Abstract：
Zebrafish are a dynamic research model in the domains of neuropsychopharmacology, biological psychiatry and behaviour. Working with larvae ≤4 days post-fertilisation (dpf) offers an avenue for high-throughput investigation whilst aligning with the 3Rs principles of animal research. The light/dark assay, which is the most widely used behavioural assay for larval neuropharmacology research, lacks experimental reliability and standardisation. This study aimed to formulate a robust, reproducible and standardised light/dark behavioural assay using 4 dpf zebrafish larvae. Considerable between-batch and inter-individual variability was found, which we rectified with a normalisation approach to ensure a reliable foundation for analysis. We then identified that 5-min light/dark transition periods are optimal for locomotor activity. We also found that a 30-min acclimation in the light was found to produce significantly increased dark phase larval locomotion. Next, we confirmed the pharmacological predictivity of the standardised assay using ethanol which, as predicted, caused hyperlocomotion at low concentrations and hypolocomotion at high concentrations. Finally, the assay was validated by assessing the behavioural phenotype of hyperactive transgenic (adgrl3.1-/-) larvae, which was rescued with psychostimulant medications. Our standardised assay not only provides a clear experimental and analytical framework to work with 4 dpf larvae, but also facilitates between-laboratory collaboration using our normalisation approach.
摘要：
斑马鱼已成为神经精神药理学领域的动态研究模型，生物学精神病学和行为。受精后（dpf）≤4天的幼虫工作为高通量研究提供了途径，同时符合动物研究的3Rs原则。光/暗测定，这是幼虫神经药理学研究中最常用的行为测定法，缺乏实验可靠性和标准化。这项研究旨在制定一个强有力的，使用4dpf斑马鱼幼虫进行可重复和标准化的光/暗行为测定。发现批次之间和个体之间存在相当大的差异，我们用标准化方法进行了纠正，以确保有可靠的分析基础。然后我们确定5分钟的光/暗过渡期对于运动活动是最佳的。我们还发现，在光线下适应30分钟会产生明显增加的暗相幼虫运动。接下来,我们证实了使用乙醇的标准化测定的药理学预测性，正如预测的那样，在低浓度时引起运动过度，在高浓度时引起运动过度。最后,通过评估过度活跃的转基因（adgrl3.1-/-）幼虫的行为表型来验证该测定，用精神兴奋剂药物抢救。我们的标准化测定不仅提供了一个清晰的实验和分析框架，用于4个dpf幼虫，而且还使用我们的标准化方法促进了实验室之间的合作。
