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Knockdown of elements in calcium and cAMP pathway in clock cells affects the periodicity of adult locomotor activity rhythm. (A) Left: Representative record of adult locomotor activity under DD: following knockdown in PDF neurons of IP3R (a), dunce (b), rutabaga (c), CREB (d), and for pdf-GAL4 driver alone control (e); Right: corresponding Autocorrelation analyses with value of free-running period (h) and rhythmicity index (RI) indicated. (B) Left: Representative record of adult locomotor activity in DD: following knockdown in all clock cells of cacophony (a), IP3R (b), dunce (c), CREB (d), and for tim-GAL4 driver alone control (e). (C) Free-running periodicity (a) and rhythmicity index (RI) values (b) for genotypes shown in (A), plus those obtained for knockdown in PDF neurons of cacophony, Ca-alpha1D and CAMKII. In (a) each circle indicates individual flies tested; average is indicated by horizontal line; ÃÃÃ p<0.001; ÃÃ p<0.01; Ã p<0.05 (two-tailed Student t-test versus control, with confidence interval of 95%). In (b) Average (± SEM) RI for genotypes shown in A-C(a), ÃÃ p<0.01 (two-tailed Student t-test versus control, with confidence interval of 95%). Numbers indicate total number of flies tested. (D) Free-running periodicity (a) and rhythmicity index (RI) values (b) for genotypes shown in (B), plus those obtained for knockdown of Ca-alpha-1D, CAMKII, rutabaga, in all clock cells, represented as in (C). ÃÃÃ p<0.001; Ã p<0.05 (two-tailed Student t-test versus control, with confidence interval of 95%). In all experiments the GAL4 driver was used in combination with UAS-dcr2 to potentiate effectiveness of RNAi knockdown. https://doi.org/10.1371/journal.pgen.1007433.g007
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Circadian clocks impose daily periodicities to animal behavior and physiology. At their core, circadian rhythms are produced by intracellular transcriptional/translational feedback loops (TTFL). TTFLs may be altered by extracellular signals whose actions are mediated intracellularly by calcium and cAMP. In mammals these messengers act directly on T...
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In many animals the circadian rhythm of locomotor activity is controlled by an endogenous circadian clock. Using custom made housing and video tracking software in order to obtain high spatial and temporal resolution, we studied the statistical properties of the locomotor activity of wild type and two clock mutants of Drosophila melanogaster. We sh...
Citations
... In addition to transcriptional regulation, other points of control such as chromatin remodeling, mRNA stability, alternative splicing, and post-translational modifications such as phosphorylation have been described as playing important roles in circadian mechanisms (3,(17)(18)(19)(20). The role of the signaling molecule cAMP has also been studied: in several organisms, this second messenger exhibits a circadian expression pattern and seems to play an important role in establishing circadian period length and phase in mammals and insects (21)(22)(23). ...
In Neurospora crassa, caffeine and other methylxanthines are known to inhibit phosphodiesterase (PDE) activity, leading to augmented cAMP levels. In this organism, it has also been shown that the addition of these drugs significantly lengthens the circadian period, as seen by conidiation rhythms. Utilizing in vivo bioluminescence reporters, pharmacological inhibitors, and cAMP analogs, we revisited the effect of methylxanthines and the role of cAMP signaling in the Neurospora clockworks. We observed that caffeine, like all tested methylxanthines, led to significant period lengthening, visualized with both core-clock transcriptional and translational reporters. Remarkably, this phenotype is still observed when phosphodiesterase (PDE) activity is genetically or chemically (via 3-isobutyl-1-methylxanthine) abrogated. Likewise, methylxanthines still exert a period effect in several cAMP signaling pathway mutants, including adenylate cyclase (cr-1) and protein kinase A (PKA) (Δpkac-1) mutants, suggesting that these drugs lead to circadian phenotypes through mechanisms different from the canonical PDE-cAMP-PKA signaling axis. Thus, this study highlights the strong impact of methylxanthines on circadian period in Neurospora, albeit the exact mechanisms somehow remain elusive. IMPORTANCE Evidence from diverse organisms show that caffeine causes changes in the circadian clock, causing period lengthening. The fungus Neurospora crassa is no exception; here, several methylxanthines such as caffeine, theophylline, and aminophylline cause period lengthening in a concentration-dependent manner. Although methylxanthines are expected to inhibit phosphodiesterase activity, we were able to show by genetic and pharmacological means that these drugs exert their effects through a different mechanism. Moreover, our results indicate that increases in cAMP levels and changes in PKA activity do not impact the circadian period and therefore are not part of underlying effects of methylxanthine. These results set the stage for future analyses dissecting the molecular mechanisms by which these drugs dramatically modify the circadian period.
... For example, Kim and colleagues showed, using in vitro cellular models, that Ca 2+ -dependent signaling pathways mediate the resetting of the circadian clock by immediate early induction of Per genes via rapid CLOCK/BMAL1 heterodimerization and nuclear localization [28,235]. This partly explains how intracellular Ca 2+ generates and maintains circadian rhythms in the mouse SCN and behavioral rhythms in flies [224,[236][237][238]. As with Ca 2+ , Cu 2+ was also found to induce nighttime phase shifts of the SCN clock in a mitogen-activated protein kinase (MAPK)-dependent manner [239]. ...
The circadian clock is a fundamental biological timing mechanism that generates nearly 24 h rhythms of physiology and behaviors, including sleep/wake cycles, hormone secretion, and metabolism. Evolutionarily, the endogenous clock is thought to confer living organisms, including humans, with survival benefits by adapting internal rhythms to the day and night cycles of the local environment. Mirroring the evolutionary fitness bestowed by the circadian clock, daily mismatches between the internal body clock and environmental cycles, such as irregular work (e.g., night shift work) and life schedules (e.g., jet lag, mistimed eating), have been recognized to increase the risk of cardiac, metabolic, and neurological diseases. Moreover, increasing numbers of studies with cellular and animal models have detected the presence of functional circadian oscillators at multiple levels, ranging from individual neurons and fibroblasts to brain and peripheral organs. These oscillators are tightly coupled to timely modulate cellular and bodily responses to physiological and metabolic cues. In this review, we will discuss the roles of central and peripheral clocks in physiology and diseases, highlighting the dynamic regulatory interactions between circadian timing systems and multiple metabolic factors.
... Therefore, it is possible that the effects observed at night in the mutant might be driven principally by the clock network, whereas the effects observed during the day are not. A recent study also confirmed that knocking-down cac in the clock caused arrhythmicity, an effect that was mimicked by expressing cac RNAi in the LNvs (Palacios-Muñoz and Ewer, 2018). Similar results were obtained here, using both the cac H18 mutant and clock wide knockdown of cac, two different genetic manipulations resulting in reduced rhythmicity ( Fig. 3-4). ...
Schizophrenia exhibits up to 80% heritability. A number of genome wide association studies (GWAS) have repeatedly shown common variants in voltage-gated calcium (Cav) channel genes CACNA1C, CACNA1I and CACNA1G have a major contribution to the risk of the disease. More recently, studies using whole exome sequencing have also found that CACNA1B (Cav2.2 N-type) deletions and rare disruptive variants in CACNA1A (Cav2.1 P/Q-type) are associated with schizophrenia. The negative symptoms of schizophrenia include behavioural defects such as impaired memory, sleep and circadian rhythms. It is not known how variants in schizophrenia-associated genes contribute to cognitive and behavioural symptoms, thus hampering the development of treatment for schizophrenia symptoms. In order to address this knowledge gap, we studied behavioural phenotypes in a number of loss of function mutants for the Drosophila ortholog of the Cav2 gene family called cacophony (cac). cac mutants showed several behavioural features including decreased night-time sleep and hyperactivity similar to those reported in human patients. The change in timing of sleep-wake cycles suggested disrupted circadian rhythms, with the loss of night-time sleep being caused by loss of cac just in the circadian clock neurons. These animals also showed a reduction in rhythmic circadian behaviour a phenotype that also could be mapped to the central clock. Furthermore, reduction of cac just in the clock resulted in a lengthening of the 24 h period. In order to understand how loss of Cav2 function may lead to cognitive deficits and underlying cellular pathophysiology we targeted loss of function of cac to the memory centre of the fly, called the mushroom bodies (MB). This manipulation was sufficient to cause reduction in both short- and intermediate-term associative memory. Memory impairment was accompanied by a decrease in Ca2+ transients in response to a depolarizing stimulus, imaged in the MB presynaptic terminals. This work shows loss of cac Cav2 channel function alone causes a number of cognitive and behavioural deficits and underlying reduced neuronal Ca2+ transients, establishing Drosophila as a high-throughput in vivo genetic model to study the Cav channel pathophysiology related to schizophrenia.
... Remarkably, inhibiting signaling mediated by pigment dispersing factor (PDF), a neuropeptide analogous to mammalian VIP, causes the phases of Ca 2+ oscillations in M and E cells to be synchronous (Liang et al., 2016) and the pattern of behavior to be unimodal or arrhythmic (Hyun et al., 2005). In addition, in Drosophila, [Ca 2+ ] i varies during the course of the day in the prothoracic gland (PG), a peripheral clock that regulates the circadian rhythm of adult eclosion (Morioka et al., 2012;Palacios-Munoz and Ewer, 2018). A study carried out in organotypic PG cultures shows that the phase of the Ca 2+ oscillations is set by inputs from the brain (Morioka et al., 2012), which are probably mediated by prothoracicotropic hormone, a neuropeptide that transmits time information from the central clock to the PG (Selcho et al., 2017). ...
... Interestingly, Ca 2+ signaling is also involved in driving rhythmic behaviors and rhythms of gene expression. In Drosophila, genetic manipulations that decrease Ca 2+ levels or reduce the expression of proteins such as Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) in circadian pacemaker neurons, lengthen the periodicity of the circadian rhythms of locomotor activity and of adult eclosion (Harrisingh et al., 2007;Palacios-Munoz and Ewer, 2018). Similarly, a mouse bearing a mutation in the CaMKII gene that abolishes all kinase activity exhibits a longer freerunning period of locomotor activity and a desynchronization between the molecular rhythms of the left and right nuclei of the SCN (Kon et al., 2014). ...
In animals, circadian clocks impose a daily rhythmicity to many behaviors and physiological processes. At the molecular level, circadian rhythms are driven by intracellular transcriptional/translational feedback loops (TTFL). Interestingly, emerging evidence indicates that they can also be modulated by multiple signaling pathways. Among these, Ca2+ signaling plays a key role in regulating the molecular rhythms of clock genes and of the resulting circadian behavior. In addition, the application of in vivo imaging approaches has revealed that Ca2+ is fundamental to the synchronization of the neuronal networks that make up circadian pacemakers. Conversely, the activity of circadian clocks may influence Ca2+ signaling. For instance, several genes that encode Ca2+ channels and Ca2+-binding proteins display a rhythmic expression, and a disruption of this cycling affects circadian function, underscoring their reciprocal relationship. Here, we review recent advances in our understanding of how Ca2+ signaling both modulates and is modulated by circadian clocks, focusing on the regulatory mechanisms described in Drosophila and mice. In particular, we examine findings related to the oscillations in intracellular Ca2+ levels in circadian pacemakers and how they are regulated by canonical clock genes, neuropeptides, and light stimuli. In addition, we discuss how Ca2+ rhythms and their associated signaling pathways modulate clock gene expression at the transcriptional and post-translational levels. We also review evidence based on transcriptomic analyzes that suggests that mammalian Ca2+ channels and transporters (e.g., ryanodine receptor, ip3r, serca, L- and T-type Ca2+ channels) as well as Ca2+-binding proteins (e.g., camk, cask, and calcineurin) show rhythmic expression in the central brain clock and in peripheral tissues such as the heart and skeletal muscles. Finally, we discuss how the discovery that Ca2+ signaling is regulated by the circadian clock could influence the efficacy of pharmacotherapy and the outcomes of clinical interventions.
... Within the CNN, PDFR is expressed by DN1as, some DN1ps, DN2s, some DN3s, some LNds, the 5th s-LNv, and the s-LNvs (Hyun et al., 2005;Mertens et al., 2005;Shafer et al., 2008) (Figure 2a). Once activated, PDFR elicits an increase in cyclic adenosine monophosphate (cAMP) and regulated calcium oscillations, which serve as signaling molecules to tune circadian clocks (Im and Taghert, 2010;Klose et al., 2016;Liang et al., 2016Liang et al., , 2017Liang et al., , 2019Mertens et al., 2005;Palacios-Muñoz and Ewer, 2018;Shafer et al., 2008). Using PDF signaling as a model, we explore the different potential neuronal responses to G-protein coupled receptor signaling. ...
... Although circadian synaptic plasticity is mediated by Rac-and Rhotype GTPases in regulating rhythmic behavior (Petsakou et al., 2015), there is no known role for Rap1 protein in circadian behavior. However, knock down of EPAC protein in the prothoracic gland leads to longer eclosion rhythms in DD conditions (Palacios-Muñoz and Ewer, 2018), suggesting some role in regulating circadian clocks. Since loss of PDF signaling leads to an initial shortening of rhythmic behavior, it is unlikely that PDF signaling through cAMP acts on target neurons through the EPAC pathway. ...
Circadian clocks are biochemical time-keeping machines that synchronize animal behavior and physiology with planetary rhythms. In Drosophila, the core components of the clock comprise a transcription/translation feedback loop and are expressed in seven neuronal clusters in the brain. Although it is increasingly evident that the clocks in each of the neuronal clusters are regulated differently, how these clocks communicate with each other across the circadian neuronal network is less clear. Here, we review the latest evidence that describes the physical connectivity of the circadian neuronal network . Using small ventral lateral neurons as a starting point, we summarize how one clock may communicate with another, highlighting the signaling pathways that are both upstream and downstream of these clocks. We propose that additional efforts are required to understand how temporal information generated in each circadian neuron is integrated across a neuronal circuit to regulate rhythmic behavior.
... On the other hand, it must be noted that i[Ca 2+ ] and cAMP levels oscillate in a circadian manner in the SCN neurons (O'Neill and Reddy, 2012). Thus, it has been suggested that cAMP and Ca 2+ signaling may not only contribute in regulating timekeeping, but also that they are regulated by the cellular clock (i.e., Ca 2+ and cAMP signaling is both output from, as well as input into the core clock pathway) (Palacios-Muñoz and Ewer, 2018). ...
Background
Stromal interaction molecule 1 (STIM1) is one of the main components of the store operated Ca²⁺ entry (SOCE) signaling pathway. Individuals with mutated STIM1 present severely hypomineralized enamel characterized as amelogenesis imperfecta (AI) but the downstream molecular mechanisms involved remain unclear. Circadian clock signaling plays a key role in regulating the enamel thickness and mineralization, but the effects of STIM1-mediated AI on circadian clock are unknown.
Objectives
The aim of this study is to examine the potential links between SOCE and the circadian clock during amelogenesis.
Methods
We have generated mice with ameloblast-specific deletion of Stim1 (Stim1fl/fl/Amelx-iCre+/+, Stim1 cKO) and analyzed circadian gene expression profile in Stim1 cKO compared to control (Stim1fl/fl/Amelx-iCre–/–) using ameloblast micro-dissection and RNA micro-array of 84 circadian genes. Expression level changes were validated by qRT-PCR and immunohistochemistry.
Results
Stim1 deletion has resulted in significant upregulation of the core circadian activator gene Brain and Muscle Aryl Hydrocarbon Receptor Nuclear Translocation 1 (Bmal1) and downregulation of the circadian inhibitor Period 2 (Per2). Our analyses also revealed that SOCE disruption results in dysregulation of two additional circadian regulators; p38α mitogen-activated protein kinase (MAPK14) and transforming growth factor-beta1 (TGF-β1). Both MAPK14 and TGF-β1 pathways are known to play major roles in enamel secretion and their dysregulation has been previously implicated in the development of AI phenotype.
Conclusion
These data indicate that disruption of SOCE significantly affects the ameloblasts molecular circadian clock, suggesting that alteration of the circadian clock may be partly involved in the development of STIM1-mediated AI.
... Knockdown of the IP 3 R in PG cells slowed down the periodicity of adult emergence rhythm, whereas increasing cytosolic Ca 2+ by reducing SERCA function shortened the periodicity of the rhythm. Mild effects of IP 3 R knockdown were also observed on the periodicity of adult locomotor rhythms [18]. ...
Identification of mutations in the gene encoding the inositol 1,4,5-trisphosphate receptor Type 1 (IP3R1) as causative for Spinocerebellar Ataxia 15, 29 and Gillespie Syndrome emphasize the importance of understanding how the IP3R impacts neuronal function and physiology. Here we discuss how cellular changes due to IP3 mediated Ca²⁺release affect systemic physiology in Drosophila and mouse. The relevance of these findings to recently identified human neurological conditions are also discussed.
... For example, manipulation of clock neurons seems to be particularly affected by varying Ca 2þ levels. Intracellular Ca 2þ levels oscillate in clock neurons even in the absence of neuronal firing, while periodic Ca 2þ influx based on circadian variations in membrane potential is critical for circadian pacemaker function (Lundkvist, Kwak, Davis, Tei, & Block, 2005;Noguchi et al., 2017;Palacios-Muñoz & Ewer, 2018). Channelrhodopsin shows by comparison little Ca 2þ permeability. ...
Optogenetics has revolutionized the field of neuroscience. Within the last decades the development and use of optogenetics gained enormous importance for the identification of functional synaptic connections. Employing optogenetic tools in anatomically defined pathways offers a straightforward strategy to demonstrate neuronal sufficiency, even during state-dependent activity within a neuronal network. Hunger, thirst, fatigue or motivation each impact an animal’s behavior and determine the internal states that tune neuronal pathways to generate context-appropriate actions. In particular, higher order brain processes, such as learning and memory formation, are often state-dependent and here optogenetics can provide the means to identify and investigate the neuronal pathways involved. Our aim with this article is to focus on the possibilities and limitations of optogenetic tools for dissecting the neuronal circuits underlying learning and memory formation in Drosophila, while emphasizing what these approaches can tell us about neuronal circuit function in general.
... We further provide molecular and genetic evidence that NMD targets specific isoforms of CrebB to sustain circadian locomotor rhythms. Daily rhythmic oscillations in cAMP levels are observed in a diversity of species (Eckel-Mahan et al., 2008;Fukuhara et al., 2004;O'Neill et al., 2008;Palacios-Munoz et al., 2018) and linked to the circadian clocks in part via the ATF/CREB family of transcriptional regulators (Ginty et al., 1993;Koyanagi et al., 2011;Obrietan et al., 1999;Scheving et al., 1998). In mammals, these transcription factors promote per transcription (Shimizu et al., 2007;Travnickova-Bendova et al., 2002), and the CLOCK-ARNTL (BMAL) heterodimer in mice negatively regulates ATF5 expression (Lemos et al., 2007). ...
Post-transcriptional regulation underlies the circadian control of gene expression and animal behaviors. However, the role of mRNA surveillance via the nonsense-mediated mRNA decay (NMD) pathway in circadian rhythms remains elusive. Here, we report that Drosophila NMD pathway acts in a subset of circadian pacemaker neurons to maintain robust 24 h rhythms of free-running locomotor activity. RNA interference-mediated depletion of key NMD factors in timeless-expressing clock cells decreased the amplitude of circadian locomotor behaviors. Transgenic manipulation of the NMD pathway in clock neurons expressing a neuropeptide PIGMENT-DISPERSING FACTOR (PDF) was sufficient to dampen or lengthen free-running locomotor rhythms. Confocal imaging of a transgenic NMD reporter revealed that arrhythmic Clock mutants exhibited stronger NMD activity in PDF-expressing neurons than wild-type. We further found that hypomorphic mutations in Suppressor with morphogenetic effect on genitalia 5 (Smg5 ) or Smg6 impaired circadian behaviors. These NMD mutants normally developed PDF-expressing clock neurons and displayed daily oscillations in the transcript levels of core clock genes. By contrast, the loss of Smg5 or Smg6 function affected the relative transcript levels of cAMP response element-binding protein B (CrebB ) in an isoform-specific manner. Moreover, the overexpression of a transcriptional repressor form of CrebB rescued free-running locomotor rhythms in Smg5-depleted flies. These data demonstrate that CrebB is a rate-limiting substrate of the genetic NMD pathway important for the behavioral output of circadian clocks in Drosophila.
... Studies on insects have shown that the rhythmic release of a steroid hormone called ecdysone can alter emergence rhythm in fruit flies Drosophila (Selcho et al. 2017;Palacios-Muñoz and Ewer 2018), development time in tobacco hornworm Manduca sexta (L.) (Njihout and Williams 1974;Rountree and Bollenbacher 1986) and regulates the expression of several developmental genes (Giebultowicz et al. 1984). Therefore, it is possible that genes involved in various signaling pathways govern developmental changes by genetic and environment mediated interactions in European wheats (Worland 1996) and Drosophila (Mensch et al. 2008). ...
The metabolic, physiological, and behavioral processes in most of the living organisms exhibit an approximate 24-h rhythmic, self-sustained circadian (L. circa = about, dies = day) oscillations. These oscillations, apart from passively persisting in synchrony with external cues, exist in their absence as well, conferring adaptive advantage by following environmental cycle (extrinsic adaptation) and physiological harmony (intrinsic advantage), and tackling the adverse seasonal changes. Interestingly, these circadian perturbations generated at cellular level as rhythms are pretty much hierarchically similar across unicellular organisms to complex mammals. The biological clocks (master clock along with peripheral clocks) are able to influence biological fitness, life-history traits, physiology and behavior of organism. Given that the circadian clocks influence various biological processes, its evolutionary significance remained the subject of several laboratory studies with the view that imposing artificial selection for various clock properties such as phase, period, etc., might lead to the co-evolution of associated other clock-controlled evolutionary traits. In this review, we have presented key hypotheses pertaining to circadian rhythms, where by its presence to the owner can confer adaptive advantage intrinsically and extrinsically and enable endogenous clock to perform better in synchrony with the environment.
