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1.
Sleep ; 47(2)2024 Feb 08.
Article in English | MEDLINE | ID: mdl-37930351

ABSTRACT

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.


Subject(s)
Circadian Clocks , Drosophila Proteins , Animals , Drosophila/physiology , Circadian Clocks/genetics , Circadian Rhythm/genetics , Sleep/physiology , Drosophila Proteins/genetics
2.
Elife ; 122023 10 31.
Article in English | MEDLINE | ID: mdl-37906092

ABSTRACT

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.


Subject(s)
Sleep Deprivation , Sleep, Slow-Wave , Animals , Drosophila , Drosophila melanogaster/physiology , Sleep/physiology , Wakefulness/physiology
3.
Proc Biol Sci ; 290(2006): 20230149, 2023 09 13.
Article in English | MEDLINE | ID: mdl-37700655

ABSTRACT

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.


Subject(s)
Drosophila melanogaster , Drosophila , Animals , Circadian Rhythm , Cryptochromes , Earth, Planet , Mammals
4.
J Exp Biol ; 223(Pt 11)2020 06 04.
Article in English | MEDLINE | ID: mdl-32291322

ABSTRACT

Even though the rhythms in adult emergence and locomotor activity are two different phenomena that occur at distinct life stages of the fly life cycle, previous studies have hinted at similarities in certain aspects of the organisation of the circadian clock driving these two rhythms. For instance, the period gene plays an important regulatory role in both rhythms. In an earlier study, we have shown that selection on timing of adult emergence behaviour in populations of Drosophila melanogaster leads to the co-evolution of temperature sensitivity of circadian clocks driving eclosion. In this study, we investigated whether temperature sensitivity of the locomotor activity rhythm evolved in our populations separately from the adult emergence rhythm, with the goal of understanding the extent of similarity (or lack thereof) in circadian organisation underlying the two rhythms. We found that in response to simulated jetlag with temperature cycles, late chronotypes (populations selected for predominant emergence during dusk) indeed re-entrained faster than early chronotypes (populations selected for predominant emergence during dawn) to 6 h phase delays, thereby indicating enhanced sensitivity of the activity/rest clock to temperature cues in these stocks (entrainment is the synchronisation of internal rhythms to cyclic environmental time cues). Additionally, we found that late chronotypes show higher plasticity of phases across regimes, day-to-day stability in phases and amplitude of entrainment, all indicative of enhanced temperature-sensitive activity/rest rhythms. Our results highlight remarkably similar organisation principles between circadian clocks regulating emergence and activity/rest rhythms.


Subject(s)
Circadian Clocks , Drosophila melanogaster , Animals , Circadian Clocks/genetics , Circadian Rhythm , Cues , Drosophila , Drosophila melanogaster/genetics , Light , Photoperiod , Temperature
5.
Chronobiol Int ; 37(4): 469-484, 2020 04.
Article in English | MEDLINE | ID: mdl-32079418

ABSTRACT

It is a common notion that phases-of-entrainment of circadian rhythms are adaptive, in that they enable organisms to time their behavior to specific times of the day to enhance their fitness. Therefore, understanding mechanisms that bring about such phases-of-entrainment is crucial to chronobiologists. Our previous studies have shown that selection for morning and evening phasing of adult emergence in Drosophila melanogaster populations leads to divergent coevolution of free-running periods of both adult emergence and activity/rest rhythms, such that early (morning) and late (evening) adult emergence chronotypes have shorter and longer circadian periods, respectively. However, there is little evidence to support the notion that phases-of-entrainment in these fly stocks is indeed driven by non-parametric mechanisms. Extending from a previous hypothesis based on anecdotal evidence for parametric mechanisms being in play, we explore the extent of non-parametric and parametric effects of light on circadian clocks of early and late chronotypes. We systematically tested predictions of the non-parametric model of entrainment, sketched the Circadian Integrated Response Characteristic (CIRC) of our stocks, assessed the effect of light pulses on amplitude of the behavior and the effect of duration of light pulse on phase-shifts of the clock. We demonstrate that, in addition to the differences in clock period, divergent CIRCs contribute to entrainment of the activity/rest rhythm. The differences in CIRC could be explained by differential transient amplitude responses and duration responses of the clock's phase between the early and late chronotypes. Our study thus highlights the role of amplitude responses and phase-shifts due to long durations of light in entrainment of circadian rhythms of D. melanogaster.


Subject(s)
Circadian Clocks , Circadian Rhythm , Animals , Drosophila , Drosophila melanogaster , Photoperiod
6.
J Biol Rhythms ; 35(2): 145-157, 2020 04.
Article in English | MEDLINE | ID: mdl-31994435

ABSTRACT

A crucial property of circadian clocks is the ability to regulate the shape of an oscillation over its cycle length (waveform) appropriately, thus enhancing Darwinian fitness. Many studies over the past decade have revealed interesting ways in which the waveform of rodent behavior could be manipulated, one of which is that the activity bout bifurcates under environments that have 2 light/dark cycles within one 24-h day (LDLD). It has been observed that such unique, although unnatural, environments reveal acute changes in the circadian clock network. However, although adaptation of waveforms to different photoperiods is well studied, modulation of waveforms under LDLD has received relatively less attention in research on insect rhythms. Therefore, we undertook this study to ask the following questions: what is the extent of waveform plasticity that Drosophila melanogaster exhibits, and what are the neuronal underpinnings of such plasticity under LDLD? We found that the activity/rest rhythms of wild-type flies do not bifurcate under LDLD. Instead, they show similar but significantly different behavior from that under a long-day LD cycle. This behavior is accompanied by differences in the organization of the circadian neuronal network, which include changes in waveforms of a core clock component and an output molecule. In addition, to understand the functional significance of such variations in the waveform, we examined laboratory selected populations that exhibit divergent eclosion chronotypes (and therefore, waveforms). We found that populations selected for predominant eclosion in an evening window (late chronotypes) showed reduced amplitude plasticity and increased phase plasticity of activity/rest rhythms. This, we argue, is reflective of divergent evolution of circadian neuronal network organization in our laboratory selected flies.


Subject(s)
Behavior, Animal , Drosophila melanogaster/genetics , Drosophila melanogaster/physiology , Evolution, Molecular , Neurons/physiology , Photoperiod , Animals , Circadian Clocks/genetics , Circadian Rhythm , Female , Male , Movement/radiation effects , Nerve Net , Neuronal Plasticity
7.
J Biol Rhythms ; 34(6): 596-609, 2019 12.
Article in English | MEDLINE | ID: mdl-31608742

ABSTRACT

Circadian rhythms in adult eclosion of Drosophila are postulated to be regulated by a pair of coupled oscillators: one is the master clock that is light sensitive and temperature compensated and the other that is a slave oscillator whose period is temperature sensitive and whose phase is reflected in the overt behavior. Within this framework, we reasoned that in populations of Drosophila melanogaster that have been artificially selected for highly divergent phases of eclosion rhythm, there may be changes in this network of the master-slave oscillator system, via changes in the temperature-sensitive oscillator and/or the coupling of the light- and temperature-sensitive oscillators. We used light/dark cycles in conjunction with different constant ambient temperatures and 2 different amplitudes of temperature cycles in an overall cool or warm temperature and analyzed phases, gate width, and normalized amplitude of the rhythms in each of these conditions. We found that the populations selected for eclosion in the morning (early flies) do not vary their phases with change in temperature regimes, whereas the populations selected for eclosion in the evening (late flies) show phase lability of up to ~5 h. Our results imply a genetic correlation between timing of behavior and temperature sensitivity of the circadian clock.


Subject(s)
Biological Clocks/genetics , Circadian Clocks , Circadian Rhythm , Drosophila melanogaster/genetics , Drosophila melanogaster/physiology , Temperature , Animals , Photoperiod
8.
J Biol Rhythms ; 34(5): 551-561, 2019 10.
Article in English | MEDLINE | ID: mdl-31307268

ABSTRACT

Research on circadian rhythms often requires researchers to estimate period, robustness/power, and phase of the rhythm. These are important to estimate, owing to the fact that they act as readouts of different features of the underlying clock. The commonly used tools, to this end, suffer from being very expensive, having very limited interactivity, being very cumbersome to use, or a combination of these. As a step toward remedying the inaccessibility to users who may not be able to afford them and to ease the analysis of biological time-series data, we have written RhythmicAlly, an open-source program using R and Shiny that has the following advantages: (1) it is free, (2) it allows subjective marking of phases on actograms, (3) it provides high interactivity with graphs, (4) it allows visualization and storing of data for a batch of individuals simultaneously, and (5) it does what other free programs do but with fewer mouse clicks, thereby being more efficient and user-friendly. Moreover, our program can be used for a wide range of ultradian, circadian, and infradian rhythms from a variety of organisms, some examples of which are described here. The first version of RhythmicAlly is available on Github, and we aim to maintain the program with subsequent versions having updated methods of visualizing and analyzing time-series data.


Subject(s)
Circadian Clocks , Circadian Rhythm , Software/economics , Animals , Data Analysis , Drosophila , Software/supply & distribution
9.
J Biol Rhythms ; 34(3): 231-248, 2019 06.
Article in English | MEDLINE | ID: mdl-30939971

ABSTRACT

The stability of circadian clock mechanisms under cyclic environments contributes to increased Darwinian fitness by accurately timing daily behavior and physiology. Earlier studies on biological clocks speculated that the timing of behavior and its accuracy are determined by the intrinsic period (τ) of the circadian clock under constant conditions, its stability, the period of the external cycle (T), and resetting of the clock by environmental time cues. However, most of these previous studies suffered from certain limitations, the major ones being a narrow range of examined τ values and a non-uniformity in the genetic background across the individuals tested. We present data that rigorously test the following hypotheses by employing Drosophila melanogaster fruit flies with τ ranging from 17 to 30 h in a uniform genetic background. We tested whether 1) precision (day-to-day stability of τ) is greater for clocks with τ close to 24 h; 2) accuracy (i.e., day-to-day stability of the phase relationship (ψ), where ψ is the duration between a phase of the rhythm and a phase of the external cycle) is greater for clocks with τ close to 24 h; 3) Ψ is delayed with an increase in τ; and 4) Ψ becomes more advanced with an increase in length of zeitgeber cycle (T). We show that precision is not always maximum for ~24-h clocks, but that accuracy is greatest when τ approximates T. Further, flies exhibit a delayed phase relationship with increasing τ and an advanced phase relationship under long T-cycles as compared with shorter T-cycles. We also describe relationships between activity and rest durations and how our observations fit predictions from models of circadian entrainment. Overall, we confirm that accuracy and phase of entrained rhythm are governed by both intrinsic clock period and the length of the external cycle; however, we find that the relationship between intrinsic period and precision does not fit previous predictions.


Subject(s)
Circadian Clocks , Drosophila melanogaster/physiology , Photoperiod , Animals , Circadian Rhythm , Drosophila melanogaster/genetics , Light , Male , Motor Activity
10.
Mol Cell Proteomics ; 18(6): 1171-1182, 2019 06.
Article in English | MEDLINE | ID: mdl-30923041

ABSTRACT

Hematopoiesis is the process of differentiation of precursor blood cells into mature blood cells that is controlled by a complex set of molecular interactions. Understanding hematopoiesis is important for the study of hematological disorders. However, a comprehensive understanding of how physiological and genetic mechanisms regulate blood cell precursor maintenance and differentiation is lacking. Owing to simplicity and ease of genetic analysis, the Drosophila melanogaster lymph gland (LG) is an excellent model to study hematopoiesis. Here, we quantitatively analyzed the LG proteome under genetic conditions that either maintain precursors or promote their differentiation in vivo, by perturbing expression of Asrij, a conserved endosomal regulator of hematopoiesis. Using iTRAQ-based quantitative proteomics, we determined the relative expression levels of proteins in Asrij-knockout and overexpressing LGs from 1500 larval dissections compared with wild type. Our data showed that at least 6.5% of the Drosophila proteome is expressed in wild type LGs. Of the 2133 proteins identified, 780 and 208 proteins were common to previously reported cardiac tube and hemolymph proteomes, respectively, resulting in the identification of 1238 proteins exclusive to the LG. Perturbation of Asrij levels led to differential expression of 619 proteins, of which 27% have human homologs implicated in various diseases. Proteins regulating metabolism, immune system, signal transduction and vesicle-mediated transport were significantly enriched. Immunostaining of representative candidates from the enriched categories and previous reports confirmed 73% of our results, indicating the validity of our LG proteome. Our study provides, for the first time, an in vivo proteomics resource for identifying novel regulators of hematopoiesis that will also be applicable to understanding vertebrate blood cell development.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Hematopoiesis , Lymph Nodes/metabolism , Membrane Proteins/metabolism , Proteomics , Animals , Mitochondria/metabolism , Molecular Sequence Annotation , Proteome/metabolism , Reproducibility of Results
11.
J Biol Rhythms ; 31(2): 125-41, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26833082

ABSTRACT

Although association of circadian clock properties with the timing of rhythmic behaviors (chronotype) has been extensively documented over several decades, recent studies on mice and Drosophila have failed to observe such associations. In addition, studies on human populations that examined effects of clock gene mutations/polymorphisms on chronotypes have revealed disparate and often contradictory results, thereby highlighting the need for a suitable model organism to study circadian clocks' role in chronotype regulation, the lack of which has hindered exploration of the underlying molecular-genetic bases. We used a laboratory selection approach to raise populations of Drosophila melanogaster that emerge in the morning (early) or in the evening (late), and over 14 years of continued selection, we report clear divergence of their circadian phenotypes. We also assessed the molecular correlates of early and late emergence chronotypes and report significant divergence in transcriptional regulation, including the mean phase, amplitude and levels of period (per), timeless (tim), clock (clk) and vrille (vri) messenger RNA (mRNA) expression. Corroborating some of the previously reported light-sensitivity and oscillator network coupling differences between the early and the late populations, we also report differences in mRNA expression of the circadian photoreceptor cryptochrome (cry) and in the mean phase, amplitude and levels of the neuropeptide pigment-dispersing factor (PDF). These results provide the first-ever direct evidence for divergent evolution of molecular circadian clocks in response to selection imposed on an overt rhythmic behavior and highlight early and late populations as potential models for chronotype studies by providing a preliminary groundwork for further exploration of molecular-genetic correlates underlying circadian clock-chronotype association.


Subject(s)
Circadian Clocks , Circadian Rhythm , Drosophila melanogaster/genetics , Drosophila melanogaster/physiology , Animals , Circadian Clocks/genetics , Circadian Rhythm/genetics , Cryptochromes/genetics , Drosophila Proteins/metabolism , Gene Expression Regulation , Light , Neuropeptides/metabolism , Phenotype , Photoperiod
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