Investigating fear memory encoding and retrieval in mice

All mice were handled for 1 h per day for two weeks prior to the behavioral tests. The behavioral session was conducted in a fear conditioning box (46001, UGO BASILE S.r.l, Italy). Freezing was defined as a complete absence of movement, except for respiration. Scoring of the freezing response duration was started after one second of sustained freezing behavior [34 (link)]. The freezing was measured using the Anymaze (version 6.0, Stoelting) software based on a threshold of change in video image pixels. The mice were delivered with 10 kHz and 1 kHz tones and the percentage of freezing time was averaged for the tone each day.
The changing index of freezing was calculated using the equation: $Thechangingindex=\left(T_{lightonfreezing}-T_{lightofffreezing}\right)/\left(T_{lightonfreezing}+T_{lightofffreezing}\right).$
The discrimination index of fear memory was calculated using the equation: $Thediscriminationindex=\left(T_{10kHzfreezing}-T_{1kHzfreezing}\right)/\left(T_{10kHzfreezing}+T_{1kHzfreezing}\right).$
At the encoding stage (day 1), after 5 min of habituation in context A, the mice received 5 pairings of auditory tone (70 dB SPL 10 kHz or 1 kHz pure tone, 20 s duration) with electric foot shocks (1 mA, 2 s duration, and overlapped with the last 2 s of tone). The time interval between each trial was 70 s. In Fig. 1E, F, and S1C, 10 min before the test, 0.5 µl of pharmacological agent was administered simultaneously bilaterally at a rate of 0.125 µl every 10 s by cannula. The mice were administered either saline, mecamylamine (MCM, 10 µM, 20 µM Aladdin, 826-39-1), or atropine alone (10 µM, 20 µM Aladdin, 5908-99-6). In Fig. 2, each tone is accompanied by optogenetic inhibition or activation. In Fig. 2G, 30 min before the test, mice were intraperitoneally injected with saline and nicotine (10 µM, Sigma Aldrich), and each tone is accompanied by optogenetic inhibition.
At test 0 (day 2), to label tone-responsive ACx neurons, the mice were delivered three times of 10 kHz tone or three times 1 kHz tone.
At test 1a or test 1b (day 2 or day 3), the mice were tested for freezing behavior in response to tests 1a (new fear-unpaired 1 kHz or 10 kHz tone) and 1b (fear-paired 10 kHz or 1 kHz) with a time interval of 2 h in different contexts B and C. The mice were delivered three (Figs. 1, 2, 4, 5 and Figs. S1, 2G, H, 4) or four (Fig. 3, S2E, F) representations of both 10 kHz and 1 kHz tones. In groups with four representations of tones, the first and third tones were accompanied by optogenetic activation or inhibition. In Fig. S2A–C, the mice were only given 10 times optogenetic activations. For test 1, each group of mice was randomly assigned and then equally received 1 kHz or 10 kHz tone in different contexts B and C, and after 90 min, the mice were carefully perfused.
All contexts were different in shape, background, and floor texture.