Experimental Trial Procedure

Once in the experimental rig, animals were allowed to procedurally acclimate and settle on the bottom of the tank, as evidenced by the animal remaining motionless for at least 5 min. Once an animal habituated, it received a sequence of approximately 90 flashes. For this study, we used a 10-s inter-stimulus interval (ISI), which does not cause attenuation due to learning, fatigue, or a combination of both (Otis and Gilly, 1990). With an ISI of 10 s, the total sequence duration lasted for 15 min per body region and each region was tested during different sessions. The duration and ISI were tested in preliminary trials and found to be appropriate for the purposes of this study. The rationale was to reduce testing sessions and have only one per body region since 15 min were sufficient.

Each flash stimulation was considered an individual trial. The purpose was to elicit the sub-jet-threshold startle response. Each animal received 90 trials for each of the three body regions, for a total of 270 trials for each of the eight animals, thus 2,160 trials in total. One body region per animal was tested at a time (we counterbalanced the order of the body regions tested per animal).

For details on video-recording, scoring, image analysis, and statistical analysis see Supplementary Material.