Figure 1.

Effect of methyllycaconitine (MLA) on the stages of heroin conditioned place preference (CPP) in male Wistar rats. (a) Acquisition: rats were tested for innate preference (habituation), and then pseudo-randomly assigned to two groups with comparable mean preference scores. The MLA treatment group received MLA (◻, 4 mg/kg, s.c.) 20 min prior to a conditioning dose of heroin (1 mg/kg, s.c.), paired with the drug-paired compartment, and prior to saline, in the unpaired compartment, alternating over four consecutive days. The control group received saline (○, 1 mL/kg, s.c.) instead of MLA. A post-conditioning preference test was conducted the day after the last conditioning session (test day 9), in which rats had free access to the CPP apparatus compartments for 15 min. Preference scores indicate the time spent in the heroin-paired compartment in seconds minus 450 (half the total time). **p < 0.01, two-way analysis of variance (ANOVA) with Bonferroni post-hoc test, n = 12 per treatment group. (b) Expression: 12 rats (◊, ○, □) were conditioned to acquire heroin CPP as in (a) and were tested for preference for the drug paired side (test day 9). Rats marked ◊ were sacrificed prior to further testing. Rats marked ○ and □ in the habituation and test day 9 were subsequently administered either saline (•) or MLA (■) in the expression test (test day 12). Three days after the post conditioning test, rats were pseudo-randomly assigned to a treatment group and given either saline (•) or MLA (◻, 4 mg/kg, s.c.) 20 min prior to an additional 15-minute preference test (test day 12). *p < 0.05, habituation vs test day 9, n = 12 (paired t-test); n = 4 per treatment group for expression (test day 12; paired t-test). (c) Reinstatement: left: rats were tested for initial preference (◊, habituation), then conditioned to acquire heroin CPP as in (a), followed by 9 days of extinction (saline injections only, paired with alternate compartments on different days). All rats acquired heroin CPP (♦, test day 9) and showed no significant preference in the post-extinction test (∆, test day 23, *p < 0.05, two-way ANOVA with Bonferroni post-hoc analysis vs habituation, n = 37). Right: on the reinstatement test day 26, rats were assigned to one of four treatment groups with comparable mean preference scores from habituation (◊), post-conditioning (♦, test day 9) and post-extinction (∆, test day 23): saline control (○, Saline+Saline, n = 10), heroin reinstatement (•, Saline+Heroin, n = 8), MLA control (□, MLA+Saline, n = 9) or MLA reinstatement (◻, MLA+Heroin, n = 10), where MLA (4 mg/kg, s.c.) or saline was administered 20 min prior to a priming dose of heroin (1 mg/kg, s.c.) or saline (1 mL/kg, s.c.). Rats were then placed free-roaming in an extended preference test (30 min); data from the second 15 min bin are presented. ***p < 0.005, two-way ANOVA with Bonferroni post-hoc analysis vs heroin reinstatement. Data are expressed as mean ± standard error of the mean (SEM).