Ketamine Blocks Morphine-Induced Conditioned Place Preference and Anxiety-Like Behaviors in Mice

Abstract

Patients suffering from opioid use disorder often relapse during periods of abstinence, which is posited to be caused by negative affective states that drive motivated behaviors. Here, we explored whether conditioning mice with morphine in a conditioned place preference (CPP) training paradigm evoked anxiety-like behavior during morphine abstinence. To do this, mice were conditioned with morphine (10 mg/kg, i.p.) for 5 days. Twenty-four hours following conditioning, anxiety levels were tested by measuring time in the open arms of the elevated plus-maze. The next day, mice were placed in the three-compartment chamber to measure morphine-induced CPP. Our results show that following morphine conditioning, mice spent significantly less time in the open arm of the elevated plus-maze and expressed robust morphine CPP on CPP test day. Furthermore, we found that an acute treatment with (R,S)-ketamine (10 mg/kg, i.p.), a medication demonstrating promise for preventing anxiety-related phenotypes, 30 min before testing on post-conditioning day 1, increased time spent in the open arm of the elevated plus-maze in saline- and morphine-conditioned mice. Additionally, we found that the second injection of ketamine 30 min before CPP tests on post-conditioning day 2 prevented morphine-induced CPP, which lasted for up to 28 days post-conditioning. Furthermore, we found that conditioning mice with 10% (w/v) sucrose using an oral self-administration procedure did not evoke anxiety-like behavior, but elicited robust CPP, which was attenuated by ketamine treatment 30 min before CPP tests. Overall, our results suggest that the ketamine-induced block of morphine CPP may not be attributed solely to alleviating negative affective states, but potentially through impaired memory of morphine-context associations.

Keywords: anxiety; conditioned place preference; ketamine; morphine; negative affect; opioid use disorder; psychedelics.

Figure 1

Morphine conditioning in a conditioned place preference (CPP) paradigm elicits anxiety-like behaviors during 24 h abstinence. (A) Timeline and drug regimen of the behavioral procedure. Animals underwent 2 days of habituation (H), followed by 5 days of saline or morphine (10 mg/kg, i.p.) conditioning (C), before being subjected to tests measuring anxiety-like behaviors using an elevated plus maze (EPM) 24 h post-conditioning. Twenty-four hours post EPM tests, CPP tests were performed. Animals were injected with saline 30 min before EPM and CPP tests. (B) A summary showing that morphine conditioning over 5 days produces robust locomotor sensitization (F_(4,152) = 17.1, p < 0.0001, two-way repeated-measures ANOVA, Bonferroni post hoc test). (C) A summary showing that morphine (Mor)-conditioned mice spent significantly less time in the open arms of the elevated plus-maze compared to saline (Sal)-conditioned mice 24 h following the last conditioning day (t₍₃₈₎ = 3.35, p = 0.002, student’s t-test). (D) A summary showing that morphine conditioning produced reliable CPP (t₍₃₈₎ = 5.61, p < 0.0001, student’s t-test). (E) Correlation of the % time in the open arm of the elevated plus-maze and CPP score in saline- or (F) morphine-conditioned mice. *p < 0.05, **p < 0.01.

Figure 2

Acute (R,S)-ketamine injection produces anxiolytic-like behaviors in mice 24 h after conditioning and blocks morphine-induced CPP. (A) Timeline and drug regimen of the behavioral procedure. Saline or (R,S)-ketamine (10 mg/kg, i.p.) was injected 30 min before EPM test with the second injection taking place 30 min before the first CPP test. (B) A summary showing that morphine conditioning over 5 days (C1–C5) produces robust locomotor sensitization (F_(4,56) = 12.55, p < 0.0001, two-way repeated-measures ANOVA, Bonferroni post hoc test). (C) A summary showing that (R,S)-ketamine significantly increased the time spent in the open arms of the elevated plus-maze in both saline (Sal)- and morphine (Mor)-conditioned mice (F_(3,52) = 22.2, p < 0.0001, one-way ANOVA, Bonferroni post hoc test; animals not receiving (R,S)-ketamine are the same data as shown in Figure 1C). (D) A summary showing that morphine produced reliable CPP at post-conditioning day 2, which was blocked by (R,S)-ketamine injected 30 min before testing (F_(3,52) = 14.04, p < 0.0001, one-way ANOVA, Bonferroni post hoc test; saline and morphine groups are the same animals as shown in Figure 1D). (E) A summary showing the activity counts in the CPP chamber during habituation (baseline) and the CPP test in saline (Sal)- or morphine (Mor)-conditioned mice treated with saline or (R,S)-ketamine 30 min before testing (F_(3,52) = 0.447, p = 0.72, two-way repeated-measures ANOVA). *p < 0.05, **p < 0.01.

Figure 3

(R,S)-ketamine administration during early abstinence is sufficient to prevent the prolonged retention of morphine-induced CPP at post-conditioning day 28. (A) Timeline and drug regimen of the behavioral procedure. (R,S)-ketamine (10 mg/kg, i.p.) was injected 30 min before the EPM test on post-conditioning day 1 (PC1) and again on the first CPP test on post-conditioning day 2 (PC2; i.e., each mouse received a ketamine injection before the EPM test and a second ketamine injection the next day before the CPP test). The second CPP test was run on PC28. (B) A summary showing that morphine produced reliable CPP 28 days post-conditioning, which was blocked by (R,S)-ketamine (column factor: F_(3,38) = 10.25, p < 0.0001, two-way repeated-measures ANOVA, Bonferroni post hoc test; PC2 data is the same data shown in Figure 2D). Abbreviation: EPM, elevated plus maze; CPP, conditioned place preference. *p < 0.05, **p < 0.01.

Figure 4

Ketamine administration attenuates sucrose-induced conditioned place preference. (A) Timeline and sucrose regimen of the behavioral procedure. Following sucrose oral self-administration in the three-compartment apparatus, mice underwent EPM testing on post-conditioning day 1 (PC1). Twenty-four hours later, mice received no injection or (R,S)-ketamine (10 mg/kg, i.p.) 30 min before the conditioned place preference (CPP) test on post-conditioning day 2 (PC2). (B) A summary showing the milliliters of water or sucrose consumed for each training session in the least preferred chamber. Groups conditioned with sucrose (i.e., sucrose (sucr.) and sucrose + ketamine (sucr. + ket.) groups) drank significantly more than groups conditioned with water (i.e., water (Wat.) and water+ketamine (Wat. + Ket.) groups; F_(15,175) = 462.1, p < 0.0001, two-way repeated-measures ANOVA, Bonferroni post hoc test). (C) A summary showing that conditioning with sucrose had no effect on anxiety-like behaviors as both water- and sucrose-conditioned mice displayed similar % time in the open arm of the EPM (t₍₁₇₎ = 0.184, p = 0.856, student’s t-test). (D) A summary showing that oral self-administration of sucrose produced CPP at PC2, which was blocked by (R,S)-ketamine treatment (F_(3,35) = 6.31, p = 0.0015, one-way ANOVA, Bonferroni post hoc test). (E) A summary showing that ketamine injections 30 min before the CPP test did not impact entrance counts in the CPP apparatus (Sal. + Sal. vs. Sal. + Ket.: t₍₃₁₎ = 0.295, p = 0.770; Mor. + Sal. vs. Mor. + Ket.: t₍₂₁₎ = 1.13, p = 0.272; Wat. + Sal. vs. Wat. + Ket.: t₍₁₆₎ = 0.874, p = 0.395; Sucr. + Sal. vs. Sucr. + Ket.: t₍₁₉₎ = 1.43, p = 0.168, student’s t-test). (F) A summary showing that ketamine injections 30 min before the CPP test did not impact exploratory counts in the CPP apparatus (Sal. + Sal. vs. Sal. + Ket.: t₍₃₁₎ = 1.42, p = 0.166; Mor. + Sal. vs. Mor. + Ket.: t₍₂₁₎ = 0.045, p = 0.964; Wat. + Sal. vs. Wat. + Ket.: t₍₁₆₎ = 1.26, p = 0.226; Sucr. + Sal. vs. Sucr. + Ket.: t₍₁₉₎ = 1.80, p = 0.088, student’s t-test). *p < 0.05, **p < 0.01.