2.4.2. Y-Maze Test

Spatial memory and the response to novelty in zebrafish was assessed using the Y-maze task [26]. The position in the Y-maze task was considered an index of memory [27]. The apparatus consisted of a Y-maze glass aquarium with three arms (25 cm long, 8 cm wide, and 15 cm high), filled with 3 L of the same water used in the home aquarium. Visible geometric shapes such as squares, circles, and triangles were placed on the external Y-maze walls. The Y-maze arms were arbitrarily assigned: (i) the start arm, where fish began to investigate (always open); (ii) the novel arm, which was obstructed during the first trial, but open during the second trial; and (iii) the other arm (constantly open). The Y-maze center (neutral zone) was not counted in the analysis. The assignment comprised of two trials separated by 1 h to evaluate the response to novelty and spatial memory. During the first trial (training, 5 min), 60 min of REO treatment, the fish could investigate only two arms of the Y-maze (the start and the other arm), whereas the third arm (the novel arm) was obstructed. For the second trial, each fish was individually introduced into the start arm and free access to all three arms for 5 min to assess the response to novelty. Behavioral activity was analyzed using ANY-Maze ® software (Stoelting CO, Wood Dale, IL, USA) and with a Logitech HD Webcam C922 Pro Stream camera (Logitech, Lausanne, Switzerland) placed above the Y-maze tank. The following measures were recorded: time spent in each arm to assess short-term spatial memory and, for the locomotory activity total, distance traveled (m) and turn angle (◦) were assessed.
