Zebrafish Y-Maze Spatial Alternation

The zebrafish Y-maze is an automated three-arm aquatic maze used to assess spatial working memory, exploratory behavior, and turn preference in adult zebrafish through spontaneous alternation and forced-choice alternation paradigms. The apparatus consists of three transparent acrylic arms of equal length joined at 120-degree angles, each equipped with a motorized guillotine gate controlled by ConductMaze, distinct visual cues (colored panels or geometric patterns) on the arm walls, and position-tracking capability via overhead camera. Spontaneous alternation in the Y-maze is a well-validated measure of spatial working memory that does not require food deprivation or extensive training, making it one of the most widely used assays in zebrafish neurobehavioral research for screening genetic mutations, pharmacological agents, and neurotoxicant exposures.

In the spontaneous alternation protocol, all three gates open simultaneously and the fish freely explores the maze for a defined session period (typically 8-10 minutes). Successive arm entries are recorded, and an alternation is scored when the fish visits all three arms in three consecutive entries (e.g., ABC, BCA, CAB). Alternation percentage is calculated as the number of alternations divided by the maximum possible alternations (total arm entries minus 2) multiplied by 100, with chance performance at 22.2%. In the forced-choice (delayed) alternation protocol, the fish is confined to a sample arm by closing the other two gates, then after a configurable delay the gates open for a choice trial, and re-entry into the previously visited arm versus the novel arm is recorded. This delayed variant allows systematic manipulation of retention interval to probe memory decay.

ConductMaze automates all gate operations with sub-second pneumatic actuator precision, tracks the fish centroid in real time via overhead camera with arm entry defined by a configurable threshold crossing (typically 60% of arm length from center), and automatically scores spontaneous alternation sequences, arm entry counts, and per-arm dwell times. The software detects and flags thigmotaxis (wall-hugging) behavior within each arm, computes turn bias ratios (left vs right turns from each arm), and generates heat maps of spatial occupancy. For the forced-choice paradigm, ConductMaze manages the full trial sequence including sample phase, delay period with gate closure, choice phase, and inter-trial interval, enabling high-throughput testing of multiple fish per session with full data export.