Figure 6. Restraint stress or intra-VTA microinjection of orexin A reinstated extinguished cocaine-CPP in WT, but not in Cnr1 KO mice or in mice pretreated with an OX1R or CB1R antagonist.

(a) The protocol for inducing, extinguishing, and reinstating cocaine-CPP. Four stages were analysed: preconditioning¹, conditioning³⁵, extinction and reinstatement. Mice received daily pairing of cocaine (i.p. 20 mg kg⁻¹) on days 2–4 to develop cocaine CPP, followed by a 3-day forced extinction training by daily paring of saline with the cocaine-paired chamber on days 6–8. (b–e) On day 10, (b–d) a 30-min restraint stress or (e) bilateral intra-VTA microinjection (i.vta.) of 0.1 nmol orexin A for 30 min was applied before the CPP test to reinstate extinguished cocaine CPP. The CPP test was conducted before (day 1, PRE) and after (day 5, CON) cocaine-conditioning, after extinction (day 9, extinction) and after restraint stress/i.vta. orexin A (day 10, reinstatement). Antagonists were given 30 min before restraint stress by either i.p. injection or bilateral i.vta. microinjection. The degree of cocaine seeking was determined by the CPP score as described in Methods section. (b–d) CPP scores at various stages (b) in WT (n=10, black bars), Cnr1^+/− (n=10, grey bars) or Cnr1^−/− (n=8, open bars) mice, or in mice (c) i.p. or (d) i.vta. pretreated with vehicle (i.p.: n=7, i.vta: n=6, black bars), SB-334867 (i.p.: n=9, i.vta: n=6, left slashed bars), AM 251 (i.p.: n=7, i.vta: n=6, grey bars) or THL (n=6, right slashed bars) before receiving restraint stress. (e) CPP scores at various stages in mice, after extinction, i.vta. microinjection of orexin A (n=7, black bars), saline (n=7, open bars), orexin A+AM 251 (n=4, grey bars) or orexin A+vehicle (n=4, right slashed grey bars). *P<0.05, **P<0.01, ***P<0.01 versus the PRE stage ; ^#P<0.05, ^##P<0.01, ^###P<0.001 versus (b–d) the WT, or the vehicle or (e) the saline group (two-way ANOVA with Bonferroni post hoc test). Data are expressed as mean±s.e.m.