1) Place the mouse in a cylindrical black start chamber in the middle of the maze. After 10 s have elapsed lift the chamber, switch the buzzer on and guide the mouse gently to the escape box. Do not force the animal to get into the hole as this may be too stressful. If the mouse does not want to get into the target hole, place it gently to the side of the hole and pull gently the base of the tail in the direction away from the hole, which induces the mouse to move in the opposite direction of the tail pull and enter the hole.21 If this does not work, place the mouse directly into the escape box.
2) Once the mouse is inside the box, buzzer, light and/or fan should be turned off.
3) The mouse should stay in the escape box for 2 min.
4) Before the acquisition phase begins, the maze must be cleaned using a solution with 1% Incidin solution (Incidin extra N, Lahmann Rauscher) to avoid olfactory cues. In addition to cleaning, the maze should be rotated around its central axis after each trial in order to control for possibly remaining odour cues.
5) Place the mouse in the cylindrical black start chamber in the middle of the maze. After 10 s have elapsed lift the chamber, switch the buzzer on and allow the mouse explore the maze for 3 minutes.
6) During these 3 minutes number of primary errors, total errors and primary latency should be measured by the experimenter.
7) The trial will end when the mouse enters the goal tunnel or after 3 min have elapsed. Immediately after the mouse enters the tunnel, the buzzer should be turned off and the mouse is allowed to stay in the tunnel for 1 min.
8) If the mouse does not reach the goal within 3 minutes the experimenter should guide the mouse gently to the escape box (see Step 1) and leave the mouse inside for 1 min.
Mice may go out of the escape box during the time it should stay in it. You may cover the escape hole once the mouse is inside for 1 minute or just place mice gently in the escape box whenever it goes out.
9) Place the mouse in its home cage until the next trial.
10) Repeat steps 4-9 until the animal has had the desired number of trials for that day. Animals will receive 4 trials per day with an inter-trial interval (ITI) of 15 minutes during 4 days.
11) The ITI of 15 minutes should be used to test the next 3 to 4 mice.
Reference memory (short- and long-term retention): Probe trial
12) On day 5, 24 h after the last training day, the probe trial should be conducted. The target hole must be closed. Rotate the maze so that the target hole is closed and re-adjust the maze so that the holes are in the same position as during the training days.
13) Place the animal in the middle of the maze under the cylindrical black start chamber and after 10 s have elapsed lift the chamber, switch the buzzer on and allow the mouse explore the maze.
14) Remove the mouse after a fixed interval (90 s). The probe trial is done in order to determine if the animal remembers where the target goal was located. Number of pokes (errors) in each hole and latency and path length to reach the virtually target hole are measured.
15) To assess long-term retention a second probe trial will be applied on day 12, without any training session between day 5 and day 12. Repeat steps 12-15 (to study reference memory probe trials should be performed at least 24 hours after the last training trial; if the probe trial is given immediately after the last training trial recent recall is being measured).