A cost-effective method to assess light/dark preference and activity in zebrafish larvae

The light-dark test is a widely used behavioral assay for studying light/dark preferences in zebrafish (Danio rerio) and rodents. Currently, options for conducting this experiment in larval zebrafish are limited and expensive, restricting its accessibility. Here, we present a simple, cost-effective strategy that can be implemented in any laboratory environment. This approach uses infrared video-tracking technology to monitor zebrafish larvae during alternating light and dark phases, allowing for continuous monitoring of larval behavior without the need for sophisticated or expensive equipment. We evaluated two different protocols (40-min habituation in dark followed by 20-min habituation in light; and 30-min habituation in the light) at two developmental stages, specifically 7-days post-fertilization (dpf) and 14 dpf. After the habituation period, larvae were exposed to multiple cycles of light and darkness, enabling the assessment of locomotor activity and light/dark preferences. Behavioral parameters such as distance traveled and movement activity during light versus dark phases were measured using freely available open-source software, and data were analyzed using custom-written Python codes. The results demonstrated erratic movements of the larvae during the first protocol, while typical larval behaviors in the light and dark were observed during the protocol using 30-min habituation, highlighting the importance of an appropriate habituation phase when conducting the LDT. This method can be used to investigate behavioral activity in larval zebrafish, retaining high experimental rigor while significantly reducing costs, making it accessible to a broader range of zebrafish research laboratories.明暗箱实验（light-dark test，LDT）是广为应用的行为学检测方法，用于研究斑马鱼（Danio rerio）与啮齿类动物的明暗偏好。目前，针对斑马鱼幼体开展该实验的可选方案匮乏且成本不菲，极大限制了其推广应用。本研究提出一种简便高效、经济可行的实验策略，可在各类实验室条件下实施。该方案采用红外视频追踪技术，在交替的明暗周期内对斑马鱼幼体进行监测，无需借助复杂或高价设备即可实现幼体行为的连续观测。我们针对受精后7天（7-dpf）与受精后14天（14-dpf）两个发育阶段，评估了两种实验范式：其一为先在黑暗环境中进行40分钟习惯化训练，随后在光照环境中进行20分钟习惯化训练；其二为直接在光照环境中进行30分钟习惯化训练。习惯化阶段结束后，将幼体暴露于多轮明暗交替环境中，以此评估其运动活性与明暗偏好行为。通过免费开源软件采集幼体在光照与黑暗阶段的移动距离、活动强度等行为参数，并采用自主编写的Python代码完成数据分析。结果显示，第一种实验范式下幼体运动表现异常，而采用30分钟习惯化期的范式则观测到典型的斑马鱼幼体明暗行为特征，凸显了开展明暗箱实验（LDT）时设置适宜习惯化阶段的重要性。该方法可用于研究斑马鱼幼体的行为活动，在保证较高实验严谨性的同时大幅降低实验成本，能够让更多斑马鱼研究实验室得以使用该技术。