Posts in Category: papers

Sensory processing during sleep in Drosophila Melanogaster

One of the most puzzling aspects of sleep is that it cannot happen without depriving us of our full conscious experience. Whatever the function of sleep is, it cannot be achieved without disconnecting our brains from the external world. A full conscious state and sleep are not compatible, it seems, to the point that one of the definitions of consciousness is that “it is all that fades away when we are in dreamless sleep”.

The fact that the brain has to surrender to the tyranny of sleep is also the main reason why scientists believe (in a rather dogmatic fashion) that sleep is “of the brain, by the brain for the brain“. Yet, even during sleep parts of our brains retain some ability to process external information. In the 1960s, Oswald et al formally showed that sleeping humans could wake up in response to some salient stimuli, such as their names being called, but not in response to stimuli of identical strength but no salience, such as other people’s names or their names played in reverse.

This finding has been confirmed and extended over the decades in the scientific literature, providing evidence that it applies to even more complex nuances of saliency, such as an angry tone of voice. The videos below suggest that scientific literature is certainly less comprehensive and (less amusing) than the phenomenon in its entirety.

Pets wake up to food odours and food-related noises.
And the human brain is certainly able of very deep sensory processing!

Even though we have numerous scientific and anecdotal evidence that animals and humans can wake up to salient sensory stimuli during sleep, hardly anything is known about the biological underpinning of this phenomenon. And here: enter Drosophila melanogaster! What better animal model than flies to dissect this amazing brain property?

In a paper titled “Sensory processing during sleep in Drosophila melanogaster” published in Nature, we introduce flies as the ideal animal model to dive into the biology of how a brain can simultaneously be asleep and respond to external stimuli.

Postdoc Alice French took the lead on this amazing project to show that even flies can recognise salient stimuli in their sleep and react accordingly, modulating their response based on their internal state. We initially expanded the robotic platform we had previously built in the lab, called ethoscopes, which allows us to monitor and interfere with flies using inexpensive @Raspberry_Pi computers. Alice wanted to build a robotic component able to challenge single flies with specific odour but only while they were asleep, to record whether they would wake up or not. She obviously started with…. LEGO!

In our first prototype, we built a robot able to operate a LEGO valve so to send a puff of air to the sleeping fly. LEGO valves were a good start because we needed 500 of them.

The system worked and we went from those early all-LEGO prototypes (left) to the final 3D printed product (right).

Using this ethoscope module we could challenge sleeping flies with different odours and check whether they would respond differently to some of them. We found they did! Flies would respond to 5% acetic acid for instance, but not to 10% acetic acid. Not only that, the valence of the odour could be modulated by internal states. Flies that had received a little starvation were increasing their response specifically to food-related odours. When we gave alcohol to flies, on the other hand, we found drunk Drosophilae were less responsive to odours in general showing somehow a deeper sleep state.

Now, flies are arguably the best animal model to study circuit neuroscience these days. We have a full connectome of the fly brain and countless genetic tools that allow us to turn neurons on and off. So that is what we did. We started turning neurons on and off in the fly brain, looking for some that would modulate their ability to sense stimuli during sleep. We found them!

Image

We actually found the whole circuit, connecting the “fly nose” all the way to the sleep centers in the brain. And when we used thermogenetics to switch those neurons on or off with infrared radiation, we could interfere with that process and make the flies more or less responsive.

In short, we have shown that flies can recognise and respond to odours during sleep, waking up only to those that they consider salient. We also show that this phenomenon is plastic and modulated by internal states, with animals being more likely to wake to food odours after a little starvation. We also described a blueprint for a neuronal circuit that connects the peripheral olfactory receptor neurons all the way to known sleep-regulating centres in the fly brain. We explore three prototypical gate-points that modulate subconscious processing of olfactory information during sleep: two at the periphery and one in the central brain.

The story is important and of general interest for at least three reasons:

  1. for the groundbreaking implications it has on the consciousness field, introducing flies as a model to study
    subconscious processing of information, and providing an experimental paradigm that allows to empirically face some key questions of the field;
  2. for the implications it has on the sleep community, describing the neuronal circuit regulating sensory processing during sleep, a neuronal feature that is poorly understood in any other animal model. Our description of the circuit regulating sensory processing during sleep is the most accurate to date and the work also potential future medical significance, for instance in the study of altered states of consciousness, such as coma;
  3. for the implications it has on the larger neuroscience community, describing how a circuit modulates the processing of sensory information to distinguish valence.

Drosophila has been employed to study arousal threshold many times before. There are many studies in which flies can be used to gauge sleep “depth” by using quantitative mechanical stimuli, such as simple vibration or touch. Our study is the first one to study a more puzzling property: how do we recognise qualitative stimuli during sleep? How do we recognise our own name while unconscious?

The video abstract below provides more information on the contents and the implications of the work.

The full reference to the paper is:

The work was supported by BBSRC and H2020-Marie Curie funding. The lead author of the study is Dr. Alice French.

Nature has featured the paper with a dedicated News & Views by Wahne Li and Alex Keene.

Imperial wrote a little PR piece.

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.
Go to pubmedDownload PDFlink to the website

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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