Development and Validation of a Programmable Conditioning Arena for Zebrafish Learning and Memory Studies

Abstract

Learning and memory can be studied through measurable behavioral changes, but such studies require controlled stimulus delivery and quantitative analysis. This work presents the development and validation of a programmable conditioning arena for adult zebrafish (Danio rerio) learning and memory studies. The system integrates a dual-chamber 3D printed arena, LED matrix visual stimulation, relay-controlled electrical stimulation, in frared video recording, and Arduino-based control. Fish position was extracted from video recordings and used to calculate a performance index (PI), where positive values indicate greater occupancy of the dark region. The platform was tested in wild-type ORAB zebrafish and two mutant strains, PCMTD1-KO and Shank3-KO. Wild-type zebrafish showed a significant baseline-to-T0 increase in PI, whereas PCMTD1-KO and Shank3-KO did not. Among wild-type fish that learned at T0, females showed more persistent post-training dark-region preference than males. These results support the system’s use for quantitative zebrafish learning and memory studies.