Figure 5. The time of day of morphine conditioning does not influence morphine-induced CPP. A, CPP experimental groups. B, Time line of CPP procedure. Mice were allowed to acclimate to the chambers for 2 d during habituation (Hab). During conditioning (days 3–7), mice were injected with saline (sal) or morphine (mor; 10 mg/kg, i.p.) and paired with the least preferred side at either ZT8, ZT13, or ZT21 CPP tests were performed 24 h postconditioning. C, Summary showing activity counts for saline-treated animals during conditioning days (C1–C5) at each time point measured (ZT8, ZT13, or ZT21) [saline (Sal)-ZT8: n = 7; saline (Sal)-ZT13: n = 8; saline (Sal)-ZT21: n = 10; F(8,88) = 0.766, p = 0.63; two-way repeated measures ANOVA with Bonferroni post hoc test]; *p < 0.05. D, Summary showing activity counts for morphine-treated animals during conditioning days (C1–C5) at each time point measured (ZT8, ZT13, or ZT21; morphine (Mor)-ZT8: n = 7; morphine (Mor)-ZT13: n = 10; morphine (Mor)-ZT21: n = 10; F(8,96) = 1.48, p = 0.17; two-way repeated measures ANOVA with Bonferroni post hoc test]; *p < 0.05. E, Summary showing activity counts for saline (Sal)-treated or morphine (Mor)-treated animals during conditioning days (C1–C5) at each time point measured (ZT8, ZT13, or ZT21); [saline (Sal)-ZT8: n = 7; morphine (Mor)-ZT8: n = 7; saline (Sal)-ZT13: n = 8; morphine (Mor)-ZT13: n = 10; saline (Sal)-ZT21: n = 10; morphine (Mor)-ZT21: n = 10; F(20,184) = 2.08, p = 0.006, two-way repeated measures ANOVA with Bonferroni post hoc test]; *p < 0.05. F, Summary showing activity counts for saline (Sal)-treated or morphine (Mor)-treated animals mice on conditioning day 5 at each time point measured [saline (Sal)-ZT8: n = 7; morphine (Mor)-ZT8: n = 7; saline (Sal)-ZT13: n = 8; morphine (Mor)-ZT13: n = 10; saline (Sal)-ZT21: n = 10; morphine (Mor)-ZT21: n = 10; F(5,46) = 17.6, p < 0.0001, one-way ANOVA with Bonferroni post hoc test]; *p < 0.05. G, Summary showing that the time of day of morphine conditioning does not influence morphine-induced CPP [saline (Sal)-ZT8: n = 14; morphine (Mor)-ZT8: n = 17; saline (Sal)-ZT13: n = 8; morphine (Mor)-ZT13: n = 10; saline (Sal)-ZT21: n = 10; morphine (Mor)-ZT21: n = 10; F(2,63) = 0.267, p = 0.767; two-way ANOVA with Bonferroni post hoc test]; *p < 0.05.