The online control of movements by the inferior olive

Our ability to move in a controlled and directed manner allows us to seek food, escape from predators, or to dance the polka. Helping us achieve this are mechanisms in the brain that do the necessary quality control: they keep track of every movement, and make changes in the nervous system when they detect that the intended behaviour is not performed correctly. Patients that lack this ability have poor balance, tremors, and an inability to perform rapid movements.
Central in this process is the olivocerebellar system (OCS), a brain area found in all higher vertebrates. It is thought that it receives information from the brain on what the muscles have been instructed to do; it then forms a prediction of what the feedback from the senses should be for that movement. By comparing the actual sensory feedback with its prediction, it determines whether there is a mismatch, and thus whether changes need to be made. However, in spite of decades of research, this is still a largely untested hypothesis. This is because in traditional model systems (primates and rodents) it has been extremely difficult to directly measure this brain area's activity during behaviour. This means it is still unclear how the OCS knows what the animal is doing, and how it compares that with what the animal intended to do.
This proposal aims to understand this important process by using some of the latest tools in neuroscience, and exploiting the unique advantages of the larval zebrafish. This model is ideal for neural activity recordings during behaviour: since it is transparent, we can use it for non-invasive imaging of brain activity. This allows us to literally see how the OCS tracks the animal's movements, and how it responds to situations in which the expected and actual sensory feedback do not match. We first need to see how the OCS responds to sensory stimuli that could be used as sources of information on what the animal is doing. Building on preliminary data, we will use a cutting-edge, custom-built microscope to image neural activity in awake, behaving animals in a virtual environment, and expose them to different stimuli: visual (reporting on the visual effect of the animal moving in its environment), tactile (the result of water flowing past the animal's body as it swims), and vestibular (picking up the force caused by the animal accelerating as it swims). We will also use this setup to see how the OCS responds to situations in which there is a mismatch between the animal's behaviour and the sensory feedback it receives. These experiments reveal 1) which specific sources of information the OCS uses to monitor behaviour, 2) how the neuronal responses map to specific OCS subareas, and 3) how deviations from expected sensory feedback are reflected in neural activity. Next, we will put to test whether our observations are actually important to the animal's behaviour. On our microscope we will simply use light to manipulate the activity of the OCS through the power of optogenetics. By inhibiting specific cells, at specific timepoints, we can prevent the OCS from presenting the patterns of activity observed during the mismatch between behaviour and sensory feedback; conversely, by activating them we can 'play back' these same patterns. We can then test whether they are necessary and sufficient to drive the corresponding adjustments in behaviour that they are associated with.
This ambitious project aims to drastically enhance our understanding of the OCS. We will see how it tracks behaviour, develop new reagents, and directly test the behavioural relevance of our findings. By using the larval zebrafish we will be able to get answers that have so far been elusive, and since it is one of the simplest animals with an OCS, our work will help further the "3Rs" agenda through partial replacement. Furthermore, a greater fundamental understanding of the OCS will help future studies to understand and remedy the many conditions affecting this brain area.

The olivocerebellar system (OCS) integrates information from across the brain and the spinal cord to assess the execution of each movement, and implements changes if the actual movement does not match the intended result. Crucial to this process is the detection of a mismatch between the intended and actual movement by the inferior olive (IO). In spite of its central importance to behaviour, it is still unclear how this error signal is encoded.
A limitation in existing model systems is the inability to comprehensively map IO patterns of activity in awake, behaving animals. This proposal will overcome this limitation by using the powerful genetic toolkit of the transparent larval zebrafish, gaining novel insights into this highly conserved structure.
We will use a closed-loop virtual reality setup to expose awake, behaving larval zebrafish to a battery of sensory stimuli, and visualise the corresponding patterns of IO activity using a custom-built, cutting-edge multiphoton microscope. Building on preliminary data, we will relate these to behavioural output and quantify IO responses to mismatches between the behaviour and the expected sensory feedback. This will reveal the encoding of the sensorimotor error signal, and how the IO is subdivided into functional clusters. We will screen existing collections of transgenic lines, and produce highly specific "split-Gal4" lines using CRISPR/Cas9-mediated transgenesis, in order to genetically target these IO clusters. We will next use these genetic reagents to either remove, or optogenetically inhibit or activate each functional group in a spatially and temporally precise manner. This approach allows us to abolish or 'mimic' the effects of the sensorimotor error and test whether this has the predicted effect on subsequent behaviour. In conclusion: by using some of the latest tools, and making optimal use of the advantages of the larval zebrafish, we will enhance our understanding of the role of the OCS in motor control.

This proposal will address the important question of how animals monitor their own behaviour, and make adjustments in the nervous system when they do not perform the intended movements correctly. We will use some of the latest tools in neuroscience, including neural activity imaging and optogenetics, to probe a specific brain area involved in this process. Our work will create positive impact through innovation, collaborative working, capacity building, training, and engagement.

Innovation: pathway for partial replacement
It is the BBSRC's intention to have all researchers "Use the simplest possible (or least sentient) species of animal as appropriate to the experiment in question." This proposal will aid this effort by promoting a clear pathway for neuroscientists to replace the use of rodents, cats, and non-human primates in research.

Collaborative working: international partnerships
One of the ways UKRI aims to support innovation is by fostering the development of collaborative research programmes. As part of the project, we will collaborate with researchers at the National Institute of Genetics (NIG), Japan. This effort will establish research links between the NIG and St Andrews, and will help foster future collaborative projects between our institutions.

Capacity building: research reagents and imaging facilities
We will generate new research reagents that are likely to be useful to the many other zebrafish researchers working on neuroscience, the mechanisms of disease, cell biology, regeneration, and other fields. We will share our results as soon as possible for maximum benefit to the research community. Furthermore, for this project, we will build a cutting-edge custom-built multiphoton laser scanning microscope that will allow us to achieve our experimental aims. We propose to turn this microscope into a self-sustaining, multi-user imaging/optical manipulation facility at the completion of the project. The microscope will be a vital addition to the optical facilities at St Andrews, benefitting a range of biomedical research projects: from developmental biology to regenerative biology, and from ALS research to coral biomineralisation. Furthermore, the facility will be advertised through the organisational frameworks of the St Andrews Centre for Biophotonics, Scottish Universities Life Sciences Alliance, and Scottish Microscopy Group, which will ensure access for researchers from across Scotland.

Training: project postdoctoral researcher, technician, and undergraduate and Master's students
One of the ways advanced research provides benefit to society is by increasing the skills of the people associated with the work. This project uses the latest tools in neuroscience, and provides excellent training opportunities for the project postdoctoral researcher and technician, and students performing research projects as part of their degrees.

Engagement: science festival & primary education
Our research lends itself particularly well to be presented to a lay audience, as the output can be presented in striking visualisations. The findings are also relatable and their biomedical importance apparent. With the help of the well-resourced and highly experienced PE team, we intend to organise two activities we think leverage these qualities best: a performance at an annual science festival, and an undergraduate student-led project to develop activities that could be used as part of the Scottish primary educational curriculum for excellence.