Role of 5-HT2A receptors in the effects of ayahuasca on ethanol self-administration using a two-bottle choice paradigm in male mice

This rodent study (2022) assessed the effects of ayahuasca on the expression of ethanol self-administration using a two-bottle choice test. Treatment with ayahuasca blocked the expression of ethanol self-administration, decreasing ethanol intake and preference during re-exposure tests. Pretreatment with the 5-HT_2A receptor antagonist M100907 blocked the effects of ayahuasca on ethanol drinking without significantly attenuating ethanol self-administration.

Abstract

“Rationale: Ayahuasca has been proposed as a potential treatment of alcohol (ethanol) use disorder (AUD). The serotonin 5-HT_2A receptor agonist N,N-dimethyltryptamine (DMT) is the main psychoactive component of ayahuasca, suggesting that its therapeutic effects may be mediated by 5-HT_2A receptors.

Objectives: The aim of the present study was to investigate the effects of ayahuasca on the expression of ethanol self-administration using a two-bottle choice procedure and the role of 5-HT_2A receptors in those effects.

Methods: Male mice had intermittent access to ethanol (10% v/v) in a two-bottle choice procedure for 30 days. Animals were then submitted to 3 treatment phases, each followed by ethanol re-exposure tests. During the treatment phase, every 3 days, animals received i.p. injections of either vehicle or the 5-HT_2A receptor antagonist M100907 (M100, 1 mg/kg) followed by an i.g. (gavage) administration of vehicle or ayahuasca (100 mg/kg) and were exposed to the self-administration apparatus with no ethanol availability. During re-exposure tests, animals were submitted to the same conditions as during acquisition, with no treatments prior to those sessions.

Results: Treatment with ayahuasca blocked the expression of ethanol self-administration, decreasing ethanol intake and preference during re-exposure tests. Pretreatment with M100 blocked the effects of ayahuasca on ethanol drinking without significantly attenuating ethanol self-administration.

Conclusions: Treatment with ayahuasca during alcohol abstinence blocked the expression of alcohol self-administration in mice, and 5-HT_2A receptor activation is critical for those effects to emerge. Our findings support the potential for ayahuasca and other 5-HT_2A receptor agonists as adjunctive pharmacotherapies for the treatment of AUD.”

Authors: Yasmin A. Serra, Thaisa Barros-Santos, Alexia Anjos-Santos, Natali D. Kisaki, Caio Jovita-Farias, Joao P. C. Leite, Maria C. E. Santana, Joao P. S. A. Coimbra, Nailton M. S. de Jesus, Agnieszka Sulima, Paulo C. R. Barbosa, Elena L. A. Malpezzi-Marinho, Kenner C. Rice, Alexandre J. Oliveira-Lima, Lais F. Berro & Eduardo A. V. Marinho

Summary

Abstract

Male mice were exposed to ethanol in a two-bottle choice procedure for 30 days, and then received ayahuasca or M100907 as a treatment. The ethanol self-administration apparatus was then exposed to the same conditions as during acquisition.

Ayahuasca treatment decreased ethanol intake and preference in mice, and 5-HT2A receptor activation was critical for those effects to emerge.

Introduction

Alcohol use disorder is a chronic, relapsing disease with high relapse rates. Current pharmacotherapies and behavioral treatments may assist patients in reducing ethanol use or facilitating ethanol abstinence, but relapse rates remain high.

Ayahuasca, a hallucinogenic beverage, blocks the abuse-related behavioral effects of ethanol, including behavioral sensitization and reinstatement of conditioned place preference. Ayahuasca also decreases drug use and craving in drug-dependent individuals.

Ayahuasca is a brew frequently prepared from the decoction of plants Banisteriopsis caapi and Psychotria viridis. It contains DMT, which is a serotonin 5HT2A/2C receptor agonist with higher affinity for 5-HT2A receptors.

In this study, we used a two-bottle choice procedure to investigate the effects of ayahuasca on ethanol self-administration in male mice. We also investigated the role of 5-HT2A receptors in the therapeutic effects of ayahuasca.

Animals

Three-month-old Swiss male mice were used, and they were group-housed for most of the study, except for self-administration sessions, during which they were single-housed for 15 h every other day.

Drugs and compounds

Ayahuasca was obtained from a member of the Santo Daime Church and lyophilized to obtain concentrations of its main constituents. The 5-HT2A receptor antagonist R( +)-MDL100,907 HCl was synthesized at the National Institutes of Health. Ayahuasca (100 mg/kg, 10 mg/ml) and M100 (1 mg/kg, 0.1 mg/ml) were diluted in saline (0.9%) and administered intragastrically and intraperitoneally, respectively. Ethanol (95%, Merck®) was made available for drinking and diluted in water at a 10% volume solution.

Two‑bottle choice ethanol self‑administration and experimental design

Mice were given 10% ethanol intermittently in 3 phases: acquisition, treatment, and re-exposure. Food was available ad libitum during all sessions.

Acquisition

90 animals were housed in polypropylene boxes for 15 h each odd day for 15 days, and were given access to two bottles containing 10 mL of 10% ethanol solution. The animals’ ethanol intake was measured every other day.

Treatment and re‑exposure

Animals were subjected to 3 treatment periods, each followed by 3 ethanol re-exposure sessions. The treatment periods were 9 days long, with 3 treatments every 3 days, and each treatment period was followed by a 6-day ethanol re-exposure phase.

Animals were weighed daily and water and ethanol consumption was measured at the end of each session. Liquid loss was less than 0.1 mL/day and ethanol consumption was calculated in g/kg for each session using the following formula.

Statistical analysis

All variables were checked for normality and homogeneity of variances, and ANOVAs were performed with or without repeated measures. Multiple comparisons were performed using Tukey’s post hoc tests, and a probability of p 0.05 was considered a statistically significant difference.

Results

Figure 2 shows the total amount of ethanol solution or water solution consumed during each phase of the study. The amount consumed increased with increasing time and decreased with increasing ayahuasca treatment. During the last week of the acquisition phase, all 4 groups showed significantly higher intake of ethanol solution compared to water. During the first re-exposure test, all 4 groups showed significantly higher intake of water solution compared to ethanol. The control group continued to show a significantly higher ethanol solution consumption compared to water during the second and third re-exposure tests, while the group treated with ayahuasca showed a significantly lower ethanol solution consumption compared to the control group.

A mixed-measures ANOVA of the total ethanol intake in grams per kilogram across the experimental phases showed only a significant effect of time. Ayahuasca treatment significantly reduced ethanol intake compared to the control group during the second and third re-exposure tests.

The preference for the ethanol bottle across the experimental phases was significantly affected by time, ayahuasca treatment, and time vs ayahuasca vs M100 treatments. The ayahuasca-treated group showed a significantly lower ethanol preference compared to itself during the last acquisition period. Ayahuasca treatment resulted in a significantly lower preference for ethanol compared to control group and M100 treatment compared to M100-Veh and M100-Aya, during all re-exposure tests.

Only a significant main effect of time was observed for total water consumption and weight across the experimental protocol.

Discussion

Ayahuasca use in naturalistic settings is associated with lower self-reported current consumption of ethanol, even after adjusting for religious or social group effects. Pre-clinical studies have shown that ayahuasca may reduce ethanol intake and ethanol choice during re-exposure to ethanol.

Ayahuasca inhibits ethanol-induced behavioral sensitization and conditioned place preference in mice at doses that do not affect locomotor activity. However, ayahuasca also induces rewarding effects per se in the conditioned place preference model. Ayahuasca treatment increased water intake during re-exposure tests compared to control group, and this increase was accompanied by a decrease in ethanol solution consumption. However, ayahuasca did not affect water intake during the first re-exposure test.

Our results contradict recent results by Nolli et al. (2020) showing that ayahuasca did not block ethanol intake in a two-bottle choice procedure in rats. The main difference in the two protocols was the treatment administration, and the main hypothesis was that ayahuasca may modify the incentive salience of drug-associated environments.

DMT, a serotonin 5HT2A/2C receptor agonist, is the main psychoactive component of ayahuasca, and it is believed to mediate the therapeutic effects of ayahuasca on ethanol self-administration. However, pretreatment with a 5-HT2A receptor antagonist before ayahuasca administration prevented ayahuasca from decreasing ethanol intake and preference.

Ayahuasca may affect the 5-HT2A/2C receptor occupancy ratio in the VTA, which may contribute to its effects on ethanol self-administration. Additionally, the extract of Banisteriopsis caapi alone may also block ethanol-induced conditioned place preference in mice.

A 5-HT2A receptor antagonist (M100-Veh) attenuated ethanol intake and preference in rats, but did not significantly decrease ethanol intake, emphasizing that other mechanisms also are involved in the reinforcing effects of ethanol.

Ayahuasca treatment during ethanol abstinence blocked the expression of ethanol self-administration in male mice, and 5-HT2A receptor activation was critical for those effects to emerge. The dose of ayahuasca used in the study was 100 mg/kg, and further studies are warranted.