Mecamylamine

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.

The experimenter was blinded to genotype during both recordings and subsequent data analysis. L3 larvae were dissected under extracellular saline as described^{59 (link)} with the only modification being a red filter applied to both the dissecting light and compound microscope to minimize DmCRY degradation before and during recordings. Thick-walled borosilicate glass electrodes (GC100F-10; Harvard Apparatus) were fire-polished to resistances of 10–15 MΩ. Recordings were made using the Multiclamp 700B amplifier controlled by pCLAMP (v.10.4) and the Digidata 1440A analogue-to-digital converter (Molecular Devices). Only cells with an input resistance of ≥500 MΩ were used. Traces were filtered at 10 kHz and sampled at 20 kHz. The extracellular saline solution contained the following: 135 mM NaCl, 5 mM KCl, 4 mM MgCl₂.6H₂O, 2 mM CaCl₂.2H₂O, 5 mM TES and 36 mM sucrose, pH 7.15. The intracellular patch solution contained the following (in mM): 140 mM potassium-d-gluconate, 2 mM MgCl₂.6H₂O, 2 mM EGTA, 5 mM KCl and 20 mM HEPES, pH 7.4. KCl and CaCl₂ were from Thermo Fisher Scientific; sucrose was from BDH; all of the remaining chemicals were from Sigma-Aldrich. Mecamylamine (1 mM) was applied to all preparations to isolate the aCC motoneurons from excitatory cholinergic synaptic input. For recordings supplemented with additional FAD or riboflavin (Sigma-Aldrich), dilutions were made up in intracellular saline and kept in the dark.

The effects of iROE on mechanical hyperalgesia caused by plantar incision were further investigated to ascertain whether they were involved in α₁ and α₂ adrenergic, cholinergic (nicotinic and muscarinic), and opioid receptors. Forty-two rats were randomly allocated to seven groups (n = six rats per group), including one iROE group that served as a control and the other six groups that included rats that were given iROE and study drugs (yohimbine 2 mg/kg, dexmedetomidine 50 μg/kg, prazosin 1 mg/kg, atropine 5 mg/kg, mecamylamine 1 mg/kg, and naloxone 5 mg/kg). Two hours after the plantar incisions, normal saline or study drugs were administered intraperitoneally. After 10 min, 300 mg/kg iROE was administered intraperitoneally. Previous study suggested the application of drugs to investigate the possible involvement of the aforementioned receptor systems [9 (link),15 (link),16 (link),17 (link),18 (link)]. All study drugs were provided by Sigma-Aldrich.