fewest synapses within the network. In addition, neurons without internal molecular clocks mediated strong synaptic connections between those that did, suggesting that 'clockless' cells still play an integral role in circadian timekeeping. With this research, Shafer, Gutierrez et al. provide unexpected insights into the organisation of the master body clock. Better understanding the networks that underpin circadian rhythms will help to grasp how and why these are disrupted in obesity, depression and Alzheimer's disease.

PHASE: An Open-Source Program for the Analysis of DrosophilaPhase, 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 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.

The Regulation of Drosophila Sleep.

Sleep is critical for diverse aspects of brain function in animals ranging from invertebrates to humans. Powerful genetic tools in the fruit fly Drosophila melanogaster have identified - at an unprecedented level of detail - genes and neural circuits that regulate sleep. This research has revealed that the functions and neural principles of sleep regulation are largely conserved from flies to mammals. Further, genetic approaches to studying sleep have uncovered mechanisms underlying the integration of sleep and many different biological processes, including circadian timekeeping, metabolism, social interactions, and aging. These findings show that in flies, as in mammals, sleep is not a single state, but instead consists of multiple physiological and behavioral states that change in response to the environment, and is shaped by life history. Here, we review advances in the study of sleep in Drosophila, discuss their implications for understanding the fundamental functions of sleep that are likely to be conserved among animal species, and identify important unanswered questions in the field.

Open-source computational framework for studying Drosophila behavioral phase.

This protocol describes a standardized method for analyzing Drosophila behavioral rhythmicity under light dark cycles, temperature ramps, and free running conditions. The protocol constitutes a step-by-step guide from generation of appropriate Drosophila genetic crosses to behavioral experiments. We also provide an open-source computational framework using R for the analysis of the phase of behavior using circular statistics. An extended method for complete use is also provided. For complete details on the use and execution of this protocol, please refer to Fernandez et al. (2020).

Why the young sleep longer.

A transcription factor helps young flies to sleep longer by delaying the maturation of a neural network that controls sleep.

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.