Fear conditioning

Mice were tested for contextual and cued fear learning and memory at 3 mpi following a standard protocol [66]. In the fear conditioning test, hippocampus-dependent and -independent learning and memory can be distinguished. Contextual fear conditioning is known to be hippocampus-dependent, whereas cued fear conditioning is known to be hippocampus-independent and amygdala-dependent [67, 68]. Briefly, mice were placed into a sound-attenuating chamber. After a 120 s baseline period without any stimuli, a tone (80 dB, 2800 Hz) was played for 30 s, which co-terminated with a 2 s shock (0.5 mA). This was followed by a 90 s period with no stimuli before the tone-shock pairing was repeated again. In total, mice received 2 tone-shock pairings. The chambers were thoroughly cleaned with 0.5%acetic acid between animals. Twenty-four hours later, mice were tested for contextual and cued fear memory recall. For contextual memory recall, mice were placed into the same chamber for a period of 5 min, with no cues presented. For cued memory recall, the chambers were changed to remove any contextual cues (floors were covered with a solid, white panel, the roof and walls were changed to a black triangle, and 10%isopropanol was used for cleaning). Animals had a baseline period of 90 s with no stimuli; at 90 s, the same tone played for a total of 3 min.

All trials were recorded and analyzed with Video Freeze software (PMED-VFC-NIR-M, Med Associates, Inc., Fairfax, VT) and automatic outputs were generated to analyze average motion and time freezing (defined as a minimum of 30 frames, or 1 s, of total movement cessation). Percent time freezing was analyzed as measure of fear memory and assessed with a 1-way ANOVA between PFF and Mono groups, or with a 1-way repeated measures ANOVA when looking over time within a trial. Because we did not observe any significant differences between PFF and Mono groups at 3 mpi, we did not repeat fear conditioning testing at 6 mpi.