Examinando por Autor "Ewer, John"
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Ítem Extracting temporal relationships between weakly coupled peptidergic and motoneuronal signaling: Application to Drosophila ecdysis behavior(Plos, 2021) Piñeiro, Miguel; Mena, Wilson; Ewer, John; Orio, PatricioNeuromodulators, such as neuropeptides, can regulate and reconfigure neural circuits to alter their output, affecting in this way animal physiology and behavior. The interplay between the activity of neuronal circuits, their modulation by neuropeptides, and the resulting behavior, is still poorly understood. Here, we present a quantitative framework to study the relationships between the temporal pattern of activity of peptidergic neurons and of motoneurons during Drosophila ecdysis behavior, a highly stereotyped motor sequence that is critical for insect growth. We analyzed, in the time and frequency domains, simultaneous intracellular calcium recordings of peptidergic CCAP (crustacean cardioactive peptide) neurons and motoneurons obtained from isolated central nervous systems throughout fictive ecdysis behavior induced ex vivo by Ecdysis triggering hormone. We found that the activity of both neuronal populations is tightly coupled in a cross-frequency manner, suggesting that CCAP neurons modulate the frequency of motoneuron firing. To explore this idea further, we used a probabilistic logistic model to show that calcium dynamics in CCAP neurons can predict the oscillation of motoneurons, both in a simple model and in a conductance-based model capable of simulating many features of the observed neural dynamics. Finally, we developed an algorithm to quantify the motor behavior observed in videos of pupal ecdysis, and compared their features to the patterns of neuronal calcium activity recorded ex vivo. We found that the motor activity of the intact animal is more regular than the motoneuronal activity recorded from ex vivo preparations during fictive ecdysis behavior; the analysis of the patterns of movement also allowed us to identify a new post-ecdysis phase.Ítem Orcokinin neuropeptides regulate reproduction in the fruit fly, Drosophila melanogaster(Elsevier, 2021) Silva, Valeria; Palacios-Muñoz, Angelina; Volonté, Mariano; Frenkel, Lía; Ewer, John; Ons, SheilaIn animals, neuropeptidergic signaling is essential for the regulation of survival and reproduction. In insects, Orcokinins are poorly studied, despite their high level of conservation among different orders. In particular, there are currently no reports on the role of Orcokinins in the experimental insect model, the fruit fly, Drosophila melanogaster. In the present work, we made use of the genetic tools available in this species to investigate the role of Orcokinins in the regulation of different innate behaviors including ecdysis, sleep, locomotor activity, oviposition, and courtship. We found that RNAi-mediated knockdown of the orcokinin gene caused a disinhibition of male courtship behavior, including the occurrence of male to male courtship, which is rarely seen in wildtype flies. In addition, orcokinin gene silencing caused a reduction in egg production. Orcokinin is emerging as an important neuropeptide family in the regulation of the physiology of insects from different orders. In the case of the fruit fly, our results suggest an important role in reproductive success.Ítem Reciprocal Relationship Between Calcium Signaling and Circadian Clocks: Implications for Calcium Homeostasis, Clock Function, and Therapeutics(Frontiers, 2021) Cavieres-Lepe, Javier; Ewer, JohnIn 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.Ítem Role of eclosion hormone during ecdysis behavior in drosophila melanogaster(Universidad de Valparaíso, 2023-09) Silva Moeller, Valeria; Ewer, JohnLos insectos son el grupo más exitoso y diverso del reino animal. Una característica que podría contribuir a su éxito es su exoesqueleto (también conocido como cutícula), que los protege y evita su desecación. Sin embargo, su estructura rígida limita su crecimiento y, para crecer, el insecto debe mudar periódicamente. Durante este proceso, el insecto secreta una nueva cutícula y desecha la del estadío anterior mediante el comportamiento innato y vital llamado ecdisis. Este comportamiento está controlado a través de la acción secuencial y compleja de varias hormonas y neuropéptidos, incluyendo la Ecdysis Triggering Hormone (ETH) y la Eclosion Hormone (EH), que actúan sobre sistema nervioso central (CNS). En este estudio, nos centramos en el papel de EH en la ecdisis, identificando los blancos de EH y determinando su función en el control de la ecdisis. Se cree que el receptor de EH (EHR) está codificado por el gen CG10738 de la mosca del vinagre, Drosophila. Aquí mostramos que las células epitraqueales (que expresan ETH y son blancos directos de EH) también expresan CG10738. Además, los mutantes de CG10738 no liberan ETH en respuesta a estímulo in vitro con EH sintético, lo que demuestra que este gen codifica para el receptor de EH. Utilizando inmunocitoquímica en combinación con EHR-GAL4 y los hemidrivers "split”-GAL4, encontramos que EHR y ETHR (receptor de ETH) se co-expresan en neuronas que también expresan los neuropeptidos EH, CCAP (péptido cardíaco crustáceo), kinina, FMRFamidas, MIPs (péptidos mioinhibidores) y bursicon, que se sabe que participan en el control de la ecdisis. Además, EHR también se expresa en motoneuronas y en muchas otras neuronas cuya identidad y función en la ecdisis son desconocidas. Para elucidar el papel de las células que expresan EHR, analizamos las consecuencias de su ablación o silenciamiento en diferentes etapas de desarrollo utilizando UAS-reaper y UAS- 2xKir2.1, respectivamente. Encontramos que ambas manipulaciones provocaron fallos en la ecdisis larval, similares al fenotipo de los mutantes de Eh y ehr. También observamos fallos en la ecdisis en la etapa de pupa y en la eclosión. Curiosamente, la activación de todas las células EHR en la eclosión utilizando UAS-TrpA1 también provocó fallos en la ecdisis, quedando las moscas atrapadas dentro del pupario. Luego, interrumpimos la expresión del gen CG10738 en subconjuntos de células que expresan EHR utilizando un RNAi específico y encontramos que la expresión de EHR en células que también expresan la proteína dimmed, sinaptobrevina neuronal, breathless, eth o amontillado es necesaria para el éxito del comportamiento de la ecdisis las diferentes etapas. Además observamos que algunas neuronas sensoriales requerían la expresión de EHR exclusivamente para la ecdisis pupal pero no así para otras ecdisis. Finalmente, evaluamos el patrón de actividad neuronal de los blancos de EH utilizando el sensor de calcio GCaMP. Al estimular exógenamente la ecdisis en preparaciones ex vivo del sistema nervioso central utilizando ETH sintético, pudimos observar que, aunque todas las neuronas fueron expuestas a este neuropéptido simultáneamente, diferentes subgrupos de células que expresan EHR respondieron en momentos diferentes con patrones de actividad específicos, lo que demuestra la complejidad de este comportamiento. Estos resultados sugieren en conjunto que las células blanco de EH son necesarias y desempeñan un rol crítico durante este complejo comportamiento de la ecdisis en Drosophila. Además, esta investigación contribuye a la comprensión de cómo los neuropéptidos actúan y controlan los comportamientos animales.Ítem The circadian clock gates Drosophila adult emergence by controlling the timecourse of metamorphosis(National Academy Of Science, 2021) Mark, Brandon; Bustos-González, Liliana; Cascallares, Guadalupe; Conejera, Felipe; Ewer, JohnThe daily rhythm of adult emergence of holometabolous insects is one of the first circadian rhythms to be studied. In these insects, the circadian clock imposes a daily pattern of emergence by allowing or stimulating eclosion during certain windows of time and inhibiting emergence during others, a process that has been described as “gating.” Although the circadian rhythm of insect emergence provided many of the key concepts of chronobiology, little progress has been made in understanding the bases of the gating process itself, although the term “gating” suggests that it is separate from the developmental process of metamorphosis. Here, we follow the progression through the final stages of Drosophila adult development with single-animal resolution and show that the circadian clock imposes a daily rhythmicity to the pattern of emergence by controlling when the insect initiates the final steps of metamorphosis itself. Circadian rhythmicity of emergence depends on the coupling between the central clock located in the brain and a peripheral clock located in the prothoracic gland (PG), an endocrine gland whose only known function is the production of the molting hormone, ecdysone. Here, we show that the clock exerts its action by regulating not the levels of ecdysone but that of its actions mediated by the ecdysone receptor. Our findings may also provide insights for understanding the mechanisms by which the daily rhythms of glucocorticoids are produced in mammals, which result from the coupling between the central clock in the suprachiasmatic nucleus and a peripheral clock located in the suprarenal gland.Ítem The Drosophila foraging gene plays a vital role at the start of metamorphosis for subsequent adult emergence(Taylor & Francis, 2021) Anreiter, Ina; Allen, Aaron M.; Vasquez, Oscar E.; To, Lydia; Douglas, Scott J.; Alvarez, Javier V.; Ewer, John; Sokolowski, Marla B.The foraging (for) gene has been extensively studied in many species for its functions in development, physiology, and behavior. It is common for genes that influence behavior and development to be essential genes, and for has been found to be an essential gene in both fruit flies and mammals, with for mutants dying before reaching the adult stage. However, the biological process underlying the lethality associated with this gene is not known. Here, we show that in Drosophila melanogaster, some but not all gene products of for are essential for survival. Specifically, we show that promoter 3 of for, but not promoters 1, 2, and 4 are required for survival past pupal stage. We use full and partial genetic deletions of for, and temperature-restricted knock-down of the gene to further investigate the stage of lethality. While deletion analysis shows that flies lacking for die at the end of pupal development, as pharate adults, temperature-restricted knock-down shows that for is only required at the start of pupal development, for normal adult emergence (AE) and viability. We further show that the inability of these mutants to emerge from their pupal cases is linked to deficiencies in emergence behaviors, caused by a possible energy deficiency, and finally, that the lethality of for mutants seems to be linked to protein isoform P3, transcribed from for promoter 3.Ítem The impact of the gut microbiome on memory and sleep in Drosophila(The Company Of Biologists, 2021) Silva , Valeria; Palacios-Muñoz, Angelina; Okray, Zeynep; Adair, Karen L.; Waddell, Scott; Douglas, Angela E.; Ewer, JohnThe gut microbiome has been proposed to influence diverse behavioral traits of animals, although the experimental evidence is limited and often contradictory. Here, we made use of the tractability of Drosophila melanogaster for both behavioral analyses and microbiome studies to test how elimination of microorganisms affects a number of behavioral traits. Relative to conventional flies (i.e. with unaltered microbiome), microbiologically sterile (axenic) flies displayed a moderate reduction in memory performance in olfactory appetitive conditioning and courtship assays. The microbiological status of the flies had a small or no effect on anxiety-like behavior (centrophobism) or circadian rhythmicity of locomotor activity, but axenic flies tended to sleep for longer and displayed reduced sleep rebound after sleep deprivation. These last two effects were robust for most tests conducted on both wild-type Canton S and w1118 strains, as well for tests using an isogenized panel of flies with mutations in the period gene, which causes altered circadian rhythmicity. Interestingly, the effect of absence of microbiota on a few behavioral features, most notably instantaneous locomotor activity speed, varied among wild-type strains. Taken together, our findings demonstrate that the microbiome can have subtle but significant effects on specific aspects of Drosophila behavior, some of which are dependent on genetic background.