Posts in Category: papers

Video tracking and analysis of sleep in Drosophila melanogaster

Nat Protoc. 2012 Apr 26;7(5):995-1007.
Video tracking and analysis of sleep in Drosophila melanogaster.
Giorgio F. Gilestro

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.

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Ethoscopes: An Open Platform For High-Throughput Ethomics

PLOS Biology, 19 Oct 2017; 15(10): e2003026
Ethoscopes: An Open Platform For High-Throughput Ethomics
Quentin Geissmann, Luis Garcia Rodriguez, Esteban J. Beckwith, Alice S. French, Arian R Jamasb, and Giorgio F Gilestro

We present ethoscopes, machines for high-throughput analysis of behaviour 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 behaviour using a supervised machine learning algorithm; can deliver behaviourally-triggered stimuli to flies in a feedback-loop mode; are highly customisable and open source. Ethoscopes can be built easily 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.

Online paper on PLoS Biology

Supplementary material.

Supplementary material 1 – webGL model of the ethoscope.
Supplementary material 2 – instruction booklet for the LEGOscope.
Supplementary material 3 – instruction booklet for the PAPERscope.
Supplementary Video 1 – Introduction to the ethoscope platform.
Supplementary Video 2 – The optogenetics component of the optomotor in action.

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Regulation of sleep homeostasis by sexual arousal







eLife 2017 Sep 12;6;e27445
Regulation of sleep homeostasis by sexual arousal
Esteban J. Beckwith, Quentin Geissmann, Alice S. French, and Giorgio F. Gilestro

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.

Online published paper

Supplementary Material

Interactive supplementary videos
Supplementary movies as raw dataset DOI

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eLife insight: Sleep: To rebound or not to rebound — Stahl BA, Keene AC

What is the paper about?

Why we sleep remains an unresolved mystery of biology. Why do humans have to spend one-third of their lifetime in a status of profound unconsciousness which leaves them vulnerable and endangered? What do we gain from it? We still do not possess an answer to this question but we assume that it must be something tremendously important, also considered that sleep appears to be a necessity not just in humans but in all animals – including fruit flies. A particularly intriguing evolutionary conserved feature of sleep is what we call “sleep homeostasis”, that is: the innate modulation of sleep pressure based on previous sleep amount. If we have a good long nap, we may have a harder time falling asleep at night; conversely, if we pull an all-nighter partying on Sunday night, we are going to have a hard time at the office on the following morning. That is sleep homeostasis.

Is sleep homeostasis an unmodifiable, sovereign need in the animal or can it somehow be suppressed? Previous studies showed that migratory birds may be able to resist the temptation to sleep while flying above the ocean. Similarly, male pectoral sandpipers, a type of Arctic bird, can forego sleep in favour of courtship during the three weeks time window of female fertility. Could we find a similar behaviour in a genetically amenable animal model, like fruit flies?

In a “blind date” experiment, we forced interaction in a restricted space between socially naive, young, male fruit flies and receptive females. The interaction between the two led to an uninterrupted passionate courtship lasting the entire 24 hour period (and to one – and, in some case, more – events of copulations). Surprisingly, not only did male flies forego sleep when prompted with a receptive female counterpart, they also suppressed their natural sleep homeostasis and never recovered for the sleep lost courting. In a second set of experiments, we forcefully kept flies awake by employing robots that would automatically disturb the flies whenever they would fall asleep. At the end of the sleep deprivation treatment, flies would normally recover the lost sleep by having an extra nap. However, raising the sexual arousal of male flies by simply exposing them to the female pheromone, abolished their homeostatic need.

Ours is a study on the fundamental biological underpinnings of sleep. Our goal is to show that sleep is not a disconnected, uncontrollable phenomenon but a biological drive that can, in some conditions, be overcome. The study is particularly directed at other researchers and provides an important caveat not to be forgotten when conducting sleep experiments: it possible to create an internal state in the animal that will heavily affect sleep regulation, without interfering with sleep regulatory circuits. A researcher may be artificially activating neurons that make an animal stressed, anxious, angered, or in love and all of these neurons will ultimately have an effect on sleep. Yet, they shall not be classified directly as “sleep neurons” or we will end up with a false map of where sleep neurons really are.

Video tracking and analysis of sleep in Drosophila melanogaster

Nat Protoc. 2012 Apr 26;7(5):995-1007.
Video tracking and analysis of sleep in Drosophila melanogaster.
Gilestro GF.

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.
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pySolo: a complete suite for sleep analysis in Drosophila

Bioinformatics. 2009 Jun 1; 25: 1466-1467
pySolo: a complete suite for sleep analysis in Drosophila
Giorgio F. Gilestro, Chiara Cirelli

pySolo is a multi-platform 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 and the entire project is leased under the GNU General Public License.
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Widespread Changes in Synaptic Markers as a Function of Sleep and Wakefulness in Drosophila

Science. 2009 Apr 3;324(5923):109-12
Widespread Changes in Synaptic Markers as a Function of Sleep and Wakefulness in Drosophila
Gilestro GF, Tononi G, Cirelli C

Sleep is universal, strictly regulated, and necessary for cognition. Why this is so remains a mystery, though recent work suggests a link between sleep, memory, and plasticity. 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.

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Redundant mechanisms for regulation of midline crossing in Drosophila

PLoS ONE. 2008;3(11):e3798. Epub 2008 Nov 24.
Redundant mechanisms for regulation of midline crossing in Drosophila.
Gilestro GF.

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 of 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.

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Regulation of commissural axon pathfinding by slit and its Robo receptors

Annu Rev Cell Dev Biol. 2006;22:651-75.
Regulation of commissural axon pathfinding by slit and its Robo receptors.
Barry J. Dickson, Giorgio F. Gilestro

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.
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Proteolytic processing converts the repelling signal Sema3E into an inducer of invasive growth and lung metastasis

Cancer Res. 2005 Jul 15;65(14):6167-77.
Proteolytic processing converts the repelling signal Sema3E into an inducer of invasive growth and lung metastasis
Christensen C, Ambartsumian N, Gilestro GF, Thomsen B, Comoglio P, Tamagnone L, Guldberg P, Lukanidin E.

We have previously shown that the expression of a semaphorin, known as a repelling cue in axon guidance, Sema3E, correlates with the ability to form lung metastasis in murine adenocarcinoma cell models. Now, besides providing evidence for the relevance of SEMA3E to human disease by showing that SEMA3E is frequently expressed in human cancer cell lines and solid tumors from breast cancer patients, we show biological activities of Sema3E, which support the implication of Sema3E in tumor progression and metastasis. In vivo, expression of Sema3E in mammary adenocarcinoma cells induces the ability to form experimental lung metastasis, and in vitro, the Sema3E protein exhibits both migration and growth promoting activity on endothelial cells and pheochromocytoma cells. This represents the first evidence of a metastasis-promoting function of a class 3 semaphorin, as this class of genes has hitherto been implicated in tumor biology only as tumor suppressors and negative regulators of growth. Moreover, we show that the full-size Sema3E protein is converted into a p61-Sema3E isoform due to furin-dependent processing, and by analyzing processing-deficient and truncated forms, we show that the generation of p61-Sema3E is required and sufficient for the function of Sema3E in lung metastasis, cell migration, invasive growth, and extracellular signal-regulated kinase 1/2 activation of endothelial cells. These findings suggest that certain breast cancer cells may increase their lung-colonizing ability by converting the growth repellent, Sema3E, into a growth attractant and point to a type of semaphorin signaling different from the conventional signaling induced by full-size dimeric class 3 semaphorins.

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Plexin-B3 is a functional receptor for semaphorin 5A

EMBO Rep. 2004 Jul;5(7):710-4. Epub 2004 Jun 25.
Plexin-B3 is a functional receptor for semaphorin 5A
Artigiani S, Conrotto P, Fazzari P, Gilestro GF, Barberis D, Giordano S, Comoglio PM, Tamagnone L.

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.
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