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Clock Mechanics Report Summary

Project ID: 648709
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - Clock Mechanics (Mechanosensation and the circadian clock: a reciprocal analysis)

Reporting period: 2015-09-01 to 2017-02-28

Summary of the context and overall objectives of the project

What is the problem/issue being addressed?
A signature property of Life on Earth is that it is rhythmic. In virtually all living organisms, so called circadian clocks pick up, and repeat, the fundamental beat of life, which is caused by the earth’s daily rotation about its own axis. Circadian clocks are autonomous agents, they keep ticking away without any external stimulus and thereby create their own nights and days. In order to synchronise, or entrain, themselves to the world they are living in, however, clocks require so called Zeitgebers, i.e., sensory stimuli, which are coupled to the underlying geophysical cycle, the most prominent one here being probably the continual succession of light and darkness; but also temperature oscillations serve as Zeitgebers and can synchronise the clock. In the fruit fly Drosophila one other sensory modality has recently been linked to circadian entrainment, namely mechanosensory pathways. This project makes a first attempt to probe the roles of mechanosensory organs for timekeeping. Circadian functions, however, are not restricted to dedicated neural circuits in the brain. Next to these brain clocks peripheral clocks exist, which are thought to be act (at least partly) independent of the brain clocks. Circadian rhythmicity and clock functions, finally, also occur in mechanosensory organs themselves. This project does therefore not only study how mechanosensory pathways contribute to the computation (or sensation) of time, but also how circadian oscillators, e.g. those within mechanosensory cells, modulate mechanosensation.

Why is it important for society?
This project studies some question at the leading edge of Life Science. As with all basic research, its greatest benefits for society arise from the excellence of its scientific execution and the universality of its topic. Circadian clocks and circadian systems are ubiquitous. They orchestrate the physiology of virtually all organisms and they tick in virtually all our cells. We know a lot about circadian clocks; e.g. how they respond to light and temperature changes in Drosophila but we do not know how mechanical stimuli contribute to their function or only how exactly circadian systems compute one “standard” time from using different clocks distributed across the body. Here excellent basic research can be first step forward. As an immediate result it will show us what else we don’t know or don’t understand. It will thereby help us to know which questions we should ask. Given the universality of circadian clocks and their multiple contribution to human health and disease, research projects like ours can help understand, and fight, clock-related diseases and increase human health and wellbeing.

What are the overall objectives?
This project addresses the topic of circadian biology from a unique and novel angle. This angle is the overlap between mechanosensory and circadian systems, which has recently been discovered. As such the project will also have to address the wider issues of circadian clock function. How do mechanosensory stimuli interact with the other canonical stimuli, e.g. light and temperature? This immediately leads to the general question of sensory integration in the circadian clock, or in other words, the sensorineural computation of time. So, the overall objectives of this project are two-fold (i) to understand what mechanosensory stimuli contribute to entrainment of circadian clocks and (ii) how circadian systems modulate mechanosensory systems.
Together, our project activities will enable a better understanding of the complex multi-sensory processing that underlies an animal’s sense of time.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Reflecting its dual nature, which explores the mutual relation between mechanosensation and the circadian system, the initial project aims, and efforts, fell into two major classes:
(Aims, class 1)
(Mechano)Sensory input to the clock – novel vibrational setups
The initial project period was dedicated to devising new tools for the project’s further lines of research. To explore mechanosensory entrainment in greater depth we designed a new experimental apparatus that can deliver an adjustable mechanical stimulus to individual flies in an open field arena. Vibrational stimuli are delivered by eccentric rotating mass (ERM) motors coupled to individual arenas with each arena being vibrationally isolated within a 3 x 3 array. Video tracking is used to monitor the activity of individual flies in each arena providing higher resolution data than infra-red ‘beam-breaking’ activity monitors. A customisable software package, the Drosophila Arousal Tracking (DART) system, was employed to control the video recording, stimulus delivery and tracking.
A working prototype has been completed after a few iterative design changes, primarily affecting how each individual arena is coupled to (or isolated within) the 3 x 3 array. The DART software package can be used for both stimulus delivery and fly tracking over shorter time scales, in order to optimize it for demonstrating re-entrainment to vibrational stimuli we are currently modifying it to be used over longer timescales (of up to three weeks). These modifications will address memory management and tracking stability. The 2D approach, which requires higher video resolutions and larger arenas reduces the number of flies that can be analysed in one experiment. We are therefore establishing a simpler 1D approach in parallel, which reduces the computational requirements of the video recording/tracking and increases the throughput of the experiment by allowing more flies to be analysed in an individual experiment. Together, the novel 2D video-based approach, the simpler 1D approach as well as modifications of the previously designed infrared beam break- based setup will form the broad fundament of the project’s future experimental course.
These experimental activities have been led by the project employed molecular- and neurobiological PDRA.

(Mechano)Sensory input to the clock – novel vibrational setups
One of the major project activities in this reporting period have been dedicated to establishing a versatile computational shell, within which circadian clock function, its multisensory integration and locomotor outputs, can be modelled. We have been making substantial progress on this front and one of the first experimental tests of multisensory integration has recently been published as cover story in Cell Reports (Harper et al. 2016). Our modelling efforts have zoomed in on a combination of Weakly Coupled Oscillator Models (WCOMs) with Hidden Markov Models (HMMs).
The field of circadian neuroscience (and also this project) is using the rhythmicity of one major clock output, locomotor activity, as a proxy for circadian rhythmicity itself. That means that one concrete goal of recording the flies’ locomotor activity is, for example, to deduce the phase of the underlying circadian oscillator (the phase of ‘the circadian clock’). While this approach is a scientifically feasible one and has also led to important discoveries, it also poses considerable problems:
(i) The waveforms of the underlying circadian oscillator and the locomotor activity are different from each other. While, e.g. the molecular clock (i.e. the transcription/translation feedback oscillators) are reasonably well described by a sinusoidal waveform, the locomotor patterns observed across all studies and animals are more complex. The field has tried to bypass this discrepancy by using particular behavioural ‘landmarks’ (e.g. certain morning or evening activity peaks) to extract the behavioural clock phase. However, the waveforms of average activity rhythm vary across experimental conditions, genetic backgrounds and even individual flies. Without a confident representation of the actual behavioural waveform, it is impossible to extract continuous clock phase data from locomotor data. The simple equation of clock phase with peak activity phase reduces temporal resolution and introduces distortions. This problem becomes particularly pressing when analysing novel behavioural patterns, which, for example, result from specific environmental or genetic manipulations, which render conventionally used landmarks useless (see also Harper et al. 2016).
(ii) Using the locomotor behaviour as a mere output of the clock and as an experimentally accessible proxy for the state of the clock neglects the fact that locomotor activity, e.g by stimulating proprioceptive chordotonal organs, is also very likely acting as a sensory input to the clock.

Our modelling approaches, which have been driven by a project-associated PhD student and a computational PDRA are working on a solution to these limitations. To achieve this we are also avoiding the field-typical averaging across multiple individuals, which is meant to reduce some intrinsic noise but which also acts to introduce undesirable distortions and artefacts, next to reducing temporal resolution. Our high resolution approaches (MB5, 2D-DART + 1D-DART) enable the acquisition of high-quality data from individual flies, which are used to optimise our heuristic loops between theory and experiment.

(Aims, class 2)
Circadian control of mechanosensation -

Based on the observation that mechanosensory input from chordotonal organs (ChOs) can entrain the central circadian pacemakers, the existence of an opposing pathway was also posited. We chose to investigate the second antennal segment, the Johnston’s organ (JO), as it contains the largest collection of ChOs. ChOs are required for the sensation of biologically important stimuli that evoke tractable behaviours. The external nature of the JO and the attached arista also permits using laser Doppler vibrometry (LDV) measurements to quantify the biophysical properties of ChO mechanotransducers. To study clock gene cycling and circadian modulation of ChO mechanotransduction we performed real time RT-qPCR on RNA isolated from JO tissue sampled every 2 hours over a 24-hour period to establish mRNA cycling of canonical clock genes. Oscillations of both clock genes and mechanosensory genes was investigated in the same samples. Subsequent experiments will investigate if any potential cycling of clock gene mRNAs in the JO acts as an independent oscillator or if it receives input from central clock pacemakers. The results of these preliminary studies will be prepared for publication soon.
On the functional side, preliminary biophysical measurements of the antenna have been carried out at two different time points chosen for their contrasting levels of activity. At the first chosen time point, ZT04, flies are largely inactive and at the second time point, ZT10, flies are at their most active. We quantified antennal mechanics to determine the best frequency, frequency selectivity and energy injections at different circadian time points. In one of the next sets of experiments will determine if any change in biophysical properties is controlled by the circadian clock and if these changes are biologically relevant. Detailed force-step analyses, finally will reveal the proximate mechanisms, by which any possible circadian modulation of JO mechanotransduction takes place.
These experimental activities have been led by the project employed molecular- and neurobiological PDRA.

Information about dissemination and communication activities
The growing progress in the project’s activities has been disseminated and communicated on various occasions during the time covered by the first reporting period.
Communication activities have included presentations at (in chronological order):
(1) Winter UK Clock Club, Edinburgh, 10th December 2015. The UK clock club meetings are vital meetings to present ideas to, and discuss with, the prestigious UK chronobiological community. The insights gathered at this meeting at the start of the project was highly fruitful for its first period.
(2) A self-convened and organized circadian symposium at the ICN 2016, 12th International Congress of Neuroethology in Montevideo, Uruguay. The symposium A brain within the brain: Sensory integration and network logic of the Drosophila circadian clock assembled emerging leaders in the field of circadian research to present their findings and views on where the field is going with the explicit goal to introduce a novel approach to circadian research, which puts greater emphasis on the complexity, potential conflicts and integration of the multisensory input pathways into the clock, specifically including mechanosensory pathways, which are the major focus of this project.
(3) Members of the ERC team also attended the Summer UK clock club meeting in Warwick, 4th July 2016 to stay in contact with the circadian community.
(4) The team of ERC-project contributors consisting of the PI, the project-employed postdoc Dr Jason Somers and the project-associated PhD student Ross Harper attended, presented and discussed their preliminary findings also at an eminent Drosophila neurobiology meeting, i.e. the 16th European Neurobiology of Drosophila Conference, Chania, Greece (September 2-6, 2016). These “Neurofly” meetings are important occasions to get feedback and insights from the leading neurobiologists in the field of Drosophila research.
(5) Members of the ERC team also attended the Winter UK clock club meeting in Oxford, 10th January 2017 to discuss the project and the field with the circadian community.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

In line with the ultimate mission of an ERC consolidator grant, our project builds on a seminal series of own discoveries with the declared goal to advance the understanding, and methodologies, of the wider field of circadian neuroscience. The project activities during the first reporting period have been heavily focused on developing novel computational and experimental tools to explore the interrelation of circadian clocks and mechanosensory systems. We have realized early on that any progress in this question will require entirely novel theoretical and experimental approaches and that its successful computational implementation will also require to widen the scope to other sensory modalities. In a nutshell, the circadian clock has to be taken more seriously as a minibrain in its own right, which carries out complex sensory integrations when computing its various output signals. The findings reported in Harper et al. 2016 give a first appreciation of this complexity. A second publication (Harper et al.) is currently under review. The advances achieved during the first reporting period have helped us here greatly to build a novel experimental and theoretical platform for the study of circadian systems, which, when completed, will be of great value for the wider field of chronobiology and neurobiology. As our approach aims at a more comprehensive and ‘holistic’ approach to ‘the clock’ we have extended our scope towards the sensory integration in between different Zeitgeber stimuli (e.g. light and temperature) and the potential role of mechanosensory signals therein (e.g. mediating activity feedback). Also, we have discovered a second powerful insect model, which allows for testing the interrelation between circadian behaviour and mechanosensory systems in molecular and neurobiological detail. These are disease-bearing mosquito species, the behaviour of which displays an extreme form of circadian rhythmicity and is heavily dependent on mechanosensory systems. Mosquitoes will be used as secondary model system next to the main model Drosophila. It is a benefit of our current methodological landscape that (in an associated PhD project) we have managed to successfully transfer virtually all experimental paradigms for the study of mechanosensory behaviour, and mechanotransduction, from Drosophila to mosquitoes. We expect this aspect of our project to allow for a more detailed analysis of the circadian basis of mechanosensation and the mechanosensory basis of clock function.
Many pathologies have been linked to, and correlated with, dysfunctions of the circadian clock in humans but the underlying causalities have remained unclear. Our project, the novel insights and general paradigms it will provide, are likely to benefit a wide range of biomedical and neuroscientific stakeholders.
Record Number: 198476 / Last updated on: 2017-05-19
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