Ayahuasca and Its DMT- and β-carbolines - Containing Ingredients Block the Expression of Ethanol-Induced Conditioned Place Preference in Mice: Role of the Treatment Environment

Alcohol (ethanol, Eth) use disorder remains a major global health problem and current treatments are only partially effective, motivating investigation of new approaches. Earlier clinical and observational work has suggested that ayahuasca (Aya), a brew made from Banisteriopsis caapi (Bc) and Psychotria viridis (Pv) that contains β-carbolines and N,N-dimethyltryptamine (DMT), may reduce substance use and craving, but it is unclear whether those effects reflect pharmacology of the plants themselves or contextual/religious factors associated with ayahuasca use. Ryabinin and colleagues set out to determine whether Aya and its constituent plant extracts have rewarding or anti-rewarding effects in mice, and whether those effects depend on DMT-containing versus non-DMT components. Using the conditioned place preference (CPP) paradigm, they tested (1) whether Aya, an extract of B. caapi (EBc) or an extract of P. viridis (EPv) produce CPP on their own, (2) whether pretreatment with those preparations alters the development of ethanol (Eth)-induced CPP, and (3) whether post-conditioning treatment in either the Eth-paired or saline-paired environment influences the expression of established Eth-induced CPP. The study therefore addresses both pharmacological contributions of DMT versus β-carbolines and the role of treatment context.

Male Swiss mice (three months old, 35–40 g) were group housed and maintained under standard temperature and light–dark conditions. All procedures were approved by the institutional animal care committee. Aya was obtained from a Santo Daime source, lyophilised and analysed by LC-MS/MS alongside lyophilised extracts of B. caapi and P. viridis; quantified constituents included DMT, tetrahydroharmine, harmine and harmaline. Harmine and harmaline standards were purchased and tetrahydroharmine and DMT were synthesised for the assays. Drugs and doses: ethanol (Eth) was given intraperitoneally (i.p.) at 1.8 g/kg (10 ml/kg). Aya and EBc were administered orally by gavage at 10 ml/kg, with Aya dose levels of 30, 100 and 300 mg/kg and EBc doses scaled to reflect harmine content (132, 440 and 1320 mg/kg). EPv (DMT-containing extract) was given i.p. at 3.75, 12.5 and 37.5 mg/kg because oral DMT is inactivated by monoamine oxidase A (MAO-A). Conditioned place preference (CPP) apparatus and protocol: the apparatus comprised two distinct conditioning compartments connected by a neutral choice compartment. An unbiased CPP design was used following habituation and a pre-conditioning test. Conditioning consisted of eight daily sessions across Days 3–10, pairing Aya, EBc, EPv or Eth with one compartment and saline (Veh) with the other; timing between treatment and confinement varied by compound (e.g. 5 min for Eth, 30 min for Aya/EBc, 20 min for EPv). Post-conditioning testing was performed 24 hours after conditioning. For post-conditioning treatment experiments, mice received alternate-day administrations of Veh or treatment in either the Eth-paired or Sal-paired compartment for 8 days (Days 12–19), were reexposed to Eth (Day 20), and were tested drug-free on Day 21. Behavioural sessions were recorded with ANY-maze and CPP score was defined as the difference in time spent in the drug-paired minus non-drug-paired compartments. Experimental structure: separate cohorts (n = 8 per group) were used across multiple experiments: Experiment 1 tested whether Aya, EBc or EPv induce CPP; Experiment 2 tested whether pretreatment with these compounds blocks development of Eth-induced CPP; Experiments 3–5 tested the influence of treatment environment (Eth-paired vs Sal-paired compartment) on the ability of Aya, EBc or EPv to alter expression of established Eth-induced CPP. Statistical analysis: data were checked for normality and homogeneity (Shapiro–Wilk, Levene). Within-group comparisons used paired t-tests; CPP scores were analysed with Student’s t-test where appropriate and with one- or two-way ANOVA (repeated measures when relevant) followed by Bonferroni post hoc tests. A two-tailed p < 0.05 threshold defined statistical significance. Reported group sizes were typically n = 8.

Experiment 1 — CPP induced by Aya, EBc or EPv alone: Pre-conditioning tests showed no initial compartment preference across groups. For Aya, two-way repeated-measures ANOVA indicated a significant interaction between time (pre- vs post-conditioning) and dose [F(2,21) = 5.99, p < 0.01]; Bonferroni post hoc tests revealed that the 100 mg/kg (intermediate) dose induced a significant increase in CPP score in the post-conditioning test relative to pre-conditioning, whereas 30 and 300 mg/kg did not. EBc did not induce CPP at any tested dose: ANOVA showed no significant effects of time, dose or interaction. For EPv, ANOVA showed a main effect of dose [F(2,21) = 3.77, p < 0.05] but no time × dose interaction and post hoc testing found no significant pre–post differences, so EPv did not produce clear CPP under the conditions used. Experiment 2 — Effects of pretreatment on development of Eth-induced CPP: All groups showed no pre-conditioning compartment bias. In the ethanol conditioning paradigm, vehicle-pretreated mice developed robust Eth-induced CPP (increase in post-conditioning CPP score). Pretreatment with Aya (both 100 and 300 mg/kg) blocked the development of Eth-induced CPP: two-way RM ANOVA showed an interaction between time and Aya treatment [F(2,21) = 3.12, p < 0.05], and Bonferroni post hoc tests indicated an absence of the Eth-induced increase in CPP score in Aya-pretreated groups. By contrast, EBc pretreatment did not prevent Eth-induced CPP: two-way RM ANOVA revealed a strong effect of time [F(1,21) = 26.18, p < 0.0001] but no EBc treatment or interaction effect. Similarly, EPv pretreatment did not alter the development of Eth-induced CPP; vehicle controls but not EPv groups showed post-conditioning increases, indicating EPv pretreatment was ineffective at blocking development. Experiments 3–5 — Role of treatment environment on expression of established Eth CPP: After Eth conditioning and a treatment phase (alternate-day administrations in either the Eth-paired or Sal-paired compartment), mice were reexposed to Eth and tested drug-free. For Aya, a combined analysis of animals treated in Eth- versus Sal-paired compartments showed a significant interaction between treatment environment and Aya treatment [F(2,42) = 4.15, p < 0.05]. Bonferroni post hoc testing indicated that 300 mg/kg Aya administered in the Eth-paired environment produced lower CPP scores in the post-treatment test than the same dose administered in the Sal-paired environment; no significant differences were observed for vehicle or 100 mg/kg Aya groups. The authors report that treatment with Aya in the Eth-paired compartment blocked expression of Eth-induced CPP for both doses tested, whereas in the Sal-paired compartment only the intermediate dose blocked expression. For EBc, the extracted results are less fully reported in the methods/results text, but the Discussion states that a high EBc dose administered in the Eth-paired environment blocked expression of Eth CPP, and that an intermediate EBc dose administered in the Sal-paired compartment also blocked expression. For EPv, post-treatment analyses showed that both 12.5 and 37.5 mg/kg EPv produced significant decreases in CPP score compared to vehicle in the post-treatment test when delivered in the Eth-paired compartment [one-way ANOVA F(2,21) = 4.55, p < 0.05]. EPv delivered in the Sal-paired compartment likewise reduced expression of Eth CPP [one-way ANOVA F(2,21) = 6.3, p < 0.01]. A combined analysis of Experiments 5a and 5b found a significant main effect of EPv treatment [F(2,42) = 10.66, p < 0.001], with no significant effect of treatment environment or interaction, indicating that EPv reduced expression of Eth CPP regardless of whether it was administered in the Eth-paired or Sal-paired compartment. Locomotor activity: the authors report that locomotor measures increased in the Eth-paired compartment after conditioning but that during the post-treatment test there were no significant between-group differences in locomotor activity scores. They therefore argue that changes in locomotion do not account for the observed CPP effects.

Ryabinin and colleagues interpret their findings as showing that ayahuasca has dose-dependent rewarding properties and can modulate ethanol reward, and that both plant constituents contribute to modulation of established ethanol reward in a context-dependent manner. Specifically, an intermediate dose of Aya produced CPP on its own, while a high dose did not, suggesting a non-linear dose–response. Pretreatment with Aya blocked the development of ethanol-induced CPP, whereas EBc and EPv alone did not, implying that the full ayahuasca formulation (both DMT and β-carbolines together) is required to prevent development of ethanol reward. In contrast, chronic treatment after ethanol conditioning reduced the expression of established ethanol CPP for Aya, EBc and EPv when administered in the ethanol-paired environment; when administered in a saline-paired (neutral) environment, intermediate—but not high—doses of Aya and EBc, and both doses of EPv, blocked expression. The authors propose a pharmacological mechanism involving serotonin 5-HT2A and 5-HT2C receptors. They note that DMT is a 5-HT2A/2C agonist with greater affinity for 5-HT2A, while β-carbolines (e.g. harmine, tetrahydroharmine) interact with serotonin systems, including reuptake inhibition and receptor binding. The hypothesis is that lower to intermediate DMT exposure preferentially activates 5-HT2A receptors to elevate nucleus accumbens dopamine and produce reward, whereas higher DMT doses produce substantial 5-HT2C activation that counteracts 5-HT2A-driven increases in dopamine and prevents CPP. This model may explain why intermediate Aya doses were rewarding but high doses were not, and why i.p. EPv (which delivers more DMT centrally than oral Aya) failed to induce CPP at the doses tested. The authors also acknowledge additional mechanisms for β-carbolines—MAO-A inhibition, DAT inhibition, DYRK1A inhibition and imidazoline I2 binding—that might explain EBc effects on expression of ethanol CPP independent of 5-HT2A/2C receptor actions. Regarding contextual influences, the researchers emphasise that the treatment environment modulated the therapeutic-like effects of Aya and EBc: administration in the ethanol-paired compartment more consistently blocked CPP expression than administration in the saline-paired compartment, suggesting that context (analogous to ritual or clinical setting) can shape outcomes. For EPv, treatment effects were significant regardless of environment. The authors report that locomotor changes do not explain the CPP effects, citing lack of group differences in locomotor activity during post-treatment tests. Limitations acknowledged by the authors include not having directly probed molecular mechanisms and potential differences introduced by route of administration (oral for Aya/EBc, i.p. for EPv). They call for further studies to vary EBc/EPv ratios and administration routes to clarify mechanisms and to define safe, effective therapeutic strategies. The authors suggest that an optimal 5-HT2A/2C receptor occupancy ratio might provide therapeutic efficacy without abuse liability, and they note the clinical and ritual relevance of the interaction between pharmacology and treatment environment.