Increased Visits to Opposing Arms of a Plus Maze as a Behavioral Indicator of Salinity Stress in Zebrafish

Zebrafish (Danio rerio) are widely used in behavioral neuroscience and ecotoxicology to study stress-related responses. Traditional plus maze paradigms rely on arm preference, with arms differing in perceived safety. Here, we propose an alternative approach using a plus maze with identical arms, where stress is assessed based on movement patterns rather than preference. Fish explored the maze for 20 min under baseline conditions, followed by exposure to hyperosmotic stress (5‰ salinity). Behavior was quantified using automated video tracking, including locomotor activity, spontaneous alternation, and transition patterns between arms. Salinity stress did not significantly affect overall locomotor activity or spontaneous alternation. However, it markedly altered movement structure. The proportion of circular transitions decreased significantly. Most notably, transitions between adjacent arms declined, while transitions between opposing arms increased to nearly 50% of total entries. The runs test analysis further indicated an increased tendency toward repetitive movement sequences under stress conditions. These behavioral changes were associated with significantly increased whole-body cortisol levels. The findings identify a shift toward opposing-arm transitions as a potentially sensitive and previously underappreciated behavioral correlate of stress in zebrafish. This metric may complement conventional behavioral endpoints and offers particular promise for automated high-throughput applications in ecotoxicology and preclinical research.