Fig. 4. Conditioned place preference despite an adverse stimulus. Fish were punished by 3 s removal from the tank each time they entered the treatment-paired side: (A) punished versus unpunished/restricted control fish; (B,D) nicotine-treated and paired control fish; (C,E) ethanol-treated and paired control fish. (A) Fish that were punished by removal from the tank for 3 s made significantly fewer returns to the treatment side compared with unpunished/restricted fish (two-sample t-test ^*P<0.01). (B,C), Number of returns made to the drug-paired side in the face of restriction or punishment. Data were subject to two-way repeat-measures ANOVA analysis followed by post-hoc, paired or two-sample, t-test, as appropriate, followed by Bonferroni adjustment. Following Bonferroni adjustment comparisons were significant at the P<0.01 level. (B) Fish that had been conditioned for 4 weeks with 30 µmol l^–1 nicotine made more returns to the drug-paired side than control fish when either restricted (two-sample t-test, P=0.03) or punished (two-sample t-test, ^*P<0.01). 3 s removal from the tank caused a significant reduction in returns made by control fish (paired t-test, restricted compared with punished, ^**P<0.01) but not nicotine-treated fish. Repeat-measures two-way ANOVA analysis showed there to be a significant interaction between drug treatment and punishment (punishment plus drug interaction F_1,34=8.74, P=0.006). (C) 3 s removal from the tank caused a significant reduction (paired t-test, restricted compared with punished, ^*P<0.01) in number of returns made by both control fish and fish that had been conditioned for 4 weeks with 175 mmol l^–1 ethanol. Fish that had been conditioned for 4 weeks with 175 mmol l^–1 ethanol made significantly more returns to the drug-paired side when punished (two-sample t-test ^**P<0.01) but not restricted. Repeat measures two way ANOVA analysis showed there to be a significant interaction between drug treatment and punishment (punishment plus drug interaction F_1,34=7.24, P=0.011). (D,E) Significantly increased drug seeking despite punishment persisted over 21 days of abstinence (two-sample t-test, ^*P<0.05 drug-treated compared with control).