CPA was conducted using a three-compartment place conditioning apparatus (Med Associates, USA) with distinct visual and tactile context, and the procedure was similar to that described previously (14, 37, 38). The chamber and drop pan were thoroughly cleaned with 75% ethanol before each behavioral session. The CPA procedure entailed naloxone-precipitated morphine withdrawal aversion following chronic morphine treatment. As shown in the left panel of Fig. 1B, the CPA procedure included four phases: pre-test (day 1), drug treatment (days 2 to 6), conditioning (days 7 to 10), and post-test (day 11).

On pre-test day (day 1), mice were given an initial preference testing to assess their baseline place preference. Mice showing strong unconditioned preference (>75% of the session time) or aversion (<25% of the session time) for any compartment were eliminated from the study. All mice with unbiased preferences were randomly divided into four groups: saline + saline, saline + naloxone, morphine + saline, and morphine + naloxone.

On drug treatment days (days 2 to 6), morphine treatment in morphine + saline and morphine + naloxone groups was commenced. Mice in saline + saline and saline + naloxone groups were treated with saline.

On conditioning days (days 7 to 10), for each mouse, morphine withdrawal was paired with the compartment that it spent more time in (baseline place preference side) during the pre-test period. On days 7 and 9, each mouse in the morphine + naloxone group was injected with naloxone (0.3 mg/kg, intraperitoneally) 2 hours after receiving morphine injection (40 mg/kg, intraperitoneally) to induce enhanced withdrawal and confined in its morphine withdrawal–paired compartment for 20 min. On alternating days (8 and 10), the mouse in the morphine + naloxone group was injected with saline injection (0.1 ml, intraperitoneally) 2 hours after receiving morphine injection (40 mg/kg, intraperitoneally) and confined in its saline-paired compartment for 20 min. To confirm that the CPA seen in the current study was induced by naloxone-precipitated morphine withdrawal aversion but not by spontaneous withdrawal after morphine administration or naloxone alone, we compared the effects of morphine-naloxone treatment on the acquisition of CPA with three control groups (saline + saline, saline + naloxone, and morphine + saline). All CPA procedures with three control groups were the same as described above, except drug treatment with saline + saline, saline + naloxone, or morphine + saline instead of morphine + naloxone.

On post-test day (day 11), each mouse was placed in the same apparatus for 15 min to assess place aversion response. CPA score was defined as the time in the naloxone-paired compartment in the post-test minus that in the pre-test.

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