Advancing understanding of anaesthesia and analgesia in the zebrafish

The zebrafish (Danio rerio) is used widely as a model for understanding biological processes in development and various human diseases. Features of embryonic and larval zebrafish (embryo-larvae, <10 days post fertilisation, or dpf) that make them highly attractive as a research model include their optical transparency for visualising biological processes and ease of genetic manipulation. Consequently, each year millions of embryo-larval zebrafish are used in laboratories across the globe. Many of these animals undergo anaesthesia, however, very little information is available to guide appropriate anaesthetic choice, and even less information is available on analgesics (pain relievers). Our project will address these knowledge gaps and has 3 main aims. Aim 1) Will establish the concentrations of anaesthetics needed to induce sedation and euthanasia, and the concentrations of analgesics needed to provide pain relief. For this we will use functional brain imaging (via fluorescent dyes that reveal the brain's neural activity) to test the responsiveness of animals to stimulation whilst under treatment. Electrophysiology (direct recording of the brain's electrical activity) will be used to establish concentrations of anaesthesia required for euthanasia. We will also gather data to understand if traditional indicators of sedation in fish are appropriate (e.g. loss of balance), and measure compound uptake to support interpretation of compound exposure effect concentrations. Aim 2) Will determine if any of the anaesthetics inhibit movement, if any of the anaesthetics and analgesics are aversive, and if any of the analgesics reduce the effects of noxious stimulation (e.g. heat, CO2). For this we will assess behaviour such as the effect of treatment on locomotion, and the avoidance of areas within a test arena where an anaesthetic or analgesic has been deposited. In addition, we will measure the ability of analgesics to decrease the behavioural impact of noxious stimuli as an indicator of their pain relieving properties. Aim 3) Will provide a better understanding of the mechanisms of action of the chosen anaesthetics and analgesics, and of sedation and pain perception in the embryo-larval zebrafish. For this we will undertake functional brain imaging in genetically-modified animals in which we are able to visualise activity in specific types of neuron in the brain. This will allow us to understand which brain regions and cell types respond to treatment with each agent, and which regions/cell types are involved in the sedative process. We will also apply this approach to animals subjected to noxious stimulation to better understand pain perception in fish. This project will allow scientists to select the most scientifically and ethically appropriate anaesthetic and analgesic for use in their work and will provide a better understanding of how these agents work, as well as advancing knowledge on the processes of sedation and pain perception in fish. Beneficiaries will include veterinary and aquarium fish industries as these data will help refine practices such as how best to monitor anaesthesia in fish, and inform on treatments most appropriate for animal transportation. Government bodies such as the UK Home Office, who oversee the scientific use of animals, will benefit through the provision of high quality reference data to use in decision making on animal experimentation practices, and charities ,such as those associated with neurological disorders (e.g. epilepsy, Alzheimer's, Parkinson's), will be provided with further validation data on an alternative (and more cost effective) model species. The public and society in general will benefit from an improvement in animal welfare standards, a greater understanding of sedation and pain perception, and an increase in our understanding of the biology of a species increasingly being used in experimental, including biomedical, research.

Each year millions of embryonic and larval (embryo-larval) zebrafish are used globally in laboratories, many of which undergo anaesthesia. Despite this, little information is available to guide anaesthetic or analgesic choices for fish, with even less data available for embryo-larvae. This knowledge gap is extensive, spanning an absence of basic information on appropriate dosing concentrations, to a lack of knowledge of the central circuitry involved in sedation or nociception. This project will fill these knowledge gaps via 3 main aims. Aim 1 will establish the efficacy of several anaesthetics/analgesics using functional brain imaging and electrophysiology. For the functional imaging we have developed a technique using zebrafish with pan-neuronally-expressed GCaMP6s that allows us to quantify brain-region specific responses to stimulation under conditions of anaesthesia and analgesia. Concentrations required to induce euthanasia will be confirmed using electrophysiology. Aim 2 will determine which agents inhibit neuromuscular activity, are aversive, or reduce avoidance of noxious stimuli using automated behavioural assessment. This includes quantifying the avoidance of each agent using a tank arena place preference assay for aversion, and the effect of analgesics on the amelioration of noxious simulation for nociception. Aim 3 is focussed on understanding mechanisms of action and central sedative and nociceptive processing in fish. For this we will undertake functional imaging in circuit-specific transgenic reporter lines and supplement this with immunohistochemistry to identify specific circuits responding to treatment with these agents. The same approach will be used following noxious stimulation in order to define the central circuitry involved in nociception, which is largely unknown in fish. This project will provide data to refine millions of animal experiments, and generate important mechanistic information on anaesthetic and analgesic action in fish.

Each year, millions of embryonic and larval (embryo-larvae) zebrafish are used in laboratories worldwide, the majority of which are subject to anaesthesia. Despite this, very little is known about the effects of anaesthetics, or analgesics, in fish. The aim of this project is to improve understanding of anaesthesia and analgesia in embryo-larval zebrafish, generating data to refine millions of animal experiments, and providing mechanistic information relevant across multiple stakeholder groups. Our impact strategy includes 3 specific deliverables focussed on disseminating detailed anaesthetic and analgesic protocols, hosting an international workshop on fish anaesthesia and analgesia, and creating an on-line interactive brain atlas detailing mechanisms of action (MoAs) of each anaesthetic and analgesic agent, and the brain circuitry involved in sedation and nociception in embryo-larval zebrafish. These are designed to maximise access to all stakeholders likely to benefit from this work. This project will, for the first time, allow academic and industry scientists using zebrafish to make scientifically-informed choices about anaesthetics and analgesics for use in their research, and as such should help to change organisational culture and working practices. Although focused on the most widely used model (zebrafish) these data will also provide a solid foundation for protocol refinement in other fish species. The data generated will also provide fundamental information on sedation and nociception in fish, and equip neuroscientists with data on the relevance of the embryo-larval zebrafish for studies into brain function and the effects of neuroactive substances. The latter extends to biomedical charities focussed on research into neurological disorders, a number of which are already interested in using the zebrafish as a more cost-effective and ethical alternative for their research (e.g. Epilepsy research UK, The Michael J Fox Foundation for Parkinson's, and Paul G. Allen Family Foundation for Alzheimer's). The veterinary and ornamental fish industries use anaesthetics extensively (and in some case analgesics) for surgery, transportation, tagging etc. Benefit will stem from the generation of translational data as a starting point to refine protocols in their species of interest, clarification of the accuracy of routine methods for monitoring anaesthetic depth and the likely effectiveness of post-operative analgesia in fish. This project has the very real possibility to transform evidence-based policy and legislation at a national and international level. For example, regulatory bodies such as the UK Home Office will for the first time have easily accessible reference data to inform policy, and data will be widely disseminated, for example, through international working groups such as that on Pain management in zebrafish for the Federation of European Laboratory Animal Science Associations (Dr. Sneddon is convener). The innovative approaches used here, particularly the functional brain imaging, are visually impactful and we expect them to capture the public interest, helping to raise the profile of zebrafish research for environmental and human health applications and promote the aims of the BBSRC. In addition, the very real ethical advantages, by refining animal experimentation, will be beneficial for society in general as will potential gains by improving the efficiency of biomedical research, providing more cost effective and higher throughput experimental approaches. Finally, the collaborative, multidisciplinary approach means a high degree of inter-institutional knowledge and skills exchange, and the employed researcher will benefit from a high degree of expert guidance, skills training and impact engagement across an unusually high number of stakeholder groups. Consequently, this project will have a long-lasting and transformative effect on zebrafish-based research at a local, national and international level.