zAcademy: an open-source programmable conditioning arena reveals sex- and genotype-dependent avoidance learning in adult zebrafish.

Zebrafish are widely used to study learning and memory. Here, we describe zAcademy, a programmable conditioning arena released in full as open hardware and open software. zAcademy combines a 3D-printed dual-chamber arena, bottom-mounted LED matrices that generate visual patterns under software control, graphite rod electrodes gated by an Arduino-controlled relay, an overhead infrared camera, and a position-based analysis workflow. In one 180-min session, adult zebrafish were classically conditioned to associate a checkerboard light pattern beneath the arena (conditioned stimulus, CS) with a mild shock (unconditioned stimulus, US). Conditioned avoidance was assessed in six unreinforced CS-alone probes: one immediately after training to test learning, the remaining five at 30-min intervals to test memory. Spatial preference was quantified as a performance index (PI; occupancy of the dark zone). Wild-type fish shifted toward the dark zone immediately after training, whereas shank3 KO fish did not. The wild-type response was carried primarily by females rather than males. In addition, among initial responders, females sustained elevated PI at more later probes. zAcademy provides an inexpensive, fully documented, and readily modified platform for comparing experience-dependent behavioral change across genotypes and sexes.