Advancing understanding of anaesthesia and analgesia in the zebrafish

斑马鱼(Danio Rerio)被广泛用作了解发育中的生物过程和各种人类疾病的模型。胚胎和幼体斑马鱼(胚胎幼体，受精后10天，或DPF)的特征使它们作为研究模型非常有吸引力，包括它们用于可视化生物过程的光学透明度和易于遗传操作。因此，全球每年都有数以百万计的胚胎幼体斑马鱼被用于实验室。然而，这些动物中的许多都接受了麻醉，但指导适当的麻醉剂选择的信息很少，关于镇痛剂(止痛药)的信息更少。我们的项目将解决这些知识差距，并有3个主要目标。目的1)将建立诱导镇静和安乐死所需的麻醉剂浓度，以及提供止痛所需的镇痛剂浓度。为此，我们将使用脑功能成像(通过荧光染料揭示大脑的神经活动)来测试动物在接受治疗时对刺激的反应。电生理学(直接记录大脑的电活动)将被用来确定安乐死所需的麻醉浓度。我们还将收集数据，以了解传统的鱼类镇静指标是否合适(例如，失去平衡)，并测量复合摄取，以支持对复合暴露效应浓度的解释。目标2)将确定是否有任何麻醉剂抑制运动，是否有任何麻醉剂和镇痛剂是令人厌恶的，以及是否有任何镇痛剂减少有害刺激(例如，热、二氧化碳)的影响。为此，我们将评估行为，如治疗对运动的影响，以及避免在测试场地内放置麻醉剂或止痛剂的区域。此外，我们将测量镇痛剂减少有害刺激对行为影响的能力，以此作为其止痛特性的指标。目的：更好地了解所选麻醉剂和镇痛剂的作用机制，以及斑马鱼胚胎的镇静和痛觉。为此，我们将在转基因动物中进行脑功能成像，在这种成像中，我们能够可视化大脑中特定类型神经元的活动。这将使我们了解哪些大脑区域和细胞类型对每个药物的治疗有反应，以及哪些区域/细胞类型参与镇静过程。我们还将把这种方法应用于受到有害刺激的动物，以更好地了解鱼类的痛觉。该项目将使科学家能够选择最科学和道德上最合适的麻醉剂和止痛剂用于他们的工作，并将提供更好的了解这些药剂是如何工作的，以及促进关于鱼类的镇静和疼痛感知过程的知识。受益者将包括兽医和水族鱼行业，因为这些数据将有助于改进做法，如如何最好地监测鱼类的麻醉，并告知最适合动物运输的治疗方法。英国内政部等监管动物科学使用的政府机构将受益于提供高质量的参考数据，用于动物实验实践的决策，慈善机构，如与神经疾病(如癫痫、阿尔茨海默氏症、帕金森氏症)相关的机构，将获得关于替代(更具成本效益的)模式物种的进一步验证数据。公众和整个社会将受益于动物福利标准的提高，对镇静和疼痛感知的更多了解，以及我们对越来越多地用于实验研究的物种生物学的了解，包括生物医学研究。

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.