Rapid Fabrication of Highly Customizable Test Chambers for Behavioral Experiments with Small Aquatic Model Organisms

Behavioral biotests with larval zebrafish and small aquatic invertebrates are predominantly conducted using digital video recording combined with specialized animal tracking software to extract quantitative locomotor and behavioral endpoints. Such assays commonly rely on commercially available polystyrene multi-well test plates, which, despite their convenience, present several limitations: fixed well geometry, meniscus-induced shadow artifacts at well edges, absence of customization options, and suboptimal optical transmittance for orthogonal sidelight illumination with infrared light. Here, we describe a rapid and low-cost technique for fabricating highly customizable test chambers for behavioral experiments with small aquatic model organisms. The method employs infrared laser cutting of biocompatible poly(methyl methacrylate) thermoplastic followed by thermal bonding under controlled compression in a standard laboratory oven. The speed and low cost of fabrication enable the production of large numbers of test plates with considerable flexibility in chamber geometry, including complex miniaturized maze configurations. As a proof of concept, we demonstrate how test chamber geometry influences both baseline locomotor activity and stimulated sensory-motor responses in larval zebrafish. Relatively little is currently understood about how larval zebrafish-with their tractable and well-characterized nervous systems-perceive and respond to spatial environmental features or how chamber geometry modulates common behavioral responses. The fabrication technique described here provides researchers with a flexible and accessible tool for developing novel behavioral biotests with broad applications across ecology, ecotoxicology, and experimental neurobiology.