Ibogaine Blocks Cue- and Drug-Induced Reinstatement of Conditioned Place Preference to Ethanol in Male Mice

Alcohol use disorder (AUD) remains a major global public health problem and current pharmacotherapies are only partially effective, with a minority of affected individuals seeking treatment. Psychedelic compounds have been proposed as potential treatments for substance use disorders, and previous clinical and preclinical work has suggested benefits of agents such as LSD, psilocybin and ayahuasca for alcohol-related outcomes. Ibogaine, an alkaloid extracted from Tabernanthe iboga, has shown promise in reducing ethanol self-administration in rodents and in retrospective reports from people with substance use disorders, but its effects on ethanol-induced conditioned place preference (CPP) have not been directly tested. Henriques and colleagues designed the present study to determine whether repeated oral ibogaine treatment alters the reinstatement of ethanol-induced CPP in male mice. Two modes of reinstatement were tested: a priming injection reinstatement in which animals received an ethanol injection and had free access to the CPP apparatus, and a context-paired re-exposure reinstatement in which animals were confined to the ethanol-paired compartment after an ethanol injection and then tested drug-free 24 hours later. The investigators also examined whether ibogaine alone produces CPP at the doses tested, to establish whether therapeutic doses have intrinsic rewarding effects.

Male Swiss mice aged approximately three months and weighing 35–40 g were used. Animals were group housed under controlled temperature and a 12 h light/dark cycle, with food and water ad libitum. All procedures were approved by the institutional animal care committee. Ethanol (absolute ethanol diluted in water) was administered intraperitoneally at 1.8 g/kg (10 ml/kg). Ibogaine (12-Methoxybogainamide) was delivered orally (gavage) at 10 or 30 mg/kg, diluted in water with 50 μL Tween 10 as vehicle; vehicle (Veh) controls received the same vehicle solution. The conditioned place preference (CPP) apparatus comprised two distinct conditioning compartments connected by a central choice compartment. CPP score was defined as time spent in the drug-paired compartment minus time in the saline-paired compartment; total distance travelled was also recorded. The behavioural protocol included habituation (Days 1–2), a pre-conditioning test (Day 3), an 8-day conditioning phase (Days 4–11) with alternating saline and drug sessions, a post-conditioning test (Day 12), an 8-day treatment phase (Days 13–20) during which animals received either oral ibogaine or vehicle paired with the ethanol compartment and saline paired with the opposite compartment, and a post-treatment (extinction) test (Day 21). Reinstatement procedures differed by subgroup: for priming reinstatement animals received an i.p. ethanol injection (1.8 g/kg) and were tested with free access to the apparatus 5 min later; for context-paired re-exposure animals received an i.p. ethanol injection and were confined to the ethanol-paired compartment for 10 min, with a drug-free test 24 h later. Two experiments were reported. Experiment 1 tested whether ibogaine (10 or 30 mg/kg, p.o.) or ethanol (1.8 g/kg, i.p.) produces CPP (group sizes reported as n = 8 per group). Experiment 2 evaluated the effects of ibogaine treatment on reinstatement of ethanol-induced CPP; group sizes for treatment groups are reported in the extracted text as n = 24 per group for several groups, and subgroups for the reinstatement tests are reported as n = 16 per group for the priming-reinstatement cohort and n = 8 per group for the context-reexposure cohort. The extracted text contains inconsistencies about total sample size across groups, so the exact overall N per experiment cannot be unambiguously reconstructed from the provided material. Statistical analysis involved checks for normality (Shapiro–Wilk) and homogeneity of variances (Levene's test), followed by one-way or two-way analysis of variance (ANOVA) with repeated measures where appropriate. Bonferroni post-hoc tests were used for multiple comparisons and a p value < 0.05 was considered statistically significant. CPP score was the primary outcome; distance travelled was analysed as a control for locomotor confounds.

Experiment 1: CPP induction. Two-way repeated-measures ANOVA revealed a significant interaction between treatment (saline, ethanol, ibogaine) and time (pre- vs post-conditioning) for CPP score [interaction reported F(3,28) = 6.564; p = 0.0017]. Post-hoc testing showed that mice conditioned with ethanol displayed a significant increase in CPP score relative to their pre-conditioning test (p = 0.0061) and compared with the saline (SAL) group at post-conditioning (p = 0.0009). Mice conditioned with ibogaine (10 or 30 mg/kg) did not show a significant CPP relative to pre-conditioning or to the SAL group, and had significantly lower CPP scores than the ethanol group at post-conditioning (IBO10: p = 0.0038; IBO30: p = 0.0019). Total distance travelled did not differ by treatment or time in the pre- vs post-conditioning tests, indicating no locomotor confound in these comparisons. Experiment 2: extinction and reinstatement. Analysis of pre- vs post-conditioning again showed a significant interaction between treatment and time (reported p = 0.0013), and all ethanol-conditioned groups (EtOH-VEH, EtOH-IBO10, EtOH-IBO30) demonstrated robust acquisition of CPP (pre vs post p < 0.0001 for each group and post-conditioning vs SAL p < 0.001 for each group). After the 8-day treatment phase, a post-treatment (drug-free) test indicated no significant differences among groups (one-way ANOVA F(3,92) = 0.6296; p = 0.5977), consistent with extinction of place preference following treatment. For priming injection reinstatement (subgroup reported as n = 16 per group), one-way ANOVA indicated a significant group effect (reported p = 0.0005). Re-exposure to ethanol reinstated CPP in the VEH-EtOH group relative to SAL-SAL (p = 0.012). By contrast, treatment with ibogaine at 10 and 30 mg/kg blocked ethanol-induced reinstatement: EtOH-IBO10 and EtOH-IBO30 did not differ from the SAL-SAL group and showed significantly lower CPP scores than the VEH-EtOH group (IBO10: p = 0.017; IBO30: p = 0.0004). For context-paired re-exposure reinstatement (drug-free test 24 h after ethanol confined to the ethanol-paired compartment), one-way ANOVA again showed a significant group effect [F(3,26) = 5.339; p = 0.0053]. VEH-EtOH exhibited reinstatement relative to SAL-SAL (p = 0.0078). Both ibogaine doses (10 and 30 mg/kg) prevented this ethanol-and-context-induced reinstatement: EtOH-IBO10 and EtOH-IBO30 did not differ from SAL-SAL and were reduced relative to VEH-EtOH (IBO10: p = 0.02; IBO30: p = 0.04). Across the experiment, measures of distance travelled showed no significant differences in any of the analysed phases (pre vs post, post-treatment, priming reinstatement test, or post-context re-exposure test), arguing against changes in general locomotion as an explanation for the CPP effects.

Henriques and colleagues interpret these results as the first demonstration that repeated oral ibogaine treatment blocks both drug-primed and context-cued reinstatement of ethanol-induced conditioned place preference in mice, at doses that do not produce CPP on their own. The investigators highlight that the anti-reinstatement effects were evident 24 hours or more after the final ibogaine treatment and were seen in both a reinstatement test conducted with ethanol on board (priming) and a drug-free test following ethanol re-exposure in the ethanol-paired compartment. To account for the observed effects, the authors discuss ibogaine's complex pharmacology. They note ibogaine acts at multiple targets implicated in ethanol reward and cue-related reinstatement: it has agonist activity at 5-HT2 receptors with higher affinity for 5-HT2C than 5-HT2A, agonist activity at κ-opioid receptors, and antagonist activity at NMDA receptors. Activation of 5-HT2C receptors on ventral tegmental area GABA interneurons can reduce dopaminergic neuron firing and nucleus accumbens dopamine levels, which may blunt ethanol reward. κ-Opioid receptor agonism could reduce the incentive salience of ethanol-predictive cues, while NMDA receptor antagonism has been shown to disrupt the attribution of incentive salience to reward-paired stimuli and to block expression of ethanol CPP. The authors further suggest that ibogaine's reported facilitation of memory retrieval might interact with these pharmacological effects to alter conditioned memory strength and cue salience, thereby preventing reinstatement. The study team situates the findings within prior work showing that ibogaine and related compounds reduce ethanol self-administration in rodents and are associated with reduced withdrawal and craving in retrospective human reports. They acknowledge that ibogaine's therapeutic actions likely reflect a combined activity across multiple neurotransmitter systems and that further studies are needed to identify the precise receptor subtypes and mechanisms responsible for the anti-reinstatement effects. Finally, the investigators note the clinical relevance of their findings, recommending controlled clinical trials to evaluate the safety and efficacy of psychoactive substances such as ibogaine for treatment of AUD, while recognising that further preclinical mechanistic work is required.