This animal study (n=71) investigated the effects of ayahuasca (35.2 µg DMT) on alcohol withdrawal in mice and found that it exerted an anxiolytic effect and attenuated the behavioral sensitization to alcohol. Furthermore, it prevented alcohol-induced changes on 5-HT1a receptor and prodynorphin levels in the hippocampus and reduced ethanol effects in the dynorphin/prodynorphin in the striatum.

Abstract

“Introduction: Alcohol use disorder needs more effective treatments because relapse rates remain high. Psychedelics, such as ayahuasca, have been used to treat substance use disorders. Our study aimed to evaluate the effects of ayahuasca on ethanol-induced behavioral sensitization (EIBS).

Methods: Swiss mice received 2.2 g/kg ethanol or saline IP injections every other day across nine days (D1, D3, D5, D7, and D9), and locomotor activity was evaluated 10 min after each injection. Then, animals were treated daily with ayahuasca (corresponding to 1.76 mg/kg of N,N-dimethyltryptamine, DMT) or water by oral gavage for eight consecutive days. On the seventh day, mice were evaluated in the elevated plus maze. Then, mice were challenged with a single dose of ethanol to measure their locomotor activity. Dopamine receptors, serotonin receptors, dynorphin, and prodynorphin levels were quantified in the striatum and hippocampus by blot analysis.

Results: Repeated ethanol administration resulted in EIBS. However, those animals treated with ayahuasca had an attenuated EIBS. Moreover, ayahuasca reduced the anxiogenic response to ethanol withdrawal and prevented the ethanol-induced changes on 5-HT1a receptor and prodynorphin levels in the hippocampus and reduced ethanol effects in the dynorphin/prodynorphin ratio levels in the striatum.

Discussion: These results suggest a potential application of ayahuasca to modulate the neuroplastic changes induced by ethanol.”

Authors: Carolina Aparecida Faria Almeida, Antonio Alves Pereira Junior, Jéssica Gonçalves Rangel, Bruna Pinheiro Pereira, Karla Cristinne Mancini Costa, Vitor Bruno, Gabriela Oliveira Silveira, Carla Speroni Ceron, Mauricio Yonamine, Rosana Camarini, Raphael Caio Tamborelli Garcia, Tania Marcourakis & Larissa Helena Torres.

Summary

Ayahuasca has been used to treat substance use disorders, such as alcohol use disorder. This study aimed to evaluate the effects of ayahuasca on ethanol-induced behavioral sensitization, and found that ayahuasca reduced the anxiogenic response to ethanol withdrawal and prevented the ethanol-induced changes on 5-HT1a receptor and prodynorphin levels in the hippocampus.

Introduction

Ethanol abuse is associated with high morbidity and mortality, and is responsible for 3 million deaths per year worldwide. Alcohol use disorder is most prevalent in the Americas and Europe.

Ethanol withdrawal symptoms may lead to anxiety, depression, and irritability, which can be treated with psychosocial and pharmacological therapies. Relapse occurs in more than half of the patients with AUD.

Ayahuasca is a psychoactive beverage prepared by decoction of leaves of Psychotria viridis and Banisteriopsis caapi vines. It is used in religious rituals and has been shown to have beneficial effects in the treatment of depression, anxiety, and addiction.

DMT and THH are partial agonists at 5-HT1a, 5-HT1b, 5-HT2a, and 5-HT2c receptors, whereas ethanol interferes with the dopaminergic, serotonergic, and opioid systems, leading to increased serotonin receptor expression and increased levels of dynorphin.

The ethanol-induced behavioral sensitization (EIBS) is a phenomenon associated with neuroadaptation induced by ethanol and can be measured by the enhancement of locomotor activity following chronic or intermittent ethanol exposure. AYA can modulate dopamine, serotonin, dynorphin and prodynorphin levels.

2.1. Animals

72 adult male Swiss mice were housed in polypropylene cages in a room with constant temperature and a 12:12 h light/dark cycle. They were used for behavioral experiments between 7:30 am and 12 pm.

2.3.1. UPLC-ESI-MS/MS analysis

Analyses were performed using a Waters UPLC Acquity System coupled to a Quattro Premier tandem MS (Mass Spectrometer) with electrospray ionization (ESI) operated in positive ion mode. The m/z transitions used for quantification are indicated with an asterisk.

2.3.2. Sample preparation

Sample preparation consisted of diluting the samples in ammonium formate buffer with formic acid to a final ratio of 1:5000 in three steps.

2.4.1. Apparatus

The locomotor activity of each animal was measured in the open-field apparatus, and the elevated plus maze. The grooming and head dipping behaviors were analyzed manually, and the results were based on visual monitoring and manual scoring of each episode.

2.4.2. Behavioral experiments

Mice were habituated in the open field for 3 days, then tested for ethanol sensitization. The effect of AYA on ethanol sensitization was evaluated by 3 phases.

Mice were treated with AYA or filtered water by gavage for 30 min, and then placed in the open field for 10 min. They were then tested for ethanol-induced sensitization expression by placing them in the elevated plus maze for 5 min.

During the experiment, animals received saline, water, ethanol or aYA. The ethanol group received ethanol during the acquisition, water during withdrawal phase and treatment, and ethanol during the challenge.

2.6. Western blot

The protocol was based on the one described by Torres et al. . The striatum and hippocampus were dissected and the homogenates were prepared in ice-cold buffer containing 50 mM Tris HCl (pH 7.4), 1.0 mM PMFS, and 10 g/mL protease inhibitor cocktail. All membranes were washed with Tris-buffered saline containing 0.1 % Tween 20, incubated with a peroxidase-conjugated secondary antibody for 2 h at room temperature, and then quantified using ImageQuant LAS 500.

2.7. Statistical analysis

The behavioral sensitization protocol was analyzed by repeated measures of analysis of variance followed by Bonferroni post-hoc test, and the elevated plus maze test was analyzed by factorial ANOVA followed by Bonferroni post-hoc test.

3.2. Effect of AYA on EIBS

During habituation, mice showed no significant differences in locomotor activity. However, during the acquisition phase, mice from the ethanol group showed increased locomotor activity when compared with saline group on all days.

3.3. Effect of AYA on elevated plus maze test

A study showed that the time spent in the open arms was significantly different between the groups during the acquisition phase of EIBS and the withdrawal phase. The animals from the ET-AYA-ET group spent more time in the open arms when compared with the other groups.

The frequency of grooming was increased in the ET-SAL-ET group compared to the other groups, and there was no significant difference on head dipping measures among groups.

3.5. Western blot analysis

In the striatum, D1, SALAYA-ET, ET-SAL-ET, and ET-AYA-ET groups had decreased levels of D1, whereas no differences were observed in D2 levels. The dynorphin/prodynorphin ratio was decreased in the SAL-SAL-ET group, and increased in the ET-SAL-ET group.

Discussion

A clinical study found that AYA was effective in treating drug addiction, and that it reduced ethanol-induced behavioral sensitization, anxiolytic effects, and changes in 5-HT1a receptor and prodynorphin levels.

This study was conducted with ayahuasca dose based on the DMT levels, and the dose used was similar to the dose used in clinical studies that evaluated the effect of ayahuasca on depression, anxiety, and panic syndrome.

Stress from repeated saline injections can induce behavioral sensitization to ethanol, and ayahuasca can attenuate this effect. Ayahuasca is also able to reduce the expression of ethanol sensitization in mice after eight days of IP treatment with 100 mg and 300 mg AYA/kg.

Anxiety is a common symptom of the withdrawal phase of addicts. Ayahuasca tea has been shown to decrease anxiety symptoms in rats and mice undergoing ethanol withdrawal, but long-term administration of AYA does not seem to have any effect on memory and anxiety.

AYA treatment did not prevent the decrease of D1 levels in the striatum induced by acute and repeated ethanol administration, suggesting that AYA does not inhibit EIBS expression by changing D1 receptors.

A postmortem study showed that dynorphin levels were elevated in the hippocampus of alcohol-dependent individuals, and that dynorphin was also increased in the amygdala and the prefrontal cortex. Dynorphin may also play a role in memory and motivation.

An eight-day AYA treatment decreased the expression of EIBS and attenuated the effects of ethanol on the opioid system and 5-HT1A.

CRediT authorship contribution statement

Carolina Aparecida Faria Almeida, Antonio Alves Pereira-Junior, Jessica GOnçalves Rangel, Karla Cristinne Mancini Costa, Vitor Bruno, Rosana Camarini, Raphael Caio Tamborelli Garcia, Tania Marcourakis, Larissa Helena Torres, conceptualized, implemented, validated, analyzed, and visualized the project.

Acknowledgments

The authors gratefully acknowledge the support of several organizations, including FAPEMIG, CAPES, CNPq, and the Secretaria Nacional de Cuidados e Prevenç ao aos Drogas – Minist ero da Cidadania.