Sharing new non-invasive circadian phenotyping methods

Life on Earth has evolved under a rhythmically changing cycle of day and night. As a result, virtually all organisms have evolved internal biological clocks with a period of around 24h, enabling them to anticipate and adapt to predictable changes in their environment. Circadian clocks regulate virtually every aspects of our physiology and behaviour - including activity, sleep and body temperature.

Circadian rhythms are widely studied in mouse models, typically using home cage activity. However, measuring sleep and body temperature require surgical implantation of electrodes or internal devices. Our group has developed new methods of measuring sleep and body temperature rhythms in mice, based upon non-invasive sensors placed above the home cage that detect movement or heat signatures. We have already shown that these methods can reliably measure sleep and body temperature, providing an alternative to invasive methods.

This proposal will share our expertise in these methods with a network of five research labs in Berlin. All five groups work on circadian rhythms, but in different areas ranging from immunology and cancer to neurodegenerative disease. By establishing our methods in a single location, we can therefore encourage the use of our new refined methods in a large number of end users. To achieve these aims we will organise a series of practical workshops in Oxford, where collaborators from Berlin will be able to learn about the systems, see first-hand how we are using them and experience the opportunity to put their own systems together and see the data they produce. These workshops will allow us to develop better online training material that will also help others use these methods.

Finally, as part of this work we will also develop new methods to analyse and share data. Using these methods we are able to measure circadian rhythms every minute of every day. However, measuring activity, sleep or body temperature rhythms over days produces huge amounts of data. This data is only of helpful if important features can be extracted from it. In many disorders, circadian rhythms become disrupted. For example, sleep patterns are affected in many different conditions, including as a result of infection, cancer and neurodegenerative disease. In this proposal, we will work with mathematical experts to develop new ways of analysing this data. In addition, we will store our data - along with key information about the experiments - on website databases that allow other researchers free to access it. By sharing data in this manner, needless replication of animal studies can be avoided, and other researchers can use this data to develop new analysis methods and even conduct new studies without the need to use further animals.

This project will transfer expertise in two new refined methods of studying mouse physiology and behaviour developed in Oxford to a network of collaborators based in Berlin. Our lab has established and validated new non-invasive approaches to assess sleep and body temperature in laboratory mice. These methods provide major welfare refinements in comparison with existing invasive methods.

In this project we will share our expertise with these new methods with collaborators in Berlin. By establishing these techniques in a central facility, this will benefit five separate circadian research groups working on a range of different mouse models, including neuropsychiatry, immunology, oncology and RNA biochemistry.

To enable our collaborators to use these methods, we will run a series of hands-on workshops in Oxford, where end-users will learn how these systems work, see them in action, and build their own systems from scratch. Attendees will also learn how to analyse the data obtained from these systems. These workshops will enable attendees to set these systems up in their own lab, encouraging the wider use of these methods in the circadian community. Moreover, they will provide an ideal opportunity to develop a range of online training material to enable the wider community to more easily implement these methods.

Finally, we will work with mathematical experts in circadian data analysis to improve our data analysis pipelines, with a particular focus on the detection of circadian disruption - a hallmark of many disorders. We will also encourage clearly annotated behavioural data deposition, which will improve transparency and reproducibility of circadian studies. This will provide a major resource to this user community, enabling data sharing to reduce - and in some cases even replace - the use of new animals.

By working with colleagues in Berlin, we are able to establish our phenotyping methods within a central facility benefitting five separate circadian groups, achieving a greater potential uptake compared with working with just a single end-user. As these groups work across a diverse range of biomedical research this will provide 3Rs benefits in multiple disease models and experimental conditions as part of ongoing research programmes. Once established, this system will continue to be used by the named collaborators, and wider uptake will be encouraged to maximise the 3Rs impact. Based upon our experience in this project, we will also develop online resources to help other users establish these systems.

The measurement of activity, sleep and body temperature require different experimental methods, typically requiring duplicate cohorts of mice. As a result, the use of combined non-invasive measurements has an immediate potential to reduce the number of mice used. As well as providing a non-invasive alternative, real-time longitudinal monitoring of activity, sleep and body temperature provides an ideal way of deviations from healthy physiology. These measures provide refined biomarkers of disease severity and animal welfare, which can also be used to establish the efficacy of therapeutic interventions and establish refined endpoints.

By developing meta-data standards for circadian data deposition, we can also ensure that the 3Rs are embedded at an early stage. We will use existing data repositories to ensure the continued legacy of this project (e.g. BioDare 2). Depositing data and analysis tools provides a key resource that can potentially replace animal use for some studies (e.g. allowing existing data to be analysed with new methods). Open data can also reduce the number of animals used, by removing the need to needlessly replicate studies as well as facilitating experimental design (e.g. providing data to estimate expected effect sizes).