Figure 4. Median split analyses demonstrate significant interactions between initial chamber preference and subsequent CPP acquisition and stress-induced reinstatement, and significant effects of guanfacine.

Two-way ANOVAs conducted within sex between split subgroup and test day found significant interactions in both male and female mice. Data were analyzed by split subgroups across pre-treatment conditions, but are presented for male (a,b) and female (c,d) mice separately for saline (a,c) and guanfacine (b,d) pre-treatment for ease of viewing. Further, the yellow box highlights the significant findings regarding stress-induced reinstatement. (a) In male mice, only nicotine treatment in the initially preferred chamber subsequently leads to significant stress-induced reinstatement of nicotine CPP when the swim stress is preceded by a control saline injection. (b) This reinstatement is not significant when male mice in the same split subgroup (i.e. nicotine treatment in the initially preferred chamber) receive guanfacine (0.15 mg/kg, i.p.) prior to the swim stress. (c) In contrast, only female mice with nicotine treatment in the initially non-preferred chamber demonstrate significant stress-induced reinstatement of nicotine CPP with a saline pre-swim treatment, (d) and not with a guanfacine pre-swim treatment. Additionally, these two reinstatement preference scores (FNS Lo-Pre vs. FNG Lo-Pre) are significantly different from each other. *p < 0.05 compared to pretest day within nicotine dose using Dunnett’s tests. Brackets indicate p < 0.05 compared to same test day in different split group using Tukey’s multiple comparison test. #p < 0.05 for comparison to corresponding day, also marked by #, across saline and guanfacine pre-treatment groups by Tukey’s test. MCS = male, control conditioning group, saline pre-treatment prior to stress. MNS = male, nicotine (0.5 mg/kg, s.c.) conditioning group, saline pre-treatment. MCG = male, control conditioning group, guanfacine (0.15 mg/kg, i.p.) pre-treatment. MNG = male, nicotine (0.5 mg/kg, s.c.) conditioning group, guanfacine pre-treatment. FCS = female, control conditioning group, saline pre-treatment. FNS = female, nicotine (0.75 mg/kg, s.c.) conditioning group, saline pre-treatment. FCG = female, control conditioning group, guanfacine (0.15 mg/kg, i.p.) pre-treatment. FNG = female, nicotine (0.75 mg/kg, s.c.) conditioning group, guanfacine pre-treatment. Lo-Pre = initial non-preference for future drug-chamber, Hi-Pre = initial preference for future drug-chamber.