This assay can help answer key questions in the spatial memory field, such as how animals navigate towards a recently-learned goal location. The main advantage of this paradigm is that it elicits etiologically important goal-directed navigation behavior that is triggered by well-controlled sensory stimulus, and it does not require prior training of the mice. Begin by attaching visual cues to the periphery of the platform, which should be at least two centimeters away from the edge to prevent mice from reaching them.
Attach the shelter to the platform. Then, position a light-emitting diode, or LED, at the entrance of the shelter to provide a local visual landmark cue. Next, to isolate the mouse from external distal visual cues, assemble an octagonal wall extending 40 centimeters above and 15 centimeters under the platform surface around the arena, positioned 10 centimeters away from the edge of the platform.
Position an ultrasound speaker 50 centimeters above the center of the platform to deliver aversive auditory stimuli and connect a sound card and amplifier to a speaker. Then, place a translucent screen 64 centimeters above the arena to back-project overhead aversive visual stimuli using a projector. Calibrate the projector light so that there is five to 10 lugs at the center of the platform surface.
Place infrared lights and an infrared camera with a loss pass filter above the platform to prevent protector flicker in the acquired video. Then, place the behavioral apparatus inside a sound-and light-proof box to prevent external cues from affecting the experiment. Finally, use commercially available software to track the position of the mouse online.
Begin by projecting the visual stimuli onto the screen positioned above the arena. Track the position of the mouse online and predefine a region of interest in the arena corresponding to the location of the previously-defined visual stimulus in order to center the visual stimulus above the animal. Next, test the auditory stimulus consisting of a train of three frequency-modulated upsweeps from 17 to 20 kilohertz over three seconds lasting nine seconds in total.
Begin by meticulously cleaning the platform surface with 70%ethanol to remove unwanted olfactory cues. Rinse the shelter with water and 70%ethanol, then wash once again with water to lesson the ethanol odor. Thoroughly dry both the platform and the shelter as the mouse may avoid entering the shelter if it is wet.
Vary the location of the shelter in the platform for each trial by rotating the platform before the experiment. Next, bring the mouse to the experiment room in its home cage, and place the cage beside the testing platform for a 10-minute acclimatization period. Add two grams of bedding from the mouse's home cage to the inside of the shelter to serve as a local olfactory cue.
Remove the mouse from its home cage and avoid retrieving by the tail. Either cup it or let it climb onto an enrichment item that can be lifted. Then, gently place the mouse in the center of the arena and start the video acquisition.
Allow for a seven-minute habituation period, during which no aversive stimulus is presented. Ensure that the mouse has enough time to find and enter the shelter at least once, which is when all four limbs are inside the shelter. Next, automatically or manually trigger the delivery of the visual stimulus centered above the mouse once the animal reaches a predefined region of interest.
To elicit more than one escape response in the same session, allow the mouse to voluntarily leave the shelter. After the assay is finished, return the animal to its home cage. Finally, clean the platform and the shelter before testing the next animal.
Mice exposed to auditory or visual stimuli initiated fast escape responses with short latencies between the onset of stimulation and initiation of flight. The mean latency to escape from the stimulus was significantly longer for the auditory stimulation than the visual. Further, navigation to shelter was accurate and flight trajectories were close to a straight line across types of stimuli.
After a seven-minute acclimatization period to the maze with no shelter, the presentation of a long visual stimulus produced freezing behavior instead of escape. Once mastered, this technique can be done in 90 minutes if it's performed properly. While attempting this procedure, it is important to remember to present the visual stimulus above or above in the head of the mouse, and not while the mouse is investigating the edge of the platform, as it will probably miss the stimulus.
After its development, this technique paved the way for researchers in the field of instinctive behavior to explore the control of learned knowledge over instinctive defensive behavior in mice. After watching this video, you should have a good understanding of how to elicit defensive behavior in mice and how to use them to study spatial navigation during escape responses.
