RESUMEN
Living organisms synchronize their biological activities with the earth's rotation through the circadian clock, a molecular mechanism that regulates biology and behavior daily. This synchronization factually maximizes positive activities (e.g., social interactions, feeding) during safe periods, and minimizes exposure to dangers (e.g., predation, darkness) typically at night. Beyond basic circadian regulation, some behaviors like sleep have an additional layer of homeostatic control, ensuring those essential activities are fulfilled. While sleep is predominantly governed by the circadian clock, a secondary homeostatic regulator, though not well-understood, ensures adherence to necessary sleep amounts and hints at a fundamental biological function of sleep beyond simple energy conservation and safety. Here we explore sleep regulation across seven Drosophila species with diverse ecological niches, revealing that while circadian-driven sleep aspects are consistent, homeostatic regulation varies significantly. The findings suggest that in Drosophilids, sleep evolved primarily for circadian purposes. The more complex, homeostatically regulated functions of sleep appear to have evolved independently in a species-specific manner, and are not universally conserved. This laboratory model may reproduce and recapitulate primordial sleep evolution.
Asunto(s)
Evolución Biológica , Ritmo Circadiano , Drosophila , Sueño , Especificidad de la Especie , Animales , Sueño/fisiología , Drosophila/fisiología , Ritmo Circadiano/fisiología , Homeostasis , Relojes Circadianos/fisiología , Masculino , FemeninoRESUMEN
Understanding how the brain encodes behaviour is the ultimate goal of neuroscience and the ability to objectively and reproducibly describe and quantify behaviour is a necessary milestone on this path. Recent technological progresses in machine learning and computational power have boosted the development and adoption of systems leveraging on high-resolution video recording to track an animal pose and describe behaviour in all four dimensions. However, the high temporal and spatial resolution that these systems offer must come as a compromise with their throughput and accessibility. Here, we describe coccinella, an open-source reductionist framework combining high-throughput analysis of behaviour using real-time tracking on a distributed mesh of microcomputers (ethoscopes) with resource-lean statistical learning (HCTSA/Catch22). Coccinella is a reductionist system, yet outperforms state-of-the-art alternatives when exploring the pharmacobehaviour in Drosophila melanogaster.
Asunto(s)
Escarabajos , Neurociencias , Animales , Drosophila melanogaster , Encéfalo , Aprendizaje AutomáticoRESUMEN
Summary: High-throughput analysis of behaviour is a pivotal instrument in modern neuroscience, allowing researchers to combine modern genetics breakthrough to unbiased, objective, reproducible experimental approaches. To this extent, we recently created an open-source hardware platform (ethoscope; Geissmann Q, Garcia Rodriguez L, Beckwith EJ et al. Rethomics: an R framework to analyse high-throughput behavioural data. PLoS One 2019;14:e0209331) that allows for inexpensive, accessible, high-throughput analysis of behaviour in Drosophila or other animal models. Here we equip ethoscopes with a Python framework for data analysis, ethoscopy, designed to be a user-friendly yet powerful platform, meeting the requirements of researchers with limited coding expertise as well as experienced data scientists. Availability and implementation: Ethoscopy is best consumed in a prebaked Jupyter-based docker container, ethoscope-lab, to improve accessibility and to encourage the use of notebooks as a natural platform to share post-publication data analysis. Ethoscopy is a Python package available on GitHub and PyPi. Ethoscope-lab is a docker container available on DockerHub. A landing page aggregating all the code and documentation is available at https://lab.gilest.ro/ethoscopy.
RESUMEN
Host behavioural changes are among the most apparent effects of infection. 'Sickness behaviour' can involve a variety of symptoms, including anorexia, depression, and changed activity levels. Here, using a real-time tracking and behavioural profiling platform, we show that in Drosophila melanogaster, several systemic bacterial infections cause significant increases in physical activity, and that the extent of this activity increase is a predictor of survival time in some lethal infections. Using multiple bacteria and D. melanogaster immune and activity mutants, we show that increased activity is driven by at least two different mechanisms. Increased activity after infection with Micrococcus luteus, a Gram-positive bacterium rapidly cleared by the immune response, strictly requires the Toll ligand spätzle. In contrast, increased activity after infection with Francisella novicida, a Gram-negative bacterium that cannot be cleared by the immune response, is entirely independent of both Toll and the parallel IMD pathway. The existence of multiple signalling mechanisms by which bacterial infections drive increases in physical activity implies that this effect may be an important aspect of the host response.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/microbiología , Bacterias Gramnegativas , Bacterias Grampositivas , Inmunidad Innata , LigandosRESUMEN
During sleep, most animal species enter a state of reduced consciousness characterized by a marked sensory disconnect. Yet some processing of the external world must remain intact, given that a sleeping animal can be awoken by intense stimuli (for example, a loud noise or a bright light) or by soft but qualitatively salient stimuli (for example, the sound of a baby cooing or hearing one's own name1-3). How does a sleeping brain retain the ability to process the quality of sensory information? Here we present a paradigm to study the functional underpinnings of sensory discrimination during sleep in Drosophila melanogaster. We show that sleeping vinegar flies, like humans, discern the quality of sensory stimuli and are more likely to wake up in response to salient stimuli. We also show that the salience of a stimulus during sleep can be modulated by internal states. We offer a prototypical blueprint detailing a circuit involved in this process and its modulation as evidence that the system can be used to explore the cellular underpinnings of how a sleeping brain experiences the world.
Asunto(s)
Drosophila melanogaster/fisiología , Percepción/fisiología , Sensación/fisiología , Sueño/fisiología , Animales , Drosophila melanogaster/genética , Masculino , Neuronas/fisiología , Odorantes/análisis , Percepción Olfatoria/genética , Percepción Olfatoria/fisiología , Estimulación Física , Sensación/genética , Sueño/genética , Olfato/genética , Olfato/fisiologíaRESUMEN
In Drosophila melanogaster, brain imaging with single-cell resolution reveals new physiological correlates of sleep and wakefulness, possibly defining different stages of sleep with functional relevance.
Asunto(s)
Drosophila melanogaster , Sueño , Animales , Encéfalo , VigiliaRESUMEN
Sleep appears to be a universally conserved phenomenon among the animal kingdom, but whether this notable evolutionary conservation underlies a basic vital function is still an open question. Using a machine learning-based video-tracking technology, we conducted a detailed high-throughput analysis of sleep in the fruit fly Drosophila melanogaster, coupled with a lifelong chronic and specific sleep restriction. Our results show that some wild-type flies are virtually sleepless in baseline conditions and that complete, forced sleep restriction is not necessarily a lethal treatment in wild-type D. melanogaster. We also show that circadian drive, and not homeostatic regulation, is the main contributor to sleep pressure in flies. These results offer a new perspective on the biological role of sleep in Drosophila and, potentially, in other species.
Asunto(s)
Conducta Animal/fisiología , Ritmo Circadiano/fisiología , Sueño/fisiología , Animales , Drosophila melanogasterRESUMEN
The recent development of automatised methods to score various behaviours on a large number of animals provides biologists with an unprecedented set of tools to decipher these complex phenotypes. Analysing such data comes with several challenges that are largely shared across acquisition platform and paradigms. Here, we present rethomics, a set of R packages that unifies the analysis of behavioural datasets in an efficient and flexible manner. rethomics offers a computational solution to storing, manipulating and visualising large amounts of behavioural data. We propose it as a tool to bridge the gap between behavioural biology and data sciences, thus connecting computational and behavioural scientists. rethomics comes with a extensive documentation as well as a set of both practical and theoretical tutorials (available at https://rethomics.github.io).
Asunto(s)
Conducta Animal/fisiología , Programas Informáticos , Animales , Ritmo Circadiano/fisiología , Biología Computacional/métodos , Bases de Datos Factuales , Drosophila/fisiología , Femenino , Masculino , MetadatosRESUMEN
Here, we present the use of ethoscopes, which are machines for high-throughput analysis of behavior in Drosophila and other animals. Ethoscopes provide a software and hardware solution that is reproducible and easily scalable. They perform, in real-time, tracking and profiling of behavior by using a supervised machine learning algorithm, are able to deliver behaviorally triggered stimuli to flies in a feedback-loop mode, and are highly customizable and open source. Ethoscopes can be built easily by using 3D printing technology and rely on Raspberry Pi microcomputers and Arduino boards to provide affordable and flexible hardware. All software and construction specifications are available at http://lab.gilest.ro/ethoscope.
Asunto(s)
Conducta Animal/fisiología , Drosophila melanogaster/fisiología , Etología/instrumentación , Algoritmos , Animales , Etología/métodos , Aprendizaje Automático , Microcomputadores , Impresión Tridimensional , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
In all animals, sleep pressure is under continuous tight regulation. It is universally accepted that this regulation arises from a two-process model, integrating both a circadian and a homeostatic controller. Here we explore the role of environmental social signals as a third, parallel controller of sleep homeostasis and sleep pressure. We show that, in Drosophila melanogaster males, sleep pressure after sleep deprivation can be counteracted by raising their sexual arousal, either by engaging the flies with prolonged courtship activity or merely by exposing them to female pheromones.
Asunto(s)
Nivel de Alerta , Conducta Animal , Drosophila melanogaster/fisiología , Homeostasis , Sueño , Animales , Cortejo , Masculino , Feromonas/metabolismoRESUMEN
In the past decade, Drosophila has emerged as an ideal model organism for studying the genetic components of sleep as well as its regulation and functions. In fruit flies, sleep can be conveniently estimated by measuring the locomotor activity of the flies using techniques and instruments adapted from the field of circadian behavior. However, proper analysis of sleep requires degrees of spatial and temporal resolution higher than is needed by circadian scientists, as well as different algorithms and software for data analysis. Here I describe how to perform sleep experiments in flies using techniques and software (pySolo and pySolo-Video) previously developed in my laboratory. I focus on computer-assisted video tracking to monitor fly activity. I explain how to plan a sleep analysis experiment that covers the basic aspects of sleep, how to prepare the necessary equipment and how to analyze the data. By using this protocol, a typical sleep analysis experiment can be completed in 5-7 d.
Asunto(s)
Drosophila melanogaster/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Sueño , Grabación en Video/métodos , Animales , Conducta Animal/fisiología , Ritmo Circadiano , Actividad Motora , Programas InformáticosRESUMEN
SUMMARY: pySolo is a multiplatform software for analysis of sleep and locomotor activity in Drosophila melanogaster. pySolo provides a user-friendly graphic interface and it has been developed with the specific aim of being accessible, portable, fast and easily expandable through an intuitive plug-in structure. Support for development of additional plug-ins is provided through a community website. AVAILABILITY: Software and documentation are located at (http://www.pysolo.net). pySolo is a free software released under the GNU General Public License.
Asunto(s)
Drosophila melanogaster/fisiología , Actividad Motora/fisiología , Sueño/fisiología , Programas Informáticos , Animales , Biología Computacional/métodos , Interfaz Usuario-ComputadorRESUMEN
Sleep is universal, strictly regulated, and necessary for cognition. Why this is so remains a mystery, although recent work suggests that sleep, memory, and plasticity are linked. However, little is known about how wakefulness and sleep affect synapses. Using Western blots and confocal microscopy in Drosophila, we found that protein levels of key components of central synapses were high after waking and low after sleep. These changes were related to behavioral state rather than time of day and occurred in all major areas of the Drosophila brain. The decrease of synaptic markers during sleep was progressive, and sleep was necessary for their decline. Thus, sleep may be involved in maintaining synaptic homeostasis altered by waking activities.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Sueño/fisiología , Sinapsis/fisiología , Vigilia/fisiología , Animales , Relojes Biológicos , Western Blotting , Encéfalo/fisiología , Ritmo Circadiano , Femenino , Homeostasis , Masculino , Microscopía Confocal , Modelos Animales , Proteínas Qa-SNARE/metabolismo , Privación de Sueño , Sinapsinas/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
During development, all neurons have to decide on whether to cross the longitudinal midline to project on the contralateral side of the body. In vertebrates and invertebrates regulation of crossing is achieved by interfering with Robo signalling either through sorting and degradation of the receptor, in flies, or through silencing of its repulsive activity, in vertebrates. Here I show that in Drosophila a second mechanism of regulation exists that is independent from sorting. Using in vitro and in vivo assays I mapped the region of Robo that is sufficient and required for its interaction with Comm, its sorting receptor. By modifying that region, I generated new forms of Robo that are insensitive to Comm sorting in vitro and in vivo, yet still able to normally translate repulsive activity in vivo. Using gene targeting by homologous recombination I created new conditional alleles of robo that are sorting defective (robo(SD)). Surprisingly, expression of these modified proteins results in phenotypically normal flies, unveiling a sorting independent mechanism of regulation.
Asunto(s)
Sistema Nervioso Central/embriología , Proteínas de Drosophila/fisiología , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Receptores Inmunológicos/fisiología , Animales , Tipificación del Cuerpo , Células COS , Movimiento Celular , Sistema Nervioso Central/citología , Sistema Nervioso Central/crecimiento & desarrollo , Chlorocebus aethiops , Drosophila , Embrión no Mamífero , Proteínas RoundaboutRESUMEN
Commissural axons grow along complex pathways toward, across, and beyond the midline of the central nervous system. Taking commissural axons in the vertebrate spinal cord and the Drosophila ventral nerve cord as examples, we examine how commissural axon pathfinding is regulated by the Slit family of guidance cues and their Robo family receptors. We extract several principles that seem likely to apply to other axons and other contexts, such as the reiterative use of the same guidance molecules in distinct pathfinding decisions, the transcriptional specification of a pathway, the posttranscriptional regulation of growth along the pathway, and the possible role of feedback mechanisms to ensure the fidelity of pathfinding choices. Such mechanisms may help explain how a relatively small number of guidance molecules can generate complex and stereotyped wiring patterns. We also highlight the many gaps in our understanding of commissural axon pathfinding and question some widely accepted views. We hope that this review encourages further efforts to tackle these questions, in the expectation that this system will continue to reveal the general principles of axon pathfinding.
Asunto(s)
Axones/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Secuencia de Aminoácidos , Animales , Drosophila melanogaster/citología , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Receptores Inmunológicos/química , Proteínas RoundaboutRESUMEN
Semaphorins are a large family of molecular cues implicated in neural development and in a variety of functions outside the nervous system. Semaphorin 5A (Sema5A) is a transmembrane semaphorin, containing seven thrombospondin type-1 repeats, which was recently found to control axon guidance. Here we show that plexin-B3 is a high-affinity receptor specific for Sema5A. We further demonstrate that plexin-B3 activation by Sema5A mediates functional responses in plexin-B3-expressing cells (either fibroblasts, epithelial and primary endothelial cells). In addition, Sema5A can trigger the intracellular signalling of the hepatocyte growth factor/scatter factor receptor, Met, associated in a complex with plexin-B3. We thus conclude that Sema5A is able to elicit multiple functional responses through its receptor plexin-B3.
Asunto(s)
Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Animales , Axones/metabolismo , Western Blotting , Células COS , Línea Celular , Membrana Celular/metabolismo , Movimiento Celular , Relación Dosis-Respuesta a Droga , Endotelio Vascular/embriología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Humanos , Inmunoprecipitación , Cinética , Proteínas de la Membrana/metabolismo , Ratones , Células 3T3 NIH , Proteínas del Tejido Nervioso/fisiología , Moléculas de Adhesión de Célula Nerviosa/fisiología , Unión Proteica , Proteínas Proto-Oncogénicas c-met/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores de Superficie Celular/metabolismo , Semaforinas , Transducción de SeñalRESUMEN
Ligand-dependent downregulation of tyrosine kinase receptors is a critical step for modulating their activity. Upon ligand binding, hepatocyte growth factor (HGF) receptor (Met) is polyubiquitinated and degraded; however, the mechanisms underlying HGF receptor endocytosis are not yet known. Here we demonstrate that a complex involving endophilins, CIN85 and Cbl controls this process. Endophilins are regulatory components of clathrin-coated vesicle formation. Through their acyl-transferase activity they are thought to modify the membrane phospholipids and induce negative curvature and invagination of the plasma membrane during the early steps of endocytosis. Furthermore, by means of their Src-homology 3 domains, endophilins are able to bind CIN85, a recently identified protein that interacts with the Cbl proto-oncogene. Cbl, in turn, binds and ubiquitinates activated HGF receptor, and by recruiting the endophilin-CIN85 complex, it regulates receptor internalization. Inhibition of complex formation is sufficient to block HGF receptor internalization and to enhance HGF-induced signal transduction and biological responses. These data provide further evidence of a relationship between receptor-mediated signalling and endocytosis, and disclose a novel functional role for Cbl in HGF receptor signalling.